Synthesis and biological activity of furoxan derivatives against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2016.07.039

Tuberculosis (TB) remains a serious health problem responsible to cause millions of deaths annually. The scenario becomes alarming when it is evaluated that the number of new drugs does not increase proportionally to the emergence of resistance to the current therapy. Furoxan derivatives, known as nitric oxide (NO) donors, have been described to exhibit antitubercular activity. Herein, a novel series of hybrid furoxan derivatives (1,2,5-oxadiazole 2-N-oxide) (compounds 4a-c, 8a-c and 14a-c) were designed, synthesized and evaluated in?vitro against Mycobacterium tuberculosis (MTB) H₃₇Rv (ATCC 27294) and a clinical isolate MDR-TB strain. The furoxan derivatives have exhibited MIC₉₀values ranging from 1.03 to 62?μM (H₃₇Rv) and 7.0–50.0?μM (MDR-TB). For the most active compounds (8c, 14a, 14b and 14c) the selectivity index ranged from 3.78 to 52.74 (MRC-5?cells) and 1.25–34.78 (J774A.1?cells). In addition, it was characterized for those compounds logP_o/wvalues between 2.1 and 2.9. All compounds were able to release NO at levels ranging from 0.16 to 44.23%. Among the series, the phenylsulfonyl furoxan derivatives (compounds 14a-c) were the best NO-donor with the lowest MIC₉₀values. The most active compound (14c) was also stable at different pHs (5.0 and 7.4). In conclusion, furoxan derivatives were identified as new promising compounds useful to treat tuberculosis.

Full text of DOI:10.1016/j.ejmech.2016.07.039

Accepted Manuscript
Synthesis and biological activity of furoxan derivatives against Mycobacterium
tuberculosis
Guilherme Felipe dos Santos Fernandes, Paula Carolina de Souza, Leonardo
Biancolino Marino, Konstantin Chegaev, Stefano Guglielmo, Loretta Lazzarato,
Roberta Fruttero, Chung Man Chin, Fernando Rogério Pavan, Jean Leandro dos
Santos
PII:
S0223-5234(16)30600-6
10.1016/j.ejmech.2016.07.039
EJMECH 8757
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 April 2016
Revised Date: 16 June 2016
Accepted Date: 19 July 2016
Please cite this article as: G.F.d.S. Fernandes, P.C. de Souza, L.B. Marino, K. Chegaev, S. Guglielmo,
L. Lazzarato, R. Fruttero, C.M. Chin, F.R. Pavan, J.L.d. Santos, Synthesis and biological activity of
furoxan derivatives against Mycobacterium tuberculosis, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.07.039.
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ACCEPTED MANUSCRIPT

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Synthesis and biological activity of furoxan derivatives against
Mycobacterium tuberculosis
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Guilherme Felipe dos Santos Fernandes,a Paula Carolina de Souza,a Leonardo Biancolino
Marino,a Konstantin Chegaev,b Stefano Guglielmo,b Loretta Lazzarato,b Roberta
Fruttero,b Chung Man Chin,a Fernando Rogério Pavan,a Jean Leandro dos Santos.a#
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a School of Pharmaceutical Sciences, UNESP – Univ Estadual Paulista, Araraquara,
14800903, Brazil.
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b Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino,
Turin, 10124, Italy
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#Address correspondence to Jean Leandro dos Santos, santosjl@fcfar.unesp.br

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Abstract
Tuberculosis (TB) remains a serious health problem responsible to cause millions of
deaths annually. The scenario becomes alarming when it is evaluated that the number of
new drugs does not increase proportionally to the emergence of resistance to the current
therapy. Furoxan derivatives, known as nitric oxide (NO) donors, have been described to
exhibit antitubercular activity. Herein, a novel series of hybrid furoxan derivatives (1,2,5-
oxadiazole 2-N-oxide) (compounds 4a-c, 8a-c and 14a-c) were designed, synthesized and
evaluated in vitro against Mycobacterium tuberculosis (MTB) H37Rv (ATCC 27294) and
a clinical isolate MDR-TB strain. The furoxan derivatives have exhibited MIC90 values
ranging from 1.03 to 62 µM (H37Rv) and 7.0 to 50.0 µM (MDR-TB). For the most active
compounds (8c, 14a, 14b and 14c) the selectivity index ranged from 3.78 – 52.74 (MRC-
5 cells) and 1.25 – 34.78 (J774A.1 cells). In addition, it was characterized for those
compounds logPo/w values between 2.1 – 2.9. All compounds were able to release NO at
levels ranging from 0.16 – 44.23%. Among the series, the phenylsulfonyl furoxan
derivatives (compounds 14a-c) were the best NO-donor with the lowest MIC90 values.
The most active compound (14c) was also stable at different pHs (5.0 and 7.4). In
conclusion, furoxan derivatives were identified as new promising compounds useful to
treat tuberculosis.
Keywords: furoxan; tuberculosis; phenotypic screening; Mycobacterium tuberculosis;
antituberculosis agents.
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1. Introduction
Tuberculosis, caused mainly by Mycobacterium tuberculosis (MTB), is the infectious
disease responsible for the largest number of deaths in the world, exceeding even human
immunodeficiency virus (HIV). The latest surveys conducted by World Health
Organization (WHO) in 2014 showed 9.6 million of new cases around the world and 1.5
million of deaths annually [1]. The emergence of drug resistant strains, including
multidrug resistant (MDR), extremely drug resistant (XDR) and the recently cases of
totally drug resistant (TDR) increase the challenges to eliminate TB worldwide.
Furthermore, WHO estimates that one third of the world population are infected by latent
TB [2], whose treatment is unavailable due to the lack of new drugs [3–5].
The current treatment against MTB have shown limitations which include: high toxicity
[6–10], drug-drug interactions [11], long-term therapy and low efficacy against resistant
strains. After a gap of 50 years without any new antitubercular drugs, bedaquiline
(SIRTURO®; Janssen, Beerse, Belgium) was approved by the United States Food and
Drug Administration (FDA) for the treatment of MDR-TB; however, resistant strains to
this drug are already reported [12]. After bedaquiline, there was a noteworthy increase in
the number of papers describing compounds with potent antitubercular activity [13–16].
In order to find new antitubercular drugs, we have established a phenotypic-based
screening program with more than five thousand compounds present in our current
library. From these data, we have identified (hydroxybenzylidene)isonicotinohydrazide
derivatives active against MTB. Specifically, the compound (E)-N'-(4-
hydroxybenzylidene)isonicotinohydrazide (I) (Fig.1) have exhibited MIC90 value of 1.0
µM against MTB H37Rv and selective index against VERO and J774A.1 cell lines

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superior to 100. Notwithstanding, this molecule did not show antitubercular activity
against MDR strains, presenting MIC90 values superior to 62 µM.
In this work, using the molecular hybridization approach, we designed new analogues of
(E)-N'-(4-hydroxybenzylidene)isonicotinohydrazide (I) containing the furoxan moiety
[17] (Fig. 1). Furoxan derivatives represent an important class of compounds that exhibit
a variety of biological activities, such as, antimycobacterial [18], antichagasic [19] and
antileishmanicidal [20]. The wide spectrum of biological activities of furoxan derivatives
have been associated to its ability to generate nitric oxide after biotransformation [21,22].
NO is an important mediator produced by macrophages during MTB infection and has an
essential role to eliminate MTB [23]. It has been demonstrated that NO can disrupt
bacterial DNA, proteins, signaling mediators, and/or induction of macrophage apoptosis
[24]. Nitric oxide is also increased in macrophages during the infection and its inhibition
promotes MTB growth [25]. MTB infected mice treated with nitric oxide synthase
inhibitors exhibited higher mortality rates and pathological tissue damages compared to
control group without treatment [26].
Not only endogenous, but also exogenous sources of NO have demonstrated effectiveness
to reduce the number of bacilli. Some works have demonstrated that low levels of NO-
donors can kill the mycobacteria [27–29]. These data suggested that strategies aiming to
raise NO levels seem to be promising as antitubercular therapy. Therefore, in a
continuing effort to develop new drug candidates to treat TB infection, we report herein
the synthesis, NO-donor release, experimental logP values, antitubercular and cytotoxic
activities of furoxan derivatives (4a-c, 8a-c, and 14a-c) (Fig. 1). The antimycobacterial
activity against a clinical isolate of MDR strain (resistant to isoniazid, rifampicin,

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streptomycin and ethambutol) was characterized for the most active compounds.
Moreover, for the most potent compound, we also studied the chemical stability at
different pHs (1.0; 5.0; 7.4 and 9.0).
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Fig. 1. Design of the hybrid furoxanyl N-acylhydrazone derivatives.
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2. Results
2.1. Chemistry
The synthetic routes for the preparation of furoxan derivatives (4a-c, 8a-c, and 14a-c)
derivatives are summarized in Scheme 1 and 2.
Compounds 2, 6, and 12 were synthesized according to a previously described
methodology [20,30–32]. The 2-, 3- or 4-hydroxybenzaldehyde was reacted with
compounds 2, 6, and 12 in dichloromethane medium, using 1,8-

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diazabicyclo[5.4.0]undec-7-ene (DBU) as base, to provide the furoxan derivatives 3a-c,
7a-c, and 13a-c, in yields varying between 20% and 67 %.
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Scheme 1. Reagents and conditions: (a) 1,2-dichloroethane, H2SO4 60%, NaNO2, 50 °C,
30 min; (b) 2, 3 or 4- hydroxybenzaldehyde, 1,8-diazabicycloundec-7-ene (DBU),
anhydrous dichloromethane, r.t., 2 h; (c) isonicotinic hydrazide, ethanol, acetic acid, r.t.,
12 h; (d) acetic acid, hydrochloric acid, dichloromethane, NaNO2, r.t, 12 h.
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The last step to obtain all furoxan derivatives (4a-c, 8a-c, and 14a-c) involves the
coupling reaction between aldehyde function present in the furoxan derivatives and
isonicotinic hydrazide in order to obtain the target compounds in excellent yields varying
between 83% and 95% (Scheme 1 and 2). All chemical structures were established by
1
infrared (IR) spectroscopy, elemental analysis and H and 13C nuclear magnetic
1
resonance (NMR). The analysis of H NMR spectra of all acyl hydrazone derivatives
(compounds 4a-c, 8a-c, and 14a-c) have shown a single signal referring to ylidenic
hydrogen attributed to the E-diastereomer [33–36]. All compounds were also analyzed by

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high-performed liquid chromatography (HPLC), and their purity was confirmed to be
greater than 98.5%.
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Scheme 2. Reagents and conditions: (a) monochloroacetic acid, NaOH, H2O, 110 °C, 3
h; (b) hydrogen peroxide 30%, acetic acid, r.t., 24 h; (c) fuming nitric acid, acetic acid,
110 °C, 1 h; (d) 2, 3 or 4- hydroxybenzaldehyde, 1,8-diazabicycloundec-7-ene (DBU),
anhydrous dichloromethane, r.t., 2 h; (e) isonicotinic hydrazide, ethanol, acetic acid, r.t.,
12 h.
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2.2. Antitubercular activity
The antitubercular activity of hybrid furoxan derivatives (4a-c, 8a-c, and 14a-c) and
intermediates (3a-c, 7a-c, and 13a-c) were determined against Mycobacterium
tuberculosis H37Rv ATCC 27294 and a clinical isolate MDR strain resistant to isoniazid,
rifampicin, streptomycin and ethambutol. Among the furoxan intermediates, only
compounds from the phenylsulfonyl (13a-c) series were active against MTB; the MIC90

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for these compounds ranged from 2.89 to 26.01 µM, while the methyl (3a-c) and phenyl
(7a-c) series presented MIC90 values superior to 88 µM.
In the assays, hybrid furoxan derivatives (4a-c, 8a-c, and 14a-c) showed similar
biological activity than those exhibited for intermediates (3a-c, 7a-c, and 13a-c).
Phenylsulfonyl (14a-c) series were the most activity compounds with MIC90 ranging
from 1.03 to 8.60 µM. Furthermore, the para isomer (compound 8c) from the phenyl
series was also active against MTB with MIC90 value of 11.82 µM. Interestingly, in the
presence of a nitric oxide scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-
oxide - PTIO) all compounds have shown MIC90 superior to 62 µM. This data
demonstrates the importance of NO to antitubercular activity for these derivatives.
The four more active compounds (8c and 14a-c) were also evaluated against a clinical
isolate MDR strain and have showed MIC90 values ranging from 7.0 to 50.0 µM (Table
2). Compounds 4a-c and 8a-b showed MIC90 superior to 62 µM (Table 1) and were not
considered promising for the determination of cytotoxicity.
2.3. Determination of cytotoxicity
Cytotoxicity studies were performed using two different cell lines: MRC-5 and J774A.1.
The selectivity index (SI) represents the ratio between IC50 and MIC90. For this assay, the
furoxans intermediates (7c and 13a-c) have exhibited cytotoxic effect and low selectivity
index against J774A.1 cell line. On the other hand, hybrid furoxan (8c and 14a-c) have
shown IC50 values ranging from 34.4 to 623.4 µM (MRC-5) and 4.30 to 408.97 µM
(J774A.1), respectively. For these compounds, SI values ranged from 3.78 to 52.74
(MRC-5) and 1.25 to 34.78 (J774A.1) (Table 1).

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2.4. Nitric oxide release
The nitrite production resulted from the oxidative reaction of nitric oxide, oxygen and
water for the hybrid compounds (4a-c, 8a-c, and 14a-c) was quantified through Griess
-
reaction [37–39]. The results, expressed as percentages of nitrite (NO2 ; mol/mol), are
summarized in Table 1. Isosorbide dinitrate (DNS), used as a positive control, induced
7.5% of nitrite formation. All furoxan derivatives (4a-c, 8a-c, and 14a-c) were able to
induce nitrite formation at levels ranging from 0.16% to 43.55%.
2.5. Partition Coefficient study
The partition coefficients were characterized by HPLC method [40] for all hybrid
furoxan derivatives. The logPo/w values of the furoxan derivatives were positive and the
values ranged from 1.2 to 2.9 (Table 1).
2.6. In vitro stability study
Chemical hydrolysis was performed for the most active compound (14c) in order to
characterize chemical stability at different pHs (1.0; 5.0; 7.4 and 9.0). At extreme pHs
(1.0 and 9.0) the compound was unstable. After 1 hour, at pH 1.0, compound 14c have
undergone 90% of degradation; while, for pH 9.0 the compound was reduced by 50%.
However, at pH 5.0 and 7.4, the compound 14c have shown better stability. After 6h, it
was not detected significant chemical degradation at pH 5.0; while a reduction of 15% at
pH 7.4 was observed. After 24h, a reduction of 20% of compound 14c was quantified at
pHs 5.0 and 7.4 (Fig. 2).

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Fig. 2. In vitro chemical stability. Hydrolytic profile of compound 14c in buffer (pH 1.0;
5.0; 7.4 and 9.0) (data are represented as means ± SEMs and expressed as %).
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Table 1 Antitubercular activity of compounds against Mycobacterium tuberculosis H37Rv; cytotoxicity against MRC-5 and J774A.1
cell lines (IC50); selectivity index (SI); NO release data and experimental LogP d.
a
-
MIC90 (µM) MIC90 (µM),
IC50 (µM) for
IC50 (µM) for
% NO2 (mol/mol), b, c
Compounds
SI1
SI2
LogP d
– H37Rv
H37Rv, PTIO
MRC-5
J774A.1
L-Cys, 50 × 10-4 M
Intermediate furoxans
> 62.0
> 62.0
> 62.0
> 62.0
> 62.0
> 62.0
20.23
20.89
26.01
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0
-
-
-
-
-
-
-
-
-
3a
3b
0
3c
-
-
0
-
-
25.10 ± 0.07
23.10 ± 0.40
19.44 ± 0.70
21.19 ± 4.12
27.05 ± 3.83
24.45 ± 3.94
7a
-
-
7b
-
-
7c
7.4
5.6
2.2
0.4
2
13a
13b
13c
0.1
Hybrid furoxans
> 62.0
> 62.0
> 62.0
> 62.0
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.35 ± 1.71
0.16 ± 2.13
2.02 ± 1.36
11.22 ± 0.5
1.4
1.3
1.3
2.7
4a
4b
4c
8a
-
-
-

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> 62.0
11.82
8.60
-
-
-
-
-
6.87 ± 0.66
7.33 ± 1.77
44.23 ± 0.81
38.49 ± 4.05
43.55 ± 4.26
2.9
2.9
2.2
2.3
2.1
8b
8c
> 62.0
> 62.0
> 62.0
> 62.0
623.44
34.40
30.10
43.01
52.74
3.78
408.97
10.75
4.30
34.78
1.25
3.00
11.98
14a
14b
14c
1.61
14.13
20.29
1.03
10.75
RIF
INH
DNS
0.5
0.11
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0
-
-
-
0
7.17 ± 0.54
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a
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Determined using the REMA methodology [41] in the presence of an equimolar
concentration of the PTIO reagent (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-
oxide), a nitric oxide scavenger.
b Mean ± standard error of the mean.
c
Determined by Griess reaction, after incubation for 1 h at 37 °C in pH 7.4 buffered
water, in the presence of a 1:50 molar excess of L-cysteine.
d Determined by partition coefficient (n-octanol/water), HPLC method [40].
e
Abbreviations: DNS, isosorbide dinitrate (DNS possesses two ONO2 groups that may
release NO); SI1, ratio between IC50 for MRC-5 and MIC90; SI2, ratio between IC50 for
J774A.1 and MIC90; RIF, rifampicin and INH, isoniazid (reference drugs); dash (-) means
not determined.
Table 2 Antitubercular activity of the most activity compounds against a clinical isolate
MDR-TB strain (MIC90).
Compound
8c
MIC90 (µM) – MDR-TB a
24.3
50.0
21.3
7.0
14a
14b
14c
RIF
Res
Res
INH
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209
210
a Resistance to isoniazid, rifampicin, streptomycin and ethambutol [42]. Res, resistant.
3. Discussion

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From a phenotypic-based screening containing more than five thousand compounds
present in our current library, we have identified the (E)-N'-(4-
hydroxybenzylidene)isonicotinohydrazide (I) (Fig. 1), (MIC90 = 1 µM), as a promising
scaffold for molecular modifications (supplementary material). In our drug design, we
have used the molecular hybridization between this compound and furoxan derivatives.
The furoxan derivatives were selected due to its anti-mycobacterial effect [18], related in
parts, to its ability to release NO after biotransformation [22].
In vivo, NO is produced as a result of cytokines and chemokines stimulation [43]. The
antimycobacterial effects of NO were firstly demonstrated in murine macrophage
infected with bacilli [44]. Accurately, iNOS -/- mutated mice have exhibited higher
susceptibility to MTB infection and early death compared to non-mutated mice [45].
Exogenous NO has been shown as a useful strategy to kill the bacilli. It is established that
compounds, such as pretomanid, can kill the MTB by induce NO intracellular after
metabolism [46]. Furthermore, different NO-donors such as diethylenetriamine nitric
oxide adduct (DETA/NO) [46] and S-nitrosothiols [47] are also examples of NO donors
presenting antitubercular activity. Nitric oxide, as well as others reactive nitrogen
intermediates, can alter mycobacterial DNA by generating abasic sites and strand breaks.
Additional NO mycobacterial-induced toxicity include interaction with proteins resulting
in enzymatic inactivation and/or structural modifications [48].
In this work, it was characterized that furoxan derivatives methyl (4a-c) and phenyl (8a-
b) did not exhibit activity against MTB. For these compounds, it was found MIC90 values
superior to 62 µM. However, three isomers from the phenylsulfonyl furoxan series: ortho
(14a), meta (14b) and para (14c) have shown promising activity against MTB with

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MIC90 values below 8.6 µM. Moreover, a phenyl furoxan derivative (8c) also exhibited a
promising MIC90 value of 11.82 µM. The MIC90 values of these four compounds (14a-c;
8a) were greater than several first and second line antitubercular drugs, such as
pyrazinamide (>48 µM), cycloserine (245 µM) and kanamycin (3.4 µM) [49]. We also
evaluated the furoxan intermediates (3a-c, 7a-c and 13a-c) against MTB H37Rv, however,
these compounds showed MIC90 values superior to 20.0 µM.
Our data suggest a direct effect in the pattern of substitution in the furoxan ring and the
antitubercular activity. For phenylsulfonyl series (14a-c), the most active compounds
were those in that N-acylhydrazone was substituted at para position (14c), followed by
meta (14b) and ortho (14a) substitution. A similar effect can be observed in the phenyl
series, wherein the para substituted derivative (8c) was the most potent compound among
its regioisomers.
We also measured the levels of nitrite in the medium as an indirect method to quantify
NO release by compounds. The results demonstrated that NO release is dependent of the
presence of a large excess of -cysteine (1:50), since conditions without this aminoacid
L
were not able to release NO (results not shown) [37]. All furoxan compounds were
capable to generate nitrite in the medium at values ranging from of 0.16% to 44.23%. Our
findings appoint that the antitubercular activity seems to be related in part, to the ability
to release nitric oxide by the furoxan subunit. It was observed that phenylsulfonyl series
(14a-c) showed the best antitubercular activity and generated high levels of nitric oxide,
while the methyl series (4a-c), with low NO-release profile, demonstrated inferior
antitubercular activity. Moreover, we evaluated the antitubercular activity of these four
promising compounds in the presence of an equimolar concentration of 2-phenyl-4,4,5,5-

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tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a nitric oxide radical scavenger [50], in
order to verify the importance of nitric oxide for antitubercular activity. The results
showed that in the presence of PTIO, the four promising compounds have shown MIC90
values higher than those found in the assay without the PTIO reagent, confirming the
influence that nitric oxide plays in the antitubercular activity of these compounds.
The ability to release NO by furoxan derivatives is directly related to the substitution in
the carbon atom at 3 position (C-3), neighboring to N-oxide function [20,39]. Furoxan
derivatives with electron-withdrawing substituents at position C-3 (i.e., phenylsulfonyl-
substituted (14a-c) derivatives) was able to release NO at high levels than methyl (4a-c)
or phenyl (8a-c) series.
The most promising compounds (8c; 14a-c) identified in the primary screening were also
evaluated against MRC-5 and J774A.1 cell in order to characterize their respective
cytotoxicity. These cells were selected because MRC-5 is widely used for phenotypic
screening of drugs to be regarded as a normal cell derived from lung human and J774A.1
is a macrophage murine cell. Compounds (8c; 14a-c) have demonstrated IC50 against
MRC-5 at values ranging from 30.10 to 623.44 µM and SI values between 3.78 and
52.74. Regarding the J774A.1 cell line, it was found IC50 values ranged from 4.30 to
408.97 µM with SI values ranging from 1.25 to 34.78. Phenylsulfonyl derivatives (14a-c)
were more cytotoxic than phenyl furoxan derivative (8c) against both cell lines; however,
among the phenylsulfonyl derivatives it was not observed a relationship between NO-
donor release and cytotoxic effect. The furoxan intermediates (3a-c, 7a-c and 13a-c) have
shown cytotoxicity with IC50 ranging from 2.2 to 113.2 µM and SI values between 0.1
and 2 against J774A.1 cell.

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After the initial screening, we selected the four promising compounds (8c; 14a-c) to be
evaluated against a clinical isolate MDR strain. This strain was phenotypically and
genotypically characterized and it exhibited resistance to isoniazid, rifampicin,
streptomycin and ethambutol [42]. Specifically, for this MDR strain it was characterized
a mutation in the inhA gene, responsible for encode the NADH-dependent enoyl-ACP
reductase of the FAS II system [51]. Compounds (8c; 14a-c) showed MIC90 values
ranging from 7.0 to 50.0 µM, being the compound 14c (MIC90 7.0 µM) the most
promising among the series. These data suggest that furoxan moiety improved the
antitubercular activity against MDR-TB, considering that compound (I) did not showed
antitubercular activity against MDR-TB strains.
Therefore, we selected the most promising compound (14c) to analyze its chemical
stability using an in vitro assay. We carried out the stability study under four conditions,
(pHs 1.0, 5.0, 7.4 and 9.0) in order to mimic the acidic stomach (pH 1.0), the macrophage
phagolysosome (pH 4.5 – 6.2) [52–54], the neutral plasma (pH 7.4) environments and a
basic condition (pH 9.0), respectively. Compound 14c were unstable at pH 1.0 and 9.0
being degraded around 90% and 50% after the first hour, respectively. Despite of that, it
was not detected significant chemical degradation at pH 5.0 (0%) and 7.4 (15%) after 6
hours. After 24h, a reduction of 20% was observed at both pHs 5.0 and 7.4, showing a
relative stability of compound 14c in these pHs values. The degradation rates of
compound 14c were calculated by HPLC-MS/MS and the degradation products were not
characterized (supplementary material).
Moreover, it is well established that the permeability through the peptidoglycan-
arabinogalactan-mycolic core in the MTB is a great limitation for antitubercular drug

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development [16]. Therefore, the lipophilicity, mostly expressed as logPo/w (the logarithm
of the partition coefficient in a specific solvent (Poctanol/Pwater)), is an important physico-
chemical property that must be evaluated for new compounds during drug discovery
[55,56]. Recently, we have identified that for the most active antitubercular compounds
described in the literature between 2012-2014 (MIC90 inferior to 7), cLogP values ranged
from 2 to 6 [16]. Hybrid furoxan derivatives reported here showed logPo/w values ranging
from 1.3 to 2.9 (Table 1). We did not find a direct relationship between logP and
antitubercular activity for all compounds; however, for those more active (8c and 14a-c)
we have observed logPo/w values superior to 2.1, comparable to those values reported in
literature [16].
4. Conclusion
In conclusion, a novel series of hybrid furoxan derivatives was synthesized and
characterized. The furoxan derivatives have demonstrated nitric oxide release properties
at levels ranging from 0.16% to 44.23%. Among the nine hybrid furoxan derivatives,
compounds (8c and 14a-c) showed MIC90 values ranging from 1.03 to 11.82 µM and SI
ranging from 3.78 to 52.74 (MRC-5) and 1.25 to 34.78 (J774A.1). Moreover, the four
selected compounds (8c and 14a-c) presented activity against a clinical isolate MDR-TB
strain with MIC90 values ranging from 7.0 to 50.0 µM. In vitro hydrolysis studies have
demonstrated that compound 14c is stable at pH 5.0 and 7.4 until 6 h. The results
described here pointed out compounds 8c and 14a-c as novel lead compounds for the
treatment of TB infection, including against resistant strain.

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5. Experimental section
5.1. Chemistry
Melting points (mp) were measured using an electrothermal melting point apparatus
(SMP3; Bibby Stuart Scientific) in open capillary tubes. Infrared spectroscopy (KBr disc)
were performed on an FTIR-8300 Shimadzu spectrometer, and the frequencies are
expressed per cm-1. The NMR for 1H and 13C of all compounds were scanned on a Bruker
Fourier with Dual probe 13C/1H (300-MHz) NMR spectrometer and a Bruker Ascend
(600-MHz) NMR spectrometer using dimethyl sulfoxide (DMSO-d6) as solvent.
Chemical shifts were expressed in parts per million (ppm) relative to tetramethylsilane.
The signal multiplicities are reported as singlet (s), doublet (d), doublet of doublet (dd),
and multiplet (m). Elemental analyses (C, H and N) were performed on a Perkin-Elmer
model 2400 analyzer, and the data were within ± 0.4% of the theoretical values. The
compounds were separated on a chromatography column with silica gel (60 Å pore size,
35-75-µm particle size) and the following solvents were used as mobile phase:
dichloromethane, hexane, ethyl acetate and petroleum ether. The reaction progress of all
compounds was monitored by thin-layer chromatography (TLC), which was performed
on 2.0- by 6.0-cm2 aluminum sheets precoated with silica gel 60 (HF-254; Merck) to a
thickness of 0.25 mm and revealed under UV light (265 nm). All compounds were
analyzed by HPLC, and their purity was confirmed to be greater than 98.5%. Reagents
and solvents were purchased from commercial suppliers and used as received.
Compounds 2, 6, and 12 were synthesized according to a previously described
methodology (Scheme 1 and 2) [20,30–32]. Isonicotinohydrazide were purchased
commercially.

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5.2. General procedure for the synthesis of compounds 4a-c, 8a-c, 14a-c and 23a-c
A solution of compound 3a-c, 7a-c or 13a-c (0.87 mmol) in 10 mL of ethanol and 3
drops of hydrochloric acid was stirred at for 20 min at room temperature (r.t.). Next,
isonicotinohydrazide (0.106 g, 0.87 mmol) was added, and the mixture was stirred at r.t.
for 12 h. The reactions were monitored by TLC (98:2, ethyl acetate : methanol). The
solvent was concentrated under reduced pressure, and 8 mL of ice water was added in
order to precipitate the desired products. If necessary, the samples could be further
purified through column chromatography (silica gel), using ethyl acetate-methanol (98:2)
as the mobile phase to give the compounds 4a-c, 8a-c and 14a-c with variable yields (83
to 95%).
5.2.1. (E)-4-(2-((2-isonicotinoylhydrazono)methyl)phenoxy)-3-methyl-1,2,5-oxadiazole
2-oxide (4a
)
White powder; yield, 83%; mp, 196 to 198°C. IR Vmax (cm-1; KBr pellets): 3.203 (N-H),
3.030 (C-H aromatic), 1.689 (C=O amide), 1,639 (C=N imine), 1,485 (N-O furoxan),
1
1,448 (CH3), 1.284 (C-N aromatic), 1.143 (C-O ether). H NMR (300 MHz, DMSO-d6)
δ: 12.08 (1H; s), 8.78 (2H; d; J = 5.8), 8.60 (1H; s), 7.98 (1H; d; J = 8.8), 7.81 (2H; d; J =
6.0), 7.57 (2H; m), 7.47 (1H; t; J = 16.1), 2.24 (3H; s) ppm. 13C NMR (75 MHz, DMSO-
d6) δ: 163.53, 161.69, 150.54, 150.39, 143.50, 140.27, 131.91, 128.29, 127.22, 125.46,
121.66, 121.55, 107.48, 7.07 ppm. Calculated analysis (%) for C16H13N5O4: C: 56.6; H:
3.8; N: 20.6. Found: C: 56.7; H: 3.8; N: 20.5.

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5.2.2. (E)-4-(3-((2-isonicotinoylhydrazono)methyl)phenoxy)-3-methyl-1,2,5-oxadiazole
2-oxide (4b
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White powder; yield, 89%; mp, 149 to 154°C. IR Vmax (cm-1; KBr pellets): 3.217 (N-H),
3.049 (C-H aromatic), 1.633 (C=O amide), 1,548 (C=N imine), 1,446 (N-O furoxan),
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1,413 (CH3), 1.305 (C-N aromatic), 1.159 (C-O ether). H NMR (300 MHz, DMSO-d6)
δ: 8.78 (2H; d; J = 5.9), 8.49 (1H; s), 7.82 (3H; d; J = 5.9), 7.69 (1H; d; J = 7.6), 7.60
(1H; t; J = 15.7), 7.51 (1H; d; J = 8.4), 2.16 (3H; s) ppm. 13C NMR (75 MHz, DMSO-d6)
δ: 163.20, 161.82, 153.01, 151.59, 150.40, 147.64, 140.35, 136.28, 130.78, 125.59,
121.59, 117.72, 107.61, 6.99 ppm. Calculated analysis (%) for C16H13N5O4: C: 56.6; H:
3.8; N: 20.6. Found: C: 56.5; H: 3.8; N: 20.5.
5.2.3. (E)-4-(4-((2-isonicotinoylhydrazono)methyl)phenoxy)-3-methyl-1,2,5-oxadiazole
2-oxide (4c
)
White powder; yield, 85%; mp, 214 to 217°C. IR Vmax (cm-1; KBr pellets): 3.236 (N-H),
3.078 (C-H aromatic), 1.666 (C=O amide), 1,604 (C=N imine), 1,485 (N-O furoxan),
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1,408 (CH3), 1.305 (C-N aromatic), 1.155 (C-O ether). H NMR (300 MHz, DMSO-d6)
δ: 12.11 (1H; s), 8.78 (2H; d; J = 5.7), 8.50 (1H; s), 7.85 (2H; d; J = 8.7), 7.82 (2H; d; J =
13
5.9), 7.51 (2H; d; J = 8.6), 2.14 (3H; s) ppm. C NMR (75 MHz, DMSO-d6) δ: 162.76,
161.68, 154.06, 150.35, 147.80, 140.41, 131.96, 129.05, 121.53, 119.98, 107.59, 6.97
ppm. Calculated analysis (%) for C16H13N5O4: C: 56.6; H: 3.8; N: 20.6. Found: C: 56.7;
H: 3.8; N: 20.6.

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5.2.4. (E)-4-(2-((2-isonicotinoylhydrazono)methyl)phenoxy)-3-phenyl-1,2,5-oxadiazole
2-oxide (8a
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White powder; yield, 85%; mp, 209 to 211°C. IR Vmax (cm-1; KBr pellets): 3.184 (N-H),
3.045 (C-H aromatic), 1.674 (C=O amide), 1,600 (C=N imine), 1,435 (N-O furoxan),
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1.300 (C-N aromatic), 1.149 (C-O ether), 769 (aromatic). H NMR (300 MHz, DMSO-
d6) δ: 12.06 (1H; s), 8.74 (2H; d; J = 6.0), 8.65 (1H; s), 8.14 (2H; d; J = 7.9), 8.03 (1H; d;
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J = 7.8), 7.75 (2H; d; J = 6.0), 7.63 (5H; m), 7.50 (1H, t; J = 15.9) ppm. C NMR (75
MHz, DMSO-d6) δ: 162.31, 161.39, 150.50, 150.08, 142.69, 139.99, 131.72, 130.75,
128.91, 127.30, 127.23, 126.47, 125.57, 121.77, 121.43, 121.24, 107.92 ppm. Calculated
analysis (%) for C21H15N5O4: C: 62.8; H: 3.7; N: 17.4. Found: C: 62.9; H: 3.7; N: 17.3.
5.2.5. (E)-4-(3-((2-isonicotinoylhydrazono)methyl)phenoxy)-3-phenyl-1,2,5-oxadiazole
2-oxide (8b
)
White powder; yield, 92%; mp, 210 to 213°C. IR Vmax (cm-1; KBr pellets): 3.324 (N-H),
3.068 (C-H aromatic), 1.660 (C=O amide), 1,604 (C=N imine), 1,483 (N-O furoxan),
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1.323 (C-N aromatic), 1.157 (C-O ether), 769 (aromatic). H NMR (300 MHz, DMSO-
d6) δ: 8.78 (2H; d; J = 5.6), 8.51 (1H; s), 8.10 (2H; d; J = 7.1), 7.94 (1H; s), 7.82 (2H; d; J
= 5.7), 7.72 (1H; m), 7.62 (5H, m) ppm. 13C NMR (75 MHz, DMSO-d6) δ: 154.31,
145.39, 130.29, 123.46, 123.24, 121.39, 120.27, 119.00, 118.61, 113.94, 100.65 ppm.
Calculated analysis (%) for C21H15N5O4: C: 62.8; H: 3.7; N: 17.4. Found: C: 62.8; H: 3.7;
N: 17,4.

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5.2.6. (E)-4-(4-((2-isonicotinoylhydrazono)methyl)phenoxy)-3-phenyl-1,2,5-oxadiazole
2-oxide (8c
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White powder; yield, 88%; mp, 198 to 202°C. IR Vmax (cm-1; KBr pellets): 3.250 (N-H),
3.066 (C-H aromatic), 1.651 (C=O amide), 1,610 (C=N imine), 1,438 (N-O furoxan),
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1.332 (C-N aromatic), 1.205 (C-O ether), 756 (aromatic). H NMR (300 MHz, DMSO-
d6) δ: 8.78 (2H; d; J = 5.8), 8.51 (1H; s), 8.07 (2H; d; J = 6.6), 7.88 (2H; d; J = 8.7), 7.82
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(2H; d; J = 5.9), 7.63 (5H; m) ppm. C NMR (75 MHz, DMSO-d6) δ: 153.62, 149.33,
126.38, 124.00, 123.26, 121.41, 118.97, 113.81, 112.77, 100.72 ppm. Calculated analysis
(%) for C21H15N5O4: C: 62.8; H: 3.7; N: 17.4. Found: C: 62.9; H: 3.7; N: 17.3.
5.2.7. (E)-4-(2-((2-isonicotinoylhydrazono)methyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-
oxadiazole 2-oxide (14a
)
White powder; yield, 85%; mp, 199 to 202°C. IR Vmax (cm-1; KBr pellets): 3.280 (N-H),
3.068 (C-H aromatic), 1.651 (C=O amide), 1,645 (C=N imine), 1,454 (N-O furoxan),
1.354 (C-N aromatic), 1.161 (S=O sulfone), 1.083 (C-O ether), 744 (aromatic). 1H NMR
(300 MHz, DMSO-d6) δ: 8.78 (2H; d; J = 5.1), 8.50 (1H; s), 8.07 (2H; d; J = 7.9), 7.93
(1H; t; J = 7.3), 7.85 (4H; m), 7.78 (1H; t; J = 7.7), 7.71 (1H; d; J = 7.5), 7.60 (1H; t; J =
7.9), 7.51 (1H; d; J = 8.9) ppm. 13C NMR (75 MHz, DMSO-d6) δ: 154.08, 150.83,
145.10, 139.80, 132.57, 129.15, 128.56, 123.07, 122.31, 120.90, 118.31, 113.97, 113.84,
110.05, 103.58 ppm. Calculated analysis (%) for C21H15N5O6S: C: 54.2; H: 3.2; N: 15.1.
Found: C: 54.3; H: 3.2; N: 15.0.

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5.2.8. (E)-4-(3-((2-isonicotinoylhydrazono)methyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-
oxadiazole 2-oxide (14b
)
White powder; yield, 95%; mp, 202 to 204°C. IR Vmax (cm-1; KBr pellets): 3.182 (N-H),
3.003 (C-H aromatic), 1.680 (C=O amide), 1,604 (C=N imine), 1,444 (N-O furoxan),
1.357 (C-N aromatic), 1.166 (S=O sulfone), 1.083 (C-O ether), 742 (aromatic). 1H NMR
(300 MHz, DMSO-d6) δ: 8.79 (2H; d; J = 6.0), 8.48 (1H; s), 8.07 (2H; d; J = 7.3), 7.95
(1H; t; J = 13.8), 7.83 (4H; m), 7.77 (1H; m), 7.71 (1H; d; J = 7.6), 7.61 (1H; t; 15.7),
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7.51 (1H; d; J = 9.3) ppm. C NMR (75 MHz, DMSO-d6) δ: 154.08, 150.87, 145.12,
142.66, 139.79, 132.59, 129.16, 128.27, 123.08, 122.32, 120.94, 118.33, 113.99, 113.85,
110.08, 103.61 ppm. Calculated analysis (%) for C21H15N5O6S: C: 54.2; H: 3.2; N: 15.1.
Found: C: 54.1; H: 3.2; N: 15.1.
5.2.9. (E)-4-(4-((2-isonicotinoylhydrazono)methyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-
oxadiazole 2-oxide (14c
)
White powder; yield, 92%; mp, 194 to 197°C. IR Vmax (cm-1; KBr pellets): 3.238 (N-H),
3.068 (C-H aromatic), 1.664 (C=O amide), 1,610 (C=N imine), 1,450 (N-O furoxan),
1.359 (C-N aromatic), 1.165 (S=O sulfone), 1.082 (C-O ether), 750 (aromatic). 1H NMR
(300 MHz, DMSO-d6) δ: 8.78 (2H; d; J = 6.0), 8.50 (1H; s), 8.05 (2H; d; J = 8.6), 7.88
(2H; d; J = 8.8), 7.82 (2H, d; J = 6.0), 7.77 (3H, t; J = 15.6), 7.53 (2H, d; J = 8.7) ppm.
13C NMR (75 MHz, DMSO-d6) δ: 153.98, 150.42, 146.23, 142.65, 139.97, 132.66,
129.12, 128.58, 124.62, 122.33, 121.32, 120.89, 113.83, 112.44, 103.63 ppm. Calculated
analysis (%) for C21H15N5O6S: C: 54.2; H: 3.2; N: 15.1. Found: C: 54.3; H: 3.2; N: 15.0.

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5.3.
Biological activity
5.3.1. Determination of Minimal Inhibitory Concentration (MIC90)
The antitubercular activity of all compounds was determined through the REMA
methodology according procedures described by Palomino and coworkers [57]. Stock
solutions of the tested compounds were prepared in DMSO and diluted in Middlebrook
7H9 broth (Difco) supplemented with 10% OADC enrichment (dextrose, albumin, and
catalase) using a Precision XS™ (BioTek®), to obtain final drug concentration ranging
from 0.09 – 25 µg/mL. Rifampicin and isoniazid were used as a control drugs. A
suspension of the MTB H37Rv ATCC 27294 or the clinical isolate MDR-TB strain was
cultured in Middlebrook 7H9 broth supplemented with 10% OADC and 0.05 % Tween
80. The culture was frozen at −80 °C in aliquots. The concentration was adjusted to 2 x
105 UFC/mL and 100 µL of the inoculum was added to each well of a 96-well microtiter
plate together with 100 µL of the compounds. Samples were set up in three independent
assays. The plate was incubated for 7 days at 37 °C. After 24 h, 30 µL of 0.01 %
resazurin in distilled water was added. The fluorescence of the wells was read using a
Cytation™ 3 (BioTek®) in which were used excitations and emissions filters at
wavelengths of 530 and 590 nm, respectively. The MIC90 value was defined as the lowest
drug concentration at which 90 % of the cells are infeasible relative to the control.
5.3.2. Cytotoxicity assay
In vitro cytotoxicity assays (IC50) were performed on MRC-5 (ATCC® CCL-171) and
J774A.1 (ATCC® TIB-67), as described by Pavan and colleagues [58]. The cells were

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routinely maintained in complete medium (DMEM) supplemented with 10 % of fetal
bovine serum (FBS) plus amphotericin B (2 mg/L) and gentamicin (50 mg/L) at 37 °C, in
a humidified 5 % CO2 atmosphere. After reaching confluence, the cells were detached,
counted and adjusted to 1 x 105 cells/mL. The cells were seeded in 200 µL of complete
medium in 96-well plates. The plates were incubated under the same conditions for 24 h
to allow cell adhesion prior to drug testing. From the stock solutions described above the
compounds were diluted using a Precision XS™ (BioTek®), to obtain final drug
concentration ranging from 0.09 – 250 µg/mL. Then, the cells were exposed to
compounds for 24 h and 30 µL of 0.01 % resazurin in distilled water was added. The
fluorescence of the wells was read using a Cytation™ 3 (BioTek®) in which were used
excitations and emissions filters at wavelengths of 530 and 590 nm, respectively. The
IC50 value was defined as the highest drug concentration at which 50 % of the cells are
viable relative to the control. Samples were set up in three independent assays.
5.3.3. Nitric oxide release
Nitric oxide has a short half-life, therefore, the quantification of NO metabolites like
nitrite and nitrate is a useful method to quantify this molecule in the medium [59,60]. The
amount of NO released was indirectly detected by Griess reaction through the
measurement of nitrites in the medium. A volume of 98 µL of a phosphate buffer (pH
7.4) solution containing 5 mM of -cysteine was added in triplicate in a 96-well, flat-
L
bottomed, polystyrene microtiter plate. After loading the plate with the phosphate buffer
(98 µL), it was added 2 µL of solution containing the appropriate compound diluted in
DMSO (3 mL). The final concentration of the compound in each well was 1 x 10-4 M.

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After 1 h of incubation at 37 °C, it was added 100 µL of the Griess reagent (4 g of
sulfanilamide, 0.2 g of N-naphthylethylenediamine dihydrochloride, 85 % phosphoric
acid [10 mL] in distilled water [final volume, 100 mL]). After 10 min at room
temperature, the absorbance was measured at 540 nm using a BioTek® microplate reader
spectrophotometer. Standard sodium nitrite solutions (0.5 to 100 nmol/mL) were used to
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construct the calibration curve. The yields of nitrite are expressed as % NO2 (mol/mol).
No production of nitrite was observed in the absence of L-cysteine [37–39].
5.3.4. Partition coefficient (n-octanol/water) measured by HPLC method
The partition coefficient was characterized using the HPLC method according procedures
described by OECD Guidelines for the Testing of Chemicals [40]. The equipment used
was a Shimadzu HPLC model CBM 20-A (Shimadzu®) equipped with UV-VIS detector
(model SPD-20A), quaternary pumping system mobile phase (model LC-20AT), solvent
degasser (model DGU-20As) and a Agilent® Eclipse XDB C-18 column (250mm x
4,6mm; 5µm). For HPLC method it was used an isocratic flow [methanol:water (75:25)]
at 1.0 mL/min. The volume injected was 20.0 µL and the wavelength in the detector was
210 nm. The following substances were used as standards to construct the curve log K x
log P: acetanilide, benzonitrile, nitrobenzene, toluene, naphthalene, biphenyl and
phenanthrene. The capacity factor (logK) of the hybrid compounds was determined from
their retention times and interpolated in linearity curve log K x log P.
5.3.5. In vitro stability study

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In vitro hydrolysis was performed by HPLC method. The compound 14c was separated
using a Phenomenex Luna reverse-phase C18 (2)-HTS column (2.5-µm particle, 2 by 50
mm). The isocratic flow was 50:50 (water : 0,1 % formic acid-acetonitrile, v/v) and the
flow rate was 0.25 mL/min. The HPLC was coupled to a API 2000 triple quadrupole
mass spectrometer equipped with a heated electrospray ionization interface (H-ESI)
operated in the positive ionization mode at capillary voltage 5200 V, source temperature
350 °C, nitrogen gas flow 65 units.
For hydrolysis, an appropriate solution of compound 14c was diluted in acetonitrile at
1000 µM. Then, this solution was diluted to 10 µM using four different PBS buffer
(phosphate-buffered saline) in order to provide the following pHs: 1.0; 5.0; 7.4 and 9.0.
During the assay, all samples were maintained at constant agitation using a shaker (400
rpm) at 37 °C. Aliquots were taken from the solution at the following times: 0, 1, 4, 6,
and 24 h. The injection volume in the HPLC was 5 µL. All analyses were conducted in
triplicate, and the results were expressed as the averages of the concentrations in
percentages (
±
standard error of the mean [SEM]).
Acknowledgments
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP grants 2013/14957-5; 2014/02240-1; 2014/24811-0; 2014/11586-9), Programa
de Estágio no Exterior (PROPG-UNESP 2012) and PADC-UNESP.
The authors would like to thank professor Alberto Gasco for his contribution to this
work.