Doridosine derivatives: binding at adenosine receptors and in vivo effects

European Journal of Pharmacology, 243 (1993) 135-139

135

© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00

EJP 53331

Doridosine derivatives: binding at adenosine receptors and in vivo effects P a o - L u h T a o a,., M a o - H s i u n g Y e n a W o e i - S h y o n g Shyu a a n d J i - W a n g C h e r n b a Department of Pharmacology, P.O. Box 90048-504, and b Institute of Pharmacy and Medical Laboratories, National Defense Medical Center, Taipei, Taiwan, ROC

Received 28 April 1993, revised MS received 2 August 1993, accepted 3 August 1993

Doridosine, an adenosine analogue, causes, in vivo, hypotension, reduction of heart rates, muscle relaxation and anti-inflammatory effects through adenosine A 1 and A 2 receptors. A series of doridosine derivatives was synthesized in a search for compounds with more selective adenosine A 1 receptor activity. These derivatives were characterized for binding to the respective adenosine receptors and for their cardiovascular effects. We used competition binding studies with highly selective radioligands: [3H]cyclohexyladenosine for adenosine A 1 and [3H]CGS 21680 for adenosine A 2 binding assays. The results for eight doridosine derivatives revealed that 1-cyclopropylisoguanosine (BN-063) and 1-allylisoguanosine (AZ-108-1) were more selective for the adenosine A 1 receptor. In vivo, both BN-063 and AZ-108-1 caused significant bradycardia but no obvious effect on blood pressure. The bradycardia was almost completely blocked by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, a specific adenosine A 1 receptor antagonist). Doridosine (1-methylisoguanosine) derivatives; Competition binding studies; Adenosine A 1 receptors; Adenosine A 2 receptors; Bradycardia; (Rat)

1. Introduction Doridosine (1-methylisoguanosine)was isolated early in 1980 from an aqueous enthanolic extract of the sponge Tedania digitata (Quinn et al., 1980; Cook et al., 1980). It causes a reduction in arterial pressure and slowing of heart rate in mammals that is qualitatively similar to those caused by adenosine but with an unusually long duration of action (Baird-Lambert et al., 1980; Davies et al., 1980). Recent appreciation of the importance of the purine nucleoside effect on the cardiovascular system mediated via the adenosine receptors (Daly, 1982; Jacobson et al., 1992) led us to further investigate the effect of this naturally occurring nucleoside on adenosine receptors. In the present studies, eight doridosine derivatives (table 1) were synthesized by modification of the structure at N-1 by reacting 5-aminoimidazole-4-carboxamide-1-/3-ribofuranoside with alkyl and aryl isothiocyanate (Chern et aI., 1991). Competition experiments were carried out to determine the adenosine A1 and

* Corresponding author. Tel. 886-2-367-3431, fax 886-2-365-7901.

A z receptor binding affinities for each derivative. The cardiovascular effects were determined by measuring the blood pressure and heart rate responses to intravenous injection of doridosine derivatives in anesthetized rats.

2. Materials and methods 2.1. M e m b r a n e preparations

Rat cortex (for adenosine A 1 receptor binding) or striatum (for adenosine A 2 receptor binding) was homogenized with a Kinematica polytron (setting 5, for 20 s) in 20 volumes of ice-cold 0.32 M sucrose buffered with 50 m M Tris-HC1, p H 7.4. This m e m b r a n e homogenate was centrifuged at 1000 x g for 10 min at 4°C and the P1 pellet which contains nuclei was removed. The supernatant was centrifuged at 22,000 x g for 20 min at 4°C. The resulting pellet (P2 m e m b r a n e ) was resuspended in 50 m M Tris buffer containing 2 I U / m l adenosine deaminase (Sigma) to 20 m g / m l original tissue weight and incubated at 37°C for 30 min to inactivate endogenous adenosine. The m e m b r a n e homogenate was recentrifuged and the final pellet was frozen at - 7 0 ° C until assay.

136

2.2. Binding studies Binding assays were carried out in triplicate in 13 × 100 m m disposable borosilicate glass test tubes. Adenosine A 1 receptor binding was measured in adenosine deaminase pretreated rat cortical P2 membranes using [3H]cyclohexyladenosine in the presence of 1 0 / z M 2-chloroadenosine to define specific binding. Assays were run at 24°C for 2 h with 100-200 /zg of protein of adenosine deaminase-treated tissue in a final volume of 1 ml of 50 m M Tris-HCl buffer and 10 m M MgCI 2, p H 7.4; [3H]cyclohexyladenosine was included at a final concentration of 1 nM for competition binding studies. Adenosine A 2 receptor binding was measured in adenosine deaminase-pretreated striatal P2 membranes. An aliquot of striatal m e m b r a n e s (100200/xg protein) was put in incubation buffer (50 m M Tris-HC1 and 10 m M MgCI2, p H 7.4) with 5 nM [3H]CGS 21680 for competition binding studies. All assays were conducted at 24°C for 90 min in a final volume of 1 ml. Non-specific binding was defined in the presence of 20 /xM 2-chloroadenosine. Binding reactions were terminated by filtration through Whatman G F / B filters under reduced pressure. The filters were washed twice with 5 ml ice-cold buffer and placed in scintillation vials. Bound radioactivity was determined by using conventional liquid scintillation spectroscopy techniques at an efficiency of 40-50%. For all the competition studies, 13-17 concentrations of inhibitor were included in the incubation

buffer. The data represent means + S.E.M. for a minim u m of two separate observations. K i values were determined by using the non-linear curve fitting program L I G A N D written by Munson and Rodbard (1980).

2.3. Pharmacological studies Adult Sprague-Dawley rats (250-300 g body weight) of either sex were anesthetized with urethane (1.2 g / k g i.p.). Femoral artery and femoral vein were cannulated for blood pressure monitoring and for drug administration respectively. H e a r t rate was obtained with a tachometer from the blood pressure pulses (Grass Co., USA). Blood pressure and heart rate were recorded with a Grass 7PD polygraph (Quincy, Grass Co., USA). Once the blood pressure reached steady state, 1-cyclopropyl-isoguanosine (BN-063, 0.05-0.5 m g / k g ) a n d / o r 1-allyl-isoguanosine (AZ-108-1, 0.5-2 m g / k g ) was administered intravenously in a series of doses.

2.4. Materials [3H]Cyclohexyladenosine (specific activity 34.4 C i / mmol) and [3H]CGS 21680 (specific activity 48.6 C i / mmol) were purchased from NEN. The scintillation liquid, Ready Safe, was purchased from Beckman, USA. All other chemicals used were of reagent grade and were purchased from Sigma Chemical Co., USA.

TABLE 1 Adenosine A 1 and A 2 receptor binding affinities for doridosine and its derivatives. Values are means+ S.E.M.

R,x N ~ 2 1 N

H3C~.N~2..N

H3C~.N

~

N

o¢. 27 HO OH Compound 1. Doridosine (R = methyl) 2. BN-063 (R = cyclopropyl) 3. AZ-108-1 (R = allyl) 4. BN-083 (R = isopropyl) 5. AZ-144 (R = cyclohexyl) 6. AZ-079-4 (R = benzyl) 7. AZ-181 8. BN-154

7

8 A 1binding

(Ki, nM)

36 + 3 62+ 4 124+ 5 828 + 7 1,132 + 95 855 + 13 > 1 ~M > 1 /zM

A 2 binding (Ki, nM) 3,625 + 1,067 23,200+ 212 40,2505:1,662 43,167 5:5,414 39,125 d: 1,361 25,775 _+ 230 -

A 2/A 1 ratio 100 374 325 52 35 30 -

137

3. Results

A 0

3.1. Competition binding studies Table 1 shows the Ki values of doridosine and doridosine derivatives at adenosine A~ and A 2 receptors. 1-Cyclopropyl-isoguanosine (BN-063) and 1-allylisoguanosine (AZ-108-1) had greater A 2 / A , K~ ratios (more than 300) than other test compounds, indicating they have greater selectivity towards the adenosine A~ receptors.

-40 ,a

""

-80

-,~ - 1 2 0

3.2. Pharmacological studies <

Since binding studies showed that BN-063 and AZ108-1 had a relatively high selectivity at adenosine A~ receptors, the compounds were further investigated to determine whether they act as adenosine A~ receptor agonists or antagonists. Fig. 1 shows that both BN-063

-160

I1

I

I

I

I

0

30

60

90

120

]3

r.I~D_______.D----~

0

0

-4o -80

A

2OO] ~.: ~1 1OO

BN-063

DPCPX

(0.5 mg/kg)

(0.5 ~/kg) ~

BN-063

(0.5 mg/kg) after 15 mia ,]~

-120 -160

O

-200 <~ - 2 4 0 0

200 100

30

6O

9O

120

Time (min)

5 mln

Fig. 2. Time course of bradycardia induced by intravenous administration of BN-063 (A) and AZ-108-1 (B) at various doses in anesthetized rats. In (A), the doses of BN-063 were: rq : 0.05 mg/kg; o: 0.I mg/kg; o: 0.5 mg/kg. In (B), the doses of AZ-108-1 were: rq: 0.5

mg/kg; o: 1,0 mg/kg; e: 2.0 mg/kg.

B AZ-108-1 (2.0 mg/kg)

~00]

,~

DPCPX (0.5 ag/kg) ~]j

Mtar

AZ-108-1 (2.0 mg/kg) 15 ain ,~

0.: ~ 100 0 3001

~

5 mln

Fig. 1. A representative experiment evaluating the antagonism of DPCPX (0.5 m g / k g i.v.) on bradycardia induced by BN-063 (0.5 m g / k g i.v.) (A) and AZ-108-1 (2.0 m g / k g i.v.) (B) in anesthetized rats.

(0.5 mg/kg) and AZ-108-1 (2.0 mg/kg) caused an initial rapid drop in heart rate which then slowly returned to a level near baseline and remained at this level for at least 2 h. In addition, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective adenosine A 1 receptor antagonist, almost completely blocked the bradycardia induced by BN-063 and AZ-108-1. The dose-response curves for the bradycardiac effect are shown in fig. 2. Both BN-063 and AZ-108-1 only caused an initial transient fall in blood pressure (fig. 1). Hence, in these in vivo pharmacological studies, BN-063 and AZ-108-1 demonstrated characteristic adenosine A~ receptor agonist actions.

138

4. Discussion Adenosine is an endogenous substance that plays an important role in mediating many physiological effects. These effects are primarily attributed to interactions with two subtypes of cell-surface adenosine receptors, A 1 and A 2, that either inhibit (adenosine A I receptor) or stimulate (adenosine A 2 receptor) the activity of adenylate cyclase (Daly, 1982; Jacobson et al., 1992). In view of the important role of the purine receptors in the control and regulation of the cardiovascular system, doridosine was prepared and identified as a mixed adenosine A z and A 2 receptor agonist (Daly, 1982). This discovery led to further synthesis of several analogues of doridosine for pharmacological evaluation (Bartlett et al., 1981). Most structural modifications of this molecule are related to the N-1 position of the base moiety. Previous works by others had indicated that the substituent at the N-1 position cannot be larger than a methyl group (Grozinger et al., 1983). However, in our present studies, we found that when the methyl group was replaced by a cyclopropyl, yielding compound 2 (BN-063), the affinity of this compound toward adenosine A1 receptors was less than half that of doridosine. However, BN-063 showed an improved A 2 / A ~ ratio (374)which is 3-fold better than that of the parent compound. Similarly, when the methyl group was replaced by an allyl, such as in compound 3 (AZ-108-1), adenosine A~ receptor agonist selectivity was again increased. Hydrophobicity is known to play an important role in the binding of a substrate or inhibitor to the active site of an enzyme or receptor; such a hydrophobic region adjacent to the active site of enzyme or receptor has been observed for purine nucleoside phosphorylase (Ealick et al., 1990) and the a~-adrenoceptor (Cotecchia et al., 1988). Exploitation of hydrophobic regions in the active site of pharmacological receptors has commonly led to the discovery of potent ligand-binding agents (Russo et al., 1991; Chern and Rong, 1991). Incorporation of a double bond into a molecule generally increases greatly the molecular hydrophobicity. Thus, doridosine derivatives without the 2',3'-dihydroxy group, such as 2',3'-didehydro-2',3'-dideoxydoridosine (compound 7) and 2',3'-dideoxydoridosine (compound 8) were prepared (Chern et al., 1992) which might provide better affinity to the adenosine receptors. Unfortunately, neither of these two compounds showed any significant pharmacological activity on intravenous administration of 4 mg to rats and exhibited poor binding to the adenosine receptors. Previous approaches to the development of selective adenosine A1 receptor agonists have focused on modifications at the N-6 position of the base moiety of adenosine. However, the results described herein indicated that 1-cyclopropyl isoguanosine is a potent and

selective adenosine A 1 receptor agonist. Although the chemical properties of doridosine (1-methylisoguanosine) and its analogues are similar to those of guanosine with poor solubility, doridosine analogues can induce a profound pharmacological action via the adenosine receptors. Thus, the initial results obtained from the present investigation suggest that the N-1 position of isoguanosine might provide an alternative site for structural modification aimed at the development of selective adenosine A~ receptor agonists.

Acknowledgements This investigation was supported by research grants from the National Science Council of the Republic of China (No. NSC800412-B-016-140R).

References Baird-Lambert, J., J.F. Marwood, L.P. Davies and K.M. Taylir, 1980, 1-Methylisoguanosine an orally active marine natural product with skeletal muscle and cardiovascular effects, Life Sci. 26, 1069. Bartlett, R.T., A.F. Cook, M.J. Holman, W.W. McComas, E.F. Nowoswait, M.S. Poonian, J.A. Baird-Lambert, B.A. Baldo and J.F. Marwood, 1981, Synthesis and pharmacologicalevaluation of a series of analogues of 1-methylisoguanosine,J. Med. Chem. 24, 947. Chern, J.-W. and J.-G. Rong, 1991, 1,2,4-Benzothiadiazine 1,1-dioxide. V: Synthesis of built-in hydroxyguanidinetricycles as potential anticancer agents, Tetrahedron Lett. 32, 2935. Chern, J.-W., G.-S. Lin, C.-S. Chen and LB. Townsend, 1991, Nucleosides. 3: Reactions of AICA-ribosidewith isothiocyanates. A convenient synthesis of isoguanosine and xanthosine derivatives, J. Org. Chem. 56, 4213. Chern, J.-W., G.-S. Lin and C.-S. Chen, 1992, Nucleosides. 4: Synthesis of 2',3'-didehydro-2',3'-dideoxydoridosineand 2',3'-dideoxydoridosine as potential antihypertensive agents, J. Chin. Chem. Soc. 39, 343. Cook, A.F., R.T. Bartlett, R.P. Gregson and R.J. Quinn, 1980, 1-Methylisoguanosine, a pharmacologically active agent from a marine sponge, J. Org. Chem. 45, 4020. Cotecchia, S., D.A. Schwinn, R.R. Randall, R.J. Lefkowitz, M.G. Caron and B.K. Kobilka, 1988, Molecular cloning and expression of the cDNA for the hamster al-adrenergic receptor, Proc. Natl. Acad. Sci. USA 85, 7159. Daly, J.W., 1982, Adenosine receptors: targets for future drugs, J. Med. Chem. 25, 197. Davies, L.P., K.M. Taylor, R.P. Gregson and R.J. Quinn, 1980, Stimulation of guinea-pig brain adenylate cyclase by adenosine analogues with potent pharmacological activity in vivo, Life Sci. 26, 1079. Ealick, S.E., S.A. Rule, D.C. Carter, T.J. Greenhough, Y.S. Babu, W.J. Cook, J. Habash, J.R. Helliwell, J.D. Stoeckler, R.E. Parks, Jr., S.-F. Chen and C.E. Bugg, 1990, Three-dimentional structure of human erythrocyticpurine nucleoside phosphorylase at 3.2 A resolution, J. Biol. Chem. 265, 1812. Grozinger, K., K.R. Freter, P. Farina and A. Gladczuk, 1983, Synthesis of 7- and a-substituted 1-methylisoguanosine from 4(5)amino-5(4)-cyanoimidazole, Eur. J. Med. Chem. Chim. Ther. 18, 221.

139 Jacobson, K.A., P.J.M. Van Galen and M. Williams, 1992, Adenosine receptors: pharmacology, structure-activity relationships, and their potential, J. Med. Chem. 35, 407. Munson, P.J. and D. Rodbard, 1980, LIGAND: a versatile computerized approach for characterization of ligand binding systems, Anal. Biochem. 107, 220. Quinn, R.J., R.P. Gregson, A.F. Cook and R.T. Bartlett, 1980,

Isolation and synthesis of l-methylisoguanosine, a potent pharmacologically active constituent from the marine sponge Tedania digitata, Tetrahedron Lett. 21,567. Russo, F., G. Romeo, S. Guccione and A. De Blasi, 1991, Pyrimido[5,4-b]indole derivatives. 1. A new class of potent and selective t~l-adrenoceptor ligands, J. Med. Chem. 34, 1850