Structure-based design of pteridine reductase inhibitors targeting African sleeping sickness and the leishmaniases

Article abstract of DOI:10.1021/jm901059x

Pteridine reductase (PTR1) is a target for drug development against Trypanosoma and Leishmania species, parasites that cause serious tropical diseases and for which therapies are inadequate. We adopted a structure-based approach to the design of novel PTR1 inhibitors based on three molecular scaffolds. A series of compounds, most newly synthesized, were identified as inhibitors with PTR1-species specific properties explained by structural differences between the T. brucei and L. major enzymes. The most potent inhibitors target T. brucei PTR1, and two compounds displayed antiparasite activity against the bloodstreamformof the parasite. PTR1 contributes to antifolate drug resistance by providing amolecular bypass of dihydrofolate reductase(DHFR) inhibition.Therefore, combining PTR1 andDHFRinhibitors might improve therapeutic efficacy. We tested two new compounds with known DHFR inhibitors. A synergistic effect was observed for one particular combination highlighting the potential of such an approach for treatment of African sleeping sickness.

Full text of DOI:10.1021/jm901059x

J. Med. Chem. 2010, 53, 221–229 221
DOI: 10.1021/jm901059x
Structure-Based Design of Pteridine Reductase Inhibitors Targeting African Sleeping Sickness
and the Leishmaniases†
Lindsay B. Tulloch,‡ Viviane P. Martini,‡,§ Jorge Iulek,‡,§ Judith K. Huggan,# Jeong Hwan Lee,# Colin L. Gibson,#
Terry K. Smith,#, Colin J. Suckling,# and William N. Hunter*,‡
‡Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, U.K.,
§
´
Universidade Estadual de Ponta Grossa, Departamento de Quımica, Av. Carlos Cavalcanti, 4748 Uvaranas, 84030-000, Ponta Grossa,
Paranaꢀ, Brazil, and #WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow,
G1 1XL, U.K. Present address: BMS, University of St. Andrews, North Haugh, St. Andrews, Fife, KY16 9ST, U.K.
Received July 21, 2009
Pteridine reductase (PTR1) is a target for drug development against Trypanosoma and Leishmania species,
parasites that cause serious tropical diseases and for which therapies are inadequate. We adopted a
structure-based approach to the design of novel PTR1 inhibitors based on three molecular scaffolds.
A series of compounds, most newly synthesized, were identified as inhibitors with PTR1-species specific
properties explained by structural differences between the T. brucei and L. major enzymes. The most
potent inhibitors target T. brucei PTR1, and two compounds displayed antiparasite activity against the
bloodstream form of the parasite. PTR1 contributes to antifolate drug resistance by providing a molecular
bypass of dihydrofolate reductase (DHFR) inhibition. Therefore, combining PTR1 and DHFR inhibitors
might improve therapeutic efficacy. We tested two new compounds with known DHFR inhibitors.
A synergistic effect was observed for one particular combination highlighting the potential of such an
approach for treatment of African sleeping sickness.
Introduction
from the host.12 A key enzyme of folate metabolism is
dihydrofolate reductase (DHFR, EC 1.5.1.3, Figure 1), a
target for the antifolates methotrexate (MTX), pyrimeth-
amine (PYR), and trimethoprim (TMP).13 A mechanism that
contributes to trypanosomatid resistance to typical antifolates
is amplification of the gene encoding the NADPH-dependent
pteridine reductase 1 (PTR1, EC 1.5.1.33), an enzyme unique
to these parasites.14 PTR1 catalyzes reduction of biopterin to
dihydrobiopterin (H2B), H2B to tetrahydrobiopterin (H4B,
Figure 1b), and since it can reduce other pterins/folates, it
provides a bypass for DHFR inhibition.7,14 PTR1overexpres-
sion promotes antifolate resistance in T. cruzi,15 and a gene
knockout in L. major is lethal unless a supplement of reduced
biopterin is provided.16 Even in the presence of reduced
biopterin the modified parasites display increased susceptibi-
lity to antifolates.14,16 These observations suggest that dual
DHFR-PTR1 inhibition may provide a successful treatment
for trypanosomatid infections. Potent DHFR inhibitors are
already known, and we worked on design of novel PTR1
inhibitors concentrating on the enzyme from T. brucei
(TbPTR1), since this organism causes the disease with greatest
unmet medical need, HAT.
Antifolates are exploited to treat malaria, bacterial infec-
tions, various cancers, rheumatoid arthritis, and psoriasis.1,2
However, despite such widespread applications, they are
ineffective against the protozoan parasites Trypanosoma and
Leishmania species, the causal agents of neglected diseases
such as human African trypanosomiasis (HAT,a Sleeping
Sickness) and the different forms of leishmaniasis. This is
surprising because these parasites are folate and pterin auxo-
trophs, totally reliant on pteridine salvage from their hosts.3,4
In mammals, biopterin and reduced derivatives are cofac-
tors for aromatic amino acid hydroxylations, the biosynthesis
of neurotransmitters and nitric oxide signaling,5 and oxida-
tion of glycerol ethers.6 Although a role in trypanosomatids is
less clear, biopterins are essential for metacyclogenesis and
implicated in resistance to reactive oxygen and nitrogen
species in Leishmania.7-10 More is known about folates in
trypanosomatid biology, where they contribute to DNA and
protein synthesis and cellular methylation (Figure 1a).11 In
mammals, folate cofactors contribute to the same processes
but in addition also to purine biosynthesis, an aspect of
metabolism lost in trypanosomatids who acquire purines
We identified three scaffolds to support the design of new
PTR1 inhibitors based on structural data for TbPTR1 and the
L. major enzyme (LmPTR1).17-20 With rounds of molecular
modeling and design, chemical synthesis, enzyme assays, and
structure determination of 14 TbPTR1-ligand complexes we
developed potent inhibitors for both LmPTR1 and TbPTR1.
The inhibitors display selectivity with respect to the ortho-
logues, and the structural basis of such discrimination is des-
cribed. The inhibitors are cytotoxic to cultured bloodstream
form (BSF) T. brucei with micromolar potency. Strikingly,
†Coordinates and diffraction data have been deposited with the
Protein Data Bank under accession codes 3BMC, 3BMN, 3BMO,
3BMQ, 3JQ6, 3JQ7, 3JQ8, 3JQ9, 3JQA, 3JQB, 3JQC, 3JQD, 3JQE,
3JQF, 3JQG.
*To whom correspondence should be addressed. Phone: þ44 1382
385745. Fax: þ44 1382 385764. E-mail: w.n.hunter@dundee.ac.uk.
a Abbreviations: BSF, bloodstream form; DHFR, dihydrofolate re-
ductase; HAT, human African trypanosomiasis; Lm, Leishmania major;
MTX, methotrexate; PTR1, pteridine reductase; PYR, pyrimethamine;
TMP, trimethoprim; Tb, Trypanosoma brucei.
r
2009 American Chemical Society
Published on Web 11/16/2009
pubs.acs.org/jmc

222 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Tulloch et al.
Figure 1. (a) Folate cycle and cellular processes supported in trypanosomatids. Enzymes are shown next to the reactions that they catalyze. The
dimeric bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS, PDB code 2H2Q) used by trypanosomatids is shown in the center
of the cycle with DHFR and TS domains colored blue and red, respectively. The PTR1 tetramer is shown bottom left next to the DHFR catalyzed
reactions, which PTR1 can also catalyze. T. brucei, unlike L. major, lacks serine hydroxymethyl transferase (SHMT), and the glycine cleavage
complex (GCC) accomplishes synthesis of CH2-H4F.10,41 (b) Two-stage reduction of biopterin to H2B and H4B catalyzed by PTR1.
potency is improved when one of the new PTR1 inhibitors is
used in combination with MTX.
the nicotinamide and Phe97. Here, the ribose and a phos-
phate of the cofactor, Ser95, and two catalytically important
residues, Asp161 and Tyr174, are positioned to interact with
ligands (Figure 3a).18
Results and Discussion
The structures of substrate/product complexes of PTR1
are highly similar. The pterin N8 accepts a hydrogen bond
from Tyr174 OH, and O4 accepts hydrogen bonds from
Arg14 and water, which bridges to the cofactor pyrophos-
phate (Figure 3b). The orientation of substrate/product
pteridines is distinct from that of MTX, which is flipped
180° such that MTX N8 interacts with the cofactor pyrophos-
phate via a water bridge and N4 donates a hydrogen bond
to Tyr174 OH (Figure 3a). The pteridines of MTX and
substrates/products have the potential to donate or accept
eight hydrogen bonds, and the difference in the orientations
of the folate and MTX pterins appears driven to maximize
PTR1 Structure and Organization of the Active Site. PTR1
is a tetrameric short-chain oxidoreductase with a single R/β-
domain subunit constructed around a seven-stranded paral-
lel β-sheet sandwiched between two sets of R-helices, a
Rossmann fold repeat (Figure 2).19 An elongated active site
is formed primarily by a single subunit but with one end
created by the C-terminus of a partner subunit. A feature of
the short-chain oxidoreductase family is the presence of a
flexible substrate-binding loop which links β6 to R6, posi-
tioned on one side of the active site (Figure 2). NADPH
contributes to the formation of the catalytic center between

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 223
Residues 207-215 in TbPTR1 form the substrate-binding
loop (Figure 2). In LmPTR1 this loop is conformationally
labile.20 The rms fit for all atoms for this stretch of residues is
˚
3.0 A when comparing substrate and MTX complexes. In
TbPTR1 the loop appears more rigid, the rms fit for the
˚
folate and MTX complexes is 0.6 A over these residues, yet it
retains enough flexibility to accommodate ligands in either
the substrate-like or MTX-like orientation (Figure 3c). In
TbPTR1, the position of R6 puts Trp221 near the pABA
group on one side of the substrate, with Pro210 on the other
side (Figure 3a). The presence of the tryptophan/proline
combination in TbPTR1 reduces the size of the pABA
binding region and introduces a significant chemical change
in this area of the active site compared to LmPTR1 which
presents a histidine/aspartate combination at the corres-
ponding positions.17
Scaffold Identification and Molecular Design. The pterin
orientations of MTX and folate, with their distinctive hydro-
gen-bonding and π-stacking interactions with PTR1, pro-
vide frameworks from which to derive novel inhibitors, and
three scaffolds for ligand design were identified. The MTX-
like framework provided scaffold I (Figure 3d, Table 1) with
modification at C6 and C7 providing capacity to generate
additional interactions and improve affinity for the target.
A substrate-like pyrrolo[2,3-d]pyrimidine framework was
selected as scaffold II (Figure 3d, Table 2), with C7 and C8 as
suitable branch points. The simplest framework that should
retain extensive interactions with the enzyme is 2,4-diamino-
pyrimidine with a hydrogen bond acceptor group at C6. This
provided scaffold III (Figure 3d, Table 3).
The scaffolds formed the basis for molecular modeling. By
varying substituents at the branch points, we sought to
enhance interactions with PTR1 or to generate ideas for
compounds that might allow us to investigate the relative
importance of certain active site features with respect to
ligand affinity. Consideration was given to the types of
substituents commonly exploited in medicinal chemistry
and to synthetic tractability.22,23 The majority of sought com-
pounds required the development of new synthetic proto-
cols.23 The synthesis of two compounds are reported here.
Potential inhibitors were assayed to determine inhibition
properties against PTR1 and the most potent prioritized
for structural characterization. On the basis of this first
round of results, further modeling, syntheses, enzymatic
assays, and structural characterization were carried out to
investigate the structure-activity relationship around each
scaffold and to improve affinity for the target. A series of
high-resolution crystal structures of TbPTR1 with the new
inhibitors and folate were determined. Supporting Informa-
tion carries experimental details, crystallographic results
(Tables S1-S3), and figures (S1-S3) depicting ligands and
interactions within the PTR1 active site.
Scaffold I Is More Effective against LmPTR1 Than
TbPTR1. Scaffold I compounds 1 and 2 are LmPTR1
inhibitors.17 1 displays Ki = 0.24 μM, but it is not as effective
as MTX (Ki = 0.039 μM, Table 1). By comparison, 2 is a
relatively poor inhibitor of LmPTR1 (Ki = 3.4 μM). Scaffold
I appears less effective for inhibition of TbPTR1 (Table 1).
The complex structures of 1 and 2 with TbPTR1 reveal that
they both adopt the MTX orientation (Figures 4a and S1).
The pteridine of 2 binds with the phenyl substituent placed in
a hydrophobic section of the active site, forming an edge-to-
face contact with Trp221. However, there is a potential clash
of the amine substituent at C7 with Pro210. The structure of
Figure 2. PTR1 subunit architecture and position of the active site.
(a) Side view of the subunit of the ternary complex with cofactor and
folate. R6, β6, and the substrate binding loop are colored red. The
cofactor and folate are depicted as blue and black sticks, respec-
tively. (b) Orthogonal view to (a) in the orientation used for all other
molecular images. Trp221 is represented as stick model on R6.
hydrogen-bonding capacity. The position of hydrogen bond
donors/acceptors at the 4- and 8-positions determines the
orientation such that an acceptor is placed to interact with a
water molecule that in turn interacts with pyrophosphate.
The orientation of the MTX pteridine matches five of these
acceptor/donor groups with those of substrates and products
(Figure 3c). N8 of biopterin/folate is an acceptor, but in H2B
and H2F it is a donor. Since the partner for interaction is the
hydroxyl of Tyr174, able to function as donor or acceptor,
then N8 can match with MTX N4. The two remaining
hydrogen bonds that might be formed involve groups acces-
sible to solvent (O4 and N4 for substrates; N4 and N5 for
MTX), and therefore, whether they are donor or acceptor is a
moot point. The proximity of the phosphate to MTX N1
suggests that, as observed when MTX binds DHFR,21 the
inhibitor is protonated (Figure 3c). For both ligands the
aminobenzoate group is directed toward Trp221 with the
glutamate extruding from the active site, exposed to solvent
(Figure 3a,b).

224 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Tulloch et al.
Figure 3. (a) TbPTR1 in complex with MTX.17 Cys168 is modified by addition of dimethylarsinoyl. Atoms are colored as follows: N, blue; O,
red; As, purple; P, orange; S, yellow; C of PTR1 and NADPH, gray; C of ligands, pale-yellow. An exception is made for the side chain of Phe97,
which for the purpose of clarity, since it is directly over the ligand binding position, is shown in thin dark gray lines. Hydrogen bonds are
depicted as dashed lines, and water molecules are shown as red spheres. (b) TbPTR1 with folate. In several of the new structures, as seen here,
Cys168 is oxidized to sulfenic acid and a number of others are modified by dithiothreitol (DTT, Supporting Information). (c) MTX and folate
in the orientation adopted when bound to PTR1. Hydrogen bond donor and acceptor groups are designated D and A, respectively. For
comparative purposes the structure of pyrimethamine (PYR), a potent DHFR inhibitor, is also shown. (d) Three scaffolds based on the pterins
of MTX and folate. For scaffold II, X = O or S. For scaffold III, X = CH2 or S.
the complex reveals that in three of the subunits that con-
stitute the asymmetric unit, the β6-R6 loop adopts a con-
formation in which the polypeptide is further away from the
catalytic center thereby accommodating bulkier substituents
on this scaffold. In the fourth subunit of the asymmetric unit
the loop is completely disordered. The isopropyl substituents

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 225
Table 3. Scaffold III
Table 1. Scaffold Ia
Ki (μM)
compd
R
X
TbPTR1
LmPTR1
14
15
16
17
18
NH2
NH
S
>35
>35
5.4
>27
>27
∼27
0.60
2.7
Ki (μM)
c-C3H5
compd
MTX
R1
R2
TbPTR1 LmPTR1
C6H4CH3
CH2C6H5
CH2C6H4OCH3 (para)
CH2N(CH3)C6H5
CONHCH(COOH)
CH2CH2COOH
CH (CH3)2
H
0.152
0.039
S
3.2
18
S
1
2
3
CH(CH3)2 3.3
0. 24
3.4
conformations in the other two subunits. A similar observa-
tion has been made for MTX-based inhibitors of LmPTR1.24
When the MTX orientation is adopted, the β6-R6 loop is
pushed away from the active site by the dimethyl substituents
in a similar manner as when 2 binds. In the substrate-like
orientation the dimethyl substituents at C7 of 3 would clash
with the nicotinamide and Phe97, therefore restricting access
to the active site (data not shown). As a result, N2 can only
interact with Ser95 and the cofactor pyrophosphate via a
water bridge in a fashion similar to that of TMP when it
binds LmPTR1.19
C6H5
CH3
NH2
(CH3)2
1.2
>35
12
a The Ki values of MTX for TbPTR1 and LmPTR1 are from ref 15.
Table 2. Scaffold II
Scaffold II Is More Effective against TbPTR1 Than
LmPTR1. We first investigated if there was scope for modifi-
cation at the 2-amino group and at N9 of this scaffold.
Modeling suggested that inhibition would only result if the
compounds adopted a new orientation in the active site (data
not shown). The addition of a tertiary butylcarbonyl at the
2-amino group resulted in a complete loss of inhibition likely
due to steric clash with the nicotinamide and Ser95. In similar
fashion, the placement of an allyl group at N9 abrogated
binding probably because of steric clash with Tyr174. These
positions are inappropriate for further development.
Scaffold II compounds adopt the substrate-like orientation
when they bind TbPTR1 (Figure 4b,c and Figure S2A-G).
Compound 4 is a poor inhibitor (Ki > 27 μM, Table 2), and
the substitution of O4 by S4 (5) had no effect (Table 2). The
addition of a nitrile at C7 (6) improved potency against
TbPTR1 (Ki = 5.8 μM) through additional van der Waals
interactions with the β6-R6 loop of TbPTR1. The addition
of 7-phenylethyl (7) dramatically improved inhibition against
TbPTR1 (Ki = 0.96 μM). Although the phenyl group is placed
near Trp221 (Figure 4b), the two-carbon link is too short to
allow formation of π-πinteractions between these aromatic side
chains.
Modifications of scaffold II at C8, designed to occupy a
solvent filled cavity between the catalytic center and Met163,
Cys168 together with His267 of the neighboring subunit,
proved highly significant (Table 2). While the 7-carbonitrile
is maintained, addition of 8-bromo (8) improved potency
against TbPTR1 slightly (Ki = 3.9 μM) and was useful from
a synthetic chemistry perspective. Replacement of the bro-
mine with a phenyl group (9) significantly improved inhibi-
tor potency (Ki = 0.71 μM) and provided additional points
from which to branch out. The addition of a 4-ethylphenyl
group (10) improved inhibition further against TbPTR1
Ki (μM)
compd
R1
R2
X
TbPTR1 LmPTR1
4a
5
6a
7
H
H
O
S
>35
>35
5.8
>27
>27
>27
>27
>27
>27
16.4
3.4
H
H
CN
H
O
O
O
O
O
O
O
O
H
CH2CH2C6H5
0.96
3.9
8
Br
C6H5
CN
CN
CN
CN
9
0.71
0.50
0.36
0.29
0.40
10a
11
12a
13
C6H4CH2CH2
C6H4OCH3
C6H4CHO (meta) CN
C7H5O2 CN
4.2
2.6
a Crystal structures of these compounds complexed to PTR1 were not
determined.
at C6 and C7 of 1 emphasize this point with the β6-R6 loop
˚
positioned even further from the active site (∼4 A), leaving
the compound more exposed to solvent. This, together with
a degree of steric clash, likely contributes to reduced affi-
nity for TbPTR1. C7 is therefore a poor branch point for
modification of scaffold I. A major factor in the different
affinities of the scaffold I inhibitors against LmPTR1 and
TbPTR1 is likely due to the different conformations of the
β6-R6 loop. In LmPTR1, the flexible β6-R6 loop is more
able to accommodate inhibitors extending from C7 with
TbPTR1 less adaptable in this respect.
In compound 3 N8 is a hydrogen bond donor, and this has
a dramatic effect on the mode of binding. The TbPTR1-3
complex reveals the inhibitor in a substrate-like orientation
in two of the subunits of the asymmetric unit but displaying
static disorder involving both substrate-like and MTX-like

226 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Tulloch et al.
modeling suggests that the carbonyl may interact with
Asp161 if the latter were protonated. Increasing the size
of the substituent at C8 from a phenyl (9) to 1,3-methylene-
dioxy (13) improved inhibitor potency slightly against
TbPTR1 (Ki = 0.40 μM, Table 2), likely because of addi-
tional van der Waals interactions formed between the ligand
with Met163 and Cys168 and perhaps with a small contribu-
tion from a weak hydrogen bond formed by the side chain of
Asp161 and the ligand (Figure 4c, the average O
O
3 3 3
˚
separation is 3.8 A over the four copies per asymmetric unit).
Scaffold II proved more effective against TbPTR1 than
LmPTR1 (Table 2). An explanation is that the more flexible
β6-R6 loop of LmPTR1 and relatively open binding site are
unable to interact closely with these inhibitors, leaving them
exposed to solvent. In TbPTR1 a less flexible β6-R6 loop
together with the hydrophobic Met163 and Cys168 may
provide less competition with solvent and more van der
Waals interactions to stabilize the inhibitor complexes.
Scaffold III. The simplest of the scaffold III series, com-
pound 14, was a weak inhibitor of LmPTR1 and TbPTR1
(Table 3), forming similar interactions with TbPTR1 and the
cofactor as the pterin of folate and MTX (Figures 4d and S2).
The addition of cyclopropyl at N6 (15) had little effect, and
the structure indicates that the substituent was unable to
form hydrophobic interactions with the protein. Increasing
the size of the substituent, to methylphenyl, coupled with
replacement of N6 by S6 (16) improved activity against
TbPTR1 and LmPTR1 (Table 3). This may have been partly
due to the substitution of the hydrogen-bond-donating N6 to
a hydrogen-bond-accepting S6, adding an interaction with
Arg14, and partly to the size of the hydrophobic substituent,
which provided favorable interactions with the β6-R6 loop.
Increasing the chain length by one carbon (17) increased
flexibility, facilitating interactions with a hydrophobic re-
gion of the β6-R6 loop, and inhibition of TbPTR1 was
moderately improved (Ki = 3.2 μM). Inhibition of LmPTR1
was, however, increased dramatically (Ki = 0.60 μM) likely
because of contacts that the inhibitor forms with His241 in
LmPTR1 (data not shown). The methylphenyl substituent
does not appear to be a suitable position for further modifi-
cation, since 18 reduces inhibition against both TbPTR1 and
LmPTR1 (Table 3).
Testing Inhibitors against BSF T. brucei. Having derived
new, potent inhibitors of PTR1, we sought to determine their
effect on cultured parasites. We tested two of the most potent
TbPTR1 inhibitors (11 and 13) together with MTX and
PYR against BSF T. brucei (Figure 5a). MTX and PYR
(Figure 3c) were selected for this purpose because they are
both potent inhibitors of DHFR. No effort was made to
reduce the high levels of folate commonly used in media
(HML9 þ 10% fetal calf serum) to culture T. brucei, and our
results therefore represent a highly stringent test of com-
pound efficacy against the parasites.
MTX displayed an ED50 of 2.7 ( 0.1 μM (Figure 5a), a
value approximately 10-fold higher than the Ki against
PTR1,18 1000-fold higher than the Ki against DHFR.25
The DHFR inhibitor PYR was less effective with an ED50
of 26.6 ( 0.7 μM, greater than 100-fold higher than the Ki
against a trypanosomatid DHFR.26 The inefficiency of PYR
against T. brucei may reflect poor uptake, inability to
compete with high folate levels in the culture media, and/or
the ability of T. brucei to use PTR1 as a bypass of DHFR
inhibition. The combination of MTX and PYR does not act
synergistically (Figure S4A), and these compounds are likely
Figure 4. Scaffold representatives in the TbPTR1 active site as
revealed by crystallographic analyses: (a) 2; (b) 7; (c) 13; (d) 17. In
the PTR1-17 complex, Cys168 displays two rotamers. The inhibi-
˚
tor complex structures were determined between 2.4 and 1.6 A
resolution (Tables S1-S3).
(Ki = 0.50 μM). A 4-methoxyphenyl group (11) improved
inhibition again (Ki = 0.36 μM) with the oxygen forming an
additional hydrogen bond to solvent within the active site.
The strongest inhibition of TbPTR1 was achieved with the
addition of a 3-formylphenyl (12, Ki = 0.29 μM). We were
unable to derive a structure of the PTR1-12 complex, but

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 227
Figure 5. Trypanocidal activity of PTR1 and DHFR inhibitors. (a) Dose-response plots for cultured T. brucei parasites subjected to
increasing concentration of inhibitor. Points are mean values of three separate determinations conducted in quadruplicate (n = 12), std dev
e 5%. (b) Changes in 13 ED50 values in combination with varying concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5 μM) of MTX. Values are the mean (
std dev (n = 4).
competing with respect to binding DHFR. The PTR1 in-
hibitors 11 and 13 had limited efficacy against BSF T. brucei
with ED50 values of 274 ( 7.5 and 123 ( 3.3 μM, respectively
(Figures 5a and S4B). The degree of uptake and off-target
effects may contribute to the concentrations required to
produce lethal doses. However, 13 acts synergistically with
MTX (the area under the slope is less than half the product of
the two ED50 values (Figure 5b)).
In conclusion, we identified and exploited three molecular
scaffolds for the generation of novel inhibitors of PTR1
targeting two species of important human pathogens. The
inhibitors display PTR1-species specific properties, which
can be explained by differences, in particular of the β6-R6
substrate-binding loops, between the active sites of LmPTR1
and TbPTR1. Scaffolds I and III are the most effective
against LmPTR1, while scaffold II is the most effective
against TbPTR1. Testing against BSF, T. brucei indicates
that two of the scaffold II compounds, 11 and 13, are lethal to
the parasites although with modest ED50 values. However,
when 13 is tested in combination with MTX, there is an
improvement in efficacy, which highlights the potential of
therapy for African sleeping sickness by combination of such
DHFR and PTR1 inhibitors.
The enzymes for assay were prepared at 15 mg mL-1 in 50 mM
Tris-HCl, 250 mM NaCl, 20% (v/v) glycerol, pH 7.5. For
crystallization, TbPTR1 was at 22 mg mL-1 in 20 mM Tris-
HCl, pH 7.5. Aliquots were flash frozen in liquid N2 and stored
at -20 °C.
Inhibitor Sourcing and Assay. MTX, PYR, and compounds 1,
2, 3, 4, and 14 were purchased from Sigma Aldrich, and 15 was a
gift from Novartis. Compounds 16 and 17 were prepared by the
method of Davies et al.27 Synthetic routes to new compounds
except 10 and 18 have been described;21 details of the prepara-
1
tion of 10 and 18 follow. H NMR and mass spectrometry of
inhibitors that were assayed confirmed purity at >95%.
6-[(40-Methoxybenzyl)sulfanyl]-2,4-pyrimidinediamine (18). To
a suspension of 2,6-diamino-4-pyrimidinethiol (0.6 g, 2.5 mmol)
and sodium hydroxide (0.16 g, 4.0 mmol) in ethanol (15 mL) and
water (10 mL) was added 1-(bromomethyl)-4-methoxybenzene
(0.6 g, 2.55 mmol). The reaction mixture was stirred at room
temperature for 46 h. The precipitate was collected by filtration
and washed with water (10 mL) and n-hexane (10 mL) to afford
the required product as a white solid (0.44 g, 1.68 mmol, 67%; mp
189-191 °C). 1H NMR (400 MHz, DMSO-d6, 25 °C, TMS): δ
3.72 (3H, s; OCH3), 4.25 (2H, s; SCH2), 5.81 (1H, s; C5-H),
6.86 (4H, d þ s, 3J(H,H) = 8.6 Hz, C03-H and NH2), 7.11 (2H,
3
brs, NH2), 7.32 (2H, d, J(H,H) = 8.6 Hz, C20-H). 13C NMR
(100 MHz, DMSO-d6, 25 °C, TMS): δ 32.63 (SCH2), 55.06
(OCH3), 90.89 (C5), 113.9 (2C, ArCH), 128.54 (ArC), 130.16
(2C, ArCH), 158.16 (C6), 158.48 (ArC), 161.94 (C2), 163.16 (C4).
IR (KBr) 3335, 3146, 1656, 1512, 1303, 1249, 1102, 972, 754,
614 cm-1. HREIMS found m/z 262.0889, C12H14N4OS requires
262.0888 (Mþ).
Methods
Protein Purification and Storage. Recombinant LmPTR1 and
TbPTR1were expressed and purified by established methods.17,18

228 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Tulloch et al.
[(3E)-4-Nitro-3-butenyl]benzene. To a solution of 1-nitro-
4-phenyl-2-butanol28 (2.94 g, 15 mmol) in dichloromethane
(20 mL) at 0 °C was added methanesulfonyl chloride (1.7 g,
15 mmol) followed triethylamine (3.02 g, 30 mmol). The mixture
was warmed to room temperature and stirred for 20 min, then
poured into water (15 mL) and extracted with dichloromethane
(20 mL). The organic extract was washed with aqueous satu-
rated sodium bicarbonate (20 mL ꢀ 3) and then dried with
anhydrous magnesium sulfate and concentrated. The residue
was purified by column chromatography (silica gel, ethyl ace-
tate/n-hexane = 1:9) to afford the product as a yellow oil which
was stored at -20 °C (1.45 g, 8.2 mmol, 55%). 1H NMR
4-phenylbutanaloxime (0.2 g, 0.7 mmol) was heated for 15 h at
reflux with Dowex-50 (Hþ form, 0.33 g) in water (25 mL). The
reaction mixture was then diluted with methanol (50 mL) and
the Dowex resin filtered. The methanol was evaporated under
vacuum and the precipitated was filtered and washed with water
(15 mL) to give the product as a brown solid (0.16 g, 0.63 mmol,
90%). Spectroscopic data are detailed below.
(b) For the Nef reaction method, to an aqueous solution of
sodium hydroxide (0.2 g, 5.0 mmol) in water (5 mL) was added
2,6-diamino-5-[10-(nitromethyl)-30-phenylpropyl]-4(3H)-pyri-
midinone (0.25 g, 0.82 mmol) at room temperature. The mixture
was stirred for 2 h, and then it was slowly added to an aqueous
solution of sulfuric acid (97%, 0.69 g, 7.0 mmol) in water (5 mL)
at 0 °C. The resulting mixture was stirred at 0 °C for 1 h and at
room temperature for 1 h. The color of the mixture changed to
gray. Concentrated ammonium acetate was added at 0 °C to
adjust the pH to 7. The precipitated solid was collected and
purified by column chromatography (silica gel, ethyl acetate/
methanol=9:1) to give the product as a dark-brown solid (0.09 g,
0.35 mmol, 43%; mp 155-157 °C). 1H NMR (400 MHz,
DMSO-d6, 25 °C, TMS): δ 2.81-2.85 (2H, m, CH2CH2Ph),
2.88-2.92 (2H, m, CH2CH2Ph), 5.99 (2H, s, NH2), 6.31 (1H, d,
3J(H,H)=1.5 Hz, C6-H), 7.13-7.28 (5H, m, ArH), 10.16 (1H,
brs, NH), 10.59 (1H, s, OCNH). 13C NMR (100 MHz, DMSO-
d6, 25 °C, TMS): δ 28.34 (ArCH2CH2), 36.37 (ArCH2CH2),
98.92 (C4a), 113.50(C5), 118.07 (C6), 125.60 (1C, ArCH),
128.20, 128.37 (4C, ArCH), 142.51 (1C, ArC), 151.37 (C7a),
152.27 (C2), 159.48 (C4). IR (KBr) 3340, 3925, 1631, 1433, 1343,
3
(400 MHz, CDCl3, 25 °C, TMS): δ 2.65 (2H, dq, J(H,H)=
7.7, 1.4 Hz, PhCH2CH2), 2.90 (2H, t, 3J(H,H) = 7.7 Hz,
PhCH2CH2), 7.02 (1H, dt, 3J(H,H) = 13.4, 1.4 Hz, CH =
CHNO2), 7.25-7.42 (6H, m, CHdCHNO2 and Ph). 13C
NMR (100 MHz, CDCl3, 25 °C, TMS): δ 30.23 (CH2CH2Ph),
34.08 (CH2CH2Ph), 126.77 (1C, ArCH), 128.44, 128.87 (4C,
ArCH), 139.75 (CHdCHNO2), 140.16 (1C, ArC), 141.55
(CHdCHNO2). IR (neat) 3028, 2929, 1953, 1734, 1648, 1523
(CNO2), 1454, 1351, 1089, 953 (CHdCH), 840, 751, 700 cm-1
.
2,6-Diamino-5-[10-(nitromethyl)-30-phenylpropyl]-4(3H)-pyri-
midinone. It was necessary to synthesize this as an intermediate
en route to some targeted compounds. To a mixture of [(3E)-
4-nitro-3-butenyl]benzene (1.27 g, 7.2 mmol) in a mixture of
water (15 mL) and ethyl acetate (15 mL) at room temperature
was added 2,6-diamino-4(3H)-pyrimidinone (0.9 g, 7.2 mmol).
The resulting mixture was stirred in an oil bath at 90 °C for 24 h.
The organic layer was separated, washed with brine (30 mL),
dried, and concentrated. The resulting solid was dried in vacuo
(120 mmHg) to give the product as a yellow solid (2.06 g, 6.8
mmol, 94%; mp 143-145 °C). 1H NMR (400 MHz, DMSO-d6,
25 °C, TMS): δ 1.67-1.76 (1H, m, CH2CH2Ph), 2.08-2.18
(1H, m, CH2CH2Ph), 2.43-2.64 (2H, m, CH2CH2Ph), 3.42
(1H, brs, CHCH2CH2Ph), 4.77-5.07 (2H, m, CH2NO2), 5.95
(2H, brs, C6-NH2), 6.08 (2H, brs, C2-NH2), 7.13-7.41 (5H, m,
ArH), 9.85 (1H, brs, NH). 13C NMR (100 MHz, DMSO-d6,
25 °C, TMS): δ 31.83 (CHCH2CH2), 32.98 (CHCH2CH2), 35.09
(CHCH2CH2), 77.65 (CH2NO2), 84.08 (C5), 125.55 (1C, ArC),
127.99, 128.22 (4C, ArCH), 142.34 (1C, ArC), 153.55 (C6), 161.93
(C2), 162.81 (C4). IR (KBr) 3471, 3401, 3183, 2859, 1625, 1595, 1536
(CNO2), 1493, 1450, 1378, 697 cm-1. HRFABMS found m/z
301.1410, C14H17N5O3 requires 302.1413 (MHþ).
1137, 786, 698, 620 cm-1
. HREIMS found 254.1168,
C14H14N4O requires 254.1169 (Mþ).
Inhibition Assay. Compounds were dissolved at 100 mM in
DMSO and screened against LmPTR1 and TbPTR1 with H2B
(10 mM in 0.1 M NaOH) as the substrate. The IC50 values
were determined from dose-response plots using SigmaPlot29
adjusted with Morrison’s equation for tight-binding inhibi-
tion.30 Ki values were derived from the IC50 values using the
equation for competitive inhibition as published.17
Cytotoxicity Studies. BSF T. brucei brucei strain Lister 427,
were cultured at 37 °C and 5% CO2 in HMI-9 medium supple-
mented to maintain neomycin drug pressure to express T7
polymerase and the tetracycline repressor.31 The Alamar blue
viability test32 established ED50 values against BSF T. brucei
(strain 427) for MTX, PYR, 11, and 13. For combination
exposures, ED50 values were determined for PYR (Figure
S4A), 11 (Figure S4B), or 13 in the presence of sub-ED50
concentrations of MTX (2.5, 2.0, 1.5, 1.0, and 0.5 μM).
TbPTR1-Ligand Cocrystallization. Crystals were grown by
vapor diffusion in hanging drops consisting of 1.5 μL of protein
solution (TbPTR1 at 6-10 mg mL-1, 1 mM NADPH, 1 mM
substrate or inhibitor, 1% (v/v) DMSO, and 20 mM DTT) and
an equal volume of the reservoir (1.5-3 M sodium acetate,
10-50 mM sodium citrate in the pH range 4.5-6.0). Crystals
grew to 0.5 mm ꢀ 0.3 mm ꢀ 0.1 mm over a few days. Crystals
grown in excess of 2.6 M sodium acetate were flash-cooled in a
stream of N2 to -173 °C directly from the mother liquor. Those
obtained in less than 2.6 M sodium acetate were cryoprotected
with either 3 M sodium acetate or 30% (v/v) glycerol.
X-ray Data Collection and Structure Determination. X-ray
data were collected in-house using a Rigaku Micromax 007
X-ray generator equipped with an Raxis IVþþ detector, at station
14.1 of the Synchrotron Radiation Source (SRS), Daresbury,
U.K., and beamlines BM14, ID23-1, ID23-2, and ID29 of the
European Synchrotron Radiation Facility (ESRF), Grenoble,
France (Supporting Information Tables S1-S3). Data were
processed with the CCP4 software suite.33 X-ray images were
integrated and scaled with MOSFLM34 and SCALA35 or XDS
and XSCALE.36 Structures were solved by molecular replace-
ment37 using TbPTR1 as the starting model.17 Several rounds of
restrained refined were carried out using REFMAC538 together
with inspection of electron and difference density Fourier maps,
(1E/Z)-2-(20,40-Diamino-60-oxo-10,60-dihydro-50-pyrimidinyl)-
4-phenylbutanaloxime. To a suspension of 2,6-diamino-5-[10-(nitro-
methyl)-30-phenylpropyl]-4(3H)-pyrimidinone (0.82 g, 2.7 mmol)
and tin(II) chloride (0.77 g, 4.0 mmol) in tetrahydrofuran (70 mL)
was added thiophenol (1.2 mL) and triethylamine (1.8 mL). The
reaction mixture was stirred at room temperature for 1 h. The
remaining tin(II) chloride was removed by filtration, and the liquid
portion was concentrated under reduced pressure (400 mmHg).
The residue was purified by column chromatography (silica gel,
ethyl acetate/n-hexane=1:1 to ethyl acetate/methanol=1:1) to
afford a white solid (0.45 g, 1.56 mmol, 58%; mp >250 °C) as an
1
E/Z mixture (3:1 by H NMR). The E-isomer was purified for
characterization. 1H NMR (400 MHz, DMSO-d6, 25 °C, TMS): δ
1.94-2.01 (2H, m, CH2CH2Ph), 2.42-2.48 (2H, m, CH2CH2Ph),
3.45 (1H, q, J=7.4 Hz, CHCH2CH2Ph), 5.77 (2H, brs, C6-NH2),
6.03 (2H, brs, C2-NH2), 7.13-7.27 (5H, m, ArH), 7.58 (1H, d,
3J(H,H)=6.9 Hz, CHdNOH), 9.83 (1H, brs, OCNH), 10.60 (1H,
s, CHdNOH). 13C NMR (100 MHz, DMSO-d6, 25 °C, TMS): δ
(E-isomer) 35.47 (CHCH2CH2), 32.45 (CHCH2CH2), 33.37 (CH-
CH2CH2), 152.10 (CHNO), 86.45 (C5), 125.45 (1C, ArC), 128.07,
128.17 (4C, ArC), 153.51 (C6), 161.81 (C2), 161.99 (C4). IR (KBr)
3371, 3104, 1621, 1596, 1503, 1430, 1374, 1020, 987, 787, 694,
559 cm-1. HRFABMS found 288.1462, C14H17N4O2 requires
288.1460 (MHþ).
2-Amino-5-(20-phenylethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]-
pyrimidin-4-one (10). (a) For cyclization with Dowex-50, Hþ-
(1E/Z)-2-(20,40-diamino-60-oxo-10,60-dihydro-50-pyrimidinyl)-

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 229
model manipulation, and identification of solvent ions and
ligands with COOT.39 Figures were generated with PyMol.40
(18) Gourley, D. G.; Schuttelkopf, A. W.; Leonard, G. A.; Luba,
J.; Hardy, L. W.; Beverley, S. M.; Hunter, W. N. Pteridine
reductase mechanism correlates pterin metabolism with drug resis-
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Acknowledgment. We thank Novartis Animal Health Inc,
Basel, Switzerland, for the gift of 15. Supported by awards were
from BBSRC (Structural Proteomics of Rational Targets,
BBS/B/14434) and The Wellcome Trust (Grant Nos. 082596
and 083481, Senior Fellowship Grant 064771 to T.K.S.). We
thank the SRS Daresbury and ESRF Grenoble for synchrotron
beam time and support. J.I. and V.P.M. thank CAPES for a
postdoctoral fellowship BEX 2000/04-0 and M.Sc. fellowship,
respectively. The University of Strathclyde provided student-
ship support for J.K.H. and J.H.L.
€
(19) Schuttelkopf, A. W.; Hardy, L. W.; Beverley, S. M.; Hunter, W. N.
Structures of Leishmania major pteridine reductase complexes
reveal the active site features important for ligand binding and to
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(20) Mpamhanga, C. P.; Spinks, D; Tulloch, L. B.; Shanks, E. J.;
Robinson, D. A.; Collie, I. T.; Fairlamb, A. H.; Wyatt, P. G.;
Frearson, J. A.; Hunter, W. N.; Gilbert, I. H.; Brenk, R. One
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Supporting Information Available: Figures of all ligands in a
TbPTR1 active site that have been characterized; tables of
crystallographic details; graphs detailing the trypanocidal acti-
vity of pyrimethamine or compound 11 in combination with
MTX. This material is available free of charge via the Internet at
http://pubs.acs.org.
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chains. J. Med. Chem. 1999, 42, 5095–5099.
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Suckling, C. J.; Clements, C.; Harvey, A. L.; Hunter, W. N.;
Tulloch, L. B. Diversity oriented syntheses of fused pyrimidines
designed as potential antifolates. Org. Biomol. Chem. 2009, 7,
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(24) Cavazzuti, A.; Paglietti, G.; Hunter, W. N.; Gamarro, F.; Piras, S.;
Loriga, M.; Allecca, S.; Corona, P.; McLuskey, K.; Tulloch, L.;
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reductase inhibitors to guide antiparasite drug development. Proc.
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