Derivatives of the triazoloquinazoline adenosine antagonist (CGS15943) are selective for the human A3 receptor subtype

Article abstract of DOI:10.1021/jm960482i

The adenosine antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5- c]quinazolin-5-amine (CGS15943) binds to human A₃ receptors with high affinity (K(i) = 14 nM), while it lacks affinity at rat A₃ receptors. Acylated derivatives of t

Full text of DOI:10.1021/jm960482i

4
142
J . Med. Chem. 1996, 39, 4142-4148
Expedited Articles
Der iva tives of th e Tr ia zoloqu in a zolin e Ad en osin e An ta gon ist (CGS15943) Ar e
Selective for th e Hu m a n A Recep tor Su btyp e
3
Yong-Chul Kim, Xiao-duo J i, and Kenneth A. J acobson*
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes,
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810
X
Received J uly 5, 1996
The adenosine antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS15943)
binds to human A
receptors. Acylated derivatives of the 5-amino group and other modifications were prepared
in an effort to provide A subtype selectivity. Affinity was determined in radioligand binding
assays at rat brain A and A2A receptors using [ H]-(R)-PIA ([ H]-(R)-N -(phenylisopropyl)-
3
receptors with high affinity (K
i
) 14 nM), while it lacks affinity at rat A
3
3
3
3
6
1
3
3
adenosine) and [ H]CGS 21680 ([ H]-2-[[4-(2-carboxy ethyl)phenyl]ethylamino]-5′-(N-ethyl-
3
carbamoyl)adenosine), respectively. Affinity was determined at cloned human A receptors
using [1 I]AB-MECA (N -(4-amino-3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine). A series
25
6
of straight chain alkyl amides demonstrated that the optimal chain length occurs with the
5
4
1
-N-propionyl derivative, 3, which had a K
0- and 14-fold selective vs rat A and A2A receptors, respectively. The 5-N-benzoyl derivative,
values of 680 and 273 nM at rat A and A2A receptors, respectively, and 3.0
receptors. A 5-N-phenylacetyl derivative, 12, was 470-fold selective for human
value of 0.65 nM. A conjugate of Boc-γ-aminobutyric acid was
also prepared but was nonselective. Conversion of the 5-amino group of CGS15943 to an oxo
function resulted in lower affinity but 15-fold selectivity for human A receptors.
i 3
value of 7.7 nM at human A receptors, and was
1
0, displayed K
i
1
nM at human A
vs rat A
3
A
3
1
receptors with a K
i
3
In tr od u ction
screening of libraries to identify structurally novel
1
1,15-18
leads.
Diverse classes of heterocycles, non-xan-
Four subtypes of adenosine receptors (A1, A2A, A2B,
and A3) have been cloned and studied pharmacologi-
thine adenosine antagonists, have already been found
to bind to A1 and A2A receptors and more recently to A3
adenosine receptors. We recently reported that two
1
cally. Since the recent discovery of the adenosine A3
2
receptor and the cloning of its species homologues,
much effort has been made in order to characterize the
receptor biochemically and pharmacologically. Princi-
pally, the development of selective agonist ligands has
aided in this effort. The A3 receptor mediates processes
1
6
chemical classes, the flavonoids and dihydropyri-
1
8
dines, are amenable to modification, resulting in
ligands selective for the recently discovered A3 receptor.
3
1
7
The flavonoid I (MRS 1067) and the dihydropyridine
1
8
derivative II (MRS 1097) (Figure 1) are selective
human A3 receptor antagonists.
2
4
of inflammation, hypotension, and mast cell degranu-
5
lation. This receptor apparently also has a role in the
The non-xanthine adenosine antagonist 1 (CGS15943,
central nervous system. The A3 selective agonist IB-
MECA induces behavioral depression and upon chronic
administration protects against cerebral ischemia. A3
selective agonists at high concentrations were also found
9
-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-
6
1
9,20
amine)
is very potent and slightly selective at human
7
2
1,29
brain A2A receptors with a Ki value of 1.7 nM.
It
had been under development as an antiasthmatic agent,
until unanticipated skin sensitivity was observed.12 This
antagonist was reported to be inactive in binding at rat
8
to induce apoptosis in HL-60 human leukemia cells.
These and other findings have made the A3 receptor a
promising therapeutic target.3 Selective antagonists for
the A3 receptor are sought as potential antiinflamma-
1
0
A3 receptors. Nevertheless, in light of the considerable
species variability in affinity of non-adenosine deriva-
tives at A3 receptors, the affinity of 1 (CGS15943) was
determined at cloned human A3 receptors and was found
to be relatively high. In this study we have prepared a
series of derivatives of 1 and found that N-acylation
greatly enhances affinity and selectivity for human A3
receptors.
9
-11
tory or possibly antiischemic agents in the brain.
Thousands of analogues of the classical adenosine
antagonists, the xanthines, have been prepared, result-
ing in high degrees of selectivity for A1 or A2A receptor
1
2-14
subtypes.
However, the xanthines have not pro-
vided fruitful leads for A3 receptor antagonists, since
they are generally much weaker in binding at the A3
subtype than at A1 or A2A subtypes.
9
,10
Thus, the search
Resu lts
for A3 receptor antagonists has been directed toward the
Syn th esis. The structures of the triazoloquinazoline
derivatives tested for affinity in radioligand binding
assays at adenosine receptors are shown in Table 1.
Several of the derivatives, e.g. the bromo derivative, 13,
and the 5-oxo derivative, 15, had been reported previ-
*
Correspondence to: Dr. K. A. J acobson, Bldg. 8A, Rm. B1A-19,
NIH, NIDDK, LBC, Bethesda, MD 20892-0810. Tel: (301) 496-9024.
Fax: (301) 480-8422. E-mail: kajacobs@helix.nih.gov.
X
Abstract published in Advance ACS Abstracts, October 1, 1996.
S0022-2623(96)00482-7
This article not subject to U.S. Copyright. Published 1996 by the American Chemical Society

Derivatives of the Triazoloquinazoline Adenosine Antagonist
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4143
1
7
F igu r e 1. Structures and affinities (K
dihydopyridine II (MRS 1097), and the nonselective antagonist triazoloquinazoline 1 (CGS15943).
i
3
value in µM) of novel A adenosine receptor antagonists the flavonoid I (MRS 1067), the
1
8
19,28,29
Ta ble 1. Affinities of Triazoloquinazoline Derivatives in Radioligand Binding Assays at A1, A2A, and A3 Receptors
Ki (nM), IC50 (nM), or % inhibition
compd
R1
R2
R3
rA1a
rA2Ab
hA3c
rA1/hA3
rA2A/hA3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
(CGS15943)
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
21 ( 3d
52.2 ( 2.6
283 ( 42
32.5 ( 9.4
28.9 ( 3.7
205 ( 20
190 ( 16
91.3 ( 13.2
11.3 ( 2.4
680 ( 82
200
3.3 ( 1.7d
36.3 ( 3.2
106 ( 30
37.6 ( 5.6
48.8 ( 10.5
88.8 ( 20.5
92.0 ( 8.0
135 ( 38
14.4
13.8 ( 2.4
13.9 ( 2.5
7.66 ( 3.03
14.6 ( 2.8
21.5 ( 6.2
244 ( 6
COCH3
3.8
40
2.2
1.3
0.84
2.3
2.6
14
(MRS1186)
COCH2CH3
CO(CH2)2CH3
CO(CH2)3CH3
COC(CH3)3
COOC(CH3)3
CO(CH2)3NH-Boc
CO(CH2)3NH2
COPh
2.6
2.3
0.36
1.1
4.1
0.18
90
82.5 ( 23.3
32.9 ( 1.7
80.8 ( 7.4
3.03 ( 1.73
23.8 ( 5.2
0.65 ( 0.25
64.0 ( 13.1
856 ( 156
260 ( 87
2.8
0.14
220
8.4
0 (MRS1177)
1
2 (MRS1220)
3
4
5
6
273 ( 42
310 ( 151
52.0 ( 8.8
531d
CO(3-I-Ph)
COCH2Ph
H
13
80
305 ( 51
470
1570d
COPh
<10%e
<10%e
4380
6960
H
3950 ( 250
1850 ( 420
15
1.0
17
3.8
(CH2)2CH3
1813 ( 720
a
3
b
Displacement of specific [ H]-(R)-PIA binding in rat brain membranes, expressed as Ki ( SEM in nM (n ) 3-5). Displacement of
3
c
125
specific [ H]CGS 21680 binding in rat striatal membranes, expressed as Ki ( SEM in nM (n ) 3-6). Displacement of specific [ I]AB-
d
MECA binding at human A3 receptors expressed in HEK-293 cells, in membranes, expressed as Ki ( SEM in nM (n ) 3-4). IC50 values
1
9
e
from Francis et al.
Percent displacement of specific binding at concentration 1 µM.
1
9
ously by Francis et al. to bind to A1 and A2A receptors.
The 5-oxo derivative19 was prepared in the present
study by an alternate route using an acid treatment of
commercially available 1 (Figure 2). Various N-acyl
derivatives were synthesized from 1 using standard
different species are compared. The species differences
in ligand affinity for triazoloquinazolines at A and A
1
2A
receptors are minor, whereas those at A receptors are
3
2
,26
significant (Figure 1).
Thus, our ratios of affinity are
indicative of selectivities in human, but not rat tissue.
1
9,20
acylation methodologies (Figure 2). Compound 13
The primary amine and 2-furanyl moieties in 1 have
was prepared by first protecting the amino group as the
tert-butyloxycarbonyl derivative, followed by bromina-
tion using N-bromosuccinimide and acidic deprotection.
The yields and chemical characterization of these
compounds are reported in Table 2.
Bin d in g a t Ad en osin e Recep tor s. Ki values were
determined in radioligand binding assays at rat cortical
A1 receptors vs [ H]-(R)-PIA ([ H]-(R)-N -(phenyliso-
been considered most important for binding to rat A1
1
9
and rat A2A receptors. In an effort to investigate the
effects on binding at human A3 receptors, various
structural modifications of these groups were made. The
structure-activity relationships (SAR) for binding at
adenosine receptors indicated that a variety of N-acyl
groups are tolerated at the 5-amino position (Table 1).
3
3
6
1
9
3
As with the parent compound 1, the affinity of the
N-acylated derivatives (2-12) at A2A receptors tended
propyl)adenosine) or rat striatal A2A receptors vs [ H]-
3
CGS 21680 ([ H]-2-[[4-(2-carboxyethyl)phenyl]ethyl-
amino]-5′-(N-ethylcarbamoyl)adenosine.2
2,23
Affinity at
to equal or surpass the affinity at A receptors. The
1
2
4
cloned human A3 receptors expressed in HEK-293 cells
substituent R1 was varied from acetyl to valeryl in a
1
25
6
was determined using [ I]AB-MECA (N -(4-amino-3-
125I]iodobenzyl)-5′-(N-methyl carbamoyl)adenosine).25
In this study, affinities at adenosine receptors from
homologous series (2-5). Also several acyl congeners
containing either bulky tert-butyl or derivatized aro-
matic moieties were synthesized to investigate steric
[

4
144 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Kim et al.
Ta ble 2. Yields and Chemical Characterization of Triazoloquinazoline Derivatives
compd no.
methoda
% yield
mp (°C)
MS
formula
analysis
2
3
4
5
6
7
8
9
0
1
2
4
6
A
A
A
A
B
A
C
96
84
96
73
70
98
49
83
73
50
35
28
50
245
224
232
220
200
285
209
252
239
243
233
266
178
CI: 328
CI: 342
CI: 356
FAB: 370
CI: 370
CI: 386
CI: 471
FAB: 371
CI: 390
CI: 515
EI: 403
FAB: 469
EI: 328
C15H10N5O2Cl‚0.16CH2Cl2
C16H12N5O2Cl
C17H14N5O2Cl‚0.2MeOH
C18H16N5O2Cl‚0.1EtOAc
C18H16N5O2Cl
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
b
C18H16N5O3Cl
C22H23N6O4Cl‚0.11CH2Cl2
C17H15N6O2Cl‚C2H1O2F3‚0.15CH2Cl2
C20H12N5O2Cl‚0.6CH2Cl2
C20H11N5O2ClI‚0.75MeOH
C21H14N5O2Cl‚0.38EtOAc
C20H11N5O2BrCl
1
1
1
1
1
A
C
B
C16H13N5O2Cl
b
a
A, symmetric anhydride method; B, acid chloride method; C, carbodiimide method. b High-resolution mass in EI or FAB+ mode (m/ z)
determined to be within acceptable limits. 14: calcd, 467.9863; found, 467.9871. 16: calcd, 328.0727; found, 328.0726.
A
B
F igu r e 2. Synthesis of N-acylated and various other deriva-
tives of 1 (CGS15943).
and electrostatic effects on the competitive binding at
each of the adenosine receptors.
Among the straight acyl chain derivatives, the N-
propionyl derivative, 3, had the highest binding affinity
(Ki ) 7.7 nM) and selectivity (40-fold vs rat A1, 14-fold
vs rat A2A) at human A3 receptors. Within this series,
the affinity at A3 receptors varied only 3-fold with
varying chain lengths, while at A2A receptors a greater
variation was observed. The affinities of compounds 6
and 7 indicated that bulky groups at position R1 were
less well tolerated than straight alkyl chains in human
A3 receptor binding. The pivaloyl derivative, 6, was 32-
fold less potent at A3 receptors than the propionyl
derivative, 3, from which it differed only in the presence
of two methyl groups at a branched carbon. The change
from an amide, 6, to the corresponding urethane, 7, had
no effect on affinity at A1 and A2A receptors, while the
affinity at A3 receptors was enhanced ∼3-fold. The
competitive binding curves of compounds 3, 6, and 7 at
human A3 receptors are shown in Figure 3A.
F igu r e 3. Representative competition curves comparing
inhibition of binding at adenosine receptors. (A) Inhibition by
125
compounds 3 (circles), 6 (squares), and 7 (triangles) of [ I]AB-
MECA binding at human A receptors, expressed in HEK-293
3
cells. (B) Inhibition by compound 12 of [1 I]AB-MECA binding
25
at human A
membranes, n
3
receptors (filled circles, transfected HEK-293 cell
3
H
) 0.75), [ H]-(R)-PIA binding at rat brain A
1
3
(filled diamonds, n
H
) 0.89), and [ H]CGS 21680 binding at
A2A (filled squares, n ) 0.84) receptors (in brain membranes).
H
receptors, while increasing potency at rat A1 and rat
A2A receptors.
Among aromatic acyl derivatives, the N-benzoyl de-
rivative, 10, showed higher binding affinity (Ki ) 3.0
nM) at the human A3 receptor than 1, while affinities
at rat A1 and rat A2A receptors were significantly
A Boc-γ-amino butyric acid conjugate, 8, of 1 was
slightly selective for human A3 receptors. The removal
of the Boc group resulting in an acyl chain containing
the primary amine functionality, 9, which is positively
charged at pH 7.4, reduced potency at human A3
9
decreased. At rat A receptors, compound 10 was much
3
less potent (data not shown), with only 23% of radioli-
gand binding displaced at 3 µM. Substitution of the
phenyl ring of the N-benzoyl group with a 3-iodo

Derivatives of the Triazoloquinazoline Adenosine Antagonist
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4145
substituent, 11, caused a marked reduction in affinity
only at human A3 receptors. However, the less sterically
hindered N-phenylacetyl compound, 12, showed yet
higher affinity (Ki ) 0.65 nM) and selectivity (470-fold
vs rat A1 receptors) for human A3 receptors than 10.
The competitive binding curves of compound 12 at rat
A1 and A2A receptors and at human A3 receptors are
shown in Figure 3B.
fold selectivity vs rat A1 receptor and 80-fold selectivity
vs rat A2A receptors. The displacement of radioligand
binding at rat A3 receptors by 10 and 12 was only 23%
(3 µM) and 12% (1 µM), respectively, showing that there
are marked differences of affinities between human A3
and rat A3 receptors.
Compound 14, the benzoyl derivative of compound 13,
however, had greatly diminished affinity at adenosine
receptors; thus the A3 receptor selectivity enhancing
effects induced by N-acylation and by 5-bromination are
not additive.
Two 5-bromofuranyl derivatives were prepared. Com-
pound 13, which was used as the synthetic precursor
in the preparation of tritiated 120 and reported to be
1
9
N-Acylations with various alkanecarboxylic acids
resulted in less selective ligands than the aryl carboxylic
acid derivatives. A structure-activity relationship
analysis of various length of acyl chain substituents (2-
much less potent at rat A1 and rat A2A receptors,
showed appreciable potencies at human A3 receptors.
Compound 14 was prepared with an expectation of
improving selectivity and affinity with respect to com-
pounds 10 and 13. Unfortunately, this combination of
functionalities displayed a dramatic loss of binding
activity at all three adenosine receptor subtypes.
7
) resulted in the interesting finding that compound 3
displayed the optimum acyl chain length for A3 receptor
binding.
There appears to be a hydrophobic pocket in the
Finally, two 5-oxo compounds were also prepared and
5
receptor in the vicinity of the N -amino group, since
tested. Compound 15 was previously reported to be
1
9
hydrophobic N-acyl and N-aryl substituents could en-
hance potency. In contrast, removal of the hydrophobic
Boc group from 8 resulted in the positively charged
congener 9 that displayed a lower affinity at A3 recep-
much less potent than 1 at A1 and A2A receptors, and
we found it to be 15-17-fold A3 receptor selective.
6
However, compound 16, the N -propyl derivative of 15
displaced radioligand binding with lower affinity at the
A3 adenosine receptor subtype.
1
5
tors. According to molecular modeling, in the receptor
6
bound states the N region of adenosine corresponds to
5
Discu ssion
the position of N of 1. In the present study, the
introduction of an aryl ring in that region enhanced
Until the present there have been few reports of leads
for the development of selective antagonists for the A3
6
selectivity, as was found in the case of N -benzylad-
3
enosine derivatives. Compound 11 was synthesized to
receptor,1
7,18
especially having high potency. We have
6
test further the hypothesis of overlap with the N region
1
1
screened chemical libraries and known adenosine
6
of adenosine, since the N -3-iodobenzyl substituent, vs
1
0
receptor ligands for potential A3 receptor antagonists.
Initially we reported that the triazoloquinazoline 1,
which was reported as the first potent, non-xanthine,
but nonselective adenosine A1 and A2A receptor antago-
nist, did not have appreciable binding activity at rat A3
6
unsubstituted N -3-benzyl, has been shown to enhance
A3 receptor affinity and selectivity. Nevertheless, in the
present series, the iodo substituent offered no advan-
5
tage.
N
is also proposed to correspond to the N3-
1
5
position of xanthines, at which large hydrophobic
substituents are tolerated, consistent with our find-
ings.
1
0
receptors. Nevertheless, in this study, it proved to be
highly potent at human A3 receptors (Ki ) 13.8 nM),
suggesting that it may serve as a lead compound in this
species. This finding encouraged us to investigate
structure-activity relationships of 1 derivatives for the
development of highly potent and selective antagonists
1
0
The selective ligands introduced here should be useful
as antagonists in characterizing and probing the physi-
ological role of human A3 receptors. They may also
provide affinity probes such as radioligands for human
A3 receptors. These selective agents must now be
studied in functional assays.
at human but not rat A3 receptors. There are dramatic
species differences2
,26
in the affinity of antagonists
binding at A3 receptors. The overall identity of protein
sequences between the A3 adenosine receptors of rat and
human was reported as 72%,1 which is relatively low,
consistent with the high variability in antagonist affin-
ity.
Exp er im en ta l Section
,2
Ma ter ia ls. Compound 1, (R)-PIA, and 2-chloroadenosine
were purchased from Research Biochemicals International
(
(
Natick, MA). All acylating agents were obtained from Aldrich
St. Louis, MO).
The present findings indicated that certain less polar
derivatives of 1 displayed increased affinity at the
human A3 receptor, and this enhanced binding affinity
could be distinguished from effects at rat A1 and rat A2A
receptors. Compound 13 and 15, which were previously
reported to have poor binding affinities at rat A1 and
A2A receptors, showed binding affinities to human
A3 receptors in the sub-micromolar range; thus some
degree of selectivity was present. The most significant
findings in the present study are that compounds 10
and 12, containing the N-benzoyl and N-phenylacetyl
substituents, respectively, are highly potent and selec-
tive for human A3 receptors. A competitive binding
assay of 10 showed high affinity at human A3 receptors
Syn th esis. Proton nuclear magnetic resonance spectros-
copy was performed on a Varian GEMINI-300 spectrometer
and spectra were taken in DMSO-d or CDCl . Unless noted,
6
3
chemical shifts are expressed as ppm downfield from tetra-
methylsilane. Chemical-ionization (CI) mass spectrometry
was performed with a Finnigan 4600 mass spectrometer and
electron-impact (EI) mass spectrometry with a VG7070F mass
spectrometer at 6 kV. Elemental analysis was performed by
Atlantic Microlab Inc. (Norcross, GA) or Galbraith Laborato-
ries, Inc. (Knoxville, TN). All melting points were determined
with a Unimelt capillary melting point apparatus (Arthur H.
Thomas Co., PA) and were uncorrected. All triazoloquinone
derivatives showed one spot on TLC (MK6F silica, 0.25 mm,
glass backed, Whatman Inc., Clifton, NJ ). Where needed
evaluation of purity was done on a Hewlett-Packard 1090
HPLC system using OD-5-60 C18 analytical column (150 mm
× 4.6 mm, Separation Methods Technologies, Inc., Newark,
DE) in two different linear gradient solvent systems. One
(
Ki ) 3.0 nM), with 220-fold selectivity vs rat A1
receptors and 90-fold selectivity vs rat A2A receptors.
Compound 12 displayed a Ki value of 0.65 nM, with 470-

4
146 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Kim et al.
solvent system (A) was 0.1 M TEAA/CH
in 20 min with flow rate 1 mL/min. The other (B) was H
3
CN, 30:70 to 10:90,
O/
7.91 (1H, d, J ) 8.8, H-7), 8.48 (1H, d, J ) 2.0, H-10), 9.15
(NH, broad s).
2
MeOH, 40:60 to 10:90, in 20 min with flow rate 1 mL/min.
Peaks were detected by UV absorption using a diode array
detector.
Gen er a l P r oced u r e for P r ep a r a tion of 5-N-Acyl De-
r iva tives of 1. Meth od A (Sym m etr ica l An h yd r id e). To
a stirred solution of 1 (10 mg, 0.035 mmol), anhydride (0.105
mmol) and (dimethylamino)pyridine (0.5 mg, 0.004 mmol) in
5-[(4-am in obu tyr yl)am in o]-9-ch lor o-2-(2-fu r an yl)[1,2,4]-
tr ia zolo[1,5-c]qu in a zolin e (9). A solution of 8 (3 mg, 6.4
µmol) in 1 mL of 50% TFA in CH
min and evaporated under reduced pressure. Crystallization
of the residue with MeOH/CH Cl gave 3.2 mg of 9 (83% as
TFA salt) as a white solid: 1H NMR (DMSO-d
) δ 1.87-1.97
(2H, m, H NCH CH CH CO), 2.82 (2H, t, J ) 7.32, H NCH
CH CH CO), 2.88-2.94 (2H, m, H NCH CH CH CO), 6.78-
2 2
Cl was left to stand for 10
2
2
6
2
2
2
2
2
2
-
1
0
.5 mL of anhydrous DMF was added triethylamine (73 µL,
.525 mmol) at room temperature. The mixture was stirred
2
2
2
2
2
2
2
6.80 (1H, m, H-4′), 7.34 (1H, d, J ) 3.05, H-3′), 7.77 (NH ,
broad s), 7.93-8.02 (3H, m), 8.40 (1H, d, J ) 1.2, H-10), 11.17
(NH, s).
for 48 h and then evaporated to dryness under reduced
pressure. The residue was purified by preparative silica gel
TLC (CH
2
Cl
2
/MeOH, 50:1 ∼ 75:1) to afford the desired
5-(Ben zoyla m in o)-9-ch lor o-2-(2-fu r a n yl)[1,2,4]tr ia zolo-
[1,5-c]qu in a zolin e (10): 1H NMR (CDCl
) δ 6.64-6.66 (1H,
compounds (2-5, 7, and 10).
3
Meth od B (Acid Ch lor id e). To a stirred solution of 1 (10
mg, 0.035 mmol) and anhydrous pyridine (40 µL, 0.5 mmol)
m, H-4′), 7.35 (1H, d, J ) 2.9, H-3′), 7.61-7.68 (3H, m), 7.68-
7.70 (1H, m, H-5), 7.77 (1H, dd, J ) 2.0, 8.8, H-8), 8.04-8.10
(3H, m), 8.52 (1H, d, J ) 2.0), 9.75 (NH, broad s).
2 2
in 1.5 mL of anhydrous CH Cl was added acyl chloride (0.105
mmol) at 0 °C. The mixture was stirred at room temperature
for 24-48 h and then treated with same procedure as method
A for purification of the desired compounds (6 and 12).
Meth od C (Ca r bod iim id e). A solution of 1 (10 mg, 0.035
mmol), the carboxylic acid component (0.210 mmol), 1-(3-
dimethylamino)propyl)-3-ethylcarbodiimide (41 mg, 0.210
mmol), 1-hydroxybenzotriazole (28 mg, 0.210 mmol), 4-(di-
methylamino)pyridine (0.5 mg, 0.004 mmol), and triethylamine
9-Ch lor o-2-(2-fu r a n yl)-5-[(3-iod oben zoyl)a m in o][1,2,4]-
1
tr ia zolo[1,5-c]qu in a zolin e (11): H NMR (CDCl
3
) δ 6.64-
6.66 (1H, m, H-4′), 7.30-7.36 (3H, m), 7.70 (1H, broad s, H-5′),
7.77 (1H, dd, J ) 2.0, 10.7, H-8), 7.92-8.10 (2H, broad m),
8.39 (1H, m), 8.52 (1H, d, J ) 2.0, hH-10), 9.62 (NH, broad s).
(
9-Ch lor o-2-(2-fu r a n yl)-5-[(p h en yla cetyl)a m in o][1,2,4]-
1
tr ia zolo[1,5-c]qu in a zolin e (12): H NMR (CDCl
3
) δ 4.38
(2H, s, CH CO), 6.62-6.65 (1H, m, H-4′), 7.24-7.26 (1H, m,
2
(
74 µL, 0.530 mmol) in 2 mL of anhydrous DMF/CH
2
Cl
2
(1:1
H-3′), 7.35-7.44 (5H, m), 7.67-7.68 (1H, m, H-5′), 7.74 (1H,
dd, J ) 2.2, 9.0, H-8), 7.93 (1H, d, J ) 8.8, H-7), 8.49 (1H, m,
H-10), 9.10 (NH, broad s).
5-Am in o-2-[2-(5-br om ofu r an yl)]-9-ch lor o[1,2,4]tr iazolo-
[1,5-c]qu in a zolin e (13): A solution of 7 (0.01 g, 0.026 mmol)
and N-bromosuccinimide (0.005 g, 0.028 mmol) in 2 mL of
v/v) was stirred at room temperature for 48 h. The mixture
was treated with same procedure as method A for purification
of desired compounds (8 and 11).
5
-(Acet yla m in o)-9-ch lor o-2-(2-fu r a n yl)[1,2,4]t r ia zolo-
1
[
1,5-c]qu in a zolin e (2): H NMR (CDCl
CH CO), 6.63-6.65 (1H, m, H-4′), 7.30 (1H, d, J ) 2.9, H-3′),
.68 (1H, broad s, H-5′), 7.73 (1H, dd, J ) 1.9, 8.8, H-8), 7.86
1H, d, J ) 8.8, H-7), 8.48 (1H, d, J ) 2.9, H-10), 8.99 (NH,
broad s).
-Ch lor o-2-(2-fu r a n yl)-5-(n -p r op ion yla m in o)[1,2,4]-
3
) δ 2.78 (3H, s,
3
3
AcOH/CHCl (1:1) was stirred for 1 h at room temperature.
7
(
The mixture was poured into 10 mL of saturated NaHCO
solution, and the product was extracted with 10 mL of CHCl
3
3
three times. The combined CHCl
brine, dried over anhydrous Na SO
ness under reduced pressure. The residue was purified by
preparative silica gel TLC (CHCl /MeOH, 80:1) to afford 2-[2-
3
solution was washed with
9
2
4
, and evaporated to dry-
1
tr ia zolo[1,5-c]qu in a zolin e (3): H NMR (CDCl
3
) δ 1.34 (3H,
t, J ) 7.8, CH CH CO), 3.10 (2H, q, J ) 7.8, CH
3
2
3
CH CO),
2
3
6
.63-6.65 (1H, m, H-4′), 7.31 (1H, d, J ) 3.9, H-3′), 7.68 (1H,
(5-bromofuranyl)]-5-[(tert-butoxycarbonyl)amino]-9-chloro[1,2,4]-
triazolo[1,5-c]quinazoline (0.012 g, 99%) as a white solid: MS
d, J ) 1.9, H-5′), 7.73 (1H, dd, J ) 2.0, 8.8, H-8), 7.88 (1H, d,
J ) 8.8, H-7), 8.48 (1H, d, J ) 2.0, H-10), 9.01 (NH, broad s).
+
1
(CI, NH
3
) 466 (M + 1); H-NMR (CDCl
3
3 3
) δ 1.63 (9H, s, (CH ) -
5
-(n -Bu tyr ylam in o)-9-ch lor o-2-(2-fu r an yl)[1,2,4]tr iazolo-
OCO), 6.58 (1H, d, J ) 3.6, H-4′), 7.29 (1H, d, J ) 3.6, H-3′),
7.73 (1H, dd, J ) 2.3, 8.8, H-8), 7.98 (1H, d, J ) 9.0, H-7),
8.46 (1H, d, J ) 2.3, H-10), 8.53 (NH, broad s). To a solution
1
[
7
3
1,5-c]qu in a zolin e (4): H NMR (CDCl
3
) δ 1.10 (3H, t, J )
CO), 1.84-1.91 (2H, m, CH CH CH CO),
.03 (2H, t, J ) 7.4, 7.3, CH CH CH CO), 6.63-6.65 (1H, m,
.4, 7.3, CH
3
CH
2
CH
2
3
2
2
3
2
2
2 2
of this compound in 2 mL of CH Cl was added TFA (0.05 mL,
H-4′), 7.31 (1H, d, J ) 3.4, H-3′), 7.68 (1H, d, J ) 1.8, H-5′),
0.67 mmol), and the mixture was stirred for 2 h at room
temperature. The reaction mixture was treated with same
workup procedure above. A preparative silica gel TLC (n-
7
8
.73 (1H, dd, J ) 2.3, 8.8, H-8), 7.88 (1H, d, J ) 8.8, H-7),
.48 (1H, d, J ) 2.3, H-10), 8.97 (NH, broad s).
9
-Ch lor o-2-(2-fu r a n yl)-5-(n -p e n t a n oyla m in o)[1,2,4]-
hexane/CHCl
3
/MeOH, 1:1:0.1) of the crude product gave 13 (4.5
1
+
tr ia zolo[1,5-c]qu in a zolin e (5): H NMR (CDCl
t, J ) 6.8, 7.8, CH CH CH CH CO), 1.48-1.55 (2H, m,
CH CH CH CH CO), 1.77-1.85 (2H, m, CH CH CH CH CO),
.05 (2H, t, J ) 7.82, 6.8, CH CH CH CH CO), 6.63-6.65 (1H,
m, H-4′), 7.31 (1H, d, J ) 2.9, H-3′), 7.68 (1H, broad s, H-5′),
.73 (1H, dd, J ) 2.0, 8.8, H-8), 7.88 (1H, d, J )8.8, 7-H), 8.48
1H, d, J ) 2.9, 10-H), 8.98 (NH, broad s).
-Ch lor o-2-(2-fu r an yl)-5-[(tr im eth ylacetyl)am in o][1,2,4]-
3
) δ 1.01 (3H,
mg, 48%) as a white solid: MS (CI, NH
3
) 366 (M + 1), 383
+
1
3
2
2
2
(M + 18); H-NMR (CDCl
3
) δ 5.94 (NH
2
, broad s), 6.56 (1H,
3
2
2
2
3
2
2
2
d, J ) 3.8, H-4′), 7.25 (1H, d, J ) 3.8, H-3′), 7.63-7.65 (2H,
m, H-8 + H-7), 8.41 (1H, d, J ) 2.1, H-10).
3
3
2
2
2
5-(Ben zoylam in o)-2-[2-(5-br om ofu r an yl)]-9-ch lor o[1,2,4]-
tr ia zolo[1,5-c]qu in a zolin e (14). The furanyl group in com-
7
(
1
pound 10 was brominated by the same method as above: H-
9
3
NMR (CDCl ) δ 6.64-6.66 (1H, m, H-4′), 7.35 (1H, d, J ) 2.9,
1
tr ia zolo[1,5-c]qu in a zolin e (6): H NMR (CDCl
s, (CH CCO), 6.63-6.65 (1H, m, H-4′), 7.31 (1H, d, J ) 3.4,
H-3′), 7.68-7.69 (1H, m, H-5′), 7.74 (1H, dd, J ) 2.5, 8.9, H-8),
.01 (1H, d, J ) 8.9, H-7), 8.49 (1H, d, J ) 2.5, H-10), 9.39
NH, broad s).
-[(ter t-Bu toxyca r bon yl)a m in o]-9-ch lor o-2-(2-fu r a n yl)-
1,2,4]tr ia zolo[1,5-c]qu in a zolin e (7): H NMR (CDCl
.63 (9H, s, (CH COCO), 6.65-6.66 (1H, m, H-4′), 7.35 (1H,
d, J ) 3.5, H-3′), 7.69 (1H, broad s, H-5′), 7.74 (1H, dd, J )
.6, 8.9, H-8), 8.01 (1H, d, J ) 8.9, H-7), 8.49 (1H, d, J ) 2.6,
H-10), 8.56 (NH, broad s).
-[[4-[(ter t-Bu t oxyca r bon yl)a m in o]bu t yr yl]a m in o]-9-
ch lor o-2-(2-fu r a n yl)[1,2,4]t r ia zolo[1,5-c]q u in a zolin e
3
) δ 1.47 (9H,
H-3′), 7.61-7.68 (2H, m), 7.68-7.70 (1H, m), 7.77 (1H, dd, J
) 2.0, 8.8, H-8), 8.04-8.10 (3H, m), 8.52 (1H, d, J ) 2.0, H-10),
9.75 (NH, broad s); HPLC retention time: 4.6 min (>95%
purity) using solvent system A, 12.5 min (>95% purity) using
solvent system B.
3 3
)
8
(
5
9-Ch lor o-2-(2-fu r a n yl)[1,2,4]tr ia zolo[1,5-c]qu in a zolin -
5(6H)-on e (15). A solution of 1 (0.075 g, 0.263 mmol) in 8.0
mL of AcOH and 2.0 mL of H O in a sealed tube was heated
2
for 72 h at 100 °C. The solution was coevaporated with toluene
under reduced pressure, and the residue was purified by
1
[
1
3
) δ
3
)
3
2
preparative silica gel TLC (CHCl
3
/MeOH, 15:1) to afford 15
5
(0.065 g, 86%) as a white solid: mp >310 °C; MS (CI NH )
3
+
+
1
287 (M + 1), 304 (M + 18); H-NMR (DMSO-d ) δ 6.73-
6
1
(
8): H NMR (CDCl
2H, pen, J ) 6.8, NHCH
.3, NHCH CH CH CO), 3.32 (2H, q, J ) 6.4, NHCH
CO), 4.81 (NH, broad s), 6.63-6.65 (1H, m, H-4′), 7.27-7.32
1H, m, H-3′), 7.67-7.68 (1H, m, H-5′), 7.71-7.75 (1H, m, H-8),
3
) δ 1.44 (9H, s, (CH
CH CH CO), 3.13 (2H, t, J ) 6.8,
2
CH CH -
3
)
3
COCONH), 2.03
6.74 (1H, m, H-4′), 7.26 (1H, d, J ) 3.5, H-3′), 7.46 (1H, d, J )
8.8, H-7), 7.77 (1H, dd, J ) 2.5, 8.9, H-8), 7.97 (1H, s, H-5′),
8.16 (1H, d, J ) 2.5, H-10), 12.47 (NH, s).
(
7
2
2
2
2
2
2
2
2
9-Ch lor o-2-(2-fu r a n yl) 6-n -p r op yl[1,2,4]tr ia zolo[1,5-c]-
qu in a zolin -5-on e (16). To a suspension of 15 (0.021 g, 0.073
(

Derivatives of the Triazoloquinazoline Adenosine Antagonist
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4147
mmol) in 2 mL of anhydrous THF was added a suspension of
NaH (6 mg, 60% in mineral oil, prewashed with n-hexane, 0.15
mmol) in 2 mL of anhydrous THF followed by HMPA (0.21
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mL, 12 mmol) under N
mixture was stirred vigorously for 30 min (H
-Bromopropane (28 µL, 0.3 mmol) was added, and the reaction
2
atmosphere at room temperature. The
2
gas evolved).
(
3
2) Linden, J . Cloned adenosine A receptors - pharmacological
1
properties, species -differences and receptor functions. Trends
Pharmacol. Sci. 1994, 15, 298-306.
mixture was refluxed for 6 h. After cooling, the precipitate
was removed by filtration through a small volume of silica gel
bed and the filtrate was evaporated. The residue was purified
(3) J acobson, K. A.; Kim, H. O.; Siddiqi, S. M.; Olah, M. E.; Stiles,
3
G.; von Lubitz, D. K. J . E. A adenosine receptors: design of
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by preparative silica gel TLC (n-hexane/EtOAc, 2:1) to afford
1
1
6 (0.012 g, 50%) as a white solid: H-NMR (CDCl
3
) δ 1.11
CH
N), 6.60-6.61
(
(
3H, t, J ) 7.5, 7.5, CH
3
CH
2
CH
2
N), 1.85-1.93 (2H, m, CH
N), 4.35 (2H, t, J ) 8.0, 7.5, CH
3
2
-
cells in adenosine A
Br. J . Pharmacol. 1995, 115, 945-952.
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receptor-mediated hypotension in the rat.
CH
2
3
CH CH
2
2
(
1H, m, H-4′), 7.32 (1H, d, J ) 3.4, H-3′), 7.37 (1H, d, J ) 9.3,
H-7), 7.66 (1H, broad s, H-5′), 7.68 (1H, dd, J ) 2.39, 9.0, H-8),
.52 (1H, d, J ) 2.5, H-10); HPLC retention time 3.5 min
3
Eur. J . Pharmacol. 1996, 298, 293-297.
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8
(
(
>95% purity) using solvent system A, 9.1 min (>95% purity)
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A -adenosine receptors: Mediation of behavioral depressant
3
using solvent system B.
P h a r m a cology: Ra d ioliga n d Bin d in g Stu d ies. Binding
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3
of [ H]-(R)-PIA to A
1
receptors from rat cerebral cortex
(7) von Lubitz, D. K. J . E.; Lin, R. C. S.; Popik, P.; Carter, M. F.;
3
J acobson, K. A. Adenosine A receptor stimulation and cerebral
membranes and of [ H]CGS 21680 to A2A receptors from rat
3
striatal membranes was performed as described previously.6
,8
ischemia. Eur. J . Pharmacol. 1994, 263, 59-67.
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(
Adenosine deaminase (3 units/mL) was present during the
preparation of the brain membranes, in a preincubation of 30
min at 30 °C and during the incubation with the radioligands.
Binding of [125I]AB-MECA in membranes prepared from
Induction of apoptosis in HL-60 human promyelocytic leukemia
cells by selective adenosine A
3
receptor agonists. Biochem.
Biophys. Res. Commun. 1996, 219, 904-910.
(9) Kim, H. O.; J i, X. D.; Melman, N.; Olah, M. E.; Stiles, G. L.;
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HEK-293 cells stably expressing the human A
3
receptor
3
xanthine derivatives at rat A adenosine receptors. J . Med.
(
Receptor Biology, Inc., Baltimore, MD), or from CHO cells
Chem. 1994, 37, 3373-3382.
7,25
stably expressing the rat A
assay medium consisted of a buffer containing 50 mM Tris,
1
3
receptor, was as described.1 The
(
10) van Galen, P. J . M.; van Bergen, A. H.; Gallo-Rodriquez, C.;
Melman, N.; Olah, M. E.; IJ zerman, A. P.; Stiles, G. L.; J acobson,
K. A. A binding-site model and structure-activity-relationships
2
+
0 mM Mg , and 1 mM EDTA, at pH 8.0. The glass
incubation tubes contained 100 µL of the membrane suspen-
for the rat A
3
-adenosine receptor. Mol. Pharmacol. 1994, 45,
1
101-1111.
sion (0.3 mg of protein/mL, stored at -80 °C in the same
1
25
(11) Siddiqi, S. M.; J i, X. D.; Melman, N.; Olah, M. E.; J ain, R.; Evans,
P.; Glashofer, M.; Padgett, W. L.; Cohen, L. A.; Daly, J . W.; Stiles,
G. L.; J acobson, K. A. A survey of non-xanthine derivatives as
adenosine receptor ligands. Nucleoside Nucleotide 1996, 15,
693-718.
buffer), 50 µL of [ I]AB-MECA (final concentration 0.3 nM),
and 50 µL of a solution of the proposed antagonist. Nonspecific
binding was determined in the presence of 200 µM NECA.
All nonradioactive compounds were initially dissolved in
DMSO and diluted with buffer to the final concentration,
where the amount of DMSO never exceeded 2%.
Incubations were terminated by rapid filtration over What-
man GF/B filters, using a Brandell cell harvester (Brandell,
Gaithersburg, MD). The tubes were rinsed three times with
(
12) J acobson, K. A.; van Galen, P. J . M.; Williams, M. Adenosine
receptorsspharmacology, structure activity relationships, and
therapeutic potential. J . Med. Chem. 1992, 35, 407-422.
(
13) Belardinelli, L.; Shryock, J . C.; Zhang, Y.; Scammells, P. J .;
Olsson, R.; Dennis, D.; Milner, P.; Pfister, J .; Baker, S. P. 1,3-
dipropyl-8-[2-(5,6-epoxy)norbornyl] xanthine, a potent, specific
and selective A
1
adenosine receptor antagonist in the guinea-
3
mL buffer each.
pig heart and brain and in DDT(1)MF-2 cells. J . Pharmacol. Exp.
Ther. 1995, 275, 1167-1176.
At least five different concentrations of competitor, spanning
orders of magnitude adjusted appropriately for the IC50 of
3
(
14) Baraldi, P. G.; Cacciari, B.; Spalluto, G.; Borioni, A.; Viziano,
M.; Dionisotti, S.; Ongini, E. Current developments of A2a
adenosine receptor antagonists. Curr. Med. Chem. 1995, 2, 707-
each compound, were used. IC50 values, calculated with the
nonlinear regression method implemented in the InPlot pro-
gram (Graph-PAD, San Diego, CA), were converted to apparent
7
22.
2
7
(15) van Rhee, A. M.; Siddiqi, S. M.; Melman, N.; Shi, D.; Padgett,
W. L.; Daly, J . W.; J acobson, K. A. Tetrahydrobenzothiophenone
derivatives as a novel class of adenosine receptor antagonists.
J . Med. Chem. 1996, 39, 398-406.
K
i
values using the Cheng-Prusoff equation and K
d
values
of 1.0 nM, 14 nM for [ H]-(R)-PIA and [ H]CGS 21680, and
.59 nM for binding of [125I]AB-MECA at human A
receptors,
3
3
0
3
respectively. Most Hill coefficients of tested compounds were
in the range 0.8-1.1.
Ab b r evia t ion s: AcOH, acetic acid; Boc, tert-butoxycar-
bonyl; CGS 21680, 2-[[4-(2-carboxyethyl)phenyl]ethylamino]-
(16) J i, X. D.; Melman, N.; J acobson, K. A. Interactions of flavonoids
and other phytochemicals with adenosine receptors. J . Med.
Chem. 1996, 39, 781-788.
(
17) Karton, Y.; J iang, J .-l.; J i, X. D.; Melman, N.; Olah, M. E.; Stiles;
G. L.; J acobson, K. A. Synthesis and biological activities of
5
′-N-(ethylcarbamoyl)adenosine; CGS15943, 9-chloro-2-(2-
flavonoid derivatives as A
Med. Chem. 1996, 39, 2293-2301.
(18) van Rhee, A. M.; J iang, J .-l.; Melman, N.; Olah, M. E.; Stiles,
G. L.; J acobson, K. A. Interaction of 1,4-dihydropyridine and
3
adenosine receptor antagonists. J .
furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine; CHO cells,
Chinese hamster ovary cells; CI, chemical ionization; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide; EDTA,
ethylenediaminetetraacatate; HEK cells, human embryonic
pyridine derivatives with adenosine receptors: selectivity for A
3
1
25
receptors. J . Med. Chem. 1996, 39, 2980-2989.
19) Francis, J . E.; Cash, W. D.; Psychoyos, S.; Ghai, G.; Wenk, P.;
Friedmann, R. C.; Atkins, C.; Warren, V.; Furness, P.; Hyun, J .
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1020.
kidney cells; HMPA, hexamethylphosphotriamide; [ I]AB-
(
1
25
6
MECA, [ I]-N -(4-amino-3-iodobenzyl)-5′-(N-methylcarbam-
oyl)adenosine; K , equilibrium inhibition constant; MS, mass
i
spectrum; NECA, (N-ethylcarbamoyl)adenosine; (R)-PIA, (R)-
6
N -(phenylisopropyl)adenosine; SAR, structure-activity rela-
(20) J arvis, M. F.; Williams, M.; Do, U. H.; Sills, M. A. Characteriza-
tion of the binding of a novel non-xanthine adenosine antagonist
tionship; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC,
thin layer chromatography; Tris, tris(hydroxymethyl)amino-
methane.
3
radioligand, [ H]CGS 15943, to multiple affinity states of the
adenosine A
9, 49-54.
21) J i, X.-D.; Stiles, G. L.; Galen, P. J . M. v.; J acobson, K. A.
Characterization of human striatal A -adenosine receptors using
radioligand binding and photoaffinity labeling. J . Recept. Res.
992, 12, 149-169.
22) Schwabe, U.; Trost, T. Characterization of adenosine receptors
1
receptor in the rat cortex. Mol. Pharmacol. 1991,
3
(
(
Ack n ow led gm en t. We thank Dr. Mark E. Olah and
Prof. Gary L. Stiles (Duke University Medical Center,
Durham, NC) and Dr. Garth Brown of DuPont NEN
2
1
1
25
(
North Billerica, MA) for providing samples of [ I]AB-
3
6
in rat brain by (-) [ H]N -phenylisopropyladenosine. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1980, 313, 179-187.
MECA.

4
148 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
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Kim et al.
(
(27) Cheng, Y. C.; Prusoff, W. H. Relationship between the inhibition
3
Williams, M. [ H]CGS 21680, an A
2
selective adenosine receptor
i
constant (K ) and the concentration of inhibitor which causes
agonist directly labels A receptors in rat brain tissue. J .
2
50 percent inhibition (IC50) of an enzyme reaction. Biochem.
Pharmacol. 1973, 22, 3099-3108.
Pharmacol. Exp. Ther. 1989, 251, 888-893.
(
24) Salvatore, C. A.; J acobson, M. A.; Taylor, H. E.; Linden, J .;
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J ohnson, R. G. Molecular cloning and characterization of the
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