Synthesis and pharmacological evaluation of pyrazine N-acylhydrazone derivatives designed as novel analgesic and anti-inflammatory drug candidates

Article abstract of DOI:10.1016/j.bmc.2010.06.002

In this paper, we report the synthesis and pharmacological evaluation of pyrazine N-acylhydrazone (NAH) derivatives (2a-s) designed as novel analgesic and anti-inflammatory drug candidates. This series was planned by molecular simplification of prototype

Full text of DOI:10.1016/j.bmc.2010.06.002

Bioorganic & Medicinal Chemistry 18 (2010) 5007–5015
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological evaluation of pyrazine N-acylhydrazone
derivatives designed as novel analgesic and anti-inflammatory drug candidates
Yolanda Karla Cupertino da Silva a, Cristina Villarinho Augusto b, Maria Letícia de Castro Barbosa b,c
,
Gabriela Muniz de Albuquerque Melo a, Aline Cavalcanti de Queiroz a, Thays de Lima Matos Freire Dias a,
Walfrido Bispo Júnior a, Eliezer J. Barreiro b,c, Lídia Moreira Lima b,c, , Magna Suzana Alexandre-Moreira
a,
*
*
a LaFI—Laboratório de Farmacologia e Imunidade, Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Alagoas, Maceió, AL, Brazil
b LASSBio —Laboratório de Avaliação e Síntese de Substâncias Bioativas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, PO Box 68024, 21944-971,
Rio de Janeiro, RJ, Brazil
c Pós-graduação em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper, we report the synthesis and pharmacological evaluation of pyrazine N-acylhydrazone
(NAH) derivatives (2a–s) designed as novel analgesic and anti-inflammatory drug candidates. This series
was planned by molecular simplification of prototype 1 (LASSBio-1018), previously described as a non-
selective cyclooxygenase inhibitor. Derivatives 2a–s were evaluated in several animal models of pain
and inflammation, standing-out compound 2o (2-N0-[(E)-(3,4,5-trimethoxyphenyl) methylidene]-2-pyr-
azinecarbohydrazide; LASSBio-1181), that was also active in a murine model of chronic inflammation
(i.e., adjuvant-induced arthritis test in rats) and can be considered a new analgesic and anti-inflammatory
lead for drug development.
Received 30 March 2010
Revised 29 May 2010
Accepted 2 June 2010
Available online 8 June 2010
Keywords:
Antinociceptive
Anti-inflammatory
Bioactive N-acylhydrazone
Arthritis
Published by Elsevier Ltd.
1. Introduction
combined effect of these cells is still insufficient, a chronic
inflammatory state ensues, involving the formation of granulo-
Lipid mediators play pivotal roles in immune regulation, in self-
defense response and in the maintenance of homeostasis in living
systems. They are produced by multi-step enzymatic pathways,
which are initiated by the de-esterification of membrane phospho-
lipids by phospholipase A2 or sphingomyelinase, and include pros-
taglandins (PGs), leukotrienes (LTs) and platelet-activating factor
(PAF). These mediators exert their biological effects by binding to
cognate receptors, which are members of the G protein-coupled
receptor (GPCR) superfamily and play a fundamental role in
inflammatory and immune responses.1
Inflammation is an adaptive response that is triggered by nox-
ious stimuli and conditions, including infection and tissue injury,
being characterized by symptoms such as pain, redness, heat,
and swelling. If the acute inflammatory response fails to elimi-
nate the pathogen, the inflammatory process persists and ac-
quires new characteristics. The neutrophil infiltrate is replaced
by macrophages, and, in case of infection, also by T cells. If the
mas and lymphoid cells.2
The treatment of acute inflammatory state is commonly based
on the use of non-steroidal anti-inflammatory drugs (NSAIDs),
which are also effective as analgesics, being frequently prescribed
to treat various forms of pain, particularly chronic musculoskeletal
pain. Such drugs exert their anti-inflammatory and analgesic activ-
ities by inhibiting the cyclooxygenase (COX), an enzyme that cata-
lyzes the incorporation of two oxygen atoms into arachidonic acid
to form PG endoperoxides, which are converted into various types
of PGs, depending on the cell or tissue, by terminal PG synthases.
Two COX isoenzymes have been identified: a constitutive form,
COX-1, and an inducible form, COX-2.1–3
Recently, the N-acylhydrazone moiety was considered as a sin-
gle molecular framework and as a privileged structure employed to
the design of several new lead-compounds with diverse pharmaco-
logical activities, including analgesic and anti-inflammatory
effects.4
In this report, we describe the design, synthesis and pharmaco-
logical evaluation of pyrazine N-acylhydrazone (NAH) derivatives
(2a–s) designed as novel analgesic and anti-inflammatory drug
candidates, planned by molecular simplification of prototype 1
(LASSBio-1018), previously described as non-selective COX inhibi-
tor (Fig. 1).5
LASSBio, http://www.farmacia.ufrj.br/lassbio/.
* Corresponding authors. Tel.: +55 8233363444/2125626644; fax: +55
8232212501 (M.S.A.M.).
E-mail addresses: lidia@pharma.ufrj.br (L.M. Lima), suzanamagna@yahoo.com.br
(M.S. Alexandre-Moreira).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.06.002

5008
Yolanda Karla Cupertino da Silva et al. / Bioorg. Med. Chem. 18 (2010) 5007–5015
Figure 1. Design concept of pyrazine N-acylhydrazone derivatives 2a–s.
The molecular simplification process was based on the exclu-
The careful analysis of the 1H NMR spectra of compounds 2a–s
indicated the diastereoselectivity of the condensation step as de-
picted by the presence of only one imino-hydrogen (N@CH) signal
for each N-acylhydrazone derivative (d 8.32–8.82), which was
attributed to the (E)-diastereomer.7
sion of subunits A and B (Fig. 1) present in the lead-compound 1
(LASSBio-1018), aiming to reduce log P value and consequently
improving aqueous solubility (Fig. 1). The resulting pyrazine pat-
tern 2 was designed in order to keep the vicinal relationship of
N1 with the carbonyl group of N-acylhydrazone moiety; preserving
all other structural features of 1. A congeneric series of analogues
was designed by replacing the para-isopropylphenyl moiety of 2a
by four other substituents, as follows: Type 1: non-substituted aro-
matic rings [i.e., 2b, 2c, 2d and 2e]; type 2: phenyl substituted by
electron-donors or electron-withdrawing groups [i.e., 2f, 2g, 2h,
2i, 2j and 2p]; type 3: polyoxygenated aromatic rings [i.e., 2k, 2l,
2m, 2n and 2o]; and type 4: heteroaromatic rings [i.e., 2q, 2r and
2s].
3. Results and discussion
All compounds (2a–s) were evaluated in the screening dose of
100
lmol/kg (po) employing chemical and thermal models of
acute pain and inflammatory tests, using thalidomide (TNF-
a
inhibitor), indomethacin (COX-1 selective inhibitor) and celecoxib
(COX-2 selective inhibitor) as drug standards. Morphine was used
as standard in the hot-plate test. The lead-compound 1 (LASSBio-
1018; 100 lmol/kg, po) was also used as standard in all assays, ex-
2. Chemistry
cept CFA-induced arthritis. Celecoxib was administered concomi-
tantly with thalidomide to evaluate an occasional synergistic
effect.
The analgesic activity of pyrazine N-acylhydrazone derivatives
2a–s was initially evaluated employing the acetic acid-induced
abdominal writhing model in mice.8 As shown in Table 1, all deriv-
atives produced marked inhibition of acetic acid-induced writhing
response. Compounds 2o (97.1%) and 2q (86.3%) proved to be sig-
nificantly more active than standard celecoxib, indomethacin, tha-
lidomide and 1.
The obvious synthetic route planned to achieve the pyrazine N-
acylhydrazone derivatives 2a–s is depicted in Scheme 1. The key
intermediate 3 was obtained in 87% yield by hydrazinolysis of
methyl pyrazine-2-carboxylate (4), using hydrazine monohydrate
98% in ethanol. With the hydrazide intermediate 3 in hands, the
pyrazine N-acylhydrazone derivatives 2a–s were obtained, in good
yields, by condensing the hydrazide intermediate 35,16 with the
corresponding aromatic aldehydes (W-ArCHO) in ethanol, using
hydrochloric acid as catalyst.6
O
O
O
N
N
NH2
N
N
N
Ar
N
N
CH3
N
H
N
H
O
i
ii
H
(4)
(3)
(2a-s)
Ar = 4-isopropylphenyl (2a); phenyl (2b); 2-naphthyl (2c); 9-antracenyl (2d); 4-
phenylbenzene (2e); 4-fluorophenyl (2f); 4-trifluoromethylphenyl (2g); 4-nitrophenyl
(2h); 4-hydroxyphenyl (2i); 2-hydroxyphenyl (2j); 3,5-di(tert-butyl)-4-hydroxyphenyl
(2p); 1,3-benzodioxole (2k); 4-hydroxy-3-methoxyphenyl (2l); 3-hydroxy-4-
methoxyphenyl (2m); 3,4-dimethoxyphenyl (2n); 3,4,5-trimethoxyphenyl (2o); 4-oxo-
4H-2-chromene (2q); 4-pyridinyl (2r) and 2-pyridinyl (2s).
Scheme 1. Reagents and conditions: (i) Hydrazine monohydrate 98%, EtOH, rt, 2 h, 87%; (ii) W-ArCHO, EtOH, HCl cat., rt, 2 h, 69–96%.

Yolanda Karla Cupertino da Silva et al. / Bioorg. Med. Chem. 18 (2010) 5007–5015
5009
Table 1
Table 2
Effect of pyrazine N-acylhydrazone derivatives 2a–s, 1 (LASSBio-1018), thalidomide,
Effect of pyrazine N-acylhydrazone derivatives 2a–s, 1 (LASSBio-1018), thalidomide,
indomethacin and celecoxib (100 mol/kg, po) on formalin (2.5%) test in mice
celecoxib and indomethacin (100
l
mol/kg, po) on the 0.6% acetic acid-induced
l
abdominal constrictions in mice, for a period of 25 min
Substance
n
Phase 1
mean S.E.M.
Phase 2
mean S.E.M.
% of inhibition
mean S.E.M.
Substance
N
Writhing number
mean S.E.M.
% of inhibition
mean S.E.M.
Control
Thalidomide
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
61.7 6.2
44.6 6.0
47.4 6.1
46.1 9.5
59.0 3.4
49.6 5.0
70.1 5.5
55.1 11.5
38.8 5.0
46.8 7.9
45.9 5.3
49.0 4.4
65.8 6.3
64.1 6.9
65.8 7.1
192.4 32.4
63.8 10.5**
61.0 7.3**
52.9 10.4**
117.2 10.6*
64.3 19.5**
64.4 11.1**
84.9 6.6**
61.4 13.5
—
Control
Thalidomide
10
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
55.0 2.3
—
66.7 5.5**
68.4 3.7**
72.4 5.4**
39.0 5.5*
66.5 10.1**
66.5 5.7**
60.8 7.0**
68.0 7.0
14.7 1.1***
23.7 2.3***
26.9 2.6***
14.8 2.2***
12.5 2.6***
17.8 2.9***
28.4 1.1***
17.4 1.5***
36.5 3.1***
23.5 2.0***
14.0 4.8***
25.0 2.7***
32.8 4.1***
21.3 1.1***
14.8 3.4***
12.0 1.0***
29.2 1.5***
19.3 2.8***
14.5 3.0***
1.60 0.5***
15.6 1.6***
7.5 2.3***
32.8 7.1***
13.5 3.5***
73.3 5.8***
56.9 4.2***
51.1 8.4***
73.1 4.0***
77.3 4.7***
67.6 5.3***
42.4 6.2***
68.4 2.7***
33.6 5.7***
57.3 3.7***
74.5 8.7***
54.5 4.9***
40.4 7.5***
61.4 2.0***
73.1 6.2***
78.2 1.9***
47.0 3.4***
64.8 5.0***
Celecoxib
Celecoxib
T + Ca
T + Ca
Indomethacin
1
Indomethacin
1
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
104.2 18.2*
106.7 4.5*
53.1 10.4**
158.6 30.3
106.8 24.1*
161.5 16.9
161.2 20.6
102.2 17.8*
131.0 16.5
169.3 20.2
139.6 33.4
26.8 12.7**
140.5 16.1
90.8 13.5**
79.9 14.9**
79.3 6.0**
45.75 9.5
46.9 3.2*
72.3 5.4**
27.7 15.1
44.4 12.5*
16.7 8.4
***
11.9 4.2
16.2 8.3
38.2 5.6
49.9 5.2
52.8 6.0
58.1 10.8
61.0 5.2
51.4 5.5
57.3 5.9
54.2 6.5
56.8 3.9
46.7 9.2*
31.8 8.6
14.7 9.7
29.1 16.3
86.0 6.6**
26.8 8.4
***
73.6 5.5
97.1 0.9***
71.6 3.0***
86.3 4.2***
40.4 13.0***
75.5 6.4***
52.7 7.0**
58.4 7.8**
56.4 6.1**
Data are expressed as mean S.E.M. 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 <0.05.
P <0.01.
Data are expressed as mean S.E.M. 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 <0.05.
a
a
Thalidomide (50
mol/kg, po).
lmol/kg, po) administered concomitantly with celecoxib
Thalidomide (50
mol/kg, po).
lmol/kg, po) administered concomitantly with celecoxib
(50
l
(50
l
In the formalin test,9 which consists of two different phases—a
neurogenic phase, generated by the activation of nociceptive neu-
rons; and a second phase (called inflammatory phase), induced by
activation of ventral horn neurons at the spinal cord level—10
Table 3
Time course effect of pyrazine N-acylhydrazone derivatives 2a–s (100 lmol/kg, po), 1 (LASSBio-1018) (100 lmol/kg, po), morphine (15 lmol/kg, ip), thalidomide (100 lmol/kg,
po) and celecoxib (100
l
mol/kg, po) in the hot-plate test in mice
Substance
n
0 min mean S.E.M.
60 min mean S.E.M.
90 min mean S.E.M.
120 min mean S.E.M.
150 min mean S.E.M.
Control
Morphine
Thalidomide
Celecoxib
T + Ca
1
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6.1 0.6
6.9 0.4*
4.4 0.5
4.0 0.3
3.1 0.4
3.3 1.0
5.3 0.8
6.7 1.4
3.4 0.7
4.4 0.8
2.8 0.5
6.1 0.7
3.9 0.6
2.9 0.8
2.9 0.6
4.7 0.6
3.3 0.5
6.2 0.8
3.1 0.4
3.7 0.8
3.9 0.6
3.1 0.5
3.0 0.3
4.1 0.3
3.3 0.7
4.9 0.5
12.8 0.4*
48 0.7
5.5 0.6
4.1 0.3
4.6 0.8
3.3 0.6
4.4 1.1
2.8 0.9
3.7 1.0
2.7 0.5
4.6 0.9
3.1 0.9
3.1 0.6
3.1 0.5
4.0 1.1
2.4 0.5
2.6 0.7
2.0 1.0
4.8 1.0
4.0 0.9
2.7 0.7
2.5 0.9
3.5 0.3
2.4 1.0
5.9 0.8
10.3 0.8*
7.5 1.1*
5.7 0.8
6.9 0.7
5.0 1.1
4.7 0.8
4.2 1.3
3.5 0.8
6.2 0.8
3.5 0.8
5.4 0.8
3.9 0.5
5.4 1.1
3.4 0.7
5.2 0.4
3.0 0.6
3.2 0.7
4.0 1.2
4.2 0.9
4.8 0.9
3.4 0.9
3.5 0.6
3.2 0.6
4.4 1.1
5.6 0.5
9.7 0.7*
6.8 0.9
4.4 0.7
5.4 1.3
5.4 0.8
5.2 0.8
4.0 0.6
4.4 0.7
5.6 1.3
4.0 0.7
4.8 0.8
2.4 0.7
4.9 1.1
5.3 0.9
6.4 1.3
4.4 0.7
3.4 0.9
1.9 0.4
6.3 1.1
4.3 0.6
3.4 0.4
6.2 1.2
5.4 0.7
4.5 0.8
5.1 0.4
9.7 0.9*
5.9 0.6
3.8 0.8
2.9 0.4
4.3 0.4
4.6 1.2
5.2 0.7
4.5 1.1
6.6 1.8
4.5 1.1
3.1 0.7
4.0 0.5
4.9 1.2
4.0 0.7
5.2 1.2
3.2 0.7
4.4 0.7
4.7 0.6
3.8 0.8
5.3 1.0
5.4 1.5
4.2 1.2
4.5 0.8
3.0 0.6
2p
2q
2r
2s
Data are expressed as mean S.E.M. 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 <0.05.
a
Thalidomide (50 lmol/kg, po) administered concomitantly with celecoxib (50 lmol/kg, po).

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Yolanda Karla Cupertino da Silva et al. / Bioorg. Med. Chem. 18 (2010) 5007–5015
compounds, that is, 2a, 2b, 2e, 2f, 2h, 2k, 2o, 2q, 2r and 2s, were
active in the second phase, indicating their ability to inhibit noci-
ception associated with inflammatory response (Table 2). Only
compound 2j was able to inhibit the first phase (i.e., neurogenic
phase) of the formalin test (Table 2). Considering that peripher-
ally-acting drugs, such as AspirinÒ and glucocorticoids, inhibit
the second phase of formalin test, without significant response in
the neurogenic phase,10 as observed for standards thalidomide,
indomethacin and celecoxib (Table 2), these results suggest an
anti-inflammatory profile for compounds 2a, 2b, 2e, 2f, 2h, 2k,
2o, 2q, 2r and 2s.
In order to analyze the effects of pyrazine N-acylhydrazone
derivatives 2a–s on other components of inflammatory response,
these compounds were studied using capsaicin-induced ear edema
in mice. In this model, topical application of capsaicin in the ear of
mice produces neurogenic acute inflammatory responses, such as
axon reflex vasodilatation, plasma leakage and erythema.13,14
As demonstrated in Table 5, compounds 2d, 2g, 2l, 2n, 2o and
2q were able to produce an antiedematogenic activity. However,
the pyrazine N-acylhydrazone derivatives 2o (LASSBio-1181) and
Table 5
Effect of pyrazine N-acylhydrazone derivatives 2a–s, 1 (LASSBio-1018), thalidomide,
indometacin and celecoxib (100 lmol/kg, po) in capsaicin-induced ear edema in mice
In order to investigate an occasional central antinociceptive
activity for compounds 2a–s, they were evaluated in the hot-plate
test11 using morphine (15
l
mol/kg, ip), thalidomide (100
lmol/kg,
Substance
n
% of edema
mean S.E.M.
% of inhibition
mean S.E.M.
po), celecoxib (100
l
mol/kg, po) and 1 (100 mol/kg, po) as stan-
l
dards. As shown in Table 3, the administration of pyrazine N-acy-
lhydrazone derivatives 2a–s did not increase significantly the
reaction time to the nociceptive stimuli in the hot-plate test, while
morphine induced a marked increase in the latency of the animals
at 60 min (12.8 0.4 s), 90 min (10.3 0.8 s), 120 min (9.7 0.7 s)
and 150 min (9.7 0.9 s), indicating that compounds 2a–s proba-
bly do not show any central antinociceptive activity.
To confirm the anti-inflammatory profile of pyrazine N-acy-
lhydrazone derivatives 2a–s, as suggested by the results obtained
in the formalin test, the zymosan-induced peritonitis assay was
performed.12
Control
Thalidomide
10
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
58.5 7.5
46.6 6.8
25.5 3.5**
24.9 4.2**
29.7 6.8*
30.7 5.0
31.2 9.8
40.6 7.2
37.8 2.7
26.5 6.5**
32.9 13.2
39.1 7.6
30.2 2.3*
34.0 6.4
33.7 1.6
33.9 9.9
32.3 8.6
21.3 6.7*
42.8 3.4
29.5 5.9**
17.6 5.3**
39.5 6.3
26.1 6.7**
45.7 6.9
32.2 10.7
—
25.2 8.8
Celecoxib
56.4 5.9**
57.4 7.2**
49.3 11.6*
47.5 8.7
T + Ca
Indomethacin
1
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
46.6 16.8
30.5 12.4
35.4 4.7
54.7 11.1**
47.7 20.0
33.1 13.0
48.3 4.0*
41.9 11.0
42.3 2.7
As depicted in Table 4, the pyrazine N-acylhydrazone deriva-
tives, with exception of compounds 2a, 2d, 2g, 2h and 2q, were
able to inhibit cell migration in zymosan-induced peritonitis mod-
el, especially compound 2k, which presented 57.2% of inhibition.
42.0 16.9
44.7 11.3
63.5 11.5*
27.0 5.7
49.6 10.1**
70.0 9.1**
32.3 10.8
55.3 11.5**
32.3 6.9
Table 4
Effect of pyrazine N-acylhydrazone derivatives 2a–s, 1 (LASSBio-1018), thalidomide,
indomethacin and celecoxib (100
mice
lmol/kg, po) on the zymosan-induced peritonitis in
44.9 18.2
Data are expressed as mean S.E.M. 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.
Substance
N
Cell Number ꢀ 106/mL
% of inhibition
mean S.E.M.
mean S.E.M.
Control
Saline
Thalidomide
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
25.5 1.5
2.6 0.6
—
—
*
P <0.05.
**
P <0.01.
Thalidomide (50
lmol/kg, po).
9.8 1.3*
19.9 2.1
4.8 1.8**
5.3 1.3**
29.3 4.0
23.7 4.8
18.9 2.3*
15.1 2.5*
27.3 4.1
22.4 1.6*
20.0 1.6*
29.8 1.3
28.6 3.4
15.3 3.9*
12.4 2.3*
10.9 1.0*
16.7 2.2*
12.6 2.0*
12.4 1.7*
15.6 2.2*
14.2 1.4*
28.2 5.9
15.1 1.8*
22.7 4.4
61.3 6.4*
24.1 6.7
80.9 6.9**
79.4 5.0**
0.0 0.0
a
lmol/kg, po) administered concomitantly with celecoxib
Celecoxib
(50
T + Ca
Indomethacin
1
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
19.4 13.5
21.7 8.8*
40.8 9.3*
0.0 0.0
14.4 5.2*
24.5 6.3*
0.0 0.0
0.0 0.0
43.1 12.9*
51.2 9.0*
57.2 4.0*
34.7 8.4*
50.4 7.8*
51.3 6.8*
38.8 8.7*
44.4 5.6*
0.0 0.0
12.7 12.7*
42.9 15.4
Data are expressed as mean S.E.M. 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.
Figure 2. Effect of compound 2o (LASSBio-1181) and thalidomide (100 lmol/kg,
*
P <0.05.
po) 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
**
P <0.01.
Thalidomide (50
lmol/kg, po).
a
lmol/kg, po) administered concomitantly with celecoxib
(50
**
*
significance in comparison with control groups. P <0.01 and P <0.05.

Yolanda Karla Cupertino da Silva et al. / Bioorg. Med. Chem. 18 (2010) 5007–5015
5011
2l (LASSBio-1182) can be pointed as the most active derivatives
when compared to standards thalidomide, celecoxib, indometha-
cin and 1.
a Buckner funnel and recovered as yellow needles in 87% yield,
mp 158–159 °C. The melting point and 1H NMR data for compound
3 are in agreement with previous reports.16
The set of results herein described (e.g., writhing test, formalin
and hot-plate models, zymosan-induced peritonitis and capsaicin-
induced ear edema) enabled 2o to be selected as the most impor-
tant compound with anti-inflammatory and analgesic profiles
among the pyrazine N-acylhydrazone derivatives 2a–s. Therefore,
prototype 2o (LASSBio-1181) was further evaluated in a chronic
inflammatory model, using complete Freund’s adjuvant (CFA)-in-
duced arthritis test in rats.15 As illustrated in Figure 2, in the
1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 4.56 (br, 2H, NH2);
8.69 (dd, 1H, J = 1.4 and 2.4 Hz, H5); 8.82 (d, 1H, J = 2.4 Hz, H6);
9.12 (d, 1H, J = 1.4 Hz, H3); 10.11 (s, 1H, CONH). 13C NMR
(50 MHz, DMSO-d6, TMS) d (ppm): 143.7 (C5); 144.0 (C3); 145.4
(C2); 147.8 (C6); 162.0 (C@O). IR (mmax, KBr) m
(cmꢁ1): 3306,
3242, 3054, 1679, 1576, 1307, 858. Anal. Calcd for C5H6N4O: C,
43.48; H, 4.38; N, 40.56. Found: C, 43.56; H, 4.36; N, 40.26.
CFA-induced arthritis model, the treatment with 2o (100
l
mol/
5.1.2. General procedure for the preparation of pyrazine-2-N-
acylhydrazones (2a–s)
Kg, po) and thalidomide (100 mol/Kg, po) for seven days resulted
l
in statistically significant inhibition of paw edema on the 16th,
17th and 21st days, indicating their ability to control a chronic
inflammatory process.
The corresponding aromatic or heteroaromatic aldehyde
(0.72 mmol) was added to a solution of pyrazine-2-carbohydrazide
(3) (100 mg; 0.72 mmol) in absolute ethanol (6 mL) containing one
drop of 37% hydrochloric acid. The mixture was stirred at room
temperature for 2 h until extensive precipitation was visualized.
Afterwards, the solvent was partially concentrated at reduced
pressure and the resulting mixture was poured into cold water.
After neutralization with 10% aqueous sodium bicarbonate solu-
tion, the precipitate formed was filtered out and dried under vac-
uum producing the desired N-acylhydrazone derivatives 2a–s, as
described below.
4. Conclusion
We have designed and synthesized nineteen pyrazine N-acy-
lhydrazone derivatives 2a–s structurally planned by molecular
simplification of the lead-compound 1 (LASSBio-1018). Derivatives
2a–s were found to have antinociceptive and anti-inflammatory
activities, especially 2o (2-N0-[(E)-(3,4,5-trimethoxyphenyl)meth-
ylidene]-2-pyrazinecarbohydrazide; LASSBio-1181). This com-
pound presented a better pharmacological profile than prototype
1 and was also active in a murine model of chronic inflammation
(i.e., adjuvant-induced arthritis test in rats). Therefore, compound
2o (LASSBio-1181) can be considered a new analgesic and anti-
inflammatory lead for drug development.
5.1.2.1.
N0-[(1E)-(4-(1-Methylethyl)phenyl)methylidene]pyra-
zine-2-carbohydra-zide (2a; LASSBio-1244). The title compound
was obtained as a white solid by the condensation of 3 with 4-
(1-methylethyl)benzaldehyde in 77% yield, mp 232–235 °C. 1H
NMR (200 MHz, CDCl3, TMS) d (ppm): 1.27 (d, 6H, J = 6.8 Hz,
CH3); 2.93 (m, 1H, ArCH(CH3)2); 7.29 (d, 2H, J = 7.9 Hz, H30 and
H50); 7.75 (d, 2H, J = 7.9 Hz, H20 and H60); 8.32 (s, 1H, N@CH);
8.56 (dd, 1H, J = 1.4 and 2.4 Hz, H5); 8.82 (d, 1H, J = 2.4 Hz, H6);
9.52 (d, 1H, J = 1.4 Hz, H3); 10.67 (s, 1H, CONH). 13C NMR
(50 MHz, CDCl3, TMS) d (ppm): 23.8 (CH3); 34.2 ArCH(CH3)2);
127.0 (C30 and C50); 128.1 (C20 and C60); 131.0 (C10); 142.5 (C5);
144.0 (C3); 145.0 (C2); 147.8 (C6); 150.0 (N@CH); 152.3 (C40);
5. Experimental
5.1. General
Reactions were routinely monitored by thin-layer chromatogra-
phy (TLC) in silica gel (F245 Merck plates) and the products visual-
ized with iodine or ultraviolet lamp (254 and 365 nm). 1H and 13C
nuclear magnetic resonance (NMR) spectra were determined in
DMSO-d6 or CDCl3 solutions using a Bruker AC-200 or a Varian
UNITY-300 spectrometer. Peak positions are given in parts per mil-
lion (d) using tetramethylsilane as internal standard, and coupling
constant values (J) are given in Hertz. Signal multiplicities are rep-
resented by: s (singlet), d (doublet), t (triplet), q (quadruplet), qu
(quintuplet), m (multiplet) and br (broad signal). Infrared (IR) spec-
tra were obtained using an ABB FTLA2000-100 IR spectrometer.
Samples were examined as potassium bromide (KBr) disks. Ele-
mental microanalyses were obtained on an Elemental Analyzer
(Flash EA 1112 Series, Thermo Scientific) from vacuum-dried sam-
ples. The analytical results for C, H, and N, were within 0.4% of the
theoretical values. Melting points were determined using a Quimis
instrument and are uncorrected. All described products showed 1H
and 13C NMR spectra according to the assigned structures. All or-
ganic solutions were dried over anhydrous sodium sulphate and
all organic solvents were removed under reduced pressure using
a rotatory evaporator.
158.8 (C@O). IR (mmax, KBr) m
(cmꢁ1): 3298, 3080, 2963, 2871,
1679, 1268, 828. Anal. Calcd for C15H16N4O: C, 67.15; H, 6.01; N,
20.88. Found: C, 67.28; H, 6.12; N, 20.77.
5.1.2.2. N0-[(1E)-Phenylmethylidene]pyrazine-2-carbohydrazide
(2b; LASSBio-1243). The title compound was obtained as a white
solid by the condensation of 3 with benzaldehyde in 87% yield,
mp 227–230 °C. The melting point and 1H NMR data are in agree-
ment with those previously published.16,18 1H NMR (200 MHz,
DMSO-d6, TMS) d (ppm): 7.47 (br, 3H, H30, H40 and H50); 7.73 (br,
2H, H20 and H60); 8.64 (s, 1H, N@CH); 8.79 (dd, 1H, J = 1.1 and
2.1 Hz, H5); 8.92 (d, 1H, J = 2.1 Hz, H6); 9.26 (d, 1H, J = 1.1 Hz,
H3); 12.28 (s, 1H, CONH). 13C NMR (50 MHz, DMSO-d6, TMS) d
(ppm): 127.8 (C30 and C50); 129.4 (C20 and C60); 130.9 (C40);
134.7 (C10); 143.9 (C5); 144.6 (C3); 145.2 (C2); 148.4 (C6); 150.5
(N@CH); 160.1 (C@O). IR (mmax, KBr) m
(cmꢁ1): 3296, 3035, 1679,
1271, 845, 759, 693. Anal. Calcd for C12H10N4O: C, 63.71; H, 4.46;
N, 24.76. Found: C, 63.69; H, 4.42; N, 25.10.
5.1.2.3. N0-[(1E)-Naphthalen-2-ylmethylidene]pyrazine-2-car-
bohydrazide (2c; LASSBio-1190). The title compound was ob-
5.1.1. Pyrazine-2-carbohydrazide (3)
tained as yellow needles by the condensation of
3 with
naphthalene-2-carbaldehyde in 82% yield, mp 190–193 °C. 1H
NMR (200 MHz, DMSO-d6, TMS) d (ppm): 7.65 (m, 3H, H50, H60
and H70); 8.02 (m, 3H, H30, H40 and H80); 8.82 (br, 2H, H10 and
N@CH); 8.94 (dd, 1H, J = 1.0 and 2.0 Hz, H5); 9.31 (d, 1H,
J = 2.0 Hz, H6); 9.38 (d, 1H, J = 1.0 Hz, H3); 12.36 (s, 1H, CONH).
13C NMR (50 MHz, DMSO-d6, TMS) d (ppm): 124.6 (C30); 126.1
(C70); 126.9 (C50); 127.9 (C60); 128.0 (C40); 129.4 (C80); 130.0
Hydrazine monohydrate 98% (1.77 mL; 1.81 g; 36.2 mmol) was
added to a 5 mL ethanolic solution of methyl 2-pyrazinecarboxy-
late (4) (0.5 g; 3.62 mmol), and the reaction mixture was stirred
for 2 h, at room temperature, when TLC indicated the end of reac-
tion. The solvent was removed under reduced pressure, followed
by addition of a water/ice mixture (1:1) to the obtained residue,
promoting extensive precipitation. The solid was filtered through

5012
Yolanda Karla Cupertino da Silva et al. / Bioorg. Med. Chem. 18 (2010) 5007–5015
(C8a0); 131.0 (C20); 131.4 (C10); 134.1 (C4a0); 143.9 (C5); 144.7
(C3); 145.1 (C2); 148.4 (C6); 150.1 (N@CH); 160.0 (C@O). IR (mmax
KBr)
(cmꢁ1): 3302, 3044, 1701, 1268, 770. Anal. Calcd for
16H12N4O: C, 69.55; H, 4.38; N, 20.28. Found: C, 69.46; H, 4.36;
5.1.2.8. N0-[(1E)-(4-Nitrophenyl)methylidene]pyrazine-2-carbo-
hydrazide (2h; LASSBio-1194). The title compound was obtained
as a yellow solid by the condensation of 3 with 4-nitrobenzalde-
hyde in 93% yield, mp >250 °C. The melting point and 1H NMR data
are in agreement with those previously published.16,18 1H NMR
(200 MHz, DMSO-d6, TMS) d (ppm): 7.99 (d, 2H, J = 8.9 Hz, H20
and H60); 8.32 (d, 2H, J = 8.9 Hz, H30 and H50); 8.76 (s, 1H, N@CH);
8.81 (dd, 1H, J = 1.4 and 2.4 Hz, H5); 8.95 (d, 1H, J = 2.4 Hz, H6);
,
m
C
N, 20.26.
5.1.2.4. N0-[(1E)-Anthracen-9-ylmethylidene]pyrazine-2-carbo-
hydrazide (2d; LASSBio-1192). The title compound was obtained
as a yellow solid by the condensation of 3 with anthracene-9-carb-
aldehyde in 74% yield, mp 248–250 °C. 1H NMR (200 MHz, DMSO-
d6, TMS) d (ppm): 7.63 (m, 4H, H20, H30, H60 and H70); 8.16 (d, 2H,
J = 8.2 Hz, H40 and H50); 8.74 (br, 1H, H100); 8.53 (m, 3H, H10, H80
and N@CH); 8.97 (dd, 1H, J = 1.4 and 2.4 Hz, H5); 9.34 (d, 1H,
J = 2.4 Hz, H6); 9.88 (d, 1H, J = 1.4 Hz, H3); 12.60 (s, 1H, CONH).
13C NMR (50 MHz, DMSO-d6, TMS) d (ppm): 125.5 (C90); 125.6
(C10 and C80); 126.1 (C30 and C60); 127.8 (C20 and C70); 129.6 (C40
and C50); 130.4 (C4a0 and C5a0); 130.5 (C100); 131.5 (C1a0 and
C8a0); 143.9 (C5); 144.7 (C3); 145.1 (C2); 148.5 (C6); 149.9
9.29 (d, 1H, J = 1.4 Hz, H3); 12.60 (s, 1H, CONH). IR (
(cmꢁ1): 3303, 3029, 1686, 1527, 1368, 1270, 853. Anal. Calcd for
12H9N5O3: C, 53.14; H, 3.34; N, 25.82. Found: C, 53.48; H, 3.22;
mmax, KBr) m
C
N, 25.72.
5.1.2.9. N0-[(1E)-(4-Hydroxyphenyl)methylidene]pyrazine-2-car
bohydrazide (2i; LASSBio-1185). The title compound was ob-
tained as a yellow solid by the condensation of 3 with 4-hydroxy-
benzaldehyde in 87% yield, mp >250 °C. The melting point and 1H
NMR data are in agreement with those previously published.16,18
(N@CH); 160.1 (C@O). IR (
m
max, KBr)
m
(cmꢁ1): 3201, 3051, 1670,
1H NMR (200 MHz, DMSO-d6, TMS)
d (ppm): 6.85 (d, 2H,
1282, 869, 729. Anal. Calcd for C20H14N4O: C, 73.61; H, 4.32; N,
17.17. Found: C, 73.26; H, 4.29; N, 16.81.
J = 8.5 Hz, H30 and H50); 7.56 (d, 2H, J = 8.5 Hz, H20 and H60); 8.53
(s, 1H, N@CH); 8.76 (dd, 1H, J = 1.4 and 2.4 Hz, H5); 8.90 (d, 1H,
J = 2.4 Hz, H6); 9.25 (d, 1H, J = 1.4 Hz, H3); 9.97 (s, 1H, OH); 12.06
5.1.2.5.
N0-[(1E)-Biphenyl-4-ylmethylidene]pyrazine-2-carbo-
(s, 1H, CONH). IR (mmax, KBr) m
(cmꢁ1): 3300, 3274, 3002, 1678,
hydrazide (2e; LASSBio-1191). The title compound was obtained
as a white solid by the condensation of 3 with biphenyl-4-carbal-
dehyde in 88% yield, mp >250 °C. 1H NMR (300 MHz, DMSO-d6,
TMS) d (ppm): 7.38 (t, 1H, J = 7.2 Hz, H400); 7.48 (dd, 2H, J = 7.2
and 7.4 Hz, H300 and H500); 7.71 (d, 2H, J = 7.4 Hz, H200 and H600);
7.77 (d, 2H, J = 8.4 Hz, H20 and H60); 7.82 (d, 2H, J = 8.4 Hz, H30
and H50); 8.67 (s, 1H, N@CH); 8.78 (dd, 1H, J = 1.2 and 2.4 Hz,
H5); 8.91 (d, 1H, J = 2.4 Hz, H6); 9.26 (d, 1H, J = 1.2 Hz, H3);
1268, 846. Anal. Calcd for C12H10N4O2: C, 59.50; H, 4.16; N,
23.13. Found: C, 59.68; H, 4.24; N, 23.39.
5.1.2.10. N0-[(1E)-(2-Hydroxyphenyl)methylidene]pyrazine-2-
carbohydrazide (2j; LASSBio-1186). The title compound was ob-
tained as a beige solid by the condensation of 3 with 2-hydroxy-
benzaldehyde in 95% yield, mp 198–200 °C. The melting point
and 1H NMR data are in agreement with those previously pub-
lished.16,18 1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 6.90 (d,
1H, J = 6.5 Hz, H30); 6.95 (dd, 1H, J = 7.5 and 7.8 Hz, H50); 7.31
(dd, 1H, J = 6.5 and 7.8 Hz, H40); 7.51 (d, 1H, J = 7.5 Hz, H60); 8.78
(dd, 1H, J = 1.4 and 2.4 Hz, H5); 8.81 (s, 1H, N@CH); 8.91 (d, 1H,
J = 2.4 Hz, H6); 9.26 (d, 1H, J = 1.4 Hz, H3); 11.26 (s, 1H, OH);
12.24 (s, 1H, CONH). IR (mmax, KBr) m
(cmꢁ1): 3285, 3013, 1677,
1275, 835, 764, 699. Anal. Calcd for C18H14N4O: C, 71.51; H, 4.67;
N, 18.53. Found: C, 71.60; H, 4.61; N, 18.47.
5.1.2.6. N0-{(1E)-[4-Fluorophenyl]methylidene}pyrazine-2-car-
bohydrazide (2f; LASSBio-1270). The title compound was ob-
12.59 (s, 1H, CONH). IR (mmax, KBr) m
(cmꢁ1): 3416, 3291, 3052,
tained as
a
beige solid by condensation of
3
with 4-
1670, 1266, 856, 761. Anal. Calcd for C12H10N4O2: C, 59.50; H,
fluorobenzaldehyde in 96% yield, mp 210–212 °C. The melting
point and 1H NMR data are in agreement with those previously
published.16,18 1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 7.30
4.16; N, 23.13. Found: C, 59.58; H, 4.12; N, 23.40.
5.1.2.11. N0-[(1E)-1,3-Benzodioxol-5-ylmethylidene]pyrazine-2-
carbohydrazide (2k; LASSBio-1180). The title compound was ob-
tained as a yellow solid by the condensation of 3 with 1,3-benzo-
dioxole-5-carbaldehyde in 84% yield, mp 227 °C. 1H NMR
(200 MHz, DMSO-d6, TMS) d (ppm): 6.10 (s, 2H, CH2); 6.99 (d,
1H, J = 7.8 Hz, H50); 7.16 (dd, 1H, J = 1.4 and 7.8 Hz, H60); 7.31 (d,
1H, J = 1.4 Hz, H20); 8.54 (s, 1H, N@CH); 8.78 (dd, 1H, J = 1.4 and
2.4 Hz, H5); 8.92 (d, 1H, J = 2.4 Hz, H6); 9.25 (d, 1H, J = 1.4 Hz,
H3); 12.21 (s, 1H, CONH). 13C NMR (50 MHz, DMSO-d6, TMS) d
(ppm): 102.2 (CH2); 105.7 (C20); 109.1 (C50); 124.3 (C60); 129.1
(C10); 143.9 (C5); 144.6 (C3); 145.3 (C2); 148.3 (C6); 148.6
3
3
(dd, 2H, JH,H = 8.5 Hz, JH,F = 8.9 Hz, H30 and H50); 7.79 (dd, 2H,
3JH,H = 8.5 Hz, 4JH,F = 5.4 Hz, H20 and H60); 8.64 (s, 1H, N@CH);
8.79 (dd, 1H, J = 1.4 and 2.4 Hz, H5); 8.92 (d, 1H, J = 2.4 Hz, H6);
9.26 (d, 1H, J = 1.4 Hz, H3); 12.29 (s, 1H, CONH). 13C NMR
2
(50 MHz, DMSO-d6, TMS) d (ppm): 115.9 (d, JC,F = 22 Hz, C30 and
3
C50); 129.4 (d, JC,F = 9 Hz, C20 and C60); 130.7 (C10); 143.3 (C5);
144.0 (C3); 144.6 (C2); 147.8 (C6); 148.7 (N@CH); 159.5 (C@O);
1
163.3 (d, JC,F = 246 Hz, C40). IR (
m
max, KBr)
m
(cmꢁ1): 3297, 3022,
1678, 1295, 1146, 835. Anal. Calcd for C12H9FN4O: C, 59.01; H,
3.71; N, 22.94. Found: C, 58.97; H, 3.49; N, 22.89.
(N@CH); 149.9 (C30); 150.2 (C40); 159.9 (C@O). IR (
mmax, KBr) m
5.1.2.7. N0-{(1E)-[4-(Trifluoromethyl)phenyl]methylidene}pyra-
zine-2-carbo-hydrazide (2g; LASSBio-1193). The title compound
was obtained as a beige solid by the condensation of 3 with 4-(tri-
fluoromethyl)benzaldehyde in 77% yield, mp 240–243 °C. 1H NMR
(200 MHz, DMSO-d6, TMS) d (ppm): 7.82 (d, 2H, J = 7.8 Hz, H30 and
H50); 7.94 (d, 2H, J = 7.8 Hz, H20 and H60); 8.72 (s, 1H, N@CH); 8.80
(dd, 1H, J = 1.4 and 2.4 Hz, H5); 8.94 (d, 1H, J = 2.4 Hz, H6); 9.28 (d,
1H, J = 1.4 Hz, H3); 12.49 (s, 1H, CONH). 13C NMR (50 MHz, DMSO-
d6, TMS) d (ppm): 124.1 (q, 1JC,F = 276 Hz, CF3); 125.7 (q, 3JC,F = 4 Hz,
C30 and C50); 127.8 (C20 and C60); 130.3 (q, 2JC,F = 32 Hz, C40); 138.1
(C10); 143.3 (C5); 144.2 (C3); 144.4 (C2); 148.0 (C6); 148.1
(cmꢁ1): 3278, 3068, 1682, 1257, 866. Anal. Calcd for C13H10N4O3:
C, 57.78; H, 3.73; N, 20.73. Found: C, 57.60; H, 3.67; N, 20.64.
5.1.2.12. N0-[(1E)-(4-Hydroxy-3-methoxyphenyl)methylidene]
pyrazine-2-carbo-hydrazide (2l; LASSBio-1182). The title com-
pound was obtained as a beige solid by the condensation of 3 with
4-hydroxy-3-methoxybenzaldehyde in 95% yield, mp >250 °C. 1H
NMR (200 MHz, DMSO-d6, TMS) d (ppm): 3.81 (s, 3H, CH3); 6.97
(d, 1H, J = 8.2 Hz, H50); 7.05 (d, 1H, J = 8.2 Hz, H60); 7.28 (s, 1H,
H20); 8.48 (s, 1H, N@CH); 8.78 (dd, 1H, J = 1.4 and 2.4 Hz, H5);
8.91 (d, 1H, J = 2.4 Hz, H6); 9.25 (d, 1H, J = 1.4 Hz, H3); 9.37(s, 1H,
OH); 12.12 (s, 1H, CONH). 13C NMR (50 MHz, DMSO-d6, TMS) d
(ppm): 55.6 (CH3); 111.9 (C20); 112.5 (C50); 120.6 (C60); 127.0
(C10); 143.3 (C5); 144.0 (C3); 144.8 (C2); 146.9 (C6); 147.7
(N@CH); 159.8 (C@O). IR (mmax, KBr) m
(cmꢁ1): 3298, 3056, 1683,
1272, 1116, 840. Anal. Calcd for C13H9F3N4O: C, 53.07; H, 3.08; N,
19.04. Found: C, 53.18; H, 3.14; N, 19.19.

Yolanda Karla Cupertino da Silva et al. / Bioorg. Med. Chem. 18 (2010) 5007–5015
5013
(N@CH); 150.0 (C30); 150.1 (C40); 159.3 (C@O). IR (
(cmꢁ1): 3368, 3265, 3095, 1673, 1274, 864. Anal. Calcd for
13H12N4O3: C, 57.35; H, 4.44; N, 20.58. Found: C, 57.28; H, 4.40;
m
max, KBr)
m
5.1.2.17. N0-[(1E)-(4-Oxo-4H-chromen-3-yl)methylidene]pyra-
zine-2-carbohydra-zide (2q; LASSBio-1188). The title compound
was obtained as a yellow solid by the condensation of 3 with 4-
oxo-4H-chromene-3-carbaldehyde in 69% yield, mp 240–242 °C.
1H NMR (300 MHz, DMSO-d6, TMS) d (ppm): 7.54 (dt, 1H, J = 1.2
and 8.5 Hz, H60); 7.71 (dd, 1H, J = 1.2 and 8.5 Hz, H80); 7.85 (dt,
1H, J = 1.7 and 8.5 Hz, H70); 8.12 (dd, 1H, J = 1.7 and 8.5 Hz, H50);
8.76 (s, 1H, N@CH); 8.77 (dd, 1H, J = 1.5 and 2.5 Hz, H5); 8.84 (s,
1H, H20); 8.89 (d, 1H, J = 2.5 Hz, H6); 9.23 (d, 1H, J = 1.5 Hz, H3);
C
N, 20.77.
5.1.2.13. N0-[(1E)-(3-Hydroxy-4-methoxyphenyl)methylidene]
pyrazine-2-carbohy-drazide (2m; LASSBio-1183). The title com-
pound was obtained as a yellow solid by the condensation of 3
with 3-hydroxy-4-methoxybenzaldehyde in 95% yield, mp 221–
223 °C. 1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 3.84 (s, 3H,
CH3); 6.85 (d, 1H, J = 8.2 Hz, H50); 7.08 (d, 1H, J = 8.2 Hz, H60);
7.32 (s, 1H, H20); 8.51 (s, 1H, N@CH); 8.77 (dd, 1H, J = 1.4 and
2.4 Hz, H5); 8.90 (d, 1H, J = 2.4 Hz, H6); 9.25 (d, 1H, J = 1.4 Hz,
H3); 9.60 (s, 1H, OH); 12.10 (s, 1H, CONH). 13C NMR (50 MHz,
DMSO-d6, TMS) d (ppm): 55.6 (CH3); 109.2 (C50); 115.5 (C20);
122.5 (C60); 125.5 (C10); 143.3 (C5); 144.0 (C3); 144.8 (C2); 147.7
12.42 (s, 1H, CONH). IR (mmax, KBr) m
(cmꢁ1): 3284, 3097, 1687,
1642, 1334, 849, 765. Anal. Calcd for C15H10N4O3: C, 61.22; H,
3.43; N, 19.04. Found: C, 61.01; H, 3.48; N, 18.88.
5.1.2.18.
N0-[(1E)-Pyridin-4-ylmethylidene]pyrazine-2-carbo-
hydrazide (2r; LASSBio-1189). The title compound was obtained
as a yellow solid by the condensation of 3 with pyridine-4-carbal-
dehyde in 56% yield, mp >250 °C. 1H NMR (200 MHz, DMSO-d6,
TMS) d (ppm): 7.66 (d, 2H, J = 5.6 Hz, H20 and H60); 8.63 (s, 1H,
N@CH); 8.65 (d, 2H, J = 5.6 Hz, H30 and H50); 8.80 (dd, 1H, J = 1.4
and 2.1 Hz, H5); 8.94 (d, 1H, J = 2.1 Hz, H6); 9.28 (d, 1H,
J = 1.4 Hz, H3); 12.56 (s, 1H, CONH). 13C NMR (50 MHz, DMSO-d6,
TMS) d (ppm): 121.7 (C20 and C60); 141.8 (C10); 143.9 (C5); 144.8
(C3); 144.9 (C2); 148.0 (C6); 148.6 (N@CH); 150.9 (C30 and C50);
(C6); 148.1 (N@CH); 149.3 (C30); 150.4 (C40); 159.2 (C@O). IR (mmax
KBr)
(cmꢁ1): 3439, 3264, 3086, 1673, 1286, 862. Anal. Calcd for
,
m
C13H12N4O3: C, 57.35; H, 4.44; N, 20.58. Found: C, 57.29; H, 4.43;
N, 20.81.
5.1.2.14. N0-[(1E)-(3,4-Dimethoxyphenyl)methylidene]pyrazine-
2-carbohydrazide (2n; LASSBio-1184). The title compound was
obtained as a beige solid by the condensation of 3 with 3,4-dime-
thoxybenzaldehyde in 71% yield, mp 187–189 °C. 1H NMR
(200 MHz, DMSO-d6, TMS) d (ppm): 3.80 (s, 3H, C40-OCH3); 3.82
(s, 3H, C30-OCH3); 7.03 (d, 1H, J = 8.2 Hz, H50); 7.20 (dd, 1H, J = 1.7
and 8.2 Hz, H60); 7.39 (d, 1H, J = 1.7 Hz, H20); 8.52 (s, 1H, N@CH);
8.76 (dd, 1H, J = 1.4 and 2.4 Hz, H5); 8.90 (d, 1H, J = 2.4 Hz, H6);
9.24 (d, 1H, J = 1.4 Hz, H3); 12.18 (s, 1H, CONH). 13C NMR
(50 MHz, DMSO-d6, TMS) d (ppm): 56.0 (C30–OCH3); 56.1 (C40–
OCH3); 109.0 (C50); 112.0 (C20); 122.9 (C60); 127.3 (C10); 143.9
(C5); 144.5 (C3); 145.2 (C2); 148.3 (C6); 149.6 (N@CH); 150.7
160.5 (C@O). IR (mmax, KBr) m
(cmꢁ1): 3380, 3054, 1696, 1272,
841. Anal. Calcd for C11H9N5O: C, 58.14; H, 3.99; N, 30.82. Found:
C, 58.22; H, 3.97; N, 30.43.
5.1.2.19.
N0-[(1E)-Pyridin-2-ylmethylidene]pyrazine-2-carbo-
hydrazide (2s; LASSBio-1269). The title compound was obtained
as a yellow solid by the condensation of 3 with pyridine-2-carbal-
dehyde in 69% yield, mp 208–210 °C. 1H NMR (200 MHz, DMSO-d6,
TMS) d (ppm): 7.43 (m, 1H, H40); 7.86 (m, 1H, H50); 8.01 (d, 1H,
J = 7.9 Hz, H60); 8.61 (d, 1H, J = 4.4 Hz, H30); 8.67 (s, 1H, N@CH);
8.79 (dd, 1H, J = 1.4 and 2.4 Hz, H5); 8.92 (d, 1H, J = 2.4 Hz, H6);
9.27 (d, 1H, J = 1.4 Hz, H3); 12.52 (s, 1H, CONH). 13C NMR
(50 MHz, DMSO-d6, TMS) d (ppm): 120.7 (C60); 125.1 (C40); 137.5
(C50); 143.9 (C5); 144.8 (C3); 145.1 (C2); 148.4 (C6); 150.1
(N@CH); 150.5 (C30); 153.7 (C10); 160.6 (C@O).
(C30); 151.6 (C40); 160.0 (C@O). IR ( (cmꢁ1): 3424,
mmax, KBr) m
3050, 1688, 1314, 867. Anal. Calcd for C14H14N4O3: C, 58.73; H,
4.93; N, 19.57. Found: C, 58.59; H, 4.96; N, 19.71.
5.1.2.15. N0-[(1E)-(3,4,5-Trimethoxyphenyl)methylidene]pyra-
zine-2-carbohydra-zide (2o; LASSBio-1181). The title compound
was obtained as a yellow solid by the condensation of 3 with
3,4,5-trimethoxybenzaldehyde in 73% yield, mp 168–170 °C. 1H
NMR data are in agreement with those previously published.17
1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 3.71 (s, 3H, C40-
OCH3); 3.84 (s, 6H, C30-OCH3 and C50-OCH3); 7.03 (s, 2H, H20 and
H60); 8.55 (s, 1H, N@CH); 8.78 (dd, 1H, J = 1.1 and 2.3 Hz, H5);
8.92 (d, 1H, J = 2.3 Hz, H6); 9.25 (d, 1H, J = 1.1 Hz, H3); 12.24 (s,
1H, CONH). 13C NMR (50 MHz, DMSO-d6, TMS) d (ppm): 56.6
(C30-OCH3 and C50-OCH3); 60.7 (C40-OCH3); 105.2 (C20 and C60);
130.1 (C10); 140.2 (C40); 143.9 (C5); 144.6 (C3); 145.3 (C2); 148.3
IR (mmax, KBr) m
(cmꢁ1): 3431, 3093, 1700, 1270, 861, 774. Anal.
Calcd for C11H9N5O: C, 58.14; H, 3.99; N, 30.82. Found: C, 58.17; H,
3.97; N, 30.51.
5.2. Biological assays
5.2.1. Reagents
Acetic acid (Merck), arabic gum (Sigma–Aldrich), morphine sul-
phate (Dimorf-Cristalia-BR), Zymosan A (Sigma–Aldrich), complete
Freund’s adjuvant (CFA) (Sigma–Aldrich), celecoxib (Merck Sharp &
Dome), indomethacin (Merck Sharp & Dome) and thalidomide (Sig-
ma–Aldrich) were obtained from commercial sources. A solution of
2.5% formalin was prepared with formaldehyde (Merck) in saline
(NaCl 0.9%). In all experiments, Indometacin, celecoxib, thalido-
mide, LASSBio-1018 (1) and compounds 2a–s were used as suspen-
sions in arabic gum.
(C6); 150.4 (N@CH); 153.8 (C30 and C50); 160.1(C@O). IR (mmax
KBr)
(cmꢁ1): 3192, 3015, 1690, 1295, 849. Anal. Calcd for
15H16N4O4: C, 56.96; H, 5.10; N, 17.71. Found: C, 56.68; H, 5.14;
,
m
C
N, 17.69.
5.1.2.16. N0-[(1E)-(3,5-Di-tert-butyl-4-hydroxyphenyl)methyli-
dene]pyrazine-2-carbohydrazide (2p; LASSBio-1187). The title
compound was obtained as a white solid by the condensation of
3 with 3,5-di-tert-butyl-4-hydroxybenzaldehyde in 87% yield, mp
>250 °C. 1H NMR data are in agreement with those previously pub-
lished.19 1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 1.41 (s, 18H,
CH3); 7.19 (s, 1H, OH); 7.48 (s, 2H, H20 and H60); 8.54 (s, 1H,
N@CH); 8.78 (dd, 1H, J = 1.0 and 2.4 Hz, H5); 8.91 (d, 1H,
J = 2.4 Hz, H6); 9.24 (d, 1H, J = 1.0 Hz, H3); 12.03 (s, 1H, CONH).
5.2.2. Animals
Experiments were conducted using Swiss mice obtained from
the BIOCEN—UFAL breeding unit, weighing 20–30 g each, males
or females, adult, with 6–8 weeks of age, distributed in groups
up to 6–8 animals for treatment. Wistar rats (130–200 g), males
or females, 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 num-
ber: 006443/2005-78).
IR (mmax, KBr) m
(cmꢁ1): 3631, 3257, 3041, 2954, 1677, 1293, 859.
Anal. Calcd for C20H26N4O2: C, 67.77; H, 7.39; N, 15.81. Found: C,
67.70; H, 7.38; N, 15.70.

5014
Yolanda Karla Cupertino da Silva et al. / Bioorg. Med. Chem. 18 (2010) 5007–5015
5.2.3. Writhing test
capsaicin injection. Control group received 10 mL/kg of
vehicle (arabic gum) (po) and another group was treated concom-
This test was performed as described by Collier and co-work-
ers.8 Acetic acid (0.6%, v/v) was administered ip in a volume of
0.1 mL/10 g. The number of writhes, a response consisting of con-
traction of an abdominal wall, pelvic rotation followed by hind
limb extension, was counted during continuous observation for
20 min beginning 5 min after the acetic acid injection. The pyra-
zine N-acylhydrazone derivatives 2a–s, indomethacin, celecoxib
and thalidomide were administered at the dose of 100 lmol/kg
(po), 40 min before the acetic acid injection. Control group received
10 mL/kg of vehicle (arabic gum) (po) and another group was trea-
ted concomitantly with 50
itantly with 50
l
mol/kg of thalidomide (po) and 50
lmol/kg of
celecoxib (po). Capsaicin (250
lg) was injected into the inner sur-
face of the right ear of mice 40 min after oral administration of
the tested compounds. The left ear received acetone, delivered
in the same manner. Thirty minutes after capsaicin application,
mice were killed and both ears were removed. Circular sections
were taken using a 6 mm diameter cork borer and weighed. The
weight gain caused by capsaicin was measured by subtracting
the weight of untreated left ear sections from that of treated right
ear sections. Ear edema was quantified as the difference in weight
between the challenged and the unchallenged ear. Inhibition per-
centage was calculated using (C ꢁ T)/C ꢀ 100(%), where C and T
indicate non-treated (vehicle) edema and drug-treated edema,
respectively.
lmol/kg of thalidomide (po) and
50 mol/kg of celecoxib (po). Antinociceptive activity was ex-
l
pressed as inhibition percentage of the usual number of writhing
observed in control animals.
5.2.4. Formalin-induced pain in mice
The formalin test was performed as described by Hunskaar and
5.2.8. Induction of arthritis by complete Freund’s adjuvant
(CFA)
Hole.9 Animals received 20
lL of a 2.5% formalin solution (0.92%
formaldehyde in saline) in the ventral surface of the right hind
paw. They were observed from 0 to 5 min (neurogenic phase)
and from 15 to 30 min (inflammatory phase) after injection and
the time they spent licking the injected paw was recorded and con-
sidered as indicative of nociception. The pyrazine N-acylhydrazone
derivatives 2a–s, indomethacin, celecoxib and thalidomide were
The arthritic syndrome was induced in rats (130–170 g) accord-
ing to the method proposed by Newbould, with some modifica-
tions.15 Complete Freund’s adjuvant (CFA) (0.1 mL) was injected
into the right paw of rats. After 13 days, animals with pronounced
arthritis were separated into groups of 6, so that each group had
approximately similar mean arthritic scores. Compounds 2o and
administered at the dose of 100
lmol/kg (po), 40 min before for-
thalidomide were administered at the dose of 100 lmol/kg (po)
malin injection. Control group received 10 mL/kg of vehicle (arabic
gum) (po) and another group was treated concomitantly with
from the 14th to the 21st day after immunization. The control
and arthritic groups were treated with 10 mL/kg of vehicle (arabic
gum) (po) during the same period. The paw volumes were mea-
sured 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 paw
volumes of the positive control group.
50
lmol/kg of thalidomide (po) and 50 lmol/kg of celecoxib (po).
5.2.5. Hot-plate test
Mice were treated according to the method described by Eddy
and Leimbach.11 Each mouse was placed on the hot-plate set at
54 1.0 °C and the time of paw licking was recorded before and
30 min after oral administration of the tested compounds. The pyr-
azine N-acylhydrazone derivatives 2a–s, celecoxib and thalido-
5.2.9. Statistical analysis
Data obtained from animal experiments are represented by
mean standard error of the mean (mean S.E.M.). Statistical dif-
ferences between the treated and the control groups were evalu-
ated by test t of Student or ANOVA in the tutorial PrismaÒ.
mide were administered at the dose of 100
group received 10 mL/kg of vehicle (arabic gum) (po) and another
group was treated concomitantly with 50 mol/kg of thalidomide
(po) and 50 mol/kg of celecoxib (po). Morphine was also used
as a drug standard at the dose of 15 mol/kg (ip). Analgesia was
lmol/kg (po). Control
l
*
**
***
l
Values were considered significant if P <0.05, P <0.01 and P
l
<0.001.
defined as an increase in the latency of paw licking, and the latency
times were compared with the values obtained for control. Sixty
seconds were taken as the cut-off time to avoid mouse tissue
damage.
Acknowledgments
The authors thank CNPq (BR), CAPES (BR), FAPEAL (BR), FAPERJ
(BR) and INCT-INOFAR (BR, 573.564/2008-6) for fellowships and
financial support.
5.2.6. Zymosan-induced peritonitis
Peritoneal inflammation was induced according to the method
described by Doherty et al.12 A solution of Zymosan A (Sigma–Al-
drich) (2 mg/mL) was prepared in saline (NaCl 0.9%) and injected
into the peritoneal cavity of mice (0.5 mL). Six hours after injection
of Zymosan A, the animals were killed by cervical dislocation and
the peritoneal cavity was washed with 3 mL of cold Hank’s. The
pyrazine N-acylhydrazone derivatives 2a–s, indomethacin, cele-
coxib and thalidomide were administered at the dose of
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5.2.7. Capsaicin-induced mouse ear edema
The capsaicin-induced ear edema model was performed as
described by Mantione and Rodriguez.13 The pyrazine N-acylhyd-
razone derivatives 2a–s, indomethacin, celecoxib and thalidomide
were administered at the dose of 100 lmol/kg (po), 40 min before

Yolanda Karla Cupertino da Silva et al. / Bioorg. Med. Chem. 18 (2010) 5007–5015
5015
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