Fragment-based design of mycobacterium tuberculosis inha inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c00007

Tuberculosis (TB) remains a leading cause of mortality among infectious diseases worldwide. InhA has been the focus of numerous drug discovery efforts as this is the target of the first line pro-drug isoniazid. However, with resistance to this drug becoming more common, the aim has been to find new clinical candidates that directly inhibit this enzyme and that do not require activation by the catalase peroxidase KatG, thus circumventing the majority of the resistance mechanisms. In this work, the screening and validation of a fragment library are described, and the development of the fragment hits using a fragment growing strategy was employed, which led to the development of InhA inhibitors with affinities of up to 250 nM.

Full text of DOI:10.1021/acs.jmedchem.0c00007

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Article
Fragment-based design of Mycobacterium tuberculosis InhA inhibitors
Mohamad Sabbah, Vitor Mendes, Robert G Vistal, David M Dias, Monika Záhorszká,
Katarína Mikušová, Jana Korduláková, Anthony G Coyne, Tom L. Blundell, and Chris Abell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00007 • Publication Date (Web): 02 Apr 2020
Downloaded from pubs.acs.org on April 5, 2020
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Fragment-based design of Mycobacterium
tuberculosis InhA inhibitors.
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Mohamad Sabbah*║,a Vitor Mendes║,b Robert G. Vistal,a David M. G. Dias,a Monika
Záhorszká,c Katarína Mikušová,c Jana Korduláková,c Anthony G. Coyne,a Tom L. Blundell,b
and Chris Abell*,a
a Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2
1EW, United Kingdom.
b Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge,
CB2 1GA, United Kingdom.
c Department of Biochemistry, Faculty of Natural Sciences, Comenius University in
Bratislava, Mlynská dolina, Ilkovičova 6, 84215, Bratislava, Slovakia
Keywords:
Tuberculosis, Mycobacterium tuberculosis, fragment-based drug discovery, fragment-
growing, InhA
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Abstract
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Tuberculosis (TB) remains a leading cause of mortality amongst infectious diseases worldwide.
InhA, an enoyl ACP-reductase, has been the focus of numerous drug discovery efforts as this
is the target of the first line pro-drug isoniazid. However, with resistance to this drug becoming
more common the aim has been to find new clinical candidates that directly inhibit this enzyme
and that do not require activation by the catalase peroxidase KatG, thus circumventing the
majority of the resistance mechanisms. In this work, the screening and validation of a fragment
library is described and development of the fragment hits using a fragment growing strategy
was employed which led to the development InhA inhibitors with affinities of up to 250 nM.
Graphical abstract
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Introduction
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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains
the deadliest infectious disease worldwide, with approximately 10.4 million affected cases and
1.7 million deaths per year.1 The emergence of a number of multidrug-resistant TB (MDR-TB)
and extensively drug resistant (XDR) strains as well as a deadly synergy with HIV1 present
many challenges. Despite recent successes in identifying new chemical entities to combat TB
and the prospect of new regimens that will shorten TB treatment from the current six months,
there remains an urgent need to find new drugs to fight this disease.
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Mycolic acids, synthesized by the fatty acid synthase complex II (FAS II), are essential
components of the unique cell envelope of mycobacteria. The essentiality of this pathway is
explored clinically by isoniazid (INH), a first-line drug, and ethionamide (ETH), a second-line
drug used to treat MDR-TB. Both of these drugs target the FAS II enzyme InhA, an NADH
dependent enzyme involved in the reduction of long-chain fatty acids. Both INH and ETH are
pro-drugs that need to be activated within the mycobacteria to form a covalent adduct with the
nicotinamide adenine dinucleotide (INH-NAD adduct), which binds strongly into the catalytic
site of InhA.2 Resistance to these two pro-drugs can be predominantly explained by mutations
in the activating enzymes (KatG for INH and EthA for ETH) or in the upstream promoter
region of InhA, and less commonly in InhA itself due to the essentiality of the enzyme.2 Many
recent drug discovery efforts have focused on several of the enzymes of this pathway such as
KasA3, Pks134, 5, Ag85C6, 7 and FadD328 with InhA receiving most of the attention.9-13
InhA has been a major target for TB drug discovery with many novel and diverse potent
inhibitors which includes triclosan derivatives, pyridomycin, 4-hydroxy-2-pyridones,
thiadiazoles, proline series, pyrrolidine carboxamide which have been reported in the literature.
These inhibitors show direct inhibition of InhA without the need for activation, thus
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circumventing the most common resistance mechanism to the current drugs targeting this
enzyme.13
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In this work, fragment-based drug discovery (FBDD) was used as an alternative
approach to previous reported drug discovery methods for InhA, where the aim is to find novel,
potent inhibitors of the enzyme. FBDD is now established as a powerful tool to discover small-
molecule ligands or fragments as hits, which are further elaborated into lead compounds for
drug development and this approach has been widely used in the TB field..14, 15 Researchers at
the University of Dundee recently reported the screening of a fragment library against InhA,
however, further development of the fragment hits has not yet been reported.16 Herein, a three-
stage biophysical screening cascade: thermal shift, ligand-based NMR and X-ray
crystallography led to the identification of several fragment hits. Using a fragment growing
strategy supported by enzymatic assays, X-ray crystallography and molecular docking, one
fragment, which displays a novel binding mode in the X-ray crystal structure, was further
elaborated into a new class of nanomolar inhibitors of InhA.
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Results and discussion
Fragment screening cascade
Differential scanning fluorimetry (DSF) was used to screen an in-house library of 800 rule-of-
three-compliant fragments. A saturating concentration of the co-factor in oxidized form (1 mM
NAD+) was used in all the experiments to ensure that the co-factor binding site would be
occupied by NAD+ and that fragments would not bind there. InhA was shown to have a melting
temperature (Tm) of 53˚C in the presence of 1 mM NAD+. Triclosan, a known inhibitor of
InhA17, was used as a positive control, and presented a thermal shift (∆Tm) of +3.0˚C with a
melting temperature of 56˚C at a concentration of 0.5 mM in the presence of NAD+. Upon
screening of the fragment library, a hit was defined where the melting point was increased by
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at least +3˚C at a concentration of 5 mM (Figure 1a). A total 42 fragment hits were identified
using DSF, representing a hit rate of 5.2 % (Table S1).
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Figure 1. a) Histogram of fragments (5 mM) found to increase the melting point (∆Tm) of InhA
(10 µM) in presence of NAD+ (1 mM) by fluorescence-based thermal-shift assay. b) Chemical
structures of the X-ray hits obtained. c) X-ray structure of fragment 1 (teal) bound in an InhA-
NAD+ complex (grey), showing cavity surface. The substrate binding regions of InhA are
divided into three sites - site I, site II and site III. Fragment 1 occupies the catalytic site I and a
part of the hydrophobic pocket site II and Y158 adopts an “in” conformation. d) Overlaid X-
ray crystal structures of fragments 1 (green) and 3 (magenta), binding to InhA in the presence
of NAD+ with Y158 in an open conformation for fragment 1 (cyan, PDB Code 6SQ5) and in a
new, previously unseen, conformation for fragment 3 (magenta, PDB Code 6SQ9).
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In order to validate the hits obtained in the thermal shift assay, ligand-based NMR
techniques, CPMG (Carr-Purcell-Meiboom-Gill, which examines the T2 relaxation in the
presence of the protein), , WaterLOGSY (which examines the transfer of magnetization from
the bulk water molecules in the protein to the ligand) and STD (Saturated transfer difference,
which involved the selective saturation of protein resonances and subsequent transfer of
magnetisation) were used.18-20 The fragments were screened at a concentration of 1 mM, in
presence of NAD+ (0.5 mM), with 20 µM InhA using three techniques. Eighteen fragments
showed interactions with InhA in at least two ligand-based NMR techniques and these were
considered as confirmed hits. The validated hit rate was 42 % in which 83% (15 fragments)
contained carboxylic acids (Table S1).
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The eighteen fragment hits validated by NMR were then soaked into InhA crystals and
X-ray diffraction data was collected. Of these fragments, five showed sufficiently clear electron
density in the X-ray crystal structures that allowed them to be modelled with confidence
(Figure 1b and S1).
The binding pocket of InhA inhibitors that are substrate-competitive can be divided into
three distinct sites that have different properties, site I being the catalytic site, site II a
hydrophobic region that binds fatty acid chains and site III a solvent exposed site that is known
as the size-limiting region (Figure 1c).21 All of the obtained X-ray crystal structures of the
fragments contain NAD+, and the compounds occupy the same area as the InhA substrate
covering regions of sites I and II. The X-ray data collection and refinement statistics are
reported in Table S2. In the X-ray crystal structures of all the initial fragments, with the
exception of compound 3, Y158 adopts a previously described conformation known as “Y158-
in” (Figure S2). The “Y158-in” conformation has previously been observed with InhA
inhibitors (triclosan, 4-hydroxypyridones and pyrrolidine carboxamides) where the hydroxy
group of Y158 is oriented toward the inhibitor in site I. The aromatic group of the fragments
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occupies the hydrophobic site II of InhA pocket (Figure 1c and S2).13 For compounds 1, 4 and
5, the carboxylic acid portion of the molecules superimposes completely and forms hydrogen
bonds with the hydroxyl group of the ribose and Y158 while compound 3 only forms hydrogen
bonds with the ribose hydroxyl group (Figure S2). Compound, 3, presented a unique mode of
binding as the Y158 residue showed a distinct conformation from all other X-ray crystal
structures in both this study and in the literature (Figure 1d). This compound was observed to
be sandwiched between the residues Y158 and F149 forming π interactions with both (Figure
S2). For compound 2, even though the carboxylic acid group forms similar interactions as
compounds 1, 4 and 5 it sits in a different position with its benzo-moiety facing away from the
ribose and occupying a similar area of the InhA site as the imidazole group of compound 5.
The identified fragment hits were further evaluated by an InhA enzymatic assay using
the 2-trans-octanoyl-CoA as a substrate.22 The fragments were screened in triplicate at a final
concentration of 2 mM using triclosan as a positive control. However, none of the fragments
displayed any inhibitory activity against InhA at a concentration of 2 mM.
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Fragment growing
The examination of the X-ray structures of the fragments 1-5 supported the feasibility
of a fragment growing approach. The carboxylate group in site I provided a good handle for
elaboration towards the site II of the InhA pocket (Figures 1c). Based on the novelty of the
fragment hits and the vectors available to grow the fragments, fragment 1 was selected for
further elaboration.
The X-ray crystal structure of triclosan was overlaid with the structure of fragment 1
and it showed a similar binding mode to the fragment in the enzyme-NAD+ complex (Figure
2a). The carboxylate of fragment 1 occupies the same region of the triclosan phenyl-ether
linker.17 The carboxylic acid in fragment 1 (P1) was replaced by an isosteric sulfonamide
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replacement, as a linker to synthesize compound 6 (Figure 2B). Docking experiments with
Glide XP 23 (Schrodinger LLC), using the X-ray crystal structure of InhA with fragment 1 as a
model, showed that the sulfonamide vector also provided the right geometry to grow the
fragments into the InhA site III while also forming hydrogen bonds with Y158 and NAD+
(Figure 3a and 3b), with compound 6 showing a significant improvement of the activity (54%
inhibition (100 µM)). All the synthesized compounds were tested at a concentration of 100 µM
using the InhA enzymatic assay. The IC50 were determined only for the compounds showing
an inhibitory activity greater than 90% under these screening conditions.
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Figure 2: A) X-ray crystal structure of fragments 1 (PDB code 6SQ5) overlayed with triclosan
(PDB code 2B35) bound to the InhA-NAD+ complex with “Y158-in” conformation. B)
Fragment growing strategy from fragment 1 to compound 6. Inhibition percentage of InhA
using an enzymatic assay for each compound is shown.
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The phenyl ring was replaced by a piperidine saturated ring which could be useful to
grow fragment 1 into site III. This modification led to loss of potency in compound 7 (15%
inhibition at 100 µM, Table 1). The introduction of a pyridine ring also displayed a lower
percentage inhibition (17%) for compound 8. Therefore, the phenyl ring of compound 6 was
maintained and analogues of compound 6 were prioritised.
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Table 1. Structure and activities of compounds 7-20 evaluated by an enzymatic assay
SO2NHR1
CF3
Inh %a
(IC50)
Inh %
(IC50)
Compound
R1
Compound
R1
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> 90 (9)b
0
COOMe
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0
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O
NHBoc
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0
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OH
CONH2
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> 90 (4)b
0
COOMe
aLigand efficiency LE: Compound 14 = 0.29, compound 19 = 0.31c
a
b
Inh% : inhibition percentage at 100 µM concentration. IC50 : measured in µM
by enzymatic assay from an average of three experiments. IC50 values were
c
determined for compounds that showed >90% inhibition at 100 µM. LE was
calculated using the equation LE = (1.37 × pIC50)/HA, where HA means heavy
atom, i.e., a non-hydrogen atom
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As shown in Table 1, compounds 9 and 10 did not add any improvement where a percentage
inhibition of 0% and 11% was measured respectively. Subsequent deprotection of compound
10 to afford the aniline 11, also did not show any improvement in the percentage inhibition
(6%). This was also observed with the electron-donating methoxy group in compound 12. The
benzyl ester 13 (% Inhibition = 24%) showed a better potency than the previous analogues
although this was still low.
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Interestingly a 10-fold increase of the potency from compound 6 was observed with the
synthesis of benzylamine analogue 14 (Table 1). This compound showed an improved InhA
inhibition (>90%) with an IC50 of 9 µM and a ligand efficiency (LE) of 0.29, which is at least
a 200-fold more potent compound compared to the initial fragment hit 1 (IC50 > 1000 µM).
(a)
Method A: NEt3, DCM
Method B: Pyridine, rt
SO2Cl2, DMF
90oC, 3 h
CF3
SO2NHR1
CF3
CF3
SO2Cl
27
N
R1 =
NH
COOMe
NHBoc
6
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9
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10
COOMe
NHMe
NHCOMe
NH2
OMe
11
17
13
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CONH2
COOH
18
(b)
O
S
HN
O
S
HN
O
O
CF3
CF3
R
TFA, DCM, rt
R
14 R = 4-CH2NH2
19 R = 3-CH2NH2
28 R = 4-CH2NHBoc
29 R = 3-CH2NHBoc
O
O
O
O
CF3
S
HN
CF3
S
HN
TFA, DCM, rt
NHBoc
NH
20
30
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Scheme 1: Synthesis of compounds substituted on the sulphonamide group
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Subsequently, compounds 15 to 20 were synthesized as analogues of compound 14 to
explore whether the primary amine could be replaced by other functional groups (Table 1). In
compounds 15 and 16, the amine of compound 14 was respectively mono-methylated and
acetylated. The amine was replaced by a carboxylic acid and an acetamide group in the
analogues 17 and 18. All these modifications on this position of the methyleneamine of
compound 14 led to a loss of potency with an InhA percentage inhibition between 0 and 25%
at a ligand concentration of 100 µM. Inhibition of InhA was slightly improved (9 µM compared
to 4 µM) by moving the methylamine to the meta-position, compound 19 and gave an IC50 of
4 µM and a LE of 0.31. The docking pose of compound 19 showed the amine group forms a
hydrogen bond with the phosphate of NAD+ and forcing the phenyl ring to move further away
from the phosphates towards M161 (Figure 3c and 3d). This changes the orientation of the
sulfonamide now only forming an hydrogen bond with Y158 and it further alters the position
of the trifluoromethylphenyl moiety when compared to compound 6 (Figure 3).
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Figure 3: Docking poses for compounds 6 (A) and 19 (C) superposed with fragment 1 (green).
Hydrogen bonds for each for compound 6 and 19 and shown as red dashes respectively in (B)
and (D) with distances in Å. The compounds 6 and 19 were docked into the structure for
fragment 1 (PDB code 6SQ5).
The activity was lost in compound 20 in which the amine is part of a closed 6-membered
ring. The development of compounds 15-20 showed that the phenylmethanamine group was
crucial to maintain affinity and was maintained for further development of a potent InhA
inhibitor. Subsequently, the sulfonamide with the phenylmethanamine scaffold on P3 (Table
1) was maintained and further modification on P2 as was examined (Table 2).
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Further Elaboration strategies
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The trifluoromethyl styrene moiety in compound 19 could potentially be metabolized
to styrene oxide, and as a result this scaffold was replaced to improve the drug-like properties.
From the X-ray crystallography of the parent fragment 1 and from molecular docking, P2 of
compound 19 should mainly occupy the hydrophobic site II of InhA (Figure 3c). However,
docking studies of derivatives of 1 showed that the trifluoromethyl styrene moiety could bind
to different hydrophobic areas of site II (Figure 3a and 3c). Hydrophobic functional groups
were chosen as modifications at P2 of compound 19 (Table 2). The tert-butylphenyl and chloro-
naphthalene groups in compound 21 and 22 were synthesized based on similarities with
respectively fragments 2 and 4. However both compounds 21 and 22 showed a drop of
percentage inhibition to 40% and 17% respectively at a screening concentration of 100 µM.
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O
NHBoc
O
NH2
NHBoc
O
O
S
O
O
S
TFA, DCM, rt
Pyridine, rt
R2
R2
S
HN
HN
H2N
R2
Cl
32
31
R2 =
Cl
S
21
22
23
F
O
S
S
26
25
24
Scheme 2: Exploration of SAR based on compound 19.
Table 2. Structure and activities of compounds 21-26 evaluated by InhA enzymatic assay
O
NH2
O
S
HN
R2
Inh %a
b
µ )
M
(IC50
Compound
R2
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> 90
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a Inh% : inhibition percentage at 100 µM concentration. b IC50 : measured in µM by enzymatic
assay from an average of three experiments. IC50 values were determined for compounds that
showed >90% inhibition at 100 µM. LE was calculated using the equation LE = (1.37 ×
c
pIC50)/HA, where HA means heavy atom, i.e., a non-hydrogen atom
The introduction of a 5-chloro-3-methylbenzothiophene in compound 23 improved the
InhA inhibition IC50 to a value of 310 nM with a ligand efficiency of 0.40. This is a 12-fold
jump in potency in comparison to compound 19, (Table 2). The replacement of the 5-chloro
with a 5-fluoro substituent in compound 24 slightly improved the potency to 250 nM. The
halogens and methyl substitutions prove to be essential for the activity as a significant drop in
activity to 6 and 22 µM were observed respectively for compounds 25 and 26 (Table 2). This
shows the importance of these functionalities in order to maintain a potency.
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Figure 4. a) X-ray crystal structure and interaction map of compound 23 (PDB Code 6SQL).
NAD+ is shown in white while compound 23 in shown in teal. Red disks represent hydrogen
bonds, blue disks depict π-π interactions, yellow disks sulphur-π. Interactions were calculated
using Intermezzo plugin for Pymol (Ochoa B., et al. unpublished). b) Substrate binding region
of InhA divided into three sites. The X-ray structure of fragment 1 (violet) (PDB code 6SQ5)
is overlaid with compound 23, showing cavity surface.
The X-ray crystal structure for compound 23 was obtained (Figure 4a) and it shows, as
predicted by docking (Figure S3), that the sulfonamide group forms an hydrogen bond with
Y158 and has the correct orientation for the phenylmethanamine moiety to explore site III
forming π-interactions with the backbone of the protein at G96 and a sulphur-π interaction with
M161 (Figure 4a and 4b). Furthermore, the amine in the meta-position of the phenyl ring
interacts with the phosphates of NAD+, but also with a highly coordinated water that further
interacts with the sulfonamide group and the NAD+ phosphate (Figure 4a). The benzothiophene
group forms π-interactions with F149 and Y158, sulphur-π interactions with M103, M155 and
M199 while exploring a different hydrophobic area on site II, (Figure 4b). The position of the
phenylmethanamine group in relation to the sulfonamide is crucial for the binding affinity. In
the earlier SAR examined in Table 1 when this was changed to a secondary amine (compounds
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15 and 20) or an aniline (compound 11) there little or no binding observed. This could be due
to the increased sterics observed in compound 15 and 20 which would not allow the H-bonding
interactions. With compound 11 the intramolecular H-bonding interactions through the water
would not be possible due to the direct attachment of the NH2 group to the benzene ring.
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Evaluation of the effects of InhA inhibitors on mycolic acid synthesis in M. tuberculosis
H37Ra
The IC50 of tested inhibitors against InhA protein were promising and these compounds
(14, 19 and 23-26) were screened to see whether they can inhibit the growth of M. tuberculosis.
Further analysis of the effect on the synthesis of mycolic acids by metabolic labeling24, 25 of
the model strain M. tuberculosis H37Ra with 14C acetate was also explored. A 48 h cultivation
in the presence of 200 µM of the compounds in the media led to 84 % growth inhibition for
compound 19; 40 and 33% growth inhibition for compounds 14 and 23, respectively; 38 %
growth inhibition for compound 24 and isoniazid; and 23 % growth inhibition for compounds
25 and 26. However, while the presence of isoniazid caused complete abolition of synthesis of
mycolic acids, only slight inhibition was observed in the case of compound 19 and no inhibition
in the case of the other screened compounds (Figure 5a).
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Figure 5. a) TLC of the metabolic labeling experiments of M. tuberculosis H37Ra with C
acetate treated with the compounds 14, 19 and 23-26 for the analysis of mycolic acids
inhibition. Fatty acid methyl esters – FAME; mycolic acid methyl esters – MAME and
isoniazid – INH. b) TLC of the metabolic labeling experiments of M. tuberculosis H37Ra with
14C acetate treated with the compounds 14, 19 and 23-26 for the analysis of the lipid inhibition.
Trehalose monomycolates – TMM; trehalose dimycolates – TDM; phosphatidylethanolamine
– PE; cardiolipin - CL and isoniazid – INH.
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The analysis of lipid profiles revealed that treatment with compound 19 led to the
accumulation of TMM and to the decrease of amount of TDM (Figure 5b). None of other tested
inhibitors affected the amounts of TMM and TDM in mycobacterial cells suggesting that
despite the potent IC50 values, the tested compounds possibly do not target InhA inside
mycobacterial cells and further experiments are needed in order to clarify this.
Conclusions
Fragment-based drug discovery is a robust and now widely used approach to identify
drug-like molecules and this methodology has led to the development of a number of drugs
which have been approved by the FDA. In this work we identified several fragment hits using
a screening cascade consisting of DSF, ligand-based NMR and X-ray crystallography. The
initial fragment hits revealed a ligand having a unique binding mode and forcing Y158 to adopt
a new conformation that “sandwiches” the compound between the residues F149 and Y158.
However, the fragment hits had no detectable inhibitory activity. Using the available structural
information, potent and novel nanomolar inhibitors of InhA were developed applying a
fragment-growing approach. The systematic exploration of chemical space in P3 and P1 after
fixing P2 with a sulfonamide and helped by molecular docking led to the development of
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potency and increase of ligand effiency. The introduction of a benzothiophenene at P2 and the
phenylmethanamine at P3 led to the development of compound 23 and this was shown to be a
potent inhibitor of InhA. However, disappointingly compound 23 was shown to be inactive
against M. tuberculosis.
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In the past decade the TB drug discovery field has seen a shift from target-based
approaches back to whole-cell phenotypic screens due to the problems faced by compounds
derived from target-based approach in translating their activity into cell-based assays15.
However, the number of lead compounds coming from these large-scale screens has been lower
than expected. The compounds in this study illustrate the current challenge of obtaining new
antitubercular compounds. The strong InhA inhibitory activity of compounds described herein
is not reflected in antitubercular activity, nor do we observe a significant reduction in the
amount of mycolic acids produced by any of the compounds suggesting that the compounds
do not penetrate the cells in sufficient amounts, are being extruded by efflux mechanisms or
are metabolized. Further work is required to clarify the nature of the problem and to improve
the cellular activity of the lead compounds.
Experimental section
Protein purification, crystallization data collection and refinement
Mycobacterium tuberculosis InhA was purified as described previously.26 Briefly, E. coli
BL21(DE3) containing an hexahistidine-SUMO tagged InhA construct in pET28a was grown
to mid-exponential growth phase (OD610 = 0.8) in LB media (Invitrogen) containing 30 mg L-
1
kanamycin at 37 ºC. Gene expression, was induced by adding Isopropyl β–D-1-
thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM and the temperature lowered
to 18ºC. Cells were lysed in 50mM HEPES pH 7.5, 0.5 M NaCl, 10% glycerol (w/v), 20 mM
Imidazole and recombinant InhA was purified with a HiTrap IMAC Sepharose FF column
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(GE-Healthcare), equilibrated in the same buffer. Elution was performed with 500 mM
Imidazole. The recovered protein was dialysed into 50 mM HEPES pH 7.5, 0.5 M NaCl and
10% glycerol (w/v) and SUMO tag was cleaved overnight at 4 ºC by adding Ulp1 Protease at
1:100 ratio. SUMO tag, Ulp1 protease and uncleaved SUMO-InhA were removed using the
same column, equilibrated with 50 mM HEPES pH 7.5, 0.5 M NaCl, 10% glycerol (w/v) and
20mM Imidazole. Flow through containing InhA was collected, concentrated and loaded in a
Superdex 200 column equilibrated with 50mM HEPES pH7.5 150 mM NaCl and 10% glycerol
(w/v). Fraction purity was determined by SDS-page. The purest fractions were pooled,
concentrated to ~12 mg.ml-1, flash frozen in liquid nitrogen and stored at -80 ºC.
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InhA was crystallized in the presence of 2 mM NAD, at 18 ºC using the sitting drop vapor
diffusion method by mixing 1 µL of InhA at 12 mg.mL-1 in a 1:1 ratio with reservoir solution
containing 0.1 M HEPES pH 7.0, 0.1 M sodium acetate and 25-30% PEG 400. Fragment
soaking was performed at by mixing compound solution at 200 mM in 100% DMSO with
mother liquor to a final concentration of 20 mM. Crystals were soaked in these solutions
overnight. Compound 24 and 2-trans-octanoyl-CoA were co-crystalized by mixing InhA 10
mg.mL-1 with 0.5 mM NAD+ and 0.5 mM compound 24 or 5 mM 2-trans-octanoyl-CoA in a
1:1 ratio with reservoir solution containing 14% PEG 4k, 200 mM ammonium acetate, 100
mM HEPES pH 7 for compound 23 and 10% PEG 8k, 100 mM TRIS pH 8.5 for 2-trans-
octanoyl-CoA. A cryogenic solution was prepared by adding 30% glycerol to mother liquor
and crystals were briefly transferred to this solution, flash frozen in liquid nitrogen and stored
for data collection. All datasets were collected at stations i03 and i04-1 at Diamond Light
Source (Oxford, UK). Data collection and refinement statistics are summarized in (Table S2).
Diffraction data were indexed, integrated and reduced using autoPROC from Global Phasing
Limited 27. Molecular replacement was performed with Phaser 28 using PDB structure 2B35 as
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a search model. Refinement was carried out iteratively with PHENIX 29 and Coot 30, and ligand
and water fitting was performed with Coot. 30
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Molecular docking
Glide XP23 (extra precision) included in the Schrodinger software package (Schrodinger, LLC,
New York, NY, 2015 and 2016) was employed for docking of compounds using the X-ray
crystal structure with fragment 1 as the receptor. Epik31, included in the same software package
was used to prepare ligands. Docking was performed using default XP settings, with flexible
ligand sampling and post-docking minimization.
Differential scanning fluorimetry
Differential scanning fluorimetry was used to screen a library of 800 fragments. The assay was
performed in a 96 well plate format using a CFX Connect (Bio-Rad). Each well contained a
solution of 10 µM InhA, 1mM NAD+, Hepes (50mM), NaCl (150 mM), pH 7.5, 5x Sypro
Orange, 5% DMSO and fragments at 5mM. A positive control with 0.5 mM Triclosan was
included in all plates. Controls were made in quadruplicates for all the experiments.
Enzymatic assay
InhA activity was followed by a colorimetric assay that measured the oxidation of NADH at
340 nm in the presence of 2-trans-octanoyl-CoA as described before in a buffer that contained
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30 mM PIPES pH 7.5, 50 mM NaCl and 0.1 mM EDTA. InhA (100 nM). This was pre-
incubated for 10 min at room temperature with 0.25 mM NADH and varying concentrations of
the compounds at a with 1% (v/v) DMSO in 150 µL reaction volume. The reaction was started
by the addition of 2-trans-octanoyl-CoA at a final concentration of 1.5 mM prepared as
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described previously. 22 Reactions were followed for 20 min using a plate reader (CLARIOstar
- BMG LABTECH).
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Analysis of effects of tested compounds on mycolic acid synthesis in M. tuberculosis
H37Ra
The culture of M. tuberculosis H37Ra was grown statically at 37°C in Middlebrook 7H9 broth
(Difco) supplemented with albumin-dextrose-catalase and 0.05% Tween 80. When the culture
reached the value of O.D.(600 nm) 0.290, it was divided into 10 mL aliquots and tested
compounds dissolved in DMSO were added in 200 µM final concentrations, while isoniazid
that served as a control inhibitor was at 40 µM. The final concentration of DMSO in each
culture was at 1 %. After 24 h of cultivation with shaking (120 rpm) in the presence of tested
inhibitors, 14C acetate (specific activity 106 mCi/mmol, ARC) was added in the final
concentration of 0.5 μCi/mL and the cells were cultivated for next 24 h.
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Lipids were extracted from whole cells as described earlier with minor modifications.
Briefly, 3 mL chloroform/methanol (1:2) were added to cells harvested from 4 mL culture
aliquots, the mixtures were incubated at 56°C for 1.5 h with intense mixing, centrifuged at
1000 x g and the extracts were collected in glass tubes. This was followed by the extraction
with chloroform/methanol (2:1) at the same conditions. Both extracts were combined together,
dried under nitrogen and subjected to biphasic wash with chloroform/methanol/water (4:2:1)
as described earlier.25 The bottom organic phase was dried and dissolved in
chloroform/methanol (2:1), 250 μL per 1 unit of O.D.(600) of harvested cells. 5 μL of each
lipid sample were loaded on the thin-layer chromatography (TLC) silica gel plates F254
(Merck), lipids were separated in chloroform/methanol/water [20:4:0.5] and detected by
autoradiography.
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Fatty acid methyl esters (FAME) and mycolic acids methyl esters (MAME) were
prepared from whole cells harvested from 4 ml culture aliquots as previously described.32 Dried
extracts were dissolved in chloroform/methanol (2:1) in the ratio 250 μL per 1 unit of (O.D. =
600) of harvested cells and 5 μL of each sample were loaded on TLC plates. Different forms
of methylesters were separated by chromatography in n-hexane/ethyl acetate [95:5], 3x and
detected by autoradiography.
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Synthetic chemistry
General Experimental Methods. Solvents were distilled prior to use and dried by standard
methods. Unless otherwise stated, 1H and 13C NMR spectra were obtained in CDCl3, MeOD,
or DMSO solutions using either a Bruker 400 MHz AVANCE III HD Smart Probe, 400 MHz
QNP cryoprobe, or 500 MHz DCH cryoprobe spectrometer. Chemical shifts (δ) are given in
ppm relative to the residual solvent peak (CDCl3: 1H, δ = 7.26 ppm; 13C, δ = 77.16 ppm), and
the coupling constants (J) are reported in hertz (Hz).
Reactions were monitored by TLC and LCMS to determine consumption of starting materials.
Flash column chromatography was performed using an Isolera Spektra One/Four purification
system and the appropriately sized Biotage SNAP column containing KP-silica gel (50 μm).
Solvents are reported as volume/volume eluent mixture where applicable.
High resolution mass spectra (HRMS) were recorded using a Waters LCT Premier Time of
Flight (TOF) mass spectrometer or a Micromass Quadrapole-Time of Flight (Q-TOF)
spectrometer.
Liquid chromatography mass spectrometry (LCMS) was carried out using an Ultra
Performance Liquid Chromatographic system (UPLC) Waters Acquity H-class coupled to a
Waters SQ Mass Spectrometer detector. Samples were detected using a Waters Acquity TUV
detector at 2 wavelengths (254 and 280 nm). Samples were run using an Acquity UPLC HSS
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column and a flow rate of 0.8 mL/min. The eluent consisted of 0.1% formic acid in water (A)
and acetonitrile (B); gradient, from 95% A to 5% A over a period of 4 or 7 min. All final
compounds had a purity greater than 95% as determined by LCMS analysis.
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(E)-2-(3-(trifluoromethyl)phenyl)ethene-1-sulfonyl chloride (27). Sulfuryl chloride (3.8 mL,
46 mmol) was added dropwise at 0 °C to a solution of anhydrous DMF (10 mL) under N2.
After the addition was completed, the mixture was warmed to r.t. and stirred further for 0.5 h.
1-(trifluoromethyl)-3-vinylbenzene (1.71 mL, 11 mmol) was then added in three portions and
the reaction mixture was gradually heated at 90°C for 3h. The reaction mixture was cooled and
then poured onto the crushed ice and the separated oily layer was extracted with Et2O and dried.
1
Evaporation of the solvent gave the desired compound 27 (77% yield) as a yellow solid. H
NMR (400 MHz, CDCl3) δ 7.94 – 7.48 (m, 3H), 4.43 – 4.25 (m, J = 14.2, 5.5 Hz, 1H), 3.85 –
3.71 (m, 1H). LC/MS [mass of sulfonic acid -H] found 251.1.
(E)-N-phenyl-2-(3-(trifluoromethyl)phenyl)ethene-1-sulfonamide (6). To a solution of
compound 27 (50 mg, 0.185 mmol) in DCM (5.0 mL), dry triethylamine (0.031 mL, 0.22
mmol) was added aniline (17 mg, 0.18 mmol). The reaction mixture stirred for 3 hours at room
temperature. The reaction was quenched with saturated solution of NaHCO3, followed by
extraction with DCM. The organic phase was dried on anhydrous MgSO4, concentrated under
reduced pressure and the crude product purified by silica flash column chromatography
o
(column gradient of 20% to 50% EtOAc in Pet. Ether 40-60 C) to yield the desired product
(32 mg, 53% yield) as a white powder. 1H NMR (400 MHz, CDCl3) δ 7.63 (s, 1H), 7.58 – 7.45
(m, 2H), 7.35 – 7.22 (m, 3H), 7.14 (t, J = 7.2 Hz, 1H), 6.92 (d, J = 15.5 Hz, 1H). HRMS (ESI)
calcd. for [C15H12F3NO2S + H]+: 328.0619, Found: 328.0608. LC/MS [M-H]- found 326.1,
purity >99%.
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(E)-N-(piperidin-4-yl)-2-(3-(trifluoromethyl)phenyl)ethene-1-sulfonamide (7). Using the same
procedure as compound 6, tert-butyl-4-aminopiperidine-1-carboxylate (0.100 g, 0.37 mmol)
and 27 (0.100 mg, 0.37 mmol) yielded the Boc-protected intermediate tert-butyl-(E)-4-((2-(3-
(trifluoromethyl)phenyl)vinyl)sulfonamido)piperidine-1-carboxylate (78 mg, 0.37 mmol, 78%
yield) as a white powder. The crude product was purified by silica flash column
chromatography (column gradient of 30% to 60% EtOAc : Pet. ether 40-60 oC 1H NMR (400
MHz, CDCl3) δ 7.72 (s, 1H), 7.66 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 7.7 Hz, 1H), 7.49 (d, J =
15.5 Hz, 1H), 6.86 (d, J = 15.4 Hz, 1H), 4.90 (d, J = 7.5 Hz, 1H), 3.94 (m, 2H), 3.52 – 3.26 (m,
1H), 3.02 – 2.69 (m, 2H), 1.92 (m, 2H), 1.51 – 1.43 (m, 2H), 1.42 (s, 9H). LC/MS [M-H] found
433.2, purity >99 %.
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To a solution of the Boc intermediate in dichloromethane (2 mL), trifluoroacetic acid (1 mL)
was added dropwise and the mixture stirred for 0.5 hours. The reaction was quenched with
saturated solution of NaHCO3. The organic phase was seperated, and the solvent removed
under reduced pressure to yield the product (45 mg, 0.18 mmol, 75% yield) as yellow oil. 1H
NMR (400 MHz, DMSO-d6) δ 1H NMR (400 MHz, DMSO-d6) δ 8.15 (s, 1H), 8.04 (d, J = 7.8
Hz, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 1.7 Hz, 1H), 3.19 (m,
2H), 2.88 (m, 1H), 2.00 – 1.88 (m, 1H), 1.61 (m, 1H). HRMS (ESI) calcd. for [C14H17F3N2O4S
+ H]+: 335.1041, Found: 335.1053. LCMS [M+H]+ found 335.1, purity >99 %.
(E)-N-(pyridin-3-yl)-2-(3-(trifluoromethyl)phenyl)ethene-1-sulfonamide (8). To a solution of
compound 27 (50 mg, 0.18 mmol) in DCM (5.0 mL), dry Et3N (0.03 mL, 0.220 mmol) was
added 3-aminopyridine (17 mg, 0.18 mmol). The reaction mixture was stirred for 3 hours at
room temperature. The reaction was quenched with NaHCO3, followed by extraction with
DCM. The organic phase was dried on anhydrous MgSO4, concentrated under reduced pressure
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and the crude product purified by silica flash column chromatography (column gradient of 20%
to 50% EtOAc : Pet. Ether 40-60 oC) to yield the desired product (39 mg, 64% yield) as a white
powder. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (d, J = 3.3 Hz, 1H), 8.70 (dd, J = 4.8, 1.5 Hz,
1H), 8.25 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 8.03 (ddd, J = 8.2, 2.6, 1.5 Hz, 1H), 7.93 (d, J =
15.3 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.77 – 7.64 (m, 2H), 7.59 (dd, J = 8.2, 4.0 Hz, 1H), 1.23
(s, 1H); LCMS: no molecular ion peak observed, purity 97% ; HRMS (ESI) calcd. for
[C14H11N2O2F3S + H]+: 329.0572, Found: 329.0563.
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Methyl (E)-4-((2-(3-(trifluoromethyl)phenyl)vinyl)sulfonamido)benzoate (9). Using the same
procedure as with compound 6, methyl 3-aminobenzoate (0.168 g, 1.108 mmol) and compound
27 (0.300 g, 1.108 mmol). The crude product was purified by silica flash column
chromatography (column gradient of 20% to 50% EtOAc : Pet. ether 40-60 oC) which yielded
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the desired product 9 (0.281 g, 0.729 mmol, 66% yield) as a yellow powder. H NMR (400
MHz, CDCl3) δ 7.88 (d, J = 2.0 Hz, 1H), 7.82 (dd, J = 7.7, 1.3 Hz, 1H), 7.65 (d, J = 7.7 Hz,
2H), 7.63 – 7.54 (m, 1H), 7.55 – 7.46 (m, 2H), 7.41 (t, J = 7.9 Hz, 1H), 7.16 (s, 1H), 6.88 (d, J
= 15.4 Hz, 1H), 3.91 (s, 3H), 3.50 (s, 1H); HRMS (ESI) calcd. for [C17H14F3NO4S+H]+:
386.0674, Found: 386.0688; LCMS: [M-H]- found 384.1, purity 94%.
tert-butyl-(E)-(4-((2-(3-(trifluoromethyl)phenyl)vinyl)sulfonamido)phenyl)carbamate
(10).
Using the same procedure as compound 6, tert-butyl (4-aminophenyl)carbamate (77 mg, 0.37
mmol) and compound 27 (100 mg, 0.37 mmol). The crude product was purified by silica flash
column chromatography (column gradient of 30% to 60% EtOAc : Pet. ether 40-60 oC). This
yielded the desired product (50 mg, 0.050 mmol, 31% yield) as a clear oil. 1H NMR (400 MHz,
CDCl3) δ 7.64 (d, J = 6.0 Hz, 2H), 7.57 (d, J = 8.2 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.43 (d, J
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= 15.5 Hz, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.20 – 7.11 (m, 2H), 6.90 – 6.80 (m, 2H), 6.54 (s,
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1H), 1.49 (s, 9H); LCMS: [M-H]- found: 441.0, purity > 99%.
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(E)-N-(4-aminophenyl)-2-(3-(trifluoromethyl)phenyl)ethene-1-sulfonamide (11). Using the
same procedure as compound 7, compound 10 (50 mg, 0.11 mmol) was deprotected to yield
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the desired product (26 mg, 0.070 mmol, 62% yield) as a brown oil. H NMR (500 MHz,
DMSO-d6) δ 8.10 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 7.1 Hz, 1H), 7.63 (t, J = 7.8
Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 7.44 (t, J = 7.6 Hz, 2H), 7.35 (d, J = 11.8 Hz, 1H), 6.87 (d, J
= 8.7 Hz, 1H), 6.46 (d, J = 8.7 Hz, 1H); LCMS: [M-H]- found 341.1, purity >99%.
(E)-N-(4-methoxyphenyl)-2-(3-(trifluoromethyl)phenyl)ethene-1-sulfonamide (12). Using the
same procedure as compound 6, 4-methoxyaniline (37 mg, 0.28 mmol) and compound 27 (50
mg, 0.18 mmol) The crude product was purified by silica flash column chromatography
(column gradient of 30% to 70% EtOAc : Pet. ether 40-60 oC). This yielded the desired product
(10 mg, 0.03 mmol, 15% yield) as a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H),
8.13 (s, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.86 – 7.57 (m, 2H), 7.19 – 7.02 (m, 2H), 6.98 – 6.80
(m, 2H), 6.71 – 6.58 (m, 1H), 6.55 – 6.24 (m, 1H), 3.68 (s, 2H); HRMS (ESI) calcd. for
[C16H14NO3F3S + H]+: 358.0725, Found: 358.0735. LCMS [M-H]- found 356.1, purity 96%
Methyl-(E)-2-(4-((2-(3-(trifluoromethyl)phenyl)vinyl)sulfonamido)phenyl)acetate (13). Using
the same procedure as compound 7, 2-(4-aminophenyl)acetate (46 mg, 0.28 mmol) and
compound 27 (50 mg, 0.18 mmol) yielded the desired product methyl (11 mg, 0.025 mmol,
15% yield) as a white powder. The crude product was purified by silica flash column
chromatography (column gradient of 30% to 60% EtOAc: Pet. ether 40-60 oC). 1H NMR (400
MHz, Chloroform-d) δ 7.65 (d, J = 6.6 Hz, 2H), 7.59 (d, J = 7.9 Hz, 1H), 7.54 – 7.46 (m, 2H),
7.23 (d, J = 8.5 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 15.4 Hz, 1H), 6.73 (s, 1H), 3.67
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(s, 3H), 3.58 (s, 2H); HRMS (ESI) calcd. for [C18H16NO4F3S+H]+: 400.0830, Found: 400.0821
LCMS: [M-H]- found 398.1, purity >99%.
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tert-butyl-(E)-(4-((2-(3-(trifluoromethyl)phenyl)vinyl)sulfonamido)benzyl)carbamate
(28).
Using the same procedure as compound 7, 4-[(N-Boc)aminomethyl]aniline (82 mg, 0.369
mmol) and compound 27 (100 mg, 0.369 mmol) yielded the Boc intermediate (138 mg, 0.302
mmol, 82% yield) as white crystals. The crude product was purified by silica flash column
chromatography (column gradient of 30% to 60% EtOAc : Pet. ether 40-60 °C). 1H NMR (400
MHz, Chloroform-d) δ 7.65 (d, J = 6.3 Hz, 2H), 7.60 (d, J = 7.8 Hz, 1H), 7.56 – 7.43 (m, 2H),
7.23 (d, J = 8.3 Hz, 2H), 7.19 – 7.12 (m, 2H), 6.86 (d, J = 15.5 Hz, 1H), 6.63 (s, 1H), 4.83 (s,
1H), 4.26 (d, J = 5.7 Hz, 2H), 1.44 (s, 9H); LCMS [M-H]- found 455.1; HRMS (ESI) calcd. for
[C21H23N2O4F3S+Na]+: 479.1223, Found: 479.1232.
(E)-N-(4-(aminomethyl)phenyl)-2-(3-(trifluoromethyl)phenyl) ethene-1-sulfonamide (14). To a
solution of the Boc intermediate 28 in dichloromethane (2 mL), trifluoroacetic acid (1 mL) was
added dropwise and the mixture stirred for 0.5 hours. The reaction was quenched with
NaHCO3, the organic phase isolated and the final product concentrated under reduced pressure
to yield the desired product (84 mg, 0.24 mmol, 78% yield) as small yellow crystals. 1H NMR
(500 MHz, DMSO-d6) δ 8.13 (s, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.64
– 7.56 (m, 2H), 7.50 (d, J = 15.5 Hz, 1H), 7.36 – 7.30 (m, 2H), 7.23 – 7.13 (m, 2H), 3.88 (s,
2H), 2.33 (s, 1H); HRMS (ESI) calcd. for [C16H13F3NO2S + H]+: 340.0614, Found: 340.0600;
LCMS: [M-H]- found 355.1, purity >99%.
(E)-N-(4-((methylamino)methyl)phenyl)-2-(3-(trifluoromethyl)phenyl)ethene-1-sulfonamide
(15). Using the same procedure as compound 6, 4-((methylamino)methyl)aniline (25 mg, 0.18
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mmol) and compound 27 (50 mg, 0.18 mmol) yielded the desired product (36 mg, 0.10 mmol,
53% yield) as yellow crystals. The crude product was purified by silica flash column
chromatography (column gradient of 30% to 60% EtOAc : Pet. ether 40-60 oC). 1H NMR (400
MHz, DMSO-d6) δ 8.04 (d, J = 7.7 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.73 – 7.54 (m, 3H), 7.33
– 7.12 (m, 1H), 7.00 (d, J = 8.3 Hz, 2H), 6.55 (d, J = 8.3 Hz, 2H), 5.25(s, 1H), 4.05 (s, 2H),
2.60 (s, 3H); HRMS (ESI) calcd. for [C17H17F3N2O2S+H]+: 371.1041, Found: 371.1031;
LCMS: [M+H]+ found 370.9, purity 87%.
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(E)-N-(4-((2-(3-(trifluoromethyl)phenyl)vinyl)sulfonamido)benzyl)acetamide (16). Using the
same procedure as compound 6, N-(4-aminobenzyl)acetamide (30 mg, 0.18 mmol) and
compound 27 (50 mg, 0.18 mmol) yielded the desired product (20 mg, 0.050 mmol, 27% yield)
as a colourless oil. The crude product was purified by silica flash column chromatography
(column gradient of 30% to 60% EtOAc : Pet. ether 40-60 oC). 1H NMR (500 MHz, DMSO-
d6) δ 10.08 (s, 1H), 8.24 (t, J = 5.9 Hz, 1H), 8.15 (s, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.76 (d, J =
7.8 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.56 (d, J = 15.5 Hz, 1H), 7.50 (d, J = 15.5 Hz, 1H), 7.14
(t, J = 2.6 Hz, 3H), 4.15 (d, J = 5.9 Hz, 2H), 1.82 (s, 3H); HRMS (ESI) calcd. for
[C18H17F3N2O3S+H]+: 399.0990, Found: 399.1003 LCMS: [M-H]- found 397.1, purity >99%.
(E)-4-((2-(3-(trifluoromethyl)phenyl)vinyl)sulfonamido) benzoic acid (17). Compound 9 (258
mg, 0.67 mmol) was stirred overnight in 10% LiOH solution in methanol:water (1:1, 4 mL).
Methanol was removed under reduced pressure then cold water added to the mixture. 1N
hydrochloric acid was used to precipitate the product, which was filtered and dried to give the
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desired product (174 mg, 0.47 mmol, 70% yield) as a yellow powder. H NMR (400 MHz,
MeOD) δ 7.90 (t, J = 1.9 Hz, 1H), 7.85 (s, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.74 (dt, J = 7.6, 1.4
Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.53 (d, J = 15.4 Hz, 1H), 7.46 (ddd,
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J = 8.1, 2.3, 1.2 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 15.4 Hz, 1H); HRMS (ESI) calcd.
for [C16H12F3NO4S+H]+: 372.0517, Found: 372.0502 LCMS: [M-H]- found 370.0, purity
>99%.
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(E)-4-((2-(3-(trifluoromethyl)phenyl)vinyl)sulfonamido)benzamide (18). To a solution of
compound 17 (145 mg, 0.39 mmol), HOBt hydrate (66 mg, 0.43 mmol) and EDCl (82 mg, 0.43
mmol) in THF (3 mL), N,N diisopropylethylamine (101 mg, 0.78 mmol) was added and the
mixture stirred for 10 mins at room temperature. 2M ammonia in methanol (0.195 mL, 0.39
mmol) was added and the reaction was stirred overnight. NaHCO3:H2O solution (1:1, 3 mL)
was added, the reaction stirred for 2 hours. The precipitate was filtered to yield the desired
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product 18 (149 mg, 0.389 mmol, 100% yield) as a cream coloured powder. H NMR (400
MHz, MeOD) δ 7.86 (s, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.73 (t, J = 2.0 Hz, 1H), 7.69 (d, J = 7.9
Hz, 1H), 7.62 – 7.53 (m, 2H), 7.52 (s, 1H), 7.44 – 7.39 (m, 1H), 7.37 (d, J = 7.9 Hz, 1H), 7.22
(d, J = 15.5 Hz, 1H), 2.40 (s, 1H); HRMS (ESI) calcd. For [C16H13F3N2O3S+H]+: 371.0677,
Found: 371.0681; LCMS: [M-H]- found 369.0, purity 95%.
Tert-butyl (E)-(3-((2-(3 (trifluoromethyl)phenyl)vinyl)sulfonamido)benzyl)carbamate (29). To
a solution of compound 27 (50 mg, 0.55 mmol) in pyridine (5 mL), tert-butyl(3-
aminobenzyl)carbamate (123 mg, 0.55 mmol) was added and the reaction mixture stirred under
a nitrogen atmosphere at room temperature for 1 hr. The crude product was concentrated under
reduced pressure and purified by silica flash column chromatography (column gradient of 30%
to 60% EtOAc : Pet. ether 40-60 oC) to yield the desired product 29 (175 mg, 0.38 mmol, 69%
yield) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.68 – 7.56 (m, 3H),
7.57 – 7.46 (m, 2H), 7.31 – 7.22 (m, 1H), 7.17 (d, J = 9.0 Hz, 2H), 7.05 (d, J = 7.5 Hz, 1H),
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