Prenylcoumarin with Rev-export inhibitory activity from Cnidii Monnieris Fructus

Article abstract of DOI:10.1016/j.bmcl.2010.04.081

By use of the fission yeast expressing the model fusion protein comprised of GST, SV40 T antigen NLS, GFP, and Rev-NES in the bioassay, the prenylcoumarin osthol (1) was disclosed as the new Rev-export inhibitor from the MeOH extract of Cnidii Monnieris Fructus. Furthermore, 1 was also found to inhibit export the genuine Rev in HeLa cells by indirect fluorescent antibody technique. By the competitive experiment using the biotinylated probe 3, osthol (1) was revealed to inhibit nuclear export of Rev through a NES non-antagonistic mode. Structure-activity relationship analysis of several analogs of 1 clarified that both prenyl side chain and double bond adjacent to the lactone carbonyl residue play an important role in the Rev-export inhibitory potency of 1.

Full text of DOI:10.1016/j.bmcl.2010.04.081

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3717–3720
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Prenylcoumarin with Rev-export inhibitory activity from Cnidii Monnieris
Fructus
Satoru Tamura a, Toshiaki Fujitani a, Masafumi Kaneko b, Nobutoshi Murakami a,b,
*
a Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
b PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
By use of the fission yeast expressing the model fusion protein comprised of GST, SV40 T antigen NLS,
GFP, and Rev-NES in the bioassay, the prenylcoumarin osthol (1) was disclosed as the new Rev-export
inhibitor from the MeOH extract of Cnidii Monnieris Fructus. Furthermore, 1 was also found to inhibit
export the genuine Rev in HeLa cells by indirect fluorescent antibody technique. By the competitive
experiment using the biotinylated probe 3, osthol (1) was revealed to inhibit nuclear export of Rev
through a NES non-antagonistic mode. Structure–activity relationship analysis of several analogs of 1
clarified that both prenyl side chain and double bond adjacent to the lactone carbonyl residue play an
important role in the Rev-export inhibitory potency of 1.
Received 10 February 2010
Revised 15 April 2010
Accepted 19 April 2010
Available online 20 May 2010
Keywords:
Osthol
Rev-export inhibitor
Anti-HIV
Ó 2010 Elsevier Ltd. All rights reserved.
Cnidium monnieri Cusson
Prenylcoumarin
The acquired immunodeficiency syndrome (AIDS) is a life-
threatening disease caused by HIV-1.1 Replication of HIV-1 entails
an ordered pattern of the viral gene expression, which is depen-
dent on the viral regulatory protein, Rev.2 Rev acts to increase cyto-
plasmic accumulation of the viral mRNAs by transport from the
nucleus to the cytoplasm with the aid of the cargo protein
CRM1.3,4 Since this process is essential for viral replication, inhibi-
tion for export of Rev is an attractive strategy for therapeutic
intervention.5
In this context, we have been devoted to exploring Rev-export
inhibitors from medicinal plants to find out the several active prin-
ciples.6–8 In addition, their mechanism of actions were compared
with the first low-molecular inhibitor, leptomycin B (LMB),4 by
using the biotinylated probe derived from LMB. Further investiga-
tion on search for new Rev-export inhibitors resulted in isolation of
the prenylcoumarin, osthol (1) from Cnidii Monnieris Fructus.
Herein, we describe not only the bioassay-guided isolation, identi-
fication, and biological property but also the structural require-
ment for the Rev-export inhibitory potency with respect to 1.
To search for the Rev-export inhibitors, the potency was
evaluated by using the fission yeast Schizosaccharomyces pombe,4
which express the model fusion protein consisting of glutathione
S-transferase (GST), SV40 T antigen nuclear localization signal
(NLS), green fluorescent protein (GFP), and the nuclear export
signal (NES) of Rev. Rev was shown to export from the nucleus
to the cytoplasm by recognition of CRM1, the receptor of Rev-
NES. In brief, the fusion protein imported by NLS is subjected to
interruption of nuclear export to cluster in the nucleus in the
presence of inhibitors.
As a result of screening about 400 extracts from medicinal
plants by using this bioassay,9 the MeOH extract of Cnidii Monnieri
Fructus (fruits of Cnidium monnieri Cusson) was revealed to be a
promising candidate. After this extract was successively parti-
tioned between EtOAc and H2O, n-BuOH and H2O, the resulting
EtOAc extract was found to exhibit the most potent activity among
the three extracts. Subsequently, the EtOAc extract was subjected
to successive separation by SiO2 column and reversed phase HPLC
under the guidance of bioassay to give the active principle (1, 0.39%
from the crude drug) with IC50 of 9.8 lM in the bioassay by the fis-
sion yeast. Intensive analysis of 1H and 13C NMR, IR, FAB-MS, and
FAB HRMS spectra unambiguously identified the active principle
as the prenylcoumarin, osthol (1).10
Furthermore, we evaluated inhibitory effect of osthol (1) for
nuclear export of genuine HIV-Rev in HeLa cells, in which the
HA-tagged Rev plasmids11 were transfected, by indirect fluorescent
antibody technique.12 In this precision bioassay, Rev protein is dis-
tributed in both cytoplasm and nucleus in the absence of 1, while
treatment with 1 at a concentration of 5 lM brings about localiza-
tion of Rev in only the nuclei as shown in Figure 1. In addition,
osthol (1) inhibits nuclear export of Rev in HeLa cells with IC50 of
* Corresponding author. Present address: Kyoto Pharmaceutical University. Tel.:
+81 75 595 4634; fax: +81 75 595 4768.
1.6
lM. This result suggests that osthol (1) securely inhibits export
of genuine Rev protein from the nucleus to the cytoplasm.
E-mail address: murakami@poppy.kyoto-phu.ac.jp (N. Murakami).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.081

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S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3717–3720
Figure 1. Osthol (1), Rev-export inhibitor from Cnidii Monnieris Fructus.
Subsequently, the mechanism of action of osthol (1) was com-
pared with that of LMB (2), the first low-molecular inhibitor for
Rev-export, by use of our synthesized probe 3 designed from 2.
Previously, LMB (2) was found to be bound to Cys-529 of CRM1,
the receptor of NES, by a covalent bond.13 Furthermore, the probe
3 was revealed to capture CRM1, whereas the potency almost com-
pletely disappeared in the presence of the competitive NES antag-
onistic inhibitors, valtrate and 10-acetoxychavicol acetate (ACA) as
well as LMB. After prior addition of osthol (1) to HeLa cells, the
Figure 3. Postulation leading to synthesis of 7-demethoxyanalog 4.
d-lactone moiety. In the case of 1, the plausible cysteine adduct
i was assumed to revert by way of the cation intermediate ii
due to the electron-donating methoxyl group at C-7, which would
lead to interrupt labeling of CRM1 by the probe 3 in the co-pres-
ence of osthol (1) (Fig. 3). To examine this possibility, we synthe-
sized the demethoxyanalog 4 and evaluated it for Rev-export
inhibitory activity.
whole was incubated with probe 3 (1.0 lM). Lysate of the har-
vested cells was treated with streptavidin beads, thereafter the
proteins attached to the beads were detected by an immunoblot-
ting technique.14
As shown in Figure 2, pre-treatment of osthol (1) even at a con-
The synthesis of the demethoxyanalog 4 was conducted as de-
picted in Scheme 1. Introduction of the 1-formyl-1-methylethyl
group to salicylaldehyde (5) was carried out by 2-bromo-2-methyl-
propanal in the presence of potassium t-butoxide and 18-crown-6 to
provide dial 6, which was subjected to Wittig olefination by using
methyltriphenylphosphonium bromide (PH3PCH3Br) and sodium
hexamethyldisilazide (NaHMDS) to give diene 7. Claisen rearrange-
mentof the diene 7 followedby acylation using acryloylchloride and
centration of 100 lM obviously presents the band due to CRM1
and this behavior is analogous to treatment of only probe 3. On
the other hand, the band of CRM1 completely disappears by pre-
treatment of LMB. Taking these experimental outcomes together
with the IC50 values of 1 (1.6 lM) and 3 (1.2 lM) into account,
osthol (1) was intensively presumed to inhibit nuclear export of
Rev through a NES non-antagonistic mode. Since osthol (1) was
previously reported as an anti-HIV principle,15 inhibition for nucle-
ar export of Rev was revealed to be one of the mechanism of
actions related to anti-HIV activity of 1.
triethylamine gave
a,b-unsaturated ester 9 via 8. Ring-closing
metathesis of the ester 9 by the second generation of Grubbs’ cata-
lyst16 furnished the desired demethoxyanalog 4 (Scheme 1).17
In HeLa cells, the synthesized analog 4 was shown to inhibit ex-
port of Rev with IC50 of 6.4 lM. This biological outcome indicates
that the methoxyl group at C-7 enhances Rev-export inhibitory
Previously, LMB was found to form the stable Michael adduct
with N-acetyl-
-lactone portion.13 In addition, the NES antagonistic Rev-export
inhibitors, valtrate and ACA found by our group, similarly afforded
the adducts on treatment with N-acetyl-
-cysteine methyl ester.6,7
On the contrary, osthol (1) gave no cysteine adducts under
the same treatment in spite of possessing the ,b-unsaturated
L-cysteine methyl ester at the a,b-unsaturated
c
activity. Despite the moderately potent activity in comparison with
L
a
Scheme 1. Synthesis of 7-demethoxyanalog 4. Reagents and conditions: (a) 2-
bromo-2-methylpropanal, t-BuOK, 18-crown-6, CH3CN, rt, 76%; (b) Ph3PCH3Br,
NaHMDS, THF, rt; (c) 120 °C, DMF; (d) acryloyl chloride, Et3N, CH2Cl2, rt, 50%, three
steps; (e) second generation Grubbs’ catalyst, CH2Cl2, reflux, 65%.
Figure 2. Comparative analysis of mechanism of action of osthol (1) and 7-
demethoxyanalog 4.

S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3717–3720
3719
M)
Table 1
Inhibitory activity of osthol (1) and its analogs for Rev-export in HeLa cells
Compound
IC50 (l
Osthol (1)
1.6
4
6.4
10
11
12
>25*
>25*
>25*
Figure 4. Congeners used for structural requirement of biological potency of 1.
*
The two analogs (10 and 11) exhibited no inhibitory activity, while the 3,4-
dihydroanalog 12 showed only 2.6% of inhibition for Rev-export at a concentration
of 25 M.
osthol (1), the demethoxyanalog 4 exhibited nearly the same
biological behavior as 1 in the competitive experiment with the
biotinylated probe 3 (Fig. 2). Thus, osthol (1) was clarified to inhibit
Rev-export through the NES non-antagonistic mode different from
LMB, valtrate, and ACA.
l
revealed to be particularly involved in the Rev-export inhibitory
potency of osthol (1).
Finally, we analyzed the structural requirement of the biological
potency of 1 by use of the two commercially available coumarins
(10 and 11) and the synthesized analog 12 lacking the double bond
conjugated to the lactone carbonyl residue (Fig. 4). The synthesis of
3,4-dihydroanalog is illustrated in Scheme 2. Namely, 2-hydroxy-
4-methoxybenzaldehyde (13) was subjected to Wittig two-carbon
homologation by (carbetoxymethylene)triphenylphosphorane to
In summary, utilization of the fission yeast, expressing the mod-
el fusion protein comprised of GST, SV40 T antigen NLS, GFP, and
Rev-NES, in the bioassay disclosed osthol (1) as the new Rev-export
inhibitor from the MeOH extract of Cnidii Monnieris Fructus. Fur-
thermore, 1 was also found to inhibit export the genuine Rev in
HeLa cells by indirect fluorescent antibody technique. By the com-
petitive experiment using the biotinylated probe 3, osthol (1) was
revealed to inhibit nuclear export of Rev through the NES non-
antagonistic mode. Based on structure–activity relationship analy-
sis of several analogs of 1, both prenyl side chain and double bond
adjacent to the lactone carbonyl residue was shown to be crucial
for the biological potency of 1.
Previously osthol (1) was reported to exhibit anti-HIV activity,
therefore inhibition for Rev-export was established as one of the
mechanism of actions related to anti-HIV activity of 1 from the
present study. Up to date, we have presented valtrate, ACA, and
(10E,12Z)-9-hydroxyoctadeca-10,12-dienoic acid as the Rev-export
inhibitors from the medicinal plant resources. The three active
principles inhibit Rev-export in the bioassay using HeLa cells with
provide a,b-unsaturated ester 14. Successive hydrogenation under
a H2 atmosphere and installation of the 1-formyl-1-methylethyl
group for the ester 14 afforded aldehyde 15. Conversion of the for-
myl function in 15 to the terminal olefin followed by Claisen rear-
rangement of the resulting allyl ether 16 gave prenylated phenol
17. Saponification and subsequent lactonization by EDCIꢀHCl and
HOBt furnished the planned 3,4-dihydroanalog 12 (Scheme 2).18
Table 1 summarizes inhibitory activity of osthol (1) and the four
analogs for export of Rev in the bioassay using HeLa cells. As de-
scribed above, the demethoxyanalog 4 retains the activity in com-
parison with 1, whereas the potency of the other analogs are
significantly reduced. With regard to the three analogs 10–12, they
showed no or little inhibition for Rev-export at a concentration of
IC50 of 2.5, 3.6, and 7.2 lM, respectively. Hence, osthol (1) should
25 lM. Judging from these biological scores, both prenyl function
and double bond conjugated to the lactone carbonyl residue were
be noted by not only difference in structural feature but also
remarkable Rev-export potency as compared with these precedent
inhibitors.
Acknowledgments
We wish to thank Professor Minoru Yoshida in RIKEN Advanced
Science Institute for giving the fission yeast Schizosaccharomyces
pombe. We also thank Professor Tominori Kimura in College of
Pharmaceutical Sciences, Ritsumeikan University for providing
the plasmid of HA-Rev. This work was supported in part by
Grants-in-Aid for Scientific Research from the Japan Society for
the Promotion of Science. The authors are grateful to the Shorai
Foundation for Science and Technology for financial support.
References and notes
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9. After picking of an aliquot of colony of S. pombe on the agar, the yeasts were
transferred and cultured in the thiamine-free MM-medium to induce the
fusion protein for 24 h at 37 °C. Then the cells were seeded in 96-well
microplates along with the test samples in the medium containing 1% DMSO
and incubated at 37 °C for further 3 h. Distribution of the GST-NLS-GFP-
RevNES-fused protein was monitored with a fluorescence microscope.
Scheme 2. Synthesis of 3,4-dihydroanalog 12. Reagents and conditions: (a)
Ph3P@CHCOOEt, THF, rt, 89%; (b) H2, Pd–C, MeOH, rt, 95%; (c) 2-bromo-2-
methylpropanal, t-BuOK, 18-crown-6, CH3CN, rt, 68%; (d) Ph3PCH3Br, NaHMDS,
THF, rt; (e) DMF, 120 °C, 41%, two steps; (f) 1 N aq. NaOH, rt; (g) EDCIꢀHCl, HOBt,
CH2Cl2, rt, 95%, two steps.

3720
S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3717–3720
10. Harkar, S.; Razdan, T. K.; Waight, E. S. Phytochemistry 1984, 23, 419.
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HCl, 0.1% NonidetP40, 0.15 mM NaCl, 2 M 2-mercaptoethanol, 1% protease
inhibitor cocktail-DMSO) was added and the mixture was sonicated for 10 min
at 0 °C. After centrifugation at 15,000 rpm for 30 min, the supernatant was
treated with 50 lL of 50% (v/v) beads immobilized with streptavidin in TBS
lysis buffer under rotation at 4 °C overnight. The beads were rinsed thrice by
12. HeLa cells (1.0 ꢁ 105 cells) were maintained on coverslips in 24-well
microplate with 1 mL of Dulbecco’s MEM medium supplemented with 10%
FBS at 37 °C in 5% CO2 for 24 h. Transfection of pCG-HA-Rev (plasmid encoding
the lysis buffer, then the bound proteins were eluted by SDS–PAGE sample
HA-tagged Rev protein) and pCRRE/
D
Rev (plasmid encoding Gag protein)
buffer (50 lL) under boiling at 95 °C for 5 min. Each eluate was separated by 5–
plasmids into HeLa cells were performed using PolyFect transfection reagent
kit (QIAGEN) for 16 h according to the manufacturer’s instructions. After the
cells were washed, each solution of tested sample at an appropriate
concentration in the medium containing 1% DMSO was inoculated and the
whole was incubated at 37 °C for further 12 h. Cells were rinsed with cold
D-PBS (ꢂ) twice and fixed with 4% formaldehyde/ D-PBS (ꢂ) for 20 min. Then
the cells were defatted with MeOH under shaking for 10 min and washed with
cold D-PBS (ꢂ) thrice. After treatment with 10% FBS in Dulbecco’s MEM
medium for 30 min, the samples were incubated with anti-HA antibody
(Roche) for 45 min followed by incubation with FITC-labeled anti-mouse IgG
antibody (Vector) for 45 min. Localization of the HA-tagged Rev protein in the
cells was examined with a fluorescence microscope, then image analysis was
conducted by Scion image software (Scion) to determine Rev-export inhibitory
activity.
20% SDS–PAGE, then the proteins were transferred to PVDF membrane and the
blot was blocked with 5% milk in TBS-T at 4 °C overnight. The membrane was
incubated with primary antibody to CRM1 (Santa Cruz Biotech) at room
temperature for 1 h. The bound antibodies were detected by treatment with
horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham
Pharmacia Biotech) at room temperature for 1 h, then the blots were
visualized using enhanced chemiluminescence.
15. Zhou, P.; Takaishi, Y.; Duan, H.; Chen, B.; Honda, G.; Itoh, M.; Takeda, Y.;
Kodzhimatov, O. K.; Lee, K. H. Phytochemistry 2000, 53, 689.
16. Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999, 1, 953.
17. Compound 4: an amorphous white powder, IR m H
max (KBr) cmꢂ1: 1734, 1609, 1
NMR (300 MHz, CDCl3) d: 7.70 (1H, d, J = 9.5 Hz, 4-H), 7.40, 7.32 (1H both, br d,
J = 7.3 Hz, 5- and 7-H), 7.20 (1H, dd, J = 7.3, 7.3 Hz, 6-H), 6.42 (1H, d, J = 9.5 Hz,
3-H), 5.34 (1H, t, J = 7.3 Hz, 20-H), 3.56 (2H, d, J = 7.3 Hz, 10-H), 1.76 (6H, s, 40-
and 50-H), FAB-MS (m/z): 215 (M+H)+, HR FAB-MS (m/z): calcd for C14H14O2+H;
215.1072, found: 215.1098.
13. Kudo, N.; Matsumori, N.; Taoka, H.; Fujiwara, D.; Schreiner, E. P.; Wolff, B.;
Yoshida, M.; Horinouchi, S. Proc, Natl. Acad. Sci. U.S.A. 1999, 96, 9112.
14. In culture dishes (3 cm id), HeLa cells (2.0 ꢁ 105 cells) were cultured in 1 mL of
Dulbecco’s MEM medium containing with 10% fetal bovine serum at 37 °C in
18. Compound 12: an amorphous white powder, IR m
max (KBr) cmꢂ1: 1715, 1H NMR
(300 MHz, CDCl3) d: 6.96 (1H, d, J = 8.4 Hz, 5-H), 6.61 (1H, d, J = 8.4 Hz, 6-H),
5.18 (1H, br t, J = 7.3 Hz, 20-H), 3.83 (3H, s, OMe), 3.39 (2H, d, J = 7.3 Hz, 10-H),
2.92, 2.74 (2H both, m, 3- and 4-H), 1.79, 1.66 (3H both, br s, 40- and 50-H), FAB-
MS (m/z): 247 (M+H)+, HR FAB-MS (m/z): calcd for C15H18O3+H; 247.1334,
found: 247.1332.
5% CO2 for 24 h. After the whole was washed, the cells were treated with 1 lM
concentration of biotinylated LMB probe 3 in 1 mL of the medium containing
1% DMSO for 3 h. For competitive experiments, osthol (1), 7-demethoxyanalog
4, and LMB (Cosmo Bio) were injected 1 h prior to addition of 3, respectively.
The cells were harvested, then 0.2 mL of TBS lysis buffer (pH 7.5, 20 mM Tris–