FABAD J. Pharnı. Sci., 25, 113-119, 2000
BİLİMSEL TARAMALAR/ SCIENTIFIC REWIEVS
Antioxidant Activities of Indole and Benzimidazole Derivatives
Sibel SÜZEN*0, Zeynep ALAGÖZ', M. Orhan PÜSKÜLLÜ'
Antioxidant Activities of lndole and Benzimidazole Derivatives
Summary : There is a possible role of free radicals such as superoxide anion, hydroxyl anion and hydrogen peroxide in the etiology of important diseases such as atherosclerosis, nephritis and carcinogenesis. The diıtioxidants and anti- oxidant enzyme systems are the major protective systems of the organism. However, environmental chemicals can de- crease the antioxidant level of the organism and thus lead to cancer and various diseases. Hence, synthetic antioxidant compounds are required /rom external sources. There are a number of studies which report that indole and ben- zimidazole derivatives have antioxidant activity, and this plays an important role in the immune system. Herein have been reported the recent developments of the antioxidant ac- tivities af indole and benzirnidazole derivatives.
Keywords : Antioxidant, Free radical, Indole, Ben-
Revised Accepted
zimidazole 13.07.1999 25.01.2000 25.01.2000
INTRODUCTION
Free radicals are chemically reactive species that can attack and damage several biomolecules such as DNA and enzymes. Oxidative damage to DNA by reactive oxygen species is a continuous problem that cells must guard against to survive. Reactive oxygen species include singlet oxygen, superoxide anion, hydrogen peroxide and hydroxyl radical.
Damage to some enzyrnes can also have a major negative impact on the cel!sl,2.
Because free radicals are chemical constituents that
İndol ve Benzimidazol Türevi Bileşiklerin Antioksidan Aktiviteleri
Özet: Arterosikleroz, nefrit ve kanser gibi önemli hastalıklann etiyolojisinde süperoksit anyon, hidroksil anyonu ve hidrojen peroksit gibi serbest radikallerin rolü bulunmaktadır. An- tioksidanlar ve antioksidan enzimler organiwıanın en büyük koruma sistemleridir. Buna rağmen çevresel kimyasallar or~
ganizmanın antioksidan düzeyinde düşmeye neden olarak kan- ser ve çesitli hastalıklara yol açabilirler. fndol ve benzinıidazol türevi bileşiklerin antioksidan aktiviteye sahip olduğu ve immun sistemde önemli rol oynadığını gösteren çalışmalar vardır. Bu makalede indol ve benzirnidazol türevi bileşiklerin
antioksidan aktiviteleri ile ilgili yeni gelişmeler yer almaktadır.
Anahtar kelimeler : Antioksidan, Serbest radikal, fndal,
Benzinıidazol
have an unpaired electron in their orbit, they are highly reactive. Inspired oxygen is harmful since up to 5% of the oxygen taken in is converted to oxygen- free radicals. The addition of a single electron to ox- ygen produces the superoxide anion radical that is catalytic-reduced by superoxide dismutase. Al- though H2ü2 is not itself a free radical, it can be tox- ic at high concentrations and, more importantly, it can be reduced to the hydroxyl radical which is the most toxic of the oxygen based radicals and it wreaks havoc within cells, particularly with macro- molecules3A. This mechanism can be explained by the Fenton reaction (Figure 1).
* Ankara Üniversitesi, Eczacılık Fakültesi, Farmasötik Kimya Anabilim Dalı, 06100 Tandoğan, Ankara
° Correspondence
Süzen, Ala göz, Püsküllü
Fenton Reaction Figure l. Fenton Reaction.
F +3
e
+
OH +OHPharrnacological agents that maintain normal intra- cellular CaZ+ levels and inhibit cellular oxidative stress may be effective in blocking abnorrnal neuro- nal apoptosis. It is known that free radicals and ox- idative stress-induced neuronal celi death have been implicated in various neurological d.isorders in- cluding neurodegenerative diseases. On the other hand the role of nitric oxide (Nü), is discovered only a decade ago, is now a central feature of mod- els describing a variety of body functionsS,6.
it is known that lipid peroxidation is a free radical chain reaction7 which causes the degradation of celi membranes. Most products of lipid peroxidation are known to have mutagenic and/ or cancerogenic propertiesB. Active oxygen species are produced by different mechanisms9,!0. Cytochrome P450 de- pendent enzymes which metabolise xenobiotics and certain endogenous substances have very important roles in the production of active oxygen speciesn Free radicals are implicated as causative agents in various forms of tissue destruction. Considerable circumstantial evidence suggests that oxygen-based free radicals generated in the blood flow return to formerly ischemic brain areas and are mainly re- sponsible for the neurodegeneration that follows pe- riods of cerebral ischemialZ.
IMPORTANT REACTIVE OXYGEN SPECIES Well over 90% of the oxygen that enters human cells is used for the production of energy by mit- ochondrial cytochrome oxidase. During this pro- cess, four electrons are added to each Üz molecule resulting in the formation of two molecules of wa- ter. An estimated 1-4 % of the Üz taken into cells, however, forms a partially reduced Üz species, the reactive oxygen species, some of which contain an unpaired electron and are therefore referred to as free radica!sl3. For Üz to oxidise a molecule directly it would have to accept a pair of electrons in 02
°
The two electrons forming a covalent bond in a molecule would not meet this criterion since they have spins opposite to one another, Thus the re- action of Üz with non-radical species which con- stitutes most molecules in cells is severely limited by this spin restriction.
1. Superoxide Anion Radical (SAR)
Within biological systems, 02 usually accepts elec- trons one at a time resulting in superoxide anion radical, hydrogen peroxide and hydroxyl radical as intermediates. The intermediates have various de- grees of reactivity with non-radical species. The ac- ceptance of a single electron generates the SAR. it is generally held !hat SAR is not highly reactive to- wards biological substrates in an aqueous environ- ment. Moreover, once formed, SAR quickly under- goes dismutation to generale hydrogen peroxide.
2. Hydrogen Peroxide
HzOz itself is not especially toxic unless it is in high concentrations within cells. Besides the reactive oxy- gen species, several other enzymes that generate Hz02 also exist in human tissues. HzOz readily dif- fuses through cellular membranes and can thereby distribute to sites distant from where it was gener- ated. it is reduces to hydroxyl radical.
3. Hydroxyl Rad.ical
The hydroxyl radical is not the only destructive spe- cies but its formation is well documented due to its ability to oxidise adjacent molecules. There is uni- versal agreement that, once formed, the hydroxyl radical reacts rapidly with any molecule because of its high reactivity. Hydroxyl rad.ical readily dam- ages nuclear and mitochondria DNA, membrane lip- ids and carbohydrates.
Besides these reactive oxygen species singlet oxygen
<10z) which is formed under photooxidative condi- tions and peroxyl radical (L00°) can be considered as a toxic species.
ANTIOXIDANT ACTMTY OF INDOLE DERIVATIVES
Melatonin, which is an irnportant indole derivative,
FABAD J. Phnrm. Sci., 25, 113-119, 2000
is a hormone secreted by the pineal glandin re- sponse to photoperiods and influences many im- portant biological processes14. It is a hydroxyl rad- ical scavenger and antioxidant, and plays an im- portant role in the immune system15. Melatonin (Figure 2) has been suggested to be a very effective antioxidant in the brain16. This neurohormone may be useful in the prevention of neurodegenerative diseases. The action of melatonin is highly pleio- tropic. It involves firstly direct effects, via specific binding sites in various peripheral tissues and cells of vertebrates, including immunomodulation; sec- ondly, systemic influences on the cytoskeleton and calmodulin mediated Nü formation, and thirdly, antioxidative protection, perhaps alsa in the context of protection in plants and unicells.
Many new lines of evidence implicate both super- oxide anion radical and biogenic amine neu- rotransmitters in the pathological mechanisms !hat underlie neuronal damage caused by metam- phetamine, glutamate-mediated oxidative toxicity, ischemia-reperfusion, and other neurodegenerative brain disorders. Oxidation of 5-hydroxytryptamine (5-HT, serotonin) (Figure 2) by an oxygen gener- ating system such as the xanthine produces tryp- tamine-4,5-dione. When the reaction is carried out in the presence of intraneuronal nucleophile glu- tathione, tryptamine-4,5-dione is scavenged to give 7-(S-glutathionyl)tryptamine-4,5-dione, whereas the putative carbocation intermediate is scavenged to give 4-(S-glutathionyl)-5-hydroxytryptaminel7.
This raises the possibility !hat tryptamine-4,5-dione and perhaps other putative intraneuronal me- tabolites formed by the oxygen/H20 2/ oxo-iron me- diated oxidations of serotonin might be endotoxins that contribute to neurodegeneration in brain re- gions innervated by serotonergic neurons caused by ischemia-reperfusion, and other neurodegenerative brain disorders.
CH2CH2NHCOCH3
cı-ı
10~
UNJ
Melatonin Serolonin
Figure 2. Chemical formula of melatonin and se- rotonin
The "nitric oxide-pathway" is presumed to be in- volved in acetylcholine and serotonin mediated vas- odilatation. In addition, both the serotonin induced transient and persistent vasodilator responses in the forearm vascular bed are abolished by the receptor antagonist [lH]-indol-3-carbonic acid tropine ester HCl18. in another study it was found !hat the effect ofa naturally occurring heterocyclic amine 3-amino- l,4-dimethyl-5H-pyrido[4,3-b]indol (Figure 3) might function as a tropic factor in contrast to its neuro- toxic effect in higher doses19_
NH
1
~ N
CHı
NH2
Figure 3. 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol Zhang et aı20 isolated 5-hydroxyindole derivatives from saff!ower. Antioxidant activities of the com- pounds were measured by the ferric thiocyanate method and compounds were found to have rel- atively strong antioxidant activity. A new anti- nephritic drug 6-lH-indol-3-yl-methyl)-5-methoxy-3- (2-methylpropyl)-2-(lH) - pyrazinone - 4 - oxide showed scavenging ability against hydroxyl radica12ı.
lndole-3-acetic acid is a plant hormone whose deg- radation is a key determinant of planı growth and development22 . Hyperoxides degrades pre- dominantly non-enzymatically to oxindol-3-yl car- binol (Figure 4) but in part enzymatically into indol- 3-yl methanol via a peroxidase eyde in which indol- 3-yl acetic acid acts as an electron donar.
W C H , C O O H
NH
(a)
~CHıOH
~NH_J,O
(b)
Figure 4. (a) Indole-3-acetic acid, (b) Oxindol-3-carbinol Lipid peroxidation of mitochondrial and celi mem- brane structure is the final step in the oxygen rad- ical-induced damage observed at reperfusion of kid- neys after ischaemia. Comparison of the ability of an indeno-indol compound with !hat of alpha- tocopherol to inhibit lipid peroxidation showed !hat the compound was 100 times more efficient than al- pha-tocopherol (Figure 5)23_
Süzen~ Ala göz~ Püsküllü
Figure 5. Chemical formula of alpha-tocopherol ANTIOXIDANT ACTIVITY OF BENZIMIDAZOLE DERIVATIVES
Substances such as irnidazoles, benzimidazoles and also quinolines (Figure 6), whose chemical structure includes a heterocyclic nitrogen, are known to inter- fere with microsomal oxidation and, in some cases, with the metabolisrn of drugs24.
(a)
~',/
~ .. LJJI
NH(b)
co
N"'
(c)
Figure 6. (a) lmidazole, (b) Benzirnidazole, (c) Quin- oline
Recently a retinoid type compound, 2-[(5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphtalene)] - 5- carboxybenzimidazole (BITN) (Figure 7) was found effective on hepatic cytochrome P450 dependent ethoxyresorufin 0-deethylase (EROD) and pen- toxyresorufin 0-depentylase (PROD) enzyme activ- ities25. These enzymes activate polycyclic hydro- carbons, aromatic arnines and aliphatic halogenated hydracarbons to their ultirnate mutagenic or can- cerogenic forms, and are effective in producing re- active oxygen species such as superoxide, hydroxyl radical and H 20 2. This compound has a greater ac- tivity that all trans retinoic acid. Also sulfur con- taining benzirnidazoles (thiabendazole, 5-hydroxy- thiabendazole) and sulfur-free derivatives (ben- zimidazole and carbendazirn) have an effect on monooxygenase activities26. Thiabendazole, cam- bendazole and carbendazim led to a significant dose-dependent increase in both EROD activity and cytochrome P4501Al/2 proteins and m RNA ex- pres·sion.
c~ V,...~~~COOH
CH~
~COOH ..~
~ NH
CH, CHı Cll_ı
,,) (b)
Figure 7. (a) Retinoic acid, (b) BITN
2-Phenyl benzirnidazole (Figure 8) and the common sunscreen consistent 2-phenylbenzimidazole-5- sulfonic acid function in DNA damage27 when they are exposed to UV-B (290-320 nm) radiation or nat- ura! sunlight. Although neither compound binds specifically to DNA, both are active and induce the formation of piperidine-labile cleavage sites that map almost exclusively to the positions of guanine residues. This characteristic behaviour irnplies that electron transfer from DNA to the photoexcited sen- sitizer is the predominent mechanism in this con- formation. A sirnilar resctivity toward denaturated
aı;ıd native DNA has been reported for riboflavin and certain pterin derivatives. The pattern of attack on single-stranded DNA, where all guanines are modified to a simi!ar extent, is typica! of photo- oxidation by singlet oxygen. The involvement of singlet oxygen is consistent with the effect of scav- engers on the radiation.
RYjıN"'·~
~N~~
R: H 2-Phenyl benzimidazole
R: S O 3H 2-Phenyl benzimidazole-5-sulfoniı.: acid
· Figure 8. 5-substituted 2·phenyl benzimidazole An investigation ofa number of benzimidazole class preparations, distinguished by a position of amino- methyl substitutes, has been carried out by Mil el
af28. it has been shown that having one substitute in position 2 causes the formation of inter-molecular cross-links in DNA and chromatin. It has been sug- gested that an UV-formation of superoxide radica!
in the presence of these molecules might be a reason of DNA-macromolecule destruction.
Adult worms of Acanthocheilonema vitae were found to be susceptible to the reactive oxygen inter- mediates (ROI) generated by the xanthine-xanthine oxidase (X-XO) system. The damage caused by this system was completely abolished by superoxide dismutase (SOD) and catalase but not by mannitol.
The results, therefore, suggest that superoxide an- ions oxygen and hydrogen peroxide alone or in
FABAD J Pharm. Sci., 25, JJ3-119, 2000
combination might be toxic to the fariid. A. vitae showed an ability to protect itself against the ox- idants in the presence of an active enzyme. SOD and catalase were present in high levels of activities and appeared to costitute the major defence system.
The role of glutathione peroxidase (GPx) was found less important due to the weak activities of glu- tathione peroxidase (GPx) was found less important due to the weak activities of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH). A. vitae also released SOD, catalase and GPx in the medium, which appear useful in pro- tecting the flariid against ROI generated by the host in the immediate surroundings of the parasite. Anti- filarial agents, diethylcarbamazine (DEC) and 2,2'- dicarbomethoxylamino-5,5'-dibenzimidazolyl ke- tone (Figure 9) appreciably inhibited catalase and GPx of A. vitae. Inhibition of these enzymes appears to render the parasite prone to H202 toxicity, lead- ing to death. No adverse effect on antioxidant en- zymes of liver and lungs were recorded as a result of exposure to 2,2'-dicarbomethoxylamino-5,5'- dibenzimidazolyl ketone suggesting the nontoxic nature of the compound. On the other hand the ef- fect of 2,2'-dicarbomethoxylamino-5,5' di- benzimidazolyl ketone on the metabolism of re- active oxygen species has been measured and el- evated levels of antioxidant enzymes and reduced glutathione were observed38. 2,2' - Dicarbometh- oxylamino-5,5'-dibenzimidazolyl ketone markedly inhibited catalase and glı..tathione peroxidase31 in another stud y.
o o NHD~Nll il H
3
CO~HN--{ j j >-NHCOCH3N ~ c --"' N
o il
Figure 9. Chemical formula of 2,2'- dicarbomethoxylamino-5,5'-dibenzimidazolyl ketone To understand the mode of anthelminthic action of thiabendazole and methyl-[5-[[ 4-(2-pyrimidinyl)-1- piperazinyl] carbonyl]-lH- benzimidazole - 2- yl]carbamate (Figure 10) against Nippostrongylus brasiliensis, their effect on the metabolism of re- active oxygen species in the parasite as well as in rat intestine was examined by Srivastava et af32. Both compounds produced a significant depression in the level of superoxide dismutase and reduce glu-
tathione. Release of antioxidant enzymes was also found to be appreciably lowered.
0 - - 0 N H
?ı
C
N\_ N ' i N \ _ J j \ N-C il \ .--"' N )-NHCOCH3Figure 10. The chemical formula of methyl-[5-[[4(2- pyrimidinyl)-1-piperazinyl]carbonyl]-lH-
benzimidazole-2-yl]carbamate
In vitro potencies of a series of benzimidazoles bear- ing the carboxyl group both in ring and chain sys- tems on NADPH- dependent lipid peroxidation was observed33_ Significant inhibition on lipid per- oxidase levels was observed at ıo-3 and 10-4 M con- centrations. The results showed that the compounds depending on the tissue have variable effects on cy- tochrome P450 which are alsa known to be express- es and regulated differentially in various tissues by numerous xenobiotics34.
Benzimidazoles which are known to be patent in- hibitors far hepatic mixed-function oxidases35, are the compounds related with P450 depending on the substitution and the chemical nature of sub- stitutents. The ability of imidazole derivatives to in- hibit some cytochrome P450 dependent monooxy- genase activities had been studied36. The results showed that the ability of compounds to interact with cytochrome P450 depends to a large extent on their lipophilic character. The derivatives of ben- zimidazoles containing additional fused and non- fused aromatic groupings were effective inhibitors of aryl hydrocarbon hydroxylase and aminopyrine N-demethylase activities in hepatic microsomes36_
These results indicate that the precise structure of benzimidazole derivatives can determine the bind- ing type observed in oxidised microsomes. lt is pos- sible therefore, that more than a single mode of in- hibition of monooxygenase activity is occurring and inhibition, as well as binding type, is dependent on structure.
CONCLUSION
Organic nitrogen compounds are found in ali living
Süzen/ Alagöz/ Püsküllü
organisms in the form of proteins, genetic material, hormones, vitamins, ete.
Drug possessing antioxidant and free radical scav- enging properties are considered far preventing and/ or treatment of such diseases which are direct- ly related to the lack of the antioxidant capacity of the organism. Investigations of this type seem es- pecially relevant considering the great increase in longevity that humans are experiencing. These re- cent developments about the antioxidant activity of indole and benzimidazole derivatives draw atten- tion to necessity and importance of the studies to- wards the understanding of possible antioxidant ac- tivity of these compounds.
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