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

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 Dmitrijs Zhulenkovs a,b,⇑, Zhanna Rudevica a, Kristaps Jaudzems c, Maris Turks d, Ainars Leonchiks a a

Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia University of Latvia, Raina bulv. 19, Riga LV-1586, Latvia 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 b c

a r t i c l e

i n f o

Article history: 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

a b s t r a c t 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. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction 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 healthcare-associated infections.1 These Gram-positive organisms are among the most commonly isolated organisms in hospitals in Europe, North America, Oceania, and Africa.2 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

Abbreviations: HTS, high-throughput screening; SAR, structure–activity relationship; 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: [email protected] (D. Zhulenkovs).

and vancomycin, are becoming more abundant every year, prompting new health threats and having a significant impact on 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, nosocomial and community-acquired methicillin-resistant S. aureus (MRSA) strains and several other staphylococcal species have emerged as a serious problem in medicine. 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 factors 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 bacterial cells. Sortases belonging to classes A and B are the most abundant in Firmicutes. In these cells, sortase A provides an important 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

http://dx.doi.org/10.1016/j.bmc.2014.09.011 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

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 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 (srtA) are unable to retain and display LPXTG-bearing proteins at the bacterial cell surface. As a consequence, SrtA mutants are significantly less virulent and incapable of establishing acute infection. In contrast, SrtA is not indispensable 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 resistance 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 mechanism 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-terminal polyglycine sequence or by a water molecule in the absence of the peptide substrate.21 The full-length S. aureus sortase A comprises 206 amino acid residues. A N-terminal signal sequence necessary for the transmembrane 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 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 molecules 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 druglike 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. Methods 2.1. Expression of recombinant sortase A Recombinant and catalytically active sortase A with the N-terminal deletion of residues 1–59 and possessing C-terminal hexahistidine sequence (SrtADN59-6His) was used for the HTS and NMR screenings. SrtADN59-6His was prepared according to a

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 resuspended 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. The 15N isotope labeling of recombinant SrtADN59-6His for 2D 15 N-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 following the same procedures. A wild-type SrtADN59 catalytic domain without hexahistidine sequence was used for the determination of the inhibitor–enzyme complex structure. The 15N and 15N/13C isotope labeling of recombinant SrtADN59 was accomplished by cultivating BL21 (DE3) cells in M9 minimal medium containing 15N-ammonium salt and 13Cglucose as the sole nitrogen and carbon sources, respectively. The expression and the purification were carried out following a previously 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 recombinant protein was purified through Sepharose Fast Flow SP (GE Healthcare) and Source Q15 (GE healthcare) sequential chromatographic 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 containing 100 mM NaCl and 1 mM DTT, was used for the final purification. The fractions containing the protein were collected and concentrated to 10 mg/mL. 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.

2.2. FRET-based high-throughput screening of the chemical library A diverse, drug-like library containing 50,240 screening compounds was ordered from the entire 1.6-million-compound collection 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 sortase 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 determined 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 performed in a volume of 100 lL containing 50 mM Tris–HCl, 5 mM CaCl2, 150 mM NaCl, pH 7.5, 10 lM S. aureus SrtA, 20 lM fluorescent

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

3

D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

peptide substrate dabcyl-QALPETGEE-edans, and the prescribed concentrations of the test compounds or positive controls. The peptide substrate without the recombinant SrtA was incubated in the same manner and used as a negative control. The reactions 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 initiation of the reaction. The relative inhibition activity was determined as %I = 100%  (Fsample/Fcontrol  100%), where Fsample is the fluorescence intensity of the well containing the corresponding test compound and Fcontrol is the fluorescence of the positive control reaction without inhibition.24,26 The Z0 factor, which is a standard statistical parameter that characterizes the reproducibility and robustness of HTS assays, was calculated 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 (without SrtA, n = 192 wells), and SD is the standard deviation.31 For the IC50 determination, 10 lM S. aureus sortase A was preincubated in the reaction buffer with increasing concentrations of the candidate inhibitors (x  y lM) for 1 h at 37 °C prior to the addition of the dabcyl-QALPETGEE-edans substrate. The total fluorescence 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.

inhibitor. The reactivity towards the cysteine side chain was confirmed through a reaction in the NMR tube, in which 14.2 mM cysteine 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, 15Nlabeled 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 100 random conformers. The 20 conformers with the lowest residual 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. 2.6. Chemistry 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. Solvents were distilled prior to use and, if required, dried over standard 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 on silica gel (60 Å, 40–63 lm, ROCC). 1H and 13C NMR spectra were

2.3. NMR spectroscopy All of the NMR experiments were performed at 298 K on a 600MHz Varian Unity Inova spectrometer equipped with an HCN triple-resonance pulsed-field-gradient cold probe. The following 2D and 3D spectra were recorded for structure determination: 15 N–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). 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) spectra were recorded for assignment of the inhibitor resonances. Spectra were processed using NMRPipe34 or Bruker Topspin 3.2 and analyzed using CARA.35 2.4. NMR screening of the selected hits The binding of the inhibitor to the active site of SrtA was studied 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. The reversibility of the binding was determined by measuring a 2D 15N–1H HSQC spectrum after dialysis of the SrtA–inhibitor complex against NMR buffer, resulting in a 100-fold dilution of the

Table 1 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 Inhibitor-to-protein H-bond restraints

1680 548 457 152 514 9 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 (°)

0.0072 ± 0.0001 0.562 ± 0.017

PARALLHDG27 energies [kcal/mol] Total van der Waals Electrostatic

5661 ± 118 636 ± 27 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– 206) All heavy atoms (inhibitor 1) Ramachandran plot statistics (PROCHECK)41 Most favored regions (%) Additional allowed regions (%) Generously allowed regions (%) Disallowed regions (%)

0.75 ± 0.09 1.24 ± 0.09 1.39 ± 0.26 77.6 21.3 0.9 0.2

* Except for the top six entries, average values and standard deviations for the 20 energy-minimized conformers are given.

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

4

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-deuterated 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. The purity of all final compounds was >95% as determined by analytical UPLC-MS/DAD on reverse phase (column Waters ACQUITY 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—acetonitrile, 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 chloride (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(6fluorobenzoyl))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 temperature. 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]isothiazol-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 filtered. 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 colorless crystalline powder. Mp 201–202 °C. RP-UPLC (C18): tR = 2.51 min, purity >95%. IR (KBr) m, cm1: 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. General procedure A for the synthesis of benzo[d]isothiazol3(2H)-one—adamantane amine conjugates 1b–m 2.6.2.1. N-(3-Hydroxy-5,7-dimethyladamantan-1-yl)-2-(3oxobenzo[d]isothiazol-2(3H)-yl)acetamide (1c). N-EthylN0 -(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (0.15 g, 0.78 m Mol, 1.2 equiv) and 3-amino-5,7-dimethyladamantan-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 >95%. IR (KBr) m, cm1: 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., 2 J = 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-(3oxobenzo[d]isothiazol-2(3H)-yl)propanamide (1f). Compound 1f was obtained by general procedure A using 7a and 3-amino-5,7dimethyladamantan-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, 3 J = 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, 3 J = 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. 2.6.2.4. N-(3,5-Dimethyladamantan-1-yl)-3-(3-oxobenzo[d]isothiazol-2(3H)-yl)propanamide (1e). Compound 1e was obtained by general procedure A using 7a and 3,5-dimethyladamantan-1amine (9a). Yield: 21%. RP-UPLC (C18): tR = 4.56 min, purity >95%. 1 H 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,

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx 3

J = 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.

2.6.2.5. 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-dimethyladamantan-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, 3 J = 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)2C(adam.)). HRMS for [C22H27FN2O2S+H+]: m/z calcd 403.1850; found 403.1857. 2.6.2.6. N-(3,5-Dimethyladamantan-1-yl)-3-(3-oxobenzo[d]isothiazol-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. 2.6.2.7. N-(3-Hydroxy-5,7-dimethyladamantan-1-yl)-3-(4-fluoro-3-oxobenzo[d]isothiazol-2(3H)-yl)propanamide (1k). Compound 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. 2.6.2.8. 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-aminoadamantan-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.

2.6.2.9. N-(Adamantan-1-yl)-3-(4-fluoro-3-oxobenzo[d]isothiazol-2(3H)-yl)propanamide (1i). Compound 1i was 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.

5

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 3aminoadamantane-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, 3 J = 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. 2.6.2.11. N-Adamantan-1-yl-3-(3-oxobenzo[d]isothiazol-2(3H)yl)propanamide (1d). Compound 1d was obtained by general procedure A using 7a and adamantane-1-amine (9d). Yield: 31%. RPUPLC (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, 3 J = 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. 2.6.2.12. Methyl 3-(3-(3-oxobenzo[d]isothiazol-2(3H)-yl)propanamido)adamantane-1-carboxylate (1h). Compound 1h was obtained by general procedure A using 7a and methyl 3-aminoadamantane-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, 3 J = 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-CH2CO), 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. 2.6.3. 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, 1.07 equiv) were successively added to a solution of 2-(3oxobenzo[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. Crystallization of the crude product from a mixture consisting from ethyl acetate and hexanes provided 1n (61 mg, 22%). Mp 242–244 °C. RP-UPLC (C18): tR = 4.83 min, purity >95%. IR (KBr) m, cm1: 3250, 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,

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

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. 2.6.3.2. N0 -(3-(3-Oxobenzo[d]isothiazol-2(3H)-yl)propanoyl)adamantane-1-carbohydrazide (1o). Compound 1o was obtained by general procedure B using 7a and adamantane1-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, 3 J = 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.

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 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 measured 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. 2.9. Associated content

2.6.3.3. N0 -(3-(4-Fluoro-3-oxobenzo[d]isothiazol-2(3H)-yl)propanoyl)adamantane-1-carbohydrazide (1p). Compound 1p was obtained by general procedure B using 7b and adamantane1-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. 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-1amine hydrochloride (12HCl) 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, cm1: 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.)); 13 C 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. 2.7. Antibacterial activity test

2.9.1. Accession codes 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-molecule 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 inhibition 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 analogs were characterized (enzymatic inhibition, bactericidal properties, 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. 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

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 series of solutions with concentrations ranging from 0.1 lg/mL to 500 lg/mL were prepared in Mueller Hinton for the Staphillococci and Luria Bertani broth for E. coli using logarithmic-phase culture with OD600 equal to 0.1 (corresponding to 1.2  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 published 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.

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

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 relative 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 dose of 100 lM in triplicate to select 41 compounds that demonstrated 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 variability, 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 experiments were performed. Proton 1D and 2D 15N–1H HSQC NMR experiments were performed to confirm the binding of the compounds to the active site of the sortase A enzyme through chemical shift perturbation of the protein resonances. The published chemical shifts for SrtA44 were transferred to in-house spectra, and the site-specific chemical shift changes upon addition of the hit compounds 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 biochemical 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

7

below protein saturation, indicating slow-exchange complex formation on the NMR time scale, whereas two compounds induced NMR resonance shifts indicative of fast-exchange complex formation. The reversibility of inhibitor binding was determined by measuring an HSQC spectrum after dialysis of the SrtA–inhibitor complexes against NMR buffer (resulting in at least 100-fold dilution of the inhibitor). The binding of all 28 hits that showed complex 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 compounds 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 demonstrated 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. 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.

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

8

D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

Table 2 SAR study of the analog compounds Compound

c Log P*

Structure

SrtA inhibition, IC50# (lM)

MIC (lg/mL) [lM] S. aureus

S. epidermidis

E. coli

NIH 3T3 growth inhibition, IC50# (lM)

O

1a (selected hit)

S

N HN

O

3.253

6.11 ± 0.34

1 [2.92]

0.5 [1.46]

>500 [>1495]

1.27 ± 0.06

4.291

4.99 ± 0.46

32 [86.37]

16 [43.18]

>500 [>1349]

4.09 ± 0.48

3.074

3.87 ± 0.36

8 [20.69]

8 [20.69]

>500 [>1293]

1.33 ± 0.19

3.385

3.81 ± 0.17

8 [22.44]

2 [5.61]

>500 [>1402]

5.95 ± 0.14

4.423

4.62 ± 0.44

8 [20.8]

8 [20.8]

>500 [>1300]

4.23 ± 0.26

3.206

7.06 ± 0.27

16 [39.95]

16 [39.95]

>500 [>1248]

7.52 ± 0.19

2.168

6.44 ± 0.31

16 [42.96]

32 [85.91]

>500 [>1342]

7.13 ± 0.56

3.076

5.08 ± 0.38

16 [38.59]

8 [19.29]

>500 [>1206]

12.11 ± 0.36

O

1b

S

N HN

O

O

S

N HN

1c

O

OH

O

1d

S

N

O NH

O

1e

S

N

O NH

O S

N

O NH

1f

OH O

S

N

O NH

1g

OH

O S 1h

N

O NH

COOMe

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

9

D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

F

O S

1i

N

O 3.528

4.01 ± 0.48

32 [85.45]

4 [10.68]

>500 [>1335]

0.90 ± 0.08

4.566

4.07 ± 0.29

16 [39.74]

1 [2.48]

>500 [>1242]

9.89 ± 0.68

3.349

3.96 ± 0.23

16 [38.23]

16 [38.23]

>500 [>1194]

2.17 ± 0.22

2.311

6.26 ± 0.16

64 [163.91]

64 [163.91]

>500 [>1280]

1.34 ± 0.15

3.219

5.06 ± 0.35

16 [36.99]

8 [18.49]

>500 [>1156]

8.02 ± 0.17

2.852

3.80 ± 0.10

16 [41.51]

4 [10.38]

>500 [>1297]

14.39 ± 0.92

O

2.931

4.22 ± 0.12

8 [20.02]

4 [10.01]

>500 [>1251]

12.19 ± 1.07

O

3.074

3.39 ± 0.26

16 [38.32]

8 [19.16]

>500 [>1197]

1.67 ± 0.11

1.667

12.78

64 [286.67]

64 [286.67]

16 [71.67]

21.82

NH

F

O

S

1j

N

O NH

F

O S

N

O NH

1k

OH

F

O S

O

N

NH

1l

OH F

O S

O

N

NH

1m

COOMe

O S

1n

N

H H N N

O

O

O

S

N

H N N H

1o

F

1p

O

O

S

N

O H N N H

O 7a

S

N

O OH

(continued on next page)

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

10

D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

Table 2 (continued) Compound

c Log P*

Structure

NH 3 Cl 9d

NH3

MIC (lg/mL) [lM] S. aureus

S. epidermidis

E. coli

NIH 3T3 growth inhibition, IC50# (lM)

1.440

No inhibition

>500 [>2663]

>500 [>2663]

>500 [>2663]

2290.95

1.302

No inhibition

>500 [>2034]

>500 [>2034]

>500 [>2034]

2290.95

2.816

No inhibition

>500 [>1541]

>500 [>1541]

>500 [>1541]

262.68 ± 10.31

NnA

16.91 ± 1.86

32 [113.24]

16 [56.62]

>500 [>1769]

9.71 ± 0.44

NnA

736 

>500 [>2958]

>500 [>2958]

>500 [>2958]

7.91 ± 0.26

Cl

9e

SrtA inhibition, IC50# (lM)

COOMe O N 11

O HN

O 1-(3,4-Dichlorophenyl)-3(dimethylamino)-1propanone24

Cl

N

Cl Phenyl vinyl sulfone

29

O S O

*

 

c Log P 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 inhibitors needed more than 4 h. 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 optimization. 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 biochemical 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-oxo2,3-dihydro-1,2-benzothiazol-2-yl)-acetamide (1a) was selected as the best hit from the library screening (Table 2). 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 elucidate the aspects of the binding and to determine the reactive groups participating in the process of irreversible adduct formation. 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 Ha (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

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. 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, 15 13 CBCA(CO)NH, N-resolved NOESY-HSQC, C-(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 calculated 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 (Experimental 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 identical 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, while in other conformers a p–p stacking interaction with W194 is observed. The adamantyl moiety is buried in a hydrophobic cleft,

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

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 b6/b7 loop as well as residues T180, I182 and I199 from b7 and b8.

realized and in total 15 new analogs 1b–p were synthesized (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 complex. Therefore, this part of the molecule had to be left intact during the optimization with the exception of substitutions in the benzo part. In particular, the benzo[d]isothiazol-3(2H)-one positions 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 possess 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 adamantane, 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 modifications 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]isothiazol3(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-(3oxobenzo[d]isothiazol-2(3H)-yl)propionic acid (7a)45 and its 4-fluoro analog 7b were synthesized by analogy to previously reported methods (Scheme 1)46 but the known 2-(3-oxobenzo[d]isothiazol2(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-carbohydrazide (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 additionally 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

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

12

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 atoms are colored grey, blue and red, respectively, except for the C atoms of the inhibitor that are colored cyan and Ca 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 p–p stacking interactions with R197 and W194, respectively. The figure was generated using The PyMOL Molecular Graphics System, version 1.5.0.4; Schrödinger, LLC.

Scheme 1. Reagents and conditions: (a) H2NCH2CH2COOMeHCl, CDI, NEt3; (b) Br2; (c) LiOH.

particular, the IC50 for the inhibition of the sortase A activity 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 values 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

to form a covalent bond with C184, and it is likely that other interactions are less important. To confirm this indication, we synthesized compound 11 (Scheme 3), in which the benzo[d]isothiazol3(2H)-one moiety of 1a was replaced by an isoindolin-1-one. Compound 11 did not exhibit any activity in the enzymatic assay and showed no binding to the enzyme by NMR analysis. If the ring opening of benzo[d]isothiazol-3(2H)-one and the formation of the disulfide 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 exhibit considerable inhibitory activity that proves the proposed mode

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

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.

Scheme 3. Synthesis of amide 11 as a 1-carba analog of the selected hit 1a.

of action of compounds containing benzo[d]isothiazol-3(2H)-one ring system. Furthermore, we tested the activity of the benzo[d]isothiazol-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. 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 comprising 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 carbon 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

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 comparison to the compounds with shorter linkers (1a). Also, no significant improvement was observed upon introduction of an additional H-bond acceptor and donor (1n, 1o and 1p vs 1d). (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). 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 acetylhydrazide 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]isothiazol3(2H)-one sulfur atom that could enhance reactivity towards

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

14

D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

nucleophiles. Simultaneously, the introduction of fluorine 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 capability of the compounds to cross the cell membrane and therefore the toxicity towards microorganisms is reduced. Some of the modifications provided simultaneous improvements to all of the parameters of the inhibitor. The analog compound 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 compound still exhibited significant inhibition of Gram-positive staphylococci. Moreover, the cytotoxicity of 1n was significantly decreased in mammalian cell culture. Additionally, none of compounds 1a–p exhibited antibacterial activity against Gram-negative E. coli.

4. Discussion In this study, we identified a novel class of potent S. aureus sortase 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 binding 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 reversibility 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 according to both the end-point product accumulation and the HSQC NMR assay. An experimental NMR structure of the enzyme–inhibitor complex was obtained and used for the subsequent design of analogs, of which 15 compounds were synthesized. The new compounds exhibited combinations of (1) substitutions in the adamantyl moiety, (2) substitutions in the benzo[d]isothiazol-3(2H)-one heterocycle, and (3) variations in the linker (Table 1). A comprehensive 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

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 suggestion is supported by the fact that 3-(3-oxobenzo[d]isothiazol2(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. The bactericidal properties of the compounds are a crucial parameter for the lead selection. It is accepted that sortase A inhibitors possessing low bactericidal activity might be better candidates for clinical use in the future. Sortases are not vitally important enzymes for bacteria in culture, and the absence of bactericidal properties can greatly reduce the selective pressure of the inhibitor molecules, thereby minimizing the risk of the development of bacterial resistance.16,17 The selected hit 1a was found to exhibit strong bactericidal activity in both S. aureus and S. epidermidis 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 selection of the possible modifications, provided a possibility of improving the specificity of the analogs. These compounds exhibit a lower ability to inhibit bacterial proteins other than sortases. The inhibitory 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 analogs. This may be explained by the optimized orientation of the reactive heterocycle within the active site provided by the modifications. Interestingly, in comparison to 1a–p, a less lipophilic 7a with reduced capabilities to cross the cell membrane demonstrated 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 larger 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 bactericidal 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 cytotoxicity against mammalian fibroblasts in vitro, their cytotoxicity was comparable to that of the known inhibitors 1-(3,4-dichlorophenyl)-3-(dimethylamino)-1-propanone24 and phenyl vinyl sulfone29 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 moieties, which orient the molecules within the active site of sortase, as adamantyl-moieties orient compound 1a and its analogs. In contrast, 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

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

in the heterocycle towards the thiol-group of the cysteine residue of the sortase protein that affects IC50 values. Relatively high cytotoxicity of the irreversible inhibitors is a consequence of their ability to inhibit enzymes other than the target protein. Despite this fact, it is accepted that irreversible inhibitors 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 pharmacokinetic 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. Taking into the consideration the improved in vitro activity and the reduced MIC values and cytotoxicity, compounds 1n (N0 -(2-(3oxobenzo[d]isothiazol-2(3H)-yl)acetyl)adamantane-1-carbohydrazide) and 1o (N0 -(3-(3-oxobenzo[d]isothiazol-2(3H)-yl)propanoyl) adamantane-1-carbohydrazide) can be considered as promising lead candidates. The majority of the synthesized and tested compounds demonstrate an unfavorable selectivity index, that is, the ratio between IC50 value of cytotoxicity and IC50 value of SrtA inhibition. The compounds 1n and 1o have a more advantageous selectivity index and can be considered as a starting point for developing of novel anti-virulence agents. Additional research may involve further adjustments of the structure and the subsequent evaluation of the lead compounds’ abilities to decrease the virulence of staphylococci. Convenient in vivo models of pulmonary infections have been previously described and successfully applied to assay the anti-staphylococcal properties of the chemical compounds.56–58 These assays could be further used to characterize compounds’ efficacy in vivo and suitability for therapeutic applications. Collectively, the presented data highlight the potential of new sortase A inhibitors that can be used for the treatment of staphylococcal infections. Although, the reactive benzisothiazolinone derivates showed substantial cytotoxicity, our preliminary hit-to-lead optimization demonstrated that the parameters of the inhibitors can be significantly improved. Thus, compounds 1n and 1o demonstrated a ten-fold decrease in cytotoxicity and higher MIC values in comparison with the selected hit 1a. To the best of our knowledge, although the bactericidal activity of N0 -heteroarylidene-1-adamantylcarbohydrazides59 has been demonstrated previously, the high SrtA inhibitory activity of adamantantane-1-carbohydrazide derivatives has not been reported. Since only preliminary hit-to-lead optimization was performed within the framework of the present study, further research is required to obtain more details regarding the minimal pharmacophore and to overcome the limitations of the optimized inhibitors 1b–p, that is, their comparatively high cytotoxicity and bactericidal activity. In contrast, because the properties of the currently selected compound are comparable to those of known inhibitors, the developed molecule may be able to serve as a starting point for further improvements. Acknowledgments We acknowledge Prof. Gottfried Otting for helpful discussions. We thank Nicholas Donaldson, James Welke and Lucio Rodriguez for their valuable contribution to the preparation of the

15

manuscript. This work was supported by the European Regional Development Fund [2010/0312/2DP/2.1.1.1.0/10/APIA/VIAA/032]. References and notes 1. Sievert, D. M.; Ricks, P.; Edwards, J. R.; Schneider, A.; Patel, J.; Srinivasan, A.; Kallen, A.; Limbago, B.; Fridkin, S. Infect. Control Hosp. Epidemiol. 2013, 34, 1. 2. Vincent, J. L.; Rello, J.; Marshall, J.; Silva, E.; Anzueto, A.; Martin, C. D.; Moreno, R.; Lipman, J.; Gomersall, C.; Sakr, Y.; Reinhart, K. JAMA 2009, 302, 2323. 3. Diekema, D. J.; Pfaller, M. A.; Schmitz, F. J.; Smayevsky, J.; Bell, J.; Jones, R. N.; Beach, M. Clin. Infect. Dis. 2001, 32, S114. 4. Rybak, J. M.; Barber, K. E.; Rybak, M. J. Expert Opin. Pharmacother. 2013, 14, 1919. 5. Woodford, N.; Livermore, D. M. J. Infect. 2009, 59, S4. 6. Chuang, Y. Y.; Huang, Y. C. Lancet Infect. Dis. 2013, 13, 698. 7. Fitzgerald, J. R.; Sturdevant, D. E.; Mackie, S. M.; Gill, S. R.; Musser, J. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8821. 8. Fitzgerald, J. R.; Sturdevant, D. E.; Mackie, S. M.; Gill, S. R.; Musser, J. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8821. 9. Kaplan, S. L.; Hulten, K. G.; Gonzalez, B. E.; Hammerman, W. A.; Lamberth, L.; Versalovic, J.; Mason, E. O., Jr. Clin. Infect. Dis. 2005, 40, 1785. 10. Tenover, F. C.; Goering, R. V. J. Antimicrob. Chemother. 2009, 64, 441. 11. Navarre, W. W.; Schneewind, O. Microbiol. Mol. Biol. Rev. 1999, 63, 174. 12. Marraffini, L. A.; Dedent, A. C.; Schneewind, O. Microbiol. Mol. Biol. Rev. 2006, 70, 192. 13. Marraffini, L. A.; Dedent, A. C.; Schneewind, O. Microbiol. Mol. Biol. Rev. 2006, 70, 192. 14. Spirig, T.; Weiner, E. M.; Clubb, R. T. Mol. Microbiol. 2011, 82, 1044. 15. Tsompanidou, E.; Denham, E. L.; Sibbald, M. J.; Yang, X. M.; Seinen, J.; Friedrich, A. W.; Buist, G.; van Dijl, J. M. PLoS One 2012, 7, e44646. 16. Mazmanian, S. K.; Liu, G.; Ton-That, H.; Schneewind, O. Science 1999, 285, 760. 17. Mazmanian, S. K.; Liu, G.; Jensen, E. R.; Lenoy, E.; Schneewind, O. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5510. 18. Cossart, P.; Jonquieres, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5013. 19. Maresso, A. W.; Schneewind, O. Pharmacol. Rev. 2008, 60, 128. 20. Marraffini, L. A.; Dedent, A. C.; Schneewind, O. Microbiol. Mol. Biol. Rev. 2006, 70, 192. 21. Ilangovan, U.; Ton-That, H.; Iwahara, J.; Schneewind, O.; Clubb, R. T. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6056. 22. Ilangovan, U.; Ton-That, H.; Iwahara, J.; Schneewind, O.; Clubb, R. T. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6056. 23. Mazmanian, S. K.; Liu, G.; Ton-That, H.; Schneewind, O. Science 1999, 285, 760. 24. Maresso, A. W.; Wu, R.; Kern, J. W.; Zhang, R.; Janik, D.; Missiakas, D. M.; Duban, M. E.; Joachimiak, A.; Schneewind, O. J. Biol. Chem. 2007, 282, 23129. 25. Oh, K. B.; Kim, S. H.; Lee, J.; Cho, W. J.; Lee, T.; Kim, S. J. Med. Chem. 2004, 47, 2418. 26. Suree, N.; Yi, S. W.; Thieu, W.; Marohn, M.; Damoiseaux, R.; Chan, A.; Jung, M. E.; Clubb, R. T. Bioorg. Med. Chem. 2009, 17, 7174. 27. Zhulenkovs, D.; Leonchiks, A. EEB 2010, 8, 97. 28. Zhulenkovs, D.; Jaudzems, K.; Zajakina, A.; Leonchiks, A. Biochem. Eng. J. 2014, 82, 200–209. 29. Frankel, B. A.; Bentley, M.; Kruger, R. G.; McCafferty, D. G. J. Am. Chem. Soc. 2004, 126, 3404. 30. Ton-That, H.; Liu, G.; Mazmanian, S. K.; Faull, K. F.; Schneewind, O. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12424. 31. Zhang, J. H.; Chung, T. D.; Oldenburg, K. R. J. Biomol. Screen. 1999, 4, 67. 32. Huang, X.; Aulabaugh, A.; Ding, W.; Kapoor, B.; Alksne, L.; Tabei, K.; Ellestad, G. Biochemistry 2003, 42, 11307. 33. Zwahlen, C.; Legault, P.; Vincent, S. b. J. F.; Greenblatt, J.; Konrat, R.; Kay, L. E. J. Am. Chem. Soc. 1997, 119, 6711. 34. Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.; Bax, A. J. Biomol. NMR 1995, 6, 277. 35. Keller, R. The Computer Aided Resonance Assignment Tutorial, 1st ed.; CANTINA Verlag: Germany, 2004; ISBN: 3-85600-112-3; http://cara.nmr-software.org/ downloads/3-85600-112-3.pdf. 36. Herrmann, T.; Guntert, P.; Wuthrich, K. J. Mol. Biol. 2002, 319, 209. 37. Herrmann, T.; Guntert, P.; Wuthrich, K. J. Biomol. NMR 2002, 24, 171. 38. Guntert, P.; Mumenthaler, C.; Wuthrich, K. J. Mol. Biol. 1997, 273, 283. 39. Brunger, A. T. Nat. Protoc. 2007, 2, 2728. 40. Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.; GrosseKunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Acta Crystallogr. D. Biol. Crystallogr. 1998, 54, 905. 41. Laskowski, R. A.; Macarthur, M. W.; Moss, D. S.; Thornton, J. M. J. Appl. Crystallogr. 1993, 26, 283. 42. Schillaci, D.; Petruso, S.; Raimondi, M. V.; Cusimano, M. G.; Cascioferro, S.; Scalisi, M.; La Giglia, M. A.; Vitale, M. Biofouling 2010, 26, 433. 43. Sunassee, S. N.; Veale, C. G.; Shunmoogam-Gounden, N.; Osoniyi, O.; Hendricks, D. T.; Caira, M. R.; de la Mare, J. A.; Edkins, A. L.; Pinto, A. V.; da Silva Junior, E. N.; vies-Coleman, M. T. Eur. J. Med. Chem. 2013, 62, 98. 44. Ilangovan, U.; Iwahara, J.; Ton-That, H.; Schneewind, O.; Clubb, R. T. J. Biomol. NMR 2001, 19, 379. 45. Slawik, T. Pharmazie 1991, 46, 777. 46. Lai, H.; Dou, D.; Aravapalli, S.; Teramoto, T.; Lushington, G. H.; Mwania, T. M.; Alliston, K. R.; Eichhorn, D. M.; Padmanabhan, R.; Groutas, W. C. Bioorg. Med. Chem. 2013, 21, 102. 47. Dou, D.; Alex, D.; Du, B.; Tiew, K. C.; Aravapalli, S.; Mandadapu, S. R.; Calderone, R.; Groutas, W. C. Bioorg. Med. Chem. 2011, 19, 5782.

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011

16

D. Zhulenkovs et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

48. Naik, M. T.; Suree, N.; Ilangovan, U.; Liew, C. K.; Thieu, W.; Campbell, D. O.; Clemens, J. J.; Jung, M. E.; Clubb, R. T. J. Biol. Chem. 2006, 281, 1817. 49. Lai, H.; Dou, D.; Aravapalli, S.; Teramoto, T.; Lushington, G. H.; Mwania, T. M.; Alliston, K. R.; Eichhorn, D. M.; Padmanabhan, R.; Groutas, W. C. Bioorg. Med. Chem. 2013, 21, 102. 50. Joice, A. C.; Harris, M. T.; Kahney, E. W.; Dodson, H. C.; Maselli, A. G.; Whitehead, D. C.; Morris, J. C. Int. J. Parasitol. Drugs Drug Resist. 2013, 3, 154. 51. Swinney, D. C. Nat. Rev. Drug Disc. 2004, 3, 801. 52. Swinney, D. C. Curr. Opin. Drug Discov. Devel. 2009, 12, 31. 53. Robertson, J. G. Biochemistry 2005, 44, 5561. 54. Robertson, J. G. Curr. Opin. Struct. Biol. 2007, 17, 674.

55. Johnson, D. S.; Weerapana, E.; Cravatt, B. F. Future Med. Chem. 2010, 2, 949. 56. Yanagihara, K.; Okada, M.; Fukuda, Y.; Imamura, Y.; Kaneko, Y.; Ohno, H.; Higashiyama, Y.; Miyazaki, Y.; Tsukamoto, K.; Hirakata, Y.; Tomono, K.; Kadota, J.; Tashiro, T.; Murata, I.; Kohno, S. Chemotherapy 2004, 50, 260. 57. Yanagihara, K.; Seki, M.; Izumikawa, K.; Higashiyama, Y.; Miyazaki, Y.; Hirakata, Y.; Tomono, K.; Mizuta, Y.; Tsukamoto, K.; Kohno, S. Int. J. Antimicrob. Agents 2006, 28, 212. 58. McElroy, M. C.; Cain, D. J.; Tyrrell, C.; Foster, T. J.; Haslett, C. Infect. Immun. 2002, 70, 3865. 59. El-Emam, A. A.; Alrashood, K. A.; Al-Omar, M. A.; Al-Tamimi, A. M. Molecules 2012, 17, 3475.

Please cite this article in press as: Zhulenkovs, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.011