Structure-activity relationships and molecular modeling of 3,5-diacyl- 2,4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm980093j

The structure-activity relationships of 6-phenyl-1,4-dihydropyridine derivatives as selective antagonists at human A₃ adenosine receptors have been explored (Jiang et al. J. Med. Chem. 1997, 39, 4667-4675). In the present study, related pyridine derivatives have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. K(i) values in the nanomolar range were observed for certain 3,5-diacyl-2,4- dialkyl-6-phenylpyridine derivatives in displacement of [¹²⁵I]AB-MECA (N⁶-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine) at recombinant human A₃ adenosine receptors. Selectivity for A₃ adenosine receptors was determined vs radioligand binding at rat brain A₁ and A(2A) receptors. Structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. A 4-phenylethynyl group did not enhance A₃ selectivity of pyridine derivatives, as it did for the 4-substituted dihydropyridines. At the 2-and 4-positions ethyl was favored over methyl. Also, unlike the dihydropyridines, a thioester group at the 3-position was favored over an ester for affinity at A₃ adenosine receptors, and a 5-position benzyl ester decreased affinity. Small cycloalkyl groups at the 6-position of 4-phenylethynyl-1,4- dihydropyridines were favorable for high affinity at human A₃ adenosine receptors, while in the pyridine series a 6-cyclopentyl group decreased affinity. 5-Ethyl 2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5- carboxylate, 38, was highly potent at human A₃ receptors, with a K(i) value of 20 nM. A 4-propyl derivative, 39b, was selective and highly potent at both human and rat A₃ receptors, with K(i) values of 18.9 and 113 nM, respectively. A 6-(3-chlorophenyl) derivative, 44, displayed a K(i) value of 7.94 nM at human A₃ receptors and selectivity of 5200-fold. Molecular modeling, based on the steric and electrostatic alignment (SEAL) method, defined common pharmacophore elements for pyridine and dihydropyridine structures, e.g., the two ester groups and the 6-phenyl group. Moreover, a relationship between affinity and hydrophobicity was found for the pyridines.

Full text of DOI:10.1021/jm980093j

3186
J . Med. Chem. 1998, 41, 3186-3201
Str u ctu r e-Activity Rela tion sh ip s a n d Molecu la r Mod elin g of
3,5-Dia cyl-2,4-d ia lk ylp yr id in e Der iva tives a s Selective A3 Ad en osin e Recep tor
An ta gon ists
An-Hu Li, Stefano Moro, Neli Melman, Xiao-duo J i, and Kenneth A. J acobson*
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes,
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810
Received February 11, 1998
The structure-activity relationships of 6-phenyl-1,4-dihydropyridine derivatives as selective
antagonists at human A3 adenosine receptors have been explored (J iang et al. J . Med. Chem.
1997, 39, 4667-4675). In the present study, related pyridine derivatives have been synthesized
and tested for affinity at adenosine receptors in radioligand binding assays. Ki values in the
nanomolar range were observed for certain 3,5-diacyl-2,4-dialkyl-6-phenylpyridine derivatives
in displacement of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyladenosine)
at recombinant human A3 adenosine receptors. Selectivity for A3 adenosine receptors was
determined vs radioligand binding at rat brain A1 and A2A receptors. Structure-activity
relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the
2- and 4-alkyl substituents) were probed. A 4-phenylethynyl group did not enhance A3
selectivity of pyridine derivatives, as it did for the 4-substituted dihydropyridines. At the 2-
and 4-positions ethyl was favored over methyl. Also, unlike the dihydropyridines, a thioester
group at the 3-position was favored over an ester for affinity at A3 adenosine receptors, and a
5-position benzyl ester decreased affinity. Small cycloalkyl groups at the 6-position of
4-phenylethynyl-1,4-dihydropyridines were favorable for high affinity at human A3 adenosine
receptors, while in the pyridine series a 6-cyclopentyl group decreased affinity. 5-Ethyl 2,4-
diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate, 38, was highly potent at
human A3 receptors, with a Ki value of 20 nM. A 4-propyl derivative, 39b, was selective and
highly potent at both human and rat A3 receptors, with Ki values of 18.9 and 113 nM,
respectively. A 6-(3-chlorophenyl) derivative, 44, displayed a Ki value of 7.94 nM at human A3
receptors and selectivity of 5200-fold. Molecular modeling, based on the steric and electrostatic
alignment (SEAL) method, defined common pharmacophore elements for pyridine and
dihydropyridine structures, e.g., the two ester groups and the 6-phenyl group. Moreover, a
relationship between affinity and hydrophobicity was found for the pyridines.
In tr od u ction
receptor selective probes. Furthermore, there are major
species differences in the affinity of antagonists, e.g.,
typically many antagonists have 1-3 orders of magni-
tude greater affinity at human vs rat A3 receptors.
Selective antagonists have been reported for adeno-
sine A1, A2A, and A3 receptors,1 which are members of
the G-protein-coupled superfamily characterized by
seven transmembrane helical domains (TMs). Activa-
tion of the A3 receptor has been linked to several second
messenger systems, such as stimulation of phospholi-
pases C2 and D3 and inhibition of adenylyl cyclase.1
Antagonists for the A3 adenosine receptor are sought
as potential antiinflammatory, antiasthmatic, or anti-
ischemic agents.4-8 The pharmacology of the A3 recep-
tor is unique within the class of adenosine receptors.4,9-10
Most strikingly, xanthines such as caffeine and theo-
phylline, which have provided versatile leads for an-
tagonists at the other adenosine receptor subtypes, are
much less potent at the A3 receptor. The most promis-
ing leads have arisen from library screening, resulting
in the identification of 1,4-dihydropyridines,11-13 tri-
azoloquinazolines,14 flavonoids,15 a triazolonaphthyri-
dine,16 and a thiazolopyrimidine16 as prototypical A3
We previously reported that is was possible to sepa-
rate the antagonism of L-type calcium channels from
adenosine receptor antagonism among the 1,4-dihydro-
pyridines, through the introduction of 6-aryl and either
4-phenylethynyl or 4-styryl substitution.11 These groups
not only eliminated affinity for the L-type calcium
channels but also greatly enhanced selectivity for the
A3 receptor subtype. For example, a dihydropyridine
derivative, 3,5-diethyl 2-methyl-6-phenyl-4-[2-phenyl-
(E)-vinyl]-1,4-(()-dihydropyridine-3,5-dicarboxylate (Fig-
ure 1, 1),11 has been found to inhibit binding of radio-
ligand at the human A3 receptor with an affinity of 108
nM, while the same derivative was inactive at ion
channels and other receptor sites. MRS 1191, 3-ethyl
5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(()-di-
hydropyridine-3,5-dicarboxylate (Figure 1, 2),12 com-
petitively antagonized the effects of N6-(3-iodobenzyl)-
5′-N-methylcarbamoyladenosine (IB-MECA, Figure 1,
3), an A3 receptor selective agonist,17 on inhibition of
adenylyl cyclase mediated by the recombinant human
* Correspondence to: Dr. K. A. J acobson, Chief, Molecular Recogni-
tion Section, LBC, NIDDK, NIH, Bldg. 8A, Rm. B1A-19, Bethesda, MD
20892-0810. Tel.: (301) 496-9024. Fax: (301) 480-8422. E-mail:
kajacobs@helix.nih.gov.
S0022-2623(98)00093-4 This article not subject to U.S. Copyright. Published 1998 by the American Chemical Society
Published on Web 07/21/1998

Selective A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3187
Resu lts
Syn t h esis. Novel 1,4-dihydropyridines (1, 2, and
7-22) and closely related pyridine derivatives (23-48)
synthesized and tested for affinity in radioligand bind-
ing assays at adenosine receptors are shown in Tables
1 and 2, respectively. As in the previous studies,17,18
the dihydropyridine (Schemes 1 and 2) and pyridine
(Scheme 3) analogues were prepared as shown. The
Hantzsch condensation, Scheme 1, which involved con-
densing three components, a 3-amino-2-propenoate ester
(49, Scheme 4), an aldehyde (50, for example, Scheme
5), and a â-ketoester (51, Schemes 6 and 7), was used
for the 1,4-dihydropyridines. The corresponding py-
ridines were prepared through oxidation of the dihy-
dropyridines using tetrachloroquinone (Scheme 3). Syn-
thesis of the 3-amino-3-phenyl-2-propenoate esters, 49,
was performed by refluxing the corresponding benzoyl
acetate ester and ammonium acetate in ethanol (Scheme
4). An aldehyde containing a dimethylacetal group,
50d , was prepared from the corresponding olefin, 53,
through sequential oxidation with potassium perman-
ganate and sodium periodate (Scheme 5).41 Ketoesters
and ketothioesters were prepared as shown in Scheme
6.23 Alternately, a more versatile route through acyla-
tion of the cyclic compound 57, Meldrum’s acid,42
followed by opening of the ring with an alcohol or thiol40
and decarboxylation was also adopted for compounds
51 (Scheme 7). A formyl group was introduced at the
4-position of the dihydropyridines and pyridines via
protected dimethyl acetal derivatives, 14 and 30 (Scheme
2). The acid deprotection using a sulfonate ion-
exchange resin was carried out successfully on a dihy-
dropyridine or pyridine derivative. Yields and charac-
terization are shown in Table 3.
P h a r m a cology. A. P oten cy a n d Selectivity of
1,4-Dih yd r op yr id in es a t Hu m a n A3 Recep tor s. 1,4-
Dihydropyridine analogues bearing small alkyl groups
(methyl, ethyl, or propyl) at the 4-position (7-13, 20-
22) displayed affinity at the human A3 receptor of
between 1 and 7 µM and, at best, moderate selectivity
vs rat A1 and A2A receptors (Table 1). Among small
4-alkyl groups, there was not a clear pattern of effect
on the adenosine receptor affinity.
The receptor affinities of a series of 2-methyl-1,4-
dihydropyridines (1, 2, and 7-19) were compared.
Ester groups at the 3- and 5-positions were varied. At
the 3-position, a propyl, 8, vs ethyl ester, 7, had no effect
on the affinity at A1 and A2A receptors, while the affinity
at A3 receptors increased 3-fold. A 3-thioethyl ester, 10,
had the same A3 receptor affinity as the oxygen ana-
logue, 9, and affinity at A1 and A2A receptors was
marginally decreased. A 3-(2-methoxyethylthio)ester,
11, had 4-fold increased A2A receptor affinity vs the
ethylthio analogue, 10, and affinity at A1 and A3
receptors was slightly decreased. A 5-benzyl ester, in
the dihydropyridine series in which the 4-position
substituent is a styryl or phenylethynyl group, has been
reported to enhance A3 receptor selectivity.12,13 Among
4-ethyl-1,4-dihydropyridine 3-thioesters, the potency-
enhancing effect of a 5-benzyl ester was not evident (13
vs 10), thus the effects of 4- and 5-position substituents
are highly interdependent.
F igu r e 1. Structures of key A3 adenosine receptor selective
antagonists and agonists. Ki values in micromolar were
reported in refs 16-20.
A3 receptor. Dihydropyridine antagonists of A3 recep-
tors have also proven selective in chick cardiac myo-
cytes, in which the activation of A3 receptors induces
protective anti-ischemic effects.18 MRS 1191 was also
shown to be A3 receptor-selective in the rat hippocam-
pus,19 in which it was demonstrated that A3 receptor
activation suppresses the effects of activation of presyn-
aptic A1 receptors on inhibition of neurotransmitter re-
lease. MRS 1191 was also utilized to demonstrate that
presynaptic A3 receptor activation antagonizes metabo-
tropic glutamate autoreceptors.20 Most of the dihydro-
pyridine adenosine A3 receptor antagonists have been
designed through a classical medicinal chemical ap-
proach to rational drug design. An alternate approach
that has been explored is called the “functionalized
congener approach”,21,22 in which an easily derivatized
functional group is incorporated at the end of a strategi-
cally designed and attached chain substituent.22
In the previous studies,11-13 a few pyridine derivatives
were synthesized but no advantage for A3 receptor
selectivity was evident. In the present study, we have
used the 1,3-diacylpyridine nucleus, obtained through
oxidation of the corresponding 1,4-dihydropyridine, as
a template for probing structure-activity relationships
(SAR) at adenosine receptors and have identified com-
binations of substituents resulting in high selectivity
for human A3 receptors. Thus, it has been possible to
compare the structural requirements for the two related
classes of compounds and to propose a basis for the
major differences in a molecular model of the ligand
binding site of the human A3 receptor. Most notably,
bulky substituents at the 4-position and a 5-benzyl
ester, which are affinity-enhancing in dihydropy-
ridines,12,13 are not well tolerated in the pyridine series
for A3 receptor binding. At other positions, structural
parallels occur between corresponding dihydropyridine
and pyridine analogues.
In the 5-ethyl ester series, homologation of the 4-posi-
tion substituent from ethyl to propyl (10 vs 12) had no

3188 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Li et al.
Ta ble 1. Affinities of 1,4-Dihydropyridine Derivatives in Radioligand Binding Assays at A1, A2A, and A3 Receptors
Ki (µM) or % inhibitiond
a
b
c
compd
R2
R3
R4
R5
R6
rA1
rA2A
hA3
rA1/hA3
7e
8
9
10
11
12
13
14
15
1e
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OCH2CH3
OCH2CH2CH3
OCH2CH3
SCH2CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3 Ph
CH2CH3 Ph
CH2CH3 Ph
CH2CH3 Ph
CH2CH3 Ph
25.9 ( 7.3
35.9 ( 15.3
28.9 ( 4.8
7.24 ( 2.13
2.11 ( 0.35
2.27 ( 0.64
2.01 ( 0.55
4.58 ( 0.35
2.17 ( 0.25
1.65 ( 0.40
15.3 ( 3.9
3.6
8.2
9.6
17.4 ( 3.1
21.9 ( 3.3
21.8 ( 7.8
35.4 ( 4.5
54.8 ( 18.8
12.5 ( 2.5
18
SCH2CH2OCH3 CH2CH3
36 ( 14% (10-4
)
>10
>20
>50
>5
>6
55
SCH2CH3
SCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
CH2CH2CH3 CH2CH3 Ph
48 ( 5% (10-4
45 ( 2% (10-4
32 ( 5% (10-4
26 ( 6% (10-4
5.93 ( 0.27
)
)
)
)
29 ( 10% (10-4
14.3 ( 4.2
)
)
CH2CH3
CH(OCH3)2
CHO
Ph-CHdCH- CH2CH3 Ph
(trans)
Ph-CtC-
Ph-CtC-
Ph-CtC-
Ph-CtC-
Ph-CtC-
CH3
CH2Ph
Ph
CH2CH3 Ph
CH2CH3 Ph
d (10-4
)
32 ( 15% (10-4
4.77 ( 0.29
15.6 ( 5.4
0.108 ( 0.012
2e
16
17
18
19
20
21
22
CH3
CH3
CH3
CH3
CH3
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
SCH2CH3
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
Ph
40.1 ( 7.5
d (10-4
d (10-4
d (10-4
)
)
)
0.0314 ( 0.0028f
0.0277 ( 0.0024 >3000
0.0225 ( 0.0030 >3000
0.0505 ( 0.0210
0.229 ( 0.014
2.83 ( 0.20
0.907 ( 0.044
2.09 ( 0.04
1300
cyclopropyl 22 ( 1% (10-4
)
)
cyclobutyl
cyclopentyl 17.1 ( 4.3
cyclohexyl
36 ( 8% (10-4
7.16 ( 1.56
20% (10-4
d (10-4
29.1 ( 9.9
d (10-4
340
>400
>30
>50
>20
22 ( 2% (10-4
)
)
)
)
CH2CH3
CH2CH3
CH2CH2CH3 SCH2CH3
CH2CH3 Ph
CH2CH3 Ph
CH2CH3 Ph
20 ( 4% (10-4
34 ( 7% (10-4
)
CH2CH3
CH2CH3
26 ( 19% (10-4
)
)
a
Displacement of specific [3H]R - PIA binding in rat brain membranes, expressed as Ki ( SEM in µM (n ) 3-5), or as a percentage of
specific binding displaced at the indicated concentration (M). Displacement of specific [3H]CGS 21680 binding in rat striatal membranes,
b
expressed as Ki ( SEM in µM (n ) 3-6), or as a percentage of specific binding displaced at the indicated concentration (M). c Displacement
of specific [125I]AB-MECA binding at human A3 receptors expressed in HEK cells, in membranes, expressed as Ki ( SEM in µM (n )
3-4). Displacement of e 10% of specific binding at the indicated concentration (M). e values taken from van Rhee et al.11 and J iang et
d
al.13
effect on A3 receptor affinity. Either a dimethyl acetal,
14, or the corresponding aldehyde, 15, at the 4-position
had decreased adenosine receptor affinity. As reported
previously, the 4-styryl, 1, or 4-phenylethynyl substitu-
ent, 2, in the dihydropyridine series greatly enhanced
A3 receptor selectivity.
The adenosine receptor affinities of a series of 2-
methylpyridines, including 3-alkyl esters (23-25 and
30-35) and 3-alkylthioesters (26-29), were examined.
At the 3-position, a thioester, 26, had a 4-fold greater
affinity at A3 receptors than the oxygen analogue, 25,
and affinity at both A1 and A2A receptors was decreased
approximately 4-fold. A 3-propyl, 24, vs 3-ethyl ester,
23, also resulted in a major increase in A3 receptor
affinity (21-fold), with little effect on affinity at A1 and
The 6-phenyl substituent, previously found to be
optimal for A3 receptor affinity when unsubstituted,
could be replaced with cycloalkyl rings of varying size
in a series of 4-phenylethynyl-5-benzyl esters (16-19).
A cyclopentyl substituent, in 18, resulted in nearly the
same degree of A3 receptor affinity as the 6-phenyl
analogue, 2. A 6-cyclohexyl substituent, in 19, did not
provide as high an affinity in A3 receptor binding, but
smaller rings resulted in very high affinity and selectiv-
ity. The 6-cyclopropyl and 6-cyclobutyl analogues, 16
and 17, displayed Ki values at A3 receptors of 28 and
23 nM, respectively.
A2A receptors. A 2-methyl-3-(2-methoxyethylthio)ester,
27, was 4-fold less potent at A3 receptors than the
corresponding ethylthio analogue, 26. Unlike compound
2, a dihydropyridine, a 5-benzyl ester in the pyridine
3-thioester series greatly reduced A3 receptor affinity
and selectivity (29 vs 25). A 3-carboxylic acid, 37, was
nonselective in binding.
Similar to the affinity-increasing effect of homologa-
tion of 3-esters, at the 4-position, an ethyl, 25, vs methyl
group, 23, resulted in a 25-fold increase in A3 receptor
affinity. However, further extension of the 4-alkyl group
was not beneficial for affinity at human A3 receptors. A
3-ethylthio-4-propyl analogue, 28, was 5-fold less potent
at A3 receptors than the corresponding 4-ethyl analogue,
26; thus ethyl appeared to be the optimal 4-substituent
among small alkyl groups. Consistent with this obser-
vation, the presence of a 4-phenylethynyl (33-35) or
4-styryl group (32), unlike the case of dihydropyridines,
greatly reduced affinity at A3 receptors, thus resulting
in nonselectivity. Among 4-(phenylethynyl)pyridines,
the presence of a 6-cyclobutyl or 6-cyclopentyl group,
34 or 35, respectively, did not affect the A3 receptor
affinity observed for the corresponding 6-phenyl deriva-
tive, 33. The A3 adenosine receptor affinity of a
Effects of alkyl group modification at the 2-position
were also probed. A 2-ethyl vs 2-methyl substituent
afforded a slight increase in A3 receptor affinity (21 vs
10) while slightly diminishing affinity at A1 and A2A
receptors. A 2-propyl analogue, 22, was only half as
potent at A3 receptors as the corresponding 2-ethyl
analogue, 21.
B. P oten cy a n d Selectivity of P yr id in es a t Hu -
m a n A3 Recep tor s. In the pyridine series (23-48,
Table 2), unlike the dihydropyridines, the analogues
having small alkyl substituents at the 4-position tended
to reach greater potency at human A3 receptors than
those analogues bearing the 4-phenylethynyl or 4-styryl
group (32-35).

Selective A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3189
Ta ble 2. Affinities of Pyridine Derivatives in Radioligand Binding Assays at A1, A2A, and A3 Receptors
a
b
c
compd
R2
R3
R4
R5
R6
rA1
rA2A
hA3
rA1/hA3
e
23
24
25
26
27
28
29
30
31
32
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OCH2CH3
OCH2CH2CH3 CH3
OCH2CH3
SCH2CH3
SCH2CH2OCH3 CH2CH3
SCH2CH3
SCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
7.41 ( 1.29
5.05 ( 0.54
3.36 ( 0.60
14.8 ( 3.5
28.4 ( 9.1
24.5 ( 8.5
4.47 ( 0.46
1.7
23
19
0.215 ( 0.022
CH2CH3
CH2CH3
3.69 ( 1.25 0.176 ( 0.038
14.9 ( 4.1
0.0429 ( 0.0088
340
36 ( 11% (10-4) 7.98 ( 1.36 0.165 ( 0.012
>500
>700
>40
2.5
CH2CH2CH3 CH2CH3
CH2CH3 CH2Ph
CH(OCH3)2 CH2CH3
CHO CH2CH3
Ph-CHdCH- CH2CH3
(trans)
Ph-CtC-
Ph-CtC-
Ph-CtC-
CH3
CH2CH3
CH2CH3
CH2CH3
29 ( 6% (10-4
20 ( 8% (10-4
1.95 ( 0.43
9.56 ( 4.09
2.49 ( 0.47
)
)
7.53 ( 2.70 0.194 ( 0.051
12.8 ( 2.9 2.61 ( 0.96
2.88 ( 0.61 0.783 (0.154
2.56 ( 0.13 1.98 ( 0.21
2.40 ( 0.22 2.80 ( 1.78
4.8
0.85
33
34
35
36
37
CH3
CH3
CH3
CH2CH3
CH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
OH
SCH2CH3
SCH2CH3
SCH2CH3
SCH2CH3
SCH2CH3
SCH2CH3
CH2Ph
CH2Ph
CH2Ph
CH2CH3
CH2CH3
CH2CH3
Ph
11.6 ( 4.8
43(2% (10-4) 2.75 ( 0.78
27.6 ( 12.0 2.41 ( 0.59
4.2
>40
15
85
3.3
>3000
940
830
93
610
16
cyclobutyl d (10-4
)
cyclopentyl 56.2 ( 20.8
22.9 ( 5.0
13.4 ( 4.2
3.85 ( 0.79
0.121 ( 0.008
Ph
Ph
Ph
10.3 ( 1.7
4.25 ( 0.65
7.09 ( 0.97 1.28 ( 0.55
38 (MRS1476) CH2CH3
39a CH2CH3
39b (MRS1523) CH2CH3
41 ( 6% (10-4
7.77 ( 1.83
)
6.13 ( 1.28 0.0200 ( 0.0019
CH2CH2CH3 Ph
d (10-5
)
0.00829 ( 0.00115
CH2CH2CH3 CH2CH2CH3 Ph
15.6 ( 6.9
2.05 ( 0.44 0.0189 ( 0.0041
10.0 ( 3.0 0.188 ( 0.061
8.91 ( 0.97 0.0134 ( 0.0015
40
41
42
43
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH2OH Ph
17.4 ( 5.29
CH2CH3
CH2CH3
CH2CH3
3-Cl-Ph
8.20 ( 2.96
cyclopentyl 55.3 ( 14.7
26.1 ( 6.2
15.7 ( 4.4
24.1 ( 7.9
3.38 ( 1.87
SCH2CH2CH3 CH2CH3
SCH2CH2CH3 CH2CH3
Ph
8.22 ( 1.21
41.4 ( 11.9
16.7 ( 3.0
0.0159 ( 0.0054
520
44 (MRS1505) CH2CH3
45 (MRS1486) CH2CH2CH3 SCH2CH3
CH2CH2CH3 3-Cl-Ph
0.00794 ( 0.00319 5200
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
Ph
Ph
Ph
Ph
2.82 ( 0.82 0.0333 ( 0.0107
500
600
1200
>500
46
47
48
(CH2)2OCH3 SCH2CH3
(CH2)3CH3 SCH2CH3
cyclobutyl
10.1 ( 2.1
12.6 ( 1.7
d (10-4
22% (10-4
)
0.0168 ( 0.0020
0.0350 ( 0.0091
0.145 ( 0.044
40.3 ( 7.4
)
SCH2CH3
30 ( 1% (10-4
)
a
Displacement of specific [3H]R-PIA binding in rat brain membranes, expressed as Ki ( SEM in µM (n ) 3-5), or as a percentage of
specific binding displaced at the indicated concentration (M). Displacement of specific [3H]CGS 21680 binding in rat striatal membranes,
expressed as Ki ( SEM in µM (n ) 3-6), or as a percentage of specific binding displaced at the indicated concentration (M). c Displacement
of specific [125I]AB-MECA binding at human A3 receptors expressed in HEK cells, in membranes, expressed as Ki ( SEM in µM (n )
b
d
3-4). Displacement of e 10% of specific binding at the indicated concentration (M).
Sch em e 1. Synthesis of Substituted 1,4-Dihydropyridines Using the Hantzsch Reaction
4-dimethoxy acetal pyridine derivative, 30, vs the cor-
responding 4-ethyl derivative, 25, was substantially
decreased, while the change in affinity at A2A receptors
was <2-fold. The corresponding aldehyde, 31, was
weaker than the acetal, 30, at both A1 and A3 receptors.
The substituent at the 2-position also modulated
adenosine receptor affinity. Among pyridine 3-ethyl
esters, a 2-ethyl vs 2-methyl substituent afforded a 37-
fold increase in A3 receptor affinity (36 vs 23), with little
effect on affinity at A1 and A2A receptors. For 3-eth-
ylthioesters in the pyridine series, a 2-ethyl vs 2-methyl
substituent afforded a 2-fold increase in A2A and A3
receptor affinity (38 vs 26), and A1 receptor affinity was
somewhat decreased. The corresponding 2-propyl py-
ridine analogue, 45, was intermediate in potency at A3
receptors. Due to the apparent favorable effect of alkyl

3190 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Li et al.
Sch em e 2. Synthesis of a 1,4-Dihydropyridine
Containing an Aldehyde Group at the 4-Position
optimized ligand at rat A3 receptors, since the 4-propyl
group was more favorable for affinity than the 4-ethyl
group, and indeed 140-fold selectivity vs rat A1 receptors
was achieved. This derivative was highly potent at both
human and rat A3 receptors, with Ki values of 18.9 and
113 nM, respectively. In rat this corresponds to selec-
tivities of 140- and 18-fold vs A1 and A2A receptors,
respectively.
Molecu la r Mod elin g. By molecular modeling stud-
ies using RHF/AM1 semiempirical calculations and the
steric and electrostatic alignment (SEAL) method, we
offer a rationale for the high affinity of pyridine com-
pounds at human A3 receptors. To propose structural
analogies between these two classes of A3 ligands, we
modeled the 1,4-dihydropyridine derivative 2 (MRS
1191), both R- and S-enantiomers, and the pyridine
derivative 38. As reported above, 38 is among the most
potent and selective pyridine-based ligands at human
A3 receptors.
groups larger than methyl at the 2-position, a variety
of such substituents were examined. A 2-methoxyethyl
group, in 46, favored affinity at human A3 receptors.
2-n-Butyl (47) and 2-cyclobutyl (48) groups resulted in
2-fold and 7-fold, respectively, lower A3 receptor affinity
than the corresponding 2-ethyl analogue, 38. The most
favorable 2-substituent, ethyl, was used in a series of
analogues which probed optimization of 5- and 6-sub-
stituents (38-42). A 5-propyl ester, 39a , was 2-fold
more potent than the corresponding 5-ethyl ester, 38;
thus propyl appeared to be the favored 5-substituent
among small alkyl esters. A 3-propyl thioester, 43,
favored A3 receptor affinity. A 5-(2-hydroxyethyl) ester,
40, was synthesized for the purpose of increasing water
solubility; however, affinity at human in A3 receptors
was significantly decreased. At the 6-position, phenyl,
38, 3-chlorophenyl, 41, and cyclopentyl, 42, substituents
were compared. A3 receptor affinity decreased in the
order 3-chlorophenyl ) phenyl . cyclopentyl. 5-Propyl
2,4-diethyl-3-propylsulfanylcarbonyl-6-(m-chlorophenyl)-
pyridine-5-carboxylate, 44, containing the optimized
substitution was prepared and found to have a Ki value
of 7.94 nM at human A3 receptors.
C. P oten cy a n d Selectivity of 1,4-Dih yd r op y-
r id in es a n d P yr id in es a t Ra t A3 Recep tor s. At
recombinant rat A3 receptors the binding affinities of
selected derivatives were found to be in the micromolar
range (Table 4). Generally, the pyridine derivatives
reached higher affinity than the 1,4-dihydropyridines.
Among 1,4-dihydropyridines, a 4-propyl, 12, or 5-benzyl
ester group, 13, slightly increased A3 receptor affinity
vs the corresponding 4,5-diethyl analogue, 10. Similar
effects were observed at the human A3 receptor. Unlike
at human A3 receptors, the presence of the 4-phenyl-
ethynyl group in 17 was only slightly potency-enhanc-
ing. Like at human A3 receptors, a 2-ethyl group in 21
enhanced potency at rat A3 receptors by severalfold over
the corresponding 2-methyl derivative, 10. A 2-propyl
group, in the dihydropyridine 22, offered no advantage
for A3 affinity over the 2-ethyl substituent.
Pyridine and 1,4-dihydropyridine structures have
high conformational flexibility, in particular regarding
correspondence of the two ester groups at 3- and
5-positions. We have explored the conformational spaces
for the three compounds, (R)-MRS 1191, (S)-MRS 1191,
and 38. A complete random search conformational
analysis was performed, and after sampling and mini-
mization procedures, four energetically important con-
formers were selected for each studied compound (Table
5). The four conformers correspond to the structures
in which the positions of the carbonyl of the 3- and
5-ester groups are up and/or down with respect to the
plane of the pyridine or 1,4-dihydropyridine ring. The
first important consideration is that the two enanti-
omers of MRS 1191 present conformers with different
structures, in particular considering the spatial orienta-
tion of the two ester groups, as shown from the
structural data collected in Table 5. The optimized
structures for two of the conformers used in the overlay
are shown in Figure 2.
Our hypothesis was that the receptor binding proper-
ties of 1,4-dihydropyridine and pyridine derivatives
were due to recognition at a common region inside the
receptor binding site and, consequently, a common
electrostatic potential profile. The electrostatic poten-
tial profile is a function of the stereochemistry of the
chiral center at the 4-position and the chemical struc-
ture of the conformer considered. To study the overall
similarity between these two classes of A3 receptor
ligands and to establish a quantitative comparison of
the electrostatic potential fields, we have not used exact
atomic matches, but rather the steric and electrostatic
alignment (SEAL) method.25 The SEAL methodology
has been developed to optimize the alignment of two
three-dimensional structures using atomic charges and
steric volume as factors. In this SEAL analysis we
compared the 32 possible superimposition combinations
between dihydropyridine/pyridine pairs for all 12 con-
formers listed in Table 5. From these structural align-
ments the best fit occurred between the (R) 3-down,5-
up conformer of MRS 1191 and the 3-down,5-up
conformer of 38, as illustrated in Figure 2. None of the
(S) conformers overlap the pyridine structure with
interaction energy comparable with the (R) 3-down,5-
up conformer of MRS 1191. If there exists a correlation
Among pyridine derivatives binding at rat A3 recep-
tors, unlike at human A3 receptors, a 4-propyl group,
in 28, caused a 2-fold increase in affinity with a Ki value
of 0.65 µM, vs the corresponding 3-ethyl analogue, 26.
5-Benzyl esters or substitutions with 4-phenylethynyl
and 6-cyclobutyl groups, 29 and 34, did not significantly
alter the affinity of 26. However, a 2-ethyl substitution
increased the binding affinity at rat A3 receptors vs 26
by 4-fold, providing a Ki value of 0.41 µM. A 2-propyl
pyridine analogue, 45, was near equipotent; however,
a 2-butyl analogue, 47, was considerably less potent at
rat A3 receptors.
5-Propyl 2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-
phenylpyridine-5-carboxylate, 39b, was prepared as an

Selective A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3191
Sch em e 3. Synthesis of Pyridine Derivatives
Sch em e 4. Synthesis of â-Amino-R,â-unsaturated
Esters
Sch em e 5. Synthesis of a Protected Aldehyde for
Incorporation at the 4-Position of 1,4-Dihydropyridines
degree of similarity (Figure 3). The first two regions
(negative electrostatic potential) are located around the
carbonyl of the 3- and 5-ester groups. The second set
designates the 1- and 6-positions corresponding to two
hydrophobic regions (positive electrostatic potential).
The last region corresponds to the π-systems of both
pyridine and 1,4-dihydropyridine structures (negative
electrostatic potential). Using both SEAL and electro-
static potential field analysis, it is possible to describe
a pharmacophore map for these two classes of A3
antagonists. First of all, the stereochemistry of the
chiral center seems to be important for the recognition
process.11 Compound 38 and (R)-MRS 1191 present a
high degree of similarity of the molecular charge
between thermodynamic binding constants and match-
ing of the electrostatic potential fields of different
antagonists, we can speculate that the (R) enantiomer
of MRS 1191 could be more potent than the (S) enan-
tiomer in A3 receptor binding. We, therefore, calculated
the electrostatic contours for both the (R) 3-down,5-up
conformer of MRS 1191 and the 3-down,5-up conformer
of 38, as shown in Figure 2B. The electrostatic potential
maps present complicated topologies; however, it is
possible to identify several regions that show a high

3192 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Sch em e 6. Synthesis of â-Ketoesters via Acylacetyl Esters
Li et al.
Sch em e 7. Synthesis of â-Ketoesters via Meldrum’s
Acid
acids in the binding cavity. A strong steric control
around the 4-position of both 1,4-dihydropyridine and
pyridine structures is suggested. In fact, bulky 4-posi-
tion substituents, which are affinity-enhancing in the
1,4-dihydropyridines, are not well tolerated in the
pyridine series. From a structural point of view, chang-
ing the C4-hybridization from sp3 to sp2, corresponding
to the transformation of a 1,4-dihydropyridine to the
corresponding pyridine, would change the C5-C4-R4
angle from 68.1° to 0.2°. A hydrophobic pocket is likely
present around the 6-position where a phenyl ring
would bind. Another important consideration is that
the modification of the chemical properties of the
nitrogen at the 1-position, from sp3 to sp2, and conse-
quently the changing of the acid-base behavior of these
compounds, does not prevent ligand recognition. These
results can be summarized in a pharmacophore map as
shown in Figure 4.
Moreover, a relationship was found for pyridine
derivatives between affinity and hydrophobicity, rep-
resented by the log P value (Figure 5), such that A3
affinity in general increases with increasing log P
values. Of course, we have to consider this correlation
within the limitations of the specific steric requirements
of the receptor binding site. Accordingly, the calculated
log P values for the dihydropyridine 12, which contains
a propyl group in place of ethyl in the 4-position, are
higher with respect to 38 (5.02 and 4.88, respectively)
but the Ki value is 2 orders of magnitude lower (2.17
and 0.0200 µM, respectively). In fact, as already
mentioned, bulky substituents at the 4-position are not
distributions. Two important hydrophilic interactions,
probably hydrogen-bonding interactions, could be in-
volved between the two ester groups and polar amino

Selective A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3193
Ta ble 3. Yields and Analysis of Dihydropyridine and Pyridine
Ta ble 4. Affinities of
Derivatives
4-Phenylethynyl-6-phenyl-1,4-dihydropyridine Derivatives in
Radioligand Binding Assays at Rat A3 Receptors
no.
formula
analysis
yield (%)
Ki(µM)
8
9
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
20H25NO4‚0.25H2O
20H25NO4
20H25NO3S
21H27NO4S
21H27NO3S‚0.1H2O
25H27NO3S
21H27NO6
19H21NO5
28H27NO4‚0.5C3H6O
29H29NO4‚0.2H2O
30H31NO4
31H33NO4
20H25NO4
21H27NO3S
22H29NO3S
20H23NO4
20H23NO4
20H23NO3S
21H25NO4S
21H25NO3S
25H25NO3S
21H25NO6
19H19NO5‚0.4C7H8
29H27NO4
30H29NO4
20H23NO4
18H19NO4‚0.1C7H8
21H25NO3S
22H27NO3S
23H29NO3S
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
H,N,Ca
HRMSc
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
HRMSd
HRMSe
C,H,N
C,H,N
C,H,N
C,H,N
HRMSf
C,H,N
C,H,N
C,H,N
HRMSg
C,H,N
C,H,N
H,N,Cb
C,H,N
C,H,N
C,H,N
HRMSh
C,H,N
C,H,N
C,H,N
HRMSi
HRMSj
C,H,N
63
55
81
68
54
47
30
82
24
35
16
52
58
72
45
78
85
61
65
78
82
59
55
83
34
96
56
39
85
71
54
58
52
79
65
51
65
53
64
a
compound
rA3
rA1/rA3
rA3/hA3
10
11
12
13
14
15
16
17
18
19
20
21
22
24
25
26
27
28
29
30
31
34
35
36
37
38
39a
39b
40
41
42
43
44
45
46
47
48
2 (MRS 1191)
1.42 ( 0.19
4.60 ( 0.38
3.10 ( 0.78
2.80 ( 0.28
1.75 ( 0.18
2.52 ( 0.88
2.73 ( 0.14
1.47 ( 0.34
0.650 ( 0.070
1.80 ( 0.32
1.90 ( 0.42
0.410 ( 0.048
0.183 ( 0.033
0.113 ( 0.012
2.87 ( 0.48
0.440 ( 0.033
2.80 ( 0.22
0.294 ( 0.006
0.814 ( 0.037
0.590 ( 0.040
2.26 ( 0.05
28
7.7
45
2.3
1.4
1.7
78
2.8
1.3
34
3.4
0.69
0.79
21
22
6.0
15
33
0.83
18
10
12
13
17
21
22
26
28
>20
>20
>40
>30
>30
10
>100
>50
>50
>100
42
140
6.1
19
>20
28
29
34
38
39a
39b (MRS 1523)
40
41
42
43
44
45
47
50
28
18
100
18
64
Displacement of specific [125I]AB-MECA binding at rat A3
a
receptors stably expressed in CHO cells2,32 (n ) 3-5).
All of these factors can modify affinities and selectivities
of pyridine compounds in comparison to the original
properties of 1,4-dihydropyridines. The critical limita-
tion in the quantitative interpretation of the SAR in the
1,4-dihydropyridine series is the limited chemical and
pharmacological information about the two pure enan-
tiomers. Since mainly racemic 1,4-dihydropyridines
have been studied in adenosine receptor binding, cor-
relating the experimental results with any structural
properties is complicated. In the present study, we have
reported that opportune combinations of substituents
on the pyridine ring resulted in highly selective ligands
for human A3 receptors. The discovery that pyridine
derivatives can also bind to human A3 receptors could
also be useful in modeling the mode of receptor binding
of the 1,4-dihydropyridine compounds. Therefore, it was
of interest to determine a hypothesis for a common mode
of action of the pyridine and 1,4-dihydropyridine deriva-
tives.
In the present study, we have compared the SAR of
1,4-dihydropyridines and the corresponding pyridine
derivatives at human A3 receptors and at other adenos-
ine receptors. Common substituents among A3 adenos-
ine receptor-selective analogues in each series, such as
3,5-diesters, the 6-phenyl group, and small alkyl groups
at the 2-position suggest a common receptor binding site
for both substituted 1,4-dihydropyridine and pyridine
derivatives. We have proposed a quantitative basis for
this hypothesis using molecular modeling, although
differences in the binding requirements of 1,4-dihydro-
pyridines and pyridines have been found. For example,
6-cycloalkyl groups only in dihydropyridines but not
pyridines favor A3 receptor affinity. Unlike the dihy-
dropyridine derivatives, the 4-styryl and the 4-phenyl-
ethynyl substituents are disfavored in A3 adenosine
receptor binding of pyridines. At the 4-position of
pyridines, an ethyl group was favored at human A3
receptors, and a propyl group favored at rat A3 recep-
tors. At the 2-position, elongation of the 2-methyl
21H25NO4S
21H24ClNO3S
20H29NO3S
22H27NO3S‚0.1H2O
23H28ClNO3S
22H27NO3S
22H27NO4S
C23H29NO3S
23H27NO3S‚0.6H2O
C
a
Elemental analysis for compound 14. C, calculated: 64.76;
found: 66.58. H, calculated: 6.99; found: 6.25. b Elemental analy-
sis for compound 39a . C, calculated: 68.54; found: 70.16. The
following compounds were shown to be pure on analytic TLC (silica
gel 60, 250 µm) EtOAc-petroleum ether ) 10:90 (v/v), unless
noted. c Compound 15, Rf ) 0.87; EI calcd for C18H20NO4 (M+
-
d
CHO) 314.1392, found 314.1432. Compound 24, Rf ) 0.44; EI
calcd for C20H23NO4 (M+) 341.1627, found 341.1635. e Compound
25, Rf ) 0.35; EI calcd for C20H23NO4 (M+) 341.1627, found
341.1615. f Compound 30, EtOAc-petroleum ether ) 20:80 (v/v),
Rf ) 0.36; EI calcd for C21H25NO6 (M+) 387.1682, found 387.1674.
Compound 36, Rf ) 0.46; EI calcd for C20H23NO4 (M+) 341.1627,
g
h
found 341.1631. Compound 42, Rf ) 0.51; EI calcd for C20H29NO3S
(M+) 363.1868, found 363.1858. i Compound 46, Rf ) 0.27; EI calcd
for C22H27NO4S (M+) 401.1661, found 401.1666. Compound 47,
j
Rf ) 0.54; EI calcd for C23H29NO3S (M+) 399.1868, found 399.1867.
well tolerated in the pyridine series. Finally, values of
calculated log P of 38 and MRS 1191, 5.29 and 4.98,
respectively, are similar, as the compounds are similar
in A3 affinity.
Discu ssion
Pyridine derivatives represent one of the possible
important in vivo metabolites of 1,4-dihydropyridine
compounds.24 The oxidation process produces three
important chemical modifications in the 1,4-dihydropy-
ridine structure: (a) the loss of the chiral center and
consequently a change in the spatial position of the
substituent in 4-position; (b) the formation of a stable
aromatic system; and (c) the decrease of the pKa value.

3194 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Li et al.
Ta ble 5. Values for Enthalpy of Formation (∆Hf) and Spatial Arrangement (Dihedral Angle Values) of the 3- and 5-Ester Groups for
the Energetically Important Conformers Calculated for 38, (R)-MRS 1191, and (S)-MRS 1191
∆Hf,
compd
no.
kcal/mola
3-COORb
5-COORb
C2-C3-C-O (deg)c
C6-C5-C-O (deg)c
38
1
2
3
4
5
6
7
8
9
-82.4
-81.9
-82.4
-81.9
-10.1
-10.3
-8.9
v
v
V
V
v
v
V
V
v
v
V
V
v
V
v
V
v
V
v
V
v
V
v
V
79.5
78.6
-91.0
-89.6
12.6
-88.9
90.7
-92.0
93.0
-83.6
112.5
-84.3
113.0
-100.8
60.3
(R)-MRS 1191
(S)-MRS 1191
10.7
-89.5
-134.0
146.8
148.1
-25.5
-21.9
-8.7
-8.8
10
11
12
-9.3
-9.7
-104.3
-10.3
61.8
a
b
Values calculated using RHF/AM1 Hamiltonian. v and V designations have been assigned using the chemical structural arrangement
shown in Figure 2. c C and O atoms refer to the carbonyl moiety of the ester group.
F igu r e 2. Top: Optimized geometries for the conformers 3-down,5-up of compound 38 and (R)-MRS 1191. Bottom: Alignments
generated by SEAL for the conformers 3-down,5-up of compound 38 and (R)-MRS 1191, viewed from the top (A) and from the
front (B).
substituent (but not beyond a 3-carbon chain) was found
to enhance the affinity at A3 receptors. There is
evidence (46) that an ether group within this chain is
tolerated at human A3 receptors. Also, a 3-thioester
group, which in dihydropyridine derivatives either
enhanced A2A adenosine receptor affinity13 or had little
effect on the adenosine receptor affinity (present study),
in the pyridine series substantially enhanced A3 recep-
tor affinity and selectivity. Thus, compound 26, con-
taining a 2-methyl group, proved to be 340-fold selective
for the human A3 receptor. The corresponding 2-ethyl
analogue, 38, was >3000-fold selective for human A3
vs rat A1 receptors. The 5-benzyl ester did not enhance
A3 receptor affinity of pyridines as it did for 4-(phenyl-
ethynyl)dihydropyridines. In the dihydropyridine se-
ries, electron-withdrawing groups at the para and meta
positions of a 5-benzyl ester provided A3 receptor
selectivity of many thousand-fold, i.e., the affinity at A1
and A2A receptors was essentially negligible, and the
affinity at A3 receptors vs 2 was either maintained or

Selective A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3195
F igu r e 3. Comparison between the isopotential surfaces of (R)-MRS 1191 and compound 38 (red ) 5 kcal/mol, and blue ) -5
kcal/mol).
F igu r e 5. Hydrophobicity structure-activity relationship
found for the pyridine derivatives. The graph reports the
correlation between the calculated log P values and the
experimental value of log Ki of different pyridine compounds.
human A3 receptor structures, using molecular model-
F igu r e 4. Scheme of the hypothetical pharmacophore map
for compound 38. In brackets, the A3 receptor transmembrane
helical domains putatively involved in the recognition of the
pyridine moiety are shown.
ing techniques, is in progress in our laboratory to
explain the different SARs found for these two receptors.
In conclusion, the dihydropyridines and now the
corresponding pyridines have served as structural scaf-
folds for enhancement of selectivity at human A3 recep-
tors.17 An unsolved problem in the studies of dihydro-
pyridines as A3 receptor antagonists is the resolution
of enantiomers, since compounds 1-3 have as yet been
characterized only as racemic compounds. The present
study circumvents the need to resolve enantiomers,
since we have demonstrated that derivatives highly
selective for human A3 receptors may be obtained simply
through oxidation of various 6-phenyl-1,4-dihydropy-
ridines to the corresponding nonchiral pyridines. Func-
tional studies and further optimization of the SAR in
order to design radioligands and other affinity probes
of the A3 receptor and to increase hydrophilicity are now
appropriate. While the dihydropyridines such as MRS
1191 have been shown to be competitive antagonists,39
enhanced.13 Among the most selective compounds at
human A3 receptors in the previous study was the
4-nitrobenzyl analogue, 3. In the present study the
most potent pyridine derivatives at human A3 receptors
were 44, 39a > 41 ) 43, 46 > 38.
A persistent problem during the development of
selective A3 receptor antagonists has been species
differences in affinity.4 Affinity at rat A3 receptors is
generally orders of magnitude lower than at human A3
receptors. This study has demonstrated that pyridine
derivatives display considerable affinity and selectivity
at rat A3 receptors (Table 4). Thus, certain compounds
in this series having high affinity in both species, such
as 39b, are likely to be of use as pharmacological probes
across species. A detailed comparison between rat and

3196 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Li et al.
1.29 (t, J ) 7.8 Hz, 3 H), 1.39 (m, 2 H), 1.49 (m, 2 H), 2.32 (s,
3 H), 2.92 (q, J ) 7.8 Hz, 2 H), 3.92 (q, J ) 7.8 Hz, 2 H), 4.19
(t, J ) 6.0 Hz, 1 H), 5.98 (s, br, 1 H), 7.27-7.31 (m, 2 H), 7.38-
7.41 (m, 3 H). MS (CI/NH3): m/z 391 (M+ + NH4, base), 373
(M+). MS (EI): m/z 330 (M+ - CH2CH2CH3, base), 314 (MH+
- OEt - Me), 284 (M+ - COSEt).
5-Ben zyl 2-Meth yl-4-eth yl-6-p h en yl-3-(eth ylsu lfa n yl-
ca r bon yl)-1,4-(()-d ih yd r op yr id in e-5-ca r boxyla te (13). 1H
NMR: δ 0.92 (t, J ) 7.8 Hz, 3 H), 1.29 (t, J ) 7.8 Hz, 3 H),
1.55-1.64 (m, 2 H), 2.32 (s, 3 H), 2.92 (q, J ) 7.8 Hz, 2 H),
4.24 (t, J ) 6.0 Hz, 1 H), 4.96 (AB, J ) 12.6 Hz, 2 H), 5.86 (s,
br, 1 H), 6.98-7.00 (m, 1 H), 7.22-7.40 (m, 9 H). MS (CI/
NH3): m/z 439 (M+ + NH4, base), 421 (M+), 360 (M+ - SEt).
3,5-Dieth yl 2-Meth yl-6-p h en yl-4-(d im eth oxym eth yl)-
1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (14). 1H NMR:
δ 0.91 (t, J ) 6.9 Hz, 3 H), 1.33 (t, J ) 6.9 Hz, 3 H), 2.33 (s, 3
H), 3.38 (s, 3 H), 3.39 (s, 3 H), 3.93 (q, J ) 6.9 Hz, 2 H), 4.14
(d, J ) 6.0 Hz, 1 H), 4.22 (q, J ) 6.9 Hz, 2 H), 4.48 (d, J ) 6.0
Hz, 2 H), 5.84 (s, br, 1 H), 7.31-7.35 (m, 2 H), 7.38-7.40 (m,
3 H). MS (CI/NH3): m/z 407 (M+ + NH4), 390 (M+ + 1), 358
(M+ - OMe, base).
3-Eth yl 5-Ben zyl 2-Meth yl-4-p h en yleth yn yl-6-cyclo-
p r op yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r b oxyla t e (16).
1H NMR: δ 0.59 (m, 1 H), 0.88-1.03 (m, 2 H), 1.18-1.28 (m,
1 H), 1.32 (t, J ) 7.8 Hz, 3 H), 2.31 (s, 3 H), 2.73-2.83 (m, 1
H), 4.17-4.35 (m, 2 H), 5.09 (s, 1 H), 5.29 (AB, J ) 12.9 Hz, 2
H), 5.56 (s, br, 1 H), 7.22-7.47 (m, 10 H). MS (EI): m/z 441
(M+), 412 (M+ - CH2CH3,), 368 (M+ - CO2Et), 350 (M+ - CH2-
Ph), 306 (M+ - CO2CH2Ph), 91 (+CH2Ph, base).
3-Eth yl 5-Ben zyl 2-Meth yl-4-p h en yleth yn yl-6-cyclobu -
tyl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (17). 1H
NMR: δ 1.32 (t, J ) 6.9 Hz, 3 H), 1.79-2.29 (m, 6 H), 2.37-
2.40 (m, 1 H), 2.38 (s, 3 H), 4.21-4.27 (m, 2 H), 5.07 (s, 1 H),
5.26 (AB, J ) 12.6 Hz, 2 H), 6.10 (s, br, 1 H), 7.21-7.46 (m,
10 H). MS (EI): m/z 455 (M+), 426 (M+ - CH2CH3,), 382 (M+
- CO2Et), 364 (M+ - CH2Ph), 320 (M+ - CO2CH2Ph), 91 (+-
CH2Ph, base).
3-Eth yl 5-Ben zyl 2-Meth yl-4-p h en yleth yn yl-6-cyclo-
p en tyl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (18). 1H
NMR: δ 1.23-1.37 (m, 4 H), 1.32 (t, J ) 6.9 Hz, 3 H), 1.70
(m, 4 H), 2.00 (m, 1 H), 2.35 (s, 3 H), 4.24 (m, 2 H), 5.09 (s, 1
H), 5.27 (AB, J ) 12.9 Hz, 2 H), 5.90 (s, br, 1 H), 7.22-7.46
(m, 10 H). MS (EI): m/z 487 (M+ + NH4), 470 (M+ + 1).
3-Eth yl 5-Ben zyl 2-Meth yl-4-p h en yleth yn yl-6-cyclo-
h exyl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (19). 1H
NMR: δ 1.13-1.38 (m, 6 H), 1.32 (t, J ) 6.9 Hz, 3 H), 1.65-
1.89 (m, 5 H), 2.35 (s, 3 H), 4.22 (q, J ) 6.9 Hz, 2 H), 5.09 (s,
1 H), 5.27 (AB, J ) 12.6 Hz, 2 H), 5.99 (s, br, 1 H), 7.21-7.46
(m, 10 H). MS (EI): m/z 483 (M+), 454 (M+ - CH2CH3,), 400
(M+ - C6H11), 410 (M+ - CO2Et), 392 (M+ - CH2Ph), 348 (M+
- CO2CH2Ph), 91 (+CH2Ph, base).
this remains to be demonstrated for the highly A3
receptor-selective pyridine derivatives reported here.
Exp er im en ta l Section
Syn t h esis. Ma t er ia ls a n d In st r u m en t a t ion . Ethyl
3-aminocrotonate (49c), aldehydes (50, except for 50d ), ethyl
acetoacetate (51a ), ethyl propionylacetate (51b), tetrachloro-
1,4-benzoquinone (52), acrolein dimethyl acetal (53), ethyl
benzoylacetate, 2,2,6-trimethyl-4H-1,3-dioxin-4-one (55), ben-
zyl acetate, N-isopropylcyclohexylamine, all acid chlorides (56,
except 56f, obtained by the reaction of the precursor acid with
thionyl chloride), 2,2-dimethyl-1,3-dioxane-4,6-dione (57),
ethanethiol, propanethiol, and Dowex 50×8-200 were pur-
chased from Aldrich (St. Louis, MO). 2-Methoxyethanethiol
was prepared by a reported method.40 All other materials were
obtained from commercial sources.
Proton nuclear magnetic resonance spectroscopy was per-
formed on a Varian GEMINI-300 spectrometer, and all spectra
were obtained in CDCl3. Chemical shifts (δ) relative to
tetramethylsilane are given. 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.
Gen er a l P r oced u r e for P r ep a r a tion of Su bstitu ted 1,4-
Dih yd r op yr id in e (8-14, 16-22, Sch em e 1). Equimolar
amounts (0.5-1.0 mmol) of the appropriate â-enaminoester
(49), aldehyde (50), and â-ketoester (51) were dissolved in 2-5
mL of absolute ethanol. The mixture was sealed in a Pyrex
tube and heated, with stirring, to 80 °C for 18-24 h. After
the mixture was cooled to room temperature, the solvent was
evaporated and the residue was purified by preparative TLC
(silica 60; 1000 or 2000 µm; Analtech, Newark, DE; petroleum
ether-ethyl acetate (4:1-9:1)). The products were shown to
be homogeneous by analytical TLC and were stored at -20
°C.
3-P r op yl 5-Eth yl 2,4-Dim eth yl-6-p h en yl-1,4-(()-d ih y-
d r op yr id in e-3,5-d ica r boxyla te (8). 1H NMR: δ 0.91 (t, J
) 6.9 Hz, 3 H), 1.00 (t, J ) 6.9 Hz, 3 H), 1.13 (d, J ) 6.9 Hz,
3 H), 1.72 (m, 2 H), 2.30 (s, 3 H), 3.88-4.00 (m, 3 H), 4.15 (m,
2 H), 5.69 (s, br, 1 H), 7.28-7.31 (m, 2 H), 7.39-7.42 (m, 3 H).
MS (CI/NH3): m/z 361 (M+ + NH4), 344 (M+ + 1). MS (EI):
m/z 343 (M+), 328 (M+ - CH3, base), 314 (M+ - CH2CH3), 284
(M+ - OPr).
3,5-Dieth yl 2-Meth yl-4-eth yl-6-p h en yl-1,4-(()-d ih yd r o-
p yr id in e-3,5-d ica r boxyla te (9). 1H NMR: δ 0.87-0.92 (m,
6 H), 1.31 (t, J ) 6.9 Hz, 3 H), 1.52 (m, 2 H), 2.32 (s, 3 H), 3.90
(m, 2 H), 4.03 (t, J ) 5.9 Hz, 1 H), 4.20 (m, 2 H), 5.71 (s, br, 1
H), 7.30-7.40 (m, 5 H). MS (CI/NH3): m/z 361 (M+ + NH4,
base), 344 (M+ + 1), 314 (M+ - C2H5). MS (EI): m/z 314 (M+
- CH2CH3, base), 298 (M+ - OCH2CH3).
5-Eth yl 2-Meth yl-4-eth yl-6-p h en yl-3-(eth ylsu lfa n ylca r -
bon yl)-1,4-(()-d ih yd r op yr id in e-5-ca r boxyla te (10). 1H
NMR: δ 0.90-0.96 (m, 6 H), 1.29 (t, J ) 7.8 Hz, 3 H), 1.57 (m,
2 H), 2.33 (s, 3 H), 2.93 (q, J ) 7.8 Hz, 2 H), 3.94 (q, J ) 6.9
Hz, 2 H), 4.03 (t, J ) 4.8 Hz, 1 H), 4.19 (q, J ) 6.0 Hz, 2 H),
5.81 (s, br, 1 H), 7.30-7.32 (m, 2 H), 7.40-7.42 (m, 3 H). MS
(CI/NH3): m/z 377 (M+ + NH4, base), 314 (M+ - OEt), 298
(M+ - SEt). MS (EI): m/z 330 (M+ - CH2CH3, base), 314 (M+
- OEt), 298 (M+ - SEt), 286 (M+ - CO2Et).
3,5-Dieth yl 2-Eth yl-6-p h en yl-4-m eth yl-1,4-(()-d ih yd r o-
p yr id in e-3,5-d ica r boxyla te (20). 1H NMR: δ 0.90 (t, J )
6.9 Hz, 3 H), 1.12 (d, J ) 6.9 Hz, 3 H), 1.19 (t, J ) 6.9 Hz, 3
H), 1.32 (t, J ) 6.9 Hz, 3 H), 2.50 (m, 1 H), 2.90 (m, 1 H),
3.89-3.98 (m, 3 H), 4.22 (m, 2 H), 5.73 (s, br, 1 H), 7.30-7.31
(m, 2 H), 7.40-7.42 (m, 3 H). MS (CI/NH3): m/z 361 (M+
+
NH4), 344 (M+ + 1). MS (EI): m/z 343 (M+), 328 (M+ - CH3,
base), 298 (M+ - OEt).
5-Eth yl 2,4-Dieth yl-6-ph en yl-3-(eth ylsu lfan ylcar bon yl)-
1,4-(()-d ih yd r op yr id in e-5-ca r boxyla te (21). 1H NMR: δ
0.89 (m, 6 H), 0.93 (t, J ) 6.9 Hz, 3 H), 1.19 (t, J ) 7.8 Hz, 3
H), 1.58 (m, 2 H), 2.69 (m, 2 H), 2.92 (q, J ) 7.8 Hz, 2 H), 3.92
(q, J ) 6.9 Hz, 2 H), 4.02 (t, J ) 6.0 Hz, 1 H), 5.94 (s, br, 1 H),
5-Eth yl 2-Meth yl-4-eth yl-6-p h en yl-3-[(2-m eth oxy-(eth -
ylsu lfa n ylca r bon yl)]-1,4-(()-d ih yd r op yr id in e-5-ca r boxy-
la te (11). 1H NMR: δ 0.91 (t, J ) 7.8 Hz, 3 H), 0.92 (t, J )
7.8 Hz, 3 H), 1.60 (m, 2 H), 2.32 (s, 3 H), 3.14 (t, J ) 6.9 Hz,
2 H), 3.38 (s, 3 H), 3.55 (t, J ) 6.9 Hz, 2 H), 3.93 (q, J ) 7.8
Hz, 2 H), 4.20 (t, J ) 6.0 Hz, 1 H), 5.91 (s, br, 1 H), 7.28-7.32
7.32 (m, 2 H), 7.41 (m, 3 H). MS (CI/NH3): m/z 391 (M+
+
NH4, base), 374 (M+ + 1), 312 (M+ - SEt). MS (EI): m/z 373
(M+), 344 (M+ - CH2CH3), 328 (M+ - OEt, base), 312 (M+
SEt).
-
(m, 2 H), 7.38-7.42 (m, 3 H). MS (CI/NH3): m/z 405 (M+
+
5-Eth yl 2-P r op yl-4-eth yl-6-p h en yl-3-(eth ylsu lfa n ylca r -
bon yl)-1,4-(()-d ih yd r op yr id in e-5-ca r boxyla te (22). 1H
NMR: δ 0.90-0.96 (m, 6 H), 0.99 (t, J ) 7.8 Hz, 3 H), 1.29 (t,
J ) 7.8 Hz, 3 H), 1.53-1.66 (m, 4 H), 2.66 (m, 2 H), 2.92 (q, J
) 6.9 Hz, 2 H), 3.95 (q, J ) 7.8 Hz, 2 H), 4.20 (t, J ) 6.0 Hz,
NH4, base), 387 (M+).
5-Eth yl 2-Meth yl-4-p r op yl-6-p h en yl-3-(eth ylsu lfa n yl-
ca r bon yl)-1,4-(()-d ih yd r op yr id in e-5-ca r boxyla te (12). 1H
NMR: δ 0.90 (t, J ) 7.8 Hz, 3 H), 0.92 (t, J ) 7.8 Hz, 3 H),

Selective A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3197
1 H), 5.85 (s, br, 1 H), 7.30-7.32 (m, 2 H), 7.41-7.43 (m, 3 H).
6.96-6.98 (m, 2 H), 7.22-7.28 (m, 3 H), 7.38-7.40 (m, 3 H),
7.55-7.58 (m, 2 H). MS (CI/NH3): m/z 420 (M+ + 1, base).
MS (CI/NH3): m/z 405 (M+ + NH4), 388 (M+ + 1), 326 (M+
-
SEt).
3,5-Dieth yl 2-Meth yl-4-(d im eth oxym eth yl)-6-p h en ylp y-
r id in e-3,5-d ica r boxyla te (30). 1H NMR: δ 1.00 (t, J ) 6.9
Hz, 3 H), 1.41 (t, J ) 6.9 Hz, 3 H), 2.62 (s, 3 H), 3.33 (s, 6 H),
4.07 (q, J ) 6.9 Hz, 2 H), 4.41 (d, J ) 6.9 Hz, 2 H), 5.76 (s, 1
H), 7.40-7.42 (m, 3 H), 7.53-7.55 (m, 2 H). MS (CI/NH3): m/z
388 (M+ + 1). HRMS: calcd for C21H25NO6 387.1682, found
387.1674.
3,5-Dieth yl 2-Meth yl-4-for m yl-6-p h en ylp yr id in e-3,5-d i-
ca r boxyla te (31). 1H NMR: δ 1.06 (t, J ) 7.8 Hz, 3 H), 1.43
(t, J ) 6.9 Hz, 3 H), 2.94 (s, 3 H), 4.17 (q, J ) 7.8 Hz, 2 H),
4.42 (d, J ) 6.9 Hz, 2 H), 7.43-7.45 (m, 3 H), 7.55 (m, 2 H),
8.63 (s, 1 H). MS (CI/NH3): m/z 342 (M+ + 1).
3-Eth yl 5-Ben zyl 2-Meth yl-4-p h en yleth yn yl-6-cyclobu -
tylp yr id in e-3,5-d ica r boxyla te (34). 1H NMR: δ 1.37 (t, J
) 7.8 Hz, 3 H), 1.81-1.98 (m, 2 H), 2.11-2.19 (m, 2 H), 2.37-
2.47 (m, 2 H), 2.61 (s, 3 H), 3.70 (m, 1 H), 4.43 (q, J ) 7.8 Hz,
2 H), 5.39 (s, 2 H), 7.28-7.40 (m, 10 H). MS (EI): m/z 454
(M+ + 1).
Syn th esis of Ald eh yd e Gr ou p Con ta in in g Dih yd r op y-
r id in e (15) (Sch em e 2). Dihydropyridine 14 (14 mg) and a
catalytic amount of Dowex 50×8-200 resin were stirred in a
mixture of acetone (2 mL) and water (0.5 mL) at room
temperature for 48 h. The resin was filtered off, and the
filtrate was dried with anhydrous MgSO4. The solvent was
removed, and the residue was purified with preparative TLC
(silica 60; 1000 µm; Analtech, Newark, DE; petroleum ether-
ethyl acetate (3:1)) to give 10 mg of the desired product (15),
yield: 82%.
3,5-Diet h yl 2-Met h yl-6-p h en yl-4-for m yl-1,4-(()-d ih y-
d r op yr id in e-3,5-d ica r boxyla te (15). 1H NMR: δ 0.89 (t, J
) 6.9 Hz, 3 H), 1.32 (t, J ) 6.9 Hz, 3 H), 2.37 (s, 3 H), 3.94 (q,
J ) 6.9 Hz, 2 H), 4.24 (d, J ) 6.9 Hz, 2 H), 4.90 (s, 1 H), 5.81
(s, br, 1 H), 7.35 (m, 2 H), 7.41 (m, 3 H), 9.66 (s, 1 H). MS
(CI/NH3): m/z 361 (M+ + NH4), 344 (M+ + 1), 314 (M+ - CHO,
base). MS (EI): m/z 343 (M+), 314 (M+ - CHO, base), 298
(M+ - OEt). HRMS: calcd for C18H20NO4 (M+ - CHO)
314.1392, found 314.1432.
3-Eth yl 5-Ben zyl 2-Meth yl-4-p h en yleth yn yl-6-cyclo-
p en tylp yr id in e-3,5-d ica r boxyla te (35). 1H NMR: δ 1.37
(t, J ) 7.8 Hz, 3 H), 1.54-1.58 (m, 2 H), 1.78-1.88 (m, 6 H),
2.57 (s, 3 H), 3.04 (m, 1 H), 4.43 (q, J ) 7.8 Hz, 2 H), 5.41 (s,
Gen er a l P r oced u r e for Oxid a tion of 1,4-Dih yd r op y-
r id in es in t o Cor r esp on d in g P yr id in e Der iva t ives
(Sch em e 3). Equimolar amounts of the 1,4-dihydropyridines
(8-22, 53a -i, ∼0.2 mmol) and tetrachloro-1,4-benzoquinone
(52) in THF (2-4 mL) were mixed and refluxed overnight.
After the mixture was cooled to room temperature, the solvent
was removed, and the residue was purified by preparative TLC
(silica 60; 1000 µm; Analtech, Newark, DE; petroleum ether-
ethyl acetate (9:1-19:1)) to give the desired products.
3-P r op yl 5-Eth yl 2,4-Dim eth yl-6-p h en ylp yr id in e-3,5-
d ica r boxyla te (24). 1H NMR: δ 0.97-1.06 (m, 6 H), 1.81 (m,
2 H), 2.37 (s, 3 H), 2.61 (s, 3 H), 4.11 (t, J ) 6.9 Hz, 2 H), 4.35
(t, J ) 6.9 Hz, 2 H), 7.40-7.43 (m, 3 H), 7.56-7.57 (m, 2 H).
2 H), 7.29-7.44 (m, 10 H). MS (EI): m/z 467 (M+), 376 (M+
-
CH2Ph), 91 (+CH2Ph, base).
3,5-Dieth yl 2-Eth yl-4-m eth yl-6-p h en ylp yr id in e-3,5-d i-
ca r boxyla te (36). 1H NMR: δ 1.00 (t, J ) 6.9 Hz, 3 H), 1.33
(t, J ) 7.8 Hz, 3 H), 1.42 (t, J ) 6.9 Hz, 3 H), 2.36 (s, 3 H),
2.86 (q, J ) 7.8 Hz, 2 H), 4.12 (q, J ) 6.9 Hz, 2 H), 4.45 (q, J
) 6.9 Hz, 2 H), 7.40-7.43 (m, 3 H), 7.58-7.60 (m, 2 H). MS
(EI): m/z 341 (M+), 312 (M+ - CH2CH3, base), 296 (M+ - OEt),
284 (MH+ - 2Et), 269 (MH+ - CO2Et). HRMS: calcd for
C
20H23NO4 341.1627, found 341.1631.
2-Meth yl-4-eth yl-5-eth oxyca r bon yl-6-p h en ylp yr id in e-
MS (EI): m/z 341 (M+), 312 (M+ - CH2CH3, base), 296 (M+
-
3-ca r boxylic Acid (37). 1H NMR: δ 0.97 (t, J ) 7.8 Hz, 3
H), 1.24 (t, J ) 7.8 Hz, 3 H), 2.61 (s, 3 H), 2.71 (q, J ) 7.8 Hz,
2 H), 4.46 (J ) 7.8 Hz, 2 H), 7.40-7.45 (m, 3 H), 7.55-7.59
(m, 2 H). MS (CI/NH3): m/z 314 (M+ + 1). MS (EI): m/z 312
(M+ - 1), 296 (M+ - OH), 284 (M+ - Et, base).
OCH2CH3), 282 (M+ - OPr). HRMS: calcd for C20H23NO4
341.1627, found 341.1635.
3,5-Dieth yl 2-Meth yl-4-eth yl-6-p h en ylp yr id in e-3,5-d i-
ca r boxyla te (25). 1H NMR: δ 0.97 (t, J ) 6.9 Hz, 3 H), 1.24
(t, J ) 7.8 Hz, 2 H), 1.43 (t, J ) 6.9 Hz, 3 H), 2.61 (s, 3 H),
2.71 (q, J ) 7.8 Hz, 2 H), 4.09 (q, J ) 6.9 Hz, 2 H), 4.46 (q, J
) 6.9 Hz, 2 H), 7.40-7.43 (m, 3 H), 7.55-7.58 (m, 2 H). MS
5-Eth yl 2,4-Dieth yl-3-(eth ylsu lfa n ylca r bon yl)-6-p h en -
ylp yr id in e-5-ca r boxyla te (38). 1H NMR: δ 0.98 (t, J ) 7.8
Hz, 3 H), 1.23 (t, J ) 7.8 Hz, 3 H), 1.34 (t, J ) 6.9 Hz, 3 H),
1.41 (t, J ) 7.8 Hz, 3 H), 2.73 (q, J ) 7.8 Hz, 2 H), 2.87 (q, J
) 7.8 Hz, 2 H), 3.14 (q, J ) 7.8 Hz, 2 H), 4.10 (q, J ) 6.9 Hz,
2 H), 7.41-7.44 (m, 3 H), 7.58-7.61 (m, 2 H). MS (CI/NH3):
m/z 372 (M+ + 1, base).
5-P r op yl 2,4-Dieth yl-3-(eth ylsu lfa n ylca r bon yl)-6-p h en -
ylp yr id in e-5-ca r boxyla te (39a ). 1H NMR: δ 0.65 (t, J ) 7.8
Hz, 3 H), 1.23 (t, J ) 7.8 Hz, 3 H), 1.34 (t, J ) 7.8 Hz, 3 H),
1.41 (t, J ) 7.8 Hz, 3 H), 1.34-1.44 (m, 2 H), 2.73 (q, J ) 7.8
Hz, 2 H), 2.87 (q, J ) 7.8 Hz, 2 H), 3.14 (q, J ) 7.8 Hz, 2 H),
3.99 (t, J ) 6.9 Hz, 2 H), 7.40-7.44 (m, 3 H), 7.59-7.62 (m, 2
H). MS (CI/NH3): m/z 404 (MH+ + NH4), 386 (M+ + 1, base).
(EI): m/z 341 (M+), 312 (M+ - CH2CH3, base), 296 (M+
-
-
OCH2CH3), 284 (MH+ - 2Et), 268 (M+ - CO2Et), 240 (MH+
Et - CO2Et). HRMS: calcd for C20H23NO4 341.1627, found
341.1615.
5-Eth yl 2-Meth yl-4-eth yl-3-(eth ylsu lfa n ylca r bon yl)-6-
p h en ylp yr id in e-5-ca r boxyla te (26). 1H NMR: δ 0.97 (t, J
) 6.9 Hz, 3 H), 1.23 (t, J ) 7.8 Hz, 3 H), 1.41 (t, J ) 7.8 Hz,
3 H), 2.61 (s, 3 H), 2.74 (q, J ) 7.8 Hz, 2 H), 3.14 (q, J ) 7.8
Hz, 2 H), 4.09 (q, J ) 6.9 Hz, 2 H), 7.40-7.44 (m, 3 H), 7.56-
7.59 (m, 2 H). MS (CI/NH3): m/z 375 (M+ + NH4), 358 (M+
1, base). MS (EI): m/z 357 (M+), 312 (M+ - OEt), 296 (M+
SEt, base), 268 (M+ - COSEt).
+
-
5-P r op yl 2-E t h yl-4-p r op yl-3-(et h ylsu lfa n ylca r b on yl)-
6-p h en ylp yr id in e-5-ca r boxyla te (39b). 1H NMR: δ 0.66 (t,
J ) 7.8 Hz, 3 H), 0.95 (t, J ) 7.8 Hz, 3 H), 1.34 (t, J ) 7.8 Hz,
3 H), 1.41 (t, J ) 7.8 Hz, 3 H), 1.40 (m, 2 H), 1.63 (m, 2 H),
2.66 (t, J ) 7.8 Hz, 2 H), 2.86 (q, J ) 7.8 Hz, 2 H), 3.13 (q, J
) 7.8 Hz, 2 H), 3.98 (t, J ) 6.9 Hz, 2 H), 7.39-7.44 (m, 3 H),
7.58-7.62 (m, 2 H). MS (CI/NH3): m/z 400 (M+ + 1, base).
5-Eth yl 2-Meth yl-4-eth yl-3-[2-m eth oxyl-(eth ylsu lfa n yl-
car bon yl)]-6-ph en ylpyr idin e-5-car boxylate (27). 1H NMR:
δ 0.97 (t, J ) 7.8 Hz, 3 H), 1.23 (t, J ) 7.8 Hz, 3 H), 2.62 (s, 3
H), 2.74 (q, J ) 7.8 Hz, 2 H), 3.36 (t, J ) 6.0 Hz, 2 H), 3.42 (s,
3 H), 3.67 (t, J ) 6.0 Hz, 2 H), 4.09 (q, J ) 7.8 Hz, 2 H), 7.39-
7.42 (m, 3 H), 7.55-7.58 (m, 2 H). MS (CI/NH3): m/z 388 (M+
+ 1), 296 (M+ - CH3OCH2CH2S).
5-Hydr oxyleth yl 2,4-Dieth yl-3-(eth ylsu lfan ylcar bon yl)-
6-p h en ylp yr id in e-5-ca r boxyla te (40). 1H NMR: δ 1.24 (t,
J ) 7.8 Hz, 3 H), 1.34 (t, J ) 7.8 Hz, 3 H), 1.42 (t, J ) 7.8 Hz,
3 H), 2.75 (q, J ) 7.8 Hz, 2 H), 2.87 (q, J ) 7.8 Hz, 2 H), 3.15
(q, J ) 7.8 Hz, 2 H), 3.48 (m, 2 H), 4.13 (t, J ) 4.8 Hz, 2 H),
7.45-7.49 (m, 3 H), 7.60-7.63 (m, 2 H). MS (CI/NH3): m/z
404 (M+ + NH4 - 1), 388 (M+ + 1).
5-E t h yl 2,4-Diet h yl-3-(et h ylsu lfa n ylca r b on yl)-6-(m -
ch lor op h en yl)p yr id in e-5-ca r boxyla te (41). 1H NMR: δ
1.07 (t, J ) 7.8 Hz, 3 H), 1.23 (t, J ) 7.8 Hz, 3 H), 1.34 (t, J )
7.8 Hz, 3 H), 1.41 (t, J ) 7.8 Hz, 3 H), 2.72 (q, J ) 7.8 Hz, 2
H), 2.86 (q, J ) 7.8 Hz, 2 H), 3.14 (q, J ) 7.8 Hz, 2 H), 4.16 (q,
5-Eth yl 2-Meth yl-4-p r op yl-3-(eth ylsu lfa n ylca r bon yl)-
6-p h en ylp yr id in e-5-ca r boxyla te (28). 1H NMR: δ 0.95 (t,
J ) 6.9 Hz, 3 H), 0.97 (t, J ) 6.9 Hz, 3 H), 1.41 (t, J ) 7.8 Hz,
3 H), 1.63 (m, 2 H), 2.61 (s, 3 H), 2.68 (t, J ) 7.8 Hz, 2 H), 3.14
(q, J ) 6.9 Hz, 2 H), 4.08 (q, J ) 6.9 Hz, 2 H), 7.41 (m, 3 H),
7.56 (m, 2 H). MS (CI/NH3): m/z 372 (M+ + 1). MS (EI): m/z
326 (M+ - OCH2CH3), 310 (M+ - SEt, base), 282 (M+
-
COSEt).
5-Ben zyl 2-Meth yl-4-eth yl-3-(eth ylsu lfa n ylca r bon yl)-
6-p h en ylp yr id in e-5-ca r boxyla te (29). 1H NMR: δ 1.18 (t,
J ) 7.8 Hz, 3 H), 1.40 (t, J ) 7.8 Hz, 3 H), 2.60 (s, 3 H), 2.70
(q, J ) 7.8 Hz, 2 H), 3.12 (q, J ) 7.8 Hz, 2 H), 5.04 (s, 2 H),

3198 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Li et al.
J ) 7.8 Hz, 2 H), 7.35-7.41 (m, 1 H), 7.46-7.50 (m, 1 H), 7.62
(s, 1 H). MS (CI/NH3): m/z 406 (M+ + 1).
7.47 (m, 3 H), 7.54-7.57 (m, 2 H). MS (CI/NH3): m/z 208 (M+
+ 1, base), 192 (MH+ - NH2).
5-Eth yl 2,4-Dieth yl-3-(eth ylsu lfa n ylca r bon yl)-6-cyclo-
p en tylp yr id in e-5-ca r boxyla te (42). 1H NMR: δ 1.18 (t, J
) 7.8 Hz, 3 H), 1.27 (t, J ) 7.8 Hz, 3 H), 1.38 (t, J ) 7.8 Hz,
3 H), 1.39 (t, J ) 7.8 Hz, 3 H), 1.63 (m, 2 H), 1.92 (m, 7 H),
2.58 (q, J ) 7.8 Hz, 2 H), 2.76 (q, J ) 7.8 Hz, 2 H), 3.91 (q, J
) 7.8 Hz, 2 H), 4.40 (q, J ) 7.8 Hz, 2 H). HRMS: calcd for
Eth yl 3-Am in o-3-(m -ch lor op h en yl)-2-p r op en oa te (49f).
1H NMR: δ 1.30 (t, J ) 6.9 Hz, 3 H), 4.18 (q, J ) 6.9 Hz, 2 H),
4.95 (s, 1 H), 7.35-7.44 (m, 3 H), 7.54 (s, 1 H). MS (CI/NH3):
m/z 226 (C11H1235ClNO2, M+ + 1, base), 227 (M+, C11H12
37ClNO2).
-
Eth yl 3-Am in o-3-cyclop en tyl-2-p r op en oa te (49g). 1H
NMR: δ 1.27 (t, J ) 6.9 Hz, 3 H), 1.54-1.81 (m, 6 H), 1.89-
1.94 (m, 2 H), 2.50 (m, 1 H), 4.11 (q, J ) 6.9 Hz, 2 H), 4.60 (s,
1 H). MS (CI/NH3): m/z 184 (M+ + 1, base).
C
20H29NO3S 363.1868, found 363.1858.
5-Eth yl 2,4-Dieth yl-3-p r op ylsu lfa n ylca r bon yl-6-p h en -
ylp yr id in e-5-ca r boxyla te (43). 1H NMR: δ 0.98 (t, J ) 7.8
Hz, 3 H), 1.07 (t, J ) 7.8 Hz, 3 H), 1.23 (t, J ) 7.8 Hz, 3 H),
1.34 (t, J ) 7.8 Hz, 3 H), 1.76 (m, 2 H), 2.73 (q, J ) 7.8 Hz, 2
H), 2.87 (q, J ) 7.8 Hz, 2 H), 3.12 (q, J ) 7.8 Hz, 2 H), 4.10 (q,
J ) 7.8 Hz, 2 H), 7.42-7.43 (m, 3 H), 7.58-7.61 (m, 2 H). MS
(CI/NH3): m/z 386 (M+ + 1, base).
P r opyl 3-Am in o-3-(m-ch lor oph en yl)-2-pr open oate (49h ).
1H NMR: δ 0.98 (t, J ) 6.9 Hz, 3 H), 1.69 (m, 2H), 4.09 (q, J
) 6.9 Hz, 2 H), 4.96 (s, 1 H), 7.32-7.45 (m, 3 H), 7.54 (s, 1 H).
MS(CI/NH3): m/z 240 (C12H1435ClNO2, M+ + 1, base). MS
(EI): m/z 239 (M+), 223 (M+ - NH2), 180 (M+ - PrO), 153
(M+ - 1 - CO2Pr, base).
5-P r op yl 2,4-Dieth yl-3-p r op ylsu lfa n ylca r bon yl-6-(m -
ch lor op h en yl)p yr id in e-5-ca r boxyla te (44). 1H NMR: δ
0.72 (t, J ) 7.8 Hz, 3 H), 1.07 (t, J ) 7.8 Hz, 3 H), 1.23 (t, J )
7.8 Hz, 3 H), 1.34 (t, J ) 7.8 Hz, 3 H), 1.46 (m, 2 H), 1.77 (m,
2 H), 2.72 (q, J ) 7.8 Hz, 2 H), 2.86 (q, J ) 7.8 Hz, 2 H), 3.13
(t, J ) 6.9 Hz, 2 H), 4.04 (t, J ) 6.9 Hz, 2 H), 7.37 (m, 2 H),
P r ep a r a t ion of 2,2-Dim et h oxyla cet a ld eh yd e (50d ,
Sch em e 5).41 Literature procedure was followed with some
modifications. Potassium permanganate (16 g, 100 mmol) in
300 mL of water was added slowly to a vigorously stirred ice-
cooled suspension of 10.2 g (100 mmol) of acrolein dimethyl
acetal in 120 mL of water. The speed of addition was
controlled to keep the temperature as near to 5 °C as possible.
Soon after the stirring stopped, the mixture formed a gel. After
2 h of standing, the mixture was heated at 95 °C for 1 h and
then filtered. Upon cooling, the filtrate was treated with 240
g of anhydrous K2CO3. The organic phase was separated, and
the aqueous phase was extracted with ethyl acetate (80 mL ×
5). Organic phases were combined and dried with anhydrous
MgSO4. After the solvent was removed, a colorless oil (6.84
g, yield: 50%) remained and was used directly for the next
reaction, identified as d l-glycer a ld eh yd e d im eth yl a ceta l
(54). 1H NMR: δ 2.44 (s, br, 1 H), 2.73 (s, br, 1 H), 3.48 (s, 6
H), 3.69-3.73 (m, 3 H), 4.36 (d, J ) 6.0 Hz, 1 H). MS (CI/
NH3): m/z 154 (M+ + NH4, base).
Compound 54 (2.11 g, 15.5 mmol) was dissolved in a mixture
of dichloromethane (100 mL) and water (5 mL) and cooled to
0 °C. While stirring, sodium periodate (7.5 g, 35 mmol) was
carefully added in three portions within 30 min. After an
additional 1 h of stirring at room temperature, anhydrous
MgSO4 (14 g) was added to the reaction mixture, and stirring
was continued for an additional 0.5 h. The reaction was then
filtered. Removal of the solvent left 1.38 g of the desired
product, 50d , yield: 85%. 1H NMR: δ 3.46 (s, 6 H), 4.50 (d, J
) 1.8 Hz, 1 H), 9.48 (d, J ) 1.8 Hz, 1 H).
7.48 (m, 1 H), 7.62 (s, 1 H). MS (CI/NH3): m/z 434 (M+(C23H28
35ClNO3S) + 1, base), 404 (M+ - C2H5), 358 (M+ - PrS).
-
5-Eth yl 2-P r op yl-4-eth yl-3-(eth ylsu lfa n ylca r bon yl)-6-
p h en ylp yr id in e-5-ca r boxyla te (45). 1H NMR: δ 0.99 (t, J
) 6.9 Hz, 6 H), 1.23 (t, J ) 7.8 Hz, 3 H), 1.41 (t, J ) 7.8 Hz,
3 H), 1.82 (m, 2 H), 2.72 (q, J ) 6.9 Hz, 2 H), 2.81 (q, J ) 6.9
Hz, 2 H), 3.14 (q, J ) 7.8 Hz, 2 H), 4.10 (q, J ) 7.8 Hz, 2 H),
7.40-7.44 (m, 3 H), 7.57-7.60 (m, 2 H). MS (EI): m/z 385
(M+), 340 (M+ - OEt), 324 (M+ - SEt), 296 (M+ - COSEt).
5-Eth yl 2-(2-Meth oxyleth yl)-4-eth yl-3-(eth ylsu lfa n yl-
ca r bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (46). 1H NMR:
δ 0.99 (t, J ) 7.8 Hz, 3 H), 1.23 (t, J ) 7.8 Hz, 3 H), 1.41 (t, J
) 7.8 Hz, 3 H), 2.73 (q, J ) 7.8 Hz, 2 H), 3.11-3.18 (m, 4 H),
3.37 (s, 3 H), 3.85 (t, J ) 7.8 Hz, 2 H), 4.10 (q, J ) 7.8 Hz, 2
H), 7.42-7.44 (m, 3 H), 7.58-7.61 (m, 2 H). MS (CI/NH3): m/z
402 (MH+, base). HRMS: calcd for C22H27NO4S 401.1661,
found401.1666.
5-E t h yl 2-Bu t yl-4-et h yl-3-(et h ylsu lfa n ylca r b on yl)-6-
p h en ylp yr id in e-5-ca r boxyla te (47). 1H NMR: δ 0.93 (t, J
) 7.8 Hz, 3 H), 0.99 (t, J ) 7.8 Hz, 3 H), 1.23 (t, J ) 7.8 Hz,
3 H), 1.28-1.39 (m, 2 H), 1.41 (t, J ) 7.8 Hz, 3 H), 1.77 (m, 2
H), 2.72 (q, J ) 7.8 Hz, 2 H), 2.83 (t, J ) 7.8 Hz, 2 H), 3.13 (q,
J ) 7.8 Hz, 2 H), 4.10 (q, J ) 7.8 Hz, 2 H), 7.40-7.43 (m, 3
H), 7.58-7.60 (m, 2 H). MS(CI/NH3): m/z 400 (M+ + 1, base).
MS (EI): m/z 400 (M+ + 1), 371 (MH+ - Et), 338 (M+ - SEt,
base). HRMS: calcd for C23H29NO3S 399.1868, found 399.1867.
5-Eth yl 2-Cyclobu tyl-4-eth yl-3-(eth ylsu lfan ylcar bon yl)-
6-p h en ylp yr id in e-5-ca r boxyla te (48). 1H NMR: δ 1.00 (t,
J ) 7.8 Hz, 3 H), 1.21 (t, J ) 7.8 Hz, 3 H), 1.42 (t, J ) 7.8 Hz,
3 H), 1.86-1.95 (m, 1 H), 1.95-2.05 (m, 1 H), 2.17-2.56 (m, 2
H), 2.51-2.64 (m, 2 H), 2.70 (q, J ) 7.8 Hz, 2 H), 3.13 (q, J )
7.8 Hz, 2 H), 3.79 (m, 1 H), 4.11 (q, J ) 7.8 Hz, 2 H), 7.42-
7.44 (m, 3 H), 7.67-7.69 (m, 2 H). MS (CI/NH3): m/z 398 (M+
+ 1, base).
Syn t h esis of â-Ket oest er s 51c,d ,g,j,k ,u (Sch em e 6).
â-Ketoester 51c and â-ketothioesters 51d and 51g were
prepared by the reaction of 2,2,6-trimethyl-4H-1,3-dioxin-4-
one (55) and an alcohol or a thiol (eq 1). Equimolar amounts
(for example, 3 mmol) of compound 55 and an alcohol or a thiol
were heated with a little toluene (1-2 mL) at 100 °C in a
sealed tube overnight. After the mixture was cooled to room
temperature, the solvent was removed under reduced pressure
and the residue was chromatographed to give the desired
products in satisfactory yields (64% for 51c, 97% for 51d , and
67% for 51g).
Gen er a l P r oced u r e for P r ep a r a tion of â-Am in o-r,â-
u n sa tu r a ted Ester s (Sch em e 4). A â-ketoester (3 mmol)
and ammonium acetate (4.5 mmol) were mixed in 5 mL of
absolute ethanol and refluxed at 80 °C for 24 h. The solvent
was removed, and the residue was chromatographed to give
the desired compounds in moderate yields.
P r op yl Acetoa ceta te (51c). 1H NMR: δ 0.94 (t, J ) 6.9
Hz, 3 H), 1.66 (m, 2 H), 2.27 (s, 3 H), 3.46 (s, 2 H), 4.09 (t, J
) 6.9 Hz, 2 H).
S-Eth yl 3-Oxoth iobu tyr a te (51d ). 1H NMR: δ 1.28 (t, J
) 7.8 Hz, 3 H), 2.27 (s, 3 H), 2.94 (q, J ) 7.8 Hz, 2 H), 3.67 (s,
2 H).
Eth yl 3-Am in o-3-p h en yl-2-p r op en oa te (49a ). 1H NMR:
δ 1.30 (t, J ) 6.9 Hz, 3 H), 4.18 (q, J ) 6.9 Hz, 2 H), 4.97 (s,
1 H), 7.41-7.53 (m, 3 H), 7.54-7.57 (m, 2 H).
S-(2-Meth oxyleth yl) 3-Oxoth iobu tyr a te (51g). 1H NMR:
δ 2.27 (s, 3 H), 3.15 (t, J ) 6.0 Hz, 2 H), 3.37 (s, 3 H), 3.55 (t,
J ) 6.0 Hz, 2 H), 3.69 (s, 2 H). MS (CI/NH3): 194 (M+ + NH4,
base), 176 (M+).
Ben zyl 3-Am in o-3-ph en yl-2-pr open oate (49b). 1H NMR:
1
3
δ 4.97 (s, /4 H), 5.05 (s, /4 H), 5.18 (s, 2 H), 7.29-7.56 (m, 10
H). MS (CI/NH3): m/z 272 (M+ + NH4), 254 (M+ + 1, base).
P r opyl 3-Am in o-3-ph en yl-2-pr open oate (49d). 1H NMR:
δ 0.98 (t, J ) 7.8 Hz, 3 H), 1.70 (m, 2 H), 4.09 (t, J ) 7.8 Hz,
2 H), 4.99 (s, 1 H), 7.39-7.44 (m, 3 H), 7.54-7.57 (m, 2 H).
MS (CI/NH3): m/z 206 (M+ + 1, base).
â-Ketoesters 51j and 51k were prepared by a route shown
in eq 2. N-Isopropylcyclohexylamine (0.786 g, 5.5 mmol) and
n-BuLi (2.2 mL, 5.5 mmol, 2.5 N in hexanes) were mixed at 0
°C in 15 mL of THF for 15 min. The temperature was then
lowered to -78 °C. Benzyl acetate (0.752 g, 0.72 mL, 5 mmol)
was then added slowly into this system, and the mixture was
stirred for 10 min at the same temperature to form an enolate.
Hyd r oxyleth yl 3-Am in o-3-p h en yl-2-p r op en oa te (49e).
1H NMR: δ 3.87 (m, 2 H), 4.28 (m, 2 H), 5.02 (s, 1 H), 7.43-

Selective A3 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3199
Cyclohexanecarbonyl chloride (0.806 g, 0.74 mL, 5.5 mL, for
51k ) or cyclopentanecarbonyl chloride (0.729 g, 0.67 mL, 5.5
mmol, for 51j) was added dropwise to this enolate solution
within 10 min. After 15 min of stirring, the reaction mixture
was allowed to warm to room temperature and poured into
10 mL of 1 N HCl. The organic phase was separated, and the
aqueous phase was extracted with ether (10 mL × 3). The
combined organic phases were washed with 1 N NaHCO3 (10
mL) and water (10 mL) and then dried with anhydrous MgSO4.
The solvent was removed, and the residue was chromato-
graphed (silica 60, petroleum ether-ethyl acetate (9:1)) to give
130 mg of 51k (yield: 10%) or 569 mg of 51j (yield: 46%).
Ben zyl 3-Oxo-3-cyclop en tylp r op ion a te (51j). 1H NMR:
δ 1.19-1.81 (m, 8 H), 2.76-2.85 (m, 1 H), 3.55 (s, 2 H), 5.11
(s, 2 H), 7.31-7.36 (m, 5 H).
S-Eth yl 3-Oxo-3-cyclobu tylth iop r op ion a te (51n ). 1H
NMR: δ 1.27 (t, J ) 7.8 Hz, 3 H), 1.85 (m, 1 H), 1.93-2.05 (m,
1 H), 2.14-2.31 (m, 4 H), 2.92 (q, J ) 7.8 Hz, 2 H), 3.42 (m, 1
H), 3.61 (s, 2 H). MS (CI/NH4): m/z 204 (M+ + NH4, base),
187 (M+ + 1).
S-Eth yl 3-Oxo-5-m eth oxyth iova ler a te (51p ). 1H NMR:
δ 1.28 (t, J ) 7.8 Hz, 3 H), 2.80 (t, J ) 6.0 Hz, 2 H), 2.93 (q,
J ) 7.8 Hz, 2 H), 3.34 (s, 3 H), 3.65 (t, J ) 6.0 Hz, 2 H), 3.71
(s, 2 H). MS (CI/NH4): m/z 208 (M+ + NH4, base), 191 (M+
1).
+
Eth yl 3-Oxo-3-cyclop en tylp r op ion a te (51q). 1H NMR:
δ 1.28 (t, J ) 7.8 Hz, 3 H), 1.59-1.71 (m, 2 H), 1.76-1.88 (m,
2 H), 2.98 (m, 1 H), 3.49 (s, 2 H), 4.19 (q, J ) 7.8 Hz, 2 H). MS
(CI/NH4): m/z 202 (M+ + NH4, base).
P r op yl Ben zoyla ceta te (51r ). 1H NMR: δ 0.95 (t, J ) 6.9
Hz, 3 H), 1.64-1.71 (m, 2 H), 3.39 (s, 2 H), 4.12 (t, J ) 6.9 Hz,
2 H), 7.47-7.97 (m, 5 H). MS (CI/NH4): m/z 224 (M+ + NH4,
base), 206 (M+).
Ben zyl 3-Oxo-3-cycloh exylp r op ion a te (51k ). 1H NMR:
δ 1.20-1.51 (m, 5 H), 1.66-1.96 (m, 5 H), 2.25-2.38 (m, 1 H),
3.51 (s, 2 H), 5.19 (s, 2 H), 7.37 (m, 5 H).
Eth yl m -Ch lor oben zoyla ceta te (51s). 1H NMR: δ 1.26
(t, J ) 6.9 Hz, 3 H), 3.91 (s, 2 H), 4.22 (t, J ) 6.9 Hz, 2 H),
7.36-7.84 (m, 3 H), 7.93 (s, 1 H). MS (CI/NH4): m/z 244
(C11H1135ClO3, M+ + NH4, base), 227 (C11H1135ClO3, M+ + 1).
P r op yl m -Ch lor oben zoyla ceta te (51t). 1H NMR: δ 0.90
(t, J ) 7.8 Hz, 3 H), 1.64 (m, 2 H), 3.98 (s, 2 H), 4.12 (t, J )
6.9 Hz, 2 H), 7.36-7.84 (m, 3 H), 7.93 (s, 1 H). MS (CI/NH4):
m/z 258 (C11H1135ClO3, M+ + NH4, base), 241 (C11H1135ClO3,
M+ + 1).
Com p u ta tion a l Meth od ologies. Compound 38, (R)-MRS
1191, and (S)-MRS 1191 models were constructed using the
“Sketch Molecule” of SYBYL.26 These structures were fully
minimized using MOPAC software27 (RHF method and AM1
Hamiltonian,28 keywords: PREC, GNORM)0.1, EF).
Conformational analysis of 38, (R)-MRS 1191, and (S)-MRS
1191 derivatives was performed on all rotatable bonds of the
pyridine or 1,4-dihydropyridine substituents using the random
search procedure of SYBYL. The optimized geometries of the
resulting conformers were calculated using MOPAC software
(RHF method and AM1 Hamiltonian, keywords: PREC,
GNORM)0.1, EF).
To prepare compound 51u , a transesterification reaction was
used (eq 3). Ethyl benzoyl acetate (1.92 g, 10 mmol) and
ethylene glycol (0.621 g, 10 mmol) in toluene (10 mL) were
heated with stirring for 24 h. The solvent was removed, and
the residue was chromatographed (silica 60, petroleum ether-
ethyl acetate (3:1)) to give 0.946 g of the desired product,
yield: 45%.
Hyd r oxyleth yl Ben zoyla ceta te (51u ). 1H NMR: δ 2.52
(s, br, 1 H), 3.4 (m, 2 H), 4.08 (s, 2 H), 4.35 (t, J ) 7.8 Hz, 2
H), 7.43-7.53 (m, 2 H), 7.60-7.65 (m, 1 H), 7.93-7.96 (m, 2
H).
Syn th esis of â-Ketoester s 51e,f,h ,i,l-n ,p -t via Mel-
d r u m ’s Acid s (Sch em e 7).42 The preparation of S-ethyl
3-oxothiovalerate (51e) is provided as an example. 2,2-
Dimethyl-1,3-dioxane-4,6-dione (57, 0.721 g, 5 mmol) and
propionyl chloride (0.509 g, 5.5 mmol) were dissolved in 10
mL of dry CH2Cl2. At 0 °C, 0.81 mL (0.791 g, 10 mmol) of
pyridine (in the cases of aromatic acid chlorides, using 4-(di-
methylamino)pyridine instead of pyridine) was then added
dropwise. The reaction temperature was kept at 0 °C for 1 h
and then raised to room temperature for an additional h. The
reaction mixture was washed with 1 N HCl (10 mL) and water
(5 mL) and then dried with anhydrous MgSO4. Removal of
the solvent left the desired product (58e), which was directly
used for the next reaction without purification.
Compound 58e (670 mg, 3.35 mmol) and ethanethiol (0.621
g, 10 mmol) were mixed in 10 mL of toluene. This mixture
was heated at 80 °C in a flask with an effective flux condenser
for 24 h. The solvent and excess ethanethiol were removed,
and the residue was chromatographed (silica 60, petroleum
ether-ethyl acetate (9:1)) to give the desired product, 282 mg,
yield: 53%.
Partial atomic charges for the calculation of the electrostatic
potential maps were obtained using RHF/3-21G(*)//RHF/AM1
ab initio level29 of Gaussian 94.30 Atomic charges were
calculated by fitting to electrostatic potential maps (CHELPG
method).31 Electrostatic contours were generated using stan-
dard procedures within SYBYL.
All calculations were performed on a Silicon Graphics Indigo
2 R8000 workstation.
Steric and electrostatic alignment (SEAL) method25 was
used to optimize the superimposition between pyridine and
1,4-dihydropyridine derivatives using PowerFit v.1.0 pro-
gram.32 Starting with 100 random orientations, SEAL utilized
the steric volume and the atomic partial charges of two
molecular structures in a determination of their optimal
alignment. SEAL setup parameters for pyridine and 1,4-
S-Eth yl 3-Oxoth iova ler a te (51e). 1H NMR: δ 1.07 (t, J
) 6.9 Hz, 3 H), 1.28 (t, J ) 6.9 Hz, 3 H), 2.58 (q, J ) 6.9 Hz,
2 H), 2.94 (q, J ) 6.9 Hz, 2 H), 3.66 (s, 2 H). MS (CI/NH4):
m/z 178 (M+ + NH4), 161 (M+ + 1).
S-Eth yl 3-Oxoth ioca p r oa te (51f). 1H NMR: δ 0.92 (t, J
) 7.8 Hz, 3 H), 1.28 (t, J ) 7.8 Hz, 3 H), 1.62 (m, 2 H), 2.53 (t,
J ) 6.9 Hz, 2 H), 2.93 (q, J ) 7.8 Hz, 2 H), 3.65 (s, 2 H). MS
(CI/NH4): m/z 192 (M+ + NH4, base), 175 (M+ + 1).
Ben zyl 3-Oxo-3-cyclop r op ylp r op ion a te (51h ). 1H NMR:
δ 0.90-0.96 (m, 2 H), 1.08-1.13 (m, 2 H), 1.98-2.05 (m, 1 H),
3.62 (s, 2 H), 5.20 (s, 2 H), 7.30-7.39 (m, 5 H). MS (CI/NH4):
m/z 236 (M+ + NH4, base), 219 (M+ + 1).
dihydropyridine alignments included R ) 0.5, WS ) 1, WE
)
1, and specification for the Gaussian attenuation function.
log P values (the log of the octanol-water partition coef-
ficient), a hydrophobicity indicator, were empirically calculated
using the atom fragment method developed by Ghose, Pritch-
ett, and Crippen33 and implemented in ChemPlus v.1.0.34
ChemPlus is an extension of Hyperchem for Windows. Both
SEAL alignments and log P calculations were performed on a
PC Pentium 166 MHz.
P h a r m a cology. Ra d ioliga n d Bin d in g Stu d ies. Binding
of [3H]R-N6-phenylisopropyladenosine ([3H]R-PIA) to A1 recep-
tors from rat cerebral cortex membranes and of [3H]-2-[4-[(2-
carboxyethyl)phenyl]ethylamino]-5′-N-ethylcarbamoyladeno-
sine ([3H]CGS 21680) to A2A receptors from rat striatal
membranes was performed as described previously.35,36 Ad-
enosine 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-methylcar-
bamoyladenosine ([125I]AB-MECA) to membranes prepared
Ben zyl 3-Oxo-3-cyclobu tylp r op ion a te (51i). 1H NMR:
δ 1.59-2.37 (m, 6 H), 3.37 (m, 1 H), 3.45 (s, 2 H), 5.17 (s, 2 H),
7.34-7.37 (m, 5 H). MS (CI/NH4): m/z 250 (M+ + NH4, base),
233 (M+ + 1).
S-Eth yl 3-Oxoth ioh ep ta n oa te (51l). 1H NMR: δ 0.91 (t,
J ) 7.8 Hz, 3 H), 1.28 (t, J ) 7.8 Hz, 3 H), 1.51-1.62 (m, 4 H),
2.55 (t, J ) 7.8 Hz, 2 H), 2.93 (q, J ) 7.8 Hz, 2 H), 3.65 (s, 2
H). MS (CI/NH4): m/z 206 (M+ + NH4, base).
S-P r op yl 3-Oxoth iova ler a te (51m ). 1H NMR: δ 0.98 (t,
J ) 6.9 Hz, 3 H), 1.07 (t, J ) 7.8 Hz, 3 H), 1.62 (m, 2 H), 2.58
(q, J ) 6.9 Hz, 2 H), 2.91 (t, J ) 7.8 Hz, 2 H), 3.67 (s, 2 H). MS
(CI/NH4): m/z 175 (M+ + 1).

3200 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Li et al.
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from HEK-293 cells stably expressing the human A3 receptor,
clone HS-21a (Receptor Biology, Inc., Baltimore MD) or to
membranes prepared from CHO cells stably expressing the
rat A3 receptor was performed as described.9,37 The assay
medium consisted of a buffer containing 10 mM Mg2+, 50 mM
Tris, and 1 mM EDTA, at pH 8.0. The glass incubation tubes
contained 100 µL of the membrane suspension (0.3 mg 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 100 µM N6-phenylisopropyladenosine (R-
PIA).
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 What-
man 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 pro-
gram (Graph-PAD, San Diego, CA), were converted to apparent
Ki values using the Cheng-Prusoff equation38 and Kd values
of 1.0 nM ([3H]R-PIA); 14 nM ([3H]CGS 21680); 0.59 nM and
1.46 nM ([125I]AB-MECA at human and rat A3 receptors,
respectively).
Abbr evia tion s: [125I]AB-MECA, [125I]N6-(4-amino-3-iodo-
benzyl)-5′-N-methylcarbamoyladenosine; CGS 21680, 2-[4-[(2-
carboxyethyl)phenyl]ethyl-amino]-5′-N-ethylcarbamoyladeno-
sine; CHO cells, Chinese hamster ovary cells; DMAPN,
N-(dimethylamino)pyridine; DMSO, dimethyl sulfoxide; DPPA,
diphenylphosphoryl azide; EDAC, 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide; HEK cells, human embryonic kidney
cells; IB-MECA, N6-(3-iodobenzyl)-5′-N-methylcarbamoylad-
enosine; Ki, equilibrium inhibition constant; log P, log of the
octanol-water partition coefficient; MRS 1191, 3-ethyl 5-ben-
zyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(()-dihydropyridine-
3,5-dicarboxylate; MRS 1476, 5-ethyl 2,4-diethyl-3-(ethylsul-
fanylcarbonyl)-6-phenylpyridine-5-carboxylate; R-PIA, R-N6-
phenylisopropyladenosine; SAR, structure-activity relationship;
SEAL, steric and electrostatic alignment; TBAF, tetrabuty-
lammonium fluoride; Tris, tris(hydroxymethyl)aminomethane.
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Ack n ow led gm en t. We thank Gilead Sciences (Fos-
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Prof. Gary L. Stiles and Dr. Mark E. Olah (Duke
University, Durham, NC) for providing samples of
[
125I]I-AB-MECA and cells expressing recombinant rat
A3 receptors and Nancy Forsythe for technical as-
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