Structural design, synthesis and substituent effect of hydrazone-N-acylhydrazones reveal potent immunomodulatory agents

Article abstract of DOI:10.1016/j.bmc.2018.02.047

4-(Nitrophenyl)hydrazone derivatives of N-acylhydrazone were synthesized and screened for suppress lymphocyte proliferation and nitrite inhibition in macrophages. Compared to an unsubstituted N-acylhydrazone, active compounds were identified within initial series when hydroxyl, chloride and nitro substituents were employed. Structure-activity relationship was further developed by varying the position of these substituents as well as attaching structurally-related substituents. Changing substituent position revealed a more promising compound series of anti-inflammatory agents. In contrast, an N-methyl group appended to the 4-(nitrophenyl)hydrazone moiety reduced activity. Anti-inflammatory activity of compounds is achieved by modulating IL-1β secretion and prostaglandin E2 synthesis in macrophages and by inhibiting calcineurin phosphatase activity in lymphocytes. Compound SintMed65 was advanced into an acute model of peritonitis in mice, where it inhibited the neutrophil infiltration after being orally administered. In summary, we demonstrated in great details the structural requirements and the underlying mechanism for anti-inflammatory activity of a new family of hydrazone-N-acylhydrazone, which may represent a valuable medicinal chemistry direction for the anti-inflammatory drug development in general.

Full text of DOI:10.1016/j.bmc.2018.02.047

Accepted Manuscript
Structural design, synthesis and substituent effect of hydrazone-N-acylhydra-
zones reveal potent immunomodulatory agents
Cássio S. Meira, José Maurício dos Santos Filho, Caroline C. Sousa, Pâmela S.
Anjos, Jéssica V. Cerqueira, Humberto A. Dias Neto, Rafael G. da Silveira,
Helena M. Russo, Jean-Luc Wolfender, Emerson F. Queiroz, Diogo R.M.
Moreira, Milena B.P. Soares
PII:
S0968-0896(18)30050-6
https://doi.org/10.1016/j.bmc.2018.02.047
BMC 14233
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
11 January 2018
16 February 2018
23 February 2018
Please cite this article as: Meira, C.S., Filho, J.M.d., Sousa, C.C., Anjos, P.S., Cerqueira, J.V., Neto, A.D., da Silveira,
R.G., Russo, H.M., Wolfender, J-L., Queiroz, E.F., Moreira, D.R.M., Soares, M.B.P., Structural design, synthesis
and substituent effect of hydrazone-N-acylhydrazones reveal potent immunomodulatory agents, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.047
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Structural design, synthesis and substituent effect of hydrazone-
N-acylhydrazones reveal potent immunomodulatory agents
a
b
a
Cássio S. Meira , José Maurício dos Santos Filho , Caroline C. Sousa , Pâmela S. Anjos
a
a
b
c
,
Jéssica V. Cerqueira , Humberto A. Dias Neto , Rafael G. da Silveira , Helena M.
d
d
d
a
Russo , Jean-Luc Wolfender , Emerson F. Queiroz , Diogo R. M. Moreira , Milena B.
a,e*
P. Soares
a
ꢀ
ꢁꢂꢃꢄꢅꢆꢇꢀꢈꢉꢊꢋꢊꢌꢊꢍꢇꢎꢍꢈꢏꢐꢑꢍꢇꢒꢍꢈꢋꢓ, 40296-710, Salvador, BA, Brazil.
b
UFPE, Centro de Tecnologia e Geociências, Laboratory of Design and Synthesis Applied to
®
Medicinal Chemistry-SintMed , CEP 50740-521 Recife, PE, Brazil.
c
IFG, Campus Ceres, CEP 76300-000, Ceres, GO, Brazil.
d
School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne,
CMU –Rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland.
e
ꢂꢔꢈꢊꢕꢍꢇꢖꢔꢇꢗꢋꢍꢊꢔꢘꢈꢍꢑꢍꢙꢋꢐꢇꢔꢇꢚꢔꢕꢐꢛꢋꢐꢇꢂꢔꢑꢌꢑꢐꢕꢆꢇꢜꢍꢉꢛꢋꢊꢐꢑꢇꢝꢞꢍꢇꢃꢐꢟꢐꢔꢑ, 41253-190, Salvador, BA,
Brazil.
ꢠꢂꢍꢕꢕꢔꢉꢛꢍꢈꢖꢋꢈꢙꢇꢐꢌꢊꢡꢍꢕꢢꢇ ꢀꢁꢂꢃꢄꢅꢆꢇꢀꢈꢉꢊꢋꢊꢌꢊꢍꢇꢎꢍꢈꢏꢐꢑꢍꢇꢒꢍꢈꢋꢓ, CEP 40296-710, Salvador, BA,
Brazil. (+55)71-31762292; email: milena@bahia.fiocruz.br

Abstract
4
-(Nitrophenyl)hydrazone derivatives of N-acylhydrazone were synthesized and screened for
suppress lymphocyte proliferation and nitrite inhibition in macrophages. Compared to an
unsubstituted N-acylhydrazone, active compounds were identified within initial series when
hydroxyl, chloride and nitro substituents were employed. Structure-activity relationship was
further developed by varying the position of these substituents as well as attaching
structurally-related substituents. Changing substituent position revealed a more promising
compound series of antiinflammatory agents. In contrast, an N-methyl group appended to the
4
-(nitrophenyl)hydrazone moiety reduced activity. Antiinflammatory activity of compounds
is achieved by modulating IL-1βꢇꢉꢔꢘꢕꢔꢊꢋꢍꢈꢇꢐꢈꢖꢇꢛꢕꢍꢉꢊꢐꢙꢑꢐꢈꢖꢋꢈꢇE2ꢇꢉyꢈꢊꢡꢔꢉꢋꢉꢇꢋꢈꢇmꢐꢘꢕꢍꢛꢡꢐꢙꢔꢉꢇ
and by inhibiting calcineurin phosphatase activity in lymphocytes. Compound SintMed65
was advanced into an acute model of peritonitis in mice, where it inhibited the neutrophil
infiltration after being orally administered. In summary, we demonstrated in great details the
structural requirements and the underlying mechanism for antiinflammatory activity of a new
family of hydrazone-N-acylhydrazone, which may represent a valuable medicinal chemistry
direction for the antiinflammatory drug development in general.
Keywords: Anti-inflammatory agents, hydrazones, methylation, peritonitis, substituent
effects.

1
. Introduction
Inflammatory responses are vital physiological processes in combating infections, as
well as in recovering injury caused by foreign bodies and tissue damage; however,
dysregulated or prolonged inflammatory responses can also induce tissue injury, giving rise
[1,2]
to a number of inflammatory diseases.
In response to an extracellular stimulus,
macrophages can produce proinflammatory mediators, such as nitric oxide (NO) controlled
by inducible nitric oxide synthase (iNOS), which is highly expressed upon activation of
nuclear factor-κꢗꢇ(N -κꢗ)in response to many stimuli, including tumor necrosis factor alpha
[3]
(
ꢚN α) and interferon-ꢙꢐmmꢐꢇ(ꢀ Nγ), among others. In addition, during an inflammatory
response, activated macrophages overexpress cyclooxygenases (COX1 and COX2), which
are key enzymes responsible for the conversion of arachidonic acid into prostaglandin E2
(
PGE2), a proinflammatory mediator involved in all processes leading to the classic signs of
[4]
inflammation: redness, swelling and pain. Therefore, chemical inhibition of COX and iNOS
can be useful in the management of inflammatory diseases.
The N-acylhydrazone group is a unique moiety very often employed into heterocyclic
compounds drug design, which endow molecules with a better thermal stability as well as
[
5-7]
hydrolytic and chemical stability in comparison to amide group , in addition to the
[
8-11]
feasibility of varying chemical composition by substituent variation.
Among the
pharmacological effects of N-acylhydrazone-derived compounds, their anticancer and anti-
[
12,13]
inflammatory effects are well described.
More specifically, N-acylhydrazone derivatives
[14-16]
[17]
have been described as potential inhibitors of COX
, iNOS
and phosphodiesterases.
[
18,19]
Lengthening the alkyl chain in the chemical structure of N-acylhydrazone-derived
compounds has been identified as a strategy for drug design and optimization. This can be

achieved by inserting alkyl and alkene groups, such as the naturally-occurring cinnamic
moiety. Hence, this strategy results in a plethora of diverse structures, along with interesting
[20-24]
features, i.e., planar structure, larger steric bulk and hydrophobic character.
On the basis
of these reports, the introduction of a side chain similar to the cinnamic moiety associated to
the N-acylhydrazone functionality depicts a potential strategy for drug design. In an effort to
develop novel anti-inflammatory agents, our research group envisaged the introduction of a
4
-(nitrophenyl)hydrazone group on aryl-N-acylhydrazones, giving a class of hydrazone-N-
acylhydrazones (HAH), where a nitro group was placed to enhance the electron-withdrawing
[
25,26]
characteristics (Figure 1).
Figure 1: Design concept for the hydrazone-N-acylhydrazones (HAH) series.
Once the 4-(nitrophenyl)hydrazone derivatives of N-acylhydrazone were designed, an
efficient synthetic route was implemented ꢍꢈꢇꢊꢡꢔꢇꢙꢕꢍꢌꢈꢖꢉꢇꢍꢟꢇꢊꢡꢔꢇꢛꢕꢍbꢑꢔm’ꢉꢇꢕꢔꢊꢕꢍꢉyꢈꢊꢡꢔꢊꢋꢘꢇ
analysis (Scheme 1), leading to a series of novel compounds, which was studied by screening
substituents attached to the aryl ring located near to the acyl group (Scheme 2). A set of
para-substituents was selected by physical-ꢘꢡꢔmꢋꢘꢐꢑꢇꢘꢕꢋꢊꢔꢕꢋꢐꢢꢇσ-Hammett constant, ranging
p
from -0.83 for N,N-dimethylamino to +0.78 for nitro, lipophilicity and hydrogen bond
acceptor/donor, allowing us to study the substituent effect on the activity. Encouraged by the
initial screening, structure-activity relationship (SAR) for active compounds was further
developed by varying the substituent position and employing structurally-related substituents.

In addition to studying in great details the substituent effect of N-acylhydrazones for anti-
inflammatory activity, we demonstrated the underlying mechanism of action as well as the
efficacy of the most active compound, denoted here as SintMed65, in an acute peritonitis
model of inflammation in mice.
2. Results and Discussion
2
.1. Chemistry
Once established the chemical template for the compounds to undergo a thorough
anti-inflammatory investigation, our research group examined possible synthetic routes that
could give access to them as expeditious as possible. The retrosynthetic analysis depicted in
Scheme 1 arises as an excellent option, since it leads to accessible starting materials, consists
of well-established reactions and can be achieved using current methodologies developed at
®
SintMed . This approach starts with the disconnection of the N-acyl-hydrazone moiety, an
almost conspicuous strategy, since it originates an arylhydrazide, the usual substrate for N-
acylhydrazone formation, whose synthesis can easily be accomplished in two steps from
commercial benzoic acids. Taking this path, the other constituent molecule is a 4-nitrophenyl
ketohydrazone, whose preparation can be reached out by applying a Japp-Klingermann
reaction involving 4-nitrophenyl diazonium salt and acetoacetic acid as reactants. Bottom
line, all requirements to a fast and successful accomplishment of the planned work were
completely fulfilled.

Scheme 1: Retrosynthetic strategy for the hydrazone-N-acylhydrazones (HAH) synthesis.
Based on this general strategy, the preparation of the targeted hydrazone-N-
acylhydrazone compounds SintMed(54-74) followed the synthetic route shown in Scheme 2.

Scheme 2: Synthetic route for the hydrazone-N-acylhydrazones (HAH) preparation.
First, the diazotization of the 4-nitroaniline 1 provided the corresponding diazonium
salt 2, which reacted with the in situ generated acetoacetic acid 4 from the hydrolysis of its
analogous ethyl acetoacetate 3, as required by the Japp-Klingermann methodology. The 4-
nitrophenyl ketohydrazone 5 was isolated in 95% yield. The arylhydrazides of interest could
easily be prepared from commercial acids, whose refluxing Fisher esterification was carried
out in methanol and sulfuric acid as catalyst. The methyl substituted aryl carboxylates were
then converted into hydrazides by reaction with hydrazine hydrate at 70 °C, according to
®
[8,9]
current methodologies in use at SintMed .
The condensation between the aryl hydrazides and the 4-nitrophenyl ketohydrazone 5
was accomplished under mild conditions in the presence of cerium(III) chloride heptahydrate
®
[8]
as catalyst. This methodology was recently developed at SintMed
, and is essential to the
excellent outcomes observed for the HAH series, since the compound 5 is a vinylogous-like

ketone, which does not withstand traditional acid conditions. Reacting both substrates at 65
C in the presence of 10 mol % CeCl ·7H O and ethanol, total conversion was observed in
°
3
2
two hours without any decomposition or side products, as confirmed by TLC, and NMR
analysis of crude products. More remarkable is the stereoselectivity observed from the NMR
examination of the crude products, which indicates the presence exclusively of the E-isomer.
[8,9]
As previously demonstrated,
the presence of isomeric mixtures of any nature leads to
1
signal splitting on the ꢜꢇNꢒꢃꢇꢉꢛꢔꢘꢊꢕꢐꢆꢇꢛꢐꢕꢊꢋꢘꢌꢑꢐꢕꢑyꢇꢔꢐꢉyꢇꢊꢍꢇbꢔꢇꢍbꢉꢔꢕvꢔꢖꢇꢟꢍꢕꢇꢊꢡꢔꢇꢡyꢖꢕꢐꢓꢍꢈꢔ’ꢉꢇ
amino hydrogen (NHN), ꢊꢡꢔꢇꢡyꢖꢕꢐꢓꢍꢈꢔ’ꢉꢇꢋmꢋꢈꢍꢇ(NNCH), the amido group
(
CONH), and the methyl linked to the imino portion (NCCH ) of the NAH. Most
3
compounds of the series HAH exhibited only one signal for each of the mentioned groups,
confirming the more stable E-isomer. Typical signals appear at 11.5 ppm (NHN), 10.7
ppm (CONH), 7.74 ppm (NNCH), and 2.27 ppm (NCCH ) (See Supporting
3
Information). Nevertheless, in some cases, e.g. ortho-substituted, and strong electron
withdrawing groups bearing derivatives, the signal splitting can be observed. This particular
[27,28]
behaviour was investigated by Barreiro et al.
, who found out that it is due to the
rotamery associated to specific structural features of such molecules, a conclusion in
[8,9]
complete accordance with our previous reports.
The expected stereochemical features of
the HAH derivatives could be confirmed by crystallographic data collected for compound
SintMed65 (Figure 2), which is an all-E,E-planar structure, held by the electronic
delocalization through the molecule. This result also supports the rotameric nature of the
signal splitting observed in some cases, including SintMed65.

Figure 2: ORTEP plot of X-ray crystal structure for SINTMED65 compound. Atomic
displacement parameters are shown at 50 % probability level.
Due to the outstanding biological results obtained with the HAH series, the key
derivatives had their structures modified through the nitrogen methylation of the hydrazone
(
NHN) moiety, providing compounds SintMed75-77. In order to avoid a possible double
methylation of the targeted molecules, a secondary synthetic route was applied, so that the 4-
nitrophenyl ketohydrazone 5 was methylated in the presence of methyl iodide and potassium
carbonate in acetone to give compound 6 in 94 % yield, prior to undergoing the condensation
with the appropriate hydrazides (Scheme 3).
Scheme 3: Synthetic route for the N-methylated hydrazone-N-acylhydrazones.

2
.2. Pharmacological evaluation
Nitric oxide production was estimated by measuring nitrite concentrations by the
Griess method in the supernatants of J774 macrophages cultures 24 h after activation with
LPS and IFN. The percentage of inhibition was determined at 20 µM compound
concentration as a primary screen, and compounds with % of inhibition > 30 % were
considered active (Table 1). Assessment of cell viability was carried out by Alamarblue 72 h
after drug exposure. Based on CC50 values, compounds were not cytotoxic at 20 µM.
Table 1: In vitro screening on cell viability, lymphocyte proliferation and nitric oxide
inhibition.
O
H
N
N
N
N
Ar
H
O N
2
Inhibition (%) of
nitrite
production at 20 proliferation
bµM (MeanS.D.) (IC50 S.E.M., µM)
a
Cell viability
Lymphocyte c
Ar / Codes
(CC S.E.M.,
5
0
µM)
>
>
100
31.4±2
>12
SintMed54
100
7.5±0.2
18.7±5
>12
>12
H3CO
SintMed55
7
8.7±6.2
F3C
SintMed56

>
100
4.8±3
>12
>12
>12
SintMed57
7
5±8
12.8±1
25.6±3
H2N
SintMed58
>100
N
SintMed59
6
5
3.9±5.0
1±1.5
23.0±3
43.8±1
39.1±7
4.2±0.2
Cl
SintMed60
1.51±0.007
0.98±0.03
HO
SintMed61
>100
O N
2
SintMed62
>
100
6.6±0.9
23.2±5
5.1±0.9
>12
HO
OCH3
SintMed63
O
>100
O
SintMed64

O2N
1
7.8±3.1
75.3±0.5
21.9±2
0.27± 0.02
6.0±0.5
>12
NO2
SintMed65
>
100
CH3
SintMed66
>
100
100
9.3±0.9
CH3
SintMed67
>
5.3±0.1
39.4±1.2
56.8±2
59.7±4
>12
F
Cl
I
SintMed68
SintMed69
SintMed70
SintMed71
>
100
>12
4
2
0±4.1
2.98±0.5
1.64±0.01
9.5±1.3
OH
>100
5.5±3
>12
O
SintMed72
2
9.8±2.9
39.3±0.5
4.9±0.5
NH2

SintMed73
N.D.
>100
14.1±0.2
N
H
SintMed74
Dexa
91.1 ±1.9
4.1±0.05
69.8±2
-
0.001±0.0005
GV
-
a
b
J774 cell viability assessed by Alamarblue 72 h after drug incubation and expressed as CC50. Percent inhibition
c
determined 24 h after incubation with compounds and LPS plus IFN-γ. Proliferation of lymphocytes induced by
concanavalin A (con A), determined by thymidine uptake 72 h after drug incubation and expressed as IC50 Values
represents the mean±S.E.M. and were calculated from three independent experiments performed. Abbreviations: IC50
=
inhibitory concentration at 50%; CC50 = cytotoxic concentration at 50%; Dexa = Dexamethasone; GV = gentian
violet; S.E.M. = standard error of mean.
Unsubstituted hydrazone-N-acylhydrazone SintMed54 inhibited nitric oxide
production. From the screening of 4-substituted HAH derivatives, in comparison to the
unsubstituted compound, we observed that attachment of methoxy (SintMed55),
trifluoromethyl (SintMed56), tert-butyl (SintMed57), amino (SintMed58), N,N-
dimethylamino (SintMed59) or chlorine (SintMed60) substituents diminished the activity,
with a remarkably deleterious effect exerted by methoxy and tert-butyl groups, both bearing
potent electron donating effect and no ability of donating or/and accepting hydrogen bonds.
On the other hand, hydroxy (SintMed61) and nitro (SintMed62) substituents increased the
activity, setting the general parameters ruling the influence of substituents on the biological
activity. On the basis of these findings, we decided to expand the series of related
compounds, exploiting ꢖꢋvꢔꢕꢉꢔꢇꢔꢑꢔꢘꢊꢕꢍꢈꢋꢘꢇꢛꢕꢍꢛꢔꢕꢊꢋꢔꢉꢆꢇꢐꢉꢇwꢔꢑꢑꢇꢐꢉꢇꢕꢋꢈꢙꢇꢛꢍꢉꢋꢊꢋꢍꢈꢉ’ꢇmꢍꢖꢋꢟꢋꢘꢐꢊꢋꢍꢈꢉ.ꢇ
The most promising compound from the initial series was the 4-hydroxyphenyl-
derivative SintMed61 (43.8±1 % of inhibition). Through the preparation and screening of the
closely related analogues of this structure, SintMed63 (3-methoxy-4-hydroxyphenyl
substituted) and SintMed64 (1,3-dioxole substituted), it became obvious that variation
around the 4-hydroxy group is not tolerated, since the activity decreased for both associated
compounds. In another attempt to improve activity, variation to the 2-position of the phenyl

ring was then explored, affording 2-hydroxyphenyl (SintMed71) and 2-phenoxyphenyl
SintMed72) compounds. Changing the position of the hydroxy group did not abolish
(
activity; in fact, compound SintMed71 was found to be more potent than SintMed61, while
the 2-phenoxy (SintMed64) compound was twice less active. Considering that both phenoxy
and methoxy substituents decreased activity in comparison to the hydroxy-derived
compounds, this suggests that a hydrogen bond donor substituent is required for activity.
Due to the promising results when a substituent was attached at the 2-position, we
carried out the screening of novel substituents. We observed that o-tolyl (SintMed66) or m-
tolyl (SintMed67) compounds did not lead to active compounds. Albeit weak, the o-amino
(
SintMed73) compound inhibited in approximately twice the nitric oxide production in
comparison to the p-amino (SintMed58) compound. Replacing the o-amino by an o-
phenylamino (SintMed74) substituent, however, caused a marked loss in activity, indicating
that limiting hydrogen-bond acceptor potential is deleterious for the activity. Screening
halogen substituents led to a more interesting set of SAR information. Whereas 4-chloro
derivative displayed weak effect, the 2-chloro derivative (SintMed69) was active. In order to
exploit the effect of the electronic properties of different halogens, the 2-position was
modified, introducing a fluorine (SintMed68), disclosing an inactive compound. On the other
hand, the change of a 2-chloro for a 2-iodine substituent (SintMed70) produced a potent
compound, demonstrating an inhibition value for nitric oxide production close to that of 2-
hydroxyphenyl derivative SintMed71. These results suggest a crucial ortho-substituent effect
for the activity of compounds. Extending our screening based on the observed result for the
4
-nitrophenyl derivative SintMed62 (39.1±7 of inhibition), we designed compound
SintMed65, whose structure bears two nitro groups at the positions 3 and 5 of the phenyl
ring, a modification that disclosed the most potent derivative among all investigated

molecules (73.5±0.5% of nitric oxide inhibition). This evidenced the role of the nitro group in
the activity.
As noted above with nitrophenyl compounds, electron-withdrawing substituents
would not be indispensable for activity. When other electron-withdrawing substituents were
attached, the resulting 4-trifluoromethylphenyl compound SintMed56 and 2-fluorophenyl
compound SintMed68 were inactive. We first thought that a bioreduction of nitro into amino
species may take place, being responsible for compound activity; however, a 4-amino
compound lacks activity. One plausible explanation is that biological reduction of nitro group
generates an N-(hydroxy)amino species; in agreement to this, we have the fact that hydroxy
substituents confer activity for compounds. Considering the weak activity observed for the
unsubstituted compound (which also contains a 4-nitro substituent at phenylhydrazone
moiety), the position of nitro group is crucial for activity.
We next assessed the ability of compounds in inhibiting lymphocyte proliferation
upon activation by concanavalin A (Con A). Compounds with IC50 values < 3.0 µM were
considered active (Table 1). Unsubstituted compound SintMed54 did not inhibit lymphocyte
proliferation. From the screening of 4-substituted compounds, we observed that, in
comparison to unsubstituted compound, attaching a hydroxy (SintMed61) or a nitro
(
SintMed62) substituent produced active compounds, while the attachment of other
substituents did not lead to active compounds. Indeed, both 4-nitro (SintMed62) and 3,5-
dinitro (SintMed65) compounds were the most active inhibitors of lymphocyte proliferation.
From the screening of 2-substituted compounds, we found that attaching a hydroxy
(
SintMed71) or iodine (SintMed70) substituents resulted in active compounds. In general,
the most potent nitric oxide inhibitors also presented antiproliferative activity in lymphocytes.
It was interesting to observe that, while o-hydroxyl (SintMed71) or o-iodine (SintMed70)
derived compounds presented similar inhibitory activity in reducing nitric oxide production,

hydroxyphenyl-derived compounds (SintMed61, SintMed71) were approximately twice
more potent than iodine (SintMed70) compound. This indicates that hydrogen bond donor
substituents have a superior effect on activity than halogen substituents.
To gain insights regarding the 4-(nitrophenyl)hydrazone moiety for activity, we
investigated the effect of N-methylation. To this end, we selected the two most active
compounds (SintMed65, SintMed71) and one inactive compound (SintMed58) for
modification. The synthesis of the respective N-methyl derived compounds (Scheme 3) was
of high-yields, affording pure compounds after recrystallization in appropriate solvent. The
results, summarized in Table 2, show that in contrast to non-methylated compounds,
insertion of a methyl group did not increase nitric oxide inhibitory activity. Regarding IC50
for lymphocyte proliferation, we observed a 2- to 3-fold reduction in the activity. While this
result indicates the importance of a free hydrazone group for activity, it also points out that
the aryl-acylhydrazone moiety is sensitive for molecular modification, possible because it
represents a pharmacophoric group.
Table 2: Comparison of activity between HAH and its N-methyled derivatives.
H
N
O
CH3
N
O
N-methylation
N
N
N
N
Ar
N
N
Ar
H
H
O2N
SintMed (58, 65, 71)
2
O N
SintMed (75-77)
a
Inhibition (%) of
nitrite production
Cell viability
c
Lymphocyte proliferation
Ar / Codes
(CC S.E.M.,
5
0
at 20 µM
(IC S.E.M., µM)
5
0
µM)
b
(
MeanS.D.)
5
1.7±4.0
3.6±2
>12
H2N
SintMed75

O2N
3
3
9.0±3.1
1.5±0.9
51.0±2.5
41.0±2
1.11±0.07
NO2
SintMed76
3.12±0.4
OH
SintMed77
a,b,c
See footnote in Table 1.
SintMed65 was the most active compound in inhibiting nitric oxide production by
macrophages, as well as in inhibiting lymphocyte proliferation, and therefore it was selected
to evaluate its mechanisms of action. To exclude that SintMed65 effects were related to
cytotoxic effects, in the next experiments we evaluated it immunomodulatory effect in
concentrations up to 12 uM, that showed no toxicity in macrophages and lymphocytes cells
(
data not shown).
In the presence of SintMed65 (2.5–10 µM), a significant and concentration-
dependent inhibition of nitric oxide production was observed (Figure 3A). Furthermore, we
ꢔvꢐꢑꢌꢐꢊꢔꢖꢇꢋꢊꢉꢇꢔꢟꢟꢔꢘꢊꢇꢍꢈꢇꢊꢡꢔꢇꢉyꢈꢊꢡꢔꢉꢋꢉꢇꢍꢟꢇꢛꢕꢍꢋꢈꢟꢑꢐmmꢐꢊꢍꢕyꢇꢘyꢊꢍkꢋꢈꢔꢉꢇꢚN αꢆꢇꢀL-1βꢇꢐꢈꢖꢇꢀL-6
(
Figure 3). A significant and concentration-dependent inhibition of IL-1βꢇꢐꢈꢖꢇꢀL-6 was
ꢍbꢉꢔꢕvꢔꢖꢆꢇwꢡꢋꢑꢔꢇꢟꢍꢕꢇꢚN αꢆꢇSintMed65 compound only inhibited its secretion when added at
1
0 µM. In various cell types, IL-1βꢇꢋꢉꢇꢐꢇꢛꢍꢊꢔꢈꢊꢇꢋꢈꢖꢌꢘꢔꢕꢇꢍꢟꢇꢂꢁX2ꢇꢔxꢛꢕꢔꢉꢉꢋꢍꢈ, an enzyme that
[
29,30]
controls the biosynthesis of PGE2, an important inflammatory mediator.
Therefore,
considering that IL-1βꢇꢉꢔꢘꢕꢔꢊꢋꢍꢈꢇwas inhibited by SintMed65, we decided to investigate this
pathway. PGE2 production and COX2 gene transcription were evaluated in LPS-activated
[31]
J774 cells, according to standard protocols. As observed in Figure 3, SintMed65 reduced
COX2 expression and PGE2 biosynthesis in a concentration-dependent manner.

A
D
B
C
8
0
5000
2000
1500
1000
500
0
#
4000
#
60
#
#*
3000
#*
40
#*
#*
2000
#*
20
#*
1000
*
*
*
*
C-
*
0
*
0
C-
DEXA
10
5.0
2.5
C-
C-
DEXA
10
5,0
2,5
C-
C-
DEXA
10
5.0
2.5
SintMed65 (M)
LPS + IFN-
SintMed65 (M)
LPS + IFN-
SintMed65 (M)
LPS + IFN-
E
F
8
00
2.5
0.8
0.6
0.4
0.2
0.0
2.0
#
600
#*
#*
1.5
#*
400
#*
#*
#*
#*
#*
#*
1.0
#*
200
0.5
*
*
*
*
*
C-
0
0.0
C-
DEXA
10
5.0
2.5
C-
C-
INDO
20
10
2.5
C-
C-
DEXA
20
10
2.5
SintMed65 (M)
LPS + IFN-
SintMed65 (M)
LPS
SintMed65 (M)
LPS
Figure 3: Immunomodulatory effects of N-acylhydrazone SintMed65 on pro-inflammatory
mediators. (A-D) Peritoneal macrophages of BALB/c mouse were stimulated in vitro with
LPꢝꢇ(0.5ꢇμꢙ/mL)ꢇꢐꢈꢖꢇꢀ N- (10 ng/mL) and treated with different concentrations of
SintMed65 (10ꢆꢇ5ꢇꢐꢈꢖꢇ2.5ꢇμꢒ)ꢇꢍꢕꢇꢖꢔxꢐmꢔꢊꢡꢐꢉꢍꢈꢔꢇ(DEXA;ꢇ10ꢇμꢒ). Supernatants were
collected for nitrite quantification by Griess method and cytokines by ELISA. (E and F) J774
mꢐꢘꢕꢍꢛꢡꢐꢙꢔꢉꢇwꢔꢕꢔꢇꢉꢊꢋmꢌꢑꢐꢊꢔꢖꢇwꢋꢊꢡꢇLPꢝꢇ(0.1ꢇμꢙ/mL)ꢇꢐꢈꢖꢇꢊꢕꢔꢐꢊꢔꢖꢇwꢋꢊꢡꢇꢖꢋꢟꢟꢔꢕꢔꢈꢊꢇꢘꢍꢈꢘꢔꢈꢊꢕꢐꢊꢋꢍꢈꢉꢇ
of SintMed65 (10ꢆꢇ5ꢇꢐꢈꢖꢇ2.5ꢇμꢒ)ꢇꢍꢕꢇDEXAꢇ(10ꢇμꢒ)ꢇꢍꢕꢇꢀꢈꢖꢍmꢔꢊꢡꢐꢘꢋꢈꢇ(ꢀNDꢁ;ꢇ20ꢇμꢒ).ꢇPꢎE2ꢇ
determination was done by enzyme immunoassay (EIA) and COX2 gene expression by qPCR
2
4 h after challenge. Values represent the mean±S.E.M. of one of two experiments
performed. One-way ANOVA and Newman-Keuls test: *P< 0.05 compared to stimulated and
untreated cells; #P < 0.05 compared to dexamethasone-treated cells or indomethacin-treated
cells.
Next, we evaluated the underlying effects of SintMed65 compound on lymphocyte
proliferation induced by Con A or anti-CD3/anti-CD28 (Figure 4).

B
A
60000
30000
#
#
4
0000
20000
10000
0
#*
#*
20000
*
*
*
#*
*
#*
0
C-
C- DEXA 2.5
0.6
0.15
C-
C- DEXA 2.5
0.6
0.15
SintMed65 (M)
SintMed65 (M)
conA
anti-CD3/anti-CD28
D
C
100
*
3
*
*
80
60
40
20
0
2
1
0
#*
*
*
*
*
C-
10
5
2.5
C-
C-
CsA
12
4
1
SintMed65 (M)
SintMed65 (M)
conA
Figure 4: N-acylhydrazone SintMed65 inhibits lymphocyte proliferation by arresting cell
cycle and inhibiting calcineurin activity. Proliferation of lymphocytes stimulated with Con A
(
panel A) or anti-CD3/anti-CD28 beads (panel B). Proliferation rates were assessed through
H-thymidine incorporation. Lymphocytes stimulated with conA, incubated for 48 h, stained
3
for PI (2 µg/ml) and analyzed by flow cytometry (panel C). Cellular calcineurin activity in
lymphocytes determined by colorimetric assay (panel D). Error bars represent the
meanꢝ.E.ꢒ.ꢇDꢔxꢐꢇ=ꢇDꢔxꢐmꢔꢊꢡꢐꢉꢍꢈꢔꢆꢇ1.0ꢇμꢒ.ꢇꢂꢉAꢇ=ꢇꢂyꢘꢑꢍꢉꢛꢍꢕꢋꢈꢇAꢆꢇ12ꢇμꢒ.ꢇꢁꢈꢔ-way
ANOVA and Newman-Keuls test: *P< 0.05 compared to stimulated and untreated cells; #P <
0
.05 compared to dexamethasone-treated cells or cyclosporin A-treated cells.
In both assays, SintMed65 exhibited a significant and concentration-dependent
suppression of lymphoproliferation. As a potential immunotherapeutic agent, it was also
important to determine the effect of SintMed65 on cytokine secretion. To this end, cytokine
production by splenocytes stimulated with Con A and treated with SintMed65 was

investigated (Figure 5). Compared to untreated and stimulated cultures, treatment with
SintMed65 decreased the secretion of IL-2, IL-4, IL-10, IL-17A and IFN-γin a
concentration-dependent manner. Under the same conditions, dexamethasone also promoted
a significant decrease in cytokine production (Figure 5).
A
B
C
1
500
000
2500
600
400
200
0
2000
#
*#
1
*#
*#
1500
*#
*
1000
*
5
00
*#
*
*
*
500
*#
*
0
*
0
*
C-
C- Dexa 10
5
2.5
C-
C- Dexa 10
5
2.5
C-
C- Dexa 10
5
2.5
SintMed65 (M)
SintMed65 (M)
SintMed65 (M)
conA
conA
conA
D
E
500
300
400
200
300
*
2
00
*#
*#
*#
100
0
*#
*#
100
*#
*#
C-
*
0
*
C- Dexa 10
5
2.5
C-
C- Dexa 10
5
2.5
SintMed65 (M)
SintMed65 (M)
conA
conA
Figure 5: N-acylhydrazone SintMed65 modulates cytokine production by activated
lymphocytes. Lymphocytes were stimulated with con A and treated with different
concentrations of SintMed65 (10ꢆꢇ5ꢇꢐꢈꢖꢇ2ꢆ5ꢇμꢒ)ꢇꢍꢕꢇꢖꢔxꢐmꢔꢊꢡꢐꢉꢍꢈꢔꢇ(Dꢔxꢐ;ꢇ1ꢇμꢒ).ꢇꢂyꢊꢍkꢋꢈꢔꢉꢇ
were determined by ELISA in the supernatant collected 48 h after stimulus. Dexa =
Dꢔxꢐmꢔꢊꢡꢐꢉꢍꢈꢔꢇꢐꢊꢇ10ꢇμꢒꢆꢇꢔxꢘꢔꢛꢊꢇꢟꢍꢕꢇꢀL-2ꢆꢇwꢡꢋꢘꢡꢇꢋꢊꢇwꢐꢉꢇꢌꢉꢔꢖꢇꢐꢊꢇ1.0ꢇμꢒ.ꢇVꢐꢑꢌꢔꢉꢇꢕꢔꢛꢕꢔꢉꢔꢈꢊꢇꢊꢡꢔꢇ
mean±S.E.M. of one of two experiments performed. One-way ANOVA and Newman-Keuls
test: *P< 0.05 compared to stimulated and untreated cells; #P < 0.05 compared to
dexamethasone-treated cells.
We further investigated whether SintMed65 compound inhibits lymphocyte
proliferation by blocking cell cycle progression. As observed in Figure 5C, treatment with
SintMed65 induced cell cycle arrest on G0/G1 phase in comparison to untreated and
stimulated control. TCR activation increases the intracellular calcium concentration on T
lymphocytes, activating calcineurin, a phosphatase that dephosphorylates NFAT transcription

[
32,33]
factor.
Notably, NFAT is involved in the transcriptional activation of genes encoding
[
34]
several cytokines important for lymphocyte expansion and differentiation. We found that
SintMed65 treatment significantly reduced cellular calcineurin phosphate activity in Con A-
activated lymphocytes after 72 h of culture (Figure 5D). These data suggest that SintMed65
effect on lymphocyte proliferation is likely due to its cell cycle blockage and calcineurin
inhibition.
Finally, we evaluated SintMed65 compound in an in vivo model of carrageenan-
induced mouse peritonitis. Indomethacin, a COX inhibitor, was used as reference drug. In
comparison to untreated group (vehicle), pre-treatment for 1 h of SintMed65 at 80 and 20
mg/kg by oral route caused a reduction in the number of neutrophils of 30.6 and 21.4 %,
respectively (Figure 6). Under the same conditions, treatment with indomethacin (80 mg/kg)
caused a reduction of 35.9 %.
200
150
*
*
*
100
50
*
0
Naive
Vehicle
INDO
80
20
SintMed65 (mg/Kg)
Figure 7: SintMed65 is an oral antiinflammatory agent in a model of peritonitis. Male
BALB/c mice (n=6/group) were treated orally by gavage 1 h before challenge with
carrageenan (1 %; v/v). Indomethacin (INDO) at 80 mg/kg was used as reference
antiinflammatory by intraperitoneal route. Exudate was collected 4 h after the injection of
carrageenan. Values represent the mean±S.E.M. of one experiment of two experiments

performed. One-way ANOVA and Newman-Keuls test: *P< 0.05 compared to vehicle group;
P < 0.05 compared to indomethacin group.
#
3. Conclusions
We demonstrated that the structural design of 4-(nitrophenyl)hydrazone derivatives of
N-acylhydrazone led to a new family of potent antiinflammatory and immunosuppressor
agents. By investigating a number of substituents, as well as structural modification, we
observed that activity was affected by the nature and position of the attached substituent,
revealing a key role of substituent effect for activity. We have found that this family of
compounds inhibits nitric oxide production by activated macrophages, which is achieved by
suppressing IL-1βꢇꢉꢔꢘꢕꢔꢊꢋꢍꢈꢆꢇwꢋꢊꢡꢇꢉꢌbꢉꢔqꢌꢔꢈꢊꢇꢕꢔꢖꢌꢘꢊꢋꢍꢈꢇꢋꢈꢇꢂꢁX2ꢇꢔxꢛꢕꢔꢉꢉꢋꢍꢈꢇꢐꢈꢖꢇPꢎE2ꢇ
biosynthesis. Moreover, these hydrazone-N-acylhydrazones were able to inhibit lymphocyte
proliferation and cytokine production, showing a potent immunomodulatory action.
Furthermore, oral administration of SintMed65 compound significantly decreased
inflammatory cell migration in a peritonitis model induced by carrageenan.
4. Materials and Methods
4
4
.1. Chemistry
.1.1. General methods
All solvents, carboxylic acids, and other reactants were purchased from Merck,
Sigma-Aldrich and Vetec, and were used without further purification. Esters were obtained
by Fischer esterification of commercial carboxylic acids. Hydrazide derivatives were

synthesised from the corresponding carboxylic esters by reaction with hydrazine hydrate,
[
8,9]
following literature methods currently applied in our laboratory.
Reactions progresses
were monitored by thin layer chromatography (TLC), performed onto glass-backed plates of
silica gel 60 F254 with gypsum, and all compounds were detected by ultraviolet light (254
nm) as well as by iodine vapour. Melting points were determined with a Gehaka PF1500
capillary apparatus, and are uncorrected. NMR spectra were recorded at 400 MHz for
hydrogen and 100 MHz for carbon nuclei, using a Varian UNMRS 400 spectrometer, as well
as at 300 MHz for hydrogen and 75 MHz for carbon nuclei, using a Varian Unitplus 300, in
DMSO-d with chemical shift values (δ) in parts per million (ppm) and coupling constants (J)
6
in Hertz (Hz), all measured at 25 °C. Structural assignments for known compounds were
made by comparison of recorded spectra with literature data experiments, while new
compounds were assigned by 2D experiments, and described according to the IUPAC
numbering rules. IR spectra were recorded on a Tensor27 FTIR spectrometer from Bruker
with the samples being analysed as KBr pellets. High resolution mass spectrometry (HRMS)
analysis of the pure compounds was performed with an UHPLC-ESI-HRMS on a Micromass
LCT Premier time-of-flight mass spectrometer from Waters with an electrospray ionization
(
ESI) interface (Waters, Milford, MA, USA) equipped with an electrospray interface and
coupled to an Acquity UHPLC system (Waters, Milford MA, USA). UHPLC-ESI-HRMS
analysis was performed using the following parameters: ESI used capillary voltage 2800 V,
cone voltage 40 V, MCP detector voltage 2400 V, source temperature 120°C, desolvation
temperature 300°C, cone gas flow 20 L/h, and desolvation gas flow 800 L/h. Detection was
performed in positive ion mode (PI) with a m/z range of 100–1300 Da and a scan time of 0.5
s in the W-mode. The MS was calibrated using sodium formate, and leucine encephalin
(
Sigma-Aꢑꢖꢕꢋꢘꢡꢆꢇꢝꢊꢔꢋꢈꢡꢔꢋmꢆꢇꢎꢔꢕmꢐꢈy)ꢇwꢐꢉꢇꢌꢉꢔꢖꢇꢐꢉꢇꢐꢈꢇꢋꢈꢊꢔꢕꢈꢐꢑꢇꢕꢔꢟꢔꢕꢔꢈꢘꢔꢇꢐꢊꢇ2μꢙ/mLꢇꢐꢈꢖꢇ
ꢋꢈꢟꢌꢉꢔꢖꢇꢊꢡꢕꢍꢌꢙꢡꢇꢐꢇLꢍꢘkꢇꢝꢛꢕꢐy™ꢇꢛꢕꢍbꢔꢇꢐꢊꢇꢐꢇꢟꢑꢍwꢇꢕꢐꢊꢔꢇꢍꢟꢇ10ꢇμL/mꢋꢈꢇwꢋꢊꢡꢇꢊꢡꢔꢇꢡꢔꢑꢛꢇꢍꢟꢇꢐꢇꢉꢔꢘꢍꢈꢖꢇ

LC pump. The separation was performed on an Acquity BEH C18 UPLC column (1.7 µm, 50
2.1 mm i.d.; Waters, Milford, MA, USA) using a linear gradient, solvent system: A) 0.1%
×
formic acid–water, B) 0.1% formic acid–acetonitrile; gradient: 5–36% B in 1 min, then 36%
B to 36% B in 2 min, 36% B to 95% B in 1.5 min; flow rate 1.1 mL/min. The temperature
was set at 40°C. The injected volume was kept constant (1 µL; solution at the concentration
of 0.5 mg/mL). Elemental analyses were performed in a Perkin Elmer2400 Series L
elemental analyzer.
4
.1.2. Synthesis of α-oxopropane-4-nitrophenylhydrazone (5)
In a 100 mL round bottom flask, a solution 0.8028 g of potassium hydroxide 85 % was
prepared in 22 mL of water at room temperature, followed by the addition of 1.20 mL (10.12
mmol) of ethyl acetoacetate 3, and the mixture was kept stirring for 24 h. After this time, the
solution was chilled using an ice/salt bath until the temperature was below 5 °C. In a second
flask, a suspension of 1.4000 g (10.12 mmol) of p-nitroaniline 1 in 10.0 mL water was placed
in an ice/salt bath until the temperature reached 5 °C. A cold solution of 4.0 mL of
concentred hydrochloride acid and 4.0 mL water was poured to the suspension, dissolving the
solid almost completely. Under stirring, a cold solution of 0.8392 g (12.16 mmol) of sodium
nitrite in 2.0 mL water was added dropwise to the acid solution of p-nitroaniline. The rate of
ꢐꢖꢖꢋꢊꢋꢍꢈꢇwꢐꢉꢇꢕꢔꢙꢌꢑꢐꢊꢔꢖꢆꢇꢉꢍꢇꢊꢡꢐꢊꢇꢊꢡꢔꢇꢕꢔꢐꢘꢊꢋꢍꢈ’ꢉꢇꢊꢔmꢛꢔꢕꢐture did not rise above 5 °C. The
resulting diazonium salt solution was kept under such conditions, while the initial solution of
potassium acetoacetate was acidified with a mixture of 1.0 mL of concentrated hydrochloric
acid and 3.0 mL water at 0 °C, followed by the addition of 1.6630 g (20.20 mmol) of sodium
acetate. Finally, the cold solution of the acetoacetic acid was added in portions to the solution
of diazonium salt under vigorous stirring, observing the immediate formation of a yellow
precipitate. Once the addition was finished, the reaction mixture was stirred for more 30

minutes at 0 °C, and the ice bath was removed, allowing the medium to reach the room
temperature. After 30 minutes, 20 mL water was poured into the reaction mixture, which was
refluxed at 50 °C for 2 h. After cooling to room temperature, the mixture was allowed to
stand in the refrigerator overnight prior to vacuum filtration and washing with cold water.
Compound 5 was obtained as yellow powder. Yield 95%; mp 221.9-222.7 °C; R
f
0.21
-1
(
AcOEt/Hexanes 3:7); IR (KBr, νmax cm ): 3252, 3150 (NH), 3078 (Ar CH), 2924 (CH),
1
3
1
4
675 (C=O), 1587 (C=N); H NMR (400 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ8.34ꢇ(ꢖꢆꢇ2ꢜꢆꢇ J = 9.6 Hz,
3
-NO
2
Ph H-3,5), 7.48 (s, 1H, N=CH), 7.43 (d, 2H, J = 8.8 Hz, 4-NO
2
Ph H-2,6), 2.50 (s, 3H,
Ph C-4),
Ph C-3,5), 113.1 (2C, 4-
1
3
CH
40.7 (1C, 4-NO
NO Ph C-2,6), 24.3 (1C, CH
3
); C NMR (100 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ196.7ꢇ(1ꢂꢆꢇꢂ=ꢁ)ꢆꢇ148.9ꢇ(1ꢂꢆ4-NO
Ph C-1), 138.6 (1C, N=CH), 125.9 (2C, 4-NO
).
2
1
2
2
2
3
4
7
.1.3. General procedure for the synthesis of hydrazone-N-acylhydrazones SintMed(54-
4)
To a stirred suspension of α-oxopropane-4-nitrophenylhydrazone (5) (0.48 mmol) and the
appropriate hydrazide (0.48 mmol) in 4.0 mL of ethanol, cerium (III) chloride heptahydrate
(
10 mol%) was added and the reaction mixture was stirred at 65 °C during 1-3 h.ꢇꢃꢔꢐꢘꢊꢋꢍꢈ’ꢉꢇ
completion was monitored by TLC, using appropriate eluent system. Once concluded, the
heating was put away, and the reaction mixture was allowed to reach room temperature,
giving yellow to orange precipitates. After cooling, the solids were filtered off under vacuum,
1
and washed with cold ethanol. H NMR analysis of all products confirmed their purity.
Recrystallization from ethanol afforded the samples for biological purposes, which were
dried in an Abderhalden’ꢉꢇapparatus to remove traces of solvent. Yields, melting points,
spectroscopic and spectrometric data are listed below for each new compound.

4
.1.3.1. N'-[(1E)-3-(4-nitrophenylhydrazono)]-(2E)-propan-2-ylidenebenzohydrazide
SintMed54)
Yellow solid, yield 90%; mp 294.5-295.9 °C (dec); R
(
-1
f
0.60 (AcOEt); IR (KBr, νmax cm ):
3
1
1
7
7
374, 3222, 3197, 3142 (NH), 3063, 3026 (CH Ar), 2970, 2922 (CH), 1654 (s, CO),
1
610 (C=C), 1597 (C=N); H NMR (400 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ11.5ꢇ(ꢉꢆꢇ1ꢜꢆꢇNꢜ)ꢆꢇ10.8ꢇ(ꢉꢆꢇ
3
3
H, CONH), 8.15 (d, 2H, J = 8.8 Hz, 4-NO
2
Ph H-3,5), 7.86 (d, 2H, J = 6.0 Hz, Ph H-2,6),
3
3
.74 (s, 1H, N=CH), 7.57 (d, 1H, J = 7.2 Hz, Ph H-4), 7.50 (t, 2H, J = 7.2 Hz, Ph H-3,5),
3
13
.16 (d, 2H, J = 8.8 Hz, 4-NO
2
Ph H-3,5), 2.27 (s, 3H, CH
C=N), 149.9 (1C, 4-NO
Ph C-4), 133.6 (1C, Ph C-1), 131.5 (1C, Ph C-4), 128.1 (4C, Ph C-
3
); C NMR (100 MHz, DMSO-
d6ꢆꢇδꢇꢛꢛm)ꢢꢇ163.9 (1C, C=O), 152.1 (1C, CH
N=CH), 139.0 (1C, 4-NO
,3,5,6), 126.0 (2C, 4-NO
TOF-MS for C16
3
2
Ph C-1), 142.1 (1C,
2
2
2
Ph C-3,5), 111.6 (2C, 4-NO
2
Ph C-2,6), 11.2 (1C, CH ); UHPLC-
3
-
H
15
N
5
O
3
calcd (found)/Error: 324.1094 (324.1097 [M-H] )/-0.9 ppm;
+
3
26.1254 (326.1253, [M+H] )/0.3 ppm.
4
.1.3.2. N'-[(1E)-3-(4-nitrophenylhydrazono)]-(2E)-propan-2-ylidene-4-
methoxybenzohydrazide (SintMed55)
-1
Yellow solid, yield 84%; mp 291.9-292.4 °C; R 0.50 (AcOEt); IR (KBr, νmax cm ): 3226,
f
3
199 (NH), 3068, 3024 (CH Ar), 2965 2932 (CH ), 1680 (CO), 1607 (C=C), 1569
1
(
(
C=N); H NMR (400 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ11.5ꢇ(ꢉꢆꢇ1ꢜꢆꢇNꢜ)ꢆꢇ10.7ꢇ(ꢉꢆꢇ1ꢜꢆꢇꢂꢁNꢜ)ꢆꢇ8.15ꢇ
3
3
d, 2H, J = 8.8 Hz, 4-NO
2
Ph H-3,5), 7.87 (d, 2H, J = 8.4 Hz, 4-CH
3
OPh H-2,6), 7.74 (s, 1H,
OPh H-
3
3
N=CH), 7.16 (d, 2H, J = 8.8 Hz, 4-NO
2
Ph H-2,6), 7.03 (d, 2H, J = 8.0 Hz, 4-CH
3
1
3
3
,5), 3.83 (s, 3H, OCH
1C, C=O), 162.0 (1C, 4-CH
42.3 (1C, N=CH), 138.9 (1C, 4-NO
NO Ph C-3,5), 125.5 (1C, 4-CH OPh C-1), 113.4 (2C, 4-CH
3
), 2.27 (s, 3H, CH
OPh C-4), 152.3 (1C, CH
Ph C-4), 130.1 (2C, 4-CH
OPh C-3,5), 111.5 (2C, 4-
3
); C NMR (100 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ163.5ꢇ
C=N), 149.9 (1C, 4-NO Ph C-1),
OPh C-2,6), 126.0 (2C, 4-
(
3
3
2
1
2
3
2
3
3

NO
2
Ph C-2,6), 55.3 (1C, OCH
3
), 11.1 (1C, CH
3
); UHPLC-TOF-MS for C17
H
17
N
5
4
O calcd
-
+
(
found)/Error: 354.1212 (354.1202, [M-H] )/2,8 ppm; 356.1371 (356.1359, [M+H] )/ 3.4
ppm.
4
.1.3.3. N'-[(1E)-3-(4-nitrophenylhydrazono)]-(2E)-propan-2-ylidene-4-
trifluoromethylbenzohydrazide (SintMed56)
-1
Orange solid, yield 82%; mp 303.4-304.2 °C (dec); R 0.48 (AcOEt); IR (KBr, νmax cm ):
f
1
3
298, 3234 (NH), 3028 (CH Ar), 2968 (CH), 1640 (CO), 1599 (C=C), 1566 (C=N); H
NMR (400 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ11.5 and 11.3 (s, 1H, NH), 11.3 and 11.0 (s, 1H,
3
CONH), 8.15 (d, 2H, J = 9.2 Hz, 4-NO
2
Ph H-3,5), 8.04 (br s, 2H, 4-CF
3
Ph H-3,5), 7.87 (d,
Ph H-
3
3
2
2
H, J = 6.8 Hz, 4-CF
3
Ph H-2,6), 7.76 (s, 1H, N=CH), 7.17 (d, 2H, J = 6.4 Hz, 4-NO
2
1
3
,6), 2.28 (s, 3H, CH
C=N), 149.8 (1C, 4-NO
37.6 (1C, 4-CF Ph C-4), 128.9 (2C, 4- CF
Ph C-2,6), 122.5 (1C, 4-CF Ph C-1), 111.6 (2C, 4-NO
3
); C NMR (100 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ163.0ꢇ(1ꢂꢆꢇꢂ=ꢁ)ꢆꢇ154.1ꢇ
Ph C-1), 141.9 (1C, N=CH), 139.1 (1C, 4-NO Ph C-4),
Ph C-3,5), 126.0 (2C, 4-NO Ph C-3,5), 125.2
Ph C-2,6), 11.4 (1C, CH );
(
1C, CH
3
2
2
1
3
3
2
(
2C, 4-CF
3
3
2
3
1
9
F NMR (376 MHz, DMSO-d
6
,  ppm): -61.4 (s, 3F, CF
3
); UHPLC-TOF-MS for calcd
-
+
(
found)/Error: 392.0985 (392.0970, [M-H] )/3.8 ppm; 394.1149 (394.1127, [M+H] )/5.6
ppm.
4
.1.3.4. N'-[(1E)-3-(4-nitrophenylhydrazono)]-(2E)-propan-2-ylidene-4-tert-
butylbenzohydrazide (SintMed57)
-1
Yellow solid, yield 93%; mp 300.3-301.4 °C; R 0.45 (AcOEt); IR (KBr, νmax cm ): 3226,
f
1
3
143 (NH), 3067 (CH Ar), 2964, 2869 (CH ), 1665 (CO), 1597 (C=C), 1571 (C=N); H
3
NMR (400 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ11.5ꢇ(ꢉꢆꢇ1ꢜꢆꢇNꢜ)ꢆꢇ10.8ꢇ(ꢉꢆꢇ1ꢜꢆꢇꢂꢁNꢜ)ꢆꢇ8.15ꢇ(ꢖꢆꢇ2ꢜꢆꢇ J
3
t
=
8.8 Hz, 4-NO Ph H-3,5), 7.79 (d, 2H, J = 8.0 Hz, 4- BuPh H-2,6), 7.74 (s, 1H, N=CH),
2

3
t
3
7
3
1
.51 (d, 2H, J = 8.4 Hz, 4- BuPh H-3,5), 7.16 (d, 2H, J = 8.8 Hz, 4-NO Ph H-2,6), 2.26 (s,
2
t
13
H, CH
3
), 1.31 (s, 9H, Bu); C NMR (100 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ163.9ꢇ(1ꢂꢆꢇꢂ=ꢁ)ꢆꢇ
t
54.4 (1C, 4- BuPh C-4), 152.8 (1C, CH
3
C=N), 149.9 (1C, 4-NO
2
Ph C-1), 142.2 (1C,
t
t
N=CH), 138.9 (1C, 4-NO
26.0 (2C, 4-NO
1C, C(CH ), 30.8 (3C, C(CH
2
Ph C-4), 130.9 (1C, 4- BuPh C-1), 127.9 (2C, 4- BuPh C-2,6),
t
1
2
Ph C-3,5), 124.9 (2C, 4- BuPh C-3,5), 111.6 (2C, 4-NO
2
Ph C-2,6), 34.6
(
(
3
)
3
3
)
3
), 11.1 (1C, CH
3
); UHPLC-TOF-MS for C20 calcd
H
23
N
5
O
3
-
+
found)/Error: 380.1700 (380.1723, [M-H] )/-6.0 ppm; 382.1877 (382.1879, [M+H] )/-0.5
ppm.
4
.1.3.5. N'-[(1E)-3-(4-nitrophenylhydrazono)]-(2E)-propan-2-ylidene-4-
aminobenzohydrazide (SintMed58)
-1
Yellow solid, yield 90%; mp 302.1-302.7 °C (dec); R 0.35 (AcOEt); IR (KBr, νmax cm ):
f
3
1
8
474, 3381, 3223, 3192, 3139 (NH), 3064, 3026 (CH Ar), 1648 (s, CO), 1599 (C=C),
1
568 (C=N); H NMR (400 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ11.4ꢇ(ꢉꢆꢇ1ꢜꢆꢇNꢜ)ꢆꢇ10.3ꢇ(ꢉꢆꢇ1ꢜꢆꢇꢂꢁNꢜ)ꢆꢇ
3
3
.14 (d, 2H, J = 9.2 Hz, 4-NO
2
Ph H-3,5), 7.74 (s, 1H, N=CH), 7.66 (d, 2H, J = 8.0 Hz, 4-
3
3
NH
2
Ph H-2,6), 7.15 (d, 2H, J = 9.2 Hz, 4-NO
2
Ph H-2,6), 6.60 (d, 2H, J = 8.8 Hz, 4-NH
2
Ph
1
3
H-3,5), 5.79 (s, 2H, NH
2
), 2.25 (s, 3H, CH
1C, C=O), 152.3 (1C, 4-NH
1C, N=CH), 138.8 (1C, 4-NO
,5), 119.4 (1C, 4-NH Ph C-1), 112.4 (2C, 4-NH
1C, CH ); UHPLC-TOF-MS for C16
3
); C NMR (100 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ163.8ꢇ
(
(
2
Ph C-4), 150.6 (1C, CH
Ph C-4), 130.0 (2C, 4-NH
Ph C-3,5), 111.5 (2C, 4-NO
calcd (found)/Error: 339.1256 (339.1206, [M-
3
C=N), 150.0 (1C, 4-NO
Ph C-2,6), 126.0 (2C, 4-NO
Ph C-2,6), 10.9
2
Ph C-1), 142.6
2
2
2
Ph C-
3
2
2
2
(
3
H
16
N
6
O
3
-
+
H] )/14.7 ppm; 341.1346 (341.1362, [M+H] )/-4.7 ppm.
4
.1.3.6. N'-[(1E)-3-(4-nitrophenylhydrazono)]-(2E)-propan-2-ylidene-4-(N,N-
dimethylamino)benzohydrazide (SintMed59)

-1
Yellow solid, yield 90%; mp 311.5-312.9 °C; R 0.52 (AcOEt); IR (KBr, νmax cm ): 3394,
f
3
190, 3134 (NH), 3062, 3018 (CH Ar), 2914, 2864 (CH), 1651 (CO), 1608 (C=C), 1569
1
(
(
C=N); H NMR (400 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ11.4ꢇ(ꢉꢆꢇ1ꢜꢆꢇNꢜ)ꢆꢇ10.4ꢇ(ꢉꢆꢇ1ꢜꢆꢇꢂꢁNꢜ)ꢆꢇ8.16ꢇ
3
3
d, 2H, J = 8.8 Hz, 4-NO
2
Ph H-3,5), 7.78 (d, 2H, J = 8.8 Hz, 4-NMe
2
Ph H-2,6), 7.74 (s, 1H,
Ph H-
3
3
N=CH), 7.16 (d, 2H, J = 9.2 Hz, 4-NO
2
Ph H-2,6), 6.74 (d, 2H, J = 9.2 Hz, 4-NMe
2
1
3
3
,5), 3.00 (s, 6H, NMe
1C, 4-NMe Ph C-4), 150.8 (1C, CH
38.8 (1C, 4-NO Ph C-4), 129.6 (2C, 4-NMe
Ph C-1), 111.5 (2C, 4-NO Ph C-2,6), 110.6 (2C, 4-NMe
); UHPLC-TOF-MS for C18 calcd (found)/Error: 367.1514
2
), 2.26 (s, 3H, CH
C=N), 149.9 (1C, 4-NO
Ph C-2,6), 125.9 (2C, 4-NO
Ph C-3,5), 39.5 (2C,
3
); C NMR (100 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ152.4ꢇ
Ph C-1), 142.5 (1C, N=CH),
Ph C-3,5), 119.5
(
2
3
2
1
2
2
2
(
1C, 4-NMe
2
2
2
NMe
2
), 10.9 (1C, CH
3
H
20
N
6
O
3
-
+
(
367.1519, [M-H] )/-1.4 ppm; 369.1696 (369.1675, [M+H] )/5.7 ppm.
4
.1.3.7. N'-[(1E)-3-(4-nitrophenylhydrazono)]-(2E)-propan-2-ylidene-4-
chlorobenzohydrazide (SintMed60)
-1
Yellow solid, yield 91%; mp 292.9-294.7 °C (dec); R 0.73 (AcOEt); IR (KBr, νmax cm ):
f
3
291, 3226 (NH), 3062 (CH Ar), 2970, 2924 (CH ), 1677 (CO), 1635 (C=C), 1596
1
(
(
C=N); H NMR (400 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ11.5ꢇ(ꢉꢆꢇ1ꢜꢆꢇNꢜ)ꢆꢇ10.9ꢇ(ꢉꢆꢇ1ꢜꢆꢇꢂꢁNꢜ)ꢆꢇ8.14ꢇ
3
3
d, 2H, J = 8.8 Hz, 4-NO
2
Ph H-3,5), 7.88 (br d, 2H, J = 6.8 Hz, 4-ClPh H-2,6), 7.73 (s, 1H,
3
3
N=CH), 7.56 (d, 2H, J = 8.4 Hz, 4-ClPh H-3,5), 7.16 (d, 2H, J = 8.8 Hz, 4-NO
2
Ph H-2,6),
1
3
2
.26 (s, 3H, CH
CH C=N), 149.8 (1C, 4-NO
1C, 4-ClPh C-4), 132.3 (1C, 4-ClPh C-1), 130.0 (2C, 4-ClPh C-2,6), 128.2 (2C, 4-ClPh C-
3
); C NMR (100 MHz, DMSO-d6ꢆꢇδꢇꢛꢛm)ꢢꢇ163.0ꢇ(1ꢂꢆꢇꢂ=ꢁ)ꢆꢇ153.7ꢇ(1ꢂꢆꢇ
3
2
Ph C-1), 142.0 (1C, N=CH), 139.1 (1C, 4-NO Ph C-4), 136.3
2
(
3
,5), 126.0 (2C, 4-NO
2
Ph C-3,5), 111.6 (2C, 4-NO
2
Ph C-2,6), 11.3 (1C, CH ); UHPLC-TOF-
3
-
MS for C16
H
14
N
5
ClO
3
calcd (found)/Error: 358.0707 (358.0707, [M-H] )/0.0 ppm; 360.0856
+
(
360.0863, [M+H] )/-1.9 ppm.