Design, synthesis and evaluation of novel indole-2-carboxamides for growth inhibition of Mycobacterium tuberculosis and paediatric brain tumour cells

Article abstract of DOI:10.1039/d0ra10728j

The omnipresent threat of tuberculosis (TB) and the scant treatment options thereof necessitate the development of new antitubercular agents, preferably working via a novel mechanism of action distinct from the current drugs. Various studies identified th

Full text of DOI:10.1039/d0ra10728j

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Design, synthesis and evaluation of novel indole-2-
carboxamides for growth inhibition of
Cite this: RSC Adv., 2021, 11, 15497
Mycobacterium tuberculosis and paediatric brain
tumour cells†
a
Shahinda S. R. Alsayed,
Shichun Lun,b Anders W. Bailey,c Amreena Suri,c
Chiang-Ching Huang,d Mauro Mocerino,
Alan Payne,e
e
cf
bg
a
*
*
*
Simone Treiger Sredni,
William R. Bishai
and Hendra Gunosewoyo
The omnipresent threat of tuberculosis (TB) and the scant treatment options thereof necessitate the
development of new antitubercular agents, preferably working via a novel mechanism of action distinct from
the current drugs. Various studies identified the mycobacterial membrane protein large 3 transporter (MmpL3)
as the target of several classes of compounds, including the indole-2-caboxamides. Herein, several
indoleamide analogues were rationally designed, synthesised, and evaluated for their antitubercular and
antitumour activities. Compound 8g displayed the highest activity (MIC ¼ 0.32 mM) against the drug-sensitive
(DS) Mycobacterium tuberculosis (M. tb) H37Rv strain. This compound also exhibited high selective activity
towards M. tb over mammalian cells [IC50 (Vero cells) ¼ 40.9 mM, SI ¼ 128], suggesting its minimal
cytotoxicity. In addition, when docked into the MmpL3 active site, 8g adopted a binding profile similar to the
indoleamide ligand ICA38. A related compound 8f showed dual antitubercular (MIC ¼ 0.62 mM) and cytotoxic
activities against paediatric glioblastoma multiforme (GBM) cell line KNS42 [IC50 (viability) ¼ 0.84 mM].
Compound 8f also showed poor cytotoxic activity against healthy Vero cells (IC50 ¼ 39.9 mM). Compounds
9a and 15, which were inactive against M. tb, showed potent cytotoxic (IC50 ¼ 8.25 and 5.04 mM, respectively)
and antiproliferative activities (IC50 ¼ 9.85 and 6.62 mM, respectively) against KNS42 cells. Transcriptional
analysis of KNS42 cells treated with compound 15 revealed a significant downregulation in the expression of
the carbonic anhydrase 9 (CA9) and the spleen tyrosine kinase (SYK) genes. The expression levels of these
genes in GBM tumours were previously shown to contribute to tumour progression, suggesting their
involvement in our observed antitumour activities. Compounds 9a and 15 were selected for further
evaluations against three different paediatric brain tumour cell lines (BT12, BT16 and DAOY) and non-
neoplastic human fibroblast cells HFF1. Compound 9a showed remarkable cytotoxic (IC50 ¼ 0.89 and 1.81
mM, respectively) and antiproliferative activities (IC50 ¼ 7.44 and 6.06 mM, respectively) against the two tested
atypical teratoid/rhabdoid tumour (AT/RT) cells BT12 and BT16. Interestingly, compound 9a was not cytotoxic
when tested against non-neoplastic HFF1 cells [IC50 (viability) ¼ 119 mM]. This suggests that an indoleamide
scaffold can be fine-tuned to confer a set of derivatives with selective antitubercular and/or antitumour activities.
Received 22nd December 2020
Accepted 10th April 2021
DOI: 10.1039/d0ra10728j
rsc.li/rsc-advances
infecting around 10 million people and claiming more than one
million lives worldwide every year.1 In 2019 before the COVID-19
1. Introduction
pandemic, the inexorable TB burden together with the currently
estimated one-quarter latent infections of global population
Mycobacterium tuberculosis (M. tb), the causative agent of
tuberculosis (TB), is a highly contagious airborne pathogen
aCurtin Medical School, Faculty of Health Sciences, Curtin University, Bentley, Perth,
WA 6102, Australia. E-mail: Hendra.Gunosewoyo@curtin.edu.au
bCenter for Tuberculosis Research, Department of Medicine, Division of Infectious
Disease, Johns Hopkins School of Medicine, 1550, Orleans Street, Baltimore,
Maryland, 21231-1044, USA. E-mail: wbishai1@jhmi.edu
eSchool of Molecular and Life Sciences, Curtin University, Perth, WA 6102, Australia
fDepartment of Surgery, Northwestern University, Feinberg School of Medicine,
Chicago, IL 60611, USA. E-mail: ssredni@northwestern.edu
gHoward Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase,
Maryland, 20815-6789, USA
cDivision of Pediatric Neurosurgery, Ann and Robert H. Lurie Children's Hospital of
† Electronic supplementary information (ESI) available. See DOI:
Chicago, Chicago, IL 60611, USA
10.1039/d0ra10728j
dDepartment of Biostatistics, Zilber School of Public Health, University of Wisconsin,
Milwaukee, WI 53205, USA
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ranked TB as the number one cause of mortality/morbidity from
a single infectious agent (surpassing HIV/AIDS).1 Due to the
poor patient compliance to the onerous multi-drug rst-line
regimen, administered for at least 6 months, and the co-
epidemic with HIV, vicious drug-resistant TB strains have
emerged, perpetuating TB as a global health threat.2,3 The
insufficiency and limited efficacy of the drug options of multi-
and extensively drug resistant TB (MDR- and XDR-TB, respec-
tively) accompanied with their high cost, toxicity and drawn-out
duration of therapy (up to 2 years) further exacerbate the TB
resistance crisis.4 Accordingly, it is imperative to revitalise the
anti-TB drug discovery efforts and identify novel lead
compounds inhibiting cellular targets different from the ones
targeted by the current anti-TB drugs. This will presumably
synergise their efficacy in combination with current agents in
combating drug-sensitive (DS) and drug-resistant (DR) M. tb.
The mycobacterial membrane protein large 3 (MmpL3) is
a vital transporter currently considered as one of the most
druggable TB targets.5 It is responsible for the translocation of
mycolic acids (MAs) precursor, trehalose monomycolate (TMM),
across the plasma membrane.6–11 MAs are the major lipid
component of the mycobacterial outer membrane and the main
driving force behind its hydrophobicity and impermeability.12–14
We previously illustrated the detailed biosynthetic machinery of
MAs and their assembly into the mycobacterial cell envelope.15
Aer the ipping and release of TMM from cytoplasm to peri-
plasm via MmpL3, the cell wall core components serve as
acceptors anchoring MAs and forming an intricate protective
lipid bilayer. This coating insulates M. tb against exogenous
substances, including antibiotics, and the host immune
system.15 The indispensable role of MmpL3 in M. tb growth and
survival through shuttling TMM across the cytoplasmic
membrane was veried when the downregulation of mmpl3
expression led to an abrogation of cell division and rapid cell
death. This was preceded by an intracellular accumulation of
TMM and signicant reduction of cell wall mycolation.16,17
A conspicuous trend of “high lipophilicity ¼ high activity”
was entrenched in many of the MmpL3 inhibitors reported to
date. Recent literature revealed that ve structurally distinct
MmpL3 inhibitors indeed directly interact with MmpL3, while
three of them additionally impact the proton motive force
(PMF) in M. tb.18 Of particular interest are the indoleamides in
which NITD-304 (1, Fig. 1) and its analogous 4,6-diuoro
derivative NITD-349 were identied as preclinical agents for
treating MDR-TB.7 We also identied the N-(1-adamantyl)-4,6-
dimethylindole-2-carboxamide (2) as potent anti-TB agent
(MIC ¼ 0.012 mM) with no cytotoxicity to healthy cells (IC50 > 200
mM).19 Subsequent efforts, mainly directed towards non-
tuberculous mycobacteria (NTM), identied the N-(1-
adamantyl)-indole-2-carboxamide (3) and the N-rimantadine-
4,6-dimethylindole-2-carboxamide (4) as highly potent anti-TB
compounds with minimum inhibitory concentration (MIC)
values of 0.68 and 0.88 mM, respectively (Fig. 1).20
Fig. 1 Potent anti-TB compounds reported in the literature.
many indole-2-carboxamide derivatives, for instance compound
3 (EC50 ¼ 0.98 mM).26 Interestingly, in the same year, Franz et al.
reported the potent anti-TB activity of compound 3 (MIC ¼ 0.68
mM).20 Similarly, rimonabant, a cannabinoid receptor 1 (CB1)
inverse agonist, was found to inhibit the growth of M. tb via
targeting MmpL3.9 The aforementioned two examples demon-
strate the polypharmacological prole of various small mole-
cules, a phenomenon that has been harnessed for drug
repurposing, whereby new indications of existing agents/drugs
can be identied.27,28 This is well exemplied by the current
repositioning of drugs in the COVID-19 era.29 Towards this end,
we were interested in exploring the antitumour activity that
might be harboured in compound 3. We were particularly
drawn into the paediatric brain tumours that are categorised as
the highest-grade (grade IV) tumours according to the World
Health Organisation (WHO). Paediatric gliomas represent the
most common type of brain tumour and the leading cause of
cancer-related death in children.30,31 Glioblastoma multiforme
(GBM) is a grade IV glioma that originate from the supportive
cells of the brain and the spinal cord, known as the glial
cells.32–34 GBMs are among the most malignant, invasive and
aggressive primary brain tumours which are usually refractory
to current treatment, wherefore they remain largely incurable
and are nearly universally fatal.35,36 The mainstays of GBM
treatment include gross total resection when feasible followed
by focal radiotherapy combined with chemotherapy.37 Many
chemotherapeutic agents have been tested including temozo-
lomide which is used in the treatment of adult GBM, but no
clear benet or remarkable improvement on survival has been
achieved yet in paediatric GBM, likely due to its non-selective
alkylating actions.38 The dismal prognosis of GBM necessi-
tates the discovery and development of new, more effective
therapies for these tumours.
To date, several groups have reported the dual anti-TB and
antineoplastic activities for a variety of scaffolds, including
indole-2-carboxamides.21–25 Our group has also demonstrated
the selective cannabinoid receptor 2 (CB2) agonistic activity of
In the present study, we describe the design, synthesis, and
in vitro biological evaluation of novel indole-2-carboxamide
analogues as antitubercular agents. Upon assessing the drug-
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like properties in silico using ACD Labs/Percepta, the designed other N-rimantadine analogue 4 prepared in the same report
compounds were predicted to cross the blood brain barrier was also obtained in a similar low yield (29%).20 However, upon
(BBB). Accordingly, all synthesised compounds as well as employing EDC$HCl and HOBt coupling conditions in the
compound 3 were also screened for their cytotoxic and anti- current study, the N-rimantadine indoleamide derivatives were
proliferative activities against the KNS42 tumor cell line generated in signicantly higher yields (66–99%).
(paediatric GBM). We subsequently performed a transcriptional
Likewise, amide coupling of 4,6-diuoroindole-2-carboxylic
analysis on the KNS42 cells treated with one of the most potent acid 7h with benzylamine or phenylhydrazine rendered
compounds in order to determine the potential mechanism of amides 9a,b in yields 98% and 93%, respectively. We used
action of our indoleamide analogues. The top two potent fragment-based drug design in compound 9a in which we
compounds were then subjected to further evaluations against incorporated the benzyl group from the previously reported
a panel of non-GBM high-grade paediatric brain tumor cell lines anti-TB compound 5 (ref. 39) into the indoleamide scaffold
as well as non-tumor cells as control.
(Fig. 1 and 2). In Scheme 1, we also reacted the 4,6-diuor-
oindole 7h with 1,10-carbonyldiimidazole (CDI) and the result-
ing N-acylimidazole intermediate was treated in situ with
ammonia (nucleophilic substitution reaction) to yield the
amide 10. The nal imide derivatives 11a,b were then formed
via reacting 10 with either 3-uorobenzoyl chloride or 3-chlor-
obenzoyl chloride, respectively, in pyridine.
Similar to 9a,b and 11a,b, we further probed the effect of
extending the linker tethering the indole scaffold and the ada-
mantane ring by introducing a carbonyl piperazine fragment as
seen in the reported anti-TB derivative 6 (Fig. 1 and 2).40 The
diamides 15 and 20 were synthesised following the protocol
delineated in Scheme 2. The synthesis of compound 15 was
accomplished in three steps starting from coupling N-(tert-
butoxycarbonyl) piperazine (N-Boc piperazine, 12) with the
carboxylic acid 7h to give the amide analogue 13. Subsequent N-
Boc deprotection using triuoroacetic acid (TFA) yielded the key
indoleamide analogue 14 which was then converted to the nal
diamide 15 through a standard EDC-mediated coupling with 1-
adamantanecarboxylic acid. On the other hand, compound 20,
entailing a phenyl group as a linker in place of piperazine, was
2. Results and discussion
2.1. Chemistry
The design of nal compounds 8a–h, 9a,b, 11a,b, 15 and 20 is
depicted in Fig. 2. The indoleamides 8a–h and 9a,b were
prepared in a one-pot amide coupling reaction as outlined in
Scheme 1. First, since the N-rimantadine-indoleamide skeleton
proved to have potent anti-TB activity,20 we were interested in
exploring the effect of different substitutions at various posi-
tions on the indole ring on the anti-TB activity. Towards this,
commercially available indole-2-carboxylic acids 7a–h were
coupled with rimantadine hydrochloride. The amide coupling
was carried out in the presence of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC$HCl),
hydroxybenzotriazole hydrate (HOBt) and N,N-diisopropyle-
thylamine (DIPEA) to deliver the requisite rimantadine-derived
indoles 8a–h. Compound 8a was previously prepared using
N,N0-dicyclohexylcarbodiimide (DCC) and 4-dimethylamino-
pyridine (DMAP) coupling reagents in a poor yield (31%).20 The
Fig. 2 A diagram summarising the design strategy of the target indoleamides.
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Scheme 1 General synthetic routes for compounds 8a–h, 9a,b, and 11a,b.
obtained in a four-step pathway. The amino group of the aniline
16 was initially protected using di-tert-butyl dicarbonate (Boc)2O
to form the N-Boc derivative 17. An amide linkage was formed
thereaer connecting both 17 with the adamantane moiety,
providing the crude N-Boc intermediate 18. Next, cleaving the
Boc group under acidic conditions (TFA) furnished the penul-
timate amidoadamantyl compound 19. Finally, amide coupling
of 19 with the carboxylic acid 7h generated the desired dicar-
boxamide derivative 20.
2.2. Antitubercular activity
Target compounds 8a–h, 9a,b, 11a,b, 14, 15 and 20 were
screened in vitro against the wild-type M. tb strain (H37Rv) to
obtain their respective MIC values (Table 1). Compounds 3 and
4 together with the two front-line anti-TB drugs, isoniazid (INH)
and ethambutol (EMB) were used as positive controls. Franz
et al. assessed the activity of both N-(1-adamantyl)-indole-2-
carboxamide (3) and the N-rimantadine-4,6-dimethylindole-2-
Scheme 2 General synthetic routes for compounds 15 and 20.
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Table 1 In vitro anti-TB activity in addition to cytotoxic and antiproliferative activity against KNS42 tumour cell line of compounds 8a–h, 9a,b,
11a,b, 14, 15, and 20 as well as reference compounds INH, EMB and compounds 3, 4, 6
H37Rv, MICa
H37Rv, MICb
(mM)
KNS42 viability,
IC50 (mM)
KNS42 prolif.,
c
Cpd
R
X
(mg mLꢀ1
)
IC50d (mM)
Clog Pe
8a
8b
8c
8d
8e
H
—
—
—
—
—
—
—
—
CH2
NH
CO
CO
—
2
1
2
1
6.20
2.84
5.67
2.97
2.80
0.62
0.32
0.70
>112
>111
>101
>96
>10
>10
>10
>10
>10
>10
>10
>10
>10
9.85
>10
>10
>10
>10
6.62
6.07
6.09
6.09
6.57
6.89
7.04
7.64
6.50
3.84
2.42
4.49
5.06
1.54
3.80
4-OCH3
5-OCH3
5-CH3
5-Cl
6-Br
4,6-Dichloro
4,6-Diuoro
H
H
3-F
3-Cl
H
4.17
4.94
>10
3.13
0.84
>10
5.66
8.25
4.45
2.16
6.65
1.34
5.04
1
8f
0.25
0.125
0.25
>32
>32
>32
>32
>32
>32
8g
8h
9a
9b
11a
11b
14
15
>121
>75
Adamantane-
—
1-carbonyl
20
—
—
—
—
—
—
—
—
>32
>71
0.29
4.89
0.68 (ref. 20)
0.88 (ref. 20)
0.031 (ref. 40)
0.003 (ref. 42)
>10
NDf
NDf
0.33
NDf
NDf
NDf
>10
NDf
NDf
>10
NDf
NDf
NDf
5.29
ꢀ0.67
0.12
4.11
7.07
INH
EMB
3
4
6
0.04 (ref. 6)
1 (ref. 6)
0.2 (ref. 20)
0.31 (ref. 20)
—
—
—
4.25
5.90
ICA38
—
a
The lowest concentration of drug causing at least 90% reduction of bacterial growth by the microplate alamarBlue assay (MABA). The reported
MIC values are an average of three individual measurements, in mg mLꢀ1
.
The reported H37Rv MIC values converted to mM. Compound dose
b
c
d
required to achieve 50% inhibition of KNS42 cell viability, reecting cytotoxicity. Compound dose required to achieve 50% inhibition of
KNS42 cell proliferation. e Calculated using ChemDraw 16.0. f Not determined.
carboxamide analogue (4) against several mycobacterial strains, values, with compounds 8f–h exhibiting potencies (MIC ¼ 0.32–
including H37Rv M. tb strain (MIC ¼ 0.68 and 0.88 mM, 0.70 mM) higher than compound 4.
respectively).20 Within this context, we previously conducted
Upon re-evaluating the activity of the unsubstituted indole
a thorough antimycobacterial screening for several indole-2- derivative 8a, it displayed an MIC value of 6.20 mM, which was
carboxamide derivatives.6,19,41–43 We found that the methyl lower than the published one (MIC > 15.5 mM (ref. 20)). The 4-
groups located at the indole moiety in compound 2 present methoxyindole analogue 8b showed a two-fold enhancement of
a potential metabolic liability (susceptible to metabolic oxida- activity compared to the 5-methoxy counterpart 8c (MIC ¼ 2.84
tion). Accordingly, in order to explore the effects of various and 5.67 mM, respectively). To further probe the effect of mon-
substituents on the indole ring, we synthesised several indo- osubstitution at position 5, more lipophilic methyl and chloro
leamide derivatives 8a–h bearing a rimantadine substituent in groups were installed. The observed activities of compounds
place of the adamantane ring. All of the tested N-rimantadine 8d,e (MIC ¼ 2.97 and 2.80 mM, respectively) were 2-fold higher
indoleamides had single digit micro- and submicro-molar MIC than the respective 5-methoxy analogue 8c. This could be
attributed to the higher lipophilicity of 8d,e (Clog P ¼ 6.57 and
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6.89, respectively) compared to 8c (Clog P ¼ 6.09). These results pyridines were previously found to exert their anti-TB activity via
suggest the lipophilicity-driven bioactivity of this series. inhibiting M. tb cytochrome b subunit of the cytochrome bc1
Nevertheless, the fact that the 5-substituted indoles 8d,e are complex (QcrB).44,45 We, thereupon, appended a benzyl moiety
equipotent to the 4-substituted analogue 8b, despite the lower to the 4,6-diuoroindoleamide architecture. Disappointingly,
lipophilicity of the latter compared to the former derivatives, the tested benzylamide analogue 9a was bere of anti-TB
reects the substitution preference for certain positions on the activity (MIC > 112 mM). We also presumed that incorporating
indole ring. Interestingly, the presence of a single bromo group an NH or a carbonyl (C]O) group, instead of CH2, between the
at position 6 of the indole ring (8f: MIC ¼ 0.62 mM) led to an carboxamide moiety and the phenyl group would result in the
approximately 10- and 4.5-fold increase in the activity in formation of more hydrogen bonds with the hydrophilic resi-
comparison to the unsubstituted and 5-halosubstituted indoles dues in the S4 hydrophilic subsite of MmpL3,9 leading to an
8a,e, respectively.
improved binding and better activity. As reported previously,46
Next, we replaced the two methyl groups in compound 4 with when we introduced a C]O group between the 4,6-diuor-
metabolically stable halogens 8g,h. It was pleasing to nd that oindoleamide and the adamantane moiety, the obtained imide
compound 8g possessing 4,6-dichloro substituents on the derivative displayed moderate anti-TB activity (MIC ¼ 22 mM).
indole core exhibited nearly a three-fold rise in activity (MIC ¼ Unfortunately, this NH or C]O extension in compounds 9b and
0.32 mM) relative to compound 4 and a comparable potency to 11a,b, did not show any improved anti-TB activity (MIC > 96
INH (MIC ¼ 0.88 and 0.29 mM, respectively). Of note is the fact mM).
that compound 8g is the most lipophilic compound in our study
It is noteworthy that the estimated lipophilicities of
(Clog P ¼ 7.64) that was translated into the highest activity as compounds 11a,b (Clog P ¼ 4.49 and 5.06, respectively) are
well which once again signies the apparent inuence of lip- higher than that of 9a,b (Clog P ¼ 3.84 and 2.42, respectively)
ophilicity on the anti-TB activity. The 4,6-diuoro analogue 8h which did not seemingly impact the anti-TB activity. Of interest
(MIC ¼ 0.70 mM) was more active than 4 and 2-fold less potent is the fact that two N-phenyl indole-2-carboxamide analogues
than the 4,6-dichloroindole counterpart 8g. The consequential exhibited good anti-TB activities in our previous work (MIC ¼
impact of the substitution pattern in the indole ring on activity, 1.7 and 3.8 mM).19 This in turn suggests that introducing an
depicted earlier in compounds 8d,e versus 8b, was recapitulated extra spacer to the amide linker tethering the indole ring and
in compound 8h versus 8f in which both compounds exhibited the phenyl moiety is unfavourable. In the same report, on the
similar activities, although 8f is more lipophilic than 8h (Clog P contrary, we found that adding a methylene spacer between the
¼ 7.04, 6.50, respectively). Importantly, the potency rendered by amide nitrogen and a cyclohexyl ring elicited almost the same
all the rimantadine analogues was comparable to or signi- high potency as the desmethylene analogue (MIC ¼ 0.88 and
cantly higher than the rst-line anti-TB drug EMB (MIC ¼ 4.89 0.93 mM, respectively).19 Evidently, the potent anti-TB activity
mM).
manifested by the N-rimantadine indoleamide analogues 8a–h
Similar to the other MmpL3 inhibitors, the inherent high in the present study corroborated the notion that incorporating
lipophilicity of the foregoing series (Clog P ¼ 6.07–7.64) likely an extra spacer next to the cycloaliphatic motifs is tolerated.
endowed them with facilitated diffusion through the lipid-rich
On the other hand, the nitrobenzothiazinone derivative 6,
bilayer of M. tb where they presumably interacted with bearing a carbonyl piperazine moiety (Fig. 1), was reported to
MmpL3 and elicited potent anti-TB activity. Nonetheless, it is have a potent anti-TB activity (MIC ¼ 0.031 mM (ref. 40)). The
worth noting that the anti-TB activities of the N-(rimantadine)- 1,3-benzothiazin-4-ones (BTZs) are a potent class of anti-
indole-2-carboxamides in our study are generally lower than the mycobacterial agents that kill M. tb in vitro, ex vivo and in TB
previously evaluated N-(1-adamantyl)-indole-2-carboxamide mouse models via targeting decaprenylphosphoryl-b-D-ribose
counterparts. Indeed, this was counterintuitive due to the 20-epimerase (DprE1) enzyme. Inhibition of the DprE1 enzy-
high lipophilicity of the rimantadine derivatives compared to matic activity compromises the formation of a key precursor in
the corresponding 1-adamantane analogues.43 The SAR hall- the synthesis of cell wall arabinans, namely decap-
marks of these analogues, in which substitutions at the 4- and/ renylphosphoryl arabinose, resulting in cell lysis and bacterial
or 6-positions of the indole ring was optimal for activity, death.47 Introducing a 4-carbonyl piperazine segment into the
together with the apparent impact of lipophilicity on the anti-TB BTZ framework was well tolerated and led to compounds with
activity, were consistent with our previous ndings.19,42,43 enhanced aqueous solubility compared to other BTZ analogues
Therefore, in our subsequent evaluations in which we used the in earlier reports.40 This prompted us to integrate the carbonyl
fragment-based drug design technique, 4,6-diuoroindole 7h piperazine fragment into the N-(adamantyl)indoleamide struc-
was the scaffold of choice, to which we attached different ture core to verify its tolerability within this template. Initially,
fragments from previously reported anti-TB agents with diverse we examined the activity of the piperazinylindole intermediate
mechanism of actions 5 and 6 (Fig. 1).
14 which displayed an MIC value > 121 mM. This dramatic loss
First, Moraski et al. called attention to the remarkable anti- in activity could be ascribed to its diminished lipophilicity
TB potency of the N-benzyl imidazo[1,2-a]pyridine-3- (Clog P ¼ 1.54) and the absence of the amide NH which is
carboxamide
5
(MIC
¼
0.37–1.9 mM in three different a crucial element for the hydrogen bonding with Asp645 in
media).39 This compound also maintained its excellent potency MmpL3.9 The NH necessity was further emphasised when we
when tested against a panel of single DR strains in addition to increased the lipophilicity of 14 via attaching an adamantane
several MDR and XDR clinical isolates.39 Imidazo[1,2-a] ring, furnishing the 1,4-dicarbonyl piperazine 15 which was
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Table 2 In vitro cytotoxicity (Vero cells) and molecular modelling results of compounds 8f and 8g
Docking scorec
Cpd
8f
MIC (mM)
0.62
IC50a (mM)
39.9
SIb
(kcal molꢀ1
)
No. of H-bonds
Distance
Amino acids
Ligand atoms
64
ꢀ13.9
2
2
1
2.51
2.66
2.55
2.62
2.54
Asp645
Asp645
Asp645
Asp645
Asp645
Amide NH
Indole NH
Amide NH
Indole NH
Amide NH
8g
0.32
40.9
128
NDd
ꢀ14.4
ICA38
0.003 (ref. 42)
NDd
ꢀ22.7
a
Cytotoxicity against Vero cells expressed as the half maximal inhibitory concentration of the drug by MABA. b Selectivity index (SI) ¼ IC50(Vero)/
c
d
MIC(H37Rv). The lowest binding energy corresponding to the top-ranked pose. Not determined.
devoid of activity (MIC > 75 mM) despite having a Clog P value of minimal primary cytotoxicity of these derivatives against
3.80 that is higher than that of 14.
healthy mammalian cells.
A similar trend was observed in the analogous dicarbox-
Taking into consideration that MmpL3 is the most relevant
amide 20, in which a phenyl motif, in lieu of piperazine, was potential enzymatic target of our analogues, molecular docking
introduced in between the indole and adamantane rings. simulations were carried out to evaluate the possible binding
Despite its higher lipophilicity (Clog P ¼ 5.29) and that the mechanism of the rimantadine-based indoles within the
amide NH was preserved, compound 20 was inactive (MIC > 71 MmpL3 active site (Table 2; Fig. 3 and 4). First, the co-
mM). Hence, it could be surmised that stretching the linker crystallised ligand ICA38 was re-docked into the MmpL3
between the indole scaffold and the adamantane moiety via ring binding site to validate the docking protocol as we reported
insertion is detrimental to the anti-TB activity, suggesting some before (Fig. 3).46 In consonance with the previous ndings,9 the
steric requirements of the S4 subsite of MmpL3. The abolition indole moiety and the spirocarbocyclic group of ICA38 were
of anti-TB activity pertaining to overextending the middle linker both lodged in the bulky S3 and S5 hydrophobic subsites,
resonates with our previous ndings.46 Overall, whilst a positive respectively, forming hydrophobic contacts with the
correlation between lipophilicity and anti-TB activity was surrounding residues. The amide linker was positioned in the
discernible in compounds 8a–h, the rest of the tested analogues S4 hydrophilic subsite, wherein the amide NH of ICA38 formed
did not conform with this trend.
a hydrogen bond with Asp645. This Asp645 is a crucial part of
Motivated by the potent in vitro anti-TB activities of the the two Asp–Tyr pairs implicated in proton relay. Upon binding,
rimantadine-derived indole derivatives 8a–h, two compounds 8f ICA38 inhibits the MmpL3 by occupying S3–S5 subsites in the
and 8g (MIC ¼ 0.62, 0.32 mM, respectively) were selected for proton transportation channel, disrupting the key components
further cytotoxicity assessment and their selectivity index (SI) of the S4 subsite, namely the two Asp–Tyr pairs (Fig. 3), thereby
were subsequently calculated (Table 2). We were pleased to nd blocking the PMF for substrate translocation.9
that both compounds displayed high IC50 values against Vero
Both compounds 8f and 8g, typifying the rimantadine class,
cells (IC50 ¼ 39.9 and 40.9 mM, respectively). These values were oriented inside the MmpL3 binding pocket in a manner
correspond to SI values of 64 and 128, respectively, indicating resembling ICA38 (Fig. 4). The most favourable docking pose
(i.e. lowest binding energy) retained the key interactions
Fig. 3 Re-docking of ICA38 (Cyan) in the MmpL3 active site (A), showing the S3–S5 subsites. The ICA38 top pose was oriented almost at the
same position as the original ICA38 co-crystallised ligand (purple). The two key Asp–Tyr pairs, implicated in proton relay, located in the S4 subsite
are marked in orange hashed ovals. The putative binding interactions of ICA38 with the MmpL3 binding pocked is represented in 2D on the right
panel (B).
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Fig. 4 Superposition of the top ranked docking pose of 8f (A) and 8g (B) (brown) and the co-crystallised ligand ICA38 (purple), showing the
putative binding mode of both compounds in the MmpL3 active site.
observed in ICA38. The S3 hydrophobic subsite accommodated (Table 2). Nonetheless, both 8f and reference compound 3
the indole nucleus, while the rimantadine moiety was showed weak antiproliferative activities against KNS42 (IC50
>
embedded in the bulky S5 hydrophobic subsite, overlapping 10 mM). Compounds 8b, 8c, 8e and 8h showed potent antitu-
with the spirocyclic group of ICA38. Meanwhile, the amide NH bercular activity and reduction of KNS42 cell viability [MIC (M.
in 8f and 8g was inserted in the S4 hydrophilic subsite, forming tb) and IC50 (KNS42) < 6 mM].
˚
a hydrogen bond with Asp645 (distance ¼ 2.51 and 2.55 A,
On the other hand, compound 9a, which was bere of
respectively). An additional hydrogen bond was formed between antitubercular activity, displayed good inhibitory activities
the indole NH of 8f and 8g with Asp645 (distance ¼ 2.66 and against KNS42 cells in the viability and proliferation assays with
˚
2.62 A, respectively). They also showed high binding affinity to IC50 values of 8.25 and 9.85 mM, respectively. Similarly,
the MmpL3 active site (docking score
¼ ꢀ13.9 and compound 15 exhibited potent cytotoxic and antiproliferative
ꢀ14.4 kcal molꢀ1, respectively). The similarities between the activity against KNS42 cells (IC50 ¼ 5.04 and 6.62 mM, respec-
binding modes of 8f and 8g and the MmpL3 inhibitor ICA38 tively). Additionally, compounds 9b, 11a, 11b and 14 displayed
suggest that this potent class of N-rimantadine indoleamides high cytotoxic activities (IC50 ¼ 1.34–6.65 mM) and poor anti-
likely inhibit the same target via disrupting the two Asp–Tyr proliferative activity (IC50 > 10 mM) against KNS42 cells.
pairs that play a pivotal role in the proton translocation. It is
worth mentioning that a recent study used a combination of in
vitro and whole-cell-based approaches to determine whether
a structurally distinct panel of MmpL3 inhibitors directly target
2.4. Transcriptional analysis of KNS42 cells and compound
15-induced alterations to gene expression
the MmpL3 or dissipate the PMF from which this transporter
derives its energy (indirect mechanism). They provided
evidence that all compounds, including the indole-2-
carboxamides, directly inhibit MmpL3, whether or not they
exert an additional impact on the PMF that potentiate their
activity.18
To shed light on the potential mechanism of action of our
indole-2-carboxamides as antitumour agents, we characterised
the gene transcriptional response of the KNS42 cells upon
exposure to compound 15, using DNBSEQ Eukaryotic Stranded
Transcriptome Resequencing. Of note, compound 15 was
chosen in this respect due to its potent activity against KNS42
cells in both viability and proliferation assays (IC50 ¼ 5.04 and
6.62 mM, respectively). When we analysed the differential
expression of the genes, in comparison to the control cells
(untreated), we found that compound 15 downregulated the
2.3. Cytotoxic and antiproliferative activity against
paediatric GBM cells
All synthesised nal compounds in addition to reference expression of 9 genes (fold change $ 8) with statistical signi-
compound 3 were initially evaluated for their cytotoxicity and cance (P < 0.05). Two genes, namely the carbonic anhydrase 9
antiproliferative activities against paediatric KNS42 GBM cell (CA9) and the spleen tyrosine kinase (SYK) stood out as poten-
line (Table 1). Compound 3, which previously showed potent tial targets of this compound, whereby it exerts its antitumour
antitubercular activity against M. tb H37Rv strain (MIC ¼ 0.68 activity. Several reports previously showed that the expression
mM (ref. 20)), displayed potent cytotoxicity against KNS42 cells levels of CA9 and SYK in GBM tumours are positively correlated
with sub-micromolar inhibitory activity on cell viability (IC50
¼
to tumour growth/progression and negatively correlated to
0.33 mM). The cytotoxicity of compound 3 was previously eval- survival rates.48–50 Therefore, downregulating the expression of
uated against human THP-1 cells in which it was non-cytotoxic these two genes could be conducive to the antitumour effects
up to 50 mM.20 Similarly, compound 8f demonstrated excellent observed in compound 15.
dual antitubercular and antitumour activity [MIC (H37Rv) ¼
First, we found that the expression level of CA9 was down-
0.62 mM, IC50 (KNS42 viability) ¼ 0.84 mM]. Importantly, regulated by 15-fold (p < 0.01) upon treating the KNS42 cells
compound 8f also exhibited minimal cytotoxicity against with compound 15. CA9 is a hypoxia-responsive gene which is
healthy mammalian Vero cells with an IC50 value of 39.9 mM used as a hypoxia marker and a regulator of the pH of tumour
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Table 3 In vitro cytotoxicity and antiproliferation activity of compounds 9a and 15 against different paediatric brain tumour cell lines (BT12, BT16
and DAOY) and non-neoplastic human fibroblasts (HFF1)
BT12 viability,
BT12 prolif.,
BT16 viability,
BT16 prolif.,
DAOY viability,
DAOY prolif.,
HFF1 viability,
HFF1 prolif.,
Cpd
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
9a
15
0.89
3.16
7.44
>10
1.81
5.89
6.06
>10
>10
>10
>10
>10
119
19.35
65
17.41
cells.51 Hypoxia is a common feature of the majority of malig- GBM cell proliferation and invasiveness in vivo. In addition,
nant tumours, in which extensively expanding and proliferating blocking the activity of SYK genetically or by inhibitors in
tumour tissues outgrow their blood supply resulting in tumour cells prolonged the life of treated mice and increased
hampered oxygen diffusion and hypoxic milieus.52 CA9 expres- their survival.49 Of note, our indoleamide derivative 15, which
sion is strongly induced by hypoxia, therefore it can serve as signicantly downregulated the expression of SYK protein,
a
marker that signies aggressive rapidly proliferating displayed the most potent antiproliferative activity in our set of
tumours.50 Hence, CA9 expression was found to be correlated to analogues. This is in fact consistent with the Moncayo et al.
tumour progression in various human cancers.53 Importantly, ndings. Overall, the evident downregulation of CA9 and SYK
strong CA9 immunoreactivity was previously detected in HGGs, expression induced by compound 15 suggest that our observed
including GBM, whereas the normal brain cells showed no antitumour effects could be correlated to modulating the
expression of CA9.53 This tumour-exclusive expression pattern activity of these two targets.
establishes CA9 as a feasible antitumour target. Indeed, Pro-
escholdt et al. found that CA9 is expressed in the GBM tumours
in all of the patients they investigated, whereby the expression
levels were generally correlated with the overall prognosis.50
Their survival analysis revealed that high expression levels of
CA9 was associated with poor survival in GBM patients (15
months) compared to GBM patients with low CA9 expression
(34 months). Therefore, high CA9 expression is considered
a prognostic marker for poor survival in GBM patients.48,50
Selective gene silencing of CA9 in GBM cells resulted in
a profound reduction of cell attachment and invasion as well as
a strong enhanced susceptibility to adjuvant chemotherapy and
radiation treatment.50 This was substantiated by Boyd et al.'s
recent ndings, in which concurrently using a CA9 inhibitor
with temozolomide inhibited the growth of GBM cells and
induced cell cycle arrest in vitro.51 This combination was also
efficacious in extending the survival of GBM-bearing mice.
Taken together, the signicant downregulation of CA9 gene in
KNS42 cells induced by compound 15 suggest the involvement
of CA9 in the cytotoxic and antiproliferative activities observed
in this compound.
On the other hand, compound 15 also repressed the
expression of the SYK by 9-fold with statistical signicance (p <
0.05). SYK is a non-receptor protein tyrosine kinase which is an
essential component of the signalling machinery in the
immune system.54 It is mainly expressed in hematopoietic cells,
including macrophages, B cells, neutrophils, monocytes, and
natural killer cells.55 SYK plays a key role in the oncogenesis and
tumour promotion of various cancers, including gliomas.55
Indeed, Moncayo and colleagues have recently found that SYK is
overexpressed in the malignant gliomas, including GBM.49
Their in vitro experiments showed that inhibiting or knocking
down SYK activity resulted in blocking the proliferation and
migration of GBM cell lines. Importantly, these ndings were
mirrored in vivo in nude mice injected with human GBM cells
overexpressing SYK.49 In this respect, SYK inhibition reduced
2.5. Cytotoxic and antiproliferative activities of the top
potent compounds against different non-GBM paediatric
brain tumour cells and non-neoplastic broblasts
Both compounds 9a and 15 were selected for further in vitro
viability and proliferation inhibition studies against different
grade IV paediatric brain tumour cell lines and non-neoplastic
mammalian cells (Table 3). Both compounds retained their
potent cytotoxic activities against two atypical teratoid/rhabdoid
tumour (AT/RT) cell lines BT12 and BT16. Remarkably,
compound 9a showed approximately 9- and 5-fold higher cyto-
toxic activity, respectively, when tested against the two fore-
named cell lines compared to KNS42 [IC50 ¼ 0.89 (BT12), 1.81
(BT16) and 8.25 (KNS42) mM]. Compound 9a also exhibited high
antiproliferative activities against BT12 and BT16 cells [IC50
¼
7.44 and 6.06 mM, respectively]. Despite the potent anti-
proliferative activity of compound 15 against KNS42, this
activity was not maintained against the two tested AT/RT cell
lines (IC50 > 10 mM). On the other hand, both compounds
demonstrated a drop in both cytotoxic and antiproliferative
activities when tested against the medulloblastoma cell line
DAOY (IC50 > 10 mM). To our delight, compound 9a displayed
negligible cytotoxicity when tested against the non-neoplastic
human broblast cell line HFF1 (IC50 ¼ 119 mM), suggesting
its selective cytotoxicity with an SI value up to 134 against
tumour cells. Compound 15 displayed moderate cytotoxicity
against HFF1 cells (IC50 ¼ 19.35 mM), giving low SI values.
The physicochemical properties of compound 9a were pre-
dicted using ACD/Labs Percepta 2016 Build 2911 (13 Jul 2016).
We evaluated the compliance of this compound with Lipinski's
rule of ve (RO5) in order to evaluate its drug-likeness.56
Compound 9a showed zero violations to the RO5, indicating the
drug-like attributes of this compound, including its prospective
in vivo bioavailability. Importantly, this compound is also ex-
pected to cross the BBB, whereupon it can elicit its antitumour
activity. Overall, the aforementioned results establish
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compound 9a as a promising antitumour agent against various non-neoplastic cells and in silico drug-like prole of the N-benzyl
aggressive paediatric brain cancer cells, whilst being safe indoleamide 9a establish this compound as a potential thera-
towards healthy cells.
peutic agent that merits further investigation.
These ndings foreground the indoleamides as a new class
of compounds that could be ne-tuned to modulate their dual
anti-TB and antitumour activities. In addition, our gene
3. Conclusions and future directions
In summary, we established a body of preliminary SAR for the N- expression results lay the foundation for future studies to
rimantadine indoleamides 8a–h as anti-TB agents, based on identify more indoleamide analogues as antitumour agents that
compound 4 which was obtained from a previous screening20 can modulate the expression of CA9 and SYK genes. Unlike the
against several mycobacterial strains. The SAR outcomes of this conventional target identication approaches which employ
class were in harmony with our published ndings,19,42,43 high- determining the activity of a small molecule at a receptor or
lighting the signicance of both the substitution pattern of the enzyme level, the transcriptional approach has the advantage of
indole moiety and the lipophilic nature of the substituents. In identifying the compound's impact on the absolute concentra-
this study, a subsequent massive drop in the anti-TB activity tion of a target protein.57 Future efforts will focus on conrming
ensued from the structural modications implemented in if the observed antitumour activity of the indoleamide scaffold
compounds 9a,b, 11a,b, 14, 15 and 20. Although lipophilicity is in pediatric brain cancer cells is the direct consequence of
a key driver for the anti-TB efficacy in 8a–h, it is not the sole inhibiting CA9 and/or SYK.
parameter inuencing the potency in our study. The most active
anti-TB compound identied in our study 8g exhibited limited
cytotoxic activity against Vero cells. A docking analysis was
performed on compounds 8g and 8f, exemplifying the binding
4. Experimental
4.1. Chemistry
mode of the N-rimantadine indoleamide series. The similarity
4.1.1. General. All indole-2-carboxylic acids 7a–h and
between the binding fashion of both compounds and ICA38 in rimantadine hydrochloride were purchased from Fluorochem,
the same MmpL3 binding pocket support the high in vitro anti- while the benzylamines were purchased from Sigma-Aldrich. N-
TB potency of the amidorimantadine class. Since the indolea- Boc-piperazine and 4-aminobenzoic acid were purchased from
1
mide architecture was previously reported to have antituber- AK Scientic. H NMR and 13C NMR spectra were recorded on
cular and antitumour activities,22,23 all compounds in the a Bruker Avance III spectrometer at 400 and 100 MHz, respec-
present study were tested for their antitumour activity against tively, with TMS as an internal standard. Standard abbreviations
the paediatric GBM cell line KNS42. Some of the tested indicating multiplicity were as follows: s ¼ singlet, d ¼ doublet,
compounds demonstrated potent anti-TB activity and cytotox- dd ¼ doublet of doublets, t ¼ triplet, td ¼ triplet of doublets, q
icity towards KNS42 cells. Compound 8f displayed the most ¼ quadruplet, dq ¼ doublet of quartets, m ¼ multiplet and br ¼
potent dual activity while showing minimal cytotoxicity against broad. HRMS experiments were done on a Thermo Scientic Q-
non-neoplastic mammalian cells. Additionally, several Exactive Orbitrap mass spectrometer. TLC was carried out on
compounds, which were devoid of anti-TB activity, showed Analtech silica gel TLC plates (200 microns, 20 ꢁ 20 cm). Flash
potent cytotoxicity and/or antiproliferative activities against the chromatography was performed using a Teledyne Isco Combi-
KNS42 cell line.
Flash Rf system with RediSep columns or manually using Sili-
Differential expression analysis of the genes in the KNS42 cells Cycle SiliaFlash® P60 Silica Gels [40–63 mm (230–400 mesh)].
in response to compound 15 versus untreated cells revealed that The preparative HPLC (Shimadzu) employed a Phenomenex
this compound repressed the expression of CA9 (p < 0.01) and Luna® Omega 5 mm Polar C18 100A (150 ꢁ 21.2 mm) column,
SYK (p < 0.05) genes by 15-fold and 9-fold, respectively. The with detection at 254 and 280 nm on a Shimadzu SPD-20A
expression levels of these genes in GBM tumours were previously detector, ow rate ¼ 25.0 mL minꢀ1. Method 1: 40–100%
shown to be directly correlated to the tumour growth and acetonitrile/H2O in 15 min; 100% acetonitrile in 10 min; 100–
progression and inversely correlated to survival rates.48–50 Indeed, 40% acetonitrile/H2O in 10 min. Method 2: 60–100% acetoni-
knocking down the activity of each of these two genes was also trile/H2O in 10 min; 100% acetonitrile in 15 min; 100–60%
found to inhibit the growth of GBM cells in vitro.49,51 Accordingly, acetonitrile/H2O in 10 min. Method 3: 80–100% acetonitrile/
the cytotoxic and antiproliferative effects of compound 15 could H2O in 10 min; 100% acetonitrile in 15 min; 100–80% aceto-
be attributed to downregulating the activity of these two genes. nitrile/H2O in 10 min. Both solvents contained 0.05 vol% of
Two compounds 9a and 15, which potently reduced cell viability TFA. Purities of nal compounds were established by analytical
and proliferation of KNS42 cells, were further evaluated against HPLC, which was conducted on Waters HPLC system (2487 dual
three paediatric brain tumour cell lines and non-neoplastic wavelength absorbance detector, 1525 binary pump, and 717
human broblasts. Compound 9a stood out as the most prom- plus autosampler) with a Phenomenex Luna® 5 mm C18(2) 100
˚
ising antitumour agent, displaying excellent cytotoxic and anti- A (150 ꢁ 4.6 mm) column. Analytical HPLC method: ow rate ¼
proliferative activities against two AT/RT cell lines (BT12 and 1 mL min; gradient elution over 30 min. Gradient 1: 20–100%
BT16) in addition to the GBM KNS42 cell line. The cytotoxicity of acetonitrile/H2O in 15 min; 100% acetonitrile in 10 min; 100–
9a was nullied against non-neoplastic human cells, suggesting 20% acetonitrile/H2O in 5 min. Gradient 2: 50–100% acetoni-
its selective activity towards tumour cells. Overall, the high trile/H2O in 15 min; 100% acetonitrile in 10 min; 100–50%
activity against brain tumour cells, negligible cytotoxicity towards acetonitrile/H2O in 5 min. Again, 0.05 vol% of TFA was
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incorporated in both solvents. Final compounds were already (DMSO-d6) d 11.49 (s, 1H), 7.83 (d, J ¼ 9.6 Hz, 1H), 7.33 (d, J ¼
>95% pure aer column chromatography or either puried by 1.6 Hz, 1H), 7.11–6.97 (m, 2H), 6.50 (d, J ¼ 7.5 Hz, 1H), 3.88 (s,
preparative HPLC or recrystallised from DMF to attain the 3H), 3.81 (overlapping dq, J ¼ 7.0 Hz, 1H), 1.94 (s, 3H), 1.69–1.47
needed purity as determined by the above-stated method.
(m, 12H), 1.06 (d, J ¼ 6.9 Hz, 3H); 13C NMR (DMSO-d6) d 160.9,
4.1.2. General procedure for amide coupling (method A). 154.0, 138.2, 131.0, 124.6, 118.6, 105.8, 100.8, 99.6, 55.4, 52.6,
To a solution of the appropriate carboxylic acid (1 mmol) in 38.6, 37.2, 36.7, 28.3, 14.5; HRMS (ESI) m/z calcd for C22H28N2O2
anhydrous dimethylformamide (DMF, 10 mL), EDC$HCl (2 ([M + H]+) m/z 353.2224; found 353.2217.
mmol), HOBt (2 mmol), the corresponding amine (1.3 mmol)
N-(1-(Adamantan-1-yl)ethyl)-5-methoxy-1H-indole-2-
and DIPEA (3–6 equiv.) were added and the reaction mixture carboxamide (8c). 5-Methoxyindole-2-carboxylic acid (7c) and
was stirred at room temperature (rt) until the disappearance of rimantadine hydrochloride were used to synthesise the title
the starting material (usually 60–72 h). Aer this time water (50 compound employing method A. White solid, yield: 99%. 1H
mL) was added, and the mixture was extracted with EtOAc (3 ꢁ NMR (DMSO-d6) d 11.34 (s, 1H), 7.81 (d, J ¼ 9.5 Hz, 1H), 7.31 (d, J
50 mL). The combined organic layers were washed with water (5 ¼ 8.9 Hz, 1H), 7.13 (d, J ¼ 1.5 Hz, 1H), 7.06 (d, J ¼ 2.3 Hz, 1H),
ꢁ 50 mL), brine (1 ꢁ 25 mL), dried over anhydrous Na2SO4, 6.83 (dd, J ¼ 8.9, 2.4 Hz, 1H), 3.83 (overlapping dq, J ¼ 7.0 Hz,
ltered, and concentrated under reduced pressure. The residue 1H), 3.76 (s, 3H), 1.94 (s, 3H), 1.72–1.48 (m, 12H), 1.08 (d, J ¼
was puried by ash chromatography or manual column using 7.0 Hz, 3H); 13C NMR (DMSO-d6) d 161.0, 154.1, 132.8, 132.1,
dichloromethane/methanol (DCM/MeOH) gradient. The 127.9, 114.6, 113.5, 103.0, 102.4, 55.7, 52.6, 38.6, 37.1, 36.7, 28.3,
purities of nal compounds 8a,b,d,g,h as well as 9a were >95% 14.5; HRMS (ESI) m/z calcd for C22H28N2O2 ([M + H]+) m/z
aer column chromatography as demonstrated by analytical 353.2224; found 353.2223.
HPLC. Compounds 8c, 9b, 15, and 20 were further puried by
preparative HPLC, whereas compounds 8e,f were recrystallised (8d). This compound was obtained from 5-methylindole-2-
from DMF to be procured in the requisite purity. carboxylic acid (7d) and rimantadine hydrochloride according
N-(1-(Adamantan-1-yl)ethyl)-5-methyl-1H-indole-2-carboxamide
4.1.3. General procedure for imide preparation (method to method A. White solid, yield: 66%. 1H NMR (DMF-d7) d 11.40
B). To a solution of 4,6-diuoro-1H-indole-2-carboxamide (10, 1 (s, 1H), 7.86 (d, J ¼ 9.6 Hz, 1H), 7.43 (d, J ¼ 8.4 Hz, 1H), 7.40 (d, J
mmol) in pyridine (5 mL), the appropriate benzoyl chloride ¼ 0.6 Hz, 1H), 7.27 (dd, J ¼ 2.0, 0.6 Hz, 1H), 7.04 (dd, J ¼ 8.4,
derivative (1.5 mmol) was added, and the reaction mixture was 1.4 Hz, 1H), 3.94 (overlapping dq, J ¼ 7.0 Hz, 1H), 2.38 (s, 3H),
reuxed for 16 h. Aer cooling, 2 M HCl solution (25 mL) was 1.93 (s, 3H), 1.75–1.53 (m, 12H), 1.12 (d, J ¼ 7.0 Hz, 3H); 13C
added and the mixture was extracted with diethyl ether (1 ꢁ 25 NMR (DMF-d7) d 161.6, 136.0, 133.2, 129.2, 128.7, 125.7, 121.5,
mL) and EtOAc (3 ꢁ 25 mL). The organic layers were collected, 112.6, 102.7, 53.2, 39.1, 37.6, 37.2, 29.1, 21.4, 14.4; HRMS (ESI)
washed with brine (1 ꢁ 25 mL), dried over anhydrous Na2SO4, m/z calcd for C22H28N2O ([M + H]+) m/z 337.2274; found
ltered, concentrated under reduced pressure, and puried by 337.2269.
ash chromatography using DCM/MeOH gradient. The ob-
tained product was further puried by preparative HPLC to (8e). The title compound was synthesised from 5-chloroindole-
achieve >95% purity. 2-carboxylic acid (7e) and rimantadine hydrochloride employ-
N-(1-(Adamantan-1-yl)ethyl)-5-chloro-1H-indole-2-carboxamide
4.1.4. General procedure for N-Boc deprotection (method ing method A. White solid, yield: 98%. 1H NMR (DMSO-d6)
C). To a solution of the N-Boc protected amine (2 mmol) in d 11.68 (s, 1H), 7.97 (d, J ¼ 9.5 Hz, 1H), 7.67 (d, J ¼ 2.0 Hz, 1H),
10 mL DCM, 4 mL TFA was added and the reaction mixture was 7.43 (d, J ¼ 8.7 Hz, 1H), 7.21 (d, J ¼ 0.8 Hz, 1H), 7.16 (dd, J ¼ 8.7,
stirred at rt for 12 h. The mixture was then concentrated in vacuo 2.1 Hz, 1H), 3.81 (overlapping dq, J ¼ 7.0 Hz, 1H), 1.93 (s, 3H),
and NaHCO3 solution was added for neutralisation, followed by 1.67–1.46 (m, 12H), 1.06 (d, J ¼ 7.0 Hz, 3H); 13C NMR (DMSO-d6)
extraction with DCM (3 ꢁ 50 mL). The combined organic pha- d 160.7, 135.2, 133.9, 128.6, 124.6, 123.7, 120.9, 114.3, 102.8,
ses were washed with brine (1 ꢁ 25 mL), dried over anhydrous 52.8, 38.6, 37.1, 36.7, 28.3, 14.4; HRMS (ESI) m/z calcd for
Na2SO4, ltered, and concentrated under reduced pressure. The
residue was puried by ash chromatography using DCM/
MeOH gradient.
C
21H25ClN2O ([M + H]+) m/z 357.1728; found 357.1732.
N-(1-(Adamantan-1-yl)ethyl)-6-bromo-1H-indole-2-carboxamide
(8f). This compound was obtained from 6-bromoindole-2-
N-(1-(Adamantan-1-yl)ethyl)-1H-indole-2-carboxamide (8a). The carboxylic acid (7f) and rimantadine hydrochloride according
title compound was obtained from indole-2-carboxylic acid (7a) to method A. White solid, yield: 91%. 1H NMR (DMSO-d6)
and rimantadine hydrochloride employing method A and its 1H d 11.66 (s, 1H), 7.97 (d, J ¼ 9.5 Hz, 1H), 7.63–7.52 (m, 2H), 7.28
NMR data matched the one reported in the literature.20 White (d, J ¼ 1.4 Hz, 1H), 7.16 (dd, J ¼ 8.6, 1.7 Hz, 1H), 3.83 (over-
solid, yield: 91%. 1H NMR (DMSO-d6) d 11.50 (s, 1H), 7.86 (d, J ¼ lapping dq, J ¼ 6.9 Hz, 1H), 1.94 (s, 3H), 1.75–1.38 (m, 12H), 1.08
9.5 Hz, 1H), 7.60 (d, J ¼ 7.9 Hz, 1H), 7.42 (dd, J ¼ 8.2, 0.6 Hz, (d, J ¼ 7.0 Hz, 3H); 13C NMR (DMSO-d6) d 160.7, 137.5, 133.3,
1H), 7.23 (d, J ¼ 1.4 Hz, 1H), 7.19–7.13 (m, 1H), 7.05–6.99 (m, 126.6, 123.8, 123.1, 116.3, 115.1, 103.3, 52.8, 38.6, 37.1, 36.7,
1H), 3.83 (overlapping dq, J ¼ 7.0 Hz, 1H), 1.94 (s, 3H), 1.72–1.44 28.3, 14.4; HRMS (ESI) m/z calcd for C21H25BrN2O ([M + H]+) m/z
(m, 12H), 1.08 (d, J ¼ 7.1 Hz, 3H).
401.1223; found 401.1224.
N-(1-(Adamantan-1-yl)ethyl)-4,6-dichloro-1H-indole-2-
N-(1-(Adamantan-1-yl)ethyl)-4-methoxy-1H-indole-2-
carboxamide (8b). The title compound was prepared from 4- carboxamide (8g). This compound was prepared from 4,6-
methoxyindole-2-carboxylic acid (7b) and rimantadine hydro- dichloroindole-2-carboxylic acid (7g) and rimantadine hydro-
chloride employing method A. Yellow solid, yield: 95%. 1H NMR chloride employing method A. White solid, yield: 87%. 1H NMR
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(DMSO-d6) d 12.01 (d, J ¼ 1.7 Hz, 1H), 8.13 (d, J ¼ 9.5 Hz, 1H), 240.6, 12.1 Hz), 160.0, 156.7 (dd, J ¼ 250.4, 15.7 Hz), 139.1 (dd, J
7.45–7.39 (m, 2H), 7.19 (d, J ¼ 1.7 Hz, 1H), 3.82 (overlapping dq, ¼ 15.4, 12.6 Hz), 136.8 (d, J ¼ 7.1 Hz), 131.5 (d, J ¼ 3.2 Hz), 131.0
J ¼ 6.9 Hz, 1H), 1.91 (s, 3H), 1.66–1.45 (m, 12H), 1.06 (d, J ¼ (d, J ¼ 8.0 Hz), 125.3 (d, J ¼ 2.8 Hz), 119.8 (d, J ¼ 21.2 Hz), 115.9
7.0 Hz, 3H); 13C NMR (DMSO-d6) d 160.2, 137.2, 134.1, 128.0, (d, J ¼ 23.1 Hz), 113.5 (dd, J ¼ 22.0, 0.5 Hz), 103.8, 96.3 (dd, J ¼
126.7, 125.3, 119.8, 111.5, 101.2, 52.9, 38.6, 37.1, 36.7, 28.3, 14.4; 30.0, 23.1 Hz), 95.2 (dd, J ¼ 26.0, 4.6 Hz); HRMS (ESI) m/z calcd
HRMS (ESI) m/z calcd for C21H24Cl2N2O ([M + H]+) m/z 391.1338; for C16H9F3N2O2 ([M + H]+) m/z 319.0689; found 319.0686.
found 391.1339.
N-(3-Chlorobenzoyl)-4,6-diuoro-1H-indole-2-carboxamide
N-(1-(Adamantan-1-yl)ethyl)-4,6-diuoro-1H-indole-2-
(11b). This compound was synthesised from 7h and 3-chlor-
carboxamide (8h). 4,6-Diuoroindole-2-carboxylic acid (7h) and obenzoyl chloride following method B. White solid, yield: 30%.
rimantadine hydrochloride were used to afford the title 1H NMR (DMSO-d6) d 12.30 (s, 1H), 11.35 (s, 1H), 7.95 (s, 1H),
compound following method A. White solid, yield: 85%. 1H 7.85 (d, J ¼ 7.8 Hz, 1H), 7.72 (s, 1H), 7.70 (d, J ¼ 1.0 Hz, 1H), 7.57
NMR (DMSO-d6) d 11.93 (s, 1H), 7.96 (d, J ¼ 9.5 Hz, 1H), 7.38 (s, (t, J ¼ 7.9 Hz, 1H), 7.07 (dd, J ¼ 9.3, 1.2 Hz, 1H), 6.95 (td, J ¼ 10.4,
1H), 7.02 (dd, J ¼ 9.4, 1.7 Hz, 1H), 6.86 (td, J ¼ 10.4, 2.0 Hz, 1H), 1.8 Hz, 1H); 13C NMR (DMSO-d6) d 166.7, 160.6 (dd, J ¼ 240.5,
3.82 (overlapping dq, J ¼ 6.9 Hz, 1H), 1.94 (s, 3H), 1.76–1.45 (m, 12.2 Hz), 160.1, 156.7 (dd, J ¼ 250.4, 15.6 Hz), 139.1 (dd, J ¼
12H), 1.07 (d, J ¼ 7.0 Hz, 3H); 13C NMR (DMSO-d6) d 160.3, 159.6 15.4, 12.6 Hz), 136.6, 133.5, 132.7, 131.5 (d, J ¼ 2.5 Hz), 130.8,
(dd, J ¼ 238.1, 12.1 Hz), 156.2 (dd, J ¼ 248.5, 15.5 Hz), 138.0 (dd, 128.8, 127.8, 113.5 (d, J ¼ 21.9 Hz), 103.8, 96.3 (dd, J ¼ 29.9, 23.1
J ¼ 15.2, 13.3 Hz), 133.4 (d, J ¼ 3.2 Hz), 113.6 (d, J ¼ 21.6 Hz), Hz), 95.2 (dd, J ¼ 25.9, 4.6 Hz); HRMS (ESI) m/z calcd for C16
-
98.9, 95.4 (dd, J ¼ 29.6, 23.3 Hz), 95.0 (dd, J ¼ 25.9, 4.3 Hz), 52.8, H9ClF2N2O2 ([M + H]+) m/z 335.0393; found 335.0393.
38.6, 37.1, 36.7, 28.3, 14.4; HRMS (ESI) m/z calcd for
tert-Butyl
4-(4,6-diuoro-1H-indole-2-carbonyl)piperazine-1-
C
21H24F2N2O ([M + H]+) m/z 359.1929; found 359.1924.
N-Benzyl-4,6-diuoro-1H-indole-2-carboxamide (9a).
carboxylate (13). A mixture of 4,6-diuoroindole 7h (1.5
This mmol), N-Boc-piperazine 12 (1.8 mmol), EDC$HCl (1.8 mmol),
compound was obtained from 4,6-diuoroindole-2-carboxylic and DMAP (1.8 mmol) in a 1 : 1 mixture of tetrahydrofuran
acid (7h) and benzylamine employing method A. White solid, (THF) and DCM (10 mL each) was stirred at rt for 72 h. The
yield: 98%. 1H NMR (DMSO-d6) d 12.05 (s, 1H), 9.13 (t, J ¼ reaction mixture was then quenched with saturated aqueous
5.9 Hz, 1H), 7.38–7.28 (m, 5H), 7.28–7.20 (m, 1H), 7.04 (dd, J ¼ solution of ammonium chloride (50 mL) and extracted with
9.4, 1.3 Hz, 1H), 6.87 (td, J ¼ 10.4, 1.9 Hz, 1H), 4.52 (d, J ¼ 6.0 Hz, DCM (3 ꢁ 25 mL) and ethyl acetate (3 ꢁ 25 mL). The combined
2H); 13C NMR (DMSO-d6) d 160.8, 159.7 (dd, J ¼ 237.0, 12.0 Hz), organic layers were washed with brine (1 ꢁ 25 mL), dried over
156.2 (dd, J ¼ 248.7, 15.6 Hz), 139.8, 138.1 (dd, J ¼ 15.2, 13.2 anhydrous Na2SO4, ltered, and concentrated under reduced
Hz), 133.1 (d, J ¼ 3.3 Hz), 128.8, 127.7, 127.3, 113.6 (d, J ¼ 21.7 pressure. The obtained residue was used for the next reaction
Hz), 98.9, 95.7 (dd, J ¼ 29.6, 23.3 Hz), 95.1 (dd, J ¼ 25.9, 4.4 Hz), without further purication; buff solid, yield: 78%.
42.7; HRMS (ESI) m/z calcd for C16H12F2N2O ([M + H]+) m/z
(4,6-Diuoro-1H-indol-2-yl)(piperazin-1-yl)methanone (14). The
crude product 13 was N-Boc deprotected employing method C
287.0987; found 287.0990.
4,6-Diuoro-N0-phenyl-1H-indole-2-carbohydrazide
(9b). to afford the title compound which was >95% pure aer ash
Compound 7h and phenylhydrazine were reacted to deliver the chromatography. Buff solid, yield: 90%. 1H NMR (DMSO-d6)
title compound following method A. White solid, yield: 93%. 1H d 7.03 (dd, J ¼ 9.4, 1.4 Hz, 1H), 6.77 (d, J ¼ 0.6 Hz, 1H), 6.71 (td, J
NMR (DMSO-d6) d 12.11 (s, 1H), 10.45 (d, J ¼ 2.0 Hz, 1H), 7.99 (d, ¼ 10.3, 2.0 Hz, 1H), 3.64 (s, 4H), 2.73 (s, 4H); 13C NMR (DMSO-
J ¼ 1.7 Hz, 1H), 7.38 (s, 1H), 7.16 (dd, J ¼ 8.4, 7.4 Hz, 2H), 7.04 d6) d 161.5, 159.5 (dd, J ¼ 238.3, 12.1 Hz), 156.0 (dd, J ¼ 248.8,
(dd, J ¼ 9.4, 1.4 Hz, 1H), 6.92 (td, J ¼ 10.4, 2.0 Hz, 1H), 6.78 (d, J 15.5 Hz), 137.6 (dd, J ¼ 15.2, 13.2 Hz), 131.5 (d, J ¼ 3.3 Hz), 113.2
¼ 7.7 Hz, 2H), 6.73 (t, J ¼ 7.3 Hz, 1H); 13C NMR (DMSO-d6) (d, J ¼ 21.7 Hz), 99.9, 95.6 (dd, J ¼ 29.6, 23.3 Hz), 94.8 (dd, J ¼
d 161.1, 159.8 (dd, J ¼ 237.4, 12.1 Hz), 156.2 (dd, J ¼ 248.9, 15.6 25.9, 4.4 Hz), 46.1 (br, 4C).
Hz), 149.8, 138.3 (dd, J ¼ 15.3, 13.0 Hz), 131.4 (d, J ¼ 3.3 Hz),
(Adamantan-1-yl)(4-(4,6-diuoro-1H-indole-2-carbonyl)
129.3, 119.2, 113.6 (d, J ¼ 21.7 Hz), 112.7, 99.2, 95.9 (dd, J ¼ 29.6, piperazin-1-yl)methanone (15). This compound was prepared
23.3 Hz), 95.1 (dd, J ¼ 25.9, 4.3 Hz); HRMS (ESI) m/z calcd for following method
A entailing compound 14 and 1-ada-
C
15H11F2N3O ([M + H]+) m/z 288.0943; found 288.0939.
4,6-Diuoro-1H-indole-2-carboxamide (10). The
mantanecarboxylic acid. White solid, yield: 75%. 1H NMR
title (DMSO-d6) d 12.02 (s, 1H), 7.04 (dd, J ¼ 9.3, 1.3 Hz, 1H), 6.92 (d, J
compound was prepared following the procedure published in ¼ 1.3 Hz, 1H), 6.88 (dd, J ¼ 10.4, 1.6 Hz, 1H), 3.70 (s, 8H), 1.98 (s,
our previous report and the 1H NMR data matched the one 3H), 1.92 (s, 6H), 1.69 (q, J ¼ 12.1 Hz, 6H); 13C NMR (DMSO-d6)
therein.46 Buff solid, yield: 98%. 1H NMR (DMSO-d6) d 11.97 (s, d 175.1, 161.6, 159.7 (dd, J ¼ 238.5, 12.0 Hz), 156.1 (dd, J ¼ 248.8,
1H), 8.03 (s, 1H), 7.44 (s, 1H), 7.22 (s, 1H), 7.01 (dd, J ¼ 9.4, 15.4 Hz), 137.7 (dd, J ¼ 15.2, 13.2 Hz), 131.2 (d, J ¼ 3.3 Hz), 113.3
1.9 Hz, 1H), 6.86 (td, J ¼ 10.5, 2.0 Hz, 1H).
(d, J ¼ 21.6 Hz), 100.5, 95.7 (dd, J ¼ 29.6, 23.3 Hz), 94.9 (dd, J ¼
25.9, 4.3 Hz), 45.1 (br, 4C), 41.4, 38.8, 36.5, 28.4; HRMS (ESI) m/z
4,6-Diuoro-N-(3-uorobenzoyl)-1H-indole-2-carboxamide
(11a). The title compound was prepared from 7h and 3-uo- calcd for C24H27F2N3O2 ([M + H]+) m/z 428.2131; found
robenzoyl chloride employing method B. White solid, yield: 428.2130.
32%. 1H NMR (DMSO-d6) d 12.31 (s, 1H), 11.31 (s, 1H), 7.79–7.69
4-((tert-Butoxycarbonyl)amino)benzoic acid (17). To a solution
(m, 3H), 7.64–7.55 (m, 1H), 7.54–7.46 (m, 1H), 7.07 (dd, J ¼ 9.3, of 4-aminobenzoic acid (16, 3 mmol) in a 1 : 2 mixture of water
1.3 Hz, 1H), 6.95 (td, J ¼ 10.4, 2.0 Hz, 1H); 13C NMR (DMSO-d6) (10 mL) and dioxane (20 mL), di-tert-butyl dicarbonate (Boc2O, 6
d 166.6 (d, J ¼ 2.6 Hz), 162.2 (d, J ¼ 244.7 Hz), 160.6 (dd, J ¼ mmol) and triethylamine (Et3N, 6 mmol) were added and the
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reaction mixture was stirred at rt for 48 h. Three quarters of the
solvent were then evaporated in vacuo and the residue was
acidied with 3 M aqueous HCL solution. The formed precipi-
tate was ltered off, washed with water, and dried. The 1H NMR
data was in agreement with the reported one.58 White solid,
yield: 86%. 1H NMR (DMSO-d6) d 9.71 (s, 1H), 7.83 (d, J ¼ 8.5 Hz,
2H), 7.55 (d, J ¼ 8.4 Hz, 2H), 1.48 (s, 9H).
4.2.2.2. Screening. Aer extensive optimization at various
concentrations and time-points, the compounds were screened
in the GBM cell line (KNS42) to assess the impact on prolifer-
ation and viability (described below). The cells were treated with
0.5, 1, 2.5, 5, 7.5 and 10 mM of each compound and assessed
aer 72 hours of treatment, whereupon their IC50 values were
calculated. The top potent compounds with IC50 values <10 mM
were further screened in multiple paediatric brain cancer cell
lines, BT-12, BT-16 and DAOY in addition to the non-neoplastic
cell line HFF1 to assess their impact on proliferation and
viability (described below). The cells were treated with 0.5, 1,
2.5, 5, 7.5 and 10 mM of each compound and assessed aer 72 h
of treatment.
4.2.2.3. Cell proliferation assay. All cell lines were seeded in
96-well plates, at 2000 cells per well, except KNS42 which was
seeded at 4000 cell per well. Aer incubating overnight in
normal growth media, the media was aspirated off and 100 ml of
treatment media was added. Media containing 0.1% DMSO was
used as a control. Aer 72 h of incubation, the MTT prolifera-
tion assay reagents were added as previously described.61 The
absorbance of the assay (570 nm/600 nm) was measured using
the CLARIOstar (BMG Lab Tech, USA) and exported into excel,
where the data was normalized by removing the background
absorbance. The data was analysed and IC50 values were
calculated using PRISM 8 (GraphPad, USA).
4.2.2.4. Cell viability assay. All cell lines were seeded at 2000
cells per well, except KNS42 which was seeded at 4000 cell per
well, in 96-well plates. Aer an overnight incubation in normal
growth media, the media was aspirated off and 100 ml of treat-
ment media was added. 0.1% DMSO was used in the control
media. Aer 72 h of incubation, the PrestoBlue Viability
(ThermoFisher Scientic, USA) was added, per the manufac-
turer's instructions. The uorescence of the assay (560 nm/590
nm) was measured using the CLARIOstar (BMG Lab Tech, USA)
and exported into Excel, where the data was normalized by
removing the background uorescence. The data was analysed
and IC50 values were calculated using PRISM 8 (GraphPad,
USA).
4.2.2.5. Transcriptional analysis of KNS42 cells. The KNS42
cell line was maintained in Roswell Park Memorial Institute
(RPMI) medium supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin/streptomycin and incubated at 37 ꢂC in
5% CO2. The cells were treated with 10 mM of compound 15. The
cells were washed with 1ꢁ PBS, scraped with a cell scraper and
centrifuged to collect cell pellets aer 72 hours of treatment.
RNA was extracted from the cell pellets using TRIzol reagent
(Thermo Fisher, USA) and following the protocol from Cold
Spring Harbor Laboratory Press.62 The RNA pellet was resus-
pended in 50 ml of sterile diethylpyrocarbonate (DEPC) treated
water. Concentration of the RNA was measured using the
standard Qubit RNA Broad-Range Assay kit (Thermo Fisher,
USA) according to the manufacturer's instructions. The RNA
samples were submitted to BGI Americas for DNBSEQ Eukary-
otic Stranded Transcriptome Resequencing.
tert-Butyl
(4-((adamantan-1-yl)carbamoyl)phenyl)carbamate
(18). Compound 17 and 1-adamantylamine were reacted
according to method A and the residue, obtained aer evapo-
rating the EtOAc extract, was used in the next step without
further purication; white solid, yield: 84%.
N-(1-Adamantyl)-4-aminobenzamide (19). The crude product
18 was N-Boc deprotected following method C to deliver the title
compound. Brown solid, yield: 96%. 1H NMR (DMSO-d6) d 7.51
(dd, J ¼ 8.5, 1.8 Hz, 2H), 7.02 (s, 1H), 6.50 (dd, J ¼ 8.5, 1.9 Hz,
2H), 5.50 (s, 2H), 2.03 (s, 9H), 1.64 (s, 6H); 13C NMR (DMSO-d6)
d 166.4, 151.7, 129.2, 123.0, 112.8, 51.5, 41.6, 36.6, 29.4.
N-(4-((Adamantan-1-yl)carbamoyl)phenyl)-4,6-diuoro-1H-
indole-2-carboxamide (20). The indole 7h was coupled with 19
following method A to afford the title compound. White solid,
1
yield: 25%. H NMR (DMSO-d6) d 12.21 (s, 1H), 10.42 (s, 1H),
7.89–7.80 (m, 4H), 7.58 (d, J ¼ 1.5 Hz, 1H), 7.49 (s, 1H), 7.08 (dd,
J ¼ 9.3, 1.4 Hz, 1H), 6.94 (td, J ¼ 10.4, 2.0 Hz, 1H), 2.08 (s, 6H),
2.06 (s, 3H), 1.67 (s, 6H); 13C NMR (DMSO-d6) d 165.9, 160.1 (dd,
J ¼ 239.4, 12.1 Hz), 159.5, 156.4 (dd, J ¼ 249.2, 15.6 Hz), 141.4,
138.5 (dd, J ¼ 15.3, 12.9 Hz), 132.7 (d, J ¼ 3.3 Hz), 131.4, 128.6,
119.5, 113.6 (d, J ¼ 21.8 Hz), 100.5, 96.0 (dd, J ¼ 29.7, 23.2 Hz),
95.2 (dd, J ¼ 25.9, 4.4 Hz), 51.9, 41.4, 36.6, 29.4; HRMS (ESI) m/z
calcd for C26H25F2N3O2 ([M + H]+) m/z 450.1988; found
450.1987.
4.2. Biology
4.2.1. Anti-TB activity. MIC was determined using Micro-
plate alamarBlue assay (MABA) as previously reported.59,60
MABA format was also used in the cytotoxicity evaluation on
Vero Cells.6
4.2.2. Antitumour activity
4.2.2.1. Cell culture. The four well-established paediatric
brain tumour cell lines were all derived from humans and were
used to assess the effects on proliferation and viability when
treated with the indole-2-carboxamides. KNS42 (glioblastoma
multiforme – GBM), BT-12 and BT-16 (atypical teratoid rhab-
doid tumour – AT/RT) cell lines were gis from Dr Hashizume,
Northwestern University, whereas DAOY cells (medulloblas-
toma – MB) were obtained from ATCC. The human broblasts
HFF1 (obtained from ATCC) were used as non-neoplastic
controls. The KNS42, BT-12 and BT-16 cell lines were main-
tained in Roswell Park Memorial Institute (RPMI) medium
supplemented with 10% fetal bovine serum (FBS). The DAOY
cell line was maintained in Eagle's minimal essential medium
(EMEM) supplemented with 10% FBS. The HFF1 were main-
tained in Dulbecco's modied Eagle's medium (DMEM) sup-
plemented with 15% FBS. All media contained 1% penicillin/
ꢂ
streptomycin. The cells were incubated at 37 C in 5% CO2.
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11 A. E. Grzegorzewicz, H. Pham, V. A. Gundi, M. S. Scherman,
E. J. North, T. Hess, V. Jones, V. Gruppo, S. E. Born,
J. Kordulakova, S. S. Chavadi, C. Morisseau, A. J. Lenaerts,
R. E. Lee, M. R. McNeil and M. Jackson, Nat. Chem. Biol.,
2012, 8, 334–341.
12 P. J. Brennan, Tuberculosis, 2003, 83, 91–97.
13 C. Hoffmann, A. Leis, M. Niederweis, J. M. Plitzko and
H. Engelhardt, Proc. Natl. Acad. Sci. U. S. A., 2008, 105,
3963–3967.
4.3. Molecular modelling
In silico molecular docking analysis was undertaken using the
Molecular Operating Environment MOE soware version
2008.10 (Chemical Computing Group, Montreal, Canada). The
crystal structure of MmpL3 in complex with ICA38 (6AJJ) was
retrieved from the protein data bank (PDB) and the docking
protocol was similar to our previous report.46
Conflicts of interest
14 J. Liu, C. E. Barry III, G. S. Besra and H. Nikaido, J. Biol.
Chem., 1996, 271, 29545–29551.
The authors declare no conict of interest.
15 S. S. R. Alsayed, C. C. Beh, N. R. Foster, A. D. Payne, Y. Yu and
H. Gunosewoyo, Curr. Mol. Pharmacol., 2019, 12, 27–49.
16 G. Degiacomi, A. Benjak, J. Madacki, F. Boldrin, R. Provvedi,
G. Palu, J. Kordulakova, S. T. Cole and R. Manganelli, Sci.
Rep., 2017, 7, 43495.
17 W. Li, A. Obregon-Henao, J. B. Wallach, E. J. North, R. E. Lee,
M. Gonzalez-Juarrero, D. Schnappinger and M. Jackson,
Antimicrob. Agents Chemother., 2016, 60, 5198–5207.
18 W. Li, C. M. Stevens, A. N. Pandya, Z. Darzynkiewicz,
P. Bhattarai, W. Tong, M. Gonzalez-Juarrero, E. J. North,
H. I. Zgurskaya and M. Jackson, ACS Infect. Dis., 2019, 5,
1001–1012.
Acknowledgements
SSRA acknowledges Curtin University for the support through
Curtin International Postgraduate Research Scholarship
(CIPRS). WRB is grateful for the support of NIH grants AI 37856
and HL 133190. HG is thankful for the ARC Discovery Early
Career Researcher Award DE160100482.
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