Phenylhydrazides as inhibitors of Leishmania amazonensis arginase and antileishmanial activity

Article abstract of DOI:10.1016/j.bmc.2019.07.022

Searching for new substances with antileishmanial activity, we synthesized and evaluated a series of α,α-difluorohydrazide and α,α-difluoramides against Leishmania amazonensis arginase (LaArg). Four α,α-difluorohydrazide derivatives showed activity against LaArg with K_i in the range of 1.3–26 μM. The study of the kinetics of LaArg inhibition showed that these substances might act via different inhibitory mechanisms or even by a combination of these. The compounds were tested against L. amazonensis promastigotes and the best result was obtained to the compound 4 (EC₅₀ of 12.7 ± 0.3 μM). In addition, in order to obtain further insight into the binding mode of such compounds, molecular docking studies were performed to obtain additional validation of experimental results. Considering these results, it is possible to conclude that α,α-difluorohydrazide derivatives are a promising scaffold in the development of new substances against the etiological agent of leishmaniasis by targeting LaArg.

Full text of DOI:10.1016/j.bmc.2019.07.022

Accepted Manuscript
Phenylhydrazides as inhibitors of Leishmania amazonensis arginase and anti-
leishmanial activity
Evanoel Crizanto de Lima, Frederico S. Castelo-Branco, Claudia C. Maquiaveli,
André B. Farias, Magdalena N. Rennó, Nubia Boechat, Edson R. Silva
PII:
S0968-0896(19)30316-5
https://doi.org/10.1016/j.bmc.2019.07.022
BMC 15006
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
8 March 2019
3 July 2019
10 July 2019
Please cite this article as: Crizanto de Lima, E., Castelo-Branco, F.S., Maquiaveli, C.C., Farias, A.B., Rennó, M.N.,
Boechat, N., Silva, E.R., Phenylhydrazides as inhibitors of Leishmania amazonensis arginase and antileishmanial
activity, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.07.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Phenylhydrazides as inhibitors of Leishmania amazonensis arginase
and antileishmanial activity
a
b
c
Evanoel Crizanto de Lima Frederico S. Castelo-Branco Claudia C. Maquiaveli , André
,
,
B. Farias Magdalena N. Rennó Nubia Boechat Edson R. Silvac*
d
d
b*
,
,
,
a
Laboratório de Catálise e Síntese de Substâncias Bioativas, Universidade Federal do Rio
de Janeiro Campus Macaé Professor Aloísio Teixeira, Estrada do Imburo s/n - Ajuda de
Baixo, Macaé, RJ, CEP 27979-000, Brazil
b
c
Departamento de Sintese de Fármacos, Instituto de Tecnologia em Fármacos,
Farmanguinhos – FIOCRUZ, Rio de Janeiro, RJ, 21041-250, Brazil
Universidade de São Paulo Faculdade de Zootecnia e Engenharia de Alimentos,
Departamento de Medicina Veterinária, Laboratório de Farmacologia e Bioquímica
(LFBq), Av. Duque de Caxias Norte, 225, Pirassununga, SP, 13635-900, Brazil
d
Instituto de Biodiversidade e Sustentabilidade NUPEM/UFRJ, Universidade Federal do
Rio de Janeiro, Campus Macaé Professor Aloísio Teixeira, Av. São José do Barreto,
7
64, Macaé, RJ, 27965-045, Brazil
*
Corresponding authors e-mail: boechat@far.fiocruz.br; edsilva@usp.br

Abstract: Searching for new substances with antileishmanial activity, we synthesized
and evaluated a series of α,α-difluorohydrazide and α,α-difluoramides against
Leishmania amazonensis arginase (LaArg). Four α,α-difluorohydrazide derivatives
showed activity against LaArg with K in the range of 1.3 - 26 μM. The study of the
i
kinetics of LaArg inhibition showed that these substances might act via different
inhibitory mechanisms or even by a combination of these. The compounds were tested
against L. amazonensis promastigotes and the best result was obtained to the compound
4
(EC of 12.7 ± 0.3 μM). In addition, in order to obtain further insight into the binding
50
mode of such compounds, molecular docking studies were performed to obtain additional
validation of experimental results. Considering these results, it is possible to conclude
that α,α-difluorohydrazide derivatives are a promising scaffold in the development of new
substances against the etiological agent of leishmaniasis by targeting LaArg.
Keywords: Leishmania, Polyamines, Arginase, difluorohydrazide,
Introduction
Leishmaniasis is a neglected tropical disease that affects 12 million people in the
1
world. Enzymes in the polyamines pathway in trypanosomatids have been studied and
2
proved to be potential antileishmanial targets. Polyamines are involved in the synthesis
of trypanothione, a physiologic antioxidant of trypanosomatids that is used to scavenge
oxygen (ROS) and nitrogen free radicals (NO) produced by host macrophages.3,4 In
Leishmania, the arginase is the first enzyme of polyamine biosynthesis that converts L-
5
arginine into L-ornithine plus urea and is involved in Leishmania pathogenesis. The
inhibition of the polyamine pathway leads to mitochondrial dysfunction, kDNA
disorganization, and parasite death.6,7 Arginase gene knockout or arginase gene mis-
localization mutation showed the importance of arginase in Leishmania infection.9
Inhibitors of Leishmania arginase were first described against Leishmania
mexicana and Leishmania amazonensis.10,11 Isolated natural compounds also showed
high inhibition of L. amazonensis arginase.12–14 Indeed, such inhibitors proved to have a
higher potency on parasite arginase in comparison with the mammalian enzyme, which
8

shows that this target can be selectively modulated.12 Otherwise, only one synthetic
compound (2-(5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-
yl)hydrazinecarbothioamide) was described as a selective L. amazonensis arginase
inhibitor.15 Arginase inhibitors first described against human and rat arginases also
showed high affinity for L. mexicana arginase.10,16
The second enzyme of the polyamine pathway is ornithine decarboxylase (ODC),
which converts the arginase product L-ornithine into putrescine and carbon dioxide. ODC
was the target of eflornithine (Fig. 1), an antiparasitic drug used against Trypanosoma
brucei species, a trypanosomatid that causes sleeping sickness.17,18
Despite the eflornithine being shown as inhibitor of ODC, one important structural
aspect of this compound is the presence of a difluoromethyl group, which differentiates
it from ornithine. In fact, fluorine is an important substituent in medicinal chemistry due
to its capability to make hydrogen bonds, increase the lipophilicity of the compound and
alter the electronic aspects of the molecule because of its high electronegativity.19
Because of these chemical characteristics, and in an attempt to develop new arginase
inhibitors, we screened a series of α,α-difluoridrazides (4-9) and α,α-difluoramides (10-
1
2) against L. amazonensis arginase. Such substances are structurally related to
eflornithine in the presence difluoromethyl groups and nitrogen atoms capable of making
electrostatic interactions like drug eflornithine (Fig. 1). Eflornithine is a weak inhibitor
2
0
of human arginase (Ki = 3.9 mM) and can be a good start to design selective parasite
arginase inhibition.

Figure 1. Design of the α,α-difluorohydrazides (4-9) and α,α-difluoramides (10-12)
evaluated.
2
2
. Results
.1 Chemistry
The α,α-difluorohydrazides (4-9) and difluoramides (10-12) were prepared by
acetylation of isatin or 5-methyl isatin with acetic anhydride under reflux and subsequent
chemoselective deoxofluorination with (Diethylamino)sulfur trifluoride (DAST) in
dichloromethane. Finally, the oxindole ring was opened by a nucleophilic attack of
different hydrazides or amines to furnish the final products 4-12 (Scheme 1). Compounds
8
and 9 are first described in this study, and 4-7 and 10-12 were previously reported.21,22

Scheme 1. Synthesis of compounds 4-12.
2
.2 Kinetics of arginase inhibition and antileishmanial activity
A trial was run with compounds 4-12 at 100 µM against recombinant L.
1
1
amazonensis arginase (LaArg) overexpressed in Escherichia coli. The compounds 4, 5,
8
and 9 showed parasite arginase inhibitory activity (> 70%), compound 6 inhibits only
%, whereas compounds 10-12 did not inhibit the arginase.
8
The IC to the active compounds showed that 4 (IC = 12 ± 2 µM) and 5 (IC =
5
0
50
50
1
2 ± 3 µM), had better IC values than those obtained with 8 (IC = 38 ± 2 µM) and 9
50 50
(
IC = 37 ± 6 µM). Kinetic analysis showed that compound 4 inhibited arginase via
50
competition with the substrate while 5, 8 and 9 showed a mixed mechanism (Fig. 2). The
K values of 4 and 5 were, respectively, 5.1 ± 1.4 µM and 1.3 ± 0.8 µM. The promastigote
i
growth inhibition by the arginase inhibitors compounds was determined and only
compound 4 showed a dose that kills 50% of the parasite under 100 µM (EC = 12.7 ±
5
0
0
.3 µM) Table 1 summarizes the IC , Ki and EC values obtained with the arginase
50 50
inhibitors.

Antileishmanial activity was determined for the best arginase inhibitor to verify
the activity against whole parasite. In a trial performed at 100 μM only compound 4
showed a good inhibition growth. Then, the effective concentration that reduces half of
the parasite growth (EC ) was determined for the compound 4 that showed an EC =
5
0
50
1
2.7 ± 0.3 μM against L. amazonensis promastigotes (Table 1).
Figure 2. Dixon (A) and Cornish-Bowden plot (B) for arginase inhibitors. The
concentrations of L-arginine used were 100 mM (●), 50 mM (■) and 25 mM (▲). Each
point drawn represents the mean of three independent experiments (n = 3) performed in
duplicate. The difference of slopes between of Cornish-Bowden plot (B) for 4 did not
differ (p=0.33), while slopes for 5 are different (p<0.05).

Table 1. Characterization of arginase inhibition: concentration that inhibit half of enzyme
activity (IC ), Ki (dissociation constant for enzyme-inhibitor complex), Kis (dissociation
5
0
constant for enzyme-substrate-inhibitor complex) and concentration that inhibit half of
promastigote growth (EC ) were determined.
5
0
F F
R1
R2
O
NH
O
IC50
Ki
(µM)
Kis
(µM)
Inhibition Promastigote
Compounds
R1
H-
R2
(
µM)
12 ± 2
12 ± 3
mode
Competitive
Mixed
EC (µM)
50
1
2.7 ± 0.3
4
5
6
7
5.1 ± 1.4
-
>
>
>
>
>
100
100
100
100
H
3
C-
1.3 ± 0.8
26 ± 6
H-
>100
-
-
-
-
-
-
H
3
C-
> 100
8
9
H-
H-
H-
38 ± 2 ***, ### 26 ± 1
37 ± 6 ***, ### 32 ± 3
11 ± 1
6.5 ± 0.2
-
Mixed
Mixed
-
100
-
1
1
0
1
inactive
inactive
-
-
-
-
H-
-
-
-
1
2
H
3
C-
inactive
inactive
-
-
-
-
Amphotericin B
-
-
-
0.33 ± 0.04
Values of IC50, EC50, Ki and Kis were expressed as a Mean ± SEM (standard error of mean).
#
##
*
**p<0.001 vs compound 4 and p<0.001 vs compound 5.
“
-“= not determined

2
.3 Docking of the competitive inhibitor
To obtain more insight into the binding mode of 4, molecular docking studies were
performed for the prediction of possible interactions to understanding the experimental
results. The X-ray crystal structure of Leishmania mexicana arginase (LmArg)
1
6
determined by D’Antonio et al. was obtained from the Protein Data Bank (PDB Code:
4
IU1) and was used for the molecular docking study. Initially, the redocking was
ꞷ
performed with LmArg with the hydroxylated substrate analog nor-N -hydroxy-L-
arginine (nor-NOHA), which is the most effective physiological inhibitor of arginase and
exhibited an RMSD value of 1.3 Å.
The L-arginine substrate (Fig. 3-A, 3-B) and nor-NOHA inhibitor (Fig. 3-C, 3-D)
interact with the active site of LmArg through a hydrogen bond and van der Waals
interactions. Redocking the LmArg-nor-NOHA complex showed that the hydroxyl group
2
+
of nor-NOHA coordinates to the Mn ion; the inhibitor interacts with Asp194, Ser150,
Thr257 and Asn143 residues (Fig. 3-C, 3-D); and the L-arginine presented an unfavorable
interaction with manganese (Fig. 3-B).
The docking analysis indicated that compound 4 interacted with the residues
His139, Ala192, and Asp194: the His139 makes a π-π stacking interaction with
phenylhydrazine moiety; Ala192 showed
a
hydrophobic interaction with
phenylacetamide while Asp194 makes H-bonds with the two nitrogen atoms in the
phenylhydrazine moiety, with the nitrogen of acetamide group, and it also interacts with
fluorine (Fig. 3-E, 3-F). The ChemScore values calculated for L-arginine, nor-NOHA,
and 4 in the complex in the active site of LmArg were obtained, with -28.21 kJ/mol, -
3
6.43 kJ/mol, and -19.49 kJ/mol, respectively.

Figure 3: The binding modes and molecular interactions of the (A) L-arginine (carbon in
cyan), (C) substrate analog nor-NOHA (carbon in gray) and (E) compound 4 (carbon in
magenta) in the active site of Leishmania mexicana arginase (PDB Code 4IU1). The
+
2
binuclear manganese cluster (Mn ) is represented by a pair of crosses in the monomer.
The important residues in the active site of LmArg and ligands: (B) L-arginine,
conventional hydrogen bond (green); unfavorable bump (red), van der Walls (green-
cyan), (D) LmArg-nor-NOHA, van der Walls (green-cyan), conventional hydrogen bond
(green), carbon-hydrogen bond (aquamarine), covalent bond (violet-purple) and (F)
compound 4, van der Walls (green-cyan), conventional hydrogen bond (green),
halogen/fluorine (cyan), pi-pi stacked (magenta), pi-alkyl (pink). Hydrogen bonds are
shown in yellow (A, C and E). The images were generated using PyMOL version 1.7.2.1
and BIOVIA Discovery Studio v.16.1.0.15350.

The docking study was also performed to obtain information about the interaction
forms of compound 4 in human arginase-1 (HsArg) (Fig. 4). The results of the
interactions in both enzymes were similar, however, with the human enzyme compound
4
suggests unfavorable interaction. This result can be indicating that compound 4 does
not interact with human arginase 1. The sequence was obtained from the Protein Data
Bank (PDB Code: 4HWW) and was used for the molecular docking study. The redocking
was performed with amino-6-borono-2-(2-(piperidin-1-yl)ethyl) hexanoic acid and
exhibited an RMSD value of 1.8 Å. The X-ray crystal structure of Homo sapiens arginase
(
HsArg) determined by Van Zandt. 23
Figure 4: Superposition and molecular interactions of compound 4 in Leishmania
mexicana and Human Arginase. (A) Superposition of compound 4 (carbono in grey) in
the active site of Leishmania mexicana Arginase (PDB Code 4IU1) and compound 4
(
(
carbono in yellow) in the active site of Homo sapiens Arginase-1 (PDB Code 4HWW).
B) Compound 4 in the active site of Leishmania mexicana, van der Walls (dotted lines
in light green), conventional hydrogen bond (dotted lines in green), halogen/fluorine
dotted lines in cyan), pi-pi stacked (dotted lines in magenta), pi-alkyl (dotted lines in
(
pink). (C) Compound 4 in the active site of Homo sapiens: van der Walls (dotted lines
in light green), conventional hydrogen bond (dotted lines in green), halogen/fluorine
(dotted lines in cyan), pi-pi stacked (dotted lines in magenta), pi-donor hydrogen bond
(dotted lines in light blue), pi-anion (dotted lines in orange) and unfavorable bump
(dotted lines in red).The images were generated using PyMOL version 2.4.0a0 (The
PyMOL Molecular Graphics System, Schrödinger, LLC) and BIOVIA Discovery

Studio v.16.1.0.15350 (Dassault Systèmes BIOVIA, Discovery Studio Modeling
Environment, Release 2017, San Diego: Dassault Systèmes, 2016).
3
. Discussion
Leishmania amazonensis arginase inhibitors were described in natural products as
polyphenols, which lead to the identification of the catechol as the pharmacophoric
group.11,12,24,25 Subsequently, we have synthesized a compound containing a
thiosemicarbazide as the pharmacophoric group needed to inhibit LaArg.15,26 In this
study, we have tested a series of α,α-difluorohydrazide derivatives and identified the
phenylhydrazide moiety as essential to target LaArg in the low micromolar range.
Phenylhydrazide is essential for inhibition of LaArg arginase as shown for compounds 4,
5
, and 8. The compounds 4 and 5 showed the lower IC (12 µM) that did not differ
50
significantly (p>0.05) (Table 1). On the other hand, when the para-phenyl was replaced
by –CF (8) or the hydrazide group was changed to amino (9) the arginase activity
3
decreased (IC ~38 µM). When phenylhydrazine was replaced with 2-hydrazinopyridine
5
0
in compounds 6 and 7, the arginase inhibition deeply decreased (IC > 100 µM). The
5
0
substitution of phenylhydrazine to aminophenyl (10 and 11) or 2-aminopropane (12) led
to a complete loss of arginase inhibition.
Despite the structures of compounds 4, 5, 8 and 9 being similar, the mechanisms of
inhibition were different and may indicate a cleft in the active site that could
accommodate compounds 5, 8, and 9 together with L-arginine. In particular, 8 and 9
showed lower Kis and could bind together with the substrate L-arginine in the active site.
Kis lower than Ki (Table 2), indicate that the affinity to the complex enzyme-substrate
(ES) is higher than the free enzyme (E). On the other hand, compound 5 (Ki < Kis), could
bind and prevent substrate interaction and it has Kis higher than Ki. This information can
be interesting because Leishmania can upregulate the transport of L-arginine27 and
increase levels of the arginase substrate and enhance the enzyme inhibition for compound

8
and 9 in a higher level of L-arginine. This result also may indicate that the substrate L-
arginine changes the structural conformation of La-Arg, decreasing the Kis of compound
with complex ES by approximately five times (K = 32 µM and K = 6.5 µM). In spite
9
i
is
of the compounds 8 and 9 not having inhibited parasite growth, this result can be used for
understanding of the structural changes that can occur with the conformation of the target
enzyme in the presence of the inhibitor and substrate. This could be used to design mixed
inhibitors with improved activity against the parasite based on the phenylhydrazide
moiety.
Previously, we have reported that isomers of natural compounds change the
mechanism of LaArg inhibition when using chlorogenic and cryptochlorogenic acids that
are respectively characterized as competitive and noncompetitive inhibitors. The same
results were observed with the antileishmanials verbascoside and isoverbascoside, which
are competitive and noncompetitive La-Arg inhibitors, respectively.14,25
1
6
D’Antonio et al. showed a simulated annealing omit map in which the hydroxyl
group of nor-NOHA displaces the metal-bridging hydroxide ion observed in the
2
+
+2
2+
unliganded enzyme and coordinates with Mn . The metal-bridging Mn -Mn activated
a water molecule to generate a hydroxy ion that attaches to the guanidine moiety of
substrate L-arginine and when it was used nor-NOHA, a hydroxylated L-arginine,
arginase was inhibited.16 The binuclear manganese cluster is required for stabilization
2
+
and orientation of the catalytic nucleophile and the dialysis of Mn from native arginase
I or the substitution of ligands to either manganese in arginase I variants results in up to
2
0,000-fold reduction in Kcat due to the disruption of the metal cluster.28–30 The binuclear
manganese cluster evolved with the optimal structure and chemical properties to orient
and activate the metal-bridging hydroxide ion for nucleophilic attack at substrate L-
arginine by this enzyme.30

The docking analysis to L. amazonensis arginase showed that compound 4 interacts
with amino acids His139, Ala192 and Asp194. Recently, we have shown that Asp194 and
2
5
Ser150 were possibly involved in LaArg inhibition by caffeic and rosmarinic acids and
1
4
with the interaction of verbascoside with His139, Asp194 and Ser 150. The amino acids
His139 and Asp194 are conserved in arginase family enzymes that are involved in the
2
+
2+
31,32
coordination of the binuclear manganese cluster (Mn -Mn ) in the active site.
In
fact, the amino acids Ser150 and Asp194 were also shown in the crystal structure of
LmArg16 with synthetic competitive inhibitors10 ABH (2(S)-amino-6-boronohexanoic
3
3
34
ω
acid) , BEC (S-(2-boronoethyl)-L-cysteine) and norNOHA (N -Hydroxy-nor-l-
arginine).35 However, Ala192, which showed an interaction with phenylacetamide in
compound 4, is not conserved in human arginase: in the equivalent position, human
arginase has an aspartic acid. It was also observed that compound 4 showed unfavorable
interaction with manganese in the active site of the human arginase. Additionally, the 4
was the only compound that inhibited the growth of promastigote forms. These results
can be used as support to design selective drugs to the treatment of leishmaniosis based
in the phenylhydrazide group.
4
. Conclusion
Four phenylhydrazides (4, 5, 8 and 9) inhibited arginase in the low micromolar range,
while 2-hydrazinopyridine (6 and 7) showed weak activity against parasite enzyme.
Compounds 8 and 9 showed IC higher than 4 and 5 because of the presence of a
5
0
trifluoromethyl substituent in the phenylhydrazine moiety (8) or absence of hydrazine (9).
Docking analysis of compound 4 showed interactions with important amino acids
targeted by other arginase inhibitors. Compound 4 showed activity against L.
amazonensis promastigotes with similar potency that inhibited arginase of the parasite.
These results indicate that α,α-difluorophenylhydrazide moiety is a good starting point

for the design of novel Leishmania arginase inhibitors with the aim of the development
of potential antileishmanial compounds.
5
. Experimental section
5
.1 Chemistry
5
.1.1. N-acetylation of isatins
Compounds 2a and 2b were prepared as previously reported.21
5
.1.2. N-acetylindoline-2,3-diones deoxofluorination
Compounds 3a and 3b were prepared as previously reported.21
5
.1.3. Synthesis of α.α-difluorohydrazides 4-7
Synthesis of α.α-difluorohydrazides 4-7 was performed as previously reported.21
5
.1.4. Synthesis of α.α-difluorohydrazides 8
General procedure. Into a round-bottom flask coupled to a condenser was added 0.6
mmol of the corresponding N-acetyl-3,3-difluoroindolin-2-one (3a or 3b) and 3 mL of
acetonitrile. The mixture was stirred at room temperature for 5 min. Then, 1 equivalent
of neat phenylhydrazine was added to the medium. The flask was then attached to a
condenser and the system kept under stirring at reflux for 12 h. At the end of this period,
the formed precipitate was vacuum filtered and washed with cold acetonitrile.
5
.1.4.1.
N-(2-(2-(2-(4-(trifluoromethyl)phenyl)hydrazinyl)-1,1-difluoro-2-
oxoethyl)phenyl) acetamide (8)
1
H NMR (DMSO, 400 MHz, δ ppm): 2.04 (s, CH CO), 6.77 (d, J = 8.4, 1HAr),
3
7
1
.37 (t, J = 7.6, 1HAr), 7.47 (d, J = 8.5, 2HAr), 7.58 (t, J = 7.5, 1HAr), 7.64 (d, J = 7.7,
1
9
HAr), 7.73 (d, J = 7.8, 1HAr), 8.61 (s, NH), 9.29 (s, NH), 11.09 (s, NH); F NMR
(DMSO, 377 MHz, δ ppm): – 59.24, – 100.32; ESI-MS(–): m/z 386;
CHN: Calc. for C H F N O : C, 52.72; H, 3.64; N, 10.85; found: C, 52.52; H,
1
7
14
5
3
2
3
5
.63; N, 10.48
.1.5. Synthesis of 2-(2-acetamidophenyl)-N-benzyl-α,α-difluoroacetamide 9

To a bound-bottom flask was added the amount of the corresponding N-acetyl-3,3-
difluoroindolin-2-one and 140 equivalents of acetonitrile. The mixture was allowed to stir
at room temperature for 5 minutes. Then, 1 equivalent of benzylamine was added to the
medium. The system was kept under stirring overnight. At the end of this period, the
formed precipitate was vacuum filtered and washed with cold acetonitrile. The product
was air dried and stored under an argon atmosphere with protection from light.
1
H NMR (DMSO, 400 MHz, δ ppm): 2.01 (s, CH CO), 4.36 (d, J = 9.9, CH ), 7.22-7.33
3
2
1
9
(
m, 6HAr), 7.52-7.59 (m, 2HAr), 7.76 (d, J = 7.6, 1HAr), 9.48 (s, NH), 9.63 (s, NH); F
NMR (DMSO, 377 MHz, δ ppm): – 100.51; ESI-MS(+): m/z 319.1 (48%), 341.0
(
100%); CHN: Calc. for C H F N O : C, 64.14; H, 5.07; N, 8.80; found: C, 63.80; H,
17 16 2 2 2
5
.16; N, 8.70
5
.1.6. Synthesis of α.α-difluoramides 10-12
Synthesis of α.α-difluoramides 10-12 was performed as previously reported.22
5
.2 Arginase inhibition and kinetics
Recombinant arginase inhibition and kinetics study were performed as previously
1
1
11
reported. Briefly, arginase was overexpressed in E. coli, and purified. The IC were
5
0
determined in a reaction buffer containing 50 mM CHES and 50 mM of L-arginine pH
,5 with variable concentration of compounds. The activity of arginase was quantified
through measure of urea production with colorimetric method of Berthelot.36
9
5
.3 Promastigotes culture test
The MHOM/BR/1973/M2269 strain of L. amazonensis was used throughout this
study. Promastigotes were grown in M199 medium supplemented with 10% fetal bovine
serum, 5 ppm of hemin, 50 μg/mL streptomycin and 100Upenicillin. Cells were grown
5
until they reached stationary phase. Initially, 5.0 x10 cells were incubated for 72 h with
the inhibitors of the parasite arginase that were added to the culture medium in a
concentration that ranged from 100 μM to 0.78 μM. Amphotericin B was used as a control
for cell growth inhibition in a concentration that ranged from 10 μM to 0.00001. All tests

were performed in 1.5 mL microtubes to a final volume of 1.4 mL. After 72 h of
incubation with arginase inhibitors compounds, surviving cells were washed two times
with 1 mL of Hanks Balanced Salt Solution (GIBCO® HBSS). Soon after that, the cells
were incubated for 13 h with the tetrazolium dye MTT 3- (4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (Sigma) that was added to the culture medium to a final
concentration of 0,5 mg/mL. MTT was transformed by the surviving cells producing
formazan crystals that were dissolved with 500 μL of DMSO. The concentration of the
formazan was determined colorimetrically using the Epoch 2 Microplate
Spectrophotometer (Biotech, Instruments, Winooski, Vermont USA) at 570 nm. EC50
values were calculated based on a sigmoidal model (Log EC ) using a normalized
5
0
variable slope in the GraphPad Prism software (version 8 for Windows, San Diego,
California, USA). One independent assay was performed six times.
5
.5 Molecular docking and bioavailability properties
The structures of the compounds studied were designed, minimized, conformationally
analyzed, and optimized, and properties were calculated using the Spartan’10 version
1
.0.1. program (Wavefunction, Inc). The calculations were performed for all structures
using Conformer Distribution with Molecular Mechanics and MMFF, and 50 conformers
were examined for Equilibrium Geometry with Semi-Empirical and RM1, for Energy
with Hartree-Fock 6-31G* in the vacuum. The crystal structure of Leishmania mexicana
ꞷ
arginase (LmArg) in complex with the hydroxylated substrate analog nor-N -hydroxy-L-
arginine (nor-NOHA), which are the most potent arginase inhibitors, was crystallized and
solved by X-ray diffraction.16 The 3D structure of LmArg in complex with inhibitor
cocrystallized was initially obtained from the Protein Data Bank with PDB Code 4IU1
1
6
(
resolution of 1.95 Å, method X-ray diffraction). We used the crystal structure of LmArg
instead of a homology model of arginase from L. amazonensis since only two residues,
not involved in the binding site, were different resulting in a 99.4% amino acid sequence
identity. The pdb files were prepared, and the water molecules were removed.
Subsequently, the molecular docking studies were done using the GOLD software v.
3
7
2+
5
.5. The Mn metal ions are cofactor and have been maintained in coordination with

the metalloenzyme arginase. The parameters for docking were determined by analysis of
poses obtained by redocking of the nor-NOHA in LmArg. The best redocking solutions
will be those with the lowest root-mean-square deviation (RMSD) value. Therefore, the
docking was performed with Chemscore function with a 20 Å of distance from oxygen
(ID 1051) of the catalytic residue Ser150. Automated docking studies were then
performed using the genetic algorithm GOLD (Genetic Optimization for Ligand
Docking), and twenty GA runs were performed for each molecule. The GOLD score was
chosen as fitness function and the standard default settings were used in all calculations.
The visual inspection of intermolecular interactions and figures were performed using
with PyMOL version 1.7.2.1 (The PyMOL Molecular Graphics System, Schrödinger,
LLC) and BIOVIA Discovery Studio v.16.1.0.15350 (Dassault Systèmes BIOVIA,
Discovery Studio Modeling Environment, Release 2017, San Diego: Dassault Systèmes,
2
016). Default cutoff values of 2.5 Å for hydrogen bonds and 4 Å for Van der Waals
interactions were employed.
5
.6 Statistical analyses
The data for IC50 of the parasite arginase inhibition was evaluated using a one-way
analysis of variance (ANOVA) followed by a Tukey post-test. All data was analyzed
using GraphPad Prism software (version 8 for Windows, San Diego, California, USA).
The differences between the means were considered significant at p<0.05.
Acknowledgment
This research was supported by grant #17/06917-4, São Paulo Research
Foundation (FAPESP), by Carlos Chagas Filho Foundation for Support Research of the
State of Rio de Janeiro (FAPERJ), CNPq (Conselho Nacional de Desenvolvimento
Científico e Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior), and FUNEMAC (Fundação Educacional de Macaé) for fellowships of
Boechat, da Silva, Lima and Farias.
References
1
.
WHO | Leishmaniasis. WHO. 2019. https://www.who.int/leishmaniasis/en/.

Accessed June 27, 2019.
2
3
4
5
.
.
.
.
Colotti G, Ilari A. Polyamine metabolism in Leishmania: from arginine to
trypanothione. Amino Acids. 2011;40(2):269-285. doi:10.1007/s00726-010-0630-
3
Bocedi A, Dawood KF, Fabrini R, et al. Trypanothione efficiently intercepts nitric
oxide as a harmless iron complex in trypanosomatid parasites. FASEB J.
2
010;24(4):1035-1042. doi:10.1096/fj.09-146407
Fairlamb AH, Cerami A. Metabolism and Functions of Trypanothione in the
Kinetoplastida. Annu Rev Microbiol. 1992;46(1):695-729.
doi:10.1146/annurev.mi.46.100192.003403
Balã Na-Fouce R, Calvo-Álvarez E, Álvarez-Velilla R, Prada CF, Pérez-Pertejo Y,
Reguera RM. Role of trypanosomatid’s arginase in polyamine biosynthesis and
pathogenesis.
Mol
Biochem
Parasitol.
2011;181:85-93.
doi:10.1016/j.molbiopara.2011.10.007
6
7
8
9
.
.
.
.
Cruz EM, Da Silva ER, Maquiaveli CC, et al. Leishmanicidal activity of Cecropia
pachystachya flavonoids: Arginase inhibition and altered mitochondrial DNA
arrangement.
Phytochemistry.
2013;89:71-77.
doi:10.1016/j.phytochem.2013.01.014
Vannier-Santos MA, Menezes D, Oliveira MF, de Mello FG. The putrescine
analogue 1,4-diamino-2-butanone affects polyamine synthesis, transport,
ultrastructure and intracellular survival in Leishmania amazonensis. Microbiology.
2
008. doi:10.1099/mic.0.2007/013896-0
Roberts SC, Tancer MJ, Polinsky MR, Gibson KM, Heby O, Ullman B. Arginase
Plays a Pivotal Role in Polyamine Precursor Metabolism in Leishmania
CHARACTERIZATION OF GENE DELETION MUTANTS*. 2004.
doi:10.1074/jbc.M402042200
da Silva MFL, Zampieri RA, Muxel SM, Beverley SM, Floeter-Winter LM.
Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is
Important for Parasite Infectivity. Costa FTM, ed. PLoS One. 2012;7(3):e34022.
doi:10.1371/journal.pone.0034022
1
1
0. Riley E, Roberts SC, Ullman B. Inhibition profile of Leishmania mexicana
arginase reveals differences with human arginase I. Int J Parasitol.
2
011;41(5):545-552. doi:10.1016/j.ijpara.2010.12.006
1. Da Silva ER, Maquiaveli CDC, Magalhães PP. The leishmanicidal flavonols

quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase.
Exp Parasitol. 2012. doi:10.1016/j.exppara.2012.01.015
1
1
2. Manjolin LC, Dos Reis MBG, Do Carmo Maquiaveli C, Santos-Filho OA, Da
Silva ER. Dietary flavonoids fisetin, luteolin and their derived compounds inhibit
arginase, a central enzyme in Leishmania (Leishmania) amazonensis infection.
Food Chem. 2013;141(3):2253-2262. doi:10.1016/j.foodchem.2013.05.025
3. Dos Reis MBG, Manjolin LC, Maquiaveli CC, Santos-Filho OA, Da Silva ER.
Inhibition of Leishmania (Leishmania) amazonensis and rat arginases by green tea
EGCG, (+)-catechin and (-)-epicatechin: A comparative structural analysis of
enzyme-inhibitor
interactions.
PLoS
One.
2013;8(11).
doi:10.1371/journal.pone.0078387
1
1
1
4. Maquiaveli CC, Lucon-Júnior JF, Brogi S, et al. Verbascoside Inhibits
Promastigote Growth and Arginase Activity of Leishmania amazonensis. J Nat
Prod. 2016;79(5):1459-1463. doi:10.1021/acs.jnatprod.5b00875
5. Da Silva ER, Boechat N, Pinheiro LCS, et al. Novel Selective Inhibitor of
Leishmania (Leishmania) amazonensis Arginase. Chem Biol Drug Des.
2
015;86(5):969-978. doi:10.1111/cbdd.12566
6. D’Antonio EL, Ullman B, Roberts SC, et al. Crystal structure of arginase from
Leishmania mexicana and implications for the inhibition of polyamine
biosynthesis in parasitic infections. Arch Biochem Biophys. 2013;535(2):163-176.
doi:10.1016/J.ABB.2013.03.015
1
1
7. Bacchi C, Nathan H, Hutner S, McCann P, Sjoerdsma A. Polyamine metabolism:
a
potential therapeutic target in trypanosomes. Science (80- ).
1
980;210(4467):332-334. doi:10.1126/SCIENCE.6775372
8. Grishin N V, Osterman AL, Brooks HB, Phillips MA, Goldsmith EJ. X-ray
Structure of Ornithine Decarboxylase from Trypanosoma brucei:ꢀ The Native
Structure and the Structure in Complex with α-Difluoromethylornithine,.
Biochemistry. 1999;38(46):15174-15184. doi:10.1021/bi9915115
9. Cama E, Pethe S, Boucher J-L, et al. Inhibitor Coordination Interactions in the
Binuclear Manganese Cluster of Arginase. Biochemistry. 2004;43:8987-8999.
doi:10.1021/bi0491705
1
2
0. Selamnia M, Mayeur C, Robert V, Blachier F. α-Difluoromethylornithine (DFMO)
as a potent arginase activity inhibitor in human colon carcinoma cells. Biochem
Pharmacol. 1998;55(8):1241-1245.

2
2
2
2
1. Silva Castelo-Branco F, Crizanto De Lima E, Luiz De Oliveira Domingos J, et al.
New hydrazides derivatives of isoniazid against Mycobacterium tuberculosis:
Higher potency and lower hepatocytotoxicity. Eur J Med Chem. 2018;146:529-
5
40. doi:10.1016/j.ejmech.2018.01.071
2. Boechat N, Kover WB, Bastos MM, et al. N-Acyl-3,3-difluoro-2-oxoindoles as
versatile intermediates for the preparation of different 2,2-difluorophenylacetic
derivatives. J Braz Chem Soc. 2008;19(3):445-457. doi:10.1590/S0103-
5
0532008000300011
3. Van Zandt MC, Whitehouse DL, Golebiowski A, et al. Discovery of (R)-2-Amino-
-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic Acid and Congeners As Highly
6
Potent Inhibitors of Human Arginases I and II for Treatment of Myocardial
Reperfusion Injury. J Med Chem. 2013;56(6):2568-2580. doi:10.1021/jm400014c
4. Dos Reis MBG, Manjolin LC, Maquiaveli CDC, Santos-Filho OA, Da Silva ER.
Inhibition of Leishmania (Leishmania) amazonensis and rat arginases by green tea
EGCG, (+)-catechin and (-)-epicatechin: A comparative structural analysis of
enzyme-inhibitor
interactions.
PLoS
One.
2013;8(11).
doi:10.1371/journal.pone.0078387
2
2
5. da Silva ER, Brogi S, Grillo A, et al. Cinnamic acids derived compounds with
antileishmanial activity target Leishmania amazonensis arginase. Chem Biol Drug
Des. 2019;93(2):139-146. doi:10.1111/cbdd.13391
6. Di Costanzo L, Pique ME, Christianson DW. Crystal Structure of Human Arginase
I Complexed with Thiosemicarbazide Reveals an Unusual Thiocarbonyl μ-Sulfide
Ligand in the Binuclear Manganese Cluster. J Am Chem Soc. 2007;129(20):6388-
6
389. doi:10.1021/ja071567j
2
7. Castilho-Martins EA, Laranjeira da Silva MF, dos Santos MG, Muxel SM, Floeter-
Winter LM. Axenic Leishmania amazonensis Promastigotes Sense both the
External and Internal Arginine Pool Distinctly Regulating the Two Transporter-
Coding Genes. El-Sayed NM, ed. PLoS One. 2011;6(11):e27818.
doi:10.1371/journal.pone.0027818
2
2
8. Christianson DW. Arginase:ꢀ Structure, Mechanism, and Physiological Role in
Male and Female Sexual Arousal. Acc Chem Res. 2005;38(3):191-201.
doi:10.1021/ar040183k
9. Scolnick LR, Kanyo ZF, Cavalli RC, Ash DE, Christianson DW. Altering the
Binuclear Manganese Cluster of Arginase Diminishes Thermostability and

Catalytic Function †.; 1997. https://pubs.acs.org/sharingguidelines. Accessed
January 30, 2019.
3
3
0. Cama E, Emig FA, Ash DE, Christianson DW. Structural and Functional
Importance of First-Shell Metal Ligands in the Binuclear Manganese Cluster of
Arginase I,. Biochemistry. 2003;42(25):7748-7758. doi:10.1021/bi030074y
1. Reczkowski RS, Ash DE. EPR evidence for binuclear manganese(II) centers in rat
liver
arginase.
J
Am
Chem
Soc.
1992;114(27):10992-10994.
doi:10.1021/ja00053a064
3
3
2. Dismukes GC. Manganese Enzymes with Binuclear Active Sites.
https://pubs.acs.org/sharingguidelines. Accessed January 30, 2019.
3. Baggio R, Elbaum D, Kanyo ZF, et al. Inhibition of Mn2+2-Arginase by Borate
Leads to the Design of a Transition State Analogue Inhibitor, 2(S)-Amino-6-
boronohexanoic Acid.
doi:10.1021/ja971312d
J
Am Chem Soc. 1997;119(34):8107-8108.
3
3
4. Kim NN, Cox JD, Baggio RF, et al. Probing Erectile Function:ꢀ S-(2-Boronoethyl)-
l-Cysteine Binds to Arginase as a Transition State Analogue and Enhances Smooth
Muscle Relaxation in Human Penile Corpus Cavernosum,. Biochemistry.
2
001;40(9):2678-2688. doi:10.1021/bi002317h
5. Custot J, Moali C, Brollo M, et al. The New α-Amino Acid Nω-Hydroxy-nor-l-
arginine:ꢀ a High-Affinity Inhibitor of Arginase Well Adapted To Bind to Its
Manganese Cluster.
doi:10.1021/ja970285o
J
Am Chem Soc. 1997;119(17):4086-4087.
3
3
6. FAWCETT JK, SCOTT JE. a Rapid and Precise Method for the Determination of
Urea. J Clin Pathol. 1960;13(2):156-159. doi:10.1136/jcp.13.2.156
7. Jones G, Willett P, Glen RC, Leach AR, Taylor R. Development and validation of
a genetic algorithm for flexible docking. J Mol Biol. 1997;267(3):727-748.
doi:10.1006/JMBI.1996.0897