Novel orally active analgesic and anti-inflammatory cyclohexyl-N-acylhydrazone derivatives

Article abstract of DOI:10.3390/molecules20023067

The N-acylhydrazone (NAH) moiety is considered a privileged structure, being present in many compounds with diverse pharmacological activities. Among the activities attributed to NAH derivatives anti-inflammatory and analgesic ones are recurrent. As part of a research program aiming at the design of new analgesic and anti-inflammatory lead-candidates, a series of cyclohexyl-N-acylhydrazones 10-26 were structurally designed from molecular modification on the prototype LASSBio-294, representing a new class of cycloalkyl analogues. Compounds 10-26 and their conformationally restricted analogue 9 were synthetized and evaluated as analgesic and anti-inflammatory agents in classical pharmacologic protocols. The cyclohexyl-N-acylhydrazones 10-26 and the cyclohexenyl analogue 9 showed great anti-inflammatory and/or analgesic activities, but compound 13 stood out as a new prototype to treat acute and chronic painful states due to its important analgesic activity in a neuropathic pain model.

Full text of DOI:10.3390/molecules20023067

Molecules 2015, 20, 3067-3088; doi:10.3390/molecules20023067
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Novel Orally Active Analgesic and Anti-Inflammatory
Cyclohexyl-N-Acylhydrazone Derivatives
Tiago Fernandes da Silva 1,2, Walfrido Bispo Júnior 3, Magna Suzana Alexandre-Moreira 3,
Fanny Nascimento Costa 4,5, Carlos Eduardo da Silva Monteiro 6, Fabio Furlan Ferreira 4,
Regina Cely Rodrigues Barroso 5, François Noël 6, Roberto Takashi Sudo 6,
Gisele Zapata-Sudo 6, Lídia Moreira Lima 1,2,6,* and Eliezer J. Barreiro 1,2,6,
*
1
Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio®),
Instituto Nacional de Ciência e Tecnologia de Fármacos e Medicamentos (INCT-INOFAR),
Universidade Federal do Rio de Janeiro, CCS, Cidade Universitária, P.O. Box 68024,
Rio de Janeiro-RJ 21941-971, Brazil; E-Mail: tiagosilfer@yahoo.com.br
2
3
Programa de Pós-Graduação em Química, Instituto de Química,
Universidade Federal do Rio de Janeiro, Rio de Janeiro-RJ 21941-909, Brazil
LaFI—Laboratório de Farmacologia e Imunidade, Instituto de Ciências Biológicas e da Saúde,
Universidade Federal de Alagoas, Maceió-AL 57072-900, Brazil;
E-Mails: walfridobispojr@hotmail.com (W.B.J.); suzana.magna@gmail.com (M.S.A.-M.)
Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC (UFABC) Av. dos
Estados 5001, Santo André-SP 09210-580, Brazil; E-Mails: fannycosta@yahoo.com.br (F.N.C.);
fabio.furlan@ufabc.edu.br (F.F.F.)
4
5
6
Physics Institute, UERJ, Rio de Janeiro-RJ 20550-900, Brazil; E-Mail: cely_barroso@hotmail.com
Programa de Pesquisa em Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas,
Universidade Federal do Rio de Janeiro, Rio de Janeiro-RJ 21941-902, Brazil;
E-Mails: cadusilvarj@ig.com.br (C.E.S.M.); fnoel@pharma.ufrj.br (F.N.);
rtsudo@farmaco.ufrj.br (R.T.S.); gsudo@farmaco.ufrj.br (G.Z.-S.)
* Authors to whom correspondence should be addressed; E-Mails: lidialima@ufrj.br (L.M.L.);
ejbarreiro@ccsdecania.ufrj.br (E.J.B.); Tel.: +55-021-3938-6503 (L.M.L.);
Tel./Fax: +55-021-3938-6478 (E.J.B.).
Academic Editor: Derek J. McPhee
Received: 19 December 2014 / Accepted: 3 February 2015 / Published: 12 February 2015
Abstract: The N-acylhydrazone (NAH) moiety is considered a privileged structure, being
present in many compounds with diverse pharmacological activities. Among the activities

Molecules 2015, 20
attributed to NAH derivatives anti-inflammatory and analgesic ones are recurrent. As part
3068
of a research program aiming at the design of new analgesic and anti-inflammatory
lead-candidates, a series of cyclohexyl-N-acylhydrazones 10–26 were structurally designed
from molecular modification on the prototype LASSBio-294, representing a new class of
cycloalkyl analogues. Compounds 10–26 and their conformationally restricted analogue 9
were synthetized and evaluated as analgesic and anti-inflammatory agents in classical
pharmacologic protocols. The cyclohexyl-N-acylhydrazones 10–26 and the cyclohexenyl
analogue 9 showed great anti-inflammatory and/or analgesic activities, but compound 13
stood out as a new prototype to treat acute and chronic painful states due to its important
analgesic activity in a neuropathic pain model.
Keywords: molecular simplification; N-acylhydrazone; anti-inflammatory; neuropathic
pain; privileged structure; adenosine receptor
1. Introduction
Defined as a minimum common subunit present in the structure of several drug candidates,
privileged structures are characterized by their ability to be recognized by plural and distinct receptors.
This concept has been widely used in the literature for the design of kinase-protein and serine-protease
inhibitors, and ligands of G-protein coupled receptors (GPCR) and ionic channels [1]. Recently, based
on several works with bioactive N-acylhydrazone (NAH) derivatives [2–7], the privileged structure
nature of this bioactive framework was proposed [5].
H C
3
O
O
O
N
O
O
N
H
H C
3
N
OH
OH
N
H C
3
H
O
(1) Antiproliferative
Activity
Tubulin inhibitor 91% at
30 μM
O
(2) Anti-HIV-1 activity (HIV-1
Reverse Transcriptase
Ribonuclease H Inhibitor:
H C
3
OH
CH
3
CH
O
O
3
IC = 0.4 μM)
Cl
Cl
50
O
O
N
3
N
Privileged Structure
N
S
H
O
N
H
CH
O
N
CH
N
3
3
(3) Anti-Mycobacterium
tuberculosis activity
MIC = 12.5 μg/mL
N
N
(7) Antipsychotic activity
CH
PDE10A inhibiton
IC = 0.010 μM
50
H
H
H
N
N
O
N-Acylhydrazone (NAH)
N
N
O
O
N
H C
3
CH
H
3
CH
3
H C
3
CH
3
(4) Anti-inflammatory activity Inhibition
of NF-kB activity in SW1353 cells
O
OH
(6) Vasodilatory
IC = 7.7 μg/mL
50
+2
O
CH
activity (L-type Ca
3
N
channel blocker and
guanylate cyclase
inhibitor:
CH
3
3
N
O
CH
H
NO
2
O
IC = 3.1 μM
50
(5) Analgesic, anti-inflammatory and antioxidant activities
CB1 receptor binding 71% at 100 μM
Figure 1. Examples of N-acylhydrazones with different mechanism of action and
pharmacological activity.

Molecules 2015, 20
3069
Compounds 1–7 containing the NAH subunit have been described to possess antitumor
(compound 1) [6], antiviral (compound 2) [8], antibacterial (compound 3) [9], anti-inflammatory
(compound 4) [4], analgesic (compound 5) [10,11], vasodilatory (compound 6) [12] or antipsychotic
(compound 7) [13] activity (Figure 1).
As part of a research program aiming at the design of new analgesic and anti-inflammatory
lead-candidates, especially useful in the treatment of neuropathic pain, molecular changes in the
prototype (E)-N'-((thiophen-2-yl)methylene)benzo[d][1,3]dioxole-5-carbohydrazide (LASSBio-294, 8)
were performed in order to improve its analgesic and anti-inflammatory profile. LASSBio-294 has
been described as a potent positive inotropic agent with modest vasodilatory [14], analgesic and
anti-inflammatory activities [15]. Structural modification in the derivative 8 was planned applying the
concept of molecular simplification [16,17], resulting in the replacement of the 1,3-benzodioxole
system by a cyclohexane moiety (Figure 2).
O
S
sp2
E
N
N
H
(9)
O
S
O
S
sp3
sp2
Molecular
Simplification
E
E
N
N
O
O
N
N
H
H
(10)
LASSBio-294 (8)
Isosteric
Substitutions
O
OH
HN
N
HN
N
OH
O
N
(11)
(12)
(14)
(15)
(13)
O
O
O
O
O
Fe
O
O
(16)
(19)
(17)
(18)
X
(20-26)
Figure 2. Design concept of novel cyclohexyl-N-acylhydrazones 10–26 from the prototype
LASSBio-294 and its cyclohexenyl analogue 9.
Considering the changes in the hybridization of the carbon atom linked to the carbonyl function in
the cyclohexyl-N-acylhydrazone derivatives 10–26, compared to the prototype LASSBio-294 (8), the
cyclohexenyl analogue 9 was also designed. Aiming to construct a congeneric series, the 2-thienyl ring

Molecules 2015, 20
3070
was replaced by several isosteric rings (compounds 10–19) and the resulting phenyl nucleus was
subsequently substituted by different monovalent substituents [18] (compounds 20–26; Figure 2). The
election of these substituents considered their electronic effects and their lipophilicity, in accordance
with Hammett and Hansch parameters, as illustrated in Figure 2.
2. Results and Discussion
2.1. Chemistry
The cyclohexyl-N-acylhydrazones 10–26 and the cyclohexenyl analogue 9 were synthesized using
classical methodology as depicted in Scheme 1. Compounds were obtained in good yields by exploring
a linear synthesis in two and three steps, based on functional group interconversion and acid-catalyzed
condensation of hydrazide intermediates 28 or 30 with aldehydes, previously selected in accordance
with the design concept illustrated in Figure 1. LASSBio-294 (8) and LASSBio-322 (33) [19,20] were
used in order to compare the biological results with the analogues 10–26 and 9; and to establish the
contribution of 1,3-benzodioxole and phenyl subunits to the analgesic and anti-inflammatory activities.
O
O
O
N
Ar
NH2
b)
a)
N
N
O
H
H
(27)
(28)
(10-26)
O
S
O
O
N
c)
d)
N
OH
NHNH2
(30)
H
(9)
(29)
O
S
O
N
e)
N
H
H
(33)
(32)
O
S
N
O
O
O
N
e)
H
(8)
O
(34)
10: Ar= 2-thienyl
19: Ar= ferrocenyl
20: Ar= 4-nitrophenyl
21: Ar= 4-carboxyphenyl
22: Ar= 4-bromophenyl
23: Ar= phenyl
24: Ar= 4-isopropylphenyl
25: Ar= 4-methoxyphenyl
26: Ar= 4-dimethylaminophenyl
11: Ar= 1H-imidazole-2-yl
12: Ar= 1H-imidazole-5-yl
13: Ar= 4-pyridinyl
14: Ar= 4-hydroxy-3-methoxyphenyl
15: Ar= 3-hydroxy-4-methoxyphenyl
16: Ar= 1,3- benzodioxolyl
17: Ar= 3,4,5-trimethoxyphenyl
18: Ar= 3,4-dimethoxyphenyl
Reagents and conditions: (a) NH2NH2·H2O 64%, EtOH, reflux, 48 h, 79%; (b) aromatic aldehyde,
EtOH, room temperature, 30–90 min, 46%–99%; (c) (1) SOCl2, DMF (cat.), room temperature, 4 h;
(2) NH2NH2·H2O 64%, CH2Cl2, 0 °C, 2 h; (d) 2-thiophenecarboxaldehyde, EtOH, room
temperature, 30 min, 51%; (e) as previously described [19,20].
Scheme 1. Synthesis route to NAH compounds.

Molecules 2015, 20
3071
Considering the possibility that N-acylhydrazones (-CONHN=CHR) may exist as syn/anti amide
1
conformers and either as Z/E geometrical isomers (-HC=N-) [21], a careful analysis of the H-NMR
spectra of the compounds was performed.
1
The H-NMR spectra of cyclohexyl-N-acylhydrazone derivative 10, recorded at room temperature
using solvents with different polarity (i.e., DMSO-d6 and CDCl3), showed duplication of the singlet
signals relative to amide and imine hydrogens (-CONHN=CH-) at 11 ppm in DMSO-d6. Curiously, the
analysis of the same sample by reversed-phased HPLC, using different mobile phases, revealed only
one peak at the chromatogram, indicating the presence of only one species. These data indicate that the
mixture found in the 1H-NMR analysis does not correspond to possible geometric isomers (Z and E) of
imine group (-N=CH-). In order to completely discard the possibility of diastereoisomers and to
identify the possible presence of conformers of the amide bond, the 1H-NMR spectrum (300 MHz) of
LASSBio-1515 (10) was recorded at 90 °C. This spectrum showed coalescence of the duplicated
singlet signals seen in the 1H-NMR spectrum at room temperature (Figure 3). These results allowed us
to discard the diastereoisomer possibility and reinforced the existence of conformers.
1
Figure 3. H-NMR (300 MHz) spectra of the NAH derivative LASSBio-1515 (10) in
DMSOd6 at 25 °C (A) and 90 °C (B).
The possibility of chair conformers was also investigated by examining the JJ-coupling in the
1
500 MHz H-NMR of LASSBio-1515 (10). It was clear that both duplicated signals relative to the
tertiary hydrogen had H-coupling constants of approximately 11 Hz (Figure 4). Data in the literature
was found for axial-axial H-coupling [1Haxial-2Haxial or 1Haxial-6Haxial] of cyclohexyl derivatives, J1Ha/2Ha
or J1-Ha/6Ha of around 8 to 14 Hz, while J1He/2Ha or J1-He/6Ha [1Hequatorial-2Haxial or Hequatorial-6Haxial] have
1
J-couplings below 5 Hz [22]. Therefore, the result for LASSBio-1515 (10) indicated that the mixtures

Molecules 2015, 20
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1
of conformers at H-NMR are amide rotamers and not chair conformers. This type of rotamers was
1
previously reported for NAH compounds [21,23,24]. The same H-NMR spectra analysis performed
with compounds 8, 9 and 33, revealed that the presence of rotamers seems to be dependent of the
hybridization nature of carbon linked to the carbonyl-amide group. The rotamers were not seen for the
cyclohexenyl derivative 9 or even for phenyl analogue 33 and LASSBio-294 (8).
Figure 4. 1H-NMR (500 MHz, CDCl3) spectrum of NAH derivative 10 with J-coupling of
the tertiary hydrogen of the cyclohexyl functionalized system.
Aiming to determine unambiguously the relative configuration and conformation of the
cyclohexyl-N-acylhydrazone derivative 10 X-ray powder diffraction was performed [25]. The results
indicated that in the solid phase the compound is an E diastereoisomer (-N=CH-), having equatorial
substitution of the cyclohexyl ring, with an antiperiplanar conformation of the hydrogen amide and
oxygen atom of the carbonyl group (Figure 5).
Figure 5. View of representative NAH derivative LASSBio-1515 (10) by X-ray powder
diffraction Red = oxygen (O); blue = nitrogen (N); yellow = sulfur (S), black = carbon (C);
and white = hydrogen (H).

Molecules 2015, 20
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2.2. Pharmacological Activities
The anti-inflammatory and analgesic activities of the cyclohexyl-N-acylhydrazones 10–26 and
the cyclohexenyl analogue 9 were determined using classical murine models, exemplified by
carrageenan-induced peritonitis, acetic acid-induced writhing and formalin-induced pain [26–28]. The
results were compared with the standard drug indomethacin and with the prototype LASSBio-294 (8)
and its simplified analogue LASSBio-382 (33). All compounds were tested by oral administration
using a screening dose of 100 µmol/Kg.
As illustrated in Table 1 the replacement of the 1,3-benzodiozole system (compound 8) by a
cyclohexyl subunit (compound 10) resulted in the improvement of the anti-inflammatory and analgesic
activities. The conformationally restricted cyclohexenyl analogue 9 was significantly less active than
the cyclohexyl derivative 10 in the carrageenan-induced peritonitis model and showed similar
antihipernociceptive profile in the acetic acid-induced writhing test (Table 1). The attempts to replace
the 2-thienyl ring (in 10) by isosteric ones, like a 4-pyridinyl (compound 13), 1H-imidazole-2-yl
(compound 11) and the 1H-imidazole-5-yl regioisomer (compound 12) resulted in loss of activity in
these models for compounds 11 and 12, while derivative 13 showed a similar pharmacological profile
as the lead 10 (Table 1) The isosteric replacement of the 2-thienyl unit in 10 by a phenyl group
(compound 23) decreased the anti-inflammatory activity, although the analgesic profile in the writhing
test was preserved (Table 1). The substitution of the phenyl ring of compound 23 by monovalent isosteric
groups (e.g., 10–26) improves the anti-inflammatory and analgesic activities, with the regioisosmers
14 and 15 and the 4-dimethylamine (26) and 4-bromo (22) derivatives standing out. The ferrocenyl
derivative 19, designed in accordance with literature reports showing its bioisosteric relationship with
the phenyl group [29], showed an improvement in the analgesic profile (Table 1) in the acetic acid-induced
writhing test and also in the 1st phase of the formalin model.
In the formalin test, a neurogenic phase associated with the direct effect on sensory C fibers (1st
phase) is followed by an inflammatory response related to analgesic mediators (2nd phase) [30]. With
this in mind, compounds 19, 22 and 26, which were active in the neurogenic phase of the formalin test,
were selected to be studied in the hot plate test [31]. These compounds were inactive in this model
(data not shown), discarding the hypothesis of a central antinociceptive activity and reinforcing the
peripheral analgesic profile of these bioactive N-acylhydrazones.
Considering recent works reporting the ability of the prototype 8 (LASSBio-294) to be a ligand of
the adenosine receptor, subtype A2A [32], the cyclohexyl-N-acylhydrazones 10–26 and the cyclohexenyl
analogue 9 were investigated using a specific binding assay for the A2A receptor (Table 1). As shown
in Table 1, LASSBio-294 presented a Ki value of 8.2 µM. However, its simplified analogues 9, 10–26
and 33 were unable to bind to the A2A adenosine receptor, suggesting the pharmacophoric characteristic
of 1,3-benzodiozole scaffold.
Bearing in mind that neuropathic pain represents a great challenge, and considering that its treatment
needs more specific and effective drugs, the analgesic activity of the cyclohexyl-N-acylhydrazones in
a murine neurophatic pain model was investigated. For this purpose, the spinal nerve ligation (SNL)
model in rats [33] was selected, since involvement of inflammatory and analgesic components has
been reported in this neuropathic pain model [34]. Compound 19 (LASSBio-1517), which presented
a high analgesic activity in the acetic acid induced writhing test, 17 (LASSBio-1513) and 13

Molecules 2015, 20
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(LASSBio-1514), that showed comparable analgesic and anti-inflammatory activities (Table 1), were
evaluated in the SNL model.
Table 1. Effect of N-acylhydrazones (10 or 30 µM in binding assay and 100 µmol/kg p.o.
in the in vivo assays), dipyrone or indomethacin (100 µmol/kg, p.o in vivo assays).
Biological Tests
Carrageenan
-induced
peritonitis
% of
Acetic acid-
induced
Formalin-induced
pain test
Binding A2a
writhing test
% of
% of
Phase I % of Phase II % of
Compounds
(R1)
(Ar)
inhibition a
inhibition b
65.1 ± 4.0 **
dipyrone
inhibition b
82.5 ± 6.2 **
indomethacin
inhibition b
inhibition b
57.4 ± 5.8 **
indomethacin
Dipyrone or
Indomethacin
(8)
23.5 ± 7.9
-
indomethacin
1,3-
2-thienyl
2-thienyl
(Ki = 8.2µM)
17% at 10 µM
7% at 10 µM
13% at 30 µM
36% at 10 µM
0% at 10 µM
0% at 10 µM
15% at 30 µM
-
55.3 ± 4.8 **
40.3 ± 2.3 **
21.4 ± 5.4 **
79.2 ± 2.7 **
39.8 ± 4.9 **
37.6 ± 1.9 **
81.9 ± 2.2 **
74.3 ± 4.4 **
68.2 ± 4.9 **
64.7 ± 3.0 **
76.3 ± 3.8 **
63.7± 3.2 **
46.9 ± 6.7 **
42.8 ± 1.9 **
68.2 ± 13.6 **
57.2 ± 8.5 **
77.3 ± 3.2 **
87.9 ± 3.4 **
20.9 ± 5.7 *
37.4 ± 4.9
12.3 ± 6.1
-
42.0 ± 4.7 **
23.2 ± 4.1
LASSBio-294
(33)
benzodioxolyl
phenyl
LASSBio-382
(9)
cyclohexenyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
2-thienyl
-
LASSBio-1780
(10)
2-thienyl
32.9 ± 11.5
5.2 ± 3.2
61.5 ± 2.9 **
15.4 ± 10.6
5.7 ± 2.4
LASSBio-1515
(11)
1H-imidazole-5-yl
1H-imidazole-2-yl
4-pyridinyl
LASSBio-1600
(12)
69.2 ± 7.9 **
65.7 ± 9.5 **
90.7 ± 2.5 **
55.6 ± 5.3 **
56.0 ± 11.9 **
78.3 ± 8.4 **
67.3 ± 8.5 **
97.6 ± 0.9 **
47.6 ± 7.0 **
14.2 ± 5.8
36.9 ± 8.5
36.9 ± 8.5
31.5 ± 8.3
31.3 ± 6.7
26.9 ± 5.8
13.1 ± 7.8
47.3 ± 4.7 **
31.3 ± 8.6
LASSBio-1602
(13)
43.0 ± 5.7 **
38.3 ± 5.8 **
63.1 ± 7.7 **
25.6 ± 5.9
LASSBio-1514
(14)
3-hydroxy-4-
methoxyphenyl
4-hydroxy-3-
LASSBio-1688
(15)
LASSBio-1689
(16)
methoxyphenyl
1,3- benzodioxolyl
33% at 30 µM
LASSBio-1691
(17)
3,4,5-trimethoxyphenyl 10% at 30 µM
38.5 ± 6.8 **
75.97 ± 4.4 **
27.1 ± 12.0
25.9 ± 6.9
LASSBio-1513
(18)
3,4-dimethoxyphenyl
ferrocenyl
9% at 10 µM
5% at 30 µM
0% at 10 µM
LASSBio-1509
(19)
LASSBio-1517
(20)
4-nitrophenyl
LASSBio-1603

Molecules 2015, 20
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Table 1. Cont.
Biological Tests
Carrageena
Acetic acid-
induced
Formalin-induced
pain test
Binding A2a
n-induced
peritonitis
% of
writhing test
% of
% of
Phase I % of
Phase II % of
Compounds
(R1)
(Ar)
inhibition a
inhibition b
inhibition b
inhibition b
inhibition b
(21)
LASSBio-1516
(22)
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
4-carboxylic-phenyl
4-bromophenyl
phenyl
0%, at 10 µM
3% at 10 µM
8% at 10 µM
12% at 10 µM
0% at 10 µM
0% at 10 µM
58.6 ± 5.3 **
70.7 ± 3.1 **
28.7 ± 4.4 **
78.6 ± 3.3 **
67.4 ± 4.9 **
63.9 ± 4.0 **
87.4 ± 6.0 **
62.2 ± 2.1 **
57.3 ± 9.5 **
64.9 ± 3.3 **
86.3 ± 4.0 **
68.3 ± 13.4 **
17.5 ± 5.9
54.7 ± 6.9 **
53.4 ± 11.1 **
20.3± 8.4
77.0 ± 3.9 **
NA
LASSBio-1511
(23)
LASSBio-1601
(24)
4-isopropylphenyl
4-methoxyphenyl
13.4 ± 8.2
38.5 ± 6.8 *
70.1 ± 5.3 **
54.4 ± 4.9 **
LASSBio-1508
(25)
30.1 ± 6.0
LASSBio-1510
(26)
4-
38.9 ± 13.1 *
LASSBio-1512
dimethylaminophenyl
a The % inhibition of specific binding displaced by 10 or 30µM competing compound. b The asterisks denote
the significance levels in comparison with control groups (* p < 0.05, ** p < 0.01 at ANOVA). NA: not active.
As demonstrated in Figure 6A, oral treatment for seven days with 100 µmol/kg of compounds 17
(LASSBio-1513) and 13 (LASSBio-1514) prevented the mechanical allodynia, while 19 (LASSBio-1517)
was inactive. Derivative 13 (LASSBio-1514) was also able to increase the latency time in the thermal
hyperalgesia component of the SNL model, revealing an important antihyperalgesic activity (Figure 6B).
The better profile observed with acylhydrazones 13 and 17 on SNL model may be explained by their
associative analgesic and anti-inflammatory activities (Table 1). Although the results presented in this
study did not clarify the mechanism of action of these bioactive cyclohexyl-N-acylhydrazones, the
results suggest that these compounds could be considered as new lead-candidates to treat acute and
chronic painful states.
A)
40
#
Sham/DMSO
SNL/DMSO
#
30
20
10
0
*
SNL/Tramadol
SNL/(13)
SNL/(17)
SNL/(19)
0
7
10
14
Days after surgery
Figure 6. Cont.

Molecules 2015, 20
3076
14
12
10
8
B)
Sham/ DMSO
SNL/DMSO
#
#
*
SNL/Tramadol
*
SNL/(13)
6
SNL/(17)
SNL/(19)
4
2
0
0
7
10
14
Days after surgery
Figure 6. Spinal nerve ligation (SNL) model in rats. (A) Treatment of mechanical allodynia
for 7 days orally at a dose of 100 μmol·kg−1 (tramadol 33.3 μmol·kg−1), n = 6 animals.
(B) Treatment of thermal hyperalgesia for 7 days orally at a dose of 100 μmol·kg−1
(tramadol 33.3 μmol·kg−1), n = 6 animals. * p < 0.05 versus day 0. # p < 0.05 versus day 7.
One-way ANOVA, followed by the Newman–Keuls test. p values lower than 0.05 were
considered significant.
3. Experimental Section
3.1. Chemistry
3.1.1. General Methods
NMR spectra were determined in deuterated chloroform or dimethyl sulfoxide containing ca. 1%
tetramethylsilane as an internal standard, using a 200/50 MHz Bruker DPX-200, 250/62.5 MHz Bruker
DPX-250, 400/100 MHz Varian 400-Mr, 300/75 MHz Varian Unity-300 and 500/125 MHz Varian
VNMRSYS-500 spectrometer. The progress of all reactions was monitored by thin layer
chromatography, which was performed on 2.0 cm × 6.0 cm aluminum sheets pre-coated with silica gel
60 (HF-254, Merck) to a thickness of 0.25 mm, or by infrared (IR) spectra obtained with Nicolet-550
Magna spectrophotometer by using potassium plates. The developed chromatograms were viewed
under ultraviolet light at 254 nm. Merck silica gel (70–230 mesh) was used for column
chromatography. Elemental analyses were carried out on a Thermo Scientific Flash EA 1112 Series
CHN-Analyzer. Melting points were determined with a Quimis 340 apparatus and are uncorrected. All
1
13
described products showed H and C NMR spectra according to the assigned structures. All organic
solutions were dried over anhydrous sodium sulfate and all organic solvents were removed under
reduced pressure in rotatory evaporator. HPLC for purity determinations were conducted using
Shimadzu LC-20AD with a SHIM-PACK CLC-ODS analytical column (4.6 mm × 6250 mm) or
Kromasil 100-5C18 (4.6 mm × 6250 mm) and a Shimadzu SPD-M20A detector at 254 nm wavelength.
The solvent system for HPLC purity analyses was 70:30 acetonitrile:phosphate buffer solution at pH 7.
The isocratic HPLC mode was used, and the flow rate was 1.0 mL/min.

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3.1.2. General Procedure for the Preparation of the 3,4-Methylenedioxybenzoylacylhydrazone
LASSBio-294 (8) and the Methylenedioxybenzoylacylhydrazone LASSBio-382 (33)
The methodology used in the synthesis of compounds 8 (LASSBio-294) and 33 (LASSBio-332)
was previously reported by our research group [17,19,20] and was reproduced in this work.
3.1.3. Procedure for the Synthesis of Cyclohex-1-enecarbohydrazide Intermediate 30
A solution of cyclohex-1-enecarboxylic acid (29, 0.3 g, 2.38 mmol) and thionyl chloride (2 mL) in
the presence of one drop of DMF was stirred at room temperature for 4 h until reaction completion.
The solution was concentrated to dryness under reduced pressure to obtain a solid residue, which was
solubilized in dichloromethane (10 mL) for the next step without further purification. To a reaction
flask containing 80% hydrazine monohydrate (1.15 mL, 23.80 mmol) in dichloromethane (10 mL) was
added dropwise the first solution at 0 °C for 2 h. At the end of the reaction, the solution was concentrated
under reduced pressure. The solid 30 formed was filtered off and washed with n-hexane.
3.1.4. Procedure for the Synthesis of (E)-N'-(Thiophen-2-ylmethylene)cyclohex-1-enecarbohydrazide
(LASSBio-1780, 9)
The cyclohex-1-enecarbohydrazide 30 previously obtained was immediately used without further
purification or characterization. After solubilization in ethanol (21 mL) thiophene-2-carboxaldehyde
(2.38 mmol) was added. The mixture was stirred at room temperature for 30 min, until TLC indicated
the end of the reaction. The resulting suspension was poured onto ice and the resulting precipitate was
filtered out and washed with n-hexane. Compound 9 (LASSBio-1780) was obtained as a yellow solid
in 51% yield, m.p. 201–203 °C. 1H-NMR (400 MHz) DMSO-d6 (ppm): 11.17 (s, 1H, CONH), 8.55 (s,
1H, N=CH), 7.61 (d, 1H, J = 5 Hz), 7.38 (d, 1H, J = 5 Hz), 7.13 (dd, 1H, J = 5 Hz, J = 2 Hz), 6.57 (m,
1H), 2.21–1.17 (m, 4H, cyclohexenyl), 1.71–1.21 (m, 4H, cyclohexenyl). 13C-NMR (50 MHz) DMSO-d6
(ppm): 164.45 (C=O), 141.77 (1C), 139.36 (1C), 133.16 (1C), 132.62 (C1), 130.27 (1C), 128.46 (1C),
127.72 (1C), 23–25 (2C), 21–22 (2C). IR (KBr)νmax (cm−1): 3215 (NH); 2932 (CH3); 1660 (C=C); 1626
(C=O); 1596 (C=N). % purity = 96.93% by HPLC-C18 (acetonitrile/water 7:3) (Rt = 3.89 min).
3.1.5. Procedure for the Preparation of Cyclohexanecarbohydrazide (28)
Hydrazine hydrate (64%, 1.36 mL, 28.13 mmol) was added to a solution of methyl
cyclohexanecarboxylate (27, 1.00 g, 7.03 mmol) in absolute ethanol (5 mL). The reaction mixture was
kept under reflux for 48 h, whenupon infrared (IR) spectroscopy indicated the end of the reaction.
Next, the ethanol was almost totally evaporated under reduced pressure. Ice was added to the residue
and the resulting precipitate was filtered out affording the title compound in 79% yield, as white
crystals, m.p. 154–156 °C. 1H-NMR (200 MHz, DMSO-d6, TMS) (ppm): 8.85 (s, 1H, CONH), 4.04 (s,
13
2H, NH2), 2.01 (m, 1H, cyclohexyl), 1.65 (m, 5H, cyclohexyl), 1.35 (m, 5H, cyclohexyl). C-NMR
(50 MHz, DMSO-d6, TMS) (ppm): 174.92 (NC=O), 42.35 (1C, cyclohexyl), 29.19 (2C, cyclohexyl),
25.43 (1C, cyclohexyl), 25.33 (2C, cyclohexyl). IR (KBr) ν (cm−1): 3311, 3195 (NH); 2930 (CH);
1629 (C=O).

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3.1.6. General Procedure for Preparation of Cyclohexylacylhydrazones 10–26
The corresponding aromatic or heteroaromatic aldehyde (2.11 mmol) was added to a solution of
cyclohexanecarbohydrazide (28, 0.3 g; 2.11 mmol) in absolute ethanol (21 mL). The mixture was
stirred for 2 h at room temperature. At the end of the reaction, the volume of ethanol was partially
concentrated at reduced pressure and the resulting mixture was poured into cold water. The precipitate
was filtered out, dried under vacuum and then the solid was washed with n-hexane and/or
recrystallized from ethanol to give the desired cyclohexyl-N-acylhydrazones 10–26.
(E)-N'-(Thiophen-2-ylmethylene)cyclohexanecarbohydrazide (LASSBio-1515, 10): LASSBio-1515
(10) was obtained as orange crystals in 92% yield by condensation of cyclohexane-carbohydrazide
(28) with thiophene-2-carboxylaldehyde, m.p. 209–211 °C. The data for this compound is in
1
agreement with previous reports [35]. H-NMR (300 MHz) DMSO-d6 (ppm): 10.74 (s, 1H, CONH),
8.37 (s, 1H, N=CH), 7.55 (d, 1H, J = 5 Hz), 7.33 (d, 1H, J = 5 Hz), 7.09 (dd, 1H, J = 5 Hz, J = 1 Hz),
1,77 (m, 1H), 1.76 (m, 5H, cyclohexyl), 1.39 (m, 5H, cyclohexyl). 13C-NMR (50 MHz) DMSO-d6
(ppm): 176.60 and 171.57 (NC=O), 141.18 (1C), 137.29 (HC=N), 130.30 (3C). IR (KBr) νmax (cm−1):
3185 (NH); 2926 (CH3); 1656 (C=O); 1590 (C=N). % purity = 96.55% by HPLC-C18
(acetonitrile/water 7:3) (Rt = 4.75 min).
(E)-N'-((1H-imidazol-2-yl)methylene)cyclohexanecarbohydrazide (LASSBio-1602, 11): LASSBio-1602
(11) was obtained as a white solid in 57% yield by condensation of cyclohexane-carbohydrazide (28)
1
with 1H-imidazole-2-carbaldehyde, m.p. > 220 °C. H-NMR (200 MHz) DMSO-d6 (ppm): 12.60 and
12.35 (s, NH, imidazole), 11.18 and 10.95 (s, 1H, CONH), 8.09 and 7.87 (s, 1H , HC=N), 7.21 (m,
2H), 2.86 and 2.19 (m, 1H), 1.72 (m, 5H, cyclohexyl), 1.32 (m, 5H, cyclohexyl). 13C-NMR
(50 MHz) DMSO-d6 (ppm): 177.75 and 172.54 (NC=O), 143.08 (HC=N), 134.46 (1C), 137.87 (2C),
43.26 (1C), 29.39 (2C), 25.75 (3C). IR (KBr) νmax (cm−1): 3188 (NH); 2925 (CH3); 1649 (C=O). Anal.
Calcd. for C11H16N4O: C, 60.26; H, 6.90; N, 25.55. Found: C, 59.97; H, 6.95; N, 25.13.).
% purity = 98.49% by HPLC-C18 (acetonitrile/water 6:4) (Rt = 2.98 min).
(E)-N'-((1H-Imidazol-5-yl)methylene)cyclohexanecarbohydrazide (LASSBio-1600, 12): LASSBio-1600
(12) was obtained as a white solid in 46% yield by condensation of cyclohexane-carbohydrazide (28)
with 1H-imidazole-5-carbaldehyde, m.p. > 220 °C. 1H-NMR (200 MHz) DMSO-d6 (ppm): 12.36 (NH,
imidazole), 11.05 and 10.80 (s, 1H, CONH), 8.13 and 7.91 (s, 1H, HC=N), 7.69 (s, 1H), 7,42 (s, 1H),
3,13 and 2,19 (m, 1H), 1.72 (m, 5H, cyclohexyl), 1.32 (m, 5H, cyclohexyl).13C-NMR (50 MHz)
DMSO-d6 (ppm): 171.83 and 177.20 (NC=O), 137.51 (HC=N), 136.23 (1C), 132.14 (1C), 121.44
(1C), 43.34 (1C), 29.52 (2C), 25.82 (3C). IR (KBr) νmax (cm−1): 3188 (NH); 2925 (CH3); 1649 (C=O).
Anal. Calcd. for C11H16N4O: C, 60.26; H, 6.90; N, 25.55. Found: C, 60.01; H, 7.23; N, 26.00.
(E)-N'-(Pyridin-4-ylmethylene)cyclohexanecarbohydrazide (LASSBio-1514, 13): LASSBio-1514 (13)
was obtained in 79% yield as yellow crystals by condensation of cyclohexane-carbohydrazide (28)
with 4-pyridinecarboxaldehyde, m.p. 151–153 °C. The compound’s data was in agreement with
1
previous reports [35]. H-NMR (200 MHz) DMSO-d6 (ppm): 11.55 and 11.46 (s, 1H, CONH), 8.63
(m, 2H), 8.34 and 8.17(s, 1H, N=CH), 7.59 (m, 2H), 3.10 and 2.19 (m, 1H), 1.72 (m, 5H, cyclohexyl),

Molecules 2015, 20
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13
1.32 (m, 5H, cyclohexyl). C-NMR (50 MHz) DMSO-d6 (ppm): 177.28 and 172.08 (NC=O), 150.22
(2C), 143.46 and 139.82 (HC=N), 141.66 (1C), 120.84 (2C), 43.40 (1C), 28.89 (2C), 25.31 (3C). IR
(KBr) νmax (cm−1): 3478 (NH); 2934 (CH3); 1664 (C=O); 1599 (C=N); 1397 (Py). Anal. Calcd. for
C13H17N3O.H2O: C, 62.63; H, 7.68; N, 16.85. Found: C, 62.22; H, 7.61; N, 16,69.
(E)-N'-(4-Hydroxy-3-methoxybenzylidene)cyclohexanecarbohydrazide (LASSBio-1689, 14): LASSBio-1689
(14) was obtained in 90% yield as white crystals by condensation of cyclohexane-carbohydrazide (28)
1
with 4-hydroxy-3-methoxybenzaldehyde, m.p. 198–200 °C. H-NMR (200 MHz) DMSO-d6 (ppm):
11.43 and 11.10 (s, 1H, CONH), 9.47 (s, 1H, OH), 8.00 (s, 1H, N=CH), 7.24 (d, 1H, J = 8 Hz), 7.04
(d, 1H, J = 8 Hz), 6.82 (d, 1H, J = 8 Hz), 3.80 (s, 3H, CH3), 3.10 and 2.19 (m, 1H), 1.72 (m, 5H,
cyclohexyl), 1.32 (m, 5H, cyclohexyl).13C-NMR (50 MHz) DMSO-d6 (ppm): 176.66 and 171.44
(NC=O), 148.76 (1C), 148.02 (1C), 146.50 and 142.53 (N=CH), 125.90 (1C), 121.81 (1C), 115.63 (1C),
109.39 (1C), 55.58 (1C), 42.89 (1C), 29.09 (2C), 25.73 (3C). IR (KBr) νmax (cm−1): 3528–2850 (OH);
3528 (NH); 2930 (CH3); 1641 (C=O); 1281, 1204 (OCH3). % purity = 99.87% by HPLC-C18
(acetonitrile/water 7:3) (Rt = 3.48 min).
(E)-N'-(3-Hydroxy-4-methoxybenzylidene)cyclohexanecarbohydrazide (LASSBio-1688, 15): LASSBio-1688
(15) was obtained in 92% yield as white crystals by condensation of cyclohexane-carbohydrazide (28)
1
with 3-hydroxy-4-methoxybenzaldehyde, m.p. 192–194 °C. H-NMR (200 MHz) DMSO-d6 (ppm):
11.51 and 11.11 (s, 1H, CONH), 9.29 (s, 1H, OH), 8.00 (s, 1H, N=CH), 7.19 (d, 1H, J = 8 Hz), 6.99
(d, 2H, J = 8 Hz), 3.80 (s, 3H, CH3), 3.10 and 2.19(m, 1H), 1.72 (m, 5H, cyclohexyl), 1.32 (m, 5H,
13
cyclohexyl). C-NMR (50 MHz) DMSO-d6 (ppm): 176.71 and 171.48 (NC=O), 149.61 (1C), 146.83
(N=CH), 142.59 (1C), 127.33 (1C), 120.03 (1C), 112.30 (1C), 111.90 (1C), 55.62 (1C), 43.91 (1C),
29.08 (2C), 25.41 (3C). IR (KBr) νmax (cm−1): 3234–2850 (OH); 3234 (NH); 2930 (CH3); 1656 (C=O)
1259, 1204 (OCH3). % purity = 98.51% by HPLC-C18 (acetonitrile/water 6:4) (Rt = 3.97 min).
(E)-N'-(Benzo[d][1,3]dioxol-5-ylmethylene)cyclohexanecarbohydrazide
(LASSBio-1691,
16):
LASSBio-1691 (16) was obtained in 98% yield as a white solid by condensation of
cyclohexane-carbohydrazide (28) with benzo[d][1,3]dioxole-5-carbaldehyde, m.p. 206–208 °C. The
compound’s data was in agreement with previous reports [36]. 1H-NMR (200 MHz) DMSO-d6 (ppm):
11.18 and 10.98 (s, 1H, CONH), 8.08 and 7.84 (s, 1H, N=CH), 7.22 (s, 1H), 7.09 (d, 1H, J = 8 Hz),
6.94 (d, 1H, J = 8 Hz), 6.11 (s, 2H, CH2), 3.10 and 2.19 (m, 1H), 1.72 (m, 5H, cyclohexyl), 1.32 (m,
5H, cyclohexyl). 13C-NMR (50 MHz) DMSO-d6 (ppm): 177.31 and 172.07 (NC=O), 149.41 (1C),
148.47 (1C), 146.28 and 142.54 (N=CH), 129.42 (1C), 123.53 (1C), 108.94 (1C), 105.58 (1C), 102.01
(1C, O-CH2-O), 43.40 (1C), 29.56 (2C), 25,83 (3C). IR (KBr) νmax (cm−1): 3228 (NH); 2942 (CH3);
1659 (C=O) 1247 (OCH3). % purity = 95.33% by HPLC-C18 (acetonitrile/water 7:3) (Rt = 4.68 min).
(E)-N'-(3,4,5-Trimethoxybenzylidene)cyclohexanecarbohydrazide (LASSBio-1513, 17): LASSBio-1513
(17) was obtained in 99% yield as a white solid by condensation of cyclohexane-carbohydrazide (28)
1
with 3,4,5-trimethoxybenzaldehyde, m.p. 215–217 °C. H-NMR (200 MHz) DMSO-d6 (ppm): 11.27
and 11.11 (s, 1H, CONH), 8.65 and 8.10 (s, 1H, N=CH), 7.22 (s, 2H), 3.81 (s, 9H), 3.10 and 2.19 (m,
1H), 1.72 (m, 5H, cyclohexyl), 1.32 (m, 5H, cyclohexyl). 13C-NMR (50 MHz) DMSO-d6 (ppm):
177.42 and 172.21 (NC=O), 153.69 (2C), 146.49 and 142.24 (HC=N), 139.60 (1C), 130.60 (1C),

Molecules 2015, 20
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106.21 (2C), 60.71 (1C), 56.49 (2C), 43.40 (1C), 29.56 (2C), 25.73 (3C). IR (KBr) νmax (cm−1): 3230
(NH); 2925 (CH3); 1660 (C=O); 1236 (O-CH3). Anal. Calcd. for C17H24N2O4: C, 63.73; H, 7.55; N,
8.74. Found: C, 63.54; H, 7.42; N, 8.51.
(E)-N'-(3,4-Dimethoxybenzylidene)cyclohexanecarbohydrazide (LASSBio-1509, 18): LASSBio-1509
(18) was obtained in 94% yield as a white solid by condensation of cyclohexane-carbohydrazide (28)
with 3,4-dimethoxybenzaldehyde, m.p. 193–195 °C. 1H-NMR (200 MHz) DMSO-d6 (ppm): 11.15 and
10.98 (s, 1H, CONH), 8.09 and 7.89 (s, 1H, N=CH), 7.27 (d, 1H, J = 2 Hz), 7.14 (dd, 1H, J = 8 Hz, J =
2 Hz), 6.99 (d, 1H, J = 8 Hz), 3.82 (s, 6H, OCH3), 3.10 na 2.19 (m,1H), 1.72 (m, 5H, cyclohexyl), 1.32
13
(m, 5H, cyclohexyl). C-NMR (50 MHz) DMSO-d6 (ppm): 177.24 and 172.00 (NC=O), 161.08 and
152.10 (1C), 150.90 (1C), 146.47 and 142.82 (HC=N), 127.79 (1C), 122.04 (1C), 112.14 (1C), 108.83
(1C), 55.98 (2C), 43.40 (1C), 29.57 and 28.92 (2C), 25.97 (3C). IR (KBr) νmax (cm−1): 3223 (NH); 2926
(CH3); 1659 (C=O); 1603 (C=N); 1266, 1210(C-O). Anal. Calcd. for C16H22N2O3: C, 66.18; H, 7.64; N,
9.65. Found: C: 66.17; H: 7.48; N: 9.40.
(E)-N'-Ferrocenylcyclohexanecarbohydrazide (LASSBio-1517, 19): LASSBio-1517 (19) was obtained
in 99% yield as a brown solid by condensation of cyclohexane-carbohydrazide (28) with
1
ferrocenecarboxaldehyde, m.p. > 220 °C. H-NMR (200 MHz) DMSO-d6 (ppm): 11.01 and 10.72 (s,
1H, CONH), 7.98 and 7.83 (s, 1H, N=CH), 4.57 (s, 2H), 4.42 (s, 2H), 4.19 (s, 5H), 2.86 and 2.19 (m,
1H), 1.72 (m, 5H, cyclohexyl), 1.32 (m, 5H, cyclohexyl). 13C-NMR (50 MHz) DMSO-d6 (ppm):
176.09 (NC=O), 142.67 (HC=N), 79.42 (1C), 69.83 (2C), 68.79 (5C), 67.28 (2C), 43.40 (1C), 29.51
(2C), 25.89 (3C). IR (KBr) νmax (cm−1): 3189 (NH); 2929 (CH3); 1652 (C=O). Anal. Calcd. for
C18H22FeN2O: C, 63.92; H, 6.56; N, 8.28. Found: C, 63.54; H, 6.37; N 8.28.
(E)-N'-(4-Nitrobenzylidene)cyclohexanecarbohydrazide (LASSBio-1603, 20): LASSBio-1603 (20)
was obtained in 83% yield as white crystals by condensation of cyclohexane-carbohydrazide (28) with
4-nitrobenzaldehyde, m.p. 205–206 °C. 1H-NMR (200 MHz) DMSO-d6 (ppm): 11.59 and 11.17 (s, 1H,
CONH), 8.29 (d, 2H, J = 8 Hz), 8.07 (s, 1H, N=CH), 7.94 (d, 2H, J = 8 Hz), 3.10 and 2.19 (m, 1H),
1.72 (m, 5H, cyclohexyl), 1.32 (m, 5H, cyclohexyl). 13C-NMR (50 MHz) DMSO-d6 (ppm): 177.82 and
172.61 (NC=O), 148.33 (1C), 143.99 (N=CH), 141.03 (1C), 128.33 (2C), 124.63 (2C), 43.42 (1C),
29.47 (2C), 25.89 (3C). IR (KBr) νmax (cm−1): 3186 (NH); 2930 (CH3); 1668 (C=O) 1520, 1336 (NO2).
Anal. Calcd. for C14H17N3O3: C, 61.08; H, 6.22; N, 15.28. Found: C, 60.81; H, 6.10; N. 14.97.
(E)-4-((2-(Cyclohexanecarbonyl)hydrazono)methyl)benzoic acid (LASSBio-1516, 21): LASSBio-1516
(21) was obtained in 83% yield as a white solid by condensation of cyclohexane-carbohydrazide (28)
with 4-formylbenzoic acid, m.p. > 220 °C. 1H-NMR (200 MHz) DMSO-d6 (ppm): 11.37 and 11.17 (s,
1H, CONH), 8.22 and 8.03 (s, 1H, N=CH), 7.97 (d, 2H, J = 8 Hz), 7.76 (d, 2H, J = 8 Hz), 3.39 (s,
OH), 3.10 and 2.19 (m, 1H), 1.72 (m, 5H, cyclohexyl), 1.32 (m, 5H, cyclohexyl). 13C- NMR (50 MHz)
DMSO-d6 (ppm): 177.65 and 172.42 (NC=O), 167.45 (COOH), 145.24 and 141.81 (HC=N), 139.09
(1C), 132.03 (1C), 130.34 (2C), 127.43 (2C), 43.40 (1C), 29.51 (2C), 25.89 (3C). IR (KBr) νmax
(cm−1): 3211–2855 (OH); 3211 (NH); 2925 (CH3); 1669 (C=O). Anal. Calcd. for C15H18N2O3: C,
65.68; H, 6.61; N, 10.21. Found: C, 65.46; H, 6.55; N, 9.88.

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(E)-N'-(4-Bromobenzylidene)cyclohexanecarbohydrazide (LASSBio-1511, 22): LASSBio-1511 (22)
was obtained in 98% yield as an orange solid by condensation of cyclohexane-carbohydrazide (28)
with 4-bromobenzaldehyde, m.p. 215–216 °C. 1H-NMR (200 MHz) DMSO-d6 (ppm): 11.36 and 11.14
(s, 1H, CONH), 8.15 and 7.94 (s, 1H, N=CH), 7.62 (m, 4H,), 3.10 and 2.19 (m, 1H), 1.72 (m, 5H,
cyclohexyl), 1.32 (m, 5H, cyclohexyl). 13C-NMR (50 MHz) DMSO-d6 (ppm): 177.51 and 172.29
(NC=O), 145.19 and 141.69 (HC=N), 134.31 (1C), 132.29 (2C), 129.29 (2C), 123.52 (1C), 43.40 (1C),
29.51 (2C), 25.89 (2C), 18.92 (1C). IR (KBr) νmax (cm−1): 3188 (NH); 2925 (CH3); 1662 (C=O);
1601(C=N); 1138 (Ar-Br). Anal. Calcd. for C14H17BrN2O: C, 54.58; H, 5.54; N, 9.06. Found: C, 54.39;
H, 5.50; N 8.92.
(E)-N'-Benzylidenecyclohexanecarbohydrazide (LASSBio-1601, 23): LASSBio-1601 (23) was obtained
in 99% yield as a white solid by condensation of cyclohexane-carbohydrazide (28) with benzaldehyde,
m.p. 165 °C. The compound’s data was in agreement with previous reports [36]. 1H-NMR (200 MHz)
DMSO-d6 (ppm): 11.28 and 11.08 (s, 1H, CONH), 8.18 and 7.98 (s, 1H, HC=N), 7.66 (m, 2H), 7.43
(m, 3H), 3.10 and 2.19 (m, 1H), 1.72 (m, 5H, cyclohexyl), 1.32 (m, 5H, cyclohexyl). 13C-NMR
(50 MHz) DMSO-d6 (ppm): 176.92 and 171.68 (NC=O), 145.90 and 142.28 (HC=N), 135.72 (1C),
129.79 (1C), 128.84 (2C), 126.89 (2C), 43.88 (1C), 29.01 (2C), 25.66 (3C). IR (KBr) νmax (cm−1): 3177
(NH); 2927 (CH3); 1663 (C=O); 1610 (C=N); 792 and 684 (Aryl). Anal. Calcd. for C14H18N2O: C,
63.92; H, 6.56; N, 8.28. Found: C, 63.44; H, 6.37; N, 8.28.
(E)-N'-(4-Isopropylbenzylidene)cyclohexanecarbohydrazide (LASSBio-1508, 24): LASSBio-1508
(24) was obtained in 98% yield as a yellow solid by condensation of cyclohexane-carbohydrazide (28)
1
with 4-isopropylbenzaldehyde, m.p. 155–156 °C. H-NMR (200 MHz) DMSO-d6 (ppm): 11.22 and
11.03 (s, 1H, CONH), 8.17 and 7.94 (s, 1H, N=CH), 7.56 (d, 2H, J = 8 Hz), 7.29 (d, 2H, J = 8 Hz),
3.10 and 2.19 (m, 1H), 2.90 (m, 1H), 1.72 (m, 5H, cyclohexyl), 1.32 (m, 5H, cyclohexyl), 1.20 (s, 6H).
13C-NMR (50 MHz) DMSO-d6 (ppm): 177.35 and 172.10 (NC=O), 150.90 and 150.71 (1C), 146.47
and 142.82 (HC=N), 132.70 (1C), 127.52 and 127.33 (2C), 127.25 and 127.25 (2C), 43.42 (1C), 33.88
(1C), 29.57 and 28.92 (2C), 25.88 (3C), 24.20 (2C). IR (KBr) νmax (cm−1): 3231 (NH); 2930 (CH3);
1662 (C=O); 1601 (C=N). Anal. Calcd. for C17H24N2O:C, 74.96; H, 8.88; N, 10.30. Found: C, 74.82;
H, 8.80; N, 10.14.
(E)-N'-(4-Methoxybenzylidene)cyclohexanecarbohydrazide (LASSBio-1510, 25): LASSBio-1510 (25)
was obtained in 99% yield as a white solid by condensation of cyclohexane-carbohydrazide (28) with
4-methoxybenzaldehyde, m.p. 161–162 °C. The compound’s data was in agreement with previous
reports [36]. 1H-NMR (200 MHz) DMSO-d6 (ppm): 11.15 and 10.96 (s, 1H, CONH), 8.11 and 7.92 (s,
1H, N=CH), 7.59 (d, 2H, J = 8 Hz), 7.02 (d, 2H, J = 8 Hz), 3.68 (s, 3H, CH3), 3.10 and 2.19 (m,1H),
1.72 (m, 5H, cyclohexyl), 1.32 (m, 5H, cyclohexyl). 13C-NMR (50 MHz) DMSO-d6 (ppm): 176.60 and
171.35 (NC=O), 160.56 (1C), 145.71 and 142.06 (HC=N), 129.90 (1C), 128.37 (2C), 114.27 (2C),
55.19 (1C), 42.80 (1C), 28.97 (2C), 25,27 (3C). IR (KBr) νmax (cm−1): 3184 (NH); 2929 (CH3); 1654
(C=O); 1608 (C=N), 1252, 1202 (C-O). Anal. Calcd. for C15H20N2O2: C, 69.20; H, 7.74; N, 10.76.
Found: C, 69.23; H, 7.54; N, 10.46.

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(E)-N'-(4-(Dimethylamino)benzylidene)cyclohexanecarbohydrazide (LASSBio-1512, 26): LASSBio-1512
(26) was obtained in 89% yield as an orange solid by condensation of cyclohexane-carbohydrazide (28)
1
1
with 4-dimethylaminobenzaldehyde, m.p. 191 °C–192 °C. H-NMR (200 MHz) DMSO-d6 (ppm): H
RMN (200 MHz) DMSO-d6 δ (ppm): 11.06 and 11.64 (s,1H, CONH), 7.95 and 7.82 (s, 1H, N=CH),
7.49 (d, 2H, J = 8 Hz), 6.69 (d, 2H, J = 8 Hz), 2.91(s, 6H, CH3), 3.10 and 2.19 (m, 1H), 1.72 (m, 5H,
cyclohexyl), 1.32 (m, 5H, cyclohexyl).13C-NMR (50 MHz) DMSO-d6 (ppm): 176.46 and 171.24
(NC=O), 159.84 (1C), 146.81 and 143.16 (HC=N), 129.53 (2C), 121.78 (1C), 112.41 (2C), 43.40 (1C),
40.00 (2C), 29.51 (2C), 25.89 (3C). IR (KBr) νmax (cm−1): 3222 (NH); 2926 (CH3); 1658 (C=O); 1607
(C=N); 1362 (Ar-N(CH3)2). Anal. Calcd. for C16H23N3O: C, 70.30; H, 8.48; N, 15.37. Found: C, 70.44;
H, 8.35; N, 15.70.
3.2. X-ray Crystallography
In this work, X-ray powder diffraction data and a simulated annealing algorithm implemented in the
DASH software program [37] were used to determine the crystal structure of LASSBio-1515, on the
basis of previous procedures [38,39]. Thus, a Rietveld refinement of the final crystal structure was
conducted using the Topas Academic v. 5 software [40]. Data pertaining to the X-ray crystallographic
determination of LASSBio-1515 have been deposited in the Cambridge Crystallographic Data Centre
with CCDC ID: 929463. These data can be obtained free of charge from the Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
3.3. Binding Assay
A binding assay was performed between NAH compounds (10 μM or 30 μM) and A2A receptors of
rats [41]. [3H]CGS21680 (10 nM) was used as A2A agonist radioligand and 30 µM NECA was used to
estimate the nonspecific binding. The data were expressed as percent inhibition of control specific binding
using the equation: % inhibition =100 − [(measured specific binding/control specific binding) ×100].
3.4. Antinociceptive and Anti-Inflammatory Pharmacological Evaluation
3.4.1. Animals
Swiss mice weighing 20–30 g (from the BIOCEN-UFAL) were housed in group cages and
maintained on a 12 h light/12 h dark cycle. Animals had free access to food and water at all times.
Experiments were carried out according to a protocol approved by the Animal Welfare Committee of
Federal University of Alagoas (UFAL) (Number: 026681/2009-23), and in according with the ethical
guidelines for investigation of experimental pain in conscious animals.
3.4.2. Reagents
Acetic acid (Merck, Rio de Janeiro, Brasil), arabic gum (Sigma-Aldrich, Rio de Janeiro, RJ, Brasil),
morphine sulphate (Dimorf-Cristalia-BR, São Paulo, Brasil), dipyrone (Sigma-Aldrich) and
indomethacin (Sigma Aldrich) were obtained from commercial sources. A solution of formalin 2.5%
was prepared with formaldehyde (Merck) in saline (NaCl 0.9%).

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3.4.3. Acetic Acid-Induced Writhing Test
This test was performed as described [27]. Acetic acid (0.6%, v/v) was administered i.p. in a volume
of 0.1 mL/10 g. The number of writhes, a response consisting of contraction of an abdominal wall,
pelvic rotation followed by hind limb extension, was counted during continuous observation for 20 min
beginning from 5 min after the acetic acid injection. Dipyrone and compounds (all 100 µmol/kg, oral
administration) were administered 60 min before the acetic acid injection. Antinociceptive activity was
expressed as inhibition percent of the usual number of writhing observed in control animals.
3.4.4. Formalin-Induced Nociception
The procedure used was essentially the same as that described previously [26]. Animals received
20 mL of 2.5% formalin solution (0.92% formaldehyde in saline) in the ventral surface of the right
hind paw. Animals were observed from 0 to 5 min (neurogenic phase) and from 15 to 30 min
(inflammatory phase) and the time that they spent licking the injected paw was recorded and
considered as indicative of nociception. Animals received indomethacin or compounds (100 µmol/kg,
oral administration) 40 min beforehand. Control animals received vehicle (Arabic gum).
3.4.5. Hot-Plate Test
Mice were treated according to the method described by Kuraishi et al. [42]. Animals (n = 6) were
placed on a hot-plate set at 55 ± 1 °C. Reaction time was recorded when the animals licked their fore
and hind-paws and jumped at 30, 60, 90 and 120 min after oral administration of 100 µmol/kg of
compounds or reference drug (morphine, 15 µmol/kg. i.p.). Baseline was considered as the mean of
reaction time obtained at 30 and 60 min before administration of derivatives or morphine and was
defined as normal reaction of animal to the temperature.
3.4.6. Carrageenan-Induced Peritonitis
Peritoneal inflammation was induced according to the method described by Ferrandiz and Alcaraz [28].
A solution of carrageenan 1% (Sigma-Aldrich) was prepared in saline (NaCl 0.9%) and injected into
the peritoneal cavity of mice (250 µL/animal). Four h after injection of carrageenan, the animals were
killed by cervical dislocation and the peritoneal cavity was washed with 3 mL of cold Hank’s.
Compounds and indomethacin were administered at the dose of 100 µmol/kg (p.o.), 30 min before
carrageenan injection. Control group received 10 mL/kg of vehicle (Arabic gum, p.o.). The number of
cells was quantified by optical microscope, using 100 × lens.
3.4.7. Statistical Analysis
Data obtained from animal experiments are represented by mean ± standard error of the mean
(Mean ± S.E.M.). Statistical differences between the treated and the control groups were evaluated by
ANOVA in the Prisma® tutorial. Values were considered significant if * p < 0.05 and ** p < 0.01.

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3.5. Thermal Sensitization and Mechanical Allodynia Induced by Spinal Nerve Ligation
3.5.1. Animals
Male Wistar rats (Rattus norvegicus) weighing 180 to 220 g were used at the start of surgery. The
animals were maintained in a temperature (25 °C) and humidity (50%–60%) controlled room on a 12 h
light: 12 h dark cycle. The Institutional Animal Care and Use Committee at Universidade Federal do
Rio de Janeiro (UFRJ) approved the procedures in this study. Animals were acclimated to the
laboratory for at least 30 min before experimental initiation. The study was approved by the Animal
Care and Use Committee at Universidade Federal do Rio de Janeiro (UFRJ; Rio de Janeiro, Brazil)
3.5.2. Surgery
Peripheral neuropathy was induced in rats by spinal nerve ligation [33]. Briefly, rats were
anesthetized with ketamine (100 mg·kg−1 i.p.) and xylazine (5 mg·kg−1 i.p.), the skin was sterilized
with 0.5% chlorhexidine. A small incision to the skin overlying L5-S1 was made followed by retraction
of the paravertebral musculature from the vertebral transverse processes. The L6 transverse process
was partially removed, exposing the L4 and L5 spinal nerve. The L5 spinal nerve was tightly ligated
using 6-0 silk suture. For sham animals (animals false operated), the same process was carried out, but
the spinal nerve was not ligated.
3.5.3. Behavioral Tests
The withdrawal latency was assessed by applying a radiant heat source to the hind paw of the
animals [43]. The light beam was interrupted when the animal lifted the hind paw, allowing
measurement the time of paw remained in contact with the heat source. Withdrawal latency was
determined using a Dynamic Plantar Anesthesiometer (model 37450, UgoBasile SRL, Varese, Italy).
The latency to evoke paw-withdrawal was determined with a cut-off value of 30 s. Control latency was
determined using the average response of three measurements. Withdrawal threshold to pressure
applied in the hind paw, expressed in grams, was measured using a digital analgesic meter (model
EFF301, Insight, Sao Paulo, Brazil) [44]. The stimulation of the paw was repeated five times.
Withdrawal threshold was determined with a cut-off value of 120 g to avoid potential tissue injury in
the absence of response.
3.5.4. Experimental Design
Forty two Wistar rats were randomly divided into sham and spinal nerve ligation (SNL) groups,
which were subdivided into six groups: SNL/DMSO, SHAM/DMSO, SNL/(13), SNL/(17), SNL/(19)
and SNL/tramadol. At seven days post-surgery, SNL animals were treated by oral administration with
compounds during seven or fourteen days. Thermal hyperalgesia and mechanical allodynia were
measured at the 10th and 14th day after surgery. In other group, thermal hyperalgesia and mechanical
allodynia were measured at the 10th, 14th, 17th and 21th days after surgery.

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3.5.5. Statistical Analysis
Values are expressed as means ± standard error of means (SEM) and one-way ANOVA followed by
the Newman–Keuls test for multiple comparisons, using GraphPad Prism (version 5.0; GraphPad
Software, Inc., San Diego, CA, USA). p values of <0.05 were considered significant.
4. Conclusions
A series of cyclohexyl-N-acylhydrazones were designed as simplified analogues of the prototype
LASSBio-294, synthesized and evaluated as analgesic and anti-inflammatory agents. The
configuration and conformation of the privileged structure N-acylhydrazone (NAH) were determined
from compound 10. Employing X-ray powder diffraction, the configuration E (N=CH) and the
antiperiplanar conformation of the amide subunit (-CONH-) of the NAH framework were
unequivocally determined. These compounds 10–26 and their conformational restricted analogue 9
showed great anti-inflammatory and/or analgesic activity, with compound 13 (LASSBio-1514)
standing out due to its important analgesic activity in a neuropathic pain model.
Acknowledgments
The authors would like to thank INCT-INOFAR (BR, 573.564/2008-6 and E-26/170.020/2008),
FAPERJ (BR), FAPEAL (BR), CNPq (BR) and CAPES (BR) for fellowship and financial support.
Author Contributions
T.F.S performed the synthesis and structure characterization; W.B.J. and M.S.A-M. performed the
analgesic and antiinflamatory evaluation; F.N.C; F.F.F. and R.C.R.B. performed X-ray experiments;
C.E.S.M; R.T.S. and G.Z-S. performed the neuropathic pain experiment; F.N. performed the binding
assay; L.M.L and E.J.B. designed the research. All the authors contributed in some way to the writing
of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 8–26 are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).