Thiazole and thiadiazole analogues as a novel class of adenosine receptor antagonists

Article abstract of DOI:10.1021/jm0003945

Novel classes of heterocyclic compounds as adenosine antagonists were developed based on a template approach. Structure-affinity relationships revealed insights for extended knowledge of the receptor-ligand interaction. We replaced the bicyclic heterocycl

Full text of DOI:10.1021/jm0003945

J . Med. Chem. 2001, 44, 749-762
749
Th ia zole a n d Th ia d ia zole An a logu es a s a Novel Cla ss of Ad en osin e Recep tor
An ta gon ists
J acqueline E. van Muijlwijk-Koezen,* Hendrik Timmerman, Roeland C. Vollinga,‡
J acobien Frijtag von Drabbe Ku¨nzel,‡ Miriam de Groote,‡ Sven Visser, and Adriaan P. IJ zerman‡
Department of Pharmacochemistry, Division of Medicinal Chemistry, Leiden/ Amsterdam Center for Drug Research,
Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, and Gorlaeus Laboratories,
Division of Medicinal Chemistry, Leiden/ Amsterdam Center for Drug Research, Universiteit Leiden,
P.O. Box 9502, 2300 RA Leiden, The Netherlands
Received September 7, 2000
Novel classes of heterocyclic compounds as adenosine antagonists were developed based on a
template approach. Structure-affinity relationships revealed insights for extended knowledge
of the receptor-ligand interaction. We replaced the bicyclic heterocyclic ring system of earlier
described isoquinoline and quinazoline adenosine A3 receptor ligands by several monocyclic
rings and investigated the influence thereof on adenosine receptor affinity. The thiazole or
thiadiazole derivatives seemed most promising, so we continued our investigations with these
two classes of compounds. The large difference between a pyridine and isoquinoline ring in
binding adenosine A1 and A3 receptors showed the importance of the second ring of the
isoquinoline ligands. We prepared several N-[4-(2-pyridyl)thiazol-2-yl]benzamides, and these
compounds showed adenosine affinities in the micromolar range. Most surprising in the series
of the N-[4-(2-pyridyl)thiazol-2-yl]amides were the retained adenosine affinities by introduction
of a cylopentanamide instead of the benzamide. A second series of compounds, the thiadia-
zolobenzamide series of compounds, revealed potent and selective adenosine receptor antago-
nists, especially N-(3-phenyl-1,2,4-thiadiazol-5-yl)-4-hydroxybenzamide (LUF5437, 8h ) showing
a Ki value of 7 nM at the adenosine A1 receptor and N-(3-phenyl-1,2,4-thiadiazol-5-yl)-4-
methoxybenzamide (LUF5417, 8e) with a Ki value of 82 nM at the adenosine A3 receptor.
4-Hydroxybenzamide 8h is the most potent adenosine A1 receptor antagonist of this new class
of compounds. Structure-affinity relationships showed the existence of a steric restriction at
the para-position of the benzamide ring for binding adenosine A1 and A3 receptors. The electronic
nature of the 4-substituents played an important role in binding the adenosine A3 receptor.
Cis- and trans-4-substituted cyclohexyl derivatives were made next to the 4-substituted
benzamide analogues. We used them to study the proposed specific interaction between the
adenosine A1 receptor and the 4-hydroxy group of this class of thiadiazolo compounds, as well
as a suggested special role for the 4-methoxy group in binding the A3 receptor. Both the
adenosine A1 and A3 receptor slightly preferred the trans-analogues over the cis-analogues,
while all compounds showed low affinities at the adenosine A2A receptor. Our investigations
provided the potent and highly selective adenosine A1 antagonist N-(3-phenyl-1,2,4-thiadiazol-
5-yl)-trans-4-hydroxycyclohexanamide (VUF5472, 8m ) showing a Ki value of 20 nM. A third
series of compounds was formed by urea analogues, N-substituted with thiazolo and thiadiazolo
heterocycles. The SAR of this class of compounds was not commensurate with the SAR of the
previously described quinazoline urea. On the basis of these findings we suggest the existence
of a special interaction between adenosine receptors and a region of high electron density
positioned between the thia(dia)zole ring and phenyl(pyridyl) ring. Molecular electrostatic
potential contour plots showed that for this reason the ligands need either a thiadiazole ring
instead of a thiazole or a 2-pyridyl group instead of a phenyl. The derived novel classes of
antagonists will be useful for a better understanding of the molecular recognition at the
adenosine receptors.
In tr od u ction
adenosine A1 and A3 receptor subtypes are coupled to
the Gi protein, inhibiting adenylate cyclase, A2A and A2B
subtypes stimulate this enzyme via Gs.
Extracellular adenosine regulates several physiologi-
cal functions by activation of specific cell membrane
receptors. The combination of pharmacological studies
and molecular cloning revealed the existence of four
distinct adenosine receptor subtypes which are identi-
fied and classified as A1, A2A, A2B, and A3. Whereas the
The xanthine core structure has served as the basis
for numerous selective antagonists for adenosine A1,
A
2A, and A2B receptors.1,2 The development of selective
antagonists for the A3 receptor has relied on chemically
rather diverse structural leads. Triazolonaphthpyri-
dine,3,4 thiazolopyrimidine,3 1,4-dihydropyridines,5-7
pyridines,8 pyridinium salts,9 pyrans,10 triazoloquinazo-
lines,11,12 flavonoids,13 and triazolopyrimidines14 have
* To whom correspondence should be addressed. Tel: (+31) 20
4447604. Fax: (+31) 20 4447610. E-mail: muylwyk@chem.vu.nl.
‡ Universiteit Leiden.
10.1021/jm0003945 CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/18/2001

750 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
van Muijlwijk-Koezen et al.
Sch em e 1
Ch em istr y
Compounds 2 and 5-13 were prepared from p-anisoyl
chloride and phenyl-substituted heterocyclic amines,
using different circumstances, depending on (1) the
deactivating properties of the heterocyclic ring and (2)
the reactivity of the resulting amide (Scheme 1). In case
of strong deactivation of the amine, such as in the
synthesis of isoquinoline derivative 5 or pyrimidine
derivative 11, high temperature and excess of acid
chloride were needed, whereas the more reactive amines
used in the synthesis of 6, 7, or 10 gave good results at
low temperature. 2-Amino-4-phenyltriazine and 4-amino-
2-phenylpyrimidine were not sufficiently reactive to-
ward acid chlorides, even after deprotonation with a
butyllithium reaction could not be achieved, probably
due to delocalization of the negative charge. In case of
9 and 12 the benzamides were more reactive than the
corresponding primary amines and the N,N-dibenzoyl
analogues were easily obtained. Adjusting the reaction
conditions of method C (Scheme 4) by using an excess
of the amine and dilution of the reaction mixture led to
the desired compound 12. Hydrolysis of the dibenzoyl
analogue with sodium methoxide in dioxane yielded the
desired compound 9. The amines 14 and 15 were
prepared from the corresponding nitriles as described
in previous studies16,17 (Scheme 2).
F igu r e 1. Template approach in the development of novel
classes of adenosine analogues, i.e. replacement of the bicyclic
ring system by a simple monocylic heterocycle. Affinities at
a
adenosine receptors are expressed as Ki values in µM. Data
b
c
from ref 16. Data from ref 17. 45% displacement of specific
[3H]DPCPX binding at human A2B receptors, stably expressed
in COS-7 cells (n ) 2).
all been identified as adenosine A3 receptor antagonists
through the screening of chemical libraries.
Recently we reported on a series of isoquinoline and
quinazoline analogues as adenosine A3 receptor an-
tagonists.15-17 In these studies we showed that higher
adenosine A1 and A3 receptor affinities resulted from
spacer-coupled aromatic groups on the 1-position of the
isoquinoline ring.15 An amide16 or urea17 moiety as
spacer between the heterocyclic ring and aromatic group
on position 1 provided an increase in adenosine A3
receptor affinity; in case the aromatic group is phenyl
a substitution of this nucleus led to A3-selective antago-
nists (Figure 1). We also found that an aromatic group
at position 2 of the quinazoline ring increased adenosine
A3 receptor affinity.17
2-Amino-4-(2-pyridyl)thiazole 16 was prepared ac-
cording to a literature procedure,18 and the amides
17a -j were synthesized by a reaction of amine 16 with
the appropriate carboxylic acids in the presence of
dicyclohexylcarbodiimide and DMAP (Scheme 3). Dif-
ferent methods were used for the synthesis of thiadi-
azole derivatives 8a -l (Scheme 4). In methods A and B
carboxylic acids were reacted with 5-amino-3-phenyl-
1,2,4-thiadiazole, while in methods C and D the acid
chlorides were used.
In the present study, we describe the development of
a new class of heterocyclic compounds as adenosine
receptor antagonists, based on a template approach (see
also Figure 1). We replaced the bicyclic heterocyclic ring
system by several monocyclic rings and investigated its
influence on adenosine receptor affinity. Thiazole or
thiadiazole derivatives appeared most promising; hence
we focused our investigations on these two classes of
compounds. We prepared several substituted N-thia-
diazole- and N-thiazolebenzamide derivatives and in-
vestigated the influence of benzamide substituents on
the adenosine receptor affinities.
4-Hydroxybenzamide 8h was prepared by cleavage of
the methyl ether of 8e under the influence of sodium
cyanide (Scheme 5). The phenol 8h in turn was alkyl-
ated by bromoethyl acetate and subsequently hydro-

Novel Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 751
striatum with [3H]CGS 2168027 (Tables 1 and 2) or
[3H]ZM 241385 (Tables 3-5) as radioligands. The af-
finity at adenosine A3 receptors was determined on
membranes from HEK 293 cells, stably expressing the
Sch em e 2
human A3 receptor, using [125I]AB-MECA as radio-
28,29
ligand.
The results are shown in Tables 1-5.
Tem p la te Ap p r oa ch . We have demonstrated earlier
that A3 receptor potency and selectivity could be achieved
in isoquinoline and quinazoline derivatives through a
combination of an aromatic group at the 2-position and
an aromatic group coupled by a spacer at the 4-position
of the quinazoline ring or at the appropriate 3- and
1-positions of an isoquinoline ring.15-17 In the present
study we applied a template approach in the develop-
ment of novel classes of adenosine receptor ligands and
replaced the bicyclic ring system by simpler monocyclic
heterocycles (Figure 1).
For synthetic reasons we preferred the phenyl instead
of the pyridinyl derivatives; earlier investigations had
revealed that 2-(2-pyridinyl)-, 2-(4-pyridinyl)-, and 2-
phenyl-substituted quinazoline derivatives showed com-
parable adenosine receptor affinities.17 To check whether
this also held in the benzamide series, we prepared the
phenyl-substituted analogues 2 and 4 of VUF8507 (1)16
and N-[2-(2-pyridinyl)quinazolin-4-yl]benzamide (3),16
respectively, and tested their adenosine receptor binding
(Table 1).
Sch em e 3
Isoquinoline as well as quinazoline analogues showed
comparable adenosine A1 and A2A receptor binding for
2-pyridinyl and phenyl substituents. However, the af-
finity at the human adenosine A3 receptor was the same
for quinazolines 3 and 4, but N-(3-phenylisoquinolin-1-
yl)benzamide 2 showed a 6-fold decrease in adenosine
A3 receptor affinity compared to pyridinyl analogue 1.
In the molecular modeling section we will deal with this
difference.
We altered the central heterocyclic ring to gain more
insight into the structural requirements for binding
adenosine receptors (compounds 5-13). We chose sev-
eral monocyclic ring systems and kept a phenyl ring and
an N-4-methoxybenzamide (as in earlier studies) as
substituents. For comparison, we prepared phenyl-
substituted bicyclic heterocycles 5 and 6. The various
compounds and their adenosine receptor affinities are
listed in Table 2.
Isoquinoline 5 showed negligible affinity at the ad-
enosine A2a receptor but has a Ki value of 3 µM at the
adenosine A1 receptor and a Ki value of 79 nM at the
adenosine A3 receptor. The 4-fold lower adenosine A3
receptor affinity of 5 compared to VUF850416 should be
determined by the lack of a nitrogen atom in the
3-substituent. Quinazoline 6 is also an adenosine A3
receptor-selective compound with a Ki value of 27 nM,
i.e. 66-fold selectivity over A1 and more than 350-fold
lyzed, yielding the carboxylic acid 8i. Methyl ethers 8k ,l
were cleaved using alumina tribromide in the presence
of ethanethiol,19,20 yielding hydroxy derivatives 8m ,n .
Other cleavage methods were explored, such as the use
of BBr3 at low temperature,21 the application of this
Lewis acid in the presence of 15-crown-5 and KI,22 or
the use of silylated compounds,23,24 without success.
Compound 18g was prepared by oxidation of 4-methoxy-
3-methylbenzaldehyde25 to the corresponding carboxylic
acid (19) which was easily converted into the acid
chloride (Scheme 6).
Urea analogues 20-22 were prepared by coupling the
aminothiazole or aminothiadiazole derivatives with
substituted phenyl isocyanates in dry acetonitrile
(Scheme 7).
over A2A
.
Replacement of the bicyclic heterocycle of 5 by a
monocyclic thiazole moiety yielded LUF5433 (7), which
showed remarkable adenosine receptor affinities. This
thiazole derivative possessed reasonable affinities at all
three receptor subtypes, with Ki values of 76 nM at the
adenosine A1 receptor, 2 µM at the adenosine A2A
receptor, and 0.7 µM at the adenosine A3 receptor. With
this compound as a lead we developed a new class of
adenosine receptor antagonists (next section).
Resu lts a n d Discu ssion
Bin d in g Stu d ies. All synthesized compounds were
tested in radioligand binding assays to determine their
affinities for the adenosine A1, A2A, and A3 receptors.
The affinities at adenosine A1 receptors were deter-
mined using membranes from rat brain cortex, with
[3H]DPCPX as radioligand.26 The affinities at adenosine
A2A receptors were obtained on membranes from rat

752 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
van Muijlwijk-Koezen et al.
Sch em e 4
Sch em e 5
In analogy to compounds 5 and 7, we replaced the
quinazoline of 6 by a monocyclic thiadiazole moiety,
resulting in LUF5417 (8e). Like thiazole 7, thiadiazole
8e showed affinities at adenosine receptors from the
micromolar to the nanomolar range. The 8 times
increased adenosine A3 receptor affinity of 8e, compared
to thiazole 7, is remarkable. This can be ascribed to the
same effect as was seen in compounds 5 and 6. Again,
we will return to this in the molecular modeling section.
We continued our investigations on monocyclic het-
erocycles with two other five-membered rings and three
six-membered ring systems. The choice of ring systems
depended on synthetic accessibility. Dihydropyrazole 9
and pyrazole analogue 10 showed negligible affinity at
all three adenosine subtypes. Different six-membered
rings were used in compounds 11-13. Compound 11
contains a pyrimidine ring, 12 a pyridyl ring, and 13 a
phenyl ring. The pyrimidine analogue, although lacking
a nitrogen between the two substituents, possessed
substantial affinities at the adenosine A1 and A3 recep-
tors with Ki values of 0.2 and 0.8 µM, respectively. The
pyridine analogue 12 was slightly A3-selective and

Novel Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 753
Ta ble 1. Affinities of Isoquinoline and Quinazoline Analogues
in Radioligand Binding Assays at Adenosine Receptors
Sch em e 6
Sch em e 7
a
Displacement of specific [3H]DPCPX binding in rat brain
cortical membranes, expressed as Ki ( SEM in µM (n ) 3) or
percentage displacement of specific binding at a concentration of
b
10 µM (n ) 2). Displacement of specific [3H]CGS 21680 binding
in rat striatal membranes, expressed as percentage displacement
of specific binding at
a concentration of 10 µM (n ) 2-3).
c Displacement of specific [125I]AB-MECA binding at human ad-
enosine A3 receptors expressed in HEK 293 cells, expressed as Ki
d
( SEM in µM (n ) 3). As published in ref 16.
affinity and decreased adenosine A1 receptor affinity.
The methoxy group has a prominent role in different
classes of adenosine A3 receptor antagonists,3,4,14-17
indicating that it could play an important role in the
molecular recognition of the adenosine A3 receptor.
The 3,4-dichlorobenzamide analogue 17f, 3-chloro-
benzamide 17g, and 4-nitrobenzamide 17h showed
affinities comparable to that of the unsubstituted benz-
amide 17a toward adenosine A1 and A3 receptors.
m-Chloro-substituted compound 17g showed also A2A
affinity with a Ki value of 1.3 µM. Increasing the size of
the ether group of 17e resulting in isopropyl ether 17i
diminished both the adenosine A1 and A3 receptor
affinities by 2-fold.
Furthermore we used a cyclopentyl group instead of
the phenyl ring. This moiety showed A1 selectivity in
other classes of adenosine receptor ligands (DPCPX,
N-0840, CPA, and CCPA).29-33 Compound 17j was also
an adenosine A1 receptor-selective ligand but showed
also increased affinities at the adenosine A2A and A3
receptors compared to benzamide 17a . This is surpris-
ing, since N-[3-(2-pyridyl)isoquinolin-1-yl]alkyl amides
were hardly active at adenosine receptors with Ki values
in the range of 10-50 µM,16 whereas a benzamide
instead of an alkylamide as in VUF8507 led to 55- and
10-fold increased adenosine A3 and A1 affinity, respec-
tively. Thus the SAR of this class of N-[4-(2-pyridyl)-
thiazol-2-yl]amides is not the same as the SAR of the
N-[3-(2-pyridyl)isoquinolin-1-yl]amides. This observa-
tion has a precedent in the SAR of pyran derivatives
and dihydropyridines not being parallel.10 On the other
hand, the p-methoxy-substituted compound 17e was as
expected the most active compound at the adenosine A3
receptor within the series of benzamides.
showed a Ki value of 8.7 µM. This analogue can hardly
be compared with the known class of 3,5-diacyl-2,4-
dialkylpyridine analogues as adenosine A3 receptor
antagonists,8 because of the large difference in substitu-
tion pattern of the compounds. Analogue 13, lacking
heteroatoms in the central ring, had a comparably low
A3 receptor affinity as 12, with a Ki value of 10 µM.
Comparison of 5 and 12 showed that the additional ring
of 5 largely increased adenosine A3 receptor affinity. It
might be that substituents at positions 3 and 4 of the
pyridine ring can fulfill the same role in binding.
Th ia zolylben za m id e An a logu es. Based on 1 and
7, several substituted N-[4-(2-pyridyl)thiazol-2-yl]-
benzamides were synthesized to explore the structure-
activity relationships (SARs) at the benzamide ring. In
Table 3 the affinities of compounds 17a -j at the
adenosine receptors are summarized.
The unsubstituted N-[4-(2-pyridyl)thiazol-2-yl]benz-
amide 17a showed affinities in the micromolar range
at the three adenosine receptors (A1 > A3 > A2A).
Introduction of a halogen atom at the para-position
(17b,c) decreased the A2A affinity and did not influence
the adenosine A3 receptor affinity. However, the 4-chloro-
substituted benzamide 17b possessed an 8-fold in-
creased affinity at the adenosine A1 receptor compared
to 17a , with a Ki value of 200 nM. Introduction of a
methyl group (17d ) does not influence adenosine recep-
tor affinities, whereas the stronger electron-donating
methoxy analogue (17e) increased adenosine A3 receptor
Th ia d ia zolylben za m id e An a logu es. We continued
our investigations on the monocyclic thiadiazole ad-
enosine ligands. In addition to the 4-methoxybenzamide

754 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
van Muijlwijk-Koezen et al.
Ta ble 2. Affinities of 4-Methoxybenzamide Analogues in Radioligand Binding Assays at Adenosine Receptors
a
Displacement of specific [3H]DPCPX binding in rat brain cortical membranes, expressed as Ki ( SEM in µM (n ) 3) or percentage
displacement of specific binding at a concentration of 10 µM (n ) 2). Displacement of specific [3H]CGS 21680 binding in rat striatal
b
membranes, expressed as percentage displacement of specific binding at a concentration of 10 µM (n ) 2-3) or Ki ( SEM in µM (n ) 3).
c Displacement of specific [125I]AB-MECA binding at human adenosine A3 receptors expressed in HEK 293 cells, expressed as Ki ( SEM
in µM (n ) 3) or percentage displacement of specific binding at a concentration of 10 µM (n ) 2).
8e we prepared other benzamides 8a -i to explore the
structure-affinity relationships (Table 3).
Unsubstituted benzamide 8a showed adenosine A1
and A2A receptor affinities comparable with 8e but a
20-fold decreased adenosine A3 receptor affinity. This
is compatible with our earlier observation on the
importance of the methoxyphenyl moiety.
8a ,b,d ,e. Introduction of more or less lipophilic substit-
uents or changing their electronic nature did not influ-
ence the relatively high adenosine A1 receptor affinity
(Ki values 30-40 nM). However, larger substituents
such as iodo in 8c and nitro in 8f decreased the
adenosine A1 receptor affinity tremendously.
Compounds 8a -f showed little affinity for the ad-
enosine A2A receptor with the exception of the 4-meth-
oxy-substituted thiadiazolobenzamide 8e (Ki ) 2 µM).
At the adenosine A1 receptor small substituents on
the para-position were allowed, as in compounds

Novel Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 755
Ta ble 3. Affinities of Thiazole and Thiadiazole Derivatives in Radioligand Binding Assays at Adenosine Receptors
a
Displacement of specific [3H]DPCPX binding in rat brain cortical membranes, expressed as Ki ( SEM in µM (n ) 3) or percentage
displacement of specific binding at a concentration of 10 µM (n ) 2). Displacement of specific [3H]ZM241385 binding in rat striatal
b
membranes, expressed as percentage displacement of specific binding at a concentration of 10 µM (n ) 2-3) or Ki ( SEM in µM (n ) 3).
c Displacement of specific [125I]AB-MECA binding at human adenosine A3 receptors expressed in HEK 293 cells, expressed as Ki ( SEM
in µM (n ) 3) or percentage displacement of specific binding at a concentration of 10 µM (n ) 2). As published in ref 15. e As published
d
in ref 16. f As published in ref 41.
At the adenosine A3 receptor a quite different SAR was
established. In addition to the steric restriction (an iodo
atom or nitro moiety at the para-position appeared to
be too large) electronic effects play an important role
in the molecular recognition. The A3 receptor potency
order of 4-substituents is Cl < H < Me < OMe. The
strong electron-donating methoxy substituent afforded
a high Ki value of 82 nM. This SAR is similar as seen
in adenosine A3 receptor antagonizing N-isoquinoline-
benzamides16 and N1-isoquinolinebenzamidines15 and
also to the SAR present in the adenosine A3 receptor
agonist class of N6-(phenylcarbamoyl)adenosine-5′-N-
ethyluronamides.34
N-(3-Phenyl-1,2,4-thiadiazol-5-yl)-3-methyl-4-meth-
oxybenzamide (8g) was proof that the introduction of
an extra 3-methyl next to the 4-methoxybenzamide 8e

756 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
van Muijlwijk-Koezen et al.
Ta ble 4. Affinities of N-(3-Phenyl-1,2,4-thiadiazol-5-yl)cyclohexanamide Derivatives in Radioligand Binding Assays at Adenosine
Receptors
a
Displacement of specific [3H]DPCPX binding in rat brain cortical membranes, expressed as Ki ( SEM in µM (n ) 3) or percentage
displacement of specific binding at a concentration of 10 µM (n ) 2). Displacement of specific [3H]ZM241385 binding in rat striatal
b
membranes, expressed as percentage displacement of specific binding at a concentration of 10 µM (n ) 2-3) or Ki ( SEM in µM (n ) 3).
c Displacement of specific [125I]AB-MECA binding at human adenosine A3 receptors expressed in HEK 293 cells, expressed as Ki ( SEM
in µM (n ) 3) or percentage displacement of specific binding at a concentration of 10 µM (n ) 2).
is adverse for binding to all three receptors. We also
prepared compound 8i, bearing the same oxyacetate
substituent as in I-ABOPX, the only xanthine derivative
with appreciable affintity for human adenosine A3
receptors.35 However, at all three receptors 8i had
decreased receptor affinities compared to the unsubsti-
tuted thiadiazole 8a .
The cyclohexyl group of 8j proved less favorable than
the phenyl group of 8a in binding adenosine receptors,
which disagrees with the SAR of 17a ,j. This suggests
that either a cyclopentyl group fits the adenosine
receptor pocket better or the thiadiazole ligands behave
differently, compared to the thiazole analogues. Intro-
duction of a 4-trans-methoxy substituent increased
adenosine A3 receptor affinity. It also increased the
adenosine A1 and A2A receptor affinities as in the
thiadiazolobenzamides (8e vs 8a ), unlike the methoxy
group in the benzamide analogues (17e vs 17a ). Intro-
duction of a 4-cis-methoxy substituent (8l) led to similar
changes, but to a lesser extent than observed for the
trans-analogue. Apparently, adenosine A1 and A3 recep-
tors slightly prefer the 4-trans-methoxycyclohexanamide
over the cis-analogue.
The 4-trans-hydroxy analogue 8m had an adenosine
A3 receptor affinity comparable to that of the 4-trans-
methoxy analogue 8k , a decreased A2A receptor affinity,
and a slightly increased A1 receptor affinity. The cis-
analogue 8n showed lower affinity again at all three
adenosine receptors. From Table 4 we concluded that
all three receptor subtypes prefer 4-trans-substituted
cyclohexanamides and, second, that a hydroxyl group
is (slightly) favored over a methoxy group for binding
the adenosine A1 receptor. Furthermore, these investi-
gations provided another potent adenosine A1 receptor
antagonist (VUF5472, 8m ). This 4-trans-hydroxycyclo-
We also tested the 4-hydroxyl-substituted benzamide
8h . This compound showed an increase in affinities at
all three receptor subtypes when compared to 8a and
appeared to be a highly potent and selective adenosine
A1 receptor antagonist. N-(3-Phenyl-1,2,4-thiadiazol-5-
yl)-4-hydroxybenzamide (LUF5437, 8h ) had a Ki value
of 7.3 nM at the A1 receptor and possessed 18- and 78-
fold selectivity over the A3 and A2A receptors, respec-
tively. A specific role for the p-hydroxyl group in binding
to the A1 receptor seems therefore likely, whereas the
p-methoxy group plays an important role in binding to
the A3 receptor. We therefore further investigated the
influence of these substituents on binding adenosine
receptors by preparing N-(3-phenyl-1,2,4-thiadiazol-5-
yl)cyclohexanamide 8j and its 4-substituted derivatives
8k -n . In the thiazolo series the cyclopentanamide
analogue 17j showed relatively high adenosine receptor
affinities (better than benzamide 17a ), and therefore we
chose also a cycloalkyl group to direct the hydroxy or
methoxy substituent, as cis- and trans-isomers, respec-
tively. The results are summarized in Table 4.

Novel Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 757
Ta ble 5. Affinities of Thiazole and Thiadiazole Urea
Derivatives in Radioligand Binding Assays at Adenosine
Receptors
the amide analogue 17a and the thiadiazole urea
analogue 20a . Introduction of a 4-methoxy substituent
diminished adenosine receptor affinities. Finally, we
studied the influence of the substituent on the 4-position
of the thiazole ring and prepared compound 22. A
comparison of analogues 22 and 21a revealed that a
2-pyridyl moiety at position 4 slightly improved binding
to the adenosine A3 receptor compared to a phenyl ring
and is apparently essential for the adenosine A1 recep-
tor. Furthermore, compounds 22 and 20a differ in the
heterocyclic ring system only, and a comparison of the
thiazole and thiadiazole analogues revealed that the
lack of the nitrogen atom in 22 is detrimental toward
the adenosine A1 receptor, while also decreasing the
adenosine A3 receptor affinity.
Molecu la r Mod elin g. The binding data in Tables 1
and 5 show that for binding the adenosine A1 and A3
receptor ligands needed an additional nitrogen atom
either in the heterocyclic central ring (as in thiadiazole
rather than thiazole) or in the substituent at the
4-position of the thiazole ring (2-pyridyl instead of
phenyl).
a
Displacement of specific [3H]DPCPX binding in rat brain
cortical membranes, expressed as Ki ( SEM in µM (n ) 3) or
percentage displacement of specific binding at a concentration of
10 µM (n ) 2). Displacement of specific [3H]ZM241385 binding
b
In earlier studies17 it had been shown that in low-
energy conformations of 3-(2-pyridyl)isoquinoline ana-
logues, the nitrogen of the pyridyl group pointed “up-
ward”, i.e. the pyridyl group was turned away from the
nitrogen of the isoquinoline ring. Therefore we built the
structures of compounds 20a and 21a in Spartan,36 and
after conformational optimization we calculated their
molecular electrostatic potentials (MEPs) semiempiri-
cally and ab initio. Indeed, also in this case, the low-
energy conformation of 21a has a pyridyl group in which
the nitrogen atom is pointed ”upward” (Figure 2). Both
compounds showed comparable MEPs in the ab initio
calculations as well as in the semiempiric study, indi-
cating that the high electron density present in the
plane of the thia(dia)zole or 2-pyridyl ring may function
as proton acceptor for amino acid residues of the
adenosine receptor (arrows in Figure 2). This could then
also be the reason for the loss of adenosine receptor
affinities of 22 compared to 20a and 21a and of 2
compared to 1 and 4.
in rat striatal membranes, expressed as percentage displacement
of specific binding at a concentration of 10 µM (n ) 2-3) or Ki (
SEM in µM (n ) 3). c Displacement of specific [125I]AB-MECA
binding at human adenosine A3 receptors expressed in HEK 293
cells, expressed as Ki ( SEM in µM (n ) 3) or percentage
displacement of specific binding at a concentration of 10 µM (n )
2).
hexanamide analogue showed a Ki value of around 20
nM and a 100-fold selectivity versus the adenosine A3
receptor and even more than 500-fold versuss the A2A
receptor.
Th ia zolyl- a n d Th ia d ia zolylu r ea An a logu es. Ear-
lier studies in a series of isoquinoline and quinazoline
adenosine A3 receptor antagonists have proven that a
urea moiety as spacer increased adenosine A3 receptor
affinity compared to an amide moiety.17 We therefore
applied the template approach again and replaced the
quinazoline ring of VUF5574 by a thiazole or thiadiazole
moiety and investigated a urea series of compounds
(Figure 1).
First, we prepared N-phenyl-N′-(3-phenyl-1,2,4-thia-
diazol-5-yl)urea (20a ) and determined its affinities at
the three adenosine receptors (Table 5). Unsubstituted
urea analogue 20a had a Ki value of 1 and 4 µM at the
adenosine A1 and A3 receptor, respectively, and pos-
sessed only 48% displacement at a concentration of 10
µM at the adenosine A2A receptor. Second, we prepared
the o- and p-methoxy-substituted phenylurea analogues
according to the positive contributions of these substit-
uents toward adenosine A3 receptor affinity in the
N-phenyl-N′-(2-phenylquinazolin-4-yl)urea17 and vari-
ous benzamide series, respectively. The SAR of this class
of thiadiazole ureas differed from the SAR of the
quinazoline urea analogues to a large extent. The
4-methoxyphenyl analogue 20b showed a decrease in
the adenosine A1 and A2A receptor affinities and did not
affect the adenosine A3 receptor affinity, compared to
unsubstituted urea 20a . Introduction of a 2-methoxy
substituent as in 20c decreased the binding at all three
receptor subtypes substantially.
We have shown that our template approach yields
thiazolo and thiadiazolo analogues as novel classes of
adenosine receptor antagonists, and subsequently sev-
eral potent c.q.-selective ligands were developed. These
compounds contribute to a better understanding of the
structural requirements necessary for the molecular
recognition by adenosine A1 and A3 receptors.
Exp er im en ta l Section
Abbr evia tion s: APT, attached proton test; CGS 15943,
9-fluoro-2-(2-furyl-5,6-dihydro[1,2,4]triazolo[1,5-c]quinazin-5-
imine; CHO cells, Chinese hamster ovary cells; CI, chemical
ionization; COSY, correlated spectroscopy; DCC, dicyclohexyl-
carbodiimide; DEPT, distortionless enhancement by polariza-
tion transfer; DMAP, N,N-(dimethylamino)pyridine; DMSO,
dimethyl sulfoxide; DPCPX, 1,3-dipropyl-8-cyclopentylxan-
thine; EA, ethyl acetate; [3H]CGS 21680, [3H]-2-[[4-(2-carboxy-
ethyl)phenyl]ethylamino]-5′-N-(ethylcarbamoyl)adenosine; [3H]-
DPCPX, [3H]-1,3-dipropyl-8-cyclopentylxanthine; [3H]ZM
241385; HEK cells, human embryonic kidney cells; [125I]-
AB-MECA,
[
125I]-N6-(4-amino-3-iodobenzyl)-5′-(N-methyl-
We also prepared N-phenyl-N′-[4-(2-pyridyl)thiazol-
2-yl]urea analogues 21a ,b. Unsubstituted analogue 21a
had adenosine receptor affinities comparable to that of
carbamoyl)adenosine; Ki, equilibrium inhibition constant;
L-249313, 6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-
[1,2,4]triazolo[5,1-a][2,7]naphthpyridine; L-268605, 3-(4-meth-

758 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
van Muijlwijk-Koezen et al.
Syn th esis. Reaction scales were used as mentioned in the
general methods, unless otherwise specified. 1H and 13C NMR
spectra were recorded on a J EOL J NM-FX 200 spectrometer
equipped with a PG 200 computer operating in the Fourier
transform mode, with tetramethylsilane as an internal stand-
ard or on a Bruker AC 200 (1H NMR: 200 MHz; 13C NMR:
50.29 MHz). 2D-NMR (H-H and C-H) COSY techniques were
frequently used to support interpretation of 1D spectra. The
multiplicity of the carbon signals was determined by DEPT
or APT spectra or by a combination of a normal decoupled
carbon spectrum and a CH correlation. The symbols used are
(p) for primary, (s) for secondary, (t) for tertiary and (q) for
quaternary carbon signals. Mass spectra were measured on a
Finnigan MAT TSQ-70 mass spectrometer, equipped with an
electrospray interface (EI). Spectra were collected by constant
infusion of the analyte dissolved in 80/20 methanol water
(sometimes with 1% acetic acid). Experiments were done in
positive ionization mode. Melting points were determined with
a Bu¨chi capillary melting point apparatus and are uncorrected
or measured on a Electrothermal IA9200 apparatus. Reactions
were routinely monitored by thinlayer chromatography on
Merck silica gel F254 plates and spots were visualized with UV
light at 254 nm or iodine or aqueous potassium permanganate
staining. Elemental analyses were performed by the analytical
department of Organic and Molecular Inorganic Chemistry at
the University of Groningen (The Netherlands) or at the
Leiden University Gorlaeus Laboratories (The Netherlands)
and are within (0.4% of theoretical values unless otherwise
specified.
Met h od A: Gen er a l P r oced u r e for t h e Syn t h esis of
8b,c. 0.5 g (2.8 mmol) of 5-amino-3-phenyl-1,2,4-thiadiazole,
0.52 g of DMAP (4.2 mmol), 4.2 mmol of the apparent benzoic
acid and 0.87 g (4.2 mmol) of DCC were dissolved in 35 mL of
dry dioxane. After stirring at 60 °C for 16 h, the yellow mixture
was filtered and concentrated in vacuo. Recrystallization from
a mixture of methanol and ethanol yielded white crystals.
Meth od B: 8a . 0.5 g (2.8 mmol) of 5-amino-3-phenyl-1,2,4-
thiadiazole was dissolved in 20 mL of dry dioxane, containing
0.39 mL (2.8 mmol) of triethylamine. A solution of 0.5 mL (4.3
mmol) in 5.0 mL of dry dioxane was added and the solution
stirred at 80 °C for 16 h. After filtration, the solution was
concentrated in vacuo and recrystallized from methanol.
F igu r e 2. Molecular electrostatic potential energy isosurfaces
of low-energy conformations of compounds 20a and 21a . Color
coding is in the range from -60 (deepest red) to 60 kcal/mol
(deepest blue).
Meth od C: Gen er a l P r oced u r e for th e Syn th esis of 6,
8d ,e,j-l, 9, a n d 10. 1.0 g (5.6 mmol) of 5-amino-3-phenyl-1,2,4-
thiadiazole was dissolved in 2.5 mL of dry pyridine in a flame-
dried 10-mL three-neck flask. This mixture was cooled to 0
°C and 8.2 mmol of acid chloride was added dropwise. The clear
solution solidified. After 0.5 h, the solid was dissolved in a
mixture of 50 mL of ethyl acetate and 50 mL of water. The
H2O layer was discarded and the ethyl acetate layer washed
subsequently with 50 mL of 1 M HCl, 50 mL of 5% NaHCO3
solution and 50 mL of brine. The organic layer was dried on
MgSO4 and concentrated under reduced pressure. The remain-
ing oil was stirred with 50 mL of n-pentane and a white solid
resulted. Recrystallization from methanol resulted in white
crystals.
Meth od D: Gen er a l P r oced u r e for th e Syn th esis of 5,
8f,g, a n d 11-13. 0.50 g (2.8 mmol) of 5-amino-3-phenyl-1,2,4-
thiadiazole was dissolved in 1.0 mL of dry pyridine in a flame-
dried 25-mL three-neck flask. This mixture was cooled to 0
°C and 3.2 mmol of acid chloride in 14 mL of dry pyridine was
added dropwise. After stirring at room temperature for 1 h
the mixture was refluxed for 5 h. After cooling to room
temperature, a precipitate was formed which was filtrated and
washed 3 times with methanol. Addition of methanol to the
filtrate yielded a second batch of solidified product.
oxyphenyl)-5-amino-7-oxothiazolo[3,2]pyrimidine; MEP, mo-
lecular electrostatic potential; MRS1191, 3-ethyl-5-benzyl-
2-methyl-6-phenyl-4-(phenylethynyl)-1,4-(()dihydropyridine-
3,5-dicarboxylate; MRS1220, 9-chloro-2-(2-furyl)-5-phenyl-
acetylamino[1,2,4]triazolo[1,5-c]quinazoline; N-0840, N6-cy-
clopentyl-9-methyladenine; VUF8504, 4-methoxy-N-[3-(2-
pyridinyl)isoquinolin-1-yl]benzamide; VUF5574, N-(2-meth-
oxyphenyl)-N′-[2-(3-pyridyl)quinazolin-4-yl]urea; XAC, 8-[4-
(((((2-aminoethyl)amino)carbonyl)methoxy)oxy)phenyl]-1,3-
dipropylxanthine.
Ma ter ia ls. DMF was distilled and stored under linde type
4 Å molecular sieves. Dioxane, triethylamine, pyridine, and
DMSO were distilled over CaH2 and stored under linde type
4 Å molecular sieves as well. All other solvents used were
analytical grade. 5-Amino-3-phenyl-1,2,4-thiadiazole, cyclo-
hexanoic acid, N,N-dimethylaminopyridine (DMAP), 3-and
4-chlorobenzoic acid, p-anisoyl chloride, p-anisic acid, p-toluic
acid, 3,4-dichlorobenzoic acid, 4-methoxyphenyl isocyanate and
4-nitrobenzoic acid were purchased from ACROS. 3-Amino-5-
phenylpyrazole, 3-methyl-4-methoxybenzaldehyde, and 4-meth-
oxycyclohexanoic acid were commercially available from Ald-
rich. Alumina tribromide, 4-iodobenzoic acid, 4-isopropoxyben-
zoic acid, and 2-amino-4-(4-chlorophenyl)thiazole were pur-
chased from Merck, n-butyllithium and phenyl isocyanate from
Fluka, and 3-amino-4,5-dihydro-1-phenylpyrazole, 2-amino-4-
phenylthiazole and 1-(2-aminophenyl)pyrrole from Lancaster.
Thionyl chloride was purchased from Riedel de Haen and
4-amino-6-phenylpyrimidine from SPECS. Compounds 2 and
4 were prepared as described previously16 and 2-amino-4-(2-
pyridyl)thiazole (16) was prepared by the method as described
by Taurins and Blaga.18
N-(3-P h en ylisoqu in olin -1-yl)ben za m id e (2): yield 28%
1
yellow-white crystals; mp 162-164 °C; Rf (MeOH) ) 0.8; H
3
NMR (CDCl3) δ 7.27-8.12 (m, 12H), 8.48 (d, 2H, J BA ) 7.8
Hz, 2H, benzamide-2H), 8.98 (d, 3J 87 ) 8.0.3 Hz, 1H, H8), and
14.76 (s, 1H, NH). Anal. (C22H16N2O) C, H, N.
N-(2-P h en ylq u in a zolin -4-yl)b en za m id e (4): yield 32%
white crystals; mp 166 °C (lit.37 mp 168-169 °C); 1H NMR
(DMSO-d6) δ 7.31-7.84 (m, 9H, H5 + H6 + H7 + H8 +

Novel Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 759
benzamide 2H + benzamide 3H + benzamide 4H), 7.93-8.22
(m, 5H, phenyl-2H + phenyl-3H + phenyl-4H), and 11.05 (s,
1H, NH). Anal. (C21H15N3O) C, H, N.
N-(3-P h en yl-1,2,4-th iadiazol-5-yl)-4-n itr oben zam ide (8f):
method D; recrystallization of the combined solids from
dichloromethane/methanol yielded 33% needlelike white crys-
tals; mp >300 °C; Rf value TLC (CH2Cl2) 0.33; 1H NMR
(DMSO-d6) δ 7.50-7.56 (m, 3H, phenyl-3H + phenyl-4H),
8.20-8.22 (m, 2H, benzamide-2H), 8.37-8.42 (m, 4H, phenyl-
2H + benzamide-3H), 10.60 (bs,1H, NH). Anal. (C15H10N4O3S‚
0.3CH2Cl2) C, H, N.
N-(3-P h en yl-1,2,4-th ia d ia zol-5-yl)-4-m eth oxy-3-m eth yl-
ben za m id e (8g): method D; purification by column chroma-
tography (EA/tol as eluent) followed by recrystallization from
methanol yielded 11% white needles; mp 160 °C; Rf value TLC
(EA/tol) 0.90; 1H NMR (CDCl3) δ 2.16 (s, 3H, CH3), 3.84 (s,
3H, OCH3), 6.73-6.78 (d, 1H, benzamide-5H), 7.34-7.37 (m,
3H, phenyl-3H + phenyl-4H), 7.67 (s, 1H, benzamide-2H),
7.71-7.75 (m, 1H, benzamide-6H), 8.10-8.15 (m, 2H, phenyl-
2H), 10.64 (bs, 1H, NH). Anal. (C17H15N3O2S) C, H, N.
N-(3-P h en ylisoqu in olin -1-yl)-4-m eth oxyben za m id e (5):
method D; yield 47% white crystals; mp 144-146 °C; 1H NMR
3
(CDCl3) δ 3.88 (s, 3H, OCH3), 6.98 (d, 2H, J AB ) 8.8 Hz, 2H,
benzamide-3H), 7.24-7.88 (m, 9H, H5 + H6 + H7 +phenyl-
2H + phenyl-3H + phenyl-4H + NH), 8.42-8.52 (m, 3H, H8
+ benzamide-2H), and 8.80-8.98 (m, 1H, H4). Anal. (C23
-
H
18N2O2‚0.2H2O) C, H, N.
N -(2-P h e n ylq u in a zolin -4-yl)-4-m e t h oxyb e n za m id e
(6): method C used for 2.20 mmol of 4-amino-2-phenylquinazo-
line; yield 38% white powder; mp 165-168 °C; Rf (EA) ) 0.6;
3
1H NMR (DMSO-d6) δ 3.87 (s, 3H, OCH3), 7.12 (d, 2H, J AB
)
8.8 Hz, 2H, benzamide-3H), 7.54-7.70 (m, 4H, H5 + H6 +
H7 + H8), 7.95-8.18 (m, 5H, phenyl-2H + phenyl-3H +
phenyl-4H), 8.45 (d, 2H, J BA ) 8.6 Hz, 2H, benzamide-2H),
3
and 11.21 (s, 1H, NH). Anal. (C22H17N3O2) C, H, N.
N -(3-P h e n yl-1,2,4-t h ia d ia zol-5-y l)-4-h y d r o x y b e n z-
a m id e (LUF 5437, 8h ). 0.5 g (1.6 mmol) of 8e was dissolved
in 5.0 mL of dry DMSO in a flame-dried pressure tube. 0.40 g
(8.2 mmol) of NaCN was added and a nitrogen flow was let in
the tube before closing. The solution was stirred at 165 °C for
90 h. The resulting black solution was poured on 30 mL of ice
and acidified (carefully in a hood with good ventilation) with
6 N HCl. The black precipitate was collected by filtration and
subsequently washed with water and n-pentane. A short
Kieselgel column was used for purification with CH2Cl2 as
eluent. 0.08 g of 8e was recovered (pure). After this ethyl
acetate was used as eluent and the product was isolated as a
white/yellow solid, which was recrystallized from CCl4/
toluene: yield white crystals 70% (corrected for recovery of
N-(4-P h en ylth ia zol-2-yl)-4-m eth oxyben za m id e
(7,
LUF 5433). 47.0 mg (0.27 mmol) of 2-amino-4-phenylthiazole
was dissolved in 5.0 mL of dry DMF and added to a solution
of 155.0 mg (1.0 mmol) of p-anisic acid in 0.5 mL of dry DMF
in a 2.2-mL Eppendorf cup. A solution of 210.0 mg (1.0 mmol)
of DCC and 15.0 mg (0.12 mmol) of DMAP in 1.0 mL of DMF
was added. After stirring at room for 4 days, the precipitate
was removed and the solution concentrated under reduced
pressure. Recrystallization of the residue from PE (40/60) and
methanol resulted in 21% white crystals: mp 172 °C; 1H NMR
3
(DMSO-d6) δ 3.85 (s, 3H, OCH3), 7.08 (d, 2H, J AB ) 8.9 Hz,
3
benzamide-3H), 7.33 (d, 1H, J 43 ) 7.2 Hz, phenyl-4H), 7.44
3
3
(dd, 1H, J 34 ) 7.2 Hz, J 32 ) 7.6 Hz, phenyl-3H), 7.7 (s, 1H,
3
1
thiazolyl-5H), 7.9 (d, 2H, J 23 ) 7.9 Hz, phenyl-2H), 8.13 (d,
8e); mp 117 °C; H NMR (CD3OD) δ 6.93 (d, 2H, J ) 8.6 Hz,
3
2H, J BA ) 8.9 Hz, benzamide-2H), and 12.6 (s, 1H, NH); MS
benzamide-3H), 7.45 (m, 3H, phenyl-3H + phenyl-4H), 8.00
(d, 2H, J ) 8.9 Hz, benzamide-2H), 8.24 (m, 2H, phenyl-2H);
MS (EI) m/z 298 (MH+, 100%). Anal. (C15H11N3O2S‚0.2CCl4)
C, H, N.
(EI) m/z 311 (MH+, 100%). Anal. (C17H14N2O2S) C, H, N.
N-(3-P h en yl-1,2,4-th iadiazol-5-yl)ben zam ide (8a): method
B; crystallization from CCl4/MeOH yielded 35% white crystals;
1
mp 145 °C; Rf value TLC (2.5% EA in CH2Cl2) 0.56; H NMR
2-(4-{[(3-P h en yl-1,2,4-th ia d ia zol-5-yl)a m in o]ca r bon yl}-
p h en oxy)a cetic Acid (8i). 100.0 mg of 8h was dissolved in
5.0 mL of dry dioxane in a flame-dried pressure tube under
nitrogen gas. 200 µL of triethylamine and 200 µL of bromoethyl
acetate were added. This light yellow solution was stirred
overnight at ambient temperature. A precipitate was formed.
A mixture of 20 mL of water and 20 mL of ethyl acetate was
added. The ethyl acetate layer was collected, washed with a 1
M HCl solution (20 mL) and a brine solution (20 mL), dried
on MgSO4 and concentrated under reduced pressure. The
residue was dissolved in 10 mL of ethanol (96%) and 10 mL of
1 M KOH (aq) was added. After 10 min 6 M HCl was added to
acidify the solution. A precipitate formed and a mixture of 20
mL of ethyl acetate and 10 mL of water was added. The ethyl
actetate layer was collected and washed with brine (20 mL).
After drying and concentrating the ethyl acetate layer, a white
“solid” oil remained (yield 100%), which was recrystallized
twice from toluene with a little acetone: yield white needlelike
crystals, 10% after recrystallization; mp >265 °C; 1H NMR
(DMSO-d6) δ 4.82 (s, 2H, CH2), 7.10 (d, 2H, J ) 8.2 Hz,
benzamide-3H), 7.52 (m, 3H, phenyl-3H + phenyl-4H), 8.17
(d, 2H, J ) 8.2 Hz, benzamdie-2H), 8.21 (m, 2H, phenyl-2H);
MS (EI) m/z 356 (MH+, 100%). Anal. (C17H13N3O4S) C, H, N.
N-(3-P h en yl-1,2,4-th iadiazol-5-yl)cycloh exan am ide (8j):
method C; crystallization from CCl4/MeOH yielded 10% off-
white crystals; mp 250 °C dec; Rf value TLC (EA/tol 4:1) 0.65;
1H NMR (CDCl3) δ 1.24-2.14 (m, 10H, chex), 2.70-2.82 (m,
1H, chex-H1), 7.44-7.61 (m, 3H, phenyl-3H + phenyl-4H), 8.52
(dd, 2H, 3J ) 7.1 Hz, phenyl-2H), and 13.48 (bs, 1H, NH).
Anal. (C15H17N3OS) C, H, N.
N -(3-P h e n yl-1,2,4-t h ia d ia zol-5-yl)-t r a n s-4-m e t h oxy-
cycloh exa n a m id e (8k ): method C; purification by column
chromatography (EA/CH2Cl2 1:7 as eluent) yielded the pure
isomer; recrystallization from PE60-80/EA yielded 48% white
crystals (corrected for cis/ trans-mixture of starting material);
mp 177 °C; Rf value TLC (2.5% EA/CH2Cl2) 0.31; 1H NMR (400
MHz, DMSO-d6) δ 1.12-1.21 (m, 2H, H3-ax), 1.46-1.56 (m,
2H, H2-ax), 1.93-1.96 (m, 2H, H2-eq), 2.05-2.09 (m, 2H, H3-
(DMSO-d6) δ 7.50-7.73 (m, 6H, phenyl-3H + phenyl-4H +
benzamide-3H + benzamide-4H), 8.16-8.32 (m, 4H, phenyl-
2H + benzamide-2H); MS (EI) m/z 282 (MH+, 100%). Anal.
(C15H11N3OS‚0.03CCl4) C, H, N.
N -(3-P h e n y l-1,2,4-t h i a d i a z o l-5-y l)-4-c h lo r o b e n z -
a m id e (8b): method A; yield white crystals 38%; mp 258 °C;
1
Rf value TLC (2.5% EA in CH2Cl2) 0.72; H NMR (DMSO-d6)
δ 7.48-7.56 (m, 3H, phenyl-3H + phenyl-4H), 7.66 (ddd, 2H,
J ) 8.6; 2.8; 1.7 Hz, benzamide-3H), 8.15-8.22 (m, 4H, phenyl-
2H + benzamide-2H); MS (EI) m/z 316 (M(35Cl)H+, 100%), 318
(M(37Cl)H+, 20%). Anal. (C15H10ClN3O2S) C, H, N.
N-(3-P h en yl-1,2,4-th iadiazol-5-yl)-4-iodoben zam ide (8c):
method A; yield white crystals 34%; mp 269 °C; Rf value TLC
(2.5% EA in CH2Cl2) 0.63; 1H NMR (DMSO-d6) δ 7.51-7.54
(m, 3H, phenyl-3H + phenyl-4H), 7.90-8.02 (m, 4H, phenyl-
2H + benzamide-2H), 8.18-8.22 (m, 2H, benzamide-3H); MS
(EI) m/z 408 (MH+, 100%). Anal. (C15H10IN3OS) C, H, N.
N -(3-P h e n y l-1,2,4-t h ia d ia zo l-5-y l)-4-m e t h y lb e n z-
a m id e (8d ): method C; yield needlelike white crystals 17%;
mp 177 °C; Rf value TLC (EA/tol) 0.69; 1H NMR (CDCl3) δ
2.38 (s, 3H, CH3), 7.22 (ddd, 2H, J ) 8.25; 2.04;2.06 Hz,
benzamide-3H), 7.24-7.40 (m, 3H, phenyl-3H + phenyl-4H),
7.71 (ddd, 2H, J ) 8.25; 2.06; 2.05 Hz, benzamide-2H), 8.11-
8.16 (m, 2H, phenyl-2H), 10.58 (bs,1H, NH). Anal. (C12H13N3-
OS) C, H, N.
N -(3-P h e n yl-1,2,4-t h ia d ia zol-5-yl)-4-m e t h oxyb e n z-
a m id e (LUF 5417, 8e): method C; yield needlelike white
crystals 77%; mp 177 °C; Rf value TLC (CH2Cl2) 0.36; 1H NMR
(CDCl3) δ 3.84 (s, 3H, CH3), 6.88 (ddd, 2H, J ) 8.93; 2.06;2.06
Hz, benzamide-3H), 7.38-7.43 (m, 3H, phenyl-3H + phenyl-
4H), 7.88 (ddd, 2H, J ) 8.93; 2.06; 2.06 Hz, benzamide-2H),
8.12-8.16 (m, 2H, phenyl-2H). 13C NMR (CDCl3) δ 55.56 (CH3),
114.44 (benzamide-3C), 122.53 (benzamide-1C), 127.78 +
128.63 + 129.88 + 130.26 (benzamide-2C + phenyl-3,4-C),
132.45 (phenyl-1C), 16402 (benzamide-4C), 165.08 (amide-C),
166.97 (thiadiazolyl-3C), 175.88 (thiadiazolyl-5C); MS (EI) m/z
312 (MH+, 100%). Anal. (C16H13N3O2S) C, H, N.

760 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
van Muijlwijk-Koezen et al.
3
eq), 2.52-2.56 (m, 1H, H1-ax), 3.06-3.13 (m, J axax ) 15 Hz,
N-(6-P h en ylp yr id in -2-yl)-4-m et h oxyb en za m id e (12):
1H, H4-ax), 3.26 (s, 3H, OCH3), 7.35-7.42 (m, 3H, phenyl-3H
+ phenyl-4H), 7.87-7.93 (m, 1H, phenyl-2H), 8.10-8.14 (m,
1H-phenyl-2H), 12.77 (bs, 1H, NH). Anal. (C16H19N3O2S) C,
H, N.
N-(3-P h en yl-1,2,4-th ia d ia zol-5-yl)-cis-4-m eth oxycyclo-
h exa n a m id e (8l): method C; purification by column chroma-
tography (EA/CH2Cl2 1:7 as eluent) yielded the pure isomer;
recrystallization from PE60-80/EA yielded 32% (corrected for
cis/ trans-mixture of starting material); mp 221 °C; Rf value
method D used for 3.00 mmol of 2-amino-6-phenylpyridine;
1
yield 22% white crystals; mp 130 °C; H NMR (CDCl3) δ 3.82
(s, 3H, OCH3), 6.93 (d, 2H, 3J AB ) 7 Hz, benzamide-3H), 7.36-
7.47 (m, 4H, H5 + phenyl-3H + phenyl-4H), 7.76 (dd, 1H, 3J 45
3
) 8.0 Hz, J 43 ) 8.2 Hz, H4), 7.85-7.93 (m, 4H, benzamide-
2H, phenyl-2H), 8.26 (d, 3J 34 ) 8.2 Hz, 1 H, H3), and 8.55 (bs,
1H, NH). Anal. (C19H16N2O2) C, H, N.
N-(Bip h en -3-yl)-4-m eth oxyben za m id e (13): method D
used for 1.20 mmol of 3-aminobiphenyl; reflux period 8 h; yield
1
1
TLC (2.5% EA in CH2Cl2) 0.50; H NMR (400 MHz, DMSO-
70% white powder; mp 137-138 °C; H NMR (CDCl3) δ 3.86
3
d6) δ 1.42-1.48 (m, 2H, H3-ax), 1.60-1.64 (m, 2H, H2-eq),
1.74-1.83 (m, 2H, H2-ax), 1.86-1.91 (m, 2H, H3-eq), 2.58-
2.65 (m, 1H, H1), 3.39 (dd, 3J eqeq ) 2.1 Hz, 1H, H4-eq), 3.22 (s,
3H, OCH3), 7.38-7.45 (m, 3H, phenyl-3H + phenyl-4H), 8.09-
8.17 (m, 2H, phenyl-2H), 12.83 (bs, 1H, NH). Anal. (C16H19
N3O2S) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 8m ,n . 100 mg
of methoxy derivative 8k or 8l was added to a mixture of 0.80
g of AlBr3 and 5.0 mL of EtSH and stirred for 3 h at room
temperature. After quenching with 10 mL of water the solution
was acidified with 3 drops of HCl (concentrated) and three
times extracted with 15 mL of ethyl acetate. The combined
organic layers were dried on Na2SO4, concentrated under
reduced pressure, and purified by column chromatography
(EA/CH2Cl2 1:1 as eluent).
(s, 3H, OCH3), 6.97 (d, 2H, J AB ) 8 Hz, benzamide-3H) and
7.38-7.87 (m, 11H, H2 + H4 + H5 + H6 + phenyl-2H +
phenyl-3H + phenyl-4H + benzamide-2H). Anal. (C20H17NO2)
C, H, N.
-
Gen er a l P r oced u r e for 17a -j. N-[4-(2-P yr id yl)th ia zol-
2-yl]ben za m id e Hyd r och lor id e (17a ). 1.0 mmol of the
appropriate benzoic acid was dissolved in 0.5 mL of dry DMF
in a 2.2-mL Eppendorf vial. 45.0 mg (0.25 mmol) of 2-amino-
4-phenylthiazole in 0.5 mL of dry DMF was added, together
with a solution of 0.21 g (1.0 mmol) of DCC in 0.5 mL of dry
DMF and 14.0 mg (0.1 mmol) of DMAP. This was stirred for
4 days with a small stirring bar. The precipitate was removed
by centrifugation, washed with 1.0 mL of DMF, and the
combined DMF solutions were concentrated in vacuo. The
residue was dissolved in 10 mL of ethyl acetate (clear solution)
and a stream of HCl gas was bubbled through the solution. A
white precipitate formed, which was collected by centrifuga-
tion. The solid was recrystalized twice from EA/CCl4/MeOH:
N -(3-P h e n yl-1,2,4-t h ia d ia zol-5-yl)-t r a n s-4-h yd r oxy-
cycloh exa n a m id e (8m ): yield 78%; mp 236 °C; 1H NMR
(DMSO-d6) δ 1.05-1.30 (m, 2H, H3-ax), 1.40-60 (m, 2H, H2-
ax), 1.75-1.95 (m, 4H, H2-eq + H3-eq), 2.12-2.48 (m, 1H, H1-
1
yield off-white powder 49%; mp 176 °C; H NMR (CD3OD) δ
ax), 3.30-3.50 (m, J axax ) 15 Hz, 1H, H4-ax), 4.64 (d 3J ) 6
3
7.53-7.70 (m, 3H, benzamide-3H + benzamide-4H), 7.96 (ddd,
1H, J ) 6.0; 6.0; 3.1 Hz, pyridyl-5H), 8.06 (dd, 2H, J ) 7.2;
1.4 Hz, benzamide-2H), 8.42 (s, 1H, thiazolyl-5H), 8.57-8.64
(m, 2H, pyridyl-3H + pyridyl-4H), 8.78 (d, 1H, J ) 5.8 Hz,
pyridyl-6H); MS (EI) m/z 282 (MH+ (-HCl), 100%). Anal.
(C15H11N3OS‚HCl) C, H, N.
Hz, 1H, OH), 7.50-7.60 (m, 3H, phenyl-3H + phenyl-4H),
8.60-8.70 (m, 2H, phenyl-2H), 13.08 (bs, 1H, NH). Anal.
(C15H17N3O2S) C, H, N.
N-(3-P h en yl-1,2,4-th ia d ia zol-5-yl)-cis-4-h yd r oxycyclo-
h exa n a m id e (8n ): yield 47%; mp 175 °C; Rf value TLC (EA/
CH2Cl2) 0.43; 1H NMR (DMSO-d6) δ 1.43-1.70 (m, 6H, H3-ax
+ H2-ax + H2-eq), 1.81-1.96 (m, 2H, H3-eq), 2.50-2.64 (m,
1H, H1), 3.46-3.50 (m, 1H, H4-eq), 3.74 (d, 3J ) 7 Hz, 1H,
OH), 7.50-7.55 (m, 3H, phenyl-3H + phenyl-4H), 8.14-8.19
(m, 2H, phenyl-2H), 13.04 (bs, 1H, NH). Anal. (C15H17N3O2S)
C, H, N.
N-(4,5-Dih yd r o-1-p h en ylp yr a zol-3-yl)-4-m eth oxyben z-
a m id e (9). Method C yielded the dibenzoyl analogue, which
was dissolved in 5 mL of dioxane. Subsequently 1.5 mL of 1
M NaOMe was added and the mixture stirred at room
temperature for 48 h. Purification by column chromatography
(EA/n-hexane 3:1) yielded 3% yellow crystals: 1H NMR (CDCl3)
δ 3.22 (t, J ) 10 Hz, 2H, CH2), 3.81-3.93 (s + t, J ) 7.3 Hz,
5H, CH2 + OCH3), 6.73-6.88 (m, 5H, phenyl-2H, phenyl-4H,
benzamide 3H), 7.19 (m, 2H, phenyl-3H), and 7.74 (d, 2H, 3J BA
) 8.6 Hz, benzamide-2H). Anal. (C17H17N3O2) C, H, N.
N-(5-P h en ylp yr a zol-3-yl)-4-m et h oxyb en za m id e (10).
Method C was used for 2.08 mmol of 3-amino-5-phenylpyrazole
and 2.28 mmol of anisoyl chloride. The white solid contained
the desired product as well as the diamide and was purified
by column chromatography (EA/tol 1:1). Recrystallization from
methanol of the second fraction yielded 13% of 31 as white
needles: mp >300 °C; 1H NMR (CDCl3) δ 3.84 (s, 3H, OCH3),
7.04-7.06 (m, 3H, benzamide-3H + pyrazol-4H), 7.37-7.46 (m,
3H, phenyl-3H + phenyl-4H), 7.72-7.76 (m, 2H, phenyl-2H),
8.0 (d, 2H, 3J BA ) 8.7 Hz, benzamide-2H), 10.70 (bs, 1H, NH),
and 12.92 (bs, 1H, pyrazol-NH). Anal. (C17H15N3O2‚0.2CH3OH)
C, H, N.
N-[4-(2-P yr id yl)t h ia zol-2-yl]-4-ch lor ob en za m id e H y-
1
d r och lor id e (17b): yield white crystals 59%; mp 245 °C; H
NMR (CD3OD) δ 7.60 (ddd, 2H, J ) 8.6; 2.4; 2.1 Hz, benz-
amide-3H), 7.97 (ddd, 1H, J ) 6.2; 6.2; 2.7 Hz, pyridyl-5H),
8.06 (ddd, 2H, J ) 8.9; 2.4; 2.1 Hz, benzamide-2H), 8.42 (s,
1H, thiazolyl-5H), 8.57-8.64 (m, 2H, pyridyl-3H + pyridyl-
4H), 8.78 (ddd, 1H, J ) 5.5; 1.0; 1.0 Hz, pyridyl-6H); MS (EI)
m/z 316 (M(35Cl)H+ (-HCl), 100%), 318 (M(37Cl)H+ (-HCl),
28%). Anal. (C15H10ClN3OS‚HCl‚0.2CCl4) C, H, N.
N-[4-(2-P yr id yl)th ia zol-2-yl]-4-iod oben za m id e Hyd r o-
ch lor id e (17c): yield off-white powder 63%; mp 212 °C; 1H
NMR (CD3OD) δ 7.81 (ddd, 2H, J ) 8.6; 2.8; 2.1 Hz, benz-
amide-2H), 7.89 (m, 1H, pyridyl-5H), 7.98 (ddd, 2H, J ) 8.6;
2.8; 2.1 Hz, benzamide-3H), 8.41 (s, 1H, thioazolyl-5H), 8.56-
8.64 (m, 2H, pyridyl-3H + pyridyl-4H), 8.78 (ddd, 1H, J ) 5.5;
1.0; 1.0 Hz, pyridyl-6H); MS (EI) m/z 408 (MH+) (-HCl, 100%).
Anal. (C15H10IN3OS‚HCl‚0.2CCl4) C, H, N.
N-[4-(2-P yr id yl)th ia zol-2-yl]-4-m eth ylben za m id e Hy-
d r och lor id e (17d ): yield white crystals 78%; mp 221 °C; 1H
NMR (CD3OD) δ 2.44 (s, 3H, CH3), 7.38 (d, 2H, J ) 7.9 Hz,
bnzamide-3H), 7.90-8.00 (m, 1H, pyridyl-5H), 7.94 (d, 2H, J
) 8.2 Hz, benzamide-2H), 8.39 (s, 1H, thiazolyl-5H), 8.52-
8.64 (m, 2H, pyridyl-3H + pyridyl-4H), 8.76 (d, 1H J ) 5.4
Hz, pyridyl-6H); MS (EI) m/z 296 (MH+(-HCl), 100%). Anal.
(C16H13ClN3OS‚HCl‚0.2CCl4) C, H, N.
3
3
N-[4-(2-P yr id yl)th ia zol-2-yl]-4-m eth oxyben za m id e Hy-
d r och lor id e (17e): yield slightly yellow crystals 67%; mp 197
1
°C; H NMR (CD3OD) δ 3.90 (s, 3H, CH3), 7.09 (ddd, 2H, J )
8.9; 3.1; 2.1 Hz, benzamide-3H), 7.97 (m, 1H, pyridyl-5H), 8.04
(ddd, 2H, J ) 8.9; 3.1; 2.1 Hz, benzamide-2H), 8.40 (s, 1H,
thiazolyl-5H), 8.62 (m, 2H, pyridyl-3H + pyridyl-4H), 8.78 (dm,
1H, J ) 5.8 Hz, pyridyl-6H); MS (EI) m/z 312 (MH+ (-HCl)
100%). Anal. (C16H13N3O2S) C, H, N.
N-(6-P h en ylp yr im id in -4-yl)-4-m eth oxyben za m id e (11):
method D used for 0.292 mmol of 4-amino-6-phenylpyrimidine
and 0.704 mmol of p-anisic acid; reflux period 12 h; preparative
TLC (EA/CH2Cl2/TEA1:1 0.01) yielded 26% white solid; mp 160
3
°C; 1H NMR (CDCl3) δ 3.85 (s, 3H, OCH3), 6.98 (d, 2H, J AB
)
9 Hz, benzamide-3H), 7.40-7.50 (m, 3H, phenyl-3H + phenyl-
N-[4-(2-P yr idyl)th iazol-2-yl]-3,4-dich lor oben zam ide Hy-
3
1
4H), 7.85 (d, 2H, J BA ) 9 Hz, benzamide-2H), 8.02-8.12 (m,
d r och lor id e (17f): yield white crystals 72%; mp 257 °C; H
2H, phenyl-2H), 8.61 (bs, 1H, NH), 8.75 (s, 1H, pyrimidine-
NMR (CD3OD) δ 7.75 (d, 1H, J ) 8.6 Hz, benzamide-5H), 7.97
(ddd, 1H, J ) 6.2; 5.8; 2.7 Hz, pyridyl-5-H), 8.00 (dd, 1H, J )
8.6, 2.1 Hz, benzamide-6H), 8.22 (d, 1H, J ) 2.1 Hz, pyridyl-
H), and 8.88 (s, 1H, pyrimidine-H). Anal. (C18H15N3O2) C, H,
N.

Novel Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 761
2H), 8.42 (s, 1H, thiazolyl-5H), 8.56-8.63 (m, 2H, pyridyl-3H
+ pyridyl-4H), 8.78 (ddd, 1H, J ) 5.8; 1.0; 1.0 Hz, pyridyl-
6H); MS (EI) m/z 350 (M(35Cl, 35Cl)H+ (-HCl), 100%), 352
(M(35Cl, 37Cl)H+ (-HCl), 70%), 354 (M(37Cl, 37Cl)H+ (-HCl),
18%). Anal. (C15H9Cl2N3OS‚HCl‚0.1CCl4) C, H, N.
1H, J ) 7.2 Hz, N-penyl-4H), 7.03-7.38 (m, 3H, N-phenyl-3H
+ pyridyl-5H), 7.50 (dm, 2H, J ) 8.6 Hz, N-phenyl-2H), 7.64
(s, 1H, thiazolyl-5H), 7.88 (ddd, 1H, J ) 7.55; 7.55; 1.8 Hz,
pyridyl-4H), 7.99 (dm, 1H, J ) 7.9 Hz, pyridyl-3H), 8.57 (dm,
1H, J ) 5.2 Hz, pyridyl-6H); MS (EI) m/z 297 (MH+, 100%).
Anal. (C15H12N4OS) C, H, N.
N-[4-(2-P yr id yl)t h ia zol-2-yl]-3-ch lor ob en za m id e H y-
1
d r och lor id e (17g): yield white crystals 62%; mp 188 °C; H
N -(4-Me t h oxyp h e n yl)-N ′-[4-(2-p yr id yl)t h ia zol-2-yl]-
u r ea (21b): recystallization from methanol yielded 66% white
crystals; mp 258 °C; 1H NMR (DMSO-d6) δ 3.72 (s, 3H, CH3),
6.90 (d, 2H, J ) 8.6 Hz, phenyl-3H), 7.30 (m, 1H, pyridyl-5H),
7.39 (d, 2H, J ) 0.86 Hz, phenyl-2H), 7.71 (s, 1H, thiazolyl-
5H), 7.82-7.96 (m, 2H, pyridyl-3H + pyridyl-4H), 8.58 (m, 1H,
pyridyl-6H), 8.74 (s, 1H, NH), 10.66 (bs, 1H, NH); MS (EI) m/z
327 (MH+, 100%), 296 (MH+ - OCH3, 95%). Anal. (C16H14N4O2S)
C, H, N.
NMR (CD3OD) δ 7.58 (dd, 1H, J ) 8.2; 7.6 Hz, benzamide-
5H), 7.78 (ddd, 1H, J ) 7.9; 1.9; 1.9 Hz, benzamide-4H), 7.98
(ddd, 1J , J ) 6.4; 5.7; 2.7 Hz, pyridyl-5H), 8.00 (m, 1H,
benzamide-6H), 8.06 (t, 1H, J ) 1.7 Hz, pyrdiyl-2H), 8.43 (s,
1H, thiazolyl-5H), 8.57-8.68 (m, 2H, pyridyl-3H + pyridyl-
4H), 8.78 (ddd, 1H, J ) 5.5; 1.4; 1.4 Hz, pyridyl-6H); MS (EI)
m/z 316 (M(35Cl)H+ (-HCl), 100%), 318 (M(37Cl)H+ -HCl),
60%). Anal. (C15H10ClN3OS‚HCl) C, H, N.
N-[4-(2-P yr id yl)th ia zol-2-yl]-4-n itr oben za m id e Hyd r o-
ch lor id e (17h ): recrystallization of the HCl salt from metha-
N-P h en yl-N′-(4-p h en ylth ia zol-2-yl)u r ea (22): yield white
crystals 80%; mp 214 °C; 1H NMR (DMSO-d6) δ 7.03 (t, 1H, J
) 7.2 Hz, N-phenyl-4H), 7.28-7.54 (m, 8H, N-phenyl-2H +
N-phenyl-3H + thiazolphenyl-3H + thiazolphenyl-4H + thi-
azolyl-5H), 7.88 (d, 2H, J ) 7.6 Hz, thiazolphenyl-2H), 8.91
(s, 1H, NH2), 10.68 (bs, 1H, NH); MS (EI) m/z 296 (MH+,
100%). Anal. (C16H13N3OS) C, H, N.
1
nol yielded 53% white crystals; mp 261 °C; H NMR (CD3OD
+ 20% DMSO-d6) δ 7.94 (m, 1H, pyridyl-5H), 8.30 (ddd, 2H, J
) 9.3; 2.4; 2.1 Hz, benzamide-2H), 8.44 (ddd, 2H, J ) 8.9; 2.4;
2.1 Hz, benzamide-3H), 8.45 (s, 1H, thiazolyl-5-H), 8.56-8.62
(m, 2H, pyrdiyl-3H + pyridyl-4H), 8.80 (dm, 1H, J ) 6.1 Hz,
pyrdiyl-6H); MS (EI) m/z 327 (MH+ (-HCl), 100%. Anal.
(C15H10N4O3S‚HCl) C, H, N.
Molecu la r Mod elin g. Calculations were performed with
SPARTAN version 5.0 (Wavefunction, Inc., Irvine)36 running
on a Silicon Graphics O2 workstation. Default values in the
Merck force field38 were used in molecular mechanics mini-
mizations. Conjugate gradient energy minimizations were
continued until the rms energy derivative was less than 0.01
kcal/mol‚Å. The Monte Carlo method was used for conforma-
tional analysis (step size 15°). The semiempirical molecular
orbital program AM139 with the AM1 Hamiltonian was used
to optimize the conformation of the compounds. The keyword
MMOK was used because the structures contain an amide
moiety. The 3-21G* Gaussian basis set40 (Hartree-Fock, closed
shell) was used for ab initio calculations of the energy and the
molecular electrostatic potential. The electrostatic potentials
were sampled over the entire accessible surface of the mol-
ecules (corresponding roughly to a van der Waals contact
surface). The most negative electrostatic potential is red, and
the most positive electrostatic potential is blue.
P h a r m a cology. Ma t er ia ls: [3H]CGS 21680 and [3H]-
DPCPX were commercially available from DuPont Nemours,
(‘s Hertogenbosch, The Netherlands). [3H]ZM 241385 was
purchased from TOCRIS (Bristol, U.K.). [125I]AB-MECA was
prepared as described by Olah et al.28 Adenosine deaminase
was from Boehringer Mannheim (Mannheim, Germany). HEK
293 cells stably expressing the human adenosine A3 receptor
were a gift from Dr. K.-N. Klotz (University of Wu¨rzburg,
Germany).
Meth od s for r ecep tor bin d in g: Binding of [3H]DPCPX
to adenosine A1 receptors on rat cerebral cortex membranes
and of [3H]CGS 21680 to adenosine A2A receptors from rat
striatal membranes was performed as described previously.26,27
Binding of [3H]ZM 241385 to adenosine A2A receptors was
performed in test tubes containing an aliquot of rat striatal
membranes (100-200 µg of protein/mL) in incubation buffer
(50 mM Tris-HCl, adjusted to pH 7.4 at 25 °C) with ap-
proximately 2 nM [3H]ZM 241385 in a final volume of 1 mL
After incubation at 25 °C for 120 min the binding reaction was
terminated by filtration through Whatman GF/B filters under
reduced pressure (200 mbar) using a Brandell cell harvester
(Brandell Gaithersburg, MD). Filters were washed twice with
ice-cold buffer (5 mL) and placed in scintillation vials. Bound
radioactivity was determined using conventional liquid scintil-
lation spectroscopy techniques in a LKB1219 counter.
N-[4-(2-P yr id yl)t h ia zol-2-yl]-4-isop r op oxyb en za m id e
Hyd r och lor id e (17i): crystallization from methanol yielded
1
48% white needlelike crystals; mp 181 °C; H NMR (CD3OD)
δ 1.37 (d, 6H, J ) 5.8 Hz, CH3), 4.75 (quin, 1H, J ) 5.8 Hz,
CH), 7.06 (ddd, 2H, J ) 8.9; 2.1; 2.1 Hz, benzamide-3H), 7.95
(ddd, 1H, J ) 6.2; 6.2; 3.1 Hz, pyridyl-5H), 8.02 (ddd, 2H, J )
8.9; 2.1; 2.1 Hz, benzamide-2H), 8.38 (s, 1H, thiazoly-5H),
8.53-8.63 (m, 2H, pyridyl-3H + pyridyl-4H), 8.78 (dm, 1H, J
) 5.8 Hz, pyridyl-6H); MS (EI) m/z 340 (MH+ (-HCl), 100%).
Anal. (C18H17N3O2S‚HCl) C, H, N.
N-[4-(2-P yr id yl)th ia zol-2-yl]cyclop en ta n a m id e Hyd r o-
ch lor id e (17j): yield white crystals 66%; mp 233 °C; 1H NMR
(DMSO-d6 + 20% CD3OD) δ 1.50-1.96 (m, 8H, cyclopentyl-
2H + cyclopentyl-3H), 3.00 (m, 1H, cyclopentyl-1H), 7.73 (m,
1H, pyridyl-5H), 8.23-8.37 (m, 2H, pyridyl-3H + pyridyl-4H),
8.42 (s, 1H, thiazolyl-5H), 8.72 (d, 1H, J ) 5.5 Hz, pyridyl-
6H); MS (EI) m/z 274 (MH+ (-HCl), 100%. Anal. (C14H15N3-
OS) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 20a ,b, 21a ,b,
a n d 22. 90.0 mg (0.5 mmol) of 5-amino-3-phenyl-1,2,4-thia-
diazole or 2-amino-4-phenylthiazole was dissolved in 2.0 mL
of acetonitrile. This slightly turbid mixture was filtered and
the clear solution was transferred in a 2.2-mL Eppendorf vial.
100 µL (0.9 mmol) of isocyanate was added and the solution
was warmed at 56 °C for 2 h. Overnight a white solid
precipitated from the reaction mixture, which was collected
by centrifugation. The dry solid was recrystallized from PE
(60/80) and methanol or absolute ethanol.
N-P h en yl-N′-(3-p h en yl-1,2,4-th ia d ia zol-5-yl)u r ea (20a ):
yield white crystals 38%; mp 218 °C; 1H NMR (CD3OD) δ 7.10
(t, 1H, J ) 7.6 Hz, N-phenyl-4-H), 7.34 (t, 2H, J ) 7.9 Hz,
N-phenyl-3H), 7.41-7.53 (m, 5H, N-phenyl-2H + thiazol-
phenyl-3H + thiazolphenyl-4H), 8.16-8.21 (m, 2H, thiazol-
phenyl-2H); MS (EI) m/z 297 (MH+, 100%). Anal. (C15H12N4-
OS) C, H, N.
N-(4-Met h oxyp h en yl)-N′-(3-p h en yl-1,2,4-t h ia d ia zol-5-
yl)u r ea (20b): yield white crystals 43%; mp 210 °C; 1H NMR
(CDCl3 + 10%CD3OD) δ 3.81 (s, 3H, OCH3), 6.90 (d, 2H, J )
8.6 Hz, N-phenyl-3H), 7.40 (d, 2H, J ) 8.6 Hz, N-phenyl-2H),
7.43-7.49 (m, 3H, thiazolphenyl-3H + thiazolphenyl-4H),
8.13-8.20 (m, 2H, thiazolphenyl-2H); MS (EI) m/z 327 (MH+,
100%). Anal. (C16H14N4O2S) C, H, N.
Binding of [125I]AB-MECA to membranes of HEK 293 cells
stably expressing the human adenosine A3 receptor was
determined as described.28,29
N-(2-Met h oxyp h en yl)-N′-(3-p h en yl-1,2,4-t h ia d ia zol-5-
yl)u r ea (20c): yield white crystals 37%; mp 178-179 °C; H
NMR (CDCl3 + 10%CD3OD) δ 3.71 (s, 3H, OCH3), 6.95-7.15
(m, 4H, N-phenylH), 7.43-7.49 (m, 3H, thiazolphenyl-3H +
thiazolphenyl-4H), 8.13-8.20 (m, 2H, thiazolphenyl-2H). Anal.
(C16H14N4O2S‚0.2CH3OH) C, H, N.
1
Ack n ow led gm en t. This research was financially
supported by Byk Gulden (Zwanenburg, The Nether-
lands).
N-P h en yl-N′-[4-(2-p yr id yl)th ia zol-2-yl]u r ea (21a ): yield
white crystals 69%; mp 218 °C; 1H NMR (DMSO-d6) δ 7.10 (t,

762 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
van Muijlwijk-Koezen et al.
(23) J ung, M. E.; Lyster, M. A. Quantitative dealkylation of alkyl
ethers via treatment with trimethylsilyl iodide. A new method
for ether hydrolysis. J . Org. Chem. 1977, 42, 3761-3764.
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