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European Journal of Medicinal Chemistry 45 (2010) 6106e6113

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Short communication

New amino acid esters of salicylanilides active against MDR-TB and othermicrobes

Martin Krátký a, Jarmila Vin�sová a,*, Vladimír Buchta b,c, Kata Horvati d, Szilvia Bösze d, Ji�rina Stola�ríková e

aDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech RepublicbDepartment of Clinical Microbiology, Faculty of Medicine and University Hospital, Charles University, Sokolská 581, 500 12 Hradec Králové, Czech RepubliccDepartment of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech RepublicdResearch Group of Peptide Chemistry, Eötvös Lórand University, Hungarian Academy of Science, Pázmány Péter Sétány 1/A, Budapest, H-1117, Hungarye Laboratory for TBC, Regional Institute of Public Health in Ostrava, Partyzánské nám�estí 7, 702 00 Ostrava, Czech Republic

a r t i c l e i n f o

Article history:Received 30 June 2010Received in revised form13 September 2010Accepted 16 September 2010

Keywords:TuberculosisMultidrug-resistant tuberculosisSalicylanilideAmino acid esterAntibacterial activityAntifungal activity

* Corresponding author. Heyrovského 1203, 500Republic. Tel.: þ420 495067343; fax: þ420 49506716

E-mail address: jarmila.vinsova@faf.cuni.cz (J. Vin�s

0223-5234/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.ejmech.2010.09.040

a b s t r a c t

Eleven halogenated (S)-2-(phenylcarbamoyl)phenyl 2-acetamido-3-phenylpropanoates (3ae3k) weredesigned and synthesized as potential antimicrobial agents. They were evaluated in vitro against somemycobacterial, bacterial and fungal strains. These compounds were active against drug-sensitive andatypical mycobacterial strains with general MIC values from 0.25 to 16 mmol/L. The most activecompounds were (S)-4-chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl 2-acetamido-3-phenyl-propanoate (3i) and (S)-4-bromo-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl 2-acetamido-3-phe-nylpropanoate (3k) which exhibited activity against MDR and XDR-TB strains with MICs from 1 to2 mmol/L. 3k was shown to be less cytotoxic with higher IC50. Some compounds exhibited low MICs onGram-positive bacteria (MICs � 0.98 mmol/L) and on fungi (MICs � 3.9 mmol/L).

� 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction

Salicylanilides (2-hydroxy-N-phenylbenzamides) have been thesubject of interest in medicinal chemistry for many years [1],mainly due to their antibacterial [1,2], antiprotozoal [1,3] andantifungal activity [1,4]. They showed a wide-range of biologicalactivities including potential anticancer efficacy [5e8], influence oninterleukins production [9e12], anti-inflammatory activity [13],activity against different viruses [14e18], or effects on ion channels[19].

Increasing emergence of drug-resistant tuberculosis is alarming[20,21]. It is estimated that one third of the world’s population iscurrently infected with Mycobacterium tuberculosis, and each year8e9 million new cases develop. Every year almost 500,000 peopleare infected with multidrug-resistant TB (MDR-TB) and there areestimated 40,000 new cases of extensively drug-resistant TB (XDR-TB) annually [22].

05 Hradec Králové, Czech6.ová).

son SAS. All rights reserved.

Multidrug-resistant M. tuberculosis was defined as resistant toisoniazid (INH) and rifampicin (RIF), but it may include resistanceto more antituberculotics [23]. XDR-TB was defined as a resistanceto any fluoroquinolone and to at least one of three injectable drugscapreomycin, kanamycin or amikacin, in addition to MDR-TB. XDR-TB is very often untreatable. HIV co-infection with MDR-TB inimmunocompromised patients is a serious challenge for theresearch since it is demanded a new type of drugs or prodrugs bya new mechanism of action [20].

Waisser et al. [24,25] described a good antimycobacterialactivity of substituted salicylanilides against drug-sensitiveM. tuberculosis and some atypical mycobacterial strains (Mycobac-terium avium, Mycobacterium kansasii) with MICs for di- and tri-halogenated salicylanilides in the range of 1e32 mmol/L for all thetested strains.

Designing of new salicylanilide derivatives based on esterifica-tion is a known trend. First recent article dealing with antimicrobialesters of salicylanilides was published in 2006 [26]. Hydrophobicityis one of the factors influencing biological activity of salicylanilide.While the presence of phenolic hydroxyls seems to be necessary forthe activity, it could have irritative properties. Temporary maskingof this group by esterification and resulting changes in physico-chemical properties could be advantageous in a high activity, an

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113 6107

improved solubility, a better bioavailability, passing through themycobacterial cell wall and a lower toxicity. When amino acids areused for esterification, they could facilitate the possibility of tar-geting drugs as a drug delivery system [4,26,27].

The worldwide epidemic of antibiotic resistance is touching allpeople, because antibiotic-resistant bacteria are increasingly seento be just as virulent as their sensitive counterparts. For example,the global emergence of methicillin-resistant Staphylococcus aureus(MRSA) has arguably been the biggest setback in the history ofantimicrobial therapy. MRSA has caused serious problems in theempirical use of the major commonly applied antibiotics. Thesituation in Gram-negative bacteria is no less serious, too [28].

The conditions in the fungal kingdom are quite similar. Fungican become resistant to each of clinically used antifungal drugs byspecific mechanisms. The phenomenon of multidrug-resistancewas described analogously to bacteria [29].

Salicylanilide esters showed antimycobacterial activity in therange from 0.5 to 62.5 mmol/L [4,27,30,31]. Salicylanilides, theiresters and some structures containing them have shown andconfirmed recently their good antibacterial activity, especially onGram-positive strains in the concentration of 0.25 mg/mL andhigher [2,32e38]. About the mechanism of action e salicylanilideswere identified as inhibitors of the two-component regulatorysystems of bacteria by a mechanism related to the effect onuncoupling oxidative phosphorylation, but other injurious effectson prokaryote cells were described, too [2,31,32,36]. Recently, itwas also found in the studies that salicylanilides are selectiveinhibitors of interleukin-12p40 production playing a specific role inthe initiation, expansion and control of the cellular response to TBinfection [10,12]. The antifungal activity of salicylanilide esters issignificant for acetates and a-amino acids esters with MICs � 0.49and �1.95 mmol/L, respectively [4,30].

Therefore we designed and synthesized a new ester seriesof halogenated salicylanilides and N-acetyl-L-phenylalanine aspotential antibacterial and antifungal agents, primarily againstM. tuberculosis includingMDR-TB strains and atypicalmycobacteria.

2. Chemistry

The starting salicylanilides were selected according to theirpreviously described high in vitro antimycobacterial activity [24] andthe N-protected amino acid N-acetyl-L-phenylalanine due to prelim-inary publications about good influence ofN-Cbz-L-phenylalanine onantimycobacterial and antifungal activity of salicylanilides [4,27].

The syntheses of salicylanilide N-acetyl-L-phenylalanine estersconsist of two steps. At first, starting halogenated salicylanilides (1)were prepared routinely by the reaction of substituted salicylicacids and appropriate anilines in the presence of PCl3 in chloro-benzene. The reaction was carried out in a microwave reactorwhich led to an increase of the yield and shortening the reactiontime [39].

Scheme 1. Esterification of salicylanilides by N-acetyl-L-phenylalanine (R1 of esters ¼ 4-Cl, 5�20 �C.

Esters of salicylanilides (3) with lipophilic amino acid N-acetyl-L-phenylalanine (2) were obtained by the activation with N,N0-dicyclohexylcarbodiimide (DCC) in dry N,N-dimethylformamide(DMF) (Scheme 1). This method, which was taken from peptidechemistry, has been recently published by our group [30].

All newly prepared compounds were characterised by themelting point, IR and NMR spectroscopy, elementary analysis andoptical activity.

3. Pharmacology

3.1. Minimum inhibitory concentration assays

3.1.1. In vitro antimycobacterial susceptibility testingAll the prepared compounds were tested in vitro for their anti-

mycobacterial activity in the Laboratory for Tuberculosis in Ostravaagainst M. tuberculosis 331/88 (H37Rv) (dilution of the strain was10�3) and moreover for some non-tuberculous INH-resistantstrains e M. avium (330/88, dilution 10�5) and M. kansasii (235/80,dilution 10�4). One strain was clinically isolated (M. kansasii 6509/96 in the dilution 10�5), other strains were obtained from the CzechNational Collection of Type Cultures. The micromethod for thedetermination of the minimum inhibitory concentration (MIC) wasused.

The most active compounds 3g, 3i and 3kwere evaluated againstMDR-TB in the similar conditions using six M. tuberculosis strains(dilution 10�3) with different resistance patterns: 357/2005, 362/1998, 53/2009, Praha 1, Praha 131 (XDR-TB strain), and 9449/2007.

3.1.2. In vitro antibacterial susceptibility testingThe broth microdilution test M27-A [40] was used for the inves-

tigationof invitroantibacterial activityofesters (3) against fourGram-positive and four Gram-negative strains: S. aureus, methicillin-resis-tant S. aureus, Staphylococcusepidermidis,Enterococcus sp.;Escherichiacoli, Klebsiella pneumoniae, EBSL-positive K. pneumoniae, and Pseu-domonas aeruginosa. The method used was microdilution panelbouillon method with twofold dilution of the compounds.

3.1.3. In vitro antifungal susceptibility testingThe broth microdilution test M27-A was used for the investi-

gation of in vitro antifungal activity of esters (3) against four yeaststrains: Candida albicans, Candida tropicalis, Candida krusei, Candidaglabrata, and four moulds: Trichosporon beigelii, Aspergillus fumi-gatus, Absidia corymbifera, and Trichophyton mentagrophytes. Themethod used was microdilution panel bouillon method withtwofold dilution of the compounds.

3.2. In vitro cytotoxicity assay

Cytotoxicity was determined on human peripheral bloodmononuclear cells (PBMC). After incubation the cell viability was

-Cl, 4-Br; R2 ¼ 3-Cl, 4-Cl, 3,4-diCl, 4-Br, 4-CF3). Reagents and conditions: (a) DMF, DCC,

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e61136108

determined by MMT-assay [41]. Cytotoxicity was calculated afterdetermination of optical density and IC50 values were determinedfrom the dose-response curves.

Values of SI indicate rate between IC50 and MIC after 14 days ofincubation of M. tuberculosis 331/88 and all MDR-TB strains.

4. Results

4.1. Chemistry

Eleven new salicylanilide esters were prepared in two stepsfrom salicylic acids and anilines to form salicylanilides, which wereesterified by the using of DCC. This method was described recently[4,30]. The yields of esters (3) ranged from 43 to 82%.

4.2. Antimycobacterial activity

Esters 3 were evaluated against four mycobacterial strains(Table 1). All compounds inhibited tested strains with MICs in therange from 0.25 to 32 mmol/L. The most susceptible strain wasM. tuberculosis, least M. avium. Compounds 3g, 3i and 3k exhibitedan excellent activity against M. tuberculosis (MICs � 1 mmol/L) and3i and 3k towards two strains of M. kansasii with MICs up to2 mmol/L. All compounds have shown a very good MIC value. Threecompounds presented the same or a better activity than INH and allesters were more efficient than para-aminosalicylic acid (PAS). N-Acetyl-L-phenylalanine alone is quite inactive in all assays (data notshown).

Based on anti-TB activities, three esters were selected for nextassessment against MDR-TB strains. All compounds were effectivein the lowest used concentration of 1 mmol/L and the maximal levelwas in one case 4 mmol/L and therefore they exhibited a very goodefficacy (Table 2). 3i was evaluated as the most active compound,closely followed by 3k. All these esters inhibited the growth ofXDR-TB strain Praha 131 with MICs of 1e2 mmol/L.

4.3. Antibacterial and antifungal assays

Prepared esters (3) were evaluated for their in vitro antibacterialand antifungal activity.

Tables 3 and 4 bring the results. Three compounds (3a, 3e, and3g) showed no inhibition of growth in antibacterial assessment inthe concentration of �125 mmol/L. Tested compounds expressedonly a weak or no effect against Gram-negative bacteria, whenP. aeruginosa and EBSL-positive K. pneumoniae were completelyunaffected by these derivatives in the concentration of 125 mmol/Land lower; E. coli was affected only by 3d and 3f (MIC 31.25 and15.62 mmol/L, respectively) and K. pneumoniae by 3d (62.5 mmol/L).Some of derivatives had a very good MIC value against Gram-positive cocci with MIC of 0.98 mmol/L, including MRSA andS. epidermidis strains (3f, 3i; MIC 0.98 mmol/L), or Enteroccocus (3d;3.9 mmol/L). The most susceptible strain was S. aureus.

In general, these esters have shown a significant antifungalactivity. Table 4 includes only compounds with at least one MIClower than125 mmol/L (3a and 3g displayed no inhibition activity inthe MIC value of 125 mmol/L). The higher activity against one ormore fungi presented in summary 3c, 3d, 3f and 3k. Candida strainswere less susceptible than moulds e the lowest MICs were 15.62and 3.9 mmol/L, respectively. The major resistance demonstratedC. glabrata, on the other hand, the most susceptible are A. cor-ymbifera and T. mentagrophytes. Eight compounds exhibited activ-ities against fluconazole-resistant A. fumigatus and A. corymbifera inthe range from 3.9 to 125 mmol/L and 3k showed only a slightlypoorer activity for T. mentagrophytes (3.91/7.81 mmol/L).

4.4. Cytotoxicity evaluation

Cytotoxicity was determined only for three most anti-mycobacterial effective esters 3g, 3i and 3k (Table 5). All threecompounds presented very good SI values for drug-sensitiveM. tuberculosis andwith a partly exception of 3i good SI for MDR-TB.If SI value �10, then it is considered to be promising for a furtherscreening [42]. The lowest SI for resistant strains could be caused bythe lowest investigated concentration of 1 mmol/L.

5. Discussion

The prepared esters are showing the high antituberculousactivity (the lowest MICs 0.25 mmol/L). Generally, this series is moreactive then starting salicylanilides [24]. Except 3j which is lessactive and 3c, 3d, where the MIC values are analogous, esters aremore active on M. tuberculosis than salicylanilides, in some casesMIC was decreased eight-fold (e.g. 3e, 3g, 3i). The effect of esteri-fication on the activity againstM. avium is ambivalente in six casesit resulted in increasing and in five cases in decreasing activity e

and toward M. kansasii 235/80 there were five esters more activeand two less active than salicylanilides.

Structureeactivity relationships against M. tuberculosis. On thesalicylic ring, relatively independent on the aniline ring substitution,4-bromo/chloro substitutions are the most convenient for the anti-mycobacterial activity; the lowest is the contribution of 5-Cl (butthese derivatives are still comparable or better than non-substitutedones). The optimal moieties on the positions 3 and 4 of the anilinering are 4-CF3 (in combinationwith 4-substituted salicylic part of themolecule) and3,4-diCl. Themonosubstitutionbychlorineonposition3 or 4 provides a minimal benefit when compared with other ones.

The lipophility influences the activity, but the relationship is notdirect. In general, the most lipophilic derivatives 3g (logP 5.12), 3i(4.92), 3k (5.19) and partly 3e (4.83) showed the highest anti-mycobacterial activity, but the effects of substituents and theirpositions are very important, too. The LogP values, that are thelogarithm of the partition coefficient for octan-1-ol/water, calcu-lated using the program CS ChemOffice Ultra version 11.0 (Cam-bridgeSoft, USA), range from 4.56 to 5.19.

The strategy to esterify salicylanilidesbyN-acetyl-L-phenylalanineto develop new antituberculotics is with the respect to MICs moreconvenient than by acetic acid, especially againstM. tuberculosis andM. kansasii [30], lipophilicN-Cbz-a-amino acids [27] and comparablewith a slight superiority in some cases with carbamates [31].

TheMDR-TB results (MICs 1e2 mmol/L) of 3g, 3k and 3i postulatethem to be promising candidates for other testing for MDR-TBtreatment and they are comparable, although the lowest testedconcentrationwas 1 mmol/L, to salicylanilide carbamates [31],whichwere published recently on the website LeadDiscovery [43]. SI ofesters 3 is favourable than N-Cbz-a-amino acid ester [27] and forM. tuberculosis 331/88 than carbamates [31]. Whenwe compared 3iand 3k, the second compound has almost the same antitubercularefficacy as 3i, and SI is higher e these results make 3k optimal fornext investigation as a promising antimycobacterial agent.

For the activity against bacteria, in general, it seems that the mostconvenient substituents are 40-CF3 (3i, 3k) and 40-Br (3f). Whencompared substituents on salicylic ring, favourable are derivativeswith5-Cl than4-Clwith3ibeinganexception.Thereforesomeof theseesters could be potential agents against Gram-positive infections.

The antifungal activity, generally worse than antibacterial andantimycobacterial activity when compared on the base of MICs, ispositively modulated by the presence of 4-chloro substitution of theanilinemoiety (3b,3d) and4-bromosubstitutionof salicylic ring (3k).However, the antifungal potential of esters 3 (expressed as MICs) isnot such hopeful when compared with the antimycobacterial and

Table 1Antimycobacterial activity of esters 3.

MIC [mmol/L]

R1 R2 M. tuberculosis M. avium M. kansasii M. kansasii

331/88 330/88 235/80 6509/96

14 d 21 d 14 d 21 d 7 d 14 d 21 d 7 d 14 d 21 d

3a 4-Cl 3-Cl 2 4 8 32 8 8 8 2 8 83b 5-Cl 3-Cl 2 4 4 8 4 4 8 2 4 83c 4-Cl 4-Cl 4 4 8 16 4 4 4 2 4 83d 5-Cl 4-Cl 4 4 8 16 4 4 4 4 8 163e 4-Cl 4-Br 2 2 4 8 4 4 4 4 4 83f 5-Cl 4-Br 2 4 4 8 2 4 4 4 4 83g 4-Cl 3,4-diCl 0.5 1 8 8 1 2 4 1 2 43h 5-Cl 3,4-diCl 2 2 8 16 4 4 4 1 2 43i 4-Cl 4-CF3 0.25 0.5 4 4 0.5 1 2 1 2 23j 5-Cl 4-CF3 8 16 16 32 4 4 4 4 8 83k 4-Br 4-CF3 0.25 0.5 4 4 0.5 2 2 1 2 2INH e e 0.5 0.5e1 >250 >250 >250 >250 >250 2 4 4e8PAS e e 62.5 62.5 32 125 125 1000 >1000 32 125 500

Bold values represent lowest values of minimal inhibition concentration (MIC).M. avium strain 330/88 resistant to INH, RIF, ofloxacin and ethambutol; INH e isoniazid; PAS e para-aminosalicylic acid.

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113 6109

antifungal activity. The introduction of N-acetyl-L-phenylalanine acylis slightly less advantageous than esterification of salicylanilideswithN-Cbz-a-amino acids [4] and significantly less advantageous thanwith acetic acid [30].

In conclusion, salicylanilides N-acetyl-L-phenylalanine esterswere found to be a new highly active group promising awide-rangeantimicrobial activity.

6. Experimental protocols

6.1. Chemistry

6.1.1. General methodsAll used reagents and solvents were purchased from commercial

sources (SigmaeAldrich). Commercial grade reagents were usedwithout a further purification. Reactions were monitored by thinlayer chromatography plates coated with 0.2 mm silica gel 60 F254(Merck), which were visualized by UV irradiation (254 nm). All

Table 2Activity of selected esters against MDR strains.

MIC [mmol/L]

M. tuberculosis M. tuberculosis M. tuberculosis

357/2005 53/2009 Praha 1

14 d 21 d 14 d 21 d 14 d 21 d

3g 2 2 1 2 1 23i 1 2 1 1 1 23k 2 2 1 2 1 2INH 14.6 14.6 14.6 14.6 14.6 14.6

Bold values represent lowest values of minimal inhibition concentration (MIC).MDR-TB M. tuberculosis strains: 357/2005 and 362/1998 (both resistant to INH, RIF, rifarifabutine, streptomycin, ethambutol); Praha 1 (resistant to INH, RIF, rifabutine, streptstreptomycin, ethambutol, ofloxacine, gentamicin and amikacin; XDR-TB strain); 9449/2

melting points were determined on a Melting Point machine B-540(Büchi) apparatus using open capillaries and they are uncorrected.Optical activities were measured on polarimeter ADP 220 BS (Bel-lingham Stanley Ltd.). Infrared spectra (KBr pellets) were recordedon FT-IR spectrometer Nicolet 6700 FT-IR in the range of400e4000cm�1. TheNMRspectraweremeasured inCDCl3orDMSO-d6 solutions at ambient temperature onaVarianMercuryeVxbb300(300MHz for 1H and 75.5MHz for 13C; Varian Comp. Palo Alto, USA).The chemical shifts d are given in ppm, related to tetramethylsilaneas an internal standard. The coupling constants (J) are reported inHz.Elemental analysis (C, H, N) were performed on an automaticmicroanalyser CHNS-O CE instrument (FISONS EA 1110, Italy).

6.1.2. Synthetic procedure for (S)-2-(phenylcarbamoyl)phenyl2-acetamido-3-phenylpropanoates

The halogenated (S)-2-(phenylcarbamoyl)phenyl 2-acetamido-3-phenylpropanoates were prepared by a two-step synthesisdescribed previously by our group [30].

M. tuberculosis M. tuberculosis M. tuberculosis

Praha 131 362/1998 9449/2007

14 d 21 d 14 d 21 d 14 d 21 d

2 2 1 2 2 41 2 1 1 2 22 2 1 1 1 2

14.6 14.6 14.6 14.6 58.3 58.3

butine, streptomycin, ethambutol, and ofloxacine); 53/2009 (resistant to INH, RIF,omycin, ethambutol and clofazimine); Praha 131 (resistant to INH, RIF, rifabutine,007 resistant to INH, RIF, rifabutine, and streptomycin.

Table 3Antibacterial activity of prepared esters.

MIC/IC80 [mmol/L]

SA MRSA SE EF EC KP

24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h

3b 31.25 125 125 125 62.5 125 125 >125 >125 >125 >125 >1253c 62.5 >125 125 >125 125 >125 125 >125 >125 >125 >125 >1253d 1.95 15.62 1.95 31.25 1.95 31.25 3.9 >125 31.25 >125 62.5 1253f 0.98 1.95 0.98 7.81 0.98 1.95 7.81 125 15.62 125 >125 >1253h 125 >125 125 >125 125 >125 125 >125 125 >125 >125 >1253i 0.98 1.95 0.98 7.81 0.98 3.9 31.25 >125 >125 >125 >125 >1253j 125 >125 125 >125 125 >125 125 >125 >125 >125 >125 >1253k 3.9 31.25 7.81 125 15.62 62.5 15.62 125 250 500 >500 >500

Bold values represent lowest values of minimal inhibition concentration (MIC).SA: Staphylococcus aureus CCM 4516/08; MRSA: methicillin-resistant Staphylococcus aureusH 5996/08; SE: Staphylococcus epidermidisH 6966/08; EF: Enterococcus sp. J 14365/08; EC: E. coli CCM4517; KP: Klebsiella pneumoniae D 11750/08.

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e61136110

At first, starting halogenated salicylanilides (1) were preparedroutinely by the modified reaction of substituted salicylic acids andappropriate anilines (in equivalent amounts; Scheme 1) in thepresence of PCl3 (0.5 equiv.) in chlorobenzene. The reaction wascarried out with vigorously stirring in a microwave reactor (530 W,600 rpm, MicroSYNTH Milestone) for 20 min to reflux. Thisprocedure led to an increase of the yield and shortening the reac-tion time from several hours to minutes. The reaction mixture wasfiltered while hot, let stand at 20 �C and then at 4 �C for 24 h. Thecrude product was filtered off and once ormore times recrystallizedfrom aqueous ethanol to obtain the pure product.

Salicylanilide esters (3) with N-acetyl-L-phenylalanine (2) wereobtained by the activation with N,N0-dicyclohexylcarbodiimide(DCC) in N,N-dimethylformamide (DMF) (Scheme 1). The N-pro-tected amino acid (2) and substituted salicylanilide (1) (both0.001 mol) were dissolved in dry DMF (15 mL). This solution wascooled to �15 �C and DCC in a mild excess (0.0011 mol) was addedin three portions during 1 h. Next the mixture was stirred for 3 h atthe same temperature and stored at þ4 �C for 48 h. The precipitateof by-productN,N0-dicyclohexylureawas filtered off and the solventwas evaporated in vacuo. The remnant was dissolved in a smallamount of ethyl acetate and the insoluble portion (another N,N0-dicyclohexylurea) was filtered off. The filtratewas again evaporatedin vacuo. The crude product 3 was purified by the crystallization(commonly once or twice) from ethyl acetateehexane.

6.1.2.1. (S)-4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl 2-acet-amido-3-phenylpropanoate (3a). White solid; yield 63%; mp143e145 �C; ½a�23D �27.8 (c 0.72; ethyl acetate). IR (KBr pellet): 3297,3062, 2933,1775 (CO ester), 1651,1594,1544,1481,1427,1372,1320,1200, 1105, 874, 779, 698, 681. 1H NMR (300 MHz, DMSO): d 10.76(1H, s, NH), 8.50 (1H, bs, NH), 7.92 (1H, t, J¼ 1.8 Hz, H20), 7.78 (1H, d,J ¼ 2.6 Hz, H3), 7.67 (1H, dd, J ¼ 8.7 Hz, J ¼ 2.6 Hz, H5), 7.59 (1H, d,J¼ 8.2Hz, H6), 7.37 (1H, t, J¼ 8.0 Hz, H50), 7.28e7.14 (7H,m, H60, H40,H200, H300, H400, H500, H600), 4.61 (1H, m, CH), 3.17 (1H, dd, J ¼ 14.1 Hz,J¼ 4.6Hz, CH2), 2.88 (1H, dd, J¼ 14.0Hz, J¼ 10.0Hz, CH2),1.76 (3H, s,CH3). 13C NMR (75 MHz, DMSO): d 170.1, 169.8, 162.9, 146.5, 140.4,137.3,133.2,131.6,131.4,130.6,130.4,129.5,129.1,128.3,126.8,125.2,123.8, 119.5, 118.4, 53.9, 36.2, 22.3. Anal. Calcd. for C24H20Cl2N2O4(471.33): C, 61.16; H, 4.28; N, 5.94. Found: C, 60.95; H, 4.41; N, 5.85.

6.1.2.2. (S)-5-Chloro-2-(3-chlorophenylcarbamoyl)phenyl 2-acet-amido-3-phenylpropanoate (3b). White solid; yield 43%; mp177.5e179 �C; ½a�23D �27.1 (c 0.7; ethyl acetate). IR (KBr pellet): 3300,3065, 2930, 1777 (CO ester), 1651, 1594, 1542, 1493, 1426, 1372, 1198,1109, 827, 780, 700, 503.1HNMR(300MHz,DMSO): d10.63 (1H, s,NH),8.52 (1H, bs, NH), 7.91 (1H, t, J¼ 1.9Hz,H20), 7.88 (1H, d, J¼ 8.2Hz,H3),7.41e7.35 (3H, m, H4, H6, H50), 7.28e7.15 (7H, m, H40, H60, H200, H300,H400, H500, H600), 4.63 (1H, m, CH), 3.17 (1H, dd, J ¼ 14.2 Hz, J ¼ 4.8 Hz,

CH2), 2.89 (1H, dd, J ¼ 14.0 Hz, J ¼ 10.0 Hz, CH2), 1.76 (3H, s, CH3). 13CNMR (75 MHz, DMSO): d 170.0, 169.8, 163.4, 148.4, 139.9, 137.6, 135.5,133.2, 131.2, 130.6, 129.5, 129.1, 128.3, 126.8, 126.6, 124.0, 120.3, 119.4,117.8, 53.9, 36.2, 22.3. Anal. Calcd. for C24H20Cl2N2O4 (471.33): C, 61.16;H, 4.28; N, 5.94. Found: C, 61.34; H, 4.39; N, 6.01.

6.1.2.3. (S)-4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl 2-acet-amido-3-phenylpropanoate (3c). White solid; yield 52%; mp164e166 �C; ½a�23D �26.8 (c 0.77; ethyl acetate). IR (KBr pellet): 3305,3066, 3031, 2927, 1751 (CO ester), 1651, 1595, 1526, 1493,1403,1371,1313, 1197, 1107, 1015, 823, 697, 504. 1H NMR (300 MHz, DMSO):d 10.64 (1H, s, NH), 8.49 (1H, bs, NH) 7.90 (1H, d, J ¼ 2.6 Hz, H3),7.78e7.73 (2H, m, H20, H60), 7.64 (1H, dd, J ¼ 8.6 Hz, J ¼ 2.6 Hz, H5),7.47e7.37 (2H, m, H30, H50), 7.29e7.20 (6H, m, H6, H200, H300, H400,H500, H600), 4.60 (1H, m, CH), 3.16 (1H, dd, J ¼ 14.0 Hz, J ¼ 4.6 Hz,CH2), 2.85 (1H, dd, J ¼ 9.3 Hz, J ¼ 4.5 Hz, CH2), 1.76 (3H, s, CH3). 13CNMR (75 MHz, DMSO): d 170.1, 169.8, 162.7, 146.5, 138.0, 137.3,133.2, 131.5, 130.4, 129.5, 129.2, 129.1, 128.3, 126.7, 125.2, 122.4,121.5, 53.9, 36.2, 22.3. Anal. Calcd. for C24H20Cl2N2O4 (471.33): C,61.16; H, 4.28; N, 5.94. Found: C, 61.02; H, 4.50; N, 5.71.

6.1.2.4. (S)-5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl 2-acet-amido-3-phenylpropanoate (3d). White solid; yield 43%; mp147.5e150 �C; ½a�23D �16.3 (c 0.4; ethyl acetate). IR (KBr pellet): 3406,3030, 2930, 1765 (CO ester), 1657, 1629, 1602, 1536, 1400, 1384, 1314,1200, 1093, 829, 745, 700, 510. 1H NMR (300 MHz, DMSO): d 10.59(1H, s, NH), 8.50 (1H, bs, NH), 7.89 (1H, d, J ¼ 8.6 Hz, H3), 7.76e7.72(2H, m, H20, H60), 7.52 (1H, dd, J ¼ 8.3 Hz, J ¼ 2.1 Hz, H4), 7.44e7.40(2H, m, H30, H50), 7.38 (1H, d, J ¼ 2.0 Hz, H6), 7.31e7.18 (5H, m, H200,H300, H400, H500, H600), 4.54 (1H, m, CH), 3.17 (1H, dd, J ¼ 14.0 Hz,J ¼ 4.7 Hz, CH2), 2.85 (1H, dd, J ¼ 9.8 Hz, J ¼ 3.4 Hz, CH2), 1.76 (3H, s,CH3). 13C NMR (75 MHz, DMSO): d 170.0, 169.8, 163.3, 148.4, 138.0,137.3, 136.4, 135.4, 131.1, 129.5, 129.2, 129.1, 128.3, 126.9, 126.8, 122.5,121.5, 53.9, 36.2, 22.3. Anal. Calcd. for C24H20Cl2N2O4 (471.33): C,61.16; H, 4.28; N, 5.94. Found: C, 61.44; H, 4.22; N, 6.17.

6.1.2.5. (S)-2-(4-Bromophenylcarbamoyl)-4-chlorophenyl 2-acetamido-3-phenylpropanoate (3e). White solid; yield 82%; mp 170.5e171.5 �C;½a�23D �22.4 (c 0.67; ethyl acetate). IR (KBr pellet): 3303, 3064, 2928,1767 (CO ester), 1654, 1592, 1529, 1489, 1394, 1314, 1197, 1105, 1072,1011, 824, 748, 698, 506. 1H NMR (300 MHz, DMSO): d 10.63 (1H, s,NH), 8.49 (1H, bs, NH) 7.90 (1H, d, J ¼ 2.7 Hz, H3), 7.71e7.66 (2H, m,H20, H60), 7.56 (1H, dd, J¼ 8.6 Hz, J¼ 2.6 Hz, H5), 7.29e7.18 (8H,m, H6,H30, H50, H200, H300, H400, H500, H600), 4.63 (1H, m, CH), 3.16 (1H, dd,J ¼ 14.3 Hz, J ¼ 4.5 Hz, CH2), 2.85 (1H, dd, J ¼ 9.0 Hz, J ¼ 4.9 Hz, CH2),1.76 (3H, s, CH3). 13C NMR (75MHz, DMSO): d 170.1,169.8,162.8,146.5,138.4, 137.3, 133.2, 131.5, 130.4, 129.5, 129.2, 129.1, 128.3, 126.8, 125.2,122.8, 121.9, 53.9, 36.2, 22.3. Anal. Calcd. for C24H20BrClN2O4 (515.78):C, 55.89; H, 3.91; N, 5.43. Found: C, 55.59; H, 3.69; N, 5.62.

Table

4Antifunga

lev

aluationof

prepared

esters.

MIC/IC80[mmol/L]

CA

CT

CK

CG

TBAF

AC

TM

24h

48h

24h

48h

24h

48h

24h

48h

24h

48h

24h

48h

24h

48h

24h

48h

3b>12

5>12

5>12

5>12

5>12

5>12

5>12

5>12

512

5>12

5>12

5>12

562

.5>12

562

.5>12

53c

>12

5>12

5>12

5>12

5>12

5>12

5>12

5>12

53.9

3.9

31.25

125

3.9

15.62

31.25

62.5

3d31

.25

>12

5>12

5>12

531

.25

>12

5>12

5>12

515

.62

62.5

>12

5>12

57.81

7.81

15.62

31.25

3e12

5>12

5>12

5>12

512

5>12

5>12

5>12

512

5>12

512

5>12

512

5>12

5>12

5>12

53f

>12

5>12

5>12

5>12

5>12

5>12

5>12

5>12

515

.62

62.5

62.5

>12

515

.62

62.5

7.81

15.62

3h12

5>12

512

5>12

5>12

5>12

5>12

5>12

512

5>12

512

5>12

512

5>12

562

.5>12

53i

>12

5>12

5>12

5>12

512

5>12

5>12

5>12

512

5>12

512

5>12

5>12

5>12

562

.5>12

53j

>12

5>12

5>12

5>12

562

.5>12

5>12

5>12

512

5>12

562

.5>12

562

.5>12

512

5>12

53k

31.25

500

125

250

15.62

125

125

500

15.62

31.25

15.62

31.25

7.81

15.62

3.9

7.81

FLU

0.06

0.12

0.12

>12

53.91

15.62

0.98

3.91

0.24

0.48

>12

5>12

5>12

5>12

51.95

3.91

Boldva

lues

representlowestva

lues

ofminim

alinhibitionco

ncentration(M

IC).

CA:Ca

ndidaalbicans

ATC

C44

859;

CT:

Cand

idatrop

icalis

156;

CK:Ca

ndidakrusei

E28;

CG:Ca

ndidaglab

rata

20/I;TB

:Tricho

sporon

beigelii11

88;AF:

Aspergillu

sfumigatus

231;

AC:Absidia

corymbifera

272;

TM:Tricho

phyton

men

tagrop

hytes44

5;FLU:fluco

nazole.

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113 6111

6.1.2.6. (S)-2-(4-Bromophenylcarbamoyl)-5-chlorophenyl 2-acetami-do-3-phenylpropanoate (3f). White solid; yield 61%; mp167e168.5 �C; ½a�23D �11.7 (c 0.9; dimethyl sulfoxide). IR (KBrpellet): 3292, 3241, 3083, 1750 (CO ester), 1653, 1602, 1521, 1488,1391, 1370, 1313, 1197, 1143, 1074, 1013, 895, 815, 749, 697. 1H NMR(300 MHz, DMSO): d 10.57 (1H, s, NH), 8.49 (1H, bs, NH), 7.73e7.66(3H, m, H3, H20, H60), 7.55e7.49 (3H, m, H4, H30, H50), 7.30 (1H, d,J¼ 2.1 Hz, H6), 7.24e7.17 (5H, m, H200, H300, H400, H500, H600), 4.61 (1H,m, CH), 3.16 (1H, dd, J ¼ 14.0 Hz, J ¼ 4.7 Hz, CH2), 2.88 (1H, dd,J ¼ 13.6 Hz, J ¼ 10.3 Hz, CH2), 1.76 (3H, s, CH3). 13C NMR (75 MHz,DMSO): d 170.0, 169.8, 163.3, 148.4, 138.4, 137.3, 136.4, 131.8, 131.1,129.5, 129.2, 128.9, 128.3, 126.8, 123.3, 122.8, 121.9, 53.9, 36.2, 22.3.Anal. Calcd. for C24H20BrClN2O4 (515.78): C, 55.89; H, 3.91; N, 5.43.Found: C, 56.08; H, 3.80; N, 5.51.

6.1.2.7. (S)-4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl 2-aceta-mido-3-phenylpropanoate (3g). White solid; yield 69%; mp160e162 �C; ½a�23D �14.8 (c 0.73; ethyl acetate). IR (KBr pellet): 3308,3251, 3083, 1748 (CO ester), 1647, 1578, 1543, 1513, 1471, 1379, 1304,1196, 1149, 1104, 1030, 907, 788, 694, 652. 1H NMR (300 MHz,DMSO): d 10.77 (1H, s, NH), 8.50 (1H, bs, NH), 8.10 (1H, d, J ¼ 1.3 Hz,H20), 7.85 (1H, d, J ¼ 2.7 Hz, H3), 7.80 (1H, d, J ¼ 2.7 Hz, H50),7.71e7.60 (3H, m, H5, H6, H60), 7.28e7.17 (5H, m, H200, H300, H400, H500,H600), 4.64 (1H, m, CH), 3.17 (1H, dd, J¼ 14.0 Hz, J¼ 4.8 Hz, CH2), 2.84(1H, dd, J ¼ 9.5 Hz, J ¼ 4.3 Hz, CH2), 1.76 (3H, s, CH3). 13C NMR(75MHz, DMSO): d 170.1, 169.8, 162.9, 146.5, 138.5, 137.3, 136.4, 131.7,130.8,130.4,129.5,129.2,128.9,128.3,126.9,126.8,125.6,122.9,121.2,53.9, 36.2, 22.3. Anal. Calcd. for C24H19Cl3N2O4 (505.78): C, 56.99; H,3.79; N, 5.54. Found: C, 56.77; H, 3.94; N, 5.76.

6.1.2.8. (S)-5-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl 2-aceta-mido-3-phenylpropanoate (3h). White solid; yield 43%; mp145e146.5 �C; ½a�23D �32.9 (c 0.73; ethyl acetate). IR (KBr pellet):3373, 3302, 3082, 1954, 1760 (CO ester), 1657, 1591, 1514, 1474, 1375,1305, 1199, 1156, 1082, 1053, 906, 818, 741, 668. 1H NMR (300 MHz,DMSO): d 10.72 (1H, s, NH), 8.50 (1H, bs, NH), 8.11 (1H, d, J ¼ 2.2 Hz,H20), 7.85 (1H, d, J ¼ 8.3 Hz, H3), 7.52 (1H, dd, J ¼ 8.3 Hz, J ¼ 2.1 Hz,H4), 7.43e7.37 (3H,m, H6, H50, H60), 7.28e7.17 (5H,m, H200, H300, H400,H500, H600), 4.64 (1H, m, CH), 3.17 (1H, dd, J¼ 14.0 Hz, J¼ 4.8 Hz, CH2),2.84 (1H, dd, J ¼ 14.3 Hz, J ¼ 4.8 Hz, CH2), 1.76 (3H, s, CH3). 13C NMR(75MHz, DMSO): d 170.0,169.8,163.5,148.4,138.6,137.3,135.7,131.2,130.8, 129.5, 129.2, 128.9, 128.3, 126.9, 126.8, 125.8, 123.3, 121.9,119.3,53.9, 36.2, 22.3. Anal. Calcd. for C24H19Cl3N2O4 (505.78): C, 56.99; H,3.79; N, 5.54. Found: C, 56.68; H, 3.83; N, 5.42.

6.1.2.9. (S)-4-Chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl2-acetamido-3-phenylpropanoate (3i). White solid; yield 59%; mp132e134 �C; ½a�23D �17.9 (c 0.7; ethyl acetate). IR (KBr pellet): 3289,3030, 2925, 1764 (CO ester), 1650, 1603, 1537, 1413, 1374, 1317, 1102,1067, 1017, 881, 823, 739, 696. 1H NMR (300 MHz, DMSO): d 10.87(1H, s, NH), 8.50 (1H, bs, NH), 7.95e7.90 (2H, m, H30, H50), 7.88 (1H,d, J ¼ 2.7 Hz, H3), 7.80 (1H, d, J ¼ 2.6 Hz, H6), 7.75e7.65 (3H, m, H5,H20, H60), 7.26e7.17 (5H, m, H200, H300, H400, H500, H600), 4.60 (1H, m,CH), 3.16 (1H, dd, J ¼ 14.0 Hz, J ¼ 4.7 Hz, CH2), 2.84 (1H, dd,J ¼ 13.3 Hz, J ¼ 4.0 Hz, CH2), 1.75 (3H, s, CH3). 13C NMR (75 MHz,DMSO): d 170.1, 169.8, 163.2, 146.5, 142.6, 137.3, 133.2, 131.7, 130.4,129.5, 129.0, 128.3, 126.8, 126.6, 126.2, 125.2, 124.3, 119.9, 53.9, 36.2,22.3. Anal. Calcd. for C25H20ClF3N2O4 (504.89): C, 59.47; H, 3.99; N,5.55. Found: C, 59.73; H, 4.14; N, 5.65.

6.1.2.10. (S)-5-Chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl2-acetamido-3-phenylpropanoate (3j). White solid; yield 57%; mp155e156.5 �C; ½a�23D �29.9 (c 0.67; ethyl acetate). IR (KBr pellet):3365, 3289, 2929, 1753 (CO ester), 1680, 1650, 1602, 1534, 1408,1373, 1320, 1114, 1067, 1018, 909, 839, 741, 701. 1H NMR (300 MHz,

Table 5Cytotoxicity and SI for selected esters.

R1 R2 PBMC IC50 (mmol/L) SI for M. tuberculosis 331/88 SI for M. tuberculosis (MDR-TB strains)

3g 4-Cl 3,4-diCl 44 88 22e443i 4-Cl 4-CF3 12.2 48.8 6.1e12.23k 4-Br 4-CF3 48.7 194.8 24.1e48.7

SI (selectivity index) ¼ IC50/MIC; MICs were chosen after 14 d of M. tuberculosis 331/88 and all MDR-TB incubation .

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e61136112

DMSO): d: 10.82 (1H, s, NH), 8.49 (bs, 1H, NH), 7.95e7.91 (2H, m,H30, H50), 7.90 (1H, d, J ¼ 2.9 Hz, H3), 7.74e7.68 (2H, m, H20, H60),7.53 (1H, dd, J ¼ 8.3 Hz, J ¼ 2.0 Hz, H4), 7.32 (1H, dd, J ¼ 2.0, H6),7.28e7.17 (5H, m, H200, H300, H400, H500, H600), 4.63 (1H, m, CH), 3.16(1H, dd, J ¼ 14.2 Hz, J ¼ 4.8 Hz, CH2), 2.85 (1H, dd, J ¼ 9.8 Hz,J¼ 2.4 Hz, CH2),1.75 (3H, s, CH3). 13C NMR (75MHz, DMSO): d 170.0,169.8, 163.7, 148.4, 142.2, 137.6, 135.6, 131.2, 129.5, 129.2, 129.1,128.3, 126.7, 126.6, 126.5, 126.2, 124.0, 119.3, 53.9, 36.2, 22.3. Anal.Calcd. for C25H20ClF3N2O4 (504.89): C, 59.47; H, 3.99; N, 5.55.Found: C, 59.51; H, 3.86; N, 5.59.

6.1.2.11. (S)-4-Bromo-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl2-acetamido-3-phenylpropanoate (3k). White solid; yield 55%; mp155e157 �C; ½a�23D �20.1 (c 0.67; ethyl acetate). IR (KBr pellet): 3349,3261, 3023, 2941,1747 (CO ester),1665,1644,1604,1530,1408,1372,1322, 1204, 1163, 1066, 1018, 973, 833, 747, 699. 1H NMR (300 MHz,DMSO): d 10.86 (1H, s, NH), 8.49 (1H, bs, NH), 7.95e7.90 (3H, m, H3,H30, H50), 7.82 (1H, d, J ¼ 2.5 Hz, H6), 7.75e7.69 (2H, m, H20, H60),7.58 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.6 Hz, H5), 7.27e7.16 (5H, m, H200, H300,H400, H500, H600), 4.60 (1H, m, CH), 3.16 (1H, dd, J¼ 13.9 Hz, J¼ 4.6 Hz,CH2), 2.85 (1H, dd, J¼ 13.8 Hz, J¼ 10.7 Hz, CH2),1.75 (3H, s, CH3). 13CNMR (75 MHz, DMSO): d 170.0, 169.8, 163.1, 147.0, 142.6, 137.3,134.6, 131.8, 129.5, 129.1, 128.3, 126.7, 126.6, 126.4, 126.2, 125.5,124.5, 119.6, 53.9, 36.2, 22.3. Anal. Calcd. for C25H20BrF3N2O4(504.89): C, 54.66; H, 3.67; N, 5.10. Found: C, 54.83; H, 3.88; N, 5.40.

6.2. Pharmacology

6.2.1. In vitro antimycobacterial susceptibility testingAll the prepared compounds were tested in vitro for their anti-

mycobacterial activity in the Laboratory for Tuberculosis in Ostravaagainst M. tuberculosis 331/88 (H37Rv) (dilution of the strain was10�3) andmoreover for some non-tuberculous INH-resistant strainseM. avium (330/88, resistant to INH, RIF, ofloxacin and ethambutol;dilution 10�5) andM. kansasii (235/80, dilution 10�4). One strainwasclinically isolated (M. kansasii 6509/96 in the dilution 10�5); otherstrains were obtained from the Czech National Collection of TypeCultures. The micromethod for the determination of the minimuminhibitory concentration (MIC) was used. Antimycobacterial activi-ties were determined in the �Sula semisynthetic medium (SEVAC,Czech Republic). The tested compounds were added to the mediumas solutions in dimethylsulfoxide (DMSO). The following concen-trations were used: 1000, 500, 250, 125, 62, 32, 16, 8, 4, 2, 1, 0.5, and0.25 mmol/L. TheMICswere determined after incubation at 37 �C for14 and 21 days, forM. kansasii additionally for 7 days. MIC (mmol/L)was the lowest concentration at which the inhibition of mycobac-terial growth occurred. As reference compounds were chosenisoniazid and PAS.

The most active compounds 3g, 3i and 3k were evaluatedagainst MDR-TB in the similar conditions using six M. tuberculosisstrains (dilution 10�3) with different resistance patterns: 357/2005,362/1998, 53/2009, Praha 1, Praha 131 (XDR-TB strain), and 9449/2007. All strains are resistant to INH, RIF, rifabutine and strepto-mycin, in some cases is present other resistance. The followingconcentrations were used: 1000, 500, 250, 125, 62, 32, 16, 8, 4, 2,and 1 mmol/L.

6.2.2. In vitro antibacterial susceptibility testingThe broth microdilution test M27-A [40] was used for the

investigation of in vitro antibacterial activity of esters (3) againstfour Gram-positive and four Gram-negative strains: S. aureus,methicillin-resistant S. aureus, S. epidermidis, Enterococcus sp.; E.coli, K. pneumoniae, EBSL-positive K. pneumoniae, and P. aeruginosa.These assays were performed in the Department of Medical andBiological Sciences at the Faculty of Pharmacy, Charles University,Hradec Králové. The method used was microdilution panel bouillonmethod with twofold dilution of the compounds in Mueller-Hintonbouillon (HiMedia Laboratories, India). The esters were dissolved inDMSO and the final concentrations of the compounds ranged from500 to 0.49 mmol/L. Drug-free controls were included. Theminimum inhibitory concentrations (MICs) were defined as 80%(IC80) or higher reduction of growth in comparisonwith the control.The determination of results was performed visually and photo-metrically. The values of MICs were determined after 24 and 48 h ofincubation in the darkness at 35 �C in a humid atmosphere.

6.2.3. In vitro antifungal susceptibility testingThe brothmicrodilution testM27-Awasused for the investigation

of in vitro antifungal activity of esters (3) against four yeast strains: C.albicans, C. tropicalis, C. krusei, C. glabrata, and four moulds: T. beigelii,A. fumigatus, A. corymbifera, and T. mentagrophytes. Fluconazole wasused as a reference drug. The method used was microdilution panelbouillon method with twofold dilution of the compounds in RPMI-1640 with glutamine (Sevapharma, Czech Republic) buffered to pH7.0 with 0.165 M of 3-morpholinopropane-1-sulphonic acid (Sigma,Germany). Other conditions and testing workplacewere the same asfor antibacterial assay, only for T. mentagrophytes the final MICsweredetermined after 72 and 120 h of incubation.

6.2.4. In vitro cytotoxicity assayCytotoxicity was determined on human peripheral blood

mononuclear cells. The cells were cultured in RPMI-1640 mediumwith 10% fetal calf serum, 2 mM L-glutamine and 160 mg/mL ofgentamicin. Cell cultures were maintained at 37 �C, 5% CO2 inwater-saturated atmosphere. Cells were plated into 96-well platewith initial cell number of 1.5e2.0 � 105 cells per well. After 24 hincubation cells were treated with compounds in 100 mL serum freemedium overnight. Controls were included. Four parallelmeasurements were performed.

After incubation the cell viability was determined by MMT-assay[41]. 45mLMTT-solution (2mg/mL)was added to eachwell [44]. After4 h of incubation cells were centrifuged for 5 min (2000 rpm) andsupernatant was removed. The obtained formazan crystals weredissolved in 50 or 100 mL DMSO and optical density (OD) wasmeasured at 540 and 620 nm using ELISA Reader (iEMS Reader,Labsystems, Finland). OD620 values were subtracted from OD540

values. Cytotoxicity was calculated using the following equation:cytotoxicity (%) ¼ [1 � (ODtreated/ODcontrol)] � 100; where ODtreated

andODcontrol correspond to theoptical densities of the treated and thecontrol cells, respectively. Two independent experiments werecarried outwithparallelmeasurements. IC50 valueswere determinedfrom the dose-response curves (defined using MicrocalTM Origin1software, version 6.0).

M. Krátký et al. / European Journal of Medicinal Chemistry 45 (2010) 6106e6113 6113

Values of SI indicate rate between IC50 and MIC after 14 days ofincubation of M. tuberculosis 331/88 and all MDR-TB strains.

Acknowledgments

This work was financially supported by GAUK 27610/2010, theResearch project MSM0021620822, IGA NS 10367-3 and SVV-2010-261-001.

References

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