Fluorosulfonyl- and bis-(β-chloroethyl)amino-phenylamino functionalized pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives: Irreversible antagonists at the human a3 adenosine receptor and molecular modeling studies

Article abstract of DOI:10.1021/jm010818a

A series of pyrazolotriazolopyrimidines was previously reported to be highly potent and selective human A₃ adenosine receptor antagonists (Baraldi et al. J. Med. Chem. 2000, 43, 4768-4780). A derivative having a methyl group at the N⁸ pyrazole combined with a 4-methoxyphenylcarbamoyl moiety at N⁵ position, displayed a K_i value at the hA₃ receptor of 0.2 nM. We now describe chemically reactive derivatives which act as irreversible inhibitors of this receptor. Electrophilic groups, specifically sulfonyl fluoride and nitrogen mustard (bis-(β-chloroethyl)-amino) moieties, have been incorporated at the 4-position of the aryl urea group. Membranes containing the recombinant hA₃ receptor were preincubated with the compounds and washed exhaustively. The loss of ability to bind radioligand following this treatment indicated irreversible binding. The most potent compound in irreversibly binding to the receptor was 14, which contained a sulfonyl fluoride moiety and a propyl group at the N⁸ pyrazole nitrogen. The bis-(β-chloroethyl)amino derivatives displayed a much smaller degree of irreversible binding than the sulfonyl fluoride derivatives. A computer-generated model of the human A₃ receptor was built and analyzed to help interpret these results. The model of the A₃ transmembrane region was derived using primary sequence comparison, secondary structure predictions, and three-dimensional homology building, using the recently published crystal structure of rhodopsin as a template. According to our model, sulfonyl fluoride derivatives could dock within the hypothetical TM binding domain, adopting two different energetically favorable conformations. We have identified two amino acids, Ser247 and Cys251, both in TM6, as potential nucleophilic partners of the irreversible binding to the receptor.

Full text of DOI:10.1021/jm010818a

J . Med. Chem. 2001, 44, 2735-2742
2735
F lu or osu lfon yl- a n d Bis-(â-ch lor oeth yl)a m in o-p h en yla m in o F u n ction a lized
P yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e Der iva tives: Ir r ever sible
An ta gon ists a t th e Hu m a n A3 Ad en osin e Recep tor a n d Molecu la r Mod elin g
Stu d ies
Pier Giovanni Baraldi,*,† Barbara Cacciari,† Stefano Moro,‡ Romeo Romagnoli,† Xiao-duo J i,§
Kenneth A. J acobson,§ Stefania Gessi,| Pier Andrea Borea,| and Giampiero Spalluto
Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Ferrara, Via Fossato di Mortara 17-19,
I-44100 Ferrara, Italy, Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Trieste, Piazzale Europa 1,
I-34127 Trieste, Italy, Molecular Modeling Section, Dipartimento di Scienze Farmaceutiche, Universita` di Padova,
Via Marzolo 5, I-35131 Padova, Italy, Molecular Recognition Section, Laboratory of Bioorganic Chemistry,
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892,
and Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, Universita` degli Studi di Ferrara,
Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy
Received J anuary 11, 2001
A series of pyrazolotriazolopyrimidines was previously reported to be highly potent and selective
human A3 adenosine receptor antagonists (Baraldi et al. J . Med. Chem. 2000, 43, 4768-4780).
A derivative having a methyl group at the N8 pyrazole combined with a 4-methoxyphenylcar-
bamoyl moiety at N5 position, displayed a Ki value at the hA3 receptor of 0.2 nM. We now
describe chemically reactive derivatives which act as irreversible inhibitors of this receptor.
Electrophilic groups, specifically sulfonyl fluoride and nitrogen mustard (bis-(â-chloroethyl)-
amino) moieties, have been incorporated at the 4-position of the aryl urea group. Membranes
containing the recombinant hA3 receptor were preincubated with the compounds and washed
exhaustively. The loss of ability to bind radioligand following this treatment indicated
irreversible binding. The most potent compound in irreversibly binding to the receptor was
14, which contained a sulfonyl fluoride moiety and a propyl group at the N8 pyrazole nitrogen.
The bis-(â-chloroethyl)amino derivatives displayed a much smaller degree of irreversible binding
than the sulfonyl fluoride derivatives. A computer-generated model of the human A3 receptor
was built and analyzed to help interpret these results. The model of the A3 transmembrane
region was derived using primary sequence comparison, secondary structure predictions, and
three-dimensional homology building, using the recently published crystal structure of rhodopsin
as a template. According to our model, sulfonyl fluoride derivatives could dock within the
hypothetical TM binding domain, adopting two different energetically favorable conformations.
We have identified two amino acids, Ser247 and Cys251, both in TM6, as potential nucleophilic
partners of the irreversible binding to the receptor.
In tr od u ction
inhibition of adenylate cyclase3 and stimulation of
phospholipases C10 and D.11 In addition, activation of
the rat A3 adenosine receptors causes the release of
histamine from mast cells,12 which leads to processes
such as inflammation and hypotension. It has been
suggested that the blockade of the A3 adenosine receptor
may be considered as a potential lead toward the
treatment of asthma and inflammation.13,14 For these
reasons, and with the aim of obtaining efficient probes
for studying this receptor subtype, there has been an
active search for potent and selective antagonists. In
the past few years, many classes of heterocyclic com-
pounds have been synthesized as A3 adenosine antago-
nists, with some showing significant selectivity for the
A3 adenosine receptor subtype.15 In particular, our
group recently reported a class of 5-[[(4-substituted-
phenyl)amino]carbonyl]amino-8-alkyl-2-(2-furyl)-pyra-
zolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as
potent and selective human A3 adenosine receptor
antagonists. These were ineffective on a rat model, due
to the well-known differences in sequence homologies
(72%) (Table 1).16,17
In the last 20 years, much effort has been made
toward the goal of identifying ligands that interact
selectively with the four adenosine receptor subtypes,
A1, A2A, A2B, and A3.1,2 In particular, while A1 and A2A
subtypes have been pharmacologically characterized
through the use of chemical probes, A2B and A3 subtypes
are still in need of additional probes.3
The A3 subtype, recently cloned from various species
(human, rat, and rabbit),4-9 seems to be involved in a
variety of physiological functions. The principal second
messengers activated by adenosine A3 receptors are
* Address correspondence to Prof. Pier Giovanni Baraldi, Diparti-
mento di Scienze Farmaceutiche, Via Fossato di Mortara 17-19, I-44100
Ferrara, Italy. Phone: +39-(0)532-291293. Fax: +39-(0)532-291296.
E-mail: pgb@dns.unife.it.
† Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di
Ferrara.
‡ Universita` di Padova.
§ National Institute of Diabetes and Digestive and Kidney Diseases,
NIH.
| Dipartimento di Medicina Clinica e Sperimentale-Sezione di Far-
macologia, Universita` degli Studi di Ferrara.
Universita` degli Studi di Trieste.
10.1021/jm010818a CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/14/2001

2736 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Baraldi et al.
Ta ble 1. Structures and Binding Affinity at hA1, hA2A, hA2B, and hA3 Adenosine Receptors of Selected Compounds Previously
Reported
hA1
hA2A
hA2B
hA3
compd
R
(Ki, nM)a
(Ki, nM)b
(Ki, nM)c
(Ki, nM)d
hA1/hA3
5485
hA2A/hA3
6950
hA2B/hA3
1305
1
CH3
1097
(928-1297)
1026
1390
(1220-1590)
1040
261
(226-301)
245
0.20
(0.17-0.24)
0.60
2
3
4
C2H5
1710
1496
925
1733
175
408
2570
946
(785-1341)
(830-1310)
(188-320)
(0.51-0.70)
n-C3H7
n-C4H9
1197
140
2056
0.80
(1027-1396)
296
(120-155)
80
(1637-2582)
303
(0.63-0.1.00)
0.32
250
(269-326)
(65-92)
(260-352)
(0.27-0.34)
a
b
Displacement of specific [3H]-DPCPX binding at human A1 receptors expressed in CHO cells (n ) 3-6). Displacement of specific
[3H]-SCH58261 binding at human A2A receptors expressed in CHO cells (n ) 3-6). c Displacement of specific [3H]-DPCPX binding at
human A2B receptors expressed in HEK-293 cells (n ) 3-6). Displacement of specific [3H]MRE-3008F20 binding at human A3 receptors
d
expressed in CHO cells (n ) 3-6). Data are expressed as geometric means, with 95% confidence limits.
A121-24 and A2A25 adenosine receptor subtypes, as clearly
reported in the literature.
Also for the A3 receptor, this approach has been used
both for agonists (adenosine derivatives)26 and antago-
nists (dihydropyridine derivatives).27 While the agonists
showed a good binding profile in a rat model,26 at the
human A3 adenosine receptors only racemic dihydro-
pyridine derivatives have been tested, and the percent-
age of irreversibility reached only 56% at 100 nM
concentration.27 Thus, there is a need for improved
irreversibly binding A3 antagonists.
In addition, the introduction of two different alkylat-
ing moieties could help to identify the nature of nucleo-
philes on the receptor which may interact with these
derivatives, and consequently this may help identify the
nature of some amino acid residues present within the
binding site. In fact, while a fluorosulfonyl group is
highly reactive28 and could interact indifferently with
any kind of nucleophile (e.g., -NH2, OH, SH), it is well-
known that nitrogen mustard, which is less reactive
than fluorosulfonyl moiety,29 reacts only with nitrogen
nucleophiles (e.g., -NH2, -NH-C(NH)dNH2).
F igu r e 1. Rational design for the synthesis of irreversible
human A3 adenosine antagonists.
It is evident from the binding data summarized in
Table 1 that affinity increased when the 4-methoxy-
phenyl carbamoyl moiety at the 5 position and small
alkyl chains (methyl (1), ethyl (2), propyl (3), butyl (4))
at the N8 pyrazole nitrogen were combined simulta-
neously. These derivatives are the most potent and
selective human A3 adenosine receptor antagonists ever
reported, with affinity at the human A3 adenosine
receptor in the subnanomolar range (0.2-0.8 nM) and
selectivities versus other receptor subtypes ranging from
200 to 7000.17
Beginning with these experimental observations, we
proposed the design of new compounds in this series by
replacing the 4-methoxy substituent on the phenyl ring
with alkylating moieties such as fluorosulfonyl and bis-
(â-chloroethyl)amino groups (Figure 1), as potential
irreversibly binding human A3 adenosine antagonists
(9-16).
Ch em istr y
Potentially irreversible human A3 receptor antago-
nists (9-16) were prepared following, as previously
reported,17 the general synthetic pathway for the prepa-
ration of urea derivatives, as summarized in Schemes
1 and 2.
Fluorosulfonyl derivatives (10, 12, 14, 16) were
prepared starting from the previously reported amino
compounds, 5-8, which reacted with the commercially
available phenyl isocyanate 17, to afford the phenyl-
carbamoyl derivatives, 18-21. After purification by
flash chromatography, the resulting compounds, 18-
21, reacted at -10 °C with fluorosulfonic acid leading
to the final derivatives, 10, 12, 14, and 16.25e The final
products were purified by crystallization from EtOAc-
light petroleum (Scheme 1).
The use of irreversible antagonists represents a useful
approach to the study of receptor structure and function,
such as ligand binding site mapping,18 physiological
receptors functioning,19 and determining the relation-
ship between receptor occupancy and a tissue or cellular
response.20 A similar approach has been successful with

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2737
Sch em e 1a
Ta ble 2. Irreversible Inhibition of Radioligand
([125I]AB-MECA) Binding to Human A3 Adenosine Receptors in
CHO Cell Membranes, Following a 1 h Incubation with
Compounds (9-16) at Concentrations of 1, 10, and 100 nM
ligand
inhibition (% of control)a
R
R1
1 nM
10 nM
100 nM
22 ( 5
NDb
18 ( 17
65 ( 4
16 (14
79 ( 8
9
CH3
N(CH2CH2Cl)2
SO2F
N(CH2CH2Cl)2
SO2F
0
11 ( 6
10 CH3
11 C2H5
12 C2H5
NDb
0
NDb
0
4.1 ( 5.1
30 ( 5
11 ( 9
70 ( 3
7.0 ( 4.9 9.0 ( 7.2
40 ( 11 73 ( 7
13 n-C3H7 N(CH2CH2Cl)2 7.8 ( 10
14 n-C3H7 SO2F
15 n-C4H9 N(CH2CH2Cl)2
16 n-C4H9 SO2F
a
Reagents: (i) PhNCO (17), THF, reflux, 18 h; (ii) FSO3H, -10
47 ( 7
0
31 ( 6
°C, 2 h.
Sch em e 2a
a
n ) 3-7. b Not determined due to the insolubility of the sample
at 1 mM in DMSO.
antagonist (9-16), followed by a thorough washing, to
remove free antagonist.
The data summarized in Table 2 showed that com-
pounds containing a fluorosulfonyl moiety (10, 12, 14,
16) proved to be irreversible antagonists with different
degrees of potency at the human A3 adenosine receptor,
while the corresponding nitrogen mustard derivatives
(9, 11, 13, 15) were unable to bind covalently at this
receptor subtype.
In the fluorosulfonyl series, derivative 10 could not
be evaluated for irreversible receptor binding due to its
low solubility in DMSO, which was less than 1 mM.
Other fluorosulfonyl derivatives were sufficiently soluble
in DMSO to prepare stock solutions for the binding
studies. (The final concentration of DMSO in buffer
must be maximum 1%.) The three fluorosulfonyl com-
pounds (12, 14, 16) showed good irreversible properties
at concentrations of 1-100 nM. In particular, high
efficiency was observed at a concentration of 100 nM,
with the percentage of inhibition being 65-79%. This
effect was quite evident also at a concentration of 10
nM, with irreversibility in the range of 30-70%. The
maximal irreversible inhibition was observed with the
derivative having a propyl chain at the N8 position (14,
100 nM, 79%).
In addition, the presence of a small alkyl chain on
the N8 position (Table 1) conferred at the compound an
optimized structural feature required for the interaction
with human A3 adenosine receptor, as previously dem-
onstrated.16,17
In contrast, derivatives with a nitrogen mustard
group on the phenyl ring (9, 11, 13, 15) showed much
less significant irreversible activity at the human A3
adenosine receptor. In fact, only at a concentration of
100 nM was slight irreversibility observed (9-22%).
With the aim to evaluate the selectivity of these
derivatives vs other adenosine receptor subtypes, the
most effective irreversible compound (14) was also
tested in competitive binding assays at human A1 and
a
Reagents: (i) ClCOOCCl3, Et3N, dioxane, reflux, 5 h; (ii) 5-8,
THF, reflux, 18 h.
The mustard derivatives (9, 11, 13, 15) were prepared
in a similar manner by reaction of the amino compounds
5-8 with freshly prepared 4-bis-(â-chloroethyl)ami-
nophenyl isocyanate, 22. The latter compound was
prepared starting from the corresponding aniline de-
rivative,30 23, by reaction with diphosgene (1.2 equiv)
in the presence of triethylamine (1.2 equiv) in dry
dioxane.31 After 5 h the mixture was concentrated at
reduced pressure, and the presence of isocyanate was
confirmed by infrared spectroscopy, which showed a
characteristic peak at 2250 cm-1. The crude residue was
utilized for coupling reactions without further purifica-
tion (Scheme 2).
Resu lts a n d Discu ssion
The reactive pyrazolo-triazolo-pyrimidine derivatives
(9-16) were tested for irreversible binding at the human
A3 adenosine receptors in CHO cells using the radioli-
gand [125I]AB-MECA (Table 2).32,33
Prior to radioligand binding, membranes containing
hA3 receptors were preincubated with the reactive

2738 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Baraldi et al.
F igu r e 2. Side view of the A3-14 (right side) and A3-13 (left side) complex models. The side chains of the two important residues
in proximity to the docked ligand molecules are highlighted and labeled.
A2A receptors expressed in CHO cells. At the human A1
receptor, no significant displacement of binding (<5%)
was observed for compound 14 at 100 nM, a concentra-
tion which effectively irreversibly inhibited most of the
hA3 receptors. However, at the human A2A receptor, a
Ki for binding of 14 was determined to be 50 ( 5 nM.
The irreversibility of binding of 14 at the human A2A
receptor was not evaluated. This result potentially
compromises use of these antagonists as selective hA3
receptor probes; however, the lower affinity at the A2A
receptor would suggest that there likely exists a con-
centration range of selectivity for irreversible inhibition
of the hA3 adenosine receptor subtype.
most stable ligand conformations of both irreversible
antagonists are shown in Figure 2.
Interestingly, the receptor-ligand complex containing
derivative 14 appeared more energetically stable by
approximately 15 kcal/mol. In derivative 13, the pres-
ence of the (bis-(â-chloroethyl)amino) moiety (derivative
13), characterized by a high degree of conformational
flexibility, increased the steric hindrance inside the
binding cavity, decreasing the relative stability of the
complex. This may be the reason for the low efficacy of
the nitrogen mustard derivatives as irreversible an-
tagonists.
As shown in Figure 2, we identified a hypothetical
binding site of both 14 and 13 surrounded by TMs 3, 5,
6, and 7 following the Monte Carlo/annealing sampling.
Both the sulfonyl fluoride and nitrogen mustard moi-
eties were close to TM6, pointing toward the extracel-
lular environment. Two residues, Ser247 and Cys251,
were tentatively implicated in irreversible binding. The
side chain of Ser247 (TM6) was 3.6 Å from the S atom
of the sulfonyl fluoride and 4.9 Å from one of the two
CR of the â-chloroethyl moiety. Moreover, the side chain
of Cys251 (TM6) was 4.2 Å from the S atom of the
sulfonyl fluoride, and 5.0 Å from one of the two CR of
the â-chloroethyl moiety. As already anticipated, Cys251
(TM6) would be a more likely candidate for the irrevers-
ible binding, based solely on its specific chemical
reactivity. However, it is useful to emphasize the
proximity of the Ser247 to the putative ligand binding
site. A major structural difference among the hypotheti-
cal binding sites of adenosine receptor subtypes is that
the A3 receptor does not contain the histidine residue
in TM6 common to all A1 (His251 in h_A1) and A2
(His250 in h_A2A) receptors. This histidine has been
shown to participate in both agonist and antagonist
binding to the A2A receptors. In the A3 receptor, this
histidine in TM6 is replaced with a serine residue, i.e.,
Ser247, a candidate for the site of irreversible binding,
due to its proximity to the electrophilic group.
This large difference in receptor interaction between
the fluorosulfonyl and mustard series can be explained
with two different hypotheses. The inactivity of the
mustard derivatives can be attributed to the absence
in the binding site of a nitrogen nucleophile (e.g., NH2),
which is the only one able to interact with the aziri-
dinium salt generated by the bis-(â-dichloroethyl)amino
function, or to the spatial orientation of this group
within the binding site, which does not permit the
nucleophiles to properly bind.
A molecular modeling study was performed to eluci-
date the hypothetical binding mode of the new A3
irreversible antagonists and to determine which amino
acid residues in the TM region might bond covalently
with the ligand. Very little structural information was
available concerning the details of ligand/GPCR inter-
actions. However, a 2.8 Å resolution crystal structure
of bovine rhodopsin was very recently published.34 Using
a homology approach and this structure as a template,
we built an improved model of the transmembrane
region of the human A3 receptor. This model may be
considered a further refinement of the hypothetical
binding site model of the A3 receptor antagonists previ-
ously proposed.35 Details of the model building are given
in the Experimental Section.
We selected derivative 14, which contained a sulfonyl
fluoride moiety and a propyl group at the N8 pyrazole
nitrogen, and the corresponding nitrogen mustard ana-
logue 13 as reference ligands in our docking studies. The
Nevertheless, it is important to note that these new
irreversible antagonists share a common question with
all other irreversibly binding compounds: is it necessary

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2739
(d, 1H, J ) 2, 5-H Furane); 8.15 (s, 1H, N8-CH); 8.67 (bs, 1H,
CO-NH-Ph); 11.16 (bs, 1H, 5-NH-CO). Anal. (C18H14N8O2) C,
H, N.
to find a nucleophile residue in proximity to the docked
conformation or can they bind provisionally elsewhere
on the receptor, on the way down to the TM site, and
remain long enough to form a covalent bond? In this
study we do not answer this question, but we can
speculate that in the proximity of the TM binding
domain there are at least two residues tentatively
implicated in the irreversible binding.
5-[[(P h en yl)a m in o]ca r bon yl]a m in o-8-eth yl-2-(2-fu r yl)-
p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (19): yield
94%, yellow solid; mp 180 °C (EtOAc-light petroleum); IR
(KBr) 3250-2985, 1675, 1610, 1510, 1460 cm-1 1H NMR
;
(DMSO-d6) δ 1.67 (t, 3H, J ) 7, CH3-CH2-N8); 4.47 (q, 2H, J )
7, CH3-CH2-N8); 6.62 (dd, 2H, J ) 2, J ) 4, 4-H Furane); 7.16-
7.42 (m, 6H, C6H5 and 3-H Furane); 7.72 (d, 1H, J ) 2, 5-H
Furane); 8.26 (s, 1H, N8-CH); 8.61 (bs, 1H, CO-NH-Ph); 11.16
(bs, 1H, 5-NH-CO). Anal. (C19H16N8O2) C, H, N.
Con clu sion s
In summary, a new series of pyrazolo-triazolo-pyri-
midines, bearing chemoreactive moieties (4-fluorsulfo-
nyl- and 4-bis-(â-chloroethyl)amino-) as irreversible
antagonists for the human A3 adenosine receptor, has
been reported. The results from an irreversible binding
assay demonstrate that one of the reported compounds,
5-[[(4-fluorosulfonyl-phenyl)amino]carbonyl]amino-8-n-
propyl-2-(2-furyl)-pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]py-
rimidine (14), irreversibly inhibited the binding of
5-[[(P h en yl)a m in o]ca r bon yl]a m in o-8-n -p r op yl-2-(2-fu -
r yl)-p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (20):
yield 98%, yellow solid; mp 155 °C (EtOAc-light petroleum);
1
IR (KBr) 3240-2985, 1674, 1615, 1510, 1470 cm-1; H NMR
(DMSO-d6) δ 1.00 (t, 3H, J ) 7, CH3-CH2-CH2-N8); 2.02-2.13
(m, 2H, CH3-CH2-CH2-N8); 4.36 (t, 2H, J ) 7, CH3-CH2-CH2-
N8); 6.62 (dd, 2H, J ) 2, J ) 4, 4-H Furane); 7.16-7.49 (m,
6H, C6H5 and 3-H Furane); 7.69 (d, 1H, J ) 2, 5-H Furane);
8.24 (s, 1H, N8-CH); 8.61 (bs, 1H, CO-NH-Ph); 11.16 (bs, 1H,
5-NH-CO). Anal. (C20H18N8O2) C, H, N.
[
125I]AB-MECA by 79% at a 100 nM concentration. In
5-[[(P h en yl)am in o]car bon yl]am in o-8-n -bu tyl-2-(2-fu r yl)-
p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (21): yield
92%, white solid; mp 145 °C (EtOAc-light petroleum); IR
contrast, all the derivatives bearing the 4-bis-(â-chlo-
roethyl)amino function failed to irreversibly bind the
hA3 adenosine receptors. These observations were ana-
lyzed by molecular modeling studies, demonstrating
that Cys251 or Ser247 are the most likely sites for
covalent binding. These hypotheses may now be tested
using a site directed mutagenesis approach.
In conclusion, these results provide new pharmaco-
logical probes for improving our understanding of the
pathophysiological role of the human A3 adenosine
receptor, yielding new information about the structural
requirements necessary for the receptor recognition.
(KBr) 3240-2980, 1675, 1610, 1500, 1470 cm-1 1H NMR
;
(DMSO-d6) δ 0.96 (t, 3H, J ) 7, CH3-CH2-CH2-CH2-N8); 1.30-
1.41 (m, 2H, CH3-CH2-CH2-CH2-N8); 1.98-2.06 (m, 2H, CH3-
CH2-CH2-CH2-N8); 4.39 (t, 2H, J ) 7, CH3-CH2-CH2-CH2-N8);
6.62 (dd, 1H, J ) 2, J ) 4, 4-H Furane); 7.16-7.42 (m, 6H,
C6H5 and 3-H Furane); 7.67 (d, 1H, J ) 2, 5-H Furane); 8.23
(s, 1H, N8-CH); 8.62 (bs, 1H, CO-NH-Ph); 11.16 (bs, 1H, 5-NH-
CO). Anal. (C21H20N8O2) C, H, N.
5-[[(4-Bis-â-clor oeth yla m in o-p h en yl)a m in o]ca r bon yl]-
a m in o-8-m eth yl-2-(2-fu r yl)-p yr a zolo[4,3-e]1,2,4-tr ia zolo-
[1,5-c]p yr im id in e (9): yield 82%, yellow solid; mp 165 °C
(EtOAc-light petroleum); IR (KBr) 3230-2990, 1670, 1615,
1510, 1460 cm-1 1H NMR (DMSO-d6) δ 3.60-3.73 (m, 8H,
;
N(CH2CH2Cl)2); 4.21 (s, 3H, CH3-N8); 6.62 (dd, 1H, J ) 2, J )
4, 4-H Furane); 6.66 (d, 2H, J ) 9, CO-NH-Ph); 7.24 (d, 1H, J
) 4, 3-H Furane); 7.51 (d, 2H, J ) 9, 4-Ph- N(CH2CH2Cl)2);
7.66 (d, 1H, J ) 2, 5-H Furane); 8.22 (s, 1H, N8-CH); 8.60 (bs,
1H, CO-NH-Ph); 10.86 (bs, 1H, 5-NH-CO). Anal. (C22H21N9O2-
Cl2) C, H, N.
Exp er im en ta l Section
Ch em istr y. Gen er a l. Reactions were routinely monitored
by thin-layer chromatography (TLC) on silica gel (precoated
F254 Merck plates). Products were visualized with iodine or
potassium permanganate solution. Infrared spectra (IR) were
measured on a Perkin-Elmer 257 spectrophotometer. 1H NMR
were determined in CDCl3 or DMSO-d6 solutions with a
Bruker AC 200 spectrometer. Peaks positions were given in
parts per million (δ), downfield from tetramethylsilane as
internal standard, and J values were given in hertz. Light
petroleum ether referred to the fractions boiling at 40-60 °C.
Melting points were determined on a Buchi-Tottoli instrument
and were uncorrected. Chromatography was performed using
Merck 60-200 mesh silica gel. All products reported showed
IR and 1H NMR spectra in agreement with the assigned
structures. Organic solutions were dried over anhydrous
magnesium sulfate. Elemental analyses were performed by the
microanalytical laboratory of Dipartimento di Chimica, Uni-
versity of Ferrara, and were within (0.4% of the theoretical
values for C, H, and N.
Gen er a l P r oced u r es for th e P r ep a r a tion of 5-[[(Su b-
stitu ted-ph en yl)am in o]car bon yl]am in o-8-alkyl-2-(2-fu r yl)-
p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (9, 11, 13,
15, 18-21). The amino precursor (5-8) (10 mmol) was
dissolved in freshly distilled THF (15 mL), and the appropriate
isocyanate (17, 22) (13 mmol) was added. The mixture was
refluxed under argon for 18 h. Then the solvent was removed
under reduced pressure, and the residue was purified by flash
chromatography (EtOAc-light petroleum 4-6) to afford the
desired compounds 9, 11, 13, 15, 18-21.
5-[[(4-Bis-â-clor oeth yla m in o-p h en yl)a m in o]ca r bon yl]-
a m in o-8-et h yl-2-(2-fu r yl)-p yr a zolo[4,3-e]1,2,4-t r ia zolo-
[1,5-c]p yr im id in e (11): yield 85%, white solid; mp 185 °C
(EtOAc-light petroleum); IR (KBr) 3235-2985, 1675, 1620,
1511, 1450 cm-1; 1H NMR (DMSO-d6) δ: 1.67 (t, 3H, J ) 6.8,
CH3-CH2-N8); 3.62-3.74 (m, 8H, N(CH2CH2Cl)2); 4.46 (q, 2H,
J ) 6.8, CH3-CH2-N8); 6.62 (dd, 1H, J ) 2, J ) 4, 4-H Furane);
6.71 (d, 2H, J ) 9, CO-NH-Ph); 7.24 (d, 1H, J ) 4, 3-H Furane);
7.51 (d, 2H, J ) 9, 4-Ph-N(CH2CH2Cl)2); 7.66 (d, 1H, J ) 2,
5-H Furane); 8.25 (s, 1H, N8-CH); 8.59 (bs, 1H, CO-NH-Ph);
11.04 (bs, 1H, 5-NH-CO). Anal. (C23H23N9O2Cl2) C, H, N.
5-[[(4-Bis-â-clor oeth yla m in o-p h en yl)a m in o]ca r bon yl]-
am in o-8-n -pr opyl-2-(2-fu r yl)-pyr azolo[4,3-e]1,2,4-tr iazolo-
[1,5-c]p yr im id in e (13): yield 78%, white solid; mp 195 °C
(EtOAc-light petroleum); IR (KBr) 3230-2985, 1674, 1625,
1
1520, 1445 cm-1; H NMR (DMSO-d6) δ 1.01 (t, 3H, J ) 6.8,
CH3-CH2-CH2-N8); 1.97-2.08 (m, 2H, CH3-CH2-CH2-N8); 3.62-
3.76 (m, 8H, N(CH2CH2Cl)2); 4.35 (t, 2H, J ) 6.8, CH3-CH2-
CH2-N8); 6.62 (dd, 1H, J ) 2, J ) 4, 4-H Furane); 6.71 (d, 2H,
J ) 9, CO-NH-Ph); 7.24 (d, 1H, J ) 4, 3-H Furane); 7.51 (d,
2H, J ) 9, 4-Ph- N(CH2CH2Cl)2); 7.66 (d, 1H, J ) 2, 5-H
Furane); 8.23 (s, 1H, N8-CH); 8.59 (bs, 1H, CO-NH-Ph); 10.85
(bs, 1H, 5-NH-CO). Anal. (C24H25N9O2Cl2) C, H, N.
5-[[(4-Bis-â-clor oeth yla m in o-p h en yl)a m in o]ca r bon yl]-
a m in o-8-n -bu tyl-2-(2-fu r yl)-p yr a zolo[4,3-e]1,2,4-tr ia zolo-
[1,5-c]p yr im id in e (15): yield 80%, pale yellow solid; mp 190
°C (EtOAc-light petroleum); IR (KBr) 3235-2980, 1672, 1620,
5-[[(P h en yl)am in o]car bon yl]am in o-8-m eth yl-2-(2-fu r yl)-
p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (18): yield
90%, yellow solid; mp 165-170 °C (EtOAc-light petroleum);
1
1
IR (KBr) 3255-2990, 1680, 1620, 1510, 1450 cm-1; H NMR
1510, 1450 cm-1; H NMR (DMSO-d6) δ 0.99 (t, 3H, J ) 6.8,
(DMSO-d6) δ 4.20 (s, 3H, N8-CH3); 6.61 (dd, 2H, J ) 2, J ) 4,
4-H Furane); 7.11-7.37 (m, 6H, C6H5 and 3-H Furane); 7.76
CH3-CH2-CH2-CH2-N8); 1.23-1.47 (m, 2H, CH3-CH2-CH2-CH2-
N8); 1.89-2.04 (m, 2H, CH3-CH2-CH2-CH2-N8); 3.64-3.74 (m,

2740 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Baraldi et al.
8H, N(CH2CH2Cl)2); 4.39 (t, 2H, J ) 6.8, CH3-CH2-CH2-CH2-
N8); 6.61 (dd, 2H, J ) 2, J ) 4, 4-H Furane); 6.71 (d, 2H, J )
9, CO-NH-Ph); 7.24 (d, 1H, J ) 4, 3-H Furane); 7.51 (d, 2H, J
) 9, 4-Ph-N(CH2CH2Cl)2); 7.66 (d, 1H, J ) 2, 5-H Furane);
8.23 (s, 1H, N8-CH); 8.59 (bs, 1H, CO-NH-Ph); 10.84 (bs, 1H,
5-NH-CO). Anal. (C25H27N9O2Cl2) C, H, N.
separated by filtering the assay mixture through Whatman
GF/B glass-fiber filters using a Micro-Mate 196 cell harvester
(Packard Instrument Company). The filter bound radioactivity
was counted on Top Count (efficiency 57%) with Micro-Scint
20. The protein concentration was determined according to the
Bio-Rad method38 with bovine albumin as reference standard.
h A3 Ir r ever sible Bin d in g Assa y. Binding of [125I]AB-
MECA to membranes prepared from CHO cells stably express-
ing the human A3 receptor was performed as described.32,33
The assay medium consisted of a buffer containing 10 mM
Mg2+, 50 mM Tris, and 1 mM EDTA, at pH 7.4. The glass
incubation tubes were filled with 100 µL of the membrane
suspension (1.0 mg of protein/mL, stored at -80 °C in the same
buffer), 20 µL of [125I]AB-MECA (final concentration 0.6 nM),
and 50 µL of a solution of the proposed antagonist. Nonspecific
binding was determined in the presence of 100 µM NECA.
Adenosine deaminase (3 units/mL) was present during the
preparation of the membranes, in a preincubation of 30 min
at 30 °C and during the incubation with the radioligands.
All nonradioactive compounds (9-16) were initially dis-
solved in DMSO and diluted with buffer to the desired final
concentartion. The amount of DMSO never exceeded 1%.
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, each
with 3 mL of buffer.
For studies of irreversible binding, membranes were incu-
bated with the indicated (Table 1) concentration of ligand (9-
16) for 1 h at room temperature. Following the incubation,
the membrane suspension was centrifuged at 28 000 rpm for
30 min, and the pellet was resuspended in fresh buffer and
kept at 4 °C for 30 min.
Membranes were washed a total of 9 times by sequential
resuspension and centrifugation with buffer (50 mM Tris, 10
mM Mg2+, and 1 mM EDTA) at pH 7.4 containing 0.02%
CHAPS. Adenosine deaminase (3 units/mL) was present
during all steps. The final pellet was suspended in a fixed
volume of the above buffer solution for the A3 receptor binding
assay as described.32,33
Gen er a l P r oced u r es for th e P r ep a r a tion of 5-[[(4-
F lu or osu lfon yl-p h en yl)a m in o]ca r bon yl]a m in o-8-a lk yl-2-
(2-fu r yl)-pyr azolo[4,3-e]1,2,4-tr iazolo[1,5-c]pyr im idin e (10,
12, 14, 16). The appropriate phenyl-carbamoyl derivative (18-
21) (0.086 mmol) were reacted with fluorosulfonic acid (0.1 mL)
at -10 °C for 2 h. Then the mixture was quenched with ice
water (1 mL), and the precipitate was filtered off to afford the
desired compounds (10, 12, 14, 16) as a solid in a good yield.
5-[[(4-F lu or osu lfon yl-p h en yl)a m in o]ca r bon yl]a m in o-
8-m et h yl-2-(2-fu r yl)-p yr a zolo[4,3-e]1,2,4-t r ia zolo[1,5-c]-
p yr im id in e (10): yield 78%, yellow solid; mp 170 °C (EtOAc-
light petroleum); IR (KBr) 3230-2995, 1670, 1615, 1500, 1320,
1470, 1160 cm-1; 1H NMR (DMSO-d6) δ 4.14 (s, 3H, CH3-N8);
6.76 (dd, 1H, J ) 2, J ) 4, 4-H Furane); 7.29 (d, 1H, J ) 4,
3-H Furane); 7.89 (d, 2H, J ) 9, CO-NH-Ph); 7.99 (d, 1H, J )
2, 5-H Furane); 8.14 (d, 2H, J ) 9, 4-Ph-SO2F); 8.80 (s, 1H,
N8-CH); 10.15 (bs, 1H, CO-NH-Ph); 11.06 (bs, 1H, 5-NH-CO).
Anal. (C18H13N8SO4F) C, H, N.
5-[[(4-F lu or osu lfon yl-p h en yl)a m in o]ca r bon yl]a m in o-
8-eth yl-2-(2-fu r yl)-p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p y-
r im id in e (12): yield 72%, pale yellow solid; mp 165 °C
(EtOAc-light petroleum); IR (KBr) 3235-2990, 1675, 1620,
1510, 1330, 1470, 1160 cm-1; 1H NMR (DMSO-d6) δ 1.5 (t, 3H,
J ) 6.8, CH3-CH2-N8); 4.42 (q, 2H, J ) 6.8, CH3-CH2-N8); 6.76
(dd, 2H, J ) 2, J ) 4, 4-H Furane); 7.29 (d, 1H, J ) 4, 3-H
Furane); 7.96 (d, 2H, J ) 9, CO-NH-Ph); 7.99 (d, 1H, J ) 2,
5-H Furane); 8.14 (d, 2H, J ) 9, 4-Ph-SO2F); 8.86 (s, 1H, N8-
CH); 10.19 (bs, 1H, CO-NH-Ph); 11.04 (bs, 1H, 5-NH-CO).
Anal. (C19H15N8SO4F) C, H, N.
5-[[(4-F lu or osu lfon yl-p h en yl)a m in o]ca r bon yl]a m in o-
8-n -p r op yl-2-(2-fu r yl)-p yr a zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]-
p yr im id in e (14): yield 75%, white solid; mp 155 °C (EtOAc-
light petroleum); IR (KBr) 3235-2990, 1673, 1615, 1515, 1325,
1
1465, 1150 cm-1; H NMR (DMSO-d6) δ 0.87 (t, 3H, J ) 6.8,
Com p u ta tion a l Ap p r oa ch . All calculations were per-
formed on a Silicon Graphics O2 R10000 workstation.
The human A3 receptor model was built and optimized using
MOE (2000.02) modeling package39 based on the approach
described by Moro et al.40 Briefly, transmembrane domains
were identified with the aid of Kyte-Doolittle hydrophobicity
and Emin surface probability parameters.41 Transmembrane
helices were built from the sequences and minimized individu-
ally. The minimized helices were then grouped together to form
a helical bundle that matched the overall characteristics of
the recently published crystal structure of bovine rhodopsin
(PDB ID: 1F88). The helical bundle was minimized using the
Amber94 all-atoms force field,42 until the rms value of the
conjugate gradient (CG) was <0.01 kcal/mol/Å. A fixed dielec-
tric constant of 4.0 was used throughout these calculations.
All pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives
were fully optimized without geometric constraints using RHF/
AM1 semiempirical calculations.43 Vibrational frequency analy-
ses were used to characterize the minima stationary points
(zero imaginary frequencies). The software package Gaussi-
an98 was utilized for all quantum mechanical calculations.44
The ligands were docked into the hypothetical TMs binding
site using the DOCK docking program, part of the MOE suite.39
This program incorporates the use of manual and automatic
docking procedures in combination with molecular mechanics
within the Simulations module of MOE. The docking method
employed enables nonbonded van der Waals and electrostatic
interactions to be simultaneously monitored during the dock-
ing, and several possible conformations for the ligand were
evaluated interactively. “Flexible” ligand docking was then
used to define the lowest energy position of each ligand using
a Monte Carlo/annealing based automated docking protocol.
This uses a random iterative algorithm to sample changes in
torsion angles and atomic positions while simultaneously
recalculating internal and interaction energies. The automated
CH3-CH2-CH2-N8); 1.90-1.95 (m, 2H, CH3-CH2-CH2-N8); 4.35
(t, 2H, J ) 6.8, CH3-CH2-CH2-N8); 6.76 (dd, 2H, J ) 2, J ) 4,
4-H Furane); 7.29 (d, 1H, J ) 4, 3-H Furane); 7.89 (d, 2H, J )
9, CO-NH-Ph); 8.01 (d, 1H, J ) 2, 5-H Furane); 8.13 (d, 2H, J
) 9, 4-Ph-SO2F); 8.85 (s, 1H, N8-CH); 10.12 (bs, 1H, CO-NH-
Ph); 11.04 (bs, 1H, 5-NH-CO). Anal. (C20H17N8SO4F) C, H, N.
5-[[(4-F lu or osu lfon yl-p h en yl)a m in o]ca r bon yl]a m in o-
8-n -bu t yl-2-(2-fu r yl)-p yr a zolo[4,3-e]1,2,4-t r ia zolo[1,5-c]-
p yr im id in e (16): yield 77%, white solid; mp 145 °C (EtOAc-
light petroleum); IR (KBr) 3225-2980, 1670, 1610, 1525, 1335,
1465, 1155 cm-1 1H NMR (DMSO-d6) δ 0.97 (t, 3H, J ) 7,
;
CH3-CH2-CH2-CH2-N8); 1.29-1.34 (m, 2H, CH3-CH2-CH2-CH2-
N8); 1.98-2.06 (m, 2H, CH3-CH2-CH2-CH2-N8); 4.43 (t, 2H, J
) 7, CH3-CH2-CH2-CH2-N8); 6.65 (dd, 2H, J ) 2, J ) 4, 4-H
Furane); 7.29 (d, 1H, J ) 4, 3-H Furane); 7.85 (d, 2H, J ) 9,
CO-NH-Ph); 8.04 (d, 1H, J ) 2, 5-H Furane); 8.10 (d, 2H, J
) 9, 4-Ph-SO2F); 8.78 (s, 1H, N8-CH); 10.11 (bs, 1H, CO-NH-
Ph); 11.88 (bs, 1H, 5-NH-CO). Anal. (C21H19N8SO4F) C, H, N.
Biology. h A1 a n d h A2A Bin d in g Assa ys. Binding of [3H]-
DPCPX to CHO cells transfected with the human recombinant
A1 adenosine receptor was performed as previously described.36
Displacement experiments were performed for 120 min at
25 °C in 0.20 mL of buffer containing 1 nM [3H]-DPCPX, 20
µL of diluted membranes (50 µg of protein/assay), and at least
six to eight different concentrations of examined compounds.
Nonspecific binding was determined in the presence of 10 µM
of CHA, and this is always e10% of the total binding.
Binding of [3H]-SCH58261 to CHO cells transfected with
the human recombinant A2A adenosine receptors (50 µg of
protein/assay) was performed according to Varani et al.37 In
competition studies, at least six to eight different concentra-
tions of compounds were used, and nonspecific binding was
determined in the presence of 50 µM NECA for an incubation
time of 60 min at 25 °C. Bound and free radioactivity were

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2741
(14) Ramkumar, V.; Stiles, G. L.; Beaven, M. A.; Ali, H. The A3
adenosine receptor is the unique adenosine receptor which
facilitates release of allergic mediators in mast cells. J . Biol.
Chem. 1993, 268, 16887-16890.
(15) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Merighi, S.; Varani,
K.; Borea, P. A.; Spalluto G. A3 Adenosine receptor ligands;
history and perspectives. Med. Res. Rev. 2000, 20, 103-128
(16) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, G.; Klotz,
K.-N.; Leung, E.; Varani, K.; Gessi, S.; Merighi, S.; Borea, P.
A.. Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as
highly potent and selective human A3 adenosine receptor
antagonists. J . Med. Chem. 1999, 42, 4473-4478.
(17) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto,G.; Moro,
S.; Klotz, K. N.; Leung, E.; Varani, K.; Gessi, S.; Merighi, S.;
Borea, P. A. Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine de-
rivatives as highly potent and selective human A3 adenosine
receptor antagonists: Influence of the chain at N8 pyrazole
nitrogen. J . Med. Chem. 2000, 43, 4768-4780.
(18) Dickinson, K. E. J .; Heald, S. L.; J effs, P. W.; Lefkowitz, R. J .;
Caron, M. G. Covalent labeling of the â-adrenergic ligand-
binding site with the par-(bromoacetamidyl)benzylcarazolol. A
highly potent â-adrenergic affinity label. Mol. Pharmacol. 1985,
27, 499-506.
docking procedure then selected the best structures and
applied the totality of the binding site. During the docking,
all torsion angles of the side chains on the ligand were allowed
to vary. This docking procedure was followed by another
sequence of CG energy minimization to a gradient threshold
of < 0.1 kcal/mol/Å. Energy minimization of the complexes was
performed using AMBER94 all-atom force field.
The interaction energy values were calculated as follows:
∆E(complex) ) E(complex) - (E(L) + E(receptor)). These energies are not
rigorous thermodynamic quantities, but can only be used to
compare the relative stabilities of the complexes. Conse-
quently, these interaction energy values cannot be used to
calculate binding affinities, since changes in entropy and
solvation effects are not taken into account.
Abbr evia tion s: DMSO, dimethyl sulfoxide; DPCPX, 1,3-
dipropyl-8-cytclopentylxanthine; THF, tetrahydrofurane; CHO
cells, Chinese hamster ovary cells; EDTA, ethylenediaminotet-
raacetate; NECA, 5′-(N-ethylcarbamoyl)adenosine; TLC, thin-
layer chromatography; mp, melting point; EtOAc, ethyl ac-
etate; IR, infrared spectra; NMR, nuclear magnetic resonance;
CDCl3, deuterated chloroform; CHAPS, 3-[(3-chloroamidopro-
pyl)-dimethylammino]-1-propanesulfonate; [125I]AB-MECA,
(19) Minneman, K. P. R1-Adrenergic receptor subtypes, inositol
phosphates, and sources of cell Ca2+. Pharmacol. Rev. 1988, 40,
87-119.
[
125I]1-[6-[[(4-amino-3-iodophenyl)methyl]amino]-9H-purin-9-
(20) Nelson, C. A.; Muther, T. F.; Pitha, J .; Baker, S. P. Differential
recovery of beta adrenoreceptor antagonist and agonist high
affinity binding sites in the guine pig lung after irreversible
blockade. J . Pharmacol. Exp. Ther. 1986, 237, 830-836.
(21) (a) Scammels, P. J .; Bakes, S. P.; Belardinelli, L.; Olsson, R. A.
Substituted 1,3-dipropylxanthines as irreversible antagonists of
A1 adenosine receptors. J . Med. Chem. 1994, 37, 2704-2712.
(b) J acobson, K. A.; Barone, S.; Kammula, U.; Stiles, G. L.
Electrophilic derivatives of purines as irreversible inhibitors of
A1-adenosine receptors. J . Med. Chem. 1989, 32, 1043-1051. (c)
Boring, D. L.; J i, X.-D.; Zimmet, J .; Taylor, K. E.; Stiles, G. L.;
J acobson, K. A. Trifunctional agents as a design strategy for
tailoring ligand properties: Irreversible inhibitors of A1 adeno-
sine receptors. Bioconjugate Chem. 1991, 2, 77-88.
(22) (a) Srinivas, M.; Shryock, J . C.; Scammels, P. J .; Baker, S. P.;
Belardinelli, L. A novel irreversible antagonist of the A1-
adenosine receptor. Mol. Pharmacol. 1996, 50, 196-205. (b)
Stiles, G. L.; J acobson, K. A. High affinity acylating antagonists
for the A1 adenosine receptor: identification of binding subunit.
Mol. Pharmacol. 1988, 34, 724-728. (c) J acobson, K. A.; Olah,
M. E.; and Stiles, G. L. Trifunctional ligands: A radioiodinated
high affinity acylating antagonist for the A1 adenosine receptor.
Pharmacol. Commun. 1992, 1, 145-154.
yl]-1-deoxy-N-methyl-â-D-ribofuranuronamide; MRE3008-F20,
5-[[(4-methoxyphenyl)amino] carbonyl]amino-8-propyl-2-(2-fu-
ryl)-pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine; SCH58261,
5-amino-2-(2-furyl)-7-(2-phenylethyl)pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidine.
Ack n ow led gm en t. This work was supported by
MURST (cofin. 2000, ex 40%, prot. n. MM03237197_004)
and MEDCO Res., Triangle Park, NC.
Refer en ces
(1) Fredholm, B. B.; Abbracchio, M. P.; Burnstock, G.; Daly, J . W.;
Harden, T. K.; J acobson, K. A.; Leff, P.; Williams, M. Nomen-
clature and classification of purinoceptors. Pharmacol. Rev.
1994, 46, 143-156.
(2) Olah, M. E.; Stiles, G. L. Adenosine receptor subtypes: charac-
terization and therapeutic regulation. Annu. Rev. Pharmacol.
Toxicol. 1995, 35, 581-606.
(3) J acobson, K. A.; Suzuki, F. Recent developments in selective
agonists and antagonists acting at purine and pyrimidine
receptors. Drug. Dev. Res. 1996, 39, 289-300.
(23) (a) Srinivas, M.; Shryock, J . C.; Dennis, D. M.; Baker, S. P.;
Belardinelli, L. Differential A1 adenosine receptor reserve for
two action of adenosine on guinea pig myocytes. Mol. Pharmacol.
1997, 52, 683-691. (b) Dennis, D.; J acobson, K. A.; Belardinelli,
L. Evidence of spare A1-adenosine in guinea pig atrioventricular
node. Am. J . Physiol. 1992, 262, H661-671. (c) Zhang, J .;
Belardinelli, L.; J acobson, K. A.; Otero, D. H.; Baker, S. P.
Persistent activation of A1 adenosine receptors by an acylating
adenosine derivative and its receptor reserve in DDT1 MF-2 cells
and heart. Mol. Pharmacol. 1997, 52, 491-498.
(24) (a) J acobson, K. A.; Fischer, B.; J i, X.-D. A “cleavable trifunc-
tional” approach to receptor affinity labeling: regeneration of
binding to A1-adenosine receptors, Bioconjugate Chem. 1995, 6,
255-263. (b) Beauglehole, A. R.; Baker, S. P.; Scammels, P. J .
Fluorosulfonyl-substituted xanthines as selective irreversible
antagonists for the A1-adenosine receptor. J . Med. Chem. 2000,
43, 4973-4980.
(25) (a) J acobson, K. A.; Barrington, W. W.; Pannell, L. K.; J arvis,
M. F.; J i, X.-D.; Williams, M.; Hutchison, A. J .; Stiles, G. L.
Agonist-derived molecular probes for A2-adenosine receptors. J .
Mol. Recognit. 1989, 2, 170-178. (b) J acobson, K. A.; Stiles, G.
L.; J i, X.-D. Chemical modification and irreversible inhibition
of striatal A2a-adenosine receptors. Mol. Pharmacol. 1992, 42,
123-133. (c) J i, X.-D.; Gallo-Rodriguez, C.; J acobson, K. A. 8-(3-
Isothiocyanatostyryl)caffeine is a selective irreversible inhibitor
of striatal A2-adenosine receptors. Drug Dev. Res. 1993, 29, 292-
298. (d) Niiya, K.; J acobson, K. A.; Silvia, S. K.; Olsson, R. A.
Covalent binding of a selective agonist irreversibly activates
guinea pig coronary artery A2 adenosine receptors. Naunyn-
Schmeideberg’s Arch. Pharmacol. 1993, 347, 521-526. (e) Shry-
ock, J . C.; Snowdy, S.; Baraldi, P. G.; Cacciari, B.; Spalluto, G.;
(4) Meyerhof, W.; Muller-Brechlin, R.; Richter, D. Molecular cloning
of a novel putative G-protein coupled receptor expressed during
rat spermiogenesis. FEBS Lett. 1991, 284, 155-160.
(5) Sajjadi, F. G.; Firestein, G. S. cDNA cloning and sequence
analysis of the human A3 adenosine receptor. Biochim. Biophys.
Acta 1993, 1179, 105-107.
(6) Salvatore, C. A.; J acobson, M. A.; Taylor, H. E.; Linden, J .;
J ohnson, R. G. Molecular cloning and characterization of the
human A3 adenosine receptor. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 10365-10369.
(7) Linden, J . Cloned adenosine A3 receptors: Pharmacological
properties, species differences and receptor functions. Trends
Pharmacol. Sci. 1994, 15, 298-306.
(8) Zhao, Z. H.; Ravid, S.; Ravid, K. Chromosomal mapping of the
mouse A3 adenosine receptor gene, adora3. Genomics 1995, 30,
118-119.
(9) Hill, R. J .; Oleynek, J . J .; Hoth, C. F.; Kiron, M. A.; Weng, W.
F.; Wester, R. T.; Tracey, W. R.; Knight, D. R.; Buchholz, R.;
Kennedy, S. P. Cloning, expression and pharmacological char-
acterization of rabbit A1 and A3 receptors. J . Pharmacol. Exp.
Ther. 1997, 280, 122-128.
(10) Abbracchio, M. P.; Brambilla, R.; Kim, H. O.; von Lubitz, D. K.
J . E.; J acobson, K. A.; Cattabeni, F. G-protein-dependent activa-
tion of phospholipase-C by adenosine A3 receptor in rat brain.
Mol. Pharmacol. 1995, 48, 1038-1045.
(11) Ali, H.; Choi, O. H.; Fraundorfer, P. F.; Yamada, K.; Gonzaga,
H. M. S.; Beaven, M. A. Sustained activation of phospholipase-D
via adenosine A3 receptors is associated with enhancement of
antigen-ionophore-induced and Ca2+-ionophore-induced secretion
in a rat mast-cell line. J . Pharmacol. Exp. Ther. 1996, 276, 837-
845.
(12) van Schaick, E. A.; J acobson, K. A.; Kim, H. O.; IJ zerman, A.
P.; Danhof, M. Haemodynamic effects and histamine release
elicited by the selective adenosine A3 receptor agonists 2-Cl-IB-
MECA in conscious rats. Eur. J . Pharmacol. 1996, 308, 311-
314.
Monopoli, A.; Ongini, E.; Baker, S. P.; Belardinelli, L. A2A
-
adenosine receptor reserve for coronary. Circulation 1998, 98,
711-718.
(26) J i, X.-D.; Gallo-Rodriguez, C.; J acobson, K. A. A selective agonist
affinity label for A3 adenosine receptors. Biochem. Biophys. Res.
Commun. 1994, 203, 570-576.
(13) Forsythe, P.; Ennis, M. Adenosine, mast cells and asthma.
Inflammation Res. 1999, 48, 301-307.

2742 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Baraldi et al.
(27) Li, A.-H.; Chang, L.; J i, X.-D.; Melman, N.; J acobson, K. A.
Functionalized congeners of 1,4-dihydropyridines as antagonist
molecular probes for A3 adenosine receptors. Bioconjugate Chem.
1999, 10, 667-677.
(28) (a) Bruncel, E. Mechanistic aspects of the chemistry of fluoro-
sulfates. Mech. React. Sulfur Compounds 1970, 5, 71-74. (b)
Fukuda, H, Frank, F. S.; Truce, W. E. Synthesis of â-disulfones
from sulfonyl fluorides and organometallic compounds. J . Org.
Chem. 1963, 28, 1420.
(29) Panthananickal, A.; Hansch, C.; Leo, A.; Quinn, F. R. Structure-
activity relationships in antitumor aniline mustards. J . Med.
Chem. 1978, 21, 16-26.
(30) Bardos, T. J .; Datta-Gupta, N.; Hebborn, P.; Triggle, D. J . A
study of comparative chemical and biological activities of al-
kylating agents. J . Chem. Soc. 1965, 8, 167-174.
(37) Varani, K.; Gessi, S.; Dionisotti, S.; Ongini, E.; Borea, P. A. [3H]-
SCH 58261 Labelling of functional A2A adenosine receptors in
human neutrophil membranes. Br. J . Pharmacol. 1998, 123,
1723-1731.
(38) Bradford, M. M. A rapid sensitive method for the quantification
of microgram quantities of protein utilizing the principle of
protein dye-binding. Anal. Biochem. 1976, 72, 248-254.
(39) Molecular Operating Environment (MOE 2000.02), Chemical
Computing Group, Inc.: 1255 University St., Suite 1600, Mon-
treal, Quebec, Canada, H3B 3X3.
(40) Moro, S.; Guo, D.; Camaioni, E,; Boyer, J . L.; Harden K. T.;
J acobson, K. A. Human P2Y1 receptor: molecular modeling and
site-directed mutagenesis as tools to identify agonist and
antagonist recognition sites. J . Med. Chem. 1998, 41, 1456-
1466.
(41) Kyte, J .; Doolittle, R. F. A simple method for displaying the
hydrophobic character of a protein. J . Mol. Biol. 1982, 157, 105-
132.
(31) Kurita, K.; Iwakura, Y. Trichloromethyl-Chloroformate as a
phosgene equivalent: 3-isocyanatopropanoyl chloride. Org. Synth.
Coll. Vol. VI 1988, 715-718.
(42) Weiner, S. J .; Kollman, P. A.; Nguyen, D. T.; Case, D. A. An
all-atom force field for simulation of protein and nucleic acids.
J . Comput. Chem. 1986, 7, 230-252.
(43) Dewar, M. J . S. E.; Zoebisch, G.; Healy, E. F. AM1: A New
General Purpose Quantum Mechanical Molecular Model. J . Am.
Chem. Soc. 1985, 107, 3902-3909.
(32) 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
flavonoid derivatives as A3 adenosine receptor antagonists. J .
Med. Chem. 1996, 39, 2293-2301.
(33) Olah, M. E.; Gallo-Rodriguez, C.; J acobson, K. A.; Stiles, G. L.
[
125I]AB-MECA,
a high affinity radioligand for the rat A3
(44) Frisch, M.; Trucks, G. W.; Schlegel, H.; Scuseria, G.; Robb, M.;
Cheeseman, J .; Zakrzewski, V.; Montgomery, J .; Stratmann, R.
E.; Burant, J . C.; Dapprich, S.; Millam, J .; Daniels, A.; Kudin,
K.; Strain, M.; Farkas, O.; Tomasi, J .; Barone, V.; Cossi, M.;
Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.;
Ochterski, J .; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma,
K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman,
J . B.; Cioslowski, J .; Ortiz, J . V.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin,
R. L.; Fox, D. J .; Keith, T.; Al-Laham, M. A.; Peng, C. Y.;
Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.;
J ohnson, B. G.; Chen, W.; Wong, M. W.; Andres, J . L.; Head-
Gordon, M.; Replogle, E. S.; Pople, J . A., Gaussian 98 (revision
A.6); Gaussian, Inc.: Pittsburgh, PA, 1998.
adenosine receptor. Mol. Pharmacol. 1994, 45, 978-982.
(34) Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C. A.; Mo-
toshima, H.; Fox, B. A.; Trong, I. L.; Teller, D. C.; Okada, T.;
Stenkamp, R. E.; Yamamoto, M.; Miyano, M. Crystal structure
of rhodopsin: a G protein-coupled receptor. Science 2000, 289,
739-745.
(35) Moro, S.; Li, A. H.; J acobson, K. A. Molecular modeling studies
of human A3 adenosine antagonists: structural homology and
receptor docking. J . Chem. Inf. Comput. Sci. 1998, 38, 1239-
1248.
(36) Varani, K.; Merighi, S.; Gessi, S.; Klotz, K. N.; Leung, E.; Baraldi,
P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, G.; Borea, P. A. [3H]-
MRE3008-F20: a novel antagonist radioligand for the pharma-
cological and biochemical characterization of human A3 adenos-
ine receptors. Mol. Pharmacol. 2000, 57, 968-975.
J M010818A