Design, synthesis, structure-activity relationship and mechanism of action studies of a series of 4-chloro-1-phthalazinyl hydrazones as a potent agent against Leishmania braziliensis

Article abstract of DOI:10.1016/j.ejmech.2017.01.022

With the aim to identify a potential drug candidate to treat cutaneous leishmaniasis, a series of 1-phthalazinyl hydrazones were synthesized and tested against Leishmania braziliensis parasite, one of the main responsible of this disease in the world. A structure-activity relationship permitted to identify two phthalazines containing nitroheterocyclic moiety 3l and 3m as promising new lead compounds. These compounds showed a significant antileishmanial activity against promastigote form of L. braziliensis, with EC₅₀values in sub-micromolar and nanomolar ranges. The phthalazine 3l also displayed a selective and excellent activity against the clinically relevant intracellular amastigotes form, with a EC₅₀value in sub-micromolar range (0.59 μM), without affecting the viability of the host cells. Oxidative stress was identified as the possible mode of action of the most active phthalazine. Considering their significant antileishmanial activity and ease synthesis, the phthalazine containing nitroheterocyclic represents a promising agent against Leishmania braziliensis for the rational design of new leads.

Full text of DOI:10.1016/j.ejmech.2017.01.022

Accepted Manuscript
Design, synthesis, structure-activity relationship and mechanism of action studies of
a series of 4-chloro-1-phthalazinyl hydrazones as a potent agent against Leishmania
braziliensis
Angel H. Romero, Rafael Medina, Anamaría Alcala, Yael García-Marchan, Jorge
Núñez-Duran, Jacques Leañez, Ali Mijoba, Carlos Ciangherotti, Xenón Serrano-
Martín, Simón E. López
PII:
S0223-5234(17)30030-2
10.1016/j.ejmech.2017.01.022
EJMECH 9173
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 October 2016
Revised Date: 12 January 2017
Accepted Date: 13 January 2017
Please cite this article as: A.H. Romero, R. Medina, A. Alcala, Y. García-Marchan, J. Núñez-Duran, J.
Leañez, A. Mijoba, C. Ciangherotti, X. Serrano-Martín, S.E. López, Design, synthesis, structure-activity
relationship and mechanism of action studies of a series of 4-chloro-1-phthalazinyl hydrazones as a
potent agent against Leishmania braziliensis, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.01.022.
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ACCEPTED MANUSCRIPT
Promastigote Amastigote
R
H
Ar(Het-Ar) EC50 (μM) EC50 (μM)
0.12 0.59
H
3.20

1
ACCEPTED MANUSCRIPT
Design, Synthesis, Structure-Activity Relationship and Mechanism of
Action Studies of a Series of 4-chloro-1-phthalazinyl hydrazones as a potent
agent against Leishmania braziliensis
a*
b
c
c
Angel H. Romero , Rafael Medina , Anamaría Alcala ,Yael García-Marchan , Jorge Núñez-
c
c
c
d
c
Duran , Jacques Leañez , Ali Mijoba, Carlos Ciangherotti, Xenón Serrano-Martín and
a*
Simón E. López
a
Laboratorio de Química Medicinal y Heterociclos, Departamento de Química, Universidad Simón Bolívar,
Valle de Sartenejas, Baruta, Caracas 1080-A, Apartado 89000, Venezuela.
b
Laboratorio de Bioquímica, Estructura y Función de Proteínas, Área de Salud, Instituto de Estudios Avanzados
(IDEA), Caracas, Venezuela.
c
Laboratorio de Biología y Quimioterapia de Parasitosis Tropicales, Área de Salud, Instituto de Estudios
Avanzados (IDEA), Caracas, Venezuela.
d
Laboratorio de Neuropéptidos, Unidad de Farmacología, Facultad de Farmacia, Universidad Central de
Venezuela, Apartado 47206, Los Chaguaramos, 1041-A, Caracas, Venezuela.
a
*
Corresponding authors. E-mail:angel.romero12@ucv.ve; slopez@usb.ve
Phone: +582126052699
ABSTRACT
With the aim to identify a potential drug candidate to treat cutaneous leishmaniasis, a series
of 1-phthalazinyl hydrazones were synthesized and tested against Leishmania braziliensis
parasite, one of the main responsible of this disease in the world. A structure-activity
relationship permitted to identify two phthalazines containing nitroheterocyclic moiety 3l and
3
m as promising new lead compounds. These compounds showed a significant
antileishmanial activity against promastigote form of L.braziliensis, with EC values in sub-
50

2
ACCEPTED MANUSCRIPT
micromolar and nanomolar ranges. The phthalazine 3l also displayed a selective and
excellent activity against the clinically relevant intracellular amastigotes form, with a EC50
value in sub-micromolar range (0.59 µM), without affecting the viability of the host cells.
Oxidative stress was identified as the possible mode of action of the most active phthalazine.
Considering their significant antileishmanial activity and ease synthesis, the phthalazine
containing nitroheterocyclic represents a promising agent against Leishmania braziliensis for
the rational design of new leads.
Keywords: Leishmania braziliensis, antileishmanial activity, superoxide dismutase,
promastigotes, phthalazine, nitroheterocycle.
1
. Introduction
Leishmaniasis is a neglected tropical disease caused by at least twenty different species of the
genus Leishmania protozoan that are transmitted to humans by the bitte of infected female
phlebotomine sandflies [1-5]. There are three main forms of the disease: visceral
leishmaniasis (VL); mucocutaneous leishmaniasis (MCL) and cutaneous leishmaniasis (CL),
being the last one the most common form of the disease [5]. More than 550 million people
are at risk of infection with Leishmania ssp. in at least 102 countries worldwide, with an
annual incidence of more than 14 million people infected, leading to 50.000 deaths [6].
According to World Health Organization (WHO), the majority of cases and deaths caused by
Leishmaniasis are associated to visceral and cutaneous leishmaniasis, registering between 0.2
and 0.4 million cases of visceral leishmaniasis, and from 0.7 to 1.2 million cases of cutaneous
leishmaniasis each year [7]. In particular, Leishmania braziliensis is the responsible of nearly
9
0% of all CL cases registered annually in South America, and absence or incomplete
treatment promotes the subsequent development of muco-cutaneous leishmaniasis, causing
permanent lesions in the mouth, nose or genital mucosa [8]. In recent decades, the number of

3
ACCEPTED MANUSCRIPT
cases of Leishmaniasis is increasing globally due to Leishmania/HIV co-infection [9],
international travels and migration [10,11]. Currently, pentavalent antimonials such as
Pentostam (sodium stibogluconate) and glucantime (N-methylglucamine antimonate) are the
first line of antileishmanial drug in many areas [12]. Nevertheless, the therapy is expensive,
requiring repeated parenteral administration, higher dosages cause severe toxic side effect
and in many cases have resulted to be ineffective [13]. Furthermore, large-scale clinical
resistance to pentavalent antimonials has been registered in endemic areas [12a, 14]. As a
consequence to the several disadvantages of pentavalent antimonials, a second line drugs
including amphotericin B and miltefosine (Figure 1) has emerged as an alternative for
treating of visceral and cutaneous leishmaniasis [12a, 15], although their use also has been
limited because of its high toxicity and effectiveness against different forms of Leishmania,
including Leishmania braziliensis.
O
NH
O
O
OH
Na
H
HO
HO
OH
Na
OH
OH
O
OH
O Sb
HO
HO
O
O
O
H HO
O
Sb
O
O
HO
Sb
OH
O
O
OH
HN
O
O
Na
sodium st ib ogluconate
meglumine antimoniate
OH
OH
O
O
OH
HO
O
OH OH
OH OH
O
OH
O
amphotericinB
HO
OH
NH
2
O
P
O
O
N
O
miltefosine
Figure 1. Drugs used to treatment of Leishmaniasis.

4
ACCEPTED MANUSCRIPT
Then, considering toxicity issues, increasing failure rates of current treatments, increasing
parasite resistance and the lack of effective clinical agents against cutaneous leishmaniasis,
there is a great and urgent need the development of effective and safe new drugs for treating
of leishmaniasis in their different clinical forms. In this sense, an enormous variety of potent
and active new heterocycles have emerged during the last decade against different leishmania
spp., including quinolines [16-17], quinazolines [18-23] and phthalazines [24].
2
. Results and discussion
2
.1. Design and synthesis of the target compounds
To design new potential drugs, it is important to understand the essential metabolic
biochemical pathways or crucial parasite-specific enzyme. Trypanothione reductase, cysteine
protease, dihydrofolate reductase, glyceraldehyde 3-phosphate dehydrogenase, superoxide
dismutase (Fe-SOD) and oxidative stress have emerged as interesting targets [25]. In this
investigation, we focused our attention on iron superoxide dismutase (Fe-SOD) and oxidative
stress for the design of our new antileishmanial compounds. Fe-SOD enzyme plays an
important role in the defence of trypanosomatids against oxidative agents and it is not present
in humans. Some evidences in trypanosomatids have shown that parasitic protozoan survival
is closely related to the ability of that enzyme to suppress toxic free radical damage
originated by their host [26]. The enzymatic activity of Fe-SOD is directly associated to the
iron metal contained into active site of the enzyme and it represents an attractive target for the
development of new agents based in complexation with iron metal. In recent years, Sanchez-
Moreno´s group has designed new phthalazines and benzo[g]phthalazines functionalized in
positions 1 and 4 of the pyridazine ring with flexible alkylamine side chains, which have
shown a significant inhibition of the SOD enzyme of T.cruzi, L.infantum and L.braziliensis
parasites. The inhibitory activity of these compounds showed a direct correlation with their

5
ACCEPTED MANUSCRIPT
respective anti-trypanosomatids activity against L.infantum, L.braziliensis and T.cruzi [24,
7].
On the other hand, the induction of oxidative stress by using of nitroheterocyclic
2
compounds has been little explored againts Leishmania parasites. This target is more
common in the design of antichagasic agents and it generates unstable highly toxic reduced
oxygen species from redox-cycling metabolism that damages proteins, lipids and DNA,
leading to parasite death [28-29]. Many oxidative stress inductors are based in a nitro-
heterocyclic segment [29]. Thus, it results very interesting to develop an antileishmanicidal
agent that acts by both mechanism because the inactivation of SOD enzyme could reduce
significantly the defences of parasite against the toxic radical species generated by the
possible bio-reduction of the nitro-compound. On these bases, we consider in our design a
nucleus of phthalazine, related to the SOD inhibition, connected to a segment of aryl or
heteroarylhydrazone, crucially important in the induction of oxidative stress when the
aryl/heteroaryl moiety is represented by a nitrofuryl or nitro-thiophene group. Interestingly,
crystal structures of mononuclear complexes with transition metals such as mercury (II),
molybdenum (V) and others [30] have been found for some 1-phthalazinyl-arylhydrazones.
Therefore, it is possible to think that the designed phthalazines 3a-t may form a complex with
the iron (II) metal, being a potential Fe-SOD inhibitor in Leishmania parasites (Figure 2).
Ar (Het-Ar)
N
N
+2
FeL4
N
N
Cl
Figure 2. Possible complex between Fe and 4-chloro-1-phthalazinyl hydrazones.

6
ACCEPTED MANUSCRIPT
2.2. Synthesis section
The preparation of title phthalazines 3a-t involved a two-step facile procedure from
commercially available starting materials according to experimental protocols reported in the
literature, with a few modifications [31]. The general synthesis of the phthalazinyl-
hydrazones 3a-t is depicted in Scheme 1. Thus, step 1 involves a SNAr reaction between 1,4-
dichlorophthalazine 1 and hydrazine monohydrate under reflux to provide the desired 1-
chloro-4-hydrazinylphthalazine 2 in good yield (83 %). Next, the sequence involves an acid
catalyzed condensation of compound 2 with the corresponding aromatic aldehyde or
acetophenone to give their respective substituted phthalazines 3a-m and 3n-t. This last
reaction displayed excellent yields, ranging from 85 to 97 %, with exception of derivatives 3s
and 3t (see Table 1). In particular, this reaction required extended times when
acetophenones were used. The experimental details and the spectroscopic data for the
intermediary 2 and phthalazine derivatives 3a-t are shown in the supporting information.
Ar (Het-Ar)
N
HN
N
N
Cl
3a-m
NH2
b
Cl
Cl
HN
Cl
a
N
N
N
N
H C
Ar
3
c
N
HN
2
1
N
N
Cl
3n-s
Scheme 1. Synthesis of phthalazin-hydrazone derivatives 3a-t. Conditions: (a) N H .xH O,
2
4
2
Ethanol, reflux, 2 h; (b) Aryl/heteroaldehyde, HCl (1M), 60ºC, 10 min.; (c) Acetophenone,
HCl (1M), 60 ºC, 2-10 h.

7
ACCEPTED MANUSCRIPT
In general, based in the chemical shift found for the ylidene proton of the compounds 3a-t
1
in the H NMR spectra and that information reported for some of their derivatives and
analogues, it is possible to conclude that most of the prepared phthalazinyl-hydrazones present
a E-configuration around the imine (H-C=N) double bond, with exception of the derivative 3i
that has a Z-configuration [32]. A detailed analysis concerning to the assignment of E or Z
configuration for the synthesized compounds is shown in the supplementary material.
Table 1. Yields in the last step for the synthesis of the phthalazine derivatives 3a-t.
R
N
Ar(Het-Ar)
N
NH
2
N
NH
N
NH
N
R=H, Me
Cl
Cl
2
3a-t
a
Compd No. Ar (Het-Ar)
R
Yield (%)
m.p. (°C)
Diastereoselection
b
3
a
H
96
173-174 (Lit. 175-176)
184-186
E
3
b
F
H
H
H
H
H
92
94
91
88
90
E
3
c
O
2
N
255-257
E/Z (94/6)
E/Z (96/4)
E/Z (93/7)
E
3
d
Br
220-222
3
e
Cl
O
203-205
3
f
235-238
2
N
3
g
H
H
87
88
217-218
196-198
E
E
Br
Cl
3
h
b
3
i
H
H
H
87
91
89
157-160 (Lit. 162-163)
246-248
Z
E
E
OH
3
j
2
O N
OCH
3
O
3
k
170-172

8
ACCEPTED MANUSCRIPT
O N
2
O
S
H
93
230-232
E/Z(93/7)
E/Z (93/7)
3
l
2
O N
3
m
H
85
220-222
3
n
o
p
HO
CH
CH
CH
3
3
3
97
79
88
212-214
234-236
182-184
E
E
E
3
3
2
O N
Br
3
q
CH
3
93
217-219
E
2
O N
3
r
s
CH
3
3
73
50
142-144
162-164
E
E
3
Cl
CH
Cl
Cl
3
t
CH
3
42
166-168
E
Cl
a
Geometric configuration and mixture of diastereomers for each phthalazine 3a-t was predicted by a detailed comparative
1
analysis between the chemical shift of ylidene proton found in their corresponding H NMR spectra and the information
b
available in literature [32]. Melting points are taken from ref. [31a] for these reported compounds.
2
.3. Biological evaluations
2
.3.1. In vitro antileishmanial activity and structure-activity relationships
The aim of this study was to determine the importance of phthalazinyl and
aryl/heteroarylhydrazone moieties as potential pharmacophores in the design of our new
antileishmanial agents. To reach this goal, twenty phthalazines 3a-t coupled to different
aromatic systems (i.e., substituted arylidenes and heteroarylidenes) were initially tested in
vitro against L. braziliensis parasite, evaluating its effect on promastigotes growth to a
specific concentration of 20 µM for each compound. In this first screening, the derivatives 3c,
3
l and 3m showed the most significant ability to inhibit the promastigote proliferation of
L.braziliensis, with PGI values of 58.14 %, 86.49 % and 88.46 %, respectively (see Table 2).
A weak antileishmanial activity was observed for the rest of compounds with low PGI values

9
ACCEPTED MANUSCRIPT
(
<40) at 20 µM (EC >20 µM) (Table 2), which demonstrates the important role of the nitro-
50
substitution in the antileishmanial activity of the tested compounds. After the first screening,
the concentration response data for the most active derivatives 3l and 3m was fitted by a
nonlinear regression model, and the concentration that induces 50% inhibition was calculated
as the effective concentration EC on L.braziliensis promastigotes. These derivatives 3l and
50
3
m affected the viability of L. braziliensis promastigotes, with EC values of 0.12 and 3.20
50
µM, respectively (Table 3). Both derivatives 3l and 3m were more potent than miltefosine
against promastigotes of L.braziliensis by a factor of 208 and 8, respectively. This result is
interesting due to that miltefosine is the first oral drug used for visceral leishmaniasis
treatment and it has been considered a major breakthrough in antileishmanial chemotherapy
[
33].
Table 2. Preliminary evaluation on the L.braziliensis promastigotes growth, theoretical ZDO
atomic charges on the ylidene carbon and absorption maxima (λmax) for the tested
phthalazinyl-hydrazones 3a-t.
a,b
b
c
d
Entries Compound
PGI (%)
8.46
EC50 (µM)
>20
ZDOC=N
-0.0449
-0.0505
-0.0863
-0.0546
-0.0516
-0.0791
-0.0563
-0.0572
0.0300
λmax (nm)
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
372
369
420
372
368
391
367
368
364
389
376
475
496
17.78
58.14
17.21
16.75
10.58
5.89
>20
<20
>20
>20
>20
3g
3h
3i
>20
7.03
>20
33.85
11.86
40.86
86.49
88.46
>20
1
0
1
2
3
3j
>20
-0.0651
-0.0441
-0.0924
-0.0849
1
1
1
3k
3l
≥20
0.12
3.20
3m

1
0
ACCEPTED MANUSCRIPT
14
15
16
17
18
19
20
21
22
3n
22.91
>20
>20
>20
>20
>20
>20
>20
<20
------
-0.0248
372
430
3o
19.08
10.62
9.46
-0.0651
-0.0404
-0.0554
-0.0329
-0.0466
-0.0497
N.D
3p
374
3q
390
3r
36.95
37.47
19.98
67.78
0.02
374
3s
3t
375
376
e
Miltefosine
N.D
-------
f
Control (-)
--------
a
b
Proliferation growth inhibition (PGI), doses=20 µM. The results are the means of three independent experiments with a SD
c
d
e
less than 6% in all cases. ZDO charge on ylidene carbon. λ was measured in methanol as solvent
.
Miltefosine was used
max
f
as reference drug. Untreated control. N.D.: not determined.
A detailed analysis of the structure-activity relationship for the evaluated phthalazines
3
a-t permitted us to establish the following remarks:
a) The substitution of a specific arylidene group by a nitroheteroarylidene group linked to
hydrazone scaffold increases significantly the antileishmanial activity of the synthesized
phthalazine (compare 3l and 3m with the rest of compounds).
b) The substitution of oxygen by sulphur in the heterocyclic ring reduces almost 27 times the
antileishmanial activity, reflecting the importance of the furyl group in the mentioned
biological activity over thiophene ring in our phthalazin-derivatives.
c) The nitro-substitution at the para-position of arylidene system increases the
antileishmanial activity with respect to their arylidene phthalazin-analogues.
d) Replacement of the hydrogen atom (3a-m) by the methyl group (3n-t) around the imine
double bond (C=N) of synthesized phthalazines decreases the antileishmanial activity in
most of the analogues cases (compare 3a, 3c, 3d, 3f vs. 3r, 3o, 3p, 3q, Table 2).
e) Localization of the nitro group at the orto-position or meta-position of arylidene system
(3f, 3j and 3q) represents a significant loss of biological activity.

1
1
ACCEPTED MANUSCRIPT
f) The introduction of halogens (F, Cl, Br) (3d-f, 3g-h, 3p-t) and electron-donating groups
3j, 3n) in the arylidene system makes phthalazines less active.
(
These evidences suggest that the antileishmanial activity of the studied phthalazin-
hydrazones is associated to the highly conjugated aryl/heteroaryl hydrazone scaffold due to
that only the nitro-derivates 3c, 3l and 3m exhibited the most significant biological response.
The specific localization of the nitro group at the position “4” and “5” of aryl and heteroaryl
moieties of phthalazine derivative, respectively, facilitates an optimal π-electron conjugation
from hydrazinyl group to nitro group. Thus, in order to understand this correlation between
high conjugation and biological activity, we decide to calculate the ZDO atomic charges on the
ylidene carbon of all phthalazines due to that this carbon atom is an important bridge for the π-
electron delocalization from hydrazinyl group to aryl or heteroaryl group. A high negative
character on the ylidene carbon indicates that there is a significant π-electron conjugation
between the mentioned segments, which can be to facilitate by a strong electron-deficient
heteroaryl or aryl moieties. The theoretical values of ZDO atomic charges were performed
using the semi-empirical method AM1 for the optimized geometry of all investigated
derivatives and the results are shown in Table 2. From data, it is clear to appreciate a direct
relationship between the electronic nature of the ylidene carbon and the antileishmanial
activity, where the most active compounds (3c, 3l and 3m) presented the most negative ZDO
atomic charge on the above mentioned carbon, which confirms the important role of an nitro-
heteroaryl highly conjugated in the antileishmanial activity of our phthalazine compounds.
This theoretical evidence permits to explain the higher potency found for the compound 3l
(major ZDO) in comparison with the compounds 3c and 3m. The high conjugated nature of the
active nitro-derivatives (3c, 3l and 3m) was experimentally confirmed by their highest λmax
values from 420 to 496 nm (see Table 2). Thus, the significant importance of the π-electron
delocalization of the nitro-derivative in the antileishmanial activity suggests that the most

1
2
ACCEPTED MANUSCRIPT
active derivatives can be involved in an electron transfer reaction of the redox metabolism of
the parasite, which is facilitated by the partial stabilization of the radical intermediaries formed
that provides this high π-electron delocalization in the phthalazine system. This type of
mechanism has been widely reported for antichagasic nitro-drugs such as nifurtimox and
benznidazole on T.cruzi [34] and more recently on L. donovani [29]. In the mechanism of
action section is shown a preliminary study that demonstrates the potential of 3l as inductor of
oxidative stress.
On the other hand, the poor antileishmanial activity found for the rest of synthesized nitro-
derivatives such as 3f, 3j, 3o and 3q can be explained by their moderate negative ZDO charge,
by comparison with 3c, 3l and 3m (see Table 2).
The relative major importance of the ylidene hydrogen (H-C=N) with respect to the methyl
moiety (CH -C=N) seems to be associated to the higher negative charge character on the
3
ylidene carbon of the phthalazines 3a-m (R=H) than on the carbon of their analogues 3n-t
(R=Me) (compare ZDO of the entries 3 and 15 of Table 2).
As we expected, compounds with the lower negative ZDO charge on the ylidene carbon
showed the lowest antileishmanicidal activity. Among these derivatives, it can be mentioned
those functionalized with electron donating groups (3i and 3n), halogens (3b, 3d-e, 3g-h, 3p
and 3s-t), unsubstituted (3a and 3r) and meta-nitro group (3f and 3o). However, derivatives 3i
and 3k particularly exhibited a moderate inhibition on promastigote growth, with PGI values
of 33.85 and 40.86, respectively. This last suggest that the moderate antileishmanial of these
two compounds can be associated to a non-electron dependent mechanism, such as a
Leishmania SOD inhibition. This hypothesis was considered and examined theoretically by a
docking computational study on SOD enzyme, where 3i and 3k exhibited a better interaction
into SOD binding site than the compounds 3c, 3l and 3m (see table 6, molecular docking

1
3
ACCEPTED MANUSCRIPT
studies section). This result should not be surprising due to that these phthalazines represent a
potential tridentate ligand which can form a stable complex with different metals [30].
NH
NH
NH
NH
NH2
H N
NH2
H N
2
2
N
O
O
N
N
H
´
Pentamidine
Berenil
H
N
O N
2
H
N
NH2
N
NH2
S
X
H
OH
As
N
N
N
S
HO
O
N
X=O, S
N
H N
N
N
OH
N
N
2
H
HO
Adenosine
M elarsoprol
Cl
Phthalazines proposed
Figure 3. Substrates of the P2 transporter. Remarked circles indicate the group involved in
the interaction with the membrane [37].
Finally, in order to know the importance of the phthalzine nucleus in our derivatives, the
antileishmanial activity was analyzed for a series of analogues containing a nitrofurylaryl-
hydrazinyl scaffold linked to a nucleus of phthalazine (our compounds), quinoline [35],
melanine [36] and aliphatic unit (nifurtimox) [29,36]. The comparative analysis demonstrates
that the electron-deficient nature of these conjugated nucleus (phthalazine, quinoline and
melanine) play an important role in antileishmanial activity due to the significant
antileishmanial response found in their respective compounds. While a no-conjugated
nucleus (a saturated aliphatic unit) connected to the nitroheterocyclic moiety such as in
nifurtimox displayed a moderate antileishmanial activity. This last suggest that the presence
of the phthalazine (or quinoline, melanine) generates a system highly conjugated from
phthalazin ring to the nitro group of heteroarylidene moiety, which seems to be crucial in the
antileishmanial activity. Although it is important to mention that probably the phthalazine
nucleus is not responsible of the biological response because in most of the tested compounds

1
4
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was observed a poor antileishmanial activity. In addition, due to the structural similarity
between the phthalazin-hydrazinyl scaffold and the amidine moiety (see Figure 3), an
important substrate of P2 that is present in the cell membrane of the trypanosomatids and
facilitate the accumulation of the compound into pathogen [37], the phthalazine nucleus can
play an important role in the transportation of the agent within the parasite.
2
.3.2. Cytoxicity assays
The cytoxicity of compounds 3l, 3m and miltefosine was evaluated using macrophages of
a BMDM cell line derived from narrow mouse [38], and the results are listed in Table 3. In
particular, derivative 3l showed a moderate in vitro toxicity with a LD value of 14 µM,
5
0
however that compound displayed an excellent selectivity index (SI) value of 116.7 (see
Table 3), being much more destructive to the parasitic form than the host cells. Additionally,
the selective index confirms the apparent advantage found in vitro for the compound 3l with
respect to reference drug miltefosine. Contrary to 3l, compound 3m resulted to be highly
toxic on this host at low concentration (LC <10 µM). Then, in this case, the nitro-thiophene
5
0
moiety can be responsible of the significant toxicity found for the compound 3m. The
moderate and high toxicity found for the two active derivatives on macrophages and the
known mutagenic effect caused by the nitro-derivatives on mammalian cells [39], suggests
that genotoxicity assays are necessary for future investigations.
Table 3. Effect of phthalazines 3l, 3m and miltefosine on BMDM macrophages and EC50
values (µM) on L.braziliensis promastigotes.
a
a
Macrophages BMDM LC (µM) Promastigotes EC (µM)
Selectivity index
50
50
3
l
14.00 ± 0.25
<10.00
0.12 ± 0.01
116.7
<3.0
5.0
3m
3.20 ± 0.92
b
Miltefosine
125.00 ± 4.70
25.00 ± 1.40

1
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a
b
The results are the means of three independent experiments with a SD less than 6% in all cases. Miltefosine was used as
reference drug.
2
.3.3. Intracellular amastigote evaluation
Going one step further in the activity study, leishmanicidal effect of phthalazine 3l on
amastigote stage was carried out through an in vitro infection model consisting of
macrophages BMDM infected by L. braziliensis amastigote. Using this model, a 40%
maximum infection was obtained, similar to previous studies [40]. The effect of 3l was
evaluated at 96 hours post-treatment and the results were shown graphically in the Figure 4. It
should be noted that phthalazine 3l also is significantly active against amastigote stage of
Leishmania braziliensis, evidenced by the monotonic decrease in the number of amastigotes
and infected macrophages to the tested concentrations, leading to a EC value of 0.59 µM for
5
0
the compound 3l. It is important to emphasize that this effect is highly selective against
intracellular amastigotes without affecting the viability of host cells (BMDM macrophages).
This antiamastigote response found for the compound 3l is extremely important because this
stage is the main responsible of the clinical manifestations of leishmaniasis. Furthermore, the
significant selectivity found for 3l on promastigotes and amastigotes with respect to the
macrophages cells, as well as its excellent potency, permits to consider to 3l as a promising
antileishmanial lead. Table 4 shows the selectivity indexes of 3l on the promastigotes and
amastigotes stages of L.braziliensis, as well as their specificity.
Table 4. Effect of phthalazine 3l on BMDM macrophages, EC50 values (µM) on
promastigote and amastigote of L.braziliensis, selectivity and specificity indexes.
Macrophages BMDM Promastigote
Amastigote
Selectivity Selectivity Specificity
a
b
c
LC (µM)
EC (µM)
EC (µM)
index
116.7
index
23.7
index
4.9
5
0
50
50
3
l
14.00 ± 0.25
0.12 ± 0.01
0.59 ± 0.13
a
Selectivity: LC50 of macrophages/EC50 of promastigotes of L. braziliensis.

1
6
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a
b
Selectivity: LC50 of macrophages/EC50 of amastigotes of L. braziliensis.
Specificity: is the ratio between amastigote EC50 and intracellular promastigote EC50
.
Figure 4. Evaluation of phthalazine 3l on intracellular amastigotes of L. braziliensis. Dose-
response curve, BMDM macrophages were infected with promastigotes of L.braziliensis
(1:10) and incubated with 3l for 96 h. % macrophagues total (solid circle line), % infected
macrophages (solid square line) and Nº amastigotes x macrophages (solid triangle line). Each
experiment was carried out by triplicate.
As last topic in this section, a detailed analysis concerning to the antileishmanial activity
against amastigote found for derivatives of phthalazine (our derivative), quinoline [35] and
melanine [36] linked to the nitrofurylhydrazinyl permits us to conclude that the nucleus of
phthalazine and quinoline can represent an important moiety in the specificity on amastigote
form due to their significant response in ranges nanomolar. In this sense, it results very
interesting to explore other mechanism of action related with the system macrophage-
amastigote such as the generation of nitric oxide (NO) (see section mechanism action
studies).

1
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2
.4. Mechanism Action Studies
In order to validate our hypothesized mechanism of actions, the inhibition of the SOD
parasite enzyme and induction of oxidative stress were evaluated preliminarily for the most
active compound 3l. In addition, other two possible targets such as the generation of nitric
oxide from macrophages induced by the action of the drug and the dihidrofolate reductase
inhibition were explored.
The oxidative stress mechanism was evaluated by the measurement of the mitochondrial
dehydrogenase activity on L.braziliensis promastigote in the absence and presence of the
compound 3l. It is well known that oxidative stress has important effect in the mitochondrial
dehydrogenase activity and its measurement at short time permits us to estimate qualitatively
whether an oxidative stress mechanism is generated in the system [41a-c]. Free radical attack
generated by oxidative stress occurs directly at complexes in the mitochondrial respiratory
chain. Specifically, complexes I and III are also thought to be major sites for the production
of superoxide and other reactive oxygen species [41d] and the high reactivity of these species
can damage macromolecules within mitochondria, including lipids, proteins and DNA [41e].
Thus, this mitochondrial DNA damage affects to the complex I and/or III function, inducing
to an increase in the electron reduction of O to superoxide ion [41d, 41f-g]. On these bases,
2
the percentage of mitochondrial dehydrogenase activities (Pmdh) with respect to untreated
control and reference drug was assessed using the colorimetric MTT assay performed at very
short times, between 30 min. and 180 min. of incubation, according to procedure described
for Leishmania parasite [41h]. The results are illustrated in Figure 5. In particular, only the
phthalazine 3l affect the mitochondrial dehydrogenase activity producing a decrease in a
time-dependent manner, while the reference drug and untreated control did not show change
in the mitochondrial dehydrogenase response. This evidence demonstrates the significant
action of the compound 3l on the mentioned enzyme at very short time of incubation, which

1
8
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suggests that the significant reduction of MTT in these times (inclusive 30 min) is induced by
the generation of radical species from metabolized compound 3l and not by cell deaths, due
to that practically no cell death was observed at these short times.
3
l
1
50
00
MILF
Untreated Control
1
50
0
Incubation time (min)
Figure 5. Variation of the percentage of mitochondrial dehydrogenase activities (Pmdh),
produced by the compound 3l exposed to an incubation time, compared to untreated control
and miltefosine (MILF).
In order to explore more about the oxidative stress mechanism and the significant
antiamastigote activity found for the compound 3l, we decided to study an interesting target
such as the generation of nitric oxide induced by action of the phthalazine 3l and analyze its
effect on the parasite stage. In particular, the mitochondrial respiratory chain is particularly
−
−
sensitive to both NO and ONOO mediated damage. Protein oxidation and nitration result in
altered function of many metabolic enzymes in the mitochondrial electron-transport chain,
including nicotinamide adenine dinucleotide dehydrogenase, cytochrome c oxidase, and
adenosine triphosphate synthase [42a]. Some investigations have identified the generation of
reactive nitrogen species as nitric oxide (NO) by macrophage as a cytotoxic molecules
responsible of the intracellular parasite damage [42b-c]. Based in this idea, the correlation

1
9
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between the antiamastigote effect of the compound 3l and the generation of NO by the
macrophage in the infected macrophage system was studied in this section, and the data
obtained are shown in Figure 6. The corresponding PGI (%) values are included in order to
establish a relationship with nitric oxide production. This assay was performed using three
concentration in turn to EC on intracellular amastigote, including 0.08, 0.80 (∼ EC ) and 8.0
50
50
µM for the compound 3l. From Figure 6, it should be noted that the infected macrophages
treated with the compound 3l showed an increase of nitric oxide in the culture compared to
untreated infected macrophage control, being the effect significantly concentration-
dependent. This in vitro evidence indicates that the compound induces the NO production in
the infected macrophage, permitting to explain the significant antiamastigote effect of the
compound 3l. In addition, a direct relationship between the antiamastigote activity and nitrite
production was observed, which suggests that definitively cell amastigote death is associated
to the NO production from infected macrophage. This NO generation may be induced by
phthalazine metabolization into macrophage.
3e-006
2e-006
1e-006
0
Mo infected
* #
&
*
Mo infected + 3l 0.08 µM
Mo infected + 3l 0.8 µM
Mo + 3l 8.0 µM
*
** #
**
Figure 6. Effect of the compound 3l at increasing concentration from 0.8 to 8.0 µM on the
production of nitrite (nitric oxide metabolite) for the system macrophages infected with
Leishmania braziliensis. Mo, untreated macrophages infected. **p<0.01, ***p<0.001
#
compared to untreated macrophages infected. p<0.01 compared to 3l 0.08 µM group.
&
p<0.05 compared to 3l 0.8 µM group.

2
0
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In second place, the effect of 3l was explored on the SOD activity from L.braziliensis
promastigote as described in the literature using xantine oxidase. This assay was evaluated
using three different concentrations in turn to the EC on the promastigote stage, including
5
0
0
.012, 0.12 (EC ) and 1.2 µM of the compound 3l. The results clearly revealed that no
50
changes were observed between the parasites treated with the compound 3l with respect to
untreated control, which permits us to discard this mechanism of action, at least for the
compound 3l on Leishmania parasite.
In order to obtain more information about the Fe-SOD activity of the tested compounds
and confirm the experimental results, a tentative molecular modelling study on their mode of
interaction with the enzyme was performed. In this sense, the closest enzyme available of
SOD in trypanosomatids is the SOD of T.cruzi (PDB ID code: 2GCP), which was taken from
the Protein Data Bank and the molecular docking was evaluated for the most active
derivatives 3c, 3l and 3m, and the potential SOD inhibitors 3i and 3k. In the molecular
docking study, the affinity of the most active derivatives 3l and 3m toward SOD enzyme was
readily poor due to their lower values of docking energy into binding site (6.06 and 7.67,
respectively) (see Figure 7), while the phthalazine 3i and 3k showed a more significant
affinity with the SOD enzyme than the mentioned nitro-derivatives with values of free energy
affinity of 8.90 and 9.73, respectively. These results are completely expected due to that the
electron-deficient character of the nitro-substitution can affect the complexation capacity of
these nitro-derivatives with iron metals. Furthermore, the presence of the hydroxyl and furyl
moieties in the structure of 3i and 3k, respectively, reinforce the potential complexing ability
of these derivatives as a tridentate ligand. This fact permits us to correlate the moderate
biological activity found for the compounds 3i and 3k with the inhibition of the SOD
enzyme. On the other hand, the moderate or poor interaction ligand-SOD into active site for
our phthalazines can be explained by the localization of the phthalazines outside binding site

2
1
ACCEPTED MANUSCRIPT
cavity of the SOD enzyme, which affects therefore the interaction phthalazine-iron metal (see
Figure 7). In order to complement the molecular modelling, the distance between the most
active compounds 3l and 3m and iron metal into binding site was calculated. It showed that
the nitrogen atom N3 of 3l and 3m (nitrogen bonded to C4 of phthalazine nucleus) was
located further away by about 8.21 and 7.90 Aº, respectively. In addition, the phthalazines 3i
and 3k also showed similar distances Fe-ligand by about 7.32 and 8.69 Aº. The compound 3c
did not exhibit poses in the binding site of SOD enzyme. The localization of the tested
phthalazines outside SOD binding site seems to be associated to that the cavity of binding site
is very small with respect to the molecular size of the tested phthalazines. It is very important
2
to explain that the high sp character of the side chains in the studied phthalazines affect its
molecular flexibility, which may thus affects the localization of the ligand (3l or 3m) inside
the active site. In this sense, in the previous experimental and theoretical studies reported for
the benzo[g]phthalazines and phthalazines analogues [27] was found that the monosubstituted
derivatives (minor molecular size) exhibited a better in vitro inhibition of the SOD enzyme
than the disubstituted compounds, which was completely correlated to its significant free
energy affinity ligand-enzyme and proximity ligand-iron metal in the molecular docking.
These theoretical evidences permit us to conclude that some molecular factors such as the
molecular size and molecular flexibility play an important role in the interaction ligand-
enzyme into binding site of the SOD enzyme. In this sense, the high electron-deficient of the
phthalazine-hydrazone 3l (associated to the nitro group) and its little molecular flexibility
may help to explain the no in vitro SOD inhibitory activity found for the compound 3l.
Finally, Table 5 shows the main H-bond and hydrophobic interactions of the compounds 3c,
3
i, 3k, 3l and 3m into binding site of the SOD enzyme.

2
2
ACCEPTED MANUSCRIPT
Table 5. Docking Statistics of the compounds with TR and SOD, and their data of PGI.
a
c
Molecules
SOD
PGI (%)
b
ESOD
H-bond Hydrophobic Interactions
d
3
c
N.F.
-8.90
-9.73
-6.06
-7.67
N.F.
31His
38Lys
NF
N.F.
41.86
64.15
59.14
13.51
11.54
3
i
35Tyr
3
k
31His,35Tyr, 162Trp
31His,35Tyr, 162Trp
31His, 35Tyr
3
l
3
m
35Tyr
a
b
In this studio were used the diasteremers most stable for each derivates.The energy unit is Kcal/mol. Docking
c
energies on Superoxido Dismustase (SOD), Proliferation growth inhibition (PGI) on promastiogotes of L.braziliensis,
d
For the compound 3c no poses were found in active site of SOD enzyme of T.cruzi parasite. N.F.: not found.
Figure 7. Localization of iron metal and phthalazines 3l (yellow colour) and 3m (orange
colour) into binding site of SOD.
Finally, in order to discard another inhibitory action of our promising compound 3l, its
inhibitory effect on an important Leishmania enzyme such as the dihydrofolate reductase
(DHFR) was explored. This enzyme is crucial in the thymine pathway formation that has an
important role in the biosynthesis of ADN of the parasite [25e]. Susceptibility to our
compound and miltefosine, as well as the antifolate drugs methotrexate as positive control,

2
3
ACCEPTED MANUSCRIPT
was assessed in the absence and presence of folinic acid at different concentration 5 nM, 50
nM and 500 nM as described in literature [43], and the data obtained was shown in Figure 8.
Folinic acid is an important substrate of the DHFR enzyme. From Figure 8, it should be noted
that the presence of folinic acid clearly does not have effect on the promastigote growth when
the parasite are treated with the compound 3l and the reference drug miltefosine, while the
parasites treated with the antifolate methotrexate drug showed a significant increase at
increasing concentration of folinic acid. Thus, these evidences permit us to discard
completely to the dihidrofolate reductase inhibition as possible mode of action of the
phthalazine 3l on the antileishmanicidal activity.
100
80
60
40
3l
2
0
MILF
MTX
0
0
100
200
300
400
500
d,l-FNA (nM)
Figure 8. In vitro inhibitory effect of 3l, methotrexate (MTX) and miltefosine (MILT) for
promastigote of L.braziliensis in the absence or presence of d,l-folinic acid (FNA). Results
are presented as the PGI (%) in the absence or presence of increasing concentration of d,l-
FNA. Results correspond to averages of three independent experiments with less than 10%
SD.
In summary, our preliminary in vitro mechanistic assays and in silico studies permit us to
suggest that the therapeutic effect of 3l may be mainly associated to the induction of
oxidative stress generated by potential bio-reduction of nitro group of 3l and the production

2
4
ACCEPTED MANUSCRIPT
of nitric oxide induced in infected macrophage cells. However, additional more specific
assays related to oxidative stress induced by the compound 3l are required for future
investigations in order to know the real nature of this possible mechanism of action. Other
possible mechanism of action such as inhibition of the enzymes SOD and DHFR were
discarded for the most active derivative 3l.
As last topic, employing the software ChemBioDraw Ultra suite [44a], the selected
phthalazines 3a-t were subjected to the Lipinski’s rule of five analysis (drug- likeness) [44b],
which helps to predict and explain biological behaviour of small molecules such as
phthalazine compound synthesized. The result showed that the active derivative followed the
Lipinski rule, with a molecular weight <500 g/mol, a log P <5 around 2,94 and 3,47 for 3l
and 3m, respectively, and these have only 6 or 7 possible interaction of hydrogen (see Table
8
). In particular, the most active derivates 3l and 3m exhibited the lowest Log P, by which
these compounds should present a better passive diffusion than the rest of derivates into cells,
explaining their significant biological response. Therefore, this hypothesis permits to explain
the significant anti-leishmanicidal activity of the compounds 3l and 3m in comparison with
3
c. Finally, the synthesized phthalazines present theoretically a minor solubility at acidic pH
due to the generation of protonated species, which could help to its absorption in the upper
digestive tract for in vivo assays.
Table 6. Calculated theoretical parameters of the Lipinski´s ruler.
Entry Product Molecular Log P
Log P
pH 3.0
3.73
n-OHNH
n-ON
Lipinski´s
Weight
282.73
300.72
327.73
361.62
316.03
327.73
pH 7.4
4.36
4.52
3.89
5.19
4.92
3.89
donors
Acceptors
Violations
1
2
3
4
5
6
3a
3b
3c
3d
3e
3f
1
1
1
1
1
1
4
5
6
4
4
6
0
0
0
0
0
0
3.86
3.69
4.62
4.35
3.69

2
5
ACCEPTED MANUSCRIPT
7
8
9
3g
3h
3i
361.62
316.03
298.73
357.06
272.05
317.03
333.01
312.75
341.75
375.65
341.75
296.75
364.01
364.01
5.19
4.92
3.97
3.82
2.98
1.64
2.20
3.54
3.65
4.75
3.65
3.92
5.04
5.04
4.62
4.35
3.34
3.62
2.41
1.64
2.20
3.30
3.65
4.58
3.65
3.69
4.93
4.93
1
1
2
1
1
1
1
2
1
1
1
1
1
1
4
4
5
7
5
7
6
5
6
4
6
4
4
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
2
3
4
5
6
7
8
9
0
3j
1
1
1
1
1
1
1
1
1
2
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3
. Conclusion
New monoarylhydranizyl-phthalazines 3a-t were synthesized through two step of
reaction and tested as antileishmanial agents against L. braziliensis. Among the tested
compounds, the derivatives 3l and 3m have shown a significant in vitro activity against
promastigotes of L.braziliensis strain at nano and sub-micromolar levels of EC , inclusive
5
0
superior to the drug standard Miltefosine. In particular, the derivative 3l demonstrated to have
low toxicity on macrophage cells with a good selectivity index of 117. This derivative 3l also
exhibited a specific anti-leishmanicidal activity against clinically relevant intracellular
amastigotes of L.braziliensis, without affecting the viability of the host cells. A structure-
activity relationship analysis suggests that a highly conjugated hydrazinyl-nitroheteroaryl
group represents an interesting pharmacophore, being the main responsible of the biological
activity observed for the investigated phthalazines 3a-t. The significant antilesihmanicidal
activity found for the derivative 3l can be explained by an oxidative stress mechanism
induced by metabolized compound 3l into the pathogen. Finally, the results demonstrate that

2
6
ACCEPTED MANUSCRIPT
the compound 3l represents a promising candidate against L.braziliensis and additional assays
related to toxicity on human cell lines such as HEPG2, genotoxicity assays and in vivo
experiments are required to estimate its real potential.
4
. Experimental
4.1. Chemistry
1
Melting points are recorded with a micro melting point apparatus und uncorrected. H
13
NMR, C NMR and NOESY spectra are recorded at JEOL Eclipse Plus 400 and JEOL
Eclipse Plus 270. Proton chemical shifts were given in relative to tetramethylsilane (δ 0.00
ppm) in DMSO-d . Multiplicity is indicated as follows: s (singlet), d (doublet), t (triple), m
6
(multiplet), dd (doublet of doublets), br s (broad singlet), and coupling constant are given in
Hz. Carbon chemical shifts are internally referenced to the deuterated solvent signals in
CDCl (δ 77.00 ppm). Absorption maxima (λmax) was measured in methanol as solvent on a
3
Varian Cary 1E spectrophotometer (Accuracy:± 2 nm). IR spectra were recorded on
NICOLET Magna IR 760 using KBr tablets. Elemental analyses of the synthesized
compounds were performed using a Perkin Elmer 2400 CHN analyser: results fell in the
range of 0.4% of the required theoretical values. Reagents were obtained from Aldrich or
Merck companies, and used without previous purification.
4
.1.1.- Procedure for the synthesis of 1-chloro-4-hydrazinphthalazine (2): This compound
was prepared by a modified procedure previously reported in literature [31a-c]. 1,4-
dichlorophthalazine 1 (2.01 g; 10 mmol) was added to a hot solution of hydrazine hydrate (4
mL, 84 mmol) in 20 mL of ethanol, and the mixture was stirred and heated under reflux for
4
5 minutes. The reaction was cooled at room temperature and the resulting solid was

2
7
ACCEPTED MANUSCRIPT
collected by filtration, washed with cold water (3x20 mL) and dried under vacuum to give a
white solid (1.63 g; 83%).
1
-chloro-4-hydrazin-phthalazine 2. White solid, 1.63 g (yield 83%); m.p. 200 ºC (decomp.)
-1
(
Lit. 200ºC decomp.) [31a,c]. FT-IR (ν, cm ): 3250 (st. N-H), 3164 (st. N-H), 1642 (st.
1
C=N), 1580-1564-1519 (st. C=C). H NMR (400 MHz, DMSO-d ) : δ 8.29 (d, 1H, J=7.7
6
1
3
Hz); 8.10-7.90 (m, 3H), 7.35 (s, 3H, NH-NH ). C NMR (100 MHz, DMSO-d ): δ 133.2
2
6
(
2C); 132.8; 125.3; 125.1 (2C); 123.1 (2C). Anal. Calcd for C H ClN : C, 49.37; H, 3.63; N,
8 7 4
2
8.79. Found: C, 49.17; H, 3.56; N, 28.50.
4
.1.2.- General procedure for the synthesis of 1-(2-benzylidenehydrazyl)-4-
chlorophthalazines 3a-m
These derivatives were prepared following a similar procedure reported in literature [31c].
The corresponding aryl or heteroarylaldehyde (0.6 mmol) was added drop wise to a solution
of 2 (100 mg, 0.5 mmol) in HCl (5 mL, 1M). The mixture was stirred and heated at 60 ºC for
1
5 minutes, cooled at room temperature and neutralized with sodium hydroxide solution
(10%) until to pH 7. The resulting solid was filtered, washed with cold water (3x15 mL) and
dried under vacuum to give yellow or orange solids. In general, products were purified by
recrystallization from ethanol or mixture ethanol/DMF depending of product 3a-m, obtaining
1
in the most case prepared the pure diastereoisomer (E or Z) according to their respective H-
NMR spectra. In a few cases (3c, 3d, 3e, 3f, 3l and 3m), it was obtained a diastereoisomeric
mixture and further purifications by recrystallization and chromatography using different
solvent mixture did not permit to separate the two diastereoisomers. These cases were
reported as a mixture (E/Z) and the proportion of the isomers was determined taking into
account the differences of intensity between the ylidene proton signal around 8.47-8.58 ppm

2
8
ACCEPTED MANUSCRIPT
1
corresponding to the E-isomer and the signal appeared to 8.75-8.85 of the Z-isomer in H-
NMR spectra.
4
.1.2.1.- 1-(2-benzylidenehydrazyl)-4-chlorophthalazine 3a. Recrystallization solvent
ethanol. Yellow solid, 141 mg (yield 96%), 100% E-diastereoisomer, m.p. 173-174 ºC (Lit.
-
1
1
1
1
75-176ºC) [31c]. FT-IR (ν, cm ): 3391 (st. N-H), 2920-2890 (st. C-H), 1618 (st. C=N),
1
578-1562-1537 (st. C=C). H NMR (400 MHz, DMSO-d ): δ 12.21 (s, 1H, N-H); 8.47 (s,
6
1
3
H, N=C-H); 8.37 (d, 1H, J=5.8 Hz); 8.00 (d, 2H, J=4.8 Hz); 7.88 (m, 3H); 7.43 (m, 3H). C
NMR (100 MHz, DMSO-d ): δ 150.3; 143.7; 136.1; 132.6; 131.2; 129.2; 129.5 (2C); 128.2
6
(
2C); 128.0; 126.1; 123.9; 123.5; 122.4. DEPT-135 (100 MHz, DMSO-d ): δ 136.0; 132.6;
6
1
3
31.2; 129.5 (2C); 128.2 (2C); 128.0; 123.5; 122.4. Anal. Calcd for C H ClN : C, 63.72; H,
15 11 4
.92; N, 19.82. Found: C, 63.66; H, 3.85; N, 19.73.
4
.1.2.2.- 1-(2-(4-fluorobenzylidene)hydrazyl)-4-chlorophthalazine 3b. Recrystallization
solvent ethanol. Yellow solid, 131 mg (yield 92%), 100% E-diastereoisomer, m.p. 184-186
-1
ºC. FT-IR (ν, cm ): 3392 (st. N-H), 2920-2890 (st. C-H), 1619 (st. C=N), 1585-1525 (st.
1
C=C). H NMR (400 MHz, J Hz, DMSO-d ): δ 12.22 (s, 1H, N-H); 8.46 (s, 1H, N=C-H);
6
8
.36 (d, 1H, J=8.0 Hz); 8.07 (dd, 2H, J =8.8 Hz; J =8.8 Hz); 7.87 (m, 3H); 7,27 (dd, 2H,
1 2
1
3
J =8.8 Hz; J =9.2 Hz). C NMR (100 MHz, J Hz, DMSO-d ): δ 165.1; 162.6; 150.3; 147.6;
1
2
6
1
1
43.6; 136.1; 132.5; 130.6 (JC-C-C-F=8.1 Hz) (2C); 128.0; 123.5; 123,2 (J =170.2 Hz);
C-F
22.7; 116.6 (J =22.83) (2C). DEPT-135 (100 MHz, DMSO-d ): δ 136.1; 132.5; 130.6
C-F
6
1
9
(
2C); 128.0; 123.5; 123.2; 116.6 (2C). F NMR (70 MHz, DMSO-d ): δ -109.35. Anal.
6
Calcd for C H ClFN : C, 59.91; H, 3.35; N, 18.63. Found: C, 59.84; H, 3.30; N, 18.57.
1
5
10
4
4
.1.2.3.- 1-(2-(4-nitrobenzylidene)hydrazyl)-4-chlorophthalazine 3c. Recrystallization
solvent: ethanol/DMF (8/2). Orange solid, 154 mg (yield 94%), isomer mixture (E/Z) (94/6),
-1
m.p. 255-257 ºC. FT-IR (ν, cm ): 3400 (st. N-H), 2920-2890 (st. C-H), 1626 (st. C=N),