Design, Synthesis, and Characterization of N-Oxide-Containing Heterocycles with in Vivo Sterilizing Antitubercular Activity

Article abstract of DOI:10.1021/acs.jmedchem.7b01332

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is the infectious disease responsible for the highest number of deaths worldwide. Herein, 22 new N-oxide-containing compounds were synthesized followed by in vitro and in vivo evaluation of their antitubercular potential against Mtb. Compound 8 was found to be the most promising compound, with MIC₉₀ values of 1.10 and 6.62 μM against active and nonreplicating Mtb, respectively. Additionally, we carried out in vivo experiments to confirm the safety and efficacy of compound 8; the compound was found to be orally bioavailable and highly effective, leading to a reduction of Mtb to undetectable levels in a mouse model of infection. Microarray-based initial studies on the mechanism of action suggest that compound 8 blocks translation. Altogether, these results indicate that benzofuroxan derivative 8 is a promising lead compound for the development of a novel chemical class of antitubercular drugs.

Full text of DOI:10.1021/acs.jmedchem.7b01332

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Article
Design, Synthesis and Characterization of N-oxide-containing
Heterocycles with In vivo Sterilizing Antitubercular Activity
Guilherme Felipe dos Santos Fernandes, Paula Carolina De Souza, Elsa Moreno-Viguri, Mery Santivañez-
Veliz, Rocio Paucar, Silvia Pérez-Silanes, Konstantin Chegaev, Stefano Guglielmo, Loretta Lazzarato,
Roberta Fruttero, Chung Man Chin, Patricia Bento Da Silva, Marlus Chorilli, Mariana Cristina Solcia,
Camila Maríngolo Ribeiro, Caio Sander Paiva Silva, Leonardo Biancolino Marino, Priscila Longhin
Bosquesi, Debbie M. Hunt, Luiz Pedro Sorio de Carvalho, Carlos Alberto De Souza Costa, Sanghyun
Cho, Yuehong Wang, Scott Gary Franzblau, Fernando Rogerio Pavan, and Jean Leandro Leandro Santos
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01332 • Publication Date (Web): 02 Oct 2017
Downloaded from http://pubs.acs.org on October 2, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Research
Wang, Yuehong; University of Illinois at Chicago, Institute for Tuberculosis
Research
Franzblau, Scott; University of Illinois, Institute for Tuiberculosis Research
Pavan, Fernando; São Paulo State University (UNESP), Biological Sciences
Santos, Jean Leandro; Universidade Estadual Paulista Julio de Mesquita
Filho, Faculdade de Ciências Farmacêuticas Fac, Fármacos e Medicamentos
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Design, Synthesis and Characterization of Nꢀoxideꢀcontaining
Heterocycles with In vivo Sterilizing Antitubercular Activity
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a,b,c*
b,g*
Guilherme Felipe dos Santos Fernandes,
Paula Carolina de Souza,
Elsa Morenoꢀ
c
c
c
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Viguri, Mery SantivañezꢀVeliz, Rocio Paucar, Silvia PérezꢀSilanes, Konstantin
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Chegaev, Stefano Guglielmo, Loretta Lazzarato, Roberta Fruttero, Chung Man Chin,
b
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Patricia Bento da Silva, Marlus Chorilli, Mariana Cristina Solcia, Camila Maríngolo
b
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Ribeiro, Caio Sander Paiva Silva, Leonardo Biancolino Marino, Priscila Longhin
b
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Bosquesi, Debbie M. Hunt, Luiz Pedro S. de Carvalho, Carlos Alberto de Souza Costa,
g
g
g
b#
Sang Hyun Cho, Yuehong Wang, Scott Gary Franzblau, Fernando Rogério Pavan,
a,b#
Jean Leandro dos Santos,
*
These authors contributed equally to this work.
a
São Paulo State University (UNESP), Institute of Chemistry, Araraquara, 14800060, Brazil
b
c
São Paulo State University (UNESP), School of Pharmaceutical Sciences, Araraquara, 14800903, Brazil
Universidad de Navarra, Department of Organic and Pharmaceutical Chemistry, Instituto de Salud
Tropical, Pamplona, 31008, Spain
d
Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Turin, 10124, Italy
e
Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, 1 Midland
Road, London NW1AT, United Kingdom
f
São Paulo State University (UNESP), School of Odontology, Araraquara, 14801903, Brazil
g
Institute of Tuberculosis Research, University of Illinois at Chicago, Chicago, 60607, USA.
#
Address correspondence to Jean Leandro dos Santos, santosjl@fcfar.unesp.br and Fernando Rogério
Pavan, fernandopavan@fcfar.unesp.br.
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ABSTRACT
Tuberculosis, caused by the Mycobacterium tuberculosis (Mtb), is the infectious disease
responsible for the highest number of deaths worldwide. Herein, 22 new Nꢀoxideꢀ
containing compounds were synthesized followed by in vitro and in vivo evaluation of
their antitubercular potential against Mtb. Compound 8 was found to be the most
promising compound, with MIC values of 1.10 and 6.62 ꢁM against active and nonꢀ
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replicating Mtb, respectively. Additionally, we carried out in vivo experiments to confirm
the safety and efficacy of compound 8; the compound was found to be orally bioavailable
and highly effective leading to the reduction of the number of Mtb to undetected levels in
a mouse model of infection. Microarrayꢀbased initial studies on the mechanism of action
suggest that compound 8 blocks the process of translation. Altogether, these results
indicated benzofuroxan derivative 8 to be a promising lead compound for the
development of a novel chemical class of antitubercular drugs.
Keywords: Mycobacterium tuberculosis, tuberculosis, antitubercular drug, Nꢀoxide,
furoxan, benzofuroxan.
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INTRODUCTION
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) in humans, is
considered to be responsible for the highest number of deaths caused by infectious
diseases worldwide in 2015. The World Health Organization (WHO) reported 9.6 million
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new cases and 2 million deaths worldwide in the same year. The high mortality rate of
TB has even surpassed the deaths caused by human immunodeficiency virus (HIV).
According to an estimation, oneꢀthird of the world population is infected with a latent
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form of TB, where the treatment is often ineffective owing to a lack of drugs with an
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ability to act in the dormant state of mycobacteria.
Furthermore, increased dissemination of multidrugꢀresistant (MDR), extensively drugꢀ
resistant (XDR), and totally drugꢀresistant (TDR) strains poses a huge challenge to be
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overcome throughout the world in the fight against TB.
For the treatment, the WHO recommends a combination of isoniazid (INH), rifampicin
RMP), ethambutol (EMB), and pyrazinamide (PZA) for six months. In the cases
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involving resistance, the treatment can be extended up to 28 months and include the use
of secondꢀline drugs, such as fluoroquinolones, aminoglycosides, Dꢀcycloserine (DCS),
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and linezolid (LZD) among others.
The current treatment suffers from several
limitations, including prolonged standard regimen, high rate of treatment discontinuation,
adverse effects, toxicity, drug–drug interactions, and a lack of effectiveness against the
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,12–16
latent mycobacteria.
Over the past few years, limited but significant progress in the development of drug
candidates against TB has been achieved. After a gap of more than 50 years without new
drugs approved for TB, the United States Food and Drug Administration (FDA) approved
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®
bedaquiline (Bdq, SIRTURO, Janssen; Beerse, Belgium) in 2012 for the treatment of
MDRꢀTB. The literature in the past 5 years has witnessed significant advances in the
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development of other compounds with potent antitubercular activity.
In this regard,
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several drug candidates were moved toward clinical trials, such as sutezolid, posizolid,
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delamanid, and pretomanid.
Nevertheless, Mtb strains, resistant to the new
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compounds, have already been reported,
reinforcing the urgency to develop more
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potent and a larger number of novel drugs for the treatment of TB.
We have previously reported a series of furoxan derivatives with potent activity against
Mtb, including MDR strains. Specifically, the compound, (E)ꢀ4ꢀ(4ꢀ((2ꢀ
isonicotinoylhydrazono)methyl)phenoxy)ꢀ3ꢀ(phenylsulfonyl)ꢀ1,2,5ꢀoxadiazole 2ꢀoxide
(Figure 1) could inhibit 90% growth of Mtb H37Rv strain at a concentration of 1 ꢁM. In
addition, we characterized that the antitubercular activity of the aboveꢀmentioned furoxan
derivative was related to its ability to generate nitric oxide (NO) following
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biotransformation.
Motivated by preliminary promising results obtained with the furoxan derivatives, we
designed new heterocyclic analogs containing the Nꢀoxide subunit, including amideꢀ
furoxans (series 1), benzofuroxans (series 2), and quinoxaline, 1,4ꢀdiꢀNꢀoxide (QdNO)
(series 3, Figure 1). Furoxans, benzofuroxans, and QdNO derivatives represent important
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scaffolds in medicinal chemistry due to their wide spectrum of biological activities,
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including antitubercular activity. The antimycobacterial activity of these compounds is
attributed to the generation of reactive oxygen species (ROS) following their
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biotransformation.
Specifically for quinoxalines, earlier studies have reported that
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these compounds lead to increased levels of ROS under hypoxic conditions, which could
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contribute to interesting properties against latent TB.
The ROS play a crucial role in the pathogenesis of TB. Several studies have demonstrated
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the relationship between the levels of ROS produced by immune cells and the
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susceptibility of patients to several species of the Mycobacterium genus.
Furthermore,
ROS exert manyꢀfold effects during TB. For example, increased ROS levels can lead to
an inhibition of Mtb growth, damage to cellular components, such as lipids, proteins, and
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nucleic acids, and activate macrophageꢀmediated inflammatory activity.
The high
levels of ROS can also induce apoptosis of macrophages (host for tubercle bacilli),
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thereby preventing the growth and replication of the bacilli. Therefore, the design of
new compounds that could act by increasing the levels of ROS and perturbing
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mycobacterial redox homeostasis seems to be a promising strategy for combating TB.
In a continuing effort to develop new drug candidates for the treatment of TB, we herein
describe the design, synthesis, and biological activities of a series of heterocyclic
compounds containing Nꢀoxide as antitubercular compounds.
RESULTS AND DISCUSSION
Chemistry
Twentyꢀtwo novel compounds containing the Nꢀoxide subunit were synthesized
according to the synthetic methodologies presented in Schemes 1, 2, and 3.
The amideꢀfuroxan derivatives (1, 2) were synthesized according to the previously
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described methods.
The furoxan derivative (2) was allowed to react with 2ꢀ, 3ꢀ , or 4ꢀ
hydroxybenzaldehyde in dichloromethane medium, using 1,8ꢀdiazabicyclo[5.4.0]undecꢀ
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7ꢀene (DBU) as the base, leading to the formation of the furoxan derivatives containing
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an aldehyde group (3a–c). Then, a condensation reaction of the aldehyde derivatives
with isonicotinohydrazide was performed in ethanolic medium catalyzed by an acid to
generate the hybrid furoxan derivatives (4a–c, Scheme 1).
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The benzofuroxan derivative containing an aldehyde group (7) was obtained according to
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a previously reported methodology. The compound was allowed to react with different
aromatic hydrazides through the same condensation reaction described above leading to
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the formation of benzofuroxan derivatives (8–17, Scheme 2). The H and C nuclear
magnetic resonance (NMR) spectra of these compounds displayed protons and carbon
signals from the benzofuroxan nucleus as broad peaks, indicating a benzofuroxan
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tautomerism.
The dioxolanꢀbenzofuroxan derivative (18) was obtained from the reaction between
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compound (7) and ethylene glycol. The quinoxaline derivatives were obtained through a
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variation of the Beirut reaction,
wherein the dioxolan–benzofuroxan derivative (18)
reacted with the appropriate nitrile derivatives in dichloromethane medium. Potassium
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carbonate (K CO ) was utilized as a catalyst leading to the generation of quinoxaline
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derivatives (19–26). Compound (28) was synthesized from quinoxaline (19), which was
submitted to a cyclic acetal hydrolysis followed by its reaction with isonicotinohydrazide
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through a condensation reaction (Scheme 3).
All compounds were characterized by elemental analysis, infrared (IR) spectroscopy,
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mass spectrometry, and H and C NMR. Furthermore, all compounds were analyzed by
highꢀpressure liquid chromatography (HPLC) and their purity was confirmed to be
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superior to 98.5%. Experimental logP values and melting points were determined for the
final compounds.
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Biological studies
The antitubercular activity of compounds containing Nꢀoxide (4a–c, 8–17, 19–26, and
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8) was performed using Mtb H Rv ATCC 27294 strain. The resazurin microtiter assay
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(REMA) was employed as described previously.
The results were expressed as
minimum inhibitory concentration (MIC ) and compounds showing MIC values below
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0 ꢁM were selected for further characterization. The potential cytotoxicity was
evaluated using human lung fibroblast, MRC–5 according to a previously reported
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methodology
and the results were expressed as IC values. The selectivity index (SI)
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of the tested compounds was calculated through the ratio between IC and MIC . The
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compounds that reported SI ≥ 10 were considered promising for further studies according
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to the cutoff value established.
Potential anaerobic activity of the best compounds was
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evaluated using the method described by Cho et al., 2007.
The analysis of the spectrum of biological activity of the compounds was performed
through determination of MIC90 values against Escherichia coli (ATCC 25922) and
Staphylococcus aureus (ATCC 29213) by measuring the optical density (OD) at 570 nm
(
OD ) after 16 h and against Candida albicans (ATCC 10231) at OD nm after 48 h.
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The compounds were also tested against Mtb H Rv isogenic strains monoresistant to
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RMP (ATCC 35838); INH (ATCC 35822); streptomycin (SM, ATCC 35820);
capreomycin (CAP), moxifloxacin (MOX), and BDQ (strains from the University of
Illinois at ChicagoꢀInstitute for Tuberculosis Research) by microdilution technique
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multiple antigen blot assay (MABA). Following this step, we selected the best
compound (compound 8) for further studies.
Due to the ability of Mtb to survive inside the macrophages, we decided to investigate
whether compound 8 could inhibit the growth of Mtb H37Rv strain in J774A.1
macrophage cell line. Furthermore, timeꢀkill experiments were performed for up to 15
days to evaluate the bactericidal profile of compound 8.
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Some earlier reports
indicated that the growth of mycobacteria can be affected by the
presence of ions, nutrients, and pH of the medium. With this regard, different conditions
of culture medium were analyzed by MABA. These included (a) adjusting the culture
medium to pH 6.0; (b) including 4% bovine serum albumin (BSA); and (c) the
supplementation of 10% fetal bovine serum (FBS). The slightly acidic pH (pH 6.0) was
selected as the pH compatible to the growth of Mtb and corresponding to the time of
fusion of Mtbꢀcontaining phagosomes with lysosomes. One of the physiological functions
of albumin (synthesized in the liver) is to transport of poorly soluble molecules of both
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endogenous and exogenous origin.
Albumin binding constitutes an essential
pharmacological parameter that affects the mechanism of action of antibiotics in
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humans. We utilized FBS, as it serves as a growth factor for mammalian cells and might
interfere with the antitubercular action of some compounds.
Preliminary ADMET (abbreviation for absorption, distribution, metabolism, and
excretion) studies were performed for compound 8 using the following in vitro assays:
chemical stability assay, plasma protein binding assay, caco–2 permeability assay,
cytochrome P450 inhibition assay, and hepG2 cytotoxicity. Additionally, we
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characterized the mutagenicity of compound 8 through the micronucleus assay using the
mouse peripheral blood reticulocytes.
To ensure a greater stability and improved solubility, compound 8 was evaluated in an in
vivo assay using a pharmaceutical formulation. The nanostructured lipid system (ME)
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was synthesized as described by our group previously, with the following composition:
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0% oil phase (cholesterol), 10% surfactant (a mixture of soy phosphatidylcholine,
sodium oleate, and Eumulgin® HRE 40 [polyoxyl 40 castor oilꢀhydrogenated]; 3:6:8),
and 80% aqueous phase (phosphate buffer, pH 7.4). The compounds studied were
incorporated at the desired concentration for the in vivo experiments by mass
solubilization at the respective volume and sonicated for 3 min in the batch mode at 15%
amplitude. The ME containing compound 8 was analyzed for tolerability and oral
bioavailability following the treatment of female BALB/C mice infected with Mtb.
Microarrays have been used to define successfully the mechanism of action (MOA) of
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antitubercular compounds. Therefore, a microarray analysis was performed to obtain an
unbiased view of the MOA of compound 8.
In Vitro Antimycobacterial Activity
The ability of Mtb to remain dormant serves as the predominant factor that contributes to
precluding sterilization with antibiotic therapy and promotes the development of
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antibiotic resistance.
Therefore, an ideal antitubercular drug should (i) reduce the
duration of treatment; (ii) be active against resistant strains; (iii) not interfere with other
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Recently, our research group has identified the compound, (E)ꢀ4ꢀ(4ꢀ((2ꢀ
isonicotinoylhydrazono)methyl)phenoxy)ꢀ3ꢀ(phenylsulfonyl)ꢀ1,2,5ꢀoxadiazole 2ꢀoxide as
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6
a promising antitubercular drug candidate. To optimize its antimycobacterial activity,
we designed novel heterocyclic compounds containing Nꢀoxide analogs of the parent
compound, which comprised the following heterocyclic moieties: amide–furoxan (series
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1
, 4a–c), benzofuroxan (series 2, 8–17), and quinoxaline 1,4ꢀdiꢀNꢀoxide (series 3, 19–26,
28). The amide–furoxan derivatives (4a–c) were selected to evaluate whether the
replacement of the phenylsulfonyl group from the parent compound by an amide group
would increase the antitubercular activity and/or decrease the cytotoxicity. The three
amide–furoxan regioisomers (4a–c) displayed an improved antitubercular activity than
26
the parent compound. The compounds from series 1 exhibited MIC values around 0.4
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0
ꢁM against actively growing Mtb H Rv strain (Table 1).
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The benzofuroxan moiety (series 2, 8–17) was selected due to its possible ability to
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generate ROS after its metabolism. The structural design of the series under the study
was based on the isosteric replacement of substituents attached at the para position on the
phenyl ring. These included hydrogen, nitro, tertꢀbutyl, amino, and hydroxyl groups.
Furthermore, we evaluated the substitution of the phenyl ring by a pyridine ring owing to
the presence of heterocyclic pyridine ring in the structure of several antituberculosis
68
drugs and bioactive compounds, such as INH, BDQ, and ethionamide. As expected, the
replacement of the phenyl ring of compound 9 by a pyridine ring in compound 8 led to a
sevenꢀfold increase in the antituberculosis activity (MIC = 1.1 ꢁM, Table 1).
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0
Among the benzofuroxan series, our group could identify the compound, (E)ꢀ6ꢀ((2ꢀ
isonicotinoylhydrazono)methyl)benzo[c][1,2,5]oxadiazole 1ꢀoxide (8) as the lead
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benzofuroxan derivative with MIC values of 1.1 and 6.6 ꢁM for actively growing and
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dormant Mtb, respectively. The observed nearly equimolar effects against replicating and
nonꢀreplicating Mtb attributed to the small difference in MIC values is considered as an
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attractive characteristic. Such differences are rarely observed but highly desired and
beneficial.
Moreover, our data revealed that the presence of bulky groups, such as tertꢀbutyl (10,
MIC = 3.9 ꢁM) and electronꢀwithdrawing groups, such as nitro (11, MIC = 5.3 ꢁM),
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90
led to an improvement in antituberculosis activity of the analyzed compounds.
Compound 9, unꢀsubstituted phenyl, presented a MIC90 value of 8.3 ꢁM, while the
hydroxyl regioisomers (12–14) presented MIC90 values greater than 62 ꢁM. Amino
derivatives (15–17) also exhibited a reduction in the antitubercular activity in comparison
to the nonꢀsubstituted compounds 9; however, these compounds reported MIC values
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(
12.3– 27.9 ꢁM) lower than those containing a hydroxyl substitution (Table 1).
With respect to the quinoxaline, 1,4ꢀdiꢀNꢀoxide series, we evaluated the influence of
electronꢀwithdrawing and electronꢀdonating groups on the phenyl ring for its
antitubercular activity. We also performed an isosteric substitution of the phenyl ring by
furan (25) and thiophen (26) moieties. For quinoxalineꢀphenyl derivatives (19–24), the
MIC values ranged from 12.0 to 30.8 ꢁM, implying a contribution of the presence of
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substitution at the para position of the phenyl ring to the antituberculosis activity. Since
compound 19 was devoid of any substituents, it displayed a lower potency among the
phenyl quinoxaline derivatives with MIC = 30.8 ꢁM. On the other hand, compound 22,
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with a methoxyl group in the para position, displayed the lowest MIC value among the
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phenyl quinoxaline series. Nevertheless, we could not observe a clear and accurate
structure–activity relationship regarding the electronic properties of the substituents.
For compounds 25 and 26, the isosteric replacement of the phenyl group by a furan or
thiophen ring led to a significant increase in the antitubercular activity. Compounds 25
and 26 exhibited MIC90 values of 5.2 and 12.1 ꢁM, respectively. Furthermore, we
synthesized a quinoxaline derivative containing an Nꢀacylhydrazone subunit (28); the
antitubercular activity of this compound decreased in comparison to the previous
quinoxalines exhibiting MIC value of 39.7 ꢁM.
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9
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Further in Vitro Biological Profiling of Selected Compounds
We evaluated the cytotoxicity of the final compounds against MRC–5 cell lines. This cell
line is derived from healthy human lung fibroblasts and is widely utilized for the
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phenotypic screening of antitubercular drugs.
The data obtained in the cytotoxicity studies of the amide–furoxan series (4a–c) indicated
a high selectivity of these compounds against Mtb. All three regioisomers exhibited high
IC values (>854.0 ꢁM), thereby leading to high SI values, which ranged from 2,033 to
5
0
3
,205 (Table 1).
Regarding the cytotoxicity studies of the benzofuroxan series, we observed IC values
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ranging from 25 to 841 ꢁM in MRC–5 cell lines. The most active benzofuroxan
derivative (8) presented an IC value of 519 ꢁM; however, the compound 17 was found
50
to be less cytotoxic among the benzofuroxan series with an IC50 value of 841 ꢁM.
Compounds 11 to 14 did not display promising antitubercular activity (MIC > 62 ꢁM)
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and consequently were excluded from the cytotoxicity studies. Rather, compound 8 was
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selected for further experiments, as it possessed the highest potency against Mtb and the
lowest cytotoxicity.
Compounds from quinoxaline series were significantly more cytotoxic in MRC–5 cells
with their IC values ranging from 13 to 67 ꢁM, thereby resulting in low SI (<6.8) values
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and making them less attractive candidates as antitubercular agents.
The data obtained from MIC in different conditions (Table 2) depicted no significant
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differences between MIC90 under normal and others conditions. These conditions
included (a) adjusting the pH of the culture medium to 6.0; (b) including 4% BSA; and
(
c) supplementation of 10% FBS.
Antimicrobials are classified as containing narrow, intermediate, or broad spectrum of
activity. Tuberculosis is a chronic infection, the treatment of which requires a drug with
narrow spectrum, such as INH. All amide–furoxan and benzofuroxan (8, 11, 14, and 17)
derivatives were checked against S. aureus, E. coli, and C. albicans. No antimicrobial
activity was detected up to a maximum concentration of 200 ꢁM.
In terms of crossꢀresistance, compound 8 displayed equipotent activity (<twoꢀfold change
in MIC ) against all drugꢀsensitive and monoresistant strains of Mtb tested, suggesting a
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novel MOC or inhibition of a shared target distinct binding site by the compound (Table
). In contrast, amide–furoxan series (4a–c) was inactive against half of the
3
monoresistant strains tested and therefore was not selected for further studies.
We utilized J774A.1, a macrophage cell line, to study the intracellular inhibition of Mtb
using the drugs under analysis. Our group observed that compound 8 exhibited a high
intracellular inhibition at all concentrations tested (around 90%). However, similar to
RMP, we could not verify the doseꢀdependent inhibition at different concentrations
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(Figure 2). In timeꢀkill kinetic experiments, compound 8 was observed to be bactericidal
with an early bactericidal effect. Noteworthy, compound 8 could sterilize the cultures
after 48 h of exposure (about of 6.7 log10, Figure 3).
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Preliminary ADMT Studies of Compound 8
On the basis of the promising biological results described above for compound 8, we
conducted stability and ADMT studies to assess its drugꢀlike properties, such as
absorption, distribution, metabolism, and excretion. To analyze the chemical stability of
the compound, we conducted an in vitro assay under two pH conditions (5.5 and 7.4) to
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mimic the environment of a macrophage phagolysosome (pH 4.5–6.2)
and the neutral
plasma (pH 7.4). At pH 5.5 and 7.4, the compound 8 exhibited a good stability (Figure
4); however, a reduction of 20% was detected at both pH values after 10 h. Interestingly,
the concentration of compound 8 increased to 67 and 75% at pH 5.5 and 7.4, respectively,
after 24 h. The degradation rates of compound 8 were calculated by HPLCꢀUV; however,
we did not characterize the degradation products. These results indicated that the
compound 8 exhibited a high stability in the physiological pH range in the target areas of
bacteria, namely, blood and phagolysosome.
The in vitro ADMT properties for compound 8 are listed in Table 4. The benzofuroxan
derivative reported 46.5% unbound fraction when an in vitro plasma protein binding
assay was conducted. The findings of the study with compound 8 on the inhibition of
cytochrome P450 isoforms depicted no potential for inhibition, presenting IC50 values
greater than 15.0 µM in all isoforms tested. The studies in the Caco–2 cell line
demonstrated a good permeability profile. The cytotoxicity study conducted in HepG2
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cells reported an IC of 16.0 µM and an SI of 14.5. We also performed the micronucleus
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assay using mouse peripheral blood reticulocytes for compound 8 to evaluate its intrinsic
mutagenic activity. The results indicated that compound 8 was not genotoxic at all
concentrations tested (Figure 5).
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Tolerability, Oral Bioavailability, and Efficacy of Compound 8 in Mice
In vivo oral bioavailability, toxicology, infection, and treatment studies were performed
to ensure the safety and efficacy of compound 8.
For toxicology studies, the mice were monitored daily for 10 days, receiving one daily
oral dose (by gavage, 200 mg/kg body weight), and the behavior parameters (hippocratic
screening) were evaluated. No significant variation in the behavior of mice was observed
during the period of 10 days. Changes in the weight of organs (heart, lungs, spleen,
kidneys, and liver) were evaluated using analysis of variance (ANOVA) and Dunnett’s
test, establishing a p-value < 0.05 as the significant level. No statistically significant
difference was observed between the drugꢀtreated and control groups. To probe the
potential liver damage, the levels of liver transaminases were checked in the plasma that
reported no significant differences for alanine aminotransferase (Figure 6a), aspartate
aminotransferase (Figure 6b), and alkaline phosphatase (Figure 6c) between the treated
and control groups. Similarly, we evaluated the levels of urea in blood samples to assess
the potential changes in kidney function. Here, a significant difference was observed for
the group treated with RMPꢀME, when compared to the control group (Figure 6d). The
study on the oral bioavailability of compound 8 displayed an inhibition of Mtb growth in
mice plasma (Table 5).
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Histology of liver (Figure 7) and kidneys (Figure 8) revealed similar morphology in all
the groups, implying that no gross abnormalities were caused by the treatments.
The efficacy of compound 8 was analyzed by infecting the mice with Mtb H37Rv strain
followed by subjecting the infected animals to the treatment with compound 8 or vehicle.
The homogenized lung samples were plated at a dilution of 1:100 to 1:10,000, at which
no growth of Mtb colonies was observed. Therefore, the homogenized lung samples were
reꢀinoculated on agar plates in undiluted form and at 1:10 dilution; however, again, no
colony was observed (Figure 9). Control experiments with vehicles, RPM, and RPMꢀCE
behaved as expected (Figure 9).
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Mode of Action Studies
Although the antitubercular activity of the benzofuroxan derivative 8 was originally
attributed to its potential to generate and release nitric oxide (NO), we believe this to be
no longer the case, since the compound could not release NO in the Griess assay (a
chemical test to analyze nitrite ions in a solution, data not shown). An alternative
mechanism was proposed as mentioned below.
Microarray analysis of Mtb treated with compound 8 or vehicle control revealed a
significant upꢀregulation in the majority of ribosomal genes as well as all genes encoding
subunits of the ATP synthase (Figure 10 and Supplementary Table S1). This included
EFꢀG, which induces GTPꢀdependent translocation of nascent peptide chains from the Aꢀ
to the Pꢀsite in the ribosome and EFꢀTu, which promotes GTPꢀdependent binding of
aminoacylated tRNAs to the Aꢀsite in ribosomes. No upꢀregulation of heat shock proteins
was observed, which in fact demonstrated a downꢀregulation of hsp, htrA, and hspR
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(Supplementary Table S1). Similarly, upꢀregulation of ribosomal proteins was observed
upon treatment of Mtb with the inhibitors of protein synthesis. Our data indicated an upꢀ
regulation of a large number of operons as well as single genes encoding ribosomal
proteins, which in turn suggested that compound 8 could affect protein synthesis by
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inhibiting the ribosome. Boshoff and coworkers (2004) divided the protein synthesis
inhibitors into two classes on the basis of their effect on the expression of heat shock
proteins (either no effect or upꢀregulation). Compound 8 did not increase the abundance
of transcripts encoding heat shock proteins, and in fact, decreased the levels of three
transcripts (hsp, htrA, and hspR). The results of the study on the inhibition of protein
synthesis indicated compound 8 to behave more like an inhibitor of initiation of
translation, such as tetracyclines, rather than an inhibitor of protein synthesis that leads to
misꢀtranslation, such as aminoglycosides. Further studies are required to define the exact
MOA of compound 8, including its binding site and inhibition of the Mtb ribosome, ATP
synthase, or other targets.
CONCLUSIONS
Twentyꢀtwo new Nꢀoxide containing compounds were synthesized followed by in vitro
and in vivo evaluation of their antitubercular activity against Mtb. The amide–furoxan
series (4a–c) was observed to be the most promising compounds with MIC90 values
around 0.40 ꢁM against actively replicating Mtb and SI values ranging from 2033.3 to
3
204.7. The benzofuroxan series (8–17) also presented promising antitubercular activity,
especially compound 8, which reported MIC value of 1.1 and 6.6 ꢁM against actively
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growing and nonꢀreplicating Mtb, respectively. Compound 8 also displayed high activity
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in the macrophage model of infection. In addition, in vivo studies employing the mouse
model of infection demonstrated the sterilizing activity for compound 8. No detectable
Mtb was observed in the lungs of mice treated, while control mice displayed expected
number of colony forming units (CFUs). Altogether, these findings highlighted the
benzofuroxan derivative 8 as a novel lead compound for designing antitubercular drug
with sterilizing activity superior to rifampicin in the mouse model of infection.
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EXPERIMENTAL SECTION
Chemistry
Melting points (mp) were measured using an electrothermal melting point apparatus
(
SMP3; Bibby Stuart Scientific) or in a Mettler FP82+FP80 apparatus (Greifense,
Switzerland). Infrared spectroscopy (KBr disc) were performed on a FTIRꢀ8300
Shimadzu or a Nicolet Nexu FTIR Thermo® spectrometer, and the frequencies are
ꢀ1
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expressed in cm . The NMR for H and C of all compounds were performed on a
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Bruker Fourier with Dual probe C/ H (300ꢀMHz) NMR spectrometer or Bruker 400
TM 13
1
Ultrashield
C/ H (400ꢀMHz) NMR spectrometer using deuterated chloroform
(
CDCl ) or dimethyl sulfoxide (DMSOꢀd ) as solvent and the chemical shifts were
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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 microanalysis (C, H and N) was performed on a PerkinꢀElmer
model 2400 analyzer or a CHNꢀ900 Elemental Analyzer (LECO, Tres Cantos, Spain) and
+
the data were within ± 0.4% of the theoretical values. HRMS (ESI ) data were acquired
using a Bruker Maxis Impact® quadrupole time of flight tandem mass spectrometer (Qꢀ
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TOF MS/MS) and the mass spectra values are reported as m/z. The compounds were
purified on a chromatography column with silica gel (60 Å pore size, 35ꢀ75ꢀµm particle
size) and the following solvents were used as mobile phase: methanol, ethyl acetate,
dichloromethane, hexane and petroleum ether in a flow rate of approximately 20 mL/min.
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The reaction progress of all compounds was monitored by thinꢀlayer chromatography
2
(
TLC), which was performed on 2.0ꢀ by 6.0ꢀcm aluminum sheets precoated with silica
gel 60 (HFꢀ254; 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%. HPLC conditions: Shimadzu HPLC model CBM 20ꢀA (Shimadzu®)
equipped with UVꢀVIS detector (model SPDꢀ20A), quaternary pumping system mobile
phase (model LCꢀ20AT), solvent degasser (model DGUꢀ20As) and a Agilent® Eclipse
XDB Cꢀ18 column (250mm x 27 4,6mm; 5ꢁm). For HPLC method it was used an
isocratic flow [methanol:water (75:25)]. Reagents and solvents were purchased from
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commercial suppliers. Compounds 3aꢀc,
7, 18, 19 and 28 were prepared according
to previously described methods.
General procedure for the synthesis of compounds 4aꢀc and 8ꢀ17
A solution of compound 3aꢀc or 7 (0.87 mmol) in 10 mL of ethanol and 3 drops of glacial
acetic acid was stirred at room temperature for 20 min. Next, the appropriate aromatic
hydrazide (0.106 g, 0.87 mmol) was added and the mixture was stirred for 12 h. The
solvent was concentrated under reduced pressure and ice was added in order to precipitate
the desired products. If necessary, the samples could be further purified by silica gel
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column chromatography using ethyl acetateꢀmethanol (98:2 v/v) as eluent to give the
appropriate compound 4aꢀc, and 8ꢀ17 with variable yields.
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(E)ꢀ3ꢀcarbamoylꢀ4ꢀ((2ꢀ((2ꢀisonicotinoylhydrazono)methyl)phenoxy)methyl)ꢀ1,2,5ꢀ
ꢀ
1
oxadiazole 2ꢀoxide (4a). White powder; yield, 51%; mp, 226 to 229°C. IR Vmax (cm ;
KBr pellets): 3433 (NH amide), 3203 (NꢀH), 1669 (C=O amide), 1587 (C=N imine),
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1
1456 (NꢀO furoxan), 1344 (CꢀN aromatic), 1145 (CꢀO ether). H NMR (300 MHz,
DMSOꢀd , δ ppm) δ: 12.10 (1H; s; NꢀH), 8.79 (2H; d;), 8.51 (1H; s), 7.91 (1H; dd), 7.84
6
13
(
2H; d), 7.46 (1H; m), 7.25 (1H, d), 7.11 (1H, t), 5.54 (2H; s) ppm. C NMR (75 MHz,
DMSOꢀd , δ ppm) δ: 161.55, 156.36, 155.65, 155.24, 150.30, 144.04, 140.47, 131.84,
6
1
25.85, 122.59, 121.87, 121.59, 113.36, 110.51 ppm. Anal. Calcd. (%) for C H N O :
16
12
6
5
+
C: 52.18; H: 3.28; N: 22.82. Found: C: 52.25; H: 3.29; N: 22.90. HRMS: m/z (ESI ) =
+
4
05.0918 [M + Na] .
(E)ꢀ3ꢀcarbamoylꢀ4ꢀ((3ꢀ((2ꢀisonicotinoylhydrazono)methyl)phenoxy)methyl)ꢀ1,2,5ꢀ
ꢀ1
oxadiazole 2ꢀoxide (4b). White powder; yield, 59%; mp, 159 to 163°C. IR Vmax (cm ;
KBr pellets): 3439 (NH amide), 3201 (NꢀH), 1712 (C=O amide), 1591 (C=N imine),
2
1
1485 (NꢀO furoxan), 1327 (CꢀN aromatic), 1147 (CꢀO ether). H NMR (300 MHz,
DMSOꢀd , δ ppm) δ: 12.09 (1H; s; NꢀH), 8.78 (2H; d), 8.46 (1H; s), 7.81 (3H; d), 7.41
6
1
3
(
3H; m), 5.48 (2H; s) ppm. C NMR (75 MHz, DMSOꢀd , δ ppm) δ: 156.96, 150.22,
6
147.97, 147.44, 142.63, 140.97, 132.70, 127.87, 122.48, 113.82, 113.14, 109.30, 105.24,
1
02.74 ppm. Anal. Calcd. (%) for C H N O : C: 52.18; H: 3.28; N: 22.82. Found: C:
16 12 6 5
+
+
5
2.27; H: 3.29; N: 22.90. HRMS: m/z (ESI ) = 405.0922 [M + Na] .
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(
E)ꢀ3ꢀcarbamoylꢀ4ꢀ((4ꢀ((2ꢀisonicotinoylhydrazono)methyl)phenoxy)methyl)ꢀ1,2,5ꢀ
ꢀ1
oxadiazole 2ꢀoxide (4c). White powder; yield, 67%; mp, 235 to 239°C. IR Vmax (cm ;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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KBr pellets): 3.367 (NH amide), 3255 (NꢀH), 1708 (C=O amide), 1612 (C=N imine),
2
1
1
413 (NꢀO furoxan), 1255 (CꢀN aromatic), 1122 (CꢀO ether). H NMR (300 MHz,
DMSOꢀd , δ ppm) δ: 11.95 (1H; s; NꢀH), 8.77 (2H; d;), 8.43 (1H; s), 7.80 (2H; d), 7.71
6
1
3
(2H; d), 7.14 (2H; d), 5.48 (2H; s) ppm. C NMR (75 MHz, DMSOꢀd , δ ppm) δ:
6
1
1
5
62.96, 162.33, 159.91, 156.34, 150.86, 149.52, 141.14, 129.65, 128.08, 122.14, 115.84,
11.05 ppm. Anal. Calcd. (%) for C H N O : C: 52.18; H: 3.28; N: 22.82. Found: C:
16 12
6
5
+
+
2.20; H: 3.28; N: 22.84. HRMS: m/z (ESI ) = 405.0913 [M + Na] .
(E)ꢀ6ꢀ((2ꢀisonicotinoylhydrazono)methyl)benzo[c][1,2,5]oxadiazole
1ꢀoxide
(8).
ꢀ1
Yellow powder; yield, 85%; mp, 230 to 232 °C. IR Vmax (cm ; KBr pellets): 3324 (NꢀH),
3
045 (NH amide), 1690 (C=O amide), 1600 (C=N imine), 1409 (NꢀO furoxan), 1273 (Cꢀ
2
1
N aromatic). H NMR (300 MHz, DMSOꢀd , δ ppm) δ: 12.44 (1H; s), 8.80 (2H; d; J =
6
13
5
.7 Hz), 8.51 (1H; s), 8.03 (3H; m), 7.84 (2H; d; J = 6.0 Hz) ppm. C NMR (75 MHz,
DMSOꢀd , δ ppm) δ: 162.4, 150.6, 146.6, 140.2, 133.7, 130.8, 130.4, 130.1, 129.4, 121.8,
6
1
14.3 ppm. Anal. Calcd. (%) for C H N O : C: 55.13; H: 3.20; N: 24.73. Found: C:
13 9 5 3
+
+
5
5.22; H: 3.21; N: 24.78. HRMS: m/z (ESI ) = 306.0602 [M + Na] .
(
E)ꢀ6ꢀ((2ꢀbenzoylhydrazono)methyl)benzo[c][1,2,5]oxadiazole 1ꢀoxide (9). Yellow
ꢀ1
powder; yield, 80%; mp, 220 to 221 °C. IR Vmax (cm ; KBr pellets): 3389 (NꢀH), 3041
(NH amide), 1641 (C=O amide), 1533 (C=N imine), 1487 (NꢀO furoxan), 1300 (CꢀN
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aromatic). H NMR (300 MHz, DMSOꢀd , δ ppm) δ: 12.24 (1H; s), 8.52 (1H; s), 7.94
6
1
3
(
5H; m), 7.58 (3H; m) ppm. C NMR (75 MHz, DMSOꢀd , δ ppm) δ: 163.4, 145.0,
6
1
32.9, 132.1, 128.5, 127.8, 118.4, 113.5 ppm. Anal. Calcd. (%) for C H N O : C:
14 10 4 3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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26
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56
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58
59
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59.57; H: 3.57; N: 19.85. Found: C: 59.65; H: 3.56; N: 19.88.
(E)ꢀ6ꢀ((2ꢀ(4ꢀ(tertꢀbutyl)benzoyl)hydrazono)methyl)benzo[c][1,2,5]oxadiazole 1ꢀoxide
ꢀ
1
(10). Yellow powder; yield, 89%; mp, 189 to 191 °C. IR Vmax (cm ; KBr pellets): 3341
(
NꢀH), 3068 (NH amide), 1659 (C=O amide), 1532 (C=N imine), 1486 (NꢀO furoxan),
2
1
1
287 (CꢀN aromatic). H NMR (300 MHz, DMSOꢀd , δ ppm) δ: 12.16 (1H; s), 8.51 (1H;
6
1
3
s), 7.93 (4H; m), 7.56 (3H; d; J = 8.4 Hz), 1.32 (9H; s) ppm. C NMR (75 MHz, DMSOꢀ
d6, δ ppm) δ: 160.3, 155.0, 144.7, 130.2, 127.7, 125.3, 118.3, 113.3, 56.0, 34.8, 30.9 ppm.
Anal. Calcd. (%) for C H N O : C: 63.89; H: 5.36; N: 16.56. Found: C: 63.95; H: 5.37;
1
8
18
4
3
N: 16.59.
(E)ꢀ6ꢀ((2ꢀ(4ꢀnitrobenzoyl)hydrazono)methyl)benzo[c][1,2,5]oxadiazole 1ꢀoxide (11).
ꢀ1
Yellow powder; yield, 78%; mp, 261 to 262 °C. IR Vmax (cm ; KBr pellets): 3373 (NꢀH),
3
073 (NH amide), 1673 (C=O amide), 1525 (C=N imine), 1400 (NꢀO furoxan), 1357 (Cꢀ
2
1
N aromatic). H NMR (300 MHz, DMSOꢀd , δ ppm) δ: 12.50 (1H; s), 8.52 (1H; s), 8.39
6
13
(2H; d; J = 8.8 Hz), 8.17 (2H; d; J = 8.8 Hz), 7.95 (3H; m) ppm. C NMR (75 MHz,
DMSOꢀd , δ ppm) δ: 162.2, 149.4, 146.2, 138.7, 129.4, 123.7 ppm. Anal. Calcd. (%) for
6
C H N O : C: 51.38; H: 2.77; N: 21.40. Found: C: 51.41; H: 2.77; N: 21.42.
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(E)ꢀ6ꢀ((2ꢀ(2ꢀhydroxybenzoyl)hydrazono)methyl)benzo[c][1,2,5]oxadiazole
1ꢀoxide
ꢀ1
(
12). Yellow powder; yield, 79%; mp, 240 to 241 °C. IR Vmax (cm ; KBr pellets): 3573
(
OꢀH), 3332 (NꢀH), 3100 (NH amide), 1638 (C=O amide), 1532 (C=N imine), 1484 (Nꢀ
2
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0
1
O furoxan), 1341 (CꢀN aromatic). H NMR (300 MHz, DMSOꢀd , δ ppm) δ: 12.12 (1H;
6
s), 11.64 (1H; s), 8.51 (1H; s), 7.95 (4H; m), 7.45 (1H; t; J = 7.5 Hz), 6.99 (2H; m) ppm.
1
3
C NMR (75 MHz, DMSOꢀd , δ ppm) δ: 164.8, 158.7, 145.7, 134.0, 129.0, 119.1, 118.4,
6
117.3, 116.3, 113.7 ppm. Anal. Calcd. (%) for C H N O : C: 56.38; H: 3.38; N: 18.79.
1
4
10
4
4
Found: C: 56.41; H: 3.37; N: 18.80.
(E)ꢀ6ꢀ((2ꢀ(3ꢀhydroxybenzoyl)hydrazono)methyl)benzo[c][1,2,5]oxadiazole
1ꢀoxide
ꢀ1
(
13). Yellow powder; yield, 81%; mp, 265 to 266 °C. IR Vmax (cm ; KBr pellets): 3578
(OꢀH), 3309 (NꢀH), 3118 (NH amide), 1643 (C=O amide), 1532 (C=N imine), 1371 (Nꢀ
2
1
O furoxan), 1293 (CꢀN aromatic). H NMR (300 MHz, DMSOꢀd , δ ppm) δ: 12.15 (1H;
6
s), 9.80 (1H; s), 8.50 (1H; s), 7.99 (3H; m), 7.33 (3H; m), 7.00 (1H; d; J = 6.9 Hz) ppm.
1
3
C NMR (75 MHz, DMSOꢀd , δ ppm) δ: 163.4, 157.4, 144.9, 134.3, 129.6, 119.0, 118.3,
6
1
14.6, 113.3 ppm. Anal. Calcd. (%) for C H N O : C: 56.38; H: 3.38; N: 18.79. Found:
1
4
10
4
4
C: 56.41; H: 3.38; N: 18.82.
(
(
(
E)ꢀ6ꢀ((2ꢀ(4ꢀhydroxybenzoyl)hydrazono)methyl)benzo[c][1,2,5]oxadiazole
1ꢀoxide
ꢀ1
14). Yellow powder; yield, 85%; mp, 280 to 281 °C. IR Vmax (cm ; KBr pellets): 3573
OꢀH), 3332 (NꢀH), 3096 (NH amide), 1638 (C=O amide), 1523 (C=N imine), 1371 (Nꢀ
2
1
O furoxan), 1280 (CꢀN aromatic). H NMR (300 MHz, DMSOꢀd , δ ppm) δ: 12.02 (1H;
6
s), 10.21 (1H; s), 8.47 (1H; s), 8.01 (2H; m), 7.83 (2H; d; J = 8.7 Hz), 6.87 (3H; d; J = 8.7
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8
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3
Hz) ppm. C NMR (75 MHz, DMSOꢀd , δ ppm) δ: 163.0, 161.0, 152.8, 144.0, 136.3,
6
1
5
30.0, 128.8, 123.4, 118.2, 115.1, 114.8 ppm. Anal. Calcd. (%) for C H N O : C:
14 10 4 4
6.38; H: 3.38; N: 18.79. Found: C: 56.39; H: 3.39; N: 18.78.
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11
12
13
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(
E)ꢀ6ꢀ((2ꢀ(2ꢀaminobenzoyl)hydrazono)methyl)benzo[c][1,2,5]oxadiazole 1ꢀoxide (15).
ꢀ1
Yellow powder; yield, 77%; mp, 193 to 194 °C. IR Vmax (cm ; KBr pellets): 3501 (NH
2
amine), 3319 (NꢀH), 3100 (NH amide), 1668 (C=O amide), 1536 (C=N imine), 1477 (Nꢀ
2
1
O furoxan), 1346 (CꢀN aromatic). H NMR (300 MHz, DMSOꢀd , δ ppm) δ: 11.98 (1H;
6
s), 8.44 (1H; s), 7.93 (3H; m), 7.59 (1H; d; J = 6.7 Hz), 7.22 (1H; t; J = 7.7 Hz), 6.76
1
3
(1H; d; J = 8.3 Hz), 6.58 (1H; d; J = 7.0 Hz), 6.45 (2H; s) ppm. C NMR (75 MHz,
DMSOꢀd , δ ppm) δ: 165.6, 152.8, 150.3, 143.8, 132.6, 128.5, 118.2, 116.5, 114.6, 112.7
6
ppm. Anal. Calcd. (%) for C H N O : C: 56.57; H: 3.73; N: 23.56. Found: C: 56.64; H:
14
11
5
3
3
.74; N: 23.58.
(E)ꢀ6ꢀ((2ꢀ(3ꢀaminobenzoyl)hydrazono)methyl)benzo[c][1,2,5]oxadiazole 1ꢀoxide (16).
ꢀ1
Yellow powder; yield, 82%; mp, 213 to 215 °C. IR Vmax (cm ; KBr pellets): 3501 (NH
2
amine), 3232 (NꢀH), 3100 (NH amide), 1638 (C=O amide), 1532 (C=N imine), 1416 (Nꢀ
2
1
O furoxan), 1312 (CꢀN aromatic). H NMR (300 MHz, DMSOꢀd , δ ppm) δ: 12.31 (1H;
6
s), 8.86 (1H; s), 8.53 (1H; s), 8.10 (3H; m), 7.87 (3H; s), 7.64 (1H; t; J = 7.9 Hz), 7.57
1
3
(1H; d; J = 7.3 Hz) ppm. C NMR (75 MHz, DMSOꢀd , δ ppm) δ: 162.9, 159.6, 150.5,
6
1
48.9, 145.2, 134.1, 129.7, 128.9, 126.3, 124.6, 120.7, 114.7 ppm. Anal. Calcd. (%) for
C H N O : C: 56.57; H: 3.73; N: 23.56. Found: C: 56.59; H: 3.73; N: 23.59.
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(E)ꢀ6ꢀ((2ꢀ(4ꢀaminobenzoyl)hydrazono)methyl)benzo[c][1,2,5]oxadiazole 1ꢀoxide (17).
ꢀ
1
Yellow powder; yield, 84%; mp, 218 to 200 °C. IR Vmax (cm ; KBr pellets): 3478 (NH
2
amine), 3319 (NꢀH), 3118 (NH amide), 1627 (C=O amide), 1500 (C=N imine), 1380 (Nꢀ
2
0
1
2
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0
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8
9
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4
5
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7
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9
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1
O furoxan), 1252 (CꢀN aromatic). H NMR (300 MHz, DMSOꢀd , δ ppm) δ: 8.44 (1H; s),
6
13
7
.92 (3H; m), 7.69 (2H; d; J = 8.6 Hz), 6.60 (2H; d; J = 8.6 Hz), 5.87 (2H; s) ppm. C
NMR (75 MHz, DMSOꢀd , δ ppm) δ: 165.3, 152.6, 142.9, 129.7, 118.9, 112.6 ppm.
6
Anal. Calcd. (%) for C H N O : C: 56.57; H: 3.73; N: 23.56. Found: C: 56.58; H: 3.74;
1
4
11
5
3
N: 23.57.
General procedure for the synthesis of compounds 20ꢀ26
Compound (18) (0.3 g; 1.06 mmol) was dissolved in dichloromethane (15 mL) and then
cooled by placing it on ice bath. Next, was added the appropriate nitrile (0.15 g; 1.06
mmol) and potassium carbonate (0.18g; 1.32 mmol) in small portions. The reaction
mixture was stirred at 40 ºC for 96 hours. After the reaction time, the solvent was
evaporated under reduced pressure and the obtained solid was dissolved in 50 mL of
ethyl acetate and washed with water. The organic phase was dried with anhydrous
magnesium sulfate and the solvent was evaporated giving a yellow solid. The obtained
solid was purified by silica gel column chromatography using hexane and ethyl acetate
(
70:30 v/v) as eluent to give the appropriate compound 20ꢀ26 with variable yields.
3ꢀcyanoꢀ6ꢀ(1,3ꢀdioxolanꢀ2ꢀyl)ꢀ2ꢀ(pꢀtolyl)quinoxaline 1,4ꢀdioxide (20). Yellow powder;
ꢀ1
yield, 9%; mp, 179 to 180 °C. IR Vmax (cm ; KBr pellets): 3090 (CꢀH aromatic), 2233
1
(
CN nitrile), 1328 (NꢀO), 1073 (CꢀO ether). H NMR (400 MHz, DMSOꢀd , δ ppm) δ:
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8.55 (2H; t; J = 18.7 Hz), 8.12 (1H; d; J = 8.8 Hz), 7.63 (2H; d; J = 8.0 Hz), 7.43 (2H; d;
1
3
J = 7.9 Hz), 6.10 (1H; s), 4.09 (4H; m), 2.43 (3H; s) ppm. C NMR (75 MHz, DMSOꢀd ,
6
δ ppm) δ: 143.1, 143.0, 141.1, 136.7, 132.2, 130.1, 129.2, 129.1, 124.7, 121.0, 117.7,
101.1, 65.3, 21.1 ppm. Anal. Calcd. (%) for C H N O : C: 65.32; H: 4.33; N: 12.03.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
9
15
3
4
Found: C: 65.36; H: 4.32; N: 12.05.
2ꢀ(4ꢀchlorophenyl)ꢀ3ꢀcyanoꢀ6ꢀ(1,3ꢀdioxolanꢀ2ꢀyl)quinoxaline 1,4ꢀdioxide (21). Yellow
ꢀ1
powder; yield, 13%; mp, 177 to 178 °C. IR Vmax (cm ; KBr pellets): 3091 (CꢀH
1
aromatic), 2235 (CN nitrile), 1330 (NꢀO), 1093 (CꢀO ether). H NMR (400 MHz,
DMSOꢀd , δ ppm) δ: 8.56 (2H; t; J = 16.0 Hz), 8.12 (1H; dd; J = 26.5 Hz), 7.75 (4H; q; J
6
13
=
25.4 Hz), 6.11 (1H; s), 4.09 (4H; m) ppm. C NMR (75 MHz, DMSOꢀd , δ ppm) δ:
6
144.8, 142.1, 138.8, 137.1, 136.0, 132.1, 130.8, 128.8, 126.5, 121.1, 117.7, 65.3 ppm.
Anal. Calcd. (%) for C H ClN O : C: 58.47; H: 3.27; N: 11.36. Found: C: 58.67; H:
1
8
12
3
4
3
.36; N: 11.53.
3
ꢀcyanoꢀ6ꢀ(1,3ꢀdioxolanꢀ2ꢀyl)ꢀ2ꢀ(4ꢀmethoxyphenyl)quinoxaline 1,4ꢀdioxide (22).
ꢀ1
Yellow powder; yield, 26%; mp, 151 to 152 °C. IR Vmax (cm ; KBr pellets): 3085 (CꢀH
1
aromatic), 2236 (CN nitrile), 1335 (NꢀO), 1095 (CꢀO ether). H NMR (400 MHz,
DMSOꢀd , δ ppm) δ: 8.55 (2H; m), 8.05 (1H; dd), 7.71 (2H; d; J = 8.8 Hz), 7.17 (2H; d; J
6
1
3
=
8.7 Hz), 6.09 (1H; s), 4.08 (4H; m), 3.87 (3H; s) ppm. C NMR (75 MHz, DMSOꢀd , δ
6
ppm) δ: 161.2, 144.6, 142.9, 138.8, 132.4, 132.0, 120.6, 119.3, 118.0, 117.7, 113.9,
1
01.1, 65.2, 55.4 ppm. Anal. Calcd. (%) for C H N O : C: 62.46; H: 4.14; N: 11.50.
19 15 3 5
Found: C: 62.51; H: 4.15; N: 11.52.
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ꢀcyanoꢀ6ꢀ(1,3ꢀdioxolanꢀ2ꢀyl)ꢀ2ꢀ(4ꢀfluorophenyl)quinoxaline 1,4ꢀdioxide (23). Yellow
ꢀ
1
powder; yield, 30%; mp, 164 to 165 °C. IR Vmax (cm ; KBr pellets): 3077 (CꢀH
0
1
2
3
4
5
6
7
8
9
0
1
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0
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7
8
9
0
1
aromatic), 2234 (CN nitrile), 1330 (NꢀO), 1097 (CꢀO ether). H NMR (400 MHz,
DMSOꢀd , δ ppm) δ: 8.56 (2H; t; J = 17.5 Hz), 8.15 (1H; d; J = 10.4 Hz), 7.81 (2H; t; J =
6
1
3
1
4.1 Hz), 7.49 (2H; t; J = 17.7 Hz), 6.10 (1H; s), 4.09 (4H; m) ppm. C NMR (75 MHz,
DMSOꢀd , δ ppm) δ: 162.2, 143.2, 142.3, 139.1, 136.7, 133.0, 132.9, 132.3, 124.0, 121.0,
6
1
1
17.7, 115.7, 101.0, 65.3 ppm. Anal. Calcd. (%) for C H FN O : C: 61.19; H: 3.42; N:
18 12 3 4
1.89. Found: C: 61.18; H: 3.41; N: 11.90.
3
ꢀcyanoꢀ6ꢀ(1,3ꢀdioxolanꢀ2ꢀyl)ꢀ2ꢀ(4ꢀ(trifluoromethoxy)phenyl)quinoxaline 1,4ꢀdioxide
ꢀ
1
(24). Yellow powder; yield, 3%; mp, 186 to 187 °C. IR Vmax (cm ; KBr pellets): 3077
1
(
CꢀH aromatic), 2236 (CN nitrile), 1333 (NꢀO), 1165 (CꢀO ether). H NMR (400 MHz,
DMSOꢀd , δ ppm) δ: 8.57 (2H; t; J = 15.4 Hz), 8.15 (1H; d; J = 8.9 Hz), 7.89 (2H; d; J =
6
1
3
8.7 Hz), 7.66 (2H; d; J = 8.4 Hz), 6.11 (1H; s), 4.10 (4H; m) ppm. C NMR (75 MHz,
DMSOꢀd , δ ppm) δ: 149.9, 143.3, 141.9, 139.1, 136.8, 132.7, 132.4, 131.0, 126.7, 121.3,
6
1
21.0, 120.9, 117.7, 101.0, 65.3 ppm. Anal. Calcd. (%) for C H F N O : C: 54.42; H:
19 12 3 3 5
2
.88; N: 10.02. Found: C: 54.47; H: 2.88; N: 10.06.
3
ꢀcyanoꢀ6ꢀ(1,3ꢀdioxolanꢀ2ꢀyl)ꢀ2ꢀ(furanꢀ2ꢀyl)quinoxaline 1,4ꢀdioxide (25). Orange
ꢀ1
powder; yield, 26%; mp, 177 to 179 °C. IR Vmax (cm ; KBr pellets): 3087 (CꢀH
1
aromatic), 2240 (CN nitrile), 1355 (NꢀO), 1107 (CꢀO ether). H NMR (300 MHz,
DMSOꢀd , δ ppm) δ: 8.57 (1H; dd; J = 5.2, 3.6 Hz), 8.49 (1H; d; J = 8.9 Hz), 8.24 (2H; d;
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J = 3.5 Hz), 8.01 (1H; dd; J = 8.9, 1.6 Hz), 6.95 (1H; dd; J = 3.6, 1.8 Hz), 6.08 (1H; d; J
1
3
=
3.1 Hz), 4.09 (4H; m) ppm. C NMR (75 MHz, DMSOꢀd , δ ppm) δ: 146.8, 144.7,
6
1
42.3, 140.2, 138.5, 135.6, 133.4, 132.3, 130.0, 120.9, 117.7, 116.8, 113.2, 111.1, 101.2,
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65.3 ppm. Anal. Calcd. (%) for C H N O : C: 59.08; H: 3.41; N: 12.92. Found: C:
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6
11
3
5
5
9.12; H: 3.42; N: 12.94.
3ꢀcyanoꢀ6ꢀ(1,3ꢀdioxolanꢀ2ꢀyl)ꢀ2ꢀ(thiophenꢀ2ꢀyl)quinoxaline 1,4ꢀdioxide (26). Brown
ꢀ1
powder; yield, 15%; mp, 171 to 173 °C. IR Vmax (cm ; KBr pellets): 3092 (CꢀH
1
aromatic), 2241 (CN nitrile), 1357 (NꢀO), 1084 (CꢀO ether). H NMR (300 MHz,
DMSOꢀd , δ ppm) δ: 8.60 (1H; dd; J = 5.3, 3.6 Hz), 8.51 (1H; d; J = 8.8 Hz), 8.45 (1H;
6
dt; J = 4.2, 1.2 Hz), 8.13 (1H; dd; J = 5.1, 1.0 Hz), 8.03 (1H; dd; J = 8.9, 1.7 Hz), 7.43
1
3
(1H; dd; J = 5.0, 4.2 Hz), 6.09 (1H; d; J = 3.8 Hz), 4.09 (4H; m) ppm. C NMR (75
MHz, DMSOꢀd , δ ppm) δ: 145.1, 142.5, 138.1, 137.7, 136.8, 135.6, 132.5, 130.2, 127.0,
6
1
26.0, 120.7, 117.9, 112.6, 101.2, 65.1 ppm. Anal. Calcd. (%) for C H N O S: C:
16 11 3 4
56.30; H: 3.25; N: 12.31. Found: C: 56.31; H: 3.25; N: 12.30.
REFERENCES
(1)
World Health Organization. Global Tuberculosis Report 2016; 2016.
World Health Organization. Global Tuberculosis Report 2013; 2013.
(
2)
3)
(
Rustad, T. R.; Harrell, M. I.; Liao, R.; Sherman, D. R. The Enduring Hypoxic
Response of Mycobacterium Tuberculosis. PLoS One 2008, 3 (1), e1502.
(4)
Patel, K.; Jhamb, S. S.; Singh, P. P. Models of Latent Tuberculosis: Their Salient
Features, Limitations, and Development. J. Lab. Physicians 2011, 3 (2), 75–79.
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