N-cycloalkyl derivatives of adenosine and 1-deazaadenosine as agonists and partial agonists of the A1 adenosine receptor

Article abstract of DOI:10.1021/jm9911231

A number of cycloalkyl substituents (from C-3 to C-8) have been introduced on the 6-amino group of adenosine, 1-deazaadenosine, and 2'- deoxyadenosine, bearing or not a chlorine atom at the 2-position, to evaluate the influence on the A₁ and A(

Full text of DOI:10.1021/jm9911231

250
J . Med. Chem. 2000, 43, 250-260
N-Cycloa lk yl Der iva tives of Ad en osin e a n d 1-Dea za a d en osin e a s Agon ists a n d
P a r tia l Agon ists of th e A1 Ad en osin e Recep tor
Sauro Vittori,† Anna Lorenzen,‡ Christina Stannek,‡ Stefano Costanzi,† Rosaria Volpini,† Adriaan P. IJ zerman,§
J akobien K. Von Frijtag Drabbe Kunzel,§ and Gloria Cristalli*,†
Dipartimento di Scienze Chimiche, Universita` di Camerino, 62032 Camerino, Italy, Pharmakologisches Institut,
Im Neuenheimer Feld 366, 69120 Heidelberg, Germany, and Division of Medicinal Chemistry, Leiden/ Amsterdam Center for
Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
Received J uly 23, 1999
A number of cycloalkyl substituents (from C-3 to C-8) have been introduced on the 6-amino
group of adenosine, 1-deazaadenosine, and 2′-deoxyadenosine, bearing or not a chlorine atom
at the 2-position, to evaluate the influence on the A1 and A2A affinity of steric hindrance and
lipophilicity. Furthermore, the guanosine 5′-triphosphate (GTP) shift and the maximal induction
of guanosine 5′-(γ-thio)triphosphate ([35S]GTPγS) binding to G proteins in rat brain membranes
were used to determine the intrinsic activity of these nucleosides at the A1 adenosine receptor.
All compounds of the ribose-bearing series proved to be full agonists, the 1-deaza derivatives
showing affinities for the A1 receptor about 10-fold lower than the corresponding adenosines.
On the other hand, all the 2′-deoxyribose derivatives bind to the A1 receptor with affinities in
the high nanomolar range, with the 2-chloro substituted compounds showing slightly higher
affinities than the 2-unsubstituted counterparts. In terms of the potencies, the most potent
compounds proved to be those bearing four- and five-membered rings. Both GTP shifts and
[35S]-GTPγS experiments showed that most of the 2′-deoxyadenosine derivatives are partial
agonists. The 2′-deoxyadenosine derivatives which were identified as partial agonists consist-
ently detected fewer A1 receptors in the high-affinity state than full agonists. However, it is
worthwhile noting that there was not a simple linear relationship between receptor occupancy
and activation. These results indicate that a critical density of A1 adenosine receptors in the
high-affinity state is required for G protein activation.
In tr od u ction
Partial agonists, compounds whose intrinsic activity
is less than that of a full agonist,7,8 could have several
advantages compared to full agonists including less
pronounced cardiovascular effects and more selective
actions. In addition, partial agonists may induce less
receptor downregulation and desensitization.
A variety of studies have demonstrated, on the basis
of biochemical and pharmacological experiments, that
most adenosine actions are mediated by at least four
extracellular receptors: A1, A2A, A2B, and A3. Further-
more, adenosine is ubiquitous in mammalian cells, and
it is structurally and metabolically related to bioactive
nucleotides, methylating agents, and various co-
enzymes.1,2
The fact that adenosine and adenosine-related mol-
ecules are involved in the regulation of many aspects
of cellular metabolism makes drugs with a purinergic
mechanism an expanding therapeutic target.1,3 How-
ever, to fully evaluate these prospects, subtype selective
agonists and antagonists with high affinity and potency
are required.2-5 Moreover, differences of adenosine
receptor distribution and activation upon various cells
and tissue types may also be exploited to achieve
selective responses by drugs acting on adenosine recep-
tor sybtypes.6
In recent years, various modifications of purine and
ribose moiety of N-cyclopentyladenosine (CPA) have
been studied in order to investigate partial agonists.9-16
It appeared from the results of these studies that partial
agonists for adenosine A1 receptors have been obtained
by substituting the 8-position of CPA,9,12-15 by removing
the 2′- or 3′-hydroxyl group from the ribose ring,10,11 and
by replacing the 5′-hydroxyl group with methylseleno-
or methylthio-group.16
We have previously examined the effects of several
deaza analogues of adenosine on rat brain membranes
and on human platelets in order to investigate the role
of the purine nitrogens in the binding to adenosine
receptors.17 It was found that in this series 1-deaza-
adenosine displayed the highest affinity and potency for
A1 adenosine receptor subtype. Moreover, 3-deaza-
adenosine behaved as a very weak ligand for this
subtype and 7-deaza- and 1,3-dideazaadenosine proved
to be inactive. Furthermore, 1-deazaadenosine deriva-
tives has been shown to inhibit adenosine deaminase
(ADA)18,19 and platelet aggregation induced by ADP.20
On the other hand, the major drawbacks to the
potential therapeutic use of adenosine agonists are to
date the strong hypotensive and cardiac depressant
effects.
* To whom correspondence should be addressed. Tel.: 39-0737-
402252. Fax: 39-0737-402252. E-mail: cristall@camserv.unicam.it.
† Dipartimento di Scienze Chimiche, Camerino.
On this basis, a series of 1-deaza analogues of N-[R-
(-)-1-methyl-2-phenethyl]adenosine (R-PIA), N-cyclo-
‡ Pharmakologisches Institut, Heidelberg.
§ Division Medicinal Chemistry, Leiden.
10.1021/jm9911231 CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/31/1999

N-Cycloalkyl Derivatives of Adenosine as A1 Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 251
F igu r e 1. Structures of all the nucleosides evaluated in this paper.
hexyladenosine (CHA), and CPA were synthesized and
evaluated in radioligand binding studies for their af-
finity at A1 and A2A receptors.20 The N-substituted
1-deazaadenosines largely retained the A1 binding af-
finity of their adenosine counterparts, but lost some of
their A2 affinity, resulting in A1 vs A2 selective com-
pounds.
The recent discoveries that adenosine derivatives can
behave as partial agonists for the adenosine A1 receptors
prompted us to investigate the effect on A1 and A2A
binding affinity of the isosteric substitution of 1-nitrogen
by a -CH- in 6-cycloalkyladenosine derivatives.
In this paper, the synthesis of a series of 1-deaza-
adenosine analogues with the 6-amino group bearing
cycloalkyl substituents (from C-3 to C-8) and with or
without a chlorine atom at the 2-position is reported,
starting from 2,6-dichloro-1-deazapurine riboside (5-
chloro-7-nitro-â-D-ribofuranosyl-3H-imidazo[4,5-b]pyri-
dine, 1).17
starting from 2,6-dichloro-9-(2,3,5-O-triacetyl-â-D-ribo-
furanosyl)purine (4),21 to evaluate the influence on the
A1 and A2A affinity of steric hindrance and lipophilicity.
Finally, a number of 2′-deoxyadenosines, bearing the
same series of cycloalkylamino substituents in 6-posi-
tion, have been prepared starting from 2,6-dichloro-9-
(2-deoxy-3,5-di-O-p-toluoyl-â-D-erythro-pentofuranosyl)-
9H-purine (8)23 to assess the modulation of affinity and
potency by the size of the cycloalkyl ring and by the
presence of a chlorine atom on C-2, when these modi-
fications are combined with removing the 2′ hydroxyl
group.
All these cycloalkylamino derivatives (Figure 1) were
tested in vitro for their affinity for the adenosine A1 and
A2A receptors, and for their GTP shift, i.e., the difference
in affinity for the adenosine A1 receptor in rat brain in
the presence and absence of GTP. Furthermore, the
intrinsic activity of the nucleosides at the adenosine A1
receptor was evaluated by measuring the maximal
induction of [35S]GTPγS binding to G proteins in rat
brain membrane preparations.
The same cycloalkyl rings have been introduced in
the 6-position of adenosine and 2-chloroadenosine,

252 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Vittori et al.
Sch em e 1. Synthesis of N-Cycloalkyl-1-deazaadenosine
Sch em e 3. Synthesis of N-Cycloalkyl-2′-deoxyadenosine
Derivativesa
Derivativesa
a
R ) see Figure 1. Reagents and conditions: (i) R-NH2, EtOH,
a
∆; (ii) EtOH, Pd/C, H2.
R ) see Figure 1. Reagents and conditions: (i) R-NH2, EtOH;
(ii) NH3/MeOH; (iii) MeOH, H2, Pd/C.
Sch em e 2. Synthesis of N-Cycloalkyladenosine
Derivativesa
of nucleosides 6a -f can be achieved by reacting the
commercially available 6-chloropurine riboside (7) with
the appropriate cycloalkylamine in EtOH at reflux for
5 h22 (Scheme 2).
The series of 2′-deoxyadenosine derivatives was ob-
tained starting from 2,6-dichloro-9-(2-deoxy-3,5-di-O-p-
toluoyl-â-D-erythro-pentofuranosyl)-9H-purine (8).23 Treat-
ment with the appropriate amine at room temperature
for 4 h gave the fully protected 2′-deoxy-2-chloro-N-
cycloalkyladenosines 9a -f, which were isolated and
characterized. Methanolic ammonia was added to the
residue in order to achieve removing of toluoyl groups
at room temperature to give 10a -f in high yields.
Finally, the dechlorinated nucleosides 11a -f were
obtained, as usual, by catalytic hydrogenolysis with 10%
Pd/C in ethanol and 2 N NaOH. Compounds 11c and
11d were already obtained in very low yield by a less
convenient route starting from 2′-deoxy-3′,5′-di-O-acetyl-
6-iodopurine-9-â-D-riboside.11
a
R ) see Figure 1. Reagents and conditions: (i) R-NH2, EtOH;
(ii) NH3/MeOH; (iii) MeOH, Pd/C, H2.
P h a r m a cology. R ibose Der iva t ives. The com-
pounds described in Schemes 1 and 2 were tested first
in radioligand binding experiments to assess their
affinities for the rat adenosine A1 and A2A receptors, and
the results are reported in Tables 3 and 4.
A1 receptor affinities were determined on rat cortical
membranes using the antagonist [3H]DPCPX as radio-
ligand. Affinities for the A2A receptor were established
on rat striatal membranes using the agonist [3H]CGS
21680.
The new 1-deazapurine nucleosides exhibited an
appreciable A1 affinity, the most active compound being
the 2-chloro-N-cyclobutyl-1-deazaadenosine (2b, Ki ) 41
nM) (Table 3). The 2-chloro derivatives are, in general,
slightly more active than the corresponding dechlori-
nated compounds, with the highest affinity shown by
molecules bearing four- and five-membered rings. All
these nucleosides exhibited a moderate A1 vs A2A
selectivity, the highest shown by molecules bearing
cycloalkyl substituents with a low number of carbons.
In comparison with the 1-deaza analogues, the purine
nucleosides exhibited higher affinity and selectivity at
A1 receptors, the most active compounds being the
N-cyclobutyl- and N-cyclopentyladenosine derivatives
5b, 6b, 5c, and 6c (Table 4).
Resu lts a n d Discu ssion
Ch em istr y. The ribose derivatives 3a -f and 6a -f
were synthesized, according to Schemes 1 and 2, start-
ing from 2,6-dichloro-1-deazapurine and 2,6-dichloro-
purine nucleosides, respectively.
Reaction of 5,7-dichloro-3-â-D-ribofuranosyl-3H-imid-
azo[4,5-b]pyridine (1)17 with the appropriate cycloalkyl-
amine, in a steel bomb at 120 °C, gave the N7-
substituted-(5-chloro-3-â-D-ribofuranosyl)-3H-imidazo-
[4,5-b]pyridines 2a -f. Catalytic hydrogenolysis of the
chlorinated compounds with 10% Pd/C in ethanol and
2 N NaOH afforded the corresponding derivatives 3a -f
(Scheme 1).
Treatment of 2,6-dichloro-9-(2,3,5-tri-O-acetyl-â-D-
ribofuranosyl)purine (4)21 with the appropriate cyclo-
alkylamine, at room temperature for 4 h, gave a mixture
of mono-, di-, and triacetylated 2-chloro-N-substituted
derivatives; to accelerate the deacetylation process and
to avoid side reactions, the crude material was treated
with methanolic ammonia at room temperature to
obtain the desired nucleosides 5a -f. Catalytic hydro-
genolysis of the chlorine atom in the 2-position with 10%
Pd/C in ethanol and 2 N NaOH afforded the corre-
sponding compounds 6a -f. Alternatively, the synthesis

N-Cycloalkyl Derivatives of Adenosine as A1 Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 253
Ta ble 1. Preparation of Compounds Reported in Schemes 1-3
compd
R
time (h)
16
chromatography solvent
yield (%)
45
mp (°C)
164-167
2a
cC3H5
CHCl3-CH3OH
(87:13)
2b
2c
cC4H7
cC5H9
cC6H11
cC7H13
cC8H15
cC3H5
cC4H7
cC5H9
cC6H11
cC7H13
cC8H15
cC3H5
cC4H7
cC5H9
cC6H11
cC7H13
cC8H15
16
24
24
24
24
24
24
24
24
48
48
72
72
76
76
92
92
CH3Cl-cC6H12-CH3OH
47
35
48
27
22
70
91
80
92
74
80
94
86
88
78
90
85
209-211
168-170
188-190
150 (dec)
165-169
(70:20:10)
CHCl3-CH3OH
(97:3)
2d
CHCl3-CH3OH-NH3
(85:14:1)
2e
CHCl3-cC6H12-CH3OH
(70:20:10)
2f
CHCl3-cC6H12-CH3OH
(70:20:10)
5a
CHCl3-CH3CN-CH3OH
(82:9:9)
169-171 dec
lit.24 113-116
181-183
5b
CHCl3-CH3CN-CH3OH
(80:10:10)
5c
CHCl3-CH3CN-CH3OH
(84:8:8)
190-196
108-111
181-186
189-192
157-158
140-142
125-128
150-152
178-180
176-178
5d
CHCl3-CH3CN-CH3OH
(84:8:8)
5e
CHCl3-CH3CN-CH3OH
(86:7:7)
5f
CHCl3-CH3CN-CH3OH
(86:7:7)
10a
10b
10c
10d
10e
10f
CHCl3-CH3CN-CH3OH
(90:5:5)
CHCl3-CH3CN-CH3OH
(90:5:5)
CHCl3-CH3CN-cC6H12
(80:10:10)
CHCl3-CH3CN-CH3OH
(90:5:5)
CHCl3-CH3CN-CH3OH
(90:5:5)
CHCl3-CH3CN-CH3OH
(90:5:5)
Ta ble 2. Preparation of Compounds in Schemes 1-3
compd
R
time (h)
16
chromatography solvent
yield (%)
69
mp (°C)
3a
cC3H5
CHCl3-C6H6-CH3CN-CH3OH
(60:20:10:10)a
102-104
3b
3c
cC4H7
cC5H9
cC6H11
cC7H13
cC8H15
cC3H5
cC4H7
cC5H9
cC6H11
cC7H13
cC8H15
cC3H5
cC4H7
cC5H9
cC6H11
cC7H13
cC8H15
16
5
CHCl3-C6H6-CH3CN-CH3OH
62
70
67
46
47
40
78
70
68
60
50
80
72
60
82
80
82
150-157
94-96
(62:20:10:8)a
AcOEt-CH3OH
(95:5)
3d
5
AcOEt-CH3OH
134-137
155-160
172-165
(95:5)
3e
24
24
10
10
16
16
24
24
8
CHCl3-C6H6-CH3CN-CH3OH
(60:20:10:10)a
3f
CHCl3-C6H14-CH3OH
(78:20:2)a
6a
CHCl3-CH3CN-CH3OH
171-174
(80:10:10)
lit.22b 184-187
130-132
6b
CHCl3-CH3CN-CH3OH
(76:12:12)
lit.22c 121-123
120-122
6c
CHCl3-CH3CN-CH3OH
(84:8:8)
lit.22a 77-81
186-188
6d
CHCl3-CH3CN-CH3OH
(84:8:8)
lit.22a 187-188
234-245
6e
CHCl3-CH3CN-CH3OH
(83:8.5:8.5)
lit.22a 93-97
238-246
6f
CHCl3-CH3CN-CH3OH
(84:8:8)
lit.22a 167-168
155-157
11a
11b
11c
11d
11e
11f
CHCl3-cC6H12-CH3CN-CH3OH
(76:10:7:7)
8
CHCl3-cC6H12-CH3CN-CH3OH
(76:10:7:7)
163-165
8
CHCl3-cC6H12-CH3CN-CH3OH
158-160
(76:10:7:7)
lit.11 151
8
CHCl3-CH3CN-CH3OH
(86:7:7)
158-160
lit.11 158-159
178-180
14
14
CHCl3-cC6H12-CH3CN-CH3OH
(80:10:5:5)
CHCl3-CH3CN-CH3OH
(86:7:7)
85-89

254 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Vittori et al.
Ta ble 3. A1 and A2A Adenosine Receptor Affinities of
2,N-Substituted 1-Deazaadenosine Derivatives
Ta ble 5. Intrinsic Activity of Selected 2,N-Substituted
1-Deazaadenosine and Adenosine Derivatives
A1 selectivity
[
35S]GTPγSa
compd
R
R1 Ki A1 (nM)a Ki A2A (nM)b (Ki A2A/Ki A1)
compd
R
R1
Cl
X
EC50
RIAb
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
2f
cC3H5 Cl 151 ( 55
cC3H5 326 ( 91
cC4H7 Cl 41.0 ( 19
cC4H7 109 ( 42
cC5H9 Cl 49.0 ( 10
cC5H9 100 ( 12
cC6H11 Cl 268 ( 146
cC6H11 169 ( 49
cC7H13 Cl 247 ( 133
cC7H13 364 ( 92
9640 ( 2260
16400 ( 3480
3110 ( 1510
6550 ( 1740
2840 ( 340
10100 ( 3820
5210 ( 1590
5120 ( 1570
4870 ( 940
9200 ( 2030
20000 ( 8750
13700 ( 2430
3370 ( 2660
64
50
76
59
58
101
19
30
19
25
2a
cC3H5
CH
704
(538-922)
1530
88.0 ( 2.1
H
3a
2c
3c
2e
3e
5a
6a
5c
6c
5e
6e
cC3H5
cC5H9
cC5H9
cC7H13
cC7H13
cC3H5
cC3H5
cC5H9
cC5H9
cC7H13
cC7H13
H
CH
CH
CH
CH
CH
N
91.8 ( 2.3
87.8 ( 2.8
96.0 ( 1.2
98.7 ( 1.0
94.9 ( 2.1
100.2 ( 3.3
103.3 ( 1.3
100
H
(1280-1820)
Cl
H
408
(289-575)
475
H
H
(400-563)
Cl
H
6580
H
(5270-8230)
6360
(3840-10500)
cC8H15 Cl 595 ( 32
cC8H15
34
25
665
3f
CPA
H
539 ( 119
5.07 ( 1.90
Cl
H
57.3
Displacement of [3H]DPCPX from rat cortical membranes
a
(55.5-59.2)
709
expressed as Ki (nM). Displacement of [3H]CGS 21680 from rat
b
N
(564-891)
striatal membranes expressed as Ki (nM).
Cl
H
N
30.0
Ta ble 4. A1 and A2A Adenosine Receptor Affinities of
(25.7-35.1)
13.3
(10.1-17.5)
2,N-Substituted Adenosine Derivatives
N
101.6 ( 2.7
100.8 ( 1.8
101.5 ( 3.7
Cl
H
N
431
(371-502)
86.4
N
(69.4-108)
a
Binding of [35S]GTPγS to rat brain membranes expressed as
b
EC50 (nM). RIA ) relative intrinsic activity (%).
(CCPA, 5c) was taken as 100% of the effect. The efficacy
of all the tested compounds was almost identical (rang-
ing from 90 to 100%), regardless of their affinities (Table
5). Therefore, these agonists were all considered to be
full agonists.
These data confirmed that the nitrogen atom at the
1-position of the purine ring is important, but not
critical, for adenosine receptor affinity, whereas intrinsic
activity is only marginally affected. The different size
of cycloalkyl substituents at the 6-position of purine and
1-deazapurine systems modulates the affinity, with the
highest affinity in both series shown by molecules
bearing four- and five-membered rings (2b, Ki A1 ) 40
nM, 2c, Ki A1 ) 50 nM and 6b, Ki A1 ) 3.92 nM, 6c, Ki
A1 ) 5.07 nM, respectively; Tables 3 and 4).
A1 selectivity
compd
5a
6a
5b
6b
R
R1 Ki A1 (nM)a Ki A2A (nM)b (Ki A2A/Ki A1)
cC3H5 Cl 12.6 ( 5.16 2060 ( 1150
cC3H5 26.7 ( 4.74 510 ( 490
cC4H7 Cl 5.30 ( 3.38 460 ( 20
cC4H7 3.92 ( 2.79 420 ( 110
163
19
87
107
86
665
19
29
29
161
35
78
H
H
5c (CCPA) cC5H9 Cl 7.43 ( 4.56 640 ( 220
6c (CPA) cC5H9
5d cC6H11 Cl 31.7 ( 3.1
6d (CHA) cC6H11
5e
6e
5f
6f
H
5.07 ( 1.90 3370 ( 2660
590 ( 130
H
21.0 ( 11.6 600 ( 130
cC7H13 Cl 37.9 ( 3.1 1100 ( 160
cC7H13
cC8H15 Cl 122 ( 71
cC8H15 53.5 ( 4.23 4180 ( 360
H
19.6 ( 2.3 3150 ( 950
4230 ( 930
H
Displacement of [3H]DPCPX from rat cortical membranes
a
expressed as Ki (nM). Displacement of [3H]CGS 21680 from rat
b
Deoxyr ib ose Der iva t ives. The 2′-deoxyadenosine
derivatives were evaluated for their A1 receptor affinity
on rat cortical membranes, both in the presence and
absence of GTP, using the antagonist [3H]DPCPX as
radioligand (Table 6). The Ki values of this series of
nucleosides for the A1 adenosine receptor were all in the
high nanomolar range, with the exception of 25 µM for
compound 11a .
striatal membranes expressed as Ki (nM).
In contrast to the 1-deaza analogues, the non-
chlorinated compounds show, in general, higher affinity
than the corresponding 2-chloro derivatives. At A2A
receptors, the new nucleosides proved to be weak
agonists, and the presence of the chlorine atom does not
seem to significantly affect the affinity.
KH and KL values for binding to the high- and low-
affinity state of the receptor and the percentage of
receptors in the high-affinity state (%RH) were also
calculated and reported in Table 6.
In addition, the potencies of the same nucleosides to
stimulate G protein activation via the A1 adenosine
Some compounds of the 1-deaza derivative series (2a ,
3a , 2c, 3c, 2e, and 3e) and the corresponding purine
analogues (5a , 6a , 5c, 6c, 5e, and 6e) were evaluated
for their relative intrinsic activity (RIA). The level of
maximum stimulation of [35S]GTPγS binding induced
by the full agonist 2-chloro-N-cyclopentyladenosine

N-Cycloalkyl Derivatives of Adenosine as A1 Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 255
Ta ble 6. A1 Adenosine Receptor Binding and G Protein Activation by 2,N-Substituted 2′-Deoxyadenosine Derivatives
[3H]DPCPX without GTPa
[3H]DPCPX + 1 mM GTPa
[
35S]GTPγSb
compd
R
R1 Ki A1 (nM)
KH
KL
KL:KH
%RH
Ki
SHIFT
EC50
RIAc
CCPA cC5H9 Cl
0.50
0.065
11.2
183 ( 28
58.6 ( 0.9
46.4
(43.5-49.5)
44.7
(40.9-48.8)
14300
96.8 ( 11.2
32.6
(24.6-43.2)
16.0
(14.6-17.5)
6180
100
(0.37-0.68) (0.039-0.11) (9.13-13.7)
0.43 0.052 10.1
(0.34-0.54) (0.038-0.070) (7.88-12.9)
724
(315-1667)
25200
(9060-70000)
245
(217-276)
524
(464-591)
154
(140-170)
212
(190-236)
497
(425-580)
412
(363-468)
369
(315-435)
769
(565-1050)
659
(494-880)
586
CPA cC5H9
cC3H5 Cl
cC3H5
cC4H7 Cl
cC4H7
cC5H9 Cl
cC5H9
cC6H11 Cl
cC6H11
cC7H13 Cl
cC7H13
cC8H15 Cl
cC8H15
H
201 ( 23
58.8 ( 1.2
107 ( 10.8
21.6 ( 6.0
6.1 ( 1.9
14.9 ( 1.5
45.1 ( 2.2
8.6 ( 0.2
48.8 ( 6.2
5.2 ( 0.5
41.0 ( 4.8
3.0 ( 0.1
27.3 ( 4.6
2.2 ( 0.3
16.0 ( 1.4
99.9 ( 1.6
10a
11a
10b
11b
10c
11c
10d
11d
10e
11e
10f
11f
115
6030
55.1 ( 11.6 44.2 ( 5.2
0
20.8 ( 0.9
(61.0-215) (4870-7450)
(11300-18000)
134000
(106000-170000)
3600
(3260-3970)
23600
(23000-24100)
1320
(1160-1500)
10200
(8610-12000)
2560
(2320-2830)
16700
(14200-19600)
1090
(5310-7200)
H
0
33.3
(25.2-44.0) (1600-2320)
121 8300
(85.6-170) (6560-10500)
18.5
(17.5-19.7)
39.3
(38.4-40.1) (3270-3800)
61.7 1590
(50.3-75.6) (1440-1750)
92.5 7330
(80.5-106) (5970-8990)
30.7
(26.1-36.1)
124
1920
58.0 ( 4.0 48.1 ( 2.0
69.1 ( 4.1 50.6 ( 1.7
43.2 ( 0.9 44.0 ( 0.4
90.4 ( 4.3 52.6 ( 0.7
25.9 ( 1.9 35.7 ( 1.3
79.9 ( 7.0 51.9 ( 0.1
24.6 ( 1.2 25.4 ( 2.0
59.6 ( 1.1 45.1 ( 2.0
45.9 ( 11.5 14.5 ( 3.0
50.4 ( 7.8 45.9 ( 3.0
3710
(1330-10400)
5110
(3950-6610)
1560
(1150-2120)
5760
(4670-7100)
19.5 ( 0.4
H
33.8 ( 2.4
800
(724-883)
3520
8.3 ( 1.0
H
42.1 ( 1.5
0
H
9640
(5880-15800)
40.1 ( 2.9
752
(623-906)
7410
0
(1010-1180)
20400
(19400-21300)
1410
(1270-1550)
9310
H
13700
(10500-17700)
24.9 ( 1.7
(95.8-162) (5740-9580)
23.6
(14.1-39.5)
84.9
1020
(975-1060)
4190
0
0
H
(489-702)
(75.6-95;4) (3510-4990)
(8670-10000)
a
Displacement of [3H]DPCPX from rat cortical membranes expressed as Ki, KH (affinity for the high-affinity state), or KL (affinity for
the low-affinity state) (all given in nM). %RH denotes the percentage of receptors in the high-affinity state. Binding of [35S]GTPγS to rat
b
brain membranes expressed as EC50 (nM). c RIA ) relative intrinsic activity with reference to CCPA, which was set as 100%.
receptor were determined in [35S]GTPγS binding assay
on rat brain membranes. In the right side of Table 6,
the EC50 values and the relative intrinsic activities
(RIA) with respect to CCPA of the 2′-deoxyadenosine
derivatives are reported. CCPA and CPA are used as
controls. None of the tested compounds proved to be full
agonists, the RIA values ranging from 0 to 42%.
Concerning structure-activity relationships, these
results showed that there is not a relevant effect of the
N-substituent ring size on affinity, although in both
series the N-cyclopentyl derivatives 10c and 11c showed
the highest affinity (Table 6).
Moreover, the 2-chloro substituted compounds showed
in general slightly higher affinities, in comparison with
the unsubstituted counterparts, in binding both to the
high- and the low-affinity state of the A1 receptors. It
is worthwhile noting that this profile is similar to that
showed by the 1-deazaadenosine derivatives (2a -f and
3a -f) and opposite to that shown by the corresponding
adenosine analogues (5a -f and 6a -f).
2′-position led to partial agonists at A1 adenosine
receptor (Table 6, right column), whereas the isosteric
substitution of 1-nitrogen by a -CH- affected only the
ligand potencies, but not intrinsic activities (Table 5).
Regarding the EC50 values from [35S]GTPγS binding
experiments, it is evident that in 2-chloro-2′-deoxy-
adenosine derivatives the potency increased with the
size of the N-substituent (from cyclopropyl 10a to
cyclopentyl 10c), whereas it showed the reverse ten-
dency for the non-chlorinated nucleosides, with an
optimum in both series for cC4 and cC5.
Regarding the intrinsic activities (Figure 2a), the
presence of a chlorine atom on C-2 was favorable only
in the case of the N-cyclopropyl substituted derivative
(10a , RIA ) 20.8% vs 11a , RIA ) 0%), all the other
2-chloro derivatives being less efficacious than the
corresponding 2-unsubstituted analogues. The highest
intrinsic activities are displayed by 2-dechlorinated
compounds bearing a cyclopentyl or cyclohexyl ring on
the 6-amino group (11c, RIA ) 42.1% and 11d , RIA )
40.1%). The GTP shift values showed exactly the same
trend (Figure 2b). Both the GTP shifts and [35S]GTPγS
binding experiments are equally indicative of full or
partial agonism at A1 adenosine receptors. We identified
a highly significant linear correlation between GTP
shifts obtained from receptor binding experiments and
intrinsic activities of CPA, CCPA, and the 2′-deoxy-
However, even though the affinity values of the 2′-
deoxyadenosine derivatives (modified on the sugar) are
comparable to those of the 1-deazaadenosine analogues
(modified on the purine moiety), these modifications of
adenosine structure have a very different effect on the
potencies of the corresponding compounds in G protein
activation. In fact, the lack of a hydroxyl group in the

256 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Vittori et al.
F igu r e 3. Relationship between binding to A1 adenosine
receptors in the high-affinity state and relative intrinsic
activity. The detection of A1 receptors by 2′-deoxyribose
adenosine derivatives, CPA, and CCPA was determined from
inhibition studies of [3H]DPCPX binding in rat brain mem-
branes to high-affinity sites in the absence of GTP (%RH).
Relative intrinsic activities (RIA) were measured as the
maximum stimulation of [35S]GTPγS binding in rat brain
membranes by ligands in relation to the full agonist CCPA.
age of receptor binding to the high-affinity state, but
they do not stimulate [35S]GTPγS binding.
In Figure 3 is reported the relationship between
intrinsic activity and the percentage of receptors de-
tected in the high-affinity state. The fact that this
relationship led to an exponential curve means that with
40% of receptor binding in the high-affinity state a
compound is almost inactive in G protein activation, as
assessed by stimulation of [35S]GTPγS binding. With
50% binding to high-affinity state receptors, the intrin-
sic activity is 50%, whereas with 60% binding to the
high-affinity state the intrinsic activity is 100%. These
results argue against a simple 1:1 relationship between
receptor occupancy and activation, and they indicate
that below a critical density of receptors in the high-
affinity state no stimulation can be achieved.
Further experiments were carried out to investigate
whether the 2′-deoxyadenosine derivatives acted at the
same A1 receptor site as CCPA and whether they also
exhibit antagonistic characteristics, as must be expected
for partial agonists. Therefore, [35S]GTPγS binding was
determined using a 100 µM concentration of nucleosides
in the absence or presence of a maximally stimulating
concentration of CCPA (1 µM). As shown in Figure 4,
most of the compounds induced stimulation of [35S]-
GTPγS binding somewhat above basal levels. In the
presence of CCPA, some of the 2′-deoxyribose deriva-
tives showed clear inhibitory effects, confirming the
partial agonist behavior. In particular, the N-cyclopentyl
derivatives 10c and 11c, with an RIA in stimulation of
about 8% and 42%, respectively, caused a reduction of
CCPA-induced [35S]GTPγS binding of about 50% and
15%, respectively. Therefore, even compounds which
show very low or no stimulatory intrinsic activity are
not inactive at A1 receptors. They inhibit agonist-
induced G protein activation, in good agreement with
their antagonist-like receptor binding mode, which is
characterized by low GTP shifts, low KL:KH ratios, and
with some precaution, detection of lower percentages
of receptors in the high-affinity state.
F igu r e 2. Structure-activity relationships for 2′-deoxyribose
derivatives of adenosine. Panels a-c show the influence of C
atom number in the cyclic N-substituent on relative intrinsic
activity (RIA, determined by stimulation of [35S]GTPγS bind-
ing; a), on GTP shifts in displacement of [3H]DPCPX binding
to rat brain membranes in the absence or presence of 1 mM
GTP, respectively (b), and on detection of receptors in the high-
affinity state in the absence of GTP (%RH; c).
adenosine derivatives from G protein activation studies
(r ) 0.9872, P < 0.001). As a further estimate of intrinsic
activity, the ratios between KL and KH values for binding
to the low- and high-affinity state of the receptor were
calculated (Table 6), based on the assumption that
agonists, but not antagonists, differentiate between G
protein-coupled receptors in the high-affinity state and
uncoupled receptors in the low-affinity state. In fact, we
found a higly significant linear correlation between KL:
KH ratios in receptor binding and relative intrinsic
activities determined in [35S]GTPγS binding experi-
ments (r ) 0.9796, P < 0.001).
The agonist intrinsic activity appears not directly
related to how many of the receptors are “seen” by the
ligand in the high-affinity state (Figure 2c). In fact, the
non-chlorinated compounds 11b-f show a high percent-

N-Cycloalkyl Derivatives of Adenosine as A1 Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 257
F igu r e 4. Stimulation of basal [35S]GTPγS binding and inhibition of CCPA-stimulated [35S]GTPγS binding by 2′-deoxyribose
adenosine derivatives. [35S]GTPγS binding to rat brain membranes (2 µg) was measured in the absence (open column and gray
columns) or presence (black columns) of 1 µM CCPA. 2′-Deoxyribose derivatives (100 µM concentration) stimulate basal [35S]-
GTPγS binding (gray columns) and inhibit the stimulatory effect of CCPA (black columns).
rated, and the residue was chromatographed on a silica gel
column eluting with the suitable mixture of solvents (Table
1) to give 2a -f as chromatographically pure solids. Compounds
2c and 2d were already described elsewhere.20
2a : 1H NMR (Me2SO-d6) δ 0.60 (m, 2H, H cyclopropyl), 0.80
(m, 2H, H cyclopropyl), 2.61 (m, 1H, H-1 cyclopropyl), 3.62 (m,
2H, CH2-5′), 3.95 (m, 1H, H-4′), 4.14 (m, 1H, H-3′), 4.57 (m,
1H, H-2′), 5.88 (d, 1H, J ) 6.1 Hz, H-1′), 6.60 (s, 1H, H-6),
7.68 (bs, 1H, NH), 8.36 (s, 1H, H-2). Anal. (C14H17ClN4O4) C,
H, N.
Con clu sion
Three series of adenosine analogues, all bearing
cycloalkyl substituents on the 6-amino group, have been
synthesized and evaluated to determine their affinities
and intrinsic activities at A1 adenosine receptor. All
compounds of the ribose-bearing series proved to be full
agonists, the 1-deaza derivatives showing affinities for
the A1 receptor about 10-fold lower than the correspond-
ing adenosine analogues. On the other hand, all the 2′-
deoxyribose derivatives bind to the A1 receptor with
affinities in the high nanomolar range. Both GTP shifts
and [35S]GTPγS experiments showed that most of the
2′-deoxyadenosine derivatives are partial agonists and
they detected fewer A1 receptors in the high-affinity
state than full agonists.
Hence, it has been proved that modifications of purine
moiety and of sugar in the adenosine structure have
very different effects on the potencies of the correspond-
ing compounds in G protein activation. In fact, the lack
of a hydroxyl group at the 2′-position led to partial
agonists at A1 adenosine receptor, whereas the isosteric
substitution of N-1 nitrogen by a carbon affected only
the ligand potencies, but not intrinsic activities.
Finally, it has been clearly demonstrated that there
was not a simple linear relationship between receptor
occupancy and activation and that below a critical
density of A1 adenosine receptors in the high-affinity
state no activation of G protein can be achieved.
2b: 1H NMR (Me2SO-d6) δ 1.75, 2.08, and 2.35 (m, 2H each,
H cyclobutyl), 3.62 (m, 2H, CH2-5′), 3.94 (m, 1H, H-4′), 4.15
(m, 1H, H-3′), 4.36 (bs, 1H, H-1 cyclobutyl), 4.58 (m, 1H, H-2′),
5.88 (d, 1H, J ) 6.1 Hz, H-1′), 6.34 (s, 1H, H-6), 7.52 (bs, 1H,
NH), 8.37 (s, 1H, H-2). Anal. (C15H19ClN4O4) C, H, N.
1
2e: H NMR (Me2SO-d6) δ 1.50-1.70 (m, 10H, H cyclohep-
tyl), 1.93 (m, 2H, cycloheptyl), 3.60 (m, 2H, CH2-5′), 3.95 (m,
1H, H-4′), 4.14 (m, 2H, H-3′ and H-1 cycloheptyl), 4.57 (m, 1H,
H-2′), 5.87 (d, 1H, J ) 6.0 Hz, H-1′), 6.37 (s, 1H, H-6), 6.99 (d,
1H, J ) 8.8 Hz, NH), 8.34 (s, 1H, H-2). Anal. (C18H25ClN4O4)
C, H, N.
2f: 1H NMR (Me2SO-d6) δ 1.51-1.84 (m, 14H, H cyclooctyl),
3.60 (m, 2H, CH2-5′), 3.95 (m, 1H, H-4′), 4.13 (m, 2H, H-3′ and
H-1 cyclooctyl), 4.57 (m, 1H, H-2′), 5.86 (d, 1H, J ) 6.0 Hz,
H-1′), 6.37 (s, 1H, H-6), 6.99 (d, 1H, J ) 8.7 Hz, NH), 8.34 (s,
1H, H-2). Anal. (C19H27ClN4O4) C, H, N.
P r ep a r a tion of 7-Cycloa lk yla m in o-(â-D-r ibofu r a n osyl)-
3H-im id a zo-[4,5-b]p yr id in es (3a -f). To a solution of 2a -f
(0.5 mmol) in 40 mL of ethanol and 1 mL of 2 N NaOH was
added 0.050 g of 10% Pd/C, and the mixture was shaken with
hydrogen at the pressure and for the time listed in Table 2.
The catalyst was removed, and the filtrate was concentrated
to dryness. The residue was chromatographed on a silica gel
column eluting with the suitable mixture of solvents to give
3a -f (Table 2) as chromatographically pure solids. Compounds
3c and 3d were already described elsewhere.20
3a : 1H NMR (Me2SO-d6) δ 0.57 (m, 2H, H cyclopropyl), 0.78
(m, 2H, H cyclopropyl), 2.59 (m, 1H, H-1 cyclopropyl), 3.62 (m,
2H, CH2-5′), 4.00 (m, 1H, H-4′), 4.15 (m, 1H, H-3′), 4.71 (m,
1H, H-2′), 5.91 (d, 1H, J ) 6.4 Hz, H-1′), 6.64 (d, 1H, J 6,5 ) 5.7
Hz, H-6), 7.27 (bs, 1H, NH), 7.95 (d, 1H, J 5,6 ) 5.7 Hz, H-5),
8.29 (s, 1H, H-2). Anal. (C14H18N4O4) C, H, N.
3b: 1H NMR (Me2SO-d6) δ 1.71, 2.06, and 2.34 (m, 2H each,
H cyclobutyl), 3.61 (m, 2H, CH2-5′), 3.99 (m, 1H, H-4′), 4.14
(m, 1H, H-3′), 4.37 (bs, 1H, H-1 cyclobutyl), 4.70 (m, 1H, H-2′),
5.90 (d, 1H, J ) 6.6 Hz, H-1′), 6.33 (d, 1H, J 6,5 ) 5.7 Hz, H-6),
7.11 (d, 1H, J ) 7.0 Hz, NH), 7.95 (d, 1H, J 5,6 ) 5.7 Hz, H-5),
8.29 (s, 1H, H-2). Anal. (C15H20N4O4) C, H, N.
Exp er im en ta l Section
Ch em istr y. Melting points were determined with a Bu¨chi
apparatus and are uncorrected. 1H NMR spectra were obtained
with a Varian VX 300 MHz spectrometer. Thin-layer chroma-
tography (TLC) was carried out on precoated TLC plates with
silica gel 60 F-254 (Merck) and preparative TLC on precoated
Whatman 60A TLC plates. For column chromatography, silica
gel 60 (Merck) was used. Microanalytical results are within
(0.4% of theoretical values.
P r epar ation of 7-Cycloalkylam in o-5-ch lor o-3-(â-D-r ibo-
fu r a n osyl)-3H-im id a zo[4,5-b]p yr id in es (2a -f). A mixture
of 0.43 g (1.34 mmol) of 5,7-dichloro-3-(â-D-ribofuranosyl)-3H-
imidazo[4,5-b]pyridine (1)17 and 10 mL of the appropriate
amine was heated in a steel bomb at the temperature and for
the time listed in Table 1. The reaction mixture was evapo-

258 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Vittori et al.
1
3e: H NMR (Me2SO-d6) δ 1.51-1.71 (m, 10H, H cyclohep-
6f: 1H NMR (Me2SO-d6) δ 1.40-1.90 (m, 14H, H cyclooctyl),
3.60 (m, 2H, CH2-5′), 3.97 (m, 1H, H-4′), 4.15 (m, 2H, H-3′),
4.48 (m, 1H, H-1 cyclooctyl), 4.62 (m, 1H, H-2′), 5.89 (d, 1H, J
) 6.2 Hz, H-1′), 7.72 (d, 1H, J ) 7.6 Hz, NH), 8.20 (s, 1H,
H-2), 8.36 (s, 1H, H-8). Anal. (C18H27N5O4) C, H, N.
tyl), 1.93 (m, 2H, cycloheptyl), 3.60 (m, 2H, CH2-5′), 4.00 (m,
2H, H-4′ and H-1 cycloheptyl), 4.15 (m, 1H, H-3′), 4.71 (m, 1H,
H-2′), 5.89 (d, 1H, J ) 6.6 Hz, H-1′), 6.36 (d, 1H, J 6,5 ) 5.7 Hz,
H-6), 6.56 (d, 1H, J ) 8.6 Hz, NH), 7.84 (d, 1H, J 5,6 ) 5.7 Hz,
H-5), 8.27 (s, 1H, H-2). Anal. (C18H26N4O4) C, H, N.
3f: 1H NMR (Me2SO-d6) δ 1.53-1.84 (m, 14H, H cyclooctyl),
3.60 (m, 2H, CH2-5′), 3.92 (m, 1H, H-4′), 4.12 (m, 2H, H-3′ and
H-1 cyclooctyl), 4.71 (m, 1H, H-2′), 5.89 (d, 1H, J ) 6.6 Hz,
H-1′), 6.36 (d, 1H, J 6,5 ) 5.7 Hz, H-6), 6.56 (d, 1H, J ) 8.6 Hz,
NH), 7.84 (d, 1H, J 5,6 ) 5.7 Hz, H-5), 8.27 (s, 1H, H-2). Anal.
(C19H28N4O4) C, H, N.
P r ep a r a t ion of 2-Ch lor o-6-cycloa lk yla m in o-9-(â-D-
r ibofu r a n osyl)-9H-p u r in es (2-Ch lor o-N-cycloa lk yla d en -
osin es 5a -f). A mixture of 0.3 g (0.67 mmol) of 2,6-dichloro-
9-(2,3,5-tri-O-acetyl-â-D-ribofuranosyl)-9H-purine (4)21 and 2
mL of the appropriate amine was stirred at room temperature
for 4 h. The exceeding amine was evaporated in vacuo,
methanol saturated at 0 °C with ammonia was added to the
residue, and the mixture was stirred at room temperature for
the time reported in Table 1. The reaction mixture was
evaporated, and the residue was chromatographed on a silica
gel column eluting with the suitable mixture of solvents (Table
1) to give 5a -f as chromatographically pure solids. Compounds
5a 24 and 5c25 were already described elsewhere.
5b: 1H NMR (Me2SO-d6) δ 1.71 (m, 2H, H cyclobutyl), 2.01-
2.38 (m, 4H, H cyclobutyl), 3.61 (m, 2H, CH2-5′), 3.95 (m, 1H,
H-4′), 4.13 (m, 1H, H-3′), 4.51 (m, 1H, H-2′), 4.61 (m, 1H, H-1
cyclobutyl), 5.83 (d, 1H, J ) 5.9 Hz, H-1′), 8.41 (s, 1H, H-8),
8.65 (m, 1H, NH). Anal. (C14H18ClN5O4) C, H, N.
P r epar ation of 2-Ch lor o-6-cycloalkylam in o-9-(2-deoxy-
3,5-d i-O-p -t olu oyl-â-D-er yth r o-p en t ofu r a n osyl)-9H -p u -
r in es (9a -f). A mixture of 0.35 g (0.65 mmol) of 2,6-dichloro-
9-(2-deoxy-3,5-di-O-p-toluoyl-â-D-erythro-pentofuranosyl)-9H-
purine (8)23 and 3 mL of the appropriate amine was stirred at
room temperature for 4 h. The exceeding amine was evapo-
rated in vacuo, and the residue was used for the next step
without further purification.
Analytical samples of 9a -f were obtained by preparative
TLC eluting with cC6H12-EtOAc (65:35).
9a : 1H NMR (Me2SO-d6) δ 0.65 (m, 2H, H cyclopropyl), 0.76
(m, 2H, H cyclopropyl), 2.38 (s, 3H, CH3), 2.42 (s, 3H, CH3),
2.81 (m, 1H, H-2′), 3.00 (m, 1H, H-1 cyclopropyl), 3.20 (m, 1H,
H-2′′), 4.58 (m, 3H, CH2-5′ and H-4′), 5.77 (m, 1H, H-3′), 6.47
(t, 1H, J ) 6.5 Hz, H-1′), 7.34 (m, 4H, H-Ph), 7.84 (d, 2H, J
) 8.1 Hz, H-Ph), 7.97 (d, 2H, J ) 8.1 Hz, H-Ph), 8.39 (s, 1H,
H-8), 8.52 (bs, 1H, NH). Anal. (C29H28ClN5O5) C, H, N.
9b: 1H NMR (Me2SO-d6) δ 1.71 (m, 2H, H cyclobutyl), 1.97-
2.33 (m, 4H, H cyclobutyl), 2.40 (s, 3H, CH3), 2.52 (s, 3H, CH3),
2.81 (m, 1H, H-2′), 3.24 (m, 1H, H-2′′), 4.58 (m, 2H, CH2-5′),
4.86 (m, 2H, H-4′ and H-1 cyclobutyl), 5.80 (m, 1H, H-3′), 6.46
(t, 1H, J ) 7.2 Hz, H-1′), 7.34 (m, 4H, H-Ph), 7.90 (m, 4H,
H-Ph), 8.40 (s, 1H, H-8), 8.69 (m, 1H, NH). Anal. (C30H30
ClN5O5) C, H, N.
-
5d : 1H NMR (Me2SO-d6) δ 1.00-2.00 (m, 10H, H cyclohexyl),
3.63 (m, 2H, CH2-5′), 3.97 (m, 1H, H-4′), 4.15 (m, 2H, H-3′ and
H-1 cyclohexyl), 4.52 (m, 1H, H-2′), 5.82 (d, 1H, J ) 5.5 Hz,
H-1′), 8.15 (d, 1H, J ) 8.0 Hz, NH), 8.40 (s, 1H, H-8). Anal.
(C16H22ClN5O4) C, H, N.
9c: 1H NMR (Me2SO-d6) δ 1.47-1.91 (m, 6H, H cyclopentyl),
1.96 (m, 2H, H cyclopentyl), 2.38 (s, 3H, CH3), 2.42 (s, 3H,
CH3), 2.79 (m, 1H, H-2′), 3.22 (m, 1H, H-2′′), 4.37-4.68 (m,
4H, CH2-5′, H-4′, and H-1 cyclopentyl), 5.78 (m, 1H, H-3′), 6.47
(t, 1H, J ) 6.6 Hz, H-1′), 7.35 (m, 4H, H-Ph), 7.84 (d, 2H, J
) 8.1 Hz, H-Ph), 7.97 (d, 2H, J ) 8.1 Hz, H-Ph), 8.38 (bs,
2H, H-8 and NH). Anal. (C31H32ClN5O5) C, H, N.
1
5e: H NMR (Me2SO-d6) δ 1.35-1.78 (m, 10H, H cyclohep-
tyl), 1.90 (m, 2H, cycloheptyl), 3.61 (m, 2H, CH2-5′), 3.95 (m,
1H, H-4′), 4.15 (m, 2H, H-3′ and H-1 cycloheptyl), 4.52 (m, 1H,
H-2′), 5.96 (d, 1H, J ) 5.8 Hz, H-1′), 8.29 (d, 1H, J ) 8.1 Hz,
NH), 8.40 (s, 1H, H-8). Anal. (C17H24ClN5O4) C, H, N.
5f: 1H NMR (Me2SO-d6) δ 1.41-1.88 (m, 14H, H cyclooctyl),
3.60 (m, 2H, CH2-5′), 3.95 (m, 1H, H-4′), 4.13 (m, 2H, H-3′),
4.26 (m, 1H, H-1 cyclooctyl), 4.52 (m, 1H, H-2′), 5.83 (d, 1H, J
) 6.1 Hz, H-1′), 8.29 (d, 1H, J ) 8.1 Hz, NH), 8.39 (s, 1H,
H-8). Anal. (C18H26ClN5O4) C, H, N.
9d : 1H NMR (Me2SO-d6) δ 1.47-1.91 (m, 8H, H cyclohexyl),
1.96 (m, 2H, H cyclohexyl), 2.38 (s, 3H, CH3), 2.42 (s, 3H, CH3),
2.80 (m, 1H, H-2′), 3.20 (m, 1H, H-2′′), 4.00 (m, 1H, H-1
cyclohexyl), 4.58 (m, 3H, CH2-5′ and H-4′,), 5.80 (m, 1H, H-3′),
6.47 (t, 1H, J ) 6.3 Hz, H-1′), 7.35 (m, 4H, H-Ph), 7.84 (d,
2H, J ) 8.2 Hz, H-Ph), 7.97 (d, 2H, J ) 8.1 Hz, H-Ph), 8.25
(d, 1H, J ) 8.8 Hz, NH), 8.40 (s, 1H, H-8). Anal. (C32H34ClN5O5)
C, H, N.
P r ep a r a tion of 6-Cycloa lk yla m in o-9-(â-D-r ibofu r a n o-
syl)-9H-p u r in es (N-Cycloa lk yla d en osin es 6a -f). To a
solution of 5a -f (0.5 mmol) in 40 mL of ethanol and 1.5 mL of
2 N NaOH was added 0.050 g of 10% Pd/C, and the mixture
was shaken with hydrogen at 45 psi for the time reported in
Table 2. The catalyst was removed, and the filtrate was
concentrated to dryness. The residue was chromatographed
on a silica gel column eluting with the suitable mixture of
solvents to give 6a -f (Table 2) as chromatographically pure
solids. Compounds 6a -f were already synthesized starting
from 6-chloropurine riboside (7).22 However, they were not
1
9e: H NMR (Me2SO-d6) δ 1.41-1.78 (m, 10H, H cyclohep-
tyl), 1.90 (m, 2H, H cycloheptyl), 2.38 (s, 3H, CH3), 2.42 (s,
3H, CH3), 2.81 (m, 1H, H-2′), 3.21 (m, 1H, H-2′′), 4.18 (m, 1H,
H-1 cycloheptyl), 4.59 (m, 3H, CH2-5′ and H-4′), 5.79 (m, 1H,
H-3′), 6.47 (t, 1H, J ) 7.0 Hz, H-1′), 7.35 (m, 4H, H-Ph), 7.84
(d, 2H, J ) 8.2 Hz, H-Ph), 7.97 (d, 2H, J ) 8.1 Hz, H-Ph),
8.29 (d, 1H, J ) 8.7 Hz, NH), 8.40 (s, 1H, H-8). Anal. (C33H36
ClN5O5) C, H, N.
-
9f: 1H NMR (Me2SO-d6) δ 1.55 (m, 12H, H cyclooctyl), 1.77
(m, 2H, H cyclooctyl), 2.38 (s, 3H, CH3), 2.42 (s, 3H, CH3), 2.81
(m, 1H, H-2′), 3.21 (m, 1H, H-2′′), 4.25 (m, 1H, H-1 cyclooctyl),
4.59 (m, 3H, CH2-5′ and H-4′), 5.79 (m, 1H, H-3′), 6.47 (t, 1H,
J ) 7.1 Hz, H-1′), 7.34 (m, 4H, H-Ph), 7.83 (d, 2H, J ) 8.2
Hz, H-Ph), 7.97 (d, 2H, J ) 8.1 Hz, H-Ph), 8.30 (d, 1H, J )
8.6 Hz, NH), 8.42 (s, 1H, H-8). Anal. (C34H38ClN5O5) C, H, N.
1
characterized by H NMR spectra. Compounds 6c and 6d are
also commercially available.
6a : 1H NMR (Me2SO-d6) δ 0.64 (m, 2H, H cyclopropyl), 0.74
(m, 2H, H cyclopropyl), 3.07 (m, 1H, H-1 cyclopropyl), 3.60 (m,
2H, CH2-5′), 3.97 (m, 1H, H-4′), 4.15 (m, 1H, H-3′), 4.61 (m,
1H, H-2′), 5.9 (d, 1H, J ) 6.1 Hz, H-1′), 8.03 (bs, 1H, NH),
8.25 (s, 1H, H-2), 8.37 (s, 1H, H-8). Anal. (C13H17N5O4) C, H,
N.
P r epar ation of 2-Ch lor o-6-cycloalkylam in o-9-(2-deoxy-
â-D-er yth r o-p en tofu r a n osyl)-9H-p u r in es (2′-Deoxy-2-ch lo-
r o-N-cycloa lk yl Ad en osin es 10a -f). To the crude mixture
of 9a -f 20 mL of methanol saturated at 0 °C with ammonia
was added, and the reaction was allowed to stand at room
temperature for the time reported in Table 1. After evaporation
under vacuum, the residue was chromatographed on a silica
gel column eluting with the suitable mixture of solvents (Table
1) to give 10a -f as chromatographically pure solids. Com-
pound 10d was already described elsewhere.26
6b: 1H NMR (Me2SO-d6) δ 1.67 (m, 2H, H cyclobutyl), 2.01-
2.35 (m, 4H, H cyclobutyl), 3.59 (m, 2H, CH2-5′), 3.97 (m, 1H,
H-4′), 4.15 (m, 1H, H-3′), 4.61 (m, 1H, H-2′), 4.73 (m, 1H, H-1
cyclobutyl), 5.89 (d, 1H, J ) 5.9 Hz, H-1′), 8.18 (m, 2H, H-2
and NH), 8.38 (s, 1H, H-8). Anal. (C14H19N5O4) C, H, N.
1
6e: H NMR (Me2SO-d6) δ 1.35-1.98 (m, 12H, H cyclohep-
tyl), 3.60 (m, 2H, CH2-5′), 3.97 (m, 1H, H-4′), 4.14 (m, 2H, H-3′),
4.29 (m, 1H, H-1 cycloheptyl), 4.61 (m, 1H, H-2′), 5.88 (d, 1H,
J ) 5.9 Hz, H-1′), 7.69 (d, 1H, J ) 7.9 Hz, NH), 8.19 (s, 1H,
H-2), 8.35 (s, 1H, H-8). Anal. (C17H25N5O4) C, H, N.
1
10a : H NMR (Me2SO-d6) δ 0.66 (m, 2H, cyclopropyl), 0.75
(m, 2H, H cyclopropyl), 2.30 (m, 1H, H-2′), 2.64 (m, 1H, H-2′′),
2.99 (m, 1H, H-1 cyclopropyl), 3.57 (m, 2H, CH2-5′), 3.82 (m,

N-Cycloalkyl Derivatives of Adenosine as A1 Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 259
1H, H-4′), 4.40 (m, 1H, H-3′), 6.28 (t, 1H, J ) 6.8 Hz, H-1′),
8.37 (s, 1H, H-8), 8.47 (bs, 1H, NH). Anal. (C13H16ClN5O3) C,
H, N.
followed the protocol of J arvis et al.31 Ki values are the means
of three independent experiments.
Ch a r a cter iza tion of Bin d in g of Ad en osin e Der iva tives
to High - a n d Low -Affin ity Sta tes of th e A1 Ad en osin e
R ecep t or . Rat brain membranes were prepared as de-
scribed.32 [3H]DPCPX binding (0.2 nM) to rat brain membranes
was performed in 50 mM Tris-HCl buffer pH 7.4 in a total
volume of 500 µL containing 40 µg of membrane protein, 0.5
units/mL adenosine deaminase, 0.02% 3-([3-cholamidopropyl]-
dimethylammonio)-1-propanesulfonate (CHAPS), and increas-
ing concentrations of unlabeled CPA, CCPA, or 2′-deoxy-
adenosine derivatives (10a -f, 11a -f). The assays were per-
formed both in the absence and presence of 1 mM GTP.
Incubations were carried out for 3 h at 25 °C and were
terminated by rapid filtration through Whatman GF/B glass
fiber filters. Tubes and filters were washed twice with 4 mL
of 50 mM Tris-HCl buffer pH 7.4, containing 0.02% CHAPS.
All results are from three to seven independent experiments.
10b: 1H NMR (Me2SO-d6) δ 1.71 (m, 2H, H cyclobutyl),
2.00-2.40 (m, 5H, H cyclobutyl and H-2′), 2.63 (m, 1H, H-2′′),
3.58 (m, 2H, CH2-5′), 3.89 (m, 1H, H-4′), 4.40 (m, 1H, H-3′),
4.60 (m, 1H, H-1 cyclobutyl), 6.26 (t, 1H, J ) 6.9 Hz, H-1′),
8.39 (s, 1H, H-8), 8.61 (m, 1H, NH). Anal. (C14H18ClN5O3) C,
H, N.
1
10c: H NMR (Me2SO-d6) δ 1.48-1.79 (m, 6H, H cyclopen-
tyl), 1.94 (m, 2H, H cyclopentyl), 2.29 (m, 1H, H-2′), 2.64 (m,
1H, H-2′′), 3.57 (m, 2H, CH2-5′), 3.87 (m, 1H, H-4′), 4.40 (m,
1H, H-3′ and H-1 cyclopentyl), 6.28 (t, 1H, J ) 6.8 Hz, H-1′),
8.31 (s, 1H, NH), 8.37 (s, 1H, H-8). Anal. (C15H20ClN5O3) C,
H, N.
10e: 1H NMR (Me2SO-d6) δ 1.43-1.78 (m, 10H, H cyclohep-
tyl), 1.87 (m, 2H, H cycloheptyl), 2.28 (m, 1H, H-2′), 2.63 (m,
1H, H-2′′), 3.56 (m, 2H, CH2-5′), 3.86 (m, 1H, H-4′), 4.17 (m,
1H, H-1 cycloheptyl), 4.38 (m, 1H, H-3′), 6.26 (t, 1H, J ) 7.3
Hz, H-1′), 8.24 (d, 1H, J ) 8.4 Hz, NH), 8.36 (s, 1H, H-8). Anal.
(C17H24ClN5O3) C, H, N.
10f: 1H NMR (Me2SO-d6) δ 1.40-1.90 (m, 14H, H cyclooctyl),
2.30 (m, 1H, H-2′), 2.63 (m, 1H, H-2′′), 3.57 (m, 2H, CH2-5′),
3.87 (m, 1H, H-4′), 4.25 (m, 1H, H-1 cyclooctyl), 4.39 (m, 1H,
H-3′), 6.27 (t, 1H, J ) 6.8 Hz, H-1′), 7.34 (m, 4H, H-Ph), 7.83
(d, 2H, H-Ph), 7.97 (d, 2H, H-Ph), 8.25 (d, 1H, J ) 8.4 Hz,
NH), 8.36 (s, 1H, H-8). Anal. (C18H26ClN5O3) C, H, N.
[
35S]GTP γS Bin d in g to Ra t Br a in Mem br a n es. Stimula-
tion of [35S]GTPγS binding to rat brain membranes by ad-
enosine A1 receptor activation was carried out as described
previously.8,15 The intrinsic activities of compounds under
study are given as relative intrinsic activities (RIA) with
reference to CCPA as a standard full agonist with its intrinsic
activity set as 100%. Potencies and intrinsic activities of
agonists are from 3 to 12 independent experiments for each
compound.
Da ta An a lysis. Apparent Ki values for A1 or A2A receptor
binding were calculated from displacement curves of [3H]-
DPCPX and [3H]CGS 21680 by nonlinear regression with
Prism (Graph Pad, San Diego, CA). In a more detailed study
of binding of 2′-deoxyadenosine derivatives to high- and low-
affinity states of the A1 receptor, competition curves were
analyzed with SCTFIT.33 All competition curves were analyzed
according to one- and two-state models. Ki values are affinities
calculated assuming only a single affinity state. KH values are
the affinities for the high-affinity state, KL values are the
affinities for the low-affinity state of the A1 adenosine receptor
calculated from nonlinear curve fitting according to a two-state
model. Curves were fitted to a one-state model only if fitting
to a two-state model did not improve the fit significantly (P <
0.05). Ki, KH, and KL values are geometric means and are
reported with 95% confidence limits from three to seven
independent experiments for each compound. Percentages of
A1 receptors in the high-affinity state (%RH) are given as the
arithmetic means with standard errors (SEM). GTP shifts were
calculated from the ratios of Ki values in the presence and in
the absence of GTP and are arithmetic means ( SEM. EC50
values for stimulation of [35S]GTPγS binding were calculated
with Sigma Plot and are reported as geometric means with
95% confidence limits. Relative intrinsic activities are arith-
metic means ( SEM.
P r ep a r a t ion of 6-Cycloa lk yla m in o-9-(2-d eoxy-â-D-
er yth r o-p en tofu r a n osyl)-9H-p u r in es (2′-Deoxy-N-cyclo-
a lk yla d en osin es 11a -f). To a solution of 10a -f (0.5 mmol)
in 40 mL of ethanol and 1 mL of 2 N NaOH was added 0.1 g
of 10% Pd/C, and the mixture was shaken with hydrogen at
45 psi and for the time listed in Table 2. The catalyst was
removed, and the filtrate was concentrated to dryness. The
residue was chromatographed on a silica gel column eluting
with the suitable mixture of solvents to give 11a -f (Table 2)
as chromatographically pure solids. Compounds 11c and 11d
were already described elsewhere.11
1
11a : H NMR (Me2SO-d6) δ 0.64 (m, 2H, cyclopropyl), 0.74
(m, 2H, H cyclopropyl), 2.29 (m, 1H, H-2′), 2.74 (m, 1H, H-2′′),
3.08 (m, 1H, H-1 cyclopropyl), 3.57 (m, 2H, CH2-5′), 3.90 (m,
1H, H-4′), 4.43 (m, 1H, H-3′), 6.37 (t, 1H, J ) 6.2 Hz, H-1′),
7.97 (d, 1H, J ) 4.1 Hz, NH), 8.25 (s, 1H, H-2), 8.35 (s, 1H,
H-8). Anal. (C13H17N5O3) C, H, N.
11b: 1H NMR (Me2SO-d6) δ 1.80 (m, 2H, H cyclobutyl),
2.04-2.35 (m, 5H, H cyclobutyl and H-2′), 2.69 (m, 1H, H-2′′),
3.59 (m, 2H, CH2-5′), 3.90 (m, 1H, H-4′), 4.42 (m, 1H, H-3′),
4.73 (m, 1H, H-1 cyclobutyl), 6.36 (t, 1H, J ) 6.6 Hz, H-1′),
8.08 (d, 1H, J ) 1.9 Hz, NH), 8.19 (s, 1H, H-2), 8.36 (s, 1H,
H-8). Anal. (C14H19N5O3) C, H, N.
11e: 1H NMR (Me2SO-d6) δ 1.25-1.75 (m, 10H, H cyclohep-
tyl), 1.89 (m, 2H, H cycloheptyl), 2.28 (m, 1H, H-2′), 2.73 (m,
1H, H-2′′), 3.57 (m, 2H, CH2-5′), 3.89 (m, 1H, H-4′), 4.31 (m,
1H, H-1 cycloheptyl), 4.41 (m, 1H, H-3′), 6.35 (t, 1H, J ) 6.2
Hz, H-1′), 7.66 (d, 1H, J ) 8.5 Hz, NH), 8.20 (s, 1H, H-2), 8.34
(s, 1H, H-8). Anal. (C17H25N5O3) C, H, N.
11f: 1H NMR (Me2SO-d6) δ 1.40-1.85 (m, 14H, H cyclooctyl),
2.28 (m, 1H, H-2′), 2.75 (m, 1H, H-2′′), 3.60 (m, 2H, CH2-5′),
3.89 (m, 1H, H-4′), 4.41 (m, 1H, H-3′ and H-1 cyclooctyl), 6.36
(t, 1H, J ) 6.0 Hz, H-1′), 7.62 (d, 1H, J ) 8.5 Hz, NH), 8.10 (s,
1H, H-2), 8.33 (s, 1H, H-8). Anal. (C18H27N5O3) C, H, N.
Biologica l Assa ys. Ad en osin e Recep tor Su btyp e Se-
lectivity. A1 selectivity of derivatives was assessed essentially
as described.16 Membranes from rat brain cortex were prepared
as described by Lohse et al.,27 but incubated with adenosine
deaminase before storage, as described by Pirovano et al.28
Inhibition of [3H]DPCPX binding to A1 adenosine receptors in
rat cortical membranes was performed according to Lohse et
al.29 For determination of affinity to A2A adenosine receptors,
membranes from rat striatum were used. Membranes from rat
striata were prepared as described by Bruns et al.30 and
pretreated with adenosine deaminase before storage. The
method of binding of the A2A-selective agonist [3H]CGS 21680
Ack n ow led gm en t. This work has been presented
in a preliminary form at the 1999 annual meeting of
the Italian chapter of the Purine Club and was sup-
ported by EC Contract No. BMH4-98-3474 and by a
grant from the University of Camerino. We thank M.
Brandi, F. Lupidi, and G. Rafaiani for technical as-
sistance.
Su p p or tin g In for m a tion Ava ila ble: Elemental analyses
of new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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