Inhibition of nucleoside transport proteins by C8-alkylamine- substituted purines

Article abstract of DOI:10.1021/jm049303k

4-Nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein ENT1. Here we report on the synthesis and the biological evaluation of compounds that are less polar than NBTI. Compound screening in our laboratory indicate

Full text of DOI:10.1021/jm049303k

J. Med. Chem. 2005, 48, 321-329
321
Inhibition of Nucleoside Transport Proteins by C8-Alkylamine-Substituted
Purines
Reynier A. Tromp, Ronald F. Spanjersberg, Jacobien K. von Frijtag Drabbe Ku¨nzel, and Adriaan P. IJzerman*
Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Leiden University,
P.O. Box 9502, 2300RA Leiden, The Netherlands
Received August 24, 2004
4-Nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein
ENT1. Here we report on the synthesis and the biological evaluation of compounds that are
less polar than NBTI. Compound screening in our laboratory indicated that introduction of an
alkylamine substituent at the C8-position of N6-cyclopentyladenosine (CPA, 2) led to an
increment in affinity for the transport protein. It was investigated whether this would also
apply for NBTI derivatives. Two series of C8-alkylamine-substituted compounds were prepared,
one in which the nitro group was absent (46-58) and another in which the ribose moiety was
replaced by a benzyl group (72-75). Comparison of the biological data of these compounds
with 6-benzylthioinosine (4, Ki ) 53 nM) and 9-benzyl-6-(4-nitrobenzylsulfanyl)purine (59, Ki
) 135 nM) confirmed the hypothesis. The Ki values improved upon elongation of the alkylamine
chain from methylamine to n-hexylamine with an optimum for n-pentylamine (50, Ki ) 2.3
nM). Substitution with 2-methylbutylamine (52), cyclopropylamine (53), cyclopentylamine (54,
72), and cyclohexylamine (55, 73) revealed that the presence of a bulky group enhanced the
affinity. The presence of tertiary amines obtained by substitution with pyrrolidine, piperidine,
and morpholine gave only poor results. For both series substitution with cyclopentylamine
was most effective. Compound 54 (LUF5942) proved the most active, showing a comparable
affinity (Ki ) 0.64 nM) to NBTI but a significantly lower polar surface area.
Introduction
Nucleosides, such as adenosine, rely on active trans-
port via specialized proteins to cross the cell membrane
as a consequence of their hydrophilic nature.1 Two
classes of these nucleoside transport proteins exist. The
first, the concentrative nucleoside proteins, or CNTs,
depends on sodium ions and drives the nucleoside flow
against its concentration. Subtypes of this family are
CNT1, CNT2, and CNT3.2 The second family, the
Figure 1. 4-Nitrobenzylthioinoisine (NBTI).
equilibrative nucleoside transport proteins, or ENTs,
drives the nucleoside flow following its concentration
gradient and includes the subtypes ENT1 and ENT2
and the more recently discovered ENT3 and ENT4.3
Differences in the subtypes is expressed in different
substrate specificity.
In our present research we are exploring new com-
pounds that can act as ENT1 inhibitors but are less
polar than NBTI itself. Both the nitro group and the
ribose moiety of NBTI (1) add greatly to its affinity, and
thus removal of these highly polar fragments would
lower a compound’s polarity but concomitantly also
decrease its affinity. However, screening of compounds
prepared earlier in our laboratory revealed that, when
N6-cyclopentyladenosine (CPA, 2), a highly potent refer-
ence agonist for adenosine A1 receptors, was substituted
at the 8-position of the purine ring with n-propylamine
(3), the compound’s potency for ENT1 was increased by
a factor of 11 (Figure 2). This improvement of affinity
by C8-substitution with alkylamines could compensate
for this loss in potency when omitting either the ribose
moiety or the nitro group, resulting in compounds that
are less polar but still show affinity in the low nano-
molar range.
Both nucleoside transport protein classes have been
recognized as a target for the treatment of several
diseases. They may be able to modulate the uptake of
nucleoside analogues that act as antiviral or anticancer
agents into target tissues.2,3 Inhibition of the nucleoside
transport protein ENT1 is an approach to combat
ischemic heart diseases and stroke and has also been
found to protect host tissue in chemotherapy.4-6 Here,
the basic principle is that blockage of the transport
protein leads to an increased extracellular concentration
of adenosine. This results in a more profound occupancy
of the adenosine A1 receptor through which adenosine
exerts its physiological effects, e.g. counteracting pain.7,8
A highly potent inhibitor is 4-nitrobenzylthioinosine
[NBTI (1), Figure 1]. However, its highly polar nature
hinders its oral bioavailability and passage through the
blood-brain barrier.
Here, we describe the synthesis and biological evalu-
ation of C8-alkylamine-substituted analogues of 6-ben-
zylthioinosine (4).9 We also prepared a number of C8-
alkylamine-substituted analogues of 9-benzyl-6-(4-
nitrobenzylsulfanyl)purine10 (59), since substitution of
the ribose group for a benzyl moiety also reduces the
* To whom correspondence should be addressed. Tel: +31-71-
5274651. Fax: +31-71-5274565. E-mail: ijzerman@lacdr.leidenuniv.nl.
10.1021/jm049303k CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/14/2004

322 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Tromp et al.
Scheme 2a
Figure 2. C8-Substitution of N6-cyclopentyladenosine (CPA)
with n-propylamine improved its affinity for the ENT1 nucleo-
side transport protein.
Scheme 1a
a Reagents: (a) benzyl bromide, NaH, DMF, 0 °C to room
temperature, overnight; (b) 1 M HCl, reflux, 2 h; (c) Br2, Na2HPO4,
H2O, dioxane, rt, 7 d; (d) alkylamine, dioxane, H2O, microwave,
175 °C, 5 h; (e) (1) phosphorus pentasulfide, pyridine, reflux, 7 h;
(2) H2O, 50 °C, 1 h; (f) 4-nitrobenzyl bromide, K2CO3, DMF, rt,
overnight.
crowave conditions. Attempts to introduce primary
amines under the conditions used for the synthesis of
7-19 failed. Therefore, mixtures of bromide 63 and
either cyclopentylamine or cyclohexylamine in dioxane/
water were heated for 5 h at 175 °C to provide 64 and
65. Pyrrolidine and morpholine containing 66 and 67
could be obtained under both regular and microwave
conditions (2.5 h at 150 °C). Thiols 68-71 were syn-
thesized by heating 64-67 in pyridine in the presence
of phosphorus pentasulfide.12 Benzylation13 of the sulfur
using 4-nitrobenzyl bromide provided 72-75.
Polar Surface Area. The polar surface area (PSA)
was calculated as a measure to quantify the polarity of
the compounds described in this study. The PSA is
defined as the van der Waals surface of a molecule
occupied by nitrogen and oxygen and the hydrogen
atoms that are attached to these atoms. Literature
values for the upper limit for good intestinal absorption
range from 120 to 140 Å2.17-19 To allow for passage
through the blood-brain barrier, a compound’s PSA
value should be lower than 60-90 Å2.18,20 Variations in
the given limits result from differences in the calculation
methods.
The polar surface area of the molecules presented in
this report were calculated using Spartan 5.0 for SGI,21
in combination with an in-house developed application
called PolSurf 1.0. The PSA value of NBTI (1) was
determined to be 154 Å2 based on the conformation (syn)
of the crystal structure.22 The value for the anti-
conformation was calculated to be 163 Å2. The difference
results from the overlap of the N3 of the purine ring with
the ribose moiety in the case of the syn-conformation
leading to a lower van der Waals surface compared to
that of the anti-conformation in which both the 5′-OH
and the N3 are exposed. The same holds for parent
compound 4, giving PSA values of 108 and 118 Å2 for
a Reagents: (a) Br2, Na2HPO4, H2O, dioxane, rt, 7 d; (b)
alkylamine, dioxane (/H2O), 80 °C, 3 d; (c) 1) phosphorus penta-
sulfide, pyridine, reflux, 7 h; (2) H2O, 50 °C, 1 h; (d) benzylbromide,
K2CO3, DMF, rt, overnight; (e) NH3, MeOH, 0 °C to room
temperature, overnight.
number of both hydrogen-bond donors and acceptors,
important in terms of cell permeability and uptake into
the central nervous system.
Chemistry. The synthetic route toward the C8-
alkylamino-substituted benzylthioinosines (46-58) was
based on a route designed for the synthesis of amine-
substituted inosines.11 The synthesis of reference com-
pound 6-benzylthioinosine (4) has been described be-
fore.9
First, acetylated inosine (5) was brominated to pro-
vide 6 as described by Roelen et al. (Scheme 1).11 Next,
the alkylamine groups were introduced by stirring the
respective amine in dioxane for 3 days at elevated
temperatures in the presence of 6, giving 7-19. The
thiol function was introduced by refluxing the purines
with phosphorus pentasulfide in pyridine, yielding
thioinosines 20-32.12 Benzylation13 (33-45) and sub-
sequent deacetylation14 provided compounds 46-58.
The C8-substituted analogues of 9-benzyl-6-(4-ni-
trobenzylsulfanyl)purine 5910 were prepared by follow-
ing the same route as the one described above. N9-
Benzylhypoxanthine (62) was prepared from commercial-
ly available 6-chloropurine (60) in two steps (Scheme
2).15,16 After bromination, which afforded 63, the intro-
duction of the alkylamines was performed under mi-

Inhibition of Nucleoside Transport Proteins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 323
Table 1. Affinities and PSA Values for the Novel NBTI Analoguesa
compd
R
R′
R′′
PSA (Å2)b
Ki (nM)
4
ribose
ribose
ribose
ribose
ribose
ribose
ribose
ribose
ribose
ribose
ribose
ribose
ribose
ribose
benzyl
benzyl
benzyl
benzyl
benzyl
H
H
108 (syn)/118 (anti)
53 ((3)
46
47
48
49
50
51
52
53
54
55
56
57
58
59
72
73
74
75
NHMe
NHEt
H
125
124
124
124
122
122
122
124
123
120
121
120
132
86
28 ((11)
H
9.5 ((0.7)
5.3 ((1.4)
5.4 ((1.9)
2.3 ((0.1)
3.7 ((0.9)
3.6 ((1.0)
3.0 ((0.8)
0.64 ((0.31)
0.94 ((0.28)
410 ((262)
744 ((43)
33%c
NH(n-Pr)
H
NH(n-Bu)
NH(n-Pent)
NH(n-Hex)
NH(2-Me-Butyl)
NH(c-Pr)
H
H
H
H
H
NH(c-Pent)
NH(c-Hex)
pyrrolidino
piperidino
morpholino
H
NH(c-Pent)
NH(c-Hex)
pyrrolidino
morpholino
H
H
H
H
H
NO2
NO2
NO2
NO2
NO2
135 ((30)d
29 ((8)
55 ((15)
1519 ((523)
51 ((15)
100
99
97
109
a Binding study: [3H]NBTI (KD ) 0.59 nM) as radioligand and human erythrocyte membranes (n ) 3). Ki values are shown with SEM
in parentheses. b See the Experimental Section for details on the calculation of the polar surface area (PSA). The given PSA values for
46-58 concern the syn-conformer. PSA values for the anti-conformer were up to 3 Å2 higher. c Percentage of displacement at a concentration
of 10 µM. d Taken from the literature.10
its syn- and anti-conformation, respectively (Table 1).
The PSA values for compounds 46-58 and 72-75 are
depicted in Table 1 together with their biological data.
For ribose-containing 46-58, the PSA values represent
those obtained for the syn-conformer. Values for the
anti-conformation were determined to be only slightly
higher (up to 3 Å2) compared to those of the syn-
conformation. The low variation between the PSA
values for the syn- or the anti-conformation in case of
the C8-amino-substituted compounds results from the
fact that either the exocyclic alkylamine at C8 is exposed
while the N3 overlaps with the ribose moiety or visa
versa.
Biological Studies. Compounds were tested in a
radioligand binding assay using human erythrocyte
membranes as the source of the nucleoside transport
protein and [3H]NBTI as the radioligand (KD value 0.59
( 0.07 nM). Compounds that inhibited radioligand
binding for 50% or more at a single concentration of 10
µM were further analyzed over a range of concentra-
tions. Their IC50 values were converted to Ki values,
which are represented in Table 1.
The presence of n-propyl- (48) and n-butylamine (49)
resulted in affinities of 5.4 and 5.3 nM, repectively.
n-Pentylamine (50) yielded a Ki value of 2.3 nM, only
slightly higher than NBTI (1, KD ) 0.59 nM), but
compound 50 is considerably less polar. Further elonga-
tion of the alkyl chain did not lead to further improve-
ment in affinity as shown by n-hexylamine substituted
51 (Ki ) 3.7 nM).
Introduction of a bulkier alkylamine proved to give a
higher affinity as was shown by 2-methylbutylamine-
substituted 52 (Ki ) 3.7 nM), which proved to be more
potent than n-butylamine 49. This was confirmed by
compounds 53-55, carrying cycloalkylamines. In com-
parison with ethylamine-substituted 47, the presence
of cyclopropylamine at the C8-position (53) gave rise to
a 3-fold increase in affinity (Ki ) 3.0 nM). Larger alkyl
rings further improved the potency. Introduction of
cyclopentylamine (54) showed a similar affinity as NBTI
(1) with a Ki value of 0.64 nM, while its polarity is
considerably lower, with a PSA value of 123 Å2 (versus
154 Å2 for NBTI), well within the limits for intestinal
absorption as suggested by Clark.19 Cyclohexylamine-
substituted 55 proved almost equipotent with a Ki value
of 0.94 nM.
C8-Substitution with pyrrolidine (56) and piperidine
(57) indicated that the presence of tertiary amines at
the C8-position greatly decreased the affinity for ENT1,
showing Ki values of 410 and 744 nM, respectively. The
presence of an oxygen in the morpholine function
present in 58 even further decreased the affinity in
comparison with 57, showing only 33% displacement at
10 µM. A possible explanation for these large differences
in affinity may be the steric hindrance between the C8-
substituents of 56-58 and the ribose moiety. While the
actual binding conformation to ENT1 is not clear, the
The most active compounds of both series (54 and 72)
were also tested for their affinity at the adenosine A1
receptor. CHO cells expressing the human adenosine
A1 receptor were used with [3H]DPCPX as the radio-
ligand. Radioligand displacement was determined at a
single concentration of 1 µM.
Results and Discussion
Parent compound benzylthioinosine (4) showed a Ki
value of 53 nM (Table 1). Substitution at the C8-position
of the purine ring with methylamine (46) led to an
almost 2-fold improvement in affinity (Ki ) 28 nM),
while the Ki value of ethylamine-substituted 47 (9.5 nM)
was four times lower than that of parent compound 4.

324 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Tromp et al.
tuted parent compound (4). In general, a bulkier alky-
lamine resulted in a more potent compound. However,
C8-substitution with secondary amines, resulting in
tertiary amines, greatly decreased a ligand’s affinity for
ENT1. This may result from steric hindrance between
the amine substituent and the ribose moiety. Cyclopen-
tylamine-substituted 54 gave the best affinity, with a
Ki value of 0.64 nM, an 80-fold increase compared to
unsubstituted 4. In addition, a high selectivity toward
the nucleoside transport protein was observed over the
human A1 receptor. With a Ki value comparable to that
of NBTI, but considerably less polar, compound 54 may
have more favorable characteristics in aspects of ab-
sorption and distribution.
Figure 3. The crystal structure11 of 8-(cyclopentylamino)-N6-
ethyladenosine, the C6-ethylamine substituted derivative of 54.
The amine hydrogen of the cyclopentylamine substituent at
C8 (see arrow) is situated above the ribose ring.
Experimental Section
Column chromatography was performed on Baker silica gel
(0.063-0.200 mm). For TLC analysis, Schleicher and Schuell
F1500/LS 254 silica plates were used. Spots were visualized
with ultraviolet light. Microwave reactions were performed in
an Emrys Optimizer. 1H NMR and 13C NMR were recorded
with a Bruker AC 200 spectrometer at room temperature
unless indicated otherwise. Tetramethylsilane was used as
internal standard; δ values are reported in ppm and J in Hz.
Melting points were determined with a Bu¨chi melting point
apparatus and are uncorrected. High-resolution mass spec-
troscopy was performed on a PE-Sciex API Qstar instrument.
Elemental analysis was accomplished on a PE-2400 series II
CHNS/O analyzer.
Analytical HPLC for compound 72 was performed on a RP-
18 column, 5.0 µm, 125 × 4 mm. Mobile phase: system A )
10 mM AcOH in MeOH/H2O (75/25 v/v), flow rate 0.6 mL/min;
system B ) gradient between (A) MeCN/H2O (1/9 v/v), 10 mM
AcOH, and 5 mM SDS and (B) MeCN/H2O (9/1 v/v), 10 mM
AcOH, and 5 mM SDS; gradient elution t ) 0 min (40% B), t
) 15 min (100% B), t ) 18 min (100% B), flow rate 0.6 mL/
min.
crystal structure11 of a derivative of 54 that carried an
n-ethylamine substituent at C6 instead of a benzyl
thioether was found to have the anti-conformation
(Figure 3). Here the hydrogen attached to the exocyclic
amine at C8 is positioned directly above the ribose
moiety close to both the ring oxygen and the 5′-OH. The
steric hindrance that occurs when the hydrogen is
replaced by an alkyl group as in 56-58 will not only
lead to a different orientation of the C8-substituent but
also have an effect on the orientation of the purine ring
with respect to the ribose moiety.
In the case of the N9-benzylthiohypoxanthine deriva-
tives 72-75, the presence of cyclopentylamine (72) or
cyclohexylamine (73) also improved the affinity com-
pared to parent compound 54, although to a lesser
extent than found for the ribose-containing compounds.
Where for 59 an improvement in affinity of over 80-fold
with respect to unsubstituted 4 was found, compound
72 (Ki ) 29 nM) was just over 4.5 times more potent
than parent compound 59. As for the ribose compounds,
cyclohexylamine-substituted 73 (Ki ) 55 nM) was less
active than its cyclopentylamine analogue 72. Substitu-
tion with pyrrolidine (74) again resulted in a lower
affinity compared to the parent compound (59), showing
a Ki value of 1519 nM. In contrast to ribose-containing
58, the presence of a morpholino function in combination
with a benzyl group at the N9-position (75) increased
the affinity with respect to unsubstituted 59, giving a
Ki value of 51 nM.
The polar surface areas of the molecules were calculated
using Spartan 5.0 for SGI,2121 in combination with an in-house
developed application called PolSurf 1.0
(A copy of PolSurf and its C source code can be obtained
from the corresponding author). First Spartan was used to
build the molecule and to optimize its 3D-structure by molec-
ular mechanics (Merck force field) followed by semiempirical
AM1 single point energy calculation. The electrostatic poten-
tials were calculated over the entire accessible surface of the
molecules (roughly equal to the van der Waals contact surface).
Subsequently PolSurf was applied to convert the raw data from
the Spartan “input” and “proparc” files into the polar surface
area of the molecule.
The crystal structure11 (CSD code TASWEP) as depicted in
Figure 3 was retrieved from the Cambridge Structural Data-
base23 using ConQuest24 and visualized with PC Spartan Pro.25
General Procedure A.11 To a solution of 2′,3′,5′-tri-O-
acetyl-8-bromoinosine (6) in dioxane (10 mL per mmol) was
added the appropiate alkylamine (14 equiv). After stirring for
3 days at 80 °C the reaction mixture was evaporated to
dryness. The residue was dissolved in dry pyridine and
evaporated to dryness (two times). To a solution of the
resulting residue in pyridine (3.5 mL per mmol) were added
acetic acid anhydride (0.85 mL per mmol) and a catalytic
amount of DMAP. The mixture was stirred for 4 h before
methanol was added (1.5 mL per mmol). After evaporation of
the solvents, the residue was dissolved in dichloromethane (20
mL per mmol) and the organic layer was washed with 10%
NaHCO3 (10 mL per mmol) and water (10 mL per mmol). The
organic layer was dried (MgSO4), filtered, and concentrated
to dryness. The remaining pyridine was removed by coevapo-
ration with toluene and dichloromethane. The product was
purified by column chromatography (eluent: CH2Cl2/MeOH,
100/0f95/5, v/v).
To study their selectivity, compounds 54 and 72 were
tested for their affinity toward the human adenosine
A1 receptor. Both compounds showed only poor displace-
ment of the radioligand in the binding assay, i.e., 12%
at 1 µM for each of them, indicating good selectivity for
the ENT1 nucleoside transport protein over the human
adenosine A1 receptor.
Conclusions
In this report the synthesis and biological evaluation
of new NBTI analogues were addressed. In our search
for compounds that are less polar than NBTI, the nitro
functionality was omitted. To compensate for the loss
in affinity, the C8-position of the purine ring was
substituted with alkylamines. Compounds with affini-
ties at the low nanomolar level and PSA values close to
the limits given for blood-brain barrier passage were
obtained. Substitution with primary amines resulted in
improved affinities in comparison with the unsubsti-
General Procedure B.12 Per mmol of purine (3× coevapo-
rated with dry pyridine) in 25 mL of dry pyridine was added

Inhibition of Nucleoside Transport Proteins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 325
1.75 g of phosphorus pentasulfide, and the resulting mixture
was refluxed for 7 h. After evaporation of the solvent, the resi-
dual solvent was removed by coevaporation with MeOH (2×).
Water was added and the resulting mixture was stirred for 1
h at 50 °C. After extraction with EtOAc (3 × 15 mL), the com-
bined organic layers were dried (Na2SO4) and filtered, and the
solvent was evaporated. The product was purified by column
chromatography (eluent: EtOAc/MeOH, 97/3f95/5 v/v).
General Procedure C.13 Per mmol of substituted purine
in 7.5 mL of dry DMF was added 1 equiv of K2CO3 and 1.2
equiv of (4-nitro-) benzyl bromide. After stirring overnight at
room temperature, 10 mL of water was added, and the mixture
was extracted with EtOAc (3 × 10 mL). The combined organic
layers were washed with brine (15 mL), dried (Na2SO4), and
filtered, and the solvent was evaporated. The product was
purified by column chromatography (eluent: EtOAc/PE 40-
60, 1/1f6/4 v/v).
3 × CH3CO), 3.35-3.68 (m, 2H, CH2N), 4.87-4.41 (m, 2H,
H-4′, 1 × H-5′), 4.49-4.63 (m, 1H, 1 × H-5′), 5.03 (t, 1H, J )
5.9, CH2NH), 5.49 (dd, 1H, J ) 4.4, J ) 6.2, H-3′), 5.80 (t, 1H,
J ) 6.2, H-2′), 6.12 (d, 1H, J ) 6.2, H-1′), 7.97 (s, 1H, H-2),
11.24 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-(n-hexylamino)inosine (12). This
compound was prepared according to general procedure A with
n-hexylamine: yield 43%; yellowish oil; 1H NMR (CDCl3) δ 0.89
(t, 3H, J ) 6.6, CH3CH2), 1.21-1.48 (m, 6H, 3 × CH2), 1.60-
1.74 (m, 2H, CH2CH2N), 2.05, 2.12, 2.15 (3 × s, 3 × 3H, 3 ×
CH3CO), 3.37-3.67 (m, 2H, CH2N), 4.27-4.43 (m, 2H, H-4′, 1
× H-5′), 4.49-4.63 (m, 1H, 1 × H-5′), 5.03 (t, 1H, J ) 5.8,
CH2NH), 5.51 (dd, 1H, J ) 4.2, J ) 6.0, H-3′), 5.80 (t, 1H, J )
6.2, H-2′), 6.13 (d, 1H, J ) 6.2, H-1′), 7.98 (s, 1H, H-2), 11.12
(br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-[(2-methylbutyl)amino]inosine
(13). This compound was prepared according to general
procedure A applying 2-methylbutylamine: yield 27%; color-
less oil; 1H NMR (CDCl3) δ 0.87-0.99 (m, 6H, 2 × CH3), 1.05-
1.28 (m, 1H, CH3CHH), 1.39-1.62 (m, 1H, CH3CHH), 1.69-
1.90 (m, 1H, CH3CH), 2.06, 2.11, 2.15 (3 × s, 3 × 3H, 3 ×
CH3CO), 3.17-3.34 (m, 0.5H, CHHNH), 3.36-3.46 (m, 1H,
CHHNH), 3.47-3.66 (m, 0.5H, CHHNH), 4.28-4.61 (m, 3H,
H-4′, H-5′), 5.07 (t, 1H, J ) 5.9, NH), 5.46-5.55(m, 1H, H-3′),
5.79-5.88 (m, 1H, H-2′), 6.07-6.15 (m, 1H, H-1′), 7.99 (s, 1H,
H-2).
2′,3′,5′-Tri-O-acetyl-8-(cyclopropylamino)inosine (14).
This compound was prepared according to general procedure
A with cyclopropylamine (1.7 mL per mmol) and H2O (3.5 mL
per mmol): yield 57%; yellowish solid; 1H NMR (CDCl3) δ
0.51-0.68 (m, 2H, CH2CHN), 0.80-0.98 (m, 2H, CH2CHN),
2.05, 2.14, 2.15 (3 × s, 3 × 3H, 3 × CH3CO), 2.88-3.05 (m,
1H, CHN), 4.29-4.40 (m, 2H, H-4′, 1 × H-5′), 4.50 (ABX, 1H,
J ) 4.8, J ) 12.8, 1 × H-5′), 5.41 (br s, 1H, CH2CHNH), 5.50
(dd, 1H, J ) 4.8, J ) 5.9, H-3′), 5.75 (t, 1H, J ) 6.2, H-2′),
6.10 (d, 1H, J ) 6.2, H-1′), 8.05 (s, 1H, H-2), 11.21 (br s, 1H,
NH).
General Procedure D.14 Per mmol of purine was added
25 mL of MeOH saturated with NH3 at 0 °C. After stirring
overnight the solvent was evaporated and the product purified
by column chromatography (eluent: EtOAc/MeOH, 95/5f90/
10 v/ v). The appropriate fractions were collected and evapo-
rated to dryness. Residual EtOAc was removed by dissolving
the product in CH2Cl2 followed by evaporation (two times).
General Procedure E. Per mmol of purine 63 were added
6 mL of dioxane and 2.4 mL of water followed by 7.5 equiv of
the required amine. The mixture was heated in an Emrys
Optimizer microwave for 5 h at 175 °C (64 and 65) or 2.5 h at
150 °C (66 and 67). After removal of the solvents by evapora-
tion, dioxane was added and the mixture evaporated to
dryness. The product was purified by column chromatography
(eluent: CH2Cl2/MeOH, 99/1f90/10 v/v).
6-Benzylthioinosine (4). This compound was prepared
according to a literature procedure.9
2′,3′,5′-Tri-O-acetylinosine (5). This compound was pre-
pared according to a literature procedure.11
2′,3′,5′-Tri-O-acetyl-8-bromoinosine (6). This compound
was prepared according to a literature procedure.11
2′,3′,5′-Tri-O-acetyl-8-(cyclopentylamino)inosine (15).
This compound was prepared according to a literature proce-
dure.11
2′,3′,5′-Tri-O-acetyl-8-(methylamino)inosine (7). This
compound was prepared according to general procedure A with
methylamine (40% in H2O, 15 mL per mmol): yield 65%; white
2′,3′,5′-Tri-O-acetyl-8-(cyclohexylamino)inosine (16).
This compound was prepared according to general procedure
A with cyclohexylamine: yield 28%; colorless oil; 1H NMR
(CDCl3) δ 1.07-1.84 (m, 8H, 2 × NHCHCHH, 3 × CH2), 2.06,
2.09, 2.14 (3 × s, 3 × 3H, 3 × CH3CO), 2.07-2.25 (m, 2H, 2 ×
NHCHCHH), 3.87-4.08 (m, 1H, CH2CHNH), 4.29-4.41 (m,
3H, H-4′, H-5′), 4.50 (ABX, 1H, J ) 4.7, J ) 12.8, 1 × H-5′),
4.73 (d, 1H, J ) 7.5, CH2CHNH), 5.53 (dd, 1H, J ) 4.6, J )
5.9, H-3′), 5.88 (t, 1H, J ) 5.9, H-2′), 6.03 (d, 1H, J ) 5.9, H-1′),
7.91 (s, 1H, H-2), 11.31 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-pyrrolidinoinosine (17). This com-
pound was prepared according to general procedure A with
pyrrolidine: yield 65%; white foam; 1H NMR (CDCl3) δ 1.72-
2.09 (m, 4H, 2 × CH2CH2N), 2.06, 2.08, 2.12 (3 × s, 3 × 3H, 3
× CH3CO), 3.52-3.75 (m, 4H, 2 × CH2N), 4.29-4.37 (m, 2H,
H-4′, 1 × H-5′), 4.48 (ABX, 1H, J ) 6.0, J ) 13.7, 1 × H-5′),
5.86 (dd, 1H, J ) 5.1, J ) 5.9, H-3′), 5.98 (d, 1H, J ) 4.9, H-1′),
6.40 (dd, 1H, J ) 4.9, J ) 5.9, H-2′), 7.97 (s, 1H, H-2), 11.44
(br s, 1H, NH).
1
solid; H NMR (CDCl3) δ 2.04, 2.15, 2.16 (3 × s, 3 × 3H, 3 ×
CH3CO), 3.11 (d, 3H, J ) 4.9 CH3N), 4.26-4.41 (m, 2H, H-4′,
1 × H-5′), 4.54-4.68 (m, 1H, 1 × H-5′), 5.09 (q, 1H, J ) 4.9,
CH3NH), 5.46 (dd, 1H, J ) 3.8, J ) 6.2, H-3′), 5.71 (t, 1H, J )
6.6, H-2′), 6.18 (d, 1H, J ) 6.8, H-1′), 7.97 (s, 1H, H-2), 11.55
(br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-(ethylamino)inosine (8). This com-
pound was prepared according to a literature procedure.11
2′,3′,5′-Tri-O-acetyl-8-(n-propylamino)inosine (9). This
compound was prepared according to general procedure A with
n-propylamine (11.5 mL per mmol) and H2O (5 mL per
mmol): yield 58%; yellowish foam; 1H NMR (CDCl3) δ 0.96 (t,
3H, J ) 7.3, CH3CH2), 1.70 (sextet, 2H, J ) 7.3, CH3CH2),
2.05, 2.13, 2.15 (3 × s, 3 × 3H, 3 × CH3CO), 3.35-3.67 (m,
2H, CH2N), 4.27-4.42 (m, 2H, H-4′, 1 × H-5′), 4.51-4.63 (m,
1H, 1 × H-5′), 5.06 (t, 1H, J ) 5.9, CH2NH), 5.48 (dd, 1H, J )
4.0, J ) 5.8, H-3′), 5.79 (t, 1H, J ) 6.2, H-2′), 6.12 (d, 1H, J )
6.6, H-1′), 7.92 (s, 1H, H-2), 11.55 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-piperidinoinosine (18). This com-
2′,3′,5′-Tri-O-acetyl-8-(n-butylamino)inosine (10). This
compound was prepared according to general procedure A with
n-butylamine: yield 47%; yellowish foam; 1H NMR (CDCl3) δ
0.96 (t, 3H, J ) 7.3, CH3CH2), 1.29-1.69 (m, 2H, CH3CH2),
1.57-1.75 (m, 2H, CH2CH2N), 2.05, 2.12, 2.15 (3 × s, 3 × 3H,
3 × CH3CO), 3.37-3.68 (m, 2H, CH2N), 4.27-4.41 (m, 2H,
H-4′, 1 × H-5′), 4.48-4.63 (m, 1H, 1 × H-5′), 5.00 (t, 1H, J )
5.9, CH2NH), 5.49 (dd, 1H, J ) 4.4, J ) 5.9, H-3′), 5.79 (t, 1H,
J ) 6.2, H-2′), 6.11 (d, 1H, J ) 6.6, H-1′), 7.91 (s, 1H, H-2),
11.40 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-(n-pentylamino)inosine (11). This
compound was prepared according to general procedure A with
n-pentylamine: yield 81%; yellow foam; 1H NMR (CDCl3) δ
0.90 (t, 3H, J ) 6.6, CH3CH2), 1.21-1.47 (m, 4H, 2 × CH2),
1.56-1.79 (m, 2H, CH2CH2N), 2.05, 2.12, 2.15 (3 × s, 3 × 3H,
pound was prepared according to general procedure A with
1
piperidine: yield 60%; white solid; H NMR (CDCl3) δ 1.51-
1.82 (m, 6H, (CH2)3CH2N), 2.07, 2.09, 2.13 (3 × s, 3 × 3H, 3 ×
CH3CO), 3.14-3.38 (m, 4H, 2 × CH2N), 4.28-4.38 (m, 2H,
H-4′, 1 × H-5′), 4.51 (ABX, 1H, J ) 6.2, J ) 13.9, 1 × H-5′),
5.84-5.96 (m, 2H, H-1′, H-3′), 6.25 (dd, 1H, J ) 4.8, J ) 6.2,
H-2′), 8.07 (s, 1H, H-2), 11.50 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-N-morpholinoinosine (19). This
compound was prepared according to general procedure A with
1
morpholine: yield 40%; white solid; H NMR (CDCl3) δ 2.06,
2.10, 2.14 (3 × s, 3 × 3H, 3 × CH3CO), 3.21-3.49 (m, 4H, 2 ×
CH2N), 3.77-3.95 (m, 4H, 2 × CH2O), 4.26-4.55 (m, 3H, H-4′,
H-5′), 5.85-5.94 (m, 2H, H-1′, H-3′), 6.29 (dd, 1H, J ) 4.6, J
) 6.0, H-2′), 8.10 (s, 1H, H-2), 11.52 (br s, 1H, NH).

326 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Tromp et al.
2′,3′,5′-Tri-O-acetyl-8-(methylamino)thioinosine (20).
This compound was prepared according to general procedure
B: yield 63%; yellow oil; 1H NMR (CDCl3) δ 2.03, 2.14, 2.15 (3
× s, 3 × 3H, 3 × CH3CO), 3.15-3.78 (d, 3H, J ) 4.8, CH3N),
4.28-4.44 (m, 2H, H-4′, 1 × H-5′), 4.53-4.66 (m, 1H, 1 × H-5′),
5.45 (dd, 1H, J ) 4.0, J ) 6.2, H-3′), 5.59 (q, 1H, J ) 4.8, NH),
5.66-5.76 (m, 1H, H-2′), 6.17 (d, 1H, J ) 6.6, H-1′), 8.11 (s,
1H, H-2).
2′,3′,5′-Tri-O-acetyl-8-(cyclopentylamino)thioinosine
(28). This compound was prepared according to general
procedure B: yield 22%; brownish oil; 1H NMR (CDCl3) δ 1.41-
1.70 (m, 8H, 4 × CH2), 2.05, 2.06, 2.15 (3 × s, 3 × 3H, 3 ×
CH3 CO), 4.29-4.63 (m, 4H, H-4′, H-5′, CHNH), 5.15 (d, 1H J
) 5.6, CHNH), 5.49 (t, 1H, J ) 5.9, H-3′), 5.81 (t, 1H, J ) 5.9,
H-2′), 6.04 (d, 1H, J ) 5.9, H-1′), 8.01 (s, 1H, H-2), 11.77 (br s,
1H, NH).
2′,3′,5′-Tri-O-acetyl-8-(cyclohexylamino)thioinosine (29).
2′,3′,5′-Tri-O-acetyl-8-(ethylamino)thioinosine (21). This
compound was prepared according to general procedure B:
yield 67%; yellow solid; 1H NMR (CDCl3) δ 1.34 (t, 3H, J )
7.3, CH3CH2), 2.03, 2.13, 2.15 (3 × s, 3 × 3H, 3 × CH3CO),
3.56-3.78 (m, 2H, CH2N), 4.28-4.42 (m, 2H, H-4′, 1 × H-5′),
4.59 (dd, 1H, J ) 4.0, J ) 12.1, 1 × H-5′), 5.29 (t, 1H, J ) 4.4,
NH), 5.45 (dd, 1H, J ) 4.0, J ) 6.6, H-3′), 5.72 (t, 1H, J ) 6.6,
H-2′), 6.12 (d, 1H, J ) 6.6, H-1′), 8.01 (s, 1H, H-2).
This compound was prepared according to general procedure
1
B: yield 41%; yellow solid; H NMR (CDCl3) δ 1.05-1.85 (m,
8H, 2 × NHCHCHH, 3 × CH2), 2.05, 2.11, 2.15 (3 × s, 3 ×
3H, 3 × CH3CO), 1.98-2.24 (m, 2H, 2 × NHCHCHH), 4.02-
4.23 (m, 1H, CH2CHNH), 4.28-4.58 (m, 3H, H-4′, H-5′), 5.07
(d, 1H, J ) 8.0, CH2CHNH), 5.48 (dd, 1H, J ) 4.4, J ) 5.9,
H-3′), 5.81 (dd, 1H, J ) 5.9, J ) 6.6, H-2′), 6.05 (d, 1H, J )
6.6, H-1′), 7.99 (s, 1H, H-2), 11.79 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-(n-propylamino)thioinosine (22).
2′,3′,5′-Tri-O-acetyl-8-pyrrolidinothioinosine (30). This
compound was prepared according to general procedure B:
yield 55%; brownish oil; 1H NMR (CDCl3) δ 1.78-2.09 (m, 4H,
2 × CH2CH2N), 2.03, 2.05, 2.11 (3 × s, 3 × 3H, 3 × CH3CO),
3.60-3.82 (m, 4H, 2 × CH2N), 4.21-4.53 (m, 3H, H-4′, H-5′),
5.80 (t, 1H, J ) 5.1, H-3′), 5.98 (d, 1H, J ) 5.1, H-1′), 6.40 (t,
1H, J ) 5.1, H-2′), 8.05 (s, 1H, H-2), 11.74 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-piperidinothioinosine (31). This
compound was prepared according to general procedure B:
yield 82%; brown oil; 1H NMR (CDCl3) δ 1.56-1.83 (m, 6H,
(CH2)3CH2N), 2.05, 2.08, 2.14 (3 × s, 3 × 3H, 3 × CH3CO),
3.22-3.49 (m, 4H, 2 × CH2N), 4.26-4.55 (m, 3H, H-4′, H-5′),
5.79-5.88 (m, 2H, H-1′, H-3′), 6.25 (t, 1H, J ) 5.5, H-2′), 8.10
(s, 1H, H-2), 12.05 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-N-morpholinothioinosine (32). This
compound was prepared according to general procedure B:
yield 84%; yellow oil; 1H NMR (CDCl3) δ 2.07, 2.09, 2.15 (3 ×
s, 3 × 3H, 3 × CH3CO), 3.27-3.42 (m, 2H, 2 × CHHN), 3.44-
3.59 (m, 2H, 2 × CHHN), 3.78-3.99 (m, 4H, 2 × CH2N), 4.25-
4.51 (m, 3H, H-4′, H-5′), 5.81-5.90 (m, 2H, H-1′, H-3′), 6.30
(dd, 1H, J ) 4.8, J ) 5.9, H-2′), 8.10 (s, 1H, H-2), 12.15 (br s,
1H, NH).
This compound was prepared according to general procedure
1
B: yield 60%; brown oil; H NMR (CDCl3) δ 0.97 (t, 3H, J )
7.3, CH3CH2), 1.59-1.81 (m, 2H, CH3CH2), 2.03, 2.12, 2.15 (3
× s, 3 × 3H, 3 × CH3CO), 3.42-3.75 (m, 2H, CH2N), 4.26-
4.45 (m, 2H, H-4′, 1 × H-5′), 4.58 (dd, 1H, J ) 4.0, J ) 12.1,
1 × H-5′), 5.36 (t, 1H, J ) 5.5, NH), 5.46 (dd, 1H, J ) 4.4, J )
6.6, H-3′), 5.76 (t, 1H, J ) 6.6, H-2′), 6.14 (d, 1H, J ) 6.6, H-1′),
8.05 (s, 1H, H-2), 11.67 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-(n-butylamino)thioinosine (23).
This compound was prepared according to general procedure
1
B: yield 61%; yellow oil; H NMR (CDCl3) δ 0.94 (t, 3H, J )
7.3, CH3CH2), 1.30-1.50 (m, 2H, CH3CH2), 1.57-1.75 (m, 2H,
CH2CH2NH), 2.04, 2.12, 2.15 (3 × s, 3 × 3H, 3 × CH3CO),
3.46-3.77 (m, 2H, CH2N), 4.26-4.44 (m, 2H, H-4′, 1 × H-5′),
4.58 (dd, 1H, J ) 4.0, J ) 12.1, 1 × H-5′), 5.30 (t, 1H, J ) 5.5,
NH), 5.45 (dd, 1H, J ) 4.0, J ) 6.2, H-3′), 5.76 (dd, 1H, J )
6.2, J ) 6.6, H-2′), 6.13 (d, 1H, J ) 6.6, H-1′), 8.04 (s, 1H,
H-2), 11.63 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-(n-pentylamino)thioinosine (24).
This compound was prepared according to general procedure
1
B: yield 45%; yellow-brown foam; H NMR (CDCl3) δ 0.89 (t,
3H, J ) 6.6, CH3CH2), 1.23-1.43 (m, 4H, 2 × CH2), 1.56-
1.75 (m, 2H, CH2CH2N), 2.03, 2.12, 2.15 (3 × s, 3 × 3H, 3 ×
CH3CO), 3.42-3.76 (m, 2H, CH2N), 4.22-4.44 (m, 2H, H-4′, 1
× H-5′), 4.57 (ABX, 1H, J ) 4.4, J ) 11.7, 1 × H-5′), 5.34 (t,
1H, J ) 5.1, CH2NH), 5.46 (dd, 1H, J ) 3.7, J ) 5.9, H-3′),
5.74 (dd, 1H, J ) 5.9, J ) 6.6, H-2′), 6.13 (d, 1H, J ) 6.6, H-1′),
8.07 (s, 1H, H-2), 11.47 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-(methylamino)thioino-
sine (33). This compound was prepared according to general
procedure C: yield 61%; white solid; 1H NMR (CDCl3) δ 2.01,
2.13, 2.15 (3 × s, 3 × 3H, 3 × CH3CO), 3.13 (d, 2H, J ) 5.1,
CH3N), 4.61 (m, 2H, H-4′, 1 × H-5′), 4.61 (ABX, 1H, J ) 3.7,
J ) 11.7, 1 × H-5′), 4.62 (s, 2H, CH2S), 5.36 (t, 1H, J ) 5.1,
NH), 5.47 (dd, 1H, J ) 3.4, J ) 6.2, H-3′), 5.73 (dd, 1H, J )
6.2, J ) 6.9, H-2′), 6.24 (d, 1H, J ) 6.9, H-1′), 7.21-7.34 (m,
3H, CH arom), 7.43-7.49 (m, 2H, CH arom), 8.52 (s, 1H, H-2).
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-(ethylamino)thioino-
sine (34). This compound was prepared according to general
2′,3′,5′-Tri-O-acetyl-8-(n-hexylamino)thioinosine (25).
This compound was prepared according to general procedure
1
B: yield 58%; yellow oil; H NMR (CDCl3) δ 0.89 (t, 3H, J )
6.6, 1 × CH3CH2), 1.25-1.44 (m, 6H, 3 × CH2), 1.55-1.77 (m,
2H, CH2CH2NH), 2.04, 2.12, 2.15 (3 × s, 3 × 3H, 3 × CH3 CO),
3.44-3.77 (m, 2H, CH2N), 4.26-4.44 (m, 2H, H-4′, 1 × H-5′),
4.58 (dd, 1H, J ) 4.4, J ) 11.7, 1 × H-5′), 5.29 (t, 1H, J ) 5.1,
NH), 5.45 (dd, 1H, J ) 4.4, J ) 6.6, H-3′), 5.73 (t, 1H, J ) 6.6,
H-2′), 6.13 (d, 1H, J ) 6.6, H-1′), 8.02 (s, 1H, H-2), 11.68 (br s,
1H, NH).
1
procedure C: yield 52%; white solid; H NMR (CDCl3) δ 1.30
(t, 3H, J ) 7.3, CH3CH2), 2.03, 2.09, 2.15 (3 × s, 3 × 3H, 3 ×
CH3CO), 3.46-3.69 (m, 2H, CH2N), 4.24-4.58 (m, 3H, H-4′,
H-5′), 4.62 (s, 2H, CH2S), 5.24 (t, 1H, J ) 5.5, NH), 5.51 (dd,
1H, J ) 3.7, J ) 5.9, H-3′), 5.84 (t, 1H, J ) 6.2, H-2′), 6.17 (d,
1H, J ) 6.6, H-1′), 7.22-7.36 (m, 3H, CH arom), 7.38-7.47
(m, 2H, CH arom), 8.50 (s, 1H, H-2).
2′,3′,5′-Tri-O-acetyl-8-[(2-methylbutyl)amino]thioin-
osine (26). This compound was prepared according to general
1
procedure B: yield 44%; yellow oil; H NMR (CDCl3) δ 0.87-
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-(n-propylamino)thioi-
nosine (35). This compound was prepared according to
general procedure C: yield 59%; yellowish solid; 1H NMR
(CDCl3) δ 0.97 (t, 3H, J ) 7.3, CH3CH2), 2.03, 2.07, 2.15 (3 ×
s, 3 × 3H, 3 × CH3CO), 3.36-3.66 (m, 2H, CH2N), 4.25-4.42
(m, 1H, H-4′, 1 × H-5′), 4.56 (ABX, 1H, J ) 3.7, J ) 11.7, 1 ×
H-5′), 4.62 (s, 2H, CH2S), 5.30 (t, 1H, J ) 5.9, NH), 5.52 (dd,
1H, J ) 4.0, J ) 5.9, H-3′), 5.87 (t, 1H, J ) 6.2, H-2′), 6.16 (d,
1H, J ) 6.2, H-1′), 7.17-7.34 (m, 3H, CH arom), 7.38-7.49
(m, 2H, CH arom), 8.50 (s, 1H, H-2).
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-(n-butylamino)thioino-
sine (36). This compound was prepared according to general
procedure C: yield 66%; off-white solid; 1H NMR (CDCl3) δ
0.95 (t, 3H, J ) 7.3, CH3CH2), 1.30-1.50 (m, 2H, CH2CH3),
1.55-1.75 (m, 2H, CH2CH2N), 2.03, 2.07, 2.15 (3 × s, 3 × 3H,
3 × CH3CO), 3.39-3.69 (m, 2H, CH2N), 4.27-4.43 (m, 2H,
H-4′, 1 × H-5′), 4.56 (ABX, 1H, J ) 3.7, J ) 11.7, 1 × H-5′),
1.00 (m, 6H, 2 × CH3), 1.07-1.31 (m, 1H, CH3CHH), 1.37-
1.60 (m, 1H, CH3CHH), 1.64-1.92 (m, 1H, CH3CH), 2.04, 2.10,
2.15 (3 × s, 3 × 3H, 3 × CH3CO), 3.27-3.70 (m, 2H, CH2N),
4.26-4.45 (m, 2H, H-4′, 1 × H-5′), 4.55 (dd, 1H, J ) 3.7, J )
12.1, 1 × H-5′), 5.30 (t, 1H, J ) 5.5, NH), 5.46 (dd, 1H, J )
4.4, J ) 6.2, H-3′), 5.77 (t, 1H, J ) 6.2, H-2′), 6.11 (d, 1H, J )
6.2, H-1′), 8.01 (s, 1H, H-2), 11.76 (br s, 1H, NH).
2′,3′,5′-Tri-O-acetyl-8-(cyclopropylamino)thioinosine
(27). This compound was prepared according to general
1
procedure B: yield 49%; yellow oil; H NMR (CDCl3) δ 0.58-
0.70 (m, 2H, 2 × CHH), 0.81-0.94 (m, 2H, CH2CH2NH), 2.05,
2.14, 2.16 (3 × s, 3 × 3H, 3 × CH3 CO), 2.98-3.13 (m, 1H,
CHN), 4.26-4.41 (m, 2H, H-4′, 1 × H-5′), 4.46-4.60 (m, 1H, 1
× H-5′), 5.46 (dd, 1H, J ) 4.4, J ) 5.9, H-3′), 5.65-5.77 (m,
2H, H-2′, CHNH), 6.13 (d, 1H, J ) 5.8, H-1′), 8.17 (s, 1H, H-2),
11.75 (br s, 1H, NH).

Inhibition of Nucleoside Transport Proteins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 327
4.62 (s, 2H, CH2S), 5.25 (t, 1H, J ) 5.4, NH), 5.53 (dd, 1H, J
) 4.4, J ) 5.9, H-3′), 5.87 (t, 1H, J ) 5.9, H-2′), 6.15 (d, 1H, J
) 5.9, H-1′), 7.17-7.34 (m, 3H, CH arom), 7.41-7.49 (m, 2H,
CH arom), 8.50 (s, 1H, H-2).
5.95-6.03 (m, 2H, H-1′, H-3′), 6.59 (dd, 1H, J ) 4.8, J ) 5.8,
H-2′), 7.20-7.33 (m, 3H, CH arom), 7.40-7.48 (m, 2H, CH
arom), 8.51 (s, 1H, H-2).
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-piperidinothioinosine
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-(n-pentylamino)thioin-
osine (37). This compound was prepared according to general
procedure C: yield 95%; yellowish oil; 1H NMR (CDCl3) δ 0.85-
0.94 (m, 3H, CH3CH2), 1.23-1.45 (m, 4H, 2 × CH2), 1.60-
1.73 (m, 2H, CH2CH2N), 2.05, 2.07, 2.14 (3 × s, 3 × 3H, 3 ×
CH3CO), 3.38-3.67 (m, 2H, CH2N), 4.27-4.41 (m, 2H, H-4′, 1
× H-5′), 4.58 (dd, 1H, J ) 4.4, J ) 11.7, 1 × H-5′), 4.62 (s, 2H,
CH2S), 5.03 (t, 1H, J ) 5.1, CH2NH), 5.53 (dd, 1H, J ) 3.7, J
) 5.9, H-3′), 5.87 (t, 1H, J ) 5.9, H-2′), 6.16 (d, 1H, J ) 5.9,
H-1′), 7.18-7.33 (m, 3H, CH arom), 7.45 (dd, 2H, J ) 1.5, J )
8.0, CH arom), 8.50 (s, 1H, H-2).
(44). This compound was prepared according to general
procedure C: yield 48%; colorless oil; 1
H NMR (CDCl3) δ 1.57-
1.84 (m, 6H, 3 × CH2), 2.06, 2.07, 2.13 (3 × s, 3 × 3H, 3 ×
CH3CO), 3.19-3.45 (m, 4H, 2 × CH2N), 4.29-4.41 (m, 2H,
H-4′, 1 × H-5′), 4.52 (ABX, 1H, J ) 6.2, J ) 13.9, 1 × H-5′),
4.62 (s, 2H, CH2S), 5.84 (d, 1H, J ) 4.8, H-1′), 6.00 (t, 1H, J )
5.9, H-3′), 6.38 (dd, 1H, J ) 4.8, J ) 5.9, H-2′), 7.17-7.34 (m,
3H, CH arom), 7.44 (dd, 2H, J ) 1.8, J ) 8.0, CH arom), 8.58
(s, 1H, H-2).
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-morpholinothioino-
sine (45). This compound was prepared according to general
procedure C: yield 66%; colorless oil; 1H NMR (CDCl3) δ 2.06,
2.07, 2.14 (3 × s, 3 × 3H, 3 × CH3CO), 3.24-3.29 (m, 2H, 2 ×
CHHN), 3.42-3.57 (m, 2H, 2 × CHHN), 3.77-3.97 (m, 4H, 2
× CH2O), 4.27-4.39 (m, 2H, H-4′, 1 × H-5′), 4.43-5.56 (m,
1H, 1 × H-5′), 4.62 (s, 2H, CH2S), 5.86 (d, 1H, J ) 4.6, H-1′),
6.01 (t, 1H, J ) 5.7, H-3′), 6.44 (dd, 1H, J ) 4.6, J ) 5.7, H-2′),
7.17-7.34 (m, 3H, CH arom), 7.45 (dd, 2H, J ) 1.8, J ) 8.0,
CH arom), 8.61 (s, 1H, H-2).
6-Benzyl-8-(methylamino)thioinosine (46). This com-
pound was prepared according to general procedure D: yield
83%; white solid; 1H NMR (MeOD) δ 3.02 (s, 3H, CH3), 3.76-
3.89 (m, 2H, H-5′), 4.15 (dd, 1H, J ) 1.8, J ) 4.0, H-4′), 4.27
(dd, 1H, J ) 1.8, J ) 5.5, H-3′), 4.61 (s, 2H, CH2S), 4.70 (dd,
1H, J ) 5.5, J ) 7.7, H-2′), 6.61 (d, 1H, J ) 7.7, H-1′), 7.17-
7.33 (m, 3H, CH arom), 7.42 (dd, 2H, J ) 2.2, J ) 8.0, CH
arom), 8.39 (s, 1H, H-2). Anal. (C18H21N5O4S‚0.15CH2Cl2) C,
H, N, S.
6-Benzyl-8-(ethylamino)thioinosine (47). This compound
was prepared according to general procedure D: yield 96%;
white solid; 1H NMR (CDCl3/MeOD 1/1, v/v) δ 1.21 (t, 3H, J )
7.3, CH3CH2), 3.34-3.56 (m, 2H, CH2N), 3.77 (ABX, 1H, J )
1.1, J ) 12.4, 1 × H-5′), 3.92 (ABX, 1H, J ) 2.0, J ) 12.4, 1 ×
H-5′), 4.18-4.24 (m, 1H, H-4′), 4.30 (dd, 1H, J ) 1.6, J ) 5.5,
H-3′), 4.61 (s, 2H, CH2S), 4.70 (dd, 1H, J ) 5.5, J ) 7.1, H-2′),
5.90 (d, 1H, J ) 7.1, H-1′), 7.18-7.34 (m, 3H, CH arom), 7.40-
7.48 (m, 2H, CH arom), 8.39 (s, 1H, H-2). Anal. (C19H23N5O4S‚-
0.05CH2Cl2) C, H, N, S.
6-Benzyl-8-(n-propylamino)thioinosine (48). This com-
pound was prepared according to general procedure D: yield
78%; white solid; 1H NMR (MeOD) δ 0.97 (t, 3H, J ) 7.3, CH3-
CH2), 1.56-1.77 (m, 2H, CH3CH2), 3.34-3.50 (m, 2H, CH2N),
3.74-3.86 (m, 2H, H-5′), 4.10-4.17 (m, 1H, H-4′), 4.21-4.29
(m, 1H, H-3′), 4.61 (s, 2H, CH2S), 4.69 (dd, 1H, J ) 5.5, J )
7.7, H-2′), 6.13 (d, 1H, J ) 7.7, H-1′), 7.13-7.31 (m, 3H, CH
arom), 7.36-7.45 (m, 2H, CH arom), 8.37 (s, 1H, H-2). Anal.
(C20H25N5O4S) C, H, N, S.
6-Benzyl-8-(n-butylamino)thioinosine (49). This com-
pound was prepared according to general procedure D: yield
87%; white solid; 1H NMR (MeOD) δ 0.95 (t, 3H, J ) 7.3, CH3-
CH2), 1.31-1.51 (m, 2H, CH3CH2), 1.55-1.73 (m, 2H, CH2-
CH2N), 3.34-3.58 (m, 2H, CH2N), 3.77-3.83 (m, 2H, H-5′),
4.11-4.16 (m, 1H, H-4′), 4.26 (dd, 1H, J ) 1.8, J ) 5.5, H-3′),
4.61 (s, 2H, CH2S), 4.68 (dd, 1H, J ) 5.5, J ) 7.7, H-2′), 6.14
(d, 1H, J ) 7.7, H-1′), 7.15-7.31 (m, 3H, CH arom), 7.37-
7.46 (m, 2H, CH arom), 8.37 (s, 1H, H-2). Anal. (C21H27N5O4S)
C, H, N, S.
6-Benzyl-8-(n-pentylamino)thioinosine (50). This com-
pound was prepared according to general procedure D: yield
83%; white foam; 1H NMR (MeOD) δ 0.89 (t, 3H, J ) 6.9,
CH3CH2), 1.28-1.43 (m, 4H, 2 × CH2), 1.61-1.76 (m, 2H, CH2-
CH2N), 3.32-3.60 (m, 2H, CH2N), 3.74-3.87 (m, 2H, H-5′),
4.12-4.18 (m, 1H, H-4′), 5.51 (dd, 1H, J ) 1.8, J ) 5.5, H-3′),
4.60 (s, 2H, CH2S), 4.69, (dd, 1H, J ) 5.5, J ) 7.3, H-2′), 6.14
(d, 1H, J ) 7.3, H-1′), 7.12-7.31 (m, 3H, CH arom), 7.40 (dd,
2H, J ) 1.5, J ) 8.0, CH arom), 8.36 (s, 1H, H-2). Anal.
(C22H29N5O4S‚0.1CH2Cl2) C, H, N, S.
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-(n-hexylamino)thioino-
sine (38). This compound was prepared according to general
1
procedure C: yield 86%; white solid; H NMR (CDCl3) δ 0.88
(t, 3H, J ) 6.6, CH3CH2), 1.25-1.45 (m, 4H, CH2CH3), 1.53-
1.77 (m, 4H, CH2CH2N), 2.03, 2.07, 2.15 (3 × s, 3 × 3H, 3 ×
CH3CO), 3.37-3.69 (m, 2H, CH2N), 4.27-4.42 (m, 2H, H-4′, 1
× H-5′), 4.56 (ABX, 2H, J ) 3.7, J ) 11.7, 1 × H-5′), 4.62 (s,
2H, CH2S), 5.25 (t, 1H, J ) 5.4, NH), 5.53 (dd, 1H, J ) 3.7, J
) 5.9, H-3′), 5.85 (t, 1H, J ) 5.9, H-2′), 6.15 (d, 1H, J ) 5.9,
H-1′), 7.17-7.37 (m, 3H, CH arom), 7.45 (dd, 2H, J ) 1.5, J )
8.0 CH arom), 8.50 (s, 1H, H-2).
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-[(2-methylbutyl)amino]-
thioinosine (39). This compound was prepared according to
1
general procedure C: yield 64%; yellow oil; H NMR (CDCl3)
δ 0.87-0.99 (m, 6H, 2 × CH3), 1.07-1.31 (m, 1H, CH3CHH),
1.36-1.58 (m, 1H, CH3CHH), 1.64-1.86 (m, 1H, CH3CH), 2.04,
2.05, 2.15 (3 × s, 3 × 3H, 3 × CH3CO), 3.18-3.33 (m, 0.5H,
CHHNH), 3.36-3.46 (m, 1H, CHHNH), 3.49-3.65 (m, 0.5H,
CHHNH), 4.25-4.43 (m, 2H, H-4′, 1 × H-5′), 4.48-4.59 (m,
1H, 1 × H-5′), 4.62 (s, 2H, CH2S), 5.28 (m, 1H, NH), 5.49-
5.58 (m, 1H, H-3′), 5.86-5.95 (m, 1H, H-2′), 6.12-6.18 (m, 1H,
H-1′), 7.16-7.34 (m, 3H, CH arom), 7.41-7.49 (m, 2H, CH
arom), 8.49 (s, 1H, H-2).
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-(cyclopropylamino)thio-
inosine (40). This compound was prepared according to
general procedure C: yield 64%; white solid; 1H NMR (CDCl3)
δ 0.59-0.69 (m, 2H, CH2), 0.82-0.93 (m, 2H, CH2), 2.02, 2.12,
2.14 (3 × s, 3 × 3H, 3 × CH3CO), 2.88-3.01 (m, 2H, CH2N),
4.27-4.39 (m, 2H, H-4′, 1 × H-5′), 4.46-4.58 (m, 1H, 1 × H-5′),
4.64 (s, 2H, CH2S), 5.47-5.56 (m, 1H, H-3′), 5.57 (d, 1H, J )
1.5, NH), 5.78 (t, 1H, J ) 6.2, H-2′), 6.14 (d, 1H, J ) 6.2, H-1′),
7.17-7.35 (m, 3H, CH arom), 7.41-7.50 (m, 2H, CH arom),
8.51 (s, 1H, H-2).
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-(cyclopentylamino)thio-
inosine (41). This compound was prepared according to
general procedure C: yield 63%; colorless oil; 1H NMR (CDCl3)
δ 1.41-1.80 (m, 6H, NHCHCHH, 2 × CH2), 2.03, 2.05, 2.15 (3
× s, 3 × 3H, 3 × CH3CO), 2.08-2.12 (m, 2H, NHCHCHH),
4.27-4.56 (m, 4H, H-4′, H-5′, CHNH), 4.62 (s, 2H, CH2S), 5.08
(d, J ) 6.8, 1H, NH), 5.57 (dd, 1H, J ) 4.8, J ) 5.7, H-3′), 5.97
(t, 1H, J ) 5.7, H-2′), 6.05 (d, 1H, J ) 5.7, H-1′), 7.21-7.34
(m, 3H, CH arom), 7.45 (dd, 2H, J ) 1.8, J ) 8.1, CH arom),
8.49 (s, 1H, H-2).
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-(cyclohexylamino)thio-
inosine (42). This compound was prepared according to
general procedure C: yield 59%; colorless oil; 1H NMR (CDCl3)
δ 1.12-1.84 (m, 8H, 2 × NHCHCHH, 3 × CH2), 2.04, 2.05,
2.15 (3 × s, 3 × 3H, 3 × CH3CO), 2.01-2.12 (m, 2H, 2 ×
NHCHCHH), 3.81-4.03 (m, 1H, CH2CHNH), 4.29-4.57 (m,
3H, H-4′, H-5′), 4.62 (s, 2H, CH2S), 5.01 (d, J ) 7.7, 1H, NH),
5.57 (dd, 1H, J ) 4.8, J ) 5.5, H-3′), 5.97 (t, 1H, J ) 5.5, H-2′),
6.07 (d, 1H, J ) 5.5, H-1′), 7.18-7.33 (m, 3H, CH arom), 7.45
(dd, 2H, J ) 1.8, J ) 8.0, CH arom), 8.48 (s, 1H, H-2).
2′,3′,5′-Tri-O-acetyl-6-benzyl-8-pyrrolidinothioino-
sine (43). This compound was prepared according to general
procedure C: yield 42%; colorless oil; 1H NMR (CDCl3) δ 1.90-
2.09 (m, 4H, 2 × CH2N), 2.05, 2.06, 2.13 (3 × s, 3 × 3H, 3 ×
CH3CO), 3.62-3.78 (m, 4H, 2 × CH2N), 4.26-4.37 (m, 1H,
H-4′, 1 × H-5′), 4.45-4.57 (m, 1H, 1 × H-5′), 4.61 (s, 2H, CH2S),
6-Benzyl-8-(n-hexylamino)thioinosine (51). This com-
pound was prepared according to general procedure D: yield
61%; colorless glass; 1H NMR (MeOD) δ 0.88 (m, 3H, CH3CH2),

328 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Tromp et al.
1.21-1.48 (m, 6H, 3 × CH2), 1.57-1.74 (m, 2H, CH2CH2N),
3.33-3.56 (m, 2H, CH2N), 3.74-3.87 (m, 2H, H-5′), 4.14 (d,
1H, J ) 1.8, J ) 3.7, H-4′), 4.27 (dd, 1H, J ) 1.8, J ) 5.5,
H-3′), 4.60 (s, 2H, CH2S), 4.68 (dd, 1H, J ) 5.5, J ) 7.7, H-2′),
6.14 (d, 1H, J ) 7.7, H-1′), 7.13-7.30 (m, 3H, CH arom), 7.36-
7.44 (m, 2H, CH arom), 8.36 (s, 1H, H-2). Anal. (C23H31N5O4S‚-
0.25CH2Cl2) C, H, N, S.
2H, J ) 1.5, J ) 8.0, CH arom), 8.52 (s, 1H, H-2). Anal.
(C21H25N5O4S‚0.2CH2Cl2) C, H, N, S.
9-Benzyl-6-(4-nitrobenzylsulfanyl)purine (59). This com-
pound was prepared according to a literature procedure.10
9-Benzyl-6-chloropurine (61). This compound was pre-
pared according to a literature procedure.15
9-Benzyl-hypoxanthine (62). This compound was pre-
6-Benzyl-8-[(2-methylbutyl)amino]thioinosine (52). This
pared according to a literature procedure.16
compound was prepared according to general procedure D:
9-Benzyl-8-bromohypoxanthine (63). This compound
was prepared according to a literature procedure.11 After
workup the product was washed with CH2Cl2 and dried in
vacuo to provide 62 as an off-white solid: yield 83%; white
solid; 1H NMR (DMSO-d6) δ 5.37 (s, 2H, CH2N), 7.20-7.42 (m,
5H, CH arom), 8.11 (s, 1H, H-2), 12.53 (br s, 1H, NH).
1
yield 87%; white solid; H NMR (MeOD) δ 0.86-0.97 (m, 6H,
2 × CH3), 1.03-1.29 (m, 1H, CH3CHH), 1.39-1.63 (m, 1H,
CH3CHH), 1.69-1.89 (m, 1H, CH3CH), 3.13-3.50 (m, 2H,
CH2N), 3.73-3.82 (m, 2H, H-5′), 4.11-4.17 (m, 1H, H-4′), 4.25
(dd, 1H, J ) 1.5, J ) 5.5, H-3′), 4.61 (s, 2H, CH2S), 4.67 (dd,
1H, J ) 5.5, J ) 7.7, H-2′), 6.15 (d, 1H, J ) 7.7, H-1′), 7.13-
7.30 (m, 3H, CH arom), 7.40 (dd, 2H, J ) 1.8, J ) 7.7, CH
arom), 8.36 (s, 1H, H-2). Anal. (C22H29N5O4S‚0.05CH2Cl2) C,
H, N, S.
9-Benzyl-8-(cyclopentylamine)hypoxanthine (64). This
compound was prepared according to general procedure E:
1
yield 67%; white solid; H NMR (DMSO-d6) δ 1.43-1.96 (m,
8H, 4 × CH2), 4.06-4.19 (m, 1H, CHN), 5.22 (s, 2H, PhCH2N),
6.46 (d, 1H, J ) 6.6, CHNH), 7.12-7.22 (m, 2H, CH arom),
7.24-7.38 (m, 3H, CH arom), 8.71 (s, 1H, H-2), 12.01 (br s,
1H, NH).
6-Benzyl-8-(cyclopropylamino)thioinosine (53). This
compound was prepared according to general procedure D:
1
yield 58%; white solid; H NMR (MeOD) δ 0.56-0.66 (m, 2H,
2 × CHH), 0.76-0.85 (m, 2H, 2 × CHH), 2.78-2.90 (m, 2H,
CH2N), 3.72-3.88 (m, 2H, H-5′), 4.11 (dd, 1H, J ) 1.8, J )
3.7, H-4′), 4.23 (dd, 1H, J ) 1.8, J ) 5.5, H-3′), 4.61 (dd, 1H,
J ) 5.5, J ) 7.7, H-2′), 4.62 (s, 2H, CH2S), 6.14 (d, 1H, J )
7.7, H-1′), 7.15-7.32 (m, 3H, CH arom), 7.41-7.47 (m, 2H, CH
arom), 8.41 (s, 1H, H-2). Anal. (C20H23N5O4S‚0.2CH2Cl2) C, H,
N, S.
9-Benzyl-8-(cyclohexylamine)hypoxanthine (65). This
compound was prepared according to general procedure E:
1
yield 67%; off-white solid; H NMR (CDCl3) δ 0.96-2.03 (m,
10H, 5 × CH2), 3.79-4.02 (m, 2H, CHNH, NHCH), 5.18 (s,
2H, CH2N), 7.18-7.43 (m, 5H, CH arom), 8.02 (s, 1H, H-2),
11.54 (br s, 1H, NH).
9-Benzyl-8-pyrrolidinohypoxanthine (66). This com-
pound was prepared according to general procedure A with
omittance of the acetylation step (yield 74%) or general
procedure E (yield: 56%): white solid; 1H NMR (CDCl3) δ
1.84-1.95 (m, 4H, 2 × CH2CH2N), 3.50-3.62 (m, 4H, 2 ×
CH2N), 5.41 (s, 2H, CH2N), 7.07-7.15 (m, 2H, CH arom), 7.25-
7.39 (m, 3H, CH arom), 7.92 (s, 1H, H-2), 11.79 (br s, 1H, NH).
9-Benzyl-8-morpholinohypoxanthine (67). This com-
pound was prepared according to general procedure A with
omittance of the acetylation step (yield 55%) or general
procedure E (yield 88%): white solid; 1H NMR (CDCl3) δ 3.20
(t, 4H, J ) 4.7, 2 × CH2N), 3.76 (t, 4H, J ) 4.7, 2 × CH2O),
5.29 (s, 2H, CH2N), 7.15-7.41 (m, 5H, CH arom), 8.01 (s, 1H,
H-2), 11.72 (br s, 1H, NH).
9-Benzyl-8-(cyclopentylamine)-6-mercaptopurine (68).
This compound was prepared according to general procedure
B: yield 8%; white solid; 1H NMR (CDCl3/MeOD 3/1, v/v) δ
1.29-1.69 (m, 6H, 2 × CHHCHNH, 2 × CH2), 4.32-4.52 (m,
1H, CHNH), 5.24 (s, 2H, CH2N), 7.13-7.42 (m, 5H, CH arom),
7.98 (br s, 1H, H-2).
9-Benzyl-8-(cyclohexylamine)-6-mercaptopurine (69).
This compound was prepared according to general procedure
B: yield 44%; pink solid; 1H NMR (CDCl3/MeOD 1/1, v/v) δ
1.03-2.04 (m, 10H, 5 × CH2), 3.83-4.05 (m, 1H, NHCH), 5.27
(s, 2H, CH2N), 7.12-7.39 (m, 5H, CH arom), 8.02 (s, 1H, H-2),
11.54 (br s, 1H, NH).
6-Benzyl-8-(cyclopentylamino)thioinosine (54). This
compound was prepared according to general procedure D:
1
yield 80%; white solid; H NMR (MeOD) δ 1.50-1.84 (m, 6H,
2 × CH2, 2 × CHHCH), 1.94-2.13 (m, 2H, 2 × CHHCH), 3.76-
3.85 (m, 2H, H-5′), 4.12 (dd, 1H, J ) 1.8, J ) 3.7, H-4′), 4.25
(dd, 1H, J ) 1.8, J ) 5.5, H-3′), 4.29-4.44 (m, 1H, CHNH),
4.62 (s, 2H, CH2S), 4.65 (dd, 1H, J ) 5.5, J ) 7.7, H-2′), 6.15
(d, 1H, J ) 7.7, H-1′), 7.15-7.32 (m, 3H, CH arom), 7.42 (dd,
2H, J ) 1.8, J ) 8.0, CH arom), 8.37 (s, 1H, H-2). Anal.
(C22H27N5O4S‚0.05CH2Cl2) C, H, N, S.
6-Benzyl-8-(cyclohexylamino)thioinosine (55). This com-
pound was prepared according to general procedure D: yield
1
93%; white solid; H NMR (MeOD) δ 1.08-1.87 (m, 8H, 2 ×
NHCHCHH, 3 × CH2), 1.93-2.09 (m, 2H, 2 × NHCHCHH),
3.73-3.97 (m, 3H, H-5′, CH2CHNH), 4.11-4.17 (m, 1H, H-4′),
4.26 (dd, 1H, J ) 1.8, J ) 5.8, H-3′), 4.61 (s, 2H, CH2S), 4.67
(dd, 1H, J ) 5.8, J ) 7.7, H-2′), 6.14 (d, 1H, J ) 7.7, H-1′),
7.14-7.32 (m, 3H, CH arom), 7.38-7.44 (m, 2H, CH arom),
8.36 (s, 1H, H-2). Anal. (C22H27N5O4S‚0.25CH2Cl2) C, H, N, S.
6-Benzyl-8-pyrrolidinothioinosine (56). This compound
was prepared according to general procedure D: yield 90%;
1
white solid; H NMR (CDCl3/MeOD 1/9, v/v) δ 1.95-2.09 (m,
4H, 2 × CH2CH2N), 3.66-3.80 (m, 5H, 2 × CH2N, 1 × H-5′),
3.88 (ABX, 1H, J ) 2.6, J ) 12.4, 1 × H-5′), 4.14 (dd, 1H, J )
2.6, J ) 4.4, H-4′), 4.38 (dd, 1H, J ) 1.8, J ) 5.5, H-3′), 4.61
(s, 2H, CH2S), 5.19 (dd, 1H, J ) 5.1, J ) 7.3, H-2′), 6.05 (d,
1H, J ) 7.3, H-1′), 7.21-7.32 (m, 3H, CH arom), 7.42 (dd, 2H,
J ) 1.5, J ) 8.0, CH arom), 8.40 (s, 1H, H-2). Anal.
(C21H25N5O4S) C, H, N, S.
9-Benzyl-8-pyrrolidino-6-mercaptopurine (70). This com-
pound was prepared according to general procedure B: yield
53%; white solid; 1H NMR (CDCl3/ MeOD 5/1, v/v) δ 1.81-
2.02 (m, 4H, 2 × CH2CH2N), 3.55-3.72 (m, 4H, 2 × CH2N),
5.46 (s, 2H, CH2N), 7.02-7.14 (m, 2H, CH arom), 7.27-7.40
(m, 3H, CH arom), 8.00 (s, 1H, H-2).
6-Benzyl-8-piperidinothioinosine (57). This compound
was prepared according to general procedure D: yield 86%;
1
colorless glass; H NMR (CDCl3/MeOD 1/9, v/v) δ 1.57-1.84
9-Benzyl-8-morpholino-6-mercaptopurine (71). This
compound was prepared according to general procedure B:
yield 54%; yellowish solid; 1H NMR (CDCl3/ MeOD 3/2, v/v) δ
3.23-3.31 (m, 4H, 2 × CH2N), 3.71-3.79 (m, 4H, 2 × CH2O),
5.32 (s, 2H, CH2N), 7.14-7.22 (m, 2H, CH arom), 7.29-7.39
(m, 3H, CH arom), 8.02 (s, 1H, H-2).
(m, 6H, 3 × CH2), 3.24-3.50 (m, 4H, 2 × CH2N), 3.80 (ABX,
1H, J ) 2.9, J ) 12.4, H-5′), 4.08-4.15 (m, 1H, H-4′), 4.40
(dd, 1H, J ) 2.2, J ) 5.5, H-3′), 4.61 (s, 2H, CH2S), 5.19 (dd,
1H, J ) 5.5, J ) 7.3, H-2′), 5.81 (d, 1H, J ) 7.3, H-1′), 7.14-
7.32 (m, 3H, CH arom), 7.37-7.46 (m, 2H, CH arom), 8.48 (s,
1H, H-2). Anal. (C22H27N5O4S‚0.2CH2Cl2) C, H, N, S.
6-Benzyl-8-morpholinothioinosine (58). This compound
was prepared according to general procedure D: yield 94%;
colorless glass; 1H NMR (MeOD) δ 3.27-3.40 (m, 2H, CH2N),
3.45-3.58 (m, 2H, CH2N), 3.73 (ABX, 1H, J ) 2.9, J ) 12.4, 1
× H-5′), 3.81-3.94 (m, 5H, 2 × CH2O, 1 × H-5′), 4.13 (dd, 1H,
J ) 2.9, J ) 4.5, H-4′), 4.38 (dd, 1H, J ) 2.2, J ) 5.1, H-3′),
4.62 (s, 2H, CH2S), 5.17 (dd, 1H, J ) 5.1, J ) 7.3, H-2′), 5.87
(d, 1H, J ) 7.3, H-1′), 7.14-7.33 (m, 3H, CH arom), 7.42 (dd,
9-Benzyl-8-(cyclopentylamine)-6-(4-nitrobenzyl)-6-mer-
captopurine (72). This compound was prepared according to
general procedure C: yield 21%; yellow solid; 1H NMR (CDCl3)
δ 1.18-1.64 (m, 6H, 2 × CH2, 2 × CHHCHNH), 1.89-2.08
(m, 2H, 2 × CHHCHNH), 4.12 (d, 1H, J ) 7.3, CHNH), 4.25-
4.42 (m, 1H, CHNH), 4.70 (s, 2H, CH2S), 5.19 (s, 2H, CH2N),
7.17 (dd, 2H, J ) 1.8, J ) 7.3, CH arom), 7.29-7.40 (m, 3H,
CH arom), 7.64 (d, 2H, J ) 8.8, CH arom), 8.12 (d, 2H, J )

Inhibition of Nucleoside Transport Proteins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 329
(2) Gray, J. H.; Owen, R. P.; Giacomini, K. M. The Concentrative
Nucleoside Transport Famliy, SLC28. Pflugers Arch.sEur. J.
Physiol. 2004, 447, 728-734.
8.8, CH arom), 8.51 (s, 1H, H-2); HPLC (system A) tR ) 8.00
min, (system B) tR ) 11.23 min.
9-Benzyl-8-(cyclohexylamine)-6-(4-nitrobenzyl)-6-mer-
captopurine (73). This compound was prepared according to
general procedure C: yield 19%; yellow solid; 1H NMR (CDCl3)
δ 0.96-1.64 (m, 8H, 3 × CH2, 2 × CHHCHNH), 1.83-1.97
(m, 2H, 2 × CHHCHNH), 3.82-4.01 (m, 1H, NHCH), 4.08 (d,
1H, J ) 8.0, CHNH), 4.70 (s, 2H, CH2S), 5.20 (s, 2H, CH2N),
7.12-7.24 (m, 2H, CH arom), 7.28-7.42 (m, 3H, CH arom),
7.64 (d, 2H, J ) 8.8, CH arom), 8.13 (d, 2H, J ) 8.8, CH arom),
8.51 (s, 1H, H-2). Anal. (C25H26N6O2S‚0.3CH2Cl2) C, H, N, S.
9-Benzyl-6-(4-nitrobenzyl)-8-pyrrolidino-6-mercaptopu-
rine (74). This compound was prepared according to general
procedure C: yield 83%; yellow solid; 1H NMR (CDCl3) δ 1.85-
1.93 (m, 4H, 2 × CH2CH2N), 3.56-3.63 (m, 4H, 2 × CH2CH2N),
4.71 (s, 2H, CH2S), 5.46 (s, 2H, CH2N), 7.02-7.09 (m, 2H, CH
arom), 7.24-7.37 (m, 3H, CH arom), 7.65 (d, 2H, J ) 8.8, CH
arom), 8.13 (d, 2H, J ) 8.8, CH arom), 8.50 (s, 1H, H-2). Anal.
(C23H22N6O2S‚0.2CH2Cl2) C, H, N, S.
(3) Baldwin, S. A.; Beal, P. R.; Yao, S. Y. M.; King, A. E.; Cass, C.
E.; Young, D. A. The Equilibrative Nucleoside Transport Family,
SLC29. Pflugers Arch.sEur. J. Physiol. 2004, 447, 735-743.
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Nucleoside Transporters: Molecular Biology and Implications for
Therapeutic Developments. Mol. Med. Today 1999, 5, 216-224.
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Nucleoside Transport and Its Significance for the Anticancer
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Activity Relationships and Potential Therapeutic Applications.
Curr. Med. Chem. 1997, 4, 35-66.
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Periphery: Release and Regulation of Pain. Prog. Neurobiol.
2003, 69, 313-340.
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Potential Analgesic Target in Inflammatory and Neuropathic
Pains. CNS Drugs 2000, 13, 77-85.
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of Photoaffinity Labels. J. Org. Chem. 1992, 57, 5968-5976.
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Sundermann, B.; von Frijtag Drabbe Ku¨nzel, J. K.; IJzerman,
A. P. Inhibition of Nucleoside Transport by New Analogues of
Nitrobenzylthioinosine: Replacement of the Ribose Moiety by
Substituted Benzyl Groups. J. Med. Chem. 2004, 47, 5441-5450.
(11) Roelen, H.; Veldman, N.; Spek, A. L.; von Frijtag Drabbe Ku¨nzel,
J.; Mathoˆt, R. A. A.; IJzerman, A. P. N6,C8-Disubstituted
Adenosine Derivatives as Partial Agonists for Adenosine A1
Receptors. J. Med. Chem. 1996, 39, 1463-1471.
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of Some Purine 8,2′-(NR-Methylhydrazino) and 8,2′-(NR-Methy-
loxamido) Cyclonucleosides. J. Org. Chem. 1981, 46, 5176-5181.
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mide by a Photoreaction. Chem. Pharm. Bull. 1986, 34, 3653-
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1998, 71, 1973-1991.
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and Hypoxanthines and their Biological Properties. Nucleosides
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a Million Crystal Structures and Rising. Acta Crystallogr. 2002,
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1999.
9-Benzyl-8-morpholino-6-(4-nitrobenzyl)-6-mercaptopu-
rine (75). This compound was prepared according to general
1
procedure C: yield 78%; white solid; H NMR (Bruker DMX
600 MHz, DMSO-d6, 60 °C) δ 3.28 (t, 4H, J ) 4.8, 2 × CH2N),
3.63 (t, 4H, J ) 4.8, 2 × CH2O), 4.77 (s, 2H, CH2S), 5.38 (s,
2H, CH2N), 7.17-7.19 (m, 2H, CH arom), 7.26-7.34 (m, 3H,
CH arom), 7.74 (d, 2H, J ) 8.8, CH arom), 8.15 (d, 2H, J )
8.8, CH arom), 8.57 (s, 1H, H-2). Anal. (C23H22N6O3S‚0.1CH2-
Cl2) C, H, N, S.
Erythrocytes and Membrane Preparation. Whole hu-
man blood (Blood Bank, Leiden University Medical Center)
was stirred in lysis buffer (1/2 v/v, 10 mM MgCl2 in 10 mM
Tris-HCl, pH 8.0 at 25 °C) for 1 h. After homogenization it
was centrifuged for 50 min at 19 000 rpm. The supernatant
was removed and the pellet was dissolved in ice-cold water
and centrifuged again for 50 min. This procedure was repeated
two more times. After removal of the last supernatant, 25 mL
of buffer (50 mM Tris-HCl, pH 7.4 at 25 °C) was added to the
final pink pellet. This suspension was homogenized and the
ghosts were collected. Aliquots were stored at -80 °C until
further use.
[3H]NBTI Binding Assay. Saturation and displacement
equilibrium NBTI binding to membranes prepared from hu-
man erythrocytes (ghosts) was determined at 25 °C based on
a method previously described.26
Adenosine A1 Receptor. The affinity for the A1 receptor
was determined on CHO cells expressing the human receptors,
using [3H]DPCPX as the radioligand. Membranes containing
10 µg of protein were incubated in a total volume of 400 µL of
50 mM Tris/HCl (pH 7.4) and [3H]DPCPX (final concentration
1.6 nM) for 1 h at 25 °C in a shaking water bath. Nonspecific
binding was determined in the presence of 10 µM CPA. The
incubation was terminated by filtration over Whatman GF/B
filters under reduced pressure with a Brandell harvester.
Filters were washed three times with ice-cold buffer and placed
in scintillation vials. Emulsifier Safe (3.5 mL) was added, and
after 2 h radioactivity was counted in an LKB rack â scintil-
lation counter.
Data Analysis. Ki values were calculated using a nonlinear
regression curve-fitting program (GraphPad Prism, GraphPad
Software Inc., San Diego, CA). The KD value of the radioligand,
[3H]DPCPX, was 1.6 nM.
Acknowledgment. This work was financially sup-
ported by Gru¨nenthal GmbH (Aachen, Germany).
Supporting Information Available: 13C NMR spectra for
7, 9-14, 16-58, 63-67, and 69-75; melting points for 46-
58 and 72-75; (HR)MS data of 20, 21, 46-58, and 72-75;
and details on elemental analyses for 46-58 and 73-75. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(26) IJzerman, A. P.; Voorschuur, A. H. The Relationship Between
Ionization and the Affinity of Nucleoside Transport Inhibitors.
Naunyn-Schmiedeberg’s Arch. Pharmacol. 1990, 342, 336-341,
and references therein.
References
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Baldwin, S. A. Molecular Mechanisms of Nucleoside and Nucleo-
side Drug Transport. Curr. Top. Membr. 2001, 50, 329-378.
JM049303K