Biotransformations: A Survey of the Biotransformations of Drugs and Chemicals in Animalsby D. R. Hawkins
Now in its seventh outstanding volume, Biotransformations has become established as a unique and important source for those involved in the discovery and development of new compounds. It broadly covers the scientific literature for the period 1987 to 1994. The series provides a complete survey of the biotransformations, in vertebrates, of the following:
Now in its seventh outstanding volume, Biotransformations has become established as a unique and important source for those involved in the discovery and development of new compounds. It broadly covers the scientific literature for the period 1987 to 1994. The series provides a complete survey of the biotransformations, in vertebrates, of the following: Pharmaceuticals; agrochemicals; food additives; environmental chemicals; industrial chemicals. Biotransformations provides a ready way of accessing information on the known pathways for the biotransformation of structurally-related compounds. Key functional groups provide an index-related procedure for retrieving information on compounds of interest. A further index allows the retrieval of examples of specific biochemical reactions which may have wider application. Each volume corresponds roughly with the scientific literature published during a calendar year. Each volume contains a review chapter which discusses examples of novel biotransformations, species differences, stereochemical aspects and mechanisms of toxicity associated with specific biotransformations.
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A Survey of the Biotransformations of Drugs and Chemicals in Animals
By D. R. Hawkins
The Royal Society of ChemistryCopyright © 1996 The Royal Society of Chemistry
All rights reserved.
The purpose of this chapter is to highlight some of the biotransformation studies included in this volume which report particularly interesting aspects. This includes novel biotransformations, stereoselective and stereospecific processes, and examples where mechanisms of toxicity have been attributed to specific biotransformation pathways. It is hoped that increasing the awareness of recent key information on biotransformations will ensure that it is utilized in making further advances in developing our knowledge of the subject.
1 NOVEL PATHWAYS
The metabolism of diphenylguanidine (1) and its dipentafluorophenyl derivative has been investigated in both rabbit and rat liver 9000g supernatants and microsomes (p. 383). The ring hydroxylation of diphenylguanidine occurred to give the metabolite (2) while introduction of fluorine substituents blocked this hydroxylation and resulted in N-hydroxylation to give a metabolite which was shown to exist in both the oxime (3) and hydroxylamine (4) forms.
The major pathways of metabolism of pioglitazone (5) in the rat involve modifications of the ethyl substituent on the pyridine group (p. 254). Metabolites were isolated from urine and bile of rats which had been administered single oral doses. Four metabolites were formed by oxidation of the ethyl group and each of these was also excreted as a conjugate. One metabolite was the benzylic alcohol (6) and its sulfate conjugate and two others were carboxylic acids (7 and 8) which were also further metabolized to taurine conjugates. A very unusual metabolite was the phenol (9) and its sulfate conjugate. There is no obvious mechanism for the formation of this metabolite and it was proved that it was not formed from a desethyl impurity in the test compound.
The major human metabolites of the antidepressant nefazodone (10) are as expected derived by N-dealkylation of the N-alkylpiperazine function and hydroxylation of the ethyl group (p. 257). A major unknown metabolite was detected in human plasma and sufficient amounts of the same component obtained from dog plasma and human liver S9 incubations for identification as the triazoledione (11). This is an unusual metabolite derived by oxidative replacement of the ethyl substituent.
The main pathways for metabolism of tributyltin chloride (12) in rats include dealkylation and C-oxidation (p. 435). Dibutyltin and monobutyltin were detected in tissues and two oxidized compounds (13) and (14) were only present in urine. Studies with diphenyltin dichloride (15) showed the major urine metabolites in rats to be phenyltin and triphenyltin. Formation of the latter is of interest since triorganotins are usually more toxic than the lower substituted compounds.
N-Acetylation and hydroxylation are the major biotransformation pathways of sulfamethazine (16) in rats (p. 285). However, there is a marked sex difference in that the intrinsic clearance in males was about twice that in females. A major metabolite in female urine was the N-acetyl derivative (17). The main pathways in male rats resulted in formation of the two hydroxylated metabolites (18) and (19). Inhibition experiments with monoclonal antibodies suggested that the male-specific cytochrome P-450 2C11 plays an important role in formation of these metabolites.
Both 1,2-dihydronaphthalene (20) and 1,2-dihydroanthracene (21) undergo hydroxylation and dehydrogenation on incubation with rat liver microsomes (p. 26). Hydroxylation occurred at the two saturated carbons but the regional stereoselectivity was found to be dependent upon the nature of inducing agents used in pretreatment of rats prior to isolating microsomes. The unsaturated compounds were shown to be formed enzymatically by direct dehydrogenation rather than by dehydration of the hydroxylated metabolites.
An interesting biotransformation of atropine (22) and scopolamine (23) which is species-dependent is formation of the unsaturated derivatives (24) and (25) (p. 139). The reaction occurs to the greatest extent in guinea-pigs and in vitro experiments with tissue homogenates and liver preparations indicated that ATP and cytosol were essential. The yield was greatly enhanced by the addition of sulfate and it was postulated that the process involves formation of a sulfate conjugate followed by non-enzymic elimination of sulfuric acid.
Selenium compounds are being investigated as cancer chemoprevention agents. The fate of selenite and methylated selenium compounds has been investigated in rats (p. 436). Inorganic selenium was excreted in urine as mono-, di- and tri-methylselenium. Dimethyl selenoside (26) was excreted in urine mainly as dimethylselenium (27). Trimethylselenonium (28) was excreted mainly unchanged but with small amounts of dimethylselenium. Evidence was obtained that methylated selenium compounds underwent both methylation and demethylation.
1.3 Ring cleavage
A major metabolite of the anxiolytic drug (29) has been isolated from rat faeces and identified as the ring-cleaved product (30) (p. 356). This reductive cleavage of the oxadiazole ring could be medicated by the action of gut flora.
Two major novel metabolites of the calcium antagonist SM-6586 (31) have been isolated from the bile of rats after oral doses (p. 259). It is of interest that one metabolite (32) retained the dihydropyridine structure and the intact methyl ester function. This metabolite could be formed by oxidative N-dealkylation of the side-chain and subsequent decarboxylation. The second metabolite (33) was formed by loss of the side-chain and cleavage of the oxadiazole ring to give an unusual cyanamide function.
The major pathways for the metabolism of risperidone (34) in rat and dog include C-oxidation in the tetrahydropyridopyrimidinone ring, N-dealkylation and scission of the benzisoxazole ring (p. 311). The latter process was believed to occur by the action of intestinal microflora and resulted in formation of metabolites containing the phenolic aryl ketone function such as (35). Some minor human metabolites have also been identified as compounds formed by isoxazole ring cleavage (p. 313) including the phenol (35). The metabolites were present in faeces again indicating the involvement of gut flora.
The metabolism of the HIV-I reverse transcriptase inhibitor (36) was investigated initially using rat liver slices (p. 307). From these experiments several metabolites were identified with one resulting from cleavage of the oxazole ring, namely the phenol (37). Other metabolites were formed by modification of the ethyl side-chain to give benzylic alcohols, ketones and the unsaturated compound (38). These metabolites also exhibited some biological activity.
The in vivo formation of thioamides as a result of ring-cleavage of thiabendazole derivatives has been investigated in mice (p. 256). The presence of thioamides in mouse urine for three compounds (39)–(41) was established by GC–MS after derivatization with p-nitrophenacyl bromide. Only small amounts (0.25–1.25% of the dose) of the thioamides were detected although it is likely that further metabolism of these would occur. The thioamides are postulated as being toxic metabolites of these compounds and possibly responsible for the observed nephro- or hepato-toxicity.
As expected N- and O-demethylation are the major biotransformation pathways for the antidepressant venlafaxine (42) (p. 172). Hydroxylation at position 4 in the cyclohexyl ring also occurred and resulting from this a novel cyclized metabolite (43) was formed which represented about 6% of an oral dose in rat and rhesus monkey urine. This is presumably formed from an N-hydroxymethyl intermediate.
Novel cyclized metabolites of the antiarrrhythmic agent actisomide (44) have been identified in dog, rhesus monkey and man (p. 343). These metabolites were formed by initial loss of an N-isopropyl group (45) followed by an intramolecular rearrangement resulting in cleavage of the pyrimidone ring and formation of the pyrrolidone (46).
Previous investigations on the metabolism of R,R-labetolol (47) have shown that glucuronide conjugates of the parent drug and a ring-hydroxylated metabolite were major components in rat, dog and monkey urine. Other metabolites detected were not identified. More recent studies have confirmed that hydroxylation in both aromatic rings occurs, and the catechol metabolite (48) also underwent cyclization to give a novel N -alkylindole analogue (49) which was excreted as a glucuronide (p. 178, 179).
N-Oxidation is a major pathway for the metabolism of pinacidil (50) in several species including man, rat, rabbit, dog and mouse. However, there are some notable species differences in metabolism and also some novel metabolites (p. 274, 275). A unique major conjugated metabolite was detected in rabbit urine only, which was assigned the structure of a glucuronide of the N-hydroxypyridonimine (51). This metabolite was formed on incubation of the N-oxide with rabbit liver slices. The N-oxide was produced after hydrolysis of the conjugate with β-glucuronidase or alkali. A minor glucuronide conjugate, also only detected as a rabbit metabolite, was identified as an N-glucuronide of the tautomeric pyridonimine form of the parent drug (52). The UV absorption spectrum was similar to that of (51) providing further evidence for a pyridonimine conjugate rather than an alternative quaternary N-glucuronide. A major metabolite in rhesus monkey urine was the glucuronide of a hydroxylated compound (53). This metabolite was formed in smaller amounts by rabbit and dog but was not apparently produced by man, rat or mouse.
Incubation of the aminoimidazole (54) with human and rabbit liver microsomes both resulted in formation of a glucuronide conjugate but which were chromatographically different and had different susceptibility to hydrolysis by β-glucuronidase and different NMR spectra (p. 318). The spectroscopic data indicated that the rabbit conjugate involved the exocyclic amino group (55) and the human conjugate the N3-imidazole nitrogen (56).
The facile formation of an N-glucuronide conjugate involving a tetrazole ring is demonstrated by studies with the antihypertensive agent (57) (p. 261). In rats almost 90% of an oral or intravenous dose was excreted in faeces. Rapid and extensive biliary excretion occurred and the single major component in bile was the N2-β-glucuronide (58) which was also the major component in plasma. The same conjugate was also a major metabolite in rhesus monkeys although other metabolites were also formed.
The formation of N-glucuronides of various model compounds containing five-membered ring nitrogen heterocycles has been investigated in vitro using liver microsomes from various species (p. 252). Generally, there was low reactivity for nitrogens adjacent to a substituted carbon such as the substituted imidazole (59). The highest reaction rate was observed with the tetrazole (60) followed by the 1,2,3-triazole (61). For the latter compound there was a species difference with monkey and rat showing a preference for formation of the N1- and N2-glucuronides respectively.
The antiviral agent (62) contains an unusual cyanamide group. Studies with rats and mice showed that most of an oral dose was excreted unchanged in urine (p. 260). The most important urinary metabolite (10–20% of the dose) was the urea (63) resulting from hydrolysis of the cyano group. A polar metabolite was also detected in mouse urine and identified as a mesionic ribose conjugate (64).
Microbial and fungal cultures are increasingly being investigated as systems to synthesize potential mammalian metabolites as reference compounds. Fungal systems are particularly useful since they also form conjugated metabolites. In experiments with furosemide (65) the fungus Cunninghamella elegans was shown to form the Phase I metabolite (66) (p. 128). An ester glucuronide of furosemide is a known human urinary metabolite and although this metabolite was not produced by the fungus a similar polar metabolite was detected. This metabolite was hydrolysed by β-glucuronidase and β-glucosidase and subsequently the structure of the aglycone and conjugating moiety were confirmed as furosemide and glucose. This example illustrates that the enzyme preparations used to characterize conjugates are not necessarily specific.
In the presence of glutathione 3-oxohexobarbital (68) is converted into 1,5-dimethylbarbituric acid (70) and a novel cyclohexenone glutathione adduct (71) (p. 279). Administration of hexobarbital (67) and the epoxide (69), which could be a metabolite of the former, to rats resulted in excretion of the barbituric acid in urine. However, after administration of hexobarbital the glutathione conjugate (71) was excreted in the bile of rats indicating that it was formed via the allylic hydroxylation and the intermediate (72).
Valproic acid (73) is an example of a compound which although having a very simple structure leads to a complicated array of metabolites. Previous studies have identified the formation of unsaturated metabolites which have been implicated in the observed toxicity of the compound. Some recent studies using the unsaturated derivative (74) have now identified the formation of glutathione conjugates in rats and three conjugates (76)–(78) were isolated from rat bile (p. 83). The route to these involved epoxidation and a CoA-dependent pathway via the intermediate (75). The pentenoic acid metabolite (74) is related to the hepatotoxic isomeric acid (79). The formation of thiol conjugates of these acids has been compared to evaluate whether the acid metabolite of valproic acid might be associated with its hepatotoxicity (p. 83). Three major metabolites of (79) in rat bile were identified as isomeric glutathione conjugates of (80). By contrast no thiol conjugates of the keto-acid (81) were identified after administration of the valproic acid metabolite. The only metabolites detected were the known conjugates of the dienoic acid (82). There are therefore basic differences in the metabolism of these two structurally related acids.
Biotransformation of pyrethroids usually involves ester hydrolysis, oxidation and conjugation as the major pathways. Studies with the insecticide (83) containing a cyclopentenone group showed the formation of two polar metabolites after administration of oral doses to rats (p. 200). Both of these were identified as new types of S-linked conjugates namely the mercapturic acid (84) formed via conjugation of glutathione with the alkyne function and the sulfonic acid (85). It is believed that the mercapturic acid is the first example of the in vivo addition of glutathione to a triple bond. It was demonstrated that co-administration of Na235SO4 to rats resulted in incorporation of 35S into the sulfonic acid metabolite and proposed that this involved addition of sulfite, produced by microbial reduction to sulfate, to a cyclopentadienone.
An unusual series of sulfonic acid metabolites are also formed from the herbicide S-23121 (86) in rats (p. 154). Three of these metabolites (87)–(89) were phenols derived by O-dealkylation and addition of sulfite to the double bond. These polar metabolites were isolated from faeces and it was found that the sulfonic acid group was also radiolabelled when sodium [35S]sulfate was co-administered.
Several major sulfonic acid metabolites of cis (90) and trans (91) isomers of tetramethrin have been isolated from rat faeces after oral doses and represented a total of about 16% of an oral dose (p. 49). It is of interest that several of these metabolites contain the intact ester function. Ester hydrolysis was greater for the trans -isomer compared with the cis-isomer. The sulfonic acids such as (92) and (93) from both isomers resulting from addition of sulfite to the double bond of the tetrahydrophthalimide group are presumably formed by the same mechanism proposed for the compound S-23121 above.
The experimental drug nitecapone (94) possess an unusual substituted arylalkene structure. Studies in rodents showed that the major metabolites were formed by reduction of the alkene and ketone functions combined with conjugation of a phenolic group with glucuronic acid or sulfate (p. 117, 119). One novel urinary metabolite was the sulfonic acid (95) formed by addition of sulfite to the alkene function. This metabolite had previously been identified as a dog urinary metabolite but was apparently not a human metabolite. There was no information or discussion on the mechanism of formation of this metabolite.
Excerpted from Biotransformations by D. R. Hawkins. Copyright © 1996 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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