A cytotoxic principle of the brown alga Sargassum tortile and structures of chromenes

0031-9422/92 S5.00+0.00 0 1992Pergamon Press plc

Phyfochemistry, Vol. 31, No. 4, pp. 1209-1213, 1992

Printedin GreatBritain.

A CYTOTOXIC

PRINCIPLE OF THE BROWN ALGA SARGASSUM TORTILE: AND STRUCTURES OF CHROMENES

ATSUSHI NUMATA,* SHOICHI KANBARA, CHIKA TAKAHASHI, RYOKO FUJIKI, MICHIKO YONEDA, YOSHIHIDE USAMI and EIICHI FUJITA

Osaka University of Pharmaceutical Sciences, 2-10-65 Kawai, Matsubara, Osaka 580, Japan (Receiued28 Augusf 1991) Key Word Index-Sargassum tortile; Sargassaceae; whole seaweed; cytotoxicity; benzoquinones; chromenes.

fractionation of a methanoiic extract of the marine. alga Sargass~ turtile has led to the isolation and characterization of eight compounds which include the new chromenes sargaol, sargasal-II, and sargadiols-I and -11 in addition to sargasal-I, hydroxysargaquinone, kjellmanianone, and fucosterol. Among them, hydroxysargaquinone and sargasals-I and -11 demonstrated significant and marginal cytotoxicity against cultured P-388 lymphocytic leukemia cells, respectively. A~t~ct-Bioassay-dir~t~

INTRODUCTION

We reported recently on the isolation of dihydroxysargaquinone as a cytotoxic principle of the brown alga Sargass~ tortile C. Agardh [ 11. Further investigation on the cytotoxic fractions of the methanolic extract of the same seaweed has led to the isolation of four new chromenes as well as four known compounds. RESULTS AND DISCUSSION

Partition fractions of hexane, carbon tetrachloride, and chloroform from the methanolic extract of the fresh S. tortile were each purified by bioassay-directed fractionation employing CC on Sephadex LH-20 and silica gel to afford the new chromenes, sargaoi (l), sargadiols -1 (2) and -11(3), and sargasal-II (4), together with the known sargasal-I (3, hydroxysar~quinone (61, kjellmanianone (t), and fucosterol (8). Among them, hydroxys~~quinone (6) and sargasalsd (5) and -11(4) exhibited significant and marginal cytotoxicities against cultured P-388 lymphocytic leukemia cells, respectively, while other compounds showed moderate activity (Table 1). Compound 5 was identified by direct comparison with an authentic sample. Although this compound was previously prepared by oxidation of sargatriol with periodic acid [2], this is the first report of its occurrence in nature. ~om~unds 7 and 8 were identihed by comparison of physical and spectral data with those already published [3, 41. The isolation and structure assignment of the cytotoxic compound 6 from this alga has already been reported by us in 1987 KS], although recently this compound was isolated from the brown alga Desmarestia menziesii by Rivera et al. and the structure published [6]. The ‘H and 13C NMR spectra of 6 showed the signals of a (2’E, 6’E,lO’E,14’E)-hydroxygeranylgeranyl side chain,

*Author to whom correspondence should be addressed.

together with signals attributable to a 2-alkyld-methyl1,4-benzoquinone (Tables 2 and 3). The position of the hydroxyl group was deduced from the coupling relationship of the hydroxy-bearing methine with the C-10’ vinylic proton and also the correlation observed in the XCORFE ZD-NMR spectrum (Table 4), which is a modified technique of long-range rH-i3C HETCOR 2DNMR spectrum [7J. The NMR spectral assignments of 6 partly revised are shown in Tables 2 and 3. Sargaol (1) had the molecular formula C,,H,,Oz as deduced from the HR mass spectrum ([Ml’ at m/z 394.2878), and its IR spectrum showed the presence of a hydroxyl group and an aromatic ring. The NMR spectral data of 1 (Tables 2 and 3) exhibited the presence of two meta-coupled aromatic protons (H-5 and H-7), two olefinic protons (H-3 and H-4) coupled to each other, two methyl protons (H-9 and H-10) on an aromatic nucleus and a quaternary sp3-hybridized carbon, and three quaternary carbons (C-2, C-6 and C-8a) bearing an oxygen function. In the XCORFE spectrum of 1 (Table 4), C-3, C-4 and C-7 were shown to correlate with H-10, H-5, and H-5 and H-9, respectively, and C-8a with H-5, H-7, and H-9. In addition, the HR mass spectral fragment ion at m/z 175.0754 showed the presence of a 2,8-dimethyl2H-1-benzopyran-6-01 moiety in this molecule. The presence of a side chain containing three prenyl units was indicated by the signals of three olefinic protons, six methylene and four allylic methyl groups in the NMR spectra (Tables 2 and 3) as well as the EI mass spectral fragments at m/z 137 and 69, resulting from cleavage of the 5’/6’ and Y/10’ bonds, respectively. Only one methyl resonance appeared above 620 in the t3C NMR spectrum and, therefore, the prenyl side chain double bonds are all E-oriented [8]. Collection of the foregoing evidence led to structure 1 for sargaol. The general spectral features of sargadiol-I (2), C2,H3s03 ([Ml’ at m/z 410.2828), closely resembled those of 1 except that the C-6’ methylene signals (6, 2.05 m, 6, 26.8). in 1 were replaced by hydroxy-Bering

1209

1210

A. NUMATAet al.

HO

4 1 2

R H OH

HO

R

HO

0

6

Table 1. Cytotoxicity ofcompounds

l-8 against tumour

C&S

Compound 1

2 3 4 5 6 I 8 Etoposide (standard)

Cell line P-388 (ED,,pgml-‘) 20.8 14.0 16.8 5.1 5.8 0.7 15.3 17.2 0.24

methine signals (6, 4.43 q, 6, 66.0) (Tables 2 and 3). Confirmation for the position of the hydroxyl group came from the coupling relationship of the hydroxyl-bearing methine proton and the C-7’ vinylic proton in the ‘HNMR spectrum as well as the HR mass spectral fragment ion ([C17H2102 +H]+) at m/z 258.1619, indicating cleavage of the C-S/C-6 bond. This evidence allowed assignment of structure 2 to sargadiol-I. Sargadiol-II (3) has the same molecular formula as that of 2 as deduced from the HR mass spectrum. A close

insepection of the ‘H NMR signals of both 2 and 3 (Table 2) revealed that signals of one allylic methyl group and one trisubstituted double bond in 2 were missing from 3 and replaced by those of a methyl group on a quaternary sp3-carbon and a disubstituted double bond, respectively. In addition, one of five allylic methylene signals in 2 was replaced by a doubly allylic methylene signal in the spectrum of 3. The XCORFE spectrum of 3 (Table 4) showed the correlation between the doubly allylic methylene carbon (C-5’) and the allylic methyl protons (H-16’) at 61.55. Besides, one carbon (C-7’) of the disubstituted double bond was shown to correlate with the methylene (H-5’) and methyl protons (H-15’), the latter correlating with the quaternary sp3-carbon (C-8’) bearing a hydroxy group. The EI mass spectrum of 3 showed [C, ,H, ,O,] + and [C&H, i]’ fragments at m/z 175 and 83, resulting from cleavage of the C-2/C-l’ and C-V/C-9 bonds, respectively. The coupling constant (J= 16 Hz) of the C-6’/C-7’ olefinic protons proved the geometry to be E. The above evidence led to assignment of structure 3 for sargadiol-II. Sargasal-II (4) had a molecular formula of C,,H,,O, ([M] + at m/z 270.1263). The presence of a 2&dimethyl2H-1-benzopyran-6-01 moiety in this molecule was demonstrated by the ‘H and i3C NMR spectra (Tables 2 and 3) and EI mass spectral fragment ion at m/z 175. The remainder of the NMR spectra consisted of signals of diand trisubstituted double bonds, an allylic methyl and a formyl group. These data and the HR mass spectral

Cytotoxic principle from Sargassum tortile

1211

Table 2. ‘H NMR spectral data of compounds 1-4 and 6 (300 MHz, CDCI,, B-values, TMS as int. standard) H

1

2

3

4

6

3 4 5 7 9 10 1’ 2 3’ 4 5’ 6 7’ 8’ 9 10’ 11’ 12 13 14’ 15’ 16’ 17 18’ 19 20 6-OH

5.58 d (10) 4.24 d (10) 6.32 d (3) 6.47 d (3) 2.14s 1.36 s 1.72 ?n 2.11 m 5.11 bF t (8)

5.57 d (9.3) 6.25 d (9.3) 6.32 d (3) 6.47 d (3) 2.14 s 1.36 s 1.70 m 2.16 m 5.26 br t (7)

5.57 d (10) 6.24 d (10) 6.32 d (3) 6.48 d (3) 2.13 s 1.36 s 1.68 m 2.14m 5.14 br t (7.6)

5.65 d (9.4) 6.37 d (9.4) 6.35 d (3) 6.52 d (3) 2.22 s 1.58 s 6.19 d (14.8) 6.67 dd (14.8, 11.3) 6.79 dq (11.3, 0.8)

6.47 dt (2.5, 1.6)

1.96 m 2.05 m 5.09 br t (7)

2.13 m 4.43 q (8) 5.15 d (8)

2.66 d (4.6) 5.54 m 5.51 d (16)

9.42 s 1.80 d (0.8)

1.96 m 2.05 m 5.09 br t (7)

2.00 m 2.07 m

1.56 m 2.02 m 5.09 br t (7.6)

1.68 s 1.59 S 1.58 s 1.58 s

1.67 s 1.60s 1.63 s 1.57 s

1.67 s 1.59s 1.27 s 1.55 s

4.35 br s

4.31 br s

4.70 br s

5.08 br

t

(8)

6.54 dg (2.5, 1.1) 2.06 d (1.1)

3.13 br d (7.2) 5.15 br t (7.2) 2.10m 2.16 m 5.22 br t (7) 2.15 d (6.5) 4.43 td (6.5, 8) 5.17 d (8) 2.01 m 2.08 m 5.10 br t (7) 1.684 s 1.60s 1.682 s 1.66 s 1.63 s

4.34 br s

J(Hz) in parentheses

fragment ion at m/z 95.0490 (C,H,O+) indicated the presence of a 4-formyl-1,3-pentadienyl side chain. The above data were interpreted in terms of structure 4, in which the geometry of the side chain double bond was deduced from the coupling constants (J1S.2P= 14 Hz and I - 11 Hz) of the olefinic protons and the chemical kg(i9.42) of aldehyde in the ‘H NMR spectrum [9 10). It has been reported that on standing at room temperature or boiling in an organic solvent, some 2geranyl~ranyl-6-methyl-1,4-~nzoquinone derivatives were converted into the corresponding chromenes whose NMR spectra indicated signals due to a mixture of two isomers at the newly-generated chiral centre (C-2). Although the chromenes obtained herein did not indicate NMR signals due to a mixture of two epimers at C-2, they may be artifacts derived from the corresponding geranylgeranylbenzoquinones, which are possibly natural products. Actually, compounds 1,4 and 5 showed no optical activity.

EXPERWNTAL Seaweed material. Sargassum tort&, collected at the coast of Tanabe Bay, Japan in October 1985, was identified by Dr Y. Nabeshima (Osaka Prefectural Fisheries Experimental Station). A voucher specimen is deposited at Osaka University of Pharmaceutical Sciences. Extraction and isolation. As reported previously [l], the MeOH extract (586 g) of the fresh alga (8.8 kg) was partitioned to

afford hexane (58.9 g), Ccl, (60.8 g) and CHCI, (29.0 g) frs. The Ccl, fr. was sepd into 8 frs by CC on Sephadex LH-20 eluted with MeOH-CHCl, (1: 1). Fr. 5 (8 g) was subjected to CC on silica gel and eluted with a gradient of CH,Cl,, CHCl, and CHCl,-MeOH (99: 1). The CH,Cl, eluate afforded 6 (40 mg). The first half of the CHCl,-MeOH eluate was further chromatographed on silica gel. Elution with CHCl, and CHCi,-MeOH (99: 1) gave 2 (30 mg) and 3 (21 mg), respectively. The hexane partition fr. was passed through a Sephadex LH-20 column using MeOH-CHCI, (1: 1) as the eluent and sepd into 8 frs. Fr. 6 (5 g) was chromatographed on silica gel with elution by CH,Cli and CHCI,, the latter eluate affording 8 (1.7g). The CH,Cl, eluate yielded l(20 mg) after another series of separations by CC on silica gel with CH,Cl, followed by HPLC (Shim-Pack PREPSIL) with CHCl,-MeOH (99:l) as the eluent. The CHCI, partition fr. was subjected to CC on Sephadex LH-20 with CHCl,-MeOH (1: 1) to give 9 frs. Fr. 8 (2.6 g) was chromatographed on silica gel with a MeOH-CHCl, gradient as the eluent and the CHCl,-MeOH (99: 1) eluate afforded 7 (80 mg). Fr. 5 (7.8 g) was repeatedly chromatography on silica gel with MeOH-CHCI, gradient as the eluent and the CHCI,-MeOH (99 : 1)eluate afforded 4 (2.5 mg) and 5 (2 mg) after purification by HPLC [CHCl,-MeOH (99: l)]. (3E,7E,11E)-2,8-Dimethyl-2-(4,8,12-t~imethylt~~deca-3,7,1l-t~ienyl)-2H-chrome+6-o/ (saryaol) (1). Pale blown oil. UV ,l,“z” nm (loge): 210 (4.39), 230 (4.39), 265 (3.77), 272 (3.74), 330 (3.58). IR Y2:1X cm-‘: 3590, 3350, 1665, 1590. EIMS m/z (rel. int.): 394 [M]’ (ll), 175 [chromenol part]+ (100), 137 [C,0H17]+ (2) 69 [CsH,]’ (5). HR-MS m/z: 394.2878 EM]’ (C,,H,,O, requires: 394.2870), 175.0754 (C,rH,rO, requires: 175.0758).

l212

A.

&kKUATA et ai.

Table 3. i3C NMR spectral data of compounds l-4 and 6* (75.4 MHz CDCl,, S-values, TMS as int. standard)

Table 4. Long range ‘Ii-‘%

correlations of compounds 1, 3 and 6

C 1 2 3 4 4a 5 6 7 8 8a 9 10 1’ 2 3 4’ 5’ 6 7 8 9 10 11’ 12’ 13’ 14’ 15’ 16 17 18 19 20

1

77.9 q 130.8 t 122.9 r 121.4 q 110.3t 148.8 q 117.1 t 126.4 q 145.0 q 15.5 p 25.9 p 40.9 s 22.6 s 124.2 r 136.0 q 39.7 s 26.8 S= 124.1 t 135.2 q 39.7 s 26.6 s’ 124.4 c 131.3 q 25.7 p 17.7 p 16.Op 15.9 p

2

77.7 q 130.5 t 123.0 t 121.3 q 110.3 t 148.7 q 117.1 t 126.3 q 144.8 q 15.5 p 26.0 p 40.8 s 22.8 s 128.4 t 131.8 $ 48.1 s 66.0 t 127.2 t 138.4 q 39.5 s 26.4 .s 124.0 t 131.6 q” 25.7 p 17.7 p 16.6 p 16.1 p

3

77.8 q 130.6 t 122.9 t 121.3 q 110.3t 148.6 q 117.1 t 126.3 q 144.9 q 15.5 p 25.9 p 40.8 s 22.7 s 125.1 t 134.0 q 42.5 s 126.1 t 138.1 t 73.1 q 42.5 s 23.0 s 142.4 t 131.8 q 25.7 p 17.7 p 28.2 p 16.0 p

4

76.1 q 127.5 t 123.3 t 121.2 q 110.7t 149.1 q 117.6 t 126.3 q 144.9 t 15.5 p 25.9 p 144.9 t 124.2 t 147.7 t 138.3 q 194.9 t 9.6 p

6 187.9 q 148.4 q 132.3 t 188.0 q 133.1 E 146.0 q 16.0 p

27.6 s 118.5 t 139.6 q 39.4 s 26.4 s 127.9 t 132.1 q 48.1 s 65.9 t 127.4 t 138.1 q 39.6 s 26.5 s 124.0 t 131&q 25.7 p 17.7 p 16.6p 16.3~ 16.0 p

*Signal assignments were based on ‘H-homonuclear decoupling and iH-‘%Z HETCOR spectra, and in case of 1,3 and 6 also on XCORFE spectra. Letters, p, s, t and q, indicate, respectively, primary, secondary, tertiary and quaternary carbons, assigned by DEPT. aInterch~geable within the same column.

(3E,7E,l lE)-2-(6-hydroxy-4,8,12-trimethyltridecu-3,7,1 l-t& eny[)-2,8-Dimethyl-2H-chromen-6-ol (sargadioCI) (2). Pale brown oil, [LY]~+ 1.5” (CHCI,; ~0.78). WA::” mn (loge): 208 (4.35), 230 (4.31), 265 (3.77), 272 (3.76), 337 (3.46). IR v,$!!ci’ cm-‘: 3574 3530,3340,1660,1590. EIMS m/z (rel. int.): 410 [M]’ (l), 393 [M -OH]+ (2), 258 [MH-Ci,H,,O]+ (3), 175[chromenol part]+ (lOO), 69 [&Ha]+ (45). HR-MS m/z: 410.2827 [Ml’ (C,,H,sO, requires: 410.28183, 393.2785 EM -OH]+ (C,,H,,O, requires: 393.2791), 258.1619 (C,,H,,O, requires: 258.1619). 175.0765 (C,,H,,02 requires: 175.0758). (3E,6E,l lE~-2-(8-~yd~oxy-4,8,12-rr~~r~y~~~~cu-3,~11 -trieny~-2,8-~imet~y~-2H-c~romen-6-o~ (snrgadiof--II) (3). Pale brown oil, [a]:: + 1.8” (CHCl,; c 0.98). UVC:” nm (loge): 210 (4.34), 230 (4.33), 260 (3.83), 275 (3.80) 335 (3.52). IR v;::‘~ cn- i: 3590, 3530, 3340, 1670, 1597. EIMS m/z (rcl. int.): 410 [M]’ (O.S),392 [M-HH,OIC (lo), 270 (ll), 255 (lo), 175 [chromenol part]+ (lOO), 83 [C,H,,]+ (37). HR-MS m/z: 410.2815 [Ml’ (C2,H3a03 requires: 410.2818), 392.2705 [M-H,O] + (Ca,H,,O, requires: 392.2713), 175.0750 [chromenol part]+ (CirHirOa requires: 175.0758).

H C

1

3

6

2 3 4 4a 5 6 7 8 8a 1’ 2 3 4 5’ 6 7 8 9 10 11’ 12’ 13’ 14’ 15 16 17 18 19 20

3, 4, IO, 1’ 10 5 3 7

4, 10 f0 5

1’ 1’

5, 9 9

5, 9 9 5. 7 10 f’, 3’ 5’. 16 5 16 5 s, 1.5 15’ 15 9 13’ 13’ 14 II’, 13

7

5, 7, 9 10 1’, 3’ 5’, 16 16 3’, 6, 16 5 6’,15 9’,15 7’, lo’, 15 9, 11’ 13’ 13’ ll’, 14 11’. 13 7 3, 5

3’. 5’

l’, 4, 20 20’ 5 4 5, 19 s’, 19 6 8’ 18 18’ 13, 18’ 12 16’. 17 16 14: 17’ 14’. 16 10 6’. 8 2

(lE,3 E)-2-(4-~ormyf-l,3-pentadienyl)-2,8-Dimethyl-2H-chromewSot (sargasaZ-II) (4). Pale brown oil. UV A:? mn (logs): 215 (3.97), 228 (3.97), 275 (3.97), 322 (3.55). IR v$jFi3cm-‘: 3590, 3333,1674,1636,1600. EIMS m/z (rel. int.): 270 [M]’ (18), 241 [M -CHO]+ (2), 175 [chrom~nol part]’ (lOO), 95 [C6H70]+ (7). HR-MS m/z: 270.1263 CM]’ (C,,H,,O, requires: 270.1255), 241.1243 [M-CHO]’ (C,,H,,O, requires: 241.1228), 175.0755 (C, iH, ,O, requires: 175.0758), 95.0490 (C$I,O requires: 95.0495). (3E)-2-(4-formy[-3-pentenyl)-2,8-Dimethy2H-chromen-(i-o[ (sargasal-I) (5). Pale brown oil. UV 1.g:” nm (logs): 208 (3.99), 225 (3.89) 265 (3.40), 275 (3.40), 325 (2.80). IRvE:” cm--‘: 3590, 3270,1680,1600. ‘H NMR (CDCl,, 300 MHz): 6 1.40 (3H, s, Hlo), 1.70 (3H, d, J= 1.6 Hz, H-5’), 1.85 (2H, m, H-l’), 2.13 (3H, s, H-9),2.51 (2H.m, H-2),4.31 (lH,s, OH), 557(1H,d, J=9.3 HZ,, H-3), 6.31 (lH, d, J=9.3 Hz, H-4), 6.34 (lH, d, J=3.0 Hz, H-5), 6.49(lH,d, J=3.0 I-k, H-7), 6.51 (lH, br t, J=7.4 Hz, H-3),9.36 (lH, s, H-6’). EIMS m/z (rel. irk): 272 [M]’ (121,175 [chromcnol part]+ (100). HR-MS m/z: 272.1406 [M]’ (C,,H,,O, requires: 272.1410) 1750766 (C,,H,,O, requires: 175.0758). (2E,6E,lOE,14E)-2-(9-hydroxy-3.7,11,15-tetr~methy~~exadeca-2,6,10,14-tetraenyl)-6-Metky1-1,4-benzoquinone (hydroxysargaquinone) (6). Pale brown oil, [a];’ f2’ (CHCI,; c 1.9). UVI:$‘” mn (log E): 212 (4.03), 253 (4.04), 295 (3.01). 325 (2.84). IRCF” cm-‘: 3500, 3300, 1640. EIMS m/z (rel. int.): 410 [M]’ (3) 394 [M-163+ (11X 257 CM-CmH,,O]+ (31) 175 [C1iHI,O,]’ (lOO), 69 [CsH,]” (23). HR-MS m/z: 410.2810

Cytotoxic principle from Sargassum to&e [Ml’ (CZ~H~,& requires: 410.2818), 394.2873 (C,,H,,O, requires: 394.2870), 175.0758 (C, ,H, ,O, requires: 175.0758). Kjellmanianone (7). Needles, mp 137-139”. UV1::$ nm (logs): 242.5 (3.88). IR1:C,‘3 cn-‘: 3550, 1740, 1700. ‘HNMR (300 MHz, CDCI,): 62.74, 3.18 (each lH, dd, J= 18, 1 Hz, H-4), 3.80 (3H, s, H-8), 3.82 (lH, s, OH), 3.94 (3H, s, H-7), 5.35 (lH, t, J = 1 Hz, H-2). HR-MS m/z: 186.0534 [M]’ (C,H,,O, requires: 186.0530). Fucosterol(8). Needles, mp 123-124”. EIMS m/z (rel. int.): 412 [M]’ (12), 314 (100). Biological assay. The in vitro cytotoxicity assay of test materials against the P-388 lymphocytic leukemia cells was carried out according to the procedure of ref. [7]. For significant activity, an ED,, value of g4 pgml-’ is required.

REFERENCES

1. Numata, A., Kambara, S., Takahashi, C., Fujiki, R., Yoneda, M., Fujita, E. and Nabeshima, Y. (1991) Chem. Pharm. Bull. 39, 2129. 2. Kikuchi, T., Mori, Y., Yokoi, T., Nakazawa, S., Kuroda, H.,

Masada, Y., Kitamura, K. and Kuriyama, K. (1983) Chem. Pharm. Bull. 31, 106. 3. Nakayama, M., Fukuoka, Y., Nozaki, H., Matsuo, A. and Hayashi, S. (1980) Chem. Letters 1243.

4. McInnes, A. G., Walter, J. A. and Wright, J. L.C. (1980) Org. Magn. Reson. 13, 302. 5. Numata, A., Kambara,

6. 7.

Acknowledgements-We are grateful to Dr Y. Nabeshima (Osaka Prefectural Fisheries Experimental Station) for the identification of the seaweed, and to Miss M. Danjyo, this university, for MS measurements.

PHYTO

31:4-I

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8.

9. 10.

S., Takahashi, C. and Fujita, E. (1987) Abstr. 107th Annu. Meet. Pharm. Sot., Kyoto, p. 354. Rivera, P., Podesta, F., Norte, M., Cataldo, F. and Gonzalez, A. G. (1990) Can. J. Chem. 68, 1399. Numata, A., Yang, P., Takahashi, C., Fujiki, R., Nabae, M. and Fujita, E. (1989) Chem. Pharm. Bull. 37, 648. Englert, G. (1975) Helu. Chim. Acta 58, 2367. Patel, D. J. (1969) Nature 221, 825. Chan, K. C., Jewel], R. A., Nutting, W. H. and Rapoport, H. (1968) J. Org. Chem. 33, 3382.