Inhibition of Mycobacterium tuberculosis InhA: Design, synthesis and evaluation of new di-triclosan derivatives

Article abstract of DOI:10.1016/j.bmc.2020.115744

Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. ‘Direct inhibitors’ of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC₅₀ of 5.6 μM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC₉₉ of 12.9 μM against M. bovis.

Full text of DOI:10.1016/j.bmc.2020.115744

Bioorganic&MedicinalChemistry28(2020)115744
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of Mycobacterium tuberculosis InhA: Design, synthesis and
evaluation of new di-triclosan derivatives
⁎
Tom Armstronga, Malcolm Lamonta, Alice Lanneb, Luke J. Alderwickb, Neil R. Thomasa,
a Biodiscovery Institute, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom
b Institute of Microbiology and Infection, School of Bioscience, University of Birmingham, Birmingham B15 2TT, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Keywords:
Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In
addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB.
Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and
identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the
enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. ‘Direct inhibitors’ of InhA are
attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-
triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been
synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-
chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) ex-
hibiting an IC50 of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclo-
propylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with
an MIC99 of 12.9 µM against M. bovis.
InhA
Triclosan
Triazole
Isoniazid
Mycobacterium tuberculosis
TB
1. Introduction
the last 40 years (delamanid, pretomanid and bedaquiline).5,6
Mycobacteria’s dense and complex cell wall represents one of the
Tuberculosis (TB) is one the leading global causes of mortality and is
currently the world’s deadliest infectious disease. In addition to the
estimated 1.3 million deaths from TB in 2018, a further 0.25 million
Human Immunodeficiency Virus-positive (HIV) individuals died as a
result of co-infection with Mycobacterium tuberculosis.1
main reasons for the hardiness of the bacteria. The relative imperme-
ability of the Mycobacterial cell wall can primarily be attributed to its
mycolic acid component. This waxy layer consists of long chain
(~C50-90) α-branched, β-hydroxylated fatty acids. This barrier not only
hinders the passage of small molecules into the cell, but also provides
protection from the host’s immune response.7
The current anti-TB treatment course relies on four drugs: isoniazid
(INH), rifampicin (RIF), ethambutol (ETH) and pyrazinamide (PZA).
The increasing prevalence of multi-drug resistant TB (MDR-TB) threa-
tens to undermine the efficacy of this treatment regimen. MDR-TB in-
fections are characterised as those displaying resistance to INH and RIF.
In 2018, the WHO reported over 484,000 new MDR-TB infections.
Additionally, 13,000 were classified as extensively drug resistant (XDR-
TB) meaning they were also insensitive to any fluoroquinolone and at
least one of the second-line injectable drugs.2–4 This emerging re-
sistance is further exacerbated by the lack of novel drugs coming
through the pipeline, with only three new drugs entering the clinic in
The frontline drug INH elicits its biological effects through disrup-
tion of the mycolic acid biosynthetic pathway, via inhibition of the
enzyme InhA.8 InhA is a nicotinamide adenine dinucleotide (NADH)-
dependent enoyl acyl carrier reductase which catalyses the chemose-
lective reduction of its 2-trans-enoyl-ACP substrate. The enzyme has an
identical amino acid sequence (and hence structure) in M. tuberculosis
and M. bovis, the main causative agents of tuberculosis in humans and
dairy cattle respectively. INH is actually a pro-drug which requires
activation before it can inhibit mycolic acid biosynthesis (Fig. 1).
Initially, the catalase peroxidase enzyme, KatG converts INH to its
Abbreviations: ETH, ethambutol; GOLD, Genetic Optimisation for Ligand Docking; HIV, human immunodeficiency virus; HPLC, high-performance liquid chro-
matography; INH, isoniazid; MDR-TB, multi-drug resistant tuberculosis; NAD, nicotinamide adenine dinucleotide (oxidised form); NADH, nicotinamide adenine
dinucleotide (reduced form); PZA, pyrazinamide; RIF, rifampicin; RuAAC, ruthenium-catalysed azide alkyne cycloaddition, TB, tuberculosis; TCS, triclosan; XDR-TB,
extensively drug-resistant tuberculosis
⁎ Corresponding author.
E-mail address: neil.thomas@nottingham.ac.uk (N.R. Thomas).
https://doi.org/10.1016/j.bmc.2020.115744
Received 10 July 2020; Received in revised form 24 August 2020; Accepted 27 August 2020
Availableonline08September2020
0968-0896/©2020PublishedbyElsevierLtd.

T. Armstrong, et al.
Bioorganic&MedicinalChemistry28(2020)115744
Fig. 1. (A) The structure of key anti-TB drug INH and the pathway through which it elicits its biological effects. (B) Crystal structure of the INH-NAD adduct bound to
InhA showing how the adduct blocks the substrate binding cavity (PDB: 1ZID).
homologous proteins from other bacteria.19 Inspection of the crystal
structure reveals that the TCS2 molecule lies only ~4.2 Å away from
the TCS1 moiety (Fig. 3). This suggests that through modification of the
B-ring, it may be possible to obtain TCS derivatives which are capable
of occupying both binding sites through a single molecule, potentially
producing a more potent and selective inhibitor for InhA, that also
benefits from a lower entropic cost to binding.
In this article, we report the in silico design, synthesis and char-
Fig. 2. The structure of TCS and the denotation of the A and B rings.
acterization of a series of novel TCS derivatives bearing a 1,5-triazole
group attached to the B-ring. These compounds were designed to oc-
cupy both of the TCS binding sites observed in the 1P45 crystal struc-
ture. Biological evaluation was performed using an isolated InhA en-
zyme assay and whole-cell screening against M. bovis.
corresponding isonicotinyl radical species. Upon activation, INH forms
a covalent adduct with the InhA co-factor which in turn blocks the
active site, preventing substrate binding.9,10 A single KatG point mu-
tation, S315T, is sufficient to render INH inactive and has been im-
plicated in up to 95% of INH resistant clinical isolates examined.11,12
With this in mind, the development of ‘direct InhA inhibitors’ re-
presents a compelling approach for the discovery of new anti-TB
drugs.13–16 Direct inhibitors refer to compounds which, unlike INH, do
not require prior activation to exert their inhibitory effects. These in-
hibitors would circumvent the current resistance mechanisms and could
be used to treat resistant infections. One such direct inhibitor is the
small molecule, broad spectrum antibiotic triclosan (TCS, Fig. 2). TCS is
a moderate, reversible inhibitor of InhA and has formed the basis of a
number of studies, using its scaffold in the development of more potent
inhibitors.17,18
The first step in designing TCS analogues which could anchor into
both binding sites was identifying a suitable linker to connect the two
fragments. It was vital that the linker did not disrupt the experimentally
observed binding mode for TCS1, while at the same time the linker
geometry had to direct the attached fragment back into the TCS2
binding region. To this end, it was thought that a disubstituted 1,2,3-
triazole ring would be an appropriate motif. It was hoped that the rigid
nature of the triazole scaffold would direct any substituent group back
into the TCS2 binding site. This notion was explored using the Genetic
Optimisation for Ligand Docking (GOLD) platform.20 Two test com-
pounds, a 1,4 and a 1,5-triazole, were designed and both were docked
into the InhA active site (PDB: 1P45). The binding poses obtained are
shown below in Fig. 4.
As demonstrated in Fig. 4, when the two fragments are connected
through a 1,5-triazole unit (2), the TCS-like binding mode is retained,
and the secondary fragment occupies the same space as the TCS2
Crystallographic studies have highlighted an unusual binding trait
for the TCS:InhA complex. Two molecules of TCS are able to occupy the
InhA active site (PDB: 1P45, Fig. 3), something not replicated in
2

T. Armstrong, et al.
Bioorganic&MedicinalChemistry28(2020)115744
Fig. 3. X-ray structure showing two TCS molecules bound to the InhA active site. Residues are shown with orange carbons, NAD+ is shown with teal carbons, TCS are
shown with yellow carbons. H-bonds are shown by dashed-black lines. (PDB: 1P45).
moiety. The 1,4-triazole (1) did not generate a suitable structure and
the B-ring modification resulted in a perturbation of the TCS binding
mode, including loss of the key hydrogen bonding network shown in
Fig. 3. This is of particular importance as this H-bonding network is
conserved amongst all potent direct InhA inhibitors, with the exception
of the methyl-thiazole compound class.19,21–23
Fig. 4. The structures of two basic TCS-triazole compounds and their docking poses generated by GOLD, this shows the overlap between the designed compounds and
the two TCS moieties found in the active site. (PDB: 1P45).
3

T. Armstrong, et al.
Bioorganic&MedicinalChemistry28(2020)115744
Fig. 5. Design strategy for novel TCS-based InhA inhibitors.
With this information in hand, a range of compounds bearing a
single aromatic ring attached through the 1,5-triazole motif were also
designed (Fig. 5). Previous studies have demonstrated that the B-ring Cl
atoms are not required for potent InhA inhibition and so they were
removed in an attempt to control the lipophilicity of the target com-
pounds.18,24
before etherification using NaH and propargyl bromide, giving ether 21
in good yield over two steps. The phenol functionality was then un-
masked using 6 M HCl to furnish the alkyne-bearing TCS fragment 22 in
a near quantitative yield.
The selective formation of 1,5-triazoles can be achieved through the
use of a ruthenium catalysed azide-alkyne cycloaddition (RuAAC), this
methodology has seen an increasing numbers of examples in the lit-
erature over the last decade.26,27 With 22 and 16 in hand, the 1,5-
triazole motif was assembled via a RuACC, using Cp*RuCl(PPh3)2 in
1,4-dioxane (Scheme 3).
The synthesis of biaryl azide 16 is shown in Scheme 1. The initial
biaryl scaffold was assembled through an SNAr reaction using 1-fluoro-
4-nitrobenzene and 4-chloro-2-methoxyphenol, furnishing 13 in near
quantitative yields. Sequential BBr3 mediated ether demethylation and
nitro reduction with Zn/NH4Cl gave amine 15 in a good yield over two
steps. Finally, functional group interconversion under the mild condi-
tions first reported by Barral et al. provided the target azide 16 in a
good yield.25
The RuAAC reaction furnished 2 in an acceptable yield, following
reverse phase high performance liquid chromatography (HPLC) pur-
ification. The identity of the product was confirmed as a 1,5-triazole
using the NMR methods previously discussed by Creary et al.28 Fol-
lowing the synthesis of 2, the aforementioned truncated analogues were
synthesised from the azide 16 and alkyne fragments (23–32, synthesis
detailed in supplementary information), using the same RuAAC con-
ditions, giving the desired 1,5-triazoles in moderate yields after HPLC
(Scheme 4).
The synthesis of alkyne-bearing TCS fragment 22 is shown in
Scheme 2. The biphenyl scaffold 17 was assembled in a similar fashion
to the corresponding nitro compound 13, in a good yield. Cleavage of
the methyl ether was performed using AcOH and HBr at elevated
temperatures, generating 18 in a modest yield. This method was used
due to the instability of the benzaldehyde group that was observed
when demethylation was attempted with BBr3. The newly exposed
phenol was then reprotected using MOMCl in a good yield. Benzalde-
hyde 19 was reduced to its corresponding benzyl alcohol using NaBH4
An isolated enzyme assay was used to assess the inhibitory prop-
erties of the compounds synthesised. Initial screening was performed at
50 µM with 150 nM of InhA from M. tuberculosis using a standard UV
absorbance assay that has been widely used in the identification of
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 130 °C, 18 h, 95%; (b) BBr3, CH2Cl2, 0 °C - > r.t., N2, 3 h, 89%; (c) Zn, NH4Cl, MeOH, r.t., 18 h, 75%; (d)
tBuONO, TMSN3, ACN, 0 °C - > r.t., 3 h, 80%.
4

T. Armstrong, et al.
Bioorganic&MedicinalChemistry28(2020)115744
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, 130 °C, 18 h, 92%; (b) AcOH, HBr, 140 °C, 18 h, 38%; (c) MOMCl, DIPEA, CH2Cl2, r.t., 18 h, 86%; (d) NaBH4,
MeOH, 0 °C - > r.t., 4 h, quant.; (e) NaH, propargyl bromide, DMF, 0 °C - > r.t., 18 h, 79%; (f) 6 M HCl (aq), MeOH, 70 °C, 2 h, 90%.
novel InhA inhibitors. Assays were performed using 2-trans-octenoyl
CoA as a mimic for the enzyme’s natural substrate.29 All compounds
evaluated were of above 90% purity as determined by analytical HPLC.
The inhibitory activity observed for compounds 2–12 is summarised
in Table 1, these results indicate a number of the compounds tested
showed moderate inhibition of InhA at 50 µM. Moving from H- > Me-
> iPr (3- > 5) results in increased inhibition, however, the introduction
of a tBu (6) leads to a significant reduction in enzyme inhibition, sug-
gesting that such a bulky group results in major clashes with the pro-
tein. This loosely correlates with the fitness scores, with compound 6
showing a lower fitness score than 5. The possibility that this region is
size sensitive is further supported by the fact that compounds 8, 10 and
12, all of which bear relatively small R groups, show moderate in-
hibition. The most potent compound was 2 which showed a total re-
duction in enzyme activity at 50 µM.
5.6
0.8 µM (n = 3). This represents a modest improvement on TCS
(IC50 = 9.2
1.3 µM, n = 3) recorded in experiments conducted in
parallel and widely reported in the literature.30
Whole-cell evaluation of all 12 compounds was undertaken to in-
vestigate the correlation between isolated enzyme activity and whole-
cell potency. Compounds were initially assessed for growth inhibition
against Mycobacterium bovis at a fixed concentration of 40 µM. Results
are shown in Table 2.
Disappointingly, the isolated assay potency of 2 towards InhA did
not correlate to whole-cell potency. This could possibly be attributed to
inability to pass through the dense mycobacterial cell wall, or other
factors such as efficient transport by efflux pumps.31,32 It is possible that
reintroducing the ortho Cl atom to the B-ring (giving a compound with a
cLogP 6.61) could result in greater whole-cell potency, although the
previous QSAR study by Sivaraman et al.24 indicate that this chlorine is
not critical, this is something that will be explored going forward. Of
the 12 compounds tested, 11 showed the greatest potency, with 99%
growth inhibition at 40 µM. This compound was subject to further
evaluation to determine its MIC99 (Fig. 7).
The predicted binding mode for 2 is shown in Fig. 6. This binding
pose shows how the ‘front’ TCS fragment is able to engage the NAD+
co-factor and Tyr158 through the key hydrogen bonding network. The
1,5-triazole architecture directs the second TCS fragment back towards
the substrate binding loop, where a number of hydrophobic contacts are
Compound 11 displayed an MIC99 of 12.9
5.0 µM (6.2 µg mL−1
,
made. Further evaluation of
2
showed it exhibits an IC50 of
n. = 3). Clearly there is a disconnect between the activity of 11 in
Scheme 3. Reagents and conditions: (a) Cp*RuCl(PPh3)2, 1,4-dioxane, 60 °C, N2, 18 h, 17%.
5

T. Armstrong, et al.
Bioorganic&MedicinalChemistry28(2020)115744
Scheme 4. Reagents and conditions: (a)
Cp*RuCl(PPh3)2, 1,4-dioxane, 60 °C, N2,
18 h, 5–33%.
Table 1
Table 2
Inhibitory data for compounds tested using an isolated enzyme assay, con-
ducted at 50 µM. GOLD fitness scores are also included for each compound.
Inhibition values are an average of duplicate assays.
Inhibition data for all compounds tested against Mycobacterium bovis at 40 µM,
results are shown as an average of 3 assays.
Compound
Inhibition/%
cLogP
Compound
GOLD Fitness Score
Inhibition/%
2
3
20
3
5.80
5.11
5.43
5.89
6.11
4.37
5.03
5.61
6.09
6.07
5.95
4.9838
2
3
87.7
65.2
73.5
77.8
74.0
69.0
76.7
73.2
75.6
80.7
83.6
70.8
100
12
38
72
6
4
−6
15
20
0
5
4
6
5
7
6
8
40
4
7
13
44
0
9
8
10
11
12
TCS
19
99
23
70
9
10
11
12
TCS
47
11
42
92
permeator and to cause disruption to both lipid and protein biosynth-
esis.18 Further work will be required to elucidate the main biological
target(s) of 11.
isolated enzyme assays and its performance in the whole-cell screening
tests. This suggests that 11 has other off-target sites within the bacteria
which result in significant potency. At high concentrations TCS has
been reported to act as a mitochondrial uncoupling, cell membrane
As previously mentioned, TCS has formed the basis for a number of
SAR studies towards potent inhibitors of InhA. The most common
Fig. 6. Predicted binding mode for 2. Residues are shown with orange carbons, NAD+ is shown with teal carbons, 2 is shown with yellow carbons. H-bonds are
shown by dashed-black lines. (PDB: 1P45).
6

T. Armstrong, et al.
Bioorganic&MedicinalChemistry28(2020)115744
giving a direct inhibitor of Mycobacterial InhA with higher affinity and
selectivity than TCS. Enzyme assays on purified M. tuberculosis InhA
were used to evaluate the compounds synthesised, with the most potent
showing an IC50 of 5.6
0.8 µM which is similar to that of TCS.
Whole-cell evaluation against M. bovis showed the most potent com-
pound was 11 which displayed an MIC of 12.9
5.0 µM, despite only
showing 11% inhibition at 50 µM in isolated enzyme assays.
Author Contributions
T.A. synthesised all compounds, performed enzyme assays, analysed
data and wrote the manuscript. M.L. expressed & purified InhA and
performed enzyme assays. A.L. performed whole-cell screening assays
and analysed data. L.J.A. supervised the whole-cell screening assays.
N.R.T. came up with the initial drug design concept, supervised the
synthetic research, contributed to writing and editing the manuscript.
Fig. 7. MIC99 curve for 11.
modification is replacement of the A-ring Cl atom with various hydro-
phobic groups. Comprehensive B-ring modifications remain relatively
unexplored.33–35 Two examples of previously disclosed direct InhA in-
hibitors are shown in Fig 8.35,36 Compound 2 is similar in concept to
33, recently reported by Rodriguez et al., which sought to merge two
TCS molecules in a macrocyclic arrangement. Compounds 33 and 2
have similar IC50 values, which would be expected as they both target
the same binding sites.
Funding
T.A. is supported by the Wellcome Trust Antimicrobials and
Antimicrobial Resistance (AAMR) doctoral training programme
Birmingham/Nottingham [203974/Z/17/A].
M.L. is supported by a Biotechnology and Biological Sciences
Research Council doctoral training studentship [Grant Number BB/
M008770/1].
In terms of MIC99 data, in this study, 11 showed significant potency
in whole-cell assays with an MIC99 value of 12.9 µM, this value is ap-
proaching the MIC99 of 34 (6.6 µM). It is possible that, following the
elucidation of 11′s target, its potency could be further improved to the
match or exceed the activity of 34.
A.L. is supported by the Wellcome Trust Antimicrobials and
Antimicrobial Resistance (AAMR) doctoral training programme
Birmingham/Nottingham [215154/Z/18/Z].
The rational in silico design, synthesis and characterization of a
novel series of triazole-bearing TCS derivatives is reported. These
compounds were designed to exploit the large volume of the InhA ac-
tive site and occupy both TCS binding sites observed in the PDB: 1P45
crystal structure. Docking results suggested that these compounds
would be able to occupy both of the TCS binding regions, possibly
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Fig. 8. The structures of previously disclosed TCS-based inhibitors of InhA and their biological activities.
7

T. Armstrong, et al.
Bioorganic&MedicinalChemistry28(2020)115744
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