Synthesis and evaluation of heterocycle structures as potential inhibitors of Mycobacterium tuberculosis UGM

Article abstract of DOI:10.1016/j.bmc.2020.115579

In this study, we screen three heterocyclic structures as potential inhibitors of UDP-galactopyranose mutase (UGM), an enzyme involved in the biosynthesis of the cell wall of Mycobacterium tuberculosis. In order to understand the binding mode, docking simulations are performed on the best inhibitors. Their activity on Mycobacterium tuberculosis is also evaluated. This study made it possible to highlight an “oxazepino-indole” structure as a new inhibitor of UGM and of M. tuberculosis growth in vitro.

Full text of DOI:10.1016/j.bmc.2020.115579

Journal Pre-proofs
Synthesis and evaluation of heterocycle structures as potential inhibitors of
Mycobacterium tuberculosis UGM
Carine Maaliki, Jian Fu, Sydney Villaume, Albertus Viljoen, Clément
Raynaud, Sokaina Hammoud, Jérôme Thibonnet, Laurent Kremer, Stéphane
P. Vincent, Emilie Thiery
PII:
DOI:
Reference:
S0968-0896(20)30409-0
https://doi.org/10.1016/j.bmc.2020.115579
BMC 115579
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
27 March 2020
26 May 2020
29 May 2020
Please cite this article as: Maaliki, C., Fu, J., Villaume, S., Viljoen, A., Raynaud, C., Hammoud, S., Thibonnet, J.,
Kremer, L., Vincent, S.P., Thiery, E., Synthesis and evaluation of heterocycle structures as potential inhibitors of
Mycobacterium tuberculosis UGM, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.bmc.2020.115579
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Synthesis and evaluation of heterocycle structures as potential inhibitors of
Mycobacterium tuberculosis UGM
a
b
b
c
c
a
Carine Maaliki , Jian Fu , Sydney Villaume , Albertus Viljoen , Clément Raynaud , Sokaina Hammoud ,
a
c,d
b
a,
Jérôme Thibonnet , Laurent Kremer , Stéphane P. Vincent , Emilie Thiery 
a
Laboratoire Synthèse et Isolement de Molécules Bioactives (SIMBA, EA 7502), Université de Tours, Faculté de Pharmacie, Parc de Grandmont, 31 Avenue
Monge, 37200 Tours, France
b
Department of Chemistry, University of Namur, Rue de Bruxelles 61, 5000 Namur, Belgium
Institut de Recherche en Infectiologie de Montpellier (IRIM), CNRS UMR 9004, Université de Montpellier, 34293 Montpellier, France
INSERM, IRIM, 34293 Montpellier France
c
d
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
In this study, we screen three heterocyclic structures as potential inhibitors of UDP-galactopyranose
mutase (UGM), an enzyme involved in the biosynthesis of the cell wall of Mycobacterium tuberculosis.
In order to understand the binding mode, docking simulations are performed on the best inhibitors. Their
activity on Mycobacterium tuberculosis is also evaluated. This study made it possible to highlight an
“oxazepino-indole” structure as a new inhibitor of UGM and of M. tuberculosis growth in vitro.
Available online
Keywords:
Heterocycles
2
009 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis
UDP-galactopyranose mutase
Inhibitor
Docking

Corresponding author. e-mail: emilie.thiery@univ-tours.fr

1
. Introduction
within the infected host, where it can persist in a non-replicating
state for several decades in granulomas. The survival strategies
developed by Mt are essentially linked to the presence of an
unusual cell wall, which consists of two major layers (Figure 1).
The highly impermeable outer layer is composed of mycolic acids
consisting of 70-90 carbon-containing fatty acids. The inner layer
consists of peptidoglycan. These two layers are covalently tethered
Tuberculosis (TB) is the world's deadliest infectious disease,
responsible for 1.8 million deaths every year. According to the
WHO report "Antibacterial agents in clinical development"
published in 2017, inadequate new treatment options exist for
1
antibiotic-resistant TB. The emergence of drug-resistant strains of
Mycobacterium tuberculosis (Mt) decreases the efficacy of
treatment, which requires a combination of at least three
antibiotics as first-line therapy. In the case of multi- and
extensively-drug-resistant (MDR-TB and XDR-TB) strains,
complex, prolonged, costly and highly toxic multidrug second-line
therapy is required and only 30-50 % of patients are treated
successfully. In more than 70 years, only two antibiotics for the
treatment of drug-resistant TB reached the market, and seven are
3
via the connecting polysaccharide arabinogalactan (AG). AG
itself comprises three regions: i) a disaccharide ‘linker’ attached to
the peptidoglycan, ii) the galactofuran [(→6)-β-D-Galf-(1→5)- β-
D-(Galf)]n which is attached to the linker unit, and iii) a complex
arabinan linked to the galactofuran and representing the site of
attachment of mycolic acids. These are oriented perpendicular to
the plane of the membrane, providing a barrier responsible for the
natural resistance of Mt to many antibiotic classes, and contribute
to the physiopathological aspects characterizing TB. In addition,
within this lipid environment are intercalated several glycolipids
with exotic structures, such as the phthiocerol dimycocerosate,
phenolic glycolipids, trehalose dimycolate (TDM) or sulfolipids.
The role of these lipids in signaling events, pathogenesis, immune
1
currently being evaluated in clinical trials. The development of
new strategies and new molecular scaffolds is, therefore, necessary
to counter the increasing threat of antimicrobial resistance and to
propose new therapeutic options for TB treatment.2
Mt has a complex lifestyle involving several developmental
4
response and even in coughing has been established.
stages. Its success results from its remarkable capacity to survive
Cell wall
Examples of Mt cell wall inhibitors
Acyl lipids &
proteins
O
N
H2N NH
isoniazid
Mycolic acids
S
N
H2N
ethionamide
NO2
O
S
R
O2N
R
N
H
N
F3C
Arabinogalactan
O
NO2
benzothiazinones dinitrobenzamides
OH
H
N
N
H
Ethambutol OH
Peptidoglycan
Cyctoplasmic
membrane
Figure 1. Schematic structure of the M. tuberculosis cell envelope. Structures and sites of action of several anti-TB drugs targeting
the cell wall are shown. Chemical entities inhibiting AG biosynthesis are in pink.
dinitrobenzamide derivatives inhibit biosynthesis of arabinan
7
Therefore, the integrity of both the mycolic acid-
arabinogalactan-peptidoglycan skeleton (mAGP) and the outer
mycomembrane leaflet of extractable lipids hinges on the integrity
of the arabinan moiety of AG. In addition to its crucial structural
role, arabinan exhibits also specific immunomodulatory activities
although these functions have mostly been connected to the
arabinan part of lipoarabinomannan that shares structural features
(Figure 1).
Several enzymes are involved in the biosynthesis of the
galactan moiety of the cell wall but marketed antitubercular agents
targeting this polysaccharide are currently lacking. One such
enzyme is the UDP-galactopyranose (UDP-Galp) mutase (UGM),
which catalyzes the interconversion of UDP-galactopyranose
UDP-Galp) into UDP-galactofuranose (UDP-Galf) (Scheme 1),
subsequently used by the Galf transferases GlfT1 and GlfT2 to
polymerize the galactofuran subunit of arabinogalactan
Interestingly, UGM, which is absent in humans, is essential for the
growth of mycobacteria, therefore representing a privileged and
(
5
with mAGP.
8
During the past two decades, intensive efforts conducted to the
discovery of new leads for TB drug development using either
target-based or cell-based approaches and the molecular
6
9
mechanisms of action of several anti-TB drugs were deciphered.
validated therapeutic target.
Several major anti-TB agents disrupt the biosynthesis of cell wall
components. For instance, isoniazid and ethionamide are key
inhibitors of mycolic acid biosynthesis, while ethambutol and the
recently identified chemical classes the benzothiazinones and

biosynthesis17 while tetrenolin18 and freelyngine19 display
antibiotic activities.
OH
UDP-Galactose OH
OH
O
acceptor
HO
HO
O
mutase
Galf transferase
O
O
Galactan
acceptor
HO
HO
O
UDP
HO
HO
O
OH
UDP
OH
To generate a first representative set of butenolides 2a-f, we
developed a new stereoselective synthetic strategy of (E)--
substituted -methyl (Z)-γ-alkylidene butenolides (Scheme 2). As
HO
Galp-UDP
Galf-UDP
Galf-UDP
Scheme 1. Isomerization of UDP-Galp by UGM
2
0
and elaboration of galactan.
previously reported for -iodopropenoic acid derivatives, the
first step is based on the cross-coupling-heterocyclization reaction
sequence between terminal alkynes and (E)-2,3-dibromobutenoic
acid in order to obtain -bromo -methyl (Z)-γ-isobutylidene
furan-2-one 1 (Scheme 2). The presence of the bromide in the 
position allows the modification of the furanone moiety via Suzuki
coupling, providing access to a wide panel of -substituted furan-
2-one 2 (Scheme 2, Table 1).
Until recently, the search for UGM inhibitors has mainly
focused on the preparation of substrate analogues. However,
screening studies have also shown that heterocyclic molecules can
1
0
1
1
exhibit strong interactions with the catalytic site of the enzyme.
Recently, various heterocyclic compounds, including flavonoids,
1
2
1
3
14
acylhydrazones and thiazol-2-amines were shown to inhibit Mt
UGM.
Br
RB(OH)2, Pd(PPh3)4, Na2CO3
Toluene/H2O/EtOH, 80 °C, 6 h
R
Br
CuI, K2CO3
Herein, we present the screening of novel heterocyclic
compounds for Mt UGM inhibition. We explored the relative
levels of UGM inhibition by the three scaffolds represented in
Figure 2. Indeed, butenolides and indole derivatives are important
pharmacophores that have not been explored for UGM inhibition
yet. To evaluate the binding mode of the best inhibitors, molecular
docking experiments are described. The in vitro anti-bacterial
activities of the best UGM inhibitors are also reported.
O
O
+
CO2H
O
or
DMF, 60 °C, overnight
O
+
Br
RBF3K , PdCl2dppf, Cs2CO3
Toluene/H2O, 80 °C, 2 h
1
, 74 %
2a-f, 61-84 %
Scheme 2. Synthesis of -substituted -methyl γ-alkylidene
butenolides.
The inhibition data for compounds 1 and 2a-f are
reported in Table 1. All butenolides were tested at 1 mM using the
FP-based assay. However, none of them displayed a satisfactory
inhibition level, encouraging us to explore two other targeted
scaffolds.
R3
I
R
O
R4
R2
R2
O
N
R1
N
O
O
R1
oxazepino indoles
Table 1. The Mt UGM inhibition data for the butenolide series
butenolides
indoles
Figure 2. Three heterocyclic structures studied for Mt UGM
Entry
Compound
Mt UGM Inhibition[a] [%]
inhibition.
Br
O
O
1
7.3
2
. Results and discussion
1
F
2
.1. Initial screening
2
3
9.0
O
O
Two distinct biochemical assays have been developed to
2
a
evaluate the binding affinity of small molecules towards purified
Mt UGM. An HPLC-based assay allows the monitoring of the
conversion of the substrate, UDP-Galf, into UDP-Galp using Mt
UGM in its active reduced form. Percentages of inhibition are
usually described with this assay. The concentration of UDP-Galf
19.2
O
O
1
0a
(
25 µM) was chosen to be close to its K
m
(23 µM for MtUGM).
2
b
A higher-throughput fluorescence polarization (FP) assay has
OMe
also been developed and exploited on the non-reduced form of the
1
5
enzyme. The latter is based on the competition between the
screened ligand(s) and a fluorescent probe and can be performed
in multi-well plates.
4
7.4
O
O
Our methodology consisted first to screen chemical libraries by
FP at inhibitor concentrations of 100 µM and 1 mM (only the
values at 1mM are displayed in Tables 1-3). When the percentage
of inhibition was greater than 30% at 1mM, the affinity of the
2
c
N
O
5
6
3.0
5.2
inhibitors (K
d
’s) was determined using the FP assay.16
O
2
d
Being much more demanding, the HPLC assay was only used
for the very best hits.
O
Butenolides and their derivatives represent a large family of
natural products. Since the 1970’s, many furan heterocycles have
been isolated with a wide range of biological activities. As
examples, xerulin and derivatives are inhibitors of cholesterol
O
O
2e

F
CO2Et
5
6
N
I
0
-
-
O
7
b
7
0
O
O
CO2H
2
f
7.4
N
a
8a
FP inhibition assay conditions: [inhibitor]= 1 mM, non-
I
reduced enzyme, [Mt UGM]= 580 nM, [fluorescent probe]= 18
MeO
nM.
CO2H
7
8
15.0
46.2
-
N
8
c
We next examined the indole series of molecules. These
heterocycles are present in many bioactive molecules, including
I
O
O
2
1
antituberculous agents. According to procedures previously
described in the literature,22 ethyl 3-iodo-1H-indole-2-
carboxylates 5 were prepared from commercially available
compounds 4 in the presence of N-iodosuccinimide (Scheme 3).
The popargylation of compounds 4 and 5 led to compounds 6 and
2
CO H
220±2
N
8
d
a
FP inhibition assay conditions: [inhibitor]= 1 mM, non-
reduced enzyme, [Mt UGM]= 580 nM, [fluorescent probe]= 18
nM FP assay conditions: [inhibitors]= 0-1 mM, non-reduced
7
, respectively. Saponification of carboxylated indoles 7 yielded
b
the corresponding acids 8. Molecules 4a-d, 6, 7 and 8a-d were
selected for preliminary UGM inhibition assays because if a hit is
discovered, they offer the possibility to be further derivatized for
a structure activity relationship (SAR) study.
enzyme, [Mt UGM]= 580 nM, [fluorescent probe]= 18 nM.
Finally, we prepared a series of tricyclic indoles based on
2
2
synthetic protocols described in the literature (Scheme 4). The
iodocyclisation of indoles 8 in the presence of silver nitrate,
diiodine and sodium carbonate in tetrahydrofuran led to the
oxazinoindole compounds 9. The functionalization of vinyl iodine
by Sonogashira coupling made it possible to generate products 10.
NaH, Br
DMF, 0°C - rt, 4 h
I
R'
R
R
R
NIS
NaH, Br
CO2Et
CO2Et
CO2Et
N
DMF, rt, 3 h
N
DMF, 0°C - rt, 4 h
N
H
H
4
5
6, R' = I
, R' = H
7
a, R = H
b, R = F
c, R = OMe
d, R = [1,3]dioxolo
1
. KOH(12 %)
Hex-1-yne
Pd(OAc)2, PPh3
CuI, NEt3
EtOH, 45°C, 3 h
I
AgNO3
I
I
2. HCl (1M)
R
I , Na CO
3
O
O
2
2
CO2H
I
N
THF, rt, overnight
N
O
DMF, rt, overnight
N
O
R
CO2H
N
8
R'
9
10
R'
R'
I
8
a, R, R' = H
b, R = F, R' = H
d, R = [1,3]dioxolo, R' = H
e, R = H, R' = Ph
c, R = OMe, R' = H
f, R = H, R' = Me
Scheme 3. Synthesis of indole derivatives.
Scheme 4. Synthesis of tricyclic indole derivatives.
The inhibition data for compounds 9 and 10 are reported in
The UGM inhibitory activity of a selection of eight indoles was
Table 3. Compound 9a displayed a poor inhibitory activity for Mt
UGM (FP assay) for the enzyme (Entry 1). The functionalization
of the indole cycle by a fluorine lead to a decrease in activity
(Entry 2). The same effect is observed when a methyl or a phenyl
is present on the vinylic pattern (Entries 3 and 4). In contrast,
compounds 10 showed good inhibitory activities and affinities for
evaluated (Table 2). The tested compounds were very poor
inhibitors (Entries 1-7), except product 8d (Entry 8) which reduced
the activity of Mt UGM to 46 % (entry 8). However, 8d showed
d
low affinity for Mt UGM (K = 220 µM). As compared to the other
molecules in this series, the presence of both the dioxolane ring
and the free carboxylic acid on the indole scaffold appears
important for UGM inhibition (Entries 3-4 and 6-8).
Mt UGM (Entries 5-7). K values for molecules 10a, 10c and 10d
d
were found in the same range (58-66 µM). To make sure that these
molecules are not false positive, we evaluated them by the HPLC
assay. More significant inhibitory differences could be measured:
molecule 10c displayed a 95% inhibition level as compared to 73%
for 10a and 60% for 10d. Such differences between these assays
are not surprising as the FP assay is conducted with the non-
reduced enzyme against a fluorescent probe whereas the HPLC
uses the reduced UGM against the natural substrate UDP-Galf.
Table 2. The Mt UGM inhibition data for the indole series
[
b]
Inhibition
Mt UGM [%]
K
d
[μM]
Entry
1
Compound
[a]
Mt UGM
CO2Me
5.1
-
N
Table 3. The Mt UGM inhibition data for the “oxazino-indole”
series
Bn
3
CO2Et
2
3
N
2.0
-
-
[b]
Inhibition Mt
UGM [%]
K
d
[μM] Mt
H
4a
Entry
1
Compound
UGM
I
I
CO2Et
11.5
O
N
H
5a
N
O
61.6[a]
2000
I
O
O
I
9a
4
CO2Et
23.1
-
N
H
5d

I
F
modelling was carried out with a tricyclic “oxazepino indole”
bearing a carboxylic acid (molecule 11a, Figure 3). The binding
mode remains the same as for 10a and 10c with characteristic
contacts in the uridine pocket with residues Tyr366, Leu141,
Thr162, Tyr161 and Tyr191. However, a clear interaction with
Arg292 and the carboxylate could be observed. Such an attractive
interaction could induce a better affinity for Mt UGM. We thus
concentrated efforts on the synthesis of compound 11a.
O
N
O
19.1[a]
2
3
4
-
-
-
I
9b
9e
I
O
N
O
22.8[a]
Ph
I
I
Tyr 366
Arg180
O
N
O
14.9[a]
Leu 141
NH
NH2+
His 294
I
9f
H2N
NH
OH
3.4
FAD
N
3.8
Thr 162
2.3
HO
I
O
O
N
3.5
N
O
I
Tyr 161
N
O
O
N
HO
3.2
5
6
7
72.9±3.3[c]
66±1.5
61.3±1.4
58.3±1.2
N
3.7
O
HN
O
Val 158
Phe 157
O
+H2N
OH
10a
HN
NH2
NH
Leu 66
N
O
H2N
HN
I
O
N
H
MeO
O
His 89
Ph
Asn 284
N
O
Arg 292
95.5±0.5[c]
60.2±3.6[c]
Tyr 191
Phe 102
Figure 4. Interaction map of 10c with Mt UGM.
1
0c
I
O
O
O
2.3. Synthesis and evaluation of new “oxazepino-indole”
compounds
N
O
The promising results obtained with molecules 10 prompted us
to explore further this design by incorporating a polar carboxylic
acid to improve the water solubility and find evidence of
hydrophobic/hydrophilic effects in the association of 10 with
UGM. Compounds 11a and 11c were respectively prepared under
Sonogashira conditions from iodoalkenes 9a and 9c (Scheme 5).
The reaction was performed at room temperature or at 50 °C under
microwave irradiation. Compounds 11 were partially degraded on
silica gel, which explains the low yields.
1
0d
a
inhibition assay conditions: [inhibitor]= 1 mM, non-reduced
enzyme, [Mt UGM]= 580 nM, [fluorescent probe]= 18 nM. FP
assay conditions: [inhibitors]= 0-1 mM, non-reduced enzyme, [Mt
UGM]= 580 nM, [fluorescent probe]= 18 nM. HPLC inhibition
assay conditions: [inhibitor]= 0.5 mM, [Mt UGM]= 25 nM, [UDP-
Galf]= 25 µM.
b
c
2
.2. Docking of “oxazepino indole” compounds with Mt UGM
I
I
R
R
O
Pd(OAc)2, PPh3
CuI, NEt3, DMF
O
To evaluate their binding modes, the best inhibitory candidates
N
+
CO2H
N
O
O
rt, overnight
or
MW 50 °C, 2 h
(10a and 10c, Figure 3) were subjected to docking simulations. All
modelling calculations were performed by using Mt UGM crystal
structures in its closed conformation (PDB code: 4RPG).10a The
UDP-galactose binding pocket of UGM consists of a galactose
sub-pocket close to the FAD cofactor, a pyrophosphate sub-pocket
where two arginine residues (Arg 292 and 180) can be found and
a more hydrophobic uridine binding pocket.
I
9a, R= H
c, R= OMe
11a, R= H, 12 %
11c, R= OMe, 14 %
9
HO C
2
Scheme 5. Synthesis of compounds 11a and 11c.
The inhibition and FP assays (Table 4) indicated that both 11a
and 11c display a good affinity for Mt UGM and a strong
inhibitory activity. These levels of affinity are comparable to the
best heterocyclic UGM inhibitors reported to date that have been
I
I
I
MeO
O
O
O
N
O
N
N
O
O
1
0e,11,12,13,15,23
found in the low micromolar range (Figure 5).
CO2H
OH
O
F
F
S
O
HO2C
HN
N
N
1
0a
10c
11a
N
OH
N
H
Cl
Cl
H
HO
HO
OUDP
Cl
Figure 3. Molecules subjected to docking simulations
F
F
I
Kd (M) Mt UGM= 3±1
Kd (M) Mt UGM= 53±2
Kd (M) Mt UGM= 8
For molecules 10a and 10c, only one binding mode could be
observed: the tricyclic indole core strongly interacts with the
residues of the uridine sub-pocket while the alkynyl chain lies
within the pyrophosphate pocket without making noticeable
contacts (Figure 4 and Supplementary information). The methoxy
group in 10c does not significantly change the position of the
molecule in the cavity as compared to 10a and makes a contact
with asparagine 284. In order to optimize interactions, the
Figure 5. Example of the best inhibitors of Mt UGM10e,13,15
Table 4. The Mt UGM inhibition data for the compounds 11a and
1c
1

I
coupling constants (J in Hz). 13C NMR was recorded at 75 MHz
on the same instrument, using the CDCl solvent peak at (δ
7.16 ppm) as reference. F NMR was recorded at 282 MHz on
R
O
3
C
=
N
O
1
9
7
the same instrument. HRMS was obtained with a LCMS-IT-TOF
mass spectrometer under conditions of ESI. IR spectra were
recorded on a Perkin-Elmer Spectrum One spectrophotometer.
Melting points were uncorrected.
HO2C
Inhibition[a]
Mt UGM [%]
[b]
K
d
[μM]
Entry
1
Compound
Mt UGM
11a, R= H
11c, R= OMe
84.0
83.5
56.8±1.2
33.8±1.2
4
.1.2. Preparation of butenolides compounds.
2
[
a
A sealed tube was loaded with (E)-2,3-dibromobut-2-enoic acid
(3 g, 12.3 mmol, 1 equiv.) and potassium carbonate (3.4 g, 24.6
mmol, 2 equiv.) in DMF (30 mL). The mixture is degassed with
argon for 10 min. 3-Methylbut-1-yne (6 mL, 61.5 mmol, 5 equiv.)
and copper iodide (2.3 g, 12.3 mmol, 1 equiv.) were added. The
tube was filled with argon and sealed. The solution was stirred at
inhibitor]= 1 mM, non-reduced enzyme, [Mt UGM]= 580
b
nM, [fluorescent probe]= 18 nM.
FP assay conditions:
inhibitors]= 0-1 mM, non-reduced enzyme, [Mt UGM]= 580 nM,
fluorescence probe]= 18 nM.
[
[
2
.4. Antitubercular activity
6
0 °C overnight, then hydrolyzed with aqueous saturated solution
of NH Cl (100 mL) and filtered on Celite®. The filtrate was
extracted with AcOEt (300 mL). The organic phase was washed
with aqueous saturated solution of NH Cl (50 mL x 3), saturated
solution of NaCl (50 mL), dried over anhydrous MgSO , filtered
and solvents were evaporated under vacuum. The residue was
The anti-tubercular activities of the best inhibitors of UGM (Kd
70 μM) were then tested by determination of the minimal
inhibitory concentration (MIC) against M. tuberculosis mc 6230
Table 5). All compounds have MICs below or equal to 50 µg/mL,
thus highlighting their potent anti-mycobacterial activity.
Compounds 10a and 10d share MIC values comparable to the best
UGM inhibitors reported so far (Entries 1 and 3).
4
<
2
4
(
4
purified by recrystallization in CH
compound.
2 2
Cl to afford the expected
1
2,23,24
(Z)-3-Bromo-4-methyl-5-(2-methylpropylidene)furan-2(5H)-
one (1): C , MW = 231.09 g/mol, yield = 74 %, white
9 2
H11BrO
Table 5. MIC values of Mt UGM inhibitors
-1
solid, mp = 93-95 °C. IR (ATR)  (cm ) = 2964, 2868, 1760,
I
1
R
1674, 1222, 995, 963, 870. H NMR (300 MHz, CDCl ) δ (ppm)
O
3
=
5.27 (d, J = 9.7Hz, 1H), 3.00 (dsept, J = 9.7 Hz, 6.8 Hz, 1H),
N
O
1
3
2
.12 (s, 3H), 1.10 (d, J = 6.8 Hz, 6H). C NMR (75 MHz, CDCl
3
)
δ (ppm) = 165.2 (C=O), 151.3 (C), 147.6 (C), 121.2 (CH), 110.1
(
C
C), 26.2 (CH), 22.6 (2CH
H12 BrO [M+H] : 233.00002; found: 232.99946.
9 2
3
), 11.7 (CH
3
). HRMS (ESI) calcd. for
R'
8
1
+
Entry
Compound
MIC[a] [μg/mL]
General procedure for Suzuki coupling, conditions A
In a Schlenk tube under argon, boronic acid (1.3 mmol, 1.2 equiv.),
sodium carbonate (1M in H O, 1.3 mL, 1 mmol, 1.2 equiv.) and
tetrakis(triphenylphosphine)palladium(0) (100 mg, 0.087 mmol,
’
1
2
3
4
5
10a, R= H, R = H
10c, R= OMe, R = H
10d, R= [1,3]dioxolo, R’= H
2
11a, R= H, R = CO H
6.2
50
3.1
50
50
’
2
’
1
0 mol%) were added to a solution of (Z)-3-bromo-4-methyl-5-(2-
’
2
11c, R= OMe, R = CO H
methylpropylidene)furan-2(5H)-one (1) (250 mg, 1.08 mmol, 1
equiv.) in toluene and ethanol (6:4, 10 mL/6 mL). The resulting
mixture was stirred for 8 hours at 80 °C, cooled at room
temperature and filtered on Celite®. The solvents were removed
from the filtrate under the vacuum and water (10 mL) was added
to the resulting residue. The aqueous phase was extracted with
diethyl ether (3 x 20 mL). The combined organic phases were
[a]
The concentrations tested varied over a discrete 2-fold range:
.5, 3.1, 6.2, 12.5, 25, 50, 100 µg/ml. MIC determinations were
performed in duplicate on three independent occasions, with zero
variation between experiments for the five compounds tested.
1
3
. Conclusion
4
washed with brine (25 mL), dried over anhydrous MgSO , filtered
This study revealed a new tricyclic structure with good affinity
and solvents were evaporated under vacuum. The residue was
purified by column chromatography on silica gel with petroleum
ether/EtOAc as eluent to afford expected compound.
for Mt UGM and potent antitubercular activity and opens the door
for subsequent SAR studies to generate derivatives with increased
activity against drug susceptible and drug-resistant Mtb strains.
(
Z)-3-(4-Fluorophenyl)-4-methyl-5-(2-methylpropylidene)
furan-2(5H)-one (2a): C15 , MW = 246.28 g/mol, yield =
67 %, white solid, mp = 87-89 °C. IR (ATR)  (cm ) = 2967,
4
4
4
. Experimental section
H
15FO
2
-1
.1. Chemistry
1
2
870, 1743, 1663, 1590, 1508, 1224, 979, 837. H NMR (300
MHz, CDCl ) δ (ppm) = 7.52 (dd, J = 8.8 Hz, 5.4 Hz, 2H), 7.14 (t,
J = 8.8 Hz, 2H), 5.26 (d, J = 9.6 Hz, 1H), 3.08 (dsept, J = 9.6 Hz,
.1.1. General methods
3
All reactions were carried out under argon atmosphere in dried
1
9
6
.7 Hz, 1H), 2.22 (s, 3H), 1.13 (d, J = 6.7 Hz, 6H). F NMR (282
glassware. Tetrahydrofuran was dried and freshly distilled from
sodium and benzophenone. Dry DMF and catalysts were
purchased from Sigma-Aldrich . H NMR spectra were recorded
1
3
3 3
MHz, CDCl ) δ (ppm) = - 112.0. C NMR (75 MHz, CDCl ) δ
(ppm) = 169.2 (C=O), 162.8 (d, J = 248 Hz, C-F), 148.3 (C), 146.9
(C), 131.0 (d, J = 8 Hz, 2CH), 126.2 (d, J = 3 Hz, C), 125.6 (C),
1
1
2
®
1
®
on a Bruker Avance 300 (300 MHz) NMR spectrometer, using
3
20.2 (CH), 115.8 (d, J = 22 Hz, 2CH), 26.4 (CH), 22.8 (2CH ),
CDCl
internal reference, were as follows (in order): chemical shift (δ in
ppm relative to CHCl ), multiplicity (s, d, t, q, quint, m, br for
singlet, doublet, triplet, quartet, quintuplet, multiplet, broad) and
3 3 H
as solvent. Data, reported using CHCl (δ = 7.26 ppm) as
+
1.1 (CH
3 2
). HRMS (ESI) calcd. for C15H16FO [M+H] :
47.11288; found: 247.11222.
3

+
(
Z)-3-([1,1'-Biphenyl]-4-yl)-4-methyl-5-(2 methylpropylidene)
furan-2(5H)-one (2b): C21 , MW = 304.39 g/mol, yield = 66
, white paste. IR (ATR)  (cm ) = 2963, 1760, 1598, 1583,
17 3
HRMS (ESI) calcd. for C17H O [M+H] : 269.11722; found:
269.11698.
H
20
O
2
-1
%
4
.1.3. Preparation of new oxazinoindoles 11a and 11c
Aryl iodide (260 mg, 0.6 mmol), alkyne (0.9 mmol),
1
1
572, 1479, 1452, 1383, 1265, 1172, 921, 805, 755, 735, 698. H
NMR (300 MHz, CDCl ) δ (ppm) = 7.74-7.72 (m, 1H), 7.62-7.58
m, 3H), 7.55-7.51 (m, 2H), 7.49-7.43 (m, 2H), 7.36 (m, 1H), 5.27
d, J = 9.6 Hz, 1H), 3.11 (dsept, J = 9.6 Hz, 6.8 Hz, 1H), 2.27 (s,
3
(
(
triphenylphosphine (15 mg, 10% mol), CuI (11 mg, 10% mol), and
triethylamine (120 μL, 0.9 mmol) were combined with DMF (4.0
mL) in schlenk sealing tube. The resulting reaction mixture was
stirred under argon for overnight at room temperature or for 2 h on
MW at 50 °C. The solvent was removed from the reaction mixture
under the vacuum and the resulting crude product was purified by
flash chromatography on silica gel (petroleum ether/AcOEt =
100:0 to 50:50).
1
3
3
=
(
H), 1.14 (d, J = 6.7 Hz, 6H). C NMR (75 MHz, CDCl
169.2 (C=O), 148.4 (C), 147.3 (C), 141.7 (C), 140.8 (C), 131.5
C), 126.3 (C), 129.1(CH), 129.0 (2CH), 128.0 (CH), 127.9 (CH),
3
) δ (ppm)
1
2
27.7 (CH), 127.5 (CH), 127.4 (2CH), 120.1 (CH), 26.4 (CH),
21 2
2.9 (2CH ), 11.2 (CH ). HRMS (ESI) calcd. for C21H O
3
3
+
[M+H] : 305.15361; found: 305.15286.
(
Z)-3-(6-Methoxynaphthalen-2-yl)-4-methyl-5-(2-
(E)-7-(10-Iodo-8-methoxy-1-oxo-1H-[1,4]oxazino[4,3-a]indol-
3(4H)-ylidene)hept-5-ynoic acid (11a): C H INO , MW =
methylpropylidene)furan-2(5H)-one (2c): C20
20 3
H O , MW =
1
9
16
5
3
08.38 g/mol, yield = 61 %, white solid, mp = 144-146 °C. IR
465.24 g/mol, yield = 12 %, yellow solid, mp = 177-179 °C. IR
-1
-1
(ATR)  (cm ) = 2964, 1749, 1664, 1628, 1595, 1483, 1217, 988,
(ATR)  (cm ) = 3050, 2891,1744, 1696, 1645, 1508, 1412, 1378,
1
1
8
80, 809 H NMR (300 MHz, CDCl
3
) δ (ppm) = 7.96 (d, J = 1.4
1308, 1227, 1194, 1076, 922, 737. H NMR (300 MHz, CDCl ) δ
3
Hz, 1H), 7.79 (dd, J = 8.6 Hz, 3.5 Hz, 2H), 7.61 (dd, J = 8.7 Hz,
(ppm) = 7.62 (d, J = 8.2 Hz, 1H), 7.50 (dd, J = 6.8 Hz, 1.0 Hz, 1H),
7.42 (d, J = 8.4 Hz, 1H), 7.31 (dd, J = 6.8 Hz, 1.0 Hz, 1H), 5.65
(m, 1H), 5.15 (d, J = 0.8 Hz, 2H), 2.59-2.51 (m, 4H), 1.95 (quint,
1
3
=
.8 Hz, 1H), 7.19-7.13 (m, 2H), 5.26 (d, J = 9.6 Hz, 1H), 3.94 (s,
H), 3.12 (dsept, J = 9.6 Hz, 6.8 Hz, 1H), 2.29 (s, 3H), 1.14 (d, J
1
3
13
6.8 Hz, 6H). C NMR (75 MHz, CDCl
3
) δ (ppm) = 169.5
J = 6.9 Hz, 2H). C NMR (75 MHz, CDCl ) δ (ppm) = 177.4 (C),
3
(
1
1
C=O), 158.5 (C), 148.5 (C), 146.5 (C), 134.4 (C), 130.1 (CH),
154.3 (C), 151.4 (C), 136.9 (C), 131.3 (C), 130.0 (CH), 124.3
(CH), 122.9 (CH), 121.0 (C), 110.6 (CH), 97.1 (C), 96.0 (CH),
74.3 (C), 69.4 (C), 40.8 (CH ), 32.7 (CH ), 23.6 (CH ), 19.2 (CH ).
HRMS (ESI) calcd. For C H INO [M+H] : 436.0046, found
18 15 4
436.0034.
28.7 (C, CH), 127.1 (CH), 126.8 (CH), 126.6 (C), 125.4 (C),
19.6 (CH), 119.4 (CH), 105.7 (CH), 55.5 (CH ), 26.4 (CH), 22.9
), 11.2 (CH ). HRMS (ESI) calcd. for C20
09.14907; found: 309.14832.
3
2
2
2
2
+
+
(2CH
3
3
H
21
O
3
[M+H] :
3
(
2
Z)-4-Methyl-5-(2-methylpropylidene)-3-(pyridin-4-yl)furan-
(5H)-one (2d): C14 , MW= 229.28 g/mol, yield = 84 %,
yellow oil. IR (ATR)  (cm ) = 3054, 2961, 2869, 1754, 1664,
(E)-7-(10-Iodo-1-oxo-1H-[1,4]oxazino[4,3-a]indol-3(4H)-
H
15NO
2
ylidene)hept-5-ynoic acid (11c): C H INO , MW = 435.00
1
8
14
4
-1
g/mol, yield = 14 %, yellow solid, mp = 157-159 °C. IR (ATR) 
1
-1
1
577, 1437, 1297, 1192, 1119, 1032, 972, 878, 694. H NMR (300
MHz, CDCl ) δ (ppm) = 8.74 (d, J = 1.5 Hz, 1H), 8.62 (dd, J = 4.9
Hz, 1.4 Hz, 1H), 8.03 (dt, J = 8.0 Hz, 1.9 Hz, 1H), 7.45 (ddd, J =
.0 Hz, 4.9 Hz, 0.6 Hz, 1H), 5.35 (d, J = 9.6 Hz, 1H), 3.09 (dsept,
(cm ) = 3066, 2929, 1741, 1705, 1638, 1510, 1433, 1313, 1281,
1
3
1236, 1195, 1080, 953, 917, 834, 809, 739. H NMR (300 MHz,
CDCl ) δ (ppm) = 7.30 (d, J = 9.1 Hz, 1H), 7.12 (dd, J = 9.1 Hz,
3
8
2.4 Hz, 1H), 6.89 (d, J = 2.3 Hz, 1H), 5.67-5.64 (m, 1H), 5.11 (d,
1
3
J = 9.6 Hz, 6.8 Hz, 1H), 2.29 (s, 3H), 1.14 (d, J = 6.7 Hz, 6H). C
NMR (75 MHz, CDCl ) δ (ppm) = 168.7 (C), 149.5 (CH), 149.4
CH), 148.4 (C), 148.2 (C), 136.6 (CH), 126.5 (C), 123.6 (CH),
J = 1.0 Hz, 2H), 3.90 (s, 3H), 2.59-2.51 (m, 4H), 1.95 (quint, J =
1
3
3
7.0 Hz, 2H). C NMR (75 MHz, CDCl ) δ (ppm)= 177.3 (C),
3
(
156.4 (C), 154.2 (C), 151.5 (C), 132.2 (C), 131.8 (C), 120.9 (CH),
1
23.5 (C), 121.3 (CH), 26.5 (CH), 22.7 (2CH
3
), 11.2 (CH
3
).
120.4 (CH), 111.7 (CH), 103.2 (CH), 97.0 (C), 95.8 (CH), 74.4
(C), 68.2 (C), 55.9 (CH ), 40.9 (CH ), 32.6 (CH ), 23.6 (CH ), 19.2
+
HRMS (ESI) calcd. for C14
30.11699.
H16NO [M+H] : 230.11756; found:
2
3
2
2
2
+
2
(CH ). HRMS (ESI) calcd. For C H INO [M+H] : 436.00403,
2
18 15
4
found : 436.0034.
(
Z)-4-Methyl-5-(2-methylpropylidene)-[3,3'-bifuran]-2(5H)-
one (2e): C13 , MW= 218.25 g/mol, yield = 64 %, white solid,
mp = 64-66 °C. IR (ATR)  (cm ) = 3155, 3134, 2958, 2870,
14 3
H O
-1
4
.2. Docking
1
8
756, 1669, 1545, 1467, 1304, 1205, 1157, 1021, 964, 931; 830,
1
00, 740, 644, 601. H NMR (300 MHz, CDCl
3
) δ (ppm) = 8.07
Molecular docking studies were carried out using GOLD v 5.3.25
(bs, 1H), 7.50 (t, J = 1.8 Hz, 1H), 6.82 (dd, J = 1.8 Hz, 0.8 Hz,
GOLD is based on a genetic algorithm and allows to perform
docking of flexible ligands inside proteins with partial flexibility
in the neighborhood of the active site. The crystal structure used
as macromolecular receptor was Mt GM in closed form with the
substrate bound (PDB code: 4RPG). Prior to docking calculation,
water molecules and the bound substrate UDP-Galp were removed
from the crystal structure. The inhibitors docked conformations
were obtained using the score function ChemPLP. Examination
of the structures of the complex were carried out using PyMOL
software.
1
2
H), 5.21 (d, J = 9.6 Hz, 1H), 3.06 (dsept, J = 9.6 Hz, 6.7 Hz, 1H),
1
3
3
.24 (s, 3H), 1.11 (d, J = 6.7 Hz, 6H). C NMR (75 MHz, CDCl )
δ (ppm) = 168.8 (C), 148.6 (C), 143.8 (C), 143.4 (CH), 142.9 (CH),
19.4 (CH), 119.1 (C), 115.8 (C), 108.9 (CH), 26.4 (CH), 22.9
1
+
(2CH
3
), 11.1 (CH
3 15 3
). HRMS (ESI) calcd. for C13H O [M+H] :
2
19.10212; found: 219.10100.
2
6
(
Z)-3-(Benzofuran-2-yl)-4-methyl-5-(2-methylpropylidene)
furan-2(5H)-one (2f): C17 , MW = 268.31 g/mol, yield = 62
, white solid, mp = 87-89 °C. IR (ATR)  (cm ) = 2961, 2928,
865, 1750, 1669, 1443, 1297, 1216, 1123, 1037, 995, 925, 824,
16 3
H O
-1
%
2
7
1
3
51, 659. H NMR (300 MHz, CDCl ) δ (ppm)= 7.63 (dd, J = 7.4
4
.3. Mt UGM inhibitory activity
Hz, 1.0 Hz, 1H), 7.56 (s, 1H), 7.50 (dd, J = 7.4 Hz, 0.8 Hz, 1H),
7
1
2
.33 ( td, J = 7.3 Hz, 1.4 Hz, 1H), 7.25 ( td, J = 7.4 Hz, 1.2 Hz,
H), 5.36 (d, J = 9.7 Hz, 1H), 3.09 (dsept, J = 9.6 Hz, 6.7 Hz, 1H),
.55 (s, 3H), 1.14 (d, J =6.7 Hz, 6H). C NMR (75 MHz, CDCl )
3
UGM preparation: A vector construct (pET-29b) containing
the gene encoding for UGM from Mt was provided by Prof. Laura
1
3
L. Kiessling. The overexpression and UGM purification followed
δ (ppm) = 167.3 (C=O), 155.0 (C), 148.7 (C), 148.6 (C), 145.2 (C),
28.2 (C), 125.5 (CH), 123.4 (CH), 122.0 (CH), 121.3 (CH), 116.7
C), 111.2 (CH), 108.4 (CH), 26.6 (CH), 22.8 (2CH ), 11.4 (CH ).
12
our previously published procedure.
1
(
3
3

HPLC assay: Inhibition of UGM was performed following the
Multimode Detector Beckman-Coulter device (λexcitation = 485
nm,λemission = 535 nm).
2
7
procedure already described by Liu et al. as well as by our
2
8
group. All assays were performed at room temperature using a
phosphate buffer (NaH PO 100 mM, pH 7.4), and fresh solutions
2
4
of sodium dithionite which provide reductive conditions. The
activity of the enzyme (in the presence and in the absence of an
inhibitor) is evaluated by measuring the conversion of UDP-α-Galf
into UDP-α-Galp. The enzyme (60 nM Mt UGM) in phosphate
buffer was first pre-incubated for 5 min, then reduced with sodium
dithionite (final concentration 12.5 mM) and incubated for specific
time at room temperature, in absence and presence of inhibitor.
The substrate UDP-α-Galf (final concentration 25 μM) was added
and allowed the reaction to proceed at five different times. The
4.4. In vitro anti-tubercular activity
Antitubercular evaluations were performed against the
2
29
avirulent, pantothenate-auxotrophic Mt mc 6230 strain cultured
in 7H9 (Middlebrook) broth supplemented with oleic-albumin-
dextrose-catalase enrichment (OADC) and 109 µM pantothenic
acid (complete 7H9 medium) at 37°C without agitation. MIC
determination was done using the broth dilution method. Briefly,
a log-phase (OD600 ~ 1) culture was diluted to an OD600 = 0.05 in
complete 7H9 medium and deposited in all the wells of a 96 well
microtiter plate (for the first row 200 µl/well, for all other rows
2
reaction was stopped by quenching the samples with liquid N . The
conversion of UDP-α-Galf into UDP-α-Galp was monitored by
HPLC (Waters 600 E with a C18 Atlantis T3 column, 5 μM 4.6 x
1
00 µl/well). The tested compounds were then directly added (2 µl
per well of a 10 mg/ml stock solution) to the first row wells. Serial
-fold dilutions were then done starting from the first row. As a
2
0
50 mm, elution with 50 mM triethylamine acetic acid pH 6.8,
.5% CH CN; UV detection at 262 nm and flow rate 1 ml/min).
2
3
measure to minimize evaporation of media, plates were wrapped
in plastic. They were then placed in a 37°C incubator and observed
after 7 days. Control wells included a control for the vehicle that
compounds were dissolved in (DMSO), in which bacterial growth
was not inhibited (as for untreated wells) and wells containing a
drug with known antitubercular activity (INH), in which bacterial
growth was inhibited at ~ 30 ng/ml in line with the reported MIC
of this drug. The MIC was defined as the lowest concentration of
compound at which no visible bacterial growth (change in
turbidity) was observed.
FP assay; The assay described by Kiessling et al. was strictly
followed, including the synthesis of the fluorescent probe (UDP-
fluorescein). To determine the binding affinity of UDP-
1
6
fluorescein towards Mt UGM, serial dilutions of dialyzed UGM
-5
(
final concentration: 1x10 to 10 μM) were incubated with 18 nM
of the fluorescent probe in 50 mM sodium phosphate buffer, pH
.0 at room temperature. Final volumes were 30 μl in 384 well
3
0
7
black microtiter plates and the measurements were realized in
triplicate. Fluorescence polarization was analyzed using DTX880
6
4
1
7
. (a) P. J. Brennan, D. C. Crick, Curr. Top. Med. Chem. 2007, 7, 475–
88. (b) A. Nzila, Z. Ma, K. Chibale, Future Med. Chem. 2011, 3,
413–1426.
Acknowledgments
This study was supported by the Fondation pour la Recherche
Médicale (grant DEQ20150331719 to L.K.), the Association Gregory
Lemarchal and Vaincre la Mucoviscidose (grant RIF20180502320 to
C.R.) The University of Namur (PhD grant to SV) and China
Scholarship Council (PhD grant to JF). We acknowledge the French
Ministry for Research and Innovation for the financial support and Dr.
Frédéric Montigny (Analysis Department, Tours University) for
HRMS.
. (a) V. Makarov, G. Manina, K. Mikusova, U. Möllmann, O.
Ryabova, B. Saint-Joanis, N. Dhar, M. R. Pasca, S. Buroni, A. P.
Lucarelli, A. Milano, E. De Rossi, M. Belanova, A. Bobovska, P
Dianiskova, J. Kordulakova, C. Sala, E. Fullam, P. Schneider, J. D.
McKinney, P. Brodin , T. Christophe, S. Waddell, P. Butcher, J.
Albrethsen, I. Rosenkrands, R. Brosch, V. Nandi, S. Bharath, S.
Gaonkar, R. K. Shandil, V. Balasubramanian, T. Balganesh, S. Tyagi,
J. Grosset, G. Riccardi, S. T. Cole, Science 2009, 324, 801–804. (b) T.
Christophe, M. Jackson, H. K. Jeon, D. Fenistein, M. Contreras-
Dominguez, J. Kim, A. Genovesio, J.-P. Carralot, F. Ewann, E. H.
Kim, S. Y. Lee, S. Kang, M. J. Seo, E. J. Park, H. Škovierová, H.
Pham, G. Riccardi, J. Y. Nam, L. Marsollier, M. Kempf, M.-L. Joly-
Guillou, T. Oh, W. K. Shin, Z. No, U. Nehrbass, R. Brosch, S. T. Cole,
P. Brodin, PLoS Pathog 2009, 5, e1000645.
Supplementary Material
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Graphical Abstract
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☒
The authors declare that they have no known
competing financial interests or personal
Synthesis and evaluation of heterocycle
structures as potential inhibitors of
Mycobacterium tuberculosis UGM
Leave this area blank for abstract info.
a
b
b
c
c
a
a
Carine Maaliki , Jian Fu , Sydney Villaume , Albertus Viljoen , Clément Raynaud , Sokaina Hammoud , Jérôme Thibonnet ,
c,d
b
a,
Laurent Kremer , Stéphane P. Vincent , Emilie Thiery 
a
Laboratoire Synthèse et Isolement de Molécules Bioactives (SIMBA, EA 7502), Université de Tours, Faculté de Pharmacie, Parc de Grandmont, 31 Avenue Monge, 37200 Tours, France. b Department of
Chemistry, University of Namur, Rue de Bruxelles 61, 5000 Namur, Belgium. c Institut de Recherche en Infectiologie de Montpellier (IRIM), CNRS UMR 9004, Université de Montpellier, 34293 Montpellier,
France. d INSERM, IRIM, 34293 Montpellier France
Declaration of interests
relationships that could have appeared to influence
the work reported in this paper.

☐
The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests: