Polyprenylated Acylphloroglucinols With Different Carbon Skeletons From the Fruits of Garcinia multiflora

Eleven new polycyclic polyprenylated acylphloroglucinols (PPAPs, 1–11) and three new monocyclic polyprenylated acylphloroglucinols (MPAPs, 12–14), together with ten known analogues were isolated from the fruits of Garcinia multiflora. These PPAPs belong to three types including the bicyclic polyprenylated acylphloroglucinols (BPAPs), the caged PPAPs, and the complicated PPAPs. Their structures and absolute configurations were determined through HRESIMS, NMR spectroscopy data, electronic circular dichroism (ECD) calculations, and gauge-independent atomic orbital (GIAO) NMR calculations with DP4+ analyses. Moreover, compounds 2 and 7 exhibited moderate cytotoxicity against three human cancer lines (MCF-7, T98, and HepG2) with IC50 values ranging from 9.81 ± 1.56 to 17.00 ± 2.75 μM.


INTRODUCTION
The plants of Guttiferae and Hypericaceae family mainly including the genus Garcinia and Hypericum are well-known for producing structurally diverse and biologically polycyclic polyprenylated acylphloroglucinols (PPAPs). Previous phytochemical studies indicated that more than 500 PPAPs have been isolated the plants of Guttiferae family with diverse structural scaffolds including the bicyclic polyprenylated acylphloroglucinols (BPAPs), the caged PPAPs, and the complicated PPAPs. BPAPs comprise approximate 60% of PPAPs which share a bicyclo [3.3.1]nonane-2,4,9-trione core (Yang et al., 2018). Depending on the relative position of the acyl group connected to the phloroglucinol core, BPAPs are categorized into type A or B. Type A BPAPs contain a C-1 acyl group which is next to C-8 quaternary center, while type B BPAPs contain a C-3 acyl group (Ciochina and Grossman, 2006). BPAPs have attracted noticeable attention from both natural product and medicinal chemists due to their fascinating chemical structures and intriguing biological activities (Phang et al., 2020).

NMR Calculations
The calculated NMR data were acquired using the Gauge-Including Atomic Orbitals (GIAO) method at the mPW1PW91/6-311+G (2 d,p) level in CHCl 3 with the IEFPCM model (the detailed NMR calculations are described in the Supplementary information).

ECD Calculations
The ECD calculation was conducted using time-dependent density functional theory (TD-DFT) in methanol by the IEFPCM model (the detailed ECD calculations are described in the Supplementary information).

Antiproliferative Activity Bioassay
The antiproliferative activities against HepG2, T98, and MCF-7 cell lines of isolated compounds were measured by the CCK-8 method using cisplatin as the positive control, according to the protocol described previously (Teng et al., 2019).
Compound 2 was isolated as a white amorphous powder. The molecular formula was established as C 38 H 50 O 6 based on a  (Tables 1 and 2) of 2 closely matched those of 1, except for the presence of a 3,4-dihydroxybenzoyl group at C-1, replacing a benzoyl group at C-1 in 1, which was supported by HMBC correlations H-12 and H-16 to C-10. The relative configuration of 2 was established as the same as that of 1 by ROESY spectrum (Supplementary Figure S138, Supplementary information) and 13 C NMR data. The absolute configuration of 2 was finally assigned as (1S, 5R, 7R, and 23R) by comparing the experimental and calculated ECD spectra (Supplementary Figure S155, Supplementary information). Thus, the structure of compound 2 was elucidated, as shown in Figure 1, and named as garcimultinone E.
Compound 3 was obtained as a white amorphous powder and showed a pseudo molecular ion peak at m/z 587.3733 [M + H] + (calcd 587.3731) in the HRESIMS, corresponding to the molecular formula C 38 H 50 O 5 . The 1 H and 13 C NMR data of 3 closely resembled those of hyperattenin C with the only difference being the presence of isogeranyl located at C-5 in 3, instead of geranyl located at C-5 in hyperattenin C . The assignment was further corroborated by the HMBC correlations from H 2 -22 to C-5 and C-9 ( Figure 2). The relative configuration of 3 was determined by ROESY spectrum (Supplementary  Frontiers in Chemistry | www.frontiersin.org October 2021 | Volume 9 | Article 756452 Figure S138, Supplementary information) and the 13 C NMR data ( Table 1) analysis. Compared to 1 and 2, the chemical shift of C-7 (δ C 48.9) and C-38 (δ C 27.0) were shifted downfield. These findings suggested that 3 belonged to endo-BPAPs, which was confirmed by ROESY correlations of H 2 -32/H 3 -37 and H-7/H 3 -38. By comparison of NMR data of 3 with those of hyperattenin C and otogirinin D (Ishida et al., 2010) indicated that the chemical shifts of C-18, 19, 20, and 21 of 3 were consistent with those of hyperattenin C, suggesting the β-orientation of H-18. Many PPAPs with the isogeranyl group have been isolated from G. multiflora (Wang et al., 2018;Chen et al., 2019b;Teng et al., 2019). Owing to the characteristics of structural flexibility of the isogeranyl group, it is difficult to solve the absolute configuration of C-23 of the isogeranyl group by conventional structural elucidation methods. Thus, the relative configuration of compound 3 except C-23 was determined, named as garcimultinone F. The relative configuration of C-23 of 3 and its absolute configuration are discussed later together with compounds 5-7.
Compound 4 was obtained as a colorless oil and showed a pseudo molecular ion peak at m/z 549.3209 [M + H] + (calcd 549.3211) in the HRESIMS, corresponding to the molecular formula C 34 H 44 O 6 . The 1 H NMR and 13 C NMR spectrum of 4 and hyperattenin D were highly similar, except for the additional methoxy and prenyl groups and the absence of ethoxy and geranyl groups in 4 . HMBC correlations (Supplementary Figure S137, Supplementary information) from MeO to C-17 and H 2 -22 to C-5 and C-9 suggested that methoxy and prenyl groups were located at C-17 and C-5, respectively. The relative configuration of 4 was established as the same as that of hyperattenin D by ROESY spectrum ( Figure 3) and 13 C NMR data ( Table 1). The absolute configuration of 4 was defined to be 1R, 5S, 7S, 17S, and 18S by the ECD calculation ( Figure 4). Thus, the structure of compound 4 was elucidated, as shown in Figure 1, and named as garcimultinone G.
Compound 5 was obtained as colorless oil. The molecular formula of 5 was deduced to be C 38 H 50 O 5 based on its negativeion HRESIMS data at m/z 585.3587 [M -H] − (calcd for C 38 H 49 O 5 , 585.3586). Comparison of the NMR spectroscopic data (Tables 1, 2) of 5 with those of garcimultine A implied that they possessed a similar structure, except for the presence of an oxygenated tertiary carbon C-24 (δ C 74.2) and a methyl [δ H 1.19 (3H, s); δ C 29.8] in 5, replacing the terminal double bond Δ 24(26) in garcimultine A. These findings suggested that 5 was a Δ 24(26)hydrate of garcimultine A (Liu et al., 2017b), which was ascertained by HMBC cross-peaks ( Figure 2) from H 3 -25/H 3 -26 to C-23 (δ C 46.9) and C-24 (δ C 74.2). Compound 5 was defined as endo-BPAPs based on the analyses of ROESY spectrum ( Figure 3) and 13 C-NMR data ( Table 1). Therefore, the relative configuration of 5 except C-23 was defined and named as garcimultinone H.
Compound 6 isolated as a colorless oil and gave the molecular formula C 38 H 50 O 4 as revealed by its HRESIMS at m/z 571.3778 [M + H] + (calcd for C 38 H 51 O 4 , 571.3782). Comparison of the NMR data of 6 and hypersampsone T indicated that their structures were highly similar, except for the C-5 substituent. Obviously, isogeranyl NMR signals of 6 replaced those for a prenyl group in hypersampsone T (Tian et al., 2016). This deduction was further confirmed by HMBC correlations from H 2 -22 to C-4, C-5, C-9, C-23, and C-24 (Supplementary Figure  S138, Supplementary information). Thus, the relative FIGURE 4 | Calculated and experimental ECD spectra of 1, 4, 5, 8, 9, 11, and 12. Frontiers in Chemistry | www.frontiersin.org October 2021 | Volume 9 | Article 756452 8 configuration of 6 except C-23 was defined and named as garcimultinone I.
Compound 7 were isolated as a colorless oil and gave the molecular formula C 38 H 50 O 6 , as revealed by its HRESIMS at m/z 603.3676 [M + H] + (calcd C 38 H 51 O 6 , 603.3641), indicating 32 mass units more than 6. Comparison of NMR data (Tables 1, 2) of 6 and 7 showed many similarities with two major differences. Firstly, the presence of a 3,4-dihydroxybenzoyl group at C-1 in 7 instead of a benzoyl group at C-1 in 6 was observed in NMR data, which was further confirmed by HMBC spectrum (Supplementary Figure S138, Supplementary information). Secondly, the chemical shifts of C-7 and CH 3 -38 were both shifted upfield 6.2 and 11.2 ppm, respectively, compared to 6, suggesting to be exo-BPAPs which was further supported by ROESY spectrum. Thus, the relative configuration of 7 except C-23 was established, and named as garcimultinone J.
Compound 9 was obtained as a white amorphous powder. The HRESIMS data at m/z 587.37262 [M + H] + of 9 together with 13 C-NMR and DEPT indicated molecular formula of C 38 H 50 O 5 . The NMR data of 9 was highly similar to those of 8 (Tables 1, 3), except for the presence of a prenyl group attached to C-23 in 9, instead of E-3-methyl-3-hydroperoxy-but-1-enyl group in 8. The key HMBC correlations from Me-30 and Me-31 to C-28 (δ C 123.1) and C-29 (δ C 131.9) confirmed this conclusion. Compound 9 was also determined as endo-type B BPAPs based on the chemical shift of C-7 (δ C 48.9) and C-38 (δ C 26.8) and ROESY correlation between Me-37 and H 2 -32. The NMR difference between compounds 8 and 9 mainly lied in the chemical shift and splitting of H-18 [(δ H 4.03, t, J 10.8 Hz) in 9 (δ H 4.68, dd, J 10.8, 7.8 Hz) in 8], suggesting the orientation of H-18 in 9 was opposite to 8, which was further confirmed by ROESY correlation of Me-37/Me-21. As with the above discussion of compounds 3 and 5-7, the relative configurations of compounds 8 and 9 could be determined as (1S*, 5R*, 7R*, 18R*, and 23R*) and (1S*, 5S*, 7R*, 18S*, and 23S*), respectively. Owing to the change of the priority order of functional groups at C-23, the relative configuration of C-23 of 8 changed from S* to R*. The calculated ECD spectrum of (1R, 5R, 7S, 18R, and 23R)-9b was in good agreement with the experimental ECD spectrum of 9, establishing the absolute configuration of 9 as 1R, 5R, 7S, 18R, and 23R ( Figure 4). Owing to the similarity of experimental ECD curves between 8 and 9, the absolute configuration of 8 was defined as 1R, 5S, 7S, 18S, and 23S. Thus, the structures of 8 and 9 were established as depicted in Figure 1, and named as garcimultinones K and L, respectively.
Compound 10 was obtained as a white amorphous powder. The HRESIMS data at m/z 571.37780 [M + H] + of 10, together with 13 C-NMR and DEPT indicated molecular formula of C 38 H 50 O 4 . Its NMR data were highly similar to those of isogarcinol (Marti et al., 2010;Gustafson et al., 1992), except for the presence of a benzoyl group in 10, instead of 3,4dihydroxybenzoyl group in isogarcinol. Thus, 10 was a 13,14didehydoxy of isogarcinol, which was further confirmed by the HMBC correlations (Figure 2). In a previous report, 13,14didehydoxyisogarcinol was isolated from the fruit of G. multiflora (Chen et al., 2009), which has been corrected to 13,14-didehydoxy-7-epi-isogarcinol (Yang et al., 2018). The relative configuration of 10 was deduced as the same as that of isogarcinol from the ROESY spectrum (Supplementary Figure   Frontiers in Chemistry | www.frontiersin.org October 2021 | Volume 9 | Article 756452 9 S139, Supplementary information). In the experimental ECD spectrum, compound 10 showed positive Cotton effect (CE) at 220 nm and negative CE at 270 nm, establishing the absolute configuration of 10 as the same as that of isogarcinol (Socolsky and Plietker, 2015). This deduction was further confirmed by ECD calculations (Supplementary Figure S159, Supplementary information). Thus, the structures of 10 were established as 13,14-didehydoxyisogarcinol and named as garcimultinone M. Compared to 8 and 9, compound 10 display the different side chain orientations of the bicyclo[3.3.1]nonane moiety. However, it is noteworthy that the corresponding compounds show the same CD spectrum. Compounds 8-9 are the presence of an enolized C-2 via ether ring closure and C-4 keto form, while compound 10 is the presence of C-2 keto form and an enolized C-4 via ether ring closure. These findings could imply that the position of an enolized 1,3-diketo group in the core structure might affect the molecular conformation and hence the ECD curves (Le et al., 2016;Sukandar et al., 2020).
Compound 13 was obtained as a white amorphous powder. The molecular formula of 13 was established as C 38 H 50 O 4 by the 13 C NMR, DEPT, and HRESIMS data at m/z [M + H] + 571.3780 (calcd for C 38 H 51 O 4 , 571.3782). Comparison of the NMR data 13 with those of 12 indicated the presence of an isogeranyl group at C-2 in 13 as opposed to a prenyl group at C-2 in 12. However, the chemical shifts of C-2 and C-8 were shifted downfield from C-2 (δ C 52.7) and C-8 (δ C 41.0) in 12 to C-2 (δ C 56.5) and C-8 (δ C 44.2) in 13, suggesting that 8-prenyl and 2-isogeranyl groups were taken as cis relationship . ROESY correlations H-17/H 2 -22/Me-38 supported this deduction (Supplementary Figure S139, Supplementary information). The relative configuration of C-23 remained undetermined. Thus, the ECD calculations for (2R, 8R, and 23R)-13a, (2R, 8R, and 23S)-13b, and their enantiomers 13a9 and 13b9 were carried out. These result showed that the calculated ECD curves of 13a9 and 13b9 were in good agreement with experimental ECD data (Supplementary Figure S160, Supplementary information). Thus, the absolute configuration of 13 except C-23 was established as (2S and 8S), and compound 13 was named as garcimultinone P.
All isolated compounds were evaluated for their inhibitory effects against the human T98, HepG2, and MCF-7 cancer cell lines by the CCK-8 method. Compounds 2 and 7 displayed evident antiproliferative activity against three tested cell lines (Supplementary Table S1, Supplementary information). The IC 50 values of antiproliferative activities of compound 2 on T98, HepG2, and MCF-7 cancer cell lines were 13.23 ± 4.24, 13.53 ± 0.17, and 9.81 ± 1.56 μM, respectively. Compound 7 showed antiproliferative activity against T98, HepG2, and MCF-7 cancer cell lines with IC 50 values of 17.00 ± 2.75, 12.84 ± 1.59, and 15.68 ± 1.65 μM, respectively. However, the other compounds were inactive showing IC 50 values in excess of 20 μM. According to the structure type and biological activity of the isolated compounds, it could be preliminarily inferred that 3,4dihydroxybenzoyl substituents in the structures of BPAPs are important for their anticancer activities (Yang et al., 2018).

CONCLUSION
In summary, the phytochemical investigation of the fruits of G. multiflora resulted in the isolation and structure elucidation of 24 structurally diverse polyprenylated acylphloroglucinols (PAPs) including 11 new PPAPs (1-11) and 3 new MPAPs (12-14). These PPAPs belong to three types including the bicyclic polyprenylated acylphloroglucinols (BPAPs, 1-10 and 15-18), the caged PPAPs (11 and 19-22), and the complicated PPAPs (23 and 24). Interestingly, most of PAPs are linked with the isogeranyl or its derivatives. The BPAPs with 3,4dihydroxybenzoyl were found to exhibit effectively antiproliferative activity. These findings indicated that the fruits of G. multiflora are an important source of structural diversity PAPs, which deserve further study.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
YC and GY conceived, designed the experiments, were responsible for structure elucidation, and revised the manuscript. HT carried out the isolation of compounds, NMR, and ECD calculation, and wrote the original draft. QL contributed to cytotoxicity testing. ZM carried out the isolation of compounds. XL and WX carried out the experiments and data analyses. All authors have read and approved the published version of the manuscript.