Phloroglucinol derivatives from Hypericum empetrifolium with antiproliferative activity on endothelial cells

Phytochemistry 77 (2012) 218–225

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Phloroglucinol derivatives from Hypericum empetrifolium with antiproliferative activity on endothelial cells Sebastian Schmidt a, Guido Jürgenliemk a, Helen Skaltsa b, Jörg Heilmann a,⇑ a b

Universität Regensburg, Pharmaceutical Biology, Universitätsstr. 31, D-93053 Regensburg, Germany Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, Panepistimiopolis, Zografou, GR-15771 Athens, Greece

a r t i c l e

i n f o

Article history: Received 30 September 2011 Received in revised form 17 November 2011 Available online 23 January 2012 Keywords: Hypericum empetrifolium Hypericaceae Acylphloroglucinols Anti-angiogenic activity NMR guided fractionation

a b s t r a c t Five acylphloroglucinols substituted with monoterpenoids (empetrifelixin A–D and empetrikajaforin), three known monocyclic acylphloroglucinols and one monocyclic acylphloroglucinol were isolated from a petrol ether extract of Hypericum empetrifolium after fractionation by flash chromatography on silica gel, RP-18 and subsequent purification by preparative HPLC (RP-18). Their structures were elucidated by 1D, 2D NMR techniques and HREIMS. To determine a possible anti-angiogenic activity, inhibition of cell proliferation was measured using a human microvascular endothelial cell line (HMEC-1). Subconfluent grown HMEC-1 cells were treated with all compounds isolated in sufficient amounts and stained with crystal violet. Highest activity was observed for empetrifelixin A and empetrifelixin D showing a concentration dependent inhibition of cell proliferation with IC50 values of 6.5 ± 0.1 and 7.3 ± 0.4 lM, respectively. Empetrifelixin A also showed activity in a cell migration assay with HMEC-1 cells in low micromolar concentrations. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Acylphloroglucinols are typical secondary metabolites accumulated in the families Hypericaceae (Avato, 2005) and Clusiaceae. The phloroglucinol core of these derivatives is often substituted by several prenyl or geranyl moieties. Both were able to undergo cyclization and oxidation processes resulting in bi- (Henry et al., 2006; Winkelmann et al., 2001) or tricyclic (Chen et al., 2010; Winkelmann et al., 2000) as well as complex caged compounds (Hu and Sim, 1999; Ishida et al., 2010). Among both families the genera Garcinia, Clusia and Vismia can be characterized by the dominance of phloroglucinols with an aromatic acyl moiety, whereas the genus Hypericum mainly (with several exceptions) accumulates derivatives exhibiting aliphatic acyl moieties. A further interesting, but rare, structural variation is the occurrence of phloroglucinols with monoterpenoid substitution (An et al., 2002; Hashida et al., 2008). The structural diversity of phloroglucinols resulted in various pharmacological activities in vitro and in vivo. Several representatives have been reported to exert significant antibacterial activities especially against gram-positive bacteria (Rocha et al., 1995; Shiu and Gibbons, 2006). Also, a moderate to strong cytotoxicity against different tumor cell lines has been observed (Hashida et al., 2008; Hu and Sim, 1999; Momekov et al., 2008). Further derivatives have been proven to exhibit anti-oxidative effects (Heilmann et al., ⇑ Corresponding author. Tel.: +49 9419434759; fax: +49 9419434990. E-mail address: [email protected] (J. Heilmann). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.11.014

2003). Hyperforin, the main acylphloroglucinol of St. John’s wort (Hypericum perforatum L.), has been reported to show inhibition of key enzymes of the arachidonic acid cascade (Albert et al., 2002) or transcription factors (Kraus et al., 2010). Moreover, it is supposed to contribute to the antidepressant efficacy of St. John’s wort extracts (Butterweck, 2003). Recently, a potent anti-angiogenic activity of hyperforin has been described (Schempp et al., 2005) and derivatives with higher stability have been semi-synthesized and tested (Martínez-Poveda et al., 2010). The aim of the present study was the identification of acylphloroglucinols from Hypericum empetrifolium (Hypericaceae) and to evaluate their anti-proliferative in vitro activity on an endothelial cell line. This in vitro activity is likely a first hint for characterization of an anti-angiogenic activity. The most active compound was also tested due to its ability to reduce cell migration in a migration (scratch) assay. H. empetrifolium is primarily found in Turkey and Greece and used in the traditional medicine of both countries. Tuzlacy (2006) reported on the treatment of kidney stones and gastric ulcers in Turkey, whereas Vokou et al. (1993) described the external use as a wash to speed wound-healing, heal scalds, and treat outbreaks of herpes. An extract of H. empetrifolium has been reported to exert analgetic and anti-inflammatory activity (Trovato et al., 2001). Consequently, Crockett et al. (2008) isolated two acylphloroglucinols (7, 8) with moderate to potent in vitro activity against COX-1, COX-2 or 5-LOX. A following phytochemical investigation on the title plant focused on the presence of hypericin derivatives (Alali et al., 2009).

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S. Schmidt et al. / Phytochemistry 77 (2012) 218–225

As several members of the acylphloroglucinol family show a free hydroxyl in the vicinity of the acyl moiety our approach for the systematic identification and isolation of these compounds was guided by 1H NMR. The protons of the hydroxyl groups, which are involved into a strong hydrogen bond with the acyl substituent are characteristically downfield shifted to dH P 11 ppm and thus easily detectable in a fraction. 2. Results and discussion All compounds 1–9 (Fig. 1) were isolated by detection of their downfield shifted proton signals (dH > 11 ppm) except compound

9" 10"

HO 15 14

12

9

5

11

16

1

4"

8"

2"

HO

1'

2

OH

3

OH

H

4

4"

13

1"

2"

12 16

9

7

11

1 3

8

10 9

7

O

5

OH 4'

3

1'

HO

2' 3'

OH

8 11

9

OH

O

1

6

7 OH O

HO OH

OH

6"

10"

O

5

4'

5"

5 OH O

15

O

3'

8" 7" 3"

6

2'

OH

11

HO

OH

O

O

9"

HO

O 5'

3'

O

O

7"

2'

1'

OH

1"

3"

O 4'

1 3

7

6"

5"

6 (dH 9.72 ppm) in 1H NMR spectrum and isolated as viscous oils. The HREIMS spectrum of 1 revealed a [M]+ at m/z 468.3237 pointing to a molecular formula of C30H44O4 (calcd. 468.3240). The 13C NMR spectrum showed 30 carbon signals, which could be sorted by HSQC into eight methyl, six methylene, six methine, and 10 quaternary carbons. The presence of a phloroglucinol skeleton was deduced from the carbon shift values of C-1–C-6 (Table 1). Among these signals, three downfield shifted carbons dC 163.9 ppm (C-3), 160.6 ppm (C-5), 157.6 ppm (C-1) substantiated the trihydroxylation. Two quaternary carbons (dC 107.7 ppm C-2; 106.3 ppm C-4) pointed to substitution with aliphatic side chains and one signified a methine at C-6 (dC 98.0 ppm; dH 6.06 ppm, s).

O Fig. 1. Structures of compounds 1–9.

OH

OH 5' 1'

2' 3'

O

4'

OH 5' 1'

8 OH O

2'

3' 4'

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S. Schmidt et al. / Phytochemistry 77 (2012) 218–225

Table 1 13 C NMR data of compounds 1–9 (150 MHz, 298 K, in CDCl3). C

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 10 20 30 40 50 100 200 300 400 500 600 700 800 900 1000

157.6 107.7 163.9 106.3 160.6 98.0 21.6 121.7 139.5 39.6 16.2 26.3 123.6 132.0 25.6 17.7 211.4 38.8 19.5 19.6

157.6 108.4 163.7 106.3 160.5 98.1 21.6 121.7 139.5 39.6 16.1 26.3 123.6 132.0 25.6 17.7 211.3 45.3 26.9 11.7 17.0 134.0 120.2 26.8 42.5 24.4 30.8 23.3 86.1 24.4 24.0

162.6 109.8 159.0 104.8 160.1 99.5 22.4 121.9 139.3 39.6 16.2 26.3 123.6 131.9 25.6 17.6 210.6 39.2 19.2 19.2

162.5 109.8 159.1 105.3 160.1 99.5 22.4 121.9 139.2 39.6 16.2 26.3 123.6 131.9 25.6 17.6 210.4 45.9 26.8 11.9 16.7 134.0 120.4 26.6 44.1 24.2 30.9 23.3 84.8 24.2 24.1

160.7 105.5 164.9 105.7 161.5 92.9 21.5 121.7 139.6 39.7 16.2 26.3 123.6 132.0 25.6 17.7 210.6 45.5 26.4 11.7 17.8 49.5 85.5 37.3 44.7 27.9 27.3 47.7 19.7 19.0 14.0

163.1 104.4 164.5 95.1 164.5 95.1 65.0 118.6 139.1 25.8 18.2

159.9 104.1 162.6 105.6 160.6 95.4 21.6 121.4 140.1 39.6 16.2 26.2 123.5 132.1 25.6 17.6 210.5 39.2 19.2 19.2

159.9 104.8 162.6 105.6 160.7 95.4 21.6 121.4 140.1 39.6 16.2 26.2 123.5 132.1 25.6 17.7 210.3 45.9 26.9 11.9 16.6

160.3 104.8 163.5 105.5 161.7 95.5 29.1 77.3 151.0 32.1 109.2 26.4 123.7 132.2 25.6 17.7 210.4 45.9 26.9 11.9 16.6

134.1 120.2 26.8 42.6 24.5 30.9 23.3 86.1 24.3 23.9

134.0 120.4 26.6 44.1 24.2 30.9 23.3 84.8 24.3 24.1

209.9 45.9 26.8 11.9 16.5

The 13C and 2D NMR data (COSY, HSQC and HMBC) displayed typical signals and correlations of a geranyl side chain (C-7–C-16) and an isobutyryl group (C-10 –C-40 , Table 1). Residual 1H and 13C signals showed the characteristic shifts and signal pattern of a limonene substituent (Tables 1 and 2, Aggarwal et al., 2002), except one aliphatic quaternary carbon (dC 86.1 ppm, C-800 ), which is shifted downfield due to linked oxygen. Thus, a connection of the C-800 in limonene via one hydroxyl group of the acylphloroglucinol can be deduced. The attachment of the geranyl group to C-4 was determined based on HMBC correlations as protons of the methylene group H-7 (dH 3.38 ppm, d, J = 7.4 Hz) showed long-range correlations to C-4 (dC 106.3 ppm) and C-3 (dC 163.9 ppm). A proton singlet at dH 13.91 ppm belonging to a hydrogen bonded proton showed HMBC correlations to C-3 as well as to the carbonyl carbon (dC 211.4 ppm, C-10 ) of the acyl moiety. Consequently, the isobutyryl side chain must be sited at meta-position to the geranyl substituent. As the limonene substituent in 1, but also in 2–4 showed no HMBC correlations to the acylphloroglucinol core its position has to be deduced from comparison of the 1H shifts of the protons belonging to the hydroxyl groups at C-1/C-3/C-5, their HMBC correlation to the respective carbons as well as NOE interactions of the limonene to the different side chains in the ROESY spectrum. According to the 1H/13C data of compound 7 (Tables 1 and 3) and their unambiguous assignment by 2D spectra, HO at C-5 is significantly upfield shifted (dH 5.97 ppm, bs) in comparison to HO at C-1 (dH 8.31 ppm, bs). As shown for compound 8 (Tables 1 and 3), this is irrespective of the variation in the neighbored aliphatic acyl moiety (dH 5.96 ppm, bs vs. dH 8.20 ppm, bs). In compound 1, the proton resonating in the downfield region vanished strongly indicating the substitution of the limonene at C-1. The residual proton resonating at dH 6.04 ppm (s) showed a HMBC correlation to C-5 (dC 160.6 ppm), which resonated at identical shift value as in the unsubstituted 7 (Table 1). In contrast, C-1 is shifted upfield

(+2.3 ppm) due to the terpenoid substitution. This confirmed the substitution at C-1 and accordingly NOE interactions in the ROESY showed that 1 can be unambiguously determined to be 4-geranyl1-O-(p-menthen-800 -yl)-2-(20 -methylpropionyl)-phloroglucinol, which was named empetrifelixin A. Due to its HREIMS spectrum which showed an increase by 14 mass units to m/z 482.3389 referring to a molecular formula of C31H46O4 (calcd. for 482.3396), compound 2 might differ from 1 only in an additional methylene group. In the 1H and 13C NMR spectra, the downfield-shifted methine carbon (dC 45.3 ppm, C-20 ) and its corresponding proton (dH 3.87 ppm, sext, J = 6.7 Hz) suggested together with additional signals of a methylene group (dC 26.2 ppm, dH 1.75 ppm (m)/1.42 ppm (m), each 1H) the variation in the acyl side chain to be a 2-methylbutyryl instead of a isobutyryl group (Tables 1 and 2). This was confirmed by comprehensive 2D NMR analysis. Hence, compound 2 was identified due to its spectroscopic data as 4-geranyl-1-O-(p-menthen-800 -yl)-2-(20 methylbutyryl)-phloroglucinol and named empetrifelixin B. The molecular formula of compounds 3 and 4 were determined to be C30H44O4 and C31H46O4, indicating that both are positional isomers of 1 and 2, respectively. This was confirmed by very similar 1H NMR (Table 1), 13C NMR (Table 2) and 2D NMR data (1H, 13C HSQC, 1H, 13C HMBC and 1H, 1H COSY). The main difference was the presence of a downfield shifted HO- in both 1H NMR spectra (dH 7.57 and 7.61 ppm, bs each) which was correlated to C-3 (dC 159.0 and 159.1 ppm) in HMBC. Additionally, both methyl groups (H3-900 , H3-1000 ) of the limonene substituent in 3 and 4 showed (in contrast to 1 and 2) NOE interactions with H2-7 (dH 3.34 ppm, 2H each, 3 and 4) and H-8 (dH 5.16 ppm, 1H each, 3 and 4) of the geranyl side chain. As the hydrogen bonded HO proton at C-1 is also present (dH 12.14 and 12.12 ppm, each bs, each 1H, 3 and 4), the limonene is consequently linked to C-5 (dC 160.1 ppm). Thus, compound 3 was identified as 4-geranyl-5-O-(p-menthen-800 -yl) -2-(20 -methylpropionyl)-phloroglucinol and named as empetrifelixin C. Likewise, compound 4 is 4-geranyl-5-O-(p-menthen-800 -yl)-2-(20 -methylbutyryl)-phloroglucinol and named empetrifelixin D. For further characterization of the limonene substituted acylphloroglucinols 1–4 also their CD spectra were recorded and deposited in the Supplementary material. The HREIMS of 5 established its molecular formula as C31H46O4 (found 482.3396 [M]+, calcd. 482.3396) and thus it has the same molecular weight compared to empetrifelixin B. The 1H and 13C NMR data (Tables 1–3) of the acylphloroglucinol core and the geranyl side chain were also very similar. In contrast, the residual 10 carbons resonating in the 13C spectrum, two quaternary (dC 49.5 ppm, C-100 ; 47.7 ppm, C-700 ), two methine (dC 44.7 ppm C-400 ; dC 85.5 ppm, C-200 ), three methylene (dC 37.3 ppm, C-300 ; 27.9 ppm, C-500 ; 27.3 ppm, C-600 ), three methyl (dC 19.7 ppm, C-800 ; 19.0 ppm, C-900 ; 14.0 ppm, C-1000 ) carbons, pointed to a different monterpene as substituent and showed the typical bornane signals except for C-200 , which is downfield shifted due to binding to an oxygen atom. The structure elucidation of 5 as 1-O-(bornan-200 yl)-4-geranyl-2-(20 -methylbutyryl)-phloroglucinol was done by extensive 1D and 2D NMR measurements applying the same strategy as pointed out for compounds 1–4. Especially helpful for confirmation of the bornane substituent linkage to the hydroxyl group at C-1 were the NOE interactions of H-20 (dH 3.96 ppm, sext, J = 6.8 Hz) with H2-500 (dH 1.80 and 1.25 ppm, m), H2-600 (dH 2.10 and 1.42 ppm, m), H3-900 (dH 0.96 ppm, s) and H3-1000 (dH 0.96 ppm, s) of the bornane substructure. Compound 5 was termed empetrikajaforin. Acylphloroglucinols 6–8 have been isolated previously (Bohlmann and Suwita, 1978, 1979; Rios and Delgado, 1992) for example from Helichrysum species (Asteraceae, compound 6) or Esenbeckia (Rutaceae, compounds 7 and 8). Compounds 7 and 8 have been previously isolated by Crockett et al. (2008) from H.

221

S. Schmidt et al. / Phytochemistry 77 (2012) 218–225 Table 2 1 H NMR data of compounds 1–4 (600 MHz, 298 K, in CDCl3, d in ppm, J in Hz). C

1

2

6 7 8 10 11 12 13 15 16 20 30

6.06 3.38 5.28 2.07 1.80 2.10 5.05 1.67 1.59 3.95 1.17

40 50 200 300

1.17 (3H, d, 6.7) 5.37 1.89 2.01 2.25 1.35 1.86 1.99 1.65 1.38 1.45

400 500 600 700 900 1000 1-OH 3-OH 5-OH

(1H, (2H, (1H, (2H, (3H, (2H, (1H, (3H, (3H, (1H, (3H,

(1H, (m) (m) (1H, (m) (m) (2H, (3H, (3H, (3H,

s) d, 7.4) t, 6.7) m) s) m) t, 6.6) s) s) sept, 6.7) d, 6.7)

6.06 3.38 5.29 2.07 1.82 2.10 5.05 1.67 1.59 3.87 1.42 1.75 0.85 1.12 5.37 1.88 2.01 2.25 1.35 1.86 1.98 1.65 1.38 1.44

bs)

m)

m) s) s) s)

13.91 (s) 6.04 (s)

3 (1H, (2H, (1H, (2H, (3H, (2H, (1H, (3H, (3H, (1H, (m) (m) (3H, (3H, (1H, (m) (m) (1H, (m) (m) (2H, (3H, (3H, (3H,

4

s) d, 7.3) t, 7.2) m) s) m) t, 6.7) s) s) sext, 6.7)

6.21 3.34 5.16 2.07 1.81 2.11 5.05 1.67 1.59 3.88 1.16

(1H, (2H, (1H, (2H, (3H, (2H, (1H, (3H, (3H, (1H, (3H,

s) d, 6.9) t, 6.6) m) s) m) t, 6.6) s) s) sept, 6.7) d, 6.7)

t, 7.4) d, 6.8) bs)

1.16 (3H, d, 6.7) 5.38 (1H, bs) 1.91 (m) 2.07 (m) 2.04 (1H, m) 1.37 (m) 1.88 (m) 2.00 (2H, m) 1.65 (3H, s) 1.39 (3H, s) 1.42 (3H, s) 12.14 (bs) 7.57 (bs)

m)

m) s) s) s)

13.92 (s) 6.06 (s)

6.20 (1H, s) 3.34 (2H, d, 6.9) 5.16 (1H, t, 7.0) 2.08 (2H, m) 1.81 (3H, s) 2.11 (2H, m) 5.06 (1H, t, 6.6) 1.67 (3H, s) 1.60 (3H, s) 3.74 (1H, sext, 6.6) 1.40 (m) 1.82 (m) 0.90 (3H, t, 7.3) 1.14 (3H, d, 6.7) 5.38 (1H, bs) 1.91 (m) 2.07 (m) 2.04 (1H, m) 1.38 (m) 1.89 (m) 2.01 (2H, m) 1.65 (3H, s) 1.39 (3H, s) 1.42 (3H, s) 12.12 (bs) 7.61 (bs)

Table 3 1 H NMR data of compounds 5–9 (600 MHz, 298 K, in CDCl3, d in ppm, J in Hz). C

5

6

7

8

9

4 6 7

– 5.75 (1H, s) 3.38 (2H, d, 7.2)

5.92 (1H, s) 5.92 (1H, s) 4.49 (2H, d, 6.7)

– 5.83 (1H, s) 3.38 (2H, d, 6.9)

– 5.82 (1H, s) 3.38 (1H, d, 7.0)

8 10

5.28 (1H, t, 7.2) 2.08 (2H, m)

5.44 (1H, t, 6.7) 1.79 (3H, s)

5.26 (1H, t, 7.1) 2.09 (2H, m)

5.26 (1H, t, 6.9) 2.09 (2H, m)

11

1.81 (3H, s)

1.73 (3H, s)

1.81 (3H, s)

1.82 (3H, s)

12 13 15 16 20 30

2.10 5.05 1.67 1.59 3.96 1.46 1.80 0.87 1.17 4.38 1.12 2.45 1.77 1.25 1.80 1.42 2.10 0.92 0.96 0.96

2.11 5.05 1.67 1.59 3.87 1.17

2.11 5.05 1.68 1.60 3.74 1.40 1.83 0.90 1.16

– 5.86 2.69 3.11 4.35 2.13 2.21 4.91 5.06 2.18 5.14 1.69 1.62 3.77 1.40 1.83 0.91 1.16

40 50 200 300 400 500 600 800 900 1000 1-OH 3-OH 5-OH 8-OH

(2H, (1H, (3H, (3H, (1H, (m) (m) (3H, (3H, (1H, (m) (m) (1H, (m) (m) (m) (m) (3H, (3H, (3H,

14.57 (s) 6.11 (s)

m) t, 6.8) s) s) sext, 6.8)

t, 7.4) d, 6.9) m)

3.70 1.40 1.84 0.91 1.16

(1H, sext, 6.4) (m) (m) (3H, t, 7.4) (3H, d, 6.7)

(2H, (1H, (3H, (3H, (1H, (3H,

m) t, 6.7) s) s) sept, 6.7) d, 6.7)

1.17 (3H, d, 6.7)

(2H, (1H, (3H, (3H, (1H, (m) (m) (3H, (3H,

m) t, 6.6) s) s) sext, 6.6)

t, 7.3) d, 6.7)

(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (2H, (1H, (3H, (3H, (1H, (m) (m) (3H, (3H,

s) m) d, 7.1) t, 7.1) m) m) s) s) m) t, 6.4) s) s) sext, 6.6)

t, 7.4) d, 6.7)

m)

s) s) s) 9.72 (bs) 9.72 (bs)

empetrifolium. As the NMR data of these compounds are not fully recorded in the literature and for good comparison with the NMR data of the new compounds they are also presented in Tables 1 and 3.

8.31 (bs) 11.60 (bs) 5.97 (bs)

8.20 (bs) 11.75 (bs) 5.96 (bs)

7.73 (bs) 12.55 (bs) 7.73 (bs) 2.45 (s)

The 1H and 13C NMR spectra of compound 9 showed similarities to compound 8 except of the signals for the geranyl side chain atoms at -7 through 11-positions (Tables 1 and 3). The signals of C-8/H-8 resonating in the region of a double bond (118–

222

S. Schmidt et al. / Phytochemistry 77 (2012) 218–225

122 ppm/5.20–5.40 ppm) are up-field shifted to the area of hydroxylated methines (77.3/4.35 ppm) indicating the presence of a hydroxyl group at this position. Furthermore, the methyl singlet of C-11/H-11 (16.2/1.82 ppm in 8) was replaced by signals characteristic for a methylidine (109.2/5.06 and 4.91 ppm). As the signal of C-9 is still that of a quaternary carbon, but +10 ppm down-field shifted to 151.0 ppm, the 1H and 13C NMR data gave evidence that modification of the geranyl side chain by hydroxylation at C-8 and displacement of the double bond from C-8/C-9 to C-9/C-11 has been taken place. This was confirmed by extensive 2D NMR analysis and 9 was consequently identified as 3-(2-hydroxy-7-methyl-3-methyleneoct-6-enyl)-1-(20 -methylbutyryl)-phloroglucinol, a derivative of 8 (3-geranyl-1-(20 -methylbutyryl)-phloroglucinol) what is a new natural compound. The compound was named empetrikathiforin. As we were able to detect not only major, but also several minor acylphloroglucinol in the extract and resulting fractions, we recommend the 1H NMR guided approach for the systematic screening of acylphloroglucinols forming a hydrogen bond with the acyl moiety as a routine method complementary to bioactivity-guided principles. H. empetrifolium has been taxonomically placed in section Coridium Spach (Robson, 1981). This section comprises six species, together with H. amblycalyx Coust. & Gand., H. jovis Greuter, H. coris L:, H. asperuloides Czern. & Turcz and H. ericoides L. Phytochemical studies on this section are relatively scarce and revealed the presence of two acylphloroglucinol derivatives from H. amblycalyx (Winkelmann et al., 2003) and six from H. jovis (Athanasas et al., 2004). Up to now, all acylphloroglucinol isolated from this section showed a linear di- or tricyclic structure. Nevertheless, this is the first report of acylphloroglucinols substituted with monoterpenoids in this section and pointed again to the need of a comprehensive phytochemical investigation of all Hypericum species (Crockett and Robson, 2011) to get valid information on the distribution of acylphloroglucinols in the different Hypericum sections to assess their value as chemotaxonomical markers. As isolated amounts of acylphloroglucinols 5, 6 and 9 were not sufficient for the pharmacological testing only compounds 1–4 as well as 7 and 8 were evaluated in the proliferation assay with HMEC-1. All compounds showed remarkable activity in 6–20 lM range (Table 4) with compounds 1 and 4 being the most active (6.5 ± 0.1 and 7.3 ± 0.4 lM, respectively). In a proliferation assay using bovine endothelial cells the most prominent acylphloroglucinol hyperforin showed slightly higher, but comparable activity exhibiting an IC50 value of 2.1 ± 0.7 lM (Martínez-Poveda et al., 2010). Moreover, hyperforin and other structurally related derivatives showed potent anti-angiogenic activity in several in vitro and in vivo systems (Lorusso et al., 2009; Martínez-Poveda et al., 2010; Schempp et al., 2005) showing that these compounds are interesting anti-angiogenic leads. As hyperforin (Wolfender et al., 2003) and highly complex acylphloroglucinols showed often instability in aqueous solution the search for simpler and more stable

templates with acylphloroglucinol skeleton and anti-angiogenic activity is an interesting challenge. The promising activity of 1 prompted us to test this compound also in a cell migration (scratch) assay. Cell migration is one of the requirements for angiogenesis, the process of blood vessel formation, during tumor growth. Incubation of the HMEC-1 cells with 1, 10 and 20 lM of compound 1 impressively showed (Fig. 2) that after 19 h migration of the cells into the scratched gap is already reduced at the lowest concentration and inhibited at 10 and 20 lM. Further experiments will focus on the quantification of this activity and the evaluation of several further acylphloroglucinols to receive systematic information on structural requirements for potent reduction of migration. 3. Experimental 3.1. General experimental procedures Optical rotations were recorded using a UniPol L1000 polarimeter (Schmidt + Haensch). UV spectra were obtained in MeOH on a Cary 50 Scan spectrophotometer (Varian). The solvents used were of spectroscopical grade (Merck). 1H, 13C NMR, [1H, 1H]-COSY, [1H, 1 H]-NOESY and [13C, 1H]-HMBC/HSQC 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 measured in chloroform-d1 99.8% (Deutero GmbH) and referenced against undeuterated solvent. Shift values (dH and dC) were always given in ppm, coupling constants in Hz. High resolution mass spectra (HREIMS) were measured on a Finnigan MAT SSQ 710 A at 70 eV. HRESIMS was measured on an Agilent 6540 UHD QTOF. CD spectra were acquired on a J-710 spectropolarimeter (JASCO), c = 0.02 mg/ml (MeOH) at room temperature using a quartz cuvette of 1.0 cm. Semi-preparative HPLC separations were performed on a Varian ProStar 210 Solvent Delivery Module equipped with a Varian ProStar 335 Photodiode Array Detector. As RP-18 HPLC columns a Varian Dynamax Pursuit XRs (250  10.0 mm, 5 lm) and a Knauer Eurosphere column (250  16.0 mm, 7 lm) were used. All solvents used were of HPLC grade (Merck). Flash chromatography was performed on an Armen Instrument Spot System, with prepacked normal phase columns: SVP D40–Si60 15–40 lm–90 g, SVF D26 Si60 15–40 lm–30 g (Merck Chimie S.A.S.). Furthermore, LiChroprepÒ RP-18 (25–40 lm, for CC, Merck) and silica gel 60 (63–200 lm for CC, Merck) were also used as stationary phases for the flash chromatography. All solvents used were of p.a. 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 controls. Acylphloroglucinols were detected by fluorescence quenching at 254 nm as well as by dark-blue colored spots at 366 nm and by a brown color after spraying with anisaldehyde/sulfuric acid reagent (anisaldehyde: 2 ml, concentrated sulfuric acid: 10 ml, glacial acetic acid: 16 ml, MeOH: 170 ml). 3.2. Plant material

Table 4 Anti-proliferative activity of acylphloroglucinols from H. empetrifolium in a human microvascular endothelial cell line (mean value ± S.D.). Compound

IC50 (lM)

1 2 3 4 7 8 Xanthohumol

6.5 ± 0.1 8.2 ± 0.3 11.0 ± 1.2 7.3 ± 0.4 21.0 ± 0.4 14.3 ± 1.5 11.4 ± 1.1

The aerial parts of H. 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 is deposited in ATHU (Dept. of Pharmacognosy, Univ. of Athens) with the identification number Skaltsa & Lazari 131-09. 3.3. Extraction and isolation Air-dried and powdered aerial parts of H. empetrifolium Willd. (922 g) were extracted by maceration with petroleum ether (PE,

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Fig. 2. Cell migration assay (see Section 3) with HMEC-1 cells. (A) Negative control (untreated cells), (B) positive control (M199), (C–E) incubation with 1, 10 and 20 lM of 1. Photos were recorded after 19 h using a Carl Zeiss Cell Observer consisting of a Zeiss Axio Observer with Software AxioVision 4.8.1.

1.5 l) yielding 11 g residue after evaporation. The PE-extract was fractionated by flash chromatography on silica gel using a linear gradient of hexane–EtOAc (0% ? 100% EtOAc 0–60 min, 30 ml/ min, detection at 270 nm) as mobile phase. One hundred and thirteen fractions of 20 ml each were obtained. Based on TLC and NMR similarities identical fractions 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 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 per collected single fraction) to give three subfractions (PE-3.1 to -3.3). Further fractionation of PE-3.1 (1.4 g, 0–800 ml) on silica gel with flash chromatography using a hexane–EtOAc gradient (0% ? 10% EtOAc 0–60 min, 10% ? 100% EtOAc 60–70 min, 20 ml/min, 15 ml per collected single fraction) yielded six further subfractions (PE3.1.1 to -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 within 5 min, 100% EtOAc for further 10 min, 20 ml/min, 15 ml per collected single fraction) resulted in four fractions (PE-3.1.2.1 to -3.1.2.4). Final purification of PE3.1.2.2 (28.4 mg, 75–105 ml) with RP-HPLC on a Varian Dynamax Pursuit XRs afforded pure 1 (4.4 mg, tR 20.5 min) and 2 (9.8 mg, tR 24 min) using a MeOH–MeCN gradient (70% ? 98% MeCN 0– 20 min, 98% MeCN 20–40 min, 3 ml/min) and 5 (0.5 mg, tR 35.8 min) which was purified by further RP-HPLC on a Varian Dynamax Pursuit using 100% MeCN (3 ml/min). Isolation of compounds 3 (2.5 mg, tR 26 min) and 4 (2.2 mg, tR 30 min) from PE3.1.3 (193 mg, 810–915 ml) was done on RP-18 material (same conditions as already used for fractionation of PE-3.1.2) yielding four fractions (PE-3.1.3.1 to -3.1.3.4). Further purification of PE3.1.3.2 (23.2 mg, 75–180 ml) was done on a Varian Dynamax Pursuit XRs 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 using a 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 collected single fraction) yielding six fractions (PE-4.1 to -4.6). Fractionation of PE-4.2 (287.2 mg, 940–1480 ml) with flash chromatography on RP-18 with a mixture of MeCN–MeOH 80:20 for 30 min (15 ml/ min, 15 ml per collected single fraction) yielded four subfractions (PE-4.2.1 to -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 6 (0.5 mg, tR 11.8 min). PE-6 (1.3 g, 820–980 ml) was separated with flash chromatography on silica gel using a hexane–EtOAc gradient (0% ? 50% EtOAc 0–80 min; 50% ? 100% EtOAc 80–105 min; washing with 100% MeOH 105–120 ml, 20 ml/min, 20 ml per collected single fraction) to give five fractions (PE-6.1 to -6.5). Fractionation of PE-6.2 (840.1 mg, 1020–1480 ml) with flash-chromatography on RP-18 using MeCN–MeOH 80:20 (v/v) as mobile phase for 30 min (10 ml/min, 10 ml per collected single fraction) resulted in four subfractions (PE-6.2.1 to -6.2.4). PE-6.2.1 (116 mg, 30–40 ml) was chromatographed on a Knauer Eurosphere using a H2O–MeCN gradient (60% ? 98% MeCN 0–30 min, 7 ml/min) to give 7 (19.5 mg, tR 19.5 min), 8 (48.1 mg, tR 21 min) and 9 (2.0 mg, tR 18.5 min). 3.4. Proliferation assay The proliferation assay was performed using a SV-40T transfected human microvascular endothelial cell line (HMEC-1, Ades et al., 1992). Cells were incubated at 37 °C under a 5% CO2/95% air atmosphere at constant humidity. HMEC-1 were seeded in 96-well microplates (100 ll, 1.5  103 cells/well) in Endothelial Cell Growth Medium (ECGM) with 10% FCS, Supplement Mix and Antibiotics (all from Provitro). After 24 h, medium in a reference plate was removed and these cells were stained with crystal violet solution for 10 min serving baseline. Cells in other plates were treated with increasing concentrations of each test compound (solved in DMSO as stock solution). After 72 h incubation, cells were also stained as previously described. After washing with distilled water, 100 ll dissolving buffer was added and absorbance was measured with a Tecan SpectraFluor Plus at 540 nm. The IC50 values ±S.D. in lM were calculated with GraphPad software (from three independent experiments, each concentration in hexaplicates). Xanthohumol was used as positive control. Pure solvent (0.1% DMSO) was used as negative control. 3.5. Cell migration assay Assay was done according the protocol of Koltermann (2007) (modified). HMEC-1 (from passage nine) were seeded in 24-well microplates and grown as monolayers on collagen G until they reach confluence. Afterwards, cells were wounded in a standard

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line across the well with the tip of a micropipette. The wounded monolayers were washed twice with PBS to remove floating cellular debris. Immediately, cells were refed with ECGM containing 10% FCS. Endothelial cells were either left untreated (negative control) or treated with compound 1 in three different concentrations (1, 10 and 20 lM). M199 without FCS was used as a positive control. After 19 h the area of cell-free wound was recorded using a Carl Zeiss Cell Observer consisting of a Zeiss Axio Observer with Software AxioVision 4.8.1. 3.6. Empetrifelixin A (4-geranyl-1-O-(p-menthen-800 -yl)-2-(20 methylpropionyl)-phloroglucinol (1)) Yellow oil (4.4 mg); ½a20 D = +8 (c 0.10, MeOH); UV (MeOH) kmax (log e) 295 (4.01); 1H NMR data in Table 2, 13C NMR spectroscopic data in Table 1; HREIMS m/z 468.3237 [M]+ (calcd. for C30H44O4, 468.3240). 3.7. Empetrifelixin B (4-geranyl-1-O-(p-menthen-800 -yl)-2-(20 methylbutyryl)-phloroglucinol (2)) Yellow oil (9.8 mg); ½a20 D = 22 (c 0.10, MeOH); UV (MeOH) kmax (log e) 295 (4.06); 1H NMR data in Table 2, 13C NMR data in Table 1; HREIMS m/z 482.3389 [M]+ (calcd. for C31H46O4, 482.3396). 3.8. Empetrifelixin C (4-geranyl-5-O-(p-menthen-800 -yl)-2-(20 methylpropionyl)-phloroglucinol (3)) Yellow oil (2.5 mg); ½a21 D = +26 (c 0.10, MeOH); UV (MeOH) kmax (log e) 290 (4.08); 1H NMR data in Table 2, 13C NMR data in Table 1; HREIMS m/z 468.3237 [M]+ (calcd. for C30H44O4, 468.3240). 3.9. Empetrifelixin D (4-geranyl-5-O-(p-menthen-800 -yl)-2-(20 methylbutyryl)-phloroglucinol (4)) Yellow oil (2.2 mg); ½a21 D = +42 (c 0.10, MeOH); UV (MeOH) kmax (log e) 290 (4.15); 1H NMR data in Table 2, 13C NMR data in Table 1; HREIMS m/z 482.3399 [M]+ (calcd. for C31H46O4, 482.3396). 3.10. Empetrikajaforin (1-O-(bornan-200 -yl)-4-geranyl-2-(20 methylbutyryl)-phloroglucinol (5)) Yellow oil (0.5 mg); ½a20 D = +128 (c 0.05, MeOH); UV (MeOH) kmax (log e) 235 (3.82), 280 (3.19) sh; 1H NMR data in Table 3, 13C NMR data in Table 1; HREIMS m/z 482.3396 [M]+ (calcd. for C31H46O4, 482.3398). 3.11. Empetrikathiforin (3-(2-hydroxy-7-methyl-3-methyleneoct-6enyl)-1-(20 -methylbutyryl)-phloroglucinol (9)) Yellow oil (2.0 mg); ½a22 D = +90 (c 0.10, MeOH); UV (MeOH) kmax (log e) 290 (4.25); 1H NMR data in Table 3, 13C NMR data in Table 1; HRESIMS m/z 363.2168 [M+H]+ (calcd for C21H31O5, 363.2166). Acknowledgments Thanks are due to Prof. Dr. T. Constantinidis (University of Athens, Faculty of Biology) for identification of the plant material. Dr. V. Saroglou (University of Athens, Department of Pharmacognosy and Chemistry of Natural Products) is gratefully acknowledged for the exhaustive extraction of the plant material with petrol ether. Thanks go to Dr. E. Ades and Mr. F.J. Candal of 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 measuring the NMR as well as J. Kiermeier and W. Söllner for recording the MS spectra (all Central Analytics of NWF IV, University of Regensburg).

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