Novel Selective Inhibitor of Leishmania (Leishmania) amazonensis Arginase

Article abstract of DOI:10.1111/cbdd.12566

Arginase is a glycosomal enzyme in Leishmania that is involved in polyamine and trypanothione biosynthesis. The central role of arginase in Leishmania (Leishmania) amazonensis was demonstrated by the generation of two mutants: one with an arginase lacking

Full text of DOI:10.1111/cbdd.12566

Received Date : 23-Oct-2014
Revised Date : 09-Feb-2015
Accepted Date : 25-Mar-2015
Article type : Research Article
Novel selective inhibitor of Leishmania (Leishmania) amazonensis arginase
Edson R. da Silva1*, Nubia Boechat2, Luiz C. S. Pinheiro2, Monica M. Bastos2, Carolina C.
P. Costa2, Juliana C. Bartholomeu1, Talita H. da Costa1
1
Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de
Alimentos, Universidade de São Paulo, Pirassununga, SP 13635-900, Brazil.
2
Departamento de Síntese de Fármacos, Instituto de Tecnologia em Fármacos,
Farmanguinhos - FIOCRUZ, Rio de Janeiro, RJ, 21041-250, Brazil.
*corresponding author e-mail: edsilva@usp.br
Phone number +55 (19) 3565-4371
Departamento de Medicina Veterinária
Faculdade de Zootecnia e Engenharia de Alimentos
Universidade de São Paulo
13635-900 Pirassununga, SP, Brazil.
Abstract
Arginase is a glycosomal enzyme in Leishmania that is involved in polyamine
and trypanothione biosynthesis. The central role of arginase in L. (L.) amazonensis was
demonstrated by the generation of two mutants: one with an arginase lacking the
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12566
This article is protected by copyright. All rights reserved.

glycosomal addressing signal and one in which the arginase coding gene was knocked
out. Both of these mutants exhibited decreased infectivity. Thus, arginase seems to be a
potential drug target for Leishmania treatment. In an attempt to search for arginase
inhibitors, twenty-nine derivatives of the [1,2,4]triazolo[1,5-a]pyrimidine system were
tested against L. (L.) amazonensis arginase in vitro. The [1,2,4]triazolo[1,5-a]pyrimidine
scaffold containing R1 = CF3 exhibited greater activity against the arginase rather than
when the substituent R1 = CH3 in the 2-position. The novel compound 2-(5-methyl-2-
(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)hydrazinecarbothioamide (30) was
the most potent, inhibiting arginase by a non-competitive mechanism, with the Ki and
IC50 values for arginase inhibition estimated to be 17 ± 1 µM and 16.5 ± 0.5 µM,
respectively. These results can guide the development of new drugs against
leishmaniasis based on [1,2,4]triazolo[1,5-a]pyrimidine derivatives targeting the
arginase enzyme.
Keywords: triazolopyrimidine; trifluoromethyl; arginase; polyamines; trypanothione;
Leishmania
1. Introduction
Leishmaniasis is considered by the World Health Organization (WHO) as one of
the world’s most neglected diseases (WHO 2010). Because there are different causative
species (1, 2) and various clinical manifestations of the disease, the treatment of
leishmaniasis is complicated. Pentavalent antimonial compounds (3), amphotericin B (1,
2), pentamidine (4), miltefosine (5) have been used in the treatment of leishmaniasis.
The misuse of those drugs, incomplete treatment, inappropriate prescription guidelines
and the occurrence of serious adverse effects have led to the emergence of drug-
resistant Leishmania parasites (6-8).
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An important approach to the rational development of new drug candidates is
the selection of a specific target with which the candidate compounds can interact.
Knowledge of parasite-host relationships is essential to the development of new drugs
against parasites. The arginase enzyme of Leishmania has been shown to be a promising
target, and simple molecules have achieved a good inhibition of arginase (9-12).
The metabolism of the amino acid L-arginine during Leishmania infection is
crucial because it is required for both the parasite and host (10, 13). Parasites sense the
internal/external pool of L-arginine and regulate the expression of two genes coding for
transporters in response (14). The amino acid L-arginine is hydrolyzed by arginase (E.C.
3.5.3.1), yielding L-ornithine and urea in the first step of polyamine biosynthesis in
Leishmania. The L-ornithine used in the synthesis of polyamines plays an important role
in cellular processes such as growth and the antioxidant activity mediated by
trypanothione (13, 15, 16). The Leishmania arginase is a glycosomal enzyme (17, 18),
and this compartmentalization is important for L-ornithine production and optimal
infectivity (19).
The deletion of the arginase gene demonstrated the importance of this enzyme
for Leishmania major, which becomes auxotrophic for polyamines (20). The Leishmania
arginase exhibits significant differences from human arginase that can be exploited in
the design of specific inhibitors (11, 12, 21). Thus, arginase seems to be a
pharmacological target of interest for the development of new anti-leishmaniasis drugs.
Many heterocyclic systems such as quinolines (22), isoquinoline (23), indoles
(24), purines (25), pyrazolopyrimidines (26), pyrazolopyridines (27) and thienopyridine
(28, 29) have been developed in an attempt to find new drugs to treat leishmaniasis.
Iyamu and co-workers reported chloroquine as the first example of an anti-malarial
agent displaying competitive inhibition of arginase (28, 29).
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Recently, we described a series of twenty-six [1,2,4]triazolo[1,5-a]pyrimidine
derivatives (4–7, 9–16, 18–29, 31-32), which were planned as bioisosteres of
chloroquine. This series was evaluated in vitro against Plasmodium falciparum and
showed good to excellent activities (28). As triazolopyrimidine is bioisostere of quinoline,
chloroquine is an arginase inhibitor, and arginase is a possible target for leishmaniasis,
we decided to test twenty nine [1,2,4]triazolo[1,5-a]pyrimidines against the arginase
from L. amazonensis.
Different arylamines and aliphatic amines were incorporated into the
[1,2,4]triazolo[1,5-a]pyrimidine scaffold to investigate the importance of the substituent
at the 7-position. The trifluoromethyl moiety, one of the most widespread fluorine-
containing functional groups in bioactive molecules, is a highly electronegative
substituent that can exert a significant electronic influence on neighboring groups. It is
also one of the most lipophilic groups known, making it useful for improving the
targeting of molecules to the active sites of enzymes (28). Therefore, we tested a
[1,2,4]triazolo[1,5-a]pyrimidine scaffold with 2-CF3 (4-11, 13-15, 17, 19-30 and 32),
2-CH3 (12, 16 and 18) and 2-H (31) to compare the importance of these substituents to
the enzymatic activity. Compounds 8 and 17 were not synthesized in our previous work
and now were obtained. The literature has described structural units such as N(C=N)N,
N(C=S)N or C(C=N)N, as required for leishmanicidal activity (Figure 1) (28).
Additionally, expecting that the introduction of a thiosemicarbazide structural unit could
enhance the leishmanicidal activity, we synthesized the new compound 30 (Figure 2).
2. Materials and Methods
2.1 Chemistry
The synthetic route used to produce [1,2,4]triazolo[1,5-a]pyrimidine derivatives
4–7, 9–16, 18–29, 31, 32 was previously described (28) and was reproduced for the
synthesis of the compounds 8, 17 and 30 (Figure 3).
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2.2 Experimental
1H-, 13C- and 19F-Nuclear Magnetic Resonance (NMR) spectra were obtained at
400.00 MHz, 100.00 MHz and 376.00 MHz, respectively, on a Bruker Avance instrument
equipped with a 5 mm probe, using tetramethylsilane as the internal standard. Chemical
shifts (δ) are reported in ppm, and coupling constants (J) are reported in Hertz. Fourier
transform infrared (FT-IR) absorption spectra were recorded on a Shimadzu mode IR
Prestige-21 spectrophotometer by reflectance in KBr. GC/MS experiments were
conducted using a model 6,890 N gas chromatograph (Agilent, Palo Alto, CA, USA)
equipped with a 7,683 B autosampler coupled with a model MS 5,973 N single
quadrupole mass spectrometer (Agilent). Electron-ionization mass spectra (EI-MS, scan
ES+ Capillary (3.0 kV) / cone (30 V) / extractor (1 V) / RF lens (1.0 V) / source
temperature (150 °C) / desolvation temperature (300 °C)) were recorded using a
Micromass/Waters Spectrometer (model: ZQ-4000). The GC was equipped with a HP-
5MS capillary column 30 m in length and 0.25 mm in diameter, with a 0.25 μm film
thickness. The initial temperature was 50 °C, and was then increased at 10 °C/min to
300 °C, where it was held for 10 minutes. The helium flow rate was 0.5 mL/min. Melting
points (m.p.) were determined with a Büchi model B-545 apparatus. TLC was performed
using silica gel F-254 glass plates (20 × 20 cm). All other reagents and solvents were of
analytical grade.
2.3
General
procedure
for
preparing
5-Methyl-7-
arylamine/hydrazinecarbothioamide[1,2,4]triazolo[1,5-a]pyrimidines 8, 17 and
30.
A mixture of a 7-chloro[1,2,4]triazolo[1,5-a]pyrimidine derivative 3c and the
appropriate amine or thiosemicarbazide (1 equivalent) in ethanol (10 mL) was stirred at
room temperature for 6–18 h. The reaction mixture was concentrated and poured into
50 mL of ice-cold water. The precipitate was collected by filtration and washed with
water.
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7-(4-Chlorophenylamine)-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine
(8). Yield: 80%; m.p. 104-106 °C. IR (KBr, cm−1): 3468; 3047; 2887; 2306; 1664;
1612, 1589; 1487; 1365; 1338; 1325; 1200, 1190, 1170; 1157; 1089; 893; 819; 763.
1H-NMR (400 MHz, DMSO-d6, δ in ppm): 2.64 (s; 3H; CH3); 6.66 (s, 1H, H-6); 7.52 (d, J
= 8.5 Hz, 2H, H-2'; H-6'); 7.62 (d; J = 8.5 Hz; 2H; H-3'; H-5'). 13C-NMR (100 MHz,
DMSO-d6, δ in ppm): 21,11; 93,60; 120,35 (q; J = 269 Hz; C-9); 128,56; 131,63;
134,56; 135,89; 151,42; 152,18; 157,37 (q; J = 40 Hz; C-2); 162,07. 19F-NMR (376
MHz, DMSO-d6, δ in ppm): - 67.45 (s; 3F). GC/ME (70 eV) m/z (%): 327 (100); 328
(M+1); 329; 312; 258; 190; 177; 111.
5-methyl-7-(4-trifluorophenylamine)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine
(17). Yield: 80%; m.p. 100-103 °C. IR (KBr, cm−1): 3633; 3005; 2357; 1631; 1608;
1
1570; 1516; 1431; 1373; 1323; 1296; 1195; 1149; 848; 806. H-NMR (400 MHz,
DMSO-d6, δ in ppm): 2.54 (s; 3H; CH3); 6.71 (s; 1H; H-6); 7,67 (d; J = 8.5 Hz; 2H; H-
2'; H-6'); 7,81 (d; J = 8.5 Hz; 2H; H-3'; H-5'). 13C-NMR (100 MHz, DMSO-d6, δ in ppm):
25.25; 92.82; 121.01 (q, J = 268 Hz, C-9); 125.59 (q, J = 269 Hz, C-18); 125.75;
128.14; 129.61 (q, J = 33 Hz, C-15); 141.53; 147.97; 156.85 (q, J = 39 Hz, C-
2);157.40; 169.10. 19F-NMR (376 MHz, DMSO-d6, δ in ppm):- 67.22 (s; 3F); - 63.90 (s;
3F). GC/ME (70 eV) m/z (%): 361 (100); 362 (M+1); 346; 292; 190; 177; 145.
2-(5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-
yl)hydrazinecarbothioamide (30). Yield: 98%; m.p. 195-197 °C. IR (KBr, cm−1): 1620;
1585; 1512; 1188; 1157; 1000; 756. 1H-NMR (400 MHz, DMSO-d6, δ in ppm): 2.50 (s;
3H; CH3); 6.30 (s; 1H; H-6); 7.88 (s; 1H; NH2); 8.17 (s; 1H; NH2); 9.87 (s; 1H; NH);
10.81 (s; 1H; NH). 13C-NMR (100 MHz, DMSO-d6, δ in ppm): 24.87; 90.11; 119.45 (q; J
= 268 Hz; C-9); 148.58; 154.40 (q, J= 40 Hz, C-2); 155.60; 166.45; 182.10. 19F-NMR
(376 MHz, DMSO-d6, δ in ppm): - 64,47 (s; 3F). EI/MS m/z (%):290 (100).
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2.4 Expression and purification of arginase
Recombinant ARG-L was expressed and purified as previously describe (30).
2.5 Inhibitor screening and determination of IC50
Arginase inhibition, IC50 and the mode of inhibition were determined as previously
described (31, 32). Compounds were screened for inhibition using 1 mM of each
prepared in MeOH at 10 mM. The inhibition experiments were performed at pH 9.5 in 50
mM CHES buffer, 50 mM L-arginine (pH 9.5) and enzyme at 0.006 µM. The samples were
incubated in a water bath at 37 °C for 15 minutes. Urea was quantified as described by
Berthelot (33). Briefly, the catalytic activity of the arginase reactions was stopped by
transferring 10 µL of reaction mixture into 750 µL of reagent A (20 mM phosphate
buffer, pH 7, containing 60 mM salicylate, 1 mM sodium nitroprusside and >500 IU of
urease). This mixture was incubated at 37oC for 5 minutes. Next, 750 µL of reagent B
(sodium hypochlorite 10 mM and NaOH 150 mM) were added, and then the samples
were incubated at 37oC for 10 min. The positive and negative controls were performed
under the same conditions in the absence of inhibitor. At least three independent
experiments were performed with duplicate samples.
The IC50 (the concentration that inhibits 50% of the catalytic activity of the
enzyme) was determined by varying the inhibitor concentration with a 1:10 dilution
factor. The experiments were performed in duplicate in at least three independent
experiments until a non-linear regression coefficient R2 ≥ 0.85 was obtained. The
reactions were performed under the same conditions described above. A sigmoidal model
(log IC50) was used to determine the IC50 in Origin 8.0 software.
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2.6 Determination of the constants Ki, Ki' and the mechanism of
inhibition
All reactions were performed in 50 mM CHES buffer, pH 9.5, containing variable
concentrations of the substrate L-arginine (12.5, 25, 50 and 100 mM) at pH 9.5.
Inhibitors were tested at three different concentrations close to the IC50. All reactions
were performed in duplicate in at least three independent experiments.
The inhibitor association constant (Ki) was determined by plotting the reciprocal
initial velocity of catalysis (1/Vo) as a function of inhibitor concentration (34). For non-
competitive inhibition, y = 0 was used to determine the values of the constants Ki and
Ki’.
2.7 Cell Cytotoxicity
The cytotoxicity of new compounds was determined as previous described (28).
3. Results and Discussion
3.1 Arginase inhibition by trifluoromethyltriazolopyrimidine
The compounds synthesized with different substituents in the 2-, 5-, and 7-
positions of the [1,2,4]triazolo[1,5-a]pyrimidine scaffold were previously tested with
HepG2 cells and were shown to induce no toxicity (28), and in this study they were
tested at 1 mM against the arginase enzyme from Leishmania (Leishmania) amazonensis
(Table 1).
Twelve compounds (5, 7-8, 11-13, 15, 17, 21, 26, 29 and 30) inhibited
arginase in the range of 23-79% at 1 mM. The [1,2,4]triazolo[1,5-a]pyrimidine scaffold
containing R1 = CF3 at the 2-position exhibited greater activity against the arginase
enzyme than when the substituent R1 = CH3.. This finding can be confirmed by
comparing the derivatives 11 and 12 (3',5'-diCl); 15 and 16 (3',5'-diOCH3); 17 and 18
(4'-CF3). The trifluoromethylated derivative 11 inhibited 71% of arginase activity and
was approximately 3 times more active than analog 12. Compounds 15 (39%) and 17
(52%) became inactive when the CF3 groups were replaced by CH3 in derivatives 16 and
18.
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Derivatives 22-25, 31, and 32 without aromatic amines at the 7-position were
inactive with the exception of the pyrrolidinyl derivative 25, which exhibited a low level
of inhibition (10%).
Compound 30 (Figure 4) with the structural units thiosemicarbazide inhibited
the arginase activity by 79% corresponding our expectations. However, compounds 5,
7-8, 11-13, 15, 17, 21, 26 and 29 also showed activity despite lacking these structural
units. Compound 30 showed selectivity for parasite enzyme because it did not inhibit rat
liver arginase, used as model of mammalian arginase. The compound 11 did not inhibit
arginase at 100 µM and we did not consider it in kinetics analysis.
3.2 Kinetic analysis of arginase inhibition by compound 30
Based on the results of the experiments with trifluoromethyl[1,2,4]triazolo[1,5-
a]pyrimidine derivatives at 1 mM described above, we designed, synthesized and tested
the novel compound 30 against the arginase enzyme from L. (L.) amazonensis.
Compound 30 has a CF3 group in the 2-position and a hydrazinecarbothioamide in the 7-
position, and is the most active compound of the series tested here. Compound 30
showed an IC50 of 16.5 ± 0.5 µM calculated by a nonlinear regression of the dose
response curve (Figure 5). The mechanism of inhibition was determined by varying the
concentration of inhibitor and substrate in the reaction. The Dixon and Cornish-Bowden
plots (28) reveal that compound 30 inhibits the arginase of L. (L.) amazonensis by a
non-competitive mechanism (Ki = Ki’) (Figure 6). Taking 1/V or S/V (y-axis) as zero in
the equations obtained by linear regression for the Dixon and Cornish-Bowden plots, the
values of the constants Ki were estimated as 17 ± 1 µM. This result shows that
compound 30 binds with equal affinity to the free enzyme or the enzyme-substrate
complex. The compound 30 showed an IC50 closed related with a natural arginase
inhibitor orientin (IC50 = 16 ± 2 µM) (32) and quercetin (IC50 = 4.30 ± 0.03 µM) (30).
Quercetin was used as a control drug and showed an IC50 of the 4.0 ± 0.5 µM.
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3.3 Toxicity
The toxicity of the active compound 30 was evaluated and showed MDL50 >1000
µg/mL (> 3.4 mM) against BGM cell line. The compounds 8 and 17 showed a MDL50 of
the 0.6 and 0.4 mM, respectively, against a human hepatocyte cell line (HepG2). The
toxicity of others compounds was determined previous (28).
Conclusions
These results show that the [1,2,4]triazolo[1,5-a]pyrimidine scaffold is an
effective starting point for the inhibition of the arginase enzyme and that the CF3 group
increases the activities of these derivatives. The compound 30 with thiosemicarbazide
structural units were crucial to enhance the inhibition of activity. However, compounds
5, 7-8, 11-13, 15, 17, 21, 26 and 29 have also exhibited activity but lack these
structural units. The novel compound 30 is a selective and non-competitive inhibitor of
the arginase from L. (L.) amazonensis. These data suggest that these inhibitors of the
enzyme arginase from L. amazonensis can be used as lead compounds for the design of
new agents against Leishmania.
Acknowledgements
This research was supported by Sao Paulo Research Foundation (FAPESP/grant
#09-08715-3), Rio de Janeiro Research Foundation (FAPERJ) and the Technological
Development Program on Products for Health (PDTIS). The authors thank the
Coordination of Improvement of Higher Education (CAPES) and the National Council of
R&D of Brazil (CNPq) for the fellowships granted. We thank Thiago S. Silva, Alcione S.
Carvalho and Karen D. B. Dutra for synthesizing the triazolopyrimidines, and Antoniana
Ursine Krettli e Anna Caroline Campos Aguiar (Rene Rachou Research Center/CPqRR
Oswaldo Cruz Foundation, Minas Gerais, Brazil) to perform toxicity assays.
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Table 1. Preliminary screening of arginase inhibition by [1,2,4]triazolo[1,5-a]pyrimidine
derivatives 4–32.
Arginase
Inhibition
(1 mM)
Compounds
R1
R2
R3
4
CF3
CH3
0%
29%
1%
HN
5
6
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
NH
7
51%
42%
3%
8
9
10
11
0%
Cl
71%
Cl
NH
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Cl
Cl
12
13
14
15
16
17
18
19
CH3 CH3
23%
47%
5%
Cl
Cl
NH
NH
CF3
CF3
CF3
CF3
CH3
CH3
39%
0%
CH3 CH3
CF3
CH3
52%
0%
CH3 CH3
F
CF3
CH3
8%
NH
F
N
20
21
22
CF3
CF3
CF3
CH3
CH3
CH3
0%
57%
0%
NH
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23
24
CF3
CF3
CH3
CH3
0%
0%
25
26
CF3
CF3
CH3
CH3
10%
47%
N
27
28
29
30
31
32
CF3
CF3
CF3
CF3
H
CH3
CH3
CH3
CH3
CH3
CH3
N
0%
0%
38%
79%
0%
CF3
0%
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.