Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D473/00—Heterocyclic compounds containing purine ring systems
  • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
  • C07D473/18—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 one oxygen and one nitrogen atom, e.g. guanine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D473/00—Heterocyclic compounds containing purine ring systems
  • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
  • C07D473/32—Nitrogen atom
  • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine

Definitions

• Novel compounds and a method of using them to antagonize adenosine receptors are provided wherein the compounds are represented by the general formula:
• R 2 is selected from the group consisting of cycloalkyl radicals having from 3 to 8, preferably 3 to 7, ring carbon atoms, alkyl radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 13, preferably 6 to 10, carbon atoms, aralkyl radicals having from 7 to 14, preferably 7 to 10, carbon atoms, and heteroatom- and halogen-substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of nitrogen, phosphorus, sulfur and oxygen;
• R 1 may be hydrogen or R 2
• R 3 is selected from the group consisting of hydrogen, halogen, amine, carboxy, thio, sufonate, sulfonamide, sulfone, sulfoxamide, phenyl, alkyl-substituted amine, cycloalkyl-substituted amine, alkyl radicals having from 1 to 10 carbon atoms, and cycloalkyl radicals having from 3
• R 4 is selected from the group consisting of benzyl, phenyl, and alkyl groups comprising from 1 to 4 carbon atoms, wherein said alkyl group can be substituted with oxygen, for example ethers and alcohols.
• R 5 is selected from the group consisting of hydrogen; hydroxy; sulfonate; halogen; alkoxy and cycloalkoxy groups comprising 1 to 6 carbon atoms, wherein said alkoxy and cycloalkoxy groups can be substituted with phenyl; and amine, wherein said amine can be substituted with alkyl, cycloalkyl, or phenyl.
• Adenosine receptors have been divided into two subtypes, based on adenylate cyclase activity: A 1 (R i ) receptors mediate inhibition and A 2 (R a ) receptors mediate stimulation of adenylate cyclase activity.
• N 6 -substituted adenosine analogs like N 6 -R-phenyl isopropyl adenosine (R-PIA) have very high affinity for A 1 adenosine receptors, but at A 2 receptors 5'-N-ethylcarboxamido-adenosine (NECA) is more potent than N 6 -substituted analogs .
• NECA 5'-N-ethylcarboxamido-adenosine
• Alkylxanthines such as caffeine and theophylline, are the best known antagonists at adenosine receptors.
• Adenine was generally believed to have no effect on adenosine receptor-controlled systems. However, it was found that at low concentrations adenine displays specific competitive antagonism of adenosine-induced cyclic Amp accumulation in a human fibroblast cell line. Methylation of adenine at the 9-position increases potency about 4-fold in this assay. At higher concentration, both compounds show non-specific inhibitory activity.
• R 2 is selected from the group consisting, of cycloalkyl radicals having from 3 to 8, preferably 3 to 7, ring carbon atoms, alkyl radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 13, preferably 6 to 10, carbon atoms, aralkyl radicals having from 7 to 14, preferably 7 to 10, carbon atoms, and heteroatom- and halogen-substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of nitrogen, phosphorus, sulfur and oxygen;
• R 1 may be hydrogen or R 2
• R 3 is selected from the group consisting of hydrogen, halogen, amine, carboxy, alkyl radicals having 1 to 10 carbon atoms, cycloalkyl radicals having from 3 to 8, preferably 5 to 6, ring carbon atoms, thio, sulfonate, sulfonamide, sulfon, sulfoxamide, phenyl, alkyl-substitued amine
• R 4 is selected from the group consisting of benzyl, phenyl, and alkyl groups comprising from 1 to 4 carbon atoms, wherein said alkyl group can be substituted with oxygen, for instance ethers and alcohols.
• R 5 is selected from the group consisting of hydrogen; hydroxy; sulfonate; halogen; aljcoxy and cycloalkoxy groups comprising 1 to 6 carbon atoms, wherein said alkoxy and cycloalkoxy groups can be substituted with phenyl; and amine, wherein said amine can be substituted with phenyl and alkyl and cycloalkyl groups comprising 1 to 6 carbon atoms.
• R 1 is hydrogen; wherein R 2 is endo-2-Norbornyl or cyclopentyl; wherein R 3 is bromine, chlorine, amino, hydrogen, thio, cyclopentyl or cyclopentylamine; wherein R 4 is methyl, ethyl, 2-hydroxy- ethyl, phenyl, or 2-hydroxyethoxy methyl; and wherein R 5 is hydrogen, hydroxy or chlorine.
• N 6 -(endo-2-Norbornyl)-8-Azido-9-MA 0.5g, 1.75 mmole
• the solution in presence of 10% palladium on charcoal (1g), was shaken with H 2 at 35 atm overnight.
• the suspension was filtered and evaporated to a small volume, and then poured through a C-18 column (HPLC) to give 0.36g 80% yield of N 6 -(endo-2-norbornyl)-8-Amino-9-MA.
• N 6 (endo-2-Norbornyl)-9-Bromo-9-MA (0.15g, 0.62 mmole) in 12 ml acetic acid was added sodium acetate (0.5g) and 1.2 ml acetic anhydride. The mixture was allowed to reflux overnight. The mixture was then evaporated under vacuo and purified on a chromatotron using CHCl 3 , stepping to 2% ethanol, and finally to 4% ethanol on 2 mm plate giving 90 mg, 75% yield of N 6 -(endo-2-Norbornyl)-8-0xo-9-MA.
• N 6 -(endo-2-Norbornyl)-8-Cyclopentylamine-9-MA To a solution of N 6 -(endo-2-Norbornyl)-8-Bromo-9-MA (0.5g, 1.55 mmols) in 20 ml ethanol was added 20ml of cyclopentylamine; the reaction mixture was refluxed overnight. The mixture was then evaporated under vacuo and passed through a C-18 column (HPLC) to.give 0.32g, 77% yield of N 6 - (endo-2-Norbornyl)-8-Cyclopentylamine-9-MA.
• N 6 (endo-2-Norbornyl)2-Chloropurine was first prepared as follows: A mixture of 2,6-dichloropurine (5.0g, 26.45 mmoles) endo-2-aminobornane hydrochloride (5.0g, 33.86 mmoles) and triethyl amine (10 ml) in absolute ethanol was refluxed for 48 hours. The solution was then cooled to room temperature and evaporated in vacuo to a white solid. The white solid was washed with water and dried to yield 6.0g, 84% yield of N 6 -(endo-2-Norbornyl)2-Chloropurine used as is with no further purification for next step.
• N 6 -(endo-2-Norbornyl)-8-bromo-9-MA (1.25g, 3.7 mmoles) and POC1 3 was refluxed for 1 hour. Then the phosphorous oxychloride was removed in vacuo and the yellow solid was passed through a C-18 column (HPLC) to give 0.96g, 84% yield of N 6 -(endo-2-Norbornyl)-8-chloro-9-MA.
• N 6 -substituted 9-methyladenines were assayed as adenosine antagonists in A 1 and A 2 test systems ( ⁇ kena, et al, FEBS Lett. 215(2), 203-208, 1987).
• compounds were tested as inhibitors of the binding of N 6 -R-[ 3 H]-Phenylisopropyladenosine in rat brain membranes and for their ability to prevent R-PIA-induced inhibition of adenylate cyclase in rat fat cell membranes.
• compounds were tested as antagonists of NECA-stimulated adenylate cyclase in membranes of human platelets and rat PC12 cells.
• a 1 receptors influence inhibition of adenylate cyclase in fat, brain and heart cells; whereas A 2 receptors stimulate adenylate cyclase in endothelial and smooth muscle cells.
• a 2 receptors stimulate adenylate cyclase in endothelial and smooth muscle cells.
• N 6 -Methylcylopentyl-9-MA 45 56 9.0 2.5 6. Cyclohexyl-9-MA 7.4 21 0.65 0.94 7. N 6 -Hethyl -9-MA 150 130 220 > 100 8. N 6 -3-PentyI -9-MA 11 53 7.6 3.3 9. N 6 - PhenyI -9-MA 21 107 10 25
• in vitro assays were conducted utilizing model tissues that are thought to contain homogenous populations of either the A 1 or A 2 adenosine receptors.
• Four examples were characterized by their ability to antagonize competitively the action of adenosine agonists in eliciting two responses: the reduction in force of contraction of guinea pig atrium (A 1 ); and the decrease in the contractile tone of the guinea pig taenia caecum (A 2 ).
• the left atria from male guinea pigs were isolated, suspended between two punctate electrodes, and placed in a 20 ml organ bath that contained Krebs-Hensileit solution that was continuously gassed with 95% O 2 + 5% CO 2 and maintained at 31oC. The resting tension was one gram. The atria were stimulated electrically at 1 Hz, 1 msec duration pulses at supramaximal voltage. The force of contraction was recorded isometrically.
• Taenia from the guinea pig caecum were cut into lengths of 1.5-2 cm.
• the tissues were suspended in a 20 ml organ bath containing de Jalon's solution that was gassed with 95% O 2 + 5% CO 2 and maintained at 31oC.
• the resting tension was 1.5 g.
• the contractile response was measured isotonically. Tissues were contracted with 10 M 5-methyl-furmethide and allowed sufficient time to reach a stable contraction before addition of adenosine agonists.
• 8-PT did antagonize the effects of agonists at low concentrations.
• the lack of competitive antagonism by the other compounds suggests that the latter compounds do not interact appreciably with A 2 -adenosine receptors and are, thus, selective for A 1 adenosine receptors.
• N 6 -3-Pentyl-9-MA, N 6 -Cyclopentyl-£-MA, N 6 -(endo-2-Norbornyl)-9-MA and N 6 -4-(2-thienyl)-3-butyl)-9-MA all were found to be competitive antagonists at adenosine receptors in the atria.
• N 6 -3-Pentyl-9-MA and N 6 -1-(2-thienyl)-2-butyl-9-MA also produced increases in basal force of contraction in the atria.
• Affinity constants (pK B ) for the present compounds determined using known methods are summarized in Table 2 below:
• Rats were anesthetized with urethan and blood pressure was monitored via a carotid cannula. Drug injections were made intravenously through a jugular cannula. Blood pressure, EGC, and heart rate were recorded on a Grass polygraph.
• Adenosine produced a dose dependent decrease in blood pressure and heart rate, with a concommitant increase in the P-R interval of the ECG.
• Administration of N 6 -(endo Norbornyl)-9-methyladenine attenuated the effects of subsequently administered adenosine on all parameters measured.
• adenosine causes heart block; this effect was also substantially reduced by the agonist. Due to the short duration of action and direct route of administration of adenosine, it is often difficult to determine whether adenosine decreased blood pressure by causing peripheral vasodilation or by reducing cardiac output.
• NECA (5'-N-ethylcarboxamide adenosine), which is longer-acting and selective for A 2 adenosine receptors, was used as an adenosine receptor agonist.
• Prior administration of N-0861 attenuated the effects of NECA on the heart while minimally affecting the NECA-induced decrease in blood pressure.
• N 6 -endo-2-Norbornyl)-9-methyladenine is a cardioselective adenosine receptor antagonist in vivo and support the data above showing selectively of the N-6 substituted 9-methyladenines of the invention as A 1 adenosine receptor antagonists.
• bovine brains were obtained fresh from a local slaughterhouse.
• the caudate nuclei were dissected out and homogenized in Buffer A (50 mm Tris; 1 mm Na 2 -EDTA; 5 mm KCl; 1 mm MgCl 2 ; 2 mm CaCl 2 ; pH 7.4) using a Brinkman Polytron.
• the homogenate was centri- fuged at 40,000 ⁇ g for 20 minutes and washed once.
• the pellet was resuspended in Buffer A, incubated at 37oC for 15 minutes, then centrifuged.
• the pellet was washed once more, resuspended to a protein concentration of 5-10 mg/ml in Buffer A and frozen at -70oC until use.
• the A 2 assays also contained 50 nM cyclopentyl-adenosine to block the binding of [ 3 H]-NECA to A 1 receptors (Bruns et al, 1986) and 1 unit/ml adenosine deaminase to degrade endognous adenosines. Varying concentrations of test compounds were incubated with the appropriate radioligand and membrane source for 1 hr at room temperature.
• N 6 -substituted adenines are antagonists of A 2 -adenosine receptor-mediated stimulation of adenylate cyclase in A 2 -adenosine receptors and antagonists of A 1 -adenosine receptor-mediated inhibition of adenylate cyclase.
• These compounds are useful in reversal of adenosine-mediated lipolysis, reversal of adenosine-mediated deleterious cardiovascular effects (conduction defects, hypotension), reversal of adenosine-mediated vascular actions in kidney, bronchodilation, antiarrhythmic action, reversal of adenomediated relaxation of smooth muscle, anti-narcoleptic action, CNS stimulation, and blockade of adenosine mediated inhibition of neurotransmitter release.

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• 1990
  • 1990-01-16 AU AU49414/90A patent/AU626983B2/en not_active Ceased
  • 1990-01-16 KR KR1019900702209A patent/KR910700253A/ko not_active Application Discontinuation
  • 1990-01-16 WO PCT/US1990/000210 patent/WO1990009178A1/en not_active Application Discontinuation
  • 1990-01-16 EP EP19900902050 patent/EP0457773A4/en not_active Withdrawn

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