Methylthilation: a new pathway of drug metabolism

77PS - September i 981

2,';0

Methylthiolation: a new pathway of drug metabolism W. G. Stillwell ht~timtefi~r Lipid Research. Buylor G~llegeof Medh'ine. Homton, TX 77030, U,?i.A.

Methylthiolation, the process leading to the formation of methylthio metabolites of foreign compounds, is one of the many novel biochemical pathways of metabolism which have been diseovered during the past several years. This pathway of xenobiotic metabolism, which results in the incorporation of a methylthio (-SCH,) group into a molecule, has been ~tudied in several laboratories, and numerous compounds. including drugs, berbicidcs, aromatic hydrocarbons and environmental contaminants, have been found to undergo this m~;tabolic conversion '--+. The formation of xenobiotic thioether derivatives, including glutathionc, cysteine and N-acetylcysteine conjugates (mcrcapturic acids), is generally considered a pathway for detoxificalion of reactive intermediates. For example, epoxides can be metabolized to glutathionc conjugates and appear as N-acetyicysteioc, cysteine or methylthio derivatives in urine. Thus in vivo studies of thioether excretion may indicate thai formaliop of a c.emically reactive mctabolite has occurred. A/ternatively, the formation of methylthio derivatives may represent new metabolic pathways ++'hose products have at present unknown biological signifi-

nitro group by nucleophilic substitution has occurred, or where reaction with an epoxide imcrmediate has been indicated (Table l), l-Allyl-3,5-diethyi-6-chlorouracil is converted into a methylthio metabolite in which the chlorine atom is replaced by a thiomethyl substituenP. Biotransformation of the anti-inflammatory agent, 2acctamido-4-chloromethyllhiazole, to a methylthio metabolite by replacement of chlorine has been reported T. Bromazepam is converted into a methylthi0 metabolit¢ in which substitution is at the a-carbon of the pyridine ring 1. A metabolite of the nitrofuran. 3-( 5-nitro-2-furyl)-2-(2-furyl)acrylamide, has been reported in which the

cailce.

Bromazepam

The introduction of a methylthio group into an aromatic compound was first described by the Millers and their associates s. Their studies with rats showed that esters of hydroxylamines formed in vivo were transformed into metabolites containing a methylthio group. Methylthio metabolites of the carcinogens methylaminoazobenzene. 2-acetylamim)fluorene, and related compounds, were imlatcd as alkaline-degradatkm products from the liver proteins of rats fed with thuse compounds. in the majority of examples reported in the literature, the formation of mcthylthio derivatives is associated with glutathion¢ conjugation and mereapturic acid excretion. Methylthio metabolites have been found where replacement of a halogen or OI05 - 6147/811001ll- 0OtMI/Sl}2.~0

nitro group is replaced by a methylthio moietya. in these examples the introduclion of a sulfur atom is likely to be the result of a nucleophilic attack on the electrondeficient carlton atom (C-CI, C = N, C-NO:), and metabolic activation of the substrate may not be necessary for reaction with the sulfur to occur. In other examples initial metabolism to a reactive intermediate, for example an arene oxide, is followed by nucleophilie attack by the sulfur atom. Included in this group are the aromatic compounds naphthalene, bromobenzene, and tetrachlorobiphenyl, Naphthalene provides an interesting example of methylthiolation reactions:. Ten methylthio metabolites have been identified as urinary products of naphthalene in the rat. One of the metabolites, l-hydroxy-2-methylthio-1,2dihydronaphthalene, is the methylthio analog of the trans-l,2-dihydrodiol of naphthalene (1/],2mdihydroxy-l, 2dihydronaphthalene). Two metabolites contain two methylthio groups and two hydroxy groups substituted on a tetrahydronaphthalene, while two contain one methylthio group and three hydroxy g r o u p on a tetrahydronaphthalene structun:, These observations indicate that, in addition to naphthalene-1,2-oxide, several

T A B L E I. Repn.~ntativc compound~ fl~rming melh~lthiL~ mciabolite~

Compound l-Allyl-). 5-diethyl~chl0r0ur4cil

Formula 0 H~C2~ N,'%.C ~H$

Metabolite(s) 0 H$C2_NvM~C2I~

o L . I~-c,

O~-N~SC.3

I H2C-CH=CH2

CI-H2C[ - ~ .~ 2-Acetami0o-4chl0romethylthiaz01e "s ~.coc.3

B~'

I H2C-CH-CH2

H3GS-H2C"r"--N I1 t].

-s"N.coc.3

Br

-LJ-$c.3 OH

~ S C H

Naphthalene

$CHs

3

H

8r

©

Bromobenzene

~.,j- SCH~

Cl Ct

SCN3

CI CI

2.5, 2'. $'-telr~hlor obiphenyl Cl

Cl

Cl

Cl HNCOCH3

AcelanlinoOhen ~SCH ON

3

Cl

Cl

7"1PS - September 1981 other epoxide metabolites, including anri1,2:3,4-naphthalene dioxide, are intermediates in the metabolism of naphthalene. Bromobenzene is als~ metabolized in rats to a methylthio

aromatic analogs. Under these conditions. identification of methyhhio metabolites is a valuable tool in assessing the structure of reactive intermediates in metabolic pathways. metabolite that is a hydroxyl substituted Oxidation of methylthio-comaining cyclohexadiene; this indicates Ihat an metabolites has been reported for several arene oxide is the precursor of this compounds '.=.' ". The S-oxidution of the metabolite ~. Metabolism of tetra- methyhhio group results in formation of chlorobiphenyl to a series of sulfur- metabolites of increamd polanty which containing products, including methylthio may he important in the subsequent elimiderivatives, has been described'. Meclo- nation of these comlxmnds m the organqualone is metabolized to several ism. Methylsulfinyl (-.~D(.'H=) and meth~lmethylthio-containing metabolites in the sulfimyl (-SO=CH=) mctalxflitc.~ arc frerat =°. One of these metabolites contains quently found in addition to methyhhio one methylthio group and one hydroxyl derivatives. For example, methylthio. group substituted on the dihydro- methylsulfinyl, and meth)-Isulfonyl quinazolinone ring of the parent com- metabolites have been reported from rats pound, indicating that an epoxide is a treated with bromazepam'. Biotransformetabolic intermediate. Clozapine is mation of 2-acetamido-4-chlorometh.~lmetabolized in the human to both hydroxy thiazole to methylthio, mcthyisulfinyl, and and methykhio derivatives in which the methylsulfonyl metabolites has been aromatically-bound chlorine has been reported ~. Methylthio and methylsulhmyl replaced by these substituents". The for- metabolites have been described from mice mation of these metabolites probably treated with 2,2'.5,5'-tetrachloroinvolves a reactive arene oxide intermedi- biphenyP: 2,4.5'-trichlorobiphenyl is also ate with elimination of the elements of metabolized in mice into two isomeric HCI; however, nucleophilic displacement methylsulfones which accumulate in the of chlorine by sulfur may ~',so occur. lungs ts. Methylsulfonyl metabolites of Methylthio metabolites of quinc;,es have DDE (2,2-b/s(4-chlorophenyl)- 1, l-dinot yet been identified but in a study, of the chloroethane) have also been identifed in metabolism of biphenyl in the rat a minor various animals. Methylsulfinyl and metabolite was identified as 3 - methylthio methylsulfonyl derivatives are also found - 4,4' - dihydroxybiphenyP =. The biological as urinary metabolites unaccompanied bt route of formation is probably through the methylthio metabolites. For example, 4,4'-quinone. Recently, the 3-substituted propachlor is metabolized to a meth.~lsulmethylthio derivative of acetaminophen lone; the methylthio derivative ha~ not has been identified in urine of dogs and been reported =. humans treated with acetaminophen or phenacetin 'a, In this case the reactive Origin of the melhylthio group intermediate may be the proposed Several pathways have been progx)sed acetimidoquinone. for the formation of methylthio The study of the thioether metabolites of metabolites. Direct attachment of a aromatic compounds is complicated by the methylthio group, involving methionine or fact that metabolites of the dihydrohy- N-acetylmethionine, was proposed by the droxy .series readily revert to the aromatic Millers and their associates. In the,~c compounds by dehydration (often accom- studies mcthylthio metabolites of panied by migration). It was first recog- 2-acetylan)inofluorcne and other strucnized by Boyland and Sims in 1958 that the turally-related coml~mnds ~cre i~'.datcd aromatic N-acetyleysteine derivative of as alk:dinc-dcgradation products from naphthalene, isolated in earlier work, was the liver proteins of rats administered formed as a product by dehydration of the the~ co,~q~unds. Administration of hydroxydihydro N-acetylcysteine metabo- radio-labeled methionine (=ti or uS) conlife. In it later study, Jeffrey and Jerina firmed that the mcthylthio group ~as showed that rearrangement of the sulfur derived front methionine s. group can accompany dehydration of ! A second mechanism has been proposed hydroxy - 2 - S - glutathionyl - i,2 - di- by sever;d groups in which the precursors hydronaphthalene and related c o m - of the methylthio-containing metabolites p o u n d s u. In our laboratory we have found are the corresponding cysteine or that very careful isolation, derivatization, N-acetylcysteinc conjugates. It was shown and chromatographic procedures must be in animal studies that administration of the used in order to detect the non-aromatic cysteine or N-acetylcystcine conjugates of metaboliles and prevent dehydration to the selected compounds leads to the excretion

~$1 of melh.vhhiolatcd h.~ S-adcm,...vlmclhionin¢ to h,rm thc meth)hlu,~ cthers. A rclatcd palh~ a.~ ha', been prolxr,cd b t MIO and co-worker., in which the meth.~lthio mctalxdite~ round a.. urina~ product,, of halogcnatcd aromatic ¢omlxmnds arc thought to be d e m ed from the correstxmding glutathione conjugates. Thc side-chain containing the gh~tathionc mohzt~ t.. propo~ed to be succe~..,~ch comerted in the ii~er and kidney to a series o| intermcdiate(thc L3qein.~lgl.~cine. c~stcme, mcrcapt,,1~.ru~ate, mcrcaptolactate, and mercaptoacetate coniugatesl im.'luding the thio~ metalxdite ~hich i.. transmeth~lated b~ 5-adeno...~lmethioninc'L Mio and co~ o r k e ~ ha~e i~)]aled the xanou_., .,tdechain metal~.)lites, including methxllhlo dematites, from the urmc ot ammal~ treated ~ith 1,3.5-trichh)robcnzcne. 1.3 dichh~robenzenc, and bromobcnzene ~: Recentl}, hosteler, another mccham~m has been proposed in v~hich the conjugated products of glutathlone excreted in the bile of cxl~rtmental antmal.~ are clea~ed in th=' gastrointestinal tract. The clea~ age proces~ is thought to be catal.x~'d b~ a C-S I}a,~." enz}me of the intestinal micro-organt-,mx or intestinal tissues.=~. Metabolism ~tudies t~ ith both germ-free and Neom}cln treate~ animals shox~ that the tnte,,tinal mtcroflora must be pre,.,,:nt h,r th¢in i n , , formation ot n)cth}l,,ulfim}l met,~b, qitc- trom propachlor '~ and meth~lthio mchii~qttc., from naphthalene ==. [ h e w findings ~upport the ~,iev. th:lt the intestinal mic=otlor,t and an enlcrohepatic circulation an." intol,.ed ill the comcrsion ot xenobtotlc glutathlonc conjugates to melh~ Ithio products. Further studies in germ-tree animals arc needed t,, as,~s the role of bactteri,d metabolism in the formation of meth}hhio nleta~)litc,, from glutathione or N-acct.~k'.~.,,teine conjag;tie... The splitting o i either c~,qeine or glulathione conjugates results in the tormalkm of thiols t~hich then ~ould require

T I P S - S e p t e m b e r 1981

252

(Eggslein, M and Liebich, It. M.. L.ds). pp. subsequent methylation in order to fc~rm tO the thiols which in turn are methylated to 378-3q0. Tubingen.FDR methylthio derivatives, ftowever, it is poss- form methylthio ethers. 12 Hallamp,K., Itorning, M. G. and Homing, E. C. ible that methylation of the sulfur atom (19781./. Chromatogr. 16fi,478-,49n 13 Klulch,A., Lcvin.W., Cha,g, R. I,.. Vane. F.and may occur prior to cleavage. The resultant Reading list I Tateishi, M. and Shimizu, tl. (19761Xenobioti¢~ Conney, A. H. (1978)Clin. Pharmactd. Ther. 24. sulfonium salt may then undergo enzym0, 43 I--439 287-293 atic or non-enzymatic cleavage m the 2 Bakke, J. E., Fell, V, J. and Price, C. E. (1976) 14 Jeffrey, A. M. and Jcrina, D. M. (1975)./. Am. methyithio metabolite, h has been shown Biomed. Ma.tv Speclrom. 3, 226-229 Chem. Sac. 97, 4427-4428 in several laboratories that the methyl 3 Stillwell,W, G., Bouwsma, O. J.. Thenol. J.-P, 15 Bergman,A., Brandk I. and Jansson,B. (IO79) Horning, M. G., Griffin,G. W., Ishikawa.K. and group o ¢ ,!e methylthio ether derivatives To.vicol. ,4ppl. I)harmal'ol. 48. 213-22(I Takaku, M. (1978) Res. Conmmn. Chem. Pathol. 16 Taleishi, M., Suzuki,S. and Shimizu,H. ( I878)./. of seveia; xenohioties is derived from Pharma¢ol. 20, 509--530 and retc~nces ciled Biol. Chem. 253. 81454-18859 methionine or S-adenosylmethiomne. therein 17 Kilamura, S., Sumino K. and Mio, "1".(1978) Thiol methyltransfemse (EC 2.1.!.91, an 4 Mio, T,, Sumino. K. and Mizutani. T. (1976) Proc. Third .~¢¢ting of the ./apanese So('iety for enzyme present in the microsomes, Chem. Pharm. Bull 24, 1958-1960 Medical Mat~s Spectrometry. 3. 83-95 (in Japan5 DcBraun, J. R., Miller. E. C. and Miller. J. A, catalyses methyl transfer from es¢) (1970) Cancer Res, 30, 577-595 lg Bakke.J. E.. Guslafsson,J.-A. and Guslafg'am,B. S.adenosylmethionine to a variety of 6 Kaul, R.. Hemp¢l, B. and Sch/ifer. W. [19761 E. ( 1980)Science 24, 433-435 aliphalic sulfhydryl compounds including A~nebi:. Forsch. 26, 489-499 19 Stillwell.W. G.. Bouwsma.O. J. and Homing, M. drugs and xenobiotics. An additional drug 7 Chalfield. D. 1t. and HumeL W. H. [19731 G. (1978)Res.Comm. Chem. PathoL Pharmatx~l. metabolizing enzyme, the Ihiopurine Biochem, 2. 134,879-884 22.329-343 8 0 u , T.. Tatsumi. K. and Yoshimura, H. (19771 20 Pazimifm,P. A.. Sladek.S. L. and Wein,~hilboura. methyltransferase, that cat;flyses Biochem. Biophys. Res. Cbmm, 75,401-405 R. M. ( 1980)Clin. Pharn~acol. ?'her, 28, 356-367 thiopurine meth.~ lation has recently been 9 Mizutani, T., Yamamolo, K. and Tajima, K studied ~. Thib e ~ m e found in the eryth(19781 Bmchen|, Biophys. Re.~. Comm. 82, rocyte and the thiol S-methyltransferase W. G. Stilh~¢n obtained her Ph.D. in biochemistry at 805-810 found in the microsomal fraction of liver, the Universio' of Houston in 197L Since then she luts IO Sfillw¢ll.W. G. (1977) Pharmacologist 19, 169 i i Stock. B. and Spiieller, G. ( 19771 in Mass Spec- been engaged in sn~diex of drug metabolism al the I~slikidney, and lung of the rat may both be trometO, and Combined Techniques in Medicine. rote for Lipid Research. Baylor College of .~#edicine. important in the enzymatic methylation of Cliniod Chemistry and Clinical Biochemistry HonMon. Texas. xenobiotic metabolites. Methylthio ether derivatives and their sulfoxidalion products represent a new cl&ss of xenobiotie metabolites whose pharmacological and toxicological properties have not been determined. However, in recent studies it was shown that the major compounds which accumulated in the bronchial mucosa of lungs from mice treated with t4C-labeled chlorobiphenyls were the methylsulfonyl metabolites of the compounds injected". A number of sulfur containing compounds including certain Department of Plo,sioh~gy. hasriturode Ci~nctas Biota&liens, Universidade de Sdo Paulo. Sfto Paulo. 05508. drugs, pesticides and industrial chemicals Brasit. have toxic effects. Several of these chemicals a l ~ bring about a decrease in the Hyperosmotic infusions have been suc~,,~s- sal of shock is better than that observed cytochrome P-450 enzymes. It may well be fully used for many years as adjuvant or after the mere replacement of shed of interest to investigate the possible interim treatment for hypovolemic shock', volumcL A recent reporP shows that if the osmotbiological effects of methylthio derivatives because they reportedly cause increased which have been identified in metabolic myocardial contractilitys, widespread pre- ic strength of a NaCI infusion is raised to studies. capillary vasodilation a and plasma volume 2400 mOsm I-', a relatively small volume expansion, due to an osmotic shift of fluid of infusion (4 ml kg -') produces permaCondusion into the vascular compartmenP. Published nent reversal of shock and indefinite surviAlthough thioether co,~iugation i,glu- reports cover a variety of infused solutes val from severe haemorrhage in dogs tathione, cysteine, and N-aeetylcysteine (glucose, mannitol, NaCI, NaHCOa). at (blood loss equivalent to 43% of total conjugates) has been recognized for many variable osmotic strength (6(10-1800 blood volume). This response is obtained years as a pathway of drug dctoxication, it mOsm I-'). Infused volumes ranged from even though no blood replacement is is only recenq-/that metbylthio derivatives 1.5 to 46 ml kg-'. Such infusions increase cffected and no other treatment given. Fig, I have been identified as products of arterial pressure, cardiac output, mesen- illustrates the experimental protocol. The xenobiotic metabolism. Methylthio teric flow and oxygen consumption; acid single intravenous infusion of hyperderivatives have been found predomin- base equilibrium is restored toward the osmotic NaCI reverts arterial pressure to antly in those cases where the glutathione normal rangeL This reversal of shock pro- a stable 100 mm Hg and base excess levels (a pathway is important in the metabolism of duced by hyperosmotic infusions is invari- reliable indicator of acid base equilibrium) foreign compounds, indicating that these ably described as transient, fading in a mat- from metabolic acidosis, caused by blood metabolites are derived from the cortes- ter of minutes. Thus, in all clinical trials and loss, to near normality (Fig. 2). The fate of ponding glutathione or eysteine in most experimental tests, such infusions control dogs treated with equivalent volderivatives. Several mechanisms have been are followed by replacement of lost or umes of i~smotic NaCI is also shown: proposed for the cleavage of the conjugates removed blood. In clinical trials, the rever- there is no appreciable recovery of arterial

Hyperosmotic infusions and the treatment of hypovolemic shock M. Rocha-e-Silva, Jr

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