Bi-, Tri-, and Polycyclic Acylphloroglucinols from - American Chemical

Article pubs.acs.org/jnp

Bi‑, Tri‑, and Polycyclic Acylphloroglucinols from Hypericum empetrifolium Sebastian Schmidt,† Guido Jürgenliemk,† Thomas J. Schmidt,‡ Helen Skaltsa,§ and Jörg Heilmann*,† †

Pharmaceutical Biology, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany Institut für Pharmazeutische Biologie und Phytochemie, Westfälische Wilhelms-Universität Münster, Hittorfstraße 56, D-48149 Münster, Germany § Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis, Zografou, GR-15771 Athens, Greece ‡

S Supporting Information *

ABSTRACT: The 1H NMR-guided fractionation of a petroleum ether extract of Hypericum empetrifolium led to the isolation of four new bicyclic (1−4), four known bicyclic (5−8), three new tricyclic (9−11), and three new polycyclic acylphloroglucinols (12/13 and 14) possessing a monoterpenoid citran moiety. Compounds 12/13 were isolated as a mixture of two inseparable structural isomers. The compounds showed in vitro antiproliferative activity against human microvascular endothelial cells (HMEC-1) with IC50 values in the range 9.2 ± 2.0 to 29.6 ± 3.5 μM.

A

protons, which are involved in hydrogen bonding with the acyl substituent, are characteristically shifted downfield to δ ≥ 11 ppm and thus are readily detectable in a fractionated sample.

cylphloroglucinols are prominent secondary metabolites of the genus Hypericum (Hypericaceae). The shared phloroglucinol core of these derivatives is usually substituted by one or more prenyl or geranyl moieties. Both types of substituents are susceptible to cyclization and oxidation processes resulting in bi-1,2 or tricyclic3,4 molecules, as well as complex caged compounds.5,6 The structural diversity among acylphloroglucinols leads to various pharmacological activities in vitro and in vivo. As reported by the Gibbons group, several representative members of this class of compounds display significant antibacterial activity especially against Gram-positive bacteria.7,8 Two additional properties of pharmacological interest are the cytotoxic and antiproliferative effects of acylphloroglucinols. For example, hyperforin and its derivatives have been reported to show potent antiangiogenic effects.9,10 Furthermore, a recent study demonstrated that simple monoterpenoid-substituted acylphloroglucinols isolated from Hypericum empetrifolium WILLD. exhibit strong antiproliferative activity and inhibition of cell migration when present at low micromolar concentrations.11 The aim of the present study was to comprehensively characterize the acylphloroglucinol composition of H. empetrifolium (Hypericaceae) and to evaluate the antiproliferative in vitro activities of the isolated compounds on an endothelial cell line (HMEC-1). Because several members of the acylphloroglucinol family possess a free hydroxy group close to the acyl moiety, 1H NMR spectroscopy was utilized for the systematic identification and isolation of these compounds. The hydroxy © 2012 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The 1H NMR-guided fractionation of a petroleum ether extract afforded four new bicyclic acylphloroglucinol derivatives, empetrikarinens A (1) and B (2) and empetrikarinols A (3) and B (4), and three new tricyclic acylphloroglucinol derivatives, empetriferdinans A (9) and B (10) and empetriferdinol (11). Three new polycyclic acylphloroglucinols containing a monoterpenoid citran moiety were named empetrifranzinans A/B (12/13) and C (14). The molecular formula of 1 was determined to be C20H26O4 from the HRESIMS data, showing a pseudomolecular ion at m/ z 329.1761 [M − H]−. The 13C NMR (Table 1) and HSQC spectra revealed the presence of eight quaternary, five methine, two methylene, and five methyl carbons. The most deshielded signal at δC 210.4 (C-1′) belonged to the carbonyl of an isobutyryl group (C-1′−C-4′) linked to a phloroglucinol skeleton (C-4a−C-8a) at C-8 (δC 105.2). The carbon resonance at δC 166.2 (C-7) could be assigned to an oxygenated aromatic carbon, with the hydroxy proton giving rise to a sharp singlet in the 1H NMR spectrum (Table 1, δH 13.83, HO-7). HMBC analysis of the 7-OH proton indicated Received: March 28, 2012 Published: October 2, 2012 1697

dx.doi.org/10.1021/np300237n | J. Nat. Prod. 2012, 75, 1697−1705

Journal of Natural Products

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Figure 1. Structures of compounds 1−14.

corresponding proton (δH 3.75, sext, J = 6.8 Hz) as well as additional signals of a methylene group (δC 26.8, δH 1.41 m/ 1.86 m, each 1H) suggested that the acyl side chain is a 2methylbutyryl moiety rather than an isobutyryl group (Table 1). This was confirmed by a detailed 2D NMR analysis. Hence, compound 2 was identified by spectroscopic analysis as 1-[5,7dihydroxy-2-methyl-2-(4-methylpent-3-enyl)chromen-8-yl]-2methylbutan-1-one and trivially named empetrikarinen B. The HREIMS of 3 indicated a molecular formula of C20H28O5 (found m/z 348.1938 [M]+, calcd 348.1937). The 1 H and 13C NMR data were similar to the known compound 7, but with an additional OH group at C-3 (δC 66.4) giving rise to the downfield-shifted signals of C-2/C-4 (δC 80.6/25.5, Table 1). The long-range coupled carbons C-1″/C-4a/C-9 (δC 37.4/ 97.9/19.0, Table 1) were shifted slightly upfield, as confirmed by HMBC and COSY spectroscopy. NOE interactions between H-3 and H-1″/H-2″ allowed assignment of the relative configuration at C-3. Hence, compound 3 was unequivocally assigned by its spectroscopic data as 1-[(2R*,3S*)-3,5,7trihydroxy-2-methyl-2-(4-methylpent-3-enyl)chroman-8-yl]-2methylpropan-1-one and trivially named empetrikarinol A.

that it was hydrogen bonded to the carbonyl (C-1′) and thus supported the peri position to the isobutyryl moiety. A second hydroxy group at C-5 (δC 157.2) showed an upfield shift in the 1 H NMR spectrum (δH 5.38, bs HO-5). The signal patterns and shift values of the remaining carbon and proton signals were similar to those of the bicyclic compound 1-[5,7-dihydroxy-2methyl-2-(4-methylpent-3-enyl)chroman-8-yl]-2-methylpropan-1-one (7) previously isolated from H. amblycalyx.12 The main difference between the two compounds was an additional C-3−C-4 double bond evident from the resonances of two methine carbons (δC 123.2, C-3; 116.8, C-4) rather than two methylene carbons and of the corresponding protons H-3 (δH 5.41, d, J = 10.0 Hz) and H-4 (δH 6.59, d, J = 10.0 Hz). Compound 1 was determined to be 1-[5,7-dihydroxy-2-methyl2-(4-methylpent-3-enyl)chromen-8-yl]-2-methylpropan-1-one and given the trivial name empetrikarinen A. The HREIMS data of 2 showed a pseudomolecular ion at m/ z 343.1917 [M − H]−, indicating a molecular formula of C21H27O4 (calcd. for 343.1915). Thus, compound 2 was proposed to differ from 1 only in the presence of an additional methylene group. In the 1H and 13C NMR spectra, the deshielded methine carbon at C-2′ (δC 46.0) and its 1698



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Table 1. 1H and 13C NMR Data of Compounds 1−4 in CDCl3 1 δH mult (J in Hz)

2 3 4

80.9 123.2 116.8

5.41 d (10.0) 6.59 d (10.0)

4a 5 6 7 8 8a 9 1′ 2′ 3′

101.5 157.2 96.1 166.2 105.2 156.8 26.5 210.4 39.2 19.2

4′ 5′ 1″

19.5

1.18 d (6.7)

41.5

2″

23.1

1.69 1.86 2.07 2.15 5.10

3″ 4″ 5″ 6″ 5-OH 7-OH a

2

δC

C/H

123.6 132.1 25.6 17.5

5.88 s

1.44 s 3.85 sept (6.7) 1.18 d (6.7)

m m m m t (7.2)

1.67 s 1.57 s 5.38 bs 13.83 s

δC 80.9 123.3 116.8 101.5 157.1 96.1 166.2 105.8 156.8 26.5 210.3 46.0 26.8 11.8 17.1 41.6 23.2 123.6 132.1 25.6 17.6

3

δH mult (J in Hz)

80.6 66.4 25.5

5.41 d (10.0) 6.59 d (10.0)

5.88 sa

1.44 sa 3.75 1.41 1.86 0.91 1.16 1.68 1.87 2.08 2.15 5.09

δC

sexta (6.8) m m ta (7.4) da (6.9) m m m m ta (7.1)

1.67 s 1.57 sa 5.40 bs 13.87 sa

97.9 160.2 96.2 165.5 105.2 155.7 19.0 210.3 39.3 19.2

4

δH mult (J in Hz) 3.84 t (5.8) 2.48 dd (16.5, 6.4) 2.76 dd (16.5, 5.3)

5.86 s

1.27 s 3.73 sept (6.7) 1.06 d (6.7)

19.6

1.06 d (6.7)

37.4

1.58 m 1.66 m 2.02 m

22.0 123.4 132.5 25.6 17.6

80.5 66.4 25.5 98.1 160.5 96.2 165.3 105.7 155.7 19.3 210.4 46.1 26.9 11.7 16.5 37.3 22.0

4.97 t (7.0) 1.57 s 1.48 s 5.91 bs 13.78 s

δC

123.3 132.5 25.6 17.6

δH mult (J in Hz) 3.94 ta (5.3) 2.62 dda (16.6, 5.8) 2.86 dda (16.6, 5.3)

5.96 sa

1.39 sa 3.75 1.40 1.80 0.89 1.15 1.68 1.75 2.12

sexta (6.6) m m ta (7.4) da (6.6) m m m

5.08 ta (7.0) 1.67 s 1.58 sa 6.48 bs 13.99 sa

Signals broadened or duplicated due to the presence of an epimer.

Compounds 7 and 8 have been previously isolated from Hypericum amblycalyx and were identified by matching NMR, MS, and other physical data.12 The molecular formulas of compounds 9 and 10 were determined to be C20H28O4 and C21H30O4, respectively, based on their HRESIMS data. The 13C NMR spectrum of 9 shows 20 signals, which could be sorted by HSQC into eight quaternary, three methine, four methylene, and five methyl carbons. The downfield-shifted resonances of C-1′ (δC 210.5) and C-6 (δC 165.2) revealed the presence of a similar chroman ring linkage to that shown for 7. The other aromatic carbon shifts correspond to those of 7, with the exception of C-8a (δC 100.8), which was slightly deshielded (Table 3) in contrast to its corresponding carbon C-4a (δC 99.5) in the bicyclic derivative. The presence of a methine (C-9a) instead of the methylene C-3 in compound 7 suggested the presence of a tricyclic ring system. This was confirmed by the long-range coupling of H2-9 (δH 2.25, dd, J = 13.3, 15.9 Hz/δH 2.64, dd, J = 5.0, 15.9 Hz, each 1H) to C-8a, to the quaternary carbons C-4a (δC 79.1) and C-1 (δC 33.4), and to the methine C-9a (δC 47.0, δH 1.63, dd, J = 5.0, 13.3 Hz). Additionally, both methyl groups H3-11 (δH 1.02, s) and H3-12 (δH 0.93, s) showed long-range HMBC correlations to C-9a and to the C-2 (δC 41.3). NOEs between H3-13 and H3-12 revealed that both are on the same side of the molecule, whereas H-9a (1.63, dd, J = 5.0, 13.3) showed a strong NOE only to H3-11. Compound 9 was thus identified as 1-[(4aR*,9aR*)-6,8-dihydroxy-1,1,4a-trimethyl2,3,4,4a,9,9a-hexahydro-1H-xanthen-5-yl]-2-methylpropan-1one and named empetriferdinan A. Similarly, compound 10 is 1-[(4aR*,9aR*)-6,8-dihydroxy-1,1,4a-trimethyl-2,3,4,4a,9,9a-

The molecular formula of compound 4 was calculated to be C21H30O5 on the basis of the HRESIMS data showing a pseudomolecular ion at m/z 361.2024 [M − H]− (calcd 361.2020). The 1D and 2D NMR data resemble those of compound 3. The difference of 14 mass units in compound 4 is attributed to an extra methylene group (δC 26.9, δH 1.40 m/ 1.80 m) in the acyl side chain. Both the chemical shift of the deshielded carbon C-2′ (δC 46.1) and the multiplicity of its related proton (δH 3.75, sext, J = 6.6 Hz) verified the conversion of the isobutyryl to a 2-methylbutyryl group (Table 1). Thus, compound 4 was identified as a derivative of 3, namely, 1-[(2R*,3S*)-3,5,7-trihydroxy-2-methyl-2-(4-methylpent-3-enyl)chroman-8-yl]-2-methylbutan-1-one and henceforth called empetrikarinol B. The known bicyclic compounds 5 and 6 were identified by comparison of their 1H NMR spectra to published data (Table 2).13 Their structures differ from those of compounds 1−4 and 7 and 8 with respect to the regiochemistry of ring closure, resulting in two free hydroxy groups adjacent to the acyl substituent. In 5, the HO at C-5 (δH 13.55, bs) forms a hydrogen bond to the C-1′ carbonyl (δC 210.1) group. In contrast, the 1H NMR spectrum of 7 shows a deshielded HO at C-7 (δH 13.83, s), which hydrogen bonds to the carbonyl at C1′ (δC 210.5). The corresponding carbon shift of δC > 165, rather than δC ≤ 164, as well as the correlations in the HMBC spectrum confirmed this fact. The 13C NMR data for compound 5 have been reported,14 whereas the carbon data for 6 have not (Table 2). Because HRMS data, the UV spectra, and optical rotation data for both compounds have not been reported, we report the full characterization herein. 1699



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Table 2. 1H and 13C NMR Data of Compounds 5−8 in CDCl3 5 C/H

δH mult (J in Hz)

2 3

77.7 30.2

4 4a 5 6 7 8 8a 9 1′ 2′ 3′

15.8 101.9 164.0 103.1 157.3 95.6 160.1 24.0 210.1 39.1 19.2

4′ 5′ 1″ 2″ 3″ 4″ 5″ 6″ 5-OH 7-OH a

δC

19.2 39.3 22.2 123.9 131.8 25.6 17.6

1.75 m 1.82 m 2.56 m

5.72 s 1.28 s 3.86 sept (6.7) 1.17 d (6.4) 1.17 d (6.4) 1.61 m 2.06 m 5.08 bt 1.67 s 1.59 s 13.55 bs 6.14 bs

6 δC

7

δH mult (J in Hz) 77.8 30.2

15.8 101.9 163.9 103.7 157.4 95.6 160.0 24.0 209.9 45.8 26.9 11.9 16.7 39.4 22.2 123.9 131.8 25.6 17.6

5.72 s 1.29 s sext (6.2) m m t (7.0) d (6.3) m m m

1.67 s 1.60 s 13.61 bs 6.19 bs

8


1.75 m 1.83 m 2.56 m

3.73 1.40 1.84 0.91 1.15 1.61 2.06 5.08

δC

16.0 99.5 159.6 95.4 165.3 105.5 156.8 23.7 210.5 39.2 19.1

1.77 m 1.87 m 2.58 m

5.93 s

1.35 s 3.85 sept (6.7) 1.16 d (6.7)

19.7

1.16 d (6.7)

39.8 22.5 123.6 132.1 25.6 17.5

1.71 m 2.08 m 5.10 t (7.1) 1.69 s 1.61 s 5.37 bs 13.83 s

δC


16.5 99.7 159.8 95.4 165.1 106.0 156.9 24.0 210.5 46.0 26.8 11.9 16.6 39.6 22.5 123.6 132.1 25.6 17.6

1.77 m 1.87 m 2.58 m

5.94 sa

1.36 sa 3.77 1.40 1.80 0.89 1.14 1.70 2.09 5.09

sexta (6.8) m m ta (7.4) da (6.8) m m ta (6.8)

1.68 s 1.60 sa 5.60 bs 13.92 sa


shifts (Table 4). 1H and 13C NMR data (Table 4) for 12/13 were similar to those reported for petiolin K, a compound recently isolated from Hypericum pseudopetiolatum var. kiusianum.15 In this acylphloroglucinol derivative, a menthane substituent is linked to the acylphloroglucinol via one −C−C− and two −C−O−C− bridges, forming a citran moiety.15 Nevertheless, the interesting methyl substitution of the acylphloroglucinol core in petiolin K is absent in 12/13 and is, instead, replaced by a typical aromatic proton resonating as a singlet at 6.03/6.05 (H-4, 1H each), respectively. Interestingly, only compound 13 showed HMBC and NOE correlations identical to those reported for petiolin K and, thus, was identified as the new compound 1-(1,9-epoxy-3-hydroxy-6,6,9trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-2yl)-2-methylpropan-1-one. In compound 12, the characteristic NOE interactions between the methyl groups H3-3′, H3-4′ of the acyl substituent, and H3-10″ were absent and were replaced by NOEs between the former methyl groups and H3-8″ and H3-9″. Consequently, the connectivity of the substituent to the acyl phloroglucinol in compound 12 is via the ether linkages C1−O−C-7″ and C-5−O−C-3″ instead of C-1−O−C-3″ and C5−O−C-7″ as in 13. Compound 12 was accordingly identified as the new compound 1-(1,9-epoxy-3-hydroxy-6,6,9-trimethyl6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-4-yl)-2-methylpropan-1-one and trivially named empetrifranzinan A. For compound 14, the molecular formula was established as C21H28O4 from the pseudomolecular ion peak at m/z 345.2050 [M + H]+ (calcd for C21H29O4, 345.2060). The 1D and 2D NMR data matched those of compound 12. The difference of 14 mass units between compounds 14 and 12/13 is caused by an additional methylene group (δC 27.8, δH 1.41 m/1.80 m) in the former due to the presence of a 2-methylbutyryl group as

hexahydro-1H-xanthen-5-yl]-2-methylbutan-1-one and named empetriferdinan B. This report is the first description of the isolation and characterization of these compounds. The HRESIMS data of 11 established its molecular formula as C21H30O5 (found 361.2019 [M − H]−, calcd 361.2020), and thus it differs from empetriferdinan B (10) by the inclusion of one additional oxygen atom. The 13C NMR spectrum showed a deshielded carbon at δC 77.9 that was identified by HSQC spectroscopy as a methine group (C-2, δH 3.45, t, J = 4.5 Hz) instead of a methylene. Evidence for the deshielding effect of a hydroxy group linked to this carbon was provided by the downfield shift of C-1 (δH 38.4) and C-3 (δC 28.1). Carbons C4/C-9a/C-11/C-12 (δC 37.6/45.8/27.2/14.2, Table 3) were shifted slightly upfield due to the proximity of the hydroxy group and showed the anticipated long-range couplings in the HMBC spectrum. In the NOESY spectrum, an NOE was observed between H-2 and H-9a, indicating the configuration at C-2. Thus, 11 was identified as 1-[(2R*,4aR*,9aR*)-2,6,8trihydroxy-1,1,4a-trimethyl-2,3,4,4a,9,9a-hexahydro-1H-xanthen-5-yl]-2-methylbutan-1-one and trivially named empetriferdinol. Duplicated 1H and 13C NMR patterns, existing in a ratio of approximately 3:1 (derived from the 1H and 13C NMR signal intensities), but with the presence of only one pseudomolecular ion at m/z 331.1911 [M + H]+ in the HRESIMS, indicated that 12/13 exist as a mixture of two isomeric forms, which were later shown to be structural isomers with the shared molecular formula of C20H26O4. Both sets of signals in the 13C NMR spectrum showed identical multiplicities (eight quaternary carbons, three methylenes, four methines, and five methyl groups) assigned by HSQC and HMBC spectroscopy. However, the carbons resonated at slightly different chemical 1700



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Table 3. 1H and 13C NMR Data of Compounds 9−11 (in CDCl3) 9 C/H

10

δH mult (J in Hz)

1 2

33.4 41.3

3

19.6

4

39.6

4a 5 6 7 8 8a 9

79.1 105.5 165.2 95.4 159.7 100.8 17.3

9a 10a 11 12 13 1′ 2′ 3′

47.0 156.2 31.9 20.5 19.6 210.5 39.2 19.3

4′ 5′ 6-OH 8-OH a

δC

19.4

1.35 1.52 1.58 1.69 1.68 2.03

δC 33.4 41.3

m m m m m m

5.93 s

2.25 dd (15.9, 13.3) 2.64 dd (15.9, 5.0) 1.63 dd (13.3, 5.0) 1.02 s 0.93 s 1.27 s 3.80 sept (6.7) 1.15 d (6.7)

11

δH mult (J in Hz)

19.6 39.7 79.1 106.0 165.1 95.4 159.7 101.0 17.4 47.0 156.3 31.9 20.5 19.7 210.5 45.9 26.8

1.15 d (6.7)

11.8 16.9

13.79 s 5.42 bs

1.35 1.52 1.58 1.70 1.68 2.03

m m m m m m

5.94 sa

2.25 dda (16.0, 13.4) 2.64 dda (16.0, 4.9) 1.64 ma 1.03 s 0.93 s 1.28 s 3.69 sexta (6.7) 1.38 m 1.82 m 0.88 ta (7.4) 1.13 da (6.7) 13.82 sa 5.48 bs

δC

δH mult (J in Hz) 38.4 77.9 28.1 37.6 78.0 106.0 165.1 95.7 159.8 100.5 17.3 45.8 156.0 27.2 14.2 19.7 210.3 45.9 26.6 11.9 16.7

3.45 ta (4.5) 1.64 1.90 1.83 2.07

m m m m

5.94 s

2.35 dda (15.5. 13.7) 2.67 dda (16.0, 5.0) 1.64 ma 1.13 sa 0.90 s 1.28 s 3.66 sexta (6.6) 1.38 m 1.81 m 0.88 ta (7.4) 1.12 da (6.5) 13.82 sa 5.61 bs


thesis. Based on the biosynthetic key mechanisms of terpenes21,22 the reactions depicted in Figure 2 are proposed. The initial geranylation of the acylphloroglucinol core via geranyldiphosphate is followed by epoxidation of the double bonds between C-2″,3″ and C-6″,7″. Nucleophilic attack of the phenolic OH groups results in epoxide cleavage and the formation of a hydroxy group. Subsequent dehydration restores the double bond in the geranyl moiety. In agreement with the key mechanistic details for the enzymatically catalyzed cyclization reactions of terpenoids by terpene synthases and the formation of different carbon skeletons,22 the generation of a carbocation by the protonation of a double bond and the subsequent cyclization are proposed as the following steps. Finally, a reductase (e.g., NADPH, FADH, or FMNH dependent) mediates the reduction of the double bond to produce compounds 12 and 14. In vitro assays measuring antiproliferative effects on endothelial cells are considered the first step in discovering potent antiangiogenic agents.23 Recently, the most prominent acylphloroglucinol, hyperforin from H. perforatum, was assessed by this protocol. Its evaluation in a proliferation assay using bovine endothelial cells revealed that hyperforin had an IC50 value of 2.1 ± 0.7 μM.10 Further characterization by several in vitro assays, such as tube formation and cell migration assays, proved that this molecule possesses promising antiangiogenic activity, which was subsequently demonstrated in vivo.10 Evaluation of the antiproliferative effect of compounds 1−11 against HMEC-1 cells revealed similar IC50 values ranging between 13.3 ± 3.8 μM for 8 and 29.6 ± 3.5 μM for compound

an acyl side chain. Thus, 14 was unambiguously identified as 1(1,9-epoxy-3-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-4-yl)-2-methylbutan-1-one and was named empetrifranzinan C. To assign the absolute configuration of C-4a and C-9a in compound 9, an ECD spectrum was recorded, and the spectrum was simulated using time-dependent density functional theory (TD-DFT). Neither the spectrum simulated for the 4aR,9aR-enantiomer nor that for the 4aS,9aS-enantiomer matched sufficiently well with the experimental ECD spectrum (Supporting Information). The reasons for the inability of this well-established computational method16,17 to reproduce the experimental spectrum are unclear and currently under investigation. The occurrence of an apparent exciton coupling phenomenon (negative−positive couplet of the UV maximum at 295 nm), pointing toward existence of a bichromophore,18 might indicate aggregation in the form of dimers or oligomers in solution. The ECD spectra of all compounds isolated in sufficient amounts (2, 4, 6−10) showed similar Cotton effects (Supporting Information), suggesting that the absolute configurations at the stereocenters in the bi- or tricyclic ring systems are identical. Acylphloroglucinols containing a citran moiety, with a menthane substructure C- and O-connected to the aromatic core, have been isolated from the genera Hypericum (Hypericaceae) and Clusia (Clusiaceae).19 Additionally, this structural element has been also found in chalcones.20 Nevertheless, no rational hypothesis has been advanced to explain its biosyn1701



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Table 4. 1H and 13C NMR Data of Compounds 12/13 (3:1) and 14 (in CDCl3) 12 δH mult (J in Hz)

13 δC

δH mult (J in Hz)

C/H

δC

1 2 3 4 5 6 1′ 2′

159.0 106.4 164.9 97.1 6.05 s 162.2 107.4 209.6 38.1 3.93 sept (6.7) 18.5 1.15 d (6.7)

158.2 105.3 165.7 98.7 162.9 106.5 209.7 39.0

20.1 1.18 d (6.7)

19.6

1.18 d (6.7)

27.6 2.75 m 34.9 1.86 m 2.17 m 75.9 37.5 1.45 m 1.83 m 21.8 0.88 m 1.31 m 46.2 2.08 ddd (11.6, 5.2, 2.8) 86.4 24.4 1.11 s 29.9 1.58 s 28.7 1.39 s 13.54 s

27.5 34.8

2.83 m 1.86 m 2.17 m

3′ 4′

19.1

6.03 s

3.77 sept (6.7) 1.18 d (6.7)

5′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 3OH a

76.4 37.4 21.9 46.0 84.9 24.2 29.6 28.7

1.45 m 1.83 m 0.88 m 1.31 m 2.04 ddd (11.5, 5.2, 2.6) 1.09 s 1.53 s 1.44 s 13.80 s

5 (Table 5). The moderate to weak activities of the bi- and tricyclic compounds are lower in comparison to those of both

14 δC

Table 5. Antiproliferative Activities of 1−14 in a Human Microvascular Endothelial Cell Line (Mean Value ± SD)

δH mult (J in Hz)

compound

159.1 106.9 165.0 97.1 6.05 s 162.3 107.5 209.3 45.1 3.78 sexta (6.8) 25.9 1.37 ma 1.85 ma 12.2 0.94 ta (7.4) 17.6 1.15 da (6.8) 27.6 2.75 ma 34.9 1.86 m 2.19 ma 75.9 37.5 1.45 m 1.85 m 21.8 0.89 m 1.31 m 46.3 2.08 ddda (11.5, 4.9, 2.8) 86.6 24.4 1.11 s 29.9 1.58 s 28.7 1.39 s 13.62 sa

1 2 3 4 5 6 7 8 9 10 11 12/13 14 xanthohumol

IC50 (μM) 24.4 15.0 23.4 18.4 29.6 21.1 16.5 13.3 24.4 16.6 29.2 11.7 9.2 11.4

± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.2 1.4 1.0 1.7 3.5 0.3 1.4 3.8 0.3 1.6 1.3 1.8 2.0 1.1

hyperforin and the monocyclic monoterpenoid-substituted acylphloroglucinols.11 In line with this observation, the acylphloroglucinols containing a citran moiety, 12/13 and 14, which can also be interpreted as monocyclic, monoterpenoidsubstituted compounds, exhibited slightly stronger activities, with IC50 values of 11.7 ± 1.8 and 9.2 ± 2.0 μM, respectively. These values are in the same range as the recently described activities of menthene- and bornane-substituted derivatives.11 Given that the proliferation assay revealed only preliminary results and did not exclude cytotoxic effects, current investigations are focusing on the relevance of nonspecific cytotoxicity in HMEC-1 and other cell lines as well as on the structural elements required for antiproliferative activity. The molecular targets for the reported antiangiogenic effects of acylphloroglucinols will be investigated also.


Figure 2. Proposed biosynthesis of compounds 12 and 14. 1702



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using the same functional and basis set, in which 30 electronic transitions were taken into account. The resulting output for electronic transition energies (E in eV) and rotator strengths (R, dipole length in cgs) was used to simulate ECD spectra for both conformers by applying a Gaussian shape function with a bandwidth at 1/e height σ = 0.15 eV. The spectra for both individual conformers were also combined in an average spectrum representing a 1:1 conformational equilibrium of A and B. No scaling or shifts on the energy/wavelength scale were applied. The resulting spectra are depicted in the Supporting Information. Plant Material. The aerial parts of Hypericum empetrifolium WILLD. (Hypericaceae) were collected at Mt. Pelion, Central Greece, in June 2009. The plant was identified by Prof. Theophanis Constantinidis, University of Athens, Faculty of Biology. A voucher specimen was deposited in ATHU (Dept. of Pharmacognosy, Univ. of Athens) with the identification number Skaltsa & Lazari 131-09. Extraction and Isolation. Air-dried and powdered aerial parts of H. empetrifolium WILLD. (922 g) were extracted by maceration with petroleum ether (PE, 1.5 L), yielding 11 g of residue after evaporation. The PE extract was fractionated by flash chromatography on silica gel using a linear gradient of n-hexane/EtOAc (0% → 100% EtOAc 0−60 min, 30 mL/min, detection at 270 nm) as mobile phase, and 113 fractions (20 mL/fraction) were obtained. Identical fractions, as determined by TLC and NMR similarities, were combined to give a total of 11 fractions (PE-1−11). PE-3 (2.9 g, 380−580 mL) was subjected to flash chromatography (silica gel) and eluted with a gradient of n-hexane/EtOAc (0% → 50% EtOAc 0−40 min, washing with 100% EtOAc for 10 min, and 100% MeOH for another 10 min, 20 mL/min, 10 mL/fraction) to give three subfractions (PE-3.1−3.3). Further fractionation of PE-3.1 (1.4 g, 0− 800 mL) on silica gel with flash chromatography using an n-hexane/ EtOAc gradient (0% → 10% EtOAc 0−60 min; 10% → 100% EtOAc 60−70 min, 20 mL/min, 15 mL/fraction) yielded six additional subfractions (PE-3.1.1−3.1.6). Flash chromatography of PE-3.1.2 (233 mg, 735−795 mL) with RP-18 using a MeOH/EtOAc gradient (100% MeOH 0−15 min, 0% → 100% EtOAc for 5 min, 100% EtOAc for further 10 min, 20 mL/min, 15 mL/fraction) resulted in four fractions (PE-3.1.2.1−3.1.2.4). Final purification of PE-3.1.2.1 (8.2 mg, 0−75 mL) with RP-HPLC on a Varian Dynamax Pursuit XRs afforded a mixture (3:1) of 12/13 (0.5 mg, tR 9.5 min) using a MeOH/MeCN gradient (70% → 98% MeCN 0−20 min, 98% MeCN 20−40 min, 3 mL/min) and 14 (0.5 mg, tR 11.5 min) with 100% MeCN. Flash chromatography of PE-3.1.3 (193.4 mg, 795−915 mL) with RP-18 using a MeOH/EtOAc gradient (100% MeOH 0−15 min, 0% → 100% EtOAc for 5 min, 100% EtOAc for further 10 min, 20 mL/min, 15 mL/fraction) resulted in four fractions (PE-3.1.3.1−3.1.3.4). Fractionation of PE-3.1.3.1 (14.5 mg, 15−60 mL) with RP-HPLC on a Varian Dynamax Pursuit XRs afforded pure 5 (0.8 mg, tR 10 min) using a MeOH/MeCN gradient (60% → 98% MeCN 0−20 min, 98% MeCN 20−40 min, 3 mL/min) and 6 (1.4 mg, tR 11 min), which was purified by further RP-HPLC on a Varian Dynamax Pursuit using a MeOH/MeCN gradient (70% → 98% MeCN 0−20 min, 98% MeCN 20−30 min; 3 mL/min). PE-4 (1.2 g, 580−740 mL) was separated with flash chromatography on silica gel (n-hexane/EtOAc gradient: 0% → 50% EtOAc 0− 85 min, 50% → 100% EtOAc 85−100 min, washing with 100% MeOH 100−120 min, 20 mL/min, 20 mL/fraction), yielding six subfractions (PE-4.1−4.6). Fractionation of PE-4.2 (287.2 mg, 940−1480 mL) with flash chromatography on RP-18 with an 80:20 mixture of MeCN/ MeOH for 30 min (15 mL/min, 15 mL/fraction) yielded four subfractions (PE-4.2.1−4.2.4). PE-4.2.1 (107 mg, 45−90 mL) was subjected to a Varian Dynamax Pursuit XRs using a H2O/MeCN gradient (80% → 98% MeCN 0−30 min, 3 mL/min) to give 1 (1.0 mg, tR 21.1 min), 7 (2.4 mg, tR 21.8 min), 9 (1.6 mg, tR 23.5 min), 2 (2.4 mg, tR 24.2 min), 8 (9.8 mg, tR 24.8 min), and 10 (4.2 mg, tR 26.9 min). PE-7 (394 mg, 980−1140 mL) was separated with flash chromatography on silica gel using an n-hexane/EtOAc gradient (10% → 100% EtOAc 0−40 min, washing with 100% MeOH 40−55 min, 15 mL/min, 15 mL/fraction) to give four subfractions (PE-7.1−

From a chemosystematic point of view there is an obvious intersection of the acylphloroglucinol spectrum of Hypericum empetrifolium WILLD with that of Hypericum amblycalyx COUST. & GAND. and Hypericum jovis GREUTER, three species endemic or common in Greece, which supports their collective systematic classification to the section Coridium SPACH of the genus Hypericum.24 In contrast to H. empetrifolium, the acylphloroglucinol profiles of H. amblycalyx and H. jovis are not comprehensively described, and thus the extent of intersection as well as the complexity of the acylphloroglucinol spectrum still needs investigation for both species. Also belonging to the section Coridium25 are H. ericoides L., H. asperuloides CZERN. EX TURCZ., and Hypericum coris L., for which phytochemical studies remain incomplete.26,27

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General Experimental Procedures. Optical rotations were recorded using a UniPol L1000 polarimeter (Schmidt + Haensch). ECD spectra were acquired on a J-710 spectropolarimeter (JASCO); c = 0.01 mg/mL (acetonitrile) at room temperature with a quartz cuvette of 1.0 cm path length. 1H, 13C, and 2D NMR spectra were measured at 298 K on a Bruker AVANCE 600 spectrometer (operating at 600.25 MHz for 1H and 150.93 MHz for 13C). All spectra were recorded in CDCl3 (99.8%, Deutero GmbH) and referenced against undeuterated (1H)/deuterated (13C) solvent. 1H and 13C NMR spectra of all compounds are provided in the Supporting Information. Shift values (δH and δC) are always given in ppm, and J values in Hz. UV spectra were obtained in MeOH on a Cary 50 Scan spectrophotometer (Varian). All solvents were spectroscopicy grade (Merck). High-resolution mass spectra were measured on a Finnigan MAT SSQ 710 A spectrometer at 70 eV (HREIMS, positive mode) or recorded on an Agilent 6540 UHD (HRESIMS, positive and negative mode). Semipreparative HPLC separations were performed on a Varian ProStar 210 solvent delivery module equipped with a Varian ProStar 335 photodiode array detector. A Varian Dynamax Pursuit XRs (250 × 10.0 mm, 5 μm) and a Knauer Eurosphere column (250 × 16.0 mm, 7 μm) were used for RP-18 HPLC. All solvents used were HPLC grade (Merck). Flash chromatography was performed on an Armen Instrument Spot System with prepacked normal-phase columns: SVP D40, Si60 15−40 μm, 90 g; SVF D26, Si60 15−40 μm, 30 g (Merck Chimie S.A.S.). LiChroprep RP-18 (25−40 μm for CC, Merck) and Geduran Si-60 (63−200 μm for CC, Merck) columns were used as stationary phases for flash chromatography. All solvents used were p.a. (pro analysi) grade (Merck). Silica gel 60 F254 precoated aluminum sheets and silica gel 60 RP-18 F254s precoated sheets (both from Merck) were used for TLC. Acylphloroglucinols were detected by fluorescence quenching at 254 nm, by dark-blue-colored spots at 366 nm, and by the green (1, 2), yellow (9, 10, 11), and brown colors for the remaining compounds (3−8 and 12−14) after spraying with anisaldehyde/sulfuric acid reagent (anisaldehyde, 2 mL; concentrated H2SO4, 10 mL; HOAc, 16 mL; MeOH, 170 mL). ECD Spectra Simulation. A molecular model of the 4aR, 9aR- and the 4aS, 9aS-enantiomeric form of compound 9 was generated using the molecular modeling package MOE (Molecular Operations Environment, rel. 2010:11, Chemical Computing Group) and submitted to a stochastic conformational search using the MMFF94x force field. The settings for this search included an energy window of 2 kcal/mol and a termination criterion of 100 unsuccessful attempts to find a new (different) conformer within this energy window. Two lowenergy geometries (A and B) were thus found. Both conformers were energy-minimized using the RB3LYP/6-31G(d,p) functional using Gaussian W03.28 The energy difference between the conformers (A − B) was calculated to be 0.16 kcal/mol, indicating that B is somewhat more stable but that both should be present in significant amounts in a conformational equilibrium at ambient temperature. Both conformers were therefore submitted to a time-dependent SCF calculation16,29 1703



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well microplates (100 μL, 1.5 × 103 cells/well) in endothelial cell growth medium with 10% FCS, supplement mix, and antibiotics (all from Provitro). After 24 h (time of cell adhesion for seeded cells), the medium in a reference plate was removed and the cells were stained with crystal violet solution for 10 min, providing a baseline value before the start of proliferation. Cells in other plates were treated with increasing concentrations of each test compound (0.1−30 or 0.2−60 μM, as stock solutions dissolved in DMSO). After 72 h incubation, cells were stained as previously described. The cells were washed with distilled H2O, 100 μL of dissolving buffer was added, and the absorbance was measured with a Tecan SpectraFluor Plus at 540 nm. A negative control in the absence of drug (pure solvent, 0.1% DMSO, in hexaplicates) was included in every 96-well plate and normalized to 100% proliferation after 72 h. The inhibitory effects of the phloroglucinols were calculated as % proliferation compared to the no-drug control. The IC50 values ± SD in μM were calculated with GraphPad software (from three independent experiments, with each concentration in hexaplicate). Xanthohumol was used as a positive control.

7.4). Fractionation of PE-7.2 (244.5 mg, 75−435 mL) with flash chromatography on RP-18 using 100% MeOH as the mobile phase for 30 min, switching to 100% EtOAc within 15 min, and final washing with 100% EtOAc for a further 10 min (15 mL/min, 15 mL/fraction) resulted in four subfractions (PE-7.2.1−7.2.4). PE-7.2.1 (84.7 mg, 15− 45 mL) was chromatographed on a Varian Dynamax Pursuit XRs using a H2O/MeCN gradient (60% → 98% MeCN 0−35 min, 3 mL/min) to give 11 (3.0 mg, tR 14.5 min), 3 (6.9 mg, tR 21 min), and 4 (14.3 mg, tR 24.2 min). Empetrikarinen A (1), 1-[5,7-dihydroxy-2-methyl-2-(4-methylpent-3enyl)chromen-8-yl]-2-methylpropan-1-one: yellow oil; [α]19D +34 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 285 (4.29) nm; 1H NMR and 13C NMR see Table 1; HRESIMS m/z 329.1761 [M − H]− (calcd for C20H25O4, 329.1758). Empetrikarinen B (2), 1-[5,7-dihydroxy-2-methyl-2-(4-methylpent-3enyl)chromen-8-yl]-2-methylbutan-1-one: yellow oil; [α]21D −8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 290 (4.34) nm; 1H NMR and 13C NMR see Table 1; HRESIMS m/z 343.1917 [M − H]− (calcd for C21H27O4, 343.1915). Empetrikarinol A (3), (1-[(2R*,3S*)-3,5,7-trihydroxy-2-methyl-2-(4methylpent-3-enyl)chroman-8-yl]-2-methylpropan-1-one: yellow oil; [α]21D −24 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 290 (4.42) nm; 1H NMR and 13C NMR see Table 1; HREIMS m/z 348.1938 [M]+ (calcd for C20H28O5, 348.1937). Empetrikarinol B (4), 1-[(2R*,3S*)-3,5,7-trihydroxy-2-methyl-2-(4methylpent-3-enyl)chroman-8-yl]-2-methylbutan-1-one: yellow oil; [α]21D +60 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 295 (4.43) nm; 1H NMR and 13C NMR see Table 1; HRESIMS m/z 361.2024 [M − H]− (calcd for C21H29O5, 361.2020). 1-[5,7-Dihydroxy-2-methyl-2-(4-methylpent-3-enyl)chroman-6-yl]-2methylpropan-1-one (5): yellow oil; [α]19D −36 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 295 (4.00) nm, 1H NMR and 13C NMR see Table 2; HRESIMS m/z 333.2065 [M + H]+ (calcd for C20H29O4, 333.2060). 1-[5,7-Dihydroxy-2-methyl-2-(4-methylpent-3-enyl)chroman-6-yl]-2methylbutan-1-one (6): yellow oil; [α]19D −24 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 295 (3.66) nm; 1H NMR and 13C NMR see Table 2; HRESIMS m/z 347.2218 [M + H]+ (calcd for C21H31O4, 347.2217). Empetriferdinan A (9), 1-((4aR*,9aR*)-6,8-dihydroxy-1,1,4a-trimethyl-2,3,4,4a,9,9a-hexahydro-1H-xanthen-5-yl)-2-methylpropan-1-one: yellow oil; [α]20D +40 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 295 (4.19) nm; 1H NMR and 13C NMR see Table 3; HRESIMS m/z 333.2066 [M + H]+ (calcd for C20H29O4, 333.2060). Empetriferdinan B (10) 1-((4aR*,9aR*)-6,8-dihydroxy-1,1,4a-trimethyl-2,3,4,4a,9,9a-hexahydro-1H-xanthen-5-yl)-2-methylbutan-1-one: yellow oil; [α]21D +44 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 295 (4.28) nm; 1H NMR and 13C NMR see Table 3; HRESIMS m/z 345.2074 [M − H]− (calcd for C21H29O4, 345.2071). Empetriferdinol (11) 1-((2R*,4aR*,9aR*)-2,6,8-trihydroxy-1,1,4a-trimethyl-2,3,4,4a,9,9a-hexahydro-1H-xanthen-5-yl)-2-methylbutan-1-one: yellow oil; [α]21D +12 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 295 (4.18) nm; 1H NMR and 13C NMR see Table 3; HRESIMS m/z 361.2019 [M − H]− (calcd for C21H29O5, 361.2020). Empetrif ranzinan A/B (12/13) 1-(1,9-epoxy-3-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-4-yl)-2-methylpropan-1-one/1-(1,9-epoxy-3-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-2-yl)-2-methylpropan-1-one: yellow oil; UV (MeOH) λmax (log ε) 230 (4.25) 295 (4.27) nm; 1H NMR and 13C NMR see Table 4; HRESIMS m/z 331.1911 [M + H]+ (calcd for C20H27O4, 331.1904). Empetrif ranzinan C (14) 1-(1,9-epoxy-3-hydroxy-6,6,9-trimethyl6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-4-yl)-2-methylbutan-1one: yellow oil; [α]22D −48 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 230 (4.37) 295 (4.42) nm; 1H NMR and 13C NMR see Table 4; HRESIMS m/z 345.2050 [M + H]+ (calcd for C21H29O4, 345.2060). Proliferation Assay. The proliferation assay was performed using an SV-40T transfected human microvascular endothelial cell line (HMEC-1).30 Cells were incubated at 37 °C under a 5% CO2/95% air atmosphere at constant humidity. HMEC-1 cells were seeded in 96-

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 941 943 4759. Fax: +49 941 943 4990. E-mail: joerg. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks are due to Prof. Dr. T. Constantinidis (University of Athens, Faculty of Biology) for identification of the plant material. The exhaustive extraction of the plant material with petroleum ether by Dr. V. Saroglou (University of Athens, Department of Pharmacognosy and Chemistry of Natural Products) is gratefully acknowledged. Special thanks are given to Dr. E. Ades and Mr. F. J. Candal of the CDC (USA) and Dr. T. Lawley of Emory University (USA) for providing the HMEC-1 cells. We thank F. Kastner, A. Schramm, and G. Stühler for recording the NMR spectra and Mr. J. Kiermeier and Mr. W. Söllner for recording the mass spectra (all Central Analytics of NWF IV, University of Regensburg). Mrs. E. Aplada (Biologist-M.Sc.) is gratefully acknowledged for photographing the plant.

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DEDICATION Dedicated to Professor Adolf Nahrstedt, Institut für Pharmazeutische Biologie und Phytochemie (Universität Münster, Germany), for 40 years of outstanding research and teaching in the field of natural products. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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Bi-, Tri-, and Polycyclic Acylphloroglucinols from - American Chemical

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