RESEARCH ARTICLES /BİLİMSEL ARAŞTIRMALAR
in Vitro Antimicrobial Activities of Some 5(6)- Substituted-2-Hydroxymethylbenzimidazoles and Their Platinum (il) Complexes
Öztekin ALGÜL*, Ufuk ABBASOGLU**, Fatma GÜMÜŞ*0
ln Vitro Antimicrobial Activities of Some 5(6)- Substituted-2-Hydroxymethylbenzimidazoles and Their
Platinum (il) Complexes
Summary : in this study, in order to evaluate the in vitro an- timicrobial.activity differences of the ligands and their Pt(II) complexes, and the role of the coordination metal ion, in vit- ro antimicrobial activities of some 5(6)- nonl or chloro/ or methly substituted -2-hydroxymethylbenzimidazoles and their Pt(ll) complexes were tested against some gram- positive and gram-negative bacteria and yeast like fungi.
The test results have shown that most of the compounds test- ed had MIC values of 50 µg lmL against the microorganisms used and tlıe poor antibacterial and antifungal activities of the conıpounds can not be considered noteworthy. A com- parative study of the MIC values indicated that, in general, there were no reasonable differences between the in vitro antimicrobial activity values of the ligands and their Pt(II) complexes against the tested microorganisms.
Key words: 2-Hydroxymethylbenzimidazale, Platinum (Il) . complexes, in vitro antimicrobial activities Received
Revised Accepted
15.06.1999 15.09.1999 15.09.1999
INTRODUCTION
Much attention has been paid to platinum com- plexes, due to their probable antitumor activities after Rosenberg's discovery of the antitumor properties of cis-Pt(NH3)ıC12 (cisplatin) in 19691.
Althongh cisplatin is a cli'nically important anti- tumor drug used in the treatınent of testicular, ovarium, bladder, head, and neck cancers2, its wide- spread use is limited by inherent resistance by ac- quired drug resistance and its major side effects3-4_
Bazı 5(6)-Sübstitute -2 -Hidroksimetilbenzimidazoller ve
bunların Platin (il) Komplekslerinin İn vitro Antimikrobiyal Aktiviteleri
Özet : Bu çalışmada, ligandlar ve bunların Pt(ll) komp- lekslerinin antimikrobiyal aktivite farklarını ve ko- ordinasyon metal iyonunun rolünü değerli:ndirmek amacı ile
bazı 5( 6 )-non/-kloro/-metil sübstitüe-2-hidroksinıetil
benzimidazoller ve bunların platin(!!) komplekslerinin bazı granı-pozitif, gram-negatif bakterilere ve maya benzeri fun- guslara karşı in vitro antimikrobiyal aktiviteleri denendi.
Test sonuçları, bileşiklerin büyük bir bölümünün kullanılan
mikroorganizmalara karşı 50 µglmL MfK değerine sahip ol-
duklarını ve bileşiklerin zayıf antibakteriyel ve antifungal aktivitelerinin önenıli kabul edilemeyeceğini gösterdi. MiK
değerlerinin karşılaştırmalı çalışması, genellikle, ligandlar ve bunların platin(!!) komplekslerinin test edilen mik- roorganizmalara karşı in vitro antimikrobiyal ak- tivitelerinde önemli bir fark olmadığını gösterdi .
Anahtar kelimeler: 2-hidroksimetilbenzimidazol, Platin (il) kompleksleri, in vitro antimikrobiyal aktivite
The need for cisplatin analogs which are less toxic and have a broader spectrum of activity has led to the synthesis of a large number of platinum com- plexes over the past two decades. Replacement of the NH3 groups by cyclic amines generally reduces the toxicity of the platinum complexess.
The benzimidazole nucleus is found in a variety of naturally occuring compounds such as vitamin B12 and its derivaties, and it is structurally similar to purin bases. Furthermore, benzimidazoles are
*
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University 06330 Etiler Ankara/ TüRKİYE.**
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Gazi University 06330 Etiler Ankara/ TÜRKİYE.°
CorrespondenceAlgül, Abbasoğlu, Gümüş
known to exhibit a wide variety of pharmacological properties including antimicrobial6, antitumor activ- ity7·8. and inhibition of nucleic acid synthesis9.
It was reasoned that, by combining the intrinsic an- titumor ·activity of certain benzimidazoles with that of platinum, it might be possible to obtain com- pounds with ·superior chemotherapeutic index in terms of increase bioavailability, higher cytotoxicty and lower side effects !han cisplatin. We have cho- sen Pt(II) complexes of 5(6)-substituted-2- hydroxymethlybenzimidazoles for this purpose.
in a previous paperlD, we described the synthesis and characterization of complexes of the structure, cis-[Pt(L)zC12]•2H20, where L is 5(6)-non/or- chlorosubstitued - 2 - hydroxymethylbenzimidazole.
We also evaluated the ability of these compounds to damage ONA or interfere with ONA replication by determining their cytotoxic effects on the repair pro- ficient E.coli strain AB1157 (Rec A + ) and repair deficient mutanı AB1157 (Rec K ). We have also characterized the ONA binding properties of the compounds and the affinity of ONA modified by these compound to the HMG- domain proteinll.
The ONA platinated with these compounds was specifically recognized by HMGl.
in this study, in order to evaluate the in vitro anti- microbial activity differences of the ligands and
'
their Pt(II) complexes, and the role of the co- ordination metal ion, in vitro antimicrobial activities ofa series of 5(6)-H/ or -Cif or -CH3-substitued-2- hydroxymethylbenzimidazole and their Pt(II) com- plexes mentioned above were screened.
MATERIAL AND METHODS
Synthesis of the ligands and their Platinum(II) com- plexes ıo
All ligands were synthesized according to the Phil- lips method12. Ali the Pt(II) complexes were syn- thesized by using K2PtC14 and the corresponding li- gands.
The chemical structures of Pt(II) complexes of the benzimidazole derivatives were characterized by their elemen tal analyses da ta and their IR and 1 H- NMR spectra comparing with those of the ligands.
Chemical structures of ali the ligands and their Pt(II) complexes are presented at Figurel.
Microbiological Methods
The ligands and their Pt(II) complexes, mixtures of the ligands with K2PtC14 were assayed for the anti- microbial activity studies. Tetracycline and clo- trimazole were used as standard.
CH20H
H-NAN---1-PtCI,
\
0 : R=H (Compound 1) L2: R=Cl (Compound 2) ı}: R= CH3 ccompound 3)
Q
R
2
R=H (Compound 4) R=CI (Compound 5)
Figure 1. Chemical Structure of the Ligands and Their Pt(II) Complexes.
.2Hı0
(Compound 6)
Test Microorganisms and Culhıre Media
In vitro antibacterial activities of the synthesized compounds and tetracycline were tested against some gram-positive (Staphylococcus aureus ATCC 25923, Streptococcus faecalis ATCC 19433) and gram-negative (Escherichia cali ATCC 25922, Pseu- domonas aeruginosa ATCC 27853) bacteria by mi- crodilution method13 in Muller-Hinton Broth and in vitro antifungal activity of the compounds and clo- trimazole were tested against some yeast-like fungi (Candida albicans, Candida stellatoidea, Candida parapsilosis, Candida pseudotropicalis) by micro- dilution method14 in Sabouraud Liquid Medium.
in vitro Antiınicrobial Assays
0.1 mL Mueller-Hinton Broth and Sabouraud Liquid Medium were dropped into each well of the micro- titer plates. Each of the compounds tested and the standarts, tetracycline and clotrimazole, were dis- solved in DMSO (Dimethyl sulfoxide) at 1600 µg/mL concentrations. The two-fold dilutions of the com- pounds (400, 200, .... ,3.124 µg/mL) were made by dispersing the stock solutions to the remaining wells. Suspensions of microorganisms at.106 CFU / mL
concentration were inoculated to the two-fold di- luted solution of the compounds.
Minimum inhibitory concentrations (MIC's) were defined as the lowest concentration of the com- pounds that inhibited visible growth of micro- organisms after incubation at 36 °C far 18 hours lor the bacteria and 48 hours far the yeast-like fungi in the incubator. it was determined that the solve.nt had no actimicrobial activity against any of the test microorganisms. MIC values of the compounds are presented in Table 1.
RESULTS AND DISCUSSION
Far comparison, the ligands and their Pt (il) com- plexes and mixtures of the ligands with K2PtC14 were assa yed in the antimicrobial activity studies.
The MIC values of 50 µg/mL were observed far the bacteria tested far almost ali the tested materials.
The MIC values far the test~d materials are shown in Table 1. We also faund that the mixture of tetra- cycline with K2PtC14 presented MIC values of 200 µg/mL far all the bacteria tested. This suggested that tetracycline may form coordination bond with Table 1 · in vitro Antimicrobial Activity of the Tested Compounds
MiNiMUM INHIBITORY CONCENTRATION (MIC) (µglmL)
Comp. No Compd Eco/i P.aeruginosa S.aureııs S.faeca//s C.a/bicans C.slEl/atoidea C.parapsilosis C.pseudotropicalis
1 L1 50 50 50 50 50 100 100 100
2 L2 50 50 50 50 50 100 100 100
3 L3 50 50 50 50 50 100 100 100
4 [ML1Cl,]2H,O 50 50 50 50 100 100 100 50
5 rML 2el,]2H,O 50 50 50 50 100 100 100 50
6 [ML3cl,] 100 50 50 50 100 100 100 100
L1+K,PtCI, 50 50 50 50 50 50 50 50
L2+K,PtCI, 50 50 50 50 50 50 50 50
L3+K,,PtCL 50 50 50 50 50 50 50 50
K,PtCI, 100 50 50 50 50 50 50 50
Tetracycline+ 200 200 200 200 - - - -
KzPtCl4
Clotrimazole+ - - - - 200 200 200 200
KzPtCl4
T etracycfıne 12.5 25 25 6.25 - - - -
Clobimazole - - - - 3.125 3.125 3.125 3.125
Algül, Abbasoğlu, Gümüş
K2PtC14 in medium, and lose their ligand prop- erties.
Lack of the differences in the antibacterial activities between !he ligands and their Pt(II) complexes have indicated that observed weak antibacterial activities (MIC values of 50 µg/mL) may not be due to the li- gand properties alone. Additional comments may be made that substituents at position 5 do not have substantial role in the activity of these molecules.
According to !he antifungal activity test results, ali the ligands were more effective on C albicans than their Pt(II) complexes. Compounds 4 and 5 had higher activity against C pseudotropicalis than ali the ligands and Compound 6. The mixture of the ligands with K2PtCl4 presented higher activity against ali yeast-like fungi except C albicans, when compared to !he ligands alone and their Pt(II) com- plexes except for Compounds 4 and 5 for the ac- ticvity on C pseudotropicalis . These increase in the activity may be due to K2PtCl4 in !he mixtures.
The MIC values of Clotrimazole was 3.125 µg/mL for the yeast-like fungi tested. However, the MIC · value for the mixture of Clotrimazole with K2PtC14 was 200 µg/mL. It was suggested that K2PtC14 may be coordinated with the nitrogen of imidazole ring in the clotrimazole structure and !his results in the loss of the activity.
Although, !he affinity of platinum for sulfur donor ligands makes D.M;SO unsuitable for use in bio- logical studies and the results of biologically related experiments employing this solvent, must be viewed with caution15, as previously used in !he lit- erature we used DMSO as the dissolving reagent due to solubility problems of the Pt(II) complexes in the other solvents. For this reason, our findings have only suggestive importance for further in- vestigations.
in conclusion, the test results have shown that most of the compounds tested had MIC values of 50 µg/mL against the microorganisms used and the poör anti- bacterial and antifungal activities of !he compounds
can not be considered noteworthy. The results also suggest that ligand properties of the compounds may not play substantial roles on !he antimicrobial activity of these compounds against the micro- organisms used.
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