Unprecedented NES non-antagonistic inhibitor for nuclear export of Rev from Sida cordifolia

Article abstract of DOI:10.1016/j.bmcl.2010.01.165

Bioassay-guided separation from the MeOH extract of the South American medicinal plant Sida cordifolia resulted in isolation of (10E,12Z)-9-hydroxyoctadeca-10,12-dienoic acid (1) as an unprecedented NES non-antagonistic inhibitor for nuclear export of Rev. This mechanism of action was established by competitive experiment by the biotinylated probe derived from leptomycin B, the known NES antagonistic inhibitor. Additionally, structure-activity relationship analysis by use of the synthesized analogs clarified cooperation of several functionalities in the Rev-export inhibitory activity of 1.

Full text of DOI:10.1016/j.bmcl.2010.01.165

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1837–1839
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Unprecedented NES non-antagonistic inhibitor for nuclear export of Rev
from Sida cordifolia
Satoru Tamura a, Masafumi Kaneko b, Atsushi Shiomi a, Guang-Ming Yang a, Toshiaki Yamaura a,
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:
Bioassay-guided separation from the MeOH extract of the South American medicinal plant Sida cordifolia
resulted in isolation of (10E,12Z)-9-hydroxyoctadeca-10,12-dienoic acid (1) as an unprecedented NES
non-antagonistic inhibitor for nuclear export of Rev. This mechanism of action was established by com-
petitive experiment by the biotinylated probe derived from leptomycin B, the known NES antagonistic
inhibitor. Additionally, structure–activity relationship analysis by use of the synthesized analogs clarified
cooperation of several functionalities in the Rev-export inhibitory activity of 1.
Received 11 December 2009
Revised 26 January 2010
Accepted 30 January 2010
Available online 6 February 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
(10E,12Z)-9-hydroxyoctadeca-10,12-dienoic
acid
Rev-export inhibitor
NES non-antagonistic inhibitor
Anti-HIV
Sida cordifolia
The acquired immunodeficiency syndrome (AIDS) is a life-
threatening disease caused by HIV-1 virus.1 In recently, the HIV
pandemic has remained one of the most serious threat to world-
wide public health.2 Replication of HIV-1 entails an ordered pat-
tern of the viral gene expression dependent on the viral
regulatory protein, Rev. Namely, the Rev protein facilitated nucle-
ar-cytoplasm export of mRNA responsible for production of the vir-
al proteins and is regarded as an essential factor for viral
replication.3 Recently, transport of Rev was shown to be mediated
by the receptor protein, chromosomal region maintenance 1
(CRM1), through tight interaction to a specific leucine-rich nuclear
export signal (NES) of Rev.4 Since export of Rev was crucial for viral
proliferation, an inhibition for this function of Rev is an attractive
strategy for therapeutic intervention.5 Thus, we have been engaged
in exploration for Rev-export inhibitors from medicinal plants. Pre-
viously, we revealed valtrate and 10-acetoxychavicol acetate as ac-
tive principles with anti-HIV activity. Moreover, they were shown
to inhibit export of Rev due to NES antagonistic mode as well as the
known inhibitor leptomycin B,6 the metabolite of actinomyces.7,8
On the other hand, no NES non-antagonistic Rev-export inhibitors
have been found out in spite of promising potentiality for anti-HIV
leads with novel mechanism of actions. This circumstance
prompted us to explore unprecedented NES non-antagonistic
inhibitors for nuclear export of Rev from medicinal plants. Here,
we deal with not only biological behavior of (10E,12Z)-9-hydroxy-
octadeca-10,12-dienoic acid (1), the first NES non-antagonistic
inhibitor for Rev-export, isolated from the South American medic-
inal plant Sida cordifolia but also structural requirement for biolog-
ical potency of 1 by structure–activity relationship analysis among
the synthesized analogs.
In the course of search for Rev-export inhibitors, we utilized fis-
sion yeast4 expressing the fusion protein, which consists of gluta-
thione S-transferase (GST), SV40 T-antigen nuclear localization
signal (NLS), green fluorescent protein (GFP), and Rev-NES.7 The fu-
sion protein possesses fairly different sequence of amino acids and
conformation except for the region around Rev-NES. Accordingly,
the bioassay using this fission yeast is intensively anticipated to
bring about the inhibitors with NES antagonistic mode. Based on
this consideration, we designed to enroll another bioassay, in
which distribution of the whole Rev protein tagged with HA in
HeLa cells was monitored by indirect fluorescent antibody tech-
nique,8,9 to search for NES non-antagonistic inhibitors for nuclear
export of Rev. In brief, the desired NES non-antagonistic inhibitor
interrupted only nuclear export of HA-Rev in HeLa cells.
* Corresponding author at present address: Kyoto Pharmaceutical University.
Tel.: +81 75 595 4634; fax: +81 75 595 4768.
E-mail addresses: murakami@poppy.kyoto-phu.ac.jp, murakami@phs.osaka-u.
ac.jp (N. Murakami).
As a result of screening about 400 extracts from medicinal
plants by combination of the two bioassays, the MeOH extract of
the South American medicinal plant S. cordifolia (powder of the
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.165

1838
S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1837–1839
ar export of Rev with NES non-antagonistic mode was revealed to
be one of the mechanism of actions related to anti-HIV activity of 1.
Subsequently, structure–activity relationship was examined to
elucidate crucial partial structures for Rev-export inhibitory activ-
ity of 1. Namely, we designed two analogs 3 and 4 containing the
shortened alkyl chains between the hydroxyl and carboxylic
groups, the hydroxycarboxylic acid 5 lacking the nona-1,3-diene
moiety, the hydroxydiene 6 devoid of the 1-carboxyhexyl portion,
and the fully saturated analog 7 along with the commercially avail-
able deoxoanalog 8 (Fig. 3).
The synthesis of these analogs was executed as shown in
Scheme 1. Condensation of the known aldehydes (12a and
b),13,14 prepared according to the described procedures, with bis-
(tri-n-butylstannyl)-ethylene (13) in the presence of n-BuLi affor-
ded vinylstannyl alcohols (14a and b). Respective Stille coupling
between 14a or 14b, and (Z)-1-iodo-1-heptene 1515 followed by
saponification furnished the desired two analogs (3 and 4).16 In
the same manner, (10E,12Z)-9-hydroxyoctadeca-10,12-dienoic
acid (1) was also synthesized from 11 in a racemic form in 29.4%
overall yield for four steps. Our synthetic route of 1 was shorter
than the previous protocols in spite of similarity in the overall
yields.17–19
Figure 1. NES non-antagonistic inhibitor for nuclear export of Rev from S. cordifolia.
whole plants) was found out as a promising candidate. After this
extract was successively partitioned 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, ODS column chromatography, and reversed phase HPLC
under the guidance of bioassay to give the active principle exhibit-
ing 54.7% of inhibition for nuclear export of Rev at the concentra-
tion of 10 lg/mL. On the basis of comparison of spectroscopic data
such as 1H, 13C NMR, and IR with those reported10 as well as the
molecular formula of C18H40O3 determined by HR FAB-MS, this ac-
tive principle was identified to be (10E,12Z)-9-hydroxyoctadeca-
10,12-dienoic acid (1) (Fig. 1). To examine optical purity of 1, the
optical rotation of the corresponding methyl ester 1a was com-
pared with that reported after conversion by trimethylsilyldia-
zomethane treatment in MeOH. However, optical rotation of 1a
is +1.5° far from the reported datum11 of S-1a, +8.3° in measure-
ment under the same condition (c 0.3, CHCl3). In addition, HPLC
analysis by chiral column presented 1 to be a mixture of R- and
S-isomers in a ratio of 40:60. On the other hand, both optical pure
R- and S-isomers separated by chiral HPLC exhibited as potent
activity as the isolated 1, indicative of no participation of the con-
figuration at C-9 in the biological potency.
Treatment of aldehyde 12c20 with methylmagnesium bromide
afforded secondary alcohol 16, which was subjected to alkaline
hydrolysis to provide the analog 5. On the other hand, Sonogashira
coupling between 3-butyn-2-ol (17) with iodide 15 and the follow-
ing Red-AlÒ reduction gave the analog 6 by way of ene-yne alcohol
18. Successive treatment of the aldehyde 12c with ethynylmagne-
sium chloride followed by introduction of 1-heptenyl function by
Sonogashira coupling afforded conjugated ene-yne alcohol 20.
The alcohol was subjected to hydrogenation in the presence of
Pd–C under a H2 atmosphere and subsequent alkaline hydrolysis
by aq NaOH to furnish the analog 7.
Table 1 summarizes inhibitory activity of 1 and the analogs (3–
8) for nuclear export of Rev in HeLa cells. Among the six analogs,
the three analogs 4, 5, and 8 exhibited about two or threefold less
potent activity than 1, while the other analogs (3, 6, and 7) showed
less than 50% inhibition for Rev-transport even at the concentra-
Next, we performed comparative analysis of the mechanism of
action of (10E,12Z)-9-hydroxyoctadeca-10,12-dienoic acid (1) in
the competitive experiment using our biotinylated probe 27 de-
rived from leptomycin B, the NES antagonistic inhibitor. As de-
picted in Figure 2, pre-incubation of 1 prior to addition of 2
resulted in little reduction of intensity of CRM1 captured by the
probe 2 even at the concentration of 100 lM. Consequently, 1
tion of 30 lM. Based on these findings, the carboxyl moiety, the
was clarified to inhibit nuclear export of Rev with NES non-antag-
onistic mode, completely distinct from the known NES antagonistic
inhibitors, leptomycin B, valtrate, and 10-acetoxychavicol acetate.
Since (10E,12Z)-9-hydroxyoctadeca-10,12-dienoic acid (1) was
previously reported as an anti-HIV principle,12 inhibition for nucle-
Figure 2. Comparative analysis of mechanism of action between 1 and leptomycin
B by use of biotinylated probe.
Figure 3. Analogs of 1 for structure–activity relationship analysis.

S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1837–1839
1839
Scheme 1. Synthesis of analogs of 1. Reagents and conditions: (a) NaOMe, MeOH; (b) (COCl)2, dimethylsulfoxide, Et3N, ꢀ78 °C, 85% for two steps; (c) H2, Pd–C, 2,6-lutidine,
73%; (d) O3, CH2Cl2 then Me2S, 97%; (e) 13, nBuLi, THF, 53% (n = 1), 50% (n = 3), 55% (n = 6); (f) 15, PdCl2(CH3CN)2, DMF, 60% (n = 1), 62% (n = 3), 67% (n = 6); (g) K2CO3, aq
MeOH, 95% for 3 (n = 1), 98% for 4 (n = 3), quant. for 1 (n = 6); (h) MeMgBr, THF; (i) NaOH, aq MeOH, 70% from 12c; (j) 15, PdCl2(CH3CN)2, CuI, piperidine, 74% for 18, 62% for
20; (k) Red-Al, THF, 67%; (l) ethynylmagnesium chloride, THF, 68%; (m) H2, Pd–C, MeOH, 78%; and (n) NaOH, aq MeOH, 80%.
Table 1
References and notes
Inhibitory activity of 1 and analogs for nuclear export of Rev
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Trideca analog (3)
Pentadeca analog (4)
C1–C10 analog (5)
C8–C18 analog (6)
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ipate in inhibition for nuclear export of Rev, cooperatively.
In conclusion, bioassay-guided separation from the MeOH ex-
tract of the South American medicinal plant S. cordifolia resulted
in isolation of (10E,12Z)-9-hydroxyoctadeca-10,12-dienoic acid
(1) as the unprecedented NES non-antagonistic inhibitor for nu-
clear export of Rev. This mechanism of action was established
by the competitive experiment with the biotinylated probe 2 de-
rived from leptomycin B, the known NES antagonistic inhibitor.
Additionally, structure–activity relationship analysis by using
the synthesized analogs clarified cooperation of the carboxyl
moiety, the conjugated diene portion, the distance between these
functions, and the 9-hydroxyl group in the Rev-export inhibitory
activity of 1.
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16. Compound 3: a white amorphous powder. IR (KBr): 3310, 1758, 1642 cmꢀ1
.
1H
NMR (500 MHz, CDCl3) d 6.53 (1H, dd, J = 15.2, 11.0 Hz, 6-H), 5.97 (1H, dd,
J = 11.0, 10.5 Hz, 7-H), 5.65 (1H, dd, J = 15.2, 7.0 Hz, 5-H), 5.47 (1H, dt, J = 10.5,
7.0 Hz, 8-H), 4.25 (1H, m, 4-H), 2.51 (2H, t, J = 7.0 Hz, 2-H), 2.16 (2H, m, 9-H),
1.25–1.94 (8H, m, 3-, 10-, 11-, 12-H), 0.89 (3H, t, J = 6.9 Hz, 13-H). FAB-MS m/z:
227 [M+H]+. FAB-HRMS m/z: Calcd for C13H23O3: 227.1647. Found: 227.1650.
Compound 4: a white amorphous powder. IR (KBr): 3280, 1756, 1642 cmꢀ1 1H
.
NMR (500 MHz, CDCl3) d 6.48 (1H, dd, J = 15.5, 11.5 Hz, 8-H), 5.97 (1H, dd,
J = 11.5, 10.5 Hz, 9-H), 5.65 (1H, dd, J = 15.5, 7.5 Hz, 7-H), 5.45 (1H, dt, J = 10.5,
7.9 Hz, 10-H), 4.18 (1H, m, 6-H), 2.36 (2H, t, J = 7.6 Hz, 2-H), 2.18 (2H, m, 11-H),
1.26–1.71 (12H, m, 3-, 4-, 5-, 12-, 13-, 14-H), 0.89 (3H, t, J = 6.7 Hz, 15-H). FAB-
MS m/z: 255 [M+H]+. FAB-HRMS m/z: Calcd for C10H19O3: 255.1960. Found:
255.1964.
Acknowledgments
This work was supported in part by Grants-in-Aid for Scien-
tific Research (Grant No. 19590100) from the Ministry of Educa-
tion, Science, Culture and Sports. The authors are grateful to the
Shorai Foundation for Science and Technology for financial
support.
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