Discovery and structure-activity relationship studies of irreversible benzisothiazolinone-based inhibitors against Staphylococcus aureus sortase A transpeptidase

Article abstract of DOI:10.1016/j.bmc.2014.09.011

Gram-positive bacteria, in general, and staphylococci, in particular, are the widespread cause of nosocomial and community-acquired infections. The rapid evolvement of strains resistant to antibiotics currently in use is a serious challenge. Novel antimicrobial compounds have to be developed to fight these resistant bacteria, and sortase A, a bacterial cell wall enzyme, is a promising target for novel therapies. As a transpeptidase that covalently attaches various virulence factors to the cell surface, this enzyme plays a crucial role in the ability of bacteria to invade the host's tissues and to escape the immune response. In this study we have screened a small molecule library against recombinant Staphylococcus aureus sortase A using an in vitro FRET-based assay. The selected hits were validated by NMR methods in order to exclude false positives and to analyze the reversibility of inhibition. Further structural and functional analysis of the best hit allowed the identification of a novel class of benzisothiazolinone-based compounds as potent and promising sortase inhibitors.

Full text of DOI:10.1016/j.bmc.2014.09.011

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and structure–activity relationship studies of irreversible
benzisothiazolinone-based inhibitors against Staphylococcus aureus
sortase A transpeptidase
a
c
d
a
Dmitrijs Zhulenkovs a,b, , Zhanna Rudevica , Kristaps Jaudzems , Maris Turks , Ainars Leonchiks
⇑
a Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia
b University of Latvia, Raina bulv. 19, Riga LV-1586, Latvia
c Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia
d Faculty of Material Science and Applied Chemistry, Riga Technical University, P. Valdena 3, Riga LV-1007, Latvia
a r t i c l e i n f o
a b s t r a c t
Article history:
Gram-positive bacteria, in general, and staphylococci, in particular, are the widespread cause of nosoco-
mial and community-acquired infections. The rapid evolvement of strains resistant to antibiotics cur-
rently in use is a serious challenge. Novel antimicrobial compounds have to be developed to fight
these resistant bacteria, and sortase A, a bacterial cell wall enzyme, is a promising target for novel ther-
apies. As a transpeptidase that covalently attaches various virulence factors to the cell surface, this
enzyme plays a crucial role in the ability of bacteria to invade the host’s tissues and to escape the immune
response. In this study we have screened a small molecule library against recombinant Staphylococcus
aureus sortase A using an in vitro FRET-based assay. The selected hits were validated by NMR methods
in order to exclude false positives and to analyze the reversibility of inhibition. Further structural and
functional analysis of the best hit allowed the identification of a novel class of benzisothiazolinone-based
compounds as potent and promising sortase inhibitors.
Received 18 June 2014
Revised 18 August 2014
Accepted 5 September 2014
Available online xxxx
Keywords:
Sortase A
High-throughput screening
Structure–activity relationship
Inhibitor
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
and vancomycin, are becoming more abundant every year,
prompting new health threats and having a significant impact on
Gram-positive bacteria are responsible for a large proportion of
the serious infections observed worldwide and are among the top
ten most frequently isolated organisms associated with health-
care-associated infections.1 These Gram-positive organisms are
among the most commonly isolated organisms in hospitals in
Europe, North America, Oceania, and Africa.2
overall healthcare costs.4,5 Bacteria with acquired resistance
against beta-lactam antibiotics have been identified in the majority
of cases of staphylococcal infections.6–10 Since the 1990s, nosoco-
mial and community-acquired methicillin-resistant S. aureus
(MRSA) strains and several other staphylococcal species have
emerged as a serious problem in medicine.
The infections caused by multidrug-resistant Gram-positive
bacteria represent a major public health problem worldwide and
are consistently associated with high mortality rates. In particular,
Staphylococcus aureus is the leading cause of the bloodstream,
respiratory tract, skin, and soft tissue infections observed in almost
all geographic areas of the world,3 and strains of this pathogen that
are resistant to the commonly used therapies, such as methicillin
All Gram-positive pathogens display surface proteins, which
play an important role in adhesion to tissues, invasion of host cells,
and evasion of the immune response.11 These virulence factors
comprise protein A, microbial surface components that recognize
adhesive matrix molecules and other proteins that are covalently
attached to the cell-wall peptidoglycans,12 and these virulence fac-
tors are covalently linked to the bacterial cell wall by enzymes
belonging to the sortase family.
To date, a total of six classes of sortases have been found in bac-
terial cells. Sortases belonging to classes A and B are the most
abundant in Firmicutes. In these cells, sortase A provides an impor-
tant virulence function by regulating the attachment of a broad
range of extracellular proteins. Class A sortases (SrtA) from various
Gram-positive microorganisms possess a high degree of homology
Abbreviations: HTS, high-throughput screening; SAR, structure–activity rela-
tionship; SDS–PAGE, sodium dodecyl sulfate poly-acrylamide gel electrophoresis;
FRET, fluorescence resonance energy transfer; MIC, minimal inhibitory
concentration.
⇑
Corresponding author. Tel.: +371 67808007.
E-mail address: dmitry@biomed.lu.lv (D. Zhulenkovs).
http://dx.doi.org/10.1016/j.bmc.2014.09.011
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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2
D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
in terms of their primary structure.13 Class B sortases (SrtB), in
turn, are often involved in more specific processes, such as the
transpeptidation of heme receptors or the polymerization of the
proteins in bacterial pili.14
slightly modified previously published method.27 Briefly, after
the IPTG induction of the Escherichia coli BL21 (DE3) transformed
strain, the cell pellet was collected by centrifugation and resus-
pended in lysis buffer, and the recombinant protein was purified
by affinity chromatography on a Ni-NTA column (Qiagen). The
enzyme was eluted with an imidazole gradient and the fractions
containing the protein were further purified by the gel filtration.
A Superdex 75 (GE Healthcare) column, which was equilibrated
with 10 mM sodium phosphate (pH 7.0) buffer containing
100 mM NaCl and 1 mM DTT, was used for the final purification.
In staphylococci, the list of proteins possessing the C-terminal
LPXTG sorting signal comprises approximately 20 surface proteins,
which explains the significance of sortase A in bacterial infectivity.
The list of polypeptides possessing the C-terminal sorting signal
include several factors that are involved in pathogenicity, such as
protein A (Spa), fibronectin-binding proteins A (FnbpA) and B
(FnbpB), clumping factors A (ClfA) and B (ClfB), serine-aspartic acid
repeat proteins (SdrC, SdrD, and SdrE), plasmin-sensitive protein
(Pls), and staphylococcal surface proteins (Sas).15 S. aureus strains
lacking the srtA gene (srtAꢀ) are unable to retain and display
LPXTG-bearing proteins at the bacterial cell surface. As a conse-
quence, SrtA mutants are significantly less virulent and incapable
of establishing acute infection. In contrast, SrtA is not indispens-
able for microbial growth and viability in cell culture; thus,
enzyme inhibitors that act as anti-infective agents have limited
selective pressure toward the development of bacterial drug resis-
tance and the emergence of new antibiotic-resistant strains.16,17
Therefore, sortase A is a very promising target for the development
of novel antibacterial compounds that can avoid the problem of
resistance against conventionally used antibiotics.18,19
Staphylococcus aureus sortase A is the best-studied sortase
enzyme to date. Its structure is available and the reaction mecha-
nism is well known.20 The transpeptidation reaction does not
depend on a presence of ATP or other energy carriers. The enzyme
recognizes a specific LPXTG motif and acts as a cysteine protease,
cleaving the substrate between threonine and glycine residues
within the recognition site. As a consequence, an acyl-enzyme
intermediate is being formed with the N-terminal fragment of
the cleaved substrate. The acyl-enzyme is then attacked either by
a second peptide substrate with a flexible and nucleophilic N-ter-
minal polyglycine sequence or by a water molecule in the absence
of the peptide substrate.21
The 15N isotope labeling of recombinant SrtA N59-6His for 2D
D
15N-1H HSQC screening was accomplished by cultivating BL21
(DE3) cells in M9 minimal medium containing 15N-ammonium salt
as the sole nitrogen source and the purification was performed fol-
lowing the same procedures.
A wild-type SrtA
catalytic domain without hexahistidine
D
N59
sequence was used for the determination of the inhibitor–enzyme
complex structure. The 15N and 15N/13C isotope labeling of recom-
binant SrtA
was accomplished by cultivating BL21 (DE3) cells
N59
D
in M9 minimal medium containing 15N-ammonium salt and 13C-
glucose as the sole nitrogen and carbon sources, respectively. The
expression and the purification were carried out following a previ-
ously published protocol.28 Briefly, after the IPTG induction of the
E. coli BL21(DE3) transformed strain, the cell pellet was collected
by centrifugation and resuspended in lysis buffer, and the recombi-
nant protein was purified through Sepharose Fast Flow SP (GE
Healthcare) and Source Q15 (GE healthcare) sequential chromato-
graphic steps. The resulting purified protein was dialyzed against
50 mM Tris–HCl (pH 7.5) buffer containing 150 mM NaCl and
1 mM DTT and concentrated to 10 mg/mL. After chromatographic
purification, as described above, an optional gel filtration step
was introduced to ensure the purity of the protein for the NMR
applications. A Superdex 75 (GE Healthcare) column, which was
equilibrated with 10 mM sodium phosphate (pH 7.0) buffer con-
taining 100 mM NaCl and 1 mM DTT, was used for the final purifi-
cation. The fractions containing the protein were collected and
concentrated to 10 mg/mL.
The full-length S. aureus sortase A comprises 206 amino acid
residues. A N-terminal signal sequence necessary for the trans-
membrane transport and anchoring of the protein is followed by
a single catalytic domain (residues 60–206). The catalytic core
without the signal polypeptide can be expressed recombinantly
without any loss of the enzymatic activity.22,23
The purified unlabeled and labeled proteins were analyzed
using matrix-assisted laser desorption-ionization time-of-flight
(MALDI-TOF) mass spectroscopy and SDS–PAGE Coomassie Blue
staining.
The possibility of producing a soluble and active recombinant
catalytic domain of sortase A with hydrolytic activity allows us to
perform efficient screening of compounds that interfere with the
enzymatic activity using a fluorimetric in vitro assay based on
the application of FRET-substrates.24,25 Such assays are suitable for
the high-throughput screening of large numbers of candidate mole-
cules and have been successfully used to identify novel and potent
sortase inhibitors, some of which have demonstrated significant
inhibitory activity in the micromolar and even sub-micromolar
ranges.24,26
In this study, a small-molecule library comprising 50,240 drug-
like compounds was screened to identify novel S. aureus sortase A
inhibitors. Subsequent optimization of one of the hits based on an
NMR structure of its complex with SrtA yielded compounds with
improved inhibitory potency and lower cytotoxicity.
2.2. FRET-based high-throughput screening of the chemical
library
A diverse, drug-like library containing 50,240 screening com-
pounds was ordered from the entire 1.6-million-compound collec-
tion of Enamine (Enamine Ltd, Ukraine). The selected compounds
were filtered according to Molecular property (Lipinski’s ‘Rule of
5’) and MedChem filters to exclude compounds undesirable for
drug discovery and high-throughput screening and compounds
with reactive groups and toxicophores. All of the compounds were
pre-plated in heat-sealed 384-well plates as 10 mM solutions in
dimethyl sulfoxide (DMSO).
The library was screened at a single dose of 100 lM (final 1%
DMSO) in black 384-well plates (Greiner Bio-One). Two known sor-
tase inhibitors, phenyl vinyl sulfone29 and 1-(3,4-dichlorophenyl)-
3-(dimethylamino)propan-1-one,24 were used as the positive
controls. The inhibitory activity of all of the compounds was deter-
mined by quantifying the increase in fluorescence intensity upon
cleavage of FRET-peptide dabcyl-QALPETGEE-edans, which was
used as the sortase substrate. A previously published method30
was used with slight modifications. Briefly, the reactions were per-
2. Methods
2.1. Expression of recombinant sortase A
Recombinant and catalytically active sortase A with the N-ter-
minal deletion of residues 1–59 and possessing C-terminal hexa-
formed in a volume of 100
lL containing 50 mM Tris–HCl, 5 mM
histidine sequence (SrtA N59-6His) was used for the HTS and
D
CaCl2, 150 mM NaCl, pH 7.5, 10
l
M S. aureus SrtA, 20 M fluorescent
l
NMR screenings. SrtA N59-6His was prepared according to a
D
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D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
peptide substrate dabcyl-QALPETGEE-edans, and the prescribed
concentrations of the test compounds or positive controls.
The peptide substrate without the recombinant SrtA was incu-
bated in the same manner and used as a negative control. The reac-
tions were conducted for 24 h at 37 °C, and the fluorescence
emitted with an excitation wavelength of 350 nm and an emission
wavelength of 495 nm after substrate cleavage was recorded.
End-point determination of product formation was used as a
criterion for the primary screening. This determination was made
by measuring the total product fluorescence 24 h after the initia-
tion of the reaction. The relative inhibition activity was determined
as %I = 100% ꢀ (Fsample/Fcontrol ꢁ 100%), where Fsample is the fluores-
cence intensity of the well containing the corresponding test com-
pound and Fcontrol is the fluorescence of the positive control
reaction without inhibition.24,26
inhibitor. The reactivity towards the cysteine side chain was con-
firmed through a reaction in the NMR tube, in which 14.2 mM cys-
teine was added to a solution containing 7.1 mM inhibitor in NMR
buffer, 5% D2O, and 7.1% DMSO-d6.
2.5. Determination of the inhibitor–enzyme complex structure
The NMR sample used for the structure determination of the
SrtA–inhibitor complex contained a 1.6 mM solution of 13C, 15N-
labeled SrtA in NMR buffer, 2.4 mM inhibitor, 5% D2O, and 2.4%
DMSO-d6. The three-dimensional (3D) structure of the inhibitor–
SrtA complex was determined using the UNIO-ATNOS/CANDID
2.0.236,37 and CYANA 2.138 software packages based on NOE distance
restraints, hydrogen bond restraints and chemical shift-derived
dihedral angle restraints. The structure calculation involved seven
iterations of automated NOE assignment with the routine CANDID36
followed by 10,000 steps of torsion angle dynamics starting from
100random conformers. The 20 conformerswith the lowestresidual
restraint violations were refined in explicit water using the program
CNS 1.2.39,40 Table 1 presents an overview of the restraints used and
the structural statistics.
The Z0 factor, which is a standard statistical parameter that char-
acterizes the reproducibility and robustness of HTS assays, was cal-
culated from a single 384-well plate using the following equation:
Z0 = 1 ꢀ 3 ꢁ (SDpos ꢀ SDneg)/(Mpos ꢀ Mneg) ꢁ 100, where Mpos is the
mean fluorescence value of the positive controls (with SrtA,
n = 192 wells), Mneg is the mean value of the negative controls (with-
out SrtA, n = 192 wells), and SD is the standard deviation.31
For the IC50 determination, 10
cubated in the reaction buffer with increasing concentrations of
the candidate inhibitors (x ꢀ y M) for 1 h at 37 °C prior to the
l
M S. aureus sortase A was prein-
2.6. Chemistry
l
All non-aqueous reactions were carried out under an inert
atmosphere of argon in oven-dried glassware unless otherwise
noted. Commercial reagents were used without purification. Sol-
vents were distilled prior to use and, if required, dried over stan-
dard drying agents (THF from metallic sodium, DMSO, DMF and
Et3N form CaH2). Analytical thin layer chromatography (TLC) was
performed on Merck precoated analytical plates, 0.25 mM thick,
silica gel 60F254. Preparative flash chromatography was performed
addition of the dabcyl-QALPETGEE-edans substrate. The total fluo-
rescence was recorded at 1-min intervals for a period of 1 h, and
the progress curves were constructed. The initial velocities of the
biphasic reactions were obtained through nonlinear regression,
as previously described.28,32 The IC50 values were determined by
fitting the obtained data to a default four-parameter variable-slope
sigmoidal function in SigmaPlot 12.5 using a nonlinear least
squares algorithm.
on silica gel (60 Å, 40–63 l
m, ROCC). 1H and 13C NMR spectra were
2.3. NMR spectroscopy
Table 1
All of the NMR experiments were performed at 298 K on a 600-
MHz Varian Unity Inova spectrometer equipped with an HCN tri-
ple-resonance pulsed-field-gradient cold probe. The following 2D
and 3D spectra were recorded for structure determination:
15N–1H HSQC, HNCA, CBCA(CO)NH, 15N-resolved NOESY-HSQC
(70 ms mixing time), 13C(aliphatic)-resolved NOESY-HSQC (70 ms
mixing time), and 13C(aromatic)-resolved NOESY-HSQC (70 ms
mixing time).
Input for structure calculation and structural statistics of the NMR structure of
inhibitor 1–SrtA complex
Quantity
Value*
NOE upper distance limits
Intra-residual (|i ꢀ j| = 0)
Short-range
Medium-range
Long-range
1680
548
457
152
514
9
Inhibitor-to-protein
H-bond restraints
Additionally, an (F1)-15N,13C-filtered 13C(aliphatic)-resolved
NOESY-HSQC33 (100 ms mixing time) spectrum was recorded for
the identification of NOEs between the modified cysteine side
chain and other protein groups and F1,F2-13C,15N-filtered 2D
NOESY (100 ms mixing time) and TOCSY (60 ms mixing time) spec-
tra were recorded for assignment of the inhibitor resonances. Spec-
tra were processed using NMRPipe34 or Bruker Topspin 3.2 and
analyzed using CARA.35
40
Residual NOE violations
Number P0.1 Å
Maximum (Å)
35
5
0.44 0.11
R.m.s.d. from idealized geometry
Bond lengths (Å)
Bond angles (°)
PARALLHDG27 energies [kcal/mol]
0.0072 0.0001
0.562 0.017
Total
van der Waals
Electrostatic
ꢀ5661 118
ꢀ636 27
2.4. NMR screening of the selected hits
ꢀ6157 106
R.m.s.d. from mean coordinates (Å)
Backbone (residues 63–119,130–158,176–183,198–206)
All heavy atoms (residues 63–119,130–158,176–183,198– 1.24 0.09
206)
All heavy atoms (inhibitor 1)
The binding of the inhibitor to the active site of SrtA was stud-
ied using NMR spectroscopy. 2D 15N–1H HSQC NMR experiments
were used to confirm binding through chemical shift perturbation.
To a sample containing 0.3 mM SrtA in the NMR buffer (20 mM
sodium acetate, 50 mM NaCl, and 10 mM CaCl2, pH 6.0, 6% D2O)
0.25, 0.5, 1, and 2 lL of a 100 mM inhibitor solution in DMSO-d6
was added in a stepwise manner, and a spectrum was recorded
after each addition.
0.75 0.09
1.39 0.26
Ramachandran plot statistics (PROCHECK)41
Most favored regions (%)
Additional allowed regions (%)
Generously allowed regions (%)
Disallowed regions (%)
77.6
21.3
0.9
0.2
The reversibility of the binding was determined by measuring a
2D 15N–1H HSQC spectrum after dialysis of the SrtA–inhibitor com-
plex against NMR buffer, resulting in a 100-fold dilution of the
*
Except for the top six entries, average values and standard deviations for the 20
energy-minimized conformers are given.
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D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
recorded on a Bruker 300 MHz and Varian 400 MHz spectrometers
in CDCl3 or DMSO-d6. The proton signals for residual non-deuter-
ated solvents (d 7.26 for CDCl3 and d 2.50 for DMSO-d6) and carbon
signals (d 77.1 for CDCl3 and d 39.5 for DMSO-d6) were used as an
internal references for 1H NMR and 13C NMR spectra, respectively.
Chemical shifts (d) values are reported in ppm and coupling
constants J in Hz. HRMS spectra (ESI+) were performed using a
Q-TOF Micromass and elemental analyses on a Carlo-Erba EA1108
analyzer. Yields refer to chromatographically and spectroscopically
homogeneous materials.
2.6.2. General procedure A for the synthesis of benzo[d]isothiazol-
3(2H)-one—adamantane amine conjugates 1b–m
2.6.2.1.
N-(3-Hydroxy-5,7-dimethyladamantan-1-yl)-2-(3-
(1c). N-Ethyl-
oxobenzo[d]isothiazol-2(3H)-yl)acetamide
N0-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI)
(0.15 g, 0.78 m Mol, 1.2 equiv) and 3-amino-5,7-dimethyladaman-
tan-1-ol (9b) (0.15 g, 0.76 m Mol, 1.06 equiv) were successively
added to a solution of 2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetic
acid (8) (0.15 g, 0.72 m Mol, 1.0 equiv) in anhydrous DMF (5 mL)
at ambient temperature. The resulting reaction mixture was stirred
at ambient temperature for 70 h. The solvent was evaporated
under reduced pressure and the residue was partitioned between
water (20 mL) and ethyl acetate (10 mL). The aqueous phase was
additionally extracted with ethyl acetate (3 ꢁ 7 mL). The combined
organic layer was successively washed with 5% aqueous solution of
citric acid (7 mL), 5% aqueous solution of NaHCO3 (8 mL), brine
(10 mL), dried over anhydrous Na2SO4, filtered and evaporated
under reduced pressure. Crystallization of the crude product from
a mixture consisting from ethyl acetate and hexanes provided 1c
(47 mg, 17%). Mp 192–193 °C. RP-UPLC (C18): tR = 4.76 min, purity
The purity of all final compounds was >95% as determined by
analytical UPLC-MS/DAD on reverse phase (column Waters ACQUI-
TY UPLC BEH C18: 2.1 ꢁ 50 mM, 1.7
lm) using a Waters Acquity™
UPLC with the binary system: A—0.01% TFA in water, B—acetoni-
trile, 5% B ? 95% B in 7 min., B ? 5% in 1.5 min; total analysis time:
8.5 min; flow rate 0.4 mL/min; detection: (a) PDAek detector
(wavelength: 320 nm), (b) MS (ESI+), tandem quadrupole detector.
2.6.1. 3-(4-Fluoro-3-oxobenzo[d]isothiazol-2(3H)-yl)propanoic
acid (7b)
A solution of 6,60-disulfanediylbis(2-fluorobenzoic acid) (4b)
(2.23 g, 6.5 m Mol) in a mixture of THF (10 mL) and DMF (6 mL)
was added to a stirred suspension of carbonyldiimidazole (CDI)
(2.21 g, 13.6 m Mol) in THF (15 mL). The resulting reaction mixture
was refluxed for 20 min until it became limpid. Then it was cooled
to ambient temperature and b-alanine methylester hydrogen chlo-
ride (2.00 g, 14.3 m Mol) and NEt3 (4.50 mL, 32.3 m Mol) were
added and the resulting mixture was stirred for 48 h at ambient
temperature. Then the reaction mixture was evaporated to dryness
and water (80 mL) was added. The resulting mixture was stirred
for 10 min., filtered and washed on the filter with water
(2 ꢁ 40 mL) and t-butylmethylether (20 mL). The drying of the
resulting solid in air for 16 h and under the vacuum (0.5 Torr) for
3 h provided intermediate dimethyl 3,30-((2,20-disulfanediylbis(6-
fluorobenzoyl))bis(azanediyl))dipropionate (5b) (3.00 g, 90%).
A solution of Br2 (1.88 g, 11.8 m Mol) in CH2Cl2 (15 mL) was
added to a solution of 5b (3.00 g, 5.9 m Mol) in CH2Cl2 (65 mL)
and the reaction mixture was stirred for 30 min at ambient tem-
perature. Then, NEt3 (1.65 mL, 11.8 m Mol) was added and the
resulting reaction mixture was refluxed for 30 min. The reaction
mixture was cooled to ambient temperature and washed with a
solution of Na2SO3 (0.2 g) in 1 M aqueous solution of NaH2PO4
(pH 4.5). The organic phase was additionally washed with water
(2 ꢁ 20 mL), brine (20 mL), dried over anhydr. Na2SO4, filtered
and evaporated to yield methyl 3-(4-fluoro-3-oxobenzo[d]isot-
hiazol-2(3H)-yl)propanoate (6b) (2.13 g, 71%) as yellowish foam.
An aqueous 1 M solution of LiOH (16.0 mL, 16.0 m Mol) was
cooled to 5–7 °C and then added to a cooled (7–10 °C) suspension
of 6b (2.13 g, 8.3 m Mol) in dioxane (17 mL). The reaction mixture
was stirred for 1 h at 2–5 °C (the temperature at which the mixture
does not freeze yet) until complete dissolution of the precipitate.
An aqueous 10% solution of HCl (10 mL) was added. The resulting
suspension was additionally diluted with water (150 mL) and fil-
tered. The precipitate was washed on the filter with water
(2 ꢁ 20 mL), Et2O (20 mL) and dried in air for 16 h. This provided
the target compound 7b (1.51 g, 75%; 48% over 3 steps) as a color-
less crystalline powder. Mp 201–202 °C. RP-UPLC (C18): tR = 2.51
>95%. IR (KBr) m
, cmꢀ1: 3430, 3070, 2950, 2910, 2865, 1675, 1645,
1600, 1560, 1490, 1350, 1240, 1205, 1069. 1H NMR (DMSO-d6,
300 MHz) d, ppm: 7.95 (d, 1H, 3J = 7.6 Hz, H–C(Ar)), 7.87 (s, 1H,
NH), 7.86 (d, 1H, 3J = 7.6 Hz, H–C(Ar)), 7.69 (t, 1H, 3J = 7.6 Hz, H–
C(Ar)), 7.43 (t, 1H, 3J = 7.6 Hz, H–C(Ar)), 4.53 (s, 1H, OH), 4.37 (s,
2H, CO–CH2–N), 1.72–1.66 (m, 2H, H–C(adam.)), 1.54, 1.47 (2d,
4H, AB syst., 2J = 12.4 Hz, C(adam.)), 1.25, 1.17 (2d, 4H, AB syst.,
2J = 11.7 Hz, C(adam.)), 1.05–0.97 (m, 2H, H–C(adam.)), 0.84 (s,
6H, (Me)2-C(adam.)). 13C-NMR (DMSO-d6, 75.5 MHz) d, ppm:
165.4, 164.8, 141.4, 131.8, 125.5, 125.2, 123.4, 121.6, 68.3, 54.4,
50.4, 49.2, 47.5, 46.0, 45.8, 33.4, 29.2. HRMS for [C21H26N2O3S+
H+]: m/z calcd 387.1737; found 387.1740. Anal. Calcd for C21H26N2
O3S: C 65.26, H 6.78, N 7.25. Found 65.15, H 6.88, N 7.31.
2.6.2.2.
N-(3-Hydroxy-5,7-dimethyladamantan-1-yl)-3-(3-
Compound
oxobenzo[d]isothiazol-2(3H)-yl)propanamide (1f).
1f was obtained by general procedure A using 7a and 3-amino-5,7-
dimethyladamantan-1-ol (9b). Yield: 36%. RP-UPLC (C18): tR = 4.62
min, purity >95%. 1H NMR (300 MHz, CDCl3) d, ppm: 8.02 (d, 1H,
3J = 7.9 Hz, H–C(Ar)), 7.64–7.53 (m, 2H, H–C(Ar)), 7.43–7.37 (m, 1H,
H–C(Ar)), 5.66 (br s, 1H, H–N), 4.15 (t, 2H, 3J = 6.3 Hz, CH2–CH2–CO),
2.61 (t, 2H, 3J = 6.3 Hz, CH2–CH2–CO), 1.83 (s, 2H, H–C(adam.)), 1.74
(m, 1H, H–C(adam.)), 1.61–1.49 (m, 4H, H–C(adam.)), 1.42–1.28 (m,
4H, H–C(adam.), 1.13–1.03 (m, 2H, H–C(adam.)), 0.89 (s, 6H,
(Me)2-C(adam.)). HRMS for [C22H28N2O3S+H+]: m/z calcd 401.1893;
found 401.1901.
2.6.2.3. N-(3-Hydroxyadamantan-1-yl)-3-(3-oxobenzo[d]isothia
zol-2(3H)-yl)propanamide (1g). Compound 1g was obtained by
general procedure A using 7a and 3-aminoadamantan-1-ol (9c).
Yield: 33%. RP-UPLC (C18): tR = 3.17 min, purity >95%. 1H NMR
(CDCl3, 300 MHz) d, ppm: 8.02 (d, 1H, 3J = 7.9 Hz, H–C(Ar)), 7.64–
7.53 (m, 2H, H–C(Ar)), 7.42–7.37 (m, 1H, H–C(Ar)), 5.61 (br s, 1H,
H–N), 4.16 (t, 2H, 3J = 6.2 Hz, CH2–CH2–CO), 2.61 (t, 2H,
3J = 6.2 Hz, CH2–CH2–CO), 2.24 (m, 2H, H–C(adam.)), 1.96 (s, 2H,
H–C(adam.)), 1.92–1.82 (m, 4H, H–C(adam.)), 1.78–1.60 (m, 5H,
H–C(adam.)), 1.58–1.44 (m, 2H, H–C(adam.)). HRMS for [C20H24N2
O3S+H+]: m/z calcd 373.1580; found 373.1572.
min, purity >95%. IR (KBr) m
, cmꢀ1: 3880–3425 (br s), 2950, 2925,
2725, 2670, 2595, 2535, 1725, 1620, 1475, 1430, 1355, 1340,
1225, 1205, 1180. 1H NMR (400 MHz, DMSO-d6) d, ppm: 12.48 (s,
1H), 7.77 (dd, 1H, J = 8.0, 0.8 Hz), 7.66 (dt, 1H, J = 5.2, 8.0 Hz),
7.16 (ddd, J = 10.4, 8.0, 0.8 Hz), 3.98 (t, 2H, J = 6.6 Hz), 2.66 (t, 2H,
J = 6.6 Hz). 13C NMR (100.6 MHz, DMSO-d6) d, ppm: 172.3, 161.4
(d, 3 Hz), 159.8 (d, 259 Hz), 143.3 (d, 4 Hz), 133.3 (d, 8 Hz), 118.1
(d, 5 Hz), 112.1 (d, 15 Hz), 111.4 (d, 19 Hz), 39.2, 33.6. HRMS for
[C13H8FNO3S+H+]: m/z calcd 242.0282; found 242.0293.
2.6.2.4. N-(3,5-Dimethyladamantan-1-yl)-3-(3-oxobenzo[d]isot-
hiazol-2(3H)-yl)propanamide (1e). Compound 1e was obtained
by general procedure A using 7a and 3,5-dimethyladamantan-1-
amine (9a). Yield: 21%. RP-UPLC (C18): tR = 4.56 min, purity >95%.
1H NMR (CDCl3, 300 MHz) d, ppm: 8.02 (d, 1H, 3J = 7.9 Hz, H–
C(Ar)), 7.64–7.53 (m, 2H, H–C(Ar)), 7.65–7.53 (m, 1H, H–C(Ar)),
7.43–7.38 (m, 1H, H–C(Ar)), 5.71 (br s, 1H, H–N), 4.16 (t, 2H,
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D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
3J = 6.2 Hz, CH2–CH2–CO), 2.62 (t, 2H, 3J = 6.2 Hz, CH2–CH2–CO),
1.92–1.75 (m, 3H, H–C(adam.)), 1.61–1.50 (m, 4H, H–C(adam.)),
1.42–1.25 (m, 4H, H–C(adam.)), 1.08 (m, 2H, H–C(adam.)), 0.90
(s, 6H, (Me)2-C(adam.)). HRMS for [C22H28N2O2S+H+]: m/z calcd
385.1944; found 385.1938.
2.6.2.10. Methyl 3-(3-(4-fluoro-3-oxobenzo[d]isothiazol-2(3H)-
yl)propanamido)adamantane-1-carboxylate (1m). Compound
1m was obtained by general procedure A using 7b and methyl 3-
aminoadamantane-1-carboxylate (9e). Yield: 23%. RP-UPLC (C18):
tR = 4.27 min, purity >95%. 1H NMR (CDCl3, 300 MHz) d, ppm:
7.58–7.51 (m, 1H, H–C(Ar)), 7.29 (d, 1H, 3J = 8.1 Hz, H–C(Ar)),
6.99 (dd, 1H, 3J = 8.4, 8.1 Hz, H–C(Ar)), 5.46 (s, 1H, H–N), 4.11 (t,
2H, 3J = 6.2 Hz, CH2–CH2–CO), 3.64 (s, 3H, Me-OCO), 2.59 (t, 2H,
3J = 6.2 Hz, CH2–CH2–CO), 2.18 (m, 1H, H–C(adam.)), 2.12 (m, 1H,
H–C(adam.)), 2.00–2.87 (m, 4H, H–C(adam.)), 1.82–1.81 (m, 3H,
H–C(adam.)), 1.63 (s, 5H, H–C(adam.)). HRMS for [C22H25FN2O4
S+H+]: m/z calcd 433.1592; found 433.1605.
2.6.2.5. N-(3,5-Dimethyladamantan-1-yl)-3-(4-fluoro-3-oxoben
zo[d]isothiazol-2(3H)-yl)propanamide (1j). Compound 1j was
obtained by general procedure A using 7b and 3,5-dimethylada-
mantan-1-amine (9a). Yield: 29%. RP-UPLC (C18): tR = 5.71 min,
purity >95%. 1H NMR (DMSOd6, 300 MHz) d, ppm: 7.77 (d, 1H,
3J = 8.3 Hz, H–C(Ar)), 7.69–7.62 (m, 1H, H–C(Ar)), 7.44 (s, 1H, H–
N), 7.15 (dd, 1H, 3J = 8.1, 7.9 Hz, H–C(Ar)), 3.92 (t, 2H, 3J = 6.4 Hz,
CH2–CH2–CO), 2.43 (t, 2H, 3J = 6.4 Hz, CH2–CH2–CO), 1.74 (m, 2H,
H–C(adam.)), 1.59–1.49 (m, 4H, H–C(adam.)), 1.32–1.20 (m, 4H,
H–C(adam.)), 1.08 (m, 3H, H–C((adam.)), 0.79 (s, 6H, (Me)2-
C(adam.)). HRMS for [C22H27FN2O2S+H+]: m/z calcd 403.1850;
found 403.1857.
2.6.2.11. N-Adamantan-1-yl-3-(3-oxobenzo[d]isothiazol-2(3H)-
yl)propanamide (1d). Compound 1d was obtained by general pro-
cedure A using 7a and adamantane-1-amine (9d). Yield: 31%. RP-
UPLC (C18): tR = 4.80 min, purity >95%. 1H NMR (CDCl3, 300 MHz)
d, ppm: 8.02 (d, 1H, 3J = 7.9 Hz, H–C(Ar)), 7.63–7.52 (m, 2H, H–
C(Ar)), 7.42–7.36 (m, 1H, H–C(Ar)), 5.37 (s, 1H, H–N), 4.16 (t, 2H,
3J = 6.4 Hz, CH2–CH2–CO), 2.58 (t, 2H, 3J = 6.4 Hz, CH2–CH2–CO),
2.04 (m, 2H, H–C(adam.)), 1.95–1.94 (m, 5H, H–C(adam.)), 1.65
(m, 8H, H–C(adam.)). HRMS for [C20H24N2O2S+Na+]: m/z calcd
379.1456; found 379.1443.
2.6.2.6. N-(3,5-Dimethyladamantan-1-yl)-3-(3-oxobenzo[d]isot-
hiazol-2(3H)-yl)acetamide (1b). Compound 1b was obtained by
general procedure A using 8 and 3,5-dimethyladamantan-1-amine
(9a). Yield: 23%. RP-UPLC (C18): tR = 6.65 min, purity >95%. 1H NMR
(CDCl3, 300 MHz) d, ppm: 8.06 (d, 1H, 3J = 7.5 Hz, H–C(Ar)), 7.68–
7.56 (m, 2H, H–C(Ar)), 7,46–7,41 (m, 1H, H–C(Ar)), 5.94 (s, 1H,
H–N), 4.39 (s, 2H, CO–CH2–N), 1.82 (m, 3H, H–C(adam.)), 1,67–
1.57 (m,4H, H–C(adam.)), 1.38–1.23 (m, 4H, H–C(adam.)), 1.18–
1.08 (m, 2H, H–C(adam.)), 0.82 (s, 6H, (Me)2-C(adam.)). HRMS for
[C21H26N2O2S+H+]: m/z calcd 371.1788; found 371.1794.
2.6.2.12. Methyl 3-(3-(3-oxobenzo[d]isothiazol-2(3H)-yl)propa-
namido)adamantane-1-carboxylate (1h). Compound 1h was
obtained by general procedure A using 7a and methyl 3-aminoada-
mantane-1-carboxylate (9e). Yield: 29%. RP-UPLC (C18): tR = 4.25 -
min, purity >95%. 1H NMR (CDCl3, 300 MHz) d, ppm: 8.02 (d, 1H,
3J = 7.9 Hz, H–C(Ar)), 7.63–7.52 (m, 2H, H–C(Ar)), 7.41–7.36 (m,
1H, H–C(Ar)), 5.56 (s, 1H, H–N), 4.15 (t, 2H, 3J = 6.2 Hz, CH2-CH2-
CO), 3.63 (s, 3H, Me-OCO), 2.58 (t, 2H, 3J = 6.2 Hz, CH2–CH2–CO),
2.17 (m, 2H, H–C(adam.)), 2.01 (m, 2H, H–C(adam.)), 1.99–1.87
(m, 4H, H–C(adam.)), 1.82–1.73 (m, 4H, H–C(adam.)), 1.66–1.56
(m, 2H, H–C(adam.)). HRMS for [C22H26N2O4S+H+]: m/z calcd
415.1686; found 415.1677.
2.6.2.7.
N-(3-Hydroxy-5,7-dimethyladamantan-1-yl)-3-(4-flu-
oro-3-oxobenzo[d]isothiazol-2(3H)-yl)propanamide (1k). Com-
pound 1k was obtained by general procedure A using 7b and
3-amino-5,7-dimethyladamantan-1-ol (9b). Yield: 25%. RP-UPLC
(C18): tR = 4.56 min, purity >95%. 1H NMR (CDCl3, 300 MHz) d,
ppm: 7.58–7.51 (m, 1H, H–C(Ar)), 7.29 (d, 1H, 3J = 8.1 Hz, H–
C(Ar)), 6.99 (dd, 1H, 3J = 8.1, 7.5 Hz, H–C(Ar)), 5.57 (br s, 1H, H–
N), 4.11 (t, 2H, 3J = 6.1 Hz, CH2–CH2–CO), 2.58 (t, 2H, 3J = 6.1 Hz,
CH2–CH2–CO), 1.84 (s, 2H, H–C(adam.)), 1.63–1.51 (m, 5H, H–
C(adam.)), 1.41–1.25 (m, 4H, H–C(adam.)), 1.14–1.04 (m, 2H,
H–C(adam.)), 0.90 (s, 6H, (Me)2-C(adam.)). HRMS for [C22H27FN2O3
S+H+]: m/z calcd 419.1799; found 419.1810.
2.6.3. General procedure B for the synthesis of benzo[d]
isothiazol-3(2H)-one—adamantane-1-carbohydrazide
conjugates 1n, 1o, 1p
2.6.3.1. N0-(2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetyl)adaman
tane-1-carbohydrazide (1n). EDCI (0.16 g, 0.83 m Mol, 1.15 equiv)
and adamantane-1-carbohydrazide (9f) (0.15 g, 0.77 m Mol,
2.6.2.8. N-(3-Hydroxyadamantan-1-yl)-3-(4-fluoro-3-oxobenzo
[d]isothiazol-2(3H)-yl)propanamide (1l). Compound 1l was
obtained by general procedure A using 7b and 3-aminoadaman-
tan-1-ol (9c). Yield: 17%. RP-UPLC (C18): tR = 3.01 min, purity >
95%. 1H NMR (CDCl3, 300 MHz) d, ppm: 7.57–7.51 (m, 1H, H–
C(Ar)), 7.29 (d, 1H, 3J = 8.1 Hz, H–C(Ar)), 6.98 (m, 1H, H–C(Ar)),
5.60 (s, 1H, H–N), 4.11 (t, 2H, 3J = 6.1 Hz, CH2–CH2–CO), 2.58
(t, 2H, 3J = 6.1 Hz, CH2-CH2-CO), 2.24 (m, 2H, H–C(adam.)), 1.96
(m, 1H, H–C(adam.)), 1.93–1.83 (m, 4H, H–C(adam.)), 1.67 (s, 6H,
H–C(adam.)), 1.59–1.44 (m, 2H, H–C(adam.)). HRMS for [C20H23
FN2O3S+H+]: m/z calcd 391.1486; found 391.1497.
1.07 equiv) were successively added to
a solution of 2-(3-
oxobenzo[d]isothiazol-2(3H)-yl)acetic acid (9) (0.15 g, 0.72 m Mol,
1.00 equiv) in anhydrous DMF (5 mL) at ambient temperature. The
resulting reaction mixture was stirred at ambient temperature for
21 h. The solvent was evaporated under reduced pressure and the
residue was partitioned between water (10 mL) and ethyl acetate
(20 mL). The aqueous phase was additionally extracted with ethyl
acetate (3 ꢁ 5 mL). The combined organic layer was successively
washed with 5% aqueous solution of citric acid (7 mL), 5% aqueous
solution of NaHCO3 (7 mL), brine (10 mL), dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure. Crystalli-
zation of the crude product from a mixture consisting from ethyl
acetate and hexanes provided 1n (61 mg, 22%). Mp 242–244 °C.
2.6.2.9.
N-(Adamantan-1-yl)-3-(4-fluoro-3-oxobenzo[d]isot-
Compound 1i was
RP-UPLC (C18): tR = 4.83 min, purity >95%. IR (KBr) m
, cmꢀ1: 3250,
hiazol-2(3H)-yl)propanamide (1i).
3040, 2905, 2850, 1725, 1650, 1600, 1530, 1450, 1350, 1225; 1H
NMR (DMSO-d6, 300 MHz) d, ppm: 10.03 (s, 1H, NH), 9.42 (s, 1H,
NH), 7.98 (d, 1H, 3J = 7.8 Hz, H–C(Ar)), 7.88 (d, 1H, 3J = 7.8 Hz, H–
C(Ar)), 7.70 (t, 1H, 3J = 7.8 Hz, H–C(Ar)), 7.44 (t, 1H, 3J = 7.8 Hz, H–
C(Ar)), 4.53 (s, 2H, CO–CH2–N), 2.02–1.92 (m, 3H, H–C(adam.)),
1.89–1.85 (m, 1H, H–C(adam.)), 1.83–1.78 (m, 5H, H–C(adam.)),
1.72–1.60 (m, 6H, H–C(adam.)). 13C NMR (DMSO-d6, 75.5 MHz) d,
ppm: 175.8, 165.8, 164.8, 141.4, 131.9, 125.6, 125.3, 123.3, 121.7,
obtained by general procedure A using 7b and adamantan-1-amine
(9d). Yield: 21%. RP-UPLC (C18): tR = 4.84 min, purity >95%. 1H
NMR (CDCl3, 300 MHz) d, ppm: 7.57–7.50 (m, 1H, H–C(Ar)), 7.28
(d, 1H, 3J = 8.1 Hz, H–C(Ar)), 6.98 (dd, 1H, 3J = 8.3, 8.1 Hz, H–
C(Ar)), 5.34 (s, 1H, H–N), 4.12 (t, 2H, 3J = 6.1 Hz, CH2–CH2–CO),
2.56 (t, 2H, 3J = 6.1 Hz, CH2–CH2–CO), 2.05 (m, 2H, H–C(adam.)),
1.95 (m, 6H, H–C(adam.)), 1.65 (m, 7H, H–C(adam.)). HRMS for
[C20H23FN2O2S+H+]: m/z calcd 375.1537; found 375.1548.
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6
D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
44.0, 38.4, 38.2, 36.0, 27.5. HRMS for [C20H23N3O3S+Na+]: m/z calcd
408.1358; found 408.1358. Anal. Calcd for C20H23N3O3S: C 62.32, H
6.01, N 10.90. Found 62.19, H 6.22, N 11.03.
seeded into 96-well microplates at an initial density of 1 ꢁ 104
cells per well in DMEM culture medium with 10% FCS. After 24 h
of incubation, the cells were exposed to the chemical compounds
at a graded series of concentrations for 48 h. The cell medium
2.6.3.2.
noyl)adamantane-1-carbohydrazide (1o).
N0-(3-(3-Oxobenzo[d]isothiazol-2(3H)-yl)propa-
Compound 1o
was removed, and 100 lL of fresh culture medium without phenol
red and 1 mM MTT solution was added to each well. After 3 h of
incubation, an equal volume of 1% SDS–HCl solution was added,
and the plates were incubated for 4 h. The absorbance was mea-
sured at 570 nm with a TECAN microplate reader. The results are
expressed as IC50 values, which were calculated from the dose
response curve through nonlinear regression using the SigmaPlot
12.5 software. The IC50 values were defined as the concentration
of the test compound that inhibits NIH 3T3 cell proliferation by
50% with respect to the untreated control.
was obtained by general procedure B using 7a and adamantane-
1-carbohydrazide (9f). Yield: 27%. RP-UPLC (C18): tR = 4.76 min,
purity >95%. 1H NMR (CDCl3, 300 MHz) d, ppm: 9.39 (br s, 1H, H–
N(hydraz.)), 8.38 (s, 1H, H–N(hydraz.)), 8.00 (d, 1H, 3J = 7.9 Hz,
H–C(Ar)), 7.62–7.51 (m, 2H, H–C(Ar)), 7.41–7.35 (m, 1H, H–
C(Ar)), 4.21 (t, 2H, 3J = 6.6 Hz, CH2–CH2–CO), 2.80 (t, 2H,
3J = 6.6 Hz, CH2–CH2–CO), 2.02 (m, 3H, H–C(adam.)), 1.95–1.80
(m, 6H, H–C(adam.)), 1.77–1.62 (m, 6H, H–C(adam.)). HRMS for
[C21H25N3O3S+H+]: m/z calcd 400.1689; found 400.1698.
2.9. Associated content
2.6.3.3. N0-(3-(4-Fluoro-3-oxobenzo[d]isothiazol-2(3H)-yl)pro-
panoyl)adamantane-1-carbohydrazide (1p).
Compound 1p
2.9.1. Accession codes
was obtained by general procedure B using 7b and adamantane-
1-carbohydrazide (9f). Yield: 34%. RP-UPLC (C18): tR = 3.98 min,
purity >95%. 1H NMR (DMSO-d6, 300 MHz) d, ppm: 9.70 (s, 1H,
H–N(hydraz.)), 9.26 (s, 1H, H–N(hydraz.)), 7.78 (d, 1H, 3J = 8.1 Hz,
H–C(Ar)), 7.70–7.63 (m, 1H, H–C(Ar)), 7.16 (dd, 1H, 3J = 8.1,
7.9 Hz, H–C(Ar)), 3.98 (t, 2H, 3J = 6.9 Hz, CH2–CH2–CO), 2.57 (t,
2H, 3J = 6.6 Hz, CH2–CH2–CO), 1.96 (m, 2H, H–C(adam.)), 1.80 (m,
6H, H–C(adam.)), 1.66 (m, 6H, H–C(adam.)). HRMS for [C21H24FN3
O3S+H+]: m/z calcd 418.1595; found 418.1607.
The chemical shifts and coordinates along with experimental
restraints for the structure of inhibitor 1–SrtA complex have been
deposited in the Biological Magnetic Resonance Bank (accession
number 19826) and in the Protein Data Bank (ID code 2mLM).
3. Results
This study included a primary HTS screening of a small-mole-
cule library based on a biochemical assay and a subsequent HSQC
NMR screening of the selected hits. The NMR screening allowed the
exclusion of the false positives and the characterization of the inhi-
bition type of all of the candidates. The best hit (1a) was selected
for further investigation, and an NMR structure of the enzyme–
inhibitor complex was obtained. Possible property-improving
modifications were introduced into the candidate compound to
perform a preliminary hit-to-lead optimization. The derived ana-
logs were characterized (enzymatic inhibition, bactericidal proper-
ties, cytotoxicity), and a molecule with improved properties was
selected. A schematic presentation of the overall concept of the
research study is provided in Figure 1.
2.6.3.4. N-(Adamantan-1-yl)-2-(1-oxoisoindolin-2-yl)acetamide
(13).
Compound 13 was obtained by general procedure A
using 2-(1-oxoisoindolin-2-yl)acetic acid (11) and adamantan-1-
amine hydrochloride (12ꢂHCl) in the presence of 1 equiv of DMAP.
Yield: 31%. Mp 188–189 °C. RP-UPLC (C18): tR = 4.52 min, purity
>95%. IR (film)
m
, cmꢀ1: 3290, 3060, 2910, 2850, 1690, 1665,
1545, 1455, 1425, 1297; 1H NMR (CDCl3, 400 MHz) d, ppm: 7.87
(d, 1H, 3J = 7.6 Hz, H–C(Ar)), 7.57 (dt, 1H, 3J = 7.6, 4J = 1.2 Hz, H–
C(Ar)), 7.50–7.46 (m, 2H, H–C(Ar)), 5.83 (s, 1H, NH), 4.54 (s, 2H,
–CH2–), 4.15 (s, 2H, –CH2–), 2.08–2.02 (bs, 3H, H–C(adam.)),
2.00–1.94 (m, 6H, H–C(adam.)), 1.69–1.60 (m, 6H, H–C(adam.));
13C NMR (CDCl3, 100.6 MHz) d, ppm: 169.1, 167.2, 141.5, 131.8
(2C), 128.1, 123.9, 122.9, 52.2, 51.2, 48.1, 41.5, 36.2, 29.4; HRMS
for [C20H24N2O2+H+]: m/z calcd 325.1911; found 325.1927.
3.1. High-throughput screening of sortase A inhibitors
A biochemical assay, which was successfully implemented for
the characterization of sortase enzymatic activity and is based on
the application of the dabcyl-QALPETGEE-edans substrate,30 was
optimized for HTS screening. The translation of the assay to the
2.7. Antibacterial activity test
The minimal inhibitory concentration (MIC) was defined as the
concentration of an antimicrobial agent that completely inhibits
cell growth during 24 h of incubation at 37 °C. The MIC values of
the test compounds that inhibited the cell growth of Gram-positive
Staphylococcus aureus (ATCC 25904) and Staphylococcus epidermidis
(ATCC 12228), and Gram-negative Escherichia coli (ATCC 25922)
were determined through the broth dilution micromethod.42 A ser-
ies of solutions with concentrations ranging from 0.1
lg/mL to
500 g/mL were prepared in Mueller Hinton for the Staphillococci
l
and Luria Bertani broth for E. coli using logarithmic-phase culture
with OD600 equal to 0.1 (corresponding to ꢃ1.2 ꢁ 108 CFU/mL in
a 96-well plate). The plate was incubated at 37 °C for 24 h, and
the growth was assayed using a microplate reader by monitoring
the absorption at 600 nm. All of the experiments were performed
in triplicate and repeated three times.
2.8. MTT cytotoxicity test
The cytotoxicity of the selected hits was tested on the Swiss
mouse embryo fibroblast cell line NIH 3T3 using a previously pub-
lished method43 with slight modifications. Briefly, the cells were
Figure 1. Schematic presentation of the library screening and further optimization
of the properties of the selected hit.
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D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
384-plate format yielded a Z0 of 0.82. Therefore, the system was
stable and suitable for the screening of inhibitor candidates.
For the primary end-point screening, the threshold for the rela-
tive sortase inhibition in the presence of the compounds being
tested was set to 65%. The relative inhibitory activity (%I) of the
50,240 molecules (Enamine) in the library was assayed at a single
below protein saturation, indicating slow-exchange complex for-
mation on the NMR time scale, whereas two compounds induced
NMR resonance shifts indicative of fast-exchange complex forma-
tion. The reversibility of inhibitor binding was determined by mea-
suring an HSQC spectrum after dialysis of the SrtA–inhibitor
complexes against NMR buffer (resulting in at least 100-fold dilu-
tion of the inhibitor). The binding of all 28 hits that showed com-
plex formation under slow-exchange conditions was irreversible
because no significant changes in the HSQC spectra were observed
after dialysis, with the exception of small peak shifts due to slightly
altered solution conditions. Furthermore, binding of most of the
irreversible inhibitors resulted in the appearance of several new
peaks corresponding to residues in the b6/b7 loop and indicating
that this loop was stabilized. Thus, 11 of the 41 compounds did
not demonstrate any binding to the sortase protein, two com-
pounds showed reversible binding, and 28 compounds exhibited
irreversible binding. Therefore, the NMR screening allowed the
exclusion of the false-positive hits selected by the biochemical
assay. In addition, further investigation of the compounds demon-
strated that two candidates are not stable in aqueous solution and
undergo hydrolysis reaction, which resulted in a reduction in the
number of hits to 26.
dose of 100 lM in triplicate to select 41 compounds that demon-
strated a relative inhibition of the assay reaction in the range of
65–100%. The selected molecules were subsequently subjected to
a secondary screening under the same conditions to limit their var-
iability, and all of the compounds demonstrated the same %I.
3.2. NMR screening of selected HTS hits
To elucidate the aspects of the inhibition caused by the HTS hits
and to evaluate the reversibility of the binding, further NMR exper-
iments were performed. Proton 1D and 2D 15N–1H HSQC NMR
experiments were performed to confirm the binding of the com-
pounds to the active site of the sortase A enzyme through chemical
shift perturbation of the protein resonances. The published chem-
ical shifts for SrtA44 were transferred to in-house spectra, and the
site-specific chemical shift changes upon addition of the hit com-
pounds were analyzed.
The data produced by the NMR analyses allowed the exclusion
of nonspecific inhibitors that demonstrated no binding according
to the NMR spectra but yielded a reduced fluorescence in the bio-
chemical assay reactions. In total, 30 of the 41 hits were confirmed
as SrtA active site binders. Twenty-eight compounds gave rise to a
second set of HSQC resonances when added at concentrations
Because the NMR experiments were performed in the same way
and under the same conditions for all of the compounds, the
potency of the binding could be compared through the evaluation
of the quantities of the accumulated enzyme molecules with the
shifted signals within certain time after adding the compounds.
Thus, seven of the most potent inhibitors were able to modify
about 50% of the sortase protein within the first hour of the
Figure 2. Binding of selected hit 1a to SrtA monitored by 2D [15N,1H]-HSQC NMR spectroscopy. (A) Spectrum of SrtA in absence of inhibitor. (B) Spectrum of SrtA after an
addition of 0.15 mM (0.5 equiv) inhibitor shows the appearance of a second set of peaks corresponding to the inhibitor-bound form (e.g., see boxed peaks corresponding to
residues G90, A118, G119 and N188 located within approx. 4–10 Å from the active site) as well as new peaks corresponding to residues located within or in contact with the
b6/b7 loop (T156, I158, D165, V166). (C) Spectrum of SrtA in presence of 0.3 mM (1.0 equiv) inhibitor shows only the inhibitor-bound form. (D) Spectrum of the sample from
(C) after dialysis against NMR sample buffer shows almost no differences with respect to the spectrum in (C) indicating that inhibitor binds to SrtA irreversibly.
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8
D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 2
SAR study of the analog compounds
Compound
Structure
cLogP* SrtA
inhibition,
MIC (
l
g/mL) [
l
M]
NIH 3T3 growth
inhibition,
S. aureus
S. epidermidis
E. coli
#
#
IC50
(
lM)
IC50
(lM)
O
N
S
O
O
>500
[>1495]
1a (selected hit)
3.253
4.291
6.11 0.34
1 [2.92]
0.5 [1.46]
1.27 0.06
HN
HN
HN
O
N
S
>500
[>1349]
1b
4.99 0.46
32 [86.37]
16 [43.18]
4.09 0.48
O
N
S
O
>500
[>1293]
1c
3.074
3.87 0.36
8 [20.69]
8 [20.69]
1.33 0.19
OH
O
N
O
S
>500
[>1402]
1d
3.385
3.81 0.17
8 [22.44]
2 [5.61]
5.95 0.14
NH
O
N
O
S
>500
[>1300]
1e
4.423
4.62 0.44
8 [20.8]
8 [20.8]
4.23 0.26
NH
O
N
O
S
>500
[>1248]
NH
1f
3.206
7.06 0.27
16 [39.95]
16 [39.95]
7.52 0.19
OH
O
N
O
S
>500
[>1342]
NH
1g
2.168
6.44 0.31
16 [42.96]
32 [85.91]
7.13 0.56
OH
O
N
O
S
>500
[>1206]
NH
1h
3.076
5.08 0.38
16 [38.59]
8 [19.29]
12.11 0.36
COOMe
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D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
F
O
N
O
S
>500
[>1335]
1i
3.528
4.01 0.48
32 [85.45]
4 [10.68]
0.90 0.08
NH
F
F
O
N
O
S
>500
[>1242]
1j
4.566
4.07 0.29
16 [39.74]
1 [2.48]
9.89 0.68
NH
O
N
O
S
>500
[>1194]
NH
1k
3.349
2.311
3.219
3.96 0.23
6.26 0.16
5.06 0.35
16 [38.23]
16 [38.23]
2.17 0.22
1.34 0.15
8.02 0.17
OH
F
O
N
O
S
>500
[>1280]
NH
1l
64 [163.91] 64 [163.91]
OH
F
O
N
O
S
>500
[>1156]
NH
1m
16 [36.99]
8 [18.49]
COOMe
O
O
N
H
N
H
N
>500
[>1297]
S
1n
2.852
2.931
3.80 0.10
16 [41.51]
4 [10.38]
14.39 0.92
O
O
N
O
S
H
>500
[>1251]
1o
4.22 0.12
8 [20.02]
4 [10.01]
12.19 1.07
N
H
N
O
O
F
O
N
O
S
>500
[>1197]
H
N
1p
7a
3.074
1.667
3.39 0.26
16 [38.32]
8 [19.16]
1.67 0.11
N
H
O
N
O
12.78
64 [286.67] 64 [286.67]
16 [71.67] 21.82
S
OH
(continued on next page)
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10
D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 2 (continued)
Compound
Structure
cLogP* SrtA
inhibition,
MIC (
l
g/mL) [
l
M]
NIH 3T3 growth
inhibition,
S. aureus
S. epidermidis
E. coli
#
#
IC50
(
lM)
IC50
(lM)
NH3
NH3
Cl
No
inhibition
>500
[>2663]
>500
[>2663]
9d
9e
1.440
1.302
>500 [>2663]
2290.95
2290.95
Cl
No
inhibition
>500
[>2034]
>500
[>2034]
>500 [>2034]
>500 [>1541]
COOMe
O
N
O
No
inhibition
>500
[>1541]
>500
[>1541]
11
2.816
262.68 10.31
HN
O
Cl
Cl
1-(3,4-Dichlorophenyl)-3(dimethylamino)-1-
propanone24
>500
[>1769]
N
NnA
NnA
16.91 1.86 32 [113.24] 16 [56.62]
9.71 0.44
7.91 0.26
O
S
O
>500
[>2958]
>500
[>2958]
29
Phenyl vinyl sulfone
736
>500 [>2958]
*
cLogP were determined for all newly synthesized compounds using ChemBioDrawUltra 13.0 software.
Frankel, et al. (2004).29
#
Data are given as the mean of three independent experiments SD.
co-incubation. The majority of the compounds required 1–4 h to
modify approximately 50% of the sortase, and the weakest inhibi-
tors needed more than 4 h.
of 17139 Da, which agrees with the theoretical calculation of
17142 Da.
Together, these data confirmed the ability of the selected hit 1a
to form a covalent bond between the sulfur atom of the active site
cysteine residue and the benzothiazol group of 1a.
Many of the selected hits belonged to the previously known
thiol-enzyme inhibitor classes, such as nitriles, thioamides, and
Michael acceptors, and were therefore excluded from further opti-
mization. The summary of the data obtained from the biochemical
assay and NMR experiments revealed that the best compound was
the one that (1) exhibited a high inhibitory effect in the biochem-
ical assay, (2) demonstrated the ability to bind an active center of
the enzyme, as confirmed through the NMR experiments, and (3)
demonstrated a high velocity of binding and adduct accumulation
according to the NMR data (Figure 2). Taking into the consideration
the above-mentioned parameters, N-(adamantan-1-yl)-2-(3-oxo-
2,3-dihydro-1,2-benzothiazol-2-yl)-acetamide (1a) was selected
as the best hit from the library screening (Table 2).
To investigate the orientation of the selected hit 1a within the
substrate binding pocket in detail, the 3D structure of the enzyme–
inhibitor complex was determined by NMR. The protein chemical
shifts in the presence of the selected hit 1a were assigned and NOE
distance restraints were collected using the following spectra: HNCA,
CBCA(CO)NH,
15N-resolved
NOESY-HSQC,
13C-(aliphatic)-
resolved NOESY-HSQC, 13C(aromatic)-resolved NOESY-HSQC, and
(F1)-15N,13C-filtered 13C(aliphatic)-resolved NOESY-HSQC. The
chemical shifts of the bound inhibitor were assigned using
(F1,F2)-13C,15N-filtered 2D NOESY and TOCSY spectra. The calcu-
lated structure was based on 1680 upper distance limits, of which
9 were long range contacts between the modified cysteine side chain
(mostly, the adamantyl moiety) and other protein groups (Experi-
mental section. Determination of the inhibitor–enzyme complex
structure). The conformation of the inhibitor was not very well
defined (Fig. 4A), however the interacting groups were almost iden-
tical among the conformers. The possible explanation is that the
inhibitor interacts with residues in the b6/b7 and b7/b8 loops that
are partially disordered and able to adapt to different conformations
of the inhibitor. Superposition with the structure of the SrtA-LPAT
substrate analogue complex showed that the inhibitor binding site
overlapped with that of the LPAT peptide (Fig. 4B). A detailed
analysis of the molecular interactions (Fig. 4C) revealed that
depending on the conformer one or both carbonyl groups of the
inhibitor might form a hydrogen bond with R197, and the phenyl
group interacts in some conformers with residues L97 and A118,
3.3. NMR characterization of the selected hit 1a
It was confirmed by the screening data that the selected hit 1a
was an irreversible inhibitor of sortase activity able to bind to the
active site. Additional NMR experiments were performed to eluci-
date the aspects of the binding and to determine the reactive
groups participating in the process of irreversible adduct forma-
tion. Because sortases possess a reactive cysteine residue in the
active site, a model reaction between cysteine (2) and the selected
hit 1a was performed, and proton 1D and 2D TOCSY NMR spectra
were collected to analyze the products of the reaction (Fig. 3).
The resulting product 3 is characterized by low-field shift of the
a
H
(C/A) and Hb (D/B) peaks as well as a new low-field peak (I) that
shows a correlation (dashed line) to the J-methylene protons in the
2D TOCSY spectrum. Additionally, MALDI-TOF-MS analysis of the
enzyme-compound 1a complex was carried out and yielded a mass
while in other conformers a
p–p stacking interaction with W194 is
observed. The adamantyl moiety is buried in a hydrophobic cleft,
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D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
Figure 3. Reaction product 3 between the selected hit 1a and cysteine 2 analyzed by 2D TOCSY.
where it interacts with residues V166, V168, and L169 of the
realized and in total 15 new analogs 1b–p were synthesized
b6/b7 loop as well as residues T180, I182 and I199 from b7 and b8.
(Schemes 1 and 2).
3.4. Preliminary hit-to-lead optimization
3.5. Synthesis of the analogs of the selected hit 1a
Based on the three-dimensional structure of the SrtA–inhibitor
complex, we defined several molecular features of the inhibitor
that seemed critical for ensuring inhibitor binding and selectivity.
These were (1) the benzo[d]isothiazol-3(2H)-one heterocycle, (2)
the adamantyl moiety, and (3) the linker connecting the previous
two features. The benzo[d]isothiazol-3(2H)-one heterocycle is
essential for the formation of a covalent protein–inhibitor com-
plex. Therefore, this part of the molecule had to be left intact dur-
ing the optimization with the exception of substitutions in the
benzo part. In particular, the benzo[d]isothiazol-3(2H)-one posi-
tions 4, 5, and 6 did not show tight contacts with the protein,
and thus, the replacement of the hydrogens with larger groups
might provide additional interactions between the inhibitor and
the protein. The adamantyl moiety is buried in a hydrophobic cleft,
where it interacts with residues I182 and I199 as well as with
V166, V168, L169 of the b6/b7 loop, which has been shown to pos-
sess an increased mobility. Thus, the b6/b7 loop could adapt to the
inhibitor and hence it was difficult to predict the most-optimal
substituent and different hydrophobic groups had to be tested
experimentally to identify the one that would provide the best
interactions. Lastly, the linker provides two functions: positioning
of the benzo[d]isothiazol-3(2H)-one heterocycle and the adaman-
tane, and hydrogen-bonding with the R197 side chain. Therefore,
the influence of the linker length could be explored simultaneously
with the incorporation of the donors for hydrogen bonding with
the R197 side chain. Guided by these observations, several modifi-
cations to the molecular structure of the selected hit 1a were
A structural design of the target compounds was determined by
the necessity to combine ‘cysteine-philic’ benzo[d]isothiazol-
3(2H)-one moieties and bulky adamantyl substituents. Amide or
hydrazide bond forming reaction was chosen as the final step of
the synthesis. The required carboxylic acid coupling partners 2-(3-
oxobenzo[d]isothiazol-2(3H)-yl)propionic acid (7a)45 and its 4-flu-
oro analog 7b were synthesized by analogy to previously reported
methods (Scheme 1)46 but the known 2-(3-oxobenzo[d]isothiazol-
2(3H)-yl)acetic acid (8) was obtained by a recently published
procedure.47 Amidation of 7a,b and 8 with commercially available
amino-adamantane derivatives 9a–e and adamantane-1-carbohyd-
razide (9f) (Olainfarm) in the presence of EDCI provided target
compounds 1b–p (Scheme 2).
The importance of the benzo[d]isothiazol-3(2H)-one core for
the hit molecule 1a and the synthesized analogs 1b–p was addi-
tionally established by reductio ad impossibilem. Thus, compound
13 can be regarded as a 1-carba analog of 1a. It was obtained from
the commercially available acid 10 and amino adamantane 9d by
standard amide coupling chemistry as discussed above (Scheme 3).
3.6. Structure–activity relationship (SAR) study of the derived
compounds
To investigate whether the modifications of the selected hit 1a
suggested by the NMR structural studies were able to improve the
properties of the derived compounds, an extended characterization
of the synthesized compounds 1b–p was performed (Table 1). In
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D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 4. NMR structure of the SrtA–inhibitor 1a complex. (A) Superposition of the 10 lowest-target function conformers representing the NMR structure of SrtA complex
with inhibitor 1a. The heavy atoms of the inhibitor are connected with thin sticks. (B) Electrostatic potential surface representation of SrtA in complex with the inhibitor 1a
(cyan sticks), superimposed with the LPAT substrate analogue (magenta sticks; PDB code 2KID) SrtA complex. Negatively charged areas are colored red and positively charged
areas are blue. (C) Stereoview of SrtA active site after modification by inhibitor 1a. The inhibitor and near-by side chains of SrtA are shown with sticks and labeled. C, N and O
a
atoms are colored grey, blue and red, respectively, except for the C atoms of the inhibitor that are colored cyan and C atoms of SrtA residues identified in Figure 2 that are
colored magenta. Hydrogen atoms are omitted for clarity. Dashed yellow lines indicate hydrogen-bonding and
The figure was generated using The PyMOL Molecular Graphics System, version 1.5.0.4; Schrödinger, LLC.
p–p stacking interactions with R197 and W194, respectively.
Scheme 1. Reagents and conditions: (a) H2NCH2CH2COOMeꢂHCl, CDI, NEt3; (b) Br2; (c) LiOH.
particular, the IC50 for the inhibition of the sortase A activity
to form a covalent bond with C184, and it is likely that other inter-
actions are less important. To confirm this indication, we synthe-
sized compound 11 (Scheme 3), in which the benzo[d]isothiazol-
3(2H)-one moiety of 1a was replaced by an isoindolin-1-one. Com-
pound 11 did not exhibit any activity in the enzymatic assay and
showed no binding to the enzyme by NMR analysis. If the ring open-
ing of benzo[d]isothiazol-3(2H)-one and the formation of the disul-
fide bridge between the inhibitor and the thiol moieties of the
enzyme were not essential for the biological activity, the selected
hit 1a and its 1-carba analog 11 would exhibit similar bactericidal
and cytotoxic properties. In contrast, the compound 11 did not exhi-
bit considerable inhibitory activity that proves the proposed mode
in vitro was determined, and the minimal inhibitory concentration
(MIC) was ascertained to evaluate the antibacterial activity of the
compounds in bacterial culture. In addition, the cytotoxic activity
of the molecules was evaluated on NIH 3T3 mice fibroblast cells
through the determination of the IC50 with respect to the inhibition
of cell proliferation.
In general, the structural modifications introduced to the
selected hit 1a had little effect on SrtA inhibition potency (IC50 val-
ues vary between 3.39 and 7.06 lM), but more strongly affected
MIC and cytotoxicity. This implies that the SrtA inhibitory activity
for this class of compounds is primarily depending on the ability
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D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
13
Scheme 2. Synthesis of the 15 novel analogs of the selected hit 1a. Reagents and conditions: (a) EDCI.
of more hydrophobic dimethyl/hydroxyl, or more polar
hydroxyl/methoxycarbonyl substituents in the adamantyl
moiety.
(2) Compounds with extended linkers comprising 4–6 atoms
(1d, 1n, 1o and 1p) showed increased activities in compari-
son to the compounds with shorter linkers (1a). Also, no sig-
nificant improvement was observed upon introduction of an
additional H-bond acceptor and donor (1n, 1o and 1p vs 1d).
Scheme 3. Synthesis of amide 11 as a 1-carba analog of the selected hit 1a.
(3) Introduction of
a fluorine in the 4-position of the
benzo[d]isothiazol-3(2H)-one heterocycle had no significant
effect on SrtA inhibition (1i vs 1d, 1j vs 1e, 1l vs 1g, 1m vs
1h, 1p vs 1o).
of action of compounds containing benzo[d]isothiazol-3(2H)-one
ring system. Furthermore, we tested the activity of the benzo[d]isot-
hiazol-3(2H)-one building block, which was used for syntheses (7a)
and observed that sortase inhibition activity was only two times
lower in comparison to compound 1a, thus further emphasizing
the importance of the benzo[d]isothiazol-3(2H)-one heterocycle.
Additionally, the adamantane building blocks (9d and 9e) showed
no activity against SrtA.
As noted above, the introduced modifications had a stronger
effect on cytotoxicities than on inhibitory activities. The analysis
of the cytotoxicity yielded the following conclusions:
(1) Introduction of substituents in the adamantyl moiety
showed no significant effect on cytotoxicity in most cases,
except for the introduction of two methyl groups (1b vs
1a, 1j vs 1i) and a methoxycarbonyl group (1d vs 1h, 1i vs
1m).
(2) Compounds containing linkers that incorporated an acety-
lhydrazide or propionohydrazide moiety showed significantly
lower cytotoxicities in mammalian cell cultures and slightly
higher MIC values in bacterial cultures (1a vs 1n–o).
(3) Introduction of the fluorine in the 4-position of the
benzo[d]isothiazol-3(2H)-one heterocycle usually resulted
in higher cytotoxicity in NIH 3T3 culture (1i vs 1d, 1k vs 1f,
1l vs 1g, 1m vs 1h) except for 1j/1e pair. This may be explained
by an increased positive charge on the benzo[d]isothiazol-
3(2H)-one sulfur atom that could enhance reactivity towards
Despite the small variations in potency, the following SAR trends
become apparent by comparing the inhibitory activity of 1b–p:
(1) Introduction of substituents in the adamantyl moiety can
either increase or decrease the inhibitory activity depending
on the linker length and other substituents in the molecule.
Thus, compounds 1b and 1c possessing methyl and hydroxyl
substituents in the adamantyl moiety and the linker com-
prising 3 atoms (2 carbons and 1 nitrogen) yielded improved
activities in comparison to 1a. An opposite effect was
observed, when the linker length was extended by one car-
bon atom (1e, 1f, 1g, 1h vs 1d). Next, compounds with an
additional 4-fluoro group showed similar (1j and 1k vs 1i)
or slightly lower activities (1l, 1m vs 1i) upon introduction
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D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
nucleophiles. Simultaneously, the introduction of fluorine
demonstrated that the ring opening was crucial for this binding. This
reactive moiety is known to inhibit other thiol-enzymes,49,50 which
explains the cytotoxicity of hit 1a and its analogs, which are able to
react with enzymes within bacterial and mammalian cells. This sug-
gestion is supported by the fact that 3-(3-oxobenzo[d]isothiazol-
2(3H)-yl)propanoic acid (7a) is slightly less toxic for the mammalian
and bacterial cells than compounds 1a–p (Table 1). Since more
hydrophobic compounds 1a–p possess improved transmembrane
crossing capabilities, their toxicity should be increased if it is caused
by inter-cellular non-specific interactions.
into the heterocycle either did not alter the MIC or slightly
increased it in the bacterial cultures. There could be two
possible reasons for this: (1) the increased reactivity towards
the nucleophiles promotes the non-specific interactions with
the components of the bacterial medium, thus decreasing the
number of molecules, which inhibit the bacterial enzymes
within the cell, (2) the presence of polar substituent in the
benzo[d]isothiazol-3(2H)-one moiety decreases the capabil-
ity of the compounds to cross the cell membrane and there-
fore the toxicity towards microorganisms is reduced.
The bactericidal properties of the compounds are a crucial
parameter for the lead selection. It is accepted that sortase A inhib-
itors possessing low bactericidal activity might be better candi-
dates for clinical use in the future. Sortases are not vitally
important enzymes for bacteria in culture, and the absence of bac-
tericidal properties can greatly reduce the selective pressure of the
inhibitor molecules, thereby minimizing the risk of the develop-
ment of bacterial resistance.16,17 The selected hit 1a was found to
exhibit strong bactericidal activity in both S. aureus and S. epidermi-
dis cultures, which was significantly greater than the activity of
compound 7a, which lacks the adamantyl moiety. The introduction
of the chemical modifications to hit 1a decreased the bactericidal
activity for all of the analogs 1b–p (Table 1). This finding may be
explained by the fact that the accurate experimental structure of
the enzyme–compound 1a complex, which was used for the selec-
tion of the possible modifications, provided a possibility of improv-
ing the specificity of the analogs. These compounds exhibit a lower
ability to inhibit bacterial proteins other than sortases. The inhib-
itory activity of the compounds 1b–p was higher than the activity
of the selected hit 1a and compound 7a, which also evidence the
impact of the structural alterations on the selectivity of the ana-
logs. This may be explained by the optimized orientation of the
reactive heterocycle within the active site provided by the modifi-
cations. Interestingly, in comparison to 1a–p, a less lipophilic 7a
with reduced capabilities to cross the cell membrane demon-
strated significantly higher bactericidal activity in E. coli culture.
This might be explained by its ability to inhibit the enzymes in
the periplasmic space, which, in turn, are not inhibited by the lar-
ger 1a–p molecules.
Some discrepancy between molecular structure and the MIC
values was observed. Although, there is an obvious correlation
between the presence of adamantyl moiety and the increased bac-
tericidal activity, other alterations of hit 1a did not demonstrate
such a clear correlation. Therefore, the collected data allow to
assume that bactericidal properties may depend on a combination
of various factors such as cell permeability, compound hydrolysis
in the presence of medium components, cell metabolites and
secreted enzymes and the reactions with cysteine side chains of
the oligopeptides that are present in the medium.
Although all of the compounds exhibited substantial cytotoxic-
ity against mammalian fibroblasts in vitro, their cytotoxicity was
comparable to that of the known inhibitors 1-(3,4-dichloro-
phenyl)-3-(dimethylamino)-1-propanone24 and phenyl vinyl sul-
fone29 under the same conditions (Table 1). Moreover, the MIC
value of 1-(3,4-dichlorophenyl)-3-(dimethylamino)-1-propanone
was comparable with the MIC values of the compounds 1a–p.
Simultaneously, 1a–p exhibited significantly greater inhibitory
activity than both reference compounds. The improved activity
may be explained by the optimized orientation of the reactive
group within the active site of sortase for the compounds 1a–p.
Both reference compounds possess hydrophobic aromatic moie-
ties, which orient the molecules within the active site of sortase,
as adamantyl-moieties orient compound 1a and its analogs. In con-
trast, benzo[d]isothiazol-3(2H)-one heterocycle of 1a–p provides
additional interactions with the environment within the active
site, which in turn optimizes orientation of the reactive sulfur atom
Some of the modifications provided simultaneous improve-
ments to all of the parameters of the inhibitor. The analog com-
pound 1n demonstrated an improved SrtA enzymatic inhibitory
activity when compared to the original hit 1a (Table 2). In contrast,
the MIC values of the molecule 1n were approximately ten-fold
higher than that of the original compound, however this com-
pound still exhibited significant inhibition of Gram-positive staph-
ylococci. Moreover, the cytotoxicity of 1n was significantly
decreased in mammalian cell culture. Additionally, none of com-
pounds 1a–p exhibited antibacterial activity against Gram-nega-
tive E. coli.
4. Discussion
In this study, we identified a novel class of potent S. aureus sor-
tase A inhibitors (Table 1). The compounds exhibited high sortase
A inhibition efficiency in vitro with IC50 values in the range of
3.39–7.06 lM. These values are comparable to the IC50 of the other
efficient inhibitors that have been reported to date.24
We combined a biochemical HTS screening with a less efficient
albeit more robust secondary NMR screening to identify hits from
the 50,240 compounds in a drug-like small-molecule library.
Although other researchers have mostly relied on a biochemical
assay for the selection of hits,24,26 we found that approaches based
on the application of FRET-substrates generate a high number of
false positives. Thus, of the 41 compounds selected by the HTS
based on the fluorimetric activity assay, 11 demonstrated no bind-
ing to a sortase molecule during the HSQC experiments. Although
these compounds can exhibit high rates of sortase inhibition
according to the biochemical experimental data, they do not bind
to the active site of the enzyme, and may in turn interfere with
the assay itself, for example, by binding to and/or precipitating
the FRET-substrate or binding Ca2+ ions, which are crucial for
sortase activity.48 Our approach allowed the exclusion of such hits
prior to time-consuming structural and SAR studies of the
candidates. Simultaneous HSQC experiments provided insights
into the enzyme–inhibitor binding process, such as the reversibil-
ity of the interactions and an approximate relative potency, based
on the accumulation of the adduct over time.
Based on the results of the screening tests, compound 1a was
selected as a novel molecule that possesses high inhibitory activity
accordingto both the end-point productaccumulationand the HSQC
NMR assay. An experimental NMR structure of the enzyme–inhibi-
tor complex was obtained and used for the subsequent design of
analogs, of which 15 compounds were synthesized. The new com-
pounds exhibited combinations of (1) substitutions in the adaman-
tyl moiety, (2) substitutions in the benzo[d]isothiazol-3(2H)-one
heterocycle, and (3) variations in the linker (Table 1). A comprehen-
sive SAR study was performed to characterize the derived
compounds.
We have found that 3-oxobenzo[d]isothiazol-2(3H)-yl moiety is
responsible for the covalent binding to the side chains of cysteine
residue within the sortase active site, and our experiments
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D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
15
in the heterocycle towards the thiol-group of the cysteine residue
of the sortase protein that affects IC50 values.
manuscript. This work was supported by the European Regional
Development Fund [2010/0312/2DP/2.1.1.1.0/10/APIA/VIAA/032].
Relatively high cytotoxicity of the irreversible inhibitors is a
consequence of their ability to inhibit enzymes other than the tar-
get protein. Despite this fact, it is accepted that irreversible inhib-
itors are able to widen therapeutic margins, since significantly
lower doses of the drugs are required for efficacy.51,52 A number
part of the enzymes inhibited by currently marketed drugs are
being modified irreversibly. It is possible to adjust the specificity
of such compounds to achieve the balance between their toxicity
and efficacy, sufficient for therapeutic applications. Irreversible
inhibitors as drug candidates have various advantages, such as
high biochemical activity, lower sensitivity towards pharmacoki-
netic parameters and increased duration of the therapeutic
effect.53–55 Although a majority of the identified library hits were
irreversible inhibitors, only two compounds demonstrated the
reversible binding. However, both reversible hits were significantly
less efficient and just slightly less toxic than the selected hit 1a and
its analogs. Additional modifications of these molecules did not
yield any improvements to their properties (data not shown).
Therefore, we have focused on the further optimization of the
selected irreversible hit 1a.
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