ELECTROCHEMICAL OXIDATION OF SOME BIOLOGICALLY IMPORTANT HETEROCYCLES AND THEIR PRECURSORS

Abstract

Nitrogen and nitrogen-sulphur containing hetero cyclic molecules are found extensively in biological systems and have been chosen by nature to be involved in many of the fundamental reactions of living organisms. Due to the extensive delocalized it -electron system these compounds are generally good electron donors and/or acceptors which allows very many biologically important heterocyclic molecules to be studied by various electrochemical techniques. Because of the involvement of these molecules in biological electron transfer processes, it is likely that many of the electrochemical reaction routes and intermediates will be similar to the physiological electron transfer processes There is, however, sufficient superficial similarity between the electrochemical and biological reactions, which is not duplicated in other chemical systems. Therefore electrochemical studies can yield an enormous amount of evidence regarding the mechanistic pathways of bio logical electron transfer reactions. A group of com pounds of this type consists of purines in which the enzymatic and electrochemical oxidations are very similar. In view of such an importance of electrochemical investigations in providing biologically significant information, the electrochemical behaviour of some biologically important heterocycles and their precursors, have been examined at pyrolytic graphite electrode. The work embodied in this thesis has been divided into the following chapters: CHAPTER -I General Introduction CHAPTER-II Electrochemical Oxidation of Ethyl 3-oxo-3phenyl-2-phenylhydrazonopropionate. This chapter of the thesis describes the electro chemical oxidation of ethyl 3-oxo-3-phenyl-2-phenylhydrazono propionate (I), a precursor for the synthesis /COC6H5 NH —N =c' \cOOC2H5 (I) of biologically important compounds, at pyrolytic graphite electrode (PGE) in phosphate buffers and B.R.buffers of pH range 3.0-11.0. The experimental evidences suggested that the compound was oxidised in a single 2e, 2H+ pH dependent step. The Ep versus pH plot exhibited two breaks at around pH 5.8 and 8.0 which corresponded topK and pK of compound I. The peak current values were practically constant in the entire pH range. The peak current function ip/v was independent of scan rate in the range 50-400 mVs- . The ratio of ipa/ipc was practically constant at different scan rates and was close to 0.80. The electrooxidation under cyclic voltammetric conditions indicated the presence of_ an ECE system., spectral studies indicated the formation of different products at pH 3.0 and 7.0. Products of electrooxidation afc pH 3.0 were isolated and character ised by TLC, IR and gas chromatography as phenol and ethyl 3-phenyl-2,3- dioxopropionate. At pH 7.0, the formation of phenyldiimide was proposed together with phenol and ethyl 3-phenyl-2,3-dioxopropionate. CHAPTER-III : Mechanism of Electrooxidation of 6- Thiouric Acid at Pyrolytic Graphite Electrode. The third chapter of the thesis deals with the electrochemical oxidation of 6-thiouric acid (II), an important purine derivative formed as major meta bolite from 6-thiopurine. A detailed electrochemical investigation of this compound exhibited an interesting behaviour in phosphate buffers of pH range 2.0-11.0. Altogether different route was followed by this compound (II) when compared with uric acid oxidation under similar experimental conditions. 6-Thiouric acid exhibited a single pH dependent voltammetric oxidation peak. The peak current was linearly dependent on concentration of the depolariser upto about 0.7mM and at higher concenOH H (III) In cyclic sweep voltammetry, at a scan rate of 200 mVs-1, two well defined oxidation peaks were observed. Cathodic peak, in the reversed scan, was visible only at pH < 6.0. The peak current versus concentration plot could be approximated as a straight line below concen tration 0.25mM, whereas at higher concentrations the peak current values were more or less constant suggest ing strong adsorption of 2-thioxanthine at the surface of PGE. The increase in peak current function with logarithm of sweep rate further supported the involve ment of adsorption. The number of electrons involved in oxidation were determined for both the peaks independ ently and found to be four and one respectively. The observed spectral changes indicated the formation of UV absorbing intermediate generated during the course of electrolysis. The final products of electrooxidation of 2-thioxanthine were characterised at pH 3.18 and 7.0 .TLC of the electrolysed solution indicated the formation of two products, one being urea as was confirmed by comparison with an authentic sample. The second component was identified as 2-thio-4,5,6-trioxopyrimidine. SH H H x (ID trations the values of peak current .were more or less constant. This behaviour indicated the involvement of adsorption in the electrode reaction. The formation of disulphide linkage was established in one of the products of electrooxidation. The mechanism proposed was supported by spectral studies, coulometric studies and product characterisation. The reason of difference in electrochemical behaviour of uric acid and 6-thiouric acid is believed to be due to mercapto group which can easily undergo oxidation loosing le , 1H to give the free radical which combines with another free radical to give a dimer. CHAPTER -IV : Electrochemical Behaviour of 2-Thioxanthine at PGE . In the fourth chapter of this thesis electro oxidation of 2-thioxanthine (III) is reported at pyro lytic graphite electrode in the pH range 2.0-11.0. A plausible mechanism has also been suggested to ration alize the observed behaviour. CHAPTER - V : Elucidation of the Oxidation Chemistry of 2-amino-4-phenylthiazole Using Elec±rochemical Techniques The anodic oxidation of aromatic amino compounds by electrochemical methods has been the subject of investigation for a long time leading to the formation of dimer or polymeric compounds. In this chapter, a detailed electrooxidation behaviour of a heterocyclic amino compound, viz., 2-amino-4-phenylthiazole (IV) is presented. In linear and cyclic sweep voltammetry, NH2 (IV) 2-amino-4-phenylthiazole exhibited a well defined anodic peak. The peak potential of this peak was dependent on pH and was found to shift cathodically with increase in sweep rate. The peak current function (ip/v ) was found to be constant with scan rate indicating the irreversible nature of the electrode reaction. Controlled potential electrolysis of compound IV at PGE gave n=2.0 + 0.15 in the entire pH range studied. The pKa value was determined by plotting absorbance at \ aqainst max 3 pH and was found to be 4.6. 2-Amino-4-phenylthiazole exists in two different forms (cationic and neutral species) below and above pKa. Cyclic voltammograms monitored during the electrolysis of solution clearly indicate that electrooxidation of IV gives an electroactive product, which undergoes reduction in the entire pH range 2.0-11.0. Thin layer chromatography confirmed the formation of a single product which exhibited a strong absorption band at 1530 cm in IR spectrum clearly indicating the presence of -N=N- linkage in the molecule. The mass spectral studies supported the azo structure of the product. A tentative mechanism is also proposed to account for the experimental results obtained. CHAPTER -VI :Studies on the Electrooxidation Behaviour of 2-Mercaptobenzothiazole. The last chapter of the thesis describes the electrochemical investigation of 2-mercaptobenzothiazole (V) , an important compound in pharmacology and rubber 8 industry. This compound undergoes electrooxidation at PGE in two well defined peaks. In the reverse sweep two cathodic peaks were also noticed. The peak potentials of all the peaks were linearly dependent on pH. Involve ment of adsorption at the electrode surface was confirmed by the change in peak current with concentration and increase in peak current function (ip/v ' ) with sweep rate. Two pKa values of compound V indicated that different forms of this compound exist in solution at different pH. Cyclic voltammograms and UV spectra were recorded during the course of controlled potential electrolysis to obtain the information about electroactive intermediates or products generated during the course of electrode reaction. The products of electro oxidation corresponding to both the peaks have been isolated and identified. Formation of dimeric disulphide and monosulphide was confirmed. A plausible mecha- nism has also been suggested for the oxidation of this compound. Chapter I GENERAL INTRODUCTION Electrochemistry, an important branch from analyti cal viewpoint, which deals with the interactions at elec trode-solution interphase, has been extensively employed to investigate the oxidation/reduction behaviour of electroactive ( capable of accepting or donating electrons) molecules or compounds for a long long time. It finds various applications in the medicinal and industrial chemistry besides being a source of energy in batteries and fuel cells [1] . Elucidation of reaction mechanism of the' electrode process is considered to be as one of the most significant advances in electrochemistry by its various techniques such as polarography and cyclic voltammetry. Though d.c. polarography was the first tool to be employed in majority of investigations earlier but since the information obtain able from it provided limited basis for characterisation of the nature of the charge transfer process, the use of more powerful technique ,cyclic voltammetry for the purpose of electrochemical studies, has also been implemented. Cyclic voltammetry, the most frequently chosen perturbation technique, is considered as the most effec tive and versatile one to examine the nature and the pathway of an electrochemical reaction in detail and also for detection and characterisation of coupled reac tions. The past two decades have witnessed the application of this technique from infancy to a state of incipient 10 maturity. Cyclic voltammetry provides important qualita tive as well as quantitative information about the ulti mate products of electrode reaction. In recent reports from this laboratory [2-5] it has been demonstrated that cyclic voltammetry in combination with controlled poten tial coulometry and spectroelectrochemistry affords a better deal of information about an electroactive inter mediate .generated chemically or electrolytically at elec trode surface, in order to explore the exact mechanistic pathway of electrode reaction. The electrochemical investigation of biologically important compounds can be carried out under an extra ordinary large number of conditions. Its importance is further enhanced, if the studies are carried out under conditions close to physiological system. For instance, an investigation at pH close to 7.0 provides more relevant information about the behaviour at physiological pH [6]. Thus, studies concerning redox chemistry of biologically important compounds using electrochemical techniques and related methodologies have the potential for providing valuable information to elucidate the mechanistic pathway ,f the electrode process. Studies on electrochemical behaviour of biologically important catecholamines,nitrofurazones[ 7-9], antiherpes uridine [10], antibiotic cephalothin [11], benzodiazepines [12], phenothiazines [13] etc. represent this example. Electrochemical studies can not only provide mechanistic information of funda- Vc Ol 11 mental interest but can also give valuable insight into the chemical aspects of enzymic and in-vivo redox reac tions of such compounds. An excellent example of virtually identical mechanisms of electrochemical and enzymic oxida tions of uric acid, 9-methyl uric acid and guanine has been reported in the literature by Dryhurst and coworkers [14-16] . Nitrogen containing heterocyclic molecules, which are most abundant in living organisms, are of much interest from the oxidation/reduction point of view due to the presence of extensive delocalized tt electrons which in turn make these compounds good electron donors and/or acceptors. Keeping this view in mind certain biologically important heterocyclic molecules, having nitrogen and/or sulphur s heterocyclic atoms, and their precursors have been selected for the detailed electrochemical investigations with a hope that the extensive studies of these molecules would shed a considerable light on their fundamental redox behaviour. Hydrazones are well known precursors of very many biologically important N-heterocyclic compounds and have found wide applications in synthetic and medicinal chemi stry. Various derivatives of hydrazones, viz., alkylamino, dialkylamino and arylamino acetylhydrazones were found to have significant analgesic and antiinflammatory activity [17]. Benzoquinolizines [A] containing hydrazono group have been patented as a remedy for lowering blood pressure a 12 and some of its substituted analogs have been claimed RNNHR2 as antiulcer agents having antisecretory activity [18]. The effect of cytostatic and immunosuppressive methyl hydrazones [B] was studied in vitro by Mauer and cowork ers [19] on myleco and lymphopoiesis. Aminoguanidine t CH=NNMeCH2CH2Cl2 (B) derivatives of hydrazones and their salts were employed as antifouling agents [20] for marine use. Phosphorous acid hydrazide on treatment with various ketones and aldehydes gave hydrazones which inhibited the growth of leukemia [21]. Various aliphatic and aromatic hydra zones containing N,N,N',N'-bis (1,2-ethanedyl) phosphoric diamide moiety have been shown to possess anticancer activity [22]. Some arylhydrazones of arylhalides and arylhydrazides of carboxylic acid were found to be anti microbial [23]. Garg et al. [24] synthesised a large number of potential antidiabetic 3-methyl-4-arylhydrazono13 2-isoxazolin-5-ones. Chloro substituted arylhydrazones of mesoxalic acid seminitrile hydrazide [c] have been titively tested for fungicidal activity in-vitro against pos: i.\ H0H-.C •NHN =C(CN)CONHNH2 (0 18 fungal species and the results were encouraging [25]. It was observed that 2-chloro-, 3-chloro-, 4-chloro-, and 3,4-dichlorophenylhydrazono compounds completely inhibited the reproduction of Trichophyton and Epidermophyton. Hydrazones of alkylglycidyl acetoacetates (D) and fluoro aralkyl/diaryl ketones have also been reported CH2CH =NNHPh (D) to be active against various fungal species [26,27]. Hydrochloride salts of l-nitro-l-arylhydrazono-2-substituted aminoethanes [E] have been satisfactorily used in controlling brown rust of wheat, powdery mildew of V ^ >-NHN = (E) CCH2N X N02 cucumber and phytophthora of tomatoes [28]. Antiviral tivity, against influenza Hz N^ virus, ac poliomyelitis H virus and group-4 adenovirus in various cell culture systems, was shown by arylhyrazones of triphenylphosphonium salts [29]. Many hydrazones have been successfully employed for the detection of metal cations [30] and as powerful chelating agents [31]. Various chromium and cobalt com plexes of hydrazones [32] have been used in dye industry for woolen and nylon fibres. Due to the aforesaid importance of hydrazones, an important precursor of antineoplastic pyrazoles [33], viz., ethyl 3-oxo- 3-phenyl-2-phenylhydrazonopropionate has been studied electrochemically at pyrolytic graphite electrode in Britton-Robinson and phosphate buffer media. The results of these studies are presented in Chapter II of this thesis. Purines, N-heterocyclic molecules, are building blocks of DNA and RNA, and play an important role in medicinal chemistry. Many of their derivatives are effect ively used to cure various ailments. Purines when substi tuted with a -SH group have been foundto play a significant role in medicinal chemistry. 6-Thiopurine is one of the most important drugs available for the treatment of var ious leukemic and neoplastic conditions [34]. A large number of purines and purine nucleosides, having -SH moiety have shown significant and reproducible activity against various rodent neoplasms [35]. 6-Mercaptopurines are used extensively in the treatment of human acute 15 lymphotic leukemia (ALL) [36]. A number of derivatives of 6-thiopurine have been evaluated against Adenocarsinoma 755, a tumor usually sensitive to purine antagonists. 6-Thiopurine ribonucleoside showed significantly higher therapeutic index [37]. Out of a series of6-(4-carboxyalkyl thio ) purines, the best member 6-(4-carboxybutyl thio) purine(F) has been used in human for in-vivo activity S(CH2UCOOH (F) [38]. Bioavailability and antileukemic effect of 6-thio purine may be decreased by the coadministration of cortimoxazole [39]. Besides anticancerous activity, 6-thio purine has also been found to be active against various other diseases. 6-Mercaptopurine and 6-thioguanine dis played antiviral activity against leukemogenic Rauscher and Friend viruses [40]. It has been effectively used as immunosuppressant for allergic encephalomyelitis in inbred chicken lines [41], as antimetabolite to inhibit experimental tumor in mice [42,43], and as a model to study muscular dystrophy in rat's skeletal muscle fibres [44]. The action of 6-thiopurine involves most of the parameters that are likely to be encountered with an enzyme inhibitor in-vivo including activation (lethal 16 synthesis), multiple enzyme inhibition, cell membrane permeability, resistance, latentiation, and cell selecti vity [45]. 6-Thiouric acid is an important purine deriva tive found as a major metabolite of 6-thiopurine in bact eria, mice and man [46]. Chapter third of the thesis describes the results of the electrochemical oxidation of 6-thiouric acid at the PGE. Another purine derivative, xanthine ( 2,6-dioxopurine) discovered by Marcet [47] as a constituent of the bladder stones, has also been found in tea [48], cow's milk, human urine, and other miscellaneous biological sources [49,50]. Various xanthine derivatives are success fully used for the treatment of various skin diseases [51,52] and asthmatic conditions [53,54]. Xanthines are the degradation products of the primary nucleic acid purines i.e., adenine and guanine [55] and are also found in retinal fluid of rabbit [56]. Literature survey revea led that most of the electrochemical studies on xanthines are restricted to N-methylated derivatives, due to their high therapeutic value in various ailments [57-59], and essentially nothing is known about their thio derivatives. 2-Thioxanthine, being an important compound in cancer chemotherapy [60], has been subjected to electrochemical investigations, and results are summarised in Chapter fourth of this thesis. The nitrogen and sulphur containing heterocyclic systems are of considerable interest due to their signi- 17 ficant pharmacological and industrial applications. A large number of such compounds have been found to be anaesthetics [61], antituberculous [62],anticancerous [63], and insecticides [64], etc. Thiazoles , another important class of nitrogen and sulphur containing heterocyclic systems, have been the subject of exhaustive chemical scrutiny since long. Penicillin, thyamine, and sulphathiazole are some of the most important com- pounds which contain thiazole nucleus [65-67]. A wide variety of arylthiazoles, with antineo plastic activity, were synthesised by Garg and Sharma [68]. 2-Amino-2-thiazoline and its copper complexes have showed antiinflammatory activity [69]. 2-Benzyl-4- (4-pyridyl) thiazole and its derivatives are useful immunoregulators in controlling transplant rejection and treating autoimmune and chronic inflammatory diseases [70]. Hydrazinothiazolopyridine and 2-phenyl-4-methyl- 5-carboxythiazole have also been used as remedy for this ailment [71,72], the activity of the latter being com parable to that of phenyl butazone. Guanidinothiazole and its derivative, viz., 2-guanidino-4-(2-methyl-4-imidazolyl \ thiazole dihydrobromide are useful in treatment of hyperaciditv and peptic ulcers [73,74]. Gastric antiulcer and cytoprotective properties of 2-(3,4-dimethoxyphenyl)-5- 18 methyl thiazolidin-4-ones were comparable to that of cimetidine in rats [75]. Alkyl and naphthyl derivatives have been reported to inhibit gastric secretion by anta gonising histamine hydrogen receptor [76]. A series of substituted 5-oxo-5H-thiazolo [2,3-b] quinazoline carboxylic acids were evaluated by LeMahieu and coworkers [77] and it has been reported that compound R = isopropyl group, exhibited significant antiallergic activity [G ] N'-[5-Arylazo-4-phenyl-2-thiazolyl ]-N-arylsulphonyl ureas (H) showed hypoglycemic activity [78]. 2-Aminothiazole-5- sulphonamide (I) showed diuretic activity [79]. NH2N S02NH2 (I) Amongst the antituberculous thiazole derivatives, 2-aryl- 3_(4-methoxybenzamido)-5-carboxymethyl-4-thiazolidinones and organometallic complexes of thiazole with Rh and Pt have been found to possess significant cytostatic 19 and antitumor activity against Mycobacterium tuberculo sis [80,81]. 2,4-Diamino-5-phenylthiazole has been claimed as a potent analpetic and good antidote for barbiturate poisoning [82]. A large number of thiazoles have been found to show antimicrobial activity, viz., 4-phenyl-2-guanidylthiazole and various derivatives (alkyl and naphthyl) of thiazoles, the latter being active against Aspergillus fumigatus, Aspergillus niger, Staphylococcus aureus, and Escherchia coli [83]. 2-Amino- 4-nitrothiazole possesses antitrichomal activity [84] and 2-amino-4- methylthiazole -5-carboxylic acid exhi bited protective effect against experimental alcohol poisoning [85]. Certain thiazoles act as covulcanizates [86] and photographic sensitizers [87] in rubber and photographic industries respectively. Thiazole ring is resistant to oxidation/reduction reactions in normal conditions, although in drastic conditions the oxidation of methylthiazoles by peracids is reported to give N-oxide derivatives [88]. A survey of literature revealed that amino group in benzenoid derivatives readily undergoes electrooxidation to give a mixture of products. The electrochemical oxidation study on 2-amino-4-phenylthiazole was undertaken to investigate the effect of thiazole ring on the oxidation of amino group and the results are presented in Chapter V of this thesis. Benzothiazole, a condensed ring system of thiazole 20 and benzene, is of great significance in various fields of chemistry. Heavy metal complexes of 2-mercaptobenzothiazole were investigated with regard to their effect on Mycobacterium tuberculosis, Trichophyton gypseum, Torulopsis minor and Staphylococcus aureus [89], 2-Mercaptobenzothiazole itself inhibits Staphylococci or the Pleuropneumonia organisms [90]. Singh and Vaid [91] synthesised and tested the antiinflammatory acti vity of some 2-(4-butyl-3,5-dimethylpyrazole -l-yl)-6- substituted benzothiazoles and 4-butyl-l-(6-substituted- 2-benzothiazolyl)-3-methyl pyrazol-5-one and found them to show significant activity. 2-Alkylthiobenzothiazole inhibited the growth of Mycobacterium tuberculosis [92] and its antibacterial activity was found to be comparable to isonicotinic acid hydrazide [93]. Anti viral activity of 2-mercaptobenzothiazole was tested in cell culture by Rada et al. [94] and found it to be effective against Vaccinia virus, New Castle disease virus (NDV) and Western Equine encephalomyelitis (WEE). A series of benzamido- and 2-acetamido benzothiazole derivatives were synthesised and screened for insecticidal activity against Periplanata americana and Culex pipiens [95]. The chloro- and nitro-derivatives were found to be most effective in both cases. Tetrahydrobenzothiazoles, their salts and enantiomers were pre pared for the treatment of central nervous system dis orders and/or circulation problems [96]. 21 In rubber industry, 2-mercaptobenzothiazole (caotax) is successfully employed as vulcanization agent[97,98] and the process was found to proceed simultaneously by free radical and polar mechanisms [99]. It is also used in manufacturing nontoxic gas masks [100], litho graphic printing plates [101], and pressure sensitive adhesive tapes [102]. 2-Mercaptobenzothiazole is also professionally used as corrosion inhibitor in various industries [103-105]. To extend the studies on thiazoles, 2-mercaptobenzothiazole was selected for the detailed electrochemical investigations. The experimental obser vations and results are summarised in the concluding chapter of this thesis.