Design, synthesis and anti-staphylococcus activity of hydroxynaphthalenecarboxanilides

SUMMARY

Several series of ring-substituted hydroxynaphthalenecarboxanilides were prepared by microwave- assisted synthesis. Primary in vitro antimicrobial screening of all the compounds against three clinical isolates of methicillin-resistant Staphylococcus aureus demonstrated promising activity some of these compounds. Generally it can be concluded that compounds substituted in the anilide part by lipophilic and/or electron-withdrawing moieties proved to have significant biological activity.

Keywords: hydroxynaphthalenecarboxanilides, microwave synthesis, in vitro antibacterial activity, methicillin-resistant Staphylococcus aureus, structure-activity relationships.

Objectives

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most common clinically relevant bacterial pathogen isolated almost all over the world. Recent studies have shown that, despite antibacterial therapy, MRSA infections are still associated with serious clinical consequences, especially treatment failure, higher morbidity and mortality (up to 40%) [1], prolonged hospitalization (1.5- fold longer length of stay than for susceptible strains) [2], increased health care costs ($21,577 versus $11,668 for susceptible strains) [2], etc. The emergence of MRSA (and vacomycin-resistant S. aureus as well) makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the re-engineering and repositioning of some old drug families to achieve effective control of these bacteria[3].

The discovery of salicylanilides dates back to phenol and research for new antiseptics. Step by step, to improve disinfectant properties, a large number of substituted compounds was prepared culminating in the synthesis of niclosamide (2‘,5-dichloro-4‘-nitrosalicylanilide) in 1955 as a potent molluscicide and taenicide drug [4]. Nowadays, salicylanilides are known as a class of aromatic compounds possessing a wide range of interesting biological activities, such as anthelmintic [5], antibacterial/antimycobacterial [6-11], antifungal [6,9,12], antiviral [13,14], and herbicidal [9,15,16] among others. Multiple mechanisms are responsible for the activity of these compounds [8,13,14,17-19], although the appropriate mechanism of action causing the overall biological activities of these compounds has not been explained so far.

Promising results of biological screening of some salicylanilides [6-12] inspired us to prepare and evaluate ring-substituted hydroxynaphthalenecarboxanilides [20-24]. The design of these compounds is based on ring analogy with salicylanilides (2-hydroxy-N-phenylbenzamide). Primary in vitro screening of the synthesized compounds was performed against S. aureus ATCC 29213 (vancomycin-susceptible, methicillin- susceptible) and three clinical isolates of MRSA (vancomycin-susceptible, methicillin-resistant).

Materials and methods

Chemistry: Hydroxynaphtalenecarboxylic acid (5.3 mM, Sigma-Aldrich) was suspended in dry

chlorobenzene (30 mL) at ambient temperature and phosphorus trichloride (2.7 mM), and the corresponding substituted aniline (5.3 mM, Sigma-Aldrich) was added dropwise. The reaction mixture was transferred to the microwave reactor, where the synthesis was performed (1st phase: 10 min, 100 °C, 100 W; 2nd phase: 15 min, 120 °C, 500 W; 3rd phase: 20 min, 130 °C, 500 W). Then the mixture was cooled to 50 °C, and then the solvent was removed to dryness under reduced pressure. The residue was washed with hydrochloride acid and water. The crude product was recrystallized from EtOH [21,22,24]. The prepared compounds were confirmed by IR, NMR and HR-MS spectrometry. Physicochemical characteristics/molecular descriptors such as lipophilicity (expressed as log P) and electronic parameters (expressed as Hammett's z parameters) of all the compounds were predicted using ACD/Percepta ver. 2012 (Advanced Chemistry Development, Toronto, ON, Canada, 2012).

In vitro antibacterial testing: The synthesized compounds were evaluated for in vitro antibacterial activity against representatives of multidrug-resistant bacteria, clinical isolates of methicillin- resistant Staphylococcus aureus (MRSA) 63718, SA 630 and SA 3202 that were obtained from the National Institute of Public Health, Prague, Czech Republic. Staphylococcus aureus ATCC 29213 was used as a reference and quality control strain. Ampicillin (Sigma-Aldrich) was used as the standard. Prior to testing, each strain was passaged onto nutrient agar (Oxoid, Hampshire, UK) with 5% of bovine blood, and bacterial inocula were prepared by suspending a small portion of bacterial colony in sterile phosphate buffered saline (pH 7.2-7.3). The cell density was adjusted to 0.5 McFarland units using a densitometer (Densi La Meter, LIAP, Riga, Latvia). The final inoculum was made by 1:20 dilution of the suspension with the Mueller- Hinton broth (MH broth). The compounds were dissolved in DMSO (Sigma), and the final concentration of DMSO in the MH broth (Oxoid) did not exceed 2.5% of the total solution composition. The final concentrations of the evaluated compounds ranging from 256 gg/mL to 0.008 gg/mL. The broth dilution micro-method modified according to NCCLS guidelines [25] in MH broth was used to determine the minimum inhibitory concentration (MIC). Drug-free controls, sterility controls and controls consisted of MH broth and DMSO alone were included. The determination of results was performed visually after 24 h of static incubation in the darkness at 37 °C in an aerobic atmosphere. The MICs were defined as the lowest concentration of the compound at which no visible bacterial growth was observed.

Results

Several series of ring-substituted hydroxynaphthalenecarboxanilides were prepared by microwave-assisted synthesis, see Scheme 1 [21,22,24], and thus preparation of the target compounds was carried out in only one step with excellent yields for short time.

Scheme 1 - Synthesis of ring-substituted hydroxynaphthalenecarboxanilides.

The discussed compounds were evaluated for their anti-Staphylococcus activity. Some of tested compounds showed the anti-MRSA activity against the tested strains comparable or higher than the standard ampicillin (>46 gM). Within series 3- and 6-hydroxynaphthalene-2-carboxanilides, 3-hydroxy-N-(2- methoxyphenyl)-naphthalene-2-carboxamide [21] and 6-hydroxy-N-[3-(trifluoromethyl)phenyl]naphthalene- 2-carboxamide showed biological activity (MIC = 55 gM and MIC = 48 gM respectively) against S. aureus as well as all the MRSA strains. The physicochemical characteristics of these compounds are log P = 4.61, z_2-OCH3 = -0.28 and log P = 4.17, z_3-CF3 = 0.43, respectively. Among 2-hydroxynaphthalene-1-carboxanilides [22] derivatives substituted by 2-NO2 (log P = 4.45, z = 0.77), 3-NO2 (log P = 4.50, z = 0.71), 4-Br (log P = 5.31, z = 0.23) and 4-CF3 (log P = 5.27, z = 0.51 gM) demonstrated activity, especially against MRSA, that was comparable with or higher (MICs ranged from 26 to 52 gM) than that of the standard ampicillin. It is important to note that within series of ring-substituted 1-hydroxynaphthalene-2-carboxanilides no antiStaphylococcus activity was observed, although these derivatives demonstrated promising activity against a wide spectrum of mycobacterial strains [23].

Conclusions

Based on the results and structure-activity relationships, it can be generally stated that anti- MRSA activity is dependent on position of phenolic moiety on naphthalene scaffold (i.e. acidity involved) and it seems to be also positively influenced by higher lipophilicity of compounds with simultaneous substitution of the anilide ring by electron-withdrawing moieties. Thus they can be considered as lead compounds for the subsequent design of novel antimicrobial agents.

Acknowledgments

This study was supported by the IGA VFU Brno 96/2012/FaF, 37/2014/FaF, 52/2014/FaF.

REFERENCES

1. Kaku N., Yanagihara K., Morinaga Y., Yamada K., Harada Y., Migiyama Y., Nagaoka K., Matsuda J., Uno N., Hasegawa H., Miyazaki T., Izumikawa K., Kakeya H., Yamamoto Y., Kohno S. Influence of antimicrobial regimen on decreased in hospital mortality of patients with MRSA bacteremia //J. Infect. Chemother. - 2014. - V. 20. - №. 6. - P. 350-355.
2. Lodise T. P., McKinnon P. S. Clinical and economic impact of methicillin resistance in patients with Staphylococcus aureus bacteremia //Diagn. Microbiol. Infect. Dis. - 2005. - V. 52 - №. 2. - P. 113122.
3. Wilcox M. H. MRSA new treatments on the horizon: Current status //Injury. - 2011. - V. 42. - №. Special Issue 5. - P. S42-S44.
4. Anand N., Sharma, S. Salicylanilides, in: Approaches to Design and Synthesis of Antiparasitic Drugs //Pharmacochem. Libr. - 1997. - V. 25. - P. 239-257.
5. Bartram D. J., Leathwick D. M., Taylor M. A., Geurden T., Maeder S. J. The role of combination anthelmintic formulations in the sustainable control of sheep nematodes //Vet. Parasitol. - 2012. - V. 186 - №. 3-4. - P. 151-158.
6. Vinsova J., Imramovsky A., Buchta V., Ceckova M., Dolezal M., Staud F., Jampilek J., Kaustova J. Salicylanilide acetates: Synthesis and antibacterial evaluation //Molecules. - 2007. - V. 12 - №. 1. - P. 1-12.
7. Imramovsky A., Vinsova J., Monreal-Ferriz J., Dolezal R., Jampilek J., Kaustova J., Kunc F. New antituberculotics originated from salicylanilides with promising in vitro activity against atypical mycobacterial strains //Bioorg. Med. Chem. - 2009. - V. 17. - №. 10. - P. 3572-3579.
8. Cheng T. J. R., Wu Y. T., Yang S. T., Lo K. H., Chen S. K., Chen Y. H., Huang W. I., Yuan C. H., Guo C. W., Huang L. Y., Chen K. T., Shih H. W., Cheng Y. S. E., Cheng W. C., Wong C. H. High- throughput identification of antibacterials against methicillin-resistant Staphylococcus aureus (MRSA) and the transglycosylase //Bioorg. Med. Chem. - 2010. - V. 18 - №. 24. - P. 8512-8529.
9. Otevrel J., Mandelova Z., Pesko M., Guo J., Kralova K., Sersen F., Vejsova M., Kalinowski D., Coffey A., Csollei J., Richardson D. R, Jampilek J. Investigating the spectrum of biological activity of ring- substituted salicylanilides and carbamoylphenylcarbamates //Molecules. - 2010. - V. 15. - №. 11. - P. 8122-8142.
10. Imramovsky A., Pesko M., Kralova, K., Vejsova M., Stolarikova J., Vinsova J., Jampilek J. Investigating spectrum of biological activity of 4- and 5-chloro-2-hydroxy-N-[2-(arylamino)-1- alkyl-2-oxoethyl]benzamides //Molecules. -2011. - V. 16. - №. 3. - P. 2414-2430.
11. Pauk K., Zadrazilova I., Imramovsky A., Vinsova J., Pokorna M., Masarikova M., Cizek A., Jampilek J. New derivatives of salicylamides: Preparation and antimicrobial activity against various bacterial species //Bioorg. Med. Chem. - 2013. - V. 21. - №. 21. - P. 6574-6581.
12. Imramovsky A., Vinsova J., Monreal-Ferriz J., Buchta V., Jampilek J. Salicylanilide esters of N- protected amino acids as novel antimicrobial agents //Bioorg. Med. Chem. Lett. - 2009. - V. 19. - №. 2. - P. 348-351.
13. Liu Y., Donner P. L., Pratt J. K., Jiang W. W., Ng T., Gracias V., Baumeister S., Wiedeman P. E., Traphagen L., Warrior U., Maring C., Kati W. M., Djuric S. W., Molla A. Identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of HCV NS5B polymerase //Bioorg. Med. Chem. Lett. - 2008. - V. 18. - №. 11. - P. 3173-3177.
14. Wu C. J., Jan J. T., Chen C. M., Hsieh H. P., Hwang D. R., Liu H. W., Liu C. Y., Huang H. W., Chen S. C., Hong C. F., Lin R. K., Chao Y. S., Hsu J. T. Inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide //Antimicrob. Agents Chemother. - 2004.- V. 48. - №. 7. - P. 2693-2696.
15. Imramovsky A., Pesko M., Monreal-Ferriz J., Kralova K., Vinsova J., Jampilek J. Photosynthesisinhibiting efficiency of 4-chloro-2-(chlorophenylcarbamoyl)phenyl alkylcarbamates //Bioorg. Med. Chem. Lett. - 2011. - V. 21. - №. 15. - P. 4564-4567.
16. Kralova K., Perina M., Waisser K., Jampilek J. Structure-activity relationships of N- benzylsalicylamides for inhibition of photosynthetic electron transport //Med. Chem. - 2014 - V. 10, in press.
17. Macielag M. J., Demers J. P., Fraga-Spano S. A., Hlasta J. D., Johnson G. S., Kanojia M. R., Russell K. R., Sui Z., Weidner-Wells A. M., Werblood H., Foleno B. D., Goldschmidt R. M., Loeloff M. J., Webb G. C., Barrett J. F. Substituted salicylanilides as inhibitors of two-component regulatory systems in bacteria //J. Med. Chem. - 1998. - V. 41. - №. 16. - P. 2939-2945.
18. Kauppi A. M., Nordfelth R., Uvell H., Wolf-Watz H., Elofsson M. Targeting bacterial virulence: Inhibitors of type III secretion in Yersinia //Chem. Biol. - 2003. - V. 10. - №. 3. - P. 241-249.
19. Kratky M., Vinsova J., Novotna E., Mandikova J., Wsol V., Trejtnar F., Ulmann V., Stolarikova J., Fernandes S., Bhat S., Liu J. O. Salicylanilide derivatives block Mycobacterium tuberculosis through inhibition of isocitrate lyase and methionine aminopeptidase //Tuberculosis. - 2012. - V. 92. - №. 5. - P. 434-439.
20. Gonec T., Bobal P., Pesko M., Guo J., Kralova K., Kos J., Coffey A, Kollar P, Jampilek J. Investigating the spectrum of biological activity of substituted quinoline-2-carboxamides and their isosteres //Molecules. - 2012. - V. 17. - №. 1. - P. 613-644.
21. Kos J., Zadrazilova I., Pesko M., Keltosova S., Gonec T., Bobal P., Kollar P., Cizek A., Kralova K., Jampilek J. Antibacterial and herbicidal activity of ring-substituted 3-hydroxynaphthalene-2- carboxanilides //Molecules. - 2013. - V. 18. - №. 7. - P. 7977-7997.
22. Gonec T., Kos J., Zadrazilova I., Pesko M., Govender R., Kollar P., Imramovsky A., O'Mahony J., Coffey A., Cizek A., Kralova K., Jampilek J. Antibacterial and herbicidal activity of ring-substituted 2-hydroxynaphthalene-1-carboxanilides //Molecules. - 2013. - V. 18. - №.8. - P. 9397-9419.
23. Gonec T., Kos J., Zadrazilova I., Pesko M., Keltosova S., Kollar J., Cizek A., Kralova K., Jampilek J. Antimycobacterial and herbicidal activity of ring-substituted 1-hydroxynaphthalene-2- carboxanilides //Bioorg. Med. Chem. - 2013. - V. 21. - №. 21. - P. 6531-6541.
24. Gonec T., Kos J., Nevin E., Govender R., Pesko M., Kushkevych I., Kollar P., O'Mahony J., Kralova K., Coffey A., Jampilek J. Preparation and biological properties of ring-substituted naphthalene-1- carboxanilides //Molecules 2014. - V. 19. - №. 7. - P. 10386-10409.
25. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, approved standard-ninth edition //Document M07-A9 PK, CLSI, Wayne, PA, USA, 2012.