The First Guaian-12-oic Acid Glucopyranosyl Ester and Other Constituents from Picris rhagadioloides (L.) Desf. (Asteraceae)

The first guaian-12-oic acid glucopyranosyl ester was isolated from the roots of Picris rhagadioloides (L.) Desf. (Asteraceae), in addition to five costuslactone-type guaianolides, four germacranolides and three phenolic compounds. This is the first time that the known phenolics syringaldehyde and syringaresinol, as well as the known sesquiterpene lactones glucozaluzanin C, cichorioside C and hypochoeroside A have been described from a Picris species. The compounds were characterized on the basis of physicochemical, 1D- and 2D-NMR spectroscopic, and mass spectrometric data.


Introduction
The genus Picris (Asteraceae, tribe Lactuceae) comprises about 40 species, which are distributed throughout Eurasia and extend to tropical Africa, Australia and New Zeland. Phytochemical investigations of several Picris taxa have revealed mainly sesquiterpene lactones, including a remarkable number of guaianolides, three germacranolides and two eudesmanolides, along with some other secondary metabolites [1][2][3]. Guaianolides represented by costuslactone-and lactucin-type compounds are the most common constituents of this taxon. Following our recent chemotaxonomic work [2,3] on Picris species, we report here the isolation and characterization of sesquiterpenoids and phenolics from roots of the hitherto not studied Picris rhagadioloides (L.) Desf. (syn. Picris sprengeriana (L.) Poir.), an annual herb of up to 50 cm height with yellow ligules, distributed thoughout Europe, Asia Minor and North Africa.

Results and Discussion
The dried roots of the plant were extracted with ethanol and the extract was chromatographed on a silica gel column to give fractions which were submitted to preparative TLC and/or semipreparative RP-18 HPLC yielding the known phenolics eugenyl-4-O-β-glucopyranoside, syringaldehyde and syringaresinol, as well as the new guaian-12-oic acid glucopyranosyl ester (1) and the known sesquiterpene lactones 9α-hydroxy-3-deoxyzaluzanin C (2) in a mixture with its 11β,13dihydroderivative (3), glucozaluzanin C (4), its 11β,13-dihydroderivative (5) in a mixture with picriside B (10), ixerin F (6), sonchuside A (7), cichorioside C (8), and hypochoeroside A (9) ( Figure  1). Except for 9, the known compounds were characterized by direct comparison of their spectra and retention time values with those of compounds previously isolated in our earlier studies [2][3][4][5]. The identity of compound 9, first isolated from Hypochoeris radicata L., was established by comparison of its spectral data and the optical rotation value with those reported [6]. Here, the first detailed 1 H-NMR evidence for the structure of this compound is provided (Table 1). In the original publication [6] only four 1 H-NMR signals in pyridine-d 5 were given:  [7]. They are also added to Table 1. The signal assignments were confirmed by COSY and NOESY correlations.
Compound 1 was isolated as an amorphous solid. Its molecular formula was deduced to be C 21 H 30 O 8 from the positive ESI MS which showed ion peaks at m/z 433 [M + Na] + and 843 [2M + Na] + and by high-resolution mass spectral measurements. 1 H-and 13 C-NMR spectra of 1 (Table 2) exhibited signals for a glucopyranosyl unit and a sesquiterpene moiety. The signals could be assigned unambigously from a combination of COSY, HETCOR and HMBC spectra, and previously reported data of related metabolites. The chemical shifts of C-1 and C-2 signals of the sugar unit at δ 96.52 and 74.30, respectively, and the anomeric proton doublet (J = 7.7 Hz) at δ 6.49 indicated the presence of an ester β-glucosidic linkage.This conclusion was supported by the HMBC correlation between the glucose anomeric proton (H-1) signal and the aglycone carbonyl (C-12) signal. After subtraction of the six carbon resonances of the glucopyranosyl unit, the remaining 15 signals, including six signals assigned to olefinic carbons and one to an ester carbonyl, were attributable to a sesquiterpene framework (C 15 H 20 O 3 ), indicating the presence of six degrees of unsaturation, i.e. three double bonds, one carbonyl functionality and two ring systems. The 1 H-NMR spectrum displayed signals assignable to three exocyclic methylene protons, an oxygenated methine proton, three further methine protons and four pairs of endocyclic methylene protons suggesting the presence of a hydroxylated 4(15), 10(14), 11(13)-guaiatrien-12-oic acid residue. The COSY spectrum established the spin system H-5→ H-1→ H 2 -2→ H-3 and allylic couplings of H-3 and H-5 with H-15a/H-15b, thus allowing the assignment of the hydroxyl group at C-3. The resonances of C-1 to C-5 and C-10 are similar to those of dehydrocostuslactone (1α, 5α,7α(H)-guaia-4(15), 10(14), 11(13)-trien-6,12-olide) derivatives having a hydroxyl group at C-3 [8,9], while the resonances of C-11 to C-13 are in good agreement with those published for the acrylic acid ester moieties at C-7 of eudesmane-type sesquiterpenoids [10,11].
The relative stereochemistry of 1 was resolved by a combination of NOESY data and coupling constant values, based on the assumption that H-7 is α, as established for closely related guaianolides of known absolute stereostructure. In the NOESY spectrum H-5 correlated with H-7 and H-1, suggesting that these protons are on the α face of the molecule. No effect was observed between H-1 and H-3. The spectrum further confirmed proximities of H-6β to H-8β; H-9β to H-14a (δ 4.86), and H-2β to H-3β/H-14b (δ 4.83). The configurations of H-5α and H-7α were also confirmed on the basis of the values of J 5α,6β (11.0 Hz), J 6β,7α (11.0 Hz) and J 7α,8β (11.0 Hz) were indicative of anti-diaxial relationships of the respective protons. Concerning the configuration of the hydroxyl group at C-3 in epimeric 3α-hydroxy-and 3β-hydroxydehydrocostuslactone derivatives, recently isolated from leaves of artichoke [12], a notable difference in the chemical shifts of H-1 was observed in pyridine-d 5 (δ 3.43 vis 2.74, respectively). The H-5 signal was less deshielded. We also noted that the presence of the hydroxyl group at C-3 induced a downfield shift of the resonance of H-1 (δ 3.43), thus supporting the 3α-OH stereochemistry. On the basis of these findings the structure of 1 was established as 3αhydroxy-1α,5α,7α(H)-guaia-4(15),10(14),11(13)-trien-12-oic acid β-glucopyranosyl ester, a new natural product.  To our knowledge, this represents the first time a guaian-12-oic acid esterified with glucose has been encountered in plants. The co-occurrence of guaiane-type sesquiterpenoids, which have different configurations of hydroxyl groups at C-3, in the same taxon, is rather unusual, albeit precedented in naturally occurring guaianolides from Cynara scolymus L. [12] and Centaurea canariensis Brouss. (var. subexpinnata Burch.) [13]. Only a few further examples of costuslactone derivatives with 3αhydroxyl groups have been reported in the literature, mainly from Saussurea species [14 -17].

Conclusions
Our chemical study of the roots of Picris rhagadioloides has led to the isolation of the new guaiane derivative 1, nine known sesquiterpene lactones (2 -10) and three known phenolics. The production of compound 1 and its distribution within the hitherto not studied Picris species might be of taxonomic importance. Moreover, P. rhagadioloides seems to be characterized by the occurrence of the costuslactone-type of guaianolides (2 -6) and the germacranolides 7 -10, accummulated mainly as glycosides. This is the first time that the sesquiterpene lactones 4, 8 and 9, and the phenolics syringaldehyde and syringaresinol have been isolated from a taxon of the genus Picris. The germacranolides 8 and 9, known as constituents of some other representatives of the tribe Lactuceae, are rare natural products. Compound 8 has been reported from Cichorium intybus L. [7], Taraxacum bessarabicum (Hornem.) Hand.-Mazz. [18] and Hypochoeris radicata L. [6], while compound 9 has been previously found in the latter species.