Guaianolide Sesquiterpene Lactones from Centaurothamnus maximus

Centaurothamnus maximus (family Asteraceae), is a leafy shrub indigenous to the southwestern Arabian Peninsula. With a paucity of phytochemical data on this species, we set out to chemically characterize the plant. From the aerial parts, two newly identified guaianolides were isolated: 3β-hydroxy-4α(acetoxy)-4β(hydroxymethyl)-8α-(4-hydroxy methacrylate)-1αH,5αH, 6αH-gual-10(14),11(13)-dien-6,12-olide (1) and 15-descarboxy picrolide A (2). Seven previously reported compounds were also isolated: 3β, 4α, 8α-trihydroxy-4-(hydroxymethyl)-lαH, 5αH, 6βH, 7αH-guai-10(14),11(13)-dien-6,12-olide (3), chlorohyssopifolin B (4), cynaropikrin (5), hydroxyjanerin (6), chlorojanerin (7), isorhamnetin (8), and quercetagetin-3,6-dimethyl ether-4’-O-β-d-pyranoglucoside (9). Chemical structures were elucidated using spectroscopic techniques, including High Resolution Fast Atom Bombardment Mass Spectrometry (HR-FAB-MS), 1D NMR; 1H, 13C NMR, Distortionless Enhancement by Polarization Transfer (DEPT), and 2D NMR (1H-1H COSY, HMQC, HMBC) analyses. In addition, a biosynthetic pathway for compounds 1–9 is proposed. The chemotaxonomic significance of the reported sesquiterpenoids and flavonoids considering reports from other Centaurea species is examined.


Introduction
Centaurothamnus maximus Wagentz and Dittri (Asteraceae) is a branched shrub that grows to a height of ca. 1.5 m [1]. Stems are densely white-tomentose with alternating leaves that are lanceolate to elliptic (2-6 cm wide by 8-15 cm long), silvery below and green above. Thistle-like magenta flowers 3-4 cm long at the end of the branches have a faint sweet scent [2]. Centaurothamnus is a monotypic genus from Centaurea and endemic The appearance of a sharp singlet signal at δH 2.11 (3H, s, H-1) together with new ester carbonyl at δC 171.9 in 1, along with an absence of these signals in 5, indicates the presence of an acetoxy group instead of hydroxyl group at C-4. While it is possible that such an acetate functionality is a chemical artifact (e.g., drying and/or extraction), the fact that such derivatizations have been phytochemically studied in the same manner from the
The appearance of a sharp singlet signal at δ H 2.11 (3H, s, H-1) together with new ester carbonyl at δ C 171.9 in 1, along with an absence of these signals in 5, indicates the presence of an acetoxy group instead of hydroxyl group at C-4. While it is possible that such an acetate functionality is a chemical artifact (e.g., drying and/or extraction), the fact that such derivatizations have been phytochemically studied in the same manner from the same genus and other genera suggests that these natural products are in fact plant-derived metabolites.
The relative configurations of both 1 and 2 were the same when compared with the corresponding guaianolides isolated from Asteraceae [23,24]. NOESY correlations ( Figure 3) between H-1, H-3, H-5, and H-9 are in an α orientation, and NOESY correlations between H-6, H-8, and H-15 are in a β orientation. Accordingly, the structure of 2 was established as a new derivative of picrolide A and named 15-descarboxy picrolide A.

Proposed Biosynthetic Pathway of the Isolated Compounds
Generally, the terpenoids biosynthesis in plants can arise in dissimilar subcellular compartments, the cytosol, mitochondria, and/or plastids [26,27]. Biosynthetically, farnesyl diphosphate (FPP) is considered the main precursor for biosynthesis of a vast array of sesquiterpene. Cyclization of FPP into (+) germacrene A is catalyzed by (+) germacrene A synthase (GAS) [28]. The latter is converted to the corresponding acid, germacrene A acid, through hydroxylation and oxidation reactions catalyzed by cytochrome P 450 germacrene A oxidase (GAO). Germacrene A acid is then hydroxylated at C-6 to produce 6-hydroxygermacrene A acid as an unstable intermediate by the action of another cytochrome P 450 (+) costunolide synthase (COS). Costunolide is obtained from this intermediate that undergoes spontaneous non-enzymatic lactonization of the hydroxyl group at C-6 with the carboxylic group at C-12 ( Figure 4) [29][30][31]. Costunolide is considered a branching point precursor for producing germacranolides, eudesmanolides, and guaianolides as the three major sesquiterpene lactones groups. Thus, 4,5 epoxidation of costunoilde is hypothesized to be the first committed step in guaianolide biosynthesis through the conversion of costunolide to parthenolidecatalyzed by parthenolide synthase (TpPTS) [31]. The opening of the epoxide through an intramolecular attack of the double bond generates the three-cyclic skeleton as a guaianolide-type intermediate that is responsible for generating a large variety of guaianolides ( Figure 4) [29,32,33]. The guaianolides in Asteraceae have a specific biosynthetic pathway with unique conformations that differ from guaianolides in the family Apiaceae. The lactone ring in Apiaceae is either 6β, 8α or 6β, 8β, whereas in Asteraceae, it has only been seen as 6α, 8β [23,24]. The hydroxylase enzymes activate an enzymatic hydroxylation of the guaianolide-type intermediate at C-3, C-8, and C-15, thereby producing compound 3. Compound 4 was biosynthetically proposed by incorporation of chloride atoms at C-15 of compound 3, which catalyzed by Flavin adenine dinucleotide (FADH 2 )-dependent halogenases as the type of halogenating enzymes of compounds activated for electrophilic attack (Figure 4). On the other hand, the C-8 position is easily hydroxylated by the enzyme CYP71BL1 and acts as an active site to accept acyloxy moiety via the P 450 acetyltransferase enzyme [34]. The generation of the side chain at C-8 is proposed via esterification of the hydroxyl group at C-8 with acrylic moiety, followed by methylation and hydroxylation of the side chain to generate compound 6. The latter is considered the main precursor of compounds 1, 2, 5, and 4 through specific biochemical pathways (Figure 4). Compound 7 is similar to 4 in chlorination of 6 at C-15, which is activated by FADH2-dependent halogenases. The acetylation of 6 at C-4 is believed to be mediated by an acetyl transferase, which catalyzes the transfer of an acetyl group from acetyl-CoA as a donor molecule to produce an acetylated analogue 1. Compound 2, however, may be obtained via condensation reaction of compound 6 at C-15 with a simple aromatic moiety, such as hydroquinone, which may be produced from shikimic acid as precursor. The dehydrogenase enzyme may be converting the primary alcoholic group at C-15 in compound 6 into a formyl group that also converted into a methyl one by reductase enzyme, and a double bond between C-4 and C-15 is formed by losing one molecule of water to produce compound 5 (Figure 4).
Flavonoids are products of a shikimic acid and the acetate pathway by condensation of 4-hydroxy cinnamoyl-coenzyme A, referred to as 4-coumaroyl coenzyme A, with unit 9 of malonyl coenzyme A. The plant utilizes a shikimic acid pathway for deriving a polyketide intermediate that forms a chalcone skeleton. The chalcone skeleton serves as a key intermediate in the biosynthesis of several classes of flavonoids [35][36][37]. The proposed biosynthetic pathway of compounds 8 and 9 is shown in Figure 5.
Based on the above studies, it appears that guaianolide sesquiterpene lactones isolated from C. maximus are similar to those reported from C. aegyptiaca, and C. linifolia, which have overlapping biosynthetic pathways and are characterized by their potential to produce chlorinated guaianolides as well as methoxylated flavonoid derivatives.

Plant Material
The wild aerial parts of C. maximus were collected in March 2015 from Al Udayn, Ibb, Yemen. The plant was kindly identified by Prof. Dr. Abdulnaser Al Gifri of the Biology Department at Education College, Aden University, Yemen. A voucher specimen (P 610) was deposited in the Pharmacy Department at the University of Sciences and Technology, Ibb, Yemen.

Conclusions
Two new guaianolide sesquiterpene lactones (1-2) were characterized from C. maximus, as well as five known guaianolide sesquiterpene (3-7), including two chlorinated guaianolides (4 and 7), together with two known flavonoids (8)(9). Compounds 3, 6 and 9 were firstly isolated from genus Centaurothamnus, while, 4 and 8 were isolated for the first time from C. maximus. Biosynthetically, costunolide is considered the branching point precursor for producing germacranolides, eudesmanolides, and guaianolides, however, 4,5 epoxidation of costunoilde is hypothesized to be the first committed step in the Asteraceae family for guaianolide biosynthesis through the conversion of costunolide to parthenolide. By opening of the epoxide through an intramolecular attack of the double bond, the threecyclic skeleton is generated as a guaianolide type intermediate and can go on to generate a variety of enzymatically mediated guaianolides. Guaianolide biosynthesized in the family Asteraceae have a specific biosynthetic pathway with the lactone ring having a 6α, 8β conformation. Based on chemosystematic analysis, guaianolide sesquiterpenes from C. maximus exhibit chemical overlap with Centaurea aegyptiaca, and C. linifolia, confirming their placement in one section. Additional data on guaianolide sesquiterpenes and flavonoids from other Centaurea species will be required to further elucidate intergeneric relationships.