Sesquiterpene lactones and a myoinositol from glandular trichomes of Viguiera quinqueremis (Heliantheae; Asteraceae)

Phytochemistry 57 (2001) 267±272

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Sesquiterpene lactones and a myoinositol from glandular trichomes of Viguiera quinqueremis (Heliantheae; Asteraceae) Otmar Spring a,*, Reinhard Zipper a, Sabine Reeb b, Bernhard Vogler b, Fernando B. Da Costa c a

Institut fuÈr Botanik, UniversitaÈt Hohenheim, D-70599 Stuttgart, Germany Institut fuÈr Chemie, UniversitaÈt Hohenheim, D-70599 Stuttgart, Germany c Faculdade de CieÃncias FarmaceÃuticas de RibeiraÄo Preto, Universidade de SaÄo Paulo, Av. do Cafe s/n , 14040-903, RibeiraÄo Preto, SP, Brazil b

Received 17 July 2000; received in revised form 17 November 2000 This paper is dedicated to Professor W. Kraus on the occasion of his 70th birthday

Abstract The extract of the ¯oral parts of Viguiera quinqueremis a€orded, in addition to known compounds, six new sesquiterpene lactones as well as a new myoinositol derivative. All compounds were detected in glandular trichomes which were collected micromechanically from the anther appendages and were analyzed by HPLC. Structure identi®cation was performed by 1H NMR measurements including LC NMR and LC MS experiments. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Viguiera quinqueremis; Heliantheae; Asteraceae; Chemotaxonomy; Sesquiterpene lactones; Myoinositols

1. Introduction

2. Results and discussion

Viguiera quinqueremis Blake is part of a group of approximately 14 species that Blake (1918) merged in the series Bracteatae of section Paradosa, the South American branch of the large and polyphyletic genus Viguiera. The taxa of Bracteatae are endemic to Brazil and Paraguay and so far relatively few attempts were made to establish their phytochemical patterns. Up to now, four heliangolides of the 1-keto-2,3-unsaturated-3,10-epoxytype were identi®ed from V. oblongifolia (Bohlmann et al., 1984; Tamayo-Castillo et al., 1990) and V. robusta (Da Costa et al., 1996), respectively, while V. nervosa was reported to lack sesquiterpene lactones (Tamayo-Castillo et al., 1990). V. quinqueremis is a perennial herb with sessile, mostly alternate and linear-oblong leaves. Microscopic studies of vegetative plant parts revealed the absence of glandular trichomes that are typical for the sequestration of sesquiterpene lactones, while numerous such glands were present on the anther appendages. It was the aim of the current study to identify the chemical constituents of these trichomes.

HPLC analyses of extracts from glandular trichomes of anther appendages of V. quinqueremis plants from a population collected near Diamantina, State of Minas Gerais, Brazil, showed the presence of at least 15 peaks with UV-spectra and chromatographic behavior typical for sesquiterpene lactones. Comparison with retention times of reference compounds from our previous chemotaxonomic studies on Helianthinae (Spring and Buschmann, 1996) together with spectral data obtained by LC 1 H NMR and LC MS measurements led to the identi®cation of the known heliangolides 1±6 (De Vivar et al., 1976; Ohno and Mabry, 1980; Delgado et al., 1982; Gao and Mabry, 1986) and 11±13 (Buschmann and Spring, 1995; Spring et al., 1982, 1989) (Fig. 1). Preparative HPLC was used to purify the remaining unidenti®ed compounds for detailed spectroscopic measurements. The structures were elucidated by extensive MS and NMR studies, including 1 H±1H COSY, HSQC and 2D-NOESY experiments. Low sample amounts prohibited the determination of the absolute con®guration of the discussed compounds and the chemical formulae are given in analogy to known compounds. 1 H NMR and COSY spectra of compounds 7 and 8 (Table 1) were mostly similar to those of the niveusin A

* Corresponding author. Tel.: +49-711-459-3811; fax: +49-711579-3355. E-mail address: spring@uni- hohenheim.de (O. Spring).

0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00019-X

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Scheme 1. Possible reaction of 1,2-unsaturated tifruticin type sesquiterpene lactones (e.g. 12) with nucleophiles leading to 1-O-substitued derivatives (e.g. 9) and to 3,10-epoxy-isomers (e.g. 3 and 7) after spontaneous reaction from the keto alcohol to the hemiketal form.

Fig. 1. Compounds isolated from Viguiera quinqueremis. The absolute con®guration of the discussed compounds was not determined and the chemical formulae are given in analogy to known compounds.

derivatives 3 and 4, respectively. However, the signal of the hydroxyl function at C-1 (e.g.  2.97 d, J=7.6 Hz in 3) was missing in the spectra of 7 and 8. Instead, a signal at  3.42 (s, 3H), suggested the presence of a methoxy group at this position in both compounds. This was con®rmed by APCI MS data of 7 and 8 which showed m/z=411 [M+H]+, indicative of C21H30O8, and 409 [M+H]+, indicative of C21H28O8, respectively. The substitution of a hydroxyl by a methoxy group also explained the more lipophilic migration behavior of the two compounds in HPLC separations when compared with the hydroxylated derivatives 3 and 4. This raised questions about their occurrence as natural constituents or as artifacts arising from addition of MeOH to the tifruticin precursors 12 and 13 when MeOH was used in the puri®cation process (Scheme 1). Attempts were made to clarify this by extracting glandular trichomes and performing the subsequent HPLC separation using solvents with and without MeOH. CH3CN extracts separated in 30% CH3CN and in 50% MeOH, respectively, led to HPLC diagrams that clearly showed minor peaks with retention times and UV spectra typical of 7 and 8. The same peaks in similar amounts appeared in samples extracted with MeOH. These results support the natural occurrence of the methoxy derivatives. On the other hand, current

experiments (data not shown) with the tifruticins 12 and 13 puri®ed from Helianthus annuus indicate that in these instances spontaneous methoxylation at C-1 occurs upon storage in MeOH over several weeks. It therefore cannot be ruled out that both ways of origin contributed to the existence of 7 and 8. The 1H NMR and COSY spectra of compound 9 (Table 1) showed the characteristic signals of a 1,15-dihydroxy-4, 11(13)-unsaturated heliangolide with a methylbutyrate side chain. Since the protons at C-2 showed coupling with only one neighboring hydrogen (H-1), C-3 was assumed to be quaternary. A 3,10-epoxy-substitution was excluded due to the chemical shift of H-7 ( 3.41 instead of ca.  4.1 as in niveusin-type compounds). This led to the assumption of a keto form at C-3, similar as in compound 12 and 13. MS data (APCI) showed m/z= 397 [M+H]+ indicative of C20H28O8, thus con®rming the proposed structure of 9. The relative stereochemistry of the substituents was deduced from 2D-NOESY experiments which showed e€ects between H-7/H-8, H7/H-2b, H-6/H-9a and H15a,b/H-1. This indicated the b-orientation of the ester at C-8 and the a-position of the hydroxyl at C-1. Compound 9 therefore has to be regarded as the keto derivative of niveusin A methylbutyrate (3). The puri®cation of this isomer in a stable form was unexpected to us, since similar changes in the constitution of niveusin B (6) and of niveusin C derivatives, previously observed during HPLC separation, led to NMR spectra (measured in CDCl3) of only the hemiketal form which appears to be stabilized in aprotic solvents (Spring et al., 1991). The 1H NMR spectrum of compound 10 (Table 1) was identical with that of 1,2-anhydridoniveusin A (11) (Spring et al., 1989), except for the side chain signals which indicated the presence of a methylbutyrate instead of an angelate. The b-orientation of the side chain at C-8 was deduced from the NOE between H-7 and H-8. MS data (APCI) showed m/z=379 [M+H]+ indicative of C20H26O7, thus con®rming the suggested structure. The

O. Spring et al. / Phytochemistry 57 (2001) 267±272

269

Table 1 1 H NMR spectral data of compounds 7±10, 14 and 15 from Viguiera quinqueremis (300 MHz, CDCl3,  CDCl3 ˆ 7.27 ppm)a H

7

8

9

10

14

15

1 2a 2b 3a 3b 5 6 7 8a 8b 9a 9b 13a 13b 14 15a 15b 20 30 a 30 b 40 50 OMe

4.07 dd (6.7, 10.4) 2.62 dd (6.7, 14) 2.08 dd (10.4, 14)

4.05 m 2.62 dd 2.07 dd

4.52 m 2.02 m 1.78 m

5.79 d (5.6) 6.35 d (5.6)

5.03 m 4.81 dt (6, 9.7)

5.03 m 4.78 dt (5.8, 10.2)

5.89 5.46 4.13 5.60

t (4.8) bd (4.8) bs m

5.93 5.49 4.16 5.62

5.72 6.04 3.40 5.43

d (9.4) dd (3.2, 9.4) bs ddd (3.7, 5.2, 10)

5.88 5.95 3.51 5.12

bd (6.7) ddd (1.0,3.2,6.7) dddd (1.0,2.1,2.3,3.2) ddd (1.0,3.2, 4)

2.18 1.75 6.27 5.63 1.55 4.22 4.12 2.26 1.60 1.44 0.86 1.04 3.42

dd (10.2, 15) dd (5.2, 15) d (2.1) d (1.8) s bd (11.7) bd (11.7) m m m dd (7) d (7) s

b

2.52 1.91 6.35 5.80 1.34 4.12 4.12 2.31 1.63 1.45 0.90 1.09

dd (10, 15.1) dd (5.2, 15.1) d (2.1) d (1.8) s m m m m m dd (7) d (7)

2.41 2.30 6.32 5.72 1.41 4.43 4.17 2.32 1.61 1.45 0.86 1.10

dd (4, 15) dd (3.2, 15) d (2.3) d (2.1) s bd (12.2) bd (12.2) m m m dd (7) d (7)y

2.79 2.35 5.00 5.11 2.98 2.83 2.40 5.82

dd (6, 11) dd (9.7, 11) bd (9.5) t (9.5) m ddd (2, 5.2, 14) m (1.5, 14) m

2.76 2.14 5.08 5.11 2.97 2.90 2.38 5.82

dd (5.8, 11) dd (10.2, 11) bd (9.5) t (9.5) m ddd (2, 5.2, 14.1) m (1.6, 14.1) m

6.38 5.64 1.56 1.81

d (3.5) d (3) bs bs

6.34 5.63 1.55 1.83

d (3.4) d (3) bs bs

2.38 1.65 1.50 0.92 1.15

m m m dd (7) d (7)

bt (3.9) bt (3.9) bs m

b

6.31 5.67 1.58 4.25 4.15

d (2.1) d (1.8) s bd bd

6.12 qq (1.5, 7.3) 1.98 dq (1.5, 7.3) 1.79 dq (1.5) 3.45 s

6.14 qq (1.5, 7.3) 1.99 dq (1.5, 7.3) 1.84 dq (1.5)

a Calculated coupling constants (J [Hz]) for stereochemical considerations: comp. 7, 8: 1a-OMe!1,2a=7; 1,2b=10; 1b-OMe!1,2a=2; 1,2b=4; comp. 10: 5,6=4.5; 6,7=3; 7,8=2.3; 8,9a=4; 8,9b=5; comp. 14, 15: 2b-OH!2,3a=2.7; 2,3b=3.8; 2a-OH!2,3a=4.6; 2,3b=11.5. b Signal obscured.

signal of H-7 ( 3.51 m) was up®eld by ca. 0.5 ppm compared with the niveusin-type compounds 3±8. According to the lowest conformation found by searching the conformational space with the GMMX option of PCMODEL, this is most likely due to inverse shift e€ects of the 3, 10-epoxy group and the 1,2-double bond with respect to H-7. These force ®eld calculations (see Experimental) also support the û-orientation of the side chain when comparing theoretical coupling constants with those experimentally determined (Table 1). Coupling constants for H-7/13 (J=3 and 3.5 Hz) of 14 (Table 1) indicated the presence of a 1(10), 4-E,E-germacradienolide. COSY experiments revealed the sequence of H-7 to H-9 and H-7 to H-5. The methylbutyrate side chain had to be attached to H-9 due to its chemical shift. H-1 ( 5.03) was coupled to the H-14 methyl ( 1.56 bs) and to a complex signal at  4.81 (ddd) which was consistent with the presence of a hydroxyl group at H-2. The suggested structure was con®rmed by MS data (APCI) which showed m/z=349 [M+H]+ indicative of C20H28O5. The relative stereochemistry at C-9 and C-2 was deduced from an NOE between H-7 and H-9 which indicated ûorientation of the ester at C-9. On the other hand, NOEs between H-2 and both methyl groups (H-14 and H-15), and the absence of NOEs between H-2, H-7 and H-9 indicated that the C-2 hydroxyl was a-oriented. This was substantiated by force ®eld calculations in the following manner: For either orientation (a or b hydroxyl)

four principal conformational options exist with respect to the orientation of the methyl groups at C-4 and C-10 (Scheme 2). However, only the two conformations are likely to explain the observed NOEs of H-2, where both methyl groups are oriented opposite to the hydroxyl (Scheme 2: conformation A for a-hydroxyl and conformation D for b-hydroxyl). Considering the energetic

Scheme 2. Possible conformations of 14 with respect to the orientation of C-2 hydroxyl and the methyl groups at C-4 and C-10. 2a±OH: R1=OH, R2=H, R=methylbutyrate; 2b-OH: R1=H, R2=OH, R=methylbutyrate.

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status (see Experimental) of the 8b-O-methylbutyrate derivative, the assumption of a 2b-hydroxyl leads to an equilibrium between all four previously described conformations (A±D). This would cause more NOEs than observed. On the other hand, for the 2a-hydroxyl, only one (A) out of these conformations has to be considered which fully explains the measured NOEs. Finally, the theoretically derived coupling constants of the 2a-hydroxyl form are in best agreement with those experimentally determined (see Table 1). The 1H NMR spectrum of 15 (Table 1) was identical with that of 14 in all parts, except for the side chain which showed the typical signals of an angelate. The proposed structure was con®rmed by MS data (APCI) which showed m/z=347 [M+H]+ (consistent for C20H26O5). HSQC spectra of compound 9, 10 and 14 allowed the assignment of the proton bearing carbon signals (Table 2). Compound 16 was isolated from the less polar fractions. The 1H NMR data (Table 3) showed the presence of three side chains, one angelate and two methylbutyrates, but no signals typical of a sesquiterpene lactone skeleton. Instead, six protons with signals between  3.65 and 5.61 indicated the pattern of an inositol. The positions of the side chains were deduced from HMBC in combination with COSY spectra. The carbonyl carbon of the angelate residue showed coupling to a triplet at  5.18 (H6) which was further coupled to the signals at  3.65 t (H5) and  3.91 dd (H-1). The sequence of the remaining protons followed from COSY experiments and HMBC measurements indicated the connection of the two methylbutyrate residues through coupling with the protons at  Table 2 Carbon data (HSQC NMR) of compounds 9, 10 and 14 from Viguiera quinqueremis [500 MHz, CDCl3,  CDCl3 ˆ 7:27 ppm (1H)/77.0 ppm (13C)] C

9

10

14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50

72.2 35.4 n.o.a n.o 128.1 73.7 46.0 73.2 38.8 n.o. n.o. n.o. 123.1 31.8 67.3 n.o. 41.3 26.3 11.5 16.4

126.8 139.8 n.o. n.o. 134.7 73.5 47.7 76.4 43.8 n.o n.o n.o 124.6 31.2 66.5 n.o. 41.2 26.2 11.5 16.4

133.6 69.2 48.4 n.o. 129.1 74.9 52.8 44.0 70.6 n.o. n.o. n.o. 121.0 20.1 18.4 n.o. 41.2 26.1 11.4 16.8

a

n.o., signals not observed due to HSQC; sample amounts were insucient for HMBC.

5.61 t (H-2) and  4.88 dd (H-3), respectively. The relative stereochemistry of the substituents was deduced from coupling constants and supported by force ®eld calculations (Table 3) leading to the suggested structure which was ®nally con®rmed by MS data (APCI) which showed m/z=431 [M+H]+ indicative of C21H34O9. 2.1. Chemotaxonomic aspects Comparison of the sesquiterpene lactone pattern of V. quinqueremis with that of other members of its section revealed similarity in the overall dominance of heliangolides. However, the 1-keto-2,4-unsaturated budlein derivatives which were the exclusive constituents of V. oblongifolia (Bohlmann et al., 1984) and V. robusta (Da Costa et al., 1996) are only present to a minor extent in V. quinqueremis. The lack of sesquiterpene lactones reported from V. nervosa (Tamayo-Castillo et al., 1990) is most likely due to the low amount of glandular trichomes present in plant samples which were dominated by vegetative plant parts. Reexamination of the ¯owering parts of this species appears to be necessary. The isolation of a myoinisitol from glandular trichomes of V. quinqueremis is so far a unique feature of this taxon within the whole

Table 3 1 H NMR (500 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) data of compound 16 from Viguiera quinqueremisa Position 1 2 3 4 5 6

C (carbon signal) 69.5 70.6 70.7 71.7 73.0 74.8

OAng 10 20 30 40 50

169.2 127.1 140.3 20.5 16.0

OMebut 100 200 300

175.7 41.2 26.4

400 500

11.4 16.4

OMebut 1000 2000 3000 4000 5000 a

176.0 41.0 26.7 11.5 16.9

H (proton signal) 3.91 5.61 4.88 3.98 3.65 5.18

dd (3, 10) t (3) dd (3, 10) t (10) t (10) t (10)

6.18 qq (1.5, 7) 2.02 dq (1.5, 7) 1.93 bs

2.49 1.73 1.54 0.97 1.22

tq (7, 7, 7) ddq (7, 7, 14) ddq t (7) d (7)

2.37 1.67 1.45 0.90 1.14

tq ddq ddq t d

Calculated coupling constants (J [Hz]) for stereochemical considerations: 16: 1,2=3.4; 2,3=3.1; 3,4=9.2; 4,5=9.6; 5,6=8.9; 1,6=9.6.

O. Spring et al. / Phytochemistry 57 (2001) 267±272

genus. Within the Heliantheae, inositols have only been reported from Hymenoxys texana and H. biennis (Gao et al., 1990a,b; Spring et al.; 1994). 3. Experimental 3.1. Extraction of plant material V. quinqueremis Blake was collected near Diamantina, along km 473±475 on BR-259 highway (S 18 250 , W 43 430 , altitude 1370 m), State of Minas Gerais, Brazil, in April 1998 by F.B. Da Costa and determined by J.N. Nakajima and E.E. Schilling (voucher specimens are deposited at the herbarium SPFR, RibeiraÄo Preto, SP under the collection number FBC No. 64). For HPLC (Hypersil ODS, 5 mmm; 4250 mm; 30% MeCN, 1.3 ml/min or 50% MeOH, 1 ml/min; UV detection simultaneously at 225 and 265 nm or with diode array detection; dimethylphenol as int. standard). For screening of compounds and for solvent experiments with respect to the natural occurrence of compound 7 and 8, glandular trichomes were collected mechanically from the anther appendages as previously described (Spring, 1991). For isolation of compounds, air-dried ¯ower heads (84 g) were extracted with CH2Cl2. The solvent was evaporated, the residue was redissolved in MeOH, diluted with water (1:1 v/v) and centrifuged in order to remove insoluble parts. The clear supernatant was applied to HPLC (conditions as given above) and compounds were separated in a gradient of aqueous MeOH (40±60% in 20 min, 1 ml/min). When necessary, 30% MeCN (1.3/per min) was used for repuri®cation of fractions on the same type of column. Prior to preparative separation of speci®c compounds, an aliquot of the crude extract (equivalent to ca. 2 g plant material) was used for LC 1H NMR and LC MS analysis which were carried out under identical HPLC conditions (MeOH gradient) in order to allow direct correlation of the obtained data. LC 1H NMR spectra were measured on a Varian Unity Inova; 500 MHz, detector type 9050 equipped with a NMR ¯ow cell of 60 ml detectable volume. Routine NMR experiments were carried out on 300 MHz Varian Unity Inova. LC MS experiments were performed on a Finnigan TSQ 700 under atmosphere pressure chemical ionization (APCI), positive mode. Calculations were done with PCMODEL, Ver. 7, Serena Software, Bloomington using the MMX force ®eld. Conformational search was done with the GMMX option. Conformations are considered within a 3 Kcal limit from the lowest energy. Coupling constants are derived from the Altona equations which are implemented in the program. They are averaged over the Boltzmann distribution of their corresponding conformer. HPLC retention times of compounds from V. quinqueremis in 50% MeOH (RRT1) and in 30% MeCN (RRT2) relative to dimethylphenol (retention time ca. 13

271

min in both solvents): 1, 0.56/0.59; 2, 0.53/0.56; 3, 0.62/ 0.46; 4, 0.57/0.42; 5, 1.17/0.82; 6, 1.12/0.79; 7, 1.37/0.95; 8, 1.25/0.90; 9, 0.46/0.31; 10, 0.60/0.54; 11, 0.59/0.53; 12, 0.72/0.60; 13, 0.68/0.51; 14, 1.96/1.63; 15, 1.86/1.60; 16, 1.60/1.48 (RRT1/RRT2). 3.1.1. 1-Methoxy-3,15-dihydroxy-3,10-epoxy-8 -Omethylbutanoyl-4,11(13)-germacradien, 6,12-olide (7) C21H30O8, APCI +; grad. 40±60% MeOH in 20 min: 411 [M+H]+, 379 [411 MeOH]+, 361 [379 H2O]+, 259 [361 methylbutyrate]+, 85 [C5H9O]+. 3.1.2. 1-Methoxy-3,15-dihydroxy-3,10-epoxy-8 -Oangeloyl-4,11(13)-germacradien, 6,12-olide (8) C21H28O8, APCI+; grad. 40±60% MeOH in 20 min: 409 [M+H]+, 377 [411 MeOH]+, 359 [377 H2O]+, 259 [359 angelate]+, 83 [C5H7O]+. 3.1.3. 1, 10 ,15-Trihydroxy-3-oxo-8 -O-methylbutanoyl-4,11(13)-germacradien, 6,12-olide (9) C20H28O8, APCI+; grad. 40±60% MeOH in 20 min: 397 [M+H]+, 379 [M+H H2O]+, 277 [379 methylbutyrate]+. 3.1.4. 3,15-Dihydroxy-3,10-epoxy-8 -O-methylbutanoyl-1,4,11(13)-germacratrien, 6,12-olide (10) C20H26O7, APCI+; grad. 40±60% MeOH in 20 min: 379 [M+H]+, 361 [M+H H2O]+, 259 [361 methylbutyrate]+, 85 [C5H9O]+. 3.1.5. 2-Hydroxy-9 -O-methylbutanoyl-1(10),4,11 (13)-germacratrien, 6,12-olide (14) C20H28O5, APCI+; grad. 40±60% MeOH in 20 min: 349 [M+H]+, 247 [M+H methylbutyrate]+, 229 [247 H2O]+, 85 [C5H9O]+. Force ®eld calculations: 2a±OH: lowest energy calculated: 41.6 Kcal/mol; 23 conformations including side chain conformations within 3 Kcal/ mol. 2b-OH: 44.53 Kcal/mol, 76 conformations including side chain conformations within 3 Kcal/mol. 3.1.6. 2-Hydroxy-9 -O-angeloyl-1(10),4,11(13)germacratrien, 6,12-olide (15) C20H26O5, APCI+; grad. 40±60% MeOH in 20 min: 347 [M+H]+, 247 [M+H angelate]+, 229 [247 H2O]+, 83 [C5H7O]+. 3.1.7. 6-Angeloyl-2,3-dimethylbutanoyl-myoinositol (16) C21H34O9, APCI +; grad. 40±60% MeOH in 20 min: 431 [M+H]+, 413 [431 H2O]+, 331 [431 angelate]+, 85 [C5H9O]+, 83 [C5H7O]+. Acknowledgements We wish to thank Dr. J.N. Nakajima, Universidade Federal de UberlaÃndia, Brazil, and Dr. E.E. Schilling,

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