Anticancer Meroterpenoids from Centrapalus pauciflorus leaves: Chromone- and 2,4-Chromadione-Monoterpene Derivatives

Eight previously undescribed chromones, named pauciflorins F–M and two 5-methyl-2,4-chromadione derivatives named as pauciflorins N and O, were isolated from the methanol extract of the leaves of Centrapalus pauciflorus (Willd.) H.Rob. together with the known (+)-spiro-ethuliacoumarin. The structures were determined via extensive spectroscopic analyses, including HRESIMS, 1D NMR (1H, 13C JMOD), and 2D NMR (HSQC, HMBC, 1H–1H COSY, and NOESY) experiments. Through an MTT assay, seven isolated compounds were tested for their antiproliferative properties against human adherent breast (MCF-7, MDA-MB-231), cervical (HeLa, SiHa), and ovarian (A2780) cancer cell lines. Pauciflorin F was effective against MCF-7 breast cancer cells, its activity (IC50 5.78 μM) was comparable to that of the reference agent cisplatin (IC50 5.78 μM).


■ INTRODUCTION
Meroterpenoids are a group of terpenoid-containing hybrid natural products with unique structural architectures and impressive pharmacological properties.Meroterpenoids can be classified based on their nonterpenoid moiety into polyketideterpenoids, shikimate-terpenoids, and alkaloid-terpenoids.−4 Notably, meroterpenoids derived from chromane/chromene can be condensed with hemi-, mono-, sesqui-, and diterpenoid units; their presence has been observed in various organisms, such as tunicates (Botryllus), 5 brown macroalgae (Sargassum, Cystoseira), 6 and Rhododendron (Ericaceae), 7 Sarcandra (Chloranthaceae), 8 and Mimosa (Fabaceae) 9 plant species.−12 These intriguing compounds have been identified in genera such as Nassauvia, 13 Triptilion, 14 and Polyachyrus from the Nassauvieae tribe, Gerbera 11 and Mutisia 15 from the Mutisieae tribe, and Bothriocline genus 16 from the Vernonieae tribe and reported to accumulate as both coumarin-and chromon-based meroterpenoids.The biosynthesis of these compounds involves the acetate−malonate pathway, where 5-methylcoumarins and 5-methylchromones serve as the main building blocks for chromone-, coumarin-based meroterpenoids, which is catalyzed by polyketide synthase enzymes. 17Chromane/ chromene meroterpenoids have displayed cytotoxic activity against different tumor cell lines, as well as antioxidant and antimicrobial activities.Furthermore, they have been reported to inhibit various enzymes, including protein tyrosine phosphatase 1B (PTP1B), butyrylcholinesterase (BChE), β-(National Research Institute for Chemical Technology, NARICT), Zaria, Nigeria.A voucher specimen was deposited in NARICT under the number Narict/Biores/321 and in the Herbarium of the Department of Pharmacognosy, University of Szeged, Szeged, Hungary, No. 897.
NP-VLC was conducted on fraction A using a gradient system of cyclohexane−EtOAc (98:2 to 80:20) as eluent.Two subfractions were obtained A/I and A/II.Subfraction A/I was purified further using RP-HPLC, affording nine fractions A/I/ 1−9.Further purification of fraction A/I/9 on NP-HPLC with n-hexane−EtOAc (95:5) as mobile phase furnished compound 7 (0.9 mg, R t 7.70 min).
Pauciflorin F (1). Colorless oil; [α] D 27 + 89.1 (c 0.1, CHCl 3 ); 1 H and 13 C NMR data, see Tables 1 and 2 13 C NMR data, see Tables 1 and 2 Determination of Antiproliferative Properties.The cell culturing and evaluation of the antiproliferative effects of the isolated compounds against a panel of human cancer cell lines derived from gynecological origin were conducted using a methodology described previously. 20
The presence of hydroxyl group (δ H 5.47 s) at position C-6 was deduced from the downfield shift of the aromatic carbon C-6 (δ C 151.0).The chemical shift assignments of the ring system of compound 1 agreed with the published data for nassauvia chromones. 13Additionally, the relative configuration    + 357.1697) in the positive-ion HRESIMS.The 1 H and 13 C NMR JMOD assignments of compound 3, obtained via the analysis of the 1 H− 1 H COSY, HSQC, and HMBC spectra, showed that chemical shifts of compound 3 were very similar to those of compound 2 with differences observed only in the resonances of the C-7′−C-9′ region of the molecule (Tables 1 and 2).The 9′-methylcarboxylate group of 2 was replaced by a methyl group [δ H 1.28 d (J = 7.0 Hz), δ C 17.3] in compound 3, as confirmed using the HMBC correlation observed between H 3 -9′ and C-6′ (δ C 38.7), C-7′ (δ C 36.0), and C-8′ (δ C 176.5).The key NOESY correlations were observed between H 3 -9 (δ H 2.83 s) and H-6 (δ H 7.06 d), as well as between H 3 -10′ (δ H 1.57 s) and H-5′ (δ H 4.41 m), revealing the stereostructure of compound 3, as shown in Figure 1.However, the configuration of C-7′ could not be determined based on NMR studies.
Pauciflorin I (4) was isolated as a colorless oily substance with an optical rotation of [  13 C NMR JMOD data (Tables 1 and 2) of 4 showed a similar structural pattern as that of compound 3, with only a difference in the functionality at C-9′ (δ C-9′ 63.7 for 4, and 17.3 for 3) and a less extent in the neighboring carbons.The 9′-methyl group (δ H 1.28 s) of + 343.1540).Analysis of the 1 H NMR and 13 C NMR JMOD spectra of 5 revealed the presence of the same dihydropyranochromone ring system, which was substituted with two methyl groups (C-9 and C-10′) and a vinyl group (C-1′−C-2′), as observed in compounds 1−4.However, compound 5 exhibited the presence of a C 9 monoterpene unit, while the C-9′ position was missing.This observation was supported by the sequence of correlated protons in the 1 7′).Furthermore, the HMBC correlation of H 2 -7′ (δ H 2.62 m) and methoxy group (δ H 3.72 s) with C-8′ (δ C 173.4) confirmed the presence of a carboxymethyl group at C-7′.The NOESY cross-peaks between H-5′ (δ H 4.40 m) and H-10′ (δ H s) showed the same stereochemistry of 5 as that of compounds 1−4.These findings were consistent with the proposed structure of pauciflorin J (5), as depicted in Figure 1.
Pauciflorin K ( 6) was isolated as a colorless oily substance with an optical rotation of [ 5.07 m), and the C-6′−C-9′ side chain at C-5′ (Tables 1 and  2).This side chain was identified as an isobutenyl group, as evidenced by the HMBC correlations of H-5′ (δ H  1 and 2).The side chain connected at C-5′ consists of two quaternary carbons (δ C 131. + 373.1646) in the positive-ion HRESIMS.The NMR characteristics of compound 8 revealed similarities to 1 regarding the aromatic part of the molecule and compound 3 regarding the monoterpene moiety.The 1 H and 13 C NMR chemical shift assignments, performed using the HSQC, HMBC, and 1 H− 1 H COSY spectra, confirmed the presence of a 5-methyl-6-hydroxychromone and a monoterpene carboxylic acid methyl ester adduct structure in compound 8 (Tables 1 and 2).
Compound 9 was isolated as colorless oil and identified based on its 1D and 2D NMR spectroscopic data as (+)-spiroethuliacoumarin (9).This compound was previously isolated from Ethulia conyzoides. 23The complete structure and the relative stereochemistry of compound 9 were established by Mahmoud et al. through X-ray crystallography.
Pauciflorin N (10) was obtained as a white amorphous powder with an optical rotation of + 341.1384).The 1 H and 13 C JMOD NMR spectra of compound 10 showed a structural pattern similar to that of compound 9 (Table 3).
In the 1 H− 1 H COSY spectrum, identical spin systems were identified for both compounds, concluding that compounds 9 and 10 are stereoisomers.Notable differences in chemical shifts were observed in their 1 H and 13 C NMR spectra in the carbon resonances around the spiro C-3 stereocenter (C-3, C-4, C-1′, C-4′, C-6′, and C-10′) and the corresponding proton resonances (H-1′cis, H-2′, H-4′β, H-6′, and H 3 -10′).The NOESY correlations of 10 between H 3 -10′ and H-5′, H-6′, H-4′α, between H-6′ and H-7′, and between H-2′ and H-4′β agreed those published for compound 9. 21 These correlations confirmed the α-position of H 3 -10′, H-5′, and H-6′, as well as β-position of vinyl and 9′-methyl groups.The only possible difference in compounds 9 and 10 is likely the opposite stereochemistry of C-3.This is supported by the considerable difference in the chemical shifts of the 10′-methyl group (δ H 9: 1.01 s vs 10: 1.22 s) and the key NOESY correlation between H 3 -9 and H 3 -10′.Such correlation is only possible when the αoriented H 3 -10′ methyl group is connected to the spirostructure opposite to that of compound 9.In the 3D model of 10, the distance between H 3 -9 and H 3 -10′ protons was 2.9 Å (Figure S73).Consequently, the structure of pauciflorin N was elucidated, as presented in the structural formula of compound 10.
Pauciflorin O (11) was isolated as a white amorphous solid material with an optical rotation value of [α] D 26 + 80.1 (c 0.1, CHCl 3 ).Its molecular formula was found to be C 20 H 20 O 4 from the protonated molecular ion observed at m/z 325.1431 [M + H] + (calcd for C 20 H 21 O 4 + 325.1434) detected in the MS spectrum.HRESIMS, 1 H and 13 C NMR JMOD data indicated that the molecule has 11 degrees of unsaturation.In the 13 C NMR spectrum, the presence of two keto groups (δ C 191.6 and 201.1) and one ester functionality (δ C 167.1) were detected beside an aromatic nucleus and two other double bonds�one monosubstituted (δ C 114.9, 139.9) and the other tetrasubstituted (δ C 130.9, 155.7).These structural elements contribute to nine unsaturations, which indicates the presence of two additional rings in the molecule: the 2,4-chromandione part and another ring in the terpene segment.(11).
The occurrence of chromone-monoterpene-based meroterpenoids in plants is sparely reported in the literature.Gerdelavin B was isolated from the Chinese Asteraceae species Gerbera delavayi, 11 and additional 5-methyl-chromone−monoterpene adducts were obtained from Gerbera piloselloides. 10 Ptaerobliquol, belonging to the same structural type, was found in Ptaeroxylon obliquum (Rutaceae). 24Nassauvia chromones, similar to pauciflorins F−M (1−8), were obtained from Triptilion spinosum, featuring a tricyclic, 5-methylchromonecontaining ring system with methyl and vinyl groups in position C-3′, but with a long aliphatic chain attached at C-5′. 14 5-Methyl-2,4-chromadione−monoterpenes are rare in nature.Previously, only the isolation of (+)-spiro-ethuliacoumarin (9) had been reported from the Egyptian plant Ethulia conyzoides. 23In addition, a compound encoded as ZINC31161132 with a 5-methyl-2,4-chromadione−monoterpene structure, was virtually screened for antituberculosis activity using a pharmacophor model. 25s regards, the biosynthesis of the compounds, a common biosynthetic origin can be supposed for the co-occurring 4hydroxy-5-methylcoumarin, 2-hydroxcy-5-methylchromone, and 5-methyl-2,4-chromadione derivatives (Figure 4).The aromatic parts are derived though the acetate-malonate pathway, 17 while the monoterpene parts form from geranyldiphosphate (GDP).Claisen cyclization of the polyketide precursor affords the aromatic rings by enzymatic route catalyzed by polyketide reductase (PKR), and O-heterocyclic rings are formed in the next steps (enolization, Michael-type nucleophilic attack, etc.).The connection of the aromatic parts with GDP includes C-alkylation, oxidative clavage, cyclization and lactonization. 26Figure 4 shows the putative biogenetic pathway proposed for the main structural types of the isolated compounds represented by 6, 9 and 11.
Seven isolated compounds were assayed for antiproliferative action against human adherent cancer cell lines of gynecological origin using the MTT method.Pauciflorin F (1) exhibited considerable activity against MCF-7 breast cancer cells, with an IC 50 value comparable to that of the clinically used drug cisplatin.However, its activity was less pronounced against ovarian (A2780) and triple-negative breast (MDA-MB-231), while no relevant effect was detected on cervical cancer cell lines (HeLa and SiHa).Despite sharing structural similarities, the remaining investigated compounds, namely pauciflorins G, H, J, M, and O (2, 3, 5, 8, and 11) and (+)-spiro-ethuliacoumarin (9), did not exhibit substantial activity against the tested cancer cell lines.

of compound 1 Figure 1 .
Figure 1.Structures of the Compounds Isolated from C. pauciflorus.

Table 4 .
Antiproliferative Properties of the Isolated Compounds 1−3, 5, 8, 9, and 11 a Inhibition values lower than 20% are considered negligible and not given numerically.b Not determined.c Results from ref 20.