N6-cyclopentyl-2-(3-phenylaminocarbonyltriazene-1-yl)adenosine (TCPA), a very selective agonist with high affinity for the human adenosine A1 receptor

Article abstract of DOI:10.1021/jm021074j

Four subtypes of adenosine receptors are currently known, that is, A₁, A_2A, A_2B, and A₃ receptors. Interestingly, quite substantial species differences exist especially between human and rat A₃ receptors. As a result, ligands such as CCPA, which are very selective for the rat A₁ receptor versus the human A₃ receptor, are substantially less selective when the human A₁ and A₃ receptors are compared. New 2-substituted and 2,N⁶-disubstituted adenosines were synthesized, and their affinities for the human adenosine A₁, A_2A, A_2B, and A₃ receptors were determined. Although large substituents on the C2-position are generally thought to yield adenosine A_2A receptor selective ligands, the reported series of 2-triazeno-substituted adenosines had a very high affinity for the A₁ receptor. For example, 2-(3-phenylaminocarbonyltriazene-1-yl)adenosine had an affinity of 6.1 ± 1.3 nM for the human adenosine A₁ receptor. Introduction of a diphenethyl substituent at the N⁶-position of this compound resulted in a high-affinity agonist, 3.1 ± 0.9 nM, for the human adenosine A₁ receptor with 316- and 45-fold selectivity versus the human A_2A and human A₃ receptors, respectively. The most selective, high-affinity human adenosine A₁ receptor agonist was the disubstituted compound N⁶-cyclopentyl-2-(3-phenylaminocarbonyltriazene-1-yl)adenosine (TCPA). TCPA had an affinity of 2.8 ± 0.8 nM for the human adenosine A₁ receptor and was 75-fold and 214-fold selective versus the human A_2A and human A₃ receptors, respectively. In addition, TCPA was a full agonist and inhibited the forskolin-induced cAMP production of CHO cells stably transfected with the human adenosine A₁ receptor with an IC₅₀ of 1.5 ± 0.5 nM.

Full text of DOI:10.1021/jm021074j

1492
J . Med. Chem. 2003, 46, 1492-1503
N6-Cyclop en tyl-2-(3-p h en yla m in oca r bon yltr ia zen e-1-yl)a d en osin e (TCP A), a
Ver y Selective Agon ist w ith High Affin ity for th e Hu m a n Ad en osin e A1
Recep tor
Margot W. Beukers,*,† Martin J . Wanner,‡ J acobien K. Von Frijtag Drabbe Ku¨nzel,† Elisabeth C. Klaasse,†
Adriaan P. IJ zerman,† and Gerrit-J an Koomen‡
Division of Medicinal Chemistry, Leiden/ Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502,
2300 RA Leiden, The Netherlands, and Laboratory of Bioorganic Chemistry, Institute of Molecular Chemistry,
University of Amsterdam, Nieuwe Achtergracht 129, 1018 WS Amsterdam, The Netherlands
Received October 21, 2002
Four subtypes of adenosine receptors are currently known, that is, A1, A2A, A2B, and A3 receptors.
Interestingly, quite substantial species differences exist especially between human and rat A3
receptors. As a result, ligands such as CCPA, which are very selective for the rat A1 receptor
versus the human A3 receptor, are substantially less selective when the human A1 and A3
receptors are compared. New 2-substituted and 2,N6-disubstituted adenosines were synthesized,
and their affinities for the human adenosine A1, A2A, A2B, and A3 receptors were determined.
Although large substituents on the C2-position are generally thought to yield adenosine A2A
receptor selective ligands, the reported series of 2-triazeno-substituted adenosines had a very
high affinity for the A1 receptor. For example, 2-(3-phenylaminocarbonyltriazene-1-yl)adenosine
had an affinity of 6.1 ( 1.3 nM for the human adenosine A1 receptor. Introduction of a
diphenethyl substituent at the N6-position of this compound resulted in a high-affinity agonist,
3.1 ( 0.9 nM, for the human adenosine A1 receptor with 316- and 45-fold selectivity versus the
human A2A and human A3 receptors, respectively. The most selective, high-affinity human
adenosine A1 receptor agonist was the disubstituted compound N6-cyclopentyl-2-(3-phenylami-
nocarbonyltriazene-1-yl)adenosine (TCPA). TCPA had an affinity of 2.8 ( 0.8 nM for the human
adenosine A1 receptor and was 75-fold and 214-fold selective versus the human A2A and human
A3 receptors, respectively. In addition, TCPA was a full agonist and inhibited the forskolin-
induced cAMP production of CHO cells stably transfected with the human adenosine A1 receptor
with an IC50 of 1.5 ( 0.5 nM.
In tr od u ction
tyladenosine (CCPA, 2735 times over human A2A and
51 times over human A3),12 and the selective agonist
for the human adenosine A2A receptor CGS21680 (11
times over human A1 and 4 times over human A3, 2-[[4-
(2-carboxyethyl)phenethyl]amino]adenosine-5′-N-ethyl-
carboxamide), on the other hand, display considerable
affinity for the human A3 receptor.12 Hence, more
selective ligands for both the human adenosine A1 and
the human adenosine A2A receptor are required. The
objective of this study is to generate agonists for the
human adenosine A1 receptor with higher overall se-
lectivity over the other human adenosine receptors.
Although substitution at the C2-position is generally
thought to yield selective agonists for adenosine A2A
receptors, some 2-substituted agonists show preference
for the adenosine A1 receptor. Introduction of small
groups such as chlorine at the C2-position in combina-
tion with the N6-cyclopentyl moiety (CCPA) resulted in
the most selective agonist for the human adenosine A1
receptor known to date as shown in this study and by
Klotz et al.12
Four adenosine receptors have been cloned from
several species including human. They are named
adenosine A1, A2A, A2B, and A3 receptors. Numerous
ligands for adenosine receptors have been synthesized
and biologically evaluated. For a recent review on
adenosine receptors and their ligands see Fredholm et
al.1 Traditionally, selective adenosine A1 receptor ago-
nists were obtained by monosubstitution of the N6-
position,2 whereas introduction of substituents such as
alkylamino,3 alkynyl,4,5 alkoxy,6,7 or N′-aralkylidenehy-
drazino8,9 at the C2-position yielded selective A2A recep-
tor agonists. To date, no selective agonists are available
for the A2B receptor. With the discovery in 1991 of the
adenosine A3 receptor, the fourth member of the ad-
enosine receptor family, new challenges to synthesize
selective ligands arose.10,11
The most selective agonist for the human adenosine
A3 receptor, N6-(4-aminobenzyl)-5′-methylcarboxami-
doadenosine (ABMECA), is substituted on the N6- and
5′-positions. This ligand is very selective for the human
A3 receptor versus the other human receptors (70 times
over A1 and 166 times over A2A). The selective agonist
for the human adenosine A1 receptor, 2-Cl-N6-cyclopen-
In a previous study we have reported on N6-substi-
tuted, 2-NO2 analogues of adenosine.13 The presence of
the NO2 group at the C2-position together with a
cyclopentyl substituent at the N6-position resulted in a
selective ligand for the rat adenosine A1 receptor.
* Corresponding author (telephone +31-71-5274607; fax +31-71-
5274565; e-mail beukers@chem.leidenuniv.nl).
† Leiden University.
To further improve selectivity we have taken 2-ni-
trosoadenosine as a starting point in our search for
‡ University of Amsterdam.
10.1021/jm021074j CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/18/2003

TCPA, a Selective Agonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1493
Sch em e 1. Synthesis of Carbamoyl Triazenes 8a -l, 11, 12, and 16a -d a
a
Reagents and conditions: (i) 1.0 equiv of NaN3, DMF, -18 °C; (ii) H2, Pd/C, EtOAc; (iii) NaIO4, EtOAc, H2O; (iv) aq NH3/MeOH; (v)
CH3CN/HOAc 10:1; room temperature, 5 h; (vi) 11: benzoyl hydrazide, CH3CN/HOAc 10:1, room temperature, 18 h, 54%; (vii) 12: phenyl
carbazate, CH3CN/MeOH/HOAc 10:2:1, room temperature, 18 h, 32%; (viii) amine, DMF, DiPEA, room temperature; (ix) DCM/HOAc
10:1, room temperature, 18 h (71%); (x) aniline, room temperature, 8 h (83%); (xi) aq NH3/MeOH (61%).
selective agonists for the human adenosine A1 receptor.
These data show that proper substitution at the C2-
position yields selective agonists for this receptor. In
contrast to small substituents such as chlorine and NO2,
the compounds we present here contain large substit-
uents at the C2-position. Combined substitution at C2
and N6 further improves this selectivity.
the adenosine 2-position. In particular, the key precur-
sor 6-chloro-2-nitropurine riboside 1 is readily obtained
from inosine, via nitration of 6-chloropurine riboside
triacetate at the purine 2-position using the tetrabutyl-
ammonium nitrate/trifluoroacetic anhydride combina-
tion (Scheme 1).14 Azidation of the 6-position in com-
pound 1 followed by redox conversion of the nitro to the
nitroso group produced crystalline 2.15 The exceptional
reactivity of this nitroso functionality allows condensa-
tion with a variety of nitrogen nucleophiles. The first
example shown is the acetic acid-catalyzed Mills reac-
Resu lts a n d Discu ssion
Ch em istr y. 2-Nitrosoadenosine is a valuable precur-
sor for the attachment of nitrogen-based side chains to

1494 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Sch em e 2. Synthesis of Semicarbazidesa
Beukers et al.
(Scheme 1). A condensation reaction of 2 with both
hydrazides proceeded in high yield, but removal of the
acetate protecting groups with ammonia resulted in
fragmentation of the triazene. Because deprotection
under milder conditions (e.g., KCN in methanol) did not
prevent this type of decomposition, the deprotection step
was avoided by using unprotected 2-nitrosoadenosine
10. This nitroso compound was prepared in a three-step
process from 2 by protection of the nitroso functional-
ity: Diels-Alder reaction with cyclopentadiene, deac-
ylation, and retro-Diels-Alder.15 Condensation of 10
with benzoylhydrazide and phenyl carbazate directly
produced the anticipated triazenes 11 and 12.
2-Triazenyl-functionalized adenosines with an ad-
ditional substituent at the N6-position were synthesized
from 6-chloro-2-nitropurine riboside 1 (Scheme 1). Ami-
nation of 1 with aniline, cyclopentylamine, and 2,2-
diphenylethylamine gave N6-substituted adenosines
13a -c, respectively.13 Nitroso derivatives 14a -c were
produced in good yield following the nitro to nitroso
reduction/oxidation sequence already applied for the
synthesis of 2. Acetic acid-catalyzed condensation be-
tween 14a -c and semicarbazide 6a or 4-phenylsemi-
carbazide 6d followed by removal of the acetates gave
N6-substituted triazenes 16a -d . In particular, for the
synthesis of TCPA the condensation step to 15c was low
yielding as a result of nitroso to hydroxylamine reduc-
tion induced by 4-phenylsemicarbazide. An improved
synthetic procedure was developed for TCPA by triazene
formation from the nitroso derivative 14b (R2 ) cyclo-
pentyl) with p-nitrophenyl carbazate18 instead of 4-phe-
nylsemicarbazide (Scheme 1). The electron-withdrawing
p-nitrophenyl substituent further reduces the reduction
potential of the hydrazine without noticeably affecting
its nucleophilic properties. Reaction of 17 with aniline
followed by aminolysis of the acetate protecting groups
produced TCPA (16c) in an acceptable 24% yield over
six steps.
a
Reagents and conditions: (i) NH2NH2‚H2O, DCM, -60 °C to
room temperature; (ii) (PhO)2CO, neat, 100-150°C; (iii) NH2-
NH2‚H2O, DCM, rT, 18 h; (iv) (PhO)2P(O)N3, DiPEA, DCM, room
temperature; (v) PhOH, CH3CN, reflux, 18 h, then NH2NH2.HBr,
Et3N, room temperature, 2 h.
tion of 2 with aniline, producing 2-phenyldiazoadenosine
3 in good yield.
Elongation of the two-atom side chain was first
examined by condensation of 2 with phenylhydrazine.
The strong reducing properties of this type of electron-
rich hydrazines, however, completely converted the
nitroso group in 2 to hydroxyamine 5, whereas conden-
sation products could not be isolated. Introduction of
electron-withdrawing substituents such as trifluoro-
methyl and cyano in the phenyl ring of the hydrazine
did not give any improvement.
Satisfactory results were obtained with 4-phenylsemi-
carbazide 6d as a nucleophile in an acetic acid-catalyzed
condensation reaction, producing phenylcarbamoyl-
substituted triazene 7d in 63% yield. Attachment of a
carbonyl group directly to the hydrazine obviously
decreases the reducing properties of the hydrazine
without affecting its nucleophilicity. Only a few ex-
amples of reactions between nitrosobenzenes and semi-
carbazides are known in the literature, and it appears
that the favorable reactivity of 2 makes this reaction a
success.16,17 Removal of the acetates from the ribose in
7d was readily accomplished with a mixture of methanol
and aqueous ammonia.
A series of 4-substituted semicarbazides 6a -l was
synthesized from hydrazine hydrate and the corre-
sponding isocyanates, as is shown in Scheme 2. Because
thiazole-2-isocyanate is not commercially available, 4-(2-
thiazolyl)semicarbazide 6l was prepared from 2-ami-
nothiazole in two steps. Furylsemicarbazide 9 could be
obtained from furan-2-carboxylic acid via Curtius rear-
rangement. Condensation of the 4-substituted semicar-
bazides 6a -l with 2 gave triazenes 7a -l in 43-63%
yield, and deprotection with aqueous ammonia fur-
nished the corresponding triazenes 8a -l (60-93%
yield). 4-Furylsemicarbazide 9 formed the only excep-
tion: instead of the anticipated condensation reaction,
a 4+2 hetero-Diels-Alder reaction occurred between the
furan ring and the nitroso group, leading to extensive
decomposition. This type of cycloaddition has been
described before in the literature.15
Sta bility Stu d ies. Because some alkyl- and aryltria-
zenes are known to decompose slowly in solution, we
examined the stability of the carbamoyl-substituted
triazenes. No decomposition of N6-unsubstituted tria-
zene 8d and N6-cyclopentyl-substituted triazene 16c
(TCPA) was found in a 9:1 mixture of 0.05 M phosphate
buffer in D2O (pD 7.8) and DMSO-d6 at 25 °C over a
period of 5 days, as was monitored by 1H NMR. The
stability of these compounds might be due to the
presence of electron-withdrawing substituents on both
sides of the triazene linker. Heating 8d and 16c at 80
°C in aqueous DMSO was necessary before formation
of aniline and 2,6-diaminopurine riboside 18 or 2-amino-
CPA 19 occurred, as is shown in Scheme 3. In addition,
the photostability of the triazene functionality in these
compounds was examined. After DMSO-d6 solutions of
8d and 16c had been exposed to daylight for several
days or to irradiation at 350 nm, no decomposition was
observed (1H NMR analysis).
Interestingly, NMR analysis of aqueous solutions of
compound 4 shows the presence of a Z/E ) 41/59
mixture in diffuse daylight; the compound was tested
as such. E to Z isomerization of the NdN double bond
has never been observed for the triazenes. In, for
example, NMR spectroscopy, only the most stable E-
isomer is observed.
Two additional carbonyl-substituted hydrazines, ben-
zoylhydrazide and phenyl carbazate, were examined

TCPA, a Selective Agonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1495
Sch em e 3. Stability Studies
8d was selective for the human adenosine A1 receptor
with a 4-fold selectivity for A1 versus A2A and an 18-
fold selectivity for A1 versus A3.
On the basis of these data, the side chain of 8d was
investigated in greater detail as shown in Table 2.
Besides the phenyl substituent (as in 8d ) also several
other substituents R1 as well as the unsubstituted (8a )
compound were tested for their affinity for the human
adenosine receptors. Within this series the phenyl (8d )
and the cyclohexyl (8c) compounds had the highest
affinity for the adenosine A1 receptor, 6.1 and 5.8 nM,
respectively. The unsubstituted compound (8a ) had a
strongly reduced affinity of 140 nM for this receptor.
Introduction of an ethyl, benzyl, or thiazole substituent
led to a 4-8-fold decrease in affinity. Substitution of
the phenyl ring with either a chlorine or methoxy group
resulted in a slight 2-4-fold decrease in affinity. Com-
pound 8j with a trifluoromethyl group at the meta
position of the phenyl ring was insoluble in our ligand
binding assay buffers. Replacement of one of the nitro-
gen atoms of 8d with oxygen resulted in the ester
analogue 12. This alteration was detrimental to the
affinity of this compound for the adenosine A1 receptor.
Biologica l Eva lu a tion . All compounds were tested
in radioligand binding assays to determine their affinity
for the human adenosine receptors. The tritiated an-
tagonist [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-
DPCPX) was used for human adenosine A1 and A2B
receptors, whereas [3H]-7-amino-2-(2-furyl)-5-[2-(4-hy-
droxyphenyl)ethyl]amino[1,2,4]triazolo[1,5-a ][1,3,5]-
triazene ([3H]ZM241385) was used as a tritiated an-
tagonist for human adenosine A2A receptors. Due to the
lack of commercially available radiolabeled antagonists,
we used I125-labeled N6-(4-amino-3-iodobenzyl)-5′-me-
thylcarboxamidoadenosine ([125I]IABMECA) as a radio-
labeled agonist for human adenosine A3 receptors. As
we used an antagonist radioligand to determine the
affinities of the agonists for the adenosine A1 and A2A
receptors, the Ki values represent a combined value for
both the high- and low-affinity states of the receptors.
The Ki values for the adenosine A3 receptor, on the other
hand, represent only the high-affinity state as we used
an agonist radioligand to study this receptor.
A different pattern was obtained with the adenosine
A2A receptor. This receptor had no preference for either
a phenyl, ethyl, cyclohexyl, benzyl, or thiazole group at
the R1 position, whereas the unsubstituted compound
had a 4-fold lower affinity compared to the phenyl-
substituted ligand. Substitution of the phenyl group of
8d was allowed at the ortho position with either a
chlorine or a methoxy group or at the para position with
a methoxy group and resulted in a 5-fold drop in affinity
at most. In contrast, introduction of a chlorine substitu-
ent at the meta or para position was not allowed. The
ester compound 12 had a very low affinity for the
adenosine A2A receptor.
The adenosine A3 receptor was relatively insensitive
to the nature of the R1 substituent, with a maximal 2-6-
fold change in affinity. Substitution of the phenyl ring
was also allowed and resulted in maximal changes in
affinity of 3-fold. Even the replacement of nitrogen with
oxygen in the spacer as in 12 was tolerated by this
receptor.
None of the investigated C2-substituted compounds
displayed marked affinity for the adenosine A2B recep-
tor. The highest affinity was obtained with thiazolyl-
carbamoyltriazenyladenosine (8l) and resulted in a
displacement of 79% of radiolabeled [3H]DPCPX at a
concentration of 10 µM. At a concentration of 1 µM 8l
was able to displace 42% of the radioligand, suggesting
that the affinity of 8l is ∼1 µM. The affinity of 8l is
comparable to the affinity of NECA (2.2 ( 0.6 µM; see
also Table 2), the reference agonist for the adenosine
A2B receptor.19
Substitution of adenosine at the N6-position together
with a halogen residue at the C2-position has yielded
selective high-affinity agonists, such as CCPA, for the
adenosine A1 receptor. However, substituents other than
halogens at this position are generally detrimental for
binding to the adenosine A1 receptor.20 In addition,
C2,N6-disubstituted adenosines with an alkyl chain at
the N6-position have a lower affinity compared to the
equivalent C2 monosubstituted compounds.21 Introduc-
tion of a cyclopentyl group at the N6-position, however,
Our encouraging results to achieve selective adeno-
sine A1 receptor ligands by introducing an NO2 sub-
stituent at the C2-position of adenosine prompted us to
explore the C2-position in further detail. The NO2
substituent was converted to 2-nitrosoadenosine to
obtain a suitable precursor for the attachment of
nitrogen-based side chains (Table 1).
Whereas the adenosine A3 receptor was relatively
insensitive to the elongation of the side chain, the A1,
A2A, and, to a lesser degree, A2B receptors preferred
compound 8d with the phenylcarbamoyltriazenyl chain.
Extension of the side chain from nitroso (10), via
diazophenyl (4) and benzoyltriazenyl (11), to phenyl-
carbamoyltriazenyl (8d ) led to a progressive increase
in affinity for the adenosine A1 receptor. Within this
series the introduction of an extra nitrogen atom, to
obtain 8d , was most effective and resulted in a 311-fold
increase in affinity as compared to 11. A different
pattern was obtained for the other three adenosine
receptors. Neither the adenosine A2B receptor nor the
adenosine A3 receptor was very sensitive (5-fold affinity
change at most) to these C2 substituents. On the other
hand, the nitroso-substituted (10) and the benzoyltria-
zenyl-substituted (11) analogues had a weak affinity for
the adenosine A2A receptor, whereas the diazophenyl-
substituted (4) compound had a slightly higher affinity.
In analogy with the adenosine A1 receptor, the major
affinity increase stemmed from the elongation of the
spacer with an extra nitrogen atom (8d ). As a result,

1496 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Beukers et al.
Ta ble 1. Affinity of 2-Nitrosoadenosine and Analogues for the Human Adenosine A1, A2A, A2B, and A3 Receptors As Determined in
Radioligand Binding Studies
Ki ( SEM in nM (n ) 3) or % displacement at 10 µM (n ) 2)
compd
R
A1
A2A
A3
A2B
14%
11%
8%
10
4
11
8d
OdN-
40%
43%
720 ( 60
180 ( 10
1000 ( 160
25 ( 3
580 ( 50
110 ( 40
190 ( 30
110 ( 20
phenyl-NdN-
phenyl-CO-NH-NdN-
phenyl-NH-CO-NH-NdN-
1900 ( 2100
6.1 ( 1.3
54%
Ta ble 2. Affinities for Human Adenosine Receptors As Determined in Radioligand Binding Studies
Ki ( SEM in nM (n ) 3) or % displacement at 10 µM (n ) 2)
compd
R1
R2
A1
A2A
A3
A2B
8a
8b
8c
8d
8e
8f
8g
8h
8i
H
ethyl
cyclohexyl
phenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
140 ( 34
45 ( 17
5.8 ( 1.8
6.1 ( 1.3
15 ( 11
15 ( 4
88 ( 12
19 ( 10
25 ( 3
209 ( 62
113 ( 21
63 ( 17
9%
15%
6%
54%
21%
64%
30%
15%
18%
insoluble
9%
6%
79%
10%
9%
10%
10%
25 ( 3
110 ( 20
39 ( 6
4-Cl-phenyl
3-Cl-phenyl
2-Cl-phenyl
4-MeO-phenyl
2-MeO-phenyl
3-CF3-phenyl
benzyl
4.4%
29.9%
45 ( 19
25 ( 1
125 ( 49
insoluble
25 ( 11
38.9%
25 ( 1
17.7%
59 ( 25
23 ( 8
179 ( 33
73 ( 11
20 ( 10
20 ( 3
insoluble
42 ( 15
43%
81 ( 13
8j
insoluble
147 ( 18
577 ( 138
610 ( 340
175 ( 57
90 ( 12
8k
12
8l
16a
16b
16c, TCP A
16d
CCP A
NECA
O-phenyla
thiazole
H
phenyl
phenyl
24 ( 1
phenyl
phenyl
cyclopentyl
Ph2CHCH2
204 ( 136
430 ( 132
2.8 ( 0.8
3.1 ( 0.9
6.4 ( 1.8
12 (9.6-15)b
24.3%
210 ( 20
980 ( 110
639 ( 55
60 ( 10
600 ( 230
140 ( 20
281 ( 5622
11 ( 0.8
phenyl
2200 ( 600
a
b
O-phenyl instead of NH-phenyl. The Ki value of NECA for the human adenosine A1 receptor was performed in duplicate.
partially restores the affinity of these disubstituted
compounds for the adenosine A1 receptor.20 These data
suggest that the binding sites of C2 and N6 substituents
might partially overlap. To test this hypothesis, we
analyzed carbamoyltriazenyladenosine with a phenyl
group at the N6-position (16a ) and compared the affinity
of this compound with that of compound 8d , in which
the phenyl group is present on the triazenyl chain
rather than the N6-position. Compound 16a had a
reduced affinity for the adenosine A1 (33-fold) as well
as the adenosine A2A receptor (>400-fold), whereas the
affinity for the adenosine A3 receptor was hardly af-
fected (2-fold). These data suggest that the C2 and N6
substituents may occupy different binding sites. More-
over, compound 8a , which lacks a phenyl group at both
the C2- and N6-positions, has an affinity comparable to
that of 16a for the adenosine A1 receptor, hence sug-
gesting that disubstitution may be allowed for the
adenosine A1 receptor. Indeed, introduction of a phenyl
group at the N6-position of 8d yielded compound 16b
with an affinity comparable to that of compound 16a
for the adenosine A1 receptor. Substitution of the N6-
position with a big substituent, diphenylethyl, further
demonstrates that the binding sites of N6 and C2
substituents are different. This compound, 16d , had a
high affinity for the adenosine A1 receptor, whereas the
affinity for the A2A receptor dropped considerably com-
pared to that of 8d . The affinity of 16d for the A3
receptor was comparable to those of 8d and 16a . To
exploit the preference of this disubstituted compound
for the adenosine A1 receptor, we also introduced the
A1 selective cyclopentyl group at the N6-position (16c,
TCPA). TCPA had indeed a high, 2.8 nM, affinity for
the adenosine A1 receptor. As anticipated on the basis
of radioligand binding data in the rat,20 the affinity of
TCPA for the adenosine A2A receptor was somewhat
higher compared to that of the diphenylethyl-substitut-
ed compound (16d ). The 5-fold drop in affinity for the

TCPA, a Selective Agonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1497
Ta ble 3. Comparison of the Relative Potency and Selectivity of
TCPA versus CCPA and NECA for the Human Adenosine A1,
A
2A, and A3 Receptorsa
relative potency
selectivity
compd A1 receptor A2A receptor A3 receptor A1/A2A A1/A3
TCPA
CCPA
NECA
1 (2.8)
0.4 (6.4)
0.2 (12)
1 (210)
0.3 (639)
3.5 (60)
1 (600)
2.1 (281)
55 (11)
75
100
5
214
44
0.9
a
The potency of TCPA for the three human adenosine receptors
is set at 1. Actual Ki value in nM is presented in parentheses.
adenosine A3 receptor, as compared to 8d , improved the
selectivity of TCPA for the adenosine A1 receptor. The
disubstituted compounds had virtually no affinity for
the adenosine A2B receptor.
F igu r e 1. Representative curves of the inhibition of forskolin-
induced (10 µM) cAMP production by CCPA (0) and TCPA (9)
in CHO cells stably expressing the adenosine A1 receptor. The
amount of cAMP formed is shown. Basal cAMP production was
0.6 pmol/2 × 105 cells. The amount of cAMP formed by 10 µM
forskolin amounted to 10.1 pmol/2 × 105 cells. After inhibition
by CCPA and TCPA, cAMP levels were reduced to 3.9 and 3.8
pmol/2 × 105 cells, respectively.
species that was studied. Thus, 2-chloro-N6-(3-iodoben-
zyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA) is
a very selective agonist for the rat adenosine A3 receptor
versus the rat adenosine A1 (2485-fold) and the rat
adenosine A2A receptor (1424-fold).23 However, consider-
ing the human receptors Cl-IB-MECA is only 10-fold
(versus A1) and 191-fold (versus A2A) selective.24 In this
study we determined the affinity of a series of C2-
substituted and C2,N6-disubstituted analogues for the
human adenosine receptors. When the affinities for all
four human adenosine receptors are compared, TCPA
turns out to have the best overall selectivity of the
agonists known to date.
The cyclopentyl- and diphenylethyl-disubstituted com-
pounds, 16c and 16d , respectively, are the most selec-
tive ligands for the human adenosine A1 receptor known
to date due to the improved selectivity with respect to
the adenosine A3 receptor. As can be deduced from Table
2, these compounds are 214- and 45-fold selective for
the adenosine A1 receptor, respectively. N6-Cyclopen-
tyladenosine, on the other hand, has affinities of 10.2
( 1.3 nM for the human A1 receptor and 281 ( 56 nM
for the human A3 receptor, resulting in a 28-fold
selectivity for the adenosine A1 receptor versus the A3
receptor. 2-Chloro-N6-cyclopentyladenosine (CCPA) has
affinities of 6.4 ( 1.8, 639 ( 55, and 281 ( 56 nM22 for
the human adenosine A1, A2A, and A3 receptors, respec-
tively (see Table 2). Hence, CCPA is 44-fold selective
for the human adenosine A1 receptor versus the human
A3 receptor. Although CPA and CCPA are more selective
toward the human A2A receptor than TCPA and 16d ,
the latter compounds are still 75- and 316-fold selective
for A1 versus A2A. As we used an agonist radioligand to
study ligand binding to the adenosine A3 receptor, and
an antagonist radioligand to study binding to the
adenosine A1 and A2A receptors, the selectivity toward
the adenosine A3 receptor will be underestimated. As a
reference, we therefore present the values for the
prototypic agonist NECA in Table 2. In our hands,
Con clu sion s
The 2-NO2 analogue of adenosine was converted to
2-nitrosoadenosine to obtain a suitable precursor for the
attachment of nitrogen-based side chains. Extension of
the nitroso group to diazophenyl, benzoyltriazenyl, and
phenylcarbamoyltriazenyl resulted in a compound (8d )
with low nanomolar affinity for the human adenosine
A1 and A2A receptors. Several analogues of 8d were
made, and biological evaluation of these compounds
demonstrated that the phenyl substituent was the most
appropriate group to achieve a high-affinity adenosine
A1 receptor ligand. To further improve the slight prefer-
ence of 8d for the adenosine A1 receptor, N6,C2-disub-
stituted compounds were synthesized. The introduction
of diphenylethyl at the N6-position yielded a very
selective compound for the human adenosine A1 receptor
with 316-fold preference over A2A and 45-fold preference
over A3. The disubstituted N6-cyclopentyl-2-phenylcar-
bamoyltriazene analogue (16c, TCPA) was very selec-
tive, 75-fold, with respect to the human adenosine A2A
receptor. Moreover, TCPA turned out to be the most
selective agonist for human adenosine A1 receptors
versus human adenosine A3 receptors known to date.
Its selectivity for A1 over A3 was 214-fold compared to
the 44-fold selectivity of the reference A1 agonist CCPA.
This selectivity is most likely underestimated due to the
fact that we used an agonist radioligand to study ligand
binding to the adenosine A3 receptor, whereas an
antagonist radioligand was used to study binding to the
NECA had Ki values for the human adenosine A1, A2A
,
A2B, and A3 receptors of 12 nM (9.6-15 nM; n ) 2) and
60 (10, 11 ( 0.8, and 2200 ( 600 nM, respectively. A
comparison of the relative potency and selectivity of
TCPA versus CCPA and NECA for the human adenos-
ine A1, A2A, and A3 receptors is presented in Table 3.
To verify that TCPA behaves as an agonist, cAMP
experiments were performed. Figure 1 shows the inhibi-
tion of forskolin-induced cAMP formation in CHO cells
stably expressing the adenosine A1 receptor by TCPA
and CCPA. Both compounds inhibited the cAMP forma-
tion to a similar extent with IC50 values of 1.5 ( 0.5
and 1.3 ( 1.1 nM for TCPA and CCPA, respectively.
Hence, in our cAMP assay TCPA is a full agonist like
CCPA.
The selectivity of ligands for the adenosine receptors
was challenged by two discoveries. First of all, the
discovery of the adenosine A3 receptor in 199110,11
showed that many ligands with a high affinity for either
the human adenosine A1 receptor (e.g., CPA or CCPA)
or the human adenosine A2A receptor (e.g., CGS21680)
had a relatively high affinity for the human adenosine
A3 receptor as well. Second, the affinity of ligands for
the adenosine A3 receptor depended strongly upon the

1498 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Beukers et al.
4-(3-Tr iflu or om eth ylp h en yl)sem ica r ba zid e 6j: yield,
2.00 g (91%), mp 98-99 °C; 1H NMR (DMSO-d6) δ 9.02 (s, 1H,
N-H), 8.11 (broad, 1H, Ar), 7.76 (m, 1H, Ar), 7.61 (s, 1H,
N-H), 7.46 (t, 1H, J ) 7.8 Hz, Ar), 7.25 (d, 1H, J ) 7.8 Hz,
Ar), 4.41 (broad, 2H, NH2).
adenosine A1 and A2A receptors. In addition, TCPA
turned out to be a full agonist, just like CCPA, and
inhibited cAMP production in CHO cells stably trans-
fected with the adenosine A1 receptor with an IC50 of
1.5 ( 0.5 nM.
4-Ben zylsem ica r ba zid e 6k : yield, 1.19 g (72%), mp 110-
1
112 °C; H NMR (DMSO-d6) δ 7.2-7.35 (m, 5H, Ar), 7.03 (s,
Exp er im en ta l Section
1H, N-H), 6.83 (broad, 1H, N-H), 4.26 (d, 1H, J ) 6.0 Hz,
CH2Ph), 4.15 (broad, 2H, NH2).
Ch em ica ls a n d Solven ts. All reagents and solvents were
used as commercially available, unless indicated otherwise.
Adenosine deaminase was from Boehringer Mannheim (Mann-
heim, Germany), and CGS15943 was a gift from Dr. M.
Williams and Dr. J . Watthey (Ciba-Geigy, Summit, NJ ). CPA
was obtained from RBI Research Chemicals (Natick, MA), and
R-PIA was obtained from Sigma (St. Louis, MO).
Ch r om a togr a p h y. Thin-layer chromatography (TLC) was
carried out using silica-coated plastic sheets (Merck silica gel
60 F254). Spots were visualized under UV (254 nm). Flash
chromatography refers to purification using the indicated
eluents and J anssen Chimica silica gel 60 (0.030-0.075 mm).
In str u m en ts a n d An a lysis. Proton nuclear magnetic
resonance (1H NMR) spectra and carbon nuclear magnetic
resonance (13C NMR; APT) spectra were determined in CDCl3
at 300 K using a Bruker ARX 400 (400 and 100 MHz,
respectively) spectrometer. Mass spectra and accurate mass
measurements were performed on a J EOL J MS-SX/SX 102 A
tandem mass spectrometer using fast atom bombardment
(FAB) or electron impact (EI). A resolving power of 10000 (10%
valley definition) for high-resolution electron impact or FAB
mass spectrometry was used. Melting points were measured
with a Leitz melting point microscope.
Gen er a l P r oced u r e for th e Syn th esis of Sem ica r ba -
zid es 6b-k . The appropriate isocyanate (10.0 mmol) was
added to a mixture of hydrazine hydrate (0.485 mL, 10.0 mmol)
and DCM (distilled from P2O5, 25 mL) at -60 °C. The bath
was removed and the suspension was stirred for 4 h at room
temperature, cooled in ice, and filtered. Additional product was
obtained by concentrating the mother liquor. Noncrystalline
semicarbazides were obtained by evaporation of the volatiles.
4-Eth ylsem ica r ba zid e 6b: yield, 0.91 g (88%), obtained
as a pure oil after evaporation; 1H NMR (DMSO-d6) δ 6.15
(broad, 1H, N-H), 6.0 (broad, 1H, N-H), 3.6 (broad, 2H, NH2),
3.26 (m, 2H, CH2), 1.1 (t, 3H, J ) 7.2 Hz, CH3).
4-Cycloh exylsem ica r ba zid e 6c: yield, 1.35 g (86%), crys-
tallized from ethyl acetate after evaporation of the DCM, mp
100-105°C; 1H NMR (DMSO-d6) δ 7.40 (s, 1H, N-H), 5.98 (d,
1H, J ) 7.1 Hz, N-H), 4.05 (m, 1H, CHNH), 3.60 (broad, 2H,
NH2), 1.0-1.8 (m, 10H, cyclohexyl).
4-(2-Th ia zolyl)sem ica r ba zid e 6l. A solution of N-(phen-
oxycarbonyl)-2-aminothiazole (1.1 g, 5 mmol, prepared from
2-aminothiazole25) and hydrazine hydrate (0.485 mL, 10 mmol)
in DCM (25 mL, distilled from P2O5) was stirred at room
temperature for 18 h. Methanol (5 mL) was added, and
insoluble material was removed by hot filtration. Evaporation
of the filtrate and crystallization of the residue from DCM/
ether gave 6e (0.54 g (68%), mp 162-165 °C; 1H NMR (DMSO-
d6) δ 10.2 (broad, 1H, N-H), 8.1 (broad, 1H, N-H), 7.34 (d,
1H, J ) 3.5 Hz, thiazole), 7.06 (d, 1H, J ) 3.5 Hz, thiazole),
4.25 (broad, 2H, NH2).
4-(2-F u r yl)sem ica r ba zid e 9. Diphenylphosphoryl azide
(2.14 g, 10 mmol) was added to a solution of furan-2-carboxylic
acid (1.12 g, 10 mmol) and DiPEA (1.918 mL, 11 mmol) in
DCM (20 mL, distilled from P2O5). Extractive workup and
crystallization from diethyl ether/light petroleum gave the
moderately stable furan-2-carbonyl azide (1.23 g, 90%), mp
58-59 °C; 1H NMR (DMSO-d6) δ 8.12 (m, 1H, furan), 7.47 (m,
1H, furan), 6.78 (m, 1H, furan). A solution of this azide (0.411
g, 3.0 mmol) and phenol (0.564 g, 6.0 mmol) in CH3CN (10
mL, anhydrous) was refluxed under N2 during 18 h. The
reaction mixture was cooled to room temperature and hydra-
zine‚HBr (0.678 g, 6 mmol) and triethylamine (1.6 mL, 12
mmol) were added. After 4 h of stirring at room temperature,
the solvent was evaporated and the residue purified by
chromatography (ethyl acetate/methanol 9:1) to give 9 (0.180
1
g, 43%) as a solid, mp >150 °C (dec); H NMR (DMSO-d6) δ
8.85 (broad, 1H, N-H), 7.50 (broad, 1H, N-H), 7.26 (m, 1H,
furan), 6.38 (m, 1H, furan), 5.93(m, 1H, furan), 4.35 (broad,
2H, NH2).
Gen er a l P r oced u r e for th e Syn th esis of Aceta te-
P r otected Tr ia zen es 7a -l. To a solution of 2-nitroso-2,3,5-
tri-O-acetyladenosine 2 (42 mg, 0.10 mmol) in a mixture of
acetonitrile (2 mL) and acetic acid (0.2 mL) was added
semicarbazide 6a -l (0.20 mmol), and the mixture was stirred
at room temperature during 4 h. The reaction mixture was
diluted with ethyl acetate (10 mL) and stirred with aqueous
NaHCO3 (5%, 10 mL) for 30 min. Extractive workup and flash
chromatography with 4% methanol in ethyl acetate produced
protected triazenes 7a -l as glass. The products were analyzed
4-P h en ylsem ica r ba zid e 6d : yield, 1.22 g (81%), mp 122-
123 °C; 1H NMR (DMSO-d6) δ 8.75 (s, 1H, N-H), 7.40 (s, 1H,
N-H), 7.2-7.8 (m, 5H, Ar), 4.35 (broad, 2H, NH2).
4-(4-Ch lor op h en yl)sem ica r ba zid e 6e: yield 1.65 g (89%),
mp 190 °C (dec); 1H NMR (DMSO-d6) δ 8.78 (s, 1H, N-H),
7.58 (m, 2H, Ar), 7.48 (s, 1H, N-H), 7.30 (m, 3H, Ar), 4.37
(broad, 2H, NH2).
4-(3-Ch lor op h en yl)sem ica r ba zid e 6f: yield, 0.95 g (51%),
mp 107-108 °C after recrystallization from DCM/ether; 1H
NMR (DMSO-d6) δ 8.85 (s, 1H, N-H), 7.81 (s, 1H, Ar), 7.55
(s, 1H, N-H), 7.41 (m, 1H, Ar), 7.25 (t, 1H, J ) 8.1 Hz, Ar),
6.97 (m, 1H, Ar), 4.39 (broad, 2H, NH2).
1
by H NMR and directly used in the deprotection step.
2′,3′,5′-Tr i-O-a cetyl-2-(3-a m in oca r bon yltr ia zen e-1-yl)-
a d en osin e 7a : yield, 30 mg (63%) after chromatography with
1
10% methanol in ethyl acetate; H NMR (DMSO-d6) δ 12.25
(broad, 1H, N-H), 8.37 (s, 1H, H-8), 7.61, (broad, 2H, NH2),
6.5-7.3 (broad, 2H, NH2), 6.21 (d, 1H, J ) 5.1 Hz, H-1′), 6.03
(m, 1H, H-2′), 5.66 (m, 1H, H-3′), 5.2-5.45 (m, 3H, H-4′, H-5′),
2.16, 2.15, and 2.07 (all 3H, s, acetates).
2′,3′,5′-Tr i-O-a cetyl-2-(3-eth yla m in oca r bon yltr ia zen e-
1-yl)a d en osin e 7b: yield, 25.7 mg (51%) after chromatography
with 7% methanol in ethyl acetate; 1H NMR δ 12.3 (broad,
1H, N-H), 8.00 (s, 1H, H-8), 7.04 (s, 1H, N-H), 6.7-7.2 (broad,
2H, NH2), 6.34 (m, 1H, H-2′), 6.09 (d, 1H, J ) 4.4 Hz, H-1′),
5.72 (m, 1H, H-3′), 4.3-4.5 (m, 3H, H-4′, H5′), 3.47 (m, 2H,
CH2CH3), 2.13, 2.09, and 2.02 (all 3H, s, acetates), 1.26 (t, 3H,
J ) 7.2 Hz, CH3).
4-(2-Ch lor op h en yl)sem ica r ba zid e 6g: yield, 1.39 g (75%),
mp 110-112 °C after recrystallization from ether/light petro-
1
leum; H NMR (DMSO-d6) δ 9.15 (s, 1H, N-H), 8.30 (m, 1H,
Ar), 7.81 (s, 1H, N-H), 7.44 (m, 1H, Ar), 7.29 (m, 1H, Ar),
6.98 (m, 1H, Ar), 4.70 (broad, 2H, NH2).
4-(4-Meth oxyp h en yl)sem ica r ba zid e 6h : yield, 1.59 g
(88%), mp 145-146 °C; 1H NMR (DMSO-d6) δ 8.45 (s, 1H,
N-H), 7.41 (d, 2H, J ) 8.7 Hz, Ar), 7.29 (s, 1H, N-H), 8.82
(d, 2H, J ) 8.7 Hz, Ar), 4.31 (s, 2H, NH2), 3.71 (s, 3H, CH3O).
4-(2-Meth oxyp h en yl)sem ica r ba zid e 6i: yield, 1.60 g
(88%), mp 155-157 °C; 1H NMR (DMSO-d6) δ 8.90 (s, 1H,
N-H), 8.17 (d, 1H, J ) 7.0 Hz, Ar), 7.62 (s, 1H, N-H), 6.8-
7.2 (m, 3H, Ar), 4.52 (broad, 2H, NH2).
2′,3′,5′-Tr i-O-a cetyl-2-(3-cycloh exyla m in oca r bon yltr ia -
zen e-1-yl)a d en osin e 7c: yield, 32.9 mg (59%) after chroma-
tography with 4% methanol in ethyl acetate; H NMR δ 11.9
(s, 1H, N-H), 8.02 (s, 1H, H-8), 7.0 (broad, 2H, NH2), 6.70
(broad, 1H, N-H), 6.23 (m, 1H, H-2′), 6.16 (d, 1H, J ) 5.4 Hz,
H-1′), 5.63 (m, 1H, H-3′), 4.3-4.5 (m, 3H, H-4′, H5′), 3.80 (m,
1H, CHNH), 2.14, 2.07, and 2.04 (all 3H, s, acetates), 1.0-1.9
(m, 10H, cyclohexyl).
1

TCPA, a Selective Agonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1499
2′,3′,5′-Tr i-O-acetyl-2-(3-ph en ylam in ocar bon yltr iazen e-
1-yl)a d en osin e 7d : yield, 30.0 mg (54%); H NMR (DMSO-
6.9-7.4 (broad, 2H, NH2), 5.9 (broad, 1H, OH), 5.85 (d, 1H, J
) 6.8 Hz, H-1′), 5.45 (d, 1H, J ) 6.4 Hz, OH), 5.22 (d, 1H, J )
4.0 Hz, OH), 4.69 (m, 1H, H-2′), 4.13 (m, 1H, H-3′ or H-4′),
1
d6) δ 12.8 (broad, 1H, N-H), 10.1 (broad, 1H, N-H), 8.36 (s,
1H, H-8), 7.65 (m, 4H, Ar-Hortho, NH2), 7.35 (t, 2H, J ) 7.7,
Ar-Hmeta), 7.09 (t, 1H, J ) 7.6 Hz, Ar-Hpara), 6.21 (d, 1H, J )
5.5 Hz, H-1′), 5.99 (m, 1H, H-2′), 5.67 (m, 1H, H-3′), 4.3-4.5
(m, 3H, H-4′, H-5′), 2.10, 2.06, and 1.99 (all 3H, s, acetates).
2′,3′,5′-Tr i-O-a cetyl-2-[3-(4-ch lor op h en yl)a m in oca r bo-
n yltr ia zen e-1-yl]a d en osin e 7e: yield, 29.5 mg (50%); 1H
NMR (DMSO-d6) δ 12.8 (broad, 1H, N-H), 10.0 (broad, 1H,
N-H), 8.36 (s, 1H, H-8), 7.69 (m, 2H, Ar), 7.66 (s, 2H, NH2),
7.40 (d, 2H, J ) 8.7 Hz, Ar), 6.21 (d, 1H, J ) 5.3 Hz, H-1′),
5.98 (m, 1H, H-2′), 5.67 (m, 1H, H-3′), 4.3-4.5 (m, 3H, H-4′,
H-5′), 2.10, 2.06, and 1.99 (all 3H, s, acetates).
2′,3′,5′-Tr i-O-a cetyl-2-[3-(3-ch lor op h en yl)a m in oca r bo-
n yltr ia zen e-1-yl]a d en osin e 7f: yield, 32.0 mg (54%); 1H
NMR δ 12.1 (broad, 1H, N-H), 8.8 (broad, 1H, N-H), 8.03 (s,
1H, H-8), 7.1-7.7 (m, 4H, Ar), 6.0-6.2 (m, 4H, H-1′, H-2′, NH2),
5.72 (m, 1H, H-3′), 4.3-4.5 (m, 3H, H-4′, H-5′), 2.07, 2.05, and
2.04 (all 3H, s, acetates).
2′,3′,5′-Tr i-O-a cetyl-2-[3-(2-ch lor op h en yl)a m in oca r bo-
n yltr ia zen e-1-yl]a d en osin e 7g: yield, 30.0 mg (51%); 1H
NMR δ 13.2 (broad, 1H, N-H), 9.03 (broad, 1H, N-H), 8.27
(d, J ) 7.7 Hz, Ar), 8.08 (s, 1H, H-8), 7.0-7.3 (m, 6H, Ar, NH2),
6.22 (d, 1H, J ) 5.1 Hz, H-1′), 5.94 (m, 1H, H-2′), 5.65 (m, 1H,
H-3′), 4.3-4.5 (m, 3H, H-4′, H-5′), 2.08, 2.07, and 2.03 (all 3H,
s, acetates).
2′,3′,5′-Tr i-O-a cetyl-2-[3-(4-m eth oxyp h en yl)a m in oca r -
bon yltr ia zen e-1-yl]a d en osin e 7h : yield, 25.4 mg (43%); 1H
NMR δ 11.5 (broad, 1H, N-H), 8.7 (broad, 1H, N-H), 7.99 (s,
1H, H-8), 7.50 (m, 2H, Ar), 7.02 (d, 2H, J ) 9.0 Hz, Ar), 6.5
(broad, 2H, NH2), 6.22 (broad, 1H, H-1′), 5.98 (m, 1H, H-2′),
5.71 (m, 1H, H-3′), 4.3-4.5 (m, 3H, H-4′, H-5′), 3.81 (s, 3H,
OCH3), 2.08, 2.04, and 2.02 (all 3H, s, acetates).
4.04 (m, 1H, H-4′ or H-3′), 3.5-3.7 (m, 2H, H-5′); [found M+
+
1, 354.1560; C11H16N9O5 requires M, 354.1587]. Anal. Calcd
(C11H15N9O5‚1.0H2O) C, H, N.
2-(3-Eth yla m in oca r bon yltr ia zen e-1-yl)a d en osin e 8b:
1
yield, 18.0 mg (93%), mp 172-173 °C; H NMR (DMSO-d6) δ
12.40 (s, 1H, N-H), 8.34 (s, 1H, H-8), 7.98 (s, 1H, N-H), 7.65
(s, 2H, NH2), 6.05 (broad, 1H, OH), 5.86 (d, 1H, J ) 7.2 Hz,
H-1′), 5.45 (d, 1H, J ) 6.4 Hz, OH), 5.22 (d, 1H, J ) 3.7 Hz,
OH), 4.72 (m, 1H, H-2′), 4.14 (m, 1H, H-3′ or H-4′), 4.06 (m,
1H, H-4′ or H-3′), 3.5-3.7 (m, 2H, H-5′), 3.24 (m, 2H, CH2-
CH3), 1.14 (t, 3H, J ) 7.2 Hz, CH3); [found M+ + 1, 382.1560;
C
13H20N9O5 requires M, 382.1587]. Anal. Calcd (C13H19N9O5‚
2.1H2O) C, H, N.
2-(3-Cycloh e xyla m in oca r b on ylt r ia ze n e -1-yl)a d e n o-
sin e 8c: yield, 23.5 mg (92%), mp 164-166 °C; 1H NMR
(DMSO-d6) δ 12.35 (s, 1H, N-H), 8.35 (s, 1H, H-8), 7.92 (d,
1H, J ) 7.1 Hz, N-H), 7.68 (s, 2H, NH2), 6.20 (broad, 1H, OH),
5.85 (d, 1H, J ) 7.4 Hz, H-1′), 5.46 (d, 1H, J ) 6.5 Hz, OH),
5.23 (d, 1H, J ) 3.4 Hz, OH), 4.79 (m, 1H, H-2′), 4.11 (m, 1H,
H-3′ or H-4′), 4.09 (m, 1H, H-4′ or H-3′), 3.5-3.7 (m, 3H,
CHNH, H-5′), 1.1-1.9 (m, 10H, cyclohexyl); [found M+ + 1,
436.2057; C17H26N9O5 requires M, 436.2056]. Anal. Calcd
(C17H25N9O5‚1.2H2O) C, H, N.
2-(3-P h en yla m in oca r bon yltr ia zen e-1-yl)a d en osin e 8d :
1
yield, 18.0 mg (78%), mp 164-165 °C; H NMR (DMSO-d6) δ
12.75 (broad, 1H, N-H), 10.08 (broad, 1H, N-H), 8.37 (s, 1H,
H-8), 7.70 (s, 2H, NH2), 7.60 (m, 2H, Ar-Hortho), 7.38 (t, 2H, J
) 7.5 Hz, Ar-Hmeta), 7.09 (t, 1H, J ) 7.5 Hz, Ar-Hpara), 6.4
(broad, 1H, OH), 5.88 (d, 1H, J ) 7.2 Hz, H-1′), 5.49 (d, 1H, J
) 6.3 Hz, OH), 5.26 (d, 1H, J ) 3.4 Hz, OH), 4.77 (m, 1H,
H-2′), 4.18 (m, 1H, H-3′ or H-4′), 4.07 (m, 1H, H-4′ or H-3′),
3.6-3.8 (m, 2H, H-5′); [found M+ + 1, 430.1569; C17H20N9O5
requires M, 430.1587]. Anal. Calcd (C17H19N9O5‚2.2H2O) C, H,
N.
2′,3′,5′-Tr i-O-a cetyl-2-[3-(2-m eth oxyp h en yl)a m in oca r -
1
bon yltr ia zen e-1-yl]a d en osin e 7i: yield, 32.1 mg (55%); H
NMR δ 12.0 (broad, 1H, N-H), 8.70 (broad, 1H, N-H), 8.22
(d, 1H, J ) 8.3 Hz, Ar), 8.01 (s, 1H, H-8), 7.5 (broad, 2H, NH2),
6.8-7.1 (m, 3H, Ar), 6.19 (broad, 1H, H-1′), 5.97 (m, 1H, H-2′),
5.70 (m, 1H, H-3′), 4.4-4.5 (m, 3H, H-4′, H-5′), 3.83 (s, 3H,
OCH3), 2.08, 2.06, and 2.03 (all 3H, s, acetates).
2′,3′,5′-Tr i-O-a cetyl-2-[3-(3-tr iflu or om eth ylp h en yl)a m i-
n ocar bon yltr iazen e-1-yl]aden osin e 7j: yield, 35.0 mg (56%);
1H NMR δ 12.5 (broad, 1H, N-H), 9.90 (broad, 1H, N-H), 8.12
(m, 1H, Ar), 7.96 (m, 3H, H-8, NH2), 7.2-7.5 (m, 3H, Ar), 6.27
(d, 1H, J ) 5.1 Hz, H-1′), 5.93 (m, 1H, H-2′), 5.62 (m, 1H, H-3′),
4.4-4.5 (m, 3H, H-4′, H-5′), 2.09, 2.08, and 2.05 (all 3H, s,
acetates).
2′,3′,5′-Tr i-O-a cetyl-2-(3-ben zyla m in oca r bon yltr ia zen e-
1-yl)a d en osin e 7k : yield, 40.6 mg (71%); 1H NMR δ 11.7
(broad, 1H, N-H), 7.95 (s, 1H, H-8), 7.41 (s, 1H, N-H), 7.2-
7.3 (m, 5H, Ar), 6.70 (broad, 2H, NH2), 6.31 (m, 1H, H-2′), 6.04
(d, 1H, J ) 4.4 Hz, H-1′), 5.68 (m, 1H, H-3′), 4.66 (m, 2H, CH2-
Ph), 4.2-4.5 (m, 3H, H-4′, H5′), 2.04, 2.00, and 1.99 (all 3H, s,
acetates).
2′,3′,5′-Tr i-O-a cet yl-2-[3-(2-t h ia zolyl)a m in oca r b on yl-
tr ia zen e-1-yl]a d en osin e 7l: yield, 27.0 mg (48%) after chro-
matography with 4% methanol in ethyl acetate; 1H NMR
(DMSO-d6) δ 12.6 (broad, 1H, N-H), 10.8 (broad, 1H, N-H),
8.40 (s, 1H, H-8), 7.71 (s, 2H, NH2), 7.49 (broad, 1H, thiazole),
7.27 (broad, 1H, thiazole), 6.20 (d, 1H, J ) 4.4 Hz, H-1′), 5.96
(m, 1H, H-2′), 5.73 (m, 1H, H-3′), 4.40 (m, 1H, H-4′), 4.32-4.5
(m, 2H, H-5′), 2.11, 2.07, and 1.99 (all 3H, s, acetates).
Dep r otection of 7a -l to 8a -l, Gen er a l Meth od . Aque-
ous ammonia (2 mL of a 25% solution) was added to a solution
of triacetate 7a -l in methanol (2 mL). After 18 h of stirring
at room temperature, the solvents were removed in vacuo and
the residue was coevaporated with methanol (4 mL). The
remaining solid was dried in vacuo (0.1 mbar, 40 °C) for 2 h
and triturated with methanol (2 mL), providing the yellow
ribosides 8a -l.
2-[3-(4-Ch lor op h en yl)a m in oca r bon yltr ia zen e-1-yl]a d -
1
en osin e 8e: yield, 17.0 mg (73%), mp 170-172 °C; H NMR
(DMSO-d6) δ 12.80 (s, 1H, N-H), 10.13 (s, 1H, N-H), 8.37 (s,
1H, H-8), 7.73 (s, 2H, NH2), 7.63 (m, 2H, Ar), 7.43 (d, 2H, J )
8.7 Hz, Ar), 6.41 (broad, 1H, OH), 5.88 (d, 1H, J ) 7.1 Hz,
H-1′), 5.48 (d, 1H, J ) 6.3 Hz, OH), 5.26 (broad, 1H, OH), 4.75
(m, 1H, H-2′), 4.19 (m, 1H, H-3′ or H-4′), 4.07 (m, 1H, H-4′ or
H-3′), 3.6-3.8 (m, 2H, H-5′); [found M+ + 1, 464.1166;
C
17H19N9O5Cl requires M, 464.1198]. Anal. Calcd (C17H18N9O5-
Cl‚2.2H2O) C, H, N.
2-[3-(3-Ch lor op h en yl)a m in oca r bon yltr ia zen e-1-yl]a d -
1
en osin e 8f: yield, 18.1 mg (78%), mp 159-161 °C; H NMR
(DMSO-d6) (two rotamers are present; only the major rotamer
is shown) δ 12.74 (s, 1H, N-H), 10.03 (s, 1H, N-H), 8.39 (s,
1H, H-8), 7.75 (s, 2H, NH2), 7.0-7.6 (m, 4H, Ar), 6.46 (d, 1H,
J ) 10.2 Hz, OH), 5.88 (d, 1H, J ) 7.2 Hz, H-1′), 5.48 (d, 1H,
J ) 6.3 Hz, OH), 5.26 (d, 1H, J ) 3.3 Hz, OH), 4.80 (m, 1H,
H-2′), 4.19 (m, 1H, H-3′ or H-4′), 4.10 (m, 1H, H-4′ or H-3′),
3.6-3.8 (m, 2H, H-5′); [found M+ + 1, 464.1186; C17H19N9O5-
Cl requires M, 464.1198]. Anal. Calcd (C17H18N9O5Cl‚0.5 H2O)
C, H, N.
2-[3-(2-Ch lor op h en yl)a m in oca r bon yltr ia zen e-1-yl]a d -
1
en osin e 8g: yield, 18.0 mg (76%), mp 160-161°C; H NMR
(DMSO-d6 δ 12.88 (s, 1H, N-H), 9.43 (s, 1H, N-H), 8.40 (s,
1H, H-8), 7.95 (d, 1H, J ) 7.3 Hz, Ar), 7.70 (s, 2H, NH2), 7.2-
7.6 (m, 3H, Ar), 5.88 (d, 1H, J ) 6.8 Hz, H-1′), 5.81 (broad,
1H, OH), 5.46 (broad, 1H, OH), 5.20 (broad, 1H, OH), 4.70 (m,
1H, H-2′), 4.18 (m, 1H, H-3′ or H-4′), 4.12 (m, 1H, H-4′ or H-3′),
3.4-3.7 (m, 2H, H-5′); [found M+ + 1, 464.1194; C17H19N9O5-
Cl requires M, 464.1198]. Anal. Calcd (C17H18N9O5Cl‚1.2H2O)
C, H, N.
2-[3-(4-Met h oxyp h en yl)a m in oca r bon ylt r ia zen e-1-yl]-
a d en osin e 8h : yield, 17.4 mg (87%), mp 159-161°C; 1H NMR
(DMSO-d6) (two rotamers are present; only the major rotamer
is shown) δ 12.68 (s, 1H, N-H), 9.93 (s, 1H, N-H), 8.37 (s,
1H, H-8), 7.4-7.7 (broad, 4H, Ar-Hortho, NH2), 6.95 (d, 2H, J
) 8.9 Hz, Ar-Hmeta), 6.4 (broad, 1H, OH), 5.88 (d, 1H, J ) 7.2
2-(3-Am in oca r bon yl)tr ia zen e-1-yl)a d en osin e 8a : yield,
1
18.0 mg (86%), mp 186-188 °C; H NMR (DMSO-d6) δ 12.54
(broad, 1H, N-H), 8.35 (s, 1H, H-8), 7.64 (broad, 2H, NH2),

1500 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Beukers et al.
Hz, H-1′), 5.5 (broad, 1H, OH), 5.3 (broad, 1H, OH), 4.79 (m,
1H, H-2′), 4.18 (m, 1H, H-3′ or H-4′), 4.09 (m, 1H, H-4′ or H-3′),
3.76 (s, 3H, OCH3), 3.4-3.7 (m, 2H, H-5′); [found M+ + 1,
460.1673; C18H22N9O6 requires M, 460.1693]. Anal. Calcd
(C18H21N9O6‚2.2H2O) C, H, N.
2-[3-(2-Met h oxyp h en yl)a m in oca r b on ylt r ia zen e-1-yl]-
a d en osin e 8i: yield, 15.1 mg (60%), mp 162-164 °C; 1H NMR
(DMSO-d6) δ 12.65 (s, 1H, N-H), 9.03 (s, 1H, N-H), 8.41 (s,
1H, H-8), 8.04 (d, 1H, J ) 7.8 Hz, Ar), 6.9-7.1 (broad, 4H,
3H-Ar, OH), 5.91 (d, 1H, J ) 6.6 Hz, H-1′), 5.49 (m, 1H, OH),
5.21 (d, 1H, J ) 4.3 Hz, OH), 4.67 (m, 1H, H-2′), 4.14 (m, 1H,
H-3′ or H-4′), 3.99 (m, 1H, H-4′ or H-3′), 3.89 (s, 3H, OCH3),
3.4-3.7 (m, 2H, H-5′); [found M+ + 1, 460.1700; C18H22N9O6
requires M, 460.1693]. Anal. Calcd (C18H21N9O6‚1.7H2O) C, H,
N.
1H, H-2′), 4.17 (m, 1H, H-3′ or H-4′), 3.97 (m, 1H, H-4′ or H-3′),
3.5-3.7 (m, 2H, H-5′); [found M+ + 1, 431.1421; C17H19N8O6
requires M, 431.1428]. Anal. Calcd (C17H18N8O6‚0.3H2O) C, H,
N.
N6-P h en yl-2′,3′,5′-t r i-O-a cet yl-2-n it r oa d en osin e 13a .
Aniline (0.548 mL, 6.0 mmol) was added to a solution of 1
(0.915 g, 2.0 mmol) in DMF (4 mL). After 1 h of stirring, water
(∼10 mL) was slowly added to precipitate the product. Filtra-
tion, washing with methanol, and drying in vacuo gave pure
13a (1.01 g, 98%), as a yellow solid: mp 95-99 °C; 1H NMR δ
8.20 (s, 1H, H-8), 8.01 (s, 1H, N-H), 7.87 (d, 2H, J ) 8.0 Hz,
Ar-Hortho), 7.45 (t, 2H, J ) 8.0 Hz, Ar-Hmeta), 7.21 (t, 1H, J )
8.0 Hz, Ar-Hpara), 6.27 (d, 1H, J ) 5.5 Hz, H-1′), 5.77 (m, 1H,
H-2′), 5.63 (m, 1H, H-3′), 4.45-4.50 (m, 3H, H-4′, H-5′), 2.19,
2.14, and 2.10 (all 3H, s, acetates).
N6-Cyclopen tyl-2′,3′,5′-tr i-O-acetyl-2-n itr oaden osin e 13b.
Cyclopentylamine (99 µL, 1.02 mmol) and N,N-diisopropyl-
ethylamine (0.262 mL, 1.5 mmol) were added to a solution of
1 (0.457 g, 1.0 mmol) in DMF (2 mL) at -18 °C. The bath was
removed, and after 1 h of stirring at room temperature, the
reaction mixture was purified by extractive workup (ether/
water) to give crude 13b, which was used without purification
in the next step: 1H NMR δ 8.60 (broad, 1H, N-H), 8.05 (s,
1H, H-8), 6.20 (m, 1H, H-2′), 6.15 (d, 1H, J ) 5.5 Hz, H-1′),
5.62 (m, 1H, H-3′), 4.7 (broad, CH-NH), 4.3-4.6 (m, 3H, H4′,
H5′), 2.15, 2.10, and 2.07 (all 3H, s, acetates), 1.3-1.9 (m, 4H,
cyclopentyl).
N6-(2,2-Dip h en yleth yl)-2′,3′,5′-tr i-O-a cetyl-2-n itr oa d e-
n osin e 13c. 2,2-Diphenylethylamine (0.197 g, 1.0 mmol) was
reacted with 1 (1 mmol) as is described for 13b to give crude
13c, which was used without purification in the next step: 1H
NMR δ 8.02 (s, 1H, H-8), 7.2-7.4 (m, 10H, Ar), 6.23 (m, 1H,
H-2′), 6.19 (d, 1H, J ) 5.5 Hz, H-1′), 5.72 (m, 1H, N-H), 5.62
(m, 1H, H-3′), 4.3-4.6 (m, 6H), 2.17, 2.11, and 2.08 (all 3H, s,
acetates).
2-[3-(3-Tr iflu or om eth ylph en yl)am in ocar bon yltr iazen e-
1-yl]a d en osin e 8j: yield, 19.0 mg (68%), mp 169-170 °C; 1H
NMR (DMSO-d6) (two rotamers are present in a ratio of 4:1;
only the major rotamer is shown) δ 13.30 (s, 1H, N-H), 10.32
(s, 1H, N-H), 8.39 (s, 1H, H-8), 7.99 (m, 1H, Ar), 7.88 (broad,
2H, NH2), 7.4-7.8 (broad, 3H, Ar), 6.52 (broad, 1H, OH), 5.89
(d, 1H, J ) 7.2 Hz, H-1′), 5.48 (d, 1H, J ) 6.3 Hz, OH), 5.28
(broad, 1H, OH), 4.79 (m, 1H, H-2′), 4.18 (m, 1H, H-3′ or H-4′),
4.11 (m, 1H, H-4′ or H-3′), 3.89 (s, 3H, OCH3), 3.4-3.7 (m, 2H,
H-5′); [found M+ + 1, 498.1454; C18H19N9O5F3 requires M,
498.1461]. Anal. Calcd (C18H18N9O5F3‚1.5H2O) C, H, N.
2-(3-Ben zyla m in oca r bon yltr ia zen e-1-yl)a d en osin e 8k :
1
yield, 30.0 mg (95%), mp 170-171 °C; H NMR (DMSO-d6) δ
12.51 (s, 1H, N-H), 8.57 (m, 1H, N-H), 8.34 (s, 1H, H-8), 7.67
(s, 2H, NH2), 7.37 (m, 5H, C6H5), 6.09 (d, 1H, J ) 8.9 Hz, OH),
5.83 (d, 1H, J ) 7.3 Hz, H-1′), 5.43 (d, 1H, J ) 6.5 Hz, OH),
5.20 (d, 1H, J ) 3.6 Hz, OH), 4.68 (m, 1H, H-2′), 4.43 (m, 2H,
CH2Ph), 4.03 (m, 1H, H-3′ or H-4′), 3.97 (m, 1H, H-4′ or H-3′),
3.3-3.5 (m, 2H, H-5′); [found M+ + 1, 444.1705; C18H22N9O5
requires M, 444.1744]. Anal. Calcd (C18H21N9O5‚1.6H2O) C, H,
N.
N6-P h en yl-2′,3′,5′-tr i-O-a cetyl-2-n itr osoa d en osin e 14a .
Nitropurine 13a (0.514 g, 1.0 mmol) was hydrogenated with
Pd/C (30 mg, 10%) in ethyl acetate (20 mL) during one night
under 1 atm of H2. The catalyst was removed by hot filtration
over hyflow, and the filtrate was immediately oxidized with a
solution of sodium periodate (0.321 g, 1.5 mmol) in water (10
mL) at 0 °C. The biphasic mixture was stirred vigorously
during 1 h at this temperature, and after separation of the
organic layer, the nitroso derivative 14a (0.51 g, purity ∼ 90%)
was obtained as a yellow glass: 1H NMR (concentration-
dependent mixture of monomer and dimer; a sample of 1 mg
in 0.5 mL of CDCl3 shows predominantly the monomer) δ 8.31
(s, 1H, H-8), 7.93 (s, 1H, N-H), 7.86 (d, 2H, J ) 7.6 Hz, Ar),
7.45 (t, 2H, J ) 7.6 Hz, Ar), 7.19 (m, 1H, Ar), 6.44 (d, 1H, J )
5.5 Hz, H-1′), 5.90 (m, 1H, H-2′), 5.69 (m, 1H, H-3′), 4.4-4.5
(m, 3H, H-4′, H-5′), 2.20, 2.15, and 2.10 (all 3H, s, acetates).
N 6-Cyclop e n t yl-2′,3′,5′-t r i-O-a ce t yl-2-n it r osoa d e n o-
sin e 14b. Nitro compound 13b (0.10 g, 0.23 mmol) was
reduced and oxidized as described for 14a to give nitroso
derivative 14b, which was used without purification in the
next step: 1H NMR δ 8.00 (broad, 2H, H-8, N-H), 6.33 (m,
1H, H-1′), 5.7 (m, 1H, H-2′), 5.5 (m, 1H, H-3′), 4.9 (broad, CH-
NH), 4.3-4.6 (m, 3H, H4′, H5′), 2.18, 2.11, and 2.10 (all 3H, s,
acetates), 1.3-1.9 (m, 4H, cyclopentyl).
2-[3-(2-Th ia zolyl)a m in oca r b on ylt r ia zen e-1-yl]a d en o-
sin e 8l: yield, 17.1 mg (81%), mp 184-185 °C; 1H NMR
(DMSO-d6) δ 13.15 (broad, 1H, N-H), 12.50 (broad, 1H, N-H),
8.38 (s, 1H, H-8), 7.80 (s, 2H, NH2), 7.52 (d, 1H, J ) 3.4 Hz,
thiazole), 7.26 (d, 1H, J ) 3.4 Hz, thiazole), 7.0 (broad, 1H,
OH), 5.88 (d, 1H, J ) 7.5 Hz, H-1′), 5.46 (d, 1H, J ) 6.4 Hz,
OH), 5.28 (d, 1H, J ) 3.4 Hz, OH), 4.78 (m, 1H, H-2′), 4.18
(m, 1H, H-3′ or H-4′), 4.14 (m, 1H, H-4′ or H-3′), 3.75-4.0 (m,
2H, H-5′); [found M+ + 1, 437.1120; C14H17N10O5S requires M,
437.1104]. Anal. Calcd (C14H16N10O5S‚2.9H2O) C, H, N, S.
2-(3-Ben zoyltr ia zen e-1-yl]a d en osin e 11. A mixture of
2-nitrosoadenosine (29.6 mg, 0.10 mmol) and benzoylhydrazide
(20.4 mg, 0.15 mmol) was stirred in a mixture of acetonitrile
(2 mL) and acetic acid (0.2 mL) during 4 h at room tempera-
ture. The suspension was heated to dissolve solid material,
cooled in ice, and filtered. Recrystallization from ethanol
produced pure 11 (22.2 mg, 54%) as a yellow solid: mp 158-
1
162°C; H NMR (DMSO-d6) δ 13.5 (s, 1H, N-H), 8.45 (s, 1H,
H-8), 8.06 (d, 2H, J ) 7.5 Hz, Ar-Hortho), 7.69 (s, 2H, NH2),
7.5-7.7 (m, 3H, Ar), 5.92 (d, 1H, J ) 6.0 Hz, H-1′), 5.50 (d,
1H, J ) 6.1 Hz, OH), 5.20 (d, 1H, J ) 4.8 Hz, OH), 5.14 (m,
1H, OH), 4.65 (m, 1H, H-2′), 4.17 (m, 1H, H-3′ or H-4′), 3.97
(m, 1H, H-4′ or H-3′), 3.5-3.7 (m, 2H, H-5′); [found M+ + 1,
415.1478; C17H19N8O5 requires M, 415.1478]. Anal. Calcd
(C17H18N8O5‚2.4H2O) C, H, N.
N6-(2,2-Dip h en yleth yl)-2′,3′,5′-tr i-O-a cetyl-2-n itr osoa d -
en osin e 14c. Nitro compound 13c (crude, from 1 mmol of 1)
was reduced and oxidized as described for 14a to give nitroso
derivative 14c, which was used without purification in the next
step: 1H NMR δ 8.12 (s, 1H, H-8), 7.0-7.3 (m, 10H, Ar), 6.23
(d, 1H, J ) 5.5 Hz, H-1′), 5.90 (m, 1H, H-2′), 5.65 (m, 1H, H-3′),
4.3-4.6 (m, 6H), 2.20, 2.15, and 211 (all 3H, s, acetates).
N 6-P h e n yl-2-(3-a m in oca r b on ylt r ia ze n e -1-yl)a d e n o-
sin e 16a . The general procedure described for the synthesis
of 7a -l was used. Starting from 14a (0.1 mmol) and 6a (0.2
mmol), 32.2 mg of triacetate 15a (58%) was isolated after
chromatography with 5% methanol in ethyl acetate. The
acetates were removed by stirring a solution of 15a in
methanol (2 mL) with aqueous ammonia (2 mL of a 25%
solution) for 18 h at room temperature. The solvents were
2-(3-P h en yloxyca r bon yltr ia zen e-1-yl)a d en osin e 12. A
solution of 2-nitrosoadenosine (21.0 mg, 0.071 mmol) and
phenyl carbazate (12.0 mg, 0.080 mmol) in a mixture of
acetonitrile (2 mL), methanol (0.5 mL), and acetic acid (0.2
mL) was stirred during 24 h at room temperature. The
suspension was diluted with dichloromethane and purified by
flash chromatography with dichloromethane/methanol 80:20
as eluent. Evaporation of the solvents and trituration of the
residue with ether gave 12 (11.2 mg, 32%) as a yellow solid:
mp >180 °C (dec); 1H NMR (DMSO-d6) δ 13.6 (broad, 1H,
N-H), 8.44 (s, 1H, H-8), 7.67 (s, 2H, NH2), 7.2-7.4 (m, 5H,
Ar), 5.92 (d, 1H, J ) 6.0 Hz, H-1′), 5.50 (d, 1H, J ) 6.2 Hz,
OH), 5.19 (d, 1H, J ) 4.8 Hz, OH), 5.14 (m, 1H, OH), 4.63 (m,

TCPA, a Selective Agonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1501
removed in vacuo, and the residue was triturated with
evaporation of the solvents (bath temperature < 30 °C) gave
crude 17. Chromatography (silica, EtOAc) gave moderately
pure 17 as a yellow glass (0.930 g, 46% based on 1); H NMR
methanol (∼2 mL), providing pure 16a (19.0 mg, 76%): mp
1
1
187-88 °C; H NMR (DMSO-d6) δ 12.50 (s, 1H, N-H), 10.21
(s, 1H, N-H), 8.55 (s, 1H, H-8), 8.00 (d, 2H, J ) 8.1 Hz, Ar),
7.35 (m, 2H, Ar), 7.2 (broad, 2H, NH2), 7.09 (m, 1H, Ar), 5.94
(d, 1H, J ) 7.0 Hz, H-1′), 5.81 (m, 1H, OH), 5.50 (d, 1H, J )
6.3 Hz, OH), 5.26 (d, 1H, J ) 3.9, OH), 4.75 (m, 1H, H-2′),
4.17 (m, 1H, H-3′ or H-4′), 4.07 (m, 1H, H-4′ or H-3′), 3.5-3.8
(m, 2H, H-5′); [found M+ + 1, 430.1591; C17H20N9O5 requires
M, 430.1587]. Anal. Calcd (C17H19N9O5‚1.1H2O) C, H, N.
(DMSO-d6) δ 12.0 (s, 1H, N-H), 8.32 (d, 2H, J ) 9.0 Hz, ArH),
7.96 (s, 1H, H-8), 7.49 (d, 2H, J ) 9.0 Hz, ArH), 6.93 (d, 1H,
J ) 7.3 Hz, N-H), 6.20 (d, 1H, J ) 3.9 Hz, H1′), 5.83 (m, 1H,
H-2′), 5.74 (m, 1H, H-3′), 4.65 (broad, 1H, CHNH), 4.45 (m,
3H, H4′, H5′), 2.09, 2.07 and 2.04 (all 3H, s, acetates), 1.5-
1.8 (m, 8H, cyclopentyl).
A solution of 17 (0.335 g, 0.50 mmol) was stirred with aniline
(0.456 mL, 5.0 mmol) in anhydrous acetonitrile (10 mL) during
8 h. Evaporation of the solvent and chromatography (silica,
ethyl acetate) gave 15c (0.259 g, 0.416 mmol, 83%) as a yellow
glass: 1H NMR δ 14.5 (broad, 1H, N-H), 9.7 (broad, 1H,
N-H), 8.51 (s, 1H, H-8), 7.93 (broad, 1H, N-H), 7.60 (broad,
2H, Ar), 7.36 (t, 2H, J ) 7.7 Hz, Ar), 7.13 (m, 1H, Ar), 6.16
(m, 1H, H-1′), 5.98 (m, 1H, H-2′), 5.75 (m, 1H, H3′), 4.49 (broad,
1H, CHNH), 4.3-4.5 (m, 3H, H4′, H5′), 2.12, 2.06, 2.04 (all
3H, s, acetates), 1.5-1.8 (m, 8H, cyclopentyl). The acetates
were removed by stirring a solution of 15c (0.238 g, 0.38 mmol)
in methanol (10 mL) with aqueous ammonia (10 mL of a 25%
solution) for 18 h at room temperature. The solvents were
removed in vacuo, and the residue was triturated with
methanol (∼5 mL), providing 16c (0.116 g, 0.23 mmol, 61%)
as a yellow solid.
N6-P h en yl-2-(3-p h en yla m in oca r b on ylt r ia zen e-1-yl]-
a d en osin e 16b. The general procedure described for the
synthesis of 7a -l was used. Starting from 14a (1.0 mmol) and
4-phenylsemicarbazide 6d (2.0 mmol), a yield was obtained of
0.153 g of 15b (24%) after chromatography with ethyl acetate.
The acetates were removed by stirring a solution of 15b in
methanol (5 mL) with aqueous ammonia (5 mL of a 25%
solution) for 18 h at room temperature. The solvents were
removed in vacuo, and the residue was triturated with
methanol (∼2 mL), providing 16b (0.062 g, 51%) as a yellow
solid: mp 190-191 °C; 1H NMR (DMSO-d6) δ 12.97 (s, 1H,
N-H), 10.28 (s, 1H, N-H), 10.05 (s, 1H, N-H), 8.56 (s, 1H,
H-8), 8.00 (broad, 2H, Ar), 7.41 (broad, 2H, Ar), 7.37 (m, 4H,
Ar), 7.10 (m, 2H, Ar), 6.34 (m, 1H, OH), 5.96 (d, 1H, J ) 7.2
Hz, H-1′), 5.87 (d, 1H, J ) 6.0 Hz, OH), 5.52 (m, 1H, OH),
4.84 (m, 1H, H-2′), 4.22 (m, 1H, H-3′ or H-4′), 4.13 (m, 1H,
H-4′ or H-3′), 3.6-3.9 (m, 2H, H-5′); [found M+ + 1, 506.1907;
Sta bility Stu d ies. A solution of 8d or 16c (0.5 mg) in
DMSO-d6 (50 µL) was diluted with phosphate buffer in D2O
(450 µL, pD 7.8, 0.05 M) and kept in an NMR tube at 25 °C.
1H NMR spectra were obtained at 2 h intervals for 5 days. No
changes in the spectrum were observed.
C
23H24N9O5 requires M, 506.1901]. Anal. Calcd (C23H23N9O5‚
1.0H2O) C, H, N.
N6-Cyclop en tyl-2-(3-p h en yla m in oca r bon yltr ia zen e-1-
yl)a d en osin e 16c (TCP A). The general procedure described
for the synthesis of 7a -l was used. Starting from 14b (98 mg,
0.23 mmol) and 4-phenylsemicarbazide 6d (0.4 mmol), a yield
was obtained of 0.024 g of 15c (0.039 mmol, 17% in four steps
starting from 1) after chromatography with ethyl acetate. The
acetates were removed by stirring a solution of 15c in
methanol (2 mL) with aqueous ammonia (2 mL of a 25%
solution) for 18 h at room temperature. The solvents were
removed in vacuo, and the residue was triturated with
methanol (∼2 mL), providing 16c (9.5 mg, 48%) as a yellow
solid: mp 164-166 °C; 1H NMR (DMSO-d6) δ 12.81 (s, 1H,
N-H), 10.17 (s, 1H, N-H), 8.37 (s, 1H, H-8), 8.21 (s, 1H, N-H),
7.59 (broad, 2H, Ar), 7.39 (t, 2H, J ) 7.7 Hz, Ar), 7.11 (m, 1H,
Ar), 6.65 (m, 1H, OH), 5.88 (d, 1H, J ) 7.4 Hz, H-1′), 5.47 (d,
1H, J ) 5.8 Hz, OH), 5.26 (m, 1H, OH), 5.18 and 4.68 (m, 1H,
CHNH, rotamers), 4.82 (m, 1H, H-2′), 4.19 (m, 1H, H-3′ or
H-4′), 4.10 (m, 1H, H-4′ or H-3′), 3.65-3.9 (m, 2H, H-5′), 1.98
(m, 2H, cyclopentyl), 1.70 (m, 2H, cyclopentyl), 1.58 (m, 4H,
cyclopentyl); [found M+ + 1, 498.2207; C22H28N9O5 requires
M, 498.2213]. Anal. Calcd (C22H27N9O5‚0.5H2O) C, H, N.
N6-(2,2-Dip h en yleth yl)-2-(3-p h en yla m in oca r bon yltr ia -
zen e-1-yl)a d en osin e 16d . The general procedure described
for the synthesis of 7a -l was used. Starting from 14c (obtained
from 1.0 mmol 1) and 4-phenylsemicarbazide 6d (1.4 mmol),
a yield was obtained of 98 mg of 15d (13% in four steps based
on 1) after chromatography with ethyl acetate. The acetates
were removed by stirring a solution of 15d in methanol (4 mL)
with aqueous ammonia (4 mL of a 25% solution) for 18 h at
room temperature. The solvents were removed in vacuo, and
the residue was triturated with methanol (∼2 mL), providing
16d (54.3 mg, 69%) as a yellow solid: mp 138-140 °C; 1H NMR
(DMSO-d6) δ 12.92 (s, 1H, N-H), 10.13 (s, 1H, N-H), 8.31 (s,
1H, H-8), 8.25 (s, 1H, N-H), 7.61 (broad, 2H, Ar), 7.0-7.4 (m,
13H, Ar), 6.49 (broad, 1H, OH), 5.86 (d, 1H, J ) 7.3 Hz, H-1′),
5.46 (d, 1H, J ) 6.1 Hz, OH), 5.26 (m, 1H, OH), 4.77 (m, 1H,
H-2′), 4.67 and 4.61 (m, 1H, CHNH, rotamers), 4.18 (m, 3H,
CH2CH, H-3′ or H-4′), 4.08 (m, 1H, H-4′ or H-3′), 3.65-3.8 (m,
2H, H-5′); [found M+ + 1, 610.2512; C31H32N9O5 requires M,
610.2527]. Anal. Calcd (C31H31N9O5‚1.1H2O) C, H, N.
Hyd r olysis of 8d . A solution of 8d in a mixture of DMSO-
d6 (450 µL) and D2O (50 µL) was heated in an NMR tube at
80 °C. After 2 h, complete conversion was observed to aniline
and 2-aminoadenosine (18): 1H NMR δ 7.93 (s, 1H, H-8), 7.01
(m, 2H, aniline), 6.58 (t, 2H, J ) 7.3 Hz, aniline), 6.53 (t, J )
7.3 Hz, aniline), 5.70 (d, 1H, J ) 6.4 Hz, H-1′), 4.51 (m, 1H,
H-2′), 3.64 (dd, 1H, J ) 12.2 Hz, J ) 3.2 Hz, H-5a′), 3.54 (dd,
1H, J ) 12.2 Hz, J ) 3.2 Hz, H-5b′).
Hyd r olysis of 16c. Hydrolysis of 16c was performed as
described for 8d to give 2-amino-CPA 19 and aniline: 1H NMR
δ 7.89 (s, 1H, H-8), 7.01 (m, 2H, aniline), 6.58 (t, 2H, J ) 7.3
Hz, aniline), 6.53 (t, J ) 7.3 Hz, aniline), 5.70 (d, 1H, J ) 6.4
Hz, H-1′), 4.50 (m, 1H, H-2′), 3.9-4.1 (m, 3H, H-3′, H-4′,
CHNH), 3.64 (dd, 1H, J ) 12.3 Hz, J ) 3.2 Hz).
Cell Cu ltu r es. Radioligand binding studies were performed
on stably transfected cell lines expressing human adenosine
receptors. CHO cells expressing the human adenosine A1
receptor were obtained from Dr. A. Townsend-Nicholson. These
cells were cultured at 37 °C in a 5% CO2 atmosphere in a 1:1
mixture of DMEM/F12, 2 mM Glutamax (a stable analogue of
glutamine), 10% newborn calf serum with 50 IU/mL penicillin,
and 50 µg/mL streptomycin. Dr. S. Rees kindly provided CHO
cells expressing the human A2A or human A2B receptor. These
cells were cultured at 37 °C in a 5% CO2 atmosphere in a 1:1
mixture of DMEM/F12, 2 mM Glutamax (a stable analogue of
glutamine), 10% newborn calf serum, 1 mg/mL G418 with 50
IU/mL penicillin, and 50 µg/mL streptomycin. HEK293 cells
expressing human adenosine A3 receptors were from Dr.
K.-N. Klotz. These cells were cultured at 37 °C in a 7% CO2
atmosphere in DMEM, 2 mM Glutamax (a stable analogue of
glutamine), 10% newborn calf serum, 0.5 mg/mL G418, with
50 IU/mL penicillin, and 50 µg/mL streptomycin.
Confluent cells expressing the human A1, A2A, or A2B
receptor or semiconfluent cells expressing the human A3
adenosine receptor were trypsinized and centrifuged for 10 min
at 1000 rpm. The cell pellet of CHO cells expressing the
adenosine A2B receptor was resuspended in 140 mM NaCl, 5
mM KCl, and 5 mM glucose in 20 mM Tris-HCl adjusted to
pH 7.4 at room temperature. The other cell pellets were
resuspended in 50 mM Tris-HCl, pH 7.4, at room temperature
and homogenized on ice for 5 s at position 8 with an Ystral.
The homogenate was centrifuged for 45 min at 12700 rpm in
an SW-30 rotor at 4 °C. The resulting pellet was resuspended
Im p r oved Syn th esis of TCP A (16c) via 17. A mixture of
p-nitrophenyl carbazate18 (0.455 g, 2.31 mmol) and 14b
(prepared from 3.0 mmol 13b) was stirred in a mixture of DCM
(25 mL) and acetic acid (2.5 mL) during 18 h. Aqueous workup
(saturated NaHCO3, diethyl ether), drying (Na2SO4), and

1502 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Beukers et al.
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in 50 mM Tris-HCl, pH 7.4, at room temperature. Adenosine
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-80 °C.
Ra d ioliga n d Bin d in g Stu d ies. Stock solutions of ligands
were made in DMSO. The final concentration of DMSO in the
assay did not exceed 1%. [3H]DPCPX and [125I]IAB-MECA
were obtained from Amersham, and [3H]ZM241385 was from
Tocris Cookson Ltd. (Northpoint, U.K.). IC50 values were
estimated by GraphPad Prism software (GraphPad, San Diego,
CA). IC50 values obtained from the competition curves were
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Ad en osin e A1 Recep tor . Membranes, containing 40 µg of
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bath. Nonspecific binding was determined in the presence of
10 µM CPA. The incubation was terminated by filtration over
Whatman GF/B filters under reduced pressure with a Brandell
harvester. Filters were washed three times with ice-cold buffer
and placed in scintillation vials. Emulsifier Safe, 3.5 mL, was
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ZM241385 (final concentration ) 2.0 nM) during 2 h at 25 °C
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in the presence of 100 µM CPA. The incubation was terminated
by filtration over Whatman GF/B filters under reduced pres-
sure with a Brandell harvester. Filters were washed four times
with ice-cold buffer and placed in scintillation vials. Emulsifier
Safe, 3.5 mL, was added, and after 2 h, radioactivity was
counted in an LKB rackbeta scintillation counter.
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temperature with [3H]DPCPX (final concentration ) 6.0 nM)
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The incubation was terminated by filtration over Whatman
GF/B filters under reduced pressure with a Millipore manifold.
Filters were washed eight times with 2 mL of ice-cold buffer
and placed in scintillation vials. Emulsifier Safe, 3.5 mL, was
added, and after 2 h, radioactivity was counted in an LKB
rackbeta scintillation counter.
Ad en osin e A3 Recep tor . Membranes, containing 20-40
µg of protein, were incubated in a total volume of 100 µL of 50
mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, and 0.01% CHAPS,
pH 7.4, at room temperature with [125I]IAB-MECA (final
concentration ) 0.10 nM) during 1 h at 37 °C in a shaking
water bath. Nonspecific binding was determined in the pres-
ence of 100 µM R-PIA. The incubation was terminated by
filtration over Whatman GF/B filters under reduced pressure
with a Brandell harvester. Filters were washed three times
with ice-cold buffer and placed in vials. Radioactivity was
counted in a gamma counter.
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competition with [3H]cAMP for binding to protein kinase A.26
The data reflect three independent experiments, performed in
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San Diego, CA) was used.
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