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Ent-kaurane diterpenoids from the liverwort Jungermannia infusca
Short Reports Phflochrmlsiry,
1977.Vol. 16.pp. 489490. Pergamon ha.
EA’T-KAURANE
Rimed in England
DITERPENOIDS
FROM THE LIVERWORT
JUNGERMANNIA
INFUSCA
AKMKO MATSUO, JUNROKODOMA,Mrmu~u NAKAYAMA and SHincm HAYASIU Department of Chemistry, Faculty of !%ence. Hiroshima University, Hiroshima 730, Japan
(Rec&ed 24 .%prmrbcr 1976)
Key Word I&x-.rungmMnnb 61Mca; Hepatieae; (16R)-enr-kauran-lS-o_one;ear-kaurcn-15-one; ear-15ahydroxykaurene; chemotaxonomy.
We have, recently, investigated the sesquiterpenoids of several liverworts and character&d enantiomeric forms of those ftom the higher plants [ 1J. Regarding the diterpenoids of the liverworts, them are few repor& i.e. of the isolation of enr-man001 [2], five ent-kaurana type diterpenoids [3,4] and three ent-pimarane diterpenoids [S]. The present paper deala with the isolation and structural elucidation of three em-kaurane diterpenoids from Junginjkua (Mitt) Steph. a leafy liverwort in the Jungermanniaceae+ The neutral fraction of extract from the liverwort gave three diterpenoids (l-3) which were isolated along with a mixture of diterpene acetates.
(16R)-ent-Kauran-I5-one (1). The middle polar compound (R, O.Sl), C,,H,,O (M’ 288), mp 14%147”, [alo -7@‘, was characterized by spectral data as a saturated tetracyclic diterpenoid containing three tertiary methyls (v 1390,137s cm-‘; d Q.83,0.90,1.10, each 3H, s), a secondary methyl (S 1.11, 3H, d. J = 6) and a partial structure of cyclopentanone (v 1735 cm-r). The compound was idemiSed as (16R)-ent-kauran-15one ((Q~~$ncidence of the physical constants and spectral
3R-H 4R=Ac ent-K-en-IS-one (2) The least polar ditespenoid (Rr 0.58), C,,H, 0 (M+ 286), mp 99-loo”, [a], -2#P, was also atsum b8 to be a kaurane type ketone containing three tertiary methyls (v 1377, 137Ocm-r; d 0.83,0.90, 1.10, each 3H, s) and a partial structure ofa cyclopentanone conjugated with an exomethylene (v 1725, 1645,
935 cm-‘; 6 5.25, 5.95, each HI, brs; A’?“= 233nm, a 8&oo).On hydrogenation over Adams cat$st in ethyl acetate it gave 1. Thus, the compound was identified as em-kauren-15-one (2). ent-l5a-Hydroxykaurerte (3). The most polar compound (R, 0.42), CzOH,,O (M+ 288), mp 99-l@O”, IaID-7O”, was isolated as a major component. The spectral data exhibited the presence of a secondary hydroxyl group (v 3600, 3450 cm- ’ ; S 3.97, lH, br), three tertiary methyls (v 1393, 1375 cm-‘; b 0.82, 0.87, 1.03, each 3H, s) and an exometbykne (v 1665,895 cm-‘; 6 4.87, 5.fQ each H-I, br.s). In addition, the compound was treated with Ac,O in pyridine to give an acetate (4), C,,H,,O, (M+-330); v 1740, 1665, 89Ocm-r; d 0.82, 0.87, 1.05.2.12 (each 3H, s), 4.86 (H-I, br), 4.86, 5.07 (each 1H. br.s), and it was oxidixed with Jones reagent to give the a&unsaturated ketone 2 Thus, it was identified as Lauren-15-oL In order to determine the configuration of the hydroxyl group located on C-15 in the enr-kaurene skeleton, the alcohol (3) was treated with HCl to afford 1 through a ready garryfolinbcuauchichine rearrangement [7,8]. Thus the stereostructure of the alcohol is d-1 Sa-hydroxykaurene (3). Although the three enr-kaurane diterpenoids have been reported as reaction products. this is their fit isolation as natural products. Their biosynthetic simplicity [9] fitsinwiththeiro ccumna in a very ancient group of plants. EXPERIMENTAL Mp’s are uncorrected. IR and PMR spectra were determined in CCl, or CHCl,, and the mass spectra on a single focusing instrument under the following conditions: 7OeV ionization chamber voltagt, 80 pA total emissios l&l0 V accelerating volw and 200” ionization chamber temperature The optical rotations were measured with an automatic polarimeter in CHCl,. MaterM Md t.~lution The liverwort (1.7 kg) collected at the suburbs of Owaae City in Mie Prefecturewas dried and digested with MeOH to obtain a crude extract (37.9g) A neutral part (23.7 g) of the estract was subjected to Si gel-column chromate graphy followed by preparative TLC with !Si gel in hexaneC,H, (1:2) to give 3 crystalliae compounds, 1 (R, O.Sl), 2 JR, 0.58) and 3 (R 0.42), which were furtherpurified by mcq&&zation from Etd H. Catalyzichydrogenaticn0/2r01.Unsaturatedkctonc(2,15 mg) was hydrogenated over PtO, (3mg) in EtOAc (7mQ at room temp_ and the reaction mixture, on working up, gave a crystalline product from EtOH: C,,H,,O (M’ 288), mp 14f-149”, [a], -77”.
489 PHYTO VOL. 16 No.
4-F
Short Reports
490
Oxidurionof 3 to 2. The alcohol (3, 20 mg) in Me,CO (3 ml) was treated with an exm of Jones reagent at 0” for 5 min. The product extracted with Et,0 was submitted to preparative TLC to give the u&msaturatal ketone (2,15 mg) which was crystallized from EtOH as needles: C,,H,,O (M+ 286), mp 99100”, [a], - 146”. Aceryfurfon43 to 4. The diterpenoid (3,lO ma) was treated with Ac,O (0.5 ml) in dried pyridine (0.5 ml) at room temp for IS hr. The product 4 (7 mg) CssHs*Or (M* 330) had mp 115 116”, [a], - 100”. Reo~~gemmt 63 to 1. The unsaturated alcohol (3.20 mg) in MeOH (4 ml) and ether (2 ml) was treated with cone NCl(0.8 ml) at room temp. for 3 hr. The reaction mixture was taken up into Et,O, and the ethereal solution was washed, dried and concd tp give a major product (1, 15mg) which was submitted to preparative TLC and recrystallixation: C,,H,,O (M+ 288). mp 148-149”. [a], -78”.
RKFKRmcEs 1. Hayashi, S. and Matsuo, A (1976) Chemfsrry31. 518 and referencescited therein. 2. Matsuo, A., Nakayama, M, Ono, J. and Hay&i, S. (1972) 2. Nattujorsch. 27R 1437. 3. Hunock S and Vevle, 0. (1970) 2. NarwjZvsA 25 B, 227. 4. ConnoIly. J. D. and Thornton, I. M. S. (1973) 1. Chem Sot. Perkfn I, 736. 5. Matsuo, A., Utq S, Nakayama, M, Hayashi, S, Yamasaki, IC.,Rasal, R and Tanaka, 0. (1976) Tetrahe&m Letters 2451. 6. MacMillan, J. and Walker, E R H. (1972) /. Chn Sot. Perkfn I, 986; Connolly, J. D. and Thornton, I. M. S (1973) J. Ckem Sot. Perktn I, 736. 7. Barnes, M. F. and MacMillan, J. (1967) J. C/tern &c.(C) 361. 8. D&ding, A S and Hartman, J. A (1956) 1. Am Chn Sot. -1216.
9. Devon, T. K. and Scott, A I. (1972) Handbook d Natvafly Occvrfng Compounds,Vol. 2 Academic Press, New York
Acknowf&emeurs-The authors thank Mr. Kosaku Yamada Ujiyamada High School, for collection and identillcation of the liverwort, and they am grateful to Dr. J. D. Connolly, University of Glasgow for the PMR spectrum of (16R)-eur-kauren-15or ~arealsoductotheJapan~MinistryolEducationand the Ito Science Foundation for f’mancialsupport of this work.
PhyrochemMry,
1977. Vol 16. pp &IO-492 Pqxmon
Rcu
Printed in England
IN VITRO CYCLIZATION OF SQUALENE 2,IEPOXIDE TO a-AMYRIN FROM BRAMBLE CELL SUSPENSION CULTURES
BY MICROSOMES
JOHNW. ELDER*,PIERREBnNvnNIsIgt and PAUL Fommewt ‘Department of Chemistry, Fairtield University, Fairfield, Conn. 06430, U.S.A.; tbtstitut de Botanique, Laboratoire de Biochimie VCgttale, E.R.A. au C.N.R.S. no 487, 28, rue Goethe, 67983 Strasbourg Cedex, France (Receiued21 Julv 1976) Key Word Idcx-R&u
fmricosus; Rosaceae; squalene 2,3-epoxidea-amyrin cyclase.
Ahatract-Squalene 2,3-epoxide incubated with microsomes from bramble cell suspension cultures is shown to be converted into a-amyrin.
INTRODUCl’lON In 1955, Ruxicka and his coworkers proposed the bie synthesis of a- and /I-amyrin, along with other triterpenes and steroids from squalene Cl]. The in vitro incorporation of mevalonate and squalene f3cpoxide into /I-amyrin has been demonstrated [2-51. In the case of a- amyrin, in aiuo experiments have shown incorporation of CO*, acetate and mevalonate into a-amyrin or its derivatives C&8]. Recently incorporation of mevalonate into a- and jl-amyrin by chopped preparations of plant parts has been shown [9]. In vitro biosynthesis of a-amyrin has not been reported ; however, Corey and Dean imply that leas than 5 % of the radioactivity found in impure j3-amyrin from in t&o incorporation of squalena epoxide might be due to a-amyrin but offer no proof [S, lo]. In many cases, a-amyrin seems to constitute a minor component of the pentacyclic triterpene mixture
and this, added to the dilBculty to separate the two amyrins, rendered very difficult the study of in vitro biosynthesis of a-amyrin. As we recently showed that a-amyrin constituted more than 70 ‘A of the pentacyclic triterpene fraction in bramble cells [ 111.we decided therefore to study the in oitro biosynthesis of this compound. Wewishtoreporthereanunambiguousincorporation of squaIene epoxide into a-amyrin by microsomes from bramble cells suspension cultures. llEstJLTs AND Dl!3CUSSION The 4,4-dimcthyl steryl acetate fraction from bramble celI suspension cultures has been resolved by argentation chromatography into 24-methylene cycloartenyl acetate (R, 0.20X cycloartenyl acetate (R, 0.45) and pentacyclic triterpene acetates (R, 0.80). CC-MS analysis showed that this latter fraction contained @myrin
1977.Vol. 16.pp. 489490. Pergamon ha.
EA’T-KAURANE
Rimed in England
DITERPENOIDS
FROM THE LIVERWORT
JUNGERMANNIA
INFUSCA
AKMKO MATSUO, JUNROKODOMA,Mrmu~u NAKAYAMA and SHincm HAYASIU Department of Chemistry, Faculty of !%ence. Hiroshima University, Hiroshima 730, Japan
(Rec&ed 24 .%prmrbcr 1976)
Key Word I&x-.rungmMnnb 61Mca; Hepatieae; (16R)-enr-kauran-lS-o_one;ear-kaurcn-15-one; ear-15ahydroxykaurene; chemotaxonomy.
We have, recently, investigated the sesquiterpenoids of several liverworts and character&d enantiomeric forms of those ftom the higher plants [ 1J. Regarding the diterpenoids of the liverworts, them are few repor& i.e. of the isolation of enr-man001 [2], five ent-kaurana type diterpenoids [3,4] and three ent-pimarane diterpenoids [S]. The present paper deala with the isolation and structural elucidation of three em-kaurane diterpenoids from Junginjkua (Mitt) Steph. a leafy liverwort in the Jungermanniaceae+ The neutral fraction of extract from the liverwort gave three diterpenoids (l-3) which were isolated along with a mixture of diterpene acetates.
(16R)-ent-Kauran-I5-one (1). The middle polar compound (R, O.Sl), C,,H,,O (M’ 288), mp 14%147”, [alo -7@‘, was characterized by spectral data as a saturated tetracyclic diterpenoid containing three tertiary methyls (v 1390,137s cm-‘; d Q.83,0.90,1.10, each 3H, s), a secondary methyl (S 1.11, 3H, d. J = 6) and a partial structure of cyclopentanone (v 1735 cm-r). The compound was idemiSed as (16R)-ent-kauran-15one ((Q~~$ncidence of the physical constants and spectral
3R-H 4R=Ac ent-K-en-IS-one (2) The least polar ditespenoid (Rr 0.58), C,,H, 0 (M+ 286), mp 99-loo”, [a], -2#P, was also atsum b8 to be a kaurane type ketone containing three tertiary methyls (v 1377, 137Ocm-r; d 0.83,0.90, 1.10, each 3H, s) and a partial structure ofa cyclopentanone conjugated with an exomethylene (v 1725, 1645,
935 cm-‘; 6 5.25, 5.95, each HI, brs; A’?“= 233nm, a 8&oo).On hydrogenation over Adams cat$st in ethyl acetate it gave 1. Thus, the compound was identified as em-kauren-15-one (2). ent-l5a-Hydroxykaurerte (3). The most polar compound (R, 0.42), CzOH,,O (M+ 288), mp 99-l@O”, IaID-7O”, was isolated as a major component. The spectral data exhibited the presence of a secondary hydroxyl group (v 3600, 3450 cm- ’ ; S 3.97, lH, br), three tertiary methyls (v 1393, 1375 cm-‘; b 0.82, 0.87, 1.03, each 3H, s) and an exometbykne (v 1665,895 cm-‘; 6 4.87, 5.fQ each H-I, br.s). In addition, the compound was treated with Ac,O in pyridine to give an acetate (4), C,,H,,O, (M+-330); v 1740, 1665, 89Ocm-r; d 0.82, 0.87, 1.05.2.12 (each 3H, s), 4.86 (H-I, br), 4.86, 5.07 (each 1H. br.s), and it was oxidixed with Jones reagent to give the a&unsaturated ketone 2 Thus, it was identified as Lauren-15-oL In order to determine the configuration of the hydroxyl group located on C-15 in the enr-kaurene skeleton, the alcohol (3) was treated with HCl to afford 1 through a ready garryfolinbcuauchichine rearrangement [7,8]. Thus the stereostructure of the alcohol is d-1 Sa-hydroxykaurene (3). Although the three enr-kaurane diterpenoids have been reported as reaction products. this is their fit isolation as natural products. Their biosynthetic simplicity [9] fitsinwiththeiro ccumna in a very ancient group of plants. EXPERIMENTAL Mp’s are uncorrected. IR and PMR spectra were determined in CCl, or CHCl,, and the mass spectra on a single focusing instrument under the following conditions: 7OeV ionization chamber voltagt, 80 pA total emissios l&l0 V accelerating volw and 200” ionization chamber temperature The optical rotations were measured with an automatic polarimeter in CHCl,. MaterM Md t.~lution The liverwort (1.7 kg) collected at the suburbs of Owaae City in Mie Prefecturewas dried and digested with MeOH to obtain a crude extract (37.9g) A neutral part (23.7 g) of the estract was subjected to Si gel-column chromate graphy followed by preparative TLC with !Si gel in hexaneC,H, (1:2) to give 3 crystalliae compounds, 1 (R, O.Sl), 2 JR, 0.58) and 3 (R 0.42), which were furtherpurified by mcq&&zation from Etd H. Catalyzichydrogenaticn0/2r01.Unsaturatedkctonc(2,15 mg) was hydrogenated over PtO, (3mg) in EtOAc (7mQ at room temp_ and the reaction mixture, on working up, gave a crystalline product from EtOH: C,,H,,O (M’ 288), mp 14f-149”, [a], -77”.
489 PHYTO VOL. 16 No.
4-F
Short Reports
490
Oxidurionof 3 to 2. The alcohol (3, 20 mg) in Me,CO (3 ml) was treated with an exm of Jones reagent at 0” for 5 min. The product extracted with Et,0 was submitted to preparative TLC to give the u&msaturatal ketone (2,15 mg) which was crystallized from EtOH as needles: C,,H,,O (M+ 286), mp 99100”, [a], - 146”. Aceryfurfon43 to 4. The diterpenoid (3,lO ma) was treated with Ac,O (0.5 ml) in dried pyridine (0.5 ml) at room temp for IS hr. The product 4 (7 mg) CssHs*Or (M* 330) had mp 115 116”, [a], - 100”. Reo~~gemmt 63 to 1. The unsaturated alcohol (3.20 mg) in MeOH (4 ml) and ether (2 ml) was treated with cone NCl(0.8 ml) at room temp. for 3 hr. The reaction mixture was taken up into Et,O, and the ethereal solution was washed, dried and concd tp give a major product (1, 15mg) which was submitted to preparative TLC and recrystallixation: C,,H,,O (M+ 288). mp 148-149”. [a], -78”.
RKFKRmcEs 1. Hayashi, S. and Matsuo, A (1976) Chemfsrry31. 518 and referencescited therein. 2. Matsuo, A., Nakayama, M, Ono, J. and Hay&i, S. (1972) 2. Nattujorsch. 27R 1437. 3. Hunock S and Vevle, 0. (1970) 2. NarwjZvsA 25 B, 227. 4. ConnoIly. J. D. and Thornton, I. M. S. (1973) 1. Chem Sot. Perkfn I, 736. 5. Matsuo, A., Utq S, Nakayama, M, Hayashi, S, Yamasaki, IC.,Rasal, R and Tanaka, 0. (1976) Tetrahe&m Letters 2451. 6. MacMillan, J. and Walker, E R H. (1972) /. Chn Sot. Perkfn I, 986; Connolly, J. D. and Thornton, I. M. S (1973) J. Ckem Sot. Perktn I, 736. 7. Barnes, M. F. and MacMillan, J. (1967) J. C/tern &c.(C) 361. 8. D&ding, A S and Hartman, J. A (1956) 1. Am Chn Sot. -1216.
9. Devon, T. K. and Scott, A I. (1972) Handbook d Natvafly Occvrfng Compounds,Vol. 2 Academic Press, New York
Acknowf&emeurs-The authors thank Mr. Kosaku Yamada Ujiyamada High School, for collection and identillcation of the liverwort, and they am grateful to Dr. J. D. Connolly, University of Glasgow for the PMR spectrum of (16R)-eur-kauren-15or ~arealsoductotheJapan~MinistryolEducationand the Ito Science Foundation for f’mancialsupport of this work.
PhyrochemMry,
1977. Vol 16. pp &IO-492 Pqxmon
Rcu
Printed in England
IN VITRO CYCLIZATION OF SQUALENE 2,IEPOXIDE TO a-AMYRIN FROM BRAMBLE CELL SUSPENSION CULTURES
BY MICROSOMES
JOHNW. ELDER*,PIERREBnNvnNIsIgt and PAUL Fommewt ‘Department of Chemistry, Fairtield University, Fairfield, Conn. 06430, U.S.A.; tbtstitut de Botanique, Laboratoire de Biochimie VCgttale, E.R.A. au C.N.R.S. no 487, 28, rue Goethe, 67983 Strasbourg Cedex, France (Receiued21 Julv 1976) Key Word Idcx-R&u
fmricosus; Rosaceae; squalene 2,3-epoxidea-amyrin cyclase.
Ahatract-Squalene 2,3-epoxide incubated with microsomes from bramble cell suspension cultures is shown to be converted into a-amyrin.
INTRODUCl’lON In 1955, Ruxicka and his coworkers proposed the bie synthesis of a- and /I-amyrin, along with other triterpenes and steroids from squalene Cl]. The in vitro incorporation of mevalonate and squalene f3cpoxide into /I-amyrin has been demonstrated [2-51. In the case of a- amyrin, in aiuo experiments have shown incorporation of CO*, acetate and mevalonate into a-amyrin or its derivatives C&8]. Recently incorporation of mevalonate into a- and jl-amyrin by chopped preparations of plant parts has been shown [9]. In vitro biosynthesis of a-amyrin has not been reported ; however, Corey and Dean imply that leas than 5 % of the radioactivity found in impure j3-amyrin from in t&o incorporation of squalena epoxide might be due to a-amyrin but offer no proof [S, lo]. In many cases, a-amyrin seems to constitute a minor component of the pentacyclic triterpene mixture
and this, added to the dilBculty to separate the two amyrins, rendered very difficult the study of in vitro biosynthesis of a-amyrin. As we recently showed that a-amyrin constituted more than 70 ‘A of the pentacyclic triterpene fraction in bramble cells [ 111.we decided therefore to study the in oitro biosynthesis of this compound. Wewishtoreporthereanunambiguousincorporation of squaIene epoxide into a-amyrin by microsomes from bramble cells suspension cultures. llEstJLTs AND Dl!3CUSSION The 4,4-dimcthyl steryl acetate fraction from bramble celI suspension cultures has been resolved by argentation chromatography into 24-methylene cycloartenyl acetate (R, 0.20X cycloartenyl acetate (R, 0.45) and pentacyclic triterpene acetates (R, 0.80). CC-MS analysis showed that this latter fraction contained @myrin