Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Design, synthesis and biological evaluation of 4-(alkyloxy)-6-methyl2H-pyran-2-one derivatives as quorum sensing inhibitors Suzie Park a, , Han-Shin Kim b, , Kiwon Ok a, YunHye Kim a, Hee-Deung Park b,⇑, Youngjoo Byun a,c,⇑ a

College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Sejong 339-700, South Korea School of Civil, Environmental and Architectural Engineering, Korea University, Anam-Dong, Seongbuk-Gu, Seoul 136-713, South Korea c Biomedical Research Center, Korea University Guro Hospital, 148 Gurodong-ro, Guro-gu, Seoul 152-703, South Korea b

a r t i c l e

i n f o

Article history: Received 26 March 2015 Revised 7 May 2015 Accepted 21 May 2015 Available online xxxx Keywords: Quorum sensing Biofilm inhibition Pyrone Pseudomonas aeruginosa

a b s t r a c t Novel pyrone-derived quorum sensing (QS) ligands to inhibit the binding of OdDHL to the LasR of Pseudomonas aeruginosa were designed, synthesized and evaluated. Among the analogs, the most potent compound 8 exhibited strong in vitro inhibitory activities against biofilm formation and down-regulated OdDHL/LasR-associated genes by 35–67%. The binding mode of 8 in silico was highly similar to that of the crystal ligand OdDHL in the active site of LasR. Ó 2015 Elsevier Ltd. All rights reserved.

Quorum sensing (QS), a cell-to-cell communication system of bacteria, enables bacteria to survive, adapt rapidly to changed environments, and compete against other microorganisms for nutrients.1–3 QS system is strictly controlled by small diffusible signaling molecules, the so-called auto-inducers. These auto-inducers freely diffuse across the cell and activate QS system at a threshold concentration by the increased density of bacteria. When the threshold concentration is reached, auto-inducers bind to the cognate receptor in the bacterial cells and activate signal pathways to mediate gene expressions that are involved in the pathogenicity and biofilm formation.4 When bacteria infect a host, including human beings, the QS system of bacteria is activated to respond against the host immune system by expressing disease-causing genes.5,6 The primary auto-inducers of QS system in Gram-negative bacteria is N-acyl-L-homoserine lactones (AHLs) that are produced by LuxItype proteins. AHLs bind to LuxR-type receptors and activate LuxI/R-signaling cascades, thus leading to the transcription of QSassociated genes that are critical for virulence of pathogens.7 Pseudomonas aeruginosa (P. aeruginosa), a ubiquitous Gramnegative bacterium in natural and medical settings, utilizes the QS system to coordinate biofilm formation and cell aggregation, potentially leading to the pathological virulence of humans.8,9 Despite advances in antimicrobial therapy for the last decades, ⇑ Corresponding authors. Tel.: +82 2 3290 4861; fax: +82 2 928 7656 (H.-D.P.); tel.: +82 44 860 1619; fax: +82 44 860 1607 (Y.B.). E-mail addresses: [email protected] (H.-D. Park), [email protected] (Y. Byun).   Contributed equally.

treatment of infections are still limited due to the increased resistance of the bacterium to commonly used antibiotic agents.10 P. aeruginosa infection is one of the important causes of morbidity and mortality in immune-compromised patients affected with cystic fibrosis, neutropenic cancer or AIDS.11 Biofilm formation by the activation of QS system further increases intrinsic resistance to antibiotic treatments and enhances the virulence of P. aeruginosa.12 The virulence of P. aeruginosa can be controlled by regulating the transcription of the QS-related genes. P. aeruginosa contains major transcriptional regulatory QS systems including LasI/LasR, RhlI/RhlR, and PQS/MvfR. In the LasI/LasR system, LasI produces N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) as auto-inducer that is recognized by LasR. In the RhlI/RhlR system, N-butanoyl homoserine lactone (BHL) is produced by RhlI and binds to the RhlR protein. In the PQS/MvfR system, the Pseudomonas quinolone signal (PQS) affects the MvfR system that are influenced by the expression of both LasR and RhlR. These 3 transcriptional systems have been considered as attractive targets for controlling the virulence of P. aeruginosa.13–15 A number of QS-modulating ligands have been designed and synthesized by mimicking the natural auto-inducers such as OdDHL, BHL or PQS.16–27 According to structure–activity relationship (SAR) studies of the synthesized QS ligands, the hydrophilic group (e.g., lactone, quinolone) and the lipophilic alkyl chains are essential for the QS-modulating activity.28–33 Inhibitors that block the complex formation between auto-inducers and the cognate receptors could downregulate the transcription of QS-associated genes and diminish or halt the virulence of P. aeruginosa.34,35

http://dx.doi.org/10.1016/j.bmcl.2015.05.054 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Park, S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.05.054

2

S. Park et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx

O O

O

O

O

CH2 (CH 2)n CH3

O

N H

O OdDHL

Pyrone analogs

Figure 1. Design strategy of pyrone analogs as QS inhibitors based on OdDHL.

O

O

CH 3(CH 2 )n CH 2-X (1.2 eq)

O

O OH

o

K2 CO3 (2.0 eq), DMF, 60 C

OCH2 (CH2 )n CH 3

1 : n=3 2 : n=4 3 : n=5 4 : n=6 5 : n=7 6 : n=8 7 : n=9 8 : n=10 9 : n=11 10 : n=12

Scheme 1. Synthesis of 4-(alkyloxy)-6-methyl-2H-pyran-2-one analogs.

Figure 2. A static biofilm assay showing the inhibitory activities of the compounds 1–10 against the P. aeruginosa biofilm formation.

Figure 3. RT-qPCR results showing the inhibitory activities of 8 against the expression of P. aeruginosa QS genes.

Recent X-ray crystal structures of LasR with OdDHL elucidated the binding mode of OdDHL, which provided a basis of structurebased drug design of QS ligands.36 Hydrophilic homoserine lactone moiety and long alkyl chain of OdDHL interacts with hydrophilic

amino acids such as Trp 60, Tyr 56, and Ser 129 and hydrophobic amino acids such as Leu 48, Leu 50 and Phe 54, respectively. Based on the binding mode of OdDHL and SAR studies of the OdDHL analogs, we designed novel pyrone-based QS inhibitors.

Please cite this article in press as: Park, S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.05.054

3

S. Park et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx Table 1 SYBYL SurflexX docking results of the pyrone analogs (1–10) with P. aeruginosa LasR protein (PDB: 2UV0) Compound

Total score

9 8 10 7 6 5 OdDHL 3 4 2 1

12.3707 12.1877 11.7384 10.5038 10.332 9.7161 9.6693 9.6332 9.6257 9.1408 8.3201

D score 186.334 177.155 193.708 167.82 154.046 153.386 167.331 119.75 130.308 130.125 106.865

PMF score 94.7691 95.4088 73.0762 71.9434 57.7602 95.0601 116.043 44.8171 45.8577 82.4936 47.4311

G score 290.794 244.589 278.788 235.291 216.433 212.542 320.118 155.505 189.955 174.797 145.549

Chem score 44.0491 43.0088 45.3358 40.7011 36.7713 37.3997 35.7574 29.615 31.7645 33.7743 26.6101

Similarity 0.658 0.660 0.587 0.613 0.507 0.605 0.694 0.450 0.442 0.482 0.397

Figure 4. (a) Overlay of compound 8 with OdDHL. (b) Hydrogen-bonding interactions between the docked pose of compound 8 and amino acids in the active site of LasR.

The designed compounds replaced the lactone ring of OdDHL with the pyrone ring with varying alkyl chain lengths, as shown in Figure 1. The designed pyrone analogs were synthesized by reacting 4hydroxy-6-methyl-2H-pyran-2-one with appropriate alkyl halides under basic conditions, as outlined in Scheme 1. Reaction of 4-hydroxy-6-methyl-2H-pyran-2-one with appropriate alkyl halides in DMF at 60 °C for 6 h afforded the corresponding 4-(alkyloxy)-6methyl-2H-pyran-2-one products. The products were obtained in high purity by silica gel column chromatography. The chemical structures of the synthesized compounds were confirmed by 1H

NMR, 13C NMR, and ESI-MS. According to 1H NMR spectra, the distinct chemical shifts and peak patterns including two singlet peaks of the pyrone ring around 6.0 ppm and 5.0 ppm and the triplet peaks of OCH2 around 4.0 ppm were observed. 13C NMR spectra exhibited indicative peaks around 165 ppm for the carbonyl carbon (C@O) and 68 ppm for that of the alkoxy group (CH2-O). Molecular ion peak of the final compounds were detected in [M+H]+ form from the positive mode of HR-ESI mass spectrometer. The inhibitory activities of the synthesized compounds against the biofilm formation were evaluated using a static biofilm assay and the results were summarized in Figure 2. The purpose of the

Please cite this article in press as: Park, S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.05.054

4

S. Park et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx

assay was to select the compounds to inhibit the biofilm formation of P. aeruginosa. As shown in Figure 2, the compounds with longer alkyl chains were more potent than those with shorter alkyl chains, as predicted from the molecular modeling docking studies. In particular, compound 8 with a dodecyloxy (C12) group was the most potent among the tested compounds, implying that the inhibition of the biofilm formation (11–47% compared with the control) was proportional to the lipophilicity of the compounds in a series of pyrone analogs. The compounds 9–10 with longer alkyl chains than 8 also inhibited biofilm formation (6–43% compared with 0 lM of treatment). The results showed that the pyrone analogs with >9 alkyl chains significantly inhibited biofilm formation. We measured the biofilm inhibition of compound 8 at low concentrations (0.1 lM, 1 lM, and 10 lM). The result showed that biofilm formation was reduced by 6–19% with the treatment of compound 8 (see Supplementary data). Furanone C-30, a positive control, also reduced biofilm formation by 13–31%. Although compound 8 was not as potent as Furanone C-30, it inhibited biofilm formation in a concentration-dependent manner. We also tested the effect of compound 8 on P. aeruginosa growth at several concentrations (0, 1, and 10 lM). The growth pattern of P. aeruginosa was not significantly different among the three cultures. (see Supporting information) Furthermore, the most potent compound 8 was tested for regulation of expression of representative P. aeruginosa QS genes. The QS genes included auto-inducer receptor genes (lasR, rhlR and mvfR), synthase genes (lasI, rhlI, pqsC, pqsD, pqsH and phnB), and virulence factor related genes (lasA, lasB, rhlA, rhlB, phzC1 and phzE1). As shown in Figure 3, fifteen QS-inducible genes expressions were quantified in P. aeruginosa cells treated with 10 lM of 8 or in the absence of 8. All of the selected QS-inducible genes by the treatment of 8 were significantly down-regulated by 35–67%, as compared with those without 8. However, the expression of the proC housekeeping gene was not affected by 10 lM of 8, but was the same as without 8. The result suggested that the compound 8 was an effective QS inhibitor in P. aeruginosa. We carried out molecular docking studies and predicted the potential binding modes of the synthesized compounds. The process of ligand preparation and optimization was performed by Sanitize preparation protocol in SYBYL-X 2.1.1 (Tripos Inc., St Louis). For docking studies, the structure of P. aeruginosa LasR in complex with OdDHL (PDB ID: 2UV0) was downloaded from RCSB.36 The docking studies of all prepared ligands were performed by Surflex-Dock module in SYBYL-X 2.1.1.37 According to the in silico binding energies and Dock scores shown in Table 1, the pyrone analogs with the longer alkyl chains showed higher in silico binding affinity to the LasR, as compared to those with shorter alkyl chains. The best-docked poses of dodecanyl pyrone compounds 8 and 9 exhibited the similar binding patterns to OdDHL in the active site of LasR with a combination of hydrophilic and hydrophobic interactions (Fig. 4a). The hydrophilic pyrone moiety interacts with Trp 60, Tyr 56 and Ser 129 via hydrogenbonding interactions (Fig. 4b). The amino acid residues involved in the LasR–OdDHL interactions are Trp 60, Tyr 56, Ser 129 and Asp 73. Compound 8 had a high similarity score of 0.660,38 which is the best among the compounds except OdDHL itself. The introduction of an amide bond between the pyrone ring and the dodecanyl chain instead of the ether linkage might improve the binding affinity of compound 8 by creating a hydrogen-bonding interaction with Asp 73 which is observed in the crystal structure of the LasR– OdDHL complex. To investigate the potential binding of compound 8 to LasR, we evaluated its inhibition of biofilm formation in P. aeruginosa transformed by a plasmid overexpressing LasR (PA14-pUCPLasR). Furanone C-30, a synthetic potent QS inhibitor, was used as a positive control. As shown in Figure 5, PA14-pUCPLasR did not exhibit any biofilm formation inhibition with the addition of compound 8

Figure 5. Quantification of PA14 biofilm for PA14-pUCP18 and PA14-pUCPLasR by compound 8 (0–100 lM) and Furanone C-30 (0–100 lM). Error bars indicate the standard deviations of 8 measurements. ⁄P