Polycyclic Polyprenylated Acylphloroglucinol Congeners Possessing Diverse Structures from Hypericum henryi

Article abstract of DOI:10.1021/acs.jnatprod.5b00057

Polycyclic polyprenylated acylphloroglucinols (PPAPs) are a class of hybrid natural products sharing the mevalonate/methylerythritol phosphate and polyketide biosynthetic pathways and showing considerable structural and bioactive diversity. In a systematic phytochemical investigation of Hypericum henryi, 40 PPAP-type derivatives, including the new compounds hyphenrones G-Q, were obtained. These compounds represent 12 different structural types, including four unusual skeletons exemplified by 5, 8, 10, and 17. The 12 different core structures found are explicable in terms of their biosynthetic origin. The structure of a known PPAP, perforatumone, was revised to hyphenrone A (5) by NMR spectroscopic and biomimetic synthesis methods. Several compounds exhibited inhibitory activities against acetylcholinesterase and human tumor cell lines. This study deals with the structural diversity, function, and biogenesis of natural PPAPs.

Full text of DOI:10.1021/acs.jnatprod.5b00057

Article
pubs.acs.org/jnp
Polycyclic Polyprenylated Acylphloroglucinol Congeners Possessing
Diverse Structures from Hypericum henryi
Xing-Wei Yang,†,⊥,∥ Ming-Ming Li,†,∥ Xia Liu,†,⊥ Daneel Ferreira,‡ Yuanqing Ding,§ Jing-Jing Zhang,†,⊥
Yang Liao,†,⊥ Hong-Bo Qin,*,† and Gang Xu*,†
†State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650201, People’s Republic of China
§
‡Department of Biomolecular Sciences, Division of Pharmacognosy, and National Center for Natural Products Research, Research
Institute of Pharmaceutical Science, School of Pharmacy, University of Mississippi, University, Mississippi 38677, United States
⊥University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
S
* Supporting Information
ABSTRACT: Polycyclic polyprenylated acylphloroglucinols
(PPAPs) are a class of hybrid natural products sharing the
mevalonate/methylerythritol phosphate and polyketide biosynthetic
pathways and showing considerable structural and bioactive
diversity. In a systematic phytochemical investigation of Hypericum
henryi, 40 PPAP-type derivatives, including the new compounds
hyphenrones G−Q, were obtained. These compounds represent 12
different structural types, including four unusual skeletons
exemplified by 5, 8, 10, and 17. The 12 different core structures
found are explicable in terms of their biosynthetic origin. The
structure of a known PPAP, perforatumone, was revised to
hyphenrone A (5) by NMR spectroscopic and biomimetic synthesis
methods. Several compounds exhibited inhibitory activities against
acetylcholinesterase and human tumor cell lines. This study deals
with the structural diversity, function, and biogenesis of natural PPAPs.
olycyclic polyprenylated acylphloroglucinols (PPAPs),
which possess highly oxygenated and various acylphlor-
Hypericum.1 Distributed in temperate regions throughout the
world, Hypericum species have been used in traditional
medicines in many countries.6 The best known PPAP-type
molecule is hyperforin (1), which exhibits cognitive-enhancing
and memory-facilitating properties as well as potential neuro-
protective effects against Alzheimer’s disease.7 In recent years,
the fascinating chemical structures and intriguing biological
activities of PPAPs have attracted widespread attention in terms
of organic synthesis, phytochemistry, and biological evalua-
tion.1,2
P
oglucinol-derived core structures decorated with prenyl
substituents, are a group of structurally fascinating and
synthetically challenging natural products that collectively
exhibit a broad range of biological activities, such as tumor
inhibitory, antimicrobial, anti-HIV, antioxidant, and antidepres-
sant activities.1,2 Biogenetically, PPAPs are derived from a
“mixed” mevalonate/methylerythritol phosphate and polyketide
biosynthetic pathway. Their acylphloroglucinol core structure is
produced by a characteristic polyketide-type biosynthesis
involving the condensation of one acyl-CoA and three
malonyl-CoA units.1,3 Prenylation of this core moiety affords
monocyclic polyprenylated acylphloroglucinols (MPAPs),
which may be further cyclized to PPAP-type metabolites with
diverse carbon skeletons.1,3
Approximately 300 members of the PPAP family have been
reported, of which the majority are bicyclic polyprenylated
acylphloroglucinols (BPAPs) with a bicyclo[3.3.1]nonane-
2,4,9-trione core, as exemplified by hyperforin and adhyperfor-
in.1,4 The adamantane-type PPAPs with “diamond-like” caged
cores consist of about 70 members, with the first member,
plukenetione A, being isolated in 1996.5 Notably, the reported
natural PPAPs have all been characterized from plants of the
family Clusiaceae, and nearly half were isolated from the genus
As part of a systematic search for new and bioactive natural
PPAPs from plants in the genus Hypericum,8 40 PPAPs were
characterized from Hypericum henryi H. Lev & Vaniot
́
(Clusiaceae), a traditional Chinese medicinal plant used for
the treatment of hepatitis and “dampness-heat” disease.9
Previous phytochemical investigations on this species have
led to the characterization of several xanthones and PPAPs,
including four PPAP-type derivatives (hyphenrones A−D)
possessing three unusual skeletons.8c,10 The compounds
obtained in the present study, including the new hyphenrones
G−Q (4, 7, 9, 17, 18, 23, 29, 30, and 32−34), are comprised of
Received: January 21, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Scheme 1. Proposed Biosynthetic Pathways to the PPAPs from H. henryi: From Polyketides to Acylphloroglucinols, to Diverse
a
PPAP-Type Derivatives (groups I−III)
a
Key: blue, newly formed rings in known skeletons; red, newly formed ring in novel skeleton.
Scheme 2. Biogenetic Pathways to BPAPs in Group I, Including the Oxidation, Ring Cleavage, and Cycloaddition of BPAPs into
Three Unusual Carbon Skeletons Exemplified by 5, 8, and 10
12 different structural types including four unusual skeletons as
exemplified by 5, 8, 10, and 17. Their structures were
determined by spectroscopic methods and single-crystal X-ray
diffraction analysis. By combining NMR comparison and
biomimetic synthesis verification, the structure of perforatu-
mone,11 a known PPAP with a seven-membered carbon
skeleton, was shown to be identical to hyphenrone A,
possessing an eight-membered carbocyclic core, reported by
our group previously.8c Interestingly, these 12 diverse carbon
skeletons are presumably all derived from a common
biosynthetic pathway via different cyclizations of the less
complex MPAPs. Characterization of intermediate compounds,
present in trace amounts, has indicated that these PPAP profiles
are generated via three major biosynthetic pathways and may be
divided into three groups (I−III) according to their different
scaffolds.
RESULTS AND DISCUSSION
■
The MeOH extract of H. henryi was subjected to purification
using silica gel column and preparative thin-layer chromatog-
B
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1
Table 1. 13C (150 MHz) and H (600 MHz) NMR Spectroscopic Data of 4, 5, 7, and 9
a
a
b
c
4
5
7
9
no.
δC, type
δH mult. (J in Hz)
δC, type
δH mult. (J in Hz)
δC, type
δH mult. (J in Hz)
δC, type
δH mult. (J in Hz)
1
2
3
4
5
6
71.4, C
62.5, CH
196.9, C
95.5, C
4.47 s
63.6, CH
198.1, C
95.7, C
4.50 s
67.5, CH
201.5, C
96.0, C
4.28 d (2.6)
207.1, C
97.2, C
209.4, C
54.9, C
206.3, C
56.7, C
206.8, C
57.3, C
208.8, C
56.6, C
33.0, CH2
1.86 m
36.5, CH2
1.57 overlap
1.57 overlap
37.2, CH2
β 1.62 overlap
α 1.58 overlap
1.56 m
39.0, CH2
β 1.80 dd (15.8, 7.5)
α 1.75 overlap
1.71 m
1.25 overlap
1.02 m
7
41.8, CH
47.0, C
39.9, CH
48.4, C
40.7, CH
48.7, C
45.0, CH
55.1, C
8
9
108.5, C
218.0, C
46.6, CH
16.5, CH3
25.3, CH2
171.9, C
206.3, C
42.4, CH
18.9, CH3
18.8, CH3
172.0, C
205.9, C
49.6, CH
16.0, CH3
26.7, CH2
174.2, C
166.8, C
37.6, CH
20.7, CH3
31.9, CH2
10
11
12
13
2.95 m
2.61 sept (6.8)
1.07 d (6.8)
1.04 d (6.8)
2.48 m
2.60 m
0.90 d (6.8)
1.58 overlap
0.98 d (6.8)
1.65 overlap
1.29 m
1.25 d (7.2)
1.64 m
14
17
11.8, CH3
24.0, CH2
0.87 t (7.8)
11.5, CH3
30.4, CH2
0.90 t (7.5)
2.96 dd (15.0, 7.0)
2.60 dd (15.0, 7.0)
4.93 t (7.0)
13.2, CH3
32.1, CH2
0.94 t (7.4)
2.46 dd (15.0, 7.0)
2.58 dd (15.0, 7.0)
5.01 t (7.0)
29.7, CH2
2.86 dd (15.0, 7.0)
2.57 dd (15.0, 7.0)
4.95 t (7.0)
2.86 dd (15.0, 7.8)
2.54 overlap
18
19
20
21
22
116.2, CH
136.0, C
113.5, CH
138.8, C
114.9, CH
138.9, C
115.1, CH
140.2, C
4.90 t (7.8)
26.1, CH3
18.2, CH3
31.2, CH2
1.63 s
1.60 s
2.39 m
25.8, CH3
18.2, CH3
27.5, CH2
1.65 s
25.9, CH3
18.1, CH3
28.2, CH2
1.65 s
26.4, CH3
18.3, CH3
30.4, CH2
1.66 s
1.65 s
1.65 s
1.66 s
2.52 dd (15.0, 7.0)
2.12 dd (15.0, 7.0)
4.71 t (7.0)
2.55 dd (15.0, 7.5)
2.10 dd (15.0, 7.5)
4.72 t (7.5)
2.50 dd (14.3, 7.2)
2.20 dd (14.3, 7.2)
4.73 t (7.2)
23
24
25
26
27
119.1, CH
134.0, C
5.26 t (7.0)
117.5, CH
136.3, C
118.7, CH
136.3, C
118.8, CH
137.4, C
26.3, CH3
17.9, CH3
28.7, CH2
1.71 s
1.54 s
2.09 m
25.8, CH3
18.1, CH3
28.8, CH2
1.61 s
25.8, CH3
18.2, CH3
29.2, CH2
1.53 s
26.3, CH3
18.2, CH3
31.5, CH2
1.62 s
1.68 s
1.66 s
1.66 s
2.00 overlap
1.74 m
2.04 overlap
1.88 m
5.14 m
2.02 brd (14.3)
1.86 m
28
29
30
31
32
33
122.2, CH
133.7, C
4.91 t (7.0)
122.8, CH
134.8, C
4.90 t (7.0)
123.7, CH
135.1, C
123.7, CH
135.9, C
5.12 m
26.0, CH3
18.1, CH3
15.7, CH3
37.1, CH2
1.68 s
26.0, CH3
18.0, CH3
17.8, CH3
38.6, CH2
1.75 s
26.1, CH3
18.1, CH3
16.3, CH3
38.9, CH2
1.77 s
26.1, CH3
18.1, CH3
15.5, CH3
44.8, CH2
1.77 s
1.54 s
1.59 s
1.64 s
1.64 s
1.00 s
1.08 s
1.09 s
0.80 s
1.75 overlap
1.59 overlap
2.25 m
1.46 m
1.31 m
1.96 m
1.87 m
4.99 (brd, 7.0)
1.53 m
1.40 m
2.01 m
1.92 m
4.97 t (6.6)
β 2.54 overlap
α 2.35 dd (14.7, 2.6)
34
24.0, CH2
21.6, CH2
22.3, CH2
141.0, C
190.0, C
1.85 m
35
36
37
38
124.3, CH
131.9, C
4.96 t (7.0)
122.5, CH
132.4, C
123.9, CH
132.6, C
10.09 s
26.1, CH3
17.9, CH3
1.65 s
25.7, CH3
1.66 (s)
1.63 (s)
25.9, CH3
17.9, CH3
1.65 s
1.63 s
1.58 s
18.0, CH3
a
b
c
Recorded in CDCl3. Recorded in acetone-d6. Recorded in methanol-d4.
raphy, coupled with RP-18 silica gel HPLC, and yielded a total
of 40 compounds based on 12 different structural types. These
diverse carbon skeletons were all derived from the less complex
MPAPs by different cycloadditions, such as aldol condensations
and Diels−Alder additions. Their structures were determined
by spectroscopic methods coupled with single-crystal X-ray
diffraction analysis of 11, 13, 19, 26, and 38, representing five
different carbon frameworks. According to their different
scaffolding constructs, these 40 isolates could be divided into
three groups (I−III) as shown in Scheme 1. The four core
structures in group I, represented by 1, 5, 8, and 10, are BPAPs
or related PPAPs, with 10 possessing a complex 6/6/5/8/5
pentacyclic system with six stereogenic centers derived from the
normal BPAPs. The adamantane-type PPAPs, derived via
linkage of C-3/C-27 or C-3/C-28 of normal endo-BPAPs, are
included in group II and consist of four different scaffolds as
exemplified by 24, 26, 29, and 33. The other four types, as
illustrated by 17, 19, 21, and 23, were assigned to group III.
Group I is perhaps the most interesting of the three groups
due to the structural novelty of constituents. In fact, the carbon
frameworks of 5, 8, and 10 are all unusual skeletons derived
from normal BPAPs possessing the bicyclo[3.3.1]nonane-2,4,9-
trione core. Biosynthetically, 5−7 are presumably formed by
cleavage of the C-1/C-9 bond of hemiketal derivatives such as 3
via a retro-aldol mechanism and subsequent formation of a five-
membered lactone moiety. The structures of 8 and 9 possess
unusual 5/8/5 fused ring systems and may be derived from 5
and 7, respectively, via oxidative cleavage of the C-35/C-36
C
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double bond followed by formation of the cyclopentene moiety
by aldol condensation. The structure of 10, featuring a
pentacyclic 6/6/5/8/5 fused ring system, is presumably formed
via an intermolecular Diels−Alder cycloaddition of 6 as a key
step (Scheme 2).
Scheme 3. Structural Revision of Perforatumone and the
Biomimetic Transformation of 3 via 5 into 11
Hyphenrone G (4) was obtained as a colorless gum. Its
molecular formula was established by its 13C NMR and
HRTOFMS data (m/z 589.3871, [M + Na]+) as C36H54O5, 14
mass units more than that of 3-hydroxyhyperforin-3,9-hemi-
ketal (3).12 Comparison of the 1H and 13C NMR spectroscopic
data of 4 (Table 1) with those of 3 indicated that the isopropyl
group in 3 should be replaced by a sec-butyl group (C-11, δC
46.6; C-12, δC 16.5; C-13, δC 25.3; and C-14, δC 11.8) in 4. The
correlations of Me-14 (δH 0.87) with C-11 and C-13 and of
Me-12 (δH 0.90) with C-11 and C-10 (δC 218.0) in the HMBC
spectrum were indicative of such a difference (Figure 1). Other
from MeOH. The X-ray data indicated that the absolute
configurations of C-1, C-3, C-5, C-7, and C-8 in 11 and 13
were consistent with those of hyperforin (1) and 10 established
previously.1,8c In addition, the 16 PPAPs in group I are closely
related biosynthetically (Scheme 2). Compounds 1 and 2
should be the precursors of the 2,35-etherocyclic derivatives 12
and 13,12 the 4,23-etherocyclic derivatives 14−16,10c,13 and the
hemiketal derivatives 3 and 4. Compounds 5−7 are derived
from 3 and 4 and yielded 8−11 via the transformations shown
in Scheme 2. Thus, the absolute configuration of these PPAPs
should be consistent with one another.
The adamantane-type PPAPs with “diamond-like” caged
cores were assigned to group II. Historically, adamantane-type
compounds have attracted much interest from medicinal
chemists since the early 1960s, when amantadine was
introduced to the clinic for the treatment of influenza.14 In
fact, the adamantyl structural moiety is present in a number of
compounds in current clinical use and in many more
compounds that are in development as potential agents for
the treatment of iron overload disease, cancer, malaria, and
tuberculosis.15 From a medicinal chemistry perspective, the
adamantyl group can be used either as a scaffold for the
development of therapeutic agents, e.g., memantine and
amantadine, or as a modifier of the pharmacokinetics of a
compound.14,15 Group II comprises 17 adamantane-type
PPAPs (24−40) including the five new hyphenrones M−Q
(29, 30, 32−34) shown in Scheme 4. Thus far, about 70 natural
adamantane-type PPAPs based on four different carbon
skeletons have been reported. They may be divided into
adamantine tricyclo[3.3.1.1]decane (24−28) and homo-ada-
mantane tricyclo[4.3.1.1]undecane (29−40) types.2h,5,16 Both
types are derived biosynthetically from the endo-BPAPs with
the bicyclo[3.3.1]nonane core via the strongly nucleophilic
enolic C-3 cyclizing onto C-27 and C-28, respectively. Two
secondary skeletons exemplified by 26 and 33 are derived from
C-18/C-29 cyclization in the adamantane- and homo-
adamantane-type PPAPs, respectively (Scheme 4).5,16
1
Figure 1. Key HMBC and H−1H COSY correlations of 4 and 7.
parts of 4 were identical to those of 3 by analysis of the 2D
NMR spectroscopic data. Similarly, hyphenrones H (7) and I
(9) shared the same carbon skeletons and relative config-
urations of hyphenrones A (5) and C (8),8c respectively, by
analysis of their HREIMS and NMR data (Table 1). The
structural novelty of 7 (Figure 1) and 9 is hence attributable to
the presence of a sec-butyl group at C-10, instead of the
isopropyl group in 5 and 8.
In addition, perforatumone, isolated from H. perforatum in
2004, was reported to possess a novel PPAP skeleton with a
seven-membered carbon core.11a The reported H and 13C
1
NMR spectroscopic data of this compound are identical to
those of 5 (Table 1), indicating that one of the two structures
must have been assigned incorrectly. The deshielded C-1 (δC
62.5) and H-1 (δH 4.47, s) resonances indicate that this
methine functionality is more likely adjacent to two carbonyl
groups (C-2 and C-10) in 5, rather than one carbonyl group in
perforatumone, hence supporting the structural assignment of
5.8c In order to clarify the structure of these two compounds, a
biomimetic synthesis of hyphenrone F (11) from 3 via
compound 5 was performed as shown in Scheme 3. A
piperidine-induced retro-aldol reaction of 3 afforded 5 in 40%
yield. When subjected to reaction with triethylamine in MeOH
at 50 °C, 5 was converted into 11 via hydrolysis and
subsequent decarboxylation of the intermediate β-keto
carboxylic acid. Single-crystal X-ray diffraction analysis of 11
confirmed its structure unequivocally and indicated that the
structure of perforatumone is indeed identical to that of
hyphenrone A (5).
Hyphenrone M (29) was assigned the molecular formula
1
C33H42O6 from its 13C NMR and HREIMS data. The H and
13C NMR spectroscopic data of 29 (Tables 2 and 3) resembled
those of sampsonione A, a homo-adamantane-type PPAP.16a
Instead of the olefinic quaternary carbon at δC 133.0 (C-19)
and a methylene (δC 33.7, C-17) in sampsonione A, an
oxygenated tertiary carbon at δC 71.9 and an olefinic methine
(δC 126.4) appeared in 29, assuming hydroxylation of C-19 and
formation of a Δ17,18 double bond. This assumption was
supported by the correlations of H-17 (δH 6.45, J = 16.2 Hz)
Definition of the absolute configurations of PPAPs is
challenging because they are usually isolated as gums or oils.
However, crystals of 11 (CCDC 962625) and 13 (CCDC
962624) suitable for X-ray diffraction analysis were obtained
D
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Scheme 4. Proposed Biosynthetic Pathway of Group II PPAPs: Cyclization of endo-BPAPs Generating Adamantane- and homo-
Adamantane-Type PPAPs
with C-2 (δC 204.9), C-3, (δC 68.9), and C-4 (δC 110.5) and of
both Me-20 (δH 1.19) and Me-20 (δH 1.16) with C-18 (δC
143.3) and C-19 in the HMBC spectrum (Figure 2). Likewise,
the structure of hyphenrone N (30) was shown to be a
homologue of 29, with the C-5 prenyl group in the latter being
replaced by a geranyl group in 30 (Tables 2 and 3). The 2D
NMR data showed that other structural units of 29 and 30 are
the same as those of sampsonione A.
The molecular formula of hyphenrone O (32) was
determined as C38H48O5 by analysis of its 13C NMR (Table
2) and HRESIMS (m/z 539.2767, [M + Na]+) data. The
1H−1H COSY and HMBC spectroscopic data (Figure 2)
suggested that 32 has the same carbon scaffold as hyper-
sampsone O (31)16g and that it carries a C-5 geranyl side chain.
In the ROESY spectrum, the correlations of Me-33 (δH 1.42)
with H-27α (δH 2.22), of H-27α with Me-31 (δH 1.12), and of
Me-30 (δH 1.30) with H-17 (δH 5.54) and H-28 (δH 2.69),
coupled with the rigid caged skeleton, defined the relative
configurations of 32.
On the basis of analysis of its MS and 1D and 2D NMR data
(Tables 2 and 3), hyphenrone P (33) was shown to possess the
same backbone and relative configuration as hypersampsone
E.17 The structural novelty of 33 involves the presence of a C-5
prenyl rather than a geranyl group. Hyphenrone Q (34) is the
C-18 epimer of 33, as exemplified by the NOE association from
H-6 (δH 2.47) to H-28 (δH 2.10) and from Me-30 (δH 1.02) to
H-28 and H-18 (δH 1.45) in the ROESY spectrum of 34
(Tables 2 and 3).
The four different caged core structures in group II are all
derived from a common biosynthetic pathway. Therefore, it is
reasonable to assume that the absolute configurations of these
caged PPAPs are similar. This was confirmed by refinement of
the Cu Kα data of the crystals of 26 [CCDC 962622, the Hooft
parameter is 0.10(6) for 2577 Bijvoet pairs] and 38 [CCDC
962623, the Flack parameter is 0.01(13)],18 indicating
(1R,3R,5R,7R,27S) and (1R,3R,5R,7S,28R) absolute configu-
rations for the adamantane- and homo-adamantane-type PPAPs,
respectively.
The seven members (17−23) in the miscellaneous group III
are derived from direct cyclizations of PPAPs rather than via
formation of the BPAPs (Scheme 5) and are constructed from
four core structures as exemplified by 17, 19, 21, and 23.
Hyphenrones J (17) and K (18), featuring an unprecedented
six-membered ring connected to the phloroglucinol core,
presumably are derived biosynthetically from 19 (CCDC
963510) via rearrangements involving the formation and
cleavage of a cyclopropane intermediate. Compounds 21 and
22, featuring an octahydrospiro[cyclohexan-1,5′-indene] core,
are considered to be the intramolecular cyclization products of
19.19 Hyphenrone L (23), with a hexacyclic core, representing
the most complex PPAP, is likely derived from a pentapreny-
lated MPAP via successive intramolecular [2 + 4] cyclo-
additions.20
Hyphenrone J (17) was obtained as a light yellow gum,
exhibited an ion peak at m/z 507.3093 [M + Na]+ in its
HRESIMS, and was assigned a molecular formula of C30H44O5,
the same as hypercalin C (19).19a Analysis of the 1D and 2D
NMR data (Table 4) revealed that 17 is a derivative of an
MPAP and shares the same acylphloroglucinol core as 19.
However, a difference was found in the C-3 side chain from the
observed long-range correlations in the HMBC spectrum from
the C-7 methine proton at δH 3.28 to three carbons (C-2, δC
191.1; C-3, δC 112.6; and C-4, δC 177.9) of the acylphlor-
oglucinol core, suggesting that the C-7 methine was linked to
C-3 in 17 rather than a methylene in 19 (Figure 3). In addition
to the HMBC correlations of Me-10 (δH 1.23) to C-7, C-9, and
C-11, the 1H−1H COSY correlations of H-7/H-8/H-13/H-12/
H-11 led to the arbitrary assignment of the six-membered
carbocyclic in 17. The strong ROESY correlations of Me-10/H-
7 and H-7/H-13 established the cofacial orientations for H-7,
H-13, and Me-10. Therefore, the structure of 17 was elucidated
as possessing an unprecedented six-membered carbocyclic ring
E
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Table 2. 13C (150 MHz, δ in ppm) NMR Spectroscopic Data of 29, 30, and 32−34
a
b
b
b
b
no.
29
30
32
33
34
1
82.2, C
204.9, C
68.9, C
81.4, C
203.8, C
68.2, C
83.0, C
200.2, C
77.5, C
82.6, C
205.7, C
73.0, C
82.0, C
204.5, C
72.4, C
2
3
4
110.5, C
60.9, C
110.3, C
60.1, C
203.4, C
69.0, C
204.1, C
68.5, C
203.5, C
68.4, C
5
6
33.0, CH2
44.7, CH
48.8, C
32.5, CH2
44.1, CH
48.1, C
36.1, CH2
42.8, CH
48.7, C
35.5, CH2
42.9, CH
48.0, C
35.6, CH2
42.7, CH
48.4, C
7
8
9
211.5, C
196.1, C
134.7, C
130.5, CH
129.0, CH
133.0, CH
126.4, CH
143.3, CH
71.9, C
210.1, C
195.3, C
136.6, C
130.1, CH
128.6, CH
132.6, CH
125.0, CH
143.8, CH
70.9, C
205.0, C
193.7, C
135.8, C
129.6, CH
129.1, CH
132.9, CH
75.3, CH
121.3, CH
139.1, C
26.8, CH3
18.5, CH3
29.5, CH2
120.1, CH
139.1, C
40.7, CH2
16.4, CH3
25.8, CH2
54.8, CH
82.7, C
206.2, C
193.8, C
135.9, C
129.2, CH
129.1, CH
133.2, CH
34.5, CH2
57.0, CH
29.6, CH
23.8, CH3
22.5, CH3
29.9, CH2
120.1, CH
135.3, C
26.2, CH3
18.0, CH3
26.3, CH2
56.1, CH
45.2, C
206.0, C
10
11
12,16
13,15
14
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
193.7, C
135.9, C
129.2, CH
129.1, CH
133.1, CH
33.9, CH2
58.0, CH
29.6, CH
23.3, CH3
23.0, CH3
30.3, CH2
120.1, CH
135.3, C
29.2, CH3
29.6, CH3
28.9, CH2
122.3, CH
137.2, C
26.3, CH3
18.0, CH3
26.7, CH2
53.4, CH
85.3, C
30.0, CH3
29.8, CH3
28.6, CH2
122.0, CH
137.7, C
40.7, CH2
16.2, CH3
26.3, CH2
52.9, CH
84.7, C
26.2, CH3
18.0, CH3
23.5, CH2
56.5, CH
46.8, C
27.4, CH3
34.0, CH3
25.2, CH3
22.4, CH3
27.4, CH3
33.7, CH3
25.1, CH3
22.3, CH3
27.3, CH2
125.1, CH
131.7, C
25.9, CH3
17.7, CH3
30.7, CH3
25.1, CH3
25.6, CH3
22.9, CH3
27.2, CH2
125.0, CH
131.8, C
25.9, CH3
17.7, CH3
27.0, CH3
26.7, CH3
25.2, CH3
22.6, CH3
28.8, CH3
15.9, CH3
25.4, CH3
22.6, CH3
a
b
Recorded in methanol-d4. Recorded in acetone-d6.
connected to the phloroglucinol core. Biosynthetically, 17
seems to be derived from the same precursor as 19. However, it
more likely originates from 19 via a rearrangement involving
the formation and cleavage of a cyclopropane intermediate,
since the carbon skeleton of the C10 unit connected to C-3 is
not consistent with the isoprene rule (Scheme 5). Hyphenrone
K (18) was shown to be an analogue of 17 by replacement of
the isopropyl moiety in 17 with a sec-butyl group in 18 from the
MS and NMR data obtained (Table 4).
indicated by the NOE correlation between Me-28 and H-34
(δH 2.17) in the ROESY spectrum.
Considering the fact that several cytotoxic PPAPs have been
isolated previously8 and that these types of metabolites are
associated with neurodegenerative diseases, such as Alzheimer’s
disease,7 the inhibitory activities of the aforementioned
compounds were examined against acetylcholinesterase
(AChE) and the five human tumor cell lines HL-60, A-549,
SMMC-7721, MCF-7, and SW-480 (Table 6). Compounds 17
and 19 exhibited cytotoxic activities (IC50 1.7−8.9 μM) against
the cancer cell lines using the MTT method.21 In the AChE
inhibition assay using the Ellman method,22 compounds 13, 17,
26, and 40 exhibited AChE inhibitory activities (IC50 37.2, 25.4,
26.4, and 9.8 μM, respectively). Notably, the PPAPs that
possess a lactone moiety (5 and 7−10) exhibited significant
negative inhibitory activities (approximately −100% for each)
at 50 μM.
The molecular formula of hyphenrone L (23) was
determined to be C38H48O4 from its 13C NMR and HREIMS
(m/z 591.3444, [M + Na]+) data, 68 mass units more than that
of garcibracteatone.20 Comparison of the NMR spectroscopic
data (Table 5) of 23 with those of garcibracteatone indicated an
extra prenyl group (δC 27.8, C-34; 126.9, C-35; 132.3, C-36;
26.0, C-37; and 18.4, C-38) to be present and linked to the C-
27 methine δC 54.8 (C-27). The correlations of H-27 (δH
1
1.76)/H-34 (δH 2.17 and 1.60)/H-35 (δH 4.90) in the H−1H
PPAPs are a class of promising molecules for drug
development, especially the BPAPs and adamantane-type
PPAPs. The presence of multiple prenyl groups and the
derived complex ring systems in the structures of PPAPs are
rare among plant secondary metabolites. Generally, polyketide
COSY spectrum, as well as the correlations from Me-28 (δH
1.43) to C-27 and C-26 (δC 46.8) in the HMBC spectrum,
confirmed such a difference (Figure 3). The relative
configuration of the C-27 stereogenic center in 23 was
F
DOI: 10.1021/acs.jnatprod.5b00057
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 3. 1H (600 MHz, δ in ppm) NMR Spectroscopic Data
of 29, 30, and 32−34
and selectivity. Furthermore, the intriguing structures of these
PPAPs and their biogenetic relationship also suggested there
are complex postmodification processes including oxidation,
rearrangement, and cyclization in plants. Although many
studies about the biosynthesis of PPAPs have been reported,3
more in-depth research works are still needed to uncover the
interesting pathway of this special group of metabolites.
a
b
b
b
b
no.
29
30
32
33
34
6
a 2.33 dd
(15.0, 6.4)
a 2.36 dd
(15.4, 6.4)
a 2.58 dd
(15.0, 7.5)
a 2.49
overlap
a 2.49
overlap
b 2.21 brd
(15.0)
b 2.24 brd
(15.4)
b 2.36 brd
(15.0)
b 2.40 brd
(14.7)
b 2.47 brd
(15.0)
7
1.76 brt
(7.5)
1.83 brt
(6.4)
2.24 brt
(7.5)
2.22 m
2.25 brt
(7.9)
EXPERIMENTAL SECTION
■
12,
16
13,
15
7.40 d (8.0) 7.46 d (8.0) 7.08 d (8.0) 7.11 d (8.0) 7.04 d (8.0)
7.29 t (8.0) 7.30 t (8.0) 7.31 t (8.0) 7.38 t (8.0) 7.38 t (8.0)
7.39 t (8.0) 7.42 t (8.0) 7.50 t (8.0) 7.49 t (8.0) 7.47 t (8.0)
General Experimental Procedures. Melting points were
obtained on an X-4 micro melting point apparatus. Optical rotations
were measured on a JASCO P-1020 polarimeter. UV spectra were
recorded on a Shmadzu UV-2401PC spectrometer. IR spectra were
recorded on a Bruker FT-IR Tensor-27 infrared spectrophotometer
with KBr disks. 1D and 2D NMR spectra were recorded on a Bruker
DRX-600 spectrometer using TMS as an internal standard. Unless
otherwise specified, chemical shifts (δ) are expressed in ppm with
reference to the solvent signals. ESIMS and HREIMS data were
acquired on Waters Xevo TQS and Waters AutoSpec Premier P776
mass spectrometers, respectively. X-ray data were generated using a
Bruker Apex Duo instrument. Semipreparative HPLC was performed
on an Agilent 1100 HPLC with a Zorbax SB-C18 (9.4 × 250 mm)
column. Silica gel (100−200 and 200−300 mesh, Qingdao Marine
Chemical Co., Ltd., Qingdao, People’s Republic of China) and MCI
gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan)
were used for column chromatography. Fractions were monitored by
TLC (GF 254, Qingdao Marine Chemical Co., Ltd.), and spots were
visualized by heating silica gel plates immersed in H2SO4 in ethanol.
Plant Material. The aerial parts of H. henryi were collected at
Hongtudi in Dongchuan Prefecture, Yunnan Province, People’s
Republic of China, in April 2011. The plant was identified by Dr. E.
D. Liu, and a voucher specimen (201104H01) has been deposited at
the Kunming Institute of Botany.
14
17
6.45 d
(16.2)
6.56 d
(16.2)
5.54 d (9.4) β 2.47
overlap
β 2.56 dd
(12.8, 7.1)
α 1.80 dd
(13.2, 6.4)
α 2.05 m
1.45 m
18
5.99 d
(16.2)
6.04 d
(16.2)
4.98 d (9.4) 2.00 m
19
20
21
22
1.63 m
1.67 m
1.19 s
1.16 s
2.74 dd
(14.6, 8.0)
2.43 dd
(14.6, 8.0)
1.15 s
1.14 s
2.78 dd
(14.3, 7.8)
2.46 dd
(14.3, 7.8)
1.58 s
1.84 s
2.53 dd
(15.4, 7.5)
2.49 dd
(15.4, 7.5)
0.95 d (6.4) 1.00 d (6.4)
0.89 d (6.4) 0.94 d (6.4)
2.56 dd
(15.0, 7.5)
2.51 overlap 2.49 dd
(15.0, 7.5)
5.29 brt
2.57 overlap
23
25
5.39 brt
(8.0)
5.39 brt
(7.8)
5.27 brt
(7.5)
5.26 brt
(7.5)
(7.5)
1.71 s
2.08 m
2.02 m
1.63 s
2.02, m
1.69 s
1.69 s
26
27
1.65 s
1.65 s
1.65 s
1.65 s
2.07 dd
(16.6, 8.5)
2.08 overlap 2.22 dd
2.02 m
1.93 m
(15.4,
11.6)
Extraction and Isolation. The aerial parts of H. henryi (18.0 kg)
were powdered and percolated with MeOH (2 × 20 L) at room
temperature for 24 h and filtered, and the solvent was evaporated in
vacuo. The crude extract (1.8 kg) was subjected to silica gel column
chromatography eluted with CHCl3 to afford a PPAP-rich fraction
(125.6 g). This fraction was separated on an MCI-gel column
(MeOH−H2O from 8:2 to 10:0) to obtain six fractions (Fr. A−F). Fr.
A (18.5 g) was chromatographed on a silica gel column, eluted with
petroleum ether−acetone (from 50:1 to 10:1), to yield three fractions
(Fr. A1−A3). Fr. A1 (8.3 g) was further purified by preparative HPLC
(MeOH−H2O, 98:2) to afford 3 (4.8 g) and 4 (1.1 g). In the same
way, 5 (120 mg) and 7 (86 mg) were obtained from Fr. A2 (2.5 g). Fr.
B (14.9 g) was subjected to silica gel column chromatography, eluted
with petroleum ether−EtOAc (from 20:1 to 10:1), to yield Fr. B1−B3.
On further purification by preparative HPLC (MeOH−H2O, 95:5),
Fr. B1 (3.5 g) afforded 1 (3 mg) and 2 (2 mg). Fr. B2 (850 mg) was
separated by semipreparative HPLC (MeCN−H2O, 92:8) to afford 6
(3 mg) and 11 (86 mg). Using preparative TLC (petroleum ether−
acetone and petroleum ether−EtOAc), combined with semiprepar-
ative HPLC with different solvent systems (MeOH−H2O and
MeCN−H2O), Fr. C (7.4 g) afforded compounds 8 (26 mg), 9 (8
mg), 10 (5 mg), 12 (130 mg), 13 (38 mg), 23 (58 mg), 24 (35 mg),
25 (30 mg), and 39 (24 mg). Similarly, compounds 14 (1.0 g), 15
(430 mg), 16 (260 mg), 31 (8 mg), 32 (12 mg), 33 (6 mg), 34 (11
mg), 37 (29 mg), 38 (32 mg), and 40 (32 mg) were obtained from Fr.
D (16.8 g), compounds 17 (42 mg), 18 (14 mg), 19 (1.7 g), 20 (130
mg), 26 (140 mg), 27 (104 mg), 35 (28 mg), and 36 (15 mg) were
from Fr. E (19.3 g), and compounds 21 (540 mg), 22 (140 mg), 28
(37 mg), 29 (14 mg), and 30 (46 mg) were from Fr. F (13.4 g).
1.98 dd
(16.6, 7.5)
1.98 dd
(17.3, 7.6)
2.04 m
1.92 m
2.20 m
1.86 m
2.10 m
28
2.65 d (7.5) 2.68 d (7.6) 2.69 dd
(11.7, 8.6)
1.30 s
30
31
32
33
34
1.43 s
1.51 s
1.34 s
1.16 s
1.44 s
1.50 s
1.37 s
1.18 s
0.97 s
0.99 s
1.36 s
1.35 s
1.02 s
0.83 s
1.38 s
1.35 s
1.12 s
1.36 s
1.42 s
2.08 overlap 2.06, m
2.02 overlap
35
37
38
5.09 t (6.8) 5.07 t (7.0)
1.63 s
1.57 s
1.64 s
1.57 s
a
b
Recorded in methanol-d4. Recorded in acetone-d6.
Figure 2. Key HMBC and H−1H COSY correlations of 29 and 32.
1
Hyphenrone G (4): colorless gum; [α]26 +26 (c 0.4, MeOH); UV
D
and prenylation synthases are widespread in plants and often
coexist in certain species, as evidenced by the presence of
natural prenylated flavones and xanthones.23 Notably, the
presence of the complex class of PPAP metabolites is hitherto
restricted to plants in the Clusiaceae, which indicated the
prenyltransferase in these plants should possess high activity
(MeOH) λmax (log ε) 204 (4.13) nm; IR (KBr) νmax 3441, 2967, 2926,
1
2856, 1776, 1741, 1676, 1450, 1378, 1238, 1117 cm−1; H and 13C
NMR data, see Table 1; FABMS m/z 565 [M − H]−; HRTOFMS m/
z 589.3871 [M + Na]+ (calcd for C36H54O5Na, 589.3868).
Hyphenrone H (7): colorless gum; [α]20 −4 (c 0.1, MeOH); UV
D
(MeOH) λmax (log ε) 203 (4.12) nm; IR (KBr) νmax 2969, 2926, 2856,
G
DOI: 10.1021/acs.jnatprod.5b00057
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Journal of Natural Products
Article
Scheme 5. Proposed Biosynthetic Pathway of Group III PPAPs: Direct Cyclization of MPAPs Generating Four Different
Carbon Scaffolds, Involving 17 and 18 with a Novel Carbon Skeleton
1
1813, 1759, 1736, 1711, 1629, 1452, 1380, 1125, 1034, 845, 785 cm−1;
1H and 13C NMR data, see Table 1; ESIMS m/z 589 [M + Na]+;
HRTOFMS m/z 589.3857 [M + Na]+ (calcd for C36H54O5Na,
589.3868).
2928, 1737, 1700, 1629, 1449, 1384, 1237, 1122, 1062, 689 cm−1; H
and 13C NMR data, see Tables 2 and 3; ESIMS m/z 607 [M + Na]+;
HREIMS m/z 584.3499 [M]+ (calcd for C38H48O5, 584.3502).
Hyphenrone P (33): colorless gum; [α]22 −83 (c 0.1, MeOH); UV
D
Hyphenrone I (9): white powder; [α]21 +22 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 204 (4.51), 245 (4.26) nm; IR (KBr) νmax 2961,
2929, 2872, 1736, 1703, 1686, 1448, 1390, 1370, 1238, 1124, 688
cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 525 [M
+ Na]+; HREIMS m/z 502.3077 [M]+ (calcd for C33H42O4,
502.3083).
D
(MeOH) λmax (log ε) 205 (4.10), 254 (4.02) nm; IR (KBr) νma1x 2967,
2930, 1810, 1756, 1724, 1665, 1453, 1385, 1111, 1036 cm−1; H and
13C NMR data, see Table 1; ESIMS m/z 545 [M + Na]+; HRTOFMS
m/z 545.3242 [M + Na]+ (calcd for C33H46O5Na, 545.3242).
Hyphenrone Q (34): colorless gum; [α]22D −4 (c 0.2, MeOH); UV
(MeOH) λmax (log ε) 204 (4.25), 246 (4.03) nm; IR (KBr) νmax 2959,
2927, 2876, 1737, 1704, 1687, 1629, 1448, 1392, 1235, 1104 cm−1; 1H
and 13C NMR data, see Tables 2 and 3; ESIMS m/z 525 [M + Na]+;
HREIMS m/z 502.3092 [M]+ (calcd for C33H42O4, 502.3083).
Crystallographic Data of 11. C34H54O4, M = 526.77,
orthorhombic, a = 9.3781(2) Å, b = 12.6568(3) Å, c = 27.0482(6)
Å, α = β = γ = 90°, V = 3210.53(12) Å3, T = 100(2) K, space group
P212121, Z = 4, μ(Cu Kα) = 0.537 mm−1, 12 272 reflections measured,
4892 independent reflections (Rint = 0.0366). The final R1 values were
0.0794 (I > 2σ(I)). The final wR(F2) values were 0.2097 (I > 2σ(I)).
The final R1 values were 0.0798 (all data). The final wR(F2) values
were 0.2106 (all data). The goodness of fit on F2 was 1.186. Flack
parameter = 0.2(3). The Hooft parameter is 0.11(7) for 1815 Bijvoet
pairs. Crystallographic data for 11 have been deposited at the
Cambridge Crystallographic Data Center (deposition number CCDC
962625). Copies of the data can be obtained free of charge via www.
ccdc.cam.ac.uk.
Hyphenrone J (17): light yellow gum; [α]23 −49 (c 0.2, MeOH);
D
UV (MeOH) λmax (log ε) 206 (4.00), 224 (3.98), 356 (3.88) nm; IR
(KBr) νmax 3419, 2965, 2928, 2859, 1727, 1637, 1585, 1519, 1439,
1
1377, 1311, 1261, 1092,1030, 803 cm−1; H and 13C NMR data, see
Table 4; ESIMS m/z 507 [M + Na]+; HRTOFMS m/z 507.3093 [M +
Na]+ (calcd for C30H44O5Na, 507.3086).
Hyphenrone K (18): light yellow gum; [α]23 +66 (c 0.1, MeOH);
D
UV (MeOH) λmax (log ε) 207 (4.09), 225 (4.04), 349 (3.91) nm; IR
(KBr) νmax 3421, 2965, 2930, 2874, 1730,1636, 1585, 1520, 1456,
1
1377, 1311, 1261, 1117,1079, 802 cm−1; H and 13C NMR data, see
Table 4; ESIMS m/z 521 [M + Na]+; HRTOFMS m/z 499.3429 [M +
H]+ (calcd for C31H47O5, 499.3423).
Hyphenrone L (23): light yellow gum; [α]23 −12 (c 0.1, MeOH);
D
UV (MeOH) λmax (log ε) 206 (4.31), 253 (3.98) nm; IR (KBr) νmax
3439, 2966, 2925, 1736, 1708, 1668, 1630, 1601, 1451, 1378, 1328,
1
1260, 1117, 761 cm−1; H and 13C NMR data, see Table 5; TOFMS
m/z 591 [M + Na]+; HRTOFMS m/z 591.3444 [M + Na]+ (calcd for
C38H48O4Na, 591.3450).
Hyphenrone M (29): colorless gum; [α]20 −82 (c 0.2, MeOH);
Crystallographic Data of 13. C36H54O5, M = 566.79, monoclinic,
a = 9.3216(4) Å, b = 13.2025(6) Å, c = 13.8291(6) Å, α = γ = 90°, β =
107.9680(10)°, V = 1618.92(12) Å3, T = 100(2) K, space group P21, Z
= 2, μ(Cu Kα) = 0.592 mm−1, 19 174 reflections measured, 5414
independent reflections (Rint = 0.0463). The final R1 values were
0.0678 (I > 2σ(I)). The final wR(F2) values were 0.1822 (I > 2σ(I)).
The final R1 values were 0.0679 (all data). The final wR(F2) values
were 0.1827 (all data). The goodness of fit on F2 was 1.079. Flack
parameter = 0.09(19). The Hooft parameter is 0.11(5) for 2319
Bijvoet pairs (CCDC 962624).
D
UV (MeOH) λmax (log ε) 202 (4.24), 245 (3.91) nm; IR (KBr) νmax
3432, 2972, 2929, 1721, 1690, 1629, 1448, 1389, 1245, 1155, 1020
cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 557 [M
+ Na]+; HREIMS m/z 534.2962 [M]+ (calcd for C33H42O6,
534.2981).
Hyphenrone N (30): light yellow gum; [α]23D −89 (c 0.2, MeOH);
UV (MeOH) λmax (log ε) 205 (4.15), 246 (3.85) nm; IR (KBr) νmax
3433, 2969, 2927, 2876, 1722, 1690, 1448, 1389, 1250, 1154, 1106,
693 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 625
[M + Na]+; HREIMS m/z 602.3613 [M]+ (calcd for C38H50O6,
602.3607).
Crystallographic Data of 19. C30H44O5, M = 484.65,
orthorhombic, a = 10.0575(3) Å, b = 16.0634(4) Å, c = 17.3900(4)
Å, α = β = γ = 90°, V = 2809.49(13) Å3, T = 100(2) K, space group
P212121, Z = 4, μ(Cu Kα) = 0.604 mm−1, 16 676 reflections measured,
Hyphenrone O (32): colorless gum; [α]23D −9 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 205 (4.30), 245 (3.99) nm; IR (KBr) νmax 2970,
H
DOI: 10.1021/acs.jnatprod.5b00057
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
1
Table 4. 13C (150 MHz) and H (600 MHz) NMR
Table 5. H (600 MHz) and 13C (150 MHz) NMR
a
Spectroscopic Data of 17 and 18
Spectroscopic Data of 23
a
a
17
18
δH mult. (J in
δH mult. (J in
no.
δC, type
Hz)
no.
δC, type
Hz)
no.
δC, type
δH mult. (J in Hz)
δC, type
δH mult. (J in Hz)
1
2
3
4
5
71.1, C
205.1, C
69.9, C
215.9, C
71.5, C
21 38.4, C
1
107.4, C
191.1, C
112.6, C
177.9, C
58.5, C
108.0, C
190.6, C
112.5, C
179.0, C
58.7, C
22 27.1, CH3 1.34 s
23 30.2, CH3 1.13 s
24 33.9, CH2 β 1.54 m
2
3
4
5
6
7
8
α 1.88 dd
(7.8,10.8)
196.4, C
42.2, CH
31.8, CH2
196.7, C
6
7
8
9
91.6, C
25 61.2, CH 1.79 m
26 46.8, C
3.28 dd (13.2, 3.8) 42.2, CH
3.28 dd (13.2, 3.8)
α 1.98 m
199.9, C
137.4, C
α 1.96 m
β 1.29 m
31.8, CH2
27 54.8, CH 1.76 m
28 14.5, CH3 1.43 s
29 33.4, CH2 2.23 m
2.08 m
β 1.28 m
127.9, CH 7.69 d (7.5)
9
73.7, C
73.5, C
10 127.6, CH 7.35 t (7.5)
11 134.7, CH 7.56 t (7.5)
12 124.8, CH 7.43 d (7.5)
13 152.2, C
10
11
27.9, CH3
40.4, CH2
1.23 s
28.0, CH3
40.5, CH2
1.22 s
α 1.90 m
β 1.69 m
α 1.79 m
β 1.61 m
2.11 m
α 1.86 m
β 1.66 m
α 1.80 m
β 1.60 m
2.10 m
30 124.7, CH 5.03 m
31 132.9, C
12
13
27.3, CH2
46.4, CH
27.4, CH2
14 24.4, CH2 2.44 dd (9.8,
15.5)
32 25.9, CH3 1.68 s
2.19 m
15 120.5, CH 4.98 m
16 134.8, C
33 18.1, CH3 1.62 s
34 27.8, CH2 2.17 m
1.60 m
46.5, CH
150.7, C
C-14 150.6, C
C-15 108.9, CH2 4.71 s
4.69 s
108.8, CH2 4.71 s
4.68 s
17 26.2, CH3 1.66 s
18 17.9, CH3 1.57 s
19 29.6, CH2 β 2.15 m
35 126.9, CH 4.90 m
36 132.3, C
16
17
18
19
20
21.1, CH3
206.4, C
1.73 s
21.1, CH3
205.8, C
1.73 s
37 26.0, CH3 1.67 s
38 18.4, CH3 1.64 s
α 1.93 dd
(8.8,12.0)
35.6, CH
19.2, CH3
19.3, CH3
4.10 sept (6.8)
1.08 d (6.8)
1.09 d (6.8)
42.1, CH
17.2, CH3
27.3, CH2
4.00 m (6.8)
1.08 d (6.8)
1.70 m
20 58.6, CH 2.60 t (8.8)
a
Recorded in methanol-d4.
1.35 m
21
22
23
24
25
26
27
28
29
30
31
12.3, CH3
39.5, CH2
0.87 t (6.8)
2.56 m
Table 6. Cytotoxicities of Selected PPAPs against Five
Cancer Cell Lines (IC50 μM)
39.0, CH2
2.58 m
119.7, CH 4.80 t (7.9)
134.3, C
119.9, CH 4.81 t (7.9)
134.1, C
a
compound
HL-60
SMMC-7721
A-549
>40
MCF-7
SW480
26.0, CH3
18.0, CH3
39.3, CH2
1.50 s
26.0, CH3
18.0, CH3
38.8, CH2
1.51 s
5
18.6
19.8
12.2
4.7
>40
30.1
25.5
18.2
3.5
>40
>40
24.1
11.6
4.9
>40
>40
>40
>40
7.0
1.55 s
1.56 s
9
24.6
16.0
12.6
3.0
2.59 overlap
2.58 overlap
10
13
17
19
23
24
26
27
28
30
120.0, CH 4.86 t (7.5)
134.4, C
120.1, CH 4.86 t (7.6)
134.2, C
1.7
26.1, CH3
18.0, CH3
1.52 s
1.56 s
26.1, CH3
18.0, CH3
1.50 s
1.55 s
2.3
7.4
5.9
7.2
8.9
14.0
23.6
16.5
14.5
17.1
14.5
1.1
20.8
16.6
16.5
14.2
20.8
11.8
6.8
19.3
19.8
14.2
13.3
24.9
13.9
6.0
27.7
27.7
14.1
17.6
28.4
14.4
15.4
<0.008
>40
>40
17.4
19.1
>40
16.0
16.3
a
Recorded in acetone-d6.
b
DDP
b
Taxol
<0.008
<0.008
<0.008
<0.008
a
b
Other isolates with IC50 > 40 μM for all cell lines are not listed. cis-
Platin and taxol were used as positive controls.
13.2739(2) Å, α = γ = 90°, β = 93.8980(10)°, V = 3239.64(9) Å3, T =
100(2) K, space group C2, Z = 2, μ(Cu Kα) = 0.631 mm−1, 21 434
reflections measured, 5565 independent reflections (Rint = 0.0464).
The final R1 values were 0.0557 (I > 2σ(I)). The final wR(F2) values
were 0.1602 (I > 2σ(I)). The final R1 values were 0.0567 (all data).
The final wR(F2) values were 0.1618 (all data). The goodness of fit on
F2 was 1.052. Flack parameter = 0.2(2). The Hooft parameter is
0.10(6) for 2577 Bijvoet pairs (CCDC 962622).
Crystallographic Data of 38. C30H36O4, M = 460.59, monoclinic,
a = 10.75090(10) Å, b = 7.76550(10) Å, c = 14.8907(2) Å, α = γ =
90°, β = 99.69°, V = 1225.44(3) Å3, T = 100(2) K, space group P21, Z
= 2, μ(Cu Kα) = 0.642 mm−1, 15 304 reflections measured, 4218
independent reflections (Rint = 0.0261). The final R1 values were
1
Figure 3. Key HMBC and H−1H COSY correlations of 17 and 23.
5061 independent reflections (Rint = 0.0345). The final R1 values were
0.0345 (I > 2σ(I)). The final wR(F2) values were 0.0899 (I > 2σ(I)).
The final R1 values were 0.0346 (all data). The final wR(F2) values
were 0.0901 (all data). The goodness of fit on F2 was 1.087. Flack
parameter = 0.04(13). The Hooft parameter is 0.07(4) for 2131
Bijvoet pairs (CCDC 963510).
Crystallographic Data of 26. 2(C33H40O5)·3(CH4O), M =
1129.43, monoclinic, a = 18.0066(3) Å, b = 13.5854(2) Å, c =
I
DOI: 10.1021/acs.jnatprod.5b00057
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
0.0296 (I > 2σ(I)). The final wR(F2) values were 0.0786 (I > 2σ(I)).
The final R1 values were 0.0297 (all data). The final wR(F2) values
were 0.0787 (all data). The goodness of fit on F2 was 1.030. Flack
parameter = 0.01(13). The Hooft parameter is 0.01(3) for 1773
Bijvoet pairs (CCDC 962623).
General Procedure for the Retro-Aldol Reaction of 3. To a
solution of 3 (0.1 mmol, 1.0 equiv) in MeCN (0.5 mL) were added
H2O (0.2 mmol, 2.0 equiv) and piperidine (0.03 mmol, 0.3 equiv).
The mixture was stirred at room temperature for 12 h under argon,
after which it was filtered through a short pad of MgSO4 and washed
with dry CH2Cl2. The solvent was removed under vacuum, and the
residue was chromatographed on silica gel (ether−EtOAc, 30:1) to
give 5 (22.3 mg, 40%) as a colorless oil.
General Procedure for the Synthesis of 11. A mixture of 5 (10
mg, 0.018 mmol), MeOH (0.5 mL), and triethylamine (0.05 mL) was
stirred at 50 °C for 24 h. The volatiles were removed under vacuum,
and the residue was purified by flash silica gel column chromatography
(ether−EtOAc, 15:1) to afford 11 as colorless crystals (3.7 mg, 39%).
Acetylcholinesterase Inhibitory Assay. The acetylcholinester-
ase (AChE) inhibitory activity of the compounds was assayed by the
spectrophotometric method developed by Ellman et al.21 with slight
modifications. S-Acetylthiocholine iodide, S-butyrylthiocholine iodide,
5,5′-dithiobis(2-nitrobenzoic) acid (DTNB, Ellman’s reagent), and
acetylcholinesterase derived from human erythrocytes were purchased
from Sigma Chemical. Compounds were dissolved in DMSO. The
mixture containing 110 μL of phosphate buffer (pH 8.0), 10 μL of test
compound (50 μM), and 40 μL of acetylcholinesterase (0.04 U/100
μL) was incubated for 20 min (30 °C). The reaction was initiated by
the addition of 20 μL of DTNB (6.25 mM) and 20 μL of
acetylthiocholine for AChE inhibitory activity, respectively. The
hydrolysis of acetylthiocholine was monitored at 405 nm after 30
min. Tacrine was used as a positive control. All the reactions were
performed in triplicate. The percentage inhibition was calculated as
follows: % inhibition = (E − S)/E × 100 (E is the activity of the
enzyme without test compound and S is the activity of enzyme with
test compound). Sample limitations precluded the testing of
compounds 6 and 18.
AUTHOR INFORMATION
Corresponding Authors
*Tel and Fax: +86-871-65217971. E-mail: xugang008@mail.
kib.ac.cn (G. Xu).
■
*E-mail: qinhongbo@mail.kib.ac.cn (H. B. Qin).
Author Contributions
∥X. W. Yang and M. M. Li contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work was supported financially by the National Natural
Sciences Foundation of China (20972167), the Young
Academic Leader Raising Foundation of Yunnan Province
(No. 2009CI073), the National Science and Technology
Support Program of China (2013BAI11B02), the foundation
from Chinese Academy of Sciences to G.X., and the Program of
“One Hundred Talented People” to H.B.Q.
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S
* Supporting Information
1
1
The original MS, H and 13C NMR, HSQC, H−1H COSY,
HMBC, and ROESY NMR spectra for all the new compounds
and crystallographic files for 11, 13, 19, 26, and 38 in CIF
format. This material is available free of charge via the Internet
at http://pubs.acs.org.
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DOI: 10.1021/acs.jnatprod.5b00057
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