Isolation and anticancer, anthelminthic, and antiviral (HIV) activity of acylphloroglucinols, and regioselective synthesis of empetrifranzinans from Hypericum roeperianum

Article abstract of DOI:10.1016/j.bmc.2015.08.028

From the ethno-medicinally used leaves of Hypericum roeperianum we isolated a new tricyclic acylphloroglucinol (1), a new tetracyclic acylphloroglucinol (2), and a new prenylated bicyclic acylphloroglucinol (3) together with four known prenylated (4-7) an

Full text of DOI:10.1016/j.bmc.2015.08.028

Accepted Manuscript
Isolation and anticancer, anthelminthic, and antiviral (HIV) activity of acyl-
phloroglucinols, and regioselective synthesis of empetrifranzinans from Hyper-
icum roeperianum
Serge Alain Tanemossu Fobofou, Katrin Franke, Giuseppina Sanna, Andrea
Porzel, Enrica Bullita, Paolo La Colla, Ludger A. Wessjohann
PII:
S0968-0896(15)30011-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.08.028
BMC 12532
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
18 June 2015
19 August 2015
25 August 2015
Please cite this article as: Fobofou, S.A.T., Franke, K., Sanna, G., Porzel, A., Bullita, E., Colla, P.L., Wessjohann,
L.A., Isolation and anticancer, anthelminthic, and antiviral (HIV) activity of acylphloroglucinols, and regioselective
synthesis of empetrifranzinans from Hypericum roeperianum, Bioorganic & Medicinal Chemistry (2015), doi:
http://dx.doi.org/10.1016/j.bmc.2015.08.028
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Bioorganic & Medicinal Chemistry
Isolation and anticancer, anthelminthic, and antiviral (HIV) activity of
acylphloroglucinols, and regioselective synthesis of empetrifranzinans from
Hypericum roeperianum
Serge Alain Tanemossu Fobofoua , Katrin Frankea, , Giuseppina Sannab , Andrea Porzela, Enrica Bullitab,
Paolo La Collab, and Ludger A. Wessjohanna, 
aDepartment of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany
b Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria, 09042 Monserrato (CA), Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
From the ethno-medicinally used leaves of Hypericum roeperianum we isolated a new
tricyclic acylphloroglucinol (1), a new tetracyclic acylphloroglucinol (2), and a new
prenylated bicyclic acylphloroglucinol (3) together with four known prenylated (4-7) and
three known tetracyclic acylphloroglucinol derivatives (8-10). Structure elucidation was
based on UV, IR, [α]25D, 1D- and 2D-NMR experiments. Furthermore, empetrifranzinans A
(8) and C (9) were synthesized regioselectively in only two steps. The isolated compounds
were evaluated for their cytotoxicity against PC-3 and HT-29 cancer cell lines as well as
antibacterial and anthelmintic activities. They were also tested in cell-based assays for
cytotoxicity against MT-4 cells and for anti-HIV activity in infected MT-4 cells.
Significant anthelmintic activity against Caenorhabditis elegans was exhibited by
compound 7 (3-geranyl-1-(2’-methylbutanoyl)-phloroglucinol), which might provide a new
lead.
Available online
Keywords:
Hypericaceae;
H. riparium
natural products;
madeleinol;
1H NMR profile guided isolation;
acylphloroglucinol synthesis;
empetrifranzinans;
metabolic profiling
2009 Elsevier Ltd. All rights reserved.
1. Introduction
present in the plant.10 Due to the great scientific interest and
economic value associated with H. perforatum, many studies
Plants of the genus Hypericum (Hypericaceae) are used
worldwide as traditional medicine against a multitude of
diseases. In China, H. sampsonii Hance is used for the treatment
of disorders such as backache, burns, diarrhea, snakebite, and
swellings.1 H. laricifolium Juss., with the common name
“Romerillo” has been used in traditional Ecuadorian medicine as
a diuretic and for provoking menstruation.2 In the folk medicine
of Papua New Guinea, leaves of H. papuanum Ridl. are applied
to treat sores.3 In Cameroonian traditional medicine, Hypericum
plants are multipurpose remedies commonly used for the
treatment of tumors, skin infections, epilepsy, infertility, venereal
diseases, gastrointestinal disorders, intestinal worms, and viral
diseases.4,5,6 In Turkey, H. empetrifolium Willd. is used against
kidney stones and gastric ulcers, in Greece as an anthelmintic and
diuretic.7 H. perforatum L. (St. John’s wort), the most popular
species of the genus Hypericum, is well known in Europe and
North America for the treatment of mild to moderate
depression.8,9 It is believed that this multitude of biological
together with other species of the same genus have been carried
out throughout the world.8,11 The most common secondary
metabolites found within the genus Hypericum include
phloroglucinols,7,8,11,12
xanthones,13
naphthodianthrones,14
flavonoids,8,9 and coumarins.15 Acylphloroglucinols, of which
hyperforin from H. perforatum is an outstanding example, are
among the most relevant bioactive compounds isolated from
Hypericum.16,17 Their structures and biological activities have
attracted wide attention in medicinal and synthetic chemistry
fields since the isolation of hyperforin in 1975.17,18,19 They
possess,
among
others,
cytotoxic,17
antidepressant,10
antibacterial,12 and anti-inflammatory activities.7 The phloro-
glucinol core of these compounds is often substituted by prenyl
or geranyl moieties that are susceptible to cyclization and
oxidation processes, affording bi-11,20 or tricyclic11 derivatives as
well as complex caged compounds.21
Previously we have reported dimeric coumarins and
flavonoids from the stem bark of H. riparium.6 As part of this
effects occur through the action of various active compounds
———
 Prof. Dr. Ludger Wessjohann. Tel.: +49-345-5582-1301; fax: +49-345-5582-1309; e-mail: wessjohann@ipb-halle.de
 Dr. Katrin Franke. Tel.: +49-345-5582-1313; fax: +49-345-5582-1309; e-mail: kfranke@ ipb-halle.de

continuing effort to study and compare the metabolomes of
different Hypericum species6,7,8 and to find potential lead
compounds, we have selected the leaves of H. riparium A.Chev.
(synonym of H. roeperianum Schimp. ex. A.Rich according to
The Plant List, http://www.theplantlist.org/, Hypericaceae) for
chemical investigations, which led to the isolation of
acylphloroglucinols 1-10. The biological activities exhibited by
the CHCl3 extract of H. roeperianum as well as its chemical and
chemotaxonomic significance fostered our effort in the isolation,
structure elucidation, and more detailed study of specific
biological activities of compounds 1-10 from H. roeperianum as
well as the regioselective synthesis of compounds 8 and 9.
7"
8"
9"
OH
6"
9"
OH
6"
5"
4"
8"
OHax
7"
9"
5"
H
H
2"
6"
10''
3"
O
1'
4"
1"
6
3"
2"
8"
1"
1"
6
2"
7"
5
3
"
4''
10"
4'
10"
5"
6
HO
O
HO
O
O
1'
HO
O
O
4'
5'
2'
1
1
1
5
4
5
4
2'
2'
1'
2
2
4
3'
3'
2
3
3
3
3
'
OH
O
OH
O
OH O
OH
4'
3
1
2
empetrifranzinan D
madeleinol B
empetriferdinol
madeleinol A
OH
O
OH
O
HO
HO
HO
OH
O
OH
O
OH
O
OH
4
5
empetrikarinol A
empetrikarinol B
OH
3-geranyl-2,4,6-trihydroxybenzophenone
HO
HO
OH
O
OH
O
OH
7
6
3-geranyl-1-(2'-methylbutanoyl)-phloroglucinol
3-geranyl-1-(2'-methylpropanoyl)-phloroglucinol
9"
2"
6"
10''
1"
6
8"
7"
3"
4''
5"
O
1'
O
1'
O
O
O
4'
2'
O
O
5'
5
1
2'
4
2
3'
3'
4'
3
O
OH
O
OH
OH
10
8
9
empetrifranzinan A
empetrifranzinan C
empetrifranzinan B
Figure 1. Structures and trivial names of compounds 1-10, empetriferdinol and empetrikarinol A

2. Material and methods
2.3. Extraction and isolation
2.1. General experimental procedures
The air-dried and powdered leaves (400 g) of Hypericum
roeperianum were sequentially extracted with CHCl3 (3 x 5 l)
and MeOH (3 x 2 l) for one day under shaking to give the
respective extracts, namely the CHCl3 (23.74 g) and MeOH (6.10
g) extracts, after evaporation under reduced pressure. The MeOH
extract from the leaves of H. roeperianum used for UPLC-PDA
analysis, anti-bacterial and anti-HIV assays, was obtained using
an extraction procedure previously described for Hypericum
species.8
Acetonitrile (HPLC grade, LiChrosolv) was obtained from
Merck KGaA, Germany; double distillated water was used for
HPLC analysis. HPLC was performed on a VARIAN PrepStar
instrument equipped with a VARIAN ProStar PDA detector.
Column chromatography was run on silica gel (Merck, 63-200
and 40-63 µm) and Sephadex LH-20 (Fluka), while TLC was
performed on precoated silica gel F254 plates (Merck). Spots were
visualized with a UV lamp at 254 and 366 nm, or by spraying
with vanillin-H2SO4-MeOH followed by heating at 100 °C. UV
spectra were obtained on
a
JASCO V-560 UV/VIS
The CHCl3 extract (23.48 g) from the leaves was subjected to
silica gel column chromatography, eluted with step gradients of
n-hexane-EtOAc and EtOAc-MeOH. Fractions were collected
using a fraction collector (Retriever II). They were combined on
the basis of their TLC profiles into 6 main fractions (Fr. 1A-Fr.
6A): Fr. 1A (170.8 mg), Fr. 2A (12.36 g), Fr. 3A (8.66 g), Fr. 4A
(1.76 g), Fr. 5A (1.9 g), Fr. 6A (81.6 mg). Fr. 2A (12.36 g)
obtained from n-hexane-EtOAc (90:10) was chromatographed on
Sephadex LH-20 column (69 x 3 cm), eluted with CH2Cl2-MeOH
(1:1), to give eight sub-fractions (Fr.2A1-Fr. 2A8).
spectrophotometer. IR (ATR) spectra were recorded using a
Thermo Nicolet 5700 FT-IR spectrometer, in MeOH or CHCl3.
Optical rotation [α]25 was measured using a JASCO P-2000
D
digital polarimeter. 1H and 13C NMR spectra were recorded on an
Agilent DD2 400 NMR spectrometer at 400 and 100 MHz and on
an Agilent VNMRS 600 NMR spectrometer at 600 and 150
MHz, respectively. 2D NMR spectra were recorded on an Agilent
VNMRS 600 NMR spectrometer using standard pulse sequences
implemented in Agilent VNRMJ software. The 1H chemical
shifts are referenced to internal TMS (δ 0.0); 13C chemical shifts
are referenced to internal CDCl3 (δ77.0) and CD3OD (δ 49.0),
respectively.
Fr. 2A3 (1.71 g) was subjected to column chromatography
over silica gel eluted with n-hexane containing increasing
amounts of EtOAc (from 100:0 to 50:50 with 5% increment ) and
100% MeOH to yield 13 fractions (Fr. 2A3a-Fr. 2A3m). A portion
of Fr. 2A3k (79 mg), obtained from n-hexane-EtOAc (80:20), was
purified through a RP18 column (5 µm, 120 x 2 mm, flow rate 17
mL/min, detection 210 nm) by semi-preparative HPLC on a
Varian PrepStar eluted with H2O-MeCN (10 100% MeCN 0-20
min, 100 10% MeCN 20-25 min, 10% MeCN 27-30 min) to
afford compounds 6 (30 mg, tR15.17 min) and 7 (34.2 mg, tR
15.65 min). An initial approach to separate Fr. 2A3c, a mixture of
citran acylphloroglucinols plus other impurities as revealed by
The low resolution electrospray (ESI) mass spectra were
performed on
a
SCIEX API-3200 instrument (Applied
Biosystems, Concord, Ontario, Canada) combined with a HTC-
XT autosampler (CTC Analytics, Zwingen, Switzerland). The
samples were introduced via autosampler and loop injection.
The negative ion high resolution ESI mass spectrum of
compound 3 was obtained from an Orbitrap Elite mass
spectrometer (Thermofisher Scientific, Bremen, Germany)
equipped with an HESI electrospray ion source (spray voltage 4
kV; capillary temperature 275 °C, source heater temperature 40
°C; FTMS resolution 60.000). Nitrogen was used as sheath gas.
The sample solutions were introduced continuously via a 500 μl
Hamilton syringe pump with a flow rate of 5 μl/min. The data
were evaluated by the Xcalibur software 2.7 SP1.
1
the H-NMR profile, by CC or HPLC as described above failed.
Fr. 2A3c (193 mg) was then first separated by silica preparative
thin layer chromatography (PTLC) and eluted with a mixture of
toluene-EtOAc-HOAc (98:2:0.5), to afford two fractions after
extraction and evaporation: Fr. 2A3c1 (Rf = 0.32) and Fr. 2A3c2 (Rf
= 0.5). Fr. 2A3c1 (115.6 mg) was suspended in MeCN and
filtered. The resulting filtrate (53 mg) was further suspended in
MeCN and filtered through a Chromabond C18 ec (Macherey-
Nagel, 1 mL/100 mg), and finally purified through a RP18
column (5 µm, 120 x 2 mm, flow rate 17 mL/min, detection 210
nm) by semi-preparative HPLC on a Varian PrepStar eluted with
H2O-MeCN (30 100% MeCN 0-20 min, 100 30% MeCN 20-
25 min, 30% MeCN 27-30 min) to yield 2 (2.3 mg, tR 16.03 min),
8 (4.6 mg, tR 14.51 min), 9 (6.3 mg, tR 15.76 min), and 10 (4.5
mg, tR 15.11 min).
The negative ion high resolution ESI mass spectra of
compounds 1-2 and 4-10 were obtained from a Bruker Apex III
Fourier transform ion cyclotron resonance (FT-ICR) mass
spectrometer (Bruker Daltonics, Billerica, USA) equipped with
an Infinity cell, a 7.0 Tesla superconducting magnet (Bruker,
Karlsruhe, Germany), an RF-only hexapole ion guide and an
external electrospray ion source (Agilent, off axis spray).
Nitrogen was used as drying gas at 150 C. The sample solutions
were introduced continuously via a syringe pump with a flow rate
of 120 l/h. The data was acquired with 512k data points, zero
filled to 2048k by averaging 16 scans and evaluated using the
Bruker XMASS software (Version 7.0.8).
Fr. 4A (1.76 g), obtained from n-hexane-EtOAc (90:10 and
50:50) and EtOAc-MeOH (100:0), was further chromatographed
on Sephadex LH-20 column (69 x 3 cm), eluted with CH2Cl2-
MeOH (1:1), to give five fractions (Fr. 4A1-Fr. 4A5). Fr. 4A3
(407 mg) was dissolved in MeCN and filtered through a
Chromabond C18 ec (1 mL/100 mg). A portion of the resulting
filtrate was purified using RP18 HPLC (5 µm, 120 x 2 mm, flow
rate 17 mL/min, detection 210 nm) on a Varian PrepStar
instrument and eluted with H2O-MeCN (30 100% MeCN 0-20
min, 100 30% MeCN 20-25 min, 30% MeCN 27-30 min) to
give 1 (1.9 mg, tR 8.04 min), 4 (2 mg, tR 12.29 min), 5 (2.7 mg, tR
13.30 min), and also 6 (3 mg, tR13.77 min) and 7 (3 mg, tR 14.63
min). The remaining portion (67.5 mg) of the filtrate was purified
using RP18 HPLC (10 µm, 250 x 10 mm, flow rate 8.9 mL/min,
detection 210 nm) on the same instrument as above and also
2.2. Plant material
The leaves of Hypericum roeperianum Schimp. ex A.Rich.
were collected in August 2011 at Mount Bamboutos (Mbouda) in
the West Region of Cameroon. The plant was identified as H.
riparium A.Chev. by Mr. Nana Victor, a botanist at the National
Herbarium of Cameroon where a voucher specimen (No.
33796/HNC) is deposited. H. riparium A.Chev is presently
treated as synonym of H. roeperianum Schimp. ex A.Rich. H.
roeperianum is commonly found on Mount Bamboutos and other
highlands in the south of Cameroon.

eluted with H2O-MeCN (30 100% MeCN 0-20 min, 100 30%
MeCN 20-25 min, 30% MeCN 27-30 min) to afford 3 (2.9 mg, tR
13.82 min).
and the residue was chromatographed through a silica gel column
(n-hexane: EtOAc 70:30) to give compound 13 as yellow oil
(2.32 g, 11.04 mmol, 27%, Rf = 0.42 in n-hexane:EtOAc
(70:30)). 1H NMR (CD3OD) δ: 12.22 (brs, 1H), 5.91 (s, 2H), 5.17
(brs, 2 x OH), 3.91 (sext, J = 6.6 Hz, 1H), 1.84 (m, 1H), 1.40 (m,
1H), 1.16 (d, J = 6.6 Hz, 3H), 0.93 (t, J = 7.45 Hz, 3H). 13C NMR
δ: 211.6, 165.40, 165.36, 115.2, 95.9, 46.5, 27.9, 16.9, 12.2.
Positive ion ESI-FTMS: [M-H]- at m/z 209.0818 (calcd. for
2.3.1. Madeleinol A (1), 1-((2R*,4aR*,9aR*)-2,6,8-trihydroxy-
1,1,4a-trimethyl-2,3,4,4a,9,9a-hexahydro-1H-xanthen-5-yl)-2-
methylpropan-1-one: Yellow oil; [α]25D -5.6 (c 0.35, MeOH); UV
(MeOH), λmax (log ε): 292 (2.06) nm; IR (ATR) υmax (cm-1): 3277,
2965, 2935, 2870, 1603, 1504, 1419, 1380, 1233, 1127, 1022,
824, 752, 696, 666; 1H NMR data see Table 1; 13C NMR data see
Table 1; negative ion ESI-FTICR-MS: [M-H]- at m/z 347.1859
-
C11H13O4 , 209.0819).
Synthesis of empetrifranzinan A (8): To 196 mg (1 mmol) of
12 and citral (183 mg, 1.2 mmol) in DMF (10 mL) was added
ethylene diamine diacetate (EDDA, 0.036 g, 0.2 mmol) at room
temperature. The reaction mixture was heated at 100 °C for 10 h.
After completion of the reaction as indicated by ESI-MS and
TLC, the reaction mixture was cooled to room temperature.
Water (30 mL) was added and the mixture was extracted with
ethyl acetate (30 mL x 3). The combined organic extracts were
freed from volatiles in a rotavap in vacuo, and the crude product
(yellowish brownish oil, 397 mg) was purified by column
chromatography on silica gel, eluted with n-hexane:EtOAc (95:5)
to afford 8 as yellow compound (163.4 mg, 0.5 mmol, 50 %, Rf =
-
(calcd. for C20H27O5 , 347.1864).
2.3.2. Empetrifranzinan D (2), 1-(1,9-epoxy-3-hydroxy-6,6,9-
trimethyl-6aS*,7R*,8,9R*,10,10a-hexahydro-6H-
benzo[c]chromene-2-yl)-2-methylbutan-1-one: White yellowish
amorphous compound; [α]25 +28.7 (c 0.27, CHCl3); UV
D
(MeOH), λmax (log ε): 294 (2.30) nm; IR (ATR) υmax (cm-1): 2967,
2924, 2873, 1614, 1577, 1454, 1368, 1230, 1124, 924, 851, 831,
1
793, 757; H NMR data see Table 1;13C NMR data see Table 1;
negative ion ESI-FTICR-MS: [M-H]-at m/z 343.1916 (calcd. for
-
C21H27O4 , 343.1915).
1
0.31 in n-hexane:EtOAc (95:5)). H and 13C NMR see Table S1;
2.3.3. Madeleinol B (3), 1-[3,5,7-trihydroxy-2-methyl-2-(4-
methylpent-3-enyl)chroman-8-yl]-2-methylpropan-1-one: Yellow
oil; [α]25D +6.8 (c 0.10, MeOH); UV (MeOH),λmax (log ε): 291
(1.77) nm; IR (ATR) υmax (cm-1): 3306, 2966, 2928, 2873, 1614,
1511, 1422, 1380, 1234, 1141, 1094, 1048, 997, 827, 756, 698;1H
NMR data see Table 1;13C NMR data see Table 1; negative ion
positive ion HRESI-FTMS: [M+H]+ at m/z 331.1905 (calcd. for
+
C20H27O4 , 331.1904).
Synthesis of empetrifranzinan C (9): To 210.23 mg (1 mmol)
of compound 13 and citral (183 mg, 1.2 mmol) in DMF (10 mL)
was added ethylene diamine diacetate (EDDA, 0.036 g, 0.2
mmol) at room temperature. The reaction mixture was heated at
100 °C for 10 h. After completion of the reaction as indicated by
ESI-MS and TLC, the reaction mixture was cooled to room
temperature. Water (30 mL) was added and the mixture was
extracted with ethyl acetate (50 mL x 3). The combined organic
extracts were freed from solvent in a rotavap in vacuo, and the
crude product (yellow oil, 431.8 mg) was purified by column
chromatography on silica gel, eluted with n-hexane:EtOAc (95:5)
to afford compound 9 as yellow oil (173.9 mg, 0.5 mmol, 50%,
Rf = 0.33 in n-hexane:EtOAc (95:5)). 1H and 13C NMR see Table
S2; positive ion HRESI-FTMS: [M+H]+ at m/z 345.2067 (calcd.
-
ESI-FTMS: [M-H]- at m/z 347.1857 (calcd. for C20H27O5 ,
347.1864).
2.4. Synthesis
Synthesis of 1-(2,4,6-trihydroxyphenyl)-2-methylpropanone
(12): Anhydrous phloroglucinol (11, 10.0 g, 79.30 mmol) was
suspended in nitrobenzene (80 mL). 40 mL of carbon disulfide
(40 mL, CS2) was added at room temperature under stirring.
AlCl3 (48.35 g, 362.61 mmol) was added in two portions. The
reaction mixture was stirred at room temperature for 30 min to
give a pale brownish solution. Isobutyl chloride (10 mL) was
added and the reaction mixture was heated at 65 °C for 21 h to
give a dark mixture, which was poured onto an ice-water bath.
Concentrated HCl was added until pH 1-2 was reached, and the
aqueous phase (400 mL) was filtered and extracted with
chloroform (200 mL x 3). The chloroform of the extract was
evaporated using a rotavap and the resulting residue was
lyophilized to give a crude product (6.36 g), which was
chromatographed through a silica gel column, eluted with n-
hexane: EtOAc (25:75 and 40:60) to give compound 12 as yellow
oil (4.89 g, 24.9 mmol, 31%, Rf = 0.39 in n-hexane: EtOAc
(75:25)). 1H NMR (CD3OD) δ: 5.86 (s, 2H), 4.96 (brs, 2 x OH),
4.01 (sept, J = 6.6 Hz, 1H), 1.16 (d, J = 6.6 Hz, 6H). 13C NMR δ:
211.7, 165.7, 104.6, 95.8, 39.8, 19.6. Positive ion ESI-FTMS:
+
for C21H29O4 , 345.2060).
2.5. Biological assays
2.5.1. Cells and viruses
Cell lines were purchased from American Type Culture
Collection (ATCC). The absence of mycoplasma contamination
was checked periodically by the Hoechst staining method.
Cell lines supporting the multiplication HIV-1 virus were
CD4+ human T-cells containing an integrated HTLV-1 genome
(MT-4). IIIB laboratory strain of HIV-1, were obtained from the
supernatant of the persistently infected H9/IIIB cells (NIH 1983).
+
[M+H]+ at m/z 197.0805 (calcd. for C10H13O4 , 197.0808).
2.5.2. Cytotoxicity assays
Synthesis of 1-(2,4,6-trihydroxyphenyl)-2-methylbutanone
(13): Anhydrous phloroglucinol (5.23 g, 41.47 mmol) was
suspended in nitrobenzene (40 mL). 40 mL of carbon disulfide
(40 mL, CS2) was added at room temperature under stirring.
AlCl3 (22.67 g, 170.02 mmol) was added in two portions. The
reaction mixture was stirred at room temperature for 30 min to
give a pale brownish solution. 2-Methylbutyryl chloride (5g,
41.47 mmol) was added and the reaction mixture heated at 65 °C
for 21 h to give a dark mixture, which was poured onto an ice-
water bath (400 mL). Concentrated HCl was added to reach pH
1-2. The aqueous phase was filtered and successively extracted
with chloroform (250 mL x 3) and ethyl acetate (250 mL x 3).
These unified extracts were freed from solvent using a rotavap
The human prostate cancer cell line PC-3 and the colon cancer
cell line HT-29 were maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 1% L-alanyl-L-
glutamine (200 mM) and 1,6% hepes (1 M). ca. 5 x 102 PC-3
cells and ca. 1.5 x 103 HT-29 cells were seeded overnight into
96-well plates and exposed to a serial dilution of each compound
(10 µM and 10 nM) and extract (50 and 0.50 µg/mL) for three
days. Cytotoxicity was determined utilizing modified XTT
method (0.25 mg/mL XTT, 6.5 µM PMS) as described by
Scudiere et al.22
Exponentially growing MT-4 cells were seeded at an initial
density of 1 x 105 cells/mL in 96-well plates in RPMI-1640

medium, supplemented with 10% fetal bovine serum (FBS), 100
units/mL penicillin G and 100 μg/mL streptomycin. Cell cultures
were then incubated at 37 °C in a humidified, 5% CO2
atmosphere, in the absence or presence of serial dilutions of test
compounds. Cell viability was determined after 96 hrs at 37 °C
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) method.23
substituted
(2-methylpropanoyl)phloroglucinol
nucleus,11,26
exhibiting a highly deshielded hydrogen-bonded singlet at δ
13.83 (1H, s, 3-OH), a broad signal accounting for one hydroxyl
group at δ 5.86 (1H, bs, 5-OH), one aromatic proton at δ 5.95
(1H, s, H-4), one methine septet at δ 3.77 (1H, sept, J = 7.0 Hz,
H-2’), and two methyl groups at δ 1.15 (6H, d, J = 7.0 Hz, H-
3’/H-4’). Additionally, many signals are observed between 3.46-
0.91 ppm, including signals of one oxygenated methine at δ 3.46
(1H, dd, J = 11.4, 4.0 Hz, H-6”), one methine at δ 1.63 (1H, m,
H-2”), two geminal protons at δ 2.68 (1H, dd, J = 16.2, 5.3 Hz,
H-1”) and δ 2.36 (1H, dd, J = 16.2, 13.2 Hz, H-1”), and three
singlet methyl groups (δ 1.13, C-8”;δ 0.91, C-9”; δ 1.28, C-10”).
The carbon signals corresponding to the acylphloroglucinol
moiety include one carbonyl group at (δ 210.4, C-1’), three
deshielded oxygenated aromatic carbons (δ 165.2, C-3; δ 160.0,
C-5; δ 156.0, C-1), two quaternary aromatic carbons (δ 105.4, C-
2; δ 100.6, C-6), and one aromatic methine (δ 95.6, C-4). An
2.5.3. Antibacterial assays
These assays were performed as described by the European
Committee on Antimicrobial Susceptibility testing.24
2.5.4. Anthelmintic assays
This assay was performed as described by Thomsen et al.25
2.5.5. Anti-HIV-1 assay
inspection of the H NMR and 13C NMR spectra and degree of
1
Activity against HIV-1 was based on the inhibition of virus-
induced cytopathogenicity in MT-4 cell acutely infected with a
multiplicity of infection (m.o.i.) of 0.01. Briefly, 50 μL of RPMI
containing 1x104 MT-4 cells was added to each well of flat-
bottom microtitre trays, containing 50 μL of RPMI without or
with serial dilutions of test compounds. Then, 20 μL of a HIV-1
suspension containing 100 CCID50 were added. After a 4-day
incubation at 37 °C, cell viability was determined by the MTT
method.23
unsaturation of compound 1 suggests a tricyclic ring system
because no additional double bond signal to the benzene ring is
observed. Moreover, a characteristic signal of one oxygenated
quaternary aliphatic carbon is observed in the 13C NMR spectrum
at δ 78.1 (C-3”). The H and 13C NMR signals of compound 1
1
resemble those reported for empetriferdinol [C21H30O5, m/z 362,
[α]25 +12 (c 0.1, MeOH)],27 except for the signals of the acyl
D
side-chain at C-2. The difference of 14 mass units in compound 1
is attributed to the fact that a 2-methylbutanoyl moiety is absent
in 1 and is, instead, replaced by a 2-methylpropanoyl group.
ROESY, COSY, HSQC, and HMBC experiments allowed for the
determination of the tricyclic structure of 1. Key observations in
the HMBC spectrum were the correlation of Me-10” (δ 1.28) to
C-3” (δ 78.1), C-2” (δ 45.8), and C-4” (δ 35.7) and the
correlation of H-2” (δ 1.63) to C-6 (δ 100.6), C-10” (δ 19.7), C-
7” (δ 38.4), C-6” (δ 78.0), C-1” (δ 17.3), C-8” (δ 27.2), C-9” (δ
14.2). Pertinent correlations are observed from H-1” (δ 2.36 and
δ 2.68) to C-7”, C-5, C-6, C-2”, C-3”, and C-1; from H-6” to C-
8” and C-9”; from H-4”(δ 1.82 and δ 2.08) to C-6”, C-5”, C-10”
and C-2”; from H-9” to C-6”, C-2”, C-7”, and C-8”; from H-4 to
C-2, C-3, C-5, C-6 as well as from H-3’ to C-1’, C-2’, and C-4’.
The COSY spectrum (Fig. 2) reveals correlations between H-6”
and H-5”, H-5” and H-4”, H-1” and H-2”, H-2’ and H-4’ as well
as H-2’ and H-3’. ROESY interactions between H-6” and H-2”,
between H-6” and Me-8” as well as between Me-10” and Me-9”
indicate the relative configuration of compound 1. Compound 1,
which is reported herein for the first time, was therefore
characterized to be 1-[(2R*,4aR*,9aR*)-2,6,8-trihydroxy-1,1,4a-
trimethyl-2,3,4,4a,9,9a-hexahydro-1H-xanthen-5-yl]-2-
2.5.6. Linear regression analysis
The extent of cell growth/viability and viral multiplication at
each drug concentration tested was expressed as a percentage of
the untreated controls. Concentrations resulting in 50% inhibition
(CC50 or EC50) were determined by linear regression analysis.
3. Results and discussion
Column chromatography on silica gel and Sephadex LH-20 as
well as semi-preparative HPLC led to the isolation of ten
acylphloroglucinol derivatives (1-10) from the CHCl3 extract of
the leaves of H. roeperianum including one new tricyclic
acylphloroglucinol (1), one new tetracyclic acylphloroglucinol
(2), and one new prenylated bicyclic acylphloroglucinol (3) along
with four known prenylated (4-7) and three known tetracyclic
acylphloroglucinol derivatives (8-10). Their structures were
established on the basis of spectroscopic evidence and
comparison with the literature data. As acylphloroglucinol
derivatives show characteristic deshielded proton signals around
9-14 ppm in the NMR spectrum, the isolation of compounds 1-10
1
was guided by H-NMR metabolite profiles of fractions. The
methylpropan-1-one, trivially named madeleinol
A
(1).
geranylated phloroglucinol derivatives 6 and 7 were isolated as
the major constituents from the leaves of H. roeperianum. These
two compounds highly dominate the UPLC-PDA and H NMR
Madeleinol is a dedication to the principal investigator’s mother
named Madeleine.
1
metabolite profiles (Figures S1, S2, and S23; see supporting
information) of the crude MeOH extract of the leaves of H.
riparium. MeOH was identified as the best solvent of the
solvents tested for extracting the whole metabolome of the plant.
Compound 2 was isolated as a slightly yellowish amorphous
and optically active substance, [α]25D +28.7 (c 0.27, CHCl3). The
HR-ESI-FTICR-MS indicates a quasi-molecular ion peak at m/z
343.1916 ([M-H]-) consistent with the molecular formula
1
C21H28O4, corresponding to 8 degrees of unsaturation. The H
Compound 1 was obtained as an optically active yellow oil,
[α]25D -5.6 (c 0.35, MeOH). Its molecular formula was established
as C20H28O5 by means of HR-ESI-FTICR-MS (347.1859,
[M-H]-), indicating 7 double bond equivalents. The IR spectrum
indicates the presence of hydroxyl (3277 cm-1) and carbonyl
(1602 cm-1) groups. The 13C NMR (Table 1) spectrum of
compound 1 exhibits 20 signals, which were sorted by HSQC
and DEPT into eight quaternary, four methine, three methylene,
and five methyl carbons. The analysis of 1H NMR and 13C NMR
(Table 1) and 13C NMR (Table 1) data of compound 2 are
1
indicative of an acylphloroglucinol derivative.11,26,27,28 The H
NMR exhibits one signal of an aromatic methine singlet (δ 6.03,
H-4) and duplicate signals of one highly deshielded hydrogen-
bonded singlet (δ 13.87, 3-OH),12,27 one methine sextet (δ 3.66, J
= 7.5 Hz, H-2’), one methyl doublet (δ 1.16, J = 7.5 Hz, H-5’),
one methyl triplet (δ 0.96, J = 7.5 Hz, H-4’), and two methine
multiplets (δ 1.84, H-3’A;δ 1.39, H-3’B). These observations are
1
(Table 1) suggests an acylphloroglucinol derivative.11,26 The H
in agreement with the presence of
a
substituted (2-
methylbutanoyl)phloroglucinol derivative.12,27 The 13C NMR
NMR spectrum of compound 1 reveals signals typical of a

spectrum (Table 1) of compound 2 displays signals of 21 carbon
atoms, which were sorted by DEPT and HSQC experiments into
eight quaternary, four methine, four methylene, and five methyl
carbons. The carbon signals corresponding to the
acylphloroglucinol moiety include one carbonyl group (δ 209.7,
C-1’) three deshielded (oxygenated) aromatic carbons (δ 158.2,
C-1; δ 165.8, C-3; δ 162.8, C-5), two quaternary aromatic
carbons (δ 106.0, C-2; δ 106.6, C-6), and one aromatic methine
of one -C-C- bridge in the structure of 2 and enables the linkage
of the 4 rings. The signals of two oxygenated aliphatic carbon
atoms (δ 76.5, C-3” and δ 84.9, C-7”) in the 13C NMR spectrum
and some important HMBC correlations, including from Me-8”
to C-6”, C-7”, and C-9” as well as from Me-10” to C-2”, C-3”,
and C-4”, corroborate the presence of two –C-O-C- bridges in the
molecule. The COSY spectrum shows correlations between H-2’
and H-3’A/B; H-2’ and Me-5’, H-3’A/B and Me-4’, H-1” and H-
2”A/B, H-1” and H-6”, H-4”A/B and H-5”A/B, as well as
between H-5”A/B and H-6”. The 1D and 2D-NMR data of 2 as
well as the key ROESY correlation observed between Me-10”
and Me-4’/Me-5’ tell us that it is a new derivative of
empetrifranzinan B (10, C20H26O4, m/z 330).27 The difference of
14 mass units in compound 2 agrees with the absence of a 2-
methylpropanoyl group, replaced by a 2-methylbutanoyl group in
the structure of 2. Given all the spectroscopic data above,
compound 2 was then characterized as 1-(1,9-epoxy-3-hydroxy-
6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-
1
(δ 98.7, C-4).12,27 The H and 13C NMR data of compound 2 are
similar to those of petiolin K26 and empetrifranzinan A-C (Fig.
1)27 recently reported in the literature. In these interesting
naturally occurring tetracyclic compounds, a menthane moiety is
linked to the acylphloroglucinol core via one -C-C- and two -C-
O-C- bridges, forming a citran moiety.26,27 Signals of a menthane
skeleton are observed in the 1H NMR spectrum (Table 1) of 2 as
one methine broad singlet (δ 2.83, H-1”) and six methine
multiplets (δ 2.20, H-2”A; δ 1.88, H-2”B; δ 1.84, H-4”A; δ 1.49,
H-4”B; δ 1.33, H-5”A; δ 0.88, H-5”B).27 In addition, the 1H NMR
spectrum reveals three methyl groups as singlets (δ 1.09, H-8”;δ
1.53, H-9”;δ 1.44, H-10”). The HMBC spectrum (Fig. 2) of 2
reveals important correlations from H-1” to C-1, C-5, C-6, C-2”,
C-3”, C-5”, C-6”, and C-7” in good agreement with the presence
benzo[c]chromene-2-yl)-2-methylbutan-1-one, which is a new
acylphloroglucinol derivative and named empetrifranzinan D (2),
based on similar compounds empetrifranzinans A-C (8-10).
Table 1.13C NMR (δ) and 1H NMR data [δ, multiplicity, J (Hz)] for compounds 1-3 (CDCl3)
1a
2a
3b
Position
1
2
3
13C
156.0
105.4
165.2
1H
13C
158.2
106.0
165.8
1H
13C
155.7
105.4
165.7
1H
4
95.6 5.95 s
98.4 6.03 s
96.3 5.97 s
5
160.0
162.8
159.8
6
100.6
106.6
97.9
1’
2’
3’
210.4
39.3 3.77 sept (7.0)
19.3 1.15 d (7.0)
209.7
210.4
39.4 3.85 sept (6.6)
19.2 1.18 d (6.6)
45.4 3.66 sext (7.5)
26.8 1.39 m
1.84 m
4’
5’
1’’
19.5 1.15 d (7.0)
12.0 0.96 t (7.5)
17.0 1.16 d (7.5)
27.5 2.83 bs
19.7 1.18 d (6.6)
17.3 2.36 dd (16.2, 13.2)
2.68 dd (16.2, 5.3)
45.8 1.63 m
25.2 2.60 dd (16.5, 6.6)
2.89 dd (16.5, 5.5)
66.4 3.95 dd (6.6,5.5)
2’’
34.8 1.88 m
2.20 m
3’’
4’’
78.1
76.5
37.5 1.49 m
2.84 m
80.7
37.5 1.71 m
1.77 m
37.5 1.82 dd (12.7, 4.0)
2.08 dt (12.7, 3.5)
28.1 1.65 m
1.90 m
5’’
22.0 0.88 m
22.0 2.15 m
1.33 m
6’’
7’’
78.0 3.46 dd (11.4, 4.0)
38.4
46.0 2.04 ddd (7.5, 5.3, 2.2)
84.9
123.4 5.09 t (7.0)
132.6
8’’
9’’
10’’
3-OH
5-OH
27.2 1.13 s
14.2 0.91 s
19.7 1.28 s
13.83 s
24.2 1.03 s
29.6 1.53 s
28.8 1.44 s
13.87 s
25.7 1.69 s
17.6 1.60 s
19.0 1.38 s
13.81 s
5.86 bs
5.43 s
a 13C NMR (100 MHz), 1H NMR (400 MHz)
b 13C NMR (150 MHz), 1H NMR (600 MHz)

8"
9"
7"
9"
OH
6"
5"
H
8" H
9
"
7"
2"
HO
10"
CH3
"
H
2"
6"
4
4"
1"
1"
6
2"
7"
3
3"
3"
4''
8"
10"
CH3
6
5
HO
H
O 4'
1'
O
H
O
HO
H
O
4'
5'CH3
1
5
1
1
1H-1H COSY
HMBC
2
2'
1'
1'
3'
2
3
3'
3
'
OH
O
OH
O
OH
O
4'
ROESY
1
2
3
Figure 2. Selected 2D NMR correlations for compounds 1-3
The structures of the polycyclic compounds 8, 9, and 10 (Fig.
1) were similarly elucidated by comparing their spectroscopic
(including 2D NMR) and physicochemical data with those
disclosed in the literature, and they were respectively identified
to be empetrifranzinan A (8), empetrifranzinan C (9), and
empetrifranzinan B (10), recently reported as antiproliferative
constitutents of H. empetrifolium.27 Acylphloroglucinols
containing a citran moiety in their structures, with a menthane
substructure C- and O-connected to the aromatic ring, have
previously been reported from Guttiferae sensu stricto, isolated
from the genera Hypericum and Clusia.26,27
MeOH),27 whereas for 3 a value of [α]25D +6.8 (c 0.1, MeOH) was
determined. Therefore, compound 3 is presumably a stereoisomer
of empetrikarinol A, which is reported herein for the first time
and named madeleinol B (3).
The structures of remaining known phloroglucinol derivatives
were assigned by comparing their spectroscopic and physical
data with those previously reported in the literature. These
compounds were identified as empetrikarinol B (4) isolated from
H. empetrifolium,27 3-geranyl-2,4,6-trihydroxybenzophenone (5)
isolated from Tovomita krukovii (Guttiferae),31 3-geranyl-1-(2’-
methylpropanoyl)-phloroglucinol (6),7 and 3-geranyl-1-(2’-
methylbutanoyl)-phloroglucinol (7).7 Compound 6 and 7 were
reported as constituents of H. empetrifolium7,12 and H.
punctatum.32
In order to provide additional evidence for the proposed
regiomeric structures (2, 8, 9, and 10) alongside 2D NMR
spectroscopic data and to obtain more material for our biological
activity testing, we performed the total and regioselective
synthesis of compounds 8 and 9 in two efficient steps (Scheme
1). The key synthetic route used provided only one regiomer each
(8 and 9) with 50 % yield. The synthetic strategy involved
Friedel-Craft acylation of phloroglucinol followed by an
ethylenediamine diacetate-catalyzed (EDDA) cyclization by a
domino aldol-type/electrocyclization/H-shift/hetero-Diels-Alder
reaction of acylphloroglucinols and citral or trans,trans-
farnesal.29 This reaction was previously applied for the
regioselective synthesis of acylphloroglucinols bearing citrans,
like the petiolin D regioisomer.29 The spectroscopic data (Tables
S1 and S2, see suppl.) of synthetic compounds 8 and 9 are
identical to those of empetrifranzinan A (8) and empetrifranzinan
C (9) isolated from H. roeperianum (this paper) and also
previously reported from H. empetrifolium.27
Table 2. Cytotoxicity and antiviral activity of compounds
(µM) and MeOH extract (µg/mL) obtained from H.
a
roeperianum leaves (1-10) against HIV-1IIIB
.
MT-4
HIV-1 IIIB
Hypericum
roeperianum
CC50 (µM)b
11.0 µg/mL
41.0
EC50 (µM)c
> 11.0 µg/mL
> 41.0
Leaf extract
1
> 100
84.0
> 100
> 84.0
2
3
45.0
> 45.0
4
69.0
> 69.0
5
> 100
26.7
> 100
> 26.7
6
7
18.7
> 18.7
8
45.0
> 100
40.0
> 45.0
> 100
0.002
9
10
Compound 3 was isolated as an optically active yellow oil,
[α]25 +6.8 (c 0.10, MeOH). Its molecular formula was
Efavirenz
D
a Data represent mean values for three independent determinations. Variation
among duplicate samples (SD) was less than 15%.
determined to be C20H28O5 from its HR-ESI-FTMS, which shows
the base peak at m/z 347.1857 ([M-H]-), consistent with 7 degrees
of unsaturation. The IR spectrum shows the presence of hydroxyl
(3306 cm-1) and carbonyl (1614 cm-1) groups. As in case of 1, the
1H (Table 1) and 13C NMR (Table 1) data of compound 3 are in
b Cytotoxic concentration (CC): Compound concentration (µM) required to
reduce the viability of mock-infected MT-4 cells by 50%, as determined by
the MTT method. Efavirenz is the reference drug. For extracts: µg/mL.
c
Effective concentration (EC): Compound concentration (µM) required to
agreement with the presence of
a (2-methylpropanoyl)-
achieve 50% protection of MT-4 cells from the HIV-1 induced
cytopathogeneticy, as determined by the MTT method. For extracts: µg/mL.
phloroglucinol moiety in the molecule.26,30 Additional signals of a
prenyl chain,27,28 including one methine triplet (δ 5.09, C-6”), one
methylene multiplet (δ 2.15, C-5”), and two methyl singlets (δ
Compounds 1-10 were tested in a cell-based assay against the
human immunodeficiency virus type-1 (HIV-1), using efavirenz
as the reference inhibitor. Cytotoxicity against uninfected MT-4
cells was evaluated in parallel with the antiviral activity. As
reported in Table 2, none of the isolated compounds shows
significant anti-HIV activity at concentrations below those
cytotoxic for exponentially growing MT-4 cells.
1
1.69, C-9”; δ 1.60, C-8”) are revealed in the H NMR spectrum.
This is supported by HMBC correlations (Fig. 2) from Me-8” to
C-6”, C-7”, and C-9” as well as from Me-9” to C-6”, C-7”, and
C-8”. The molecular formula and 1D and 2D NMR data of
compound 3 are similar to those reported for empetrikarinol A,27
except the ROESY spectrum which shows in 3 a key interaction
between H-2” (δ 3.95, dd 6.6, 5.5 Hz) and Me-10” (δ 1.38) in
accordance with an equatorial position of H-2’’. Furthermore,
empetrikarinol A shows an optical rotation of [α]21D -24 (c 0.1,
Compounds 1-10 were also tested for antibacterial activity
against representative human pathogenic Gram negative

(Escherichia coli DSM 1103) and Gram positive (Staphylococcus
aureus DSM 2569, and Enterococcus faecalis DSM 2570)
bacteria. Ciprofloxacin was used as reference compound. None
of the tested compounds shows significant inhibitory activity
(MIC > 1 mg/L). Olympicin A, which differs from compound 7
only by the position of the geranyl group shifted onto the 2-OH
group (in olympicin A) of the phloroglucinol core, showed potent
antibacterial activity.12 The lack of activity of compounds 6 and 7
in comparison to olympicin A could be due to different protocols
and strains used or to the aforementioned structural differences.
(Fig. 4). Compounds 1-10 were found not active at 10 nM.
Compound 7, one of the two major constituents (6 and 7) of the
leaves, shows the highest cytotoxic effects among the tested
acylphloroglucinols (Fig. 3 and 4). An initial explanation or
presumption of the difference of activity observed between the
prenylated compounds 6-7 and 3-4 may be the length of the acyl
chain as (2-methylbutanoyl)phloroglucinols are more potent than
(2-methylpropanoyl)phloroglucinols in the present study.
However, a contrary effect is observed between the two pairs of
citran acylphloroglucinols 8-9 and 2, 10. Another activity-
relevant feature is the prenyl side chain; the loss of a prenyl side
chain can decrease the activity. For instance, the tricyclic
acyphloroglucinol 1, which is derived biosynthetically from
prenylated phloroglucinols, does not cause any growth inhibition
of the cell lines. Cytotoxic and antiproliferative phloroglucinols,
including hyperforin, have been already reported from the genus
Hypericum.17,27,30
Figure 3. Cytotoxic activities of the CHCl3 extract (leaves) and compounds
1-10 against the human prostate cancer cell line PC-3. The results are
expressed as percentage of inhibition ± SD. Compounds were tested at two
different concentrations of 10 µM and 10 nM while extracts were tested at 50
µg/mL and 0.5 µg/mL. Digitonin at 125 µM was used as positive control
(100% growth inhibition). 0.05% DMSO was used as negative control.
Figure 5. Anthelmintic activities of the CHCl3 extract (dried leaves, 500
µg/mL) and compounds 1, 4-7 (100 µg/mL) against Caenorhabditis elegans.
The results are expressed as percentage of death worms ± SD. 2% DMSO (3
± 3 %) was used as negative control while ivermectin 10 µg/mL (99 ± 1 %)
was used as positive control. Thiabendazole is a reference anthelmintic drug.
Some natural phloroglucinol derivatives, including aspidin
and desaspidin from Dryopteris filix-mas (Dryopteridaceae) as
well as kosins from Hagenia abyssinica (Rosaceae), have also
been reported to show anthelmintic properties.25,33,34 One of the
objectives of the United Nations Millennium Development Goals
is to halt or reverse the incidence of infections caused by
neglected tropical diseases like helminths,35 as billions of people
suffer from helminthic diseases worldwide resulting in many
thousands of deaths annually.36 Although some effective
anthelmintic drugs are available, recent treatment failures have
occurred apparently due to the development of genetic resistance
in nematodes.37 Thus, there is a need for new and inexpensive
drugs able to act longer before the resistance sets in. As described
by Thomsen et al.25,37 the non-parasitic nematode Caenorhabditis
elegans can be used as a model organism for inexpensive and
rapid initial screening for the detection of compounds active
against parasitic helminths. Preliminary anthelmintic activities of
the CHCl3 extract from the leaves of H. roeperianum and
compounds 1, 4-7 against the model organism Caenorhabditis
elegans (Bristol N2 wild type strain) were determined in a
modified microtiter plate assay by enumeration of living and
dead nematodes using a microscope.25 At a test concentration of
100 µg/mL, compound 7 shows a nematode death percentage of
37 ± 12% (Fig. 5) while the reference drug thiabendazole exhibits
a percentage of death of 19 ± 11%. Compounds 1, 4-6 are
inactive. They show a percentage of death ranging from 0.7 to
5.1 %. All the tested compounds are inactive at 12.5 µg/mL
Figure 4. Cytotoxic activities of the CHCl3 extract (leaves) and compounds
1-10 against the human colon cancer cell line HT-29. The results are
expressed as percentage of inhibition ± SD. Compounds were tested at two
different concentrations of 10 µM and 10 nM while extracts were tested at 50
µg/mL and 0.5 µg/mL. Digitonin at 125 µM was used as positive control
(100% growth inhibition). 0.05% DMSO was used as negative control.
The CHCl3 extract as well as compounds 1-10 were screened
for cytotoxicity against HT-29 and PC-3 cancer cell lines. The
CHCl3 extract (from the leaves) exhibits cytotoxic activities
indicated by a growth inhibition of the cell lines of 84 and 82%,
respectively, at a concentration of 50 µg/mL. No inhibition was
observed at 0.5 µg/mL. As shown in figure 3, the isolated
compounds 7-10 show weak cytotoxic effects (growth inhibition
of 20-40%) against PC-3 at a tested concentration of 10 µM
while compounds 1-6 are inactive (less than 20% of growth
inhibition). They were all inactive (inhibition of less than 20%) at
10 nM. Compounds 4, 7, 9-10 show weak cytotoxic activities
(growth inhibition of 20-51 %) against the cancer cell line HT-29

(results not shown). The known compound 7, one of the two
major constituents of the extract, may be responsible for the
observed anthelmintic activity of the CHCl3 extract. It hints to a
potential anthelmintic lead structure, which may be structurally
modified on its acyl side chain in order to investigate its structure
activity relationship. An initial observation of the structures of 6
and 7 and their anthelmintic activities allows us to speculate that
longer acyl side chains may give more active compounds.
However, such alterations also enhance the lipophilicity and thus
the bioavailability. Thus, it is yet unclear if the improved
activities are based on better target binding or on better
bioavailability.
moieties were successfully synthetized in only two steps. Their
anthelmintic (1, 4-7), cytotoxic (1-10) and anti HIV (1-10)
activities were evaluated. Compounds 4, 7, 9-10 exhibit weak
cytotoxic effects against cancer cell lines. The crude CHCl3
extract and compound 7 show anthelmintic activity against
Caenorhabditis elegans. This may explain the use of Hypericum
species in Cameroonian traditional medicine against intestinal
worm infections. Even though this is a preliminary screening, the
force of our study lies on the ability of Hypericaceae natural
products to provide yet unexplored structures with the potential
for anthelmintic, cytotoxic or anti-HIV activity. Also,
acylphloroglucinols have significance as chemotaxonomic
markers in Hypericum species.38,39 Based on this and similar
studies on Hypericum, it will be interesting to evaluate the
activity profiles of more species and compare them by
established metabolomics approaches for chemotaxonomic
significance as well as for differences or specificities in bio-
markers and bioactivities.
4. Conclusions
In summary, we isolated and identified new constituents of H.
roeperianum for the first time, namely compounds 1-10, which
include some very minor compounds and three new natural
products. Two acylphloroglucinols (8 and 9) bearing citran
key step
Cl
citral
R
OH
HO
OH
HO
O
O
O
O
EDDA
O
R
DMF
100 °C, 10 h
50%
PhNO2
R
OH
CS2/AlCl3
65 °C, 21 h
OH
OH
Phloroglucinol
(11)
empetrifranzinan A (8) R = CH3
empetrifranzinan C (9) R = CH2CH3
12 R = CH3
13 R = CH2CH3
Scheme 1. Regioselective synthesis of acylphloroglucinol derivatives 8 and 9
.
13. Tanaka, N.; Kashiwada, Y.; Kim, S.–Y.; Sekiya, M.; Ikeshiro, Y.;
Takaishi, Y. Phytochemistry 2009, 70, 1456.
Acknowledgments
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Mass Spectrom. 2009, 23, 885.
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J.–Z.; Wang, H.; Yang, L.–X.; Xu, G. J. Nat. Prod. 2013, 76,
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Pham, H.D.; Giannis, A. Angew. Chem. Int. Ed. Engl. 2007, 46,
5219.
This research was supported by the German Academic
Exchange Service (DAAD) through a grant to Serge Alain
Fobofou Tanemossu. We are indebted to Dr. Jürgen Schmidt for
HR-ESI-MS measurements. We thank Anja Ehrlich for carrying
out the cytotoxic assays and Nicole Hünecke for her assistance in
growing C. elegans. Dr. S.V.T. Sob is acknowledged for
providing financial support for plants collecting expeditions.
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Graphical Abstract
Isolation and anticancer, anthelmintic, and antiviral (HIV)
activity of acylphloroglucinols, and regioselective synthesis
of empetrifranzinans from Hypericum roeperianum
Leave this area blank for abstract info.
S. A. Tanemossu Fobofoua, K. Frankea, G. Sannab, A. Porzela, E. Bullitab, P. La Collab and L. A. Wessjohanna
aDepartment of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany
bDepartment of Biomedical Sciences, University of Cagliari, Cittadella Universitaria, 09042 Monserrato (CA), Italy
HO
O
OH
O
O
O
1H NMR profile guided isolation
Regioselective synthesis
R
R
One step
50 %
OH
OH
H. roeperianum