Azaaurones as Potent Antimycobacterial Agents Active against MDR- and XDR-TB

Article abstract of DOI:10.1002/cmdc.201900289

Herein we report the screening of a small library of aurones and their isosteric counterparts, azaaurones and N-acetylazaaurones, against Mycobacterium tuberculosis. Aurones were found to be inactive at 20 μm, whereas azaaurones and N-acetylazaaurones emerged as the most potent compounds, with nine derivatives displaying MIC₉₉ values ranging from 0.4 to 2.0 μm. In addition, several N-acetylazaaurones were found to be active against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical M. tuberculosis isolates. The antimycobacterial mechanism of action of these compounds remains to be determined; however, a preliminary mechanistic study confirmed that they do not inhibit the mycobacterial cytochrome bc1 complex. Additionally, microsomal metabolic stability and metabolite identification studies revealed that N-acetylazaaurones are deacetylated to their azaaurone counterparts. Overall, these results demonstrate that azaaurones and their N-acetyl counterparts represent a new entry in the toolbox of chemotypes capable of inhibiting M. tuberculosis growth.

Full text of DOI:10.1002/cmdc.201900289

Accepted Article
Title: Azaaurones as potent antimycobacterial agents active against
MDR- and XDR-TB
Authors: Andre Campaniço, Marta P Carrasco, Mathew Njoroge,
Ronnett Seldon, Kelly Chibale, João Perdigão, Isabel
Portugal, Digby F Warner, Rui Moreira, and Francisca Lopes
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Azaaurones as potent antimycobacterial agents active against
MDR- and XDR-TB
André Campaniço,a,# Marta P. Carrasco,a,# Mathew Njoroge,b Ronnett Seldon,d Kelly Chibale,c,d,e João
Perdigão,a Isabel Portugal,a Digby F. Warner,e,f,g Rui Moreira,a Francisca Lopes*,a
[a]
A. Campaniço 0000-0002-0560-4597, Dr. M. P. Carrasco 0000-0002-7775-2919, Dr. J. Perdigão, Prof. I. Portugal, Prof. R. Moreira, Prof. F. Lopes 0000-
0002-5350-147X
Instituto de Investigação do Medicamento (iMed.ULisboa)
Faculdade de Farmácia, Universidade de Lisboa
Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
E-mail: fclopes@ff.ulisboa.pt
[b]
[c]
[d]
[e]
[f]
Dr. M. Njoroge
Division of Clinical Pharmacology, Department of Medicine
Drug Discovery and Development Centre (H3D), University of Cape Town
Observatory 7925, South Africa
Prof. K. Chibale
Department of Chemistry
University of Cape Town
Rondebosch 7701, South Africa
R. Seldon, Prof. K. Chibale
Department of Chemistry
South African Medical Research Council Drug Discovery and Development Research Unit, University of Cape Town
Rondebosch 7701, South Africa
Prof. K. Chibale, Prof. D. F. Warner
Institute of Infectious Disease and Molecular Medicine
University of Cape Town
Rondebosch 7701, South Africa
Prof. D. F. Warner
Department of Pathology
SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, University of Cape Town
Rondebosch 7701, South Africa
[g]
Prof. D. F. Warner
Wellcome Centre for Infectious Diseases Research in Africa
University of Cape Town
Rondebosch 7701, South Africa
#
These authors contributed equally for this work
Corresponding author
*
Supporting information for this article is given via a link at the end of the document.
Abstract: Herein we report the screening of a small library of
aurones and their isosteric counterparts, azaaurones and N-
acetylazaaurones, against Mycobacterium tuberculosis. Aurones
were inactive at 20 µM, while azaurones and N-acetylazaaurones
emerged as the most potent compounds, with nine derivatives
displaying MIC99 values ranging from 0.4 to 2.0 μM. In addition,
several N-acetylazaaurones were found to be active against
multidrug resistant (MDR) and extensively drug resistant (XDR)
clinical M. tuberculosis isolates. The anti-mycobacterial mechanism
of action of these compounds remains to be determined, however a
preliminary mechanistic study confirmed that they do not inhibit the
mycobacterial cytochrome bc1 complex. Additionally, microsomal
metabolic stability and metabolite identification studies revealed that
N-acetylazaaurones are deacetylated to their azaaurone
counterparts. Overall, these results demonstrate that azaaurones
and their N-acetyl counterparts represent a new entry in the toolbox
of chemotypes capable of inhibiting M. tuberculosis growth.
Introduction
Tuberculosis (TB) stands as one of the most lethal infectious
diseases worldwide, ranking above HIV and malaria. It is
estimated that 10 million people developed TB in 2017, and that
TB caused more than 1.5 million deaths in the same period.[1]
The current complexity and duration of the treatment often leads
to misuse, mismanagement and poor adherence. These factors
collectively contribute to an increased disease burden and the
emergence of drug resistance.[2] Multidrug-resistant tuberculosis
(MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB)
pose serious health threats, with strains resistant to first-line and
multiple second-line antibiotics. [3] It is now estimated that MDR-
TB and XDR-TB account for 21% and 2%, respectively, of all TB
cases worldwide, [1] and these estimates are likely to increase as
resistant strains spread and proliferate.[4] It is also noteworthy
that reports suggest a new emerging threat, totally drug-resistant
tuberculosis (TDR-TB), which is resistant to every known
treatment.[5] In the past forty years, only two drugs from novel
1
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chemical classes were approved: bedaquiline, 1 (FDA) and
delamanid, 2 (EMA) (Fig. 1), both active against MDR-TB.[6]
However, these drugs present several drawbacks, including
common adverse effects such nausea, arthralgia and cardiac
our library comprising 70 aurones, azaaurones and their N-
acetyl precursors against M. tuberculosis. Azaaurones and N-
acetylazaaurones were found to be significantly more potent
than their aurone counterparts. The most potent compounds
were also tested against XDR- and MDR-TB strains. To better
understand the pharmacokinetic behaviour of the azaaurone
scaffold, preliminary in vitro ADME characterization was also
performed.
Results and Discussion
Chemistry
Aurones, 3, were previously developed by our group in order to
probe their activities against Plasmodium falciparum. The
synthesis of aurones involved the aldol condensation of
toxicity that limit their clinical usefulness. [7]
benzofuranones
5
with the appropriately substituted
benzaldehydes,[17] leading to the desired compounds with the
more stable Z configuration (Scheme 1).[14a] Microwave assisted
Suzuki-Miyaura and Buchwald-Hartwig cross-coupling reactions
were also used to decorate the aurone scaffold.[18]
Figure 1. Recent FDA approved drugs bedaquiline, 1, and
delamanid, 2, and chemical structures of aurones, 3, and
azaaurones, 4.
Azaaurone derivatives, 4, were synthesized as reported
previously and as depicted in Scheme 2.[16b] The N-acetyl
precursors, 10, were obtained as mixtures of E and Z isomers,
while hydrolysis of 10 generated the thermodynamically more
stable Z isomer of 4.
Discovering new TB drugs remains a challenging task. Despite
success in identifying active compounds through phenotypic
screenings,[8] the conversion of hits into novel optimized leads
suitable for selection as clinical candidates is hampered by the
poor efficacy in eliminating M. tuberculosis within different host
compartments, including macrophages, as well as a lack of
knowledge about the specific target(s) inhibited and/or
upregulated.[9] Furthermore, there is growing recognition that
current TB screening programmes are characterized by
significant redundancy in the nature of the compounds screened
and, consequently, in the number (and variety) of mycobacterial
targets identified.[10] Many high-throughput screening (HTS)
campaigns performed so far have identified small, hydrophobic
molecules affecting the function of essential membrane proteins
such
as
DprE1
(subunit
of
the
heteromeric
AtpE
decaprenylphosphoryl-beta-D-ribose-2’-epimerase),
(membrane ATP synthase), MmpL3 (Trehalose monomycolate
exporter) and QcrB (subunit of the cytochrome c oxireductase,
bc1 complex).[11] This highlights the prospect that highly
lipophilic hit compounds may lead to toxicity that might be linked
to inhibition of host membrane proteins or menaquinol
membrane integrity. Interestingly, bedaquiline has been found to
target the membrane protein AtpE, and its high lipophilicity (logP
ca. 7) might be related to the liver and heart toxicity displayed by
this compound.[12]
Scheme 1. (i) Al2O3, MeOH, reflux under N2, 48 hours; (ii) glacial AcOH, HCl,
rt, 4 hours; (iii) Tf2O, TEA, DCM.
Aurones, 3 (Fig. 1), are heterocyclic compounds of the flavonoid
family, with a benzofuran moiety linked to a benzylidene group
at position C-2.[13] This scaffold has been studied for a variety of
biological applications,[14] including as antimycobacterial
agents.[15] However, the structure-activity relationship (SAR)
studies of aurones are limited as a result of the narrow range of
potencies displayed by these compounds. As a strategy to
enrich the SAR, we hypothesised that the isosteric azaaurones,
4, (Fig. 1), which present stereoelectronic and hydrogen-bonding
properties significantly different from those of aurones, would
broaden the range of potencies against M. tuberculosis.
Although previously studied as antimalarial agents,[16] to the best
of our knowledge azaaurones have not been screened for
activity against M. tuberculosis. We now report the screening of
Scheme 2. (i) a) BCl3 1M in dichloromethane, chloroacetonitrile, ZnCl2, dry
1,2-DCE, reflux; b) HCl 1M, reflux; (ii) AcOH, Ac2O; (iii) NaH, dry DMF; (iv)
ArCHO, piperidine, toluene, reflux; (v) KOH (50% in H2O), MeOH, rt.
Activity against Mycobacterium tuberculosis H37Rv
Aurones, 3, azaaurones, 4, and N-acetylazaaurones, 10, were
screened against the virulent M. tuberculosis H37Rv strain, and
the corresponding MIC90 and MIC99 values are presented in
2
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Tables 1-3. Cytotoxicity for selected compounds was determined
against human embryonic kidney (HEK) 293T cells.
Table 1. In vitro antimycobacterial activities and cytotoxicity of aurone derivatives 3a-3y.
Cpd
3a
3b
3c
3d
3e
3f
R1
H
R2
H
R3
4-Br
H
R4
H
H
Br
H
H
H
H
H
H
H
H
H
H
R5
H
Br
H
H
H
H
H
H
H
H
H
H
H
MIC90 (μM)
>20
MIC99 (μM)
>20
H
H
>20
>20
H
H
H
>20
>20
H
OMe
H
H
>20
>20
OH
OH
H
H
>20
>20
H
>20
>20
3g
3h
3i
H
>20
>20
H
H
>20
>20
H
H
>20
>20
3j
H
H
>20
>20
3k
3l
H
H
>20
>20
H
H
>20
>20
3m
H
H
>20
>20
3n
H
H
H
H
>20
>20
3o
3p
3q
3r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>20
>20
>20
>20
>20
>20
>20
>20
3
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3s
3t
H
H
H
H
H
H
H
H
H
H
H
H
>20
>20
>20
>20
>20
>20
3u
3v
OH
H
H
H
>20
>20
3w
3x
Rif
OH
OH
H
H
-
H
H
H
H
>20
>20
>20
>20
-
0.00291
0.00482
Table 2. In vitro antimycobacterial activities and cytotoxicity of azaaurones derivatives 4a-4u.
MIC90
H37Rv
(μM)
MIC99
H37Rv
(μM)
R1
R2
R3
HEK293T
(µM)
Compd
4a
4b
H
H
H
H
Br
6.5
7.56
2.4
>100
>100
1.78
4c
4d
4e
4f
H
H
H
H
H
H
H
H
1.3
1.5
>100
>100
>100
>100
2.47
1.27
1.28
2.74
1.44
1.53
4g
4h
H
H
H
H
>20
>20
>20
>20
>100
>100
N(Me)2
4i
4j
H
H
H
H
>20
>20
>100
>100
1.21
1.36
4k
4l
H
H
H
H
H
H
>20
>20
3.07
>20
>20
4.09
>100
>100
>100
4m
4
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4n
4o
4p
4q
H
H
H
H
H
H
H
H
>20
15.9
7.37
>20
>20
>20
10.6
>20
>100
>100
20
>100
4r
4s
4t
H
H
H
H
H
H
H
>20
>20
>20
>20
>20
>20
>100
>100
>100
4u
H
-
>20
>20
>100
-
Rif
-
0.00291
0.00482
Table 3. In vitro antimycobacterial activities and cytotoxicity of N-acetylazaaurone derivatives 10a-y.
MIC90
H37Rrv
(μM)
MIC99
H37Rv
(μM)
HEK293T
(µM)
Cpd
R1
R2
R3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
0.31
1.32
2.32
1.26
2.71
2.65
1.44
>20
0.37
2.29
4.05
1.98
4.37
4.3
ND
ND
ND
7
10a
10b
10c
10d
10e
10f
9
ND
ND
8
3.19
>20
10g
10h
N(CH3)2
H
H
H
H
0.649
0.336
0.736
0.6
19
10i
10j
ND
5
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H
H
H
H
H
H
3.16
1.89
6.2
3.66
2.58
8.4
ND
ND
ND
10k
10l
10m
H
H
H
H
H
H
H
H
H
H
4.49
5.1
4.94
5.66
7.85
17.5
>20
ND
ND
4
10n
10o
10p
10q
10r
6.44
11.6
>20
ND
ND
H
H
H
H
H
2.65
3.19
4.93
3.13
3.34
5.61
14
10s
10t
10u
ND
ND
H
H
H
H
1.24
6.02
>20
1.39
6.87
>20
12
10v
10w
10x
Cl
Cl
ND
ND
H
H
Me
>20
>20
ND
10y
Examination of the data in Table 1 reveals that all the aurone
derivatives (3a-y) were found to be inactive against M.
tuberculosis at the maximum concentration tested, 20 µM. The
absence of active aurones in our library is consistent with other
sreening campaigns. For example, the screening of a kinase-like
inhibitor library, which contained ca. 60 aurones, identified only
three hits based on this scaffold with reproducible anti-TB
activity.[15a] In contrast, several azaaurones, 4 (Table 2), and N-
acetylazaaurones, 10 (Table 3), emerged as active
antimycobacterials, with MIC90 and MIC99 values under 10 µM,
the cut-off value used in this study to select hits for further
validation and optimization.[8c] Analysis of data presented in
Tables 2 and 3 allows the following conclusions to be drawn:
First, N-acetylazaaurones generally exhibited higher potency
than the corresponding azaaurones (e.g. 10a vs 4a, 10i vs 4i,
10k vs 4k, 10v vs 4u), a result suggesting that the more
lipophilic N-acetylazaaurones 10 may cross the mycobacterial
cell wall more efficiently than their deacetylated counterparts 4.
However, the different hydrogen-bonding properties of
compounds 4 and 10 may also affect the antimycobacterial
activity.
Second, substitution at C-4 of the B-ring is beneficial for the
antimycobacterial activity when compared with substitution at C-
3, both in N-acetylazaaurones (e.g. 10b vs 10n, 10c vs 10p, 10f
vs 10q, 10g vs 10r, 10i vs 10s, 10j vs 10t) as well as in
azaaurones (4b vs 4n, 4f vs 4q, and 4j vs 4s). The most potent
6
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10o
10p
10q
10s
10t
5.66
7.85
17.5
3.13
3.34
5.61
1.39
6.87
5
5
10
5
compounds contained electron-withdrawing and/or lipophilic
substituents at C-4 (e.g. 10a, 10i and 10j).
Third, substitution at C-5 in ring A of N-acetylazaaurones was
tolerated (e.g. 10m vs 10w).
Forth, azaaurones were shown to be non-cytotoxic against
HEK293T cells, with CC50 values >100 μM, while their N-acetyl
counterparts displayed CC50 values ranging from 4 to 20 μM,
thus indicating selectivity against M. tuberculosis.
5
>10
2.5
5
2.5
5
10u
10v
10w
5
5
1.25
5
2.5
10
Activity against Mycobacterium tuberculosis cydKO mutant
Azaaurones were reported as potential inhibitors of P.
falciparum cytochrome bc1,
a
key component of the
Activity against drug-resistant clinical M. tuberculosis
mitochondrial electron-transport chain (mtETC).[16b] Cytochrome
bc1 complex is also an essential component of the respiratory
electron transport chain required for ATP synthesis in M.
isolates
A set of selected N-acetyl azaaurones was evaluated for the
ability to suppress the growth of various patient-derived,
genotyped drug-resistant (DR), multidrug resistant (MDR) and
extensively drug resistant (XDR) M. tuberculosis clinical strains,
which are part of the iMed.ULisboa’s pathogenic mycobacterial
strain collection (Table 5). Although there are differences in the
assay methods to determine the inhibitory concentrations of the
compounds against the bacterial pathogen in the primary
screening and the poly-resistant strains, these methods have
been extensively standardized. Pleasingly, we found that
selected compounds were active against all resistant mutants,
being equipotent against wild type, MDR and XDR isolates.
tuberculosis and was identified as
a
high-confidence
[19]
antimycobacterial drug target.
The cytochrome bc1 transfers
electrons from menaquinol to the cytochrome c oxidase, a
process linked to proton translocation across the membrane and
that ultimately is the most energetically favorable respiratory
pathway in mycobacteria.[20] Hence, N-acetylazaaurones with
MIC99 < 20 μM were further assayed in order to ascertain
whether the respiratory pathway of M. tuberculosis could be the
potential target for these compounds. The assay used a M.
tuberculosis cytochrome bd oxidase deletion mutant cydKO,
which is hypersusceptible to compounds such as
imidazopyridines that target the QcrB subunit of the qcrCAB-
encoded cytochrome bc1 complex.[20a,21] No MIC shift was
observed when the cydKO mutant was tested against all
selected N-acetylazaaurones (Table 4). The lack of
hypersensitivity observed in the cydKO mutant strongly suggests
that QcrB does not constitute the primary target for N-
acetylazaaurones.[22]
Table 5. Antimycobacterial activity against drug resistant (DR, resistant to
isoniazid), multidrug resistant (MDR, resistant to isoniazid, rifampicin,
ethambutol, pyrazinamide, capreomycin, and ethionamide), and extensively
resistant (XDR, resistant to isoniazid, rifampicin, ethambutol, pyrazinamide,
streptomycin, capreomycin, amikacin, kanamycin, ofloxacin, and ethionamide)
M. tuberculosis clinical isolates.
Table 4. Antimycobacterial activity against the M. tuberculosis cydKO mutant
lacking a cytochrome bd oxidase. Data determined at day 7 and day 14 in
GAST/Fe media.
MIC (µM)
Compound
10a
XDR-TB
15.2
MDR-TB
9.15
DR-TB
15.2
H37Rv
15.2
10b
12.3
12.3
12.3
12.3
MIC99
H37Rrv
(μM)
MIC99 at day
14 (µM)
cydKO
MIC99 at day 7
(µM) cydKO
Compd
10a
10c
8.37
10.4
8.37
8.37
10e
17.7
17.7
17.7
14.8
0.37
1.25
1.25
10j
7.55
7.55
7.55
7.55
10b
10c
10d
10e
2.29
4.05
1.98
4.37
1.25
2.5
1.25
10
Rifampicin
>12.2
>12.2
0.377
0.377
1.25
2.5
1.25
2.5
In vitro metabolic studies.
A microsomal metabolic stability study was performed on
selected azaaurones and N-acetylazaaurones by incubating
compounds in mouse liver microsomes activated in the
presence of NADPH, at 37 ºC. Samples were analyzed by LC-
MS/MS to determine the percentage of compounds remaining
after 30 min of incubation.
Inspection of the results presented in Table 6 reveals that
selected N-acetylazaaurones, 10, underwent extensive NADPH-
dependent degradation, with only 10 to 20% of the parent
compounds remaining after 30 minutes of incubation. In contrast,
selected compounds 4 were shown to be significantly more
10f
10g
10i
4.3
3.19
0.736
0.6
2.5
1.25
1.25
1.25
2.5
5
2.5
5
1.25
1.25
5
10j
10k
10l
3.66
2.58
8.4
5
10m
10n
5
5
4.94
2.5
2.5
7
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stable in mouse liver microsomes. Interestingly, the most
metabolically stable compound was the 3-thienyl azaaurone 4j.
Analysis of GlaxoSmithKline corporate database revealed that
thiophene-containing compounds exhibited the highest CYP450
inhibition when compared to other 5-membered and 6-
membered heterocycles,[23] and thus the predicted EH value of
0.37 for 4j may reflect, at least partially, inhibition of this
metabolizing enzyme system.
Figure 2. Metabolism of azaaurones and N-acetylazaaurones in
mouse liver microsomes.
Conclusions
To better understand the metabolic pathway associated with
azaaurones and N-acetylazaaurones, a metabolite identification
study using LC-MS was carried out for compounds 4b, 10j and
10v. Both compounds 10j and 10v underwent NADPH-
independent deacetylation to form 4j and 4u, respectively (Fig.
2A), as revealed by the peaks at m/z M-43 in samples incubated
in the presence or absence of NADPH. Various hydrolases are
known to be present in microsomal preparations and may
contribute to this metabolic activation. Importantly, deacetylation
also occurred in pH 7.4 buffer, but at a lower extent than in
microsomes.
For the azaaurone 4b, the metabolites were tentatively identified
by comparison of the fragmentation patterns of the parent
compound with those from the incubation samples. The
fragmentation pattern of 4b showed two peaks, one at m/z 297
corresponding to the molecular ion and the other at m/z 165
corresponding to the biaryl side chain (Fig. 2B). After incubation
with microsomes, the fragment at m/z 165 shifted to m/z 181,
suggesting that hydroxylation had occurred on the side chain of
the compound.
There is an urgent need for new antimycobacterial agents
preferably with novel mechanisms of action in order to tackle
drug-resistant M. tuberculosis infection.[24] This work shows that
azaaurones and their N-acetyl counterparts represent a new
entry in the toolbox of chemotypes capable of inhibiting M.
tuberculosis growth. In particular, N-acetylazaaurones 10a, 10i
and 10j were found to be active against M. tuberculosis with
MIC99 values in the submicromolar range, with 10i showing
excellent activity against MDR and XDR-TB from clinical isolates.
The microsomal metabolic stability and metabolite identification
studies showed that N-acetylazaaurones are rapidly and
extensively deacetylated to their azaaurone counterparts.
Moreover, a preliminary mechanistic study revealed that these
compounds do not target the QcrB subunit of the mycobacterial
cytochrome bc1 complex, therefore eliminating one of several
“promiscuous” targets increasingly identified in screening
campaigns in vitro.[11h] Current work in our laboratory aims to
elucidate the precise mechanism of action. Therefore, despite
the fact that their molecular targets remain unknown,
azaaurones may serve as leads for the generation of new anti-
mycobacterial agents.
Table 6. In vitro metabolism of selected azaaurones and N-acetylazaaurones
in mouse liver microsomes.
Compd
% remaining
(30 min)
CLint
(μl/min/mg)
Predicted
EH
Experimental Section
4b
4c
49
75
78
85
20
16
9
101
38
0.73
0.50
0.46
0.37
0.85
0.68
0.90
0.88
0.89
0.87
0.94
Chemistry
General procedure for the synthesis of aurones 3a, 3b, 3c and
3d: To a solution of benzofuran-3(2H)-one (134 mg, 1 mmol) in
dry methanol (20 mL) at room temperature was added the
appropriate aldehyde (1.2 mmol) and Al2O3 (1 mmol). The
mixture was refluxed, under N2, for 48 hours. After, the solvent
was removed and the solid residue was dissolved in CH2Cl2.
The organic layer was washed with water, dried with anhydrous
Na2SO4 and concentrated under reduced pressure to give the
crude product.
General procedure for the synthesis of aurone 3e: To a solution
of 6-hydroxybenzofuran-3(2H)-one (0.57 mmol) in glacial acetic
acid (5.7 mL) at room temperature was added the appropriate
benzaldehyde (0.68 mmol) and HCl (3 drops). The reaction
mixture was stirred for 4 hours at room temperature. After, the
mixture was dropped in cold water and the precipitate formed
was filtered and washed with water.
4e
33
4j
22
10a
220
261
335
289
293
252
610
10b
10c
10e
12
11
24
<7
10j
10v
Midazolam
General procedure for the synthesis of ethers derivatives 3f: To
a solution of 6-hydroxybenzofuran-3(2H)-one (0.57 mmol) in
glacial acetic acid (5.7 mL) at room temperature was added the
appropriate aldehyde (0.68 mmol) and HCl (cat, 3 drops). The
reaction mixture was stirred for 4 hours at room temperature.
After, the mixture was dropped in cold water and the precipitate
formed was filtered and washed with water.
General procedure for the synthesis of aurones derivatives 3g,
3h, 3i, 3j, 3n, 3o, 3p, 3q, 3r, 3s, 3t and 3u via Suzuki Coupling:
To a solution of the appropriate aurone derivative (0.23 mmol) in
8
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10.1002/cmdc.201900289
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FULL PAPER
dioxane (2.3 mL) was added Pd(PPh3)2Cl2 (0.023 mmol) and
Na2CO3 1M (690 μL) followed by the proper boronic acid (0.28
mmol). The resulting mixture was degassed and stirred at 100oC
for 3 hours under N2. After cooling to room temperature, the
reaction mixture was diluted with CH2Cl2, filtered under celite
and concentrated under pressure to give the crude product.
General procedure for the synthesis of ethers derivatives 3k: To
a solution of benzofuran-3(2H)-one (0.57 mmol) in dry methanol
(10 mL) at room temperature was added the appropriate
aldehyde (0.68 mmol) and Al2O3 (0.57 mmol). The mixture was
refluxed, under N2, for 48 hours. After, the solvent was removed
and the solid residue was dissolved in CH2Cl2. The organic layer
was washed with water, dried with anhydrous Na2SO4 and
concentrated under reduced pressure to give the crude product.
General procedure for the synthesis of aurones derivatives 3l
The compounds to be tested were reconstituted to
concentration of 10 mM in DMSO. Two-fold serial dilutions of the
test compound were prepared in GAST-Fe, across a 96-well
micro titre plate, after which, 50 μL of the 1:100 diluted M.
tuberculosis culture was added to each well in the serial dilution.
The concentration range assayed was 20 – 0.039 µM. The plate
layout was a modification of the method previously described.[27]
Controls used were a minimum growth control (Rifampicin at
2xMIC), and a maximum growth control (5% DMSO in GAST-Fe).
The micro titre plate was sealed in a secondary container and
incubated at 37 °C with 5% CO2 and humidification. Relative
fluorescence (excitation 485 nM; emission 520 nM) was
measured using a plate reader (FLUOstar OPTIMA, BMG
LABTECH), at day 7 and day 14. In the absence of a MIC shift
between day 7 and day 14, day 14 data were analysed and
reported. The raw fluorescence data were archived and
analysed using the CDD Vault from Collaborative Drug
Discovery, in which, data were normalised to the minimum and
maximum inhibition controls to generate a dose response curve
(% inhibition), using the Levenberg-Marquardt damped least-
squares method, from which the MIC90 was calculated.[28] The
lowest concentration of drug that inhibits growth of more than
90 %, and 99%, of the bacterial population was considered to be
the MIC90 and MIC99 respectively.
a
and
3m
via
Buchwald
Coupling:
(Z)-2-(4-
bromobenzylidene)benzofuran-3(2H)-one (5.27) (0.23 mmol),
Pd2(dba)3 (0.0115 mmol), (R)-BINAP (0.075 mmol) and NaOtBu
(0.322 mmol) were dissolved in dry toluene (2.3 mL). The
resulting mixture was degassed and the appropriate amine
(0.276 mmol) was added. The mixture was stirred at 100oC for
15 minutes under MW conditions. After cooling to room
temperature, the reaction mixture was diluted with Et2O, filtered
under celite and concentrated under pressure to give the crude
product.
General procedure for the synthesis of Mannich Bases
derivatives 3v, 3w and 3x: To a solution of (Z)-2-benzylidene-6-
hydroxybenzofuran-3(2H)-one (0.29 mmol) in absolute ethanol
(1 mL) was added the appropriate amine (0.32 mmol) followed
by formaldehyde solution (0.32 mmol). The mixture was refluxed
for 3h30. After, the solvent was removed and the solid residue
was dissolved in CH2Cl2 and extracted with HCl 1M. The
aqueous layer was neutralized with NaHCO3 saturated solution
and extracted with CH2Cl2. The organic layer was dried with
anhydrous Na2SO4 and concentrated under reduced pressure to
give the crude product.
General procedure for the synthesis of azaaurone derivatives
10a to 10y: To a solution of the appropriate 1-acetylindolin-3-
one derivative (0.5 mmol) in toluene (5 mL) at room temperature
was added the proper aldehyde (1.2 mmol) and piperidine (1
drop). The mixture was refluxed for 4 hours. After reaction
completion, the solvent was removed to provide the crude
product.
General procedure for the synthesis of azaaurone derivatives 4a
to 4t: To a solution of the proper acetylated azaaurone
derivative (0.25 mmol) in MeOH (2.5 mL) at room temperature
was added a solution of KOH 50% in water (375 μL). The
mixture was stirred for 45 minutes. After reaction completion, the
reaction mixture was neutralized with extracted with EtOAc. The
organic layer was dried with anhydrous Na2SO4 and
concentrated under reduced pressure to give the crude product.
Antimycobacterial activity against M. tuberculosis clinical
strains
Ten selected compounds were evaluated against three clinical
M. tuberculosis isolates displaying different drug resistance
spectra. All three isolates are part of the iMed.ULisboa M.
tuberculosis strain collection, were previously characterized by
classical genotyping methods (24-loci MIRU-VNTR and
Spoligotyping) as previously described and, have further been
subjected to whole genome sequencing with the raw sequence
data made publicly available at the European Nucleotide Archive
(Supplementary Table S1).[4a, 29] Phenotypic drug susceptibility
testing was also previously performed for these strains through
the standardized BACTEC Mycobacterial Growth Indicator Tube
(MGIT) 960 system (Becton Dickinson Diagnostic Systems,
Sparks, MD, USA) using the standardized procedure according
to the manufacturer’s instructions.[30] Herein, MIC determination
was carried out using the resazurin microtitre assay (REMA), a
non-commercial drug susceptibility testing endorsed by the
WHO, coupled with microscopic examination for growth under
an inverted microscope.[31] Briefly, all compounds were 2-fold
serially diluted in a 96-well microtitre plate as to achieve a final
concentration ranging between 100.00-0.10 μg/ml in
Middlebrook 7H9-S (Middlebrook 7H9 broth supplemented with
0.1% casitone, 0.5% glycerol and 10% OADC supplement).
Positive (no drug) and negative/sterile controls were included at
each plate. The bacterial inoculum was prepared by adjusting a
bacterial suspension to a McFarland turbidity standard 1.0 and
diluted 1:20 before inoculating 100 μl to each well (final volume:
200 μl). Plates were sealed in plastic bags, incubated at 37ºC for
7 days and growth observed under a microscope before addition
of 30 μl of 0.01% resazurin, followed by an additional incubation
period of 24h. Blue to pink colour change was indicative of
bacterial growth through the reduction of resazurin (blue) to
resorufin (pink). Resazurin sodium salt (Sigma-Aldrich) solution
was prepared at a concentration of 0.01%, sterilized by filtration
Antimycobacterial screening against Mycobacterium
tuberculosis H37Rv
The minimum inhibitory concentration (MIC) was determined
using the standard broth micro dilution method, where a 10 mL
culture of M. tuberculosis pMSp12::GFP,[25] was grown to an
optical density (OD600) of 0.6 – 0.7 in GAST-Fe (glycerol–
alanine–salts) medium pH 6.6, supplemented with 0.05%
Tween-80.[26] The culture was then diluted 1:100 in GAST-Fe.
9
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10.1002/cmdc.201900289
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FULL PAPER
using a 0.2μm syringe filter and stored at 4ºC for a maximum
period of one week as recommended.[32] MIC was recorded as
the lowest concentration yielding bacterial growth. All assays
were also performed in parallel for reference strain M.
tuberculosis H37Rv (ATCC 2794) and the activity of rifampicin
(Sigma-Aldrich) also assayed in parallel. All assays were
performed in triplicate and final MIC values are herein expressed
as the result of at least two concordant values.
[1]
[2]
Global Tuberculosis Report 2018, World Health Organization, Genebra,
2018.
a) A. M. Ginsberg and M. Spigelman, Nat Med 2007, 13, 290-294; b) V.
P. Waman, S. C. Vedithi, S. E. Thomas, B. P. Bannerman, A. Munir, M.
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8, 109-118; c) C. Silva, J. Perdigao, E. Alverca, A. P. de Matos, P. A.
Carvalho, I. Portugal and L. Jordao, Microsc Microanal 2013, 19, 1159-
1169.
[3]
[4]
Reported Tuberculosis in the United States, Centers for Disease
Control and Prevention, United States of America, 2017.
Metabolic stability
The metabolic stability study was performed for selected
azaaurones and N-acetylazaaurones in triplicate by incubating
compounds in mouse liver microsomes activated in the
presence of the co-factor, NADPH, using the cosolvent
method.[33] Compounds were incubated for 30 min at 37 ºC and
then the reactions were quenched by adding cold ACN with
carbamazepine as internal standard. The samples were kept at -
20 ºC for 15 min. and then centrifuged. The supernatants were
analyzed by LC-MS/MS using Multiple Reaction Monitoring
(MRM) methods. The % remaining was calculated using the
analyte:internal standard ratios at 0 min and 30 min and
converted to intrinsic clearance and EH.[34]
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Fitzgerald, P. Govender, S. Gujja, S. Hamilton, C. Howarth, J. D.
Larimer, K. Maharaj, M. D. Pearson, M. E. Priest, Q. Zeng, N.
Padayatchi, J. Grosset, S. K. Young, J. Wortman, K. P. Mlisana, M. R.
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Metabolite identification
The compounds (10 µM) were incubated with a solution of
mouse liver microsomes and NADPH (1 mM) in phosphate
saline buffer (100 mM, pH 7.4) and MgCl2 (5 mM) for 1 h while
shaking at 37 ºC. An equal volume of acetonitrile was added at
the end of the incubation to quench the reaction. The mixture
was centrifuged at 14000 rpm for 30 min and the supernatant
was transferred to an HPLC vial. Control samples were prepared
without NADPH or microsomes. The NADPH and the
microsomes volumes were replaced by the same volume of PBS.
T0 samples were quenched with acetonitrile after microsomes
were added and placed at 8ºC for the duration of the incubation.
[6]
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Acknowledgements
This work was supported by projects Pest-OE/SAU/UI4013/2014
(Fundação para a Ciência e Tecnologia (FCT), Portugal) and
PTDC/MED-FAR/30266/2017 (FCT and FEDER). We also
acknowledge FCT for fellowships SFRH/BD/61611/2009 (MC),
SFRH/BD/131896/2017 (AC), SFRH/BPD/95406/2013 (JP) and
SFRH/BSAB/113783/2015 (FL). The University of Cape Town,
South African Medical Research Council, and South African
Research Chairs Initiative of the Department of Science and
Technology, administered through the South African National
Research Foundation are gratefully acknowledged for support
(K.C). This work was also supported by the Strategic Health
Innovation Partnerships (SHIP) initiative of the South African
Medical Research Council with funds from National Treasury
under its Economic Competitiveness and Support Package
(DFW).
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a) C. B. Cooper, J Med Chem 2013, 56, 7755-7760; b) U. H.
Manjunatha and P. W. Smith, Bioorg Med Chem 2015, 23, 5087-5097;
c) L. Ballell, R. H. Bates, R. J. Young, D. Alvarez-Gomez, E. Alvarez-
Ruiz, V. Barroso, D. Blanco, B. Crespo, J. Escribano, R. Gonzalez, S.
Lozano, S. Huss, A. Santos-Villarejo, J. J. Martin-Plaza, A. Mendoza, M.
J. Rebollo-Lopez, M. Remuinan-Blanco, J. L. Lavandera, E. Perez-
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Mattevi, M. R. Pasca, G. Riccardi, L. Ballell, K. Mikušová and L. R.
Chiarelli, ACS Infect Dis 2017, 3, 428-437.
Keywords: azaaurones • drug discovery • tuberculosis •
multidrug-resistant tuberculosis • Mycobacterium tuberculosis
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Entry for the Table of Contents
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The screening of an in-house library against M. tuberculosis H37Rv identified azaaurones as novel antimycobacterial agents. In
particular, several N-acetylazaaurones displayed MIC99 values in the submicromolar range and showed to be active against MDR
and XDR-TB clinical isolates. Although the primary mechanism of action remains undetermined, azaaurones stand as promising
leads for further optimization.
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