Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of trypanosomatidae via structure-based drug design

Article abstract of DOI:10.1021/jm000472o

In our continuation of the structure-based design of anti-trypanosomatid drugs, parasiteselective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH's provided details of how the adenosyl moiety of NAD⁺ interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH's. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N⁶-(1-naphthalenemethyl)-2′-deoxy-2′- (3-methoxybenzamido)adenosine (6e), N⁶-(substituted-naphthalenemethyl)-2′-deoxy-2′- (substituted-benzamido)adenosine analogues were investigated. N⁶(1 -Naphthalenemethyl)-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (6m), N⁶- [1-(3-hydroxynaphthalene)methyl]-2′-deoxy-2′- (3,5-dimethoxybenzamido)adenosine (7m), N⁶-[1-(3-methoxynaphthalene)methyl]-2′-deoxy-2′- (3,5-dimethoxybenzamido)adenosine (9m), N⁶-(2-naphthalenemethyl)-2′-deoxy-2′1- (3-methoxybenzamido)adenosine (11e), and N⁶-(2-naphthalenemethyl)-2′deoxy-2′- (3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2- to 3-fold improvement over 6e and a 7100- to 25000-fold improvement over the adenosine template. IC₅₀'S of these compounds were in the range 2-12 μM for T. brucei, T. cruzi, and L. mexicana GAPDH's, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure- activity relationships of this class of compounds, a library of 240 N⁶-(substituted)-2′-deoxy-2′-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N⁶ and C2′ substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH's in the low micromolar range. We also explored adenosine analogues containing 5′-amido substituents and found that 2′,5′-dideoxy-2′-(3,5-dimethoxybenzamido)-5′- (diphenylacetamido)adenosine (49) displays an IC₅₀ of 60-100 μM against the three parasite GAPDH's.

Full text of DOI:10.1021/jm000472o

2080
J . Med. Chem. 2001, 44, 2080-2093
Ad en osin e An a logu es a s Selective In h ibitor s of Glycer a ld eh yd e-3-p h osp h a te
Deh yd r ogen a se of Tr ypa n osom a tid a e via Str u ctu r e-Ba sed Dr u g Design
J erome C. Bressi,† Christophe L. M. J . Verlinde,‡ Alex M. Aronov,§ My Le Shaw,| Sam S. Shin,†
Lisa N. Nguyen, Stephen Suresh,‡,# Frederick S. Buckner, Wesley C. Van Voorhis, Irwin D. Kuntz,§
Wim G. J . Hol,‡,|,# and Michael H. Gelb*,†,|
Departments of Chemistry, Biochemistry, Medicine, and Biological Structure, University of Washington,
Seattle, Washington 98195, Biomolecular Structure Center and Howard Hughes Medical Institute, Seattle, Washington 98195,
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143
Received November 6, 2000
In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-
selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-
3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma
cruzi, Leishmania mexicana, and human GAPDH’s provided details of how the adenosyl moiety
of NAD+ interacts with the proteins, and this facilitated the understanding of the relative
affinities of a series of adenosine analogues for the various GAPDH’s. From exploration of
modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N6-
(1-naphthalenemethyl)-2′-deoxy-2′-(3-methoxybenzamido)adenosine (6e), N6-(substituted-naph-
thalenemethyl)-2′-deoxy-2′-(substituted-benzamido)adenosine analogues were investigated. N6-
(1-Naphthalenemethyl)-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (6m ), N6-[1-(3-hydroxy-
naphthalene)methyl]-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (7m ), N6-[1-(3-methoxy-
naphthalene)methyl]-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (9m ), N6-(2-naphthalene-
methyl)-2′-deoxy-2′-(3-methoxybenzamido)adenosine (11e), and N6-(2-naphthalenemethyl)-2′-
deoxy-2′-(3,5-dimethoxybenzamido)adenosine (11m ) demonstrated a 2- to 3-fold improvement
over 6e and a 7100- to 25000-fold improvement over the adenosine template. IC50’s of these
compounds were in the range 2-12 µM for T. brucei, T. cruzi, and L. mexicana GAPDH’s, and
these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To
explore more thoroughly the structure-activity relationships of this class of compounds, a
library of 240 N6-(substituted)-2′-deoxy-2′-(amido)adenosine analogues was generated using
parallel solution-phase synthesis with N6 and C2′ substituents chosen on the basis of
computational docking scores. This resulted in the identification of 40 additional compounds
that inhibit parasite GAPDH’s in the low micromolar range. We also explored adenosine
analogues containing 5′-amido substituents and found that 2′,5′-dideoxy-2′-(3,5-dimethoxy-
benzamido)-5′-(diphenylacetamido)adenosine (49) displays an IC50 of 60-100 µM against the
three parasite GAPDH’s.
In tr od u ction
or more drawbacks such as ineffectiveness against more
virulent strains, ineffectiveness against post-CNS inva-
sion, toxicity, resistance, or parenteral administration.4,5
Other diseases caused by Trypanosomatidae, which
share a similar state with respect to available drug
treatment, include Chagas’ disease (Trypanosoma cruzi)6
and leishmaniasis (Leishmania spp.).7 Clearly, there
exists a need for safer and more efficacious chemothera-
peutic agents against trypanosomatids.
According to the World Health Organization, Trypa-
nosoma brucei, the causative agent of African sleeping
sickness, infected 300 000 to 500 000 people in 1999 and
poses an escalating health threat to nearly 60 million
people of 36 countries of sub-Saharan Africa.1,2 Intro-
duced into the mammalian host via the blood-meal bite
of the tsetse fly vector, T. brucei freely replicates in the
bloodstream as it makes its way to the central nervous
system (CNS). From there, this parasitic infection
induces delirium, seizures, coma, and, if left untreated,
death.3 Existing drug therapies, including suramin,
pentamidine, melarsoprol, and difluoromethylornithine
(DFMO), are inadequate because they suffer from one
The focus of this work is on glycolysis in trypanosomes
as a target for structure-based drug design. Rigorous
studies of energy metabolism in T. brucei have estab-
lished that, unlike the insect form, the bloodstream form
lacks a functional TCA cycle and mitochondrial oxida-
tive phosphorylation and depends solely on glycolysis
with the excretion of pyruvate for energy production.8-10
As has been shown by Clarkson and Brohn, treatment
of cultured parasites with combinations of glycerol and
salicylhydroxamic acid causes inhibition of carbohydrate
catabolism and severely impedes parasite prolifera-
tion.11 Computer modeling of T. brucei’s glycolytic flux
suggests that, unlike in mammalian cells, glycosomal
* To whom correspondence should be addressed. Phone: 206-543-
7142. Fax: 206-685-8665. E-mail: gelb@chem.washington.edu.
† Department of Chemistry, University of Washington.
‡ Department of Biological Structure, University of Washington.
§ University of California, San Francisco.
| Department of Biochemistry, University of Washington.
Department of Medicine, University of Washington.
#
Biomolecular Structure Center and Howard Hughes Medical
Institute.
10.1021/jm000472o CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/22/2001

Adenosine Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2081
F igu r e 1. (Left) L. mexicana GAPDH/6m crystal structure. Circles denote the cleft recognized by the 2′-substituent, which provides
for selectivity and affinity, while the N6 region provides for affinity. In human GAPDH (right), recognition of the C2′ substituent
is precluded by steric occlusion.
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
exerts significant control over the glycolytic pathway.
Competitive inhibitors of this enzyme, when present at
concentrations 10- to 100-fold higher than their KI
values, are predicted to reduce parasite glycolytic flux
to zero.12-14 Energy metabolism in Leishmania spp. and
in T. cruzi has been studied predominantly with the
promastigote and epimastigote forms, respectively, be-
cause the intracellular forms are difficult to analyze
because of interference from host cell metabolism.
However, glycolysis is always active in these parasites
and biochemical studies with the T. cruzi axenic amastig-
ote intracellular form suggest that carbohydrate catabo-
lism is its major source of energy.15 Thus, glycolysis
inhibitors have the potential to serve as effective anti-
T. cruzi and anti-Leishmania agents.
determined to be 25, 12, and 6 µM for T. brucei, T. cruzi,
and L. mexicana GAPDH’s, respectively.
In this paper, the exploration of the C2′ and N6
regions is continued with a structure-activity relation-
ship (SAR) series of benzamides and with modifications
to the naphthalene ring, respectively. To further inves-
tigate the diversity of substituents that could be accom-
modated by these two regions, a 2.8 Å X-ray structure
of an L. mexicana GAPDH/adenosine analogue complex
(Figure 1)25 was used to generate a combinatorial library
of 240 N6-(substituted)-2′-deoxy-2′-(amido)adenosine de-
rivatives, via computational docking methodology. Ad-
ditionally, the incorporation of 5′-amido substituents
into adenosine analogues bearing either of the most
potent N6 or C2′ substituents is explored.
Ch em istr y
Guided by crystal structures of T. brucei, T. cruzi, and
L. mexicana GAPDH’s16-18 in comparison with human
GAPDH,16,19 the process of designing competitive and
selective inhibitors is facilitated. X-ray structures co-
bound with their natural substrates reveal the detailed
binding mode of the adenosyl moiety of the NAD+
cosubstrate and have led to the successful design of
adenosine analogues as selective and competitive in-
hibitors of trypanosomatid GAPDH’s.20-24 Although the
active sites of the parasite and mammalian enzymes are
highly conserved in the region that binds glyceralde-
hyde-3-phosphate, the adenosyl moiety is well removed
from the catalytic cysteine and sits in a region that is
far less conserved between parasite and mammalian
enzymes. A detailed description of the targeted region
has been described previously.21 The adenosine scaffold
provides several opportunities not only for improving
affinity but also for improving selectivity. Specifically,
a hydrophobic cleft protruding from the ribosyl C2′ is a
hallmark of the trypanosomatid GAPDH’s. This “selec-
tivity cleft” is absent from the human enzyme because
of a difference in protein backbone conformation. The
area around the adenosyl N6 exhibits an additional
surface for hydrophobic substituents in all enzymes,
though the region appears somewhat larger in the
trypanosomatid GAPDH’s. Both mentioned opportuni-
ties were exploited previously by our group, culminating
in the design of a trypanosomatid-selective inhibitor N6-
(1-naphthalenemethyl)-2′-deoxy-2′-(3-methoxybenzami-
do)adenosine (6e)24 with IC50 values that have been
There are three reported methods for the synthesis
of N6-(substituted)-2′-deoxy-2′-(benzamido)adenosine ana-
logues: (1) acylation of 2′-amino-2′-deoxy-3′,5′-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)adenosine20,26 followed
by N1 alkylation with subsequent Dimroth rearrange-
ment and removal of the Markiewicz protecting group;23,27
(2) acylation of 2′-amino-2′-deoxy-3′,5′-O-(1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl)adenosine followed by diazo-
tization-iodination of N6, C6 amination, and removal
of the Markiewicz protecting group;24 (3) diazotization-
chlorination of 2′-azido-2′-deoxy-3′,5′-O-(1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl)adenosine, removal of the
Markiewicz protecting group, amination of the C6
position, reduction of the C2′ azide, and polymer-
assisted amidation.28 Route 1 suffers from poor yields
of the alkylation and rearrangement steps and is limited
in scope to the availability, either commercially or
synthetically, of activated halide alkylating agents.
Routes 2 and 3 both suffer from poor yields at the
diazotization-halogenation step. The acylation step of
route 3 is attractive because the desired end-product can
simply be washed cleanly from the resin; however, the
resin adds two additional steps to the synthesis because
it also needs to be prepared.29,30 To more thoroughly
explore N6,2′-disubstituted adenosine analogues, we
needed to develop an economically robust route to a
difunctionalized intermediate that would allow for the
incorporation of a wide variety of substituents at both
the N6 and C2′ positions.

2082 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Bressi et al.
Sch em e 1a
a
(a) Adenosine deaminase in H2O at room temperature; (b) EtOC(O)CF3 in DMF at 60 °C; (c) Ac2O, Et3N, and DMAP in ACN at room
temperature; (d) N,N-dimethylaniline, POCl3, and Et4NCl in ACN; (e) R1NH2 and Et3N in EtOH at 60 °C; (f) NH4OH in EtOH at 60 °C;
(g) R2COOH and EDCI in CH2Cl2/DMF at room temperature.
Our route (Scheme 1) starts with the synthesis of 2′-
deoxy-2′-aminoadenosine (1)20,26 and its quantitative
conversion to 2′-deoxy-2′-aminoinosine (2) by prepara-
tive use of adenosine deaminase.31 Trifluoroacetylation
of the C2′ amine and acetylation of the C3′ and C5′
hydroxyls to yield 2′-deoxy-2′-trifluoroacetamidoinosine
(3) and 2′-deoxy-2′-trifluoroacetamido-3′,5′-O-(diacetyl)-
inosine (4), respectively, is accomplished in greater than
95% yield for each step. By use of the methodology of
Matsuda et al.,32 subsequent chlorination of the C6
position of 4 with phosphorus oxychloride to yield 9-[2′-
deoxy-2′-trifluoroacetamido-3′,5′-O-(diacetyl)]-(1-â-D-ri-
bofuranosyl)-6-chloropurine (5) is accomplished in better
than 90% yield. Starting from the commercially avail-
able arabinose adenosine and transforming through
nine steps, the intermediate 5 can be synthesized in 65%
overall yield. Amination of the C6 position, in situ
removal of the acetates, and 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide HCl (EDCI) mediated acylation of
the C2′ amino yield the desired disubstituted products
(Scheme 1; 6a -6a l, 7e-11e, 7f, 7m , 9m , 11m -34m ,
7n , 9n , 11-14n , 19-34n , 12-14a c to 12-14a l, and
19a c-34a l). Typically, amination of 5 led to a variety
of deacetylated products as determined by electrospray
ionization mass spectrometry (ESI-MS), but this was
of no adverse consequense.
enosine (39) and N6-(1-naphthalenemethyl)-5′-deoxy-5′-
aminoadenosine (40) intermediates. EDCI-mediated
acylation or polymer-assisted acylation with the ap-
propriately acylated safety-catch resin29,30 is chemose-
lective and gives the desired products N6-(1-naphtha-
lenemethyl)-5′-deoxy-5′-(cyclohexylacetamido)adenosine
(41) and N6-(1-naphthalenemethyl)-5′-deoxy-5′-(diphen-
ylacetamido)adenosine (42) in high yield (Scheme 2).
Despite the additional steps needed to prepare the resin,
employing the safety-catch acylating agent has the
added benefit of merely filtering off the solid support
to obtain the desired product in nearly quantitative yield
and in greater than 95% purity as determined by HPLC.
Synthesis of 2′,5′-dideoxy-2′,5′-(bisamido)adenosine
analogues also starts with 1 (Scheme 3). Trifluoroacet-
ylation of the 2′ amine yields 2′-deoxy-2′-(trifluoro-
acetamido)adenosine (43). From use of standard Mit-
sunobu conditions, 43 is converted to 2′,5′-dideoxy-2′-
(trifluoroacetamido)-5′-(phthalimido)adenosine (44), and
subsequent in situ hydrazine-mediated cleavage of both
the phthalimide and the trifluoroacetamide protecting
group yields the desired 2′,5′-dideoxy-2′,5′-diamino-
adenosine intermediate (45). Regioselective acylation of
the 5′ amine is accomplished with the use of the
appropriately acylated safety-catch resin at ambient
temperature to give 2′,5′-dideoxy-2′-amino-5′-(cyclohex-
ylacetamido)adenosine (46) and 2′,5′-dideoxy-2′-amino-
5′-(diphenylacetamido)adenosine (47) in greater than
95% yield. EDCI-mediated acylation of the 2′ amine
gives the final products 2′,5′-dideoxy-2′-(3,5-dimethoxy-
benzamido)-5′-(cyclohexylacetamido)adenosine (48) and
2′,5′-dideoxy-2′-(3,5-dimethoxybenzamido)-5′-(diphen-
ylacetamido)adenosine (49) (Scheme 3). Alternatively,
acylation of 46 and 47 can be accomplished by employ-
Synthesis of N6-(1-naphthalenemethyl)-5′-deoxy-5′-
(amido)adenosine analogues (Scheme 2) starts with the
conversion of commercially available 6-chloropurine-9-
riboside (37) to 6-chloropurine-9-[5′-deoxy-5′-(phthal-
imido)]riboside (38) via standard Mitsunobu conditions.
Amination at C6 followed by in situ hydrazine-mediated
cleavage of the phthalimide group yields the respective
N6-(1-naphthalenemethyl)-5′-deoxy-5′-(phthalimido)ad-

Adenosine Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2083
Sch em e 2a
a
(a) Phthalimide, Ph3P, and DIAD in THF at room temperature; (b) R1NH2 and Et3N in EtOH at 60 °C; (c) NH2NH2‚H2O in EtOH at
80 °C; (d) method a, R2COOH and EDCI in CH2Cl2/DMF at room temperature, or method b, appropriately acylated safety-catch resin in
DMF at room temperature.
Sch em e 3a
a
(a) EtOC(O)CF3 in DMF at 60 °C; (b) phthalimide, Ph3P, and DIAD in THF at room temperature; (c) NH2NH2‚H2O in EtOH at 80 °C;
(d) appropriately acylated safety-catch resin in DMF at room temperature; (e) method a, R2COOH and EDCI in CH2Cl2/DMF at room
temperature, or method b, appropriately acylated safety-catch resin in DMF at 60 °C.
ing the appropriately acetylated safety-catch resin at
elevated temperatures.28,33,34
coupled to the nucleoside, deprotection of the phenolic
groups was accomplished in methanolic ammonia.
2-Naphthalenemethylamine hydrochloride (51), 4-
methoxy-1-naphthalenemethylamine hydrochloride (55),
and 3-methoxy-1-naphthalenemethylamine hydrochlo-
ride (57) were prepared by LiAlH4 reduction of 2-naph-
thonitrile (50),35 4-methoxy-1-naphthonitrile (54), and
3-methoxy-1-naphthonitrile (56),36 respectively. By use
of the methodology of Deana et al.37,38 for the synthesis
of 2-hydroxy-1-naphthalenemethylamine, 2-methoxy-1-
naphthalenemethylamine (53) was prepared by treat-
ment of 2-methoxynaphthalene (52) with 2-chloro-N-
(hydroxymethyl)acetamide and hydrochloric acid. 3-Hy-
droxy-1-naphthalenemethylamine hydrobromide (58)
was prepared by treatment of 57 with BBr3 (Scheme
4).
Resu lts a n d Discu ssion
All inhibitors were screened against L. mexicana
GAPDH (Tables 1-5). Additionally, all of the com-
pounds were docked into the structure of L. mexicana
GAPDH, which was derived from the X-ray structure
of the complex with N6-(1-naphthalenemethyl)-2′-deoxy-
2′-(3,5-dimethoxybenzamido)adenosine (6m ) after re-
moval of the inhibitor coordinates.25 Docking studies
were performed with the program FLO in which mo-
lecular potential energy calculations with the QXP force
field are accessible through a graphical interface.40 With
this information, we attempted to rationalize the ob-
served inhibition data.
Hydroxylated benzoic acids needed for the synthesis
of compounds 6c, 6g, 6k , 6o, 6r , 6t, 6v, and 6x were
acetylated under standard conditions.39 After they were
Initially we docked the inhibitor of the crystal-
lographic complex, compound 6m , to verify whether the
program could reproduce the experimentally observed

2084 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Bressi et al.
Sch em e 4a
Ta ble 1. Inhibition of L. mexicana GAPDH by
N6-(1-Naphthalenemethyl)-2′-deoxy-2′-(benzamido)adenosine
Analoguesa
compd
R1
R2
R3
R4
IC50 (µM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
OCH3
Cl
OAc
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OAc
OH
H
H
H
H
H
H
H
H
H
OCH3
Cl
OAc
OH
H
H
H
H
OCH3
Cl
OAc
OH
OCH3
OAc
OH
OCH3
OCH3
OAc
OH
OCH3
OCH3
N(CH3)2
H
H
H
H
H
H
H
H
OCH3
Cl
OAc
OH
H
H
H
H
OCH3
OAc
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
Cl
OAc
OH
H
H
H
H
H
H
H
H
H
inactiveb
inactive
inactive
inactive
6
6
50
25
100
100
100
80
2
12
10
5
4
25
a
(a) LiAlH4 in THF at 0 °C; (b) ClCH2CONHCH2OH and H+ in
EtOH at 80 °C; (c) [HCl] in EtOH at 80 °C; (d) BBr3 in CH2Cl2 at
40 °C.
binding mode. After docking, the root-mean-square
(rms) deviation between the experimental coordinates
and the docked model was only 0.70 Å for 42 atoms,
confirming the validity of the docking protocol. This rms
deviation is similar to the value of 0.76 Å found by the
FLO authors for a diverse set of unrelated test cases.
Subsequently, all other inhibitors were docked using the
same protocol. The calculated binding energies (data not
shown) comprise ligand and protein strain energies, van
der Waals energy, electrostatic interaction energy (dis-
tance-dependent dielectric is 4Rꢀ), and a term account-
ing for hydrophobic interaction. Because there is no
explicit penalty term for desolvation of hydrophilic
groups, the calculated interaction energies allow only
for qualitative interpretation of ligand binding,41 as
clearly demonstrated in a design study of potent angio-
tensin-converting enzyme (ACE) inhibitors.
6s
6t
18
inactive
inactive
78
37
10
6u
6v
6w
6x
6y
6z
H
OCH3
OCH3
OAc
OH
H
10
25
H
a
For compounds with IC50’s e 50 µM, inhibitor concentrations
were varied, with at least five different concentrations used to
determine the IC50 values. Statistical error limits on the IC50
values have been calculated and amount to 10% or less. For
compounds with IC50’s > 50 µM, these values were extrapolated
b
from at least three different concentrations. Inactive ) inactive
at 50 µM.
Mod ifica tion s of th e C2′ Ben za m id e. To more
thoroughly explore the conformational space of the C2′
selectivity cleft, an SAR of N6-(1-naphthalenemethyl)-
2′-deoxy-2′-(benzamido)adenosine analogues was gener-
ated. This series of compounds incorporates methoxy,
chloro, acetyl, and hydroxy groups at various positions
on the benzamide ring.
unlikely because the lone pairs of the two oxygen atoms
point unfavorably toward each other. Work by Cignitti
et al. would support this observation,42 and a Cambridge
Structural Database (CSD)43 search for o-methoxy- and
o-chlorobenzamides reveals that the 26 available crystal
structures all exhibit the anti conformation. In contrast,
the anti conformation allows the methoxy and hydroxy
substituents to make a 2.7 Å hydrogen bond with the
amide nitrogen. Docking of the anti conformation of the
ortho-substituted compounds by FLO reveals that the
benzamido is pushed out of the selectivity cleft by 1.3-
1.8 Å compared to that of 6m because of steric con-
straints. The end result is that a key hydrogen bond
between the amide nitrogen and the carboxylate of Asp-
38 is significantly weakened, and the benzamide ring
buries less hydrophobic surface in the cleft. This most
likely explains the poor activity of these compounds.
Of the N6-naphthalenemethyl-2′-benzamido ana-
logues, the most drastic decreases in binding affinity
were noted with 2-substituted benzamides that had
IC50’s greater than 100 µM (Table 1). In principle, two
orientations of the benzamide fragments are possible:
syn, which places the ortho substituent adjacent to the
amide carbonyl oxygen and pointing into solvent; and
anti, which places the ortho substituent opposite the
carbonyl oxygen pointing into the selectivity cleft.
Modeling experiments suggest the syn conformation is

Adenosine Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2085
Ta ble 2. Inhibition of L. mexicana GAPDH by
N6-Naphthalenemethyl-2′-deoxy-2′-(3-methoxybenzamido)-
adenosine Analogues
benzamides that were well tolerated by L. mexicana
GAPDH (3-chlorobenzamide, 3,5-dimethoxybenzamide,
and 3,5-dichlorobenzamide), but there were only mar-
ginal improvements or, in the case of 7n , a marginal
decrease in affinities. Comparable results were also
obtained with the incorporation of the methoxy group
into the 3- and 4-positions of the naphthalene ring, and
docking experiments with FLO place all of these com-
pounds such that they essentially superimpose with 6m .
Integration of the methoxy group into the 2-position of
the naphthyl ring, yielding 8e, was predicted to drasti-
cally decrease binding affinity because the methoxy
would clash with either the adenosyl N6 or the residues
Ala-90 and Gln-91 of the protein backbone. In fact, FLO
displaced the entire molecule partially out of the binding
site. The 2-methoxynaphthalenemethyl analogue (8e)
was synthesized as proof of principle, and it was inactive
against L. mexicana GAPDH when tested up to its
solubility limit of 50 µM.
An alternative connectivity of the naphthalene ring
was explored with the incorporation of 2-naphthalene-
methyl and 2-naphthalene substituents. Compounds
11e, 11m , and 11n were well tolerated, especially 11e
and 11m with IC50’s of 2 µM. FLO docked these
compounds with their benzamido adenosine fragments
in the same position as in 6m , and the analysis showed
that the â-naphthyl ring makes favorable contacts with
the guanadinium of Trp-92. Docking of 18m (IC50 ) 60
µM), which has a â-naphthyl directly attached to N6,
elucidated poor packing of the naphthalene ring with
the aliphatic portion of Arg-92 and the side chains of
Met-39 and Leu-113, which caused a small rotation of
the purine ring and a pivoting of the C1′ of the ribose.
Mod ifica tion s of th e N6 P osition . Incorporation of
N6 and C2′ amido substituents into one adenosine
scaffold reveals that there is a degree of “cross-talk”
between these two regions. For example, N6-benzyl-
adenosine and N6-(1-naphthalenemethyl)adenosine have
IC50’s of 4 and 0.15 mM, respectively.23 Despite the 27-
fold difference in binding affinity between these two
compounds, incorporation of the C2′-(3-methoxy)benz-
amide into both of them resulted in compounds showing
only a 3-fold affinity difference; the IC50 of N6-benzyl-
2′-deoxy-2′-(3-methoxybenzamido)adenosine for L. mexi-
cana GAPDH is 16 µM,23 and the IC50 of N6-(1-naph-
thalenemethyl)-2′-deoxy-2′-(3-methoxybenzamido)adeno-
sine for L. mexicana GAPDH is 6 µM. A variety of N6-
monosubstituted adenosine analogues have been
reported that exhibited IC50’s against L. mexicana
GAPDH in the range 4-0.15 mM (N6-(2-methylbenzyl)-
adenosine, IC50 ) 0.7 mM; N6-(3-methylbenzyl)adeno-
sine, IC50 ) 0.7 mM; N6-(1,2,3,4-tetrahydro-1-naphthyl)-
adenosine, IC50 ) 0.36 mM; N6-[(2-(hydroxymethyl)-
phenylthio)benzyl]adenosine, IC50 ) 0.34 mM; N6-
(diphenylmethyl)adenosine, IC50 ) 0.24 mM).23 We
wished to see if these too would exhibit the same
increase in binding affinity as N6-benzyladenosine and
N6-(1-naphthalenemethyl)adenosine when a well-toler-
ated C2′ benzamide, 3,5-dimethoxybenzamido, was in-
corporated into the molecule.
compd
R1
R2
OH
OH
OH
OH
H
OCH3
OCH3
OCH3
H
R3
R4
R5
IC50 (µM)
7e
7f
7m
7n
8e
9e
9m
9n
10e
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
Cl
OCH3 OCH3
Cl
OCH3
OCH3
H
H
6
4
2
Cl
H
25
OCH3
inactivea
H
H
H
H
H
3
2
25
6
OCH3 OCH3
Cl
Cl
H
OCH3 OCH3
a
Inactive ) inactive at 50 µM.
With the exceptions of 6e and 6f, for which affinities
had been previously determined,24 the meta- and para-
substituted benzamides (Table 1) displayed IC50’s of 25-
100 µM against L. mexicana GAPDH. Essentially, these
compounds all had the same coordinates as 6m and FLO
preferentially oriented the meta substituents to point
into the selectivity cleft. Compounds 6m , 6o, and 6p ,
with a 3,5-disubstituted benzamido substituent, exhib-
ited a 3- to 5-fold increase in affinity for L. mexicana
GAPDH when compared to their 3-substituted ana-
logues 6e, 6g, and 6h , while 6n exhibited a 2-fold
decrease in activity. The marginal differences in the
inhibitor potency suggests that the 3-monosubstituted
benzamides can adopt either the anti or syn conforma-
tion. Compound 6z was synthesized in an attempt to
more completely fill the “selectivity cleft” and to explore
alternative conformations of the methoxy group of the
lead inhibitor 6e. Modeling suggested that replacing
methoxy with dimethylamino might accomplish that
goal, but the IC50 of 6z was 4 times worse than 6e.
Modification s of th e N6-Naph th alen em eth yl. Com-
putational modeling experiments suggested that a hy-
droxyl group incorporated into the 3-position of the
naphthyl ring or a methoxy group in the 3- or 4-position
of the naphthyl ring might be well tolerated or might
enhance binding affinity of the lead compound 6e (Table
2). The hydrogen bond provided by N6-(3-hydroxy-1-
naphthalenemethyl)-2′-deoxy-2′-(3-methoxybenzamido)-
adenosine (7e) to the backbone carbonyl of Ala-90 was
well tolerated, and the compound displayed an IC50 of
6 µM. The 3-hydroxynaphthalenemethyl substituent
was also incorporated into compounds bearing other C2′
A combination of benzyl (12m ), 2-methylbenzyl (13m ),
or 3-methylbenzyl (14m ) with C2′-(3,5-dimethoxybenz-
amide) resulted in activities in the 20-25 µM range.
The diphenylmethyl (15m ), tetrahydronaphthyl (16m ),

2086 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Bressi et al.
and 2-(2-(hydroxymethyl)phenylthio)benzyl (17m ) N6
analogues exhibited IC50’s in the range 55-75 µM. All
of these N6 substituents that exhibited some modest
binding affinity toward parasite GAPDH as monosub-
stituted adenosine analogues did exhibit some degree
of “cross-talk” when incorporated into analogues bearing
the C2′ benzamide group, yielding tighter binding
derivatives, but none better than what had previously
been obtained with the incorporation of the naphthalene
ring, 6m .
Ta ble 3. Inhibition of L. mexicana GAPDH by
N6-(Subsutituted)-2′-deoxy-2′-(benzamido)adenosine Analogues
N6-(Su b st it u t ed )-2′-d eoxy-2′-(a m id o)a d en osin e
Com bin a tor ia l Libr a r y. According to the methodology
and criteria of Aronov et al.,44 UC_Select,45 in conjunc-
tion with the Daylight version of the Available Chemi-
cals Directory (ACD), was used to select primary amines
and carboxylic acids that were devoid of undesirable
pharmaceutical and chemical properties (i.e., substitu-
ents without phosphates or sulfates). Sybyl’s CONCORD
module (version 6.5, 1999; Tripos Associates, St. Louis,
MO) was used to generate the three-dimensional coor-
dinates of substituent libraries that were then fused to
the adenosyl template. When the coordinates of 6m ,
from the L. mexicana GAPDH complex, were used, the
amine test library (consisting of approximately 460
amines) was fused to the C6 position of 6m after the
removal of the naphthalenemethyl substituent. Like-
wise, the carboxylic acid test library (consisting of
approximately 1400 acids) was fused to the C2′ position
of 6m after the removal of the dimethoxybenzamide.
DOCK 4.0146 was used as previously described44 to
evaluate the potential binding energies of the candidate
adenosine derivatives to GAPDH, and then a scoring
algorithm that takes into account differential solvation
of the adenosine substituents was applied.47 The best
scoring molecules were visually inspected using Sybyl
and Insight II (version 98.0, 1998; Molecular Simula-
tions, Inc., San Diego, CA) software packages.
Our initial hope in venturing into the exploration of
the virtual library described above was to discover
substituents that were structurally different from naph-
thylenemethyl and benzoyl and could be accommodated
in the N6 and C2′ regions of parasite GAPDH’s. This
hope was not realized because many of the top DOCK-
ranked amines had benzyl and naphthalenemethyl
substituents, or substituents that were structurally
similar to groups that had already been used to probe
the N6 and C2′ areas. The majority of top DOCK-ranked
carboxylic acids were benzoic acid derivatives. Still, we
attempted to select a diverse group of ligands while, at
the same time, including top DOCK-ranked substituents
known to enhance adenosine affinity (Table 4; 3-meth-
ylbenzyl-, 2-methylbenzyl-, naphthylenemethyl-, and
benzylamine; 4-phenylbenzoic acid,22 3,5-dimethoxyben-
zoic acid, and 3,5-dichlorobenzoic acid). This led to a
selection of 20 amines and 12 carboxylic acids (Table 4)
that were used to generate a 240-compound library of
N6-(substituted)-2′-deoxy-2′-(amido)adenosine analogues
via parallel solution-phase synthesis based on the
common intermediate 5. The solvation-corrected DOCK
scores for the 1400 carboxylic acids ranged from 10.22
to 87.13 kcal/mol, while those for the 460 amines ranged
from 14.17 to 1814.83 kcal/mol. The 12 acids and 20
amines selected for parallel synthesis were chosen from
the top 200 scoring compounds of their respective virtual
libraries for which the difference in force field scores
between the best and the worst compounds was no more
than 10 kcal/mol.
N6-(Substituted)-2′-deoxy-2′-aminoadenosine inter-
mediates were purified by preparative HPLC, and the

Adenosine Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2087
Ta ble 4. Primary Amines and Carboxylic Acids Selected from Docked ACD Answer Seta
a
Substituents are presented in qualitative order of decreasing DOCK scores. For example, reading down the column, 19 > 20 >
14 > 26.
final acylated library was spot-checked by ESI-MS and
HPLC (as described in the Experimental Section) to
ensure that the desired final products were present in
reasonable yield. The crude library was screened against
L. mexicana GAPDH at an approximate concentration
of 30 µM to yield a number of low-micromolar inhibitors
(Chart 1), but none were superior to 6m . Compounds
6m , 6a f, 28m , 29m , and 30m were isolated from their
respective crude reaction, characterized, and assayed
against L. mexicana GAPDH to give IC50’s of 2, 20, 20,
8, and 20 µM, respectively.
The relative experimental potencies of the library
compounds did not correlate well with their solvation-
corrected DOCK scores, but it should be noted that the
scores for these 240 compounds were all among the very
best scores (within 10 kcal/mol of each other) compared
to the much larger range of energies for all compounds
analyzed computationally, as noted above. Indeed, 25%
of the 240 compounds display an IC50 that was within
4- to 15-fold of our best inhibitor, 6m . Thus, the process
of using DOCK provided approximately 40 new, reason-
ably potent GAPDH inhibitors. It is presumed that
many of the poor-scoring compounds would be poor
GAPDH inhibitors and thus were not synthesized. To
put the DOCK program to a rigorous test, a large
number of compounds with poor DOCK scores would
need to be synthesized. We refrained from doing this
because we believe that we have thoroughly explored
2′,N6-disubstituted adenosines as parasite GAPDH in-
hibitors.
Mod ifica tion s of th e C5′ of N6-(1-Na p h th a len e-
m eth yl)a d en osin e a n d 2′-Deoxy-2′-(3,5-d im eth oxy-
ben za m id o)a d en osin e (Ta ble 5). Both 5′deoxy-(5′-
cyclohexylacetamido)adenosine (35)23 and 5′deoxy-(5′-
diphenylacetamido)adenosine (36)22 inhibit L. mexicana
GAPDH with IC50’s of 250 µM. We wished to investigate
if the integration of these C5′ substituents with either
the N6-naphthalenemethyl or C2′-(3,5-dimethoxybenz-
amido) substituents would increase binding affinity.
Surprisingly, N6-(1-naphthalenemethyl)-5′deoxy-(5′-cy-

2088 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Bressi et al.
respective N6 or C2′ substituents were superimposed
with the coordinates of 6m . It is unclear why compounds
41 and 42 did not benefit from the additive effect that
48 and 49 did. Solubility limitations hindered a careful
examination of the competitive and selective nature
of 49.
Ch a r t 1. Screening of
N6-(Substituted)-2′-deoxy-2′-(amido)Adenosine
Combinatorial Library against L. mexicana GAPDH
GAP DH Cr oss Sp ecies Eva lu a tion a n d Tr yp -
a n osom a tid Gr ow th In h ibition Stu d ies (Ta ble 6).
Only the more potent compounds (6e, 6m -6q, 7e, 7f,
7m , 7n , 9e, 9m , 9n , 10e, 11e, 11m , and 11n ) were
screened against T. brucei and T. cruzi GAPDH (Table
5) because these two enzymes are more difficult to
overexpress and purify, and only small quantities were
available. In general, the activity of the adenosine
analogues decreases in the order L. mexicana, T. cruzi,
and T. brucei (Table 6). This was also true for 2′,5′-
dideoxy-2′-(3,5-dimethoxybenzamido)-5′-(diphenylacet-
amido)adenosine, 49, which displayed IC50’s of 60, 82,
and 100 µM for the respective parasite GAPDH’s.
Although the homology between the adenosyl binding
regions is highly conserved across trypanosomatids, the
replacement of Ser-40 in the selectivity cleft of L.
mexicana with an asparagine in T. cruzi and T. brucei
may account for the small differences in affinity. All of
these compounds were tested against rabbit muscle
GAPDH (mammalian control) and were found to be
completely selective for the parasite enzymes up to their
solubility limit. Compound 6m was shown to be com-
petitive with NAD+ (data not shown), which is consis-
tent with the crystallographic data.
Ta ble 5. Inhibition of L. mexicana GAPDH by
5′-Deoxy-5′-amido-, N6-(Substituted)-5′-deoxy-5′-amido-, and
2′,5′-Dideoxy-2′,5′-bisamidoadenosine Analoguesa
The above-mentioned inhibitors were tested for the
ability to block the growth of bloodstream T. brucei,
mammalian stage T. cruzi grown in murine 3T3 fibro-
blasts, and murine 3T3 fibroblasts (Table 6). All cell
culture assays were performed twice with very similar
results. Suprisingly, ED50’s for the cultured parasites
were within a 2- to 4-fold range of the IC50’s measured
against GADPH in vitro, with the exception of 6n . On
the basis of the aforementioned computational studies
performed by Baker et al.,12-14 a 10- to 100-fold differ-
ence between IC50’s and EC50’s might be expected. It is
possible that these analogues accumulate in parasites
to a higher concentration than present extracellularly.
Alternatively, it is possible these compounds act on a
target other than or in addition to GAPDH. The ED50’s
of the compounds against mammalian cells were con-
sistently higher than the ED50’s against parasites. This
is consistent with the selectivity of the adenosine
analogues for parasitic GAPDH over mammalian.
Con clu sion s
New robust routes for the synthesis of N6-(substituted)-
2′-deoxy-2′-(amido)adenosine and N6-(substituted)-5′-
deoxy-5′-(amido)adenosine analogues and a route to a
new class of derivative, 2′,5′-dideoxy-2′,5′-(bisamido)-
adenosine, have been developed. The synthetic route
from the common intermediate 9-[2′-deoxy-2′-trifluoro-
acetamido-3′,5′-O-(diacetyl)]-(1-â-D-ribofuranosyl)-6-chlo-
ropurine (5) allows a library of hundreds of N6-
(substituted)-2′-deoxy-2′-(amido)andenosines to be readily
synthesized via parallel solution-phase synthesis. Uti-
lizing structure-based design methodology, we synthe-
sized a number of inhibitors that are 7000- to 25000-
fold more active (across trypanosmatid species) than
a
Inactive ) inactive at 50 µM.
clohexylacetamido)adenosine (41) and N6-(1-naphtha-
lenemethyl)-5′deoxy-(5′-diphenylacetamido)adenosine (42)
did not inhibit GAPDH when tested up to their solubil-
ity limit of 50 µM. 2′,5′-Dideoxy-2′-(3,5-dimethoxybenz-
amido)-5′-(cyclohexylacetamido)adenosine (48) and
2′,5′-dideoxy-2′-(3,5-dimethoxybenzamido)-5′-(diphenyl-
acetamido)adenosine (49) did inhibit L. mexicana
GAPDH with IC50’s of 100 and 60 µM, respectively.
Docking experiments positioned all four C5′-substituted
compounds such that their adenosyl moieties and their

Adenosine Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2089
Ta ble 6. Effect of Adenosine Analogues on Trypanosomatide GAPDH and on Growth of Cultured Parasites
T. brucei
ED50 (µM)
T. cruzi
L. mexicana
IC50 (µM)
rabbit muscle
IC50 (µM)
3T3 fibroblasts
ED50 (µM)
compd
6e
6m
6n
6o
6p
6q
7e
7f
7m
7n
9e
9m
9n
10e
11e
IC50 (µM)
IC50 (µM)
ED50 (µM)
6
2
12
10
5
4
6
4
2
25
2
2
25
5
2
2
25
a
25
7
18, 16
17, 17
5, 4
12
7
75
12
14
7
12
12
10
40
12
7
40
50
10
5
10, 12
8, 13
7, 7
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
40, 50
40, 37
35, 33
100
14
26
47
25
25
8
55
12
5
45
26
24
10
30
a
>50, >50
50, 35
36, 40
35, 34
16, 20
38, 38
12, 20
31, 32
35, 40
16, 16
10, 12
19, 36
37, 35
16, 17
>50, >50
>50, >50
22, 28
>50, >50
30, 48
>50
>50, >50
>50, >50
>55, 44
>50, >50
>50, >50
>50, >50
43, 50
>50, >50
50, >50
32, 38
41, 38
20, 33
35, 30
16, 16
12, 12
10, 17
25, 21
15, 13
a
50, 50
>50, >50
50, >50
34, 38
11m
11n
pentamidine
benznidazole
30
a
a
0.006, 0.004
a
a
a
a
0.5, 0.5
>50, >50
a
Not determined.
H), 5.64 (d, 1H, C1′-H), 8.10 (d, 1H, C2-H), 8.27 (s, 1H, C8-H),
12.51 (s, 1H, N1-H). ESI-MS: m/z 268.1 (M + H+)+.
adenosine. Additionally, the application of rational
design and combinatorial chemistry led to a number of
low-micromolar inhibitors of L. mexicana GAPDH. The
most potent GAPDH inhibitors block the growth of
bloodstream T. brucei and the intracellular stage T.
cruzi in the low-micromolar range.
Syn th esis of 2′-Deoxy-2′-tr iflu or oa ceta m id oin osin e (3).
Ethyl trifluoroacetate (5.36 mL, 45.0 mmol) was added to a
solution of 2 (4.0 g, 15.0 mmol) in anhydrous DMF (50 mL),
and the reaction was heated at 60 °C under argon for 3 h. The
reaction was evaporated via rotary evaporation at 50 °C and
then further dried in vacuo for 24 h. The resulting amorphous
solid was of sufficient purity to use in the subsequent reaction.
For characterization, a small sample was purified via flash
Exp er im en ta l Section
Gen er a l Syn t h et ic P r oced u r es. CH2Cl2 was freshly
distilled over CaH2 under argon. 6-Chloropurine-9-riboside and
anhydrous DMF were purchased from Aldrich Chemical Co.
Ltd. 4-Sulfonamidobutyryl-AM resin (loading, 0.4-1.2 mmol/
g; polymer matrix, copoly(styrene-1% DVB); 200-400 mesh)
was purchased from Novabiochem. 1H NMR spectra were
recorded on a Bruker AF 300 MHz or AM 500 MHz spectrom-
eter in DMSO-d6. Electrospray ionization mass spectra (EIS-
MS) were obtained on a Bruker Esquire LC ion trap or Kratos
Profile HV4 mass spectrometer. Reactions were monitored by
TLC on precoated silica gel 60 F254 glass plates (EM Science)
with CH2Cl2/EtOH (9:1) or CHCl3/MeOH (80:20) as eluent and
visualized at 254 nm. Medium-pressure flash column chro-
matography was carried out on silica gel 60 (230-400 mesh;
EM Science) with CHCl3/MeOH (98:2 to 80:20) eluent. Pre-
1
chromatography. H NMR: δ 3.57 and 3.68 (2 × m, 2H, C5′-
H
a,b), 4.03 (m, 1H, C4′-H), 4.34 (m, 1H, C3′-H), 5.02 (m, 1H,
C2′-H), 5.19 (br s, 1H, C5′-OH), 5.92 (br s, 1H, C3′-OH), 6.18
(d, 1H, C1′-H), 8.10 and 8.27 (2 × s, 2H, C2-H and C8-H), 9.65
(d, 1H, C2′-NH), 12.54 (s, 1H, N1-H). ESI-MS: m/z 364.1 (M
+ H+)+.
Syn th esis of 2′-Deoxy-2′-tr iflu or oa ceta m id o-3′,5′-O-(d i-
a cetyl)in osin e (4). To a mixture of 3 (4.5 g, ∼12.4 mmol) in
anhydrous ACN (250 mL) was added sequentially Ac2O (11.7
mL, 124 mmol), anhydrous Et3N (13.8 mL, 99.2 mmol), and
DMAP (128 mg, 1.0 mmol) at ambient temperature under
argon. After the mixture was stirred for 1 h at ambient
temperature, MeOH (15.0 mL) was added and then the
reaction was evaporated via rotary evaporation at 40 °C. The
resulting residue was partitioned between EtOAc (250 mL)
and H2O (250 mL), washed with H2O (2 × 250 mL), washed
with saturated NaCl (aq, 2 × 250 mL), dried over MgSO4,
filtered, and then evaporated. The crude material was purified
via flash chromatography to yield 6.2 g of 4 (93% based on 2
parative HPLC was performed on
a C18 column (Vydac
218TP1010) using one of the following elution gradients:
(method A) linear gradient of 10 mM triethylammonium
acetate (aq, pH ) 7.0) from 0 to 100% ACN over 60 min at 4.0
mL/min; (method B) linear gradient of 5% HCOOH (aq) from
0 to 100% MeOH over 60 min at 4.0 mL/min; (method C)
isocratic 10 mM triethylammonium acetate (aq, pH ) 7.0) of
0% over 15 min, then a linear gradient of 10 mM triethyl-
ammonium acetate (aq, pH ) 7.0) from 0 to 25% ACN over 45
min at 4.0 mL/min; (method D) same as method B except a
flow rate of 7.0 mL/min on a C18 column (Vydac 218TP1022)
was used. The purity of all compounds was verified by HPLC
with methods A and B on a C18 column (Vydac 218TP52) at
1.0 mL/min.
1
as the starting material). H NMR: δ 2.04 (s, 3H, C5′-OAc),
2.08 (s, 3H, C3′-OAc), 4.23 (m, 1H, C5′-Ha), 4.38 (m, 2H, C4′-H
and C5′-Hb), 5.37 (m, 1H, C3′-H), 5.44 (m, 1H, C2′-H), 6.23 (d,
1H, C1′-H), 8.10 and 8.34 (2 × s, 1H, C2-H and C8-H), 10.10
(br s, 1H, C2′-NH), 12.50 (s, 1H, N1-H). ESI-MS: m/z 448.1
(M + H+)+.
Syn th esis of 9-[2′-Deoxy-2′-tr iflu or oa ceta m id o-3′,5′-O-
(d ia cetyl)]-(1-â-D-r ibofu r a n osyl)-6-ch lor op u r in e (5). With
minor modifications, the methodology of Matsuda et al.32 was
employed. To a rapidly stirring mixture of 4 (6.0 g, 13.4 mmol),
Et4NCl (8.9 g, 53.7 mmol), and dry N,N-dimethylaniline (1.7
mL, 13.4 mmol) in anhydrous ACN (30 mL) was added POCl3
(3.1 mL, 33.6 mmol) at ambient temperature under argon. The
reaction was placed in a preheated oil bath and refluxed at
100 °C for 10 min. The reaction was then evaporated via rotary
evaporation at 40 °C, and the resulting green foam was
dissolved in CH2Cl2 (100 mL) and then stirred vigorously over
crushed ice for 15 min. The layers were separated, and the
aqueous phase was extracted with CH2Cl2 (2 × 100 mL). The
combined organic phase was then washed with H2O (2 × 100
Syn th esis of 2′-Am in o-2′-d eoxya d en osin e (1). Com-
pound 1 was synthesized via 2′-azido-2′-deoxy-3′,5′-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)adenosine as per the reliable
methodologies of Robins et al.26 and Van Calenberg et al.20
Because minor modifications to the procedure were made that
resulted in slightly improved yields, we have included the
details of the preparation in Supporting Information.
Syn th esis of 2′-Am in o-2′-d eoxyin osin e (2). Starting with
1 (4.0 g, 15.0 mmol), the procedure of Kotra et al..31 was used
1
to give 4.0 g of 2 (99.0%). H NMR: δ 3.54 and 3.63 (2 × m,
2H, C5′-Ha,b), 3.95 (m, 2H, C3′-H and C4′-H), 4.18 (m, 1H, C2′-

2090 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Bressi et al.
mL), washed with 5% NaHCO3 (aq, pH ∼7, 2 × 100 mL), dried
over MgSO4, filtered, and evaporated to dryness. The resulting
yellow foam was then purified via flash chromatography to
give 5.7 g of 5 as a white foam (92.0%). 1H NMR: δ 2.15 (s,
3H, C5′-OAc), 2.20 (s, 3H, C3′-OAc), 4.28 and 4.41 (2 × m, 2H,
C5′-Ha,b), 4.44 (m, 1H, C4′-H), 5.50 (m, 2H, C2′-H and C3′-H),
6.42 (d, 1H, C1′-H), 8.87 and 8.95 (2 × s, 1H, C2-H and C8-
H), 10.09 (d, 1H, C2′-NH). ESI-MS: m/z 448.1 (M + H+)+. ESI-
MS: m/z 466.1 (M + H+)+.
once) from the 20 × 12 matrix and were analyzed by ESI-MS
and HPLC. For carboxylic acid vectors a c, a d , a i, a j, a k , a l,
m , and n (Table 4), acylation reactions gave 80-95% conver-
sion to the desired products while a e and a f gave ap-
proximately 50% and a g and a h gave approximately 25%.
Upon completion of the reaction, the solutions were diluted
with MeOH and then evaporated to dryness in a Speed-Vac.
The crude material was dissolved in DMSO and used for in
vitro enzyme inhibition analysis.
Gen er a l P r oced u r e for th e P r ep a r a tion of N6-(Su bsti-
tu ted )-2′-Deoxy-2′-a m in oa d en osin e An a logu es (6-18). To
a solution of 5 (10.0 mg, 2.2 × 10-2 mmol) in EtOH (500 µL),
in a 4.0 mL screw cap vial with Teflon septum, was added the
appropriate amine or amine hydrochloride (6.4 × 10-2 mmol)
and then Et3N (9.0 µL, 6.4 × 10-2 mmol for free amines; 18.0
µL, 12.8 × 10-2 mmol for amine hydrochlorides). The reaction
was heated at 60 °C for 18 h and then cooled to ambient
temperature. Concentrated NH4OH (aq, 500 µL) was then
added, and the reaction was heated at 80 °C for 24 h. Without
further workup, the reaction was purified via preparative
HPLC using method A. The desired fraction was concentrated
and then lyophilized to obtain the desired products in 85-
95% yield.
N6-(1-Na p h t h a len em et h yl)-2′-d eoxy-2′-a m in oa d en o-
sin e (6). 1H NMR: δ 3.53 and 3.64 (2 × m, 2H, C5′-Ha,b), 3.98
(m, 3H, C2′-H, C3′-H, and C4′-H), 5.19 (br s, 3H, N6-CH2 and
C5′-OH), 5.50 (br s, 1H, C3′-OH), 5.68 (d, 1H, C1′-H), 7.42 (m,
2H, naphthyl-H), 7.55 (m, 2H, naphthyl-H), 7.81 (d, 1H,
naphthyl-H), 7.94 (d, 1H, naphthyl-H), 8.15 and 8.36 (2 × s,
2H, C2-H and C8-H), 8.19 (d, 1H, naphthyl-H), 8.50 (br s, 1H,
N6-H). ESI-MS (MeOH): m/z 407.2 (M + H)+.
Gen er a l P r oced u r e for th e P r ep a r a tion of N6-(Su bsti-
t u t ed )-2′-d eoxy-2′-(a m id o)a d en osin e An a logu es. EDCI
(3.7 × 10-2 mmol) was added to a solution of the appropriate
carboxylic acid (3.7 × 10-2 mmol) in anhydrous CH2Cl2 (100
µL), and the solution was stirred under argon for 30 min. A
total of 100 µL of this solution was added to a solution of N6-
(substituted)-2′-deoxy-2′-aminoadenosine (1.2 × 10-2 mmol) in
anhydrous DMF (100 µL), and the mixture was stirred for 30
min. Without further workup, the reaction mixture was
purified via preparative HPLC using method B to obtain the
desired products in 85-95% yields after removal of solvents.
For reactions involving acetylated hydroxybenzoic acids, the
solutions were evaporated to dryness. The resulting solid was
dissolved in MeOH (250 µL) and cooled to 0 °C, and then NH3-
(g) was bubbled into the solution. The reaction mixture was
stirred at ambient temperature for 15 min and then, without
further workup, was purified by preparative HPLC using
method B.
N6-(1-Na p h th a len em eth yl)-2′-d eoxy-2′-(3,5-d im eth oxy-
ben za m id o)a d en osin e (6m ). 1H NMR: δ 3.61 and 3.72
(2 × m, 2H, C5′-Ha,b), 3.75 (s, 6H, OCH3), 4.10 (m, 1H, C4′-H),
4.33 (m, 1H, C3′-H), 5.16 (br s, 2H, N6-CH2), 5.34 (m, 1H, C2′-
H), 5.60 (br s, 1H, C5′-OH), 5.79 (br s, 1H, C3′-OH), 6.23 (d,
1H, C1′-H), 6.63 (s, 1H, Ph-H), 6.98 (s, 2H, Ph-H), 7.39 (m,
2H, naphthyl-H), 7.52 (m, 2H, naphthyl-H), 7.80 (d, 1H,
naphthyl-H), 7.94 (d, 1H, naphthyl-H), 8.21 and 8.31 (2 × s,
2H, C2-H and C8-H), 8.22 (d, 1H, naphthyl-H), 8.42 (d, 1H,
C2′-NH), 8.55 (br s, 1H, N6-H). ESI-MS: (MeOH) m/z 571.2
(M + H)+.
6-Ch lor opu r in e-5′-deoxy-(5′-ph th alam ido)r iboside (38).
To a solution of 6-chloropurine-9-riboside (37, 1.0 g, 3.5 mmol),
phthalimide (615 mg, 4.2 mmol), and triphenylphosphine (1.8
g, 7.0 mmol) in anhydrous THF (10 mL) was added DIAD (1.3
mL, 7.0 mmol) at ambient temperature under argon. After the
mixture was stirred for 2 h, MeOH (10 mL) was added to the
reaction mixture that was then stirred for an additional 15
min. Without further workup, the reaction was absorbed onto
silica gel and purified via flash chromatography to give 1.2 g
1
of 38 (86.0%). H NMR: δ 3.98 (m, 2H, C5′-H), 4.18 (m, 1H,
C4′-H), 4.25 (m, 1H, C3′-H), 4.80 (m, 1H, C2′-H), 5.41 (d, 1H,
OH), 5.62 (d, 1H, OH), 6.01 (s, 1H, C1′-H), 7.82 (s, 4H,
phthalimide-H), 8.59 and 8.96 (2 × s, 2H, C2-H and C8-H).
ESI-MS: m/z 416.1 (M + H+)+.
N 6-(1-Na p h t h a le n e m e t h yl)-5′d e oxy-5′-a m in oa d e n o-
sin e (40). To a solution of 38 (100 mg, 2.4 × 10-1 mmol) and
Et3N (101 µL, 7.2 × 10-1 mmol) in EtOH (5 mL) was added
1-naphthalenemethylamine (113 µL, 7.2 × 10-1 mmol). After
the solution was refluxed for 18 h, conversion to 39 was
complete, as determined by TLC and ESI-MS, and the sample
was used without further workup. Hydrazine hydrate (50%
aq, 82 µL, 2.4 mmol) was then added to the crude reaction
mixture and was refluxed for 18 h. Without further workup
the reaction was purified via preparative HPLC method A to
1
give 91 mg of 40 (93.6%). H NMR: δ 2.79 and 2.87 (2 × m,
2H, C5′-Ha,b), 3.92 (m, 1H, C4′-H), 4.15 (m, 1H, C3′-H), 4.69
(m, 1H, C2′-H), 5.17 (br s, 3H, N6-CH2), 5.87 (d, 1H, C1′-H),
7.41 (m, 2H, naphthyl-H), 7.55 (m, 2H, naphthyl-H), 7.80 (d,
1H, naphthyl-H), 7.94 (d, 1H, naphthyl-H), 8.17 and 8.41 (2 ×
s, 2H, C2-H and C8-H), 8.22 (d, 1H, naphthyl-H), 8.44 (br s,
1H, N6-H). ESI-MS (MeOH): m/z 407.2 (M + H)+.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of N6-(1-
Na p h th a len em eth yl)-5′-d eoxy-5′-(a m id o)a d en osin e An a -
logu es. Meth od A. EDCI (3.7 × 10-2 mmol) was added to a
solution of the appropriate carboxylic acid (3.7 × 10-2 mmol)
in anhydrous CH2Cl2 (100 µL), and the mixture was stirred
under argon for 30 min. A total of 100 µL of this solution was
added to a solution of 40 (5.0 mg, 1.2 × 10-2 mmol) in
anhydrous DMF (100 µL), and the reaction mixture was stirred
for 30 min. Without further workup, the reaction mixture was
purified via preparative HPLC using method B to obtain the
desired products 41 and 42 in 75 and 90% yields, respectively.
Meth od B. A mixture of the appropriately acylated safety-
catch resin (6.0 × 10-2 mmol) and 40 (5.0 mg, 1.2 × 10-2 mmol)
was stirred under argon in anhydrous DMF (500 µL) for 18 h
at ambient temperature. The product was filtered from the
resin, and the resin was washed with DMF (500 µL), CH2Cl2/
MeOH (1:1, 2 × 500 µL), and CH2Cl2 (2 × 500 µL). The filtrate
and washings were combined and then evaporated to dryness
via rotary evaporation at 40 °C. The desired products were
obtained in >95% yield and purity as determined by HPLC.
1H NMR and ESI-MS of samples prepared via methods A and
B were identical.
N6-(1-Na p h th a len em eth yl)-5′d eoxy-(5′-cycloh exyla cet-
a m id o)a d en osin e (41). 1H NMR: δ 0.85-1.65 (band, 11H,
cyclohexyl-H), 1.99 (d, 2H, C5′-NHC(O)CH2), 3.35 and 3.45
(2 × m, 2H, C5′-Ha,b), 3.95 (m, 1H, C4′-H), 4.01 (m, 1H,
C3′-H), 4.69 (m, 1H, C2′-H), 5.18 (br s, 2H, N6-CH2), 5.28 (br
s, 1H, OH), 5.49 (br s, 1H, OH), 5.85 (d, 1H, C1′-H), 7.41 (m,
2H, naphthyl-H), 7.55 (m, 2H, naphthyl-H), 7.80 (d, 1H,
naphthyl-H), 7.94 (d, 1H, naphthyl-H), 8.12 (m, 1H, C5′-NH),
8.20 and 8.35 (2 × s, 2H, C2-H and C8-H), 8.22 (d, 1H,
naphthyl-H), 8.44 (br s, 1H, N6-H). ESI-MS: m/z 531.3
(M + H+)+.
Com bin ator ial Libr ar y. The 240 N6-(substituted)-2′-deoxy-
2′-(amido)adenosine derivatives were synthesized individually
via solution-phase parallel synthesis. Starting with 5 (50 mg
for each amine reaction, 0.11 µmol), the above procedure for
the synthesis of N6-substituted-2′-deoxy-2′-aminoadenosine
analogues was followed using the set of 20 amines shown in
Table 4. These intermediates were purified by preparative
HPLC using method D and characterized by NMR and ESI-
MS (see Supporting Information) to yield the desired inter-
mediates in 85-95% yield. Acylation of the 2′-aminoadenosine
derivatives (approximately 4.4 µmol each) was accomplished
as described above, and the reaction progress was monitored
by TLC. Two members from each carboxylic acid vector were
chosen (such that each amine vector was represented at least

Adenosine Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2091
N6-(1-Na p h t h a len em et h yl)-5′d eoxy-(5′-d ip h en yla cet -
a m id o)a d en osin e (42). 1H NMR: δ 3.42 and 3.53 (2 × m,
2H, C5′-Ha,b), 3.95 (m, 1H, C4′-H), 4.01 (m, 1H, C3′-H), 4.64
(m, 1H, C2′-H), 4.98 (s, 1H, C5′-NHC(O)CH), 5.18 (br s, 1H,
N6-CH), 5.28 (br s, 1H, OH), 5.46 (br s, 1H, OH), 5.85 (d, 1H,
C1′-H), 7.17-7.32 (band, 10H, Ph-H), 7.41 (m, 2H, naphthyl-
H), 7.55 (m, 2H, naphthyl-H), 7.80 (d, 1H, naphthyl-H), 7.94
(d, 1H, naphthyl-H), 8.20 and 8.35 (2 × s, 2H, C2-H and C8-
H), 8.21 (d, 1H, naphthyl-H), 8.44 (br s, 1H, N6-H), 8.57 (m,
1H, C5′-NH). ESI-MS: m/z 601.3 (M + H+)+.
2′-Deoxy-2′-tr iflu or oa ceta m id oa d en osin e (43). Ethyl
trifluoroacetate (135 µL, 1.1 mmol) was added to a solution of
1 (100 mg, 3.7 × 10-1 mmol) in anhydrous DMF (1.25 mL),
and the reaction mixture was heated at 60 °C under argon for
3 h. The reaction mixture was evaporated via rotary evapora-
tion at 50 °C and then further dried in vacuo for 24 h. The
resulting amorphous solid was purified via flash chromatog-
raphy to yield 130 mg of 43 (95.5%). 1H NMR: δ 3.57 and 3.68
(2 × m, 2H, C5′-Ha,b), 4.05 (m, 1H, C4′-H), 4.35 (m, 1H, C3′-
H), 5.12 (m, 1H, C2′-H), 5.57 (m, 1H, C5′-OH), 5.84 (d, 1H,
C3′-OH), 6.21 (d, 1H, C1′-H), 7.41 (br s, 2H, N6-H), 8.13 and
8.24 (2 × s, 2H, C2-H and C8-H), 9.60 (br s, 1H, C2′-NH). ESI-
MS: m/z 363.1 (M + H+)+.
2′,5′-Did eoxy-2′-(t r iflu or oa cet a m id o)-5′-p h t h a lim id o-
a d en osin e (44). To a solution of 43 (100 mg, 2.8 × 10-1
mmol), phthalimide (49.4 mg, 3.4 × 10-1 mmol), and triph-
enylphosphine (144 mg, 5.5 × 10-1 mmol) in anhydrous THF
(1.0 mL) was added DIAD (80.5 µL, 5.5 × 10-1 mmol) at
ambient temperature under argon. After the solution was
stirred for 2 h, MeOH was added to the reaction mixture (1.0
mL), which was then stirred for an additional 15 min. Without
further workup, the reaction was absorbed onto silica gel and
purified via flash chromatography to give 111 mg of 44 (81.5%).
1H NMR: δ 3.85 and 4.02 (2 × dd, 2H, C5′-Ha,b), 4.22 (m, 1H,
C4′-H), 4.49 (m, 1H, C3′-H), 5.28 (t, 1H, C2′-H), 5.91 (br s, 1H,
C3′-OH), 6.12 (d, 1H, C1′-H), 7.31 (br s, 2H, N6-H), 7.84 (s,
4H, phthalimide-H), 7.91 and 8.34 (2 × s, 2H, C2-H and C8-
H), 9.60 (d, 1H, C2′-NH). ESI-MS: m/z 492.1 (M + H+)+.
2′,5′-Did eoxy-2′,5′-(bisa m in o)a d en osin e (45). A solution
of 44 (100 mg, 2.0 × 10-1 mmol) and hydrazine hydrate (50%
aq, 94 µL, 2.8 mmol) in EtOH (5.0 mL) was refluxed for 18 h.
Without any further workup, the crude reaction mixture was
purified via preparative HPLC method C to yield 48 mg of 45
(88.9%). 1H NMR: δ 2.88 (m, 2H, C5′-H), 3.95 (m, 1H, C4′-H),
4.02 (m, 2H, C2′-H and C3′-H), 5.65 (d, 1H, C1′-H), 7.23 (br s,
2H, N6-H), 8.12 and 8.32 (2 × s, 2H, C2-H and C8-H). ESI-
MS: m/z 266.2 (M + H+)+.
the resin was washed with DMF (500 µL), CH2Cl2/MeOH (1:
1, 2 × 500 µL), and CH2Cl2 (2 × 500 µL). The filtrate and
washings were combined and then evaporated to dryness via
rotary evaporation at 40 °C to give 48 and 49 in >90% yield
and in >95% purity as determined by HPLC. 1H NMR and
ESI-MS of samples prepared via methods A and B were
identical.
2′,5′-Did eoxy-2′-(3,5-d im et h oxyb en za m id o)-5′-(cyclo-
h exyla ceta m id o)a d en osin e (48). 1H NMR: δ 0.89-1.65
(band, 11H, cyclohexyl-H), 2.04 (d, 2H, C5′-NHC(O)CH2), 3.58
(m, 2H, C5′-H), 3.78 (s, 6H, OCH3), 4.10 (m, 1H, C4′-H), 4.19
(m, 1H, C3′-H), 5.34 (m, 1H, C2′-H), 5.92 (br s, 1H, OH), 6.17
(d, 1H, C1′-H), 6.64 (s, 1H, Ph-H), 6.98 (s, 2H, Ph-H), 7.39 (br
s, 2H, N6-H), 8.19 and 8.25 (2 × s, 2H, C2-H and C8-H), 8.39
(m, 1H, C5′-NH), 8.42 (d, 1H, C2′-NH). ESI-MS: m/z 554.3
(M + H+)+.
2′,5′-Did e oxy-2′-(3′′,5′′-d im e t h oxyb e n za m id o)-5′-(d i-
p h en yla ceta m id o)a d en osin e (49). 1H NMR: δ 3.58 (m,
2H, C5′-H), 3.75 (s, 6H, OCH3), 4.07 (m, 1H, C4′-H), 4.23 (m,
2H, C3′-H), 5.03 (s, 1H, C5′-NHC(O)CH), 5.37 (m, 1H, C2′-H),
5.92 (br s, 1H, OH), 6.14 (d, 1H, C1′-H), 6.62 (s, 1H, Ph-H),
6.96 (s, 2H, Ph-H), 7.19-7.33 (band, 12H, N6-H and Ph-H),
8.09 and 8.26 (2 × s, 2H, C2-H and C8-H), 8.43 (d, 1H, C2′-
NH), 8.73 (m, 1H, C5′-NH). ESI-MS: m/z 624.3 (M + H+)+.
P r ep a r a tion of Acetyla ted Hyd r oxyben zoic Acid s. The
method of White et al. was employed.39
P r ep a r a tion of Cycloh exylm eth yl- a n d Dip h en yl-
m eth ylsu lfon a m id obu tyr yl-AM r esin . These resins were
prepared from sulfonamidobutyryl-AM resin as per the meth-
odology of Kenner et al.29 and Backes et al.30
Gen er a l P r oced u r e for th e Syn th esis of Na p h th a len e-
m eth yla m in es fr om Na p h th yln itr iles. LiAlH4 (2.7 mmol)
was added to naphthylnitrile (2.7 mmol) in anhydrous THF
(10.0 mL) at 0 °C, and the mixture was stirred for 1 h. The
reaction was quenched with MeOH and then washed with H2O
(2 × 10 mL). The organic layer was then extracted with 1 N
HCl (2 × 10 mL), and the combined extracts were left to stand
for 24 h at 4 °C to yield crystals of the desired amine
hydrochloride in 60-80% yield.
2-Na p h th a len em eth yla m in e Hyd r och lor id e (49). 1H
NMR: δ 4.18 (br s, 1H, C2-CH2), 7.55 (m, 2H), 7.62 (d, 1H),
7.89-7.99 (band, 4H), 8.46 (br s, 3H, NH3). ESI-MS: m/z 158.1
(M + H+)+.
1-(Am in om eth yl)-4-m eth oxyn a p h th a len e Hyd r och lo-
r id e (53). 1H NMR: δ 3.98 (s, 3H, OCH3), 4.41 (br s, 2H, C1-
CH2), 7.01 (d, 1H), 7.57 (m, 2H), 7.65 (t, 1H), 8.09 (d, 1H), 8.23
(d, 1H), 8.41 (br s, 3H, NH3). ESI-MS: m/z 188.1 (M + H+)+.
1-(Am in om eth yl)-3-m eth oxyn a p h th a len e Hyd r och lo-
r id e (55). 3-Methoxy-1-naphthonitrile was synthesized as
described by DeCosta et al.36 1H NMR: δ 3.89 (s, 3H, OCH3),
4.49 (d, 2H, C1-CH2), 7.32 (s, 1H), 7.38 (s, 1H), 7.45 (t, 1H),
7.53 (t, 1H), 7.89 (d, 1H), 8.02 (d, 1H), 8.42 (br s, 3H, NH3).
ESI-MS: m/z 188.1 (M + H+)+.
1-(Am in om eth yl)-2-m eth oxyn a p h th a len e Hyd r och lo-
r id e (51). To a solution of 2-methoxynaphthalene (500 mg,
3.2 mmol) in EtOH (2.5 mL) was added concentrated HCl (125
µL) and 2-chloro-N-(hydroxymethyl)acetamide (395 mg, 3.2
mmol). The reaction mixture was refluxed for 30 min and then
was cooled to ambient temperature. Additional concentrated
HCl (1.25 mL) was added, and the reaction mixture was
refluxed for 1.5 h and then was cooled to ambient temperature.
The desired product crystallized upon cooling to give 375 mg
of 34 (53.0%). 1H NMR: δ 4.06 (s, 3H, OCH3), 4.38 (d, 2H,
C1-CH2), 7.34-7.60 (band, 3H), 7.92 (d, 2H), 8.06 (d, 1H), 8.11
(d, 1H), 8.38 (br s, 3H, NH3). ESI-MS: m/z 188.1 (M + H+)+.
3-Hydr oxy-1-n a p h th a len em eth yla m in e Hyd r obr om id e
(56). To a solution of 41 (200 mg, 0.89 mmol) in CH2Cl2 (2.0
mL) was added BBr3 (422 µL, 4.5 mmol) at ambient temper-
ature. The reaction was refluxed for 1 h, cooled to ambient
temperature, and then evaporated to dryness via rotary
evaporation at 40 °C. The resulting solid was partitioned
between CH2Cl2 (25 mL) and H2O (25 mL). The organic layer
was washed with H2O (3 × 25 mL), and the aqueous layers
were combined. The aqueous layer was reduced in volume to
Gen er al P r ocedu r e for th e P r epar ation of 2′,5′-Dideoxy-
2′-a m in o-5′-(a m id o)a d en osin e An a logu es. See Method B
of General Procedure for the Preparation of N6-(1-Naphtha-
lenemethyl)-5′-deoxy-5′-(Amido)adenosine Analogues.
2′,5′-Did eoxy-2′-a m in o-5′-(cycloh exyla ceta m id o)a d en -
1
osin e (46). H NMR: δ 0.87-1.65 (band, 11H, cyclohexyl-H),
2.04 (d, 2H, C5′-NHC(O)CH2), 3.38 and 3.51 (2 × m, 2H, C5′-
H
a,b), 3.87 (m, 1H, C4′-H), 3.96 (m, 2H, C2′-H and C3′-H), 5.61
(d, 1H, C1′-H), 7.33 (br s, 2H, N6-H), 8.14 and 8.29 (2 × s, 3H,
C2-H, C8-H, and C5′-NH). ESI-MS: m/z 390.2 (M + H+)+.
2′,5′-Did eoxy-2′-a m in o-5′-(d ip h en yla cet a m id o)a d en o-
sin e (47). 1H NMR: δ 3.38 and 3.51 (2 × m, 2H, C5′-Ha,b),
3.82 (m, 2H, C3′-H and C4′-H), 3.89 (m, 1H, C2′-H), 5.02 (s,
1H, N6-CH), 5.61 (d, 1H, C1′-H), 7.18-7.32 (band, 12H, N6-H
and Ph-H), 8.16 and 8.25 (2 × s, 2H, C2-H and C8-H), 8.75
(m, 1H, C5′-NH). ESI-MS: m/z 460.2 (M + H+)+.
Gen er al P r ocedu r e for th e P r epar ation of 2′,5′-Dideoxy-
2′-a m id o-5′-(a m id o)a d en osin e An a logu es. See Method A
of General Procedure for the Preparation of N6-(1-Naphtha-
lenemethyl)-5′-deoxy-5′-(amido)adenosine Analogues. The de-
sired products 48 and 49 were obtained in 86 and 92% yields,
respectively.
Meth od B. As per the methodology of Link et al.,33
a
mixture of the activated 3,5-dimethoxybenzylsulfonamide
safety-catch resin (6.0 × 10-2 mmol) and 46 or 47 (1.2 × 10-2
mmol) was stirred under argon in anhydrous DMF (500 µL)
for 6 h at 60 °C. The product was filtered from the resin, and

2092 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Bressi et al.
(4) Croft, S. L. The Current Status of Antiparasite Chemotherapy.
Parasitology 1997, 114, S3-S15.
approximately 25 mL and was left to stand at 4 °C for 24 h to
yield crystals of 56 (71%). H NMR: δ 4.46 (d, 2H, C1-CH2),
7.18 (s, 1H), 7.20 (s, 1H), 7.37 (t, 1H), 7.46 (t, 1H), 7.75 (d,
1H), 7.98 (d, 1H), 8.28 (br s, 3H, NH3), 9.97 (s, 1H, OH). ESI-
MS: m/z 174.1 (M + H+)+.
1
(5) Tropical Disease Research: Progress 1997-1998. World Health
Organization: Geneva, 1999.
(6) Urbina, J . A. Chemotherapy of Chagas’ Disease: The How and
the Why. J . Mol. Med. (Berlin) 1999, 77, 332-338.
In h ibition Stu d ies. Assays were performed at 23 °C in a
final volume of 0.50 mL of a 0.10 M triethanolamine/HCl
(pH ) 7.5) assay buffer while the absorption at 340 nm was
monitored using a quartz cuvette. Activity for GAPDH was
measured in the direction of NADH formation. Fresh glycer-
aldehyde-3-phosphate was prepared from its diethylacetal [di-
(cyclohexylammonium) salt] as described by the manufacturer
(Sigma). All compounds tested were dissolved in DMSO-d6 (the
combinatorial library was dissolved in DMSO), and the final
concentration of DMSO-d6 in the assay was kept at 5%. The
reaction was initiated by addition of enzyme, and control
reactions were run in the absence of inhibitors but in the
presence of 5% DMSO-d6. Remaining activity was calculated
as a percent of control using the initial velocities measured
from 0 to 2 min. For compounds with IC50’s less than or equal
to 50 µM, inhibitor concentrations in the cuvette were varied,
with at least five different concentrations used to determine
the IC50 values. Statistical error limits on the IC50 values have
been calculated and amount to 10% or less. For compounds
with IC50’s greater than 50 µM, these values were extrapolated
from at least three different concentrations. Final concentra-
tion of the components in the assay buffer was as follows: 5.0
mM MgSO4, 1.0 mM EDTA, 12.8 mM KH2PO4, 1.0 mM DTT,
1.0 mM NaN3, 0.45 mM NAD+ (Sigma), and 1.87 mM glycer-
aldehyde-3-phosphate. Trypanosome and Leishmania GAPDH
were obtained as described previously.24
(7) Pepin, J .; Milord, F. The Treatment of Human African tryp-
anosomiasis. Adv, Parasitol. 1994, 33, 1-47.
(8) Opperdoes, F. R. Biochemical Peculiarities of Trypanosomes,
African and South American. Br. Med. Bull. 1985, 41, 130-136.
(9) Opperdoes, F. R. Compartmentation of Carbohydrate Metabo-
lism in Trypanosomes. Annu. Rev. Microbiol. 1987, 41, 127-
151.
(10) Clayton, C.; Hausler, T.; Blattner, J . Protein Trafficking in
Kinetoplastid Protozoa. Microbiol. Rev. 1995, 59, 325-344.
(11) Clarkson, A. B., J r.; Brohn, F. H. Trypanosomiasis: An Approach
to Chemotherapy by the Inhibition of Carbohydrate Catabolism.
Science 1976, 194, 204-206.
(12) Bakker, B. M.; Michels, P. A.; Opperdoes, F. R.; Westerhoff, H.
V. Glycolysis in Bloodstream Form Trypanosoma brucei Can Be
Understood in Terms of the Kinetics of the Glycolytic Enzymes.
J . Biol. Chem. 1997, 272, 3207-3215.
(13) Bakker, B. M.; Michels, P. A.; Opperdoes, F. R.; Westerhoff, H.
V. What Controls Glycolysis in Bloodstream Form Trypanosoma
brucei? J . Biol. Chem. 1999, 274, 14551-14559.
(14) Bakker, B. M.; Mensonides, F. I.; Teusink, B.; van Hoek, P. A.;
Michels, P.; Westerhoff, H. V. From the Cover: Compartmen-
tation Protects Trypanosomes from the Dangerous Design of
Glycolysis. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2087-2092.
(15) Engel, J . C.; Franke de Cazzulo, B. M.; Stoppani, A. O.; Cannata,
J . J .; Cazzulo, J . J . Aerobic Glucose Fermentation by Trypano-
soma cruzi Axenic Culture Amastigote-like Forms During
Growth and Differentiation to Epimastigotes. Mol. Biochem.
Parasitol. 1987, 26, 1-10.
(16) Vellieux, F. M.; Hajdu, J .; Verlinde, C. L. M. J .; Groendijk, H.;
Read, R. J .; Greenhough, T. J .; Campbell, J . W.; Kalk, K. H.;
Littlechild, J . A.; Watson, H. C.; Hol, W. G. J . Structure of
Glycosomal Glyceraldehyde-3-phosphate Dehydrogenase from
Trypanosoma brucei Determined from Laue Data. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 2355-2359.
(17) Souza, D. H.; Garratt, R. C.; Araujo, A. P.; Guimaraes, B. G.;
J esus, W. D.; Michels, P. A.; Hannaert, V.; Oliva, G. Trypano-
soma cruzi Glycosomal Glyceraldehyde-3-phosphate Dehydro-
genase: Structure, Catalytic Mechanism and Targeted Inhibitor
Design. FEBS Lett. 1998, 424, 131-135.
(18) Kim, H.; Feil, I. K.; Verlinde, C. L. M. J .; Petra, P. H.; Hol, W.
G. J . Crystal Structure of Glycosomal Glyceraldehyde-3-phos-
phate Dehydrogenase from Leishmania mexicana: Implications
for Structure-Based Drug Design and a New Position for the
Inorganic Phosphate Binding Site. Biochemistry 1995, 34,
14975-14986.
P a r a site Cu ltu r es a n d Dr u g Scr een in g Assa ys. T.
brucei (bloodstream form strain 427 from K. Stuart, Seattle
Biomedical Research Institute, Seattle, WA) was cultured in
HMI-9 medium containing 10% fetal bovine serum, penicillin,
and streptomycin at 37 °C with 5% CO2.48 Drug sensitivity of
the T. brucei strain was determined in 96-well microtiter plates
in triplicate with an initial inoculum of 5 × 103 trypomastigotes
per well. Drugs were added in serial dilutions for a final
volume of 200 µL/well. Parasite growth was quantified at 48
h
by the addition of Alamar Blue (Alamar Biosciences,
Sacramento, CA).49 Alamar Blue quantification corresponded
with microscopic counts determined with a hemacytometer.
The Tulahuen strain of T. cruzi was provided by S. Reed
(Infectious Diseases Research Institute, Seattle, WA). This
strain was stably transfected with the E. coli â-galactosidase
gene (lacZ) so that intracellular growth could be monitored
with a colorimetric assay. The mammalian stages were grown
in coculture with mouse 3T3 fibroblasts in RPMI 1640
containing 10% fetal bovine serum, penicllin, and streptomycin
at 37 °C with 5% CO2. Drug sensitivity studies with mam-
malian stage T. cruzi were carried out in triplicate using the
â-galactosidase assay as described.50
(19) Mercer, W. D.; Winn, S. I.; Watson, H. C. Twinning in Crystals
of Human Skeletal Muscle D-Glyceraldehyde-3-phosphate De-
hydrogenase. J . Mol. Biol. 1976, 104, 277-283.
(20) Van Calenbergh, S.; Van Den Eeckhout, E.; Herdewijn, P.; De
Bruyn, A.; Verlinde, C. L. M. J .; Hol, W. G. J .; Callens, M.; Van
Aerschot, A.; Rozenski, J . Synthesis and Conformational Analy-
sis of 2′-Deoxy-2′-(3-methoxybenzamido)adenosine. A Rational-
Designed Inhibitor of Trypanosomal Glyceraldehyde Phosphate
Dehydrogenase (GAPDH). Helv. Chim. Acta 1994, 77, 631-644.
(21) Verlinde, C. L. M. J .; Callens, M.; Van Calenbergh, S.; Van
Aerschot, A.; Herdewijn, P.; Hannaert, V.; Michels, P. A.;
Opperdoes, F. R.; Hol, W. G. J . Selective Inhibition of Trypano-
somal Glyceraldehyde-3-phosphate Dehydrogenase by Protein
Structure-Based Design: Toward New Drugs for the Treatment
of Sleeping Sickness. J . Med. Chem. 1994, 37, 3605-3613.
(22) Van Calenbergh, S.; Verlinde, C. L. M. J .; Soenens, J .; De Bruyn,
A.; Callens, M.; Blaton, N. M.; Peeters, O. M.; Rozenski, J .; Hol,
W. G. J .; Herdewijn, P. Synthesis and Structure-Activity
Relationships of Analogues of 2′-Deoxy-2′-(3-methoxybenzami-
do)adenosine. A Selective Inhibitor of Trypanosomal Glycosomal
Glyceraldehyde-3-phosphate Dehydrogenase. J . Med. Chem.
1995, 38, 3838-3849.
(23) Aronov, A. M.; Verlinde, C. L. M. J .; Hol, W. G. J .; Gelb, M. H.
Selective Tight Binding Inhibitors of Trypanosomal Glyceral-
dehyde-3-phosphate Dehydrogenase via Structure-Based Drug
Design. J . Med. Chem. 1998, 41, 4790-4799.
(24) Aronov, A. M.; Suresh, S.; Buckner, F. S.; Van Voorhis, W. C.;
Verlinde, C. L. M. J .; Opperdoes, F. R.; Hol, W. G. J .; Gelb, M.
H. Structure-Based Design of Submicromolar, Biologically Active
Inhibitors of Trypanosomatid Glyceraldehyde-3-phosphate De-
hydrogenase. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4273-4278.
(25) Suresh, S.; Bressi, J . C.; Kennedy, K. J .; Verlinde, C. L. M. J .;
Gelb, M. H.; Hol, W. J . G. Manuscript in preparation.
Ack n ow led gm en t. This work was supported by
Grant AI44119 from the National Institutes of Health
to Michael H. Gelb. J erome C. Bressi is the recipient of
the University of Washington Shain Fellowship. We
thank Dr. Paul A. Michels for providing the trypanos-
matid GAPDH enzymes.
Su p p or tin g In for m a tion Ava ila ble: Synthetic and ana-
lytical data for 1 and analytical data for N6-(substituted)-2′-
deoxy-2′-aminoadenosine and N6-(substituted)-2′-deoxy-2′-
(amido)adenosine analogues. This material is available free
of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Web site: http://www.who.org.
(2) Smith, D. H.; Pepin, J .; Stich, A. H. Human African trypanoso-
miasis: An Emerging Public Health Crisis. Br. Med. Bull. 1998,
54, 341-355.
(3) Despommier, D. D.; Gwadz, R. W.; Hotez, P. J . Parasitic
Diseases, 3rd ed.; Springer-Verlag: New York, 1995.

Adenosine Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2093
(26) Robins, M. J .; Wilson, J . S.; Hansske, F. Nucleic Acid Related
Compounds. 42. A General Procedure for the Efficient Deoxy-
genation of Secondary Alcohols. Regiospecific and Stereoselective
Conversion of Ribonucleosides to 2′-Deoxynucleosides. J . Am.
Chem. Soc. 1983, 105, 4059-4065.
(27) Robins, M. J .; Trip, E. M. Nucleic Acid Related Compounds. 6.
Sugar-Modified N6-(3-Methyl-2-butenyl)adenosine Derivatives,
N6-Benzyl Analogues, and Cytokinin-Related Nucleosides Con-
taining Sulfur or Formycin. Biochemistry 1973, 12, 2179-2187.
(28) Golisade, A.; Van Calenbergh, S.; Link, A. 2′-Amino-2′-deoxy-
N6-(1-naphthylmethyl)adenosine as Novel Scaffold for a Polymer-
Assisted Amidation Protocol. Tetrahedron 2000, 56, 3167-3172.
(29) Kenner, G. W.; McDermott, J . R.; Sheppard, R. C. Safety Catch
Principle in Solid Phase Peptide Synthesis. J . Chem. Soc. D
1971, 636-637.
(30) Backes, B. J .; Virgilio, A. A.; Ellman, J . A. Activation Method
To Prepare a Highly Reactive Acylsulfonamide “Safety-Catch”
Linker for Solid-Phase Synthesis. J . Am. Chem. Soc. 1996, 118,
3055-3056.
(31) Kotra, L. P.; Manouilov, K. K.; Cretton-Scott, E.; Sommadossi,
J .-P.; Boudinot, F. D.; Schinazi, R. F.; Chu, C. K. Synthesis,
Biotransformation, and Pharmacokinetic Studies of 9-(â-D-
Arabinofuranosyl)-6-azidopurine: A Prodrug for Ara-A Designed
To Utilize the Azide Reduction Pathway. J . Med. Chem. 1996,
39, 5202-5207.
(32) Matsuda, A.; Shinozaki, M.; Yamaguchi, T.; Homma, H.; Nomoto,
R.; Miyasaka, T.; Watanabe, Y.; Abiru, T. Nucleosides and
Nucleotides. 103. 2-Alkynyladenosines: A Novel Class of Selec-
tive Adenosine A2 Receptor Agonists with Potent Antihyper-
tensive Effects. J . Med. Chem. 1992, 35, 241-52.
(33) Link, A.; Van Calenbergh, S.; Herdewijn, P. Practical Method
for the Parallel Synthesis of 2′-Amido-2′-deoxyadenosines. Tet-
rahedron Lett. 1998, 39, 5175-5176.
(34) Golisade, A.; Bressi, J . C.; Calenbergh, S.; Gelb, M. H.; Link, A.
Polymer-Assisted Solution-Phase Synthesis of 2′-Amido-2′-
deoxyadenosine Derivatives Targeted at the NAD+-Binding Sites
of Parasite Enzymes. J . Comb. Chem. 2000, 2, 537-544.
(35) Kumar, H. M. S.; Reddy, B. V. S.; Reddy, P. T.; Yadav, J . S.
Efficient One-Pot Preparation of Nitriles from Aldehydes Using
N-Methyl-pyrrolidone. Synthesis 1999, 586-587.
(36) DeCosta, D. P.; Pincock, J . A. Photochemistry of substituted
1-naphthylmethyl esters of phenylacetic and 3-phenylpropanoic
acid: radical pairs, ion pairs, and Marcus electron transfer. J .
Am. Chem. Soc. 1993, 115, 2180-90.
(38) Deana, A. A.; Stokker, G. E.; Schultz, E. M.; Smith, R. L.; Cragoe,
E. J ., J r.; Russo, H. F.; Watson, L. S. 2-(Aminomethyl)phenols,
a New Class of Saluretic Agents. 5. Fused-Ring Analogues. J .
Med. Chem. 1983, 26, 580-585.
(39) White, B. B.; Barabash, E. Chem. Abstr. 1952, No. 2,581,565.
(40) McMartin, C.; Bohacek, R. S. QXP: Powerful, Rapid Computer
Algorithms for Structure-Based Drug Design. J . Comput. Aided
Mol. Des. 1997, 11, 333-344.
(41) Bohacek, R.; De Lombaert, S.; McMartin, C.; Priestle, J .;
Gruetter, M. Three-Dimensional Models of ACE and NEP
Inhibitors and Their Use in the Design of Potent Dual ACE/
NEP Inhibitors. J . Am. Chem. Soc. 1996, 118, 8231-8249.
(42) Cignitti, M.; Ramusino, M. C.; Farina, A.; Rajevic, M. Confor-
mational Properties of o-Alkoxybenzamides in Different Sol-
vents. J . Mol. Struct. 1996, 384, 9-16.
(43) Allen, F. H.; Kennard, O. 3D Search and Research Using the
Cambridge Structural Database. Chem. Des. Autom. News 1993,
8, 31-37.
(44) Aronov, A. M.; Munagala, N. R.; Ortiz de Montellano, P. R.;
Kuntz, I. D.; Wang, C. C. Rational Design of Selective Submi-
cromolar Inhibitors of Tritrichomonas foetus Hypoxanthine-
Guanine-Xanthine Phosphoribosyltransferase. Biochemistry 2000,
39, 4684-4691.
(45) Skillman, A. G.; Kuntz, I. D. Unpublished results.
(46) Ewing, T. J . A.; Kuntz, I. D. Critical Evaluation of Search
Algorithms for Automated Molecular Docking and Database
Screening. J . Comput. Chem. 1997, 18, 1175-1189.
(47) Zou, X.; Sun, Y.; Kuntz, I. D. Inclusion of Solvation in Ligand
Binding Free Energy Calculations Using the Generalized Born
Model. J . Am. Chem. Soc. 1999, 121, 8033-8043.
(48) Hirumi, H. Continuous Cultivation of Trypanosoma brucei Blood
Stream Forms in a Medium Containing a Low Concentration of
Serum Protein without Feeder Cell Layers. J . Parasitol. 1989,
75, 985-989.
(49) Raz, B.; Iten, M.; Grether Buhler, Y.; Kaminsky, R.; Brun, R.
The Alamar Blue Assay To Determine Drug Sensitivity of
African Trypanosomes (T.b. rhodesiense and T.b. gambiense) In
vitro. Acta Trop. 1997, 68, 139-147.
(50) Buckner, F. S.; Verlinde, C. L. M. J .; La Flamme, A. C.; Van
Voorhis, W. C. Efficient Technique for Screening Drugs for
Activity against Trypanosoma cruzi Using Parasites Expressing
beta-Galactosidase. Antimicrob. Agents Chemother. 1996, 40,
2592-2597.
(37) Stokker, G. E.; Deana, A. A.; DeSolms, S. J .; Schultz, E. M.;
Smith, R. L.; Cragoe, E. J ., J r.; Baer, J . E.; Ludden, C. T.; Russo,
H. F.; et al. 2-(Aminomethyl)phenols, a New Class of Saluretic
Agents. 1. Effects of Nuclear Substitution. J . Med. Chem. 1980,
23, 1414-1427.
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