Inhibition of nucleoside transport by new analogues of 4- nitrobenzylthioinosine: Replacement of the ribose moiety by substituted benzyl groups

Article abstract of DOI:10.1021/jm049735v

4-Nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein ENT1. However, its highly polar nature is unfavorable for oral absorption and/or penetration into the CNS. In the search for compounds with lower polarity th

Full text of DOI:10.1021/jm049735v

J . Med. Chem. 2004, 47, 5441-5450
5441
In h ibition of Nu cleosid e Tr a n sp or t by New An a logu es of
4-Nitr oben zylth ioin osin e: Rep la cem en t of th e Ribose Moiety by Su bstitu ted
Ben zyl Gr ou p s
Reynier A. Tromp,† Susan van Ameijde,† Claudia Pu¨tz,‡ Corinna Sundermann,‡ Bernd Sundermann,‡
J acobien K. von Frijtag Drabbe Ku¨nzel,† and Adriaan P. IJ zerman*,†
Leiden/ Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Leiden University, P.O. Box 9502,
2300RA Leiden, The Netherlands, and Gru¨nenthal GmbH, Center of Research, 52099 Aachen, Germany
Received April 7, 2004
4-Nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein
ENT1. However, its highly polar nature is unfavorable for oral absorption and/or penetration
into the CNS. In the search for compounds with lower polarity than NBTI we replaced its
ribose moiety by substituted benzyl groups. Halogen, hydroxyl, (trifluoro)methyl(-oxy), nitro,
and amine functionalities were among the substituents at the benzyl group. In general,
substitution of the benzyl group resulted in a lower affinity for ENT1. Only 2-hydroxyl
substitution showed a higher affinity. Most likely this is the result of hydrogen bonding.
Substitution at the 2-position of the benzyl group with aryl groups was also addressed.
Compared to parent compound carrying a 2-phenylbenzyl group, all synthesized analogues
gave higher affinities. Introduction of fluoro, trifluoromethyl, methoxy, and hydroxyl groups
at the phenyl group clearly showed that addition to the 4-position was preferable. Despite the
highly different character of a ribose and a benzyl group, Ki values in the low nanomolar range
were obtained for the benzyl-substituted derivatives. Compound 35, LUF5919, and compound
60, LUF5929, displayed the highest affinity (Ki ) 39 nM for both compounds), having a polar
surface area of 101 Å2 and 85 Å2, respectively.
In tr od u ction
Nucleoside transport proteins are specialized proteins
that enable hydrophilic nucleosides such as adenosine
to cross the cell membrane. These proteins are divided
into two families: (1) the concentrative nucleoside
transporters (CNT), which are Na+-dependent and drive
the nucleoside flow against their concentration gradient,
and (2) equilibrative nucleoside transporters (ENT),
which drive the nucleoside flow following their concen-
tration gradient.1-5 Inhibition of the nucleoside trans-
port proteins has been recognized as an important
strategy for the treatment of several afflictions, includ-
ing ischemic heart diseases, stroke, and host tissue
protection in chemotherapy.5-7
F igu r e 1. 4-Nitrobenzylthioinoisine (NBTI, PSA ) 154 Å2).
however, its high-polarity limits its possibilities for
intestinal absorption or its use as a CNS drug.
A parameter to predict intestinal absorption and/or
brain permeation of compounds is the so-called polar
surface area (PSA). The PSA is defined as the surface
area of a molecule occupied by nitrogen and oxygen, and
the hydrogen atoms that are attached to these atoms.
In literature the upper limit for good intestinal absorp-
tion ranges from 120 Å2 to 140 Å2.11-13 For passage over
the blood-brain barrier, the PSA value should be lower
than 60-70 Å2 or 90 Å2.12,14 Variations in the given
limits result from the differences in the methods used
for the calculation of the PSA values.
Our present work aims at the synthesis of compounds
that are significantly less polar than NBTI to improve
their oral bioavailability and to increase their potential
to cross the blood-brain barrier. Both the presence of
the ribose and the nitro group contribute highly to this
polarity. Much effort has been put in the search for a
different substituent at the 6-thiol position but has so
far not resulted in compounds that are less polar and
still retain the high activity.15,16 On the other hand
The ENTs are subdivided into equilibrative sensitive
(ENT1) and equilibrative insensitive (ENT2) proteins,
based on their sensitivity toward inhibition by 4-nitro-
benzylthioinosine (NBTI (1), Figure 1). Recently, two
novel isoforms have been discovered, ENT3 and
ENT4.2,3,8 ENT1 is distributed in the dorsal horn of the
spinal cord in close proximity to the adenosine A1
receptor. Thus, inhibition of ENT1 will result in an
increased extracellular concentration of adenosine, which
in turn will lead to a more profound occupancy of the
adenosine A1 receptor through which adenosine exerts
many of its physiological effects, e.g. counteracting
pain.9,10 NBTI (1) is a very active inhibitor of ENT1;
* To whom correspondence should be addressed. Tel: +31-71-
5274651. Fax: +31-71-5274565. E-mail: ijzerman@lacdr.leidenuniv.nl.
† Leiden University.
‡ Gru¨nenthal GmbH.
10.1021/jm049735v CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/22/2004

5442 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Tromp et al.
Sch em e 1a
a
Reagents: (a) 4-nitrobenzyl bromide, K2CO3, DMF, rt, 4 h; (b) appropiate arylic halide, NaH (1 equiv), DMF, rt, 16 h; (c) appropiate
aryl bromide, toluene, EtOH, 2 M Na2CO3 (aq), Pd(PPh3)4 (cat.), 80 °C, 1-2 d; (d) appropiate aryl bromide, H2O, TBAB, Na2CO3, Pd(PPh3)4
(cat.), µW, 150 °C, 5-10 min; (e) NBS, benzoyl peroxide, CCl4, 80 °C, 40h; (f) compound 25, BBr3, CH2Cl2, -78° to rt, 5 h; (g) compound
29, TFA, H2O, rt, 6 h; (h) compound 61, 62, or 63, BBr3, CH2Cl2, -78° to rt, 5 h.
previous reports have shown that NBTI analogues
without the ribose function are still able to inhibit the
nucleoside transport protein.7 Substitution of the ribose
group also reduces the number of both hydrogen bond
donors and acceptors, which is also important in terms
of cell permeability and uptake into the CNS. Therefore,
we chose to replace the ribose function. Here, we report
on the synthesis and the affinities of NBTI analogues
in which the ribose was substituted for a benzyl group.
Also the influence of the distance between the aryl ring
and the purine ring was investigated as well as the
presence of a pyridyl function.
Ch em ist r y. The novel NBTI analogues containing
aryl functions instead of a ribose moiety were obtained
as depicted in Scheme 1. First, commercially available
mercaptopurine 2 was substituted at the 6-position with
the 4-nitrobenzyl15 group to give 3. Substitution at N9
with the aryl functions was performed by dissolving
compound 3 in DMF, followed by addition of 1 equiv of
sodium hydride and the appropriate arylic bromide.16
To investigate the influence of the chain length between
the purine and the phenyl group, compounds 4-6 were
prepared. The effect of a pyridyl ring was explored by
the synthesis of 7-9.
As benzyl substitution provided the most active
compound (4), we subsequently investigated the influ-
ence of substitution of the benzyl function itself. Com-
pounds 10-34 (Scheme 1) were synthesized directly from
3 using the substituted benzylic halide and sodium
hydride in DMF as described for 4-9. Boc-protected
2-aminobenzyl bromide used for the preparation of
29 was synthesized as described in the literature.17
Hydroxyl-substituted 35 was obtained by demethylation
of 25 using boron tribromide.18 Compound 36 could be
prepared directly from 3 using 3-hydroxybenzyl iodide.
Removal of the Boc group of 29 with TFA afforded
amine 37.19
The result of compound 30 carrying a biphenyl group
instead of a highly polar ribose urged us to further
investigate aryl-substituted benzyl groups at the N9-
position. These compounds were prepared by application
of the Suzuki reaction.20 2-Tolylboronic acid (38) was
reacted with the appropriate arylic bromide, using
conventional or microwave-assisted heating to provide

Inhibition of Nucleoside Transport by NBTI Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5443
Sch em e 2a
a
Reagents: (a) 2-tolylboronic acid (38), H2O, TBAB, Na2CO3, Pd(PPh3)4 (cat.), µW, 150 °C, 5-10 min; (b) MsCl, Et3N, MTBE, 0 °C-rt,
3 h; (c) compound 3, NaH (1 equiv), DMF, rt, 16 h.
Ta ble 1. aAffinities of Novel NBTI Analogues for the
Nucleoside Transport Protein on Human Erythrocyte
Membranes
Ta ble 2. aAffinities of Novel NBTI Analogues Carrying
Substituted Benzyl Groups at the N9-Position
compound
R
PSA (Å2)b
Ki (nM)
4
5
6
7
8
9
benzyl
ethylphenyl
propylphenyl
pyridin-2′-ylmethyl
pyridin-3′-ylmethyl
pyridin-4′-ylmethyl
86
86
86
96
99
99
135 (( 30)
2200 (( 600)
488 (( 68)
42%c
2709 (( 1165)
2165 (( 585)
compd
R2
R3
R4
R5 R6 PSA (Å2)b
Ki (nM)
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
H
F
H
H
F
F
F
F
H
H
Cl
H
H
Cl
H
H
H
H
H
H
H
F
H
H
H
H
H
H
F
H
H
F
H
H
H
H
H
H
H
F
86
86
86
86
86
86
86
86
86
135 (( 30)
38%c
361 (( 108)
3340 (( 860)
32%c
4877 (( 1358)
1637 (( 1002)
32%c
653 (( 61)
1544 (( 89)
44%c
F
H
F
H
F
H
H
F
H
H
H
Binding study: [3H]NBTI (KD ) 0.59 nM) as radioligand and
a
H
H
H
human erythrocyte membranes (n ) 3). Ki values are shown with
b
SEM in parentheses. See the Experimental Section for details
on the calculation of the polar surface area (PSA). c Displacement
F
F
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
at a concentration of 10 µM.
86
H
Cl
H
H
Cl
H
H
OMe
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
86
86
86
85
86
86
94
97
biaryls 39-47 (Scheme 1). Next, the benzylic position
was converted into the corresponding bromide (48-56)
using NBS.21 Coupling of the biarylic bromides to purine
3 provided compounds 57-65. To avoid problems in the
bromination reaction due to the presence of two methyl
groups in 2,2′-dimethylbiphenyl, a different approach
was used for the synthesis of 69. 2-Tolylboronic acid (38)
was attached to 2-bromobenzyl alcohol (66) by a Suzuki
reaction, yielding 67 (Scheme 2). Mesylation of the
benzylic alcohol function and subsequent coupling to 3
provided 69. Hydroxyl-substituted 70-72 were obtained
by demethylation of 61-63 following the same proce-
dure as described for the synthesis of 35 (Scheme 1).18
Biologica l Stu d ies. 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 Tables 1-3.
Ca lcu la tion of th e P ola r Su r fa ce Ar ea a n d Mo-
lecu la r Mod elin g. The polar surface areas of the
molecules were calculated using Spartan 5.0 for SGI,22
in combination with an in-house developed application
called PolSurf 1.0. The PSA of NBTI (1) calculated in
this way was 154 Å2.23 The contributions of the ribose
function and the 4-nitrobenzyl group to the total PSA
value of NBTI were 77 Å2 and 45 Å2, respectively. The
PSA values for all compounds together with the biologi-
cal data are represented in Tables 1-3.
212 (( 62)
236 (( 36)
6%c
Cl
H
Cl
I
34%c
130 (( 21)
110 (( 16)
264 (( 43)
563 ( 91
47%c
422 (( 73)
281 (( 33)
2165 (( 221)
711 (( 82)
200 (( 126)
24%c
OMe
H
H
H
H
H
OMe
H
97
OMe
108
122
86
86
86
129
97
101
109
106
NHBoc H
Ph
H
H
H
H
H
CF3
H
H
H
H
OH
H
H
H
H
Me
NO2
OCF3
H
OH
H
NH2
39 (( 11)
153 (( 33)
124 (( 8)
H
H
a
Binding study: [3H]NBTI (KD ) 0.59 nM) as radioligand and
human erythrocyte membranes (n ) 3). Ki values are shown with
b
SEM in parentheses. See the Experimental Section for details
on the calculation of the polar surface area (PSA). c Displacement
at a concentration of 10 µM.
The crystal structure of NBTI23 (1) used for the
illustrations in Figure 2 was retrieved from the Cam-
bridge Structural Database24 and imported into Spar-
tan.25 The structures of 35 and 36 were drawn and
energy minimized in Spartan. The purine rings of NBTI
and 35 or 36 were superimposed after which the benzyl
ring was rotated to achieve an overlay between its
hydroxyl function and either the 2′-, 3′-, or the 5′-OH of
NBTI. The structures for NBTI and 35 with maximum
overlay for the benzyl group and the ribose moiety are
depicted in Figure 2.

5444 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Ta ble 3. aAffinities of Biaryl-Substituted NBTI Analogues
Tromp et al.
showed lower affinities than those (17, 18) that did not.
The same pattern was found for chloro-containing 19-
23. Substitution at the 3-position (20) or 4-position (21)
gave the best Ki values, whereas 2-Cl derivative 19
was less active. Also, it seems that for mono-halogen-
substituted compounds, the presence of a stronger
electron-withdrawing halogen disfavors binding, since
chlorinated 19-21 gave better results than fluorinated
10-12. In line with the above 4-iodo substituted 24
proved better than both 12 (4-F) and 21 (4-Cl) and
showed an affinity similar to that of 4.
In contrast to what was found for the halogenated
derivatives, methoxy substitution at the 2-position (25)
showed a higher affinity (110 nM) than at the 3-position
(26, Ki ) 264 nM), which in turn proved better than
substitution at the 4-position (27, Ki ) 563 nM). Again
disubstitution (28) proved less favorable.
The presence of a Boc-protected amino group (29) or
a phenyl substituent (30) at the 2-position showed lower
affinities than the 2-methoxy-substituted 25, but con-
siderably higher than the halogen-substituted com-
pounds. In view of the size of an NHBoc group or of a
phenyl ring, a large pocket seems to be present at the
binding site. 3-Trifluoromethyl derivative 31 proved the
least active of the 3-monosubstituted compounds, which
might result from the highly electronegative nature of
its substituent. 4-Methyl (32), 4-nitro (33), and 4-tri-
fluoromethoxy (34) substitution provided lower affinities
in comparison with unsubstituted 4. 4-OCF3 gave the
poorest result of all 4-substituted compounds. 4-NO2
substitution was equipotent to 4-Cl (21).
2-Hydroxy-substituted 35, obtained by demethylation
of 25, gave the highest affinity of the tested compounds,
with a Ki value of 39 nM. A possible explanation may
be that the hydroxyl function of 35 interacts in a similar
manner with ENT1 as one of the hydroxyl functions of
the ribose moiety of NBTI (1). Overlaying the structures
of NBTI23 (1) and 35 showed that the hydroxyl function
of 35 can be placed in close proximity to either the 2′-,
the 3′-, and the 5′-hydroxyl function of NBTI. This
superposition also shows that the volumes of both the
ribose and the benzyl group are quite similar (150 Å3
and 127 Å3, respectively, based on Spartan calculations).
Six matching possibilities were found, two for each of
the three hydroxyl functions of the ribose in which the
ribose and the benzyl group either overlap or not. The
cases in which the ribose and the benzyl overlapped are
depicted in Figure 2. Most probable seems to be option
compd
R
PSA (Å2)b
Ki (nM)
30
57
58
59
60
61
62
63
64
65
69
70
71
72
phenyl
2′-fluorophenyl
4′-fluorophenyl
3′-trifluoromethylphenyl
4′-trifluoromethylphenyl
2′-methoxyphenyl
3′-methoxyphenyl
4′-methoxyphenyl
2′-thiophene
86
86
85
85
85
96
96
96
86
86
85
108
109
109
281 (( 33)
111 (( 8)
93 (( 15)
86 (( 11)
39 (( 13)
225 (( 76)
221 (( 77)
104 (( 25)
129 (( 46)
89 (( 14)
126 ((15)
196 (( 78)
196 (( 96)
154 (( 58)
3′-thiophene
2′-methylphenyl
2′-hydroxyphenyl
3′-hydroxyphenyl
4′-hydroxyphenyl
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. See the Experimental Section for details
b
on the calculation of the polar surface area (PSA).
Resu lts a n d Discu ssion
Parent compound 4 showed an affinity of 135 nM
(Table 1). Elongation of the chain length between the
purine and the phenyl ring (5 and 6) resulted in lower
affinities. The presence of a pyridyl ring gave rise to
much higher Ki values compared to benzyl-substituted
4, independent of the position of the nitrogen atom.
Compound 4 was also tested for its affinity for the
adenosine receptor subtypes. It appeared to have little
affinity for the human A1, A2A, and A3 receptors (4%,
16%, and 37% radioligand displacement at 10 µM,
respectively), indicating high selectivity for the nucleo-
side transport protein.
Next the effect of substitution of the N9-benzyl group
was investigated. Therefore compounds 10-37 were
prepared (Table 2). The presence of the strong electron-
withdrawing fluoro group (10-18) decreased the affinity
substantially. Fluoro substitution at the 3-position (11)
gave the best affinity (Ki ) 361 nM), whereas 2-fluoro-
substituted 10 proved the least favorable. A similar
effect was observed for disubstituted compounds, such
that all compounds carrying a fluoro at the 2-position
F igu r e 2. Overlaying structures of NBTI and compound 35. See text for explanation. The nitrobenzyl group was omitted for
reasons of clarity.25

Inhibition of Nucleoside Transport by NBTI Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5445
B since it has been reported that the 3′-OH of NBTI is
of major importance for high affinity.7 Also, it has been
demonstrated that the presence of either 2′- or 5′-deoxy
ribose instead of an intact ribose moiety improved the
affinity of adenosine derivatives, making options A and
C less likely.26,27 When we tried to superimpose NBTI23
(1) and 36 no option was found in which the hydroxyl
group of 36 coincided with one of the hydroxyl functions
of the ribose moiety of NBTI. This might explain the
lower affinity of 3-hydroxy-substituted 36 (Ki ) 153 nM)
when compared to 35.
in polarity is likely to improve oral absorption and tissue
distribution.
Exp er im en ta l 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. 1H NMR and 13C NMR were recorded
with a Bruker AC 200 spectrometer. Tetramethylsilane was
used as internal standard; δ in ppm, J in Hz. Melting points
were determined with a Bu¨chi melting point apparatus and
are uncorrected. High-resolution mass spectroscopy was per-
formed on a PE-Sciex API Qstar instrument. Microwave
reactions were performed in an Emrys Optimizer.
An amino group at the 2-position (37) gave a 3-fold
less potent compound in comparison to 2-hydroxy-
substituted 35, resulting in a similar affinity as parent
compound 4.
The synthesis of compounds 10, 11, 13-19, 22, 24, 28, 30,
34 has been published.28 Boc-protected 2-aminobenzyl bromide
for the formation of 29 was synthesized as described in the
literature.17
The relatively good affinity of 30 was further inves-
tigated by variation of the biphenyl group. Improved Ki
values were found regardless of the nature of the
substituent in comparison to 30 (Table 3). Substitution
of the biaryl at the 2′- and 3′-position gave equipotent
compounds whereas substitution at the 4′-position
resulted in higher affinity. In contrast to the above, the
presence of electron-withdrawing fluoro (57, 58) and
trifluoromethyl (59, 60) improved the affinity in com-
parison to the unsubstituted biaryl compound 30.
Moreover, these compounds also showed better affinities
than 4. For compound 60, a 7-fold improvement was
found compared to unsubstituted 30. Methoxy-substi-
tuted 61-63 also gave lower Ki values but to a lesser
extent than 57-60. Replacement of a phenyl ring by
the smaller thiophene led to a 2- to 3-fold increased
affinity, with a Ki value of 89 nM for 3′-thiophene-
substituted 65. 2′-Methyl substitution (69) showed 2-fold
higher affinity than 30. Hydroxy substitution (70-72)
resulted in affinities comparable to those of the meth-
oxy-substituted compounds.
With respect to the polar surface area, it is to be noted
that all compounds have a much lower PSA value than
NBTI.23 In contrast to the latter, the synthesized ligands
are within the range of the limits given for oral
absorption. Moreover, taking into account the given
maximum values for possible blood-brain barrier pas-
sage, most compounds may well be able to enter the
CNS to some extent. In this respect, 60 is a particularly
interesting compound with a PSA value of only 85 Å2,
almost half of that of NBTI.
Gen er a l P r oced u r e A: Su bstitu tion a t N9. To a well-
stirred suspension of 40 mg of NaH (1 mmol, 60% dispersion
in oil) in 4 mL of DMF was added 1 mmol of 6-(4-nitroben-
zylsulfanyl)purine 3 (0.29 g). After stirring for 5 min, 1 mmol
of the appropiate arylic halide was added and the reaction was
left to stir overnight. After addition of 15 mL of water, the
aqueous layer was extracted three times with 15 mL of EtOAc.
The combined organic layers were dried (MgSO4), the solvent
was evaporated, and the product was purified by column
chromatography (eluent: mixtures of PE 40-60/EtOAc or CH2-
Cl2/MeOH).
Gen er a l P r oced u r e B: Dem eth yla tion of th e Meth oxy
Gr ou p s.18 To a solution of 1 mmol of methoxybenzyl-
substituted purine in 5 mL of dry CH2Cl2 at -78 °C was added
3 mmol of BBr3 (3 mL of a 1 M solution in CH2Cl2). The
solution was allowed to slowly warm to rt. After stirring for
6h, H2O and diethyl ether were added. The layers were
separated, and the aqueous layer was extracted once more with
diethyl ether. The combined layers were washed with brine
and dried (MgSO4), and the solvent was evaporated. The
product was purified by column chromatography (eluent:
mixture of PE 40-60/EtOAc).
Gen er a l P r oced u r e C: Cou p lin g of Ar om a tic Rin gs by
a Su zu k i Rea ction .21 To a solution of the aryl bromide (5
mmol) in 15 mL of toluene/EtOH (1/1, v/v) was added 0.17 g
(0.14 mmol) of Pd(PPh3)4, and the mixture was stirred under
nitrogen atmosphere. Then 2 M aqueous Na2CO3 (7.5 mL) and
0.80 g (6 mmol) of 2-tolylboronic acid 38 were added. The
mixture was refluxed at 80 °C for 1-2 days until reaction was
completed (TLC). After cooling to rt, the product was extracted
with EtOAc (2 × 25 mL). The organic layers were dried with
MgSO4, filtered, and concentrated. Purification was performed
by column chromatography using a mixture of PE 40-60/
EtOAc.
Gen er a l P r oced u r e D: Su zu k i Rea ction s P er for m ed
in a Micr ow a ve. The Suzuki reactions in the microwave were
performed at 150 °C. TBAB (1 equiv), 2-tolylboronic acid 38
(1.2 equiv), Na2CO3 (1.8 equiv), and H2O (2 mL) were added
to the appropriate arylic bromide. Pd(PPh3)4 was used as the
catalyst. The reaction mixtures were extracted with EtOAc,
dried (MgSO4), filtered, and concentrated. Products were
purified by column chromatography using PE 40-60/EtOAc
(99/1, v/v).
Con clu sion s
In this study the search for less hydrophilic nucleoside
transport inhibitors was addressed. Replacement of the
ribose moiety of NBTI (1) by substituted benzyl func-
tions was shown to greatly decrease the polarity. Most
of the compounds presented have PSA values below 100
Å2. Generally, the biarylmethyl-substituted compounds
proved to have higher affinities than the non-aryl-
substituted benzyl compounds. Compound 35 carrying
a 2-hydroxylbenzyl group at the N9-position proved to
be the most active compound for the first series (Ki )
39 nM, PSA ) 101 Å2). 4-Trifluoromethyl-substituted
biphenylmethyl 60 showed the highest affinity for the
N9-biarylmethyl purines (Ki ) 39 nM, PSA ) 85 Å2).
Thus, despite the highly different character of a ribose
and an (aryl)benzyl group, Ki values in the low nano-
molar range were obtained. The substantial reduction
Gen er a l P r oced u r e E: Br om in a tion a t th e Ben zylic
P osition .21 To a stirred solution of the appropriately substi-
tuted toluene in CCl4 (10 mL per mmol) were added 0.95 equiv
of NBS and benzoyl peroxide (5 mg per mmol). The reaction
mixture was refluxed at 80 °C for about 40 h and then cooled
to rt, filtered, and concentrated. The product was used without
further purification.
6-(4-Nitr oben zylsu lfa n yl)p u r in e (3). To 30 mmol of
6-mercaptopurine (2) in 25 mL of DMF were added 30 mmol
of K2CO3 and, after 5 min, 30 mmol of 4-nitrobenzyl bromide.
After stirring for 4 h, 200 mL of H2O was added, the
suspension filtered, the solid washed with EtOAc and CH2Cl2
and dried in vacuo. Yield: 98%. White powder. Mp 239 °C

5446 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Tromp et al.
(decomp), lit.: 244-247 °C,29 262 °C.30 1H NMR (DMSO-d6) δ
) 4.80 (s, 2H, CH2S), 7.76 (d, 2H, J ) 8.8, CH arom), 8.18 (d,
2H, J ) 8.8, CH arom), 8.48 (s, 1H, H-2), 8.74 (s, 1H, H-8).
13C NMR (DMSO-d6) δ ) 30.7, 123.5, 129.2, 130.2, 143.7, 146.5,
146.6, 150.5, 151.4, 156.5.
8.65 (s, 1H, H-2), 8.76 (s, 1H, H-8). 13C NMR (DMSO-d6) δ )
30.8, 45.6, 122.1, 123.6, 130.3, 131.0, 145.2, 145.2, 146.5, 148.7,
150.1, 151.7, 158.2. HRMS (ESI) m/z Found: 379.0950 [M +
H]+, Calcd: 379.0971. Elem. anal. (C18H14N6O2S‚0.15 CH2Cl2)
C, H, N, S.
9-Ben zyl-6-(4-n itr oben zylsu lfa n yl)p u r in e (4). This com-
pound was prepared according to general procedure A applying
benzyl bromide. Yield: 37%. Yellowish solid. Mp 139 °C. 1H
NMR (CDCl3) δ ) 4.73 (s, 2H, CH2S), 5.41 (s, 2H, CH2N), 7.26-
7.39 (m, 5H, CH arom), 7.66 (d, 2H, J ) 8.8, CH arom), 7.95
(s, 1H, H-2), 8.15 (d, 2H, J ) 8.8, CH arom), 8.77 (s, 1H, H-8).
13C NMR (CDCl3) δ ) 31.6, 47.3, 123.5, 127.7, 128.6, 129.0,
129.9, 130.9, 134.9, 142.8, 145.7, 146.9, 148.7, 151.8, 159.0.
HRMS (ESI) m/z Found: 378.0981 [M + H]+, Calcd: 378.1024.
Elem. anal. (C19H15N5O2S‚0.1EtOAc) C, H, N, S.
9-(2-P h en et h yl)-6-(4-Nit r ob en zylsu lfa n yl)p u r in e (5).
This compound was prepared according to general procedure
A applying (2-bromoethyl)benzene. Yield: 61%. White solid.
Mp 137 °C. 1H NMR (CDCl3) δ ) 3.17 (t, 2H, J ) 6.9, CH2Ph),
4.49 (t, 2H, J ) 6.9, CH2N), 4.71 (s, 2H, CH2S), 7.02-7.09 (m,
2H, CH arom), 7.23-7.32 (m, 3H, CH arom), 7.59 (s, 1H, H-2),
7.64 (d, 2H, J ) 8.8, CH arom), 8.14 (d, 2H, J ) 8.8, CH arom),
8.75 (s, 1H, H-8). 13C NMR (CDCl3) δ ) 31.6, 35.9, 45.4, 123.5,
127.0, 128.5, 128.7, 129.9, 131.0, 136.9, 143.0, 145.7, 146.9,
148.5, 151.6, 158.8. HRMS (ESI) m/z Found: 392.1190 [M +
H]+, Calcd: 392.1181. Elem. anal. (C20H17N5O2S) C, H, N, S.
6-(4-Nitr oben zylsu lfan yl)-9-(3-ph en ylpr opyl)pu r in e (6).
This compound was prepared according to general procedure
A applying (3-bromopropyl)benzene. Yield: 69%. White solid.
Mp 130 °C. 1H NMR (CDCl3) δ ) 2.27 (quint., 2H, J ) 7.3,
CH2CH2Ph), 2.67 (t, 2H, J ) 7.3, CH2Ph), 4.26 (t, 2H, J ) 7.3,
CH2N), 4.72 (s, 2H, CH2S), 7.12-7.34 (m, 5H, CH arom), 7.64
(d, 2H, J ) 9.1, CH arom), 7.92 (s, 1H, H-2), 8.15 (d, 2H, J )
9.1, CH arom), 8.74 (s, 1H, H-8). 13C NMR (CDCl3) δ ) 30.8,
31.4, 32.4, 43.3, 123.3, 126.1, 128.0, 128.3, 129.7, 130.9, 139.8,
142.9, 145.6, 146.7, 148.6, 151.4, 158.7. HRMS (ESI) m/z
Found: 406.1292 [M + H]+, Calcd: 406.1337. Elem. anal.
(C21H19N5O2S) C, H, N, S.
6-(4-N it r o b e n zy ls u lfa n y l)-9-(p y r id in -2-y lm e t h y l)-
p u r in e (7). This compound was prepared according to general
procedure A applying 2-(bromomethyl)pyridine hydrobromide.
Additional NaH (1 equiv) was added to the reaction mixture
after 10 min. Yield: 32%. White solid. Mp 152 °C. 1H NMR
(CDCl3) δ ) 4.72 (s, 2H, CH2S), 5.53 (s, 2H, CH2N), 7.21-7.30
(m, 2H, CH arom), 7.62-7.71 (m, 3H, CH arom), 8.15 (d, 2H,
J ) 8.8, CH arom), 8.19 (s, 1H, H-2), 8.56 (ddd, 1H, J ) 0.7,
J ) 1.5, J ) 4.8, CH arom), 8.74 (s, 1H, H-8). 13C NMR (CDCl3/
MeOD) δ ) 31.0, 47.8, 122.0, 123.0, 129.5, 137.2, 143.7, 145.4,
146.3, 147.9, 148.9, 151.3, 153.7, 158.8. HRMS (ESI) m/z
Found: 379.0989 [M + H]+, Calcd: 379.0971. Elem. Anal.
found C 57.61, H 3.41, N 21.61, S 7.91, calcd for C18H14N6O2S
C 57.13, H 3.73, N 22.21, S 8.47.
6-(4-N it r o b e n zy ls u lfa n y l)-9-(p y r id in -3-y lm e t h y l)-
p u r in e (8). This compound was prepared according to general
procedure A applying 3-(bromomethyl)pyridine hydrobromide.
Additional NaH (1 equiv) was added to the reaction mixture
after 10 min. Yield: 29%. Yellowish solid. Mp 159-160 °C.
1H NMR (CDCl3) δ ) 4.72 (s, 2H, CH2S), 5.44 (s, 2H, CH2N),
7.25-7.31 (m, 1H, CH arom), 7.55-7.68 (m, 3H, CH arom),
7.99 (s, 1H, H-2), 8.15 (d, 2H, J ) 8.8, CH arom), 8.58-8.62
(m, 1H, CH arom), 8.65 (br s, 1H, CH arom), 8.76 (s, 1H, H-8).
13C NMR (CDCl3/MeOD, 1/1) δ ) 31.1, 44.3, 123.0, 123.8, 129.5,
130.1, 131.3, 136.1, 143.0, 145.4, 146.5, 148.0, 148.1, 148.6,
151.5, 159.0. HRMS (ESI) m/z Found: 379.0948 [M + H]+,
Calcd: 379.0971. Elem. Anal. found C 57.09, H 3.17, N 21.89,
S 8.03, calcd for C18H14N6O2S C 57.13, H 3.73, N 22.21, S 8.47.
6-(4-N it r o b e n zy ls u lfa n y l)-9-(p y r id in -4-y lm e t h y l)-
p u r in e (9). This compound was prepared according to general
procedure A applying 4-(bromomethyl)pyridine hydrobromide.
Additional NaH (1 equiv) was added to the reaction mixture
after 10 min. Yield: 40%. Yellow solid. Mp 187-188 °C. 1H
NMR (DMSO-d6) δ ) 4.80 (s, 2H, CH2S), 5.77 (s, 2H, CH2N),
7.21-7.24 (m, 2H, CH arom), 7.76 (d, 2H, J ) 8.8, CH arom),
8.18 (d, 2H, J ) 8.8, CH arom), 8.50-8.54 (m, 2H, CH arom),
9-(4-Flu or oben zyl)-6-(4-n itr oben zylsu lfan yl)pu r in e (12).
This compound was prepared according to general procedure
A applying 4-fluorobenzyl bromide. Yield: 63%. Off-white solid.
1
Mp 149 °C. H NMR (CDCl3) δ ) 4.72 (s, 2H, CH2S), 5.38 (s,
2H, CH2N), 7.04 (t, 2H, J ) 8.6, CH arom), 7.26-7.33 (m, 2H,
CH arom), 7.65 (d, 2H, J ) 8.8, CH arom), 7.95 (s, 1H, H-2),
8.16 (d, 2H, J ) 8.8, CH arom), 8.76 (s, 1H, H-8). 13C NMR
(CDCl3) δ ) 31.7, 46.6, 116.0 (d, J ) 21), 123.6, 129.7 (d, J )
9), 129.9, 130.8 (d, J ) 3), 131.0, 142.6, 145.7, 146.9, 148.7,
151.9, 159.0, 162.3 (d, J ) 247). 19F NMR (CDCl3) δ ) -35.4.
HRMS (ESI) m/z Found: 396.0899 [M + H]+, Calcd: 396.0930.
Elem. anal. (C19H14FN5O2S‚0.1EtOAc) C, H, N, S.
9-(3-Ch lor oben zyl)-6-(4-n itr oben zylsu lfan yl)pu r in e (20).
This compound was prepared according to general procedure
A applying 3-chlorobenzyl chloride. Yield: 56%. Yellow solid.
1
Mp 139 °C. H NMR (CDCl3) δ ) 4.72, (s, 2H, CH2S), 5.39 (s,
2H, CH2N), 7.18-7.31 (m, 4H, CH arom), 7.66 (d, 2H, J ) 8.4,
CH arom), 7.97 (s, 1H, H-2), 8.15 (d, 2H, J ) 8.4, CH arom),
8.77 (s, 1H, H-8). 13C NMR (CDCl3) δ ) 31.7, 46.7, 123.6, 125.8,
127.8, 128.8, 129.9, 130.4, 135.0, 137.0, 139.1, 142.6, 147.0,
148.7, 155.5, 161.3. HRMS (ESI) m/z Found: 412.0606 [M +
H]+, Calcd: 412.0635. Elem. anal. (C19H14ClN5O2S‚0.25hexane)
C, H, N, S.
9-(4-Ch lor oben zyl)-6-(4-n itr oben zylsu lfan yl)pu r in e (21).
This compound was prepared according to general procedure
A applying 4-chlorobenzyl chloride. Yield: 61%. White solid.
1
Mp 170 °C. H NMR (CDCl3) δ ) 4.73 (s, 2H, CH2S), 5.39 (s,
2H, CH2N), 7.22 (d, 2H, J ) 8.8, CH arom), 7.33 (d, 2H, J )
8.8, CH arom), 7.66 (d, 2H, J ) 8.8, CH arom), 7.97 (s, 1H,
H-2), 8.15 (d, 2H, J ) 8.8, CH arom), 8.77 (s, 1H, H-8). 13C
NMR (CDCl3) δ ) 31.7, 47.7, 123.6, 129.1, 129.2, 129.9, 130.9,
144.5, 134.6, 142.6, 145.5, 148.7, 152.0, 159.3. HRMS (ESI)
m/z Found: 412.0629 [M + H]+, Calcd: 412.0635. Elem. anal.
(C19H14ClN5O2S‚0.2EtOAc) C, H, N, S.
9-(3,4-D ic h lo r o b e n zy l)-6-(4-n it r o b e n zy ls u lfa n y l)-
p u r in e (23). This compound was prepared according to
general procedure A applying 3,4-dichlorobenzyl chloride.
1
Yield: 67%. Off-white solid. Mp 171 °C. H NMR (CDCl3) δ )
4.74 (s, 2H, CH2S), 5.39 (s, 2H, CH2N), 7.15 (dd, 2H, J ) 2.2,
J ) 8.0, CH arom), 7.41 (d, 1H, J ) 2.2, CH arom), 7.43 (d,
1H, J ) 8.0, CH arom), 7.66 (d, 2H, J ) 8.8, CH arom), 8.06
(s, 1H, H-2), 8.16 (d, 2H, J ) 8.8, CH arom), 8.77 (s, 1H, H-8).
13C NMR (DMSO-d6) δ ) 30.7, 45.5, 123.4, 128.1, 129.9, 130.1,
130.4, 130.7, 130.8, 131.3, 137.2, 144.8, 146.3, 146.4, 148.4,
151.5, 158.1. HRMS (ESI) m/z Found: 446.0233 [M + H]+,
Calcd: 446.0245. Elem. anal. Calcd for C19H13Cl2N5O2S: C
51.13, H 2.94, N 15.69, S 7.18. Found: C 51.27, H 2.45, N
15.41, S 6.81.
9-(2-M e t h o x y b e n z y l)-6-(4-n i t r o b e n z y ls u lfa n y l)-
p u r in e (25). This compound was prepared according to
general procedure A applying 2-methoxybenzyl chloride.
Yield: 52%. White solid. Mp 164 °C. 1H NMR (CDCl3) δ ) 3.85
(s, 3H, CH3), 4.70 (s, 2H, CH2S), 5.39 (s, 2H, CH2N), 6.82-
6.98 (m, 2H, CH arom), 7.33 (d, 2H, J ) 7.3, CH arom), 7.63
(d, 2H, J ) 8.8, CH arom), 8.05 (s, 1H, H-2), 8.12 (d, 2H, J )
8.8, CH arom), 8.74 (s, 1H, H-8). 13C NMR (CDCl3) δ ) 31.6,
42.9, 55.3, 110.5, 120.7, 123.0, 123.5, 129.9, 130.2, 130.4, 130.9,
143.7, 145.8, 146.7, 148.8, 151.6, 157.2, 158.5. HRMS (ESI)
m/z Found: 408.1123 [M + H]+, Calcd: 408.1130. Elem. anal.
(C20H17N5O3S) C, H, N, S.
9-(3-M e t h o x y b e n z y l)-6-(4-n i t r o b e n z y ls u lfa n y l)-
p u r in e (26). This compound was prepared according to
general procedure A applying 3-methoxybenzyl chloride.
Yield: 52%. White solid. Mp 126 °C. 1H NMR (CDCl3) δ ) 3.77
(s, 3H, CH3O), 4.72 (s, 2H, CH2S), 5.38 (s, 2H, CH2N), 6.78-
6.92 (m, 3H, CH arom), 7.22-7.33 (m, 1H, CH arom), 7.65 (d,
2H, J ) 8.8, CH arom), 7.95 (s, 1H, H-2), 8.15 (d, 2H, J ) 8.8,
CH arom), 8.77 (s, 1H, H-8). 13C NMR (CDCl3) δ ) 31.5, 47.1,
55.0, 113.4, 119.7, 123.4, 129.8, 130.0, 130.8, 136.7, 142.8,

Inhibition of Nucleoside Transport by NBTI Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5447
145.6, 146.7, 148.6, 151.7, 158.8, 159.8. HRMS (ESI) m/z
Found: 408.1124 [M + H]+, Calcd: 408.1130. Elem. anal.
(C20H17N5O3S‚0.05hexane) C, H, N, S.
general procedure A applying 3-hydroxybenzyl iodide, which
was prepared by stirring 3-hydroxybenzyl alcohol overnight
in dioxane in the presence of BF3‚OEt2 (1 equiv) and KI (1
equiv).31 Yield: 56%. White solid. Mp 183-184 °C. 1H NMR
(CDCl3/MeOD, 3/1) δ ) 4.74 (s, 2H, CH2S), 5.37 (s, 2H, CH2N),
6.74-6.82 (m, 3H, CH arom), 7.18 (t, 1H, J ) 8.0, 1H, CH
arom), 7.45 (s, 1H, H-2), 7.69 (d, 2H, J ) 8.8, CH arom), 8.11
(s, 1H, H-8), 8.16 (d, 2H, J ) 8.8, CH arom). 13C NMR (CDCl3)
δ ) 31.9, 47.1, 114.4, 115.3, 118.6, 123.3, 129.7, 129.9, 136.0,
143.2, 145.5, 146.7, 148.2, 151.5, 157.3. HRMS (ESI) m/z
Found: 394.0926 [M + H]+, Calcd: 394.0974. Elem. anal.
(C19H15N5O3S‚0.1EtOAc) C, H, N.
9-(2-Am in oben zyl)-6-(4-n itr oben zylsu lfan yl)pu r in e (37).
Compound 29 (0.12 mmol) was dissolved in 2 mL of a mixture
of TFA and H2O (5/2, v/v) and stirred for 7 h at rt.19 The
solvents were evaporated, and the product was purified by
column chromatography (eluent: EtOAc). Yield: 76%. Yellow
solid. Mp 137 °C. 1H NMR (CDCl3) δ ) 4.70 (s, 2H, CH2S),
5.48 (s, 2H, CH2N), 7.26-7.46 (m, 4H, CH arom), 7.59 (d, 2H,
J ) 8.8, CH arom), 8.15 (d, 2H, J ) 8.8, CH arom), 8.36 (s,
1H, H-2), 8.76 (s, 1H, H-8). 13C NMR (CDCl3) δ ) 31.8, 44.0,
113.7, 123.1, 123.5, 126.0, 127.6, 129.8, 131.0, 131.5, 133.0,
144.6, 151.1, 160.8. HRMS (ESI) m/z Found: 393.1082 [M +
H]+, Calcd: 393.1128. Elem. anal. (C19H16N6O2S‚0.2EtOAc) C,
H, N, S.
9-(4-M e t h o x y b e n z y l)-6-(4-n i t r o b e n z y ls u lfa n y l)-
p u r in e (27). This compound was prepared according to
general procedure A applying 4-methoxybenzyl chloride.
Yield: 59%. White solid. Mp 153 °C. 1H NMR (CDCl3) δ ) 3.79
(s, 3H, OCH3), 4.72 (s, 2H, CH2S), 5.34 (s, 2H, CH2N), 6.87 (d,
2H, J ) 8.8, CH arom), 7.25 (d, 2H, J ) 8.8, CH arom), 7.65
(d, 2H, J ) 8.8, CH arom), 7.93 (s, 1H, H-2), 8.14 (d, 2H, J )
8.8, CH arom), 8.77 (s, 1H, H-8). 13C NMR (CDCl3) δ ) 31.6,
46.9, 55.1, 114.3, 123.5, 126.9, 129.3, 129.8, 131.0, 142.7, 145.7,
146.9, 148.6, 151.7, 158.8, 159.6. HRMS (ESI) m/z Found:
408.1123 [M + H]+, Calcd: 408.1130. Elem. anal. (C20H17N5O3S‚
0.15 hexane) C, H, N, S.
9-(2′-ter t-Bu tyloxoca r bon yla m in o-6-(4-n itr oben zylsu l-
fa n yl)ben zyl)p u r in e (29). This compound was prepared
according to general procedure A applying Boc-protected 2′-
amino-benzyl bromide.17 Yield 27%. Off-white solid. Mp 77 °C.
1H NMR (CDCl3) δ ) 1.56 (s, 9H, C(CH3)3), 4.71 (s, 2H, CH2S),
5.35 (s, 2H, CH2N), 7.08 (dt, 1H, J ) 1.5, J ) 7.7, CH arom),
7.27-7.38 (m, 2H, CH arom), 7.64 (d, 2H, J ) 8.8, CH arom),
7.75 (d, 1H, J ) 8.0, CH arom), 8.09 (s, 1H, H-2), 8.15 (d, 1H,
J ) 8.8, CH arom), 8.72 (s, 1H, H-8), 9.02 (br s, 1H, NH). 13C
NMR (CDCl3) δ ) 28.2, 31.6, 44.2, 80.1, 123.6, 124.2, 124.6,
126.1, 129.9, 130.2, 137.0, 142.7, 145.5, 151.3, 153.8. Elem.
anal. (C24H24N6O4S‚0.4CH2Cl2) C, H, N.
2-F lu or o-2′-m eth ylbip h en yl (39). This compound was
prepared according to general procedure D applying 2-bromo-
fluorobenzene. Reaction time: 5 min. Yield: 68%. Colorless
oil. 1H NMR (CDCl3) δ ) 2.20 (s, 3H, CH3), 7.11-7.34 (m, 8H,
CH arom).
6-(4-Nitr oben zylsu lfa n yl)-9-(3-tr iflu or om eth ylben zyl)-
p u r in e (31). This compound was prepared according to
general procedure A applying 3-trifluoromethylbenzyl bromide.
Yield: 56%. White solid. Mp 147 °C. 1H NMR (CDCl3) δ ) 4.73
(s, 2H, CH2S), 5.48 (s, 2H, CH2N), 7.46-7.61 (m, 4H, CH arom),
7.61 (d, 2H, J ) 8.8, CH arom), 7.99 (s, 1H, H-2), 8.16 (d, 2H,
J ) 8.8, CH arom), 8.77 (s, 1H, H-8). 13C NMR (CDCl3) δ )
31.6, 46.7, 123.5, 123.7 (q, J ) 272), 124.4 (q, J ) 4), 125.2 (q,
J ) 4), 129.6, 129.8, 130.8, 131.0, 131.2 (q, J ) 33), 136.1,
142.6, 145.6, 146.8, 148.6, 151.9, 159.2. 19F NMR (CDCl3) δ )
14.6. HRMS (ESI) m/z Found: 446.0923 [M + H]+, Calcd:
446.0899. Elem. anal. (C20H14F3N5O2S‚0.1hexane) C, H, N, S.
9-(4-Meth ylben zyl)-6-(4-n itr oben zylsu lfan yl)pu r in e (32).
This compound was prepared according to general procedure
A applying 4-methylbenzyl chloride. Yield: 61%. White solid.
Mp 152 °C. 1H NMR (CDCl3) δ ) 2.33 (s, 3H, CH3), 4.72 (s,
2H, CH2S), 5.36 (s, 2H, CH2N), 7.14 (d, 2H, J ) 8.8, CH arom),
7.20 (d, 2H, J ) 8.8, CH arom), 7.65 (d, 2H, J ) 8.8, CH arom),
7.94 (s, 1H, H-2), 8.14 (d, 2H, J ) 8.8, CH arom), 8.77 (s, 1H,
H-8). 13C NMR (CDCl3) δ ) 19.9, 31.7, 47.2, 123.6, 127.8, 129.7,
129.9, 131.0, 131.8, 138.5, 142.8, 145.7, 147.0, 148.8, 151.9,
157.9. HRMS (ESI) m/z Found: 392.1157 [M + H]+, Calcd:
392.1181. Elem. anal. (C20H17N5O2S‚0.1hexane) C, H, N, S.
9-(4-Nitr oben zyl)-6-(4-n itr oben zylsu lfa n yl)p u r in e (33).
This compound was prepared according to general procedure
A applying 4-nitrobenzyl bromide. Yield: 40%. White solid.
4-F lu or o-2′-m eth ylbip h en yl (40). This compound was
prepared according to general procedure D applying 4-bromo-
fluorobenzene. Reaction time: 5 min. Yield: 73%. Colorless
1
liquid. H NMR (CDCl3) δ ) 2.24 (s, 3H, CH3), 7.04-7.12 (m,
2H, CH arom), 7.17-7.30 (m, 6H, CH arom).
2′-Meth yl-3-tr iflu or om eth ylbip h en yl (41). This com-
pound was prepared according to general procedure D applying
3-bromobenzotrifluoride. Reaction time: 5 min. Yield: 72%.
Colorless oil. 1H NMR (CDCl3) δ ) 2.25 (s, 3H, CH3), 7.19-
7.28 (m, 4H, CH arom), 7.48-7.59 (m, 4H, CH arom).
2′-Meth yl-4-tr iflu or om eth ylbip h en yl (42). This com-
pound was prepared according to general procedure D applying
4-bromobenzotrifluoride. Reaction time: 5 min. Yield: 88%.
Colorless liquid. 1H NMR (CDCl3) δ ) 2.25 (s, 3H, CH3), 7.22-
7.28 (m, 4H, CH arom), 7.42 (d, 2H, J ) 8.0, CH arom), 7.66
(d, 2H, J ) 7.7, CH arom).
2-Meth oxy-2′-m eth ylbip h en yl (43). This compound was
prepared according to general procedure C applying 2-bromo-
anisole. Yield: 32%. Colorless liquid. 1H NMR (CDCl3) δ ) 2.13
(s, 3H, CH3), 3.72 (s, 3H, OCH3), 6.91-7.03 (m, 2H, CH arom),
7.11-7.36 (m, 6H, CH arom).
3-Meth oxy-2′-m eth ylbip h en yl (44). This compound was
prepared according to general procedure C applying 3-bromo-
anisole. Yield: 74%. Colorless liquid. 1H NMR (CDCl3) δ ) 2.26
(3s, 3H, CH3), 3.75 (s, 3H, OCH3), 6.86 (m, 3H, CH arom), 7.25
(m, 5H, CH arom).
4-Meth oxy-2′-m eth ylbip h en yl (45). This compound was
prepared according to general procedure C applying 4-bromo-
anisole. Yield: 58% (95% pure), colorless liquid. 1H NMR
(CDCl3) δ ) 2.25 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 6.91 (d, 2H,
J ) 8.8, CH arom), 7.19-7.24 (m, 6H, CH arom).
1
Mp 182 °C. H NMR (DMSO-d6) δ ) 4.81 (s, 2H, CH2S), 5.67
(s, 2H, CH2N), 7.57 (d, 2H, J ) 8.8, CH arom), 7.77 (d, 2H, J
) 8.8, CH arom), 8.16-8.23 (m, 4H, CH arom), 8.69 (s, 1H,
H-2), 8.77 (s, 1H, H-8). 13C NMR (DMSO-d6) δ ) 30.8, 46.1,
123.6, 124.0, 128.7, 130.3, 143.9, 145.2, 146.5, 147.2, 148.6,
151.7, 158.3. HRMS (ESI) m/z Found: 423.0828 [M + H]+,
Calcd: 423.0870. Elem. Anal. (C19H14N6O4S‚0.1CH2Cl2) C, H,
N, S.
2-(2′-Th iop h en e)-tolu en e (46). This compound was pre-
pared according to general procedure C applying 2-bromo-
thiophene. Reaction time: 2 h. Yield: 69%. Blue-ish liquid.
1H NMR (CDCl3) δ ) 2.51 (s, 3H, CH3), 7.12-7.17 (m, 2H, CH
arom), 7.24-7.32 (m, 4H, CH arom), 7.38-7.40 (m, 1H, CH
arom), 7.47-7.48 (m, 1H, CH arom).
9-(2-H y d r o x y b e n z y l)-6-(4-n i t r o b e n z y ls u lfa n y l)-
p u r in e (35). This compound was prepared from 25 according
to general procedure B. Yield: 66%. White solid. Mp 201 °C.
1H NMR (CDCl3) δ ) 4.74, (s, 2H, CH2S), 5.41 (s, 2H, CH2N),
6.75-6.88 (m, 2H, CH arom), 7.12-7.23 (m, 1H, CH arom),
7.33 (d, 1H, J ) 8.0, CH arom), 7.69 (d, 2H, J ) 8.8, CH arom),
8.15 (d, 2H, J ) 8.8, CH arom), 8.28 (s, 1H, H-2), 8.75 (s, 1H,
H-8). 13C NMR (CDCl3) δ ) 30.6, 42.6, 115.2, 119.0, 122.1,
123.6, 129.4, 129.5, 130.2, 145.4, 146.5, 148.6, 151.4, 155.2,
157.7. HRMS (ESI) m/z Found: 394.0938 [M + H]+, Calcd:
394.0973. Elem. anal. (C19H15N5O3S‚0.2CH2Cl2) C, H, N, S.
9-(3-H y d r o x y b e n z y l)-6-(4-n i t r o b e n z y ls u lfa n y l)-
p u r in e (36). This compound was prepared according to
2-(3′-Th iop h en e)-tolu en e (47). This compound was pre-
pared according to general procedure C applying 3-bromo-
thiophene. Reaction time: 2 h. Yield: 69%. Colorless liquid.
1H NMR (CDCl3) δ ) 2.31 (s, 3H, CH3), 7.22 (m, 7H, CH arom).
2′-Br om om eth yl-2-flu or obip h en yl (48). This compound
was prepared from 39 according to general procedure E. Yellow
1
oil. H NMR (CDCl3) δ ) 4.40 (s, 2H, CH2Br), 7.15-7.43 (m,

5448 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Tromp et al.
7H, CH arom), 7.52-7.57 (m, 1H, CH arom). 13C NMR (CDCl3)
δ ) 31.7, 113.6, 115.5, 124.1, 127.9, 128.4, 128.6, 129.6, 129.8,
130.6, 130.9, 131.6, 197.6.
145.7, 147.0, 148.7, 151.6, 158.7, 159.6 (d, J ) 247). 19F NMR
(CDCl3) δ ) -36.9. HRMS (ESI) m/z found 472.1246 [M + H]+,
calcd 472.1244. Elem. anal. (C25H18FN5O2S‚0.1EtOAc) C, H,
N, S.
2′-Br om om eth yl-4-flu or obip h en yl (49). This compound
was prepared from 40 according to general procedure E. Yellow
6-(4-Nitr oben zylsu lfa n yl)-9-(3′-tr iflu or om eth ylbip h e-
n yl-2-ylm eth yl)p u r in e (59). This compound was prepared
from 50 according to general procedure A. Yield: 41%. Yel-
lowish solid. Mp: 142 °C. 1H NMR (CDCl3) δ ) 4.70 (s, 2H,
CH2S), 5.35 (s, 2H, CH2S), 7.25-7.35 (m, 2H, CH arom), 7.38-
7.53 (m, 6H, CH arom), 7.57 (s, 1H, H-2), 7.64 (d, 2H, J ) 8.8,
CH arom), 8.14 (d, 2H, J ) 8.8, CH arom), 8.63 (s, 1H, H-8).13C
NMR (CDCl3) d ) 31.7, 45.2, 123.6, 123.7 (q, J ) 272), 124.4
(q, J ) 4), 125.4 (q, J ) 4), 128.7, 128.9, 129.0, 129.1, 129.9,
130.6, 131.2, 131.9, 132.1, 132.4, 140.4, 140.5, 142.5, 145.7,
147.0, 148.6, 151.7, 159.0. 19F NMR (CDCl3) δ ) 14.7. HRMS
(ESI) m/z found 522.1224 [M + H]+, calcd 522.1212. Elem.
anal. (C26H18F3N5O2S‚0.1EtOAc) C, H, N, S.
1
oil. H NMR (CDCl3) δ ) 4.41 (s, 2H, CH2Br), 7.11-7.23 (m,
3H, m, CH arom), 7.32-7.51 (m, 5H, CH arom). 13C NMR
(CDCl3) δ ) 32.0, 115.0, 115.4, 128.1, 128.6, 129.5, 130.5, 130.7,
130.9, 197.4.
2-Br om om eth yl-3′-tr iflu or om eth ylbip h en yl (50). This
compound was prepared from 41 according to general proce-
dure E. Yellow oil. 13C NMR (CDCl3) δ ) 31.5, 124.3, 125.9,
128.6, 128.8, 130.3, 131.1, 132.3, 135.2, 140.5, 140.9.
2-Br om om eth yl-4′-tr iflu or om eth ylbip h en yl (51). This
compound was prepared from 42 according to general proce-
dure E. Yellow crystals. 1H NMR (CDCl3) δ ) 4.39 (s, 2H, CH2-
Br), 7.31-7.44 (m, 2H, CH arom), 7.50-7.59 (m, 4H, CH
arom), 7.69-7.73 (d, 2H, J ) 8.0, CH arom). 13C NMR (CDCl3)
δ ) 31.6, 125.3, 128.7, 129.4, 130.2, 131.1.
6-(4-Nitr oben zylsu lfa n yl)-9-(4′-tr iflu or om eth ylbip h e-
n yl-2-ylm eth yl)p u r in e (60). This compound was prepared
from 51 according to general procedure A. Yield: 30%. Yel-
2-Br om om et h yl-2′-m et h oxyb ip h en yl (52). This com-
pound was prepared from 43 according to general procedure
1
lowish solid. Mp: 147 °C. HH NMR (CDCl3) δ ) 4.69 (s, 2H,
1
CH2S), 5.35 (s, 2H, CH2N), 7.25-7.44 (m, 6H, CH arom), 7.53
(s, 1H, H-2), 7.57 (m, 2H, CH arom), 7.64 (d, 2H, J ) 8.8, CH
arom), 8.15 (d, 2H, J ) 8.8, CH arom), 8.63 (s, 1H, H-8). 13C
NMR (CDCl3) d ) 31.6, 45.2, 123.6, 123.9 (q, J ) 272), 125.3
(q, J ) 4), 128.7, 128.8, 129.1, 129.9, 130.4, 132.3, 140.5, 142.5,
143.3, 145.7, 147.0, 148.6, 151.7, 158.9. 19F NMR (CDCl3) δ )
14.7. HRMS (ESI) m/z found 522.1220 [M + H]+, calcd
522.1212. Elem. anal. (C26H18F3N5O2S‚0.1EtOAc) C, H, N, S.
E. Orange oil. H NMR (CDCl3) δ ) 3.70 (s, 3H, OCH3), 4.35
(AB, 2H, J ) 9.9, CH2Br), 6.92-7.06 (m, 2H, CH arom), 7.17-
7.36 (m, 5H, CH arom), 7.49-7.54 (m, 1H, CH arom). 13C NMR
(CDCl3) δ ) 32.2, 55.3, 110.6, 120.5, 125.3, 127.2, 127.8, 128.1,
129.1, 130.1, 130.7, 131.1, 136.1, 156.2.
2-Br om om et h yl-3′-m et h oxyb ip h en yl (53). This com-
pound was prepared from 44 according to general procedure
E. Colorless oil. 1H NMR (CDCl3) δ ) 3.80 (s, 3H, CH3O), 4.43
(s, 2H, CH2Br), 6.89-7.03 (m, 3H, CH arom), 7.22-7.36 (m,
4H, CH arom), 7.46-7.49 (m, 1H, CH arom). 13C NMR (CDCl3)
δ ) 32.2, 55.1, 114.1, 121.2, 125.5, 127.1, 127.9, 128.4, 129.2,
130.1, 130.8, 135.0, 141.4, 141.8.
9-(2′-Met h oxyb ip h en yl-2-ylm et h yl)-6-(4-n it r ob en zyl-
su lfa n yl)p u r in e (61). This compound was prepared from 52
according to general procedure A. Yield: 45%. Yellow white
1
solid. Mp: 103 °C. H NMR (CDCl3) δ ) 3.69 (s, 3H, CH3O),
4.68 (s, 2H, CH2S), 5.35 (s, 2H, CH2N), 6.82-6.89 (m, 1H, CH
arom), 6.92 (dd, 1H, J ) 1.1, J ) 7.3, CH arom), 7.03 (dd, 1H,
J ) 1.8, J ) 7.3, CH arom), 7.20-7.25 (m, 2H, CH arom),
7.27-7.37 (m, 3H, CH arom), 7.42 (s, 1H, H-2), 7.62 (d, 2H, J
) 8.8, CH arom), 8.11 (d, 2H, J ) 8.8, CH arom), 8.65 (s, 1H,
H-8). 13C NMR (CDCl3) d ) 31.6, 45.6, 55.3, 110.8, 120.8, 123.5,
127.9, 128.5, 128.9, 129.3, 129.9, 130.8, 130.9, 133.6, 138.5,
143.2, 145.8, 146.9, 148.8, 151.5, 156.0, 158.3. HRMS (ESI)
m/z found 484.1401 [M +H], calcd 484.1443. Elem. anal.
(C26H21N5O3S) C, H, N, S.
9-(3′-Met h oxyb ip h en yl-2-ylm et h yl)-6-(4-n it r ob en zyl-
su lfa n yl)p u r in e (62). This compound was prepared from 53
according to general procedure A. Yield: 37%. Yellow crystals.
Mp: 121 °C. 1H NMR (CDCl3) δ ) 3.77 (s, 3H, CH3O), 4.69 (s,
2H, CH2S), 5.40 (s, 2H, CH2N), 6.73-6.75 (m, 1H, CH arom),
6.79-6.86 (m, 2H, CH arom) 7.24-7.38 (m, 5H, CH arom),
7.46 (s, 1H, H-2), 7.64 (d, 2H, J ) 9.1, CH arom), 8.14 (d, 2H,
J ) 8.8, CH arom), 8.67 (s, 1H, H-8). 13C NMR (CDCl3) δ )
31.5, 45.1, 55.0, 112.9, 114.2, 120.9, 123.4, 127.9, 128.4, 129.0,
129.4, 129.7, 130.2, 132.2, 140.9, 141.7, 142.7, 145.6, 151.4,
159.4. HRMS (ESI) m/z found 484.1442 [M + H], calcd
484.1443. Elem. anal. (C26H21N5O3S‚0.1EtOAc) C, H, N, S.
9-(4′-Met h oxyb ip h en yl-2-ylm et h yl)-6-(4-n it r ob en zyl-
su lfa n yl)p u r in e (63). This compound was prepared from 54
according to general procedure A. Yield: 37%. Yellow solid.
Mp: 163 °C. 1H NMR (CDCl3) δ ) 3.79 (s, 3H, CH3O), 4.68 (s,
2H, CH2S), 5.39 (s, 2H, CH2N), 6.90 (d, 2H, J ) 8.8, CH arom),
7.16 (d, 2H, J ) 8.8, CH arom), 7.26-7.31 (m, 4H, CH arom),
7.44 (s, 1H, H-2), 7.62 (d, 2H, J ) 8.8, CH arom), 8.10 (d, 2H,
J ) 8.8, CH arom), 8.66 (s, 1H, H-8). 13C NMR (CDCl3) δ )
31.6, 45.3, 55.2, 113.9, 123.5, 127.7, 128.6, 129.2, 129.9, 130.7,
132.0, 132.5, 141.7, 142.8, 145.7, 147.1, 148.8, 151.6, 159.1.
HRMS (ESI) m/z found 484.1476 [M + H], calcd 484.1443.
Elem. anal. (C26H21N5O3S‚0.15CH2Cl2) C, H, N, S.
6-(4-Nit r ob en zylsu lfa n yl)-9-[2-(2′-t h iop h en e)b en zyl]-
p u r in e (64). This compound was prepared from 55 according
to general procedure A. Yield: 36%. Off-white foam. Mp: 62
°C. 1H NMR (CDCl3) δ ) 4.70 (s, 2H, CH2S), 5.52 (s, 2H,
CH2N), 7.01 (dd, 1H, J ) 1.3, J ) 3.5, CH arom), 7.08 (dd,
1H, J ) 3.5, J ) 5.1, CH arom), 7.24-7.48 (m, 5H, CH arom),
7.51 (s, 1H, H-2), 7.64 (d, 2H, J ) 8.8, CH arom), 8.14 (d, 2H,
2-Br om om et h yl-4′-m et h oxyb ip h en yl (54). This com-
pound was prepared from 45 according to general procedure
E. Yellow liquid. 1H NMR (CDCl3) δ ) 3.80 (s, 3H, CH3O), 4.43
(s, 2H, CH2Br), 6.95 (d, 2H, J ) 8.4, CH arom), 7.20-7.38 (m,
5H, CH arom), 7.45-7.50 (m, 1H, CH arom). 13C NMR (CDCl3)
δ ) 32.4, 55.1, 113.6, 115.6, 125.6, 126.8, 127.6, 128.4, 130.0,
130.9 132.4, 135.2, 141.6, 158.9.
2-(2′-Th iop h en e)ben zyl Br om id e (55). This compound
was prepared from 46 according to general procedure E.
Colorless oil. 1H NMR (CDCl3) δ ) 4.59 (s, 2H, CH2Br), 7.06-
7.14 (m, 1H, CH arom), 7.30-7.47 (m, 5H, CH arom), 7.48-
7.53 (m, 1H, CH arom). 13C NMR (CDCl3) δ ) 32.2, 125.8,
127.3, 127.6, 128.3, 128.4, 130.3, 130.5, 131.1.
2-(3′-Th iop h en e)ben zyl Br om id e (56). This compound
was prepared from 47 according to general procedure E. Brown
1
oil. H NMR (CDCl3) δ ) 4.46 (s, 2H, CH2Br), 7.14-7.49 (m,
7H, CH arom). 13C NMR (CDCl3) δ ) 32.6, 122.5, 123.1, 124.8,
125.6, 125.7, 127.2, 127.9, 128.6, 128.6, 128.8, 129.7, 130.3,
131.1, 135.3, 136.6, 140.2.
9-(2′-F lu or obip h en yl-2-ylm eth yl)-6-(4-n itr oben zylsu l-
fa n yl)p u r in e (57). This compound was prepared from 48
according to general procedure A. Yield: 24%. White solid.
1
Mp: 156 °C. H NMR (CDCl3) δ ) 4.70 (s, 2H, CH2S), 5.36 (s,
2H, CH2N), 7.13-7.17 (m, 3H, CH arom), 7.26-7.43 (m, 5H,
CH arom), 7.48 (s, 1H, H-2), 7.64 (d, 2H, J ) 8.9, CH arom),
8.14 (d, 2H, J ) 8.9, CH arom), 8.67 (s, 1H, H-8). 13C NMR
(CDCl3) δ ) 31.7, 45.3, 115.8 (d, J ) 23), 123.6, 124.5, 124.6,
128.7, 128.8 (d, J ) 9), 128.7, 129.8 130.0, 131.0, 131.2 (d, J )
3), 133.5, 135.3, 142.9, 145.7, 147.0, 148.9, 151.7, 158.7, 159.6
(d, J ) 245). 19F NMR (CDCl3) δ ) -37.4. HRMS (ESI) m/z
found 472.1241 [M + H]+, calcd 472.1244. Elem. anal. (C25H18
FN5O2S‚0.1EtOAc) C, H, N, S.
-
9-(4′-F lu or obip h en yl-2-ylm eth yl)-6-(4-n itr oben zylsu l-
fa n yl)p u r in e (58). This compound was prepared from 49
according to general procedure A. Yield: 39%. Yellow solid.
1
Mp: 109 °C. H NMR (CDCl3) δ ) 4.70 (s, 2H, CH2S), 5.36 (s,
2H, CH2N), 7.00-7.09 (m, 2H, CH arom), 7.23-7.41 (m, 6H,
CH arom), 7.48 (s, 1H, H-2), 7.64 (d, 2H, J ) 8.8, CH arom),
8.15 (d, 2H, J ) 8.8, CH arom), 8.68 (s, 1H, H-8). 13C NMR
(CDCl3) d ) 31.6, 45.2, 115.4 (d, J ) 21), 123.5, 128.2, 128.6,
129.1, 129.9, 130.3 (d, J ) 9), 130.6, 132.4, 135.6, 140.9, 142.7,

Inhibition of Nucleoside Transport by NBTI Analogues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5449
J ) 8.8, CH arom), 8.72 (s, 1H, H-8). 13C NMR (CDCl3) δ )
31.5, 45.3, 123.4, 126.2, 127.0, 127.3, 128.5, 128.6, 129.2, 129.7,
130.7, 131.5, 133.0, 134.0, 140.2, 143.6, 145.5, 146.8, 148.6,
151.6, 158.7. HRMS (ESI) m/z found 460.0941[M + H]+, calcd
460.0901. Elem. anal. (C23H17N5O2S2‚0.1CH2Cl2) C, H, N
6-(4-Nit r ob en zylsu lfa n yl)-9-[2-(3′-t h iop h en e)b en zyl]-
p u r in e (65). This compound was prepared from 56 according
to general procedure A. Yield: 44%. Yellow solid. Mp: 134 °C.
1H NMR (CDCl3) δ ) 4.69 (s, 2H, CH2S), 5.43 (s, 2H, CH2N),
7.06 (d, 1H, J ) 4.8, CH arom) 7.18 (d, 1H, J ) 4.4, CH arom),
7.28-7.39 (m, 5H, CH arom), 7.45 (s, 1H, H-2), 7.63 (d, 2H, J
) 8.6, CH arom), 8.12 (d, 2H, J ) 8.6, CH arom), 8.69 (s, 1H,
H-8). 13C NMR (CDCl3) δ ) 31.5, 45.3, 123.2, 123.4, 126.2,
128.0, 128.2, 128.5, 129.1, 129.7, 130.5, 132.5, 136.5, 139.8,
142.6, 145.6, 148.6, 151.5. HRMS (ESI) m/z found 460.0929
[M + H]+, calcd 460.0901. Elem. anal. (C23H17N5O2S2) C, H,
N.
9-(4′-H yd r oxyb ip h en yl-2-ylm et h yl)-6-(4-n it r ob en zyl-
su lfa n yl)p u r in e (72) This compound was prepared from 63
according to general procedure B. Yield: 43%. White solid.
Mp: 194 °C. 1H NMR (DMSO-d6) δ ) 4.78 (s, 2H, CH2S), 5.45
(s, 2H, CH2N), 6.84 (d, 2H, J ) 8.8, CH arom), 6.95 (d, 1H, J
) 7.7, CH arom), 7.18-7.38 (m, 5H, CH arom), 7.74 (d, 2H, J
) 8.8, CH arom), 8.08 (s, 1H, H-2), 8.17 (d, 2H, J ) 8.8, CH
arom), 8.68 (s, 1H, H-8), 9.60 (s, 1H, PhOH). 13C NMR (DMSO-
d6) d ) 30.7, 45.1, 115.3, 123.5, 127.3, 127.6, 127.9, 130.2,
130.3, 133.2, 141.0, 144.8, 146.4, 148.5, 151.4, 156.8, 157.8.
HRMS (ESI) m/z found 470.1271 [M + H]+, calcd 470.1281.
Elem. anal. (C25H19N5O3S‚0.1CH2Cl2) C, H, N.
Er yth r ocytes a n d Mem br a n e P r ep a r a tion . 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.
2-(2′-Meth ylp h en yl)ben zyl a lcoh ol (67). This compound
was prepared according to general procedure D applying
2-bromobenzyl alcohol (66). Reaction time: 10 min. Catalyst:
1
Pd(OAc)2. Yield: 53%. White solid. H NMR (CDCl3) δ ) 2.05
(s, 3H, CH3), 4.40 (s, 2H, CH2OH), 7.11-7.29 (m, 5H, CH
arom), 7.32-7.39 (m, 2H, CH arom), 7.52-7.55 (m, 1H, CH
arom).
[3H]NBTI Bin d in g Assa y. 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.32
2-(2′-Meth ylp h en yl)ben zyl m esyla te (68). A solution of
0.45 mmol of 67 in dry MTBE (2 mL) was cooled on an ice
bath. 1.1 equiv of MsCl and 1.2 equiv of triethylamine were
added. After addition of triethylamine the ice bath was
removed and the mixture was stirred for 3h at rt. Brine (1
mL) was added and the mixture was extracted with MTBE.
The organic layers were dried (MgSO4) and concentrated. The
crude product was used without further purification. Yield:
73%. Colorless liquid. 1H NMR (CDCl3) δ ) 2.06 (s, 3H,
PhCH3), 2.64 (s, 3H, SO2CH3), 4.96 (AB, 2H, J ) 10.8, CH2-
OMs), 7.11-7.30 (m, 5H, CH arom), 7.40-7.45 (m, 2H, CH
arom), 7.51-7.57 (m, 1H, CH arom).
Ca lcu la tion of th e P ola r Su r fa ce Ar ea a n d Molecu la r
Mod elin g. The polar surface areas of the molecules were
calculated using Spartan 5.0 for SGI,22 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, to optimize its 3D-structure by molecular mechanics
(Merck force field) followed by semiempirical AM1 single point
energy calculation. In all cases default settings were used. The
electrostatic potentials were calculated over the entire acces-
sible 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.
9-(2′-Meth ylbip h en yl-2-ylm eth yl)-6-(4-n itr oben zylsu l-
fa n yl)p u r in e (69). This compound was prepared from 68
according to general procedure A. Yield: 53%. Yellowish solid.
1
Mp: 132 °C. H NMR (CDCl3) δ ) 1.96 (s, 3H, PhCH3), 4.69
(s, 2H, CH2S), 5.17 (AB, 2H, J ) 15.0, CH2N), 7.64 (d, 2H, J
) 8.9, CH arom), 8.14 (d, 2H, J ) 8.9, CH arom), 8.65 (s, 1H,
H-8). 13C NMR (CDCl3) d ) 19.8, 31.6, 45.5, 123.6, 125.9, 127.9,
128.0, 128.6, 129.1, 129.4, 129.9 130.2 130.4, 130.8, 132.6,
135.7, 139.0, 141.3, 142.9, 145.8, 147.0, 148.7, 151.6, 158.6.
HRMS (ESI) m/z found 468.1470 [M + H]+, calcd 468.1494.
Elem. anal. (C26H21N5O2S‚0.1EtOAc) C, H, N, S.
The crystal structure of NBTI23 (1, CSD code: NBTINS10)
used for the illustrations in Figure 2 was retrieved from the
Cambridge Structural Database24 using ConQuest.33 The
structures of 35 and 36 were drawn and energy minimized
(equilibrium geometry, semiempirical, AM1) in PC Spartan
Pro.25 Next, the 4-nitrobenzyl groups of NBTI, 35, and 36 were
omitted for reasons of clarity. In PC Spartan Pro, the purine
rings of NBTI and 35 or 36 were matched after which the
benzyl ring was rotated over the Ph-CH2, the PhCH2-purine
and the Ph-OH bond to achieve an overlay between its
hydroxyl function and either the 2′-, 3′-, or 5′-OH of NBTI.
The structure of NBTI was left untouched.
9-(2′-H yd r oxyb ip h en yl-2-ylm et h yl)-6-(4-n it r ob en zyl-
su lfa n yl)p u r in e (70). This compound was prepared from 61
according to general procedure B. Yield: 20%. White solid.
1
Mp: 164 °C. H NMR (CDCl3) δ ) 4.65 (s, 2H, CH2S), 5.29 (s,
2H, CH2N), 6.90-7.00 (m, 2H, CH arom), 7.08 (dd, 1H, J )
1.5, J ) 7.7, CH arom), 7.19-7.40 (m, 5H, CH arom), 7.59 (d,
2H, J ) 8.8, CH arom), 7.74 (s, 1H, H-2), 8.12 (d, 2H, J ) 8.8,
CH arom), 8.63 (s, 1H, H-8). 13C NMR (CDCl3) d ) 31.6, 45.7,
116.7, 120.7, 123.6, 126.7, 128.6, 128.8, 128.9, 129.6, 129.9,
130.8, 131.3, 133.6, 137.7, 143.6, 145.6, 147.0, 148.4, 151.6,
153.0. HRMS (ESI) m/z found 470.1272 [M + H]+, calcd
470.1287. Elem. anal. (C25H19N5O3S‚0.5EtOAc) C, H, N, S. C:
calcd. 63.14: found 62.34.
Ack n ow led gm en t. This work was financially sup-
ported by Gru¨nenthal GmbH.
Refer en ces
(1) Gray, J . H.; Owen, R. P.; Giacomini, K. M. The Concentrative
Nucleoside Transport Famliy, SLC28. Pflugers Arch. - Eur. J .
Physiol. 2004, 447, 728-734.
9-(3′-H yd r oxyb ip h en yl-2-ylm et h yl)-6-(4-n it r ob en zyl-
su lfa n yl)p u r in e (71). This compound was prepared from
62 according to general procedure B. Yield: 22%. White
solid. Mp: 206 °C. 1H NMR (DMSO-d6) δ ) 4.78 (s, 2H, CH2S),
5.44 (s, 2H, CH2N), 6.72-6.80 (m, 3H, CH arom), 6.98 (d, 1H,
J ) 6.6, CH arom), 7.18-7.37 (m, 4H, CH arom), 7.75 (d, 2H,
J ) 8.8, CH arom), 8.11 (s, 1H, H-2), 8.17 (d, 2H, J ) 8.8,
CH arom), 8.68 (s, 1H, H-8), 9.60 (br, s, 1H, PhOH). 13C
NMR (DMSO-d6) d ) 30.7, 45.0, 114.4, 115.8, 119.5, 123.6,
127.6, 127.8, 128.0, 129.5, 129.9, 130.2, 133.1, 141.0, 141.0,
144.9, 146.5, 148.6, 151.4, 157.4, 157.8. HRMS (ESI) m/z
found 470.1258 [M + H]+, calcd 470.1281. Elem. anal.
(C25H19N5O3S‚0.1CH2Cl2) C, H, N, S.
(2) 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. - Eur. J . Physiol. 2004, 447, 735-743.
(3) Hyde, R. J .; Cass, C. E.; Young, J . D.; Baldwin, S. A. The ENT
Family of Eukaryote Nucleoside and Nucleobase Transporters:
Recent Advances in the Investigation of Structure/Function
Relationships and the Identification of Novel Isoforms. Mol.
Membr. Biol. 2001, 18, 53-63.
(4) Young, J . D.; Cheeseman, C. I.; Mackey, J . R.; Cass, C. E.;
Baldwin, S. A. Molecular Mechanisms of Nucleoside and Nucleo-
side Drug Transport. Curr. Top. Membr. 2001, 50, 329-378.
(5) Baldwin, S. A.; Mackey, J . R.; Cass, C. E.; Young, J . D.
Nucleoside Transporters: Molecular Biology and Implications
for Therapeutic Developments. Mol. Med. Today 1999, 5, 216-
224.

5450 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Tromp et al.
(6) Mackey, J . R.; Baldwin, S. A.; Young, D. A.; Cass, C. E.
Nucleoside Transport and its Significance for the Anticancer
Drug Resistance. Drug Resist. Update 1998, 1, 310-324.
(7) Buolamwini, J . K. Nucleoside Transport Inhibitors: Structure-
Activity Relationships and Potential Therapeutic Applications.
Curr. Med. Chem. 1997, 4, 35-66.
(8) Sankar, N.; Machado, J .; Abdulla, P.; Hilliker, A. J .; Coe, I. R.
Comparative Genomic Analysis of Equilibrative Nucleoside
Transporters Suggest Conserved Protein Structure Despite
Limited Sequence Identity. Nucleic Acids Res. 2002, 30, 4339-
4350.
(20) For
a
recent review on aryl-aryl coupling see: Hassan, J .;
Se´vignon, M.; Gozzi, C.; Schulz, E.; Lemaire, M. Aryl-Aryl Bond
Formation one Century after the Discovery of the Ullmann
Reaction. Chem. Rev. 2002, 102, 1359-1469.
(21) Huang, C. G.; Wan, P. Photochemistry of Phenoxybenzyl Alcohols
in Aqueous Solution: Photosolvolysis vs Photorearrangement to
6H-Dibenzo[b,d]pyrans. J . Org. Chem. 1991, 56, 4846-4853.
(22) SPARTAN 5.0; Wavefunction, Inc., Irvine, CA92612; 1995.
(23) This value applies for the syn-conformation and is based on the
conformation of the crystal structure. See: Soriano-Garcia, M.;
Parthasarathy, R.; Paul, B.; Paterson, A. R. P. Structure and
Conformation of 6-(4-Nitrobenzyl)thioinosine, C17H17N5O6S, a
Potent Inhibitor of Nucleoside Transport. Acta Crystallogr. 1984,
C40, 1897-1901. The PSA value of the anti-conformation was
calculated to be 163 Å2.
(24) Allen, F. H. The Cambridge Structural Database: a Quarter of
a Million Crystal Structures and Rising. Acta Crystallogr. 2002,
B58, 380-388.
(25) PC Spartan Pro V1.0.2, Wavefunction, Inc., Irvine, CA92612;
1999.
(26) Robins, M. J .; Asakura, J . I.; Kaneko, M.; Shibuya, S.; J acobs,
E. S.; Agbanyo, F. R.; Cass, C. E.; Paterson, A. R. P. Synthesis
of Substituted-Benzyl and Sugar-Modified Analogues of 6-N-(4-
Nitrobenzyl)adenosine and their Interactions with “ES” Nucleo-
side Transport System. Nucleosides Nucleotides 1994, 13, 1627-
1646.
(27) A similar result was found for 2′-deoxy-NBTI, which gave a Ki
value of 0.29 ( 0.03 nM in our assay. Tromp, R. A.; von Frijtag
Drabbe Ku¨nzel, J . K.; IJ zerman, A. P. Unpublished results.
(28) Pu¨tz, C.; Sundermann, C.; Sundermann, B.; IJ zerman, A. P.;
Tromp, R.; von Frijtag Drabbe Ku¨nzel, J . New Analogs of
Nitrobenzylthioinosine. Patent WO 03/084875 A1; 2003.
(29) Wagner, G.; Oehle, J .; Pischel, H. Darstellung Kupplungsfa¨higer
6-Mercaptopurinederivative mit Isothiocyanatstruktur. Phar-
mazie 1974, 29, 160-164.
(30) J ohnston, T. P.; Holum, L. B.; Montgomery, J . A. Synthesis of
Potential Anticancer Agents. XVI. S-Substituted Derivatives of
6-Mercaptopurine. J . Am. Chem. Soc. 1958, 80, 6265-6271.
(31) Bandgar, B. P.; Sadavarte, V. S.; Uppalla, L. S. An Expedient
and Highly Selective Iodination of Alcohols using a KI/BF3‚Et2O
system. Tetrahedron Lett. 2001, 42, 951-953.
(32) IJ zerman, 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.
(9) Sawynok, J .; Liu, X. J . Adenosine in the Spinal Cord and
Periphery: Release and Regulation of Pain. Prog. Neurobiol.
2003, 69, 313-340.
(10) Dickenson, A. H.; Suzuki, R.; Reeve, A. J . Adenosine as a
Potential Analgesic Target in Inflammatory and Neuropathic
Pains. CNS Drugs 2000, 13, 77-85.
(11) Palm, K.; Stenberg, P.; Luthman, K.; Artursson, P. Polar Surface
Properties Predict the Intestinal Absorption of Drugs in Hu-
mans. Pharm. Res. 1997, 14, 568-571.
(12) Kelder, J .; Grootenhuis, P. D. J .; Bayada, D. M.; Delbressine, L.
P. C.; Ploemen, J . P. Polar Molecular Surface as a Dominating
Determinant for Oral Absorption and Brain Penetration of
Drugs. Pharm. Res. 1999, 16, 1514-1519.
(13) Clark, D. E. Rapid Calculation of Polar Molecular Surface Area
and its Application to the Prediction of Transport Phenomena.
1. Prediction of Intestinal Absorption. J . Pharm. Sci. 1999, 88,
807-814.
(14) van de Waterbeemd, H.; Camenisch, G.; Folkers, G.; Chretien,
J . R.; Raevsky, O. A. Estimation of Blood-Brain Barrier Crossing
of Drugs using Molecular Size and Shape, and H-Bonding
Descriptors. J . Drug Target. 1998, 6, 151-165.
(15) Paul, B.; Chen, M. F.; Paterson, A. R. P. Inhibitors of Nucleoside
Transport. A Structure-Activity Study using Human Erythro-
cytes. J . Med. Chem. 1975, 18, 968-973.
(16) Deghati, P. Y. F.; Borghini, A.; van den Nieuwendijk, A. M. C.
H.; Dissen-de Groote, M.; IJ zerman, A. P. Inhibition of Nucleo-
side Transport by New Analogues of Nitrobenzylthioinosine.
Bioorg. Med. Chem. 2003, 11, 899-908.
(17) Kline, T.; Fromhold, M.; McKennon, T. E.; Cai, S.; Treiberg, J .;
Ihle, N.; Sherman, D.; Schwan, W.; Hickey, M. J .; Warrener, P.;
Witte, P. R.; Brody, L. L.; Goltry, L.; Barker, L. M.; Anderson,
S. U.; Tanaka, S. K.; Shawar, R. M.; Nguyen, L. Y.; Langhorne,
M.; Bigelow, A.; Embuscado, L.; Naeemi, E. Antimicrobial Effects
of Novel Siderophores linked to â-Lactam Antibiotics. Bioorg.
Med. Chem. 2000, 8, 73-93.
(18) Stauffer, S. R.; Sun, J .; Katzenellenbogen, B. C.; Katzenellen-
bogen, J . A. Acyclic Amides as Estrogen Receptor Ligands:
Synthesis, Binding, Activity and Receptor Interaction. Bioorg.
Med. Chem. 2000, 8, 1293-1316.
(19) Castro-Pichel, J .; Garc´ıa-Lo´pez, M. T.; De las Heras, F. G. A
Facile Synthesis of Ascamycin and Related Analogues. Tetra-
hedron 1987, 43, 383-389.
(33) Bruno, I. J .; Cole, J . C.; Edgington, P. R.; Kessler, M.; Macrae,
C. F.; McCabe, P.; Pearson, J .; Taylor, R. New Software for
Searching the Cambridge Structural Database and Visualising
Crystal Structures. Acta Crystallogr. 2002, B58, 389-397.
J M049735V