A SURVEY OF NONXANTHINE DERIVATIVES AS ADENOSINE RECEPTOR LIGANDS

Abstract

The binding affinities at rat A₁, A_2a, and A₃ adenosine receptors of a wide range of heterocyclic derivatives have been determined. Mono-, bi-, tricyclic and macrocyclic compounds were screened in binding assays, using either [³H]PIA or [³H]CGS 21680 in rat brain membranes or [¹²⁵I]AB-MECA in CHO cells stably transfected with rat A₃ receptors. Several new classes of adenosine antagonists (e.g. 5-oxoimidazopyrimidines and a pyrazoloquinazoline) were identified. Various sulfonylpiperazines, 11-hydroxytetrahydrocarbazolenine, 4H-pyrido[1,2-a]pyrimidinone, folic acid, and cytochalasin H and J bound to A₃ receptors selectively. Moreover, cytochalasin A, which bound to A₁ adenosine receptors with K_i value of 1.9 μM, inhibited adenylyl cyclase in rat adipocytes, but not via reversible A₁ receptor binding.

Introduction

The A₁, the A_2a, the A_2b and the A₃ adenosine receptors are members of the G-protein-coupled superfamily and have now been defined both on the basis of pharmacological differences¹ and on the basis of distinct amino acid sequences.² Adenosine receptors mediate a wide variety of physiological functions^3–5. These include: Inhibition of neurotransmitter release from nerve endings, vasoconstriction in the kidney, cardiac depression and inhibition of lipolysis via A₁ receptors; vasodilatation, inhibition of platelet aggregation, and inhibition of lymphocyte function via A₂ receptors; potentiation of histamine release from mast cells⁶ resulting in hypotension⁷ via A₃ receptors. The A₁ and A₃ receptors cause inhibition of adenylyl cyclase and activation of phospholipase C. A₁ receptors also couple to activation of potassium channels and inhibition of calcium channels. The A_2a and A_2b receptors activate adenylyl cyclase.

Numerous structure-activity relationship studies at A₁ and A₂ adenosine receptors, aimed at increasing potency and selectivity of agonists and antagonists, have been published (see ref. 1). Although xanthines are the classical adenosine antagonists, numerous classes of nonxanthine antagonists,^1,8 mainly fused nitrogen-containing heterocyclic structures, have been reported. Xanthines have, however, proven to be either inactive at cloned rat A₃ receptors, or relatively inactive at the sheep and human cloned A₃ receptors. The lack of any selective antagonist for the rat A₃ adenosine receptor^9,27 prompted us to undertake a detailed examination of a variety of nonxanthine derivatives as ligands for A₃ and other adenosine receptors. Ligands selective for A₃ receptors¹⁰ have promise as agents for treating ischemia of the brain¹¹ and heart,³ inflammation,⁵ and asthma.⁵ The present study is a survey of some known and some novel classes of nonxanthine adenosine ligands of widely varying structure.

Results and Discussion

Xanthines, the best known class of adenosine antagonists, contain fused 5:6 heterocyclic rings. Caffeine and theophylline, two well known xanthine antagonists have K_i values of approximately 40 and 15 μM, respectively, at both the A₁ and A_2a receptor.¹ No significant antagonist activity has been observed for xanthines or nonxanthine derivatives at the rat A₃ adenosine receptor. Thus, an A₃ antagonist is actively being sought. In addition to screening this diverse group of compounds at the cloned A₃ receptor, they were also tested at rat A₁ and rat A_2a receptors.

Table 1 shows the results of radioligand binding competition experiments at rat brain adenosine receptors for 110 cyclic compounds. Structures of selected compounds of interest, having K_i values at A₁ receptors in the 10⁻⁶ to 10⁻⁴ M range, are shown in Figure 1. Potential antagonism of adenylyl cyclase inhibition mediated by A₁ receptors in rat adipocytes (Table 2) and by cloned rat A₃ receptors expressed in CHO cells⁹ was also examined. Several compounds that inhibited A₁ receptor binding were shown to antagonize the inhibitory effects of N⁶-phenylisopropyladenosine (R-PIA) on adenylyl cyclase in adipocyte membranes (Table 2), with K_B values of ≥1 μM, as calculated using Schild analysis (see below).⁹ Functional assays at A_2a receptors were not carried out. At rat A₃ receptors, although numerous compounds showed K_i values in the range of 10⁻⁵ to 10⁻⁴ M, two of these compounds (6 and 47), examined in the functional assay at cloned rat A₃ receptors coupled to adenylyl cyclase in CHO cells, appeared to have no antagonist activity. The dose-response curve for IB-MECA³⁹ was not shifted in the presence of 40 μM of either 6 or 47. In preliminary experiments, the amino-substituted pteroic acid derivative 68 alone inhibited forskolin-stimulated adenylyl cyclase in A₃ transfected CHO cells, indicating possible agonist properties.

Table 1.

Affinities of heterocyclic derivatives in radioligand binding assays at rat brain A₁, A_2a, and A₃ receptors^a–d.

Ki (μM) or % inhibitiond
Compound	Sourcef	Name	(A1)a	(A2a)b	(A3)c
Non-Fused Rings
1	F	N,N′-(2-Chloro-5-cyano-1,3-phenylene)dioxamic acid (Lodoxamide)	n.d.	n.d.	17%
2	T	N,N-Diethylaminoethyl-2,2-diphenylvalerate (SKF-525A, Proadifen)	11±6%d	e	49.3 ±10.3
3	A	2,6-bis[(4S)-Isopropyl-2-oxazolin-2-yl]pyridine	90.9±5.8	19±5%d	46.2 ± 0.3
4	A	6-(4-Chlorophenyl)-4,5-dihydro-2-(2-hydroxybutyl)-3(2H)-pyridazinone	75.1±20.8	e	22.4 ± 10.0
Fused Bicyclics (6:5)
5	A	(−)-5-Bromo-4-chloro-3-indolyl-β-D-glucoside	25±7%d	e	37.0 ±9.4
6	A	5-Bromo-4-chloro-3-indolyl-β-D-galactoside	18±14%d	e	18.1 ±3.8
7	R	5-Chloro-2(3H)-benzoxazolone (Chlorzoxazone)	21 ±3%	15±4%	34±5%
8	R	Calcimycin	29± 4%	e	24±8%
9	A	1-Methyl-2-phenylbenzimidazole	[23]j	[99]k	19±8% (10−5)
10	R	4-[3-[(1,1-Dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride ((±)-CGP-12177A)	23±3%	40±3%	109±5
11	Bo	2-[2-Methoxy-4-(methylsulfinyl)phenyl]-1H-imidazo[4,5-b]pyridine (Sulmazole, ARL115)	[52]j	22.7±8.6	15±0% (10−5)
12	D	1,4,6-Trimethyl-1H-pyrazolo-pyridine	[>100]j	e	22±0% (10−5)
13	D	Ethyl 4-phenylamino-1-methyl-pyrazolopyridine-5-carboxylate	[1.1]i	[2.4]i	43±2%
14	D	Ethyl 4-benzylamino-1-methyl-pyrazolopyridine-5-carboxylate	[1.0]i	[2.5]i	29±3% (10−5)
15	D	Ethyl 4-phenylethylamino-1-methyl-pyrazolopyridine-5-carboxylate	[4.3]i	[12]i	26±6%
16	D	Ethyl 4-ethoxy-1-methyl-pyrazolopyridine-5-carboxylate	[22]i	[19]i	15 ± 2%
17	M	Ethyl 1,3-dimethyl-4-(4-methoxyphenoxy)-1H-pyrazolo-[3,4-b]pyridine	9.39±0.95	11.7 ± 2.0	e
18	M	Ethyl 1,3-dimethyl-4-(4-fluorophenoxy)-1H-pyrazolo-[3,4-b]pyridine-5-carboxylate	6.95±1.10	9.24 ± 1.04	e
19	M	Ethyl 4-[3,5-dichlorophenoxy]1,3-dimethyl-1H-pyrazolo [3,4-b]pyridine-5-carboxylate	22.8 ± 6.1	e	e
20	C	5-(Hydroxymethyl)-4,5,6,7-(tetrahydro)imidazo-[4,5-c]pyridine-6-carboxylic acid	e	16 ± 2%d	86.1 ± 3.4
21	C	5-Oxo-(5,6-dihydro)imidazo[1,5-c]pyrimidine-7-carboxylic acid methyl ester	73.6±24.6	24 ± 2%d	n.d.
22	C	1-Chloro-5-oxo-(5,6-dihydro)imidazo[1,5-c]pyrimidine-7-carboxylic acid methyl ester	60.6 ± 20.7	85.1 ± 1.4	36.8 ± 1.0
23	C	1-Chloro-5-oxo-(5,6,7,8-tetrahydro)imidazo[1,5-c]pyrimidine-7-carboxylic acid methyl ester	26 ± 3%d	14 ± 7%d	n.d.
24	C	1,3-Dichloro-5-oxo-(5,6 dihydro)imidazo[1,5-c]pyrimidine-7-carboxylic acid methyl ester	10.3 ± 2.7	16.5 ± 3.0	53.0 ± 13.0
25	C	5-Oxo-(5,6,7,8-tetrahydro)imidazo[1,5-c]pyrimidine	e	22 ± 1 %d	53.5 ± 15.8
26	C	1,3-Diiodo-5-oxo-(5,6,7,8-tetrahydro)imidazo[1,5-c]pyrimidine-7-carboxylic acid methyl ester	8.48 ± 0.84	35.9 ± 5.9	n.d.
27	C	1-Iodo-5-oxo-(5,6,7,8-tetrahydro)imidazo[1,5-c]pyrimidine-7-carboxylic acid methyl ester	e	27 ± 4%d	n.d.
28	C	1-Bromo-5-oxo-(5,6,7,8-tetrahydro)imidazo[1,5-c]pyrimidine-7-carboxylic acid methyl ester	11 ±4%d	e	n.d.
29	C	6-t-Butyloxycarbonyl-8-chloro-5-oxo-(5,6,7,8-tetrahydro)imidazo[1,5-c]pyrimidine-7-carboxylic acid methyl ester	e	24±7%	n.d.
30	C	5-Oxo-(5,6,7,8-tetrahydro)imidazo[1,5-c]pyrimidine-7-carboxylic acid benzyl ester	e	35±8%d	n.d.
31	C	5-Oxo-(5,6,7,8-tetrahydro)imidazo[1,5-c]pyrimidine-7-carboxylic acid methyl ester	e	e	n.d.
32	C	7-(Methoxycarbonyl)-2-phenylmethyl-5-oxo-(5,6,7,8-tetrahydro)imidazo[1,5-c]pyrimidinium bromide	440	e	n.d.
33	E	4-Amino-5,6,-dimethyl-2-phenyl-7H-pyrrolo-[2,3-d]pyrimidine	[1.49]g	[21.3]l	15% (10−5)
34	E	4-Amino-5,6,-dimethyl-2-phenyl-7H-7-(phenyl)pyrrolo-[2,3-d]pyrimidine	[0.036]g	[14.3]l	25 ± 2% (10−5)
35	M	Ethyl 6-(4-chlorophenyl)-4-methyl pyrazolo[1,5-a] pyrimidine 3-carboxylate	12 ± 1%	e	e
36	M	5,7-bis(Trifluoromethyl)-3-cyano-2-(methylthio)pyrazolo-[1,5-a]pyrimidine	e	e	e
37	G	Anhydro-2-phenyl-6,8-diethyl-5-hydroxy-7-oxo-1,3,4-thiadiazolo[3,2-a]pyrimidinium hydroxide	[80]h	23±6%	55.6 ± 17.1
38	D	2-Phenyl-4,6-dimethyl thiazolopyrimidine-5,7-dione	[79]j	[130]k	31 ±7% (10−5)
39	C	8-(Heptatluoropropyl)adenine	e	27±3%d	99.5 ±11.5
40	C	8-(Heptafluoropropyl)guanine	e	24±1%d	77.7 ±27.1
41	R	6-[(1-Methyl-4-nitro-1H-imidazol-5-yl)thio]-1H-purine (Azathioprine)	18±1%	28±5%	116±3
42	P	8-Methyl-6-(1-piperidinlyl)-1,2,4-triazolo[4,3-b]pyridazine (MDL 257)	[>100]	[0.9]k	32±6% (10−5)
43	P	6- (Morpholinyl)-1,2,4-triazolo [4,3-b]pyridazine (MDL 850)	[22]	11.2±2.5	21±9% (10−5)
44	Mu	7,9-Dibenzyl-1-methylxanthinium chloride	20%	23±6%	25±4% (10−5)
45	G	Anhydro-1,6-dimethyl-5-hydroxy-6-propyl-1,3,4-triazolo[3,2a]pyrimidinium hydroxide	[≫250]h	e	20±10% (10−5)
46	R	1-Methylisoguanosine	n.d.	1.97±0.56	25.7 ± 0.1
Fused Bicyclics (6:6)
47	A	Esculin	27±5%d	32±5%d	101 ± 29
48	F	Disodium chromoglycate	n.d.	n.d.	19%
49	A	8-(β-D-Glucopyranosyloxy)-7-hydroxyl-6-methoxycoumarin	15±7%d	e	104±6
50	A	Hesperidine	22±3%d	e	263 ± 34
51	R	5,7-Dichloro-4-hydroxy-quinoline-2-carboxylic acid (5,7-Dichlorokynurenic acid)	22±5%	e	38±3%
52	T	1-(5-Chloronaphthalenesulfonyl) piperazine (ML-9)	e	e	96.2 ± 21.0
53	T	1-(5-Iodonaphthalenesulfonyl) piperazine (ML-7)	e	e	119 ± 67
54	T	1-(5-Isoquinolinesulfonyl) piperazine (HA-100)	e	e	24.5±10.0
55	T	1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7)	e	e	50.9 ± 15.2
56	T	1-(5-Isoquinolinesulfonyl)-3-methylpiperazine	e	e	59.0 ± 21.8
57	A	1-(5-Isoquinolinesulfonyl) homopiperazine (HA-1077)	23±9%d	e	43.3 ±13.9
58	T	1-[5-(8-Chloro-isoquinoline)sulfonyl]piperazine (HA-156)	e	e	40.4 ± 12.9
59	R	6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX)	25±2%	e	36±9%
60	R	6-Chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide (Chlorothiazide)	e	24±5%	34±4%
61	R	7-Benzyl-1-ethyl-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic acid (Amfonelic acid)	25.2±7.5	e	157±3
62	W	4H-Pyrido[1,2-a]-pyrimidin-4-one	e	e	48.3 ± 4.8
63	D	1,3-Dipropyllumazine	[20]j	24±7%	46.1±3.8
64	D	1,3-Dimethyl-7-phenyllumazine	32.9±7.1	107±8	20±1% (10−5)
65	R	Neopterin	e	e	n.d.
66	A	2, 6-Diamino-6-(hydroxymethyl)-pteridine	14±7%d	e	135 ± 21
67	A	2, 4-Diamino-6,7-diisopropyl-pteridine	2.51 ± 0.47	11.9±3.2	71 ± 14%
68	A	4-[N-(2,4-Diamino-6-pteridinylmethyl)amino]benzoic acid	e	e	48.2±7.5
69	A	4-[N-(2,4-Diamino-6-pteridinylmethyl)-N-methylamino]benzoic acid	27±1 %d	e	48.5 ± 10.0
70	A	Folic acid	e	20.3%	28.4 ± 9.9
71	A	Dihydrofolic acid	33.8±2.3	e	45.9 ± 18.0
72	A	Dipyridamole	22±17%d	54±2%	19.0 ± 0.7
Fused Bicyclic (>6)
73	W	1-Aza-8,9-benzcyclononadi-2,7-one	22.5%	e	70.5 ± 0.7
74	Br	7-Methyl-4-propyl-4,5,6,7-tetrahydro-6H-imidazo[4,5e][1,4]diazepine-5,8-dione	19±2%	16±7%	27±3% (10−5)
75	Br	7-Benzyl-1,4-dipropyl-4,5,6,7-tetrahydro-6H-imidazo[4,5e][1,4]diazepine-5,8-dione	13.4±2.1	2.18±0.12	e(10−5)
Cytochalasins (Macrocyclics)
76	A	Cytochalasin A	1.91±0.43	29±9%	see text
77	A	Cytochalasin B	27.0±3.8	e	47.5±21.8
78	A	Cytochalasin C	e	e	18±2% (10−5)
79	A	Cytochalasin D	e	e	53.7 ± 11.8
80	A	Cytochalasin E	e	10±1%	155 ± 56
81	A	Cytochalasin H	e	16±7%	23.0 ± 7.1
82	A	Cytochalasin J	e	19±8%	27.9 ± 11.9
Fused Tricyclics (5:6:5)
83	P	1,7-Dihydro-3,5-dimethylbenzo[1,2-c:5,4-c′]dipyrazole (MDL 26,020)	[8.0]m	[25.6]k	12±2% (10−5)
84	P	1,7-Diethyl-1,7-dihydro-3,5-dimethylbenzo[1,2-c:5,4-c′]dipyrazole (MDL 26,629)	[27]m	[56]k	e(10−5)
85	P	1-Hydro-3,6-dimethylbenzo-[1, 2-c: 5, 4-c′]dipyrazole (MDL 26,687A)	[74]	[17]k	55.7 ± 22.4
Fused Tricyclics (6:5:6)
86	R	Methyl 6,7-dimethoxy-4-ethyl-β-carboline-3 carboxylate (DMCM)	1.57±0.32	3.33±0.67	49.2 ± 16
87	E	4-Hydroxy-5,6,7,8-tetrahydro-9-phenyl-9H-pyrimido[4,5-b]indole	[2.07]g	1.49±0.51	15±1% (10−5)
88	W	11-Hydroxytetrahydrocarbazolenine	e	20%	21.9±6.5
89	G	Anhydro-1-cyclopropylmethyl-3-ethyl-2-hydroxy-4-oxo-pyrimido[2,1a] benzothiazolium hydroxide	[37]h	6.15±0.69	53.4 ± 16.1
90	E	4-Hydroxy-9-phenyl-9H-pyrimido[4,5-b]indole	[0.88]g	[1.44]l	18% (10−5)
Fused Tricyclics (6:6:5)
91	-	lin-Benzohypoxanthine	21.2±4.9	0.992	26±8%
92	Pf	1,3-Dimethyl-7-phenyl-6H-imidazo-(4,5-g)lumazine	e	e	n.d.
93	P	2,11-Dihydro-11-(4-morpholinyl)-6H-pyrimido [2,1-b]quinazolin-6-one (MDL 43400A)	[>100]	[160]k	12±1% (10−5)
94	Pf	1,3,6-Trimethyl-7-(3,4-dichlorophenyl)-imidazo-(4,5-g)lumazine	n.d	n.d.	15% (10−5)
95	M	Ethyl 5-chloropyrazolo[1,5-a]quinazoline-3-carboxylate	5.36±0.36	4.06±0.50	e
Fused Tricyclics (6:6:6)
96	A	Alloxazinen	n.d.	n.d.	32±3%
97	T	Roseoflavin	28.9±4.0	e	84±8
98	B	Riboflavin	12.7±2.9	e	see text
99	S	Flavin adenine dinucleolide (FAD)	33.8±12.0	18.3±2.5	e
100	S	Fluorescein	76.1±6.8	34±5%	37±2%
101	A	Carminic acid	21±9%d	e	117 ± 22
Fused Tricyclics (>6)
102	T	Doxepin	66.4±16.8	e	72±14%
103	W	R,S-6-Hydroxy-7,8,9,10-tetrahydro-6H-cyclohept-[b]indole	e	n.d.	68.2 ± 23.8
104	W	5,6,8,9,10,11-Hexahydro-7H-cyclohepta[c]-quinolin-6-one	45±7%	e	n.d.
Fused Rings (Misc.)
105	M	Cedrol	28± 2%	34±10%	50 ± 0%
106	M	Cedrene	18± 3%	e	26± 1%
107	A	Reserpine	e	25.2%	n.d.
108	T	Podophyllotoxin	31±8%d	e	79±11%
109	T	4′-Dimethylepipodophyllotoxin	18±5%d	e	70±15%
110	D	Mitragynineo	>100	75.6±28.0	55±2%

n.d.: not determined.

^aDisplacement of specific [³H]PIA binding, unless noted, in rat brain membranes expressed as K_i ± S.E.M. in μM (n = 3–5).

^bDisplacement of specific [³H]CGS 21680 binding, unless noted, in rat striatal membranes, expressed as K_i ± S.E.M. in μM (n = 3–6).

^cDisplacement of specific [¹²⁵I]AB-MECA binding, unless noted, in membranes of CHO cells stably transfected with the rat A₃-cDNA, expressed as K_i ± S.E.M. in μM (n = 3–5).

^dA percent value indicates the percent displacement of radioligand at the concentration (M) given in parentheses or at 10⁻⁴M, if none specified.

^e≤10% displacement of radioligand.

^fA = Aldrich (Milwaukee, WI); B = BioRad; Bo, Boehringer-Ingelheim (Germany); Br = Prof. Peter Bridson (Univ. Memphis); C = Dr. Louis Cohen (NIH), D = Dr. John W. Daly (NIH); E = Prof. Kurt Eger (Univ. Tübingen, Germany); F = Dr. John Fozard (Sandoz, Geneva); G = Prof. Richard Glennon (Medical College of Virginia, Richmond); M = Maybridge (Trevillett, UK); Mu = Dr. Christa Müller (Univ. Tübingen); P = Dr. Norton Peet (Marion Merrell Dow, Cincinnati OH); Pf = Prof. Wolfgang Pfleiderer (Univ. Konstanz); R = RBI (Natick MA); S = Sigma (St. Louis MO), T = Toronto Research Chemicals (Toronto); W = Dr. B. Witkop (NIH).

^gref. 33.

^href. 34.

ⁱref. 12.

^jref. 8.

^kK_B versus NECA-stimulation of adenylate cyclase in human platlets.

^lK_i versus [³H]NECA in rat striatal membranes.

^mref. 35.

ⁿref. 40.

^oref. 38.

Fig. 1.

Structures of selected adenosine receptor ligands.

Table 2.

Antagonism of A₁ receptor-mediated inhibition of adenylyl cyclase in rat adipocycte membranes.^a

Compoundb	KB ± SEM (μM)
4	600
18	17.1 ± 3.6
24	10.8 ± 3.0
26	600
61	600
64	46.7 ± 11.9
67	3.94 ± 0.79
77	81
86	1.36 ± 0.48
95	12.7 ± 2.95

^aCalculated from shift in dose-response curve to R-PIA (see Figure 3), using the Schild equation⁸ (n=3–5, or for single determination).

^bCompound 71 (50 μM) caused a slight shift of the R-PIA dose-respose curve to the left. Compound 91 (50 μM) antagonized the effects of R-PIA only weakly, thus a K_B could not be determined.

Numerous fused ring compounds and a few nonfused ring compounds were examined in binding assays in the present study. Among nonfused ring compounds examined, only compounds 3 and 4 were weak competitors at A₁ receptors. A number of fused 5:6 heterocyclic ring compounds showed moderate receptor affinity. The effects of certain pyrazolopyridines¹², compounds 17 – 19, in binding were studied. They had K_i values around 10 μM at both A₁ and A_2a receptors. No binding activity was detected at A₃ receptors. Related pyrazolopyridines, such as tracazolate, have been reported to have anxiolytic activity, and the A₁ and A_2a receptor antagonist activity of a large series of pyrazolopyridines has been reported.¹²

Imidazopyrimidine-7-carboxylic acid derivatives, compounds 24 and 26, have K_i values of 10 and 8 μM, respectively at A₁ receptors. This class of histidine derivatives has been prepared in connection with potential antimalarial activity.¹³ Compound 42 is a triazolopyridazine bronchodilator³⁶ that did not bind appreciably to adenosine receptors.

We have determined the K_i value of a nucleoside analogue, 1-methylisoguanosine, 46, at A_2a and A₃ receptors to be 2.0 μM and 25 μM, respectively. This compound was reported previously to bind to adenosine receptors as an agonist.¹⁴

The sulfonylpiperazine derivatives 52–58 displayed no binding activity at A₁ and A_2a receptors. However, they did displace radioligand binding weakly at A₃ receptors. These compounds are members of a well-known class of inhibitors of protein kinase C activity, acting in the concentration range of 0.1 – 1 μM.¹⁵

A number of fused 6:6 bicyclic heterocycles displayed affinity at adenosine receptors. The K_i value of amfonelic acid, 61, at the A₁ receptor was 25 μM. Amfonelic acid has been reported to induce seizures,¹⁶ and at that time it was not realized that the same compound binds to adenosine receptors with an affinity similar to that of caffeine, another proconvulsant agent. 4H-Pyrido[1,2-a]pyrimidin-one, 62, bound selectively to rat A₃ receptors with a K_i value of 48 μM. A phenyllumazine derivative, 64, which could be considered an analogue of the A₁/A₂ antagonist 8-phenyltheophylline,¹ bound to A₁ receptors with a K_i value of 33 μM.

Various pteridine derivatives were shown to act as antagonists at adenosine receptors.⁸ We have found that the presence of chained alkyl groups (e.g. isopropyl) at the 6- and 7- positions enhance the potency of binding to A₁ receptors in this series. Thus, compound 67 has a K_i value of 2.5 μM. Pteridine derivatives 68 and 69, related to the anticancer and antiinflammatory drug methotrexate, displaced radioligand from A₃ receptors with K_i values of 48 μM. Another related heterocycle, dihydrofolic acid, 71, bound to A₁ receptors with a K_i value of 34 μM, while curiously the more planar folic acid, 70, itself, was inactive at A₁ or A_2a receptors. The adenosine uptake blocker dipyridamole, 72, bound to A₃ receptors with a K_i value of 19 μM.

Surprisingly, several of the cytochalasins showed considerable affinity at adenosine receptors. Cytochalasin A, 76, was the most potent of the series with a K_i value of 1.9 μM at A₁ receptors. Cytochalasin A caused an increase in the amount of [¹²⁵I]AB-MECA bound in membranes of A₃-transfected CHO cells (150±16% of control at 300 μM, n = 4). In the presence of 100 μM NECA, the level of radioligand binding was 59±8% of control, thus the additional binding likely occurs at a non-A₃ receptor site. Cytochalasin B, 77, displayed a K_i value of 27 μM at A₁ receptors. The other cytochalasins, 78–82, were totally inactive in binding at A₁ receptors. None of the cytochalasins bound appreciably at A_2a receptors. Given the pharmacological difference between cytochalasins A and B and their subtle differences in molecular structure, the region of the molecule which is responsible for binding to or modulating binding to the adenosine receptors is not apparent. The cytochalasins are macrocyclic compounds that are fungal metabolites, used as tools in cytological research and in characterization of polymerization properties of actin.¹⁷

A number of benzodipyrazole derivatives, 83 and 84, studied previously at adenosine receptors³⁵ lacked affinity at A₃ receptors. A related compound, 85, had a K_i of 56 μM at A₃ receptors.

Methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), 86, a β-carboline derivative, displayed considerable affinity for adenosine receptors, with K_i values of 1.6 and 3.3 μM at A₁ and A_2a receptors, respectively. DMCM acts as a proconvulsant, by virtue of its activity as a benzodiazepine inverse agonist.¹⁸ Previously β-carboline itself and several derivatives⁸ were shown to have weak affinity at the A₁ receptor and to act as antagonists at that receptor, and this was proposed to be related to their proconvulsant activity. Since the potency of DMCM is even greater than other β-carbolines examined,⁸ it becomes an even more relevant aspect of the biological effects. DMCM was used to induce seizures in a study of the anticonvulsant effect of adenosine agonists,¹⁹ apparently without knowledge of its potency in adenosine antagonism. Another carbazole derivative, 11-hydroxytetrahydrocarbazolenine, 88,²² was A₃ selective in binding with a K_i value of 22 μM.

A series of lin-benzoxanthine derivatives was shown to have adenosine antagonist activity.²⁰ lin-Benzohypoxanthine, 91,²¹ formalistically an elongated derivative of unsubstituted hypoxanthine, showed considerable affinity at adenosine receptors, although it was nonselective. An imidazoquinazolinone derivative, 93,³⁷ lacked affinity at adenosine receptors. Another 6:6:5 fused tricyclic, compound 95, a pyrazoloquinazoline derivative, showed an affinity of 4–5 μM at A₁ and A_2a receptors, with no displacement of radioligand from A₃ receptors.

Fused 6:6:6 tricyclic derivatives were also examined. Alloxazine, 96, has been found to be 9-fold selective for A_2b (Kg 2.3 μM) vs A_2a receptors.⁴⁰ The naturally occurring flavins contain the same ring structure as alloxazine with an appended carbohydrate mioety. Riboflavin,²³ 98, a vitamin and enzyme cofactor having an attached open chain ribose moiety, and roseoflavin, 97, the homologous derivative with a shorter carbohydrate chain (C3), bound to A₁ receptors with K_i values of 13 and 29 μM, respectively, with no detectable binding at A_2a receptors. Unexpectedly, the binding of the A₃ receptor radioligand was dramatically enhanced (Figure 3), with 355±35% of control binding in membranes of A₃-transfected CHO cells at 100 μM riboflavin. Since the additional binding occurred also in the presence of 100 μM NECA (280±40% of control binding), it does not represent selective binding enhancement at A₃ receptors, as has been shown for benzoylthiophene derivatives (allosteric enhancers) at A₁ receptors.²⁴ Instead, riboflavin apparently causes enhanced binding of [¹²⁵I]AB-MECA to a nonreceptor site on the membranes. The nature of this site has not been explored, but perhaps it is related to an enzyme at which riboflavin acts as a cofactor. The adenosine conjugate FAD, 99, was somewhat weaker than riboflavin in binding to A₁ receptors, yet bound with increased affinity at A_2a receptors. Fluorescein dye, 100, also bound weakly to adenosine receptors with a K_i value of 76 μM at the A₁ subtype.

Fig. 3.

Effects on A1-agonist-induced inhibition of adenylyl cyclase in rat adipocyte membranes.

The antidepressant drug doxepin,²⁵ 102, is a dibenzoxepin derivative. This compound showed a K_i value of 66 μM at A₁ receptors. Other psychotropic drugs, such as barbiturates, were previously shown to bind weakly to adenosine receptors.²⁶

Functional assays at rat adipocyte A₁ receptors were carried out (Table 2, and Figures 3 and 4). The compounds that bound with highest affinity were examined for the ability to antagonize the inhibition of adenylyl cyclase elicited by R-PIA. It was found that DMCM, 86, and the pteridine derivative 67, were the most potent antagonists, with micromolar K_B values. The cyclized histidine derivative (a 5-oxoimidazopyrimidine) 24, the pyrazoloquinazoline 95, the pyrazolopyridine 18, and the phenyllumazine derivative 64 were also weak antagonists. lin-Benzohypoxanthine, 91, slightly antagonized the effects of R-PIA. Cytochalasin A, 76 (10 μM), shifted the R-PIA dose response curves to the left and alone inhibited adenylyl cyclase in the adipocyte membranes with an IC₅₀ of 6 μM (Figure 4). However, the inhibition by cytochalasin A was not reversed in the presence of the selective A₁-antagonist l,3-dipropyl-8-cyclopentylxanthine, 1 μM (data a not shown). Thus, cytochalasin A appears to have a direct inhibitory effect not mediated through A₁ receptors. Doxepin, 102 (50 μM), clearly shifted the R-PIA dose response curve to the left, however alone did not inhibit adenylyl cyclase. Thus doxepin is not an agonist at A₁ receptors, but it may act to enhance agonist-induced effects. If so, it is conceivable that this may occur at an allosteric site. Dihydrofolic acid, 71, produced a slight left shift of the R-PIA dose response curve, but 71 alone had no effect on adenylyl cyclase in adipocyte membranes.

Fig. 4.

Inhibition of adenylyl cyclase in rat adipocyte membranes.

Conclusions

A selective antagonist at the rat A₃ receptor is lacking. Several xanthine analogs that act as potent antagonists at rat, rabbit, and human A₁ and A₂ receptors only weakly displaced the binding of radioligand from cloned rat A₃ receptors.²⁷ The present study has not identified any effective A₃ antagonist in the rat, however, it has provided leads for future structural modification.

Sulfonylpiperazines, 52–58, and cytochalasin H and J, 81 and 82, respectively, bind to A₃ receptors without binding to the other subtypes. Moreover, compounds 2, 6, 25, 62, 68, 70, 72, and 88 also were somewhat selective in binding to A₃ receptors. The pteridine derivative, 67, was identified as a slightly A₁ selective (binding) antagonist. Compounds 26, 76, 77, 97, and 98 were also weakly A₁ selective. Compounds 11, 43, 75, and 89 were identified as slightly A_2a selective ligands.

The most significant new findings are the discovery of several new classes of adenosine antagonists (e.g. 5-oxoimidazopyrimidines and a pyrazoloquinazoline), and that nonpurine heterocycles (e.g. 68 at A₃ receptors and 76 at A₁ receptors) inhibit adenylyl cyclase, possibly through activation of adenosine receptors. It will be necessary to explore the mechanism of the inhibition of adenylyl cyclase, since action at the P site would also have this effect.³⁹ Previously it was observed¹ that only purine nucleosides were known to activate adenosine receptors.

Experimental

Compound 91 was synthesized as described by Leonard and coworkers.²¹

Cell culture and radioligand binding

CHO cells stably expressing the A₃ receptor^9,28 were grown in F-12 medium containing 10% FBS and penicillin/streptomycin (100 U/mL and 100 μg/mL respectively) at 37 °C in a 5% CO₂ atmosphere, and membrane homogenates were prepared as reported.²⁸

Binding of [¹²⁵I]4-amino-3-iodobenzyladenosine-5′-N-methyluronamide ([¹²⁵I]-AB-MECA) to the CHO cell membranes was performed as described.²⁹ Assays were performed in 50/10/1 buffer in glass tubes and contained 100 μL of the membrane suspension, 50 μL of [¹²⁵I]AB-MECA (final concentration 0.3 nM), and 50 μL of inhibitor. Inhibitors were routinely dissolved in DMSO and were then diluted with buffer; final DMSO concentrations never exceeded 1%. Incubations were carried out in duplicate for 1 hour at 37 °C, and were terminated by rapid filtration over Whatman GF/B filters, using a Brandell cell harvester (Brandell, Gaithersburg, MD). Tubes were washed three times with 3 mL of buffer. Radioactivity was determined in a Beckman gamma 5500B counter. Nonspecific binding was determined in the presence of 40 μM R-PIA. K_i -values were calculated according to Cheng-Prusoff,³⁰ assuming a K_d for [¹²⁵I]AB-MECA of 1.55 nM.²⁹

Binding of [³H]PIA (Amersham, Arlington Heights, IL) to A₁ receptors from rat brain membranes and of [³H]CGS 21680 (DuPont NEN, Boston MA) to A_2a receptors from rat striatal membranes was performed as described previously.^{31, 32} Adenosine deaminase (3 U/mL) was present during the preparation of brain membranes, in which an incubation at 30°C for 30 min was carried out, and during the incubation with radioligand. At least six different concentrations spanning three orders of magnitude, adjusted appropriately for the IC₅₀ of each compound, were used. The IC₅₀ values that were computer-generated using a nonlinear regression formula on the InPlot program (GraphPAD, San Diego CA), were converted to apparent K_i values using K_d values of 1.0 and 14 nM for [³H]PIA and [³H]CGS 21680 binding, respectively, and the Cheng-Prusoff equation.³⁰

Adenylyl cyclase measurements in rat adipocyte membranes and in A₃-transfected CHO cells were carried out as described.^8,28

Figure 2.

Enhancement of radioligand (0.4 nM) binding in A3-transfected CHO cell membranes by riboflavin, 98.

Abbreviations

AB-MECA

N⁶-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide

CGS 21680

2-[4-[(2-carboxyethyl)phenyl]ethyl8amino]-5′-N-ethylcarboxamido-adenosine

CHO

Chinese hamster ovary

DMCM

methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate

DMSO

dimethylsulfoxide

PIA

R-N⁶-phenylisopropyladenosine

Tris

tris(hydroxymethyl)aminomethane