N6,C8-distributed adenosine derivatives as partial agonists for adenosine A1 receptors.

Article abstract of DOI:10.1021/jm950267m

The synthesis and biological evaluation of N6, C8-disubstituted derivatives of adenosine as potential partial agonists for adenosine receptors is described. Via three routes, two series of compounds were prepared, viz., N6-cyclopentyladenosine derivatives 3a-e and C8-(cyclopentylamino)adenosine analogs 3e and 9a-d, respectively. The X-ray structure determination of one of these compounds, N6-ethyl-8(cyclopentylamino)adenosine (9b), was carried out (orthorhombic, space group P2(1)2(1)2(1) (No. 19) with a = 11.039(3), b = 8.708(2), and c = 24.815(12) angstrom, Z=4,R1=0.0974,R2(W) = 0.2455). Due to intramolecular hydrogen bonding, the ribose moiety of this compound is in an anti conformation. The compounds were tested in vitro in radioligand binding studies, yielding their affinities for A1 and A2a adenosine receptors. All compounds appeared A1 selective, with affinities in the high nanomolar, low micromolar range. On A1 receptors the so-called GTP shift was also determined, i.e., the ratio between the affinities measured in the presence and absence of 1 mM GTP. All GTP shifts (values between 1.1 and 3.8) were lower than the GTP shift for CPA (6.0). This GTP shift appeared indicative for partial agonism in vivo, since the N6-cyclopentyladenosine derivatives showed lower intrinsic activities than the prototypic full agonist N6-cyclopentyladenosine on the decrease in heart rate in conscious, normotensive rats.

Full text of DOI:10.1021/jm950267m

J . Med. Chem. 1996, 39, 1463-1471
1463
N6,C8-Disu bstitu ted Ad en osin e Der iva tives a s P a r tia l Agon ists for Ad en osin e A1
Recep tor s†
Harlof Roelen, Nora Veldman,‡ Anthony L. Spek,‡,| J acobien von Frijtag Drabbe Ku¨nzel, Ron A. A. Mathoˆt,§ and
Ad P. IJ zerman*
Leiden/ Amsterdam Center for Drug Research, Divisions of Medicinal Chemistry and Pharmacology, P.O. Box 9502,
2300RA Leiden, The Netherlands, and Bijvoet Center for Biomolecular Research, Vakgroep Kristal-en Structuurchemie,
Utrecht University, Padualaan 8, 3584CH Utrecht, The Netherlands
Received April 10, 1995X
The synthesis and biological evaluation of N6,C8-disubstituted derivatives of adenosine as
potential partial agonists for adenosine receptors is described. Via three routes, two series of
compounds were prepared, viz., N6-cyclopentyladenosine derivatives 3a -e and C8-(cyclopent-
ylamino)adenosine analogs 3e and 9a -d , respectively. The X-ray structure determination of
one of these compounds, N6-ethyl-8-(cyclopentylamino)adenosine (9b), was carried out (ortho-
rhombic, space group P212121 (No. 19) with a ) 11.039(3), b ) 8.708(2), and c ) 24.815(12) Å,
Z ) 4, R1 ) 0.0974, R2w ) 0.2455). Due to intramolecular hydrogen bonding, the ribose moiety
of this compound is in an anti conformation. The compounds were tested in vitro in radioligand
binding studies, yielding their affinities for A1 and A2a adenosine receptors. All compounds
appeared A1 selective, with affinities in the high nanomolar, low micromolar range. On A1
receptors the so-called GTP shift was also determined, i.e., the ratio between the affinities
measured in the presence and absence of 1 mM GTP. All GTP shifts (values between 1.1 and
3.8) were lower than the GTP shift for CPA (6.0). This GTP shift appeared indicative for partial
agonism in vivo, since the N6-cyclopentyladenosine derivatives showed lower intrinsic activities
than the prototypic full agonist N6-cyclopentyladenosine on the decrease in heart rate in
conscious, normotensive rats.
In tr od u ction
appeared to be partial agonists in a variety of in vitro
and in vivo test systems. However, their affinity toward
adenosine receptors was modest. In the present study
we have synthesized N6,C8-disubstituted adenosines.
These compounds, while retaining partial agonism,
proved to be more potent than the C8-monosubstituted
series, with an additional gain in A1 receptor selectivity.
Extracellular adenosine has significant physiological
activity. Through its interaction with adenosine recep-
tors, the compound mediates a large variety of effects,
e.g., on the cardiovascular, immune, and central nervous
systems. Three subclasses of adenosine receptors have
been identified by pharmacological and molecular bio-
logical techniques, viz., A1, A2, and A3 receptors. The
A2 receptors have been further divided into A2a and A2b
receptors. A large number of both A1 and A2a selective
ligandssboth agonists and antagonistssare available
(for a recent review and references therein, see ref 1).
This is not yet the case for A2b receptors, whereas only
A3 selective agonists have been reported so far.2
Due to the ubiquity of adenosine receptor subtypes
in the body, the desired activity profile of adenosine
receptor ligands is often confounded by serious side
effects. In particular, the profound hemodynamic dis-
turbances caused by adenosine receptor agonists have
largely precluded their use for other therapeutic targets.
In this respect, the design of partial agonists seems
worthwhile, since virtually all known agonists behave
as full agonists.
Ch em istr y
In our search for the preparation of 8-aminoalkyl-
derived N6-substituted adenosines, we developed and
evaluated three possible and different synthetic routes
as depicted in Schemes 1-3. The first one is based on
the introduction of an 8-alkylamino substituent on the
N6-derivatized 8-halogenide adenosine moiety, whereas
the second one concerns the introduction of a 6-alkyl-
amino substituent on the 8-aminoalkyl-derivatized 6-chlo-
ropurine riboside as key intermediate. Alternatively,
the desired compounds could be obtained by a selective
introduction of the alkylamino substituent on the 6,8-
dichloropurine riboside, yielding one of the above-
mentioned key intermediates, and its subsequent am-
ination.
In the first route (see Scheme 1), our attention was
focused on the preparation of key intermediate 2 by
direct chlorination of 2′,3′,5′-tri-O-acetyl-N6-cyclopent-
yladenosine (1). Starting compound 1 was easily avail-
able by substitution of 6-chloropurine riboside with
cyclopentylamine4 and subsequent acetylation of the
formed N6-cyclopentyladenosine in 78% overall yield.
Unfortunately, several attempts to convert 1 into 8-bro-
mo-substituted nucleoside by direct bromination with
Br2/H2O (pH 4.0), N-bromoacetamide, Br2/Na2HPO4/
H2O (pH 7.0), or N-bromosuccinimide met with little
success. The failure to prepare this intermediate com-
pound was due to cleavage of the glycosidic bond, as
Recently, we have reported on the synthesis and
biological activity of 8-substituted adenosines.3 Among
the 8-amino-substituted derivatives, several analogs
† Dedicated to Professor Ernst Mutschler on the occasion of his 65th
birthday.
* Address for correspondence: Ad P. IJ zerman, Ph.D., Assoc.
Professor of Medicinal Chemistry, LACDR, P.O. Box 9502, 2300RA
Leiden, The Netherlands. Tel: 31-71-5274651. Fax: 31-71-5274277.
‡ Utrecht University.
§ Division of Pharmacology, LACDR.
| Address correspondence pertaining to crystallographic studies to
this author.
X Abstract published in Advance ACS Abstracts, February 15, 1996.
0022-2623/96/1839-1463$12.00/0 © 1996 American Chemical Society

1464 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Roelen et al.
Sch em e 1a
has a beneficial effect on the overall yield of 5 (83%)
when compared with the reverse order, i.e., bromina-
tion11 and subsequent acetylation12 (overall 48%). Sub-
stitution of 5 with ethylamine or cyclopentylamine and
subsequent acetylation of the intermediate products
afforded 6a ,b in 81% and 82% yields, respectively.
Chlorination of 6a ,b with an excess of dimethylchlo-
romethyleneammonium chloride (DMCMAC)13 in chlo-
roform furnished the 6-chloro derivatives 7 and 8 in 56%
and 58% yields, respectively. Amination at the 6-posi-
tion of derivative 7 with cyclopentylamine yielded 3b,
which was in every aspect identical with the product
obtained from 2 following the route in Scheme 1.
Although this procedure was useful and resulted in
products of high quality with acceptable yields ((35%),
the preparation of each intermediate in order to obtain
the remaining target compounds 3a ,c,d made this route
quite laborious and time-consuming in comparison with
the first route described before, and further application
was abandoned. We also explored the substitution of
the 6-chloro function in 8 with the corresponding amines
used above. The formation of the adenosine derivatives
9a -d and 3e, respectively, could indeed be effected in
good yields (76-85%) in a similar way as described
above. The reaction time was slightly shorter and the
reaction temperature lower during the substitution at
the 6-position than with 8-substitution, probably caused
by decrease in steric hindrance. Elemental analyses,
NMR, and high-resolution mass spectroscopy of com-
pounds 9a -d supported their structures. Moreover, the
structure of one of them, namely, 9b, was corroborated
with X-ray single-crystal structure determination (see
Figure 1).
a
(i) m-CPBA, HCl/DMF; (ii) NCS, ClCH2CH2Cl; (iii) RNH2/H2O
or dioxane, ∆.
proved by the presence of the acetylated riboside after
workup in the first two bromination reactions, or to
inertness of the purine ring for electrophilic or radical
attack, as proved by the recovery of the starting mate-
rial after workup in the last two reactions. On the other
hand, 2 could be isolated in low yields after chlorination
with m-chloroperbenzoic acid in an anhydrous hydro-
chloric acid/DMF solution5 (29%) or with N-chlorosuc-
cinimide in dry dichloroethane6 (31%). Intended nu-
cleophilic displacement of the chlorine atom in 2 with
different monoalkylamines at room temperature re-
sulted only in removal of the acetyl groups, but heating
for 1 or more days at temperatures between 50 and 70
°C yielded smooth conversion with simultaneous deacety-
lation. After isolation and purification, the products
3a -e were obtained in good yields (75-83%). The
homogeneity and identity of these products were estab-
lished by NMR and mass spectroscopy as well as by
elemental analysis.
For the preparation of compound 2 or 8 via the third
method (Scheme 3), 8-bromoinosine derivative 5 was
used as starting material. Substitution at the 6-position
and simultaneous exchange of the 8-bromo function by
The second route (Scheme 2) started with the fully
acetylated inosine 4, obtained by a known procedure7,8
that was slightly modified. Bromination of 4 with a
saturated Br2/Na2HPO4/H2O solution (pH 7.0)9 gave the
8-bromoinosine derivative 5. Use of this bromination
procedure resulted in a substantially higher yield (85%)
than bromination with N-bromoacetamide (52%).10 More-
over, it is noteworthy that the present order of reactions
13,14
treatment with a DMCMAC solution (2 M) in CHCl3
gave the 6,8-dichloropurine derivative 1012,15 in 91%
yield after purification by column chromatography.
However, nucleophilic attack on 10 with cyclopentyl-
Sch em e 2a
a
(i) Br2, Na2HPO4/H2O; (ii) a. EtNH2/H2O, ∆, b. Ac2O/DMAP/pyridine; (iii) a. cyclopentylamine, dioxane, ∆, b. Ac2O/DMAP/pyridine;
(iv) DMCMAC/CHCl3; (v) cyclopentylamine/dioxane, ∆; (vi) RNH2/H2O or dioxane, ∆.

N6,C8-Disubstituted Adenosine Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1465
Ta ble 1. Adenosine A1 and A2a Receptor Affinities (Apparent Ki Values for the A1 Receptor in the Presence and Absence of GTP) and
GTP Shifts for the A1 Receptor of the C8-Amino Monosubstituted and C8-Amino-N6-disubstituted Adenosine Analogs (Data for the
monosubstituted derivatives and CPA are from Van der Wenden et al.3)
a
Ki A1 (µM)
compd
R1 (N6)
R2 (C8)
-CH3
-GTP
+GTP
GTP shift
Ki A2,b -GTP (µM)
3a
3b
3c
3d
3e
9a
9b
9c
9d
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
-CH3
-CH2-CH3
-(CH2)2-CH3
-(CH2)3-CH3
-H
0.26 (0.16-0.36)
0.47 (0.34-0.59)
0.35 (0.27-0.43)
0.50 ( 0.08
1.09 ( 0.08
5.61 ( 2.39
0.31 ( 0.10
1.59 ( 0.90
0.76 ( 0.21
2.42 ( 0.36
6.56 ( 0.71
5.96 ( 0.68
11.4 ( 1.1
0.98 (0.88-1.07)
1.33 (1.10-1.56)
1.05 (0.89-1.21)
1.13 ( 0.18
1.28 ( 0.08
15.7 ( 4.0
3.8 ( 1.5
2.8 ( 0.9
3.0 ( 0.8
2.3 ( 0.5
1.2 ( 0.1
2.8 ( 1.4
4.1 ( 1.5
1.1 ( 0.6
1.1 ( 0.4
7.7 ( 1.4
3.6 ( 0.4
5.8 ( 0.8
4.0 ( 0.5
-
20.8 (18.1-23.4)
10.1 (8.80-11.3)
7.67 (6.61-8.72)
12.4 ( 4.0
45.5 ( 4.0
101 (111-92)
30.2 ( 5.1
-CH2-CH3
-(CH2)2-CH3
-(CH2)3-CH3
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
-CH3
-CH2-CH3
-(CH2)2-CH3
-(CH2)3-CH3
cyclopentyl
1.26 ( 0.23
1.76 ( 0.29
0.86 ( 0.16
18.6 ( 1.9
59.4 (44.9-74.0)
35.3 (19.4-51.1)
1.85 ( 0.36c
3.97 ( 0.64c
4.96 ( 1.73c
11.5 ( 1.9c
-H
-H
-H
-H
23.8 ( 1.3
34.7 ( 2.2
45.5 ( 4.2
na
-
na
CPA (N6-cyclopentyladenosine)
0.0059
(0.0058-0.0060)
0.035
(0.030-0.040)
6.0 ( 0.9
0.58 ( 0.12
a
b
Displacement of 0.4 nM [3H]DPCPX (KD ) 0.28 nM) from rat cortical membranes (n ) 2-3). Displacement of 5.6 nM [3H]CGS
21680 (KD ) 14.5 nM) from rat striatal membranes, unless stated otherwise (n ) 2-3). c Displacement of 4 nM [3H]NECA (KD ) 15.3 nM)
in the presence of 50 nM CPA from rat striatal membranes31 (n ) 3). -, not determined; na, not active (<50% displacement at 10-4
M
ligand).
Sch em e 3a
a
(i) DMCMAC/CHCl3; (ii) a. cyclopentylamine/dioxane, 40 °C,
b. Ac2O/pyridine, c. separation by column chromatography; (iii)
RNH2/H2O or dioxane, ∆ (see Scheme 1).
used for conversion into 3a -e or 9a -d , as described
before.
Compound 9b was studied by single-crystal X-ray
analysis. Its structure is shown in Figure 1.
F igu r e 1. X-ray structure of compound 9b: small open circles,
hydrogen atoms; large filled circles, carbon atoms; open circles,
nitrogen atoms; dashed circles, oxygen atoms; dashed lines,
possible hydrogen bonds.
Biologica l Eva lu a tion
amine showed no selectivity in displacement of the
8-chloro function over the 6-chloro function in contrast
to the results presented by Sutcliffe and Robins16 and
Szekeres et al.12 when ammonia was used. The two
isomeric products (2 and 8) formed were obtained in
approximately equal amounts according to TLC analysis
and could be isolated after separation by column chro-
matography in 33% and 41% yields, respectively. On
the basis of comparison of NMR spectra or retention
times of both compounds with that of the intermediate
obtained via route in Scheme 1 and that one obtained
via route in Scheme 2, the product with higher Rf value
was identified as 2 and the one with lower Rf value as
8. The chlorinated derivatives could subsequently be
All compounds were tested in radioligand binding
assays to determine their affinities toward adenosine
A1 and A2a receptors in rat brain cortex and rat
striatum, respectively (Table 1). For A1 receptors the
tritiated antagonist [3H]-1,3-dipropyl-8-cyclopentylxan-
thine (DPCPX) was used. Displacement experiments
were performed in the absence and presence of 1 mM
GTP, allowing the determination of the so-called GTP
shift (i.e., the ratio of the apparent Ki values in the
presence and absence of GTP, respectively). This shift
is an in vitro parameter often indicative for intrinsic
activity.17 Since no radiolabeled antagonist is available
for A2a receptors, the tritiated agonist [3H]CGS21680
was used. This prohibited the determination of a GTP

1466 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Roelen et al.
Ta ble 2. Pharmacodynamics of 8-Alkylamino Derivatives of N6-Cyclopentyladenosines 3a -e in Normotensive, Conscious Ratsa
dose
(mg/kg)
heart rate
base line (bpm)
max
reduction (%)
MAP base line
(mmHg)
max
reduction (%)
compd
n
CPA
0.20
4.8
4.8
4.8
8.0
8.0
6
6
6
6
6
5
345 ( 16
334 ( 16
350 ( 15
344 ( 9
348 ( 12
329 ( 17
54 ( 3
34 ( 4
25 ( 4
20 ( 5
8 ( 5
109 ( 5
105 ( 6
100 ( 3
97 ( 4
94 ( 3
96 ( 1
61 ( 2
50 ( 5
24 ( 3
15 ( 4
11 ( 3
3 ( 2
8-(methylamino)-CPA (3a )
8-(ethylamino)-CPA (3b)
8-(propylamino)-CPA (3c)
8-(butylamino)-CPA (3d )
8-(cyclopentylamino)-CPA (3e)
2 ( 3
a
The compounds were dissolved in 20% (v/v) DMSO and administered intravenously during 15 min. Data are presented as means (
SE.
shift on A2a receptors, and all experiments on A2a
respectively. Apparently, the bulk tolerance in the “C8-
region” is greater than previously thought. The con-
comitant increases in size and lipophilicity of the C8
substituents in 3a -e, as in the monosubstituted com-
pounds in Table 1, do not influence receptor affinity very
much.
receptors were done in the absence of GTP. Compounds
3a -e were also tested in vivo. Heart rate and mean
arterial pressure were recorded of conscious, normoten-
sive, and unrestrained rats that received an intravenous
infusion of the drugs. The results are presented in
Table 2.
Interestingly, 8-substitution also lowered GTP shift
values. Full agonists such as CPA and R-PIA (data not
shown) consistently have GTP shifts of ca. 6 in our rat
membrane preparations. All N6,C8-disubstituted de-
rivatives have significantly lower values, viz., 1.1-3.8.
If this in vitro parameter reflected in vivo partial
agonism, all compounds synthesized would behave as
partial agonists. Therefore we considered it worthwhile
to further investigate this aspect. We tested the more
potent series 3a -e and N6-cyclopentyladenosine (CPA)
in conscious, normotensive rats (Table 2). Upon infu-
sion of the compounds at dosages that caused maximal
effectss3-fold higher dosages did not produce higher
effectssit was found that CPA, as the reference full
agonist, caused a severe bradycardia and hypotension.21
Heart rate was lowered from 345 bpm (preadministra-
tion levels) to ca. 160 bpm at the end of the 15 min
infusion, a reduction of 54%. Similarly, mean arterial
blood pressure (MAP) was only 43 mmHg after admin-
istration of CPA, down by 61% from 109 mmHg. All
8-substituted CPA analogs caused lower maximum
reductions in varying degree. With increasing chain
length, going from methyl to cyclopentyl substitution,
both cardiovascular parameters were less influenced.
The maximum reductions in heart rate and MAP were
highly correlated (r ) 0.96). The GTP shifts of CPA and
compounds 3a -e appear to be correlated with the
maximum reductions possible (r ) 0.98 for heart rate
vs GTP shifts, r ) 0.93 for MAP vs GTP shifts).
Recently, we have shown that the in vivo modulation
of heart rate is a sensitive pharmacologic end point for
A1 receptor activation,21 which seems to be corroborated
by this high correlation between heart rate reductions
and GTP shifts on A1 receptors.
Resu lts a n d Discu ssion
Three different synthetic routes gave access to the
target compounds 3a -e and/or 9a -d . The first one
seems useful for straightforward synthesis of 3a -e,
although the overall yields (ca. 20%) are rather low. The
second route is most suitable for the synthesis of
compounds 9a -d or 3e, despite the again moderate
overall yields (ca. 30%). Although the substitution of
the dichloropurine derivative 10 in Scheme 3 was not
regioselective, this method appeared quite efficient,
based on the fact that both intermediates (2 and 8)
formed could be individually converted after separation
into the required target compounds in higher overall
yields (ca. 46%).
In Table 1 radioligand binding data are gathered for
all synthesized disubstituted end products. For reasons
of comparison, data of previously reported C8-mono-
substituted adenosines3 are also incorporated. Substi-
tution of the exocyclic N6-amino group by cyclopentyl,
as in 3a -e, led to compounds that showed higher
affinity for the A1 receptors than the corresponding
monosubstituted ones, in both the absence and presence
of GTP. On average this increase in affinity was at least
10-fold. In contrast, A2 receptor affinities were consis-
tently lower throughout the series. This is fully in line
with the fact that N6-cyclopentyl substitution enhances
potency and selectivity for adenosine A1 receptors.1
Compounds 9a -d were somewhat less potent than the
corresponding compounds 3a -d .
Obviously, C8-substitution caused a significant drop
in affinity when compared to the reference compound
CPA (see also Table 1). A few reports deal with
8-substituted adenosines. Bruns,18 J acobson,19 and
Olsson20 concluded that substituents at this carbon
atom often lead to inactive compounds, e.g., for 8-bro-
moadenosine. It was suggested that such substituents
force the ribose ring into the syn conformation, whereas
the anti conformation is thought to be essential for
receptor binding. From the crystal structure of 9b
(Figure 1), it is apparent that the anti conformation is
compatible with 8-substitution. In this particular case
a bifurcated hydrogen bond appears to be formed
between the -NH- element in the 8-position and the 5′-
OH group in the ribose moiety (distance H‚‚‚O, 2.3 Å;
angle N-H‚‚‚O, 147°) and the oxygen atom in the ribose
ring (distance H‚‚‚O, 2.4 Å; angle N-H‚‚‚O, 127°),
Con clu sion
Our previous efforts in synthesizing partial agonists
have led to the identification of theophylline 7-riboside,22
C8-monosubstituted adenosines,3 and deoxyribose de-
rivatives of N6-substituted adenosines23 as such com-
pounds. The present series of compounds is among the
derivatives with the highest A1 receptor affinity and
“controllable” intrinsic activity. It is anticipated that
these compounds may be useful tools in pharmacology
and biochemistry. Rapid receptor downregulation and
desensitization have been demonstrated for full agonists
for adenosine receptors. Partial agonists may behave
less outspoken in this respect. Second, partial agonism

N6,C8-Disubstituted Adenosine Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1467
2′,3′,5′-Tr i-O-a ce t yl-8-ch lor o-N 6-cyclop e n t yla d e n o-
sin e (2): Meth od A. To a stirred solution of 1 (461 mg, 1.0
mmol), dried by evaporation with DMF (2 × 5 mL), in HCl/
DMF (0.5 M, 3.0 mL, 1.5 mmol) was added a solution of
m-chloroperbenzoic acid (m-CPBA; 50-60%, 1.0 g, 3.0 mmol),
dried by evaporation with DMF (2 × 5 mL), in DMF (4.0 mL).
After 1 h, an additional amount of m-CPBA (500 mg, 1.5
mmol), as solution in DMF (2.0 mL), was added and stirring
was continued for another 16 h. The mixture was concentrated
to a small volume, and the residue was purified by column
chromatography eluted with a 0-4% gradient of acetone in
CH2Cl2 to give 2 as a white foam: yield 144 mg (29%); Rf 0.55
may induce selectivity of effects due to differences in
receptor-effector coupling in various tissues. It would
therefore be worthwhile to study the effects of the
compounds described on other physiological processes
mediated by adenosine receptors, e.g., inhibition of
lipolysis and anticonvulsive activity.
Exp er im en ta l Section
Ch em ica ls a n d Solven ts. Dimethylchloromethyleneam-
monium chloride (DMCMAC) solution in CHCl3 (2 M) was
prepared from dry dimethylformamide and freshly distilled
thionyl chloride as described before.14 All other reagents were
of analytical grade.
1
(C), 0.23 (D); H NMR (CDCl3) δ 8.33 (s, 1H, H-2), 6.34 (dd,
J 1′,2′ ) 4.4 Hz, J 2′,3′ ) 6.0 Hz, 1H, H-2′), 6.10 (d, J 1′,2′ ) 4.5 Hz,
1H, H-1′), 5.93 (t, J 2′,3′ ) J 3′,4′ ) 5.9 Hz, 1H, H-3′), 5.73 (d, J )
7.7 Hz, 1H, NH, exchangeable with CD3OD), 4.64-4.54 (m,
1H, N-CH, cyclopentyl), 4.54-4.50 (m, 1H, H-5′′), 4.39-4.36
(m, 2H, H-4′/H-5′), 2.18-2.03 (m, 2H, N-CH-CHH, cyclopentyl)
coinciding with 2.16, 2.11, 2.05 (3s, 9H, 3CH3, Ac), 1.93-1.45
(m, 6H, N-CH-CHH, N-CH-CH2-CH2, cyclopentyl); 13C NMR
(CDCl3) δ 169.9, 169.4, 169.3 (3CdO, Ac), 152.9 (C-2), 152.8
(C-6, C-4), 136.6 (C-8), 118.1 (C-5), 86.9 (C-1′), 79.8 (C-4′), 71.6
(C-2′), 70.0 (C-3′), 62.5 (C-5′), 52.0 (N-CH, cyclopentyl), 32.8,
32.7 (N-CH-CH2, cyclopentyl), 23.3 (N-CH-CH2-CH2, cyclo-
pentyl), 20.1, 2 × 20.0 (3CH3, Ac); MS m/ z 497 (M + 1)+.
Meth od B. To a stirred solution of 1 (461 mg, 1.0 mmol)
in DCE (5.0 mL) was added NCS (534 mg, 4.0 mmol). After
heating at 50 °C for 24 h, precipitated succinimide was filtered
and the filtrate evaporated in vacuo to dryness. The residue
was purified by column chromatography as described before:
yield 154 mg (31%); the analytical data (TLC, 1H NMR, 13C
NMR, and MS analyses) were in every aspect identical with
those described above.
Ch r om a togr a p h y. Thin-layer chromatography (TLC) was
carried out using silica F254 preformed layers 0.1 mm thick on
a plastic backing (Schleicher and Schu¨ll DC Fertigfolien F1500
LS254) in the following mobile phases: A, CH2Cl2/CH3OH, 90/
10, v/v; B, ethyl acetate/acetone, 3/1, v/v; C, CH2Cl2/CH3OH,
96/4, v/v; D, diethyl ether/hexane, 4/1, v/v; and E, CH2Cl2/CH3-
OH, 4/1, v/v. Spots were visualized either under UV (254 or
356 nm) light or by spraying with sulfuric acid/methanol (1:4)
or molybdate reagent (H2O/concentrated H2SO4/(NH4)6-
Mo7O24‚4H2O/(NH4)2Ce(SO4)4‚2H2O, 90/10/2.5/1, v/v/w/w) and
charring at 140 °C for a few minutes. Preparative column
chromatography was performed on silica gel (230-400 mesh
ASTM), suspended in CH2Cl2.
In str u m en ts a n d An a lyses. Elemental analyses (results
within (0.4%) were done for C,H,N (Department of Mi-
croanalysis, Groningen University, Groningen, The Nether-
lands). 13C NMR spectra were measured at 50.1 MHz with a
J EOL J NM-FX 200 spectrometer equipped with a PG 200
computer operating in the Fourier-transform mode. 1H NMR
spectra were measured at 200 MHz, using the above-
mentioned spectrometer, or at 300 MHz, using a Bruker WM-
300 spectrometer equipped with an ASPECT-2000 computer
operating in the Fourier-transform mode. Chemical shifts for
1H and 13C NMR are given in ppm (δ) relative to tetrameth-
ylsilane (TMS) as internal standard. All high-resolution mass
spectra were measured on a Finnigan MAT TSQ-70 mass
spectrometer equipped with an electrospray interface (EI).
Experiments were done in positive ionization mode. Samples
were dissolved in CH2Cl2, diluted with 80/20 methanol/water
+ 1% acetic acid, and introduced by means of constant infusion
at a flow rate of 1 µL/min.
Gen er a l P r oced u r e for th e Am in a tion of Com p ou n d
2 in to 3a -e. To a solution of 2 (321 mg, 0.65 mmol) in dioxane
(10 mL) was added an excess of the corresponding amine, and
the mixture was heated at 50-80 °C for 2-5 days. The
mixture was concentrated and evaporated with toluene (2 ×
50 mL), ethanol (50 mL), and CH2Cl2/CH3OH (50 mL, 1/1, v/v).
The crude product was purified by column chromatography
with a gradient of CH3OH in CH2Cl2 (98/2 f 90/10, v/v). The
appropriate fractions were collected and concentrated to a
white solid, which was dried in vacuo at 50 °C for 24 h.
Recrystallization of small amounts from acetone/ether gave
the analytical samples.
N6-Cyclop en tyl-8-(m eth yla m in o)a d en osin e (3a ). Reac-
tion was carried out with aqueous methylamine (40%, 25 mL)
at 50 °C for 48 h: yield 185 mg (78%); mp 146-148 °C; Rf
0.48 (E); 1H NMR (DMSO-d6) δ 7.94 (s, 1H, H-2), 6.90 (q, J )
4.5 Hz, 1H, 8-NH, exchangeable with D2O), 6.68 (d, J ) 7.8
Hz, 1H, 6-NH, exchangeable with D2O), 5.91 (bt, 1H, 5′-OH,
exchangeable with D2O), 5.85 (d, J 1′,2′ ) 7.3 Hz, 1H, H-1′), 5.24
(d, J ) 6.7 Hz, 1H, 2′-OH, exchangeable with D2O), 5.15 (d, J
) 4.1 Hz, 1H, 3′-OH, exchangeable with D2O), 4.64 (AB, which
changed by addition of D2O in dd, J 1′,2′ ) 7.3 Hz, J 2′,3′ ) 5.5
Hz, 1H, H-2′), 4.60-4.47 (m, 1H, 6-NH-CH, cyclopentyl), 4.11
(m, which changed by addition of D2O in dd, J 2′,3′ ) 5.3 Hz,
J 3′,4′ ) 1.5 Hz, 1H, H-3′), 3.96 (m, 1H, H-4′), 3.62 (m, which
changed by addition of D2O in bs, 2H, H-5′/H-5′′), 2.87 (d, J )
4.6 Hz, which changed by addition of D2O in s, 3H, N-CH3,
methyl), 1.96-1.86 (m, 2H, N-CH-CHH, cyclopentyl), 1.71-
1.48 (m, 6H, N-CH-CHH-CH2, cyclopentyl); 13C NMR (DMSO-
d6) δ 152.3 (C-6 or C-4), 151.3 (C-6 or C-4), 149.3 (C-8), 149.2
(C-2), 117.1 (C-5), 86.9 (C-1′), 86.0 (C-4′), 71.1 (C-2′, C-3′), 61.8
(C-5′), 52.0 (N-CH, cyclopentyl), 33.0, 32.9 (N-CH-CH2, cyclo-
pentyl), 29.1 (N-CH3, methyl), 23.7 (N-CH-CH2-CH2, cyclo-
pentyl); MS m/ z 365 (M + 1)+; Anal. (C16H24N6O4) C,H,N.
N6-Cyclop en tyl-8-(eth yla m in o)a d en osin e (3b). Reac-
tion was carried out with aqueous ethylamine (70%, 10 mL)
at 50 °C for 48 h: yield 199 mg (81%); mp 151-153 °C; Rf
0.51 (E); MS m/ z 379 (M + 1)+. Anal. (C17H26N6O4) C,H,N.
N6-Cyclop en tyl-8-(n -p r op yla m in o)a d en osin e (3c). Re-
action was carried out with n-propylamine (5.0 mL) at 70 °C
for 72 h: yield 207 mg (80%); mp 138-141 °C; Rf 0.55 (E); MS
m/ z 393 (M + 1)+. Anal. (C18H28N6O4) C,H,N.
Syn th eses. 2′,3′,5′-Tr i-O-a cetyl-N6-cyclop en tyla d en o-
sin e (1). To a solution of dry 6-chloro-9-â-D-ribofuranosyl-
purine (1.44 g, 5.00 mmol) in dry EtOH (50 mL) were added
Et3N (0.9 mL, 6.5 mmol) and cyclopentylamine (1.0 mL, 10.1
mmol); the mixture was refluxed for 16 h. After concentration
in vacuo, the residue was evaporated with toluene (2 × 25 mL)
and subsequently dried by evaporation with pyridine (2 × 10
mL). The crude N6-cyclopentyladenosine was dissolved in
pyridine (25 mL), and Ac2O (2.83 mL, 30.0 mmol), together
with a catalytic amount of DMAP, was added. After stirring
for 3 h, the reaction was quenched by addition of MeOH (3
mL) and the mixture concentrated under reduced pressure to
dryness. The residue was dissolved in CH2Cl2 (75 mL) and
washed with an aqueous NaHCO3 solution (10%, 50 mL) and
H2O (50 mL). The organic layer was dried with MgSO4,
filtered, and concentrated. The product was purified by
column chromatography eluted with a 0-5% gradient of MeOH
in CH2Cl2 to give 1 as a white foam: yield 1.81 g (78%); Rf
0.51 (C); 1H NMR (CDCl3) δ 8.38 (s, 1H, H-8), 7.90 (s, 1H, H-2),
6.19 (d, J 1′,2′ ) 5.5 Hz, 1H, H-1′), 5.93 (t, J 1′,2′ ) J 2′,3′ ) 5.5 Hz,
1H, H-2′), 5.78 (bd, J ) 7.1 Hz, 1H, 6-NH, exchangeable with
CD3OD), 5.67 (t, J 2′,3′ ) J 3′,4′ ) 5.5 Hz, 1H, H-3′), 4.75-4.52
(m, 1H, N-CH, cyclopentyl), 4.46-4.33 (m, 3H, H-4′/H-5′/H-
5′′), 2.16-1.95 (m, 2H, N-CH-CHH, cyclopentyl) coinciding
with 2.14, 2.08, 2.00 (3s, 9H, 3CH3, Ac), 1.85-1.51 (m, 6H,
N-CH-CHH, N-CH-CH2-CH2, cyclopentyl); 13C NMR (CDCl3)
δ 169.8, 169.0, 168.8 (3CdO, Ac), 154.1 (C-6, C-4), 152.8 (C-
2), 137.5 (C-8), 119.5 (C-5), 85.6 (C-1′), 79.7 (C-4′), 72.7 (C-2′),
70.2 (C-3′), 62.7 (C-5′), 51.9 (N-CH, cyclopentyl), 32.8, 32.4 (N-
CH-CH2, cyclopentyl), 23.2 (N-CH-CH2-CH2, cyclopentyl), 20.2,
20.0, 19.9 (3CH3, Ac); MS m/ z 462 (M + 1)+.
N6-Cyclop en tyl-8-(n -bu tyla m in o)a d en osin e (3d ). Reac-

1468 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Roelen et al.
tion was carried out with n-butylamine (5.5 mL) at 80 °C for
120 h: yield 198 mg (76%); mp 110-112 °C; Rf 0.60 (E); MS
m/ z 407 (M + 1)+. Anal. (C19H30N6O4) C,H,N.
residue was dissolved in CH2Cl2 (30 mL) and washed with an
aqueous NaHCO3 solution (10%, w/v, 20 mL) and water (20
mL). The organic layer was dried over MgSO4, filtered,
concentrated, and evaporated with toluene (20 mL) and CH2-
Cl2 (10 mL). The product was purified by column chromatog-
raphy with a 0-5% gradient of CH3OH in CH2Cl2. The
appropriate fractions were collected and concentrated to a
N6-Cyclop en tyl-8-(cyclop en tyla m in o)a d en osin e (3e).
Reaction was carried out with cyclopentylamine (5.5 mL) at
80 °C for 120 h: yield 176 mg (82%); mp 121-123 °C; Rf 0.57
(E); MS m/ z 419 (M + 1)+. Anal. (C20H30N6O4) C,H,N.
2′,3′,5′-Tr i-O-a cetylin osin e (4). The preparation of this
compound has been described before.7,8
1
white foam: yield 0.59 g (82%); Rf 0.44 (A); H NMR (CDCl3)
δ 12.2 (s, 1H, 1-NH, exchangeable with CD3OD), 7.83 (s, 1H,
H-2), 6.63 (d, J ) 6.6 Hz, 1H, 8-NH, exchangeable with CD3-
OD), 6.17 (dd, J 1′,2′ ) 4.7 Hz, J 2′,3′ ) 6.2 Hz, 1H, H-2′), 6.08 (d,
J 1′,2′ ) 4.7 Hz, 1H, H-1′), 5.60 (t, J 2′,3′ ) J 3′,4′ ) 6.1 Hz, 1H,
H-3′), 4.38-4.34 (m, 1H, H-5′), 4.24-4.06 (m, 2H, H-4′/H-5′′),
2.08, 2.07, 2.05 (3s, 9H, 3CH3, Ac), 1.78-1.61 (m, 2H, N-CH-
CHH, cyclopentyl), 1.60-1.48 (m, 6H, N-CH-CHH, N-CH-CH2-
CH2, cyclopentyl); 13C NMR (CDCl3) δ 170.0, 2 × 169.3 (3CdO,
Ac), 157.9 (C-6), 151.1 (C-4), 148.1 (C-8), 142.2 (C-2), 122.3
(C-5), 85.1 (C-1′), 79.8 (C-4′), 71.2 (C-2′), 69.7 (C-3′), 62.7 (C-
5′), 54.5 (N-CH, cyclopentyl), 33.0, 32.9 (N-CH-CH2, cyclo-
pentyl), 23.4 (N-CH-CH2-CH2, cyclopentyl), 20.4, 20.2, 20.1
(3CH3, Ac); MS m/ z 478 (M + 1)+.
2′,3′,5′-Tr i-O-a cetyl-6-ch lor o-8-(eth yla m in o)p u r in e r i-
bosid e (7). To a solution of 6a (0.44 g, 1.0 mmol), dried by
evaporation with dioxane (2 × 5.0 mL), in dry CHCl3 (10.0
mL) was added a DMCMAC solution in CHCl3 (2 M, 1.5 mL,
3.0 mmol). The mixture was heated at 40 °C in an oil bath
for 24 h. Thereafter the mixture was allowed to cool with an
ice/water bath and added dropwise into a cold aqueous
NaHCO3 solution (10%, w/v, 20 mL). The aqueous layer was
extracted with two portions of CH2Cl2 (50 mL). The combined
organic layers were dried over MgSO4, filtered, and evaporated
under reduced pressure. The product was purified by column
chromatography with a 0-8% gradient of acetone in CH2Cl2.
The appropriate fractions were collected and concentrated to
a white foam: yield 0.26 g (56%); Rf 0.49 (C), 0.15 (D); 1H NMR
(CDCl3) δ 8.44 (s, 1H, H-2), 6.17 (d, J 1′,2′ ) 6.4 Hz, 1H, H-1′),
5.84 (t, J 1′,2′ ) J 2′,3′ ) 6.2 Hz, 1H, H-2′), 5.54 (t, J ) 5.6 Hz,
1H, NH, exchangeable with CD3OD), 5.51 (dd, J 2′,3′ ) 6.1 Hz,
J 3′,4′ ) 4.2 Hz, 1H, H-3′), 4.59 (dd, J 4′,5′ ) 4.4 Hz, J 5′,5′′ ) 12.2
Hz, 1H, H-5′), 4.43-4.40 (m, 1H, H-4′), 4.35 (dd, J 4′,5′′ ) 2.6
Hz, J 5′,5′′ ) 12.2 Hz, 1H, H-5′′), 3.72-3.62 (m, 2H, N-CH2,
ethyl), 2.16, 2.10, 2.04 (3s, 9H, 3CH3, Ac), 1.34 (t, J ) 7.3 Hz,
3H, N-CH2-CH3, ethyl); 13C NMR (CDCl3) δ 170.0, 2 × 169.5
(3CdO, Ac), 154.4 (C-6), 152.6 (C-4), 148.2 (C-2), 143.3 (C-8),
131.6 (C-5), 85.0 (C-1′), 80.6 (C-4′), 71.0 (C-2′), 69.9 (C-3′), 62.8
(C-5′), 38.1 (N-CH2, ethyl), 20.6, 20.4, 20.3 (3CH3, Ac), 14.8
(N-CH2-CH3, ethyl); MS m/ z 456 (M + 1)+.
2′,3′,5′-Tr i-O-a cetyl-8-br om oin osin e (5). To an aqueous
Na2HPO4 solution (10%, w/v, 75 mL) at room temperature was
added Br2 (2.0 mL), and the mixture was stirred vigorously
for 15 min until most of the bromine had dissolved. The
decanted bromine solution was added to a solution of dry 4
(1.97 g, 5.0 mmol) in dioxane (75 mL), and the mixture was
stirred for 4 days at room temperature. Then again freshly
prepared bromine solution (35 mL) was added, and the mixture
was stirred for another 3 days at room temperature. After
cooling in an ice/water bath, an aqueous NaHSO3 solution (2
N) was added dropwise until the solution became colorless.
The water layer was extracted with CH2Cl2 (3 × 75 mL). The
organic layer was washed with an aqueous NaHSO3 solution
(0.2 N, 50 mL) and water (50 mL), dried over MgSO4, filtered,
and concentrated in vacuo. The product was purified by
column chromatography eluted with a 0-5% gradient of CH3-
OH in CH2Cl2. The appropriate fractions were collected and
concentrated to a white foam: yield 2.04 g (86%); Rf 0.50 (A),
0.65 (B); 1H NMR (CDCl3) δ 13.1 (bs, 1H, 1-NH, exchangeable
with CD3OD), 8.38 (s, 1H, H-2), 6.26 (dd, J 1′,2′ ) 4.8 Hz, J 2′,3′
) 5.8 Hz, 1H, H-2′), 6.13 (d, J 1′,2′ ) 4.8 Hz, 1H, H-1′), 5.81 (t,
J 2′,3′ ) J 3′,4′ ) 5.8 Hz, 1H, H-3′), 4.55-4.29 (m, 3H, H-4′/H-5′/
H-5′′), 2.17, 2.12, 2.08 (3s, 9H, 3CH3, Ac); 13C NMR (CDCl3) δ
169.9, 169.0, 168.9 (CdO, Ac), 156.7 (C-6), 149.3 (C-4), 145.8
(C-2), 125.7 (C-8), 125.1 (C-5), 88.1 (C-1′), 79.6 (C-4′), 71.6 (C-
2′), 69.7 (C-3′), 62.4 (C-5′), 20.6, 20.1, 19.9 (CH3, Ac); MS m/ z
474 (M + 1)+.
2′,3′,5′-Tr i-O-a cetyl-8-(eth yla m in o)in osin e (6a ). To a
solution of 5 (1.95 g, 4.12 mmol) in dioxane (20 mL) was added
an aqueous ethylamine solution (50 mL, 70%, w/v), and the
solution was heated at 80 °C in an oil bath. After 48 h the
mixture was concentrated under reduced pressure and the
residue dried with and dissolved in pyridine (20 mL). To this
solution were added Ac2O (4.50 mL, 47.7 mmol) and a catalytic
amount of DMAP, and the mixture was stirred for 3 h at room
temperature. The reaction was quenched by addition of MeOH
(5 mL), and the mixture was concentrated in vacuo. The
residue was dissolved in CH2Cl2 (75 mL) and washed with an
aqueous NaHCO3 solution (10%, w/v, 50 mL) and water (50
mL). The organic layer was dried over MgSO4, filtered,
concentrated, and evaporated with toluene (50 mL) and CH2-
Cl2 (25 mL). The product was purified by column chromatog-
raphy with a 0-6% gradient of CH3OH in CH2Cl2. The
appropriate fractions were collected and concentrated to a
N6-Cyclop en tyl-8-(eth yla m in o)a d en osin e (3b) Sta r tin g
fr om 7. To a solution of 7 (228 mg, 0.5 mmol), dried by
evaporation with dioxane (2 × 5 mL), in dioxane (10 mL) was
added cyclopentylamine (5.0 mL, 51 mmol), and the solution
was heated in an oil bath at 50 °C for 48 h. The mixture was
concentrated and evaporated with toluene (2 × 50 mL), ethanol
(50 mL), and CH2Cl2/CH3OH (50 mL, 1/1, v/v). The crude
1
white foam: yield 1.45 g (81%); Rf 0.35 (A); H NMR (CDCl3)
product was purified by column chromatography with
a
δ 12.9 (bs, 1H, 1-NH, exchangeable with CD3OD), 8.02 (s, 1H,
gradient of CH3OH in CH2Cl2 (98/2 f 90/10, v/v). The
appropriate fractions were collected and concentrated to a
white solid, which was dried in vacuo at 50 °C for 24 h: yield
142 mg (75%); the analytical data (mp, TLC, 1H NMR, 13C
NMR, and MS analyses) were in every aspect identical with
those described above.
H-2), 6.12 (d, J 1′,2′ ) 6.5 Hz, 1H, H-1′), 5.82 (t, J 1′,2′ ) J 2′,3′
)
6.3 Hz, 1H, H-2′), 5.50 (dd, J 2′,3′ ) 6.2 Hz, J 3′,4′ ) 4.3 Hz, 1H,
H-3′), 5.04 (t, J ) 5.7 Hz, 1H, 8-NH, exchangeable with CD3-
OD), 4.58-4.53 (m, 1H, H-5′), 4.39-4.33 (m, 2H, H-4′/H-5′′),
3.65-3.49 (m, 2H, N-CH2, ethyl), 2.15, 2.12, 2.05 (3s, 9H, 3CH3,
Ac), 1.30 (t, J ) 7.1 Hz, 3H, N-CH2-CH3, ethyl); 13C NMR
(CDCl3) δ 169.9, 169.3, 169.2 (3CdO, Ac), 157.8 (C-6), 151.2
(C-4), 148.0 (C-8), 142.2 (C-2), 122.1 (C-5), 84.7 (C-1′), 80.0 (C-
4′), 70.7 (C-2′), 69.7 (C-3′), 62.6 (C-5′), 37.8 (N-CH2, ethyl), 20.3,
20.1, 20.0 (3CH3, Ac), 14.7 (N-CH2-CH3, ethyl); MS m/ z 438
(M + 1)+.
2′,3′,5′-Tr i-O-a cetyl-8-(cyclop en tyla m in o)in osin e (6b).
To a solution of 5 (0.71 g, 1.50 mmol) in dioxane (10 mL) was
added cyclopentylamine (2.0 mL, 20.2 mmol), and the solution
was heated at 80 °C in an oil bath. After 48 h the mixture
was concentrated under reduced pressure and the residue
dried with and dissolved in pyridine (5.0 mL). To this solution
was added Ac2O (1.0 mL, 10.6 mmol) followed by a catalytic
amount of DMAP, and the mixture was stirred for 3 h at room
temperature. The reaction was quenched by addition of MeOH
(2 mL), and the mixture was concentrated in vacuo. The
2′,3′,5′-Tr i-O-a ce t yl-6-ch lor o-8-(cyclop e n t yla m in o)-
p u r in e Ribosid e (8). To a solution of 6b (0.25 g, 0.52 mmol),
dried by evaporation with dioxane (2 × 5.0 mL), in dry CHCl3
(5.0 mL) was added a DMCMAC solution in CHCl3 (2 M, 1.2
mL, 2.4 mmol). The mixture was warmed at 40 °C in an oil
bath for 24 h. Thereafter the mixture was allowed to cool in
an ice/water bath and added dropwise into a cold aqueous
NaHCO3 solution (10%, w/v, 10 mL). The aqueous layer was
extracted with two portions of CH2Cl2 (30 mL). The combined
organic layers were dried over MgSO4, filtered, and evaporated
under reduced pressure. The product was purified by column
chromatography with a 0-6% gradient of acetone in CH2Cl2.
The appropriate fractions were collected and concentrated to
a white foam: yield 151 mg (58%); Rf 0.56 (C); 1H NMR (CDCl3)
δ 8.42 (s, 1H, H-2), 6.05 (d, J 1′,2′ ) 5.4 Hz, 1H, H-1′), 5.99 (t,

N6,C8-Disubstituted Adenosine Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1469
Ta ble 3. Crystallographic Data for 9b
J 1′,2′ ) J 2′,3′ ) 5.5 Hz, 1H, H-2′), 5.58 (t, J 2′,3′ ) J 3′,4′ ) 5.4 Hz,
1H, H-3′), 5.44 (d, J ) 7.1 Hz, 1H, NH, exchangeable with CD3-
OD), 4.53-4.35 (m, 4H, N-CH, cyclopentyl/H-4′/H-5′/H-5′′),
2.23-2.15 (m, 2H, N-CH-CHH, cyclopentyl), 2.15, 2.07, 2.04
(3s, 9H, 3CH3, Ac), 1.77-1.51 (m, 6H, N-CH-CHH-CH2,
cyclopentyl); 13C NMR (CDCl3) δ 170.0, 169.4, 169.3 (3CdO,
Ac), 154.2 (C-6), 152.4 (C-4), 148.0 (C-2), 143.3 (C-8), 131.7
(C-5), 85.6 (C-1′), 80.3 (C-4′), 71.6 (C-2′), 70.0 (C-3′), 62.8 (C-
5′), 54.7 (N-CH, cyclopentyl), 33.1, 33.0 (N-CH-CH2, cyclo-
pentyl), 23.3 (N-CH-CH2-CH2, cyclopentyl), 20.3, 2 × 20.0
(3CH3, Ac); MS m/ z 497 (M + 1)+.
Gen er a l P r oced u r e for th e Am in a tion of 8 in to 9a -d .
To a solution of 8 (270 mg, 0.5 mmol) in dioxane (10 mL) was
added an excess of the corresponding amine, and the mixture
was heated at 50-70 °C for 2-3 days. The mixture was
concentrated and evaporated with toluene (2 × 50 mL), ethanol
(50 mL), and CH2Cl2/CH3OH (50 mL, 1/1, v/v). The crude
Crystal Data
formula
C17H26N6O4‚C4H10
O
molecular weight
crystal system
space group
Z
452.55
orthorhombic
P212121 (No. 19)
4
a, b, c (Å)
11.039(3), 8.708(2), 24.815(12)
2385.4(14)
1.260
976
V (Å3)
Dcalcd (g cm-3
F(000)
)
µ
MoKR (cm-1
crystal size (mm)
)
0.9
0.03 × 0.50 × 0.63
Data Collection
T, K
295
θ
min, θmax (deg)
1.6, 24.2
0.710 73
radiation (Mo KR, graphite
monochromator) (Å)
scan type
product was purified by column chromatography with
a
ω/2θ
1.69 + 0.35 tan θ
4.35, 4.00
gradient of CH3OH in CH2Cl2 (98/2 f 90/10, v/v). The
appropriate fractions were collected and concentrated to a
white solid, which was dried in vacuo at 50 °C for 24 h.
Recrystallization of small amounts from acetone/ether gave
the analytical samples.
∆ω (deg)
horizontal, vertical aperture
(mm)
reference reflections
data set
total data
2h05, 1h2h2h, 302h
-12:0, -10:0, -27:27
8-(Cyclop en tyla m in o)-N6-m eth yla d en osin e (9a ). Reac-
tion was carried out with aqueous methylamine (40%, 20 mL)
at 50 °C for 24 h: yield 147 mg (81%); mp 128-130 °C; Rf
0.53 (E); 1H NMR (DMSO-d6) δ 7.96 (s, 1H, H-2), 6.77 (q, J )
4.7 Hz, 1H, 6-NH, exchangeable with D2O), 6.67 (d, J ) 7.0
Hz, 1H, 8-NH, exchangeable with D2O), 5.90 (d, J 1′,2′ ) 7.4 Hz,
1H, H-1′), 5.81 (t, J ) 4.3 Hz, 1H, 5′-OH, exchangeable with
D2O), 5.24 (d, J ) 6.5 Hz, 1H, 2′-OH, exchangeable with D2O),
5.15 (d, J ) 4.1 Hz, 1H, 3′-OH, exchangeable with D2O), 4.60
(AB, which changed by addition of D2O in dd, J 1′,2′ ) 7.5 Hz,
J 2′,3′ ) 5.4 Hz, 1H, H-2′), 4.20 (m, 1H, 8-N-CH, cyclopentyl),
4.10 (m, which changed by addition of D2O in dd, J 2′,3′ ) 5.4
Hz, J 3′,4′ ) 2.0 Hz, 1H, H-3′), 3.94 (m, 1H, H-4′), 3.62 (m, which
changed by addition of D2O in d, J ) 2.2 Hz, 2H, H-5′/H-5′′),
2.91 (d, J ) 4.7 Hz, which changed by addition of D2O in s,
3H, N-CH3, methyl), 1.94-1.88 (m, 2H, N-CH-CHH, cyclo-
3913
total unique data
observed data
3424 (Rint ) 0.0815)
1450 [Fo > 4.0σ(Fo)]
Refinement
no. of refined parameters
weighting schemea
final R2w, R1, S
298
w ) 1/[σ2(Fo2) + (0.1P)2]
0.2455, 0.0974, 0.947
0.001, 0.004
b
(∆/σ)av, (∆/σ)max in final cycle
min, max residual density
-0.25, 0.23
(e Å-3
)
a
b
P ) (max(Fo2,0) + 2Fc2)/3. R1 ) ∑||Fo| - |Fc||/∑|Fo|, R2w
)
[∑[w(Fo - Fc2)2]/∑[w(Fo2)2]]1/2
.
2
w/v, 20 mL) and H2O (20 mL). The organic layer was dried
over MgSO4, filtered, concentrated, and evaporated with
toluene (50 mL) and CH2Cl2 (25 mL). The residue, existing
as a mixture of two isomers, was purified by column chroma-
tography with a gradient of diethyl ether/hexane (1/1-95/5,
v/v) to give the individual products as white foams.
F a ster r u n n in g isom er 2: yield 0.47 g (33%); the analytical
data (TLC, 1H NMR, 13C NMR, and MS analyses) were
identical in all aspects with those described above.
pentyl), 1.67-1.48 (m, 6H, N-CH-CHH-CH2, cyclopentyl); 13
C
NMR (DMSO-d6) δ 152.1 (C-6 or C-4), 151.2 (C-6 or C-4), 149.1
(C-2), 148.9 (C-8), 117.4 (C-5), 86.6 (C-1′), 85.9 (C-4′), 71.0 (C-
2′, C-3′), 61.7 (C-5′), 54.3 (N-CH, cyclopentyl), 32.6, 32.5 (N-
CH-CH2, cyclopentyl), 27.5 (N-CH3, methyl), 23.8, 23.7 (N-CH-
CH2-CH2, cyclopentyl); MS m/ z 365 (M + 1)+. Anal.
(C16H24N6O4) C,H,N.
8-(Cyclop en tyla m in o)-N6-eth yla d en osin e (9b). Reac-
tion was carried out with aqueous ethylamine (70%, 8.0 mL)
at 50 °C for 48 h: yield 150 mg (80%); mp 132-133 °C; Rf
0.55 (E); MS m/ z 379 (M + 1)+. Anal. (C17H26N6O4) C,H,N.
8-(Cyclop en tyla m in o)-N6-n -p r op yla d en osin e (9c). Re-
action was carried out with n-propylamine (5.0 mL) at 50 °C
for 48 h: yield 168 mg (85%); mp 119-121 °C; Rf 0.61 (E); MS
m/ z 393 (M + 1)+. Anal. (C18H28N6O4) C,H,N.
Slow er r u n n in g isom er 8: yield 0.59 g (41%); the analyti-
cal data (TLC, 1H NMR, 13C NMR, and MS analyses) were
identical in all aspects with those described earlier starting
from compound 6b.
Cr ysta l Str u ctu r e Deter m in a tion a n d Refin em en t of
9b. A colorless, plate-shaped crystal of 9b was glued to the
tip of a Lindemann glass capillary and transferred to an Enraf-
Nonius CAD4-T diffractometer on rotating anode. Accurate
unit-cell parameters and an orientation matrix were deter-
mined by least-squares refinement of the setting angles of 25
well-centered reflections (SET4) in the range 9.0° < θ < 15.3°.
Reduced-cell calculations did not indicate higher lattice sym-
metry.24 Crystal data and details on data collection and
refinement are given in Table 3. All data were collected in
ω/2θ scan mode. Data were corrected for Lp effects and the
observed linear decay of 2% of the three periodically measured
reference reflections. The structure was solved by automated
direct methods (SHELXS8625). Refinement on F 2 was carried
out by full-matrix least-squares technique (SHELXL-9326); no
observance criterion was applied during refinement. All non-
hydrogen atoms were refined with anisotropic thermal pa-
rameters. The hydrogen atoms were refined with a fixed
isotropic thermal parameter amounting to 1.5 or 1.2 times the
value of the equivalent isotropic thermal parameter of their
carrier atoms, for the hydrogen atoms on N7, N8, O12, O13,
O15, and the methyl groups, and the other hydrogen atoms,
respectively. Weights were introduced in the final refinement
cycles. The cyclopentyl moiety was disordered. No attempt
was made to model this conformational disorder in view of the
limited quality of the data. Final positional parameters are
8-(Cyclop en tyla m in o)-N6-n -bu tyla d en osin e (9d ). Reac-
tion was carried out with n-butylamine (5.5 mL) at 50 °C for
72 h: yield 183 mg (76%); mp 100-102 °C; Rf 0.64 (E); MS
m/ z 407 (M + 1)+. Anal. (C19H30N6O4) C,H,N.
2′,3′,5′-Tr i-O-a cet yl-6,8-d ich lor op u r in e
9-â-D-R ib o-
fu r a n osid e (10). The preparation of this compound has been
described before.12
2′,3′,5′-Tr i-O-a ce t yl-8-ch lor o-N 6-cyclop e n t yla d e n o-
sin e (2) a n d 2′,3′,5′-Tr i-O-a cetyl-8-(cyclop en tyla m in o)-6-
ch lor op u r in e 9-â-D-Ribofu r a n osid e (8). Compound 10
(1.29 g, 2.88 mmol) was dried with and dissolved in dioxane
(15 mL). To this solution was added cyclopentylamine (1.14
mL, 11.5 mmol), and the mixture was warmed in an oil bath
at 40 °C for 48 h. The mixture was concentrated under
reduced pressure to dryness, and the residue was evaporated
with and dissolved in pyridine (5.0 mL). Then, Ac2O (0.5 mL,
5.3 mmol) was added, and the mixture was stirred for 24 h at
room temperature. TLC analysis (C) showed complete conver-
sion of starting material into a mixture of two products. The
mixture was concentrated in vacuo and evaporated with
toluene (2 × 25 mL). The residue was dissolved in CH2Cl2
(50 mL) and washed with an aqueous NaHCO3 solution (10%,

1470 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Roelen et al.
Ta ble 4. Final Atomic Coordinates and Equivalent Isotropic
Thermal Parameters for 9b (C17H26N6O4‚C4H10O) (esd’s in
parentheses)
ological saline containing 50 IU/mL heparin (Pharmacy Aca-
demic Hospital, Leiden, The Netherlands), which was renewed
daily.
Ca r d iova scu la r Mea su r em en t. Arterial blood pressure
was measured from the femoral catheter in the abdominal
aorta using a miniature strain gauge P10EZ transducer,
equipped with a TA1017 CritiFlo diaphragm dome (both Viggo-
Spectramed BV, Bilthoven, The Netherlands). This dome
allowed a continuous flushing of the cannula with heparinized
saline (20 IU/mL) at a rate of 500 µL/h (Harvard infusion pump
22, Plato, Diemen, The Netherlands). The pressure transducer
was placed at the level of the animal heart, when in normal
position, and connected to a polygraph amplifier console
(RMP6018, Nihon Kohden Corp., Tokyo, J apan). Heart rate
was captured from the pressure signal which was used to
trigger a tachograph. Signals were recorded on a polygraph
and concurrently converted in a CED1401 interface (Cam-
bridge Electronics Design Ltd., Cambridge, England) and fed
into a 80387 computer (Philips, Eindhoven, The Netherlands).
The data were stored on hard disk for off-line analysis. Data
acquisition and reduction were performed with Spike2 com-
puter software (Cambridge Electronics Design Ltd., Cam-
bridge, England).
P h a r m a cod yn a m ic Exp er im en ts. Conscious, free-rang-
ing rats received an intravenous infusion of the compounds,
dissolved in 20% (v/v) DMSO/water, in 5 min. Control rats
were similarly treated with vehicle only. During the experi-
ments arterial blood pressure and heart rate were continuously
monitored. After connection of the arterial catheter to the
recording equipment, the rat was allowed to accommodate to
the surroundings and the experimental conditions for 30 min.
The recording of the hemodynamic parameters was started
15 min prior to the administration, for base-line determination,
and was continued for 5.5 h. The administrations took place
between 10:00 and 11:00 a.m. to minimize the potential
influence of diurnal rhythms. During the experiment the
animal had free access to water.
atom
x
y
z
U(eq) [Ang**2]
O(1)
O(12)
O(13)
0.1215(6)
0.0738(6)
0.1332(7)
0.5856(6)
0.7682(8)
0.9294(6)
0.6161(8)
0.2168(11) 0.3335(4)
0.4616(9) 0.3705(4)
-0.0011(10) 0.3371(4)
0.1959(8)
0.3432(8)
0.4466(7)
0.3683(13) 0.3464(5)
0.3861(10) 0.3846(4)
0.2320(10) 0.3744(4)
0.1537(12) 0.3481(5)
0.4866(3)
0.3613(3)
0.4531(3)
0.5017(3)
0.044(3)
0.056(3)
0.055(3)
0.059(3)
0.073(4)
0.056(4)
0.071(4)
0.042(3)
0.044(3)
0.036(3)
0.071(5)
0.038(4)
0.044(4)
0.058(4)
0.037(3)
0.042(4)
0.034(3)
0.038(3)
0.053(4)
0.055(4)
0.105(7)
0.176(11)
0.055(4)
0.078(5)
0.174(12)
0.121(8)
0.071(5)
0.085(4)
0.121(8)
0.132(10)
0.133(9)
0.168(11)
O(15) -0.1367(7)
N(1)
N(3)
N(6)
N(7)
N(8)
N(9)
C(2)
C(4)
C(5)
C(6)
C(8)
C(11)
C(12)
C(13)
C(14)
0.4140(9)
0.3473(9)
0.2978(9)
0.1075(8)
-0.0430(8)
0.1441(7)
0.4262(11)
0.2479(9)
0.2205(10)
0.3126(12)
0.0675(10)
0.1404(10)
0.0418(8)
0.0347(9)
0.0588(10)
0.3950(3)
0.4368(3)
0.4082(3)
0.3261(9)
0.6004(9)
0.7059(9)
0.8217(8)
0.7211(9)
0.4137(4)
0.4306(4)
0.4107(4)
0.4584(4)
0.5063(5)
C(15) -0.0502(9)
C(61)
C(62)
0.6750(12) 0.5372(5)
0.3920(12) -0.0924(15) 0.3086(7)
0.3441(17) -0.207(2)
0.2749(8)
0.2106(10) 0.4463(5)
0.1494(15) 0.3945(5)
0.209(3)
0.291(2)
0.2557(14) 0.4782(5)
0.3346(10) 0.2015(3)
0.199(2)
0.295(2)
0.436(2)
0.457(3)
C(81) -0.1200(10)
C(82) -0.1727(11)
C(83) -0.2866(15)
C(84) -0.3248(14)
C(85) -0.2311(11)
O(33)
C(31)
C(32)
C(34)
0.3891(7)
0.4382(7)
0.1314(9)
0.2793(14)
0.2522(16)
0.108(2)
0.1514(7)
0.1984(8)
0.2462(6)
0.2562(10)
C(35) -0.011(2)
a
1
U(eq) )
/ of the trace of the orthogonalized U.
3
listed in Table 4. Neutral atom scattering factors and anoma-
lous dispersion corrections were taken from International
Tables for Crystallography.27 Geometrical calculations and
illustrations were performed with PLATON.28 All calculations
were performed on a DECstation 5000/125.
Ack n ow led gm en t. This work was supported in part
(A.L.S. and N.V.) by The Netherlands Foundation of
Chemical Research (SON) with financial aid from The
Netherlands Organization for Scientific Research (NWO).
Ra d ioliga n d Bin d in g Stu d ies. Adenosine A1 receptor
affinities were determined on rat cortical membranes with
[3H]-1,3-dipropyl-8-cyclopentylxanthine (DPCPX) as radio-
ligand according to a protocol published previously.29 Mea-
surements with [3H]DPCPX were performed in the presence
and absence of 1 mM GTP.
Su p p or tin g In for m a tion Ava ila ble: Further details of
the NMR spectra of compounds 3b-e and 9b-d , the structure
determination of 9b, including atomic coordinates for the
hydrogen atoms, bond lengths and angles, thermal parameters,
and a thermal motion ellipsoid plot for 9b, and details of the
elemental analyses (13 pages); listings of observed and calcu-
lated structure factor amplitudes for 9b (8 pages). Ordering
information is given on any current masthead page.
Adenosine A2a receptor affinities were determined on rat
striatal membranes with [3H]CGS 21680 as radioligand ac-
cording to J arvis et al.30
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cm of polythene tubing (i.d. 0.28 mm; Portex, Medica BV,
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0.58 mm) of polythene tubing (Portex), by an approach through
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