Optimization of phthalazin-based aryl/heteroarylhydrazones to design new promising antileishmanicidal agents: Synthesis and biological evaluation of 3-aryl-6-piperazin-1,2,4-triazolo[3,4-a] phthalazines

Article abstract of DOI:10.1039/d0nj01206h

1-Monosubstituted and 1,4-substituted phthalazins based on aryl/heteroarylhydrazinyl have demonstrated attractive antileishmanial profiles against amastigote forms of the Leishmania braziliensis parasite. Further optimization of the mentioned acyclic scaffold motivated us to design a series of 3-aryl-1,2,4-triazolo[3,4-a]phthalazines, cyclic versions of the phthalazins based on aryl/heteroarylhydrazinyl, which have not been evaluated against Leishmania parasites yet. In order to compare to phthalazine-based aryl/heteroarylhydrazones, five essential 3-aryl-6-piperazin-1,2,4-triazolo[3,4-a]phthalazines were efficiently prepared in excellent yields (73-83%) through a facile one-pot procedure from 4-chloro-1-phthalazinyl-arylhydrazones via C-H dehydrogenative cyclization using silver(i) salt. From in vitro antileismanial evaluation, compound 2d, a nitro derivative, was identified as the most promising agent with a good anti-amastigote response (IC50 = 9.37 μM) and low relative toxicity against peritoneal macrophages (LD50 = 123.93 μM). A moderate response was found against clinical amastigote isolates of L. braziliensis, although superior compared to the reference glucantime. A comparison with their phthalazin analogues based on aryl/heteroarylhydrazinyl gave evidence that the efficacy of each chemical system is determined by the nature of the functionalization next to the aryl moiety, which suggests that different mechanisms of action are involved for each chemical system. The cyclized form led to an enhancement of the antileismanial activity compared to the acyclic form, but the nitroderivatives seemed to be highly more toxic than the parent non-cyclized compounds. From the three compared phthalazine groups, 4-chloro-1-phthalazine-(5-nitrofuryl)hydrazinil with a nanomolar antileishmanial response was identified as a promising lead for further optimization, whereas compound 2d emerges as a prominent hit platform to prepare a group of derivatives based on phthalazine-1,2,4-triazolo bearing 3-nitro-phenyl at the 3-position. This journal is

Full text of DOI:10.1039/d0nj01206h

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Optimization of phthalazin-based aryl/heteroarylhydrazones to design new
promising antileishmanicidal agents: Synthesis and biological evaluation of 3-
aryl-6-piperazin-1,2,4-triazolo[3,4-a]phthalazines
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Angel H. Romero,a,b* Noris Rodríguez,b Oscar G. Ramíreza
aCátedra de Química General, Facultad de Farmacia, Universidad Central de Venezuela, Los Chaguaramos,
Caracas 1041-A, Venezuela.
bLaboratorio de Ingeniería Genética, Instituto de Biomedicina, Facultad de Medicina, Universidad Central de
Venezuela, San Luis, Caracas, Venezuela.
*Corresponding Author E-mail: angel.ucv.usb@gmail.com (A.H. Romero)
Abstract: 1-Monosubstituted and 1,4-substituted phthalazin-based on aryl/heteroarylhydrazinyl have
demonstrated attractive antileishmanial profiles against amastigote forms of Leishmania braziliensis
parasite. More optimization on mentioned acyclic scaffold motivated us to design a series of 3-aryl-1,2,4-
triazolo[3,4-a]phthalazine, a cyclic version of the phthalazin-based on aryl/heteroarylhydrazinyl which
has not been evaluated against Leishmania parasites yet. In order to compare to phthalazine-based
aryl/heteroarylhydrazones, five essential 3-aryl-6-piperazin-1,2,4-triazolo[3,4-a]phthalazine were
efficiently prepared in excellent yields (73-83%) through a facile one-pot procedure from 4-chloro-1-
phthalazinyl-arylhydrazones via a C-H dehydrogenative cyclization using silver(I) salt. From in vitro
antileismanial evaluation, compound 2d, a nitro derivative, was identified as the most promising agent
with a good anti-amastigote response (IC50 = 9.37 μM) and low relative toxicity against peritoneal
macrophage (LD50=123.93 μM). A moderate response was found against clinical amastigote isolates of L.
braziliensis, although superior compared to reference glucantime. A comparison with their phthalazin-
based on aryl/heteroarylhydrazinyl analogues put in evidence that the efficacy of each chemical system is
determined by the nature of the functionalization in turn to aryl moiety, which suggests that different
mechanism of action are involved for each chemical system. The cyclized form led an enhancement on the
antileismanial activity compared to acyclic form, but the nitroderivatives seemed to be highly more toxic
than parent non-cyclized compounds. From the three compared phthalazine groups, the 4-chloro-1-
phthalazine-(5-nitrofuryl)hydrazinil with nanomolar antileishmanial response was identified as promising
lead for further optimization, whereas the compound 2d emerges as prominent hit platform to prepare a
group of derivatives based on phthalazine-1,2,4-triazolo bearing 3-nitro-phenyl at 3-position.
Keywords: Leishmania braziliensis, phthalazine, 1,2,4-triazolo[3,4-a]phthalazine, promastigote, amastigote,
cutaneous leishmaniasis.

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1. Introduction
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Leishmaniasis is a neglected tropical disease resulting from infection of macrophages by
obligate intracellular parasites of the genus Leishmania spp., which is transmitted to human by
insects [1,2]. There are mainly three clinical manifestations for the leishmaniasis including: (i)
mucocutaneous leishmaniasis (ML), cutaneous leishmaniasis (CL), and visceral leishmaniasis
(VL), which are caused by different Leishmania species depending on geographic region [3].
Leishmaniasis is prevalent in 88 countries with 350 million people at risk of acquiring the
disease. World Health Organization revealed that between 0.6 and 1.0 million cases of CL are
registered annually [4]. Leishmania braziliensis is responsible for nearly 90% of all CL cases
annually in South America, and the absence or incomplete treatment can facilitate the subsequent
development of ML [5,6]. Treatments against cutaneous leishmaniasis is severely limited due to
that only two approved drugs (e.g. pentavalent antimonials such as pentostam and glucantime)
are currently used, have more than 60 years in the market and, they are highly toxic (affecting
heart, liver and kidneys), highly expensive, low effective and unpractical administration for
patient [7]. A second line of current drugs such as pentamidine, amphotericin B and miltefosine
also are employed when the first line failed. Moreover, resistance reports have been found for
pentavalent antimonials in endemic areas [8].
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The discovery of new potential drugs against neglected tropical disease, in this case against
Leishmaniasis, prevail as a challenge for the science due to the complexity of parasite can cause
diverse pathologies depending on the type of Leishmania, and its high adaptability that permits to
develop resistance to drugs in short time [9a]. Diverse criteria have been established in order to
identify potential antileishmanial agents. DNDi (Drug for Neglected Disease initiative) described
four stages for the final discovery of a medication against neglected tropical disease: (i) the
discovery stage that implies initial screening, hit to lead and lead optimization; (ii) the translation
stage that implies pre-clinical, phase I and phase IIa steps, (iii) the development stage that
consists of two steps, phase IIb/III and registration, and (iv) the fourth stage that involves the
implementation for the treatment access to public [9b]. To access to stages II, III and IV, it is
important to overcome the different criteria established for the hit step of stage I [9c], which
included: (i) IC50 below 10 M against form clinically relevant of any parasite (T. cruzi,
Leishmania, T. brucei, etc), (ii) selectivity index superior to 10 related to mammalian cell lines
[9c], and (iii) good drug-likeness parameters. Whereas significant exigencies are included for the

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step of hit to lead: (i) activities in vitro against relevant stage of parasite from 10 to 20-fold more
than hit stage; (ii) selectivity index superior to 50-fold more than hit stage, (iii) significant
reduction of parasitaemia (> 70%) and/or increase of life-span of animal in vivo assay, (iv) good
solubility by about 0.01 mg/mL at pH 7.4; (v) plasma concentration in the range of active
concentration after administration; (vi) relevant further toxicity assays such as genotoxicity (e.g.
Ames test) and cardiotoxicity (e.g. hEGR assay) are needed. A large number of new heterocycles
based on quinolin [10a-e], quinazoline [10f-i] and phthalazine [11] have been identified as
promising hit as leishmanicidal agent. Recently, our group identified to the 4-chloro-phthalazine
1 functionalized with highly conjugated nitroheteroarylhydrazones at 1-position of phthalazine
core as a potential lead for further improvements (Figure 1). Further optimization motivated the
design, synthesis and evaluation of disubstituted phthalazine-arylhydrazinyl 2, identifying
compounds with good anti-amastigote response against L. braziliensis parasite [11a-d]. A
isosteric form of these compounds of phthalzin-arylhydrazinyl is the 1,2,4-triazolo[3,4-
a]phthalazine, a cyclic version, which has not been proved against trypanosomal parasites yet.
The 1,2,4-triazolo[3,4-a]phthalazine system is a known multi-target agents that possess a wide
spectrum of pharmacological activities, including anti-inflammatory agents [12], anticancer drugs
[13], bromo-domain inhibitors[14], GABA receptors [15], anticonvulsants [16] and
antimicrobials [17]. Specifically, 3-substituted-6-alkylamino-1,2,4-triazolo[3,4-a]phthalazine 1
system (Figure 1) is present in the structure of some of these bioactive triazolo agents. From our
previous results, it was suggested that the high conjugation from phthalazine core to nitro-
aryl/heteroaryl through hydrazine linker is crucially important in the antileishmanial response of
the monosubstituted phthalazine 4-chlorophthalazin-arylhydrazones, while the disubstitution in
the 1,4-disusbtituted-phthalazine are essential to enhance the biological activity [11a-d].
Therefore, it results very interesting to study the biological relevance of the hydrazine linker for
the mentioned system compared to triazolo linker of the phthalazine-1,2,4-triazolo system (Figure
1). Then, based on the above mentioned, we evaluated leishmanicidal activity of a series of 1,2,4-
triazolo[3,4-a]phthalazines (as lead core) containing a N-methylpiperazine at 6-position and a
substituted aryl/heteroaryl R1 group at 3-position of the 1,2,4-triazolo core. We focused our
attention in the evaluation of phthalazine-1,2,4-triazolo bearing relevant non-toxic and active
moieties detected from active previous phthalazine-arylhydrazones mono-substituted and di-
substituted including: 4-nitrophenyl, furyl, phenyl, 4-fluorophenyl and 3-nitrophenyl (Figure 1).
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F
NO2
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N
O
N
N
Y(NO2)
N
N
N
NH
NH
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NH
N
N
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N
N
Cl
N
F
antileishmania lead 1
This work
antileishmania lead 2
Figure 1. General structure from antileishmania lead (1 and 2) to current design 6-(N-piperazin)-
3-aryl-1,2,4-triazolo[3,4-a]phthalazine lead.
A number of specific derivatives were prepared by a facile and efficient protocol based on the
intramolecular cyclization via C-H activation mediated by coinage transition metal such as
cooper (I) and silver (I). In the last decades, the metal-catalyzed intramolecular cyclization has
been employed as a competitive strategy to obtain a variety of fused-heterocyclic systems [18-
25]. Specifically, strategies based on dehydrogenation of vicinal C-H ylidenic or C-H aldehyde
bonds as well as vicinal Csp3-H bonds in N,N-disubstituted hydrazones has permitted the
synthesis of five-membered heterocycles via the formation of C-N or C-O bonds [25]. Recently,
our group reported an one-pot protocol for the synthesis of 6-(N-piperazin)-3-aryl-1,2,4-
triazolo[3,4-a]phthalazine via a copper-catalyzed dehydrogenation of ylidenic hydrogen from 4-
chloro-1-phthalazinyl-arylhydrazones [26]. The majority of the mentioned strategies are
catalyzed through redox action of transition metals such as copper(I), copper(II), palladium (II)
and iron (II and III) and, the reactions in general require of temperature above 80 ºC, prolonged
time more than 24 h and the use of a base. In last decades, a large number of silver-catalyzed
2
2-
Csp -H functionalization have been reported using silver(I)/S2O8 system under mild conditions
in absence of base and, the reactions proceeded under a Ag(I)/Ag(II) catalytic cycle [27]. Other
2
way for the silver-mediated Csp -H functionalization is to use silver (I) salts in equimolar ranges
under hard conditions and prolonged time and, the reaction is determined by a non-catalytic
redox Ag(I)/Ag(0) process [27]. This last strategy in general involves re-aromatization process in
heteroaromatic systems. From these evidences, we developed an intramolecular dehydrogenative
cyclization mediated by a silver (I) to obtain a series of 3-aryl-6-(N-methylpiperazin)-1,2,4-
triazole[3,4-a]phthalazines 2 from 4-chloro-1-phthalazinyl-arylhydrazones 1 (Table 1).

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2. Chemistry
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Initially, the reaction conditions were optimized using 4-chloro-1-phthalazinyl-arylhydrazone
1a and N-methylpiperazine as model substrates. The initial reaction conditions were extracted
from silver-protocol reported by H.J. Xu [28] for a standard Ag(I)/Ag(II) catalytic process, a
silver-strategy reported by S. Chang [29] for a Ag(I)/(Ag(0) mediation and our cooper based-
protocol [26] with a few modifications including reaction time of 48 h and temperatures from 40
to 100ºC). Silver(I) nitrate was used as standard silver catalyst or mediator and, the results were
compared with cooper (I) catalysis [26]. Optimization conditions are summarized in Table 1. In
general, a poor reaction yield was found in absence of catalyst (entries 1-2, Table 1). In presence
of copper (I)-L-proline system as catalyst and reaction conditions including 100ºC of temperature
and reaction time of 48 h, the following remarks are extracted: (i) the selection of solvent plays
an important role in the progression of the reaction, finding the best results for DMF or N-
methylpiperazine as solvents, whereas a decrease in reaction yield was found under toluene
environment (entries 3-5, Table 1); and (ii) the oxidizing is mandatory, finding good results
under aerobic conditions (entries 3-5, Table 1) and, contrary poor results under argon
environment (entry 6, Table 1). The oxygen is crucial to keep the catalytic cycle. Regarding to
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2-
silver (I) catalyst, we found that the system Ag+/S2O8 was not compatible for our chemical
2-
system under different oxidizing (e.g. S2O8 , TBHP), solvents (e.g. THF/H2O, acetone/H2O,
CH2Cl2/H2O, DMF/H2O) and temperature (40-80 ºC) conditions (entries 7-12, Table 1).
Probably, the potential radical species formed on hydrazinil moiety in our system during
Ag(I)/Ag(II) process is not sufficient stable compared to phenyl radical formed in reported
protocol, which is a strong limitation in the progress of the reaction. Discarded the applicability
of Ag(I)/Ag(II) process in our system, we decided to study the potential of Ag(I)/Ag(0)
mediation according to reported protocol [29]. As expected, the strategy based on Ag(I)/Ag(0)
process using non-stoichiometric amount of silver (I) salt displayed poor reaction yields (11-
35%) (entries 13-14, Table 1), whereas a significant increase in reaction yields was found for
stoichiometric quantities of silver (I) (entries 15-16, Table 1), being 2.5 equivalent of silver(I)
nitrate the most convenient amount to afford an optimal reaction. The selection of solvent also
was an important point. The best results were obtained under DMF milieu with a reaction yield
of 83% (entry 16, Table 1); whereas modest results were found under THF or toluene milieu
(entries 17-18, Table 1). A decrease of temperature to 80ºC reduced the reaction yield from 83%

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to 44% (compare entries 16 and 18 in Table 1). K3PO4 was selected as the most convenient base
compared to K2CO3 (compare entries 16 and 20, Table 1). Finally, the reaction operates well
under air environment for 48 h.
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Table 1. Reaction optimization conditions
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N
N
N
N
N
NH
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Catalyst
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Base, T(°C)
Solvent
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Cl
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N
Entries
Catalysts
Solventa
Oxidizing
T(ºC)
Base or additive
Y (%)c
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2
3
No Catalyst
No Catalyst
NMPz
DMF
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none
air
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100
100
K3PO4 (3 equiv.)
K3PO4 (3 equiv.)
K3PO4 (3 equiv.)
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10
73
CuI/L-proline (15%/30%)
NMPz
4
CuI/L-proline (15%/30%)
CuI/L-proline (15%/30%)
CuI/L-proline (15%/30%)
AgNO3 (20 %)
DMF
Toluene
air
air
100
100
100
40
K3PO4 (3 equiv.)
K3PO4 (3 equiv.)
K3PO4 (3 equiv.)
KOAc (1.1 eq.)
KOAc (1.1 eq.)
KOAc (1.1 eq.)
KOAc (1.1 eq.)
KOAc (1.1 eq.)
KOAc (1.1 eq.)
K3PO4 (3 equiv.)
K3PO4 (3 equiv.)
K3PO4 (3 equiv.)
K3PO4 (3 equiv.)
K3PO4 (3 equiv.)
K3PO4 (3 equiv.)
K3PO4 (3 equiv.)
K2CO3 (3 equiv.)
70
46
9
5
6
NMPz
noneb
7
THF/ H2O (1:1)
K2S2O8 (3 eq.)
0
8
AgNO3 (20 %)
Acetone/H2O (1:1)
DMF/H2O (1:1)
CH2Cl2/H2O (1:1)
CH2Cl2/H2O (1:1)
THF/H2O (1:1)
DMF
K2S2O8 (3 eq.)
40
0
9
AgNO3 (20 %)
K2S2O8 (3 eq.)
40
0
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20
AgNO3 (20 %)
K2S2O8 (3 eq.)
40
0
AgNO3 (20%)
TBHP (3 eq.)
40
0
AgNO3 (20%)
K2S2O8 (3 eq.)
80
0
AgNO3 (15%)
air
air
air
air
air
air
air
air
100
100
100
100
100
100
80
11
35
52
83
31
38
44
74
AgNO3 (50%)
DMF
AgNO3 (1.0 eq.)
AgNO3 (2.5 eq.)
AgNO3 (2.5 eq.)
AgNO3 (2.5 eq.)
AgNO3 (2.5 eq.)
AgNO3 (2.5 eq.)
DMF
DMF
THF
Toluene
DMF
DMF
100
Reaction conditions: 1a (1eq., 1 mmol), catalyst (CuI/L-proline) or AgNO3, base (3 eq., 3 mmol) or additive (3 eq., 3 mmol), N-
a
methylpiperazine (5 eq., 5 mmol), solvent (5 mL). All reactions were carried out in presence of air for 48 h. NMPz (N-
methylpiperazine). bReaction under argon. cIsolated yield of pure product after purification.

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With optimized reaction conditions in hand (entry 2, Table 1), different 3-aryl-6-piperazin-
1,2,4-triazolo[3,4-a]phthalazines 1a-g were prepared from the corresponding phthalazinyl-
arylhydrazone 2a-g (Table 2), focusing our attention on relevant functionalization found in
previous active mono- and di-substituted phthalazines [11a-b]. These starting phthalazine-
hydrazones were prepared following the reported procedure [11a]. As shown in Table 2, the
reaction mediated by silver (I) afforded better reaction yields for most of studied cases compared
to copper (I) catalyst, with a good group tolerance in general. Unfortunately, substrates
containing electron-deficient heteroaryl moieties such as 5-nitrofuryl or 5-nitrothiophene
generated a complex mixture and products containing amine moiety by nitro group was
presumably formed during the reaction according to 1H-NMR evidence. There are many reported
about the high susceptibility of nitro-substitution in aromatic systems highly electron-deficient to
suffer nucleophilic aromatic substitution in presence of aliphatic amines [30]. Moreover,
collateral products derived from ring opening may be generated from the catalyzed-reaction for
high electron-deficient ring such as 5-nitrofuryl and 5-nitrothiophene in substrates 2f and 2g,
respectively. Some recent reports have demonstrated that five membered rings such as furyl and
thiophene are susceptible to ring opening reaction under the presence of metallic-catalyst (Co,
Cu, Sc or Ir) [31-32].
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Table 2. Synthesis of 3-aryl-6-(N-methylpiperazin)-1,2,4-triazolo[3,4-a]phthalazines 2a-g.
N
N
Y
N
N
N
CuI/L-proline
Y
(15%/30%)
N
NH
N
K3PO4 (3 eq.)
N-methylpiperazine
100 °C, 48 h
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N
1a-g
2a-g
%Yielda,c %Yieldb,c
Entries
1
Y
Compound codes
2a
73
62
83
83
79
73
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3
2b
2c
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2e
2f
79
81
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O
S
traces
traces
traces
traces
NO2
NO2
2g
aReaction conditions: 1a-g (1.0 eq., 1 mmol), CuI (0.15 eq., 0.15 mmol), L-proline (0.30 eq., 0.3 mmol), K3PO4 (3.0 eq., 3 mmol),
N-methylpiperazine (5.0 eq., 5 mmol). bReaction conditions: 1a-g (1.0 eq., 1 mmol), AgNO3 (2.5 equiv., 2.5 mmol), K3PO4 (3 eq.,
3 mmol), N-methylpiperazine (5 eq., 5 mmol), solvent (5 mL). cIsolated yield of pure product after purification. N.I.: not isolated.
A putative and simplified mechanism of reaction for the formation of the product 2 (or E) from
the starting material 1 (or A) was proposed based on our previous copper protocol [27] and
reported examples of C-H functionalization mediated by Ag(I)/Ag(0) redox [29] (Scheme 1).
Initially, starting 4-chloro-1-phthalazin-aryl/heteroarylhydrazone A under basic conditions give
the intermediate ion B possessing a nitrogen at position 3 highly nucleophilic, which attacks to
adjacent ylidenic carbon via radical nucleophilic intamolecular addition to form the intermediate
ion C. C aromatized via dehydrogenation of the "pseudo ylidenic" C-H bond to lead the
intermediate D, directed by the reductive action of silver (I) releasing silver (0) metal. Finally, the
intermediate D suffers a N-amination on the C-Cl bond via SNAr to give the desired product E.
N
Y
N
N
N
Y
NH
N
N
N
E
A
Cl
N
N
-H+
Deprotonation
NH
N
N-amination
N
Y
N
N
Ag0
N
Ag+
N
N
Y
H
Y
N
H
N
N
N
N
N
Aromatization
C
Cl
B
D
Cl
Cl
Scheme 1. Plausible mechanism.

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3. Antileishmanial evaluation
5
6
7
8
9
We have focused our research in the identification of any potential molecule to treat the
American CL. In this sense, all compounds were tested against L. braziliensis parasite, the main
responsible of the mentioned disease in Latin-America (90% of all CL cases) [5]. Initially, we
evaluated the therapeutic effect of the five synthesized compounds on proliferative culture of the
promastigote stage. Results are shown in Table 3. It should be noted that four derivatives showed
excellent activities with low-micromolar IC50 values, ranging from 1.60 to 4.95 μM, being 5-13.5
fold more potent than glucantime (reference drug). It should be noted that the Y substitution on
1,2,4-triazolo ring has an important effect in the biological response of these phthalazine-
triazolos, where the 4-nitrophenyl and 4-fluorphenyl Y substituent led to an increase in the
antileishmanial activity, with low micromolar IC50 of 2.46 and 1.60 μM, respectively. The 3-
nitrophenyl and furyl substitutions generated a second line of active derivatives against
promastigote proliferation of L. braziliensis with good IC50 values of 4.73 and 4.86 μM,
respectively. The derivative containing an unsubstituted phenyl (2a) as Y group displayed a weak
antileishmanial activity at 50 μM. Therefore, we can conclude that the potency of tested
compounds based on the Y substitution increased in the following order: 4-fluorophenyl > 4-
nitrophenyl > 3-nitrophenyl > furyl > phenyl. These results demonstrated that 1,2,4-triazolo ring
fused with phthalazine ring generated active compounds against Leishmania parasite, reflecting
that the phthalazine ring play an important role in the activity of these compounds because
previous compounds based on 1,2,4-triazolo have demonstrated from a moderate to weak
antileishmanial activity [35].
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34
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36
37
38
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41
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44
45
46
47
48
49
50
51
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Table 3. In vitro growth inhibitory effects of the compounds 2a-e against promastigote and
amastigote forms of L. braziliensis.
N
N
Y
N
N
N
N
2a-e
54
Ent.
55
Compd
Promastigote IC50
Amastigote LD50 (μM)
Toxicity CC50 (μM)
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9
(μM)
Axenica
Intracellularb
Human isolatec
N.D
Macrophaged
N.D.
1
2
3
4
5
6
2a
2b
> 20.0
N.D
> 50.0
1.60 ± 0.08 (65)
2.46 ± 0.19 (16)
4.73 ± 0.32 (26)
4.89 ± 0.21
5.69±0.32 (18)
0.83 ±0.04 (47)
2.58 ±0.12 (48)
N.D.
28.93±1.21 (4)
9.01±0.81 (4)
9.37±0.72 (13)
32.02±1.56
> 50.00 (< 2)
48.18±2.12 (1)
43.24±2.23 (<3)
N.D.
103.34± 5.12
38.89±2.15
123.93±7.89
N.D.
2c
2d
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2e
Glte
32.00± 1.78 (4.3)
N.D.
12.21± 2.12 (12)
>50.0 (<3)
138.22 ± 10.11
aAxenic amastigote of L. braziliensis. Reference murine intracellular amastigote isolate of L .braziliensis. Clinical intracellular
amastigote isolate of L. braziliensis.dMurine peritoneal macrophage. eGlucantime reference drug. The results are the mean of three
independent experiments with a SD less than 5 % in all cases. Note: marked in bold parameters indicated a pronounced
antileishmania activity. N.D.: Not determined. Glt: glucantime. IC50: inhibitory concentration to inhibit 50% of cell proliferation;
LD50: lethal doses to kill the haft cell population (50%); CC50: 50% cytotoxicity concentration. Selectivity indexes are reported in
parenthesis.
b
c
To validate the therapeutic effect of the most active compounds, an in vitro evaluation at
different compound concentration was performed against axenic and intracellular amastigote
isolates. Firstly, promastigote of L. braziliensis was transformed in axenic amastigote under
specific conditions of pH=5.5 at 35 ºC for 48 hours. Then, amastigote was maintained in culture
and the therapeutic effect of the derivative 2b, 2c and 2d was evaluated at 48 h. Results are
shown in Table 3. Interestingly, compounds 2c and 2d were by about 2-fold more selective
toward axenic amastigote than toward promastigote form with low IC50 values of 0.83 and 2.58
μM, respectively. While compound 2b was significantly more active against promastigote than
axenic amastigote strain. To complement the anti-amastigote evaluation, the effect of the
mentioned compounds was assessed on reference intracellular amastigote isolates. The
intracellular amastigote model is one of the most important assays for the in vitro antileishmanial
evaluation because of the amastigote stage, that is found in mammalian host, is the main
responsible of the clinical manifestations of leishmaniasis. Traditionally, the intracellular
amastigote are produced in situ from macrophage in presence of promastigote parasite under
specific conditions (pH 5.5 at 37ºC). Nevertheless, this lab adapted strain could be more
susceptible to the action of drug compared to amastigote isolate because the machinery defence is
less activated in axenic culture as consequence of numerous passages. Then, we decided to work
with amastigote isolates from murine footpad lesion to obtain more approximated information to
the reality. This reference isolate was directly extracted from murine lesion footpad, and parasite
cells was exposed in culture media at specific conditions (pH 5.5 at 37 °C) and, treated with the

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selected compounds 2b, 2c and 2d. This protocol was previously reported by our group [9a, 10a,
36]. The effect of the compounds was evaluated at 48 h post-treatment and, the results are shown
in Table 3. As was expected, it should be noted that the amastigote intracellular isolate was from
5- to 9-fold more resistant than axenic amastigote for the tested compounds. Specifically, nitro-
compounds 2c and 2d were identified as the most potent derivatives, confirming that the nitro-
substitution has a specific role in the biological responses of these compounds. On the other
hand, compounds 2b and 2e exhibited a moderate response against amastigote intracellular
isolate, while the compound 2a no responding at 50 µM. It is important to mention that the
compound 2d was comparable in therapeutic potency against amastigote intracellular isolate and
selectivity to glucantime drug.
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To evaluate the real potentiality of the most active derivatives 2b, 2c and 2d, we performed an
in vitro evaluation using clinical amastigote intracellular isolate. The clinical human strain, called
MHOM/VE/2018/M2903/LR, was identified as L. braziliensis specie and, it was isolated from a
Venezuelan CL patient [10f]. Experimental details can be found in supplementary material.
Results are listed in Table 3. The clinical amastigote isolate was 4-fold more resistant than
reference amastigote strain for the two most active derivatives 2c and 2d, whereas not
appreciable response was observed for the compound 2b at 50 μM. Compounds 2c and 2d were
more potent than reference drug against human amastigote isolate. Despite the decreasing
antileishmanial activity of the compounds 2c and 2d against human amastigote isolate,
compound 2d, with a better activity/toxicity profile than reference drug against promastigote,
axenic amastigote and amastigote isolates, was identified as a hit platform from initial screening
step for further structural improvements to identify an antileishmanial lead based on phthalazine-
1,2,4-triazolo according to criteria of drug discovery for neglected tropical disease [9c].
To quantify the selectivity of the most active derivatives, it is important to know its effect on
macrophage cells, which are the traditional host cell of Leishmania parasite. The toxicity of the
selected compounds 2b, 2c and 2d was evaluated on peritoneal macrophages derived from
peritoneal cavity of Balb/c mice. Toxicity results (expressed in terms of CC50 parameter) are
summarized in Table 3 together with their respective selectivity indexes related to anti-
promastigote and anti-amastigote activities. In particular, the mentioned compounds showed
moderate in vitro toxicities with CC50 values from 38 to 123 μM. Specifically, compound 2d
emerged as the most promising antileishmanial agents for further in vivo assays, with selectivity

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indexes by about 48 and 13 against promastigote or against amastigote stages of L. braziliensis,
respectively, indicating that 2d is significantly more destructive on the parasite form than on the
host cells. Compound 2c is one of the most active derivatives, but its moderate toxicity
(LD50=38.89 µM) compromises its selectivity (S.I.~4 against amastigote reference strain) as
antileishmanial agent. This cytotoxic effect associated to the 4-nitrophenyl suggests that other
compounds bearing higher electron-deficient nitro-groups such as the compound 2f and 2g may
be highly toxic against host mammalian cell.
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Table 4. Comparison between anti-promastigote and anti-amastigote activities for the
compounds 2a-e, 3a-e and 4a-e.
Y
N
N
Y
N
Y
N
N
N
N
N
N
NH
NH
NH
N
N
2a-e
4a-e
N
N
Cl
3a-e
Y
Promastigote IC50 (μM)
Amastigote IC50 (μM)
Ent.
2a-e
3a-ea
>25.0
> 25.0
14.16
> 25.0
> 25.0
4a-eb
2a-e
3a-ec
4a-eb
Y
C6H5
> 25.0
1.60
2.46
4.73
4.89
>25.0
2.37
N.D.
N.D.
>50.0
4.71
1
2
3
4
5
4F- C6H5
4NO2- C6H5
3NO2- C6H5
C5H4O
28.93 N.D.
>25.0
23.70
> 25.0
9.01
9.37
31.31
N.D.
>50.0
9.23
> 50.0 >50.0
>50.0
aBiological data for 3a-e was extracted from Ref. 11b. bBiological data for 3a-e was extracted from Ref. 11d.
cAntiamastigote biological data for 3a-e was performed in the present investigation.
A comparison between the activity of these five compounds 2a-e and their corresponding
acyclic version, arylhydrazone-phthalazinyl 3a-e and disubstituted phthalazines 4a-e (see Table
4), put in evidence that the cyclic version 2a-e (IC50 ranging from 1.6 to 4.89 μM) was in general
more active than corresponding acyclic analogues 3a-e (IC50 > 14 μM) and 4a-e (IC50 > 8 μM for
the majority of compound with exception of 4b with an IC50 value of 2.37 μM) (see comparative
details in Table 4). We think that all these three phthalazine systems act under different mode of
action due to that specific and different functionalizations are essential for each system (Figure

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2): (i) the 5-nitrofuryl for the monosubstituted phthalazines 3a-e, (ii) the 4-fluorophenyl for the
disubstituted phthalazines 4a-e and, (iii) the 3-nitrophenyl for the 1,2,4-triazolo[3,4-a]phthalazine
2a-e. Figure 2 showed the most prominent candidate for each phthalazine group, and from this
data it should be noted that the 4-chloro-1-phthalazine-(5-nitrofuryl)hydrazinil 3 was identified as
a promising lead for further chemical modifications according to criteria established for the
identification of lead in the stage I of drug discovery for neglected tropical disease [9c], in
particular to improve or regulate its toxicity level. The chemical modifications may be directed to
incorporate specific substitutions, for example, on 1-position of phthalazine core by replacement
of chlorine atom. Meanwhile phthalazine-1,2-4-triazolo 2 and disubstituted phthalazine 3, with a
more moderate profile compared to phthalazine 3, emerge as promising hit for further
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improvements in order to identify an antileishmanial lead.
F
N
N
N
NO2
N
O
NO2
N
N
NH
NH
N
N
N
NH
N
N
N
N
Cl
4
2
3
F
IC50 = 4.73 M (promastigote)
IC50 = 2.37 M (promastigote)
LD50 = 2.58 M (axenic amastigote)
LD50 = 9.37 M (intracellular amastigote)
LD50 = 43.24 M (clinical amastigote)
CC50 = 123.93 M (peritoneal macrophage)
EC50 = 0.12 M (promastigote)
EC50 = 0.50 M (intracellular amastigote)
CC50 = 15.0 M (macrophage BMDM)
LD50 = 4.71 M (intracellular amastigote)
LD50 = 19.62 M (resistant amastigote)
LD50 = 35.30 M (clinical amastigote)
CC50 = 30.00 M (macrophage BMDM)
Figure 2. Comparison between the most active compounds of different phthalazine-based
derivatives 2, 3 and 4. Antileismanial biological data for phthalazine-1,2,4-triazolo 2 (in blue
color), monosusbtituted-phthalazine 3 (in green color) and disusbtituted-phthalazine 4 (in red
color). Biological information for 3 and 4 were extracted from ref. 11b and 11d, respectively.
In summary, an one-pot catalyzed strategy was developed for the synthesis of a series of 3-aryl-
6-(N-methylpiperazine)-1,2,4-triazolo[3,4-a]phthalazines
from
phthalazin-based
on
aryl/heteroarylhydrazinyl derivatives using silver(I) as mediator. Compared to the traditional
synthetic strategies, this protocol represents an attractive and competitive strategy by its
simplicity, economy and one-pot reaction procedure. From biological evaluation, two derivatives

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2b and 2d displayed a good in vitro activity against the promastigote and axenic amastigote
forms of Leishmania braziliensis with low-micromolar IC50 values (ranging from 1 to 4 μM), low
toxicities by about 103-125 μM and acceptable selectivity indexes. Compound 2d showed better
activity/toxicity profiles than glucantime drug against the tested promastigote and amastigote
strains. A structure-activity relationship study suggests that the formation of fused triazolo-
phthalazine and nitro-substitution are crucial pharmacophores in the biological activity of this
kind of compounds, although the electron-deficiency nature of that nitro-substitution must be
moderate such as a 3-nitrophenyl. Finally, the results demonstrated that compound 2d represents
a prominent antileishmanial hit platform for further chemical modifications, whereas the 4-
chloro-1-phthalazine-(5-nitrofuryl)hydrazinil was identified as a promising lead for further
biological experiments including genotoxicity evaluation, drug-likeness parameters, and in vivo
assays to estimate its real therapeutic potential within the stage I of drug discovery for neglected
tropical disease.
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Acknowledgments
The authors thank Facultad de Farmacia (Universidad Central de Venezuela, Caracas) and
Instituto de Biomedicina “Dr. Jacinto Convit” (Facultad de Medicina, Universidad Central de
Venezuela) for financial support. The investigation was supported by the Project CDCH-UCV
under number grant PG-09-8819/2013.
Conflict of Interest
Author declared not conflict of interest related to present report.
Supplementary material
1
Full chemistry and biological experimental details, H-NMR and 13C-NMR data, IR and
elemental analysis information associated with the compounds obtained herein can be found in
the online version of the Supplementary material.

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