European Journal of Pharmaceutical Sciences 10 (2000) 119–123www.elsevier.nl / locate /ejps

In vitro inhibition properties of a new group of thiobenzanilides in relationto yeasts

* ~Joanna Matysiak, Andrzej Niewiadomy , Grazyna Ma̧cik-NiewiadomyaUniversity of Agriculture, Department of Chemistry, Akademicka 15, 20-950 Lublin, Poland

Received 30 June 1999; received in revised form 21 October 1999; accepted 22 November 1999

Abstract

The antifungal potency of a series of 2,4-dihydroxythiobenzanilides was tested. MIC assessments were used for the estimation ofpotential activity in vitro against Candida, Cryptococcus, Geotrichum and Trichosporon species. The strongest fungistatic activity wasobserved for dichloro derivatives (MIC 7.82–31.21 mg/ml). The action of these compounds depends on lipophilicity, determined by thesubstitution of N-aryl moiety and the electron properties of molecules. The lipophilicity, expressed by R values, was determined in theMw

1reversed-phase system. The changes in the nature of the thioamide bond were interpreted on the basis of UV and H NMR spectra. 2000 Elsevier Science B.V. All rights reserved.

Keywords: 2,4-Dihydroxythiobenzanilides; Antifungal activity; MIC; Lipophilicity

1. Introduction and thiobenzanilides (Waisser et al., 1990, 1998a,b). Someˇ ´of them also exhibit an antifungal activity (Klimesova et

Much attention is recently being paid to the discovery al., 1996; Waisser et al., 1996). These results encouragedand development of new, more selective antifungal agents. us to carry out investigations on antifungal action in aA wide spectrum of antimycobacterial action is displayed series of thiobenzanilides.by a number of compounds containing a thiocarbamide In the search for new leading structures in the thioben-group such as the pharmacophore, like thiobenzamides zanilide group, synthesis of a series of the compounds(Waisser et al., 1995a), thiooxalmides (Waisser et al., containing thioacyl system derived from rezorcine was1995b), 2-alkylthiopyridine-4-carbothioamides (Waisser et carried out. It seems that such substitution can guarantee

ˇ ´al., 1995c; Klimesova et al., 1996), pyrazine car- achievement of hydrophilic–hydrophobic equilibrium bybothioamides (Kaliszan et al., 1978), N-(2-ben- the compounds. This acts by lytic interactions and the

ˇzothiazolyl)benzenecarbothioamides (Kunes et al., 1998) possibility of penetrating the wall and membrane structuresof microorganisms (particularly fungi). The proposedmethod of synthesis (patent in preparation) makes itpossible to obtain the derivatives containing the optionallymodified N-aromatic moiety of the general formula shownin Fig. 1.

This paper describes the antifungal estimation andQSAR analysis of a group of derivatives of 2,4-dihydrox-ythiobenzanilide substituted in the N-aromatic moiety.

Fig. 1. General structure of thiobenzanilides. 2. Materials and methods

Thin-layer chromatography (TLC) was performed on*Corresponding author. Tel.: 148-81-445-6097; fax: 148-81-533-10310-cm pre-coated HPTLC plates of RP-8, F3752. 254S

E-mail address: jmaty@ursus.ar.lublin.pl (A. Niewiadomy) (Merck); 1-ml samples of the solutes (0.5 mg/ml in

0928-0987/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0928-0987( 99 )00096-2

120 J. Matysiak et al. / European Journal of Pharmaceutical Sciences 10 (2000) 119 –123

methanol) were spotted with a Desaga AS 30 applicator. coupled thiocarbamoyl moiety. They are used for de-The chromatograms were developed over a distance of 9.5 termination of electron density changes and probablycm in horizontal ‘sandwich’ chambers of Camag for TLC. conformational equilibrium transitions caused by substitu-The chambers were saturated with the organic solvent tion.vapour for 20 min. In the studies with the 2,4-dihydrox- Calculations of dipole moments of compounds for twoythiobenzanilides, water–methanol mixtures were used as tautomeric forms –C(=S)–NH–↔–C(SH)iN– were car-the mobile phases. The concentration of the organic ried out with an ALLCHEME 2000 program.modifier in a mobile phase ranged from 50 to 85%. All With the use of the dilution method, MIC values of theTLC measurements were performed at 218C. Spots were compounds against six strains of yeasts (Table 2) havevisualised under the UV light at 254 nm. been determined. Microorganisms were multiplied on the

1H NMR spectra were recorded with a FT-NMR Tesla agar Mueller–Hinton slants containing 4% of glucose (pHBS 567 A spectrometer (100 MHz) using deuterated 5.6) and in the analogous Mueller–Hinton broths.dimethylsulphoxide (DMSO) and acetone as the solvents. The tested compounds were dissolved in methanol.Chemical shifts are given in relation to tetramethylsilane Appropriate volumes of these solutions were added to the(TMS) and interpretation of the spectra is limited only to cooled (to 458C) medium and after mixing stirring spreadthe position of the amide proton. onto the Petri plates. After solidification the plates were

4UV–Vis spectra were recorded with an UV-160 dried and then 0.02 ml of fungi culture (10 cfu) wasShimadzu spectrophotometer equipped with a QS 1.000 sprayed onto their surface. The cultures were incubated forquartz cuvette using ethanol solutions. Interpretation of the 2–10 days at 228C. At the same time the sensitivity of thespectra was confined to recording the changes in the strains to methanol was determined. The results were

Table 1Analytical data and the lipophilicity parameter obtained for 2,4-dihydroxythiobenzanilides

2 3 4 5 1Compound –R –R –R –R Formula M M. p. m (D) HPTLC UV H NMR

(8C) l (nm) (d, ppm)max–C(=S)NH– –C(SH)iN–

I H H H H C H NO S 245.30 181–183 4.2059 0.9387 2.26 295, 326 11.3013 11 2

II CH H CH H C H NO S 273.33 113–114 4.0824 0.8892 3.57 288, 321 11.283 3 15 15 2

III H H CH(CH )C H H C H NO S 301.41 139–140 4.2459 0.8119 5.36 395, 302, 329 11.283 2 5 17 19 2

IV F H H H C H FNO S 263.29 99–100 6.3951 2.7000 2.93 293, 331 11.5813 10 2

V H F H H C H FNO S 263.29 163–164 2.6903 3.4765 4.34 296, 325 11.0613 10 2

VI H H F H C H FNO S 263.29 183–184 4.6016 3.8620 2.94 297, 329 11.2413 10 2

VII F H F H C H F NO S 281.28 115–116 5.9481 3.3965 2.85 292, 330 11.4613 9 2 2

VIII Cl H H H C H ClNO S 279.74 95–96 5.5878 1.8583 2.74 292, 334 11.6713 10 2

IX H H Cl H C H ClNO S 279.74 177–178 4.2743 2.9562 3.91 298, 327 11.1313 10 2

X Cl Cl H H C H Cl NO S 314.19 175–176 6.6735 3.5246 4.63 295, 336 11.7213 9 2 2

XI Cl H H Cl C H Cl NO S 314.19 175–176 4.0432 0.9439 4.47 291, 338 11.7813 9 2 2

XII H Cl Cl H C H Cl NO S 314.19 163–164 3.6736 4.2452 4.92 294, 328 11.0213 9 2 2

XIII H Cl F H C H FClNO S 297.74 105–106 4.2057 5.0799 4.73 292, 329 11.1113 9 2

XIV Br H H H C H BrNO S 324.18 110–111 4.3680 0.8097 3.75 288, 292, 336 11.6013 10 2aXV H H I H C H INO S 371.20 198–199 0.0125 6.1891 4.40 284, 329 10.8413 10 2

XVI CH H H F C H FNO S 277.32 146–147 3.1872 3.6436 3.74 293, 334 11.463 14 12 2

XVII CH Cl H H C H ClNO S 293.78 193–194 5.2940 2.6104 4.40 284, 333 11.413 14 12 2

XVIII Cl H CH H C H ClNO S 293.78 161–162 5.6182 1.9502 4.65 293, 330 11.123 14 12 2

XIX H CF H H C H F NO S 313.30 164–165 5.5512 2.2724 3.20 300, 330 11.073 14 10 3 2

XX OCH H H H C H NO S 275.33 169–170 5.9406 2.1410 2.72 300, 334 10.653 14 13 3

XXI H H OCH H C H NO S 275.33 193–194 5.2076 3.5545 2.32 294, 328 11.353 14 13 3aXXII H OH H H C H NO S 261.30 177–178 2.6917 3.4508 1.02 300, 328 10.7213 11 3

XXIII CH H OH H C H NO S 275.33 213–214 4.8801 3.7071 1.59 289, 325 11.143 14 13 3

XXIV H H COOH H C H NO S 289.31 213–214 5.6977 6.6400 3.73 298, 338 11.0814 11 4

XXV H COOH OH H C H NO S 305.31 219–220 8.5135 7.7968 1.44 296, 323 11.2114 11 5

XXVI COOCH H H H C H NO S 303.34 187–188 8.0358 4.6277 3.70 295, 339 11.533 15 13 4

XXVII H H COCH H C H NO S 284.34 236–237 7.7285 6.4811 2.62 294, 346 11.033 15 13 3

XXVIII H H COC H H C H NO S 301.37 171–172 7.7123 6.4824 3.81 296, 334 11.022 5 16 15 3

XXIX H H CN H C H N O S 270.31 235–236 8.1500 4.7596 3.07 286, 339, 364 10.9414 10 2 2

XXX OH H NO H C H N O S 306.30 257–258 4.1340 1.3413 5.11 290, 327 10.532 13 10 2 5

XXXI H H CONH H C H N O S 288.33 245–246 7.7454 5.0308 1.41 300, 338 11.122 14 12 2 3

XXXII H H CONHCH CO H H C H N O S 346.35 141–142 5.8290 3.1612 1.23 298, 338 11.002 2 16 14 2 5

a 1Spectrum H NMR in CD COCD .3 3

J. Matysiak et al. / European Journal of Pharmaceutical Sciences 10 (2000) 119 –123 121

obtained from three independent measurements. The in- with the conventionally determined break point for suscep-vestigations were carried out in the Department of Pharma- tibility to topical antifungals, i.e., 15–25 mg/ml, and

ˇ ´ceutical Microbiology, Medical Academy, Lublin. exceptionally up to 100 mg/ml (Klimesova et al., 1996)substances X and XII inhibit in proper concentrations three(60%) and all species tested, respectively. The compounds

3. Results and discussion XX and XXXI exhibit a potent activity too, but onlyagainst one species Trichosporon cutaneum and

The chemical and physical data of the new compounds Cryptococcus neoformans, respectively, for which MIC5

are presented in Table 1. The results of in vitro screening 7.8 mg/ml. Derivatives XVI, XXII–XXV, XXVII, XXX,of 2,4-dihydroxythiobenzanilides are summed up in Table XXXII exhibit the poorest fungistatic activities with the2. MIC values of 125 to .1000 mg/ml for the five species

The data of inhibitory effects indicate that, depending on tested.the type of substitution, the obtained compounds are Considering the most active compounds, the sensitivitycharacterised by differentiated activities expressed in the of individual varieties of fungi is usually comparable andMIC values ranging from 7.8 to $1000 mg/ml. The only in the case of compounds XX, XXIX and XXXI arestrongest fungistatic activity is observed for some studied significant deviations (Trichosporon cutaneum, Cryptococ-compounds against Trichosporon cutaneum (MIC57.8 cus neoformans) observed.mg/ml). An especially high activity is exhibited by mono- Lipophility of the compounds (Table 1) was determinedand dichloro- (IX, X, XII), as well as by chloro-alkyl by HPTLC from the relationship between the R valuesM

(XVII, XVIII) derivatives for all six yeasts tested. Starting and the composition of hydro–organic mobile phase

Table 2aFungistatic activity of 2,4-dihydroxythiobenzanilides against yeasts

Compound Strain, MIC (mg/ml)

C. a. C. a. C C. n. G. c. T. c. T. sp.

I – 250.00 250.00 – 125.00 –II 62.50 125.00 62.50 125.00 – 62.50III 125.00 125.00 62.50 .125.00 – .125.00IV 125.00 250.00 125.00 250.00 – 62.50V – 62.50 62.50 62.50 31.25 –VI 125.00 125.00 62.50 125.00 – 125.00VII 62.50 125.00 125.00 125.00 31.25VIII 62.50 125.00 62.50 125.00 – 62.50IX 31.25 62.50 62.50 31.25 7.82 –X 15.63 31.25 15.63 31.25 – 15.63XI 62.50 62.50 62.50 125.00 – 15.63XII 15.63 15.63 31.21 31.21 7.82 –XIII 62.50 62.50 62.50 62.50 62.50 62.50XIV 62.50 62.50 62.50 125.00 – 62.50XV 62.50 62.50 62.50 62.50 – 31.25XVI 125.00 125.00 125.00 125.00 125.00 125.00XVII 31.25 31.25 31.25 31.25 62.50XVIII 31.25 31.25 31.25 31.25 31.25XIX 62.50 125.00 125.00 62.50 15.63 –XX 62.50 250.00 125.00 250.00 7.82 –XXI 62.50 .500.00 62.50 500.00 – 500.00XXII 250.00 250.00 250.00 250.00 – 250.00XXIII 250.00 500.00 500.00 500.00 – 500.00XXIV 500.00 500.00 125.00 500.00 – 500.00XXV 1000.00 1000.00 1000.00 .1000.00 – 1000.00XXVI 62.50 62.50 31.50 .250.00 – 62.50XXVII .500.00 .500.00 .500.00 .500.00 125.00 –XXVIII 250.00 250.00 62.50 .1000.00 – 125.00XXIX 250.00 250.00 15.63 .250 – 125.00XXX .500.00 .500.00 250.00 .500.00 250.00 –XXXI 125.00 250.00 7.82 .250.00 – 250.00XXXII – 500.00 250.00 500.00 62.50 –

a Abbreviations: C. a, Candida albicans; C. a. C, Candida albicans ATCC 10231 (strain isolated from the clinical material); C. n, Cryptococcusneoformans; G. c., Geotrichum candidum; T. c., Trichosporon cutaneum (beigen); T. sp., Trichosporon sp.

122 J. Matysiak et al. / European Journal of Pharmaceutical Sciences 10 (2000) 119 –123

(Barbaro et al., 1984; Biagi et al., 1994a,b). In all cases thelinear relationship described by the equation was obtained

R 5 af 1 R (1)M Mw

where a is the constant describing a given system and f

the molar fraction of the organic modifier. This equationpermits determination of the R value, i.e. the so-calledMw

hydrophobicity index, by extrapolation, even for sub-stances which do not migrate in water (Table 1). It is agenerally accepted way of expressing the lipophilic natureof molecules making it possible to neglect the selectiveinteractions with a modifier at the same time (Barbaro etal., 1984).

Investigating relations between lipophilicity and activityhave revealed theoretical R values determined for theMw

aqueous mobile phase with fungistatic activity of 2,4-dihydroxythiobenzanilides expressed by log MIC. For fourspecies studied the parabolic dependencies were obtained(compounds for which MIC values were not determinedaccurately were not included in the regression analyses), asexpressed the equations

Candida albicans2log MIC 5 0.050 (R ) –0.544 R 1 3.084 (n 5 25, r 5 0.703) (2)Mw Mw

Cryptococcus neoformans2log MIC 5 0.060 (R ) –0.628 R 1 3.277 (n 5 29, r 5 0.753) (3)Mw Mw

Trichosporon cutaneum2log MIC 5 0.239 (R ) –1.498 R 1 3.558 (n 5 9, r 5 0.721) (4)Mw Mw

Trichosporon sp.2log MIC 5 0.077 (R ) –0.748 R 1 3.522 (n 5 21, r 5 0.734) (5)Mw Mw

Fig. 2. Relations between the fungistatic activity of 2,4-dihydrox-ythiobenzanilides (log MIC) and the lipophilicity parameter (R ) forMwIt follows from the above that an optimal value of RMw Trichosporon sp. and Candida albicans ATCC 10231.

exists for which appropriately substituted compounds from2,4-dihydroxythiobenzanilides exhibit a maximal fungis-

log MIC 5 2 0.295 R 1 3.036 (n 5 22, r 5 0.847) (7)Mwtatic activity. The most optimal R values againstMw

Candida albicans are in the range of 4.0–5.0, againstThe presence of a linear dependence for those strainsCryptococcus neoformans 3.5–4.5, against Trichosporon

suggests the direction of further synthesis towards moresp. 4.25–4.75 (Fig. 2) and for Trichosporon cutaneum thelipophilic structures. However, it can be supposed that toowide range 2.5–4.0 exists. However, deviations frequentlyhigh an increase can cause a sudden decrease of biologicaloccur in compounds XXIV, XXVIII and XXX. Theactivity and a parabolic dependence, analogous to theactivity of compound XXXI with the –CONH substituent2previous ones can be obtained (Eqs. (2–5)). For thoseagainst Cryptococcus neoformans is also higher thanstrains, the range of optimum lipophility values would onlypredicted from the correlation of antifungal activity (ex-be shifted towards higher R values. Some compounds,pressed by means of log MIC) with lipophilicity of the Mw

most often with low lipophilicity (XXXI), exhibit signifi-molecule.cant deviations from the correlation equations. TheirFor Candida albicans ATCC 10231 and Geotrichumactivity probably depends on the power of molecule-activecandidum (Fig. 2) linear relationships between the activitycentre (receptor) interactions, but not on the transportand lipophilicity were obtained as expressed by followingdetermined by means of Requations, respectively Mw.

During the investigations of qualitative and quantitativelog MIC 5 2 0.280 R 1 3.015 (n 5 28, r 5 0.822) (6) relationships for the group of these compounds, theMw

J. Matysiak et al. / European Journal of Pharmaceutical Sciences 10 (2000) 119 –123 123

Biagi, G.L., Barbaro, A.M., Recanatini, M., 1994. Determination ofsecondary stereochemical effects caused by conformationlipophilicity by means of reversed-phase thin-layer chromatography.change resulting from the tautomeric rearrangementIII. Study of the TLC equations for a series of ionizable quinolone

–C(=S)–NH–↔–C(SH)iN– is of significant importance. derivatives. J. Chromatogr. 678, 127–137.It depends on the volume, electronic properties and Biagi, G.L., Barbaro, A.M., Sapone, A., Recanatini, M., 1994. De-location of substituents in the N-aromatic moiety. Especial- termination of lipophilicity by means of reversed-phase thin-layer

chromatography II Influence of the organic modifier on the slope ofly electron-donating substituents located on the ortho-the thin-layer chromatographic equation. J. Chromatogr. 669, 246–position make it difficult to explain interactions. Some of253.

these show a strong electron effect, probably connected Grupce, O., Petrov, I., 1984. Infrared spectra and polymorphism ofalso with the possibility of intramolecular hydrogen bonds thiobenzanilide. J. Mol. Struct. 115, 119–122.formation, others can protect only –C(=S)NH– bonds or Kaliszan, R., Foks, H., Janowiec, M., 1978. Studies on quantitative

structure–activity relationships in pyrazine carbothioamide derivatives.stiffen the molecule limiting the probability of amidePol. J. Pharmacol. Pharm. 30, 579–583.tautomery in this way (Grupce and Petrov, 1984; Sudha et

ˇ ´ ´ˇKlimesova, M., Otcenasek, M., Waisser, K., 1996. Potential antifungalal., 1985). The compounds with electron-donating sub- agents. Synthesis and activity of 2-alkylthiopyridine-4-car-stituents in the ortho-position with intramolecular hydro- bothioamides. Eur. J. Med. Chem. 31, 389–395.

ˇ ˇˇ ´ ´ ´gen bonds were characterised by high lipophilicity com- Kunes J., Szepessy S, Pour M., Waisser K., Odlerova Z., Kralova K.,Thiel W. N-(2-benzothiazolyl)benzenecarbothioamides as agents withpared to meta- and para-isomers (XXVI, XXX). Thesebiological activity. 2nd European Symposium on Antimicrobialmolecules, in which the amide system may form theAgents. Mechanisms of action and structure–activity relationships.

internal hydrogen bond often exhibit different solvation ´ ´Hradec Kralove 1998, Chech Republic. P-34:108.1 13abilities in relation to analogues. These interactions proba- Sudha, L.V., Manogarah, S., Sathyaharagana, D.N., 1985. H and C-

bly impose one on another only in some cases (X, XI, NMR study pyridylamides and thioamides. J. Mol. Struct. 129, 137–144.XVII, XVIII) causing a significant increase of R valuesMw ˇ´Waisser, K., Hrbata, J., Odlerova, Z., 1995. Relationships between thein this way. At the same time, the comparison of lipophili-chemical structure of substances and their antimycobacterial activity

ty of isomers indicates that local changes of the dipole against atypical strains. V. Thiobenzamides substituted in position 3 ormoment in the amide fragment due to the interactions of 4. Ces a Slov Farm 44, 187–189.

ˇ´ortho-substituents significantly influence the total value of Waisser, K., Odlerova, Z., Thiel, W., 1995. Relationships between thechemical structure of substances and their antimycobacterial activitydipole moment during the tautomeric rearrangement (Tableagainst atypical strains. VI. N,N-disubstituted dithiooxalamides. Fol.2) and association parameters, and as a result the chro-Pharm. Univ. Carol. 20, 13–16.

matographic parameters. ˇˇ ´ ´Waisser, K., Kimesova, M., Odlerova, Z., 1995. Relationships betweenIn conclusion, the presented results have proved that the chemical structure of substances and their antimycobacterial

2,4-dihydroxythiobenzanilides represent an important activity against atypical strains. VII. 2-Alkylthio-4-pyridinecar-bothioamides. Fol. Pharm. Univ. Carol. 19, 59–62.broad spectrum of antifungal compounds. The above

ˇ ´Waisser, K., Kimesova, M., Buchta, W., 1996. New group of potentialrelations may facilitate not only the elaboration of theantifungal agents: 2-alkylthio-4-pyridinecarbothioamides. Fol. Pharm.

model making it possible to predict some conditions of Univ. Carol. 20, 53–57.synthesis but also, after determination of R values, may ´Waisser, K., Houngbedji, N., Machacek, M., Sekera, M., Urban, J.,Mw

ˇ´indicate trends for biological investigations. Odlerowa, Z., 1990. Antimycobacterial thiobenzanilides. Collect.Czech. Chem. Commun. 55, 307–316.

´ ´ ´ ˇWaisser, K., Kubicova, L., Gregor, J., Budova, J., Androlova, A., Drsata,ˇ´J., Odlerowa, Z., 1998. Relationships between the chemical structure

References of substances and their antimycobacterial activity against atypicalstrains thiosalicylanilides. Ces a Slov Farm 47, 84–86.

ˇˇ ´ ´ ´Barbaro, A.M., Pietrogrande, M.C., Guerra, M.C., Cantelli Forti, G., Waisser, K., Kunes, J., Odlerova, Z., Roman, M., Kubicova, L., Horak,V.,Borea, P.A., Biagi, G.L., 1984. Relationship between the chromato- 1998. Antimycobacterial activity of 39- and 49-fluorothiobenzanilides.graphic behaviour of dermorphin-related oligopeptides and the com- Pharmazie 53, 193–195.position of the mobile phase in reversed-phase thin-layer chromatog-raphy: comparison of extrapolated R values. J. Chromatogr. 287,M

259–270.