3-Aminothiophene-2-acylhydrazones: Non-toxic, analgesic and anti-inflammatory lead-candidates

Article abstract of DOI:10.3390/molecules19068456

Different chemotypes are described as anti-inflammatory. Among them the N-acylhydrazones (NAH) are highlighted by their privileged structure nature, being present in several anti-inflammatory drug-candidates. In this paper a series of functionalized 3-ami

Full text of DOI:10.3390/molecules19068456

Molecules 2014, 19, 8456-8471; doi:10.3390/molecules19068456
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
3-Aminothiophene-2-Acylhydrazones: Non-Toxic, Analgesic and
Anti-Inflammatory Lead-Candidates
Yolanda Karla Cupertino da Silva 1, Christian Tadeo Moreno Reyes 2,3, Gildardo Rivera 3,
Marina Amaral Alves 2, Eliezer J. Barreiro 2, Magna Suzana Alexandre Moreira 1,* and
Lídia Moreira Lima 2,*
1
LaFI—Laboratório de Farmacologia e Imunidade, Instituto de Ciências Biológicas e da Saúde,
Universidade Federal de Alagoas, Maceió 57072-900, AL, Brazil;
E-Mail: yolandakarla@yahoo.com.br
2
Laboratório de Avaliação e Síntese de Substâncias Bioativas—LASSBio, Programa de Pesquisa em
Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de
Janeiro, PO Box 68024, Rio de Janeiro 21944-902, RJ, Brazil;
E-Mails: cristian@buscarsalud.com (C.T.M.R.); marinamaral@hotmail.com (M.A.A.);
ejbarreiro@ccsdecania.ufrj.br (E.J.B.)
3
Centro de Biotecnologia Genomica, Instituto Politecnico Nacional, Boulevard del Maestro, s/n,
88710 Reynosa, Mexico; E-Mail: gildardors@hotmail.com
* Authors to whom correspondence should be addressed;
E-Mails: suzana.magna@gmail.com (M.S.A.M.); lidia@lassbio.icb.ufrj.br (L.M.L.);
Tel./Fax: +55-21-3938-6503 (L.M.L.).
Received: 19 May 2014; in revised form: 6 June 2014 / Accepted: 5 June 2014 /
Published: 20 June 2014
Abstract: Different chemotypes are described as anti-inflammatory. Among them the
N-acylhydrazones (NAH) are highlighted by their privileged structure nature, being present
in several anti-inflammatory drug-candidates. In this paper a series of functionalized
3-aminothiophene-2-acylhydrazone derivatives 5a–i were designed, synthesized and bioassayed.
These new derivatives showed great anti-inflammatory and analgesic potency and efficacy.
Compounds 5a and 5d stand out in this respect, and were also active in CFA-induced arthritis
in rats. After daily treatment for seven days with 5a and 5d (50 µmol/Kg), by oral
administration, these compounds were not renal or hepatotoxic nor immunosuppressive.
Compounds 5a and 5d also displayed good drug-scores and low risk toxicity calculated
in silico using the program OSIRIS Property Explorer.

Molecules 2014, 19
Keywords: anti-inflammatory; toxicity; acylhydrazone; analgesic; arthritis; privileged structure
8457
1. Introduction
Inflammation is an adaptive response that is triggered by noxious stimuli and conditions, such as
infection and tissue injury, and can be classified into acute and chronic [1,2]. The role of inflammation
in the etiology of several diseases is well-documented [3,4]. Many pathways are involved in acute and
chronic inflammatory processes, among them the cyclooxygenase (COX) and p38 mitogen-activated
protein kinase (p38 MAPK). These enzymes have been studied as important targets for the design of
novel anti-inflammatory drugs [5–8]. COX and its isoenzymes (COX-1; COX-2 and COX-3) are the
molecular targets of non-steroidal anti-inflammatory drugs (e.g., indomethacin, diclofenac and
celecoxib). COX-3 is believed to be responsible for the antipyretic and analgesic effects of dipyrone
and paracetamol [9]. Distinct chemotypes are described as anti-inflammatory, and among them the
N-acylhydrazones (NAH) are especially noteworthy [10]. These privileged structures [11], exemplified
by compounds 1–4 (Figure 1), are present in several anti-inflammatory drug-candidates acting by
different mechanism of action [12–18].
Figure 1. Examples of anti-inflammatory lead-candidates presenting the privileged
N-acylhydrazone structure.
In a continuous effort to identify new anti-inflammatory drug-candidates a series of functionalized
3-aminothiophene-2-acylhydrazone derivatives 5a–i were designed by molecular modifications of the
prototype 1. These modifications aimed to preserve the N-acylhydrazone moiety (CONHN=CH) and
were based on application of ring isosterism between the pyridine and thiophene nucleus, and on
molecular simplification with consequent elimination of the methyl group of the original prototype 1
(Figure 2). The aromatic pattern of compound 5a was later modified following classical isosteric
replacement of monovalent groups and equivalent rings exchange [19] (Figure 2).

Molecules 2014, 19
Figure 2. Design concept of the 3-aminothiophene-2-acylhydrazone derivatives 5a–i.
8458
c
O
O
a
a
N
N
S
N
N
H
H
(1)
N
NH
NH2
5a
classical
isosterism
ring isosterism
molecular simplification
CH3
b
Br
NO2
OH
5b
5c
5e
5d
N(CH3)2
CO2H
S
N
5h
5i
5g
5f
2. Results and Discussion
2.1. Chemistry
The functionalized 3-aminothiophene-2-acylhydrazone derivatives 5a–i were synthesized as depicted
in Scheme 1, following previously described methodology [20–23]. Compounds were characterized by
IR, 1H- and 13C-NMR and mass spectroscopy. The purity was determined by HPLC. The stereochemistry of
the imine double bond (N=CH) was assigned as the E-isomer. This isomer was assigned based on the
1
chemical shift of the imine-hydrogen singlet signal visualized in the H-NMR spectra of NAH
derivatives 5a–i and considering similar data previously reported in the literature [21–23].
Scheme 1. Synthesis of 3-aminothiophene-2-acylhydrazones derivatives 5a–i.
O
O
O
N
Ar
S
S
S
NHNH2
N
b)
OCH3
a)
H
NH2
5a-i
NH2
NH2
Ar = Ph (5a); 4-isopropylPh (5b); 4-BrPh (5c); 4-NO2Ph (5d); 4-OHPh (5e);
4-N(CH3)2Ph (5f); 4-CO2HPh (5g); 2-pyridinyl (5h); 2-thiophene (5i)
Reagents and Conditions: (a) N2H4.H2O (80%), EtOH, reflux, 24 h, 64%; (b) ArCHO, EtOH,
HCl (cat), r.t., 1 h, 47%–95%.
2.2. In Silico Drug-Like Profile and Toxicity
In order to investigate the potential toxicity of N-acylhydrazones 5a–i and their drug-like profile,
these compounds were evaluated in silico using the Program OSIRIS Property Explorer [24].

Molecules 2014, 19
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As showed in Figure 3A,B, with exception of compounds 5g and 5d, the 3-aminothiophene-2-
acylhydrazones 5a–i were predicted to have similar druglikeness as dipyrone and dexamethasone, and
inferior to that of indomethacin. Examining the drug-score parameter, OSIRIS predicted all compound
5a–i as being better than dipyrone. Likewise, derivatives 5a, 5e, 5h and 5i were predicted to have
similar drug-score as dexamethasone, while compounds 5c, 5f and 5g were similar to indomethacin.
Despite the close structural similarity between compounds 5a–i, the in silico study predicted different
toxic profiles (Table 1). Compounds 5a, 5c, 5d, 5g and 5i were predicted with low risk of toxicity;
whereas derivatives 5b, 5e, 5f and 5h showed moderate to high theoretical toxicity risk, for at least one
of the parameters analyzed by the Osiris program.
Figure 3. Drug-like profile of 3-aminothiophene-2-carbohydrazide derivatives 5a–i calculated
by Osiris Program. (A) druglikeness; (B) drug-score values.
Table 1. In silico toxicity of 3-aminothiophene-2-carbohydrazide derivatives 5a–i calculated
using Osiris program [24,25].
Theoretical Toxicity Risks *
Compound
Mutagenic Tumorigenic Irritant Reproductive Effects
Dipyrone
3
1
1
1
1
1
1
1
1
1
3
1
3
1
1
1
1
1
1
1
3
1
1
1
1
1
1
1
3
1
1
2
1
1
1
1
3
1
1
1
1
1
1
2
1
1
1
1
Dexamethasone
Indomethacin
5a
5b
5c
5d
5e
5f
5g
5h
5i
* Theoretical toxicity risks calculated using Osiris program. The scale of side effects is low (1), medium (2),
and high (3) toxicity profile.

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2.3. Biological Assays
To investigate the antinociceptive profile of compounds 5a–i, the dose-response of these derivatives
in the acetic acid-induced abdominal constriction test was determined [26]. Mice were treated with
compounds 5a–i in doses of 100, 30, 10, 3 and 1 µmol/Kg, by oral administration, using dipyrone as
standard drug. As exemplified in Table 2, with the exception of compounds 5e and 5g, that were
inactive, the N-acylhydrazone derivatives 5a–i showed high potency and efficacy. Compounds 5a
(ID50 = 3.5 ± 0.1 µmol/Kg), 5c (ID50 = 2.3 ± 0.4 µmol/Kg), 5d (ID50 = 2.6 ± 0.5 µmol/Kg) and 5h
(ID50 = 2.5 ± 0.4 µmol/Kg) were more potent than the standard dipyrone (ID50 = 11.4 ± 4.9 µmol/Kg).
Table 2. ID50s of compounds 5a–i (100, 30, 10, 3 and 1 µmol/Kg, p.o.) and dipyrone (100,
30, 10, 3 and 1 µmol/Kg, p.o.) in the 0.6% acetic acid-induced abdominal constrictions in
mice assay, for a period of 25 min.
Compound ID50 (µmol/Kg ± S.E.M.) Emax (% ± S.E.M.)
Dipyrone
11.4 ± 4.9
3.5 ± 0.1
6.1 ± 2.2
2.3 ± 0.4
2.6 ± 0.5
>100
75.2 ± 5.8
72.6 ± 4.1
64.3 ± 8.5
76.7 ± 5.2
81.0 ± 5.7
-
5a
5b
5c
5d
5e
5f
32.8 ± 17.3
>100
74.5 ± 9.6
-
5g
5h
5i
2.5 ± 0.4
7.8 ± 3.2
84.1 ± 5.6
86.8 ± 5.2
Next, these compounds were evaluated in a formalin test [27] using a screening dose of 30 µmol/Kg
(per os). All compounds were able to reduce the nociception response in phase 1 (neurogenic) and
phase 2 (inflammatory) of formalin test. Compounds 5d and 5i produced noteworthy inhibition during
the inflammatory phase of the formalin assay (Figure 4A,B).
The anti-inflammatory activity of compounds 5a–i was evaluated using a carrageenan-induced
peritonitis test [28]. Animals were treated with 5a–i by oral administration in doses of 100, 30, 10, 3
and 1 µmol/Kg. The anti-inflammatory potency of compounds 5a–i is listed in Table 3. All
compounds, with exception of 5e and 5i promoted a significant reduction of cell recruitment, being
derivatives 5a (ID50 = 7.2 ± 1.8 µmol/Kg) and 5d (ID50 = 5.2 ± 2.0 µmol/Kg) the most promising,
showing anti-inflammatory potency and efficacy similar to the standard indomethacin (Table 3).
One the anti-inflammatory profile of 3-aminothiophene-2-acylhydrazones 5a–i was confirmed in a
murine acute inflammatory model, compounds 5a and 5d were selected in order to investigate their
activity in a chronic model of inflammation. Therefore, these compounds (50 µmol/Kg, pathway
orally) were evaluated in an arthritis model induced by complete Freund’s adjuvant (CFA) in rats [29],
and the results were compared to those obtained for the standard dexamethasone (5 µmol/Kg, pathway
orally, Figure 5). As demonstrated in Figure 5, the daily treatment for seven days with compounds 5a
and 5d was able to reduce paw edema on the 17th to 21st days of experiment, although, this reduction
was less prominent than that caused by the standard dexamethasone.

Molecules 2014, 19
Figure 4. Effect of compounds 5a–i (30 µmol/Kg, p.o.) and indomethacin (10 µmol/Kg, p.o)
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on formalin (2.5%) test in mice. (A) neurogenic phase; (B) inflammatory phase. Statistical
differences between the treated and the control groups were evaluated by ANOVA and
Dunnett tests and the asterisks denote the levels of significance in comparison with control
groups. *** p < 0.001; ** p < 0.01 and * p < 0.05.
Table 3. ID50 of compounds 5a–i (100, 30, 10, 3 and 1 µmol/Kg, p.o.) and indomethacin
(100, 30, 10, 3 and 1 µmol/Kg, p.o.) on the carrageenan-induced peritonitis in mice.
Compound
ID50 (µmol/Kg ± S.E.M.)
3.3 ± 1.0
Emax (% ± S.E.M.)
73.8 ± 5.1
69.7 ± 12.1
73.9 ± 11.7
72.7 ± 5.9
70.3 ± 5.2
-
Indomethacin
5a
5b
5c
5d
5e
5f
7.2 ± 1.8
15.9 ± 6.3
8.5 ± 4.2
5.2 ± 2.0
>100
8.8 ± 6.1
62.4 ± 5.8
75.1 ± 1.6
76.4 ± 10.1
-
5g
5h
5i
15.3 ± 12.5
20.2 ± 17.2
>100
After the 21st day of the arthritis experiment, animals were anesthetized and blood was collected to
investigate whether treatment with 5a and 5d had generated any liver or kidney toxicity. To assess
liver and renal functions the AST/ALT and urea/creatinine levels were determined on serum of
animals treated with 5a and 5d (Figures 6 and 7). No alterations were observed in the levels of
AST/ALT (Figure 6) and/or urea/creatinine (Figure 7). These data indicated that, in these conditions,
compounds 5a and 5d were neither hepatotoxic nor nephrotoxic. After the daily treatment for seven
days with 5a and 5d (50 µmol/Kg, pathway orally) the stomach of the animals was macroscopically
analyzed [30] and no signal of redness or bleeding was observed (data not shown).

Molecules 2014, 19
Figure 5. Effect of compounds 5a (50 µmol/Kg, p.o.) and 5d (50 µmol/Kg, p.o.) and
8462
dexamethasone (5 µmol/Kg; p.o.) on the CFA-induced arthritis in rats. Each point
represents the mean ± S.E.M. of six animals. Statistical differences between the treated and
the control groups were evaluated by ANOVA and Dunnett tests and the asterisks denote
the levels of significance in comparison with control groups. *** p < 0.001; ** p < 0.01
and * p < 0.05.
Figure 6. Effect of compounds 5a (50 µmol/Kg, p.o) and 5d (50 µmol/Kg, p.o) and
dexamethasone (5 µmol/Kg) on serum ALT and AST levels.Statistical differences between
the treated and the control groups were evaluated by ANOVA and Dunnett tests and the
asterisks denote the levels of significance in comparison with control groups. *** p < 0.001
and ** p < 0.01.

Molecules 2014, 19
Figure 7. Effect of compounds 5a (50 µmol/Kg, p.o.) and 5d (50 µmol/Kg, p.o.)
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and dexamethasone (5 µmol/Kg, p.o.) on serum Urea and Creatinine levels. Statistical
differences between the treated and the control groups were evaluated by ANOVA.
In order to discount an eventual immunosuppressive profile and considering that several indicators
of immunosuppression can be observed in standard nonclinical toxicology studies, such as alterations
in organ weight and hypocellularity of immune system tissues [31–33], spleens of animals treated daily
for seven days with compounds 5a, 5d and dexamethasone were also analyzed. No change in spleen
weight was observed after treatment with 5d (50 µmol/Kg, p.o.), whereas a slight alteration was detected
with 5a (50 µmol/Kg, p.o.). These results indicate that 5d doesn’t promote immunosuppression, while
the standard drug dexamethasone does (5 µmol/Kg, p.o.), as indicated by its ability to change the
spleen’s weight (Figure 8).
Figure 8. Effect of compounds 5a (50 µmol/Kg, p.o.) and 5d (50 µmol/Kg, p.o.) and
dexamethasone (5 µmol/Kg, p.o.) on weight of spleen. Statistical differences between the
treated and the control groups were evaluated by ANOVA and Dunnett tests and the
asterisks denote the levels of significance in comparison with control groups. *** p < 0.001
and * p < 0.05.

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3. Experimental
3.1. General Information
Acetic acid (Merck, São Paulo, SP, Brazil), arabic gum (Sigma-Aldrich, São Paulo, SP, Brazil),
carrageenan (Sigma-Aldrich), complete Freund’s adjuvant (CFA) (Sigma-Aldrich), dipyrone
(Sigma-Chemical, São Paulo, SP, Brazil), indomethacin (Merck Sharp & Dohme, Barueri, SP, Brazil)
and dexamethasone (Sigma-Chemical) were obtained from commercial sources. A solution of 2.5%
formalin was prepared with formaldehyde (Merck) in saline (NaCl 0.9%). (Merck). Kits were used for
biochemical dosage: AST, ALT, urea and creatinin (Doles-Brazil, Goiania, GO, Brazil). The caliper
was used to measure the paws (Mitutoyo, Santo Amaro, SP, Brazil).
3.2. General Procedure for the Preparation of 3-Aminothiophene-2-Carbohydrazide [23]
To a solution of methyl 3-aminothiophene-2-carboxylate (1 mmol) in ethanol (5 mL) was added
hydrazine monohydrate (35 mmol). The reaction mixture was maintained under reflux for 24 h,
at which time TLC (hexane‒ethyl acetate (1:1)) indicated the end of the reaction. Then the reaction
mixture was poured on ice and the resulting white precipitate was filtered out affording the title
compound. This compound was prepared by Reinecke and coworkers using a similar procedure [22].
1H-NMR (400 MHz, DMSO-d6) δ (ppm): δ 4.3 (s, 2H, NH2); 6.3 (s, 2H, Ar-NH2); 6.5 (d, 1H, H4),
13
7.4 (d, 1H, H3); 8.6 (s, 1H, NH). C-NMR (50 MHz, DMSO-d6) δ (ppm): 165 (CO); 152 (CNH2);
128 (C4); 120 (C1); 99 (C3). MS: m/z = 158 [M + H]+.
3.3. General Procedure for the Preparation of 3-Aminothiophene-2-Carbohydrazide Derivatives 5a–i
Under anhydrous conditions 3-aminothiophene-2-carbohydrazide (1.0 mmol), ethanol (5 mL), the
corresponding benzaldehyde derivative (1.0 mmol) and a catalytic amount of hydrochloric acid were
mixed together and stirred for 1 h at reflux and then the solvent was evaporated under reduced
pressure. Next the reaction mixture was added to ice and the precipitate obtained was purified by
column chromatography on silica gel with a CH2Cl2/MeOH 2% mobile phase.
3-Amino-N'-[(1(E)-phenylmethylene]-2-thiophenecarbohydrazide (5a; LASSBio-1660). This compound
was previously prepared by Huddleston and coworkers [22]. Yield: 48%; white solid; m.p. 170–173 °C;
IR (KBr) cm−1: 3049 (ν NH), 1639 (ν CO); 1H-NMR (400 MHz, DMSO-d6) δ (ppm): δ 4.45 (s, 2H, NH2),
6.68 (d, 1H, H5), 7.39 (m, 3H, H2', H3' & H4'), 7.62 (d, 1H, H4'), 7.74 (d, 2H, H1' & H2'),
8.04 (s, 1H, N=CH), 11.30 (s, 1H, NH) ppm; 96% purity by HPLC (R.T. = 4.2 min; CH3CN‒H2O (70:30));
MS: m/z = 246 [M + H]+. These data are in agreement with the previous report [27].
3-Amino-N'-[(1(E)-(4-isopropylphenyl)methylene]-2-thiophenecarbohydrazide (5b, LASSBio-1661).
Yield: 97%; white solid; m.p. 155–157 °C; IR (KBr) cm−1: 3301 (ν NH), 1638 (ν CO), 1599 (ν CN);
1H NMR (400 MHz, DMSO-d6) δ (ppm): δ 1.34 (s, 3H, CH3), 1.36 (s, 3H, CH3), 2.98 (m, 1H, H4),
4.55 (s, 2H, NH2), 6.62 (d, 1H, H3), 7.38 (d, 2H, H2' & H4'), 7.75 (d, 2H, H1' & H5'), 8.11 (s, 1H,
N=CH), 11.3 (s, 1H, NH) ppm; 97% purity in HPLC (R.T. = 7.3 min; CH3CN‒H2O (70:30)); MS:
m/z = 288 [M + H]+.

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3-Amino-N'-[(1(E)-(4-bromophenyl)methylene]-2-thiophenecarbohydrazide (5c, LASSBio-1654).
Yield: 62%; yellow solid; m.p. 230–233 °C; IR (KBr) cm−1: 3290 (ν NH), 1637 (ν CO), 1597 (ν CN);
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 6.63 (d, 2H, H2' & H3'), 7.0 (s, 2H, NH2), 7.5 (d, 2H, H1' & H4'),
7.58–7.73 (m, 2 H, 1H, H3 & H4), 8.0 (s, 1H, N=CH), 11.29 (s, 1H, NH); 13C-NMR (50 MHz,
DMSO-d6) δ (ppm): 165 (CO), 157 (C4'), 141 (C2), 134 (HC=N), 132 (C3' & C5'), 129 (C2' & C6'),
122 (H4), 119 (H3), 96 (C1'); 99% purity by HPLC (R.T. = 5.9 min; CH3CN‒H2O (70:30)); MS: m/z = 323
[M + H]+.
3-Amino-N'-[(1(E)-(4-nitrophenyl)methylene]-2-thiophenecarbohydrazide
(5d,
LASSBio-1656).
Yield: 95%; orange solid; m.p. 255–257 °C; IR (KBr) cm−1: 3369 (ν NH), 1645 (ν CO), 1586 (ν CN);
1H-NMR (400 MHz, DMSO-d6) δ (ppm): δ 4.62 (s, 2H, NH2), 6.7 (d, 1H, H5), 7.7 (d, 1H, H4),
8.0 (2, 2H, H3' & H5'), 8.1 (s, 1H, N=CH), 8.3 (m, 3H, H4, H2' & H6'), 11.6 (d, 1H, NH); 98% purity
by HPLC (R.T. = 4.1 min; CH3CN‒H2O (70:30)); MS: m/z = 291 [M + H]+.
3-Amino-N'-[(1(E)-(4-hidroxyphenyl)methylene]-2-thiophenecarbohydrazide (5e, LASSBio-1657).
Yield: 84%; yellow solid; m.p. 245–248 °C; IR (KBr) cm−1: 3289 (ν NH), 1637 (ν CO), 1581 (ν NH);
1H-NMR (400 MHz, DMSO-d6) δ (ppm): δ 3.87 (s, 2H, NH2), 6.62 (s, 1H, H5), 6.84 (d, 2H, C3' & C5'),
7.56-7.71 (m, 3H, H4, H2' & H6'), 7.92 (s, 1H, N=CH), 9.89 (s, 1H, OH), 11.02 (s, 1H, NH);
13C-NMR (50 MHz, DMSO-d6) δ (ppm): 165 (CO), 159 (C3), 157 (C4'), 142 (HC=N), 134 (C5),
129 (C3' & C5'), 126 (C4), 119 (C1'), 116 (C2' & C6'); 99% purity by HPLC (R.T. = 3.0 min;
CH3CN‒H2O (70:30)); MS: m/z = 262 [M + H]+.
3-Amino-N'-[(1(E)-(4-dimethylaminophenyl)methylene]-2-thiophenecarbohydrazide (5f, LASSBio-1653).
Yield: 73%; brown solid; m.p. 180–184 °C; IR (KBr) cm−1: 3301 (ν NH), 1629(ν CO), 1597 (ν NH);
1H-NMR (400 MHz, DMSO-d6) δ (ppm): δ 2.95 (s, 6H, N(CH3)2), 3.43 (s, 2H, NH2), 6.62 (d, 1H, H5),
6.76 (d, 2H, H3' & H5'), 7.55–7.60 (m, 3H, H4, H2' & H6'), 7.89 (s, 1H, N=CH), 10.95 (s, 1H, NH) ppm;
13C-NMR (50 MHz, DMSO-d6) δ (ppm): 165 (CO), 157 (C4'), 151 (C3), 143 (HC=N), 134 (C5),
128 (C3' & C5'), 122 (C2), 119 (C4), 112 (C2' & C6'), 97 (C1); 93% purity by HPLC (R.T. = 4.5 min;
CH3CN‒H2O (1:1)); MS: m/z = 289 [M + H]+.
3-Amino-N'-[(1(E)-(4-carboxyphenyl)methylene]-2-thiophenecarbohydrazide (5g, LASSBio-1655).
Yield: 77%; yellow solid; m.p. >300 °C; IR (KBr) cm−1: 3347 (ν NH), 1692 (ν CO), 1589 (ν CO);
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 3.7 (s, 2H, NH2), 6.62 (d, 1H, H5), 7.59 (d, 1H, H4), 7.84 (d, 2H,
C3' & C5'), 7.99 (d, 2H, C2' & C6'), 8.07 (s, 1H, N=CH), 11.45 (s, 1H, NH) ppm; 99% purity by
HPLC (R.T. = 2.8 min; CH3CN‒H2O (1:1)). MS: m/z = 290 [M + H]+.
3-Amino-N'-[(1(E)-(2-pyridinyl)methylene]-2-thiophenecarbohydrazide
(5h,
LASSBio-1659).
Yield: 53%; yellow solid; m.p. 226–228 °C; IR (KBr) cm−1: 3288 (ν NH), 1633 (ν CO), 1579 (ν NH);
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 3.5 (s, 2H, NH2), 6.64 (d, 1H, C5), 7.36 (t, 1H, H4'), 7.59 (d, 1H,
H4), 7.89 (d, 1H, H6'), 8.06 (m, 2H, H3' & H5'), 8.5 (s, 1H, N=CH), 11.42 (s, 1H, NH) ppm;
98% purity by HPLC (R.T. = 3.1 min; CH3CN‒H2O (1:1)); MS: m/z = 247 [M + H]+.

Molecules 2014, 19
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3-Amino-N'-[(1(E)-(2-thienyl)methylene]-2-thiophenecarbohydrazide (5i, LASSBio-1658). Yield: 79%;
1
yellow solid; m.p. 221–225 °C; H-NMR (400 MHz, DMSO-d6) δ (ppm): 6.60 (s, 1H, H4'), 4.65 (s,
2H, NH2), 7.09 (s, 1H, H5'), 7.37 (s, 1H, H3'), 7.55–7.60 (m, 2H, H4 & H5), 8.20 (s, 1H, N=CH),
11.16 (s, 1H, NH); 99% purity by HPLC (R.T. = 3.8 min; CH3CN‒H2O (1:1)); MS: m/z = 252 [M + H]+.
3.4. In Silico Toxicological Evaluation and Drug-Like Profile
The in silico toxicity and drug-like profile of 3-aminothiophene-2-acylhydrazone derivatives 5a–i
were calculated using the Program OSIRIS Property Explorer [24]. Data were generated on-line in the
Osiris Program, accessed by the link (http://www.organic-chemistry.org/prog/peo/) [25] and represented
by toxicity risks (mutagenic, irritant, tumorigenic and reproductive effects), druglikeness and
drug-score. Druglikeness was calculated based on equation summing up score values of the fragments
present in the molecule under investigation. The fragments were identified from a list of 5300 distinct
substructure fragments with associated druglikeness scores [25]. Drug-score was calculated combining
the druglikeness, cLogP, logS, molecular weight and toxicity risks data [25].
3.5. Analgesic and Anti-Inflammatory Murine Models
3.5.1. Animals
Experiments were conducted using Swiss mice obtained from the BIOCEN—UFAL breeding unit,
weighing 20–30 g each, males, adult, with 6–8 weeks of age, distributed in groups up to 6–8 animals
for treatment. Wistar rats (130–170 g), males, were used in the experiment of induction of arthritis.
The animals were maintained with free access to food and water and kept at 25–28 °C under
a controlled 12 h light/dark cycle. All animals were manipulated according to the norms established by
the Ethics Commission—UFAL for handling animals (Protocol number: 14/2013).
3.5.2. Acetic Acid-Induced Abdominal Constriction Test
The peripheral analgesic activity was evaluated in male mice using the acetic acid-induced writhing
test [23]. The acetic acid-induced abdominal constriction test was carried out as described previously
by Coolier, 1968 with minor modifications in groups of six animals. In order to induce pain in mouse
peritoneal cavity, 0.6% of acetic acid (10 mL/Kg) was injected intraperitoneally, 40 min after the oral
administration of 3-amino-thiophene-2-acylhydrazone derivatives 5a–i (dose = 100, 30, 10, 3 and
1 µmol/Kg) and the standard dipyrone (dose = 100, 30, 10, 3 and 1 µmol/Kg). The ID50 was calculated
by nonlinear regression. The abdominal constriction resulting from the injection of acetic acid consists
of a contraction of the abdominal region together with a stretching of the hind limbs or all limbs.
The number of abdominal constrictions was counted cumulatively over a period of 20 min,
commencing 5 min after acetic acid administration. The animals were then placed immediately to
individual in a transparent plastic box. Anti-nociception response was indicated by the reduction in the
mean of 3-amino-thiophene-2-acylhydrazone derivatives 5a–i number of abdominal constrictions in
the test groups compared to the control group. Dipyrone was used as reference drugs while control
group received vehicle (Arabic gum) that was used to dissolve compounds.

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3.5.3. Formalin Test
The method used for this test was similar to that described by Hunskaar and Hole [24] with minor
modifications. Adult Swiss mice were divided in groups of six mice each and pretreated with
3-amino-thiophene-2-acylhydrazone derivatives 5a–i (dose = 30 µmol/Kg, p.o.) or indomethacin
(dose = 10 µmol/Kg, p.o.). Forty minutes after this treatment they were administered with 20 µL of
a 2.5% solution of formalin, subcutaneously under the plantar surface of the left hind-paw. Using a
chronometer, the total time spent in licking and biting the injected paw is recorded, quantifying thus
the nociceptive behavior. Anti-nociceptive effect was determined in two phases, the early phase from
0 to 5 min and the late phase 15 to 30 min with a 10 min lag period in between both phases.
3.5.4. Carrageenan-Induced Peritonitis
Peritoneal inflammation in male mice was produced according to the method described by
Ferrándiz and Alcaraz, 1991 [25]. Carrageenan was freshly prepared (10 mg/mL) in sterile 0.9% w/v
saline, and 250 μL were injected i.p., After 4 h, the animals were killed by cervical dislocation.
The peritoneal cavity was washed with 3.0 mL cold PBS, and after a gentle manual massage, the
exudate was retrieved and its volume was measured. The number of recruit leukocytes to the
peritoneum was counted in a Newbauer chamber and results were expressed as cells × 106/mL.
The 3-amino-thiophene-2-acylhydrazone derivatives 5a–i were tested orally in doses of 100, 30, 10, 3
and 1 µmol/Kg. The carrageenan group (Arabic gum, p.o.) and the reference drug (indomethacin, 100,
30, 10, 3 and 1 µmol/Kg, p.o.) were administered 30 min before the carrageenan injection. These five
doses were perfomed in order to calculate the ID50. The ID50 was calculated by nonlinear regression.
In the negative control group, animals have just received the same dose of a vehicle (arabic gum, p.o.)
30 min before the saline injection by intraperitoneal route.
3.5.5. Arthritis Model Induced by Complete Freund’s Adjuvant (CFA) in Rats
Before the onset of arthritis, Wistar rats were randomly divided separately into five groups: the
normal control group, the AA model group, the positive control group (i.e., dexamethasone; 5 µmol/Kg,
pathway orally, daily), 5a (50 µmol/Kg, pathway orally, daily), 5d (50 µmol/Kg, pathway orally, daily),
and Complete Freund’s adjuvant (CFA) (0.2 mL/paw) was injected into the right paw of the rats.
The CFA solution was prepared as a 1 mg/mL suspension of heat-killed Mycobacterium butyricum in
0.85 mL of paraffin oil and 0.15 mL of mannide monooleate (Sigma-Aldrich) [26]. At 14 days after
the CFA injection the animals received during seven days treatment with derivatives 5a, 5d and
dexamethasone. The paw volumes were measured on the 1st, 14th, 15th, 16th, 17th, 18th, 19th, 20th
and 21st days using a digital caliper (Mitutoyo). Mean paw volumes were obtained daily for treated
groups and compared with the paw volumes of the positive control group. At the 21st day the animals
were anesthetized with a solution ketamine/diazepam (75 mg/Kg and 5 mg/Kg, i.p.), blood was
collected by cardiac puncture for biochemical measurements using an Aspartate aminotransferase
(AST), Alanine aminotransferase (ALT), Urea and Creatinin, methods kit (Doles-Brazil). After
collection deeper anesthesia was applied to euthanize the animals. The arthritic paw was collected for
histological analysis and the spleen was collected for weighing.

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3.5.6. Biochemical Measurements
The blood was collected by cardiac puncture using 50 µL of EDTA as an anticoagulant. The blood
was centrifuged after collection was centrifuged at 1500 rpm for 5 min. After centrifugation the serum
was aliquoted and used for carrying out the biochemical levels of urea, creatinine, AST and ALT.
The level of AST/ALT was performed as follows. Two hundred µL of working reagent was placed in
the wells of a 96 well plate, 20 µL of serum was added and homogenized performed as reading
spectrophotometer wavelength at 340 nm. This was done with the initial reading and repeated three
readings at intervals of 1, 2, and 3 min to calculate the Delta Abs. The formula used to calculate the dose
of ALT/AST is below:
ALT UI/L = ΔA/minute × 1746
AST UI/L = ΔA/minute × 1746
The urea was carried expressed as follows: 100 µL of reagent 1 was placed in the cavity of a 96
well plate, and 5 µL of urease and 1 µL of serum sample or standard were added. The plate was
incubated in a spectrophotometer for 5 min at 37 °C and then added to 100 µL of reagent 2. After that
the reading plate with a wavelength of 600 nm was performed. The quantity of urea was calculated
following the formula below:
80
Factor (F) =
Absorbance of the standard
Urea (mg/dL) = Absorbance test × F
The serum creatinine test was performed according to the following protocol: 200 µL of working
reagent was aliquoted into a 96-well plate, and 20 µL of serum were added. The plate was incubated
for 30 s in the spectrophotometer. After incubation took place after the initial reading and the final
reading of 60 s. The formula used to calculate the amount of creatinine in the samples is as follows:
5
Factor (F) =
(Absorbance initial − Absorbance final) standard
Creatinine (mg/dL) = (Abs ini − Abs fin) sample × F
3.5.7. Macroscopic Analyses of the Stomach
After euthanasia, the stomachs were removed for macroscopic examination of the occurrence of lesions
with daily treatment for 7 days with dexamethasone (5 µmol/Kg, pathaway orally), 5d (50 µmol/Kg,
pathway orally), and 5a (50 µmol/Kg, pathway orally), The mucosal damage was examined by means
of a magnifying glass. For each stomach, the mucosal damage was assessed according to the following
scoring system [28].
0.5 Redness
1.0 Spot ulcers
1.5 Hemorrhagic streaks
2.0 Ulcers < 3, but ≤ 5
3.0 Ulcers > 5

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3.5.8. Spleen Weight
In order to evaluate a possible immunosuppression promoted by the daily treatment for 7 days with
dexamethasone (5 µmol/Kg, orally), 5d (50 µmol/Kg, orally), and 5a (50 µmol/Kg, orally), after
euthanasia we proceeded with the removal of the spleen. The spleen was washed with PBS and
weighed on an analytical balance. Treated groups were compared to the arthritic group [31,32].
3.5.9. 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
test t of Student or ANOVA in the tutorial Prisma®. Values were considered significant if * p < 0.05,
** p < 0.01 and *** p< 0.001.
4. Conclusions
In summary a series of functionalized 3-aminothiophene-2-acylhydrazone derivatives 5a–i were
designed and synthesized. These new N-acylhydrazone derivatives showed potent analgesic and
anti-inflammatory activities, with 5a and 5d standing out in this respect. These compounds were active
in acute and chronic inflammation models. After animals’ daily treatment for seven days with 5a
and 5d, with a dose of 50 µmol/Kg by oral administration, they were unable did not present renal or
hepatic toxicity. Moreover, 5d didn’t demonstrate an immunosuppressive profile. Taken together,
these data suggest the 3-aminothiophene-2-acylhydrazones 5a and 5d are new non-toxic, analgesic and
anti-inflammatory lead-candidates. The mechanism of action of these two bioactive compounds is now
being investigated.
Acknowledgments
The authors would like to thank INCT-INOFAR (BR, 573.564/2008-6 and E-26/170.020/2008),
FAPERJ (BR), FAPEAL (BR) and CNPq (BR) for fellowship and financial support.
Author Contributions
Christian Tadeo Moreno Reyes, Gildardo Rivera and Marina Amaral Alves: Synthesis, purifications
and structural characterization; Yolanda Karla Cupertino da Silva and Magna Suzana Alexandre Moreira:
Pharmacological assays; Eliezer J. Barreiro and Lídia Moreira Lima: Intellectual conception of the
project and paper, and molecular design.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2014 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/3.0/).