Design and Synthesis of Pyrano[3,2-b]indolones Showing Antimycobacterial Activity

Article abstract of DOI:10.1021/acsinfecdis.0c00622

Latent Mycobacterium tuberculosis infection presents one of the largest challenges for tuberculosis control and novel antimycobacterial drug development. A series of pyrano[3,2-b]indolone-based compounds was designed and synthesized via an original eight-step scheme. The synthesized compounds were evaluated for their in vitro activity against M. tuberculosis strains H37Rv and streptomycin-starved 18b (SS18b), representing models for replicating and nonreplicating mycobacteria, respectively. Compound 10a exhibited good activity with MIC99 values of 0.3 and 0.4 μg/mL against H37Rv and SS18b, respectively, as well as low toxicity, acceptable intracellular activity, and satisfactory metabolic stability and was selected as the lead compound for further studies. An analysis of 10a-resistant M. bovis mutants disclosed a cross-resistance with pretomanid and altered relative amounts of different forms of cofactor F420 in these strains. Complementation experiments showed that F420-dependent glucose-6-phosphate dehydrogenase and the synthesis of mature F420 were important for 10a activity. Overall these studies revealed 10a to be a prodrug that is activated by an unknown F420-dependent enzyme in mycobacteria.

Full text of DOI:10.1021/acsinfecdis.0c00622

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Article
Design and Synthesis of Pyrano[3,2‑b]indolones Showing
Antimycobacterial Activity
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Natalia Monakhova, Jana Kordulakova, Anthony Vocat, Anna Egorova, Alexander Lepioshkin,
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Elena G. Salina, Jozef Nosek, Eva Repkova, Julia Zemanova, Helena Jurdakova, Renata Gorova,
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Jaroslav Roh, Giulia Degiacomi, Jose Camilla Sammartino, Maria Rosalia Pasca, Stewart T. Cole,
Katarína Mikusova,* and Vadim Makarov*
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ABSTRACT: Latent Mycobacterium tuberculosis infection presents one of the largest challenges for tuberculosis control and novel
antimycobacterial drug development. A series of pyrano[3,2-b]indolone-based compounds was designed and synthesized via an
original eight-step scheme. The synthesized compounds were evaluated for their in vitro activity against M. tuberculosis strains H37Rv
and streptomycin-starved 18b (SS18b), representing models for replicating and nonreplicating mycobacteria, respectively.
Compound 10a exhibited good activity with MIC99 values of 0.3 and 0.4 μg/mL against H37Rv and SS18b, respectively, as well as
low toxicity, acceptable intracellular activity, and satisfactory metabolic stability and was selected as the lead compound for further
studies. An analysis of 10a-resistant M. bovis mutants disclosed a cross-resistance with pretomanid and altered relative amounts of
different forms of cofactor F420 in these strains. Complementation experiments showed that F420-dependent glucose-6-phosphate
dehydrogenase and the synthesis of mature F420 were important for 10a activity. Overall these studies revealed 10a to be a prodrug
that is activated by an unknown F420-dependent enzyme in mycobacteria.
KEYWORDS: pyrano[3,2-b]indolone, antimycobacterial activity, latent tuberculosis
It thus seems appropriate to develop drugs with potent
efficacy against both active and latent forms of TB. To date,
there are three new molecules (Figure 1), which have good
proven in vitro activity against actively replicating and dormant
Mycobacterium tuberculosis, the infectious agent causing the
disease: bedaquiline, a first-in-class diarylquinoline compound
(FDA/2012) and two molecules from the 5-nitroimidazole
class, delamanid, and pretomanid (EMA/2014 and FDA/2019,
respectively).3−7 However, these drugs were approved only to
treat active multi-drug-resistant TB in combination therapy,
n the 21st century, tuberculosis (TB) remains a major
Ipublic health problem; it is not only one of the top 10
causes of death globally but also the leading cause of death
from a single infectious agent (ranking above HIV). According
to the WHO estimates, about 10 million people fell ill with
tuberculosis and 1.5 million people died from its active form in
2018 worldwide.1 Even though TB treatment with available
drugs is effective in many cases, drug-resistant (particularly
with respect to first-line drug rifampicin) and multi-drug-
resistant TB constitute a major health threat. Nevertheless, it is
only one of the problems of global TB management. An
additional challenge concerns latent TB: about 1.7 billion
people have this form,2 which can convert to active TB disease
under adverse social or health conditions.1 The treatment of
people with latent TB infection should be an important part of
TB course control.
Received: September 1, 2020
Published: December 22, 2020
https://dx.doi.org/10.1021/acsinfecdis.0c00622
© 2020 American Chemical Society
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Figure 1. New approved antituberculosis drugs acting in vitro on both active and dormant Mycobacterium tuberculosis.
and there are no clinical data on their use to treat latent TB
infection.8−10 Despite the unprecedented value of these drugs
for treatment of the most complicated forms of TB, it should
be noted that they have certain limitations. For example,
treatment with bedaquiline or delamanid is associated with QT
prolongation and an increased risk of death; the FDA
recommends the use of bedaquiline or a combination of
these drugs only as salvage therapy.8,9 Furthermore, delamanid
is also one of the most expensive anti-TB drugs, making it
unattractive for use in low-income countries with high rates of
TB (although the price has been reduced for South Africa).11
Pretomanid was approved only for limited use in combination
with bedaquiline and linezolid for the treatment of a specific
type of highly treatment-resistant tuberculosis of the lungs.10
Taken together, the pipeline for the discovery of safe and
inexpensive antituberculosis drugs with efficiency against both
replicating and dormant M. tuberculosis cells remains empty.
During a routine screening of our chemical library, we found
that compounds with a pyrano[3,2-b]indolone core12 have
moderate inhibitory activity against both replicating (H37Rv)
and nonreplicating [streptomycin-starved 18b (SS18b)] M.
tuberculosis cells (Figure 2, our unpublished data).
RESULTS AND DISCUSSION
■
Synthesis of Pyrano[3,2-b]indolone Derivatives. We
have developed an eight-step method for the synthesis of novel
pyrano[3,2-b]indolones according to Scheme 1. The starting
1H-indole-3-carbaldehydes (2) were obtained from the
corresponding commercially available 5-substituted 1H-indoles
(1) under the conditions of the Vilsmeier−Haack reaction.13
Compounds 2 were treated with excess acetic anhydride and
triethylamine as a catalyst to protect the indole nitrogen atom
and to obtain corresponding intermediates 3. Mild oxidation of
the formyl group of N-protected indoles 3 was successfully
carried out using meta-chloroperoxybenzoic acid (mCPBA) in
a dichloromethane medium.14 5-Substituted 1-acetyl-1H-
indole-3-yl esters of formic acid 4 thus obtained were reacted
with dimethoxymethylpiperidine to introduce the piperidin-1-
ylmethylene fragment at the 2-position of indole15 and then
with triethylamine in methanol to synthesize the correspond-
ing compounds, 5. In the next stage, the acetylation of indoles
5 at the 3-position with acetyl chloride led to acetates 6. The
treatment of 2-formyl-1H-indoles (6) by malononitrile in the
presence of triethylamine provided the major intermediates: 2-
(2,2-dicyanovinyl)-1H-indoles (7).16 The key process
cyclization and pyrano[3,2-b]indolone formationwas con-
ducted using concentrated hydrochloric acid in refluxing
acetonitrile to yield target compounds 8.12 Finally, N-alkylated
and N-acylated target compounds 9−11 were prepared by the
reaction of 2-oxo-2,5-dihydropyrano[3,2-b]indole-3-carbon-
itriles (8) with different alkyl and acyl halides in the presence
of cesium carbonate in dimethylformamide (DMF).
In addition, we synthesized two 5,8-dimodified pyrano[3,2-
b]indolones (12a,b) from 8d as shown in Scheme 2, using
methyl chloride (12a) or acetyl chloride (12b) in the presence
of cesium carbonate.
Figure 2. Compounds based on the pyrano[3,2-b]indolone core with
moderate activity against M. tuberculosis strains found during the
screening of the chemical library.
Another synthesis scheme was developed for the synthesis of
pyrano[3,2-b]indolones 17a−e with a 4-substitution pattern
(Scheme 3). The reaction of commercially available 1-
acetylindolin-3-one (13) with methyl 2-cyano-3,3-bis(methyl-
thio)acrylate 14 in the presence of sodium hydride as a strong
base gave hydroxyindole 15. Subsequent treatment of 15 with
HCl in a MeOH/dioxane medium provided cyclization into
pyrano[3,2-b]indolone (16). Finally, aminolysis of the
methylthio group of 16 with various amines led to the target
4-modified pyrano[3,2-b]indolones (17a−e).
In Vitro Antimicrobial Activity and Cytotoxicity
Studies of Novel Pyrano[3,2-b]indolones. The dormant
forms of M. tuberculosis present in latent TB infection (i.e.,
nonreplicating cells with greatly reduced metabolism) pose a
challenge in developing novel antitubercular drugs.17 Cur-
We decided to continue this work, and in this article, we
report the synthesis, biological evaluation against replicating
and nonreplicating M. tuberculosis cells, and structure−activity
relationship studies of novel pyrano[3,2-b]indolone-based
compounds containing different substitutions at the 4-, 5-,
and 8-positions of the scaffold. Moreover, we present an
evaluation of activity in two models of dormant M. tuberculosis,
intracellular activity, in vitro metabolic stability, and our efforts
toward the identification of the mechanism of action of lead
compound 10a.
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a
Scheme 1. Synthesis of 5-Substituted Pyrano[3,2-b]indolones (8−10)
a
Reagents and conditions: (a) POCl3, DMF, 5 °C; (b) Ac2O, Et3N, Δ; (c) mCPBA, CH2Cl2; (d) (1) 1-(dimethoxymethyl)piperidine, piperidine,
Δ and (2) Et3N, MeOH, Δ; (e) CH3COCl, DMF; (f) CH2(CN)2, Et3N, benzene; (g) HCl/H2O, acetonitrile, Δ; (h) Alk-Hal or Ac-Hal, Cs2CO3,
DMF.
Scheme 2. Synthesis of 5,8-Dialkylated/Acylated
Pyrano[3,2-b]indolones 12a,b from 8d
rently, there are several in vitro models of M. tuberculosis
dormancy available.18 In our work, we used a previously
validated streptomycin-starved M. tuberculosis 18b model
(SS18b).19 In the absence of streptomycin, a viable but
nonreplicating state of M. tuberculosis 18b is achieved,
corresponding to a dormancy model for high-throughput
drug susceptibility screening.20 The synthesized compounds
were evaluated for their activity against M. tuberculosis H37Rv
and SS18b strains using the resazurin reduction microplate
assay (REMA) as previously described.21 A change in the color
of resazurin from blue to pink indicates its reduction to
resorufin, which directly correlates with bacterial growth and is
a
a
Reagents and conditions: (a) methyl chloride or acetyl chloride,
Cs2CO3, DMF.
a
Scheme 3. Synthesis of 4-Amino and 4-Amino-Modified Pyrano[3,2-b]indolones 17a−e
a
Reagents and conditions: (a) NaH, MeOH; (b) HCl, MeOH/dioxane; (c) corresponding amine, MeOH, Δ.
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Table 1. Cytotoxicity (TD50), in Vitro Activity against M. tuberculosis (MIC99), and Selectivity Indexes (SI; Ratio of MIC99 to
TD50) of Pyrano[3,2-b]indolones 8−12 and 17
MIC99 (μg/mL)
H37Rv SS18b
TD50 (μg/mL)
A549, lung HepG2, liver
SI (H37Rv)
A549 HepG2
no.
R1
R2
R3
8a
8b
8c
8d
8e
8f
8g
8h
8i
Me
Et
iPr
OH
OCF3
NO2
F
Cl
Br
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.3
1.6
1.5
25.0
3.6
25.0
1.6
0.4
>100.0
0.3
1.6
0.4
1.6
1.5
>25.0
3.6
>25.0
3.2
0.8
>100.0
<0.2
3.2
0.8
8.0
8.0
8.0
8.0
>100.0
8.0
32.0
32.0
32.0
32.0
>100.0
32.0
20
5
21.33
1.28
8.89
>4
5.33
0.32
2.22
4
20
5
8j
9.4
2.4
13.4
3.4
9a
9b
9c
9d
9e
9f
6.2
6.2
32.0
16.0
5.16
>4
>4
>4
>4
2.58
>4
>4
>4
>4
nC6H13
allyl
25.0
25.0
25.0
25.0
6.2
>25.0
>25.0
25.0
25.0
12.5
>25.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
CH2CCH
CH2CN
CH2COOMe
CH(Me)
COOMe
CH(Et)
COOMe
benzyl
pCN-benzyl
CH2CONH
(pOMe-Ph)
Me
Et
allyl
CH2CCH
CH2COOMe
Me
9g
9h
16.13
>4
16.13
4
25.0
9i
H
H
12.5
12.5
32.0
8.0
2.56
0.64
9j
9k
9l
H
H
H
H
H
H
25.0
6.2
25.0
25.0
6.2
>25.0
>100.0
32.0
64.0
>100.0
16.0
32.0
4
5.16
2.56
4
2.58
1.28
10a
10b
10c
10d
10e
11
12a
12b
17a
17b
17c
17d
17e
Me
Me
Me
Me
Me
Cl
OMe
OCOMe
H
H
H
H
H
H
H
H
H
H
H
H
H
0.3
0.4
25.0
>25.0
25.0
3.1
25.0
12.5
25.0
1.5
>100.0
>100.0
>100.0
>100.0
>100.0
0.156
>100.0
>100.0
>100.0
>100.0
64.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
32.0
>100.0
>100.0
>100.0
>333
>4
>4
>4
20.56
>4
>333
4
4
4
25.0
25.0
25.0
3.1
25.0
12.5
25.0
0.8
25
25
>100.0
>100.0
0.02
0.1
10.32
4
8
4
Me
COMe
H
H
H
>8
>4
NH2
MeNH
cyclopropyl-NH
benzyl-NH
Ph(NH)CNH
>100.0
>100.0
>100.0
>100.0
>4
>4
>4
>4
H
H
isoniazid (INH)
bedaquiline (BDQ)
>100.0
75.0
>100.0
50.0
>5000
750
>5000
500
measured by fluorescence. Thus, the minimal inhibitory
concentration (MIC99) was defined as the lowest compound
concentration that prevented this change in color. The
cytotoxicity of novel pyrano[3,2-b]indolone derivatives was
tested against two human cell lines, namely, HepG2 (human
hepatocellular carcinoma cells) and A549 (lung epithelial
cells). Their cytotoxic effects [50% toxic doses (TD50)] were
measured as described in the experimental section and were
defined as the compound concentration reducing the mean cell
viability by 50%.
hydrophilic/hydrophobic properties in the phenyl fragment
of the indole ring, at the nitrogen atom, and at the 4-position
of pyrano[3,2-b]indolone in order to preliminarily identify the
crucial positions of the scaffold and the necessary substituents
at these positions for an increase in antituberculosis activity.
The in vitro antimycobacterial activity and cytotoxicity of the
novel compounds are summarized in Table 1.
During the structure−activity relationship (SAR) inves-
tigation, it was found that an increase in the length and volume
of the alkyl chain at the 8-position of the scaffold from the
hydrogen atom to the isopropyl group slightly reduced the
activity of the compounds against both M. tuberculosis H37Rv
New pyrano[3,2-b]indolone-based compounds were synthe-
sized with substituents displaying electronic, steric, or
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Figure 3. Intracellular activity of compound 10a and hit compound 11126066 in (A) MRC-5 cells and (B) THP-1 cells.
and SS18b. Unsubstituted 2-oxo-2,5-dihydropyrano[3,2-b]-
indole-3-carbonitrile 11126066, our hit compound (Figure
2), has an MIC99 value of 0.78 μg/mL toward H37Rv and
SS18b strains, whereas derivatives 8b,c with alkyl groups had
similar MIC99 values among themselves (1.5−1.6 μg/mL).
However, compound 8a with a small methyl group inhibited
H37Rv and SS18b metabolism with MIC99 values of 0.3−0.4
μg/mL but was significantly cytotoxic toward liver cells (TD50
= 0.8 μg/mL). The introduction of the 8-hydroxyl group led to
a great reduction in activity (8d, MIC99s ≥ 25 μg/mL). The
same antituberculosis activity was observed in the case of the
nitro group (8f), but the compound did not show toxicity
(TD50 > 100 μg/mL toward A459 and HepG2 cells). It is
interesting to note that the activity of compound 8e, with a
strongly electron-withdrawing trifluoromethoxy group, was
higher than that of 8f but was about 2 times lower than that of
8b−c (MIC99s 3.6 μg/mL). The structure−activity relation-
ship in a series of compounds with halogen atoms (8g−i) is
unclear. It was found that 8g with a fluorine atom had MIC
values of 1.6 and 3.2 μg/mL, and the compound with chlorine
atom 8h was more active with MIC values of 0.4 and 0.8 μg/
mL against H37Rv and SS18b, respectively. At the same time,
8i with a bromine atom was completely inactive against M.
tuberculosis H37Rv and SS18b. Compound 8j with the 8-OMe
group was more active (MIC99 values of 0.3 and <0.2 μg/mL
against strains tested) than related compound 8d, but it was
also more cytotoxic (TD50 values of 2.4−9.4 μg/mL) and thus
does not meet the criteria of a “lead compound”.
It was observed that pyrano[3,2-b]indolones with the free 8-
position and with bulky alkyl substituents at the 5-position
were inactive against both M. tuberculosis strains (9c−e, MIC99
≥ 25 μg/mL), while compounds 9a and 9b with N-methyl or
N-ethyl chains, respectively, had moderate activity with MIC99
values of 1.6 μg/mL (H37Rv) and 3.2 μg/mL (SS18b) for the
former compound and 6.2 μg/mL (both strains) for the latter.
The replacement of a hydrogen atom in the N-methyl group by
a cyano group (9f) resulted in a large activity decrease. The
same replacement by a methoxycarbonyl group (9g) slightly
increased the antituberculosis activity, but the replacement of
the second hydrogen atom in the N-methyl group by the alkyl
side chain (9h,i) greatly reduced the activity. It is interesting
that a change in just one CH2 group (9h → 9i) not only
improved the activity but at the same time increased the
toxicity. Nitrogen modification by the benzyl group indicated
the same reduction in the activity (9j), but interestingly, a
compound with p-cyanobenzyl group 9k was slightly more
active and exhibited an MIC99 value of 6.2 μg/mL but was also
more toxic toward cells tested than 9j.
Among the pyrano[3,2-b]indolones with a methyl group at
the C8 position and different substituents at the C5 position,
the introduction of alkyl substituents, such as ethyl, allyl, and
propargyl decreased the activity (10b−d, MIC99 ≥ 25 μg/mL),
except for compound 10a with a 5-methyl group, which was
the most active compound in the series against M. tuberculosis
H37Rv and SS18b strains with MIC99 values of 0.3 and 0.4 μg/
mL, respectively. Moreover, derivatives 10a−d with this type
of substitution pattern are not cytotoxic. The introduction of
the CH2COOMe group at the nitrogen atom position resulted
in a reduction of activity and an increase in toxicity: compound
10e had an MIC99 value of 3.1 μg/mL for the tested strains of
M. tuberculosis and TD50 values of 32.0 and 64.0 μg/mL for
both cell lines. Another modification of the 5,8-positions of the
scaffold simultaneously (12a,b) did not improve the activity
overall.
Structure−activity relationship studies have also shown that
large groups at position 4 reduced the antimycobacterial
activity of the compounds. Among derivatives 17a−e, only 17a
with a small amino group had moderate activity with MIC
values of 0.8 and 1.5 μg/mL against H37Rv and SS18b strains,
respectively, whereas other amino-modified compounds 17b−
e are almost or completely inactive.
An evaluation of the antimicrobial activity and cytotoxicity
of the novel pyrano[3,2-b]indolone-based compounds pointed
to 5,8-dimethyl-2-oxo-2,5-dihydropyrano[3,2-b]indole-3-car-
bonitrile (10a) as the most promising derivative with an
acceptable selectivity index of >333. We thus chose this
molecule as a lead compound and decided to examine its
activity in another in vitro dormancy model, the recently
developed “Salina model”.22,23 In this model, dormancy is
achieved by cultivating M. tuberculosis cells under potassium
limiting conditions, and the survival of the resulting non-
culturable mycobacteria following the drug treatment is
evaluated after their resuscitation in ADC-supplemented
Sauton’s medium.22 It was found that compound 10a
decreased the viability of nonculturable cells in a dose-
dependent manner, similar to that of replicating cells (Figure
S1). It efficiently killed bacteria at 5 μg/mL (a 1 log decrease
in colony-forming units), and the incubation of nonculturable
cells with 50 μg/mL compound resulted in an approximately 3
log killing effect (Figure S1), which further underlines the
potential of 10a to target dormant mycobacteria.
Intracellular Activity and Microsomal Stability of
Lead Compound 10a. To assess the activity of 10a ex vivo,
we tested its ability to protect fibroblast cells (MRC-5) against
cytotoxicity mediated by the Erdman strain of M. tuberculosis.
Treatment with pyrano[3,2-b]indolone 10a protected MRC-5
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Table 2. Metabolic Stability of 10a in Human and Mouse Microsomes
microsome origin
cmpd
human
PBTZ 169
2824
mouse
PBTZ 169
36.720
10a
NIF
CBZ
3.59
10a
NIF
CBZ
7.84
CLint, μL/min/mg protein
13.56
219.50
10.66
117.10
Figure 4. Evaluation of the resistance of M. bovis BCG mutant strains 10a-R1−10a-R6 against (A) compound 10a and (B) pretomanid. The
standard M. bovis BCG strain and selected mutants 10a-R1−10a-R6 were diluted to OD600 0.5, and from these cultures dilutions of 100, 10−1, and
10−2 were applied to the solid medium as 4 μL drops. The plates were incubated for (A) 24 or (B) 30 days at 37 °C. WT, standard strain; 10a-R1−
10a-R6, resistant strains of M. bovis BCG.
cells from cell death induced by M. tuberculosis (Figure 3A)
and seemed to reduce the intracellular bacterial load by
protecting fibroblasts from killing induced by M. tuberculosis.
These data were confirmed in another ex vivo model of
intracellular infection employing THP-1 macrophages. When
compared with hit compound 11126066, lead compound 10a
was more efficient at protecting macrophages against M.
tuberculosis (Figure 3B).
To evaluate the potential of pyrano[3,2-b]indolone deriva-
tive 10a for future in vivo studies, we tested its metabolic
stability in human or mouse CD-1 microsomes (Table 2). The
intrinsic clearance (CLint) of 10a was established and
compared to the CLint of carbamazepine (CBZ) and nifedipine
(NIF), representing the controls for low and high CLint,
respectively. Lead compound 10a is metabolically stable in the
presence of both human and mouse microsomal enzymes and
has a CLint considered to be medium but superior to
benzothiazinone PBTZ169 (macozinone),24 which is currently
in phase II of clinical development.25 These data suggest that
metabolic stability should not present a liability for the further
development of this compound.
Search for Mechanism of Action of 10a. Numerous
attempts to isolate 10a-resistant strains of M. tuberculosis
H37Rv and M. tuberculosis H37Ra failed. Finally, we were able
to select six M. bovis BCG mutants resistant to 10a. The
resistance of these mutants was confirmed by the evaluation of
their growth on a solid medium in the presence of 10a at the
MIC established for the parent strain under the same
conditions (2 μg/mL) as well as at a 10 times higher
concentration (20 μg/mL). Although M. bovis BCG mutants
10a-R1−10a-R6 appeared to grow slightly worse compared to
the wild type, all of them were resistant to 10a at both
concentrations (Figure 4A). Subsequently, we tested the
sensitivity of strains 10a-R1−10a-R2 against isoniazid,
ethambutol, benzothiazinone BTZ043, pretomanid, bedaqui-
line, rifampicin, and ethionamide by REMA assay. We
observed that sensitivity is comparable to the parent strain
for all of the tested drugs except for pretomanid, which was not
active against the mutant strains. The resistance to pretomanid
was subsequently confirmed for all of the mutant strains
(Figure 4B, Figure S2). Several studies have shown that
pretomanid-resistant strains of M. tuberculosis H37Rv and M.
bovis BCG carry mutations in the ddn gene, encoding an F420
-
dependent nitroreductase involved in the activation of the
prodrug, or in the genes involved in the synthesis or reduction
26
of a mature active form of cofactor F420
.
From a structural
point of view, this cofactor is a deazaflavin derivative with
functions comparable to those of nicotinamide cofactors.27
The synthesis of F420 is initiated by the production of the
deazariboflavin precursor, fluorescently active Fo-chromophore
7,8-didemethyl-8-hydroxy-5-deazariboflavin, by the catalytic
action of the Fo-synthase encoded by the fbiC gene.28,29
Subsequently, according to a recently revised pathway for the
F420 cofactor in prokaryotes, intermediate Fo reacts with
enolpyruvyl-diphospho-5′-guanosine by the action of fbiA-
encoded transferase enzyme to form dehydro F420-0.30 The
final steps in the production of an active form of F420 are the
reduction of dehydro F420-0 to F420-0 and the addition of two
to eight glutamyl residues, catalyzed by a bifunctional enzyme
encoded by fbiB.30,31 The reduction of the mature F420
cofactor is catalyzed by the fgd1 gene product, F420-dependent
glucose-6-phosphate dehydrogenase.32
Since we did not expect nitroreductase Ddn to be involved
in the metabolism of the drug due to the lack of a nitro group
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Figure 5. HPLC-FLD chromatograms of cell extracts prepared from M. bovis BCG and the corresponding 10a-R1−10a-R6 strains.
in the structure of 10a, we focused on the analysis of the genes
involved in the production (fbiA, fbiB, and fbiC) and
reduction (fgd1) of the F420 cofactor. We amplified the target
genes along with their promoter regions by PCR, using
chromosomal DNA isolated from control strain M. bovis BCG
and the corresponding 10a-R1−10a-R6 strains as templates.
Sequencing of the isolated PCR products revealed that strains
10a-R1, 10a-R2, 10a-R5, and 10a-R6 carry mutations in the
fbiC gene and 10a-R4 contains a mutation in the fgd1 gene
(Figure S3). Since PCR amplification of the fgd1 gene in strain
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10a-R3 was not successful and fbiA, fbiB, or fbiC did not carry
any mutation, we decided to perform whole-genome
sequencing of this strain. In silico analysis of the obtained
data revealed a 460-bp-long deletion of the chromosomal DNA
comprising the 83- and 300-bp-long 5′ regions of the genes
encoding hypothetical β-lactamase-like hydrolase (Mb0414c)
and F420-dependent glucose-6-phosphate dehydrogenase
(fgd1), respectively, as well as a 77-bp-long intergenic
sequence (Figure S3).
These findings were confirmed by the assessment of the
relative amounts of different forms of cofactor F420 in the cell
extracts of the control strain and mutants 10a-R1−10a-R6 by
high-performance liquid chromatography with fluorescence
detection (HPLC-FLD) (Figure 5).
dependent enzyme. The favorable selectivity index, intra-
cellular activity, and microsomal stability of lead compound
10a all support additional in-depth structure−activity relation-
ship studies of pyrano[3,2-b]indolones as a new class of
potential antituberculosis agents.
METHODS
■
Chemistry. All reagents and solvents were purchased from
commercial suppliers and used without further purification. 1H
and 13C spectra were recorded with a Bruker AC-300 (300
MHz, 1H) or a Bruker AC-200 (50 MHz, 13C). Chemical shifts
were measured in DMSO-d6 using tetramethylsilane as an
internal standard and reported as unit (ppm) values. The
following abbreviations are used to indicate the multiplicity: s,
singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of
doublets; brs, broad singlet; and brm, broad multiplet. Mass
spectra were recorded on Finnigan MAT INCO 50 mass
spectrometer (EI, 70 eV) with direct injection. The purity of
the final compounds was analyzed on an Agilent 1290 Infinity
II HPLC system coupled to an Agilent 6460 triple-quadrupole
mass spectrometer equipped with an electrospray ionization
source. All tested final compounds are >95% pure. Elemental
analysis (% C, H, N) was carried out with a EURO EA
elemental analyzer. Melting points were determined on an
Electrothermal 9001 (10 °C per min) and are uncorrected.
Merck KGaA silica gel 60 F254 plates were used for analytical
thin-layer chromatography. Yields refer to purified products
and are not optimized.
The profile of cofactor F420 species in the wild-type M. bovis
BCG revealed the presence of Fo and the predominant amount
of F420-5, containing five L-glutamic acid residues.33 Strains
10a-R3 and 10a-R4 with mutations in the fgd1 gene lost the
capacity to synthesize longer, active forms of the cofactor, F420
-
5, and F420-6 (Figure 5). This profile is comparable to that
obtained for delamanid-resistant strains of M. bovis BCG with
mutations found in the fgd1 gene.34,35 Mutations in the fbiC
genes in strains 10a-R1, 10a-R2, 10a-R5, and 10a-R6 led to a
complete loss of F420, as observed before (Figure 5).28
To verify the critical role of the F420-dependent redox system
in the activation of 10a, we tried to complement the mutant
strains with the wild-type copies of the respective affected
genes fgd1 and fbiC. The wild-type control M. bovis BCG and
mutant strains 10a-R1−10a-R6 were transformed with
General Procedure for the Synthesis of 5-Substituted 1H-
Indole-3-carbaldehydes (2). Dimethylformamide (DMF, 50
mmol) was cooled to 5 °C, and then phosphoryl chloride (12.0
mmol) was added dropwise with stirring. The corresponding
commercially available 5-substituted 1H-indoles (10.0 mmol)
were added to the solution in one step, and the resulting
mixture was stirred for 12 h at room temperature. The reaction
mixture was poured into ice-cold water (200 mL) and stirred
for 2 h. The precipitate was filtered, washed with water, and
dried.
General Procedure for the Synthesis of 5-Substituted 1-
Acetyl-1H-indole-3-carbaldehydes (3). A mixture of the
corresponding 5-substituted 1H-indole-3-carbaldehydes (2)
(10 mmol), acetic anhydride (70 mmol), and triethylamine (3
mmol) was refluxed for 3 h. The cooled reaction mixture was
concentrated in vacuo. The residue was treated with water and
stirred for 1 to 2 h. Crystals were collected, washed with water,
and dried.
corresponding plasmids pVV2-fgd1BCG and pVV16-fbiCBCG
,
respectively, designed for the constitutive production of the N-
terminally or C-terminally His-tagged target proteins in
mycobacteria.36,37 While the complementation was not
successful for the strains with the mutated fbiC gene, since
the constitutive expression of fbiC using pVV16 expression
system was toxic, the synthesis of recombinant Fgd1 in strains
10a-R3 and 10a-R4 led to their reversion to the sensitive
phenotype, confirming the role of F420 (Figure S4). We thus
concluded that 10a interacts with an unknown F420-dependent
oxidoreductase in M. bovis BCG and M. tuberculosis, which is
responsible for its activation.
In addition to the genetic approach, we further addressed
the possible mechanism of action of 10a for M. tuberculosis
H37Rv by metabolic labeling studies using [14C]-labeled
acetate, leucine, or uracil (Figure S5). However, no significant
effects were observed.
General Procedure for the Synthesis of 5-Substituted 1-
Acetyl-1H-indole-3-yl Esters of Formic Acid (4). To a solution
of the corresponding carbaldehydes (3) (10 mmol) in
dichloromethane (50 mL), meta-chloroperoxybenzoic acid
(mCPBA, 15 mmol) was added, and the resulting mixture
was stirred for 20 h at room temperature. The reaction
solution was washed with an aqueous solution of sodium
bicarbonate and water (three times). The organic phase was
dried over anhydrous sodium sulfate and concentrated in
vacuo. The residue was treated with diethyl ether and stored in
the refrigerator for 12 h. The precipitate was collected, washed
with diethyl ether, and dried.
General Procedure for the Synthesis of 5-Substituted 2-
(Piperidin-1-ylmethylene)-1,2-dihydro-3H-indole-3-ones (5).
To a solution of the corresponding formates (4) (10.0 mmol)
in benzene (40 mL), 1-(dimethoxymethyl)piperidine (20.0
mmol) and piperidine (1.1 mmol) were added, and the
CONCLUSIONS
■
A series of novel pyrano[3,2-b]indolone-based compounds
were synthesized, characterized physicochemically, and eval-
uated in vitro for activity against replicating and nonreplicating
M. tuberculosis. Preliminary SAR investigation of the scaffold
revealed that the hydrogen atom (i.e., unsubstituted position)
or the methyl group at the 8 position of the pyrano[3,2-
b]indolone core is optimal for antituberculosis activity. In
addition, it was established that the introduction of a methyl
group at the 5 position of the scaffold increases the activity.
Thus, compound 10a with 5,8-dimethyl groups exhibited the
most promising antituberculosis activity against H37Rv and
SS18b strains (MIC99 0.3 and 0.6 μg/mL, respectively).
Although the mechanism of action of 10a in M. tuberculosis and
close relatives such as M. bovis BCG is currently not known, it
appears to be a prodrug requiring activation by an F420
-
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resulting solution was stirred for 30 min at room temperature
and then refluxed for 3 h. The reaction mixture was
concentrated in vacuo. Methanol (30 mL) and triethylamine
(10.0 mmol) were added to the residue, and the resulting
mixture was refluxed for 2 h. The cooled reaction solution was
concentrated in vacuo, and then the residue was treated with
diethyl ether and stored in the refrigerator for 12 h. The
precipitated crystals were collected, washed with diethyl ether,
and dried.
General Procedure for the Synthesis of 5-Substituted 2-
Formyl-1H-indole-3-yl Acetates (6). To a suspension of the
corresponding indolones (5) (10 mmol) in DMF (40 mL) was
added acetyl chloride (30 mmol) dropwise, and the resulting
mixture was stirred for 2 h at room temperature. The reaction
solution was concentrated in vacuo. The residue was treated
with water and stirred for 2 h. The crystals were collected,
washed with water, and dried.
General Procedure for the Synthesis of 5-Substituted 2-
(2,2-Dicyanovinyl)-1H-indole-3-yl Acetates (7). To a suspen-
sion of the corresponding acetates 6 (10.0 mmol) in benzene
(100 mL) were added malononitrile (12.5 mmol) and
triethylamine (1.0 mmol), and the resulting mixture was
stirred for 4 h at room temperature. The precipitated crystals
were collected, washed with diethyl ether, and dried.
then the resulting mixture was stirred at 65 °C for 2 h (17d,
17e − reflux). The solid was collected, washed with methanol
and diethyl ether, and recrystallized from a DMF−methanol
mixture.
In Vitro Antimycobacterial Testing. Bacterial Strains
and Culture Conditions. M. tuberculosis strains H37Rv and
18b were grown at 37 °C with shaking in Middlebrook 7H9
(Difco) broth supplemented with 10% albumin−dextrose−
catalase (ADC) enrichment, 0.2% glycerol, 0.05% Tween 80,
and, in the case of 18b, 50 μg/mL streptomycin (STR) or on a
solid Middlebrook 7H10 medium (Difco) supplemented with
0.5% glycerol, 10% oleic acid−albumin−dextrose−catalase
(OADC), and 50 μg/mL STR.
Nonreplicating SS18b was generated as follows. Strain 18b
was grown to mid-log phase in STR-containing medium and
washed three times in phosphate-buffered saline (PBS)
containing 0.05% Tween 80 (PBST). The bacterial pellets
were resuspended in STR-free medium. SS18b cultures were
maintained at an optical density at 600 nm (OD600) of
between 0.2 and 0.5 for 2 weeks (with the addition of fresh
medium if necessary), by which time cells had stopped
replicating.
Resazurin Reduction Microplate Assay (REMA). To
determine the in vitro activity of the compounds (MIC99), a
2-week-old SS18b culture (OD600 = 0.2) and an H37Rv culture
(OD600 = 0.0002) were used in the resazurin reduction
microplate assays (REMA) as previously described.21 Briefly,
serial 2-fold dilutions (starting from 25 μg/mL (for H37Rv) or
10 μg/mL (for SS18b) of each drug were prepared in 96-well
plates containing M. tuberculosis H37Rv or SS18b in 100 μL of
7H9 medium (the medium for SS18b contained streptomycin
at 50 μg/mL). After 7 days of incubation at 37 °C, 10 μL of
0.025% resazurin was added to each well, and the plates were
incubated overnight at 37 °C again. Then, the fluorescence
level of resorufin was measured by using a TECAN Infinite
M200 PRO microplate reader (excitation and emission at 560
and 590 nm, respectively). DMSO (1%) and rifampicin (0.5
mg/mL) were used as negative and positive controls,
respectively. The minimal inhibitory concentration (MIC99)
was defined as the lowest compound concentration that
prevented the change in color from blue to pink (reduction of
resazurin to resorufin). The mean MIC99 values were
calculated from at least three independent experiments.
Determination of Cytotoxicity. To determine the cytotox-
icity of the compounds (TD50), HepG2 human hepatocellular
carcinoma cells and A549 lung epithelial cells were seeded in
96-well plates at a concentration of 4 × 104 cells/mL in
DMEM (without phenol red) supplemented with 10% fetal
calf serum (FCS). Cells were incubated in the presence of
different concentrations of compounds in a humidified
incubator with 5% CO2 at 37 °C for 3 days, and then the
cell viability was measured by fluorescence spectroscopy 6 h
after adding resazurin. The cytotoxic concentration (TD50)
was defined as the compound concentration reducing the
mean cell viability by 50%. The mean TD50 values were
calculated from at least three independent experiments.
Ex Vivo Activity. Fibroblast Assay. The ability of the drugs
to protect fibroblast cells (MRC-5) against the MTB Erdman
strain was tested as described previously.38,39 Briefly, MRC-5
cells (0.8−1.2 × 106 cells/flask) were cultured in minimum
essential media + GlutaMAX (MEM-Gibco) supplemented
with 1 mM sodium pyruvate, 1× MEM NEAA (stock
concentration 100×), and 10× heat-inactivated fetal bovine
General Procedure for the Synthesis of 8-Substituted 2-
Oxo-2,5-dihydropyrano[3,2-b]indole-3-carbonitriles (8). To
a refluxing suspension of the corresponding acetates (7) (10
mmol) in acetonitrile (250 mL) was slowly added concen-
trated hydrochloric acid (30 mL) dropwise with stirring, and
then the resulting mixture was refluxed for 3 h. The cooled
reaction mixture was partially concentrated in vacuo. The
precipitate was collected, washed with an acetonitrile/water
(1:1) mixture, water, and methanol, and recrystallized from
DMF−CH3COOH, DMF−EtOH, or DMF−H2O.
General Procedure for the Synthesis of 5,8-Disubstituted
2-Oxo-2,5-dihydropyrano[3,2-b]indole-3-carbonitriles (9−
12). To a suspension of the corresponding carbonitriles (8)
(1.00 mmol) and cesium carbonate (1.25 mmol) in DMF (5
mL) was added the corresponding alkyl halide or acyl halide
(1.50 mmol) dropwise, and then the resulting mixture was
stirred at room temperature. After 3 h of stirring, acetic acid
(5.00 mmol) and water (10 mL) were added to the reaction
mixture. The precipitated crystals were collected, washed with
a DMF−water mixture, and recrystallized from acetonitrile.
Synthesis of Methyl 2-Cyano-3-(3-hydroxy-1H-indol-2-yl)-
3-(methylthio)acrylate (15). To a suspension of NaH (0.01
mol) in methanol were added 1-acetylindolin-3-one 13 (0.01
mol) and methyl 2-cyano-3,3-bis(methylthio)acrylate 14 (0.01
mol) dropwise at room temperature, and then the resulting
mixture was refluxed for 2 h. The mixture was concentrated in
vacuo and treated with water and 1 to 2 drops of 10% HCl
solution. The precipitate was collected and recrystallized from
methanol.
Synthesis of 4-(Methylthio)-2-oxo-2,5-dihydropyrano[3,2-
b]indole-3-carbonitrile (16). A mixture of 15 (0.01 mmol)
and hydrogen chloride solution in dioxane (0.02 mmol) in
methanol was refluxed for 2 h. The cooled reaction mixture
was filtered, and the precipitate was recrystallized from a
DMF−methanol mixture.
General Procedure for the Synthesis of 4-Substituted 2-
Oxo-2,5-dihydropyrano[3,2-b]indole-3-carbonitriles (17a−
e). To a suspension of 16 (1.00 mmol) in methanol was
added the corresponding amine (1.50 mmol) dropwise, and
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serum (FBS). Cells were grown for 10−14 days at 37 °C in 5%
CO2. (The medium was changed twice a week.) On the day of
the experiment, the fibroblasts were trypsinated (trypsin-
EDTA) and then washed once with 1× phosphate-buffered
saline (PBS). Fibroblasts were then counted using tryptan blue
and adjusted to a concentration of 7.35 × 104 cells/mL in the
MEM medium. Fibroblasts (40 μL) were added to a 384 black
well plates (Corning number 3683), and the compounds (1
μL) were added to the fibroblasts. The drugs were then 2-fold
serially diluted. Fibroblasts were incubated for 2 to 3 h at 37
°C in 5% CO2 before the bacteria were added. The MTB
Erdman strain was cultured in the 7H9 complete to mid-
logarithmic phase (OD 0.5) and concentrated to an OD600 nm
of 1 (3 × 108 cells/mL) in 7H9 complete. Bacteria were
diluted to a concentration of 4 × 104 bacteria/10 μL by adding
133 μL of Erdman culture OD600 nm = 1 to 10 mL MEM.
Bacteria (10 μL) were added to the 384 fibroblast-containing
plates. The plates were incubated for 3 days at 37 °C in 5%
CO2. Plates were read using a TECAN infinite M200
microplate reader (by excitation at 560 nm and emission at
590 nm) after 1 h of incubation with 5 μL of PrestoBlue cell
viability reagent (Invitrogen). Wells containing fibroblasts
alone, fibroblasts with DMSO, and medium alone were used as
controls. The experiment was performed in duplicate.
Macrophage Assay. THP-1 cells were grown in RPMI-
1640 medium containing 10% fetal bovine serum and
incubated at 37 °C in a 5% CO2 environment. THP-1 cells
(100 000) per well were differentiated into macrophages by
incubating the cells with 100 nM phorbol myristate acetate
(PMA) for 12 h. Cell differentiation was checked by
microscopy, and PMA was removed and replaced with fresh
RPMI. M. tuberculosis Erdman bacteria were prepared at an
optical density of 1 (3 × 108 cells/mL) in Middlebrook 7H9
media. Cells were infected at a multiplicity of infection (MOI)
of 5 per macrophage and incubated for 4 h at 37 °C with 5%
CO2. The medium containing bacteria was removed, and cells
were washed twice with PBS. Test compounds (pyranoindo-
lones and RIF) dissolved in RPMI were added to the 96-well
plates and incubated for 3 days at 37 °C with 5% CO2. On day
3, macrophage survival was tested by exposing the cells to
PrestoBlue cell viability solution (Life Technology) for 1 h.
Fluorescence was recorded using a TECAN Infinite M200
microplate reader.
Microsomal Stability. Compound 10a (10 mg/mL) or
control compounds nifedipine (Sigma N7634) and carbama-
zepine (Sigma C4024) (10 mg/mL) in DMSO were diluted in
ddH2O to a final concentration of 0.2 mg/mL. An aliquot of
this solution was used for a further dilution in potassium
phosphate pH 7.4 buffer to a final concentration of 4 μg/mL
followed by incubation at 37 °C with mouse CD-1 liver
microsomes (2 mg/mL) (Invitrogen MSMC-PL) and human
liver (Invitrogen HMMC-PL) for 10 min (50 μL). The
reaction was initiated by adding an NADPH regeneration
system (Promega V9510) (50 μL) and incubating at 37 °C
with gentle agitation. At 0, 5, 15, 30, and 60 min, the reaction
was stopped by the addition of 100 μL of ice-cold acetonitrile.
Samples were then centrifuged at 16 100g (13 200 rpm) at 4
°C for 10 min, and the supernatants were analyzed using
HPLC-UV. Samples without NADPH cofactor (0 and 60 min)
were used as controls to monitor the nonenzymatic
degradation of the compounds.
7H9 liquid medium containing 0.2% glycerol, 0.05% Tween
80, and 10% ADC were plated on 7H11 agar containing 10%
OADC and 10a at final concentrations of 0, 10, and 20 μg/mL.
Compound 10a was dissolved in DMSO to give a final
concentration of 2% in the medium. Culture (200 or 1000 μL)
with OD600 = 1 was used for plating. In the latter case, the
culture was centrifuged (10 min, 3500g, 23 °C), and the
resulting pellet was suspended in 200 μL of 7H9 medium and
then spread on the plate. The plates were incubated for 22 days
at 37 °C. Out of about 1000 obtained colonies, approximately
75% had a small diameter (about 0.5 mm) and the rest were
larger (about 2 mm) and the calculated frequency of resistance
was 10−7. Twelve colonies from each phenotype were
inoculated into 5 mL of 7H9 liquid medium with 0.2%
glycerol, 10% ADC, and 0.05% Tween 80 containing 10 or
20 μg/mL compound 10a. Cultures were incubated for about
16 days at 37 °C with shaking at 130 rpm. The resistant
phenotype was confirmed for six strains, labeled 10a-R1−10a-
R6. Mutant strains 10a-R1 and 10a-R2 originated from the
small colonies, and the rest originated from the larger colonies.
To monitor the susceptibility of the selected resistant
mutants to different drugs, an REMA method was used.21 The
cultures were inoculated to the starting OD600 value of 0.02 in
the total volume of 200 μL of 7H9 medium with 0.2% glycerol,
10% ADC, and 0.05% Tween 80 and incubated for 7 days in
the presence of various concentrations of selected inhibitors in
a 96-well microtiter plate at 37 °C. Subsequently, 60 μL of
0.01% (w/v) resazurin was added to the cultures, and the
plates were incubated for 48 h at 37 °C. The MICs of the
inhibitors were determined on the basis of the visual evaluation
of the color changes from blue to pink in the individual wells.
The susceptibility of selected mutants toward 10a (from 0.1
to 20 μg/mL) and pretomanid (from 0.01 to 20 μg/mL) was
also established on 7H11 agar plates containing 10% OADC.
The drugs were dissolved in DMSO, and its final concentration
in the medium was 2%. The cultures were diluted to OD600
=
0.5, and 4 μL portions of these cultures diluted by factors of
100, 10−1, and 10−2 were applied to the plate. Subsequently, the
plates were incubated at 37 °C for about 1 month.
DNA was extracted from the wild-type M. bovis BCG and
selected resistant mutants 10a-R1−10a-R6 as described in the
Supporting Information. The fgd1, fbiA, fbiB, and fbiC genes
were PCR amplified as described in the Supporting
Information and subjected to sequencing (Microsynth).
Whole-genome sequencing (WGS, Illumina HiSeq) of the
parent M. bovis BCG and mutant strain 10a-3 was performed
and analyzed as described in the Supporting Information.
HPLC-FLD. The samples for HPLC-FLD analyses were
obtained as described,33 with minor modifications. The cells
were incubated in the buffer containing 27.5 mM sodium
acetate (pH 4.7) and 2% (v/v) acetonitrile at 95 °C for 20
min. The mixtures were centrifuged twice for 10 min at
15 000g and 23 °C. The supernatants from the second
centrifugation were used directly for quantification of the
F420 forms using a Dionex Ultimate 3000 HPLC system
(Thermo Scientific) equipped with an autosampler and a
fluorescence detector (FLD). Samples were kept at 15 °C in
the autosampler. Analyses were performed according to Choi
et al.33 with some modifications. Briefly, coenzyme F420 species
were separated on a reverse-phase Eclipse XDB-C18 column
(4.6 mm i.d. × 150 mm, 5 μm) (Agilent) maintained at 28 °C,
and the injection volume was 10 μL. The mobile phase
consisted of 27.5 mM sodium acetate (pH 4.7) containing 2%
Isolation and Characterization of Resistant Mutants
10a-R1−10a-R6. M. bovis BCG cells cultured in 10 mL of
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acetonitrile (buffer A) and 100% acetonitrile (buffer B) with
gradient elution at a flow rate of 1 mL min−1 and a run time of
31 min. The applied gradient was 0−2 min 0% B, 2−6 min
from 0 to 2% B, 6−15 min from 2 to 9% B, 15−22 min from 9
to 28% B, 22−25 min from 28 to 50% B, 25−26 min from 50
to 0% B, and 26−31 min at 0% B. For fluorescence detection,
an excitation wavelength was set at 400 nm, emission was set at
470 nm, and the flow cell temperature was set at 42 °C.
Jozef Nosek − Department of Biochemistry, Faculty of Natural
Sciences, Comenius University in Bratislava, Bratislava 842
15, Slovak Republic
́
Eva Repkova − Department of Biochemistry, Faculty of
Natural Sciences, Comenius University in Bratislava,
Bratislava 842 15, Slovak Republic
́
́
Julia Zemanova − Department of Biochemistry, Faculty of
Natural Sciences, Comenius University in Bratislava,
Bratislava 842 15, Slovak Republic
́
́
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00622.
Helena Jurdakova − Department of Analytical Chemistry,
Faculty of Natural Sciences, Comenius University in
Bratislava, Bratislava 842 15, Slovak Republic
■
sı
́
́
́
Renata Gorova − Department of Analytical Chemistry,
Faculty of Natural Sciences, Comenius University in
Bratislava, Bratislava 842 15, Slovak Republic
Jaroslav Roh − Faculty of Pharmacy in Hradec Králové,
Charles University, Hradec Králové 50005, Czech Republic
Giulia Degiacomi − Department of Biology and Biotechnology
“Lazzaro Spallanzani”, University of Pavia, Pavia 27100,
Italy
Characterization of the novel pyrano[3,2-b]indolone-
based compounds; optimized synthesis of pretomanid;
identification of mutations in resistant strains 10a-R1−
10a-R6 by PCR and WGS; construction and suscepti-
bility testing of the complementants; bactericidal activity
of lead compound 10a against replication and non-
culturable M. tuberculosis cells in the Salina model of
dormant M. tuberculosis; susceptibility testing of M. bovis
BCG and mutant strains 10a-R1−10a-R6 by the REMA
method; identification of mutations in isolated resistant
M. bovis BCG strains 10a-R1−10a-R6 by PCR and
Illumina sequencing; testing of susceptibility of M. bovis
BCG and mutant strains 10a-R1−10a-R6 complemented
with the functional copy of fgd1 provided on pVV2-
fgd1BCG plasmid against pretomanid and 10a; and
investigation of the effects of 10a and 11126066 by
macromolecular synthesis assays with M. tuberculosis
H37Rv in Sauton’s medium (PDF)
́
Jose Camilla Sammartino − Department of Biology and
Biotechnology “Lazzaro Spallanzani”, University of Pavia,
Pavia 27100, Italy; orcid.org/0000-0003-3707-3118
Maria Rosalia Pasca − Department of Biology and
Biotechnology “Lazzaro Spallanzani”, University of Pavia,
Pavia 27100, Italy; orcid.org/0000-0002-8906-4937
Stewart T. Cole − Global Health Institute, Ecole
Polytechnique Fédérale de Lausanne, Lausanne 1015,
Switzerland
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsinfecdis.0c00622
Author Contributions
∇N.M., J.K., and A.V. contributed equally to this work. N.M.
and A.L., synthesis of compounds, data collection; A.V., in vitro
and ex vivo efficiency and microsomal stability studies; A.E.,
SAR analysis; E.R. and J.K., isolation and characterization of
resistant mutants; E.S., dormancy model; J.Z. and K.M., MMS
experiments; R.G. and H.J., HPLC-FLD analysis; J.N., WGS of
M. bovis BCG and 10a-R3 strains; J.R., synthesis of
pretomanid; G.D. and J.C.S., H37Rv mutant generation and
characterization; and V.M., J.K., K.M., M.R.P., and S.T.C.,
conception and design of research and supervision. A.E., V.M.,
J.K., and K.M. wrote the manuscript with contributions from
all authors. All authors discussed the data and have given
approval to the final version to be published.
AUTHOR INFORMATION
■
Corresponding Authors
̌
́
Katarína Mikusova − Department of Biochemistry, Faculty of
Natural Sciences, Comenius University in Bratislava,
Bratislava 842 15, Slovak Republic; orcid.org/0000-
0002-0100-4877; Email: katarina.mikusova@uniba.sk
Vadim Makarov − Bach Institute of Biochemistry, Research
Center of Biotechnology of the Russian Academy of Sciences,
Moscow 119071, Russian Federation; Email: makarov@
inbi.ras.ru
Authors
Natalia Monakhova − Bach Institute of Biochemistry,
Research Center of Biotechnology of the Russian Academy of
Sciences, Moscow 119071, Russian Federation
Jana Kordulakova − Department of Biochemistry, Faculty of
Natural Sciences, Comenius University in Bratislava,
Bratislava 842 15, Slovak Republic
Anthony Vocat − Global Health Institute, Ecole Polytechnique
Fédérale de Lausanne, Lausanne 1015, Switzerland
Anna Egorova − Bach Institute of Biochemistry, Research
Center of Biotechnology of the Russian Academy of Sciences,
Moscow 119071, Russian Federation; orcid.org/0000-
0002-6708-2421
Alexander Lepioshkin − Bach Institute of Biochemistry,
Research Center of Biotechnology of the Russian Academy of
Sciences, Moscow 119071, Russian Federation
Notes
The authors declare no competing financial interest.
́
́
ACKNOWLEDGMENTS
■
This work was supported by the European Community’s
Seventh Framework Program (grant 260872). V.M. and E.G.S.
acknowledge support from the Russian Foundation of Basic
Research (grant no. 20-04-00798). K.M. and J.K. acknowledge
support by Ministry of Education, Science, Research and Sport
of the Slovak Republic (grant 0395/2016 for Slovak/Russian
cooperation in science 2015-15075/33841:1-15E0), by the
Slovak Research and Development Agency (grants DO7RP-
0015-11 and APVV-19-0189), and by the Research and
Development Operational Program funded by the European
Regional Development Fund (contract ITMS 26240120027).
R.G. and H.J. acknowledge support by the Research and
Elena G. Salina − Bach Institute of Biochemistry, Research
Center of Biotechnology of the Russian Academy of Sciences,
Moscow 119071, Russian Federation
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Article
(14) Horn, A., and Kazmaier, U. (2018) Purified mCPBA, a useful
reagent for the oxidation of aldehydes. Eur. J. Org. Chem. 20−21,
2531−2536.
Development Operational Program funded by ERDF (contract
ITMS 26240220086).
(15) Ryabova, S. Y., Trofimkin, Y. I., Alekseeva, L. M., Bogdanova,
G. A., Sheinker, Y. N., and Granik, V. G. (1990) Acetals of lactams
and acid amides. 61. Synthesis and transamination of indoxyls and
pyrrol-2-in-4-ones. Chem. Heterocycl. Compd. 26, 1238−1244.
(16) Ryabova, S. Y., Trofimkin, Y. I., Alekseeva, L. M., Kerbnikova, I.
F., Shvarts, G. Y., and Granik, V. G. (1995) Synthesis and biological
activity of dienediaminoketones of the indolin-3-one and 2-pyrrolin-4-
one series. Pharm. Chem. J. 29, 610−617.
ABBREVIATIONS
■
EMA, European Medicines Agency; FDA, Food and Drug
Administration; HIV, human immunodeficiency virus; MIC99,
minimal inhibitory concentration; OADC, oleic acid−
albumin−dextrose−catalase; REMA, resazurin reduction mi-
croplate assay; TD50, 50% toxic dose; WHO, World Health
Organization; WGS, whole genome sequencing
(17) Cano-Muniz, S., Anthony, R., Niemann, S., and Alffenaar, J. C.
̃ ̃
(2017) New approaches and therapeutic options for Mycobacterium
tuberculosis in a dormant State. Clin. Microbiol Rev. 31, No. e00060-17.
(18) Gibson, S. E. R., Harrison, J., and Cox, J. A. G. (2018)
Modelling a silent epidemic: A review of the in vitro models of latent
tuberculosis.(2018) Pathogens 7(4), DOI: 10.3390/patho-
gens7040088.
(19) Sala, C., Dhar, N., Hartkoorn, R. C., Zhang, M., Ha, Y. H.,
Schneider, P., and Cole, S. T. (2010) Simple model for testing drugs
against nonreplicating Mycobacterium tuberculosis. Antimicrob. Agents
Chemother. 54, 4150−4158.
(20) Zhang, M., Sala, C., Hartkoorn, R. C., Dhar, N., Mendoza-
Losana, A., and Cole, S. T. (2012) Streptomycin-starved Mycobacte-
rium tuberculosis 18b, a drug discovery tool for latent tuberculosis.
Antimicrob. Agents Chemother. 56, 5782−5789.
(21) Palomino, J. C., Martin, A., Camacho, M., Guerra, H., Swings,
J., and Portaels, F. (2002) Resazurin microtiter assay plate: simple and
inexpensive method for detection of drug resistance in Mycobacterium
tuberculosis. Antimicrob. Agents Chemother. 46, 2720−2722.
(22) Salina, E., Ryabova, O., Kaprelyants, A., and Makarov, V.
(2014) New 2-thiopyridines as potential candidates for killing both
actively growing and dormant Mycobacterium tuberculosis cells.
Antimicrob. Agents Chemother. 58, 55−60.
(23) Albesa-Jove, D., Chiarelli, L. R., Makarov, V., Pasca, M. R.,
Urresti, S., Mori, G., Salina, E., Vocat, A., Comino, N., Mohorko, E.,
Ryabova, S., Pfieiffer, B., Lopes Ribeiro, A. L., Rodrigo-Unzueta, A.,
Tersa, M., Zanoni, G., Buroni, S., Altmann, K. H., Hartkoorn, R. C.,
Glockshuber, R., Cole, S. T., Riccardi, G., and Guerin, M. E. (2014)
Rv2466c mediates the activation of TP053 to kill replicating and non-
replicating Mycobacterium tuberculosis. ACS Chem. Biol. 9, 1567−1575.
(24) Makarov, V., Lechartier, B., Zhang, M., Neres, J., van der Sar, A.
M., Raadsen, S. A., Hartkoorn, R. C., Ryabova, O. B., Vocat, A.,
Decosterd, L. A., Widmer, N., Buclin, T., Bitter, W., Andries, K., Pojer,
F., Dyson, P. J., and Cole, S. T. (2014) Towards a new combination
therapy for tuberculosis with next generation benzothiazinones.
REFERENCES
■
(1) World Health Organization. Global Tuberculosis Report 2019.
www.who.int/tb/publications/global_report/en/ (accessed April 13,
2020).
(2) Houben, R. M., and Dodd, P. J. (2016) The Global Burden of
Latent Tuberculosis Infection: A Re-estimation Using Mathematical
Modelling. PLoS Med. 13 (10), No. e1002152.
̈
(3) Andries, K., Verhasselt, P., Guillemont, J., Gohlmann, H. W.,
Neefs, J. M., Winkler, H., Van Gestel, J., Timmerman, P., Zhu, M.,
Lee, E., Williams, P., de Chaffoy, D., Huitric, E., Hoffner, S., Cambau,
E., Truffot-Pernot, C., Lounis, N., and Jarlier, V. (2005) A
diarylquinoline drug active on the ATP synthase of Mycobacterium
tuberculosis. Science 307, 223−227.
(4) Koul, A., Vranckx, L., Dendouga, N., Balemans, W., Van den
Wyngaert, I., Vergauwen, K., Gohlmann, H. W., Willebrords, R.,
̈
Poncelet, A., Guillemont, J., Bald, D., and Andries, K. (2008)
Diarylquinolines are bactericidal for dormant mycobacteria as a result
of disturbed ATP homeostasis. J. Biol. Chem. 283, 25273−25280.
(5) Matsumoto, M., Hashizume, H., Tomishige, T., Kawasaki, M.,
Tsubouchi, H., Sasaki, H., Shimokawa, Y., and Komatsu, M. (2006)
OPC-67683, a nitro-dihydro-imidazooxazole derivative with promis-
ing action against tuberculosis in vitro and in mice. PLOS Med. 3,
e466.
(6) Chen, X., Hashizume, H., Tomishige, T., Nakamura, I., Matsuba,
M., Fujiwara, M., Kitamoto, R., Hanaki, E., Ohba, Y., and Matsumoto,
M. (2017) Delamanid kills dormant mycobacteria in vitro and in a
guinea pig model of tuberculosis. Antimicrob. Agents Chemother. 61,
No. e02402-16.
(7) Stover, C. K., Warrener, P., VanDevanter, D. R., Sherman, D. R.,
Arain, T. M., Langhorne, M. H., Anderson, S. W., Towell, J. A., Yuan,
Y., McMurray, D. N., Kreiswirth, B. N., Barry, C. E., and Baker, W. R.
(2000) A small-molecule nitroimidazopyran drug candidate for the
treatment of tuberculosis. Nature 405, 962−966.
(8) U.S. Food and Drug Administration (FDA). Sirturo (bedaqui-
line) - full prescribing information. www.accessdata.fda.gov/
drugsatfda_docs/label/2012/204384s000lbl.pdf (accessed April 14,
2020).
EMBO Mol. Med. 6, 372−383.
̌
́
(25) Makarov, V., and MikuSova, K. (2020) Development of
macozinone for TB treatment: an update. Appl. Sci. 10, 2269.
(26) Haver, H. L., Chua, A., Ghode, P., Lakshminarayana, S. B.,
Singhal, A., Mathema, B., Wintjens, R., and Bifani, P. (2015)
Mutations in genes for the F420 biosynthetic pathway and a
nitroreductase enzyme are the primary resistance determinants in
spontaneous in vitro-selected PA-824-resistant mutants of Mycobacte-
rium tuberculosis. Antimicrob. Agents Chemother. 59, 5316−5323.
(27) Hossain, M. S., Le, C. Q., Joseph, E., Nguyen, T. Q., Johnson-
Winters, K., and Foss, F. W., Jr. (2015) Convenient synthesis of
deazaflavin cofactor Fo and its activity in F(420)-dependent NADP
reductase. Org. Biomol. Chem. 13, 5082−5085.
(9) European Medicines Agency (EMA). Delbyta (delamanid).
www.ema.europa.eu/en/medicines/human/EPAR/deltyba (accessed
April 14, 2020).
(10) U.S. Food and Drug Administration (FDA). FDA approves
new drug for treatment-resistant forms of tuberculosis that affects the
lungs. www.fda.gov/news-events/press-announcements/fda-approves-
new-drug-treatment-resistant-forms-tuberculosis-affects-lungs (ac-
cessed April 14, 2020).
́
̀
(11) Medecins Sans Frontieres. TB drug delamanid must be more
affordable and made available in more countries www.msf.org/
delamanid-must-be-more-affordable-tuberculosis (accessed June 28,
2020).
(28) Choi, K. P., Kendrick, N., and Daniels, L. (2002)
Demonstration that fbiC is required by Mycobacterium bovis BCG
for coenzyme F420 and Fo biosynthesis. J. Bacteriol. 184, 2420−2428.
(29) Decamps, L., Philmus, B., Benjdia, A., White, R., Begley, T. P.,
and Berteau, O. (2012) Biosynthesis of Fo, precursor of the F420
cofactor, requires a unique two radical-SAM domain enzyme and
tyrosine as substrate. J. Am. Chem. Soc. 134, 18173−18176.
(30) Bashiri, G., Antoney, J., Jirgis, E. N. M., Shah, M. V., Ney, B.,
Copp, J., Stuteley, S. M., Sreebhavan, S., Palmer, B., Middleditch, M.,
Tokuriki, N., Greening, C., Scott, C., Baker, E. N., and Jackson, C. J.
(12) Masterova, N. S., Ryabova, S. Y., Alekseeva, L. M., Evstratova,
M. I., Kiselev, S. S., and Granik, V. G. (2010) Novel pyrano[3,2-
b]indole derivatives: synthesis and some properties. Russ. Chem. Bull.
59, 637−641.
(13) James, P. N., and Snyder, H. R. (1959) Indole-3-aldehyde. Org.
Synth. 39, 30.
99
https://dx.doi.org/10.1021/acsinfecdis.0c00622
ACS Infect. Dis. 2021, 7, 88−100

ACS Infectious Diseases
pubs.acs.org/journal/aidcbc
Article
(2019) A revised biosynthetic pathway for the cofactor F420 in
prokaryotes. Nat. Commun. 10, 1558.
(31) Bashiri, G., Rehan, A. M., Sreebhavan, S., Baker, H. M., Baker,
E. N., and Squire, C. J. (2016) Elongation of the poly-gamma-
glutamate tail of F420 requires both domains of the F420:gamma-
glutamyl ligase (FbiB) of Mycobacterium tuberculosis. J. Biol. Chem.
291, 6882−6894.
(32) Manjunatha, U. H., Boshoff, H., Dowd, C. S., Zhang, L., Albert,
T. J., Norton, J. E., Daniels, L., Dick, T., Pang, S. S., and Barry, C. E.,
3rd. (2006) Identification of a nitroimidazo-oxazine-specific protein
involved in PA-824 resistance in Mycobacterium tuberculosis. Proc.
Natl. Acad. Sci. U. S. A. 103, 431−436.
(33) Choi, K. P., Bair, T. B., Bae, Y. M., and Daniels, L. (2001) Use
of transposon Tn5367 mutagenesis and a nitroimidazopyran-based
selection system to demonstrate a requirement for fbiA and fbiB in
coenzyme F(420) biosynthesis by Mycobacterium bovis BCG. J. Bacteriol.
183, 7058−7066.
(34) Fujiwara, M., Kawasaki, M., Hariguchi, N., Liu, Y., and
Matsumoto, M. (2018) Mechanisms of resistance to delamanid, a
drug for Mycobacterium tuberculosis. Tuberculosis 108, 186−194.
(35) Purwantini, E., Loganathan, U., and Mukhopadhyay, B. (2018)
Coenzyme F420-dependent glucose-6-phosphate dehydrogenase-
coupled polyglutamylation of coenzyme F420 in mycobacteria. J.
Bacteriol. 200, No. e00375-18.
(36) Dhiman, R. K., Schulbach, M. C., Mahapatra, S., Baulard, A. R.,
Vissa, V., Brennan, P. J., and Crick, D. C. (2004) Identification of a
novel class of w E,E-farnesyl diphosphate synthase from Mycobacte-
rium tuberculosis. J. Lipid Res. 45, 1140−1147.
(37) Jackson, M., Crick, D. C., and Brennan, P. J. (2000)
Phosphatidylinositol is an essential phospholipid of mycobacteria. J.
Biol. Chem. 275, 30092−30099.
(38) Rybniker, J., Vocat, A., Sala, C., Busso, P., Pojer, F., Benjak, A.,
and Cole, S. T. (2015) Lansoprazole is an antituberculous prodrug
targeting cytochrome bc1. Nat. Commun. 6, 7659.
(39) Rybniker, J., Chen, J. M., Sala, C., Hartkoorn, R. C., Vocat, A.,
Benjak, A., Boy-Rottger, S., Zhang, M., Szekely, R., Greff, Z., Orfi, L.,
Szabadkai, I., Pato, J., Keri, G., and Cole, S. T. (2014) Anticytolytic
screen identifies inhibitors of mycobacterial virulence protein
secretion. Cell Host Microbe 16, 538−548.
100
https://dx.doi.org/10.1021/acsinfecdis.0c00622
ACS Infect. Dis. 2021, 7, 88−100