Structure-based selectivity optimization of piperidine-pteridine derivatives as potent Leishmania pteridine reductase inhibitors

Article abstract of DOI:10.1021/jm300563f

The upregulation of pteridine reductase (PTR1) is a major contributor to antifolate drug resistance in Leishmania spp., as it provides a salvage pathway that bypasses dihydrofolate reductase (DHFR) inhibition. The structure-based optimization of the PTR1 inhibitor methyl-1-[4-(2,4-diaminopteridin-6- ylmethylamino)benzoyl]piperidine-4-carboxylate (1) led to the synthesis of a focused compound library which showed significantly improved selectivity for the parasite's folate-dependent enzyme. When used in combination with pyrimethamine, a DHFR inhibitor, a synergistic effect was observed for compound 5b. This work represents a step forward in the identification of effective antileishmania agents.

Full text of DOI:10.1021/jm300563f

Article
pubs.acs.org/jmc
Structure-Based Selectivity Optimization of Piperidine−Pteridine
Derivatives as Potent Leishmania Pteridine Reductase Inhibitors
Paola Corona,‡ Federica Gibellini,† Andrea Cavalli,§,∥ Puneet Saxena,† Antonio Carta,‡ Mario Loriga,‡
Rosaria Luciani,† Giuseppe Paglietti,‡ Davide Guerrieri,† Erika Nerini,† Shreedhara Gupta,⊥
Veronique Hannaert,⊥ Paul A. M. Michels,⊥ Stefania Ferrari,*,† and Paola M. Costi*,†
́
†Dipartimento di Scienze Farmaceutiche, Universita
̀
degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy
degli Studi di Sassari, via Muroni 23/a, 07100 Sassari, Italy
§Dipartimento di Scienze Farmaceutiche, Universita
degli Studi di Bologna, Via Belmeloro, 40126 Bologna, Italy
‡Dipartimento di Chimica e Farmacia, Universita
̀
̀
∥Drug Discovery and Development, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova, Italy
⊥Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Universite
Hippocrate 74, Postal Box B1.74.01, B-1200 Brussels, Belgium
́
catholique de Louvain, Avenue
S
* Supporting Information
ABSTRACT: The upregulation of pteridine reductase (PTR1) is a
major contributor to antifolate drug resistance in Leishmania spp., as it
provides a salvage pathway that bypasses dihydrofolate reductase
(DHFR) inhibition. The structure-based optimization of the PTR1
inhibitor methyl-1-[4-(2,4-diaminopteridin-6-ylmethylamino)benzoyl]-
piperidine-4-carboxylate (1) led to the synthesis of a focused compound
library which showed significantly improved selectivity for the parasite’s
folate-dependent enzyme. When used in combination with pyrimeth-
amine, a DHFR inhibitor, a synergistic effect was observed for
compound 5b. This work represents a step forward in the identification
of effective antileishmania agents.
able to reduce other pterins and folates. Despite the requirement
for reduced biopterin for the growth and survival of Leishmania
major, PTR1 is not a drug target on its own, likely because of the
parasite’s ability to scavenge for tetrahydrobiopterin in the
phagolysosomes of its host macrophages. For this reason,
antifolate therapy could be successfully achieved in Leishmania
only when both DHFR and PTR1 are simultaneously inhibited
by a single drug or by two drugs administered in combination. A
successful therapy should not affect the activity of human DHFR
(hDHFR). Although the overall protein fold of DHFR is
conserved, the primary sequence has diverged considerably
among various species through evolution; DHFRs from
different organisms show dramatic differences in their inhibition
by certain folate analogues.4 While human DHFR is a
monofunctional enzyme, in trypanosomatidic parasites, DHFR
and TS activities are expressed as a bifunctional enzyme,
dihydrofolate reductase−thymidylate synthase (DHFR-TS), in
which the N-terminal DHFR domain is linked to the TS
domain. LmDHFR domain share around 25% and 40%,
respectively, of identity and similarity with hDHFR; this
INTRODUCTION
■
Approximately 350 million people in the tropical and
subtropical regions of the world are at risk of contracting
forms of the parasitic disease known as leishmaniasis. Its clinical
spectrum ranges from the self-healing or scarring cutaneous
form to the disfiguring mucocutaneous leishmaniasis and the
deadly (if untreated) visceral form. The disease is caused by
protists of the genus Leishmania; to date, no satisfactory
treatment option is available due to high costs, difficulty of
administration, and the development of drug resistance.1,2
Drugs that target the folate pathway, named antifolates, have
been successfully employed against cancer, bacterial infections,
certain autoimmune diseases, and malaria, but they have no
efficacy against Leishmania despite it being a folate auxotroph.3
The main target of antifolates is the enzyme dihydrofolate
reductase (DHFR, E.C. 1.5.1.3), which carries out the
progressive reduction of folate to dihydrofolate and then
tetrahydrofolate. Reduced folates are employed as cofactors in
crucial cellular events such as DNA and protein synthesis and
methylation reactions.
Leishmania is able to overcome DHFR inhibition by
overexpressing pteridine reductase 1 (PTR1, E.C. 1.5.1.33), an
enzyme mainly involved in the reduction of biopterin (first to
dihydrobiopterin, then to tetrahydrobiopterin) but that is also
Received: April 20, 2012
© XXXX American Chemical Society
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Table 1. Inhibition Constants (Ki) and Selectivity Index (SI) of the Synthesized Compounds; MTX, 1, and 2 Are Reported for
Comparison
a
b
Already reported in ref 7. NI: no inhibition at the concentration reported in brackets.
suggests that the selective inhibition of LmDHFR with respect
to hDHFR could be possible.5,6
We previously reported the design, synthesis, and biological
an additive inhibition profile when tested in combination with
known DHFR inhibitors such as pyrimethamine (PYR).7
Compound 1 (Table 1) was shown to be a potent L. major
PTR1 (LmPTR1) inhibitor with an inhibition constant (Ki) of
evaluation of a novel PTR1 inhibitor (1, Table 1) that produced
B
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Figure 1. (left) 3D representation of the interactions between compound 1 (orange) and the enzyme’s active site residues (in yellow) (PDB ID:
3H4V). Color code: O and N atoms are in red and blue, respectively; C atoms for compound 1 are in orange, whereas of NADP+ and active site
residues are in lime and yellow, respectively. The hydrogen bond interactions are shown in broken black lines. Water molecules are shown in red CPK
representation. (right) Schematic overview of the interactions are shown where the head region of compound 1 is drawn in black, while the tail region
is in red.
100 nM and a selectivity index (Supporting Information, Table
1) over hDHFR of approximately 100. Compound 1 was shown
to bind PTR1 (PDB ID 3H4V) in the active site with an
orientation that resembled that of the substrate, dihydrobiopter-
in (PDB IDs 2BF7 and 1E92), rather than that of the archetypal
antifolate methotrexate (MTX, PDB ID 1E7W, Figure 1),
despite having a similar chemical structure.7
analysis of the crystal structure suggests the opportunity to
increase and optimize the interactions between PTR1 and the
tail region of the inhibitor. By performing a structure-based
refinement of lead 1, we aimed to achieve more productive
binding interactions between PTR1 and the inhibitor at the tail
region, thereby producing an increase in potency and a decrease
in toxicity.
Here, seven compounds were designed, synthesized, and
tested for their antileishmanial activity both against purified
LmPTR1 and on cultured promastigote forms of Leishmania
mexicana and L. major; their synergistic effect in combination
with known antifolates was evaluated. Inhibitors of PTR1 are
known to make cells more sensitive to oxidative stress by
decreasing the intracellular levels of tetrahydrobiopterin,
although the mechanistic details are still unclear. Accordingly,
the inhibition of PTR1 caused a reduction of the levels of
tetrahydrobiopterines, and the parasitic cells became more
sensitive to oxidative stress than control cells. This assay
represents further confirmation that inhibition of the target
enzyme is taking place in the cell.
The zone between N10 and Arg287′ is hydrophilic and rich in
ordered waters, suggesting that the introduction of short chains
carrying hydroxyl groups on N10 should mimic the role of the
water interactions. If a 2-hydroxy-ethyl chain is added at N10
(compound 5d, Table 1), the hydroxyl group could establish
interactions with both Arg287′ amino groups (from chain D of
the tetramer), Asp181 oxygens, and the Gly225 carbonyl group
(Figure 1). With the same aim, compound 5c (Table 1) was
designed.
Exploring the surface clearly revealed the need of an hydrogen
bond acceptor on the benzene ring due to its proximity of
His241 (Figure 1). A possible solution could be the replacement
of the benzenic ring with a pyridine ring (compound 5b, Table
1) in which the nitrogen atom could accept a hydrogen bond
from His241.
The area facing the p-amino-benzoic-acid (PABA) group on
the opposite site of His241 is particularly wide and lacking in
hydrophilic groups. The introduction of an ethyl group on this
side of the ring (compounds 5f and 5e, Table 1) could establish
a hydrophobic interaction with Phe113, Leu229, and Val230
(Figure 1). The space available for ligand binding is quite wide,
but this is a particularly flexible zone of the active site (as can be
observed by comparing the structures of PTR1 in complex with
dihydrobiopterin and MTX); thus, the enzyme should be able to
easily optimize the interaction with ligands in this region. The
replacement of the terminal ester with a carboxylic group
(compound 6a, Table 1) or an amide (compound 5a, Table 1)
can establish a hydrogen bond with the backbone oxygen of
Leu189 (Figure 1).
RESULTS AND DISCUSSION
■
Design of Compound 1 Analogues. To improve the
biological profile of compound 1, a new structural optimization
program was started using the X-ray crystal structure of
LmPTR1-1 (PDB-ID: 3H4V), and visual inspection analysis
was performed. In the LmPTR1−1 complex structure, the
inhibitor adopts an orientation similar to that observed for the
substrate, with the C7−N8 bond near Asp181 and Tyr194. The
headgroup of the inhibitor binds between Phe113 and the
nicotinamide ring of the cofactor NADPH and forms hydrogen
bonds with Ser111, Tyr194, the phosphate, and ribose
components of the cofactor and an ordered water molecule.
The tail of compound 1 forms hydrogen bonds through one-
water-molecule bridges with Tyr191, His241, and the backbone
of Leu189; it also interacts with Asp181, Leu188, Gly225,
Asp232, and Met233 (Figure 1).
Synthetic Chemistry. On the basis of the structure-based
design, we synthesized compounds 5a−f and 6a. The synthesis
of the 2,4-diaminopteridine derivatives 5a−f is shown in Scheme
1, while the preparation of the intermediate amines 4a−f is
Numerous hydrogen bonds are formed between the pteridine
headgroup of 1, the cofactor and the surrounding amino acids,
as well as ordered water molecules. On the other hand, the
C
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a
Scheme 1. Scheme of Synthesis of Compounds 5a−f and 6a
a
Reagents: (i) DMA, room temperature; (ii) aqueous NaOH.
reported in Schemes 2, 3, 4, and 5 (details are reported in
Supporting Information).
genation (Scheme 3). Compound 4e was obtained according to
the sequence of reactions outlined in Scheme 4, under similar
conditions as described above, starting with 2-ethyl-4-nitro-
benzoyl chloride (17)10 and the ester (9). Finally, the isomer
compound (4f) was prepared in an identical manner as
described above from 3-ethyl-4-nitrobenzoyl chloride (20),
which was obtained from the known parent acid (19)11
(Scheme 5) and the ester (9).
Evaluation of the Inhibition of Enzyme Activity
Inhibition and Structure−Activity Relationships. The
ability of the designed compounds to inhibit purified
LmPTR1 in vitro was compared with their ability to inhibit
the purified human folate-dependent enzymes hDHFR and
thymidylate synthase (hTS) to estimate their potency and
potential toxicity (Table 1).
In vitro evaluation of 5d validated the design strategy: the
addition of the hydroxyethyl chain led to a 3-fold improvement
in affinity toward LmPTR1 (Ki from 100 to 30 nM) and a less
marked increase in inhibitory activity toward hDHFR (Ki from
10 to 4.33 μM) with respect to compound 1; overall, the
modification caused an increase in both the potency and the
selectivity (Table 1). In 5c, a bulkier ethyl ethanoate substituent
was inserted on N10; its affinity toward LmPTR1 with respect
to the leading compound 1 did not change, but it completely
lost its inhibitory activity toward hDHFR, most likely as a
consequence of increased steric clash. Compound 5c was found
Displacement of the bromide of the known 6-
(bromomethyl)pteridine-2,4-diamine hydrobromide8 (3) with
both the appropriate substituted anilines (4a,c−f) and amino
picoline derivative (4b) was carried out in anhydrous N,N-
dimethylacetamide (DMA) at room temperature to afford the
desired target compounds 5a−f in yields of 33−65% (Scheme
1).
The acid (6a) was obtained by alkaline hydrolysis of the
amide (5a) with a 92% yield (Scheme 1).
The intermediates 4a, 4c, and 4d were synthesized starting
from the 4-nitrobenzoyl chloride (7), which was condensed with
piperidine-4-carboxamide (8) or methyl piperidine-4-carbox-
ylate (9) to give the nitro compounds 10 and 11, respectively
(Scheme 2). Reduction of the nitro compounds was
accomplished by hydrogen over 10% Pd−C to give the
aminoderivatives 4a and 12. Alkylation of the latter with ethyl
2-bromoacetate or 2-bromoethanol resulted in the (4c) and
(4d) derivatives, respectively.
Compound 4b was synthesized by condensation of 5-
nitropicolinoyl chloride (14) obtained from the parent acid
purposely prepared as described,9 with methyl piperidine-4-
carboxylate (9) at room temperature in N,N-dimethylforma-
mide (DMF) and in the presence of triethylamine (Et3N) to
give the intermediate 15, which underwent successive hydro-
D
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a
Scheme 2. Scheme of Synthesis of Compounds 4a and 4c−d
a
Reagents: (i) NaHCO3 H2O, 100 °C; (ii) H2 10% Pd−C, EtOH; (iii) Et3N, DMF, room temperature; (iv) ethyl 2-bromoacetate, N-ethyl-N-
isopropylpropan-2-amine, anhydrous DMF, under N2 70 °C; (v) 2-bromoethanol, N,N-dimethylaniline, under argon, 60 °C.
a
Scheme 3. Scheme of Synthesis of Compound 4b
a
Reagents: (i) SOCl2 90 °C; (ii) Et3N, DMF, room temperature; (iii) H2 10% Pd−C, EtOH.
a
Scheme 4. Scheme of Synthesis of Compound 4e
a
Reagents: (i) SOCl2 90 °C; (ii) Et3N, DMF, room temperature; (iii) H2 10% Pd−C, EtOH.
to be the most selective LmPTR1 inhibitor of the present series
and one of the most promising LmPTR1 inhibitors reported by
us to date.
Regarding the second set of modifications (5b, 5f, and 5e),
compounds 5f and 5e were found to be potent LmPTR1
inhibitors (Ki = 78 and 60 nM, respectively); however, none of
them was more potent than 5d (Ki = 30 nM) and more selective
than 5c, which points to N10 as the most promising position to
further explore the present class of compounds. Tail-end
modification as in compounds 6a and 5a (Table 1) did not lead
to an improvement in activity compared to compound 1.
As a general rule, for a compound to be active on LmPTR1
while possessing a good selectivity over hDHFR, a substitution
on N10 with a chain that can interact with hydrophilic residues
is required; substitutions of the phenyl ring of PABA that allow
hydrophobic interaction with the nonpolar environment of the
binding site of hDHFR should be avoided to preserve the
selectivity. This is in agreement with our previous analysis of
E
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a
Scheme 5. Scheme of Synthesis of Compound 4f
a
Reagents: (i) SOCl2 90 °C; (ii) Et3N, DMF, room temperature; (iii) H2 10% Pd−C, EtOH.
Figure 2. Dockings of the structure-based modifications of lead compound 1 (Figure 1) into the active site of LmPTR1 (PDB ID: 1E92). The ligands
(shown in orange) are surrounded by protein’s active site residues (shown in licorice, lime color). Color code: O and N atoms are in red and blue,
respectively; C atoms for compounds 5b (A), 5c (B), 5d (C), 5f (D), and 6a (E) are in orange, whereas for NADP+ and active site residues are in gray
and lime, respectively.
F
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compounds 1 and 2. Whereas compound 1 displayed marked
selectivity for LmPTR1, the presence of a methyl group on N10
in compound 2 (instead of a hydrogen atom in 1) increased the
affinity of the molecule for hDHFR (Ki = 800 nM).7 Like the
parent compound, 1, all compounds, except one, 5d, do not
inhibit hTS. This is in line with MTX profile which is more than
10000-fold less active toward hTS with respect to hDHFR.
Docking Studies. The docking studies were performed to
predict the binding modes of the interesting compounds: 5b, 5c,
5d, 5f, and 6a versus LmPTR1 with the aim to compare the
docking results with the observed inhibition data. To evaluate
the correctness of our approach, the docking protocol was first
implemented on the available crystallographic structures of
compound 1 and MTX and its ability to reproduce the same was
tested. The obtained results were found to be in agreement with
the experimentally observed binding modes, thereby indicating
the validity of the procedure.
The docking result for compound 5b showed that the
nitrogen inserted into the aromatic ring can have a possible
interaction with His241 (Figure 2A). Moreover, the rationale
behind the synthesis of compound 5c and 5d was well
corroborated by their docking results. The substitution of acetic
acid ethyl ester chain (compound 5c) made the ligand to
interact with Asp181 oxygens. However, the inclusion of the
flexibility factor of active residues side chains made Arg187′
(from chain D of the tetramer) move apart due to less
availability of space in that region (Figure 2B). Also, the 2-
hydroxy-ethyl chain substituted at N10 position of compound
5d (Figure 2C) was found to orient itself toward the Asp181
oxygens as well as the backbone carbonyl group of Gly225.
The introduction of an ethyl group on the ring helped
compound 5f to establish hydrophobic interaction with Leu229
and Val230. Because of the flexible nature of the side chains, it
could be well observed that side chain of Val230 moved in a way
(Figure 2D) so as to make enough room for ethyl group
substitution. Although the motive behind replacing the terminal
ester with carboxylic group (in compound 6a) was to gain
hydrogen bond with the backbone oxygen of Leu189, the
expected hydrogen bond could not be observed in the docking
result (Figure 2E). Furthermore, from the docking it was also
observed that the pteridine ring of the above-mentioned
compounds were able to maintain head-to-head stacking
interactions between nicotinamide ring of NADP as well as
the phenyl ring of Phe113.
Figure 3. Binding modes of folic acid (C atoms in pink) (PDB ID:
2W3M) and MTX (C atoms in green) (PDB ID: 1U72) with hDHFR
(C atoms in gray). All residues and ligands were colored by atom (N in
blue, O in red, P in orange); different colors were used for the C atoms
to highlight important residues/molecules. Only residues surrounding
the N10 position are displayed; most of the residues in the surrounding
pocket are hydrophobic: Asp21, Leu22, Thr56, Ser59, Ile60.
pteridine ring of this compound plays an important role in
defining its selectivity against hDHFR. On observing the docked
pose of 5b in the active site of LmPTR1 enzyme, the pteridine
rings gets firmly sandwiched between the phenyl ring of Phe113
and nicotinamide ring of NADP+ (Figure 4). Such π−π stacking
interaction induces the role of an anchor, giving the compound a
firm stability. Apart from this, it also indulges in hydrogen bond
formation with Ser111 residue and Tyr194. The 2-amino group
makes hydrogen bond with Ser111, whereas the N8 atom of
pteridine ring fetches H-bond from hydroxyl group of Tyr194
(Figure 4). All these interactions help the compound gain better
activity against LmPTR1.
Although, in the case of hDHFR, the pteridine ring of 5b is
able to occupy region between nicotinamide ring and phenyl
ring of Phe34, planarity among them could not be well achieved
(Figure 4). Moreover, in the case of hDHFR enzyme, the
pteridine rings are able to make only one single hydrogen bond
interaction with backbone carbonyl of Ile7 due to relatively poor
availability of any hydrogen bond donor atoms in that region.
Furthermore, insertion of nitrogen atom in the phenyl ring was
well suited for LmPTR1 as it tried to engage interaction with
His241, which in the case of hDHFR was not possible due to
absence of any such residue.
A visual inspection of the X-ray structures of the complexes of
hDHFR with folic acid or MTX (PDB IDs: 2W3M and 1U72)
was helpful in rationalizing the observed SAR. If we reasonably
assume that compound 1 can adopt a binding conformation and
a binding mode in the active site of hDHFR that is similar to the
folate substrate or to the inhibitor MTX (Figure 3), then N10
would sit in a mostly hydrophobic pocket surrounded by Asp21,
Leu22, Phe31, Thr56, Ser59, Ile60, and NADPH. It is
conceivable that the presence of a methyl group on N10
would dampen its hydrophilic nature and increase the affinity for
hDHFR. On the other hand, a bulkier or hydrophilic substituent
on N10 could reduce the affinity for hDHFR, thus increasing the
selectivity of this compound series toward the parasite’s PTR1.
For analyzing the factors contributing to the selectivity of
compound 5b against hDHFR, the docking was used to predict
the binding modes of 5b versus hDHFR. The results were
compared with the one obtained vs LmPTR1. The docking
results of compound 5b versus LmPTR1 (PDB ID: 1E92) and
hDHFR (PDB ID: 2W3M) showed that the 2,4-diamino-
Antiparasitic Activity. Compounds 5a−f were tested for
their antiparasitic activity. As expected (as PTR1 is not a drug
target on its own in Leishmania12), compound 1 and its
derivatives were able to inhibit parasite growth only weakly,
barely reaching 50% inhibition within the concentration range
tested (0.045−100.0 μg/mL). Table 2 and Figure 5 report the
growth percentage of both L. mexicana and L. major exposed to
a 50 μg/mL dose of each compound alone or in combination
with PYR at 30 μg/mL.
On their own, the compounds induce only limited variations
in the L. mexicana growth rate, ranging from 111.6% for
compound 1 to 88.5% for compound 5a. Similar results were
obtained for L. major, where the growth rates ranged from
122.2% for 5d to 99.6% for 1 (Table 2, Figure 5).
The inhibitors were also tested on L. mexicana and L. major
promastigote lines in combination with the DHFR inhibitor
PYR at a fixed concentration. In both cell lines, the compounds
were able to considerably enhance PYR activity. Growth rates
ranged from 24.4% for the combination 1 + PYR to 12.9% for
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Figure 4. Three-dimensional representation of the interactions made by compound 5b (licorice, C atoms in orange). The pteridine moiety of 5b is
able to have multiple hydrogen bond interactions (drawn in black dotted lines) with Ser111 and Tyr194 in LmPTR1 (A), whereas in the case of
hDHFR (B), the compound is unable to gain any such bindings except Ile7, thereby making it evident that the compound 5b is well suited for targeting
LmPTR1 enzyme with respect to hDHFR. Color code: O and N atoms are in red and blue, respectively; C atoms for compound 5b are in orange,
whereas for NADP+ and active site residues are in gray and lime, respectively, for both the proteins.
Table 2. Effect of Different Compounds, Administered at 50
μg/mL, Alone or Combined with PYR at 30 μg/mL on the
a
Growth of Leishmania Promastigotes
growth of L.
mexicana (%)
growth of L.
major (%)
ED50 of human MRC5
fibroblasts (μg/mL)
compd
PYR
102.5 0.3
111.6 1.4
24.4 4.1
68.0 1.2
99.6 1.8
30.2 0.53
16.2 2.5
1
23.4 3.9
1 +
PYR
5a
88.5 1.4
15.7 2.1
121.2 1.7
47.2 3.7
51.0 4.0
Figure 5. Growth of L. mexicana (in black), L. major (in light gray) in
the presence of 30 μg/mL of PYR or of 50 μg/mL of compounds, left
axis; ED50 on human fibroblasts (in dark gray), right axis. The growth
values are expressed as percentages calculated with respect to the
growth of parasites without PYR and pteridine compounds. * not
determined, i.e., at 50 μg/mL, no inhibition was observed.
5a +
PYR
b
5b
95.6 2.1
103.4 1.9
16.1 0.9
ND
5b +
PYR
17.6 0.03
5c
102.3 2.1
21.9 3.1
100.8 1.8
37.8 0.3
28% inhibition at 100 μg/
c
mL
5c +
PYR
Toxicity of the series has been addressed using cultured
MRC-5 human fibroblasts. Most of the compounds appear to be
toxic, showing a considerable inhibition of cell growth (ED50
values range from 21.8 μg/mL for 5f to 51.0 μg/mL for 5a).
Notably, no growth inhibition was apparent with 5b and 5d in
the same concentration range.
Because of the lack of toxicity, 5b was chosen for a more
precise evaluation of its synergistic activity with PYR on
Leishmania promastigote growth; 1 was also tested as a
reference (Figure 6). Parasites were treated with the compounds
alone or in combination with PYR (used at its ED30 values: 3.5
b
5d
100.1 2.1
12.9 2.6
122.2 2.2
27.4 0.4
ND
5d +
PYR
5f
90.6 1.4
19.6 1.7
104.2 1.8
23.1 0.8
21.8 0.6
5f +
PYR
a
Data are expressed as the percentage of growth compared to control
cultures to which no compound had been added. The results obtained
for both Leishmania and human cells are the mean standard
b
deviation obtained in three independent experiments. ND: not
determined, i.e., at 50 μg/mL, no inhibition was observed. Little
growth inhibition of human MRC5 fibroblasts was obtained at the
concentration range tested for 5c, so no ED50 value could be calculated
for this compound.
c
0.12 μg/mL for L. major and 1.78
0.09 μg/mL for L.
mexicana). The resulting combination indexes, calculated using
the Chou−Talalay method13 were 0.75 for 1 + PYR and 0.80 for
5b + PYR in L. major and 2.05 for 1 + PYR and 1.51 for 5b +
PYR in L. mexicana. Values lower than 1 in L. major point to a
synergistic effect. Data reported in Table 2 were obtained in
experiments that had to be performed with parasites grown in
culture medium prepared with a new, different batch of serum
than the experiments for which data are shown in Figure 6.
Although parasite growth and compound effects showed a
consistent trend, comparison of absolute numerical values is not
the combination 5d + PYR for L. mexicana and from 47.2% for
5a + PYR to 16.7% for 5b + PYR for L. major (Table 2, Figure
5). PYR on its own showed a growth rate percentage of 102.3
and 68 at 30 μg/mL, respectively, against L. mexicana and L.
major.
H
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Figure 6. Growth curve of L. major (A) and L. mexicana (B) promastigotes after exposure to compound 1 (A1 and B1) and compound 5b (A2 and
B2) alone (square dots) or in combination with PYR at its ED30 (round dots).
Figure 7. Oxidative stress effect expressed as the percentage of surviving parasites after their exposure to increasing concentrations of H2O2 [untreated
cells (light blue triangular dots), cells treated with compounds 1 (yellow square dots) and 5b (red rhomboidal dots), blue round dots represent
parasites treated with DMSO alone]. (A) Survival of L. major after 45 min of H2O2 exposure; (B) survival of L. mexicana after 45 min of H2O2
exposure.
possible; the results from the different experiments have to be
considered independently.
exposed to the same concentrations of oxidant (Figure 7).
This suggests an impaired ability of the parasites to cope with
oxidative stress and is in agreement with the inhibitors being
able to target PTR1 in the parasites.14
As PTR1 has a well-established role in the resistance of
Leishmania to oxidative stress,14 its inhibition could lead to
increased sensitivity to oxidant challenges. Promastigotes
treated with 1 and 5b at their ED50 in L. mexicana and at 100
μg/mL in L. major showed a marked reduction in cell survival
upon exposure to H2O2 compared to untreated parasites
CONCLUSIONS
■
Our structure-based design and optimization of new inhibitors
of LmPTR1 successfully led to the identification of compounds
I
dx.doi.org/10.1021/jm300563f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
the eluent in different ratios. Purification conditions and analytical data
are reported below.
with an improved binding affinity and selectivity. The
compounds were designed to achieve specific interactions with
hydrophilic regions of the active site that the precursor,
compound 1, did not exhibit. N10 appeared to be the most
promising position for derivatization to enhance the potency of
the compounds. Compounds 5c and 5d were the best-
performing compounds in terms of both potency and selectivity.
The compounds were tested on promastigote-stage cells of
the parasite and on MRC-5 human cells to evaluate their
antileishmanial activity and toxicity, respectively. The com-
pounds alone did not show appreciable growth inhibition
activity, but in combination with the known DHFR inhibitor,
PYR, they showed remarkable synergistic activity at the
concentration tested. More precisely, 5b (compared to 1 and
to the other derived compounds) showed the best combination
of high synergistic inhibitory activity and low toxicity. It showed
no toxicity at 50 μg/mL and had parasitic growth inhibition in
combination with PYR at 30 μg/mL of 82.4% and 83.9% on L.
mexicana and on L. major, respectively.
The good combination index against L. major, the lack of
toxicity on human cells, and the ability to impair cell resistance
to oxidative stress (which is crucial for these trypanosomatids
due to their life-cycle phase occurring in the acidified
phagolysosome of macrophages) highlight the potential for
these compounds to be developed into more specific clinical
agents for counteracting leishmaniasis and other neglected
parasitic diseases.
1-(4-((2,4-Diaminopteridin-6-yl)methylamino)benzoyl)-
piperidine-4-carboxamide (5a). This compound was prepared in 42%
yield by the protocol described in the general procedure starting from 3
and 1-(4-aminobenzoyl)piperidine-4-carboxamide (4a) for 7 days; the
compound was purified by flash chromatography (chloroform:metha-
1
nol = 8:2, Rf 0.28) to give a white solid; mp >300 °C. H NMR
(CDCl3/DMSO-d6): δ 8.71 (1H, s, pteridin-H), 7.65 (2H, br s, exc.
with D2O, NH2), 7.20 (2H, d, J = 8.2 Hz, aryl-H), 6.73 (2H, d, J = 8.8
Hz, aryl-H), 6.64 (1H, br s, exc. with D2O, NH), 6.34 (2H, s, exc. with
D2O, NH2), 4.49 (2H, d, J = 4.6 Hz, CH2), 4.30−4.08 (2H, m,
piperidin-H), 3.02−2.90 (2H, m, piperidin-H), 2.60−2.30 (1H, m,
piperidin-H), 1.85−1.50 (4H, m, piperidin-H). IR (nujol): ν 3401,
3181, 1645, 1611 cm−1. UV (EtOH): λmax 368, 264, 205 nm. LC/MS:
m/z 422 [M + 1].
Methyl 1-(5-((2,4-Diaminopteridin-6-yl)methylamino)picolinoyl)-
piperidine-4-carboxylate (5b). This compound was prepared in 35%
yield by the protocol described in the general procedure starting from 3
and methyl 1-(5-aminopicolinoyl)piperidine-4-carboxylate (4b) for 7
days; the compound was purified by flash choromatography
(chloroform:methanol = 8:2, Rf 0.39) to give a white solid; mp >300
1
°C. H NMR (CDCl3/DMSO-d6): δ 8.75 (1H, s, pteridine-H), 8.10
(1H, d, J = 2.4 Hz, picolin-H), 7.73 (2H, br s, exc. with D2O, NH2),
7.44 (1H, d, J = 9.0 Hz, picolin-H), 7.11 (1H, dd, J = 8.4 Hz and J = 2.4
Hz, picolin-H), 6.96 (1H, t, exc. with D2O, NH), 6.40 (2H, s, exc. with
D2O, NH2), 4.55 (2H, d, J = 4.6 Hz, CH2), 4.52−4.04 (2H, m,
piperidin-H), 3.65 (3H, s, OCH3), 3.40−2.95 (2H, m, piperidin-H),
2.65−2.50 (1H, m, piperidin-H), 2.04−1.80 (2H, m, piperidin-H),
1.78−1.50 (2H, m, piperidin-H). IR (nujol): ν 3312, 1727, 1590 cm−1.
UV (EtOH): λmax 375, 264, 204 nm. LC/MS: m/z 438 [M + 1].
Methyl 1-(4-(((2,4-Diaminopteridin-6-yl) methyl)(2-ethoxy-2-ox-
oethyl) amino) benzoyl)piperidine-4-carboxylate (5c). This com-
pound was prepared in 50% yield by the protocol described in the
general procedure starting from 3 and methyl 1-(4-(2-ethoxy-2-
oxoethylamino) benzoyl) piperidine-4-carboxylate (4c) for 7 days; the
compound was purified by flash choromatography (chloroform:me-
thanol = 9:1, Rf 0.39) to give a pale yellow solid; mp 143−146 °C.1H
NMR (CDCl3/DMSO-d6): δ 8.76 (1H, s, pteridin-H), 7.67 (2H, s, exc.
with D2O, NH2), 7.24 (2H, d, J = 8.0 Hz, aryl-H), 6.69 (2H, d, J = 8.4
Hz, aryl-H), 6.45 (2H, s, exc. with D2O, NH2), 4.81 (2H, s, CH2), 4.38
(2H, s, CH2), 4.17 (2H, q, J = 6.4, CH2), 4.22−4.02 (2H, m, piperidin-
H), 3.64 (3H, s, CH3), 3.12−2.92 (2H, m, piperidin-H), 2.65−2.50
(1H, m, piperidin-H), 1.96−1.80 (2H, m, piperidin-H), 1.66−1.46
(2H, m, piperidin-H), 1.25 (3H, t, J = 6.4, CH3).IR (nujol): ν 3318,
3160, 1731, 1606 cm−1.UV (EtOH): λmax 376, 264, 205 nm.LC/MS:
m/z 523 [M + 1].
Methyl 1-(4-(((2,4-Diaminopteridin-6-yl)methyl)(2-hydroxyethyl)-
amino)benzoyl)piperidine-4-carboxylate (5d). This compound was
prepared in 57% yield by the protocol described in the general
procedure starting from 3 and methyl 1-(4-(2-hydroxyethylamino)-
benzoyl)piperidine-4-carboxylate (4d) for 7 days; the compound was
purified by flash choromatography (chloroform:methanol = 9:1, Rf
0.21) to give a white solid; mp 250−252 °C.1H NMR (CDCl3/DMSO-
d6): δ 8.59 (1H, s, pteridin-H), 7.52 (2H, s, exc. with D2O, NH2), 7.21
(2H, d, J = 8.6 Hz, aryl-H), 6.75 (2H, d, J = 8.6 Hz, aryl-H), 6.44 (2H,
br s, exc. with D2O, NH2), 4.79 (2H, s, CH2), 4.15−3.95 (2H, m,
piperidin-H), 3.71 (3H, s, CH3), 3.63 (2H, t partially obscured, CH2),
3.26 (2H, t partially obscured, CH2), 3.10−2.90 (2H, m, piperidin-H),
2.70−2.90 (1H, m, piperidin-H), 1.95−1.50 (4H, m, piperidin-H).IR
(nujol): ν 3457, 3226, 1732, 1682, 1650, 1604 cm−1.UV (EtOH): λmax
361, 246, 205 nm.LC/MS: m/z 481 [M + 1].
EXPERIMENTAL SECTION
Chemistry. General Procedures. All commercially available
solvents and reagents were used without further purification. Melting
■
points were measured with a Kofler hot stage or Digital Electrothermal
̈
melting point apparatus and are uncorrected. Infrared spectra were
recorded as nujol mulls on NaCl plates with a Perkin-Elmer 781 IR
spectrophotometer and are expressed in ν (cm−1). UV spectra are
qualitative and were recorded in nm for solutions in EtOH with a
Perkin-Elmer Lambda 5 spectrophotometer. Nuclear magnetic
resonance (1H, 13C -NMR) spectra were determined in CDCl3,
DMSO-d6, and CDCl3/DMSO-d6 (1:3 ratio) and were recorded with a
Varian XL-200 (200 MHz) spectrometer. Chemical shifts (δ scale) are
reported in parts per million (ppm) downfield from tetramethylsilane
(TMS) used as an internal standard. Splitting patterns are designated,
as follows: s, singlet; a s, apparent singlet; d, doublet; t, triplet; q,
quadruplet; m, multiplet; br s, broad singlet; dd, double doublet. The
assignment of exchangeable protons (OH and NH) was confirmed by
the addition of D2O. MS spectra were performed with a combined
liquid chromatograph−Agilent 1100 series mass selective detector
(MSD). Analytical thin-layer chromatography (TLC) was performed
on Merck silica gel F-254 plates. Pure compounds showed a single spot
in TLC. For flash chromatography, Merck silica gel 60 was used with a
particle size 0.040−0.063 mm (230−400 mesh ASTM). Elemental
analyses were performed on a Perkin-Elmer 2400 instrument at the
Laboratorio di Microanalisi, Dipartimento di Chimica e Farmacia,
̀
Universita di Sassari, Italy, and the results were within 0.4% of
theoretical values (Table S1, Table S2 in the Supporting Information).
The purity of final products was determined by either elemental
analysis or analytical HPLC, and this was more than 95%. The
preparation of compounds 4a−f and 10−12, 15, 18, and 21 are
reported in the Supporting Information.
Methyl1-(5-((2,4-Diaminopteridin-6-yl)methylamino)-2-
ethylbenzoyl)piperidine-4-carboxylate (5e). This compound was
prepared in 65% yield by the protocol described in the general
procedure starting from 3 and methyl 1-(5-amino-2-ethylbenzoyl)-
piperidine-4-carboxylate (4e) for 7 days; the compound was purified by
flash choromatography (chloroform:methanol = :1, Rf 0.30) to give a
white solid; mp 197−200 °C.1H NMR (CDCl3/DMSO-d6): δ 8.71
(1H, s, pteridin-H), 7.64 (2H, s, exc. with D2O, NH2), 7.02 (1H, d, J =
General Method for the Preparation of 2,4-Diaminopteridine
Derivatives 5a−f. A mixture of 6-(bromomethyl)pteridine-2,4-diamine
hydrobromide (3; 0.3 mmol), prepared according to the literature
procedure,8 and an excess of the amines (4a−f; 0.6 mmol), synthesized
as described in Supporting Information, in DMA (5 mL) was stirred at
room temperature until the reaction was complete. The solvent was
removed under reduced pressure, and the residue was purified by flash
column chromatography using a mixture of chloroform/methanol as
J
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Journal of Medicinal Chemistry
Article
8.2 Hz, aryl-H), 6. 72 (1H, d, J = 8.4 Hz, aryl-H), 6.52 (1H, s, aryl-H),
6.24 (1H, t, exc. with D2O, NH), 6.17 (2H, br s, exc. with D2O, NH2),
4.47 (2H, d, J = 6.4, CH2), 3.65 (3H, s, CH3), 3.30−2.80 (4H, m, CH2
and 2H, piperidin-H), 2.65−2.35 (3H, m, piperidin-H), 2.20−1.40
(4H, m, piperidin-H), 1.13 (3H, t, J = 6.4, CH3).IR (nujol): ν 3448,
3312, 1732, 1628 cm−1.UV (EtOH): λmax 375, 260, 207 nm.LC/MS:
m/z 465 [M + 1].
percentage growth inhibition that was achieved with a single compound
was compared to the one obtained by combining each inhibitor with
PYR.
Compounds ED50 Evaluation. To estimate the concentrations at
which compounds cause 50% inhibition of growth (effective dose,
ED50) of cultured Leishmania promastigotes, the Alamar Blue
micromethod based on monitoring the reducing environment of
proliferating cells was employed as previously described.17 Inhibitor
stock solutions were in DMSO. For each compound, dilutions were
made in culture medium and added to the parasite cultures, giving a
series of concentrations starting from 100 μg/mL downward. The ED50
values for compound 5b were higher than 100 μg/mL; therefore, a
specific series of assays was performed, giving a set of concentrations
from 500 μg/mL downward. The ED50 values were calculated by linear
interpolation and were the average of three different experiments each
performed in duplicate. The optical density in the absence of
compounds was set as the 100% control, whereas the commercial
antileishmaniasis drug Amphotericin B was used as a positive control.
Synergistic Activity with PYR. The linear interpolation procedure
described above has been used to calculate the ED30 values (estimated
concentrations at which compounds cause 30% inhibition of growth) of
PYR in L. major and L. mexicana. These values have been used to assess
the leishmanicidal effect of our compounds and PYR combined: the
Alamar Blue micromethod described above was modified, and each
sample was added at a series of concentrations of our compounds (from
100 μg/mL downward) and a fixed EC30 concentration of PYR.
Samples with only PYR were taken as the 100% control. The synergistic
effect of PYR combined with the additional compound on Leishmania
growth was calculated using the combination index, following the
Chou−Talalay method.13
Methyl 1-(4-((2,4-Diaminopteridin-6-yl)methylamino)-3-
ethylbenzoyl)piperidine-4-carboxylate. (5f). This compound was
prepared in 33% yield by the protocol described in the general
procedure starting from 3 and methyl 1-(4-amino-3-ethylbenzoyl)-
piperidine-4-carboxylate (4f) for 7 days; the compound was purified by
flash choromatography (chloroform:methanol = 9:1, Rf 0.29) to give a
1
pale-yellow solid; mp 218−220 °C. H NMR (CDCl3/DMSO-d6): δ
8.74 (1H, s, pteridin-H), 7.79 (2H, s, exc. with D2O, NH2), 7.38 (2H, s,
exc. with D2O, NH2), 7.14 (1H, d, J = 2.4 Hz, aryl-H), 7.09 (1H, dd, J =
8.2 Hz and J = 2.4 Hz aryl-H), 6.52 (1H, d, J = 8.2 Hz, aryl-H), 5.83
(1H, br s, exc. with D2O, NH), 4.60 (2H, s, CH2), 4.24−4.00 (2H, m,
piperidin-H), 3.68 (3H, s, CH3), 3.10−2.80 (2H, m, piperidin-H),
2.67−2.58 (3H, m, CH2 and 1H, piperidin-H), 2.10−1.45 (4H, m,
piperidin-H), 1.13 (3H, t, J = 7.0, CH3).IR (nujol): ν 3424, 3315, 1733,
1628 cm−1.UV (EtOH): λmax 375, 260, 207 nm.LC/MS: m/z 465 [M +
1].
1-(4-((2,4-Diaminopteridin-6-yl)methylamino)benzoyl)-
piperidine-4-carboxylic Acid (6a). To a solution of 5a (0.09 g, 0.21
mmol) in 4 mL of methanol, cooled with an external ice bath, was
added an aqueous solution of 1N NaOH (0.67 mL). The mixture was
stirred for 20 min. Then stirring was continued at room temperature for
an additional 2 h and 45 min. The brown mixture was filtered to remove
impurity on suspension. The mother liquors were diluted with 2 mL of
water and made acidic with some drops of 6N HCl. After removal of the
most methanol under reduced pressure and a few hours standing in the
fridge, two portions of 10 mg of acid as a yellow−orange dust were
collected. On evaporation in the air of the solvent, a gummy residue was
taken up with acetone to give rise to 6a (80 mg) with an overall yield of
92%; mp >300 °C. 1H NMR (DMSO-d6): δ 12.15 (1H, br s, COOH),
8.84 (1H, s, pteridin-H), 7.65 (2H, br s, exc. with D2O, NH2), 7.21
(2H, d, J = 8.6 Hz, aryl-H), 6.74 (2H, d, J = 8.6 Hz, aryl-H), 6.64 (1H,
br s, exc. with D2O, NH), 6.34 (2H, s, exc. with D2O, NH2), 4.52 (2H, a
s, CH2), 4.30−4.08 (2H, m, piperidin-H), 3.02−2.90 (2H, m, piperidin-
H), 2.60−2.30 (1H, m, piperidin-H), 1.85−1.50 (4H, m, piperidin-H).
IR (nujol): ν 3385 (broad), 3181, 1637 cm−1. UV (EtOH): λmax
388,386, 334, 322, 254 nm. LC/MS: m/z 423 [M + 1].
Oxidative Stress Evaluation. Parasites were plated at 105 cells/mL
in a 24-well plate, and the compounds were added at their ED50 doses
for 48 h at 28 °C in L. mexicana experiments. In L. major experiments,
compounds 1 and 5b were added at 100 μg/mL due to the very high
ED50. Subsequently, cells were diluted to 2 × 104 cells/mL and
subjected to H2O2 treatment for 45 min. Surviving motile parasites
were counted using a Neubauer counting grid on an invertoscope. The
results are expressed as a series of percentages, taking the compound-
treated, non-H2O2 exposed sample as the 100% control for each
compound treatment. This series was compared with a series of
untreated, H2O2-exposed samples.
Toxicity Test. The human fibroblast cell line MRC-5 was used to
determine the possible toxicity of the compounds in human cells. The
culture and toxicity assays were performed using essentially the same
method as described previously.18 In brief, the fibroblasts were
cultivated at 37 °C in DMEM medium (Gibco) in the presence of
10% heat-inactivated FBS and extra glutamine in humidified incubators
with an atmosphere of 5% CO2. Cells were seeded to each well of the
microplates at a density such that, after 72 h of incubation, adhesive
cells had formed a confluent monocellular film in the control wells.
After 72 h, Alamar Blue was added and the optical density was
measured.
Docking Studies. The docking simulations were performed using
the crystallographic structure of LmPTR1 in complex with the cofactor
NADP and the substrate dihydrobiopterin (PDB ID: 1E92). The
rationale behind choosing this X-ray structure was that it displayed the
highest resolution of 2.20 Å among all the available structures of this
protein. However, to continue our study, the dihydrobiopterin
molecule was removed from the active site and our target compounds
were docked in place of it. The docking was performed using the
Lamarckin genetic algorithm, which is a subutility available in Autodock
software version 4.0.5.19 Prior to the starting of docking our target
compounds, the accuracy of the algorithm was validated by verifying its
ability to reproduce the published crystallographic binding conforma-
tions of compound 1 (PDB ID: 3H4V) and MTX (PDB ID: 1E7W).
Thereafter, an initial population of random individuals was used with a
population size of 150 individuals. A maximum number of energy
evaluations count was set to be 2500000, and the value of the highest
number of generations was kept as 27000. The number of individuals
that automatically survive into the next generation (i.e., the elitism
Enzymology. The proteins were purified as described previously.7
The folate cofactors and substrates were a gift from Merck Eprova; all
other substrates, cofactors, and reagents were purchased from Sigma.
LmPTR1, hDHFR, and hTS activities were assessed spectrophoto-
metrically as previously described.7 Kinetic studies were performed as
continuous assays executed in a Beckman DU640 spectrophotometer.
Ki values were obtained from IC50 (concentration of inhibitor causing
50% enzyme activity inhibition) plots assuming competitive inhib-
ition.15 The Ki standard errors were determined from at least two
independent experiments performed in triplicate. Compounds were
screened for their activity against LmPTR1, hDHFR, and hTS as
previously described.7 Selectivity indices (SI) were calculated as follow:
SI
= Ki hDHFR/Ki LmPTR1; SI
= Ki hTS/Ki LmPTR1.
hDHFR/LmPTR1
hTS/LmPTR1
The DMSO concentration was kept below the concentration affecting
enzyme activity (1% for LmPTR1, 5% for hDHFR, 8% for hTS).
Parasitology. Cell Culture. Promastigote forms of L. mexicana
(MHOM/BZ/84/BEL46) and L. major (MHOM/SU/73/5-ASKH)
were cultured as described earlier.16 The cultures were initiated at 105
parasites/mL, and the cells were harvested in their exponential phase of
growth at a density of 2 × 107 parasites/mL.
Parasitic Growth Inhibition. For a first evaluation of the
combinatory effect, promastigotes were exposed to high compounds
concentrations (50 μg/mL of compound) alone or in combination with
PYR at 30 μg/mL; to work with such high concentrations of
compounds, parasites were plated at 106 cells/mL in a 96-well plate,
enabling to perform the experiment on cells that were leaving the
exponential growth phase and entering the stationary phase. The
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Journal of Medicinal Chemistry
Article
value) was kept 1; the probability that a gene would mutate was set to
0.02. Moreover, the probability that 2 individuals could get crossover
was 0.8. Also, the proportional selection criteria was used, where the
average of the worst energy was calculated over a window size of 10
generations. The pseudo Solis and Wets local search method was
implemented during the docking and the maximum number of
iterations per local search was set to 300; the probability of performing
a local search on an individual in the population was used as 0.06. The
maximum number of consecutive successes or failures before doubling
or halving the local search step size (P, ρ) was 4 in both cases, where the
lower bound value on ρ was 0.01.
During the ligand binding procedure, it is very common that the
binding might induce some conformational changes in the active site
pocket. So, to hold our docking calculations accountable, side chain
flexibility criteria was also included, where the side chains of the
residues in the vicinity of the active region (Ser111, Phe113, Asp181,
Leu189, Tyr191, Tyr194, Asp232, and His241 of chain A and Arg287′
of chain D of tetramer were allowed to move, keeping their backbone
fixed.
For analyzing the factors contributing to the selectivity of compound
5b against hDHFR (PDB ID: 2W3M), all the above parameters were
kept the same while performing the docking, except owing to the
different active site topology, in this case, side chain flexibility was
included for the active site residues: Ile7, Leu22, Arg28. Phe31, Phe34,
Gln35, Ile60, Asn64, Leu67, Lys68, and Tyr121 of chain A.
This methodology allowed us to sample their flexible conformations,
keeping the ligand binding simultaneously into consideration.
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ASSOCIATED CONTENT
* Supporting Information
■
(8) Piper, J. R.; Montgomery, J. A. Preparation of 6-(Bromomethyl)-
2,4-pteridinediamine Hydrobromide and Its Use in Improved
Syntheses of Methotrexate and Related Compounds. J. Org. Chem.
1977, 42, 208−211.
S
Elemental analyses of compounds 5a−f and 6a; elemental
analyses of intermediates 4a, 10, 4b, 15, 4c, 4d, 12, 11, 4e, 18,
4f, and 21; 13C NMR spectra of compounds 5a−f and 6a. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(9) Schneider, M.; Harris, T. M. Synthesis of DL-Slaframine. J. Org.
Chem. 1984, 49, 3681−3684.
(10) Reimschneider, R.; Kassahn, H.-G. Acylderivate cyclischer
Verbindungen, V. Zur Herstellung von o-Diacetyl-benzol und 4-
Nitro-1.2-diacetyl-benzol. Chem. Ber. 1959, 92, 1705−1709.
AUTHOR INFORMATION
Corresponding Author
(11) Buhler, S.; Lagoja, I.; Giergrich, H.; Stengele, K. P.; Pfleiderer, W.
̈
■
New Types of Very Photolabile Protecting Groups Based upon the [2-
(2-Nitrophenyl)propoxy]carbonyl (NPPOC) Moiety. Helv. Chim. Acta
2004, 87, 620−659.
*For M.P.C.: phone, 0039-059-205-5143; fax, 0039-059-205-
5131; E-mail, mariapaola.costi@unimore.it. For S.F.: phone,
0039-059-205-5125; fax, 0039-059-205-5131; E-mail, stefania.
ferrari@unimore.it.
(12) Sienkievikz, N.; Ong, H. B.; Fairlamb, A. H. Trypanosoma brucei
pteridine reductase 1 is essential for survival in vitro and for virulence in
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Author Contributions
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The manuscript was written through contributions of all
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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We gratefully acknowledge the support of the Italian
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ABBREVIATIONS USED
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Michels, P. A.; Wade, R. C.; Costi, M. P. Virtual screening identification
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DHFR, dihydrofolate reductase; ED30, effective dose at which
compounds cause 30% inhibition of growth; ED50, effective dose
at which compounds cause 50% inhibition of growth; hDHFR,
human dihydrofolate reductase; hTS, human thymidylate
synthase; Ki, inhibition constant; LmPTR1, Leishmania major
pteridine reductase; MTX, methotrexate; PABA, p-amino-
benzoic acid; PTR1, pteridine reductase; PYR, pyrimethamine
L
dx.doi.org/10.1021/jm300563f | J. Med. Chem. XXXX, XXX, XXX−XXX