The Fragment-Based Development of a Benzofuran Hit as a New Class of Escherichia coli DsbA Inhibitors

Article abstract of DOI:10.3390/molecules24203756

A fragment-based drug discovery approach was taken to target the thiol-disulfide oxidoreductase enzyme DsbA from Escherichia coli (EcDsbA). This enzyme is critical for the correct folding of virulence factors in many pathogenic Gram-negative bacteria, and small molecule inhibitors can potentially be developed as anti-virulence compounds. Biophysical screening of a library of fragments identified several classes of fragments with affinity to EcDsbA. One hit with high mM affinity, 2-(6-bromobenzofuran-3-yl)acetic acid (6), was chemically elaborated at several positions around the scaffold. X-ray crystal structures of the elaborated analogues showed binding in the hydrophobic binding groove adjacent to the catalytic disulfide bond of EcDsbA. Binding affinity was calculated based on NMR studies and compounds 25 and 28 were identified as the highest affinity binders with dissociation constants (K_D) of 326 ± 25 and 341 ± 57 μM respectively. This work suggests the potential to develop benzofuran fragments into a novel class of EcDsbA inhibitors.

Full text of DOI:10.3390/molecules24203756

molecules
Article
The Fragment-Based Development of a Benzofuran
Hit as a New Class of Escherichia coli DsbA Inhibitors
Luke F. Duncan 1,† , Geqing Wang 2,† , Olga V. Ilyichova 3 , Martin J. Scanlon 3,4
,
Begoña Heras 2 and Belinda M. Abbott 1,
*
1
Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University,
Melbourne 3086, Australia; dunclf01@gmail.com
2
3
4
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University,
Melbourne 3086, Australia; G.Wang@latrobe.edu.au (G.W.); B.Heras@latrobe.edu.au (B.H.)
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade,
Parkville 3052, Australia; olga.illyichova@monash.edu (O.V.I.); martin.scanlon@monash.edu (M.J.S.)
ARC Training Centre for Fragment Based Design, Monash Institute of Pharmaceutical Sciences,
Monash University, 381 Royal Parade, Parkville 3052, Australia
*
Correspondence: B.Abbott@latrobe.edu.au
†
These authors contributed equally to this paper.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Brian J. Stockman
Received: 9 September 2019; Accepted: 15 October 2019; Published: 18 October 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A fragment-based drug discovery approach was taken to target the thiol-disulfide
oxidoreductase enzyme DsbA from Escherichia coli (EcDsbA). This enzyme is critical for the correct
folding of virulence factors in many pathogenic Gram-negative bacteria, and small molecule inhibitors
can potentially be developed as anti-virulence compounds. Biophysical screening of a library of
fragments identified several classes of fragments with affinity to EcDsbA. One hit with high mM
affinity, 2-(6-bromobenzofuran-3-yl)acetic acid (6), was chemically elaborated at several positions
around the scaffold. X-ray crystal structures of the elaborated analogues showed binding in the
hydrophobic binding groove adjacent to the catalytic disulfide bond of EcDsbA. Binding affinity was
calculated based on NMR studies and compounds 25 and 28 were identified as the highest affinity
binders with dissociation constants (KD) of 326
the potential to develop benzofuran fragments into a novel class of EcDsbA inhibitors.
±
25 and 341
±
57 µM respectively. This work suggests
Keywords: fragment-based drug discovery; Escherichia coli; antibiotic resistance; DsbA inhibition;
benzofuran; disulfide bond; bacterial virulence
1. Introduction
The rise of multidrug-resistant bacteria has rendered many of our current antibiotics ineffective.
With few antibacterial drugs in the development pipeline, antibiotic resistance has been identified as a
significant global health threat, highlighting the urgent need for new classes of antibacterial drugs
with novel mechanisms of action. Bacteria rely on the biosynthesis of virulence factors to establish
an infection in a host and cause disease. Many of these virulence factors, such as secreted toxins and
surface proteins, require folding into their native state by the thiol-disulfide oxidoreductase enzyme
DsbA. It has been shown that DsbA null mutant bacteria show pleiotropic phenotypes with defects
in secretion and motility [
to attenuated virulence, increased sensitivity to antibiotics, and reduced fitness in animal models
of infection [ ]. Chemical inhibition of DsbA thus represents an attractive approach to attenuate
bacterial virulence and could potentially lead to the development of novel antibacterial therapeutics [ ].
The highly oxidising protein DsbA catalyses disulfide bond formation between two cysteine
residues of unfolded substrates in the periplasm of Gram-negative bacteria [ ]. The structure of
1]. The loss of functional DsbA in Gram-negative bacteria generally leads
2–6
7
3,8
Molecules 2019, 24, 3756; doi:10.3390/molecules24203756
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3756
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DsbA is comprised of a thioredoxin domain with an inserted helical domain [9–11]. Like many other
thiol-disulfide oxidoreductase enzymes, the active site of DsbA is comprised of the characteristic CXXC
motif (Cys30–Pro31–His32–Cys33 in Escherichia coli DsbA) and a highly conserved cis-proline in an
adjacent loop which has been shown to play a key role in the binding of substrates (Figure 1A) [12].
Disulfide exchange between oxidised DsbA and its unfolded substrates leaves DsbA in its reduced
inactive form which is in turn oxidised by its redox partner DsbB to reprime the catalytic cycle. Adjacent
to the active site is a hydrophobic groove which serves as a site for both DsbB and unfolded substrates
to form a complex (Figure 1A).
Figure 1. Structural characterisation of benzofuran analogues binding to DsbA. (A) Structure of apo
Escherichia coli DsbA. Left panel: Crystal structure of E. coli DsbA (PDB ID: 1FVK) is presented as
a cartoon. The active site is shown in cyan. The disulfide bond is shown in yellow sticks. Inlet:
A close-up view of the active site Cis30-Pro31-His32-Cis33 motif and cis-Pro loop (Val150-cis-Pro151).
Right panel: Surface representation of E.coli DsbA in the same orientation. The hydrophobic groove
of DsbA is highlighted by a dashed line. The active site is shown in cyan. (B) Superposition of the
co-crystal structures of the 15 (pink)–E. coli DsbA (EcDsbA) (grey) complex and the 16 (green)-EcDsbA
(light blue) complex. Left panel: The residues within 4 Å of the ligands are shown as sticks and labelled
individually. Right panel: Surface representation of the EcDsbA complexes. The active site is shown
in cyan. (
(purple)-EcDsbA (grey) complexes. The surface of 23-complexed EcDsbA is shown in white. The active
site is shown in cyan. ( ) Superposition of the co-crystal structures of 25 (salmon)-EcDsbA (salmon),
C) Superposition of the co-crystal structures of 21 (orange)-EcDsbA (light orange) and 23
D
28 (blue)-EcDsbA (purple), and 26 (yellow)-EcDsbA (grey) complexes. The surface of 26-complexed
EcDsbA is shown in white. The active site is shown in cyan.

Molecules 2019, 24, 3756
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Previous work on the development of DsbA inhibitors has included small peptide mimetics of
a natural substrate and more recently small molecule inhibitors. Martin and co-workers reported
a heptapeptide inhibitor of DsbA of Escherichia coli (EcDsbA) that was an analogue of the EcDsbB
periplasmic loop [13]. The heptapeptide bound to EcDsbA (KD = 2.0
±
0.3 µM) and inhibited its
activity (IC50 = 5.1 1.1 M). The crystal structure showed that the peptide occupies the hydrophobic
±
µ
groove and forms a covalent disulfide bond with Cys30 of DsbA. Although peptides are difficult to
be developed into therapeutics due to their limitations, i.e., poor stability, membrane permeability,
and oral availability, this study demonstrates that the hydrophobic groove is a promising target site to
block DsbA function.
The development of small molecule inhibitors against the DsbA-DsbB system has been actively
pursued [14–16]. In addition to development of DsbB inhibitors, a fragment-based approach was taken
by Adams and co-workers in which a library of 1132 fragments, sourced from the Maybridge Ro3
collection, was screened against oxidised EcDsbA [17]. This approach identified several structural
classes of fragments with weak affinity to EcDsbA. A phenylthiazole fragment was synthetically
elaborated into a higher affinity compound with mid-µM inhibition, providing the first example of
small molecule inhibitors of EcDsbA. Crystal structures of EcDsbA in complex with the inhibitors
indicated binding to the hydrophobic groove adjacent to the active site [17]. Herein, the chemical
elaboration of a benzofuran fragment identified by the screening technique already described is
explored as an inhibitor of EcDsbA.
2. Results and Discussion
2.1. Chemical Synthesis and X-Ray Crystallography
We previously reported the fragment screening campaign against EcDsbA and identified 37
fragment hits which are clustered into eight different chemical classes [17]. In this work, we focus on
elaboration of benzofuran fragment hits identified in the screen using a structure-guided approach.
Assessing the identified hit fragment (6), the 6-bromo substituent was identified as an obvious site for
elaboration. This site was amenable to coupling reactions to extend the fragment from the benzene ring
of the benzofuran moiety and introduce a range of functionalities to probe for favourable interactions
with residues in the EcDsbA binding site. Hydroxy analogues of the hit fragment were also synthesised
to broaden the chemistry that could be applied for elaboration. The benzofuran precursors were
prepared in two steps through Pechmann condensation between a substituted phenol and ethyl
4-chloroacetoacetate followed by Perkin rearrangement into the corresponding benzofuran by heating
in sodium hydroxide (Scheme 1).
OH
O
O
O
O
(a)
(b)
(c)
R
R
R
R
COOH
COOMe
Cl
1
R = 7-Br
R = 6-Br
R = 7-I
R = 7-OH 66%
R = 6-OH 3%
26%
26%
38%
6
7
8
9
R = 6-Br 99%
R = 5-Br 86%
11 R = 6-Br 96%
12 R = 6-I 60 %
2
3
4
5
R = 6-I
94%
R = 6-OH 72%
10 R = 5-OH 89%
Scheme 1. Synthesis of key benzofurans 6–12 for elaboration. Reagents and conditions: (a) Ethyl
4-chloroacetoacetate, H2SO4 or MsOH, 0 ◦C, 2 h, RT, and 24 h; (b) 2 M NaOH, 80 ◦C, and 16 h;
(c) CH3OH, SOCl2, reflux, and 16 h.
The withdrawing effect of the bromine substituents resulted in poor yields of 26% for coumarins
1
and
2
, while a less electron withdrawing halogen substituent resulted in a modestly improved yield
. A higher yield was achieved for 7-hydroxycoumarin ; however, the same
was not observed for 6-hydroxycoumarin . Coumarins were rearranged to their corresponding
for the 7-iodo analogue
3
4
5
1–5

Molecules 2019, 24, 3756
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benzofurans in high to excellent yields giving bromo and hydroxy functionality at both the 5-position
and bromo, iodo, and hydroxy functionality at the 6-position. Benzofurans 10 were then used as
6
–
precursors for fragment elaboration. The carboxylic acid group was protected as the methyl ester for
the reactions where acid sensitivity was of concern.
The first series of analogues were synthesised via Buchwald–Hartwig coupling to introduce
amine functionality at C-6 through the palladium catalysed coupling of amines with the 6-bromo
starting material 11 (Scheme 2A). The methyl ester was chosen over the carboxylic acid
6 to avoid
any interactions with the basic amine coupling partners and to avoid the complication of isolating
a zwitterionic coupled product from a potentially complicated crude mixture. The aniline 13 and
p-anisidine 14 analogues were isolated in fair yields (68% and 55% respectively) and subsequent
hydrolysis of the methyl ester gave 15 and 16 as the carboxylic acids in excellent yields. The alkyl
amines of n-butylamine and i-propylamine failed to couple under the same conditions.
Scheme 2.
hydrolysis. (
formation at C-6 via Ullmann-type coupling. (
(
A
B
) Aryl amine formation at C-6 via Buchwald–Hartwig coupling and subsequent ester
) Alkyl ether formation at the 6-position via Williamson ether synthesis. ( ) Aryl ether
) Benzyl formation at C-6 via Negishi coupling and
C
D
subsequent hydrolysis. Reagents and conditions: (a) Aryl amine, K2CO3, Pd2(dba)3, XPhos, t-BuOH,
reflux, 16 h; (b) 2 M NaOH, 80 ◦C, 16 h; (c) alkyl bromide, K2CO3, CH3CN, reflux, 16 h; (d) substituted
phenol, CuI, picolinic acid, K3PO4, CH3SOCH3, 90 ◦C, 24 h. ( ) (1) Zn, LiCl, BnBr, THF, 0 ◦C, 1.5 h;
e
(2) 11, Pd(OAc)2, XPhos, THF, 50 ◦C, 18 h.
The synthesised analogues were soaked into crystals of EcDsbA and X-ray diffraction data was
collected for each analogue in order to determine their site of binding. Crystal structures of aniline
15 and anisidine 16 revealed that both ligands were bound in the hydrophobic end of the binding
groove adjacent to the catalytic disulfide bond (Figure 1B, the 2Fo-Fc electron density map and omit
map for compounds are shown in Supplementary Figure S1). Elucidation of the crystal structure of
EcDsbA in complex with aniline 15 (Figure 1B) showed a
π-stacking interaction between the benzofuran
core and His32 of the active site CPHC motif. The carboxy moiety of 15 was oriented towards the

Molecules 2019, 24, 3756
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left-hand side of the binding groove, where more polar residues are located. The phenyl ring of the
aniline moiety occupied the hydrophobic end of the binding pocket with the nitrogen atom of the
amine linker positioned in close proximity to Gln35. The side chain of Gln35 is highly flexible and
adopts two conformations in the co-structure, suggesting that 15 does not stabilize the Gln35 side
chain through the hydrogen bond. In general, the interaction of 15 with the hydrophobic groove
residues His32, Phe36, Leu40, Pro163, Gln164, Thr168, Phe174, and Met171 strongly resembles the
previously elaborated phenylthiazole series [17], suggesting a similar mode of binding for these two
chemical series. The co-structure of 16 showed the p-methoxy group of the anisidine moiety resulted
in a shift of the benzofuran core further down into the more hydrophilic region of the binding groove.
Although anisidine 16 was occupying a larger region of the EcDsbA binding groove, the shift appeared
to have interrupted the π-stacking interactions the benzofuran was making with the His32 side chain
previously (Figure 1B and Figure S1).
Alkyl ethers were next formed at the 6-position coupling alkyl bromides to the
6-hydroxybenzofuran 9 via Williamson ether synthesis (Scheme 2B). These conditions also esterified
the carboxylic acid moiety which required subsequent base hydrolysis. This coupling resulted in
the n-butyl 21, i-butyl 22, and benzyl 23 ethers which were soaked into crystals of EcDsbA for X-ray
diffraction data collection. Analysis of the electron density maps revealed that the 21 and 23 bound
in the hydrophobic binding groove of EcDsbA (Figure 1C, the 2Fo-Fc electron density map, and the
omit maps for these compounds are shown in Supplementary Figure S1). Common among each of
the ethers was a π-stacking interaction between the benzofuran moiety and His32. Interestingly, the
binding poses of alkyl ether 21 contrasted with amines 15 and 16, in that the alkyl ether moiety was
facing into the more hydrophilic region of the binding groove with the carboxylic acid located in the
hydrophobic end of the groove. The benzyl ether 23 displayed a similar orientation to that observed in
previous aromatic derivatives with the benzyl ether orienting towards the hydrophobic end of the
binding groove. The conformation of alkyl ether 21 was not considered ideal as the alkyl chain of the
ether did not appear to be making any favourable interactions with the residues in the binding site.
For this reason, aromatic groups at the 6-position were preferred to maintain the binding pose.
To test the effect of the site of elaboration, the 5-benzyl ether analogue was synthesised using
5-hydroxybenzofuran 10 (Scheme 2B). The reaction was refluxed in acetone in an attempt to achieve
milder conditions to avoid any thermal decomposition that may have been occurring previously;
however, the lower resulting yield suggested that more forcing conditions were required. Hydrolysis
of the benzyl ester gave benzofuran 24, which, after soaking into EcDsbA crystals, did not show clear
enough electron density to unambiguously determine the binding pose.
Synthesis of diaryl ether 25 was initially attempted using 6-bromobenzofuran 6 under Ullmann
coupling conditions but only moderate conversion to the desired product was observed by HPLC
after 48 h at 90 ◦C. Far superior conversion was observed using the more reactive iodobenzofuran
8
, resulting in the cross-coupled diaryl ethers 25 and 26 (Scheme 2C). Unfortunately, both products
were very difficult to isolate from the crude reaction mixture, requiring normal phase chromatography
and recrystallisation followed by reverse-phase high performance liquid chromatography. Both diaryl
ethers 23 and 25, were soaked into crystals of EcDsbA and co-crystals were diffracted to 2.0 and 1.77 Å
of resolution, respectively. Phenyl ether 25 bound to the EcDsbA groove neighbouring the active site
(Figures 1D and 2A) showing a binding pose to the benzyl ether 23 (Figure 1C) and the direct anilino
analogue 15 (Figure 1B).

Molecules 2019, 24, 3756
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Figure 2. Biophysical characterisation of compound 25. (
with 25. Simulated annealing omit σA-weighted mFo-DFc electron density map for 25 is contoured
at 2.5 and shown as blue mesh. The active site is shown in cyan. The residues whose chemical
shifts are strongly perturbed by 25 at 1 mM (chemical shift perturbations > 0.04 ppm) are coloured in
yellow and labelled. ( ) LIGPLOT analysis of the interaction between DsbA and 25. ( ) Binding of
A) Crystal structure of EcDsbA in complex
σ
B
C
25 to EcDsbA was assessed by ligand-detected NMR spectroscopy. Comparison of the 1D 1H NMR
spectrum of 25 (green) and the saturation transfer difference (STD) spectrum of 25 + DsbA (blue) shows
magnetization transfer from DsbA to the aromatic resonances of 25. waterLOGSY NMR spectrum
shows that the aromatic resonances of 25 become less negative in the presence of DsbA (purple)
compared to 25 alone (green). The CPMG NMR spectrum (
aromatic resonances in the presence of DsbA (orange) compared to 25 alone (yellow). (
the 1H-15N HSQC spectra of 15N-labelled EcDsbA showing chemical shift perturbation (CSP) upon
titration of increasing concentrations of 25 (0, 0.0625, 0.125, 0.25, 0.5, and 1 mM). The direction of the
τ
= 350 ms) of 25 shows attenuation of its
) A portion of
D
CSP is indicated by black arrows. (E) The equilibrium dissociation constant (KD) of 25 is determined by
measurement of chemical shift perturbation (∆δ) in the 1H-15N-HSQC spectra of EcDsbA as a function
of compound concentration.

Molecules 2019, 24, 3756
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The co-structure was more comparable with the direct anilino analogue 15. The phenoxy group
of 25 occupied the hydrophobic end of the binding groove with the oxygen atom of the ether linker
at a distance of 4.1 Å from Gln35. The side chain of Gln35 is conformationally flexible in the crystal
structure and therefore unlikely to form a hydrogen bond with 25 (Figure 1D). The carboxyl group of
25 is positioned more closely to the imidazole side chain of His32 (N–O distance = 3.7Å) compared to
that of 15 (N–O distance = 4.5Å) and possibly formed a weak polar interaction.
As the crystal structure of the p-anisidine analogue 16 showed that the installation of the p-methoxy
group induced a shift of the benzofuran core away from His32 (relative to anilino 15), the m-methoxy
substituted phenyl ether 26 was synthesised to observe whether the shift could be avoided. The
co-structure (Figure 1D) showed a horizontal rotation relative to the unsubstituted phenoxy 25
(comparable to 16) but notably a less pronounced shift into the left-hand side of the binding groove,
meaning the π-stacking interaction between the benzofuran core and His32 could be better retained.
The m-methoxy group of 26 may potentially form a polar interaction with side chain of Gln35, although
electron density for this compound was insufficient to unambiguously define the interaction (Figure S1).
To investigate the importance of a hydrogen bond donor/acceptor as a linker, benzyl analogue 28
was synthesised featuring a methylene linker. Benzylzinc bromide was prepared from benzyl bromide
and zinc powder [18], then coupled to the 6-bromobenzofuran 11 via Negishi coupling to afford the
product 27 (Scheme 2D). The methyl ester was hydrolysed to give the benzyl analogue 28 as the desired
carboxylic acid. The co-structure of 28 bound to EcDsbA was determined using X-ray crystallography
which showed that the methylene linker had little to no effect on the binding conformation relative to
the anilino 15 and phenoxy 25 analogues (Figure 1D).
With a series of derivatives prepared by substitution at C-6, attention was turned to the carboxy
moiety which until now had been maintained for aqueous solubility. A tetrazole isostere was
synthesised in three steps via an amide and nitrile intermediate (Scheme 3). The acid chloride of
benzofuran
6 was formed in situ then exposed to ammonia gas that formed the primary amide 29.
Refluxing of 29 in toluene with thionyl chloride dehydrated the amide to the nitrile 30 which then
underwent a 2,3-addition with sodium azide to form the tetrazole 31. Unfortunately, the tetrazole did
not produce electron density upon analysis by X-ray crystallography.
Br
Br
Br
Br
O
O
O
O
(a)
(b)
(c)
H
N
N
COOH
CONH2
CN
N
N
6
29
90%
31
75%
30
66%
Scheme 3. Synthesis of tetrazole 4.27 as a carboxy isostere. Reagents and conditions: (a) (1) PCl5,
CH2Cl2, reflux, 1 h; (2) NH3, 5 m. (b) SOCl2, toluene, reflux, 1.5 h. (c) NaN3, Et3N
3 days.
·
HCl, toluene, reflux,
Indole analogues were prepared next as a bioisostere of the benzofuran, where the hydrogen
bond acceptor of the benzofuran was replaced by a secondary amine (Scheme 4). The amino group
of the indole not only had the potential to form additional interactions with residues in the binding
groove but also offered a new moiety from which to probe a different region of the binding groove
using N-alkylation. Indole synthesis was undertaken based on the method of Suárez-Castillo and
co-workers who previously regioselectively brominated the 6-position of methyl-3-indole acetate after
installing electron withdrawing methyl carbonyl groups at N-1 and the alpha carbon adjacent to
C-3 [19]. Using commercially available ethyl 3-indole acetate as the starting material, methyl carbonyl
groups were installed using dimethyl carbonate and sodium hydride (Scheme 4). These conditions
transesterified the ethyl ester affording 32 as the dimethyl malonate and this group, and in conjunction
with the N-carbamate group, directed substitution regioselectively to the 6-position in the subsequent
bromination step to afford 33. Decarbomethoxylation was achieved using potassium hydroxide

Molecules 2019, 24, 3756
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in methanol:water (9:1) to avoid use of toxic sodium cyanide, as described by Su
árez-Castillo and
co-workers [19], resulting in the production of the 6-bromo indole analogue 34, albeit at a lower yield.
One derivative of indole 34 was synthesised by coupling phenylboronic acid to 33 via Suzuki–Miyaura
coupling, which, following the decarbomethoxylation of 35, afforded the 6-phenyl indole compound
36. Unfortunately, neither 34 nor 36 appeared to bind in the EcDsbA binding groove as shown by the
lack of electron density corresponding to the compounds after X-ray diffraction analysis of the EcDsbA
soaked crystals.
Scheme 4. Synthesis of 6-bromo and 6-phenyl indole derivatives. Reagents and conditions: (a) NaH,
(CH3)2CO3, THF, reflux, 16 h. (b) (1) Br2, CCl4, room temperature, 24 h; (2) Na2SO3, H2O, room
temperature, 24 h. (c) KOH, CH3OH:H2O (9:1), reflux, 2 h.
CH3CN:H2O (4:1), 90 ◦C, 24 h.
(d) Ph(BOH)2, CsF, Pd(dppf)Cl2,
The co-structures at this point had shown the benzofurans bind to the hydrophobic end of the
binding groove and it was considered desirable to develop a benzofuran which could bind to the more
polar region of the groove. The next series of analogues focused on elaboration from the
α-carbon in an
attempt to probe into this region. The -carbon of 11 was brominated under Wohl–Ziegler conditions
α
to afford bromide 37 (Scheme 5), which in turn was displaced using sodium azide to give the azido
compound 38. Low temperatures were necessary during this reaction to avoid the formation of an
α
-keto-ester by-product. The azide was then hydrogenated over platinum oxide to produce amine 39.
Br
O
Br
Br
Br
O
O
O
a
b
c
Br
N3
NH2
MeOOC
11
MeOOC
37
MeOOC
38
MeOOC
39
Scheme 5. Amination of 11 via bromo and azido intermediates as an amide precursor. Reagents and
conditions: (a) NBS, AIBN, CCl4, reflux, 16 h; (b) NaN3, DMF, −40 ◦C, 1 h; (c) H2, PtO2, MeOH.
A series of amides/sulfonamides were synthesised by coupling amine 39 to the relevant acyl-
and sulfonyl chlorides (Table 1). Base hydrolysis of the resulting amides/sulfonamide methyl esters
afforded alkyl amide 46, aromatic amides 47 49, and sulfonamides 50 51 in modest to high yields.
–
–
This series was evaluated for binding and crystallography as racemic mixtures to gauge whether it
was worthwhile separating each enantiomer. The impact of the amide on the binding pose of the

Molecules 2019, 24, 3756
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benzofurans was evaluated by X-ray crystallography. After soaking the amide/sulfonamide series into
crystals of EcDsbA, only the (S)-enantiomer of sulfonamide 50 produced sufficient electron density in
the binding groove. In comparison with the binding pose of the 6-aryl series, the crystal co-structure of
50 showed a 180◦ horizontal rotation of the benzofuran core and a shift towards the hydrophobic end
of the binding groove, resulting in a close proximity of the sulfonamide moiety to His32 (Figure S2). It
was not clear if this binding pose was typical for the amides/sulfonamide series, as 50 was the only
analogue to produce a crystal structure.
Table 1. Amide/sulfonamide formation and subsequent ester hydrolysis.
Entry
R
Amide Coupling Product (Yield)
Hydrolysis c (Yield)
1
40 a (58%)
46 (98%)
41 b (55%)
42 b (52%)
2
3
4
5
6
47 (96%)
48 (58%)
49 (50%)
50 (68%)
51 (92%)
43 b (88%)
44 a (60%)
45 a (15%)
a Reagents and conditions: amine, acid/sulfonyl chloride, Et3N, Et2O. b Reagents and conditions: (1) acid, PCl5, DCM,
c
reflux, 30 min; (2) amine, Et3N, DCM, 2–5 h. Reagents and conditions: 2 M methanolic NaOH, DCM:MeOH (9:1).
2.2. Binding Analysis by NMR Spectroscopy
To validate the binding of benzofuran analogues, three ligand-detected NMR methods,
saturation-transfer difference (STD) [20], Carr–Purcell–Meiboom–Gill (CPMG) [21], and water–ligand
observed via gradient spectroscopy (waterLOGSY) [22], were employed to detect protein-ligand
binding between the benzofurans and EcDsbA (Figure 2C). These experiments were designed as
a primary screening tool to select analogues to undergo further affinity evaluation and prioritise
compounds for crystal soaking. It has previously been reported that compounds which passed more
ligand-detected NMR experiments had a higher chance of obtaining the co-crystal structures [23]. In a
good agreement with this finding, eight out of nine benzofurans that passed all three ligand-detected
experiments have their co-crystal structures successfully solved; in contrast, five compounds that failed
two ligand-detected experiments only have one co-structure solved (Table 2).

Molecules 2019, 24, 3756
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Table 2. Summary of NMR binding data.
Crystal
Structure in
EcDsbA
Ligand Efficiency
Dose Response
HSQC KD (µM)
ID
Structure
wLOGSY
CPMG
STD
(kcal mol−1
HAC−1
)
2
No
Yes
Pass
Pass
Fail
Pass
Pass
-
-
-
15
Pass
-
16
21
22
Yes
Yes
Yes
Pass
Pass
Fail
Pass
Pass
Pass
Fail
Pass
Fail
2700 ± 920
1970 ± 640
-
0.16
0.20
-
23
25
Yes
Yes
Pass
Pass
Pass
Pass
Pass
Pass
820 ± 140
326 ± 25
0.20
0.24
26
28
No
Yes
Pass
Pass
Pass
Pass
Pass
Pass
639 ± 87
341 ± 47
0.20
0.24
31
36
46
47
48
49
50
No
No
No
No
No
No
Yes
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Fail
Fail
Fail
Fail
Pass
Pass
Pass
Fail
Fail
Fail
Fail
Pass
-
-
-
-
Br
O
O
-
-
N
H
HOOC
Br
O
O
-
1350 ± 420
>>1000
-
-
N
H
HOOC
OMe
Br
O
O
0.15
N
H
HOOC
Br
O
O
-
-
F
N
H
HOOC
Br
O
O
O
S
N
H
HOOC
Compounds that passed at least one ligand-detected NMR experiment were further validated
by 2D 1H-15N heteronuclear single quantum coherence (HSQC) spectroscopy. In HSQC experiments,
chemical shift perturbations (CSP) of backbone amide resonances of 15N-labelled EcDsbA were
measured upon addition of compound at 1 mM. If a compound caused CSP > 0.04 ppm for residues
present in the hydrophobic groove of DsbA (the threshold was set based on previous experiments) [17],
K
D could be determined by measuring the CSP of those residues induced by different concentrations of
compounds (Figure 2D,E). As shown in Table 2, the binding of all compounds was validated by at least
one ligand-detected NMR experiment. In accordance with the crystal structure, the majority of the
residues perturbed by 1 mM of benzofuran analogues in single-point HSQCs are located around the

Molecules 2019, 24, 3756
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hydrophobic groove which is an important binding site for DsbA substrates (Figure 2A). Although the
co-structures showed -stacking of the benzofuran analogues with His32, this residue is not assigned in
π
the apo-HSQC spectrum of EcDsbA, and therefore, CSP could not be measured for this residue. Eight
compounds produced large enough CSP (>0.04 ppm) to enable HSQC titration and KD determination.
KD values of benzofuran analogues ranged from 326 to 2000
fragment hit was not possible as the weak affinity did not allow for accurate determination of its KD.
The ligand efficiency (LE) ranged from 0.15 to 0.24 kcal mol−1 HAC−1. The strongest binding affinities
were observed for 6-phenoxy 25 and 6-benzyl 28 with KD values of 326 25 M and 341 47 M,
µM (Table 2). A comparison to
6
±
µ
±
µ
respectively. Biophysical characterisation of 25 is illustrated in Figure 2. The similar binding affinities
observed for 25 and 28 suggested that the oxygen atom of the ether linker does not contribute to the
interaction, which is consistent with what we observed in the co-crystal structures (Figures 1D and
2B). The addition of a m-methoxy 26 substituent on the 6-phenoxy group led to a 2-fold decrease in
binding affinity, while extending the phenoxy group to a benzyloxy group led to a 2.5-fold decrease.
This suggests that 26 may not engage any polar interactions with DsbA despite its close proximity
to the polar residues His32 and Gln35. A dramatic decrease was observed for alkyl ether 21 which
led to a six-fold decrease relative to 25. Alkyl substituents at C-6 flipped the compound orientation
within the hydrophobic groove and disrupt the polar interaction between the imidazole side chain of
His32 and carboxyl group of the compound present in the 25 co-structure (Figure 1C). Similarly, the
flipped orientation within the groove also makes 22 a weaker binder, although its binding to DsbA
was detected by CPMG spectroscopy, its CSPs are too small to determine KD in HSQC. Unfortunately,
aniline 15, which bound in a similar orientation to 25 and 28, did not produce interpretable data from
the HSQC titration due to poor solubility. Of the amine analogues, a KD was calculated only for
p-anisidine analogue 16 which showed a significantly weaker binding affinity. It was evident in the
co-crystal structure of p-anisidine 16 that the p-methoxy group had caused the benzofuran scaffold
to shift further along the hydrophobic groove, possibly disrupting the
π-stacking interaction with
His32 and polar interactions with Gln164 (Figure 1B). LE values showed 6-phenoxy 25 and 6-benzyl 28
were the most efficient binders. Adding functional groups (16 and 26) or extending the ring linker (23
increased the heavy atom count but did not contribute to binding affinity; therefore, leading to less
efficient binders.
)
3. Conclusions
A fragment-based drug discovery process was employed to identify weak binders of EcDsbA
and improve the binding affinity through elaboration of the fragment scaffold. A series of analogues
were synthesised derivatising the C-6 position to install ether, amino, and benzyl substituents. X-ray
crystallography showed the analogues were binding in the hydrophobic groove of EcDsbA, an
important site for binding of the cognate redox partner and unfolded substrates. Ligand-detected
NMR experiments confirmed the most promising binders, and after further evaluation by HSQC,
phenoxy 25 and benzyl 28 analogues were identified as the strongest binders with a KD of 326
±
25 and
341 47 M respectively. The results presented in this work suggest further opportunity to develop
±
µ
appropriately substituted benzofuran analogues into high affinity DsbA inhibitors.
4. Materials and Methods
4.1. General
All commercial materials were used as received without further purification, unless otherwise
specified. Purification of solvents and reagents, if required, was carried out by procedures described by
Chai and Armarego [24]. Moisture sensitive reactions were performed under an atmosphere of nitrogen
with all reactions carried out at room temperature, unless otherwise noted. Glassware was oven-dried
and cooled under nitrogen prior to use. Analytical thin layer chromatography (TLC) was performed
on Merck Kieselgel (Darmstadt, Germany) 60 F254 aluminium backed plates and visualised using a

Molecules 2019, 24, 3756
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254 nm UV lamp. Flash chromatography was performed on silica gel (Davisil® LC60Å 40–63 micron,
Columbia, SC, USA). Melting points were determined on a Reichert (Buffalo, NY, USA) ‘Thermopan’
microscope hot stage apparatus and values were corrected by a 12% increase after calibration against
known reference samples. Low-resolution electrospray ionisation (ESI) mass spectra were recorded
on a Bruker Daltronics Esquire 6000 Ion Trap mass spectrometer (Billerica, MA, USA) in methanol
or acetonitrile (0.1% formic acid for positive mode) at 300 ◦C, with a 40 eV cone voltage, and a scan
rate of 5500 m/z/s. High-resolution electrospray ionisation (ESI) mass spectrometry was carried out
using an Agilent Technologies (Santa Clara, CA, USA) Accurate Mass Q-TOF LC-MS 6530 using
Autosampler 1260 Infinity II in positive mode. The samples were analysed using a flow rate of
1 mL/min, a mass range of 100–1,000 m/z and a scan rate of 10,000 m/z/s. Analytical RP-HPLC was
performed on a Shimadzu (Kyoto, Japan) LC-20AB Prominence Liquid Chromatography system fitted
with a Phenomenex® Jupiter C18 300 Å column (250 mm
×
4.6 mm, 10 µm), using a buffered binary
system; solvent A: 0.1% trifluoroacetic acid; solvent B: acetonitrile. Gradient elution was performed
using a gradient of 90% solvent A to 90% solvent B over 20 min with a flow rate of 1 mL/min, monitored
at 254 nm. Semi-preparative RP-HPLC was performed using a Phenomenex Jupiter (Torrance, CA,
USA) C18 column with the same binary buffer system described for RP-HPLC over 60 min with a flow
rate of 2 mL/min, unless otherwise stated. The purity of biological tested compounds was >95% in all
cases, unless specified otherwise. Microwave assisted reactions were performed using a Milestone
StartSYNTH system. NMR spectra were recorded on a Bruker AV-400 and AV-500 spectrometer at
400.13 and 500.02 MHz for 13C respectively, and for 1H nuclei at 100.62 and 125.74 MHz respectively
(13C nuclei at 300 K). For 1H-NMR the residual CDCl3 peak (7.26 ppm), DMSO-d6 peak (2.50 ppm),
CD3OD peak (3.31 ppm), or acetone-d6 peak (2.05 ppm) were used as internal standards. Similarly,
13C-NMR spectra were referenced to the residual solvent; the central peak of the CDCl3 ‘triplet’
(77.0 ppm), DMSO-d6 ‘heptet’ (40.0 ppm), CD3OD ‘heptet’ (47.6), or acetone-d6 ‘singlet’ (206.4 ppm).
Chemical shifts were reported as
δ values in parts per million (ppm). The following abbreviations
have been used upon reporting spectral data: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
and br, broad.
4.2. Chemical Syntheses
General Procedure A: Base mediated ester hydrolysis. Esters were hydrolysed according
to the method of Theodorou and co-workers [25]. Esters were dissolved in a mixture of
methanol:dichloromethane (1:9) and treated with a 2 M methanolic solution of sodium hydroxide
(1–5 equiv) to give a final 0.1 M concentration of ester. The solution was stirred for 3–20 h at which
time a cloudy suspension had formed. The suspension was concentrated under reduced pressure
and the residue was dissolved in water. The solution was acidified with concentrated HCl to pH 1
then the carboxylic acids were extracted with dichloromethane or ethyl acetate ( 3). The combined
×
organic layers were dried over magnesium sulfate then concentrated under reduced pressure. Unless
otherwise stated, no purification was required.
General Procedure B: Preparation of amines via Buchwald–Hartwig coupling. t-Butyl alcohol
(25 mL/mmol) was degassed by bubbling nitrogen for 20 min. The aryl bromide, amine (1.2 equiv) and
potassium carbonate (2.3 equiv) were added and the reaction vessel was evacuated and refilled with
nitrogen. This was repeated twice more before addition of XPhos (10 mol%) and Pd2(dba)3 (5 mol%).
The reaction vessel was again purged thrice more before stirring at reflux under nitrogen for 16 h.
The reaction mixture was partitioned between water and dichloromethane. The aqueous phase was
extracted with dichloromethane (
The dichloromethane was removed under reduced pressure and the crude amine was purified by
flash chromatography.
×
3) then the combined organic phases were washed with brine ( 3).
×
General Procedure C: Preparation of ethers/esters via Williamson ether synthesis. Compound 9,
anhydrous potassium carbonate (3.3 equiv) and alkyl bromide (4.6 equiv) in dry acetone or acetonitrile
were refluxed under an atmosphere of nitrogen for 16 h. The reaction was cooled to room temperature

Molecules 2019, 24, 3756
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and concentrated to a residue which was partitioned between ethyl acetate and brine. The aqueous
phase was washed twice with ethyl acetate and the combined organic phases were dried over
magnesium sulfate. The solution was concentrated under reduced pressure to afford the product that
was purified, if necessary, by flash chromatography.
General Procedure D: Preparation of ethers via Ullmann-type coupling. Compound 9 or 10,
phenol (1.2 equiv), tri-potassium phosphate (3 equiv), copper(I) iodide (10 mol%), and picolinic acid
(20 mol%) were added to an oven-dried Schlenk tube charged with a stir bar. Dry dimethyl sulfoxide
was added via syringe under a positive pressure of nitrogen. The tube was purged with nitrogen ( 3),
×
then sealed and heated to 90 ◦C causing a colour change from green to brown. The reaction was stirred
until analytical RP-HPLC indicated no starting material (24 h), and then, was partitioned between
ethyl acetate and water. The aqueous phase was acidified with concentrated hydrochloric acid and was
extracted with ethyl acetate ( 3). The combined organic phases were dried over magnesium sulfate
×
then concentrated under reduced pressure to give the crude compound. Purification was carried out
by flash chromatography, semi-preparative RP-HPLC or recrystallization to afford the pure products.
General Procedure F: Preparation of amides via coupling of amines to acid/sulfonyl chlorides. A
solution of amine 39 and triethylamine (3 equiv) in dichloromethane was treated with acid chloride
(1.1 equiv) or sulfonyl chloride (1.1 equiv). The solution was refluxed under nitrogen until complete
consumption of starting material by TLC (5–16 h). The reaction was quenched with sodium hydrogen
carbonate then extracted with dichloromethane (×3). The organic phase was washed with brine (×3),
and then concentrated by rotary evaporation to yield the amide. Purification by flash chromatography
was undertaken if required.
General Procedure G: Preparation of amides via in-situ formation of acid/sulfonyl chloride and
subsequent coupling to amine. A solution of carboxylic acid (1–1.2 equiv) or sulfonic acid (1–1.2 equiv)
in dry dichloromethane (30 mL/mmol) was treated with phosphorous pentachloride (1.0–1.2 equiv)
and refluxed for 30 min under nitrogen. After cooling to room temperature, a solution of amine 39 and
triethylamine (3 equiv) in dry dichloromethane (6 mL/mmol) was added. The resulting solution was
refluxed until complete consumption of the amine as determined by TLC (2–5 h). The reaction was
quenched with sodium hydrogen carbonate then extracted with dichloromethane (
phase was washed with brine ( 3) then concentrated by rotary evaporation to yield the amide, which
was purified by flash chromatography if required.
×
3). The organic
×
7-Bromo-4-chloromethylcoumarin (
1
): 3-Bromophenol (5.00 g, 28.9 mmol) was added to ch◦illed 70%
sulfuric acid (30 mL) and the stirring solution was cooled in an ice bath to less than 5 C. Ethyl
4-chloroacetoacetate (3.90 mL) was added dropwise ensuring the internal temperature remained below
5 ◦C. The solution was stirred for two hours and was then warmed to room temperature and stirred
for 22 h. The resulting suspension was poured onto ice water (500 mL) and the off-white precipitate
was collected by vacuum filtration and recrystallised from ethanol:ethyl acetate (1:1) to afford the
compound in question as white needles (2.04 g, 26%); mp 242–244 ◦C. δH (500 MHz, CDCl3) 7.56 (1H, d,
J = 2.0 Hz, ArH), 7.54 (1H, d, J = 8.5 Hz, ArH), 7.47 (1H, dd, J = 7.0 Hz, 1.5, ArH), 6.58 (1H, s, CH),
4.64 (2H, s, CH2). δC (125 MHz, CDCl3) 159.4 (Cq), 154.1 (Cq), 149.0 (Cq), 127.9, 126.3 (Cq), 125.3, 120.7,
116.3, 116.1, 41.0 (CH2). LRMS (ESI): m/z 272.97 [M (79Br) + H]+, 274.96 [M (81Br) + H]+. HRMS (ESI):
294.9137 [M (79Br) + Na]+, 296.9117 [M (81Br) + H]+.
6-Bromo-4-chloromethylcoumarin (2): 4-Bromophenol (4.00 g, 23.1 mmol) was dissolved in
methanesulfonic acid (21 mL) and cooled in an ice bath to 2 ◦C. Ethyl 4-chloroacetoacetate (3.12 mL) was
added dropwise ensuring the temperature of the solution did not exceed 5 ◦C. The dark red solution
stirred at 0 ◦C for 2 h then at room temperature for one week. The reaction mixture was poured onto
ice then extracted with dichloromethane. The organic phase was washed with 1 M sodium hydroxide
( 2) to remove the unreacted phenol; then the organic phase was dried (MgSO4) and concentrated to
×
a light yellow semi-solid. Trituration with diethyl ether afforded the pure product as a light orange
solid (1.66 g, 26%); mp 164–166 ◦C. δH (400 MHz, CDCl3) 7.79 (1H, s, ArH), 7.66 (1H, d, J = 8.8 Hz,

Molecules 2019, 24, 3756
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ArH), 7.27 (1H, d, J = 8.8 Hz, ArH), 6.61 (1H, s, CH), 4.64 (2H, s, CH2). δC (100 MHz, CDCl3) 159.4 (Cq),
152.8 (Cq), 148.4 (Cq), 135.1, 126.8, 119.2, 118.9 (Cq), 117.3 (Cq), 116.9 (CH), 40.9 (CH2). LRMS (ESI):
m/z 276.9 [M (79Br) + H]+, 278.9 [M (81Br) + H]+. HRMS (ESI): 296.9128 [M (79Br) + Na]+, 296.9099
[M (81Br) + Na]+.
4-(Chloromethyl)-7-iodo-2H-chromen-2-one (
3
): 3-Iodophenol (1.00 g, 4.55 mmol) was dissolved in
L,
methanesulfonic acid (5 mL) and cooled in an ice bath to 15 ◦C. Ethyl 4-chloroacetoacetate (619
µ
4.55 mmol) was added drop-wise at a slow enough rate to keep the reaction mixture below 10 ◦C. The
reaction stirred for one hour before being removed from the ice bath. After 40 h of stirring at room
temperature the purple suspension was poured onto ice and the resulting precipitate was collected
by vacuum filtration. The crude off-white solid was recrystallised from ethanol:ethyl acetate (1:1) to
afford the compound in question as a white solid (554 mg, 38%); mp 215–217 ◦C. δH (400 MHz, CDCl3)
7.76 (1H, s, ArH), 7.67 (1H, d, J = 8.4 Hz, ArH), 7.37 (1H, d, J = 8.0 Hz, ArH), 6.59 (1H, s, CH), 4.63
(2H, s, CH2). δC (100 MHz, CDCl3) 159.3, 153.7, 149.1, 133.7, 126.6, 125.1, 116.8, 116.4, 97.9, 41.0. LRMS
(ESI): m/z 320.90 [M + H]+.
7-Hydroxy-4-chloromethylcoumarin (4): To a chilled solution of resorcinol (5.00 g, 45.4 mmol) in 70% H2SO4
(50 mL) was added, dropwise, ethyl 4-chloroacetoacetate (6.15 mL), while ensuring the temperature did
not exceed 5 ◦C. The chilled solution stirred for two hours, then 20 h at room temperature. The reaction
mixture was poured onto ice and the precipitate was collected by vacuum filtration. The light pink
semi-solid was triturated with diethyl ether then recrystallised from ethanol to give the compound
in question as a white solid (5.18 g, 57%); mp. 178–180 ◦C (lit. 185 ◦C) [26]. The mother liquor was
concentrated and purified by flash chromatography (20–40% ethyl acetate in hexanes) to afford the
compound in question as a white solid (888 mg, 9%). δH (500 MHz, DMSO-d6) 10.63 (1H, bs, s, OH),
7.67 (1H, d, J = 8.5 Hz, ArH), 6.83 (1H, d, J = 8.5 Hz, ArH), 6.74 (1H, s, ArH), 6.41 (1H, s, ArH), 4.94
(1H, s, CH2). δC (100 MHz, DMSO-d6) 161.9, 160.6, 155.8, 151.5, 127.0, 113.6, 111.5, 109.8, 103.0, 41.8.
6-Hydroxy-4-chloromethylcoumarin (5): Hydroquinone (1.60 g, 14.7 mmol) was dissolved in 73% sulfuric
acid (10 mL) by heating. The solution was cooled to 4 ◦C in an ice bath and ethyl 4-chloroacetoacetate
(1.99 mL) was added dropwise ensuring the internal temperature remained below 5 ◦C. The dark red
solution stirred in an ice bath for one hour then at room temperature for three days. The reaction was
poured onto ice and the resulting light brown solid was collected by vacuum filtration. Recrystallisation
from 1,4-dioxane afforded the compound in question as a light brown solid (100 mg, 3%); mp 220–222 ◦C
(lit. 220–222 ◦C) [27]. δH (500 MHz, DMSO-d6) 9.83 (1H, br s, OH), 7.28 (1H, d, J = 9.0 Hz, ArH), 7.13
(1H, s, ArH), 7.07 (1H, d, J = 8.8 Hz, ArH), 6.64 (1H, s, CH), 5.00 (2H, s, CH2). δC (100 MHz, DMSO-d6)
160.3 (Cq), 154.2 (Cq), 150.8 (Cq), 147.1 (Cq), 120.7, 118.2, 118.1 (Cq), 116.1, 110.1 (CH), 41.8 (CH2).
2-(6-Bromobenzofufran-3-yl)acetic acid (6): Coumarin 1 (1.77 g, 6.48 mmol) was suspended in 2 M NaOH
(37 mL) and stirred at 80 ◦C for 18 h. The resulting suspension was cooled to room temperature and
acidified with concentrated hydrochloric acid to pH 1. The white precipitate was collected by vacuum
filtration and dried under reduced pressure (1.63 g, 99%); mp 154–156 ◦C (lit. 156–157 ◦C) [28]. δH
(500 MHz, DMSO-d6) 7.90 (1H, s, ArH), 7.86 (1H, s, ArH), 7.56 (1H, d, J = 8.0 Hz, ArH), 7.42 (1H, d,
J = 8.25 Hz, ArH), 3.68 (2H, s, CH2). δC (100 MHz, DMSO-d6) 172.2 (Cq), 155.4 (Cq), 144.8, 127.7 (Cq),
126.2, 122.3, 117.3 (Cq), 114.9, 114.7 (Cq), 29.3 (CH2).
2-(5-Bromobenzofuran-3-yl)acetic acid (7): Coumarin 2 (252 mg, 0.920 mmol) was dissolved in 2 M NaOH
(5.5 mL) and stirred at 80 ◦C for 16 h. TLC indicated complete consumption of the starting material.
The solution was cooled to room temperature and acidified with concentrated hydrochloric acid. The
product precipitated and was collected by vacuum filtration as a light grey solid (203 mg, 86%); mp
142–143 ◦C (lit. 142–143 ◦C) [28]. δH (400 MHz, DMSO-d6) 7.93 (1H, s, ArH), 7.81 (1H, s, ArH), 7.55
(1H, d, J = 8.8 Hz, ArH), 7.44 (1H, d, J = 8.6 Hz, ArH), 3.70 (2H, s, CH2). δC (100 MHz, CDCl3) 176.2
(Cq), 154.0 (Cq), 144.3, 129.4 (Cq), 127.6, 122.4, 116.0 (Cq), 113.1, 112.0 (Cq), 29.2 (CH2). LRMS (ESI):

Molecules 2019, 24, 3756
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m/z 253.5 [M (79Br) + H]+, 254.9 [M (81Br) + H]+. HRMS (ESI): 252.9521 [M (79Br) + H]+, 254.9509
[M (81Br) + H]+.
2-(6-Iodobenzofuran-3-yl)acetic acid (8): Coumarin 3 (480 mg, 1.50 mmol) was suspended in 2 M NaOH
(10 mL) and stirred at 80 ◦C for 16 h. The resulting solution was cooled to room temperature and
acidified with concentrated hydrochloric acid to pH 1. The white precipitate was collected by vacuum
filtration and dried under reduced pressure (423 mg, 94%); mp 164–166 ◦C. δH (400 MHz, CDCl3)
7.86 (1H, s, ArH), 7.57–7.56 (2H, m, 2H), 7.31 (1H, d, J = 8.0 Hz, ArH), 3.73 (2H, s, CH2). δC (100
MHz, DMSO-d6) 172.2, 155.6, 144.3, 131,8, 128.0, 122.6, 120.5, 114.6, 89.0, 29.3. LRMS (ESI): m/z 300.80
[M − H]−. HRMS (ESI): 300.9397 [M − H]−.
2-(6-Hydroxybenzofuran-3-yl)acetic acid (9): A suspension of coumarin 4 (200 mg, 0.950 mmol) in 2 M
sodium hydroxide (10 mL) was stirred at 80 ◦C for 16 h. The resulting brown solution was cooled
to room temperature and acidified with concentrated hydrochloric acid to pH 1. The compound in
question precipitated as a brown solid which was collected by vacuum filtration (183 mg, 66%); mp
141–143 ◦C (lit. 142–143 ◦C) [28]. δH (500 MHz, DMSO-d6) 9.49 (1H, br s, OH), 7.65 (1H, s, ArH), 7.33
(1H, d, J = 8.4 Hz, ArH), 6.87 (1H, s, ArH), 6.73 (1H, d, J = 8.4 Hz, ArH), 3.59 (2H, s, CH2). δC (125 MHz,
DMSO-d6) 172.5 (Cq), 156.2 (Cq), 156.1 (Cq), 142.0, 120.6, 120.3 (Cq), 114.3 (Cq), 112.4, 98.1, 29.6 (CH2).
LRMS (ESI): m/z 191.0 [M − H]−.
2-(5-Hydroxybenzofufran-3-yl)acetic acid (10): Coumarin
5 (93.3 mg, 0.4458 mmol) was suspended in 2 M
sodium hydroxide (3 mL) and the resulting brown solution was stirred at 80 ◦C for 48 h. The solution
was cooled to room temperature and acidified with concentrated hydrochloric acid to pH 1, then
extracted with ethyl acetate ( 3). The combined organic phases were dried over magnesium sulfate,
×
then concentrated under reduced pressure to a light brown oil which crystallised to an off-white solid
(76.5 mg, 89%); mp 128–130 ◦C. δH (400 MHz, DMSO-d6) 9.14 (1H, br s, OH), 7.75 (1H, s, ArH), 7.31
(1H, d, J = 8.8 Hz, ArH), 6.86 (1H, s, ArH), 6.72 (1H, d, J = 8.8 Hz, ArH), 3.58 (2H, s, CH2). LRMS (ESI):
m/z 191. 0 [M − H]−.
Methyl 2-(6-bromobenzofuran-3-yl)acetate (11): Thionyl chloride (1.31 mL, 18.1 mmol) was added dropwise
to chilled methanol (6 mL) and the solution was stirred in an ice bath. After one hour,
6 (1.54 g,
4.02 mmol) was added and the suspension was refluxed for 16 h. Upon cooling to room temperature,
white crystals precipitated. The methanol was removed under reduced pressure and the resulting crude
solid was purified by flash chromatography (30–70% ethyl acetate in hexanes) to afford the product
as an oil, which crystallised to an off-white solid under reduced pressure (1.57 g, 97%); mp 70–71 ◦C.
δH (400 MHz, CDCl3) 7.66 (1H, s, ArH), 7.61 (1H, s, ArH), 7.43 (1H, d, J = 8.0 Hz, ArH), 7.38 (1H, d,
J = 8.0 Hz, ArH), 3.73 (3H, s, CH3), 3.69 (2H, s, CH2). δC (100 MHz, CDCl3) 170.8, 155.5, 143.4, 126.7,
126.1, 120.7, 117.9, 115.0, 113.1, 52.2, 29.4. HRMS (ESI): 304.9408, 306.9388 [M + K]+.
Methyl 2-(6-iodobenzofuran-3-yl)acetate (12): Thionyl chloride (932
µL) was added dropwise to chilled
methanol (10 mL) and the solution was stirred for 50 min under nitrogen. The acid
8
(1.29 g, 4.28 mmol)
was added and the suspension was heated to reflux for four hours. Upon cooling to room temperature,
a precipitate formed. The suspension was chilled in an ice bath and water was added, causing more
solid to precipitate. The precipitate was collected by vacuum filtration to give the compound in
question as an off-white solid (945 mg, 70%); mp 80–82 ◦C. δH (400 MHz, CDCl3) 7.85 (1H, s, ArH),
7.57–7.54 (2H, m, ArH), 7.31 (1H, d, J = 8.2 Hz, ArH), 3.73 (3H, s, OCH3), 3.68 (2H, s, CH2). δC (100
MHz, CDCl3) 170.8 (Cq), 155.7 (Cq), 143.2, 131.7, 127.3 (Cq), 121.1, 120.8, 113.1 (Cq), 88.1 (Cq), 52.2
(OCH3), 29.4 (CH2). LRMS (ESI): m/z 339.0 [M + Na]+. HRMS (ESI): 296.9860 [M − H2O − H]−.
Methyl 2-(6-(phenylamino)benzofuran-3-yl)acetate (13): Compound 11 (250 mg, 0.929 mmol) was coupled
to aniline (102.0 µL) according to General Procedure B. The crude orange oil was purified by flash
chromatography (5–10% ethyl acetate in hexanes) to afford the compound in question as an orange oil
(178 mg, 68%). δH (400 MHz, CDCl3) 7.52 (1H, s, ArH), 7.42 (1H, d, J = 8.4 Hz, ArH), 7.30–7.24 (3H, m,
ArH), 7.08 (2H, d, J = 8.5 Hz, ArH), 6.99 (1H, d, J = 8.4 Hz, ArH), 6.93 (1H, t, J = 7.4 Hz, ArH), 3.74 (3H,

Molecules 2019, 24, 3756
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s, OCH3), 3.68 (2H, s, CH2). δC (125 MHz, CDCl3) 171.2 (Cq), 156.3 (Cq), 143.5 (Cq), 141.9, 140.9 (Cq),
129.4, 121.8 (Cq), 120.9, 120.0, 117.5, 115.3, 113.0 (Cq), 100.9, 52.1 (CH3), 29.6 (CH2). LRMS (ESI): m/z
282.0 [M + H]+.
Methyl 2-(6-((4-methoxyphenyl)amino)benzofuran-3-yl)acetate (14): Compound 11 (100 mg, 0.372 mmol)
was coupled to p-anisidine (54.9 mg) according to General Procedure B. The crude dark orange oil was
purified by flash chromatography (dichloromethane) to afford the compound in question as a yellow
oil (57.5 mg, 50%). δH (400 MHz, CDCl3) 7.47 (1H, s, ArH), 7.37 (1H, d, J = 8.4 Hz, ArH), 7.09 (2H, d,
J = 8.9 Hz, ArH), 7.04 (1H, s, ArH), 6.89–6.83 (3H, m, ArH), 5.61 (1H, br s, NH), 3.81 (3H, s, OCH3),
3.73 (3H, s, OCH3), 3.66 (2H, s, CH2). δC (100 MHz, CDCl3) 171.2 (Cq), 156.6 (Cq), 155.2 (Cq), 143.2 (Cq),
141.3, 136.0 (Cq), 121.9, 120.5 (Cq), 119.9, 114.7, 113.3, 112.9 (Cq), 98.2, 55.5 (OCH3), 52.1 (OCH3), 29.6
(CH2). LRMS (ESI): m/z 312.1 [M + H]+.
2-(6-(Phenylamino)benzofuran-3-yl)acetic acid (15): Ester 13 (178 mg, 0.634 mmol) was hydrolysed using 2
M sodium hydroxide according to General Procedure A to afford the compound in question as a brown
solid (160 mg, 94%); mp 127–130 ◦C. δH (400 MHz, CDCl3) 7.53 (1H, s, ArH), 7.43 (1H, d, J = 8.4 Hz
,
ArH), 7.30–7.25 (3H, m, ArH), 7.09 (2H, d, J = 8.4 Hz, ArH), 6.99 (1H, d, J = 8.4 Hz, ArH), 6.95 (1H, t,
J = 7.4 Hz, ArH), 3.72 (2H, s, CH2). δC (125 MHz, CDCl3) 176.5 (Cq), 156.3 (Cq), 143.4 (Cq), 142.1, 141.0
(Cq), 129.4, 121.6 (Cq), 121.0, 120.0, 117.6, 115.3, 112.4 (Cq), 100.8, 29.5 (CH2). LRMS (ESI): m/z 265.96
[M − H]−. HRMS (ESI): 306.1621 [M + K]+.
2-(6-((4-Methoxyphenyl)amino)benzofuran-3-yl)acetic acid (16): Ester 14 (50.0 mg, 0.161 mmol) was
hydrolysed using 2 M methanolic sodium hydroxide (5 equiv, 402
µ
L) according to General Procedure
◦
A to afford the compound in question as a light brown solid (47.9 mg, 100%); mp 120–122 C. δH
(400 MHz, CDCl3) 7.49 (1H, s, ArH), 7.38 (1H, d, J = 8.4 Hz, ArH), 7.13 (3H, m, ArH), 6.93 (1H, d,
J = 8.4 Hz, ArH), 6.89–6.83 (3H, m, ArH), 3.81 (3H, s, OCH3), 3.70 (2H, s, CH2). δC (100 MHz, CDCl3)
176.2 (Cq), 156.6 (Cq), 155.4 (Cq), 143.4 (Cq), 141.6, 135.9 (Cq), 122.1, 120.4 (Cq), 120.0, 114.8, 113.4, 112.3
(Cq), 98.3, 55.6 (OCH3), 29.5 (CH2). LRMS (ESI): m/z 296.1 [M
−
H]−. HRMS (ESI): 296.0959 [M
−
H]−.
Butyl 2-(6-butoxybenzofuran-3-yl)acetate (17): Compound (200 mg. 1.041 mmol) was reacted with
9
n-butyl bromide according to General Procedure C using acetonitrile as solvent, to give the compound
in question as a yellow oil (205 mg, 65%) which did not require purification. δH (500 MHz, CDCl3) 7.52
(1H, s, ArH), 7.41 (1H, d, J = 9.0 Hz, ArH), 6.99 (1H, s, ArH), 6.88 (1H, d, J = 8.5 Hz, ArH), 4.13 (2H, t,
J = 6.8 Hz, OCH2), 4.00 (2H, t, J = 6.5 Hz), OCH2), 3.65 (2H, s, CH2), 1.79 (2H, m, CH2), 1.61 (2H, m,
CH2), 1.53 (2H, m, CH2), 1.35 (2H, m, CH2), 0.97 (3H, t, J = 7.5 Hz, CH3), 0.91 (3H, t, J = 7.3 Hz, CH3).
δC (100 MHz, CDCl3) 170.8 (Cq), 157.7 (Cq), 156.2 (Cq), 141.8, 120.9 (Cq), 119.7, 113.1 (Cq), 112.3, 96.8,
68.3 (OCH2), 64.9 (OCH2), 31.3 (CH2), 30.6 (CH2), 29.9 (CH2), 19.3 (CH2), 19.1 (CH2), 13.8 (CH3), 13.6
(CH3). LRMS (ESI): m/z 305.1 [M + H]+.
Isobutyl 2-(6-isobutoxybenzofuran-3-yl)acetate (18): Compound 9 (200 mg, 1.04 mmol) was reacted with
i-butyl bromide according to General Procedure C using acetonitrile as solvent, to give the compound
in question as a yellow oil (50.0 mg, 16%) which did not require further purification. δH (400 MHz,
CDCl3) 7.52 (1H, s, ArH), 7.41 (1H, d, J = 8.4 Hz, ArH), 6.99 (1H, s, ArH), 6.89 (1H, d, J = 8.4 Hz, ArH),
3.91 (2H, d, J = 6.8 Hz, CH2), 3.75 (2H, d, J = 6.4 Hz, OCH2), 3.67 (2H, s, CH2), 2.11 (1H, sept, J = 6.8 Hz,
CH), 1.92 (1H, sept, J = 6.8 Hz, CH), 1.04 (6H, d, J = 6.4 Hz, 2CH3), 0.90 (6H, d, J = 6.8 Hz, 2CH3). δC
(100 MHz, CDCl3) 170.8 (Cq), 157.8 (Cq), 156.2 (Cq), 141.8, 120.9 (Cq), 119.7, 113.1 (Cq), 112.3, 96.8, 75.1
(OCH2), 71.2 (OCH2), 29.9 (CH2), 28.3 (CH), 29.7 (CH), 19.3 (CH3), 19.0 (CH3). LRMS (ESI): m/z 305.1
[M + H]+.
Benzyl 2-(6-(benzyloxyl)benzofuran-3-yl)acetate (19): Compound
9 (315 mg, 1.64 mmol) was reacted with
benzyl bromide according to General Procedure C using acetone as solvent. Purification by flash
chromatography (1–10% ethyl acetate in hexanes) afforded the compound in question as a white solid
(315 mg, 52%); mp 77–79 ◦C (lit. 77–78.5 ◦C) [29]. δH (400 MHz, CDCl3) 7.53 (1H, s, ArH), 7.46–745 (2H,
m, ArH), 7.41–7.33 (9H, m, ArH), 7.07, (1H, s, ArH), 6.94 (1H, d, J = 8.4 Hz, ArH), 5.16 (2H, s, OCH2),

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5.11 (2H, s, OCH2), 3.71 (2H, s, CH2). δC (100 MHz, CDCl3) 170.5, 157.2, 156.1, 142.0, 136.9, 135.6, 128.6,
128.5, 128.3, 128.3, 128.0, 127.5, 121.2, 119.9, 112.8, 112.5, 97.3, 70.6, 66.8, 29.8. LRMS (ESI): m/z 373.1
[M + H]+.
Benzyl 2-(5-benzyloxy)benzofuran-3-yl)acetate (20): Compound 10 (75.0 mg, 0.390 mmol) was reacted
with benzyl bromide (21.3 µL) according to General Procedure C using acetone as solvent. Purification
by flash chromatography (1–30% ethyl acetate in hexanes) gave the compound in question as a white
solid (44.7 mg, 31%); mp 70–72 ◦C. δH (400 MHz, CDCl3) 7.60 (1H, s, ArH), 7.46–7.32 (11H, m, ArH),
7.06 (1H, d, J = 2.4 Hz, ArH), 7.00 (1H, dd, J = 9.0 Hz, 2.6, ArH), 5.16 (2H, s, OCH2), 5.01 (2H, s, OCH2),
3.72 (2H, s, CH2). δC (100 MHz, CDCl3) 170.4 (Cq), 155.1 (Cq), 150.3 (Cq), 143.7, 137.1 (Cq), 135.6 (Cq),
128.6, 128.5, 128.4, 128.3, 128.1 (Cq), 127.9, 127.6, 114.1, 113.0 (Cq), 112.0, 103.5, 70.8 (CH2), 66.9 (CH2),
29.9 (CH2). LRMS (ESI): m/z 373.1 [M + H]+.
2-(6-Butoxybenzofuran-3-yl)acetic acid (21): Ester 17 (205 mg, 0.673 mmol) was hydrolysed using 2
M methanolic sodium hydroxide (5 equiv, 1.68 mL) according to General Procedure A to give the
compound in question as a white solid (143 mg, 86%); mp 108–109 ◦C. δH (500 MHz, CDCl3) 7.53 (1H,
s, ArH), 7.40 (1H, d, J = 8.8 Hz, ArH), 7.00 (1H, s, ArH), 6.89 (1H, d, J = 8.4 Hz, ArH), 3.99 (2H, t, J = 6.4
Hz, OCH2), 3.71 (2H, s, CH2), 1.79 (2H, p, J = 6.8 Hz, CH2), 1.51 (2H, sextet, J = 7.6 Hz, CH2), 0.99 (3H,
t, J = 7.4 Hz, CH3). δC (100 MHz, CDCl3) 176 (Cq), 157.8 (Cq), 156.3 (Cq), 142.0, 120.6 (Cq), 119.7, 112.4,
112.3 (Cq), 96.8, 68.3 (OCH2), 31.3 (CH2), 29.4 (CH2), 19.3 (CH2), 13.8 (CH3). LRMS (ESI): m/z 247.2
[M − H]−.
2-(6-Isobutoxybenzofuran-3-yl)acetic acid (22): Ester 18 (50.0 mg, 0.164 mmol) was hydrolysed using
2 M methanolic sodium hydroxide (5 equiv, 411 µL) according to General Procedure A. The crude
compound required purification by RP-HPLC. The fractions containing the product were lyophilised
to give the compound in question as a white solid (6 mg, 15%); mp 130–132 ◦C. δH (400 MHz, CDCl3)
7.53 (1H, s, ArH), 7.40 (1H, s, J = 8.4 Hz, ArH), 6.99 (1H, s, ArH), 6.90 (1H, d, J = 8.6 Hz, ArH), 3.75 (2H,
d, J = 6.4 Hz, OCH2), 3.71 (2H, s, CH2), 2.11 (1H, sept, J = 6.4 Hz, CH), 1.05 (6H, d, J = 6.8 Hz, CH3). δC
(100 MHz, CDCl3) 157.9 (Cq), 156.3 (Cq), 142.0, 120.6 (Cq), 119.6, 112.4, 112.3 (Cq), 96.9, 75.1 (OCH2),
29.7 (CH2), 19.3 (CH3). One quaternary carbon not observed. LRMS (ESI): m/z not found.
2-(6-((Benzyloxy)benzofuran-3-yl)acetic acid (23): Ester 19 (248 mg, 0.665 mmol) was hydrolysed using
2 M sodium hydroxide (5 equiv, 1.66 mL) according to General Procedure A. The compound in question
was afforded after recrystallisation from chloroform as a crystalline white solid (124 mg, 61%); mp
153–155 ◦C. δH (500 MHz, CDCl3) 7.54 (1H, s, ArH), 7.46–7.32 (7H, m, ArH), 7.08 (1H, s, ArH), 6.98
(1H, d, J = 8.5 Hz, ArH), 5.11 (2H, s, OCH2), 3.71 (2H, s, CH2). δC (100 MHz, CDCl3) 176.3, 157.4,
156.1, 142.2, 136.9, 128.6, 128.0, 127.5, 121.1, 119.8, 112.6, 112.3, 97.5, 70.6, 29.5. LRMS (ESI): m/z 281.0
[M − H]−. HRMS (ESI): 281.0848 [M − H]−.
2-(5-(Benzyloxy)benzofuran-3-yl)acetic acid (24): Ester 20 (28.7 mg, 0.0771 mmol) was hydrolysed using 2
M methanolic sodium hydroxide (5 equiv, 193 µL) according to General Method B to give the compound
in question as a white solid (16.9 mg, 78%); mp 147–149 ◦C. δH (400 MHz, CDCl3) 7.61 (1H, s, ArH),
7.47–7.30 (6H, m, ArH), 7.10 (1H, s, ArH), 7.00 (1H, dd, J = 8.8 Hz, 2.0, ArH), 5.10 (2H, s, OCH2), 3.71
(2H, s, CH2). δC (100 MHz, CDCl3) 176.2 (Cq), 155.2 (Cq), 150.3 (Cq), 143.9, 137.1 (Cq), 128.6, 127.9,
127.6, 114.1, 112.4 (Cq), 112.1, 103.6, 70.9 (OCH2), 29.4 (CH2). One quaternary carbon not observed.
LRMS (ESI): m/z 281.0 [M − H]−. HRMS (ESI): 281.0848 [M − H]−.
2-(6-Phenoxybenzofuran-3-yl)acetic acid (25): Compound 8 (302 mg, 1.00 mmol) was coupled to phenol
according to General Procedure D. The crude compound was purified by flash chromatography (2.5%
methanol in dichloromethane) to give a red solid (61.3 mg) that was recrystallised from acetonitrile to
give the compound in question as a white solid (7.1 mg, 3%). The mother liquor was concentrated and
purified by semi-preparative RP-HPLC. The fractions containing the product were lyophilised to give
the compound in question as a white solid (19.7 mg, 7%); mp 107–109 ◦C. δH (400 MHz, CDCl3) 7.60
(1H, s, ArH). 7.50 (1H, d, J = 8.4 Hz, ArH), 7.36–7.32 (2H, m, ArH), 7.19–7.09 (2H, m, ArH), 7.03–7.00

Molecules 2019, 24, 3756
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(3H, m, ArH), 7.74 (2H, s, CH2). δC (100 MHz, CDCl3) 176.2 (Cq), 157.6 (Cq), 155.8 (Cq), 155.3 (Cq),
143.2 (Cq), 129.8, 123.2, 120.1, 118.6, 115.3, 112.4 (Cq), 102.6, 29.7 (CH2). One carbon signal obscured.
LRMS (ESI): m/z 267.2 [M − H]−. HRMS (ESI): 267.0689 [M − H]−.
2-(6-(3-Methoxyphenoxy)benzofuran-3-yl)acetic acid (26): Compound
8 (302 mg, 1.00 mmol) was coupled
to 3-methoxyphenol (149 mg) according to General Procedure D. The crude brown oil was purified
by flash chromatography (2.5% methanol in dichloromethane) and subsequent recrystallisation from
aqueous ethanol afforded the compound in question as an off-white solid (30.0 mg, 10%); mp. 91–93 ◦C.
δH (400 MHz, CDCl3) 7.62 (1H, s, ArH), 7.50 (1H, d, J = 8.5 Hz, ArH), 7.22 (1H, t, J = 8.5 Hz, ArH), 7.15
(1H, s, ArH), 7.02 (1H, d, J = 8.5 Hz, ArH), 7.66 (1H, d, J = 8.5 Hz, ArH), 6.61–6.58 (2H, m, ArH), 3.78
(3H, s, OCH3), 3.75 (2H, s, CH2). LRMS (ESI): m/z 299.11 [M + H]+. HRMS (ESI): 297.0800 [M − H]−.
Methyl 2-(6-benzylbenzofuran-3-yl)acetate (27): A three-necked flask charged with zinc powder (535 mg),
lithium chloride (433 mg), and a stir bar was flame dried under reduced pressure. The flask was cooled
to room temperature and filled with nitrogen. Dry tetrahydrofuran (5 mL) was added via cannula
and the suspension was cooled to 0 ◦C. Benzyl bromide (486
µL) was added dropwise via syringe and
the suspension stirred for 1.5 h. The suspension was then transferred via cannula to an evacuated,
flame dried flask containing compound 11 (250 mg, 0.929 mmol), Pd(OAc)2 (17.9 mg), and XPhos
(35.9 mg). The flask was filled with nitrogen then heated to 50 ◦C. The resulting black reaction mixture
was stirred for 18 h, cooled to room temperature, and then treated with 1 M HCl and allowed to stir for
1 min. The organic phase was collected and the aqueous phase was extracted with ethyl acetate ( 3).
×
The combined organic phases were dried over magnesium sulfate then concentrated under reduced
pressure to a dark yellow oil. The crude oil was purified by flash chromatography (2.5% ethyl acetate
in hexanes) to give the compound in question as a yellow oil (60.5 mg, 35%). δH (400 MHz, CDCl3)
7.57 (1H, s, ArH), 7.46 (1H, d, J = 8.1 Hz, ArH), 7.31–7.29 (2H, m, ArH), 7.22–718 (3H, m, ArH), 7.12
(1H, d, J = 8.1 Hz, ArH), 4.09 (2H, s, CH2), 3.72 (3H, s, OCH3), 3.68 (2H, s, CH2). δC (100 MHz, CDCl3)
171.1 (Cq), 155.6 (Cq), 142.6, 141.1 (Cq), 138.1 (Cq), 128.9, 128.4, 126.1, 125.7 (Cq), 123.9, 119.4, 112.9 (Cq),
111.7, 52.1 (OCH3), 42.0 (CH2), 29.5 (CH2). LRMS (ESI): m/z 303.1 [M + Na]+.
2-(6-Benzylbenzofuran-3-yl)acetic acid (28): Ester 27 (60.5 mg, 0.216 mmol) was hydrolysed using 2 M
methanolic sodium hydroxide (5 equiv, 540 µL) according to General Procedure A to give the compound
in question as a while solid (29.2 mg, 51%); mp 124–127 ◦C. δH (400 MHz, CDCl3) 7.58 (1H, s, ArH),
7.47 (1H, s, ArH), 7.32–7.28 (3H, m, ArH), 7.23–7.21 (3H, m, ArH), 7.13 (1H, d, J = 8.0 Hz, ArH), 4.10
(2H, s, CH2), 3.73 (2H, s, CH2). δC (100 MHz, CDCl3) 176.9, 155.6 (Cq), 142.8, 141.1 (Cq), 138.3 (Cq),
128.9, 128.5, 126.1, 125.5 (Cq), 124.0, 199.4, 112.2 (Cq), 111.8, 42.0 (CH2), 29.5 (CH2). LRMS (ESI): m/z
265.0 [M − H]−. HRMS (ESI): 265.0895 [M − H]−.
2-(6-Bromobenzofuran-3-yl)acetamide (29): Compound
6 (150 mg, 0.588 mmol) was dissolved in
dichloromethane (6 mL) in an atmosphere of nitrogen. Phosphorous pentachloride (122 mg, 0.588 mmol)
was added and the solution was heated to reflux for 30 min. The reaction cooled to room temperature
and ammonia gas (produced by heating an aqueous solution of ammonium hydroxide to 50 ◦C) was
bubbled through the pale-yellow solution. Within 10 s, a white solid precipitated out of solution. After
several minutes, the ammonia gas was removed from the reaction then the dichloromethane was
removed under reduced pressure. The white residue was partitioned between ethyl acetate and water.
The aqueous phase was washed with ethyl acetate (
over magnesium sulfate and concentrated under reduced pressure to afford the amide as a white solid
×
2) then the combined organic phases were dried
(135 mg, 90%); mp 140–143 ◦C. δH (400 MHz, CDCl3) 7.85–7.84 (2H, m, ArH), 7.57 (1H, d, J = 8.4 Hz
ArH), 7.53 (1H, br s, NH), 7.42 (1H, d, J = 8.4 Hz, ArH), 6.98 (1H, br s, NH), 3.47 (2H, s, CH2). δC
(100 MHz, DMSO-d6) 171.6 (Cq), 155.4 (Cq), 144.5, 127.8 (Cq), 126.1, 122.3, 117.3 (Cq), 115.7 (Cq), 115.0,
30.6 (CH2). LRMS (ESI): m/z 253.9 [M + H]+. HRMS (ESI): 275.9617 [M + Na]+.
,
2-(6-Bromobenzofuran-3-yl)acetonitrile (30): Amide 29 (110 mg, 0.431 mmol) was suspended in dry
toluene (4.5 mL) in an atmosphere of nitrogen. Distilled thionyl chloride (47.0 µL) was added and

Molecules 2019, 24, 3756
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the reaction mixture was heated to reflux. The starting material dissolved upon heating. After 1.5 h,
TLC showed a new product had formed and only a small amount of starting material remained. The
reaction was cooled to room temperature, and then the toluene and excess thionyl chloride were
removed under reduced pressure. The resulting dark orange oil was dissolved in ethyl acetate and
washed with a saturated solution of sodium hydrogen carbonate and brine ( 2). The organic phase
×
was dried (MgSO4) and concentrated to a dark orange oil under reduced pressure. The crude oil was
purified by flash chromatography (10% ethyl acetate in hexanes) to afford the nitrile as a dark pink
solid (66.9 mg, 66%); mp 88–90 ◦C. δH (400 MHz, CDCl3) 7.70 (1H, s, ArH), 7.65 (1H, s, ArH), 7.46
(1H, d, J = 8.4 Hz, ArH), 7.43 (1H, d, J = 8.4 Hz, ArH), 3.75 (2H, s, CH2). δC (100 MHz, CDCl3) 155.7(Cq),
143.2, 126.7, 125.1 (Cq), 119.9, 118.8 (Cq), 116.3 (Cq), 115.4, 110.2 (Cq), 13.0 (CH2). LRMS (ESI): m/z not
found. I.R (KBr) 2254 cm−1 (-C≡N).
5-((6-Bromobenzofuran-3-yl)methyl)-1H-tetrazole (31): Nitrile 30 (57 mg, 0.241 mmol), sodium azide
(47.1 mg), and triethylammonium chloride (99.7 mg) were suspended in dry toluene (1 mL) in an
atmosphere of nitrogen. The suspension was stirred at reflux for 3 days. The reaction was cooled
to room temperature and water was added. The aqueous phase was collected and acidified with
concentrated hydrochloric acid. Upon acidification, the compound in question precipitated as an
off-white solid which was collected by vacuum filtration (50.2 mg, 75%); mp 184–185 ◦C. δH (400 MHz,
DMSO-d6) 7.98 (1H, s, ArH), 7.90 (1H, s, ArH), 7.67 (1H, d, J = 8.4 Hz, ArH), 7.42 (1H, d, J = 8.0 Hz,
ArH), 4.39 (2H, s, CH2). δC (100 MHz, DMSO-d6) 155.5 (Cq), 144.9, 126.9 (Cq), 126.4, 121.8, 117.7 (Cq),
115.3 (Cq), 115.2, 18.3 (CH2). One quaternary carbon was not observed. LRMS (ESI): m/z 279.0 [M (79Br)
+ H]+, 280.9 [M (81Br) + H]+. HRMS (ESI): 276.9758 [M (79Br) − H]−, 278.9738 [M (81Br) − H]−.
Dimethyl 2-(1-(methoxycarbonyl)-1H-indole-3-yl)malonate (32): Based on the method of Suarez–Castillo [19].
A solution of ethyl-3-indole acetate (1.5 g, 7.38 mmol) in dry tetrahydrofuran (10 mL) was added
to a suspension of sodium hydride (1.18 g as 60% dispersion in mineral oil, 29.5 mmol) in dry
tetrahydrofuran (40 mL) under an atmosphere of nitrogen. The suspension was stirred until the
formation of gas was no longer observed (5–10 min). To the resulting green suspension was added
dimethyl carbonate (12.0 mL, 142 mmol) and the reaction mixture was stirred at reflux for 16 h,
after which time the colour became maroon. The reaction was cooled to room temperature and the
tetrahydrofuran was removed under reduced pressure. The resulting residue was partitioned between
a saturated solution of sodium hydrogen carbonate and diethyl ether. The aqueous phase was washed
with diethyl ether (
×
2) and the combined organic phases were washed with brine ( 3), dried over
×
magnesium sulfate, and concentrated to a red oil by rotary evaporation. A small portion of ether was
added and heated to boil. Upon cooling, a solid precipitated which was recrystallised from diethyl
ether and the mother liquor was purified by flash chromatography (dichloromethane) to afford the
titled product as an off-white solid (1.33 g, 59%); mp 108–110 ◦C (lit. 108–110 ◦C) [30]. δH (500 MHz,
CDCl3) 8.20 (1H, d, J = 7.5 Hz, ArH), 7.82 (1H, s, ArH), 7.57 (1H, d, J = 7.5 Hz, ArH), 7.36 (1H, t,
J = 7.5 Hz, ArH), 7.28 (1H, t, J = 7.5, ArH), 4.90 (1H, s, CH), 4.04 (3H, s, OCH3), 3.78 (6H, s, (OCH3)2).
δC (100 MHz, CDCl3) 168.0 (Cq), 151.2 (Cq), 135.3 (Cq), 129.1 (Cq), 125.1, 125.0, 123.2, 119.2, 115.3, 112.8
(Cq), 53.9 (OCH3), 53.0 (OCH3), 49.0 (CH).
Dimethyl 2-(6-bromo-1-(methoxycarbonyl)-1H-indole-3-yl)malonate (33): According to the method of
Suarez–Castillo [19]. Malonate 32 (1.25 g, 4.09 mmol) was dissolved in carbon tetrachloride (50.0 mL),
treated with bromine (1.68 mL), and stirred at room temperature for 24 h. A saturated solution of
sodium sulfite (50 mL) was added and stirred vigorously for 24 h. The aqueous phase was extracted
with dichloromethane (
×
2) and the combined organic phases were washed with brine ( 3), dried
×
over magnesium sulfate, and concentrated under reduced pressure to an orange oil. Diethyl ether
(15 mL) was added and the solution was cooled until a precipitate formed which was collected and
recrystallised from diethyl ether to afford the titled product as an off-white solid (826 mg, 52%). The
mother liquor was purified by flash chromatography (5–20% ethyl acetate in hexanes) to afford an
off-white solid after trituration with diethyl ether (157 mg, 10%); mp 110–112 ◦C (lit. 110–112 ◦C) [19].

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δH (400 MHz, CDCl3) 8.38 (1H, br s, ArH), 7.79 (1H, s, ArH), 7.45 (1H, d, J = 8.4 Hz, ArH), 7.40 (1H, d,
J = 8.4 Hz, ArH), 4.85 (1H, s, CH), 4.04 (3H, s, NCOOCH3), 3.78 (6H, s, (COOCH3)2). δC (100 MHz,
CDCl3) 167.8 (Cq), 150.8 (Cq), 135.9 (Cq), 127.9, 126.5, 125.6 (Cq), 120.6, 118.8 (Cq), 118.4, 112.7 (Cq), 54.1
(CH3), 53.1 (CH3), 49.0 (CH).
2-(6-Bromo-1H-indol-3-yl)acetic acid (34): Malonate 33 (200 mg, 0.640 mmol) was dissolved in
methanol:water (9:1) and treated with potassium hydroxide (180 mg, 3.21 mmol) and refluxed
for 18 h. The reaction was cooled to room temperature and concentrated under reduced pressure. The
resulting residue was dissolved in water and the aqueous solution was washed with ethyl acetate. The
aqueous phase was then acidified with 1 M hydrochloric acid to pH 2 and the cloudy suspension was
extracted with ethyl acetate ( 3). The combined organic layers were dried over magnesium sulfate,
×
then concentrated under reduced pressure to a light yellow solid. Recrystallisation from chloroform
afforded the titled product as a white solid (64.0 mg, 39%); mp 178–180 ◦C δH (400 MHz, DMSO-d6)
11.04 (1H, br s, NH/COOH), 7.52 (1H, s, ArH), 7.44 (1H, d, J = 8.4 Hz, ArH), 7.25 (1H, s, ArH), 7.10 (1H,
d, J = 8.4 Hz, ArH), 3.62 (2H, s, CH2). δC (100 MHz, DMSO-d6) 173.4, 137.4, 126.8, 125.6, 121.8, 120.9,
114.4, 114.3, 108.6, 31.3. LRMS (ESI): m/z 252.3 [M (79Br) − H)−, 253.9 [M (81Br) − H]−.
Dimethyl 2-(1-(methoxycarbonyl)-6-phenyl-1H-indol-3-yl)malonate (35): Bromide 33 (250 mg, 0.651 mmol),
phenylboronic acid (476 mg), caesium fluoride (297 mg), and Pd(dppf)Cl2 (24.0 mg) were dissolved in
degassed acetonitrile:water (4:1) in an atmosphere of nitrogen. The suspension was heated to 90 ◦C
and stirred for 18 h. The reaction was partitioned between ethyl acetate and brine and the organic
layer was separated, washed with brine ( 2), and dried over magnesium sulfate. The ethyl acetate was
×
removed under reduced pressure then the crude brown solid was purified by flash chromatography
(20% ethyl acetate in hexane, then 2.5–5% methanol in dichloromethane). The fractions containing
the product were concentrated under reduced pressure and triturated with diethyl ether to afford the
compound in question as a white solid (30.0 mg, 12%); mp 124–126 ◦C. δH (400 MHz, CDCl3) 8.47 (1H,
bs s, ArH), 7.85 (1H, s, ArH), 7.68 (2H, d, J = 7.2 Hz, ArH), 7.65 (1H, d, J = 8.3 Hz, ArH) 7.55 (1H, d,
J = 8.2 Hz, ArH), 7.46 (2H, t, J = 7.6 Hz, ArH), 7.36 (1H, t, J = 7.4 Hz, ArH), 4.94 (1H, s, CH), 4.05 (3H, s,
OCH3), 3.81 (6H, s, OCH3) δC (100 MHz, CDCl3) 168.0 (Cq), 151.1 (Cq), 141.5 (Cq), 138.6 (Cq), 135.9 (Cq),
128.7, 128.2 (Cq), 127.5, 127.1, 125.5, 122.8, 119.5, 113.9, 112.8 (Cq), 53.9 (CH3), 53.0 (OCH3), 49.1 (CH).
2-(6-Phenyl-1H-indol-3-yl)acetic acid (36): Malonate 35 (30 mg, 78.7
µmol) was dissolved in
methanol:water (9:1) and treated with potassium hydroxide (22.0 mg). The reaction refluxed for 16 h
before additional potassium hydroxide (8.80 mg) was added. After one hour at reflux the reaction
was cooled to room temperature and concentrated under reduced pressure. The resulting residue
was dissolved in water and the aqueous solution was washed with ethyl acetate. The aqueous phase
was then acidified with 1 M hydrochloric acid to pH 2 and the cloudy suspension was extracted with
ethyl acetate ( 3). The combined organic layers were dried over magnesium sulfate, then concentrated
×
under reduced pressure to a light yellow solid. Purification by flash chromatography (10% methanol
in dichloromethane) and subsequent recrystallisation from acetonitrile afforded the compound in
question as a white solid (5.0 mg, 25%); mp 214–216 ◦C. δH (500 MHz, Acetone-d6) 7.69 (4H, m, ArH),
7.43 (2H, t, J = 7.8, ArH), 7.36 (1H, d, 8.3, ArH), 7.34 (1H, s, ArH), 7.30 (1H, t, J = 7.4 Hz, ArH), 3.78 (2H,
s, CH2). δC (100 MHz, Acetone-d6) 172.4 (Cq), 142.5 (Cq), 134.7 (Cq), 127.7, 127.0, 126.4, 124.6, 124.4
(Cq), 119.1, 118.5, 109.6, 108.3 (Cq), 30.6 (CH2). LRMS (ESI): m/z 252.1 [M + H]+. HRMS (ESI): m/z
250.0895 [M − H]−.
Methyl 2-bromo-2-(6-bromobenzofuran-3-yl)acetate (37): Compound 11 (500 mg, 1.86 mmol),
N-bromosuccinimide (363.8 mg, 2.04 mmol) and azobisisobutyronitrile (61.0 mg, 0.370 mmol) were
suspended in carbon tetrachloride (25 mL) and the mixture was refluxed for two hours. The reaction
mixture was cooled to room temperature, filtered through a bed of Celite®, and the carbon tetrachloride
was removed by distillation. The crude compound was purified by flash chromatography (0.5%
ethyl acetate in hexanes) to afford the compound in question as an off-white solid (523 mg, 81%);

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mp 73–75 ◦C
. δH (400 MHz, CDCl3) 7.88 (1H, s, ArH), 7.69 (1H, s, ArH), 7.66 (1H, d, J =8.4 Hz, ArH),
7.45 (1H, d, J = 8.4 Hz, ArH), 5.57 (1H, s, CH), 3.84 (3H, s, OCH3). δC (100 MHz, CDCl3) 168.0, 155.9,
144.5, 126.7, 124.6, 121.9, 118.7, 116.2, 115.3, 53.5, 36.5, 29.6. LRMS (ESI): m/z 368.52 [M (79Br, 79Br) +
Na]+, 370.93 [M (79Br, 81Br) + Na]+, 372.92 [M (81Br, 81Br) + Na]+. HRMS (ESI): m/z 344.9786 [M (79Br,
79Br) − H]−.
Methyl-2-azido-2-(6-bromobenzofuran-3-yl)acetate, (38): Bromide 37 (760 mg, 2.18 mmol) was dissolved in
dry N,N-dimethylformamide (20 mL) in an atmosphere of nitrogen. The stirring solution was chilled
◦
to
−
40 C in a bath of acetonitrile/dry ice. Sodium azide (426 mg, 6.55 mmol) was added and the
colourless solution gradually became yellow. The reaction was monitored by TLC, and after 50 min
the solution was removed from the bath. Ethyl acetate and 1 M hydrochloric acid were added and
the aqueous phase was extracted with ethyl acetate (
×
2). The combined organic phases were washed
with brine ( 5), dried over magnesium sulfate, then concentrated under reduced pressure to yield the
×
product as a yellow oil (594 mg, 88%). δH (400 MHz, CDCl3) 7.72 (1H, s, ArH), 7.70 (1H, d, 1.6, ArH),
7.55 (1H, d, J = 8.4 Hz, ArH), 7.43 (1H, dd, J = 8.4 Hz, 1.6, ArH), 5.16 (1H, s, CH), 3.83 (3H, s, OCH3).
δC (100 MHz, CDCl3) 168.7 (Cq), 155.8 (Cq), 144.2, 126.9 (Cq), 124.4 (Cq), 121.2, 118.8 (Cq), 115.4, 114.4
(Cq), 57.2 (CH), 53.2 (OCH3). LRMS (ESI): m/z not found.
Methyl-2-amino-2-(6-bromobenzofuran-3-yl)acetate (39): Azide 38 (114 mg, 0.369 mmol) was dissolved in
dry methanol in an atmosphere of nitrogen. Adams catalyst (2.5% w/w) was added and the flask was
purged three times with hydrogen. The reaction stirred at room temperature under hydrogen for 5 h,
at which time TLC indicated negligible starting material. The reaction was filtered through Celite®,
then concentrated to afford the amine as a yellow oil (97.1 mg, 0.342 mmol, 92%). δH (500 MHz, CDCl3)
7.66 (1H, d, J 1.5, ArH), 7.63 (1H, s, ArH), 7.56 (1H, d, J = 8.0 Hz, ArH), 7.38 (1H, dd, J = 8.3 Hz, 1.5,
ArH), 4.84 (1H, s, CH), 3.74 (1H, s, CH3). δC (100 MHz, CDCl3) 173.4 (Cq), 155.9 (Cq), 143.0, 126.3, 125.0
(Cq), 121.3, 119.8 (Cq), 118.1 (Cq), 115.2, 52.6 (OCH3), 50.5 (CH). LRMS (ESI): m/z 283.9 [M (79Br) + H]+,
285.9 [M (81Br) + H]+.
Methyl 2-Acetamido-2-(6-bromobenzofuran-3-yl)acetate (40): Amine 39 (100 mg, 0.326 mmol) was coupled
to acetyl chloride (25.6 µL) according to General Procedure F to form the compound in question as an
off-white solid (62.0 mg, 58%); mp 166–169 ◦C. δH (400 MHz, CDCl3) 7.65 (1H, s, ArH), 7.62 (1H, s,
ArH), 7.49 (1H, d, J = 8.4 Hz, ArH), 7.37 (1H, d, J = 8.4 Hz, ArH), 6.58 (1H, d, J = 7.2 Hz, NH), 5.84
(1H, d, J = 7.5 Hz, CH), 3.76 (3H, s, OCH3), 2.02 (3H, s, CH3). δC (100 MHz, CDCl3) 170.6, 169.6, 155.7,
143.9, 126.6, 124.6, 120.9, 118.4, 116.5, 115.2, 53.0, 47.7, 22.9. LRMS (ESI): m/z 325.9 [M (79Br) + H]+,
327.9 [M (81Br) + H]+.
Methyl 2-benzamido-2-(6-bromobenzofuran-3-yl) acetate (41): Benzoic acid was coupled to amine 39
(100 mg, 0.626 mmol) according to general procedure G. The crude amide was purified by flash
chromatography (dichloromethane) to give the compound in question as a white solid (69.3 mg, 55%);
mp 161–162 ◦C. δH (500 MHz, CDCl3) 7.80–7.78 (2H, m, ArH), 7.69 (1H, s, ArH), 7.66 (1H, s, ArH), 7.56
(1H, d, J = 8.4 Hz, ArH), 7.51 (1H, t, J = 7.4 Hz, ArH), 7.43–7.39 (2H, m, ArH), 7.38 (1H, d, J = 8.4 Hz
,
ArH), 7.16 (1H, d, J = 7.1 Hz, NH), 6.04 (1H, d, J = 7.2 Hz, CH), 3.80 (3H, s, OCH3). δC (100 MHz,
CDCl3) 170.7, 166.8, 155.8, 144.1, 133.2, 132.1, 128.6, 127.1, 126.6, 124.7, 120.9, 118.4, 116.5, 115.3, 53.1,
48.2. LRMS (ESI): m/z 388.0 [M (79Br) + H]+, 390.1 [M (81Br) + H]+.
Methyl 2-(6-bromobenzofuran-3-yl)-2-(2-(4-methoxyphenyl)acetamido)acetate (42): Amine 39 (46.3 mg, 0.163
mmol) was coupled to 4-methoxyphenylacetic acid (27.1 mg) according to General Procedure G to give
the compound in question as a light yellow solid (37.0 mg, 52%); mp 114–116 ◦C. δH (400 MHz, CDCl3)
7.64 (1H, s, ArH), 7.53 (1H, s, ArH), 7.31 (2H, s, ArH), 7.15 (2H, d, J = 8.4 Hz, ArH), 6.87 (2H, d, J = 8.4
Hz, ArH), 6.39 (1H, br d, J = 7.2 Hz, NH), 5.80 (1H, d, J = 7.6 Hz, CH), 3.80 (3H, s, OCH3), 3.73 (1H, s,
OCH3), 3.55 (2H, s, CH2). δC (100 MHz, CDCl3) 170.9 (Cq), 170.4 (Cq), 159.0 (Cq), 155.7 (Cq), 143.8,
130.4, 126.5, 126.0 (Cq), 124.5 (Cq), 120.9, 118.3 (Cq), 116.3 (Cq), 115.2, 114.5, 55.3 (OCH3), 53.0 (CH),
48.0 (OCH3), 42.5 (CH2). LRMS (ESI): m/z 432.0 [M (79Br) + H]+, 434.1 [M (81Br) + H]+.

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Methyl 2-(6-bromobenzofuran-3-yl)-2-(2-(2-fluorophenyl)acetamido)acetate (43): Amine 39 (87.0 mg,
0.284 mmol) was coupled to 2-fluorophenylacetic acid (43.8 mg) according to General Procedure G to
give the compound in question as a light yellow solid (105 mg, 71%); mp 154–156 ◦C. δH (400 MHz,
CDCl3) 7.64 (1H, s, ArH), 7.57 (1H, s, ArH), 7.37 (1H, d, J = 8.4 Hz, ArH), 7.33 (1H, d, J = 8.4 Hz, ArH),
7.31–7.26 (2H, m, ArH), 7.14–7.05 (2H, m, ArH), 5.58 (1H, d, J = 7.2 Hz, NH), 5.81 (1H, d, J = 7.2 Hz,
CH), 3.74 (3H, s, OCH3), 3.64 (2H, s, CH2). δC (100 MHz, CDCl3) 170.3, 169.4, 162.1, 159.6, 155.7, 143.9,
131.6, 131.5, 129.6, 129.5, 126.5, 124.6, 124.6, 124.5, 121.5, 121.3, 120.9, 118.4, 116.2, 115.7, 115.5, 115.2,
53.0, 48.1, 36.6. LRMS (ESI): m/z 420.2 [M (79Br) + H]+, 422.0 [M (81Br) + H]+.
Methyl 2-(6-bromobenzofuran-3-yl)-2-(methylsulfonamido)acetate (44): Amine 39 (150 mg, 0.528 mmol) was
coupled to mesyl chloride (45 µL) according to General Procedure F. The crude yellow oil was purified
by flash chromatography (dichloromethane) to afford the compound in question as a light-yellow oil
(115 mg, 60%). δH (400 MHz, CDCl3) 7.69 (1H, s, ArH), 7.68 (1H, s, ArH), 7.52 (1H, d, J = 8.4 Hz, ArH),
7.39 (1H, d, J = 8.4 Hz, ArH), 5.74 (1H, d, J = 6.7 Hz, NH), 5.46 (1H, d, J = 6.7 Hz, CH), 3.77 (3H, s,
OCH3), 2.84 (3H, s, CH3). δC (100 MHz, CDCl3) 170.0 (Cq), 155.8 (Cq), 144.2, 126.9, 124.0 (Cq), 121.0,
118.7 (Cq), 116.0 (Cq), 115.4, 53.4 (CH), 51.4 (OCH3), 42.1 (CH3). LRMS (ESI): m/z 361.9 [M (79Br) + H]+,
363.9 [M (81Br) + H]+.
Methyl 2-(6-bromobenzofuran-3-yl)-2-(phenylsulfonamido)acetate (45): Amine 39 (75 mg, 245 µmol) was
coupled to benzenesulfonyl chloride according to General Procedure F. The crude yellow solid was
purified by flash chromatography (15% ethyl acetate in hexanes) to afford the compound in question as
a pale yellow solid (16.0 mg, 15%); mp 133–135 ◦C. δH (400 MHz, CDCl3) 7.74–7.72 (2H, m, ArH), 7.57
(1H, s, ArH), 4.48–7.45 (2H, m, ArH), 7.38–7.33 (3H, m, ArH), 7.30 (1H, d, J = 8.4 Hz, ArH), 5.84 (1H, d,
J = 7.6 Hz, NH), 5.30 (1H, d, J = 7.6 Hz, CH), 3.59 (3H, s, OCH3). δC (100 MHz, CDCl3) 169.5, 155.64,
144.0, 139.5, 132.8, 128.8, 127.0, 126.6, 124.1, 121.0, 118.4, 115.5, 115.1, 53.2, 51.4. LRMS (ESI): m/z 446.4
[M (79Br) + H]+, 448.2 [M (81Br) + H]+.
2-Acetamido-2-(6-bromobenzofuran-3-yl)acetic acid (46): Ester 40 (61.0 mg, 0.187 mmol) was hydrolysed
using 2 M methanolic sodium hydroxide (5 equiv, 468 µL) according to General Procedure A to give the
compound in question as a light yellow solid (57.0 mg, 98%); mp 194–196 ◦C. δH (500 MHz, Acetone-d6)
7.96 (1H, s, ArH), 7.78 (1H, s, ArH), 7.70 (1H, d, J = 8.4 Hz, ArH), 7.45 (1H, d, J = 8.4 Hz, ArH), 5.83–5.81
(1H, m, CH), 1.99 (3H, s, CH3). δC (100 MHz, Acetone-d6) 171.4 (Cq), 170.5 (Cq), 156.7 (Cq), 145.6, 127.1,
126.4 (Cq), 123.0, 118.6 (Cq), 118.3 (Cq), 115.7, 48.6 (CH), 22.6 (CH3). LRMS (ESI): m/z 309.94 [M (79Br) +
H]+, 311.82 [M (81Br) + H]+.
2-Benzamido-2-(6-bromobenzofuran-3-yl)-2-acetic acid (47): Ester 41 (69.3 mg, 0.179 mmol) was hydrolysed
using 2 M methanolic sodium hydroxide (5 equiv, 448 µL) according to General Procedure A to give the
compound in question as a white solid (64.2 mg, 96%); mp 209–212 ◦C. δH (500 MHz, DMSO-d6) 9.18
(1H, d, J = 7.1 Hz, NH), 8.13 (1H, s, ArH), 7.93–7.89 (3H, m, ArH), 7.70 (1H, d, J = 8.4 Hz, ArH), 7.54
(1H, t, J = 8.4 Hz, ArH), 7.50–7.44 (3H, m, ArH), 5.83 (1H, d, J = 7.2 Hz, CH). δC (100 MHz, DMSO-d6)
171.0 (Cq), 166.5 (Cq), 155.0 (Cq), 145.1, 133.59 (Cq), 131.6, 128.3, 127.7, 126.1 (Cq), 125.8, 122.3, 117.2
(Cq), 116.8 (Cq), 114.7, 48.2 (CH). LRMS (ESI): m/z 747.5 [2 M]+. HRMS (ESI): m/z 371.9920 [M (79Br)
−
H]−, 373,9904 [M (81Br) − H]−.
2-(6-Bromobenzofuran-3-yl)-2-(2-(4-methoxyphenyl)acetamido)acetic acid (48): Ester 42 (15 mg, 34.7
µmol)
was hydrolysed using 2 M methanolic sodium hydroxide (5 equiv, 87.0 L) according to General
µ
Procedure A to afford the compound in question as an off-white solid (8.4 mg, 58%); mp 159–161 ◦C.
δH (400 MHz, CD3OD) 7.80 (1H, s, ArH), 7.71 (1H, s, ArH), 7.48 (1H, d, J = 8.4 Hz, ArH), 7.35 (1H,
d, J = 8.4 Hz, ArH), 7.19 (2H, d, 8.6, ArH), 6.84 (2H, d, J = 8.6 Hz, ArH), 5.70 (1H, s, CH), 3.77 (3H, s,
OCH3), 3.52 (2H, s, CH2). LRMS (ESI): m/z 415.89 [M (79Br) + H]+, 417.85 [M (81Br) + H]+. HRMS (ESI):
m/z 416.0196 [M (79Br) − H]−, 418.0181 [M (81Br) − H]−.
2-(6-Bromobenzofuran-3-yl)-2-(2-(2-fluorophenyl)acetamido)acetic acid (49): Ester 43 (43.5 mg, 0.104 mmol)
was hydrolysed using 2 M methanolic sodium hydroxide (5 equiv, 259 µL) according to General

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Procedure A to afford the compound in question as a light yellow solid (20.0 mg, 50%); mp 209–211 ◦C
.
δH (500 MHz, DMSO-d6) 13.09 (1H, br s, COOH), 8.97 (1H, d, J = 7.0 Hz, NH), 8.07 (1H, s, ArH), 7.93 (1H,
s, ArH), 7.64 (1H, d, J = 8.5 Hz, ArH), 7.48 (1H, d, J = 8.5 Hz, ArH), 7.34–7.25 (2H, m, ArH), 7.15–7.10
(2H, m, ArH), 5.59 (1H, d, J = 7.0 Hz, CH), 3.60 (2H, s, CH2). δC (125 MHz, DMSO-d6) 171.4, 169.7, 161.1
(d, J = 243 Hz), 155.6, 145.3, 132.2 (d, J = 3.75 Hz), 129.13, 129.07, 126.6, 125.8, 124.6 (d, J = 3.73 Hz),
122.7, 117.8, 117.4, 115.4 (d, J = 21 Hz), 115.2, 48.3, 35.1. LRMS (ESI): m/z 404.4 [M − H]−. HRMS (ESI):
m/z 403.9985 [M (79Br) − H]−, 405.997 [M (81Br) − H]−.
2-(6-Bromobenzofuran-3-yl)-2-(methylsulfonamido)acetic acid (50): Ester 44 (115 mg, 318
µmol) was
hydrolysed using 2 M methanolic sodium hydroxide (5 equiv, 794 L) according to General Method A
µ
to afford the compound in question as a light yellow solid (75 mg, 68%); mp 140–142 ◦C. δH (400 MHz,
DMSO-d6) 8.16 (1H, d, J = 8.3 Hz, ArH), 8.06 (1H, s, ArH), 7.91 (1H, s, ArH), 7.73 (1H, d, J = 8.4 Hz,
ArH), 7.48 (1H, d, J = 8.4 Hz, ArH), 5.34 (1H, d, J = 8.3 Hz, CH), 2.92 (2H, s, CH2). δC (100 MHz,
DMSO-d6) 171.3 (Cq), 155.6 (Cq), 145.3, 126.5, 125.5 (Cq), 122.8, 117.8 (Cq), 117.7 (Cq), 155.2, 51.4 (CH),
41.6 (CH3). LRMS (ESI): m/z 345.95 [M (79Br) + H]+, 347.84 [M (81Br) + H]+. HRMS (ESI): m/z 345.9425
[M (79Br) − H]−, 347.9403 [M (81Br) − H]−.
2-(6-Bromobenzofuran-3-yl)-2-(phenylsulfonamido)acetic acid (51): Ester 45 (15.0 mg, 35.4
µmol) was
hydrolysed using 2 M methanolic sodium hydroxide (5 equiv, 88.5 L) according to General Method A
µ
to afford the compound in question as an off-white solid (13.3 mg, 92%); mp 179–182 ◦C. δH (500 MHz,
Acetone-d6) 7.83 (1H, s, ArH), 7.82–7.80 (2H, m, ArH), 7.69 (1H, s, ArH), 7.62 (1H, d, J = 8.4 Hz, ArH),
7.52 (1H, t, J = 7.4 Hz, ArH), 7.43 (2H, t, J = 7.7 Hz, ArH), 7.38 (1H, d, J = 8.4 Hz, ArH), 5.37 (1H, s, CH).
δC (125 MHz, Acetone-d6) 170.5, 156.6, 145.8, 142.0, 133.3, 129.7, 127.8, 127.0, 125.8, 123.0, 118.6, 117.8,
115.6, 52.2. HRMS (ESI): m/z 407.9707 [M (79Br) − H]−, 409.9614 [M (81Br) − H]−.
4.3. Expression and Purification of Unlabelled and 15N-Labelled EcDsbA
Unlabelled and 15N-labelled EcDsbA were prepared as previously described [17]. Briefly, both
unlabelled and 15N-labelled EcDsbA were expressed by autoinduction using appropriate media.
After harvesting the cells, periplasmic proteins were extracted by resuspending cell pellet with lysis
buffer (20 mM Tris-HCl, pH 8.0, 25 mM NaCl, 4 mg/mL colistin sulfate) and incubating for 18–24 h
with gentle stirring at 4 ◦C. After centrifugation, EcDsbA was purified from the supernatant using a
three-step purification method, including hydrophobic interaction chromatography, anion exchange
chromatography, and size exclusion chromatography. DsbA was oxidised with copper-phenanthroline
and buffer exchanged to the final storage buffer (20 mM HEPES, pH 7.0).
4.4. Protein Crystallisation and Structure Determination
EcDsbA was crystallised as previously described [17]. Briefly, 1
mixed with an equal volume of crystallisation buffer (11–13% PEG8000, 5–7.5% glycerol, 1 mM CuCl2,
100 mM, sodium cacodylate pH 6.1) and equilibrated against 500
L of reservoir buffer at 20 ◦C using
hanging drop vapour diffusion. For compound soaking, crystals were transferred into 2 L drops
µL of 30 mg/mL EcDsbA was
µ
µ
of 24% PEG8000, 22% glycerol, and 100 mM sodium cacodylate (pH 6.1) containing a compound of
interest at the final concentration of 10 mM (5% DMSO) and were incubated for 3–6 h. Crystals were
mounted on loops and flash-cooled in liquid nitrogen.
Datasets were collected at the Australian synchrotron on MX1 and MX2 beamlines using the
Blue-Ice software (Irvine, CA, USA) [31]. MX1 beamline was equipped with an ADSC Quantum 210r
detector and MX2 with an ADSC Quantum 315r detector. All datasets were indexed and integrated
with iMOSFLM [32] or XDS [33]—and scaled using AIMLESS [34,35] or SCALA [36]. Phasing was
performed by molecular replacement with Phaser [37] using the previously solved structure of EcDsbA
as a search model (PDB code 1FVK) [38]. The final structure was obtained after several rounds of
manual model building using Coot [39] and refinement in phenix.refine [40]. Data collection and
refinement statistics are summarised in Table S1. Structures, factors and coordinates have been

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deposited in the Protein Data Bank (PDB; http://www.pdb.org) under the accession codes of 6PMF,
6POQ, 6POH, 6PML, 6PVY, 6POI, and 6PVZ.
4.5. NMR Spectroscopy
For ligand-detected NMR spectroscopy, two samples (compound alone and compound + protein)
were prepared for each compound. Unlabelled EcDsbA was prepared at 10
sodium phosphate, pH 6.8, 25 mM NaCl, 100 M 4,4-dimethyl 4-silapentane-1-sulfonic acid (DSS),
and 10% 2H2O. Compound was added to the protein sample to achieve final concentration of 500
(2% 2H6-DMSO). 1H 1D spectrum was acquired for each compound at 500
M as a reference spectrum.
µM in a buffer of 50 mM
µ
µM
µ
All ligand-detected NMR experiments (STD, CPMG, and waterLOGSY) were acquired at 298 K on a
Bruker Avance III 600 MHz NMR spectrometer equipped with a 5 mm TXI CryoProbe (Bruker, Billerica,
MA, USA). STD spectra were acquired with 3 s of saturation at 1 ppm (on-resonance) and 33.3 ppm
(off-resonance). WaterLOGSY spectra were acquired for compound alone and compound + protein
with a 0.52 s acquisition time, 3 s relaxation delay, and 3 s Nuclear Overhauser effect (NOE) mixing
time. CPMG spectra for compound alone and compound + protein were acquired with a constant spin
echo delay of 1 ms and spin-lock period of 350 ms. The data was analysed using Topspin3.5 (Bruker)
and DSS was used to reference the spectra.
For the 2D 1H-15N-HSQC titration experiment, 100 M of 15N-labelled EcDsbA in a buffer of
µ
50 mM HEPES, pH 6.8, 50 mM NaCl, 2% 2H6-DMSO, and 10% 2H2O, was titrated with increasing
concentrations of compounds (0, 0.625, 0.125, 0.25, 0.5, and 1 mM). Data were acquired on a Bruker
600 MHz spectrometer equipped with a 5 mm TXI CryoProbe and a Bruker 700 MHz spectrometer
equipped with a 5 mm TXI CryoProbe. Data was processed by Topspin3.5 (Bruker) and analysed by
Sparky (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco (UCSF)).
Weighted chemical shift perturbations (CSP) that were observed upon addition of compounds to
EcDsbA were calculated using an Equation (1) in [41]:
q
CSP = ∆δH2 + (0.2 × ∆δN)2
(1)
where ∆δH and ∆δN denote the change in chemical shift of amide proton and nitrogen resonances upon
addition of the compound. Equilibrium dissociation constants (KD) were determined by fitting the plot
of CSP against compound concentrations using one site with ligand depletion model (Equation (2)):
ꢀ
q
ꢃ
ꢁ
ꢂ
CSPmax × (KD + Pt + Lt) −
(KD + Pt + Lt)2 − 4 × Pt × Lt
2 × Pt
CSP =
(2)
where CSP is the measured CSP at a given concentration, CSPmax is the CSP observed at the saturating
concentration of compound, and Pt and Lt are the total protein and compound concentrations. The
ligand efficiency (LE) is calculated using the Equation (3) in [42]:
RTln(KD)
∆G
HAC
LE = −
= −
(3)
HAC
where R is gas constant (1.9858775 cal K−1 mol−1), T is temperature (298 K), KD is equilibrium
dissociation constant, and HAC is the number of heavy (non-hydrogen) atoms.
Supplementary Materials: The following are available online: Figure S1: Electron density maps for compounds.
Figure S2: Co-crystal structure of 50. Table S1: Data collection and refinement statistics. NMR Figures: 1H and
13C-NMR characterisation of all compounds presented in the main text.
Author Contributions: Conceptualisation, investigation, and formal analysis, L.F.D., G.W., O.V.I., M.J.S., B.H., and
B.M.A.; methodology, validation, data curation, and visualisation, L.F.D., G.W., and O.V.I.; writing—original draft
preparation, L.D and G.W.; writing—review and editing, O.V.I., M.J.S., B.H., and B.M.A.; supervision, resources,
software, project administration, and funding acquisition, M.J.S., B.H., and B.M.A.

Molecules 2019, 24, 3756
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Funding: This research was supported by National Health and Medical Research Council (NHMRC) project
(grants 1099151 and 1144046). Dr Luke Duncan would like to acknowledge La Trobe University as a recipient
of La Trobe University Postgraduate Research Scholarship. Dr Begoña Heras is supported by an Australian
Research Council (ARC) Future Fellowship (FT130100580). We would also like to acknowledge the La Trobe
University-Comprehensive Proteomics Platform and the Monash Fragment Platform (MFP) for providing
infrastructure and expertise. This research was undertaken on the MX1 and MX2 beamlines at the Australian
Synchrotron, part of Australian Nuclear Science and Technology Organisation (ANSTO).
Acknowledgments: We are very grateful for the ongoing support and many helpful chemical discussions provided
by Les Deady.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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