Benzofuroxan Derivatives as Potent Agents against Multidrug-Resistant Mycobacterium tuberculosis

Article abstract of DOI:10.1002/cmdc.202000899

Tuberculosis (TB) is currently the leading cause of death related to infectious diseases worldwide, as reported by the World Health Organization. Moreover, the increasing number of multidrug-resistant tuberculosis (MDR-TB) cases has alarmed health agencies, warranting extensive efforts to discover novel drugs that are effective and also safe. In this study, 23 new compounds were synthesized and evaluated in vitro against the drug-resistant strains of M. tuberculosis. The compound 6-((3-fluoro-4-thiomorpholinophenyl)carbamoyl)benzo[c][1,2,5]oxadiazole 1-N-oxide (5 b) was particularly remarkable in this regard as it demonstrated MIC₉₀ values below 0.28 μM against all the MDR strains evaluated, thus suggesting that this compound might have a different mechanism of action. Benzofuroxans are an attractive new class of anti-TB agents, exemplified by compound 5 b, with excellent potency against the replicating and drug-resistant strains of M. tuberculosis.

Full text of DOI:10.1002/cmdc.202000899

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Very Important Paper
Benzofuroxan Derivatives as Potent Agents against
Multidrug-Resistant Mycobacterium tuberculosis
Guilherme F. S. Fernandes+,*[a, b] Débora L. Campos+,[a] Isabel C. Da Silva,[a]
João L. B. Prates,[a, b] Aline R. Pavan,[a, b] Fernando R. Pavan,*[a] and Jean L. Dos Santos*[a, b]
Tuberculosis (TB) is currently the leading cause of death related
to infectious diseases worldwide, as reported by the World
Health Organization. Moreover, the increasing number of multi-
drug-resistant tuberculosis (MDR-TB) cases has alarmed health
agencies, warranting extensive efforts to discover novel drugs
that are effective and also safe. In this study, 23 new
compounds were synthesized and evaluated in vitro against the
drug-resistant strains of M. tuberculosis. The compound 6-((3-
fluoro-4-thiomorpholinophenyl)carbamoyl)benzo[c][1,2,5]
oxadiazole 1-N-oxide (5b) was particularly remarkable in this
regard as it demonstrated MIC90 values below 0.28 μM against
all the MDR strains evaluated, thus suggesting that this
compound might have a different mechanism of action.
Benzofuroxans are an attractive new class of anti-TB agents,
exemplified by compound 5b, with excellent potency against
the replicating and drug-resistant strains of M. tuberculosis.
Introduction
second-line injectable drug] strains.[7,8] According to the WHO’s
latest report, there were an estimated 465000 incident cases of
MDR-TB and 214000 deaths due to MDR-TB in 2019. The cases
were mainly concentrated in three countries: India (27%), China
(14%), and the Russian Federation (8%).[3] The emergence of
totally drug-resistant TB strains (TDR-TB)[9,10] has worsened the
general scenario concerning the TB disease. TDR-TB refers to
those M.tb clinical strains that exhibit in vitro resistance to all
first- and second-line drugs tested.[11] This situation of TDR-TB is
critical, as there is a lack of drugs that can be used against the
MDR strains. Although the WHO does not use the term TDR-TB
specifically, such cases continue to appear[12] across the world,
mainly in Africa, Eastern Europe, China, and India.[13–15]
Tuberculosis (TB) is one of the deadliest diseases affecting
humankind. Historical evidence suggests that the common
ancestor of all the modern members of the Mycobacterium
tuberculosis (M.tb) family might have first appeared around
35000–15000 years ago.[1,2] M.tb is the main causative agent of
tuberculosis in humans. According to the World Health
Organization (WHO), TB was the leading cause of deaths due to
infectious diseases in 2019, among which 1.4 million deaths
occurred in HIV-negative people and 251000 deaths occurred
in HIV-positive people. This scenario is alarming, as in 2019,
around 10 million new cases of TB were reported.[3]
Even though the number of confirmed deaths due to TB has
been decreasing in recent years,[4–6] the scientific community
and the health agencies around the world are particularly
concerned regarding the high and rising numbers of multidrug-
resistant tuberculosis [MDR-TB; defined as resistant to at least
rifampicin (RIF) and isoniazid (INH)] and extensively drug-
resistant tuberculosis [XDR-TB; defined as MDR plus an addi-
tional resistance to at least one fluoroquinolone and one
In view of the worrying number of cases of this disease, the
WHO finally stated that “without novel anti-M.tb drugs and
regimens, it would be quite difficult to improve treatment
outcomes of this disease in the near future”; this highlights the
important role of the research and development aimed at
identifying novel drugs to fight against this disease.[4] The
current therapeutic regimen recommended by the WHO for the
treatment of TB involves the use of a series of drugs classified
as the first- and second-line treatment. The first-line drugs
include INH, RIF, ethambutol (EMB), and pyrazinamide (PZA),
and the second-line drugs include fluoroquinolones, amino-
glycosides, d-cycloserine (DCS), linezolid (LZD), and bedaquiline
(BDQ), among others.[7,16,17] However, a considerable number of
these drugs were developed several years ago and, therefore,
present certain limitations, such as prolonged standard regi-
men, high rate of treatment discontinuation, adverse effects,
toxicity, drug–drug interactions, and lack of effectiveness
against the most-resistant strains of Mycobacteria.[18–23]
[a] Dr. G. F. S. Fernandes,+ Dr. D. L. Campos,+ Dr. I. C. Da Silva,
Dr. J. L. B. Prates, Prof. A. R. Pavan, Prof. F. R. Pavan, Prof. J. L. Dos Santos
School of Pharmaceutical Sciences
São Paulo State University (UNESP)
Araraquara Jaú Highway KM 01, 14800903 Araraquara (Brazil)
E-mail: jean.santos@unesp.br
fernando.pavan@unesp.br
guilhermefelipe@outlook.com
[b] Dr. G. F. S. Fernandes,+ Dr. J. L. B. Prates, Prof. A. R. Pavan,
Prof. J. L. Dos Santos
Institute of Chemistry
São Paulo State University (UNESP)
Francisco Degni Street 55, 14800060 Araraquara (Brazil)
Although the investment in the research and development
of novel drugs against TB is far from the $2 billion a year
recommended as necessary by “The End TB Strategy” to
eliminate the disease by 2030,[24] important advances have been
achieved in recent years. After a period of over 50 years in
which no novel drugs were approved for TB, three novel drugs
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000899
This article belongs to the Special Collection “BrazMedChem 2019: Medicinal
Chemistry in Latin America”.
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have recently been approved by the regulatory agencies
(Figure 1). One of these drugs is bedaquiline (Sirturo®; Janssen
Therapeutics), which was first approved in 2012 by the U.S.
Food and Drug Administration (FDA) for the treatment of MDR-
TB. The mechanism of action of bedaquiline involves the energy
metabolism of replicating and nonreplicating mycobacteria.[25]
The other two approved drugs, delamanid and pretomanid, are
representatives of the nitroimidazole class. Delamanid (Deltyba®;
Otsuka Pharmaceutical) was first approved in 2014 by the
decided to continue the drug development process to find
ways to overcome the chemical instability and improving the
potency of the compound against the MDR-M.tb strains. There-
fore, in an attempt to identify novel anti-M.tb compounds based
on BZ8 optimization, the present study reports the synthesis
and anti-M.tb activity of novel Bfx derivatives designed to serve
as better alternatives for the treatment of MDR-TB.
European Medicines Agency (EMA) and the Japanese regulatory Results and Discussion
authority, while pretomanid (TB Alliance) was approved by the
FDA in 2019 for use in combination with BDQ and LZD for the
treatment of highly drug-resistant forms of pulmonary TB (XDR-
TB or treatment-intolerant/non-responsive MDR-TB).[26] Both
these nitroimidazoles act by inhibiting the biosynthesis of
mycolic acids.[27,28]
Drug design
The initial phase of the BZ8 optimization strategy involved
replacing the N-acylhydrazone subunit of this compound with
an amide bond (Figure 2) to increase the chemical stability of
the generated series of compounds, as certain N-acylhydrazone
subunits exhibit chemical instability at acidic pH. The exper-
imental results indicated that the derivatives containing the N-
acylhydrazone subunit were poorly stable or completely
unstable at acidic pH. This finding was consistent with the
previous works published by our research group.[31–33] Therefore,
the substitution of the N-acylhydrazone subunit was performed
to overcome this inherent chemical instability. Similar to the N-
acylhydrazone subunit-containing drug, the amide-containing
drugs are also susceptible to hydrolysis, although at a much
slower rate.
In the next step, the pyridine ring present in BZ8 was
replaced with the fluorine-substituted phenyl ring attached to a
morpholine ring at the para position relative to the amide
bond. The efficiency of the synthesized compound 5c was
evaluated against several MDR-M.tb strains. The initial evalua-
tion of the newly synthesized compound 5c was performed
against MDR strains rather than H37Rv because the objective of
this initial stage of drug design was to assess whether changes
in BZ8 would increase the potential of the compound against
resistant strains. Indeed, the novel synthesized compound
presented better results against these MDR strains compared to
BZ8, with the MIC90 values ranging between 1.81 and 5.23 μM.
This promising result against the resistant strains was sufficient
motivation to expand the series of compounds by evaluating
the different substituents attached to the fluorine-substituted
Several other drug candidates are currently in clinical phase
studies, such as SQ109, delpazolid (LCB01-0371), sutezolid,
telacebec (Q203), macozinone (PBTZ169), GSK-3036656, etc.[29]
In this context and considering The End TB Strategy to eradicate
TB, continual research and development of novel anti-TB
compounds are crucial. We, as well as other researchers, have
previously reported a series of benzofuroxan (Bfx) derivatives
with potent activity against M.tb, including the MDR strains.[30,31]
Our first study demonstrated the potential of these compounds
as antimycobacterial agents by assessing their activity and
cytotoxicity profile,[30] while the subsequent studies evaluated
the in vivo efficacy and the mode of action of these
compounds.[31] In particular, the compound (E)-6-((2-isonicoti-
noylhydrazono)methyl)benzo[c][1,2,5]oxadiazole
1-N-oxide
(BZ8; Figure 1) could reduce the burden of M.tb to undetectable
levels in a mouse model of TB infection.[31] Moreover, BZ8 was
also active against several M.tb mono-resistant strains, including
those with resistance to INH, RIF, BDQ, moxifloxacin (MOX),
capreomycin (CAP), and streptomycin (SM). The MIC90 values of
BZ8 against these strains ranged from 1.2 to 16.9 μM. Further
studies revealed that BZ8 exhibited high intracellular inhibition
(ca. 90%) and an early bactericidal effect.[31] However, the
compound has low stability in the acid medium due to the
presence of the N-acylhydrazone subunit and a poor anti-M.tb
activity against the MDR strains (MIC90 >25 μM). Therefore,
despite the excellent in vivo activity demonstrated by BZ8, we
Figure 2. Example of the expansion of the chain of rings linked to the
benzofuroxan nucleus. In the figure, compound 5d is used as a model to
exemplify the three-ring systems.
Figure 1. Recently approved drugs for the MDR-TB treatment and the lead
benzofuroxan BZ8.
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phenyl ring, including the cyclic amines and the substituted
piperazines with different aromatic/heteroaromatic rings. There-
fore, further experiments were focused on expanding the
substituting chain of the rings attached to the Bfx nucleus. First,
the 4-pyridine ring (ring A) present in BZ8 was replaced with a
fluorine-substituted phenyl ring (ring A) and a cyclic amine
(ring B) in the para position relative to the amide bond
(Figure 2).
used widely as anti-cancer and cytotoxic agents activated after
bioreduction.[39] ROS play a crucial role in the pathogenesis of
TB. For instance, enhanced levels of ROS may damage the
cellular components, such as lipids, proteins, and nucleic acids,
thereby inhibiting M.tb growth.[40–43] Moreover, the Bfx deriva-
tives may interact with the thiol (À SH) groups present in
cysteine residues, which is considered a parallel mechanism of
action underlying Bfx cytotoxicity.[44–46]
The structures of compounds 5a–5c contain different cyclic
amines attached to the fluorine-substituted central phenyl ring,
including 1-azepanyl (compound 5a), 4-thiomorpholinyl (com-
pound 5b), and 4-morpholinyl (compound 5c), while the other
compounds contain a piperazine ring (ring B) attached to the
fluorine-substituted phenyl ring (ring A; compounds 5d–5q).
Furthermore, these compounds contain other aromatic/hetero-
aromatic rings (ring C) attached at the para position of the
piperazine ring (Figure 2). Different rings, including phenyl,
substituted-phenyl, pyridines, pyrimidine, and phenylcarbonyl
rings, were selected.
The aim was to evaluate the actual contribution of the
terminal part of the molecule to the anti-M.tb activity.
Subsequently, the most active compound obtained in the first
series was selected, and in its structure, the Bfx subunit was
exchanged with other heterocycles. Several heterocycles,
including benzofurazan 11a, indole 11b, benzimidazole 11c,
tetrazole 11d, and 2-nitroaniline 11e, were evaluated. In
addition, the fluorine atom was replaced with a hydrogen atom
in the most active compound obtained in the previous series.
These changes in the most active compound produced the
second series of compounds. The aim was to explore the
influence of the Bfx subunit and the fluorine atom on the
biological activity of the final compounds (Figure 3).
Chemistry
First, a series of nitroaromatics para-substituted with cyclic
amines, 2a–2q, were synthesized. The nitroaromatic intermedi-
ates 2a–2q were obtained by exploring the nucleophilic
aromatic substitution reactions between 3,4-difluoronitroben-
zene 1 and the appropriate cyclic amine. This reaction was
performed using anhydrous acetonitrile heating at reflux and
N,N-diisopropylethylamine (DIPEA) as the non-nucleophilic base
(Scheme 1). The nitroaromatic intermediates were obtained in
good yields (60–99%). The next step was the reduction of the
aromatic nitro group into amine group, and the reaction was
performed using ammonium chloride (NH4Cl) and iron powder
as reducing agents in a mixture of ethanol/H2O, followed by
°
heating at 90 C (Scheme 1). This reduction reaction produced
the aniline intermediates 3a–3q in moderate yields (13–79%).
The synthetic intermediates 2a–2q and 3a–3q were synthe-
sized following the procedures reported previously.[47–49] The
chemical characterization data of all the synthetic intermediates
are provided in the Supporting Information.
The final step was to obtain the first series of compounds
by coupling the Bfx derivative 4 with the aniline derivatives 2a–
The Bfx derivatives represent an important scaffold in
medicinal chemistry owing to their wide spectrum of biological
activities,[34,35] including the anti-M.tb activity.[30,31] The antimyco-
bacterial activity of these compounds is attributed to the
reactive oxygen species (ROS) formed after their biotransforma-
tion.,[36][37,38] The presence of the =N(!O)oÀ subunit in the Bfx
scaffold might confer electron-accepting properties similar to
those in the nitroaromatic compounds or N-oxides, which are
Scheme 1. Synthesis of the first series of compounds. i) The corresponding
°
amine, DIPEA, acetonitrile, reflux, 6 h; ii) NH4Cl, Fe, ethanol/H2O, 90 C, 1 h; iii)
CDI, acetonitrile, 24 h.
Figure 3. Design of the new series of benzofuroxan derivatives.
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2q for the formation of amide. Bfx 4 was obtained using the
methodology described previously.[50] The reaction was per-
formed in anhydrous acetonitrile in the presence of carbon-
yldiimidazole (CDI) as the coupling agent, producing the final
compounds of the 1st series, 5a–5q, in moderate yields (10–
potent antimycobacterial activity against M.tb, with MIC90 values
ranging from 0.09 to 31.60 μM (Table 1).
Determining the structure-activity relationship (SAR) for this
series was challenging as neither steric nor electronic effects
appeared to present any clear dominant trend. Nonetheless,
certain patterns were observed and are discussed ahead. A SAR
analysis of the first series of compounds revealed that this novel
class of anti-M.tb compounds support, to a certain extent,
structural variations in the portion of the molecule linked to the
cyclic amine, as inferred from the retaining of the anti-M.tb
activity after the exchange of substituents or the bioisosteric
replacement of similar rings. This relationship could be clearly
observed in the series containing halogens linked at the para
position of the terminal phenyl ring. The compounds containing
fluorine (5g), chlorine (5h), and bromine (5i) atoms attached at
the para position exhibited MIC90 values of 3.82, 2.69, and
2.53 μM, respectively. A similar relationship could be observed
for compounds 5e and 5f, which contained methyl and
methoxy groups, respectively, linked at the para position of the
phenyl ring, with the compounds exhibiting MIC90 values of
7.96 and 6.98 μM, respectively. Nevertheless, when an amine
group was inserted in this position (compound 5j), the MIC90
value of 1.84 μM was achieved, thus indicating that the groups
acting as hydrogen bond donors are important for the bio-
logical activity. Regarding the large substituents, it was not
possible to observe a direct SAR as the compounds 5k and 5q
that contained large groups exhibited varied MIC90 values.
Compound 5k exhibited an MIC90 value of 10.97 μM, while
compound 5q exhibited an MIC90 value of 0.78 μM. A possible
explanation for this variation could be the presence of a
nitrogen donor for hydrogen bonding in compound 5q.
1
97%; Scheme 1). The H and 13C nuclear magnetic resonance
(NMR) spectra of these final compounds contained broad peaks
corresponding to the proton and carbon signals from the Bfx
subunit, indicating Bfx tautomerism.[34,38]
The synthesis of the second series of compounds was
performed by following the same methodologies as for the first
series of compounds. First, the aniline derivative 3n was
synthesized, which was then treated with different heterocycles
using CDI as the coupling agent for amide bond formation,
thereby producing the final compounds of the second series,
11a–11f, in moderate yields (20–71%; Scheme 2). The carbox-
ylic acid indole 7, benzimidazole 8, and 2-nitroaniline 10
intermediates were purchased commercially. The Bfx 6 and
tetrazole 9 intermediates were synthesized using the method-
ologies reported previously.[51,52] The final compound of the
second series, 11f was obtained in a manner similar to the
synthesis of compound 5n of the first series. However, an
aniline derivative containing a hydrogen atom, 13, was used in
place of the parent compound containing the fluorine atom,
3n. The synthetic intermediates 3n and 12 were synthesized by
following previously described procedures.[47–49]
Initial in vitro biological studies
Initially, all the first series compounds were evaluated against
the sensitive strains of M.tb H37Rv ATCC 27294 using the
resazurin microtiter assay (REMA) methodology.[32,53,54] The
results were expressed as the minimum inhibitory concentra-
tion (MIC90). The final compounds of the first series exhibited
The isosteric replacement of the phenyl ring attached to the
piperazine with different heteroaromatic rings led to similar
effects on the anti-M.tb activity. The exchange of the phenyl
ring in compound 5d with a pyridine ring in compound 5m led
to a loss in the antimycobacterial activity. However, when the
nitrogen in the pyridine ring was added to a different position
in the ring (compound 5n), the anti-M.tb activity was increased
significantly. Compound 5n was the most active in this series,
exhibiting an MIC90 value of 0.09 μM. Similarly, compound 5o
containing
a pyrimidine ring attached to the piperazine
exhibited a MIC value of 2.22 μM.
Furthermore, the piperazinyl moiety was replaced with
other isosteric rings, such as 1-azepanyl (compound 5a), 4-
thiomorpholinyl (compound 5b), and 4-morpholinyl (com-
pound 5c). The compound containing the 4-thiomorpholinyl
ring (5b) demonstrated promising anti-M.tb activity with an
MIC90 value of 0.70 μM. On the other hand, its bioisostere
containing a morpholine ring (5c) exhibited an MIC90 value of
5.22 μM, thus indicating that the bioisosteric replacement of
oxygen atom with sulfur in the cyclic amine leads to a
significant increase in the biological activity. The 1-azepanyl
derivative (5a) exhibited an MIC90 value of 2.89 μM. An attempt
to establish a structure-activity relationship between these
compounds by considering their lipophilicity was not success-
ful.
Scheme 2. Synthesis of the second series of compounds. i) The correspond-
ing carboxylic acid, CDI, acetonitrile, 24 h.; ii) 1-(2-pyridyl)piperazine, DIPEA,
°
acetonitrile, reflux, 6 h; iii) NH4Cl, Fe, ethanol/H2O, 90 C, 1 h.
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Table 1. Anti-M.tb activity of the first series against M.tb H37Rv (MIC90), cytotoxicity against the MRC-5 cell line (IC50), selectivity index (SI), clogD, and LiPE.
Compound
MIC90 [μM]
IC50 [μM]
SI
clogD
LiPE
2.89�1.55
174.47
60
2.83
2.71
0.70�0.26
5.22�2.33
11.75�3.19
57.07
81
1.95
1.37
3.66
4.20
3.91
1.27
58
11
>230
>19
7.96�4.07
6.98�3.81
>223.48
>28
4.11
3.81
1.06
1.36
>215.76
>30
3.82�1.36
>221.52
>58
3.70
1.72
2.69�1.12
>213.73
>79
4.22
0.96
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Table 1. continued
Compound
MIC90 [μM]
2.53�1.29
IC50 [μM]
SI
clogD
LiPE
1.07
>195.18
>77
4.46
1.84�0.94
>223.11
>121
2.68
3.03
10.97�3.60
>197.82
>18
4.62
0.34
1.56�0.85
18.19�9.26
0.09�0.04
2.22�1.20
>210.31
>134
3.01
1.37
2.80
2.21
2.16
3.37
4.25
3.28
127.81
7
>180.04
>2085
>229.66
>103
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Table 1. continued
Compound
MIC90 [μM]
2.45�1.01
IC50 [μM]
SI
clogD
LiPE
3.06
28.14
11
2.54
0.78�0.40
>182.28
>233
4.48
1.32
>25
N.D.
N.D.
3.68
N.D.
>25
N.D.
N.D.
4.43
N.D.
>25
N.D.
N.D.
3.62
N.D.
>25
N.D.
N.D.
2.03
N.D.
>25
N.D.
N.D.
4.09
N.D.
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Table 1. continued
Compound
MIC90 [μM]
2.5�1.08
IC50 [μM]
SI
clogD
LiPE
2.79
>240.13
>96
2.81
INH
RIF
LZD
BZ8
0.95�0.43
0.05�0.01
0.74�0.29
1.10
>729.18
>121.51
N.D.
>1695
>2430
N.D.
À 0.69
2.06
0.64
6.71
5.24
5.49
5.83
519.20
472
0.13
INH: isoniazid; RIF: rifampicin; LZD: linezolid; N.D.: not determined; cLog D: calculated distribution coefficient; LiPE: lipophilic efficiency; �: standard deviation
(SD), average of three independent assays.
Compound 5p exhibited an MIC90 value of 2.45 μM. This
compound is a retroisostere of compound 5l and contains a
phenylcarbonyl group attached in reverse to the piperazine
ring. This retroisosterism produced a significant increase in the
anti-M.tb activity of compound 5l over compound 5p. Com-
pound 5l and 5p exhibited MIC90 values of 1.56 and 2.45 μM,
respectively.
fibroblast tissue and is used widely for phenotypic screening in
antituberculosis drug discovery.[57–59] The selectivity index (SI) of
the evaluated compounds was the ratio between their IC50 and
MIC90 values. All the Bfx derivatives in the first series exhibited a
low cytotoxic profile against the MRC-5 cell line. The IC50 values
of all the final compounds were above 128 μM, reflecting high
SI values for the most potent compounds (Table 1).
Altogether, the SAR analyses revealed that, in steric terms,
the smaller cyclic amines 5a–5c were as active as the larger
aryl-piperazines 5d–5q. Nevertheless, differences could be
observed between the two sub-series. For instance, the
compounds containing electron-withdrawing groups attached
to the C-ring, such as halogens, appeared to have enhanced
anti-M.tb activity compared to the compound without any
substituents on the C ring (5d). Similarly, the compounds 5l
and 5q containing other electron-withdrawing groups, such as
acyl groups, were also potent in the anti-M.tb assay. However,
no such relationship occurred in the electron-donating groups
attached to the C-ring, such as in compounds 5e and 5f, which
contained a methyl group and a methoxy group attached at
the para position of the C ring, respectively. These compounds
were not as potent as compound 5j with an amine at the same
position. Unlike compounds 5e and 5f, compound 5j could act
as a hydrogen-bond donor, which explains the difference in the
activity between this compound and the ones with electron-
donating groups. Among the compounds containing hetero-
aromatic rings, the only compound demonstrating potency was
compound 5n, which contained 2-pyridyl as the C ring.
It is noteworthy that the most promising compounds in the
series exhibited anti-TB activity profiles superior to several
clinically used drugs that were included as positive controls in
the present study. For instance, the most active compound 5n
(MIC90 =0.09 μM) exhibited activity higher than that of INH
(MIC90 =0.95 μM) and LZD (MIC90 =0.74 μM) and statistically
similar to that of RIF (MIC90 =0.05 μM). INH, LZD, and RIF are the
drugs used widely in the treatment of susceptible and resistant
forms of TB.
After the initial in-vitro biological evaluation, the lipophilic
efficiency (LiPE) values for the compounds were determined
(Table 1). Lipophilic efficiency is calculated from the biological
activity data (anti-M.tb activity) and logD values and is used for
selecting the most promising drug candidate compounds
within a series. LiPE establishes a relationship between potency
and lipophilia to estimate the differences among the com-
pounds, considering not only the potency but also the
physicochemical properties of the compounds. The literature
recommends selecting the compounds with the highest LiPE
values within a series to advance the discovery process.[60] In
the present study, LiPE was calculated by subtracting the logD
of the molecule from the negative log10 of the potency (M)
against the desired target. The logD values were calculated
using ChemAxon’s Calculator and the MIC90 values were used as
potency in the calculation of LiPE. It is noteworthy that the LiPE
values presented here are only approximate LiPE values as the
logD values used were calculated theoretically and not
measured experimentally.
The LiPE values revealed the most promising compounds
within the series worthy of being prioritized for further develop-
ment. Compound 5n emerged as the most promising one
within the series, exhibiting not only a potent anti-M.tb activity
but the highest LiPE value as well. Therefore, compound 5n
was used in the further molecular modification steps conducted
to understand better the contribution of the other subunits of
the molecule to its anti-M.tb activity.
The second series of compounds was designed based on
the identification of the leading compound 5n as the most
promising one in the first series. The designing process involved
the isosteric replacement of the Bfx ring with other aromatic
and heteroaromatic rings to verify the importance that the
benzofuroxanyl subunit has for the compound’s anti-M.tb
The cytotoxicity was evaluated on the MRC-5 cell line, as
described previously[55,56] and the results were expressed as IC50
values. This cell line is derived from healthy human lung
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activity. Furthermore, compound 11f was designed by replac-
ing the fluorine atom attached to the phenyl ring with a
hydrogen atom to verify the contribution of the fluorine atom
to the compound’s biological activity.
Further in-vitro biological profiling of selected compounds
The promising compounds in the first series selected for further
evaluation based on their MIC90 and LiPE values were assayed
against a panel of clinical isolates, including MDR-TB and XDR-
TB.[63] These strains were phenotypically characterized and
exhibited resistance to several anti-M.tb drugs. Specifically, each
MDR strain exhibited resistance to the following drugs: MDR-1:
INH, RIF, and amikacin (AMK), MDR-2: INH and RIF, MDR-3: INH
and RIF, MDR-4: INH, RIF, and AMK, and MDR-5: INH, RIF, AMK,
and MOX. The strains were classified as MDR or XDR based on
the MIC90 values of control drugs against them.[54]
The MIC90 values of the selected compounds against these
strains varied (Table 2). For instance, most of the compounds
exhibited potent activity against the XDR-TB strains (MDR-5),
among which compounds 5a, 5b, 5n, 5o, and 5p should be
highlighted as these presented MIC90 values against the XDR-TB
strains in the range of 0.27–1.68 μM. These compounds also
exhibited potent activity against the MDR-1, MDR-2, MDR-3,
and MDR-4 strains, with MIC90 values ranging from 0.21 to
9.40 μM against all the strains, the MIC90 values exhibited by
compounds 5a, 5b, 5n, 5o, and 5p were below 10 μM,
confirming their potency against the resistant strains.
Clinical isolates differed significantly from the standard
strains of ATCC. The H37Rv strain, which is used as the reference
strain in most studies related to drug discovery, has certain
limitations, as evidenced by the genetic diversity observed in
the studies that compared its genome with those of the clinical
isolates.[64] Both in vitro and in vivo, the differences in the profile
of pathogenicity and virulence are important factors and,
therefore, it is recommended to use different strains for a study
to ensure reliable results and accurate prediction of the effect
of the compound in a population.[64–66]
The compounds 11a–11f obtained in the second series
were evaluated against M.tb H37Rv ATCC 27294. The compounds
in which the Bfx subunit was replaced with another heterocycle
exhibited a significant loss in the anti-M.tb activity. The MIC90
values for these compounds (11a–11e) were above 25 μM.
These data indicated that the N-oxide-containing subunit
present in the benzofuroxan is essential for biological activity.
This fact was confirmed when compound 5n was compared
with its analog containing the benzofurazan ring (11a). The
difference between the structures of these two compounds was
the N-oxide subunit, which was present in the Bfx 5n and
absent in the benzofurazan 11a. The simple removal of this
subunit led to a complete loss of the anti-M.tb activity. These
results suggested that the N-oxide subunit could be serving as
a pharmacophoric group and the essential warhead. Previous
studies have also demonstrated that Bfx derivatives interact
with the thiol groups (À SH) present in the cysteine residues,
often leading to enzymatic inhibition.[61,62] Therefore, we
hypothesized that the Bfx nucleus could lead to the formation
of a nitrous intermediate after biotransformation, which then
served as an electrophilic site that would lead to the formation
of an adduct after interaction with the cysteine residues (À SH)
acting as nucleophiles.
The analog without the fluorine, compound 11f, was quite
potent despite the reduction in the anti-M.tb activity in relation
to its parent compound 5n. The MIC90 value for compound 11f
was 2.5 μM, accounting for a 27-fold reduction in the activity
relative to the parent compound. The difference between these
two compounds was the fluorine atom attached to the phenyl
ring, present in compound 5n and absent in compound 11f.
The latter contained a hydrogen atom at the same position.
This result indicated that the fluorine atom, although not
essential, has a certain contribution to the anti-M.tb activity.
In the present study, compounds 5b (Lapdesf-20–5b) and
5n (Lapdesf-20-5n) were identified as the most potent against
the resistant strains and, therefore, the most promising. These
compounds demonstrated potent activity against all the
Table 2. Anti-M.tb activity of the most active compounds against the clinically isolated MDR-TB strains (MIC90).
Compound
MIC90 [μM]
MDR-1
MDR-2
MDR-3
MDR-4
MDR-5
5a
4.98�4.56
0.28�0.02
0.75�0.33
4.65�4.23
10.98�10.09
4.84�4.28
16.80�6.48
0.56�0.47
9.40�9.09
6.90�6.01
1.68�0.78
N.D.
1.38�0.84
<0.27
1.95�1.22
<0.27
2.20�0.67
<0.27
>53.43
0.92�0.47
<0.27
5b
5g
5h
5i
5j
5l
2.47�2.31
52.96�0.80
13.88�8.70
36.22�21.78
4.55�2.57
18.23�8.56
0.26�0.05
0.93�0.62
0.90�0.30
0.44�0.31
0.58�0.02
>30.38
33.39�24.64
6.26�6.16
3.55�3.32
3.88�2.78
2.18�2.15
1.68�1.32
1.92�1.55
0.41�0.01
8.67�7.84
0.47�0.32
>30.38
<0.20
16.53�11.63
2.35�2.25
1.45�0.03
6.77�5.43
1.84�1.73
<0.22
1.47�0.06
1.43�0.22
0.95�0.01
>30.38
>182.30
0.40�0.04
5.33�0.95
>25.00
>48.80
13.33�0.05
48.69�5.51
2.24�1.47
1.21�0.61
1.06�0.40
0.61�0.17
0.41�0.03
>30.38
5n
5o
5p
11f
LZD
RIF
INH
MOX
AMK
BZ8
>30.38
>182.30
71.56�24.71
2.48�1.12
0.24�0.10
>25.00
>182.30
>182.30
3.37�0.69
4.69�4.17
41.60�35.81
19.52�12.55
>25.00
>42.69
>42.69
>25.00
>25.00
INH: isoniazid; RIF: rifampicin; LZD: linezolid; MOX: moxifloxacin; AMK: amikacin; �: standard deviation (SD), average of three independent assays.
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evaluated strains. For instance, compounds 5b and 5n
presented MIC90 values of 0.27 and 1.68 μM, respectively,
against the MDR-5 strain that is resistant to INH, RIF, AMK, and
MOX (as evidenced by its phenotypic characterization). These
results suggested that both 5b and 5n act through some
mechanism of action that is different from the one used by the
reference drugs.
preliminary assays. Finally, the most active compounds also had
higher clogP values compared to the parent compound (BZ8).
These promising results against MDR-M.tb strains highlight
the identified Bfx derivatives as leading candidate compounds
for the discovery of novel drugs for the treatment of resistant
forms of TB.
The MDR-2 strain appeared to be the most sensitive to this
class of compounds as most of these compounds exhibited Conclusion
MIC90 values below 2.0 μM against this strain. On the contrary,
the MDR-1 strain appeared to be the most resistant to this class
of compounds as most of the compounds exhibited MIC90
values above 4.65 μM against this strain. Compounds 5g, 5i,
and 5l were the least potent within this series as these
compounds exhibited MIC90 values above 30 μM against the
panel of resistant strains. Compound 11f, the fluorine-free
analog of compound 5n, presented results similar to those
presented by its parent compound 5n. For instance, com-
pounds 5n and 11f presented MIC90 values of 1.84 and
1.43 μM, respectively, against the MDR-2 strain, and the MIC90
values of 0.26 and 0.44 μM, respectively, against the MDR-3
strain. Despite the similar activity of these two compounds, the
compound containing the fluorine atom, 5n, was more
promising as the fluorine atom provided greater lipophilicity to
the parental compound 5n (clogP: 2.6) compared to the
compound without the fluorine atom 11f (clogP: 2.2). In the
development of anti-TB drugs, lipophilicity is an important
parameter for consideration. Studies have reported that the
compounds with logP values within a certain range are likely to
exhibit higher activity.[67,68]
This study aimed to evaluate the chemical instability and
potency against MDR-M.tb strains of a novel series of benzofur-
oxan derivatives designed from the lead compound BZ8 already
identified in previous studies. The synthesized novel com-
pounds were more potent than the parent compound BZ8, in
general, against
a panel of MDR-M.tb strains evaluated.
Compounds 5b (Lapdesf-20-5b) and 5n (Lapdesf-20-5n) were
particularly remarkable in this regard as these demonstrated
potent activities against all the evaluated strains. Compound
5b was particularly superior, with MIC90 values below 0.28 μM
against all the evaluated MDR-M.tb strains, thus suggesting that
this compound might have a mechanism of action different
from that of the other compounds. The BZ8 optimization
process led to the discovery of a novel class of benzofuroxan
derivatives exhibiting excellent potency against MDR-M.tb
strains. Further studies are required for determining the
mechanism of action of this novel class of compounds and
assessing their in vivo activities.
Overall, eight among the 11 evaluated compounds exhib- Experimental Section
ited potent anti-M.tb activity against the evaluated MDR strains.
Among these eight compounds, 5b and 5n are noteworthy.
Compound 5b was particularly remarkable as it exhibited MIC90
values below 0.28 μM against all the MDR-M.tb strains, suggest-
ing that this compound might have a mechanism of action
different from that of all the others.
Chemistry
The melting points (mp) were measured using an electrothermal
melting point apparatus (SMP3; Bibby Stuart Scientific). The H and
1
13C NMR spectra for all the compounds were obtained using a
Bruker Fourier with Dual probe 13C/1H (300-MHz) NMR spectrometer
and Bruker Avance III HD 600 13C/1H (600-MHz) NMR spectrometer,
respectively, with deuterated chloroform (CDCl3) or dimeth-
ylsulfoxide ([D6]DMSO) as the solvent. The chemical shifts were
expressed in parts per million (ppm) relative to tetramethylsilane.
The signal multiplicities were reported as singlet (s), doublet (d),
doublet of doublet (dd), doublet of doublet of doublets (ddd),
triplet (t), and multiplet (m). The compounds were purified using a
chromatography column containing silica gel (60 Å pore size, 35–
75 μm particle size) and methanol, ethyl acetate, dichloromethane,
hexane, and petroleum ether as solvents at a flow rate of
approximately 15 mL/min. The progress of reaction for all the
compounds was monitored using thin-layer chromatography (TLC)
performed on 2.0 by 6.0 cm2 aluminum sheets precoated with a
0.25-mm-thick layer of silica gel 60 (HF-254; Merck) and observed
under UV light (265 nm). All the compounds were analyzed using
HPLC, and their purity was confirmed to be greater than 98.5%.
The HPLC conditions were as follows: 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 an Agilent® Eclipse XDB C-18
column (250 mm×274.6 mm; 5 μm). An isocratic flow [methanol/
water (70:30)] was used in the HPLC and the required reagents and
solvents were purchased from commercial suppliers. Compounds 4,
Altogether, the drug development and BZ8 optimization
process led to the identification of a novel class of Bfx
derivatives exhibiting an anti-M.tb activity profile superior to
that of the parent compound BZ8. For instance, compound 5n
exhibited anti-M.tb activity against all the evaluated MDR-M.tb
strains, with MIC90 values below 2.3 μM, while BZ8 demon-
strated poor activity against the MDR-M.tb strains (MIC90 >
25 μM). These results do not imply that BZ8 is disqualified as a
promising compound for the TB treatment as BZ8 has already
demonstrated outstanding effectiveness in several previous
in vivo studies conducted on mice infected with M.tb.[31] None-
theless, BZ8 requires optimization so that it exhibits better anti-
M.tb activity against the resistant strains. The strategy used in
the present study was to replace the N-acylhydrazone subunit
(BZ8) with an amide and the 4-pyridine ring (BZ8) with different
heterocycles and cyclic amines, producing a class of com-
pounds that are active against the MDR strains. Most of the
compounds identified in the present work were active against
the panel of the MDR-M.tb strains evaluated. In addition, the
most active compounds exhibited no cytotoxicity in the
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6, and 9 were synthesized according to previously described
methodologies.[50–52] The synthetic intermediates 2a–2q, 3a–3q,
3n, and 13 were also synthesized as described previously.[47–49] The
chemical characterization data for all the synthetic intermediates
are provided in the Supplementary Material.
150.9, 136.0 (d, JC-F =8.9 Hz, 1C), 134.6 (d, JC-F =10.5 Hz, 1C), 133.5,
132.8, 132.3, 132.0, 129.0, 119.3 (d, JC-F =24.9 Hz, 1C), 119.1, 116.5
(d, JC-F =2.5 Hz, 1C), 115.6, 113.5, 108.6 (d, JC-F =25.6 Hz, 1C), 50.3,
48.4; anal. calcd (%) for C23H20FN5O3: C 63.73, H 4.65, N 16.16; found:
C 63.71, H 4.65, N 16.17.
6-((3-Fluoro-4-(4-(p–tolyl)piperazin-1-yl)phenyl)carbamoyl)benzo
[c][1,2,5]oxadiazole 1-N-oxide (5e). Yellow solid; yield 65%; mp:
Synthesis
1
°
206–207 C; H NMR (600 MHz, [D6]DMSO): δ=10.60 (s, 1H), 8.44–
8.23 (m, J=8.4 Hz, 1H), 8.04–7.74 (m, J=8.4 Hz, 2H), 7.71 (dd, J=
14.8, 2.3 Hz, 1H), 7.48 (dd, J=8.7, 1.6 Hz, 1H), 7.12 (t, J=9.3 Hz, 1H),
7.05 (d, J=8.2 Hz, 2H), 6.90 (d, J=8.5 Hz, 2H), 3.24–3.21 (m, J=
4.2 Hz, 4H), 3.15–3.12 (m, J=4.9 Hz, 4H), 2.21 (s, 3H); 13C NMR
(151 MHz, [D6]DMSO): δ=162.9, 155.1 (d, JC-F =243.0 Hz, 1C), 148.9,
136.1 (d, JC-F =8.9 Hz, 1C), 134.6 (d, JC-F =10.6 Hz, 1C), 133.6, 133.5,
General procedure for the synthesis of compounds 5a–5q
A solution of 6-carboxybenzo[c][1,2,5]oxadiazole 1-oxide 4 (100 mg,
0.55 mmol) was stirred into 10 mL of anhydrous acetonitrile under
nitrogen, followed by the addition of CDI (90 mg, 0.55 mmol). The
reaction mixture was stirred for 90 min at room temperature and
then a solution of the corresponding amine derivative 3a–3q
(0.33 mmol, 0.6 equiv. of the carboxylic acid 4) in anhydrous
acetonitrile (1 mL) was added dropwise. The resultant mixture was
stirred at room temperature for 48 h under nitrogen atmosphere,
followed by the removal of the solvent under reduced pressure.
The obtained crude was washed with ethyl acetate. The solid
reaction product was purified using flash chromatography on silica
(EtOAc/petroleum ether, 20:80 to 70:30), producing the final
compounds 5a–5q in variable yields as described below.
132.8, 132.0, 129.4, 127.9, 119.3 (d, JC-F =24.9 Hz, 1C), 116.5 (d, JC-F
=
2.0 Hz, 1C), 115.9, 108.6 (d, JC-F =25.8 Hz, 1C), 50.3, 48.9, 20.0; anal.
calcd (%) for C24H22FN5O3: C 64.42, H 4.96, N 15.65; found: C 64.45, H
4.98, N 15.66.
6-((3-Fluoro-4-(4-(4-methoxyphenyl)piperazin-1-yl)phenyl)
carbamoyl)benzo[c][1,2,5]oxadiazole 1-N-oxide (5f). Yellow solid;
1
°
yield 55%; mp: 190–191 C; H NMR (600 MHz, [D6]DMSO): δ=10.57
(s, 1H), 8.44–8.21 (m, J=32.5 Hz, 1H), 8.03–7.73 (m, J=9.5 Hz, 1H),
7.70 (dd, J=14.8, 2.2 Hz, 1H), 7.48 (dd, J=8.7, 1.5 Hz, 1H), 7.11 (t,
J=9.3 Hz, 1H), 6.95 (d, J=9.1 Hz, 1H), 6.84 (d, J=9.1 Hz, 1H), 3.69 (s,
1H), 3.16 (d, J=5.5 Hz, 2H), 3.14 (d, J=5.3 Hz, 2H); 13C NMR
(151 MHz, [D6]DMSO): δ=162.7, 155.0 (d, JC-F =243.3 Hz, 1C), 153.1,
145.3, 136.1 (d, JC-F =8.9 Hz, 1C), 134.6 (d, JC-F =10.7 Hz, 1C), 133.5,
6-((4-(Azepan-1-yl)-3-fluorophenyl)carbamoyl)benzo[c][1,2,5]
oxadiazole 1-N-oxide (5a). Orange solid; yield 33%; mp: 232–
1
°
235 C; H NMR (600 MHz, [D6]DMSO): δ=10.55 (s, 1H), 8.64 (s, 1H),
8.18 (dd, J=9.4; 0.9 Hz, 1H), 7.99 (dd, J=9.4; 1.3 Hz, 1H), 7.62 (dd,
J=16.1; 2.4 Hz, 1H), 7.38 (dd, J=8.9; 1.9 Hz, 1H), 6.93 (dd, J=10.2;
9.0 Hz, 1H), 3.32–3.30 (m, 4H), 1.78–1.74 (m, 4H), 1.57–1.54 (m, 4H);
13C NMR (151 MHz, [D6]DMSO): δ=163.2, 152.5 (d, JC-F =239.9 Hz,
1C), 136.0 (d, JC-F =8.1 Hz, 1C), 132.7, 131.9, 130.0 (d, JC-F =10.3 Hz,
1C), 117.0, 117.0 (d, JC-F =5.3 Hz, 1C), 116.6, 116.5, 116.0, 109.1 (d,
132.8, 132.4, 132.0, 119.2 (d, JC-F =24.9 Hz, 1C), 117.6, 116.4 (d, JC-F
=
2.2 Hz, 1C), 114.2, 108.6 (d, JC-F =25.8 Hz, 1C), 55.1, 50.3, 49.8; anal.
calcd (%) for C24H22FN5O4: C 62.20, H 4.78, N 15.11; found: C 62.23, H
4.79, N 15.11.
6-((3-Fluoro-4-(4-(4-fluorophenyl)piperazin-1-yl)phenyl)
J
C-F =26.7 Hz, 1C), 51.6, 28.4, 26.6; anal. calcd (%) for C19H19FN4O3: C
carbamoyl)benzo[c][1,2,5]oxadiazole 1-N-oxide (5g). Pale brown
1
61.61, H 5.17, N 15.13; found: C 61.60, H 5.16, N 15.13.
°
solid; yield 37%; mp: 190–191 C; H NMR (600 MHz, [D6]DMSO): δ=
10.59 (s, 1H), 8.46–8.22 (m, 1H), 8.04–7.74 (m, 2H), 7.49 (dd, J=8.7;
1.6 Hz, 1H), 7.71 (dd, J=14.8; 2.2 Hz, 1H), 7,12 (t, J=9.3 Hz, 1H),
7.09–7.05 (m, 2H), 7.03–7.00 (m, 2H), 3.25–3.22 (m, 4H), 3.16–3.13
6-((3-Fluoro-4-thiomorpholinophenyl)carbamoyl)benzo[c][1,2,5]
oxadiazole 1-N-oxide (5b). Pale yellow solid; yield 13%; mp: 242–
1
°
245 C; H NMR (600 MHz, [D6]DMSO): δ=10.57 (s, 1H), 8.28 (s, 1H),
(m, 4H); 13C NMR (151 MHz, [D6]DMSO): δ=162.9, 157.0 (d, JC-F
=
8.00–7.73 (m, J=37.4 Hz, 2H), 7.68 (d, J=14.2 Hz, 1H), 7.45 (d, J=
8.3 Hz, 1H), 7.09 (t, J=9.1 Hz, 1H), 3.22 (s, 4H), 2.75 (s, 4H); 13C NMR
(151 MHz, [D6]DMSO): δ=162.8, 155.2 (d, JC-F =243.2 Hz, 1C), 137.0
(d, JC-F =8.7 Hz, 1C), 135.0, 133.9 (d, JC-F =10.7 Hz, 1C), 132.8, 132.4,
132.0, 124.8, 120.4 (d, JC-F =3.1 Hz, 1C), 116.4 (d, JC-F =1.8 Hz, 1C),
235.8 Hz, 1C), 155.4 (d, JC-F =243.0 Hz, 1C), 147.8 (d, JC-F =4.1 Hz, 1C),
136.4 (d, JC-F =8.9 Hz, 1C), 133.6 (d, JC-F =10.5 Hz, 1C), 133.3, 133.0,
132.8, 132.6, 132.0, 119.3, 117.4 (d, JC-F =7.4 Hz, 1C), 116.5 (d, JC-F
=
1.9 Hz, 1C), 116.4, 115.4 (d, JC-F =21.7 Hz, 1C), 108.6 (d, JC-F =25.7 Hz,
1C), 50.3, 49.2; anal. calcd (%) for C23H19F2N5O3: C 61.19, H 4.24, N
15.51; found: C 61.21, H 4.25, N 15.50.
108.5 (d,
JC-F =25.6 Hz, 1C), 53.0, 27.2; anal. calcd (%) for
C17H15FN4O3S: C 54.54, H 4.04, N 14.97; found: C 54.56, H 4.05, N
14.97.
6-((4-(4-(4-Chlorophenyl)piperazin-1-yl)-3-fluorophenyl)
carbamoyl)benzo[c][1,2,5]oxadiazole 1-N-oxide (5h). Off-white
6-((3-Fluoro-4-morpholinophenyl)carbamoyl)benzo[c][1,2,5]
1
°
solid; yield 10%; mp: 202–205 C; H NMR (600 MHz, [D6]DMSO): δ=
oxadiazole 1-N-oxide (5c). Orange solid; yield 40%; mp: 192–
193 C; 1H NMR (600 MHz, [D6]DMSO): δ=10.57 (s, 1H), 8.46–8.20
10.60 (s, 1H), 8.46–8.23 (m, 1H), 8.03–7.76 (m, 2H), 7.71 (dd, J=14.8;
2.3 Hz, 1H), 7.48 (dd, J=8.7; 1.8 Hz, 1H), 7.26 (d, J=9.0 Hz, 2H), 7.12
(t, J=9.3 Hz, 1H), 7.01 (d, J=9.1 Hz, 2H), 3.30–3.28 (m, 4H), 3.15–
3.12 (m, 4H); 13C NMR (151 MHz, [D6]DMSO): δ=162.9, 155.1 (d,
°
(m, J=25.6 Hz, 1H), 8.01–7.72 (m, J=9.7 Hz, 2H), 7.68 (dd, J=14.9,
2.2 Hz, 1H), 7.47 (dd, J=8.7, 1.6 Hz, 1H), 7.06 (t, J=9.3 Hz, 1H), 3.76–
3.72 (m, 4H), 3.00–2.96 (m, 4H); 13C NMR (151 MHz, [D6]DMSO): δ=
162.8, 155.0 (d, JC-F =243.2 Hz, 1C), 136.0 (d, JC-F =8.7 Hz, 1C), 134.9,
J
C-F =243.2 Hz, 1C), 149.7, 135.9 (d, JC-F =8.9 Hz, 1C), 133.6 (d, JC-F
=
10.6 Hz, 1C), 133.4, 133.0, 132.9, 132.6, 132.0, 128.7, 128.6, 119.3 (d,
133.5 (d, JC-F =10.5 Hz, 1C), 132.8, 132.3, 132.0, 124.8, 119.0 (d, JC-F
=
J
J
C-F =3.9 Hz, 1C), 117.1, 116.5 (d, JC-F =2.7 Hz, 1C), 116.4, 108.6 (d,
C-F =25.6 Hz, 1C), 50.1, 48.2; anal. calcd (%) for C23H19ClFN5O3: C
3.8 Hz, 1C), 116.4 (d, JC-F =2.3 Hz, 1C), 108.5 (d, JC-F =25.6 Hz, 1C),
66.1, 50.6; anal. calcd (%) for C17H15FN4O4: C 56.98, H 4.22, N 15.64;
found: C 56.98, H 4.20, N 15.63.
59.04, H 4.09, N 14.97; found: C 59.02, H 4.07, N 14.97.
6-((4-(4-(4-Bromophenyl)piperazin-1-yl)-3-fluorophenyl)
6-((3-Fluoro-4-(4-phenylpiperazin-1-yl)phenyl)carbamoyl)benzo
carbamoyl)benzo[c][1,2,5]oxadiazole 1-N-oxide (5i). Pale brown
[c][1,2,5]oxadiazole 1-N-oxide (5d). Yellow solid; yield 67%; mp:
1
°
203–204 C; 1H NMR (600 MHz, [D6]DMSO): δ=10.60 (s, 1H), 7.71
solid; yield 33%; mp: 199–203 C; H NMR (600 MHz, [D6]DMSO): δ=
°
10.60 (s, 1H), 8.45–8.22 (m, 1H), 8.03–7.74 (m, 2H), 7.71 (dd, J=14.7;
2.1 Hz, 1H), 7.48 (d, J=10.1 Hz, 1H), 7.37 (d, J=8.9 Hz, 2H), 7.11 (t,
J=9.3 Hz, 1H), 6.96 (d, J=9.0 Hz, 2H), 3.31–3.28 (m, 4H), 3.15–3.11
(dd, J=14.8, 2.3 Hz, 1H), 7.49 (dd, J=8.7, 1.8 Hz, 1H), 7.24 (dd, J=
8.6, 7.4 Hz, 2H), 7.12 (t, J=9.3 Hz, 1H), 7.00 (d, J=8.0 Hz, 2H), 6.81 (t,
J=7.2 Hz, 1H), 3.29 (d, J=9.8 Hz, 4H), 3.14 (d, J=9.7 Hz, 4H); 13C
NMR (151 MHz, [D6]DMSO): δ=162.7, 155.1 (d, JC-F =243.0 Hz, 1C),
(m, 4H); 13C NMR (151 MHz, [D6]DMSO): δ=163.0, 155.1 (d, JC-F
=
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243.3 Hz, 1C), 150.1, 135.9 (d, JC-F =8.9 Hz, 1C), 133.7, 133.6 (d, JC-F
=
147.6, 137.6, 136.1, 135.0 (d, JC-F =8.9 Hz, 1C), 133.6 (d, JC-F =10.6 Hz,
1C), 132.3, 132.0, 124.8, 119.4 (d, JC-F =3.8 Hz, 1C), 116.4 (d, JC-F
2.6 Hz, 1C), 113.2, 108.6 (d, JC-F =25.6 Hz, 1C), 107.2, 50.1, 44.7; anal.
calcd (%) for C22H19FN6O3: C 60.82, H 4.41, N 19.35; found: C 60.84, H
4.42, N 19.34.
10.7 Hz, 1C), 132.8, 132.0, 131.6, 131.5, 119.3 (d, JC-F =3.8 Hz, 1C),
117.5, 116.5 (d, JC-F =2.5 Hz, 1C), 116.4, 110.3, 108.6 (d, JC-F =25.8 Hz,
1C), 50.1, 48.1; anal. calcd (%) for C23H19BrFN5O3: C 53.92, H 3.74, N
13.67; found: C 53.95, H 3.76, N 13.69.
=
6-((4-(4-(4-Aminophenyl)piperazin-1-yl)-3-fluorophenyl)
6-((3-Fluoro-4-(4-(pyrimidin-2-yl)piperazin-1-yl)phenyl)carbamoyl)
carbamoyl)benzo[c][1,2,5]oxadiazole 1-N-oxide (5j). Yellow solid;
benzo[c][1,2,5]oxadiazole 1-N-oxide (5o). Pale brown solid; yield
1
yield 97%; mp: 212–215 C; H NMR (600 MHz, [D6]DMSO): δ=10.73
58%; mp: 203–204 C; 1H NMR (600 MHz, [D6]DMSO): δ=10.58 (s,
°
°
(s, 1H), 8.48–8.27 (m, 1H), 8.05–7.80 (m, 2H), 7.73 (dd, J=14.8;
2.0 Hz, 1H), 7.52 (d, J=8.6 Hz, 1H), 7.10 (t, J=9.3 Hz, 1H), 6.75 (d,
J=8.7 Hz, 2H), 6.51 (d, J=8.7 Hz, 2H), 4.61 (s, 1H), 3.11 (d, J=4.9 Hz,
4H), 3.06 (d, J=4.5 Hz, 4H); 13C NMR (151 MHz, [D6]DMSO): δ=
1H), 8.39 (d, J=4.7 Hz, 2H), 8.36–8.21 (m, 1H), 8.02–7.75 (m, 2H),
7.71 (dd, J=14.7; 2.1 Hz, 1H), 7.47 (dd, J=8.7; 1.3 Hz, 1H), 7.10 (t,
J=9.3 Hz, 1H), 6.66 (t, J=4.7 Hz, 1H), 3.91–3.87 (m, 4H), 3.07–3.03
(m, 4H); 13C NMR (151 MHz, [D6]DMSO): δ=162.7, 161.2, 158.0,
155.1 (d, JC-F =243.3 Hz, 1C), 136.1 (d, JC-F =9.0 Hz, 1C), 135.1, 133.7
162.4, 155.0 (d, JC-F =243.0 Hz, 1C), 142.4, 139.0, 136.7 (d, JC-F
=
(d, JC-F =10.6 Hz, 1C), 132.8, 132.3, 132.0, 124.8, 119.5 (d, JC-F =
3.5 Hz, 1C), 116.4 (d, JC-F =2.5 Hz, 1C), 108.6 (d, JC-F =25.5 Hz, 1C),
108.4, 50.2, 43.3; anal. calcd (%) for C21H18FN7O3: C 57.93, H 4.17, N
22.52; found: C 57.91, H 4.15, N 22.51.
6-((4-(4-Benzoylpiperazin-1-yl)-3-fluorophenyl)carbamoyl)benzo
6-((4-(4-(4-(Ethoxycarbonyl)phenyl)piperazin-1-yl)-3-fluorophen-
yl)carbamoyl)benzo[c][1,2,5]oxadiazole 1-N-oxide (5k). Orange
[c][1,2,5]oxadiazole 1-N-oxide (5p). Yellow solid; yield 30%; mp:
1
°
238–240 C; H NMR (600 MHz, [D6]DMSO): δ=10.59 (s, 1H), 8.44–
1
°
solid; yield 54%; mp: 200–202 C; H NMR (600 MHz, [D6]DMSO): δ=
8.22 (m, 1H), 8.00–7.73 (m, 2H), 7.69 (dd, J=14.6; 2.2 Hz, 1H), 7.48–
4.43 (m, 6H), 7.09 (t, J=9.3 Hz, 1H), 3.85–3.46 (m, 4H), 3.11–2.92 (m,
10.61 (s, 1H), 8.42–8.,24 (m, 1H), 8.08–7.87 (m, 2H), 7.81 (d, J=
8.7 Hz, 2H), 7.72 (dd, J=13.6 Hz, 1H), 7.49 (dd, J=8.6 Hz, 1H), 7.12
(t, J=9.3 Hz, 1H), 7.05 (d, J=8.7 Hz, 2H), 4.24 (q, J=7.0 Hz, 2H),
3.51–3,45 (m, 4H), 3.15–3.12 (m, 4H), 1.29 (t, J=7.1 Hz, 3H); 13C NMR
(151 MHz, [D6]DMSO): δ=165.6, 162.9, 153.8 (d, JC-F =243.3 Hz, 1C),
135.9 (d, JC-F =8.5 Hz, 1C), 135.8, 134.6, 133.7 (d, JC-F =10.5 Hz, 1C),
133.3, 132.6, 131.4, 130.7, 119.4 (d, JC-F =4.0 Hz, 1C), 118.7, 116.5 (d,
4H); 13C NMR (151 MHz, [D6]DMSO): δ=169.0, 162.2, 155.1 (d, JC-F
=
243.3 Hz, 1C), 135.8 (d, JC-F =7.8 Hz, 1C), 135.7, 134.8, 134.6, 133.9,
133.8 (d, JC-F =10.6 Hz, 1C), 129.6, 128.5, 127.0, 119.7 (d, JC-F =3.5 Hz,
1C), 116.4 (d, JC-F =2.6 Hz, 1C), 108.6 (d, JC-F =25.6 Hz, 1C), 50.6, 47.2;
anal. calcd (%) for C24H20FN5O4: C 62.47, H 4.37, N 15.18; found: C
62.47, H 4.35, N 15.17.
J
C-F =2.6 Hz, 1C), 116.4, 113.6, 108.7 (d, JC-F =25.8 Hz, 1C), 59.9, 50.0,
46.6, 14.3; anal. calcd (%) for C26H24FN5O5: C 61.78, H 4.79, N 13.85;
found: C 61.75, H 4.78, N 13.86.
6-((4-(4-(4-Acetylphenyl)piperazin-1-yl)-3-fluorophenyl)
carbamoyl)benzo[c][1,2,5]oxadiazole 1-N-oxide (5l). Pale brown
solid; yield 27%; mp: 225–227 C; H NMR (600 MHz, [D6]DMSO): δ=
10.59 (s, 1H), 8.44–8.23 (m, 1H), 8.04–7.86 (m, 2H), 7.83 (d, J=8.8 Hz,
2H), 7.72 (dd, J=14.7; 1.9 Hz, 1H), 7.49 (d, J=8.5 Hz, 1H), 7.12 (t, J=
9.3 Hz, 1H), 7.04 (d, J=8.9 Hz, 2H), 3.52–3.48 (m, 4H), 3.15–3.12 (m,
4H), 2.46 (s, 3H); 13C NMR (151 MHz, [D6]DMSO): δ=195.7, 162.7,
155.1 (d, JC-F =243.5 Hz, 1C), 135.9 (d, JC-F =8.9 Hz, 1C), 135.8, 135.1,
134.6, 133.7 (d, JC-F =10.6 Hz, 1C), 132.8, 132.0, 130.1, 119.4 (d, JC-F
3.7 Hz, 1C), 116.5 (d, JC-F =2.6 Hz, 1C), 116.4, 113.3, 108.6 (d, JC-F
1
°
=
=
25.5 Hz, 1C), 50.0, 46.8, 26.1; anal. calcd (%) for C25H22FN5O4: C 63.15,
H 4.66, N 14.73; found: C 63.15, H 4.65, N 14.75.
General procedure for the synthesis of compounds 11a-11f
6-((3-Fluoro-4-(4-(pyridin-4-yl)piperazin-1-yl)phenyl)carbamoyl)
A solution of the corresponding carboxylic acid 6, 7, 8, 9, or 10
(compound 6=100 mg, 0.61 mmol; compound 7=100 mg,
0.62 mmol; compound 8=100 mg, 0.61 mmol; compound 9=
100 mg, 0.52 mmol; or compound 10=100 mg, 0.54 mmol) was
stirred into 10 mL of anhydrous acetonitrile under nitrogen
atmosphere, followed by the addition of CDI (1.0 equiv. of the
corresponding carboxylic acid 6, 7, 8, 9, or 10). The reaction mixture
was stirred for 90 min at room temperature, after which a solution
of the corresponding amine derivative 3n (0.6 equiv. of the
corresponding carboxylic acid 6, 7, 8, 9, or 10) in anhydrous
acetonitrile (1 mL) was added dropwise. The mixture was stirred at
room temperature for 48 h under nitrogen, and then the solvent
was removed under reduced pressure. The obtained crude was
washed with ethyl acetate. The solid reaction product was purified
using flash chromatography on silica (EtOAc/petroleum ether,
20:80 to 70:30), producing the final compounds 11a–11f in
variable yields as described below.
benzo[c][1,2,5]oxadiazole 1-N-oxide (5m). Orange solid; yield
60%; mp: 222–225 C; 1H NMR (600 MHz, [D6]DMSO): δ=10.65 (s,
°
1H), 8.43–8.28 (m, 1H), 8.23 (t, J=5.8 Hz, 2H), 8.02–7.82 (m, 2H),
7.73 (d, J=14.6 Hz, 1H), 7.50 (d, J=8.5 Hz, 1H), 7.12 (t, J=9.2 Hz,
1H), 7.03 (d, J=6.3 Hz, 2H), 3.65–3.60 (m, 4H), 3.15–3.10 (m, 4H); 13
C
NMR (151 MHz, [D6]DMSO): δ=162.8, 155.3 (d, JC-F =243.2 Hz, 1C),
153.5, 146.3, 135.6 (d, JC-F =8.7 Hz, 1C), 133.9, 133.8 (d, JC-F =10.6 Hz,
1C), 133.0, 132.0, 124.8, 119.5 (d, JC-F =3.9 Hz, 1C), 116.5 (d, JC-F
=
3.0 Hz, 1C), 116.4, 108.1 (d, JC-F =25.4 Hz, 1C), 108.0, 49.8, 45.6; anal.
calcd (%) for C22H19FN6O3: C 60.82, H 4.41, N 19.35; found: C 60.86, H
4.44, N 19.36.
6-((3-Fluoro-4-(4-(pyridin-2-yl)piperazin-1-yl)phenyl)carbamoyl)
benzo[c][1,2,5]oxadiazole 1-N-oxide (5n). Orange solid; yield 50%;
mp: 196–197 C; 1H NMR (600 MHz, [D6]DMSO): δ=10.59 (s, 1H),
°
8.41–8.22 (m, 1H), 8.14 (dd, J=4.8; 1.2 Hz, 1H), 8.02–7.75 (m, 2H),
7.71 (dd, J=14.7; 2.1 Hz, 1H), 7.59–7.53 (m, 1H), 7.48 (dd, J=8.7;
1.4 Hz, 1H), 7.11 (t, J=9.3 Hz, 1H), 6.89 (d, J=8.6 Hz, 1H), 6.67 (dd,
J=6.8; 5.1 Hz, 1H), 3.66–3.62 (m, 4H), 3.11–3.07 (m, 4H); 13C NMR
(151 MHz, [D6]DMSO): δ=162.6, 158.9, 155.1 (d, JC-F =243.1 Hz, 1C),
N-(3-Fluoro-4-(4-(pyridin-2-yl)piperazin-1-yl)phenyl)benzo
[c][1,2,5]oxadiazole-5-carboxamide (11a). Yellow solid; yield 56%;
mp: 244–247 C; 1H NMR (600 MHz, [D6]DMSO): δ=12.03 (s, 1H),
°
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8.70 (s, 1H), 8.13 (ddd, J=4.9; 1.9; 0.6 Hz, 1H), 7.64 (m, 1H), 7.55
(ddd, J=9.5; 5.8; 2.2 Hz, 1H), 7.45 (dd, J=14.9; 2.3 Hz, 1H), 7.06 (dd,
J=8.7; 2.2 Hz, 1H), 7.01 (t, J=9.2 Hz, 1H), 6.88 (d, J=8.7 Hz, 1H),
6.85 (d, J=8.6; 0.7 Hz, 1H), 6.66 (dd, J=7.1; 5.3 Hz, 1H), 3.64–3.61
(m, 4H), 3.05–3.01 (m, 4H); 13C NMR (151 MHz, [D6]DMSO): δ=162.4,
159.0, 155.7, 154.1 (d, JC-F =242.7 Hz, 1C), 152.4, 147.6, 137.6, 135.1
(d, JC-F =10.9 Hz, 1C), 134.2 (d, JC-F =9.3 Hz, 1C)124.1, 120.7, 119.7 (d,
6-((4-(4-(Pyridin-2-yl)piperazin-1-yl)phenyl)carbamoyl)benzo
[c][1,2,5]oxadiazole 1-oxide (11f). Orange solid; yield 71%; mp:
1
°
227–229 C; H NMR (600 MHz, [D6]DMSO): δ=10.41 (s, 1H), 8.48–
8.22 (m, 1H), 8.14 (d, J=3.3 Hz, 1H), 8.06–7.74 (m, 2H), 7.65 (d, J=
8.7 Hz, 2H), 7.56 (t, J=7.1 Hz, 1H), 7.02 (d, J=8.7 Hz, 2H), 6.90 (d,
J=8.4 Hz, 1H), 6.69–6.65 (m, 1H), 3.67–3.60 (m, 4H), 3.25–3.19 (m,
4H); 13C NMR (151 MHz, [D6]DMSO): δ=164.2, 159.0, 147.8, 147.6,
137.6, 135.8, 132.5, 130.7, 130.6, 128.2, 127.8, 121.5, 115.9, 113.2,
107.3, 48.4, 44.6; anal. calcd (%) for C22H20N6O3: C 63.45, H 4.84, N
20.18; found: C 63.49, H 4.86, N 20.20.
J
C-F =4.0 Hz, 1C), 114.2 (d, JC-F =2.7 Hz, 1C), 113.2, 109.5, 107.2,
107.1, 106.7 (d, JC-F =25.6 Hz, 1C), 50.4, 44.8; anal. calcd (%) for
C22H19FN6O2: C 63.15, H 4.58, N 20.08; found: C 63.13, H 4.55, N
20.09.
N-(3-Fluoro-4-(4-(pyridin-2-yl)piperazin-1-yl)phenyl)-1H-indole-5-
Determination of minimal inhibitory concentration (MIC90)
1
°
carboxamide (11b). Off-white solid; yield 51%; mp: 211–215 C; H
According to Palomino et al.,[53] the resazurin microtiter assay
(REMA) was developed to determine the minimum inhibitory
concentration (MIC90) of the compounds against the standard
(H37Rv ATCC 27294) and clinical isolates of M. tuberculosis. Briefly,
10 mg/mL solution of the compound in DMSO was diluted in the
Middlebrook 7H9 broth supplemented with OADC (oleic acid,
albumin, dextrose, and catalase) and 0.5% glycerol, in a concen-
tration range of 0.09 to 25 μg/mL in a 96-well microplate. The
bacterial inoculum was cultured for 14 days (for standard strain) or
20 days (for clinical strains) until the optical density of McFarland
no. 1 standard was reached, which was then adjusted to 105 CFU/
mL, following which the culture was added to the microplates and
NMR (600 MHz, [D6]DMSO): δ=11.40 (s, 1H), 10.16 (s, 1H), 8.25 (s,
1H), 8.14 (dd, J=4.8; 1.9 Hz, 1H), 7.78 (dd, J=15.0; 2.3 Hz, 1H), 7.72
(dd, J=8.5; 1.6 Hz, 1H), 7.58–7.54 (m, 1H), 7.52 (dd, J=8.7; 1.8 Hz,
1H), 7.48 (d, J=8.5 Hz, 1H), 7.47- 7.46 (m, 1H), 7.08 (t, J=9.4 Hz, 1H),
6.89 (d, J=8.6 Hz, 1H), 6.67 (dd, J=7.1; 4.9 Hz, 1H), 6.59–6.58 (m,
1H), 3.66–3.63 (m, 4H), 3.09–3.06 (m, 4H); 13C NMR (151 MHz, [D6]
DMSO): δ=166.4, 159.0, 155.2 (d, JC-F =242.5 Hz, 1C), 147.6, 137.6,
135.2 (d, JC-F =8.9 Hz, 1C), 135.1 (d, JC-F =10.7 Hz, 1C), 127.0, 125.5,
120.9, 119.3 (d, JC-F =3.9 Hz, 1C), 116.0 (d, JC-F =2.7 Hz, 1C), 113.2,
111.1, 108.3 (d, JC-F =25.5 Hz, 1C), 107.2, 102.2, 50.3, 44.8; anal. calcd
(%) for C24H22FN5O: C 69.38, H 5.34, N 16.86; found: C 69.37, H 5.37,
N 16.85.
°
incubated at 37 C and 5% CO2 atmosphere for seven days.
N-(3-Fluoro-4-(4-(pyridin-2-yl)piperazin-1-yl)phenyl)-1H-benzo[d]
imidazole-5-carboxamide (11c). Off-white solid; yield 32%; mp:
Subsequently, resazurin was added, and after 24–48 h, the
fluorescence was measured at 530/590 nm. The assays were
performed in triplicate, and MIC90 was defined as the average of the
lowest concentrations [of the compound] that could inhibit the
mycobacterial growth by 90%. In order to determine the resistance
profile, a critical concentration based on a MycoTB Plate readout
was used,[54] with the MICs above this critical concentration
indicating the resistance of the strains to the tested drug. The
critical concentrations used for INH, RFP, MOX/GATI, and AMK were
3.6, 2.4, 5.0, and 6.8 μM, respectively.
1
°
202–205 C; H NMR (600 MHz, [D6]DMSO): δ=13.03 (s, 1H), 10.38 (s,
1H), 8.38 (s, 1H), 8.14 (dd, J=4.9; 1.2 Hz, 2H), 7.85 (dd, J=8.6 Hz,
1H), 7.80 (d, J=15.0 Hz, 1H), 7.68 (dd, J=8.4 Hz, 1H), 7.58–7.54 (m,
2H), 7.08 (t, J=9.3 Hz, 1H), 6.89 (d, J=8.6 Hz, 1H), 6.67 (dd, J=6.7;
5.0 Hz, 1H), 3.66–3.61 (m, 4H), 3.09–3.05 (m, 4H); 13C NMR (151 MHz,
[D6]DMSO): δ=165.8, 159.0, 155.2 (d, JC-F =242.3 Hz, 1C), 147.6,
143.8, 142.4, 135.7 (d, JC-F =8.7 Hz, 1C), 135.3 (d, JC-F =10.5 Hz, 1C),
128.7, 127.9, 122.4, 119.3 (d, JC-F =3.9 Hz, 1C), 116.3, 116.2, 116.1 (d,
J
C-F =2.4 Hz, 1C), 113.2, 108.8 (d, JC-F =26.1 Hz, 1C), 107.2, 50.3, 44.8;
anal. calcd (%) for C23H21FN6O: C 66.33, H 5.08, N 20.18; found: C
66.33, H 5.11, N 20.20.
Cytotoxicity assay
N-(3-Fluoro-4-(4-(pyridin-2-yl)piperazin-1-yl)phenyl)-4-(1H-tetra-
zol-5-yl)benzamide (11d). Off-white solid; yield 71%; mp: 220–
The cytotoxicity of the compounds was determined in a human
pulmonary fibroblast cell line (MRC-5-ATCC CCL-171) using the
microdilution method in a 96-well plate. The cells were cultured
with the DMEM supplemented with 10% fetal bovine serum,
gentamicin sulfate (50 mg/L), and amphotericin B (2 mg/L). Sub-
sequently, a concentration of 2.5×105 cell/mL was seeded in a
1
°
224 C; H NMR (600 MHz, [D6]DMSO): δ=10.46 (s, 1H), 8.19 (d, J=
8.3 Hz, 1H) 8.15 (d, J=8.6 Hz, 1H), 8.14–8.13 (m, 1H), 7.75 (dd, J=
14.8; 2.3 Hz, 1H), 7.51 (ddd, J=8.8; 7.2; 2.0 Hz, 1H), 7.51 (dd, J=8.7;
1.7 Hz, 1H), 7.11 (t, 1H), 6.90 (d, J=8.6 Hz, 1H), 6.67 (dd, J=6.9;
5.2 Hz, 1H), 3.66–3.63 (m, 4H), 3.10–3.07 (m, 4H); 13C NMR (151 MHz,
[D6]DMSO): δ=164.5, 158.9, 155.1 (d, JC-F =243.0 Hz, 1C), 147.5,
137.7, 136.7, 135.3 (d, JC-F =8.9 Hz, 1C), 134.1 (d, JC-F =10.6 Hz, 1C),
128.7, 127.3, 126.9, 119.3 (d, JC-F =3.8 Hz, 1C), 116.4 (d, JC-F =2.7 Hz,
1C), 113.3, 108.6 (d, JC-F =25.5 Hz, 1C), 107.3, 50.2, 44.8; anal. calcd
(%) for C23H21FN8O: C 62.15, H 4.76, N 25.21; found: C 62.18, H 4.78,
N 25.24.
°
microplate and incubated at 37 C and 5% CO2 atmosphere for
24 h. After the incubation, the compounds were applied in a
concentration range of 0.39–100 μg/mL. After incubation for
another 24 h, resazurin was added and then fluorescence was
measured at 530/590 nm in a Sinergy H1 microplate reader
(BioTek). The assays were performed in triplicate and IC50 was
defined as the lowest concentration [of the compound] that could
inhibit 50% of the inoculum.[56]
4-Amino-N-(3-fluoro-4-(4-(pyridin-2-yl)piperazin-1-yl)phenyl)-3-ni-
1
°
trobenzamide (11e). Yellow solid; yield 20%; mp: 199–205 C; H
NMR (600 MHz, [D6]DMSO): δ=10.23 (s, 1H), 8.71 (s, 1H), 8.14 (s,
1H), 7.97 (d, J=8.4 Hz, 1H), 7.87 (s, 2H), 7.56 (t, J=7.2 Hz, 1H), 7.46
(d, J=8.3 Hz, 1H), 7.09 (s, 1H), 7.07 (d, J=9.7 Hz, 1H), 6.69–6.65 (m,
1H), 3.65–3.62 (m, 4H), 3.09–3.06 (m, 4H); 13C NMR (151 MHz, [D6]
DMSO): δ=163.3, 159.0, 155.2 (d, JC-F =242.7 Hz, 1C), 148.0, 147.6,
137.6, 135.5 (d, JC-F =8.9 Hz, 1C), 134.4 (d, JC-F =10.8 Hz, 1C), 134.3,
125.9, 121.1, 119.2 (d, JC-F =3.5 Hz, 1C), 119.0, 116.3 (d, JC-F =2.5 Hz,
1C), 113.2, 107.2 (d, JC-F =25.5 Hz, 1C), 50.2, 44.8; anal. calcd (%) for
C22H21FN6O3: C 60.54, H 4.85, N 19.26; found: C 60.55, H 4.88, N
19.28.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
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Keywords: antitubercular agents · benzofuroxan · multidrug-
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Manuscript received: November 18, 2020
Revised manuscript received: January 6, 2021
Accepted manuscript online: January 6, 2021
Version of record online: February 9, 2021
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