Chiral resolution and stereospecificity of 6-phenyl-4-phenylethynyl- 1,4-dihydropyridines as selective A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm980688e

Racemic 6-phenyl-4-phenylethynyl-1,4-dihydropyridine derivatives have been shown to be highly selective A₃ adenosine receptor antagonists (Jiang et al. J. Med. Chem. 1997, 40, 2596-2608). Methods for resolving the optical isomers at the C4 position, involving selective crystallization or chromatographic separation of diastereomeric ester derivatives, have been developed. Optically pure glycerol and threitol derivatives were used as chiral auxiliary groups for ester formation at the 3-position, resulting in diastereomeric mixtures of dihydropyridines. Esterification of a 6-phenyl-4- phenylethynyl-1,4-dihydropyridine derivative at the 3-position with a chiral, protected glycerol moiety, (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol, allowed the selective crystallization of a pure diastereomer, 9. The ¹H NMR spectrum of 9 using the lanthanide shift reagent Eu(fod)₃ indicated optical purity, and the (4S,2'R)-configuration was assigned using X-ray crystallography. The noncrystalline (4R,2'R)-isomer 10 was also isolated and shown to be 3-fold more potent than the (4S,2'R)-isomer in binding to A₃ receptors. The 2,2-dimethyl-1,3-dioxolane moiety also served as a protected form of a diol, which showed selective reactivity versus a 5-ethyl ester in basic transesterification reactions. A racemic 5-carboxylic acid derivative could not be resolved through crystallization of diastereomeric salts. Enantiomers of 5-benzyl 3-ethyl 2-methyl-6-phenyl-4-phenylethynyl-1,4- dihydropyridine-3,5-dicarboxylate (2) were obtained via an ester derived from (4R,5R)-(-)-2,3-O-isopropylidene-D-threitol at the 3-position, which was resolved using HPLC, and each diastereomer was subsequently deprotected in acidic conditions. The resulting diols were exchanged for ethyl ester groups by base-catalyzed transesterification. The binding of pure enantiomers of 2 at A₃ adenosine receptors indicated a 35-fold stereoselectivity for the (4S)-isomer 21. A receptor docking hypothesis, using a previously derived human A₃ receptor model, shows the bulkier of the two ester groups (5-Bn) of 21 oriented toward the exofacial side and the 4-position phenylethynyl group situated between transmembrane helical domain TM6 and TM7.

Full text of DOI:10.1021/jm980688e

J . Med. Chem. 1999, 42, 3055-3065
3055
Ch ir a l Resolu tion a n d Ster eosp ecificity of 6-P h en yl-4-p h en yleth yn yl-
1,4-d ih yd r op yr id in es a s Selective A3 Ad en osin e Recep tor An ta gon ists
J i-long J iang, An-Hu Li, Soo-Yeon J ang, Louis Chang, Neli Melman, Stefano Moro, Xiao-duo J i,
Emil B. Lobkovsky,† J on C. Clardy,† 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, and Baker Laboratory, Department of Chemistry,
Cornell University, Ithaca, New York 14853
Received December 8, 1998
Racemic 6-phenyl-4-phenylethynyl-1,4-dihydropyridine derivatives have been shown to be highly
selective A3 adenosine receptor antagonists (J iang et al. J . Med. Chem. 1997, 40, 2596-2608).
Methods for resolving the optical isomers at the C4 position, involving selective crystallization
or chromatographic separation of diastereomeric ester derivatives, have been developed.
Optically pure glycerol and threitol derivatives were used as chiral auxiliary groups for ester
formation at the 3-position, resulting in diastereomeric mixtures of dihydropyridines. Esteri-
fication of a 6-phenyl-4-phenylethynyl-1,4-dihydropyridine derivative at the 3-position with a
chiral, protected glycerol moiety, (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol, allowed the
selective crystallization of a pure diastereomer, 9. The 1H NMR spectrum of 9 using the
lanthanide shift reagent Eu(fod)3 indicated optical purity, and the (4S,2′R)-configuration was
assigned using X-ray crystallography. The noncrystalline (4R,2′R)-isomer 10 was also isolated
and shown to be 3-fold more potent than the (4S,2′R)-isomer in binding to A3 receptors. The
2,2-dimethyl-1,3-dioxolane moiety also served as a protected form of a diol, which showed
selective reactivity versus a 5-ethyl ester in basic transesterification reactions. A racemic
5-carboxylic acid derivative could not be resolved through crystallization of diastereomeric salts.
Enantiomers of 5-benzyl 3-ethyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-dihydropyridine-3,5-
dicarboxylate (2) were obtained via an ester derived from (4R,5R)-(-)-2,3-O-isopropylidene-D-
threitol at the 3-position, which was resolved using HPLC, and each diastereomer was
subsequently deprotected in acidic conditions. The resulting diols were exchanged for ethyl
ester groups by base-catalyzed transesterification. The binding of pure enantiomers of 2 at A3
adenosine receptors indicated a 35-fold stereoselectivity for the (4S)-isomer 21. A receptor
docking hypothesis, using a previously derived human A3 receptor model, shows the bulkier of
the two ester groups (5-Bn) of 21 oriented toward the exofacial side and the 4-position
phenylethynyl group situated between transmembrane helical domain TM6 and TM7.
In tr od u ction
their interactions with these receptors. We observed
that at adenosine receptors, the affinity of many 1,4-
dihydropyridines, even commercial L-type calcium chan-
nel blockers, is in the micromolar range, and this lead
has been optimized in the design of selective A3 adeno-
sine receptor antagonists. The essential modifications
leading to A3 selectivity and preventing binding to
L-type calcium channels and other sites are a phenyl
ring at the 6-position and at the 4-position either a
styryl or a phenylethynyl group (e.g. 1 and 2, Figure
1). The trisubstituted analogue 5-benzyl 3-ethyl 2-meth-
yl-6-phenyl-4-phenylethynyl-1,4-(()-dihydropyridine-
3,5-dicarboxylate, 2, is 1300-fold selective for the human
A3 receptor versus A1 receptors, with a Ki value of 31
nM.
Antagonists that interact selectively with A3 adeno-
sine receptors recently have been reported.1-7 A3 ad-
enosine receptor antagonists have been proposed to have
potential as antiasthmatic, antiinflammatory, and cere-
broprotective agents.8-12 Diverse structural classes of
A3 antagonists are 1,4-dihydropyridines (e.g. MRS
1191),1,3 flavonoids (e.g. MRS 1067),4 triazoloquinazo-
lines (e.g. MRS 1220),5 triazolonaphthyridines (e.g.
L-249313),6 pyridines (e.g. MRS 1523),7 and pyridyliso-
quinolines (e.g. VUF 8504).2
We have used the 1,4-dihydropyridine1,3 nucleus as
a template for probing structure-activity relationships
(SAR) at the subtypes of adenosine receptors. Dihydro-
pyridines are privileged structures in medicinal chem-
istry; i.e. they display low-affinity binding at a variety
of receptor sites.13-17 Varying substituents on the 1,4-
dihydropyridine template can have dramatic effects on
While the stereospecificity of the binding of chiral
ligands is a general property of receptors, the enantio-
meric ratios of affinities may vary widely. At L-type
calcium channels, the potency ratios between (4R)- and
(4S)-1,4-dihydropyridines are generally 10-100-fold,
with the (4S)-enantiomer being more potent.18-20 For
most of those enantiomeric pairs of calcium channel
* Address correspondence to: Dr. K. A. J acobson, Bldg 8A, Rm B1A-
19, NIH, NIDDK, LBC, Bethesda, MD 20892-0810. Tel: (301) 496-
9024. Fax: (301) 480-8422. E-mail: kajacobs@helix.nih.gov.
† Cornell University.
10.1021/jm980688e CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/22/1999

3056 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
J iang et al.
Sch em e 1. Synthesis of Diastereomeric
5-Ethyl-6-phenyl-4-phenylethynyl-1,4-dihydropyridines
Using the Hantzsch Reaction and Resolution by
Crystallization (R1 ) CtC-Ph)a
F igu r e 1. Structures of key racemic dihydropyridine A3
adenosine receptor-selective antagonists 1 and 2 and optically
pure L-type calcium channel ligands 3 and 4 for which the
adenosine receptor affinity has been measured. Ki values in
micromolar are reported in refs 1 and 3.
a
Reagents: (a) toluene, NaOAc, 90 °C, 4 h; (b) EtOH, 80 °C; (c)
crystallization; (d) THF, 25 °C, 1 N HCl.
of 2 (Figure 1), i.e. 5-benzyl ester derivatives, involved
use of a chiral auxiliary group at the 3-ester position,
which could later be exchanged for an ethyl ester.
Analogues of 2 and other 1,4-dihydropyridines substi-
tuted with a variety of chiral auxiliaries at the 3-position
(groups A-F , Figure 2) were prepared and compared
for ease of separation of diastereomers, either by HPLC
or by crystallization. Group A (2,2-dimethyl-1,3-diox-
olan-4-ylmethyl), a protected glycerol moiety having a
single chiral center, proved useful for the fractional
crystallization of diastereomers. The synthesis and
resolution of 1,4-dihydropyridine derivatives containing
group A are shown in Scheme 1. While a group A
derivative containing a 5-ethyl ester was separable by
crystallization and led to the determination of the
absolute configuration using X-ray crystallography (be-
low), the corresponding 5-benzyl ester was unseparable.
ligands, e.g. 3a and 3b, the key factor determining
stereospecificity is a bulky ester substituent at the
5-position. At adenosine receptors, a previous study
including several pairs of enantiomers of 1,4-dihydro-
pyridines bearing 2,6-dimethyl groups (i.e. the antago-
nist niguldipine, 3, and the antagonist/agonist pair
BayK 8644, 4) demonstrated a slight preference for the
(4R)-enantiomers (2- and 8-fold, respectively, at human
A3 receptors).1 The effects of the 6-phenyl substituent
of 1,4-dihydropyridines on stereospecificity of binding
to adenosine receptors have not previously been studied.
Also, prior information on stereospecificity of dihydro-
pyridine binding concerns mainly the 4-aryl derivatives.
Since racemic 6-phenyl-4-phenylethynyl-1,4-dihydro-
pyridine derivatives have displayed exceptionally high
A3 selectivity (in some cases having no measurable
affinity at A1 and A2A adenosine receptors),3 we sought
a facile method to chemically resolve compounds in this
series and to study the biological properties of pure
stereoisomers. Such a method for resolving the enanti-
omers at the C4 position consisted of either the selective
crystallization or the HPLC purification of diastereo-
meric ester derivatives. The chiral ester moieties,
derivatives of 2,2-dimethyl-1,3-dioxolane-4-methanol
and 2,3-O-isopropylidene-D-threitol, could be selectively
replaced with an ethyl moiety in two steps: i.e. depro-
tection of a diol followed by transesterification in
ethanol.
To synthesize a diastereomeric pair for resolution by
crystallization, the Hantzsch reaction was carried out
using a chiral â-ketoester derived from a protected
glycerol moiety (5, Scheme 1). This intermediate con-
sisted of an acetoacetate ester of (S)-(+)-2,2-dimethyl-
1,3-dioxolane-4-methanol, which corresponds to the (R)-
enantiomer 5 following esterification. This â-ketoester
5 and the other components of the reaction to form the
6-phenyl-4-phenylethynyl-1,4-dihydropyridine deriva-
tive, e.g. phenylpropargyl aldehyde, 6, and â-enami-
noester 7, were dissolved in ethanol and refluxed
overnight. Proton NMR in deuterated chloroform showed
the isolated, gummy product 8 to be a 1:1 mixture of
(4S,2′R)- and (4R,2′R)-diastereomers. The signals from
each of the two ester R-methylene groups were well-
resolved in the spectrum and separated by 0.1 ppm. The
signals from the 4-H of the diastereomers were sepa-
Resu lts
Ch em istr y. The chemical synthesis and resolution
of diastereomeric mixtures of 1,4-dihydropyridine de-
rivatives are outlined in Schemes 1-5. The strategy
utilized for the successful resolution of the enantiomers

6-Phenyl-4-phenylethynyl-1,4-dihydropyridines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3057
Sch em e 2. Synthesis of Diastereomeric 5-Benzyl-6-phenyl-4-phenylethynyl-1,4-dihydropyridines Using the Hantzsch
Reaction and Resolution by HPLCa
a
Reagents: (a) EtOH, 90 °C, overnight; (b) HPLC separation.
Sch em e 3. Deprotection of Isopropylidene Group from 3-Ester Chiral Auxiliary Esters and Transesterification in
Ethanol (R1 ) CtC-Ph)a
a
Reagents: (a) THF, 25 °C, 1 N HCl; (b) 95% EtOH, 25 °C, 1.5 equiv NaOH; (c) 1 N HCl/THF (1:1), rt, overnight; (d) NaOH, 95%
EtOH, rt, 3 days.
Sch em e 4. Synthesis of Chiral â-Ketoester as a
Starting Material for the Synthesis of Compounds 21
and 23a
Sch em e 5. Attempted Resolution by Crystallization of
Diastereomeric Salts of a 1-Protected 6-Phenyl-4-
phenylethynyl-1,4-dihydropyridine (R4 ) CtC-Ph)a
a
Reagents: (a) TBAF; (b) cinchonidine or cinchonine.
the (4S,2′R)-configuration, 9. The remaining isomer,
evidently having the (4R,2′R)-configuration, 10, was
obtained from the mother liquor as an oil and was
a
Reagents: (a) PhCH2Cl, NaOH, DMSO/H2O (6:1); (b) toluene,
90 °C, overnight.
1
shown by 1H NMR to be pure. The H NMR resonances
rated by 0.03 ppm. Subsequent resolution of the enan-
tiomers (below) and addition of a chiral shift reagent
verified that these peaks corresponded to pure diaster-
eomers.
Fractional crystallization of 8 from methanol provided
a pure diastereomer of this 6-phenyl-4-phenylethynyl-
1,4-dihydropyridine derivative. This isomer was shown
by X-ray crystallography (Figure 3, see below) to be of
of the two separate isomers with and without the
lanthanide shift reagent Eu(fod)321 were catalogued and
are shown in Table 1. Each of the isolated isomers was
shown to be a pure diastereomer and with the peaks of
the other isomer not visible in the spectrum. Without
Eu(fod)3 the 4-H and R-methylene resonances were
downfield in the (4R,2′R)-isomer 10 versus the (4S,2′R)-

3058 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
J iang et al.
Ta ble 1. Proton NMR Chemical Shifts of Diastereomeric
1,4-Dihydropyridine Derivatives Resolved by Crystallization
chemical shift (ppm from TMS)a
F igu r e 2. Chiral auxiliary ester groups (3-position) used in
the resolution of 6-phenyl-4-phenylethynyl-1,4-dihydropy-
ridines; R4 ) benzyl or ethyl.
resonance
9
9 + Eu(fod)3
10
10 + Eu(fod)3
2-CH3
4-H
3-OCH2
5-OCH2
N-H
2.37
5.10
4.26
3.99
5.90
2.59
5.83
4.79
4.47
6.03
2.37
5.13
4.36
4.09
5.88
2.49
5.43
4.46
4.19
5.94
a
In CDCl3, in the presence or absence of 4 mg/mL Eu(fod)3
(europium tris(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedi-
onate)). The dihydropyridines were dissolved at a concentration
of 5 mg/mL.
tion (Figure 3), which is consistent with crystal struc-
tures determined for other 1,4-dihydropyridines in
which the 4-position substituent is not highly hin-
dered.18
The 2,2-dimethyl-1,3-dioxolane moiety also served as
a protected form of a diol, 18, obtained following
deprotection of 8 in HCl/THF (Scheme 3). This diol
showed a selective reactivity versus the 5-ester in basic
transesterification reactions. The 3-ethyl ester 19 was
obtained in this manner using sodium hydroxide in 95%
ethanol. An attempt to synthesize the corresponding
3-thioester via transesterification in ethanethiol was
unsuccessful. During the basic transesterification reac-
tion, a simple 5-alkyl ester group was expected to be
unreactive, as found previously with similar derivatives.
The resolved diastereomers 9 and 10 were also depro-
tected separately, to give 11 and 12. These diols were
then tested for biological activity.
The analogue of 2, corresponding to compound 8
except containing a 5-benzyl ester instead of ethyl, was
prepared (scheme not shown); however, it could not be
resolved by crystallization. Since the more biologically
interesting dihydropyridine derivatives contained a
bulkier ester, e.g. benzyl ester, at the 5-position, alter-
nate methods (Schemes 2-4, see below) were required
for the resolution. Groups B-D (Figure 2: B, 1,4-
dioxaspiro[4.5]decan-2-ylmethyl; C, 1-benzyl-2-pyrro-
lidinylmethyl; D, 4-isopropyl-2-oxazolidinon-3-yl), also
containing a single chiral center, were likewise incor-
porated into analogues of 2 as the 3-position substituent
(schemes not shown); however, none of these analogues
were amenable to resolution by fractional crystalliza-
tion.
F igu r e 3. X-ray crystallographic structure determined for 9,
3-(2′R)-(2,2-dimethyl-1,3-dioxolan-4-ylmethyl) 5-ethyl 4-(S)-2-
methyl-6-phenyl-4-phenylethynyl-1,4-dihydropyridine-3,5-di-
carboxylate.
isomer 9. Eu(fod)3 (4 mg/mL) caused a downfield shift
of all of the resonances. In each case the resonances of
the (4S,2′R)-isomer were shifted downfield to a greater
degree in the presence of the NMR shift reagent. For
example, the signals from the 4-H were shifted by Eu-
(fod)3 by >0.7 ppm in the (4S,2′R)-isomer and by only
0.3 ppm in the (4R,2′R)-isomer. Thus, in the presence
of Eu(fod)3 this important resonance was separated by
0.4 ppm between the two diastereomers. The resonances
of the two ester R-methylene groups, which were the
best-resolved in the spectrum measured in the absence
of Eu(fod)3, were separated by ∼0.3 ppm in the presence
of Eu(fod)3.
To establish the absolute configuration at the C4
position of compound 9, an X-ray crystallographic
structure was determined. Small needles of 9 were
grown by vapor diffusion from methanol/water solution.
X-ray diffraction studies22,23 were performed with a
Siemens P4 diffractometer (Mo KR radiation). Crystals
of 9 were found to be monoclinic (P21) with lattice
parameters a ) 11.059(2) Å, b ) 8.212(2) Å, c ) 15.629-
(3) Å, â ) 104.46(1)°, Z ) 2. The spatial coordinates of
the X-ray structure are provided as Supporting Infor-
mation. The carbonyl groups were in the cis/cis orienta-
Another approach to the resolution of these enanti-
omers is the crystallization of diastereomeric salts of
the 5-carboxylic acid derivative 27 using a chiral base
(Scheme 5). Methods for synthesizing 5-carboxylic acid

6-Phenyl-4-phenylethynyl-1,4-dihydropyridines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3059
Ta ble 2. Proton NMR Chemical Shifts of Diastereomeric 1,4-Dihydropyridine Derivatives Resolved by HPLCa
racemic mixture
16 (tR ) 32 min) (4S,2′R,3′R)
17 (tR ) 35 min) (4R,2′R,3′R)
number or
position
chemical
shift (ppm)
+Eu(fod)3
(ppm)
chemical
shift (ppm)
+Eu(fod)3
(ppm)
chemical
shift (ppm)
+Eu(fod)3
(ppm)
(CH3)2
2-CH3
a
b
c
s, 1.41
s, 2.34
m, 3.60-3.70
bs, 4.16
s. 1.42
s, 2.37
s, 3.67
m, 4.17-4.23
m, 4.32-4.40
s, 4.54
s, 5.17
m, 4.93-5.12
s, 5.93
s, 1.42
s, 2.37
s, 3.62
m, 4.17-4.22
bs, 4.35
bs, 4.51
s, 5.20
m, 4.91-5.15
s, 5.90
m, 7.20-7.37
2.55, -2.61
2.53
2.56
bs, 4.32
d
d, 4.51-4.54
d, 5.16-5.18
m, 4.89-5.13
d, 6.05-6.08
m, 7.19-7.35
4-H
2CH
NH
C6H5
5.78, 5.96
6.02, 6.07
5.89
6.06
5.64
5.99
m, 7.20-7.39
a
In CDCl3, in the presence or absence of 4 mg/mL Eu(fod)3 (europium tris(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionate)).
The dihydropyridines were dissolved at a concentration of 5 mg/mL.
derivatives of 1,4-dihydropyridines have already been
explored,18 and we have reported a method specifically
with 6-phenyl-4-phenylethynyl-1,4-dihydropyridine de-
rivatives.39 This method utilizes the 2-trimethylsilyl-
ethyl ester group at the 5-position, as in 26, combined
with the acid-sensitive 1-ethoxymethyl group. However,
the attempted crystallization of the carboxylic acid
intermediate 27 with cinconidine failed. From a 1:1
mixture in ethyl ether, only cinconidine itself precipi-
tated. Thus, it appears that the cinconidine salt does
not form, presumably due to steric hindrance. In previ-
ous examples of resolution of 4-aryldihydropyridine
salts,18 generally there was a methyl group rather than
a phenyl group at the 6-position.
None of the diastereomeric mixtures of either 5-ethyl
or 5-benzyl esters containing chiral auxiliaries A-D
(including compound 8 and the four corresponding
analogues of 2) were separable by normal-phase HPLC.
The separation of diastereomers by HPLC required
introduction of group E or F (Figure 2), each containing
two chiral centers. Group E esters ((4R,5R)-5-benzy-
loxymethyl-2,2-dimethyl-1,3-dioxolan-4-ylmethyl)24 were
chemically stable, while group F derivatives ((4S,5S)-
2-methyl-5-phenyl-2-oxazolin-4-ylmethyl) decomposed
rapidly upon storage; thus, group E was selected for
resolution of enantiomers of 2. The incorporation of
group E as a 3-position ester leading to the successful
synthesis of the pure enantiomers is shown in Schemes
2-4. Thus, the subsequent transesterification reaction
at the 3-position completed the synthesis of pure enan-
tiomers of the highly potent A3 adenosine receptor
antagonist.
(Table 1) were 5.83 and 5.43 ppm, respectively. The
chemical shift of the (4S)-form was downfield from that
of (4R)-form. The differences in chemical shift caused
by Eu(fod)3 in (4S)-9 and (4R)-10 were 0.73 and 0.30
ppm, respectively. Thus, Eu(fod)3 caused larger chemical
shift differences in the (4S)-form than in the (4R)-form.
For diastereomers 16 and 17 (Table 2), which were
structurally similar to (4S)-9 and (4R)-10, without Eu-
(fod)3, the chemical shift of the 4-hydrogen in 16 (5.17
ppm) was upfield from that of 17 (5.20 ppm). After
addition of Eu(fod)3, the chemical shift of the 4-hydrogen
in 16 (5.89 ppm) shifted downfield from that of 17 (5.64
ppm). The difference of chemical shift caused by
Eu(fod)3 in 16 (0.72 ppm) was also larger than in 17
(0.44 ppm). Thus, the NMR analysis was consistent with
the absolute configuration of the 4-position in 16 being
(S) and in 17 being (R), and by direct analogy, the
absolute configurations of enantiomers 21 and 23 were
assigned as (S) and (R), respectively.
P h a r m a cology. The structures of the 1,4-dihydro-
pyridines derivatives (2, 9-12, 21, 23, and 28) tested
for affinity in standard radioligand binding assays25-28
at adenosine receptors are shown in Table 3. Ordinarily,
a comparison of A1, A2A, and A3 affinities within one
species would be preferred. We chose the human A3
receptor for the present study instead of the rat A3
receptor since the affinity of dihydropyridine antago-
nists is only marginal at rat A3 receptors.3 In contrast,
at non-A3 adenosine receptors, the affinity of dihydro-
pyridines does not show as marked a species depen-
dence.
The (4S,2′R)-isomer of the 5-ethyl derivative 9 was
>70-fold selective for human A3 receptors versus rat A1
receptors. The corresponding (4R,2′R)-isomer 10, al-
though less selective, was approximately 3-fold more
potent at human A3 receptors, with a Ki value of 0.426
µM. Following removal of the isopropylidene groups,
there was no stereoselectivity of binding. Thus, com-
pounds 11 and 12 each bound to human A3 receptors,
with a Ki value of 0.5-0.6 µM.
The assignment of absolute configuration at the
4-position in the two diastereomers separated by HPLC,
16 and 17, and consequently by analogy the two
enantiomers of 2, i.e. 21 and 23, was based on NMR
analysis. In the absence of Eu(fod)3, the chemical shifts
of the 4-hydrogen in (4S)-9 and (4R)-10 were 5.10 and
5.13 ppm, respectively. The chemical shift of the (4S)-
form was more upfield than that of the (4R)-form. After
the chemical shift reagent Eu(fod)3 was added, the
chemical shifts of the 4-hydrogen in (4S)-9 and (4R)-10
Introduction of 3-ethyl and 5-benzyl esters in the
enantiomers of 2, compounds 21 and 23, selectively

3060 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
J iang et al.
Ta ble 3. Affinities of 1,4-Dihydropyridine Derivatives in Radioligand Binding Assays at A1, A2A, and A3 Receptors
Ki (µM) or % inhibitiond
chirality at
4-position
a
b
c
compd
R3
R5
rA1
rA2A
hA3
rA1/hA3
1800
2
OCH2CH3
racemic
OCH2Ph
40.1 ( 7.5e
d (10-4
)
)
0.0222 ( 0.0038
(0.0314 ( 0.0028)e
1.31 ( 0.57
9
S
OCH2CH3
OCH2CH3
32 ( 4% (10-4
)
d (10-4
d (10-4
>70
10
R
16.4 ( 3.1
)
0.426 ( 0.133
38
11
12
S
OCH2CH3
OCH2CH3
14.8 ( 1.0
39 ( 8% (10-4
d (10-4
)
0.52
0.65
28
11
R
6.83 ( 0.20
)
21
OCH2CH3
OCH2CH3
OCH2CH3
S
R
OCH2Ph
OCH2Ph
OCH2CH3
32 ( 12% (10-4
25 ( 11% (10-4
11.0 ( 0.1
)
)
14 ( 6% (10-4
11 ( 4% (10-4
)
)
0.0213 ( 0.0065
0.750 ( 0.151
0.0766 ( 0.0151
>400
>15
140
23
28e
racemic
26 ( 12% (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 10% of specific binding at the indicated concentration (M). e Values taken from van Rhee et al.1 or from J iang et
d
al.3
enhanced affinity at human A3 receptors for the (4S)-
derivative 21. The affinity of 21 was similar to that of
the racemic mixture 2, while the affinity of the opposite
enantiomer 23 at human A3 receptors was 35-fold less
than that of 21. The effect of the benzyl ester alone was
determined by comparison to the corresponding racemic
3,5-diethyl ester 28, which was 4-fold less potent than
2. The stereoselectivity of the weak binding of 21 and
23 at A1 and A2A receptors could not be analyzed due to
limited solubility.
Molecu la r Mod elin g. A symmetric 1,4-dihydro-
pyridine29 and related pyridine7 antagonists have been
docked in the putative ligand binding site of a rhodop-
sin-based molecular model of the human A3 receptor.29,31
To illustrate a possible conformational explanation of
the results of the present study, the enantiomers of 2
were similarly docked in this site. According to our
model, it is possible to dock both (4R)- and (4S)-
enantiomers in energetically favorable conformations,
simply by rotating the 1,4-dihydropyridine ring by 180°
about the NH-C4 axis. In this way, the bulky 4-phen-
ylethynyl substituent of both enantiomers may still be
arranged between TM6 and TM7 (see Figure 4 and
Discussion). Thus, for the (4S)-enantiomer 21, the bulky
5-benzyl ester is facing the exofacial side of the helical
bundle, while the 3-ethyl ester of the opposite enanti-
omer 23 points in the same direction.
of pharmacological information about the two pure
enantiomers. In the present study, we have explored the
stereoselectivity of A3 adenosine receptor-selective 1,4-
dihydropyridines related to the 4-phenylethynyl deriva-
tive 2.
It was necessary to resolve the analogues by crystal-
lization or by HPLC of diastereomeric derivatives. In
one synthetic approach, a free carboxylic acid was
created at the 5-position; however, attempted crystal-
lization with chiral alkaloids, such as cinconidine, as
counterion was unsuccessful. Alternately, a chiral ester
group such as a glycerol acetonide (group A) was
introduced at the 3-position, thus allowing the crystal-
lization of a pure (4S,2′R)-isomer, 9, as determined by
X-ray crystallography. The effect of the glycerol group
on receptor affinity was also of interest, since the
hydroxyl groups would be expected to enhance water
solubility; however, only moderate affinity was achieved.
After removal of the acetonide, the glycerol ester was
removable with base and could be exchanged through
basic transesterification. This allowed for greater flex-
ibility of substitution. The previous methods for resolu-
tion of 1,4-dihydropyridines via separation of diastere-
omeric esters18 relied on subsequent removal of the
chiral esterifying group by one of three methods: (1)
activation of the ester for selective reaction, (2) removal
via â-elimination, and (3) removal through hydrogenoly-
sis. Hydrogenolysis may not be used for the present
target compounds due to the unsaturated 4-substituent
which would also be reduced. A scheme not involving
â-elimination was also desired, since this method may
be needed for optional substitution at the 5-position.39
Discu ssion
Previously, analysis of the structure-activity rela-
tionships and receptor docking of the series of mainly
racemic 1,4-dihydropyridines has been limited by lack

6-Phenyl-4-phenylethynyl-1,4-dihydropyridines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3061
F igu r e 4. Overlapped compounds 21 ((4S), yellow structure) and 23 ((4R), red structure) computer-docked inside the putative
binding pocket of the human A3 receptor.29,31 Three of the seven transmembrane helices are shown as viewed from the plane of
the plasma membrane. The side chains of the important residues in proximity (e5 Å) to the docked dihydropyridine molecules
are highlighted and labeled: Leu90 (TM3), Phe182 (TM5), Ser242 (TM6), Ser247 (TM6), Asn250 (TM6), Ser271 (TM7), and His272
(TM7).
The glycerol acetonide served the purpose as a chiral
moiety which, following removal of the ketal, provided
an ester that was activated for selective reaction.
The resolution of the more potent antagonist 2 was
carried out for comparison using a similar approach,
requiring a more complex chiral auxiliary ester (group
E) and chromatographic resolution. The absolute con-
figuration of the resulting stereoisomers was determined
based on the effects of an NMR shift reagent in
comparison with the corresponding shifts of the glycerol
acetonides.
From the structural information obtained for the
(4S,2′R)-isomer 9 and from the receptor binding of both
(4S,2′R)- and (4R,2′R)-isomers, we have concluded that
the more potent isomer of the group A esters is 10, of
the (4R)-configuration at the 4-position. However, the
binding of pure enantiomers 21 and 23 at A3 adenosine
receptors indicates a 35-fold selectivity for the 4-position
(4S)-isomer. Therefore, the stereoselectivity of binding
at A3 receptors appears to be a function of the orienta-
tion of the 4-position phenylethynyl group relative to
the bulkier of the two ester groups, rather than relative
to the 6-phenyl group, which determines its absolute
(4R)- or (4S)-configuration. The substantial stereo-
selectivity of enantiomers of 2 in binding to A3 receptors
is noteworthy since stereoselectivity has not generally
been a prominent feature of antagonists at any of the
adenosine receptors.
recognition site is located in the upper region of the
helical bundle of the receptor. According to this docking
model, the pyridine or 1,4-dihydropyridine rings are
most favorably oriented perpendicular to the plane of
the lipid bilayer with the 3-ester position in the exofacial
direction. The carbonyl of the 5-ester group is in
proximity to Ser275 (TM7), and the carbonyl of the
3-ester group is close to Asn250 (TM6). Two important
hydrophilic interactions, probably hydrogen-bonding
interactions, seem to be involved between the two ester
groups and these two polar amino acids. Two hydro-
phobic pockets are likely present around the 2-position,
where an alkyl side chain would bind (Leu90, TM3; and
Phe182, TM5), and around the 6-position, where the
phenyl ring would bind (Leu90, TM3; and Ile186, TM5).
The TM7 backbone is close to the 4-position of the
pyridine ring. Consequently, a strong steric control is
suggested around the 4-position of both 1,4-dihydro-
pyridine and pyridine structures. In fact, bulky 4-posi-
tion substituents, such as the 4-phenylethynyl group,
which is affinity-enhancing in the 1,4-dihydropyridines,
are not well-tolerated in the pyridine series. From a
structural point of view, changing the C4 hybridization
from sp3
to sp2, i.e. transformation of 1,4-dihydropyri-
dine to the corresponding pyridine, would change the
C5-C4-R4 angle from 68.1° to 0.2°. As already re-
ported, in the case of 1,4-dihydropyridine derivatives,
the bulky and rigid 4-phenylethynyl substituent could
be situated between TM6 and TM7; thus, the nature of
the 4-substituent is critical for recognition of this series
inside the receptor. To maintain the critical orientation
of the 4-phenylethynyl group, energetic optimization in
the docking of the enantiomers of 2 required inverting
These results are consistent with the conformational
features of our previously published human A3 receptor
model, in which a symmetric dihydropyridine29 and
structurally related pyridine derivatives7,31 have been
docked in a common mode of binding. This putative

3062 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
J iang et al.
p yr id in e-3,5-d ica r boxyla te (9): 1H NMR: δ 0.94 (t, J ) 6.9
Hz, 3 H), 1.35 (s, 3 H), 1.44 (s, 3 H), 2.37 (s, 3 H), 3.85 (m, 1),
4.10 (m, 1 H), 4.00 (q, J ) 6.9 Hz, 2 H), 4.25 (d, J ) 4.9 Hz, 2
H), 4.40 (m, 1 H), 5.10 (s, 1 H), 5.88 (s, br, 1 H), 7.25-7.43 (m,
the 3- and 5-ester groups, such that the benzyl ester
pointed in the exofacial direction for the more potent
enantiomer 21.
As reported in Table 3, 11 and 12, which have 3- and
5-substitutents of similar size, show the same affinity
at the human A3 receptor. However, the presence of
bulky substituents at the carbonyl of the 3- and/or
5-ester groups can change the enantioselectivity of
receptor binding. In fact, secondary to the arrangement
of the substituent at the 4-position, the bulkier of the
two ester groups, independent of its position, seems to
prefer to be located at the extracellular region of the
TM domain. The (4S)-enantiomer 21 is 35-fold more
active than the (4R)-isomer 23 and is more favorably
docked with the 5-benzyl ester pointing toward the
extracellular environment. Accordingly, the 3-(1,3-di-
oxolane) substituent of the (4R)-enantiomer 10 would
be located in the upper region of the TM bundle, and
its affinity is ca. 3-fold higher than that of the (4S)-
enantiomer. Apparently, the orientation of the 6-phenyl
substituent does not seem to direct stereocontrol of
binding, which, rather, appears to be dominated by the
presence, or not, of bulky ester groups. This is a clear
example of the fact that receptor stereoselectivity is not
only due to chiral properties of the ligand but also to
the steric and electronic requirements of the receptor
cleft. Other studies are in progress to test this hypoth-
esis.
10 H). MS (EI): m/z 501 (M+), 428 (M+ - CO2Et), 386 (M+
-
(2,2-dimethyl-1,3-dioxolan-4-yl)methyl), 342 (M+ - (2,2-di-
methyl-1,3-dioxolan-4-yl)methoxycarbonyl, base). HRMS: cal-
cd for C30H31NO6 501.2151, found 501.2141.
3-(2′R)-(2,2-Dim eth yl-1,3-d ioxola n -4-ylm eth yl) 5-eth yl
4-(R)-2-m et h yl-6-p h en yl-4-p h en ylet h yn yl-1,4-d ih yd r o-
p yr id in e-3,5-d ica r boxyla te (10): 1H NMR: δ 0.98 (t, J )
6.8 Hz, 3 H), 1.38 (s, 3 H), 1.49 (s, 3 H), 2.37 (s, 3 H), 3.98 (m,
2 H), 4.09 (m, 2 H), 4.22 (m, 2 H), 4.36 (m, 1 H), 5.13 (s, 1 H),
5.88 (s, br, 1 H), 7.25-7.44 (m, 10 H). MS (EI): m/z 501 (M+),
428 (M+ - CO2Et), 386 (M+ - (2,2-dimethyl-1,3-dioxolan-4-
yl)methyl), 342 (M+ - (2,2-dimethyl-1,3-dioxolan-4-yl)methoxy-
carbonyl, base). HRMS: calcd for C30H31NO6 501.2151, found
501.2151.
P r ep a r a tion of 3-(2′R)-(2,3-Dih yd r oxy-1-p r op yl) 5-Eth -
yl 4-(S)-2-Meth yl-6-p h en yl-4-p h en yleth yn yl-1,4-d ih yd r o-
pyr idin e-3,5-dicar boxylate (11) an d 3-(R)-(2,3-Dih ydr oxy-
1-pr opyl)5-Eth yl 4-(R)-2-Meth yl-6-ph en yl-4-ph en yleth yn yl-
1,4-d ih yd r op yr id in e-3,5-d ica r b oxyla t e (12) b y Acid ic
Hyd r olysis. Optically pure diastereomer 9 (15 mg) or 10 (9
mg) was stirred in a mixture of 1 N HCl and THF (1:1) (1.0
mL) at room temperature for 4 h. The solvents were removed,
and the residue was diluted with ethyl acetate (10 mL). The
organic layer was washed with water (5 mL × 2), dried over
MgSO4, and evaporated to give a residue, which was purified
by preparative TLC (silica 60, 1000 µm; Analtech, Newark,
DE; petroleum ether-ethyl acetate (1:1)) to give 8.0 mg
(yield: 58%) of compound 11 or 5.0 mg (60%) of compound 12.
1H NMR: δ 0.95 (t, J ) 6.8 Hz, 3 H), 2.38 (s, 3 H), 3.71 (m, 2
H), 4.00 (m, 2 H), 4.18 (m, 2 H), 4.54 (m, 1 H), 5.11 (s, 1 H),
5.94 (s, br, 1 H), 7.25-7.45 (m, 10 H). MS (EI): m/z 461 (M+),
432 (M+ - Et), 388 (M+ - CO2Et), 342 (M+ - CO2CH2CH-
(OH)CH2OH, base). HRMS for 11: calcd for C27H27NO6
461.1838, found 461.1861. HRMS for 12: calcd for C27H27NO6
461.1838, found 461.1845.
Ma ter ia ls a n d Meth od s
Syn th esis. Ma ter ia ls a n d In str u m en ta tion . Phenylpro-
pargyl aldehyde (6), benzyl chloride, 2,2,6-trimethyl-4H-1,3-
dioxin-4-one, (4R,5R)-(-)-2,3-O-isopropylidene-D-threitol (24),
(S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol, diketene, and
all other chiral auxiliaries were purchased from Aldrich (St.
Louis, MO). Benzyl 3-amino-3-phenyl-2-propenoate (13) and
ethyl 3-amino-3-phenyl-2-propenoate (7) were prepared by our
previous method.7 All other materials were obtained from
commercial sources.
P r ep a r a tion of (R)-(+)-2,2-Dim eth yl-1,3-d ioxola n -4-yl-
m eth yl Acetoa ceta te (5) (Sch em e 1). (S)-(+)-2,2-Dimethyl-
1,3-dioxolane-4-methanol (1.32 g, 10 mmol) and diketene (912
mg, 11 mmol) were dissolved in toluene (10 mL). Sodium
acetate (0.2 g) was added, and the mixture was heated at 90
°C for 4 h. After cooling to room temperature, the solvent was
removed under reduced pressure, and the residue was purified
by column chromatography (silica 60; CH2Cl2) to give 0.932 g
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).
P r ep a r a tion of 3-(2′R)-(2,2-Dim eth yl-1,3-d ioxola n -4-
ylm eth yl) 5-Eth yl 4-(R a n d S)-2-Meth yl-6-p h en yl-4-p h en -
ylethynyl-1,4-dihydropyridine-3,5-dicarboxylate (8)(Scheme
1). Phenylpropargyl aldehyde (6; 130 mg, 1.0 mmol), ethyl
3-amino-3-phenyl-2-propenoate (7; 191 mg, 1.0 mmol), and
chiral ketoester (R)-5 (174 mg, 1.0 mmol) were dissolved in 3
mL of 95% ethanol. The mixture was refluxed, with stirring,
at 80 °C overnight. After cooling to room temperature, the
solvent was evaporated, and the residue was purified by
preparative TLC (silica 60, 1000 µm; Analtech, Newark, DE;
petroleum ether-ethyl acetate (3:1)) to afford 105 mg of
compound 8 as a mixture of two diastereomers (yield: 21%).
Sep a r a tion of Dia ster eom er ic Mixtu r e of 8 by Cr ysta l-
liza tion . Compound 8 (45 mg) was dissolved in hot methanol
(2 mL), and the solution was left at room temperature for
crystallization. The crystals 9 (21 mg, 47%), which were
optically pure (by NMR and HPLC) and identified as the
(4S,2′R)-isomer by X-ray crystallography, were collected by
filtration, and the mother liquor was evaporated to give 22
mg (49%) of the other isomer which was also optically pure
(by NMR and HPLC), i.e. (4R,2′R)-10.
1
of compound 5, yield: 54%. H NMR: δ 1.37 (s, 3 H), 1.44 (s,
3 H), 2.29 (s, 3 H), 3.77 (m, 1 H), 4.07 (m, 1 H), 3.52 (s, 2 H),
4.22 (m, 2 H), 4.35 (m, 1 H).
P r ep a r a t ion of 5-Ben zyl 3-(4′R,5′R)-(5-Ben zyloxy-
m eth yl-2,2-d im eth yl-1,3-d ioxola n -4-ylm eth yl) 4-(R a n d
S)-2-Meth yl-6-p h en yl-4-p h en yleth yn yl-1,4-d ih yd r op yr i-
d in e-3,5-d ica r boxyla te (15) (Sch em e 2). Phenylpropargyl
aldehyde (6; 65 mg, 0.5 mmol), benzyl 3-amino-3-phenyl-2-
propenoate (13; 127 mg, 0.5 mmol), and chiral ketoester 14
(168 mg, 0.5 mmol) were dissolved in 3 mL of 95% ethanol.
The mixture was sealed in a Pyrex tube and heated, with
stirring, to 90 °C overnight. After cooling to room temperature,
the solvent was evaporated and the residue was purified by
preparative TLC (silica 60, 1000 µm; Analtech, Newark, DE;
petroleum ether-ethyl acetate (2:1)) to afford 150 mg of
compound 15 as a mixture of two diastereomers (yield: 44%).
Ch r om atogr aph ic Separ ation of Diaster eom er s.Samples
of the mixture of diastereomers were separated by HPLC
(Hewlett-Packard 1090 liquid chromatography system) using
a
silica column (300 × 10 mm) with the mobile phase
consisting of a linear gradient of hexane/10% 2-propanol in
ethyl acetate from 90:10 to 85:15 in 50 min. Samples dissolved
in the mobile phase were injected at a flow rate of 2.5 mL/min
and with a column temperature of 50° C. Peaks were detected
by UV absorption using a diode array detector. From 40 mg
of a 50:50 mixture of diastereomers, 10 mg (25%) of 5-benzyl
3-(4′R,5′R)-(5-benzyloxymethyl-2,2-dimethyl-1,3-dioxolan-4-yl-
3-(2′R)-(2,2-Dim eth yl-1,3-d ioxola n -4-ylm eth yl) 5-eth yl
4-(S)-2-m et h yl-6-p h en yl-4-p h en ylet h yn yl-1,4-d ih yd r o-

6-Phenyl-4-phenylethynyl-1,4-dihydropyridines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3063
methyl) 4-(S)-2-methyl-6-phenyl-4-phenylethynyl-1,4-dihydro-
pyridine-3,5-dicarboxylate (16) and 10.1 mg (25%) of 5-benzyl
3-(4′R,5′R)-(5-benzyloxymethyl-2,2-dimethyl-1,3-dioxolan-4-yl-
methyl) 4-(R)-2-methyl-6-phenyl-4-phenylethynyl-1,4-dihydro-
pyridine-3,5-dicarboxylate (17) were obtained.
(-)-21 a n d (+)-23 by a Tr a n sester ifica tion Rea ction .
Optically pure compound 7 (5.0 mg) or 8 (7.3 mg) and sodium
hydroxide (3 equiv) were stirred in 95% ethanol at room
temperature for 3 days. The solvents were removed under
reduced pressure, and the residue was purified by preparative
TLC (silica 60, 1000 µm; Analtech, Newark, DE; petroleum
ether-ethyl acetate (4:1)) to give 0.4 mg of (S)-(-)-21 (yield:
11%) or 2.28 mg of (R)-(+)-23 (yield: 42%).
The diastereomeric excess of each fraction was determined
by analytical HPLC using a silica column (250 × 4.6 mm) and
a flow rate of 1.0 mL/min with the same mobile phase gradient
system and column temperature as above. The diastereomeric
purities of compounds 16 and 17 were 99.0% and 95.0%. The
specific rotations for 16 and 17, [R]D (c ) 0.5, CHCl3), were
+0.05 and +0.26, respectively.
5-Ben zyl 3-eth yl 4-(S)-2-m eth yl-6-p h en yl-4-p h en yleth -
yn yl-1,4-d ih yd r op yr id in e-3,5-d ica r boxyla te (21): 1H
NMR: δ 1.35 (t, J ) 6.9 Hz, 3 H), 2.37 (s, 3 H), 4.28 (m, 2 H),
5.04 (AB, J ) 12.6 Hz, 2 H), 5.19 (s, 1 H), 5.86 (s, br, 1 H),
7.07 (m, 2 H), 7.20-7.27 (m, 7 H), 7.37 (m, 6 H). MS (EI): m/z
477 (M+), 448 (M+ - Et), 404 (M+ - CO2Et), 384 (M+ - 1 -
Me-Ph), 356 (MH+ - Me - OCH2Ph), 342 (M+ - CO2CH2Ph),
91 (+CH2Ph, base). HRMS: calcd for C31H27NO4 477.1940,
(4S)-Dia ster eom er 16: 1H NMR: δ 1.42 (s, 6 H), 2.37 (s, 3
H), 3.67 (s, 2 H), 4.17-4.23 (m, 2 H), 4.32-4.40 (m, 2 H), 4.54
(s, 2 H), 5.17 (s, 1 H), 4.93-5.12 (m, 2 H), 5.93 (s, br, 1 H),
7.20-7.39 (m, 20 H). MS (EI): m/z 683 (M+), 668 (M+ - Me),
592 (M+ - CH2Ph), 91 (+CH2Ph, base).
found 477.1937. [R]20 ) -70 ° (c ) 0.3, MeOH).
D
1
(4R)-Dia ster eom er 17: H NMR: δ 1.42 (s, 6 H), 2.37 (s,
5-Ben zyl 3-et h yl 4-(R)-2-m et h yl-6-p h en yl-4-p h en yl-
et h yn yl-1,4-d ih yd r op yr id in e-3,5-d ica r boxyla t e (23): 1H
NMR: δ 1.35 (t, J ) 6.9 Hz, 3 H), 2.37 (s, 3 H), 4.28 (m, 2 H),
5.04 (AB, J ) 12.6 Hz, 2 H), 5.19 (s, 1 H), 5.87 (s, br, 1 H),
7.06 (m, 2 H), 7.20-7.25 (m, 7 H), 7.36-7.40 (m, 6 H). MS
(EI): m/z 477 (M+), 448 (M+ - Et), 432 (M+ - OEt), 404 (M+
- CO2Et), 386 (M+ - CH2Ph), 342 (M+ - CO2CH2Ph), 91
(+CH2Ph, base). HRMS: calcd for C31H27NO4 477.1940, found
3 H), 3.62 (s, 2 H), 4.17-4.22 (m, 2 H), 4.35 (m, 2 H), 4.51 (m,
2 H), 5.20 (s, 1 H), 4.91-5.15 (m, 2 H), 5.90 (s, br, 1 H), 7.20-
7.37 (m, 20 H). MS (CI/NH3): m/z 701 (M+ + NH4, base).
1H NMR Reson a n ces of Dia ster eom er ic 1,4-Dih yd r o-
p yr id in e Der iva tives Resolved by HP LC w ith th e La n -
th a n id e Sh ift Rea gen t Eu (fod )3. Proton NMR chemical
shifts of compounds 16 and 17 were measured in the presence
of 4 mg/mL Eu(fod)3 (europium tris(6,6,7,7,8,8,8,-heptafluoro-
2,2-dimethyl-3,5-octanedionate)). Samples were dissolved at
a concentration of 5 mg/mL CDCl3.
477.1949. [R]20 ) +70.2 ° (c ) 1.14, MeOH).
D
Ch r om a togr a p h ic P u r ity of 9-12, 21, a n d 23. Purity of
diastereomers 9-12 and enantiomers of MRS 1191, com-
pounds 21 (4S) and 23 (4R), was checked by HPLC using SMT
OD-5-60 RP C18 (250 × 4.6 mm) as an analytical column in
two solvent systems. System A consisted of a linear gradient
solvent system of 0.1 M TEAA/CH3CN from 80:20 to 20:80 in
40 min, and flow rate was 1 mL/min. System B consisted of a
linear gradient solvent system of CH3CN/H2O from 60:40 to
80:20 in 30 min, and flow rate was 1 mL/min. Peaks were
detected by UV absorption in the range of 200-400 nm using
a diode array detector. Peaks showed >95% purity for all
compounds in these systems. The retention times of 9, 10, 11,
12, 21, and 23 were 23.83, 23.73, 19.12, 19.14, 20.86, and 20.82
min, respectively, in system A; and 8.92, 8.86, 3.00, 3.00, 10.03,
and 9.96 min, respectively, in system B.
Hyd r olytic Dep r otection of Ch ir a l Com p ou n d s 16 a n d
17. Optically pure diastereomer 16 (10 mg) or 17 (10.1 mg)
was stirred in a mixture of 1 N HCl and THF (1:1) (2 mL) at
room temperature overnight. The solvents were removed under
reduced pressure, and the residue was subjected to preparative
TLC (silica 60, 1000 µm; Analtech, Newark, DE; petroleum
ether-ethyl acetate (1:1)) to give 5.5 mg of 5-benzyl 3-(2′R,3′R)-
(4-benzyloxy-2,3-dihydroxy-1-butyl) 4-(S)-2-methyl-6-phenyl-
4-phenylethynyl-1,4-dihydropyridine-3,5-dicarboxylate (20)
(yield: 58%) or 7.8 mg of 5-benzyl 3-(2′R,3′R)-(4-benzyloxy-
2,3-dihydroxy-1-butyl) 4-(R)-2-methyl-6-phenyl-4-phenyleth-
ynyl-1,4-dihydropyridine-3,5-dicarboxylate (22) (yield: 83%).
1
Com p ou n d 20: H NMR: δ 2.37 (s, 3 H), 3.60 (d, J ) 5.7
Hz, 2 H), 3.92 (m, 1 H), 3.99 (m, 1 H), 4.17 (m, 1 H), 4.50 (m,
1 H), 4.51 (s, 2 H), 5.02 (AB, J ) 12.6 Hz, 2 H), 5.15 (s, 1 H),
5.89 (s, br, 1 H), 7.05 (m, 1 H), 7.20-7.39 (m, 19 H). MS (CI/
NH3): m/z 661 (M+ + NH4, base), 644 (M+ + 1).
P r ep a r a t ion of (4R,5R)-(5-Ben zyloxym et h yl-2,2-d i-
m eth yl-1,3-dioxolan -4-yl)m eth an ol (25) (Sch em e 4).24 (4R,-
5R)-(-)-2,3-O-Isopropylidene-D-threitol (24; 1.00 g, 6.16 mmol)
and NaOH (0.444 g, 11 mmol) were stirred in a mixture of
DMSO (6 mL) and water (1 mL) at room temperature for 1 h.
Benzyl chloride (0.937 g, 7.4 mmol) was then added, and
stirring was continued for 12 h. The reaction mixture was
diluted with brine (20 mL) and extracted with ether (3 × 60
mL). The ether extract was washed with water and dried over
MgSO4. The solvent was removed, and the residue was purified
by preparative TLC (silica 60, 2000 µm; Analtech, Newark,
DE; petroleum ether-ethyl acetate (3:1)) to give 1.00 g of
1
Com p ou n d 22: H NMR: δ 2.37 (s, 3 H), 3.59 (d, J ) 4.8
Hz, 2 H), 3.91 (m, 1 H), 4.02 (m, 1 H), 4.23 (m, 1 H), 4.40 (m,
1 H), 4.51 (s, 2 H), 5.02 (AB, J ) 12.6 Hz, 2 H), 5.16 (s, 1 H),
5.90 (s, br, 1 H), 7.07 (m, 1 H), 7.19-7.39 (m, 19 H). MS (CI/
NH3): m/z 404 (M+ - CO2CH2CH(OH)CH(OH)CH2OCH2Ph).
P r ep a r a tion of 3-(2′R)-(2,3-Dih yd r oxy-1-p r op yl) 5-Eth -
yl 4-(R a n d S)-2-Meth yl-6-p h en yl-4-p h en yleth yn yl-1,4-
d ih yd r op yr id in e-3,5-d ica r boxyla te (18) by Acid ic Hy-
d r olysis (Sch em e 3). A mixture of diastereomers 8 (50 mg,
0.1 mmol) was stirred in a mixture of 1 N HCl and THF (1:1)
(4.0 mL) at room temperature for 4 h. The solvents were
removed, and the residue was diluted with ethyl acetate (10
mL). The organic layer was washed with water (5 mL × 2),
dried over MgSO4, and evaporated to give a residue, which
was purified by preparative TLC (silica 60, 1000 µm; Analtech,
Newark, DE; petroleum ether-ethyl acetate (1:1)) to give 35
mg (yield: 76%) of compound 18. The analytical data are
identical with its pure diastereomers 11 and 12.
1
product 25, yield: 64%. H NMR: δ 1.42 (s, 6 H), 3.56 (dd, J
) 6.0, 3.9 Hz, 1 H), 3.66-3.72 (m, 2 H), 3.78 (dd, J ) 7.8, 3.9
Hz, 1 H), 3.95 (m, 1 H), 4.06 (m, 1 H), 4.59 (s, 2 H), 7.29-7.39
(m, 5 H). MS (CI/NH3): m/z 270 (M+ + NH4, base).
P r ep a r a t ion of (4R,5R)-(5-Ben zyloxym et h yl-2,2-d i-
m eth yl-1,3-dioxolan -4-yl)m eth yl Acetoacetate (14)(Sch em e
4). (4R,5R)-(5-Benzyloxymethyl-2,2-dimethyl-1,3-dioxolan-4-
yl)methanol (25; 1.00 g, 3.96 mmol) and 2,2,6-trimethyl-4H-
1,3-dioxin-4-one (0.677 g, 4.76 mmol) in toluene (3 mL) were
heated at 90 °C in a sealed tube overnight. After cooling to
room temperature, the solvent was removed under reduced
pressure, and the residue was purified by preparative TLC
(silica 60, 2000 µm; Analtech, Newark, DE; petroleum ether-
ethyl acetate (3:1)) to afford 0.911 g of compound 14, yield:
68%. 1H NMR: δ 1.42 (s, 6 H), 2.26 (s, 3 H), 3.47 (s, 2 H), 3.61
(m, 2 H), 4.00-4.12 (m, 2 H), 4.18 (dd, J ) 6.0, 5.7 Hz, 1 H),
4.40 (dd, J ) 7.0, 9.2 Hz, 1 H), 4.59 (s, 2 H), 7.30-7.36 (m, 5
H). MS (CI/NH3): m/z 354 (M+ + NH4, base), 337 (M+ + 1).
P r ep a r a tion of 3,5-Dieth yl 4-(R a n d S)-2-Meth yl-6-
p h en yl-4-p h en yleth yn yl-1,4-d ih yd r op yr id in e-3,5-d ica r -
boxyla te (19) by a Tr a n sester ifica tion Rea ction (Sch em e
3). Compound 18 (30 mg, 0.065 mmol) and sodium hydroxide
(4 mg, 1.5 equiv) were stirred in 95% ethanol (1 mL) at room
temperature for overnight. The solvents were removed under
reduced pressure, and the residue was purified by preparative
TLC (silica 60, 1000 µm; Analtech, Newark, DE; petroleum
ether-ethyl acetate (4:1)) to give 6 mg (yield: 22%) of 19,
which has the same analytical data with 28 (Table 3).3
P r ep a r a tion of Op tica lly P u r e 1,4-Dih yd r op yr id in es
X-r a y Str u ctu r a l Deter m in a tion of Com p ou n d 9. Small
needle crystals of compound 9 were grown by vapor diffusion
from methanol/water solution. X-ray diffraction studies were

3064 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
J iang et al.
performed with Siemens P4 diffractometer (Mo KR radiation).
Crystals of C30H31NO6 are monoclinic (P21) with lattice pa-
rameters a ) 11.059(2) Å, b ) 8.212(2) Å, c ) 15.629(3) Å, â
) 104.46(1)°, Z ) 2. Intensities were measured with variable
speed ω/20 scans from a crystal size of 0.80 × 0.12 × 0.06 mm.
No significant variation was observed in the intensities of three
standard reflections monitored at regular intervals; no cor-
rections were necessary for absorption.
The structure was determined by direct methods [1] and
refined on F2 by full-matrix least-squares method [2] from 1895
unique reflections (20max ) 45°, Rint ) 0.029). Isotropic
hydrogen atoms were refined with a riding model; all other
atoms were assigned anisotropic displacement parameters.
23-A3 receptor complex. Cross-docking allowed possible ligand-
induced rearrangements of the 7TM bundle to be explored by
sampling 7TM conformations in the presence of the docked
ligands. Small translations and rotations were applied to each
helix relative to its original position until a new lower-energy
geometry was obtained. These manual adjustments were
followed by 25 ps of molecular dynamics (MD module of
Macromodel) performed at a constant temperature of 300 K
using a time step of 0.001 ps and a dielectric constant ) 4.0.
This procedure was followed by another sequence of CG energy
minimization to a gradient threshold of <0.1 kcal/mol/Å.
Energy minimization of the complexes was performed using
the AMBER all-atom force field in MacroModel.
Abbr evia tion s: [125I]AB-MECA, [125I]N6-(4-amino-3-iodo-
benzyl)-5′-N-methylcarbamoyladenosine; CGS 21680, 2-[[4-(2-
carboxyethyl)phenyl]ethylamino]-5′-N-ethylcarbamoyladeno-
sine; DMAP, N,N-(dimethylamino)pyridine; DMSO, dimethyl
sulfoxide; Eu(fod)3, europium tris(6,6,7,7,8,8,8,-heptafluoro-2,2-
dimethyl-3,5-octanedionate); HEK cells, human embryonic
kidney cells; IB-MECA, N6-(3-iodobenzyl)-5′-N-methylcarbam-
oyladenosine; Ki, equilibrium inhibition constant; MRS 1097,
3,5-diethyl 2-methyl-6-phenyl-4-[2-phenyl-(E)-vinyl]-1,4-(()-
dihydropyridine-3,5-dicarboxylate; MRS 1191, 5-benzyl 3-ethyl
2-methyl-6-phenyl-4-phenylethynyl-1,4-(()-dihydropyridine-
3,5-dicarboxylate; R-PIA, (R)-N6-phenylisopropyladenosine;
SAR, structure-activity relationship; TBAF, tetrabutyl-
ammonium fluoride; Tris, tris(hydroxymethyl)aminomethane;
TEAA, triethylamine-acetic acid buffer.
The final conventional R ) 5.50% on
F values of 1345
reflections having F0 > 4σ(F0).
P h a r m a cologica l Meth od s. Bin d in g a t Ad en osin e Re-
cep tor s. Binding of [3H](R)-N6-phenylisopropyladenosine ([3H]-
R-PIA; Amersham, Chicago, IL) to A1 receptors from rat
cerebral cortex membranes,25 binding of [3H]-2-[[4-(2-carboxy-
ethyl)phenyl]ethylamino]-5′-N-ethylcarbamoyladenosine ([3H]-
CGS 21680; DuPont NEN, Boston MA) to A2A receptors from
rat striatal membranes,26 and binding of [125I]N6-(4-amino-3-
iodobenzyl)-5′-N-methylcarbamoyladenosine ([125I]AB-MECA;
Amersham, Chicago, IL) to membranes prepared from HEK-
293 cells stably expressing the human A3 receptor27,28 (Receptor
Biology, Inc., Baltimore MD) were performed as described.
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.
Su p p or tin g In for m a tion Ava ila ble: Spatial coordinates
of the X-ray structure of 9. This information is available free
of charge via the Internet at http://pubs.acs.org.
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 equation30 and Kd values
of 1.0 and 14 nM for [3H]R-PIA and [3H]CGS 21680, respec-
tively, and 0.59 nM for binding of [125I]AB-MECA at human
A3 receptors.
Refer en ces
(1) van Rhee, A. M.; J iang, J . L.; Melman, N.; Olah, M. E.; Stiles,
G. L.; J acobson, K. A. Interaction of 1,4-dihydropyridine and
pyridine-derivatives with adenosine receptors - selectivity for
A3 receptors. J . Med. Chem. 1996, 39, 2980-2989.
(2) Van Muijlwijk-Koezen, J . E.; Timmerman, H.; Link, R.; Van der
Goot, H.; IJ zerman, A. P. A novel class of adenosine A3 receptor
ligands. II. Structure affinity profile of a series of isoquinoline
and quinazolinecompounds. J . Med. Chem. 1998, 41, 3994-4000.
(3) J iang, J .-l.; van Rhee, A. M.; Chang, L.; Patchornik, A.; Evans,
P.; Melman, N.; J acobson, K. A. Structure activity relationships
of 4-phenylethynyl-6-phenyl-1,4-dihydropyridines as highly se-
lective A3 adenosine receptor antagonists. J . Med. Chem. 1997,
40, 2596-2608.
(4) Karton, Y.; J iang, J . L.; J i, X. D.; Melman, N.; Olah, M. E.; Stiles,
G. L.; J acobson, K. A. Synthesis and biological-activities of
flavonoid derivatives as as adenosine receptor antagonists. J .
Med. Chem. 1996, 39, 2293-2301.
(5) Kim, Y.-C.; de Zwart, M.; Chang, L.; Moro, S.; von Frijtag Drabbe
Ku¨nzel, J . K.; Melman, N.; IJ zerman, A. P.; J acobson, K. A.
Derivatives of the triazoloquinazoline adenosine antagonist
(CGS15943) having high potency at the human A2B and A3
receptor subtypes. J . Med. Chem. 1998, 41, 2835-2841.
(6) J acobson, M. A.; Chakravarty, P. K.; J ohnson, R. G.; Norton, R.
Novel selective nonxanthine selective A3 adenosine receptor
antagonists. Drug Devel. Res. 1996, 37, 131.
(7) Li, A.-H.; Moro, S.; Melman, N.; J i, X.-d.; J acobson, K. A.
Structure activity relationships and molecular modeling of 3,5-
diacyl-2,4-dialkylpyridine derivatives as selective A3 adenosine
receptor antagonists. J . Med. Chem. 1998, 41, 3186-3201.
(8) Beaven, M. A.; Ramkumar, V.; Ali, H. Adenosine-A3 receptors
in mast-cells. Trends Pharmacol. Sci. 1994, 15, 13-14.
(9) Knight, D.; Zheng, X.; Rocchini, C.; J acobson, M. A.; Bai, T.;
Walker, B. Adenosine A3 receptor stimulation inhibits migration
of human eosinophils. J . Leukocyte Biol. 1997, 62, 465-468.
(10) von Lubitz, D. K. J . E.; Lin, R. C. S.; Popik, P.; Carter, M. F.;
J acobson, K. A. Adenosine A3 receptor stimulation and cerebral
ischemia. Eur. J . Pharmacol. 1994, 263, 59-67.
(11) J acobson, K. A. A3 adenosine receptors: Novel ligands and
paradoxical effects. Trends Pharmacol. Sci. 1998, 19, 184-191.
(12) Triggle, D. J . Drugs acting on ion channels and membranes. In
Comprehensive Medicinal Chemistry; Emmett, J . C., Ed.; Per-
gamon Press: London, 1985; Vol. 3, pp 1047-1099.
Molecu la r Mod elin g. All calculations were performed on
a Silicon Graphics Indigo 2 R8000 workstation. All (4R)- and
(4S)-enantiomer models of 1,4-dihydropyridine derivatives
were constructed using the “Sketch Molecule” of SYBYL
6.4.2.32 Semiempirical molecular orbital calculations were done
using the AM1 Hamiltonian33 as implemented in MOPAC 6.034
(keywords: PREC, GNORM ) 0.1, EF, MMOK if necessary).
The three-dimensional human A3 receptor model was built
and optimized using SYBYL 6.4.2 and Macromodel 5.0,35
respectively, based on the approach described by Moro et al.29,31
Briefly, the seven transmembrane helical domains were
identified with the aid of Kyte-Doolittle hydrophobicity36 and
36
Emini surface probability parameters. The helices were built
and energy-minimized for each transmembrane sequence. The
minimized helices were then grouped together to form a helical
bundle matching the overall characteristics of the electron
density map of rhodopsin. The helical bundle was energy-
minimized using the AMBER37 all-atom force field, until the
rms value of the conjugate gradient (CG) was <0.1 kcal/mol/
Å. A fixed dielectric constant ) 4.0 was used throughout these
calculations.
Compounds 21 and 23 were each rigidly docked into the
helical bundle using graphical manipulation with continuous
energy monitoring (Dock module of SYBYL). The manually
docked local energy-minimized receptor-ligand complexes
were subjected to an additional CG minimization run of 300
steps. Partial atomic charges for the ligands were taken from
the MOPAC output files. A fixed dielectric constant ) 4.0 was
used throughout the docking calculations. We have recently
introduced the cross-docking procedure to obtain energetically
refined structures of P2Y1 receptor-ligand complexes.38 We
applied this technique to predict the structure of both 21- and
(13) Coburn, R. A.; Wierzba, M.; Suto, M. J .; Solo, A. J .; Triggle, A.
M.; Triggle, D. J . 1,4-Dihydropyridine antagonist activities at
the calcium channel: a quantitative structure-activity relation-
ship approach. J . Med. Chem. 1988, 31, 2103-2107.

6-Phenyl-4-phenylethynyl-1,4-dihydropyridines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3065
(14) Wong, W. C.; Chiu, G.; Wetzel, J . M.; Marzabadi, M. R.;
Nagarathnam, D.; Wang, D.; Fang, J .; Miao, S. W.; Hong, X.;
Forray, C.; Vaysse, P. J .; Branchek, T. A.; Gluchowski, C.; Tang,
R.; Lepor, H. Identification of a dihydropyridine as a potent R1a
adrenoceptor-selective antagonist that inhibits phenylephrine-
induced contraction of the human prostate. J . Med. Chem. 1998,
41, 2643-2650.
(15) Sunkel, C. E.; de Casa-J uana, M. F.; Santos, L.; Gomez, M. M.;
Villarroya, M.; Gonzalez-Morales, M. A.; Priego, J . G.; Ortega,
M. P. 4-Alkyl-1,4-dihydropyridines derivatives as specific PAF-
acether antagonists. J . Med. Chem. 1990, 33, 3205-3210.
(16) Lopez, M. G.; Fonteriz, R. I.; Gandia, L.; de la Fuente, M.;
Villarroya, M.; Garcia-Sancho, J .; Garcia, A. G. The nicotinic
acetylcholine receptor of the bovine chromaffin cell, a new target
for dihydropyridines. Eur. J . Pharmacol. 1993, 247, 199-207.
(17) Goldmann, S.; Stoltefuss, J . 1,4-Dihydropyridines: Effects of
Chirality and Conformation on the Calcium Antagonist and
Calcium Agonist Activities. Angew. Chem., Int. Ed. Engl. 1991,
30, 1559-1578.
(18) Ho¨llt, V.; Kouba, M.; Dietel, M.; Vogt, G. Stereoisomers of
calcium antagonists which differ markedly in their potencies as
calcium blockers are equally effective in modulating drug
transport by P-glycoprotein. Biochem. Pharmacol. 1992, 43,
2601-2608.
(19) Rovnyak, G. C.; Kimball, S. D.; Beyer, B.; Cucinotta, G.; DiMarco,
J . D.; Gougoutas, J .; Hedberg, A.; Malley, M.; McCarthy, J . P.;
Zhang, R.; Moreland, S. Calcium entry blockers and activators:
conformational and structural determinants of dihydropyrimi-
dine calcium channel modulators. J . Med. Chem. 1995, 38, 119-
129.
(20) Wenzel, T. J .; Bettes, T. C.; Sadlowski, J . E.; Sievers, R. E. New
Binuclear Lanthanide NMR Shift Reagents Effective for Aro-
matic Compounds. J . Am. Chem. Soc. 1980, 102, 5903-5904.
(21) Sheldrick, G. M. SHELX-86. A Computer Program for Crystal
Structure Determination; University of Gottingen: Gottingen,
1986.
(22) Sheldrick, G. M. SHELX-93. Program for the Refinement of
Crystal Structures; University of Gottingen: Gottingen, 1993.
(23) Suemune, H.; Harabe, T.; Sakai, K. Syntheses of Unsaturated
Trihydroxy C-18 Fatty Acids Isolated from Rice Plants Suffering
from Rice Blast Disease. Chem. Pharm. Bull. 1988, 36, 3632-
3637.
(24) Schwabe, U.; Trost, T. Characterization of adenosine receptors
in rat brain by (-) [3H]N6-phenylisopropyladenosine. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1980, 313, 179-187.
(25) J arvis, M. F.; Schutz, R.; Hutchison, A. J .; Do, E.; Sills, M. A.;
Williams, M. [3H]CGS 21680, an A2 selective adenosine receptor
agonist directly labels A2 receptors in rat brain tissue. J .
Pharmacol. Exp. Ther. 1989, 251, 888-893.
(26) Olah, M. E.; Gallo-Rodriguez, C.; J acobson, K. A.; Stiles, G. L.
[
125I]AB-MECA,
a high affinity radioligand for the rat A3
adenosine receptor. Mol. Pharmacol. 1994, 45, 978-982.
(27) J i, X.-d.; von Lubitz, D.; Olah, M. E.; Stiles, G. L.; J acobson, K.
A. Species differences in ligand affinity at central A3-adenosine
receptors. Drug Devel. Res. 1994, 33, 51-59.
(28) Moro, S.; Li, A. H.; J acobson, K. A. Molecular modeling studies
of human A3 adenosine receptor antagonists: structural homol-
ogy and receptor docking. J . Chem. Inf. Comput. Sci. 1998, in
press.
(29) Cheng, Y. C.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes
50% inhibition (IC50) of an enzyme reaction. Biochem. Pharma-
col. 1973, 22, 3099-3108.
(30) Li, A.-H.; Moro, S.; Forsyth, N.; Melman, N.; J i, X.-d.; J acobson,
K. A. Synthesis, CoMFA analysis, and receptor docking of 3,5-
diacyl-2,4-dialkylpyridine derivatives as selective A3 adenosine
receptor antagonists. J . Med. Chem. 1999, 42, 706-721.
(31) The program SYBYL 6.3 is available from TRIPOS Associates,
St. Louis, MO; 1993.
(32) Dewar, M. J . S. E.; Zoebisch, G.; Healy, E. F. AM1: A New
General Purpose Quantum Mechanical Molecular Model. J . Am.
Chem. Soc. 1985, 107, 3902-3909.
(33) MOPAC 6.0 is available from Quantum Chemistry Program
Exchange.
(34) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W.
C. MacroModel-An Integrated Software System for Modeling
Organic and Bioorganic Molecules using Molecular Mechanics.
J . Comput. Chem. 1990, 11, 440-450.
(35) Kyte, J .; Doolittle, R. F. A simple method for displaying the
hydrophobic character of a protein. J . Mol. Biol. 1982, 157, 105-
132.
(36) Weiner, S. J .; Kollman, P. A.; Nguyen, D. T.; Case, D. A. An
all-atom force field for simulation of protein and nucleic acids.
J . Comput. Chem. 1986, 7, 230-252.
(37) Moro, S.; Guo, D.; Camaioni, E.; Boyer, J . L.; Harden, K. T.;
J acobson, K. A. Human P2Y1 receptor: molecular modeling and
site-directed mutagenesis as tools to identify agonist and
antagonist recognition sites. J . Med. Chem. 1998, 41, 1456-
1466.
(38) Li, A.-H.; Chang, L.; J i, X.-d.; Melman, N.; Olah, M.; Stiles, G.;
J acobson, K. A. Functionalized Congeners of 1,4-Dihydro-
pyridines as Molecular Probes for A3 Adenosine Receptors.
Bioconjugate Chem. 1999, 10, 667-677.
J M980688E