In vitro evaluation of 2,4-dihydroxythiobenzanilides against various moulds

European Journal of Pharmaceutical Sciences 13 (2001) 243–248 www.elsevier.nl / locate / ejps

In vitro evaluation of 2,4-dihydroxythiobenzanilides against various moulds ~ Andrzej Niewiadomy*, Joanna Matysiak, Grazyna Ma˛cik-Niewiadomy Department of Chemistry, University of Agriculture, Akademicka 15, 20 -950 Lublin, Poland Received 28 July 2000; received in revised form 13 December 2000; accepted 4 January 2001

Abstract The antimycotic potency of 2,4-dihydroxythiobenzanilide derivatives was tested. The MIC assessments by an agar dilution method were used for the estimation of potential activity in vitro against the four mould strains: Scopulariopsis brevicalis, Aspergillus niger, Aspergillus fumigatus and Penicillium sp. The strongest fungistatic activity was observed for 39-fluoro-derivative (MIC 7.82 mg / ml). It was stated that the inhibition action of these compounds depends mainly on lipophilicity of molecules. Parabolic relationships between the antimycotic activity and lipophilicity were found.  2001 Elsevier Science B.V. All rights reserved. Keywords: 2; 4-Dihydroxythiobenzanilides; Anti-moulds activity; In vitro study; MIC; Lipophilicity

1. Introduction One of the most essential problems in the chemotherapy not solved yet is treatment of systemic mycosis. Systemic candidiasis, aspergillosis, histoplasmosis and cryptococcosis are still a serious problem. All available antimycotic substances are not sufficiently effective and are characterized by high animal toxicity. The need for new effective and nontoxic antimycotics results from the spread of HIV infections which has contributed to a greater number of immunocompromised patients during the last few decades. Another problem is appearance of the fungus strains with multi-drug resistance (MDR) (Borowski, 1998). Therefore, there is still a need for new substances of great effectiveness and low toxicity. Thiobenzanilides are a promising group of compounds of a wide range of activities including antifungal (Kubicova´ et al., 1998; Waisser et al., 1998a) and antimycobacterial activities (Kunesˇ et al., 1997; Waisser et al., 1990, 1993, 1998b). The synthesis of thiobenzanilides with 2,4-dihydroxybenzenecarbothioacyl moiety and the differently modified N-aryl fragment was carried out in our laboratory. The obtained compounds are characterized by ´ ~ «o et al., inhibitory effect against dermatophytes (Rozyl 1999; Matysiak et al., 2000a), yeasts (Matysiak et al.,

*Corresponding author. Tel.: 148-81-445-6097; fax: 148-81-5333752. E-mail address: [email protected] (A. Niewiadomy).

2000b) and phytopathogenic fungi (Niewiadomy et al., 1998, 1999a). Specially high inhibitory activity in relation to dermatophytes was stated with MIC values 1.9 mg / ml in relation to Epidermophyton floccosum (Matysiak et al., 2000a). In the preliminary toxicological studies acute toxicity was determined for several compounds. The values LD 50 , depending on the kind of N-aryl ring substitution are in the range 239–840 mg / kg (manuscript in preparation). One of the most essential quantities describing physicochemical properties of the molecule in a complex way its lipophilicity (hydrophobicity). This is commonly accepted parameter used to characterize xenobiotic responsible for distribution, activity, toxicity and other processes (Ong et al., 1996). The problem consists in the choice of technique and the model system: the conventional shake-flask partition coefficient, chromatographic methods in the reversedphase system, theoretical calculations. Therefore numerous papers have been published concerning the liquid chromatographic procedures for hydrophobicity determination and the correlation of the data obtained with the logarithm of the partitioning system, log P. The ultimate goal of such efforts is the search for optimal conditions of both stationary and mobile phases with regard to hydrophobicity of a compounds to be estimated (Hsieh and Dorsey, 1995). In our laboratory lipophilicity of compounds by mean of chromatography in the reversed phase system was examined. The correlation between lipophilicity of thiobenzanilides determined by RP-18 HPLC or RP-8 HPTLC techniques and the activity of individual fungus strains was

0928-0987 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 01 )00126-9

A. Niewiadomy et al. / European Journal of Pharmaceutical Sciences 13 (2001) 243 – 248

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found (Matysiak et al., 2000a,b; Niewiadomy et al., 1999b; ´ ~ «o et al., 1999). Rozyl The paper presents the changes of fungistatic activity of 29 compounds of 2,4-dihydroxythiobenzanilides group caused by the effect of substitution in the aniline moiety. The investigations were carried out under in vitro conditions for four strains of mould fungi. The attempts were made to find the relation between activity and structure with particular regard to lipophilicity.

2. Materials and methods Compounds were obtained in S E reaction of new thioacylating reagent bis(2,4-dihydroxybenzenecarbo-

thion)thionyl with properly substituted aromatic amines (Niewiadomy et al., 1999b). Using the dilution method the minimal inhibitory concentration (MIC) of individual compounds against four fungi strains (moulds) has been determined. These were either reference strains of known sensitivity to antifungal drugs or the strains isolated directly from the clinical material. Microorganisms were multiplied on the slants developed from the Muller–Hinton agar containing 4% glucose (pH 5.6) and from the analogous Muller–Hinton broth. The tested compounds were dissolved in methanol. Different amounts of solutions were added to the precisely measured, dissolved and cooled to 458C agar medium, and then mixed and emptied onto Petri plates. The medium of more and more decreasing concentration ranging from 1000 to 3.9 mg / ml was obtained. The medium containing

Table 1 The structure and fungistatic activity of 2,4-dihydroxythiobenzanilides in relation to moulds a

Compound

I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX XXI XXII XXIII XXIV XXV XXVI XXVII XXVIII XXIX

Substituents

R Mw

–R2

–R3

–R4

–R5

H CH 3 H F H H F Cl H Cl Cl H H Br H CH 3 CH 3 Cl H OCH 3 H H CH 3 H H H OH H H

H H H H F H H H H Cl H Cl Cl H H H Cl H CF 3 H H OH H COOH H H H H H

H CH 3 CH(CH 3 )C 2 H 5 H H F F H Cl H H Cl F H I H H CH 3 H H OCH 3 H OH OH COCH 3 CN NO 2 CONH 2 CONHCH 2 CO 2 H

H H H H H H H H H H Cl H H H H F H H H H H H H H H H H H H

2.26 3.57 5.36 2.93 4.34 2.94 2.85 2.74 3.91 4.63 4.47 4.92 4.73 3.75 4.40 3.74 4.40 4.65 3.20 2.72 2.32 1.02 1.59 1.44 2.62 3.07 5.11 1.23 1.36

Tested moulds (MIC, mg / ml) Asp. f.

Asp. n.

Penic. sp

Sc. brev.

125 62.5 125 125 31.25 125 125 62.5 62.5 31.25 31.25 31.25 62.5 125 62.5 125 62.5 15.63 62.5 125 500 125 500 .1000 .500 .250 500 .250 .250

125 125 .125 250 62.5 250 125 125 62.5 62.5 62.5 31.25 62.5 125 62.5 250 125 62.5 125 125 .500 500 500 .1000 .500 250 .500 .250 500

125 125 .125 125 31.25 125 62.5 250 31.25 31.25 125 62.5 62.5 125 125 125 62.5 62.5 31.25 62.5 .500 500 500 1000 500 250 62.5 .250 500

62.5 62.5 .125 62.5 7.82 125 125 62.5 31.25 31.25 31.25 31.25 31.25 31.25 31.25 62.5 62.5 31.25 62.5 125 250 250 500 .1000 500 250 500 250 250

a Asp. f.: Aspergillus fumigatus, Asp. n.: Aspergillus niger, Penic. sp.: Penicillium sp., Sc. brev.: Scopulariopsis brevicalis. R Mw values were taken from Matysiak et al., 1999.

A. Niewiadomy et al. / European Journal of Pharmaceutical Sciences 13 (2001) 243 – 248

0.5 ml of the substance had also 5% of methanol. After solidification the plates were dried, and after spraying the 0.02 ml culture (10 4 cfu of fungi) the plates were incubated for 2–10 days at 228C. At the same time the sensitivity of the strains to methanol was determined. The presented results were obtained from three independent measurements. Lipophilicity of the compounds (R Mw ) was determined by means of the thin layer chromatography in the reversed phase system HPTLC RP-8 (Matysiak et al., 1999) (Table 1). Calculations of molecular surface area, volume and energy of compounds for two tautomeric forms –C(=S)– NH–↔–C(SH)iN– were carried out with an ALLCHEME 2000 program.

3. Results and discussion The structures of 2,4-dihydroxythiobenzanilide derivatives are given in Table 1. Most of the chemical and physical data of the tested compounds were presented earlier (Matysiak et al., 2000a,b). Some theoretically calculated descriptors are given in Table 2. The results of in vitro screening of 2,4-dihydroxythiobenzanilides against four strains of moulds are sum-

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med up in Table 1. The data of inhibitory effects indicate that depending on the type of substitution, the obtained compounds are characterised by differentiated activities against moulds expressed in MIC values ranging from 7.8 mg / ml to $1000. The strongest fungistatic activity is exhibited by 39-fluoro derivative (V) for which MIC57.8 mg / ml in relation to Scopulariopsis brevicalis. Compounds X, XII and XVIII show also potent activity. The inhibition action of these compounds is comparable and is at the level 31.25 mg / ml. Derivatives XXI–XXIX exhibit weak fungistatic activity with MIC values $250 mg / ml, except for compound XXVII against Penicillium sp. for which MIC562.5 mg / ml. Compound XXIV with MIC values $1000 mg / ml is the least effective towards all tested strains. S. brevicalis was the most susceptible strain to the studied antifungal agents and Aspergillus niger was the most resistant. Searching for the relations between lipophilicity and activity, log MIC of the tested compounds for individual strains was compared with lipophilicity expressed by R Mw . In all cases there were obtained parabolic dependences described by the equations: For Penicillium sp. strain: log MIC 5 0.0878(R Mw )2 2 0.802R Mw 1 3.707 (n 5 29, r 5 0.746)

(1)

Table 2 Theoretical descriptors: volume, surface area and energy obtained for two tautomeric forms of 2,4-dihydroxythiobenzanilides Compound

I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX XXI XXII XXIII XXIV XXV XXVI XXVII XXVIII XXIX

˚ 3) Volume (A

˚ 2) Surface area (A

Energy (kJ)

–C(=S)NH–

–C(SH)iN–

–C(=S)NH–

–C(SH)iN–

–C(=S)NH–

–C(SH)iN–

209.95 244.23 276.95 214.12 214.25 213.39 218.23 224.7 223.13 238.01 238.18 237.72 227.81 231.56 239.07 231.02 240.61 241.15 239.3 235.89 235.08 218.17 234.49 244.27 245.63 226.63 238.23 239.24 283.85

209.95 244.76 277.94 214.28 214.90 214.11 217.99 224.58 223.84 238.67 238.64 237.67 228.18 231.46 239.48 230.95 240.74 240.94 239.81 235.3 235.52 218.5 235.69 244.97 246.58 227.6 237.84 240.21 284.31

252.03 296.05 335.46 258.47 256.96 258.25 261.56 267.79 269.66 282.75 284.88 284.43 271.2 275.82 280.21 276.74 282.81 288.83 288.3 281.81 280.2 262.08 280.08 293.04 296.15 273.34 287.88 289.74 342.7

254 300.93 343.76 259.5 258.77 260.51 264.2 270.34 268.41 287.41 287.28 288.63 272.52 278.89 280.72 284.72 290.79 294.56 290.8 289.26 286.86 267.06 283.44 295.16 296.69 276.27 286.73 293.6 350.64

30.63 33.88 33.09 32.37 30.91 31.72 32.43 32.2 31.24 31.84 32.31 30.92 30.97 31.69 30.87 32.92 32.69 33.31 35.86 31.52 29.91 32.56 35.12 40.56 32.41 32.27 50.65 45.49 46.13

22.3 25.08 25.47 22.25 22.51 22.99 21.95 22.18 22.49 21.52 22.12 22.19 21.94 22.02 22.13 23.89 23.47 23.77 27.34 22.3 21.89 24.41 26.01 30.89 23.22 23.24 39.85 37.43 37.97

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For Scopulariopsis brevicalis:

For Aspergillus niger:

log MIC 5 0.077(R Mw )2 2 0.778R Mw 1 3.557

log MIC 5 0.036(R Mw )2 2 0.502R Mw 1 3.441

(n 5 28, r 5 0.737)

(2)

(n 5 28, r 5 0.768)

(3)

Fig. 1. Relations between fungistatic activity of 2,4-dihydroxythiobenzanilides (log MIC) and the lipophilicity parameter (R Mw ) for Aspergillus fumigatus and Scopulariopsis brevicalis.

A. Niewiadomy et al. / European Journal of Pharmaceutical Sciences 13 (2001) 243 – 248

For Aspergillus fumigatus: log MIC 5 0.082(R Mw )2 2 0.894R Mw 1 4.056 (n 5 27, r 5 0.826)

(4)

The regression equations did not take into account compound XXVII which confirms the parabolic dependences (activity decrease for lipophilicity R Mw .5) but decreases correlation significantly. As follows from the above dependences, lipophilicity of the most active derivatives of 2,4-didydroxythiobenzanilide group towards Aspergillus niger and Scopulariopsis brevicalis is found in the range 3.75–5.0 and towards Penicillium sp 3.2–4.7 (Eqs. (1)– (3); Fig. 1). For Aspergillus fumigatus a very narrow range of optimal lipophilicity 4.25–5.0 (Eq. (4); Fig. 1) was obtained. The analysis of these dependences indicates specific conditions in the structure of cellular walls and membranes as high lipophilicity (R Mw .5) does not promote probably fungistatic activity. The compounds are bonded in the lipid layers of cellular membranes and may not reach the activity sites. Parabolic relationships were

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also obtained for all tested strains of dermatophytes but with higher optimal values R Mw (Matysiak et al., 2000a). In the case of yeasts for most tested strains these were quadratic dependences, though in some cases linear relations were found in the studied range of lipophilicity (Matysiak et al., 2000b). Distinct deviation from the lipophylicity–activity dependence is observed for compound XXII particularly towards Aspergillus niger. This effect often occurs in the compounds of low lipophilicity whose inhibitory action is much stronger than it follows from the general equation. A different activity mechanism can be predicted for these compounds because molecules of high value dipole moments can change character of lipo-protein membrane cohesion as a result of dispersion force interferences. This also refers to the compounds with substituents which can form hydrogen bond. In the group of compounds under investigation the attention should be paid to the possibility of tautomeric rearrangement of the type –C(=S)–NH–↔–C(SH)iN– which makes the quantitative description of the structure–

Fig. 2. Two equilibrium structures: amido-thionic (a) and imido-thiol (b) for 39,49-dichloro-2,4-dihydroxythiobenzanilide (XII) and for 39-trifluoromethyl2,4-dihydroxythiobenzanilide (XIX).

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A. Niewiadomy et al. / European Journal of Pharmaceutical Sciences 13 (2001) 243 – 248

activity complicated. The existence of two equilibrium structures (Fig. 2) is confirmed mainly by means of IR spectrum analysis and their probability depends on properties and position of substituents (Matysiak et al., 1999; Petrov and Grupce, 1984). Theoretical calculation indicate a general tendency for transition to the imido-thiol form described by lower values of energy than corresponding amido-thionic structures (Table 2). Thiol structure domination can be attributed to derivatives where energetic gains resulting from intramolecular rearrangements are relatively high. The hypothesis can be made that the change of molecule structure may take place at the time of penetration into the cellular membranes and the energy evolved in this way is used during the transport. The volume changes connected with the tautomeric transition are insignificant (Table 2) and probably do not affect penetration significantly. The rearrangement is usually accompanied by the molecule surface area increase. Assuming the molecular mechanism of activity of compounds this effect is believed to be advantageous because it increases possibility of non-specific interactions with the receptor. As no close relations between activity and theoretical descriptors describing the molecule are found, it can be stated that of the studied parameters lipophilicity has the decisive influence on activity. Chromatographic studies of the derivative series enable switching synthesis to the systems of defined lipophilicity, potentially most active towards individual strains.

References Borowski, E., 1998. Molecular aspects of rational design of novel antifungal agents of low toxicity. In: Proceedings of the 2nd European Symposium on Antimicrobial Agents. Mechanisms of Action and ´ ´ Czech Republic, Structure Activity Relationships. Hradec Kralove, PL-7. Hsieh, M., Dorsey, J.G., 1995. Bioavailability estimation by reversedphase liquid chromatography: high bonding density C-18 phase for modelling biopartitioning processes. Anal. Chem. 67, 48–57. ´ L., Buchta, V., Kunes, ˇ J., Machack, ´ Kubicova, M., Waisser, K., 1998. 3and 4-Fluorothiobenzanilides as antifungal substances. In: Proceedings of the 2nd European Symposium on Antimicrobial Agents. Mecha´ ´ nisms of Action and Structure Activity Relationships. Hradec Kralove, Czech Republic, pp. 150–151, P-56.

ˇ J., Jachym, ´ ´ ´ Z., Waisser, K., 1997. Kunes, J., Jirasko, P., Odlerova, Combination of Toplis approach with the free-Wilson analysis in the study of antimycobacterial activity of 4-alkylthiobenzanilides. Collect. Czech. Chem. Commun. 62, 1503–1509. Matysiak, J., Niewiadomy, A., Ma˛cik-Niewiadomy, G., 2000a. Dependence of fungistatic activity of 2,4-dihydroxythiobenzanilides on the structure and lipophilic nature of the compounds. Eur. J. Med. Chem. 35, 393–404. Matysiak, J., Niewiadomy, A., Ma˛cik-Niewiadomy, G., 2000b. Inhibition in vitro properties of a new group of thiobenzanilides in relation to yeasts. Eur. J. Pharm. Sci. 10, 119–123. ´ ~ «o, J.K., 1999. StrucMatysiak, J., Niewiadomy, A., Zabinska, A., Rozyl ture and retention of 2,4-dihydroxythobenzanilides in a reversed-phase system. J. Chromatogr. 830, 491–496. Niewiadomy, A., Matysiak, J., Ma˛cik-Niewiadomy, G., 1999a. The relationship between fungistatic activity of thiobenzanilides and their lipophilicity expressed in term of TLC parameters. Pestycydy 1–2, 21–28. Niewiadomy, A., Matysiak, J., Macik-Niewiadomy, G., 1999. Nowe ´ thioamidy, produkt posredni do otrzymywania nowych thioamidow, P330263. ~ ´ ´ ~ «o, J.K., Senczyna, B., Niewiadomy, A., Matysiak, J., Zabinska, A., Rozyl ´´ Jozwiak, K., 1998. Reversed-phase high-performance liquid chromatography in quantitative structure–activity relationship studies of new fungicides. J. Chromatogr. 828, 431–438. Ong, S., Liu, H., Pidgeon, Ch., 1996. Immobilized-artificial-membrane chromatography: measurements of membrane partition coefficient and predicting drug membrane permeability. J. Chromatogr. 728, 113–128. Petrov, I., Grupce, O., 1984. Amide and thioamide bonds of benzanilide and thiobenzanilide in the vibrational spectra. J. Mol. Struct. 115, 481–484. ~ ´ ~ «o, J.K., Zabinska, Rozyl A., Matysiak, J., Niewiadomy, A., 1999. Reversed-phase thin-layer chromatography with different stationary phases in studies of quantitative structure–biological activity relationship of new antimycotic compounds. J. Assoc. Off. Anal. Chem. Int. 82, 1–7. ´ Waisser, K., Houngbedji, N., Machacek, M., Sekera, M., Urban, J., ´ Z., 1990. Antimycobacterial thiobenzanilides. Collect. Odlerova, Czech. Chem. Commun. 55, 307–316. ´ L., Dostal, ´ H., 1998a. Biological effects of Waisser, K., Kubicova, substances similar to salicylanilides: thiobenzanilides. Folia Pharm.Univ. Carol. 23, 59–66. ´ L., Odlerova, ´ Z., 1993. Antituberculotic 49Waisser, K., Kubicova, cyclohexylthiobenzanilides: combination of free-Wilson method in QSAR with Topliss approach. Collect. Czech. Chem. Commun. 58, 205–216. ˇ J., Odlerova, ´ Z., Roman, M., Kubicova, ´ L., Horak, ´ V., Waisser, K., Kunes, 1998b. Antimycobacterial activity of 39- and 49-fluorothiobenzanilides. Pharmazie 53, 193–195.