Synthesis of 5′-methylthio coformycins: Specific inhibitors for malarial adenosine deaminase

Article abstract of DOI:10.1021/ja0708363

Transition state theory suggests that enzymatic rate acceleration (k _cat/k_non) is related to the stabilization of the transition state for a given reaction. Chemically stable analogues of a transition state complex are predicted to convert catalytic energy into binding energy. Because transition state stabilization is a function of catalytic efficiency, differences in substrate specificity can be exploited in the design of tight-binding transition state analogue inhibitors. Coformycin and 2′-deoxycoformycin are natural product transition state analogue inhibitors of adenosine deaminases (ADAs). These compounds mimic the tetrahedral geometry of the ADA transition state and bind with picomolar dissociation constants to enzymes from bovine, human, and protozoan sources. The purine salvage pathway in malaria parasites is unique in that Plasmodium falciparum ADA (PfADA) catalyzes the deamination of both adenosine and 5′-3 methylthioadenosine. In contrast, neither human adenosine deaminase (HsADA) nor the bovine enzyme (BtADA) can deaminate 5′-methylthioadenosine. 5′-Methylthiocoformycin and 5′-methylthio-2′-deoxycoformycin were synthesized to be specific transition state mimics of the P. falciparum enzyme. These analogues inhibited PfADA with dissociation constants of 430 and 790 pM, respectively. Remarkably, they gave no detectable inhibition of the human and bovine enzymes. Adenosine deamination is involved in the essential pathway of purine salvage in P. falciparum, and prior studies have shown that inhibition of purine salvage results in parasite death. Inhibitors of HsADA are known to be toxic to humans, and the availability of parasite-specific ADA inhibitors may prevent this side-effect. The potent and P. falciparum-specti'ic inhibitors described here have potential for development as antimalarials without inhibition of host ADA.

Full text of DOI:10.1021/ja0708363

Published on Web 05/08/2007
Synthesis of 5′-Methylthio Coformycins: Specific Inhibitors
for Malarial Adenosine Deaminase
Peter C. Tyler,*,† Erika A. Taylor,‡ Richard F. G. Fro¨hlich,† and Vern L. Schramm*,‡
Contribution from the Carbohydrate Chemistry Team, Industrial Research Ltd., Lower Hutt, New
Zealand, and Department of Biochemistry, Albert Einstein College of Medicine at YeshiVa
UniVersity, 1300 Morris Park AVenue, Bronx, New York 10461
Received February 5, 2007; E-mail: p.tyler@irl.cri.nz; vern@aecom.yu.edu
Abstract: Transition state theory suggests that enzymatic rate acceleration (kcat/knon) is related to the
stabilization of the transition state for a given reaction. Chemically stable analogues of a transition state
complex are predicted to convert catalytic energy into binding energy. Because transition state stabilization
is a function of catalytic efficiency, differences in substrate specificity can be exploited in the design of
tight-binding transition state analogue inhibitors. Coformycin and 2′-deoxycoformycin are natural product
transition state analogue inhibitors of adenosine deaminases (ADAs). These compounds mimic the
tetrahedral geometry of the ADA transition state and bind with picomolar dissociation constants to enzymes
from bovine, human, and protozoan sources. The purine salvage pathway in malaria parasites is unique
in that Plasmodium falciparum ADA (PfADA) catalyzes the deamination of both adenosine and 5′-
methylthioadenosine. In contrast, neither human adenosine deaminase (HsADA) nor the bovine enzyme
(BtADA) can deaminate 5′-methylthioadenosine. 5′-Methylthiocoformycin and 5′-methylthio-2′-deoxycofor-
mycin were synthesized to be specific transition state mimics of the P. falciparum enzyme. These analogues
inhibited PfADA with dissociation constants of 430 and 790 pM, respectively. Remarkably, they gave no
detectable inhibition of the human and bovine enzymes. Adenosine deamination is involved in the essential
pathway of purine salvage in P. falciparum, and prior studies have shown that inhibition of purine salvage
results in parasite death. Inhibitors of HsADA are known to be toxic to humans, and the availability of
parasite-specific ADA inhibitors may prevent this side-effect. The potent and P. falciparum-specific inhibitors
described here have potential for development as antimalarials without inhibition of host ADA.
Introduction
and deoxyadenosine. Production of excess dATP from ac-
cumulated deoxyadenosine unbalances the deoxynucleotide pool,
Adenosine deaminase (ADA) deaminates adenosine and
deoxyadenosine to form the respective inosines and thereby
plays an essential role in purine metabolism.1 Genetic deficiency
of ADA in humans causes severe combined immunodeficiency
disease (SCID), which results from the accumulation of dATP
in B- and T-cells.2-4 Adenosine deaminase is also important in
purine salvage pathways in microorganisms and is thought to
be essential for salvage pathways in purine auxotrophs like
Plasmodium falciparum.5,6 The adenosine deaminase inhibitors
coformycin and 2′-deoxycoformycin (also known as Pentostatin)
are natural product transition state analogue inhibitors that bind
with picomolar affinity to adenosine deaminases.1,7-9 Inhibition
of adenosine deaminase results in the accumulation of adenosine
inhibits ribonucleotide reductase, and initiates apoptosis in B-
and T-cells.10 Deoxycoformycin has been approved by the FDA
for the treatment of hairy cell leukemia since 1991.11 However,
it causes renal, liver, pulmonary, and central nervous system
toxicities at high doses and is therefore limited in its therapeutic
profile.12
Recently, the ADA from P. falciparum (PfADA) has been
characterized and found to catalyze the deamination of adenos-
ine, as well as the conversion of 5′-methylthioadenosine (MTA)
to 5′-methylthioinosine (MTI) and ammonia (Figure 1).13 5′-
Methylthioinosine was not previously reported as a metabolite
in any biological system. The ability of PfADA to utilize MTA
† Industrial Research Ltd.
(7) Agarwal, R. P.; Spector, T.; Parks, J. R. E. Biochem. Pharmacol. 1977,
26, 359-367.
‡ Albert Einstein College of Medicine at Yeshiva University.
(1) Agarwal, R. P. Pharmacol. Ther. 1982, 17, 399-429.
(2) Giblett, E. R.; Anderson, J. E.; Cohen, F.; Pollara, B.; Meuwissen, H. J.
Lancet 1972, 2, 1067-1069.
(8) Cha, S.; Agarwal, R. P.; Parks, J. R. E. Biochem. Pharmacol. 1975, 24,
2187-2197.
(9) Schramm, V. L. Arch. Biochem. Biophys. 2005, 433, 13-26.
(10) Blackburn, M. R.; Kellems, R. E. AdV. Immunol. 2005, 86, 1-41.
(11) Published online at: http://www.accessdata.fda.gov/scripts/cder/onctools/
summary.cfm?ID)111.
(3) Agarwal, R. P.; Crabtree, G. W.; Parks, R. E., Jr.; Nelson, J. A.; Keightley,
R.; Parkman, R.; Rosen, F. S.; Stern, R. C.; Polmar, S. H. J. Clin. InVest.
1976, 57, 1025-1035.
(4) Ullman, B.; Gudas, L. J.; Cohen, A.; Martin, D. W., Jr. Cell 1978, 14,
365-375.
(12) Johnson, S. A. Clin. Pharmacokinet. 2000, 39, 5-26. Margolis, J.; Grever,
M. R. Semin. Oncol. 2000, 27, 9-14.
(5) Webster, H. K.; Weismann, W. P.; Pavia, C. S. AdV. Exp. Med. Biol. 1984,
165, 225-229.
(13) Ting, L. M.; Shi, W.; Lewandowicz, A.; Singh, V.; Mwakingwe, A.; Birck,
M. R.; Taylor Ringia, E. A.; Bench, G.; Madrid, D. C.; Tyler, P. C.; Evans,
G. B.; Furneaux, R. H.; Schramm, V. L.; Kim, K. J. Biol. Chem. 2005,
280, 9547-9554.
(6) Wiesmann, W. P.; Webster, H. K.; Lambros, C.; Kelly, W. N.; Daddona,
P. E. Prog. Clin. Biol. Res. 1984, 165, 325-342.
9
6872
J. AM. CHEM. SOC. 2007, 129, 6872-6879
10.1021/ja0708363 CCC: $37.00 © 2007 American Chemical Society

Specific Inhibitors of Malarial Adenosine Deaminase
A R T I C L E S
Figure 1. The reaction catalyzed by adenosine deaminase involves nucleophilic attack on adenosine by a hydroxide ion formed by a catalytic site zinc atom.
A short-lived Meisenheimer complex intermediate is protonated at the exocyclic amino group, followed by loss of NH3 to produce inosine. PfADA has
substrate specificity that includes both adenosine and 5′-MeS-adenosine, an activity not shared by mammalian ADAs.
µM ZnCl2, pH 8.0 and frozen in 20 µL aliquots at -80 °C. The protein
was equally active with or without the (histidine)6 tag, which could be
removed by combining 1 unit thrombin (GE Healthcare, Piscataway)
for every 1 mg of protein in PBS at 22 °C, incubation for 1 h, followed
by purification over an 8 mL Source 15Q column with elution by a
0-1 M NaCl gradient in the dialysis buffer.
is specific to the parasite, and human ADA does not catalyze
this reaction. Prior studies have shown that inhibition of malarial
purine salvage enzymes results in parasite death;14 however,
potent ADA inhibitors with specificity for the enzymes from
malaria parasites have not been described.
5′-Methylthiocoformycin, 5′-methylthio-2′-deoxycoformycin,
and other 5′-functionalized 2′-deoxycoformycin molecules were
synthesized and investigated for their ability to inhibit PfADA,
as compared to the ADAs from human (HsADA) and bovine
(BtADA) sources. The comparative enzymology and inhibitor
specificity of malarial and human ADAs highlight the impor-
tance of examining substrate specificity in addition to transition
state analysis in the design of species-specific inhibitors for use
as therapeutic agents.
Enzyme Inhibition Assay. Adenosine deaminase activity was
determined by monitoring the conversion of adenosine to inosine via
the change in absorbance at 267 nm in assay mixtures containing 20
mM potassium phosphate, pH 7.0, 100 µM adenosine, and 1 µM EDTA
at 30 °C.15 The kinetics for slow-onset inhibition and Ki measurement
were performed using inhibitor concentrations ranging from 10 µM to
2 nM and an enzyme concentration of 1 nM. The Ki values were
determined by fitting the initial rate and inhibitor concentrations to the
16,17
following expression of competitive inhibition:
Experimental Section
(Vo′/Vo) ) (KM + [S])/(KM + [S] + (KM[I]/Ki))
Reagents, Bacterial Strains, and Transformation. Calf spleen
ADA, human erythrocyte ADA, adenosine, potassium phosphate, and
EDTA were purchased from Sigma-Aldrich Chemical Co. LR Clonase
II and pDEST-14 vector were both obtained from Invitrogen (Carlsbad,
CA). Ni Sepharose High Performance and Source 15Q media were
obtained from GE Healthcare (Piscataway, NJ). A diastereomeric mix-
ture of 2′-deoxy-8-(R/S)-coformycin was a gift from Dr. David C. Baker.
Concentrations of inhibitors were determined spectrophotometric-
ally using the extinction coefficient of 8250 M-1 cm-1 at 282 nm.15
Enzyme concentrations for BtADA and HsADA were determined by
the manufacturer, and enzyme concentrations for PfADA was deter-
mined spectrophotometrically using the extinction coefficient of 32340
M-1 cm-1 at 280 nm, as determined by the ExPASy - ProtParam tool.
Adenosine concentrations were also determined spectrophotometric-
ally using the extinction coefficient of 14900 M-1 cm-1 at 260 nm.
Escherichia coli cells BL21 Star-(DE3) and BL21 Star cells (Invitrogen,
Carlsbad) were grown in Luria broth (LB) with 100 µg/mL ampicillin
(AMP) at 37 °C.
where Vo′ is the initial rate in the presence of inhibitor, Vo is the initial
rate in the absence of inhibitor, [I] is the inhibitor concentration, and
[S] is the substrate concentration. This expression is valid only under
the condition where the inhibitor concentration is 10 times greater than
the enzyme concentration. In conditions where inhibitor concentration
does not exceed 10 times the enzyme concentration, the effective
inhibitor concentration was obtained by the expression:
I′ ) I - (1 - Vo′/Vo)Et
where I′ is the effective inhibitor concentration, Vo′ and Vo are the initial
rates in the presence and absence of inhibitor, and Et is the total enzyme
concentration.
In cases where slow onset inhibition was observed, where the
inhibitor reached a tighter binding thermodynamic equilibrium with
the enzyme, the equilibrium dissociation constant (Ki*) was obtained
by fitting the rates to the following equation for competitive inhibition:
PfADA Gene Construction. A chemically synthesized gene encod-
ing the adenosine deaminase from P. falciparum was synthesized,
cloned into a pDONR221 vector, and sequenced by DNA 2.0 (Menlo
Park). The gene was synthesized using optimized codons for E. coli
protein expression and the addition of an N-terminal thrombin cleavable
(histidine)6 tag.
(Vo′/Vo) ) (KM + [S])/(KM + [S] + (KM[I]/Ki*))
with [I] concentrations being corrected as above.18 The KM values for
Protein Expression and Purification. The designed gene was
transferred to the pDEST-14 vector with LR Clonase II. The pDEST-
14-PfADA construct was transformed into BL21 Star competent cells,
and the protein was expressed without induction with shaking at
37 °C overnight. The cells were ruptured by passage through a French
press, the cell debris was pelleted by centrifugation, and the remaining
supernatant was purified over a 25 mL Ni Sepharose High Performance
His-tag affinity column with elution by a 0-250 mM imidazole
gradient. The enzyme was then dialyzed into 100 mM Tris-HCl, 10
(14) Kicska, G. A.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.; Schramm, V.
L.; Kim, K. J. Biol. Chem. 2002, 277, 3226-3231.
(15) Schramm, V. L.; Baker, D. C. Biochemistry 1985, 24, 641-646.
(16) Merkler, D. J.; Brenowitz, M.; Schramm, V. L. Biochemistry 1990, 29,
8358-8364.
(17) Singh, V.; Shi, W.; Evans, G. B.; Tyler, P. C.; Furneaux, R. H.; Almo, S.
C.; Schramm, V. L. Biochemistry 2004, 43, 9-18.
(18) Singh, V.; Evans, G. B.; Lenz, D. H.; Mason, J. M.; Clinch, K.; Mee, S.;
Painter, G. F.; Tyler, P. C.; Furneaux, R. H.; Lee, J. E.; Howell, P. L.;
Schramm, V. L. J. Biol. Chem. 2005, 280, 18265-18273.
9
J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007 6873

A R T I C L E S
Tyler et al.
adenosine with the enzymes are 16 ( 2 µM for the bovine enzyme,15
29 ( 3 µM for the malarial enzyme,13 and 22 ( 2 µM for the human
enzyme.19
H-5). 13C NMR: (CDCl3) δ -4.6 (SiMe), -4.3 (SiMe), 18.7
(SiC(CH3)3), 26.2 (SiC(CH3)3), 28.5 (OC(CH3)3), 41.6 (C-2′), 47.0 (C-
7), 63.5 (C-5′), 67.6 (C-8), 73.3 (C-3′), 84.3 (OC(CH3)3), 87.3 (C-1′),
88.7 (C-4′), 132.7, 134.9 (C-2), 136.2, 143.3 (C-5), 152.6. HRMS:
(MH+) calcd for C22H39N4O6Si+, 483.2639; found, 483.2643.
(8R)-6-(tert-Butoxycarbonyl)-8-(tert-butyldimethylsilyloxy)-3-(2′-
deoxy-3′,5′-di-O-toluoyl-â-D-erythro-pentofuranosyl)-3,6,7,8-tetra-
hydroimidazo[4,5-d][1,3]diazepine (3). (8R)-6-(tert-Butoxycarbonyl)-
8-(tert-butyldimethylsilyloxy)-3-(2′-cyanoethyl)-3,6,7,8-tetrahydroimi-
dazo[4,5-d][1,3]diazepine 2a (0.76 g, 1.81 mmol) was dissolved in dry
THF (15 mL), and potassium tert-butoxide (1.0 M in THF, 3.6 mL)
was added at room temperature. The mixture turned brown immediately
and was quenched by addition of glacial acetic acid (206 µL, 3.6 mmol)
and coevaporated with toluene (30 mL). The residue was suspended
in chloroform/ethyl acetate (5 mL, 1:2 v/v) by ultrasonification and
applied on a chromatography column (50 g of silica, chloroform/ethyl
acetate ) 1:2 v/v, then ethyl acetate), which gave (8R)-6-(tert-
butoxycarbonyl)-8-(tert-butyldimethylsilyloxy)-3,6,7,8-tetrahydro-imi-
dazo[4,5-d][1,3]diazepine 2b as an amorphous yellow solid (0.51 g,
1.39 mmol, 77%).
(8R)-6-(tert-Butoxycarbonyl)-8-(tert-butyldimethylsilyloxy)-3-(2′-
deoxy-5′-O-tosyl-â-D-erythro-pentofuranosyl)-3,6,7,8-tetrahydroimi-
dazo[4,5-d][1,3]diazepine (5). 4 (0.375 g, 0.777 mmol) was dissolved
in dry pyridine (7 mL, 87 mmol), and tosyl chloride (0.222 g, 1.17
mmol) was added at 0 °C. After 20 min, the mixture was warmed to
room temperature and stirred overnight. The reaction mixture was
diluted with chloroform (30 mL) and consecutively washed with water
(40 mL), citric acid (5% w/w, 3 × 50 mL), and saturated NaHCO3 (60
mL), dried (MgSO4), and evaporated in vacuo. The residue was purified
by chromatography (46 g of silica, chloroform/methanol ) 20:1 v/v),
which gave the title compound as a glass (313 mg, 63%). Rf ) 0.39
(chloroform/methanol ) 10:1 v/v), [R]20 ) +25.8 (c 3.62, CHCl3).
D
1H NMR: (CDCl3) δ 0.07 (s, 3H), 0.20 (s, 3H), 0.88 (s, 9H), 1.54 (s,
9H), 2.43 (m, 4H), 2.34-2.46 (m, 4H), 2.55-2.66 (m, 2H, 1H D2O
exchangeable), 3.07 (br d, J ) 13.5 Hz, 1H), 4.06-4.16 (m, 2H), 4.20-
4.29 (m, 1H), 4.48 (dd, J ) 4.5, 13.5 Hz, 1H), 4.60-4.70 (m, 1H),
5.12 (br d, J ) 4.5 Hz, 1H), 6.30 (t, J ) 6.7 Hz, 1H), 7.28-7.34 (m,
2H), 7.48 (s, 1H), 7.70-7.75 (m, 2H), 7.83 (s, 1H). 13C NMR: (CDCl3)
δ -4.6, -4.1, 18.7, 22.0, 26.2, 28.5, 40.6, 47.2, 67.6, 69.1, 72.2,
83.8, 83.9, 128.4, 130.4, 132.4, 132.6, 133.0, 134.6, 142.6, 145.6, 152.8.
HRMS: (MH+) calcd for C29H45N4O8SSi+, 637.2727; found, 637.2753.
(8R)-6-(tert-Butoxycarbonyl)-3-(2′-deoxy-5′-O-tosyl-â-D-erythro-
pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-
ol (6). 5 (0.31 g, 0.49 mmol) was dissolved in dry methanol (12 mL),
ammonium fluoride (0.54 g, 14.6 mmol) was added, and the mixture
was heated to reflux for 6.5 h. After the mixture was cooled to room
temperature, stirring was continued overnight. The next morning the
mixture was refluxed for an additional 6 h. The reaction mixture was
evaporated in vacuo, and the residue was purified by chromatography
(27 g of silica, chloroform/methanol ) 10:1 v/v), which gave 6 as a
2b was dissolved in dry acetonitrile (10 mL) and evaporated in vacuo,
flushed with argon, and dissolved again in dry acetonitrile (15 mL).
Addition of sodium hydride (60% in mineral oil, 72 mg) gave a visible
gas formation. After 30 min, 2-deoxy-3,5-di-O-(p-toluoyl)-R-D-erythro-
pentofuranosyl chloride 120 (0.70 g, 1.81 mmol) was added to the
reaction mixture, and a thick heterogeneous slurry was formed. After
further 40 min, the reaction mixture was filtered through flux calcined
diatomaceous earth and rinsed thoroughly with ethyl acetate. The filtrate
was evaporated to dryness in vacuo. Purification of the residue by
chromatography (60 g of silica, petroleum ether/ethyl acetate ) 2:1
v/v) gave 3 as yellowish oil (0.85 g, 1.18 mmol, 85% calcd. from 2b).
Rf ) 0.31 (petrol ether/EtOAc ) 3:2 v/v), [R]20 ) -17.4 (c 11.8,
D
1
chloroform). H NMR: (CDCl3) δ 0.05 (s, 3H, SiMe), 0.19 (s, 3H,
SiMe), 0.88 (s, 9H, SitBu), 1.54 (s, 9H, OtBu), 2.40 (s, 3H, ArMe),
2.43 (s, 3H, ArMe), 2.70 (ddd, J ) 2.2, 5.9, 14.2 Hz, 1H, H-2′), 2.84
(ddd, J ) 6.4, 8.4, 14.2 Hz, 1H, H-2′), 3.08 (br d, J ) 13.6 Hz, 1H,
H-7), 4.50 (dd, J ) 4.4, 13.6 Hz, 1H, H-7), 4.55-4.60 (m, 1H, H-4′),
4.61-4.65 (m, 2H, H-5′), 5.13 (br d, J ) 4.4 Hz, H-8), 5.70 (ddd, J )
2.2, 2.3, 6.0 Hz, 1H, H-3′), 6.42 (dd, J ) 5.9, 8.4 Hz, 1H, H-1′), 7.19-
7.30 (m, 4H, Ar), 7.57 (s, 1H, H-2), 7.85-7.99 (m, 5H, 4×Ar and
H-5). 13C NMR: (CDCl3) δ -4.6 (SiMe), -4.2 (SiMe), 18.6
(SiC(CH3)3), 22.0 (ArCH3), 26.2 (SiC(CH3)3), 28.5 (OC(CH3)3), 38.9
(C-2′), 47.2 (C-7), 64.6 (C-5′), 67.6 (C-8), 75.6 (C-3′), 82.7 (C-4′),
83.7 (OC(CH3)3), 84.4 (C-1′), 127.0, 127.2, 129.6, 130.1, 130.2,
132.3 (C-2), 133.1, 134.8, 142.7 (C-5), 144.4, 144.7, 152.8, 166.3,
166.6. HRMS: (MH+) calcd for C38H51N4O8Si+, 719.3476; found,
719.3492.
hard foam (147 mg, 58%). Rf ) 0.23 (chloroform/methanol ) 10:1
1
v/v), [R]20 ) +30.3 (c 2.15, chloroform). H NMR: (CDCl3) δ 1.53
D
(s, 9H), 2.37-2.64 (m, 6H), 3.59 (br d, J ) 13.5 Hz, 1H), 4.03 (dd, J
) 5.9, 13.5 Hz, 1H), 4.08-4.17 (m, 3H), 4.18-4.26 (m, 1H), 4.58-
4.67 (m, 1H), 5.01 (dd, J ) 2.0, 5.9 Hz, 1H), 6.31 (t, J ) 6.6 Hz, 1H),
7.27-7.32 (m, 2H), 7.59 (s, 1H), 7.67-7.73 (m, 2H), 7.80 (s, 1H).
13C NMR: (CDCl3) δ 22.0, 28.4, 40.8, 46.8, 65.8, 69.4, 71.6, 84.0,
84.2, 128.3, 130.4, 132.3, 132.6, 133.0, 134.2, 142.9, 145.6, 152.9.
HRMS: (MH+) calcd for C23H31N4O8S+, 523.1863; found, 523.1848.
(8R)-3-(2′-Deoxy-5′-methylthio-â-D-erythro-pentofuranosyl)-3,6,7,8-
tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol (7). Under argon, 6 (35
mg, 0.067 mmol) was dissolved in dry methanol (3 mL) and treated
with sodium thiomethoxide (95% w/w, 0.30 g) at room temperature.
After 2 h, the reaction mixture was evaporated in vacuo. Purification
of the residue by chromatography (12 g of silica, chloroform/methanolic
ammonia (7 M) ) 5:1 v/v, 120 mL, 4:1 v/v 100 mL) gave 7 as a white
(8R)-6-(tert-Butoxycarbonyl)-8-(tert-butyldimethylsilyloxy)-3-(2′-
deoxy-â-D-erythro-pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d]-
[1,3]diazepine (4). In a rubber sealed round-bottom flask, 3 (0.79 g,
1.10 mmol) was treated with ammonia (7 M in methanol, 75 mL) at
room temperature. After 17 h, the mixture was evaporated in vacuo,
the residue was transferred into an ace pressure tube, fresh ammonia
(7 M in methanol, 50 mL) was added, and the tube was sealed and
warmed to 45 °C for another 4 h. After evaporation in vacuo, the residue
was purified by chromatography (41 g of silica, chloroform/methanol
) 15:1 v/v), which gave 4 as colorless oil (375 mg, 71%). Rf ) 0.34
(chloroform/methanol ) 10:1 v/v), [R]20D ) +10.0 (c 5.23, chloroform).
1H NMR: (CDCl3) δ 0.06 (s, 3H, SiMe), 0.19 (s, 3H, SiMe), 0.88 (s,
9H, SitBu), 1.53 (s, 9H, OtBu), 2.25 (ddd, J ) 1.1, 5.9, 14 Hz, 1H,
H-2′), 2.94 (ddd, J ) 5.3, 8.7, 14 Hz, 1H, H-2′), 3.08 (br d, J ) 13.6
Hz, 1H, H-7), 3.74 (dd, J ) 1.3, 12.2 Hz, 1H, H-5′), 3.89 (dd, J ) 1.8,
12.2 Hz, 1H, H-5′), 4.10-4.15 (m, 1H, H-4′), 4.54 (dd, J ) 4.7, 13.6
Hz, 1H, H-7), 4.65-4.71 (m, 1H, H-3′), 5.14 (d, J ) 4.7 Hz, 1H, H-8),
6.20 (dd, J ) 5.9, 8.7 Hz, 1H, H-1′), 7.49 (s, 1H, H-2), 7.85 (s, 1H,
solid (14.5 mg, 73%). Rf ) 0.35 (chloroform/methanolic ammonia (7
1
M) ) 4:1 v/v), [R]20 ) +89 (c 0.41, methanol). H NMR: (D2O) δ
D
2.09 (s, 3H, SMe), 2.48 (ddd, J ) 4.1, 6.4, 14.1 Hz, 1H, H-2′), 2.68
(ddd, J ) 6.7, 7.2, 14.1 Hz, 1H, H-2′), 2.74 (dd, J ) 6.6, 14.1 Hz, 1H,
H-5′), 2.82 (dd, J ) 5.7, 14.1 Hz, 1H, H-5′), 3.34 (br d, J ) 13.5 Hz,
1H, H-7), 3.49 (dd, J ) 4.4, 13.5 Hz, 1H, H-7), 4.16 (ddd, J ) 3.5,
6.0, 6.2 Hz, 1H, H-4′), 4.52 (m, 1H, H-3′), 5.12 (br d, J ) 3.5 Hz, 1H,
H-8), 6.25 (t, J ) 6.8 Hz, 1H, H-1′), 7.20 (s, 1H), 7.75 (s, 1H). 13C
NMR: (D2O) δ 15.8 (SMe), 36.7 (C-5′), 39.1 (C-2′), 47.7 (C-7), 67.2
(C-8), 73.8 (C-3′), 83.2 (C-1′), 85.8 (C-4′), 128.4, 131.4 (C-2), 135.9,
151.0 (C-5). HRMS: (MH+) calcd for C12H19N4O3S+, 299.1178; found,
299.1187. Anal. Calcd for C12H18N4O3S‚1.4H2O: C, 44.54; H, 6.48;
N, 17.31; S, 9.91. Found: C, 44.42; H, 6.13; N, 17.19; S, 9.69.
(8R)-3-(2′-Deoxy-5′-propylthio-â-D-erythro-pentofuranosyl)-3,6,7,8-
tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol (8). 1-Propanethiol (0.42
(19) The human enzyme sample received from Sigma was characterized to have
a kcat of 36 ( 1 s-1 and a KM of 22 ( 2 µM.
(20) Hoffer, M. Chem. Ber. 1960, 93, 2777-2781.
9
6874 J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007

Specific Inhibitors of Malarial Adenosine Deaminase
A R T I C L E S
Scheme 1 a
mL, 4.64 mmol) was diluted in dry methanol (3 mL), and sodium
methoxide (30% w/w in methanol, 0.72 mL) was added. After 5 min
at room temperature, 6 (40 mg, 0.077 mmol) was added, and the mixture
was stirred overnight at room temperature. The next day the solution
was evaporated in vacuo and purified by chromatography (15 g of silica,
chloroform/methanolic ammonia (7 M) ) 5:1 v/v), which gave the
product as a white soapy material (17 mg, 68%). Rf ) 0.31 (chloroform/
methanolic ammonia (7 M) ) 4:1 v/v), [R]20 ) +76 (c 0.85,
D
methanol). 1H NMR: (CD3OD) δ 0.97 (t, J ) 7.3 Hz, 3H), 1.60 (sext,
J ) 7.3 Hz, 2H), 2.35 (ddd, J ) 3.9, 6.3, 13.5 Hz, 1H, H-2′), 2.47
(dd, J ) 6.9, 13.5 Hz, 1H, H-2′), 2.55 (t, J ) 7.3 Hz, 2H), 2.76
(dd, J ) 5.9, 13.8 Hz, 1H, H-5′), 2.81 (dd, J ) 5.9, 13.8 Hz, 1H,
H-5′), 3.27-3.35 (m, 1H, H-7), 3.39 (dd, J ) 4.4, 13.1 Hz, 1H,
H-7), 4.02 (ddd, J ) 3.5, 5.9, 5.9 Hz, 1H, H-4′), 4.35-4.43 (m, 1H,
H-3′), 4.98-5.05 (m, 1H, H-8), 6.27 (t, J ) 6.8 Hz, 1H, H-1′), 7.08 (s,
1H), 7.66 (s, 1H). 13C NMR: (CD3OD) δ 13.7, 24.0, 35.7, 35.9, 41.4
(C-2′), 49.1 (C-7), 68.4 (C-8), 74.5 (C-3′), 84.4 (C-1′), 87.6 (C-4′),
130.3 (C-2), 131.4, 136.8, 149.8 (C-5). HRMS: (MH+) calcd for
C14H23N4O3S+, 327.1491; found, 327.1487. Anal. Calcd for
C14H22N4O3S: C, 51.51; H, 6.79; N, 17.16. Found: C, 51.49; H, 6.77;
N, 16.94.
(8R)-3-(2′-Deoxy-5′-phenylthio-â-D-erythro-pentofuranosyl)-3,6,7,8-
tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol (9). Under argon, sodium
methoxide (30% w/w in methanol, 0.72 mL) was added to a
solution of thiophenol (0.47 mL, 4.6 mmol) in dry methanol (3 mL).
After 5 min, 6 (39 mg, 0.075 mmol) was added, and the solution
was stirred at room temperature for 24 h. Sodium thiomethoxide
(95%, 0.32 g) was added to the mixture and stirred for further 3 h 20
min. The reaction mixture was concentrated in vacuo, redissolved in
chloroform/methanol (4:1 v/v), and silica (1 g) was added and again
concentrated to dryness in vacuo and applied to a column (14 g of
silica, chloroform/methanol ) 10:1 v/v 200 mL which washed off
excess thiophenol, then 4:1 v/v 100 mL eluted the product), which gave
a Reagents: (i) KOtBu, THF, 77%; (ii) NaH, CH3CN, 85%; (iii) NH3,
MeOH, 71%; (iv) TsCl, pyridine, 60%; (v) NH4F, MeOH, reflux, 58%;
(vi) NaSMe, MeOH, 73%; (vii) PrSH, NaOMe, MeOH, 68%; (viii) PhSH,
NaOMe, MeOH, then NaSMe, 90%.
and concentrated. Chromatography (EtOAc/Pet Ether ) 1:4 v/v) gave
separately the two anomers of the title compound: first the major
compound (4.16 g, 13.7 mmol) followed by the minor (0.73 g, 2.4
mmol). Total yield 40% overall. For the major anomer, 1H NMR:
(CDCl3) δ 5.94-5.81 (1H, m), 5.34-5.12 (4H, m), 5.03 (1H, s), 4.30-
4.19 (2H, m), 4.04-3.97 (1H, m), 2.77 (2H, m), 2.18 (3H, s), 2.10
(3H, s), 2.06 (3H, s). 13C NMR: δ 170.1, 170.0, 133.9, 117.9, 104.7,
80.8, 75.5, 74.8, 68.9, 38.8, 20.9, 16.6. For the minor anomer, 1H
NMR: (CDCl3) δ 5.95-5.84 (1H, m), 5.34-5.02 (5H, m), 4.35-4.21
(2H, m), 4.12-4.05 (1H, m), 2.87 (1H, dd, J ) 14.0, 4.6 Hz), 2.78
(1H, dd, J ) 14.0, 5.5 Hz), 2.18 (3H, s), 2.12 and 2.11 (3H each, s).
13C NMR: δ 170.8, 170.2, 134.4, 117.5, 99.8, 81.6, 72.4, 71.3, 68.9,
37.1, 21.1, 20.9, 17.4. HRMS: (MH+) calcd for C13H21O6S, 305.1059;
found, 305.1072.
8-(R)-3-(2,3-Di-O-acetyl-5-S-methyl-5-thio-â-D-ribofuranosyl)-6-
tert-butoxycarbonyl-8-(tert-butyldimethylsilyloxy)-3,6,7,8-tetrahy-
droimidazo[4,5-d][1,3]diazepine (12). To a solution of 10 (3.48 g,
11.5 mmol) in acetic acid (70 mL) was added tetrakis(triphenylphos-
phine)palladium(0) (5.3 g, 4.59 mmol), and the solution was stirred
under argon at 70 °C. After 1.5 h, more tetrakis(triphenylphosphine)-
palladium(0) (1.0 g, 0.87 mmol) was added, and after another 0.5 h,
the solution was concentrated to dryness. Chromatography (EtOAc/
Pet Ether ) 1:1 v/v) afforded de-allylated material as a syrup (1.9 g,
7.2 mmol, 63%). Trichloroacetonitrile (0.75 mL, 7.5 mmol) and then
DBU (4 drops) were added to a stirred solution of this material (0.31
g, 1.17 mmol) in dichloromethane (3 mL). After 0.5 h, the solution
was diluted with hexanes and chromatographed (EtOAc/hexanes 1:3
with 0.5% Et3N) to give 0.40 g (0.98 mmol) of syrupy trichloroace-
timidate 11. A solution of this material and 2b (0.12 g, 0.328 mmol)
in dry dichloromethane (5 mL) was cooled in an ice bath, trimethylsilyl
trifluoromethanesulfonate (0.10 mL, 0.55 mmol) was added, and the
solution was allowed to warm to room temperature. After 1 h, the
solution was washed with aqueous NaHCO3, dried, and concentrated.
Chromatography (1% Et3N in EtOAc/CHCl3/hexanes 1:2:4) gave 12
compound 9 as an oil (24 mg, 90%). Rf ) 0.21 (chloroform/methanol
1
) 4:1 v/v), [R]20 ) +83 (c 0.49, methanol). H NMR: (CD3OD) δ
D
2.35 (ddd, J ) 3.4, 6.2, 13.6 Hz, 1H, H-2′), 2.51 (ddd, J ) 6.2, 7.4,
13.6 Hz, 1H, H-2′), 3.19 (dd, J ) 6.0, 13.8 Hz, 1H, H-5′), 3.24 (dd, J
) 6.1, 13.8 Hz, 1H, H-5′), 3.27-3.34 (m, 1H, H-7), 3.39 (dd, J ) 4.4,
13.2 Hz, 1H, H-7), 4.05 (ddd, J ) 3.1, 6.0, 6.1 Hz, 1H, H-4′), 4.41
(ddd, J ) 3.0, 3.2, 6.2 Hz, 1H, H-3′), 5.01 (dd, J ) 1.4, 4.4 Hz, 1H,
H-8), 6.26 (dd, J ) 6.2, 7.4 Hz, 1H, H-1′), 7.07 (s, 1H, H-5), 7.14-
7.22 (m, 1H), 7.23-7.32 (m, 2H), 7.36-7.43 (m, 2H), 7.59 (s, 1H,
H-2). 13C NMR: (CD3OD) δ 37.7 (C-5′), 41.3 (C-2′), 49.1 (C-7), 68.4
(C-8), 74.6 (C-3′), 84.7 (C-1′), 86.6 (C-4′), 127.3, 130.1, 130.4, 130.6,
131.5 (C-2), 136.8, 137.5, 149.8 (C-5). HRMS: (MH+) calcd for
C17H21N4O3S+, 361.1334; found, 361.1324. Anal. Calcd for C17H20N4O3S‚
1.7H2O: C, 52.21; H, 6.03; N, 14.33. Found: C, 52.01; H, 5.75; N,
14.50.
Allyl 2,3-Di-O-acetyl-5-S-methyl-5-thio-r,â-D-ribofuranoside (10).
Acetyl chloride (2.0 mL, 28 mmol) was added to a stirred suspension
of D-ribose (6.0 g, 40 mmol) in allyl alcohol (100 mL). After 0.5 h,
pyridine (5 mL, 61.8 mmol) was added to the clear solution, and it
was concentrated to dryness. Chromatography (CHCl3/EtOAc/MeOH
) 5:2:1 v/v) afforded allyl R,â-D-ribofuranoside (7.47 g, 39.3 mmol,
98%) as a syrup. A solution of this material in dry pyridine (60 mL)
was treated with 2,4,6-triisopropylbenzenesulfonyl chloride (14.2 g, 46.9
mmol) and then allowed to stand for 2 days. It was diluted with water
(1 L) and extracted two times with chloroform. The combined extracts
were washed consecutively with water, diluted HCl, and aqueous
NaHCO3, dried, and concentrated. Chromatography (EtOAc/Pet Ether
) 1:3 v/v) afforded 9.77 g of major product. A stirred solution of 8.84
g of this material in DMF (30 mL) was treated with sodium
thiomethoxide (4.1 g, 58.6 mmol). After 2 h, pyridine (50 mL) and
acetic anhydride (50 mL) were added, and the solution was stirred for
16 h before being partitioned between toluene and water. The organic
phase was washed with water, diluted HCl, and aqueous NaHCO3, dried,
9
J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007 6875

A R T I C L E S
Tyler et al.
Table 1. Inhibition Constants of Transition State Analogue Inhibitors of Adenosine Deaminase
BtADA
HsADA
PfADA
inhibitor
Ki (nM)
Ki* (nM)
Ki (nM)
Ki* (nM)
Ki (nM)
Ki* (nM)
coformycin
5′-MeS-coformycin (13)
2′-deoxycoformycin
5′-MeS-2′-deoxycoformycin (7)
5′-PrS-2′-deoxycoformycin (8)
5′-PhS-2′-deoxycoformycin (9)
1.1 ( 0.4
>10 000a
0.39 ( 0.12
>10 000
0.06 ( 0.01
13.9 ( 3.4
>10 000
0.5 ( 0.1
>10 000
>10 000
>10 000
0.11 ( 0.02
0.68 ( 0.07
2.66 ( 0.13
8.2 ( 2.9
2.3 ( 0.8
12 ( 1
0.08 ( 0.02
0.43 ( 0.12
0.038 ( 0.009
0.73 ( 0.22
ND
NDb
ND
0.027 ( 0.004
0.026 ( 0.005
ND
ND
ND
ND
ND
ND
>10 000
>10 000
61 ( 11
ND
a Ki values of >10 000 indicate that assays with 10 µM of the indicated inhibitor exhibited no inhibition under the conditions of the assay. b No slow
onset phase was detected.
Scheme 2 a
a Reagents: (i) HCl, AllOH; (ii) triisopropylbenzenesulfonyl chloride, py; (iii) NaSMe, DMF, then py/Ac2O; (iv) (Ph3P)4Pd(0), HOAc; (v) Cl3CCN,
DBU, CH2Cl2; (vi) 2b, TMSOTf, CH2Cl2; (vii) Bu4NF, HOAc, THF; (viii) NaSMe, MeOH.
as a syrup (0.109 g, 0.178 mmol, 54% based on the acceptor). 1H
Results and Discussion
NMR: (CDCl3) δ 7.88 (1H, s), 7.60 (1H, s), 6.03 (1H, d, J ) 5.0 Hz),
The recent determination that the adenosine deaminase from
5.80 (1H, t, J ) 5.4 Hz), 5.50 (1H, t, J ) 5.5 Hz), 5.13 (1H, d, J ) 4.4
P. falciparum has catalytic activity for both adenosine and
Hz), 4.48 (1H, dd, J ) 13.5, 4.6 Hz), 4.34 (1H, dd, J ) 13.5, 5.2 Hz),
5′-methylthioadenosine as physiological substrates13 led to
3.13 (1H, d, J ) 13.5 Hz), 2.87 (2H, m), 2.14, 2.10, 2.08 (3H each, s),
the investigation of adenosine deaminase inhibitors with
5′-methylthio and related groups. Two potent inhibitors of
human adenosine deaminase, coformycin and 2′-deoxy-
1.54 (9H, s), 0.88 (9H, s), 0.19, 0.05 (3H each, s). 13C NMR: δ 169.9,
169.7, 152.8, 142.8, 135.0, 133.2, 86.4, 83.9, 82.0, 73.9, 72.8, 67.6,
47.2, 36.9, 28.5, 26.2, 21.0, 20.8, 18.7, 17.4, -4.2, -4.6. HRMS:
coformycin, were targeted as parent compounds for this
investigation. Both inhibitors have picomolar dissociation con-
stants for human adenosine deaminase,7-9 and 2′-deoxycofor-
mycin (Pentostatin) was approved by the FDA for treatment of
hairy cell leukemia in 1991. The use of Pentostatin as a thera-
peutic agent has been limited due to its toxicity related to the
inhibition of ADA and its incorporation into DNA.12 Modifica-
tion of ADA inhibitors to eliminate binding to the human
enzyme and to eliminate 5′-phosphorylation for entry into DNA
pathways, while retaining powerful inhibition of the malarial
enzyme, would allow for therapeutic efficacy against the parasite
while eliminating the toxicity of the parent compounds.
Synthesis of 5′-Functionalized Coformycins. Previous
syntheses of coformycin and 2′-deoxycoformycin have focused
on the preparation of 8-keto-diazepine derivatives followed by
reduction of the ketone and separation of the diastereoisomeric
8-(R/S)-alcohols.21-23 The chromatographic separation of these
isomers is a synthetically inefficient and difficult route. Con-
sequently, we have concentrated on methods to synthesize the
(MH+) calcd for C27H45N4O8Ssi, 613.2727; found, 613.2739.
8-(R)-Hydroxy-3-(5-S-methyl-5-thio-â-D-ribofuranosyl)-3,6,7,8-
tetrahydroimidazo[4,5-d][1,3]diazepine (13). Tetrabutylammonium
fluoride (2 mL, 1 M in THF) was added to a solution of 12 (0.25 g,
0.408 mmol) and acetic acid (0.05 mL) in THF (2 mL), and the solution
was allowed to stand for 2 days. The solution was evaporated, and
chromatography (5% MeOH in EtOAc v/v) afforded 8-(R)-3-(2,3-di-
O-acetyl-5-S-methyl-5-thio-â-D-ribofuranosyl)-6-tert-butoxycarbonyl-
8-hydroxy-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepine as a foam
1
(0.143 g, 0.287 mmol, 70%). H NMR: (CDCl3) δ 7.86 (1H, s), 7.68
(1H, s), 6.06 (1H, d, J ) 5.2 Hz), 5.78 (1H, t, J ) 5.4 Hz), 5.48 (1H,
t, J ) 5.4 Hz), 4.98 (1H, br s), 4.35 (1H, dd, J ) 10.4, 5.2 Hz), 3.94-
3.75 (2H, m), 2.94-2.82 (2H, m), 2.16, 2.11, 2.09 (3H each, s), 1.55
(9H, s). 13C NMR: δ 170.0, 169.7, 152.7, 143.3, 133.6, 86.4, 84.4,
82.1, 73.9, 72.7, 65.7, 46.7, 36.9, 28.5, 20.9, 20.8, 17.4. Sodium
thiomethoxide (0.20 g, 2.85 mmol) was added to a stirred solution of
this material (0.14 g, 0.28 mmol) in methanol (3 mL). After 4 h, the
solution was concentrated, and chromatography of the residue gave 13
1
as a white solid (0.062 g, 0.197 mmol, 70%). H NMR: (CD3OD) δ
8.00 (1H, s), 7.16 (1H, s), 5.93 (1H, d, J ) 4.3 Hz), 5.01, (1H, dd, J
) 4.5, 2.2 Hz), 4.43 (1H, t, J ) 4.5 Hz), 4.23-4.16 (2H, m), 3.46-
3.34 (2H, m), 2.94-2.79 (2H, m), 2.15 (3H, s). 13C NMR: δ 150.9,
137.5, 131.5, 128.4, 90.2, 85.3, 76.4, 74.1, 67.7, 37.9, 17.1. HRMS:
(MH+) calcd for C12H18N4O4S, 315.1127; found, 315.1130.
(21) Chan, E.; Putt, S. R.; Showalter, H. D. H.; Baker, D. C. J. Org. Chem.
1982, 47, 3457-3464.
(22) Hawkins, L. D.; Hanvey, J. C.; Boyd, F. L., Jr.; Baker, D. C.; Showalter,
H. D. H. Nucleosides Nucleotides 1983, 2, 479-494.
(23) Jeanette, T. H.; Riordan, J. M.; Montgomery, J. A. Nucleosides Nucleotides
1986, 5, 431-439.
9
6876 J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007

Specific Inhibitors of Malarial Adenosine Deaminase
A R T I C L E S
Table 2. Kinetic Constants for HsADA, BtADA, and PfADA with
Adenosine and 5′-Methylthioadenosine as Substratesa
adenosine
5′-methylthioadenosine
kcat
KM
k
cat/KM
kcat
KM
M)
kcat/KM
1
1
1
1
organism
(s-
)
(
µM)
(M-1 s-
)
(s-
)
(
µ
(M-1 s-
)
B. taurus
H. sapiens
P. falciparum 1.8
65
16
1.1 × 106 <0.02 NAb
NA
NA
36.3 ( 1.3 22 ( 3 1.6 × 106 <0.02 NA
29
6.2 × 105 15
170 9.6 × 104
a H. sapiens values were determined here; B. taurus and P. falciparum
values and associated errors reported in references 13 and 15. b Not
applicable because no significant reaction rate is observed.
4. Selective tosylation at the primary hydroxy group afforded
tosylate 5 in moderate yield (60%). Desilylation28 then gave
diol 6, the treatment of which with sodium thiomethoxide
simultaneously displaced the tosylate and, somewhat sur-
prisingly, cleaved the Boc carbamate to provide methylthio
ether 7 in 73% yield from compound 6. The propylthio ana-
logue 8 was prepared in the same fashion in a similar yield
(68%). However, when sodium thiophenoxide was used, the
tosylate was displaced, but the Boc group remained unaffected.
The addition of sodium thiomethoxide to this reaction mix-
ture removed the Boc group and provided the phenylthio
compound 9, which was isolated in 90% yield in a one-pot
procedure.
The successful synthesis of the â-nucleoside derivative 3
above depends on the use of crystalline 2-deoxy-R-ribosyl
chloride 1 so that essentially a single anomer of ribosyl chloride
is available in solution for the SN2 reaction. There are no readily
available crystalline R-ribosyl halides available, so the 5′-
methylthiocoformycin derivative was prepared using a ribosyl
donor with a participating group at O-2 to control the anomeric
stereochemistry. We found it convenient to use a 5-methylthio-
ribose donor and after some experimentation settled on a
trichloroacetimidate donor as the most effective. Thus, D-ribose
was converted to the allyl 5-S-methylribofuranosides 10 (Scheme
2) in 40% overall yield. Deallylation and formation of the
trichloroacetimidate donor 11 were followed directly by gly-
cosylation of 2b in the presence of TmsOTf to give the protected
coformycin derivative 12 (54%). The stereochemistry of 12 was
confirmed by NMR NOESY experiments. Desilylation with
Bu4NF was complicated by degradation, but when buffered with
HOAc gave the desired alcohol in 70% yield. Since we have
found that sodium thiomethoxide will remove the Boc carbamate
from diazepines such as 12, it was convenient to use these
conditions (NaSMe, MeOH) to effect deacetylation and the Boc
removal to give 5′-methylthiocoformycin 13 in 70% yield.
Inhibition of Adenosine Deaminase by Coformycin Ana-
logues. The inhibition of human (HsADA), bovine (BtADA),
and malarial (PfADA) adenosine deaminases was examined
with the coformycins described above (Table 1, Figure 2).
Both coformycin and 2′-deoxycoformycin exhibit slow onset
inhibition of all three enzymes with the dissociation con-
stants ranging from 27 to 110 pM. None of the 5′-functional-
ized analogues inhibited the bovine or human enzymes, whereas
they all inhibited PfADA. The 5′-methylthio analogues were
the most effective inhibitors of PfADA, with slow onset
inhibition and dissociation constants in the range of 430-730
pM. The propylthio- and phenylthio-analogues also inhibited
Figure 2. Dissociation constants (Kd) for inhibition of ADAs from bovine,
human, and P. falciparum sources.
desired 8-(R)-diazepine specifically. Synthesis of the chiral
aglycone 2b has been reported,24 and we have developed an
improved synthesis of this material, the details of which will
be published elsewhere. The aglycone was conveniently stored
as the N-protected 2a, as 2b does not survive storage for even
a few days.
There have been no reports of the successful glycosylation
of 2b, but we found that a number of 5′-substituted thio-2′-
deoxycoformycin derivatives (7, 8, and 9) were accessible by
using 2-deoxy-di-O-toluoylribosyl chloride 1 as the glycosy-
lating agent, which was made from 2-deoxy-D-ribose (Scheme
1). The aglycone 2a was deprotected at N-3 with potassium
tert-butoxide in THF to give free amine 2b. This was depro-
tonated by treatment with sodium hydride followed by coupling
with the R-glycosyl chloride 1 under SN2 conditions.25-27 That
the isolated anomerically pure nucleoside analogue 3 was the
â-anomer depicted was confirmed by NOESY NMR experi-
ments on compounds 7, 8, and 9, which showed interactions
between H-1′ T H-4′, H-3′ T H-2, and H-5′ T H-2. This
product was deacylated with ammonia in methanol to give diol
(24) Van Truong, T.; Rapoport, H. J. Org. Chem. 1993, 58, 6090-6096.
(25) Kayzimierczuk, Z.; Binding, U.; Seela, F. HelV. Chim. Acta 1989, 72, 1527-
1536.
(26) Kayzimierczuk, Z.; Cotton, H. B.; Revankar, G. R.; Robins, R. K. J. Am.
Chem. Soc. 1984, 106, 6379-6382.
(27) Kondo, K. S. H.; Ishibashi, M.; Kobayashi, J. Tetrahedron 1992, 48, 7145-
7148.
(28) Zhang, W. R. M. J. Tetrahedron Lett. 1992, 33, 1177-1180.
9
J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007 6877

A R T I C L E S
Tyler et al.
Figure 3. Amino acid sequence alignment for adenosine deaminases from human, bovine, mouse and two plasmodium sources. Residues highlighted in
gray are completely conserved in sequence, those in light and dark blue are similar.31 Residues located in the active site (defined on the basis of structural
analysis from a superposition of the mouse and P. yoelii structures, PDB ID 1ADD and 2AMX, respectively, and being located within 10 Å of bound
1-deazaadenosine) are indicated in red. Those active site residues within 5 Å of the 5′-hydroxyl of 1-deazaadenosine, where identity is not conserved
between the mammalian and Plasmodium sequences, are highlighted in yellow. The insertion in the mammalian enzymes corresponding to residues 109-
119 (MmADA) causes an alteration in the structural orientation of residues 101-127, relative to the malarial enzyme, which is indicated by a black bar.
PfADA, but with no detectible slow onset phase, and these
compounds gave dissociation constants29 of 12 and 61 nM,
respectively.
conservation, especially of active site residues (Figure 3).30
An exception is in the amino acids that are near the 5′-position,
where residues Cys153 and Met155 in MmADA structurally
and sequentially align with residues Ile171 and Asp173 in
PyADA. Additionally, Tyr129 of PyADA, whose identity is
maintained with respect to the mammalian homologue (corre-
sponds to Tyr102 in MmADA), is 1 Å removed from the active
site as compared to Tyr102 (corresponding to distances of 5.6
and 4.7 Å, respectively; data not shown). These differences
cause an increased pocket size for the 5′-group in PfADA, which
The substrate specificity of the ADA enzymes with adenosine
and 5′-methylthioadenosine as reactants reveals that the mam-
malian enzymes, although more proficient at utilization of
adenosine than PfADA, are unable to utilize 5′-methylthioad-
enosine (Table 2). Comparison of the amino acid sequences of
PfADA, BtADA, and HsADA with the structurally charac-
terized ADAs from Mus musculus (MmADA) and Plas-
modium yoelii (PyADA) reveals a high level of sequence
(30) The PDB ID’s for the structurally characterized ADAs from Mus musculus
and Plasmodium yoelii are 1ADD and 2AMX, respectively.
(31) Sequences were aligned with the CLUSTALW algorithm using the Gonnet
series for amino acid alignment.
(29) Dissociation constants are defined as the final equilibrium dissociation
constant, which corresponds to Ki*, or Ki if no slow-onset inhibition is
observed.
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6878 J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007

Specific Inhibitors of Malarial Adenosine Deaminase
A R T I C L E S
is likely to be the structural change that allows for the binding
and deamination of the 5′-functionalized compounds.
and bovine enzymes. The ADA isozyme specificity highlights
the importance of examining substrate specificity in addition
to transition state features in the design of species-specific
inhibitors.
Conclusions
The substrate specificity differences between mammalian and
malarial ADAs have been exploited for the generation of
inhibitors specific for ADA from P. falciparum. The 5′-
methylthio group results in a 5-fold difference in the binding
affinity of coformycin and 5′-methylthiocoformycin to PfADA.
However, this same change causes a >20 000-fold prefer-
ence in inhibitor binding to PfADA as compared to the human
Acknowledgment. This work was supported by research grant
AI49512 from the National Institutes of Health and contract
C08X0209 from the New Zealand Foundation for Research
Science and Technology.
JA0708363
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J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007 6879