Indoline‐6‐sulfonamide inhibitors of the bacterial enzyme dape

Article abstract of DOI:10.3390/antibiotics9090595

Inhibitors of the bacterial enzyme dapE‐encoded N‐succinyl‐L,L‐diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18) hold promise as antibiotics with a new mechanism of action. Herein we describe the discovery of a new series of indoline sulfonamide DapE inhibitors from a high‐throughput screen and the synthesis of a series of analogs. Inhibitory potency was measured by a ninhydrin‐based DapE assay recently developed by our group. Molecular docking experiments suggest active site binding with the sulfonamide acting as a zinc‐binding group (ZBG).

Full text of DOI:10.3390/antibiotics9090595

antibiotics
Article
Indoline-6-Sulfonamide Inhibitors of the Bacterial
Enzyme DapE
Cory T. Reidl 1,2, Tahirah K. Heath 1, Iman Darwish 1, Rachel M. Torrez 1 , Maxwell Moore 1,
Elliot Gild 1, Boguslaw P. Nocek 3, Anna Starus 1, Richard C. Holz 4,* and Daniel P. Becker 1,
*
1
Department of Chemistry and Biochemistry, Loyola University Chicago, 1032 West Sheridan Road,
Chicago, IL 60660, USA; cory.reidl@northwestern.edu (C.T.R.); tkheath10@gmail.com (T.K.H.);
idarwish@luc.edu (I.D.); torrezrm@med.umich.edu (R.M.T.); maxmoore@scripps.edu (M.M.);
egild@luc.edu (E.G.); astarus@luc.edu (A.S.)
2
Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute,
Center for Molecular Innovation and Drug Discovery, Northwestern University,
Evanston, IL 60208-3113, USA
Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division,
Argonne National Laboratory, Argonne, IL 60439, USA; boguslaw.nocek@abbvie.com
Department of Chemistry, Colorado School of Mines, 1500 Illinois St., Golden, CO 80401, USA
Correspondence: rholz@mines.edu (R.C.H.); dbecke3@luc.edu (D.P.B.)
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 August 2020; Accepted: 9 September 2020; Published: 11 September 2020
Abstract: Inhibitors of the bacterial enzyme dapE-encoded N-succinyl-l,l-diaminopimelic acid
desuccinylase (DapE; EC 3.5.1.18) hold promise as antibiotics with a new mechanism of action.
Herein we describe the discovery of a new series of indoline sulfonamide DapE inhibitors from a
high-throughput screen and the synthesis of a series of analogs. Inhibitory potency was measured by
a ninhydrin-based DapE assay recently developed by our group. Molecular docking experiments
suggest active site binding with the sulfonamide acting as a zinc-binding group (ZBG).
Keywords: diaminopimelate desuccinylase; DapE; ninhydrin enzyme assay; indoline; sulfonamide;
enzyme inhibition; antibiotic
1. Introduction
Although momentarily eclipsed by the global COVID-19 pandemic, we continue to face a
global healthcare crisis due to the increasing resistance of bacteria to all approved antibiotics.
Invasive methicillin-resistant Staphylococcus aureus (MRSA) is a serious and growing health problem [
Newly discovered strains of MRSA show antibiotic resistance even to vancomycin, which has served
as a drug of last resort for treating systemic infections [ ]. Moreover, multi-drug resistant tuberculosis
1].
2
(TB), an infectious disease caused by the pathogen Mycobacterium tuberculosis (Mtb), continues to be a
leading cause of death from an infectious agent worldwide. In 2018, the World Health Organization
(WHO) estimated that 1.5 million of the approximately 10 million people who acquired a TB infection
succumbed to this devastating chronic illness. [
bacterial infections by antibiotic-resistant bacteria [
with new mechanisms of action. A very attractive and underexplored bacterial enzyme target is the
dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE, E.C. 3.5.1.18) [ ] that is present
3,
4] The steady increase in mortality and morbidity from
5] reveals the urgent need to discover antibiotics
6
in all Gram-negative and most Gram-positive bacteria. DapE is one of the key enzymes in the lysine
biosynthetic pathway in bacteria responsible for the biosynthesis of lysine and meso-diaminopimelate
(m-DAP) [7], that are both essential for cell-wall synthesis. The enzyme DapE catalyzes the hydrolysis
of N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) to succinate and l,l-diaminopimelic acid (l,l-DAP)
(Figure 1). The deletion of the DapE gene has been shown to be lethal to Helicobacter pylori and
Antibiotics 2020, 9, 595; doi:10.3390/antibiotics9090595
www.mdpi.com/journal/antibiotics

Antibiotics 2020, 9, 595
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Mycobacterium smegmatis, demonstrating the indispensable role of this enzyme in bacterial survival
and pathogenesis [ ]. Furthermore, the lack of a similar enzymatic pathway in humans suggests that
8,9
inhibition of DapE would provide selective toxicity to bacteria-free mechanism-based side effects in
humans, making DapE a very promising target for antibiotics with a new mechanism of action [6].
Figure 1. Hydrolysis of N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) and N6-Me-SDAP by HiDapE.
L,L-SDAP (1a) and N6-methyl SDAP analog (1b) and with the formation of hydrolysis products
succinate (2) and L,L-diaminopimelic acids (l,l-DAP) (3a,b).
To conveniently measure the inhibitory potency of test compounds against DapE, we recently
developed a ninhydrin-based assay employing the unnatural but well-tolerated substrate
N6-methyl-l,l-SDAP (1b) [10]. Enzymatic cleavage of 1b affords primary amine product 3b (Figure 1),
which can be quantified spectrophotometrically following treatment with ninhydrin. Importantly,
this assay avoids the significant background signal that would be produced by the reaction of the
primary amine in native substrate 1a with ninhydrin.
The first X-ray crystal structure of apo-DapE, lacking active-site zinc atoms, was solved in
2005 [11] and was followed by structures of mono- and di-Zn forms from Haemophilus influenzae [12
]
as well as mono- and di-Zn forms from N. meningitidis [13] In sharp distinction to the reported
open-DapE structures, some of us recently reported a new X-ray crystal structure of DapE revealing
the heretofore-unknown closed conformation of dimeric DapE containing the products of enzymatic
cleavage, succinate, and diaminopimelic acid, bound in the active site [14]. This structure uncovered
the role of His194B of the opposite peptide chain in the dimeric enzyme, which moves 10.1 Å to provide
a key H-bond in the active site, activating the substrate for enzymatic cleavage [14]. This new insight
into the enzymatic mechanism also explains the observed inactivity of monomeric DapE constructs [10].
This products-bound DapE structure has enabled further refinement of a mechanistic hypothesis for
amide bond cleavage by DapE enzymes, supported by our products-bound transition state modeling
(PBTSM) approach [14,15], which in turn will facilitate inhibitor identification.
Earlier, we screened a small, focused library of compounds containing zinc-binding groups
that led to the identification of the thiol-containing angiotensin-converting enzyme (ACE) inhibitor
captopril as a low micromolar competitive inhibitor (IC50 = 3.3 µM) of DapE from Haemophilus influenza
(HiDapE) [16]. We then reported a high-resolution (1.8 Å) X-ray crystal structure of captopril bound to
the active site of DapE from Neisseria meningitidis (NmDapE), revealing the dinuclear Zn(II) metals
bridged by the thiolate of captopril [13]. This structure has served as a model for in silico approaches
to designing potential new inhibitors of DapE [17].
2. Results
We now report a high-throughput screen of ~33,000 compounds, which resulted in the discovery
of two structurally similar indoline sulfonamide derivatives
4 and 5 (Figure 2) as inhibitors of DapE,
showing >20% inhibition at 12 M that was selected as an arbitrary cutoff. We were pleased that these
µ
two indoline hit structures provided mutual confirmation of one another. Furthermore, indolines are a
privileged scaffold in medicinal chemistry, as there are several examples of indoline-containing drugs
approved for a variety of treatments: advanced or transitional cell carcinoma of the urothelial tract

Antibiotics 2020, 9, 595
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(vinflunine) [18], glaucoma and severe anticholinergic toxicity (physostigmine) [19], and schizophrenia
in adults (lumateperone) [20 21], or are under investigation, as for benign prostatic hyperplasia
(BPH) [22]. Furthermore, sulfonamides are pervasive in agrochemicals and in pharmaceuticals
,
across therapeutic areas [23
intermediates [28].
–27], and primary sulfonamides have recently been leveraged as versatile
Figure 2. N-Acetyl-6-sulfonamide indoline screening hits from the high-throughput screen (HiTS).
2.1. Overview and Regiochemistry
Although the indoline sulfonamide hit structures were listed by the supplier as the corresponding
indoline-7-sulfonamides, it was concluded that the compounds must be 6-sulfonamides (as illustrated
in Figure 2), as there were no general methods in the literature to prepare the 7-sulfonamide hits.
Specifically, electrophilic aromatic chlorosulfonylation of 5-bromo-N-acetyl-indoline affords the product
of electrophilic aromatic substitution at the sterically preferred 6-position rather than the presumed
7-position, as clearly and definitively demonstrated by Borror [29]. Yet, widespread errors persist in the
literature regarding the regiospecificity of sulfonylation of indolines. Herein, we report the synthesis
and structure-activity relationships (SAR) of a series of 5-halo-6-indolinesulfonamides as new DapE
inhibitors assessed using our new ninhydrin-based assay.
2.2. Molecular Docking Experiments
Molecular docking employing the open crystal structure of DapE (PDB 5UEJ) was used to
determine potential binding poses of inhibitors in the active site of the enzyme. Although the easily
cloned and expressed HiDapE enzyme was used in the assay, HiDapE and NmDapE share a very
high sequence homology of 55% with no sequence gaps and bear the same active site architectures,
including metal-binding residues and substrate-binding residues necessary for hydrolytic activity.
We, therefore, decided to use the atomic-level resolution PDB 5UEJ structure of NmDapE for docking.
Docking of the indoline sulfonamide lead structures (
4
and
5) suggests these compounds bind to the
di-zinc active site indicative of competitive inhibition (Figure 3). Specifically, compound
4
docked to the
active site of DapE, which suggests coordination of the two sulfonamide oxygen atoms with the two zinc
atoms with simultaneous hydrogen bonding between the N-acetyl carbonyl and the guanidine moiety
of Arg258A. Additionally, in this conformation, the proton on the nitrogen atom of the sulfonamide
moiety is poised to form a strong hydrogen bond with the backbone carbonyl of Thr325A.
2.3. Chemistry
Bromination of the N-acetyl indoline at the 5-position was performed by exposure of
N-acetylindoline
acetate to afford 7a in an 83% yield (Scheme 1). Chlorination with N-chlorosuccinimide was more
sluggish and provided the 5-chloroindoline derivative Figure S1 7b 31] in variable yield and required
6 to N-bromosuccinimide [29,30] in the presence of a catalytic amount of ammonium
[
a tedious chromatographic purification. We, therefore, turned to a continuous flow synthesis condition
for the synthesis of 5-chloroindoline derivative 7b using conditions adapted from general flow
methods [32], in particular, those developed by Pelleter [33]. Optimized continuous flow protocols
afforded 7b at a superior reaction rate and throughput, and provided excellent purity after direct
crystallization of the product stream from water (Figure 4).

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Figure 3. Docked N-isoamyl indoline sulfonamide hit compound 4 (cyan). The majority of the active
site residues were contributed to by subunit I of the DapE dimer (green residues), but two residues
came from subunit II of the dimer (orange).
X
X
S
X
S
R1R2NH
ClSO3H
O
halogenation
O
N
N
R1
N
Et3N, CH2Cl2
N
N
Cl
O
O
O
R2
O
O
O
6
7a
X = Br
7b X = Cl
8a
X = Br
8b X = Cl
9a-n
X = Br
10a-g X = Cl
Scheme 1. Synthesis of N-acetyl-5-bromo-6-sulfonamide indoline derivatives.
EA
(wash line)
N-acetylindoline
100 psi
BPR
(2.0 M in DMF)
q = 275 mL/m
N-chlorosuccinimide
(2.0 M in DMF, 1.2 eq)
Cl
80°C
RT
N
7b
O
Figure 4. Flow chemistry apparatus for preparation of N-acetyl-5-chloroindoline 7b.
The 5-chloro and 5-bromo N-acetyl indoline analogs 7a and 7b were then subjected to
chlorosulfonation at the 6-position by heating at 65–70 ◦C in neat chlorosulfonic acid. The reaction
mixture was quenched over ice to afford the corresponding sulfonyl chlorides 8a [29] and 8b, respectively,
in good purity based on nuclear magnetic resonance (NMR) spectroscopy, which were used directly in
the next step.
A variety of primary and secondary alkyl amines were reacted with the 5-halo-6-sulfonyl chloride
derivatives 8a or 8b in the presence of triethylamine base to afford a series of sulfonamide inhibitors
9a–n and 10a–f, respectively, according to general literature precedent [29,30]. In instances where
the reacting amine was secondary, a catalytic amount of DMAP was added to the reaction solution.
The sulfonamide products were isolated by extraction followed by passage through an activated carbon
plug and recrystallization from chloroform–ethyl acetate to afford the final inhibitors in high purity.

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2.4. DapE Enzyme Inhibition and Structure–Activity Relationships
The compounds synthesized along with their DapE inhibitory potencies measured using
the N-Me-SDAP substrate are summarized in Table 1. We kept the N-acetyl group as well as
the 5-halogen moiety constant in this first series while varying the sulfonamide N-substituent.
The 5-bromo-6-isoamylsulfonamide hit
of the original hit, which was found to exhibit a modest but measurable 42% inhibition at 200
The corresponding isobutyl derivative 9a was less potent, with a 20% inhibition at 200 M. Among other
secondary sulfonamides synthesized, N-cyclohexyl derivative 9b was somewhat more potent with an
IC50 of 162 M, whereas N-benzyl and N-t-butyl derivatives 9c and 9d showed 56% and 39% inhibition
at 200 M and 100 M, respectively. Several derivatives had poor solubility, limiting the ability to
test inhibition at higher concentrations. The addition of more polar ester functionalities provided the
nearly inactive glycine ester 9e, while the -alanine ester 9f was found to be moderately potent with
an IC50 of 118 M. Valine ester 9g was more potent, with an IC50 of 82 M, whereas the phenylalanine
ester 9h was less potent, with 61% inhibition at 200 µM.
4
was synthesized and tested first to confirm the activity
µM.
µ
µ
µ
µ
β
µ
µ
Table 1. Inhibition of DapE enzyme by synthesized compounds 4, 9a–n, and 10a–f a.
IC50 (µM) or %
0
4
X
R
MW
clogP
mp (◦C)
Inhibition a
Br
389.3
2.79
190–191
42% at 200 µM
9a
9b
9c
Br
Br
Br
374.0
400.0
408.0
2.26
2.84
2.57
225.4–226.1
226–228
20% at 200 µM
162
205–208
56% at 200 µM
9d
Br
374.0
2.04
235.7–236.1
39% at 100 µM
H
N
9e
9f
Br
Br
390.0
404.0
0.79
1.1
179–182
190–193
17% at 200 µM
MeO2C
118
9g
Br
432.0
2.0
190.9–191.7
82

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Table 1. Cont.
IC50 (µM) or %
0
X
R
MW
clogP
mp (◦C)
Inhibition a
9h
Br
480.0
2.5
184.0–184.8
61% at 200 µM
9i
9j
Br
Br
386.0
372.0
2.1
1.6
212–214
238–240
133
97
9k
Br
420.0
2.6
215–218
86
5% at 20 µM,
insol. at 200 µM
9l
9m
9n
Br
Br
Br
402.1
434.1
374.0
3.0
1.5
2.0
118–120
110–111
195–196
26% at 100 µM
99
10a
10b
10c
Cl
Cl
Cl
344.1
356.1
342.1
2.8
2.9
2.2
179.1–180.4
230.0–230.5
199.7–201.4
54
58% at 200 µM
44
10d
10e
Cl
Cl
328.1
358.1
1.6
3.1
199.7–200.4
118–120
172
88
10f
Cl
390.1
1.5
93.2–94.4
>200
a
IC50 values were determined from a set of three or four % inhibition points, and percent inhibition values were
determined at a single concentration; insol. = insoluble.

Antibiotics 2020, 9, 595
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Among secondary sulfonamides examined in this work, the simplified piperidine analog 9i
was synthesized to eliminate the stereochemical complexity of dimethyl piperidine hit compound
Piperidine sulfonamide 9i exhibited an IC50 of 133 M, with the IC50 of the corresponding pyrrolidine
sulfonamide 9j, improving to 97 M. The bulkier and more rigid indoline sulfonamide 9k was
synthesized to take advantage of hydrophobic interactions in the active site and resulted in an IC50 of
86 M. Conversely, the more flexible acyclic secondary sulfonamides 9l and 9m were significantly less
5
.
µ
µ
µ
potent, whereas N,N-diethyl sulfonamide 9n revealed an IC50 of 99 µM comparable to 9j.
To decrease the molecular weight and logP, several 5-chloro derivatives were synthesized
and evaluated for DapE inhibitory potency. Gratifyingly, the 5-chloro-isoamylsulfonamide 10a
with an IC50 of 54
The 5-chloro-N-cyclohexyl derivative 10b exhibited comparable potency to the corresponding 5-bromo
derivative 9b. Pyrrolidine 5-chloro derivative 10c with an IC50 of 44 M was ~3 more potent than
the corresponding 5-bromo derivative 9i, but the trend was not maintained for 5-chloro pyrrolidine
derivative 10d with an IC50 of 172 M, relative to 5-bromo derivative 9j with an IC50 of 97 M.
M was again more potent than the 5-bromo
µM was ~5
×
more potent than the corresponding 5-bromo derivative 4.
µ
×
µ
µ
The di-n-propyl derivative 10e with an IC50 of 88
derivative 9l.
µ
Direct comparison of inhibitory data for 5-chloro and 5-bromo analogs shows that the inclusion of
a 5-chloro substituent generally increased the inhibition of DapE relative to the 5-bromo series, with the
5-chloro piperidine 10a and 5-chloro isopentyl 10c showing the two most potent IC50 values in the series
at 54 µM and 44 µM, respectively. Additionally, while some 5-bromo analogs showed solubilities at the
higher concentrations, water solubilities increased on average for the chloro derivatives, making them
more attractive as potential drug candidates.
3. Summary
In summary, a series of N-acetyl-5-halo-6-sulfonamide indolines was designed, synthesized,
and assayed for inhibition against DapE. The original lead inhibitor isopentyl sulfonamide
hit analog piperidine sulfonamide 9i were found to be the modest inhibitors, exhibiting IC50 values
of >200 M and 130 M, respectively. Significantly higher potencies were realized by varying the
4 and
µ
µ
N-substitution of the sulfonamide moiety. Moreover, replacing the aryl bromide substituent with the
corresponding chloride led to noticeably increased inhibition against DapE for nearly all analogs tested,
as well as more favorable solubility properties. In silico studies were used to rationalize the improved
potency of several key compounds. The 5-chloro analogs of the most promising 5-bromo ligands
will be synthesized in future studies for continued SAR exploration, as will as N-acyl sulfonamides
and sulfonylurea derivatives thereof. Inclusion of water-solubilizing groups, such as carboxylates,
which also bind tightly to Zn(II) atoms, and tertiary amines, which will allow the production of
ammonium salts, will be pursued in future efforts. This study represents the first reported inhibitory
data and SAR analysis of DapE with the newly reported ninhydrin-based enzymatic assay and will be
used to inform drug design strategies in the development of DapE inhibitors as potential antibiotics
with a new mechanism of action.
4. Materials and Methods
4.1. General Experimental Methods
All reagents were used as purchased without further purification unless otherwise noted.
All solvents were distilled before use. All reactions were performed under an inert atmosphere
of nitrogen. For chromatography, silica gel 60A, 40
−
75
µ
m (200
×
400 mesh) was utilized,
and aluminum-backed silica gel 200
µm plates were used for thin-layer chromatography (TLC).
1H (proton) NMR spectra were obtained employing either a 400 MHz or a 500 MHz spectrometer
with tetramethylsilane (TMS) as the internal standard. NMR spectra were processed using the Mnova
NMR software program by Mestrelab Research. The purity of all assayed compounds was confirmed

Antibiotics 2020, 9, 595
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to be
≥
95% unless otherwise noted, as determined by high-performance liquid chromatography
(HPLC) utilizing a mobile phase A comprises 5% acetonitrile B in water and a mobile phase B = 0.1%
trifluoroacetic acid (TFA) in acetonitrile, employing a gradient of 60% B increasing to 95% over 10 min,
holding at 95% B for 5 min, and then returning to 60% B and finally holding for 5 min. High-resolution
mass spectra (HRMS) spectra were measured on a Time-of-Flight (TOF) instrument utilizing the
method of electrospray ionization (ESI). High-resolution mass spectral (HRMS) data were obtained at
the Integrated Molecular Structure Education and Research Center (IMSERC, Northwestern University,
Evanston, IL, USA) on an Agilent 6210A TOF mass spectrometer in the positive ion mode coupled to
an Agilent 1200 series high-performance liquid chromatography (HPLC) system. Data were processed
using MassHunter software version B.04.00.
4.2. Molecular Docking Protocol
Molecular models were developed using the Molecular Operating Environment (MOE)
computational suite’s Builder utility, followed by minimization in the gas phase using the MMFF94X1
force field. The X-ray crystal structure of NmDapE was then uploaded into the MOE and prepared
for docking using MOE’s Structure Preparation utility. This high-resolution DapE structure from
Neisseria meningitidis (PDB 5UEJ, 1.30 Å resolution) was used as a docking receptor due to the
high quality of refinement at the dimerization loops, which play an important role in substrate
binding. The hydrogen-bonding network of the docking receptor was further optimized at pH 7.4 by
automatically sampling different tautomer/protomer states using Protonate3D, which calculates optimal
protonation states, including titration, rotamer, and “flips” using a large-scale combinatorial search.
The substrate-binding cleft of chain A, which includes the dinuclear Zn(II) metal center, was surveyed
using MOE’s Site Finder utility and populated with inactivated dummy atoms that define the docking
location. Following preparation of the NmDapE docking receptor model, an induced-fit molecular
docking using the previously generated ligand conformation database of 4 was carried out with solvent
atoms inactivated at the docking site specified by the dummy atoms populating the substrate-binding
cleft of chain A of the docking receptor. The proxy triangle method with London
50 data points, which were further refined using the induced fit method with GBVI/WSA
∆
G scoring generated
G scoring to
∆
obtain the top 30 docking results. The Amber12:EHT3 force field was used to perform these calculations.
The top ligand docking pose is shown in Figure 3.
4.3. DapE Enzyme Inhibition
DapEinhibitionwasmeasuredaccordingtothemethodpreviouslydescribed[10].Inbrief, theenzymatic
activity of DapE was measured in triplicate at 570 nm by the Ruhemann’s purple complex formed
through the reaction of the exposed primary amine and ninhydrin. To 175
(4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid)bufferatpH7.5with5 Lof1
30 ◦C, 20
L of 10 mM monomethyl SDAP ((2S,6S)-2-(3-carboxypropanamido)-6-(methylamino)heptanedioic
µL of 50 mM HEPES
µ
µMDapEstocksolutionat
µ
acid) TFA salt was added. The reaction was allowed to proceed for 10 min and quenched by heating at
100 ◦C for 1 min and subsequently cooling on ice for 1 min. To the cooled reaction, 2% ninhydrin reagent
in 100% DMSO (final volume 300
quenched by placing in ice water for 2 min, and the absorbance of an 80
µ
L) was added and subsequently heated to 80 ◦C for 15 min. This was
µL aliquot was read at 570 nm via a
microplate reader. These reactions were set as 100% standard enzyme activity of DapE.
4.4. Protein Expression and Purification
DapE enzymes were cloned, expressed, and purified according to the standard protocol as
described previously for HiDapE [12] and the DapE from Neisseria meningitidis (NmDapE) [13]. The cell
pellet was thawed, and the cells were disrupted by sonication. The resulting cell debris was pelleted by
centrifugation at 15,000 rpm for 40 min. at 4 ◦C and the supernatant was loaded onto a column packed
with HisTrap HP resin from GE Healthcare and washed with twenty-bed volumes of lysis buffer.
The His6-tagged HiDapE enzyme was then eluted with an elution buffer comprising 500 mM NaCl,

Antibiotics 2020, 9, 595
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5% glycerol, 50 mM HEPES, pH 8.0, 250 mM imidazole, and 10 mM 2-mercaptoethanol. The His6-tag
was then removed with His6-tagged tobacco etch virus (TEV) protease for 16 h at 4 ◦C in 50 mM HEPES,
pH 8.0. The cleaved DapE protein was then concentrated with a Centricon (30,000-MW cutoff; Amicon)
to a volume of 3 mL and purified on a HiLoad 16/600 Superdex 200 Prep Grade column (GE Healthcare,
Marlborough, MA, USA). The resulting solution was mixed with 5 mL of His-Trap HP resin packed on
a column to remove the remaining cleaved His6-tag, uncut protein, and the His6-tagged TEV protease,
while the eluent containing HiDapE was collected and washed with crystallization buffer (150 mM
NaCl, 20 mM HEPES pH 8.0, and 1 mM TCEP) and concentrated to a concentration of ~20 mg/mL.
4.5. Synthetic Organic Chemistry
4.5.1. 1-Acetyl-5-Chloroindoline (7b)
According to a general procedure, [31] N-chlorosuccinimide (NCS, 1.74 g, 13.0 mmol) was
added to a solution of N-acetylindoline 6 (2.00 g, 12.4 mmol) and NH4OAc (96.1 mg, 1.24 mmol)
in acetonitrile (65.1 mL), slowly and stirred at room temperature under air and monitored by TLC.
The completed reaction was partitioned between methylene chloride and washed with 10 mL water,
10 mL HCl (1M), 10 mL brine solution before drying over sodium sulfate and concentration afforded
1
the 1-acetyl-5-chloroindoline 7b with spectral data matching the literature [34]. H NMR (500 MHz
CDCl3):
δ 8.13 (d, J = 8.5 Hz, 1H), 7.15 (d, J = 9.5 Hz, 1H), 7.14 (s, 1H), 4.07 (t, J = 8.0 Hz, 2H), 3.18 (t,
J = 8.5 Hz, 2H), 2.22 (s, 3H). 13C NMR (125 MHz, CDCl3):
δ 168.8, 141.8, 133.2, 128.6, 127.6, 124.7, 124.8,
117.9, 49.1, 28.0, 24.3.
4.5.2. 1-Acetyl 5-Bromoindoline-6-Sulfonyl Chloride (8a)
Chlorosulfonic acid (18.7 mL) was added dropwise by an addition funnel to an oven-dried
round-bottom flask containing N-acetyl-5-bromoindoline 7a (2.50 g, 10.4 mmol). The neat reaction
was stirred at 60–70 ◦C for 3 h. The resulting black solution was cooled to room temperature and then
quenched by pouring slowly over ice with stirring. An off-white precipitate was isolated via vacuum
1
filtration, washed with deionized water, and dried in vacuo to provide 8a (1.83 g, 52%) [35]. H NMR
(500 MHz, Acetone-d6)
δ 8.99 (s, 1H), 7.84 (t, J = 1.2 Hz, 1H), 4.34 (t, J = 8.7 Hz, 2H), 3.42 (t, J = 8.7 Hz,
2H), 2.24 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
δ 169.2, 146.0, 142.4, 135.1, 130.3, 117.1, 113.3, 49.2, 27.4,
284 24.7.
4.5.3. 1-Acetyl-5-Chloroindoline-6-Sulfonyl Chloride (8b)
Chlorosulfonic acid (3.75 mL) was added to chloroindoline 7b (500 mg, 2.56 mmol) dropwise
via a pressure-equalizing funnel with stirring, and the solution was then heated to 60–70 ◦C for 3 h.
The resulting black solution was cooled to room temperature and carefully quenched by pouring
slowly over solid ice with stirring. The resulting light yellow precipitate was collected by vacuum
filtration, washed with water (3
chloride 8b (368 mg, 49%). H NMR (500 MHz, DMSO-d6)
2H), 3.11 (t, J = 8.5 Hz, 2H), 2.15 (s, 3H).
×
10 mL), and dried under vacuum to produce the desired sulfonyl
1
δ 8.57 (s, 1H), 7.19 (s, 1H), 4.09 (t, J = 8.5 Hz,
4.5.4. General Procedure for the Synthesis of N-acetyl 5-Bromo-6-Sulfonamide Indolines (
To a stirred solution of sulfonyl chloride 8a or 8b (1 eq, 0.148 mmol) and triethylamine (1.25 eq,
L, 0.185 mmol) in methylene chloride was added the requisite amine (1.25 eq, 0.185 mmol),
and the reaction was stirred at room temperature until the reaction was deemed complete by TLC.
The reaction was then diluted with methylene chloride, washed with water (1 ), twice with 1 M HCl,
4, 9 and 10)
26.0
µ
×
and then once with brine. The solution was dried over Na2SO4, filtered, and concentrated under
vacuum to provide the indoline sulfonamide products. Secondary amines and nitrogen heterocycles
were reacted in the presence of 4-dimethylaminopyridine (DMAP) as a catalyst (10 mol%), and in those

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cases, the reactions proceeded more slowly than reactions employing primary amines. The synthesis
of 4, 9a–n, and 10a–f were accomplished using this general procedure.
4.5.5. 1-Acetyl-5-Bromo-N-Isopentylindoline-6-Sulfonamide (4)
Light yellow solid (114 mg, 99%): mp 190–191 ◦C. 1H NMR (500 MHz, CDCl3)
δ 8.88 (s, 1H),
7.48 (s, 1H), 5.04 (s, 1H), 4.13 (t, J = 8.6 Hz, 2H), 3.29–3.21 (m, 2H), 2.91 (d, J = 6.6 Hz, 2H), 2.23 (s, 3H),
1.61 (dt, J = 13.3, 6.8 Hz, 1H), 1.38 (q, J = 7.5 Hz, 2H), 0.84 (d, J = 6.6 Hz, 6H). 13C NMR (126 MHz,
CDCl3) δ 168.9, 142.9, 137.9, 137.3, 130.7, 119.2, 112.9, 49.0, 41.7, 38.3, 27.6, 25.4, 24.1, 22.3. HRMS-ESI
[M + H]+ calculated for C15H22BrN2O3S+, 388.04564; found, 388.04563.
4.5.6. 1-Acetyl-5-Bromo-N-Isobutylindoline-6-Sulfonamide (9a)
Tan solid (93 mg, 84%): mp 215 ◦C (softens), 225.4–226.1 ◦C; 1H NMR (500 MHz, CDCl3)
δ 8.86
(s, 1H), 7.47 (s, 1H), 5.13 (t, J = 6.4 Hz, 1H), 4.13 (t, J = 8.6 Hz, 2H), 3.25 (t, J = 8.6 Hz, 2H), 2.70 (t, J = 6.6 Hz,
2H), 2.23 (s, 3H), 1.74 (dq, J = 13.4, 6.7 Hz, 1H), 0.89 (d, J = 6.7 Hz, 6H). 13C NMR (126 MHz, CDCl3)
δ
169.2, 143.1, 138.2, 137.7, 130.9, 119.2, 113.1, 51.0, 49.2, 46.1, 28.7, 27.8, 24.3, 20.2, 8.9. HRMS-ESI:
[M + H]+ calculated for C14H20BrN2O3S+, 374.02993; found, 377.02998.
4.5.7. 1-Acetyl-5-Bromo-N-Cyclohexylindoline-6-Sulfonamide (9b)
White crystalline solid (92.6 mg, 78%): mp 226–228 ◦C; 1H NMR (500 MHz, CDCl3)
δ 8.85 (s, 1H),
7.40 (s, 1H), 4.95 (d, J = 7.6 Hz, 1H), 4.07 (t, J = 8.6 Hz, 2H), 3.18 (t, J = 8.6 Hz, 2H), 3.09 (dt, J = 9.4,
4.5 Hz, 1H), 2.17 (s, 3H), 1.70 (dd, J = 10.1, 4.7 Hz, 2H), 1.56 (dd, J = 11.2, 5.3 Hz, 2H), 1.46–1.39 (m, 2H),
1.15 (q, J = 9.0 Hz, 4H). 13C NMR (126 MHz, CDCl3)
δ 168.9, 142.8, 139.6, 137.1, 130.7, 118.7, 113.1,
52.9, 49.0, 33.7, 29.7, 27.6, 25.2, 24.5, 24.1. HRMS-ESI: [M + Na]+ calculated for C16H20BrN2NaO3S−,
422.02809; found, 422.02998.
4.5.8. 1-Acetyl-N-Benzyl-5-Bromoindoline-6-Sulfonamide (9c)
Yellow solid (184 mg, 81%): mp 205–208 ◦C. 1H NMR (500 MHz, CDCl3)
δ 8.79 (s, 1H), 7.53 (s, 1H),
7.42–7.22 (m, 5H), 4.45 (s, 2H), 4.14 (t, J = 8.6 Hz, 2H), 3.26 (t, J = 8.6 Hz, 2H), 2.24 (s, 3H).
4.5.9. 1-Acetyl-5-Bromo-N-(Tert-Butyl)Indoline-6-Sulfonamide (9d)
Off-white solid (44.2 mg, 40%): mp 224.4 ◦C (softens), 235.7–236.1 ◦C; 1H NMR (500 MHz, CDCl3)
δ
8.92 (s, 1H), 7.44 (s, 1H), 5.07 (s, 1H), 4.16–4.09 (m, 2H), 3.24 (t, J = 8.6 Hz, 2H), 2.23 (s, 3H), 1.23 (s, 9H).
13C NMR (126 MHz, CDCl3)
δ 168.9, 142.9, 141.5, 137.0, 130.6, 118.3, 113.0, 54.9, 48.9, 30.2, 30.1, 27.5,
24.1. HRMS-ESI: (M + NH4) + calculated for C14H20BrN2O3S+, 392.0638; found, 392.0639.
4.5.10. 1-Acetyl-5-Bromoindolin-6-(Sulfonyl Glycine Methyl Ester) (9e)
Tan solid (105 mg, 93%): mp 179–182 ◦C; 1H NMR (500 MHz, CDCl3)
δ 8.86 (s, 1H), 7.50 (s, 1H),
5.74 (t, J = 5.6 Hz, 1H), 4.13 (t, J = 8.6 Hz, 2H), 3.82 (d, J = 5.3 Hz, 2H), 3.68 (s, 3H), 3.25 (t, J = 8.7 Hz,
2H), 2.24 (s, 3H). 13C NMR (126 MHz, CDCl3)
46.1, 44.6, 24.3, 8.9.
δ 169.2, 161.8, 143.0, 137.9, 137.7, 131.1, 119.1, 113.7, 49.2,
4.5.11. Methyl 3-((1-Acetyl-5-Bromoindoline)-6-Sulfonamido)Propanoate (9f)
◦
1
Off-white solid (117 mg, 97%): mp 190–193 C; H NMR (500 MHz, CDCl3)
δ 8.87 (s, 1H),
7.47 (s, 1H), 5.76 (d, J = 6.6 Hz, 1H), 4.11 (t, J = 8.6 Hz, 2H), 3.67 (s, 3H), 3.23 (t, J = 8.6 Hz, 2H), 3.17 (dd,
J = 12.0, 6.5 Hz, 2H), 2.50 (t, J = 5.9 Hz, 2H), 2.21 (s, 3H). 13C NMR (126 MHz, CDCl3)
δ 172.3, 168.9,
142.9, 138.1, 137.5, 130.8, 119.0, 113.1, 52.0, 49.0, 38.8, 33.9, 27.6, 24.1. HRMS-ESI: [M + Na]+ calculated
for C14H17BrN2NaO5S+, 426.9934; found, 426.9937.

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4.5.12. Methyl ((1-Acetyl-5-Bromoindolin-6-yl)Sulfonyl)Valinate (9g)
Tan solid (111 mg, 87%): mp 190.9–191.7 ◦C; 1H NMR (300 MHz, CDCl3)
δ
8.80 (s, 1H), 7.46 (s, 1H),
5.70 (d, J = 9.4 Hz, 1H), 4.12 (t, J = 9.2 Hz, 2H), 3.83 (dd, J = 9.4, 5.2 Hz, 1H), 3.54 (s, 3H), 3.24 (dt, J = 11.7,
5.4 Hz, 2H), 2.23 (s, 3H), 2.17–1.99 (m, 1H), 0.92 (dd, J = 10.5, 6.7 Hz, 6H). 13C NMR (126 MHz, CDCl3)
δ
171.3, 168.9, 142.7, 138.2, 137.4, 130.7, 118.6, 113.6, 61.5, 52.2, 48.9, 31.8, 27.6, 24.1, 18.8, 17.6. HRMS-ESI:
[M + H]+ calculated for C16H22BrN2O5S+, 433.0427; found, 433.0426.
4.5.13. Methyl ((1-Acetyl-5-Bromoindolin-6-yl)Sulfonyl)-L-Phenylalaninate (9h)
Tan solid (127 mg, 90%): mp 184.0–184.8 ◦C; 1H NMR (500 MHz, CDCl3)
δ 8.82 (s, 1H), 7.41 (s, 1H),
7.29–7.16 (m, 3H), 7.14–7.08 (m, 2H), 5.68 (d, J = 8.0 Hz, 1H), 4.28 (dt, J = 8.2, 5.8 Hz, 1H), 4.11 (td, J = 8.7,
5.1 Hz, 2H), 3.56 (s, 3H), 3.26–3.18 (m, 2H), 3.09 (dd, J = 5.9, 3.9 Hz, 2H), 2.23 (s, 3H). 13C NMR
(126 MHz, CDCl3) δ 170.8, 168.9, 142.7, 138.2, 137.3, 135.0, 130.8, 129.5, 128.6, 127.2, 118.6, 113.5, 60.4,
57.0, 52.4, 48.9, 39.5, 27.6, 24.1, 14.2. HRMS-ESI: [M + Na]+ calculated for C20H21BrN2NaO5S+, 503.0247;
found, 503.0255.
4.5.14. 1-Acetyl-5-Bromo-6-(Piperidin-1-Sulfonyl) Indoline (9i)
Off-white crystalline solid (114 mg, 70%): mp 212–214 ◦C; 1H NMR (500 MHz, CDCl3)
(s, 1H), 7.41 (s, 1H), 4.05 (t, J = 8.5 Hz, 2H), 3.25–3.20 (m, 4H), 3.17 (t, J = 8.5 Hz, 2H), 2.16 (s, 3H),
δ 8.68
1.57 (t, J = 5.6 Hz, 4H), 1.48 (q, J = 5.6 Hz, 2H). 13C NMR (126 MHz, CDCl3)
δ 169.0, 142.5, 137.7,
137.0, 131.4, 118.7, 113.8, 49.0, 46.8, 29.7, 27.5, 25.6, 24.1, 23.8. HRMS-ESI: [M + H]+ calculated for
C15H20BrN2O3S+, 387.0373; found, 387.0373.
4.5.15. 1-Acetyl-5-Bromo-6-(Pyrrolidin-1-Sulfonyl) Indoline (9j)
Light brown crystalline solid (98.8 mg, 90%): mp 238–240 ◦C; 1H NMR (500 MHz, CDCl3)
(s, 1H), 7.43 (s, 1H), 4.06 (t, J = 8.6 Hz, 2H), 3.43–3.36 (m, 4H), 3.17 (t, J = 8.6 Hz, 2H), 2.16 (s, 3H),
δ 8.61
1.92 (s, 4H). 13C NMR (126 MHz, CDCl3)
δ 169.0, 142.6, 138.2, 136.8, 131.5, 117.7, 113.8, 49.0, 48.2, 27.5,
25.8, 24.1. HRMS-ESI: [M + H]+ calculated for C14H18BrN2O3S+, 373.0216; found, 373.0216.
4.5.16. 1-Acetyl-5-Bromo-6-(Indolin-1-Sulfonyl) Indoline (9k)
Dark brown solid (52 mg, 83.4% yield), mp 215–218◦C. 1H NMR (500 MHz, CDCl3)
δ 8.86 (s, 1H),
7.47 (s, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.16 (d, J = 7.4 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 6.93 (t, J = 7.5 Hz,
1H), 4.26 (t, J = 8.4 Hz, 2H), 4.10 (t, J = 8.6 Hz, 2H), 3.19 (dt, J = 17.3, 8.5 Hz, 4H), 2.21 (s, 3H). 13C NMR
(126 MHz, DMSO-d6) δ 169.1, 138.5, 131.6, 127.6, 125.5, 123.4, 118.9, 113.8, 113.6, 50.9, 49.2, 29.9, 28.1,
27.9, 24.4. HRMS-ESI: [M + Na]+ calculated for C18H17BrN2NaO3S+, 443.0035; found, 443.0037.
4.5.17. 1-Acetyl-5-Bromo-N,N-Dipropylindoline-6-Sulfonamide (9l)
◦
1
Light brown solid (104 mg, 85%): mp 118–120 C; H NMR (500 MHz, CDCl3)
δ 8.71 (s, 1H),
7.49 (s, 1H), 4.13 (t, J = 8.6 Hz, 2H), 3.33–3.27 (m, 4H), 3.24 (t, J = 8.6 Hz, 2H), 2.24 (s, 3H), 1.62 (q, J = 7.6 Hz,
4H), 0.86 (t, J = 7.4 Hz, 6H).
4.5.18. 1-Acetyl-5-Bromo-N,N-bis(2-Methoxyethyl)Indoline-6-Sulfonamide (9m)
Tan solid (125 mg, 97%): mp 110–111 ◦C; 1H NMR (500 MHz, CDCl3)
4.23 (t, J = 8.5 Hz, 2H), 3.40 (t, J = 6.0 Hz, 4H), 3.53 (t, J = 5.5 Hz, 4H), 3.30 (s, 6H), 3.24 (t, J = 9.0 Hz, 2H),
δ 8.78 (s, 1H), 7.49 (s, 1H),
2.23 (s, 3H); 13C NMR (126 MHz, CDCl3)
δ 169.2, 142.8, 139.0, 137.2, 131.5, 118.7, 114.2, 71.7, 59.0, 48.8,
27.8, 24.3, 8.9. HRMS-ESI: [M + Na]+ calculated for C16H23BrN2NaO5S+, 457.0403; found, 457.0403.
4.5.19. 1-Acetyl-5-Bromo-N,N-Diethylindoline-6-Sulfonamide (9n)
Off-white solid (110 mg, 99%): mp 195-196 ◦C; 1H NMR (500 MHz, CDCl3)
4.13 (t, J = 8.5 Hz, 2H), 3.42 (q, J = 7.0 Hz, 4H), 3.23 (t, J = 8.0 Hz, 2H), 2.23 (s, 3H), 1.19 (t, J = 7.5 Hz,
δ 8.72 (s, 1H), 7.47 (s, 1H),

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6H); 13C NMR (126 MHz, CDCl3)
δ
169.2, 142.7, 139.6, 136.9, 131.5, 118.1, 114.0, 49.2, 42.3, 27.7, 24.3,
14.4. HRMS-ESI: [M + Na]+ calculated for C14H19BrN2NaO3S+, 397.0192; found, 397.0186.
4.5.20. 1-Acetyl-5-Chloro-N-Isopentylindoline-6-Sulfonamide (10a)
Tan solid (117 mg, 99%): mp 174 ◦C (softens), 179.1–180.4 ◦C; 1H NMR (300 MHz, CDCl3)
δ 8.84
(s, 1H), 7.28 (s, 1H), 4.90 (t, J = 6.1 Hz, 1H), 4.14 (t, J = 8.6 Hz, 2H), 3.25 (t, J = 8.6 Hz, 2H), 2.93 (td, J = 7.3,
6.2 Hz, 2H), 2.24 (s, 3H), 1.60 (dq, J = 13.3, 6.6 Hz, 1H), 1.36 (q, J = 7.1 Hz, 2H), 0.84 (d, J = 6.6 Hz, 6H).
13C NMR (126 MHz, CDCl3)
δ 167.8, 141.2, 136.2, 135.3, 126.3, 124.3, 117.8, 47.9, 40.6, 37.3, 26.7, 24.4,
23.0, 21.2. HRMS-ESI: [M + NH4]+ calculated for C15H25ClN3O3S+, 362.1305; found, 362.1301.
4.5.21. 1-Acetyl-5-Chloro-N-Cyclohexylindoline-6-Sulfonamide (10b)
White solid (95.3 mg, 79%): mp 230.0–230.5 ◦C; 1H NMR (300 MHz, CDCl3)
δ 8.9 (s, 1H), 7.28 (s, 1H),
4.90 (t, J = 6.0 Hz, 1H), 4.14 (t, J = 8.7 Hz, 2H), 3.25 (t, J = 8.7 Hz, 2H), 2.93 (dd, J = 13.7, 7.2 Hz, 2H),
2.24 (s, 3H), 1.36 (dd, J = 14.4, 6.9 Hz, 2H), 1.6 (m, 1H), 0.84 (d, J = 6.6 Hz, 6H). 13C NMR (126 MHz,
CDCl3)
δ 178.7, 168.8, 142.2, 137.9, 137.0, 127.3, 125.3, 118.4, 52.9, 48.9, 33.7, 27.7, 25.2, 24.5, 24.1.
HRMS-ESI: [M + Na]+ calculated for C16H21ClN2NaO3S+, 379.0854; found, 379.0854.
4.5.22. 1-(5-Chloro-6-(Piperidin-1-Ylsulfonyl)Indolin-1-yl)Ethan-1-One (10c)
Tan solid (104 mg, 79%): mp 199.7–201.4 ◦C; 1H NMR (500 MHz, CDCl3)
4.13 (t, J = 8.6 Hz, 2H), 3.28 (t, J = 5.4 Hz, 4H), 3.24 (t, J = 8.5 Hz, 2H), 2.23 (s, 3H), 1.63 (p, J = 5.6 Hz,
δ 8.72 (s, 1H), 7.26 (s, 1H),
4H), 1.55 (t, J = 5.8 Hz, 2H). 13C NMR (126 MHz, CDCl3)
δ 169.1, 142.1, 137.2, 135.9, 128.1, 126.6,
118.7, 49.1, 47.0, 27.9, 25.8, 24.2, 24.0. HRMS-ESI: [M + H]+ calculated for C15H20ClN2O3S+, 343.0878;
found, 343.0871.
4.5.23. 1-(5-Chloro-6-(Pyrrolidin-1-Ylsulfonyl)Indolin-1-yl)ethan-1-One (10d)
White crystalline solid (103 mg, 92%): mp 195 ◦C (softens), 199.7–200.4 ◦C; 1H NMR (500 MHz,
CDCl3) δ 8.67 (s, 1H), 7.27 (s, 1H), 4.13 (t, J = 8.6 Hz, 2H), 3.44 (td, J = 6.7, 5.4, 2.9 Hz, 4H), 3.24 (t, J = 8.5 Hz,
2H), 2.23 (s, 3H), 1.99 – 1.93 (m, 4H). 13C NMR (126 MHz, CDCl3)
δ 169.2, 142.1, 137.1, 136.4, 128.2,
126.6, 117.9, 49.2, 48.3, 27.9, 26.0, 24.3. HRMS-ESI: [M + Na]+ calculated for C14H17ClN2NaO3S+,
351.0541; found, 351.0537.
4.5.24. 1-Acetyl-5-Chloro-N,N-Dipropylindoline-6-Sulfonamide (10e)
◦
1
Light brown solid (104 mg, 85%): mp 118–120 C; H NMR (500 MHz, CDCl3)
δ 8.78 (s, 1H),
7.33 (s, 1H), 4.20 (t, J = 8.6 Hz, 2H), 3.37–3.32 (m, 4H), 3.30 (t, J = 8.5 Hz, 2H), 2.31 (s, 3H), 1.66 (h, J = 7.5 Hz,
4H), 0.92 (t, J = 7.4 Hz, 6H). 13C NMR (126 MHz, CDCl3)
δ 169.2, 142.0, 137.8, 136.9, 128.0, 126.6, 118.0,
49.9, 49.6, 49.2, 27.9, 24.3, 22.1, 19.6, 11.5, 11.4. HRMS-ESI: [M + H]+ calculated for C16H24ClN2O3S+,
359.1191; found, 359.1187.
4.5.25. 1-Acetyl-5-Chloro-N,N-bis(2-Methoxyethyl)Indoline-6-Sulfonamide (10f)
Gray solid (150 mg, 99%): mp 93.2–94.4 ◦C; 1H NMR (500 MHz, CDCl3)
4.13 (t, J = 8.6 Hz, 2H), 3.61–3.49 (m, 8H), 3.29 (s, 6H), 3.24 (t, J = 9.0 Hz, 3H), 2.23 (s, 3H). 13C NMR
δ 8.77 (s, 1H), 7.27 (s, 1H),
(126 MHz, CDCl3)
δ 169.0, 142.0, 137.2, 137.1, 127.8, 126.5, 118.5, 71.6, 58.9, 49.1, 48.6, 27.8, 24.1.
HRMS-ESI: [M + Na]+ calculated for C16H23ClN2NaO5S+, 413.0908; found, 413.0905.
5. Conclusions
In summary, we have synthesized a series of indoline-6-sulfonamide derivatives based on original
hits discovered in a high-throughput screen, and we have assayed these compounds for inhibition of
the bacterial enzyme DapE using our recently-reported ninhydrin-based assay. Docking experiments
suggest that one of the electron-rich sulfonamide oxygens may serve as a ligand for one of the two

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Lewis acidic Zn(II) atoms in the active site of DapE. This study offers promise on the path toward
drug-like small molecule inhibitors of DapE as antibiotics with a new mechanism of action.
6. Patents
Daniel P. Becker, Richard Holz, Tahirah Heath, Cory Reidl, and Anna Starus, “Indoline sulfonamide
inhibitors of DapE and NDM-1 and use of the same” US 10,385,040, issued 8-20-19.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/9/595/s1,
Figure S1: copies of spectral characterization of compounds 4, 7b, 8a, 8b, 9a–n, and 10a–f.
Author Contributions: Conceptualization, C.T.R., T.K.H., R.C.H., and D.P.B.; methodology, C.T.R., T.K.H., E.G.,
B.P.N., A.S., and D.P.B.; software, C.T.R. and B.P.N.; validation, C.T.R., T.K.H., I.D., R.M.T., M.M., and E.G.;
formal analysis, C.T.R., T.K.H., E.G., and D.P.B.; investigation, C.T.R., T.K.H., I.D., R.M.T., M.M., and E.G.; resources,
R.C.H. and D.P.B.; data curation, C.T.R., T.K.H., I.D., R.M.T., M.M., and E.G.; writing—original draft preparation,
C.T.R., T.K.H., and D.P.B.; writing—review and editing, C.T.R., T.K.H., I.D., R.M.T., M.M., R.C.H., and D.P.B.;
visualization, C.T.R., M.M., and D.P.B.; supervision, C.T.R., T.K.H., R.C.H., and D.P.B.; project administration,
D.P.B.; funding acquisition, R.C.H. and D.P.B. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported by the National Institute of Health (NIH) and the National Institute of Allergy
and Infectious Diseases (NIAID) (Contracts HHSN272200700058C and HHSN272201200026C to the Center of
Structural Genomics of Infectious Diseases), the National Science Foundation (CHE-1412443, R.C.H.), and the
Todd Wehr Foundation (R.C.H.).
Conflicts of Interest: The authors declare no conflict of interest.
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