1177
CARCINOGENIC LACTONES. I11
November 1968
was obtained and, following the detailed investigation described below, it mas assigned structure 9. lo When the lactone 1 was treated with cysteine in aqueous solution a t p H 7 and worked up under strongly acid conditions,6 the compound mp 194” described by Cavallito and Haskell was obtained in 85y0 yield. The ir spectrum contained bands indicative of a fivemembered ring lactam (1665 cm-’) and of a free carboxyl group (174012and 3300-2800 cm-l). The broad peak, 3300-2800 cm-l, has previously been cited” as evidence for the S-H group of structure 8, but the ir spectrum does not allow an unequivocal decision to be made in this regard. However, the pmr spectra in DJISO-d, and in pyridine-& exclude the possibility of the latter interpretation being correct since no peaks due to an amide proton, or to a vinylic proton as required by 8, were detectable in either solvent. Furthermore, the methyl protons appear as a sharp singlet, whereas those in structures such as 8 should show long-range coupling ( J 1-2 Hz) with the vinylic proton.14 Of particular significance is the presence in the spectra in both solvents of the ilA’BB’ system multiplet (6 2-3) of the four C-6 and C-7 hydrogens. Only structure 9 mould appear to be consistent with all of these spectroscopic data.15 It is also of interest to note that, in contrast to the deceptively simple ABX splitting patterns observed for the C-3 and C-2 protons of 9 in DI\ISO-&, the corresponding protons of its carboxylate anion (in pyridine-&) behave as a remarkably clean ,4BX system. This situation might also obtain for structure 8 if the amide proton was undergoing sufficiently rapid exchange. However, that this is not the case is demonstrated by the fact that only one exchangeable proton (COOH) can be detected in either solvent in the range 6 0-16. The one piece of data which, at first, could not be accounted for in terms of structure 9 for the cysteine-1 product was the evidence of Cavallito and HaskelP for one double bond provided by the iodine number determination.6 However, it has been shown that when iodine numbers of compounds containing sulfide linkages are determined, the addition of iodine to the sulfur atom must be taken into account. For simple sulfides such addition is quantitative. l8 The reaction of cysteine with levulinic acid is also reported to give 9.19 This is of interest since levulinic
-
(10) While this paper mas in preparation, Black” reported t h e results of his reinvestigation of this reaction, in which h e concurred with t h e original assignment of structure 8 t o t h e product isolated by Cavallito a n d Haskel1.6 For t h e product of t h e reaction of cysteine with lactone S his structural assignment, 16a, is in agreement with t h e conclusions reported in this paper. (11) D. K . Black, J . Chem. Soc., C , 1123 (1966). (12) This frequency for t h e CO stretch of t h e carboxyl group is s o m e what outside t h e normally accepted range12 (1i25-1700 cm-1) of aliphatic acids; however, t h e related compound, cysteine hydrochloride monohydrate, also shows CO stretch a t 1740 cm-1 (Nujol). (13) C. N. R. Rao, “Chemical Applications of Infrared Spectroscopy, Academic Press Inc., S e w York, N. Y., 1963, p 193. (14) I n lactone 1, t h e analogous CHs shows long-range coupling of this kind. (15) T h a t structure 9 was isomeric with 8 a n d was a possible structure for t h e Cavallito and Haskell compound was first pointed o u t in a review article by Byhora.’e Following t h e submission of our manuscript, t h e report of t h e independent investigations of Hellstrom a n d his covorkers” appeared in which they also concluded t h a t structure 9 was t h e correct one. (16) K. Syhora, Chem. Listy, 65, 311 (1969). (17) K. Hellstrom, S. 0. Almqvist, M. Aamisepp, and S. Rodmar, J . Chem. S o c . , C , 392 (1968). (18) E . E. Reid, “Organic Chemistry of Bivalent Sulfur,” Vol. 11, Chemical Publishing Co., New York, Pi. Y.. 1960, p p 47-79. (19) E. D. Bergmann and A. Kalusayner, Rec. Trau. Chim., 78, 288 (1959).
acid is the hydrolysis product of lactone 1. The possibility that hydrolysis of 1 was the first step in the formation of 9 was eliminated by the observation that under the reaction conditions, the rate of reaction of cysteine with the lactone was much more rapid than that of the competing hydrolysis. The formation of 9 as the final product of the reaction of cysteine with the A3-lactone 1 obviously involves a multiple-step pathway requiring the participation of several intermediates. I n view of our interest in identifying the biological site of alkylation by compounds exemplified by structures 1-7, and in developing a reliable chemical method for predicting their carcinogenicityJZ0it became of interest to elucidate the mechanistic details of the reaction, and, in particular, to identify the nucleophilic group effecting the initial attack on the lactone. The mechanism proposed originally6 suggested the first step to be addition of the thiol group across the A 3 double bond, and this was reiterated recently by Black” in his speculation on the mechanism. However, in the current investigation it was observed that the reaction mixtures gave positive nitroprusside tests indefinitelyz1 (provided that oxygen was excluded from the reaction), and only the crystalline product isolated from solution after acidification gave a negative test for free thiol. Our previous work on the reaction of lactone 1 with amines had shown nucleophilic attack at the carbonyl group to be a very facile process.20 This information together with a consideration of the pKa’s of the SH and + S H 3groups of cysteine,22suggested that, a t p H 7, the most probable initial step mas attack on the lactone carbonyl group by the cysteine thiol anion. That such a reaction could occur was demonstrated by the reaction, in benzene solution and in the presence of potassium carbonate, of a-toluenethiol with l to give the thiolester 10 in 60% yield.23 Unfortunately, for 1
+ CsH,CH*SH
KzC03 &
A
CH~COCH~CHZCOSCHZC~HS 10
reasons of solubility, this reaction could not be carried out in aqueous solution and when the analogous reaction of 1 with S-acetylcysteine in water was attempted, no thiol ester formation could be detected.24 This indication of the importance of the free cysteine amino group in the reactions leading to the formation of 9 was substantiated by the facile reaction a t p H 7 of isopropenyl acetate (the acyclic analog of 1) with cysteine to give X-acetylcysteine (60%) as the only isolable cysteine derivative. CHz
I1
CH,COCOCH,
CHiSH
I + qsteine + CH,COSHCHCOOH
These results prompted us to search for similar (20) J B Jones and J. h l Young, Can. J . Chem , I C , 1059 (1966). (21) T h a t this positive test % a s due t o t h e presence of a n S H i n t e r m e h a t e a n d not t o traces of cysteine was confirmed b y t h e subsequent isolation of
t h e intermediate l l a . (22) J. T. Edsall a n d J. Wyman, E d , “Biophysical Chemistry,” Vol I , Academic Press Inc , Ne\+ York, N. Y., 1958, p p 496-504 (23) Under t h e reaction conditions, vaz., reflux for 1 hr, t h e isomerization of 1 t o S becomes a strongly competing reaction. (24) Further support for t h e postulated initial attack b> SH can be drawn from t h e recent d a t a o f Hellstrom, et al.,” who noted t h a t mercaptoacetic acid reacted with 1 in H t 0 to give t h e correspondmg thiol ester, which subsequently underwent rapid hydrolysis.
\'(It.
iritcririctiiatr+ iii tlic c j iteiiic 1 t w c t i o i i niixtuic, \Vhcii thc latter iv:tb worlied up k)> c1iriini:ttogrnph~o i i :iii ion-exchange columll of pH 5 i l l p1:tcc ot tlw 11i11:tl acidificntioii procedure :in i d n-as ot)t:tiiicyl coiit:tiiiiilg the ketoamide l l a (73';; 111 pnir). ~'lJI11pOI111(~l l a caiiuld riot be purified h ( i e lic~toamide~ of tlii. t > pc' : ~ i ( ' nn-;table and cyclizc rcaciil) t o the rorre-poiidiiig 111 tlios!.p).rrolidiiioiic~;?" thu., n l i c ~ itlic, lla-co~it:ti~ii~~g oil \vu, kept for several cia? + I I I ctiloioioimi \iilutioii. e ) c1iz:iticiii iollon-ecl 117. deli) (1r:ttioii oc~~urrotl to c' l~cOcl~ 1 ?L'( ~ ()X ~f1I c 11cc ) 2 1 i , I
('fl-bll
i i a , I: b, 1: c, I:
= = =
C ' I I ~ . I:, ClII), I:, C ' J I I , I:,
= = =
Ir I{ ('Il$
'Yllc? other A '-1actOIlc' ,.tucl1etl. 4-11) drox) hex-:3-cllolc~ acid lactorie (2), 1 5 behaved aiiulogoiihl!. and in aqueou. wlution at p H 7 uiiderwent rapid. quautitative reaction with cysteine to give S-(3-osohexaiioyljcysteiIie ( l l b ) . .Uhough this product showed 1 1 0 t endeiicy toward p j rrolidinonc form:ttion it could iiot be crystallizeel. In constrast, the reaction c)f 2 with methyl cysteinatc tlic iiieth) 1 cster l l c a i :L coinpletel\. characterized. atctl the initial nucleophilic attack to be b > r the cysteine :imino grciup, thib coiiclu,ion lost much of its appcal with the rliscovery that :imino acids lacliiiig the thiol group, buch as glycine, reacted not :it all with 1 at pH 7 and oiily ;lowly at pfI !I. 1 h r iiiitler the lattc>rconditiolis the reaction mixture cont:tinetl b o ~ i i cof the cxptlctecl amide and i t 5 rivntive. ln :tdditiiin, levulinic acid, of the itartiiig lactone. J W ~ protlucccl. r 7 1h e x app:ireiitly conflictiiig &ita iirc t m t :tccoiiiiiiot l a f d hy the niechaiiism sliowi in Chart I1 i l l n-liich
attach h) the thiol groiip t o torin the thiole\tcr 12 i5 fOll(J\\-(d 1)) :I iapitl S to S :le> 1 migr:ition lendiiig t o l l a . That iuch niigr:itioii. o w u r rlipidli i i acle-
iiiiti:tl
tlocunicntctl iri the litct*:tture2f md, III gerit~ral,:ininit\ :iiitl :\mino ii :Let with thiole>tcrs t
2 0 ~ CHJCH,COCH2CH2C02H 3.Ht
A+
A
indicates cyclization of 4-oxohexanoic acid afforded a mixture of 2 and 4a which a t its room temperature equilibrium contained 40yo of the latter isomer. All attempts to obtain a pure sample of 4a, including careful distillation and chromatography, were unsuccessful owing to the facility with which 4a isomerizes to the corresponding A3 compound. On one occasion an extremely rapid distillation of the cyclization product afforded a mixture containing 6Oyo (by pmr) of 4a; however, on keeping overnight this too reverted to the equilibrium c o m p ~ s i t i o n . ~ ~ By careful and slow distillation of the lactone equilibrium mixture, a 90% yield of the more volatile component, 4-hydroxyhex-3-enoic acid lactone (2), could be obtained. The reactions (with cysteine, etc.) described earlier for this compound were carried out immediately following the distillations ; on keeping, pure samples of 2 gradually equilibrated to the 60:40 mixture of A3- and A4-lactones. The equilibration process ( 3 0 ) T h e analogous proton in 4-hydroxypent-3-enoic acid lactone (1) occurs a t 6 5.15.20 (31) I n view of t h e facility with which this isomerization occurs i t appears t h a t caution should be exercised when considering t h e carcinogenicity ascribed‘ t o this structure. I n fact, i t seems probable t h a t t h e tumor induction observed4 is due t o a compound other t h a n 4a. This conclusion is supported by the observation t h a t , a t present, l a is t h e one anomaly in t h e structureactivity relationships t h a t have emerged from t h e studies of Dickens and hi6 co\rorkers.4
1179
was markedly accelerated by the addition of p-toluenesulfonic acid. I n order to enable the reactions of lactones such as 4 to be studied, the synthesis of 3,3-dimethyl-4-hydroxyhex-4-enoic acid lactone (4b) was effected. This compound, for which isomerization to the A3-lactone is precluded, was prepared by the free-radical condensation of 3,3-dimethylacrylic acid with propionaldehyde followed by distillation in the presence of p-toluenesulfonic acid of the 3,3-dimethyl-4-oxohexanoic acid so produced. The results of attempted reactions of 4b with cysteine in aqueous solution at pH 7, with cysteine methyl ester in aqueous ethanol, and with a-toluenethiol and triethylamine in benzene were disappointing since the lactone was recovered unchanged in all cases. However, that its chemical behavior was analogous to A3lactones was established by its reaction with benzylamine to give l-benzyl-4,4-dimethyl-5-ethyl-Z-hydroxypyrrolidin-2-one (14b) in 51% yield. In contrast to the other pyrrolidinones encountered during our investigations, 14b was obtained as a crystalline solid although all attempts to recrystallize this material from a variety of solvents led to its contamination with the dehydration product, l-benzyl-4,4-dimethyl-5-ethylidenepyrrolidin-2-one. During our attempts to rationalize the ready reaction of A3-lactones 1 and 2, and isopropenyl acetate, with cysteine with the lack of reaction of the structurally similar compound 4b, it was observed that in addition to the normal carbonyl absorption, the ir spectrum of 4b showed a very strong band a t 1710 cm-I. I n contrast, for 1, 2, and isopropenyl acetate the corresponding band at 1680-1690 cm-I was a much weaker one. Unfortunately any possible empirical predictive value this observation might have had was invalidated when the series of C=COCO-containing compounds studied, was extended to vinyl acetate. The ir spectrum of this ester in the 16Z0-1S00-cm-1 region is very similar to that of 4b but on treatment with cysteine in aqueous solution a t p H 7, it gives the expected product, S-acetylcysteine. I n view of its potency as a tumor-producing agent,4 its slow rate of reaction with cysteine,’ and its structural resemblance to A3-lactones, it was decided to extend the investigation to vinylene carbonate (6). As far as could be determined, no reaction with cysteine occurred even though the pH of the solutioh dropped rapidly and carbon dioxide was evolved ; furthermore, the cysteine could be quantitatively recovered from the reaction mixture. Treatment of vinylene carbonate alone with a sodium bicarbonate buffer at p H 7 also resulted in carbon dioxide evolution and the slow formation of a fluffy precipitate. Perusal of the literature showed that vinylene carbonate is readily hydrolyzed to glycolaldehyde which may then polymerize to tetroses and eventually to caramels.32 hlthough it is possible that nucleophilic attack on vinylene carbonate by the cysteine thiol or amino group occurs,33it seems more likely that the reaction involved is simply hydrolysis to glycolaldehyde and that the
(32) A . H. Saadi and W. H. Lee, J . Chem. Soc., B. 4 (1966). (33) T h e rapid p H drop a n d COz evolution would seem t o indicate some cysteine participation.
November 1968
CARCINOGENIC LACTONES.111
(0.5 g).49 The progress of reaction was monitored by pmr and when a 507, conversion to 3,3-dimethyl-4-oxohexanoic acid had occurred (7 days), the solution was evaporated and vacuumdistilled in the presence of p-toluenesulfonic acid. Chromatography of the distillate (7 g) on Florisil (200 g, CsH6 elution) followed by redistillation yielded pure 4b (4.1 g, 257, based on 3,3-dimethylacrylic acid): bp 82-83' (9 mm); ir (CHCl,), 1795 (vs), 1710 (vs), 1105, and 993 cm-'. Anal. (C~HIPOZ)C, H. Reaction of 4-Hydroxypent-3-enoic Acid Lactone (1) with LCysteine. ( a ) With Acid Work-up.-The lactone 1 (2 g, 20 mmol) was added with stirring to a solution of L-cysteine (2.4 g, 20 mmol) in H2O (15 ml) which had previously been adjusted to pH 7 . 5 0 Since a two-phase system was formed, stirring mas continued throughout and the mixture was maintained in the range p H 6-7 by periodic titration with base.s0 Within 15 min 20 mmol of base had been added and the reaction mixture had become homogeneous; it was then kept a t room temperature overnight. Acidification (to p H 1.5) of the solution with 37% HCI, followed by evaporation under reduced pressure and trituration of the residue with 1 aYHCl (5 ml), yielded 1-aza-2-carboxy4-thia-5-methylbicyclo[3.3.0]octan-8-one(9) (3.4 g, 8570). On recrystallization from absolute EtOH, thick needles, mp 194O, were obtained which gave a negative SH (nitroprusside) test but which evolved CO, on treatment with aqueous NaHC03; ir (Nujol), 3300-2800 (acid OH), 1740 (sharp, COOH), and 1665 cm-l (broad, amide COj, pmr (DhlSO-cl6, 60 MHzj, 6 1.62 (9, 3, CH,), 2.1-2.9 (m, 4, CH,CH2CO), 3.58 (d, 2, J = 7 Hz, SCH2), 4.86 (t, 1, J = 7 Hz, SCHSCHN), and 10.5 ppm (s, 1, COOH). The spectrum obtained in D2O was similar and confirmed the presence of only one exchangeable proton; pmr (pyridine, 60 MHzj, 6 1.80 (8, 3, CH3), 2.00-2.90 (AA'BB', m, 4, CH&H,), 3.464.08 (ABX, m, 2, S C H Z ) ,5.41 (ABX, d of d, 1, J A X = 8.6 Hz, J S X = 6 Hz, CHzCHN), and 14.8 ppm (s, 1, COOH). The 100-11Hz spectra provided no additional data. Mass spectrum (70 eV) showed m e 201 (parent ion). Anal. (C,HiiSOaS) C, H, N, S. When the reaction was carried out in 1 JI potassium-sodium phosphate buffer (pH i),the product 9 was obtained in 75% yield after acidification and recrystallization. No significant reaction was observed at p H 7 with N-acetyl-Lcysteine or with glycine. (b) Without pH Control.-The lactone 1 (2.0 g, 20 mmol) was added with stirring to a solution of L-cysteine (2.4 g, 20 mmol) in H2O (25 ml) a t p H 7 5 0 as described above. Within 25 min the pH had fallen to a terminal value of 3.6 and work-up of the reaction mixture afforded 9 in only 107, yield. (c) With pH Control and with Ion-Exchange Resin Work-up. s in part a except that after -This experiment was carried out a standing overnight the neutral solution was filtered through a column of Rexyn AG-50 (100 g, Fisher) and the fractions giving a positive free SH test were collected and combined. Evaporation (bath temperature
