Chem. Res. Tonicol. 1994, 7, 297-306
297
Structure-Activity Relationships for Contact Allergenic Potential of y,y-Dimethyl-y-butyrolactoneDerivatives. 1. Synthesis and Electrophilic Reactivity Studies of a-(w-Substituted-alkyl)-y,y-dimethyl-y-butyrolactones and Correlation of Skin Sensitization Potential and Cross-Sensitization Patterns with Structure Christelle Franot,? David W. Roberts,$ Richard G . Smith,$ David A. Basketter,§ Claude Benezra,t7ll and Jean-Pierre Lepoittevin*it Laboratoire de Dermatochimie associ6 a u CNRS, Universit6 Louis Pasteur, Clinique Dermatologique, CHU, F-67091 Strasbourg, France, Port Sunlight Laboratory, Unilever Research, Quarry Road East Bebington, Wirral, Merseyside L63 3 J W , United Kingdom, and Environmental Safety Laboratory, Unilever Research, Colworth House, Sharnbrook, Bedford MK44 ILQ, United Kingdom Received June 14,1993"
A series of a-(X-substituted-methyl)-y,y-dimethyl-y-butyrolactones (series l),a series of 4 2 X-substituted-ethyl)-y,y-dimethyl-y-butyrolactones (series 21, where X is a leaving group, and the compound a-(&bromopropyl)-y,y-butyrolactone(3) were synthesized. Their reactions as electrophiles toward n-butylamine, used as a model for nucleophilic groups on skin proteins whose in vivo chemical modification leads to skin sensitization, were investigated. The compounds of series 1 were shown to react via a two-stage elimination-Michael addition sequence whereby which the elements of HX are eliminated to form a-methylene-y,y-dimethyl-y-butyrolactone, reacts more slowly with n-butylamine to give a-[(N-buty1amino)methyll-y,y-dimethyl-ybutyrolactone. The compounds of series 2 and compound 3 were shown to react with n-butylamine via a single-stage substitution reaction to give respectively a-[2-(N-butylamino)ethyl]- and a-[3-(N-butylamino)propyll-~,y-dimethyl-y-butyrolactones. Rate constants for these reactions have been determined, and it is found that substitution reactions of series 2 and compound 3 are slower than Michael addition of a-methylene-y,y-dimethyl-y-butyrolactone,which in turn is slower than the elimination reactions of series 1. The results of guinea pig skin sensitization tests on these compounds were found to be consistent with the above findings in that the compounds of series 1were found to be in general much stronger sensitizers than those of series 2 and compound 3. The results of cross-challenge tests indicate that sensitizing compounds from series 2 were cross-reactive with both series 1 and compound 3 but that compound 3 is only weakly cross-reactive with series 1. These observations indicated that for these compounds a difference of two carbon atoms between the determinant groups transferred to protein had a markedly greater effect than a difference of one carbon atom on antigenic specificity.
Introduction
(alkylation)to carrier protein in the skin at the induction andchallenge stages of skin sensitization testing deterThere is a growing interest in the development of mines the magnitude of the sensitization response and chemical structure-skin sensitization relationships which that the extent of this binding can be quantitated by the can rationalize patterns of varying sensitization potential RAI, a composite parameter made up of dose, chemical among groups of chemicals and, ideally, predict the reactivity, and hydrophobicity terms (2). sensitization potential of compounds not yet tested. The RAI model has been successfully applied to the To date, the relative alkylation index (RAI)l model has interpretation of sensitization datasets for a variety of proved the most successful approach to the quantitative classes of chemicals (3-6). These studies have all been interpretation of skin sensitization data ( I ) . In brief, the opportunistic, in that the datasets were produced for basis of the model is that the extent of covalent binding purposes other than evaluating the RAI model. Up to now, the RAI model has not been tested against datasets * Address correspondence to this author at the Laboratoire de in which there is an independently varying multiplicity of Dermatochimie, Clinique Dermatologique, CHU, F-67091 Strasbourg, different lipophilicity and reactivity values. France. We now present results of an investigation designed to t Universitb Louis Pasteur. t Port Sunlight Laboratory, Unilever Research. provide wide variations in reactivity and lipophilicity. In I Environmental Safety Laboratory, Unilever Research. this and the following paper we report our findings for 11 Accidentally deceased, January 20, 1992. Abstract published in Advance ACS Abstracts, March 15, 1994. a-(w-substituted-alkyl)-y,y-dimethyl-y-butyrolactones of 1 Abbreviations: ACD, allergic contact dermatitis; DMF, dimethylgeneral structures 1-3 as shown in Chart 1. These formamide; FCA, Freund's complete adjuvant; HMPA, hexamethylphoscompounds are structurally related to the tulipalin phoramide; HSA, human serum albumin;LDA, lithium diisopropylamide; MSIAT, modified single-injection adjuvant test; RAI, relative alkylation derivatives and sesquiterpene lactones whose chemistry index. and O S ~ ~ - ~ ~ ~ ~ I ~ ~ I 0~ 1994 ~ OAmerican ~ - O Chemical ~ ~ ~ ~ Society O ~ . ~ O I ~
298 Chem. Res. Tonicol., Vol. 7, No. 3, 1994
Franot et al.
C h a r t 1. S t r u c t u r e of Lactone Derivatives la-i, 2a-i,
and 3b
1 a-i
28-i
3b
allergenic properties have been extensively studied (7,8). Series 1-3 were chosen for study as offering the opportunity to vary physicochemical properties without changing the nature of the epitope, Le., the entity which becomes chemically bound to carrier protein in the skin. Within each series the epitope is constant, and from series to series the variation is small. The present paper deals with the effects of changes in the w-substituted alkyl chain length and the nature of the w-substituent on the chemistry of the compounds and relates these effects in a qualitative way to the pattern of sensitization potential and cross-reactivity observed. In a following paper we apply a more mathematical and statistical approach aimed at quantitative interpretation of the findings. Materials and Methods Caution: Skin contact with all lactone derivatives must be avoided. As sensitizing substances, these compounds must be handled with care. Chemistry. lH and l3C NMR spectra were recorded on a Bruker 200-MHz spectrometer in CDCls unless otherwise specified. Chemical shifts are reported in ppm (6) with respect to TMS, and CHC13 was used as internal standard (6 = 7.27 ppm). Multiplicities are indicated by s (singlet), d (doublet), t (triplet), and m (multiplet). Infrared spectra were obtained on a PerkinElmer spectrometer; peaks are reported in reciprocalcentimeters. Melting points were determined on a Buchi Tottoli 510 apparatus and are uncorrected. Dried solvents were freshly distilled before use. Tetrahydrofuran and ethyl ether were distilled from sodium benzophenone. Triethylamine and hexamethylphosphoramide (HMPA) were distilled from powdered calcium hydride. Methylene chloride was dried over P205 before distillation. All air- or moisturesensitivereactions were conducted in flame-driedglasswareunder an atmosphere of dry argon. Chromatographicpurifications were conducted on silica gel columns according to the flash chromatography technique. 2-Methyl-4-penten-2-01 (4). To acetone (30.6 mL, 422.4 mmol) in a saturated solution of NH4C1 (100 mL) were added THF (20 mL), allyl bromide (10.15 mL, 119.9 mmol), and zinc (14.11 g, 215.8 mmol) by fractions to maintain a gentle reflux. The mixture was stirred for 1 h at 25 "C. The suspension (containing zinc salts) was extracted with diethyl ether, and combined organic layers were dried over MgS04, filtered, and then evaporated under reduced pressure. The crude alcohol was purified by distillation (110 "C; 1 0 - 2 mmHg) to give 6.12 g (51% yield) of 4 as a colorless liquid: 1H NMR (CDCl3) 6 1.21 (s,6H, CH3), 1.57 ( 8 , lH, OH), 2.22 (d, 2H, CH2, J = 7.4 Hz), 5.05-5.17 (m, 2H, CH2=, J = 9.9 Hz), 5.77-5.98 (m, lH, J = 7.4 Hz, J = 9.9 Hz); IR (Cc4) v 3618, 3470 (0-H). 5,5-Dimethyldihydro-2(3H)-furanone (5). To a stirred solution of alcohol 4 (13.22 g, 131.98 mmol) in dry diethyl ether (130 mL) was added, at -78 "C, a solution of borane-methyl sulfide complex in diethyl ether (66 mL, 131.98 mmol, 2 N). The mixture was allowed to warm up to room temperature and was further stirred for 12h. A solution of chromicacid (396.38 mmol) [prepared from NazCr20~2H20(118.12 g), concentrated HzS04 (86 mL), and 460 mL of water] was added dropwise, at 0 OC, to the mixture, whichwas then heated to reflux for 1h. The ethereal layer was separated, washed with brine, and dried over MgS04.
Removal of diethyl ether and distillation (56-58 "C; 10-2mmHg) gave 12.20 g (81% yield) of 5 as a colorless liquid: lH NMR (CDCl3) 6 1.41 ( 8 , 6H, CH3), 2.04 (t, 2H, CHz, J = 8.0 Hz), 2.60 (t, 2H, CH2, J = 8.0 Hz); IR (CCq) v 1759 ( C 4 ) . Anal. Calcd for CeHloO2: C, 63.13; H, 8.83. Found C, 62.91; H, 8.93. 3 4Hydroxymethyl)-5,5-dmethyldihydro-2( BB)-furanone (6). To a solution of lithium diisopropylamine (LDA),prepared at -40 OC from diisopropylamine (3.5 mL, 24.99 mmol) in THF (90 mL) and a solution of butyllithium in THF (18.93 mL, 24.99 mmol, 1.32 M),was added dropwise at -78 "C a solution of lactone 5 (2.74 g, 23.8 mmol) in THF (90 mL). The mixture was stirred for 15 min at -78 "C, and lithium chloride (3.03 g, 71.4 mmol) was added. The solution was stirred for an additional 30 min at -78 "C, and gaseous formaldehyde (1g, 33.3 mmol, generated from paraformaldehyde at 150 "C) was passed through the reaction mixture with the aid of a dry nitrogen stream. The reaction mixture was stirred for an additional 3 h at -78 "C, hydrolyzed with a saturated solution of NH4Cl (150 mL), and extracted with ethyl acetate (3 X 50 mL). Organic layers were dried over MgSO4, solventswere removed under reduced pressure, and the crude lactone was purified by column chromatography (20% AcOEt/hexane) to give 2.37 g (69% yield) of alcohol 6 as a pale yellow liquid lH NMR (CDCl3) 6 1.41 (s,3H, CHs), 1.49 (s,3H, CHs), 1.98 (A part of an ABX system, lH, CH, JAB= 12.6 Hz, JM = 12.2 Hz), 2.18 (B part of an ABX system, lH, CH, JW = 12.6 Hz, JBX= 9.7 Hz), 2.92-2.08 (X part of two ABX systems,
m,lH,CH),3.77(ApartofanABXsystem,lH,CHO,Jm=ll.2 Hz, Jm = 6.1 Hz), 3.93 (B part of an ABX system, lH, CHO, Jm = 11.2 Hz, Jex = 4.7 Hz); '3C NMR (CDCls) 6 27.2, 28.5, 37.1, 43.3,60.9,83.2,177.8; IR (CC4) v 3620,3500 (0-H), 1756 (CEO). Anal. Calcd for C7H12O3: C, 58.31; H, 8.39. Found C, 58.06; H, 8.63. 3-(2-Methyl-2-propenyl)dihydro-2(3R)-furanone ( 8 ) . To a solution of LDA prepared at -40 "C from diisopropylamine (2.02 mL, 14.4 mmol) and butyllithium (10.02 mL, 14.4 mmol, 1.42M)inTHF (10mL) wereaddeddropwiseat-78"Casolution of y-butyrolactone (1 mL, 13.12 mmol) in THF (10 mL) and hexamethylphosphoramide (4 mL). The mixture was stirred at -78 OC for 30 min, 3-iodo-2-methylpropene (2.5 g, 13.7 mmol) (prepared from 3-chloro-2-methylpropene and NaI in acetone) was added, and the solution was stirred for an additional 20 min at -78 "C. The reaction was hydrolyzed at -78 "C with a saturated solution of ammonium chloride (20 mL) and extracted with diethyl ether (2 X 25 mL). Combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. Purification by column chromatography (10% AcOEt/hexane) gave 1.47 g (80% yield) of 8 as a colorlessliquid: lH NMR (CDCh) 6 1.74 (t, 3H, CH3,J= 0.7 Hz), 1.85-2.17 (m, 2H, CH2), 2.25-2.45 (m, lH, CH), 2.60-2.80 (m, 2H, CH&, 4.14-4.40 (m, 12 peaks, 2H, CH20), 4.74 (t, lH, CH=, J = 0.7 Hz), 4.82 (9, lH, CH=); '3C NMR (CDCl3) 6 21.8,28.1,37.5,38.4,66.4,112.3,142.1,179.2; IR (CHCl3) v 1780 (C=O). 3 4Hydroxyethyl)-5,5-dimethyldihydro-2(3H)-furanone (10). To mercuric acetate (454.7 mg, 1.43 mmol) in water (1.5 mL) wereaddeddiethylether (1.5mL) and8 (200mg, 1.43mmol), and the suspension was vigorously stirred for 30 min. A solution of sodium hydroxide (0.7 mL, 6 N) was then added followed by sodium borohydride (38.4 mg, 0.75 mmol) in sodium hydroxide (1.5 mL, 3 M). The borohydride solution was added at a rate such that the reaction mixture could be maintained at or below 25 "C with an ice bath. The reaction mixture was stirred at room temperature for 2 hand mercury removed by filtration on Celite. The solution was acidified with a solution of 10% HCl and p-toluenesulfonic acid (2 g) added. The mixture was stirred for 12 h and extracted with dichloromethane, dried over magnesium sulfate, and solvent evaporated under vacuum. Purification by column chromatography (20% AcOEt/hexane) gave 174.2 mg (77% yield) of 10 as a colorless liquid: lH NMR (CDCl3) d 1.39 (s,3H, CH3),1.47 (s,3H, CH,), 1.65-1.81 (m, lH, CHCHzO),1.80 (A part of an ABX system, lH, CHCMe20, Jm = 12.6 Hz, JM
Synthesis and Reactivity of Lactone Deriuatiues
= 12.1Hz), 2.00-2.16 (m, lH, CHCHzO), 2.35 (B part of an ABX, lH, CHCMe20, JAB= 12.6 Hz, JBX= 8.9 Hz), 2.39 (e, lH, OH), 2.87-3.05 (X part of an ABX system, m, IH, CH), 3.65-3.93 (m, 2H, CH2O); '3C NMR (CDCla) 6 26.6, 28.6, 33.2, 38.1,41.2,60.0, 82.8,179.2; IR (CHCla) Y 3684,3482 (0-H), 1752 (C=O). Anal. Calcd for C&I1403: C, 60.74; H, 8.92. Found C, 60.50; H, 9.08. 3-(3'-Bromopropy1)-5,5'-dimet hyldihydro-2(3H)-f uranone (3b). To a solution of LDA (8.76 mmol) in THF (9 mL) prepared at -40 OC from diisopropylamine(1.23 mL, 8.76 mmol) and butyllithium (2.26 mL, 8.76 mmol, 1.4 M in hexane) were added at -78 "C a solution of lactone 5 (1g, 8.76 mmol) in THF (9 mL) and HMPA (1.8 mL, 10.5 mmol). The mixture was stirred at -78 "C for 30 min, and a solution of l,&dibromopropane (0.98 mL, 9.64 mmol) in THF (9 mL) was added dropwise. The mixture was stirred at -40 OC for 1 h and hydrolyzed with a saturated solution of NH4C1. The solution was extracted with ether (3 X 10 mL), dried over magnesium sulfate, and solvent evaporated under vacuum. Purification by column chromatography (10% AcOEt/hexane) gave 784.5 mg (38% yield) of 3b as a colorless liquid (9). 1H NMR (CDCl3) 6 1.38 (a, 3H, CHs), 1.47 (a, 3H, CHa), 1.53-1.71 (m, lH, CHCHCO), 1.74 (A part of an ABX system, lH, CHCMe20, JAB= 12.6 Hz, J m = 12.0 Hz), 1.87-2.09 (m, 3H, CHzCHzBr CHCHCO), 2.27 (B part of an ABX system, 1H, CHCMezO, JAB= 12.6 Hz, JBX= 8.9 Hz), 2.68-2.82 (m, lH, CH, J m = 12.0 Hz, JBX= 8.9 Hz), 3.39-3.48 (m, 2H, CH2Br); '3C NMR (CDCl3) 6 26.8, 28.7, 29.2, 30.2, 32.9, 39.7, 41.0, 82.0, 177.6; IR (CHCl3) v 1763 (C=O).
+
3-(Chloromethyl)-5,5-dimethyldihydro-2(3H)-furanone (la). To a solution of alcohol 6 (100 mg, 0.694 mmol) and tetrachloromethane (129 mg, 0.90 mmol) in dichloromethane (2.5 mL) was added at 0 "C triphenylphosphine (473.28 mg, 1.80 mmol). Temperature was allowed to warm up to 25 "C and the mixture stirred for 20 h. Hexane (5 mL) was addedto the mixture to precipitate triphenylphosphine oxide, and the solution was filtered over Celite. The organic layer was concentrated under reduced pressure to give the crude chloride which was purified by columnchromatography(20%AcOEt/hexane),yielding 112.86 mg (quantitative yield) of compound la as pale yellow liquid 'H NMR (CDCla) 6 1.38 (s,3H, CH3), 1.47 (8,3H, CH3), 2.11 (A part of an ABX system, lH, CHCMe20, JAB= 15.5 Hz, J m = 10.8 Hz), 2.30 (B part of an ABX system, lH, CHCMe20, JAB= 15.5 Hz, JBX=9.2 Hz), 3.21-3.30 (X part of an ABX system, m, lH, CH), 3.78 (d, 2H, CH2C1, J = 5.1Hz); '3C NMR (CDC13) 6 27.4,28.6, 38.4, 43.2,43.3, 82.7, 174.6; IR (CCL) Y 1772 (C=O). Anal. Calcd for C7HllC102: C, 51.70; H, 6.82. Found C, 51.92; H, 6.61.
3-(Bromomethyl)-5,5-dimethyldihydro-2(3H)-furanone (lb). The same procedure as for the synthesis of chloride la
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 299 7.42; C1, 20.07. Found: C, 54.63; H, 7.59; C1, 19.91.
3-(2-Bromoethyl)-5,5-dimethyldihydro-2(3a)-furanone (2b). The same procedure as for the synthesis of la (3 g, 18.96 mmol) was used. The crude bromide was purified by column chromatography (10% AcOEt/hexane) to give 3.10 g (74% yield) of 2b as a colorless liquid 1H NMR (CDCl3) 6 1.40 (s,3H, CHa), 1.47 (a, 3H, CH3),1.72 (Apart of an ABX system, lH, CHCMe20,
JAB= 12.4 Hz, J m = 12.1 Hz), 2.34 (Bpart of an ABX system, lH, CHCMe20, JAB= 12.4 Hz, JBX= 8.6 Hz), 1.86-2.04 (m, lH, CHZCHZBr), 2.36-2.53 (m, lH, CHzCHZBr), 2.93-3.10 (m, lH, CH, J m = 12.1 Hz, JBX= 8.6 Hz), 3.40-3.71 (m, 2H, CHZBr); '3C NMR (CDCl3) 6 26.8, 28.7, 30.8, 33.7, 39.1, 40.9, 82.3, 177.3; IR (CHC4) Y 1762 ( ( 2 4 ) . Anal. Calcd for C&IlSBrOz: C, 43.46; H, 5.93; Br, 36.14. Found C, 43.72; H, 6.08; Br, 35.72.
3-[(Mesyloxy)methyl]-5,5-dimethyldihydro-2(3H)-furanone (IC). To a solution of mesyl chloride (0.267 mL, 3.45 mmol) in pyridine (4 mL) was slowly, added at 0 "C, a solution of alcohol 6 (314mg, 2.3 mmol) in pyridine (2 mL). The solution was stirred
at 0 OC for 2 h, neutralized with 10% HCI, and extracted with ethyl acetate. Organic layers were dried over MgSOd, solvents were evaporated and the crude compound was purified by column chromatography (50% AcOEt/hexane) to give 521.4 mg (68% yield) of IC as a white solid mp 53-55 "C; lH NMR (CDCb) 6 1.42 (8, 3H, CH3), 1.51 (8, 3H, CH3), 2,13 (A part of an ABX system, lH, CHCMe20, JAB= 12.8 Hz, J m = 12.2 Hz), 2.31 (B part of an ABX system, lH, CHCMeZO, JAB= 12.8 Hz, JBX= 9.2 Hz), 3.05 (e, 3H, CHB), 3.10-3.25 (m,X part of 2 ABXsystems, lH, CHI, 4.45 (A part of an ABX system, lH, CHO, JAB= 10.4 Hz, JU = 2.7 Hz), 2.31 (B part of an ABX system, lH, CHO, JAB = 10.4 Hz, JBX= 4.1 Hz); IR (CCL) Y 1772 (C-O), 1372 ( S - 0 ) . Anal. Calcd for CsH1,0& C, 43.23; H, 6.35. Found: C, 43.49; H, 6.54. 3 4[(Met hylsulfonyl)oxy]ethyl]-5,5-dimethyldihydro2(3a)-furanone (2c). The same procedure as for the synthesis of IC from alcohol 10 (2.2 g) was used. Purification by column chromatography (50% AcOEt/hexane) gave 2.96 g (90% yield) of 2c as a white solid mp 47-48 "C; lH NMR (CDCl3) 6 1.38 (a, 3H, CH3),1.46 (a, 3H, CHs), 1.76 (A part of an ABX system, lH, CHCMe20, JAB= 12.6 Hz, J m = 12.2 Hz), 1.80-2.33 (m, 2H, CHZCHZO), 2.35 (B part of an ABX system, lH, CHCMe20, JAB = 12.6Hz, J B X =8.1Hz), 1.80-2.02 (m, lH,CH2CH20),2.20-2.36 (m, lH, CHZCH~O), 2.83-2.99 (m, lH, CH), 3.02 (a, 3H, CHsS), 4.29-4.50 (m, 2H, CH20); l3C NMR (CDCl3) 6 26.8, 28.8, 30.4, 37.3,37.3,41.3,87.6,82.6,177.3; IR (CHCl3) Y 1759 (C=O), 1358 (S=O). Anal. Calcd for CgHleO&: C, 45.75; H, 6.83; S, 13.57. Found: C, 45.97; H, 6.97; S, 13.60.
5,S-Dimethyl-3-[(tosy1oxy)met hylldihydro-2(3m-furanone (ld). To a solution of alcohol 6 (3 g, 20.81 mmol) and
pyridine (11.76 mL) in dichloromethane (60 mL) were added at 25 "C dimethylaminopyridine(249.1mg) and dropwise a solution of tosyl chloride (5.95 mg, 31.21 mmol) in dichloromethane (10 mL). The mixture was stirred for 24 h and neutralized with a solution of 10% HCI. The organic layer was washed twice with 10%HCl and then with brine. Removal of solvents under reduced pressure and purification by column chromatography (25% AcOEt/hexane) gave 3.41 g (55%) of compound Id as a white solid: mp 53-55 "C;lH NMR (CDCh) 6 1.35 (a, 3H, CHs), 1.46 J~x=4.0H~);'3CNMR(CDCl3)627.3,28.2,31.3,39.6,42.9,82.7,(a, 3H, CHs),2.05 (A part of an ABX system, lH, CHCMe20, JAB = 12.9 Hz,J m = 12.1 Hz), 2.31 (B part of an ABX system, lH, 174.5; IR (CCL) Y 1778 (C=O). Anal. Calcd for C7H11Br02: C, 40.60; H, 5.35; Br, 38.59. Found C, 40.74; H, 5.45; Br, 40.45. CHCMe20, JAB= 12.9 Hz, JBX= 9.2 Hz), 2.46 (a, 3H, CH&, 3-(2-Chloroethyl)-5,5-dimethyldihydro-2(3H)-furanone 3.04-3.19 (X part of 2 ABX systems, m, lH, CH), 4.16 (A part of an ABX system, lH, CHO, JAB= 10.0 Hz, J m = 6.7 Hz), 4.33 (2a). The same procedure as for the synthesis of la (3 g, 18.96 (B part of an ABX system, lH, CHO, JAB= 10.0 Hz, J m = 3.9 mmol) was used. The crude chloride was purified by column Hz), 7.35 (AA' part of an AA'XX' system, 2H, ArH, J m = 8.7 chromatography (10% AcOEt/hexane) togive 2.44 g (73% yield) Hz, Jfii = 1.9 Hz, Jmi -0.3 Hz), 7.77 (XX' part of an AA'XX' of 2b as a colorless liquid lH NMR (CDCl3) 6 1.40 (a, 3H, CHs), system, 2H, ArH, J m = 8.7 Hz, JXX.= 1.9 Hz, J u t = -0.3 Hz); 1.48 (a, 3H, CHa), 1.74 (A part of an ABX system, lH, CHCMe20, '3C NMR (CDCl3, 6 21.5, 27.1, 28.5, 37.8, 41.0, 66.1, 83.0, 127.8, JAB= 12.5 Hz, J m = 12.2 Hz), 2.35 (B part of an ABX system, 129.9,132.2,145.1,174.0;IR (CC4) v 1772 (C=O), 1374 (s----O). lH, CHCMe20, JAB= 12.5 Hz, JBX= 8.8Hz), 1.78-1.96 (m, lH, Anal. Calcd for C14HlsO5S: C, 56.36; H, 6.08. Found C, 56.20; CH&HZCl), 2.32-2.46 (m, lH, CHzCH2Cl), 2.93-3.10 (m, lH, H, 6.23. CH, J m = 12.2 Hz,JBX= 8.8 Hz), 3.55-3.85 (m, 2H, CH2C1); '3C 34[[(4-Methoxyphenyl)sulfonyl]oxy]methyl]-5,5-dimethNMR (CDCl3) 6 26.5, 28.5, 33.3, 37.8, 40.7, 42.3, 82.1, 177.3; IR yldihydro-2(3H)-furanone (le). The same procedure as for (CHCl3) Y 1773 (C=O). Anal. Calcd for C&I&lO2: C, 54.40; H, using CBr4 instead of CC4 was used. The mixture was stirred for 30 min at room temperature to give 143.70 mg (quantitative yield) of compound lb as a colorless liquid lH NMR (CDCla) 6 1.42 (8, 3H, CH3), 1.52 (8, 3H, CH3), 2.10 (A part of an ABX system, lH, CHCMe20, JAB= 12.4 Hz, J m = 12.3 Hz), 2.36 (B part of an ABX system, lH, CHCMe20, JAB= 12.4 Hz, JBX= 8.7 Hz), 3.21-3.37 (X part of two ABX systems, m, lH, CH), 3.61 (A part of an ABX system, lH, CHBr, JAB= 10.5 Hz, J m = 7.3 Hz), 3.69 (B part of an ABX system, lH, CHBr, JAB= 10.5 Hz,
300 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
Franot et al.
the synthesis of Id (2.5 g) was used. The reaction time was 3 gave 2.01 g (71% yield) of 2f as a white solid: mp 56-57 "C; 1H NMR (CDCl3) 6 1.34 (5,3H, CH3), 1.44 (8,3H, CH3), 1.70 (A part days. Purification by column chromatography (35%, AcOEt/ of an ABX system, lH, CHCMe20, JAB = 12.6 Hz, Jm = 11.7 hexane) gave 2.18 g (40% yield) of compound le as pale yellow Hz), 2.26 (B part of an ABX system, lH, CHCMe20, JAB= 12.6 liquid 1H NMR (CDCl3) 6 1.38 (s,3H, CHs), 1.48 (8, 3H, CH3), Hz, JBX= 8.7 Hz), 1.69-1.89 (m, lH, CH2CH20), 2.15-2.28 (m, 2.05 (A part of an ABX system, lH, CHCMe20, JAB= 12.9 Hz, lH, CH2CH20), 2.75-2.92 (m, lH, CH),4.1Ck4.31 (m, 2H, CH20), Jm = 12.2 Hz), 2.31 (B part of an ABX system, lH, CHCMe20, 7.23-7.67 (m, 3H,ArH), 7.88-7.93 (m, 2H, ArH); l9C NMR (CDCb) JAB 12.9 Hz, JBX = 9.3 Hz), 3.04-3.19 (X part of two ABX 6 26.7,28.7,30.2, 37.4,41.3, 68.6,82.5, 127.7, 129.3, 133.9,135.8, systems, m, lH, CH), 3.89 (s,3H, CH30),4.14 (A part of an ABX 177.2; IR (CHCls) v 1759 ( C 4 ) , 1365 (S=O). Anal. Calcd for system, lH, CHO, JAB= 10.0 Hz, J A =~6.5 Hz), 4.31 (B part of Cl4HlsO&3: C, 56.36; H, 6.08; S, 10.75. Found C, 56.53; H, 6.19; an ABX system, lH, CHO, JAB= 10.0 Hz, JBX= 3.8 Hz), 7.02 S, 10.61. (AA' part of an AA'XX' system, 2H, ArH, J u = 9.6 Hz, Jut = 2.5 Hz, J u t = -0.6 Hz), 7.77 (XX' part of an AA'XX' system, 2H, 34[[(4-Chlorophenyl)sulfonyl]oxy]methyl]-5,5-dimethArH, Jm = 9.6 Hz, J X X=~ 2.5 Hz, Jmt = -0.6 Hz); l3C NMR yldihydro-2(3a)-furanone (lg). To a solution of alcohol 6 (CDCla)6 27.0,28.4,37.7,40.9,55.6,67.9,82.8,114.5,126.6,130.0, (2.5g, 17.34mmol)inTHF(25mL)wasaddedat-78OCasolution 163.9,173.9;IR (CCL) v 1773 (C=O), 1375 (S=O). Anal. Calcd of LDA in THF [prepared at -40 "C from diisopropylamine (2.43 for C14HlsO&: C, 53.49; H, 5.77; S, 10.20. Found: C, 53.75; H, mL, 17.34 mmol) in THF (25mL) and a solution of buthyllithium 5.87; S, 10.20. in hexane (12.6 mL, 1.38 M)]. The solution was stirred at -78 3-[[(Phenylsulfonyl)oxy]methyl]-5,5-dimethyldihydro"C for 30 min, and a solution of sulfonyl chloride (2 equiv) in 2(3a)-furanone (If). The same procedure as for the synthesis THF (25 mL) was added dropwise at -78 "C. The reaction of Id (2.5 g) was used. The reaction time was 12 h. Purification mixture was hydrolyzed at -78 "C with a saturated solution of by recrystallization in diethyl ether gave 2.71 g (55% yield) of NH&l and acidified with 10% HC1, and the temperature was raised to room temperature. The mixture was extracted with compound If as a white solid: mp 72-73 "C; lH NMR (CDCl3) 6 1.38 (8, 3H, CH3), 1.47 (8, 3H, CH3), 2.04 (A part of an ABX dichloromethane and dried over magnesium sulfate, and the solvent was removed under reduced pressure. Purification by system, lH, CHCMe20, JAB = 12.9 Hz, Jm = 12.3 Hz), 2.30 (B part of an ABX system, lH, CHCMe20, JAB= 12.9 Hz, JBX= column chromatography (30% AcOEt/hexane) and recrystallization in a diethyl ethedhexane mixture gave 2.93 g (53% yield) 9.4 Hz), 3.08-3.15 (X part of two ABX systems, m, lH, CH), 4.19 of compound lg as a white solid: mp 82-84 "C; lH NMR (CDCl3) (A part of an ABX system, lH, CHO, JAB= 10.1 Hz, Jm = 6.4 6 1.39 (5, 3H, CH3), 1.49 (8, 3H, CH3), 2.06 (A part of an ABX Hz), 4.34 (B part of an ABX system, lH, CHO, JAB= 10.1 Hz, system, lH, CHCMe20, JAB= 12.9 Hz, J u = 12.1 Hz), 2.31 (B JBX = 3.7 Hz), 7.56-7.60 (m, 2H, ArH, J = 7.6 Hz), 7.66-7.92 (m, part of an ABX system, lH, CHCMe20, JAB = 12.9 Hz, JBX= lH, ArH, J = 7.6 Hz), 7.90-7.92 (m, 2H, ArH, J = 7.2 Hz); 13C 9.2 Hz), 3.05-3.20 (X part of two ABX systems, m, lH, CH), 4.21 NMR (CDCls) 6 27.1, 28.5, 37.7, 41.0, 68.3, 83.0, 127.8, 129.3, (A part of an ABX system, lH, CHO, JAB= 10.0 Hz, Jm = 6.1 134.0,135.3,174.0;IR (CCL) v 1775 (C=O), 1375 (S=O). Anal. Hz), 4.35 (B part of an ABX system, lH, CHO, JAB= 10.0 Hz, Calcd for C13Hle05S: C, 54.92; H, 5.67; S, 11.28. Found C, 55.21; H, 5.75; S, 11.28. JBX= 3.9 Hz), 7.55 (AA' part of an AA'XX' system, 2H, ArH, Jm = 9.2 Hz, Jut = 2.2 Hz, JAX,= -0.4 Hz), 7.84 (XX' part of 5,5-Dimethyl-3-[ (tosyloxy)ethyl]dihydro-2(3a)-furaan AA'XX' system, 2H, ArH, J u = 9.2 Hz, JXXf = 2.2 Hz, Jm none (2d). The same procedure as for the synthesis of Id from = -0.4 Hz); 13C NMR (CDCla) 6 27.2, 28.6, 37.8, 41.1, 68.5, 83.1, alcohol 10 (1.5 g) was used. The reaction time was 12 h. 129.3, 128.7, 133.9, 140.8, 173.9; IR (CC4) v 1772 (C=O), 1376 Purification by column chromatography (25% AcOEt/hexane) (S=O). Anal. Calcd for ClsH&10$: C, 48.98; H, 4.74; S, 10.06; gave 2.22 g (75% yield) of 2d as a white solid: mp 95-96 "C; lH C1, 11.12. Found C, 48.88; H, 4.75; S, 10.29; C1, 11.17. NMR (CDCl3) S 1.35 (5,3H, CH3), 1.44 (8,3H, CH3), 1.71 (A part 5,S-Dimethyl-3-[[[(4-nitrophenyl)sulfonyl]oxy]methyllof an ABX system, lH, CHCMe20, JAB= 12.6 Hz, Jm = 12.2 Hz), 2.28 (B part of an ABX system, lH, CHCMe20, JAB= 12.6 dihydro-2(3a)-furanone(lh). The same procedure as for the synthesis of compound lg was used. Purification by column Hz, JBX= 8.8 Hz), 1.67-1.84,2.15-2.31 (m, 2H, CH2CH20),2.45 (8, 3H, CH3),2.75-2.92 (m, lH, CH), 4.08-4.28 (m, 2H, CHzO), chromatography (80% CHzCl~/hexane)gave 3.65 g (64%) of 7.36 (AA' part of an AAXX' system, 2H, ArH, J f i = 8.7 Hz, J u t compound lh as a pale yellow solid mp 119-121 "C; 1H NMR = 1.9 Hz, Jm, = -0.3 Hz), 7.79 (XX' part of an AA'XX' system, (CDCl3) 6 1.40 (5,3H, CH3), 1.50 (8, 3H, CH3), 2.09 (A part of an ABX system, lH, CHCMe20,JAB= 12.8Hz, J u = 12.2 Hz), 2.32 2H, ArH, Jm = 8.7 Hz, JXX,= 1.9 Hz, Jml = -0.3 Hz); 13C NMR (CDCls)6 21.6,26.7,28.8,30.2,37.6,41.4,68.4,82.5,127.8,129.9,(B part of an ABX system, lH, CHCMe20, JAB = 12.8Hz, J ~ x = 9.2 Hz), 3.04-3.22 (X part of two ABX systems, m, lH, CH), 132.9,145.0,177.3;IR (CHCl3) v 1759 (C=O), 1365 ( S 4 ) . Anal. 4.31 (A part of an ABX system, lH, CHO, JAB= 10.0 Hz, Jm Calcd for C15Hm05S:C, 56.36; H, 6.08; S, 10.75. Found C, 56.53; = 5.4 Hz), 4.35 (B part of an ABX system, lH, CHO, JAB= 10.0 H, 6.19; S, 10.61. (AA'partofanAA'XX'system,2H,ArH, Hz,J~x=3.6Hz),8.11 3-[[[(4-Methoxyp henyl)sulfonyl]oxy ]et hyl1-5,bdimethJ A =~9.4 Hz, Jui = 2.2 Hz, Jm = -0.3 Hz), 8.42 (XX' part of yldihydro-2(3a)-furanone (20). The same procedure as for an AA'XX' system, 2H, ArH, Jm = 9.4 Hz, J X X=~ 2.2 Hz, Jug the synthesis of Id from alcohol 10 (1.5 g) was used. The reaction = -0.3 Hz); 13C NMR (CDCl3) 6 27.2, 28.6, 37.5, 41.0, 69.0, 83.1, time was 24 h. Purification by column chromatography (40% 124.5, 129.4, 141.0, 150.9, 173.7; IR (CC4) v 1765 (C=O), 1352 AcOEt/hexane) gave 2.68 g (86% yield) of 2e as a white solid: (5410).Anal. Calcd for ClsHlaNO,S: C, 47.41; H, 4.59; N, 4.25; mp 74-75 "C; 1H NMR (CDCl3) 6 1.35 (8, 3H, CH3), 1.44 (8, 3H, S, 9.74. Found: C, 47.44; H, 4.54; N, 4.15; S, 9.75. CH3),1.70 (A part of an ABX system, lH, CHCMe20, JAB= 12.2 Hz,Jm= 12.4Hz),2.35(BpartofanABXsystem,lH,CHCMe20, 3 4[ [(4-Chlorophenyl)sulfonyl]oxy]ethyl]-5,5-dimethJAB= 12.2 Hz,JBX= 8.7 Hz), 1.66-1.84, 2.16-2.30 (m, 2H, CHzyldihydro-2(3H)-furanone (2g). To a solution of alcohol 10 (m, CH20),2.75-2.92 (m,lH,CH),3.88(s,3H,OCH3),4.06-4.26 (1.5 g, 9.48 mmol) in triethylamine (6 mL) and dichloromethane 2H, CH20),7.02 (AA' part of an AA'XX' system, 2H, ArH, J A ~ (15mL) was added at 0 "C a solution of (4-chlorophenyl)sulfonyl = 9.5 Hz, J u t = 2.5 Hz, Jm, = -0.5 Hz), 7.79 (XX' part of an chloride (3.01 g, 14.22 mmol) in dichloromethane (15 mL). The AA'XX' system, 2H, ArH, Jm = 9.5 Hz, Jxx, = 2.5 Hz, J,w = mixture was stirred at room temperature for 12 h, extracted with -0.5 Hz); "C NMR (CDC13) d 21.6, 26.7, 28.8, 30.2, 37.6, 41.4, dichloromethane, and dried over magnesium sulfate, and the 68.4, 82.5, 127.8, 129.9, 132.9, 145.0, 177.3; IR (CHCls) Y 1759 solvent was evaporated under vacuum. Purification by column (C=O), 1361 (S=O). Anal. Calcd for C15H200&3: C, 54.86; H, chromatography (20% AcOEt/hexane) gave 2.33 g (74% yield) 6.14; S, 9.77. Found: C, 55.07; H, 6.22; S, 10.01. of 2g as a white solid mp 85-86 "C; lH NMR (CDC13) 6 1.37 (8, 3H, CHs), 1.46 (s,3H, CHs), 1.72 (A part of an ABX system, lH, 34[(Phenylsulfonyl)oxy]ethyl]-5,5-dimethyldihy~2(3a)CHCMe20, JAB = 12.5 Hz, JIJC= 12.2 Hz), 2.29 (B part of an furanone (2f). The same procedure as for the synthesis of Id ABX system, lH, CHCMe20,JAB= 12.5Hz, JBX= 8.9 Hz), 1.75from alcohol 10 (1.5 g) was used. The reaction time was 12 h. 1.90,2.16-2.32 (m, 2H, CH2CH20), 2.76-2.94 (m, lH, CH),4.14Purification by column chromatography (30% AcOEt/hexane)
Synthesis a n d Reactivity of Lactone Derivatives
Chem. Res. Toxicol., Vol. 7,No. 3, 1994 301
Me), 2.75 (t,2H, J = 2.7 Hz, CHzCMeZ), 5.60 (t, lH, J = 2.7 Hz, =CH), 6.19 (t,lH, J = 2.7 Hz, =CH); 13CNMR (CDCl3) 6 28.8, 41.0, 81.5, 121.8, 136.0, 169.6; IR (CC4) v 1772 (C=O). Anal. Calcd for C7H1002: C, 66.65; H, 7.99. Found C, 66.37; H, 8.17. Relative Rate Constants. Most of the compounds' reactivities (all sulfonates and thiocyanato compounds) were compared to the reactivities of the reference substances la or 2a except the bromo derivatives, which were compared to the reactivities of the tosylates. In an NMR tube, the compound to be tested and the comparative substance (corresponding chloro derivatives or tosylates) in an equimolar amount (0.025 mmol) were dissolved in CDCls (0.5 mL). At t = 0, n-butylamine (2.47 pL, 0.025 mmol) was added and a lH NMR spectrum was recorded. At t = (48h for compounds of series 1 and 27 days for compounds of series 2), another spectrum was recorded using the same acquisition and treatment conditions. The proportion of each consumed compound was measured by integration of characteristic signals of starting materials and of the formed compound. Absolute Rateconstants. InanNMRtube, tothecompound to be tested (0.025 mmol) in CDCl3 (0.5 mL) was added n-butylamine (12 pL, 0.121 mmol). A t t = 0, a1H NMR spectrum was recorded and then at a constant interval of time (20 min during formation of the methylene lactone, then 7 h for series 1 and 24 h for series 2)until the completion of the reaction. The proportion of consumed material or formed product was measured 5~-Dimethyl-3-(thiocyanatomethyl)dihydro-2(3R)-fura- by integration of characteristic signals of the starting material or of the formed compound. none (li). To a solution of the tosyl derivative Id (500 mg, 1.7 mmol) in DMF (10 mL) was added potassium thiocyanate (186.6 In none of the competition experiments or the absolute rate mg, 2.9 mmol). The mixture was heated to reflux and stirred for constant determinations were any products other than the 12 h. After hydrolysis with water, the mixture was extracted methylene lactone (series 1)and the compounds 1-3 with X = with dichloromethane and dried over magnesium sulfate, and n-BuNH detected. solvents were removed under reduced pressure. Purification by Biological Testing. The guinea pig sensitization tests were recrystallization in a mixture of diethyl ethedhexane gave 182.6 carried out using the modified single-injection adjuvant test mg (58%)of li as a white solid mp 59-60 OC; lH NMR (CDC13) (MSIAT) (IO). Preliminary irritation tests were used to deter6 1.45 (s, 3H, CHs), 1.54 (s, 3H, CHs), 2.00 (A part of an ABX mine the concentration range suitable for induction and to ensure system, lH, CHCMe20, JAB= 12.7 Hz, Jm = 12.2 Hz), 2.53 (B that the challenge was conducted at an optimal nonirritant part of an ABX system, lH, CHCMe20, JAB= 12.7 Hz, JBX= concentration. In general, all the chemicals were tested at 8.6 Hz), 3.19-3.36 (X part of two ABX systems, m, lH, CH), 3.05 equimolar concentrations of 0.31 M, exceptions are indicated in (A part of an ABX system, lH, CHS, JAB = 13.4 Hz, JAX = 8.6 Table 3. In brief, the MSIAT protocol was as follows. SensiHz), 3.49 (B part of an ABX system, lH, CHS, JAB = 13.4 Hz, tization was induced in groups of 10albino Dunkin-Hartley guinea J~x=4.2H~);"CNMR(CDC13)626.9,28.8,34.3,40.3,42.2,83.1, pigs by a series of six 0.1-mL intradermal injections of test 111.5,174.6; IR (CC4) Y 2159 (C-S), 1774 (C=O). Anal. Calcd chemicals in 0.9% w/v sodium chloride vehicle, in combination for CJ411N02S: C, 51.87; H, 5.99; N, 7.56. Found: C, 51.72; H, with Freund's complete adjuvant (FCA), in the shoulder region. 6.03; N, 7.28. This part of the procedure was identical to that used for the 5,5-Dimethyl-3-(thiocyanatoethyl)dihydro-2(3H)-furaguinea pig maximization test (11). After a 2-week interval, the none (2i). The same procedure as for the synthesis of li from initial challengewas normally conducted by giving a 6-h occluded the tosyl derivative 2d (1.0 g) was used. Purification by column topical patch application of the test chemical in Finn chambers chromatography (30% AcOEt/hexane) gave 554 mg (79% yield) to clipped and shaved flank skin. The challenge vehicle was of 2i as a pale yellow solid: mp 38-39 OC; lH NMR (CDCls) 6 1.41 70/30 v/v acetone/poly(ethylene glycol) 400, and the challenge (8,3H, CHs), 1.49 (8,3H, CHa), 1.79 (A part of an ABX system, concentration was in all cases 0.31 M. Skin reactions were scored lH, CHCMe20, JAB= 12.6 Hz, Jm = 12.1 Hz), 2.36 (B part of for erythema (scale0-3) and the presence of edema approximately an ABX system, lH, CHCMe20, JAB= 12.6 Hz, JBX = 9.0 Hz), 24 and 48 h after removal of the patch. Subsequent cross1.94-2.12 (m,lH, CH2CH20),2.22-2.40 (m, lH, CHzCHzO),2.87challenges were made at weekly intervals on alternate flanks 3.05 (m, lH, CH), 3.10-3.34 (m, 2H, CH2S); 13C NMR (CDCls) using the same patch procedure and scoring system. For each 6 26.8, 28.7, 31.2, 31.4, 38.6, 41.2, 82.5, 111.6, 176.9; IR (CHC13) animal, erythema was scored as 0,0.5,1,2, or 3 on each occasion. Y 2157 (C-S), 1759 (C=O). Anal. Calcd forC~H13NOzS: C, 54.25; For each challenge the erythema scores for all animals at 24 and H, 6.28; N, 7.03; S, 16.09. Found: C, 54.20; H, 6.66; N, 6.64; S, 48 h were totaled and the total was expressed as a percentage of 15.78. the maximum possible score (this being 6 times the number of 5,5-Dimethyl-3-methylenedihydro-2(3H)-furanone (11). animals involved in the challenge). This figure was then used To a saturated solution of NHdCl(17.5 mL), THF (7 mL), and as the quantifier of the biological response. EhO (5 mL) were added acetone (1.03 mL, 14 mmol), methyl bromomethacrylate (1.4 g, 7 mmol), and zinc (0.55 g, 8.4 mmol). Results and Discussion The mixture was heated to reflux for 1h and filtered on Celite. The organic layer was extracted with ether (3 X 10 mL), dried Chemistry. Title compounds la-i and 2a-i (Chart 1) over MgSO4, and filtered, and solvents were removed under were prepared from the alcohol derivatives 6 and 10, reduced pressure. The residue was taken up in ether (35 mL), respectively. Lactone 6 was synthesized in three steps p-toluenesulfonic acid was added (0.52 g), and the mixture was according t o Scheme 1. Reaction of allyl bromide 3 with heated to reflux for 1 h to allow the lactone cyclization. The (12)in acetone gave the alcohol 4, which was submitted zinc ethereal layer was washed with water and evaporated under to hydroboration (BHrMe2S) and t h e n oxidation using vacuum to give the crude lactone, which was purified by column chromic acid to give t h e lactone 5 in 81% yield. Treatment chromatography (30% EhO/hexane) to give 565 mg (64% yield) of 5 with LDA and lithium chloride (3 equiv) and of lactone 11 as a colorless oil: 1H NMR (CDCl3) 6 1.42 (8, 6H,
4.34 (m, 2H, CHzO), 7.55 (AA' part of an AA'XX' system, 2H, ArH, Jm = 9.3 Hz, JMf = 2.2 Hz, Jmr= -0.4 Hz), 7.79 (XX' part of an AA'XX' system, 2H, ArH, Jm = 9.3 Hz, JW = 2.2 Hz, JAX' = -0.4 Hz); '3C NMR (CDCla) 6 26.8,28.8,30.3,37.4,41.4,68.8, 82.5, 129.3,129.7,134.4, 140.7, 177.2; IR (CHC13) v 1760 (C=O), 1370 (S=O). Anal. Calcd for C14Hl,C10& C, 50.53; H, 5.15; C1, 10.65; S, 9.63. Found: C, 50.68; H, 5.20; C1, 11.00; S, 9.51. 5,S-Dimethyl-3-[ [[(4-nitrophenyl)sulfonyl]oxy]ethyl]dihydro-2(3H)-furanone (2h). The same procedure as for the synthesis of 2g (1.5 g) was used. The reaction time was 4 h. Purification by successive recrystallization in methanol and column chromatography (50% AcOEt/hexane) gave 1.82 g (56% yield) of 2h as a pale yellow solid mp 117-118 OC; 'H NMR (CDC13)6 1.39 (s,3H, CH3),1.47 (8, 3H, CHd, 1.75 (A part of an ABX system, lH, CHCMe20,JAB= 12.6 Hz, JAX= 12.2 Hz), 2.33 (B part of an ABX system, lH, CHCMe20, JAB= 12.6 Hz, JBX = 8.8 Hz), 1.80-1.97 (m, lH, CHzCHzO), 2.15-2.29 (m, lH, CH2CH20), 2.79-2.96 (m, lH, CH), 4.24-4.44 (m, 2H, CHzO), 8.12 (AA' part of an AA'XX' system, 2H, ArH, Jm = 9.3 Hz, JAN = 2.3 Hz, Jmf= -0.3 Hz), 7.79 (XX' part of an AA'XX' system, 2H, ArH, Jm = 9.3 Hz, Jxxt = 2.3 Hz, J u t -0.3 Hz); '3C NMR (CDCls)6 26.8,28.8,30.3,37.2,41.4,69.4,82.6,124.5,129.2,141.5, 150.8,177.1;IR (CHCl3)v 1759 (C=O), 1355(S=O). Anal. Calcd for Cl4H17N07S: C, 48.97; H, 4.99; N, 4.08; S, 9.34. Found C, 48.99; H, 4.91; N, 3.94; S, 9.39.
Franot et al.
302 Chem. Res. Toxicol., Vol. 7,No.3, 1994 Scheme 1. Synthetic Pathway to Synthon 6
Scheme 4. Synthetic Pathway to Derivatives lc-f and 2c-f
-
n = l IC
MsCl
3
q
4
py n d I ne
n = 2 2c q
O
M
s
OH
n=l 6
X=Me
n = 2 IO
pyridine. DMAP,CH2CI2
n = I Id n=2 Id
X = O M c n = l le
n = 2 2e x O s O * C l
1) LDA, LiCl
X=H
n = I If n = 2 2f
OH
2) formaldehyde
6
Scheme 5. Synthetic Pathway to Derivatives lg,h and 2g,h
5
Scheme 2. Synthetic Pathway to Synthon 10
WOH
LDA,TW
X=CI
Ig
S02ArX
X=NO2 I h
S02ArX
X = N 0 2 2h
6
7
E,,,= 12.6 kcal
8
E,., = 9 2 kcal
Et,&, CHI&
X=CI
10
9
Scheme 3. Synthetic Pathway to Chloro Derivatives l a and 2a and Bromo Derivatives l b and 2b n = l la n = 2 2a
PPh3
CCll
IO
Scheme 6. Synthetic Pathway to Thiocyanato Derivatives li and 2i
qoTs - qscv KSCN, DMF
PPh3
n=l 6 n = 2 10
n = l lb n = 2 2b
CBq
*
q
B
r
subsequent trapping of the intermediate anion with gaseous formaldehyde (13)(generated by thermal decomposition of paraformaldehyde under a stream of dry nitrogen) led to alcohol 6 in 69% yield. Under same conditions, but without lithium chloride, the reaction was found to give byproducts such as dialkylated compounds. It is known, in the case of lithium enolates, that addition of lithium salts could lead to the cleavage of aggregates and thus increase the reactivity of such intermediates toward electrophiles (14). Lactone 10 was prepared (Scheme 2) in two steps from commercial y-butyrolactone 7. Treatment of 7 with LDA and subsequent trapping of the intermediate anion with 2-iodopropene (15)in the presence of HMPA led to 8 in 80% yield. Oxymercuration (16) of the double bond followed by equilibration under acidic conditions gave the more stable lactone 10 in 77 % yield. The relative energies of lactones 9 and 10 calculated using MM2(91) (17,18) indicate a difference of about 3.4 kcal in favor of 10 that should lead to a99.7/0.03 mixture of 10and 9. In practice, only 10 was obtained. Chlorides la and 2a and bromides l b and 2b were quantitatively prepared (Scheme 3) in one step from 6 and 10, respectively, by treatment with CCld or CBr4 and 2 equiv of triphenylphosphine in methylene chloride (19). Treatment of 6 and 10 with mesyl chloride in pyridine at 0 "C (Scheme 4) led to mesyl derivatives IC(68% yield) and 2c (90% yield), respectively. Attempts to prepare the tosyl derivative Id using the same procedure failed, giving only 32% of the expected compound but 25% of the chloride product la as a reaction byproduct. Compounds ld-f and 2d-f were therefore synthesized in methylene chloride from the appropriate sulfonyl chloride derivatives using pyridine as base and (dimethylaminoh
2g
n=l
li
n=2
2i
n = l Id n = 2 2d
Scheme 7. Synthetic Pathway to Bromo Derivative 3b 0
5
3b
pyridine as catalyst to achive a complete reaction. Attempts to use other bases such as triethylamine led to an increase of the percentage of chloro derivatives la and 2a. In the extreme, when sulfonyl chlorides bearing highly electron-withdrawing groups such as chloro or nitro were used, compound la was the major product of the reaction. Derivatives l g and -h were therefore prepared (Scheme 5) from the lithium alcoholate derived from 6 and subsequent treatment with the appropriately substituted sulfonyl chloride. Compounds 2g and -h were prepared in methylene chloride from the alcohol 10 and chloro- or nitrosulfonyl chloride using triethylamine as base. The moderate yields (5544%)obtained in the preparation of sulfonyl derivatives lc-h can be explained by the tendency of the target compounds to undergo an elimination reaction with formation of the exo-methylene derivative. Compounds 2c-h, less sensitive to elimination, were obtained in better yields (5646%1. The thiocyanato derivatives li and 2i were synthesized from Id and 2d, respectively, by treatment with potassium thiocyanate in DMF heated to reflux (Scheme 6). The bromo derivative 3b was prepared in one step from the lactone 5. Treatment of 5 with LDA and subsequent trapping of the intermediate anion with l,&dibromopropane (9) in the presence of HMPA led to 3b in 38 5% yield (Scheme 7). The a-methylene- y,y-dimethyl-y-butyrolactone 11was prepared from acetone and methyl bromomethacrylate
Synthesis and Reactivity of Lactone Derivatives
using a classical Reformatsky type reaction with zinc. Kinetic Studies. One of the key parameters in the induction of allergic contact dermatitis (ACD) together with the molar sensitization dose and the partition coefficient (octanol/water) is the chemical reactivity toward nucleophiles. For the purposes of RAI calculation the electrophilicity of a compound should be estimated using a model nucleophile and only the relative rate constant krel needs to be determined, with one compound of the series being used as an internal reference. There is no ideal global nucleophile which can model reactions between biological nucleophiles and all types of allergenic electrophile. On the basis of past experience (2, 3) we consider n-butylamine to be an adequate model for protein nucleophilic groups which react with S Nelectrophiles, ~ and it should also be suitable to model biological bases which can promote elimination reactions of appropriate compounds such as those of series 1. On the other hand, we recognize that n-butylamine is not a good model for protein nucleophilic groups which react with Michael acceptor electrophiles-for which a sulfur nucleophile would be a much better model. In the present study, s N 2 and elimination reactions were anticipated as being likely to be relevant to differences in behavior among the compounds studied. Although the compounds of series 1 can give rise by elimination to a Michael electrophile which can then react with protein nucleophiles, there is no variation in Michael reactivity since the same Michael electrophile is derived from all of the compounds 1. For these reasons we chose to use n-butylamine as the model nucleophile in the present study. The chlorides la and 2a were chosen as reference materials. The relative rate constant, defined as
was determined from the results of competition experiments in which a mixture of the test compound (X) and the chloride (Cl) was treated with a deficiency of butylamine and the concentrations [XI and [Cll were determined at the beginning ( t = 0) and the end ( t = -) of the experiment: krel = (l0g([Xl,/[Xlo)j/~l0g([C1l,/[C1lo)j
After preliminary studies, we decided to use lH NMR to determine the kxl/kcl ratio. A first step was to define the appropriate solvent for this kinetic study as it is wellknown that solvents can have a major influence on both reaction mechanisms and kinetics. With derivatives lai, two different mechanisms have to be considered for the reaction with nucleophiles: an s N 2 reaction or an elimination reaction (E2 or ElcB) followed by the subsequent addition of another molecule of nucleophile on the resulting unsaturated lactone. We, therefore, tested several solvents like CDCl3, CD30D, and MezSO-ds which have different properties in terms of polarity and acidity. CD30D is protic and the most polar, while the two other are aprotic solvents and CDC13 is the least polar. The reaction of an equimolar mixture of the chloro derivative l a and tosylate Id with 1 equiv of butylamine in the three deuterated solvents was examined. The variation of solvent did not seem to have a major influence on the reaction mechanism as, in the three cases, the 11 interformation of the a-methylene-y-butyrolactone mediate was observed. However, the elimination reaction
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 303 Table 1. Relative Kinetic Reactivity of Lactone Derivatives (Series 1 and 2)'
substituent X c1 Br OSOzCH3 OSOzArCH3 OSOzArOCH3 OSOzAr OSOZArCl OSOzArNOz SCN
series 1 la lb
series 2 kdkcl 2a 1 2b 4.1 IC 2c 3.2 Id 2d 2.5 le 2e 2.3 1f 2f 3.7 L3 2g 1.3 lh 2h 19.8 li 2i 3, react either by the E2 mechanism or by the (E1cB)I mechanism, in which deprotonation is the rate-determining step and elimination
304
Table 2. Isotopic Effect ( k ~Ratio) / b for the Reaction of la, -b, -e, -f, and -i with n-Butylamine
substituent
series 1 k d k D la 1.3 Br lb 3.6 OS02ArOCH3 le 0.8
c1
substituent X series 1 kHlkD OSOzAr If 3.6 OSOzArNOz lh 4.8 SCN li 1.0
Table 3. Absolute Rate Constants for Bromo Derivatives bromo derivative k (L.mol-l.s-l)a (1.66 f 0.28) X 103 lb (elimination reaction) lb (addition to elimination product) (1.52 f 0.04) X 1W (8.8 f 1.7) X 10-8 2b (1.81 f 0.06) X 10-5 3b a
Franot et al.
Chem. Res. Toxicol., Vol. 7, No. 3, 1994
Absoluterate constant of the tested compound with n-butylamine.
of the X group from the carbanion is faster than reprotonation of the carbanion. The results of series 2 are more homogeneous. The presumed mechanism with n-butylamine is an sN2reaction where reactivity depends on the leavinggroup. No reaction of the thiocyanato compound 2i a t room temperature was observed. Relative reactivities obtained increase in the order of their leaving groups as follows: SCN < C1< OSO2ArOMe < OSOzArMe < OSOzArH < OSOzMe < Br < OSOzArCl< OS02ArN02. This order corresponds well with leaving group effectiveness for other sN2 reactions. In this series, as was the case with series 1, a Hammett plot for the substituted aromatic sulfonates showed good linearity-this time with the nitro compound included-with a p value of 0.94 f 0.28 similar to Hammett values for established sN2 reactions ( p = 1.17,25"C) (20), supporting our assumption that the sN2 mechanism applies for the series 2 compounds. The quite good linearity supports the applicability of the lH NMR method used to calculate the relative reactivity. Absolute rate constants of the three bromo derivatives lb, 2b, and 3b were determined, using lH NMR in CDC13, by following the decrease of starting material and the increase of formed product during time. Plots of the natural logarithm of the bromo derivative concentration against time gave the pseudo-first-order rate constants, division of which by the butylamine concentration (0.484 mol/L) gave the second-order rate constant It. These rate constants are shown in Table 3. From the above data the relative rate constants can be put on a common basis, using the chloro compound of series 1 as the universal reference compound. These krei values are shown in Table 4. We conclude that with nucleophiles similar to n-butylamine (e.g., lysine units in proteins) for series 1 the elimination reaction to form the methylene lactone is faster than the subsequent Michaeladdition reaction of the latter. In all of the competition experiments involving series 1, formation of the methylene lactone was observed, confirming that the compounds of series 1 react as electrophiles with n-butylamine-like nucleophiles by the elimination1 Michael addition pathway, rather than by a single-step substitution reaction. Reactions between compounds of series 1 and the protein human serum albumin (HSA) follow the same pattern (21). We have found that the mechanism of the reaction between either the amine in organic milieu or HSA in aqueous medium and the lactone derivatives tested is identical. Both in the experiments reported here and in the HSA experiments it is found that the second stage, the Michael addition reaction, is the slower step. This would suggest that differences in
Table 4. Relative Rate Constants, log P Values, and Sensitization Test Results sensitization substituent X compd no. kmf log Pb score ( % ) E series 1:d lh 50 1.76 37 lb 8.94 1.86 70 78 2.13 2.73 183 49 1.51 1.28 li If Id le la 11
1.50 1.31 1.25 1.01 1 0.98
0.78 2.02 2.52 2.26 1.72 1.73
2h 2g 2b 2c 2f 2d 2e 2a 2i
0.24 0.09 0.05 0.04 0.04 0.03 0.03 0.01
