Selaginpulvilins A-D, new phosphodiesterase-4 inhibitors with an unprecedented skeleton from selaginella pulvinata

Article abstract of DOI:10.1021/ol403282f

Selaginpulvilins A-D (1-4), four new phenols with an unprecedented 9,9-diphenyl-1-(phenylethynyl)-9H-fluorene skeleton, together with four known selaginellins (5-8) were isolated from Selaginella pulvinata. Their structures were elucidated by spectroscopic analysis and chemical correlation. The structure of 1 was confirmed by single-crystal X-ray diffraction. Compounds 1-8 exhibited remarkable inhibitory activities (IC₅₀ values in the range of 0.11-5.13 μM) against phosphodiesterase-4 (PDE4), a drug target for the treatment of asthma and chronic obstructive pulmonary disease.

Full text of DOI:10.1021/ol403282f

Letter
pubs.acs.org/OrgLett
Selaginpulvilins A−D, New Phosphodiesterase‑4 Inhibitors with an
Unprecedented Skeleton from Selaginella pulvinata
Xin Liu,† Hai-Bin Luo,† Yi-You Huang, Jing-Mei Bao, Gui-Hua Tang, Yun-Yun Chen, Jun Wang,
and Sheng Yin*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, P. R. China
S
* Supporting Information
ABSTRACT: Selaginpulvilins A−D (1−4), four new phenols
with an unprecedented 9,9-diphenyl-1-(phenylethynyl)-9H-
fluorene skeleton, together with four known selaginellins (5−
8) were isolated from Selaginella pulvinata. Their structures
were elucidated by spectroscopic analysis and chemical
correlation. The structure of 1 was confirmed by single-crystal
X-ray diffraction. Compounds 1−8 exhibited remarkable
inhibitory activities (IC50 values in the range of 0.11−5.13
μM) against phosphodiesterase-4 (PDE4), a drug target for
the treatment of asthma and chronic obstructive pulmonary
disease.
he phosphodiesterases (PDEs) are an 11-membered
The air-dried powder of the whole plant of S. pulvinata (1.0
Tfamily of enzymes that catalyze the hydrolysis of the kg) was extracted with 95% EtOH at room temperature to give
a crude extract, which was suspended in H2O and successively
partitioned with petroleum ether, EtOAc, and n-BuOH.
Column chromatographic separations of the EtOAc extract
afforded compounds 1−8.
second messengers, cyclic adenosine monophosphate (cAMP)
and guanosine monophosphate (cGMP).1 Phosphodiesterase-4
(PDE4), which specifically targets cAMP, is a therapeutic target
of high interest for central nervous system (CNS), inflamma-
tory, and respiratory diseases.2 Although a number of molecules
of chemically diverse classes have been developed as PDE4
inhibitors over the decades, roflumilast is the sole PDE4
inhibitor recently approved in both the United States and
Europe for the treatment of chronic obstructive pulmonary
disease (COPD).3 However, the efficacy of roflumilast may be
restricted by the dose-limiting side effects of nausea, diarrhea,
weight loss, and headaches. Thus, the search for novel PDE4
inhibitors with stronger potency and less side effects is
compelling.
Selaginella pulvinata (Hook. et Grev.) Maxim. (Selaginella-
ceae), a qualified species listed in the Chinese Pharmacopoeia,
has been widely used in Traditional Chinese Medicine (TCM)
for the treatment of dysmenorrhea, asthma, and traumatic
injury.4 Previous investigations on this genus showed, in
addition to flavonoids, the presence of 17 phenolic pigments
featuring unique alkynyl and p-quinone methide function-
alities.5 In our efforts toward novel PDE4 inhibitors from
medicinal plants, a fraction of the ethanolic extract of S.
pulvinata showed significant inhibitory activity. Subsequent
chemical investigation led to the isolation of four new phenols
(1−4) with an unprecedented 9,9-diphenyl-1-(phenylethynyl)-
9H-fluorene skeleton, together with four known selaginellins
(5−8). Bioassay verified that compounds 1−8 had remarkable
inhibitory activities against PDE4. Herein, the isolation,
structural elucidation, biogenetic origin, and inhibitory activities
of selaginpulvilins A−D are described.
Selaginpulvilin A (1)6 was obtained as a yellow crystal. The
HRESIMS displayed a pseudo-molecular ion at m/z 511.1543
[M − H]− (calcd 511.1551), which was consistent with a
molecular formula of C34H24O5 with 23 degrees of unsatura-
tion. The IR spectrum exhibited absorption bands for OH
(3312 cm−1), CC (2207 cm−1), and benzene (1510 and
1
1607 cm−1) functionalities. The H NMR spectrum showed
signals for three p-phenyl groups (two were overlapped) [δH
6.59 (4H, d, J = 8.0 Hz), 7.12 (4H, d, J = 8.0 Hz), 6.89 (2H, d, J
= 8.3 Hz), and 6.71 (2H, d, J = 8.3 Hz)], a 1,2,4-trisubstituted
benzene ring [δH 6.72 (1H, brs), 6.78 (1H, d, J = 8.0 Hz), and
Received: November 13, 2013
Published: December 12, 2013
© 2013 American Chemical Society
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7.60 (1H, d, J = 8.0 Hz)], a 1,2,3,4-tetrasubstituted benzene
ring [δH 7.49 (1H, d, J = 7.7 Hz) and 7.67 (1H, d, J = 7.7 Hz)],
and a hydroxymethyl group [δH 4.80 (2H, s)]. The 13C NMR
spectrum of 1 exhibited 24 carbon resonances that were
classified by DEPT experiments as two superimposed 4-
hydroxylphenyl groups, a 4-hydroxylphenyl group, two multi-
substituted benzene rings, an alkynyl (δC 85.4 and 102.4), an
sp3 quaternary carbon (δC 66.1), and a hydroxymethyl (δC
63.7). The aforementioned information implied that compound
1 possessed most structural features of selaginellin.5a However,
as the five benzene rings and one alkynyl only consumed 22 of
the 23 degrees of unsaturation, the remaining unsaturation unit
required that 1 had one more ring than that of selaginellin.
The connectivities of these benzene rings, the quaternary
carbon, and the alkynyl were mainly achieved by analysis of the
HMBC correlations. Strong HMBC correlations from the
aromatic protons at δH 7.12 (4H, d, H-18/22/24/28) to the
quaternary carbon (δC 66.1, C-9) revealed the presence of two
geminal 4-hydroxylphenyl groups at C-9. The HMBC
correlations of H-5/C-4a and H-4/C-4b connected two
multisubstituted benzene rings (A and E) via the C4b−C4a
bond. Ring E was further linked to C-9 by HMBC correlation
of H-8/C-9. The hydroxymethyl was located at C-2 by HMBC
correlations from the protons at δH 4.80 (2H, s) to C-1, C-2,
and C-3. The alkynyl was linked to a 4-hydroxylphenyl group
by HMBC correlation of H-12 and H-16/C-11 to form a p-
hydroxyl phenylethynyl, which was further placed at C-1 by a
weak J 4 HMBC correlation from the hydroxymethyl to C-10.
As an additional ring was required in the structure, the still
“loose end” of C-9 and C-9a was linked to form a five-
membered ring. This was supported by the observation of a
weak J 4 HMBC correlation from H-18 to C-9a. Thus, a
fluorene core attached by two p-phenyl groups at the central
pentene ring and a para-pheylalkynyl at one of the benzene
rings as depicted in Figure 1 was proposed for 1, which was
Selaginpulvilin C (3),9 isolated as colorless oil, had a
molecular formula of C34H24O4 as determined by HRESIMS at
m/z 495.1594 [M − H]− (calcd 495.1602). The NMR spectra
of 3 were very similar to those of 1 except for the absence of
signals for the hydroxymethyl group and the presence of a
tertiary methyl (δH 2.43, δC 21.0), which indicated that the
hydroxymethyl group in 1 was replaced by a methyl group in 3.
This was further confirmed by HMBC correlations from the
methyl protons to C-1, C-2, and C-3.
Selaginpulvilin D (4),10 isolated as colorless oil, had a
molecular formula of C33H22O4 as determined by HRESIMS at
1
m/z 505.1417 [M + Na]+ (calcd 505.1410). The H and 13C
NMR data of 4 showed high similarity to those of 3 except that
the methyl group in 3 was eliminated in 4. This was supported
by the characteristic 1,2,3-trisubstited pattern of ring A [δH 7.20
(d, J = 7.5 Hz), 7.28 (t, J = 7.5 Hz), and 7.65 (d, J = 7.5 Hz)]
and further confirmed by detailed 2D analysis.
The known compounds were identified as selaginellin (5),5a
selaginellin A (6),5f selaginellin N (7),5b and selaginellin H
(8)5g by comparison of their NMR data with those in the
literature.
Selaginpulvilins A−D (1−4) represent a new class of natural
products possessing a fluorene core. Based on the co-
occurrence of compounds 1−8, the biogenetic pathway for
them was proposed (Scheme 1). Selaginellin (5), the major
Scheme 1. Proposed Biosynthetic Pathway for Compounds
1−8
component, was considered as the precusor. In brief, 5 was
modified via three different biosynthetic routes involving
oxidative decarboxylation of hydroxymethyl, oxidative cleavage
of alkynyl,11 and acid-aided protonation to form selaginellin A
(6), the intermediates II, and I, respectively. Methylation of 6
would generate 7, and hydration of II followed by esterification
could yield selaginellin H (8). The key step in this proposal, the
linkage of the C-8a and C-9 of I to produce 1, probably
involved the aromatic electrophilic substitution reaction
between ring E and a C-9 carbocation, similar to a Friedel−
Crafts reaction. Finally, 1 underwent sequential oxidation to
give 2 and 4 or underwent reduction to afford 3.
To mimic the formation of the C8a−C9 bond, selaginellin
(5) was reacted with different acids with the hope of generating
1 (Supporting Information S3). However, all efforts failed
probably due to the vulnerability of the phenolic hydroxyl
groups under acid condition. Thus, 5 was first converted to its
1
Figure 1. Key H−1H COSY () and HMBC (→) correlations of 1
(left). Single-crystal X-ray structure of 1 (right).
fully consistent with its molecular composition. Since the
connectivities achieved by weak J 4 HMBC correlations remains
ambiguous, a single-crystal X-ray diffraction analysis was carried
out for 1, which further secured the structure of 1.7
Selaginpulvilin B (2),8 isolated as yellow oil, had a molecular
formula of C34H22O5 as determined by HRESIMS at m/z
509.1386 [M − H]− (calcd 509.1394). The NMR data of 2
were very similar to those of 1, with differences only
attributable to the replacement of the hydroxymethyl group
in 1 by a formyl group in 2 (δH 10.45, δC 192.9). This was
further confirmed by HMBC correlations from the formyl
proton to C-1, C-2, and C-3.
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methylated derivative (9), which then reacted with formic acid
at 50 °C in 2 days to successfully generate the C8a−C9 linked
product, 7,14,26-trimethoxyselaginpulvilin A (10) (Scheme 2).
mechanism of action on PDE4 and their selectivity in the
PDE family are in progress.
ASSOCIATED CONTENT
* Supporting Information
■
S
Scheme 2. Chemical Correlations to Mimic Formation of the
C8a−C9 Bond
Full experimental procedures, tabulated NMR data of 1−4, and
1D and 2D NMR spectra of 1−10. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: yinsh2@mail.sysu.edu.cn.
Author Contributions
†These authors contributed equally to this paper.
Notes
Compounds 1−8 were tested for their inhibitory activity
against PDE4D2. Rolipram, a well-known PDE4 inhibitor, was
used as the positive control, which exhibited an IC50 of 0.54
μM, comparable to that reported in the literature (1.0 μM).1
Compounds 1−8 showed remarkable inhibitory activity with
IC50 values in the range of 0.11−5.13 μM, which may explain
the anti-inflammatory usage of S. pulvinata in TCM. In
particular, selaginpulvilin B (2), the most active compound
showed an IC50 of 0.11 μM, being 5-fold stronger than the
positve control (Table 1). The dose−response curves of the
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from Natural Science Foundation of China
(81102339 and 21103234) and Guangdong Natural Science
Foundation (S2011040002429 and S2011030003190) are
gratefully acknowledged. We cordially thank Prof. H. Ke in
the Department of Biochemistry and Biophysics at University
of North Carolina, Chapel Hill for his help for the expression,
purification, and enzymatic assay of PDE4D2.
Table 1. Enzymatic Activities (IC50, μM) of Compounds 1−8
against the Catalytic Domain of PDE4D2
compound
IC50 (μM)
1
0.24 0.03
0.11 0.02
0.18 0.02
0.26 0.05
0.97 0.10
1.42 0.10
0.22 0.01
5.13 0.60
0.54 0.04
2
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two most active compounds (2 and 3) are represented in
Figure 2. It was noteworthy that although compounds 5 and 6
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Figure 2. Inhibitory curves of compounds 2 and 3 against PDE4D2.
were very active, their C8a−C9 connected counterparts,
compounds 1 and 4, showed at least 5-fold stronger inhibition,
which suggested that formation of a fluorene core would
enhance the activity.
Natural PDE4 inhibitors are very rare. It is possible that the
unique skeleton of selaginpulvins A−D renders them potent
activity, which makes them promising lead structures for the
development of PDE4 inhibitors. Studies toward their
(6) Selaginpulvilin A (1). Yellow crystal; mp 294−296 °C; UV
(MeOH) λmax (log ε) 204 (4.84), 289 (4.46), 300 (4.47), 320 (4.25)
nm; IR (KBr) νmax 3312, 2207, 1607, 1510, 1449, 1357, 1241, 1174,
1
833 cm−1; H and 13C NMR data, see Tables 1 and 2 in Supporting
Information; negative ESIMS m/z 511.2 [M − H]−, 546.9 [M + Cl]−,
556.7 [M + HCOO]−; HRESIMS m/z 511.1543 [M − H]− (calcd for
C34H23O5, 511.1551).
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(7) Crystallographic data for selaginpulvilin A (1) have been
deposited at the Cambridge Crystallographic Data Centre (deposition
no. CCDC-934814). Copies of these data can be obtained free of
charge via www.ccdc.cam.ac.uk/conts/retrieving.htm.
(8) Selaginpulvilin B (2). Yellow oil; UV (MeOH) λmax (log ε) 208
(4.70), 294 (4.24), 305 (4.26), 314 (4.28), 360 (4.21) nm; IR (KBr)
νmax 3284, 2202, 1679, 1604, 1557, 1507, 1243, 1019 cm−1; 1H and 13
C
NMR data, see Tables 1 and 2 in Supporting Information; positive
ESIMS m/z 511.1 [M + H]+, negative ESIMS m/z 509.1 [M − H]−;
HRESIMS m/z 509.1386 [M − H]− (calcd for C34H21O5, 509.1394).
(9) Selaginpulvilin C (3). Colorless oil; UV (MeOH) λmax (log ε)
208 (4.76), 288 (4.36), 299 (4.41), 315 (4.18) nm; IR (KBr) νmax
1
3375, 2193, 1604, 1508, 1447, 1373, 1247, 1022, 829 cm−1; H and
13C NMR data, see Tables 1 and 2 in Supporting Information; positive
ESIMS m/z 497.2 [M + H]+, negative ESIMS m/z 495.2 [M − H]−;
HRESIMS m/z 495.1594 [M − H]− (calcd for C34H23O4, 495.1602).
(10) Selaginpulvilin D (4). Colorless oil; UV (MeOH) λmax (log ε)
205 (4.89), 288 (4.52), 298 (4.53), 316 (4.35) nm; IR (KBr) νmax
3367, 2203, 1563, 1509, 1468, 1170, 1073, 950, 863 cm−1; 1H and 13
C
NMR data, see Tables 1 and 2 in Supporting Information; positive
ESIMS m/z 483.2 [M + H]+, negative ESIMS m/z 481.2 [M − H]−;
HRESIMS m/z 505.1417 [M + Na]+ (calcd for C33H22O4Na,
505.1410).
(11) Enthaler, S. ChemCatChem 2011, 3, 1929−1934.
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