Synthesis, Absolute Configuration, and Enantiomeric Enrichment of a

Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan, Knowledge-based. Information Engineering, Toyohashi University of ...
0 downloads 0 Views 198KB Size
Download PDF
3940

J. Org. Chem. 2001, 66, 3940-3947

Synthesis, Absolute Configuration, and Enantiomeric Enrichment of a Cruciferous Oxindole Phytoalexin, (S)-(-)-Spirobrassinin, and Its Oxazoline Analog Mojmı´r Suchy´,† Peter Kutschy,*,† Kenji Monde,*,‡ Hitoshi Goto,§ Nobuyuki Harada,| Mitsuo Takasugi,| Milan Dzurilla,† and Eva Balentova´† Department of Organic Chemistry, P. J. S ˇ afa´ rik University, Moyzesova 11, 041 67 Kosˇ ice, Slovak Republic, Division of Material Science, Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan, Knowledge-based Information Engineering, Toyohashi University of Technology, Toyohashi, 441-8580, Japan, and Institute for Chemical Reaction Science, Tohoku University, Aoba-ku, Sendai 980-8577, Japan [email protected] Received January 5, 2001

Stereochemical studies of a cruciferous oxindole phytoalexin, (S)-(-)-spirobrassinin [(-)-4], and its oxazoline analogue, spirooxazoline (11), were carried out. Racemic spirobrassinin [(()-4] was synthesized by SOCl2- or MsCl-mediated cyclization of dioxibrassinin [(()-8]. Treatment of (3-hydroxyoxindol-3-yl)methylammonium chloride [(()-9] with CSCl2 and subsequent methylation of the obtained spirooxazolidinethione (()-10 afforded spirooxazoline [(()-11]. Enantioresolution of (()-4 and (()-11 was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate (12), chromatographic separation of diastereomeric amides 13, 14 or 15, 16, and their cleavage with CH3ONa. Absolute configuration of the stereogenic center in natural (S)-(-)-4 was derived from the exciton, calculated via CD methods, and unequivocally confirmed by X-ray crystallographic analyses of 1-[1′S,4′R-(-)-camphanoyl] derivatives [(-)-19 and (-)-20] of (+)- and (-)-4. Novel enantiomeric enrichment phenomena of 4 and 11 were discovered during their chromatographic separations under achiral HPLC conditions. Screening of antifungal activity against the fungus Bipolaris leersiae revealed no significant dependence of this activity on absolute configuration. Introduction In responding to biological, physical, or chemical stress, plants produce antimicrobial secondary metabolites, called phytoalexins, de novo.1 These compounds play a significant role in the induced defense mechanisms of plants against microbial microrganisms.2 An important group of stress metabolites represented by phytoalexins has been reported from the plant family cruciferae,3 which includes many important crops cultivated worldwide. A common structural feature of more than 20 * To whom correspondence should be addressed. Tel: +421-95-6222610, ext. 192; Fax: +421-95-62-22124. † P. J. S ˇ afa´rik University. ‡ Tohoku University. Tel: +81-22-217-5634; Fax; +81-22-217-5667. E-mail: [email protected]. § Toyohashi University of Technology. | Hokkaido University. (1) Purkayastha, R. P. In Handbook of Phytoalexins metabolism and Action; Daniel, M., Purkayastha, R. P., Eds.; Marcel Dekker: New York, 1995; pp 1-39. (2) (a) Bailey, J. A.; Mansfield, J. W. Phytoalexins; Blackie & Sons Ltd.: Glasgow, 1982; pp 1-334. (b) Lamb, C. J.; Ryals, J. A.; Ward, R. E.; Dixon, R. A. Biotechnology 1992, 10, 1436-1445. (c) Hain, R.; Reif, H.-J.; Krause, J.; Langebartels, R.; Kindl, H.; Vornam, B.; Wiese, W.; Schmelzer, E.; Schreier, P. H.; Sto¨cker, R. H.; Stenzel, K. Nature 1993, 361, 153-156. (d) Yoshikawa, M.; Tsuda, M.; Takeuchi, Y. Naturwissenschaften 1993, 80, 417-420. (e) Afek, U.; Carmeli, S.; Aharoni, R. Phytochemistry 1995, 39, 1347-1350. (f) Glazerbrook, J.; Zook, M.; Mert, F.; Kagan, I.; Rogers, E. E.; Crute, I. R.; Holub, E. B.; Hammerschmidt, R.; Ausubel, F. M. Genetics 1997, 146, 381-392. (3) For a recent reviews of indole phytoalexins, see: (a) Pedras, M. S. C.; Okanga, F. I.; Zaharia, I. L.; Khan, A. Q. Phytochemistry 2000, 53, 161-176. (b) Rouxel, T.; Kollman, A.; Belesdent, M.-H. In Handbook of Phytoalexins metabolism and Action; Daniel, M., Purkayastha, R. P., Eds.; Marcel Dekker: New York, 1995; pp 229-261. (c) Gross, D. J. Plant. Dis. Prot. 1993, 100, 433-442.

hitherto isolated cruciferous phytoalexins is the presence of indole or an indole-related nucleus possessing a side chain or another heterocycle containing one or two sulfur atoms.3a Typical representatives are brassinin (1), methoxybrassinin (2), and cyclobrassinin (3) isolated from Pseudomonas cichorii-inoculated Chinese cabbage (Brassica campestris).4

In the past decade, interesting biological activities of the cruciferous phytoalexins have been reported from various research groups, including antimicrobial,3a,c antitumor,5 and oviposition-stimulant6 activities. With respect to the examinations of various biological activities (4) (a) Takasugi, M.; Katsui, N.; Shirata, A. J. Chem. Soc., Chem. Commun. 1986, 1077-1078. (b) Takasugi, M.; Monde, K.; Katsui, N.; Shirata, A. Bull. Chem. Soc. Jpn. 1988, 61, 285-289.

10.1021/jo0155052 CCC: $20.00 © 2001 American Chemical Society Published on Web 04/27/2001

Stereochemical Studies of Spirobrassinin

of the cruciferous phytoalexins as well as the dietary importance of brassicaceous vegetables in cancer chemoprevention,7 it is quite important to investigate synthetic approaches to the indole phytoalexins4,8,9 and their analogues. In 1987, the first oxindole phytoalexin, spirobrassinin [(-)-4] was isolated from P. cichorii-inoculated Japanese radish (Raphanus sativus).10 However, no studies regarding the stereochemistry of 4 have been reported to date. Moreover, stereochemical studies of the other three spiroindoline[3,5′]thiazolidine-type phytoalexins, methoxyspirobrassinin (5),11 methoxyspirobrassinol (6), and methoxyspirobrassinol methyl ether (7),12 have not been carried out.3a Preliminary experiments have revealed that partially enantioenriched spirobrassinin (4) shows an enantiomeric-enrichment phenomenon during nonchiral chromatographic separation.13 This interesting feature is extremely novel in a natural product. Because of this curious stereochemical phenomenon and interest in biological studies of chiral cruciferous phytoalexins, we have begun stereochemical studies of spirobrassinin (4) and its oxazoline analogue, spirooxazoline (11). Preliminary studies have revealed that natural (-)-4 has an S configuration based on X-ray crystallography of a (1′S,4′R)-camphanoyl derivative of (+)-4.14 Herein, we describe our detailed stereochemical studies of spirobrassinin (4) and its analogue, spirooxazoline (11) by means of chiral syntheses, the exciton chirality and calculated CD methods, and X-ray crystallography, as well as their enantiomeric-enrichment phenomena and biological activities. Results and Discussion Synthesis and Enantioresolution. The common starting material for the syntheses of racemic spirobrassinin [(()-4] and spirooxazoline [(()-11] was (()-(3hydroxyoxindol-3-yl)methylammonium chloride15 [(()-9] (Scheme 1). Compound 9 was converted to racemic dioxibrassinin [(()-8], a minor phytoalexin-related metabolite isolated from cabbage, by a previously reported method.16 Sub(5) (a) Mehta, R. G.; Liu, J.; Constantinou, A.; Hawthorne, M.; Pezzuto, J. M.; Moon, R. C.; Moriarty, R. M. Anticancer Res. 1994, 14, 1209-1214. (b) Mehta, R. G.; Liu, J.; Constantinou, A.; Thomas, C. F.; Hawthorne, M.; You, M.; Gerha¨user, C.; Moon, R. C.; Moriarty, R. M. Carcinogenesis 1995, 16, 399-404. (6) Baur, E.; Sta¨dler, K.; Monde, K.; Takasugi, M. Chemoecology 1998, 8, 163-168. (7) Verhoeven, D. T. H.; Verhagen, H.; Goldbohm, R. A.; van den Brandt, P. A.; van Poppel, G. Chem. Biol. Interact. 1997, 103, 79129, and cited references. (8) Yamada, F.; Kobayashi, K.; Shimizu, A.; Aoki, N.; Somei, M. Heterocycles 1993, 36, 2783-2804. (9) Kutschy, P.; Dzurilla, M.; Takasugi, M.; To¨ro¨k, M.; Achbergerova´, I.; Homzova´, R.; Ra´cova´, M. Tetrahedron 1998, 54, 3549-3566. (10) Takasugi, M.; Monde, K.; Katsui, N.; Shirata, A. Chem. Lett. 1987, 1631-1632. (11) Gross, D.; Porzel, A.; Schmidt, J. Z. Naturforsch. 1994, 49c, 281-285. (12) Monde, K.; Takasugi, M.; Shirata, A. Phytochemistry 1995, 39, 581-586. (13) Monde, K.; Harada, N.; Takasugi, M.; Kutschy, P.; Suchy´, M.; Dzurilla, M. J. Nat. Prod. 2000, 63, 1312-1314. (14) The details of the X-ray analyses of compounds (+)-15, (-)-19, and (-)-20, see Supporting Information. Preliminary communication [compound (-)-19]: Monde, K.; Osawa, S.; Harada, K.; Takasugi, M.; Suchy´, M.; Kutschy, P.; Dzurilla, M.; Balentova´, E. Chem. Lett. 2000, 886-887. (15) Conn, W. R.; Lindwall, H. G. J. Am. Chem. Soc. 1936, 58, 12361239; for a modified procedure for the preparation of (()-9, see Supporting Information. (16) Monde, K.; Sasaki, K.; Shirata, A.; Takasugi, M. Phytochemistry 1991, 30, 2915-2917.

J. Org. Chem., Vol. 66, No. 11, 2001 3941 Scheme 1

sequent cyclization of (()-8 with thionyl chloride17 or methanesulfonyl chloride in pyridine afforded racemic (()-4. Spectroscopic data of (()-4 were fully identical to those of the natural spirobrassinin.10 In contrast, (()-9 was treated with thiophosgene to give (()-10, with the desired spiro ring system, in 92% yield. The subsequent methylation of (()-10 afforded racemic spirooxazoline [(()-11], an isosteric analogue of 4. The structure of 11 was confirmed by NMR, including NOE, DQF-COSY, HMQC, and HMBC experiments18 at the stage of (R)(+)-11 as well as other spectroscopic data. To enantioresolve (()-4 and (()-11, a chiral auxiliary method was applied. Each racemate was allowed to react with (S)-(-)-1-phenylethyl isocyanate (12) with the addition of triethylamine for reaction acceleration to produce urea derivatives, respectively (Scheme 2). As expected, the obtained diastereomeric pairs of amides (+)-13, (+)-14 and (+)-15, (+)-16 could be separated by simple silica gel chromatography. Removal of the chiral auxiliary by treatment with sodium methoxide afforded (+)-4 ([R]20D +142.7, c 0.25, CH2Cl2, 91% ee), (-)-4 ([R]20D -143.6, c 0.25, CH2Cl2, 92% ee), and (+)-11 ([R]20D +31.7, c 1.37, CHCl3, 95% ee), (-)-11 ([R]20D -34.1, c 0.89, CHCl3, 83% ee), respectively. The chiral analysis was carried out by HPLC using a Sumichiral OA-4700 chiral column (i-PrOH/dichloroethane/hexane), which was monitored by a photodiode array and on-line HPLCCD detectors.18 It should be noted that the online HPLCCD detector proved to be a powerful tool to verify the chirality of each enantiomer during the analysis. CD Studies. To predict the absolute configuration of natural (-)-spirobrassinin [(-)-4], we carried out CD studies of (+)-4, (-)-4, (+)-11, and (-)-11. CD spectra of enantiomeric pairs are completely mirror images.18 Enantiomer (-)-4 showed an exciton-type split at 221 (∆ +25.9) and 204 (∆ -21.0) nm with a moderate A value (+46.9) in the short-wavelength region and two negative Cotton effects at 308 (∆ -5.1) and 264 nm (∆ -5.9) in the long-wavelength region (Figure 1). To assign this split type to the Cotton effect, a model compound, dimethyl N-hexyldithiocarbonimidate (17), (17) Monde, K.; Takasugi, M.; Ohnishi, T. J. Am. Chem. Soc. 1994, 116, 6650-6657. (18) See Supporting Information.

3942

J. Org. Chem., Vol. 66, No. 11, 2001

Suchy´ et al.

Figure 2. A structure of natural (S)-(-)-4. Two arrows on the molecule indicate axes of transition dipole moments.

Figure 1. Experimental UV and CD spectra of (-)-4, 17, and 3,3-dimethyloxindole. Solid line: UV and CD spectra of (-)-4 (a major natural enantiomer). Dotted line: UV spectrum of 17. Alternate long and short dash line: UV spectrum of 3,3dimethyloxindole.

Scheme 2

Figure 3. UV and CD spectra of (-)-4 and (-)-11. Solid line: UV and CD spectra of (-)-11. Dotted line: UV and CD spectra of (-)-4.

(Figure 2). The CD spectrum of (-)-11 fully corresponds to the spectrum of (-)-4 (Figure 3), and (+)-11 to (+)-4, suggesting that (-)-11 also has an S configuration. To more accurately predict the absolute configuration, the theoretical CD calculation was examined. The geometries of spirobrassin (4) for s-cis/trans conformations on a S-C-S-CH3 side-chain were optimized using the Gaussian 98 program.21 Using these two conformers, both oscillator and rotational strengths in the dipole length and velocity expressions corresponding to 30 excitation energies were calculated by the Dalton program.22 The calculated UV and CD spectra of s-cis and s-trans forms

was synthesized from n-hexylamine. The UV spectrum of 17 shows a moderate absorption at 219 nm ( 8590), while 3,3-dimethyloxindole shows a stronger absorption at 205 nm ( 30000).19 These data suggest that two chromophores in (-)-4 (oxindole and NdC(SCH3)2 moiety) would induce the split of the Cotton effects in its CD spectrum.20 In the case of natural (-)-4, two axes of their transition dipole moments should be clockwise to give positive chirality leading to the S configuration of (-)-4 (19) 3,3-Dimethyloxindole was prepared by the published method: Do¨pp, D.; Weiler, H. Chem. Ber. 1979, 112, 3950-3955.

(20) (a) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy - Exciton Coupling in Organic Stereochemistry; University Science Books: Mill Valley, CA, 1983. (b) Nakanishi, K.; Berova, N. In Circular Dichroism - Principles and Application; Nakanishi, K., Berova, N., Woody, R. W., Eds.; VCH Publishers: New York, 1994; pp 361-398. (21) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98 (Revision A.7); Gaussian, Inc.: Pittsburgh, PA, 1998.

Stereochemical Studies of Spirobrassinin

J. Org. Chem., Vol. 66, No. 11, 2001 3943 Scheme 3

Figure 4. Experimental UV and CD spectra of (-)-4 and calculated UV and CD spectra of (S)-4. Solid line: Experimental UV and CD spectra of (-)-4 (a major natural enantiomer). Dotted line: Calculated UV and CD spectra of s-trans(S)-4. Alternate long and short dash line: Calculated UV and CD spectra of s-cis-(S)-4.

of (S)-4 show reasonable agreement with the experimental spectrum of (-)-4, especially at 221 and 308 nm (Figure 4).23 This comparison suggests that the configuration of (-)-4 should be S, which is consistent with the result from the exciton chirality method. Almost the same result was obtained in the calculation for (S)-11.18 X-ray Crystallography. Although all of the prepared enantiomers were crystalline compounds, none of them afforded single crystals suitable for X-ray analysis to allow determination of the absolute stereochemistry. Of the diastereomeric amides (+)-13 - (+)-16, only (+)-15 is crystalline, and the others are amorphous. Fortunately, compound (+)-15 afforded well-developed single crystals from dichloromethane/hexane, and X-ray analysis revealed its configuration at the spiro atom to be R by the internal reference method.14 Consequently, we can conclude that spirooxazoline (+)-11 has an R configuration and therefore that (-)-11 has an S configuration. The absolute configuration of natural spirobrassinin [(-)-4] was clearly solved by derivatization with (1S,4R)-(-)camphanoyl chloride (18), including a crystalline-inducing portion (Scheme 3). In addition, the use of compound 18 as an internal standard was advantageous as its absolute configuration is known. Racemic 4 was allowed to reacted with (1S,4R)(-)-18 to give two amides. Flash chromatographic separation of diastereomers (-)-19 and (-)-20 was somewhat complicated by their instability on silica gel. After a (22) Helgaker, T.; Jensen, H. J. Aa.; Jorgensen, P.; Olsen, J.; Ruud, K.; Agren, H.; Andersen, T.; Bak, K. L.; Bakken, V.; Christiansen, O.; Dahle, P.; Dalskov, E. K.; Enevoldsen, T.; Fernandez, B.; Heiberg, H.; Hettema, H.; Jonsson, D.; Kirpekar, S.; Kobayashi, R.; Koch, H.; Mikkelsen, K. V.; Norman, P.; Packer, M. J.; Saue, T.; Taylor, P. R.; Vahtras, O. Dalton - An electronic structure program, Release 1.0, 1997. (23) The caluculated CD curve of s-cis-(S)-4 is more compatible with the experimental CD curve of (-)-4. However, a major conformer calculated Gaussian 98 and MOPAC-AM1 is s-trans. See also Supporting Information.

column separation lasting more than 1 h, the original spirobrassinin (4) was recovered. Rapid separation, in less than 1 h, gave sufficient yields of (-)-19 (22%) and (-)-20 (22%). The diastereomers of (-)-19 and (-)-20 were assigned to derivatives from (+)-4 and (-)-4 by a direct comparison of products obtained from the reactions of (+)-4 and (-)-4 with 18, respectively (Scheme 3). The diastereomeric amides (-)-19 and (-)-20 afforded suitable single crystals from dichloromethane/hexane which were submitted to X-ray analysis.14 The absolute configuration of (-)-20, derived from (-)-4, was confirmed as S by the internal reference method. Thus, the natural spirobrassinin [(-)-4] has an S-configuration. Furthermore, the X-ray analysis of (-)-19 confirmed the Rconfiguration of (+)-spirobrassinin [(+)-4]14 by both the internal reference method and the Bijvoet method using the heavy-atom (sulfur) effect. These conclusions are consistent with the result predicted by the CD data. Enantiomeric Enrichment. To investigate its enantiomeric purity, natural (S)-(-)-4, was newly isolated from P. cichorii - inoculated turnip roots (B. campestris L. ssp. Rapa).13 A short separation by nonchiral HPLC gave two spirobrassinin fractions. Surprisingly, their enantiomeric excesses were considerably different (earliereluted fraction 98% ee, later-eluted fraction 83% ee) as determined by the chiral HPLC. Preliminary experiments revealed that this phenomenon, called enantiomeric enrichment, was caused by natural (S)-(-)-4 not being enantiomerically pure. In addition, partially enantioenriched (R)-(+)-4 (47% ee, 7.7 mg) showed a more dramatic change in its enantiomeric excesses during separation by nonchiral HPLC (Figure 5, I). This phenomenon of 4 was observed even in the case of a very small injection amount (0.1 mg, Figure 5, II). To investigate the related mechanisms in more detail, spirooxazoline 11 was examined. As expected, partially enantioenriched (R)-(+)-11 (48% ee) also showed the same phenomenon.18 (Figure 5, III). This phenomenon has previously been observed in several cases;24 however, this is a novel example of the enantiomeric-enrichment phenomenon in a natural product in the case of 4.25 As

3944

J. Org. Chem., Vol. 66, No. 11, 2001

Suchy´ et al.

Figure 5. Enantiomeric enrichment of spirobrassinin (4) and spirooxazoline (11) by nonchiral HPLC system. Partially enantioenriched 4 and 11 was separated by HPLC on YMC SIL06 column (300 × 10 mm) using i-PrOH/CHCl3 at speed of 3 mL/min. The peak of 4 or 11 was divided into several fractions (one fraction volume is ca. 0.5 mL). Enantioexcesses of each fraction were analyzed by the chiral HPLC analysis system. I: (+)-4 (47% ee, 7.7 mg) was injected to the HPLC (1% i-PrOHCHCl3) and a peak (tR 25.0-30.0 min) was divided into 30 fractions. II: (+)-4 (43% ee, 0.1 mg) was injected to the HPLC (1% i-PrOH-CHCl3) and a peak (tR 33.6-35.6 min) was divided into 12 fractions. III: (+)-11 (48% ee, 0.1 mg) was injected to the HPLC (5% i-PrOH-CHCl3) and a peak (tR 16.2-17.6 min) was divided into 9 fractions. (a): On-line CD detection at 308 or 307 nm. (b): UV detection at 308 or 307 nm. (c): Enantioexcesses of each fraction and total elution curve calculated from absorption areas of each enantiomer at 230 nm.

other authors have mentioned, this phenomenon is likely caused by a diastereomeric difference between homochiral and heterochiral associations on the surface of the stationary phase. At this stage, we have concluded that the enantiomeric excess of natural (S)-(-)-4 is 95% from (24) (a) Cundy, K. C.; Crooks, P. A. J. Chromatogr. 1983, 281, 1733. (b) Charles, R.; Gil-Av, E. J. Chromatogr. 1984, 298, 516-512. (c) Tsai, W.-L.; Hermann, K.; Hug, E.; Rohde, B.; Dreiding, A. S. Helv. Chim. Acta 1985, 68, 2238-2243. (d) Dobashi, A.; Motoyama, Y.; Kinoshita, K.; Hara, S. Anal. Chem. 1987, 59, 2209-2211. (e) Matusch, R.; Coors, C. Angew. Chem., Int. Ed. Engl. 1989, 28, 626-627. (f) Carman, R. M.; Klika, K. D. Aust. J. Chem. 1991, 44, 895-896. (g) Diter, P.; Taudien, S.; Samuel, O.; Kagan, H. B. J. Org. Chem. 1994, 59, 370-373. (h) Nicoud, R.-M.; Jaubert, J.-N.; Rupprecht, I.; Kinkel, J. Chirality 1996, 8, 234-243. (i) Kosugi, H.; Abe, M.; Hatsuda, R.; Uda, H.; Kato, M. Chem. Commun. 1997, 1857-1858. (25) To the best of our knowledge, only two examples of enantiomeric enrichment in the case of natural products have been reported. One is 14C-labeled nicotine,24a and the other is a urinary metabolite of the female Australian brushtail possum.24f

the average calculation of both fractions. The composite nature of 4 suggests to us a difference in biological activity between the racemate and both of the enantiomers. Therefore, the biological activities of these compounds were examined. Antifungal Activity. An examination of the antifungal activity of 4 and 11, following the previously described procedure,9 against the fungus Bipolaris leersiae revealed only moderate activity, compared to brassinin (1), with little or no difference between the enantiomers and racemates of 4 and 11 (Table 1). Conclusion Racemic spirobrassinin [(()-4] and its oxazoline analogue, spirooxazoline [(()-11], were synthesized and enantioresolved by a chiral auxiliary method. The absolute configurations of natural (-)-4 and (-)-11 were

Stereochemical Studies of Spirobrassinin

J. Org. Chem., Vol. 66, No. 11, 2001 3945

Table 1. Inhibition by 4 and 11 of Fungal Growth of Bipolaris leersiaea concentration (mmol‚L-1) compound

0.05

0.1

0.25

0.5

1

(()-4 (+)-4 (-)-4 (()-10 (+)-10 (-)-10 1

++

+++

+ + +++

++ ++ + +++

+ + + ++ ++ + +++

a Intensity of antifungal activity was scored and classified into four grades: -, normal growth; +, a small reduction in growth; ++, about half of the normal growth; +++, no growth.

determined to be S by the X-ray crystallographic analyses and their CD studies. The HPLC separation under achiral conditions of nonracemic mixtures of (R)-(+)-4 and (S)-(-)-4 as well as (R)-(+)-11 and (S)-(-)-11 showed a novel enantiomeric-enrichment phenomenon.

Experimental Section General Methods. Melting points are uncorrected. Optical rotations were measured at room temperature in either 1-cm or 1-dm tubes, using JASCO DIP-1000 and Messtechnik POLAR L-mp digital polarimeters; specific rotations are given in 10-1 deg cm2 g-1. UV and CD spectra were obtained using a JASCO V-570 and a JASCO J-720 spectrometer, respectively. IR spectra were recorded on a Zeiss IR-75 spectrometer. The NMR spectra were measured on a JEOL Lambda 400 spectrometer with TMS as an internal standard. Mass spectra were taken on a Finigan SSQ 700 spectrometer at ionization energy 70 eV. X-ray crystallographic data were collected with a Mac Science single-crystal diffractometer MXC18SH. Microanalyses were performed with a Perkin-Elmer, Model 2400 analyzer. HPLC separations were performed on a JASCO 900S instrument equipped with a JASCO MD-1515 multiwavelength detector, a JASCO-Borwin data processing system and a JASCO CD-1595 on-line CD detector using an YMC-Pack SIL-06 column (10 × 300 mm, YMC Co., Ltd.). The reaction course was monitored by thin-layer chromatography, using Kavalier Silufol plates. The preparative column chromatography (flash chromatography) was performed over the Kieselgel Merck Type 9385, 230-400 mesh. (()-Spirobrassinin [(()-4]. To a stirred solution of dioxibrassinin16 [(()-8, 400 mg, 1.48 mmol] in dry pyridine (4 mL) was added SOCl2 (320 mg, 0.195 mL, 2.68 mmol). Alternatively, MsCl (133 mg, 0.09 mL, 1.16 mmol) was added to a solution of (()-8 (100 mg, 0.37 mmol) in dry pyridine (3 mL). After being stirred for 1 h (SOCl2), or 5.5 h and setting aside for 18 h (MsCl), the reaction mixture was neutralized with 1 N HCl, and extracted with AcOEt. Organic layer was dried over Na2SO4, filtered on charcoal and concentrated in vacuo. Crystallization of the residue from acetone/cyclohexane afforded (()-4 as colorless crystals (245 mg, 66%), using SOCl2 or (45 mg, 49%), using MsCl: mp 160-162 °C, ref10 158-159 °C. Spectral data of (()-4 are identical with natural (-)spirobrassinin.10 Anal. Calcd for C11H10N2OS: C, 52.77; H, 4.03; N, 11.19. Found: C, 52.90; H, 4.21, N, 10.92. (()-Spiro[indoline-3,5′-oxazolidin]-2-one-2′-thione [(()10]. To a solution of (()-915 (645 mg, 3 mmol) in water (9 mL) was added CSCl2 (384 mg, 0.225 mL, 3.3 mmol) in CH2Cl2 (15 mL). Then, a solution of 5% Na2CO3 (12 mL) was added to the mixture under vigorous stirring within 15 min, and the reaction mixture was stirred for another 20 min. Resulting precipitate was filtrated, washed with water, to gave (()-10 (610 mg, 92%) as colorless crystals: mp 204-206 °C (acetone/ cyclohexane); IR (CHCl3) 3360, 3185, 1728 cm-1; 1H NMR (400 MHz, acetone-d6) δ 9.67 (1H, br s), 9.04 (1H, br s), 7.57 (1H, d, J ) 7.6 Hz), 7.38 (1H, ddd, J ) 7.8, 7.6, and 1.2 Hz), 7.12 (1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 6.99 (1H, d, J ) 7.8 Hz), 4.16 (1H, d, J ) 10.7 Hz), 4.10 (1H, d, J ) 10.7 Hz); 13C NMR

(100 MHz, acetone-d6, COM, DEPT) δ 189.3 (C), 174.0 (C), 143.5 (C), 132.5 (CH), 127.0 (C), 126.0 (CH), 123.9 (CH), 111.4 (CH), 85.5 (C), 51.8 (CH2); UV (EtOH) λmax () 211.4 (26900), 244.6 (20200), 302.8 (1670) nm; EIMS m/z (%) 220 (M+, 20), 191 (25). 177 (10), 164 (50), 148 (60), 89 (22), 60 (100). Anal. Calcd for C10H8N2O2S2: C, 54.53; H, 3.66; N, 12.72. Found: C, 54.62; H, 3.57 N, 12.70. (()-2′-(Methylsulfanyl)spiro{indoline-3,5′-([4′,5′]dihydrooxazol}-2-one [(()-11]. To a stirred suspension of powdered K2CO3 (307 mg, 2.25 mmol) in dry acetone (27 mL) was added (()-10 (451 mg, 2.05 mmol) and CH3I (862 mg, 0.38 mL, 6.13 mmol). After being stirred for 7.5 h, the reaction mixture was poured into cold water (300 mL) and extracted with AcOEt. The organic layer was dried over Na2SO4 and concentrated in vacuo. Crystallization from methanol afforded (()11 as white crystals (370 mg, 77%): mp 174-176 °C; IR, NMR, and EIMS data are identical with those for (R)-(+)-11. Anal. Calcd for C11H10N2O2S: C, 56.39; H, 4.30; N, 11.96. Found: C, 56.60; H, 4.57; N, 12.13. Preparation of Diastereomeric Amides (+)-13 and (+)14. To a solution of (S)-(-)-1-phenylethyl isocyanate (12, 98 mg, 0.66 mmol) in dry acetone (3 mL) was added (()-4 (150 mg, 0.6 mmol). Then, triethylamine (65 mg, 0.09 mL, 0.6 mmol) was added, and the reaction mixture was set aside for 68 h. The solvent was evaporated in vacuo, and the residue was submitted to column chromatography (CH2Cl2). The first fraction gave (+)-13 (110 mg, 46%) and second fraction afforded (+)-14, contaminated with (+)-13. Repeated chromatography of the second fraction afforded pure (+)-14 (55 mg, 23%). (+)N1-[(1S)-1-Phenylethyl]-1-[(R)-spirobrassinin]carboxamide [(+)-13]: an amorphous pale yellow solid, mp 43-46 °C; Rf (CH2Cl2) 0.24; [R]20D +92.4 (c 0.17, CH2Cl2); IR (CHCl3) 3320, 1736, 1720 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.84 (1H, br d, J ) 7.3 Hz, NH), 8.21 (1H, d, J ) 8.3 Hz,), 7.37 (6H, m), 7.28 (1H, m), 7.21 (1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 5.12 (1H, quintet, J ) 7.1 Hz), 4.69 (1H, d, J ) 15.4 Hz), 4.48 (1H, d, J ) 15.4 Hz), 2.63 (3H, s), 1.58 (3H, d, J ) 7.1 Hz); 13C NMR (100 MHz, CDCl3, COM, DEPT) δ 179.1(C), 163.9 (C), 150.6 (C), 142.9 (C), 139.2(C), 130.2 (CH), 128.8 (CH), 128.3 (C), 127.5 (CH), 126.0 (CH), 125.6 (CH), 123.7 (CH), 116.6 (CH), 75.5 (CH2), 65.5 (C), 50.1 (CH), 22.7 (CH3), 15.7 (CH3); UV (EtOH) λmax () 196.2 (48400), 216.8 (31500) nm; CD (EtOH) λext (∆) 196.8 (-17.0), 208.0 (+24.8), 230.2 (-3.6), 245.4 (+3.6), 259.2 (+3.6), 306.4 (+1.3) nm. EIMS m/z (%) 397 (M+, 10), 250 (100), 203 (20), 177 (30), 145 (10), 105 (30). Anal. Calcd for C20H19N3O2S2: C, 60.43; H, 4.82; N, 10.57. Found: C, 60.85; H, 4.43; N, 10.97. (+)-N1-[(1S)-1-Phenylethyl]-1-[(S)-spirobrassinin]carboxamide [(+)-(14)]: an amorphous pale yellow solid, mp 48-51 °C; Rf (CH2Cl2) 0.18; [R]20D +26.8 (c 0.17, CH2Cl2); IR (CHCl3) 3322, 1738, 1726 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.83 (1H, br d, J ) 7.1 Hz, NH), 8.21 (1H, d, J ) 8.1 Hz,), 7.37 (6H, m), 7.28 (1H, m), 7.21 (1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 5.12 (1H, quintet, J ) 7.1 Hz), 4.73 (1H, d, J ) 15.4 Hz), 4.50 (1H, d, J ) 15.4 Hz), 2.63 (3H, s), 1.59 (3H, d, J ) 7.1 Hz); 13C NMR (100 MHz, CDCl3, COM, DEPT) δ 179.1 (C), 164.0(C), 150.6 (C), 142.8 (C), 139.2(C), 130.2 (CH), 128.8 (CH), 128.3 (C), 127.5 (CH), 126.0 (CH), 125.6 (CH), 123.7 (CH), 116.6 (CH), 75.6 (CH2), 65.5 (C), 50.1 (CH), 22.7 (CH3), 15.7 (CH3); UV (EtOH) λmax () 195.8 (48600), 216.4 (30300) nm; CD (EtOH) λext (∆) 218.0 (-21.6), 234.2 (+22.8), 299.4 (-2.4) nm. EIMS m/z (%) 397 (M+, 10), 250 (100), 203 (20), 177 (28), 145 (11), 105 (30). Anal. Calcd for C20H19N3O2S2: C, 60.43; H, 4.82; N, 10.57. Found: C, 60.79; H, 4.48; N, 10.19. Preparation of Diastereomeric Amides (+)-15 and (+)16. Derivatization of (()-11 (178 mg, 0.8 mmol) in dry acetone (6 mL), following the above procedure gave (+)-15 and (+)16. (+)-N1-[(1S)-1-Phenylethyl]-1-[(3R)-2′-(methylsulfanyl)spiro{indoline-3,5′-[4′,5′]dihydrooxazol)}-2-one]carboxamide [(+)-(15)] was given as colorless crystals (110 mg, 36%), mp 132 °C (CH2Cl2/hexane): Rf (CH2Cl2) 0.39; [R]20D +58.0 (c 0.20, CH2Cl2); IR (CHCl3) 3340, 1742, 1710 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.70 (1H, br d, J ) 7.3 Hz, NH), 8.23 (1H, dd, J ) 8.1 and 1.0 Hz), 7.37 (6H, m), 7.28 (1H, m), 7.23 (1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 5.12 (1H, quintet,

3946

J. Org. Chem., Vol. 66, No. 11, 2001

J ) 7.1 Hz), 4.36 (1H, d, J ) 14.2 Hz), 4.11 (1H, d, J ) 14.2 Hz), 2.56 (3H, s), 1.59 (3H, d, J ) 6.8 Hz); 13C NMR (100 MHz, CDCl3, COM, DEPT) δ 176.6 (C), 165.8 (C), 150.4 (C), 142.9 (C), 140.4 (C), 131.4 (CH), 128.8 (CH), 127.5 (CH), 126.1 (C), 126.0 (CH), 125.6 (CH), 123.8 (CH), 116.8 (CH), 84.7 (C), 65.4 (CH2), 50.1 (CH), 22.7 (CH3), 14.7 (CH3); UV (EtOH) λmax () 198.6 (41600), 211.6 (36300), 216.2 (36100) nm; CD (EtOH) λext (∆) 204.4 (+57.0), 219.8 (-51.3), 236.0 (+6.9) nm. EIMS m/z (%) 381(M+, 10), 234 (100), 187 (35), 159 (28), 105 (55). Anal. Calcd for C20H19N3O3S: C, 62.97; H, 5.02; N, 11.02. Found: C, 62.75; H, 5.19; N, 10.81. (+)-N1-[(1S)-1-Phenylethyl]-1-[(3S)-2′-(methylsulfanyl)spiro{indoline-3,5′-[4′,5′]dihydrooxazol)}-2-one]carboxamide [(+)-16] was given as a white amorphous solid (140 mg, 46%), mp 26-28 °C: Rf (CH2Cl2) 0.19; [R]20D +7.0 (c 0.20, CH2Cl2); IR (CHCl3) 3340, 1750, 1710 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.69 (1H, br d, J ) 7.3 Hz, NH), 8.23 (1H, dd, J ) 8.2 and 1.0 Hz,), 7.37 (6H, m), 7.28 (1H, m), 7.23 (1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 5.11 (1H, quintet, J ) 7.1 Hz), 4.40 (1H, d, J ) 14.2 Hz), 4.14 (1H, d, J ) 14.2 Hz), 2.55 (3H, s), 1.59 (3H, d, J ) 6.8 Hz); 13C NMR (100 MHz, CDCl3, COM, DEPT) δ 176.6 (C), 165.9 (C), 150.4 (C), 142.8 (C), 140.4 (C), 131.4 (CH), 128.8 (CH), 127.5 (CH), 126.1 (C), 126.0 (CH), 125.7 (CH), 123.8 (CH), 116.8 (CH), 84.7 (C), 65.5 (CH2), 50.1 (CH), 22.7 (CH3), 14.7 (CH3); UV (EtOH) λmax () 195.8 (46600), 211.4 (34500) nm; CD (EtOH) λext (∆) 207.6 (-15.9), 227.0 (+17.8), 253.8 (-2.1) nm. EIMS m/z (%) 381(M+, 10), 234 (100), 187 (30), 159 (25), 105 (40). Anal. Calcd for C20H19N3O3S: C, 62.97; H, 5.02; N, 11.02. Found: C, 62.58; H, 4.71; N, 10.63. Enantiomers of Spirobrassinin [(R)-(+)-4, (S)-(-)-4]. To a stirred solution of (+)-13 or (+)-14 (110 mg, 0.277 mmol) in dry CH3OH (7 mL) was added a solution of CH3ONa (151 mg, 2.8 mmol) in dry CH3OH (2 mL) within 20 min at -10 °C. After being stirred at the same temperature for 40 min, the reaction mixture was diluted with water (1 mL) and neutralized with 1 N HCl. After removal of CH3OH, the product was extracted with AcOEt, and AcOEt solution was dried over Na2SO4 and evaporated in vacuo. Purification of the residue by flash chromatography (cyclohexane/acetone 2:1) afforded (R)(+)-4 [35 mg, 51% from (+)-13] and (S)-(-)-4 [31 mg, 45% from (+)-14]. (R)-(+)-Spirobrassinin [(R)-(+)-4]: Colorless needles, mp 143-145 °C (acetone/cyclohexane); [R]D20 +142.7 (c 0.25, CH2Cl2); 91% ee; CD (EtOH) λext (∆) 204.4 (21.0), 221.0 (-25.9), 240.0 (2.6), 248.8 (-1.0), 263.6 (5.9), 308.2 (5.1) nm. IR, NMR, UV and EIMS data are identical with those of natural spirobrassinin.10 Anal. Calcd for C11H10N2OS2: C, 52.77; H, 4.03; N, 11.19. Found: C, 52.63; H, 4.12, N, 11.04. (S)-(-)-Spirobrassinin [(S)-(-)-4]: Colorless needles, mp 142-144 °C (acetone/cyclohexane); [R]20D -143.6 (c 0.25, CH2Cl2); 92% ee; CD (EtOH) λext (∆) 204.4 (-21.0), 221.0 (25.9), 240.0 (-2.6), 248.8 (1.0), 263.6 (-5.9), 308.2 (-5.1) nm. IR, NMR, UV, and EIMS data are identical with those of natural spirobrassinin.10 Anal. Calcd for C11H10N2OS2: C, 52.77; H, 4.03; N, 11.19. Found: C, 52.81; H, 3.89, N, 11.35. Enantiomers of Spirooxazoline [(R)-(+)-11, (S)-(-)-11]. Deprotection of (+)-15 and (+)-16 (162 mg, 0.424 mmol) following the above protocol afforded (R)-(+)-11 [50 mg, 53% from (+)-15] and (S)-(-)-11 [45 mg, 48% from (+)-16]. (R)(+)-2′-(Methylsulfanyl)spiro{indoline-3,5′-([4′,5′]dihydrooxazol}-2-one [(R)-(+)-11]: colorless needles, mp 186-188 °C (CH3OH); [R]22D +31.7 (c 1.37, CHCl3); 95% ee; IR (CHCl3) 3455, 1738 cm-1; 1H NMR (CDCl3) δ 9.13 (1H, br s, NH), 7.35 (1H, d, J ) 7.3 Hz, H-4), 7.32 (1H, ddd, J ) 7.8, 7.6, 1.2 Hz, H-6), 7.09 (1H, ddd, J ) 7.6, 7.3, 1.0 Hz, H-5), 6.94 (1H, d, J ) 7.8 Hz, H-7), 4.37 (1H, d, J ) 13.9 Hz, H-11a), 4.14 (1H, d, J ) 13.9 Hz, H-11b), 2.55 (3H, s, SCH3); 13C NMR (COM, DEPT, CDCl3) δ 176.5 (C-2, CdO), 166.0 (C-9, CdN), 140.9 (C-7a), 131.0 (C-6), 127.9 (C-4a), 124.5 (C-4), 123.6 (C-5), 110.8 (C-7), 84.5 (C-3), 64.4 (C-11), 14.6 (SCH3). The full assignment of NMR was performed by the following data: Difference NOE spectra (CDCl3) irr. at δ 9.13 (NH) enhanced signal δ 6.94 (6.8%, H-7), irr. at δ 4.14 (H-11b) enhanced signal δ 4.37 (26.1%, H-11a), 7.35 (4.2%, H-4); DQF COSY correlation (CDCl3) H-4/H-5, H-6: H-6/H-7, H-5, H-4: H-5/H-7, H-6, H-4: H-7/H-5, H-6; HMQC correlation (CDCl3) H-4/C-4: H-6/C-6:

Suchy´ et al. H-5/C-5: H-7/C-7: H-11a/C-11: H-11b/C-11; HMBC correlation (CDCl3) H-4/C-6, C-7a: H-6/C-4, C-7a: H-5/C-4a, C-6: H-7/C-5, C-4a: H-11a/C-4a, C-9(CdN), C-2(CdO): H-11b/C4a, C-9(CdN), C-2(CdO): SCH3/C-9(CdN); UV (EtOH) λmax () 213.8 (28100), 252.4 (4880), 267.4 (sh, 2920) 301.8 (1520) nm; CD (EtOH) λext (∆) 202.4 (22.3), 221.2 (-34.1), 249.8 (5.9), 307.8 (1.9) nm; EIMS m/z (%) 234 (M+, 70), 219 (5), 206 (16), 187 (100), 159 (70), 87 (65). Anal. Calcd for C11H10N2O2S: C, 56.39; H, 4.30; N, 11.96. Found: C, 56.18; H, 4.10; N, 12.09.). (S)-(-)-2′-(Methylsulfanyl)spiro{indoline-3,5′-([4′,5′]dihydrooxazol}-2-one [(S)-(-)-11]: colorless needles, mp 180182 °C (CH3OH); [R]22D -34.1 (c 0.89, CHCl3); 84% ee; UV (EtOH) λmax () 214.2 (28300), 251.8 (4670), 267.2 (sh, 2660), 303.4 (1340) nm; CD (EtOH) λext (∆) 202.2 (-23.4), 221.0 (35.7), 249.8 (-6.2), 306.4 (-2.0) nm. IR, NMR and EIMS data are identical with (R)-(+)-11. Anal. Calcd for C11H10N2O2S: C, 56.39; H, 4.30; N, 11.96. Found: C, 56.45; H, 4.32; N, 12.24. Dimethyl N-Hexyldithiocarbonimidate (17). To a mixture of hexylamine (317 mg, 3.13 mmol), dry triethylamine (0.44 mL, 3.13 mmol), and dry pyridine (3 mL) was added carbon disulfide (239 mg, 0.19 mL, 3.13 mmol) at 0 °C. The mixture was kept at 0 °C for 2 h, treated with methyl iodide (488 mg, 0.21 mL, 3.44 mmol), and kept at 0 °C for 14 h. The reaction mixture was poured into 2 M HCl and extracted with AcOEt twice. The combined organic solution was washed with 2 M HCl, saturated NaHCO3, and water, and dried over Na2SO4. The residue obtained after solvent removal in vacuo was submitted to the silica gel flash column chromatography (hexane/ethyl acetate 10:1) to give an oily compound (methyl N-hexyldithiocarbamate, 536 mg, 90%). IR (neat) 3222, 2955, 2927, 2857, 1509, 1465, 1389, 1335, 1303, 1203, 1151, 1065, 954, 727 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.01 (1H, br s, NH), 3.73 (2H, q, J ) 7.3 Hz, NH-CH2-), 2.64 (3H, s, SCH3), 1.65 (2H, m, NHCH2CH2), 1.32 (6H, m), 0.89 (3H, t, J ) 6.7 Hz, CH3). The spectrum showed minor signals (1/2 intensity of the major signal) due to a rotamer at δ 7.79 (br s, NH), 3.43 (q, J ) 6.8 Hz, NHCH2), 2.68 (3H, s, SCH3), 1.65 (m NHCH2CH2), 1.32 (m), 0.89 (t, J ) 6.7 Hz, CH3); 13C NMR (100 MHz, CDCl3, COM, DEPT) δ 198.7 (CdS), 47.4 (CH2), 31.4 (CH2), 28.3 (CH2), 26.5 (CH2), 22.5 (CH2), 18.1 (CH3), 13.9 (CH3). The spectrum showed minor signal due to a rotamer at δ 201.7 (CdS), 46.3 (CH2), 31.2 (CH2), 28.6 (CH2), 26.4 (CH2), 22.5 (CH2), 18.8 (CH3), 13.9 (CH3); UV (EtOH) λmax 269.2 ( 12200), 252.6 (11300), 222.4 (10300) nm. EIMS m/z (%) 191 (M+, 100), 176 (3), 144 (52), 115 (87), 110 (27), 100 (9), 91 (22), 85 (49). Anal. Calcd for C8H17NS2: C, 50.21; H, 8.95; N, 7.32; S, 33.51. Found: C, 50.07; H, 8.69; N, 7.22; S, 33.50. Methyl N-hexyldithiocarbamate (302 mg, 1.58 mmol) was dissolved in CH3OH (3 mL) and treated with CH3I (2.28 g, 1 mL, 16 mmol) and K2CO3 (0.5 g, 3.62 mmol) at 0 °C. After being kept at room temperature for 3 h, the reaction mixture was filtered with Celite and evaporated. The reaction mixture was extracted with Et2O, and the organic fraction was washed with water. The residue obtained after solvent removal in vacuo was submitted to silica gel flash column chromatography (hexane) to give an oily compound 17 (159 mg, 50%). IR (neat) 2955, 2925, 2856, 1584, 1465, 1428, 1351, 1308, 1120, 1017, 963, 906, 724 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.39 (2H, t, J ) 7.1 Hz), 2.54 (3H, s), 2.36 (3H, s), 1.65 (2H, q, J ) 7.1 Hz), 1.40 (2H, m), 1.32 (4H, m), 0.89 (3H, t, J ) 7.0 Hz); 13C NMR (100 MHz, CDCl3, COM, DEPT) δ 156.5 (CdN), 53.0 (CH2), 31.6 (CH2), 30.6 (CH2), 27.1 (CH2), 22.6 (CH2), 14.5 (CH3), 14.4 (CH3), 14.0 (CH3); UV (EtOH) λmax 231.0 (sh,  7630), 219.1 (8590) nm. EIMS m/z (%) 205 (M+, 2), 190 (9), 158 (100), 85 (18), 74 (77). Anal. Calcd for C9H19NS2: C, 52.63; H, 9.32; N, 6.82; S, 31.23. Found: C, 52.50; H, 9.11; N, 6.76; S, 31.04. 1-[(1′S,4′R)-(-)-Camphanoyl] Derivatives of (R)-(+)-4 and (S)-(-)-4 [(-)-19, (-)-20]. To a stirred suspension of sodium hydride (64 mg of 60% suspension in mineral oil) in anhydrous acetonitrile (3 mL) was added (()-4 (100 mg, 0.40 mmol) and after 5 min (1S,4R)-(-)-camphanoyl chloride [(-)18] (173 mg, 0.8 mmol). After being stirred for 30 min, the reaction mixture was poured into cold water (30 mL), and the product was extracted with AcOEt. The organic solvent was dried over Na2SO4 and concentrated in vacuo, and the residue

Stereochemical Studies of Spirobrassinin was subjected to flash chromatography (cyclohexane/AcOEt 5:1, the duration of the separation must not exceed 60 min). The first fraction gave (-)-19 (37.8 mg, 22%), while the second fraction afforded (-)-20, contaminated with (-)-19. Repeated chromatography of the second fraction afforded pure (-)-20 (37.2 mg, 22%). Alternatively, (-)-19 (50%) was prepared from (R)-(+)-4, and (-)-20 (50%) from (S)-(-)-4, following the above procedure. In these cases, however, the pure products were given by crystallization of the residue obtained after evaporation of AcOEt. (-)-1-[(1′S,4′R)-Camphanoyl]-(R)-spirobrassinin [(-)-19]: colorless prisms, mp 177-179 °C (CH2Cl2/ hexane); Rf (cyclohexane/AcOEt 5:1) 0.54; [R]20D -16.9 (c 0.20, CH2Cl2); IR (CHCl3) 1787, 1772, 1718 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.52 (1H, d, J ) 8.1 Hz, H-7), 7.39 (1H, dd, J ) 7.4 and 1.3 Hz, H-4), 7.27 (1H, ddd, J ) 8.1, 7.6, and 1.3 Hz, H-6), 7.17 (1H, ddd, J ) 7.6, 7.4, and 1.0 Hz, H-5), 4.82 (1H, d, J ) 15.4 Hz, H-11a), 4.40 (1H, d, J ) 15.4 Hz, H-11b), 2.92 (1H, ddd, J ) 13.5, 9.4, and 4.4 Hz, H-6′R), 2.55 (3H, s, SCH3), 2.27 (1H, ddd, J ) 13.5, 10.8, and 4.4 Hz, H-6′β), 1.89 (1H, ddd, J ) 12.8, 10.8, and 4.4 Hz, H-5′β), 1.72 (1H, ddd, J ) 12.8, 9.4, and 4.4 Hz, H-5′R), 1.22 (3H, s, H-11′, 7′-CH3), 1.04 (3H, s, H-9′, 4′-CH3), 0.90 (3H, s, H-10′, 7′-CH3); 13C NMR (100 MHz, CDCl3, COM, DEPT) δ 177.4 (C-3′, CdO), 175.1 (C-2, CdO), 171.1 (C-8′, CdO), 163.9 (C-9, CdN), 138.2 (C7a), 129.9 (C-6), 129.4 (C-4a), 125.9 (C-5), 124.6 (C-4), 113.7 (C-7), 92.9 (C-1′), 76.0 (C-11), 65.7 (C-3), 57.3 (C-4′), 54.5 (C7′), 30.4 (C-6′), 29.5 (C-5′), 17.6 (C-11′), 16.6 (C-10′), 15.6 (SCH3), 9.7 (C-9′). The full assignment of NMR was performed by the following data: Difference NOE spectra (CDCl3, 400 MHz) irr. at δ 4.40 (H-11b), enhanced signal δ 4.82 (34.4%, H-11a), 7.39 (7.0%, H-4); irr. at δ 1.22 (H-11′, 7′-CH3), enhanced signal δ 2.27 (6.0%, H-6′β), 1.89 (4.8%, H-5′β); irr. at δ 1.04 (H-9′, 4′-CH3) enhanced signal δ 1.89 (2.5%, H-5′β), 1.72 (1.5%, H-5′R); irr. at δ 0.90 (H-10′, 7′-CH3) enhanced signal δ 7.52 (4.3%, H-7); HMQC correlations (CDCl3) H-7/C-7: H-4/ C-4: H-6/C-6: H-5/C-5: H-6′R/C-6′: H-11a/C-11: H-11b/C11: H-6′β/C-6′: H-5′R/C-5′; H-5′β/C-5′: H-SCH3/SCH3: H-11′/ C-11′: H-9′/C-9: H-10′/C-10′; HMBC correlations (400 MHz, CDCl3) H-7/C-4a, C-5, C-7a: H-4/C-3, C-5, C-6, C-7a: H-6/C4, C-7, C-7a: H-5/C-4, C-7: H-11a/C-2 (CdO), C-4a, C-9(Cd N): H-11b/C-2 (CdO), C-4a, C-9(CdN): H-6′R/C-1′, C-4′, C-5′, C-7′:H-SCH3/C-9(CdN): H-6′β/C-5′, C-7′: H-5′R/C-3′(CdO), C-6′, C-9′, C-7′: H-5β/C-3′(CdO), C-6′, C-4′, C-7′: H-11′/C-1′, C-4′, C-7′, C-10′: H-9′/C-3′ (CdO), C-4′, C-5′, C-7′: H-10′/C-1′, C-4′, C-7′, C-11′; UV (EtOH) λmax () 297.0 (sh, 1340), 269.4 (sh, 5290), 237.2 (sh, 14900), 205.6 (30700) nm; CD (EtOH) λext (∆) 203.4 (+6.2), 213.6 (-15.7), 226.4 (+2.1), 240.2 (-6.2), 258.2 (+4.3), 274.8 (+5.7), 306.2 (-1.3) nm; EIMS m/z (%) 430 (M+, 58), 402 (10), 383 (12), 357 (100), 329 (19), 249 (38). Anal.

J. Org. Chem., Vol. 66, No. 11, 2001 3947 Calcd for C21H22N2O4S2: C, 58.58; H, 5.15; N, 6.51. Found: C, 58.43; H, 5.29; N, 6.62. (-)-1-[(1′S,4′R)-Camphanoyl]-(S)spirobrassinin [(-)-20]: colorless prisms, mp 207-209 °C (CH2Cl2/hexane); Rf (cyclohexane/AcOEt 5:1) 0.46; [R]D20-39.6 (c 0.20, CH2Cl2); IR (CHCl3) 1788, 1770, 1710 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.63 (1H, d, J ) 7.8 Hz), 7.45 (1H, dd, J ) 7.6 and 0.7 Hz), 7.35 (1H, ddd, J ) 7.8, 7.6, and 1.2 Hz), 7.24 (1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 4.76 (1H, d, J ) 15.3 Hz), 4.54 (1H, d, J ) 15.3 Hz), 2.96 (1H, ddd, J ) 13.5, 9.3, and 4.4 Hz), 2.62 (3H, s), 2.35 (1H, ddd, J ) 13.5, 10.7, and 4.4 Hz), 1.96 (1H, ddd, J ) 13.0, 10.7, and 4.4 Hz), 1.80 (1H, ddd, J ) 13.0, 9.3, and 4.4 Hz), 1.29 (3H, s), 1.11 (3H, s), 0.96 (3H, s); 13C NMR (100 MHz, CDCl3, COM, DEPT) δ 177.3 (C), 175.3 (C), 171.5 (C), 163.7 (C), 137.8 (C), 130.0 (CH), 129.9 (C), 126.1 (CH), 124.5 (CH), 113.8 (CH), 92.8 (C), 75.1 (CH2), 63.8 (C), 57.3 (C), 54.5 (C), 30.6 (CH2), 29.5 (CH2), 17.6 (CH3), 16.6 (CH3), 15.7 (CH3), 9.7 (CH3); UV (EtOH) λmax () 206.8 (30100), 229.8 (sh, 16700), 267.4 (sh, 5950), 297.6 (sh, 1210) nm; CD (EtOH) λext (∆) 203.4 (-5.2), 210.8 (+3.0), 224.0 (-17.1), 243.4 (+9.9), 265.0 (-3.7), 291.4 (-2.8), 314.4 (+0.4) nm. EIMS m/z (%) 430 (M+, 58), 402 (10), 383 (12), 357 (100), 329 (19), 249 (38). Anal. Calcd for C21H22N2O4S2: C, 58.58; H, 5.15; N, 6.51. Found: C, 58.32; H, 5.37; N, 6.70.

Acknowledgment. The authors are grateful to Assoc. Professor V. Milata and Dr. J. Salon, Slovak Technical University Bratislava, for their cooperation with a new synthesis of amine (()-9. We wish to thank the Grant Agency for Science of the Slovak Republic (project No. 1/6080/99) for their financial support. This research was partially supported by the Ministry of Education, Science, Sports, and Culture, Grant-in-Aid for Encouragement of Young Scientists, 11780413, 2000. The authors would also like to thank Dr. Shuichi Osawa, Tohoku University, for his helpful discussion regarding the computational analyis of (S)-4. Supporting Information Available: Experimental procedure for the synthesis of (()-9; copies of 1H and 13C NMR spectra of (+)-13, (+)-14, (+)-16 and 2D-NMR spectra for (R)(+)-11; CD spectra of (R)-(+)-4, (S)-(-)-4, (R)-(+)-11, and (S)(-)-11; calculated UV and CD data for (S)-4 and (S)-11; the chiral HPLC analyses with the chiral column of (()-4 and (()11; X-ray data for (+)-13, (-)-19 and (-)-20. This material is available free of charge via the Internet at http://pubs.acs.org. JO0155052