Structure-activity relationships of carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase

Article abstract of DOI:10.1016/j.bmc.2010.04.003

Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding

Full text of DOI:10.1016/j.bmc.2010.04.003

Bioorganic & Medicinal Chemistry 18 (2010) 3403–3412
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of carbocyclic 6-benzylthioinosine analogues
as subversive substrates of Toxoplasma gondii adenosine kinase
Young Ah Kim a, Ravindra K. Rawal a, Jakyung Yoo a, Ashoke Sharon a, Ashok K. Jha a, Chung K. Chu a,
,
*
Reem H. Rais b, Omar N. Al Safarjalani b, Fardos N. M. Naguib b, Mahmoud H. el Kouni b
a Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602, USA
b Department of Pharmacology and Toxicology, Center for AIDS Research, University of Alabama School of Medicine, Birmingham, AL 35294, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity
against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of
the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsub-
stituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding
affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine
kinase active site to determine the probable binding mode. Experimental investigations and theoretical
calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In
agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated signifi-
Received 11 February 2010
Revised 31 March 2010
Accepted 1 April 2010
Available online 8 April 2010
Keywords:
Toxoplasmosis
Toxoplasma gondii adenosine kinase
Carbocyclic nucleoside
Molecular modeling
cant anti-toxoplasma activity (IC50 = 11.9
l
M) in cell culture without any apparent host-toxicity.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
tics of T. gondii adenosine kinase and the mammalian enzyme render
T. gondii adenosine kinase an excellent chemotherapeutic tar-
get.12,13 We have recently reported structure–activity relationship
studies of 6-benzylthioinosine,14–16 N6-benzyladenosine17 and
7-deaza-6-benzylthioinosine analogues18,19 as substrates of T. gon-
dii adenosine kinase. We have also reported that 6-benzylthioino-
sine15,16,20,21 and 7-deaza-6-benzylthioinosine analogues18,19 are
selectively toxic to T. gondii infected cells but not uninfected host
cells. In spite of the similarity in their structures, and unlike the 6-
benzylthioinosines, most of the N6-benzyladenosine analogues
were toxic to both infected and uninfected cells.17 These studies
demonstrated that the sulfur atom on the 6-position of the inosine
motif is indispensable for the selective binding to T. gondii adenosine
kinase, which results in selective toxicity against the parasite. It is
noteworthy that all of these aforementioned compounds are ribonu-
cleosides, consisting of a ribose ring as the sugar moiety.
As part of our continuing efforts to develop potent and selective
anti-toxoplasmic agents, we turned our attention to carbocyclic
nucleosides, wherein an oxygen atom of the ribose ring in 6-ben-
zylthioinosine analogues (II) is replaced by a methylene group to
make the corresponding carbocyclic nucleosides (I) (Fig. 1). One
of the advantages of the carbocyclic nucleosides is their metabolic
stability due to the absence of a typical glycosidic bond.22,23 The
carbocyclic nucleosides also have considerable effects on ring con-
formation and lipophilicity which may improve their therapeutic
potency.22,23 Specifically, we wanted to investigate the structural
effects of the carbasugar moiety on the binding of 6-benzylthioino-
sine to T. gondii adenosine kinase. Other molecular alterations were
Toxoplasma gondii is an obligate intracellular protozoan parasite
that infects a broad range of warm-blooded animals including hu-
mans.1–3 The prevalence of toxoplasmic infection is estimated to
be a billion people throughout the world.4,5 Toxoplasmic infection
is generally asymptomatic and self limiting because it is controlled
by the immune system of healthy individuals. However, T. gondii
causes severe disease in immunocompromised patients with AIDS,
organ transplant and leukemia.6,7 During pregnancy, infection with
T. gondii can be transmitted to the fetus leading to congenital toxo-
plasmosis of the fetus.8,9
Current therapeutic regimens are antifolate drugs such as the
combination of pyrimethamine and sulfadiazine, which synergisti-
cally inhibits folate metabolism.7,10,11 Unfortunately, this therapy
is not effective against T. gondii tissue cysts and should not be used
in pregnant women. Furthermore, this treatment causes side ef-
fects such as bone marrow depression and skin rashes attributed
to the sulfa drugs,7,9,10 which frequently result in discontinuation
of this therapy. Therefore, their application has been hampered
by limited effectiveness, host-toxicity and drug-resistance. Hence,
there is a continuing need to develop new and effective chemo-
therapeutic agents for long-term treatment of toxoplasmosis.
T. gondii adenosine kinase (EC.2.7.1.20) is a major enzyme of pur-
ine metabolism in the parasite. Differences between the characteris-
* Corresponding author. Tel.: +1 706 542 5379; fax: +1 706 542 5381.
E-mail addresses: dchu@rx.uga.edu, david.dr.chu@gmail.com (C.K. Chu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.003

3404
Y. A. Kim et al. / Bioorg. Med. Chem. 18 (2010) 3403–3412
Figure 1. Chemical structures of carbocyclic 6-benzylthioinosine and 6-benzylthioinosine analogues.
also accompanied by introducing substituents on the aromatic ring
of the 6-benzylthio group of this carbocyclic nucleoside. It was of
interest to ascertain how the carbasugar moiety would be accom-
modated within the T. gondii adenosine kinase active site in con-
junction with the glide XP docking studies. Herein, we report the
synthesis, structure–activity relationships and molecular modeling
studies of novel carbocyclic 6-benzylthioinosines as well as the
structural effects of the carbasugar moiety on their anti-toxoplas-
mic activities.
thiourea in ethanol provided the corresponding thione,28 which was
deprotected with aqueous trifluoroacetic acid to give a 6-mercapto-
purine derivative 8. Carbocyclic nucleoside analogues 9a–u were
synthesized via solution phase parallel synthesis (Scheme 2).29,30
The thione 8 served as a key intermediate, which was alkylated with
appropriate benzyl halide to afford target compounds 9a–u in good
yields after short column purification.
2.2. Structure–activity relationships
2. Results and discussion
2.1. Chemistry
Carbocyclic 6-benzylthioinosine (9a) and its analogues (9b–u)
were assayed for their binding affinity to purified T. gondii adeno-
sine kinase, and the results are shown in Table 1. In the previous
studies,18 replacement of the nitrogen atom at the 7-position by
CH function in the purine ring enhanced the binding affinity of
6-benzylthioinosine to T. gondii adenosine kinase, providing the
flexibility of the 6-benzylthio group movement to fit better in
the hydrophobic pocket at 6-position. This finding prompted us
to investigate the influence of sugar-based modification on binding
affinity by replacing the oxygen atom from the ribose ring by a
methylene group. Therefore, we decided to incorporate the carba-
sugar moiety, while the hydrophobic 6-benzylthio group was left
intact to preserve the selective activity against the parasite and
not the host as established in our previous studies.13,5–21
We synthesized a series of carbocyclic 6-benzylthioinosine ana-
logues for structure–activity relationships on T. gondii adenosine ki-
nase as shown in Schemes 1 and 2. Compound 2 was prepared from
D-ribose 1 in nine steps using previously developed methodology in
our laboratory, which allowed the preparation of multigram quanti-
ties of cyclopentanone 2.24–26 Compound 2 was stereoselectively re-
duced with sodium borohydride to provide the corresponding
alcohol 3 in quantitative yield. Compound 3 was treated with meth-
anesulfonyl chloride in the presence of Et3N to afford compound 4,
which was then displaced with sodium azide in DMF at 150 °C to
give carbocyclic azide 5. Reduction of compound 5 with 10% palla-
dium on carbon at 30 psi under hydrogen atmosphere,27 followed
by coupling with 5-amino-4,6-dichloropyrimidine produced com-
pound 6. The 6-chloropurine derivative 7 was prepared by cycliza-
tion of the compound 6 with ethyl orthoformate and a catalytic
amount of p-toluenesulfonic acid.24 Treatment of compound 7 with
The binding affinity (apparent Km values) of carbocyclic ana-
logue 9a with an unsubstituted benzylthio group was compared
to that of 6-benzylthioinosine as the corresponding ribonucleoside.
Compound 9a exhibited lower binding affinity (105
zyme than that of 6-benzylthioinosine (14.3 M). It appeared that
the carbasugar modification to the ribosyl group is detrimental to
lM) to the en-
l
Scheme 1. Synthesis of 6-mercaptopurine derivative 8. Reagents and conditions: (a) NaBH4, MeOH, 0 °C, 1 h, 96%; (b) MsCl, Et3N, CH2Cl2, 0 °C, 1 h, 92%; (c) NaN3, DMF, 150 °C,
6 h, 85%; (d) H2, 10% Pd/C, MeOH, 30 psi, 2 h; (e) 5-amino-4,6-dichloropyrimidine, Et3N, n-PrOH, reflux, 24 h, 68% (two steps); (f) CH(OEt)3, p-TsOH, rt, 14 h, 65%; (g) thiourea,
EtOH, reflux, 3 h; (h) TFA/H2O (2:1, v/v), 50 °C, 3 h, 52% (two steps).

Y. A. Kim et al. / Bioorg. Med. Chem. 18 (2010) 3403–3412
3405
Scheme 2. Synthesis of carbocyclic 6-benzylthioinosine analogues 9a–u. Reagents and conditions: (a) appropriate benzylhalide, NH4OH/H2O, rt, 11 h.
Table 1
more potent than the unsubstituted compound 9a. Interestingly,
meta substitutions such as m-nitro (9e, 26.6 M), m-trifluoro-
methyl (9f, 22.2 M), and m-methyl (9g, 26.8 M) exhibited al-
Binding affinities (apparent Km) of carbocyclic 6-benzylthioinosine (9a), its analogues
(9b–u) and 6-benzylthioinosine to Toxoplasma gondii adenosine kinase
l
l
l
most similar binding affinities. Therefore, it appears that there is
no difference in the binding affinity between electron-withdraw-
ing and electron-donating substituents at the meta-position.
Apparently, the nature of the substituent at the para-position has
a substantial impact on the binding affinity to the enzyme. Carbo-
R1
R5
R2
R4
R3
S
N
N
N
cyclic 6-(p-methylbenzylthio)inosine (9n, 7.5
gand displaying a 14-fold increase in the binding affinity relative to
the unsubstituted compound 9a (105 M). On the other hand,
introduction of cyano substituent at para-position resulted in a de-
creased binding affinity, 9l (p-cyano, 129 M), which was the
lM) was the best li-
N
l
HO
X
l
weakest ligand in this carbocyclic series. In the case of di-substi-
tuted carbocyclic analogues 9r–t, meta–para di-substitutions (9s,
OH OH
a
16.8
ortho–meta di-substitutions (9r, 60.7
(9t, 31.3 M). However, trisubstituted methyl group (9u, o,m,p-tri-
methyl, 63.1 M) showed decreased binding affinity in comparison
to single methyl substitution at the ortho (9d, 24.9 M), meta (9g,
26.8 M) or para (9n, 7.5 M) position.
l
M) resulted in an increase in the binding affinity relative to
Compound
X
R1
R2
R3
R4 R5
Km (lM)
l
M) or di-ortho substitutions
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
CH2
CH2
H
F
H
H
H
H
NO2
CF3
CH3
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
F
Cl
Br
NO2
CN
CO2CH3
CH3
SO2CH3
OCF3
OCH3
Cl
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
CH3
H
105.1 12.4
54.8 10.2
24.2 5.4
24.9 3.3
26.6 2.7
22.2 1.3
26.8 4.2
31.9 8.3
15.6 2.2
21.1 3.7
94.1 15.2
128.7 25.0
40.9 3.2
7.5 0.6
73.7 7.8
65.5 7.2
62.0 4.7
60.7 7.9
16.8 4.1
31.3 4.2
63.1 6.9
14.3 2.4
l
CH2 Cl
CH2 CH3
l
l
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
H
H
H
H
H
H
H
H
H
H
H
H
H
l
l
These results illustrate that the nature and position of the sub-
stituent(s) on the aromatic ring of the benzylthio group seem to
have distinct influences on the binding affinity of carbocyclic 6-
benzylthioinosine (Table 1). These results revealed a different pat-
tern of binding than that of 6-benzylthioinosine analogues.15,16
Previous structure–activity relationships studies on 6-benzylthioi-
nosines14–16 and 7-deaza-6-benzylthioinosines18,19 indicated that
compounds having the p-cyano substituent were among the best
ligands of T. gondii adenosine kinase while the carbocyclic nucleo-
side with same substituent (carbocyclic 6-(p-cyanobenzylthio)ino-
sine 9l had the weakest binding affinity. These results indicate that
the binding affinity for T. gondii adenosine kinase is sensitive to the
sugar motif conformational distribution at the enzyme binding
site. It is possible that the carbasugar moiety slightly changed
the overall orientation of these analogues inside the binding site
and thus carbocyclic nucleosides with the functionalized aromatic
group are recognized by the enzyme active site in a slightly differ-
ent manner than do the corresponding ribonucleosides.
CH2 Cl
9s
9t
CH2
CH2
H
F
Cl
H
CH3
H
9u
6-Benzyl-
thioinosine
CH2 CH3
O
H
H
a
All values represent the mean S.D. of at least two independent experiments
with three replica each as previously described.14–19
the binding. It can be, therefore, assumed that the oxygen atom of
the ribose ring is important, but not critical, for binding to the en-
zyme. Nevertheless, various substituents appended to the aromatic
ring of the benzylthio group of the carbocyclic nucleoside 9a re-
sulted in the regaining of its binding affinity. All of the carbocyclic
nucleoside analogues, with the exception of 9l (p-cyano), showed
an increased binding affinity to the enzyme as compared to the
unsubstituted compound 9a. Therefore, these substituents seem
to provide additional proper interactions with the surrounding res-
idues in the hydrophobic pocket at the 6-position. Single substitu-
tions at the ortho or meta-position resulted in an increase in the
binding affinity to the enzyme. They were approximately 2–5-fold
2.3. Evaluation of anti-toxoplasma activity
To determine whether these carbocyclic 6-benzylthioinosines
retain the anti-toxoplasmic activities of the 6-benzylthioino-
sines,15,16,20,21 the two best ligands, carbocyclic 6-(p-methylbenzyl-
thio)inosine (9n, 7.5
thio)inosine (9i, 15.6
l
M) and carbocyclic 6-(p-chlorolbenzyl-
l
M) (Table 1), were tested for their anti-toxo-
plasma efficacy and host-toxicity in tissue culture. Pyrimethamine
and sulfadiazine as chemotherapeutic agents for the treatment of
toxoplasmosis were used as references. As shown in Table 2 the
two analogues were effective against infection with the wild type
(RH) T. gondii in a dose-dependent manner, and neither of the two

3406
Y. A. Kim et al. / Bioorg. Med. Chem. 18 (2010) 3403–3412
analogues was found effective against infection with adenosine ki-
nase deficient strain (TgAKꢀ3).31 In agreement with their binding
affinities (Table 1), 9n and 9i showed potent cellular activity with
structure of the docking pose of 6-benzylthioinosine (Fig. 2c) and
carbocyclic 6-benzylthioinosine (9n) clearly reveals the adoption
of the anti-conformation and ring-mimicry. Figure 2c also shows
that the carbocyclic analogues can bind to the active site of T. gondii
adenosine kinase with a 20-endo puckered conformation. Further-
more, 6-benzylthioinosine and the p-methylbenzyl moiety of the
carbocyclic nucleoside 9n superimposes well with those of 6-ben-
zylthioinosine. From the docking and energetic results it is observed
that the compound 9n has more favorable energetics for the overall
interaction with the enzyme binding site. This is probably due to the
presence of the carbocyclic ring, which may provide better confor-
mational flexibility to the OH groups at the 2’- and 3’-postions to
interact with neighboring residues such as Asp24, Gly69 and
Asn73. Two direct protein–adenine hydrogen bonds are formed be-
tween the amide N atom of Ser70 and N3 of the purine ring as well as
IC50 values of 12 and 15
lM, respectively. Furthermore, 9n and 9i
exhibited close to, or better potency than pyrimethamine (IC50
=
16 lM) and sulfadiazine (IC50 = 27 lM). The lack of activity against
infection with the adenosine kinase deficient (TgAK3) strain of
T. gondii demonstrates that these compounds are active substrates
for T. gondii adenosine kinase in vivo as was as in vitro.
The two carbocyclic 6-benzylthioinosine analogues 9n and 9i
were also tested for their host-toxicity on human fibroblasts in cul-
ture using the MTT assay (Table 2). In this assay, neither of the two
compounds showed any substantial effect on the viability of the
uninfected host cells at concentrations up to 50 lM. Therefore,
these carbocyclic analogues can still exhibit selective toxicity
against the parasite and may hold a promise in the development
of anti-toxoplasmic agents.
c
the O of Thr140 and N7 of the purinering. The purinering is also sta-
bilized by stacking and van der Waals interactions with the side
chain of Try169. Additional interactions between the carbocyclic
analogues and the enzyme active site result from the substitutions
on the purine moiety. The 6-thiobenzyl group occupies the hydro-
phobic pocket at the 6-position and is involved in van der Waals
interaction with the surrounding residues such as Leu46, Leu142,
Phe201 and Tyr206.
2.4. Molecular modeling
T. gondii adenosine kinase31 is a key purine metabolic enzyme.
The T. gondii adenosine kinase is a 363-residue (39.3 kDa) mono-
meric protein that catalyzes the phosphorylation of adenosine to
adenosine 50-monophosphate (AMP), using ATP as a co-substrate.
Recent studies15,18,32–34 suggested that the enzyme becomes func-
tional through induced fit movement to accommodate various
classes of substrates, and leads to effective phosphate transfer
catalysis. Various co-crystal structures of T. gondii adenosine kinase
suggest that substituents at the 6-position of purine nucleoside
analogues are accommodated in a hydrophobic pocket of the en-
zyme. Our previous studies also highlighted the molecular basis
for the binding of various 6-substituted inosine analogues in the
active site of T. gondii adenosine kinase.15,18 In addition, these pre-
vious studies also demonstrated that substituted benzyl moieties
at the 6-position of the purine ring provided stronger binding
through utilization of the hydrophobic pocket of T. gondii adeno-
sine kinase.
The orientation of the 6-benzylthio substitution of compound 9l
was completely different from that of 9n (Fig. 2b). The distance be-
tween the 5’-OH group and the triphosphate chain of the b,c-meth-
ylene adenosine 50-triphosphate (AMP-PCP, a non-hydrolysable
ATP analogue) is critical for the phosphorylation. Compound 9l
looses the hydrogen bonding interaction between its 5’-OH group
and the
c-phosphate of AMP-PCP as well as that between the
amide N atom of Ser70 and the N3 of the purine. The presence of
the p-CN group not only changes the favorable orientation of p-
cyanobenzyl group but also does not favor the overall interaction
of 6-(p-cyanobenzylthio)inosine (9l) with the enzyme binding site
(glide XP score ꢀ5.17; total energy ꢀ46.7 kJ/mol, Table 3) and ulti-
mately leads to the least binding affinity.
In an effort to elucidate the structure–activity relationships ob-
served at the T. gondii and guide further SAR studies, molecular dock-
ing of the most potent inhibitor 6-(p-methylbenzylthio)inosine (9n)
and least potent 6-(p-cyanobenzylthio)inosine (9l) into adenosine
binding site of T. gondii adenosine kinase (1LII.pdb). The binding
mode of compounds 9n and 9l are depicted in Figure 2a and b,
respectively. The presence of hydrophobic methyl group on the
6-benzyl substitution favors the overall interaction (glideXP score,
ꢀ8.15; total energy, ꢀ114.2 kJ/mol; Table 3) and shows that the car-
basugar adopted an appropriate conformation. The superimposed
3. Conclusion
In conclusion, we have described the synthesis of a series of
new carbocyclic 6-benzylthioinosine analogues from D-ribose as
potent and selective anti-toxoplasmic subversive substrates. We
have successfully used parallel solution phase synthesis in incorpo-
ration of the substituted benzyl group, which allowed the rapid
synthesis and examination of structure–activity relationships of
various carbocyclic 6-benzylthioinosines. The present experimen-
tal investigations and theoretical calculations revealed that the
Table 2
The effect of carbocyclic 6-(p-methylbenzylthio)inosine (9n), carbocyclic 6-(p-chlorolbenzylthio)inosine (9i), and therapeutic compounds on host-toxicitya and percent survivalb
of wild type (RH) and adenosine kinase deficient (TgAKꢀ3) strains of Toxoplasma gondii grown in human fibroblasts in culture
Compound
9n (p-CH3)
9i (p-Cl)
Infection
Concentration (
10
lM)
IC50 (lM)
0
5
25
50
Wild type (RH)
TgAKꢀ3
None
100
100
100
100
100
100
100
100
100
100
43
99
100
73
100
100
99
101
93
2.5
100
100
12
100
100
55
100
58
100
0
0
11.9 0.4
14.5 1.3
100
100
0
100
100
25
108
53
100
100
100
0
100
100
23
108
46
102
Wild type (RH)
TgAKꢀ3
None
Wild type (RH)
None
Wild type (RH)
None
Pyrimethaminec
Sulfadiazinea
16.1 2.5
27.3 3.3
98
a
b
c
Host-toxicity of uninfected cells was measured by MTT method in at least two independent experiments each of three replica as previously described.15–21
Percent survival of parasites was measured by incorporation of [5,6-13H]uracil in at least two independent experiments of three replica each as previously described.15–21
Therapeutic compounds.

Y. A. Kim et al. / Bioorg. Med. Chem. 18 (2010) 3403–3412
3407
Figure 2. (a) Binding mode of carbocyclic 6-(p-methylbenzylthio)inosine (9n, carbon color gray) showing the polar interaction as well as orientation of the methyl group,
which is more towards the hydrophobic pocket. (b) Superimposed structure of the most active substrate carbocyclic 6-(p-methylbenzylthio)inosine (9n, carbon color gray)
and the least active carbocyclic 6-(p-cyanobenzylthio)inosine (9l, carbon color green) showing difference in the orientation of the p-methylbenzyl and p-cyanobenzyl group
inside the binding pocket. H-atoms are not shown for the sake of clarity. (c) Superimposed structure of the carbocyclic 6-(p-methylbenzylthio)inosine (9n, carbon color gray)
and 6-benzylthioinosine (carbon color green) showing the same binding mode with the nearby residue in the active site with hydrophobic surface area of Leu46, Phe201 and
Tyr206.
Table 3
4. Experimental section
XP GScore and MBAE (multi-ligand bimolecular association with energetics) calcu-
lation of carbocyclic 6-benzylthioinosine analogues after Glide XP docking and energy
minimization
4.1. Chemistry: general procedures
Compound Km
(
l
M)
XP GScore Energy difference results (
Total energy vdWa
Electrostatic
ꢀ41.77 ꢀ275.90
ꢀ10.23 ꢀ425.68
DE, kJ/mol)
[8-14C]Adenosine (55 Ci/mol) and [5,6-13H]uracil were pur-
chased from Moravek Biochemicals. RPMI-1640 medium from GIB-
CO BRL; fetal bovine serum (FBS) from HyClone Laboratories. All
other chemicals and compounds were obtained from Sigma Chem-
ical Co. or Fisher Scientific. 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. High res-
olution mass spectra were recorded on an Agilent 1100 series LC
with Waters LCT Premier™. NMR spectra were recorded on a
Varian Inova 500 spectrometer and chemical shifts are expressed
in parts per million (d) relative to tetramethylsilane as internal ref-
erence. Chemical shifts (d) 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 chromatography.
Elemental analyses were performed by Atlantic Microlab Inc., Nor-
cross, GA.
9l (p-CN)
9n (p-CH3)
128.7 25.0 ꢀ5.17
7.5 0.6 ꢀ8.15
ꢀ46.65
ꢀ114.17
a
van der Waals interaction.
oxygen atom in sugar moiety is important, but not critical, for the
binding to the enzyme because of the preference of the enzyme for
ribose rather than carbasugar ring in the pentose binding region.
The replacement of the ribose ring of the parent compound 6-ben-
zylthioinosine with a carbasugar moiety resulted in appreciable
changes in binding affinities. Nevertheless, there is significant con-
formational similarity between carbocyclic 6-benzylthioinosine
and the parent compound 6-benzylthioinosine. The molecular dock-
ing studies demonstrated the adaptation of an anti-conformation
and overall ligand-fitting into the active site of T. gondiiadenosine ki-
nase by carbocylic nucleosides. The extended functionality of the
aromatic ring of the carbocyclic nucleosides by substituents such
as p-methyl led to improvement in the binding affinity. Among the
synthesized analogues, carbocyclic 6-(p-methylbenzylthio)inosine
(9n) was the best ligand and also showed high potency in terms of
the high anti-toxoplasmic efficacy and lack of host-toxicity in cell-
based studies. These observations could provide useful guidance to-
wards the development of novel carbocyclicnucleosides as potential
anti-toxoplasmic agents.
4.1.1. (1S,2S,3R,4R)-4-(tert-Butoxymethyl)-2,3-(isopropyliden-
edioxy)cyclopentan-1-ol (3)
A solution of compound 2 (1.00 g, 4.13 mmol) in MeOH (10.3 mL)
was treated with sodium borohydride (0.312 g, 8.25 mmol) at 0 °C
and then stirred for 1 h. After the reaction mixture was quenched
with water, it was diluted with EtOAc and then washed with brine.
The organic layer was dried over Na2SO4 and concentrated to

3408
Y. A. Kim et al. / Bioorg. Med. Chem. 18 (2010) 3403–3412
dryness in vacuo. The residue was purified by silica gel column chro-
matography (n-hexane/EtOAc = 9:1) to give 3 (0.970 g, 96%) as an
column chromatography (n-hexane/EtOAc = 4:1) to give 7 (0.394 g,
65%) as an oil: ½a D27
= ꢀ41.92 (c 0.64, CHCl3); UV (MeOH) kmax
ꢁ
oil: ½a 2D6
ꢁ
= ꢀ16.67 (c 0.96, CHCl3) [lit.24
½
a 2D6
ꢁ
= ꢀ16.95 (c 1.59,
264.0 nm; 1H NMR (500 MHz, CDCl3) d 1.23 (s, 9H), 1.35 (s, 3H),
1.61 (s, 3H), 2.41 (m, 1H), 2.51 (m, 1H), 2.63 (m, 1H), 3.52 (m,
1H), 3.57 (m, 1H), 4.66 (m, 1H), 4.94 (m, 1H), 4.99 (m, 1H), 8.31
(s, 1H), 8.78 (s, 1H); 13C NMR (125 MHz, CDCl3) d 25.2, 27.5, 27.6,
33.8, 43.8, 62.1, 62.4, 73.0, 81.7, 84.3, 113.4, 118.5, 132.1, 144.3,
151.0, 151.7; HRMS [M+H]+ m/z calcd for C18H26ClN4O3 381.1693,
found 381.1693.
CHCl3)]; 1H NMR (500 MHz, CDCl3) d 1.14 (s, 9H), 1.36 (s, 3H), 1.49
(s, 3H), 1.84 (m, 2H), 2.21 (m, 1H), 2.43 (d, J = 8.5 Hz, 1H), 3.21 (dd,
J1 = 9.0 Hz, J2 = 4.5 Hz, 1H), 3.32 (dd, J1 = 8.5 Hz, J2 = 5.0 Hz, 1H),
4.24 (m, 1H), 4.45 (m, 2H); 13C NMR (125 MHz, CDCl3) d 24.2, 26.1,
27.3, 35.7, 42.1, 63.0, 72.0, 72.5, 79.6, 83.4, 110.5.
4.1.2. (1S,2S,3R,4R)-4-(tert-Butoxymethyl)-2,3-(isopropyliden-
edioxy)-1-[(methylsulfonyl)oxy]-cyclopentane (4)
4.1.6. (10R,20S,30R,40R)-9-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-mercaptopurine (8)
A solution of compound 3 (3.24 g, 13.3 mmol) in CH2Cl2
(66.0 mL) was treated with methanesulfonyl chloride (1.1 mL,
13.7 mmol) and Et3N (2.8 mL, 19.9 mmol) at 0 °C. The reaction
mixture was stirred for 1 h at 0 °C and then washed with brine.
The organic layer was dried over Na2SO4, and concentrated to dry-
ness in vacuo. The residue was purified by silica gel column chro-
matography (n-hexane/EtOAc = 8:1) to give 4 (3.94 g, 92%) as an
A solution of compound 7 (1.01 g, 2.65 mmol) and thiourea
(0.404 g, 5.30 mmol) in EtOH (13.0 mL) was heated to reflux for
3 h. The reaction mixture was concentrated to dryness and the res-
idue was purified by short column chromatography on silica gel
(CH2Cl2/MeOH = 95:5) to give a solid. The resultant compound
was dissolved in 18.0 mL of trifluoroacetic acid/H2O (2:1, v/v)
and heated at 50 °C for 3 h. After evaporation of the solvent in va-
cuo, the residue was triturated with EtOH and the resulting solid
was filtered and dried to give 8 (0.389 g, 52% for two steps) as a
oil: ½a 2D7
ꢁ
= ꢀ59.49 (c 1.13, CHCl3); 1H NMR (500 MHz, CDCl3) d
1.13 (s, 9H), 1.31 (s, 3H), 1.47 (s, 3H), 1.93 (m, 1H), 2.24 (m, 2H),
3.05 (s, 3H), 3.24 (m, 1H), 3.35 (m, 1H), 4.43 (d, J = 5.5 Hz, 1H),
4.59 (t, J = 5.5 Hz, 1H), 5.11 (m, 1H); 13C NMR (125 MHz, CDCl3) d
24.5, 26.2, 27.3, 32.8, 38.6, 41.6, 63.1, 72.9, 78.7, 80.0, 83.5, 111.2.
white solid: mp 260–262 °C (dec); ½a D24
= ꢀ63.39 (c 0.37, DMSO);
ꢁ
UV (H2O) kmax 321.0 nm; 1H NMR (500 MHz, DMSO-d6) d 1.66
(m, 1H), 2.01 (m, 1H), 2.22 (m, 1H), 3.44 (m, 2H), 3.80 (m, 1H),
4.25 (m, 1H), 4.70 (m, 1H), 8.16 (s, 1H), 8.38 (s, 1H); 13C NMR
(125 MHz, CDCl3) d 29.9, 45.8, 59.8, 63.4, 72.0, 75.4, 135.9, 142.5,
145.0, 176.2; HRMS [M+H]+ m/z calcd for C11H15N4O3S 283.0865,
found 283.0816.
4.1.3. (1R,2S,3R,4R)-1-Azido-4-(tert-butoxymethyl)-2,3-(isopro-
pylidenedioxy) cyclopentane (5)
A solution of compound 4 (3.94 g, 12.2 mmol) and sodium azide
(7.93 g, 122 mmol) in DMF (122.0 mL) was heated at 150 °C for 6 h.
Then it was diluted with water and extracted with EtOAc. The com-
bined organic layer was washed with brine and dried over Na2SO4.
After the solvent was removed in vacuo, the residue was purified
by silica gel column chromatography (n-hexane/EtOAc = 9:1) to
4.1.7. General procedure for the synthesis of carbocyclic
nucleoside analogues (9a–u)
Compound 8 (60.0 mg, 0.213 mmol) was added to each reaction
vessel of Argonaut Quest 210 Organic Synthesizer and then H2O
(4.0 mL) and concentrated NH4OH (0.02 mL) were added. To this
solution, the appropriate benzylbromides (1.3 equiv, 0.266 mmol)
were added. The reaction mixtures were stirred vigorously (up-
ward stroke = 50%, time = 4 s) for 11 h at room temperature. The
reaction vessels were drained and the collected crude material
was co-evaporated with ethanol in vacuo. The residues were puri-
fied by short column chromatography on silica gel (CH2Cl2/
MeOH = 95:5) to give the desired products as white solids.
give 5 (2.79 g, 85%) as an oil: ½a D27
ꢁ
= -48.95 (c 0.67, CHCl3); 1H
NMR (500 MHz, CDCl3) d 1.18 (s, 9H), 1.30 (s, 3H), 1.46 (s, 3H),
1.71 (m, 1H), 2.27 (m, 2H), 3.29 (m, 1H), 3.38 (m, 1H), 3.95 (m,
1H),), 4.40 (dd, J1 = 6.5 Hz, J2 = 2.0 Hz, 1H), 4.47 (dd, J1 = 6.5 Hz,
J2 = 2.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) d 24.5, 26.9, 27.5,
31.7, 45.4, 62.2, 67.0, 72.7, 82.6, 85.1, 111.3.
4.1.4. (10R,20S,30R,40R)-1-(5-Amino-6-chloropyrimidylamino)-4-
(tert-butoxymethyl)-2,3-(isopropyl-idenedioxy)cyclopentane (6)
A solution of compound 5 (1.00 g, 4.13 mmol) in MeOH (10.3 mL)
was shaken under 30 psi of a hydrogen atmosphere in the presence
of 10% Pd/C (300 mg) for 2 h. The reaction mixture was filtered
through a pad of celite and then concentrated to dryness in vacuo.
The residue was dried and used next reaction without further puri-
fication. The crude product in n-PrOH (20.7 mL) was treated with 5-
amino-4,6-dichloropyrimidine (0.745 g, 4.54 mmol) and Et3N
(2.9 mL, 20.7 mmol). The reaction mixture was heated to reflux for
24 h. After the solvent was removed in vacuo, the residue was puri-
fied by silica gel column chromatography (CH2Cl2/MeOH = 95:5) to
4.1.7.1. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)cy-
clopentan-1-yl]-6-benzylthiopurine (9a). Yield 90%; mp 110–
111 °C; ½a 2D7
ꢁ
= ꢀ48.32 (c 0.27, MeOH); UV (H2O) kmax 293.0 nm (
e
18,100, pH 2), 293.0 nm (
e
18,600, pH 7), 293.0 nm ( 18,700, pH
e
11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H), 2.28 (m, 1H),
2.49 (m, 1H), 3.73 (m, 2H), 4.08 (m, 1H), 4.60 (m, 1H), 4.71 (s,
2H), 4.96 (m, 1H), 7.27 (m, 1H), 7.32 (m, 2H), 7.49 (d, J = 7.0 Hz,
2H), 8.46 (s, 1H), 8.75 (s, 1H); 13C NMR (125 MHz, CD3OD) d 28.6,
32.1, 45.5, 60.7, 63.3, 72.3, 75.0, 127.0, 128.2, 128.8, 130.9, 137.6,
143.5, 148.6, 151.2, 160.1. Anal. Calcd for C18H20N4O3Sꢂ0.3H2O: C,
57.22; H, 5.50; N, 14.83; S, 8.49. Found: C, 56.96; H, 5.65; N,
14.75; S, 8.42.
give 6 (1.04 g, 68% for two steps) as an oil: ½a D27
= ꢀ47.85 (c 0.81,
ꢁ
CHCl3); 1H NMR (500 MHz, CDCl3) d 1.22 (s, 9H), 1.28 (s, 3H), 1.48
(s, 3H), 1.53 (m, 1H), 2.41 (m, 1H), 2.62 (m, 1H), 3.42 (dd,
J1 = 9.5 Hz, J2 = 3.5 Hz, 1H), 3.51 (m, 3H), 4.41 (m, 1H), 4.48 (m,
1H), 4.54 (m, 1H), 5.84 (d, J = 8.5 Hz, 1H), 8.06 (s, 1H); 13C NMR
(125 MHz, CDCl3) d 24.6, 27.1, 27.6, 34.2, 45.2, 58.0, 63.4, 73.8,
83.7, 87.3, 110.7, 122.4, 141.9, 149.3, 154.1; HRMS [M+H]+ m/z calcd
for C17H28ClN4O3 371.1850, found 371.1874.
4.1.7.2.
(10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(2-fluorobenzyl-thio)purine
(9b). Yield
= ꢀ43.08 (c 0.30, MeOH); UV (H2O) kmax
21,100, pH 7), 291.0 nm (
94%; mp 104–106 °C; ½a D27
ꢁ
292.0 nm (
e
21,000, pH 2), 291.0 nm (
e
e
21,100, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H), 2.29 (m,
1H), 2.49 (m, 1H), 3.74 (m, 2H), 4.08 (m, 1H), 4.60 (dd, J1 = 8.5 Hz,
J2 = 5.5 Hz, 1H), 4.75 (s, 2H), 4.96 (m, 1H), 7.13 (m, 2H), 7.31 (m,
1H), 7.60 (t, J = 7.5 Hz, 1H), 8.46 (s, 1H), 8.76 (s, 1H); 13C NMR
(125 MHz, CD3OD) d 25.3, 28.6, 45.5, 60.7, 63.3, 72.3, 74.9, 114.9
(d, J = 21.4 Hz), 124.0, 124.7 (d, J = 14.4 Hz), 129.1 (d, J = 8.1 Hz),
130.9, 131.1, 143.6, 148.7, 151.1, 159.6, 161.1 (d, J = 245.1 Hz).
4.1.5. (10R,20S,30R,40R)-9-[4-(tert-Butoxymethyl)-2,3-(isopropyl-
idenedioxy)cyclopentan-1-yl]-6-chloropurine (7)
A solution of compound 6 (0.588 g, 1.59 mmol) and p-toluene-
sulfonic acid momohydrate (15.1 mg, 0.0793 mmol) in ethyl ortho-
formate (5.3 mL) was stirred for 14 h at room temperature and
then concentrated in vacuo. The residue was purified by silica gel

Y. A. Kim et al. / Bioorg. Med. Chem. 18 (2010) 3403–3412
3409
Anal. Calcd for C18H19FN4O3Sꢂ0.2H2O: C, 54.87; H, 4.96; N, 14.22; S,
J = 8.0 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.26 (d, J = 7.5 Hz, 1H), 7.30
(s, 1H), 8.46 (s, 1H), 8.74 (s, 1H); 13C NMR (125 MHz, CD3OD) d
20.1, 28.6, 32.2, 45.5, 60.7, 63.3, 72.3, 75.0, 125.9, 127.7, 128.1,
129.4, 130.8, 137.3, 138.0, 143.4, 148.5, 151.2, 160.2. Anal. Calcd
for C19H22N4O3Sꢂ0.1H2O: C, 58.77; H, 5.76; N, 14.43; S, 8.26. Found:
C, 58.56; H, 5.83; N, 14.30; S, 8.23.
8.14. Found: C, 54.78; H, 5.01; N, 14.12; S, 8.03.
4.1.7.3. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)cy-
clopentan-1-yl]-6-(2-chlorobenzyl-thio)purine (9c). Yield 90%;
mp 100–101 °C; ½a D28
ꢁ
= ꢀ42.09 (c 0.14, MeOH); UV (H2O) kmax
292.0 nm (e 21,600, pH 2), 292.0 nm (e 21,400, pH 7), 292.0 nm
(e
21,400, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H),
4.1.7.8. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)cy-
clopentan-1-yl]-6-(4-fluorobenzyl-thio)purine (9h). Yield 89%;
2.28 (m, 1H), 2.49 (m, 1H), 3.74 (m, 2H), 4.08 (m, 1H), 4.60 (dd,
J1 = 8.5 Hz, J2 = 5.5 Hz, 1H), 4.84 (s, 2H), 4.97 (m, 1H), 7.28 (m,
2H), 7.45 (m, 1H), 7.69 (m, 1H), 8.46 (s, 1H), 8.77 (s, 1H); 13C
NMR (125 MHz, CD3OD) d 28.6, 29.9, 45.5, 60.7, 63.3, 72.3, 75.0,
126.8, 128.8, 129.2, 130.9, 131.1, 134.0, 135.3, 143.6, 148.6,
151.1, 159.7. Anal. Calcd for C18H19ClN4O3S: C, 53.13; H, 4.71; N,
13.77; S, 7.88. Found: C, 52.84; H, 4.86; N, 13.56; S, 7.74.
mp 114–116 °C; ½a D27
ꢁ
= ꢀ35.71 (c 0.21, MeOH); UV (H2O) kmax
293.0 nm (e 20,200, pH 2), 293.0 nm (e 20,200, pH 7), 292.0 nm
(
e
19,700, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H),
2.29 (m, 1H), 2.49 (m, 1H), 3.74 (m, 2H), 4.09 (m, 1H), 4.60 (dd,
J1 = 9.0 Hz, J2 = 5.5 Hz, 1H), 4.69 (s, 2H), 4.96 (m, 1H), 7.05 (t,
J = 8.5 Hz, 2H), 7.52 (m, 2H), 8.46 (s, 1H), 8.74 (s, 1H); 13C NMR
(125 MHz, CD3OD) d 28.6, 31.2, 45.5, 60.7, 63.3, 72.3, 74.9, 114.8
(d, J = 21.5 Hz), 130.7 (d, J = 8.1 Hz), 130.9, 133.9, 143.5, 148.6,
151.2, 159.8, 162.1 (d, J = 243.3 Hz). Anal. Calcd for
C18H19FN4O3Sꢂ0.2H2O: C, 54.87; H, 4.96; N, 14.22; S, 8.14. Found:
C, 54.72; H, 4.98; N, 14.12; S, 7.99.
4.1.7.4.
(10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(2-methylbenzyl-thio)purine (9d). Yield
91%; mp 92–93 °C; ½a D28
ꢁ
= -45.57 (c 0.14, MeOH); UV (H2O) kmax
294.0 nm (e 21,800, pH 2), 293.0 nm (e 21,900, pH 7), 293.0 nm
(
e
21,900, pH 11); 1H NMR (500 MHz, CD3OD) d 2.05 (m, 1H),
2.29 (m, 1H), 2.47 (s, 3H), 2.51 (m, 1H), 3.74 (m, 2H), 4.09 (m,
1H), 4.61 (dd, J1 = 8.5 Hz, J2 = 5.5 Hz, 1H), 4.73 (s, 2H), 4.96 (m,
1H), 7.19 (m, 3H), 7.46 (d, J = 8.0 Hz, 1H), 8.46 (s, 1H), 8.77 (s,
1H); 13C NMR (125 MHz, CD3OD) d 18.1, 28.6, 30.6, 45.5, 60.7,
63.3, 72.3, 75.0, 125.8, 127.5, 129.8, 130.1, 130.8, 134.7, 136.8,
143.5, 148.5, 151.2, 160.3. Anal. Calcd for C19H22N4O3Sꢂ0.2H2O: C,
58.50; H, 5.79; N, 14.36; S, 8.22. Found: C, 58.36; H, 5.98; N,
14.27; S, 8.23.
4.1.7.9. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)cy-
clopentan-1-yl]-6-(4-chlorobenzyl-thio)purine (9i). Yield 90%;
mp 107–108 °C; ½a D27
ꢁ
= ꢀ44.44 (c 0.34, MeOH); UV (H2O) kmax
293.0 nm (e 21,700, pH 2), 293.0 nm (e 21,800, pH 7), 293.0 nm
(
e
21,300, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H),
2.29 (m, 1H), 2.49 (m, 1H), 3.73 (m, 2H), 4.08 (m, 1H), 4.60 (dd,
J1 = 8.5 Hz, J2 = 5.5 Hz, 1H), 4.68 (s, 2H), 4.96 (m, 1H), 7.31 (d,
J = 7.0 Hz, 2H), 7.49 (d, J = 7.5 Hz, 2H), 8.46 (s, 1H), 8.74 (s, 1H);
13C NMR (125 MHz, CD3OD) d 28.6, 31.2, 45.5, 60.7, 63.3, 72.3,
74.9, 128.2, 130.4, 130.9, 132.7, 136.9, 143.6, 148.6, 151.2, 159.6.
Anal. Calcd for C18H19ClN4O3S: C, 53.13; H, 4.71; N, 13.77; S,
7.88. Found: C, 52.99; H, 4.79; N, 13.61; S, 7.67.
4.1.7.5.
(10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(3-nitrobenzyl-thio)purine (9e). Yield 88%;
mp 84–86 °C; ½a 2D7
ꢁ
= ꢀ39.93 (c 0.24, MeOH); UV (H2O) kmax
287.0 nm (e 29,900, pH 2), 287.0 nm (e 11,100, pH 7), 286.0 nm
(e
9900, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H), 2.28
4.1.7.10. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)cy-
clopentan-1-yl]-6-(4-bromobenzyl-thio)purine (9j). Yield 92%;
(m, 1H), 2.49 (m, 1H), 3.73 (m, 2H), 4.08 (m, 1H), 4.60 (dd,
J1 = 8.5 Hz, J2 = 5.5 Hz, 1H), 4.81 (s, 2H), 4.97 (m, 1H), 7.57 (t,
J = 8.0 Hz, 1H), 7.94 (d, J = 7.0 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H),
8.43 (s, 1H), 8.48 (s, 1H), 8.75 (s, 1H); 13C NMR (125 MHz, CD3OD)
d 28.5, 31.0, 45.5, 60.7, 63.3, 72.2, 74.9, 121.7, 123.6, 129.3, 131.0,
135.1, 140.9, 143.8, 148.2, 148.7, 151.2, 159.0. Anal. Calcd for
C18H19N5O5S: C, 51.79; H, 4.59; N, 16.78; S, 7.68. Found: C,
51.61; H, 4.77; N, 16.58; S, 7.67.
mp 152–153 °C; ½a D27
ꢁ
= ꢀ36.57 (c 0.17, MeOH); UV (H2O) kmax
293.0 nm (e 21,500, pH 2), 293.0 nm (e 21,200, pH 7), 293.0 nm
(
e
21,400, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H),
2.28 (m, 1H), 2.49 (m, 1H), 3.73 (m, 2H), 4.08 (m, 1H), 4.60 (dd,
J1 = 8.5 Hz, J2 = 5.5 Hz, 1H), 4.67 (s, 2H), 4.96 (m, 1H), 7.45 (m,
4H), 8.47 (s, 1H), 8.74 (s, 1H); 13C NMR (125 MHz, CD3OD) d 28.6,
31.3, 45.5, 60.7, 63.3, 72.3, 75.0, 120.6, 130.7, 130.9, 131.2, 137.3,
143.6, 148.6, 151.2, 159.6. Anal. Calcd for C18H19BrN4O3Sꢂ0.2H2O:
C, 47.52; H, 4.30; N, 12.32; S, 7.05. Found: C, 47.32; H, 4.26; N,
12.13; S, 6.87.
4.1.7.6.
(10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(3-trifluoro-methylbenzylthio)purine
(9f). Yield 87%; mp 118–119 °C; ½a D27
ꢁ
= ꢀ42.20 (c 0.33, MeOH);
UV (H2O) kmax 292.0 nm (
e
21,000, pH 2), 292.0 nm (
e
20,500, pH
4.1.7.11. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)cy-
clopentan-1-yl]-6-(4-nitrobenzyl-thio)purine (9k). Yield 87%;
7), 292.0 nm (
e
20,500, pH 11); 1H NMR (500 MHz, CD3OD) d
2.04 (m, 1H), 2.28 (m, 1H), 2.49 (m, 1H), 3.73 (m, 2H), 4.08 (m,
1H), 4.60 (dd, J1 = 8.5 Hz, J2 = 5.5 Hz, 1H), 4.78 (s, 2H), 4.96 (m,
1H), 7.52 (t, J = 7.5 Hz, 1H), 7.56 (d, J = 7.5 Hz, 1H), 7.79 (d,
J = 8.0 Hz, 1H), 7.84 (s, 1H), 8.47 (s, 1H), 8.75 (s, 1H); 13C NMR
(125 MHz, CD3OD) d 28.5, 31.3, 45.5, 60.7, 63.3, 72.2, 74.9, 123.6,
125.5, 128.9, 130.3 (q, J = 32.0 Hz), 131.0, 132.6, 139.7, 143.7,
148.7, 151.1, 159.3. Anal. Calcd for C19H19F3N4O3Sꢂ0.1H2O: C,
51.60; H, 4.38; N, 12.67; S, 7.25. Found: C, 51.45; H, 4.41; N,
12.57; S, 7.18.
mp 89–90 °C; ½a 2D7
ꢁ
= ꢀ46.89 (c 0.31, MeOH); UV (H2O) kmax
288.0 nm (e 27,300, pH 2), 288.0 nm (e 27,500, pH 7), 288.0 nm
(
e
27,300, pH 11); 1H NMR (500 MHz, CD3OD) d 2.03 (m, 1H),
2.28 (m, 1H), 2.48 (m, 1H), 3.73 (m, 2H), 4.08 (m, 1H), 4.59 (dd,
J1 = 9.0 Hz, J2 = 5.0 Hz, 1H), 4.81 (s, 2H), 4.96 (m, 1H), 7.76 (d,
J = 8.5 Hz, 2H), 8.19 (d, J = 8.5 Hz, 2H), 8.48 (s, 1H), 8.74 (s, 1H);
13C NMR (125 MHz, CD3OD) d 28.6, 31.0, 45.5, 60.7, 63.2, 72.2,
74.9, 123.1, 129.9, 131.0, 143.8, 146.3, 147.1, 148.8, 151.2, 158.9.
Anal. Calcd for C18H19N5O5S: C, 51.79; H, 4.59; N, 16.78; S, 7.68.
Found: C, 51.73; H, 4.72; N, 16.66; S, 7.60.
4.1.7.7.
(10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(3-methylbenzyl-thio)purine
(9g). Yield
4.1.7.12. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(4-cyanobenzyl-thio)purine (9l). Yield 92%;
89%; mp 112–113 °C; ½a D28
ꢁ
= ꢀ38.92 (c 0.16, MeOH); UV (H2O) kmax
294.0 nm (
e
22,500, pH 2), 293.0 nm (
e
22,400, pH 7), 293.0 nm (
e
mp 114–116 °C; ½a D24
ꢁ
= ꢀ37.61 (c 0.29, MeOH); UV (H2O) kmax
22,200, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H), 2.29 (m,
1H), 2.33 (s, 3H), 2.49 (m, 1H), 3.74 (m, 2H), 4.09 (m, 1H), 4.60 (dd,
J1 = 9.0 Hz, J2 = 5.5 Hz, 1H), 4.66 (s, 2H), 4.96 (m, 1H), 7.08 (d,
292.0 nm (e 21,500, pH 2), 292.0 nm (e 21,200, pH 7), 292.0 nm
(e
21,400, pH 11); 1H NMR (500 MHz, CD3OD) d 2.03 (m, 1H),
2.28 (m, 1H), 2.49 (m, 1H), 3.73 (m, 2H), 4.08 (m, 1H), 4.60 (dd,

3410
Y. A. Kim et al. / Bioorg. Med. Chem. 18 (2010) 3403–3412
J1 = 8.5 Hz, J2 = 6.0 Hz, 1H), 4.76 (s, 2H), 4.96 (m, 1H), 7.69 (m, 4H),
8.48 (s, 1H), 8.74 (s, 1H); 13C NMR (125 MHz, CD3OD) d 28.5, 31.3,
45.5, 60.7, 63.2, 72.2, 74.9, 110.6, 118.2, 129.8, 131.0, 132.0, 143.7,
144.3, 148.7, 151.1, 159.0. Anal. Calcd for C19H19N5O3Sꢂ0.6H2O: C,
55.90; H, 4.99; N, 17.15; S, 7.85. Found: C, 55.69; H, 4.84; N,
16.84; S, 7.70.
(e
22,200, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H),
2.29 (m, 1H), 2.49 (m, 1H), 3.74 (m, 2H), 3.80 (s, 3H), 4.09 (m,
1H), 4.60 (dd, J1 = 8.5 Hz, J2 = 5.5 Hz, 1H), 4.65 (s, 2H), 4.97 (m,
1H), 6.88 (d, J = 8.5 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 8.45 (s, 1H),
8.74 (s, 1H); 13C NMR (125 MHz, CD3OD) d 28.6, 31.8, 45.5, 54.3,
60.7, 63.3, 72.3, 74.9, 113.6, 129.3, 130.0, 130.9, 143.4, 148.6,
151.2, 159.1, 160.4. Anal. Calcd for C19H22N4O4S: C, 56.70; H,
5.51; N, 13.92; S, 7.97. Found: C, 56.41; H, 5.69; N, 13.82; S, 7.87.
4.1.7.13. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(4-methoxy-carbonylbenzylthio)purine
(9m). Yield 91%; mp 126–127 °C; ½a D25
ꢁ
= ꢀ38.23 (c 0.22, MeOH);
4.1.7.18. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(2,4-dichloro-benzylthio)purine (9r). Yield
UV (H2O) kmax 293.0 nm (e 23,200, pH 2), 292.0 nm (e 22,700, pH
7), 293.0 nm (
e
20,900, pH 11); 1H NMR (500 MHz, CD3OD) d
93%; mp 163–164 °C; ½a D27
ꢁ
= ꢀ39.33 (c 0.25, MeOH); UV (H2O) kmax
2.04 (m, 1H), 2.28 (m, 1H), 2.49 (m, 1H), 3.73 (m, 2H), 3.91 (s,
3H), 4.08 (m, 1H), 4.60 (dd, J1 = 8.5 Hz, J2 = 5.5 Hz, 1H), 4.76 (s,
2H), 4.96 (m, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 8.0 Hz, 2H),
8.47 (s, 1H), 8.75 (s, 1H); 13C NMR (125 MHz, CD3OD) d 28.6,
31.5, 45.5, 51.2, 60.7, 63.3, 72.2, 74.9, 128.8, 129.0, 129.3, 130.9,
143.7, 143.8, 148.7, 151.2, 159.4, 166.9. Anal. Calcd for
C20H22N4O5Sꢂ0.3H2O: C, 55.11; H, 5.23; N, 12.85; S, 7.36. Found:
C, 55.02; H, 4.97; N, 12.70; S, 7.19.
292.0 nm (
e
22,000, pH 2), 292.0 nm (
e
21,500, pH 7), 291.0 nm (
e
17,400, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H), 2.29 (m,
1H), 2.49 (m, 1H), 3.73 (m, 2H), 4.08 (m, 1H), 4.60 (dd, J1 = 8.5 Hz,
J2 = 5.5 Hz, 1H), 4.80 (s, 2H), 4.97 (m, 1H), 7.29 (m, 1H), 7.52 (m,
1H), 7.70 (d, J = 8.5 Hz, 1H), 8.47 (s, 1H), 8.77 (s, 1H); 13C NMR
(125 MHz, CD3OD) d 28.6, 29.2, 45.5, 60.7, 63.3, 72.2, 74.9, 127.0,
128.9, 130.9, 132.2, 133.6, 134.5, 134.8, 143.7, 148.7, 151.1,
159.2. Anal. Calcd for C18H18Cl2N4O3S: C, 48.99; H, 4.11; N,
12.69; S, 7.27. Found: C, 49.13; H, 4.21; N, 12.75; S, 7.19.
4.1.7.14. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(4-methylbenzyl-thio)purine (9n). Yield
4.1.7.19. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(3,4-dichloro-benzylthio)purine (9s). Yield
91%; mp 112–113 °C; ½a D27
ꢁ
= ꢀ41.04 (c 0.27, MeOH); UV (H2O) kmax
294.0 nm (
e
23,200, pH 2), 293.0 nm (
e
23,200, pH 7), 294.0 nm (
e
93%; mp 160–161 °C; ½a D27
ꢁ
= ꢀ39.73 (c 0.30, MeOH); UV (H2O) kmax
23,100, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H), 2.29 (m,
1H), 2.33 (s, 3H), 2.49 (m, 1H), 3.74 (m, 2H), 4.09 (m, 1H), 4.60 (dd,
J1 = 8.5 Hz, J2 = 5.5 Hz, 1H), 4.65 (s, 2H), 4.96 (m, 1H), 7.13 (d,
J = 8.0 Hz, 2H), 7.35 (d, J = 7.5 Hz, 2H), 8.45 (s, 1H), 8.73 (s, 1H);
13C NMR (125 MHz, CD3OD) d 19.8, 28.6, 32.0, 45.5, 60.7, 63.3,
72.3, 75.0, 128.7, 128.8, 130.8, 134.4, 136.8, 143.4, 148.6, 151.2,
160.3. Anal. Calcd for C19H22N4O3Sꢂ0.2H2O: C, 58.50; H, 5.79; N,
14.36; S, 8.22. Found: C, 58.45; H, 5.74; N, 14.19; S, 8.07.
292.0 nm (
e
24,400, pH 2), 292.0 nm (
e
24,500, pH 7), 291.0 nm (
e
19,300, pH 11); 1H NMR (500 MHz, CD3OD) d 2.04 (m, 1H), 2.28 (m,
1H), 2.49 (m, 1H), 3.73 (m, 2H), 4.08 (m, 1H), 4.60 (dd, J1 = 8.5 Hz,
J2 = 5.5 Hz, 1H), 4.69 (s, 2H), 4.96 (m, 1H), 7.46 (m, 2H), 7.70 (s, 1H),
8.48 (s, 1H), 8.76 (s, 1H); 13C NMR (125 MHz, CD3OD) d 28.6, 30.6,
45.5, 60.7, 63.3, 72.3, 74.9, 128.7, 130.1, 130.6, 130.8, 130.9, 131.7,
139.2, 143.7, 148.7, 151.1, 159.1. Anal. Calcd for C18H18Cl2N4O3S: C,
48.99; H, 4.11; N, 12.69; S, 7.27. Found: C, 48.96; H, 4.32; N, 12.55;
S, 7.13.
4.1.7.15. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(4-methyl-sulfonylbenzylthio)purine
4.1.7.20. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
(9o). Yield 88%; mp 100–102 °C; ½a D25
ꢁ
= ꢀ38.80 (c 0.45, MeOH);
cyclopentan-1-yl]-6-(2-chloro-6-fluorobenzylthio)purine
UV (H2O) kmax 291.0 nm (
e
18,800, pH 2), 291.0 nm (
e
18,900, pH
(9t). Yield 91%; mp 119–120 °C; ½a D27
ꢁ
= ꢀ37.92 (c 0.25, MeOH); UV
7), 291.0 nm (
e
18,900, pH 11); 1H NMR (500 MHz, CD3OD) d
(H2O) kmax 292.0 nm (e 22,100, pH 2), 292.0 nm (e 22,000, pH 7),
2.04 (m, 1H), 2.29 (m, 1H), 2.49 (m, 1H), 3.74 (m, 2H), 4.08 (m,
1H), 4.60 (dd, J1 = 8.5 Hz, J2 = 5.5 Hz, 1H), 4.75 (s, 2H), 4.96 (m,
1H), 7.13 (m, 2H), 7.31 (m, 1H), 7.60 (t, J = 7.5 Hz, 1H), 8.46 (s,
1H), 8.76 (s, 1H); 13C NMR (125 MHz, CD3OD) d 28.6, 31.2, 43.0,
45.5, 60.7, 63.2, 72.2, 74.9, 127.2, 129.8, 131.0, 139.4, 143.7,
145.0, 148.7, 151.2, 159.0. Anal. Calcd for C19H22N4O5S2ꢂ0.3H2O:
C, 50.05; H, 5.00; N, 12.29; S, 14.07. Found: C, 49.94; H, 4.90; N,
12.20; S, 13.79.
291.0 nm (e
15,900, pH 11); 1H NMR (500 MHz, CD3OD) d 2.06
(m, 1H), 2.29 (m, 1H), 2.50 (m, 1H), 3.74 (m, 2H), 4.09 (m, 1H),
4.61 (dd, J1 = 9.0 Hz, J2 = 5.5 Hz, 1H), 4.93 (s, 2H), 4.97 (m, 1H),
7.16 (t, J = 8.5 Hz, 1H), 7.36 (m, 2H), 8.47 (s, 1H), 8.79 (s, 1H); 13C
NMR (125 MHz, CD3OD) d 23.6, 28.6, 45.5, 60.7, 63.3, 72.3, 74.9,
114.0 (d, J = 22.5 Hz), 122.8 (d, J = 18.1 Hz), 125.4, 129.8 (d,
J = 9.6 Hz), 130.8, 135.5, 143.7, 148.7, 151.2, 159.6, 161.6 (d,
J = 248.5 Hz). Anal. Calcd for C18H18ClFN4O3S: C, 50.88; H, 4.27;
N, 13.19; S, 7.55. Found: C, 50.82; H, 4.34; N, 12.97; S, 7.33.
4.1.7.16. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(4-trifluoro-methoxybenzylthio)purine
4.1.7.21. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(2,4,6-trimethyl-benzylthio)purine (9u). Yield
(9p). Yield 90%; mp 150–151 °C; ½a D27
ꢁ
= ꢀ40.27 (c 0.26, MeOH);
UV (H2O) kmax 293.0 nm (
e
21,200, pH 2), 292.0 nm (
e
21,000, pH
90%; mp 92–94 °C; ½a D29
ꢁ
= ꢀ34.98 (c 0.17, MeOH); UV (H2O) kmax
7), 292.0 nm (
e
19,100, pH 11); 1H NMR (500 MHz, CD3OD) d
295.0 nm (e 22,700, pH 2), 294.0 nm (e 22,600, pH 7), 294.0 nm
2.04 (m, 1H), 2.29 (m, 1H), 2.49 (m, 1H), 3.73 (m, 2H), 4.08 (m,
1H), 4.60 (dd, J1 = 9.0 Hz, J2 = 5.5 Hz, 1H), 4.73 (s, 2H), 4.96 (m,
1H), 7.23 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 9.0 Hz, 2H), 8.47 (s, 1H),
8.75 (s, 1H); 13C NMR (125 MHz, CD3OD) d 28.6, 31.0, 45.5, 60.7,
63.3, 72.3, 74.9, 120.5 (q, J = 254.3 Hz), 120.7, 130.5, 130.9, 137.4,
143.6, 148.2, 148.7, 151.2, 159.5. Anal. Calcd for C19H19F3N4O4S:
C, 50.00; H, 4.20; N, 12.27; S, 7.03. Found: C, 49.73; H, 4.27; N,
12.20; S, 6.92.
(e
22,500, pH 11); 1H NMR (500 MHz, CD3OD) d 2.05 (m, 1H),
2.28 (m, 4H), 2.40 (s, 6H), 2.50 (m, 1H), 3.74 (m, 2H), 4.09 (m,
1H), 4.61 (dd, J1 = 9.0 Hz, J2 = 5.5 Hz, 1H), 4.73 (s, 2H), 4.97 (m,
1H), 6.90 (s, 2H), 8.46 (s, 1H), 8.77 (s, 1H); 13C NMR (125 MHz,
CD3OD) d 18.4, 19.7, 27.9, 28.6, 45.5, 60.7, 63.3, 72.3, 75.0, 128.6,
128.7, 130.9, 137.1, 137.2, 143.4, 148.5, 151.3, 161.3. Anal. Calcd
for C21H26N4O3Sꢂ0.4H2O: C, 59.81; H, 6.41; N, 13.29; S, 7.60. Found:
C, 59.70; H, 6.36; N, 13.22; S, 7.60.
4.1.7.17. (10R,20S,30R,40R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)-
cyclopentan-1-yl]-6-(4-methoxy-benzylthio)purine (9q). Yield
4.2. Molecular modeling
89%; mp 94–95 °C; ½a D27
ꢁ
= ꢀ44.09 (c 0.33, MeOH); UV (H2O) kmax
The X-ray crystal structure of the T. gondii adenosine kinase
294.0 nm (
e
22,600, pH 2), 294.0 nm (
e
22,800, pH 7), 294.0 nm
complex with adenosine and AMP-PCP (PDB ID: 1LII.pdb),32,35 were

Y. A. Kim et al. / Bioorg. Med. Chem. 18 (2010) 3403–3412
3411
used as a target for the modeling calculation of compounds 9n and
9l. The ligands (carbocyclic nucleoside) were docked with the ac-
tive site using the ‘xtra precision’ (XP) GLIDE algorithm.36 After suc-
cessful docking, enzyme–ligand complexes were subjected to
energy minimization (EM)38 to obtain the final binding mode. All
the calculations were performed on Schrödinger Suite 2007 (Schrö-
dinger Inc.)37 with GB/SA continuum water solvation model.
OPLS_2001 force field was used for ‘xtra precision’ docking and
then the MMFFs force field was implemented in MACROMODEL 9.138
for energy minimization.
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 concentra-
tion of 0, 5, 10, 25, and 50 lM. 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
drug free media containing [5,6-13H]uracil (5
lCi/mL) and incu-
bated for another 6 h after which the media was removed. The
fibroblasts were then released from the wells by trypsinization
with the addition of 200
l
L trypsin/EDTA (2.5ꢄ) to each well. After
10 min incubation, 1 mL of ice cold 10% trichloroacetic acid (TCA)
was added to each well. The plates were then placed on a shaker
to insure the detachment of the cells. The suspended contents of
each well was filtered through GF/A 2.4 cm glass microfiber filters
(Whatman), which were pre-washed each with 1 mL double dis-
tilled H2O and dried. After filtration, the filters were washed with
10 mL methanol, left to dry, then placed in scintillation vials con-
taining 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 trea-
ted parasites as compared to the untreated controls.14,20–26,40
Radiolabel incorporation closely correlates with parasite
growth.14,20–26,40
4.3. Evaluation of the newly designed carbocyclic 6-benzylthio-
inosine analogues as alternative substrates for purified T. gondii
adenosine kinase
Enzyme assays were run under conditions where activity was
linear with time and enzyme concentration.14–19 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 ki-
nase, 5
gondii adenosine kinase, prepared as previously described39 in a fi-
nal volume of 100 L, in the absence or presence of various concen-
l lL purified cloned T.
M [8-14C]adenosine (55 Ci/mol), 50
l
trations 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
lL of the supernatant were spot-
4.5. Host-toxicity of carbocyclic 6-benzylthioinosine analogues
ted 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: adenosine, 0.27 and AMP, 0.17. The amounts of
radioactivity in both the substrate and product were calculated
on a percentage basis using a computerized Berthold LB-284 Auto-
matic TLC-Linear Analyzers. Apparent Ki values of these analogues
were calculated from Dixon plots 1/v versus [I] by least-squares fit-
ting by computer programs written by Dr. Naguib as previously de-
scribed.14–20 The synthesis of the nucleoside 50-monophosphates
from the tested carbocyclic 6-benzylthioinosine analogues was
confirmed by HPLC and NMR analyses as previously described,15–19
indicating that these compounds are alternative substrates to
T. gondii adenosine kinase. Since these compounds are alternate
substrates of T. gondii adenosine kinase, their apparent Ki values
are equal to their apparent Km values39 as presented in Table 1.
Possible toxicity against the host cells by equivalent doses of
the various analogues used in the above experiments was mea-
sured, at least in triplicates, using a modification of the Microcul-
ture Tetrazolium (MTT) assay on uninfected monolayers of
human foreskin fibroblasts (grown for no more than 20 passages)
in RPMI-1640 medium.15–19,21 Briefly, confluent cells were incu-
bated for at least 24 h in 96-well flat bottom microtiterplates
(ꢃ105/200
lL/well) at 37 °C in 5% CO2, 95% air to allow the cells
to attach. The medium was then replaced with 200
lL of fresh
medium. The appropriate concentration of the compounds was
dissolved in 50 L of medium, and added to each well to give the
desired final concentrations. The cultures were then incubated
for 48 h after which 50 L of sterile MTT solution (2 mg/1 mL
PBS) was added to each well. MTT solution was sterilized by filtra-
tion through 0.22 m filters (Costar, Cambridge, MA). After 4 h
incubation, the medium was removed and 100 L of dimethylsulf-
l
l
l
4.4. Evaluation of carbocyclic 6-benzylthioinosine analogues as
potential anti-toxoplasmosis agents against tachyzoites in
tissue culture
l
oxide (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 micro-
titerplate reader (Themomax, Molecular Devices).
The wild type RH and the adenosine kinase deficient mutant
TgAKꢀ3strains31 of T. gondii were used in these experiments. The
adenosine kinase deficient mutant TgAKꢀ3 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 carbocyclic
analogues as anti-toxoplasmosis agents in tissue culture was mea-
sured by their ability to inhibit the replication of intracellular T.
gondii in tissue culture, using monolayers of human foreskin fibro-
blasts (CRL-1634, American Type Culture Collection), grown for no
more than 20 passages in RPMI-1640 medium.15–19,21 The viability
of intracellular parasites was evaluated by the selective incorpora-
tion of radiolabeled uracil into nucleic acids of the parasites at least
in triplicates as previously described.16–21 Briefly, confluent cells
(4–5 day incubation) were cultured for 24 h in the 24-well flat bot-
tom microtiter plates (ꢃ5 ꢄ 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 incubation, the
cultures were washed with media with 10% FBS to remove
Acknowledgment
This research was supported by the US Public Health Service
Grants AI-52838 from the National Institute of Health.
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