NO168708B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTICALLY ACTIVE N6-SUBSTITUTED 9-METHYLADENINES - Google Patents

Description

N<6->substitution can markedly increase the potency of 9-methyladenine

at A^ receptors, but have lesser effects or even reduced potency at A2 receptors. Effects of N<6->substituents on the adenosine receptor activity of the 9-methyladenines are reminiscent of effects of N<6->substituents on the activity of adenosine, and suggest that N<6->substituted 9-methyladenines bind to adenosine receptors in the same orientation as an N<6->substituted adenosine does. N<6->cyclopentyl-9-methyladenine is stronger than 9-methyladenine.

N<6->cyclopentyl and several other N<6->alkyl and N<6->cycloalkyl analogs are selective for A^ receptors, but 9-methyladenine is the most A2-receptor selective antagonist. N<6->R- and N<6->S-(1-phenyl-2-propyl)-9-methyladenines, analogs of N<6->R- and N<6->S-phenylisopropyladenosines, show stereoselectivity at both A^-

and A2 receptors…

The present invention relates to an analog method

in the preparation of a therapeutically active adenosine receptor ligand with the structural 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 6 carbon atoms.

According to the present invention, the compounds are prepared by reacting a first reactant of the general formula where Z is halogen with a second reactant of the general formula

in which R± is defined as before.

The preparation of 9-methyladenines is well known. See R. K. Robins, K. J. Dille, and B. E. Christensen, J. Ora. 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).

Adenosine receptors have been divided into two subtypes based on

on adenylate cyclase activity: (n^) receptors effect inhibition and A2 (Ra) receptors effect stimulation of adenylate cyclase activity (for overviews see ref. l.,2). Some N<6->substituted adenosine analogs such as N<6->R-l-phenyl-2-propyladenosine (R-PIA) have very high affinity for A^-adenosine receptors, while 5'-N-ethylcarboxamidoadenosine (NECA) is stronger than N <6->substituted analogs and A2 receptors. Alkyl xanthines, such as caffeine and theophylline, are the best known antagonists at adenosine receptors. Adenine was generally thought to have no effect on adenosine receptor-controlled systems. However, adenine is a specific, competitive antagonist of adenosine-induced cyclic AMP accumulation in a human fibroblast cell line with an A K of 200 pM (3). Methylation of adenine at the 9-position increases the potency approx. 4 times. A number of N<6->substituted-9-methyladenine derivatives have now been prepared and tested in three adenylate cyclase-coupled adenosine receptor systems. For A2-adenosine receptors, human platelets and rat pheochromocytoma (PC12) cells and for A^ binding site for [<3>H]N<6->R-1-phenyl-propyladenosine ([<3>H]PIA) were determined in rat brain membranes. Certain N^-substituted 9-methyladenines proved to be potent antagonists at adenosine receptors and some showed selectivity for either A± or A2 receptors.

[α-<32>P]ATP (40~ Ci/mmol)' was purchased from Amersham (Arlington Heights, IL, USA). [<3>H]N<6->R-1-phenyl-2-propyladenosine ([<3>H]PIA, 49.9 Ci/mmol) was purchased from New England Nuclear, Boston, MA.

USA). Other compounds used in this study were from standard sources as described (4).

The following manufacturing examples and pharmacological experiments illustrate the invention.

Manufacturing examples

Example 1

To prepare N<6->cyclopentyl-9-methyl-adenine, the following additional steps were taken. A mixture of 6-chloro-9-methyl-purine (0.82 g), 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 and gave 0.78 g or 74% yield with a melting point of 108 - 109°C. <1>HNMR(Me2SO-d6): S1-2(m,9H); 3,7(S,CH3); 7.6(d, NH) ; 8.1 (S, 1H); 8.2(S,1H).

Example 2

To prepare N<6->3-pentyl-9-methyl-adenine, the following steps were taken. A mixture of 6-chloro-9-methyl-purine (1.5 g), 3-pentylamine (1.3 ml), triethylamine (1.3 ml) and ethanol (60 ml) was refluxed for 24 hours. The solution was passed through a C-18 column and gave a white solid with a melting point of 107 - 109°C.

Example 3

To prepare N<6-(2-norbornanyl)-9-methyladenine, the following additional steps were taken. A mixture of 1.5 g of 6-chloro-9-methyl-purine, 1.75 g of 2-aminonorbornane, 2.9 ml of triethylamine and 60 ml of ethanol was refluxed overnight. The solution was then concentrated in vacuo and the residue was run through C-18 prep chromatography to give 1.6 g (75% yield), mp 130-131°C. <1>HNMR(Me2SO-d6): 51-2.6(m,10H); 3.8(S, CH3) ; 4.1(m, 1H) ; 7.2(S, NH) ; 7.4(S, 1H) ; 7.6(S, 1H) .

PHARMACOLOGICAL EXPERIMENTS

Platelets, rat pheochromocytoma (PC12) cells, rat fat cells and rat cerebral cortex membranes were prepared as described in Bruns, R.F., Biochem. Pharmacol. 30 325-333 (1981); Ukena, D. et al., Life Science 38 797-807 (1986); and Ukena, D. et al., Nayryn-Schmiedeberfs Arch. Pharmacol. 327 36-42,

(1984). Adenylyl cyclase activity and binding of [<3>H]PIA to cerebral cortex membranes were determined mainly as described in the indicated publications.

KB values for the compounds were also determined as described in the indicated publications. Briefly, the NECA concentration-response curves show stimulation of adenylate cyclase in PC12 cells and platelet membranes. Similar R-PIA concentration-response curves show the inhibition of isoproterenol-stimulated adenylate cyclase activity in fat cell membranes using at least 7 concentrations of adenine derivative. For agonists, EC50 and IC50 values were obtained from the concentration-response curves by linear regression after log t-log transformation. For the antagonists, KB values were calculated using the Schild equation KB = C/(CR-1), where C indicates the concentration of the competitor, and CR indicates the ratio of EC50 and IC50 values in the presence or absence of the competitor, respectively. The IC 50 of the compounds for inhibition of [<3>H]PIA binding two cerebral cortex membranes were transformed to K 2 values as described in Jacobson, K.A. et al., Proe. Nati. Acad. Pollock. USA 83 4089-4093 (1986).

RESULTS

A2 Adenosine receptors.

It has been found that the N<6->cycloalkyl analog (Example 1) is more potent than 9-methyladenine itself.

Introduction of an additional methyl group into N<6->cyclopentyl-9-methyl-adenine (Example 1) giving a tertiary carbon at the N<6->nitrogen reduces potency.

Ai Adenosine receptors.

Rat fat cells were used to assess the structure-activity relationships of ademine derivatives at adenylate cyclase-coupled A^adenosine receptors. In these cells, adenosine analogs inhibit adenylate cyclase activity and lipolysis (see London, C. et al., Proe. Nat'l Acad. Seil, USA 77 2551-2554 (1980).

It has now been found that the introduction of cycloalkyl or alkyl substituents in the N<6-> position of 9-methyladenine can markedly increase the antagonistic strength of the fat cell A^ receptor. Thus, N6-cycloalkyl-9-methyl-adenine is stronger than the parent compound 9-methyl-adenine.

The present studies were designed to test the premise that, as in the case of adenosine, N<6->substituents on 9-methyl-adenine would alter activity in the same way that N<6->substituents alter the activity of adenosines. The topography of the binding site for N<6->substituents in A± and A2 adenosine receptors has been thoroughly investigated by Ukena et al. (Can J. Physical Pharmacol (1987), FEBS Lett 209 122-128 (1986). The binding site for N<6->substituents differs significantly for receptors compared to A2 receptors. In the case of A-^ receptors, the N6-substituents can markedly increase activity . The stereoselectivity for compounds such as R-PIA and S-PIA containing chiral N<6->substituents is a well-known property of A^ receptors. In the case of A2 receptors, most N<6->substituents reduce the activity of adenosine and stereoselectivity to a lesser extent distinctly than at A]^ receptors. Certain N<6->substituents markedly increase the activity of 9-methyl-adenine at A± receptors. N<6->cycloalkyl-9-methyl-adenine (example 1) is the strongest of the N <6->substituted 9-methyl-adenines at A^ receptors.

Similarly, N<6->cycloalkyladenosines are among the most potent N6-substituted adenosines at A-^ receptors. Introduction of an additional methyl into N<6->cyclopentylmethyl-adenine which gives N<6->1-methylcyclopentyl-9-methyl-adenine reduces the activity at A± receptors.

It is known that A± receptors affect inhibition of adenylate cyclase in fat, brain and heart cells, while A2 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 A2-Adenosine Receptor Regulating Coronary Blood Flow," Biochemical Pharma- colocty. Vol. 35,' No. 15, pages 2467-2471 (1986).

To prove the selectivity of these compounds, in vitro measurements were performed using model tissues thought to contain homogeneous populations of either the A 2 or A 2 adenosine receptor. The drugs were characterized by their ability to competitively antagonize the action of adenosine agonists in the production of two reactions: the reduction in contraction force in guinea pig atrium (A3J ; and the reduction in contractile tone in guinea pig teenia caecum (A2)•

The left atria from male guinea pigs were isolated, suspended between two point electrodes and placed in a 20 ml organ bath containing KrebsHensileit solution which was continuously gassed with 95% O2 + 5% CO2 and maintained at 31°C. The resting stretch was 1 gram. Atria were electrically stimulated with 1 Hz, 1 ms duration pulses at supramaximal voltage. The contraction force was recorded isometrically.

Teania from 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 gassed with 95% O2 + 95%

C02 and kept at 31°C. The resting tension was 1.5 g. The contraction reaction was measured isotonically. Tissues were contracted with 10~<7>M 5-methylfurmetide and allowed sufficient time to reach a stable concentration before addition of adenosine agonists.

The ability of compounds to antagonize the effects of antagonists was analyzed using modified Schild plots.

Although there was some sensitization of the tissue, i.e. addition of the agonist produced a greater response in the presence of high concentrations of the present compounds, Examples 1, 2 and 3 did not competitively antagonize the effects of adenosine agonists on relaxation of taenia caecum. There was some sensitization of the tissue. This sensitizing effect is also observed using high concentrations of 8-phenyltheophylline (8-PT), a non-selective adenosine receptor antagonist. 8-PT antagonized the action of agonists at low concentrations. The lack of competitive antagonism suggests that the latter compounds do not react significantly with A2 adenosine receptors.

However, Examples 1, 2 and 3 were all found to be competitive antagonists at adenosine receptors in the atria. Example 2 also produced increases in the base contraction force of the atria. Affinity constants (pKB) for the present compounds were determined using known methods and were as follows:

These results show that the compounds prepared according to the invention show selectivity towards the A^ adenosine receptor, with N6-(endonorbornyl)-9-MA being the strongest antagonist.

In vitro selectivity for the present antagonists was confirmed by in vivo experiments on heart rate and blood pressure, the former in connection with A 2 receptors and the latter in connection with A 2 receptors.

Rats were anesthetized with urethane, and blood pressure was controlled through 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 reduction in blood thickness and heart rate with a simultaneous increase in the P-R interval of the ECG. Administration of N<6->(endonorbornyl)-9-methyladenine

attenuated the effects of subsequently administered adenosine on all measured parameters. At high doses, adenosine causes heart block; this effect was also significantly reduced with the agonist due to the short duration of action and route of administration of adenosine, and it is often difficult to determine whether adenosine reduced blood pressure by causing peripheral vasodilation or by reducing cardiac output. Therefore, NECA (5'-N-ethylcarboxamide-adenosine) was used as an adenosine agonist due to its longer duration of action and A2-adenosine receptor selectivity. Prior administration of N-0861 attenuated the effects of NECA on the heart, but minimally affected the NECA-induced reduction in blood pressure. These results demonstrate that N<6->endonorbornyl)-9-methyladenine is a cardioselective adenosine receptor antagonist in vivo and support the above data showing the selectivity of the N<6->substituted 9-methyladenine as A^adenosine receptor antagonists.

Claims (1)

Analogous procedure for the preparation of a therapeutically active adenosine receptor ligand with the structural formula: wherein Ri is selected from the group consisting of cycloalkyl radicals having from 5 to 7 ring carbon atoms, alkyl radicals having from 2 to 6 carbon atoms, characterized by reacting a first reactant with the general formula where Z is halogen with a second reactant of the general formula wherein R 1 is as previously defined.