6-Phenyl-1,4-dihydropyridine Derivatives as Potent and Selective A₃ Adenosine Receptor Antagonists

Abstract

An approach to designing dihydropyridines that bind to adenosine receptors without binding to L-type calcium channels has been described. 1,4-Dihydropyridine derivatives substituted with β-styryl or phenylethynyl groups at the 4-position and aryl groups at the 6-position were synthesized and found to be selective for human A₃ receptors. Combinations of methyl, ethyl, and benzyl esters were included at the 3- and 5-positions. Affinity was determined in radioligand binding assays at rat brain A₁ and A_2A receptors using [³H]-(R)-PIA [[³H]-(R)-N⁶-(phenylisopropyl)adenosine] and [³H]CGS 21680 [[³H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5′-(N-ethylcarbamoyl)adenosine], respectively. Affinity was determined at cloned human and rat A₃ receptors using [¹²⁵I]AB-MECA [N⁶-(4-amino-3-iodobenzyl)-5′-(N-methylcarbamoyl)-adenosine]. Structure-activity analysis indicated that substitution of the phenyl ring of the β-styryl group but not of the 6-phenyl substituent was tolerated in A₃ receptor selective agents. Replacement of the 6-phenyl ring with a 3-thienyl or 3-furyl group reduced the affinity at A₃ receptors by 4- and 9-fold, respectively. A 5-benzyl ester 4-trans-β-styryl derivative, 26, with a K_i value of 58.3 nM at A₃ receptors, was >1700-fold selective vs either A₁ receptors or A_2A receptors. Shifting the benzyl ester to the 3-position lowered the affinity at A₃ receptors 3-fold. A 5-benzyl, 3-ethyl ester 4-phenylethynyl derivative, 28, displayed a K_i value of 31.4 nM at A₃ receptors and 1300-fold selectivity vs A₁ receptors. The isomeric 3-benzyl, 5-ethyl diester was >600-fold selective for A₃ receptors. Oxidation of 28 to the corresponding pyridine derivative reduced affinity at A₃ receptors by 88-fold and slightly increased affinity at A₁ receptors.

The physiological role of A₃ adenosine receptors,^1–4 the most recently cloned receptors of the adenosine,^5,6 is under investigation, and potent and selective antagonists are needed as pharmacological probes and as potential therapeutic agents. Activation of the A₃ receptor in the rat results in hypotension⁷ by promotion of release of inflammatory mediators from mast cells.⁸ IB-MECA, a selective A₃ receptor agonist,⁴ was shown to inhibit the release of TNF-α in macrophages.⁹ A₃ adenosine receptor antagonists are being sought as potential antiasthmatic,¹⁰ antiinflammatory,¹⁰ and cerebroprotective agents.¹¹ Selective A₃ receptor agonists at high concentrations were found to induce apoptosis in a HL-60 human leukemia cell line.¹² There may also be an involvement of A₃ receptors in the cytolytic activity of antitumor killer lymphocytes.¹³

1,4-Dihydropyridine blockers of L-type calcium channels¹⁴ are used extensively in the treatment of cardiovascular disorders as dilators of coronary arteries. We have found that, in addition to binding to calcium channels, this class of 1,4-dihydropyridines tends to bind to three subtypes of adenosine receptors, i.e. A₁, A_2A, and A₃.^15,16 For example, nifedipine (Figure 1, 1) is bound by these three adenosine receptors with micromolar affinity, although it is much more potent at L-type channels. A newer generation calcium channel blocker, nicardipine,¹⁷ 2, within the family of adenosine receptors is actually selective for the A₃ subtype. The (S)-enantiomer of niguldipine, 3, which is the more potent enantiomer at L-type channels, binds to human A₃ adenosine receptors with a K_i value of 2.8 μM and is totally inactive at A₁ and A_2A receptors. Thus, with respect to adenosine receptors alone it is highly specific for the A₃ subtype.

Figure 1.

Structures of 1,4-dihydropyridines as potent calcium channel antagonists (1-3) and an adenosine receptor antagonist (4). K_i values (μM) are shown for rat brain A₁ receptors (rA₁) and for cloned human A₃ receptors (hA₃).

We have recently described approaches to designing 1,4-dihydropyridines that bind to adenosine receptors without binding to L-type calcium channels.¹⁶ Furthermore, the moderate affinity for A₃ adenosine receptors of readily available 1,4-dihydropyridines has provided leads for novel antagonists for that subtype. Inclusion of 4-trans-β-styryl and 6-phenyl substituents enhanced A₃ receptor selectivity in an additive fashion and completely abolished recognition at L-type channels. Compound 4 (3,5-diethyl 2-methyl-6-phenyl-4-[2-phenyl-(E)-vinyl]-1,4-(±)-dihydropyridine-3,5-dicarboxylate, MRS 1097) was 55-fold selective for human A₃ receptors vs rat A₁ receptors and 44-fold selective vs rat A_2A receptors. In a functional assay, compound 4 attenuated the A₃ agonist-elicited inhibitory effect on adenylyl cyclase. Furthermore, whereas nicardipine, 2, displaced radioligand ([³H]-S-(4-nitrobenzyl)-6-thioinosine) from the Na⁺-independent adenosine transporter with an apparent affinity of 5.4 μM, compound 4 displaced less than 10% of total binding at a concentration of 100 μM. The selectivity of compound 4 for adenosine receptors was further demonstrated through its inactivity in binding assays at other receptors, ion channels, and second messenger components. The present study examines combinations and compatibility of dihydropyridine substituents found previously to enhance affinity at A₃ receptors. New derivatives of exceptionally high potency and selectivity have been discovered.

Results

Synthesis.

The structures of the 1,4-dihydropyridines (4-31) and related pyridine derivatives (32–34) tested for affinity in radioligand binding assays at adenosine receptors are shown in Table 1. New dihydropyridine derivatives were synthesized using standard methodology (Scheme 1),^18–20 as reported in Table 2. The synthesis consisted of the Hantzsch condensation of a 3-amino-2-butenoate ester, 35a,b, an aldehyde, 36a-h, and a 3-ketopropionate ester derivative, 37a-i, that were dissolved in ethanol and refluxed under N₂. In order to obtain substitution at the 4-position the aldehyde component, 36a-h, was varied. Substitution at the 6-position was achieved by varying the 3-ketopropionate ester component, 37e-g,i (Scheme 2), prepared according to method of Straley and Adams.²¹ In the synthesis of compounds 29-31, the precursor, ethyl 3-aminocinnamate was prepared from the imidate hydrochloride and Meldrum’s acid (Scheme 3).²² The yields of the 6-phenyl-1,4-dihydropyridines obtained using a ≤72 h reaction time varied between 6 and 63%. All of the dihydropyridines examined in this study were racemic mixtures at position C-4.

Table 1.

Affinities of Dihydropyridine and Pyridine Derivatives in Radioligand Binding Assays at A₁, A_2a, and A₃ Receptors^a-f

					Ki (μM) or % inhibitiond
compd	R3	R4	R5	R6	rA1a	rA2Ab	hA3c	rA1/hA3
5 e	CH3	CH3	CH2CH3	CH3	32.6 ± 6.3	46.1 ± 6.8	32.3 ± 5.1	1.0
6 e	CH3	CH3	CH2Ph	CH3	6.45 ± 1.47	9.72 ± 0.63	2.78 ± 0.89	2.3
7 e	CH3	CH2CH3	CH2CH3	CH3	7.52 ± 2.79	7.89 ± 2.87	13.6 ± 2.0	0.53
8 e	CH3	PhCH=CH-(trans)	CH2CH3	CH3	16.1 ± 0.5	49.3 ± 12.5	0.670 ± 0.195	24
9	CH2CH3	PhCH=CH-(trans)	CH2CH3	CH3	4.65 ± 1.21	9.23 ± 3.60	0.887 ± 0.138	5.2
10	CH2CH3	PhCH=CH-(trans)	CH2Ph	CH3	13.7 ± 2.6	14 ± 4% (10−4)	3.13 ± 0.51	4.4
11 e	CH2CH3	CH3	CH2CH3	Ph	25.9 ± 7.3	35.9 ± 15.3	7.24 ± 2.13	3.6
12	CH2CH3	CH2CH3	CH2CH3	Ph	21.5 ± 2.7	14.5 ± 3.5	8.49 ± 1.74	2.5
13	CH2CH3	CH3	CH2Ph	Ph	26.0 ± 8.7	3.15 ± 0.96	1.75 ± 0.47	15
4 e	CH2CH3	PhCH=CH-(trans)	CH2CH3	Ph	5.93 ± 0.27	4.77 ± 0.29	0.108 ± 0.012	55
14	CH2CH3	PhCH=CH-(trans)	CH2CH3	4-CH3Ph	14.9 ± 4.9	37 ± 2% (10−4)	9.13 ± 2.43	1.6
15	CH2CH3	PhCH=CH-(trans)	CH2CH3	4-OCH3Ph	9.49 ± 1.99	d (10−4)	1.43 ± 0.37	6.6
16	CH2CH3	PhCH=CH-(trans)	CH2CH3	4-ClPh	33.0 ± 7.5	12 ± 7% (10−4)	0.785 ± 0.272	42
17	CH2CH3	PhCH=CH-(trans)	CH2CH3	4-NO2Ph	13.1 ± 1.6	40 ± 3% (10−4)	4.14 ± 0.51	3.2
18	CH2CH3	PhCH=CH-(trans)	CH2CH3	3-furyl	5.49 ± 0.25	35 ± 6% (10−4)	0.907 ± 0.307	6.1
19	CH2CH3	PhCH=CH-(trans)	CH2CH3	3-thienyl	7.52 ± 1.38	d (10−4)	0.407 ± 0.066	18
20	CH2CH3	2-OCH3PhCH=CH-(trans)	CH2CH3	Ph	25.3 ± 2.4	17 ± 12% (10−4)	0.334 ± 0.059	76
21	CH2CH3	2-NO2PhCH=CH-(trans)	CH2CH3	Ph	6.03 ± 1.39	d (10−4)	0.109±0.017	55
22	CH2CH3	4-NO2PhCH=CH-(trans)	CH2CH3	Ph	23 ± 9% (10−4)	33% (10−4)	0.0585 ± 0.0164	>1700
23	CH2CH3	4-NH2PhCH=CH-(trans)	CH2CH3	Ph	31 ± 3% (10−4)	26 ± 6% (10−4)	0.198 ± 0.047	>500
24	CH2CH3	(Ph)2C=CH-	CH2CH3	Ph	1.20 ± 0.14	12 ± 6% (10−4)	1.42 ± 0.23	0.84
25	CH2CH3	PhC≡C-	CH2CH3	Ph	11.0 ± 0.1	26 ± 12% (10−4)	0.0766 ± 0.0151	140
26	CH2CH3	PhCH=CH-(trans)	CH2Ph	Ph	35 ± 3% (10−4)	15 ± 3% (10−4)	0.0583 ± 0.0124	>1700
27	CH2CH3	4-NO2PhCH=CH-(trans)	CH2Ph	Ph	33 ± 1% (10−4)	d (10−4)	0.0724 ± 0.0377	>1300
28	CH2CH3	PhC≡C-	CH2Ph	Ph	40.1 ± 7.5	d (10−4)	0.0314 ± 0.0028f	1300
29	CH2Ph	PhCH=CH-(trans)	CH2CH3	Ph	d (10−4)	16 ± 11% (10−4)	0.142 ± 0.047	>700
30	CH2Ph	4-NO2PhCH=CH-(trans)	CH2CH3	Ph	d (10−4)	d (10−4)	0.286 ± 0.038	>400
31	CH2Ph	PhC≡C-	CH2CH3	Ph	24 ± 4% (10−4)	d (10−4)	0.169 ± 0.026	>600
32 e		CH3	CH2CH3		7.41 ± 1.29	28.4 ± 9.1	4.47 ± 0.46	1.7
33		PhCH=CH-(trans)	CH2CH3		2.49 ± 0.47	2.40 ± 0.22	2.80 ± 1.78	0.85
34		PhC≡C-	CH2Ph		11.6 ± 4.8	43 ± 2% (10−4)	2.75 ± 0.78	4.2

^aDisplacement of specific [³H]-(R)-PIA binding in rat brain membranes, expressed as K_i ± SEM in μM (n = 3–5), or as a percentage of specific binding displaced at the indicated concentration (M).

^bDisplacement of specific [³H]CGS 21680 binding in rat striatal membranes, expressed as K_i ± SEM in μM (n = 3–6), or as a percentage of specific binding displaced at the indicated concentration (M).

^cDisplacement of specific [¹²⁵I]AB-MECA binding at human A₃ receptors expressed in HEK cells, in membranes, expressed as K_i ± SEM in μM (n = 3–4).

^dDisplacement of ≤ 10% of specific binding at the indicated concentration (M).

^eValues taken from van Rhee et al.¹⁶

^fK_i value at rat A₃ receptors stably expressed in CHO cells¹ found to be 3.53 ± 0.61 μM.

Scheme 1.

General Procedure for the Synthesis of 1,4-Dihydropyridine Derivatives 9-22 and 24-31

Table 2.

Characterization of Dihydropyridine and Pyridine Derivatives

no.	Tm (°C)		formula	MS	analysis	yielda (%)
9	148–149		C21H25NO4	355(CI)	C,H,N	16.3
10	115–117		C26H27NO4	417 (CI)	C,H,N	69.5
12	106–108		C20H25NO4	343 (CI)	C,H,N	25.5
13	112–114		C24H25NO4	391 (EI)	C,H,N	62.9
14	129–131		C27H29NO4	431 (CI)	C,H,N	32.2
15	137–139		C21H33NO4	447 (CI)	C,H,N	6.1
16	149–151		C26H26NClO4	451 (EI)	C,H,N	31.2
17	oil		C26H26N2O6	462 (EI)	b	15.3
18	114–116		C24H25NO5	407 (CI)	C,H,N	25.5
19	oil		C24H25NSO4·0.50EtOH	423 (EI)	C,H,N	15.1
20	130–132		C27H29NO5	447 (EI)	C,H,N	12.0
21	175–176		C26H26N2O6	462 (EI)	C,H,N	8.0
22	195–196		C26H26N2O6	462 (EI)	C,H,N	58.2
23	oil		C26H28N2O4	432 (EI)	c	24.1
24	164–165		C32H31NO4	491 (EI)	C,H,N	8.2
25	132–134		C26H25NO4	415 (EI)	C,H,N	36.4
26	161–163		C31H29NO4	479 (EI)	C,H,N	27.9
27	oil		C31H28N2O6	524 (EI)	d	26.2
28	156–158		C31H27NO4	477 (CI)	C,H,N	31.0
29	149–151		C31H29NO4	479 (EI)	C,H,N	41.5
30	oil		C31H28N2O6	524 (EI)	e	32.9
31	124–125		C31H27NO4	477 (CI)	C,H,N	29.3
33	oil		C26H25NO4	415 (EI)	f	29.8
34	oil		C31H25NO4	475 (EI)	g	74.0

^aPurification was achieved by thin layer chromatography, silica 60, 1000 μm layer thickness, using EtOAc-petroleum ether (pe) 35–60, 20:80 (v/v) as eluent.

^b17 pure on analytical TLC (silica 60, 250 μm) EtOAc-pe = 20:80 (v/v), R_f = 0.21; CH₂Cl₂-MeOH = 40:1 (v/v), R_{f =} 0.46; EI calcd 462.1806, found 462.1791.

^c23 (C₂₆H₂₈N₂O₄) insufficient quantity for CHN, pure on analytical TLC (silica 60, 250 μm) EtOAc-pe = 20:80 (v/v), R_{f =} 0.06; CHCl₃-MeOH-CH₃COOH = 94:6:1 (v/v/v), R_{f =} 0.47; EI calcd 432.2049, found 432.2042.

^d27 pure on analytical TLC (silica 60, 250 μm) EtOAc-pe = 20:80 (v/v), R_{f =} 0.23; CH₂Cl₂-MeOH = 40:1 (v/v), R_{f =} 0.68; EI calcd 524.1938, found 524.1947.

^e30 pure on analytical TLC (silica 60, 250 μm) EtOAc-pe = 20:80 (v/v), R_{f =} 0.21; CH₂Cl₂-MeOH = 40:1 (v/v), R_{f =} 0.71; EI calcd 524.1938, found 524.1928.

^f33 pure on analytical TLC (silica 60, 250 μm) EtOAc-pe = 20:80 (v/v), R_{f =} 0.62; CH₂Cl₂-MeOH = 40:1 (v/v), R_{f =} 0.69; EI calcd 415.1772, found 415.1783.

^g34 pure on analytical TLC (silica 60, 250 μm) EtOAc-pe = 20:80 (v/v), R_{f =} 0.52; CH₂Cl₂-MeOH = 40:1 (v/v), R_{f =} 0.75; EI calcd 475.1776, found 475.1783.

Scheme 2.

General Procedure for the Synthesis of β-Keto Esters

Scheme 3.

Procedure for the Synthesis of Ethyl 3-Aminocinnamate

An aromatic nitro group on the C-4 side chain of a dihydropyridine could be reduced selectively using zinc/acetic acid (Scheme 4). This was desired as a precursor for the potential radioiodination reaction for the preparation of an A₃ receptor radioligand, as has been carried out for an agonist.²⁷

Scheme 4.

Method for Reduction of the Nitro Group on 1,4-Dihydropyridines

Oxidation of 1,4-dihydropyridines (4 and 28) to the corresponding pyridine derivatives (33 and 34, respectively) was carried out using tetrachloro-1,4-benzoquinone (chloranil, 45) in tetrahydrofuran (Scheme 5).²³

Scheme 5.

General Procedure for the Oxidation of 1,4-Dihydropyridine Derivatives Using Tetrachlorobenzoquinone, 45

Binding at Adenosine Receptors.

K_i values at A₁ and A_2A receptors were determined in radioligand binding assays in rat brain membranes vs [³H]-(R)-PIA[[³H]-(R)-N⁶-(phenylisopropyl)adenosine] or [³H]CGS 21680 [[³H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5′-(N-ethylcarbamoyl)adenosine], respectively.^24,25 Affinity at cloned human A₃ receptors expressed in HEK-293 cells²⁶ was determined using [¹²⁵I]AB-MECA [N⁶-(4-amino-3-[¹²⁵I]iodobenzyl)-5′-(N-methylcarbamoyl)adenosine].^27,28

Structure-activity relationship (SAR) analysis of 1,4-dihydropyridine derivatives at adenosine receptors has indicated that increasing the size of the substituents from small alkyl either at the C-4 position or at the 5-ester position tends to increase binding affinity at A₁, A_2A, and A₃ subtypes (Table 1).¹⁶ This was illustrated with the 5-benzyl ester, 6, and the 4-ethyl analogue, 7.¹⁶ The benzyl ester, 6, vs compound 5 provided a 12-fold enhancement of affinity at A₃ receptors. The 4-β-styryl substituent in 8 provided an even greater enhancement of A₃ receptor affinity (48-fold vs 5) and selectivity. The combination of the 5-benzyl ester and 4-β-styryl substituents in 10 was compatible in its affinity enhancement at A₁ and A₃, but not at A_2A, subtypes.

The 6-phenyl substituent enhanced affinity particularly at A₃ receptors, thus compound 11 was slightly A₃ selective. Extending the 4-methyl group to ethyl in 12 did not result in an enhancement of affinity, as it did in the 6-methyl case (compound 7 vs 5). The combination of the 5-benzyl ester and 6-phenyl substituents in 13 was additive in affinity enhancement at A_2A and A₃, but not at A₁, subtypes. The ratios of affinity of compound 13 vs the corresponding 5-ethyl ester, 11, were 11- and 4.1-fold at A_2A and A₃ receptors, respectively.

We have previously demonstrated that compound 4 is 55-fold selective for human A₃ vs rat A₁ receptors; thus the combination of the 4-β-styryl and 6-phenyl substituents is well tolerated at the A₃ receptor.¹⁶ The compatibility of the 4-β-styryl group with other aromatic substituents at the 6-position was examined in the current study. In compounds 14-17, the 6-phenyl ring was substituted at the para position with methyl, methoxy, chloro, and nitro groups. All substitutions were less potent than 6-phenyl at A₃ receptors, although the 4-chloro derivative, 16, retained 42-fold selectivity vs A₁ receptors. The 4-methoxy derivative, 15, was 85-fold less potent than 4 at A₃ receptors. Affinity at A_2A receptors was particularly sensitive to substitution of the 6-phenyl ring. Substitution of the 6-phenyl ring with 3-furyl, 18, or 3-thienyl, 19, groups reduced the affinity at A₃ receptors by 9- and 4-fold, respectively.

Substitutions of the phenyl ring of the β-styryl group, 20-23, suggested much more freedom in this region in binding at A₁ and A₃, but not A_2A, subtypes. The 4-nitro analogue, 22 (MRS 1222), was slightly more potent at A₃ receptors than 4. Phenyl substitution at the β-olefinic carbon in 24 resulted in relatively weak affinity at adenosine receptors (A₁, A₃ > A_2A). Compound 24 was 5-fold more potent than 4 at A₁ receptors and 13-fold less potent at A₃ receptors. The affinity of the dehydro equivalent (4-phenylethynyl substituent) of 4, i.e. compound 25, was very similar to its affinity at A₁ and A₃ receptors.

Triple substitutions of the simple dihydropyridines were also probed in compounds 26-31. All contained the 6-phenyl substituent and either a 4-β-styryl or a 4-phenylethynyl group. Benzyl esters at the 3- (29–31) or 5-positions (26-28) were included. A 5-benzyl ester 4-trans-β-styryl derivative, 26, with a K_i value of 58.3 nM at A₃ receptors, was >1700-fold selective vs either A₁ receptors or A_2A receptors. The enhanced selectivity was mainly the result of loss of affinity at A₁ receptors. Shifting the benzyl ester from the 5- to the 3-position as in 29 lowered the affinity at A₃ receptors 3-fold. The presence of a 4-nitro group on the β-styryl substituent, as in 27 and 30, had a negligible effect on affinity at any of the receptors studied. 3-Ethyl 5-benzyl 2-methyl-4-(phenylethynyl)-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate, 28 (MRS 1191), displayed a K_i value of 31.4 nM at A₃ receptors and 1300-fold selectivity vs A₁ receptors. Relative to the corresponding 5-ethyl ester derivative, 25, compound 28 was 2.4-fold more potent at A₃ receptors and less potent at A₁ receptors. A derivative isomeric to compound 28, the corresponding 3-benzyl 5-ethyl diester, 31, was >600-fold selective for A₃ receptors. Thus, bulky groups at combinations of either 3-, 4-, and 6-positions or 4-, 5-, and 6-positions still resulted in high A₃ receptor selectivity as a result of very low affinity at A₁ and A_2A receptors.

Oxidation of a simple 6-phenyl-1,4-dihydropyridine, 11, to the corresponding pyridine derivative increased affinity at A₁ but not A₃ receptors. The presence of additional bulky substituents, e.g. at the 4- and 5-positions, caused the effects of oxidation to be less tolerated in binding, at A₃ receptors in particular. Oxidation of 28 to the corresponding pyridine derivative, 34, reduced affinity at A₃ receptors by 88-fold, while affinity at A₁ receptors increased 4-fold.

Discussion

Among the most selective ligands in the present study were compounds 26-28, i.e. 5-benzyl esters having ≥ 1300-fold selectivity for A₃ vs A₁ adenosine receptors, and compounds 29-31, i.e. 3-benzyl esters having ≥ 400-fold selectivity. The para-substituted 4-styryl-6-phenyl derivatives 22 and 23 were also highly selective. Structure–activity analysis at A₃ adenosine receptors indicated that sterically bulky groups in 1,4-dihydropyridines are tolerated at the 3-, 4-, 5-, and 6-positions. In the present study simultaneous substitution at two or three, but not all four, of these positions was carried out. In this study large substituents at the 4-(β-styryl or phenylethynyl) and 6-positions (phenyl) were combined with bulky esters at either the 3- or 5-position, and a beneficial effect on selectivity for human A₃ receptors was observed. In combination with the 4- and 6-position substitutions mentioned above, a benzyl ester group preserved affinity (vs 4) at the A₃ receptors to a greater extent when located at the 5-position (e.g. 26) than at the 3-position (e.g. 29). Nevertheless, it is surprising that combinations of bulky substituents at the 3-, 4-, and 6-positions result in receptor affinity profiles not radically different from those combinations at the 4-, 5-, and 6-positions. This suggests that the A₃ receptor binding site is not highly sterically constrained. Also, similar enhancements of affinity at A₃ receptors by either a 4-trans-β-styryl or a 4-phenylethynyl group point to the same conclusion. Both of these groups are highly rigid, yet occupy nonidentical spatial regions.

A series of 8-styrylxanthines^30,31 were found to be subject to photoisomerization about the olefinic bond, which reduces their utility as pharmacological probes. One might expect a similar complication with the 4-styryldihydropyridine derivatives. Incorporation of a linear phenylethynyl group instead of the styryl group avoids this potential problem of isomerization.

Pyridine derivatives, which have a distinctly more planar geometry than that of the corresponding dihydropyridines, lost affinity for A₃ adenosine receptors but, remarkably, gained affinity for A₁ receptors. Numerous classes of adenosine receptor non-xanthine antagonists have been found for the A₁ receptor, and all tend to have a planar geometry.²⁹ It is possible that the A₃ receptor does not share this preference for planar antagonists.

There appears to be more flexibility of substitution of the phenyl ring of 4-β-styryl derivatives (e.g. compounds 20-23) than of the 6-phenyl substituent in A₃ receptor selective agents. Substitution of the 6-phenyl ring in the para position with a variety of electron-donating (methyl, methoxy) or -withdrawing (nitro, chloro) groups or its replacement with 3-thienyl or 3-furyl groups (e.g. compounds 14-19) all reduced the affinity at A₃ receptors.

It will now be necessary to investigate the regio- (and stereo-) selectivity of the ligand receptor interaction more fully. Many of the dihydropyridines examined in our previous study¹⁶ are asymmetric only on account of nonequivalent ester substitutions at the 3- and 5-positions. One such pair of enantiomers, (R)- and (S)- niguldipine, was studied at adenosine receptors. The difference in affinity at human A₃ receptors between (R)- and (S)-niguldipine, 3, was insignificant (K_i values of 1.9 and 2.8 μM, respectively). Thus, by analogy to the present set of mixed ethyl and benzyl esters, it is likely that there is no dramatic difference in A₃ receptor affinity between (R)- and (S)-enantiomers of 3,4- or 4, 5-disubstituted dihydropyridines (i.e. in which methyl groups occur at both the 2- and 6-positions). The effects of 6-aryl substitution on stereoselectivity of binding at adenosine receptors have yet to be explored.

At the rat A₃ receptor, the affinity of 1,4-dihydropyridines is considerably lower than at human A₃ receptors.¹⁶ For example the K_i value of 28 at the rat A₃ receptor was found to be 3.53 μM, while at the human A₃ receptor the K_i value is only 31 nM. Although compound 28 is still somewhat selective for A₃ receptors in the rat (11-fold), it is 110-fold less potent than at human A₃ receptors. This species dependence of A₃ receptor affinity has also been demonstrated for xanthines^2,4 and other classes of adenosine antagonists, such as flavonoids,²⁸ which tend to bind with lower K_i values at human vs rat A₃ receptors.

It is expected that all of the 4-(arylalkyl)-6-phenyl-1,4-dihydropyridines prepared in the present study are selective for adenosine receptors vs L-type Ca²⁺ channels, since this principle was demonstrated in a previous study for compound 4.¹⁶ Moreover, compound 4 was also shown to have negligible affinity for the adenosine transporter.¹⁶ Furthermore, it was demonstrated that dihydropyridines such as compound 4 effectively attenuate the IB-MECA elicited inhibition of adenylyl cyclase in CHO cells expressing the cloned rat A₃ adenosine receptor.¹⁶ Thus, the selective ligands introduced here should be useful as antagonists in probing the role of A₃ receptors, especially for the human homologue of the receptor. These selective agents are now suitable for study in functional assays and in investigations of the physiological role of A₃ receptors. With slight additional improvement of affinity, dihydropyridines may also provide affinity probes such as radioligands for human A₃ receptors, although it has not been determined if the binding is competitive.

Experimental Section

Materials.

Ethyl acetoacetate (37a), acetaldehyde (36a), propionaldehyde (36b), ethyl 3-aminocrotonate (35a), and trans-cinnamaldehyde (36c) were obtained from Fluka (Ronkonoma, NY). Ethyl 3-aminocrotonate (35a), 4-nitro-trans-cinnamaldehyde (36d), 2-nitro-trans-cinnamaldehyde (36e), 2-methoxy-trans-cinnamaldehyde (36f), β-phenylcinnamaldehyde (36g), phenyl propargylaldehyde (36h), benzyl acetoacetate (37b), ethyl benzoylacetate (37c), and tetrachloro-1,4-benzoquinone (45) were from Aldrich (St. Louis, MO). Compounds 4-8 and 32 were prepared as described in van Rhee et al.¹⁶ (R)-PIA and 2-chloroadenosine were purchased from Research Biochemicals International (Natick, MA). All other materials were obtained from commercial sources.

Synthesis.

Proton nuclear magnetic resonance spectroscopy was performed on a Varian GEMINI-300 spectrometer, and spectra were taken in DMSO-d₆ or CHCl₃-d. Chemical-ionization (CI) mass spectrometry was performed with a Finnigan 4600 mass spectrometer, and electron-impact (EI) mass spectrometry with a VG7070F mass spectrometer at 6 kV. Elemental analysis was performed by Atlantic Microlab Inc. (Norcross, GA). All melting points were determined with a Unimelt capillary melting point apparatus (Arthur H. Thomas Co., PA) and were uncorrected.

General Procedure for the Preparation of 1,4-Dihydropyridine-3,5-dicarboxylate Esters (9–22, 24–31).

Equimolar amounts (0.5 mmol) of the appropriate 3-amino-2-propenoate ester (35a,b), aldehyde (36a-h), and 3-ketopropionate ester (37a-i) derivative were dissolved in 5 mL of absolute ethanol. The solution was sealed in a glass tube and heated to 100 °C (for volatile aldehydes) or was refluxed under N₂ for at least 24 h, and at most 72h. The solvent was then evaporated, and products were purified either by crystallization, column chromatography (silica 60; 220–440 mesh; Fluka, Buchs, CH; 20% ethyl acetate-80% petroleum ether 35–60), or preparative TLC (silica 60; 1000 μm; Analtech, Newark, DE; 20% ethyl acetate-80% petroleum ether 35–60). All procedures were performed under nitrogen and low-light conditions to prevent oxidation of the products. The products were shown to be homogeneous by analytical TLC.

3,5-Diethyl 2,6-Dimethyl-4-[2-phenyl-(E)-vinyl]-1,4-(±)-dihydropyridine-3,5-dicarboxylate (9).

¹H NMR (CDCl₃): δ 1.29 (t, 6H, J = 6.8 Hz, 3 and 5-CH₂CH₃), 2.33 (s, 6H, 2 and 6-CH₃), 4.16 (m, 4H, 3 and 5-OCH₂), 4.61 (d, 1H, J = 5.8 Hz, 4-H), 5.60 (br, 1H, NH), 6.17 (dd, 1H, J = 5.9, 16.6 Hz, C₆H₅C=CH), 6.25 (d, 1H, J = 16.6 Hz, C₆H₅CH=C), 7.16–7.33 (m, 5H, C₆H₅). MS (CI/NH₃): m/z 356 (MH)⁺, 252 (M-C₆H₅CH=CH)⁺, base.

3-Ethyl 5-Benzyl 2,6-Dimethyl-4-[2-phenyl-(E)-vinyl]-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (10).

¹H NMR (CDCl₃): δ 1.29 (t, 3H, J = 7.0 Hz, 3-CH₂CH₃), 2.34 (s, 3H, 2-CH₃), 2.38 (s, 3H, 6-CH₃), 4.22 (m, 2H, 3-OCH₂), 4.70 (d, 1H, J = 5.8 Hz, 4-H), 5.20 (AB, 2H, J = 12.7 Hz, 5-OCH₂), 6.16 (dd, 1H, J = 5.9, 15.8 Hz, C₆H₅C=CH), 6.21 (d, 1H, J = 15.8 Hz, C₆H₅CH=C), 7.20–7.44 (m, 10H, 2 × C₆H₅). MS (CI/NH₃): m/z 418 (MH⁺), base.

3,5-Diethyl 2-Methyl-4-ethyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (12).

¹H NMR (CDCl₃): δ 0.88 (2t, 6H, J = 6.8 Hz, 3 and 5-CH₂CH₃), 1.32 (t, 3H, J = 7.0 Hz, 4-CH₂CH₃), 1.55 (m, 2H, 4-CH₂CH₃), 2.33 (s, 3H, 2-CH₃), 3.90 (m, 2H, 3-OCH₂), 4.06 (t, 1H, J = 6.5 Hz, 4-H), 4.22 (m, 2H, 5-OCH₂), 5.63 (br, 1H, NH), 7.29–7.41 (m, 5H, 6-C₆H₅). MS (CI/NH₃): m/z 361 (M + NH₄⁺), base, 344 (MH⁺).

3-Ethyl 5-Benzyl 2,4-Dimethyl,6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (13).

¹H NMR (CDCl₃): δ 1.15 (d, 3H, J = 6.6 Hz, 4-CH₃), 1.32 (t, 3H, J = 7.0 Hz, 3-CH₂CH₃), 2.31 (s, 3H, 2-CH₃), 4.02 (q, 1H, J = 6.7 Hz, 4-H), 4.21 (m, 2H, 3-OCH₂), 4.95 (AB, 2H, J = 12.7 Hz, 5-OCH₂), 5.70 (br, 1H, NH), 6.95–7.36 (m, 10H, 2 × C₆H₅). MS (EI): m/z 376 (M - CH₃)⁺, 91 (C₆H₅CH₂⁺), base.

3,5-Diethyl 2-Methyl-4-[2-phenyl-(E)-vinyl]-6-(4-toluyl)-1,4-(±)-dihydropyridine-3,5-dicarboxylate (14).

¹H NMR (DMSO-d₆): δ 0.78 (t, 3H, J = 7.0 Hz, 3CH₂CH₃), 1.20 (t, 3H, J = 7.0 Hz, 5-CH₂CH₃), 2.28 (s, 3H, toluyl-CH₃), 2.34 (s, 3H, 2-CH₃), 3.78 (q, 2H, J = 6.7 Hz, 3-OCH₂), 4.10 (m, 2H, 5-OCH₂), 4.49 (d, 1H, J = 7.3 Hz, 4-H), 6.20 (m, 2H, CH=CH), 7.18–7.36 (m, 9H, C₆H₄ and C₆H₅), 9.01 (br, 1H, NH). MS (CI/NH₃): m/e 432 (MH⁺), 328 (M - C₆H₅CH=CH⁺), base.

3,5-Diethyl 2-Methyl-4-[2-phenyl-(E)-vinyl]-6-(4-methoxyphenyl)-1,4-(R,S)-dihydropyridine-3,5-dicarboxylate (15).

¹H NMR (CDCl₃): δ 1.00 (t, 3H, J = 7.0 Hz, 3-CH₂CH₃), 1.33 (t, 3H, J = 7.0 Hz, 5-CH₂CH₃), 2.38 (s, 3H, 2-CH₃), 3.85 (s, 3H, 4′-OCH₃), 4.00 (q, 2H, J = 6.7 Hz, 3-OCH₂), 4.22 (m, 2H, 5-OCH₂), 4.74 (d, 1H, J = 6.1 Hz, 4-H), 5.76 (br, 1H, NH), 6.32 (dd, 1H, J = 6.6, 15.8 Hz, C₆H₅C=CH), 6.41 (d, 1H, J = 15.8 Hz, C₆H₅CH=C), 6.94 (d, 2H, J = 8.7 Hz, 2′- and 6′-H), 7.19–7.31 (m, 5H, C₆H₅), 7.37 (d, 2 H, J = 7.3 Hz, 3′- and 6′-H). MS (CI/NH₃): m/z 448 (MH)⁺, 344 (M - C₆H₅CH=CH)⁺, base.

3,5-Diethyl 2-Methyl-4-[2-phenyl-(E)-vinyl]-6-(4-chlorophenyl)-1,4-(±)-dihydropyridine-3,5-dicarboxylate (16).

¹H NMR (CDCl₃): δ 0.99 (t, 3H, J = 7.2 Hz, 3-CH₂CH₃), 1.29 (t, 3H, J = 7.2 Hz, 5-CH₂CH₃), 2.37 (s, 3H, 2-CH₃), 3.97 (q, 2H, J = 7.0 Hz, 3-OCH₂), 4.22 (m, 2H, 5-OCH₂), 4.75 (d, 1H, J = 6.3 Hz, 4-H), 5.71 (br, 1H, NH), 6.28 (dd, 1H, J = 6.6, 15.8 Hz, C₆H₅C=CH), 6.40 (d, 1H, J = 15.8 Hz, C₆H₅CH=C), 7.20–7.41 (m, 9H, C₆H₄ and 6-C₆H₅). MS (EI): m/z 451 (M)⁺, 422 (M - C₂H₅)⁺, 378 (M - CO₂C₂H₅)⁺, base, 348 (M - C₆H₅CH=CH)⁺.

3,5-Diethyl 2-Methyl-4-[2-phenyl-(E)-vinyl]-6-(4-nitrophenyl)-1,4-(±)-dihydropyridine-3,5-dicarboxylate (17).

¹H NMR (CDCl₃): δ 1.21 (t, 3H, J = 6.8 Hz, 3-CH₂CH₃), 1.28 (t, 3H, J = 7.0 Hz, 5-CH₂CH₃), 2.38 (s, 3H, 2-CH₃), 4.13 (q, 2H, J = 7.0 Hz, 3-OCH₂), 4.25 (m, 2H, 5-OCH₂), 4.77 (d, 1H, J = 5.9 Hz, 4-H), 5.67 (br, 1H, NH), 6.26 (dd, 1H, J = 6.6, 15.8 Hz, C₆H₅C=CH), 6.37 (d, 1H, J = 15.8 Hz, C₆H₅CH=C), 7.21–7.54 (m, 7H, 3′,5′-H and 6-C₆H₅), 8.28 (m, 2H, 2′ and 6′-H). MS (EI): m/z 462 (M)⁺, 433 (M - C₂H₅)⁺, 389 (M CO₂C₂H₅)⁺, base, 359 (M - C₆H₅CH=CH)⁺.

3,5-Diethyl 2-Methyl-4-[2-phenyl-(E)-vinyl]-6-(3-furyl)1,4-(±)-dihydropyridine-3,5-dicarboxylate (18).

¹H NMR δ 1.13 (t, 3H, J = 7.0 Hz, 3-CH₂CH₃), 1.31 (t, 3H, J = 7.0 Hz, 5-CH₂CH₃), 2.37 (s, 3H, 2-CH₃), 3.85 (s, 3H, 4′-OCH₃), 4.08 (m, 2H, 3-OCH₂), 4.21 (q, J = 7.1 Hz, 2H, 5-OCH₂), 4.73 (d, 1H, J = 6.3 Hz, 4-H), 5.73 (br, 1H, NH), 6.24 (dd, 1H, J = 6.7, 15.8 Hz, C₆H₅C=CH), 6.35 (d, 1H, J = 15.8 Hz, C₆H₅CH=C), 6.52 (m, 1H, 3′-H), 7.19–7.37 (m, 5H, C₆H₅), 7.45 (m, 1 H, 2′-H), 7.65 (s, 1H, 5′-H). MS (CI/NH₃): m/z 408 (MH)⁺, 304 (M - C₆H₅CH=CH)⁺, base.

3,5-Diethyl 2-Methyl-4-[2-phenyl-(E)-vinyl]-6-(3-thienyl)-1,4-(±)-dihydropyridine-3,5-dicarboxylate (19).

¹H NMR (CDCl₃): δ 1.05 (t, 3H, J = 7.0 Hz, 3-CH₂CH₃), 1.27 (t, 3H, J = 7.0 Hz, 5-CH₂CH₃), 2.38 (s, 3H, 2-CH₃), 4.05 (m, 2H, 3-OCH₂), 4.22 (m, 2H, 5-OCH₂), 4.73 (d, 1H, J = 6.2 Hz, 4-H), 5.88 (br, 1H, NH), 6.30 (dd, 1H, J = 6.5, 15.8 Hz, C₆H₅C=CH), 6.38 (d, 1H, J = 15.8 Hz, C₆H₅CH=C), 7.06 (m, 1H, 4′-H), 7.18–7.31 (m, 5H, C₆H₅), 7.35 (d, 1H, J = 5.2 Hz, 3′-H), 7.40 (d, 1H, J = 4.8 Hz, 5′-H). MS (EI): m/z 423 (M)⁺, 350 (M - CO₂CH₂CH₃), base, 320 (M - C₆H₅CH=CH)⁺.

3,5-Diethyl 2-Methyl-4-[2-(2-methoxyphenyl)-(E)-vinyl]-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (20).

¹H NMR (CDCl₃): δ 0.93 (t, 3H, J = 7.3 Hz, 3-CH₂CH₃), 1.33 (t, 3H, J = 6.8 Hz, 5-CH₂CH₃), 2.35 (s, 3H, 2-CH₃), 3.82 (s, 3H, 2′-OCH₃), 3.92 (m, 2H, 3-OCH₂), 4.20 (m, 2H, 5-OCH₂), 4.75 (d, 1H, J = 5.9 Hz, 4-H), 5.76 (br, 1H, NH), 6.26 (dd, 1H, J = 5.9, 15.6 Hz, C₆H₅C=CH), 6.84 (d, 1H, J = 16.6 Hz, C₆H₅-CH=C), 7.15–7.48 (m, 9H, 4-C₆H₄ and 6-C₆H₅). MS (EI): m/z 447 (M)⁺, 402 (M - OC₂H₅)⁺, 374 (M - CO₂C₂H₅)⁺.

3,5-Diethyl 2-Methyl-4-[2-(2-nitrophenyl)-(E)-vinyl]-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (21).

¹H NMR (CDCl₃): δ 0.91 (t, 3H, J = 7.3 Hz, 3-CH₂CH₃), 1.33 (t, 3H, J = 7.3 Hz, 5-CH₂CH₃), 2.37 (s, 3H, 2-CH₃), 3.96 (q, 2H, J = 7.2 Hz, 3-OCH₂), 4.27 (q, 2H, J = 7.2 Hz, 5-OCH₂), 4.78 (d, 1H, J = 5.9 Hz, 4-H), 5.93 (br, 1H, NH), 6.34 (dd, 1H, J = 5.9, 15.6 Hz, C₆H₅C=CH), 6.90 (d, 1H, J = 16.6 Hz, C₆H₅-CH=C), 7.30–7.48 (m, 8H, 4-C₆H₃ and 6-C₆H₅), 7.90 (d, 1H, J = 9.8 Hz, 3′-H). MS (EI): m/z 462 (M)⁺, 417 (M - OC₂H₅)⁺, 389 (M - CO₂C₂H₅)⁺.

3,5-Diethyl 2-Methyl-4-[2-(4-nitrophenyl)-(E)-vinyl]-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (22).

¹H NMR (CDCl₃): δ 0.89 (t, 3H, J = 7.3 Hz, 3-CH₂CH₃), 1.32 (t, 3H, J = 7.3 Hz, 5-CH₂CH₃), 2.39 (s, 3H, 2-CH₃), 3.94 (q, 2H, J = 6.8 Hz, 3-OCH₂), 4.24 (q, 2H, J = 6.8 Hz, 5-OCH₂), 4.80 (d, 1H, J = 4.9 Hz, 4-H), 5.82 (br, 1H, NH), 6.47 (dd, 1H, J = 3.9, 16.1 Hz, C₆H₅C=CH), 7.32–7.51 (m, 8H, C₆H₅CH=C and 4-C₆H₂ and 6-C₆H₅), 8.15 (d, 2H, J = 8.8 Hz, 3′- and 5′-H). MS (EI): m/z 462 (M)⁺, 417 (M - OC₂H₅)⁺, 389 (M - CO2C2H5)⁺.

3,5-Diethyl 2-Methyl-4-[2-(4-aminophenyl)-(E)-vinyl]-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (23).

Compound 23 was prepared by the catalytic reduction of compound 22 with zinc and acetic acid as described previously.³⁰ Compound 22 (23 mg, 0.05 mmol) was dissolved in 1.5 mL of glacial acetic acid. Zn powder (0.15 mmol, 10 mg) was added to the solution, and the reaction mixture was stirred with a magnetic stirring bar at room temperature. Six hours after the start of the reaction, another batch of zinc powder was added. At 9 h reaction time, TLC (silica 60; petroleum ether 35–60-EtOAc = 80:20) analysis of the reaction mixture indicated that all starting material had been converted. The reaction mixture was diluted with 30 mL of water and neutralized with saturated NaHCO₃ solution. The aqueous solution was extracted three times with 15 mL of chloroform, and the organic phase was washed once with 20 mL of water. The organic phase was separated and dried over anhydrous MgSO₄. The product was purified by preparative TLC (1000 μm of silica 60; petroleum ether 35–60-EtOAc, 90:10) to yield 5.2 mg (24%) of a slightly yellow oil, which was shown to be pure by HPLC (OD-5–60, C-18 column, Separation Methods Technologies, Inc, Newark, DE; 0.1 M triethylammonium acetate buffer-CH₃CN, 40:60 gradient to 10:90 in 20 min; 1 mL/min flow; retention time = 4.45 min), and analytical TLC (silica 60; petroleum ether 35–60-EtOAc, 80:20, R _{f =} 0.06; CHCl₃-MEOH-HOAc, 94:6:1, R _{f =} 0.47). ¹H NMR (CDCl₃): δ 0.91 (t, 3H, J = 6.8 Hz, 3-CH₂CH₃), 1.31 (t, 3H, J = 6.8 Hz, 5-CH₂CH₃), 2.34 (s, 3H, 2-CH₃), 3.67 (br, 2H, 4′-NH₂), 3.92 (m, 2H, 3-OCH₂), 4.17 (m, 2H, 5-OCH₂), 4.70 (d, 1H, J = 6.8 Hz, 4-H), 5.78 (br, 1H, NH), 6.08 (dd, 1H, J = 6.8, 15.6 Hz, C₆H₅C=CH), 6.30 (d, 1H, J = 15.6 Hz, C₆H₅CH=C), 6.60 (d, 2H, J = 8.8 Hz, 3′- and 5′-H), 7.18 (d, 2H, J = 8.8 Hz, 2′- and 6′-H), 7.32–7.42 (m, 5H, 6-C₆H₅). MS (EI): m/z 432 (M)⁺, 387 (M - OC₂H₅)⁺, 359 (M - CO₂C₂H₅)⁺.

3,5-Diethyl 2-Methyl-4-(2,2-diphenylvinyl)-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (24).

¹H NMR (CDCl₃): δ 0.84 (t, 3H, J = 7.0 Hz, 3-CH₂CH₃), 1.10 (t, 3H, J ) 7.2 Hz, 5-CH₂CH₃), 2.32 (s, 3H, 2-CH₃), 3.70–4.18 (m, 4H, 3-OCH₂ and 5-OCH₂), 4.95 (d, 1H, J = 9.9 Hz, 4-H), 5.82 (br, 1H, NH), 6.02 (d, 1H, J = 10.3 Hz, C₆H₅C=CH), 7.17–7.46 (m, 15H, 4-C₆H₅ and 4-C₆H₅ and 6-C₆H₅). MS (EI): m/z 491 (M)⁺, 446 (M - OC₂H₅)⁺, 418 (M - CO₂C₂H₅)⁺.

3,5-Diethyl 2-Methyl-4-(phenylethynyl)-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (25).

¹H NMR (CDCl₃): δ 0.95 (t, 3H, J = 7.0 Hz, 3-CH₂CH₃), 1.32 (t, 3H, J ) 6.8 Hz, 3-CH₂CH₃), 2.37 (s, 3H, 2-CH₃), 3.98 (m, 2H, 3-OCH₂), 4.27 (m, 2H, 3-OCH₂), 5.12 (s, 1H, 4-H), 5.92 (br, 1H, NH), 7.23–7.43 (⁺m, 10H, 2 × C₆H₅). MS (EI+ ): m/z 415 (M)⁺, 386 (M - C₂H₅) , 342 (M - CO₂C₂H₅) , base.

3-Ethyl 5-Benzyl 2-methyl-4-[2-phenyl-(E)-vinyl]-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (26).

¹H NMR (CDCl₃): δ 1.28 (t, 3H, J = 6.8 Hz, 3-CH₂CH₃), 2.38 (s, 3H, 2-CH₃), 4.22 (m, 2H, 3-OCH₂), 4.78 (d, 1H, J = 6.2 Hz, 4-H), 4.99 (AB, J = 12.7 Hz, 5-OCH₂), 5.76 (br, 1H, NH), 6.30 (dd, 1H, J = 6.5, 16.2 Hz, C₆H₅C=CH), 6.38 (d, 1H, J = 16.2 Hz, C₆H₅CH=C), 6.98 (m, 2H, 2′- and 6′-H), 7.21–7.38 (m, 13H, 2 × C₆H₅, 3′-+, 4′-, and 5′-H). MS (EI): m/z+ 479 (M)⁺, 388 (M - C₆H₅CH₂) , 376 (M - C₆H₅CH=CH) , 344 (M - CO₂CH₂-CH₃), 91 (C₆H₅CH₂)⁺, base.

3-Ethyl 5-Benzyl 2-Methyl-4-[2-(4-nitrophenyl)-E)-vinyl]-6-phenyl-1,4-(R,S)-dihydropyridine-3,5-dicarboxylate (27).

¹H NMR (CDCl₃): δ 1.31 (t, 3H, J = 6.9 Hz, 3-CH₂CH₃), 2.38 (s, 3H, 2-CH₃), 4.24 (m, 2H, 3-OCH₂), 4.84 (d, 1H, J = 5.8 Hz, 4-H), 5.02 (AB, J = 12.7 Hz, 5-OCH₂), 5.82 (br, 1H, NH), 6.43 (dd, 1H, J = 6.5, 16.0 Hz, NO₂C₆H₄C=CH), 6.45 (d, 1H, J = 16.0 Hz, NO₂C₆H₄CH=C), 6.96 (dd, 2H, J = 2.0, 7.7 Hz, 3′- and 5′-H), 7.19–7.39 (m, 5H, C₆H₅), 8.13 (d, 2H, J = 8.8 Hz, 2′- and 6′-H). MS (EI): m/z 524 (M)⁺, 389 (M - CO₂C₆H₅CH₂)⁺, 376 (M - NO₂C₆H₅CH=CH)⁺, 91 (C₆H₅CH₂)⁺, base.

3-Ethyl 5-Benzyl 2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (28).

¹H NMR (CDCl₃): δ 1.35 (t, 3H, J = 6.8 Hz, 3-CH₂CH₃), 2.37 (s, 3H, 2-CH₃), 4.27 (m, 2H, 3-OCH₂), 5.10 (AB, J = 12.7 Hz, 5-OCH₂), 5.19 (s, 1H, 4-H), 5.87 (br, 1H, NH), 7.08⁺ −7.39 (m, 15H, 3 × C₆H₅). MS (CI/NH₃): m/z 478 (MH) , 376 (M - C₆H₅CdC)⁺, 242 (M - CO₂C₆H₅CH₂)⁺, base.

3-Benzyl 5-Ethyl 2-Methyl-4-[2-phenyl-(E)-vinyl]-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (29).

¹H NMR (CDCl₃): δ 0.91 (t, 3H, J = 7.0 Hz, 5-CH₂CH₃), 2.38 (s, 3H, 2-CH₃), 3.92 (q, 2H, J = 6.7 Hz, 5-OCH₂), 4.81 (d, 1H, J = 5.9 Hz, 4-H), 5.18 (AB, J = 12.7 Hz, 2H, 3-OCH₂), 5.78 (br, 1H, NH), 6.31 (dd, 1H, J = 5.9, 16.0 Hz, C₆H₅C=CH), 6.36 (d, 1H, J = 16.0 Hz, C₆H₅CH=C), 7.20–7.42 (m, 10H, 2 × C₆H₅). MS (EI): m/z 406 (M - CO₂CH₂)⁺, 376 (M - C₆H₅CH=CH)⁺, 91 (C₆H₅CH₂)⁺, base.

3-Benzyl 5-Ethyl 2-Methyl-4-[2-(4-nitrophenyl)-(E)-vinyl]-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (30).

¹H NMR (CDCl₃): δ 0.88 (t, 3H, J = 6.8 Hz, 5-CH₂CH₃), 2.39 (s, 3H, 2-CH₃), 4.09 (q, 2H, J = 6.8 Hz, 3-OCH₂), 4.84 (d, 1H, J = 5.8 Hz, 4-H), 5.30 (AB, J = 12.2 Hz, 3-OCH₂), 5.87 (br, 1H, NH), 6.40 (d, 1H, J = 16.0 Hz, NO₂C₆H₄CH=C), 6.50 (dd, 1H, J = 5.9, 16.6 Hz, NO₂C₆H₄C=CH), 6.96 (dd, 2H, J = 2.0, 7.7 Hz, 3′- and 5′-H), 7.19–7.39 (m, 5H, C₆H₅), 8.14 (d, 2H, J = 7.8 Hz, 2′- and 6′-H). MS (EI): m/z 524 (M)⁺, 389 (M - CO₂C₆H₅CH₂)⁺, 376 (M - NO₂C₆H₅CH=CH)⁺, 91-(C₆H₅CH₂)⁺, base.

3-Benzyl 5-Ethyl 2-Methyl-4-(phenylethynyl)-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (31).

¹H NMR (CDCl₃): δ 0.96 (t, 3H, J = 6.8 Hz, 5-CH₂CH₃), 2.38 (s, 3H, 2-CH₃), 4.00 (q, 2H, J = 6.8 Hz, 5-OCH₂), 5.21 (s, 1H, 4-H), 5.30 (AB, J = 13.6 Hz, 3-OCH₂), 5.87 (br, 1H, NH), 7.30–7.49 (m, 15H, 3⁺× C₆H₅). MS (CI/NH₃): m/z 478 (MH)⁺, 376 (M - C₆H₅CdC) , base.

Procedure for Oxidation of 1,4-Dihydropyridine-3,5-dicarboxylate Esters (33–34).

Equimolar amounts (0.25 mmol) of the 1,4-dihydropyridine-3,5-dicarboxylate ester (4, 28) and tetrachloro-1,4-benzoquinone (45) in tetrahydrofuran (2 mL) were mixed and refluxed for up to 4 h. The solvent was then evaporated, and products were purified by preparative TLC (silica 60; 1000 μm; Analtech, Newark, DE; 20% ethyl acetate-80% petroleum ether 35–60).

3,5-Diethyl 2-Methyl-4-[2-phenyl-(E)-vinyl]-6-phenylpyridine-3,5-dicarboxylate (33).

¹H NMR (CDCl₃): δ 0.96 (t, 3H, J = 6.9 Hz, 3-CH₂CH₃), 1.26 (t, 3H, J = 6.8 Hz, 5-CH₂CH₃), 2.67 (s, 3H, 2-CH₃), 4.07 (q, 2H, J = 6.8 Hz, 3-OCH₂), 4.35 (q, 2H, J = 6.8 Hz, 5-OCH₂), 6.88 (d, 1H, J = 16.6 Hz, C₆H₅C=CH), 6.88 (d, 1H, J = 16.6 Hz, C₆H₅CH=C), 7.31–7.61 (2m, 10H, 2 × C₆H₅). MS (EI): m/e+ 415 (M)⁺, base, 370 (M - OC₂H₅)⁺, 358 (386 - CH₂=CH₂)⁺.

3-Ethyl 5-Benzyl 2-Methyl-4-(phenylethynyl)-6-phenylpyridine-3,5-dicarboxylate (34).

¹H NMR (CDCl₃): δ 1.42 (t, 3H, J = 6.8 Hz, 3-CH₂CH₃), 2.68 (s, 3H, 2-CH₃), 4.48 (q, 2H, J = 6.8 Hz, 3-OCH₂), 5.17 (s, 2H, 5- OCH₂), 7.10–7.60 (4m, 15H, 3 × C6H5). MS (EI):+ m/e 475 (M)+, 438 (M - OC₂H₅)⁺, 384 (M - C₆H₅CH₂)⁺, 356 (M - CH₂=CH₂)⁺, base.

Pharmacology. Radioligand Binding Studies.

Binding of [³H]-(R)-N⁶-(phenylisopropyl)adenosine ([³H]-(R)-PIA) to A₁ receptors from rat cerebral cortex membranes and of [³H]-2[[4-(2-carboxyethyl)phenyl]ethylamino]-5′-(N-ethylcarbamoyl)adenosine ([³H]CGS 21680) to A_2A receptors from rat striatal membranes was performed as described previously.^24,25 Adenosine deaminase (3 units/mL) was present during the preparation of the brain membranes, in a preincubation of 30 min at 30 °C, and during the incubation with the radioligands.

Binding of [¹²⁵I]-N⁶-(4-amino-3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine ([¹²⁵I]AB-MECA) to membranes prepared from HEK-293 cells stably expressing the human A₃ receptor (Receptor Biology, Inc., Baltimore, MD) or to membranes prepared from CHO cells stably expressing the rat A₃ receptor was performed as described.^27,28 The assay medium consisted of a buffer containing 50 mM Tris, 10 mM Mg²⁺, and 1 mM EDTA, at pH 8.0. The glass incubation tubes contained 100 μL of the membrane suspension (0.3 mg of protein/mL, stored at −80 °C in the same buffer), 50 μL of [¹²⁵I]AB-MECA (final concentration 0.3 nM), and 50 μL of a solution of the proposed antagonist. Nonspecific binding was determined in the presence of 200 μM NECA.

All nonradioactive compounds were initially dissolved in DMSO and diluted with buffer to the final concentration, where the amount of DMSO never exceeded 2%.

Incubations were terminated by rapid filtration over Whatman GF/B filters, using a Brandell cell harvester (Brandell, Gaithersburg, MD). The tubes were rinsed three times with 3 mL of buffer each.

At least five different concentrations of competitor, spanning 3 orders of magnitude adjusted appropriately for the IC₅₀ of each compound, were used. IC₅₀ values, calculated with the nonlinear regression method implemented in the InPlot program (Graph-PAD, San Diego, CA), were converted to apparent K_i values using the Cheng-Prusoff equation³² and K_d values of 1.0 and 14 nM for [³H]-(R)-PIA and [³H]CGS 21680, respectively, and 0.59 nM for binding of [¹²⁵I]AB-MECA at human A₃ receptors, respectively.

Abbreviations:

[¹²⁵I]AB-MECA, [¹²⁵I]

N ⁶-(4-amino-3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine

CGS 21680

2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5′-(N-ethylcarbamoyl)adenosine

CHO cells

Chinese hamster ovary cells

DMSO

dimethyl sulfoxide

EDTA

ethylenediaminetetraacetic acid

HEK cells

human embryonic kidney cells

IB-MECA

N⁶-(3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine

K _i

equilibrium inhibition constant

NECA

5′-(N-ethylcarbamoyl)adenosine

(R)-PIA

(R)-N ⁶-(phenylisopropyl)adenosine

SAR

structure-activity relationship

Tris

tris(hydroxymethyl)aminomethane