SAR and identification of 2-(quinolin-4-yloxy)acetamides as Mycobacterium tuberculosis cytochrome bc 1 inhibitors

Article abstract of DOI:10.1039/c6md00236f

A previous phenotypic screen by GSK identified 2-(quinolin-4-yloxy)acetamides as potent growth inhibitors of Mycobacterium tuberculosis (Mtb). We report the results of a preliminary structure-activity relationship (SAR) study of the compound class which has yielded more potent inhibitors. An Mtb cytochrome bd oxidase deletion mutant (cydKO) was found to be hypersensitive to most members of the compound library, while strains carrying single-nucleotide polymorphisms of the qcrB gene, which encodes a subunit of the menaquinol cytochrome c oxidoreductase (bc₁) complex, were resistant to the library. These results identify that the 2-(quinolin-4-yloxy)acetamide class of Mtb growth inhibitors can be added to the growing number of scaffolds that target the M. tuberculosis bc₁ complex.

Full text of DOI:10.1039/c6md00236f

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SAR and identification of 2-(quinolin-4-yloxy)acetamides as Mycobacterium
tuberculosis cytochrome bc1 inhibitors†
Narisa Phummarin,a Helena I. Boshoff,b Patricia S. Tsang,b James Dalton,c Siouxsie Wiles,c,d
Clifton E. Barry 3rd b and Brent R. Copp*a
a School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
b Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and
Infectious Disease, National Institutes of Health, Bethesda, Maryland, USA
c
Bioluminescent Superbugs Lab, Department of Molecular Medicine and Pathology, University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand
d Te Pūnaha Matatini, c/o University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
∗ Corresponding author. E-mail: b.copp@auckland.ac.nz; Tel: +64 9 3737599; Fax: +64 9
3737422
†The authors declare no competing interests.
Abstract.
A previous phenotypic screen by GSK identified 2-(quinolin-4-yloxy)acetamides as potent
growth inhibitors of Mycobacterium tuberculosis (Mtb). We report the results of a
preliminary structure-activity relationship (SAR) study of the compound class which has
yielded more potent inhibitors. An Mtb cytochrome bd oxidase deletion mutant (cydKO) was
found to be hypersensitive to most members of the compound library, while strains carrying
single-nucleotide polymorphisms of the qcrB gene, which encodes a subunit of the
menaquinol cytochrome c oxidoreductase (bc1) complex, were resistant to the library. These
results identify that the 2-(quinolin-4-yloxy)acetamide class of Mtb growth inhibitors can be
added to the growing number of scaffolds that target the M. tuberculosis bc1 complex.
Electronic supplementary information (ESI). Materials and methods, and characterisation
of compounds 10a-f, 11a-x and 5, 9, 12a-12aa.
1. Introduction
The current paradigm for the discovery of antibiotics that can be developed into much-needed
new treatments for tuberculosis typically starts with a whole-cell phenotypic screening
campaign.1,2 The majority of these screens use aerobic culture conditions and so by necessity,
lead to the discovery of growth inhibitors of replicating Mycobacterium tuberculosis (Mtb).
Recent efforts have also been directed towards undertaking screening using culture
conditions considered more relevant to the in vivo disease state,3,4 or towards Mtb inside host
macrophages.5 Defining the mechanism of action of screening hits obtained in such an
agnostic manner can be challenging, however with significant cost reductions, sequencing of
resistant mutants has become a productive pathway. Two recent examples of screening
campaign hits are the imidazo[1,2-a]pyridine amides (IPAs),2,3,6-8 exemplified by the clinical
candidate Q203 (1) identified during an Mtb/macrophage assay,9 and lansoprazole (2,
Prevacid), identified in a pharmaceutical re-purposing screen designed to identify compounds
that protect lung fibroblasts from Mtb-induced cytotoxicity (Fig. 1).10 Genome sequencing of
spontaneous resistant mutants generated for both 1 and 2 revealed a common target of QcrB
(Rv2196), which encodes the b subunit of the cytochrome bc1 complex, an essential
component of the electron transport chain. In the case of Q203, a mutation of T313A or
1

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DOI: 10.1039/C6MD00236F
T313I in QcrB was observed to cause resistance, while for 2, a L176P mutation was
responsible.
H
O
N
N
OCF3
Cl
N
N
1 (Q203)
CF3
H
N
O
O
S
N
N
2
H
N
N
N
N
N
O
N
O
N
Cl
3
4
Fig. 1 Structures of cytochrome bc1 oxidase inhibitors 1–4.
In addition to the chemtypes encompassed by 1 and 2, further chemically diverse scaffolds
have been identified to bind to QcrB,11 including imidazo[4,5-c]pyridine 3 and pyrazole 4,12
suggesting the target is somewhat promiscuous (Fig. 1).
GSK recently made publically available the results of a phenotypic screening campaign,
whereby a large compound library was assessed for the ability to inhibit the growth of
Mycobacterium bovis BCG and M. tuberculosis H37Rv. The curated results led to the
identification of 177 hits covering a number of different structural classes.13 Amongst this set
were five 2-(quinolin-4-yloxy)acetamides (QOAs) (5-9, Fig. 2) exhibiting favourable Mtb
growth inhibition properties with MIC90 Mtb H37Rv 0.3 – 3.3 ꢀM.
R2
H
H
N
R3
N
O
O
R1
O
R1
O
R3
N
N
5 (GSK 358607A) R1 = R2 = OMe, R3 = H
8 (GSK 124576A) R1 = Br
9 (GSK 463114A) R1 = OMe
(GSK 749336A) R1 = OMe, R2 = H, R3 = Me
(TCMDC-142552) R1 = Br, R2 = R3 = H
6
7
Fig. 2 Structures of GSK hit compounds 5–9.
The potency of activity and structural simplicity of this series makes them an attractive target,
prompting efforts to explore the structure-activity relationship of the class. During the
preparation of this manuscript, two groups have reported the results of their studies of the
QOA class, confirming compound potency, selectivity for M. tuberculosis and lack of
toxicity in a Danio rerio (zebrafish) model.14 Herein we report our efforts in optimizing the in
vitro anti-tuberculosis activity of the more potent GSK QOA analogue 5 and also identify, via
use of selective mutant strains, their cellular target as involving the cytochrome bc1 complex.
2. Results and discussion
2

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Our preliminary structure-activity relationship study of GSK 358607A (5) focused on
changes to the (i) 2-methoxyphenylacetamide moiety and (ii) the quinoline 6-position. The
target 2-(quinolin-4-yloxy)acetamide library (12a-12aa), in addition to GSK compounds 5
and 9, were prepared in three steps from commercially available materials (Scheme 1). The
synthesis began by preparing the required 4-hydroxyquinolines by heating the appropriate
aniline with ethyl acetoacetate in polyphosphoric acid to afford, after purification, 10a-f in
yields of 25-77%. Bromoacetamides 11a-x were prepared in 27-100% yields from the
acylation, at -77 oC, of various substituted anilines or alkylamines with bromoacetyl bromide.
In the final step, 2-(quinolin-4-yloxy)acetamides 5, 9 and 12a-12aa were prepared, in yields
of 16-89%, by reaction of 4-hydroxyquinolines with bromoacetamides in either acetone or
DMF solvent in the presence of K2CO3 base.
OH
R1
R1
(a)
NH2
N
10a-f
1
1
1
a
b
c
R = OMe, R = H, R = Me,
1
1
1
d
e
f
R = Cl, R = Br, R = CF3
H
N
Br
R2
(b)
O
11a-x
H
N
O
R2
R1
O
N
5, 6, 12a-aa
12a-v R1 = OMe
a R2 = Ph, b R2 = 3-OMe-Ph,
c R2 = 4-OMe-Ph, d R2 = 2,4-diOMe-Ph,
2
2
e
g
i
f
R = 3,4-diOMe-Ph, R = 3,5-diOMe-Ph,
2
2
h
R = 3,4-dioxomethylene-Ph, R = 3-Br-Ph,
2
2
j
R = 4-Br-Ph, R = 2-Cl-Ph,
k R2 = 4-Cl-Ph, l R2 = 4-Me-Ph,
m R2 = 4-Ph-Ph, n R2 = 3,4-dioxomethylene-Ph-CH2,
o R2 = 2-phenethyl, p R2 = 2-(2,5-diOMe-Ph)-ethyl,
q R2 = 2-(3,4-diOMe-Ph)-ethyl,
2
r
R = 2-(3,4-dioxomethylene-Ph)-ethyl,
2
2
s
u
t
R = 3-phenylpropyl, R = 2-methoxyethyl,
2
2
v
R = prop-2-yn, R = n-pentyl
2
12w-aa
R = 2-OMe-Ph
w R1 = H, x R1 = Me, y R1 = Cl,
1
1
z
aa
R = CF3
R = Br,
3

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Scheme 1 Synthesis of target compounds 5, 9, 12a–12aa. Reagents and conditions: (a)
ethylacetoacetate, PPA, 130 oC, 2h, yield: 25–77%; (b) bromide 11a-x, K2CO3, DMF or
acetone, 5–21 h, yield: 16–89%.
The library of analogues were tested for whole-cell growth inhibition of Mycobacterium
tuberculosis (Mtb) H37Rv under a variety of growth conditions and different readouts of
growth and minimum inhibitory concentration (MIC) values were determined at either 7 or
14 days (Table 1). Using laboratory-adapted strain Mtb H37Rv grown in Middlebrook 7H9
media with microplate Alamar Blue assay (MABA) readout after 14 days (Table 1, data
column 1) revealed that the GSK hits 5 and 9 exhibited MICs of 1.1 and 2.3 ꢀM respectively
(entries 1 and 2). Of the phenylacetamide sidechain analogues prepared, examples containing
either no substituent (12a, entry 3), or 2-chloro (12j, entry 12) or 4-methyl (12l, entry 14)
substitution were slightly more potent than GSK compounds 5 and 9. The remaining
phenylacetamide sidechain analogues (12b-12i, 12k, 12m, entries 4-11, 13, 15) were either
equipotent or less active than the original hits. In most cases introduction of conformational
flexibility in the form of 2-phenethyl (12o, 12p, 12q, 12r) or 3-phenylpropyl (12s) acetamide
sidechains yielded compounds that were poor growth inhibitors. The exception to this trend
was 12n, a 3,4-dioxomethylenebenzyl derivative, which exhibited an MIC of 0.53 ꢀM, being
slightly more potent than the unsubstituted GSK benzyl analogue 9. Acetamide sidechains
that incorporated no aryl moiety (i.e. 12t 2-methoxyethyl, 12u prop-2-yn and 12v n-pentyl,
entries 22-24) were also significantly less active than the original hit compounds. Overall,
four compounds 12a, 12j, 12l, 12n were identified as being more potent growth inhibitors
than the original GSK hit compound. In general, bulky, lipophilic substituents of limited
conformational flexibility at R2 improved the antimycobacterial activity of the compounds,
while R1 being methoxyl was critical for activity. Similar trends in overall structure-activity
relationship were observed by Pissante et al., where they arrived at conclusions regarding the
requirement for a bulky lipophilic group at R2, finding a 2-naphthyl derivative to be potently
antimycobacterial.14a
When Mtb H37Rv was grown in the same media (7H9/ADC/Tween) but using optical density
as the growth readout, there was apparent bacterial outgrowth as evidenced by time-
dependent outgrowth of the cells in the presence of the compound resulting in a dramatic
shift in apparent MIC (Table 1, data columns 2 and 3). Similar outgrowth was also observed
for M. tuberculosis BSG001 (M. tuberculosis H37Rv transformed with the bacterial
luciferase-encoding vector pMV306hsp + LuxAB + G13 + CDE)15 grown in Middlebrook
7H9 media (data not shown). This discordance between growth MIC and the Alamar Blue
MIC has been previously reported to be a characteristic of compounds that inhibit the
respiratory bc1 complex of Mycobacterium tuberculosis.12 The ability of cells to overcome
the growth inhibitory effect of these compounds could at least in part be driven by
compensatory upregulation of the alternate oxygen-dependent cytochrome bd oxidase
pathway.16 Evidence for this upregulation-based protection model is based upon the findings
of a number of groups, whereby cytochrome bd oxidase knock-out strains (ꢁcydKO) of
mycobacteria are hyper-susceptible to electron transport chain inhibitors17 including those
compounds that target bc1.8,11,12 As summarized in Table 1 (data column 4), most of the test
set of compounds did indeed exhibit enhanced MIC potency towards the Mtb ꢁcydKO strain
versus laboratory-adapted Mtb H37Rv. Based upon the magnitude of MIC enhancement
evident in Table 1, it was concluded that the majority of the analogues, and GSK compounds
5 and 9, are inhibitors of cytochrome bc1 oxidase. Further direct evidence for the bc1 oxidase
inhibiting properties of 5 and 9 and analogues 12a, 12c, 12g, 12l and 12n was obtained when
the compounds were found to exhibit reduced potency against a series of QcrB subunit
mutants of the cydKO strain of Mtb (Table 2). The seven defined Mtb cydKO qcrB amino
acid mutants, A317V, M342T, W312G, A396T, M342I, A317T and S182P, were originally
4

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generated in response to imidazo[4,5-c]pyridine 3 with homology modeling of QcrB
identifying all mutations to be located proximal to the binding site of stigmatellin.12 In the
present study, the magnitudes of the fold-resistance observed for the test compounds against
these seven cydKO mutants confirms cytochrome bc1 oxidase as a cellular target of 2-
(quinolin-4-yloxy)acetamides. Compounds that inhibit cytochrome bc1 oxidase have
previously been shown to deplete intracellular ATP levels in anaerobic cells.9 We found that
our 2-(quinolin-4-yloxy)acetamides were able to reduce ATP levels within 24 hours of
exposure under anaerobic conditions in a similar manner to TMC2073,9 (Bedaquiline, a F0F1
ATP synthase inhibitor) and 3, a known cytochrome bc1 oxidase inhibitor12 (Fig. 3) (and ESI
figures 1 – 3). ATP levels were not reduced by the cell-wall synthesis inhibitor linezolid.
Fig. 3 ATP depletion (%) under anaerobic conditions for test compounds 12c, 12d, 12g, and
12j.
Previous groups have determined the selectivity of antitubercular cytochrome bc1 oxidase
inhibitors towards Mycobacterium tuberculosis, with little or no detectable antibacterial
activity being observed towards panels of Gram-positive or Gram-negative bacteria.8,10 We
can confirm the recent report by Pissante et al.,14a where they noted the absence of
antibacterial activity of the 2-(quinolin-4-yloxy)acetamide compound class towards
Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter
baumannii. In the present study, no activity was observed towards Escherichia coli 25922,
Staphylococcus aureus XEN36, Mycobacterium smegmatis and M. abscessus (data not
shown).
The cytotoxicity of the 2-(quinolin-4-yloxy)acetamide library was determined using HepG2
cells, with the assay media containing either glucose or galactose. The use of galactose forces
the cells to rely on mitochondrial oxidative phosphorylation rather than glycolysis for
growth.18 While five of the test compounds (12b, 12g, 12k, 12o, 12w; Table 3, entries 4, 9,
13, 17, 25) were considered to be mildly cytotoxic independent of glucose/galactose-based
media, two analogues (5, 12l) exhibited mild to moderate levels of cytotoxicity (CC50 < 50
ꢀM) only towards cells grown in galactose-containing media. This latter result identifies
these two compounds, which includes GSK hit 5, as being potential inhibitors of
mitochondrial respiration.
3. Conclusions
5

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In conclusion, our preliminary structure-activity relationship investigation of the anti-
tuberculosis activity of 2-(quinolin-4-yloxy)acetamides has identified four analogues (12a,
12j, 12l, 12n) as being more potent growth inhibitors than the original GSK 358607A (5) hit
compound, exhibiting sub-micromolar MIC values. We have established that this compound
class targets QcrB, an essential component of the electron transport chain. Knowledge of the
cellular target of 2-(quinolin-4-yloxy)acetamides will now facilitate structure-based drug
design as we seek to optimize the antituberculosis potency of this series.
Author contributions
N.P. and B.R.C synthesized the compound library. H.I.B, C.E.B., J.D. and S.W. performed
antibacterial testing. P.S.T. performed cytotoxicity testing. B.R.C. wrote the manuscript with
contributions from all co-authors.
Acknowledgements
We acknowledge funding from the University of Auckland (including in part from the Vice
Chancellors Strategic Development Fund [23563]) and, in part, by the Intramural Research
Program of NIAID, NIH. We thank Dr M. Schmitz for assistance with NMR data acquisition,
Mr Tony Chen for MS data and V. de Guzman for preliminary studies.
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Table 1 In vitro activity of 5, 9, 12a-12aa against Mycobacterium tuberculosis H37Rv in various assays and media
and an Mtb cytochrome bd oxidase mutant cydKO
MIC (ꢀM)
H37Rv
H37Rv
H37Rv
cydKO
2 weekd
entry
compound
MABA
1 week b
2 week c
2 week a
1
5
9
1.11
2.32
2.21
3.57
53.92
>148
>155
>142
>142
32.69
>131
>131
>136
>125
>125
53.25
>140
>149
>125
24.71
>143
>122
>122
>127
68.59
>164
>176
158.03
>155
>74
0.14
0.59
0.12
0.57
0.14
0.18
3.14
2.04
0.19
0.37
0.17
<0.067
0.20
<0.071
0.12
<0.063
4.45
7.62
90.13
3.04
2.14
41.06
21.98
0.95
7.13
0.45
1.09
0.50
>128
0.6
2
3
12a
12b
12c
12d
12e
12f
0.62
1.21
4
6.53
141.88
2.21
5
1.11
6
2.04
24.58
>131
>131
4.26
7
24.58
24.58
2.13
8
9
12g
12h
12i
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
>125
1.94
92.20
>125
26.35
103.70
0.45
12j
0.56
12k
12l
3.36
0.45
12m
12n
12o
12p
12q
12r
12s
1.51
>125
0.53
0.53
54.22
60.90
122.00
31.69
25.79
121.55
>176
19.75
>78
142.69
121.80
>122
>=127
68.59
>164
>176
158.03
38.77
56.48
26.35
23.42
>128
0.3
12t
12u
12v
12w
12x
12y
12z
12aa
PASe
Linezolide
3e
9.30
103.70
>125
>128
0.3
>140
>125
>128
0.6
2.3
2.3
2.3
1.56
0.31
1.56
12.5
>25
MIC values are the average of two independent assays. Assay protocols are described in Ref. 8
a MIC against H37Rv grown in 7H9/ADC/Tween media. MIC determination using microplate Alamar Blue assay (MABA) after 2 weeks
post compound addition.
b MIC against H37Rv grown in 7H9/ADC/Tween media. MIC determination after 1 week post compound addition.
c MIC against H37Rv grown in 7H9/ADC/Tween media. MIC determination after 2 weeks post compound addition.
d MIC against cyd knock-out strain of H37Rv grown in 7H9/ADC/Tween media. MIC determination after 2 weeks post-compound
addition.
e Linezolid, PAS (p-aminosalicylic acid) and 3 were used as positive controls.
8

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DOI: 10.1039/C6MD00236F
Table 2 qcrB Mutants are resistant to compounds that target the bc1 complex
Fold resistance to qcrB mutant
W312G A396T M342I
Entry
compound
A317V
46.9
>250
>1250
63.9
134.3
>32.5
>130
>81
M342T
1.4
3.0
3.8
4.1
2.1
>2
>12.5
20.2
0.3
A317T
24.5
23.5
30.0
24.5
32.9
>12.5
>32.5
10.1
0.2
S182P
15.9
15.7
19.5
15.9
32.9
>16.3
>95.8
61.3
0.5
1
2
3
4
5
6
7
8
5
9
46.9
47.0
235.0
24.5
17.1
>16.3
>25
4.1
3.9
5.0
3.1
5.6
6.1
3.9
1.8
3.1
1.4
12a
12c
12g
12l
12n
3a
>2
1.0
>6.3
20.2
0.3
>12.5
20.2
0.5
>81
0.5
PASa
0.5
a Compound 3 (positive control), PAS (p-aminosalicylic acid, negative control).
9

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Table 3 In vitro cytotoxicity of 5, 9, 12a-aa against HepG2 cells during growth on galactose and glucose
Galactose
CC50 (ꢀM)
Glucose
CC50 (ꢀM)
entry
compound
1
5
9
19.6
>149
118.1
24.7
>142
>149
>155
43.0
2
3
12a
12b
12c
4
5
>142
>131
>131
>131
39.6
>142
>131
>131
>131
80.0
6
12d
12e
7
8
12f
9
12g
12h
12i
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
>125
>125
>140
71.1
>125
>125
>140
89.9
12j
12k
12l
41.5
>149
>126
>131
61.4
12m
12n
12o
12p
12q
12r
>126
86.9
83.9
>122
>122
>127
50.7
>122
>122
>127
>137
>164
>176
>158
43.9
12s
12t
>164
>176
106.3
8.64
12u
12v
12w
12x
12y
12z
>74
>74
99.2
140.1
>125
>128
>25
>125
>128
>25
12aa
3a
Antimycin Aa
0.017
>50
IC50 values are the average of two independent assays. Assay protocols are described in Ref. 18
a Compound 3 and antimycin A were used as positive controls.
10

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Graphical image and text
New antimycobacterial 2-(quinoline-4-yloxy)acetamides were prepared, and using gene deletion and
resistant mutants, we conclude that the compound class inhibits the mycobacterial cytochrome bc1
complex.