Adenosine analogues as inhibitors of Trypanosoma brucei phosphoglycerate kinase: Elucidation of a novel binding mode for a 2-Amino-N6-substituted adenosine

Article abstract of DOI:10.1021/jm000287a

As part of a project aimed at structure-based design of adenosine analogues as drugs against African trypanosomiasis, N⁶-, 2-amino-N⁶-, and N²-substituted adenosine analogues were synthesized and tested to establish structure - activity relationships for inhibiting Trypanosoma brucei glycosomal phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycerol-3-phosphate dehydrogenase (GPDH). Evaluation of X-ray structures of parasite PGK, GAPDH, and GPDH complexed with their adenosyl-bearing substrates led us to generate a series of adenosine analogues which would target all three enzymes simultaneously. There was a modest preference by PGK for N⁶-substituted analogues bearing the 2-amino group. The best compound in this series, 2-amino-N⁶-[2_-(p-hydroxyphenyl)ethyl]adenosine (46b), displayed a 23-fold improvement over adenosine with an IC₅₀ of 130 μM. 2-[[2_-(p-Hydroxyphenyl)ethyl]amino]adenosine (46c) was a weak inhibitor of T. brucei PGK with an IC₅₀ of 500 μM. To explore the potential of an additive effect that having the N⁶ and N² substitutions in one molecule might provide, the best ligands from the two series were incorporated into N⁶,N²-disubstituted adenosine analogues to yield N⁶-(2_-phenylethyl)-2-[(2_-phenylethyl)amino]adenosine (69) as a 30 μM inhibitor of T. brucei PGK which is 100-fold more potent than the adenosine template. In contrast, these series gave no compounds that inhibited parasitic GAPDH or GPDH more than 10-20% when tested at 1.0 mM. A 3.0 A? X-ray structure of a T. brucei PGK/46b complex revealed a binding mode in which the nucleoside analogue was flipped and the ribosyl moiety adopted a syn conformation as compared with the previously determined binding mode of ADP. Molecular docking experiments using QXP and SAS program suites reproduced this 'flipped and rotated' binding mode.

Full text of DOI:10.1021/jm000287a

J . Med. Chem. 2000, 43, 4135-4150
4135
Ad en osin e An a logu es a s In h ibitor s of Tr ypa n osom a br u cei P h osp h oglycer a te
Kin a se: Elu cid a tion of a Novel Bin d in g Mod e for a 2-Am in o-N6-Su bstitu ted
Ad en osin e
J erome C. Bressi, J ungwoo Choe,† Melinda T. Hough,† Frederick S. Buckner,| Wesley C. Van Voorhis,|
,†
Christophe L. M. J . Verlinde,§ Wim G. J . Hol,*,†,§,‡ and Michael H. Gelb*,
Departments of Chemistry, Biochemistry, Medicine, and Biological Structure, University of Washington, Seattle, Washington
98195, and Biomolecular Structure Center and Howard Hughes Medical Institute, Seattle, Washington 98195
Received J une 29, 2000
As part of a project aimed at structure-based design of adenosine analogues as drugs against
African trypanosomiasis, N6-, 2-amino-N6-, and N2-substituted adenosine analogues were
synthesized and tested to establish structure-activity relationships for inhibiting Trypanosoma
brucei glycosomal phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), and glycerol-3-phosphate dehydrogenase (GPDH). Evaluation of X-ray structures
of parasite PGK, GAPDH, and GPDH complexed with their adenosyl-bearing substrates led
us to generate a series of adenosine analogues which would target all three enzymes
simultaneously. There was a modest preference by PGK for N6-substituted analogues bearing
the 2-amino group. The best compound in this series, 2-amino-N6-[2′′-(p-hydroxyphenyl)ethyl]-
adenosine (46b), displayed a 23-fold improvement over adenosine with an IC50 of 130 µM. 2-[[2′′-
(p-Hydroxyphenyl)ethyl]amino]adenosine (46c) was a weak inhibitor of T. brucei PGK with
an IC50 of 500 µM. To explore the potential of an additive effect that having the N6 and N2
substitutions in one molecule might provide, the best ligands from the two series were
incorporated into N6,N2-disubstituted adenosine analogues to yield N6-(2′′-phenylethyl)-2-[(2′′-
phenylethyl)amino]adenosine (69) as a 30 µM inhibitor of T. brucei PGK which is 100-fold
more potent than the adenosine template. In contrast, these series gave no compounds that
inhibited parasitic GAPDH or GPDH more than 10-20% when tested at 1.0 mM. A 3.0 Å X-ray
structure of a T. brucei PGK/46b complex revealed a binding mode in which the nucleoside
analogue was flipped and the ribosyl moiety adopted a syn conformation as compared with the
previously determined binding mode of ADP. Molecular docking experiments using QXP and
SAS program suites reproduced this “flipped and rotated” binding mode.
In tr od u ction
In an effort to develop safer and more efficacious
therapeutic agents for the treatment of African trypa-
nosomiasis, we are focusing our efforts on trypanosome
glycolysis as a target for structure-based drug design.
It has rigorously been shown that upon going from
insect to bloodstream form, T. brucei becomes solely
dependent on glycolysis to the stage of pyruvate for its
energy production.5-7 As has been demonstrated by
Clarkson and Brohn, disrupting carbohydrate catabo-
lism in bloodstream form T. brucei significantly stifles
parasite proliferation.8 In computro modeling of parasite
glycolytic flux suggests that, unlike in the mammalian
pathway, glycosomal phosphoglycerate kinase (PGK),
glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
and glycerol-3-phosphate dehydrogenase (GPDH) all
exert a significant amount of control over flux. At [I]/KI
) 10-100, competitive inhibitors of these enzymes may
reduce glycolytic flux to zero.9-11 Because of this unique
influence on metabolic flux, it is surmised that inhibition
of these particular enzymes may add a degree of
selectivity for disrupting the parasite pathway over the
mammalian one. Further selectivity will be sought by
the design of compounds with greater affinity for the
parasite over the human enzyme.
African trypanosomiasis is a devastating parasitic
disease that affects more than 300 000 people of sub-
Saharan Africa each year.1 The causative agent of this
affliction is the protozoan Trypanosoma brucei. Upon
taking a blood meal, T. brucei is introduced to the
mammalian host via the salivary glands of the tsetse
fly vector. T. brucei flourishes in the mammalian
bloodstream, evading the immune response via anti-
genic variation, until it eventually finds its way across
the blood-brain barrier and attacks the central nervous
system.2 This parasitic assault leads to dementia,
epileptic attacks, coma, and eventually, if left untreated,
death. Prevalent chemotherapeutic agents include
Suramin, Pentamidine, Melarsoprol, and difluorom-
ethylornithine (DFMO). All of these drugs have draw-
backs such as limitations in use to the period before
central nervous system involvement or to only one
subspecies of the parasite, resistance, toxicity, or
parenteral administration.3,4
* To whom correspondence should be addressed. For M.H.G.: tel,
206-543-7142; fax, 206-685-8665; e-mail, gelb@chem.washington.edu.
Department of Chemistry.
† Department of Biochemistry.
| Department of Medicine.
§ Department of Biological Structure.
There is a wealth of crystallographic and sequence
data on parasite and mammalian PGK,12-15 GAPDH,16-19
‡ Biomolecular Structure Center and Howard Hughes Medical
Institute.
10.1021/jm000287a CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/12/2000

4136 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Bressi et al.
Sch em e 1a
lectivity of the initial amination of 6-chloro-2-fluoropur-
ine 9-(2′,3′,5′-triacetyl)riboside or involved C2 bromination
of 2′,3′,5′-triacetylguanosine (60) followed by C2 ami-
nation, C6 chlorination, and C6 amination with subse-
quent deprotection of the hydroxyls.27 We found that
N6,N2-disubstituted adenosines could be readily syn-
thesized from either 6-chloro-2-iodopurine 9-(2′,3′,5′-tri-
O-acetyl)riboside (62a ) or 6-chloro-2-iodopurine 9-ribo-
side (62b) by taking advantage of the well-documented
nucleophilic lability of the chloride of 62a in the
synthesis of 1c′ (Scheme 2).28 Treatment of either 62a
or 62b with 1.1 equiv of the amine to be substituted at
the 6-position, a cobase, and heating at 60 °C afforded
intermediates 63a -68a or 63b-68b, respectively, while
in situ treatment of these compounds with a 20-fold
excess of the amine to be substituted at the 2-position
and heating at 125 °C yielded the desired disubstituted
products 69-82. Amination of 62a invariably led to
some degree of deacetylation of the desired intermedi-
ates 63a -68a as determined by electrospray ionization
mass spectrometry (ESI-MS). Amination of 62b led to
a relatively clean intermediate for which subsequent
amination of 63b-68b gave the desired products in 10-
20% greater yields than those obtained with 62a as the
starting material.
a
Conditions: (a) R1NH2 and Et3N in EtOH at 60 °C.
and GPDH20 to use for structure-based design of com-
petitive and parasite selective inhibitors. The majority
of these structures contain the adenosyl-bearing cofactor
bound in the active site, and thus indirectly we know
the binding mode of the adenosyl moiety in all three of
our targets. With this information, we chose to use
adenosine as our template for inhibitor design. Work
in our group has already had some success with
generating N6,2′-disubstituted adenosine analogues as
inhibitors of parasitic GAPDH,21,22 and it seemed rea-
sonable to pursue a template which could be applied to
all three targets simultaneously. Additionally, all three
enzymes have common areas from which modifications
to adenosine could extend. For example, PGK, GAPDH,
and GPDH all have regions adjacent to the N6, C2′, and
C5′ positions of the adenosyl moiety that could accom-
modate modifications to the template.
We present a library of monosubstituted N6 and N2
adenosine analogues generated via solution-phase par-
allel synthesis. The binding affinity of these compounds
for parasite PGK, GAPDH, and GPDH was determined,
and the data led us to generate a small set of N6,N2-
disubstituted adenosine analogues targeted at T. brucei
PGK. It was clear, from analysis of the canonical
binding mode of ADP to the kinase, that our most potent
PGK inhibitors could not possibly adopt a binding mode
similar to the adenosyl template of the ADP substrate.
However, we obtained a 3.0 Å X-ray structure of a PGK/
2-amino-N6-substituted adenosine analogue complex
that revealed a novel binding mode. This structure was
used to test a number of molecular docking programs
to see if they could reproduce the binding mode that was
observed experimentally.
Intermediate 62a was synthesized as described by
Matsuda et al. starting from commercially available
2′,3′,5′-triacetylguanosine (60).29 We have shown that
direct diazotization-iodination of 1b′ gives the inter-
mediate 6-chloro-2-iodopurine 9-riboside (62b). The
yield is not as robust as that obtained in the route
starting with 60, but this shorter synthetic route to the
desired N6,N2-disubstituted compounds avoids the use
of toxic and corrosive POCl3. Ammoniaolysis of both
62a 29 and 62b affords 1c′ (Scheme 2).
Compounds 16a -20a and 16b-20b could also be
synthesized by employing diaminoalkyltrityl resins.
Coupling of 1a ′ or 1b′ to these resins followed by
cleavage of the alkylamino group with 30% hexafluoro-
30
2-propanol (HFIP) in CH2Cl2 yielded the desired
nucleosides in greater than 95% purity as determined
by HPLC and in greater than 90% yield (Scheme 3).
This route avoids the need for multiple crystallizations31
or HPLC purification of these polar compounds when
synthesized via Scheme 1. Alkyl-bridged bisadenosines
(16a ′-20a ′ and 16b′-20b′) were either synthesized as
described by Agathocleous et al.32 or isolated as impuri-
ties generated from the synthesis of compounds 16a -
20a and 16b-20b via Scheme 1.
Str u ctu r e-Activity Rela tion sh ip Ser ies. T. brucei
PGK, GAPDH, and GPDH have regions near the N6
position of the adenine ring of their bound adenosyl
containing substrates for which modifications to the
adenosine template could extend. For PGK, this small
hydrophilic area is filled with three conserved water
molecules. The large hydrophobic N6 region of GAPDH
has been previously described.21,22 The N6 region of
GPDH points toward the protein surface/solvent inter-
face and appears to be able to accommodate either
hydrophobic or hydrophilic substituents depending on
their orientation. The C6 amino group of the adenine
ring makes critical hydrogen bonds in both the PGK and
GAPDH structures, while it interacts with solvent in
the GPDH structure. Because of our success with N6
Resu lts a n d Discu ssion
Ch em istr y. N6-Substituted adenosines 2a -59a and
2-amino-N6-substituted adenosines 2b-59b were syn-
thesized via solution-phase parallel synthesis in one-
step conversion from the commercially available 6-chlo-
ropurine riboside (1a ′) and 2-amino-6-chloropurine
riboside (1b′), respectively, by nucleophilic aromatic
substitution with the appropriate amine at 60 °C
(Scheme 1).23,24 In a similar fashion, N2-substituted
adenosines 2c-59c were prepared from 2-iodoadenosine
(1c′) at 125 °C (Scheme 2).25 Alternatively, compounds
2c-59c could be synthesized as described by Francis
et al. by using the commercially available 2-chloroad-
enosine.26 We preferred not to use this route as we found
it a bit more cost-effective to use 1c′.
Previous attempts at synthesizing N6,N2-disubstitut-
ed adenosines by Trivedi et al. resulted in poor regiose-

Adenosine Analogues as Inhibitors of T. brucei PGK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4137
Sch em e 2a
a
Conditions: (a) N,N-dimethylaniline, POCl3, Et4NCl in ACN at 100 °C; (b) CuI, CH2I2, I2 and isoamyl nitrite in THF at 80 °C; (c) NH3
in MeOH at 60 °C; (d/f) R2NH2 in MeOCH2CH2OH at 125 °C; (e) R1NH2 and Et3N in MeOCH2CH2OH at 60 °C.
Sch em e 3a
a
Conditions: (a) trityl chloride resins, 1a or 1b in EtOH/CH2Cl2 (1:1) at 60 °C; (b) 30% CF3CH(OH)CF3 in CH2Cl2 at rt.
substituents for GAPDH and our desire to maintain the
hydrogen bonds in the PGK and GAPDH structures, we
chose to explore the attachment of substituents to the
N6 position. We attempted to pick a diverse group of
ligands for the structure-activity relationship series
(SAR) that would simultaneously accommodate all three
enzymes and, at the same time, incorporate materials
that were readily available commercially or from previ-
ous synthetic projects. Unfortunately, the screening of
our small SAR library elucidated no inhibition greater
than 10-20% against parasite GAPDH or GPDH, when
tested at 1.0 mM, with the exception of 24a , 29a , 30a ,
and 41a for which potency against GAPDH was previ-
ously determined.21
With respect to PGK, these compounds were also
fairly weak inhibitors. However, there was a general
trend for 2-amino-N6-substituted analogues to be more
active than the corresponding N6 analogues, which
lacked the C2 amino group (Table 1). Our initial SAR
led to the surprising result of 2-amino-N6-(2′′-phenyl-

4138 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Bressi et al.
Ta ble 1. Inhibition of T. brucei PGK by N6- and
Ta ble 2. Inhibition of T. brucei PGK by N6-Phenethyl-,
2-Amino-N6-Substituted Adenosine Analogues
2-Amino-N6-phenethyl-, and N2-Phenethyladenosine Analogues
IC50 (mM)
compd
R1 or R2
a
b
c
IC50 (mM)
1
2
-H
3.0
3.5
-CH3
inactivea inactive NSb
compd
R3
R4
R5
a
b
c
3
-CH2CH3
inactive
inactive
inactive
inactive
3.0
3.5
3.0
NS
1.5
37
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
H
H
H
H
H
H
H
H
inactivea 0.2
0.7
4
-CH2CH2CH3
-CH(CH3)2
CH3
H
OH
OH
H
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
2.0
inactive
5
inactive NS
OH
H
OH
H
Cl
0.25
0.13
0.25
1.9
NSb
6
-CH2(CH2)2CH3
-CH2(CH2)3CH3
-CH2CHCH2
0.9
2.0
3.0
1.5
1.5
0.5
7
inactive
NS
NS
8
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
2.5
Cl
H
2.2
9
-CH2CCH
-CH2CH2OH
NS
H
1.2
1.4
inactive
inactive
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
inactive inactive
inactive inactive
inactive inactive
inactive inactive
inactive inactive
inactive inactive
inactive inactive
H
H
Cl
Cl
H
-CH2(CH2)2OH
-CH2CH(OH)CH3
-CH2CH(OH)CH2OH
-CH2(CH2)3OH
-CH2(CH2)4OH
-CH2CH2NH2
-CH2(CH2)2NH2
Cl
OCH3
H
H
inactive 0.6
H
1.8
1.9
1.5
inactive
inactive
inactive
OCH3
H
H
H
OCH3 inactive
H
OCH3 OCH3 inactive
OCH3 OH inactive
OCH3 inactive
inactive inactive
1.5
0.85
0.25
inactive NS
H
NS
NS
0.5
inactive
4.0
inactive
H
OH
H
-CH2CH(OH)CH2NH2 inactive
inactive inactive
inactive inactive
inactive inactive
H
NH2
NO2
inactive
inactive
-CH2(CH2)3NH2
-CH2(CH2)4NH2
-cyclopentyl
inactive
inactive
3.9
inactive
inactive
3.9
inactive
inactive
1.0
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
H
H
1.5
1.5
a
b
Inactive ) inactive at 4 mM. NS ) not synthesized.
-cyclohexyl
inactive 1.5
-CH2-cyclohexyl
-cycloheptyl
2.5
2.5
inactive
3.0
Both 37b and 46b were 10-fold more selective for
parasite PGK over rabbit muscle PGK (mammalian
control).
-tetrahydrofurfuryl
-furfuryl
inactive 0.8
1.5
0.6
2.9
1.2
3.5
ns
-CH2C6H5
The preference, by PGK, for the C2 NH2 led us to
explore N2 adenosine analogues. Inspection of the avail-
able T. brucei PGK binary and ternary structures
suggested that substitutions in this area would be well-
tolerated by PGK. GPDH also has room to accommodate
substituents in this position; however, GAPDH does not
due to the snug fit of the adenosyl moiety in this region.
Despite the modeling suggestions for GAPDH, the N2
SAR was screened against all three target enzymes.
None of the analogues inhibited T. brucei GAPDH or
GPDH more than 10-20% at 1.0 mM, and a weak
inhibitor, 2-[(2′′-phenylethyl)amino]adenosine (37c), with
an IC50 of 700 µM was found for PGK (Table 1). Again,
we pursued a small SAR subset of N2-phenethyl ana-
logues and found 2-[[2′′-(p-hydroxyphenyl)ethyl]amino]-
adenosine (46c) to be marginally better than the parent
analogue with an IC50 of 500 µM (Table 2). Both 37c
and 46c were competitive with ATP, noncompetitive
with 3-PGA (data not shown), and 4-fold more selective
for parasite PGK over rabbit muscle PGK.
Attachment of the phenethyl groups on the adenine
ring to give N6,N2-disubstituted adenosine analogues,
targeted toward T. brucei PGK, yielded N6-(2′′-phenyl-
ethyl)-2-[(2′′-phenylethyl)amino]adenosine (69) with an
IC50 of 30 µM (100-fold more potent than adenosine).
The addition of a p-hydroxyl group to just one of either
of the phenethyl substituents, N6-[2′′-(p-hydroxyphenyl)-
ethyl]-2-[(2′′-phenylethyl)amino]adenosine (72) and N6-
(2′′-phenylethyl)-2-[[2′′-(p-hydroxyphenyl)ethyl]amino]-
adenosine (73), decreased activity approximately 3-fold,
-CH(CH3)C6H5
-CH2-(o-CH3-C6H4)
-CH2-(m-CH3-C6H4)
-CH2-(p-CH3-C6H4)
-CH2-(o-Cl-C6H4)
-CH2-(m-Cl-C6H4)
-CH2-(p-Cl-C6H4)
-CH2-(o-OCH3-C6H4)
-CH2-(p-OCH3-C6H4)
-CH2CH2C6H5
inactive 1.5
0.4 1.5
inactive 1.5
inactive 2.0
inactive 0.8
inactive 1.4
inactive 1.4
inactive 1.0
0.2
2.9
0.7
3.5
-CH2CH(CH3)C6H5
-CH2CH(OH)C6H5
-CH2CH2CH2C6H5
-CH2-1-naphthalene
-CH(C6H5)CH2(C6H5) inactive
-CH2CH(C6H5)2 inactive
inactive 1.4
4.0
1.9
inactive NS
inactive NS
1.8
NS
a
b
Inactive ) inactive at 4 mM. NS ) not synthesized.
ethyl)adenosine (37b) inhibiting T. brucei PGK with an
IC50 of 200 µM. It was quite clear from analysis of the
PGK/ADP structure13 that the phenethyl substituent,
along with many of the other N6 ligands which showed
activity, was not only too large to fit into the small
pocket positioned adjacent to the N6 region but also not
well-suited to the hydrophilic nature of the cavity. Still,
we pursued a small SAR subset of phenethyl analogues
and found 2-amino-N6-[2′′-(p-hydroxyphenyl)ethyl]ad-
enosine (46b) to be marginally better than the parent
analogue, 37b, with an IC50 of 130 µM (Table 2). This
was a 23-fold improvement over the adenosine template
which has an IC50 of 3000 µM. Adenosine (1a ), 2-ami-
noadenosine (1b), 37b, and 46b were competitive with
ATP and noncompetitive with 3-PGA (data not shown).

Adenosine Analogues as Inhibitors of T. brucei PGK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4139
Ta ble 3. Inhibition of T. brucei PGK by
N6,N2-Diphenethyladenosine Analogues
Ta ble 4. Inhibition of T. brucei PGK by
N,N′-Alkanediylbisadenosine Analogues
compd
R1
R2
IC50 (µM)
IC50 (µM)
69
70
71
72
73
74
75
76
77
78
79
80
81
82
-CH2CH2C6H5
-CH2CH2C6H5
30
compd
R
a ′
b′
-CH2CH2-(p-OH-C6H4) -CH2CH2-(p-OH-C6H4) inactivea
-CH2CH2-(p-NH2-C6H4) -CH2CH2-(p-NH2-C6H4) inactive
16
17
18
19
20
-CH2CH2-
inactivea
inactive
inactive
inactive
inactive
500
-CH2CH2-(p-OH-C6H4) -CH2CH2C6H5
100
-CH2CH2CH2-
-CH2CH(OH)CH2-
-CH2(CH2)2CH2-
-CH2(CH2)3CH2-
50
-CH2CH2C6H5
-CH2CH2-(p-OH-C6H4) 100
inactive
750
1000
-CH2(CH2)2CH3
-CH2(CH2)2OH
-CH2-cyclohexyl
-CH2C6H5
-CH2(CH2)2OH
-CH2-cyclohexyl
-CH2C6H5
-CH2(CH2)2OH
-CH2-cyclohexyl
-CH2C6H5
inactive
-CH2(CH2)2CH3
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
-CH2(CH2)2CH3
a
Inactive ) inactive at 1000 µM.
-tetrahydrofurfuryl
-tetrahydrofurfuryl
-tetrahydrofurfuryl
-CH2-1-naphthalene
-CH2-1-naphthalene
-CH2-1-naphthalene
adenine ring. The adenosine moiety of 46b is also
translated by approximately 1.5 Å in the adenine plane
along the C6/N1 vector of the ADP substrate. In addi-
tion, the ribosyl moiety is rotated by 180° along the N9
and C1′ bond such that it now adopts a syn orientation
to the adenine base rather than the anti orientation
found for the bound ADP (Figure 3). Analysis of all
nucleoside crystallographic data suggests that purine
nucleosides adopt both syn and anti forms with a
preference for the anti orientation.34 Inspection of the
41 purine nucleoside analogues in the Cambridge
database revealed that 14 out of 41 compounds adopt
the syn conformation suggesting that the free energy
difference between the two conformations is quite small.
a
Inactive ) inactive at 100 µM.
while the addition of a p-hydroxyl group to both of the
phenethyl substituents, N6-[2′′-(p-hydroxyphenyl)ethyl]-
2-[[2′′-(p-hydroxyphenyl)ethyl]amino]adenosine (70), led
to an inactive compound when tested at its solubility
limit of 100 µM. Compound 69 was competitive with
ATP, noncompetitive with 3-PGA (data not shown), and
completely selective for parasite PGK over rabbit muscle
PGK when tested up to its solubility limit of 100 µM.
As the substituents we chose to incorporate into our
analogues were both similar and identical in structure
(i.e. phenethyl), we generated a 3 × 3 matrix of
randomly selected ligands and synthesized the respec-
tive compounds, 74-82, to further demonstrate the
utility of the chemistry. These compounds were inactive
against T. brucei PGK when tested at 100 µM.
As an interesting aside, synthesis of compounds 16a -
20a and 16b-20b via Scheme 1 invariably led to N6,N6′-
alkanediylbisadenosines (16a ′-20a ′ and 16b′-20b′) for
which 16a ′, 17a ′, 19a ′, and 20a ′ are known inhibitors
of adenosine kinase.33 We isolated/synthesized the
bisadenosines and screened them against parasite PGK
to find that most of the C2 amino-bearing compounds
had some marginal activity and N6,N6′-1,3-propanediyl-
bis-2-aminoadenosine (17b′) had an IC50 of 50 µM (Table
4). Compound 17b′ was competitive with ATP, noncom-
petitive with 3-PGA (data not shown), and not selective
for T. brucei PGK over rabbit muscle PGK.
Str u ctu r e of th e T. br u cei P GK/2-Am in o-N6-[2′′-
(p-h yd r oxyp h en yl)eth yl]a d en osin e (46b) Com p lex.
Attempts at obtaining X-ray crystal structures of T.
brucei PGK complexed with 46c or 69 were unsuccess-
ful; however, we were able to obtain an X-ray crystal
structure of a T. brucei PGK/2-amino-N6-[2′′-(p-hydrox-
yphenyl)ethyl]adenosine (46b) complex (Figures 1 and
2). Quite remarkably, compound 46b sits in the C-
terminal ADP binding region such that the purine ring
is flipped 180°, compared to bound ADP, along an axis
that is approximately defined by N1 and N9 of the
The adenine ring of ADP, in T. brucei PGK, sits in a
narrow hydrophobic cleft flanked by side chains Ala-
218, Ala-242, and the methylenes of Lys-259 on one side
and Val-343, Leu-315, and Phe-294 on the other (Figure
1). Adenine occupies part of the cleft leaving vacant
space between Phe-294 and Lys-259. In the PGK/46b
complex, the adenine base has been translated to sit
between Phe-294 and Lys-259. In this “flipped and
rotated” binding mode, the C2′ and C3′ hydroxyls of the
ribose form hydrogen bonds with the same protein
residue, Glu-345, as the respective hydroxyls of the ADP
ribose. For the PGK/substrate complex, the C5′ oxygen
does not participate in hydrogen bonding, and modeling
suggests that, in the absence of phosphate, the proton
of the hydroxyl would not be engaged in a hydrogen-
bonding interaction with the protein. In contrast, as a
result of the flipping and rotation of the sugar, the C5′
hydroxyl of 46b participates in a hydrogen bond with
the backbone amide of Ala-242.
The N2 position of the inhibitor’s adenine ring oc-
cupies approximately the N6 of ADP (Figures 1 and 3),
with the respective amino group in both molecules
making a hydrogen bond with the backbone carbonyl
of Ala-314. This, in part, explains the importance of the
C2 amino group in the series of N6-monosubstituted
compounds. The conserved water molecules, observed
in numerous PGK structures, were not observed in the
structure of the 46b complex due to the lower resolution
of the latter structure. However, it is surmised that the

4140 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Bressi et al.
F igu r e 1. X-ray crystal structure of T. brucei PGK (a) complexed with compound 46b and depicted with a molecular surface
colored by electrostatic potential; (b) complexed with the adenosine moiety of ADP (from 2.5 Å structure) and depicted with a
molecular surface colored by electrostatic potential; (c) with stereoview of the adenosine binding region shown with the two binding
modes of 46b (dark and light purple) and adenosine. Residues are colored blue if they are conserved between human and T.
brucei enzymes and yellow if they are different (residues in parentheses correspond to human enzyme). Hydrogen bonds are
drawn as dashed line. Generated using MOLSCRIPT,49 GRASP,50 and Raster3D.51
hydrogen bond that is observed in the ADP structure,
between N6 and one of the conserved waters, can also
be made by the C2 amino group with minor rearrange-
ment of a water molecule. In this binding mode, the C6
amino of 46b is now directed almost precisely where
the C2 proton of ADP is directed (Figure 3).
Without a structure of the PGK/46c complex, it is
difficult to comment on the binding mode of this
compound that bears the same substituent as 46b.
Molecular modeling studies suggest that 46c could
indeed sit in the ADP active site in such a fashion as to
have the adenosyl moieties superimposed and such that
the p-hydroxyphenethyl substituent sits in the same
region as that of 46b in the PGK/46b complex. The
modest selectivity experienced by 46b for T. brucei PGK
may be accounted for by Lys-259 being replaced by a
leucine in the mammalian enzyme. Although this
structure has provided insight into how the 2-amino-
N6-substituted adenosine analogue binds, it is unlikely
that compound 69 could bind in a similar fashion as that
of 46b or ADP since the N2-phenethyl group has merely
exchanged places with the N6-phenethyl group for which
there is still not enough room to be accommodated.
Dock in g Exp er im en ts. Compound 46b was docked
by SAS35 (Stochastic Approximation with Smoothing)
and QXP36 in an effort to see if these programs could
reproduce the crystallographic binding mode. Although
both programs successfully found the “flipped and
rotated” binding mode of 46b when a 1.6 Å PGK crystal
structure14 was used (data not shown), only the program
SAS gave acceptable results when the 3.0 Å T. brucei
PGK structure was used.
Both protein molecules in the asymmetric unit reveal
bound inhibitor. Compound 46b exhibits stronger elec-
tron density in PGK subunit A than in subunit B, which
may be accounted for by different crystal packing
environments of the inhibitor (Figure 2). The density
of the hydroxyphenethyl group of the inhibitor in
molecule A has branches in two directions suggesting
alternative binding modes. In one binding mode, the
phenyl ring leans on the hydrophobic protein surface
formed by Phe-294 and Met-313. In the second, the ring
makes hydrophobic interactions with the methylene
chain of Lys-259 and the backbone of Ser-258. In both
cases, the N6 proton and the hydroxyl of the phenethyl
group are exposed to solvent and do not appear to
contribute to hydrogen bonding with the protein. We
considered synthesizing a molecule that could simulta-
neously take advantage of the two orientations of the
phenethyl substituent; however, it was clear from
modeling experiments that any compound that could
maintain the distinct phenethyl orientations would
suffer from severe intramolecular clashes between the
phenyl rings.
For the docking experiment using the SAS program,
50 runs of 1000 cycles were performed to dock 46b to

Adenosine Analogues as Inhibitors of T. brucei PGK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4141
F igu r e 2. Stereoview of simulated annealing omit map (2Fo - Fc, 1.0σ) around 46b in (a) subunit A with the two binding modes
of the p-hydroxyphenethyl substituents and (b) subunit B. Prepared using the programs XtalView52 and Raster3D.51
Ta ble 5. Statistics from Docking Experiments of 46b into the
3.0 Å T. brucei PGK Structure with the Program SAS35
rmsdc (Å)
min/max
cluster
rank
scorea
size of compared compared adensoine
clusterb to conf A to conf B moiety only
(kcal/mol)
1
2
3
4
5
6
7
8
-61.9/-58.9
-61.4/-56.8
-60.7/-59.5
-60.7/-57.5
-60.5/-60.4
-60.2/-60.2
-59.8/-59.8
-59.7/-58.0
-59.2/-59.2
-58.6/-57.7
8
6
2
13
2
1
1
8
1
2
1.64
0.56
0.61
0.64
1.25
2.67
2.95
0.59
1.38
2.73
1.18
1.72
1.87
1.78
1.94
2.06
1.53
2.25
F igu r e 3. Superposition of the bound conformations of
9
10
1.70
1.52
adenosine (light gray, from PGK ternary complex structure13
)
and 46b (dark gray) in T. brucei PGK. N6 positions of both
molecules are marked for comparison. Arrows indicate the
rotation and translation of the purine moiety.
a
Min/max score ) minimum and maximum energy score (kcal/
b
mol) of the docked structures in the cluster. Size of cluster )
number of docked structures with rmsd’s within 1.0 Å of each
other. c rmsd ) rmsd’s (Å) of docked 46b compared with conforma-
tion A of 46b, conformation B of 46b, and with only the adenosine
moiety of the crystallographically determined orientation of 46b.
the 3.0 Å T. brucei PGK structure, and results with
rmsd’s smaller than 1.0 Å were clustered (Table 5). The
program found the flipped binding mode of adenosine
in all top 10 ranks, and the sugar moiety was rotated
with respect to the nucleoside base in all cases except
ranks 5 and 6. The program also successfully positioned
the p-hydroxyphenethyl substituent of 46b in the two
alternative binding modes observed crystallographically,
and the results were clustered in two categories that
represented the two observed orientations of the sub-
stituent: “A-like” and “B-like” (Figure 4); rmsd com-
parison of the adenosine moiety of 46b, from the docking
experiment, showed that, in most cases, this portion of
the inhibitor had a smaller rmsd compared with that
of the complete 46b molecule from the PGK/46b struc-
ture. In the three top-ranked clusters (Table 5), the
agreement between the docked and the experimental
binding modes only varied between 0.50 and 0.64 Å.
Tr yp a n osom a tid Gr ow th In h ibition Stu d ies. Our
best inhibitor, 69, was tested for the ability to block the
growth of two strains of bloodstream T. brucei. The

4142 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Bressi et al.
F igu r e 4. Docking results of 46b to the PGK crystal structure using the program SAS: (a) binding modes of 46b observed in the
crystal structure; (b) superposition of conformation A with docking results (clusters 1, 4-6, 9, and 10, Table 5) that have “A-like”
conformations; (c) superposition of conformation B with docking results (clusters 2, 3, 7, and 8, Table 5) that have “B-like”
conformations.
Ta ble 6. Data Collection and Refinement Statistics for the T.
brucei PGK/46b Complex
Con clu sion s
Utilizing structure-based design methodologies, we
identified regions of our adenosine template for which
Data Collection
resolution (Å)
space group
3.0
modifications could be tolerated by our target enzymes.
The quasi-rational selection of ligands to be incorporated
into our molecules, based on diversity and availability,
led to our most potent compound, 69, which binds 100-
fold tighter to PGK than our adenosine template. A
crystallographic structure has revealed the detailed
interactions between 46b and T. brucei glycosomal PGK,
including an entirely different binding mode than previ-
ously seen for ADP, and has also provided a structural
rationale for the observed activities. This structure, used
in conjunction with the SAS program which successfully
reproduced the unexpected binding mode, has provided
a direction for future attempts to improve binding
affinity of this class of adenosine analogues.
P6322 (a ) b ) 186.6 Å, c ) 159.0 Å)
number of unique reflections 32896 (617652)a
completeness (%)
I/σ(I)
Rmerge (%)
98.9 (99.5)b
28.0 (5.6)b
7.0 (41.8)b
Refinement
number of protein atoms
number of ligand atoms
resolution (Å)
σ cut-off
number of reflections
Rcryst (%)
6254
58
15-3.0
F > 2σ(F)
31864
21.7
Rfree (%)
28.2c
rmsd from ideality:
bond length (Å)
angle (deg)
average B factor:
main chain (Å2)
side chain (Å2)
ligand (Å2)
0.020
1.89
29.7 (subunit A), 33.7 (subunit B)
36.4 (subunit A), 39.7 (subunit B)
35.0 (subunit A), 45.6 (subunit B)
Exp er im en ta l Section
Gen er a l Syn th etic P r oced u r es. 1H NMR spectra were
recorded on a Bruker AF-300 MHz or AM-500 MHz spectrom-
eter in DMSO-d6 unless otherwise stated. Electrospray ioniza-
a
b
Number of total reflections. Values in parentheses refer to
c
data in the last shell between 3.3 and 3.0 Å resolution. Rfree was
calculated for 5% of reflections omitted from the refinement.
tion 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 CHCl3/MeOH (80:20) as
eluent and visualized at 254 nm. Medium-pressure flash
column chromatography was carried out on silica gel 60 (230-
400 mesh; EM Science) with CHCl3/MeOH (98:2 to 80:20)
eluent. Preparative HPLC was performed on a C18 column
(Vydac 218TP1010) using one of the following elution gradi-
ents: method A, 100% 10 mM triethylammonium acetate (pH
) 7.0) for 5 min then linear gradient of 10 mM triethylam-
monium acetate (pH ) 7.0) from 0 to 15% over 60 min at 4.0
mL/min; method B, linear gradient of 5% HCOOH from 0 to
100% MeOH over 60 min at 4.0 mL/min; method C, linear
gradient of 10 mM triethylammonium acetate (pH ) 7.0) from
0 to 100% over 60 min at 4.0 mL/min. Purity of all compounds
concentration of inhibitor that caused 50% growth
inhibition of both T. brucei brucei and T. brucei rhod-
esiense was 20 µM. We also tested the activity of this
inhibitor against the trypanosome species that causes
Chagas disease, Trypanosoma cruzi. The concentration
of the inhibitor that caused 50% growth inhibition of
mammalian stages of T. cruzi grown in murine fibro-
blasts was also 20 µM. The concentration of the inhibitor
that inhibited 50% growth of the murine fibroblasts was
40 µM. All cell culture assays were performed twice with
very similar results.

Adenosine Analogues as Inhibitors of T. brucei PGK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4143
was verified by HPLC with methods B and C on a C18 column
(Vydac 218TP52) at 1.0 mL/min.
2-Am in o-N6-(4′′-h ydr oxybu tyl)aden osin e (14b): 1H NMR
δ 1.43 (p, 2H, C2”-H), 1.56 (p, 2H, C3′′-H), 3.38 (m, 4H, C1′′-H
and C3′′-H), 3.51 and 3.61 (2 × m, 2H, C5′-Ha,b), 3.89 (m, 1H,
C4′-H), 4.08 (dd, 1H, C3′-H), 4.40 (br s, 1H, C3”-OH), 4.47 (dd,
1H, C2′-H), 5.12 (d, 1H, OH), 5.38 (d, 1H, OH), 5.46 (br s, 1H,
OH), 5.70 (d, 1H, C1′-H), 5.83 (br s, 2H, C2-NH2), 7.30 (br s,
1H, N6-H), 7.91 (s, 1H, C8-H); ESI-MS m/z 355.1 (M + H+)+.
N6-(5′′-Hyd r oxyp en tyl)a d en osin e (15a ): 1H NMR δ 1.32
(p, 2H, C3′′-H), 1.43 (p, 2H, C2′′-H), 1.56 (p, 2H, C4′′-H), 3.38
(m, 4H, C1′′-H and C5′′-H), 3.53 and 3.64 (2 × m, 2H, C5′-
THF was freshly distilled over Na/benzophenone under
argon, and CH2Cl2 was freshly distilled over CaH under argon.
Adenosine (1a ), 6-chloropurine riboside (1a ′), 2-amino-6-chlo-
ropurine riboside (1b′), N6-benzyladenosine (27a ), and 2′,3′,5′-
triacetylguanosine (60) were purchased from Aldrich Chemical
Co. Ltd. N6-Methyladenosine (2a ) and N6-(2′′-hydroxyethyl)-
adenosine (10a ) were purchased from Sigma Chemical Co.
2-Aminoadenosine (1b) was purchased from R. I. Chemical.
3-Hydroxyphenethylamine hydrochloride, 3-hydroxy-4-meth-
oxyphenethylamine hydrochloride, and 4-hydroxy-3-methox-
yphenethylamine hydrochloride were purchased from Trans
World Chemical Inc. Trityl chloride resin (swelling volume in
CH2Cl2: 2-5 mL/g, loading: 0.95 mmol/g of active chloride,
polymer matrix: copoly(styrene-2% DVB), 200-400 mesh) was
purchased from Novabiochem. The diaminoalkyltrityl resins,
which were prepared for these experiments using standard
methods, can also be purchased through Novabiochem.
Gen er a l P r oced u r e for th e P r ep a r a tion of N6- a n d
2-Am in o-N6-Su bstitu ted Ad en osin e An a logu es (2a -59a
a n d 2b-59b). In parallel fashion and with minor modifica-
tions, the method of Fleysher was employed.23,24 To a suspen-
sion of 1a ′ or 1b′ (0.017 mmol) in EtOH (250 µL) were added
the appropriate amine or amine hydrochloride (0.05 mmol) and
then triethylamine (0.05 mmol for free amines or 0.10 mmol
for amine hydrochlorides). The reactions were heated at 60
°C for 18 h and then cooled to ambient temperature. The
reaction mixtures were purified via flash chromatography,
except for compounds 16a -20a and 16b-20b which were
purified via preparative HPLC using method A, to yield the
desired products in 75-95% yield.
H
a,b), 3.95 (m, 1H, C4′-H), 4.12 (dd, 1H, C3′-H), 4.36 (t, 1H,
C5′′-OH), 4.60 (dd, 1H, C2′-H), 5.20 (d, 1H, OH), 5.44 (m, 2H,
OH), 5.86 (d, 1H, C1′-H), 7.89 (br s, 1H, N6-H), 8.19 and 8.33
(2 × s, 2H, C2-H and C8-H); ESI-MS m/z 354.2 (M + H+)+.
2-Am in o-N6-(5′′-h yd r oxyp en t yl)a d en osin e (15b ): 1H
NMR δ 1.30 (p, 2H, C3′′-H), 1.43 (p, 2H, C2′′-H), 1.56 (p, 2H,
C4′′-H), 3.37 (m, 4H, C1′′-H and C5”-H), 3.50 and 3.61 (2 ×
m, 2H, C5′-Ha,b), 3.88 (m, 1H, C4′-H), 4.08 (dd, 1H, C3′-H),
4.36 (t, 1H, C5′′-OH), 4.48 (dd, 1H, C2′-H), 5.14 (d, 1H, OH),
5.39 (d, 1H, OH), 5.45 (br s, 1H, OH), 5.70 (d, 1H, C1′-H), 5.83
(br s, 2H, C2-NH2), 7.29 (br s, 1H, N6-H), 7.91 (s, 1H, C8-H);
ESI-MS m/z 369.2 (M + H+)+.
2-Am in o-N6-(cycloh exylm et h yl)a d en osin e (23b ): 1H
NMR δ 0.80-1.80 (band, 11H, cyclohexyl-H), 3.22 (br s, 2H,
N6-CH2), 3.52 and 3.63 (2 × m, 2H, C5′-Ha,b), 3.90 (m, 1H, C4′-
H), 4.08 (dd, 1H, C3′-H), 4.51 (dd, 1H, C2′-H), 5.10 (d, 1H, OH),
5.37 (d, 1H, OH), 5.43 (br s, 1H, OH), 5.70 (d, 1H, C1′-H), 5.74
(br s, 2H, C2-NH2), 7.28 (br s, 1H, N6-H), 7.89 (s, 1H, C8-H);
ESI-MS m/z 379.2 (M + H+)+.
2-Am in o-N6-cycloh ep tyla d en osin e (24b): 1H NMR (CD3-
OD) δ 1.53-2.01 (band, 12H, cycloheptyl-CH2), 3.71 and 3.87
(2 × dd, 2H, C5′-Ha,b), 4.13 (m, 1H, C4′-H), 4.28 (m, 2H, C3′-H
and C1”-H), 4.74 (dd, 1H, C2′-H), 5.76 (d, 1H, C1′-H), 7.83 (s,
1H, C8-H); ESI-MS m/z 379.3 (M + H+)+.
2-Am in o-N6-isop r op yla d en osin e (5b): 1H NMR δ 1.16 (d,
6H, C2′′-H), 3.52 and 3.61 (2 × m, 2H, C5′-Ha,b), 3.89 (m, 1H,
C4′-H), 4.07 (dd, 1H, C3′-H), 4.41 (br s, 1H, C1′′-H), 4.55 (dd,
1H, C2′-H), 5.10 (d, 1H, OH), 5.35 (d, 1H, OH), 5.47 (br s, 1H,
OH), 5.70 (d, 1H, C1′-H), 5.74 (br s, 2H, C2-NH2), 7.05 (br s,
1H, N6-H), 7.89 (s, 1H, C8-H); ESI-MS m/z 325.3 (M + H+)+.
2-Am in o-N6-(n -a m yl)a d en osin e (7b): 1H NMR (CD3OD)
δ 0.92 (t, 3H, C5′′-H), 1.37 (m, 4H, C3′′-H and C4”-H), 1.64
(m, 2H, C2′′-H), 3.48 (br s, 2H, C1′′-H), 3.75 and 3.87 (2 × dd,
2H, C5′-Ha,b), 4.14 (m, 1H, C4′-H), 4.28 (dd, 1H, C3′-H), 4.74
(dd, 1H, C2′-H), 5.77 (d, 1H, C1′-H), 7.83 (s, 1H, C8-H); ESI-
MS m/z 353.2 (M + H+)+.
2-Am in o-N6-tetr ah ydr ofu r fu r yladen osin e (25b): 1H NMR
δ 1.59-1.88 (band, 4H, C3′′- and C4′′-H), 3.41-3.80 (band, 6H,
N6-CH2, C5′-Ha,b, and C5′′-H), 3.88 (m, 1H, C4′-H), 4.02 (m,
1H, C2′′-H), 4.08 (dd, 1H, C3′-H), 4.47 (dd, 1H, C2′-H), 5.10
(d, 1H, OH), 5.36 (d, 1H, OH), 5.42 (br s, 1H, OH), 5.71 (d,
1H, C1′-H), 5.82 (br s, 2H, C2-NH2), 7.14 (br s, 1H, N6-H), 7.91
(s, 1H, C8-H); ESI-MS m/z 367.2 (M + H+)+.
2-Am in o-N6-(m -m eth ylben zyl)a d en osin e (30b): 1H NMR
δ 2.26 (s, 3H, Ph-CH3), 3.55 and 3.65 (2 × m, 2H, C5′-Ha,b),
3.90 (m, 1H, C4′-H), 4.09 (dd, 1H, C3′-H), 4.51 (dd, 1H, C2′-
H), 4.62 (br s, 2H, N6-CH2), 5.09 (d, 1H, OH), 5.34 (d, 1H, OH),
5.41 (br s, 1H, OH), 5.72 (d, 1H, C1′-H), 5.79 (br s, 2H, C2-
NH2), 6.70 (d, 1H, Ph-H), 7.15 (m, 3H, Ph-H), 7.82 (br s, 1H,
N6-H), 7.92 (s, 1H, C8-H); ESI-MS m/z 387.2 (M + H+)+.
2-Am in o-N6-(p-m eth ylben zyl)a d en osin e (31b): 1H NMR
δ 2.27 (s, 3H, Ph-CH3), 3.53 and 3.63 (2 × m, 2H, C5′-Ha,b),
3.90 (m, 1H, C4′-H), 4.11 (dd, 1H, C3′-H), 4.52 (dd, 1H, C2′-
H), 4.64 (br s, 2H, N6-CH2), 5.09 (d, 1H, OH), 5.34 (d, 1H, OH),
5.41 (br s, 1H, OH), 5.72 (d, 1H, C1′-H), 5.79 (br s, 2H, C2-
NH2), 6.75 (d, 2H, Ph-H), 7.03 (d, 2H, Ph-H), 7.87 (br s, 1H,
N6-H), 7.98 (s, 1H, C8-H); ESI-MS m/z 387.2 (M + H+)+.
2-Am in o-N6-(m -ch lor oben zyl)a d en osin e (33b): 1H NMR
δ 3.53 and 3.61 (2 × m, 2H, C5′-Ha,b), 3.89 (m, 1H, C4′-H),
4.08 (dd, 1H, C3′-H), 4.51 (dd, 1H, C2′-H), 4.61 (br s, 2H, N6-
CH2), 5.11 (br s, 1H, OH), 5.44 (br s, 2H, OH), 5.72 (d, 1H,
C1′-H), 5.86 (br s, 2H, C2-NH2), 7.21-7.39 (band, 4H, Ph-H),
7.94 (s, 1H, C8-H), 7.99 (br s, 1H, N6-H); ESI-MS m/z 407.3
(M + H+)+.
2-Am in o-N6-a llyla d en osin e (8b): 1H NMR (CD3OD) δ 3.74
and 3.87 (2 × dd, 2H, C5′-Ha,b), 4.14 (m, 3H, C4′-H and C1′′-
H), 4.29 (dd, 1H, C3′-H), 4.74 (dd, 1H, C2′-H), 5.12 and 5.24
(2 × dd, 2H, C3′′-H), 5.78 (d, 1H, C1′-H), 5.97 (m, 1H, C2′′-H),
7.85 (s, 1H, C8-H); ESI-MS m/z 323.3 (M + H+)+.
1
N6-P r op a r gyla d en osin e (9a ): H NMR δ 3.04 (br s, 1H,
C3′′-H), 3.55 and 3.66 (2 × m, 2H, C5′-Ha,b), 3.95 (m, 1H, C4′-
H), 4.14 (dd, 1H, C3′-H), 4.24 (br s, 2H, C1′′-H), 4.59 (dd, 1H,
C2′-H), 5.19 (d, 1H, OH), 5.35 (m, 1H, OH), 5.45 (d, 1H, OH),
5.89 (d, 1H, C1′-H), 8.25 (br s, 1H, N6-H), 8.27 and 8.40 (2 ×
s, 2H, C2-H and C8-H); ESI-MS m/z 306.3 (M + H+)+.
2-Am in o-N6-p r op a r gyla d en osin e (9b): 1H NMR (CD3OD)
δ 2.59 (br s, 1H, C3′′-H), 3.72 and 3.87 (2 × dd, 2H, C5′-Ha,b),
4.14 (m, 1H, C4′-H), 4.28 (dd, 1H, C3′-H), 4.30 (br s, 2H, C1′′-
H), 4.75 (dd, 1H, C2′-H), 5.78 (d, 1H, C1′-H), 7.86 (s, 1H, C8-
H); ESI-MS m/z 321.3 (M + H+)+.
2-Am in o-N6-(3′′-h ydr oxypr opyl)aden osin e (11b): 1H NMR
δ 1.71 (p, 2H, C2′′-H), 3.38 (m, 2H, C1′′-H), 3.49 (m, 2H, C3′′-
H), 3.51 and 3.61 (2 × m, 2H, C5′-Ha,b), 3.89 (m, 1H, C4′-H),
4.09 (dd, 1H, C3′-H), 4.48 (dd, 1H, C2′-H), 4.58 (br s, 1H, C3′′-
OH), 5.14 (d, 1H, OH), 5.39 (d, 1H, OH), 5.45 (br s, 1H, OH),
5.71 (d, 1H, C1′-H), 5.83 (br s, 2H, C2-NH2), 7.39 (br s, 1H,
N6-H), 7.93 (s, 1H, C8-H); ESI-MS m/z 341.2 (M + H+)+.
2-Am in o-N6-(2′′-h ydr oxypr opyl)aden osin e (12b): 1H NMR
δ 1.05 (d, 3H, C3′′-H), 3.31 and 3.40 (2 × m, 2H, C1′′-Ha,b),
3.52 and 3.63 (2 × m, 2H, C5′-Ha,b), 3.83 (m, 1H, C2′′-H), 3.89
(m, 1H, C4′-H), 4.08 (dd, 1H, C3′-H), 4.47 (dd, 1H, C2′-H), 4.90
(br s, 1H, C2′′-OH), 5.12 (d, 1H, OH), 5.38 (d, 1H, OH), 5.42
(br s, 1H, OH), 5.70 (d, 1H, C1′-H), 5.84 (br s, 2H, C2-NH2),
7.04 (br s, 1H, N6-H), 7.92 (s, 1H, C8-H); ESI-MS m/z 341.2
(M + H+)+.
2-Am in o-N6-(p -m et h oxyb en zyl)a d en osin e (36b ): 1H
NMR δ 3.53 and 3.61 (2 × m, 2H, C5′-Ha,b), 3.70 (s, 3H, Ph-
OCH3), 3.90 (m, 1H, C4′-H), 4.08 (dd, 1H, C3′-H), 4.51 (dd,
1H, C2′-H), 4.55 (br s, 2H, N6-CH2), 5.11 (d, 1H, OH), 5.36 (d,
1H, OH), 5.41 (br s, 1H, OH), 5.71 (d, 1H, C1′-H), 5.82 (br s,
2H, C2-NH2), 6.83 (d, 2H, Ph-H), 7.25 (d, 2H, Ph-H), 7.80 (br
s, 1H, N6-H), 7.91 (s, 1H, C8-H); ESI-MS m/z 403.2 (M + H+)+.
2-Am in o-N 6-(2′′-h yd r oxy-2′′-p h e n yle t h yl)a d e n osin e
(39b): 1H NMR δ 3.55 and 3.63 (2 × m, 2H, C5′-Ha,b), 3.77 (br
s, 2H, C1′′-H), 3.90 (m, 1H, C4′-H), 4.14 (dd, 1H, C3′-H), 4.59
(dd, 1H, C2′-H), 4.85 (br s, 1H, C2′′-H), 5.08 (d, 1H, OH), 5.33
(d, 1H, OH), 5.36 (m, 1H, OH), 5.62 (br s, 1H, C2′′-OH), 5.72
(d, 1H, C1′-H), 5.87 (br s, 2H, C2-NH2), 6.98 (br s, 2H, N6-H),

4144 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Bressi et al.
7.23 (t, 1H, Ph-H), 7.32 (t, 1H, Ph-H), 7.41 (d, 1H, Ph-H), 7.92
(s, 1H, C8-H); ESI-MS m/z 403.2 (M + H+)+.
Ha,b), 3.72 (m, 2H, C1′′-H), 3.96 (m, 1H, C4′-H), 4.14 (dd, 1H,
C3′-H), 4.60 (dd, 1H, C2′-H), 5.16 (d, 1H, OH), 5.36 (m, 1H,
OH), 5.41 (d, 1H, OH), 5.88 (d, 1H, C1′-H), 7.32 (m, 2H, Ph-
H), 7.55 (s, 1H, Ph-H) 7.96 (br s, 1H, N6-H), 8.21 and 8.34 (2
× s, 2H, C2-H and C8-H); ESI-MS m/z 440.1 (M + H+)+.
2-Am in o-N6-[2′′-(o,p -d ich lor op h en yl)et h yl]a d en osin e
(51b): 1H NMR δ 3.00 (t, 2H, C2′′-H), 3.55 and 3.65 (2 × m,
4H, C5′-Ha,b, and C1′′-H), 3.89 (m, 1H, C4′-H), 4.08 (dd, 1H,
C3′-H), 4.49 (dd, 1H, C2′-H), 5.08 (br s, 1H, OH), 5.34 (br s,
1H, OH,), 5.43 (br s, 1H, OH), 5.72 (d, 1H, C1′-H), 5.78 (br s,
2H, C2-NH2), 7.33-7.41 (band, 3H, N6-H and Ph-H), 7.55 (d,
2H, Ph-H), 7.90 (br s, 1H, C8-H); ESI-MS m/z 455.1 (M + H+)+.
2-Am in o-N 6-[2′′-(o-m e t h oxyp h e n yl)e t h yl]a d e n osin e
(52b): 1H NMR δ 2.90 (t, 2H, C2′′-H), 3.55 and 3.66 (2 × m,
4H, C5′-Ha,b and C1′′-H), 3.79 (s, 3H, Ph-OCH3), 3.90 (m, 1H,
C4′-H), 4.14 (dd, 1H, C3′-H), 4.60 (dd, 1H, C2′-H), 5.16 (d, 1H,
OH), 5.34 (d, 1H, OH), 5.45 (m, 1H, OH), 5.72 (d, 1H, C1′-H),
5.81 (br s, 2H, C2-NH2), 6.89 (t, 1H, Ph-H), 6.92 (d, 1H, Ph-
H), 7.20 (m, 3H, N6-H and Ph-H), 7.89 (s, 1H, C8-H); ESI-MS
m/z 417.1 (M + H+)+.
2-Am in o-N6-(3′′-p h en ylp r op yl)a d en osin e (40b): 1H NMR
(CD3OD) δ 1.93 (p, 2H, 2′′-H), 2.69 (t, 2H, 3′′-H), 3.50 (br s,
2H, 1′′-H), 3.71 and 3.86 (2 × m, 2H, C5′-Ha,b), 4.14 (m, 1H,
C4′-H), 4.28 (dd, 1H, C3′-H), 4.75 (dd, 1H, C2′-H), 5.77 (d, 1H,
C1′-H), 7.12-7.24 (band, 5H, Ph-H), 7.83 (s, 1H, C8-H); ESI-
MS m/z 401.2 (M + H+)+.
2-Am in o-N6-(1′′-n a p h th ylm eth yl)a d en osin e (41b): 1H
NMR δ 3.51 and 3.62 (2 × m, 2H, C5′-Ha,b), 3.89 (m, 1H, C4′-
H), 4.08 (dd, 1H, C3′-H), 4.51 (dd, 1H, C2′-H), 5.11 (m, 3H,
OH and C1′′-H), 5.38 (m, 2H, OH), 5.73 (d, 1H, C1′-H), 5.84
(br s, 2H, C2-NH2), 7.41-8.23 (band, 9H, N6-H, C8-H, and
naphthyl-H); ESI-MS m/z 423.2 (M + H+)+.
N6-(1′′,2′′-Dip h en eth yl)a d en osin e (42a ): 1H NMR δ 3.08
and 3.32 (2 × m, 2H, C2′′-H), 3.54 and 3.65 (2 × m, 2H, C5′-
H
a,b), 3.95 (m, 1H, C4′-H), 4.13 (dd, 1H, C3′-H), 4.59 (dd, 1H,
C2′-H), 5.20 (br s, 1H, OH), 5.39 (br s, 1H, OH), 5.48 (br s,
1H, OH), 5.52 (m, 1H, C1′′-H), 5.87 (d, 1H, C1′-H), 7.09-8.55
(band, 10H, Ph-H), 8.23 and 8.34 (2 × s, 1H, C2-H and C8-H),
8.44 (d, 1H, N6-H); ESI-MS m/z 448.2 (M + H+)+.
2-Am in o-N6-[2′′-(m -m et h oxyp h en yl)et h yl]a d en osin e
1
2-Am in o-N6-(1′′,2′′-d ip h en et h yl)a d en osin e (42b ): 1H
NMR δ 3.08 and 3.32 (2 × m, 2H, C2′′-H), 3.55 and 3.65 (2 ×
m, 2H, C5′-Ha,b), 3.89 (m, 1H, C4′-H), 4.08 (dd, 1H, C3′-H),
4.49 (dd, 1H, C2′-H), 5.11 (br s, 1H, OH), 5.36 (br s, 1H, OH),
5.44 (br s, 1H, OH), 5.56 (m, 1H, C1′′-H), 5.70 (d, 1H, C1′-H),
5.74 (br s, 2H, C2-NH2), 7.09-8.55 (band, 10H, Ph-H), 7.91
(s, 1H, C8-H), 7.92 (d, 1H, N6-H); ESI-MS m/z 463.2 (M + H+)+.
2-Am in o-N6-[2′′-(p-m eth ylph en yl)eth yl]aden osin e (44b):
1H NMR δ 2.25 (s, 3H, Ph-CH3), 2.84 (t, 2H, C2′′-H), 3.52 and
3.64 (2 × m, 4H, C5′-Ha,b and C1′′-H), 3.89 (m, 1H, C4′-H),
4.10 (dd, 1H, C3′-H), 4.50 (dd, 1H, C2′-H), 5.07 (d, 1H, OH),
5.32 (m, 1H, OH), 5.40 (d, 1H, OH), 5.72 (d, 1H, C1′-H), 5.78
(br s, 2H, C2-NH2), 7.07 (d, 2H, Ph-H), 7.14 (d, 2H, Ph-H),
7.25 (br s, 1H, N6-H), 7.89 (br s, 1H, C8-H); ESI-MS m/z 401.2
(M + H+)+.
(53b): H NMR δ 2.90 (t, 2H, C2′′-H), 3.55 and 3.66 (2 × m,
4H, C5′-Ha,b and C1′′-H), 3.79 (s, 3H, Ph-OCH3), 3.90 (m, 1H,
C4′-H), 4.14 (dd, 1H, C3′-H), 4.60 (dd, 1H, C2′-H), 5.16 (d, 1H,
OH), 5.34 (d, 1H, OH), 5.45 (m, 1H, OH), 5.72 (d, 1H, C1′-H),
5.81 (br s, 2H, C2-NH2), 6.89 (t, 1H, Ph-H), 6.92 (d, 1H, Ph-
H), 7.20 (m, 3H, N6-H and Ph-H), 7.89 (s, 1H, C8-H); ESI-MS
m/z 417.1 (M + H+)+.
2-Am in o-N 6-[2′′-(p -m e t h oxyp h e n yl)e t h yl]a d e n osin e
1
(54b): H NMR δ 2.85 (t, 2H, C2′′-H), 3.55 and 3.66 (2 × m,
4H, C5′-Ha,b, and C1′′-H), 3.72 (s, 3H, Ph-OCH3), 3.93 (m, 1H,
C4′-H), 4.14 (dd, 1H, C3′-H), 4.60 (dd, 1H, C2′-H), 5.09 (d, 1H,
OH), 5.34 (d, 1H, OH), 5.42 (m, 1H, OH), 5.72 (d, 1H, C1′-H),
5.78 (br s, 2H, C2-NH2), 6.84 (d, 2H, Ph-H), 7.18 (d, 2H, Ph-
H), 7.29 (br s, 1H, N6-H), 7.89 (s, 1H, C8-H); ESI-MS m/z 417.1
(M + H+)+.
2-Am in o-N 6-[2′′-(p -h yd r oxyp h e n yl)e t h yl]a d e n osin e
(46b): 1H NMR δ 2.76 (t, 2H, C2′′-H), 3.53 and 3.62 (2 × m,
4H, C5′-Ha,b and C1′′-H), 3.90 (m, 1H, C4′-H), 4.10 (dd, 1H,
C3′-H), 4.50 (dd, 1H, C2′-H), 5.10 (t, 1H, OH), 5.35 (d, 1H, OH),
5.48 (m, 1H, OH), 5.78 (d, 1H, C1′-H), 5.82 (br s, 2H, C2-NH2),
6.65 (d, 2H, Ph-H), 7.04 (d, 2H, Ph-H), 7.41 (t, 1H, N6-H), 7.92
(s, 1H, C8-H), 9.18 (s, 1H, Ph-OH); ESI-MS m/z 403.2 (M +
H+)+.
N 6-[2′′-(p -H yd r oxy-m -m e t h oxyp h e n yl)e t h yl]a d e n o-
sin e (56a ): 1H NMR δ 2.78 (t, 2H, C2′′-H), 3.55 and 3.66 (2 ×
m, 4H, C5′-Ha,b and C1′′-H), 3.71 (s, 3H, Ph-OCH3), 3.95 (m,
1H, C4′-H), 4.13 (dd, 1H, C3′-H), 4.60 (dd, 1H, C2′-H), 5.21 (d,
1H, OH), 5.44 (m, 2H, OH), 5.87 (d, 1H, C1′-H), 6.61 (d, 1H,
Ph-H), 6.66 (d, 1H, Ph-H), 6.78 (s, 2H, Ph-H), 7.90 (br s, 1H,
N6-H), 8.23 and 8.34 (2 × s, 1H, C2-H and C8-H), 8.72 (s, 1H,
Ph-OH); ESI-MS m/z 418.2 (M + H+)+.
2-Am in o-N6-[2′′-(m ,p -d ih yd r oxyp h en yl)et h yl]a d en o-
sin e (47b): 1H NMR δ 2.68 (t, 2H, C2′′-H), 3.39 (br s, 2H, C1′′-
H), 3.51 and 3.61 (2 × m, 2H, C5′-Ha,b), 3.89 (m, 1H, C4′-H),
4.08 (dd, 1H, C3′-H), 4.50 (dd, 1H, C2′-H), 5.20 (br s, 1H, OH),
5.42 (br s, 2H, OH), 5.77 (d, 1H, C1′-H), 5.83 (br s, 2H, C2-
NH2), 6.48 (d, 1H, Ph-H), 6.63 (m, 2H, Ph-H), 7.23 (br s, 1H,
N6-H), 7.93 (s, 1H, C8-H), 8.75 (br s, 2H, Ph-OH); ESI-MS m/z
419.2 (M + H+)+.
2-Am in o-N6-[2′′-(p-h yd r oxy-m -m eth oxyp h en yl)eth yl]-
a d en osin e (56b): 1H NMR δ 2.72 (t, 2H, C2′′-H), 3.55 and
3.66 (2 × m, 4H, C5′-Ha,b and C1′′-H), 3.71 (s, 3H, Ph-OCH3),
3.90 (m, 1H, C4′-H), 4.10 (dd, 1H, C3′-H), 4.50 (dd, 1H, C2′-
H), 5.13 (d, 1H, OH), 5.39 (d, 1H, OH), 5.44 (br s, 1H, OH),
5.71 (d, 1H, C1′-H), 5.84 (br s, 2H, C2-NH2), 6.61-6.69 (band,
2H, Ph-H), 6.80 (br s, 1H, Ph-H), 7.31 (br s, 1H, N6-H), 7.90
(s, 1H, C8-H), 8.71 (s, 1H, Ph-OH); ESI-MS m/z 433.2 (M +
H+)+.
2-Am in o-N6-[2′′-(o-ch lor op h en yl)eth yl]a d en osin e (48b):
1H NMR δ 3.07 (t, 2H, C2′′-H), 3.55 and 3.65 (2 × m, 4H, C5′-
Ha,b and C1′′-H), 3.94 (m, 1H, C4′-H), 4.13 (dd, 1H, C3′-H),
4.54 (dd, 1H, C2′-H), 5.12 (d, 1H, OH), 5.38 (d, 1H, OH), 5.45
(br s, 1H, OH), 5.76 (d, 1H, C1′-H), 5.80 (br s, 2H, C2-NH2),
7.25 (m, 2H, Ph-H), 7.45 (m, 3H, N6-H and Ph-H), 7.94 (s, 1H,
C8-H); ESI-MS m/z 421.1 (M + H+)+.
N 6-[2′′-(m -H yd r oxy-p -m e t h oxyp h e n yl)e t h yl]a d e n o-
sin e (57a ): 1H NMR δ 2.75 (t, 2H, C2′′-H), 3.55 and 3.66 (2 ×
m, 4H, C5′-Ha,b and C1′′-H), 3.70 (s, 3H, Ph-OCH3), 3.95 (m,
1H, C4′-H), 4.13 (dd, 1H, C3′-H), 4.60 (dd, 1H, C2′-H), 5.21 (d,
1H, OH), 5.44 (m, 1H, OH), 5.48 (d, 1H, OH), 5.87 (d, 1H, C1′-
H), 6.59 (d, 2H, Ph-H), 6.68 (s, 1H, Ph-H), 6.79 (d, 2H, Ph-H),
7.90 (br s, 1H, N6-H), 8.23 and 8.34 (2 × s, 1H, C2-H and C8-
H), 8.88 (s, 1H, Ph-OH); ESI-MS m/z 418.2 (M + H+)+.
2-Am in o-N6-[2′′-(m -h yd r oxy-p-m eth oxyp h en yl)eth yl]-
a d en osin e (57b): 1H NMR δ 2.72 (t, 2H, C2′′-H), 3.55 and
3.66 (2 × m, 4H, C5′-Ha,b and C1′′-H), 3.71 (s, 3H, Ph-OCH3),
3.90 (m, 1H, C4′-H), 4.10 (dd, 1H, C3′-H), 4.50 (dd, 1H, C2′-
H), 5.13 (d, 1H, OH), 5.39 (d, 1H, OH), 5.44 (br s, 1H, OH),
5.71 (d, 1H, C1′-H), 5.84 (br s, 2H, C2-NH2), 6.61 (d, 1H, Ph-
H), 6.70 (s, 1H, Ph-H), 6.80 (d, 1H, Ph-H), 7.31 (br s, 1H, N6-
H), 7.91 (br s, 1H, C8-H), 8.86 (s, 1H, Ph-OH); ESI-MS m/z
433.2 (M + H+)+.
2-Am in o-N6-[2′′-(m-ch lor oph en yl)eth yl]aden osin e (49b):
1H NMR δ 2.90 (t, 2H, C2′′-H), 3.55 and 3.65 (2 × m, 4H, C5′-
Ha,b and C1′′-H), 3.89 (m, 1H, C4′-H), 4.08 (dd, 1H, C3′-H),
4.48 (dd, 1H, C2′-H), 5.07 (d, 1H, OH), 5.33 (d, 1H, OH), 5.45
(br s, 1H, OH), 5.72 (d, 1H, C1′-H), 5.82 (br s, 2H, C2-NH2),
7.23 (t, 1H, Ph-H), 7.30 (m, 3H, N6-H and Ph-H), 7.90 (s, 1H,
C8-H); ESI-MS m/z 421.1 (M + H+)+.
2-Am in o -N 6-[2′′-(p -c h lo r o p h e n y l)e t h y l]a d e n o s in e
(50b): 1H NMR δ 2.90 (t, 2H, C2′′-H), 3.55 and 3.65 (2 × m,
4H, C5′-Ha,b and C1′′-H), 3.89 (m, 1H, C4′-H), 4.09 (dd, 1H,
C3′-H), 4.50 (dd, 1H, C2′-H), 5.08 (d, 1H, OH), 5.33 (d, 1H,
OH), 5.45 (br s, 1H, OH), 5.72 (d, 1H, C1′-H), 5.81 (br s, 2H,
C2-NH2), 7.28 (d, 2H, Ph-H), 7.32 (d, 2H, Ph-H), 7.35 (br s,
1H, N6-H), 7.91 (s, 1H, C8-H); ESI-MS m/z 421.1 (M + H+)+.
2-Am in o-N6-[2′′-(p-a m in op h en yl)eth yl]a d en osin e (58b):
1H NMR δ 2.67 (t, 2H, C2′′-H), 3.55 and 3.66 (2 × m, 4H, C5′-
Ha,b and C1′′-H), 3.89 (m, 1H, C4′-H), 4.08 (dd, 1H, C3′-H),
4.49 (dd, 1H, C2′-H), 4.85 (br s, 2H, Ph-NH2), 5.09 (d, 1H, OH),
5.36 (d, 1H, OH), 5.48 (m, 1H, OH), 5.71 (d, 1H, C1′-H), 5.81
1
N6-[2′′-(o,p-Dich lor op h en yl)eth yl]a d en osin e (51a ): H
NMR δ 3.04 (t, 2H, C2′′-H), 3.55 and 3.66 (2 × m, 2H, C5′-

Adenosine Analogues as Inhibitors of T. brucei PGK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4145
(br s, 3H, OH), 5.70 (d, 1H, C1′-H), 6.12 (s, 1H, C2-NH), 6.66
(br s, 2H, C2-NH2), 6.48 (d, 2H, Ph-H), 6.90 (d, 2H, Ph-H),
7.35 (s, 1H, N6-H), 7.90 (s, 1H, C8-H); ESI-MS m/z 402.2 (M +
H+)+.
(br s, 2H, N6
H+)+.
-H), 7.89 (s, 1H, C8-H); ESI-MS m/z 353.2 (M +
2-[(3′′-Hyd r oxyp r op yl)a m in o]a d en osin e (11c): 1H NMR
δ 1.64 (p, 2H, C2′′-H), 3.27 (m, 2H, C1′′-H), 3.46 (m, 2H, C3′′-
H), 3.51 and 3.62 (2 × m, 2H, C5′-Ha,b), 3.87 (m, 1H, C4′-H),
4.11 (dd, 1H, C3′-H), 4.56 (dd, 1H, C2′-H), 5.71 (d, 1H, C1′-H),
6.14 (br s, 1H, C2-NH), 6.73 (br s, 2H, N6-H), 7.89 (s, 1H, C8-
H); ESI-MS m/z 341.2 (M + H+)+.
N6-[2′′-(p-Nitr op h en yl)eth yl]a d en osin e (59a ): 1H NMR
δ 3.07 (t, 2H, C2′′-H), 3.54 and 3.65 (2 × m, 2H, C5′-Ha,b), 3.76
(br d, 2H, C1′′-H), 3.95 (m, 1H, C4′-H), 4.13 (dd, 1H, C3′-H),
4.59 (dd, 1H, C2′-H), 5.20 (d, 1H, OH), 5.39 (dd, 1H, OH), 5.44
(d, 1H, OH), 5.87 (d, 1H, C1′-H), 7.52 (d, 2H, Ph-H), 8.01 (br
s, 1H, N6-H), 8.14 (d, 2H, Ph-H), 8.23 and 8.34 (2 × s, 1H,
C2-H and C8-H); ESI-MS m/z 417.1 (M + H+)+.
2-Am in o-N6-[2′′-(p-n itr op h en yl)eth yl]a d en osin e (59b):
1H NMR δ 3.04 (t, 2H, C2′′-H), 3.56 and 3.65 (2 × m, 2H, C5′-
Ha,b), 3.76 (br s, 2H, C1′′-H), 3.89 (m, 1H, C4′-H), 4.08 (dd,
1H, C3′-H), 4.49 (dd, 1H, C2′-H), 5.11 (d, 1H, OH), 5.36 (d,
1H, OH), 5.44 (s, 1H, OH), 5.72 (d, 1H, C1′-H), 5.90 (br s, 2H,
C2-NH2), 7.50 (br s, 1H, N6-H), 7.55 (d, 2H, Ph-H), 7.93 (s,
1H, C8-H), 8.14 (d, 2H, Ph-H); ESI-MS m/z 432.2 (M + H+)+.
6-Ch lor o-2-iod op u r in e 9-Ribosid e (62b). As described by
Matsuda et al.29 for the iodination of 9-(2′,3′,5′-triacetyl-1-â-
D-ribofuranosyl)-2-amino-6-chloropurine (61), fresh isoamyl
nitrite (1.45 mL, 10.81 mmol) was added to a mixture of 1b′
(1.0 g, 3.32 mmol), CuI (670 mg, 3.52 mmol), CH2I2 (2.67 mL,
33.2 mmol), and I2 (842 mg, 3.32 mmol) in dry THF (10.0 mL)
at ambient temperature and under argon. The reaction
mixture was refluxed for 30 min, cooled to ambient tempera-
ture, filtered to remove insolubles, and then adsorbed onto
silica gel via rotary evaporation. The product was purified via
flash chromatography. Iodine was first eluted from the column
with 100% CHCl3 and then compound 62b was eluted with
10% MeOH in CHCl3 in 56% yield: 1H NMR δ 3.58 and 3.70
(2 × m, 2H, C5′-Ha,b), 3.98 (m, 1H, C4′-H), 4.15 (dd, 1H, C3′-
H), 4.51 (dd, 1H, C2′-H), 5.04 (t, 1H, OH), 5.25 (d, 1H, OH),
5.56 (d, 1H, OH), 5.95 (d, 1H, C1′-H), 8.87 (s, 1H, C8-H); ESI-
MS m/z 413.0 (M + H+)+.
Syn th esis of 2-Iod oa d en osin e (1c′) fr om 62b. As de-
scribed by Matsuda et al.29 for the ammoniaolysis of 62a ,
ammonia gas was condensed into a solution of 62b (100 mg,
0.24 mmol) in MeOH (10.0 mL) at 0 °C in a pressure tube.
The reaction was heated to 60 °C for 18 h and then chilled to
0 °C in order to safely open the tube. The yellow solution was
adsorbed onto silica gel via rotary evaporation and purified
via flash chromatography. Compound 1c′ was eluted with 15%
MeOH in CHCl3 to yield 87 mg of the desired product (94%):
1H NMR δ 3.54 and 3.64 (2 × m, 2H, C5′-Ha,b), 3.94 (m, 1H,
C4′-H), 4.13 (dd, 1H, C3′-H), 4.54 (dd, 1H, C2′-H), 5.04 (t, 1H,
OH), 5.20 (t, 1H, OH), 5.47 (d, 1H, OH), 5.80 (d, 1H, C1′-H),
7.74 (s, 2H, N6-H), 8.30 (s, 1H, C8-H); ESI-MS m/z 394.0 (M +
H+)+.
2-[(2′′-Hyd r oxyp r op yl)a m in o]a d en osin e (12c): 1H NMR
δ 1.04 (d, 3H, C3′′-H), 3.11 and 3.25 (2 × m, 2H, C1′′-Ha,b),
3.51 and 3.62 (2 × m, 2H, C5′-Ha,b), 3.76 (m, 1H, C2′′-H), 3.87
(m, 1H, C4′-H), 4.11 (dd, 1H, C3′-H), 4.56 (dd, 1H, C2′-H), 5.70
(d, 1H, C1′-H), 5.94 (br s, 1H, C2-NH), 6.79 (br s, 2H, N6-H),
7.90 (s, 1H, C8-H); ESI-MS m/z 341.2 (M + H+)+.
2-[(2′′,3′′-Dih yd r oxyp r op yl)a m in o]a d en osin e (13c): 1H
NMR δ 3.12-3.62 (band, 7H, C5′-Ha,b, C1′′-Ha,b, C2′′-H, and
C3′′-Ha,b), 3.87 (m, 1H, C4′-H), 4.10 (dd, 1H, C3′-H), 4.55 (dd,
1H, C2′-H), 5.70 (d, 1H, C1′-H), 5.91 (br s, 1H, C2-NH), 6.83
(br s, 2H, N6-H), 7.91 (s, 1H, C8-H); ESI-MS m/z 357.1 (M +
H+)+.
1
2-[(4′′-Hyd r oxybu tyl)a m in o]a d en osin e (14c): H NMR
δ 1.44 (m, 2H, C2”-H), 1.49 (m, 2H, C3′′-H), 3.21 (m, 2H, C1′′-
H), 3.38 (m, 2H, C4′′-H), 3.51 and 3.62 (2 × m, 2H, C5′-Ha,b),
3.87 (m, 1H, C4′-H), 4.11 (dd, 1H, C3′-H), 4.58 (dd, 1H, C2′-
H), 5.70 (d, 1H, C1′-H), 6.15 (br s, 1H, C2-NH), 6.70 (br s, 2H,
N6-H), 7.88 (s, 1H, C8-H); ESI-MS m/z 355.2 (M + H+)+.
2-[(5′′-Hyd r oxyp en tyl)a m in o]a d en osin e (15c): 1H NMR
δ 1.32 (m, 2H, C3′′-H), 1.42 (m, 2H, C2′′-H), 1.49 (m, 2H, C4′′-
H), 3.21 (m, 2H, C1′′-H), 3.38 (m, 2H, C5′′-H), 3.51 and 3.62
(2 × m, 2H, C5′-Ha,b), 3.87 (m, 1H, C4′-H), 4.11 (dd, 1H, C3′-
H), 4.58 (dd, 1H, C2′-H), 5.70 (d, 1H, C1′-H), 6.13 (br s, 1H,
C2-NH), 6.70 (br s, 2H, N6-H), 7.88 (s, 1H, C8-H); ESI-MS m/z
369.2 (M + H+)+.
2-[(3′′-Am in op r op yl)a m in o]a d en osin e (17c): 1H NMR δ
1.39 (m, 2H, C2′′-H), 2.76 (t, 2H, C3′′-H), 3.26 (m, 2H, C1′′-H),
3.52 and 3.63 (2 × m, 2H, C5′-Ha,b), 3.87 (m, 1H, C4′-H), 4.11
(dd, 1H, C3′-H), 4.55 (dd, 1H, C2′-H), 5.71 (d, 1H, C1′-H), 6.42
(t, 1H, C2-NH), 6.81 (br s, 2H, N6-H), 7.91 (s, 1H, C8-H); ESI-
MS m/z 340.2 (M + H+)+.
2-[(2′′-Hydr oxy-3′′-am in opr opyl)am in o]aden osin e (18c):
1H NMR δ 2.80 (m, 2H, C3′′-H), 3.30 (m, 2H, C1′′-H), 3.52 and
3.63 (2 × m, 2H, C5′-Ha,b), 3.80 (br s, 1H, C2′′-H), 3.86 (m,
1H, C4′-H), 4.11 (m, 1H, C3′-H), 4.55 (m, 1H, C2′-H), 5.70 (m,
1H, C1′-H), 6.20 (t, 1H, C2-NH), 6.81 (br s, 2H, N6-H), 7.92 (s,
1H, C8-H); ESI-MS m/z 356.2 (M + H+)+.
2-[(5′′-Am in op r op yl)a m in o]a d en osin e (20c): 1H NMR δ
1.33 (m, 2H, C3′′-H), 1.57 (m, 4H, C2′′-H and C4′′-H), 2.70 (m,
2H, C5′′-H), 3.20 (m, 2H, C1′′-H), 3.52 and 3.63 (2 × m, 2H,
C5′-Ha,b), 3.86 (m, 1H, C4′-H), 4.10 (m, 1H, C3′-H), 4.57 (m,
1H, C2′-H), 5.72 (m, 1H, C1′-H), 6.20 (t, 1H, C2-NH), 6.75 (br
s, 2H, N6-H), 7.91 (s, 1H, C8-H); ESI-MS m/z 368.2 (M + H+)+.
2-[(Cycloh exylm eth yl)a m in o]a d en osin e (23c): 1H NMR
δ 1.32-1.86 (band, 11H, cyclohexyl-H) 3.08 (m, 2H, N2-CH2),
3.51 and 3.61 (2 × m, 2H, C5′-Ha,b), 3.86 (m, 1H, C4′-H), 4.11
(dd, 1H, C3′-H), 4.59 (dd, 1H, C2′-H), 5.20 (br s, 3H, OH), 5.70
(d, 1H, C1′-H), 6.09 (br s, 1H, C2-NH), 6.63 (br s, 2H, N6-H),
7.92 (s, 1H, C8-H); ESI-MS m/z 379.2 (M + H+)+.
Gen er a l P r oced u r e for th e P r ep a r a tion of N2-Su bsti-
tu ted Ad en osin e An a logu es (2a -59a a n d 2b-59b) fr om
1c′. In parallel fashion and with minor modifications, the
method of Marumoto et al. was employed.25 To a solution of
1c′ (5.0 mg, 0.013 mmol) in 2-methoxyethanol (100 µL) was
added the appropriate amine or amine hydrochloride (0.25
mmol). For reactions requiring the use of a hydrochloride, i-Pr2-
EtN (44 µL, 0.25 mmol) was added. The reactions were
refluxed for 24 h and then allowed to cool to ambient temper-
ature. Without further workup, the homogeneous reaction
mixtures were purified via preparative HPLC method B,
except for compounds 10c-15c which were purified by pre-
parative HPLC method C and 16c-20c which were purified
by preparative HPLC method A, to give the desired products
in 40-85% yield.
2-(Cycloh ep tyla m in o)a d en osin e (24c): 1H NMR δ 1.36-
1.86 (band, 12H, cycloheptyl-H) 3.51 and 3.61 (2 × m, 2H, C5′-
Ha,b), 3.86 (m, 1H, C4′-H), 3.89 (m, 1H, C4′-H), 4.11 (dd, 1H,
C3′-H), 4.39 (m, 2H, C1”-H), 4.59 (dd, 1H, C2′-H), 5.20 (br s,
3H, OH), 5.70 (d, 1H, C1′-H), 5.88 (d, 1H, C2-NH), 6.62 (br s,
2H, N6-H), 7.86 (s, 1H, C8-H); ESI-MS m/z 379.2 (M + H+)+.
2-(Tetr a h yd r ofu r fu r yla m in o)a d en osin e (25c): 1H NMR
δ 1.80-1.99 (band, 4H, C3′′- and C4′′-H), 3.51 and 3.62 (2 ×
m, 4H, C5′-Ha,b and C5′′-H), 3.75 (m, 2H, C1”-H), 3.87 (m, 1H,
C4′-H), 3.95 (m, 1H, C2”-H), 4.11 (dd, 1H, C3′-H), 4.43 (m, 2H,
N2-CH2), 4.56 (dd, 1H, C2′-H), 5.72 (d, 1H, C1′-H), 6.58 (t, 1H,
C2-NH), 6.80 (br s, 2H, N6-H), 7.92 (s, 1H, C8-H); ESI-MS m/z
367.2 (M + H+)+.
1
2-(n -Bu tyla m in o)a d en osin e (6c): H NMR (DMSO-d6
+
TFD) δ 0.88 (m, 2H, C4′′-H), 1.34 (m, 2H, C3′′-H), 1.52 (m,
2H, C2′′-H), 3.32 (m, 2H, C1′′-H), 3.25 and 3.40 (2 × m, 2H,
C5′-Ha,b), 3.97 (m, 1H, C4′-H), 4.18 (dd, 1H, C3′-H), 4.54 (dd,
1H, C2′-H), 5.81 (d, 1H, C1′-H), 8.33 (s, 1H, C8-H); ESI-MS
m/z 339.2 (M + H+)+.
1
2-(F u r fu r yla m in o)a d en osin e (26c): 1H NMR δ 3.51 and
3.62 (2 × m, 2H, C5′-Ha,b), 3.75 (m, 1H, N2-CH2), 3.87 (m, 1H,
C4′-H), 4.11 (dd, 1H, C3′-H), 4.43 (t, 2H, C1′′-H), 4.56 (dd, 1H,
C2′-H), 5.72 (d, 1H, C1′-H), 6.21 (d, 1H, C3′′-H), 6.34 (t, 1H,
2-(n -Am yla m in o)a d en osin e (7c): H NMR δ 1.41 (t, 3H,
C5′′-H), 1.49 (p, 2H, C4′′-H), 1.63 (p, 2H, C3′′-H), 1.86 (p, 2H,
C2′′-H), 3.50 and 3.61 (2 × m, 2H, C5′-Ha,b), 3.86 (m, 1H, C4′-
H), 4.11 (m, 2H, C3′-H and C1”-H), 4.60 (dd, 1H, C2′-H), 5.20

4146 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Bressi et al.
C4′′-H), 6.58 (t, 1H, C2-NH), 6.80 (br s, 2H, N6-H), 7.51 (s,
1H, C5′′-H), 7.92 (s, 1H, C8-H); ESI-MS m/z 363.2 (M + H+)+.
C3′-H), 4.61 (dd, 1H, C2′-H), 5.25 (br s, 3H, OH), 5.73 (d, 1H,
C1′-H), 6.25 (m, 1H, C2-NH), 6.75 (br s, 2H, N6-H), 7.25 (m,
2H, Ph-H), 7.30 (m, 2H, Ph-H), 7.90 (s, 1H, C8-H); ESI-MS
m/z 421.1 (M + H+)+.
2-[[2′′-(p-Ch lor op h en yl)eth yl]a m in o]a d en osin e (50c):
1H NMR δ 2.82 (t, 2H, C2′′-H), 3.43 (m, 2H, C1′′-H), 3.52 and
3.62 (2 × m, 2H, C5′-Ha,b), 3.88 (m, 1H, C4′-H), 4.13 (dd, 1H,
C3′-H), 4.61 (dd, 1H, C2′-H), 5.25 (br s, 3H, OH), 5.73 (d, 1H,
C1′-H), 6.24 (m, 1H, C2-NH), 6.75 (br s, 2H, N6-H), 7.27 (d,
2H, Ph-H), 7.32 (d, 2H, Ph-H), 7.90 (s, 1H, C8-H); ESI-MS m/z
421.1 (M + H+)+.
2-[[2′′-(o,p-Dich lor oph en yl)eth yl]am in o]aden osin e (51c):
1H NMR δ 2.95 (t, 2H, C2′′-H), 3.44 (m, 2H, C1′′-H), 3.52 and
3.62 (2 × m, 2H, C5′-Ha,b), 3.88 (m, 1H, C4′-H), 4.12 (dd, 1H,
C3′-H), 4.57 (dd, 1H, C2′-H), 5.25 (br s, 3H, OH), 5.74 (d, 1H,
C1′-H), 6.32 (m, 1H, C2-NH), 6.74 (br s, 2H, N6-H), 7.34 (d,
2H, Ph-H), 7.40 (d, 2H, Ph-H), 7.90 (s, 1H, C8-H); ESI-MS m/z
455.1 (M + H+)+.
2-[[2′′-(o-Meth oxyp h en yl)eth yl]a m in o]a d en osin e (52c):
1H NMR δ 2.81 (t, 2H, C2′′-H), 3.42 (m, 2H, C1′′-H), 3.52 and
3.62 (2 × m, 2H, C5′-Ha,b), 3.79 (s, 3H, Ph-OCH3), 3.88 (m,
1H, C4′-H), 4.11 (dd, 1H, C3′-H), 4.58 (dd, 1H, C2′-H), 5.25
(br s, 3H, OH), 5.73 (d, 1H, C1′-H), 6.08 (m, 1H, C2-NH), 6.72
(m, 2H, N6-H), 6.85 (t, 1H, Ph-H), 6.94 (d, 1H, Ph-H), 7.16 (m,
2H, Ph-H), 7.90 (s, 1H, C8-H); ESI-MS m/z 417.2 (M + H+)+.
2-[[2′′-(m -Me t h o x y p h e n y l)e t h y l]a m in o ]a d e n o s in e
(53c): 1H NMR δ 2.80 (t, 2H, C2′′-H), 3.44 (m, 2H, C1′′-H),
3.52 and 3.62 (2 × m, 2H, C5′-Ha,b), 3.73 (s, 3H, Ph-OCH3),
3.88 (m, 1H, C4′-H), 4.12 (dd, 1H, C3′-H), 4.61 (dd, 1H, C2′-
H), 5.25 (br s, 3H, OH), 5.73 (d, 1H, C1′-H), 6.16 (m, 1H, C2-
NH), 6.74 (m, 4H, N6-H and Ph-H), 6.81 (m, 2H, Ph-H), 7.19
(t, 1H, Ph-H), 7.93 (s, 1H, C8-H); ESI-MS m/z 417.2 (M + H+)+.
Gen er a l P r oced u r e for th e P r ep a r a tion of N6,N2-
Disu bstitu ted Ad en osin e An a logu es (69-82) fr om 62a .
To a solution of 62a (5.0 mg, 0.009 mmol) in 2-methoxyethanol
(250 µL) were added the appropriate amine or amine hydro-
chloride (0.01 mmol) targeted for the 6-position and then
CaCO3 (60.0 mg, 0.06 mmol). The reaction was heated at 60
°C for 18 h and then allowed to cool to ambient temperature.
The amine or amine hydrochloride (0.184 mmol) targeted for
the 2-position was then added and the reaction was refluxed
for 24 h. For reactions with amine hydrochlorides targeted for
the 2-position, i-Pr2EtN (32 µL, 0.184 mmol) was added prior
to heating. After the reactions were cooled to ambient tem-
perature, water (100 µL) was added to the reaction to dissolve
any insoluble salts and, without further workup, the reactions
were purified via preparative HPLC method B to give the
desired products in 40-70% yield.
1
2-[(o-Meth ylben zyl)a m in o]a d en osin e (29c): H NMR δ
2.30 (s, 3H, Ph-CH3), 3.45 and 3.57 (2 × m, 2H, C5′-Ha,b), 3.86
(m, 1H, C4′-H), 4.09 (dd, 1H, C3′-H), 4.45 (d, 2H, N2-CH2), 4.53
(dd, 1H, C2′-H), 5.70 (d, 1H, C1′-H), 6.52 (t, 1H, C2-NH), 6.71
(br s, 2H, N6-H), 7.08 (m, 3H, Bz-H), 7.27 (d, 1H, Bz-H), 7.89
(s, 1H, C8-H); ESI-MS m/z 387.2 (M + H+)+.
2-[(m -Meth ylben zyl)a m in o]a d en osin e (30c): 1H NMR δ
2.26 (s, 3H, Ph-CH3), 3.51 and 3.62 (2 × m, 2H, C5′-Ha,b), 3.86
(m, 1H, C4′-H), 4.09 (dd, 1H, C3′-H), 4.45 (m, 2H, N2-CH2),
4.53 (dd, 1H, C2′-H), 5.70 (d, 1H, C1′-H), 6.63 (t, 1H, C2-NH),
6.71 (br s, 2H, N6-H), 6.98 (d, 1H, Ph-H), 7.13 (m, 3H, Ph-H),
7.89 (s, 1H, C8-H); ESI-MS m/z 387.2 (M + H+)+.
2-[(p-Meth ylben zyl)a m in o]a d en osin e (31c): 1H NMR δ
2.28 (s, 3H, Ph-CH3), 3.50 and 3.62 (2 × m, 2H, C5′-Ha,b), 3.86
(m, 1H, C4′-H), 4.09 (dd, 1H, C3′-H), 4.46 (m, 2H, N2-CH2),
4.57 (dd, 1H, C2′-H), 5.75 (d, 1H, C1′-H), 6.64 (t, 1H, C2-NH),
6.75 (br s, 2H, N6-H), 7.10 (d, 2H, Ph-H), 7.25 (d, 2H, Ph-H),
7.93 (s, 1H, C8-H); ESI-MS m/z 387.2 (M + H+)+.
1
2-[(o-Ch lor oben zyl)a m in o]a d en osin e (32c): H NMR δ
3.47 and 3.58 (2 × m, 2H, C5′-Ha,b), 3.86 (m, 1H, C4′-H), 4.09
(dd, 1H, C3′-H), 4.53 (dd, 1H, C2′-H), 4.57 (m, 2H, N2-CH2),
5.70 (d, 1H, C1′-H), 6.70 (t, 1H, C2-NH), 6.77 (br s, 2H, N6-H),
7.36 (m, 2H, Ph-H), 7.83 (m, 2H, Ph-H), 7.90 (s, 1H, C8-H);
ESI-MS m/z 407.1 (M + H+)+.
2-[(m -Ch lor oben zyl)a m in o]a d en osin e (33c): 1H NMR δ
3.46 and 3.58 (2 × m, 2H, C5′-Ha,b), 3.86 (m, 1H, C4′-H), 4.09
(dd, 1H, C3′-H), 4.47 (d, 2H, N2-CH2), 4.53 (dd, 1H, C2′-H),
5.70 (d, 1H, C1′-H), 6.76 (br s, 2H, N6-H), 6.82 (t, 1H, C2-NH),
7.22-7.30 (band, 4H, Ph-H), 7.90 (s, 1H, C8-H); ESI-MS m/z
407.1 (M + H+)+.
1
2-[(p-Ch lor oben zyl)a m in o]a d en osin e (34c): H NMR δ
3.46 and 3.58 (2 × m, 2H, C5′-Ha,b), 3.86 (m, 1H, C4′-H), 4.09
(dd, 1H, C3′-H), 4.44 (d, 2H, N2-CH2), 4.53 (dd, 1H, C2′-H),
5.70 (d, 1H, C1′-H), 6.73 (br s, 2H, N6-H), 6.78 (t, 1H, C2-NH),
7.31 (d, 2H, Ph-H), 7.34 (d, 2H, Ph-H), 7.90 (s, 1H, C8-H); ESI-
MS m/z 407.1 (M + H+)+.
2-[(o-Meth oxyben zyl)a m in o]a d en osin e (35c): 1H NMR
δ 3.46 and 3.58 (2 × m, 2H, C5′-Ha,b), 3.81 (s, 3H, Ph-OCH3),
3.86 (m, 1H, C4′-H), 4.09 (dd, 1H, C3′-H), 4.45 (m, 2H, N2-
CH2), 4.52 (dd, 1H, C2′-H), 5.70 (d, 1H, C1′-H), 6.33 (t, 1H,
C2-NH), 6.73 (br s, 2H, N6-H), 6.84 (t, 1H, Ph-H), 6.94 (d, 1H,
Ph-H), 7.18 (m, 2H, Ph-H), 7.89 (s, 1H, C8-H); ESI-MS m/z
403.2 (M + H+)+.
2-[(p-Meth oxyben zyl)a m in o]a d en osin e (36c): 1H NMR
δ 3.50 and 3.63 (2 × m, 2H, C5′-Ha,b), 3.70 (s, 3H, Ph-OCH3),
3.86 (m, 1H, C4′-H), 4.09 (dd, 1H, C3′-H), 4.39 (d, 2H, N2-CH2),
4.54 (dd, 1H, C2′-H), 5.70 (d, 1H, C1′-H), 6.59 (t, 1H, C2-NH),
6.71 (br s, 2H, N6-H), 6.82 (d, 2H, Ph-H), 7.23 (d, 2H, Ph-H),
7.89 (s, 1H, C8-H); ESI-MS m/z 403.2 (M + H+)+.
2-[[2′′-(p-Meth ylp h en yl)eth yl]a m in o]a d en osin e (44c):
1H NMR δ 2.25 (s, 3H, Ph-CH3), 2.77 (t, 2H, C2′′-H), 3.40 (m,
2H, C1′′-H), 3.52 and 3.62 (2 × m, 2H, C5′-Ha,b), 3.88 (m, 1H,
C4′-H), 4.12 (dd, 1H, C3′-H), 4.61 (dd, 1H, C2′-H), 5.30 (br s,
3H, OH), 5.73 (d, 1H, C1′-H), 6.13 (t, 1H, C2-NH), 6.73 (br s,
2H, N6-H), 7.08 (d, 2H, Ph-H), 7.13 (d, 2H, Ph-H), 7.90 (s, 1H,
C8-H); ESI-MS m/z 401.2 (M + H+)+.
2-[[2′′-(p-Hyd r oxyp h en yl)eth yl]a m in o]a d en osin e (46c):
1H NMR δ 2.71 (t, 2H, C2′′-H), 3.41 (m, 2H, C1′′-H), 3.51 and
3.60 (2 × m, 2H, C5′-Ha,b), 3.90 (m, 1H, C4′-H), 4.10 (dd, 1H,
C3′-H), 4.62 (dd, 1H, C2′-H), (br s, 3H, OH), 5.72 (d, 1H, C1′-
H), 6.15 (br s, 1H, C2-NH), 6.68 (d, 2H, Ph-H), 6.75 (br s, 2H,
N6-H), 7.05 (d, 2H, Ph-H), 7.92 (s, 1H, C8-H), 9.30 (s, 1H, Ph-
OH); ESI-MS m/z 403.2 (M + H+)+.
2-[[2′′-(o-Ch lor op h en yl)et h yl]a m in o]a d en osin e (48c):
1H NMR δ 2.97 (t, 2H, C2′′-H), 3.45 (m, 2H, C1′′-H), 3.52 and
3.62 (2 × m, 2H, C5′-Ha,b), 3.88 (m, 1H, C4′-H), 4.12 (dd, 1H,
C3′-H), 4.58 (dd, 1H, C2′-H), 5.25 (br s, 3H, OH), 5.75 (d, 1H,
C1′-H), 6.29 (m, 1H, C2-NH), 6.73 (br s, 2H, N6-H), 7.25 (m,
2H, Ph-H), 7.39 (m, 2H, Ph-H), 7.90 (s, 1H, C8-H); ESI-MS
m/z 421.1 (M + H+)+.
Gen er a l P r oced u r e for th e P r ep a r a tion of N6,N2-
Disu bstitu ted Ad en osin e An a logu es (69-82) fr om 62b.
To a solution of 62b (5.0 mg, 0.012 mmol) in 2-methoxyethanol
(100 µL) were added the appropriate amine or amine hydro-
chloride (0.013 mmol) targeted for the 6-position and then i-Pr2-
EtN (6.3 µL, 0.036 mmol for free amines; 8.4 µL, 0.048 mmol
for hydrochlorides). The reaction was heated at 60 °C for 18 h
and then allowed to cool to ambient temperature. The amine
or amine hydrochloride (0.24 mmol) targeted for the 2-position
was then added and the reaction was refluxed for 24 h. For
reactions with amine hydrochlorides targeted for the 2-posi-
tion, i-Pr2EtN (42 µL, 0.24 mmol) was added prior to heating.
The reactions were cooled to ambient temperature and,
without further workup, the reactions were purified via
preparative HPLC method B to give the desired products in
50-80% yield. Intermediates 63b-68b were isolated via flash
chromatography, in 90-95% yield, for the purpose of charac-
terization.
2-Iod o-N6-(2′′-p h en yleth yl)a d en osin e (63b): 1H NMR δ
2.89 (t, 2H, C2′′-H), 3.51 and 3.62 (2 × m, 4H, C5′-Ha,b and
C1′′-H), 3.93 (m, 1H, C4′-H), 4.11 (dd, 1H, C3′-H), 4.53 (dd,
1H, C2′-H), 5.03 (t, 1H, OH), 5.21 (d, 1H, OH), 5.47 (d, 1H,
OH), 5.78 (d, 1H, C1′-H), 7.17-7.30 (band, 5H, Ph-H), 8.29
(m, 2H, N6-H and C8-H); ESI-MS m/z 498.1 (M + H+)+.
2-I o d o -N 6-[2′′-(p -h y d r o x y p h e n y l)e t h y l]a d e n o s in e
(64b): 1H NMR δ 2.76 (t, 2H, C2′′-H), 3.53 and 3.62 (2 × m,
4H, C5′-Ha,b and C1′′-H), 3.93 (m, 1H, C4′-H), 4.11 (dd, 1H,
2-[[2′′-(m -Ch lor op h en yl)eth yl]a m in o]a d en osin e (49c):
1H NMR δ 2.85 (t, 2H, C2′′-H), 3.44 (m, 2H, C1′′-H), 3.52 and
3.62 (2 × m, 2H, C5′-Ha,b), 3.88 (m, 1H, C4′-H), 4.13 (dd, 1H,

Adenosine Analogues as Inhibitors of T. brucei PGK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4147
C3′-H), 4.52 (dd, 1H, C2′-H), 5.03 (t, 1H, OH), 5.21 (d, 1H, OH),
5.48 (d, 1H, OH), 5.79 (d, 1H, C1′-H), 6.66 (d, 2H, Ph-H), 7.04
(dd, 2H, Ph-H), 8.25 (t, 1H, N6-H), 8.28 (s, 1H, C8-H), 9.18 (s,
1H, Ph-OH); ESI-MS m/z 514.1 (M + H+)+.
2-Iod o-N6-[2′′-(p-a m in op h en yl)eth yl]a d en osin e (65b):
1H NMR δ 2.69 (t, 2H, C2′′-H), 3.53 and 3.62 (2 × m, 4H, C5′-
Ha,b and C1′′-H), 3.93 (m, 1H, C4′-H), 4.11 (dd, 1H, C3′-H),
4.51 (dd, 1H, C2′-H), 4.86 (s, 2H, Ph-NH2), 5.03 (t, 1H, OH),
5.21 (d, 1H, OH), 5.45 (d, 1H, OH), 5.79 (d, 1H, C1′-H), 6.48
(d, 2H, Ph-H), 6.89 (dd, 2H, Ph-H), 8.21 (t, 1H, N6-H), 8.27 (s,
1H, C8-H); ESI-MS m/z 513.1 (M + H+)+.
2-Iod o-N6-n -bu tyla d en osin e (66b): 1H NMR δ 0.88 (t, 3H,
N6-C4′′-H), 1.30 (m, 2H, N6-C3′′-H), 1.52 (m, 2H, N6-C2′′-H),
3.35 (m, 2H, N6-C1”-H), 3.51 and 3.62 (2 × m, 2H, C5′-Ha,b),
3.91 (m, 1H, C4′-H), 4.09 (dd, 1H, C3′-H), 4.51 (dd, 1H, C2′-
H), 5.04 (t, 1H, OH), 5.22 (d, 1H, OH), 5.47 (d, 1H, OH), 5.79
(d, 1H, C1′-H), 8.21 (m, 1H, N6-NH), 8.28 (s, 1H, C8-H); ESI-
MS m/z 450.1 (M + H+)+.
2-Iod o-N6-tetr a h yd r ofu r fu r yla d en osin e (67b): 1H NMR
δ 1.60-1.89 (band, 4H, C3′′-H and C4′′-H), 3.51-4.02 (band,
7H, N6-CH2, C4′-H, C5′-Ha,b, and C5′′-H), 4.10 (dd, 1H, C3′-
H), 4.51 (dd, 1H, C2′-H), 5.05 (t, 1H, OH), 5.22 (d, 1H, OH),
5.48 (d, 1H, OH), 5.80 (d, 1H, C1′-H), 8.15 (m, 1H, N6-H), 8.31
(s, 1H, C8-H); ESI-MS m/z 478.1 (M + H+)+.
N6-n -Bu tyl-2-[(3′′-h ydr oxypr opyl)am in o]aden osin e (74):
1H NMR δ 0.88 (t, 3H, N6-C4′′-H), 1.30 (m, 2H, N6-C3′′-H), 1.53
(m, 2H, N6-C2′′-H), 1.65 (m, 2H, N2-C2′′-H), 3.25-3.63 (band,
6H, N2-C1′′-H, N6-C1′′-H, and C5′-Ha,b), 3.88 (m, 1H, C4′-H),
4.09 (dd, 1H, C3′-H), 4.44 (t, 1H, C3′′-OH), 4.55 (dd, 1H, C2′-
H), 5.11 (br s, 2H, OH), 5.35 (br s, 1H, OH), 5.69 (d, 1H, C1′-
H), 6.16 (br s, 1H, C2-NH), 7.27 (br s, 1H, N6-H), 7.86 (s, 1H,
C8-H); ESI-MS m/z 397.2 (M + H+)+.
N6-n -Bu tyl-2-[(cycloh exylm eth yl)am in o]aden osin e (75):
1H NMR δ 0.88-1.71 (band, 18H, N6-C4′′-H, N6-C3′′-H, N6-
C2′′-H, and N2-cyclohexyl-H), 3.08 (m, 2H, N2-C1′′-H), 3.37 (m,
2H, N6-C1′′-H), 3.51 and 3.61 (2 × m, 2H, C5′-Ha,b), 3.86 (m,
1H, C4′-H), 4.10 (dd, 1H, C3′-H), 4.56 (dd, 1H, C2′-H), 5.10
(br s, 2H, OH), 5.35 (br s, 1H, OH), 5.69 (d, 1H, C1′-H), 6.22
(br s, 1H, C2-NH), 7.32 (br s, 1H, N6-H), 7.85 (s, 1H, C8-H);
ESI-MS m/z 435.3 (M + H+)+.
N6-n -Bu tyl-2-(ben zyla m in o)a d en osin e (76): 1H NMR δ
0.84 (t, 3H, N6-C4′′-H), 1.25 (m, 2H, N6-C3′′-H), 1.46 (m, 2H,
N6-C2′′-H), 3.33 (m, 2H, N6-C1′′-H), 3.49 and 3.61 (2 × m, 2H,
C5′-Ha,b), 3.86 (m, 1H, C4′-H), 4.09 (dd, 1H, C3′-H), 4.46 (d,
2H, N2-CH2), 4.53 (dd, 1H, C2′-H), 5.12 (br s, 2H, OH), 5.34
(br s, 1H, OH), 5.70 (d, 1H, C1′-H), 6.85 (br s, 1H, C2-NH),
7.16-7.47 (band, 6H, N6-H and Ph-H), 7.89 (s, 1H, C8-H); ESI-
MS m/z 429.2 (M + H+)+.
N6-Tetr a h yd r ofu r fu r yl-2-[(3′′-h yd r oxyp r op yl)a m in o]-
2-Iodo-N6-(1′′-n aph th ylm eth yl)aden osin e (68b): 1H NMR
δ 3.53 and 3.63 (2 × m, 2H, C5′-Ha,b), 3.92 (m, 1H, C4′-H),
4.11 (dd, 1H, C3′-H), 4.52 (dd, 1H, C2′-H), 5.04 (t, 1H, OH),
5.08 (br s, 2H, N6-CH2), 5.22 (d, 1H, OH), 5.48 (d, 1H, OH),
5.80 (d, 1H, C1′-H), 7.45 (m, 2H, naphthyl-H), 7.55 (m, 2H,
naphthyl-H), 7.83 (d, 1H, naphthyl-H), 7.93 (d, 1H, naphthyl-
H), 8.29 (d, 1H, naphthyl-H), 8.33 (s, 1H, C8-H), 8.87 (m, 1H,
N6-H); ESI-MS m/z 534.1 (M + H+)+.
1
a d en osin e (77): H NMR δ 1.69-1.87 (band, 6H, N6-C3′′-H,
N6-C4′′-H, and N2-C2′′-H), 3.29-3.78 (band, 10H, N6-CH2, N6-
C5′′-H, N2-C1′′-H, N2-C3′′-H and C5′-Ha,b), 3.86 (m, 1H, C4′-
H), 4.05 (m, 1H, N6-C2′′-H), 4.11 (dd, 1H, C3′-H), 4.45 (br s,
1H, N2-C3′′-OH), 4.56 (dd, 1H, C2′-H), 5.14 (br s, 2H, OH),
5.38 (br s, 1H, OH), 5.70 (d, 1H, C1′-H), 6.25 (br s, 1H, C2-
NH), 7.19 (br s, 1H, N6-H), 7.89 (s, 1H, C8-H); ESI-MS m/z
425.2 (M + H+)+.
N6-(2′′-P h en yleth yl)-2-[(2′′-p h en yleth yl)a m in o]a d en o-
N6-Tetr a h yd r ofu r fu r yl-2-[(cycloh exylm eth yl)a m in o]-
a d en osin e (78): 1H NMR δ 0.88-1.87 (band, 15H, N6-C3′′-H,
N6-C4′′-H, and N2-cyclohexyl-H), 3.07 (m, 2H, N2-CH2), 3.39-
3.78 (band, 6H, N6-CH2, N6-C5′′-H, N2-C1′′-H, N2-C3′′-H and
C5′-Ha,b), 3.86 (m, 1H, C4′-H), 4.05 (m, 1H, N6-C2′′-H), 4.10
(dd, 1H, C3′-H), 4.56 (dd, 1H, C2′-H), 5.11 (br s, 2H, OH), 5.37
(br s, 1H, OH), 5.69 (d, 1H, C1′-H), 6.25 (br s, 1H, C2-NH),
7.17 (br s, 1H, N6-H), 7.87 (s, 1H, C8-H); ESI-MS m/z 463.3
(M + H+)+.
1
sin e (69): H NMR δ 2.82 (t, 2H, N2-C2′′-H), 2.92 (t, 2H, N6-
C2′′-H), 3.47-3.68 (band, 6H, N2-Cl′′-H, N6-C1′′-H and C5′-
H
a,b), 3.90 (m, 1H, C4′-H), 4.13 (dd, 1H, C3′-H), 4.61 (dd, 1H,
C2′-H), 5.13 (br s, 2H, OH), 5.41 (br s, 1H, OH), 5.73 (d, 1H,
C1′-H), 6.38 (br s, 1H, C2-NH), 7.15-7.29 (band, 10H, Ph-H),
7.47 (br s, 1H, N6-H), 7.92 (s, 1H, C8-H); ESI-MS m/z 491.2
(M + H+)+.
N6-[2′′-(p-Hydr oxyph en yl)eth yl]-2-[[2′′-(p-h ydr oxyph en -
yl)eth yl]a m in o]a d en osin e (70): 1H NMR δ 2.66 (t, 2H, N2-
C2′′-H), 2.76 (t, 2H, N6-C2′′-H), 3.38-3.63 (band, 6H, N2-C1”-
H, N6-C1′′-H and C5′-Ha,b), 3.88 (m, 1H, C4′-H), 4.14 (dd, 1H,
C3′-H), 4.61 (dd, 1H, C2′-H), 5.25 (br s, 3H, OH), 5.73 (d, 1H,
C1′-H), 6.38 (br s, 1H, C2-NH), 6.62 (d, 4H, Ph-H), 7.01 (d,
4H, Ph-H), 7.45 (br s, 1H, N6-H), 7.95 (s, 1H, C8-H), 9.30 (br
s, 2H, Ph-OH); ESI-MS m/z 523.2 (M + H+)+.
N6-[2′′-(p-Am in op h en yl)eth yl]-2-[[2′′-(p-a m in op h en yl)-
eth yl]a m in o]a d en osin e (71): 1H NMR δ 2.67 (t, 2H, N2-C2′′-
H), 2.72 (t, 2H, N6-C2′′-H), 3.45-3.68 (band, 6H, N2-C1′′-H,
N6-C1′′-H and C5′-Ha,b), 3.90 (m, 1H, C4′-H), 4.13 (dd, 1H, C3′-
H), 4.61 (dd, 1H, C2′-H), 4.90 (br s, 4H, Ph-NH2), 5.13 (br s,
2H, OH), 5.40 (d, 1H, OH), 5.72 (d, 1H, C1′-H), 6.25 (br s, 1H,
C2-NH), 6.51 (d, 4H, Ph-H), 6.92 (d, 4H, Ph-H), 7.45 (br s, 1H,
N6-H), 7.91 (s, 1H, C8-H); ESI-MS m/z 521.3 (M + H+)+.
N6-[2′′(p -H yd r oxyp h en yl)et h yl]-2-[(2′′-p h en ylet h yl)-
a m in o]a d en osin e (72): 1H NMR δ 2.72 (t, 2H, N2-C2′′-H),
2.88 (t, 2H, N6-C2′′-H), 3.35-3.66 (band, 6H, N2-C1′′-H, N6-
C1′′-H and C5′-Ha,b), 3.88 (m, 1H, C4′-H), 4.14 (dd, 1H, C3′-
H), 4.60 (dd, 1H, C2′-H), 5.25 (br d, 3H, OH), 5.75 (d, 1H, C1′-
H), 6.36 (br s, 1H, C2-NH), 6.64 (d, 2H, Ph-H), 7.01 (d, 2H,
Ph-H), 7.18-7.26 (band, 5H, Ph-H), 7.45 (br s, 1H, N6-H), 7.93
(s, 1H, C8-H), 9.28 (br s, 1H, Ph-OH); ESI-MS m/z 507.2 (M +
H+)+.
N 6-Te t r a h yd r ofu r fu r yl-2-(b e n zyla m in o)a d e n osin e
(79): 1H NMR δ 1.52-1.80 (band, 4H, N6-C3′′-H and N6-C4′′-
H), 3.51-4.10 (band, 7H, N6-CH2, N6-C5′′-H, N2-CH2, C3′-H,
C4′-H, and C5′-Ha,b), 4.56 (dd, 1H, C2′-H), 5.11 (br s, 2H, OH),
5.37 (br s, 1H, OH), 5.70 (d, 1H, C1′-H), 6.98 (br s, 1H, C2-
NH), 7.11-7.38 (band, 6H, N6-H and Ph-H), 7.90 (s, 1H, C8-
H); ESI-MS m/z 457.2 (M + H+)+.
N6-(1′′-Na p h th ylm eth yl)-2-[(3′′-h yd r oxyp r op yl)a m in o]-
a d en osin e (80): 1H NMR δ 1.58 (m, 2H, N2-C2′′-H), 3.22 and
3.27 (2 × m, 4H, N2-C1′′-H and N2-C3′′-H), 3.52 and 3.62 (2 ×
m, 2H, C5′-Ha,b), 3.88 (m, 1H, C4′-H), 4.11 (dd, 1H, C3′-H),
4.38 (t, 1H, C3′′-OH), 4.58 (dd, 1H, C2′-H), 5.11 (m, 4H, N6-
CH2 and OH), 5.36 (br s, 1H, OH), 5.71 (d, 1H, C1′-H), 6.28
(br s, 1H, C2-NH), 7.41-7.96 (band, 8H, C8-H and naphthyl-
H), 8.22 (br s, 1H, N6-H); ESI-MS m/z 481.2 (M + H+)+.
N6-(1′′-Na p h th ylm eth yl)-2-[(cycloh exylm eth yl)a m in o]-
a d en osin e (81): 1H NMR δ 1.30-1.80 (band, 11H, N2-
cyclohexyl-H), 2.97 (m, 2H, N2-CH2), 3.51 and 3.62 (2 × m,
2H, C5′-Ha,b), 3.88 (m, 1H, C4′-H), 4.11 (dd, 1H, C3′-H), 4.58
(dd, 1H, C2′-H), 5.12 (m, 4H, N6-CH2 and OH), 5.38 (br s, 1H,
OH), 5.71 (d, 1H, C1′-H), 6.26 (br s, 1H, C2-NH), 7.41-7.96
(band, 8H, C8-H and naphthyl-H), 8.25 (br s, 1H, N6-H); ESI-
MS m/z 519.3 (M + H+)+.
Gen er a l P r oced u r e for th e P r ep a r a tion of Dia m in o-
a lk yl-F u n ction a lized Tr ityl Resin s. To a slurry of trityl
chloride resin (0.48 mmol) in CH2Cl2 (2.0 mL) were added
triethylamine (2.36 mmol) and a diamine (2.36 mmol). The
reactions were shaken on a platform shaker for 18 h at
ambient temperature. The resins were filtered and washed
with CH2Cl2 (3 × 3 mL), THF (3 × 3 mL), and CH2Cl2 (3 × 3
mL), and then dried in vacuo for 24 h.
N6-(2′′-P h en ylet h yl)-2-[[2′′-(p -h yd r oxyp h en yl)et h yl]-
a m in o]a d en osin e (73): 1H NMR δ 2.69 (t, 2H, N2-C2′′-H),
2.88 (t, 2H, N6-C2′′-H), 3.32-3.63 (band, 6H, N2-C1′′-H, N6-
C1′′-H, and C5′-Ha,b), 3.88 (m, 1H, C4′-H), 4.13 (dd, 1H, C3′-
H), 4.58 (dd, 1H, C2′-H), 5.25 (br s, 3H, OH), 5.74 (d, 1H, C1′-
H), 6.35 (br s, 1H, C2-NH), 6.63 (d, 2H, Ph-H), 7.01 (d, 2H,
Ph-H), 7.18-7.26 (band, 5H, Ph-H), 7.48 (br s, 1H, N6-H), 7.93
(s, 1H, C8-H), 9.20 (br s, 1H, Ph-OH); ESI-MS m/z 507.2 (M +
H+)+.
Gen er a l P r oced u r e for th e P r ep a r a tion of Dia m in o-
a lk yl Nu cleosid e-F u n ction a lized Tr ityl Resin s. To a

4148 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Bressi et al.
slurry of diaminoalkyltrityl resin (0.05 mmol) in CH2Cl2/EtOH
(1:1, 1.0 mL) were added Et3N (0.24 mmol) and either 1a ′ or
1b′ (0.24 mmol). The reactions were heated without stirring
at 60 °C for 18 h. After the reactions were cooled to ambient
temperature, the resins were filtered and washed with CH2-
Cl2/EtOH (1:1, 3 × 3 mL), EtOH (2 × 3 mL), THF (2 × 3 mL),
and CH2Cl2 (3 × 3 mL), and then dried in vacuo for 24 h.
Gen er a l P r oced u r e for th e Clea va ge of Dia m in oa lk yl
Nu cleosid e fr om Tr ityl Resin . A slurry of diaminoalkyl
nucleoside trityl resin in 30% hexafluoro-2-propanol in CH2-
(d, 2H, OH), 5.27 (br s, 1H, C2′′-OH), 5.39 (m, 2H, OH), 5.41
(d, 2H, OH), 5.88 (d, 2H, C1′-H), 7.83 (br s, 2H, N6-H), 8.22
and 8.38 (2 × s, 4H, C2-H and C8-H); ESI-MS m/z 591.2 (M +
H+)+.
N6,N6′-2′′-H yd r oxyp r op a n ed iylb is-2-a m in oa d en osin e
(18b′): 1H NMR δ 3.54 and 3.65 (2 × m, 8H, C5′-Ha,b and C1′′-
H), 3.73 (m, 1H, C2′′-H), 3.89 (m, 2H, C4′-H), 4.08 (m, 2H, C3′-
H), 4.49 (m, 2H, C2′-H), 5.03 (br s, 1H, C2”-OH), 5.11 (d, 2H,
OH), 5.37 (m, 4H, OH), 5.73 (d, 2H, C1′-H), 6.25 (br s, 4H,
C2-NH2), 7.80 (br s, 2H, N6-H), 7.98 (s, 2H, C8-H); ESI-MS
m/z 621.2 (M + H+)+.
30
Cl2 (2.0 mL) was shaken on a platform shaker for 15 min at
N6,N6′-Bu ta n ed iylbis-2-a m in oa d en osin e (19b′): 1H NMR
δ 1.62 (br s, 4H, C2′′-H), 3.39-4.65 (band, 8H, C5′-Ha,b and
C2′′-H), 3.88 (m, 2H, C4′-H), 4.08 (m, 2H, C3′-H), 4.48 (m, 2H,
C2′-H), 5.10 (br s, 2H, OH), 5.34 (br s, 2H, OH), 5.68 (br s,
2H, OH), 5.71 (d, 2H, C1′-H), 5.82 (br s, 4H, C2-NH2), 7.37 (br
s, 2H, N6-H), 7.88 (s, 2H, C8-H); ESI-MS m/z 619.3 (M + H+)+.
N6,N6′-P en tan ediylbis-2-am in oaden osin e (20b′): 1H NMR
δ 1.34 (m, 2H, C3′′-H), 1.58 (m, 4H, C2′′-H), 3.42 (m, 4H, C1′′-
H), 3.55 and 3.66 (2 × m, 4H, C5′-Ha,b), 3.88 (m, 2H, C4′-H),
4.08 (m, 2H, C3′-H), 4.48 (m, 2H, C2′-H), 5.10 (br s, 2H, OH),
5.36 (br s, 2H, OH), 5.65 (br s, 2H, OH), 5.71 (d, 2H, C1′-H),
5.78 (br s, 4H, C2-NH2), 7.34 (br s, 2H, N6-H), 7.88 (s, 2H, C8-
H); ESI-MS m/z 633.3 (M + H+)+.
In h ibition Stu d ies. All 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 monitoring absorption at 340
nm using a quartz cuvette. Activity for PGK was measured in
the direction of NAD+ formation, while activity for GAPDH
and GPDH was measured in the direction of NADH formation.
For the GAPDH assay, fresh glyceraldehyde-3-phosphate was
prepared from its diethyl acetal as described by the manufac-
turer. All compounds tested were dissolved in DMSO-d6 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 calcu-
lated as percent of control using the initial velocities measured
from 0-2 min. Inhibitor concentration in the cuvette was
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.
D-(-)-3-Phosphoglyceric acid [tri(cyclohexylammonium) salt],
adenosine-5′-triphosphate (disodium salt), D-glyceraldehyde-
3-phosphate diethyl acetal [di(cyclohexylammonium) salt],
dihydroxyacetone phosphate (lithium salt), NAD+, â-NADH,
and rabbit muscle PGK (sulfate-free lyophilized powder) were
purchased from Sigma Chemical Co. Recombinant T. brucei
PGK, GAPDH, and GPDH were expressed and purified as
described by Misset et al.37
ambient temperature. The resins were filtered and then
washed with CH2Cl2/EtOH (1:1, 3 × 3 mL) and EtOH (3 × 3
mL). The washings were combined with the initial filtrate and
stripped to dryness via rotary evaporation to give the desired
diaminoalkyl nucleosides, 16a -20a and 16b-20b, in greater
than 90% yield and in greater than 95% purity as determined
by HPLC and NMR.
2-Am in o-N6-(2′′-a m in oeth yl)a d en osin e (16b): 1H NMR
δ 2.72 (t, 2H, C2′′-H), 3.38 (br s, 2H, C1′′-H), 3.51 and 3.62 (2
× m, 2H, C5′-Ha,b), 3.89 (m, 1H, C4′-H), 4.08 (dd, 1H, C3′-H),
4.49 (dd, 1H, C2′-H), 5.71 (d, 1H, C1′-H), 5.75 (br s, 2H, C2-
NH2), 7.20 (br s, 1H, N6-H), 7.90 (s, 1H, C8-H); ESI-MS m/z
326.2 (M + H+)+.
2-Am in o-N6-(3′′-a m in op r op yl)a d en osin e (17b): 1H NMR
δ 1.62 (m, 2H, C2′′-H), 2.58 (t, 2H, C3′′-H), 3.42 (br s, 2H, C1′′-
H), 3.51 and 3.62 (2 × m, 2H, C5′-Ha,b), 3.89 (m, 1H, C4′-H),
4.08 (dd, 1H, C3′-H), 4.49 (dd, 1H, C2′-H), 5.70 (d, 1H, C1′-H),
5.74 (br s, 2H, C2-NH2), 7.30 (br s, 1H, N6-H), 7.88 (s, 1H, C8-
H); ESI-MS m/z 340.2 (M + H+)+.
N6-(3′′-Am in o-2′′-h yd r oxyp r op yl)a d en osin e (18a ): 1H
NMR δ 2.58 (dd, 2H, C3′′-H), 3.41-3.73 (band, 5H, C5′-Ha,b
,
C2′′-H and C1′′-H), 3.95 (m, 1H, C4′-H), 4.13 (dd, 1H, C3′-H),
4.59 (dd, 1H, C2′-H), 5.87 (d, 1H, C1′-H), 7.69 (br s, 1H, N6-
H), 8.20 and 8.34 (2 × s, 1H, C2-H and C8-H); ESI-MS m/z
341.2 (M + H+)+.
2-Am in o-N6-(3′′-a m in o-2′′-h yd r oxyp r op yl)a d en osin e
(18b): 1H NMR δ 2.54 (dd, 2H, C3′′-H), 3.41-3.73 (band, 5H,
C5′-Ha,b, C2′′-H and C1′′-H), 3.89 (m, 1H, C4′-H), 4.08 (dd, 1H,
C3′-H), 4.48 (dd, 1H, C4′-H), 5.70 (d, 1H, C1′-H), 5.81 (br s,
2H, C2-NH2), 7.19 (br s, 1H, N6-H), 7.91 (s, 1H, C8-H); ESI-
MS m/z 356.2 (M + H+)+.
1
2-Am in o-N6-(4′′-a m in obu tyl)a d en osin e (19b): H NMR
δ 1.36 (m, 2H, C3′′-H), 1.62 (m, 2H, C2′′-H), 2.54 (t, 2H, C4′′-
H), 3.40 (br s, 2H, C1′′-H), 3.51 and 3.62 (2 × m, 2H, C5′-Ha,b),
3.89 (m, 1H, C4′-H), 4.08 (dd, 1H, C3′-H), 4.49 (dd, 1H, C2′-
H), 5.70 (m, 3H, C1′-H and C2-NH2), 7.28 (br s, 1H, N6-H),
7.88 (s, 1H, C8-H); ESI-MS m/z 354.2 (M + H+)+.
2-Am in o-N6-(5′′-a m in op en tyl)a d en osin e (20b): 1H NMR
δ 1.27-1.38 (band, 4H, C3′′-H and C4′′-H), 1.54 (m, 2H, C2′′-
H), 2.50 (t, 2H, C4′′-H), 3.40 (br s, 2H, C1′′-H), 3.51 and 3.62
(2 × m, 2H, C5′-Ha,b), 3.89 (m, 1H, C4′-H), 4.08 (dd, 1H, C3′-
H), 4.49 (dd, 1H, C2′-H), 5.70 (m, 3H, C1′-H and C2-NH2), 7.25
(br s, 1H, N6-H), 7.88 (s, 1H, C8-H); ESI-MS m/z 368.2 (M +
H+)+.
P GK In h ibition Assa y. Final concentration of the com-
ponents in the assay buffer was as follows: 5.0 mM MgSO4,
1.0 mM EDTA, 7.1 mM NaHCO3, 2.0 mM DTT, 5.1 mM 3-PGA,
1.1 mM ATP, 0.42 mM â-NADH, T. brucei PGK, and GAPDH
(200-fold molar excess of PGK).
Syn th esis of Alk yl-Br id ged Bisa d en osin es 16a ′-20a ′
a n d 16b′-20b′. These compounds were either synthesized as
described by Agathocleous et al.32 or isolated as impurities
generated from the synthesis of compounds 16a -20a and
16b-20b via Scheme 1.
GAP DH In h ibition Assa y. Final concentration 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+, T. brucei GAPDH, and 1.87 mM
glyceraldehyde-3-phosphate.
N6,N6′-Eth an ediylbis-2-am in oaden osin e (16b′): 1H NMR
δ 3.45 and 3.60 (2 × m, 6H, C5′-Ha,b and C1′′-H), 3.88 (m, 2H,
C4′-H), 4.08 (m, 2H, C3′-H), 4.48 (m, 2H, C2′-H), 5.10 (br s,
2H, OH), 5.39 (br s, 4H, OH), 5.71 (d, 2H, C1′-H), 6.05 (br s,
4H, C2-NH2), 7.93 (s, 2H, C8-H), 8.06 (br s, 2H, N6-H); ESI-
MS m/z 591.2 (M + H+)+.
GP DH In h ibition Assa y. Final concentration of the com-
ponents in the assay buffer was as follows: 0.60 mM NAD+,
19.27 mM glycerol-3-phosphate, and T. brucei GPDH.
Cr ysta llogr a p h y. Our crystallization experiments utilized
a truncated form of PGK which is made by exchanging the
last 34 residues of T. brucei glycosomal PGK with the C-
terminal 14 amino acids of T. brucei cytosolic PGK.38 It is
believed that the highly flexible native C-terminal region of
PGK is not conducive to obtaining crystal structures. For the
crystallization of this truncated PGK with 46b, a solution of
pure protein was concentrated to 6.0-7.0 mg/mL and mixed
with a 100-fold molar excess of 46b. Crystals of the PGK/46b
complex were obtained after a few weeks from a reservoir
solution containing 0.1 M Tris/HCl (pH ) 7.5) and 2.4 M
(NH4)2SO4 by the hanging drop vapor diffusion method.
N6,N6′-P r op a n ed iylb is-2-a m in oa d en osin e (17b ′): 1H
NMR δ 1.88 (br s, 2H, C2′′-H), 3.45-3.60 (band, 8H, C5′-Ha,b
and C1′′-H), 3.86 (m, 2H, C4′-H), 4.10 (m, 2H, C3′-H), 4.50 (m,
2H, C2′-H), 5.12 (br s, 2H, OH), 5.39 (br s, 4H, OH), 5.72 (d,
2H, C1′-H), 6.01 (br s, 4H, C2-NH2), 7.79 (br s, 2H, N6-H), 7.93
(s, 2H, C8-H); ESI-MS m/z 605.3 (M + H+)+.
N6,N6′-2′′-Hyd r oxyp r op a n ed iylbisa d en osin e (18a ′): 1H
NMR δ 3.50-3.68 (band, 9H, C5′-Ha,b, C1′′-H and C2′′-H), 3.96
(m, 2H, C4′-H), 4.13 (m, 2H, C3′-H), 4.61 (m, 2H, C2′-H), 5.19

Adenosine Analogues as Inhibitors of T. brucei PGK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4149
One of these crystals (0.3 × 0.3 × 0.02 mm3) was quickly
transferred to a solution composed of 80% reservoir solution
and 20% glycerol and then cryocooled to 100 K. Data were
collected at this temperature on a MAR area detector mounted
on an SSRL beam line 9-1. Frames (exposure: 1 min; crystal
rotation: 0.5°) were processed using the software programs
DENZO39 and SCALEPACK.40 The Rmerge for all data between
30 and 3.0 Å was 7.0% with a 18.8-fold redundancy (Table 6).
The crystal unit cell indicated the possible presence of several
molecules in the asymmetric unit (VM ) 4.3 Å3/Da of protein
for 2, 2.9 Å3/Da for 3, and 2.15 Å3/Da for 4 PGK molecules in
the asymmetric unit).
of the compounds on growth of mammalian host cells (murine
3T3 fibroblasts) were determined using the Alamar blue
method.48
Ack n ow led gm en t. The presented work was sup-
ported 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 Fel-
lowship. We are grateful to Dr. Paul A. Michels for
providing the trypanosome PGK and GAPDH enzymes
and to Dr. Steven Suresh for providing the trypanosome
GPDH enzyme. We thank Farideh Ghomashchi for her
assistance with enzyme assays, Lisa N. Nguyen for her
assistance with generating the cell culture data, and the
staff of the Stanford Synchrotron Radiation Laboratory
for their support with X-ray data collection.
Several T. brucei PGK structures, which have been deter-
mined in our laboratory,13,14 were used as probes for molecular
replacement procedures incorporated in the program AmoRe.41
The signal-to-noise ratio increased when the partially opened
form PGK structure (1.6 Å resolution)14 was used instead of
the closed form PGK structure (2.8 Å resolution).13 On the
basis of this result, a new ‘open’ form probe was built by
superimposing the N- and C-terminal domains of the T. brucei
PGK structure onto that of horse PGK,12 which adopts an open
conformation. The peaks obtained from this ‘open’ form probe
gave the highest signal-to-noise ratio. The orientations ob-
tained from the rotation function were subsequently used in
the translation function. The translation function gave accept-
able results for each subunit with reasonable packing when
both subunits were placed in the crystal unit cell. After the
introduction of two molecules in the asymmetric unit, the
correlation coefficient and the R-factor were 0.315 and 0.484,
respectively.
Refinement was performed with the program X-PLOR42 and
manual rebuilding of the models was performed with the
program O.43 All data between 15.0 and 3.0 Å were used and
a bulk solvent correction was applied. Rigid body refinement
was performed to position the two molecules more precisely.
Subsequently, refinement of the proteins using strict noncrys-
tallographic symmetry was performed. Visual inspection of an
electron density map confirmed the presence of the 46b in the
active site and allowed the incorporation of two inhibitors into
the model (Figure 2). Further refinement was performed
followed by minor modifications of the protein.
Registr y Nu m ber s: 2b, 5059-59-6; 3a , 14357-08-5;
3b, 177949-65-4; 4a , 17270-22-3; 4b, 26783-33-5; 4c,
35109-89-8; 5a , 17270-23-4; 6a , 23096-10-8; 6b, 26784-
37-2; 7a , 26293-51-6; 8a , 126434-89-7; 10b, 26775-39-
3; 10c, 56720-65-1; 11a , 35662-05-6; 12a , 35662-06-7;
13a , 103842-14-4; 13b, 103842-19-9; 14a , 182950-54-5;
16a , 35662-04-5; 16a ′, 60687-64-1 16c, 77112-18-6; 17a ,
34436-52-7; 17a ′, 90275-18-6; 19a , 60687-66-3; 19a ′,
60687-65-2; 19c, 77112-19-7; 20a , 64630-73-5; 20a ′,
64630-69-9; 21a , 41552-82-3; 21b , 120524-28-9; 21c,
57972-89-1; 22a , 36396-99-3; 22b , 26783-44-8; 22c,
53296-12-1; 23a , 97826-48-7; 24a , 41552-83-4; 25a ,
36397-01-0; 26a , 4338-47-0; 26b , 26783-39-1; 27b ,
26783-32-4; 27c, 56720-66-2; 28a , 20315-90-6; 28b ,
26775-40-6; 29a , 23707-33-7; 29b , 26783-37-9; 30a ,
35940-03-5; 31a , 35940-04-6; 32a , 23707-32-6; 32b ,
26783-35-7; 33a , 23660-97-1; 34a , 23660-96-0; 34b ,
26775-37-1; 35a , 23660-98-2; 35b , 26775-34-8; 36a ,
23666-24-2; 37a , 20125-39-7; 37b , 26775-35-9; 37c,
56720-67-3; 38a , 109956-81-2; 38b , 26783-34-6; 38c,
124498-68-6; 39a , 54712-70-8; 39c, 124498-53-9; 40a ,
101565-57-5; 40c, 124499-05-4; 41a , 21924-65-2; 43a ,
98383-40-5; 43b , 98383-43-8; 44a , 101565-68-8; 46a ,
41552-94-7; 46b , 26888-84-4; 46c, 124499-08-7; 47a ,
54241-03-1; 48a , 101565-56-3; 49a , 101565-64-4; 50a ,
101565-65-5; 52a , 101565-69-9; 53a , 101565-70-2; 54a ,
101565-71-3; 54c, 124499-07-6; 55a , 36799-20-9; 55b,
26783-40-4; 58a , 89705-21-5; 58c, 161536-30-7; 69,
120524-43-8; 82, 120524-46-1.
The final model consists of 832 protein residues and two
46b inhibitor molecules. The R-factor (Rfree) dropped to 0.217
(0.282) with acceptable deviations from ideal geometry (Table
6). Analysis of main chain torsion angles using PROCHECK44
showed that 87% of the residues are located in the most
favored regions with 13% occurring in the additional allowed
regions of the Ramachandran plot.
P a r a site Cu ltu r es a n d Dr u g Scr een in g Assa ys. Blood-
stream forms of T. brucei brucei (strain 427 from K. Stuart,
Seattle Biomedical Research Institute, Seattle, WA) were
cultured in HMI-9 medium containing 10% fetal bovine serum,
penicillin, and streptomycin at 37 °C with 5% CO2.45 Blood-
stream forms of T. brucei rhodesiense (strain STIB900 from
S. Croft, London School of Hygiene and Tropical Medicine,
London, U.K.) were cultured in HMI-18 medium under the
same conditions.46 Drug sensitivity of the T. brucei strain was
determined in 96-well microtiter plates with an initial inocu-
lum of 5 × 103 trypomastigotes/well. Drugs were added in
serial dilutions for a final volume of 200 µL/well. Parasite
growth was quantified after 48 h by the addition of Alamar
blue (Alamar Biosciences, Sacramento, CA).47 Alamar blue
quantitation 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 co-culture with mouse 3T3 fibroblasts
in RPMI 1640 containing 10% fetal bovine serum, penicillin,
and streptomycin at 37 °C with 5% CO2. Drug sensitivity
studies with mammalian stage T. cruzi were carried out using
the â-galactosidase assay as described.48 The inhibitory effects
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