5-Amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one: A versatile scaffold to obtain potent and selective A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm0708376

Binding assays on human A₁, A_2A, and A₃ adenosine receptors (ARs) and functional studies on A_2B ARs revealed that various 2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-diones VIII, previously reporte

Full text of DOI:10.1021/jm0708376

5676
J. Med. Chem. 2007, 50, 5676-5684
5-Amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one: A Versatile Scaffold To Obtain
Potent and Selective A3 Adenosine Receptor Antagonists†
Federico Da Settimo,§ Giampaolo Primofiore,§ Sabrina Taliani,*,§ Anna Maria Marini,§ Concettina La Motta,§
Francesca Simorini,§ Silvia Salerno,§ Valentina Sergianni,§ Tiziano Tuccinardi,§ Adriano Martinelli,§ Barbara Cosimelli,‡
Giovanni Greco,‡ Ettore Novellino,‡ Osele Ciampi,# Maria Letizia Trincavelli,# and Claudia Martini#
Dipartimento di Scienze Farmaceutiche and Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, UniVersita` di Pisa,
Via Bonanno 6, 56126 Pisa, Italy, and Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersita` di Napoli “Federico II”,
Via D. Montesano, 49, 80131 Napoli, Italy
ReceiVed July 10, 2007
Binding assays on human A1, A2A, and A3 adenosine receptors (ARs) and functional studies on A2B ARs
revealed that various 2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-diones VIII, previously
reported as ligands at the central benzodiazepine receptor (BzR), possess nanomolar affinity at the A3 AR.
Replacement of the amide of VIII with an amidine moiety gave the 5-amino-2-phenyl[1,2,3]triazolo[1,2-
a][1,2,4]benzotriazin-1-ones IX, which maintain a nanomolar potency at the A3 AR with selectivity over
the BzR. Insertion of a p-methoxybenzoyl at the 5-amino moiety enhanced A3 AR affinity and selectivity
over the A1, A2A, and A2B ARs. The best result of our lead optimization efforts is 9-chloro-5-(4-
methoxybenzoyl)amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one (23), which displayed a Ki
of 1.6 nM at the A3 AR and no significant affinity at the other ARs or the BzR. Docking simulations on
selected ligands into a model of the A3 AR allowed us to rationalize the structure-activity relationships of
phenyltriazolobenzotriazindiones VIII and aminophenyltriazolobenzotriazinones IX at the molecular level.
Introduction
allergenic and cerebroprotective agents.13 Furthermore, the
significant overexpression of A3 AR in several types of tumor
cells14,15 has recently led Baraldi and co-workers to propose
that A3 AR antagonists, in view of the prosurvival and
antiapoptotic effect of this subtype, might sensitize tumor cells
to chemotherapeutic drugs.16,17
Adenosine is a ubiquitous homeostatic substance released
from most cells and acts as an agonist at the A1, A2A, A2B, and
A3 adenosine receptors (ARs), all of which belong to the
superfamily of the G-protein-coupled receptors.1-3 ARs from
different species show 82-92% amino acid sequence identity,
the only exception being the A3AR, which exhibits a 74%
identity between rats and humans.4-6
The best known classes of A3 AR antagonists are triazolo-
quinazolines I,18,19 imidazopurinones II,20,21 pyrazolotriazol-
opyrimidines III,17,22,23 triazoloquinoxalines IV and V,24-26
isoquinolines VI,27-29 and pyridines VII30-32 (Chart 1). All these
compounds share a nitrogen-containing polyheterocycle analo-
gous to the one featured by phenyltriazolobenzotriazindiones
VIII (PTBTs, Chart 1), recently described by us as ligands at
the central benzodiazepine receptor (BzR).33 This structural
similarity, as well as our experience in switching a series of
3-aryltriazinobenzimidazoles (ATBIs) from Bz34 to A1AR
binding,35,36 provided the rationale for the screening of available
phenyltriazolobenzotriazindiones as AR antagonists. The na-
nomolar affinity for the A3AR displayed by most of the
compounds tested prompted the design of further analogues that
hopefully may possess an improved potency at the A3AR and
selectivity over the A1 and A2A ARs, as well as the BzR.
Specifically, the first subset of phenyltriazolobenzotriazindiones
evolved toward the 5-amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]-
benzotriazin-1-ones IX (APTBTs, Chart 1) bearing the same
amidine moiety featured by the AR antagonists I and III-V.
The structures of the investigated phenyltriazolobenzotriazin-
diones 1-8 and aminophenyltriazolobenzotriazinones 9-29 are
indicated in Table 2.
Responses to activation of ARs are mediated by different
second messenger systems, such as calcium or potassium
channels (A1 AR), phospholipase C (A1 AR, A2B AR, A3 AR),
and phospholipase D (A3 AR).7 Furthermore, all four AR
subtypes are differently coupled with adenylate cyclase, which
is inhibited by activation of A1 and A3 ARs, thus decreasing
cyclic AMP (cAMP) production, and is stimulated by activation
of A2A and A2B ARs, thus increasing cAMP levels.7 Each AR
subtype is currently considered as an attractive target for
pharmacological intervention in many pathological conditions
such as renal failure, cardiac and cerebral ischemeia, central
nervous system (CNS) disorders, neurodegenerative diseases,
and inflammatory pathologies, including asthma.8,9 Accordingly,
A1 AR antagonists have a therapeutic potential as potent
diuretics for the prevention of renal failure,10 arousal stimulators,
or cognitive enhancers in Alzheimer’s disease and memory
disorders;11 A2A AR antagonists may be promising new drugs
to treat neurodegenerative disorders, such as Parkinson’s
disease;11,12 A3 AR antagonists may be employed in the acute
treatment of stroke and glaucoma and as antiasthmatic, anti-
Our lead optimization strategy was based on the following
modifications: (i) insertion of small groups (H, F, Cl, Me, OMe)
in the 9-position of the tricyclic scaffold (R1) and in the 4′-
position of the pendent phenyl ring (R2) (10-16); (ii) func-
tionalization of the amidine 5-NH2 group of compounds 9, 11,
12, 14, and 16 to yield N5-substituted benzoyl (17-25) and N5-
substituted carbamoyl derivatives (27-30). The R3 substituents
† This paper is dedicated to Yvonne C. Martin on the occasion of her
retirement from Abbott Laboratories.
* To whom correspondence should be addressed. Phone: +390502219500.
Fax: +390502219605. E-mail: taliani@farm.unipi.it.
§ Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
‡ Universita` di Napoli “Federico II”.
# Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
nologie, Universita` di Pisa.
10.1021/jm0708376 CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/10/2007

Benzotriazin-1-ones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5677
Chart 1. Structures of Known (I-VII) and New (VIII and XI) A3 AR Antagonists
Table 1. Physical Properties of 5-Amino-[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one Derivatives 9-30
compd
R1
R2
R3
yield (%)
mp (°C)a
Anal. (formula) C, H, Nb
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
H
H
H
H
F
Cl
CH3
OCH3
H
Cl
CH3
H
H
H
H
H
H
H
H
H
H
H
67
60
61
67
60
65
62
69
42
44
59
44
52
44
47
43
46
51
45
44
48
43
236-238
242-244
273-275
254-255
253-255
246-248
255-257
203-205
235-236
200-202
240-242
168-170
237-239
220-222
158-160
234-236
208-210
223-225
190-192
209-211
218-220
235-236
C15H11N5O
C15H10FN5O
C15H10ClN5O
C16H13N5O
H
C16H13N5O2
C15H10ClN5O
C15H9Cl2N5O
C16H12ClN5O
C22H15N5O2
C23H17N5O3
C22H14ClN5O2
C23H16ClN5O3
C24H19N5O3
C23H16ClN5O2
C23H16ClN5O3
C22H13Cl2N5O2
C24H18ClN5O3
C29H19N5O3
C22H16N6O2
C23H18N6O3
C22H15ClN6O2
C23H18N6O2
Cl
Cl
Cl
H
H
H
H
H
H
COC6H5
COC6H4-4-OCH3
COC6H4-3-Cl
COC6H4-4-OCH3
COC6H4-4-OCH3
COC6H4-3-Cl
COC6H4-4-OCH3
COC6H4-3-Cl
COC6H4-4-OCH3
Cl
CH3
CH3
H
H
CH3
Cl
Cl
Cl
H
H
H
H
H
H
H
H
CONHC6H5
CONHC6H4-4-OCH3
CONHC6H4-3-Cl
CONHCH2C6H5
a Calculated after recrystallization from ethanol. b Elemental analysis results for C, H, and N were within (0.4% of the calculated values.
attached to N5 were selected taking into account literature data
about the key role of NH-acyl side chains in ligand recognition
by the A3 AR.19,23,26,28
starting material is represented by the 1-(2-amino-5-substituted
phenyl)-4-(4-substituted phenyl)-5-oxo-1,2,3-triazoles 31-38,
obtained as previously reported.33 Reaction of 31-38 with
cyanogen bromide in methanolic solution at room temperature
gave the target tricyclic derivatives 9-16 (Table 1).
Chemistry
The synthetic pathways yielding the novel aminophenyltria-
zolobenzotriazinones 9-30 are outlined in Scheme 1. The
Compounds 9, 11, 12, 14, and 16 were selected to be suitably
functionalized at the 5-NH2 group by acylation or nucleophilic

5678 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Da Settimo et al.
Table 2. Binding Activity at Human A1, A2a, and A3 ARs of Compounds 1-29
Ki (nM)a
b
c
d
compd
R1
R2
R3
hA1
hA2A
hA3
1
2
3
4
5
6
7
8
H
H
H
H
H
F
Cl
CH3
OCH3
H
Cl
CH3
H
F
Cl
CH3
OCH3
H
Cl
CH3
H
H
H
Cl
339.5 ( 54.2
592.8 ( 32.9
>10000
4400.5 ( 345.2
1070.5 ( 79.8
960 ( 85
108.9 ( 15.4
890.3 ( 76.5
43.9 ( 3.9
65.2 ( 3.5
104.2 ( 14.0
46.0 ( 3.6
89.5 ( 6.4
80.2 ( 6.6
106.7 ( 7.2
19.0 ( 1.5
204.5 ( 44.5
99.0 ( 4.5
389.2 ( 26.0
>10000
>10000
91.2 ( 8.2
97.0 ( 6.0
41.9 ( 5.2
86.5 ( 11.2
49.8 ( 8.1
382.5 ( 22.9
96.2 ( 7.1
39.6 ( 3.3
34.6 ( 7.2
368.5 ( 55.2
33.2 ( 2.1
29.2 ( 4.6
51.0 ( 3.4
295.2 ( 32.1
557.3 ( 79.3
103.0 ( 5.2
101.2 ( 7.8
4.3 ( 0.4
15.0 ( 1.2
55.7 ( 3.3
1.1 ( 0.01
41.5 ( 1.3
1.6 ( 0.07
>1000
>10000
>10000
>10000
H
>10000
Cl
Cl
Cl
H
H
H
H
H
Cl
Cl
Cl
H
>10000
>10000
>10000
9
H
H
H
H
H
H
H
H
134 ( 20.5
20.1 ( 4.6
493.3 ( 18.0
50.6 ( 7.7
9.0 ( 0.6
4.7 ( 0.22
67.5 ( 4.8
2.3 ( 0.1
1030.0 ( 166.1
>10000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
COC6H5
COC6H4-4-OCH3
COC6H4-3-Cl
COC6H4-4-OCH3
COC6H4-4-OCH3
COC6H4-3-Cl
COC6H4-4-OCH3
COC6H4-3-Cl
COC6H4-4-OCH3
H
H
H
H
>10000
>10000
CH3
CH3
H
H
CH3
80.9 ( 7.1
256.0 ( 27.0
2700.0 ( 100.1
916.0 ( 49.0
>10000
>10000
H
>10000
Cl
Cl
Cl
>10000
>10000
>10000
12.3 ( 3.5
9.2 ( 0.8
97.0 ( 8.3
49.1 ( 3.3
49.0 ( 5.1
43.4 ( 2.5
>1000
116.5 ( 11.4
91.1 ( 3.2
462.2 ( 55.2
764.0 ( 13.1
>10000
4800.0 ( 348.8
460.2 ( 66.9
262.1 ( 76.9
625.0 ( 14.8
120.1 ( 9.1
337
H
H
H
H
H
H
H
H
CONHC6H5
CONHC6H4-4-OCH3
CONHC6H4-3-Cl
CONHCH2C6H5
DPCPX
NECA
0.5
14
16
73
CI-IBMECA
890
401
0.22
a
The Ki values are means ( SEM derived from an iterative curve-fitting procedure (Prism program, GraphPad, San Diego, CA). bDisplacement of
specific [3H]DPCPX binding in membranes obtained from hA1 AR stably expressed in CHO cells. cDisplacement of specific [3H]NECA binding in membranes
obtained from hA2A AR stably expressed in CHO cells. dDisplacement of specific [125I]AB-MECA binding in membranes obtained from hA3 AR stably
expressed in CHO cells.
addition (procedures i-iii, Scheme 1). In particular, the prepara-
tion of N5-benzoyl compounds 18-25 was performed by
acylation with p-anisoylbenzoyl chloride or m-chlorobenzoyl
chloride in anhydrous dichloromethane at room temperature in
the presence of pyridine (Scheme 1, procedure i; Table 1). When
9 was allowed to react with benzoyl chloride under the same
conditions, only the dibenzoyl derivative 26 was obtained
(Scheme 1, procedure i; Table 1). We modified the reaction
procedure and found that the desired compound 17 was formed
with good yields by acylation of 9 with benzoic anhydride in
freshly distilled dimethylformamide at 70 °C in the presence
of triethylamine and a catalytic amount of (dimethylamino)-
pyridine (DMAP) (Scheme 1, procedure ii; Table 1). The N5-
carbamoyl derivatives 27-30 were obtained starting from 9 by
treatment with the appropriate substituted phenyl isocyanate or
benzyl isocyanate in tetrahydrofuran solution at room temper-
ature (Scheme 1, procedure iii; Table 1).
Biology
The affinities of the new compounds toward human A1, A2A
,
and A3 ARs were evaluated by competition experiments
assessing their respective abilities to displace [3H]DPCPX, [3H]-
NECA, or [125I]AB-MECA binding from transfected Chinese
hamster ovary (CHO) cells. Experiments were performed as
described elsewhere.36 Compounds 3, 21, 23, and 25, which
showed a high A3 affinity, were also tested in functional assays
at human A2B and A3 ARs by measuring their effects on NECA-
mediated cAMP modulation in transfected CHO cells.37
A subset of compounds (9, 11, 12, 14, 17, 23, 27, and 30),
selected as the most representative of the new AR ligands, were
evaluated for their affinity at the BzR in bovine brain membranes
by competition experiments against the radiolabeled antagonist
[3H]flumazenil at a fixed concentration of 10 µM.33
Results and Discussion
The syntheses of compounds 1-8 were performed as previ-
Table 2 lists the binding affinities at the human A1, A2A, and
A3 ARs of phenyltriazolobenzotriazindiones 1-8 and ami-
ously reported.33

Benzotriazin-1-ones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5679
Scheme 1. Synthesis of Target Compounds 9-30a
a Reagents. For 9, 11, 12, 14, 16: (i) acyl chloride, pyridine in dichloromethane. For 9: (ii) benzoic anhydride, DMAP, triethylamine in DMF; (iii)
substituted phenyl isocyanate or benzyl isocyanate in THF.
Table 3. Potency of Compounds 3, 21, 23, and 25 versus hA2B and hA3
nophenyltriazolobenzotriazinones 9-30 expressed as Ki values
Adenosine Receptor Subtypes
or inhibition percentages.
IC50 a (nM) of cAMP assays
Compounds 1-8 are moderately potent at the A1 AR and
compd
for hA2B CHO cells
for hA3 CHO cells
completely inactive at the A2A AR, with nanomolar affinity at
the A3 AR. Compounds 3, 5, and 8 are also selective over the
A1 and A2A ARs. A few combinations of R1 and R2 different
from a hydrogen slightly increase the potency at the A1 AR
(compare 7 vs 1) or at the A3 AR (compare 8 vs 1).
3
21
23
25
>1000 (26%)
1527 ( 150
>1000 (15%)
>1000 (26%)
12 ( 1.2
0.58 ( 0.05
3.14 ( 0.3
11.61 ( 1.02
a The data are expressed as the mean ( SEM of four independent
experiments performed in triplicate. In parentheses are indicated the
percentage of inhibition, at 10 µM compound, of the cAMP levels,
stimulated by 100 nM NECA for A2B adenosine receptors.
A pairwise comparison of the binding data of compounds
1-8 with those of compounds 9-16 reveals that the replacement
of the amide with an amidine moiety enhances the binding to
the A1 AR and, to a much greater extent, to the A2A AR, whereas
it does not significantly improve or worsen the affinity for the
A3 AR.
As mentioned, the choice of functionalizing the 5-NH2 of
compound 9 with acyl groups relied on the well-established role
of specific NHCOR3 side chains in conferring high potency and
selectivity on A3 AR antagonists.19,23,26,28
Compound 23 stands out for its remarkable potency and
selectivity at the A3 AR (Ki values at the A1, A2A, and A3 ARs
are 2700, >10000 nM, and 1.6 nM, respectively), thus
representing the best result of our lead optimization efforts.
Analogous attempts to enhance the potency of 19 at the A3
AR by changing R1 and/or R2 were unsuccessful (compare 22
and 24 vs 19).
Insertion of a benzoyl chain into the structure of 9, to give
17, slightly lowers the affinity at the ARs. This fact might be
interpreted by hypothesizing that the attractive interactions
created by the benzoyl group at each AR are slightly outweighed
by the disadvantageous ones (e.g., solvation, steric bulk). The
introduction of a methoxy in the para position of the benzoyl
moiety of 17, to yield 18, increases the A3 AR affinity by a
23-fold and also improves selectivity over the A1 and A2A ARs.
Compared with the p-methoxy group, an m-chlorine on the same
benzoyl is less effective but still beneficial for the affinity and
selectivity at the A3 AR (compare 19 vs 17). The above gains
in A3 AR affinity might reflect a hydrogen bond and hydro-
phobic interactions established by the receptor with the p-
methoxy of 18 and the m-chlorine of 19, respectively.
Taking compound 18 as a reference structure, further slight
improvements in the potency at the A3 AR can be achieved by
modulating the nature of R1 and R2 (compare the 4′-methyl
derivative 21 and the 9-chlorine derivative 23 vs the unsubsti-
tuted counterpart 18). The simultaneous presence of a 4′-methyl
and a 9-chlorine in the same compound does not lead to additive
favorable effects on A3 AR affinity but only to a significant
drop in A1 AR affinity (compare 25 vs 18).
Finally, two benzoyl groups attached to a hexocyclic nitrogen
negatively affect A1 and A2A AR affinities while they enhance
A3 AR binding by 4-fold (compare 26 vs 9).
Replacement of the benzoyl of 17 with the phenylcarbamoyl
to give 27 does not greatly influence affinities at the ARs. The
insertion of a p-methoxy or an m-chlorine on the phenylcar-
bamoyl of compound 27 to yield 28 or 29, respectively,
increases affinity at the A3 AR only 2-fold. Homologation of
27 to give 30 does not give better results for the A3 AR affinity.
Compounds 3, 21, 23, and 25 were also tested at the A2B AR
by evaluating their inhibitory effects on NECA-mediated cAMP
accumulation in CHO cells stably expressing this receptor
subtype (Table 3). These derivatives proved to be inactive in
this assay, thus indicating a complete lack of affinity toward
the A2B AR.
Given the recent discoveries of non-nucleoside AR ago-
nists,38,39 the efficacy profile of compounds 3, 21, 23, and 25
were evaluated in cAMP functional assays at the A3 AR (Table
3). Expectedly, all these compounds displayed full antagonism,
with potencies similar to their binding affinity. Furthermore,
when tested in the absence of NECA, they did not show any

5680 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Figure 1. Compound 1 docked into the A3 AR model.40
Figure 2. Compound 9 docked into the A3 AR model.40
Da Settimo et al.
significant effect on the cAMP level even after stimulation by
forskolin, indicating neutral antagonism (data not shown).
A subset of compounds (9, 11, 12, 14, 17, 23, 27, and 30),
selected as the most representative of the entire series of new
AR ligands, were evaluated for their affinity to the BzR. None
of the compounds tested inhibited [3H]flumazenil binding by
more than 50% at the fixed concentration of 10 µM (data not
shown).
The following chemical rules summarize the affinity/selectiv-
ity data of the new AR antagonists: (i) two carbonyl groups in
positions 1 and 5 of the triazolobenzotriazine scaffold (com-
pounds 1-8) produce good A3 AR antagonists with a fair or
high selectivity toward the A1 and A2A ARs, respectively; (ii)
replacement of the CdO(5) with an amine fragment to give
the amidine derivatives 9-16 enhances affinity at the A1 AR
and, to a much greater extent, at the A2A AR, with no significant
effect at the A3 AR, leading to a number of potent and
moderately A2A-selective ligands (compounds 13, 14, and 16);
(iii) functionalization of the 5-NH2 group with acyl or carbamoyl
substituents generally affords ligands with a nanomolar potency
at the A3 AR, with a different extent of selectivity over the A1
and A2A ARs (compounds 17-30); in particular, if the acyl
group is a p-methoxybenzoyl, it is possible to achieve a high
affinity and selectivity at the A3 AR (compounds 23 and 25).
A number of our ligands (1, 9, 17-19, 23, 26, and 28) were
docked into our recently published human A3 AR model40 using
the AUTODOCK 3.0 program41 and were then submitted to
1.2 ns of molecular dynamics simulation (see Experimental
Section for details). Their overall binding disposition appeared
to be very similar to the one recently hypothesized for the
triazoloquinazolines I and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidines III.40
As shown in Figure 1, the main scaffold of compound 1
interacts with the TM3 and TM5-7 domains of the receptor,
made up mostly of lipophilic amino acids (H95 (3.37), I98
(3.40), F182 (5.43), F239 (6.44), W243 (6.48), L246 (6.51),
H272 (7.43)); the pendent phenyl is likewise surrounded by
lipophilic residues (M99 (3.41), L102 (3.44), W185 (5.46), I186
(5.47), P189 (5.50)); the 5-carbonyl oxygen receives an H-bond
from the N250 (6.55) side chain.
Replacement of the 5-oxo moiety of compound 1 with a
hexocyclic amino group produced 9 and a corresponding 3-fold
gain in potency. The docking simulations performed on
compound 9 revealed that this ligand maintains all the hydro-
phobic interactions of compound 1, with its amidine moiety
making two H-bonds with the N250 (6.55) side chain (Figure
2).
As outlined previously, the introduction of a p-methoxy group
on the benzoyl moiety of 17 to yield 18 increased the A3 AR
affinity 23-fold. This fact seems to be consistent with a putative
H-bond accepted by the p-methoxy of compound 18 from the
K152 (EL2) ammonium moiety (see Figure 3). Such an H-bond

Benzotriazin-1-ones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5681
Figure 3. Compound 18 docked into the A3 AR model.40
Figure 4. Compound 28 docked into the A3 AR model40 superimposed on the A3 AR-18 complex (colored sky blue).
might explain mutagenesis data,42 which assign an important
role to K152 (EL2) in the binding of VUF850 (V, R ) COC6H4-
4-OCH3), a highly potent A3 AR antagonist sharing with
compound 18 the same p-methoxybenzoylamino side chain.
Favorable hydrophobic interactions between the m-chlorine
of compound 19 and the F182 (5.43) phenyl ring seem to
account for the 6.7-fold higher A3 AR affinity of this ligand,
compared with its unsubstituted counterpart 17.
Compound 28, featuring a p-methoxyphenylcarbamoylamino
side chain, was 10-fold less potent than 18. Docking studies
revealed that, unlike compound 18, the p-methoxy group of 28
is unable to engage an H-bond with K152 (EL2) (Figure 4 shows
an overlay of the two corresponding docking models). It is noted
that both of the carbonyl oxygens of the two ligands receive an
H-bond from N250 (6.55) while their p-methoxyphenyl moieties
present different dispositions in the receptor binding cleft.
Compound 23 shows an A3 affinity similar to that of
compound 18, but it possesses a much higher selectivity over
the A1 AR probably because of a steric interference of the
9-chlorine atom with the A1 AR protein. As shown in Figure 5,
23 is orientated similarly to 18 within the A3 AR binding site.
Although the 9-chlorine of 23 does not seem to be involved in
strong interactions, it determines a slight translation of the ligand
toward the extracellular side of the receptor.
adopts an overall orientation within the A3 AR binding site quite
similar to those displayed by the N5-benzoyl APTBT derivatives.
According to our model, while one benzoyl group of compound
26 forms an H-bond with N250 (6.55), the other one is stabilized
by hydrophobic interactions with F182 (5.43), W185 (5.46), and
I186 (5.47).
In conclusion, the aminophenyltriazolobenzotriazinone system
proved to be a versatile scaffold to obtain potent and selective
A3 AR antagonists, provided that suitable groups are attached
to the 5-amino moiety and in the 4′-position and/or 9-position.
The best result of our lead optimization efforts was represented
by compound 23 (R1 ) Cl, R2 ) H, and R3 ) p-methoxyben-
zoyl), which displayed a Ki of 1.6 nM at the A3 AR and no
significant affinity at the other ARs or the BzR. Docking
simulations on selected ligands into a model of the A3 AR
allowed us to develop structure-activity relationships at the
molecular level, which will be useful in the design of novel A3
AR antagonists.
Experimental Section
Chemistry. Melting points were determined using a Reichert
Ko¨fler hot-stage apparatus and are uncorrected. Infrared spectra
were recorded with a Nicolet/Avatar FT-IR spectrometer in Nujol
mulls. Routine nuclear magnetic resonance spectra were recorded
in DMSO-d6 solution on a Varian Gemini 200 spectrometer
operating at 200 MHz. Evaporation was performed in vacuo (rotary
evaporator). Analytical TLC was carried out on Merck 0.2 mm
Finally, our binding hypotheses received further support from
the docking of the highly potent compound 26. In spite of the
presence of two sterically demanding benzoyl groups, this ligand

5682 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Da Settimo et al.
Figure 5. Compound 23 docked into the A3 AR model.40
precoated silica gel aluminum sheets (60 F-254). Elemental analyses
were performed by our Analytical Laboratory and agreed with
theoretical values to within (0.4%.
The 9-substituted-2-(4-substituted phenyl)[1,2,3]triazolo[1,2-a]-
[1,2,4]benzotriazin-1,5(6H)-diones 1-8 and the 1-(2-amino-5-
substituted phenyl)-4-(4-substituted phenyl)-5-oxo-1,2,3-triazole
derivatives 31-38 were prepared essentially as previously described
in ref 33.
(0.16 mL) in 3.0 mL of the same solvent. The mixture was stirred
at room temperature for 24 h, and the precipitate obtained was
collected and washed with diethyl ether. The crude product 26 was
then purified by recrystallization from ethanol (Table 1 and
Supporting Information).
General Procedure for the Synthesis of 9-Substituted-2-(4-
substituted phenyl)-5-(substituted phenylaminocarbamoyl)-
[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one Derivatives 27-
29 and 2-Phenyl-5-(benzylaminocarbamoyl)[1,2,3]triazolo[1,2-
a][1,2,4]benzotriazin-1-one 30. The appropriate substituted isocyanate
(phenyl, 4-methoxyphenyl, 3-chlorophenyl, and benzyl isocyanates)
(0.8 mmol) was added dropwise to a stirred solution of 5-amino-
2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one 9 (0.137 g,
0.50 mmol) in 25.0 mL of anhydrous tetrahydrofuran. The mixture
was heated at 50 °C for 24-48 h (TLC analysis). After the mixture
was cooled, the precipitate that formed was collected and washed
with tetrahydrofuran. The organic solution was concentrated, and
the separated precipitate was collected to yield an additional amount
of crude product. The quantities of ureido derivatives obtained from
the initial insoluble precipitate and from the organic solution were
variable, depending on the solubility of the various compounds.
Finally, the crude products 27-30 were purified by recrystallization
from ethanol (Table 1 and Supporting Information).
General Procedure for the Synthesis of 5-Amino-9-substituted-
2-(4-substituted phenyl)[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-
1-one Derivatives 9-16. Cyanogen bromide (0.090 g, 0.85 mmol)
was added in a single portion to a stirred solution of the appropriate
1-(2-amino-5-substituted phenyl)-4-(4-substituted phenyl)-5-oxo-
1,2,3-triazole derivative 31-3833 (0.85 mmol) in 7.0 mL of absolute
methanol. The mixture was allowed to react at room temperature
under a nitrogen atmosphere for 12-36 h, and the reaction was
monitored by TLC analysis. The precipitated crude products 9-16
that formed were collected and purified by recrystallization from
ethanol (Table 1 and Supporting Information).
5-Benzoylamino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]-
benzotriazin-1-one 17. A solution of triethylamine (1.1 mL, 0.76
mmol) in 1.0 mL of freshly distilled dimethylformamide was added
dropwise to an ice-cooled, stirred suspension of 5-amino-2-phenyl-
[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1-one 9 (0.140 g, 0.51
mmol), benzoic anhydride (0.340 g, 1.52 mmol), and (dimethy-
lamino)pyridine (0.007 g, 0.006 mmol) in 2.0 mL of the same
solvent. The mixture was heated at 70 °C under a nitrogen
atmosphere for 72 h. After the mixture was cooled, the precipitated
crude product 17 was collected, washed with 5% NaHCO3 aqueous
solution, and purified by recrystallization from ethanol (Table 1
and Supporting Information).
General Procedure for the Synthesis of 9-Substituted-2-(4-
substituted phenyl)-5-(substituted benzoylamino)[1,2,3]triazolo-
[1,2-a][1,2,4]benzotriazin-1-one Derivatives 18-25. A solution
of the appropriate benzoyl chloride (4-methoxybenzoyl chloride,
3-chlorobenzoyl chloride) (0.7 mmol) in 1.0 mL of anhydrous
dichloromethane was added dropwise at 0 °C and under a nitrogen
atmosphere to a stirred suspension of the appropriate amino
derivatives 9, 11, 12, 14, and 16 (0.43 mmol) and anhydrous
pyridine (0.16 mL) in 3.0 mL of the same solvent. The mixture
was stirred at room temperature for 24-48 h, the reaction being
monitored by TLC analysis. The precipitate obtained was collected,
washed with diethyl ether, and purified by recrystallization from
ethanol (Table 1 and Supporting Information).
5,5-Dibenzoylamino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]-
benzotriazin-1-one 26. A solution of benzoyl chloride (0.11 mL,
0.7 mmol) in 1.0 mL of anhydrous dichloromethane was added
dropwise at 0 °C and under a nitrogen atmosphere to a stirred
suspension of 5-amino-2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]-
benzotriazin-1-one 9 (0.120 g, 0.43 mmol) and anhydrous pyridine
Biological Methods. Materials. [3H]DPCPX, [3H]NECA, and
[
125I]AB-MECA were obtained from DuPont-NEN (Boston, MA).
Adenosine deaminase (ADA) was from Sigma Chemical Co. (St.
Louis, MO). All other reagents were from standard commercial
sources and of the highest commercially available grade. CHO cells,
stably expressing human A1, A2A, A2B, and A3 receptors were kindly
supplied by Prof. K. N. Klotz, Wurzburg University, Germany.43
Adenosine Receptor Binding Assay. Human A1 Adenosine
Receptors. Aliquots of membranes (50 µg proteins) obtained from
A1 CHO cells were incubated at 25 °C for 180 min in 500 µL of
T1 buffer (50 mM Tris-HCl, 2 mM MgCl2, 2 units/mL ADA, pH
7.4) containing [3H]DPCPX (3 nM) and six different concentrations
of the newly synthesized compounds. Nonspecific binding was
determined in the presence of 50 µM R-PIA.36 The dissociation
constant (Kd) of [3H]DPCPX A1 CHO cell membranes was 3 nM.
Human A2A Adenosine Receptors. Aliquots of cell membranes
(80 µg) were incubated at 25 °C for 90 min in 500 µL of T2 buffer
(50 mM Tris-HCl, 2 mM MgCl2, 2 units/mL ADA, pH 7.4) in the
presence of 30 nM of [3H]NECA and six different concentrations
of the newly synthesized compounds. Nonspecific binding was
determined in the presence of 100 µM NECA.36 The dissociation
constant (Kd) of [3H]NECA in A2A CHO cell membranes was 30
nM.
Human A3 Adenosine Receptors. Aliquots of cell membranes
(40 µg) were incubated at 25 °C for 90 min in 100 µL of T3 buffer
(50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, 2 units/mL ADA,
pH 7.4) in the presence of 1.4 nM [125I]AB-MECA and six different

Benzotriazin-1-ones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5683
concentrations of the newly synthesized compounds. Nonspecific
binding was determined in the presence of 50 µM R-PIA.36 The
dissociation constant (Kd) of [125I]AB-MECA in A3 CHO cell
membranes was 1.4 nM.
sequential use of steepest descent and conjugate gradient methods
for the first 1000 and the last 4000 steps, respectively.
Molecular dynamics trajectories were run using the minimized
structure as a starting input. The time step of the simulations was
2.0 fs with a cutoff of 12 Å for the nonbonded interaction, and
SHAKE was employed to keep all bonds involving hydrogen atoms
rigid. The 1.2 ns of MD simulation was carried out by applying a
decreasing constraint on the R carbon of the TMs from 30 to 5
kcal mol-1 Å-1. The final structure of the complexes was obtained
as the average of the last 600 ps of MD simulation minimized with
the conjugate gradient method until a convergence of 0.05 kcal
Å-1 mol-1 was obtained.
In reference to specific amino acids, both the sequence number
and the numbering system proposed by Ballesteros and Weinstein47
(in parentheses) were employed. For the latter, the most highly
conserved residue in each transmembrane helix (TMH) was
assigned a value of 0.50 and this number was preceded by the TMH
number. The other residues in the helix were given a locant value
relative to this.
Measurement of Cyclic AMP Levels on hA2B and hA3 CHO
Cells. Intracellular cAMP levels were measured using a competitive
protein-binding method.37 CHO cells, expressing recombinant hA2B
or hA3 ARs, were harvested by trypsinisation. After centrifugation
and resuspension in medium, cells (about 60 000) were plated in
24-well plates in 0.5 mL of medium. After 48 h, the medium was
removed and the cells were incubated at 37 °C for 15 min with 0.5
mL of DMEM in the presence of Ro20-1724 (20 µM), as a
phosphodiesterase inhibitor, and ADA (1 U/mL). The antagonistic
profile of the new compounds toward A2B AR was evaluated by
assessing their ability to inhibit 100 nM NECA-mediated accumula-
tion of cAMP. The antagonistic profile of the new compounds
toward A3 AR was evaluated by assessing their ability to counteract
100 nM NECA-mediated inhibition of cAMP stimulated by 1 µM
forskolin. Cells were incubated in the reaction medium (15 min at
37 °C) with different compound concentrations (1 nM to 10 µM)
and then treated with NECA. In parallel, aliquots of cells were
treated with the compound alone (10 µM) in the absence or in the
presence of forskolin. The reaction was terminated by the removal
of the medium and the addition of 0.4 N HCl. After 30 min, lysates
were neutralized with 4 N KOH and the suspension was centrifuged
at 800g for 5 min. For determination of cAMP production, cAMP
binding protein (isolated from calf adrenal glands) was incubated
with [3H]cAMP (2 nM) and 50 µL of cell lysate or cAMP standard
(0-16 pmol) at 4 °C for 150 min in a total volume of 300 µL.
Bound radioactivity was separated by rapid filtration through GF/C
glass fiber filters and washed twice with 4 mL of 50 mM Tris/
HCl, pH 7.4. The radioactivity was measured by liquid scintillation
spectrometry.
All compounds were routinely dissolved in DMSO and diluted
with the assay buffer to the final concentration, where the amount
of DMSO never exceeded 2%. Percentage inhibition values of
specific radiolabeled ligand binding at 1-10 µM are the mean (
SEM of at least three determinations. For IC50 compound deter-
mination, at least six different ligand concentrations were used. IC50
values, computer-generated using a nonlinear regression formula
on a computer program (Graph-Pad, San Diego, CA), were
converted to Ki values, knowing the Kd values of radioligands in
the different tissues, and using the Cheng and Prusoff equation.44
Ki values are the mean ( SEM of at least three determinations.
Computational Methods. The ligands were submitted to a
conformational search of 1000 steps. The algorithm used was the
Montecarlo method with MMFFs as the force field and a distance-
dependent dielectric constant of 1.0. The ligands were then
minimized using the conjugated gradient method until a conver-
gence value of 0.05 kcal Å-1 mol-1, using the same force field
and dielectric constant used for the conformational search.45 Then
the ligands were docked into the recently published A3 receptor40
using the AUTODOCK 3.0 program.41 The regions of interest used
by AUTODOCK were defined by considering N250 (6.55) as the
central residue of a grid of 44, 54, and 50 points in the x, y, and z
directions. A grid spacing of 0.375 Å and a distance-dependent
function of the dielectric constant were used for the energy map
calculations.
Acknowledgment. This work was financially supported by
MIUR (Cofin 2005, ex 40%). We thank Prof. Dr. Karl-Norbert
Klotz for his generous gift of transfected CHO cells expressing
human A1, A2A, A2B, and A3 receptors.
Supporting Information Available: Spectral and analytical
data of compounds 9-30. This material is available free of charge
via the Internet at http://pubs.acs.org.
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