Structure-activity relationships of thiazole and thiadiazole derivatives as potent and selective human adenosine A3 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2003.10.041

4-(4-Methoxyphenyl)-2-aminothiazole and 3-(4-methoxyphenyl)-5- aminothiadiazole derivatives have been synthesized and evaluated as selective antagonists for human adenosine A₃ receptors. A methoxy group in the 4-position of the phenyl ring and

Full text of DOI:10.1016/j.bmc.2003.10.041

Bioorganic & Medicinal Chemistry 12 (2004) 613–623
Structure–activity relationships of thiazole and thiadiazole
derivatives as potent and selective human adenosine
A3 receptor antagonists
Kwan-Young Jung,a Soo-Kyung Kim,b Zhan-Guo Gao,b Ariel S. Gross,b Neli Melman,b
Kenneth A. Jacobsonb and Yong-Chul Kima,*
aLaboratory of Drug Discovery, Department of Life Science, Kwangju Institute of Science and Technology, Gwangju 500-712, South Korea
bLaboratory of Bioorganic Chemistry, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health,
DHHS, Bethesda, MD 20892-0810, USA
Received 14 August 2003; accepted 21 October 2003
Abstract—4-(4-Methoxyphenyl)-2-aminothiazole and 3-(4-methoxyphenyl)-5-aminothiadiazole derivatives have been synthesized and
evaluated as selective antagonists for human adenosine A3 receptors. A methoxy group in the 4-position of the phenyl ring and N-acetyl or
propionyl substitutions of the aminothiazole and aminothiadiazole templates displayed great increases of binding affinity and selectivity for
human adenosine A3 receptors. The most potent A3 antagonist of the present series, N-[3-(4-methoxy-phenyl)-[1,2,4]thiadiazol-5-yl]-acet-
amide (39) exhibiting a Ki value of 0.79 nM at human adenosine A3 receptors, showed antagonistic property in a functional assay of cAMP
biosynthesis involved in one of the signal transduction pathways of adenosine A3 receptors. Molecular modeling study of conformation
search and receptor docking experiments to investigate the dramatic differences of binding affinities between two regioisomers of
thiadiazole analogues, (39) and (42), suggested possible binding mechanisms in the binding pockets of adenosine receptors.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
Unlike typical xanthine core antagonists of other
adenosine receptor subtypes, several diverse heterocyclic
compound classes of selective antagonists for A3 receptors
have been developed. Triazolonaphthyridine,11 1,4-dihy-
dropyridines,12,13 pyridines,14,15 pyrans,16 triazoloquina-
Extracellular adenosine regulates a number of physio-
logical functions by activation of specific cell membrane
receptors, classified as A1, A2A, A2B, and A3 receptors,
which belong to the super family of G-protein coupled
receptors.1,2 Adenosine A3 receptors, linked to Gi proteins
decreasing cAMP production upon receptor activation,
have been determined to show important roles in physio-
logical systems, including protective actions against
myocardial and brain ischemia,3,4 antiproliferative effects
through cell cycle arrest and the Wnt signaling pathway,5ꢀ7
and airway inflammation with high receptor expression in
lung mast cells in mice,8,9 implicating a potentialdrug target
for ischemia, cancer and asthma. Furthermore, A3 receptor
antagonists may be useful in the treatment of glaucoma.10
zolines,17,18
flavonoids,19
triazolopyrimidines,20,21
triazolopurines,22 isoquinolines and quiazolines,23 imida-
zopurinones,24 pyridopurinediones,25 have been synthe-
sized and identified as adenosine A3 receptor antagonists.
Recently, thiazole and thiadiazole analogues have been
described as the possible core skeletons of A3 receptor
antagonists with moderate affinity and selectivity.26,27 In
this study, we report new findings of great improvement
of the scaffold derivatives with subnanomolar affinity at
human adenosine A3 receptors and high subtype selectiv-
ity through an SAR (structure–activity relationship)
study combined with molecular modeling approaches.
Since the cloning of adenosine A3 receptors from several
species in the early 1990s, many efforts have been devoted
to develop agonists and antagonists of the receptors,
resulting in the design of potent and selective antagonists.
2. Results and discussion
2.1. Chemistry
Keywords: Adenosine; A3 receptor; Antagonist; Thiazole; Thiadiazole.
*Corresponding author. Tel.: +82-62-970-2502; fax: +82-62-970-
2484; e-mail: yongchul@kjist.ac.kr
The general syntheses of various regioisomers of 4-(4-
methoxyphenyl)-2-aminothiazole and 3-(4-methoxy-
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.041

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K.-Y. Jung et al. / Bioorg. Med. Chem. 12 (2004) 613–623
phenyl)-5-aminothiadiazole derivatives are depicted in
Schemes 1–3. 2-Aminothiazole analogues (3a–d) were
prepared from the cyclization of 2-thiocyanoacetophe-
nones in the presence of alumina supported ammonium
acetate as described by Kodomari et al. (Scheme 1).28
diazole compounds were subjected in the standard
acylation protocols including coupling reagents with
acids or anhydrides, or acid chlorides with pyridine as
base to afford various derivatives 11–43 to be tested in
adenosine receptor binding assays.
In order to synthesize the 5-amino[1,2,4]thiadiazole
derivative 7, 4-methoxybenzonitrile was converted to
the 4-methoxybenzamidine hydrochloride derivaive 5,
followed by a cyclization reaction using perchloro-
methyl mercaptan29 in basic aqueous solution to afford
the chlorothiadiazole intermediate 6. Substitution
reaction of 6 with ammonia and methylamine gave 7
and 8, respectively (Scheme 2).
3. Biological activity
The binding affinity of each compound at four different
adenosine receptor subtypes was determined as Ki
values in radioligand binding assays at rat cortical or
recombinant human A1 receptors versus [3H]-(R)-PIA
([3H]-(R)-N6-(phenylisopropyl)adenosine) and recombi-
nant human A2A receptors versus [3H]-CGS 21680 ([3H]
-2-[4-(2-carboxyethyl)phenyl]ethylamino-50-(N-ethylcar-
bamoyl)adenosine).31,32 Affinity at recombinant human
A3 receptors expressed in CHO cells33 was determined
Another regioisomer, 5-amino[1,3,4]thiadiazole derivative
10 was prepared from the reaction of p-anisaldehyde
with thiosemicarbazide to give intermediate 9, followed
by cyclization in the presence of ferric chloride in
aqueous solution (Scheme 3).30 The synthesized and
commercially available aminothiazole and aminothia-
using
[
125I]AB-MECA
(N6-(4-amino-3-[125I]iodo-
benzyl)-50-(N-methyl carbamoyl)-adenosine.34
The representative thiadiazole analogue reported earlier
by the IJzerman’s group27 was N-(3-phenyl-1,2,4-thi-
adiazole-5-yl)-4-methoxybenzamide (LUF5417) with Ki
values of 0.032, 2.3, and 0.082 mM at rat A1, rat A2A
,
and human A3 receptors, respectively. In the course of
SAR studies of the derivatives of thiazole and thiadia-
zole templates, we found that 4-methoxyphenyl and
aliphatic acyl group substitutions displayed large
enhancement of the affinity and selectivity for human
adenosine A3 receptors compare to the parent
compound, LUF5417. For example, 11, a 5-acetamido
analogue of phenylthiazole showed a Ki value of 18.3
nM at human adenosine A3 receptors with high selectivity
versus human A1 and A2A receptors and 16 with 4-
methoxy substitution of the phenyl group of 11, displayed
a 6-fold increase of binding affinity at A3 receptors with
a Ki value of 3 nM, while maintaining high selectivity
(Table 1). Structural variations and binding assays
following aliphatic and aromatic acyl substitutions at
the 5-amino group identified a slightly more potent
analogue, 20, which contained one more methylene unit
than 16 and displayed a Ki value of 2.4 nM. Although
most of the aromatic acyl substituted derivatives 26–35
Scheme 1. Synthesis of 2-aminothiazole skeleton.
Scheme 2. Synthesis of 5-amino[1,2,4]thiadiazole skeleton.
Scheme 3. Synthesis of 5-amino[1,3,4]thiadiazole skeleton.

K.-Y. Jung et al. / Bioorg. Med. Chem. 12 (2004) 613–623
615
Table 1. Binding affinities of thiazole and thiadiazole derivatives at adenosine receptors
Compd
R
X
Y
Ki (nM) or % inhibition at 10 mM
Human A1
Human A2A
Human A3
LUF541727
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
4-OCH3C6H4
CH3
(CH3)3CO
NCCH2
CH3
C6H5CH2
CH3
CH3
CH3
CF3
N
H
H
H
H
4-Cl
32 (rat A1)
59%
11%
50%
0%
2,300 (rat A2A
74%
31%
68%
27%
0%
)
82ꢁ4
18.3ꢁ4.2
5,100ꢁ1,130
204ꢁ17
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
50.9ꢁ23.5
4-Cl
0%
100
3.0ꢁ0.8
4-CH3O
3-CH3O
2-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
4-CH3O
35%
34%
18%
0%
22%
49%
49%
33%
69%
0%
0%
17%
0%
0%
0%
0%
0%
41%
10%
21%
11%
56%
19%
24%
7%
33%
61%
63%
16%
47%
48%
56%
40%
20%
0%
14%
27%
28%
0%
12%
18%
17%
35%
7%
4.1ꢁ1.8
82ꢁ21
530ꢁ91
CH3CH2
2.4ꢁ0.6
CH3CH2CH2
(CH3)2CH
NCCH2
(CH3)3C
(CH3)3O
C6H5
7.8ꢁ1.5
16.3ꢁ4.1
24.3ꢁ3.9
31.9ꢁ4.2
3,260ꢁ340
28.7ꢁ3.3
14.2ꢁ3.0
29.1ꢁ9.3
1,160ꢁ190
28.6ꢁ7.3
526ꢁ193
400
C6H5CH2
C6H5CH2CH2
p-CH3OC6H4CH2
p-CH3OC6H4CH2CH2
(C6H5)2CH
(C6H5)2CHCH2
2-Furan
31.5ꢁ1.3
32.3ꢁ7.1
69.3ꢁ10.0
373ꢁ74
Thiophene-2-CH2
2-Thiophene
23%
0%
CH3
C6H5CH2
CH3
C6H5CH2
CH3CH2
CH3
N
N
N
N
N
H
H
78%
73%
28%
18%
3530ꢁ600
0%
2.3ꢁ0.2
79ꢁ26
4-CH3O
4-CH3O
4-CH3O
0.79ꢁ0.18
23.8ꢁ0.3
1.13ꢁ0.14
4,690ꢁ1,380
>100,000
47%
0%
0%
43
C6H5CH2
0%
showed lower binding affinities than 16 or 20, the binding
affinities were two-digit nanomolar Ki values except for
29, 31, and 32, which possessed an extra phenyl group,
suggesting a hydrophobic binding pocket of limited
space in the receptor for the recognition of the acylamino
moieties.
16 and 20, showed a Ki value of 1.13 nM at A3 receptors,
again with high subtype selectivity. Interestingly, the
affinity of another regioisomer of 39, N-(2-(4-methoxy-
phenyl)-[1,3,4]thiadiazol-5-yl)-acetamide,
42
was
dramatically decreased to a Ki value of 4.7 mM.
In terms of species differences, we attempted to determine
the receptor binding affinity at rat A1 receptors, since
the parent compound LUF5417 displayed high affinity
at the receptors. However, most of the derivatives
displayed very weak binding affinities with Ki values
>50 mM at the rat A1 and A2A receptors. Also binding
to the rat adenosine A3 receptors was weak. Ki values
were 3.03 mM for compound 33 and >10 mM for
compounds 17, 39 and 40.
In an effort to further enhance the A3 binding affinity,
we applied the acetamido functionality to a thiadiazole
template. N-(3-Phenyl-[1,2,4]thiadiazol-5-yl)-acetamide,
37, showed an 8-fold increase of binding affinity, with a
Ki value of 2.3 nM at A3 receptors, than the corre-
sponding thiazole derivative, 11. Thus, we could assume
that 4-methoxyphenyl substitution would afford a further
increase of affinity, which encouraged us to explore
synthesizing 3-(4-methoxy-phenyl)-[1,2,4]thiadiazol-5-
ylamine, 7. An acetylated derivative of 7, that is, 39, was
characterized as a selective and potent antagonist for
human A3 receptors with subnanomolar affinity, Ki
value of 0.79 nM. The corresponding propionyl analo-
gue, 41, which was expected to be slightly more potent
than 37 based on a comparision in the thiazole series of
4. Molecular modeling
In order to explain the different binding affinities of two
regioisomers, 39 and 42, we calculated the thermo-
dynamically stable conformations. From the result of

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K.-Y. Jung et al. / Bioorg. Med. Chem. 12 (2004) 613–623
the PM3 method, the lowest HF (Heat of Formation) of
1,2,4- and 1,3,4-thiadiazole compounds, 39 and 42, were
ꢀ20.09 and 12.17 kcal/mol, respectively. Thus, the most
potent one, 39, was more thermodynamically stable
than the other, poor binding isomer 42 by >30 kcal.
The lowest energy conformers of both derivatives were
used for docking to the human adenosine A3 receptor.41
We used an automated docking procedure (FlexiDock)
with manual adjustment to determine the most energe-
tically favorable binding location and orientation of the
antagonist 39 (Fig. 1A). The result of FlexiDock applied
to 39 displayed the possibility of a few complex models
clustering within small energy differences. The complex
model with the lowest energy among several docking
models was well correlated with SAR and selectivity
data in binding to the human A3 receptor. The bound
conformation of the amide showed trans-geometry with
4 kcal higher energy than the lowest one with a cis-
amide bond obtained using the PM3 method. The high
affinity antagonist 39, a 1,2,4-thiadiazole derivative,
interacted through the H-bonding of the CO group with
Q167 in EL2, which was a unique residue in human A3
receptors in relation to other subtypes of adenosine
receptors; The homologous residue was E for A1, L for
A2 adenosine receptors (Fig. 1). This hydrophilic inter-
action appears to explain why compound 39 is selective
for the human adenosine A3 receptors. In addition,
S1815.42 formed H-bonding with the N atom of the
thiadiazole ring. The amino acid corresponding to
S1815.42 in other adenosine receptor subtypes was N.
This additional H-bonding to the thiadiazole ring, absent
with the thiazole derivatives, could contribute to the
increase of human adenosine A3 receptor affinity. The
thiadiazole and phenyl rings were surrounded by many
hydrophobic amino acids; F168EL2, F1825.43, I1865.47
,
and L2466.51. The methyl group of the 4-methoxy phenyl
ring substituent showed a hydrophobic interaction with
I1865.47 and W2436.48. S2476.52, which corresponds to H
in other adenosine receptor subtypes, and was very close
to the O of the methoxy group. The distance between the
oxygen atom of the methoxy group and the hydroxyl
group of S was 4.2 A. The methyl of the acetamide was
oriented toward EL2, indicating a hydrophobic interac-
tion of methyl with I2536.58. In proximity of the terminal
methyl group, there was S170 in EL2, suggesting that a
hydrophilic group in preference to a hydrophobic group,
aromatic ring or aliphatic chain, might increase the
binding affinity to the human adenosine A3 receptor.
Figure 1. The complex of the human adenosine A3 receptors with two
regioisomers in the putative binding site of the receptor model. (A) a
1,2,4-thiadiazole compound 39 and (B) 1,3,4-thiadiazole analogue 42.
All ligands are displayed as ball-and-stick models, and the side chains
of human A3 receptors are shown as line models. The H-bonding
between ligand and the receptor is displayed in yellow. The human A3
receptor is represented by a tube model with a different color for each
TM domain (TM3 in yellow, TM4 in green, TM5 in cyan, TM6 in
blue, TM7 in purple).
In the case of the 1,3,4 analogue 42, there was no
H-bonding in its complex when docked in the binding
site of its isomer 39, correlating with its binding data;
that is, about 6000-fold decrease of the binding affinity
for the human adenosine A3 receptor (Fig. 1B). The
total energy of its complex was 9 kcal higher than that
of the complex of the 1,2,4-analogue 39. Thus, the
molecular modeling results supported the docking of the
1,2,4-thiadiazole 39 being energetically more favorable
than the 1,3,4-derivative 42.
reliability of the docked complexes, additional mutational
experiments and more SAR data are needed, because
theoretically there are other possible models of
complexes within small energy differences.
The adenosine receptor sequence alignment indicated
that most of the amino acids in the putative binding site
within 5A of the A3-selective antagonist 39 were
Thus, the result of FlexiDock was consistent with the
current binding results. However, to confirm the

K.-Y. Jung et al. / Bioorg. Med. Chem. 12 (2004) 613–623
617
conserved among adenosine receptors. Highly
conserved amino acids were T943.36, V1785.39, F168EL2
F1825.43, W2436.48, and N2506.55. However, some vari-
able residues among the four subtypes of ARs, H953.37
Q167EL2, S170EL2, S2476.52, and I253 6.58, were located
near R, X, and Y positions of the thiazole compound.
Thus, variation of substituents in proximity to R, X and
Y positions would be expected to increase the affinity
and selectivity for the human A3 subtype through
additional H-bonding or hydrophobic interactions.
6. Experimental
,
6.1. Chemistry
,
Proton nuclear magnetic resonance spectroscopy was
performed on a Bruker AVANCE 600, JEOL JNM-LA
300WB spectrometers and spectra were taken in
DMSO-d6 or CDCl3. Unless noted, chemical shifts are
expressed as ppm downfield from tetramethylsilane as
the internal standard, and J values are given in Hz.
Mass spectroscopy was carried out on an MALDI-TOF
and FAB instrument. All thiazole and thiadiazole deri-
vatives showed one spot on thin layer chromatography
(Merck silica gel 60; F254, 0.25 mm). Elemental analyses
determined on a Flash EA 1112series (CE Instruments)
analyzer. Elemental and high-resolution mass analyses
were performed in Seoul Branch Analytical Laboratory
of Korea Basic Science Institute.
4.1. Functional assay of cAMP biosynthesis
The most potent derivative, 39, was further evaluated in
a functional assay, which measured the antagonism of
the inhibitory effect on cAMP production via Gi protein
mediated signal transduction system upon activation of
the adenosine A3 receptor. Compound 39 displayed
functional antagonistic properties with antagonism of
inhibition of cAMP production by Cl-IB-MECA, a
selective A3 receptor agonist in a concentration-dependent
manner with KB value of 1.7 nM (Fig. 2).
6.1.1. General procedure for the synthesis of 2-amino-4-
[methoxy (or 4-chloro)-phenyl]-thiazole analogues (3). 1
mmol of 2-bromo-methoxy(or 4-chloro)acetophenone, 5
mmol of KSCN/SiO2, and 18 mmol of NH4OAc/Al2O3
were added in 15 mL of dry benzene. After stirring at
90 ꢂC for 16 h, the yellow mixture was filtered and dried
under reduced pressure. The residue was purified by
silica gel column chromatography (CHCl3/MeOH, 50:1–
30:1) to afford the desired compound (3a–d). Yield 60%.
5. Conclusion
In summary, we have identified antagonists for the
human adenosine A3 receptor having subnanomolar
affinity and high selectivity versus other subtype recep-
tors through structure–activity relationship studies of
aminothiazole and aminothiadiazole as the lead
templates. An assay of cAMP production also demon-
strated functional inhibition by the most potent
antagonist 39, and speculation of a binding mode of
thiadiazole isomers was suggested by molecular docking
experiments with a human adenosine A3 receptor model
built from homology modeling of the X-ray crystal
structure of rhodopsin. The potent human adenosine A3
receptor antagonists developed in this study will be
useful to investigate biological significance of adenosine
receptors and to develop therapeutics for the treatment
of inflammatory disease, such as asthma, and glaucoma.
6.1.2. 5-Amino-3-(ꢀ40-methoxyphenyl)-1,2,4-thiadiazole
(7). Was synthesized from 4-methoxybenzonitrile 4. A
100 mL 2-necked round-bottom flask was charged with
30 mL of anhydrous MeOH, 1.0 g (7.5 mmol) of the 4-
methoxybenzonitrile 4, and 0.04 g (0.75 mmol) of
sodium methoxide. The contents of the flask were pro-
tected from moisture and stirred magnetically for 48 h.
Then, 0.4 g (7.5 mmol) of NH4Cl was added and stirring
was continued for 24 h. Unreacted NH4Cl was filtered,
and methanol was stripped from the filtrate to afford
the crude product, 4-methoxybenzamidine 5, which was
washed ether to remove unreacted 4-methoxybenzoni-
trile and used for the next step without further purific-
ation. 2.2 g (14.6 mmol) of 5 was dissolved into 50 mL
water and mixed with 1 mL of sodium dodecylsulfate (as
emulsion) and 3.2 mL (29.2 mmol) of perchloromethyl
mercaptan. To the mixture was added a solution of 1.6 g
of NaOH in 50 mL of water (0.04 M) dropwise,
maintaining the temperature under 10 ꢂC. Stirring was
continued for another 15 min. The product color was
changed to the orange-yellow, which was washed with
cold water and dried under reduced pressure. The crude
product was purified by silica gel column chromato-
graphy (hexanes–EtOAc, 30:1) to give 5-chloro-3-(40-
methoxyphenyl)-1,2,4-thiadiazole 6 (Yield 10%). 1H
NMR (600 MHz, DMSO-d6) d 3.84 (3H, s, CH3O), 7.10
(2H, d, J=7.2 Hz, phenyl-3H), 8.11 (2H, d, J=7.2 Hz,
phenyl-2H); MS [MALDI-TOF] m/z=226.7 (M).
Compound 6 was converted to 5-amino-3-(40-methox-
yphenyl)-1,2,4-thiadiazole 7 (Yield 34%) and 5-methy-
lamino-3-(40-methoxyphenyl)-1,2,4-thiadiazole 8 (Yield
74%) in ethanolic ammonia and methylamine at room
temperature, respectively. 7: 1H NMR (300 MHz,
DMSO-d6) d 3.80 (3H, s, CH3O), 7.01 (2H, d, J=9.0
Hz, phenyl-3H), 7.97 (2H, s, NH2), 8.00 (2H, d, J=9.0
Figure 2. Antagonism by compound 39 of the inhibition of cyclic
AMP production elicited by 100 nM Cl-IB-MECA in CHO cells stably
transfected with the human adenosine A3 receptors. The experiment
was performed in the presence of 10 mM rolipram and 3 units/mL
adenosine deaminase. Forskolin (10 mM) was used to stimulate cyclic
AMP levels. The level of cAMP corresponding to 100% was 220ꢁ30
pmol mLꢀ1. The KB value for compound 39 was calculated to be 1.7 nM.

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K.-Y. Jung et al. / Bioorg. Med. Chem. 12 (2004) 613–623
Hz, phenyl-2H); MS [MALDI-TOF] m/z=207.2 (M); 8:
1H NMR (300 MHz, DMSO-d6) d 2.97 (3H, d, J=4.8,
CH3), 3.84 (3H, s, CH3O), 7.02 (2H, d, J=9.0 Hz,
phenyl-3H), 8.04 (2H, d, J=9.0 Hz, phenyl-2H), 8.42
(1H, s, NH); MS [MALDI-TOF] m/z=221.2 (M).
methoxyphenyl)-thiazole 3a (0.19 mmol), 74.4 mg of
EDAC (0.388 mmol), 71.1 mg of dimethylaminopyridine
(0.582 mmol) and appropriate acid (0.388 mmol) in 3 mL
of anhydrous dichloromethane was stirred at room tem-
perature for 2 h. The solvent was removed under reduced
pressure, and the residue was partitioned between satu-
rated ammonium chloride (30 mL) and CHCl3 (3ꢃ20
mL). The organic layer was dried on Na2SO4 and
evaporated under vacuum. The residue was purified by
column chromatography on silica gel (hexanes–EtOAc,
6:1) to afford the final products as solids (30 and 32).
6.1.3.
5-Amino-2-(40-methoxyphenyl)-1,3,4-thiadiazole
(10). 10 g (0.11 mol) of thiosemicarbazide and 13.3 g
(0.11 mol) of p-anisaldehyde were dissolved in 30 mL
ethanol. The mixture was stirred at room temperature
for 7 h and filtered under reduced pressure. The residue
was washed with ethanol to remove the side-products,
and dried under vacuum oven to afford 19.83 g of 9
6.1.8. N-(4-Phenyl-thiazol-2-yl)-acetamide (11). Yield
20%; ocher yellow powder; 1H NMR (600 MHz,
CDCl3) d 1.94 (3H, s, CH3), 7.14 (1H, d, J=7.0 Hz,
phenyl-4H), 7.34 (1H, s, thiazole-3H), 7.41 (2H, dd,
J=6.5, 7.0 Hz, phenyl-3H), 7.81 (2H, d, J=6.5 Hz,
phenyl-2H), 10.67 (1H, s, NH); HRMS calcd for
C11H10N2OS (MH+) 219.0514, found 219.0511.
1
(86%). H NMR (600 MHz, DMSO-d6) d 3.85 (3H, s,
CH3O), 6.92 (2H, d, J=8.4 Hz, phenyl-3H), 7.02 (1H, s,
NCH), 7.60 (2H, d, J=8.4 Hz, phenyl-2H), 7.85 (2H,
s, NH2), 10.07 (1H, s, SCNH); MS [MALDI-TOF]
m/z=209.3 (M). A mixture of 1.67 g (8 mmol) of 9, 360
mL of water and 6.0 g (22 mmol) of ferric chloride
hexahydrate was stirred and heated at 80–90 ꢂC for one-
half hour, then filtered while hot. The filtrate was
concentrated under reduced pressure and the residue
was treated with 10% aqueous ammonia to an alkaline
solution. The insoluble material was filtered, dried and
purified by silica gel column chromatography (CHCl3–
MeOH, 20:1). Yield 5.6%. 10: 1H NMR (300 MHz,
DMSO-d6) d 3.80 (3H, s, CH3O), 7.00 (2H, d, J=9.0
Hz, phenyl-3H), 7.29 (2H, s, NH2), 7.66 (2H, d, J=9.0
Hz, phenyl-2H); MS [MALDI-TOF] m/z=207.3 (M).
6.1.9. (4-Phenyl-thiazol-2-yl)-carbamic acid tert-butyl
ester (12). Yield 19.1% orange-yellow powder; 1H
NMR (600 MHz, CDCl3) d 1.61 (9H, s, (CH3)3CO),
7.08 (1H, d, J=7.0 Hz phenyl-4H), 7.37 (1H, s, thia-
zole-3H), 7.39-7.41 (2H, dd, J=6.5, 7.0 Hz, phenyl-3H),
7.89 (2H, d, J=6.5 Hz, phenyl-2H), 11.45 (1H, s, NH).
HRMS calcd for C14H16N2O2S (MH+) 277.0932, found
277.0933.
6.1.10. 2-Cyano-N-(4-phenyl-thiazol-2-yl)-acetamide
(13). Yield 52% ochre yellow powder; 1H NMR
(600 MHz, CDCl3) d 3.1 (2H, s, CH2), 7.26 (1H, s,
thiazole-3H), 7.41–7.44 (1H, d, J=7.6 Hz, phenyl-4H),
7.47–7.50 (2H, dd, J=7.1, 7.6 Hz, phenyl-3H) 7.79 (2H,
d, J=7.1 Hz, phenyl-2H), 12.61 (1H, s, NH). HRMS
calcd for C12H9N3OS (MH+) 244.0466, found
244.0461.
6.1.4. General procedure for the preparation of N-acyl
substituted thiazole and thiadiazole derivatives. Method
A.
A mixture of appropriate aminothiazoles or
aminothiadiazoles (1 mmol), pyridine (2 mmol) and acyl
chloride (3 mmol) in 5 mL of anhydrous dichloro-
methane was stirred at room temperature for 1 h. The
solvent was removed under reduced pressure, and the
residue was partitioned between saturated ammonium
chloride solution (30 mL) and CHCl3 (3ꢃ20 mL). The
organic layer was dried on Na2SO4 and evaporated under
vacuum, and the residue was purified by silica gel column
chromatography (hexanes–EtOAc, 3:1) to afford the
product as solid (11, 14–18, 20–22, 24, 26–29, 31, 33–43).
6.1.11. N-[4-(4-Chloro-phenyl)-thiazol-2-yl]-acetamide
1
(14). Yield 86.1% white powder; H NMR (300 MHz,
DMSO-d6) d 2.16 (3H, s, CH3), 7.50 (2H, d, J=8.4 Hz,
phenyl-3H), 7.67 (1H, s, thiazole-3H), 7.90 (2H, d,
J=8.4 Hz, phenyl-2H), 12,31 (1H, s, NH); MS
[MALDI-TOF] m/z=252.2 (M). Anal. calcd for
C11H9ClN2OS: C, 52.28; H, 3.59; N, 11.08. Found: C,
51.96; H, 3.44; N, 11.27.
6.1.5. Method B. The appropriate aminothiazole (0.242
mmol) was dissolved in absolute EtOH (3 mL), and
ethyl cyanoacetate (0.726 mmol) and sodium ethoxide
(0.726 mmol) were added. The mixture was refluxed for
3 h, then, after cooling, the solvent was removed under
reduced pressure and the crude product was purified by
column chromatography on silica gel (CHCl3/MeOH,
50:1) to afford the product as solid (13 and 23).
6.1.12. N-[4-(4-Chloro-phenyl)thiazol-2-yl]-2-phenyl-acet-
1
amide (15). Yield 98.6% light green powder; H NMR
(300 MHz, DMSO-d6) d 3.76 (2H, d, J=7.5 Hz,
CH2C6H5), 7.14–7.48 (5H, m, phenyl-4H+phenyl-
3H+phenyl 2H), 7.50 (2H, d, J=8.7 Hz, phenyl-3H),
7.68 (1H, s, thiazole-3H), 7.90 (2H, d, J=8.7 Hz,
phenyl-2H), 12.52 (1H, s, NH). HRMS calcd for
C17H13ClN2OS (MH+) 329.0437, found 329.0433.
6.1.6. Method C. To the appropriate aminothiazole (0.2
mmol) in 3 mL of absolute dichloromethane was added
dimethylaminopyridine (0.3 mmol) and corresponding
anhydride (0.4 mmol). The mixture was stirred at room
temperature for 3 h and the solvent was removed under
reduced pressure. The residue was purified by column
chromatography on silica gel (hexanes–EtOAc, 6:1) to
afford the final products (12, 19 and 25).
6.1.13. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-acetamide
(16). Yield 20% light green powder; 1H NMR
(300 MHz, DMSO-d6) d 3.77 (3H, s, CH3), 6.97 (2H, d,
J=8.7 Hz, phenyl-3H), 7.41 (1H, s, thiazole-3H), 7.80
(2H, d, J=8.7 Hz, phenyl-2H), 12.19 (1H, s, NH).
HRMS cald for C12H12N2O2S (MH+) 249.0619, found
249.0627.
6.1.7. Method D. A mixture of 40 mg of 2-amino-4-(4ꢀ-

K.-Y. Jung et al. / Bioorg. Med. Chem. 12 (2004) 613–623
619
6.1.14. N-[4-(3-Methoxy-phenyl)-thiazol-2-yl]-acetamide
1
(2H, d, J=3.6 Hz, CH2), 6.98 (2H, d, J=8.4 Hz,
phenyl-3H), 7.52 (1H, s, thiazole-3H), 7.82 (2H, d,
J=8.4 Hz, phenyl-2H), 12.56 (1H, s, NH). HRMS
calcd for C13H11N3O2S (MH+) 274.0572, found
274.0583.
(17). Yield 62.1% white powder; H NMR (300 MHz,
DMSO-d6) d 2.16 (3H, s, CH3), 3.79 (3H, s, CH3O),
0
.
6.90 (1H, d, J=8.1 Hz, phenyl-6 H), 7.33 (1H, t, J=8.1
0
.
Hz, phenyl-5 H), 7.43-7.48 (2H, m, phenyl-
20H+phenyl-40H), 7.63 (1H, s, thiazole-3H), 12.26 (1H,
s, NH). HRMS calcd for C12H12N2O2S (MH+)
249.0619, found 249.0618.
6.1.21. 6.1.21.N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-2,2-
dimethyl-propionamide (24). Yield 95% white crystals;
1H NMR (300 MHz, DMSO-d6)
d 1.25 (9H, s,
6.1.15. N-[4-(2-Methoxy-phenyl)-thiazol-2-yl]-acetamide
1
C(CH3)3), 3.78 (3H, s, CH3O), 6.99 (2H, d, J=12.0 Hz,
phenyl-3H), 7.43 (1H, s, thiazole-3H), 7.85 (2H, d,
J=12.0 Hz, phenyl-2H), 11.79 (1H, s, NH); MS
[MALDI-TOF] m/z=289.6 (M). Anal. calcd for
C15H18N2O2S: C, 62.04; H, 6.25; N, 9.65. Found: C,
62.33; H, 6.34; N, 9.43.
(18). Yield 99.3% white powder; H NMR (300 MHz,
DMSO-d6) d 2.16 (3H, s, COCH3), 3.91 (3H, s, CH3O),
7.03 (1H, t, J=7.5 Hz, phenyl-50 H), 7.13 (1H, d, J=7.5
Hz, phenyl-60 H), 7.28 (1H, dd, J=7.5, 7.8 Hz, phenyl-
40 H ), 7.62 (1H, s, thiazole-3H), 8.05 (1H, d, J=7.8
Hz, phenyl-30 H), 12.18 (1H, s, NH); MS [MALDI-
TOF] m/z=248.2 (M). Anal. calcd For C12H12N2O2S:
C, 58.05; H, 4.87; N, 11.28. Found: C,57.93; H, 4.49;
N, 11.08.
6.1.22.
[4-(4-Methoxy-phenyl)-thiazol-2-yl]-carbamic
acid tert-butyl ester (25). Yield 12% orange-yellow
crystals; 1H NMR (300 MHz, CDCl3) d 1.60 (9H, s,
OC(CH3)3), 3.84 (3H, s, CH3O), 6.93 (2H, d. J=8.4 Hz,
phenyl-3H), 7.26 (1H, s, thiazole-3H), 7.80 (2H, d,
J=8.4 Hz, phenyl-2H), 11.44 (1H, s, NH). HRMS calcd
for C15H18N2O3S (MH+) 307.1038, found 307.1032.
6.1.16. 2,2,2-Trifluoro-N-[4-(4-methoxy-phenyl)-thiazol-
2-yl]-acetamide (19). Yield 20.7% white powder; 1H
NMR (300 MHz, DMSO-d6) d 3.79 (3H, s, CH3O), 7.03
(2H, d, J=9.0 Hz, phenyl-3H), 7.57 (1H, s, thiazole-
3H), 7.82 (2H, d, J=9.0 Hz, phenyl-2H), 14.12 (1H,
broad s, NH); MS [MALDI-TOF] m/z=302.1 (M).
Anal. calcd for C12H9F3N2O2S: C, 47.68; H, 3.00; N,
9.27. Found: C, 47.38; H, 2.80; N, 9.08.
6.1.23. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-benzamide
1
(26). Yield 56.1% white powder; H NMR (300 MHz,
DMSO-d6) d 3.79 (3H, s, CH3O), 6.99 (2H, d, J=8.7
Hz, phenyl-3H), 7.51–7.64 (3H, m, COC6H5-
H3+COC6H5-H4), 7.53 (1H, s, thiazole-3H), 7.87 (2H,
d, J=8.7 Hz, phenyl-2H), 8.14 (2H, d, J=8.1 Hz,
COC6H5-H2), 12.83 (1H, s, NH). HRMS calcd for
C17H14N2O2S (MH+) 311.0776, found 311.0773.
6.1.17. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-propiona-
mide (20). Yield 74.7% white powder; 1H NMR
(300 MHz, DMSO-d6) d 1.1 (3H, t, J=7.5 Hz, CH3),
2.46 (2H, q, J=7.5 Hz, CH2), 3.78 (3H, s, CH3O), 6.97
(2H, d, J=9.0 Hz, phenyl-3H), 7.42 (1H, s, thiazole-
3H), 7.83 (2H, d, J=9.0 Hz, phenyl-2H), 12.24 (1H, s,
NH); MS [MALDI-TOF] m/z=262.4 (M). Anal. calcd
for C13H14N2O2S: C, 59.52; H, 5.38; N, 10.68. Found:
C, 59.43; H, 5.52; N, 10.39.
6.1.24. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-2-phenyl-
1
acetamide (27). Yield 41.8% white powder; H NMR
(300 MHz, DMSO-d6) d 3.79 (2H, s, CH2), 3.79 (3H, s,
CH3O), 6.97 (2H, d, J=8.7 Hz, phenyl-3H), 7.24-7.35
(5H, m, C6H5-2H+C6H5-3H+C6H5-4H), 7.44 (1H, s,
thiazole-3H), 7.83 (2H, d, J=8.7 Hz, phenyl-2H), 12.47
(1H, s, NH). HRMS calcd for C18H16N2O2S (MH+)
325.0932, found 325.0931.
6.1.18.
N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-butyra-
mide (21). Yield 82.2% white powder; 1H NMR
(300 MHz, DMSO-d6) d 0.90 (3H, t. J=7.3 Hz, CH3),
1.59–1.66 (2H, m, CH2CH3), 2.41 (2H, t, J=7.3 Hz,
COCH2), 3.78 (1H, s, CH3O), 6.99 (2H, d, J=4.8 Hz,
phenyl-3H), 7.43 (1H, s, thiazole-3H), 7.82 (2H, d,
J=4.8 Hz, phenyl-2H), 12.36 (1H, s, NH); MS
[MALDI-TOF] m/z=276.1 (M). Anal. calcd for
C14H16N2O2S: C, 60.85; H, 5.84; N, 10.14. Found: C,
60.53; H, 5.91; N, 9.92.
6.1.25. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-3-phenyl-
propionamide (28). Yield 80.6% white powder; 1H
NMR (300 MHz, DMSO-d6) d 2.76 (2H, t, J=8.1 Hz,
CH2C6H5), 2.90 (2H, t, J=8.1 Hz, COCH2), 3.78 (3H,
s, CH3O), 6.99 (2H, d, J=8.7 Hz, phenyl-3H), 7.21-7.29
(5H, m, C6H5-2H+C6H5-3H+C6H5-4H), 7.43 (1H, s,
thiazole-3H), 7.80 (2H, d, J=8.7 Hz, phenyl-2H), 12.25
(1H, s, NH). HRMS calcd for C19H18N2O2S (MH+)
339.1089, found 339.1081.
6.1.19. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-isobutyra-
mide (22). Yield 71.6% white crystals; 1H NMR
(300 MHz, DMSO-d6) d 1.10 (6H, d, J=7.2 Hz,
CH(CH3)2), 2.75 (1H, m, CH(CH3)2), 3.78 (1H, s,
CH3O), 6.99 (2H, d, J=8.7 Hz, phenyl-3H), 7.43 (1H,
s, thiazole-3H), 7.82 (2H, d, J=8.7 Hz, phenyl-2H),
12.18 (1H, s, NH); MS [MALDI-TOF] m/z=276.7
(M). Anal. calcd for C14H16N2O2S: C, 60.85; H, 5.84;
N, 10.14. Found: C, 60.71; H, 5.89; N, 10.01.
6.1.26. 2-(4-Methoxy-phenyl)-N-[4-(4-methoxy-phenyl)-
thiazol-2-yl]-acetamide (29). Yield 61.1% lemon-yellow
1
powder; H NMR (300 MHz, DMSO-d6) d 3.69 (2H, s,
COCH2), 3.73 (3H, s, CH3OC6H4CH2), 3.78 (3H, s,
CH3O), 6.88 (2H, d, J=8.4 Hz, CH2C6H4OCH3-3H),
6.97 (2H, d, J=8.7 Hz, thiazole-C6H4-3H), 7.25 (2H, d,
J=8.4 Hz, CH2C6H4OCH3-2H), 7.44 (1H, s, thiazole-
3H), 7.83 (2H, d, J=8.7 Hz, thiazole-C6H4-2H), 12.32
(1H, s, NH); MS [MALDI-TOF] m/z=354.7 (M). Anal.
calcd for C19H18N2O3S: C, 64.39; H, 5.12; N, 7.90.
Found: C, 64.13; H, 5.01; N, 7.88.
6.1.20. 2-Cyano-N-[4-(4-methoxy-phenyl)-thiazol-2-yl]-
1
acetamide (23). Yield 91.2% ocher yellow powder; H
NMR (300 MHz, DMSO-d6) d 3.79 (3H, s, CH3O), 4.05

620
K.-Y. Jung et al. / Bioorg. Med. Chem. 12 (2004) 613–623
6.1.27. 3-(4-Methoxy-phenyl)-N-[4-(4-methoxy-phenyl)-
thiazol-2-yl]-propionamide (30). Yield 43.4% gray-
brown powder; H NMR (300 MHz, DMSO-d6) d 2.50
6.1.33.
[3-(4-Methoxy-phenyl)-[1,2,4]-thiadiazol-5-yl]-
1
methyl-amine (36). Yield 74% white powder; H NMR
(300 MHz, DMSO-d6) d 2.95 (3H, d, J=4.5 Hz, CH3),
3.80 (3H, s, CH3O), 7.01 (2H, d, J=9.0 Hz, phenyl-3H),
8.03 (2H, d, J=9.0 Hz, phenyl-2H), 8.43 (1H, broad s,
NH). HRMS calcd for C10H11N3OS (MH+) 222.0623,
found 222.0623.
1
(2H, t, J=7.5 Hz, CH2C6H4OCH3), 2.71 (2H, t, J=7.5
Hz, COCH2), 3.70 (3H, s, CH3OC6H4CH2), 3.78 (3H, s,
CH3O), 6.86 (2H, d, J=8.4 Hz, CH2C6H4OCH3-3H),
6.99 (2H, d, J=8.7 Hz, thiazole-C6H4-3H), 7.16 (2H, d,
J=8.7 Hz, thiazole-C6H4-2H), 7.43 (1H, s, thiazole-
3H), 7.82 (2H, d, J=8.4 Hz, CH2C6H4OCH3-2H), 12.36
(1H, s, NH); MS [MALDI-TOF] m/z=367.8(M). Anal.
calcd for C20H20N2O3S: C, 65.20; H, 5.47; N, 7.60.
Found: C, 64.87; H, 5.40; N, 7.66.
6.1.34. N- (3- Phenyl -[1,2,4]thiadiazol -5-yl) -acetamide
1
(37). Yield 67.8% white crystals; H NMR (300 MHz,
DMSO-d6) d 2.27 (3H, s, CH3), 7.51 (1H, d, J=5.1 Hz,
phenyl-4H), 7.54 (2H, d, J=4.5 Hz, phenyl-2H), 8.16
(2H, dd, J=4.5, 5.1 Hz, phenyl-3H), 13.10 (1H, broad s,
NH); MS [MALDI-TOF] m/z=219.0 (M). Anal. calcd
for C10H9N3OS: C, 54.78; H, 4.14; N, 19.16. Found: C,
54.48; H, 4.01; N, 19.02.
6.1.28. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-2,2-diphe-
nylacetamide (31). Yield 39.5% white powder; 1H
NMR (300 MHz, DMSO-d6) d 3.78 (3H, s, CH3O), 5.37
(1H, s, CH(C6H5)2), 6.99 (2H, d, J=8.7 Hz,
CH3OC6H4-3H), 7.22–7.40 (10H, m, CH(C6H5)2), 7.48
(1H, s, thiazole-3H), 7.82 (2H, d, J=8.7 Hz,
CH3OC6H4-2H), 12.67 (1H, s, NH). HRMS calcd for
C24H20N2O2S (MH+) 401.1245, found 401.1243.
6.1.35. 2-Phenyl-N-(3-phenyl-[1,2,4]thiadiazol-5-yl)-acet-
amide (38). Yield 41.2% white powder; 1H NMR
(300 MHz, DMSO-d6) d 3.91 (2H, s, CH2), 7.21–7.36
(5H, m, phenyl-2H+phenyl-3H+phenyl-4H), 7.51 (1H,
d, J=2.1 Hz, phenyl-4H), 7.54 (2H, d, J=4.5 Hz,
phenyl-2H), 8.17 (2H, dd, J=2.1, 4.5 Hz, phenyl-3H),
12.93 (1H, s, NH). HRMS calcd for C16H13N3OS
(MH+) 296.0799, found 296.0791.
6.1.29. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-3,3-diphe-
nyl-propionamide (32). Yield 79.3% light-brown
1
crystals; H NMR (300 MHz, DMSO-d6) d 3.26 (2H, d,
J=8.1 Hz, CH2), 3.78 (3H, s, CH3O), 4.61 (1H, s,
CH(C6H5)2), 6.99 (2H, d, J=8.7 Hz, CH3OC6H4-3H),
7.14–7.2 (5H, m, phenyl-2H+phenyl-3H+phenyl-4H),
7.25–7.33 (5H, m, phenyl-2H+phenyl-3H+phenyl-4H),
7.39 (1H, s, thiazole-3H), 7.78 (2H, d, J=8.7 Hz,
CH3OC6H4-2H), 12.67 (1H, s, NH); MS [MALDI-TOF]
m/z=413.8 (MS). Anal. calcd for C25H22N2O2S: C,
72.44; H, 5.35; N, 6.76. Found: C, 72.26; H, 5.62; N, 6.58.
6.1.36. N-[3-(4-Methoxy-phenyl)-[1,2,4]thiadiazol-5-yl]-
1
acetamide (39). Yield 74.2% white powder; H NMR
(300 MHz, DMSO-d6) d 2.26 (3H, s, CH3), 3.82 (3H, s,
CH3O), 7.04 (2H, d, J=8.7 Hz, phenyl-3H), 8.07 (2H,
d, J=8,7 Hz, phenyl-2H), 13.05 (1H, s, NH). HRMS
calcd for C11H11N3O2S (MH+) 250.0572, found
250.0571.
6.1.30. Furan-2-carboxylic acid [4-(4-methoxy-phenyl)-
thiazol-2-yl]-amide (33). Yield 97.8% light-brown
powder; H NMR (300 MHz, DMSO-d6) d 3.79 (3H, s,
CH3O), 6.74 (1H, dd, J=1.5, 2.1 Hz, furan-3H), 7.0
(2H, d, J=9.0 Hz, phenyl-3H), 7.51 (1H, s, thiazole-
3H), 7.71 (1H, d, J=1.5 Hz, furan-2H), 7.88 (2H, d,
J=9.0 Hz, phenyl-2H), 8.02 (1H, d, J=2.1 Hz, furan-
4H), 12.61 (1H, s, NH). HRMS calcd for C15H12N2O3S
(MH+) 301.0569, found 301.0571.
6.1.37. N-[3-(4-Methoxy-phenyl)-]1,2,4]thiadiazol-5-yl]-
1
2-phenyl-acetamide (40). Yield 81.0% white powder; H
1
NMR (300 MHz, DMSO-d6) d 3.82 (3H, s, CH3O), 3.90
(2H, s, CH2), 7.05 (2H, d, J=9.0 Hz, phenyl-3H), 7.25–
7.36 (5H, m, C6H5-2H+C6H5-3H+C6H5-4H), 8.08
(2H, d, J=9.0 Hz, phenyl-2H), 13.25 (1H, s, NH).
HRMS calcd for C17H15N3O2S (MH+) 326.0885, found
326.0889.
6.1.38. N-[3-(4-Methoxy-phenyl)-[1,2,4]thiadiazol-5-yl]-
propionamide (41). Yield 52.0% white powder; 1H
NMR (300 MHz, DMSO-d6) d 1.10 (3H, t, J=7.5 Hz,
CH3), 2.53 (2H, q, J=7.5 Hz, CH2), 3.82 (3H, s,
CH3O), 7.04 (2H, d, J=9.0 Hz, phenyl-3H), 8.08 (2H,
d, J=9.0 Hz, phenyl-2H), 12.97 (1H, s, NH). HRMS
calcd for C12H13N3O2S (MH+) 264.0728, found
264.0731.
6.1.31. N-[4-(4-Methoxy-phenyl)-thiazol-2-yl]-2-thio-
phen-2-yl-acetamide (34). Yield 11.7% light-gray
powder; H NMR (300 MHz, DMSO-d6) d 3.79 (3H, s,
CH3O), 4.03 (2H, s, CH2), 6.98–7.12 (2H, m, thiophen-
4H+thiophen-2H), 7.15 (2H, d, J=8.7 Hz, phenyl-3H),
7.42 (1H, t, J=1.5 Hz, thiophen-3H), 7.47 (1H, s,
thiazole-3H), 7.98 (2H, d, J=8.7 Hz, phenyl-2H), 12.51
(1H, s, NH). HRMS calcd for C16H14N2O2S2 (MH+)
331.0497, found 331.0492.
1
6.1.39. N-[5-(4-Methoxy-phenyl)-[1,3,4]thiadiazol-2-yl]-
1
acetamide (42). Yield 79.0% white powder; H NMR
6.1.32. Thiophene-2-carboxylic acid [4-(4-methoxy-
phenyl)-thiazol-2-yl]-amide (35). Yield 18% light-gray
powder; H NMR (300 MHz, DMSO-d6) d 3.79 (3H, s,
CH3O), 6.99 (2H, d. J=8.7 Hz, phenyl-3H), 7.26 (1H,
dd, J=2.7, 3.9 Hz, thiophenyl-3H), 7.52 (1H, s,
thiazole-3H), 7.87 (2H, d, J=8.7 Hz, phenyl-2H), 7.98
(1H, d, J=3.9 Hz, thiophenyl-2H), 8.31 (1H, d, J=2.7
Hz, thiophenyl-4H), 12.87 (1H, s, NH). HRMS calcd
for C15H12N2O2S2 (MH+) 317.0340, found 317.0347.
(300 MHz, DMSO-d6) d 2.20 (3H, s, CH3), 3.83 (3H, s,
CH3O), 7.08 (2H, d, J=9.0 Hz, phenyl-3H), 7.88 (2H,
d, J=9.0 Hz, phenyl-2H), 12.51 (1H, broad s, NH); MS
[MALDI-TOF] m/z=248.9 (M). Anal. calcd for
C11H11N3O2S: C, 53.00; H, 4.45; N, 16.86. Found: C,
52.97; H, 4.33; N, 16.98.
1
6.1.40. N-[5-(4-Methoxy-phenyl)-[1,3,4]thiadiazol-2-yl]-
1
2-phenyl-acetamide (43). Yield 62.3% white powder; H

K.-Y. Jung et al. / Bioorg. Med. Chem. 12 (2004) 613–623
621
NMR (300 MHz, DMSO-d6) d 3.81 (3H, s, CH3O), 3.90
(2H, s, CH2), 7.05 (2H, d, J=9.0 Hz, phenyl-3H), 7.26–
7.35 (5H, m, C6H5-2H+C6H5-3H+C6H5-4H), 7.84
(2H, d, J=9.0 Hz, phenyl-2H), 12.87 (1H, broad s,
NH). HRMS calcd for C18H16N2O2S (MH+) 325.0932,
found 325.0933.
mM EDTA. Nonspecific binding was determined using
10 mM Cl-IB-MECA in the buffer. The mixtures were
incubated at 25 ꢂC for 60 min. Binding reactions were ter-
minated by filtration through Whatman GF/B filters
under reduced pressure using a MT-24 cell harvester
(Brandell, Gaithersburgh, MD, USA). Filters were
washed three times with 9 mL ice-cold buffer. Radio-
activity was determined in a Beckman 5500B g-counter.
6.2. Pharmacology
[
125I]N6-(4-Amino-3-iodobenzyl)adenosine-50-N-methyl-
Intracellular cyclic AMP levels were measured with a
competitive protein binding method.36,37 CHO cells
expressing human or rat adenosine A3 receptors were
harvested following trypsinization. After centrifugation
and resuspended in medium, cells were planted in 24-
well plates in 0.5 mL medium. After 24 h, the medium
was removed and cells were washed three times with 1
mL DMEM, containing 50 mM HEPES, pH 7.4. Cells
were then treated with agonists and/or test compounds
in the presence of rolipram (10 mM) and adenosine
deaminase (3 U/mL). After 45 min forskolin (10 mM)
was added to the medium, and incubation was
continued an additional 15 min. The reaction was
terminated by removing the supernatant, and cells were
lysed upon the addition of 200 mL of 0.1 M ice-cold
HCl. The cell lysate was resuspended and stored at
ꢀ20 ꢂC. For determination of cyclic AMP production,
protein kinase A (PKA) was incubated with [3H]cyclic
AMP (2 nM) in K2HPO4/EDTA buffer (K2HPO4, 150
mM; EDTA, 10 mM), 20 mL of the cell lysate, and 30
mL 0.1 M HCl or 50 mL of cyclic AMP solution (0–16
pmol/200 mL for standard curve). Bound radioactivity
was separated by rapid filtration through Whatman
GF/C filters and washed once with cold buffer. Bound
radioactivity was measured by liquid scintillation
spectrometry.
uronamide (I-AB-MECA; 2000 Ci/mmol), [3H]-(R)-PIA
([3H]-(R)-N6-(phenylisopropyl)adenosine),
[3H]-CG0S
21680 ([3H]-2-[4-(2-carboxyethyl)phenyl]ethylamino]-5 -
(N-ethylcarbamoyl)adenosine), and [3H]cyclic AMP (40
Ci/mmol) were from Amersham Biosciences (Piscataway,
NJ). All other reagents including Cl-IB-MECA (N6-(3-
iodobenzyl)-2-chloro-adenosine-50-N-methyluronamide)
were obtained from Sigma (St. Louis, MO, USA).
CHO (Chinese Hamster Ovary) cells expressing the
recombinant human A1, A3 adenosine receptors and
HEK293 cells expressing the recombinant human
adenosine A2A receptors were cultured in Dulbecco
Modified Eagle’s Medium (DMEM) and F12 (1:1)
supplemented with 10% fetal bovine serum, 100 units/
mL penicillin, 100 mg/mL streptomycin, 2 mmol/mL
glutamine and 800 mg/mL geneticin. After homogeniz-
ation and suspension, cells were centrifuged at 500 g for
10 min, and the pellet was re-suspended in 50 mM Tris–
HCl buffer (pH 8.0) containing 10 mM MgCl2. The
suspension was homogenized with an electric homo-
genizer for 10 s, and was then re-centrifuged at 20,000g
for 20 min at 4 ꢂC. The resultant pellets were resus-
pended in buffer containing 3 U/mL adenosine deami-
nase, and the suspension was stored at ꢀ80 ꢂC. Striatal
and forebrain tissues from Wistar rats were homo-
genized in ice-cold 50 mM TrisHCl buffer, pH 7.4, using
an electric homogenizer. The homogenate was
centrifuged at 20,000g for 10 min at 4 ꢂC, and the pellet
was washed in fresh buffer. The final pellet was stored at
ꢀ80 ꢂC until the binding experiments. The protein
concentration was measured using the Bradford assay.35
6.3. Molecular modeling
All calculations were performed on a Silicon Graphics
(Mountain View, CA) Octane workstation (300 MHz
MIPS R12000 (IP30) processor). All ligand structures
were constructed with the use of the Sketch Molecule of
SYBYL 6.9.38 A conformational search of antagonists
39 and 42 was performed by grid search, rotating three
rotatable bonds in 60ꢂ increments and the amide bond
at 0ꢂ or 180ꢂ. A random search was also performed,
using the following options for all rotatable bonds; 3000
iterations, 3-kcal energy cutoffs, and no chirality check-
ing. In all cases, MMFF force field39 and charge were
applied with the use of distance-dependent dielectric
constants and the conjugate graꢀd1ient method until the
For binding to human A1 receptors, [3H]R-PIA (2 nM)
was incubated with membranes (40 mg/tube) from
CHO cells stably expressing human A1 receptors at
25 ꢂC for 60 min in 50 mM Tris–HCl buffer (pH 7.4;
MgCl2, 10 mM) in a total assay volume of 200 mL.
Nonspecific binding was determined using 10 mM of
N6-cyclopentyladenosine. For human A2A receptor
binding, membranes (20 mg/tube) from HEK-293 cells
stably expressing human A2A receptors were incubated
with 15 nM [3H]CGS21680 at 25 ꢂC for 60 min in 200
mL 50 mM Tris–HCl, pH 7.4, containing 10 mM MgCl2.
50-N-ethyluronamidoadenosine (10 mM) was used to
define nonspecific binding. Reaction was terminated
upon filtration over GF/B filters.
.
gradient reached 0.05 kcal mol
ꢀ1. Representative
.
A
conformers selected from clusters of low-energy con-
formers obtained in the conformational search were reop-
timized by semiempirical molecular orbital calculations
with the PM3 method in the MOPAC 6.0 package.40
A human adenosine A3 receptor model, which was pre-
viously built using homology modeling41 from the X-ray
structure of bovine rhodopsin with 2.8 A resolution42
was used for docking studies. For a brief explanation
of this method, multiple-sequence alignment data of
selected GPCRs were used for the construction of
For binding to recmbinant A3 receptors, each tube
contained 50 mL membrane suspension (20 mg protein),
25 mL of [125I]I-AB-MECA (0.5 nM), and 25 mL of
increasing concentrations of the test ligands in Tris–HCl
buffer (50 mM, pH 8.0) containing 10 mM MgCl2, 1

622
K.-Y. Jung et al. / Bioorg. Med. Chem. 12 (2004) 613–623
human adenosine A3 receptor TM domains..43 For the
model of the second extracellular loop, EL2, two beta-
sheet domains in rhodopsin were first aligned, including
the disulfide bond between Cys83 and Cys166, and then
other components were added or deleted. To construct
the N-terminal region and the intra- and extracellular
loops, each alignment was manually adjusted to
preserve the overall shape of the loop. N-acetyl and
N-methyl amide groups blocked the N-terminal and
C-terminal regions, respectively, to prevent electrostatic
interactions. All helices with backbone constraints and
loops were separately minimized. After combining, all
structures were reminimized initially with backbone
constraints in the secondary structure and then without
any constraints. The Amber all-atom force field44 with a
fixed dielectric constant of 4.0 was used for all calcula-
tions, terminating when the conjugate gradient reached
conformation of antagonist 42 compared to the bound
conformation of antagonist 39 was chosen and super-
imposed into the putative binding sites of several possi-
ble docking models. The energies of the two regioisomer
complexes were compared.
Acknowledgements
This work was supported by the Korea Research
Foundation Grant (KRF-2002-042-E00111 and the
Brain Korea 21 Project).
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.
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