Structure-activity relationships of 4-(phenylethynyl)-6-phenyl-1,4- dihydropyridines as highly selective A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm970091j

4-(Phenylethynyl)-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A₃ adenosine receptors, with K(i) values in a radioligand binding assay vs [¹²⁵I]AB-MECA (N⁶(4-amino-3-iodobenzyl)-5'- (N-methylcarbamoyl)adenosine) in the submicromolar range. In this study, structure-activity relationships at various positions of the dihydropyridine ring (the 3- and 5-acyl substituents, the 4-aryl substituent, and 1-methyl group) were probed synthetically. Using the combined protection of the 1- ethoxymethyl and the 5-[2-(trimethylsilyl)ethyl] ester groups, a free carboxylic acid was formed at the 5-position allowing various substitutions. Selectivity of the new analogues for cloned human A₃ adenosine receptors was determined vs radioligand binding at rat brain A₁ and A(2A) receptors. Structure-activity analysis at adenosine receptors indicated that pyridyl, furyl, benzofuryl, and thienyl groups at the 4-position resulted in, at most, only moderate selectivity for A₃ adenosine receptors. Ring substitution (e.g., 4-nitro) of the 4-phenylethynyl group did not provide enhanced selectivity, as it did for the 4-styryl-substituted dihydropyridines. At the 3-position of the dihydropyridine ring, esters were much more selective for A₃ receptors than closely related thioester, amide, and ketone derivatives. A cyclic 3-keto derivative was 5-fold more potent at A₃ receptors than a related open-ring analogue. At the 5-position, a homologous series of phenylalkyl esters and a series of substituted benzyl esters were prepared and tested. (Trifluoromethyl)-, nitro-, and other benzyl esters substituted with electron-withdrawing groups were specific for A₃ receptors with nanomolar K(i) values and selectivity as high as 37000-fold. A functionalized congener bearing an [(aminoethyl)amino]carbonyl group was also prepared as an intermediate in the synthesis of biologically active conjugates.

Full text of DOI:10.1021/jm970091j

2596
J . Med. Chem. 1997, 40, 2596-2608
Str u ctu r e-Activity Rela tion sh ip s of 4-(P h en yleth yn yl)-6-p h en yl-1,4-
d ih yd r op yr id in es a s High ly Selective A3 Ad en osin e Recep tor An ta gon ists
J i-long J iang, A. Michiel van Rhee, Louis Chang, Abraham Patchornik,† Xiao-duo J i, Patricia Evans,
Neli Melman, 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, 1997X
4-(Phenylethynyl)-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human
A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA (N6-
(4-amino-3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine) in the submicromolar range. In this
study, structure-activity relationships at various positions of the dihydropyridine ring (the 3-
and 5-acyl substituents, the 4-aryl substituent, and 1-methyl group) were probed synthetically.
Using the combined protection of the 1-ethoxymethyl and the 5-[2-(trimethylsilyl)ethyl] ester
groups, a free carboxylic acid was formed at the 5-position allowing various substitutions.
Selectivity of the new analogues for cloned human A3 adenosine receptors was determined vs
radioligand binding at rat brain A1 and A2A receptors. Structure-activity analysis at adenosine
receptors indicated that pyridyl, furyl, benzofuryl, and thienyl groups at the 4-position resulted
in, at most, only moderate selectivity for A3 adenosine receptors. Ring substitution (e.g., 4-nitro)
of the 4-phenylethynyl group did not provide enhanced selectivity, as it did for the 4-styryl-
substituted dihydropyridines. At the 3-position of the dihydropyridine ring, esters were much
more selective for A3 receptors than closely related thioester, amide, and ketone derivatives.
A cyclic 3-keto derivative was 5-fold more potent at A3 receptors than a related open-ring
analogue. At the 5-position, a homologous series of phenylalkyl esters and a series of substituted
benzyl esters were prepared and tested. (Trifluoromethyl)-, nitro-, and other benzyl esters
substituted with electron-withdrawing groups were specific for A3 receptors with nanomolar
Ki values and selectivity as high as 37000-fold. A functionalized congener bearing an
[(aminoethyl)amino]carbonyl group was also prepared as an intermediate in the synthesis of
biologically active conjugates.
Medicinal chemists are currently developing agonists
and antagonists that interact selectively with receptors
for adenosine, of which A1, A2A, A2B, and A3 subtypes
are known.1 Relatively recently identified through
cloning,2,3 the A3 adenosine receptor has provided new
therapeutic opportunities in the adenosine field, due to
its unique biological effects.4 Activation of A3 receptors
would require relatively high physiological concentra-
tions of adenosine; the Ki value of adenosine in binding
to the rat A3 receptor has been estimated to be ∼1 µM
vs 10 and 30 nM at rat A1 and A2A receptors, respec-
tively.4 Since activation may occur only under condi-
tions of severe stress, the physiological role of A3
receptors may be very different from that of A1 and A2A
subtypes, which are likely to partially activated by
adenosine under basal conditions. The A3 receptor has
a unique structure-activity relationship (SAR) profile
and tissue distribution.4 Activation of the A3 receptor
has been shown to stimulate phospholipases C5 and D6
and to inhibit adenylate cyclase.1
leukemia cell line and in human blood eosinophils in
response to a high concentration (g10 µM) of A3 selec-
tive agonists developed in our laboratory.7,8 In cultured
chick cardiac myocytes, a brief prior exposure to nano-
molar concentrations of the A3 receptor agonist 2-chloro-
N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-
IB-MECA) protected cells from damage induced by
subsequent hypoxia,9 through a phenomenon termed
“preconditioning.” High concentrations of the same
agonist induce apoptosis in rat cardiac myocytes.10 The
first cytoprotective effects of an A3 agonist were shown
following its chronic administration in gerbils in a model
of stroke, in which the agonist was highly cerebropro-
tective and depressed nitric oxide synthase.11,12 Acute
presence of the agonist during the ischemia exacerbated
damage. In astroglial cultures, A3 agonists induced
differentiation and protection at nanomolar concentra-
tions, while promoting cell death at high concentra-
tions.13 There may be an involvement of A3 receptors
in cancer.14 An A3 agonist inhibited the release of
potentially damaging TNF-R in activated macrophages;
thus A3 agonists may be protective in models of inflam-
mation.15
The varied effects of A3 receptor agonists appear to
be dual and opposite, i.e., either cytoprotective or
cytotoxic, depending on the level of receptor activation
and the system studied. Apoptosis (programmed cell
death) has been shown to occur in the HL-60 human
A3 adenosine receptor antagonists, although only
recently introduced,16-20 were previously hypothesized
to act as potential anti-asthmatic,21 anti-inflammatory,21
or cerebroprotective agents.11 The most promising leads
for A3 receptor antagonists have appeared recently
among chemically diverse non-xanthine heterocycles
(Figure 1). For example, the 1,4-dihydropyridines,
* Address correspondence to: Dr. K. A. J acobson, Chief, Molecular
Recognition 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.
† Permanent address: Poly Chem PCJ , Box 271, Ness-Ziona, Israel.
X Abstract published in Advance ACS Abstracts, J uly 1, 1997.
S0022-2623(97)00091-5 This article not subject to U.S. Copyright. Published 1997 by the American Chemical Society

SAR of 4-(Phenylethynyl)-6-phenyldihydropyridines
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2597
F igu r e 1. Structures of key A3 adenosine receptor selective antagonists reported in 1996. Ki values (µM) are reported in refs
16-20.
known commonly as potent blockers of L-type calcium
channels that are used widely in treating coronary heart
disease, have provided leads for designing adenosine
antagonists, particularly with selectivity for the A3
receptor subtype.17,18 In the present study and in two
previous studies, we have used the 1,4-dihydropyridine
nucleus as a template for probing structure-activity
relationships at adenosine receptors. By careful struc-
tural modification, it has been possible to select for
affinity at adenosine receptors and deselect for affinity
at L-type Ca2+ channels. For example, a dihydropyri-
dine derivative, 3,5-diethyl 2-methyl-6-phenyl-4-[2-
phenyl-(E)-vinyl]-1,4-(()-dihydropyridine-3,5-dicarbox-
ylate (MRS 1097, 1; Figure 1),17 has been found to
inhibit binding of radioligand at the human A3 receptor
with an affinity of 108 nM, while the same derivative
was inactive at ion channels and other receptor sites.
Furthermore, MRS 1097 antagonized the effects of IB-
MECA, an A3 receptor selective agonist, on inhibition
of adenylate cyclase via the cloned rat A3 receptor.
Affinity and selectivity for the human A3 receptor within
this series was further enhanced in the trisubstituted
analogue MRS 1191, 3-ethyl 5-benzyl 2-methyl-6-phen-
yl-4-(phenylethynyl)-1,4-(()-dihydropyridine-3,5-dicar-
boxylate (2; Figure 1,).18 Other A3 selective antagonists
that have been recently reported include a flavonoid
derivative (MRS 1067, 3),16 a derivative of the triazolo-
quinazoline CGS 15943 (MRS 1220, 4),20 a triazolonaph-
thyridine (L-249313, 5),19 and a thiazolopyrimidine (L-
268605, 6).19 Although having high affinity, L-249313
appears to bind noncompetitively to the human A3
receptor.
4-aryl substituent, and 1-methyl group). We have
discovered that substitutions of the 5-ester group pro-
vide the greatest versatility for improving A3 receptor
selectivity dramatically and achieving nanomolar po-
tency. The efficient synthesis of these new analogues
has been accomplished through a general approach
using orthogonal protecting groups at the N1 and
5-ester positions.
Resu lts
Syn th esis. The structures of the 1,4-dihydropy-
ridines and related derivatives (1, 2, and 7-43) tested
for affinity in radioligand binding assays at adenosine
receptors are shown in Table 1. The dihydropyridine
analogues were prepared by methods outlined in Schemes
1-4.
As in the previous studies,17,18 the basic synthesis of
the 1,4-dihydropyridine nucleus consisted of the Hantz-
sch condensation (reviewed in ref 22), an example of
which is shown in Scheme 1. This method involves a
three-component reaction of a 3-amino-2-propenoate
ester, such as ethyl 2-aminocrotonate (44), an aldehyde,
such as propiolaldehyde (45), and a benzoylacetate ester,
such as 46, that were dissolved in ethanol and refluxed.
In some reactions, other â-keto esters27 were used to
synthesize analogues having groups other than phenyl
in the 6-position. For example, the 6-trifluoromethyl
group was introduced in compound 4 using ethyl tri-
fluoroacetoacetate instead of 46. Good yields of 1,4-
dihydropyridines containing the 6-phenyl group were
obtained using a 72 h reaction time. Alternately, the
6-phenyl group was introduced using ethyl 3-aminocin-
namate, 50, prepared as reported previously. An ex-
ample of the latter approach is given in Scheme 2. This
allowed the introduction of the 3-keto group in the 1,4-
dihydropyridine, of which both open-ring, e.g., 38, and
In the present study, we have explored the structure-
activity relationships of known selective antagonists,
MRS 1097 and MRS 1191, containing both subtle and
drastic structural changes at various positions of the
dihydropyridine ring (its 3- and 5-acyl substituents, the

2598 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
J iang et al.
Sch em e 1. Synthesis of 5-Ester-Substituted 1,4-Dihydropyridines Using the Hantzsch Reaction and an Orthogonal
Protecting Schemea
a
R4 ) phenylethynyl.
Sch em e 2. Synthesis of Open-Chain and Ring-Constrained 3-Keto-Substituted 1,4-Dihydropyridines Using a
Variation of the Hantzsch Reaction
ring-constrained, e.g., 39, examples were prepared,
using 2,4-pentanedione or 1,3-cyclohexanedione,23 re-
spectively.
yield (<20%) by this method. Alternately, the carboxylic
acid could be esterified in very high yield (>90%) under
alkylating conditions (alkyl bromide with potassium
carbonate in acetone). Thus, esterification by means of
alkylation was preferred over the condensation reaction,
since the latter was sluggish, presumably due to the
steric hindrance at the carboxylic acid. The 1-ethoxy-
methyl group was finally removed upon heating at 45
°C for 1 h with 1 N HCl in acetone.
The specific example shown in Scheme 1 incorporates
a protecting group at the 5-ester position that allows
for subsequent substitution of this ester.28 Using the
combined protection of the 1-position with the ethoxym-
ethyl group24 and the 5-[2-(trimethylsilyl)ethyl] ester,25
a free carboxylic acid was formed at the 5-position
allowing subsequent esterification by means of alkyla-
tion or condensation (Scheme 1). These protecting
groups could then be removed separately to allow
selective derivatization of the carboxylic acid. The 5-[2-
(trimethylsilyl)ethyl] ester group was introduced at the
stage of the Hantzsch condensation, using the appropri-
ate 3-ketopropionate ester 46. The silyl group was later
deprotected using tetrabutylammonium fluoride in tet-
rahydrofuran. The carboxylic acid could then be con-
verted to an amide, a thioester, or an ester using a
carbodiimide-condensing reagent in DMF or methylene
chloride, in the presence of catalytic 4-(N-dimethylami-
no)pyridine, although esters were obtained in only low
Ring substitution of the 4-phenylethynyl group of 2
was accomplished through the preparation of the ap-
propriate aldehyde intermediates 55. These substituted
phenylpropargyl aldehyde derivatives were prepared as
shown in Scheme 3 using a modified application of the
Stephans-Castro reaction.26 A substituted iodoben-
zene, 52, was coupled to propiolaldehyde diethyl acetal,
53, using copper(I) and palladium(II) catalysis to give
the protected phenylpropiolaldehyde 54. Deprotection
of the diethyl acetal was accomplished using formic acid
in hexanes, since hydrolysis in aqueous medium led to
decomposition.

SAR of 4-(Phenylethynyl)-6-phenyldihydropyridines
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2599
Sch em e 3. Synthesis of Phenylpropiolaldehyde
Intermediates and Preparation of Ring-Substituted
4-(Phenylethynyl)dihydropyridines Using the
Stephans-Castro Methodology26 a
previously reported for the purpose of introducing a
prosthetic group in peptides and other derivatized
receptor ligands for facile radiofluorination reactions.28
P h a r m a cology. van Rhee et al.17 demonstrated that
A3 receptor selectivity could be achieved in 1,4-dihydro-
pyridines through a combination of large substituents
at the 4- and 6-positions. 4-Styryl or 4-phenylethynyl
groups together with 6-phenyl groups are particularly
suited for this pharmacological profile. In a second
study by J iang et al.,18 we have explored the structure-
activity relationships at the 6-position. In the present
study we have introduced a variety of heterocyclic
aromatic groups at the 4-position and modified other
positions. Compound 7 was reported in van Rhee et al.17
showing that the 4-phenyl group is tolerated in binding
at adenosine receptors; although lacking the 6-phenyl
group it does not provide A3 receptor selectivity. The
comparable affinity of the phenyl, 7, and thienyl, 8,
derivatives at A1 and A3 receptors indicates that other
aryl groups are acceptable at the 4-position. Unlike the
4-phenyl analogue 7, which is nonselective, elongating
the connection to the phenyl group with an acetylene
linkage in 9 provides A3 receptor selectivity. The
4-phenylacetylene substituent has been combined with
other favorable modifications of the dihydropyridines18
to result in analogues with as great as 1700-fold
selectivity for A3 vs A1 receptors.
a
X ) 3-CH3 or 4-NO2.
Most of the derivatives in Table 1 represent a classical
medicinal chemical approach to drug design. An alter-
nate approach, called the “functionalized congener ap-
proach”,35 in which an easily derivatized functional
group is incorporated at the end of a strategically
designed and attached chain substituent, was also
applied in the present study. Thus, an amine-function-
alized congener, 25, was synthesized as shown in
Scheme 4. The reaction involved alkylation of the
5-carboxylic acid 42 using a benzyl bromide intermedi-
ate, 58. This intermediate was formed in two steps from
p-toluic acid chloride, 56, which reacted with trichloro-
ethanol to form ester 57. The ester was brominated
using N-bromosuccinimide to give 58. After acidic
deprotection of the 1-ethoxymethyl protecting group of
the dihydropyridine 59, the p-(trichloroethyl ester)
group of 24h could be aminolyzed selectively using neat
ethylenediamine at room temperature. The ester groups
at the 3- and 5-positions of the dihydropyridine ring
were much less susceptible to reaction with amines. This
gave directly the desired primary amine congener 25.
An alternate route that proved unsuccessful used in-
stead of 58 an intermediate benzyl bromide already
containing a Boc-protected ethylenediamine moiety,
At the 6-position, substitution of the methyl group
with trifluoromethyl, 10, appeared to reduce the affinity
of the dihydropyridine at A1 and A2A but not A3 receptor
subtypes, resulting in 15-fold selectivity. Since we have
shown that the phenyl group is optimal at the 6-posi-
tion,18 further alkyl or haloalkyl derivatization at this
position was not carried out.
The 4-phenyl group in combination with a 6-phenyl
group, in 11, resulted in A3 receptor selectivity (4.7-fold
vs A1 receptors). Thus, analogues 12-17 were synthe-
sized to probe the effects of aromatic heterocycles
directly attached at the 4-position in combination with
the 6-phenyl group. The 2-, 3-, and 4-pyridyl analogues
12-14 were neither potent nor selective in binding.
Among the positional isomers of the nitrogen in 4-py-
ridyl derivatives, no significant differences were ob-
served at A1 receptors, and the affinity at A3 receptors
varied slightly in the order of 4- > 2- > 3-position.
Sch em e 4. Synthesis of an Amine-Functionalized Congener of the A3 Adenosine Receptor Selective Antagonists Based
on Dihydropyridinesa
a
Reagents: (a) 2,2,2-trichloroethanol; (b) N-bromosuccinimide, benzene; (c) acetone, potassium carbonate; (d) HCl; (e) ethylenediamine,
rt.

2600 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
J iang et al.

SAR of 4-(Phenylethynyl)-6-phenyldihydropyridines
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2601
Other aromatic moieties, such as in the thienyl and furyl
derivatives 15-17, were substituted at the 4-position
resulting in moderate A3 selectivity.
Several analogues of 4-styryl-1,4-dihydropyridines17
were prepared. In the case of compound 17, the
benzofuryl group may be considered a ring-constrained
version of the 4-styryldihydropyridines, such as 1, which
were shown to provide A3 selectivity. Compound 17,
with a 3-fold lower affinity, was less selective for A3
receptors than 1. J iang et al.18 showed that the styryl
ring may be substituted with a nitro, 18, or an amino,
20, group with enhancement of high selectivity for A3
vs A1 receptors. Elongation of the 3-ester group of 18,
resulting in the allyl ester 19, diminished the A3
receptor affinity by 5-fold, yet A3 receptor selectivity
remained relatively high (>300-fold).
In combination with the 6-phenyl substituent, the
4-phenylethynyl group in analogue 21 resulted in
slightly higher A3 receptor potency and selectivity (140-
vs 55-fold) than the 4-styryl group in analogue 1.18 In
comparison to the corresponding 6-trifluoromethyl ana-
logue 10, the affinity of 21 had increased by 44-fold at
A3 receptors, while at A1 and A2A receptors the differ-
ence was not significant. Therefore, ring substitutions
of the 4-phenylethynyl group (4-nitro, 22a , and 3-meth-
yl, 22b) of the 3,5-diethyl ester 21 were prepared in the
hope that these relatively minor changes carried out at
a distance from the dihydropyridine pharmacophore
would be tolerated at A3 receptors. Yet, both of these
modifications, especially 22a , diminished A3 receptor
affinity and selectivity. The effects of ring substitution
of the 4-phenylethynyl group appear to be unlike the
case of the 4-styryl analogues, in which a 4-nitro group
enhanced potency and selectivity for A3 receptors. The
4-nitro substitution of the ring of the 4-phenylethynyl
group of 22a decreased the A3 receptor affinity 48-fold
while decreasing the selectivity vs A1 receptors from
140- to 9-fold.
Appending the 3,5-diethyl ester 21 with an additional
aromatic group in the 5-position, in the form of a benzyl
ester, 2, was found previously to greatly enhance A3
receptor potency and selectivity. There is considerable
flexibility of substitution of the 3-ester group as well as
the 5-ester group in A3 receptor selective dihydropy-
ridines. For example, the 3-benzyl ester 35 is >600-
fold selective, although not as potent as the isomer
5-benzyl ester derivative 2. Thus, in the present study
the structure-activity relationships at the 3- and
5-ester positions were systematically probed.
Substituted 5-benzyl esters (24a -j) and a homologous
series of 5-phenylalkyl esters (2, 27, and 28) were
prepared and tested. The parent benzyl ester 2 was
1300-fold selective for A3 receptors with a Ki value of
31 nM. The next higher homologue, the 2-phenylethyl
derivative 27, displayed a Ki value of 146 nM. The
3-phenylpropyl ester 28 was more potent than 27, with
a Ki value of 76 nM. Since no advantage was apparent
to homologation of the benzyl ester, the ring substitution
of the preferred 5-benzyl ester was examined in detail.
The A3 affinity of the methylbenzyl esters 24a -c varied
in the order m > p, o. A p-(trifluoromethyl)benzyl ester,
24d , was specific for A3 receptors with a Ki value of 18
nM and had an estimated selectivity ratio of greater
than 5000-fold vs either A1 or A2A receptors. A 3-iodo
analogue, 24e, included for its structural similarity to

2602 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
J iang et al.
Ta ble 2. Characterization of Dihydropyridine and Pyridine Derivatives
yielda
(%)
compd
Tm (°C)
formula
C17H21NO4S
MS
335 (EI)
anal.
8
10
11
12
13
14
17
22b
23
24a
24b
24c
24d
24e
24f
24g
24h
24i
25
26
27
28
29
30
31
32a
32b
33
34
36
37
38
39
40
41
42
167-168
oil
C,H,N
b
48
35
17
21
32
26
31
55
17
76
22
28
53
53
34
41
70
58
48
15
81
95
14
38
16
15
17
6
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
21H20NF3O4
24H25NO4
23H24N2O4
23H24N2O4
23H24N2O4
26H25NO5
27H27NO4
32H29NO4
32H29NO4
32H29NO4
407 (CI)
391 (EI)
392 (EI)
392 (EI)
392 (EI)
431 (EI)
429 (EI)
491 (EI)
491 (EI)
491 (EI)
491 (EI)
545 (EI)
603 (EI)
522 (EI)
522 (EI)
652 (EI)
613 (EI)
564 (FAB, M + H)
491 (EI)
491 (EI)
505 (EI)
443 (EI)
521 (CI)
487 (EI)
(EI)
432 (CI, M + H)
387 (CI)
414 (EI)
443 (EI)
414 (EI)
385 (EI)
397 (EI)
531 (CI)
545 (CI)
445 (EI)
472 (EI)
140-145
125-128
137-140
157-163
131-134
118-123
oil
95-100
150-152
139-144
140-144
115-117
146-147
150-152
70-74
179-180
101-105
oil
oil
oil
oil
oil
146-147
oil
72-74
173-175
oil
c
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
d
e
f
g
32H29NO4
32H26F3NO4
31H26INO4
31H26N2O6
31H26N2O6
34H28Cl3NO6
33H25F6NO4
34H33N3O5
32H29NO4
32H29NO4
33H31NO4
28H29NO4
33H31NO5
29H33NSiO4
26H25NO3S
26H25NO3S
24H21NO4‚0.50H2O
26H26N2O3‚0.85H2O
28H29NO4
26H28N2O3‚0.75H2O
25H23NO3
26H23NO3‚0.75H2O
31H37NSiO5
32H39NSiO5
27H27NO5
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,N; Hn
h
i
C,H,N
C,H,N
C,H,N
j
C,H,N
k
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
l
61
29
20
13
27
85
85
85
47
oil
199-200
158-159
oil
oil
oil
m
C,H,N
C,H,N
182-183
oil
43
29H32N2O4
a
b
Purification was achieved by thin layer chromatography (silica gel 60, 1000 µm layer thickness). 10: pure on analytical TLC (silica
gel 60, 250 µm) EtOAc:petroleum ether ) 20:80 (v/v), Rf ) 0.21; CH2Cl2:MeOH ) 40:1 (v/v), Rf ) 0.46; EI calcd 462.1806, found 462.1791.
c 11: pure on analytical TLC (silica gel 60, 250 µm) CHCl3:MeOH ) 40:1 (v/v), Rf ) 0.3; EI calcd 391.1784, found 391.1774. 23 (C32H29NO4):
d
insufficient quantity for CHN, pure on analytical TLC (silica gel 60, 250 µm) EtOAc:petroleum ether ) 20:80 (v/v), Rf ) 0.35; CH2Cl2:
MeOH ) 40:1 (v/v), Rf ) 0.47; EI calcd 491.2096, found 491.2096. e 24a : pure on analytical TLC (silica gel 60, 250 µm) hexanes:EtOAc
) 2:1 (v/v), Rf ) 0.6; EI calcd 491.2097, found 491.2095. f 24b: pure on analytical TLC (silica gel 60, 250 µm) hexanes:EtOAc ) 2:1 (v/v),
g
Rf ) 0.6; EI calcd 491.2097, found 491.2094. 24c: pure on analytical TLC (silica gel 60, 250 µm) hexanes:EtOAc ) 2:1 (v/v), Rf ) 0.6;
h
EI calcd 491.2097, found 491.2109. 25: pure on analytical TLC (silica gel 60, 250 µm) CHCl3:MeOH ) 5:1 (v/v), Rf ) 0.1; FAB (M + H)
i
564.4. 26: pure on analytical TLC (silica gel 60, 250 µm) EtOAc:petroleum ether ) 20:80 (v/v), Rf ) 0.30; CH2Cl2:MeOH ) 40:1 (v/v), Rf
j
) 0.43; EI calcd 491.2096, found 491.2069. 30: pure on analytical TLC (silica gel 60, 250 µm) EtOAc:petroleum ether ) 20:80 (v/v), Rf
k
) 0.41; CH2Cl2:MeOH ) 40:1 (v/v), Rf ) 0.50; EI calcd 521.2202, found 521.2172. 32a : pure on analytical TLC (silica gel 60, 250 µm)
l
EtOAc:petroleum ether ) 20:80 (v/v), Rf ) 0.29; CH2Cl2:MeOH ) 40:1 (v/v), Rf ) 0.41; EI calcd 431.1555, found 431.1541. 40: pure on
analytical TLC (silica gel 60, 250 µm) EtOAc:petroleum ether ) 20:80 (v/v), Rf ) 0.50; CH2Cl2:MeOH ) 40:1 (v/v), Rf ) 0.58; EI calcd
m
531.2441, found 531.2418. 41: pure on analytical TLC (silica gel 60, 250 µm) EtOAc:petroleum ether ) 20:80 (v/v), Rf ) 0.54; CH2Cl2:
n
MeOH ) 40:1 (v/v), Rf ) 0.60; EI calcd 545.2597, found 545.2606. 24i: H calcd, 4.12; found, 4.94.
the N6-(3-iodobenzyl) adenosine derivatives,4 which are
selective agonists at A3 receptors, was not the most
potent analogue yet was >1000-fold selective. The
presence of one or two electron-withdrawing groups on
the benzyl ring, as in 3- and 4-nitrobenzyl esters 24f,g
and the disubstituted ester 24i, resulted in exceptionally
high potency and selectivity for A3 receptors. Compound
24f displayed an A3 receptor selectivity ratio of >11000-
fold, and compound 24g was at least 37000-fold selective
for human A3 receptors vs either A1 or A2A receptors.
The 4-[[(trichloroethyl)oxy]carbonyl] ester 24h was much
less potent at A3 receptors.
Other 5-ester derivatives were prepared and exam-
ined in receptor binding. Branching of the ester chain
was found to have variable effects on receptor affinity.
A (trimethylsilyl)ethyl ester, 31, was highly potent and
selective at A3 receptors, while a 5-tert-butyl ester, 29,
was less well tolerated at A3 receptors. Adding a
methoxy group in the R-configuration to the R-position
of the 2-phenylethyl ester, in 30 (tested as a diastere-
omeric mixture), was also not well tolerated in binding
at A3 receptors.
A 5-thioester derivative, 32a , was not well tolerated
in receptor binding, and the affinity at A1 and A3
receptors was 5- and 7-fold less, respectively, than the
ester 21. The corresponding 3-thioester derivative 32b
was more potent than the 5-thioester at A3 receptors
but was still 3.8-fold less potent than 21. Curiously the
affinity of 32b at A2A receptors was greater than at A1
A functionalized congener bearing an [(aminoethyl)-
amino]carbonyl group, 25, was also prepared as an
intermediate for the synthesis of biologically active
conjugates. Unfortunately, the affinity was much less
than anticipated. At a concentration of 100 nM, no
displacement of the radioligand was observed.

SAR of 4-(Phenylethynyl)-6-phenyldihydropyridines
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2603
Ta ble 3. Affinities of 4-(Phenylethynyl)-6-phenyl-1,4-
dihydropyridine Derivatives in Radioligand Binding Assays at
Rat A3 receptors
receptors. This represents a >20-fold affinity enhance-
ment at the A2A subtype by replacing oxygen with
sulfur.
Ki (µM)
Other structurally simple modifications of the ethyl
ester at the 5-position of the dihydropyridine ring were
also examined. A free carboxylic acid at the 5-position,
33, greatly decreased affinity at A3 but not A1 receptors.
A 5-ethylamide, 34, was nearly as potent as the corre-
sponding ethyl ester 21 at A1 receptors; however, it was
73-fold less potent at A3 receptors. Thus, it appears that
the 5-ester linkage is preferred for dihydropyridines
with high A3 receptor selectivity.
a
compd
rA3
rA1/rA3
2, MRS 1191
1.42 ( 0.19
1.52 ( 0.65
1.69 ( 0.42
3.00 ( 0.58
4.02 ( 1.26
5.07 ( 0.69
3.85 ( 0.92
13.0 ( 2.3
5.07 ( 2.00
1.12 ( 0.05
1.32 ( 0.11
1.38 ( 0.27
28
>60
>50
>30
>20
>10
>20
>7
>10
>80
>70
>70
24b, MRS 1328
24c, MRS 1326
24d , MRS 1329
24e, MRS 1330
24f, MRS 1333
24g, MRS 1334
24h , MRS 1353
24j, MRS 1355
26, MRS 1323
27, MRS 1321
28, MRS 1322
Increasing the size of the 3-position ester was also
examined. Previously, compound 35 was shown to be
highly selective for A3 receptors, suggesting that added
steric bulk on the side opposite the 6-phenyl ring might
be beneficial for selectivity by reducing affinity at the
other subtypes. Nevertheless, the homologation of the
3-ethyl ester of 2, to the propyl ester 26, diminished A3
affinity 2-fold. The effect of chain branching of the
3-ester group was also probed. As for the 5-ester series,
introduction of the bulky tert-butyl group, 36, greatly
decreased the A3 receptor potency and selectivity. Much
like the 5-ester series, the presence of a 3-amide was
not well tolerated at the A3 receptors. Thus, the
3-ethylamide 37 was 6-fold less potent than the corre-
sponding ethyl ester 21 at A1 receptors and ∼30-fold
less potent at A3 receptors. The 3-methyl ketone 38 was
also much less potent and selective at A3 receptors than
the 3-ethyl ester 21. Thus, it appears that both the 3-
and 5-ester linkages in the dihydropyridines are highly
favored in A3 receptor binding. A ring-constrained
analogue, 39, related to the 3-methyl ketone 38 was of
similar affinity vs 38 at A1 and A2A receptors yet 5-fold
more potent at A3 receptors and 28-fold selective vs A1
receptors.
During the synthesis of a number of the above
analogues (Scheme 1), the N1-position was protected
with methoxymethyl, 40, or ethoxymethyl groups, 41-
43. Although this N-alkylation tended to decrease both
water solubility and affinity (e.g., 40 and 41 vs the
corresponding 1-NH derivative 31), several of these
intermediates were tested in binding. Although affinity
generally was decreased, one of these derivatives was
nearly as potent as the N-unprotected form (43 vs 34
at A1 and A3 receptors).
a
Displacement of specific [125I]AB-MECA binding at rat A3
receptors stably expressed in CHO cells2,32 (n ) 3-5).
ligands of exceptionally high selectivity. In addition to
the 4-styryl and the 4-phenylethynyl substituents, vari-
ous other aromatic groups were placed at the 4-position.
Some differences in affinity were observed for the
heterocyclic derivatives 12-17 in comparison to the
4-phenyl derivative 11. At A3 receptors, the furyl and
benzofuryl derivatives 16 and 17 were more potent than
the phenyl derivative, although the selectivity was only
moderate. Ring substitution of the 4-styryl group
maintained high A3 receptor selectivity, but similar
substitution of the 4-phenylethynyl substituent in com-
pounds 22a ,b reduced affinity.
At the 2-position, only one homologue, the 2-ethyl
derivative 23, was prepared and found not to enhance
the affinity at A3 receptors over the 2-methyl derivative.
An effort to produce a ring-constrained analogue, 39,
in which 2- and 3-substituents are joined and which may
be useful for purposes of eventual QSAR molecular
modeling of the dihydropyridines in binding to adenos-
ine receptors, displayed only moderate potency, but this
was likely due to the presence of a 3-keto group rather
than an ester group. Nevertheless, the cyclic 3-keto
derivative 39 was 5-fold more potent at A3 receptors
than the related open-ring analogue, 38.
Functional group replacement at the 3- and 5-posi-
tions produced analogues that were less potent and
selective than the parent 1,4-dihydropyridine 3,5-di-
esters, indicating the requirement for ester groups at
these positions for binding with high affinity to A3
adenosine receptors. At A1 receptors, the ester groups
appeared to be less critical, since their replacement with
3- or 5-ethylamide groups (37 and 34) resulted in only
6- and 3-fold loss of affinity vs 21, respectively. Thioester
derivatives 32a ,b, although similar in electronic struc-
ture to the corresponding ester 21, were less potent in
binding at A3 adenosine receptors.
Affinity at rat A3 receptors of selected compounds is
shown in Table 3. As expected from previous studies
of A3 antagonists, the affinity of the dihydropyridine
derivatives in the rat was considerably less than that
at human A3 receptors. Nevertheless, certain analogues
(24b,c and 26-28) were still moderately selective (>50-
fold) for rat A3 vs rat A1/A2A receptors. The highest
potency at rat A3 receptors was observed for compound
26, with a Ki of 1.12 µM.
A homologous series of arylalkyl esters at the 5-posi-
tion, 2, 27, and 28, indicated the affinity at A3 receptors
varied in the order benzyl > 3-phenylpropyl > 2-phe-
nylethyl. Thus, for achieving selectivity there appeared
to be no advantage to homologation of the benzyl ester
at this position, since the affinity of all three analogues
was very weak at both A1 and A2A receptors. Thus, the
benzyl ester group of 2 remained the most promising
structural lead in the present study, and consequently
the most selective pharmacological probes arose from
ring substitution of the benzyl group. It was possible
Discu ssion
Although highly selective antagonists have been
reported for both A1 and A2A subtypes of adenosine
receptors,1 the development of such ligands for A3
receptors has lagged behind the other subtypes.4 In the
present study, we have probed the SAR of A3 adenosine
receptor selective 1,4-dihydropyridines based on the
17
4-styryl derivative MRS 1097,
1, and the 4-phenyl-
ethynyl derivative MRS 1191, 2,18 and have designed

2604 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
J iang et al.
to vary considerably the electronic and steric properties
surrounding this ester group. Electron-withdrawing
groups, particularly at the para- and meta positions,
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 enhanced. The most selective
compounds (human) in this study were 4-nitrobenzyl >
3-nitrobenzyl > 4-(trifluoromethyl)benzyl > 3,5-dini-
trobenzyl esters. It will be interesting to probe which
region of the human A3 receptor contains an electron-
rich binding site for this benzyl moiety. At rat A3
receptors, the most potent and selective was the 3-propyl
ester 26; however, this compound was not significantly
more potent than 2.
5-positions, especially electron-poor benzyl esters, are
most likely to result in high selectivity as A3 receptor
antagonists.
Exp er im en ta l Section
Syn th esis. 1. Ma ter ia ls. Ethyl 3-aminocrotonate (44),
phenyl propargylaldehyde (45), 2,4-pentanedione (49), and 1,3-
cyclohexanedione (51) were from Aldrich (St. Louis, MO).
Compounds 1, 2, 7, 9, 18, 20, 21, and 35 were prepared as
described in van Rhee et al. or J iang et al.17,18 (R)-PIA and
2-chloroadenosine were purchased from Research Biochemicals
International (Natick, MA). All other materials were obtained
from commercial sources.
2. Syn th esis. Proton nuclear magnetic resonance spec-
troscopy was performed on a Varian GEMINI-300 spectro-
meter, and spectra were taken in DMSO-d6, CH3OH-d4, or
CHCl3-d. Chemical-ionization (CI) mass spectrometry was
performed with a Finnigan 4600 mass spectrometer and
electron-impact (EI) mass spectrometry with a VG7070F mass
spectrometer at 6 kV. Elemental analysis was performed by
Atlantic Microlab Inc. (Norcross, GA). All melting points were
determined with a Unimelt capillary melting point apparatus
(Arthur H. Thomas Co., PA) and are uncorrected.
Gen er a l P r oced u r e for P r ep a r a tion of 1,4-Dih yd r o-
p yr id in e-3,5-d ica r boxyla te Ester s 8, 10-17, 22, 26, 29-
31, 32b, a n d 36-39. Equimolar amounts (0.5 mmol) of the
appropriate 3-amino-2-propenoate ester, aldehyde, and 3-ke-
topropionate ester derivative were dissolved in 2-5 mL of
absolute ethanol. The solution was sealed in a glass tube and
heated to 100 °C (for volatile aldehydes) or was refluxed under
N2 for at least 24 h and, at most, 72 h. The solvent was then
evaporated, and products were purified either by crystalliza-
tion, column chromatography (silica gel 60, 220-440 mesh;
Fluka, Buchs, CH; 20% ethyl acetate-80% petroleum ether,
35-60), or preparative TLC (silica gel 60, 1000 µm; Analtech,
Newark, DE; 2.5% methanol-97.5% dichloromethane). All
procedures were performed under nitrogen and low-light
conditions to prevent oxidation of the products. The products
were shown to be homogeneous by analytical TLC and were
stored at -20 °C.
3,5-Dieth yl 2-Meth yl-6-p h en yl-4-(2-p h en yleth yn yl)-1,4-
(()-d ih yd r op yr id in e-3,5-d ica r b oxyla t e (2). Equimolar
amounts (0.5 mmol) of ethyl 3-amino-2-propenoate (65 mg),
phenylpropargylaldehyde (65 mg) and benzyl benzoylacetate18
(127 mg) were dissolved in 2 mL of absolute ethanol. The
solution was sealed in a glass tube and refluxed under N2 for
24 h. The solvent was then evaporated, and products were
purified by preparative TLC (silica gel 60, 1000 µm; ethyl
acetate:petroleum ether, 2:8). The product (Rf 0.36, same
solvent) was isolated (73 mg) as white crystals, which were
recrystallized from methanol:water, 7:3. 1H NMR (CDCl3): δ
1.35 (t, 3H, 5-methyl, J ) 7.1 Hz), 2.36 (s, 6H, 2-, 6-CH3), 3.80
(s, 3H, 3-methyl), 4.23-4.31 (m, 2H, 5-methylene), 4.99 (s, 1H,
H-4), 5.71 (br, 1H, H-1), 7.24 (t, 3H, H-3′, H-4′, H-5′, J ) 3.2
Hz), 7.36 (d, 2H, H-2′, H-6′, J ) 3.6 Hz). MS (CI/NH3): m/ z
478 MH+, 376 (M - Ph-CC)+, 242 (376 - CO2CH2Ph)+, base.
UV absorbance peaks (MeOH) at 242 (λmax, ꢀ ) 20 200), 351
nm.
The indication that the 5-ester position is very flexible
for substitution in relation to A3 receptor affinity has
suggested the design of an amine-functionalized conge-
ner, 25. As in our previous studies of purines deriva-
tized with long chains for the purpose of conjugating
with other molecules while retaining the biological
potency, this derivative may prove to be a key interme-
diate in the design of much higher molecular weight
derivatives bearing “carrier” moieties, reporter groups,
prosthetic groups, etc. The presence of the amino group
also increased water solubility; unfortunately it was not
potent in binding at A3 receptors.
A persistent problem during the development of
selective A3 receptor antagonists has been species
differences.4 Which of the novel antagonists16-20
(screened for affinity principally at the human A3
receptor) may be useful for pharmacological studies in
rat and other species of interest? This study has
demonstrated that considerable selectivity for rat A3
receptors is still present (Table 3). Compound 2, which
has a ratio of selectivity in binding of only 28-fold, has
been demonstrated to antagonize agonist action at the
A3 receptor in rat hippocampal slices.33 In the latter
study A3 receptor activation antagonized the effects of
presynaptic A1 receptor activation to depress EPSPs.
Compound 2 at a concentration of 10 µM selectively
blocked the A3 receptor activity without any measurable
effect on A1 receptors. Thus, certain compounds in this
series having exceptionally high selectivity, such as 24b
and 26-28, are likely of broad utility as pharmacological
probes across species.
In conclusion, the dihydropyridines have served as a
structural scaffold on which to add substituents to
enhance the potency at the desired (A3) receptor.17
Nearly complete specificity for this subtype has now
been achieved. Although these selective A3 antagonists
have not been evaluated at the A2B receptor, it is likely
that the affinity at that subtype is also low. A lead
dihydropyridine derivative, nimodipine,17 was found to
be inactive at 100 µM in antagonizing the adenosine
agonist effects on intracellular calcium via the cloned
human A2B receptor expressed in CHO cells (IJ zerman
et al., unpublished results). Remaining challenges are
to enhance potency to the subnanomolar level and to
prepare radioligands. In the future, methods for resolv-
ing C-4 enantiomers of the present racemic compounds
will be reported. In the previous and present studies
we have probed systematically all of the positions of the
6-phenyl-1,4-dihydropyridines for the flexibility of sub-
stitution and have found that substitutions at the 4- and
3,5-Dieth yl 2,6-Dim eth yl-4-(2-th ien yl)-1,4-(()-d ih yd r o-
p yr id in e-3,5-d ica r boxyla te (8). 1H NMR (CDCl3): δ 1.27
(t, 6H, J ) 7.75 Hz, 3, 5-CH2CH3), 2.3 (s, 6H, 2-, 6-CH3), 4.17
(m, 4H, 3-, 5-OCH2), 5.35 (s, 1H, 4-H), 5.92 (br, 1H, NH), 6.8
(d, 1H, J ) 3.9 Hz, 3′-H), 6.85 (m, 1H, 4′-H), 7.06 (d, 1H, J )
4.89 Hz, 5′-H).
3,5-Diet h yl 2-Met h yl-4-(p h en ylet h yn yl)-6-(t r iflu or o-
m eth yl)-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (10).
1H NMR (CDCl3): δ 1.32 (m, 6H, 3-, 5-CH2CH3); 2.35 (s, 3H,
7-CH3), 4.20-4.38 (m, 4H, 3-, 5-OCH2), 4.84 (s, 1H, 4-H), 6.29
(br, 1H, NH), 7.25-7.35 (m, 5H, C6H5). MS (EI): m/ z 415
(M)+; 386 (M - C2H5)+, 342 (M - CO2C2H5)+, base.
3,5-Dieth yl 2-Meth yl-4,6-d ip h en yl-1,4-(()-d ih yd r op y-
r id in e-3,5-d ica r boxyla te (11). 1H NMR (CDCl3): δ 0.84 (t,
3H, J ) 6.83 Hz, 5-CH2CH3), 1.24 (t, 3H, J ) 6.84 Hz,
3-CH2CH3), 2.36 (s, 3H, 2-CH3), 3.84 (m, 2H, 5-OCH2), 4.12

SAR of 4-(Phenylethynyl)-6-phenyldihydropyridines
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2605
(q, 2H, J ) 6.83 Hz, 3-OCH2), 5.12 (s, 1H, 4-H), 5.76 (br, 1H,
NH), 7.18-7.44 (m, 10H, 4-, and 6-C6H5).
3-Eth yl 5-(3-Meth ylben zyl) 4-(P h en yleth yn yl)-6-p h en -
yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (24b). 1H
NMR (CDCl3): δ 1.34 (t, 3H, J ) 6.84 Hz, 3-CH2CH3), 2.25 (s,
3H, 2′-CH3), 2.36 (s, 3H, 2-CH3), 4.3 (m, 2H, 3-OCH2), 4.99
(AB, 2H, J ) 12.7 Hz, 5-OCH2), 5.18 (s, 1H, 4-H), 5.91 (br,
1H, NH), 6.88-7.39 (m, 14H, 4-C6H5, 5-C6H4, 6-C6H5).
3-Eth yl 5-(4-Meth ylben zyl) 4-(P h en yleth yn yl)-6-p h en -
yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (24c). 1H
NMR (CDCl3): δ 1.34 (t, 3H, J ) 6.84 Hz, 3-CH2CH3), 2.3 (s,
3H, 2′-CH3), 2.37 (s, 3H, 2-CH3), 4.3 (m, 2H, 3-OCH2), 4.99
(AB, 2H, J ) 12.7 Hz, 5-OCH2), 5.17 (s, 1H, 4-H), 5.86 (br,
1H, NH), 7.0-7.4 (m, 14H, 4-C6H5, 5-C6H4, 6-C6H5).
3-Eth yl 5-[4-(Tr iflu or om eth yl)ben zyl] 4-(P h en yleth yn -
yl)-6-p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r b oxyla t e
(24d ). 1H NMR (CDCl3): δ 1.35 (t, 3H, J ) 6.84 Hz,
3-CH2CH3), 2.37 (s, 3H, 2-CH3), 4.3 (m, 2H, 3-OCH2), 5.15 (AB,
2H, J ) 13.7 Hz, 5-OCH2), 5.18 (s, 1H, 4-H), 5.94 (br, 1H, NH),
7.13-7.45 (m, 14H, 4-C6H5, 5-C6H4, 6-C6H5).
3-Eth yl 5-(3-Iod oben zyl) 4-(P h en yleth yn yl)-6-p h en yl-
1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (24e). 1H NMR
(CDCl3): δ 1.35 (t, 3H, J ) 6.84 Hz, 3-CH2CH3), 2.37 (s, 3H,
2-CH3), 4.3 (m, 2H, 3-OCH2), 4.93 (AB, 2H, J ) 13.7 Hz,
5-OCH2), 5.16 (s, 1H, 4-H), 5.92 (br, 1H, NH), 6.9-7.4 (m, 14H,
4-C6H5, 5-C6H4, 6-C6H5).
3-Eth yl 5-(3-Nitr oben zyl) 4-(P h en yleth yn yl)-6-p h en yl-
1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (24f). 1H NMR
(CDCl3): δ 1.35 (t, 3H, J ) 6.84 Hz, 3-CH2CH3), 2.38 (s, 3H,
2-CH3), 4.3 (m, 2H, 3-OCH2), 5.15 (AB, 2H, J ) 13.7 Hz,
5-OCH2), 5.17 (s, 1H, 4-H), 5.94 (br, 1H, NH), 7.3-8.1 (m, 14H,
4-C6H5, 5-C6H4, 6-C6H5).
3,5-Dieth yl 2-Meth yl-4-(2-p yr id yl)-6-p h en yl-1,4-(()-d i-
h yd r op yr id in e-3,5-d ica r boxyla te (12). 1H NMR (CDCl3):
δ 0.81 (t, 3H, J ) 6.84 Hz, 5-CH2CH3), 1.22 (t, 3H, J ) 6.83
Hz, 3-CH2CH3), 2.37 (s, 3H, 2-CH3), 3.82 (m, 2H, 5-OCH2), 4.11
(q, 2H, J ) 6.84 Hz, 3-OCH2), 5.25 (s, 1H, 4-H), 5.88 (br, 1H,
NH), 7.08-7.56 (m, 3H, pyridyl 3′-, 4′-, 5′-H), 7.37 (m, 5H,
6-C6H5), 8.55 (d, 1H, J ) 4.89, pyridyl 6′-H).
3,5-Dieth yl 2-Meth yl-4-(3-p yr id yl)-6-p h en yl-1,4-(()-d i-
h yd r op yr id in e-3,5-d ica r boxyla te (13). 1H NMR (CDCl3):
δ 0.84 (t, 3H, J ) 6.83 Hz, 5-CH2CH3), 1.24 (t, 3H, J ) 6.84
Hz, 3-CH2CH3), 2.38 (s, 3H, 2-CH3), 3.83 (m, 2H, 5-OCH2), 4.11
(q, 2H, J ) 6.84 Hz, 3-OCH2), 5.09 (s, 1H, 4-H), 6.43 (br, 1H,
NH), 7.2-7.7 (m, 2H, pyridyl 4′, 5′-H), 7.3-7.4 (m, 5H, 6-C6H5),
8.33 (d, 1H, pyridyl 6′-H), 8.64 (s, 1H, pyridyl 2′-H).
3,5-Dieth yl 2-Meth yl-4-(4-p yr id yl)-6-p h en yl-1,4-(()-d i-
h yd r op yr id in e-3,5-d ica r boxyla te (14). 1H NMR (CDCl3):
δ 0.83 (t, 3H, J ) 6.84 Hz, 5-CH2CH3), 1.24 (t, 3H, J ) 6.84
Hz, 3-CH2CH3), 2.4 (s, 3H, 2-CH3), 3.84 (m, 2H, 5-OCH2), 4.12
(q, 2H, J ) 7.81 Hz, 3-OCH2), 5.12 (s, 1H, 4-H), 6.35 (br, 1H,
NH), 7.3-7.4 (m, 7H, 6-C6H5, pyridyl 3′-, 5′-H), 8.42 (d, 2H, J
) 5.86 Hz, pyridyl 2′-, 6′-H).
3,5-Dieth yl 2-Meth yl-4-(2-ben zofu r yl)-6-p h en yl-1,4-(()-
d ih yd r op yr id in e-3,5-d ica r boxyla te (17). 1H NMR (CDCl3):
δ 0.89 (t, 3H, J ) 6.84 Hz, 5-CH2CH3), 1.31 (t, 3H, J ) 6.84
Hz, 3-CH2CH3), 2.39 (s, 3H, 2-CH3), 3.92 (m, 2H, 5-OCH2), 4.21
(m, 2H, 3-OCH2), 5.48 (s, 1H, 4-H), 5.96 (br, 1H, NH), 6.48 (s,
1H, benzofuryl 3′-H), 7.1-7.5 (m, 9H, 6-C6H5, benzofuryl 4′-,
5′-, 6′-, 7′-H).
3-Eth yl 5-(4-Nitr oben zyl) 4-(P h en yleth yn yl)-6-p h en yl-
1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (24g). 1H NMR
(CDCl3): δ 1.36 (t, 3H, J ) 6.84 Hz, 3-CH2CH3), 2.38 (s, 3H,
2-CH3), 4.3 (m, 2H, 3-OCH2), 5.12 (AB, 2H, J ) 12.7 Hz,
5-OCH2), 5.20 (s, 1H, 4-H), 5.94 (br, 1H, NH), 7.1-8.0 (m, 14H,
4-C6H5, 5-C6H4, 6-C6H5).
3-Eth yl 5-[4-[(2,2,2-Tr ich lor oeth oxy)ca r bon yl]ben zyl]
4-(P h en yleth yn yl)-6-p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-
d ica r boxyla te (24h ). 1H NMR (CDCl3): δ 1.36 (t, 3H, J )
6.84 Hz, 3-CH2CH3), 2.38 (s, 3H, 2-CH3), 4.3 (m, 2H, 3-OCH2),
5.12 (AB, 2H, J ) 12.7 Hz, 5-OCH2), 5.20 (s, 1H, 4-H), 5.94
(br, 1H, NH), 7.1-8.0 (m, 14H, 4-C6H5, 5-C6H4, 6-C6H5).
3-Eth yl 5-[3,5-Bis(tr iflu or om eth yl)ben zyl] 4-(P h en yl-
eth yn yl)-6-p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r box-
yla te (24i). 1H NMR (CDCl3): δ 1.33 (t, 3H, J ) 6.83 Hz,
3-CH2CH3), 2.39 (s, 3H, 2-CH3), 4.3 (m, 2H, 3-OCH2), 5.08 (AB,
2H, J ) 12.7 Hz, 5-OCH2), 5.15 (s, 1H, 4-H), 5.94 (br, 1H, NH),
7.34 (s, 10H, 4-C6H5, 6-C6H5), 7.52 (s, 2H, 5-Ar), 7.75 (s, 1H,
5-Ar).
3-Eth yl 5-(3,5-Din itr oben zyl) 4-(P h en yleth yn yl)-6-ph en -
yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (24j). 1H
NMR (CDCl3): δ 1.36 (t, 3H, J ) 6.84 Hz, 3-CH2CH3), 2.39 (s,
3H, 2-CH3), 4.3 (m, 2H, 3-OCH2), 5.17 (AB, 2H, J ) 13.7 Hz,
5-OCH2), 5.16 (s, 1H, 4-H), 5.97 (br, 1H, NH), 7.37 (s, 10H,
4-C6H5, 6-C6H5), 8.24 (s, 2H, 5-Ar), 8.9 (s, 1H, 5-Ar).
3-Eth yl 5-[4-[[(2-Am in oeth yl)a m in o]ca r bon yl]ben zyl]
4-(P h en yleth yn yl)-6-p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-
d ica r boxyla te (25). Compound 24h (16.2 mg, 0.03 mmol)
was mixed with ethylenediamine (12.4 mg, 0.21 mmol). After
30 min at room temperature, the mixture was quenched with
H2O (1 mL) and extracted with CH2Cl2 (3 mL × 3). The
product was purified with preparative TLC (5:1 CHCl3:MeOH)
to yield 7.0 mg of a white solid (47.9%). 1H NMR (CD3OD): δ
1.39 (t, 3H, J ) 6.84 Hz, 3-CH2CH3), 2.40 (s, 3H, 2-CH3), 2.91
(br, 2H, -CH2NH2), 3.51 (t, 2H, J ) 5.86 Hz, -NHCH2), 4.3 (m,
2H, 3-OCH2), 5.12 (AB, 2H, J ) 12.7, Hz, 5-OCH2), 5.14 (s,
1H, 4-H), 7.14 (d, 2H, J ) 7.81 Hz, 5-Ar), 7.3-7.4 (m, 10H,
4-C6H5, 6-C6H5), 7.74 (d, 2H, J ) 7.81 Hz, 5-Ar), 7.96 (s, 1H,
NH).
3,5-Dieth yl 2-Meth yl-4-[2-(4-n itr op h en yl)eth yn yl]-1,4-
(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (22a ). 1H NMR
(CHCl3-d): δ 1.25-1.34 (m, 6H, 3-, 5-CH3), 2.49 (s, 3H, 2-CH3),
3.47-3.59 (m, 2H, 3-CH2), 4.23 (q, 2H, 5-CH2, J ) 7.8 Hz),
5.42 (s, 1H, H-4), 6.29 (d, 1H, H-1, J ) 2.9 Hz), 7.55 (d, 2H,
H-2′, H-6′, J ) 8.8 Hz), 8.21 (d, 2H, H-3′, H-5′, J ) 8.8 Hz),
8.26 (wide, 1H, H-6).
3,5-Diet h yl 2-Met h yl-4-[(3-m et h ylp h en yl)et h yn yl]-6-
p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (22b).
1H NMR (CDCl3): δ 0.95 (t, 3H, J ) 6.84 Hz, 5-CH2CH3), 1.35
(t, 3H, J ) 6.84 Hz, 3-CH2CH3), 2.29 (s, 3H, 3′-CH3), 2.36 (s,
3H, 2-CH3), 4.0 (m, 2H, 5-OCH2), 4.3 (m, 2H, 3-OCH2), 5.11
(s, 1H, 4-H), 5.92 (br, 1H, NH), 7.0-7.43 (m, 9H, 4-C6H4,
6-C6H5).
3-Eth yl 5-Ben zyl 2-Eth yl-4-(p h en yleth yn yl)-6-p h en yl-
1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (23). 1H NMR
(CDCl3): δ 1.22 (t, J ) 6.9 Hz, 3H, 2-CH2CH3), 1.34 (t, J ) 6.9
Hz, 3H, 3-CH3 CH2), 2.55, 3.01 (2m, 2H, 2-CH2CH3), 4.25 (m,
2H, 3-OCH2 ), 5.06 (AB, J ) 12.7 Hz, 2H, 5-OCH2), 5.18 (s,
1H, 4-H), 5.95 (br, 1H, NH), 7.05-7.39 (m, 15H, 3 × C6H5).
MS (EI): m/ z 491 (M)+, 462 (M - C2H5)+, 418 (M - CO2C2H5)+,
356 (M - CO2CH2C6H5)+, base.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
24a -j, 27, a n d 28. Compound 42 (0.2 mmol) was dissolved
in dry acetone (10 mL), anhydrous potassium carbonate (0.5
g) and the appropriate phenylethyl bromide or phenylpropyl
bromide (5 equiv) were added, and the mixture was refluxed
for 2-6 h. After the mixture was filtered, the solvent was
evaporated in vacuo. The residue was separated by prepara-
tive TLC (40:1 CHCl3:MeOH), and the desired N-protected
5-benzyl ester DHP was isolated. Deprotection was achieved
by dissolving the DHP in acetone (1 mL), adding excess 1 N
HCl (200 µL), and heating at 45 °C for 2-3 h. An equivolume
amount of water was added to the solution followed by
extraction with CH2Cl2 (2 mL × 2). The extracts were
combined, washed with water and brine, and dried over
sodium sulfate. The solvent was removed in vacuo, and the
residue was purified by preparative TLC (2:1 hexanes:EtOAc)
to afford the desired 5-benzyl ester dihydropyridine.
3-Eth yl 5-(2-Meth ylben zyl) 4-(P h en yleth yn yl)-6-p h en -
yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (24a ). 1H
NMR (CDCl3): δ 1.34 (t, 3H, J ) 6.84 Hz, 3-CH2CH3), 2.16 (s,
3H, 2′-CH3), 2.37 (s, 3H, 2-CH3), 4.26 (m, 2H, 3-OCH2), 5.04
(AB, 2H, J ) 12.7 Hz, 5-OCH2), 5.17 (s, 1H, 4-H), 5.92 (br,
1H, NH), 7.06-7.36 (m, 14H, 4-C6H5, 5-C6H4, 6-C6H5).
3-P r op yl 5-Ben zyl 2-Meth yl-4-(p h en yleth yn yl)-6-p h en -
yl-1,4-(()-dih ydr opyr idin e-3,5-dicar boxylate (26). 1H NMR
(CDCl3): δ 1.01 (t, J ) 6.9 Hz, 3H, 3-CH2CH3), 1.73 (m, 2H,
3-CH3 CH2), 2.37 (s, 3H, 2-CH3), 4.15 (m, 2H, 3-OCH2), 5.06
(AB, J ) 12.7 Hz, 2H, 5-OCH2), 5.20 (s, 1H, 4-H), 5.88 (br,
1H, NH), 7.07-7.37 (m, 15H, 3 × C6H5). MS (EI): m/ z 491

2606 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
J iang et al.
(M)+, 448 (M - C3H7)+, 404 (M - CO2C3H7)+, 356 (M - CO2-
3-(Eth oxycar bon yl)-2-m eth yl-4-(ph en yleth yn yl)-6-ph en -
yl-1,4-(()-d ih yd r op yr id in e-5-ca r boxylic Acid Eth yla m id e
(34). A mixture of compound 42 (75 mg, 0.17 mmol), N-
hydroxysuccinimide (22 mg, 0.17 mmol), and EDAC (34 mg,
0.17 mmol) in DMF (1 mL) was stirred at room temperature
for 4 h. Ethylamine (2.0 M in THF, 0.4 mL) was added, and
the reaction mixture was stirred overnight. The solvent was
removed, and the residue was diluted with dichloromethane
(10 mL), washed with water (5 mL × 2) and brine (5 mL × 2),
and dried with sodium sulfate. The solvent was evaporated,
and the residue was purified with a preparative TLC plate to
give 38 mg of compound 43, which was carried out for
deprotection with 1 N HCl to give 18 mg of product 34.
43. 1H NMR (CDCl3): δ 0.72 (t, J ) 6.8 Hz, 3H, 1-CH2CH3),
0.96 (t, J ) 6.8 Hz, 3H, 5-CH2CH3), 1.33 (t, J ) 6.8 Hz, 3H,
3-CH2CH3), 2.60 (s, 3H, 2-CH3), 3.08 (m, 2H, 5-NCH2), 3.62
(m, 2H, 1-OCH2), 4.23 (m, 2H, 3-OCH2), 4.45, 4.85 (AB, J )
11.7 Hz, N-CH2-O), 4.92 (s, 1H, 4-H), 4.97 (br, 1H, CONH),
7.22-7.44 (m, 10H, 2 × C6H5). MS (CI/NH3): m/ z 473 (MH)+.
34. 1H NMR (CDCl3): δ 0.85 (t, J ) 6.8 Hz, 3H, 5-CH2CH3),
1.31 (t, J ) 6.8 Hz, 3H, 3-CH2CH3), 2.37 (s, 3H, 2-CH3), 3.16
(m, 2H, 5-NCH2), 4.25 (m, 2H, 3-OCH2), 5.09 (s, 1H, 4-H), 5.37
(br, 1H, CONH), 5.64 (br, 1H, NH), 7.25-7.44 (m, 10H, 2 ×
C6H5).
3-ter t-Bu tyl 5-Eth yl 2-Meth yl-4-(ph en yleth yn yl)-6-ph en -
yl-1,4-(()-dih ydr opyr idin e-3,5-dicar boxylate (36). 1H NMR
(CDCl3): δ 0.91 (t, J ) 6.9 Hz, 3H, 5-CH2CH3), 1.52 (s, 9H,
C(CH3)3), 2.29 (s, 3H, 2-CH3), 3.98 (m, 2H, 5-OCH2), 5.05 (s,
1H, 4-H), 5.71 (br, 1H, NH), 7.21-7.40 (m, 10H, 2 × C6H5).
MS (CI/NH3): m/ z 461 (M + NH4)+, 443 (MH)+.
5-(Eth oxycar bon yl)-2-m eth yl-4-(ph en yleth yn yl)-6-ph en -
yl-1,4-(()-d ih yd r op yr id in e-3-ca r boxylic Acid Eth yla m id e
(37). 1H NMR (CDCl3): δ 0.85 (t, J ) 6.8 Hz, 3H, 3-CH2CH3),
1.18 (t, J ) 6.8 Hz, 3H, 5-CH2CH3), 2.24 (s, 3H, 2-CH3), 3.38
(m, 2H, 3-NCH2), 3.94 (m, 2H, 5-OCH2), 4.80 (s, 1H, 4-H), 5.65
(br, 1H, NH), 6.32 (br, 1H, CONH), 7.23-7.38 (m, 10H, 2 ×
C6H5). MS (EI): m/ z 414 (M)+, 385 (M - C2H5)+, base, 342
(M - CONHC2H5)+.
3-(Eth ylcar bon yl)-5-(eth oxycar bon yl)-2-m eth yl-4-(ph en -
yleth yn yl)-6-ph en yl-1,4-(()-dih ydr opyr idin e (38). 1H NMR
(CDCl3): δ 0.93 (t, J ) 7.0 Hz, 3H, 5-CH2CH3), 2.33 (s, 3H,
2-CH3), 2.48 (s, 3H, 3-COCH3), 4.00 (q, 2H, J ) 7.0 Hz,
5-OCH2), 5.04 (s, 1H, 4-H), 5.88 (br, 1H, NH), 7.26-7.43 (m,
10H, 2 × C6H5). MS (EI): m/ z 385 (M)+, 443 (M - CH3CO)+,
base.
E t h yl 5-Oxo-4-(p h en ylet h yn yl)-2-p h en yl-1,4,5,6,7,8-
h exa h yd r oqu in olin e-3-ca r boxyla te (39). 1H NMR (CDCl3):
δ 1.02 (t, J ) 6.8 Hz, 3H, 5-CH2CH3), 2.04 (t, J ) 4.9 Hz, 2H,
7-CH2), 2.46 (m, 4H, 8-, 9-CH2), 4.00 (m, 2H, 5-OCH2), 5.13 (s,
1H, 4-H), 6.14 (br, 1H, NH), 7.20-7.41 (m, 10H, 2 × C6H5).
MS (EI): m/ z 397 (M)+, 368 (M - C2H5)+, base.
Gen er a l P r oced u r e of N-H Gr ou p P r otection of Com -
p ou n d 31. Sodium hydride (60% in mineral oil, 1.5 equiv)
was added to compound 31 in a solution of DMF (1.5 mL). The
mixture was stirred for 5 min, chloromethyl methyl (or
chloromethyl ethyl) ether (1.5 equiv) was added slowly to the
solution under argon at room temperature and stirred for 2
h. The reaction was quenched by adding cold water (10 mL),
the mixture was extracted with ethyl acetate (10 mL × 2), and
the organic layer was washed with water (10 mL × 2) and
brine (10 mL × 2) and dried with sodium sulfate. The solvent
was evaporated, and the residue was purified with preparative
TLC plates to give corresponding N-protected products 40 and
41.
3-Eth yl 5-[2-(Tr im eth ylsilyl)eth yl] 1-(Meth oxym eth yl)-
2-m eth yl-4-(p h en yleth yn yl)-6-p h en yl-1,4-(()-d ih yd r op y-
r id in e-3,5-d ica r boxyla te (40). 1H NMR (CDCl3): δ -0.04
(s, 9H, Si(CH3)3), 0.63 (m, 2H, CH2Si), 1.33 (t, J ) 7.0 Hz, 3H,
3-CH2CH3), 2.54 (s, 3H, 2-CH3), 3.18 (s, 3H, OCH3), 3.97 (t, J
) 7.8 Hz, 2H, 5-OCH2), 4.27 (m, 2H, 3-OCH2), 4.37, 4.85 (AB,
J ) 11.7 Hz, N-CH2-O), 5.07 (s, 1H, 4-H), 7.21-7.40 (m, 10H,
2 × C6H5). MS (CI/NH3): m/ z 549 (M + NH4)+, 532 (MH)+.
3-Eth yl 5-[2-(Tr im eth ylsilyl)eth yl] 1-(Eth oxym eth yl)-
2-m eth yl-4-(p h en yleth yn yl)-6-p h en yl-1,4-(()-d ih yd r op y-
r id in e-3,5-d ica r boxyla te (41). 1H NMR (CDCl3): δ -0.08
(s, 9H, Si(CH3)3), 0.61 (m, 2H, CH2Si), 0.91 (t, J ) 6.9 Hz, 3H,
CH2C6H5)+, base.
3-Eth yl 5-P h en yleth yl 2-Meth yl-4-(p h en yleth yn yl)-6-
p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (27).
1H NMR (CDCl3): δ 1.36 (t, J ) 6.8 Hz, 3H, 3-CH2CH3), 2.37
(s, 3H, 2-CH3), 2.69 (t, J ) 6.8 Hz, 2H, 5-CH2C6H5), 4.15 (m,
2H, 3-OCH2), 4.30 (m, 2H, 5-OCH2), 5.11 (s, 1H, 4-H), 5.87
(br, 1H, NH); 7.10-7.41 (m, 15H, 3 × C6H5). MS (EI): m/ z
491 (M)+, 462 (M - C2H5)+, 418 (M - CO2C2H5)+, 342 (M -
CO2(CH2)2C6H5)+.
3-Eth yl 5-P h en ylp r op yl 2-Meth yl-4-(p h en yleth yn yl)-6-
p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (28).
1H NMR (CDCl3): δ 1.30 (t, J ) 6.8 Hz, 3H, 3-CH2CH3), 1.65
(m, 2H, 5-CH2CH2CH2), 2.35 (s, 3H, 2-CH3), 2.40 (m, 2H,
5-CH2C6H5), 3.98 (m, 2H, 3-OCH2), 4.26 (m, 2H, 5-OCH2); 5.14
(s, 1H, 4-H), 5.85 (br, 1H, NH), 7.20-7.41 (m, 15H, 3 × C6H5).
MS (EI): m/ z 505 (M)+, 476 (M - C2H5)+, 432 (M - CO2C2H5)+,
342 (M - CO2(CH2)3C6H5)+.
3-Eth yl 5-ter t-Bu tyl 2-Meth yl-4-(ph en yleth yn yl)-6-ph en -
yl-1,4-(()-dih ydr opyr idin e-3,5-dicar boxylate (29). 1H NMR
(CDCl3): δ 1.25 (t, J ) 7.1 Hz, 3H, 3-CH2CH3), 1.31 (s, 9H,
C(CH3)3), 2.31 (s, 3H, 2-CH3), 4.22 (m, 2H, 3-OCH2); 4.94 (s,
1H, 4-H), 5.56 (br, 1H, NH), 7.21-7.34 (m, 10H, 2 × C6H5).
MS (EI): m/ z 505 (M)+, 476 (M - C2H5)+, 432 (M - CO2C2H5)+,
342 (M - CO2(CH2)3C6H5)+.
3-(2-Meth oxy-2-p h en yleth yl) 5-Eth yl 2-Meth yl-4-(p h e-
n yleth yn yl)-6-p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r -
boxyla te (30). 1H NMR (CDCl3): δ 0.91 and 1.00 (2t, J )
6.8 Hz, 3H, 5-CH2CH3), 2.35 (s, 3H, 2-CH3), 4.00 (m, 2H,
5-OCH2), 4.35 (m, 1H, 3-CH), 4.50 (m, 2H, 3-OCH2), 5.05 (s,
1H, 4-H), 5.82 (br, 1H, NH), 7.21-7.41 (m, 10H, 2 × C6H5).
MS (CI/NH3): m/ z 539 (M ) NH4)+.
3-Eth yl 5-[2-(Tr im eth ylsilyl)eth yl] 2-Meth yl-4-(p h en yl-
eth yn yl)-6-p h en yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r box-
yla te (31). 1H NMR (CDCl3): δ 0.03 (s, 9H, Si(CH3)3), 0.79
(t, J ) 8.8 Hz, 2H, CH2Si), 1.41 (t, J ) 6.8 Hz, 3H, 3-CH2CH3),
2.40 (s, 3H, 2-CH3), 4.10 (t, J ) 8.8 Hz, 2H, 5-OCH2), 4.31 (m,
2H, 3-OCH2), 5.15 (s, 1H, 4-H), 5.87 (br, 1H, NH), 7.28-7.46
(m, 10H, 2 × C6H5).
3-Eth yl 5-Th ioeth yl 2-Meth yl-4-(ph en yleth yn yl)-6-ph en -
yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (32a ). Tri-
ethylamine (20 mg) was added to a mixture of compound 42
(90 mg, 0.2 mmol), diphenyl phosphorazate (56 mg, 0.2 mmol),
and ethanethiol (20 mg, 0.3 mmol) in DMF (1 mL) with stirring
while cooled in an ice-water bath. The bath was removed,
and the mixture was stirred at room temperature for 3 h, then
diluted with dichloromethane (20 mL), washed with water (10
mL × 2), and dried with sodium sulfate. The solvent was
evaporated, and the residue was carried out for deprotection
with 1 N HCl to give 15 mg of product. 1H NMR (CDCl3): δ
1.15 (t, J ) 6.8 Hz, 3H, 5-CH2CH3), 1.28 (t, J ) 6.8 Hz, 3H,
3-CH2CH3), 2.37 (s, 3H, 2-CH3), 2.82 (m, 2H, 5-SCH2), 4.31
(m, 2H, 3-OCH2), 5.23 (s, 1H, 4-H), 5.95 (br, 1H, NH), 7.24-
7.46 (m, 10H, 2 × C6H5).
3-Th ioeth yl 5-Eth yl 2-Meth yl-4-(ph en yleth yn yl)-6-ph en -
yl-1,4-(()-d ih yd r op yr id in e-3,5-d ica r boxyla te (32b). The
3-ketopropionate ester for this reaction was synthesized by
adding 2,2,6-trimethyl-4H-1,3-dioxin-4-one (4.6 mmol) drop-
wise to a solution of ethanethiol (4.6 mmol) in 15 mL of toluene
at 100 °C. Heating at 100 °C was continued for 5 h. The
solvent was evaporated and the residue chromatographed by
preparative TLC (3:1 hexanes:EtOAc) to yield 49.1 mg of a
yellowish oil (7.3%). 32b was then synthesized via the
Hantszch condensation method described in the general
procedure. 1H NMR (CDCl3): δ 0.98 (t, 3H, J ) 6.84 Hz,
3-CH2CH3), 1.32 (t, 3H, J ) 6.84 Hz, 5-CH2CH3), 2.37 (s, 3H,
2-CH3), 3.0 (m, 2H, 3-SCH2), 4.02 (q, 2H, J ) 6.83 Hz, 5-OCH2),
5.27 (s, 1H, 4-H), 6.06 (br, 1H, NH), 7.25-7.44 (m, 10H, 4-C6H5,
6-C6H5).
3-(Eth oxycar bon yl)-2-m eth yl-4-(ph en yleth yn yl)-6-ph en -
yl-1,4-(()-d ih yd r op yr id in e-5-ca r boxylic Acid (33). 1H
NMR (CDCl3): δ 1.32 (t, J ) 6.8 Hz, 3H, 3-CH2CH3), 2.38 (s,
3H, 2-CH3), 4.29 (m, 2H, 3-OCH2), 5.08 (s, 1H, 4-H), 5.95 (br,
1H, NH), 7.24-7.44 (m, 10H, 2 × C6H5). MS (CI/NH3): m/ z
405 (M + NH4), 388 (MH)+.

SAR of 4-(Phenylethynyl)-6-phenyldihydropyridines
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2607
OCH2CH3), 1.41 (t, J ) 6.9 Hz, 3H, 3-CH2CH3), 2.57 (s, 3H,
2-CH3), 3.13 (m, 2H, OCH2CH3), 3.95 (t, J ) 7.9 Hz, 2H,
5-OCH2), 4.12 (m, 2H, 3-OCH2), 4.41, 4.81 (AB, J ) 10.8 Hz,
2H, N-CH2-O), 5.02 (s, 1H, 4-H), 7.20-7.39 (m, 10H, 2 × C6H5).
MS (CI/NH3): m/ z 563 (M + NH4)+, 546 (MH)+.
Binding of [125I]-N6-(4-amino-3-iodobenzyl)-5′-N-methylcar-
bamoyl)adenosine ([125I]AB-MECA) to membranes prepared
from HEK-293 cells stably expressing the human A3 receptor,3
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.13,32 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 of
protein/mL, stored at -80 °C in the same buffer), 50 µL of [125I]-
AB-MECA (final concentration 0.3 nM), and 50 µL of a solution
of the proposed antagonist. Nonspecific binding was deter-
mined in the presence of 100 µM N6-(phenylisopropyl)adenos-
ine ((R)-PIA).
1-(E t h o x y m e t h y l)-3-(e t h o x y c a r b o n y l)-2-m e t h y l-4-
(p h en yleth yn yl)-6-p h en yl-1,4-(()-d ih yd r op yr id in e-5-ca r -
boxylic Acid (42). TBAF (hydrate, 208 mg, 0.8 mmol) was
added to a solution of 41 (115 mg, 0.21 mmol) in DMF (1 mL).
The mixture was stirred under argon at room temperature for
2 h, diluted with ethyl acetate (20 mL), washed with 1 N HCl
(5 mL), H2O (20 mL × 2), and brine (20 mL × 2), and dried
with magnesium sulfate. The solvent was evaporated, and the
residue was separated with preparative TLC plates to give 80
mg of product. 1H NMR (CDCl3): δ 0.93 (t, J ) 6.8 Hz, 3H,
OCH2CH3), 1.31 (t, J ) 6.9 Hz, 3H, 3-CH2CH3), 2.59 (s, 3H,
2-CH3), 3.09, 3.65 (2m, 2H, OCH2CH3), 4.27 (m, J ) 7.9 Hz,
2H, 3-OCH2), 4.39, 4.85 (2d, J ) 10.7 Hz, 2H, 3-OCH2), 4.41,
4.81 (AB, J ) 10.8 Hz, 2H, N-CH2-O), 5.02 (s, 1H, 4-H), 7.12
(br, 1H, COOH), 7.20-7.39 (m, 10H, 2 × C6H5). MS (CI/
NH3): m/ z 463 (M + NH4)+, 446 (MH)+.
1-(4-Nitr op h en yl)-2-p r op yn -1-a l (55b). 4-Nitroiodoben-
zene (1.25 g, 5 mmol) was added to 20 mL of diethylamine
and stirred vigorously. To the mixture were added 0.1 mmol
(5%) of copper(I) iodide (20 mg) and 0.1 mmol (5%) of PdIICl2-
(PPh3)2 (70 mg). 3,3-Diethoxy-1-propyne (53; 790 µL, 5.5
mmol) was then slowly added to the reaction mixture. The
reaction was monitored by TLC (silica gel, petroleum ether/
EtOAc ) 95/5) every 5 min until virtually all starting material
had reacted (∼25 min). The reaction was then quenched by
the addition of 50 mL of cold water. The product was extracted
twice with 50 mL of toluene and washed once with 50 mL of
water. The combined organic phase was dried over anhydrous
MgSO4 and the solvent evaporated in vacuo. The acetal
intermediate 54 was purified by flash column chromatography
(silica gel 60, petroleum ether/EtOAc ) 95/5), and 0.89 g (72%
yield) of a yellowish oil was obtained.
All nonradioactive compounds were initially dissolved in
DMSO and diluted with buffer to the final concentration,
where the amount of DMSO never exceeded 2%. Incubations
were terminated by rapid filtration over Whatman GF/B
filters, using a Brandell cell harvester (Brandell, Gaithersburg,
MD). The tubes were rinsed three times with 3 mL of buffer
each.
At least five different concentrations of competitor, spanning
3 orders of magnitude adjusted appropriately for the IC50 of
each compound, were used. IC50 values, calculated with the
nonlinear regression method implemented in the InPlot pro-
gram (Graph-PAD, San Diego, CA), were converted to apparent
Ki values using the Cheng-Prusoff equation41 and Kd values
of 1.0M and 14 nM for [3H]-(R)-PIA and [3H]CGS 21680,
respectively, and 0.59 nM for binding of [125I]AB-MECA at
human A3 receptors, respectively.
Abbr evia tion s: [125I]AB-MECA, [125I]-N6-(4-amino-3-iodo-
benzyl)-5′-(N-methylcarbamoyl)adenosine; CGS 21680, 2-[[[4-
(2-carboxyethyl)phenyl]ethyl]amino]-5′-(N-ethylcarbamoyl)-
adenosine; CHO cells, Chinese hamster ovary cells; DMAP,
(N,N-dimethylamino)pyridine; DMSO, dimethyl sulfoxide;
DPPA, diphenyl phosphorazidate; EDAC, 1-ethyl-3-[3-(dim-
ethylamino)propyl]carbodiimide; HEK cells, human embryonic
kidney cells; IB-MECA, N6-(3-iodobenzyl)-5′-(N-methylcarbam-
oyl)adenosine; 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, 3-ethyl 5-benzyl
2-methyl-6-phenyl-4-(phenylethynyl)-1,4-(()-dihydropyridine-
3,5-dicarboxylate; (R)-PIA, (R)-N6-(phenylisopropyl)adenosine;
SAR, structure-activity relationship; TBAF, tetrabutylam-
monium fluoride; TNF, tumor necrosis factor; Tris, tris-
(hydroxymethyl)aminomethane.
The intermediate diethyl acetal was then dissolved in
hexane (mixed isomers), and 2.2 mol equiv of formic acid was
added to the solution. The product precipitated from the
solution, was recovered by decantation, and was purified by
crystallization from chloroform; 0.30 g (57% yield) of pure red-
brown crystals (needles) of 55b was thus obtained.
1-(3-Tolu yl)-2-p r op yn -1-a l (55c). 55c was prepared in a
similar fashion as described for compound 55b from 3-iodot-
oluene (1.09 g, 5 mmol) and 3,3-diethoxy-1-propyne (790 µL,
5.5 mmol) in 15 mL of anhydrous triethylamine. Deprotection
of the aldehyde required heating to 40 °C.
2,2,2-Tr ich lor oeth yl p-Meth ylben zoa te (57). p-Toluyl
chloride (56) (Aldrich; 1.07 g, 6.92 mmol) and 2,2,2-trichloro-
ethanol (2 mL) were dissolved in 3 mL of chloroform. Tri-
ethylamine (1 mL, 7.19 mmol) was added dropwise with
stirring at room temperature. The reaction mixture was
washed with water (2×), saturated sodium bicarbonate, 0.1
N HCl, and water. The organic layer was separated and dried
leaving 1.45 g of a clear oil (100% yield). 1H NMR (CDCl3): δ
2.5 (s, 3H, -CH3), 5.0 (s, 2H, -CH2CCl3), 7.3 (d, 2H, J ) 7.82
Hz, Ar), 8.1 (d, 2H, J ) 7.81 Hz, Ar).
2,2,2-Tr ich lor oeth yl 4-(Br om om eth yl)ben zoa te (58). A
solution of 57 (0.96 g, 3.6 mmol), N-bromosuccinimide (0.71 g,
4.0 mmol), and a catalytic amount of benzoyl peroxide dis-
solved in 4 mL of benzene was refluxed for 1 h. After cooling,
the succinimide was filtered off and the filtrate chromato-
graphed with preparative TLC (95% petroleum ether, 5% ethyl
acetate) to yield 0.98 g of a yellow oil (76.8%). 1H NMR
(CDCl3): δ 4.52 (s, 2H, -CH2Br), 4.98 (s, 2H, -CH2CCl3), 7.52
(d, 2H, J ) 7.82 Hz, Ar), 8.12 (d, 2H, J ) 7.81 Hz, Ar).
P h a r m a cology: Ra d ioliga n d Bin d in g Stu d ies. Binding
of [3H]-(R)-N6-(phenylisopropyl)adenosine ([3H]-(R)-PIA) to A1
receptors from rat cerebral cortex membranes and of [3H]-2-
[[[4-(2-carboxyethyl)phenyl]ethyl]amino]-5′-(N-ethylcarbamoy-
l)adenosine ([3H]CGS 21680) to A2A receptors from rat striatal
membranes was performed as described previously.30,31 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.
Ack n ow led gm en t. We thank Gilead Sciences (Fos-
ter City, CA) and the Cystic Fibrosis Foundation (Silver
Spring, MD) for financial support. We thank Dr. Gary
L. Stiles and Dr. Mark E. Olah (Duke University,
Durham, NC) for providing samples of [125I]AB-MECA
and Reuven Muller for technical assistance.
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