Chem. Res. Toxicol. 1995,8, 22-26
22
Communications Synthesis and Photocyclization of a-Methylene-y-butyrolactone-ThymineHeterodimers Elbna Gimenez-Arnau, Stbphane Mabic, and Jean-Pierre Lepoittevin” Laboratoire de Dermatochimie associC au CNRS, UniversitC Louis Pasteur, Clinique Dermatologique, CHU, F-67091 Strasbourg, France Received August 24, 199P
Two a-methylene-y-butyrolactone-thymineheterodimers 6 and 7 with 5 and 10 carbon alkyl chains, respectively, were prepared to study the photoreactivity of lactone rings toward thymine and the influence of spacer length on photoadduct geometry. Deoxygenated solutions M) of heterodimers 6 and 7 were irradiated at 365 nm in acetone. Irradiation of compound 6 (5 carbons alkyl chain) gave a single photoadduct 8 (70% yield), while irradiation of compound 7 (10 carbons alkyl chain) gave two photoadducts 9a and 9b in a 3/2 ratio (95% yield). Compound 8 was identified a s a cis-syn-endo intramolecular [2+21 photoadduct involving the 5 ” , 6 double bond of the thymine moiety and the 3,6 exomethylenic group of the lactone. Compound 9a was identified a s a cis-syn-exo intramolecular [2+2] photoadduct involving the 5”,6” double bond of the thymine moiety and the 3,6 exomethylenic group of the lactone, and compound 9b as the corresponding cis-syn-endo intramolecular [2+21 photoadduct.
Introduction Allergic contact dermatitis (ACD)’ to plants of the Asteraceae (Compositae) family is one of the major cause of phytodermatoses in Western Europe (1) and usually affects male subjects living in the countryside. The clinical aspects of “compositae dermatitis” are variable (2), ranging from acute to chronic, and the main Asteraceae allergens are sesquiterpene lactones. These molecules, which belong to six main structural groups, are well-known skin sensitizers and all contain the a-methylene-y-butyrolactone ring, a powerful electrophile (3). This function has been shown to react readily with nucleophilic residues in proteins (4) forming antigens, whose processing by the skin immune system leads to ACD. It has also been reported that chronic actinic dermatitis (CAD) may develop after sensitization to sesquiterpene lactones (5-7) in the absence of any identifiable exogenous factors. The term CAD includes the conditions of persistent light reactivity, photosensitive eczema, photosensitivity dermatitis, and actinic reticuloid (8). On monochromator testing, there is a reduction of the minimal erythematous dose (MED) to UVB, but this sometimes also extends to longer wavelengths of light (UVA and visible light) (9). CAD, with marked photosensitivity, that expresses itself even without exposure to plant material, was observed in some patients with allergic (not photoallergic) contact sensitivity to oleoresins from members of Compositae. In this disease, it
* Address correspondence to this author at Laboratoire de Dermatochimie, Clinique Dermatologique, CHU, F-67091 Strasbourg,France. Abstract published in Advance ACS Abstracts, December 1,1994. ‘Abbreviations: allergic contact dermatitis, ACD; chronic actinic dermatitis, CAD; correlation and nuclear Overhauser spectroscopy, CONOESY, heteronuclear multiple band correlation, HMBC; heteronuclear multiquantum correlation, HMQC; minimal erythematous dose, MED; nuclear Overhauser effect, NOE. @
appears that an initial contact sensitization progresses to a generalized photosensitivity state (10). Although the mechanism of ACD to sesquiterpene lactones is beginning to be understood, the origin of the CAD syndrome is still unknown. Psoralen molecules are known to increase skin photosensitivity and have therefore, in association with UV irradiation, been used extensively in dermatology (11). Photobinding to DNA is generally considered to be the molecular basis for photobiological effects (12). On the basis of this mechanism, we have investigated whether a-methylene-y-butyrolactone rings can undergo a similar, or parallel process and generate photoadducts with pyrimidine on irradiation. Intermolecular photoreaction between thymine and even a highly photoreactive molecule such as 5-methoxypsoralenhave been shown to be difficult (13) and to result in low yields of photoadducts when performed in solution (14). To optimize the potential photoreaction, two a-methylene-y-butyrolactonethymine heterodimers 6 and 7 (Scheme 1) with alkyl chains of 5 and 10 carbons, respectively, were prepared in order to study the photoreactivity of lactone rings toward thymine and the influence of spacer length on the geometry of the photoadducts. The 5 carbon chain was selected as an “optimal” length to give a cyclic photoadduct, while the 10 carbon chain should allow any kind of conformation.
Materials and Methods Caution: Skin contact with a-methylene-y-butyrolactones must be avoided. Being skin sensitizers, these compounds must be handled with care. General Methods. ‘H and I3C NMR spectra were recorded on a Bruker AM 400-MHz spectrometer in CDC13 unless otherwise specified. Chemical shifts are reported in ppm ( 6 ) with respect to TMS,and CHCl3 was used as internal standard ( 6 = 7.27 ppm). Multiplicities are indicated by s (singlet), d
0893-228x/95/2708-0022$09.00/00 1995 American Chemical Society
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 23
Communications
Scheme 1. Synthetic Pathway and Photoreaction of Heterodimers 6 and 7 in Acetone
1
f 2 - J
*
I
II
hv, acetone
~
oY5T .-"y ",,Me
0 6:n=5 7: n = 10
(doublet), t (triplet), and m (multiplet). Infrared spectra were obtained on a Perkin-Elmer 1600 FTIR spectrometer; peaks are reported in reciprocal centimeters. Melting points were determined on a Buchi Tottoli 510 apparatus and are uncorrected. U V spectra were obtained using a Hewlett Packard HP 8452 diode-array spectrometer. Dried solvents were freshly distilled before use. Tetrahydrofuran was distilled from sodium benzophenone. All air- or moisture-sensitive reactions were conducted in flame-dried glassware under an atmosphere of dry argon. Chromatographic purifications were performed on silica gel columns according to the flash chromatography technique. Irradiation experiments were carried out a t 365 nm with a 125-W high-pressure mercury lamp (Philips HPK). Spectrophotometric or analytical grade organic solvents were used; solutions were deaerated by bubbling with argon and were irradiated in Pyrex glassware. Synthesis. (A)l-(5-Dioxolanyl-l-pentyl)thymine (4).To a solution of thymine (6.03 g, 47.8 mmol, 3.0 equiv) in freshly distilled dimethyl sulfoxide (164 mL) were added bromoacetal 2 (3.56 g, 16 mmol, 1.0 equiv) and potassium carbonate (7.49 g, 54.2 mmol, 3.4 equiv). After stirring a t room temperature for 17 h, the reaction mixture was filtered and the solvent removed under reduced pressure. The residue was suspended in water (100 mL) and extracted with chloroform (3 x 70 mL). The combined organic layers were dried over MgS04 and filtered, and after concentration under vacuum, the crude product was purified by column chromatography over silica (AcOEt)to give 3.39 g (12.63 mmol, 79% yield) of alkylated thymine as a white solid, mp 92-94 "C. 'H NMR (CDC13, 200 MHz) 6 8.47 (broad S, l H , N-H), 6.97 (d, l H , H6, J = 1.2 Hz), 4.84 (t, l H , H,, J = 4.5 Hz), 4.05-3.80 (m, 4H, -0CH2CH20-), 3.69 (t, 2H, CHzN, J = 7.2 Hz), 1.92 (d, 3H, CH3, J = 1.1Hz), 1.77-1.37 (m, 8H, (CH2)4). 13CNMR (CDC13, 50 MHz) 6 164.5, 151.0, 140.4, 110.6, 104.4,64.9 (2CHz1, 48.4,33.6,29.0,26.3,23.5,12.3. IR (CHCl3) v 3396 (N-H); 2949, 2865, 1683 (C=C). Anal. Calcd for ($3HzoN204: C, 58.20; H, 7.51; N, 10.44. Found: C, 57.83; H, 7.56; N, 9.88. (B) 1-(10-Dioxolanyl-1-decany1)thymine (5). Same procedure as for compound 4 starting from thymine (1.29 g, 10.23 mmol, 3.0 equiv) and bromoacetal 3. After reaction for 20 h and purification by column chromatography over silica (80% AcOEt, hexane), 928.6 mg (2.75 mmol, 81% yield) of alkylated thymine was obtained as a white solid, mp 90-92 "C. lH NMR (CDC13, 200 MHz) 6 8.35 (broad 8 , l H , NH), 6.97 (d, l H , H6, J = 1.2 Hz), 4.84 (t, l H , H,, J = 4.7 Hz), 4.01-3.81 (m, 4H,
8: n = 5 cis-syn-endo 9a: n = 1 0 cis-syn-exo 9b: n = IO cis-syn-endo
-0CHzCH20-),3.68 (t, 2H, CHzN, J = 7.5 Hz), 1.92 (d, 3H, CH3, J = 1.1Hz), 1.67-1.22 (m, 18H, (CHZ)~).13C NMR (CDC13, 50 MHz) 6 164.4, 151.0, 140.5, 110.5, 104.7,64.9 (2CH2),48.6,33.9, 29.5 (ZCHz), 29.4 (2CH2), 29.2 (2CHz), 26.4, 24.0, 12.4. IR (CHC13) v 3394 (N-H); 2932, 2857, 1687 (C-C). Anal. Calcd for C18H30N204: C, 63.96; H, 8.94; N, 8.28. Found: C, 63.65; H, 8.96; N, 7.92. (C) 5-[5'-(2,4''-Dioxo-5"-methyl-3,4"-~hydro-1"(2"H)-
pslrimidinyl)pentyll-3.methylen~~~~y~~one (6). To compound 4 (700 mg, 2.8 mmol) in THF (16 mL) were added 16 mL of a solvent mixture (CH30(CH&OH, 37.5 mL; H20, 20 mL; CH3COOH, 10 mL; HCl conc, 0.5 mL), a-bromomethacrylic acid (707.1 mg, 4.2 mmol, 1.5 equiv), and SnClz (1.61 g, 7.0 mmol, 2.5 equiv). The reaction mixture was heated to reflux for 4.5 h, and solvents were removed under vacuum. The residue was taken up with dichloromethane (100 mL) and washed with 1%HC1 (50 mL), with water (50 mL), with a saturated solution of NaHC03, and finally with water (50 mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure and the crude lactone purified by column chromatography over silica (AcOEt) to give 648.4 mg (2.22 mmol, 79% yield) of 6 as a white solid, mp 130-132 "C. lH NMR (CDCl3) 6 8.65 (broad s, l H , NH), 6.97 (d, l H , He, J = 1.1Hz), 6.23 (t, lH, Hc or HD,J = 2.8 Hz), 5.62 (t, l H , Hc or HD,J = 2.31 Hz), 4.49 (m, l H , Hx), 3.69 (t, 2H, C&N, J = 7.20 Hz), 3.06 (m, l H , HAor HB,detriplet B part of an ABX system, JAB= 17.2 Hz, JBX= 7.6 Hz), 2.56 (m, lH, HAor HB,detriplet A part ABX system, JAB= 17.2 Hz, JAX= 6.1 Hz), 1.92 (d, 3H, CH3, J = 1.2 Hz), 1.69-1.20 (m, 8H, (CH2)4). 13CN M R (CDCl3) 6 170.3,164.5,151.1, 140.4,134.6, 122.1,110.8, 77.3,48.3,36.1, 33.6, 28.9, 26.1, 24.6, 12.4. IR (CHCl3) Y 3396 (N-H); 2941, 2862,1758 (C=O lactone); 1682 (C=O thymine). UV (CH3CN) 1 209 ( E = 26000), 272 ( E = 13084). Anal. Calcd for C15H20N204: C, 61.63; H, 6.89; N, 9.58. Found: C, 61.48; H, 6.99; N, 9.24. (D)5410-(2",4"-Dioxo-S"-methyl-~,4-dihydrol"(2H)pyrimidinyl)decyl]-3-methylene-2-tetrahydrofuranone (7). Same procedure as for compound 6 starting from 532 mg (1.82 mmol) of compound 5. After purification by column chromatography over silica (80%AcOEt, hexane), 491.3 mg (1.35 m o l , 73.8% yield) of 7 was obtained as a white solid, mp 132-134 "C. lH NMR (CDCl3)6 8.51 (broad s, lH, NH),6.98 (d, l H , H6, J = 1.13 Hz), 6.22 (t, l H , Hc or HD,J = 2.87 Hz), 5.62 (t, lH, H, or HD, J = 2.51 Hz), 4.57-4.44 (m, l H , Hx), 3.76 (t, 2H, CHzN, J = 7.26 Hz), 3.13-2.98 (m, l H , HAor HB, detriplet B part of an ABX system, JAB= 17.2 Hz, JBX= 7.6 Hz, J = 2.6
24 Chem. Res. Toxicol., Vol. 8, No. I , 1995 Table 1. ’€3 and 13C NMR Data of Photoproduct8 6 IHC atom 6 13Ca mb CO-2 C-3 CHz-4 CH-5 CHz-6 CHZ-1’ CHz-2’ CHz-3’ CHz-4‘ CHz-5’ CO-2” NH-3” CO-4” C-5” CH-6 CH3-7”
179.85 49.14 31.74 77.21 41.16 32.56 20.77 20.03 26.54 49.56 152.22
0 0 2 2.65 dd (14.614.4) 2.38 dd (14.7h0.2) 1 4.90 dddd (10.2/4.4/4.4/2.2) 2 2.49 d (12.1) 2.37 d (12.1) 2 2.14m 1.56 m 2 1.58 m 1.25 m 2 1.54m 1.44 m 2 2.05 m 1.21 m 2 4.13 ddd (14.0/6.6/4.3)2.86 ddd (14.0B.714.2)
Communications CH3). 13C NMR (CDCl3, 100 MHz) 6 176.5, 173.5, 151.5, 77.1, 65.3,48.3,47.3,40.6,40.2,40.1,31.9,27.9,26.9,26.5,25.9,25.5, 24.8, 21.3, 21.1. IR (CHC13) v 1761 (C=O lactone); 1706 (C=O thymine). UV (CH3CN)1 278 ( E = 2527). Anal. Calcd for CZOH30N204: C, 66.28; H, 8.34; N, 7.73. Found: C, 66.06; H, 8.52; N, 7.22.
Results
Synthesis (Scheme 1). Thymine 1 was directly alkylated with bromoacetal 2 or 3 in dimethyl sulfoxide, using K2C03 as base (15),and compounds 4 and 5 were 0 obtained in 79% and 81% yield, respectively. Several 7.80 bs attempts to deprotect the aldehydic function by reaction 174.70 0 with 5%HC1 in THF (16) were not very successful. Even 43.13 0 66.26 1 3.94 s after 48 h at 50 “C, the reaction was not quantitative 20.40 3 1.50 s (about 25% of protected aldehyde remained) and some degradation products were formed. Conditions for the aBased on I3C(IH} correlation experiments. *Multiplicity deduced from DEPT experiments. CBasedon 13C{IH} and IH-lH “Reformatsky type” reaction of bromomethacrylic acid COSY correlation experiments. with aldehydes, using SnC12 as catalyst (171, are rather acidic and should allow an “in situ” deprotection. We Hz), 2.64-2.50 (m, lH, HA or HB,detriplet A part of an ABX found it more convenient to perform the “Reformatsky” system, J.Q = 17.2 Hz, J m = 6.1 Hz, J = 3.0 Hz), 1.92 (d, 3H, type reaction directly on the protected aldehydes. ComCH3, J = 1.08 Hz), 1.64-1.22 (m, 18H, (CHZ)~).13C NMR pounds 4 and 5, respectively, were treated with bro(CDC13) 6 170.4, 164.5, 151.0, 140.5, 134.8, 121.9, 110.5, 77.7, momethacrylic acid and SnCl2 under reflux for 5 h to give 48.6, 36.3, 33.6, 29.4 (6CHz), 29.3, 24.9, 12.35. IR (CHCl3) v the lactone-thymine heterodimers 6 and 7 in 80% and 3396 (N-H); 2930, 2857, 1757 (C=O lactone); 1682 (C=O 74% yield, respectively. thymine). UV (CH3CN) I 210 ( E = 28 750); 272 ( E = 13 477). Anal. Calcd for CZOH~ONZO~: C, 66.28; H, 8.34; N, 7.72. Photochemical Experiments. Deoxygenated soluFound: C, 66.57; H, 8.50; N, 7.55. tions M) of heterodimers 6 and 7 were first Photocyclization Reactions. (A) Irradiation of 545‘irradiated at 365 nm in ethanol and acetonitrile. After (2,4-Dioxo-S“-methyl-3,4’ -dihydro-l”(2H)-pyrimidin- 20 h of irradiation no photoreaction product could be yl)pentyl]-3-methylene-2-tetrahydrofuranone. A 10-3 M detected and the starting material was recovered quansolution of compound 6 (43.8 mg, 0.15 mmol) in acetone (150 titatively. In contrast, irradiation in acetone led to the mL) was irradiated (365 nm) for 4.5 h, and the reaction was complete convertion of heterodimers 6 and 7 aRer 4.5 and followed by TLC. The solvent was removed under reduced 4 h of irradiation, respectively. Irradiation of compound pressure and the crude material purified by column chroma6 with a 5 carbon alkyl chain gave one single photoadduct tography over silica (AcOEt) to give 30.2 mg (0.10 mmol, 69% 8 (70% yield), while irradiation of compound 7 with a 10 yield) of a single [2+2] photocycloaddition product 8 as a white carbon alkyl chain gave two photoadducts 9a and 9b in solid, mp 208-210 “C. The 400 MHz proton and 100 MHz carbon nuclear magnetic resonance data are listed in Table 1. a 312 ratio (95% yield) (Scheme 1). IR (CHC13) Y 1757 (C=O lactone); 1705 (C=O thymine). UV Structural Analysis of the Photoproducts. The lH (CH3CN) I 280 ( E = 5904). Anal. Calcd for ClaHzoNzO4: C, NMR spectra of compounds 8, 9a, and 9b showed the 61.63; H, 6.89; N, 8.58. Found: C, 62.09; H, 7.23; N, 8.57. absence of both the characteristic exomethylenic protons (B)Irradiation of 5-[10-(2”,4”-Dioxo-5“-methyl-3,4a t 6.2 and 5.6 ppm and the signal of the H6.t proton of dihydm-l”(2~-pyrimidinyl)decyll-3-methylene-2-tetrahythe thymine moiety at 7.0 ppm. This is suggestive of the drofuranone. A M solution of compound 7 (54.4 mg; 0.15 formation of intramolecular photoadducts between the mmol) in acetone (150 mL) was irradiated (365 nm) for 4 h, and exomethylenic function and the 5 ” , 6 double bond of the the reaction was followed by TLC. The solvent was removed thymine. U V spectra of the photoproducts compared under reduced pressure and the crude material purified by column chromatography over silica (hexane, ethyl acetate 80%) with those of the starting materials clearly showed the to give two [2+21 photocycloaddition products 9a (32.1 mg, 88.6 disappearance of the broad absorption peak at 210 nm, pmol, 59% yield) and 9b (20.0 mg, 55.2 mmol, 36% yield). characteristic of the n-n* transition of the 3,6 conjugated Photoproduct 9a. White solid, mp 106-108 “C. IH NMR double bond, and of the absorption at 272 nm, charac(CDC13,400 MHz) 6 7.48 (broad s, lH, NH), 4.60 (dddd, l H , H5, teristic of the thymine double bond. The structure and J = 6.3, 6.3, 9.9, 4.9 Hz), 4.13 (m, lH), 4.12 ( s , lH, Hv), 2.82 geometry of the photoproducts were established by a (m, lH), 2.51 (d, l H , J = 12.0 Hz), 2.24 (d, l H , Hsb, J = combination of lH and 13C NMR and assigned by lH12.0 Hz), 2.19 (dd, l H , H4a, J =13.1 Hz; J = 6.8 Hz), 2.08 (dd, l H , ,&I J = 13.1 Hz, J = 9.8 Hz), 1.70-1.20 (m, 18H, (CHZ)~), 13C correlation (HMQC and HMBC) and CONOESY. Compound 8 was identified as a cis-syn-endo (Chart 1) 1.50 (s, 3H, CH3). NMR (CDC13, 100 MHz) 6 177.9, 174.9, intramolecular [2+2] photoadduct involving the 5”,6” 151.9, 77.9, 63.1, 49.5, 48.9, 43.0, 40.2, 34.1, 32.5, 29.8, 26.9, double bond of the thymine moiety and the 3,6 exometh26.8,26.1,25.9,25.6, 25.1,23.3, 21.4. IR (CHCl3) v 1761 (C=O lactone); 1706 (C=O thymine). W (CH3CN)I 276 ( E = 3166). ylenic group of the lactone. Compound 9a was identified Anal. Calcd for CZOH~ONZO~: C, 66.28; H, 8.34; N, 7.73. as a cis-syn-exo intramolecular [2+21 photoadduct involvFound: C, 66.06; H, 8.52; N, 7.22. ing the 5”,6” double bond of the thymine moiety and the Photoproduct 9b. White solid, mp 230 “C dec. IH NMR 3,6 exomethylenic group of the lactone, while compound (CDC13,400 MHz) 6 7.33 (broad s, l H , NH); 4.52 (dddd, lH, Hg, 9b was assigned as the corresponding cis-syn-endo inJ = 6.5, 9.5, 5.2, 3.8 Hz), 3.75 (d, l H , Hy, J = 1.0 Hz), 3.62 tramolecular [2+23 photoadduct. (ddd, lH, J = 13.9, 11.8, 5.0 Hz), 2.99 (ddd, l H , J = 14.0, 12.5, Structure Assignment of Compound 8. We first 4.6 Hz), 2.96 (dd, lH, Hsa, J = 12.5, 1.1Hz), 2.36 (dd, lH, H4a, examined the cyclobutyl protons (Table 1)that appeared J = 13.3, 6.6 Hz), 2.30 (dd, lH, H4b, J = 13.3, 9.7 Hz), 1.99 (d, 1H, H6b, J = 12.5 Hz), 1.95-1.20 (m, 18H, (CHZ)~), 1.5 (s, 3H, as a doublet at 2.37 ppm JHG~ = -12.1 H~ HZ), ~a
Chem. Res. Toxicol., Vol. 8, No.1, 1995 25
Communications Chart 1. Definitions of cieltruns and synlunti Lactone-Thymine Photoadducts
methyl group at 1.55 ppm and H6b and H,y. The cis and endo configuration was assigned by comparison with NOE’s observed for compound 8 and confirmed by an NOE interaction between H4b and Hv.
Discussion 0
syn
anti
cis
trans
Chart 2. lH NMR Chemical Shifts and Main NOE Interactions Observed between Protons of Compounds 8 and 9a
8 cis-syn-endo
90 cis-syn-exo
doublet at 2.49 ppm (&,I, and a singlet at 3.94 ppm (&). This indicated a syn configuration, which, in fact, is the only possible with this compound due to the short 5 carbon alkyl chain. This syn configuration was confirmed by the NOE interactions (Chart 2) between the methyl group at 1.50 ppm and protons H6b and Hv. In the same way, the cis configuration was deduced from the NOE interactions observed between H6b and proton &a at 2.38 ppm. This NOE interaction is only compatible with a cis arrangement of the lactone with the carbonyl group in the direction of the thymine ring (Chart 2). The endo junction of the alkyl chain with the lactone ring was deduced from the NOE interaction between H3 (4.90ppm) and proton H4a. Structure Assignment of Compound 9a. The cissyn-exo conformation of this compound was assigned on the basis of the same arguments used for the structure attribution of compound 8. Cyclobutyl protons at 2.24 ppm (d, Hsb, JHGb-HGa = 12.0 HZ), 2.51 ppm (d, H d , and 4.12 ppm (s, &,) indicated a syn conformation while NOE interactions between H6b and confirmed the cis conformation (Chart 2). In this case the alkyl chain junction was found to be exo. Thus NOE interactions were observed between Hsb-&a, Hda-H4b, and H4b-H5, which is characteristic of a pseudo-axial junction of the alkyl chain. Structure Assignment of Compound 9b. The chemical shiRs and coupling constants of the cyclobutyl protons, with a doublet of doublets at 2.96 ppm (Hea, JHG~-HG= ~ 12.6 HZ, JH6a-HV = 1.0 HZ), a doublet a t 1.99 ppm (Heb), and a doublet at 3.75 ppm (Hg,), are characteristic of a syn configuration. This was confirmed by the unambiguous NOE interactions observed between the
The photocyclization of lactone-thymine heterodimers could, in theory, result in the formation of 8 different products. By analogy with psoralen photodimers (18) and psoralen-thymine photoadducts (19), they have been designated cisltrans and synlanti. The terms cisltrans and syn /anti refer, respectively, to the relative position of the cyclobutyl protons and to the relative orientation of the two rings, one to another. In our case, the situation is complicated by the formation of a “spiro”-typeadduct between the exomethylene of the lactone and the double bond of the thymine; there is thus no longer a strict definition of the terms cisltrans and synlanti. The conformation has therefore been designated as cis or trans on the basis of the relative position of the Hv and H6b protons and the Eaand H4b protons of the lactone ring (Chart 11, as determined by studying NOE interactions. The syn and anti conformations were defined by analogy with those of psoralen-thymine photodimers (19) on the basis of the relative orientation of the two molecules (determined by the carbonyl on the lactone and the carbonyl on position 4 of the thymine) and assuming a photoreaction resulting of a butenolide-type derivative with an endocyclic double bond. The third point relates to the linking of the alkyl chain to the lactone ring. As the 5 member ring is relatively flat because of ring tension, it was difficult to define pseudo-axial and pseudoequatorial positions. We therefore decided to characterize the geometry of the adducts by the terms endo and exo, describing the face to which the alkyl chain was attached. On irradiating compounds 6 and 7 in ethanol or acetonitrile, purged with argon prior to irradiation, no reaction occurred, while, in acetone, rapid photocyclization was seen. This difference can be explained by acetone’s ability to act as a photosensitizer (20), i.e., to facilitate the formation of an excited triplet state by energy transfer. However, an attempt to produce photoreaction of compound 6 in ethanol in the presence of 0.2 equiv of the strong photosensitizer benzophenone resulted in the rapid (30 min) complete degradation of the molecule. This may be due to a-hydrogen atom abstraction from ethanol by excited state benzophenone to generate the a-hydroxy radical, which then leads to degradation of 6 through free radical reactions. In all cases, we obtained cis-syn derivatives, the only difference being the endo or ex0 nature of the chain junction. In the case of derivative 6, the 5 carbon chain does not allow the formation of the anti derivatives, but the formation of the trans product might have been expected. In the case of derivative 7, the 10 carbon chain should theoretically allow the formation of all conformations, but, again, no anti or trans derivatives were seen. The syn direction of addition can be explained by the mechanism of photoreaction (19) and the overlapping of orbitals, the exomethylene bond of the lactone being highly polarized, but the cis selectivity is more difficult to understand. Even if in our case a n-n stacking as described between psoralen and thymine is not likely to occur, we have to consider if some sort of interaction leading to the exclusive formation of the cis-syn dimers
26 Chem. Res. Toxicol., Vol. 8, No. 1, 1995
could be detected. We have thus compared the UV and lH and 13C NMR spectra of compound 7 with spectra of a mixture of 1-heptylthymine and 5-decyl-3-methylene24etrahydrofiranone. No significant hypochromic effect nor modification in chemical shifts, both effects reported for psoralen-thymine interactions (Zl),was observed that could explain the stereochemistry obtained. “he production of an endo lex0 mixture during photoreaction of compound 7 can easily be explained by the quasiplanar nature of the lactone ring due to high ring tensions. The pseudo-axial and pseudo-equatorial positions are very similar in geometry; this was confirmed by the values for the coupling constants between the H4a, H4b, and H g protons. We have demonstrated the photoreactivity of a-methylene-y-butyrolactones toward a substrate such as thymine. Lactones of this type have always been considered for their electrophilic properties, which makes them excellent “Michael” acceptors, but never as photoreactive molecules. The fact that a-methylene-y-butyrolactones react with thymine to form [2+2] type photoadducts does not prove that they are able to form adducts of this type with DNA in vivo nor that this is the mechanism responsible for the chronic actinic dermatitis seen in certain patients sensitized to sesquiterpene lactones. Many factors such as bioavailability, the ability to interact with DNA, etc., remain to be explored, but this new mode of interaction of the lactones with biological material in the presence of light opens new fields of investigation for the understanding of the toxic and phototoxic reactions of these molecules present in our environment.
References (1) Paulsen E. (1992)Compositae dermatitis: a survey. Contact Dermatitis 26,76-86. (2) English, J. S. C., Norris, P., White, I. R., and Cronin, E. (1989) Variability in the clinical patterns of compositae dermatitis. Br. J. Dermatol. 121 (Suppl. 341,27. (3)Benezra, C., Ducombs, G., Sell, Y., and Foussereau, J. (1985)Plant contact dermatitis, BC Decker, Toronto, and Philadelphia. (4)Franot, C., Benezra, C., and Lepoittevin, J.-P. (1993)Synthesis and interaction studies of 13Clabeled lactone derivatives with a model protein using I3C NMR. Bioorg. Med. Chem. 1, 389-397.
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