Additional Insights into Hypericum perforatum Content: Isolation, Total

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Additional Insights into Hypericum perforatum Content: Isolation, Total Synthesis, and Absolute Configuration of Hyperbiphenyls A and B from Immunomodulatory Root Extracts Dimitri Bréard,† Guillaume Viault,† Marie-Charlotte Mezier,‡,§ Sylvain Pagie,‡,§ Antoine Bruguière,† Pascal Richomme,† Béatrice Charreau,*,‡,§ and Séverine Derbré*,† †

EA921 SONAS, SFR4207 QUASAV, UNIV Angers, Université Bretagne Loire, 49035 Angers, France Centre de Recherche en Transplantation et Immunologie (CRTI), UMR1064, INSERM, Université de Nantes, 44093 Nantes, France § CHU de Nantes, Institut de Transplantation-Urologie-Néphrologie, 44200 Nantes, France

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‡

S Supporting Information *

ABSTRACT: Phytochemical investigation of the root extracts of Hypericum perforatum led to the isolation of two biphenyl derivatives named hyperbiphenyls A and B (1 and 2) and four known xanthones (3−6). These structures were elucidated by spectroscopic and spectrometric methods including UV, NMR, and HRMS. The absolute configuration of the biphenyl derivatives was defined by two different approaches: biomimetic total synthesis of racemic hyperbiphenyl A followed by 1H and 19F NMR Mosher’s esters analysis and stereoselective total synthesis of hyperbiphenyl B, permitting assignment of the S absolute configuration for both compounds. The bioactivity of compounds 1−6 toward a set of biomolecules, including major histocompatibility complex (MHC) molecules expressed on vascular endothelial cells, was measured. The results showed that the major xanthone, i.e., 5-Omethyl-2-deprenylrheediaxanthone B (3), is a potent inhibitor of MHC that efficiently reduces HLA-E, MHC-II, and MICA biomolecules on cell surfaces. their flowering tops to produce antidepressant herbal drugs and dietary supplements.14 This herbal medicine is thus available in large amounts. On the contrary, the tiny roots are not collected. Thus, the natural products biosynthesized in the roots of cultivated SJW are not fully characterized: only xanthones and quercetin glycosides were identified in the dichloromethane (DCM) or MeOH extracts from either root material or nonelicited root cultures.14−16 All this prompted further investigation into the phytochemistry of SJW roots and the effect of its extracts and purified P2 on MHC expression in ECs. Herein, the isolation and identification of two new biphenyls, i.e., hyperbiphenyls A and B (1 and 2), together with four known xanthones, 5-O-methyl-2-deprenylrheediaxanthone B (3), kielcorin (4), 5′-demethoxycadensin G (5), and paxanthone (6), are reported (Chart 1). The absolute configurations of 1 and 2 are also elucidated using total synthesis and Mosher’s reagents. Assayed for its effect on MHC expression, the major xanthone 3 showed a strong inhibition of HLA-E and had a moderate effect on MHC-II and MICA biomolecules.

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o avoid the potential side effects of cytotoxic anticancer drugs, alternative therapeutic strategies such as immunotherapies, specifically targeting cell type or biomolecules, are emerging.1,2 These approaches require the development of dedicated cellular and molecular models to assess the bioactivity of pure compounds or mixtures.2 Our previous studies highlighted the use of human primary vascular endothelial cells (ECs) as a cellular target to determine the activity of natural products toward a panel of biomolecules involved in inflammation and immunity.3,4 In this framework, we identified polyprenylated polyphenols (P2) molecular scaffolds from Clusiaceae and Calophyllaceae that efficiently reduce both major histocompatibility complex (MHC) and MHC-like [MHC-I related chain A (MICA) and HLA class I histocompatibility antigen, alpha chain E (HLA-E)] expression.4 Plants from Clusiaceous and Calophyllaceous species are pantropical trees growing in high-biodiversity areas. Therefore, access to these zones is strictly regulated.5,6 P2 such as xanthones, biflavonoids, benzophenones, or polyprenylated polycyclic acylphloroglucinols were previously isolated from these species.7−12 Hypericaceae, particularly Hypericum species, biosynthesize the same type of compounds.13 Some species such as St John’s wort (SJW, H. perforatum) are cultivated for © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 25, 2018

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DOI: 10.1021/acs.jnatprod.8b00325 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Extracts were evaluated at 5, 25, and 50 μg/mL, and purified compound 3 was evaluated at 5, 25, and 50 μM. Data show the percentages of inhibition for the expression of the adhesion molecule VCAM-1, MHC class I, and MHC-related molecule MICA on the cell surface obtained compared to the controls. The HPLC-PDA-ESIMSn analysis of the crude cyclohexane and DCM extracts revealed the presence of two types of natural products. In the first group, most of them exhibited xanthone-type UV spectra19 and a molecular weight between 340 and 438 Da.16 In the second group, two minor compounds with unusual UV spectra (λmax at 200, 225 (sh), and 265 nm) and a lower molecular weight at 284 Da were also detected (Figure S1 and Table S1, Supporting Information). Both apolar extracts were consecutively fractionated by flash chromatography and semipreparative HPLC to afford the new compounds, hyperbiphenyls A and B (1 and 2), and four known xanthones 5-O-methyl-2-deprenylrheediaxanthone B (3), kielcorin (4), 5′-demethoxycadensin G (5), and paxanthone (6) (Chart 1). The molecular formula of 1, C18H20O3, was established on the basis of HRCIMS data (m/z 284.1409 [M]+•; calcd 284.1412) and 13C NMR data. The UV and mass spectra were similar to those of garcibiphenyls and garcibenzopyran.20,21 The 1H NMR data (Table 1) showed two meta-coupled hydrogens at δH 6.65 and 6.75, suggesting the presence of a 1,2,3,5-tetrasubstituted benzene ring. Additionally, it showed two multiplets of two hydrogens each at δH 7.43 and 7.63 and one more multiplet of one hydrogen at δH 7.33, suggesting the presence of an unsubstituted phenyl group. Two singlets at δH 1.25 and 1.36 for three hydrogens were assigned to a gemdimethyl group. A doublet of triplets at δH 3.79 suggested the presence of an oxygenated methine group attached to a methylene group, whose presence was indicated by the two doublets of doublets at δH 2.54 (17.4 and 7.7 Hz) and 2.93 (17.4 and 5.5 Hz). Moreover, two additional signals, a threeproton singlet at δH 3.91 and a one-proton doublet at 4.22, revealed the presence of a methoxy and a hydroxy group, respectively. The 13C NMR data indicated five quaternary aromatic carbons including two oxygenated ones at δC 155.0 (C-8a) and 159.3 (C-5) besides the five signals of seven aromatic methines. Indeed, two of these chemical shifts, at δC 127.6 (C-2′, C-6′) and 129.6 (C-3′, C-5′), corresponded to two CH each in the phenyl group. In the aliphatic region of the spectrum, signals were observed at δC 77.6, 69.7, 27.2, 26.0,

Chart 1. Structures of Biphenyls 1 and 2 and Xanthones 3− 6 Isolated from H. perforatum Roots

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RESULTS AND DISCUSSION H. perforatum roots were successively extracted with solvent with increasing polarity, i.e., cyclohexane, DCM, EtOAc, MeOH, and water. To rapidly decipher the biological potential of the extracts, we used a cellular enzyme-linked immunosorbent assay (ELISA) performed on cultured vascular endothelial cells as a screening step to define and compare the bioactivity of the SJW root extracts. The assay included the quantification of three cell surface molecules regulated on the cell surface upon pathological conditions:3,17 VCAM-1, an adhesion molecule, and MHC molecules (MHC class I, HLAA, -B, -C, and MICA) involved in the inflammation and immune responses, respectively. To mimic pathological inflammatory contexts, endothelial cells were treated with TNF, a positive regulator of VCAM-1 and MICA,17 or with IFNγ, a positive regulator of MHC class I, class II, and HLA-E molecules.18 As a result, consistent inhibitory effects on the expression of these molecules were observed for SJW root apolar extracts (Figure 1). Indeed, the SJW cyclohexane and DCM extracts inhibited the three biomolecules assayed with a more potent effect on MHC-I and MICA biomolecules. Although showing the same effects, the SJW MeOH extract was not further investigated, as polymeric proanthocyanidins appeared to be responsible for an unspecific inhibition.

Figure 1. Activity of SJW root extracts and the major compound 5-O-methyl-2-deprenylrheediaxanthone B (3) determined by cellular ELISA performed on cultured vascular endothelial cells. B



Article

Table 1. 1H (300 MHz) and 13C (125 MHz) NMR Spectroscopic Data in Acetone-d6 for Hyperbiphenyl A (1) and Hyperbiphenyl B (2) (Figures S2, S3, S8, S9, S30, S31, S48, S49, Supporting Information) hyperbiphenyl A (1) position

δC, type

2 3

77.6, Cq 69.7, CH

4

27.2, CH2

4a 5 6 7 8 8a 1′ 2′, 6′ 3′, 5′ 4′ 2″ 2‴ 5-OMe 3-OH

109.2, Cq 159.3, Cq 101.5, CH 141.2, Cq 108.9, CH 155.0, Cq 142.1, Cq 127.6, CH 129.6, CH 128.1, CH 20.4, CH3 26.0, CH3 55.9, CH3

hyperbiphenyl B (2) δH (J in Hz) 3.79, dt (7.7, 5.5) 2.93, dd (17.4, 5.5) 2.54, dd (17.4, 7.7)

6.75, d (1.5) 6.65, d (1.5)

7.63, 7.43, 7.33, 1.25, 1.36, 3.91, 4.22,

δC, type

δH (J in Hz)

2 3

90.9, CH 28.5, CH2

4.67, dd (9.5, 8.4) 3.10, dd (15.9, 9.5) 3.17, dd (15.9, 8.4)

3a

114.3, Cq

4 5 6 7 7a 1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4-OMe 1″-OH

157.5, Cq 102.9, CH 143.4, Cq 101.7, CH 162.8, Cq 142.4, Cq 127.8, CH 129.5, CH 128.1, CH 71.5, Cq 26.0, CH3 25.5, CH3 55.8, CH3

position

m m m s s s d (5.5)

6.71, d (1.2) 6.61, d (1.2)

7.62, m 7.42, m 7.34, m 1.23, 1.26, 3.91, 3.69,

s s s s

Scheme 1. Retrosynthesis of Racemic Hyperbiphenyls A [(±)-1] and B [(±)-2]

heterocyclic moieties. Compound 2 was readily identified as having a dihydrobenzofuran moiety since a methylene [δH 3.10 (15.9 and 9.5 Hz) and 3.17 (15.9 and 8.4 Hz)] adjacent to an oxygenated methine δ 4.67 (9.5 and 8.4 Hz) was observed instead of the chemical shifts from a dihydrobenzopyran moiety in 1. It was substituted at C-2 by a 1-hydroxy-1methylethyl moiety, as signals at δH 1.23 (3H, s, C-2″), 1.26 (3H, s, C-3″), and 3.69 (3-OH) were also observed. A positive optical rotation was observed; hence compound 2 was named (+)-hyperbiphenyl B. The absolute configuration of 2 was defined via stereoselective total syntheses of both (R)- and (S)hyperbiphenyl B and comparison of their specific rotations and retention time on chiral-phase HPLC (see below). Compounds with a dihydrobenzofuran-type structure were previously found in the fungi kingdom.24,25 5-O-Methyl-2-deprenylrheediaxanthone B (3) was identified by comparison with literature data.26 The optical rotation was recorded as zero as previously described for such compounds in H. perforatum roots16 and other Hypericum and Garcinia species.9,26 Regarding kielcorin (4),27 the trans-orientation of the 1,4-dioxane protons was deduced from their axial−axial coupling constant (3J = 7.8 Hz).28 The NMR chemical shifts of the benzylic oxymethine proton of the 1,4-dioxane ring

and 20.4, compatible with a 2,2-dimethyldihydropyran group with a hydroxy group at C-3 and the signal at δC 55.9 of the methoxy group at C-5. The COSY, HMQC, and HMBC correlations (Figures S4−S6, Supporting Information) and a comparison with literature data22 indicated that 1 is a biphenyl derivative with a 3-hydroxy-2,2-dimethyldihydropyran moiety located at C-4a and C-8a and carrying a methoxy group at C-5. Compound 1 has a negative optical rotation and was given the trivial name (−)-hyperbiphenyl A. The absolute configuration of 1 was defined via total synthesis of both enantiomers and by using Mosher’s method. It is the first time that such a biphenyl derivative has been isolated from H. perforatum. Only a 4′hydroxy-3,4,5-trimethoxy-1,1′-biphenyl was reported from the Hypericum genus, specifically from H. ref lexum.23 The 4′hydroxylated derivative of hyperbiphenyl A was previously isolated from the root extract of Garcinia linii.21 The molecular formula of 2, C18H20O3, established on the basis of HRCIMS data (m/z 284.1404 [M]+•; calcd 284.1412) is identical to that of hyperbiphenyl A (1). The 13C NMR spectrum is similar. The UV spectrum exhibited the same λmax, also suggesting similar skeletons. Direct comparison of the 1H and 13C NMR data with those of 1 (Table 1) showed that compound 2 was a biphenyl derivative, differing from 1 in the C



Article

Scheme 2. Synthesis and Resolution of Racemic Hyperbiphenyl A [(±)-1]a

Reagents and conditions:(a) PhB(OH)2, Pd(PPh3)2Cl2, K2CO3, dioxane/H2O (9:1), 100 °C, 3 h, 78%; (b) BBr3, DCM, 0 °C to rt, 3 h, 83%; (c) NaH, MOMBr, DMF, 0 °C to rt, 12 h, 79%; (d) nBuLi, THF, prenyl bromide, 0 °C to rt, 2 h, 87%; (e) HCl, MeOH, 40 °C, 4 h, 74%; (f) mCPBA, PTSA, DCM, 0 °C to rt, 1 h, 71%; (g) (i) (S) or (R)-MTPA-Cl, pyridine, rt, 12 h; (ii) HPLC separation; (iii) KOH, MeOH/H2O (3:1), rt, 12 h, 70% (2 steps) for (S)-1, 78% (2 steps) for (R)-1.

a

Scheme 3. Synthesis of Racemic Hyperbiphenyl B [(±)-2]a

Reagents and conditions: (a) NaH, BnBr, DMF, 0 °C to rt, 2 h, 97%; (b) mCPBA, DCM, rt, 1 h, 73%; (c) H2, Pd/C, K2CO3, EtOH, rt, 2 h, 68%.

a

permitted differentiation of 4 from isokielcorin. Indeed, the proton in the para-position relative to the xanthone carbonyl group in 4 is more deshielded than those in meta-position in isokielcorin (Chart S1, Supporting Information). Thus, the aromatic ring and the hydroxymethyl group are located at C-7′ and C-8′, respectively.28 As far as 5′-demethoxycadensin G (5)29 was concerned, the trans-configuration of the 1,4-dioxane protons (3J = 8.2 Hz) and the positions of the aromatic ring and hydroxymethyl group at C-7′ and C-8′, respectively, were deduced in the same way as for kielcorin.28,30 The optical activity was recorded as zero for both compounds 4 and 5, indicating that they comprised both (7′R,8′R) and (7′S,8′S) enantiomers of kielcorin (4) and 5′-demethoxycadensin G (5), respectively.31,32 Paxanthone (6) was identified by comparison with literature data.33 To determine the absolute configurations of hyperbiphenyls A (1) and B (2), the first synthesis of racemic hyperbiphenyls A [(±)-1] and B [(±)-2] via ortho-prenylated phenolic intermediate 7 as a common precursor was peformed. The stereoselective synthesis of (R)- and (S)-hyperbiphenyl B is also presented. A retrosynthetic analysis for (±)-hyperbiphenyls A and B is proposed in Scheme 1. The dihydrobenzopyran ring of (±)-hyperbiphenyl A and the dihydrobenzofuran ring of (±)-hyperbiphenyl B could be obtained after in situ opening of the epoxide generated by the oxidation of the prenyl chain of 7. Ortho-prenylated phenolic intermediate 7 could be obtained after regioselective

prenylation and methoxymethyl group deprotection of biphenyl 8, itself being derived from commercially available 1-bromo-3,5-dimethoxybenzene, 9. Reaction of 9 with phenylboronic acid in the presence of K2CO3 and PdCl2(PPh3)2 in a mixture of dioxane and water (9:1) afforded biphenyl 10 in 78% yield (Scheme 2). Deprotection of a methoxy group of 10 with BBr3 in DCM followed by reaction of 11 with NaH/methoxymethyl bromide afforded intermediate 8 in good yield. Ortho-lithiation of 8 with n-butyllithium followed by addition of prenyl bromide34 afforded the prenylated product 12 (87%), which was deprotected under acidic conditions to give phenol 7 in 74% yield. The NMR data of synthesized 7 (Figures S22 and S23, Supporting Information) were identical to those of the natural product.35 Epoxidation of 7 using meta-ChloroPeroxyBenzoic Acid (mCPBA) in the presence of p-toluenesulfonic acid (PTSA) afforded (±)-hyperbiphenyl A in 71% yield. The racemate was resolved, and the absolute configuration of hyperbiphenyl A was defined using Mosher’s method.36−38 The reaction of (+)-(S)- and (−)-(R)-α-methoxy-α-trifluoromethylphenylacetyl chloride [(+)-(S)- and (−)-(R)-MTPACl] with racemic hyperbiphenyl A [(±)-1] afforded the diastereomeric esters, which were separated by preparative HPLC (Scheme S1, Supporting Information). In addition, the absolute configuration of the four stereoisomers could be established by a comparative analysis of their 1H and 19F NMR spectra (Tables S2 and S3, Supporting Information). After D



Article

Scheme 4. Enantioselective Synthesis of (S)- and (R)-Hyperbiphenyls Ba

a

Reagents and conditions: (a) AD-mix-α, CH3SO2NH2, tBuOH/H2O (2:1), rt, 48 h, 89% for (S)-15; (b) AD-mix-β, CH3SO2NH2, tBuOH/H2O (2:1), rt, 48 h, 94% for (R)-15; (c) (i) MsCl, Et3N, DCM, rt, 6 h, (ii) K2CO3, MeOH, reflux, 1 h, 41% (2 steps) for (R)-14, 46% (2 steps) for (S)14; (d) H2, Pd/C, K2CO3, EtOH, rt, 2 h, 63% for (S)-2, 63% for (R)-2.

IFNγ to upregulate MHC molecules (MHC class I, class II, and HLA-E) in the presence of the tested compounds (10 μM). To determine their efficacy to inhibit MHC, flow cytometry was used since it allows an accurate measurement of the level of surface expression. No significant activity was observed (Figure S55, Supporting Information) except with the major 5-O-methyl-2-deprenylrheediaxanthone B (3). In the absence of stimulation with IFNγ, 3 reduced the basal level of MICA by 25% but had no effect on the other MHC biomolecules tested. The inhibitory effect of 3 was confirmed when cells were stimulated with IFNγ. The expression of MICA (24% of inhibition vs IFNγ alone) but also HLA-E (40% inhibition vs IFNγ alone) and MHC class II (HLA-DR, 25% inhibition vs IFNγ alone) was significantly reduced as compared to controls (IFNγ alone and IFNγ plus diluent) (Figure 2). In the experimental conditions, the bisphosphonate zoledronic acid (ZA) used as a control44 only inhibits the expression of MHC-I molecules (67% inhibition). Thus, the findings suggest that in pathological inflammatory conditions where MHC molecules are upregulated, 5-O-methyl-2deprenylrheediaxanthone B (3) could be an interesting lead compound to reduce vascular cell activation and to modulate immune responses. Nevertheless, the overall effect and the functional impact of the MHC regulations reported here remain to be determined. In summary, two new biphenyl derivatives, hyperbiphenyls A and B (1, 2), as well as four known xanthones (3−6) were isolated from the apolar root extracts of H. perforatum. Regarding the new compounds, their absolute configuration was determined as S by two different approaches of total synthesis, i.e., Mosher’s esters derivative analysis and stereoselective synthesis of hyperbiphenyls A and B, respectively. This it is the first report of such derivatives of prenylated biphenyls in H. perforatum. Only the presence of 4′-hydroxy3,4,5-trimethoxy-1,1′-biphenyl was previously reported in the Hypericum genus from H. reflexum. Using a dedicated ELISA bioassay to select extracts modulating the expression of MHC biomolecules, the cyclohexane extract of SJW roots and, to a lesser extent, the DCM extract were found to reduce the expression of MHC-I and MICA biomolecules. Among the isolated compounds assayed by flow cytometry, only the major 5-O-methyl-2deprenylrheediaxanthone B (3) seemed to be responsible for this biological effect. In ECs, 3 reduced the expression of HLA-

ester hydrolysis, enantiopure (S)-hyperbiphenyl A [(S)-1] and (R)-hyperbiphenyl A [(R)-1] were obtained in good yield. Chiral-phase HPLC and optical rotation confirmed that the natural product was (S)-hyperbiphenyl A (1) (Figure S53, Supporting Information). In order to synthesize the racemic hyperbiphenyl B [(±)-2], epoxidation of 7 using mCPBA in the presence of K2CO3 was performed aiming at 5-exo-tet rather than 6-endo-tet cyclization.39 However, racemic hyperbiphenyl A [(±)-1] was exclusively obtained. The reaction of 7 with NaH and benzyl bromide afforded product 13 in 97% yield, which was epoxidized with mCPBA to give compound (±)-14 in 73% yield. The Pd/C hydrogenation in the presence of K2CO3 afforded the expected racemic hyperbiphenyl B [(±)-2] in 68% yield (Scheme 3). The compound has identical 1H and 13 C NMR spectra to the natural product (Figures S38 and S39, Supporting Information). Furthermore, for defining the absolute configuration of natural hyperbiphenyl B (2), the stereoselective syntheses of (R)- and (S)-hyperbiphenyl B were carried out using the Sharpless stereoselective dihydroxylation of intermediate 13 with AD-mix-α or -β to afford diol (S)-15 or (R)-15 in 89% and 94% yields.40,41 According to the reported methodology for the stereoselective formation of the benzofuran ring,42 the secondary hydroxy group was converted into the corresponding mesylate, which was directly transformed into the target epoxide (R)-14 or (S)-14 in 41% and 46% yields, respectively.42,43 The benzyl ether was hydrogenolyzed under basic conditions to afford the target (S)-hyperbiphenyl B [(S)2] and (R)-hyperbiphenyl B [(R)-2] in 63% yield (Scheme 4). The enantiomeric excess of the Sharpless dihydroxylation was determined on (R)- and (S)-hyperbiphenyl B (i.e., 48% and 16%, respectively) by chiral-phase HPLC. Comparison of retention times showed that the natural product was (S)hyperbiphenyl B (2) (Figure S54, Supporting Information). The bioactivity of the major 5-O-methyl-2-deprenylrheediaxanthone B (3), available in a large amount after purification, was assessed by ELISA. As compared to the activity of SJW extracts, 5-O-methyl-2-deprenylrheediaxanthone B (3) displayed a strong inhibition of VCAM-1 (>50%) and MHC-I (10−25%) and to a lesser extent MICA (

Additional Insights into Hypericum perforatum Content: Isolation, Total

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