Synthesis, biological evaluation and molecular modeling studies of N6-benzyladenosine analogues as potential anti-toxoplasma agents

Article abstract of DOI:10.1016/j.bcp.2007.01.026

Toxoplasma gondii is an opportunistic pathogen responsible for toxoplasmosis. T. gondii is a purine auxotroph incapable of de novo purine biosynthesis and depends on salvage pathways for its purine requirements. Adenosine kinase (EC.2.7.1.20) is the major enzyme in the salvage of purines in these parasites. 6-Benzylthioinosine and analogues were established as "subversive substrates" for the T. gondii, but not for the human adenosine kinase. Therefore, these compounds act as selective anti-toxoplasma agents. In the present study, a series of N⁶-benzyladenosine analogues were synthesized from 6-chloropurine riboside with substituted benzylamines via solution phase parallel synthesis. These N⁶-benzyladenosine analogues were evaluated for their binding affinity to purified T. gondii adenosine kinase. Furthermore, the anti-toxoplasma efficacy and host toxicity of these compounds were tested in cell culture. Certain substituents on the aromatic ring improved binding affinity to T. gondii adenosine kinase when compared to the unsubstituted N⁶-benzyladenosine. Similarly, varying the type and position of the substituents on the aromatic ring led to different degrees of potency and selectivity as anti-toxoplasma agents. Among the synthesized analogues, N⁶-(2,4-dimethoxybenzyl)adenosine exhibited the most favorable anti-toxoplasma activity without host toxicity. The binding mode of the synthesized N⁶-benzyladenosine analogues were characterized to illustrate the role of additional hydrophobic effect and van der Waals interaction within an active site of T. gondii adenosine kinase by induced fit molecular modeling.

Full text of DOI:10.1016/j.bcp.2007.01.026

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/biochempharm
Synthesis, biological evaluation and molecular modeling
studies of N6-benzyladenosine analogues as potential
anti-toxoplasma agents
b
Young Ah Kim a, Ashoke Sharon a, Chung K. Chu a, , Reem H. Rais ,
*
Omar N. Al Safarjalani b, Fardos N.M. Naguib b, Mahmoud H. el Kouni b
a University of Georgia College of Pharmacy, Athens, GA 30602, United States
b Department of Pharmacology and Toxicology, Center for AIDS Research, University of Alabama School of Medicine,
Birmingham, AL 35294, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Toxoplasma gondii is an opportunistic pathogen responsible for toxoplasmosis. T. gondii is a
purine auxotroph incapable of de novo purine biosynthesis and depends on salvage path-
ways for its purine requirements. Adenosine kinase (EC.2.7.1.20) is the major enzyme in the
salvage of purines in these parasites. 6-Benzylthioinosine and analogues were established
as ‘‘subversive substrates’’ for the T. gondii, but not for the human adenosine kinase.
Therefore, these compounds act as selective anti-toxoplasma agents. In the present study,
a series of N6-benzyladenosine analogues were synthesized from 6-chloropurine riboside
with substituted benzylamines via solution phase parallel synthesis. These N6-benzylade-
nosine analogues were evaluated for their binding affinity to purified T. gondii adenosine
kinase. Furthermore, the anti-toxoplasma efficacy and host toxicity of these compounds
were tested in cell culture. Certain substituents on the aromatic ring improved binding
affinity to T. gondii adenosine kinase when compared to the unsubstituted N6-benzylade-
nosine. Similarly, varying the type and position of the substituents on the aromatic ring led
to different degrees of potency and selectivity as anti-toxoplasma agents. Among the
synthesized analogues, N6-(2,4-dimethoxybenzyl)adenosine exhibited the most favorable
anti-toxoplasma activity without host toxicity. The binding mode of the synthesized N6-
benzyladenosine analogues were characterized to illustrate the role of additional hydro-
phobic effect and van der Waals interaction within an active site of T. gondii adenosine
kinase by induced fit molecular modeling.
Received 10 November 2006
Accepted 12 January 2007
Keywords:
Toxoplasma gondii adenosine kinase
Toxoplasmosis
Chemotherapy
N6-Benzyladenosine analogues
Molecular modeling
# 2007 Elsevier Inc. All rights reserved.
1.
Introduction
in immunocompromised patients such as acquired immu-
nodeficiency syndrome (AIDS) [1–3]. Pyrimethamine is cur-
Toxoplasma gondii is an opportunistic pathogen responsible
for toxoplasmosis. Toxoplasmosis is known to be one of the
most prevalent parasitic infections of the central nervous
system and causes severe congenital defects in unborn
children of infected mothers as well as lethal encephalitis
rently used in combination with sulfa drugs such as
sulfadiazine in toxoplasmic infections [4,5]. This combina-
tion therapy targets enzymes of folate metabolism such as
dihydrofolate reductase [6–9]. However, current therapies
have been limited due to resistance, host toxicity and side
* Corresponding author. Tel.: +1 706 542 5379; fax: +1 706 542 5381.
E-mail address: dchu@rx.uga.edu (C.K. Chu).
0006-2952/$ – see front matter # 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2007.01.026

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
1559
Fig. 1 – (a) Superimposition of the apo T. gondii adenosine kinase (yellow) and enzyme:adenosine:ATP complex (green)
highlights the 308 domain rotation of lid domain (upper small domain) towards large domain near to substrate binding site
(space filling representation for substrates). (b) Catalytic site showing the mode of binding for the transfer of g-phosphate
from ATP to adenosine mediated by Arg136 interactions.
effects. Particularly, sulfa drugs cause severe side effects such
as skin allergy and bone marrow suppression [10,11]. To
overcome such disadvantages, it is desirable to develop novel
drug candidates based on different mechanism of action with
high degree of selectivity.
purine salvage pathways, and hence contributes more
significantly to the salvage of purines in T. gondii than any
other enzyme of the purine salvage pathways [12–16].
Secondly, structure–activity relationships [17,18], comparative
enzymatic [18–20], metabolic [19–21], molecular [22] and X-ray
structural studies [23–25] have demonstrated that there are
sufficient differences between the active sites and substrate
specificities of human and T. gondii adenosine kinases that can
be exploited to design specific ‘‘subversive substrates’’ for the
T. gondii enzyme [12,13].
T. gondii adenosine kinase is an attractive target for the
treatment of toxoplasmosis [12,13] for the following reasons.
First, T. gondii is a purine auxotroph and depends on the
salvage pathways for its purine requirements, and the salvage
of adenosine is the major source of purines in T. gondii [14,15].
Specifically, the activity of adenosine kinase (EC.2.7.1.20), the
enzyme responsible for the salvage of adenosine in this
parasite, is 10-fold higher than those of other enzymes in the
T. gondii adenosine kinase is
a 363-residue (39.3 kDa)
monomeric protein [22], that catalyzes the phosphorylation
of adenosine to adenosine 50-monophosphate (AMP), using the
Fig. 2 – (a) Binding mode of adenosine to T. gondii adenosine kinase with hydrophilic (yellow dotted H-bonds) and
hydrophobic effects (green mesh): H-bonds by 20-OH and 30-OH groups of adenosine with Asp24, Gly69 and Asn73 residues
are represented by the yellow dotted line. Hydrophobic stacking by adenine with Tyr169 and Ile22 by p–p and C–H–p
interaction, other hydrophobic effect by Leu46 and Leu142 are represented by the green mesh. (b) Active site map analysis
of enzyme shows the hydrophobic pocket (brown patch) near to N6-region of adenine, the hydrophilic region of enzyme
(white patch), adenine occupying the hydrophobic region (brown patch) and the ribose moiety in the hydrophilic region
(white patch).

1560
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
g-phosphate group of ATP as the phosphate donor [12,13]. The
overall enzyme structure consists of two domains: a large
domain and a small (lid) domain as shown in Fig. 1a. The
catalytic site for adenosine and ATP is located at the domain
interfaces. The overlay structures of apo enzyme (apo enzyme:
1LIO) and the enzyme:adenosine:ATP complex (complex: 1LII)
suggest that there is a movement of the small domain towards
the large domain (Fig. 1a), which forms the closed conformation
causing translocation of Arg136 towards the g-phosphate of
ATP to initiate catalysis (Fig. 1b). Consequently, ATP acquires
extended conformation and orientation toward the 50-OH of
adenosine for its phosphorylation as shown in Fig. 1b. The
adenosine binding pocket is composed of the ribose binding
hydrophilic region (Asp24, Gly69 and Asn73), the purine binding
region [23] and an additional hydrophobic region [18]. The
purine motif of adenosine is surrounded by some of the
hydrophobic residues such as Tyr169 and Ile22, that forms
aromatic stacking by p–p and C–H–p interaction (Fig. 2a).
Our previous structure–activity relationships and molecu-
(broad singlet). 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., Norcross, GA, USA.
2.2.2. N,N-Di-tert-butyl[(2-fluoro-4-
nitro)benzylamino]dicarboxylate (2a)
A solution of 2-fluoro-4-nitrotoluene (300 mg, 1.93 mmol) in
CCl4 (4.8 mL) was treated with N-bromosuccinimide (413 mg,
2.32 mmol) and benzoyl peroxide (46.8 mg, 0.193 mmol). The
mixture was heated to reflux for 8 h. The insoluble material
was filtered and the filtrate was washed with brine. The
organic layer was dried over MgSO4 and then concentrated.
The crude product was dissolved in THF (7.0 mL) and was
cooled to 0 8C. To this solution, di-tert-butyl iminodicarbox-
ylate (462 mg, 2.13 mmol) and sodium hydride (51.0 mg,
2.13 mmol) in THF (21.0 mL) were added. The reaction mixture
was stirred at 0 8C for 1 h followed by stirring at room
temperature for 7 h. The reaction mixture was diluted with
water and extracted with EtOAc. The organic layer was
washed with brine and then dried over MgSO4. After
concentration, the residue was purified by silica gel column
chromatography (n-hexane:EtOAc = 95:5) to give 2a (358 mg,
50% for two steps): 1H NMR (500 MHz, CDCl3) d 1.48 (s, 18H), 4.93
(s, 2H), 7.42 (t, J = 7.8 Hz, 1H), 7.92 (dd, J1 = 10 Hz, J2 = 2.5 Hz, 1H),
8.02 (dd, J1 = 8.5 Hz, J2 = 2.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) d
27.9, 43.2, 83.3, 111.1 (d, J = 27.1 Hz), 119.3, 128.7, 133.5, 147.7 (d,
J = 8.6 Hz), 152.1, 159.6 (d, J = 249.4 Hz).
lar modeling studies of
a series of 6-benzylthioinosine
analogues with T. gondii adenosine kinase [17,18] emphasized
the importance of the hydrophobic effect of the benzyl moiety
at the 6-position of the purine ring as well as the type and
position of substituents on the benzyl moiety in enhancing the
binding affinity of 6-benzylthioinosine analogues to T. gondii
adenosine kinase. These studies also furthered our under-
standing of the structural requirements for the binding of
ligands by induced fit mechanism.
In the present study, we report the synthesis of N6-
benzyladenosine analogues by solution phase parallel synth-
esis with the goal of identifying anti-toxoplasma agents. We
have also carried out systematic characterization of the
hydrophobic effect and van der Waals interaction employing
an induced fit molecular modeling approach to demonstrate
the role of additional interaction in enhancing the binding
affinity of ligands to T. gondii adenosine kinase in this series.
2.2.3. N,N-Di-tert-butyl[(2-chloro-4-
cyano)benzylamino]dicarboxylate (2b)
Compound 2b was prepared under the same procedure
outlined above for compound 2a (52% for two steps): 1H
NMR (500 MHz, CD3OD) d 1.48 (s, 18H), 4.92 (s, 2H), 7.37 (d,
J = 8.0 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.89 (s, 1H); 13C NMR
(125 MHz, CD3OD) d 26.8, 47.0, 83.2, 112.0, 116.9, 127.4, 130.7,
132.5, 132.9, 141.8, 152.1.
2.2.4. (2-Fluoro-4-nitro)benzylamine hydrochloride (3a)
A solution of compound 2a (800 mg, 2.16 mmol) in MeOH
(21.6 mL) was treated with 1N HCl/Et2O (21.6 mL). The reaction
mixture was stirred for 5 h at room temperature and then
concentrated. The residue was triturated with ether and then
filtered to give 3a (379 mg, 85%): UV (MeOH) lmax 255.0 nm; 1H
NMR (500 MHz, DMSO-d6) d 4.21 (s, 2H), 7.91 (t, J = 7.8 Hz, 1H),
8.23 (m, 2H), 8.64 (bs, 3H); 13C NMR (125 MHz, DMSO-d6) d 35.9,
111.7 (d, J = 26.6 Hz), 120.1, 129.4, 132.7, 148.9 (d, J = 9.1 Hz),
160.1 (d, J = 249.9 Hz).
2.
Materials and methods
2.1.
Reagents
[8-14C]Adenosine (55 Ci/mol) and [5,6-13H]uracil were pur-
chased from Moravek Biochemicals. RPMI-1640 medium from
GIBCO BRL; fetal bovine serum (FBS) from HyClone Labora-
tories. All other chemicals and compounds were obtained
from Sigma Chemical Co. or Fisher Scientific.
2.2.
Chemical synthesis
2.2.5. (2-Chloro-4-cyano)benzylamine hydrochloride (3b)
Compound 3b was prepared under the same procedure
outlined above for compound 3a (83%): UV (MeOH) lmax
284.0 nm; 1H NMR (500 MHz, DMSO-d6) d 4.22 (s, 2H), 7.86 (d,
J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 8.19 (s, 1H), 8.81 (bs, 3H);
13C NMR (125 MHz, DMSO-d6) d 40.2, 113.2, 117.7, 131.5, 131.7,
133.2, 134.1, 137.9.
2.2.1. General
Solutionphase synthesis was performed onArgonautQuest 210
Organic Synthesizer. 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 (d) relative to
tetramethylsilane as internal reference. Chemical shifts (d) are
reported as s (singlet), d (doublet), t (triplet), m (multiplet) or bs
2.2.6. (2-Fluoro-4-methoxy)benzylalcohol (5)
A
solution of (2-fluoro-4-methoxy)benzaldehyde (4.50 g,
29.2 mmol) in MeOH (73.0 mL) was treated with NaBH4

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
1561
(1.66 g, 43.8 mmol) at 0 8C. After 30 min, the reaction mixture
was stirred for 3 h at room temperature. The reaction mixture
was quenched with water and then concentrated. The residue
was diluted with EtOAc and then washed with 5% HCl and
brine. The organic layer was dried over MgSO4 and concen-
trated. The residue was purified by silica gel column
to give9 (1.03 g, 62% for two steps): UV (MeOH) lmax 248.0 nm; 1H
NMR (500 MHz, DMSO-d6) d 4.02 (s, 2H), 5.32 (d, J = 11.0 Hz, 1H),
5.90 (d, J = 17.5 Hz, 1H), 6.77 (dd, J1 = 17.5 Hz, J2 = 11.5 Hz, 1H),
7.48 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 8.42 (bs, 3H); 13C
NMR (125 MHz, DMSO-d6) d 42.3, 115.5, 126.7, 129.7, 134.2, 136.6,
137.7.
chromatography (n-hexane:EtOAc = 7:1 ! 4:1) to give
5
(4.38 g, 96%): 1H NMR (500 MHz, CDCl3) d 2.50 (bs, 1H), 3.78 (s,
3H), 4.62 (s, 2H), 6.60 (dd, J1 = 11.5 Hz, J2 = 2.5 Hz, 1H), 6.66 (dd,
J1 = 8.0 Hz, J2 = 2.5 Hz, 1H), 7.26 (t, J = 8.8 Hz, 1H); 13C NMR
(125 MHz, CDCl3) d 55.5, 58.8, 101.6 (d, J = 25.3 Hz), 109.7, 119.9
(d, J = 15.3 Hz), 130.3, 160.5, 161.3 (d, J = 245.1 Hz).
2.2.10. General procedure for the synthesis of N6-
benzyladenosine analogues (11a–v)
Compound 10 (100 mg, 0.349 mmol) was added to each
reaction vessel of Argonaut Quest 210 Organic Synthesizer
and then n-PrOH (10.0 mL) was added. To this solution, the
appropriate amine (or amine hydrochloride) and triethyla-
mine (or N,N-diisopropylethylamine) (0.2 mL) were added. The
reaction mixtures were stirred (upward stroke = 50%,
time = 4 s) at 100 8C (or 80 8C) for 11 h and then allowed to
cool. The reaction mixtures were concentrated and the
residues were purified by silica gel column chromatography
(CH2Cl2:MeOH = 95:5) to give the desired products as white
solid.
2.2.7. N,N-Di-tert-butyl[(2-fluoro-4-
methoxy)benzylamino]dicarboxylate (6)
A solution of (2-fluoro-4-methoxy)benzylalcohol 5 (3.21 g,
20.6 mmol) in CH2Cl2 (69.0 mL) was treated with carbon
tetrabromide (8.18 g, 24.7 mmol) and triphenylphosphine
(6.47 g, 24.7 mmol). The reaction mixture was stirred for 3 h
at room temperature. The insoluble material was filtered and
the filtrate was washed with brine. The organic layer was dried
over MgSO4 and then concentrated. The crude product was
dissolved in THF (76.0 mL) and was cooled to 0 8C. To this
solution, di-tert-butyl iminodicarboxylate (4.91 g, 22.6 mmol)
and sodium hydride (0.543 g, 22.6 mmol) in THF (228.0 mL)
were added. The reaction mixture was stirred at 0 8C for 1 h
followed by stirring at room temperature for 7 h. The reaction
mixture was diluted with water and extracted with EtOAc. The
organic layer was washed with brine and then dried over
MgSO4. After concentration, the residue was purified by silica
gel column chromatography (n-hexane:EtOAc = 95:5) to give 6
(4.02 g, 55% for two steps): 1H NMR (500 MHz, CDCl3) d 1.49 (s,
18H), 3.81 (s, 3H), 4.81 (s, 2H), 6.61 (m, 1H), 6.68 (m, 1H), 7.20 (t,
J = 8.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) d 27.9, 42.8, 55.5, 82.5,
101.3 (d, J = 25.3 Hz), 109.7, 117.4 (d, J = 14.8 Hz), 129.3, 152.4,
160.0, 160.9 (d, J = 252.8 Hz).
2.2.11. N6-Benzyladenosine (11a)
23
Yield 79%; mp 168–169 8C; ½aꢀD ꢁ 57:17 (c 0.22, MeOH); UV
(MeOH) lmax 269.0 nm; 1H NMR (500 MHz, DMSO-d6) d 3.57 (m,
1H), 3.69 (m, 1H), 3.98 (m, 1H), 4.17 (m, 1H), 4.64 (m, 1H), 4.73 (br,
2H), 5.21 (d, J = 4.5 Hz, 1H), 5.41 (m, 1H), 5.47 (d, J = 6.5 Hz, 1H),
5.91 (d, J = 6.5 Hz, 1H), 7.23 (m, 1H), 7.33 (m, 4H), 8.22 (s, 1H), 8.40
(s, 1H), 8.50 (bs, 1H); 13C NMR (125 MHz, DMSO-d6) d 43.3, 62.1,
71.1, 73.9, 86.3, 88.4, 120.2, 127.1, 127.5, 128.7, 140.4, 148.9,
152.8, 154.9. Anal. calcd. for C17H19N5O4 requires C, 57.14; H,
5.36; N, 19.60; found C, 57.28; H, 5.34; N, 19.66.
2.2.12. N6-(2-Fluorobenzyl)adenosine (11b)
25
Yield 73%; mp 191–192 8C; ½aꢀD ꢁ 64:07 (c 0.18, MeOH); UV (H2O)
lmax 267.0 nm (e 21376, pH 2), 267.0 nm (e 21341, pH 7), 267.0 nm
(e 21615, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.59 (m, 1H), 3.71
(m, 1H), 4.00 (m, 1H), 4.18 (m, 1H), 4.65 (m, 1H), 4.79 (m, 2H), 5.23
(m, 1H), 5.40 (m, 1H), 5.49 (d, J = 5.0 Hz, 1H), 5.93 (d, J = 5.0 Hz,
1H), 7.14 (m, 1H), 7.20 (m, 1H), 7.32 (m, 2H), 8.24 (s, 1H), 8.44 (s,
1H), 8.48 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 37.3, 62.1, 71.1,
74.0, 86.4, 88.4, 115.5 (d, J = 21.0 Hz), 120.3, 124.7, 127.1, 129.0 (d,
J = 8.1 Hz), 129.4, 140.5, 149.1, 152.8, 155.0, 160.5 (d, J = 242.8 Hz).
Anal. calcd. for C17H18FN5O4 requires C, 54.40; H, 4.83; N, 18.66;
found C, 54.27; H, 4.84; N, 18.50.
2.2.8. (2-Fluoro-4-methoxy)benzylamine hydrochloride (7)
Compound 7 was prepared under the same procedure outlined
above for compound 3a (86%): UV (MeOH) lmax 270.0 nm; 1H
NMR (500 MHz, DMSO-d6) d 3.81 (s, 3H), 4.00 (s, 2H), 6.88 (dd,
J1 = 9.0 Hz, J2 = 2.5 Hz, 1H), 6.94 (dd, J1 = 12.0 Hz, J2 = 2.5 Hz, 1H),
7.50 (t, J = 9.0 Hz, 1H), 8.33 (bs, 3H); 13C NMR (125 MHz, DMSO-
d6) d 35.7, 56.3, 102.0 (d, J = 24.9 Hz), 111.0, 113.1 (d, J = 15.3 Hz),
132.6, 161.5, 161.6 (d, J = 244.8 Hz).
2.2.13. N6-(2-Chlorobenzyl)adenosine (11c)
23
2.2.9. 4-Vinylbenzylamine hydrochloride (9)
Yield 75%; mp 190–191 8C; ½aꢀD ꢁ 75:56 (c 0.12, MeOH); UV (H2O)
A solution of 4-vinylbenzyl chloride (1.50 g, 9.83 mmol) in N,N-
dimethylformamide (25.0 mL) was treated with sodium azide
(1.28 g, 19.7 mmol) and was stirred for 1 h at room temperature.
The reaction mixturewasdilutedwith waterand thenextracted
withEtOAc.Theorganiclayerwaswashedwithbrine,driedover
MgSO4 andthen concentrated. The crude productwas dissolved
in benzene (10.0 mL) and then triphenylphosphine (3.87 g,
14.7 mmol) and water was added. The reaction mixture was
heated at 70 8C for 3 h and then extracted with 2N HCl. The
aqueous layer was washed with CH2Cl2, basified with NaOH,
and extracted with CH2Cl2. The combined organic layers were
concentrated, the residue was dissolved in Et2O and 3N HCl was
added dropwise. The resultingprecipitate was filteredand dried
lmax 267.0 nm (e 17633, pH 2), 267.0 nm (e 17792, pH 7), 267.0 nm
(e 18115, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.59 (m, 1H), 3.71
(m, 1H), 4.00 (m, 1H), 4.19 (m, 1H), 4.67 (m, 1H), 4.79 (m, 2H), 5.24
(m, 1H), 5.40 (m, 1H), 5.50 (d, J = 6.0 Hz, 1H), 5.94 (d, J = 4.0 Hz,
1H), 7.29 (m, 3H), 7.48 (m, 1H), 8.23 (s, 1H), 8.46 (s, 1H), 8.52 (s,
1H); 13C NMR (125 MHz, DMSO-d6) d 41.5, 62.2, 71.1, 74.0, 86.4,
88.4, 120.4, 127.6, 128.5, 128.8, 129.6, 132.2, 137.2, 140.6, 149.2,
152.8, 155.0. Anal. calcd. for C17H18ClN5O4 requires C, 52.11; H,
4.63; N, 17.87; found C, 52.00; H, 4.65; N, 17.76.
2.2.14. N6-(2-Nitrobenzyl)adenosine (11d)
25
Yield 66%; mp 202–204 8C; ½aꢀD ꢁ 62:98 (c 0.04, MeOH); UV (H2O)
lmax 266.0 nm (e 16034, pH 2), 266.0 nm (e 10095, pH 7), 266.0 nm

1562
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
(e 9614, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.59 (m, 1H), 3.70
(m, 1H), 3.99 (m, 1H), 4.18 (m, 1H), 4.65 (m, 1H), 5.02 (m, 2H), 5.23
(m, 1H), 5.37 (m, 1H), 5.49 (d, J = 6.0 Hz, 1H), 5.93 (d, J = 5.5 Hz,
1H), 7.54 (m, 2H), 7.70 (t, J = 7.5 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H),
8.20 (s, 1H), 8.47 (s, 1H), 8.58 (s, 1H); 13C NMR (125 MHz, DMSO-
d6) d 40.8, 62.1, 71.1, 74.0, 86.4, 88.4, 120.4, 125.0, 128.5, 129.4,
134.3, 135.3, 140.7, 148.6, 149.2, 152.8, 154.8. Anal. calcd. for
C17H18N6O6 requires C, 50.75; H, 4.51; N, 20.89; found C, 50.62;
H, 4.60; N, 20.60.
2.2.19. N6-(4-Chlorobenzyl)adenosine (11i)
25
Yield 76%; mp 178–180 8C; ½aꢀD ꢁ 63:69 (c 0.16, MeOH); UV (H2O)
lmax 267.0 nm (e 21391, pH 2), 268.0 nm (e 21487, pH 7), 268.0 nm
(e 21890, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.59 (m, 1H), 3.71
(m, 1H), 4.00 (m, 1H), 4.19 (m, 1H), 4.65 (m, 1H), 4.72 (m, 2H), 5.23
(m, 1H), 5.41 (m, 1H), 5.49 (d, J = 6.0 Hz, 1H), 5.93 (d, J = 6.5 Hz,
1H), 7.38 (m, 4H), 8.24 (s, 1H), 8.43 (s, 1H), 8.54 (s, 1H); 13C NMR
(125 MHz, DMSO-d6) d 42.8, 62.2, 71.1, 74.0, 86.4, 88.4, 120.3,
128.7, 129.5, 131.7, 139.6, 140.5, 149.0, 152.8, 154.9. Anal. calcd.
for C17H18ClN5O4 requires C, 52.11; H, 4.63; N, 17.87; found C,
52.10; H, 4.65; N, 17.72.
2.2.15. N6-(2-Methoxybenzyl)adenosine (11e)
25
Yield 76%; mp 155–157 8C; ½aꢀD ꢁ 64:43 (c 0.15, MeOH); UV (H2O)
lmax 267.0 nm (e 21173, pH 2), 270.0 nm (e 21878, pH 7), 270.0 nm
(e 22118, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.59 (m, 1H), 3.71
(m, 1H), 3.87 (s, 3H), 4.00 (m, 1H), 4.18 (m, 1H), 4.67 (m, 1H), 4.70
(m, 2H), 5.23 (m, 1H), 5.43 (m, 1H), 5.49 (d, J = 6.0 Hz, 1H), 5.93 (d,
J = 6.0 Hz, 1H), 6.87 (m, 1H), 7.01 (d, J = 8.5 Hz, 1H), 7.12 (m, 1H),
7.23 (m, 1H), 8.21 (s, 1H), 8.26 (s, 1H), 8.43 (s, 1H); 13C NMR
(125 MHz, DMSO-d6) d 38.8, 55.8, 62.2, 71.2, 74.0, 86.4, 88.5, 110.9,
120.4, 120.6, 127.2, 127.7, 128.2, 140.4, 149.0, 152.9, 155.3, 157.1.
Anal. calcd. for C18H21N5O5 requires C, 55.81; H, 5.46; N, 18.08;
found C, 55.69; H, 5.31; N, 17.87.
2.2.20. N6-(4-Cyanobenzyl)adenosine (11j)
26
Yield 77%; mp 118–120 8C; ½aꢀD ꢁ 64:31 (c 0.14, MeOH); UV (H2O)
lmax 267.0 nm (e 24306, pH 2), 268.0 nm (e 24776, pH 7), 268.0 nm
(e 25086, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.57 (m, 1H), 3.68
(m, 1H), 3.99 (m, 1H), 4.17 (m, 1H), 4.64 (m, 1H), 4.80 (m, 2H), 5.23
(m, 1H), 5.38 (m, 1H), 5.48 (d, J = 6.0 Hz, 1H), 5.92 (d, J = 6.0 Hz,
1H), 7.53 (d, J = 7.5 Hz, 2H), 7.79 (d, J = 8.5 Hz, 2H), 8.22 (s, 1H),
8.44 (s, 1H), 8.62 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 43.3,
62.1, 71.1, 74.0, 86.4, 88.4, 109.9, 119.4, 120.3, 128.4, 132.7, 140.6,
146.5, 149.1, 152.8, 154.9. Anal. calcd. for C18H18N6O4 requires C,
56.54; H, 4.74; N, 21.98; found C, 56.52; H, 4.72; N, 21.89.
2.2.16. N6-(3-Fluorobenzyl)adenosine (11f)
25
Yield 74%; mp 155–156 8C; ½aꢀD ꢁ 56:31 (c 0.20, MeOH); UV (H2O)
2.2.21. N6-(4-Trifluoromethylbenzyl)adenosine (11k)
25
lmax 267.0 nm (e 22491, pH 2), 268.0 nm (e 22885, pH 7), 268.0 nm
(e 23107, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.59 (m, 1H), 3.71
(m, 1H), 4.00 (m, 1H), 4.19 (m, 1H), 4.65 (m, 1H), 4.75 (m, 2H), 5.23
(m, 1H), 5.41 (m, 1H), 5.49 (m, 1H), 5.93 (d, J = 3.5 Hz, 1H), 7.07 (t,
J = 8.5 Hz, 1H), 7.18 (m, 2H), 7.37 (m, 1H), 8.25 (s, 1H), 8.44 (s, 1H),
8.55 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 43.0, 62.1, 71.1, 74.0,
86.4, 88.5, 113.9 (d, J = 21.0 Hz), 114.2 (d, J = 21.5 Hz), 120.3,
123.6, 130.6 (d, J = 8.1 Hz), 140.5, 143.7, 149.1, 152.8, 155.0, 162.7
(d, J = 241.9 Hz). Anal. calcd. for C17H18FN5O4 requires C, 54.40;
H, 4.83; N, 18.66; found C, 54.30; H, 4.78; N, 18.49.
Yield 75%; mp 151–153 8C; ½aꢀD ꢁ 52:82 (c 0.18, MeOH); UV (H2O)
lmax 267.0 nm (e 22451, pH 2), 268.0 nm (e 22387, pH 7), 268.0 nm
(e 22659, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.58 (m, 1H), 3.70
(m, 1H), 4.00 (m, 1H), 4.18 (m, 1H), 4.65 (m, 1H), 4.81 (m, 2H), 5.23
(m, 1H), 5.39 (m, 1H), 5.49 (d, J = 6.0 Hz, 1H), 5.93 (d, J = 6.5 Hz,
1H), 7.56 (d, J = 7.5 Hz, 2H), 7.69 (d, J = 7.5 Hz, 2H), 8.23 (s, 1H),
8.44 (s, 1H), 8.62 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 43.2,
62.1, 71.1, 74.0, 86.4, 88.4, 120.3, 121.6, 123.8, 125.6, 126.0, 127.8
(q, J = 30.9 Hz), 128.2, 140.6, 145.4, 149.1, 152.8, 154.9. Anal.
calcd. for C18H18F3N5O4 requires C, 50.83; H, 4.27; N, 16.46;
found C, 50.74; H, 4.20; N, 16.41.
2.2.17. N6-(3-Methylbenzyl)adenosine (11g)
26
Yield 75%; mp 151–152 8C; ½aꢀD ꢁ 56:84 (c 0.23, MeOH); UV (H2O)
2.2.22. N6-(4-Methylbenzyl)adenosine (11l)
25
lmax 267.0 nm (e 17760, pH 2), 268.0 nm (e 17780, pH 7), 268.0 nm
(e 18077, pH 11); 1H NMR (500 MHz, DMSO-d6) d 2.29 (s, 3H), 3.58
(m, 1H), 3.71 (m, 1H), 4.00 (m, 1H), 4.19 (m, 1H), 4.66 (m, 1H), 4.71
(m, 2H), 5.23 (m, 1H), 5.44 (m, 1H), 5.49 (d, J = 6.5 Hz, 1H), 5.93 (d,
J = 6.0 Hz, 1H), 7.05(d, J = 7.0 Hz,1H), 7.18(m, 3H),8.24(s, 1H), 8.41
(s, 1H), 8.47 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 21.5, 43.3, 62.2,
71.2, 74.0, 86.4, 88.5, 120.3, 124.7, 127.8, 128.2, 128.6, 137.7, 140.4,
149.0, 152.9, 155.0. Anal. calcd. for C18H21N5O4 requires C, 58.21;
H, 5.70; N, 18.86; found C, 58.18; H, 5.86; N, 18.75.
Yield 77%; mp 158–159 8C; ½aꢀD ꢁ 67:33 (c 0.11, MeOH); UV (H2O)
lmax 267.0 nm (e 22135, pH 2), 269.0 nm (e 22300, pH 7), 269.0 nm
(e 22192, pH 11); 1H NMR (500 MHz, DMSO-d6) d 2.27 (s, 3H), 3.58
(m, 1H), 3.70 (m, 1H), 3.99 (m, 1H), 4.18 (m, 1H), 4.66 (m, 1H), 4.69
(m, 2H), 5.22 (m, 1H), 5.43 (m, 1H), 5.48 (d, J = 5.5 Hz, 1H), 5.92 (d,
J = 6.5 Hz, 1H), 7.12 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 8.23
(s, 1H), 8.40 (s, 1H), 8.46 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d
21.1, 43.1, 62.2, 71.1, 74.0, 86.4, 88.5, 120.3, 127.6, 129.2, 136.1,
137.5, 140.4, 149.0, 152.8, 155.0. Anal. calcd. for C18H21N5O4
requires C, 58.21; H, 5.70; N, 18.86; found C, 58.43; H, 5.75; N,
18.90.
2.2.18. N6-(4-Fluorobenzyl)adenosine (11h)
26
Yield 77%; mp 179–181 8C; ½aꢀD ꢁ 62:21 (c 0.20, MeOH); UV (H2O)
lmax 267.0 nm (e 20123, pH 2), 268.0 nm (e 20181, pH 7), 268.0 nm
(e 20532, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.59 (m, 1H), 3.71
(m, 1H), 4.00 (m, 1H), 4.18 (m, 1H), 4.65 (m, 1H), 4.71 (m, 2H), 5.23
(m, 1H), 5.42 (m, 1H), 5.49 (d, J = 5.0 Hz, 1H), 5.93 (d, J = 5.5 Hz,
1H), 7.15 (m, 2H), 7.40 (m, 2H), 8.25 (s, 1H), 8.42 (s, 1H), 8.52 (s,
1H); 13C NMR (125 MHz, DMSO-d6) d 42.7, 62.2, 71.1, 74.0, 86.4,
88.4, 115.4 (d, J = 21.0 Hz), 120.3, 129.6, 136.7, 140.4, 149.0, 152.8,
154.9, 161.6 (d, J = 240.9 Hz). Anal. calcd. for C17H18FN5O4
requires C, 54.40; H, 4.83; N, 18.66; found C, 54.15; H, 4.84; N,
18.47.
2.2.23. N6-(4-Isopropylbenzyl)adenosine (11m)
25
Yield 78%; mp 148–150 8C; ½aꢀD ꢁ 58:14 (c 0.16, MeOH); UV (H2O)
lmax 267.0 nm (e 23879, pH 2), 269.0 nm (e 23535, pH 7), 269.0 nm
(e 23078, pH 11); 1H NMR (500 MHz, DMSO-d6) d 1.19 (d,
J = 6.5 Hz, 6H), 2.85 (m, 1H), 3.57 (m, 1H), 3.70 (m, 1H), 3.99 (m,
1H), 4.17 (m, 1H), 4.64 (m, 1H), 4.69 (m, 2H), 5.22 (m, 1H), 5.42 (m,
1H), 5.47 (d, J = 6.5 Hz, 1H), 5.92 (d, J = 6.5 Hz, 1H), 7.18 (d,
J = 8.5 Hz, 2H), 7.28 (d, J = 7.5 Hz, 2H), 8.23 (s, 1H), 8.40 (s, 1H),
8.46 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 24.4, 33.6, 43.1, 62.2,
71.1, 74.0, 86.4, 88.5, 120.3, 126.6, 127.7, 137.9, 140.4, 147.3,

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
1563
149.0, 152.8, 155.0. Anal. calcd. for C20H25N5O4 requires C, 60.14;
H, 6.31; N, 17.53; found C, 59.94; H, 6.36; N, 17.51.
(m, 1H), 5.37 (m, 1H), 5.48 (d, J = 5.5 Hz, 1H), 5.93 (d, J = 6.0 Hz,
1H), 7.35 (d, J = 7.5 Hz, 1H), 7.59 (m, 2H), 8.25 (s, 1H), 8.44 (s, 1H),
8.57 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 42.5, 62.1, 71.1, 74.0,
86.4, 88.4, 120.3, 128.0, 129.6, 130.9, 131.3, 140.6, 141.9, 149.1,
152.8, 154.8. Anal. calcd. for C17H17Cl2N5O4 requires C, 47.90; H,
4.02; N, 16.43; found C, 48.18; H, 4.06; N, 16.32.
2.2.24. N6-(4-Vinylbenzyl)adenosine (11n)
25
Yield 76%; mp 154–156 8C; ½aꢀD ꢁ 58:66 (c 0.19, MeOH); UV (H2O)
lmax 265.0 nm (e 31103, pH 2), 267.0 nm (e 30473, pH 7), 267.0 nm
(e 30601, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.57 (m, 1H), 3.69
(m, 1H), 3.99 (m, 1H), 4.17 (m, 1H), 4.64 (m, 1H), 4.72 (m, 2H), 5.23
(m, 2H), 5.40 (m, 1H), 5.48 (d, J = 5.5 Hz, 1H), 5.79 (d, J = 18.0 Hz,
1H), 5.91 (d, J = 6.0 Hz, 1H), 6.71 (dd, J1 = 17.5 Hz, J2 = 10.5 Hz, 1H),
7.33 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 8.23 (s, 1H), 8.41 (s,
1H), 8.51 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 43.2, 62.2, 71.1,
74.0, 86.4, 88.5, 114.2, 120.3, 126.5, 127.9, 136.1, 136.9, 140.3, 140.4,
149.0, 152.8, 155.0. Anal. calcd. for C19H21N5O4 requires C, 59.52;
H, 5.52; N, 18.27; found C, 59.45; H, 5.52; N, 18.22.
2.2.29. N6-(2-Fluoro-4-nitrobenzyl)adenosine (11s)
25
Yield 68%; mp 147–149 8C; ½aꢀD ꢁ 55:71 (c 0.17, MeOH); UV (H2O)
lmax 270.0 nm (e 24334, pH 2), 269.0 nm (e 24565, pH 7), 269.0 nm
(e 24614, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.58 (m, 1H), 3.70
(m, 1H), 3.99 (m, 1H), 4.17 (m, 1H), 4.65 (m, 1H), 4.86 (m, 2H), 5.23
(m, 1H), 5.35 (m, 1H), 5.49 (d, J = 6.0 Hz, 1H), 5.93 (d, J = 6.5 Hz,
1H), 7.58 (m, 1H), 8.06 (dd, J1 = 8.0 Hz, J2 = 2.5 Hz, 1H), 8.13 (dd,
J1 = 10.0 Hz, J2 = 2.5 Hz, 1H), 8.24 (s, 1H), 8.47 (s, 1H), 8.64 (s, 1H);
13C NMR (125 MHz, DMSO-d6) d 37.6, 62.1, 71.1, 74.0, 86.4, 88.4,
111.3 (d, J = 26.8 Hz), 120.1, 120.4, 130.1, 135.5, 140.8, 147.7 (d,
J = 8.6 Hz), 149.3, 152.8, 154.8, 159.7 (d, J = 247.6 Hz). Anal. calcd.
for C17H17FN6O6 requires C, 48.57; H, 4.08; N, 19.99; found C,
48.69; H, 4.01; N, 19.94.
2.2.25. N6-(4-Methoxybenzyl)adenosine (11o)
25
Yield 78%; mp 147–149 8C; ½aꢀD ꢁ 66:51 (c 0.12, MeOH); UV (H2O)
lmax 268.0 nm (e 19552, pH 2), 269.0 nm (e 19581, pH 7), 269.0 nm
(e 20045, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.59 (m, 1H), 3.70
(m, 1H), 3.73 (s, 3H), 4.00 (m, 1H), 4.18 (m, 1H), 4.66 (m, 3H), 5.23
(m, 1H), 5.44 (m, 1H), 5.49 (d, J = 4.5 Hz, 1H), 5.92 (d, J = 4.5 Hz,
1H), 6.88 (d, J = 7.0 Hz, 2H), 7.30 (d, J = 7.0 Hz, 2H), 8.25 (s, 1H),
8.41 (s, 1H), 8.44 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 42.8,
55.5, 62.2, 71.2, 74.0, 86.4, 88.5, 114.1, 120.3, 129.0, 132.5, 140.3,
149.0, 152.8, 155.0, 158.6. Anal. calcd. for C18H21N5O5 requires C,
55.81; H, 5.46; N, 18.08; found C, 56.00; H, 5.37; N, 17.90.
2.2.30. N6-(2-Chloro-4-cyanobenzyl)adenosine (11t)
25
Yield 75%; mp 148–150 8C; ½aꢀD ꢁ 60:56 (c 0.31, MeOH); UV (H2O)
lmax 268.0 nm (e 24613, pH 2), 267.0 nm (e 24963, pH 7), 267.0 nm
(e 25026, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.58 (m, 1H), 3.69
(m, 1H), 3.99 (m, 1H), 4.17 (m, 1H), 4.65 (m, 1H), 4.81 (m, 2H), 5.24
(m, 1H), 5.35 (m, 1H), 5.49 (d, J = 6.0 Hz, 1H), 5.93 (d, J = 5.0 Hz,
1H), 7.43 (m, 1H), 7.77 (m, 1H), 8.10 (d, J = 1.5 Hz, 1H), 8.23 (s, 1H),
8.48 (s, 1H), 8.63 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 41.8,
62.1, 71.1, 74.0, 86.4, 88.4, 111.6, 118.1, 120.4, 129.2, 131.6, 132.9,
133.0, 140.8, 143.5, 149.3, 152.8, 154.8. Anal. calcd. for
C18H17ClN6O4 requires C, 51.87; H, 4.11; N, 20.16; found C,
51.68; H, 4.21; N, 19.93.
2.2.26. N6-(2,4-Difluorobenzyl)adenosine (11p)
25
Yield 73%; mp 169–171 8C; ½aꢀD ꢁ 62:59 (c 0.10, MeOH); UV (H2O)
lmax 267.0 nm (e 20041, pH 2), 267.0 nm (e 19901, pH 7), 267.0 nm
(e 20057, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.59 (m, 1H), 3.71
(m, 1H), 4.00 (m, 1H), 4.18 (m, 1H), 4.65 (m, 1H), 4.74 (m, 2H), 5.23
(m, 1H), 5.39 (m, 1H), 5.49 (d, J = 6.0 Hz, 1H), 5.93 (d, J = 6.5 Hz,
1H), 7.03 (m, 1H), 7.24 (m, 1H), 7.40 (m, 1H), 8.25 (s, 1H), 8.44 (s,
1H), 8.49 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 37.0, 62.1, 71.1,
74.0, 86.4, 88.4, 104.0 (t, J = 25.8 Hz), 111.7 (dd, J1 = 20.8 Hz,
J2 = 3.8 Hz), 120.4, 123.4, 130.7, 140.6, 149.1, 152.8, 154.9, 160.4
(dd, J1 = 245.6 Hz, J2 = 12.0 Hz), 161.7 (dd, J1 = 243.4 Hz,
J2 = 12.0 Hz). Anal. calcd. for C17H17F2N5O4 requires C, 51.91;
H, 4.36; N, 17.80; found C, 51.81; H, 4.35; N, 17.70.
2.2.31. N6-(2-Fluoro-4-methoxybenzyl)adenosine (11u)
25
Yield 75%; mp 148–150 8C; ½aꢀD ꢁ 59:04 (c 0.13, MeOH); UV (H2O)
lmax 268.0 nm (e 23804, pH 2), 268.0 nm (e 23755, pH 7), 268.0 nm
(e 23563, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.57 (m, 1H), 3.68
(m, 1H), 3.75 (s, 3H), 3.99 (m, 1H), 4.17 (m, 1H), 4.64 (m, 1H), 4.69
(m, 2H), 5.23 (m, 1H), 5.40 (m, 1H), 5.48 (d, J = 5.5 Hz, 1H), 5.92 (d,
J = 6.0 Hz, 1H), 6.73 (dd, J1 = 8.0 Hz, J2 = 2.5 Hz, 1H), 6.82 (dd,
J1 = 12.0 Hz, J2 = 2.5 Hz, 1H), 7.27 (m, 1H), 8.24 (s, 1H), 8.41 (m,
2H); 13C NMR (125 MHz, DMSO-d6) d 36.9, 56.0, 62.2, 71.1, 74.0,
86.4, 88.4, 101.8 (d, J = 24.9 Hz), 110.5, 118.7 (d, J = 15.6 Hz),
120.3, 130.3, 140.4, 149.0, 152.8, 155.0, 160.1, 161.0 (d,
J = 251.8 Hz). Anal. calcd. for C18H20FN5O5ꢂ0.2H2O requires C,
52.86; H, 5.03; N, 17.12; found C, 52.85; H, 4.97; N, 17.04.
2.2.27. N6-(2,4-Dichlorobenzyl)adenosine (11q)
25
Yield 75%; mp 193–195 8C; ½aꢀD ꢁ 54:59 (c 0.23, MeOH); UV (H2O)
lmax 267.0 nm (e 17854, pH 2), 267.0 nm (e 15763, pH 7), 267.0 nm
(e 17216, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.57 (m, 1H), 3.69
(m, 1H), 3.99 (m, 1H), 4.17 (m, 1H), 4.65 (m, 1H), 4.74 (m, 2H), 5.23
(m, 1H), 5.37 (m, 1H), 5.49 (d, J = 6.0 Hz, 1H), 5.93 (d, J = 6.0 Hz,
1H), 7.29 (m, 1H), 7.38 (m, 1H), 7.65 (m, 1H), 8.23 (s, 1H), 8.46 (s,
1H), 8.55 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 41.2, 62.1, 71.1,
74.0, 86.4, 88.4, 120.4, 127.8, 129.0, 129.8, 132.4, 133.1, 136.4,
140.8, 149.1, 152.9, 154.8. Anal. calcd. for C17H17Cl2N5O4
requires C, 47.90; H, 4.02; N, 16.43; found C, 47.80; H, 3.96; N,
16.22.
2.2.32. N6-(2,4-Dimethoxybenzyl)adenosine (11v)
25
Yield 77%; mp 196–198 8C; ½aꢀD ꢁ 56:13 (c 0.18, MeOH); UV (H2O)
lmax 267.0 nm (e 20952, pH 2), 270.0 nm (e 21958, pH 7), 270.0 nm
(e 21868, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.59 (m, 1H), 3.72
(m, 1H), 3.75 (s, 3H), 3.84 (s, 3H), 4.00 (m, 1H), 4.18 (m, 1H), 4.64
(m, 3H), 5.23 (m, 1H), 5.44 (m, 1H), 5.49 (d, J = 4.0 Hz, 1H), 5.92 (d,
J = 4.0 Hz, 1H), 6.45 (d, J = 8.0 H, 1H), 6.58 (s, 1H), 7.04 (m, 1H),
8.13 (s, 1H), 8.21 (s, 1H), 8.41 (s, 1H); 13C NMR (125 MHz, DMSO-
d6) d 38.5, 55.7, 55.9, 62.2, 71.2, 74.0, 86.4, 88.5, 98.7, 104.7, 119.8,
120.4, 128.3, 140.4, 148.9, 152.9, 155.2, 158.1, 160.0. Anal. calcd.
for C19H23N5O6 requires C, 54.67; H, 5.55; N, 16.78; found C,
54.52; H, 5.54; N, 16.71.
2.2.28. N6-(3,4-Dichlorobenzyl)adenosine (11r)
24
Yield 73%; mp 184–185 8C; ½aꢀD ꢁ 57:21 (c 0.19, MeOH); UV (H2O)
lmax 267.0 nm (e 19002, pH 2), 268.0 nm (e 17314, pH 7), 268.0 nm
(e 19186, pH 11); 1H NMR (500 MHz, DMSO-d6) d 3.58 (m, 1H), 3.70
(m, 1H), 3.99 (m, 1H), 4.18 (m, 1H), 4.64 (m, 1H), 4.72 (m, 2H), 5.22

1564
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
2.3.
Molecular modeling
purified T. gondii adenosine kinase, prepared as previously
described [29] in a final volume of 100 mL, in the absence or
presence of various concentrations of the compound under
evaluation. Incubation was carried out at 37 8C and terminated
by boiling in a water bath for 2 min, followed by freezing for at
least 20 min. Precipitated proteins were removed by centrifu-
gation and 10 mL 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: adenosine, 0.27 and AMP, 0.17. The amounts of
radioactivity in both the substrate and product were calcu-
lated on a percentage basis using a computerized Berthold LB-
284 Automatic TLC-Linear Analyzers. Apparent Ki values of
these analogues were calculated from Dixon plots 1=v versus
[I] by least-squares fitting by computer programs developed by
Naguib and co-workers as previously described [17–19]. The
synthesis of the nucleoside 50-monophosphates from the
tested N6-benzyladenosine analogues was confirmed by HPLC
and NMR analyses as previously described [20], 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 values [30] as presented
in Table 1.
T. gondii adenosine kinase functions by induced fit mechanism
and involves a hinge bending motion of two domains. This
domain movement is complex, and apparently involves
further tuning of the protein structure when substrates bind
into the active site of the enzyme. As a result, our initial
modeling attempts of utilizing the apo form (1LIO.pdb) [23] of
the enzyme were far less successful than subsequent analysis
with the T. gondii adenosine kinase–adenosine complex
(1LII.pdb) [23]. The crystal coordinates of the reported
enzyme:substrate complex is missing the following residues:
1–10, 239–240, 255–269, 359–363. Residues 239–240 and 255–269
were added using the SYBYL Biopolymer module (Tripos
Associates, St. Louis, MO, USA). The terminal residues were
not added because it has been shown by mutation and
sequential deletions studies that their effect on the overall
binding affinity is negligible. Determination of the binding
mode of the N6-benzyladenosine analogues was complicated
by the fact that the size of the ligand (N6-benzyladenosine) is
very large as compared to the natural substrate adenosine. N6-
˚
Benzyladenosine analogues are almost ꢃ16 A in length
diagonally, where as the diagonal length of the adenosine is
˚
ꢃ8 A. We are almost doubling the size of the ligand while
keeping the size of the pocket the same. Therefore, the
induced fit docking method [26] and the prime module of
2.5.
Evaluation of N6-benzyladenosine analogues as
Schrodinger suit [27] were used to judge/perform the protein
¨
potential anti-toxoplasma agents against T. gondii in tissue
culture
conformational change according to larger ligand. A second
module ‘glide’ was used to dock the ligands in the active site
followed by active site residue modification and refinement by
the prime module. After successful active site modification
and refinement by prime and re-docking by glide, enzyme:-
ligand complexes were subjected to energy minimization [28].
The wild type RH and the adenosine kinase deficient mutant
TgAKꢁ3 [22] strains of T. gondii were used in these experiments.
The adenosine kinase deficient mutant TgAKꢁ3 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 anti-toxoplasmosis 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 [20,31]. The viability of intracellular parasites was
evaluated by the selective incorporation of radiolabeled uracil
into nucleic acids of the parasites at least in triplicates as
previously described [20,31]. Briefly, confluent cells (4–5 day
incubation) were cultured for 24 h in the 24-well flat bottom
microtiter plates (ꢃ5 ꢄ 105 1 mLꢁ1 wellꢁ1) and incubated at
37 8C 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 (one parasite/cell).
After 1 h 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 mM. 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 mCi/mL) and
incubated 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 mL trypsin/EDTA
All the calculations were performed on Schrodinger Suite
¨
2006 (Schrodinger Inc.) with GB/SA continuum water solvation
¨
model. OPLS_2001 and OPLS_2005 force field was used for
induced fit docking and then the same force field was
implemented into MACROMODEL 9.1 for energy minimization.
For energy minimization studies, residues further away by
˚
more than 16 A from the adenosine binding site were frozen
˚
and residues from 6 to 16 A were constrained by harmonic
˚
constraints. Only residues inside a 6 A 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. In the case
of the N6-benzyladenosine analogues, the starting conforma-
tion was obtained after performing a 5000 step Monte Carlo
conformational search under GB/SA continuum water solva-
tion model, Polak–Ribiere conjugate gradient (PRCG) as a
method for the subsequent energy minimization.
2.4.
Evaluation of the N6-benzyladenosine 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 [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 kinase, 5 mM [8-14C]adenosine (55 Ci/mol), 50 mL

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
1565
Table 1 – Binding affinities (apparent Km) of N6-benzyladenosine and its analogues to Toxoplasma gondii adenosine kinase
a
Compound
R1(ortho)
R2(meta)
R3(para)
Km (mM)
11a N6-Benzyladenosine
H
H
H
H
H
H
F
H
179.8 ꢅ 18.8
51.3 ꢅ 10.5
12.0 ꢅ 2.4
37.4 ꢅ 6.6
11.2 ꢅ 1.8
82.5 ꢅ 14.4
25.3 ꢅ 4.3
52.6 ꢅ 7.8
81.2 ꢅ 11.4
17.9 ꢅ 4.8
98.7 ꢅ 19.3
27.0 ꢅ 4.7
47.1 ꢅ 7.9
88.7 ꢅ 13.4
9.9 ꢅ 2.2
11b N6-(2-Fluorobenzyl)adenosine
11c N6-(2-Chlorobenzyl)adenosine
11d N6-(2-Nitrobenzyl)adenosine
11e N6-(2-Methoxybenzyl)adenosine
11f N6-(3-Fluorobenzyl)adenosine
11g N6-(3-Methylbenzyl)adenosine
11h N6-(4-Fluorobenzyl)adenosine
11i N6-(4-Chlorobenzyl)adenosine
11j N6-(4-Cyanobenzyl)adenosine
11k N6-(4-Trifluoromethylbenzyl)adenosine
11l N6-(4-Methylbenzyl)adenosine
11m N6-(4-Isopropylbenzyl)adenosine
11n N6-(4-Vinylbenzyl)adenosine
11o N6-(4-Methoxybenzyl)adenosine
11p N6-(2,4-Difluorobenzyl)adenosine
11q N6-(2,4-Dichlorobenzyl)adenosine
11r N6-(3,4-Dichlorobenzyl)adenosine
11s N6-(2-Fluoro-4-nitrobenzyl)adenosine
11t N6-(2-Chloro-4-cyanobenzyl)adenosine
11u N6-(2-Fluoro-4-methoxybenzyl)adenosine
11v N6-(2,4-Dimethoxybenzyl)adenosine
Adenosine
F
H
Cl
NO2
OCH3
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
–
H
H
F
H
Cl
H
CN
CF3
CH3
iPr
H
H
H
H
CH CH2
OCH3
F
H
F
12.1 ꢅ 2.0
20.2 ꢅ 6.5
35.8 ꢅ 3.2
167.8 ꢅ 25.3
53.7 ꢅ 13.6
25.5 ꢅ 3.6
27.6 ꢅ 1.7
5.2 ꢅ 0.3
Cl
H
Cl
Cl
F
NO2
CN
OCH3
OCH3
–
Cl
F
OCH3
–
a
All values represent the mean ꢅ S.D. of at least two independent experiments with three replica each as previously described [17–20].
(2.5X) 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 distilled H2O and
dried. After filtration, the filters were washed with 10 mL
methanol, left to dry, then placed in scintillation vials
containing 5 mL of Econo-Safe scintillation fluor (Research
Products International Corp., Mount Prospect, IL, USA), and
radioactivity was counted using an LS5801 Beckman scintilla-
tion 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 as compared to the
untreated controls [19,20,31,32]. Radiolabel incorporation
closely correlates with parasite growth [19,20,31,32].
20 passages) in RPMI 1640 medium [19,20,31]. Briefly, confluent
cells were incubated for at least 24 h in 96-well flat bottom
microtiterplates (ꢃ105 200 mLꢁ1 wellꢁ1) at 37 8C in 5% CO2, 95%
air to allow the cells to attach. The medium was then replaced
with 200 mL of fresh medium. The appropriate concentration
of the compounds was dissolved in 50 mL of medium, and
added to each well to give the desired final concentrations. The
cultures were then incubated for 48 h after which 50 mL of
sterile MTT solution (2 mg/1 mL PBS) was added to each well.
MTT solution was sterilized by filtration through 0.22 mm
filters (Costar, Cambridge, MA, USA). After 4 h incubation, the
medium was removed and 100 mL of dimethylsulfoxide
(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 computer-
ized microtiterplate reader (Themomax, Molecular Devices).
2.6.
Host toxicity of N6-benzyladenosine analogues
3.
Results
Possible toxicity against the host cells by the same doses of the
various analogues used in the above experiments was
measured, at least in triplicates, using a modification of the
microculture tetrazolium (MTT) assay on uninfected mono-
layers of human foreskin fibroblasts (grown for no more than
3.1.
Chemistry
The substituted benzylamines 3a–b, 7 and 9 were prepared
according to the literature procedures [33,34] as shown in

1566
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
Scheme 1 – Synthesis of substituted benzylamines 3a–b, 7 and 9. (a) N-Bromosuccinimide, CCl4, benzoyl peroxide, reflux,
8 h; (b) NH(Boc)2, NaH, THF, rt, 7 h; (c) 1N HCl/Et2O, MeOH, rt, 5 h; (d) NaBH4, MeOH, rt, 3 h; (e) CBr4, PPh3, CH2Cl2, rt, 3 h; (f)
NaN3, DMF, rt, 1 h; (g) PPh3, H2O, benzene, 70 8C, 3 h.
Scheme 1. Compounds 1a–b were treated with N-bromosuc-
cinimide in the presence of benzoyl peroxide to obtain the
benzyl bromide derivatives, which were then reacted with di-
tert-butyl iminodicarboxylate and sodium hydride to give
compounds 2a–b. The Boc groups were removed with 1N HCl/
Et2O in methanol to give the desired benzylamines 3a–b.
Compound 4 was reduced to provide the benzyl alcohol 5.
Bromination of compound 5 with carbon tetrabromide and
triphenylphosphine provided the corresponding benzyl bro-
mide followed by introduction of an amino group to give
compound 6. Subsequent deprotection of compound 6 was
performed under the acidic conditions described above for
compounds 3a–b to obtain compound 7. 4-Vinylbenzyl
chloride (8) was converted into the azide intermediate as a
crude oil and treated with triphenylphosphine followed by
hydrolysis to yield benzylamine 9.
obtained in 70–80% yields after purification. However, 11d and
11s containing a nitro group were obtained in lower yields. A
mild condition (N,N-diisopropylethylamine and lower tem-
perature) provided the desired compounds 11d and 11s with
improved yields.
3.2.
Structure–activity relationships by enzyme binding
affinity
The binding affinities of N6-benzyladenosine (11a) and its
analogues (11b–v) to T. gondii adenosine kinase were evaluated
in enzyme-based assays, along with adenosine as reference
substrate (Table 1).
The present results allowed us to examine the relation-
ships between the type and the position of the substitutions on
the aromatic ring, as well as the biological activity of these N6-
benzyladenosine analogues. Chlorine at the ortho position
(11c) exhibited higher binding affinity in comparison to the
fluorine substitution (11b). Compounds 11e (o-methoxy) and
11g (m-methyl) displayed an increase in the binding affinity to
T. gondii adenosine kinase when compared to 11d (o-nitro) and
N6-Benzyladenosine analogues 11a–v were synthesized via
solution phase parallel synthesis (Scheme 2) [35–37]. Intro-
duction of the appropriate benzylamine was achieved by
nucleophilic substitution of commercially available 6-chlor-
opurine riboside (10). Compounds 11a–c, 11e–r and 11t–v were
Scheme 2 – Synthesis of N6-benzyladenosine analogues 11a–v. (a) appropriate benzylamine, TEA, n-PrOH, 100 8C, 11 h; (b) 2-
nitrobenzylamine or 2-fluoro-4-nitrobenzylamine, DIEA, 80 8C, 11 h.

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
1567
Table 2 – The effect of different doses of N6-benzyladenosine and its analogues on host toxicitya and percentage survivalb
of wild type and adenosine kinase deficient (TgAKS3) strains of Toxoplasma gondii grown in human fibroblasts in culture
Compound
Infection
Concentration (mM)
IC50 (mM)
0
5
10
25
50
11a N6-Benzyladenosine
Wild type (RH)
TgAKꢁ3
Nonea
100
100
100
99.2
91.1
82.2
73.1
791 ꢅ 53
100
100
100
100
22.9
20.9
19.1
18.0
11b N6-(2-Fluorobenzyl)adenosine
11c N6-(2-Chlorobenzyl)adenosine
11d N6-(2-Nitrobenzyl)adenosine
11e N6-(2-Methoxybenzyl)adenosine
11f N6-(3-Fluorobenzyl)adenosine
11g N6-(3-Methylbenzyl)adenosin
11h N6-(4-Fluorobenzyl)adenosine
11i N6-(4-Chlorobenzyl)adenosine
11j N6-(4-Cyanobenzyl)adenosine
11k N6-(4-Trifluoromethylbenzyl)adenosine
11l N6-(4-Methylbenzyl)adenosine
11m N6-(4-Isopropylbenzyl)adenosine
11n N6-(4-Vinylbenzyl)adenosine
11o N6-(4-Methoxybenzyl)adenosine
11p N6-(2,4-Difluorobenzyl)adenosine
11q N6-(2,4-Dichlorobenzyl)adenosine
11r N6-(3,4-Dichlorobenzyl)adenosine
Wild type (RH)
TgAKꢁ3
100
100
100
98.3
89.5
36.0
20.4
37.8 ꢅ 27.9
10.6 ꢅ 1.7
15.0 ꢅ 0.9
8.7 ꢅ 3.2
23.2 ꢅ 3.7
8.5 ꢅ 0.3
20.6 ꢅ 2.9
25.8 ꢅ 1.3
8.3 ꢅ 1.2
22.9 ꢅ 2.9
10.5 ꢅ 1.6
22.0 ꢅ 2.4
26.2 ꢅ 1.9
7.5 ꢅ 1.0
8.2 ꢅ 2.3
9.3 ꢅ 0.8
14.3 ꢅ 1.2
100
100
100
100
100
None
78.1
68.1
64.7
Wild type (RH)
TgAKꢁ3
100
100
100
79.3
100
74.0
46.8
0.3
0.0
100
100
100
57.2
None
71.2
57.6
Wild type (RH)
TgAKꢁ3
100
100
100
97.9
100
100
72.3
17.7
5.42
100
100
100
100
100
100
None
Wild type (RH)
TgAKꢁ3
100
100
100
75.2
100
26.8
22.1
100
22.6
0.2
100
22.3
0.0
100
21.4
None
Wild type (RH)
TgAKꢁ3
100
100
100
93.6
100
100
75.2
100
100
45.7
100
100
24.7
100
100
None
Wild type (RH)
TgAKꢁ3
100
100
100
70.5
28.2
100
95.5
3.5
100
89.5
0.96
100
87.6
100
100
None
Wild type (RH)
TgAKꢁ3
100
100
100
96.3
92.5
100
33.9
100
19.9
100
99.0
44.1
None
33.8
15.7
14.6
Wild type (RH)
TgAKꢁ3
100
100
100
91.4
80.1
44.1
32.5
100
100
100
100
100
100
100
None
78.5
Wild type (RH)
TgAKꢁ3
100
100
100
72.5
100
55.7
16.7
100
53.7
2.2
100
45.8
0.0
100
None
31.9
Wild type (RH)
TgAKꢁ3
100
100
100
93.0
100
100
75.0
100
100
42.0
100
100
26.3
100
100
None
Wild type (RH)
TgAKꢁ3
100
100
100
83.8
100
86.7
33.0
100
83.4
4.3
100
80.5
1.4
100
74.2
None
Wild type (RH)
TgAKꢁ3
100
100
100
96.2
100
100
88.5
100
38.9
100
22.0
100
None
83.7
71.9
63.9
Wild type (RH)
TgAKꢁ3
100
100
100
88.1
100
98.9
83.0
50.5
27.4
100
100
100
None
89.3
84.6
76.5
Wild type (RH)
TgAKꢁ3
100
100
100
66.9
100
16.9
1.9
0.0
100
100
100
None
93.4
90.5
72.3
59.7
Wild type (RH)
TgAKꢁ3
100
100
100
67.7
33.6
2.4
0.0
100
100
100
100
None
69.6
63.5
58.8
40.5
Wild type (RH)
TgAKꢁ3
100
100
100
78.3
27.4
1.8
0.0
100
100
100
100
100
100
None
94.3
89.6
Wild type (RH)
TgAKꢁ3
100
100
100
85.0
73.3
11.8
2.7
100
100
100
100
100
85.5
100
77.2
None

1568
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
Table 2 (Continued )
Compound
Infection
Concentration (mM)
IC50 (mM)
0
5
10
25
50
11s N6-(2-Fluoro-4-nitrobenzyl)adenosine
11t N6-(2-Chloro-4-cyanobenzyl)adenosine
11u N6-(2-Fluoro-4-methoxybenzyl)adenosine
11v N6-(2,4-Dimethoxybenzyl)adenosine
Pyrimethamine
Wild type (RH)
TgAKꢁ3
100
100
100
97.8
91.0
75.5
66.4
230 ꢅ 23
21.1 ꢅ 1.7
9.5 ꢅ 0.9
8.7 ꢅ 0.6
100
100
100
100
100
100
None
96.4
83.1
Wild type (RH)
TgAKꢁ3
100
100
100
99.2
96.3
32.3
21.2
100
100
100
100
100
100
100
100
None
Wild type (RH)
TgAKꢁ3
100
100
100
73.4
39.0
6.7
100
96.5
1.1
100
95.7
100
100
100
100
None
Wild type (RH)
TgAKꢁ3
100
100
100
72.0
98.4
100
29.9
3.7
100
100
0.5
100
100
100
100
None
Wild type (RH)
None
100
100
54.8
36.3
13.8
5.9
5.3 ꢅ 1.0
7.0 ꢅ 1.9
101.3
99.9
108.2
108.6
Sulfadiazine
Wild type (RH)
None
100
100
75.7
98.2
64.5
99.7
59.2
99.8
46.7
102.5
a
Host toxicity of uninfected cells was measured by MTT method in at least two independent experiments each of three replica as previously
described [19,20,31].
b
Percentage 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 [19,20,31].
11f (m-fluoro), respectively. It appears that electron-rich
substitution at the ortho or the meta position resulted in an
increase in the binding affinity to the enzyme. Our previous
studies with 6-benzylthioinosine analogues indicated that
substitutions at the para position have a substantial impact on
the binding affinity to T. gondii adenosine kinase [18]. These
previous results prompted us to examine the effects of
substituents at the para position of the benzyl moiety of N6-
benzyladenosine (11h–o). N6-( para-Methoxybenzyl)adenosine
(11o) was the best ligand in this series as it produced an 18-fold
enhancement in the binding affinity. In the case of disub-
stituted benzyladenosine analogues (11p–v), a small disub-
stitution such as fluorine (11p) appeared to be favored. It was
also observed that the difluorinated compound 11p exhibited
improved binding affinity relative to the monofluorinated
compounds (11b, 11f and 11h).
and the Km values obtained from the enzyme binding assays
(Table 1). As expected, N6-(2-methoxybenzyl)adenosine (11e)
and N6-(4-methoxybenzyl)adenosine (11o) appeared to be
more potent in the cell culture evaluations and were also
comparable with that of sulfadiazine and/or pyrimethamine
(Table 2). On the other hand, the unsubstituted N6-benzyla-
denosine (11a) and N6-(2-fluoro-4-nitrobenzyl)adenosine
(11s), which were weak substrates for T. gondii adenosine
kinase (Table 1), exhibited lower anti-toxoplasma potency in
the cell culture assays (Table 2). Discrepancies in the order of
potency between the enzymatic assay (Table 1) and the cell-
based assay (Table 2) may be attributed to the differences in
cell permeability of these compounds.
We have also tested the host cell toxicity of N6-benzyla-
denosine (11a) and its analogues (11b–v) at concentration up
to 50 mM in an effort to delineate their selective toxicity as
potential anti-toxoplasma agents. Most of the N6-benzylade-
nosines had toxic effects on the survival of uninfected host cell
(Table 2) and therefore may be inadequate for the treatment of
toxoplasmosis. However, N6-(2,4-dimethoxybenzyl)adenosine
(11v) had no detectable host toxicity and hence exhibited
favorable potency and selectivity as an anti-toxoplasma agent.
3.3.
Evaluation of anti-toxoplasma activity
N6-Benzyladenosine (11a) and its analogues (11b–v) were
evaluated against wild type (RH) and adenosine kinase
deficient (TgAKꢁ3) strains of T. gondii to further investigate
their potency as anti-toxoplasma agents (Table 2). Pyrimetha-
mine and sulfadiazine as chemotherapeutic agents for the
treatment of toxoplasmosis were used as positive controls.
As shown in Table 2, all of the N6-benzyladenosines were
effective against infection with the wild type (RH) T. gondii in a
dose-dependent manner. Furthermore, none of these com-
pounds were found effective against infection with adenosine
kinase deficient strain (TgAKꢁ3). Therefore, these compounds
appear to be the active substrates for T. gondii adenosine
kinase in vivo as was the case in vitro (Table 1). Taken as a
whole, there was a general trend of correlation between the
degree of anti-toxoplasma efficacy in the cell-based activity
3.4.
Molecular modeling
Previous structure–activity relationship studies [17,18] and X-
ray structural reports [23,24] revealed the molecular criteria
for ligand binding to the adenosine binding site of T. gondii
adenosine kinase. Recent structural studies of adenosine
kinase complex with N6-dimethyladenosine and 6-methyl-
mercaptopurine riboside [29] showed the intermediate-con-
formation between closed (catalytic-conformation) and open
lid domain (apo-conformation), and revealed the conforma-
tional steps for catalysis. The molecular recognition analysis

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
1569
Fig. 3 – (a) Binding mode of the natural substrate, adenosine to T. gondii adenosine kinase. (b) Binding mode of N6-(4-
methoxybenzyl)adenosine (11o) showing how the induced fit mechanism is working to accommodate a larger ligand in the
active site of T. gondii adenosine kinase after conformational changes of nearby hydrophobic residues. (c) Binding mode of
N6-(2,4-dimethoxybenzyl)adenosine (11v) showing the orientation of additional 2-methoxy substituent towards middle of
the Thr140 and Leu142 residues.
from the detailed study of the reported crystal structures of
enzyme–ligand complexes [23] and structure–activity relation-
ships analysis [17,18] indicate that the following factors play
important roles in ligand binding into the adenosine binding
pocket: (a) 20-OH and 30-OH are crucial for the binding because
they form four strong H-bond with nearby residues, which
accounts for a strong electrostatic interaction and conforma-
tional determinant for the ribose moiety as shown in Fig. 2a; (b)
active site map analysis clearly showed the presence of a large
hydrophobic regions around the N6 and N7 positions of
adenine as shown in Fig. 2b; (c) adenine is surrounded with
hydrophobic residues such as Ile22, Leu46, Leu138, Leu142,
Tyr169. Our recent modeling and biological studies on 6-
benzylthioinosine analogues [18] also revealed that when the
substituted benzyl moiety moves into the pocket, the above
described hydrophobic residues near the purine motif allow a
favorable interaction by the induced fit mechanism and
strengthen binding by increasing the van der Waals interac-
tion.
Our study on the determination of binding mode for N6-
benzyladenosine analogues was complicated because the size
of the N6-benzyladenosine analogues is almost a twice the size
Fig. 4 – (a) Superimposed structure of the strong substrate N6-(4-methoxybenzyl)adenosine (11o) and the weaker substrate
N6-(4-chlorobenzyl)adenosine (11i) showing the same binding mode with the nearby residues in the active site of T. gondii
adenosine kinase. (b) The binding mode of 11i to T. gondii adenosine kinase showing a low van der Waals surface area for
hydrophobic effect with Leu46. (c) While more surface area is found in the case of 11o for hydrophobic effect with Leu46
residue.

1570
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
of the natural substrate, adenosine. As T. gondii adenosine
kinase functions via the induced fit mechanism, an advance
induced fit docking method [26] was used to allow for the
larger ligands that we synthesized in this study. The N6-
benzyladenosine analogues docked successfully into the T.
gondii adenosine kinase active site by induced fit method
[27]. The binding mode shows an anti-conformation of the
nucleoside and an 20-endo conformation of the ribose
moiety, which are the preferred conformations observed
by the co-crystal structure [23]. The benzyl moiety occupies
the hydrophobic pocket and forms a favorable van der
Waals interaction with surrounding residues. Crucial inter-
actions such as 20-OH and 30-OH interactions with Asp24, p–
p and C–H–p (Fig. 2a), which are required for ligand binding
into the adenosine pocket, were present for all N6-
benzyladenosine analogues while significant differences
in van der Waals interactions were observed for individual
compounds. These results indicate that the type and
position of the substituents on the benzyl group of the
N6-benzyladenosines significantly affect the van der Waals
interaction and ultimately lead to differences in their
binding affinities.
4.
Discussion
N6-Benzyladenosine analogues were synthesized and evalu-
ated for their binding affinity to purified T. gondii adenosine
kinase. Compound 11a, containing unsubstituted benzyl
moiety, showed a weak binding affinity with a Km value of
179.8 mM to T. gondii adenosine kinase. Compounds 11c, 11e,
11o and 11p were the best among the synthesized analogues
(Km = 12.0, 11.2, 9.9 and 12.1 mM, respectively), and provided a
15–18-fold increase in the binding affinity relative to the
unsubstituted N6-benzyladenosine (11a). Furthermore, the
anti-toxoplasma efficacy and host toxicity of these compounds
were tested in cell culture. Varying the type and position of the
substituents on the aromatic ring led to different degrees of
potency and selectivity as anti-toxoplasma agents. However,
most of the N6-benzyladenosine, in contrast to their 6-
benzylinosine counterparts [20], were toxic to the host and
therefore, are may be inadequate for the treatment of
toxoplasmosis. In spite of the host toxicity of most of the
synthesized N6-benzyladenosine analogues, N6-(2,4-dimethox-
ybenzyl)adenosine (11v) exhibited favorable anti-toxoplasma
activity without host toxicity. These results demonstrate the
necessity of testing and evaluating newly synthesized analo-
gues irrespective of their structural similarity to promising
analogues before a general conclusion can be made.
The enhanced binding of the synthesized N6-benzylade-
nosine analogue was due mainly to the hydrophobic effect
mediated by hydrophobic residues (Y169, F201, L46 and L142)
to gain favorable van der Waals interactions around benzyl
moiety of the ligands, as established by molecular modeling
studies. In addition, we analyzed the multi-ligand bimolecular
association with energetics (MBAE) calculation of N6-(4-
chlorobenzyl)adenosine (11i), N6-(4-methoxybenzyl)adeno-
sine (11o) and N6-(2,4-dimethoxybenzyl)adenosine (11v)
(Table 3) to determine whether or not differences in the
binding energies of these compounds may also be involved in
enhancing their binding with the enzyme. Compounds 11o
and 11v gained favorable van der Waals interaction (ꢁ36.1 and
ꢁ44.2 kcal/mol, respectively), which led to reduced total
binding energy difference by ꢁ34.4 and ꢁ27.1 kcal/mol,
respectively. In contrast, compound 11i had less favorable
van der Waals interaction (9.2 kcal/mol) as well as an
increased binding energy difference of 15.2 kcal/mol. These
results indicate that differences in the binding energies could
also contribute to the differences in the binding affinities of
these compounds to T. gondii adenosine kinase.
Fig. 3 shows the molecular surface around adenosine
(natural substrate), N6-(4-methoxybenzyl)adenosine (11o) and
N6-(2,4-dimethoxybenzyl)adenosine (11v), the best ligands
among the synthesizsed N6-benzyladenosine analogues, after
induced fit modeling, which explains the reason for accom-
modating the larger ligands in the adenosine binding site of T.
gondii adenosine kinase by conformational changes in nearby
hydrophobic residues. In the case of N6-(4-methoxybenzyl)a-
denosine (11o), the benzyl group position in the hydrophobic
pocket and the presence of methoxy substitution at the para
position increases the van der Waals surface interaction with
Leu46. On the other hand, N6-(2,4-dimethoxybenzyl)adeno-
sine (11v) showed the orientation of additional ortho-methoxy
substituent towards middle of the Thr140 and Leu142 residue.
Fig. 4 shows the binding modes of N6-(4-chlorobenzyl)adeno-
sine (11i) and N6-(4-methoxybenzyl)adenosine (11o), where
N6-(4-chlorobenzyl)adenosine (11i) exhibits less van der Waals
surface interaction with Leu46 than N6-(4-methoxybenzyl)a-
denosine (11o). The present results suggest the long tubular
hydrophobic region at the N6 region can accommodate the
benzyl group and in spite of similar binding mode, the benzyl
substituents play an important role to induce the favorable or
unfavorable van der Waals interaction with the surrounding
residues.
Table 3 – Comparison between multi-ligand bimolecular association with energetics (MBAE) calculation of representative
N6-benzyladenosine analogues after induced fit docking and energy minimization
Compound
Km (mM)
Complex total energy
(kcal/mol)
Energy difference results
(DE, kcal/mol)
Electrostatic
VdWa
Total
11i N6-(4-Chlorobenzyl)adenosine
11o N6-(4-Methoxybenzyl)adenosine
11v N6-(2,4-Dimethoxybenzyl)adenosine
81.2 ꢅ 11.4
9.9 ꢅ 2.2
ꢁ6279.6
ꢁ6331.5
ꢁ6317.2
ꢁ52.1
ꢁ86.1
ꢁ53.7
9.2
ꢁ36.1
ꢁ44.2
15.2
ꢁ34.4
ꢁ27.1
27.6 ꢅ 1.7
a
van der Waals interaction.

b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 1 5 5 8 – 1 5 7 2
1571
In summary, N6-benzyladenosine and its analogues were
synthesized as a novel class of anti-toxoplasma agents.
Molecular modeling studies were conducted to understand
their binding mode to T. gondii adenosine kinase. Varying the
type and position of the substituents on the aromatic ring
resulted in different degrees of the binding affinity to T. gondii
adenosine kinase as well as potency and selectivity as anti-
toxoplasma agents. Based on their structure–activity relation-
ships, additional syntheses of various other analogues as well
as biological studies are warranted.
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