Discovery of new orally effective analgesic and anti-inflammatory hybrid furoxanyl N-acylhydrazone derivatives

Article abstract of DOI:10.1016/j.bmc.2012.01.034

We report the design, the synthesis and the biological evaluation of the analgesic and anti-inflammatory activities of furoxanyl N-acylhydrazones (furoxanyl-NAH) by applying molecular hybridization approach. Hybrid compounds with IL-8-release inhibition capabilities were identified. Among them, furoxanyl-NAH, 17, and benzofuroxanyl-derivative, 24, together with furoxanyl-NAH derivative, 31, without IL-8 inhibition displayed both orally analgesic and anti-inflammatory activities. These hybrid derivatives do not have additional LOX- or COX-inhibition activities. For instance, LOX-inhibition by furoxanyl-NAH derivative, 42, emerged as a structural lead to develop new inhibitors. The lack of mutagenicity of the active derivatives 17, 31, and 42, allow us to propose them as candidates for further clinical studies. These results confirmed the success in the exploitation of hybridization strategy for identification of novel N-acylhydrazones (NAH) with optimized activities.

Full text of DOI:10.1016/j.bmc.2012.01.034

Bioorganic & Medicinal Chemistry 20 (2012) 2158–2171
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of new orally effective analgesic and anti-inflammatory hybrid
furoxanyl N-acylhydrazone derivatives
Paola Hernández a, Mauricio Cabrera a, María Laura Lavaggi a, Laura Celano b, Inés Tiscornia c,
Thiago Rodrigues da Costa d, Leonor Thomson b, Mariela Bollati-Fogolín c, Ana Luisa P. Miranda d,
d,
d,
Lidia M. Lima d, Eliezer J. Barreiro d, , Mercedes González , Hugo Cerecetto
⇑
⇑
⇑
a Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Uruguay
b Laboratorio de Enzimología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
c Cell Biology Unit, Institut Pasteur de Montevideo, Uruguay
d LASSBio-Laboratório de Avaliação e Síntese de Substâncias Bioativas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the design, the synthesis and the biological evaluation of the analgesic and anti-inflammatory
activities of furoxanyl N-acylhydrazones (furoxanyl-NAH) by applying molecular hybridization approach.
Hybrid compounds with IL-8-release inhibition capabilities were identified. Among them, furoxanyl-
NAH, 17, and benzofuroxanyl-derivative, 24, together with furoxanyl-NAH derivative, 31, without IL-8
inhibition displayed both orally analgesic and anti-inflammatory activities. These hybrid derivatives do
not have additional LOX- or COX-inhibition activities. For instance, LOX-inhibition by furoxanyl-NAH
derivative, 42, emerged as a structural lead to develop new inhibitors. The lack of mutagenicity of the
active derivatives 17, 31, and 42, allow us to propose them as candidates for further clinical studies. These
results confirmed the success in the exploitation of hybridization strategy for identification of novel
N-acylhydrazones (NAH) with optimized activities.
Received 6 November 2011
Revised 29 December 2011
Accepted 6 January 2012
Available online 2 February 2012
Keywords:
N-Acylhydrazone
Furoxane
Analgesic
Anti-inflammatory
Interleukin-8
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
two isoforms,4 one (COX-1) with a cytoprotective role5 and another
(COX-2) with a role in the inflammation, pain, and fever.6 Conse-
Inflammation is defined as a complex defensive process, in
which the body response to different injuries, characterized by
the accumulation of local fluids and leukocytes with the objective
of eliminating the noxious stimulus. In pathological conditions the
evolution of persistent tissue damage by inflammatory cells is not
suitably repaired.1 An important group of inflammatory mediators
are the eicosanoids which are able to activate and sensibilize
nociceptors, leading to hyperalgesia.2 Eicosanoids role in the gene-
sis of several pathological states, for instance chronic inflammatory
diseases, thrombosis and pain, is well established.3,4 The non-ste-
roidal anti-inflammatory drugs (NSAIDs) have been the most
widely used drugs in the treatment of pain, that is, in rheumatoid
arthritis, and in inflammatory diseases.
quently, COX-2-inhibition appears as a therapeutic key for treat-
ment of pain and inflammation. For example the COX-2 inhibitors
celecoxib7 and valdecoxib8 (1 and 2, Scheme 1a) display anti-
inflammatory activity. However cardiac toxicity, that is, with val-
decoxib and rofecoxib (3, Scheme 1a),9 has raised a cautionary flag
on its research.10 Though, COX-2 remains a very important pharma-
ceutical target for the treatment of debilitating diseases like rheu-
matoid arthritis and osteoarthritis and as a preventative agent for
colon cancer. On the other hand, COX-2 inhibitors, like etoricoxib
(4, Scheme 1a) used for the treatment of chronic rheumatoid and
osteoarthritis, exhibit less gastrointestinal toxicity than traditional
NSAIDs.10 The gastrointestinal bleeding11 and nephrotoxicity asso-
ciated with NSAIDs long term use mandates the investigation of
new analgesic and anti-inflammatory agents as a major chal-
lenge.12–14
The majority of clinically employed NSAIDs are inhibitors of the
prostaglandin synthase (cyclooxygenase, COX). The COX exists in
Recently, we have described the N-acylhydrazone (NAH) sub-
unit as the pharmacophoric framework of agents with potent
antinociceptive,15–17 antiplatelet,18–20 anti-inflammatory,21 and
cardiac stimulant22,23 activities. The relative acidity of the amide
hydrogen of the NAH function, as well as its capacity for stabilizing
free radicals give these compounds the ability of mimicking the
bis-allylic moiety of unsaturated fatty acids and amides like, that
is, arachidonic acid.24,25 We have discovered the novel orally active
⇑
Corresponding authors. Addresses: LASSBio—Laboratório de Avaliação e Síntese
de Substâncias Bioativas, Faculdade de Farmácia, Universidade Federal do Rio de
Janeiro, PO Box 68023, ZIP 21941902 Rio de Janeiro, RJ, Brazil. Tel./fax: +55 21
25626644 (E.J.B); Laboratorio de Química Orgánica, Facultad de Ciencias, Iguá 4225,
11400 Montevideo, Uruguay. Tel.: + 598 2 5258618x216; fax: +598 2 5250749
(M.G., H.C.).
E-mail addresses: ejbarreiro@ccsdecania.ufrj.br (E.J. Barreiro), megonzal@fq.
edu.uy (M. González), hcerecet@fq.edu.uy (H. Cerecetto).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.034

P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
2159
(a)
SO2CH3 H3C
SO2CH3
SO2NH2
H3C
SO2NH2
N
N
N
N
N
O
CH3
O
O
Cl
CF3
1
(celecoxib)
2
(valdecoxib)
3
(rofecoxib)
4 (etoricoxib)
(b)
H3C
Br
Br
OH
OCH3
N
CH3
NO2
S
H3C
O
N
O
N
N
H
O
N
O
N
N
N
N
H
H
H
N
N
H
H
H3C
H3C
HN
N
N
H
H
H
H
N
N
N
O
O
O
O
5
(LASSBio-125)
6
(LASSBio-294)
7
(LASSBio-208)
8
(LASSBio-346)
9 (LASSBio-132)
O
(c)
O
O
HO
OH
SO2CH3
O
O
O
O
O
O
O2S
N
O
NH2
O
O
O
N
O
N
O
N
N
N
O
O
O
O
CH3
10
11
12
Scheme 1. (a) COX-2 inhibitors. (b) Orally active NAH derivatives previously developed. (c) Furoxans developed as analgesic and anti-inflammatory agents.
prototypes 5–7 (LASSBio-125, LASSBio-294, and LASSBio-208,
respectively, Scheme 1b)26,27 with analgesic profile, in the AcOH-
induced mice model of abdominal constriction, and cardioactive
effects in the case of derivative 6.22 Other interesting NAHs have
been the derivatives belonging to the 2-methyl-imidazolyl-3-acy-
lhydrazone class which were designed by applying molecular sim-
plification on the imidazo[1,2-a]pyridine-derivative 7. These NAH
derivatives, that is, 8 (LASSBio-346, Scheme 1b), have important
antinociceptive profiles28 and anti-inflammatory activities.29 Other
interesting example, that belongs to hydrazone group, has been
compound 9 (LASSBio-132, Scheme 1b).30 This compound, de-
signed as 5-lipoxygenase (LOX) inhibitor via its free-radical scav-
enger properties,31 displayed in vivo anti-inflammatory activity
with concomitant inhibition of neutrophil accumulation in the
pleural cavity.
These approaches encourage us to design and develop hybrid
agents, which combine NAH and Fx moieties, with potential analge-
sic and anti-inflammatory properties. In the design stage we reck-
oned the COX-2-inhibitors valdecoxib and etoricoxib (2 and 4,
respectively, Scheme 1a) as chemical templates for the develop-
ment of new compounds combining in these structures molecular
moieties from NAH-parent compounds 5–9 and 3-methyl-4-furox-
anyl, 4-phenyl-3-furoxanyl or 5-benzofuroxanyl as NO-release and/
or free radical-scavenger moieties (Scheme 2). In our planning the
acyl moiety of NAH (R1) was pyridine-3-yl (like 4), benzo[d]
[1,3]dioxole-5-yl (like 5 and 6), 4H-imidazo[1,2-a]pyridine-3-yl
(like 7), 1H-imidazole-4-yl (like 8), 1-phenyl-1H-pyrazole-4-yl (like
9), furoxan-3- or -4-yl (as potential bioisoster of 5-methylisoxazole-
4-yl present in compound 2). On the other hand, the hydrazonyl
moiety of NAH (R2) was 4-(dimethylamino)phenyl (like 5), 2-thienyl
(like 6), 4-bromophenyl (like 7 and 8), 4-hydroxy-3-methoxyphenyl
(like 9), or the furoxanyl moieties. Additionally, in this position
other substituents were investigated, that is, 2-furyl, 5-nitro-2-
thienyl, 5-nitro-2-furyl, 3-hydroxy-4-methoxyphenyl, 3,4-dime-
thoxyphenyl, benzo[d][1,3]dioxole-5-yl, 2-hydroxyphenyl, and
4-carboxyphenyl.
Recently, hybrid NSAID-furoxan derivatives, that is, 10 (Scheme
1c), have been developed as promising antiarthritic drugs.32 These
hybrid compounds, designed as COX inhibitors without gastrotoxic
effects due to its potential nitric oxide (NO) release capability, have
demonstrated ability to inhibit tumor necrosis factor-alpha (TNFa)
release from both monocytes and macrophages. Interestingly,
furoxan (1,2,5-oxadiazole N-oxide, Fx) system has also exhibited
bioisosterism with the 2-(5H)furanone moiety,33 present in COX-
2-inhibitor rofecoxib. The mixture of N-oxide 11 (Scheme 1c) is
the most selective COX-2/COX-1 developed Fx. The successful use
of the Fx moiety in the generation of hybrid glucocorticoid has also
been described (compound 12, Scheme 1c).34 Apart from the
gastroprotective activity, the Fx pharmacophore is able to act as
regulator of expression- and activity- of COX and -LOX, by NO-ara-
chidonic acid release-modulation,35,36 or as a free-radical scaven-
ger moiety37 modifying the intracellular redox-balance.
Thirty two hybrid furoxanyl N-acylhydrazones (furoxanyl-NAH)
have been synthesized and their in vitro abilities to decrease the
release of the proinflammatory cytokine interleukin-8 (IL-8, CXCL8
according to the new nomenclature)38 in HT-29-NF-
jB-hrGFP cells
have been determined. The best IL-8-release inhibitors with the
lowest in vitro cytotoxicity in J774 murine macrophages were used
orally to determine analgesic and anti-inflammatory properties in
mice. Besides, the anti-inflammatory mechanisms by which the
compounds act will be discussed. In this sense we studied inhibi-
tory properties on COX-1, COX-2, LOX, and NO-releasing capability.

2160
P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
Br
Br
OH
OCH3
H3C
SO2CH3
SO2NH2
NO2
N
R
H3C
O
N
H
O
N
H
N
H
O
N
H
N
N
N
H
H
H
N
H
H3C
H3C
HN
N
N
N
N
N
N
N
2
4
-R= -Ph-4-N(CH3)2,
-R= -2-thienyl,
5
6
7
8
9
O
CH3
O
O
Cl
Hybridization
N
N
O
O
O
N
R2
R1
N
H
R1-
-R2
H3C
H3C
O
O
O
CH3
N
N
N
N
HN
N
N
N
O
N
N
N
N
N
O
O
O
O
CH3
(OR)n
OR'
N
Br
R''
H3C
X
CH3
N
N
N
N
O
O
O
O
CO2H
-X-, -R, -R', -R''= variable, n= 0 or 1
Scheme 2. Design of new hybrid-NAH as potential analgesic and anti-inflammatory agents.
Moreover, the most promising derivatives were studied for their
mutagenicities in order to confirm their potentials as candidate
drugs.
the desired intermediate (VI) in excellent yield (Scheme 3b). Deriv-
ative 44 was prepared using this intermediate. All the compounds
were characterized by 1H NMR, 13C NMR, IR and MS. Except for
derivatives 13 and 25 (see example in Supplementary data) and
according to the NMR experiments, 1H NMR45 and NOE-diff (see
examples in Supplementary data), and characteristic fragmenta-
tions in MS experiments (see example in Supplementary data the
NAH were obtained as the E-isomers around the ylidenic moiety.
In the case of compounds 13 and 25 the main stereoisomers were
the E, with ratio of E:Z 95:5 and 67:33, respectively. The purity was
established by TLC and microanalysis. Only compounds with ana-
lytical results for C, H and N, within 0.4 of the theoretical values
were considered pure enough.
2. Methods and results
2.1. Chemistry
The first compounds synthesized, where R2 were the furoxanyl
moieties, were the 3-pyridinyl derivatives 13–15 (Scheme 3a)
furoxanyl-hybrid of parent compound etoricoxib (4, Scheme 1).
Nicotinic acid was used as starting material followed by the prep-
aration of the corresponding hydrazide39,40 and finally the conden-
sation with furoxanyl aldehydes ((I), (II) or (III)). The same
procedure was used to prepare the hybrid products 16–27 (Scheme
3a) using the hydrazides27,28,40–43 derived from benzo[d][1,3]diox-
ole, 4H-imidazo[1,2-a]pyridine, 1H-imidazole, and 1-phenyl-1H-
pyrazole where the parent compounds are the NAH 5–9 (Scheme
1). When R1 was a furoxanyl moiety, derivatives 28–43 were pre-
pared according to shown in Scheme 3b. The common reagent,
hydrazide (IV), was prepared via the transformation of the alde-
hyde (I) to the corresponding methyl ester42 by treatment with
manganese dioxide and potassium cyanide in methanol followed
by the reaction with 0.9 equiv of hydrazine hydrate. The following
condensation with different aldehydes produced derivatives 28–
43 in good yields. The high reactivity of aldehyde (II) (3-formyl-
4-phenylfuroxan) did not allow us to generate hydrazide (VI) using
the same procedure employed for preparation of (IV) (i. MeOH/
KCN/MnO2; ii. NH2NH2), consequently we used the acid (V) as
the starting material and, by activation with CDI,44 we generated
2.2. Biological characterization
2.2.1. In vitro evaluation of NF-
of IL-8
jB pathway inhibition and level
The in vitro ability to decrease activation of NF-
of IL-8 was evaluated using a human pathway-specific reporter cell
system (HT-29-NF- B-hrGFP). Human intestinal epithelial cells
HT-29 stably transfected with pNF- B-hrGFP plasmid were stimu-
lated with the pro-inflammatory cytokine TNF- . The activation of
NF- B was estimated by measuring the percentage of GFP-express-
jB and the level
j
j
a
j
ing cells while the IL-8-secretion was quantified in the supernatant
by flow cytometry (see example in Supplementary data). Both
determinations were performed in the absence and presence of hy-
brid-NAH (Table 1). The compounds were evaluated at the highest
non-cytotoxic doses (100% of cell survival on HT-29-NF-
jB-hrGFP
cells). Thalidomide (Thal, Table 1) was used as reference drug.

P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
2161
R1-
(a)
Me
Me
HN
O
N
N
N
N
O
N
N
Ph
O
Me
N
O
O
13
14
16
17
19
22
25
26
N
N
N
R1
R1
N
H
Ph
N
O
O
O
O
(I), (II), or (III)
20
21
23
24
N
R1
NHNH2
N
H
EtOH anh. or toluene anh.
AcOH (cat)
O
N
N
O
O
15
18
27
N
R1
N
H
R2
(b)
O
O
N
N
NH
Me
CHO
Me
Me
NHNH2
1) MeOH/KCN/MnO2-0 O
C
R2-CHO
N
N
O
N
N
N
2) NH2NH2 (0.9 equiv.)-0 O
C
EtOH anh. or toluene anh.
AcOH (cat)
O
O
O
O
O
(I)
(IV)
R2-
O
CH3
Ph
N
N
CH3
CH3
O
N
Br
Y
N
N
N
N
O
O
O
O
28
29
30
31
32
HO
OR'
OR
NO2
X
X
X=S, 33
X=S, 35
37 R=R'=Me, 38 R=Me, R'=H, 40 Y=CO2H, 42
R-R'=CH2, 39 R=H, R'=Me, 41 Y=CF3, 43
X=O, 34 X=O, 36
CO2H
O
O
N
OHC
CO2H
Ph
CO2H 1) CDI/Et2O/r.t.
Ph
Ph
NHNH2
NH
EtOH anh./AcOH (cat)
2) NH2NH2/Et2O
N
N
N
N
N
N
O
O
O
O
O
O
44
(V)
(VI)
Scheme 3. Synthetic procedures used to prepare the new derivatives. (I): 3-methyl-4-formylfuroxan; (II): 3-formyl-4-phenylfuroxan; (III): 5-formylbenzofuroxan.
Additionally, two hydrazides, benzo[d][1,3]dioxole-5-carbohyd-
razide and 2-methyl-4H-imidazo[1,2-a]pyridine-3-carbohydraz-
ide, 45 and 46 (Table 1), respectively, and the well-known
NO-releasing compound, 3,4-bis(phenylsulfonyl)furoxan (47, Table
1), were included in order to evaluate the effect of reducing IL-8
levels. Concomitantly, the cytotoxicity against murine cells, J774
macrophages, was evaluated in order to select the best compounds
to progress to in vivo studies (Table 1). Controls of NAH without
2.2.2. In vivo evaluation of analgesia and anti-inflammation
Hybrid-NAHs, 15, 17, 20, 24, 28, 31–33, 36, 38, and 42, were se-
lected for further pharmacological evaluation on pain models and
temporal and chronic inflammation models in order to confirm
its potential as drug candidates. The evaluation of the analgesic
profile of NAH derivatives was performed using the classical acetic
acid-induced mice abdominal constrictions assay,26 administering
orally at 100 lmol/kg and using dipyrone and indomethacin (Ind)
TNF-
a
stimulation were also included with the aim to evaluate
as standard drugs (Table 2). The hybrid NAHs derived from benzof-
uroxan 15 and 24, and derived from furoxan 17 were the best
inhibitors of abdominal constrictions having values in the order
of the reference drug. The hybrid-NAHs derived from methylfuro-
xan displayed lower inhibition capabilities. Derivatives 31 and 32
exhibited better inhibition at 60 min (Table 2) compared to other
compounds. For compounds 15, 17, 24, and 31 we performed a
modified version of the classical hot-plate method,26 suitable for
measuring inflammatory nociception (hyperalgesia) through the
previous treatment of the animals with carrageenan (Fig. 1).
In spite of the poor efficacy, furoxanyl-NAHs 17 and 24 reduced
the potential pro-inflammatory activity of the compounds, finding
that hybrid-NAHs were not pro-inflammatories per se. Most of the
hybrid-NAH displayed the same profile in both biological systems
(see Supplementary data). Hybrid-NAH 15, 17, 18, 20, 24 inhibited
releasing IL-8 and activation of NF-jB in the same order. But others
affected more the release of IL-8 as Thal do, that is, 28–33, 36, 38,
40–42. The hybrid-NAH 17, derived from furoxan, was the best
in vitro IL-8 releasing inhibitor being 16 times more potent than
Thal at the same dose (400 lM). The hybrid NAHs benzofuroxan
15 and furoxan 20 also showed better ability to inhibit IL-8 produc-
tion than Thal while benzofuroxan 24 and furoxans 28, 30, 32, 33,
36, 38, 40, and 42 were as active as Thal. On the other hand, NAH
23 was an IL-8 release promotor, at an intermedium dose
in ca. 14% the
D latency time in comparison with the vehicle group,
at 1 h for 17 and 3 h for 24. Thus indicate their ability to control the
hyperalgesic stimuli in inflammatory pain models. These data were
in agreement with the in vitro results where the reduction of IL-8
(200 lM), duplicating this parameter respect to untreated cells.

2162
P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
Table 1
NF-kB pathway inhibition capabilities, IL-8 levels and J774 cytotoxicities of new hybrid-NAHs
c
c
Compd
Doses (
lM)
%GFPa
%IL-8b
IC50,J774
>400
(l
M)
Compd
Doses (
lM)
%GFPa
%IL-8b
IC50,J774 (lM)
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
100
25
50
112
114
66 10
2
6
105
138
29
4
3
2
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45f
46g
47h
100
100
400
100
100
100
100
100
100
100
400
100
400
100
400
400
50
127
122
121
150
126
122
4
2
5
1
4
2
65 20
>400
>400
>400
195
31
2
1
77
1
68 21
400
100
400
400
400
400
100
200
50
102
12
1
1
106 16
132
191
141
1
2
2
98
61
3
6
129
162
1
2
4
1
87 16
64
5
75 10
82 19
>400
>400
120
32
4
9
77 23
>400
99 14
d
38
76
6
6
56
1
111
136
55
120
97
117
149
125
55
115
118
111
6
4
2
1
1
8
1
9
2
1
1
1
1
1
—
68
1
104
126
103
71
1
3
3
>400
>400
>400
48
—
1
96
1
103 16
246
2
199
51
—
1
1
68 10
98
59 30
>400
>400
>400
>400
>400
>400
>300
>400
>400
—
6
38
1
3
100
400
—
80 21
295
42
37
3
1
1
105
—
6
—
—
77
53
—
2
2
114 31
—
—
Thale
400
200
100
116
114
126 13
6
3
57
77
5
4
>400
50
50
20
63 13
6
5
a
b
c
%GFP: percentage of GFP—expressing cells normalized against the TNF-
a
control cells treated with DMSO 60.5% ( SD).
%IL-8: percentage of IL-8 production respect to TNF-
IC50,J774: IC50 against murine macrophages J774.
—: not determined.
a control cells treated with DMSO 60.5% ( SD).
d
e
f
Thal: thalidomide.
Benzo[d][1,3]dioxole-5-carbohydrazide.
2-Methyl-4H-imidazo[1,2-a]pyridine-3-carbohydrazide.
3,4-Bis(phenylsulfonyl)furoxan. Thal, and compounds 45–47 were included as controls.
g
h
Table 2
Analgesic and anti-inflammatory profiles of new selected NAHs and controls in AcOH-induced abdominal constriction test in mice and carrageenan-induced rat paw edema assay
Compd
Constrictions inhibition at 30 mina (%)
Constrictions inhibition at 60 min (%)
d Paw volume (edema inhibitionc (%)
b
15
17
35 9⁄
—
—
0
0
42 8⁄
20
24
2
5
14
—
5
18
5
46 13⁄⁄
31 9⁄⁄
28
31
32
33
36
38
—
—
—
—
—
—
—
—
14
22
23
14
13
17
0
6
5
6
4
5
6
10
4
19 7⁄
0
0
14
4
3
8
1
42
16
Indd
44 8⁄⁄
61 4⁄⁄
—
Dipyrone
Nimesulidef
6f
36⁄,e
—
—
—
—
—
57.5⁄⁄,g
30 3⁄⁄ g
,
a
b
c
d
e
f
% of inhibition obtained by comparison with vehicle control group.
—: not determined.
Respect to untreated controls (100%). The results refer to the 3rd hour.
Ind: Indomethacin.
From Ref. 26
Evaluated at 300 lmol/kg.
g
From Ref. 21 ⁄p <0.05, ⁄⁄p <0.01. Student t-test.
8
6
4
2
10
8
Control
15
Control
17
24
31
6
4
2
0
0.5
1
2
3
0
1
2
3
4
5
Time (h)
Time (h)
Figure 1. Effect of NAHs 15, 17, 24, and 31 against carrageen-induced hyperalgesia in rats, at 100
lmol/kg, po. Each point represents the mean SEM of 8–10 experimental
values. Controls correspond to treatment with vehicle.

P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
2163
conduces to the reduction of the hyperalgesic response.46 On the
other hand, hybrid benzofuroxan 15 and methylfuroxan 31 were
unable to reduce inflammatory nociception. The furoxanyl-NAH
15 was active in vitro in the IL-8-model however it was inactive
in this in vivo assay. Probably, it could be the result of the low bio-
availability of derivative 15 in the inflammation site.
For the in vivo pharmacological evaluation of furoxanyl-NAHs
anti-inflammatory activities, the carrageenan induced rat paw ede-
ma model, a classic model of acute inflammation,21 was used. Ind,
However in the case of NAH 31 its remarkable in vivo activities were
not related to LOX-inhibition. In general, the methylfuroxan deriva-
tives were better LOX-inhibitors than the corresponding phenylf-
uroxan- or benzofuroxan-analogues (i.e., compare inhibition
capabilities between 3-pyridinyl derivatives 13, 14, and 15 or
3-imidazolyl derivatives 22, and 24). In the series of methylfuro-
xan-NAHs, 28–44, the better LOX-inhibitors were 28, 37, 40, 42,
and 43, being the acid 42 the best one. The phenyl-analogue 44 also
displayed good LOX-inhibition capability at the assayed dose. Inter-
at 100
drug and the NAH-parent compound 6 were used, at 300
kg, as controls. All compounds were administered orally
(100 mol/kg) 1 h before inflammatory challenge. The paws vol-
l
mol/kg, and nimesulide, a diarylether anti-inflammatory
estingly, the best in vitro LOX-inhibitor and a good NF-jB pathway
lmol/
inhibitor (Table 1), NAH 42, did not produce in vivo analgesia or
anti-inflammatory activity (Table 2) probably as result of poor
bioavailability.
l
umes were measured, for all the NAHs, 3 h after the subplantar
injection (Table 2) and temporally for 15, 17, and 31 (Fig. 2). The
edema was calculated as the volume variation between the carra-
geenan and saline treated paw. Between the analyzed hybrid-NAHs
the benzofuroxan 24 and the furoxan 31 were as effective on rat
paw edema prevention as the NAH-parent compound 6 (evaluated
NAHs 17, 24, and 31 were characterized for their COX-1 and
COX-2 inhibitory activity using a COX inhibitor screening assay
kit. Ind was employed as positive control (Table 4).48 Comparing
to Ind the analyzed NAH were poor COXs inhibitors and their bio-
logical in vivo activities seem to be not related with these inhibi-
tions. The NAHs were better COX-1- than COX-2-inhibitors as it
was previously observed with other NAH.21
at 300 lmol/kg). Furoxanyl-NAH 15 and 17, the major in vitro IL-8
inhibitors, are poor or unable to diminishing the edema at the 1st
and 2nd hours (15 produce only ca. 12%, Fig. 2). Contrarily furoxan
31, a slightly in vitro IL-8 inhibitor, showed good ability to reduce
edema since the 1st hour of study, ca. 23%, achieving a 25% of pro-
tection at the 4th hour of the study (Fig. 2). For the complete
in vivo studied NAHs population no clear correlation between
in vitro IL-8 inhibition and in vivo edema diminution was ob-
served. However, the methylfuroxan series, NAHs 28, 31–33, 36,
38, and 42, show certain tendency in the correlation. In this rela-
tionship compound 31 could be considered as an outlier suggesting
a different mechanism of action (see Supplementary data).
Finally, we analyzed the ability of selected hybrid-NAHs to re-
lease NO. These were evaluated in the presence of equimolar con-
centrations of cysteine (Cys) at pH 7.4 and pH 8.3 measuring the
NO production during 20 minutes (Table 5). Comparing to the
well-known NO-releasing furoxan, derivative 47,49 the new hybrid
furoxanyl-NAHs displayed lower or null ability to produce NO.
Benzofuroxan 15 and furoxans 17 and 42 release small amount
of NO under certain conditions. The release of NO could be related
to the capabilities to inhibit IL-8 production however no relation-
ship was observed between the rate of NO-releasing (Table 5)
and the percentage of inhibition on interleukin-8 release (i.e., com-
pare both values for furoxans 17 and 47). Additionally, the best hy-
brid-NAH LOX-inhibitor and good IL-8 release inhibitor, derivative
42, was the best NO-producer. On the other hand, the in vivo activ-
ities seemed to be unrelated to the NO-releasing activities.
2.3. Studying the mechanism of analgesia and anti-
inflammation
The in vivo anti-inflammatory properties of furoxanyl-NAH 24
could be explained by its in vitro inhibitory action on IL-8 release.
But for some of the rest NAH the in vivo analgesia and anti-
inflammation displayed activities are not clearly related, so more
studies were done to access to the biological targets. Consequently,
LOX-, COX-1-, and COX-2-inhibition and capability to produce NO
were studied.
2.4. Hybrid furoxanyl-NAHs as drug candidates
In order to confirm NAHs potential as drug candidates, for treat-
ment of different inflammatory diseases, some others experiments
were performed. Firstly, hybrid-NAH stability at different pH was
studied. Secondly, we studied the mutagenic potential of the com-
pounds using the Ames test.
A commercial kit of soybean lipoxygenase-1547 was used to
determine the inhibitory effect of selected NAH in the lipooxygen-
ation process promoted by hydrogen peroxide. Quercetin (Quer),
at 4 lM, was employed as positive control (Table 3). In the case of
NAH 28 the LOX-inhibition capabilities could be indicating that this
mechanism is the responsible of the in vivo analgesia (Table 2).
Table 3
In vitro LOX-inhibition for new selected NAHs and quercetin
a
Compd
%LOX Inhib a/IC50
(l
M)
Compd
%LOX Inhib / IC50
(l
M)
13
14
15
17
18
20
22
23
24
25
22 20/agb
14 15/>100
0/—c
28
31
32
33
34
36
37
38
40
41
42
43
44
55 10⁄/ꢀ50
1000
0
Control
0/—
10 4/>100
15
0/>100
800
55 11⁄/ꢀ50
0/—
17
d
0
600
31
27 28/ag
37 12/ag
0/—
58 23
d
0/—
0/—
400
200
0
1
4/—
74 10⁄⁄/35
55 5⁄/ꢀ50
53 7⁄/ꢀ50
3
Quere
35
a
% of LOX-inhibition at 50
ag: the compound formed aggregates in the buffer, pH 7.4, at doses higher than
lM doses.
0
1
2
3
4
5
b
Time (h)
50
l
M.
c
d
e
—: not determined.
Figure 2. Time course effect of NAHs on carrageenan induced rat paw edema.
Results are expressed in terms of mean SEM (n = 8–10 animals per group). Control
corresponds to treatment with vehicle.
The compound interfered in the assay measurement.
Quer: quercetin, evaluated at 4 l
M. ⁄p <0.05, ⁄⁄p <0.01 Student t-test.

2164
P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
The method of direct incubation in plate50 using culture of
Salmonella typhimurium TA98 strain was performed on one
furoxan-NAH with in vivo analgesic property, 17, one benzofuro-
xan-NAH and one furoxan-NAH with in vivo analgesic and anti-
inflammation properties, 24, and 31, and the furoxan-NAH with
in vitro LOX-inhibition ability, 42. Except for NAH 31, the com-
pounds evaluated in this test presented an important capability
Table 4
In vitro COX-1 and COX-2 inhibition assay for new selected NAHs and indomethacin
a
Compd
Dose (
lM)
%COX-1 Inhib
%COX-2 Inhiba
17
10
50
10
50
10
50
0.1
6
27.0 0.4⁄⁄⁄
28.5 0.1⁄⁄⁄
25.9 3.6⁄⁄⁄
22.3 0.9⁄⁄⁄
22.5 0.5⁄⁄⁄
25.3 0.3⁄⁄⁄
49.7 0.7⁄⁄⁄
—
13.1 2.3
11.9 1.7
19.6 2.1
6.4 2.5
24
31
17.8 1.2⁄
of inhibition of the NF-jB pathway. The influence of metabolic
22.0 1.3⁄
Inde
—
c
activation was tested by adding S9 fraction of mouse liver. Positive
controls of 4-nitro-o-phenylendiamine and 2-aminofluorene were
run in parallel. The revertant number was manually counted and
compared to the natural revertant (Table 6). The compound is
considered mutagenic when the number of revertant colonies is
at least twofold of the spontaneous revertant frequencies for at
least two consecutive dose levels.51 The maximum assayed doses
were determined according to toxic effect on S. typhimurium. The
furoxan-NAHs, 17, 31, and 42, were not mutagenic however the
benzofuroxan-NAH, 24, displayed mutagenic effects without S9
fraction and surprisingly this property disappeared after
metabolization.
53.4 9.8⁄⁄
a
b
c
% of COX-inhibition.
Ind: indomethacin.
—: not determined. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001. Student t-test.
Table 5
NO releasing rate for new selected NAHs
Compd
NO (nM/min)a
pH 7.4
pH 8.3
15
17
18
20
24
41
42
44
47
0
0
—
0
0
0.64b
0.39c
0
0
0
3. Discussion and conclusions
d
We reported the synthesis and biological evaluation of new hy-
brid furoxanyl N-acylhydrazones as orally effective analgesic and
anti-inflammatory agents. The compounds were designed as hy-
brid entities combining different aromatic residues, between oth-
ers the furoxan system, connected by N-acylhydrazone moiety.
—
0
1.04
—
0.19
0
3700e
ndf
a
b
c
d
e
f
At 25 °C and NAH:Cys ratio of 1:1.
At 37 °C.
NAH:Cys ratio of 3:1.
—: not determined.
From Ref. 49
Hybrid furoxans 17 and 20 were one of the best in vitro NF-
pathway inhibitors (Table 1) being at 400 M 15 and 1.6 times
more active than thalidomide, the latest described as a reducing
agent for IL-8 production (in vivo NF-
B pathway inhibitor).52
The corresponding parent hydrazides 45 and 46, according to
GFP expression, displayed not activity on the NF- B pathway.
jB
l
j
nd: not described.
j
The benzofuroxan 15 was also one of the best in vitro IL-8 inhibitor
being 2 times more active than thalidomide at the lower assayed
concentration. Some other hybrid N-acylhydrazones, 18, 19, 21,
In aqueous solution, at 37 °C, and at different pHs (2.0, 5.0, 7.2,
and 8.0) the studied hybrid-NAHs, 13–15, 17, 20, 21, 27-33, 36, 38,
and 42, were stables during 24 h (evidenced by chromatography)
(not shown).
30, 38, 40, and 42, at 400
lM displayed similar in vitro activity
to thalidomide while derivatives 24, 28, 29, 32, 33, 36, 37, 41,
Table 6
Number of revertants of derivatives 17, 24, 31, and 42 on TA98 S. typhimurium strain
S9
17
24
31
42
Da
NRb,c
Md
Da
NRb,c
Md
Da
NRb,c
Md
Da
NRb,c
Md
(ꢁ)
0
10
8
9
10
7
11
8
3
4
3
12
13
2
0
6
19
56
167
500
0
6
19
56
167
500
10
10
23
30
40
47
11
12
12
13
17
3
0
4
13
39
117
350
0
4
13
39
117
350
10
11
8
17
8
15
11
7
10
8
3
0
6
19
56
167
500
0
6
19
56
167
500
10
5
9
11
10
18
11
14
10
8
3
3.1
9.3
28
83
250
0
3.1
9.3
28
83
250
1
4
3
1
1
4
1
1
1
3
3
2
2
5
6
9
1
7
6
1
1
1
3
1
6
4
1
1
0
3
1
1
7
1
2
4
5
1
4
3
1
1
6
M+e
(+)
5
10
9
14
27 14
4-NPDf
AFg
Dc
NRd,e
1900 200
Dc
NRd,e
844 80
ꢁS9
20.0
+S9
10.0
a
b
c
d
e
f
D: doses in
NR: number of revertants.
lg/plate.
The results are the means of two independent experiments SD.
M: mutagenicity, according to Ref. 51 (see text).
M+: Response is considered positive because it is the second dose in which the revertant levels are at least twice the spontaneous frequencies.
4-NPD: 4-nitro-o-phenylendiamine.
AF: 2-aminofluorene.
g

P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
2165
and 43 were more effective than thalidomide even at the lowest
concentration. Even though the well NO-releasing agent, 47, inhib-
ited the IL-8 production, the inhibitory effect of our hybrid deriva-
tives on IL-8 production seems not to be related to NO-production
for further studies. However, the benzofuroxan 24 resulted muta-
genic in absence of S9 fraction which does not justify, at least ini-
tially, additional studies.
These findings prompt us to deepen our studies, modifying
doses, administration routes, and combinations with other drugs.
(Table 5). From a chemical point of view our best NF-jB pathway/
IL-8 releasing inhibitors, 15, 17, 20, 24, and 43, share with some
previously described inhibitors (48–50, Fig. 3)53 several structural
motives, that is, trifluorophenyl-substituted, and aza-heterocyclic
carboxamide/imide- or benzo[d][1,3]dioxole-arylic substituted.
The toxicity of the new derivatives against murine macrophages
was studied (Table 1) finding a wide range of behaviors. In general,
the methylfuroxan derivatives, NHA 28–44, showed lower toxici-
ties than the rest of the studied hybrid compounds. This informa-
tion together with IL-8 production inhibition was used to select
the N-acylhydrazones to be evaluated in vivo. Accordingly, we in-
cluded in the in vivo assays the best IL-8 release inhibitors 17,
and 20, with low macrophage-IC50s, the good IL-8 inhibitors 15,
24, 28, and 33, with high macrophage-IC50s, the good IL-8 inhibi-
tors 32, and 36, with low macrophage-IC50s, and the moderate
IL-8 inhibitors 38, and 42, with high macrophage-IC50s. Addition-
ally, an in vitro non IL-8 inhibitor was included in the in vivo assay,
derivative 31. Compounds 24 and 31 were the best developed hy-
brid NAHs that display both analgesic and anti-inflammatory activ-
ities when they were administered orally (Table 2). Compounds 20,
28, and 36 exhibited moderate analgesic and anti-inflammatory
properties. On the other hand, derivatives 15 and 17 at 30 min
and 32 and 38 at 60 min displayed relevant in vivo analgesic prop-
erties. Furthermore derivatives 17 and 24 were able to modestly
reduce inflammatory nociception between 0.5 and 3 h (Fig. 1)
and the analgesic property of derivative 31 was maintained be-
tween 1 to 4 h ca. 20% (Fig. 2). These data agree with the in vitro
4. Experimental
Reagents were purchased from Aldrich and used without fur-
ther purification. Melting points were performed using an Electro-
thermal Engineering Ltd melting point apparatus, and the results
were uncorrected. Infrared spectra were recorded on a Bomem
FTLA2000 spectrophotometer instrument as films on KBr discs.
1H and 13C NMR spectra were recorded in the indicated solvent
on Bruker DX 400 MHz spectrometer. Chemical shifts are quoted
in parts per million downfield from TMS and the coupling con-
stants are in Hertz. Mass spectra were recorded on a Hewlett Pack-
ard MSD 5973 (electronic impact, EI) or on a Hewlett Packard LC/
MS Series 1100 (electrospray ionization, ESI) instruments. All sol-
vents were dried and distilled prior to use. All the reactions were
carried out in a nitrogen atmosphere. Reactions were monitored
by TLC using commercially available precoated plates (Merck Kie-
selgel 60 F254 silica) and developed plates were examined under
UV light (254 nm) or as iodine vapor stains. Column chromatogra-
phy was performed using 200 mesh silica gel. To determine the
purity of the compounds, microanalyses were done on a Fisons
EA 1108 CHNS–O instrument from vacuum-dried samples and
were within 0.4 of the values obtained by calculated composi-
tions. Compounds (I)–(III), benzo[d][1,3]dioxole-5-carbohydrazide,
2-methyl-4H-imidazo[1,2-a]pyridine-3-carbohydrazide, 5-methyl-
1H-imidazole-4-carbohydrazide, and 1-phenyl-1H-pyrazole-4-car-
bohydrazide were prepared following synthetic procedures
previously reported.27,28,40–43,54–56
inhibition of the NF-jB pathway.
Apart from IL-8 inhibition, mechanism of in vivo analgesia and
anti-inflammation, additional biochemical pathways were studied
including inhibition of LOX, COX-1, and COX-2. The in vivo active
derivatives 17, 24, and 31 did not inhibit COXs at the indomethacin
level however some degree of better inhibition of COX-1 than
COX-2 were displayed (Table 4). Derivatives 17, 24, and 31 did
not inhibit LOX at the quercetin level (Table 3). In reference to
LOX-inhibition (Table 3), derivative 42 appeared as a structural
hit to develop new inhibitors of this enzyme, also hybrid-NAHs
18, 28, 43, and 44 could be considered.
4.1. Nicotinohydrazide
A mixture of nicotinic acid (3 g, 24 mmol), MeOH (60 mL) and
H2SO4 (c) (12 mL) was heated at reflux for 1.5 h. The MeOH was
distilled at reduced pressure and the residue was dissolved AcOEt
(50 mL) and washed with saturated aqueous solution of NaHCO3
(3 ꢂ 10 mL). The organic phase was dried with anhydrous Na2SO4
and evaporated under reduced pressure. The residue, methyl nico-
tinate (white solid, 2.7 g, 83%), was used in the next step without
purification. A mixture of methyl nicotinate (2.7 g, 20 mmol),
In line with these findings, furoxans 17, 31, and 42 were not
mutagenic in the Ames test (Table 6), making them excellent leads
CF3
N
N
N
N
N
N
N
N
O
O
O
O
O
Me
N
Me
HN
Me
O
O
O
O
O
O
O
O
N
N
N
N
N
O
O
N
H
N
H
N
H
N
H
N
H
N
O
O
N
N
N
N
15
17
20
24
43
F
CF3
N
O
CF3
O
NH
O
NH
N
O
O
N
N
H
CF3
N
O
Cl
N
Me
N
Me
48
49
50
Figure 3. Structural scaffolds (dotted squares) shared by some IL-8-production inhibitors and our hybrid-NAHs.

2166
P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
MeOH (4 mL) and NH2NH2ꢃH2O (98%, 0.95 mL) was heated at reflux
for 6.5 h. The white solid, nicotinohydrazide, was filtered and
washed several times with cold EtOH (1.8 g, 67%).
5%)). White solid (56%); mp: 196.3–198.1 °C. 1H NMR (DMSO-d6) d
ppm: 6.13 (2H, s), 7.07 (1H, d, J = 8.0), 7.43 (1H, s), 7.50 (1H, m),
7.58 (4H, m), 7.62 (3H, m), 7.98 (2H, m), 8.35 (1H, s), 12.14 (1H,
s). 13C NMR (DMSO-d6) d ppm: 102.5, 108.0, 108.5, 113.5, 123.5,
126.0, 127.0, 129.0, 129.5, 131.5, 132.5, 148.0, 151.0, 157.0. IR
4.1.1. General procedure for the synthesis of compounds 13–15
To a solution of nicotinohydrazide (150 mg, 1.1 mmol) in dry
EtOH (2.5 mL) was added the corresponding aldehyde ((I), (II), or
(III), 0.9 mmol) and a drop of glacial AcOH. The mixture was stirred
until the aldehyde was consumed (checked by TLC). The solid pre-
cipitated was filtered and washed with cold EtOH.
(KBr)
t
(cmꢁ1): 3219, 1658, 1565, 1565, 1504, 1262, 1173, 1117,
1083, 1041, 929, 874, 842. MS (EI) m/z (%): 352 (M+, 1), 149
(100), 121 (26), 77 (9). Calcd analysis for C17H12N4O5: C: 58.0; H:
3.4; N: 15.9. Found: C: 57.8; H: 3.3; N: 15.6.
4.2.3. (E)-N0-(5-Benzofuroxanylmethylidene)benzo[d][1,3]
dioxole-5-carbohydrazide (18)
4.1.1.1. N 0-(2-Methyl-3-furoxanylmethylidene)nicotinohydraz-
ide (13).
(E/Z proportion = 95:5). White solid (46%). E-isomer:
The solid precipitated was filtered and washed with cold tolu-
ene. Yellow solid (98%); mp: 220.4–221.8 °C. 1H NMR (DMSO-d6)
d ppm: 6.14 (2H, s), 7.07 (1H, d, J = 8.0), 7.47 (1H, s), 7.55 (1H, d,
J = 8.0), 7.85–8.12 (3H, m), 8.47 (1H, s), 12.08 (1H, s). 13C NMR
(DMSO-d6) d ppm: 102.4, 108.2, 108.6, 113.7, 118.0, 123.6, 127.1,
1H NMR (DMSO-d6) d ppm: 2.41 (3H, s), 7.63 (1H, d, J = 8.0), 8.28
(1H, d, J = 8.0), 8.48 (1H, s), 8.91 (1H, s), 9.09 (1H, s), 12.56 (1H,
s). 13C NMR (DMSO-d6) d ppm: 9.6, 112.2, 124.3, 128.9, 136.1,
137.3, 149.1, 153.3, 154.5, 162.6, 207.0. IR (KBr)
t
(cmꢁ1): 3205,
1703, 1604, 1565, 1271, 1024, 853, 795. MS (ESI) m/z (%): 248
(M++H, 100), 106 (M+ꢁ141, 1). Calcd analysis for C10H9N5O3: C:
48.6; H: 3.7; N: 28.3. Found: C: 48.5; H: 3.4; N: 27.9.
129.9, 145.0, 147.9, 150.9, 162.9. IR (KBr) t
(cmꢁ1): 3222, 1649,
1614, 1572, 1551, 1257, 1171, 1110, 1062, 1031, 929, 857. MS
(EI) m/z (%): 326 (M+, 1), 149 (100), 121 (27), 65 (50). Calcd analysis
for C15H10N4O5: C: 55.2; H: 3.1; N: 17.2. Found: C: 55.0; H: 3.1; N:
17.1.
4.1.1.2. (E)-N0-(4-Phenyl-3-furoxanylmethylidene)nicotinohyd-
razide (14).
Yellow solid (24%); mp: 159.7–162.8 °C. 1H NMR
(DMSO-d6) d ppm: 7.35 (1H, s), 7.59 (2H, d, J = 8.0), 7.98 (2H, d,
J = 8.0), 8.16 (1H, d, J = 8.0), 8.37 (1H, s), 8.81 (1H, d, J = 4.0), 9.07
(1H, s), 12.44 (1H, s). 13C NMR (DMSO-d6) d ppm: 113.3, 124.2,
126.1, 129.5, 131.8, 134.2, 135.1, 136.0, 149.2, 152.3, 153.2, 156.5,
4.3. General procedure for the synthesis of compounds 19–21
To a solution of 2-methyl-4H-imidazo[1,2-a]pyridine-3-carbo-
hydrazide (1 equiv) in dry EtOH (5 mL/mmol of hydrazide) was
added the corresponding aldehyde ((I), (II), or (III), 0.9 equiv) and
a drop of glacial AcOH. The mixture was stirred until the aldehyde
was consumed (checked by TLC). The product was purified as is
indicated below.
162.2. IR (KBr)
t
(cmꢁ1): 3242, 1667, 1587, 1557, 1298, 1022, 858,
820. MS (ESI) m/z (%): 310 (M++H, 100). Calcd analysis for
C15H11N5O3: C: 58.2; H: 3.6; N: 22.6. Found: C: 57.9; H: 3.7; N: 22.7.
0
4.1.1.3. (E)-N -(5-Benzofuroxanylmethylidene)nicotinohydraz-
ide (15).
Yellow solid (74%); mp: 195.3–198.6 °C. 1H NMR
4.3.1. (E)-2-Methyl-N0-(2-methyl-3-furoxanylmethylidene)-4H-
imidazo[1,2-a]pyridine-3-carbohydrazide (19)
(DMSO-d6) d ppm: 7.59 (1H, dd, J1 = 4.0 Hz, J2 = 4.0 Hz), 7.49–8.29
(3H, br m), 8.28 (1H, d, J = 8.0), 8.50 (1H, s), 8.79 (1H, s), 9.09
(1H, s), 12.38 (1H, s). 13C NMR (DMSO-d6) d ppm: 114.0, 119.0,
124.2, 129.3, 131.0, 136.1, 142.5, 146.2, 149.2, 153.0, 162.6. IR
The solid precipitated was filtered and washed with cold EtOH.
Yellow solid (81%); mp: 165.9–167.4 °C. 1H NMR (DMSO-d6) d
ppm: 2.26 (3H, s), 2.53 (3H, s), 7.07 (1H, t, J = 4.0), 7.45 (1H, t,
J = 4.0), 7.65 (1H, d, J = 8.0), 8.40 (1H, s), 8.91 (1H, d, J = 8.0),
12.01 (1H, s). 13C NMR (DMSO-d6) d ppm: 9.4, 16.3, 114.0, 115.0,
(KBr)
t
(cmꢁ1): 3219, 1657, 1603, 1572, 1528, 1295, 1014, 828,
800. MS (ESI) m/z (%): 284 (M+ꢃ+H, 100), 268 (M+ꢃ+H-16, 1), 178
(M+ꢃ-106, 1). Calcd analysis for C13H9N5O3: C: 55.1; H: 3.2; N:
24.7. Found: C: 55.0; H: 2.9; N: 24.4.
116.7, 127.8, 128.1, 135.6, 154.4, 159.3. IR (KBr)
t
(cmꢁ1): 3184,
1660, 1610, 1573, 1339, 1296, 1229, 1014, 865, 802. MS (EI) m/z
(%): 300 (M+, 5), 201 (2), 159 (100), 90 (19). Calcd analysis for
C13H12N6O3: C: 52.0; H: 4.0; N: 28.0. Found: C: 51.9; H: 3.9; N:
27.8.
4.2. General procedure for the synthesis of compounds 16–18
To
a
solution of benzo[d][1,3]dioxole-5-carbohydrazide
(250 mg, 1.4 mmol) in dry toluene (5 mL) was added the corre-
sponding aldehyde ((I), (II), or (III), 1.3 mmol) and a drop of glacial
AcOH. The mixture was stirred until the aldehyde was consumed
(checked by TLC). The product was purified as is indicated below.
4.3.2. (E)-2-Methyl-N0-(4-phenyl-3-furoxanylmethylidene)-4H-
imidazo[1,2-a]pyridine-3-carbohydrazide (20)
The solvent of reaction was evaporated in vacuo and the residue
was purified by column chromatography (SiO2, CH2Cl2:MeOH (0 to
10%)). White solid (12%); mp: 184.2–186.6 °C. 1H NMR (DMSO-d6)
d ppm: 2.51 (s, 3H), 7.01 (t, 1H, J = 8.1), 7.25–7.65 (m, 6H), 7.87 (d,
1H, J = 8.1), 8.24 (s, 1H), 8.84 (d, 1H, J = 8.1), 11.92 (s, 1H). 13C NMR
(DMSO-d6) d ppm: 40.0, 113.5, 115.0, 117.0, 128.0, 129.0, 129.0,
4.2.1. (E)-N0-(2-Methyl-3-furoxanylmethylidene)benzo[d][1,3]
dioxole-5-carbohydrazide (16)
The solid precipitated was filtered and washed with cold toluene.
Light yellow solid (86%); mp: 189.4–192.0 °C. 1H NMR (DMSO-d6) d
ppm: 2.39 (3H, s), 6.13 (2H, s), 7.08 (1H, d, J = 8.0), 7.45 (1H, s), 7.54
(1H, d, J = 8.0), 8.45 (1H, s), 12.25 (1H, s). 13C NMR (DMSO-d6) d ppm:
9.7, 102.3, 108.2, 108.7, 112.2, 123.7, 123.7, 148.1, 150.0, 154.6,
131.0, 132.0, 146.0, 147.0, 148.0, 156.0. IR (KBr)
t
(cmꢁ1): 3226,
1681, 1634, 1337, 1293, 1212, 1047, 845, 753. MS (EI) m/z (%):
332 (M+ 30, 4), 159 (100), 131 (10). Calcd analysis for
C18H14N6O3: C: 59.7; H: 3.9; N: 23.2. Found: C: 59.5; H: 3.7; N:
136.1, 151.2, 162.9. IR (KBr)
t
(cmꢁ1): 3200, 1648, 1570, 1613,
23.2.
1259, 1169, 1125, 1069, 1034, 929, 857, 801. MS (EI) m/z (%): 290
(M+, 2), 149 (100), 121 (15), 65 (12). Calcd analysis for
4.3.3. (E)-N0-(5-Benzofuroxanylmethylidene)-2-methyl-4H-
imidazo[1,2-a]pyridine-3-carbohydrazide (21)
C12H10N4O5: C: 49.7; H: 3.5; N: 19.3. Found: C: 49.6; H: 3.3; N: 19.2.
The solid precipitated was filtered and washed with cold EtOH.
Yellow solid (87%); mp: 263.9 (d). 1H NMR (DMSO-d6) d ppm: 2.52
(3H, s), 7.09 (1H, t, J = 4.0), 7.46 (1H, t, J = 8.0), 7.64 (1H, d, J = 8.0),
7.63–8.19 (3H, bs), 8.40 (1H, s), 8.89 (1H, d, J = 8.0), 11.88 (1H, s).
13C NMR (DMSO-d6) d ppm: 113.8, 116.7, 127.7, 135.0, 144.0,
4.2.2. (E)-N0-(4-Phenyl-3-furoxanylmethylidene)benzo[d][1,3]
dioxole-5-carbohydrazide (17)
The solvent of reaction was evaporated in vacuo and the residue
was purified by column chromatography (SiO2, CH2Cl2:MeOH (0 to

P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
2167
161.0. IR (KBr)
t
(cmꢁ1): 3200, 1647, 1620, 1344, 1297, 1231, 1012,
4.5.2. (E)-1-Phenyl-N0-(4-phenyl-3-furoxanylmethylidene)-1H-
862, 757. MS (EI) m/z (%): 336 (M+., 2), 320 (1), 159 (100), 90 (35).
Calcd analysis for C16H12N6O3: C: 57.1; H: 3.6; N: 25.0. Found: C:
57.1; H: 3.3; N: 25.3.
pyrazole-4-carbohydrazide (26)
Gray solid (80%); mp: 200.2–202.3 °C. 1H NMR (DMSO-d6) d
ppm: 7.43 (1H, t, J = 8.0), 7.59 (5H, d, J = 12.0), 7.77 (2H, d,
J = 8.0), 7.84 (2H, d, J = 8.0), 8.02 (1H, s), 8.26 (1H, s), 9.18 (1H, s),
11.96 (1H, s). 13C NMR (DMSO-d6) d ppm: 112.7, 117.0, 119.3,
126.0, 127.8, 128.3, 129.0, 129.7, 130.5, 131.8, 139.5, 147.7,
4.4. General procedure for the synthesis of compounds 22–24
156.9, 163.0. IR (KBr)
t
(cmꢁ1): 3165, 1658, 1599, 1544, 1503,
To
a solution of 5-methyl-1H-imidazole-4-carbohydrazide
1206, 1036, 874, 751. MS (EI) m/z (%): 171 (M+.-203, 100), 77
(90). Calcd analysis for C19H14N6O3: C: 61.0; H: 3.8; N: 22.4. Found:
C: 60.8; H: 3.6; N: 22.4.
(1 equiv) in dry EtOH (2.5 mL/mmol of hydrazide) was added the
corresponding aldehyde ((I), (II), or (III), 0.9 equiv) and a drop of
glacial AcOH. The mixture was stirred until the aldehyde was con-
sumed (checked by TLC). The solid precipitated was filtered and
washed with cold EtOH.
4.5.3. (E)-N0-(5-Benzofuroxanylmethylidene)-1-phenyl-1H-
pyrazole-4-carbohydrazide (27)
Yellow solid (21%); mp: 210.0–212.0 °C. 1H NMR (DMSO-d6) d
ppm: 7.42 (1H, t, J = 8.0), 7.51–7.93 (3H, br m), 7.53 (2H, t,
J = 8.0), 7.85 (2H, d, J = 8.0), 8.14 (1H, s), 8.18 (1H, s), 8.90 (1H, s),
12.04 (1H, s). 13C NMR (DMSO-d6) d ppm: 113.5, 119.4, 127.8,
4.4.1. (E)-5-Methyl-N0-(2-methyl-3-furoxanylmethylidene)-1H-
imidazole-4-carbohydrazide (22)
White solid (40%); mp: 190.1–192.3 °C. 1H NMR (DMSO-d6) d
ppm: 2.39 (3H, s), 2.49 (1H, s), 7.70 (1H, s), 8.59 (1H, s), 12.15
(1H, s), 12.58 (1H, s). 13C NMR (DMSO-d6) d ppm: 9.7, 112.3,
130.1, 139.5, 141.0, 142.2, 144.3, 162.0. IR (KBr)
t
(cmꢁ1): 3252,
1658, 1620, 1564, 1530, 1281, 1012, 882, 758. MS (EI) m/z (%):
171 (M+.-177, 100), 77 (26). Calcd analysis for C17H12N6O3: C:
58.6; H: 3.5; N: 24.1. Found: C: 58.5; H: 3.7; N: 24.3.
128.0, 134.4, 134.8, 155.0, 160.3. IR (KBr)
t
(cmꢁ1): 3200, 1652,
1599, 1538, 1305, 1039, 863, 800. MS (ESI) m/z (%): 251 (M+ꢃ+H,
100), 141 (2), 109 (7). Calcd analysis for C9H10N6O3: C: 43.2; H:
4.0; N: 33.6. Found: C: 43.4; H: 3.8; N: 33.3.
4.6. 3-Methylfuroxan-4-carbohydrazide (IV)
4.4.2. (E)-5-Methyl-N0-(4-phenyl-3-furoxanylmethylidene)-1H-
imidazole-4-carbohydrazide (23)
To a solution of (I) (1 g, 7.8 mmol) in MeOH (12 mL) stirred at
0 °C was added subsequently KCN (839 mg, 12.9 mmol) and finally
MnO2 (3.392 g, 39 mmol) in small portions over 5 min. The reac-
tion was stirred for 30 minutes at 0 °C and then it was filtered
through a Celite column and the Celite was washed three times
with CH2Cl2 (15 mL each). The organic phase was washed with
brine, dried with anhydrous Na2SO4 and evaporated in vacuo to
yield methyl 3-methylfuroxan-4-carboxylate as light brown oil
(597 mg, 48%). 1H NMR (DMSO-d6) d ppm: 2.40 (s, 3H), 4.06 (s,
3H). 13C NMR (DMSO-d6) d ppm: 8.0, 53.0, 110.0, 149.0, 159.0.
The ester was used without further purification in the next step.
To a solution of methyl 3-methyl-4-furoxancarboxylate (597 mg,
3.8 mmol) in EtOH (10 mL) stirred at 0 °C was added NH2NH2ꢃH2O
(70%, 0.13 mL). The reaction mixture was stirred for 1 h and then
the solvent was evaporated in vacuo and the residue was treated
with EtOAc (150 mL) and washed with brine. The organic phase
was dried with anhydrous Na2SO4 and evaporated in vacuo. The
product (IV), as a light brown oil (484 mg, 81%), was used without
purification in the next reactions.
White solid (14%); mp: 226.8–229.5 °C. 1H NMR (DMSO-d6) d
ppm: 2.47 (3H, s), 7.56–7.63 (3H, m), 7.67 (1H, s), 7.98 (2H, d,
J = 8.0), 8.48 (1H, s), 12.09 (1H, s), 12.59 (1H, s). 13C NMR (DMSO-
d6) d ppm: 11.0, 113.5, 126.3, 129.0, 129.2, 129.6, 131.6, 134.0,
134.2, 156.5, 160.1. IR (KBr)
t
(cmꢁ1): 3097, 1660, 1595, 1559,
1512, 1244, 1039, 863, 800. MS (ESI) m/z (%): 313 (M++H, 100).
Calcd analysis for C14H12N6O3: C: 53.8; H: 3.9; N: 26.9. Found: C:
53.7; H: 3.7; N: 26.7.
4.4.3. (E)-N0-(5-Benzofuroxanylmethylidene)-5-methyl-1H-
imidazole-4-carbohydrazide (24)
Yellow solid (76%); mp: 229.7–231.8 °C. 1H NMR (DMSO-d6) d
ppm: 2.45 (3H, s), 7.52–8.12 (4H, m), 8.55 (1H, s), 11.74 (1H, s),
12.55 (1H, s). 13C NMR (DMSO-d6) d ppm: 11.1, 113.0, 119.0,
129.1, 130.2, 133.5, 134.2, 143.9, 160.3. IR (KBr)
t
(cmꢁ1): 3137,
1657, 1603, 1518, 1295, 1015, 893, 805. MS (ESI) m/z (%): 287
(M+.+H, 100), 205 (1), 109 (3). Calcd analysis for C12H10N6O3: C:
50.3; H: 3.5; N: 29.4. Found: C: 50.1; H: 3.4; N: 29.1.
4.6.1. General procedure for the synthesis of compounds 28–43
To a solution of (IV) (1 equiv) in dry toluene (5 mL/mmol of
hydrazide) was added the corresponding aldehyde (0.9 equiv)
and a drop of glacial AcOH. The mixture was stirred until the alde-
hyde was consumed (checked by TLC). The solid precipitated was
filtered and washed with cold toluene.
4.5. General procedure for the synthesis of compounds 25–27
To
a
solution of 1-phenyl-1H-pyrazole-4-carbohydrazide
(1 equiv) in dry EtOH (5 mL/mmol of hydrazide) was added the
corresponding aldehyde ((I), (II), or (III), 0.9 equiv) and a drop of
glacial AcOH. The mixture was stirred until the aldehyde was con-
sumed (checked by TLC). The solid precipitated was filtered and
washed with cold EtOH.
4.6.1.1. (E)-3-Methyl-N 0-(2-methyl-3-furoxanylmethylidene)
furoxan-4-carbohydrazide (28).
Yellow solid (51%); mp:
150.4–152.0 °C. 1H NMR (DMSO-d6) d ppm: 2.40 (s, 3H), 2.48 (s,
3H), 8.75 (s, 1H), 11.98 (s, 1H). 13C NMR (DMSO-d6) d ppm: 7.5,
8.0, 112.0, 113.0, 138.0, 151.0, 154.0, 155.0. IR (KBr)
4.5.1. N0-(2-Methyl-3-furoxanylmethylidene)-1-phenyl-1H-
pyrazole-4-carbohydrazide (25)
t
(cmꢁ1):
(E/Z proportion = 67:33). White solid (23%). E-isomer: 1H NMR
(DMSO-d6) d ppm: 2.40 (1H, s), 7.40 (1H, t, J = 8.0), 7.56 (2H, t,
J = 8.0), 7.92 (2H, d, J = 8.0), 8.29 (1H, s), 8.42 (1H, s), 9.10 (1H, s),
12.22 (1H, s). 13C NMR (DMSO-d6) d ppm: 9.6, 112.2, 118.6,
3206, 1719, 1616, 1598, 1560, 1234, 1044, 864, 800. MS (EI) m/z
(%): 268 (M+ 6), 208 (5), 169 (11), 143 (16), 125 (2). Calcd analysis
for C8H8N6O5: C: 35.8; H: 3.0; N: 31.3. Found: C: 35.6; H: 3.1; N:
31.2.
119.4, 127.8, 130.2, 130.6, 135.5, 139.4, 140.9, 154.6. IR (KBr)
t
(cmꢁ1): 1635, 1503, 1224, 1013, 870, 821. MS (EI) m/z (%): 312
4.6.1.2. (E)-3-Methyl-N 0-(4-phenyl-3-furoxanylmethylidene)
(M+, 1), 213 (2), 171 (100), 77 (17). Calcd analysis for
furoxan-4-carbohydrazide (29).
White solid (14%); mp:
185.0–189.0 °C. 1H NMR (DMSO-d6) d ppm: 2.29 (3H, s), 7.36
(1H, d, J = 8.0), 7.64 (2H, d, J = 8.0), 7.91 (2H, d, J = 8.0), 8.46 (1H,
C14H12N6O3: C: 53.8; H: 3.9; N: 26.9. Found: C: 53.6; H: 3.8; N:
26.7.

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P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
s), 12.98 (1H, s). 13C NMR (DMSO-d6) d ppm: 8.9, 113.1, 113.4,
C9H7N5O5S: C: 36.4; H: 2.4; N: 23.6; S: 10.8. Found: C: 36.1; H:
2.1; N: 23.5; S: 11.0.
126.0, 129.0, 129.0, 129.2, 129.5, 136.3, 151.6, 154.2, 156.6. IR
(KBr)
t
(cmꢁ1): 1727, 1611, 1561, 1541, 1221, 1048, 866, 843.
MS (EI) m/z (%): 330 (M+ 2), 314 (1), 231 (2), 127 (47), 67 (100).
Calcd analysis for C13H10N6O5: C: 47.3; H: 3.0; N: 25.4. Found: C:
47.5; H: 3.1; N: 25.3.
4.6.1.9. (E)-3-Methyl-N -(5-nitro-2-furylmethylidene)furoxan-
0
4-carbohydrazide (36).
Yellow solid (55%); mp: 197.0–
198.6 °C. 1H NMR (acetone-d6) d ppm: 2.40 (s, 3H), 7.27 (d, 1H,
J = 3.5), 7.64 (d, 1H, J = 3.4), 8.64 (s, 1H), 11.78 (s, 1H). 13C NMR
(acetone-d6) d ppm: 12.0, 112.0, 113.0, 119.0, 137.0, 151.0, 152.0,
4.6.1.3. (E)-N 0-(5-Benzofuroxanylmethylidene)-3-methylfuro-
xan-4-carbohydrazide (30).
Brown solid (32%); mp: 107.9–
154.0. IR (KBr) t
(cmꢁ1): 3092, 1688, 1621, 1568, 1521, 1353,
109.3 °C. 1H NMR (DMSO-d6) d ppm: 2.41 (3H, s), 7.10–7.29 (1H,
1308, 1248, 1038, 887, 815. MS (EI) m/z (%): 281 (M+ 12), 221
(8), 182 (28), 154 (20), 67 (100), 57 (17). Calcd analysis for
C9H7N5O6: C: 38.4; H: 2.5; N: 24.9. Found: C: 38.2; H: 2.3; N: 24.6.
br s), 7.60–7.90 (2H, m), 8.72 (1H, s), 10.09 (1H, s). IR (KBr)
t
(cmꢁ1): 1616, 1468, 1250. MS (EI) m/z (%): 304 (M+ 20), 288 (5),
205 (7), 67 (100). Calcd analysis for C11H8N6O5: C: 43.4; H: 2.6;
N: 27.6. Found: C: 43.2; H: 2.7; N: 27.4.
0
4.6.1.10. (E)-N -(2-Hydroxyphenylmethylidene)-3-methylfuro-
xan-4-carbohydrazide (37).
White solid (70%); mp: 206.0–
218.0 °C. 1H NMR (acetone-d6) d ppm: 2.41 (3H, s), 6.97 (1H, t,
J = 7.5), 6.99 (1H, d, J = 8.2), 7.37–7.44 (2H, m), 8.76 (1H, s), 11.25
(1H,s), 11.72 (1H, s). 13C NMR (acetone-d6) d ppm: 8.1, 112.8,
117.3, 118.1, 119.8, 131.8, 132.5, 151.3, 153.0, 153.9, 159.2. IR
0
4.6.1.4. (E)-N -(4-Dimethylaminophenylmethylidene)-3-meth-
ylfuroxan-4-carbohydrazide (31).
Yellow solid (54%); mp:
183.0–185.0 °C. 1H NMR (acetone-d6) d ppm: 2.38 (s, 3H), 3.05 (s,
6H), 6.80 (2H, d, J = 9.0), 7.64 (2H, d, J = 9.0), 8.44 (s, 1H), 11.05
(s, 1H). 13C NMR (acetone-d6) d ppm: 8.1, 39.7, 112.1, 112.9,
(KBr)
t
(cmꢁ1): 3586, 3158, 1680, 1620, 1559, 1506, 1248, 1037,
121.8, 129.4, 151.4, 152.0, 152.7, 153.5. IR (KBr)
t
(cmꢁ1): 3100,
870, 845. MS (EI) m/z (%): 262 (M+ 44), 163 (57), 143 (6), 135
(14), 119 (53), 67 (94), 57 (100). Calcd analysis for C11H10N4O4:
C: 50.4; H: 3.8; N: 21.4. Found: C: 50.6; H: 3.6; N: 21.3.
1605, 1605, 1553, 1526, 1258, 1034, 839, 825. MS (EI) m/z (%):
289 (M+ 100), 175 (89), 146 (68), 133 (50), 118 (36). Calcd analysis
for C13H15N5O3: C: 54.0; H: 5.2; N: 24.2. Found: C: 53.8; H: 5.0; N:
23.9.
4.6.1.11. (E)-N 0-(3,4-Dimethoxyphenylmethylidene)-3-methylf-
uroxan-4-carbohydrazide (38).
White solid (55%); mp:
4.6.1.5. (E)-N 0-(4-Bromophenylmethylidene)-3-methylfuroxan-
193.0–195.2 °C. 1H NMR (DMSO-d6) d ppm: 2.27 (3H, s), 3.87
(6H, s), 7.02 (1H, d, J = 8.1), 7.23 (1H, d, J = 8.1), 7.34 (1H, s), 8.40
(1H, s), 12.50 (1H, s). 13C NMR (DMSO-d6) d ppm: 8.8, 55.7,
108.8, 112.1, 113.6, 122.9, 126.8, 149.6, 151.7, 152.2, 152.7,
4-carbohydrazide (32).
White solid (56%); mp: 175.2–
182.0 °C. 1H NMR (acetone-d6) d ppm: 2.39 (s, 3H), 7.68 (2H, d,
J = 8.5), 7.67 (2H, d, J = 8.5), 8.60 (s, 1H), 11.43 (s, 1H). 13C NMR
(acetone-d6) d ppm: 8.1, 113.0, 124.7, 129.6, 132.4, 133.9, 149.4,
153.8. IR (KBr)
t
(cmꢁ1): 3254, 2842, 1696, 1609, 1577, 1513,
152.0. IR (KBr)
t
(cmꢁ1): 3200, 1683, 1615, 1469, 1259, 1038,
1271, 1025, 881, 790. MS (ESI) m/z (%): 307 (M+, 100), 291 (67),
100 (3). Calcd analysis for C13H14N4O5: C: 51.0; H: 4.6; N: 18.3.
Found: C: 50.8; H: 4.4; N: 18.2.
896, 827, 652. MS (EI) m/z (%): 326/324 (M+ 12), 227/225 (21),
198/196 (6), 183/181 (36), 170/168 (11), 157/155 (20), 143 (17),
118 (34), 89 (77), 67 (100), 57 (5). Calcd analysis for C11H9BrN4O3:
C: 40.6; H: 2.8; N: 24.6. Found: C: 40.7; H: 2.6; N: 24.3.
4.6.1.12. (E)-N0-(Benzo[d][1,3]dioxol-5-yl-methylidene)-3-meth-
ylfuroxan-4-carbohydrazide (39).
White solid (53%); mp:
4.6.1.6. (E)-3-Methyl-N 0-(thiophen-2-ylmethylidene)furoxan-4-
197.5–202.8 °C. 1H NMR (acetone-d6) d ppm: 2.11 (1H, s), 2.39
(3H, s), 6.94 (1H, d, J = 8.0), 7.22 (1H, dd, J1 = 8.0, J2 = 1.3), 7.40
(1H, d, J = 1.3), 8.51 (1H, s), 11.29 (1H, s). 13C NMR (acetone-d6) d
ppm: 8.13, 102.3, 105.8, 108.6, 112.9, 124.8, 129.0, 149.0, 150.4,
carbohydrazide (33).
White solid (54%); mp: 179.3–183.7 °C.
1H NMR (acetone-d6) d ppm: 2.39 (s, 3H), 7.17 (dd, 1H, J1 = 4.9,
J2 = 3.8), 7.49 (d, 1H, J = 3.7), 7.67 (d, 1H, J = 5.1), 8.84 (s, 1H),
11.33 (s, 1H). 13C NMR (acetone-d6) d ppm: 12.0, 108.0, 127.0,
150.6, 151.8, 153.9. IR (KBr)
t
(cmꢁ1): 3094, 1713, 1625, 1564,
130.0, 137.0, 145.0, 158.0, 170.0. IR (KBr)
t
(cmꢁ1): 3096, 1672,
1269, 1142, 1115, 1057, 928, 1034, 849, 803. MS (EI) m/z (%):
290 (M+ 68), 191 (43), 147 (100), 134 (28), 76 (21), 67 (24). Calcd
analysis for C12H10N4O5: C: 49.7; H: 3.5; N: 19.3. Found: C: 49.5; H:
3.1; N: 19.0.
1614, 1593, 1566, 1256, 1042, 883, 843, 731. MS (EI) m/z (%):
252 (M+ 53), 153 (65), 143 (11), 109 (76), 96 (51), 83 (25), 67
(100), 57 (13). Calcd analysis for C9H8N4O3S: C: 42.8; H: 3.2; N:
22.2; S: 12.7. Found: C: 42.6; H: 2.9; N: 21.9; S: 12.5.
4.6.1.13. (E)-N 0-(3-Hydroxy-4-methoxyphenylmethylidene)-3-
4.6.1.7. (E)-N 0-(2-Furylmethylidene)-3-methylfuroxan-4-carbo-
methylfuroxan-4-carbohydrazide (40).
Yellow solid (43%);
hydrazide (34).
White solid (28%); mp: 179.5 °C (d). 1H NMR
mp: 165.3–168.7 °C. 1H NMR (DMSO-d6) d ppm: 2.30 (3H, s),
3.84 (3H, s), 6.80 (1H, d, J = 8.0), 7.10 (1H, d, J = 8.0), 7.32 (1H, s),
8.40 (1H, s), 9.62 (1H, s), 12.36 (1H, s). 13C NMR (DMSO-d6) d
ppm: 8.9, 56.0, 109.6, 113.6, 115.9, 123.1, 125.2, 148.5, 152.7,
(acetone-d6) d ppm: 2.39 (s, 3H), 6.64 (1H, dd, J1 = 1.8, J2 = 1.6), 6.97
(1H, d, J = 3.4), 7.68 (1H, s), 8.52 (1H, s), 11.33 (1H, s). 13C NMR
(acetone-d6) d ppm: 8.1, 112.5, 113.0, 114.3, 140.2, 145.8, 150.0,
152.0, 154.0. IR (KBr)
t
(cmꢁ1): 3130, 1684, 1615, 1582, 1259,
153.7. IR (KBr) t
(cmꢁ1): 3500, 3243, 2830, 1677, 1607, 1592,
1038, 879, 843. MS (EI) m/z (%): 236 (M+ 43), 220 (3), 143 (5),
137 (72), 93 (40), 81 (28), 67 (100), 57 (6). Calcd analysis for
C9H8N4O4: C: 45.8; H: 3.4; N: 23.7. Found: C: 45.5; H: 3.2; N: 23.5.
1522, 1268, 1036, 883, 849. MS (ESI) m/z (%): 293 (M+ꢃ+H, 25),
277 (6). Calcd analysis for C12H12N4O5: C: 49.3; H: 4.1; N: 19.2.
Found: C: 49.0; H: 4.1; N: 19.0.
4.6.1.8. (E)-3-Methyl-N 0-(5-nitrothiophen-2-ylmethylidene)
4.6.1.14. (E)-N 0-(4-Hydroxy-3-methoxyphenylmethylidene)-3-
furoxan-4-carbohydrazide (35).
White solid (45%); mp:
methylfuroxan-4-carbohydrazide (41).
White solid (55%);
191.0 °C (d). 1H NMR (acetone-d6) d ppm: 2.34 (3H, s), 7.56 (1H,
d, J = 4.3), 8.05 (1H, d, J = 4.3), 8.88 (1H, s), 11.76 (1H, s). 13C
NMR (acetone-d6) d ppm: 8.0, 112.5, 128.0, 129.0, 143.0, 146.0,
mp: 203.0–205.3 °C. 1H NMR (DMSO-d6) d ppm: 2.30 (3H, s), 3.82
(3H, s), 6.99 (1H, d, J = 8.0), 7.07 (1H, d, J = 8.0), 7.28 (1H, s), 8.37
(1H, s), 9.39 (1H, s), 12.42 (1H, s). 13C NMR (DMSO-d6) d ppm:
8.9, 56.0, 112.3, 112.8, 113.7, 121.4, 126.9, 147.4, 150.8, 151.1,
152.0, 153.0. IR (KBr)
t
(cmꢁ1): 3168, 1677, 1609, 1600, 1538,
1508, 1317, 1253, 1031, 883, 847, 817. MS (EI) m/z (%): 297 (M+
152.2, 153.8. IR (KBr)
t
(cmꢁ1): 3498, 3205, 2850, 1681, 1615,
17), 198 (44), 191 (2), 143 (9), 67 (100). Calcd analysis for
1576, 1511, 1277, 1029, 882, 779. MS (ESI) m/z (%): 293 (M+ꢃ+H,

P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
2169
58), 277 (47), 100 (4). Calcd analysis for C12H12N4O5: C: 49.3; H:
4.1; N: 19.2. Found: C: 49.2; H: 3.9; N: 18.9.
viability was assessed by measuring the mitochondrial-dependent
reduction of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium
bromide (MTT, Sigma) to formazan. For that a solution of MTT,
in sterile PBS 0.2% glucose, was added to the cells and incubated
for 2 h at 37 °C and 5% CO2. The supernatant was removed and
0
4.6.1.15. (E)-N -(4-Carboxyphenylmethylidene)-3-methylfuro-
xan-4-carbohydrazide (42).
Light–yellow solid (98%); mp:
224.8 °C (d). 1H NMR (DMSO-d6) d ppm: 2.30 (3H, s), 7.86 (2H, d,
J = 8.1), 8.00 (2H, d, J = 8.1), 8.59 (1H, s), 10.11 (1H, s), 12.76 (1H,
s). 13C NMR (DMSO-d6) d ppm: 8.9, 113.7, 127.9, 130.4, 132.7,
180 lL of DMSO and 20 lL MTT buffer (0.1 M glycine, 0.1 M NaCl,
0.5 mM EDTA) was added to each well. Measurements were per-
formed at 560 nm in a spectrophotometer plate reader. B- NF-kB
pathway inhibition assay: Human intestinal epithelial cells (HT-
29, ATCC) were transfected with pNF-jB-hrGFP plasmid (Strata-
gene) using LipofectAmine 2000 (Invitrogen) and selected with
138.2, 149,.8, 152.0, 154.0, 194.0. IR (KBr)
t
(cmꢁ1): 3456, 1683,
1612, 1564, 1289, 1039, 902, 842. MS (EI) m/z (%): 290 (M+ 13),
191 (47), 148 (46), 67 (100). Calcd analysis for C12H10N4O5: C:
49.7; H: 3.5; N: 19.3. Found: C: 49.5; H: 3.3; N: 19.5.
hygromycin (Sigma). After two weeks, cells were stimulated dur-
ing 24 h with a pro-inflammatory cocktail (25 ng/mL TNF-
1.25 ng/mL IL1-B and 3.75 ng/mL IFN- ) and GFP positive cells
were sorted employing a MoFlo instrument (Dako). The respon-
siveness of the cells towards different concentrations of TNF-
(25–0.2 ng/mL) was tested, the clone showing the best perfor-
mance was selected for anti-inflammatory screening assays and
a,
4.6.1.16. (E)-3-Methyl-N 0-(4-trifluoromethylphenylmethylid-
c
ene)furoxan-4-carbohydrazide (43).
White solid (52%);
mp: 95.0–97.0 °C. 1H NMR (DMSO-d6) d ppm: 2.32 (s, 3H), 7.83
(d, 2H J = 8.0), 7.97 (d, 2H J = 8.0), 8.62 (s, 1H), 12.75 (s, 1H). 13C
NMR (DMSO-d6) d ppm: 8.0, 113.6, 123.1, 126.3, 128.2, 130.9,
a
138.1, 149.0, 152.0, 154.3. IR (KBr)
t
(cmꢁ1): 3405, 3171, 1683,
was named HT-29-NF-jB-hrGFP. HT-29-NF-jB-hrGFP cells
1622, 1324, 1259. MS (EI) m/z (%): 314 (M+ 15), 215 (48), 172
(33), 145 (42), 67 (100). Calcd analysis for C12H9F3N4O3: C: 45.9;
H: 2.9; N: 17.8. Found: C: 45.7; H: 2.7; N: 17.5.
(0.5 ꢂ 106/mL) were seeded in a 96 well plate in 200
lL fresh
DMEM supplemented milieu and were incubated for 24 h at
37 °C, 5% CO2. Culture milieu was removed and the NAHs, dis-
solved in DMSO (lower than 0.5%, v/v, in the final volume of
DMEM), were added to the cells at the maximum non-cytotoxic
4.7. 4-Phenylfuroxan-3-carbohydrazide (VI)44
concentration diluted in supplemented DMEM milieu (200
volume) and the plate was incubated for 2 h at 37 °C, 5% CO2. A
solution of TNF- (3 ng/mL final concentration, diluted in com-
plete DMEM) was added and further incubated 18–24 h at 37 °C,
5% CO2. Three controls were included: 1) Cells treated only with
lL final
To a solution of (V) (74 mg, 0.38 mmol) in dry THF (5 mL), car-
bonyldiimidazole (68 mg, 0.42 mmol) and the mixture was stirred
for 1 h at room temperature. Subsequently, it was slowly added
NH2NH2ꢃH2O (70%, 0.012 mL) in dry Et2O (0.5 mL) and the mixture
was stirred for 24 h at room temperature. The solvent was evapo-
rated in vacuo and the residue was treated with EtOAc (30 mL) and
washed with saturated aqueous solution of Na2HCO3. The organic
phase was dried with anhydrous Na2SO4 and evaporated in vacuo.
The product (VI), as a light brown oil (75 mg, 90%), was used with-
out purification in the next reaction.
a
TNF-a but without NAH; 2) Cells treated only with NAH; 3) Cells
treated with different Thalidomide concentrations and TNF-
a.
Supernatants were taken, clarified by centrifugation and stored
at ꢁ80 °C for further IL-8 determination. Cells were detached by
adding 50 lL of trypsin (0.5 g/L trypsin and 0.2 g/L EDTA.4Na, Sig-
ma) to each well, incubated for 5 min at 37 °C, and the trypsin was
neutralized by adding 150 L of complete DMEM milieu. Cell sus-
pensions were transferred into cytometer tubes with PBS (150 L)
and propidium iodide (3 L of a stock solution of 200 g/mL).
l
4.7.1. (E)-N0-(4-Carboxyphenylmethylidene)-4-phenylfuroxan-
3-carbohydrazide (44)
l
l
l
To a solution of (VI) (103 mg, 0.47 mmol) in anhydrous EtOH
(2 mL), at 0 °C, was added p-formylbenzoic acid (63 mg,
0.42 mmol) and a drop of glacial AcOH. The mixture was stirred
for 72 h at room temperature. The solid precipitated was filtered
and washed with cold EtOH. Yellow solid (16 mg, 9%); mp:
287.0–289.3 °C. 1H NMR (DMSO-d6) d ppm: 7.56 (1H, t, J = 8.0),
7.64 (2H, t, J = 8.0), 7.96 (2H, d, J = 8.0), 7.98 (2H, d, J = 8.0), 8.10
(2H, d, J = 8.0), 8.42 (1H, s), 11.3 (1H, s). 13C NMR (DMSO-d6) d
ppm: 127.3, 127.4, 128.4, 129.7, 132.1, 132.6, 138.2, 147.9, 165.8.
Acquisition and analysis were performed using a CyAn™ ADP
(DAKO) flow cytometer and Summit 4.3 software. For each condi-
tion, 5000 counts were collected, gated on FSC vs SSC dot plot.
Only living cells (cells that excluded propidium iodide) were con-
sidered for results comparison and the percentage of GFP positive
were normalized against the percentage of GFP cells obtained with
the TNF-a control. C- IL-8 quantitation: the levels of the proinflam-
matory cytokine IL-8 was assessed in the cell culture supernantant
by flow cytometry using Human Simplex Kit (Bender Medsystems,
Austria) and 500 events were collected according to manufacturer
recommendations. For results calculation BMS FlowCytomix Soft-
ware version 2.2.1 was used.
IR (KBr)
t
(cmꢁ1): 3075, 2893, 1680, 1634, 1549, 1256, 1076,
865, 776. MS (EI) m/z (%): 306 (M+-NO2, 2), 289 (5), 273 (5), 247
(3), 191 (2), 121 (29), 105 (100), 94 (9), 77 (43), 69 (15). Calcd anal-
ysis for C17H12N4O5: C: 58.0; H: 3.4; N: 15.9. Found: C: 57.9; H: 3.1;
N: 15.7.
4.8.2. Murine macrophages cytotoxicity
J774 murine macrophage-like cells (ATCC, USA) were main-
4.8. Biology
tained in DMEM containing 4 mM L-glutamine, and supplemented
with 10% heat-inactivated FBS and 1% penicillin–streptomicin. J774
4.8.1. NF-
j
B pathway inhibition assay and IL-8 production
cells were seeded (1 ꢂ 105 cells/well) in 96-well microplates with
A- HT-29-NF-
jB-hrGFP cells cytotoxicity assay: HT-29-NF-
j
B-
100 lL of DMEM supplemented milieu. Cells were allowed to
hrGFP cells (0.5 ꢂ 106/mL) were seeded in a 96 well plate in
growth for 48 h in a humidified 5% CO2/95% air atmosphere at
200
l
L fresh culture milieu DMEM (10% fetal bovine serum
37 °C and, then, exposed to NAHs (25–400 M) for 48 h. MTT in
l
[FBS], 1% penicillin–streptomycin) and incubated for 24 h at
37 °C and 5% CO2. The culture milieu was removed and the NAHs,
dissolved in DMSO (lower than 0.5%, v/v, in the final volume of
DMEM), were added at the desired final concentrations diluted
sterile PBS (0.2% glucose) was added to cells to a final concentra-
tion of 0.1 mg/mL and cells were incubated at 37 °C for 3 h. After
removing the media, formazan crystals were dissolved in DMSO
(180
10.5 pH) (20 l
l
L) and MTT buffer (0.1 M glycine, 0.1 M NaCl, 0.5 mM EDTA,
L) and the absorbance at 560 nm was read using a
in fresh DMEM (200
400 M) and the cells were further incubated for 24 h at 37 °C,
5% CO2. Afterwards, the culture supernatant was removed and cell
l
L final volume) (12.5, 25, 50, 100, 200,
l
microplate spectrophotometer. Results were expressed as IC50
(compound concentration that reduced 50% control absorbance

2170
P. Hernández et al. / Bioorg. Med. Chem. 20 (2012) 2158–2171
at 560 nm). Every IC50 was the average of at least three
determinations.
calculated as the means of duplicate determinations. The data were
statistically analyzed by the Student t-test.
4.8.6. COXs inhibition assay
The inhibition of isolated ovine COX-1 and human recombinant
COX-2 was determined with 10 and 50 lM of the respective NAHs
(17, 24, and 31) by ELISA according to the manufacturer’s instruc-
tions (‘COX inhibitor screening assay’, Cayman Chemical Cat. N°
560131). Indomethacin (0.1 lM for COX-1 and 6 lM for COX-2)
was used as positive control. Results were calculated as mean of
duplicate determinations. The data were analyzed by the Student
t-test.
4.8.3. Analgesic activity determinations
Animal studies were approved by the Local Institutional Animal
Care and Research Advisory committee. Acetic acid-induced abdom-
inal constriction assay: The abdominal constriction assay induced
by acetic acid (0.6%; 0.1 mL/10 g) was performed using albino mice
of both sexes (18–23 g). Compounds were administered orally
(100
l
mol/kg) as a suspension in 5% arabic gum in saline solution
mol/kg) were used as
(vehicle). Dipyrone and indomethacin (100
l
standard drug under the same conditions. Acetic acid solution was
administered intraperitoneally 1 h after administration of NAHs
(15, 17, 20, 24, 28, 31–33, 36, 38, and 42). Ten minutes following
intraperitoneally acetic acid injection the number of constriction
per animal was recorded for 20 min. Control animals received an
equal volume of vehicle. Analgesic activity was expressed as per-
centage of inhibition of constrictions when compared with the
vehicle control group. Results were expressed as the mean SEM
of n animals per group. The data were statistically analyzed by
the Student t-test. Hot-plate test (hyperalgesia).57 Wistar rats of
both sexes (150–200 g) were used. The NAHs (15, 17, 24, and 31)
4.8.7. Nitric oxide release determinations
An Apollo 4000 electrode—Free radical analyzer Instrumental
(World Precision Instruments) an ISO-NO membrane and an ISO-
NOP sensor were used to measure NO release. The compounds
(15, 17, 18, 20, 24, 41, 42, and 44) dissolved in DMSO, were incor-
porated to the work buffer, Tris–HCl 0.1 M, DTPA 0.1 mM, pH 7.4 or
8.3. Cysteine (300 or 100 lM) was added and the NO production
was registered for 20 min at 25 or 37 °C.
4.8.8. Mutagenicity assay
were administered orally (100 lmol/kg) as a suspension in 5% ara-
The method of direct incubation in plate was performed. Cul-
ture of S. typhimurium TA98 strain in the agar minimum glucose
milieu (AMG)—agar solution, Vogel Bonner E(VB) 50ꢂ, and 40%
glucose solution—was used. First, the direct toxicity of the com-
pounds under study against S. typhimurium TA98 strain was as-
sayed. DMSO solutions of 17, 24, 31, and 42 at different doses
(starting at the highest doses without toxic effects, 250.0 or
bic gum in saline solution (vehicle). Control animals received an
equal volume of vehicle. One hour later, the animals were injected
with either 0.1 mL of 1% carrageenan solution in saline (1 mg/paw)
or sterile saline (NaCl 0.9%), into the subplantar surface of the hind
paws respectively. The thermal hyperalgesia was determined using
the modified hot-plate test. Rats were placed individually on a hot
plate with the temperature adjusted to 51 0.1 °C. The latency of
the withdrawal response of the left hind paw was determined at
0, 30, 60, 120, 180, and 240 min post-challenge. The time of max-
imum permanence permitted on the hot surface was 20 s. Hyperal-
gesia to heat was defined as a decrease in withdrawal latency and
calculated as follows: d paw withdrawal latency (s) = (left paw
withdrawal latency in time 0) ꢁ (left paw withdrawal latency in
other times). The data were statistically analyzed by the Student
t-test.
350.0
tro-o-phenylendiamine (20.0
vation) and 2-aminofluorene (10.0
l
g/plate) were assayed in triplicate. Positive controls of 4-ni-
g/plate, in the run without S9 acti-
g/plate, in the cases of S9
l
l
activation) and negative control of DMSO were run in parallel.
The influence of metabolic activation was tested by adding
500 lL of S9 fraction of mouse liver treated with Aroclor, obtained
from Moltox, Inc. (Annapolis, MD, USA). The revertant number was
counted manually. The sample was considered mutagenic when
the number of revertant colonies was at least double that of the
negative control for at least two consecutive dose levels.
4.8.4. Anti-inflammatory activity determinations
Thirty albino rats of both sexes (150–200 g) were used in this
protocol. NAHs (15, 17, 20, 24, 28, 31–33, 36, 38, and 42) were
administered in the dose of choice as a suspension in 1% tween
80 in saline (vehicle). Control animals received equal volume of
the vehicle. One hour after, the animals were then injected with
either 0.1 mL of 1% carrageenan solution in saline (0.1 mg/paw)
or sterile saline (NaCl 0.9%), into the subplantar surface of one of
the hind paws, respectively. The paw volumes were measured tem-
porally (for NAHs 15, 17, and 31) and at 3 h after the subplantar
injection using a glass plethysmometer coupled to a peristaltic
pump. The edema was calculated as the difference of volume be-
tween the carrageenan and saline-treated paw. Anti-inflammatory
activity was expressed as percentage of inhibition of the edema
when compared with vehicle control group. The data were statisti-
cally analyzed by the Student t-test.
Acknowledgements
We thank PEDECIBA-ANII for scholarship to P.H., M.C., L.C., and
CNPq-PROSUL network for fellowships to P.H. E.J.B., A.L.P.M. and
L.M.L. thanks CNPq (BR) for fellowships. We thank INCT-INOFAR
(CNPq CNPq 573.564/2008-6 and FAPERJ E-26/170.020/2008) for
partial financial support.
Supplementary data
Supplementary data (spectroscopy characterization, and biolog-
ical data (eight pages)) associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.01.034.
References and notes
4.8.5. Lipoxygenase inhibition assay
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turer’s instructions (Lipoxygenase Inhibitor Screening Assay
Kit—Cayman Chemicals Cat. N° 760700) using araquidonic acid as
substrate and H2O2 (90 mL of Tris–HCl 0.1 M, pH 7.4, and 10 mL
of H2O2 420 mM) as lipoxygenated agent. For NAH 42 IC50 was
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