N<6>-SUBSTITUTED 9-METHYLADENINES: A NEW CLASS OF ADENOSINE RECEPTOR ANTAGONISTS

Abstract: A series of N<6>-substituted adenines are disclosed to be antagonists of A2-adenosine receptor-mediated stimulation of adenylate cyclase in A2-adenosine receptors and antagonists of A1-adenosine receptor-mediated inhibition of adenylate cyclase. These compounds are useful in reversal of adenosine-mediated lipolysis, reversal of aenosine-mediated deleterious cardiovascular effects (conduction defects, hypotension), reversal of adenosine-mediated vascular actions in kidney, bronchodilation, antiarrhythmic action, reversal of adeno-mediated relaxation of smooth muscle, anti-narcoleptic action, CNS stimulation, and blockade of adenosine mediated inhibition of neurotransmitter release.

Description: N 6 -SUBSTITU ED 9-METHYLADENINES: A NEW CLASS OF ADENOSINE RECEPTOR ANTAGONISTS SUMMARY OF THE INVENTION Novel compounds and a method of using them to antagonize adenosine receptors are provided wherein the compounds are represented by the general formula: wherein 2 is selected from the group consisting of cyclo¬ alkyl 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^ may be hydrogen or R 2 , and R 3 is selected from the group consisting of hydrogen, halogen, amine, carboxy, thio, sufonate, sul- fonamide, sulfone, sulfoxamide, phenyl, alkyl-substituted amine, cycloalkyl-substituted amine, alkyl radicals having from 1 to 10 carbon atoms, and cycloalkyl radicals having from 3 to 8, preferably 5 to 6, ring carbon atoms. 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. 5 is selected from the group con¬ sisting 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. BACKGROUND OF THE INVENTION This application is a continuation-in-part of U.S. patent application Serial No. 042,383, filed April 23, 1987 entitled "N 6 -Substituted 9-Methyladenines: A New Class of Adenosine Receptor Antagonists," which is incorporated herein by reference in its entirety. Adenosine receptors have been divided into two subtypes, based on adenylate cyclase activity: τ_ (R^) receptors mediate inhibition and 2 (R a ) receptors mediate stimulation of adenylate cyclase activity. Some N 6 -substituted adeno¬ sine analogs, like N 6 -R-phenyl isopropyl adenosine (R-PIA) have very high affinity for ι_ adenosine receptors, but at A 2 receptors 5'-N-ethylcarboxamido-adenosine (NECA) is more potent than N 6 -substituted analogs , 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. DETAILED DESCRIPTION OF THE INVENTION The compounds of this invention are represented by the general formula: wherein R 2 is selected from the group consisting, of cyclo¬ alkyl 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; 1R^ may be*hydrogen or R 2 , and R 3 is selected from the group consisting of hydrogen, halogen, amine, carboxy, alkyl radicals having l to 10 carbon atoms, cycloalkyl radicals having from 3 to 8, preferably 5 to 6, ring carbon atoms, thio, sulfonate, sulfona ide, sulfon, sulfoxa ide, phenyl, alkyl-sύbstitued amine, and cycloalkyl substituted amine. R4 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 consist¬ ing 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. The preferred compounds are those wherein R^ is hydrogen; wherein R 2 is endo-2-Norbornyl or cyclopentyl; wherein R 3 is bromine, chlorine, amino, hydrogen, thio, cyclopentyl or cyclopentylamine; wherein R4 is methyl, ethyl, 2-hydroxy- ethyl, phenyl, or 2-hydroxyethoxy methyl; and wherein R 5 is hydrogen, hydroxy or chlorine. The following is a list of compounds useful in the practice of the present invention. This list is intended to be illustrative and the scope of the invention is not limited to compounds named therein: N 6 -Cyclobutyl-9-Methyl Adenine (MA) N 6 -Cyclopentyl-9-MA N 6 -Methylcyclopenty1-9-MA N 6 -Cyclohexyl-9-MA N 6 -Methyl-9-MA N 6 -3-Pentyl-9-MA N 6 -Phenyl-9-MA N 6 -2-Fluorophenyl-9-MA N 6 -Benzyl-9-MA N 6 -2-Phenethyl-9-MA N 6 -2-(3, ,5-Trimethoxyphenyl)ethyl-9-MA N 6 -2-(3-Pyridylethyl)-9-MA N 6 -2-(3-Thienylethyl)-9-MA N 6 -E-l-Phenyl-2-propyl-9-MA N 6 -S-l-Phenyl-2-propyl-9-MA N 6 -(endo-2-Norbornyl)-9-MA N 6 -l-(2-Thienyl)-2-butyl-9-MA N 6 -(exo-2-Norbornyl)-9-MA N 6 -2,2-diphenylethyl-9-MA N 6 -2-phenylethyl-9-MA N 6 -2-(2-chlorophenyl)ethyl-9-MA N 6 l-indanyl-9-MA N 6 -2-aminoethyl-9-MA N 6 -(N,N-Dimethylaminoethyl)-9-MA N 6 -R-l-phenyl-l-ethyl-9-MA N 6 -S-l-phenyl-l-ethyl-9-MA N 6 -2-thienyl-9-MA N6-(4-chloro-2-methyl phenyl)-9-MA N 6_ 2-(3-ethylindole)-9-MA N<S- :i-methyl-2-phenylethyl)-9-MA N 6_ [l-methyl-2-phenoxyethyl)-9-MA N 6 -l-carboxy-l-butyl-9-MA N 6. endo-2-norbornyl)-2-σhloro-9-MA N<5- endo-2-norbornyl)-8-cyclopentyl-9-MA N 6_ endo-2-norbornyl)-8-hydroxy-9-MA N6- endo-2-norbornyl)-8-bromo-9-MA N 6_ endo-2-norbornyl)-8-amino-9-MA N6- endo-2-norbornyl)-8-carboxy-9-MA N 6 -cyclopentyl-8-cyclopentyl-9-MA N 6_ endo-2-norbornyl)-9-[ (2-hydroxyethoxy)methyl]adenine N 6_ endo-2-norbornyl)-8-thio-9-MA N 6_ endo-2-norbornyl)-8-chloro-9-MA N 6_ endo-2-norbornyl)-8-sulfonate-9-MA sodium salt N 6_ endo-2-norbornyl)-2-hydroxy-9-MA N 6 - endo-2-norbornyl)-8-cyclopentylamine-Sf-MA N 6_ endo-2-norbornyl)-8-propylamine-9-MA N 6 - endo-2-norbornyl)-9-phenyl adenine N 6 -cyclopentyl-2-chloro-9-MA N 6 -phenyl-2-chloro-9-MA N 6 -cyclopentyl-9-phenyl adenine N 6 -R-l-phenyl-2-propyl-9-phenyl adenine N 6 -S-l-phenyl-2-propyl-9-phenyl adenine N 6 -[ (3-chloro-endo-2-norbornyl) ]-9-MA N 6 -pheny1-9-phenyl adenine 2-ethoxy-9-MA 2-propoxy-9-MA 2-butoxy-9-MA .2-isopropoxy-9-MA 2-(2-butoxy)-9-MA 2-(2-methyl propoxy) 9-MA 2-pentoxy-9-MA 2-(2-phenylethoxy) 9-MA 2-phenylamino-9-MA 9-hydroxyethyladenine N 6 -cyclopenty1-9-benzyl adenine N 6 -cyclohexyl-9-ethyl adenine The preparation of 9-methyl adenines is well known. See R. K. Robins, K. J. Dille, and B. E. Christensen, J. Orσ. Chem.. 19, 930 (1954); R. K. Robins and H. H. Lin, J. Am. Chem. Soc.. 79, 490 (1957; and J. A. Montgomery and Carroll Temple, Jr., J. Am. Chem. Soc.. 79, 5238 (1957). Preparation of N 6 -Cyclopentyl-9-Methyl Adenine To prepare N 6 -cyclopentyl-9-methyl Adenine the following additional steps were taken. A mixture of 6- chloro-9-methyl Adenine (0.82g), cyclopentylamine (0.52 ml), triethylamine (0.53 ml) and ethanol (60 ml), was refluxed for 24 hours. The solution was concentrated in vacuo to a yellow syrup. The syrup was passed through a C-18 column to give 0.78g or 74% yield of with m.p. 108-109°C. 1 HNMR- (Me 2 SO-d6): 5l-2(m,9 H) ; 3.7(S,CH 3 ); 7.6(d,NH); 8.1(S,1H); 8.2(S,1H) . Preparation of N 6 -3-Pentyl-9-Methyladenine A mixture of 6-chloro-9-methyladenine (1.5g), 3-pen- tylamine (1.3 ml), triethylamine (1.3ml) and ethanol (60 ml) , was refluxed for 24 hours. The solution was con¬ centrated and passed through a C-18 column to give a white solid having .p. 107-109 β C. Preparation of N 6 -(2-Aminonorbornyl)-9-methyl Adenine A mixture of 1.5g 6-chloro-9-methyl Adenine, 1-75 g endo- 2-aminonor-bornane, 2.9 ml triethylamine and 60 *ml ethanol was refluxed overnight. The solution was then concentrated in vacuo and the remainder was passed through C-18 prep- chromatography to give 1.6g (75% yield) m.p. 130- f 131°C. 1 HNMR(Me 2 S0-d6) : 51-2.6(m,10 H) ; 3.8(S, CH 3 ) ; 4.1(m,lH); 7.2(S,NH); 7.4(8,1H); 7.6(8,1H). Preparation of N 6 -(endo-2-Norbornyl)-8-Bromo-9-MA * To a stirred suspension of N 6 -(endo-2-norbornyl)-9-MA (6g, 24.66 mmoles) in 150 ml of 1M sodium acetate buffer (pH 3.9) was added a solution of bromine (3.0 ml) in 300 ml of 1M sodium acetate buffer (pH 3.9). The mixture was stirred overnight and the resulting precipitate was filtered and washed with water. To the residue was added silica gel and the suspension was evaporated to a powder. The powder was added to a silica gel column (150g, packed with petroleum ether) . The purine was eluted with 10% to 25% ethylacetate in petroleum ether. Evaporation of the" appropriate fractions gave 6.7g, 84% yield of N 6 -(endo-2-Norbornyl)-8- Bromo-9-MA. Preparation of N 6 -(endo-2-Norbornyl)-8-Azido-9-MA To a solution of N 6 -(endo-2-Norbornyl)-9-Bromo-9-MA (0.72g, 2.23 mmoles) in DMF was added sodium azide (0.91g, 13.98 mmoles). The mixture was heated at 70-80°C overnight. The crude was dissolved in water, extracted with ethyl acetate, and then dried over magnesium sulfate and the organic phase was evaporated in vacuo to give 0.62g, 98% yield. Preparation of N 6 -(endo-2-Norbornyl)-8-Amino-9-MA The crude product, N 6 -(endo-2-Norbornyl)-8-Azido-9-MA (0.5g, 1.75 mmole) was dissolved in ethanol. The solution, in presence of 10% palladium on charcoal (lg) , 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. Preparation of N 6 -(endo-2-Norbornyl)-8-0xo-9-MA To a mixture of 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 CHC1 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. Preparation of 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. Preparation of N 6 -(endo-2-Norbornyl)8-Bromo-2-Chloro-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. A mixture of N 6 -(endo-2-Norbornyl)-2-chloropurine (5.0g, 18.96 mmoles), triethyl ammonium hydroxide (18.9 ml), and methyl iodide (1.41 ml, 22.68 mmoles) in dichloromethane was heated to 35°C for 24 hours. The solution was then evaporated in vacuo and the syrup was crystallized in methanol to give 4.0g, 76% yield of N 6 -(endo-2-Norbornyl)2- chloro-9-MA. To a stirred solution of N 6 -(endo-2-Norbornyl)-2-chloro- 9-MA (4.3g, 14.4 mmoles) in acetate buffer (l molar acetic acid and 1 M sodium acetate mixture, 45:1 ratio respectively; pH ≡ 3.9) was added dropwise Bromine (3.12g, 19.56 mmoles) dissolved in the acetate buffer. The reaction mixture was stirred for 72 hours; the mixture was then filtered and the solid material collected was eluted from ethyl acetate/petroleum ether on silica gel column to yield 4.9g, 85% of N 6 -(endo-2-Norbornyl)8-Bromo-2-Chloro-9-MA. Preparation of N 6 -(endo-2-Norbornyl)-8-Cyclopentyl-9-MA To a vigorously stirred solution of 2g (12.2 mmoles) of 4-methylamino-5-amino-6-chloropyrimidine in CHC1 3 was added dropwise over a period of 20 minutes cyclopentane carbonyl chloride (1.6g, 12.2 mmoles). The mixture was stirred overnight and then evaporated in vacuo to a yellow syrup. The syrup was then dissolved in methanol and purified through a C-18 column (HPLC) to give 2.2g, 71% yield of 4 methylamino-6-chloro-5-cyclopentylamido-pyrimidine. 4-methylamino-6-chloro-5-cyclopentylamido-pyrimidine 2.2g, 8.6 mmoles) was refluxed in POCl 3 for approximately 2 hours. The solution was concentrated in vacuo to a syrup. The syrup was added dropwise to ice. The aqueous mixture was then extracted with chloroform. The organic layer was evaporated and the syrup was passed through a C-18 column (HPLC) giving 1.25g, 63% yield of 8-cyclopentyl-6-chloro-9- methyladenine. A mixture of 8-cyclopentyl-6-chloro-9-methyladenine (0.48g 2.0 mmoles) and endo-2-aminonorbornane hydrochloride (0.5g, 3.4 mmoles) in absolute ethanol was refluxed for 48 hours. The mixture was then evaporated in vacuo and purified through a C-18 column (HPLC) to give 0.45g, 71% yield of N 6 -(endo-2-Norbornyl)-8-cyclopentyl-9-MA. Preparation of N 6 -(endo-2-Norbornyl)-8-Chloro-9-MA A mixture of N 6 -(endo-2-Norbornyl)-8-bromo-9-MA (1.25g, 3.7 mmoles) and P0C1 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. Preparation of N 6 -(endo-2-Norbornyl)-9-[(2-hydroxyethoxy) methyl]purine. To a solution of 6-chloropurine (6g, 38.8 mmoles) in DMF was added sodium hydride 60% (0.93g) over 1.5 hour period. (2-acetoxyethoxy) ethyl bromide was then added at room temperature; the reaction mixture was allowed to stir for 2 hours under N 2 atmosphere. H 2 0 was added and the product was extracted with ethyl acetate. The organic phase was dried over MgS04, filtered, and evaporated in vacuo to give a light yellow solid 7.1g, 68% yield of 9-[(2-Acetoxy- ethoxy)methyl]-6-chloro-purine. The crude wa^used without further purification. To a solution of 9-[ (2-acetoxyethoxy)methyl]-6-chloro- purine (5.1g, 18.8 mmoles) in ethanol and triethylamine was added endo-2-aminonorbornane hydrochloride (4.0g * , 27.1 mmoles) . The mixture was refluxed in vacuo and the residue was purified by HPLC to give 4.70g, 77% yield of N 6 -(endo-2- Norbornyl)-9-[ (2-acetoxyethoxy)methyl]purine. A solution of N 6 -(endo-2-Norbornyl)-9-[ (2-acetoxyethoxy) methyl]purine (3.75g, 10.8 mmoles) in methanol was saturated with NH 3 gas under N 2 . The mixture was stirred overnight, then evaporated in vacuo to give 2.03g, 62% yield o__ N 6 -(endo-2-Norbornyl)-9-[ (2-hydroxyethoxy) ethyl]purine. The invention is further illustrated by the follow¬ ing examples which are illustrative of various aspects of the invention. These examples are not intended as limiting the scope of the invention as defined by the appended claims. PHARMACOLOGIC TESTING A series of N 6 -substituted 9-methyladenines were assayed as adenosine antagonists in k^ and A 2 test systems (ϋkena, et al, FEBS Lett. 215(2), 203-208, 1987). For activity at &1 receptors, 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. For activity at A 2 receptors, compounds were tested as antagonists of NECA-stimulated adenylate cyclase in membranes of human platelets and rat PC12 cells. It is known that &ι_ 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. (See John W. Daly, et al., "Structure- Activity Relationship for N 6 -Substituted Adenosines at a Brain A^Adenosine Receptor With A Comparison to an A 2 - Adenosine Receptor Regulating Coronary Blood Flow," Bio¬ chemical Pharmacology. Vol. 35. No. 15, pp. 2467-2471 (1986)) . The results summarized below in Table I show that N 6 substitution can markedly increase the potency of 9- methyladenine at adenosine receptors. The lower apparent affinity values (Kg, K^) identify the most potent compounds. The most pronounced effect is seen at ^ receptors. For example, N 6 -Cyclopentyl-9-methyladenine is at least 100-fold more potent than 9-methyladenine at A^ receptors. At A 2 receptors, this compound is 5-fold more potent than 9- methyladenine in the human platelet assay. Thus, this data demonstrates the activity of a novel series of adenosine antagonists. TABLE 1 Effec s Effects I nd-H) VS NECA St imulat ion K B ( M) (Adenyl ate Cyc lase) vs PIA INHIBITION (Adenylate Cyclase) 1 Adenine 760 570 > 1000 2 9 - Me t hy I aden i ne (9-MA) 24 24 112 «-• ubst i tuted 9 - me t h y I aden i nes 3. N 6 -Cyc lobutyl -0-HA 5.5 23 0.89 4. M 6 -Cyclopentyl -9-MA 4.9 25 1.3 5. N 6 -Methylcylopentyt -9-MΛ 45 56 9.0 6. Cyclohexyl -9-MA 7.4 21 0.65 7. N°-Hethyl -9-MA 150 130 220 8. 3 - Pen ty I - 9-HA 11 53 7.6 9. M β - Pheny I - 9 - MA 21 107 10 10. N 6 -2- uorophenyl -9-HΛ 11 29 17 11. N 6 - 2 - Benzyl -9-MA 57 100 49 12. M 6 - 2-Phenethy I -9-MA 170 120 >300 13. N 6 - 2 - ( 3 ,4 , 5- Tr i m- ethoxyphenylethyl ) -9-MA 23 40 122 14 M 6 -2-(3-Pyridylethyl )-9-MA 92 117 96 15 M -2- (3-Thi enyl ethyl ) -9-HA 14 25 24 16 N 6 -R- 1 -Phenyl -2-propyl -9-MA 13 25 7.2 17 M 6 -S- 1 -Phenyl -2-propyl -9-MA 23 74 23 (A) - Human P latelet Membranes (B) - Rat PC12 Membranes (C) - Rat Fat Cel l Membranes (D) - Rat Brain Membranes FURTHER FUNCTIONAL ASSAYS To test the selectivity of the compounds of the invention, in vitro assays were conducted utilizing model tissues that are thought to contain homogenous populations of either the A^ 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^) ; 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% 0 2 + 5% C0 2 and maintained at 31°C. 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% 0 2 + 5% C0 2 and maintained at 31 β C. 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. The ability of the compounds to antagonize the effects of agonists was analyzed using modified Schild plots. Although there was some sensitization of the tissue, i.e. addition of the agonist produced a larger response in the presence of high concentrations of the subject com¬ pounds, N 6 -3-Pentyl-9-MA, N 6 -Cyclopentyl-9-MA and N 6 -(endo- 2-Norbornyl)-9-MA did not competitively antagonize" the effects of adenosine agonists in relaxing the taenia caecum. Sensitization is also observed when using high concentra¬ tions of 8-phenyltheophylline (8-PT) , a non-selective adenosine receptor antagonist. 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 fbr A j adeno¬ sine receptors. * * However, 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 -l-(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: Table 2 Druσ pK B N 6 -3-Pentyl-9-MA 5.4 ± 0.14 N 6 -Cyclopentyl-9-MA 6.17 ± 0.11 N 6 -(endo-2-Norbornyl)-9-MA 6.28 ± 0.09 N 6 -l-(2-Thienyl)-2-butyl)-9-MA 5.36 ± 0.1 These results show that the above examples display selectivity towards the A adenosine receptor, with N 6 - (endo-2-Norbornyl)-9-MA being the most potent antagonist. IN VIVO ASSAY In vitro selectivity of the present antagonists was confirmed by in vivo tests on rat heart rate and blood pressure, the former associated with -^ receptors and the latter associated with A 2 receptors. Rats were anesthetized with urethan and blood pressure was monitored via a carotid cannula. Drug injec¬ tions 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 concom itant 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. At high doses, adenosine causes heart block; this effect was also substantially reduced by the agonist. Due to the short duration of action and direct route of ad¬ ministration of adenosine, it is often difficult to deter¬ mine whether adenosine decreased blood pressure by causing peripheral vasodilation or by reducing cardiac output. To overcome these problems, 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. These results show that 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 ^ adenosine receptor antagonists. FURTHER RECEPTOR AFFINITY ASSAYS Further tests to discover the affinities of test compounds at A 2 receptors were conducted. [ 3 H]-N-ethylcar- boxamido adenosine ([ 3 H-]-NECA) was used as the radioligand, bovine caudate was the source of membranes, and the assay buffer was 50 mM Tris; 10 mM MgCl 2 , pH 7.4. To provide bovine caudate nuclei, 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 x g for 20 minutes and washed once. The pellet was resuspended in Buffer A, incubated at 37°C 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 -70°C until use. The A 2 assays also contained 50 nM cyclopentyl- adenosine to block the binding of [ 3 H]-NECA to K^ 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 * radio¬ ligand and membrane source for 1 hr at room temperature. Assays were terminated by filtration over Whatman GF/B filters that had been pre-soaked with 0.1% polyethyl- eneimine using a 24 port Brandell cell hawester. The filters were washed three times with 3 ml of ice cold"buffer and transfered to plastic scintillation vials to which 4 ml of Beckman Ready-Protein scintillation cocktail was added. The tubes were shaken and counted in a Beckman 3801 scintil¬ lation counter that converted cpm to dpm. • Data were analyzed by utilizing the Ligand ® commercial computer program (Munson and Rodbard, 1980) . The results of these tests, expressed as the molar concentration of test compound needed to displace 50 percent of the [ 3 H]-CHA radioligand from rat cortical A.^ receptors, are summarized in Table 3 below: Table 3 Adenosine Antagonists Rat Cortical Sample Binding Constant No. Name Ki (M) 0861 N 6 -(endo-2-norbornyl)-9-MA 11.6 x 10~ 8 0913 N 6 -(endo-2-norbornyl)-2-chloro-9-MA 10.5 X 10" 8 0966 N 6 -2 , 2-diphenylethyl-9-MA >10" 5 0967 N 6 -2 (2-chlorophenylethyl) 9-MA >10" 5 0982 N 6 -2-Aminoethyl-9-MA >10" 5 0983 N 6 -(2 ,2-N-dimethylethyl)-9-MA >10" 5 0840 N 6 -cyclopentyl-9-MA 37.5 x 10~ 8 0984 N 6 -R-l-phenyl-l-ethyl-9-MA >10~ 5 0985 N 6 -S-l-phenyl-l-ethyl-9-MA >10" 4 0986 N 6 -S-l-phenyl-2-propyl-9-MA >10~ 5 0987 N6 2-thienyl-9-MA >10" 4 0988 N6(4-chloro-2-methylphenyl)-9-MA >10~ 5 0989 N 6 -2-(3-ethylindole)-9-MA >10" 5 0990 N 6 -2-(phenethyl)9-MA >10" 5 1001 N 6 -(endo-2-norbornyl)-8-oxo-9-MA «10" 5 1002 N 6 -2-(3,4,5-trimethoxyphenyl)ethyl-9-MA >10" 5 1003 N 6 -(endo-2-norbornyl)-8-bromo-9-MA 1.3 X 10~ 8 1004 N 6 -l-carboxy-l-butyl-9-MA >10 -4 1005 N 6 -(endo-2-norbornyl)-8-amino-9-MA 87 X 10~ 8 1006 N 6 -(endo-2-norbornyl)-8-carboxy-9-MA Sodium Salt >10 -5 1059 N 6 -(endo-2-norbornyl)9-[ (2 hydroxyethoxy) methyl]adenine 49 x 10 -8 1060 N 6 -(endo-2-norbornyl)-8-thio-9-MA 37 x 10 -8 1061 N 6 -(endo-2-norbornyl)-8-chloro-9-MA 1.5 X 10 -8 1062 N 6 -(endo-2-norbornyl)-8-sulfonate- 9-MA Sodium Salt >10 -4 1063 N 6 -(Endo-2-norbornyl)-2-oxo-9-MA 112 X 10 -8 1064 N^-(endo-2-norbornyl)-8-cyclopentyl- amine-9-MA 190 X 10 -8 0964 N 6 -(endo-2-norbornyl)-8-cyclopentyl-9-MA 24 x 10" *8 0965 N 6 -cyclopentyl-8-cyclopentyl-9-MA 14.1 x 10 -8 0978 N 6 -(exo-2-norbornyl)-9-MA 43 * x 10" 8 The compounds in Table 3 for which a solution having a concentration greater than 10~ 5 M was required to displace 50 percent of the radioligand are deemed ineffective as A^ adenosine receptor antagonists. In further experiments designed to determine the selectivity of N 6 -endo-2-Norbornyl-9-methyl adenine at A._ receptors, [ 3 H]-cyclohexyladenosine ([ 3 H]-CHA) was used as the radioligand, rat cortical membranes were the receptor source, and the assay buffer was 50 mM Tris; 2 mM MgCl 2 pH 7.4. Male Sprague Dawley rats were killed by decapitation and the brains removed. The cerebral cortices were homogenized in 50 mm Tris; 2mm MgCl (pH 7.4), and centrifuged at 40,000 x g for 10 minutes. The pellet was washed once, resuspended in Tris/MgCl 2 and incubated with 8 units/ml adenosine deaminase at 37°C for 30 minutes. The homogenate was centrifuged, washed once, resuspended to . a protein concentration of 5-10 mg/ml and frozen at -70°C until use. The results in Table 4 below show that the test compound has 170 times more affinity for &.χ receptors than for A 2 recep¬ tors. Table 4 Selectivity of N 6 -endo-2-Norbornyl-9-MA Bovine Caudate Binding Constants At A i Receptors At A 2 Receptors EilMl 4.1 X 10~ 8 M References Munson, Peter J. and Rodbard, David (1980) . "Ligand: A Versatile Computerized Approach for Characterizing Ligand- Binding Systems." Anal. Biochem. 107:220-239. Bruns, Robert F. , Lee, Gina H. , and Pugsley, Thomas A. (1986) "Characterization of the A Adenosine Receptor Labeled by 3 H-NeCA in Rat Striatal Membranes," Mol. Pharmacol. 29:331-346. These 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^-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 adeno- mediated relaxation of smooth muscle, anti-narcoleptic action, CNS stimulation, and blockade of adenosine mediated inhibition of neurotransmitter release. While particular embodiments of the invention have been described it will be understood of course that the invention is not limited thereto since many obvious modifications can be made and it is intended to include within this invention any such modifications as will fall within the scope of the appended claims.

Claims: What is claimed is: 1. Novel compounds represented by the general formula: wherein R 2 is selected from the group consisting of cycloalkyl radicals having from 3 to 8 ring carbon atoms, alkyl radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 13 carbon atoms, aralkyl radicals having from 7 to 14 carbon atoms and halogen- and heteroatom-substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of halogen, nitrogen, phosphorus, " sulfur and oxygen; R may be hydrogen or R 2 , and R 3 is selected from the group consisting of hydrogen, halogen, amine, carboxy, thio, sufonate, sulfonamide, sulfone, sulfoxamide, phenyl, alkyl- or cycloalkyl-substituted amine, alkyl radicals having 1 to 10 carbon atoms and cycloalkyl radicals having from 3 to 8 ring carbon atoms. R4 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; and R5 is selected from the group consisting of hydrogen, hydroxy, 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 members of the group consisting of phenyl, and alkyl and cycloalkyl, having 1 to 6 carbon atoms. 2. The compound of claim 1 wherein R 4 is methyl. 3. The compound of claim 2 wherein R^ is hydrogen. 4. The compound of claim 3 wherein R 2 is a cycloalkyl having from 4 to 8 carbon atoms in the ring. r 5. The compound of claim 3 wherein R 2 is phenyl or a substituted phenyl. 6. The compound of claim 3 wherein R 2 is 2-norbornyl, cyclopentyl and R 3 is selected from the group consisting of hydrogen, cyclopentyl, oxo, bromo, amino, carboxy, thio, chloro, fluoro, sulfonate, sulfona ido, cyclopentyϊamino, cyclopentyl, and physiologically acceptable salts thereof. 7. The compound of claim 3 wherein R 2 is selected from the group consisting of benzyl, phenyl, o-flύorophenyl, 3,4,5-trimethoxyphenylethyl, 3-pentyl, 2-phenylethyl, 2-(2- chlorophenylethyl) ; 1-indanyl, 2-aminoethyl, N,N-dimethyl- aminoethyl, 2-thienylbutyl, and cyclohexyl. 8. The compound of claim 3 wherein R 2 is endo-2- Norbornyl and R 4 is phenyl or (2-hydroxyethoxy)methyl. 9. The compound of claim 1 selected from the group consisting of N 6 -(endo-2-Norbornyl)-9-[ (2-hydroxyethoxy) methyl]adenine, N 6 -(endo-2-Norbornyl) -8-thio-9-methyl adenine, N 6 -(endo-2-Norbornyl)-8-chloro-9-methyl adenine, N 6 -(endo-2-Norbornyl)-2-oxo-9-methyl adenine, N 6 -(endo-2- Norbornyl)-8-cyclopentylamino-9-methyl adenine, N 6 -cyclo- pentyl-9-methyl adenine, N 6 -(endo-2-Norbornyl)-9-methyl adenine, N 6 -(endo-2-norbornyl)-8-bromo-9-MA, N 6 -cycϊopentyl- 8-cyclopentyl-9-methyl adenine, N 6 -(exo-2-norb rnyl)-9-MA, N 6 -cyclopentyl-2-chloro-9-methyl adenine, N 6 -[(3-chloro)- endo-2-norbornyl]-9-MA, N 6 -cyclopentyl-9-phenyl adenine, N 6 - (endo-2-norbornyl)-8-cyclopentyl-9-MA, N 6 -cyclopentyl-9- benzyl adenine, and N 6 -(endo-2-norbornyl)-8-amino-9-MA. 10. The compound of claim 2 wherein R5 is selected from the group consisting of hydrogen, ethoxy, methoxy, propoxy, n-butoxy, isopropoxy, 1-methylpropoxy, 2-methylpropoxy, 2- phenyl-ethoxy, methylamino, butylamino and anilino. 11. The compound of claim 3 wherein R5 is chloro. 12. The compound of claim 3 wherein R 2 is selected from the group consisting of 3-pentyl, l-phenyl-2-propyl, and phenyl. 13. The compound of claim 2 wherein R 2 is hydrogen and R 5 is selected from the group consisting of ethoxy, methoxy, propoxy, n-butoxy, isopropoxy, butyl-2-oxy, 2-methylpropoxy, pentoxy, 2-phenylethoxy, methylamino, butylamino and anilino. 14. The compound of claim 11 wherein R 2 is 2-(3- thienylethyl) . 15. The compound of claim 7 wherein R 2 is cyclohexyl. 16. The compound of claim 11 wherein R 2 is 2-(3- pyridylethyl) . 17. The method of antagonizing the A 2 -adenosine receptor-mediated stimulation of adenylate cyclase which comprises administering to a subject an effective amount of one or more of the compounds of claim 4, 6, 7, 8 or 9. 18. The method of claim 17 wherein said subject is a human. 19. The method of antagonizing the A -adenosine receptor-mediated inhibition of adenylate cyclase which comprises administering to a subject an effective amount of a compound of claim 4, 6, 7, 8 or 9. 20. The method of claim 19 wherein said subject is a human. 21. The method of antagonizing the adenosine receptor which comprises administering to a subject an effective amount of a compound selected from the group of compounds represented by the general formula wherein R 2 ^s selected from the group consisting of cycloalkyl radicals having from 3 to 8 ring carbon atoms, alkyl radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 13 carbon atoms, aralkyl radicals having from 7 to 14 carbon atoms, and halogen- and heteroatom-substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of halogen, nitrogen, phosphorus, sulfur and oxygen; R^ may be hydrogen or R , and R 3 is selected from the group consisting of hydrogen, halogen, amine, carboxy, thio, sufonate, sulfonamide, sulfone, sulfoxamide phenyl, alkyl- or cycloalkyl-substituted amine, alkyl radicals having 1 to 10 carbon atoms and cycloalkyl radicals having from 3 to 8 ring carbon atoms. 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; and R5 is selected from the group consisting of hydrogen, hydroxy, amine, halogen, alkoxy and cycloakoxy 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 members of the group consisting of phenyl, alkyl, cycloalkyl, having 1 to 6 carbon atoms. 22. The method of claim 21 wherein said subject is a human. 23. Novel compounds represented by the general formula: wherein R^ is selected from the group consisting of cycloalkyl radicals having from 3 to 7 ring carbon atoms, alkyl radicals having from 2 to 10 carbon atoms, aryl radicals having from 6 to 10 carbon atoms, aralkyl radicals having from 7 to 10 carbon atoms and heteroatom substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of halogen, nitrogen, phosphorus, sulfur and oxygen; R may be hydrogen or R lf and R 3 is an alkyl group comprising from l to 4 carbon atoms. 24. The compound of claim 23 wherein R 3 is methyl. 25. The compound of claim 24 wherein R 2 is hydrogen. 26. The compound of claim 25 wherein R is a cycloalkyl having from 4 to 6 carbon atoms in the ring. 27. The compound of claim 25 wherein R is phenyl or a substituted phenyl. 28. The compound of claim 27 wherein R^ is selected from the group consisting of phenyl, o-fluorophenyl and 3,4,5- trimethoxyphenyl. 29. The compound of claim 25 wherein R i s benzyl or 2- phenylethyl. 30. The compound of claim 25 wherein ^ is 2-(3- pyridylethyl) or 2-(3-thienylethyl) . 31. The compound of claim 25 wherein R is 3-pentyl. 32. The compound of claim 24 wherein R 2 is selected from the group consisting of methyl and 2-propyl and ^ is selected from the group consisting of cyclopentyl and phenyl. 33. The compound of claim 30 wherein R^ is 2-(3- thienylethyl) . 34. The compound of claim 32 wherein R 2 is 2-propyl and i is phenyl. 35. The compound of claim 28 wherein R^ is benzyl. 36. The compound of claim 30 wherein R is 2-(3- pyridylethyl) . 37. The method of antagonizing the A 2 -adenosine receptor-mediated stimulation of adenylate cyclase which comprises administering to a subject an effective amount of one or more of the compounds of claims 26, 28, 31, 33 or 34. 38. The method of claim 37 wherein said subject is a human. 39. The method of antagonizing the Ai-adenosine receptor-mediated inhibition of adenylate cyclase which comprises administering to a subject an effective amount of a compound of claim 26, 28, 31, 33, 34, 35 or 36. 40. The method of claim 39 wherein said subject is a human. 41. The method of antagonizing the adenosine receptor which comprises administering to a subject an effective amount of a compound selected from the group of compounds represented by the general formula wherein ^ is selected from the group consisting of cycloalkyl radicals having from 3 to 7 ring carbon atoms, alkyl .radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 10 carbon atoms, aralkyl radicals having from 7 to 10 carbon atoms and heteroatom substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of halogen, nitrogen, phosphorus, sulfur and oxygen; R 2 may be hydrogen or R, and R 3 is an alkyl group comprising from 1 to 4 carbon atoms. 42. The method of claim 41 wherein said subject is a human.

Inventors: MARCUS TED, OLSSON RAY A, MARCUS, TED, OLSSON, RAY, A.
Applicants: WHITBY RESEARCH INC, WHITBY RESEARCH INCORPORATED
Application year: 1990
Main classification: C07D473
IPC codes: C07D473