Tricyclic Acylphloroglucinols from Hypericum lanceolatum and Regioselective Synthesis of Selancins A and B

Article abstract of DOI:10.1021/acs.jnatprod.5b00673

The chemical investigation of the chloroform extract of Hypericum lanceolatum guided by ¹H NMR, ESIMS, and TLC profiles led to the isolation of 11 new tricyclic acylphloroglucinol derivatives, named selancins A-I (1-9) and hyperselancins A and

Full text of DOI:10.1021/acs.jnatprod.5b00673

Article
pubs.acs.org/jnp
Tricyclic Acylphloroglucinols from Hypericum lanceolatum and
Regioselective Synthesis of Selancins A and B
Serge A. T. Fobofou, Katrin Franke,* Andrea Porzel, Wolfgang Brandt, and Ludger A. Wessjohann*
Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany
S
* Supporting Information
ABSTRACT: The chemical investigation of the chloroform
1
extract of Hypericum lanceolatum guided by H NMR, ESIMS,
and TLC profiles led to the isolation of 11 new tricyclic
acylphloroglucinol derivatives, named selancins A−I (1−9)
and hyperselancins A and B (10 and 11), along with the
known compound 3-O-geranylemodin (12), which is
described for a Hypericum species for the first time.
Compounds 8 and 9 are the first examples of natural products
with a 6-acyl-2,2-dimethylchroman-4-one core fused with a
dimethylpyran unit. The new compounds 1−9 are rare
acylphloroglucinol derivatives with two fused dimethylpyran
units. Compounds 10 and 11 are derivatives of polycyclic
polyprenylated acylphloroglucinols related to hyperforin, the
active component of St. John’s wort. Their structures were elucidated by UV, IR, extensive 1D and 2D NMR experiments,
HRESIMS, and comparison with the literature data. The absolute configurations of 5, 8, 10, and 11 were determined by
comparing experimental and calculated electronic circular dichroism spectra. Compounds 1 and 2 were synthesized
regioselectively in two steps. The cytotoxicity of the crude extract (88% growth inhibition at 50 μg/mL) and of compounds 1−6,
8, 9, and 12 (no significant growth inhibition up to a concentration of 10 mM) against colon (HT-29) and prostate (PC-3)
cancer cell lines was determined. No anthelmintic activity was observed for the crude extract.
he scientific interest and economic value of Hypericum
perforatum (St. John’s Wort), a medicinal herb mostly
from plants including various Hypericum species and
accessions.1,2,10,11,18−20 H. lanceolatum is a medicinal plant
occurring in the mountainous region of West Cameroon
(Central Africa).21 Previous studies on this plant revealed the
presence of xanthones, a polyprenylated phloroglucinol
(isogarcinol), and terpenoids.21 In Cameroonian traditional
medicine, Hypericum plants are multipurpose remedies
commonly used for the treatment of tumors, skin infections,
epilepsies, infertility, venereal diseases, gastrointestinal disor-
ders, intestinal worms, and viral diseases.2,21,22 Recently, the
first isolation and structural elucidation of biscoumarins from
Cameroonian H. riparium were reported.2 The aim of the
present study was to isolate and characterize the chemical
constituents of H. lanceolatum and evaluate their biological
activities.
T
used for the treatment of mild to moderate depression, entailed
the investigation of bioactive metabolites from other Hypericum
species.1−3 The most common secondary metabolites found
within the genus Hypericum include phloroglucinols,4−6
xanthones,7 naphthodianthrones,8,9 flavonoids,1,10 and coumar-
ins.11 Acylphloroglucinols, of which hyperforin from H. perfo-
ratum is an outstanding example, are among the most relevant
bioactive compounds isolated from Hypericum.12−14 Their
structures and biological activities have attracted much
attention in the medicinal and synthesis chemistry fields since
the isolation of hyperforin in 1975.13 They possess, among
others, cytotoxic,13 antidepressant,15 antibacterial,6 and anti-
inflammatory activities.5 The phloroglucinol (1,3,5-trihydroxy-
benzene) core of these compounds is often substituted by
prenyl or geranyl moieties that are susceptible to cyclization
and oxidation processes, affording bi-3,16 or tricyclic3 derivatives
as well as complex cage compounds.17 Recently, a revised
structure for adhyperfirin with a C-methylated phloroglucinol
core, named hyperpolyphyllirin from H. polyphyllum, was
reported.1
RESULTS AND DISCUSSION
■
ESIMS, TLC, and particularly 1H NMR-guided fractionation of
the chloroform extract of the leaves of H. lanceolatum led to the
isolation of 11 new acylphloroglucinol derivatives, 1−11, and
the known compound 3-O-geranylemodin (12). The character-
istic deshielded proton signals around δH 9−14 in the 1H NMR
spectrum of acylphloroglucinols are caused by the presence of a
Hypericum lanceolatum Lam. (Hypericaceae) has been
prioritized for chemical investigation as part of a metabolo-
mics-driven isolation project aiming to study and compare the
metabolomes, biosynthesis, and biological activities of preny-
lated aromatics and to find new or potential lead compounds
Received: July 31, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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1
Table 1. H NMR Data [δ, multiplicity, J (Hz)] for Compounds 1−9 (400 MHz, CDCl3)
position
3
1
2
3
4
5
6
7
8
9
5.43, d (10.1) 5.44, d (9.7) 3.81, dd
(5.8, 5.3)
3.80, m
5.41, d (10.1)
5.46, d (10.1) 5.46, d (10.1) 5.55, d (10.1) 5.55, d (10.1)
4
6.59, d (10.1) 6.60, d (9.7) 2.85, dd
(17.1, 5.3)
2.76, dd
6.60, d (10.1)
3.80, t (5.3)
6.43, d (10.1) 6.43, d (10.1) 6.55, d (10.1) 6.55, d (10.1)
(17.1, 5.3)
2.60, dd
(17.1, 5.8)
2.60, dd
(17.1, 5.3)
7
8
5.46, d (10.1) 5.46, d (9.7) 5.43, d (10.1)
6.66, d (10.1) 6.66, d (9.7) 6.66 d (10.1)
5.43, d (10.1)
6.67, d (10.1)
5.09, d (3.1) 5.09, d (3.1) 4.85, s
5.83, d (3.1) 5.83, d (3.1)
4.85, s
2.87, dd
(17.1, 5.3)
2.65, dd
(17.1, 5.3)
11
12
13
14
2′
1.49, s
1.49, s
1.44, s
1.44, s
1.49, s
1.50, s
1.44, s
1.44, s
1.39, s
1.39, s
1.48, s
1.53, s
1.51, s
1.44, s
1.12, s
1.53, s
1.51, s
1.44, s
1.12, s
1.59, s
1.57, s
1.39, s
1.30, s
1.59, s
1.58, s
1.39, s
1.30, s
1.40, s
1.40, s
1.48, s
1.43, s
1.43, s
1.38, s
1.43, s
1.43, s
1.33, s
3.84, sept
(6.6)
3.74, sext
(7.0)
3.81, sept (6.8)
3.72, sext (7.0)
3.77, sext (7.0)
3.82, sept
(6.6)
3.72, sext
(6.6)
3.76, sept
(7.0)
3.64, sext
(6.6)
3′
1.19, d (6.6) 1.86, m
1.40, m
1.17, d (6.8)
1.85, m
1.86, m
1.19, d (6.6) 1.86, m
1.41, m
1.21, d (7.0) 1.86, m
1.44, m
1.41, m
1.40, m
4′
5′
7-OH
1.19, d (6.6) 0.91, t (7.5) 1.17, d (6.8)
1.16, d (7.0)
0.91, t (7.5)
1.15, d (7.0)
0.91, t (7.5)
1.16, d (7.0)
1.19, d (6.6) 0.92, t (7.5) 1.21, d (7.0) 0.93, t (7.0)
1.17, d (6.6)
8.30, s
1.19, d (6.6)
8.97, s
8.30, s
9.10, s
14.70, s
8.98, s
8-OH
9.10, s
9-OH 14.13, s
14.18, s
14.13, s
14.16, s
14.30, s
14.75, s
15.31, s
15.35, s
free hydroxy group hydrogen bonded to the carbonyl group of
the acyl moiety. This feature is readily visible in metabolite
profiles of fractions and can be used for H NMR-guided
hydroxy-2,2,8,8-tetramethyl-2H,8H-pyrano[2,3-f ]chromen-6-
yl)-2-methylpropan-1-one.
1
A difference of 14 atomic mass units between 1 (C20H24O4)
and 2 (C21H26O4) was derived from MS and NMR data. A
comparison of the 1D and 2D NMR data of 2 with those of 1
shows that the structural differences are restricted to the acyl
side chain (Tables 1 and 2). HMBC correlations of Me-4′ with
C-3′ (δC 26.8) and C-2′ (δC 46.0) and HMBC correlations of
Me-5′ with C-1′ (δC 210.5) as well as with C-3′ and C-4′
clearly showed the replacement of the 2-methylpropanoyl
substituent in 1 by a 2-methylbutanoyl side chain in 2. Hence,
the structure of compound 2 (Figure 1), selancin B, was
elucidated as 1-(5-hydroxy-2,2,8,8-tetramethyl-2H,8H-pyrano-
[2,3-f ]chromen-6-yl)-2-methylbutan-1-one.
isolation to reduce replication.23 The new compounds were
trivially named selancins A−I (1−9) and hyperselancins A and
B (10 and 11). The assigned compound names are based on
the Cameroonian word “Se”, which is used for Hypericum in the
local language of Mbouda and also means “God” in the
Bamendjida language. “Lancin” is derived from the source plant
species H. lanceolatum, whose leaves are lanceolated.
The molecular formula of 1 was established as C20H24O4
from the 13C NMR and HRESI-FTMS, which shows the
deprotonated molecular ion at m/z 327.1601 [M − H]−,
corresponding to nine indices of hydrogen deficiency. The
NMR data (Tables 1 and 2) are similar to those of
octandrenolone, a tricyclic acetophloroglucinol derivative,
isolated from Melicope erromangensis (Rutaceae), bearing two
nonsymmetrical fused 2,2-dimethyl-2H-pyran rings.24,25 The
main difference between the two compounds is that the acyl
side chain in 1 is a 2-methylpropanoyl moiety, while an acetyl
group is present in octandrenolone.25 The characteristic signals
The regioselective total synthesis of compounds 1 and 2
(racemic) was performed in two efficient steps (Scheme 1)
starting with phloroglucinol. The synthetic routes afforded only
one regiomer each (1 and 2) in 80% and 85% yield,
respectively. The synthesis strategy involved Friedel−Crafts
acylation of phloroglucinol followed by an ethylenediamine
diacetate (EDDA)-catalyzed double condensation of
acylphloroglucinols with 3-methyl-2-butenal. The NMR and
MS data of synthetic compounds 1 and 2 (racemic) are
identical to those of natural selancins A (1) and selancin B (2)
isolated from the leaves of H. lanceolatum. In order to assign the
C-2′ absolute configuration of 2, the sign of its optical rotation
was compared with that of synthetic compound (S)-2,
synthesized from (+)-(S)-2-methylbutanoic acid (Scheme 2).
1
of the 2-methylpropanoyl group are observed in the H NMR
spectrum as one methine septet at δH 3.84 (1H, sept, J = 6.6
Hz, H-2′) and two equivalent methyl groups at δH 1.19 (6H, d,
J = 6.6 Hz, Me-3′/Me-4′). The structure assigned to compound
1 (Figure 1) was confirmed based on HMBC correlations from
9-OH to C-10, C-9, and C-8a; from H-8 to C-9, C-8a, C-4b,
and C-6; from H-7 to C-8a, C-6, and C-13/C-14; from H-4 to
C-10a, C-4b, C-4a, and C-2; from H-3 to C-4a, C-2, and C-11/
C-12; and from H3-3′/H3-4′ to C-1′ and C-2′ as well as from
H-2′ to C-1′ and C-3′/C-4′. The COSY spectrum reveals three
spin systems (H-7/H-8, H-3/H-4, and H-2′/Me-3′/Me-4′),
whereas five cross-peaks are displayed in the ROESY spectrum
(H-7/H-8, H-3/H-4, H-2′/Me-3′/Me-4′, H-7/Me-13/Me-14,
and H-3/Me-11/Me-12). The structure of compound 1,
selancin A, was therefore unambiguously defined as 1-(5-
The specific rotations of (S)-2 and 2 were found to be [α]25
D
+2 (c 0.24, CHCl3) and [α]26 +13 (c 0.22, CHCl3),
D
respectively. This, supported by matching the experimental
electronic circular dichroism (ECD) spectra of natural
compound 2 and synthetic (S)-2 (Figure S48, Supporting
Information), confirms the 2′S configuration of the natural
product 2.
A variation of 18 atomic mass units (addition of H2O)
between 1 (C20H24O4) and 3 (C20H26O5) was derived from MS
B
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Figure 1. Chemical structures of compounds 1−12. a2′S configuration tentatively assigned as in 2 due to the same biosynthetic origin.6,27,38−40 b7R
configuration tentatively assigned as in 8.
Scheme 1. Regioselective Synthesis of Acylphloroglucinol Derivatives 1 and 2
Scheme 2. Total Synthesis of (+)-(S)-2
analysis. This difference was explained by the 1H NMR
spectrum (Table 1), which shows the replacement of the two
doublets (H-3 and H-4) of a 2,2-dimethyl-2H-pyran moiety in
1 by the three signals (δH 3.81, dd, J = 5.8, 5.3, H-3; δH 2.85,
dd, J = 17.1, 5.3, H-4a; and δH 2.60, dd, J = 17.1, 5.8, H-4b) of a
3-hydroxy-2,2-dimethyldihydropyran moiety in 3.26 The
connection of the 3-hydroxy-2,2-dimethyldihydropyran moiety
via C-4−C-4a was evident from HMBC correlations of H-4a/
H-4b to C-10a, C-4b, C-4a, C-3, and C-2 as well as from H-3 to
C-4a, C-11, and C-12. ROESY interactions were observed
between H-3/Me-11/Me-12 and H-4a/Me-11 as well as a weak
association between H-4b/Me-12. The ROESY interaction
between H-3/Me-11/Me-12 and the coupling constant of H-3
(δH 3.81, dd, J = 5.8 and 5.3 Hz) required 3-OH to be axial.
D
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Figure 2. ECD spectra (7R, 2′S: red, 7S, 2′R: blue) of 5 in comparison to the experimental one (black). Similarity factor = 0.8963 at 24 nm shift.
However, it is not possible to determine the C-3 absolute
configuration based on the axial orientation of 3-OH. On the
basis of the above spectroscopic evidence the structure of 3,
selancin C, was defined as 1-(5,9-dihydroxy-2,2,8,8-tetramethyl-
9,10-dihydro-2H,8H-pyrano[2,3-f ]chromen-6-yl)-2-methylpro-
pan-1-one.
and C-10 and from H-8a/H-8b to C-6. ROESY interactions
were observed between H-7/Me-13/Me-14, which required 7-
OH to be axial. On the basis of the above spectroscopic
evidence the structure of 5, selancin E, was defined as 1-(3,5-
dihydroxy-2,2,8,8-tetramethyl-3,4-dihydro-2H,8H-pyrano[2,3-
f ]chromen-6-yl)-2-methylbutan-1-one. The absolute configu-
ration of selancin E (5) was investigated by comparing its
experimental and calculated ECD spectra. The calculations
indicated that compound 5 has a 7R configuration with axial
orientation of the 7-OH group (Figures 2 and S39, Supporting
Information). However, it was not possible to unambiguously
distinguish between (7R, 2′S) and (7R, 2′R) absolute
configurations based on calculated data. Compared to the
experimental data the (7R, 2′S) diastereoisomer exhibits a
slightly better similarity factor (0.8963 at 24 nm shift) than the
(7R, 2′R) epimer (0.8580 at 23 nm shift, Figure S39). Likewise,
previously reported acylphloroglucinols as well as compound 2
possess the S configuration at C-2′.6,27 Therefore, the 2′S
configuration (Figure 1) was tentatively assigned for 5.
The HRESI-FTMS data of compound 4 exhibits the
deprotonated molecule at m/z 359.1864 [M − H]−,
corresponding to the molecular formula C21H28O5, which was
also derived from 13C NMR analysis. The 1D and 2D NMR
data resemble those of selancin C (3), except in the acyl side
chain. The difference of 14 mass units in compound 4 is
attributed to a methylene group (δC 26.8, δH 1.85 m/1.41 m,
1H each) in the acyl side chain, which implies the replacement
of the 2-methylpropanoyl unit of 3 by a 2-methylbutanoyl
moiety in 4 (Tables 1 and 2).23 Thus, the structure of
compound 4, selancin D, was identified as 1-(5,9-dihydroxy-
2,2,8,8-tetramethyl-9,10-dihydro-2H,8H-pyrano[2,3-f ]-
chromen-6-yl)-2-methylbutan-1-one. Compounds 3 and 4 are,
therefore, 3-hydroxylated derivatives of 1 and 2, respectively
(Scheme S1, Supporting Information). On the basis of this
consideration, the C-2′ absolute configuration of 4 was
tentatively assigned to be S as in 2.
The molecular formula of selancin F (6, C20H26O6), as
determined by HRESI-FTMS and 13C NMR data, differs from
that of selancin A (1, C20H24O4) by the addition of two
hydroxy groups. The NMR data of 1 and 6 are similar, except
that signals of one of the two 2,2-dimethyl-2H-pyran moieties
in 1 are substituted by those of a 3,4-dioxygenated
dimethylchromane moiety (Tables 1 and 2; Figure 1).28,29
Compound 5, which has a molecular formula of C21H28O5 as
determined from HRESI-FTMS and 13C NMR data, is a
regiomer of 4. They differ in the position of the 2,2-dimethyl-
2H-pyran and 3-hydroxy-2,2-dimethyldihydropyran moieties, as
shown by 1D (Tables 1 and 2) and 2D NMR data. As for 3 and
1
The H NMR data (Table 1) of 6 show signals of three
hydroxy groups. The chemical shifts at δH 5.83 (d, J = 3.1 Hz;
δC 84.7, C-8) and 5.09 (d, J = 3.1 Hz; δC 91.9, C-7) are
assigned to the 3,4-dioxygenated dimethylchromane unit.28,29
The 2,2-dimethyl-2H-pyran moiety of 6 was connected via C-
4−C-4a−C-10a as revealed by HMBC correlations from H-4 to
C-10a, C-4b, C-4a, and C-2. The connection of the
dioxygenated dimethylchromane moiety via C-8−C-8a−C-4b
was consequently derived from 2D NMR data analysis. Further
HMBC correlations were observed from 9-OH to C-10, C-9,
and C-8a; from Me-14 to C-7, C-6, and C-13; and from Me-13
to C-7, C-6, and C-14. The relative configuration depicted in
Figure 1 was established by 1D and 2D NMR. The ROESY
correlations observed between H-8/Me-14 and H-7/Me-13/
Me-14 suggest H-7 and Me-13 to be equatorial, while H-8 and
Me-14 are axially oriented. These observations, in addition to
the ROESY correlation observed between H-7/H-8 and the
small coupling constant (3JH7−H8 = 3.1 Hz), required H-7 and
H-8 to be cis oriented. Compound 6, selancin F, was therefore
characterized as 1-(3R*,4S*,5-trihydroxy-2,2,8,8-tetramethyl-
1
4, the H NMR data (Table 1) of 5 show three characteristic
signals (δH 3.80, t, J = 5.3, H-7; δH 2.87, dd, J = 17.1, 5.3, H-8a;
and δH 2.65, dd, J = 17.1, 5.3, H-8b) for a 3-hydroxy-2,2-
dimethyldihydropyran moiety in addition to signals of a
hydrogen-bonded hydroxy group (δH 14.30, s), a 2,2-
dimethyl-2H-pyran, and a 2-methylbutanoyl group. The
position of the OH group at C-7 was supported by HMBC
cross-peaks from Me-13 (respectively, Me-14) to C-6, C-7, and
C-14 (respectively, C-13). The key HMBC correlations from 9-
OH to C-8a; from H-7 to C-8a; and from H-8a/H-8b to C-9,
C-8a, C-4b, and C-7 established the connection of the 3-
hydroxy-2,2-dimethyldihydropyran moiety via C-8−C-8a−C-4b
on the phloroglucinol core. The linkage of the 2,2-dimethyl-
2H-pyran moiety via C-4−C-4a−C-10a was indicated by the
HMBC correlations from H-4 to C-10a, C-4b, and C-2 as well
as from H-3 to C-4a and C-2. The 2-methylbutanoyl residue
containing the carbonyl group hydrogen-bonded to the hydroxy
group at C-9 was, therefore, deduced to be linked at C-10.
Further HMBC correlations were detected from 9-OH to C-9
E
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Figure 3. Boltzmann-weighted ECD spectra of 8 (7R: red, 7S: blue) in comparison to the experimental one (black). Similarity factor = 0.9174 with a
shift of −8 nm. Weighting factors see Figure S40: 8a (48.1%), 8b (38.2%), and 8c (13.7%).
3,4-dihydro-2H,8H-pyrano[2,3-f ]chromen-6-yl)-2-methylpro-
pan-1-one.
precursor (2′S)-2 and its analogue (7R)-8 (Scheme S1,
Supporting Information).6,27
The absolute configurations of 3, 4, and 9 were not
determined since no reasonable conclusions could be drawn
solely from ECD data. However, it can be presumed that they
maintain the same configurations as their structural analogues
2, 5, or 8 due to the proposed biogenetic relationship (Scheme
S1, Supporting Information). These compounds were not in
sufficient quantities for further purification or chemical
derivatization.
Although selancin G (7, C21H28O6) was not of good purity
and rapidly decomposed, its 1H and 13C NMR signals could be
readily assigned (Tables 1 and 2). The MS and NMR data
resemble those of selancin F (6, C20H26O6). A variation of 14
atomic mass units as determined from the HRMS data is due to
the replacement of a 2-methylpropanoyl unit by a 2-
methylbutanoyl moiety (Tables 1 and 2). The chemical shifts
and coupling constants of H-7 and H-8 are the same as in 6,
which implies the same relative configuration. The 2′S
configuration was tentatively assigned for 7 as in 2 and
previously reported acylphloroglucinols.6,27 Compound 7 was
therefore assigned the structure 1-(3R*,4S*,5-trihydroxy-
2,2,8,8-tetramethyl-3,4-dihydro-2H,8H-pyrano[2,3-f ]chromen-
6-yl)-2S-methylbutan-1-one.
Hyperselancin A (10) has the molecular formula C31H44O4
based on the HRESI-FTMS (m/z 481.3303, [M + H]+) and
13C NMR data. The assignment of carbon and proton signals of
this compound was based on 1D and 2D NMR data, including
1
DEPT, HSQC, HMBC, COSY, and ROESY. The H NMR
data (Table 3) of 10 indicate signals of four olefinic protons
including two doublets of a 2,2-dimethyl-2H-pyran moiety at
δH 6.49 (1H, d, J = 10.1 Hz, H-1″) and 5.38 (1H, d, J = 10.1
Hz, H-2″) as well as two triplets at δH 4.99 (1H, t, J = 6.6 Hz,
H-11) and δH 4.92 (1H, t, J = 6.6 Hz, H-18) consistent with
two prenyl units.24,31 They also indicate signals of six methyls at
oxygenated and nonoxygenated sp3 C atoms and the four
methyl groups of the two prenyl chains. The 13C NMR data of
10 (Table 3) exhibit 31 carbon signals, including characteristic
signals of a polyprenylated polycyclic acylphloglucinol (PPAP)
containing a bicyclo[3.3.1]nonane-2,4,9-trione skeleton with
two nonconjugated carbonyl groups (δC 208.6, C-1′; 206.8, C-
9), one enolized keto group (δC 188.8, C-1; 114.1, C-8; 171.7,
C-7), three quaternary carbons (δC 81.2, C-2; 55.1, C-6; 48.2,
C-3), one methine (δC 44.7, C-4), one methylene (δC 38.4, C-
5), and two methyl groups at C-3 (δC 26.5, C-16; 22.1, C-
15).13,24,31 Signals of the 2-methylbutanoyl moiety of PPAP-
type compounds (δH 0.83, 3H, t, J = 7.5 Hz, Me-4′; 1.02, 3H, d,
J = 6.1 Hz, Me-5′; 1.94 m/1.32 m, 1H each, diastereotopic C-3′
protons; 1.84, 1H, m, H-2′) are also displayed in the 1H NMR
spectrum of 10 (Table 3).13,24,31 The COSY spectrum of 10
displays proton spin systems of H-1″/H-2″, H2-10/H-11, H-
18/H-17a/H-17b/H-4/H-5a, and H3-5′/H-2′/H-3′a/H-3′b/
H3-4′. Inspection of the HMBC spectrum of 10 permitted
the unambiguous placement of each substituent on the core
structure. ROESY correlations were observed between H-17b/
H-3′b, H-17b/H-5a, H-5a-H2-10, and H-17a/Me-15. In
addition to the COSY data, HMBC correlations from H-10
to C-6, C-7, C-9, C-11, and C-12; from H-5a (δH 2.11) to C-4,
C-6, C-7, C-9, and C-17; from H-5b (δH 2.06) to C-7, C-9, and
C-17; from H-1″ to C-7 and C-3″; and from H-2″ to C-8 and
The molecular formulas of compounds 8 and 9 were
determined as C20H24O6 and C21H26O6 based on their negative
ion HRESI-FTMS data, which reveal the [M − H]− ion peaks
at m/z 359.1573 and 373.1729, respectively. These MS
observations were supported by NMR data. Compounds 8
and 9 differ only in the acyl side chain as established by NMR
and MS data. Compound 8 is similar to 6 except that C-8 (δC
193.7) is oxidized to a carbonyl group in 8,30 as revealed by key
HMBC correlations from H-7 (δH 4.85, s) to C-8, C-6, and C-
13/C-14, as well as from Me-14 (respectively, Me-13) to C-7
(δC 88.8), C-6 (δC 83.4), and C-13 (respectively, C-14). The
ROESY spectrum suggests H-7 to be equatorial oriented
because it exhibits interactions with both Me-13 and Me-14,
thus placing the 7-OH group axial. The absolute configuration
of 8 was determined by comparing its experimental and
calculated ECD spectra (Figures 3 and S40, Supporting
Information). The calculations clearly indicated that compound
8 has a 7R configuration with axial orientation of the 7-OH
group. The energetically preferred conformation is shown in
Figure S40 (Supporting Information). Calculated spectra with
equatorial orientation of the 7-OH group do not fit with the
experimental spectrum. On the basis of spectroscopic data, the
structure of 8 (Figure 1), selancin I, was thus identified as 3R,5-
dihydroxy-2,2,8,8-tetramethyl-6-(2-methylpropanoyl)-2,3-dihy-
dro-4H,8H-pyrano[2,3-f ]chromen-4-one. Similarly, compound
9, selancin I, is 3R,5-dihydroxy-2,2,8,8-tetramethyl-6-(2S-
methylbutanoyl)-2,3-dihydro-4H,8H-pyrano[2,3-f ]chromen-4-
one. The (7R, 2′S) absolute configuration was tentatively
assigned for 9 in comparison to its potential biosynthetic
F
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1
Table 3. 13C and H NMR Data [δ, multiplicity, J (Hz)] for
C-2 (δC 81.2, C), C-3 (δC 48.2, C), C-4 (δC 47.7, CH), and C-
16/C-15 require Me-15/Me-16 at C-3. Me-5′ of the C-2 acyl
chain correlates to C-1′, C-2′, and C-3′. The chemical shift of
the highly deshielded and nonoxygenated quaternary carbon C-
2 (δC 81.2) indicates that it is surrounded by three carbonyl
groups as extensively reported for PPAP-type com-
pounds.13,24,31 The connectivity inferred by the HMBC
spectrum is compatible only with structure 10. According to
these NMR data, the structure of compound 10 resembles
therefore androforin A, scrobiculatone A, and papuaforin C,
which were previously isolated from Guttiferae plants.24,31,32
Owing to the rigid structure of the bridged ring system, the
substituents at C-2 and C-6 must be cis oriented, as extensively
reported for PPAPs.13,24,33 The C-4 relative configuration of 10
was assessed by NMR analysis. ROESY correlations observed
between H-17b/H-5a, H-5a/H2-10, and H-17a/Me-15 in-
dicated that CH2-17, Me-15, and CH2-10 are β-oriented. The
absolute configuration of compound 10 was determined by
comparing its experimental and calculated ECD spectra. The
results (Figure 4) suggest the configuration at the four
stereogenic centers to be (2S, 4S, 6R, 2′S). The most stable
conformer agrees with ROESY analysis and literature evidence
where the chemical shift of C-4 (δC 45−49) in PPAPs with an
α-oriented H-4 can be readily distinguished from that of C-4
(δC 41−44) in PPAPs with a β-oriented H-4.31−33 On the basis
of the above data, the structure of compound 10, hyperselancin
A, was established as 2,2,7,7-tetramethyl-6S-(2S-methylbutano-
yl)-8S,10R-bis(3-methylbut-2-enyl)-2,6,7,8,9,10-hexahydro-5H-
6,10-methanocycloocta[b]pyran-5,11-dione.
Hyperselancin B (11) has the molecular formula C30H42O4
based on the HRESI-FTMS (m/z 467.3156, [M + H]+) and
13C NMR data. A difference of 14 atomic mass units is observed
between 10 and 11. The 1D and 2D NMR data of 11 indicate
that signals of the 2-methylbutanoyl group in 10 are replaced
by signals of a 2-methylpropanoyl group (δH 1.14, 3H, d, J = 6.6
Hz, Me-4′; 1.03, 3H, d, J = 6.6 Hz, Me-3′; 2.15, 1H, m, H-2′) in
11. This is confirmed by HMBC correlations from Me-4′/Me-
3′ to C-1′ (δC 208.9). The ROESY data of 11 exhibit similar
correlations to those in 10, which implies the same relative
configuration. The absolute configuration of 11 was determined
to be (2S, 4S, 6R) by comparing the experimental and
calculated ECD spectra (Figure 5). The results are consistent
with NMR analysis. Analogues of hyperselancins A and B
(PPAPs), including hyperforin,1 androforins,31 hypercohins,13
a
Compounds 10 and 11 (CDCl3)
10
11
position
δC
δH
δC
δH
1
2
3
4
5
188.8,
81.2,
48.2,
47.7,
38.4,
C
C
C
187.6,
81.1,
48.0,
47.7,
38.1,
C
C
C
CH
1.36, m
CH
1.36, m
CH2 2.11, m
CH2 2.11, m
2.06, m
2.06, m
6
55.1,
171.7,
114.1,
206.8,
30.5,
C
55.0,
171.8,
114.2,
206.7,
30.5,
C
7
C
C
8
C
C
9
C
C
10
11
12
13
14
15
16
17
CH2 2.45, d (6.6)
CH2 2.45, d (6.6)
119.7,
133.6,
26.0,
CH
C
4.99, t (6.6)
119.7,
133.5,
25.9,
CH
C
5.02, t (6.6)
CH3 1.65, s
CH3 1.69, s
CH3 1.32, s
CH3 1.19, s
CH2 2.10, m
1.87, m
CH3 1.67, s
CH3 1.53, s
CH3 1.32, s
CH3 1.20, s
CH2 2.10, m
1.87, m
18.1,
18.1,
22.1,
22.1,
26.5,
26.5,
29.5,
29.4,
18
19
20
21
1′
125.0,
132.1,
25.8,
CH
4.92, t (7.0)
125.0,
132.1,
25.8,
CH
4.92, t (7.5)
C
C
CH3 1.65, s
CH3 1.53, s
C
CH3 1.65, s
CH3 1.68, s
C
18.1,
18.1,
208.6,
48.9,
208.9,
41.9,
2′
CH
1.84, m
CH
2.15, m
3′
26.7,
CH2 1.94, m
1.32, m
21.7,
CH3 1.03, d (6.6)
4′
5′
1″
11.7,
18.0,
CH3 0.83, t (7.5)
CH3 1.02, d (6.1)
20.6,
CH3 1.14, d (6.6)
115.5,
CH
CH
C
6.49, d
(10.1)
115.4,
124.0,
CH
CH
C
6.49, d
(10.1)
2″
124.0,
5.38, d
(10.1)
5.38, d
(10.1)
3″
4″
5″
82.5,
29.3,
28.2,
82.5,
29.3,
28.1,
CH3 1.51, s
CH3 1.37, s
CH3 1.51, s
CH3 1.36, s
a
Assignment aided by 1D and 2D NMR data including HMBC,
HSQC, and DEPT.
C-3″ confirmed the position of the prenyl and 2,2-dimethyl-
2H-pyran groups. HMBC correlations from Me-15/Me-16 to
Figure 4. ECD spectra (red: calculated, black: experimental) and structure of 10 with 2S, 4S, 6R, 2′S configuration in boat conformation at 4S (cyan
ring). Similarity factor = 0.9529, 9 nm shift. Blue spectrum: calculated spectrum with 2R, 4R, 6S, 2′R configuration.
G
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Figure 5. ECD spectra (red: calculated, black: experimental) and structure of compound 11 in 2S, 4S, 6R configuration with boat conformation
(cyan ring). Similarity factor = 0.9695, shift 2 nm. Blue spectrum: calculated spectrum with 2R, 4R, 6S configuration.
and papuaforins,24 have been previously reported from
Hypericum species.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using a JASCO P-2000 digital polarimeter. UV spectra
were obtained on a JASCO V-560 UV/Vis spectrophotometer. ECD
The structure of 3-O-geranylemodin (12) was established by
comparing its observed and reported spectroscopic data.20,34 3-
O-Geranylemodin is the first geranylated anthraquinone
isolated from the genus Hypericum, although simple anthraqui-
nones (e.g. emodin) and bisanthraquinones have been reported
from this genus.35,36
spectra in CHCl3 were recorded using a JASCO J-815 spectrometer.
IR (ATR) spectra were recorded using a Thermo Nicolet 5700 FT-IR
spectrometer, in MeOH or CHCl3. 1H and 13C NMR and 2D
(gDQCOSY, ROESY (mixing time 200 ms), gHSQCAD,
gHMBCAD) spectra were recorded on an Agilent DD2 400 NMR
spectrometer at 399.915 and 100.569 MHz, respectively. The 1H
NMR chemical shifts are referenced to internal TMS (δH 0.0); 13C
NMR chemical shifts are referenced to internal CDCl3 (δC 77.0). The
low-resolution electrospray (ESI) mass spectra were performed on a
SCIEX API-3200 instrument (Applied Biosystems, Concord, Ontario,
Canada) combined with an HTC-XT autosampler (CTC Analytics,
Zwingen, Switzerland). The samples were introduced via autosampler
and loop injection. The HRESI mass spectra of compounds 1−12 as
well as the corresponding MSn measurements were 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 30.000). 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.
HPLC was performed on a Varian PrepStar instrument equipped
with a Varian ProStar PDA detector using an RP18 column (10 μm,
250 × 10 mm; flow rate 8.9 mL/min; detection 210 nm) and
acetonitrile (HPLC grade, LiChrosolv, Merck KGaA, Germany) and
double distilled water as solvents. Before HPLC separation samples
were filtered using solid phase extraction through a Chromabond
C18ec SPE cartridge (Macherey-Nagel, 1 mL/100 mg). Column
chromatography (CC) 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.
Compounds 1−9 belong to the rare group of acylphloro-
glucinol derivatives bearing two fused dimethylpyran units
around a central benzene core. Only 18 compounds showing
such structural features have been reported from nature, and
they occur mainly in Clusia ellipticifolia (Clusiaceae or
Guttiferae) and Melicope erromangensis (Rutaceae).25,37 Some
of the new derivatives reported here are partially oxidized.
Compounds 8 and 9 are the first natural products that have a 6-
acyl-2,2-dimethylchroman-4-one core fused with an additional
dimethylpyran unit. Two key steps presumably govern the
biosynthesis of compounds 1 and 2: the diprenylation of the
acylphloroglucinol core intermediate followed by cyclization.
Compounds 3−9 might henceforth derive from 1 or 2 via
oxidation processes.38−40 The biosynthesis and diprenylation
mechanisms of acylphloroglucinol intermediates have been
disclosed.41
Considering that Hypericum species are used in Cameroonian
folk medicine for the treatment of various diseases including
intestinal worms, the crude MeOH extract obtained from the
whole leaf powder of H. lanceolatum was tested in an
anthelmintic assay. The anthelmintic activity against the
model organism Caenorhabditis elegans was determined in a
modified microtiter plate assay by enumeration of living and
dead nematodes using a simple microscopic view.42 At a test
concentration of 500 μg/mL, the extract showed a nematode
death percentage of only 2.52
2.20% (no activity). In
contrast, the same extract induced significant growth inhibition
of the prostate cancer cell line PC-3 (88.6 0.6%) and the
colon cancer cell line HT-29 (88.7 1.8%) at a concentration
of 50 μg/mL. No cytotoxicity (less than 2% growth inhibition)
was observed at a concentration of 0.5 μg/mL. Compounds 1−
6, 8, 9, and 12 were also screened for cytotoxicity against HT-
29 and PC-3 cancer cell lines. However, up to a concentration
of 10 μM no significant growth inhibition was determined.
These results suggest a potential synergistic cytotoxic effect of
H. lanceolatum constituents. Compounds 7 and especially 10
and 11 were not tested for any biological activity because they
readily decomposed after isolation and spectroscopic measure-
ments.
Plant Material. The leaves of H. lanceolatum Lam. were collected
in August 2011 at Mount Bamboutos (Mbouda) in the West Region of
Cameroon. The plant was identified by Mr. Nana Victor, botanist at
the National Herbarium of Cameroon, where a voucher specimen
(No. 32356/HNC) is deposited.
Extraction and Isolation. The air-dried and powdered leaves
(750 g) of H. lanceolatum were sequentially and successively extracted
with CHCl3 (3 × 3 L) and MeOH (2 × 2 L) for 1 day under shaking
to give the respective extracts, namely, the CHCl3 (68.82 g) and
MeOH (15.63 g) extracts, after solvent evaporation under reduced
pressure. For the evaluation of anthelmintic and cytotoxic activities,
the whole powdered leaves (3 g) of H. lanceolatum were extracted in
80% MeOH (30 mL) under sonification for 30 min to afford a crude
extract (140.2 mg) after filtration and solvent removal.
H
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A portion of the CHCl3 extract (42 g) was subjected to silica gel
column flash chromatography, eluted with step gradients of n-hexane−
EtOAc and EtOAc−MeOH, to afford 23 fractions of 400 mL each,
which were combined on the basis of their TLC profiles into seven
main fractions (Fr.1−Fr.7). Fr.1 (7.65 g), obtained from n-hexane−
EtOAc (100:0 and 90:10), was further chromatographed on a SiO2
column, eluted with n-hexane−EtOAc, to afford eight subfactions
(Fr.11−Fr.18). Fr.11 (1.96 g, from n-hexane−EtOAc, 90:10 and 80:20)
also afforded eight subfractions (Fr.11a−Fr.11h) after CC on silica gel,
eluted with step gradients of n-hexane−EtOAc (98:2, 95:5, 90:10,
0:100). Repeated chromatography of Fr.11a (558 mg, obtained from n-
hexane−EtOAc, 98:2) on silica gel CC and preparative TLC, as well as
final purification by reversed phase HPLC, eluted with H2O−MeCN
(30% → 100% MeCN 0−20 min, 100% MeCN 20−25 min, 100% →
30% MeCN 25−27 min, 30% MeCN 27−30 min), afforded
compounds 1 (7.79 mg, tR 6.43 min) and 2 (4.5 mg, tR 7.25 min).
Fr.11c (151.4 mg), also obtained from n-hexane−EtOAc (98:2), was
dissolved in THF and purified by semipreparative HPLC, eluted with
H2O−MeCN (70% → 100% MeCN 0−20 min, 100% MeCN 20−25
min, 100% → 70% MeCN 25−27 min, 70% → 50% MeCN 27−30
min), to yield 6 (4.04 mg, tR 9.08 min), 7 (2.9 mg, tR 9.85 min), 8 (4.4
mg, tR 9.34 min), 9 (1.8 mg, tR 10.10 min), 10 (1.8 mg, tR 17.42 min),
11 (1.78 mg, tR 16.98 min), and 12 (2.2 mg, tR 18.51 min). Fr.2 (3.1
g), obtained from n-hexane−EtOAc (90:10 and 80:20), was further
subjected to silica gel CC eluted with n-hexane containing increasing
amounts of EtOAc to yield eight subfractions (Fr.21−Fr.28). Fr.24
(252.7 mg), obtained from n-hexane−EtOAc (80:20 and 70:30), was
suspended in isopropyl alcohol, filtered by SPE, and finally purified by
semipreparative HPLC, eluted with H2O−MeCN (30% → 100%
MeCN 0−20 min, 100% MeCN 20−25 min, 100% → 30% MeCN
25−27 min, 30% MeCN 27−30 min), to afford compounds 3 (6.5 mg,
tR 3.88 min), 4 (3.5 mg, tR 4.29 min), and 5 (2.6 mg, tR 4.61 min).
Selancin A (1), 1-(5-hydroxy-2,2,8,8-tetramethyl-2H,8H-pyrano-
[2,3-f ]chromen-6-yl)-2-methylpropan-1-one: yellow oil; UV
(CHCl3) λmax (log ε) 273 (4.49), 379 (3.38) nm; IR (ATR) νmax
(cm−1) 3604, 2974, 2928, 2872, 1640, 1592, 1462, 1381, 1132, 997,
871, 709, 660; 1H NMR data see Table 1; 13C NMR data see Table 2;
negative ion ESI-FTMS m/z 327.1601 [M − H]− (calcd for
data see Table 2; negative ion ESI-FTMS m/z 359.1867 [M − H]−
−
(calcd for C21H27O5 , 359.1864).
Selancin F (6), 1-(3R*,4S*,5-trihydroxy-2,2,8,8-tetramethyl-3,4-
dihydro-2H,8H-pyrano[2,3-f ]chromen-6-yl)-2-methylpropan-1-one:
yellow oil; UV (CHCl3) λmax (log ε) 269 (4.76), 279 (4.75) nm; IR
(ATR) νmax (cm−1) 3400, 2977, 2933, 2873, 1650, 1615, 1423, 1367,
1
1127, 1058, 749, 666; H NMR data see Table 2; 13C NMR data see
Table 2; positive ion ESI-FTMS m/z 385.1625 [M + Na]+ (calcd for
C20H26O6Na+, 385.1622).
Selancin G (7), 1-(3R*,4S*,5-trihydroxy-2,2,8,8-tetramethyl-3,4-
dihydro-2H,8H-pyrano[2,3-f ]chromen-6-yl)-2S-methylbutan-1-one:
1
yellow oil; H NMR data see Table 1; 13C NMR data see Table 2;
positive ion ESI-FTMS m/z 399.1774 [M + Na]+ (calcd for
C21H28O6Na+, 399.1778). [α]D, UV, and IR were not determined
because of compound degradation.
Selancin H (8), 3R,5-dihydroxy-2,2,8,8-tetramethyl-6-(2-methyl-
propanoyl)-2,3-dihydro-4H,8H-pyrano[2,3-f ]chromen-4-one: yellow
oil; [α]25 +1 (c 0.4, CHCl3); UV (CHCl3) λmax (log ε) 265 (4.88),
D
357 (3.84) nm; ECD (CDCl3) Δε (nm) +0.2 (279) and −0.03 (321);
IR (ATR) νmax (cm−1) 3401, 2978, 2933, 2874, 1703, 1645, 1613,
1467, 1378, 1141, 1111, 747, 688, 666; 1H NMR data see Table 1; 13
C
NMR data see Table 2; negative ion ESI-FTMS m/z 359.1505 [M −
−
H]− (calcd for C20H23O6 , 359.1500).
Selancin I (9), 3R,5-dihydroxy-2,2,8,8-tetramethyl-6-(2S-methyl-
butanoyl)-2,3-dihydro-4H,8H-pyrano[2,3-f ]chromen-4-one: yellow
oil; [α]25 +10 (c 0.3, CHCl3); UV (CHCl3) λmax (log ε) 266
D
(5.12), 357 (4.04) nm; ECD (CDCl3) Δε (nm) +1.3 (271) and −0.7
(308); IR (ATR) νmax (cm−1) 3401, 2972, 2930, 2875, 1704, 1645,
1609, 1462, 1366, 1137, 1111, 749, 689, 667; 1H NMR data see Table
1; 13C NMR data see Table 2; negative ion ESI-FTMS m/z 373.1659
−
[M − H]− (calcd for C21H25O6 , 373.1657).
Hyperselancin A (10), 2,2,7,7-tetramethyl-6S-(2S-methylbutano-
yl)-8S,10R-bis(3-methylbut-2-enyl)-2,6,7,8,9,10-hexahydro-5H-6,10-
methanocycloocta[b]pyran-5,11-dione: yellow oil; [α]25 −1 (c 0.3,
D
CHCl3); UV (CHCl3) λmax (log ε) 237 (4.65), 270 (4.63) nm; ECD
(CDCl3) Δε (nm) −3.6 (274) and +1.2 (311) cm2 mmol−1; IR
(ATR) νmax (cm−1) 2973, 2929, 1722, 1600, 1461, 1369, 1129, 751,
667; 1H NMR and 13C NMR data see Table 3; positive ion ESI-FTMS
+
m/z 481.3303 [M + H]+ (calcd for C31H45O4 , 481.3312).
−
Hyperselancin B (11), 2,2,7,7-tetramethyl-8S,10R-bis(3-methyl-
but-2-enyl)-6S-(2-methylpropanoyl)-2,6,7,8,9,10-hexahydro-5H-
6,10-methanocycloocta[b]pyran-5,11-dione: yellow oil; [α]25D −9 (c
0.2, CHCl3); UV (CHCl3) λmax (log ε) 235 (4.73), 271 (4.72) nm;
ECD (CDCl3) Δε (nm) +1.4 (269), 0.5 (298), and −0.2 (328); IR
(ATR) νm1ax (cm−1) 2973, 2928, 1721, 1614, 1456, 1371, 1259, 1085,
752, 667; H NMR and 13C NMR data see Table 3; positive ion ESI-
C20H23O4 , 327.1602).
Selancin B (2), 1-(5-hydroxy-2,2,8,8-tetramethyl-2H,8H-pyrano-
[2,3-f]chromen-6-yl)-2S-methylbutan-1-one: yellow oil; [α]26 +13
D
(c 0.2, CHCl3); UV (CHCl3) λmax (log ε) 273 (4.47), 378 (3.37) nm;
ECD (CDCl3) Δε (nm) +1.8 (279) and −0.8 (317); IR (ATR) νmax
(cm−1) 3594, 2970, 2926, 2874, 1639, 1591, 1461, 1382, 1132, 880,
1
709, 660; H NMR data see Table 1; 13C NMR data see Table 2;
+
FTMS m/z 467.3147 [M + H]+ (calcd for C30H43O4 , 467.3156).
negative ion ESI-FTMS m/z 341.1756 [M − H]− (calcd for
Synthesis. The synthetic procedure and spectroscopic data for
compounds 13, 14, 15, 16, and (+)-(S)-2 are in the Supporting
Information.
−
C21H25O4 , 341.1758).
Selancin C (3), 1-(5,9-dihydroxy-2,2,8,8-tetramethyl-9,10-dihy-
dro-2H,8H-pyrano[2,3-f ]chromen-6-yl)-2-methylpropan-1-one: yel-
Synthesis of Selancin A (1), 1-(5-hydroxy-2,2,8,8-tetramethyl-
2H,8H-pyrano[2,3-f ]chromen-6-yl)-2-methylpropan-1-one. Ethyl-
ene diamine diacetate (36 mg, 0.2 mmol) was added to a solution
of 1-(2,4,6-trihydroxyphenyl)-2-methylpropanone (13, 196 mg, 1
mmol) and 3-methyl-2-butenal (252 mg, 289 μL, 3.0 mmol) in
CH2Cl2 (10 mL). The reaction mixture was stirred at room
temperature for 10 h. After evaporation of the solvent under reduced
pressure, the mixture was chromatographed over a silica gel column,
eluted with n-hexane−EtOAc (95:5), to give selancin A (1) as a yellow
oil (80%, 261 mg, 0.8 mmol, Rf = 0.5 in n-hexane−EtOAc (95:5)): 1H
NMR (CDCl3) δ 14.14 (1H, s), 6.66 (1H, d, J = 10.1 Hz), 6.59 (1H,
d, J = 10.1 Hz), 5.45 (1H, d, J = 10.1 Hz), 5.43 (1H, d, J = 10.1 Hz),
3.85 (1H, sept, J = 6.6 Hz), 1.49 (6H, s), 1.44 (6H, s), 1.19 (6H, d, J =
6.6 Hz); 13C NMR (CDCl3) δ 210.5, 161.1, 156.0, 154.8, 125.3, 124.5,
116.5, 116.3, 104.6, 102.5, 102.1, 78.2, 78.1, 39.3, 28.4, 27.9, 19.4;
positive ion ESI-FTMS [M + H]+ at m/z 329.1749 (calcd for
low oil; [α]25 +2 (c 0.3, CHCl3); UV (CHCl3) λmax (log ε) 279
D
(4.33), 354 (3.42) nm; ECD (CDCl3) Δε (nm) +0.2 (293) and −0.04
(327); IR (ATR) νmax (cm−1) 3444, 2975, 2929, 2872, 1634, 1594,
1424, 1381, 1125, 751, 717, 666; 1H NMR data see Table 1; 13C NMR
data see Table 2; negative ion ESI-FTMS [M − H]− at m/z 345.1712
−
(calcd for C20H25O5 , 345.1707).
Selancin D (4), 1-(5,9-dihydroxy-2,2,8,8-tetramethyl-9,10-dihy-
dro-2H,8H-pyrano[2,3-f ]chromen-6-yl)-2S-methylbutan-1-one: yel-
low oil; [α]25 +12 (c 0.3, CHCl3); UV (CHCl3) λmax (log ε) 279
D
(4.58), 357 (3.65) nm; ECD (CDCl3) Δε (nm) +0.9 (292) and −0.6
(322); IR (ATR) νmax (cm−1) 3444, 2972, 2929, 2874, 1613, 1594,
1423, 1379, 1123, 752, 718, 666; 1H NMR data see Table 1; 13C NMR
data see Table 2; negative ion ESI-FTMS m/z 359.1867 [M − H]−
−
(calcd for C21H27O5 , 359.1864).
Selancin E (5), 1-(3R,5-dihydroxy-2,2,8,8-tetramethyl-3,4-dihy-
+
dro-2H,8H-pyrano[2,3-f ]chromen-6-yl)-2S-methylbutan-1-one: yel-
C20H25O4 , 329.1747).
low oil; [α]25 +7 (c 0.2, CHCl3); UV (CHCl3) λmax (log ε) 287
D
Synthesis of Selancin B (2), 1-(5-hydroxy-2,2,8,8-tetramethyl-
2H,8H-pyrano[2,3-f ]chromen-6-yl)-2-methylbutan-1-one. EDDA
(36 mg, 0.2 mmol) was added to a solution of 1-(2,4,6-
trihydroxyphenyl)-2-methylbutanone (14, 210 mg, 1 mmol) and 3-
(5.04), 360 (4.12) nm; ECD (CDCl3) Δε (nm) +0.6 (281) and −0.8
(313); IR (ATR) νmax (cm−1) 3450, 2966, 2924, 2873, 1638, 1596,
1417, 1380, 1126, 754, 716, 666; 1H NMR data see Table 1; 13C NMR
I
DOI: 10.1021/acs.jnatprod.5b00673
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
methyl-2-butenal (252 mg, 289 μL, 3.0 mmol) in CH2Cl2 (10 mL).
The reaction mixture was stirred at room temperature for 10 h. After
removal of the solvent by evaporation under reduced pressure, the
reaction mixture was chromatographed over a silica gel column, eluted
with n-hexane−EtOAc (95:5), to give selancin B (2) as a yellow oil
(85%, 290.1 mg, 0.85 mmol, Rf = 0.71 in n-hexane−EtOAc (95:5)):
1H NMR (CDCl3) δ 14.18 (1H, s), 6.66 (1H, d, J = 10.1 Hz,), 6.60
(1H, d, J = 10.1 Hz,), 5.45 (1H, d, J = 10.1 Hz), 5.44 (1H, d, J = 10.1
Hz), 3.74 (1H, sext, J = 6.6 Hz), 1.86 (1H, m), 1.50 (3H, s), 1.49 (3H,
s), 1.44 (6H, s), 1.41 (1H, m), 1.17 (3H, d, J = 6.6 Hz), 0.91 (3H, t, J
= 7.5 Hz); 13C NMR (CDCl3) δ 210.4, 161.1, 156.1, 154.7, 125.3,
124.6, 116.5, 116.3, 105.2, 102.5, 102.2, 78.2, 78.1, 46.0, 28.4, 28.3,
27.8, 26.8, 16.8, 11.9; positive ion ESI-FTMS [M + H]+ at m/z
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Biological Assays. The cytotoxicity of the methanolic crude
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00673.
■
S
(19) Heinke, R.; Franke, K.; Michels, K.; Wessjohann, L.; Ali, N. A.
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Biochem. Syst. Ecol. 2015, 59, 174−176.
1H and 13C NMR spectra of isolated compounds 1−11
and synthesized compounds 1, 2, 13, 14, 16, and
(+)-(S)-2; synthesis procedures of compounds 13, 14,
15, 16, and (+)-(S)-2 and computational methods
(PDF)
(21) Wabo, H. K.; Kowa, T. K.; Lonfouo, A. H. N.; Tchinda, A. T.;
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +49 345 5582 1313. E-mail: kfranke@ipb-halle.de (K.
Franke).
*Tel: (+49) 345 5582 1301. Fax: (+49) 345 5582 1309. E-mail:
wessjohann@ipb-halle.de (L. Wessjohann).
Notes
■
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the German Academic Exchange
Service (DAAD) for a fellowship to S.A.T.F. (A/10/98628).
We are grateful to G. Hahn and A. Ehrlich from the Leibniz
Institute of Plant Biochemistry for optical measurements and
cytotoxic assays against HT-29 and PC-3 cancer cell lines,
respectively. We wish to thank Dr. T. Bruhn from the
University Wurzburg (Germany) for technical support to
̈
perform the TDDFT calculations with ORCA and for
providing the program SpecDis for the graphical analysis of
the spectra.
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