Structure-activity relationships of 7-deaza-6-benzylthioinosine analogues as ligands of Toxoplasma gondii adenosine kinase

Article abstract of DOI:10.1021/jm800201s

Several 7-deaza-6-benzylthioinosine analogues with varied substituents on aromatic ring were synthesized and evaluated against Toxoplasma gondii adenosine kinase (EC.2.7.1.20). Structure-activity relationships indicated that the nitrogen atom at the 7-position does not appear to be a critical structural requirement. Molecular modeling reveals that the 7-deazapurine motif provided flexibility to the 6-benzylthio group as a result of the absence of H-bonding between N7 and Thr140. This flexibility allowed better fitting of the 6-benzylthio group into the hydrophobic pocket of the enzyme at the 6-position. In general, single substitutions at the para or meta position enhanced binding. On the other hand, single substitutions at the ortho position led to the loss of binding affinity. The most potent compounds, 7-deaza-p-cyano-6- benzylthioinosine (IC₅₀ = 5.3 μM) and 7-deaza-p-methoxy-6- benzylthioinosine (IC₅₀ = 4.6 μM), were evaluated in cell culture to delineate their selective toxicity.

Full text of DOI:10.1021/jm800201s

3934
J. Med. Chem. 2008, 51, 3934–3945
Structure-Activity Relationships of 7-Deaza-6-benzylthioinosine Analogues as Ligands of
Toxoplasma gondii Adenosine Kinase
Young Ah Kim,† Ashoke Sharon,† Chung K. Chu,*,† Reem H. Rais,‡ Omar N. Al Safarjalani,‡ Fardos N. M. Naguib,‡ and
Mahmoud H. el Kouni‡
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, UniVersity of Georgia, Athens, Georgia 30602, and Department
of Pharmacology and Toxicology, Center for AIDS Research, UniVersity of Alabama School of Medicine, Birmingham, Alabama 35294
ReceiVed February 25, 2008
Several 7-deaza-6-benzylthioinosine analogues with varied substituents on aromatic ring were synthesized
and evaluated against Toxoplasma gondii adenosine kinase (EC.2.7.1.20). Structure-activity relationships
indicated that the nitrogen atom at the 7-position does not appear to be a critical structural requirement.
Molecular modeling reveals that the 7-deazapurine motif provided flexibility to the 6-benzylthio group as
a result of the absence of H-bonding between N7 and Thr140. This flexibility allowed better fitting of the
6-benzylthio group into the hydrophobic pocket of the enzyme at the 6-position. In general, single substitutions
at the para or meta position enhanced binding. On the other hand, single substitutions at the ortho position
led to the loss of binding affinity. The most potent compounds, 7-deaza-p-cyano-6-benzylthioinosine (IC50
) 5.3 µM) and 7-deaza-p-methoxy-6-benzylthioinosine (IC50 ) 4.6 µM), were evaluated in cell culture to
delineate their selective toxicity.
Introduction
thus depend on the salvage pathways for their purine require-
ments. Specifically, the activity of adenosine kinase in T. gondii
is 10-fold higher than those of other enzymes in the purine
salvage pathways. Therefore, T. gondii adenosine kinase
contributes significantly to the survival of the parasite.20
However, deficiency of adenosine kinase was shown not to be
lethal to the parasites, indicating that inhibition of the enzyme
will not lead to toxicity in toxoplasma. Structure-activity
relationships, comparative enzymatic, metabolic, and X-ray
structural studies of T. gondii adenosine kinase have identified
subversive substrates that are selectively metabolized to cyto-
toxic nucleotides by the T. gondii but not human adenosine
kinase.19–30
Toxoplasma gondii is an obligate intracellular protozoan
parasite belonging to the phylum Apicomplexa. T. gondii infects
a wide range of hosts, including human and warm-blooded
animals.1–3 Toxoplasmic infection in human is estimated to
affect more than a billion individuals worldwide, resulting in
an important public health problem.4,5 T. gondii infection causes
morbidity and mortality in immunocompromised individuals
such as organ transplant, leukemia, and AIDS patients, as well
as congenitally infected children. In immunocompromised
patients, reactivation of chronic T. gondii infection can cause a
severe central nervous system disease, and thus, toxoplasmic
encephalitis and focal neurological lesions are the most common
clinical manifestations.6,7 Infection acquired during pregnancy
can lead to congenital toxoplasmosis of the fetus, which results
in abortion, neonatal death, or fetal abnormalities that may be
present at birth or may develop later in life.8,9
At the present time, there is no vaccine available to prevent
human infection with this class of pathogens.10,11 Antifolate
drugs, such as the combination of pyrimethamine and sulfadi-
azine, are currently used for the treatment of toxoplasmic
infection.12–15 This therapy, however, is ineffective against
toxoplasma tissue cysts6,16 and causes side effects such as bone
marrow depression and skin rashes.15,17 In order to reduce
undesirable side effects, folic acid and spiramycin are used as
alternative regimen of sulfa drugs.11,18 However, the emergence
of drug resistance and complications associated with long-term
treatment of toxoplasmosis warrant the need for the development
of new and effective chemotherapeutic agents.
We have previously reported that 6-benzylthioinosine
analogues19,20,22,24–26 are selective subversive substrates for the
T. gondii, but not host, adenosine kinase. Certain 6-benzylth-
ioinosine analogues showed potent antitoxoplasma activity in
cell culture as well as in mouse infection model,19,22,25
demonstrating the potential usefulness of these classes of
compounds in the treatment of toxoplasmosis. Furthermore, the
6-benzylthioinosine analogues act as inhibitors of the mam-
malian nucleoside transporter ENT1 (es) in host cells,26 thus
elucidating the basis for the lack of uptake of these compounds
by uninfected host cells.23 Several N6-benzyladenosine ana-
logues also showed appreciable antiparasitic activity, however;
they exhibited host toxicity.27 In light of these findings,19,22,24–27
we have chosen the 6-benzylthio group as a more suitable
scaffold for further development of purine nucleoside analogues
as antitoxoplasmic agents.
Recently, T. gondii adenosine kinase (EC.2.7.1.20) has been
investigated as an attractive chemotherapeutic target for the
development of antitoxoplasmic agents.19,20 Several differences
between T. gondii and their host are found in purine metabolism.
Unlike their host, T. gondii lack de novo purine synthesis and
In our continuing effort to develop potent and selective
antitoxoplasmic agents, our interest has focused on base-
modified 7-deazapurine (pyrrolo[2,3-d]pyrimidine) nucleosides
in view of their significant biological properties, including
antiparasitic as well as antiviral, antimicrobial, and anticancer
activities.31–34 The base-modified nucleosides are synthetically
challenging and can generate new classes of chemotherapeutic
agents. 7-Deazapurine is an ideal mimic of purine in which the
nitrogen atom at the 7-position is replaced by a carbon atom.
* To whom correspondence should be addressed. Phone: (706) 542-5379.
Fax: (706) 542-5381. E-mail: dchu@rx.uga.edu.
†
University of Georgia.
University of Alabama School of Medicine.
‡
10.1021/jm800201s CCC: $40.75
2008 American Chemical Society
Published on Web 06/19/2008

7-Deaza-6-benzylthioinosine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3935
Scheme 1a
a Reagents and conditions: (a) 2,2-dimethoxypropane, p-TsOH, acetone, 0 °C to room temp, 1 h, 81%; (b) TBDMSCl, imidazole, CH2Cl2, room temp,
2 h, 83%; (c) CCl4, HMPT, THF, -30 °C, 1 h; (d) 6-chloro-7-deazapurine, KOH, TDA-1, THF, 16 h, 40% (two steps from 3); (e) Dowex 50WX8-200,
MeOH, room temp, 8 h, 95%; (f) appropriate benzylthiol, NaH, THF, room temp, 1 h.
Scheme 2a
a Reagents and conditions: (a) thiourea, EtOH, reflux, 3 h, 85%; (b) appropriate benzylhalide, NH4OH/H2O, room temp, 11 h.
Hence, the altered character of the heterocycle might contribute
to differential interactions at the binding site of T. gondii
adenosine kinase. Therefore, the main goal of the present studies
is to investigate whether or not the elimination of the N7 of
inosine will enhance the binding of 6-benzylthioinosines to T.
gondii adenosine kinase. A better understanding of substrate
analogues binding would aid in the rational design of drugs that
maintain activity while decreasing toxicity. Herein, we report
the synthesis of novel 7-deaza-6-benzylthioinosine analogues
and the structure-activity relationships of these analogues as
ligands of T. gondii adenosine kinase.
The phase-transfer catalyst TDA-1 increased the nucleophilicity
of the 6-chloro-7-deazapurine anion by forming a complex with
a potassium cation as reported in the literature.35 The tert-
butyldimethylsilyl and isopropylidene groups of compound 4
were removed with Dowex 50WX8-200 in methanol to give
the ꢀ-anomer 5a and R-anomer 5b in good yield. In this step,
the anomeric mixture was readily separated by silica gel column
chromatography (ꢀ/R ) 10:1, 95% yield). Finally, variation of
the key intermediate 5a was accomplished by treatment with
the appropriate benzylthiols in the presence of sodium hydride
at room temperature to give the corresponding 7-deaza-6-
benzylthioinosine analogue 6 in good yields. Some of the
substituted benzylthiols, which are not commercially available,
have been prepared according to the reported procedures.38
Unexpectedly, the yield of 4-nitrobenzyl analogue 6l was low
(∼10%) because of decomposition under this condition. We
found that the use of thione 7 as an intermediate resulted in
improved yield (Scheme 2). Treatment of triol 5a with thiourea
in ethanol gave compound 7, which was alkylated with
4-nitrobenzyl bromide39 to provide compound 6l in 76% yield.
In a similar reaction, we obtained a good result in the
transformation of the intermediate 7 to compounds 6q and 6t.
Synthesis of compound 12 was accomplished as shown in
Scheme 3. The commercially available 6-chloro-7-deazapurine
8 could be directly iodinated with N-iodosuccinimide in DMF
at the C7-position to give the 7-iodinated compound 9 utilizing
a known procedure.40 Compound 9 was coupled with 1-chloro-
2,3-O-isopropylidene-5-O-tert-butyldimethylsilyl-R-D-ribofura-
nose freshly prepared from compound 3 to give iodinated
Results and Discussion
Chemistry. Synthesis of 7-deaza-6-benzylthioinosine ana-
logues 6a-w is described in Schemes 1 and 2. D-Ribose was
converted to the isopropylidene derivative 2 with 2,2-dimetho-
xypropane in the presence of catalytic amounts of p-toluene-
sulfonic acid in 81% yield, followed by the protection with tert-
butyldimethylsilyl chloride that gave compound 3 in 83% yield.
Stereospecific glycosylation of the protected lactol 3 with
6-chloro-7-deazapurine was conducted via an R-chloro sugar
intermediate.35–37 Compound 3 was reacted with carbon tetra-
chloride and hexamethylphosphorus triamide (HMPT) in tet-
rahydrofuran at -30 °C to generate in situ 1-chloro-2,3-O-
isopropylidene-5-O-tert-butyldimethylsilyl-R-D-ribofuranose. The
resulting ribofuranosyl chloride was coupled with the potassium
salt of 6-chloro-7-deazapurine in the presence of tris[2-(2-
methoxyethoxy)ethyl]amine (TDA-1) at room temperature to
give compound 4 as an R/ꢀ mixture in 40% yield in two steps.

3936 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Kim et al.
Scheme 3a
a Reagents and conditions: (a) NIS, DMF, room temp, 2 h, 93%; (b) CCl4, HMPT, THF, -30 °C, 1 h; (c) 9, KOH, TDA-1, THF, 16 h, 44% (two steps
from 3); (d) Dowex 50WX8-200, MeOH, room temp, 8 h, 93%; (e) benzylthiol, NaH, THF, room temp, 1 h, 84%.
Table 1. Binding Affinities (Apparent Ki) of
7-Deaza-6-benzylthioinosine Analogues (6a and 12) and
6-Benzylthioinosine to Toxoplasma gondii Adenosine Kinase
the enzyme (82.6 µM) than 7-deaza-6-benzylthioinosine (6a,
27.9 µM). The decrease in binding affinity of 7-iodo-7-deaza-
6-benzylthioinosine (12) appears to be due to a poor interaction
and ligand-fit profile in the enzyme active site, as will be
discussed below.
Structure-Activity Relationships of Various Substitu-
ents on the Aromatic Ring. Structure-activity relationships
were performed by varying substituents on the aromatic ring
of 6a. The binding affinities of the 7-deaza-6-benzylthioinosine
analogues 6a-w to T. gondii adenosine kinase were compared
with that of the natural substrate adenosine (Table 2). The
a
compd
X
Y
Ki (µM)
binding affinities of compounds 6h (m-methyl, 8.5 µM), 6j (p-
chloro, 11 µM), 6m (p-cyano, 5.2 µM), 6n (p-methoxycarbonyl,
7.0 µM), 6s (p-methoxy, 5.8 µM), and 6w (o-chloro-o-fluoro,
7.2 µM) to T. gondii adenosine kinase were better than the
unsubstituted compound 6a (28 µM). Compounds 6m (p-cyano,
5.2 µM) and 6s (p-methoxy, 5.8 µM) were relatively bettrer
than adenosine (8.9 µM).
6a
12
C
C
N
H
I
28 ( 8.6
82.6 ( 4.2
37.7 ( 3.6
6-benzylthioinosine
a All values represent the mean ( SD of at least two independent
experiments with three replications each as previously described.24,25,27 Since
these compounds are alternative substrates of T. gondii adenosine kinase,
their apparent Ki values are equal to their apparent Km values.48
In general, single substitutions at the ortho position (6b-e)
led to the loss in binding affinity when compared to the
unsubstituted compound 6a. The weakest ligands in this series
were 6e (o-methyl, 227.9 µM) and 6k (p-bromo, 252.7 µM).
Notably, when a methyl group at the ortho position (6e, 227.9
µM) was moved to the meta (6h, 8.5 µM) or the para (6o, 18.5
µM) position, a substantial increase in the binding affinity was
observed. At the meta position, an electron-donating substituent
(6h, 8.5 µM) resulted in a better binding affinity than an
electron-withdrawing substituent (6f, 44.6 µM and 6g, 22.4 µM).
At the para position, a chlorine substitution (6j, 11.3 µM)
exhibited somewhat increased binding affinity in comparison
to a fluorine substitution (6i, 36.7 µM). Interestingly, the
bromine substitution (6k, 252.7 µM) displayed a dramatic
decrease in binding affinity. A similar trend was also observed
for halogen substitutions in the ortho-substituted compounds
6b-d, and the order of the potency was Cl > F . Br. In
addition to electronic effects, it appears that the relative size of
the halogen substituents might also play a role in how they were
accommodated in the active site of T. gondii adenosine kinase.
Compounds with a para substituent of similar size but different
electoronegativity [e.g., 6r (p-trifluoromethoxy 35.0 µM) vs 6s
(p-methoxy, 5.8 µM)] exhibited significantely different binding
affinities. Compounds with different size but similar electrone-
gativity [e.g., 6r (p-trifluoromethoxy, 35.0 µM) and 6m (p-
cyano, 5.2 µM)] also showed significant difference in binding
affinity. In fact, the modeling results suggest the different
binding mode of 6r (p-trifluoromethoxy) in comparison to 6s
ribonucleoside 10. In this case, the formation of R-anomer was
negligible. Subsequently, compound 10 was treated with Dowex
50WX8-200 in methanol at room temperature to afford com-
pound 11 in good yield followed by the nucleophilic displace-
ment reaction gave 7-iodo-7-deaza-6-benzylthioinosine 12 in
84% yield.
Effects of Structural Modification at the 7-Position of
the Purine Ring on the Binding Affinity of 6-Benzylthioi-
nosine. Previous structure-activity relationship21,24,27 and
crystal structure28 studies demonstrated that two hydrophobic
pockets exist on the T. gondii adenosine kinase around the C6
and N7 positions of the purine ring. The hydrophobic pocket at
N7-position is smaller than that at C6-position.24 Therefore, we
wanted to examine the effects of eliminating the N7 of inosine,
and the role of substitutions at the C6 and C7 positions on the
binding affinity of the synthesized compounds.
Table 1 shows that the binding affinity (apparent Ki values)
of 7-deaza-6-benzylthioinosine (6a, 27.9 µM) was relatively
better than 6-benzylthioinosine (37.7 µM). This finding indicated
that the nitrogen atom at the 7-position does not appear to be a
critical structural requirement for binding to T. gondii adenosine
kinase. Furthermore, since 7-iodo-7-deazaadenosine (7-iodotu-
bercidin) is a better ligand than adenosine for T. gondii
adenosine kinase,21 we were prompted to explore the effects of
adding the iodo substitution at the C7-position of 7-deaza-6-
benzylthioinosine (6a). Unexpectedly, however, 7-iodo-7-deaza-
6-benzylthioinosine (12) exhibited a lesser binding affinity to

7-Deaza-6-benzylthioinosine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3937
Table 2. Binding Affinities (Apparent Ki) of
7-Deaza-6-benzylthioinosine Analogues 6a-w and Adenosine to
Toxoplasma gondii Adenosine Kinase
Table 3. Effect of 7-Deaza-6-benzylthioinosine Analogues (6m and 6s)
and Therapeutic Compounds on Host Toxicitya and Percent Survivalb of
Wild Type (RH) and Adenosine Kinase Deficient (TgAK-3) Strains of
Toxoplasma gondii Grown in Human Fibroblasts in Culture
concentration (µM)
compd
infection
0
5
10
4.0
25
0.0
50 IC50 (µM)
0.0 5.3 ( 0.6
6m (p-CN)
wild type 100 52.5
(RH)
TgAK-3 100 98.4 100 100 100
nonea
100 100 100
94.3 82.1
0.0 0.0 4.6 ( 0.2
6s (p-OCH3)
wild type 100 49.8
(RH)
5.0
TgAK-3 100 100 100 100 100
nonea
100 100 100 100
90.6
5.8 5.9 ( 0.8
a
compd
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
R1
R2
R3
R4
R5
Ki (µM)
pyrimethaminec wild type 100 55.6 36.5 13.8
(RH)
H
F
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
28 ( 8.6
59 ( 6.1
36 ( 5.7
106 ( 17
228 ( 49
45 ( 8.9
22 ( 4.0
8.5 ( 1.4
37 ( 14
11 ( 1.1
253 ( 56
35 ( 9.5
5.2 ( 1.1
7.0 ( 1.3
19 ( 3.9
24 ( 2.1
180 ( 30
35 ( 14
nonea
100 101 100 108 108
sulfadiazinec
wild type 100 74.6 64.8 59.8 46.4 41.2 ( 1.9
Cl
Br
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
(RH)
nonea
100 98.2 99.8 99.8 102
H
NO2
CF3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
a Host toxicity of uninfected cells was measured by MTT method in at
least two independent experiments each with three replications as previously
described.22–25,27 b Percent survival of parasites was measured by incorpora-
tion of [5,6-13H]uracil in at least two independent experiments with three
replications each as previously described.22–25,27 c Therapeutic compounds.
Cl
Br
NO2
CN
CO2CH3
CH3
tBu
CHdCH2
OCF3
OCH3
F
one of the best ways to influence the binding affinity for T.
gondii adenosine kinase as indicated by previous studies.24,27
Evaluation of Antitoxoplasma Activity. The two best
ligands, 7-deaza-p-cyano-6-benzylthioinosine (6m) and 7-deaza-
p-methoxy-6-benzylthioinosine (6s), were tested for their anti-
toxoplasma efficacy and host toxicity. Their IC50 values were
compared to those obtained from the clinically used drugs
pyrimethamine and sulfadiazine (Table 3). The two analogues
were effective against infection with the wild type (RH) T.
gondii in a dose-dependent manner but not against infection
with the adenosine kinase deficient (TgAK-3) strain. In agree-
ment with their binding affinities (Table 2), 6m and 6s showed
potent cellular activity with IC50 values of 5.3 and 4.6 µM,
respectively. Furthermore, 6m and 6s exhibited close to or better
potency than pyrimethamine (IC50 ) 5.9 µM) and sulfadiazine
(IC50 ) 41.2 µM). The lack of activity against infection with
the adenosine kinase deficient (TgAK-3) strain of T. gondii
demonstrates that these compounds are active substrates for T.
gondii adenosine kinase in vivo as was the case in vitro (Table
2). It is also noted that 6m and 6s had no detectable host cell
toxicity at concentrations up to 50 µM, in spite of the absence
of the nitrogen atom in the 7-position (Table 3). This is
noteworthy, since 7-iodo-7-deazaadenosine (7-iodotubercidin)
is an extremely toxic22 and nonselective ligand of T. gondii
adenosine kinase.19,20,22
6s
6t
5.8 ( 1.0
16 ( 3.9
26 ( 7.6
29 ( 11
7.2 ( 1.3
8.9 ( 3.2
6u
6v
6w
adenosine
Cl
H
F
Cl
Cl
H
a All values represent the mean ( SD of at least two independent
experiments with three replications each as previously described.24,25,27 Since
these compounds are alternative substrates of T. gondii adenosine kinase,
their apparent Ki values are equal to their apparent Km values.48
(p-methoxy), in which the p-trifluormethoxy group of 6r orients
away from the hydrophobic pocket in comparison to 6s
(p-methoxy). These results suggest that the binding affinity to
T. gondii adenosine kinase is affected by both the electronic
and the size of the para substituents. Previously, similar
observation was made from the study of 6-benzylthioinosine
analogues.24 The best result was achieved when a cyano group
(6m, 5.2 µM) or a methoxy group (6s, 5.8 µM) was added to
the para position. In the case of disubstituted analogues, 6t-w,
di-ortho substitutions (6w, 7.2 µM) showed increased binding
affinity in comparison to the ortho-para disubstitutions (6t, 16.0
µM and 6u, 25.8 µM) or the meta-para disubstitutions (6v,
28.7 µM). Furthermore, the binding affinities of dihalogen
derivatives (6t-w) were relatively better than the mono
derivatives (6b, 59 µM and 6c, 36 µM). Compounds with fluoro
substituents (6t, 16.0 µM and 6w, 7.2 µM) were the best ligands
among the disubstituted derivatives.
These results indicate that the incorporation of the 7-dea-
zapurine motif led to improvement in the binding affinity of
the 6-benzylthioinosines. Furthermore, the functionality of the
6-benzylthio group on the 7-deazapurine moiety was extended
by the addition of substituents such as p-cyano (6m) and
p-methoxy (6s). Such substituents provided additional interac-
tions with the surrounding residues in the binding pocket of T.
gondii adenosine kinase and were conducive to enhancing the
binding affinity of the 7-deaza-6-benzylthioinosines. The present
results also confirm that this structural optimization process is
These results demonstrated that 7-deaza-6-benzylthioinosine
analogues are selectively activated by T. gondii, but not human,
adenosine kinase, which leads to the selective death of the
parasite. These findings also implied that the absence of the
nitrogen atom in the 7-position does not play a significant role
in the antitoxoplasma effects of the 6-benzylthioinosines. These
studies provided useful information to define the structural
feature required for the potency and selectivity of antitoxoplas-
mic agents and to guide further design of such agents.
Molecular Modeling. T. gondii adenosine kinase consists
of 363 residues (39.3 kDa).41 It catalyzes the phosphorylation
of adenosine to adenosine 5′-monophosphate (AMP), using the
γ-phosphate of ATP. The overall enzyme structure28 including
recent studies30 suggested an induced fit enzyme movement from
open conformation to closed conformation for effective phos-
phate transfer. Our recent induced fit molecular modeling studies

3938 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Kim et al.
Figure 1. (a) Binding mode of 7-deaza-6-benzylthioinosine (6a), showing superposition with 6-benzylthioinosine binding mode. Rigid conformation
was observed in 6-benzylthioinosine because of H-bonding with Thr140, and it enforces Leu46 (red) to adopt away-orientation in comparison to
Leu46 (green) in the 7-deaza-6-benzylthioinosine complex. (b) Binding mode of 7-deaza-p-cyano-6-benzylthioinosine (6m), showing significant
favorable orientation and position with proper intermolecular interactions (H-bonding with Asp24).
highlighted the molecular basis for the binding of various
6-substituted 9-ꢀ-D-ribofuranosylpurines in the active site of T.
gondii adenosine kinase.24,27 Our earlier structure-activity
relationship and molecular modeling studies21,24,27 suggested
that the position of the ribose ring is mediated by interaction
with hydrophilic residues (Asp24, Gly69, and Asn73) while the
purine ring orientation is governed by hydrophobic interactions
mediated by Tyr169 and Ile22 through a stacking interaction
by π-π and C-H-π, respectively. These studies also dem-
onstrated that substituted benzyl moieties at the 6-position of
the purine ring further enhance binding to T. gondii adenosine
kinase and their potential as antitoxoplasmic agents. Moreover,
the degree of binding of these compounds is influenced by the
type and position of substituent on the benzyl group. In the
present molecular modeling studies we investigated the underly-
ing molecular basis of the effects of eliminating the N7 from
6-benzylthioinosine analogues, as well as the role of substituents
at the 7-position and the benzyl group on the binding of these
compounds to the active site of T. gondii adenosine kinase.
The 7-deaza-6-benzylthioinosine analogues docked success-
fully into the T. gondii adenosine kinase active site by the
induced fit method.24,27 The binding mode showed an anti
conformation of the nucleoside with an 2′-endo puckered ribose
conformation. The benzylthiol moiety occupied the hydrophobic
pocket at the 6-position and was involved in van der Waals
interaction with the surrounding residues such as Leu46, Phe201,
Tyr206, Thr172, and Leu142. These residues played a crucial
role in terms of the binding affinity in response to different
atomic substituents on the 6-benzyl group and ultimately defined
the binding affinities of these compounds (Table 2).
Table 4. MM-GBSA Free Energy Calculation42 of 7-Deaza-6-
benzylthioinosine Analogues (6a and 12) and 6-Benzylthioinosine
MM-GBSA energy difference results
ligand energy
in complex
receptor strain
energy
compd
total
6a
12
-38.7
-25.06
-36.52
5.8
11.0
7.8
-53.1
-52.7
-62.4
6-benzylthioinosine
utilize the hydrophobic pocket at the 6-position for better binding
than their 6-benzylthioinosine counterparts. It should also be
noted that the introduction of the iodine atom at the C7-position
did not improve the binding affinity of 7-iodo-7-deaza-6-
benzylthioinosine (12). It seems likely that the decrease in the
binding affinity of compound 12 is due to poor interaction and
ligand-fit profile in the enzyme active site. Binding mode studies
suggest that the hydrophobic pockets at the C6 and C7 positions
are shared by Leu46. The present studies indicated that the
interaction of Leu46 with the iodine atom at the C7-position
was not allowed by the presence of the benzylthio group at the
C6-position. Hence, the binding affinity of 7-iodo-7-deaza-6-
benzylthioinosine (12) is decreased when compared with
7-deaza-6-benzylthioinosine (6a) (Table 1). These findings were
supported further by free energy calculations using the MM-
GBSA approach (molecular mechanics energy with an SGB
solvation model),42 which showed significant differences in
terms of the strain energy between 7-iodo-7-deaza-6-benzylth-
ioinosine (12) and 7-deaza-6-benzylthioinosine (6a) (Table 4).
Ligand strain energy and receptor strain energy for 12 were
higher than those for 7-deaza-6-benzylthioinosine (6a) (Table
4), and this clearly suggests that accommodation of 7-iodo-7-
deaza-6-benzylthioinosine (12) in the active site of T. gondii
adenosine kinase is relatively unfavorable when compared to
that of 7-deaza-6-benzylthioinosine (6a).
Molecular modeling studies of the binding mode of 7-deaza-
6-benzylthioinosine (6a) revealed the absence of H-bonding with
Thr140 (Figure 1a) and a lack of movement of the 7-deazapurine
ring toward Thr140, which were found to be present in the case
of 6-benzylthioinosine binding. The replacement of N7 with
C7 in 7-deaza-6-benzylthioinosine (6a) caused the loss of the
electrostatic interaction between the 7-postion and Thr140,
observed in 6-benzylthioinosine binding. The absence of H-
bonding with Thr140 allowed flexibility in the movement of
the bulky 6-benzylthio group to fit better in the hydrophobic
pocket at the 6-position. Therefore, it can be concluded that
7-deaza-6-benzylthioinosine analogues are more appropriate to
The binding affinity of 6a (27.9 µM) was greatly increased
by the addition of the proper substituent on the 6-benzyl group.
Figure 1b shows the significant binding mode of 7-deaza-p-
cyano-6-benzylthioinosine (6m), one of the two best ligands
(5.2 µM), where the 2′- and 3′-OH formed H-bonding with
Asp24 and the p-cyanobenzyl group occupied a favorable
position and proper orientation in the hydrophobic pocket. Figure

7-Deaza-6-benzylthioinosine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3939
Figure 2. (a) Binding mode of 7-deaza-o-methyl-6-benzylthioinosine (6e) showing the o-methyl orientation, which is away from the hydrophobic
pocket and is contributing toward unfavorable binding. (b) Binding mode of 7-deaza-p-bromo-6-benzylthioinosine (6k) superimposed with 7-deaza-
p-cyano-6-benzylthioinosine (6m, red) showing unfavorable orientation of 7-deazapurine position and 6-benzylthio group of 6k in the hydrophobic
pocket.
Table 5. MBAE (Multiligand Bimolecular Association with Energetics)
Calculation of 7-Deaza-6-benzylthioinosine Analogues after Induced Fit
Docking and Energy Minimization
kinase. 7-Deaza-p-cyano-6-benzylthioinosine (6m) and 7-deaza-
p-methoxy-6-benzylthioinosine (6s) were the best ligands and
exhibited better binding affinity than the natural substrate
adenosine. Structure-activity relationships indicated that the
presence of a nitrogen at the 7-position is not an essential
structural requirement for binding, since 7-deaza-6-benzylth-
ioinosine analogues have higher binding affinities than their
corresponding 6-benzylthioinosine counterparts. Molecular mod-
eling demonstrated that 7-deaza-6-benzylthioinosine analogues
do not form H-bonding with Thr140 which provides flexibility
in the movement of the benzylthio group for better fitting in
the 6-position hydrophobic pocket. Furthermore, 7-deaza-6-
benzylthioinosine analogues with certain substituents on the
benzyl group may better utilize the 6-position hydrophobic
pocket for binding. Ligands with high affinity to the enzyme,
6m and 6s, displayed potent antitoxoplasmic activity in cell
culture with IC50 values of 5.3 and 4.6 µM, respectively, without
significant host cell toxicity. The present studies provide
important insights into the structual basis for substrate specificity
of T. gondii adenosine kinase and provide direction for further
development of potent and less cytotoxic antitoxoplasmic agents.
energy difference results (∆E, kJ/mol)
compd
Ki [µM]
electrostatic
vdWa
total
6a
28 ( 8.6
228 ( 49.4
253 ( 56
5.2 ( 1.1
-244.7
-363.8
-399.3
-291.9
-363.3
-414.1
-244.7
-14.7
2.7
-54.2
-36.6
-75.5
-63.6
-70.6
-60.1
-84.9
6e (o-CH3)
6k (p-Br)
6m (p-CN)
6n (p-CO2CH3) 7.0 ( 1.3
6s (p-OCH3)
-9.5
-53.8
-33.9
-5.6
-14.7
5.8 ( 1.0
7.2 ( 1.3
6w (o-Cl-o-F)
a van der Waals interaction.
2 shows the unfavorable orientation of substituted 6-benzylthio
group of 7-deaza-o-methyl-6-benzylthioinosine (6e) and 7-deaza-
p-bromo-6-benzylthioinosine (6k) into the hydrophobic pocket,
which resulted in the poor binding of these compounds. The
6-benzylthio group of compound 6k was oriented away from
the hydrophobic pocket, and the overall conformation of 6k was
unsuitable in terms of its position in the active site in comparison
with 6m as shown in Figure 2b. The van der Waals binding
energy difference of 6k (-9.5 kJ/mol) was very low in
comparison to 6m (-53.8 kJ/mol), which shows that unfavor-
able interactions were mediated by the p-bromobenzyl group
in the hydrophobic pocket. Energetic correlations of a few
representative compounds are shown in Table 5 and reveal a
qualitative comparison with respect to experimental data. Table
5 clearly shows the favorable binding energy differences of 6m,
6n, 6s, and 6w and the relatively poor binding energy of 6a
and 6e. These molecular modeling studies suggest that the proper
substitution on the 6-benzylthio group renders the compound
active or inactive because of its conformational orientation and
position in the 6-position hydrophobic pocket. In addition, proper
substitution on the 6-benzylthio group affects the overall
conformation of the analogue to fit into the active site of T.
gondii adenosine kinase.
Experimental Section
General. Melting points were determined on a Mel-Temp II
apparatus and are uncorrected. Optical rotation was determined on
a Jasco DIP-370 digital polarimeter. UV spectra were obtained on
a Beckman DU-650 spectrophotometer. NMR spectra were recorded
on a Varian Inova 500 spectrometer, and chemical shifts are
expressed in parts per million (δ) relative to tetramethylsilane as
internal reference. Chemical shifts (δ) are reported as s (singlet), d
(doublet), t (triplet), q (quarter), or m (multiplet). TLC was
performed on Uniplates (silica gel) purchased from Analtech Co.
Silica gel 60 (220-440 mesh) was used for flash column chroma-
tography. 6-Chloro-7-deazapurine was purchased from Toronto
Research Chemicals, Inc. Elemental analyses were performed by
Atlantic Microlab Inc., Norcross, GA.
6-Hydroxymethyl-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-
4-ol (2). A solution of D-ribose (40.0 g, 0.266 mol) in acetone (133.0
mL) was treated with p-toluenesulfonic acid (1.90 g, 13.3 mmol)
In summary, 7-deaza-6-benzylthioinosine analogues were
synthesized and evaluated as ligands of T. gondii adenosine

3940 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Kim et al.
and 2,2-dimethoxypropane (49.0 mL, 400 mol). The reaction
mixture was stirred for 1 h at room temperature and then neutralized
with solid NaHCO3 (1.11 g, 13.3 mmol). The suspension was
filtered, and the filtrate was concentrated in vacuo. The residue was
purified by column chromatography on silica gel (n-hexane/EtOAc
) 3:1 to 1:1) to give anomeric mixture 2 (41.0 g, 81%) as a
colorless oil: 1H NMR (500 MHz, CDCl3) for ꢀ-isomer δ 1.33 (s,
3H), 1.49 (s, 3H), 3.54 (m, 1H), 3.73 (m, 1H), 3.75 (d, J ) 2.0 Hz,
1H), 4.42 (m, 1H), 4.59 (d, J ) 6.5 Hz, 1H), 4.68 (m, 1H), 4.85
(d, J ) 6.0 Hz, 1H), 5.43 (m, 1H); for R-isomer (diagnostic peaks
only) δ 1.40 (s, 3H), 1.58 (s, 3H), 4.06 (m, 1H), 4.19 (m, 1H),
4.64 (m, 1H), 4.75 (m, 1H), 3.78 (d, J ) 2.0 Hz, 1H), 3.71 (m,
1H); 13C NMR (125 MHz, CDCl3) for ꢀ-isomer δ 24.7, 26.3, 63.4,
81.6, 86.7, 87.6, 102.7, 112.2.
(125 MHz, CD3OD) δ 61.8, 70.9, 74.6, 85.6, 89.0, 99.7, 118.4,
128.6, 150.0, 151.2, 151.5; HRMS [M + H]+ m/z calcd for
C11H13ClN3O4 286.0594, found 286.0594. 5b: UV (MeOH) λmax
1
273.0 nm; H NMR (500 MHz, CD3OD) δ 3.71 (dd, J1 ) 12.5
Hz, J2 ) 4.0 Hz, 1H), 3.86 (dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H),
4.31 (m, 1H), 4.37 (d, J ) 5.8 Hz, 1H), 4.50 (t, J ) 5.0 Hz, 1H),
6.69 (d, J ) 4.0 Hz, 1H), 6.75 (d, J ) 5.0 Hz, 1H), 7.97 (d, J )
4.0 Hz, 1H), 8.59 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 61.6,
70.9, 71.6, 84.6, 85.0, 98.4, 117.7, 130.7, 149.7, 150.9, 151.2;
HRMS [M + H]+ m/z calcd for C11H13ClN3O4 286.0594, found
286.0602.
General Procedure for the Compounds 6a-k,m-p,r,s,u-w.
To a solution of compound 5a (65.0 mg, 0.228 mmol) in anhydrous
THF (5.7 mL) were added the appropriate benzylthiol (0.912 mmol)
and sodium hydride (0.456 mmol) at 0 °C. The reaction mixture
was allowed to warm to room temperature and kept for 1 h. Excess
hydride was quenched by addition of methanol, and then the
reaction mixture was concentrated in vacuo. The residues were
purified by short column chromatography on silica gel (CH2Cl2/
MeOH ) 95:5) to give the desired products as white solids.
4-Benzylthio-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
dine (6a). Yield 85%; mp 145-147 °C; [R]2D7 -64.33 (c 0.21,
EtOH); UV (H2O) λmax 299.0 nm (ε 11 908, pH 2), 296.0 nm (ε
5-O-[(1,1-Dimethylethyl)dimethysilyl]-2,3-O-(1-methyleth-
ylidene)-D-ribofuranose (3). A solution of compound 2 (8.60 g,
45.2 mmol) in anhydrous CH2Cl2 (150.7 mL) was treated with tert-
butyldimethylsilyl chloride (8.86 g, 58.8 mmol) and imidazole (9.24
g, 131 mmol) at 0 °C. The reaction mixture was allowed to warm
to room temperature and kept for 2 h. The reaction mixture was
washed with water. The organic layer was dried over Na2SO4 and
filtered. The filtrate was concentrated in vacuo, and the residue was
purified by column chromatography on silica gel (n-hexane/EtOAc
1
1
) 7:1) to give 3 (11.4 g, 83%) as a colorless oil: H NMR (500
13 827, pH 7), 296.0 nm (ε 13 841, pH 11); H NMR (500 MHz,
MHz, CDCl3) for ꢀ-isomer δ 0.00 (s, 6H), 0.79 (s, 9H), 1.19 (s,
3H), 1.35 (s, 3H), 3.62 (m, 2H), 4.21 (m, 1H), 4.37 (d, J ) 5.5 Hz,
1H), 4.56 (d, J ) 5.5 Hz, 1H), 4.65 (d, J ) 11.0 Hz, 1H), 5.14 (d,
J ) 12.0 Hz, 1H); for R-isomer (diagnostic peaks only) δ -0.08
(s, 6H), 0.75 (s, 9H), 1.25 (s, 3H), 1.41 (s, 3H); 13C NMR (125
MHz, CDCl3) for ꢀ-isomer δ -5.7, 18.2, 24.8, 25.7, 26.4, 64.7,
81.7, 86.9, 87.5, 103.4, 111.9.
CD3OD) δ 3.78 (d, J ) 12.5 Hz, 1H), 3.89 (d, J ) 9.5 Hz, 1H),
4.14 (m, 1H), 4.33 (m, 1H), 4.65 (m, 1H), 4.68 (s, 2H), 6.18 (d, J
) 6.0 Hz, 1H), 6.58 (d, J ) 4.0 Hz, 1H), 7.27 (m, 1H), 7.32 (m,
2H), 7.47 (d, J ) 7.0 Hz, 2H), 7.63 (d, J ) 3.5 Hz, 1H), 8.63 (s,
1H); 13C NMR (125 MHz, CD3OD) δ 32.3, 62.0, 71.1, 74.3, 85.7,
89.4, 99.0, 116.9, 126.5, 126.9, 128.2, 128.7, 137.8, 148.0, 149.8,
161.2. Anal. (C18H19N3O4S·0.3H2O) C, H, N, S.
4-[(2-Fluorobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6b). Yield 82%; mp 135-136 °C; [R]2D5 -76.88 (c
0.16, MeOH); UV (H2O) λmax 297.0 nm (ε 13 079, pH 2), 296.0
4-Chloro-7-[5-O-[(1,1-dimethylethyl)dimethysilyl]-2,3-O-(1-
methylethylidene)-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimi-
dine (4). A mixture of compound 3 (4.08 g, 13.4 mmol) and CCl4
(1.7 mL, 17.4 mmol) in anhydrous THF (67.0 mL) was treated
with hexamethylphosphoramide (3.2 mL, 17.4 mmol) at -78 °C.
The reaction mixture was allowed to warm up at -30 °C and stirred
for 1 h. This resulting solution of chloro sugar was cannulated into
a well-stirred mixture of 6-chloro-7-deazapurine (1.37 g, 8.93
mmol), KOH (1.00 g, 17.9 mmol), and tris[2-(2-methoxyethoxy-
)ethyl]amine (2.9 mL, 8.93 mmol) in anhydrous CH3CN (55.8 mL)
at 0 °C. The reaction mixture was allowed to warm to room
temperature and kept for 16 h. After the solvent was removed in
vacuo, the residue was purified by column chromatography on silica
gel (n-hexane/EtOAc ) 95:5 to 9:1) to give anomeric mixture of
4 (1.57 g, 40% for two steps) as a colorless oil: 1H NMR (500
MHz, CDCl3) for ꢀ-isomer δ 0.07 (s, 6H), 0.90 (s, 9H), 1.39 (s,
3H), 1.65 (s, 3H), 3.80 (dd, J1 ) 11.5 Hz, J2 ) 3.5 Hz, 1H), 3.89
(dd, J1 ) 12.0 Hz, J2 ) 3.5 Hz, 1H), 4.36 (q, J ) 3.2 Hz, 1H),
4.95 (m, 1H), 5.06 (m, 1H), 6.41 (d, J ) 3.0 Hz, 1H), 6.63 (d, J )
3.5 Hz, 1H), 7.57 (d, J ) 3.5 Hz, 1H), 8.66 (s, 1H); for R-isomer
(diagnostic peaks only) δ 6.55 (d, J ) 3.5 Hz, 1H, H-7), 6.84 (d,
J ) 4.0 Hz, 1H, H-1′), 7.56 (d, J ) 4.0 Hz, 1H, H-8), 8.53 (s, 1H,
H-2); 13C NMR (125 MHz, CDCl3) for ꢀ-isomer δ -5.4, 18.3,
25.4, 25.8, 27.3, 63.4, 80.8, 85.0, 86.1, 90.6, 100.4, 114.2, 118.2,
1
nm (ε 14 749, pH 7), 296.0 nm (ε 13 925, pH 11); H NMR (500
MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.5 Hz, 1H), 3.89
(dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.15 (m, 1H), 4.33 (m, 1H),
4.65 (t, J ) 5.8 Hz, 1H), 4.71 (s, 2H), 6.18 (d, J ) 5.5 Hz, 1H),
6.57 (d, J ) 4.0 Hz, 1H), 7.12 (m, 2H), 7.30 (m, 1H), 7.58 (m,
1H), 7.63 (d, J ) 3.5 Hz, 1H), 8.64 (s, 1H); 13C NMR (125 MHz,
CD3OD) δ 25.5, 62.0, 71.1, 74.3, 85.7, 89.4, 99.0, 114.9 (d, J )
21.5 Hz), 117.0, 124.0, 124.9 (d, J ) 14.9 Hz), 126.5, 129.0 (d, J
) 8.0 Hz), 131.1, 148.1, 149.8, 160.6, 161.1 (d, J ) 244.6 Hz).
Anal. (C18H18FN3O4S) C, H, N, S.
4-[(2-Chlorobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6c). Yield 83%; mp 146-147 °C; [R]2D5 -75.55 (c
0.22, MeOH); UV (H2O) λmax 297.0 nm (ε 13 470, pH 2), 296.0
1
nm (ε 14 815, pH 7), 296.0 nm (ε 14 193, pH 11); H NMR (500
MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.5 Hz, 1H), 3.89
(dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.15 (m, 1H), 4.33 (m, 1H),
4.66 (t, J ) 6.0 Hz, 1H), 4.81 (s, 2H), 6.19 (d, J ) 6.0 Hz, 1H),
6.58 (d, J ) 3.5 Hz, 1H), 7.27 (m, 2H), 7.45 (m, 1H), 7.63 (d, J )
4.0 Hz, 1H), 7.67 (m, 1H), 8.66 (s, 1H); 13C NMR (125 MHz,
CD3OD) δ 30.0, 62.0, 71.1, 74.3, 85.7, 89.4, 99.0, 117.0, 126.5,
126.8, 128.7, 129.2, 131.1, 134.0, 135.5, 148.1, 149.8, 160.7. Anal.
(C18H18ClN3O4S) C, H, N, S.
127.1, 150.9, 152.1; HRMS [M
C20H31ClN3O4Si 440.1772, found 440.1739.
+
H]+ m/z calcd for
4-[(2-Bromobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6d). Yield 86%; mp 158-160 °C; [R]2D5 -56.23 (c
0.29, MeOH); UV (H2O) λmax 297.0 nm (ε 13 619, pH 2), 296.0
4-Chloro-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
dine (5a) and r-Anomer (5b). A mixture of compound 4 (1.57 g,
3.57 mmol) and Dowex 50WX8-200 (0.785 g) in anhydrous MeOH
(36.0 mL) was stirred for 8 h at room temperature. The resin was
filtered off, and the filtrate was concentrated. The residue was
purified by column chromatography on silica gel (CH2Cl2/MeOH
) 95:5) to give ꢀ-anomer 5a (0.881 g, 86%) and R-anomer 5b
(0.088 g, 9%) as white solids. 5a: mp 160-161 °C [lit.35 mp
160-162 °C]; [R]D27 -46.17 (c 0.25, MeOH); UV (MeOH) λmax
274.0 nm [lit.35 UV (MeOH) λmax 273.0 nm]; 1H NMR (500 MHz,
CD3OD) δ 3.80 (dd, J1 ) 12.0 Hz, J2 ) 3.5 Hz, 1H), 3.89 (dd, J1
) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.15 (q, J ) 3.3 Hz, 1H), 4.35 (m,
1H), 4.62 (t, J ) 5.8 Hz, 1H), 6.31 (d, J ) 5.5 Hz, 1H), 6.75 (d,
J ) 4.0 Hz, 1H), 7.90 (d, J ) 4.0 Hz, 1H), 8.62 (s, 1H); 13C NMR
1
nm (ε 14 390, pH 7), 296.0 nm (ε 14 305, pH 11); H NMR (500
MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.5 Hz, 1H), 3.89
(dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.15 (m, 1H), 4.33 (m, 1H),
4.65 (t, J ) 6.0 Hz, 1H), 4.80 (s, 2H), 6.18 (d, J ) 6.0 Hz, 1H),
6.56 (d, J ) 3.0 Hz, 1H), 7.18 (t, J ) 7.8 Hz, 1H), 7.29 (t, J ) 7.8
Hz, 1H), 7.62 (m, 2H), 7.67 (d, J ) 7.5 Hz, 1H), 8.65 (s, 1H); 13
C
NMR (125 MHz, CD3OD) δ 32.7, 62.0, 71.1, 74.3, 85.7, 89.4, 99.0,
116.9, 124.3, 126.5, 127.4, 128.9, 131.1, 132.6, 137.2, 148.0, 149.8,
160.6. Anal. (C18H18BrN3O4S) C, H, N, S.
4-[(2-Methylbenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6e). Yield 83%; mp 136-137 °C; [R]2D8 -75.57 (c
0.17, MeOH); UV (H2O) λmax 298.0 nm (ε 14 757, pH 2), 297.0

7-Deaza-6-benzylthioinosine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3941
1
nm (ε 15 868, pH 7), 297.0 nm (ε 15 558, pH 11); H NMR (500
MHz, CD3OD) δ 2.46 (s, 3H), 3.79 (dd, J1 ) 12.5 Hz, J2 ) 3.0
Hz, 1H), 3.89 (dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.15 (m, 1H),
4.34 (m, 1H), 4.66 (t, J ) 4.8 Hz, 1H), 4.71 (s, 2H), 6.19 (d, J )
6.0 Hz, 1H), 6.58 (d, J ) 4.0 Hz, 1H), 7.19 (m, 3H), 7.44 (d, J )
7.5 Hz, 1H), 7.63 (m, 1H), 8.65 (s, 1H); 13C NMR (125 MHz,
CD3OD) δ 18.0, 30.8, 62.1, 71.1, 74.3, 85.8, 89.4, 99.1, 117.0,
125.8, 126.4, 127.4, 129.7, 130.1, 134.9, 136.8, 148.1, 149.9, 161.4.
Anal. (C19H21N3O4S) C, H, N, S.
4-[(4-Bromobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6k). Yield 80%; mp 159-161 °C; [R]2D5 -57.64 (c
0.16, MeOH); UV (H2O) λmax 297.0 nm (ε 14 589, pH 2), 296.0
1
nm (ε 15 973, pH 7), 296.0 nm (ε 15 930, pH 11); H NMR (500
MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.0 Hz, 1H), 3.88
(dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.14 (m, 1H), 4.33 (m, 1H),
4.65 (m, 3H), 6.18 (d, J ) 6.0 Hz, 1H), 6.58 (d, J ) 3.5 Hz, 1H),
7.42 (d, J ) 8.5 Hz, 2H), 7.47 (d, J ) 8.0 Hz, 2H), 7.64 (d, J )
4.0 Hz, 1H), 8.63 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 31.4,
62.0, 71.1, 74.3, 85.7, 89.3, 99.0, 117.0, 120.6, 126.6, 130.7, 131.2,
137.5, 148.1, 149.8, 160.6. Anal. (C18H18BrN3O4S) C, H, N, S.
4-[(3-Nitrobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6f). Yield 72%; mp 197-199 °C; [R]2D5 -54.88 (c
0.09, MeOH); UV (H2O) λmax 293.0 nm (ε 18 159, pH 2), 292.0
4-[(4-Cyanobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6m). Yield 77%; mp 184-186 °C; [R]2D5 -61.99 (c
0.15, MeOH); UV (H2O) λmax 297.0 nm (ε 14 656, pH 2), 296.0
1
nm (ε 18 677, pH 7), 292.0 nm (ε 18 299, pH 11); H NMR (500
MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.0 Hz, 1H), 3.88
(dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.14 (m, 1H), 4.32 (m, 1H),
4.64 (t, J ) 5.8 Hz, 1H), 4.80 (s, 2H), 6.19 (d, J ) 6.0 Hz, 1H),
6.60 (d, J ) 3.5 Hz, 1H), 7.57 (t, J ) 7.8 Hz, 1H), 7.65 (d, J ) 4.0
Hz, 1H), 7.93 (d, J ) 7.5 Hz, 1H), 8.13 (m, 1H), 8.42 (s, 1H),
8.65 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 31.1, 62.0, 71.1,
74.3, 85.7, 89.3, 98.9, 117.0, 121.7, 123.5, 126.7, 129.3, 135.1,
141.1, 148.2, 149.9, 159.9. Anal. (C18H18N4O6S) C, H, N, S.
1
nm (ε 15 474, pH 7), 296.0 nm (ε 14 860, pH 11); H NMR (500
MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.5 Hz, 1H), 3.88
(dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.14 (m, 1H), 4.33 (m, 1H),
4.64 (t, J ) 5.5 Hz, 1H), 4.74 (s, 2H), 6.19 (d, J ) 6.0 Hz, 1H),
6.58 (d, J ) 4.0 Hz, 1H), 7.65 (d, J ) 4.0 Hz, 1H), 7.68 (m, 4H),
8.63 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 31.5, 62.0, 71.1,
74.3, 85.7, 89.3, 98.9, 110.5, 117.0, 118.3, 126.7, 129.7, 131.9,
144.5, 148.2, 149.8, 160.0. Anal. (C19H18N4O4S) C, H, N, S.
4-[(3-Trifluoromethylbenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine (6g). Yield 82%; mp 161-163 °C; [R]2D5
-65.27 (c 0.19, MeOH); UV (H2O) λmax 297.0 nm (ε 13 117, pH
4-[(4-Methoxycarbonylbenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine (6n). Yield 79%; mp 168-170 °C; [R]2D5
-55.59 (c 0.17, MeOH); UV (H2O) λmax 297.0 nm (ε 13 962, pH
1
2), 296.0 nm (ε 14 319, pH 7), 296.0 nm (ε 14 291, pH 11); H
1
NMR (500 MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.5 Hz,
1H), 3.88 (dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.14 (m, 1H), 4.33
(m, 1H), 4.65 (t, J ) 5.5 Hz, 1H), 4.75 (s, 2H), 6.19 (d, J ) 6.0
Hz, 1H), 6.59 (d, J ) 3.5 Hz, 1H), 7.51 (m, 1H), 7.56 (m, 1H),
7.64 (m, 1H), 7.77 (d, J ) 7.5 Hz, 1H), 7.82 (m, 1H), 8.64 (s, 1H);
13C NMR (125 MHz, CD3OD) δ 31.5, 62.0, 71.1, 74.3, 85.7, 89.3,
99.0, 117.0, 123.5, 125.4, 126.6, 128.9, 129.3, 130.3 (q, J ) 32.0
2), 296.0 nm (ε 15 088, pH 7), 296.0 nm (ε 14 616, pH 11); H
NMR (500 MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.5 Hz,
1H), 3.91 (m, 4H), 4.14 (m, 1H), 4.33 (m, 1H), 4.65 (t, J ) 5.5
Hz, 1H), 4.74 (s, 2H), 6.19 (d, J ) 5.5 Hz, 1H), 6.59 (d, J ) 3.5
Hz, 1H), 7.61 (d, J ) 8.0 Hz, 2H), 7.64 (d, J ) 3.5 Hz, 1H), 7.98
(d, J ) 7.5 Hz, 2H), 8.64 (s, 1H); 13C NMR (125 MHz, CD3OD)
δ 31.7, 51.2, 62.0, 71.1, 74.3, 85.7, 89.3, 99.0, 117.0, 126.6, 128.9,
129.3, 144.0, 148.1, 149.8, 160.4, 166.9, 180.1. Anal.
(C20H21N3O6S) C, H, N, S.
Hz),
132.5,
139.9,
148.1,
149.8,
160.3.
Anal.
(C19H18F3N3O4S·0.1H2O) C, H, N, S.
4-[(3-Methylbenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6h). Yield 85%; mp 149-150 °C; [R]2D5 -58.12 (c
0.17, MeOH); UV (H2O) λmax 299.0 nm (ε 14 105, pH 2), 297.0
4-[(4-Methylbenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6o). Yield 82%; mp 123-124 °C; [R]2D5 -66.72 (c
0.22, MeOH); UV (H2O) λmax 299.0 nm (ε 14 362, pH 2), 297.0
1
1
nm (ε 15 211, pH 7), 297.0 nm (ε 14 638, pH 11); H NMR (500
nm (ε 16 169, pH 7), 297.0 nm (ε 16 026, pH 11); H NMR (500
MHz, CD3OD) δ 2.34 (s, 3H), 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.0
Hz, 1H), 3.89 (dd, J1 ) 12.0 Hz, J2 ) 2.5 Hz, 1H), 4.15 (m, 1H),
4.33 (m, 1H), 4.65 (m, 3H), 4.75 (s, 2H), 6.18 (d, J ) 6.0 Hz, 1H),
6.58 (d, J ) 3.5 Hz, 1H), 7.09 (d, J ) 7.5 Hz, 1H), 7.20 (m, 1H),
7.25 (d, J ) 8.0 Hz, 1H), 7.29 (m, 1H), 7.63 (d, J ) 4.0 Hz, 1H),
8.63 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 20.0, 32.4, 62.0,
71.1, 74.3, 85.8, 89.4, 99.1, 116.9, 125.8, 126.4, 127.6, 128.1, 129.4,
137.5, 138.0, 148.0, 149.8, 161.3. Anal. (C19H21N3O4S) C, H, N,
S.
MHz, CD3OD) δ 2.33 (s, 3H), 3.78 (dd, J1 ) 13.0 Hz, J2 ) 3.0
Hz, 1H), 3.89 (dd, J1 ) 12.0 Hz, J2 ) 2.5 Hz, 1H), 4.15 (m, 1H),
4.33 (m, 1H), 4.63 (s, 2H), 4.66 (t, J ) 6.0 Hz, 1H), 6.18 (d, J )
5.5 Hz, 1H), 6.57 (d, J ) 3.5 Hz, 1H), 7.14 (d, J ) 7.5 Hz, 2H),
7.35 (d, J ) 7.5 Hz, 2H), 7.62 (d, J ) 4.0 Hz, 1H), 8.62 (s, 1H);
13C NMR (125 MHz, CD3OD) δ 19.7, 32.2, 62.1, 71.1, 74.3, 85.7,
89.4, 99.1, 117.0, 126.4, 128.6, 128.8, 134.5, 136.7, 148.1, 149.9,
161.4. Anal. (C19H21N3O4S·0.1H2O) C, H, N, S.
4-[(4-tert-Butylbenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrro-
lo[2,3-d]pyrimidine (6p). Yield 81%; mp 83-85 °C; [R]2D5 -51.07
(c 0.23, MeOH); UV (H2O) λmax 299.0 nm (ε 12 456, pH 2), 297.0
4-[(4-Fluorobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6i). Yield 83%; mp 99-101 °C; [R]2D5 -67.30 (c
0.21, MeOH); UV (H2O) λmax 298.0 nm (ε 12 936, pH 2), 296.0
1
nm (ε 11 421, pH 7), 297.0 nm (ε 12 420, pH 11); H NMR (500
1
nm (ε 14 171, pH 7), 296.0 nm (ε 14 135, pH 11); H NMR (500
MHz, CD3OD) δ 1.33 (s, 9H), 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.5
Hz, 1H), 3.89 (dd, J1 ) 12.5 Hz, J2 ) 2.5 Hz, 1H), 4.15 (m, 1H),
4.34 (m, 1H), 4.63 (s, 2H), 4.66 (t, J ) 5.8 Hz, 1H), 6.18 (d, J )
6.0 Hz, 1H), 6.57 (d, J ) 4.0 Hz, 1H), 7.37 (m, 4H), 7.62 (d, J )
4.0 Hz, 1H), 8.62 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 30.4,
32.1, 33.9, 62.1, 71.1, 74.3, 85.7, 89.5, 99.1, 117.0, 125.1, 126.4,
MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.0 Hz, 1H), 3.89
(dd, J1 ) 12.0 Hz, J2 ) 2.5 Hz, 1H), 4.15 (m, 1H), 4.33 (m, 1H),
4.63 (s, 2H), 4.66 (t, J ) 6.0 Hz, 1H), 6.18 (d, J ) 5.5 Hz, 1H),
6.55 (d, J ) 3.5 Hz, 1H), 7.03 (m, 2H), 7.48 (m, 2H), 7.61 (d, J )
3.5 Hz, 1H), 8.62 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 31.5,
62.0, 71.1, 74.3, 85.7, 89.4, 99.0, 114.8 (d, J ) 21.9 Hz), 116.9,
126.5, 130.6 (d, J ) 8.1 Hz), 134.0, 148.0, 149.8, 160.9, 162.0 (d,
J ) 242.8 Hz). Anal. (C18H18FN3O4S) C, H, N, S.
128.4,
134.5,
148.1,
149.9,
150.0,
161.4.
Anal.
(C22H27N3O4S·0.1H2O) C, H, N, S.
4-[(4-Trifluoromethoxybenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine (6r). Yield 79%; mp 159-160 °C; [R]2D8
-57.12 (c 0.23, MeOH); UV (H2O) λmax 297.0 nm (ε 11 500, pH
4-[(4-Chlorobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6j). Yield 81%; mp 162-163 °C; [R]2D8 -76.24 (c
0.17, MeOH); UV (H2O) λmax 298.0 nm (ε 15 494, pH 2), 296.0
1
2), 296.0 nm (ε 14 378, pH 7), 296.0 nm (ε 13 504, pH 11); H
1
nm (ε 16 401, pH 7), 296.0 nm (ε 16 312, pH 11); H NMR (500
NMR (500 MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.5 Hz,
1H), 3.88 (dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.14 (m, 1H), 4.33
(m, 1H), 4.65 (t, J ) 5.8 Hz, 1H), 4.71 (s, 2H), 6.19 (d, J ) 5.5
Hz, 1H), 6.59 (d, J ) 3.5 Hz, 1H), 7.23 (d, J ) 8.5 Hz, 2H), 7.59
(d, J ) 8.5 Hz, 2H), 7.64 (d, J ) 3.5 Hz, 1H), 8.64 (s, 1H); 13C
NMR (125 MHz, CD3OD) δ 31.3, 62.0, 71.1, 74.3, 85.7, 89.4, 99.0,
117.0, 120.5 (q, J ) 254.0 Hz), 120.6, 126.5, 130.4, 137.5, 148.1,
148.2, 149.9, 160.6. Anal. (C19H18F3N3O5S) C, H, N, S.
MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.0 Hz, 1H), 3.89
(dd, J1 ) 12.0 Hz, J2 ) 2.5 Hz, 1H), 4.15 (m, 1H), 4.33 (m, 1H),
4.65 (m, 3H), 6.19 (d, J ) 5.5 Hz, 1H), 6.58 (d, J ) 4.0 Hz, 1H),
7.32 (d, J ) 8.5 Hz, 2H), 7.47 (d, J ) 7.5 Hz, 2H), 7.64 (d, J )
4.0 Hz, 1H), 8.63 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 31.4,
62.0, 71.1, 74.3, 85.7, 89.4, 99.0, 117.0, 126.5, 128.2, 130.3, 132.6,
137.0, 148.1, 149.9, 160.7. Anal. (C18H18ClN3O4S) C, H, N, S.

3942 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Kim et al.
4-[(4-Methoxybenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrro-
lo[2,3-d]pyrimidine (6s). Yield 86%; mp 163-164 °C; [R]D29
-68.73 (c 0.13, MeOH); UV (H2O) λmax 299.0 nm (ε 14 510, pH
4-[(4-Vinylbenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6q). Yield 92%; mp 175-176 °C; [R]2D5 -69.81 (c
0.16, MeOH); UV (H2O) λmax 297.0 nm (ε 15 766, pH 2), 296.0
1
1
nm (ε 17 382, pH 7), 296.0 nm (ε 17 169, pH 11); H NMR (500
2), 297.0 nm (ε 16 537, pH 7), 298.0 nm (ε 15 818, pH 11); H
MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.0 Hz, 1H), 3.89
(dd, J1 ) 12.0 Hz, J2 ) 2.5 Hz, 1H), 4.15 (m, 1H), 4.33 (m, 1H),
4.65 (m, 3H), 5.23 (d, J ) 10.5 Hz, 1H), 5.78 (d, J ) 17.5 Hz,
1H), 6.18 (d, J ) 6.0 Hz, 1H), 6.58 (d, J ) 3.5 Hz, 1H), 6.73 (dd,
J1 ) 17.5 Hz, J2 ) 10.5 Hz, 1H), 7.41 (m, 4H), 7.63 (d, J ) 3.5
Hz, 1H), 8.63 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 32.1, 62.0,
71.1, 74.3, 85.7, 89.4, 99.0, 112.6, 117.0, 126.0, 126.5, 128.9, 136.4,
136.7, 137.4, 148.0, 149.8, 161.1. Anal. (C20H21N3O4S) C, H, N,
S.
NMR (500 MHz, CD3OD) δ 3.78 (m, 4H), 3.89 (dd, J1 ) 12.0
Hz, J2 ) 3.0 Hz, 1H), 4.15 (m, 1H), 4.33 (m, 1H), 4.62 (s, 2H),
4.66 (t, J ) 6.0 Hz, 1H), 6.18 (d, J ) 6.0 Hz, 1H), 6.57 (d, J ) 3.5
Hz, 1H), 6.88 (d, J ) 9.0 Hz, 2H), 7.39 (d, J ) 8.0 Hz, 2H), 7.62
(d, J ) 4.0 Hz, 1H), 8.62 (s, 1H); 13C NMR (125 MHz, CD3OD)
δ 32.0, 54.3, 62.1, 71.1, 74.3, 85.7, 89.4, 99.1, 113.6, 117.0, 126.4,
129.5, 129.9, 148.1, 149.9, 159.1, 161.4. Anal. (C19H21N3O5S) C,
H, N, S.
4-[(2,4-Dichlorobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrro-
lo[2,3-d]pyrimidine (6u). Yield 80%; mp 160-161 °C; [R]D25
-48.77 (c 0.22, MeOH); UV (H2O) λmax 296.0 nm (ε 8008, pH 2),
295.0 nm (ε 10121, pH 7), 296.0 nm (ε 13 509, pH 11); 1H NMR
(500 MHz, CD3OD) δ 3.78 (dd, J1 ) 12.5 Hz, J2 ) 3.5 Hz, 1H),
3.89 (dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.14 (m, 1H), 4.33 (m,
1H), 4.65 (t, J ) 5.8 Hz, 1H), 4.77 (s, 2H), 6.19 (d, J ) 6.0 Hz,
1H), 6.57 (d, J ) 4.0 Hz, 1H), 7.28 (dd, J1 ) 7.5 Hz, J2 ) 1.5 Hz,
1H), 7.50 (d, J ) 2.0 Hz, 1H), 7.63 (d, J ) 4.0 Hz, 1H), 7.67 (d,
J ) 8.5 Hz, 1H), 8.65 (s, 1H); 13C NMR (125 MHz, CD3OD) δ
29.4, 62.0, 71.1, 74.3, 85.7, 89.4, 98.9, 117.0, 126.6, 127.0, 128.8,
132.1, 133.5, 134.7, 134.8, 148.2, 149.8, 160.2. Anal.
(C18H17Cl2N3O4S) C, H, N, S.
4-[(2,4-Difluorobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrro-
lo[2,3-d]pyrimidine (6t). Yield 95%; mp 143-144 °C; [R]D25
-56.75 (c 0.24, MeOH); UV (H2O) λmax 296.0 nm (ε 11 824, pH
1
2), 295.0 nm (ε 12 186, pH 7), 296.0 nm (ε 11 637, pH 11); H
NMR (500 MHz, CD3OD) δ 3.78 (dd, J1 ) 13.0 Hz, J2 ) 3.0 Hz,
1H), 3.88 (dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.14 (m, 1H), 4.33
(m, 1H), 4.65 (t, J ) 5.8 Hz, 1H), 4.69 (s, 2H), 6.18 (d, J ) 6.0
Hz, 1H), 6.57 (d, J ) 4.0 Hz, 1H), 6.91 (m, 1H), 6.98 (m, 1H),
7.63 (m, 2H), 8.64 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 24.9,
62.0, 71.1, 74.3, 85.7, 89.3, 98.9, 103.2 (t, J ) 25.8 Hz), 110.8
(dd, J1 ) 21.5 Hz, J2 ) 3.8 Hz), 117.0, 121.3 (dd, J1 ) 14.7 Hz,
J2 ) 3.9 Hz), 126.6, 132.1 (dd, J1 ) 10.2 Hz, J2 ) 5.1 Hz), 148.1,
149.8, 160.4, 161.1 (dd, J1 ) 247.6 Hz, J2 ) 12.4 Hz), 162.5 (dd,
J1 ) 246.1 Hz, J2 ) 11.4 Hz). Anal. (C18H17F2N3O4S) C, H, N, S.
7-(ꢀ-D-Ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine-4(3H)-
thione (7). To a solution of compound 5a (160 mg, 0.560 mmol)
in anhydrous EtOH (5.6 mL) was added thiourea (86.0 mg, 1.12
mmol). The reaction mixture was heated to reflux for 3 h. After
the solvent was removed in vacuo, the residue was purified by
column chromatography on silica gel (CH2Cl2/MeOH ) 95:5) to
give 7 (135 mg, 85%) as a white solid: mp 208-209 °C; [R]D28
-85.30 (c 0.47, MeOH); UV (H2O) λmax 324.0 nm (ε 22 197, pH
4-[(3,4-Dichlorobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrro-
lo[2,3-d]pyrimidine (6v). Yield 79%; mp 133-134 °C; [R]D26
-78.24 (c 0.11, MeOH); UV (H2O) λmax 297.0 nm (ε 14 268, pH
1
2), 296.0 nm (ε 14 890, pH 7), 296.0 nm (ε 14 495, pH 11); H
NMR (500 MHz, CD3OD) δ 3.77 (dd, J1 ) 12.5 Hz, J2 ) 3.5 Hz,
1H), 3.88 (dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.14 (m, 1H), 4.33
(m, 1H), 4.65 (m, 3H), 6.19 (d, J ) 5.5 Hz, 1H), 6.59 (d, J ) 4.0
Hz, 1H), 7.45 (m, 2H), 7.65 (d, J ) 4.0 Hz, 1H), 7.67 (d, J ) 1.5
Hz, 1H), 8.64 (s, 1H); 13C NMR (125 MHz, CD3OD) δ 30.8, 62.0,
71.1, 74.3, 85.7, 89.3, 99.0, 117.0, 126.6, 128.6, 130.1, 130.6, 130.7,
131.7, 139.4, 148.1, 149.8, 160.1. Anal. (C18H17Cl2N3O4S) C, H,
N, S.
1
2), 324.0 nm (ε 21 362, pH 7), 310.0 nm (ε 20 225, pH 11); H
NMR (500 MHz, DMSO-d6) δ 3.56 (m, 1H), 3.64 (m, 1H), 3.92
(m, 1H), 4.10 (m, 1H), 4.33 (m, 1H), 5.06 (t, J ) 5.3 Hz, 1H),
5.19 (d, J ) 4.5 Hz, 1H), 5.40 (d, J ) 6.0 Hz, 1H), 6.07 (d, J )
6.0 Hz, 1H), 6.72 (d, J ) 3.5 Hz, 1H), 7.64 (d, J ) 4.0 Hz, 1H),
8.14 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 61.9, 71.0, 74.9,
85.7, 87.3, 105.2, 121.0, 124.6, 143.8, 177.0. Anal. (C11H13N3O4S)
C, H, N, S.
4-[(2-Chloro-6-fluorobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine (6w). Yield 84%; mp 170-172 °C; [R]2D6
-72.80 (c 0.20, MeOH); UV (H2O) λmax 296.0 nm (ε 14 071, pH
1
2), 296.0 nm (ε 15 191, pH 7), 296.0 nm (ε 14 707, pH 11); H
NMR (500 MHz, CD3OD) δ 3.79 (dd, J1 ) 12.5 Hz, J2 ) 3.0 Hz,
1H), 3.89 (dd, J1 ) 12.0 Hz, J2 ) 2.5 Hz, 1H), 4.15 (m, 1H), 4.34
(m, 1H), 4.66 (t, J ) 5.8 Hz, 1H), 4.90 (s, 2H), 6.20 (d, J ) 6.5
Hz, 1H), 6.57 (d, J ) 3.5 Hz, 1H), 7.16 (m, 1H), 7.36 (m, 2H),
7.64 (d, J ) 3.0 Hz, 1H), 8.67 (s, 1H); 13C NMR (125 MHz,
CD3OD) δ 23.8, 62.0, 71.1, 74.3, 85.7, 89.3, 98.9, 114.0 (d, J )
22.4 Hz), 116.8, 123.0 (d, J ) 18.1 Hz), 125.3, 126.6, 129.7 (d, J
) 10.0 Hz), 135.5, 148.1, 149.9, 160.6, 161.5 (d, J ) 248.5 Hz).
Anal. (C18H17ClFN3O4S) C, H, N, S.
5-Iodo-4-chloropyrrolo[2,3-d]pyrimidine (9). 6-Chloro-7-dea-
zapurine 8 (0.430 g, 2.80 mmol) was treated with N-iodosuccinimide
(0.630 g, 2.80 mmol) in DMF (9.0 mL). The reaction mixture was
stirred for 2 h at room temperature. After the solvent was removed
in vacuo, the residue was purified by column chromatography on
silica gel (n-hexane/EtOAc ) 3:1) to give 9 (0.728 g, 93%) as a
white solid: mp 197-199 °C; UV (MeOH) λmax 305.0 nm; 1H NMR
(500 MHz, DMSO-d6) δ 7.97 (s, 1H), 8.61 (s, 1H), 12.98 (br, 1H);
13C NMR (125 MHz, DMSO-d6) δ 52.1, 116.3, 134.3, 150.9, 151.2,
152.0.
General Procedure for the Compounds (6l, 6q, and 6t). To a
solution of compound 7 (50.0 mg, 0.176 mmol) in H2O (2.2 mL)
were added concentrated NH4OH (0.03 mL) and the appropriate
benzyl halide (0.221 mmol). The reaction mixture was stirred for
11 h at room temperature. The resulting precipitates were filtered
and purified by short column chromatography on silica gel (CH2Cl2/
MeOH ) 95:5) to give the desired products as white solids.
4-Chloro-5-iodo-7-[5-O-[(1,1-dimethylethyl)dimethysilyl]-2,3-
O-(1-methylethylidene)-ꢀ-D-ribofuranosyl]-7H-pyrrolo[2,3-d]py-
rimidine (10). A mixture of compound 3 (0.200 g, 0.657 mmol)
and CCl4 (70 µL, 0.723 mmol) in anhydrous THF (6.0 mL) was
treated with hexamethylphosphoramide (0.13 mL, 0.723 mmol) at
-78 °C. The reaction mixture was allowed to warm up at -30 °C
and stirred for 1 h. This resulting solution of chloro sugar was
cannulated into a well-stirred mixture of compound 9 (0.141 g,
0.505 mmol), KOH (57.0 mg, 1.01 mmol), and tris[2-(2-methoxy-
ethoxy)ethyl]amine (0.19 mL, 0.607 mmol) in anhydrous CH3CN
(6.0 mL) at 0 °C. The reaction mixture was allowed to warm to
room temperature and kept for 16 h. After the solvent was removed
in vacuo, the residue was purified by column chromatography on
silica gel (n-hexane/EtOAc ) 9:1) to give 10 (126 mg, 44% for
two steps) as a colorless oil: [R]2D8 -66.34 (c 1.8, CHCl3); UV
4-[(4-Nitrobenzyl)thio]-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidine (6l). Yield 86%; mp 163-164 °C; [R]2D5 -71.18 (c
0.15, MeOH); UV (H2O) λmax 293.0 nm (ε 21 370, pH 2), 293.0
1
nm (ε 22 011, pH 7), 293.0 nm (ε 21 278, pH 11); H NMR (500
MHz, CD3OD) δ 3.78 (dd, J1 ) 12.0 Hz, J2 ) 3.5 Hz, 1H), 3.88
(dd, J1 ) 12.0 Hz, J2 ) 3.0 Hz, 1H), 4.14 (m, 1H), 4.32 (m, 1H),
4.64 (t, J ) 5.8 Hz, 1H), 4.80 (s, 2H), 6.19 (d, J ) 5.5 Hz, 1H),
6.59 (d, J ) 3.5 Hz, 1H), 7.65 (d, J ) 3.5 Hz, 1H), 7.76 (d, J )
9.0 Hz, 2H), 8.20 (d, J ) 8.5 Hz, 2H), 8.64 (s, 1H); 13C NMR
(125 MHz, CD3OD) δ 31.2, 62.0, 71.1, 74.3, 85.7, 89.3, 98.9, 117.0,
123.1, 126.7, 129.8, 146.5, 147.0, 148.2, 149.9, 159.9. Anal.
(C18H18N4O6S) C, H, N, S.
1
(MeOH) λmax 305.0 nm; H NMR (500 MHz, CDCl3) δ 0.10 (s,
6H), 0.90 (s, 9H), 1.36 (s, 3H), 1.63 (s, 3H), 3.79 (dd, J1 ) 11.0
Hz, J2 ) 3.0 Hz, 1H), 3.90 (dd, J1 ) 11.5 Hz, J2 ) 3.0 Hz, 1H),

7-Deaza-6-benzylthioinosine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3943
4.39 (m, 1H), 4.88 (dd, J1 ) 6.0 Hz, J2 ) 2.0 Hz, 1H), 4.92 (dd,
J1 ) 6.0 Hz, J2 ) 3.0 Hz, 1H), 6.41 (d, J ) 3.0 Hz, 1H), 7.76 (s,
1H), 8.63 (s, 1H); 13C NMR (125 MHz, CDCl3) δ -5.3, -5.2,
18.5, 25.4, 26.0, 27.4, 52.3, 63.6, 80.9, 85.5, 86.3, 90.9, 114.2,
117.4, 132.1, 150.5, 151.1, 152.6; HRMS [M + H]+ m/z calcd for
C20H30ClIN3O4Si 566.0739, found 566.0727.
in MACROMODEL 9.1 for energy minimization (EM). For EM
studies, residues further than 16 Å from the adenosine binding site
were frozen and residues from 6 to 16 Å were constrained by
harmonic constraints. Only residues inside a 6 Å sphere from the
nucleoside were allowed to move freely. EM calculations were
performed for 5000 steps or until the energy difference between
subsequent structures was 0.05 kJ/mol. The starting conformation
for all of the ligands were obtained by 5000 steps of Monte Carlo
conformational search under GB/SA continuum water solvation
model and Polak-Ribiere conjugate gradient (PRCG) method used
for the subsequent energy minimization.
4-Chloro-5-iodo-7-(ꢀ-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidine (11). Compound 11 was prepared from compound 10
(94.5 mg, 0.167 mmol) under the procedure outlined above for
compound 5 and was obtained as a white solid (64 mg, 93%): mp
192-194 °C; [R]2D3 -55.18 (c 0.32, MeOH); UV (MeOH) λmax
1
306.0 nm; H NMR (500 MHz, DMSO-d6) δ 3.58 (m, 1H), 3.67
Evaluation of the Newly Designed 7-Deaza-6-benzylthioi-
nosine Analogues as Alternative Substrates for Purified T.
gondii Adenosine Kinase. Enzyme assays were run under condi-
tions where activity was linear with time and enzyme concentra-
tion.22 Activity was determined by following the formation of
radiolabeled AMP from adenosine. The assay mixture contains 50
mM Tris-Cl, pH 7.4; 2.5 mM ATP; 5 mM MgCl2; 5 mM creatine
phosphate; creatine kinase; 5 µM [8-14C]adenosine (55 Ci/mol);
50 µL of purified T. gondii adenosine kinase; prepared as previously
described30 in a final volume of 100 µL, in the absence or presence
of various concentrations of the compound under evaluation.
Incubation was carried out at 37 °C and terminated by boiling in a
water bath for 2 min, followed by freezing for at least 20 min.
Precipitated proteins were removed by centrifugation and 10 µL
of the supernatant was spotted on silica gel TLC plates. The TLC
plates were developed in a mixture of chloroform/methanol/acetic
acid (102:12:6 v/v/v). The Rf values were 0.27 for adenosine and
0.17 for AMP. The amounts of radioactivity in both the substrate
and product were calculated on a percentage basis using computer-
ized Berthold LB-284 automatic TLC-linear analyzers. Apparent
Ki values of these analogues were calculated from Dixon plots 1/V
vs [I] by least-squares fitting by computer programs written by Dr.
Naguib as previously described.21,22,24,25,27 The synthesis of the
nucleoside 5′-monophosphates from the tested 7-deaza-6-benzylth-
ioinosine analogues was confirmed by HPLC and NMR analyses
as previously described,22,25,27 indicating that these compounds are
alternative substrates to T. gondii adenosine kinase. Since these
compounds are alternative substrates of T. gondii adenosine kinase,
their apparent Ki values are equal to their apparent Km values48 as
presented in Table 1.
Evaluation of 7-Deaza-6-benzylthioinosine Analogues as
Potential Antitoxoplasmosis Agents against Tachyzoites in
Tissue Culture. The wild type RH and the adenosine kinase
deficient mutant TgAK-3 41 strains of T. gondii were used in these
experiments. The adenosine kinase deficient mutant TgAK-3 was
used as a control to verify that the promising drugs were
metabolized by adenosine kinase in vivo as was the case in vitro.
The effects of purine analogues as antitoxoplasmosis agents in tissue
culture was measured by their ability to inhibit the replication of
intracellular T. gondii in tissue culture, using monolayers of human
foreskin fibroblasts (CRL-1634, American Type Culture Collection),
grown for no more than 20 passages in RPMI 1640 medium.23,25,27
The viability of intracellular parasites was evaluated by the selective
incorporation of radiolabeled uracil into nucleic acids of the
parasites at least in triplicate as previously described.23,25,27 Briefly,
confluent cells (4-5 days of incubation) were cultured for 24 h in
the 24-well flat bottom microtiter plates ((∼5 × 105/1 mL)/well))
and incubated at 37 °C in 5% CO2, 95% air to allow the cells to
attach. The medium was then removed, and the cells were infected
with isolated T. gondii in medium with 3% FBS (1 parasite/cell).
After 1 h of incubation, the cultures were washed with media with
10% FBS to remove extracellular parasites. FBS was maintained
at a final concentration of 10%. Compounds were dissolved in 50%
ethanol and then added to cultures of the parasite-infected cells to
give a final concentration of 0, 5, 10, 25, and 50 µM. The final
concentration of ethanol when the compounds were added to the
wells was 2.5%. After an additional 18 h incubation the medium
was replaced with 1 mL of drug-free medium containing [5,6-
13H]uracil (5 µCi/mL) and incubated for another 6 h after which
the medium was removed. The fibroblasts were then released from
(m, 1H), 3.95 (m, 1H), 4.12 (m, 1H), 4.40 (q, J ) 5.5 Hz, 1H),
5.14 (t, J ) 5.0 Hz, 1H), 5.24 (d, J ) 5.0 Hz, 1H), 5.47 (d, J ) 6.0
Hz, 1H), 6.21 (d, J ) 6.5 Hz, 1H), 8.27 (s, 1H), 8.70 (s, 1H); 13
C
NMR (125 MHz, DMSO-d6) δ 54.1, 61.7, 70.8, 74.8, 86.0, 87.5,
117.1, 134.0, 151.2, 151.5, 151.6; HRMS [M + H]+ m/z calcd for
C11H12ClIN3O4 411.9561, found 411.9567.
4-Benzylthio-5-iodo-7-(ꢀ-D-ribofuranosyl)pyrrolo[2,3-d]pyri-
midine (12). A solution of compound 11 (55.0 mg, 0.134 mmol)
in anhydrous THF (4.5 mL) was treated with benzylthiol (63 µL,
0.536 mmol) and sodium hydride (6.43 mg, 0.268 mmol) at 0 °C.
The reaction mixture was allowed to warm to room temperature
and stirred for 1 h. Excess hydride was quenched by addition of
methanol, and then the reaction mixture was concentrated to dryness
in vacuo. The residues were purified by column chromatography
on silica gel (CH2Cl2/MeOH ) 95:5) to give 12 (56 mg, 84%) as
a white solid: mp 223-224 °C; [R]2D5 -56.85 (c 0.33, DMSO);
UV (H2O) λmax 312.0 nm (ε 6106, pH 2), 312.0 nm (ε 5534, pH
1
7), 312.0 nm (ε 8631, pH 11); H NMR (500 MHz, DMSO-d6) δ
3.56 (m, 1H), 3.65 (m, 1H), 3.92 (m, 1H), 4.10 (m, 1H), 4.37 (q,
J ) 5.5 Hz, 1H), 4.65 (m, 2H), 5.11 (t, J ) 5.0 Hz, 1H), 5.20 (d,
J ) 5.0 Hz, 1H), 5.42 (d, J ) 6.5 Hz, 1H), 6.17 (d, J ) 6.0 Hz,
1H), 7.28 (m, 1H), 7.35 (m, 2H), 7.49 (m, 2H), 8.27 (s, 1H), 8.70
(s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 32.9, 53.3, 61.8, 70.9,
74.7, 85.9, 87.2, 117.2, 127.7, 129.0, 129.6, 131.4, 138.0, 149.3,
151.0, 161.1; HRMS [M + H]+ m/z calcd for C18H19IN3O4S
500.0141, found 500.0128. Anal. (C18H18IN3O4S) C, H, N.
Molecular Modeling. The enzymatic reaction of T. gondii
adenosine kinase involves the bending motion of its two domains
through induced fit movements. These domain movements are
complex and apparently involve further tuning of the protein
structure upon the binding of ATP. Therefore, the crystal structure
of the enzyme-adenosine complex (1LII.pdb),28 and not that of
the apo-form, was used for our modeling calculation. In general,
crystal structures lack hydrogen atoms, bond order, and sometimes
few amino acid residues. We used the builder module of Maestro43
to manually correct the structure of T. gondii adenosine kinase.
This involved the adding of missing residues (239-240 and
255-269), hydrogen atoms, appropriate bond order, formal charges,
and +2 for magnesium ion, as well as removing of water and fixing
of proper atom-types (according to atomic hybridization). The
manually inspected and corrected structure of T. gondii adenosine
kinase was then subjected to a single run by Glide44 protein
preparation program to optimize the structure and ensure its
chemical correctness. The determination of the binding mode of
7-deaza-6-benzylthioinosines was complicated by the fact that the
enzyme functions via induced fit mechanism and the enzyme--
adenosine complex is not perfect for the studies because our
analogues are almost ∼16 Å in length diagonally, whereas the
diagonal length of the adenosine was ∼8 Å. Therefore, we used
the induced fit docking45 and the prime module of the Schro¨dinger
Suite46 to determine the binding mode of the 7-deaza-6-benzylth-
ioinosines. After successful active-site modification and refinement
by prime and redocking by Glide, enzyme-ligand complexes were
subjected to energy minimization (EM)47 to obtain the final binding
mode.
All the calculations were performed on Schro¨dinger Suite 200646
(Schro¨dinger Inc.) with the GB/SA continuum water solvation
model. OPLS_2001 and OPLS_2005 force fields were used for
induced fit docking and then the same force fields were implemented

3944 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Kim et al.
Toxoplasmosis; Thomas, P. A., Petersen, E., Eds.; Springer: New York;
pp 314-322.
the wells by trypsinization with the addition of 200 µL of trypsin/
EDTA (2.5×) to each well. After 10 min of incubation, 1 mL of
ice cold 10% trichloroacetic acid (TCA) was added to each well.
The plates were then placed on a shaker to ensure the detachment
of the cells. The suspended contents of each well were filtered
through GF/A 2.4 cm glass microfiber filters (Whatman), which
were prewashed each with 1 mL of doubly distilled H2O and dried.
After filtration, the filters were washed with 10 mL of methanol,
left to dry, then placed in scintillation vials containing 5 mL of
Econo-Safe scintillation fluor (Research Products International Corp,
Mount Prospect, IL), and radioactivity was counted using an
LS5801 Beckman scintillation counter. The effect of the compounds
on the growth of the parasite was estimated as percentage reduction
in the uptake of radiolabeled uracil by treated parasites compared
to the untreated controls.22,23,25,27,49 Radiolabel incorporation
closely correlates with parasite growth.22,23,25,27,49
Host Toxicity of 7-Deaza-6-benzylthioinosine Analogues.
Possible toxicity against the host cells by the same doses of the
various analogues used in the above experiments was measured, at
least in triplicate, using a modification of the microculture tetra-
zolium (MTT) assay on uninfected monolayers of human foreskin
fibroblasts (grown for no more than 20 passages) in RPMI 1640
medium.22,23,25,27 Briefly, confluent cells were incubated for at least
24 h in 96-well flat bottom microtiterplates ((∼105/200 µL)/well))
at 37 °C in 5% CO2, 95% air to allow the cells to attach. The
medium was then replaced with 200 µL of fresh medium. The
appropriate concentration of the compounds was dissolved in 50
µL of medium and added to each well to give the desired final
concentrations. The cultures were then incubated for 48 h, after
which 50 µL of sterile MTT solution (2 mg/1 mL PBS) was added
to each well. MTT solution was sterilized by filtration through 0.22
µm filters (Costar, Cambridge, MA). After 4 h of incubation, the
medium was removed and 100 µL of dimethyl sulfoxide (DMSO)
was added to each well and the plates were shaken gently for 2-3
min to dissolve the formed formazan crystals. The absorbance was
measured at 540 nm using a computerized microtiterplate reader
(Themomax, Molecular Devices).
(11) Zenner, L.; Estaquier, J.; Darcy, F.; Maes, P.; Capron, A.; Cesbron-
Delaw, M. Protective Immunity on the Rat Model of Congenital
Toxoplasmosis and the Potential of Excreted-Secreted Antigens as
Vaccine Components. Parasite Immunol. 1999, 21, 261–172.
(12) Anderson, A. C. Targeting DHFR in Parasitic Protozoa. Drug
DiscoVery Today 2005, 10, 121–128.
(13) Chio, L.-C.; Queener, S. F. Identification of Highly Potent and
Selective Inhibitors of Toxoplasma gondii Dihydrofolate Reductase.
Antimicrob. Agents Chemother. 1993, 37, 1914–1923.
(14) van de Ven, E. S.; Vree, T.; Melchers, W.; Camps, W.; Galama, J. In
Vitro Effects of Sulfadiazine and Its Metabolites Alone and in
Combination with Pyrimethamine on Toxoplasma gondii. Antimicrob.
Agents Chemother. 1995, 39, 763–765.
(15) Klepser, M. E.; Lepser, T. B. Drug Treatment of HIV-Related
Opportunistic Infections. Drugs 1997, 53, 40–73.
(16) Gormley, P. D.; Pavesio, C. E.; Minnasian, D.; Ligbtman, S. Effects
of Drug Therapy on Toxoplasma Cysts in an Animal Model of Acute
and Chronic Disease. InVest. Ophthalmol. Visual Sci. 1998, 39, 1171–
1175.
(17) Katlama, C.; de Wit, S.; O’Doherty, E.; van Glabeke, M.; Clumeck,
N. Pyrimethamine-Clindamycin versus Pyrimethamine-Sulfadiazine
as Acute and Long-Term Therapy for Toxoplasmic Encephalitis in
Patients with AIDS. Clin. Infect. Dis. 1996, 22, 268–275.
(18) Grujic´, J.; Djurkovic´-Djakovic´, O.; Nikolic´, A.; Klun, I.; Bobic´, B.
Effectiveness of Spiramycin in Murine Models of Acute and Chronic
Toxoplasmosis. Int. J. Antimicrob. Agents 2005, 25, 226–230.
(19) el Kouni, M. H. Potential Chemotherapeutic Targets in the Purine
Metabolism of Parasites. Pharmacol. Ther. 2003, 99, 283–309.
(20) el Kouni, M. H. Adenosine Metabolism in Toxoplasma gondii:
Potential Targets for Chemotherapy. Curr. Pharm. Des. 2007, 13, 581–
597.
(21) Iltzsch, M. H.; Uber, S. S.; Tankersley, K. O.; el Kouni, M. H. Structure
Activity Relationship for the Binding of Nucleoside Ligands to
Adenosine Kinase from Toxoplasma gondii. Biochem. Pharmacol.
1995, 49, 1501–1512.
(22) el Kouni, M. H.; Guarcello, V.; Al Safarjalani, O. N.; Naguib, F. N. M.
Metabolism and Selective Toxicity of 6-Nitrobenzylinosine in Toxo-
plasma gondii. Antimicrob. Agents Chemother. 1999, 43, 2437–2443.
(23) Al Safarjalani, O. N.; Naguib, F. N. M.; el Kouni, M. H. Uptake of
Nitrobenzylthioinosine and Purine ꢀ-L-Nucleosides by Intracellular
Toxoplasma gondii. Antimicrob. Agents Chemother. 2003, 47, 3247–
3251.
(24) Yadav, V.; Chu, C. K.; Rais, R. H.; Al Safarjalani, O. N.; Guarcello,
V.; Naguib, F. N. M.; el Kouni, M. H. Synthesis, Biological Activity
and Molecular Modeling of 6-Benzylthioinosine Analogues as Sub-
versive Substrates of Toxoplasma gondii Adenosine Kinase. J. Med.
Chem. 2004, 47, 1987–1996.
(25) Rais, R. H.; Al Safarjalani, O. N.; Yadav, V.; Guarcello, V.; Kirk,
M.; Chu, C. K.; Naguib, F. N. M.; el Kouni, M. H. 6-Benzylthioinosine
Analogues as Subversive Substrate of Toxoplasma gondii Adenosine
Kinase: Activities and Selective Toxicities. Biochem. Pharmacol. 2005,
69, 1409–1419.
Acknowledgment. This research was supported by the U.S.
Public Health Service Grants AI-52838 from the National
Institutes of Health.
Supporting Information Available: Elemental analysis data of
compounds 6a-w, 7, and 12. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(26) Gupte, A.; Buolamwini, J. K.; Yadav, V.; Chu, C. K.; Naguib, F. N. M.;
el Kouni, M. H. 6-Benzylthioinosine Analogues: Promising Anti-
Toxoplasmic Agents as Inhibitors of the Mammalian Nucleoside
Transporter ENT1 es. Biochem. Pharmacol. 2005, 71, 69–73.
(27) Kim, Y. A.; Sharon, A.; Chu, C. K.; Rais, R. H.; Al Safarjalani, O. N.;
Naguib, F. N. M.; el Kouni, M. H. Synthesis, Biological Evaluation
and Molecular Modeling Studies of N6-Benzyladenosine Analogues
as Potential Anti-Toxoplasma Agents. Biochem. Pharmacol. 2007, 73,
1558–1572.
(28) Schumacher, M. A.; Scott, D. M.; Mathews, I. I.; Ealick, S. E.; Roos,
D. S.; Ullman, B.; Brennan, R. G. Crystal Structure of Toxoplasma
gondii Adenosine Kinase Reveal a Novel Catalytic Mechanism and
Prodrug Binding. J. Mol. Biol. 2000, 298, 875–893.
(29) Zhang, Y.; el Kouni, M. H.; Ealick, S. E. Structure of Toxoplasma
gondii Adenosine Kinase in Complex with an ATP Analog at 1.1 Å
Resolution. Acta Crystallogr. D 2006, 62, 140–145.
(30) Zhang, Y.; el Kouni, M. H.; Ealick, S. E. Substrate Analogs Induce
an Intermediate Conformational Change in Toxoplasma gondii Ad-
enosine Kinase. Acta Crystallgr. D 2007, 63, 126–134.
(1) Montoya, J. G.; Liesenfeld, O. Toxoplasmosis. Lancet 2004, 363,
1965–1976.
(2) Su, C.; Evans, D.; Cole, R. H.; Kissinger, J. C.; Ajioka, J. W.; Sibley,
L. D. Recent Expansion of Toxoplasma through Enhanced Oral
Transmission. Science 2003, 299, 414–416.
(3) Dubey, J. P. Advances in the Life Cycle of Toxoplasma gondii. Int.
J. Parasitol. 1998, 28, 1019–1024.
(4) Jones, J. L.; Kruszon-Moran, D.; Wilson, M.; McQuillan, G.; Navin,
T.; McAuley, J. B. Toxoplasma gondii Infection in the United States:
Seroprevalence and Risk Factors. Am. J. Epidemiol. 2001, 154, 357–
365.
(5) Mead, P. S.; Slutsker, L.; Dietz, V.; Caig, L. F.; Bresee, J. S.; Shapiro,
C.; Griffin, P. M.; Tauxe, R. V. Food-Related Illness and Death in
the United States. Emerging Infect. Dis. 1999, 5, 607–624.
(6) Luft, B. J.; Remington, J. S. Toxoplasmic Encephalitis in AIDS. Clin.
Infect. Dis. 1992, 15, 211–222.
(7) Porter, S. B.; Sande, M. Toxoplasmosis of the Central Nervous System
in the Acquired Immunodeficiency Syndrome. N. Engl. J. Med. 1992,
327, 1643–1648.
(31) Gerster, J. F.; Carpenter, B.; Robins, R. K.; Townsend, L. B.
Pyrrolopyrimidine Nucleosides. I. The Synthesis of 4-Substituted 7-(ꢀ-
D-Ribofuranosyl)pyrrolo[2,3-d]pyrimidines from Tubercidin. J. Med.
Chem. 1967, 10, 326–331.
(32) Suhadolnik, R. J. Pyrrolopyrimidine Nucleosides. In Nucleoside
Antibiotics; Wiley-Interscience: New York, 1970; pp 298-353.
(33) Ritch, P. S.; Glazer, R. I. Pyrrolo[2,3-d]pyrimidine nucleosides. In
DeVelopments in Cancer Chemotherapy; Glazer, R. I., Ed.; CRC Press:
Boca Raton, FL, 1984; pp 1-33.
(8) Dunn, D.; Wallon, M.; Peyron, F.; Petersen, E.; Peckham, C.; Gilbert,
R. Mother-to-Child Transmission of Toxoplasmosis: Risk Estimates
for Clinical Counseling. Lancet 1999, 353, 1829–1833.
(9) Remington, J. S.; McLeo, R.; Thuliez, P.; Desmonts, G. Toxoplasmosis
In Infectious Diseases of the Fetus and Newborn Infant, 6th ed.;
Remington, J. S., Klein, J. O., Wilson, C. B., Baker, C. J., Eds.;
Elsevier Saunders: Philadelphia, PA, 2006; pp 947-1091.
(10) Nielsen, H. V.; Innes, E. A.; Petersen, E.; Buxton, D. Strategies for
Development of Vaccines against Toxoplasma gondii. In Congenital

7-Deaza-6-benzylthioinosine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3945
(34) Olsen, D. B.; Eldrup, A. B.; Bartholomew, L.; Bhat, B.; Bosserman,
M. R.; Ceccacci, A.; Colwell, L. F.; Fay, J. F.; Flores, O. A.; Getty,
K. L.; Grobler, J. A.; LaFemina, R. L.; Markel, E. J.; Migliaccio, G.;
Prhavc, M.; Stahlhut, M. W.; Tomassini, J. E.; MacCoss, M.; Hazuda,
D. J.; Carroll, S. S. A 7-Deaza-adenosine Analog Is a Potent and
Selective Inhibitor of Hepatitis C Virus Replication with Excellent
Pharmacokinetic Properties. Antimicrob. Agents Chemother. 2004, 48,
3944–3953.
(35) Rosemeyer, H.; Seela, F. 171. Stereoselective Synthesis of Pyrrolo[2,3-
d]pyrimidine R- and ꢀ-D-Ribonucleosides from Anomerically Pure
D-Ribofuranosyl Chlorides: Solid-Liquid Phase-Transfer Glycosyla-
tion and 15N-NMR Spectra. HelV. Chim. Acta 1988, 71, 1573–1585.
(36) Ramasamy, K.; Imamura, N.; Robins, R. K.; Revankar, G. R. A Facile
and Improved Synthesis of Tubercidin and Certain Related Pyrrolo[2,3-
d]pyrimidine Nucleosides by the Stereospecific Sodium Salt Glyco-
sylation Procedure. J. Heterocycl. Chem. 1988, 25, 1893–1898.
(37) Moreau, C.; Wagner, G. K.; Weber, K.; Guse, A. H.; Potter, B. V. L.
Structural Determinants for N1/N7 Cyclization of Nicotinamide
Hypoxanthine 5′-Dinucleotide (NHD+) Derivatives by ADP-Ribosyl
Cyclase from Aplysia californica: Ca2+-Mobilizing Activity of
8-Substituted Cyclic Inosine 5′-Diphosphoribose Analogues in T-
Lymphocytes. J. Med. Chem. 2006, 49, 5162–5176.
(38) (a) For the synthesis of 4-nitrobenzylthiol: Molina, P.; Alajarin, M.;
Vilaplana, M. J.; Katritzky, A. R. One Pot Conversion of Alkyl-Halides
into Thiols under Mild Conditions. Tetrahedron Lett. 1985, 26, 469–
472. (b) For the synthesis of 4-methoxycarbonylbenzylthiol: Daines,
R. A.; Chambers, P. A.; Eggleston, D. S.; Foley, J. J.; Griswold, D. E.;
Haltiwanger, R. C.; Jakas, D. R.; Kingsbury, W. D.; Martin, L. D.;
Pendrak, I.; Schmidt, D. B.; Tzimas, M. N.; Sarau, H. M. (E)-3-[[[[6-
(2-Carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl]oxy]-2-pyridinyl]-
methyl]thio]methyl]benzoic Acid and Related Compounds: High
Affinity Leukotriene B4 Receptor Antagonists. J. Med. Chem. 1994,
37, 3327–3336. (c) For the synthesis of 4-cyanobenzylthiol and
3-nitrobenzylthiol: Bellas, M.; Tuleen, D. L.; Field, L. Organic
Disulfides and Related Substances. XXII. Substituted Benzyl 2-(n-
Decylamino)ethyl Disulfide Hydrochlorides. A Possible Neighboring-
Group Effect Involving Sulfur. J. Org. Chem. 1967, 32, 2591–2595.
(39) Tromp, R. A.; van Ameijde, S.; Pu¨tz, C.; Sundermann, C.; Sunder-
mann, B.; von Frijtag Drabbe Ku¨nzel, J. K.; Ijzerman, A. P. Inhibition
of Nucleoside Transport by New Analogues of 4-Nitrobenzylthioi-
nosine: Replacement of the Ribose Moiety by Substituted Benzyl
Groups. J. Med. Chem. 2004, 47, 5441–5450.
(40) (a) Ramzaeva, N.; Mittelbach, C.; Seela, F. 7-Halogenated 7-Deaza-
2′-deoxyinosines. HelV. Chim. Acta 1999, 82, 12–18. (b) Pudlo, J. S.;
Nassiri, M. R.; Kern, E. R.; Wotring, L. L.; Drach, J. C.; Townsend,
L. B. Synthesis, Antiproliferative, and Antiviral Activity of Certain
4-Substituted and 4,5-Disubstituted 7-[(1,3-Dihydroxy-2-propoxy)
methyl]pyrrolo[2,3-d]pyrimidines. J. Med. Chem. 1990, 33, 1984–
1992.
(41) Sullivan, W. J., Jr.; Chiang, C. W.; Wilson, C. M.; Naguib, F. N.; el
Kouni, M. H.; Donald, R. G.; Roos, D. S. Insertional Tagging of at
Least Two Loci Associated with Resistance to Adenine Arabinoside
in Toxoplasma gondii, and Cloning of the Adenosine Kinase Locus.
Mol. Biochem. Parasitol. 1999, 103, 1–14.
(42) Prime (Schrodinger Suite 2006), version 1.5; Schrodinger LLC: New
York, 2005.
(43) Maestro, version 7.5; Schrodinger LLC: New York, 2005.
(44) Glide, version 4.0; Schrodinger LLC: New York, 2005.
(45) Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R.
Novel Procedure for Modeling Ligand/Receptor Induced Fit Effects.
J. Med. Chem. 2006, 49, 534–553.
(46) Schrodinger Suite 2006; Schrodinger LLC: New York, 2005.
(47) MacroModel (Schrodinger Suite 2006), version 9.1; Schrodinger LLC:
New York, 2005.
(48) Cha, S. Kinetics of Enzyme Reactions with Competing Alternative
Substrates. Mol. Pharmacol. 1968, 4, 621–629.
(49) Pfefferkorn, E. R.; Pfefferkorn, L. C. Specific Labeling of Intracellular
Toxoplasma gondii with Uracil. J. Protozool. 1977, 24, 449–453.
JM800201S