Design, synthesis and biological evaluation of novel N-alkyl- and N-acyl-(7-substituted-2-phenylimidazo[1,2-a][1,3,5]triazin-4-yl)amines (ITAs) as novel A1 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm020911e

Prompted by pharmacophore and docking based models, we have synthesized and tested a number of N-alkyl and N-acyl-(7-substituted-2-phenylimidazo[1,2-a] [1,3,5]triazin-4-yl)amines (ITAs, 7) designed as a new class of A₁ adenosine receptor (A

Full text of DOI:10.1021/jm020911e

5030
J . Med. Chem. 2002, 45, 5030-5036
Design , Syn th esis a n d Biologica l Eva lu a tion of Novel N-Alk yl- a n d
N-Acyl-(7-su bstitu ted -2-p h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-yl)a m in es (ITAs) a s
Novel A1 Ad en osin e Recep tor An ta gon ists
Ettore Novellino,* Enrico Abignente, Barbara Cosimelli, Giovanni Greco, Manuela Iadanza, Sonia Laneri,
Antonio Lavecchia, and Maria Grazia Rimoli
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita` di Napoli “Federico II”,
Via D. Montesano, 49, 80131 Napoli, Italy
Federico Da Settimo and Giampaolo Primofiore
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy
Daniela Tuscano, Letizia Trincavelli, and Claudia Martini
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa,
Via Bonanno 6, 56126 Pisa, Italy
Received April 17, 2002
Prompted by pharmacophore and docking based models, we have synthesized and tested a
number of N-alkyl and N-acyl-(7-substituted-2-phenylimidazo[1,2-a][1,3,5]triazin-4-yl)amines
(ITAs, 7) designed as a new class of A1 adenosine receptor (A1AR) antagonists. Binding affinities
at the A1AR, A2AAR, and A3AR were determined using bovine cerebral membranes. Most of
the compounds displayed Ki values at the A1AR in the submicromolar or even in the low
nanomolar range, thus confirming the rationale leading to their synthesis. All or most of the
ligands turned out to be selective for the A1AR over the A2AAR and A3AR subtypes, respectively.
Structure-affinity relationships at the A1AR were rationalized by docking simulations in terms
of putative ligand/receptor interactions. Among the ITAs investigated, 1-[(7-methyl-2-phenyl-
imidazo[1,2-a][1,3,5]triazin-4-yl)amino]acetone (7j) exhibited the best combination of affinity
at the A1AR (Ki ) 12 nM) and selectivity over the A2AAR and A3AR subtypes (Kis > 10000
nM).
In tr od u ction
and CNS have been studied. Currently, A1AR selective
antagonists are developed as potassium-saving diuretics
with kidney-protective properties, as general cognition
enhancers for geriatric therapy, and for the treatment
of acute renal failure and of CNS disorders such as
Alzheimer desease.5-7
The activation of cell surface adenosine receptors
(ARs) is predominantly responsible for the wide variety
of effects produced by adenosine throughout several
organ systems. The combination of pharmacological
studies and molecular cloning have led to the discovery
of the existence of at least four distinct adenosine
receptors which have been identified and classified as
A1, A2A, A2B, and A3. The responses of these four ARs
are mediated by receptor-coupled G-proteins which
activate several effector systems including adenylate
cyclase, potassium and calcium channels, phospholipase
A2 or C, and guanylate cyclase.1-3
On the basis of the widespread effects attributed to
the accumulation of endogenously released adenosine,
it has long been considered that regulation of ARs has
substantial therapeutic potential.
The A1 adenosine receptor (A1AR) is the best known
and the most comprehensively studied AR subtype, and
during the last 20 years a large number of A1AR
agonists4 and antagonists5-7 have been developed. With
regards to possible therapeutic application of A1AR
antagonist, effects on the cardiovascular system, kidney,
We have recently described 3-aryl[1,2,4]triazino[4,3-
a]benzimidazol-4(10H)-one (ATBI) derivatives as a new
class of selective A1AR antagonists.8 The most potent
ATBI, compound 1, displayed a Ki value of 18 nM on
bovine cortical preparations. Pharmacophore- and dock-
ing-based modeling of 1 and other well-known A1AR
antagonists (2-6) (see Chart 1) suggested that these
ligands could interact with three H-bond sites (HB1,
HB2, and HB3) and three lipophilic pockets (L1, L2, and
L3) within the receptor binding site. The Asn254 (helix
VI) side chain CO and NH2 moieties (HB1 and HB2,
respectively) and the Ser277 (helix VII) hydroxyl (HB3
as a H-bond donor) group correspond to the H-bond
centers. The above interactions are highlighted in
Figure 1.
In pursuing our researches in this field we have
investigated N-alkyl and N-acyl 7-substituted-2-phenyl-
imidazo[1,2-a][1,3,5]triazin-4-amines (ITAs, 7a-r ), which
fulfill the pharmacophore requirements for A1AR an-
tagonism (Figure 2). These compounds were prepared
* To whom all correspondence should be addressed. Tel: +39
081678643. Fax: +39 081678644. E-mail: novellin@unina.it.
10.1021/jm020911e CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/16/2002

Novel A1 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5031
Ch a r t 1
the results of docking simulations into our model of
bovine A1AR (details are given in the Experimental
Section). Inspection of the L1 lipophilic pocket suggested
that R′ has to be hydrophobic and not exceedingly large.
However, a less lipophilic R′, such as CH2COCH3 would
enable the carbonyl oxygen to accept one or two H-bonds
from Ser277 and Thr91 (helix III) hydroxyls. Figure 3
shows the docking model of 7j (R′ ) CH2COCH3, R )
CH3) featuring the two putative H-bonds described
above.
A phenyl ring in position 2, supposed to fill the L2
site (Figure 2), was maintained fixed all through the
ITA series. This substituent gave, in fact, the most
potent ATBI (compound 1) by interacting with the L2
lipophilic pocket.
Lead optimization efforts focused initially on the
nature of R′ by keeping fixed R ) CH3. Replacements
of the 7-CH3 with a 7-C2H5, a 7-isoC3H7, or a 7-Ph, to
optimize the hydrophobic and steric interactions be-
tween R and the L3 site, were accomplished on a few
selected structures.
The present paper describes the design, the synthesis,
the biological evaluation, and the structure-activity
relationships (SARs) of compounds with a general
formula 7.
F igu r e 1. Pharmacophoric overlay of compounds 1-6. HB1,
HB2 and HB3 are H-bond sites of A1AR corresponding to
Asn254 (helix VI) and Ser277 (helix VII) side chains. L1, L2
and L3 map hydrophobic interactions. 1-6 are described in the
following references: 1,8 2,9 3,10 4,11 5,12 6.13
Ch em istr y
The imidazo[1,2-a][1,3,5]triazine derivatives 7a -r
were prepared in two steps. First, the imidazo[1,2-a]-
[1,3,5]triazines were synthesized using a synthetic
method in which the starting 2,4-diamino-6-phenyl-
[1,3,5]triazine 8 was warmed in refluxing ethanol with
2-chloroacetone 9, 1-chlorobutan-2-one 10, 1-bromo-3-
methylbutan-2-one 11, or 2-bromoacetophenone 12, to
obtain the corresponding 7-methyl-2-phenylimidazo[1,2-
a][1,3,5]triazin-4-ylamine 7a , 7-ethyl-2-phenylimidazo-
[1,2-a][1,3,5]triazin-4-ylamine 13, 7-isopropyl-2-phenyl-
imidazo[1,2-a][1,3,5]triazin-4-ylamine 14, and 2,7-di-
phenylimidazo[1,2-a][1,3,5]triazin-4-ylamine 15, respec-
tively (Scheme 1).
F igu r e 2. Pharmacophoric scheme of ITAs/A1AR interaction.
following a versatile procedure similar to that described
by us for isosteric imidazo[1,2-a]pyrimidines showing
antiinflammatory activity.14
Selection of substituents R′ and R at the N-4 and
7-position of the imidazotriazine nucleus was guided by
The correct structural assignments for these products
1
were performed with H and 13C NMR spectroscopy. In

5032 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Novellino et al.
F igu r e 3. Model of the A1AR binding site complexed with compound 7j. Only amino acids located within 5 Å from any atom of
the bound ligand are displayed. Dotted lines highlight ligand/receptor H-bonds.
Sch em e 1
Sch em e 2a
a
(a) R′Cl or R′Br, CH3CN, K2CO3, KI; (b) (MeCO)2O, 4-DMAP,
THF.
of [3H]-N6-cyclohexyladenosine ([3H]CHA) and [3H]-2-
{[4-(2-carboxyethyl)phenethyl]amino}-5′-(N-ethylcar-
bamoyl)adenosine ([3H]CGS 21680) to bovine cortical
(A1) and striatal (A2A) membranes, respectively. Affinity
at the A3AR was determined in competition assays
of [125I]-N6-(3-iodo-4-aminobenzyl)-5′-N-methylcarbox-
amidoadenosine ([125I]AB-MECA) to bovine cortical
membranes in the presence of the A1AR selective
antagonist DPCPX (20 nM).8
The results of the binding experiments are sum-
marized in Table 1. Most of the compounds display Ki
values at the A1AR in the sub-micromolar or even in
the low nanomolar range, thus confirming the rationale
leading to their synthesis. Most of the ligands turned
out selective for the A1AR over the A2AAR and A3AR
subtypes. Those binding to the A1AR with a high
potency and selectivity are 7g, 7j, and 7n . Moreover,
compounds 7g, 7l, and 7m are characterized by Ki
values which represent a 3-5.5-fold gain in affinity with
respect to that of the previously described most active
ATBI derivative 1 (Ki 18 nM).
particular, the starting compound 8 is differently sub-
stituted in the 4 and 6 positions, so it was possible, in
theory, to obtain a pair of isomeric products from the
cyclocondensation reaction of this amine with the R-
haloketo derivatives 9, 10, 11, and 12. The correct
structural assignment for products 7a , 13, 14, and 15,
was obtained by a HMBC experiment. The carbon atom
in the triazine ring that showed a correlation pathway
with the phenyl hydrogen atoms (δ 162.9) was not
correlated to the hydrogen atom into the imidazole ring
(H-6, δ 7.35). This hydrogen, however, showed a cor-
relation with the two carbon atoms (δ 152.4 and 152.3)
in the triazine ring.
The amino group of compounds 7a , 13, 14, and 15 was
subsequently allowed to react with haloalkyl or acyl
derivatives according to the condition described in the
Experimental Section, and summarized in Scheme 2,
to give the derivatives 7b-i,k -r . The compound 7j was
obtained together 7a by reaction of triazine 8 with
2-chloroacetone. The presence of carbonyl function in
compounds 7j, 7k , 7o, 7p , and 7q has been unequivo-
cally ascertained on the basis of the IR spectra.
As stated in the Introduction, our research started
with the 7-methyl derivatives 7a -k seeking the optimal
R′ substituent. In the 7-methyl series 7a -k , the parent
compound 7a showed a fair affinity at the A1AR with a
low selectivity toward A2A (2-fold) and A3 (6-fold) ARs.
The affinity, as expected, is improved by a hydrophobic
character of R′ (hypothesized to fill the L1 lipophilic
pocket as schematized in Figure 2). Indeed, introduction
of an alkyl (cyclopentyl) substituent at N-4 (7b) deter-
mined an 8-fold enhancement in A1AR potency and a
parallel gain in selectivity vs A2A and A3ARs (100-fold
and 55-fold, respectively). Bulkier alkyl and arylalkyl
substituents at this position (7c-e) dramatically de-
creased A1 and A2AARs affinity, while varying results
were obtained at the A3AR with respect to 7a : an
Resu lts a n d Discu ssion
The binding affinity of each ITA derivative at the
A1 and A2AARs were determined in competition assays

Novel A1 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5033
Ta ble 1. Binding Affinity Data of ITA Derivatives 7
Ki (nM)a
b
c
d
no.
R
R′
bA1
bA2A
bA3
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
iC3H7
Ph
H
332 ( 130
41.0 ( 9.0
511 ( 9.0
231 ( 99
3900 ( 1100
855 ( 60
4.4 ( 1.2
116 ( 75
27.2 ( 5.2
12.0 ( 3.0
>10000
3.3 ( 1.1
5.8 ( 3.1
17.0 ( 13.0
>10000
2360 ( 800
>10000
682 ( 129
4100 ( 400
>10000
2000 ( 700
2250 ( 300
526 ( 13
2100 ( 400
>10000
cyclopentyl
1-adamantyl
CH2-cyclohexyl
CH2Ph
>10000
>10000
>10000
COCH3
231 ( 60
410 ( 60
202 ( 22
11.8 ( 1.1
>10000
CO-cyclopentyl
CO-cyclohexyl
COPh
4300 ( 300
>10000
>10000
CH2COCH3
CH2COC2H5
CO-cyclopentyl
CO-cyclopentyl
CO-cyclopentyl
CH2COCH3
CH2COCH3
CH2COCH3
COCH3
>10000
>10000
>10000
2600 ( 200
>10000
20.2 ( 7.0
17.4 ( 10
1240 ( 180
>10000
2500 ( 200
>10000
C2H5
iC3H7
Ph
>10000
>10000
3050 ( 800
5800 ( 900
40.0 ( 3.3
>10000
73 ( 5
0.22 ( 0.02
Ph
23.0 ( 2.0
0.5 ( 0.03
14 ( 4
3200 ( 220
337 ( 28
16 ( 3
DPCPX
NECA
Cl-IBMECA
890 ( 61
401 ( 25
a
The Ki values are means ( SEM derived by an iterative curve-fitting procedure (program Prism, GraphPad, San Diego, CA).
Displacement of specific [3H]CHA binding in bovine cortical membranes. c Displacement of specific [3H] CGS21680 binding in bovine
b
striatal membranes. Displacement of specific [125I]AB-MECA binding in bovine cortical membranes in the presence of 20 nM DPCPX or
d
percentage of inhibition of specific binding at 1 µM concentration.
enhancement in affinity for 7c, equipotency for 7d , and
a complete loss of affinity in the case of 7e.
latter ring is exceedingly large. Unfavorable steric
effects of R′ can be likewise observed on comparing 7b
vs 7c, 7d vs 7e and 7j vs 7k . Regarding 7d and 7e, the
steric differences between the CH2-cyclohexyl and CH2-
phenyl groups seem to us essentially related to the
different valence geometry about the endocyclic C1 atom
(sp3 or sp2 hybridized, respectively).
The two most potent 7-CH3 derivatives, 7g and 7j,
were then selected as leads for structural modifications
aimed at optimizing the nature of the substituent R
hypothesized to dock into the L3 lipophilic site (Figure
2). Accordingly, the 7-CH3 in 7g and 7j was replaced
with a 7-C2H5, a 7-isoC3H7 or a 7-Ph by synthesizing
compounds 7l-n and, respectively, 7o-q.
In the CO-cyclopentyl series (7l-n ) potency remained
almost similar (7l Ki ) 3.3 nM) to that of the parent
compound 7g (Ki ) 4.4 nM), while selectivity, especially
toward A3AR, was lost. The CH2COCH3 series (7o-q)
gave even worst results, both in affinity and selectivity.
The above results suggest that the A1AR L3 cavity is
filled with optimal shape complementarity by the C2H5
or CH3 of 7l and 7j, respectively, so that bulkier
substituents R cannot fit properly in this cavity. It is
interesting to note that in the 7g, 7l-n and 7j, 7o-q
series the R groups larger than the optimal ones reduce
affinity to quite different extents (up to 4-fold or more
than 200-fold, respectively). The moderate or dramatic
impact of nonoptimal R substituents on affinity strictly
depends on the nature of R′. In other words, the effects
of R and R′ are not additive but interdependent.
In light of the docking model of 7j (Figure 3), we
speculate that the strong H-bonds accepted by the
Also the introduction of an acetyl moiety at the N-4
(7f) reduced A1 and A2AAR affinity, while a 8.5-fold gain
in potency was observed for the A3AR. Notwithstanding,
7g (R′ ) CO-cyclopentyl), 7h (R′ ) CO-cyclohexyl), and
7i (R′ ) COPh) showed an enhancement in A1AR
affinity vs 7a by 75.5-fold, 3-fold and 12-fold, respec-
tively, with a good selectivity toward A2AAR but not
A3AR. Moreover, a comparison between 7h (R′ ) CO-
cyclohexyl) and the 2-fold less potent 7d (R′ ) CH2-
cyclohexyl) suggests that the amidic CO in R′ is indeed
beneficial, provided that a sufficiently hydrophobic
moiety occupies the L1 pocket. The favorable effect of
the CO linker with respect to CH2 might be ascribed to
either an improved H-bond donor ability of the NH
group and/or to a more precise orientation of R′ into L1.
However, prompted by docking simulations, we also
prepared 7j featuring R′ ) CH2COCH3. This substituent
is rather hydrophilic but nevertheless makes two H-
bonds with the Ser277 and Thr91 side chains (see
Figure 3). The significant gain in A1AR potency (71-fold)
and selectivity achieved by inserting a methylene spacer
between the acetyl group and the N-4 (compare 7j vs
7f) confirmed our prevision. Finally, we observed a
complete loss of affinity at all the three ARs when the
acetyl moiety of 7j was substituted with a propionyl one
in 7k .
In the 7-CH3 series of ITAs, 7g (R′ ) CO-cyclopentyl,
Ki ) 4 nM) achieves the highest potency. Replacement
of the cyclopentyl of 7g with a cyclohexyl to yield 7h
produces a 25-fold drop of affinity probably because the

5034 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Novellino et al.
solution. This alkaline mixture was extracted with ethyl
acetate (3 × 20 mL). The combined extracts were dried
(Na2SO4), reduced to a small volume, and purified by flash
chromatography.
carbonyl oxygen of CH2COCH3 from Ser277 and Thr91
hydroxyls somehow “freeze” the mobility of the com-
plexed ligand. This implies that the steric hindrance
arising from insertion of a relatively large substituent
R cannot be counterbalanced through rotations and
translations of the ligand to adapt R into the L3 pocket.
Alternatively, accommodation of R into the L3 cavity
would disrupt the putative H-bonds described above. If
R′ is not engaged in H-bonds with L1 (i.e., CO-cyclo-
pentyl), the ligand can more freely move within the
binding site, thus minimizing the steric repulsion
between R and the L3 pocket.
To test our “dynamic” model of ITA/A1AR interaction,
we synthesized and assayed 7r which combines a
substituent R′ smaller than CO-cyclopentyl (COCH3)
with a large R (Ph). In this case the replacement of the
7-CH3 with a 7-Ph leads to a 37-fold improvement of
affinity (compare 7r vs 7f) reaching a Ki value of 23 nM.
The remarkable beneficial effect of 7-Ph in 7r might be
related to an “easier” positioning of the small COCH3
into the L1 cavity, which in turn facilitates the anchor-
ing of 7-Ph into the L3 site. Unfortunately, this en-
hancement in the affinity for A1AR was paralleled by
A3AR potency (Ki ) 40 nM), with a consequent decrease
in A1/A3 selectivity.
7-Me t h yl-2-p h e n ylim id a zo[1,2-a ][1,3,5]t r ia zin -4-yl-
am in e (7a) an d 1-[(7-Meth yl-2-ph en ylim idazo[1,2-a ][1,3,5]-
tr ia zin -4-yl)a m in o]a ceton e (7j). Eluant: n-hexane/ethyl
acetate (3:2 v/v). The faster running band gave pure 7j (36%,
mp 290 °C dec), the slower one gave pure 7a (43%, mp 293 °C
dec). Spectroscopic data for 7a : 1H NMR (500 MHz, CD3OD)
δ 8.40 (d, J ) 6.9 Hz, 2H, H-Ar), 7.51-7.41 (m, 3H, H-Ar),
7.35 (s, 1H, H-6), 2.34 (s, 3H, 7-Me); 13C NMR (125 MHz, CD3-
OD) δ 162.9 (C-2), 152.4, 152.3 (C-4 and C-8a), 143.9 (C-7),
138.1 (C-1′), 131.9, 129.4, 129.2, 104.1 (C-6), 14.1 (Me). Anal.
(C12H11N5) C, H, N. Spectroscopic data for 7j: 1H NMR (500
MHz, CDCl3) δ 8.46 (d, J ) 7.7 Hz, 2H, H-Ar), 7.51-7.43 (3H,
H-Ar), 7.32 (s, 1H, H-6), 4.99 (s, 2H, NCH2), 2.39 (s, 3H, 7-Me),
2.24 (s, 3H, COMe). IR (Nujol) 1685 cm-1. Anal. (C15H15N5O)
C, H, N.
7-E t h y l-2-p h e n y lim id a zo [1,2-a ][1,3,5]t r ia zin -4-y l-
a m in e (13). Eluant: n-hexane/ethyl acetate (7:3 v/v). 46%, mp
280 °C dec; 1H NMR (250 MHz, CD3OD) δ 8.50 (d, J ) 8.4 Hz,
2H, H-Ar), 7.62-7.30 (m, 4H, H-Ar and H-6), 2.72 (q, J )
7.7 Hz, 2H, CH2Me), 1.32 (t, J ) 7.7 Hz, 3H, CH2Me); 13C NMR
(125 MHz, CD3OD) δ 163.1 (C-2), 152.8, 152.6 (C-4 and C-8a),
150.3 (C-7), 138.3 (C-1′), 132.1, 129.5, 129.3, 103.2 (C-6), 23.1
(CH2), 13.4 (Me). Anal. (C13H13N5) C, H, N.
7-Isop r op yl-2-p h e n ylim id a zo[1,2-a ][1,3,5]t r ia zin -4-
yla m in e (14). Eluant: n-hexane/ethyl acetate (7:3 v/v). 48%,
mp 273 °C dec; 1H NMR (500 MHz, CD3OD) δ 8.49 (d, J ) 7.0
Hz, 2H, H-Ar), 7.52-7.49 (m, 3H, H-Ar), 7.66 (s, 1H, H-6),
3.05 (septuplet, J ) 7.0 Hz, 1H, CH), 1.38 (d, J ) 7.0 Hz, 6H,
2 × Me); 13C NMR (125 MHz, CD3OD) δ 163.1 (C-2), 152.9,
152.6, 152.3 (C-4 and C-8a and C-7), 138.1 (C-1′), 132.1, 129.5,
129.3, 104.5 (C-6), 75.0 (CH), 27.2 (2 × Me). Anal. (C14H15N5)
C, H, N.
2,7-Diph en ylim idazo[1,2-a ][1,3,5]tr iazin -4-ylam in e (15).
Eluant: n-hexane/ethyl acetate (4:1 v/v). 38%, mp 356 °C dec;
1H NMR (500 MHz, CD3OD) δ 8.51 (d, J ) 7.3 Hz, 2H, H-Ar),
8.04 (s, 1H, H-6), 7.94 (d, J ) 8.1 Hz, 2H, H-Ar), 7.55-7.45
(m, 5H, H-Ar), 7.39 (t, J ) 7.3 Hz, 1H, H-Ar); 13C NMR (125
MHz, CD3OD) δ 162.4 (C-2), 152.3, 152.0 (C-4 and C-8a), 145.7
(C-7), 138.1 (C-1′), 134.2 (C-1′′), 132.3, 129.1, 128.3, 128.0,
127.2, 126.1, 103.0 (C-6). Anal. (C17H13N5) C, H, N.
Con clu sion s
Prompted by pharmacophore- and docking-based mod-
els, we have synthesized and tested a series of N-alkyl
and N-acyl-(7-substituted-2-phenylimidazo[1,2-a][1,3,5]-
triazin-4-yl)amines (ITAs, 7) designed as a new class of
A1AR antagonists. Nonadditive, but interdependent,
effects of R and R′ substituents on A1AR affinity could
be rationalized by our docking model. The most inter-
esting compound within this series is 7j (R′ ) CH2COCH3
and R ) CH3) which exhibits the best combination of
affinity at he A1AR (Ki ) 12 nM) and selectivity over
the A2AAR and A3AR subtypes (Kis > 10000 nM).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
7b-d . K2CO3 (10 mmol) and a catalytic amount of KI were
added to a solution of amine 7a (20 mmol) and the opportune
alkyl bromide (30 mmol) in acetonitrile (50 mL). The solution
was stirred under reflux for 10-20 h with small amounts of
molecular sieves. After being cooled, the solvent was removed
under reduced pressure and the residue was treated with
NaHCO3 saturated aqueous solution. This alkaline mixture
was extracted with CHCl3 (3 × 20 mL). The combined extracts
were dried (Na2SO4), reduced to small volume and purified by
flash chromatography.
Exp er im en ta l Section
Ch em istr y. The course of reactions and purity of products
were controlled by TLC, using precoated silica gel plates
(Merck 60 F254) and chloroform/methanol 95:5 as eluant.
Preparative separation was performed in columns containing
Merck 60 silica gel (70-230 mesh ASTM) and with a Flash
40i chromatography module using prepacked cartridge system
(Biotage). Melting points were determined with a Kofler hot
stage microscope and are uncorrected.
N-Cyclop en t yl-N-(7-m et h yl-2-p h en ylim id a zo[1,2-a ]-
[1,3,5]tr ia zin -4-yl)a m in e (7b). Eluant: n-hexane/ethyl ace-
tate (3:2 v/v). 41%, mp 298 °C dec; 1H NMR (500 MHz, DMSO-
d6) δ 8.40 (m, 1H, H-Ar), 8.30 (m, 2H, H-Ar), 7.72 (s, 1H,
H-6), 7.48 (m, 2H, H-Ar), 4.68 (sextet, J ) 7.8 Hz, 1H, NCH),
2.28 (s, 3H, 7-Me), 2.15-2.02 (m, 3H, cyclopentyl), 1.82-1.62
(m, 5H, cyclopentyl). Anal. (C17H19N5) C, H, N.
N-(1-Ad a m a n tyl)-N-(7-m eth yl-2-p h en ylim id a zo[1,2-a ]-
[1,3,5]tr ia zin -4-yl)a m in e (7c). Eluant: n-hexane/ethyl ace-
tate (3:2 v/v). 46%, mp 299 °C dec; 1H NMR (500 MHz, CDCl3)
δ 8.45 (m, 1H, H-Ar), 8.35 (m, 1H, H-Ar), 7.48-7.46 (m, 3H,
H-Ar), 7.27 (s, 1H, H-6), 2.58 (m, 2H, adamantyl), 2.50 (m,
1H, adamantyl), 2.47 (m, 1H, adamantyl), 2.45 (m, 1H,
adamantyl), 2.26 (s, 3H, Me), 2.19-2.17 (m, 4H, adamantyl),
1.76 (m, 6H, adamantyl). Anal. (C22H25N5) C, H, N.
Elemental analysis was within (0.4% of the theoretical
1
values. H, 13C NMR, and HMBC experiments were recorded
using a Bruker WM-250 or AMX-500 spectrometer, equipped
with a Bruker X-32 computer. Chemical shift values are
reported in δ units (ppm) relative to TMS used as the inter-
nal standard. Infrared spectra were recorded with a PYE/
UNICAM Infracord model PU 9516 spectrophotometer in Nujol
mulls. Molecular sieves UOP Type 4A (Fluka 69834) and UOP
Type 13X (Fluka 69853), reagent grade chemicals, and solvents
were used. Compound 1115 was prepared according to pub-
lished procedure.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
7a , 7j, 13-15. A solution of 2,4-diamino-6-phenyl[1,3,5]triazine
8 (20 mmol) and the appropriate R-haloketo derivative 9, 10,
11, 12 (30 mmol) in absolute ethanol (100 mL) was stirred
under reflux for 30-40 h with small amounts of molecular
sieves UOP Type 4A and, when suitable, UOP 13X. After being
cooled, the solvent was removed under reduced pressure and
the residue was treated with NaHCO3 saturated aqueous
N-(Cycloh exylm et h yl)-N-(7-m et h yl-2-p h en ylim id a zo-
[1,2-a ][1,3,5]tr ia zin -4-yl)a m in e (7d ). Eluant: n-hexane/
ethyl acetate (3:2 v/v). 45%, mp 220 °C dec; 1H NMR (500 MHz,
CD3OD) δ 8.45 (d, J ) 6.5 Hz, 2H, H-Ar), 7.50-7.47 (m, 3H,

Novel A1 Adenosine Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5035
H-Ar), 7.43 (s, 1H, H-6), 3.60 (d, J ) 6.6 Hz, 2H, NCH2), 2.38
(s, 3H, 7-Me), 1.90-1.77 (m, 4H, cyclohexyl), 1.75-1.63 (m,
1H, cyclohexyl), 1.34-1.25 (m, 4H, cyclohexyl), 1.13-1.05 (m,
2H, cyclohexyl). Anal. (C19H23N5) C, H, N.
1-[(7-Meth yl-2-p h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-yl)-
a m in o]bu ta n on e (7k ). Eluant: n-hexane/ethyl acetate (7:3
v/v). 34%, mp 182 °C dec; H NMR (500 MHz, CDCl3) δ 8.50
1
(d, J ) 8.4 Hz, 2H, H-Ar), 7.56-7.45 (m, 3H, H-Ar), 7.35 (s,
1H, H-6), 5.06 (s, 2H, NCH2), 2.68 (q, J ) 7.9 Hz, 2H, CH2Me),
2.28 (s, 3H, 7-Me), 1.20 (t, J ) 7.9 Hz, 3H, CH2Me). IR (Nujol)
1690 cm-1. Anal. (C16H17N5O) C, H, N.
N-(7-E t h yl-2-p h en ylim id a zo[1,2-a ][1,3,5]t r ia zin -4-yl)-
cyclop en ta n eca r boxa m id e (7l). Eluant: n-hexane/ethyl
acetate (7:3 v/v). 50%, mp 239 °C dec; 1H NMR (500 MHz,
CDCl3) δ 8.18 (m, 2H, H-Ar), 7.57-7.50 (m, 4H, H-Ar and
H-6), 3.08-2.92 (m, 1H, COCH), 2.82 (q, J ) 7.8 Hz, 2H,
CH2Me), 2.12-1.79 (m, 4H, cyclopentyl), 1.70-1.59 (m, 4H,
cyclopentyl), 1.37 (t, J ) 7.8 Hz, 3H, CH2Me). Anal. (C19H21N5O)
C, H, N.
N-(7-Isop r op yl-2-p h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-
yl)cyclop en ta n eca r boxa m id e (7m ). Eluant: n-hexane/ethyl
acetate (7:3 v/v). 53%, mp 234 °C dec; 1H NMR (500 MHz,
CDCl3) δ 8.24 (d, J ) 7.0 Hz, 2H, H-Ar), 7.75-7.49 (m, 4H,
H-Ar and H-6), 3.12 (quintet, J ) 8.1 Hz, 1H, COCH), 2.18
(d, J ) 7.1 Hz, 6H, 2xMe), 2.15-1.93 (m, 5H, cyclopentyl and
CH), 1.86-1.67 (m, 4H, cyclopentyl). Anal. (C20H23N5O) C, H,
N.
N-(Ben zyl)-N-(7-m eth yl-2-p h en ylim id a zo[1,2-a ][1,3,5]-
tr ia zin -4-yl)a m in e (7e). K2CO3 (10 mmol) and a catalytic
amount of KI were added to a solution of 7a (20 mmol) and
benzyl bromide (30 mmol) in acetonitrile (50 mL). The solution
was stirred under reflux for 10 h. After being cooled, the
solvent was removed under reduced pressure and the residue
was treated with concentrated NH4OH and warmed to reflux
for 10 h. This alkaline mixture was then extracted with CHCl3
(3 × 20 mL). The combined extracts were dried (Na2SO4),
reduced to a small volume and purified by flash chromatog-
raphy (eluant: n-hexane/ethyl acetate 3:2 v/v). 48%, mp 186
°C dec; 1H NMR (500 MHz, CDCl3) δ 8.57 (d, J ) 7.6 Hz, 2H,
H-Ar), 7.52-7.43 (m, 3H, H-Ar), 7.40-7.32 (m, 2H, H-Ar),
7.31-7.27 (m, 4H, H-Ar and H-6), 5.39 (s, 2H, NCH2), 2.27
(s, 3H, 7-Me). Anal. (C19H17N5) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of N-(7-Meth yl-
2-p h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-yl)a ceta m id e (7f)
an d N-(2,7-Diph en ylim idazo[1,2-a ][1,3,5]tr iazin -4-yl)acet-
a m id e (7r ). Acetic anhydride (15 mmol) was added to a
solution of 7a or 15 (10 mmol) and 4-(dimethylamino)pyridine
(5 mmol) in anhydrous THF (20 mL). The solution was kept
at 0 °C for 1 h and was then added with NaHCO3 saturated
aqueous solution (40 mL) and extracted with CHCl3 (3 × 20
mL). The combined extracts were dried (Na2SO4), the solvent
was evaporated and the residue was treated with diethyl ether
to obtain a precipitate collected by filtration and identified as
pure 7f or 7r .
N-(7-Meth yl-2-p h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-yl)-
a ceta m id e (7f). 70%, mp 261 °C dec; 1H NMR (500 MHz,
CDCl3) δ 8.30 (m, 2H, H-Ar), 7.56-7.51 (m, 3H, H-Ar), 7.50
(s, 1H, H-6), 2.51 (s, 3H, 7-Me), 2.22 (s, 3H, COMe). Anal.
(C14H13N5O) C, H, N.
N-(2,7-Dip h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-yl)a cet-
a m id e (7r ). 20%, mp 353 °C dec; 1H NMR (500 MHz, DMSO-
d6) δ 8.54 (s, 1H, H-6), 8.42 (m, 2H, H-Ar), 7.97 (d, J ) 7.4
Hz, 2H, H-Ar), 7.60-7.55 (m, 3H, H-Ar), 7.52 (t, J ) 7.4 Hz,
2H, H-Ar), 7.40 (t, J ) 7.4 Hz, 1H, H-Ar), 2.58 (s, 3H, COMe).
Anal. (C19H15N5O) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
7g-i,k -q. K2CO3 (10 mmol) and a catalytic amount of KI were
added to a solution of amine 7a (or 13, 14, 15) (20 mmol) and
the opportune alkyl or acyl chloride (30 mmol) in acetonitrile
(50 mL). The solution was stirred under reflux for 10-20 h
with small amounts of molecular sieves. After being cooled,
the solvent was removed under reduced pressure and the
residue was treated with NaHCO3 saturated aqueous solution.
This alkaline mixture was extracted with CHCl3 (3 × 20 mL).
The combined extracts were dried (Na2SO4), reduced to small
volume, and purified by flash chromatography.
N-(7-Meth yl-2-p h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-yl)-
cyclop en ta n eca r boxa m id e (7g). Eluant: n-hexane/ethyl
acetate (7:3 v/v). 62%, mp 245 °C dec; 1H NMR (500 MHz,
CDCl3) δ 8.16 (d, J ) 5.7 Hz, 2H, H-Ar), 7.61-7.50 (m, 4H,
H-Ar and H-6), 3.21 (m, 1H, COCH), 2.45 (s, 3H, 7-Me), 2.08-
1.89 (m, 4H, cyclopentyl), 1.80-1.78 (m, 2H, cyclopentyl),
1.68-1.63 (m, 2H, cyclopentyl). Anal. (C18H19 N5O) C, H, N.
N-(7-Meth yl-2-p h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-yl)-
cycloh exa n eca r boxa m id e (7h ). Eluant: n-hexane/ethyl
acetate (7:3 v/v). 54%, mp 254 °C dec; 1H NMR (500 MHz,
CDCl3) δ 8.22 (m, 2H, H-Ar), 7.58-7.53 (m, 3H, H-Ar), 7.45
(s, 1H, H-6), 2.45 (s, 3H, Me), 2.05 (d, J ) 12.5 Hz, 2H,
cyclohexyl), 1.84 (d, J ) 12.8 Hz, 2H, cyclohexyl), 1.73 (d, J )
13.2 Hz, 1H, cyclohexyl), 1.40-1.37 (m, 4H, cyclohexyl), 1.29-
1.24 (m, 2H, cyclohexyl). Anal. (C19H21N5O) C, H, N.
N-(2,7-Dip h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-yl)cyclo-
p en ta n eca r boxa m id e (7n ). Eluant: n-hexane/ethyl acetate
1
(7:3 v/v). 45%, mp 220 °C dec; H NMR (500 MHz, CDCl3) δ
8.23 (d, J ) 7.3 Hz, 2H, H-Ar), 8.07-8.04 (m, 3H, H-Ar),
7.68-7.36 (m, 6H, H-Ar and H-6), 3.10 (quintet, J ) 7.7 Hz,
1H, COCH), 2.10-1.90 (m, 4H, cyclopentyl), 1.83-1.61 (m, 4H,
cyclopentyl). Anal.(C23H21N5O) C, H, N.
1-[(7-Eth yl-2-p h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-yl)-
a m in o]a ceton e (7o). Eluant: n-hexane/ethyl acetate (7:3 v/v).
1
25%, mp 276 °C dec; H NMR (500 MHz, CDCl3) δ 8.41 (d, J
) 7.6 Hz, 2H, H-Ar), 7.48-7.39 (m, 3H, H-Ar), 7.22 (s, 1H,
H-6), 4.93 (s, 2H, NCH2), 2.46 (q, J ) 7.5 Hz, 2H, CH2Me),
2.34 (s, 3H, COMe), 1.30 (t, J ) 7.5 Hz, 3H, CH2Me). IR (Nujol)
1685 cm-1. Anal. (C16H17N5O) C, H, N.
1-[(7-Isop r op yl-2-p h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-
yl)a m in o]a ceton e (7p ). Eluant: n-hexane/ethyl acetate (7:3
1
v/v). 24%, mp 269 °C dec; H NMR (500 MHz, CDCl3) δ 8.46
(d, J ) 7.7 Hz, 2H, H-Ar), 7.46 (m, 1H, H-Ar), 7.45 (m, 2H,
H-Ar), 7.35 (s, 1H, H-6), 5.00 (s, 2H, NCH2), 2.75 (septuplet,
J ) 6.9 Hz, 1H, CH), 2.39 (s, 3H, COMe), 1.33 (d, J ) 6.9 Hz,
6H, 2xMe). IR (Nujol) 1686 cm-1. Anal. (C17H19N5O) C, H, N.
1-[(2,7-Dip h en ylim id a zo[1,2-a ][1,3,5]t r ia zin -4-yl)a m i-
n o]a ceton e (7q). Eluant: n-hexane/ethyl acetate (7:3 v/v).
1
20%, mp 353 °C dec; H NMR (500 MHz, CDCl3) δ 8.49 (d, J
) 7.4 Hz, 2H, H-Ar), 7.57 (s, 1H, H-6), 7.55-7.52 (m, 4H,
H-Ar), 7.50-7.48 (m, 2H, H-Ar), 7.43-7.37 (m, 2H, H-Ar),
4.97 (s, 2H, NCH2), 2.29 (s, 3H, COMe). IR (Nujol) 1687 cm-1
Anal. (C20H17N5O) C, H, N.
.
Recep tor Bin d in g Assa ys. A1, A2A, a n d A3 Recep tor
Bin d in g. Displacement of [3H]CHA (31Ci/mmol) from A1AR
in bovine cortical membranes, [3H]CGS21680 (42.1Ci/mmol)
from A2AAR in bovine striatal membranes and [125I]AB-MECA
(2000Ci/mmol) from A3AR in bovine cortical membranes were
performed as described elsewhere.8
Compounds were dissolved in DMSO and added to the assay
mixture (DMSO concentration maximum 2%). Blank experi-
ments were carried out to determine the effect of solvent on
binding. Protein estimation was based on a reported method,16
after solubilization with 0.75N sodium hydroxide, using bovine
serum albumin as standard. At least six different concentra-
tions of each compound were used. The experiments (n ) 4),
carried out in triplicate, were analyzed by an iterative curve-
fitting procedure (GraphPad, Prism program, San Diego, CA),
which provided IC50, Ki, and SEM values for tested com-
pounds.
The dissociation constant (Kd) of [3H]CHA, [3H]CGS21680
and [125I]AB-MECA was 1.2, 14, and 1.02 nM, respectively.
Com p u ta tion a l Ch em istr y. All molecular modeling was
performed using the software package SYBYL17 running on a
Silicon Graphics R10000 workstation. Molecular models of
imidazotriazine derivatives were built according to SYBYL
N-(7-Meth yl-2-p h en ylim id a zo[1,2-a ][1,3,5]tr ia zin -4-yl)-
b en zen ca r b oxa m id e (7i). Eluant: n-hexane/ethyl acetate
(7:3 v/v). 42%, mp 284 °C dec; H NMR (500 MHz, CDCl3) δ
8.57 (d, J ) 8.1 Hz, 2H, H-Ar), 8.21 (d, J ) 7.3 Hz, 2H, H-Ar),
7.66-7.57 (m, 5H, H-Ar and H-6), 7.50 (t, J ) 8.1 Hz, 2H,
H-Ar), 2.50 (s, 3H, 7-Me). Anal. (C19H15N5O) C, H, N.
1

5036 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Novellino et al.
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Novel Class of Potent Adenosine Receptor Antagonists and
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(17) Sybyl Molecular Modelling System (version 6.2), Tripos Inc., St.
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standard bond lengths and valence angles. Geometry optimi-
zations were realized with the SYBYL/MAXIMIN2 minimizer
by applying the BFGS algorithm18 and setting a root-mean-
square gradient of the forces acting on each atom at 0.05 kcal/
mol Å as a convergence criterion. Molecular graphics, root-
mean-squares (rms) fit, and calculations of ring centroids of
substructures were also carried out using SYBYL.
Docking simulations were carried out starting from the
previously published bovine A1AR model,8 which was built
using as template the frog rhodopsin,19,20 a membrane protein
belonging to the G-protein coupled receptors (GPCRs) super-
family. Additional details regarding the receptor structure,
site-directed mutagenesis data and methods applied in devel-
oping the A1AR model are reported in ref 8. The model has
also been compared with the rhodopsin template recently
published by Palczewski and co-workers.21 Although slight
differences in the orientation of the helices in the transmem-
brane domain are apparent; the juxtaposition of the residues
within the putative adenosine binding pocket is well-conserved
between the two models.
The position of the docked ligand 5 (Chart 1) in the receptor
model8 was taken as a starting point. Selected ITAs (7g and
7j) were docked into the receptor model by means of the
following steps: (i) superposition on the docked compound 5
about the NH atoms plus the centroid of the pendant phenyl
ring; (ii) removal of 5 from the receptor; (iii) energy-minimiza-
tion of the resulting ligand/binding site complexes based on
the molecular mechanics Tripos force field.22
Molecular dynamics simulations of the 7j/A1AR complex
were performed with the SYBYL/DYNAMICS module under
default settings. These simulations, in which the protein
backbone was treated as a rigid aggregate, were run at 300 K
for 50 ps after reaching equilibration. Five structures extracted
at 10, 20, 30, 40, and 50 ps were selected and energy-
minimized. Figure 3, displaying the interaction between 7j and
the side chains of residues within a distance of 5 Å from any
atom of the bound ligand, refers to the last MD snapshot.
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J M020911E