New Rev-transport inhibitor with anti-HIV activity from Valerianae Radix

Article abstract of DOI:10.1016/S0960-894X(02)00624-8

Bioassay-guided separation by use of the fission yeast expressing NES of Rev, a HIV-1 viral regulatory protein, resulted in isolation of valtrate (1) as a new Rev-transport inhibitor from the nucleus to cytoplasm from Valerianae Radix. Valtrate (1) also inhibited the p-24 production of HIV-1 virus without showing any cytotoxicity against the host MT-4 cells.

Full text of DOI:10.1016/S0960-894X(02)00624-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2807–2810
New Rev-Transport Inhibitor with Anti-HIV Activity from
Valerianae Radix
Nobutoshi Murakami,a Ying Ye,a Motoyuki Kawanishi,a Shunji Aoki,a Nobuaki Kudo,b
Minoru Yoshida,b Emi E. Nakayama,c Tatsuo Shiodac and Motomasa Kobayashia,*
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
bGraduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
cResearch Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Received 14 June 2002; accepted 24 July 2002
Abstract—Bioassay-guided separation by use of the fission yeast expressing NES of Rev, a HIV-1 viral regulatory protein, resulted
in isolation of valtrate (1) as a new Rev-transport inhibitor from the nucleus to cytoplasm from Valerianae Radix. Valtrate (1) also
inhibited the p-24 production of HIV-1 virus without showing any cytotoxicity against the host MT-4 cells.
# 2002 Elsevier Science Ltd. All rights reserved.
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 dependent upon the viral regulatory
protein, Rev.2 Revacts to increase cytoplasmic accu-
mulation of the viral mRNAs, which encodes the viral
structural proteins, through the transport from the
nucleus to cytoplasm.3 As Revis critical for viral repli-
cation, inhibition of the function of Revis an attractive
strategy for therapeutic intervention.4 Recently, the
transport of Revwas shown to be mediated by the
receptor protein, chromosomal region maintenance 1
(CRM1), through the direct binding to the nuclear
export signal (NES) of Rev.5 Leptomycin B6 (2) has
been shown to inhibit the binding of the NES of Rev
(RevNES) to CRM1 and exhibits potent inhibitory
effect on the proliferation of HIV-1 virus.5,7 Further-
more, the analogous polyketide, callystatin A (3), iso-
lated from a marine sponge by our group also inhibited
Radix and a comparative analysis for the mode of
action between 1 and callystatin A (3) by use of a bio-
tinylated probe 13 (Fig. 1).
In order to search for new Rev-transport inhibitors, we
utilized a fission yeast Schizosaccharomyces pombe,5
which expresses a fusion protein consisting of gluta-
thione S-transferase (GST), SV40 T antigen nuclear
localization signal (NLS), green fluorescent protein
(GFP), and RevNES, in bioassay-directed separation.
The practical assay protocol is as follows. After inducing
the fusion protein of S. pombe in thiamine-free medium
for 24 h at 37 ꢀC, the cells were seeded in 96-well plates
along with test samples in the medium containing 1%
DMSO and incubated at 37 ꢀC for further 3 h. The dis-
tribution of the GST-NLS-GFP-RevNES-fused protein
was monitored by fluorescence microscope. Among
about 200 kinds of extracts of medicinal plants, the
extract of Valerianae Radix (the roots of Valeriana
fauriei Briquet) showed Rev-transport inhibitory
activity. Bioassay-guided separation of the extract dis-
closed valtrate (1),9 an iridoid ester previously obtained
by Thies, as a new Rev-transport inhibitor with moderate
lipophilicity.10 Valtrate (1) completely inhibited the
transport from the nucleus to cytoplasm of the fused
protein of S. pombe at the concentration of 3 mg/mL.
8
the Revtransport from the nucleus to cytoplasm. In
spite of the potent in vitro biological activity, the sig-
nificant toxic feature of the two polyketides limited their
use as chemotherapeutics. This circumstance urged us to
engage in exploring new Rev-transport inhibitors from
natural products. Here, we describe the isolation of a new
Rev-transport inhibitor, valtrate (1), from Valerianae
Recently, leptomycin B (2) was shown to link to the
Cys-529 in CRM1 by the a,b-unsaturated lactone moiety
through S–C bond formation.11 We clarified that callystatin
*Corresponding author. Tel.: +81-6-6879-8215; fax: +81-6-6879-
8219; e-mail: kobayasi@phs.osaka-u.ac.jp
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00624-8

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N. Murakami et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2807–2810
Figure 1. Chemical structures of valtrate (1), leptomycin B (2), and
callystatin A (3).
A (3) also binds to CRM1 in the same fashion. Next, we
analyzed the mode of action of valtrate (1) in comparison
with those of 2 and 3. Prior to the comparative analysis,
exploration for a feasible probe was undertaken. During
the course of our study on structure activity relationship
of 3, we found a simplified lead compound 10 with
moderate Rev-transport inhibitory activity (MIC=3.8
mM).12 Consequently, the probes were designed to connect
10 and d-biotin by using amino carboxylic acids as linkers.
In the first instance, two biotinylated probe candidates (12,
13) with linker moieties were synthesized as depicted in
Scheme 1. One linker is commercially available hydro-
chloride of glycine methyl ester (6), and the other (7)
was prepared from methyl 1-hydroxydecanoate (4) in
the following manner. Namely, treatment of 4 with
mesylchloride in the presence of Et3N afforded a mesylate,
which was submitted to azidation by NaN3 and
nBu4NBr to give an azide 5. Hydration of 5 by use of
10% Pd/C under H2 atmosphere furnished a hydro-
chloride salt of methyl 10-aminodecanoate 7 in 56%
yield for three steps. Condensation of 6 and d-biotin
Scheme 1. Synthesis of biotinylated probe (13) derived from cally-
statin A (3). Reagents and conditions: (a) MsCl, Et3N, toluene, 0 ꢀC
then NaN3, Bu4NBr, H2O, 60 ꢀC; (b) H2, 10% Pd/C, MeOH–CHCl3
n
.
(50:1), two steps 56%; (c) d-biotin, EDCI HCl, HOBt, Et3N, DMF;
(d) 0.5 N aq LiOH, dioxane, two steps quant for 8, quant for 9; (e)
.
EDCI HCl, HOBt, Et3N, DMF, 83% for 11, 86% for 12, 84% for 13.
.
with EDCI HCl, HOBt, and Et3N followed by saponi-
fication with 1 N NaOH provided a carboxylic acid 8
quantitatively. The carboxylic acid 8 was coupled with
10 under the same conditions as the condensation of 6
and biotin to furnish a biotinylated probe 12 in 86%
yield. In accordance with this protocol, the other probe
13 was synthesized from 7 via 9. At the same time,
readily accessible biotin conjugate 11 was also prepared.
Assessment of the biological scores of the three probes
revealed that the probe 13 (MIC=1 mM) completely
inhibited Rev-transport with 10-fold more potency than
the other two probes (11, 12; MIC=10 mM). Therefore,
the comparativ e analysis for the mode of action of v altrate
(1) and callystatin A (3) was conducted by using 13.13
First of all, we examined binding affinity of the bio-
tinylated probe 13 against CRM1.14 SDS-PAGE analysis
through streptavidin–biotin affinity disclosed a char-
acteristic protein band around 100 KDa (lane 2), which
was assignable to CRM1 (Fig. 2). This protein band was
not detected in the absence of 13 (lane 1) or the pre-
treatment with callystatin A (3) (lane 3). Similarly, the
addition of valtrate (1) prior to the inoculation of 13
gave rise to the same behavior as that of 3 (lane 4).
Valtrate (1) is, therefore, presumed to inhibit the Rev-
transport from the nucleus to cytoplasm through direct
binding to the Cys-529 in CRM1.
Figure 2. Analysis for the binding of valtrate (1) to CRM1 using bio-
tinylated probe (13).
In order to confirm this presumption and deduce the
reactive site of valtrate (1) with the Cys-529 in CRM1,
reaction of 1 and N-acetyl-cysteine methyl ester (14) was
examined (Fig. 3). Treatment of 1 with 14 in Tris buffer
(pH 7.5) afforded an alcohol 15.15 with concomitant
1
epoxy ring cleavage as a major reactant. The H NMR
spectrum of 15 exhibited the signals [d 2.76 (1H, d,
J=14.0 Hz), d 3.36 (1H, d, J=14.0 Hz)] due to the

N. Murakami et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2807–2810
2809
Foundation for Food Chemical Research for financial
support.
References and Notes
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5. Kudo, N.; Wolff, B.; Sekimoto, T.; Schreiner, E. P.;
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Cell Res. 1998, 242, 540.
6. Kobayashi, M.; Wang, W.; Tsutsui, Y.; Sugimoto, M.;
Murakami, N. Tetrahedron Lett. 1998, 39, 8291.
Figure 3.
methylene bearing a sulfur or an oxygen atom instead of
the methylene proton signals [d 2.91 (1H, d, J=5.0 Hz),
d 3.03 (1H, d, J=5.0 Hz)] of the epoxy portion in 1.
Location of the hydroxyl group of 15 was determined
by the isotope effect induced by exchange of an OH
group for an OD group in 13C NMR.16 In comparison
of the 13C NMR spectrum of 15 taken in CD3OH with
that in CD3OD, distinct deuterium shifts were observed
with respect to the four carbon signals (C-7, C-8, C-9,
and C-10) around C-8. In particular, the chemical shift
ascribable to C-8 showed large up-field shift by 0.10
ppm, while the other four carbons were shifted to lower
field by 0.02–0.05 ppm. Consequently, the chemical
structure of the reactant 15 was unambiguously estab-
lished as depicted in Figure 3.
7. Wolff, B.; Sanglier, J.-J.; Wang, Y. Chem. Biol. 1997, 4,
139.
8. Murakami, N.; Sugimoto, M.; Nakajima, T.; Higuchi, K.;
Aoki, S.; Yoshida, M.; Kudo, N.; Kobayashi, M. Abstracts of
Papers, 41st Symposium on the Chemistry of Natural Pro-
ducts, Nagoya, Oct., 1999; p 229. Chem. Abstr., 776311.
9. (a) Thies, P. W. Tetrahedron 1968, 24, 313. (b) Konowal,
A.; Snatzke, G.; Thies, P. W. Tetrahedron 1978, 34, 253.
10. Leo, A. J. Chem. Rev. 1993, 93, 1281 ClogP has been
taken as a calculated parameter of lipophilicity and utilized as
a convenient index to predict in vivo pharmacological potency
of medicinal leads. In general, clogPs ranging from ꢁ3 to 3 are
believed to be favorable for exerting in vivo potency. The two
polyketides possessed very large clogPs (2: 6.90, 3: 6.85), while
the clogP of 1 is 1.92. These clogPs were calculated using the
computer program (version 4.0, Bio Byte Corporation, Clar-
emont, CA 91711, USA).
Finally, anti-viral activity of valtrate (1) was assessed by
measurement of HIV-p24 antigen production in the
supernatants of the infected MT-4 cell cultures with a
commercially available HIV-antigen kit.17 Valtrate (1)
showed 44% inhibition on p-24 production at the con-
centration of 0.5 mM without showing any cytotoxicity
against the host MT-4 cells.
11. 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.
12. Murakami, N.; Kawanishi, M.; Sugimoto, M.; Matsui,
K.; Aoki, S.; Kobayashi, M. Unpublished results.
13. A colorless amorphous solid, [a]D+38.3ꢀ (c 0.05, MeOH,
27 ꢀC). IR nmax (KBr) cmꢁ1: 1726, 1697, 1680 (sh), 1650, 1575,
1263, 1244. 1H NMR (500 MHz, CDCl3/CD3OD=1:1) d: 1.02
(3H, d, J=6.4 Hz, 10-CH3), 1.06 (3H, d, J=6.2 Hz, 4-CH3),
1.09 (3H, d, J=7.2 Hz, 8-CH2CH3), 1.22–1.80 (22H, m,
CH2ꢂ11), 2.25 (2H, q, J=7.2 Hz, 8-CH2CH3), 2.35 (2H, t,
J=7.5 Hz), 2.37 (2H, t, J=7.2 Hz) (CH2CO2, 80-H2), 2.64
(1H, m, H-4), 2.74 (1H, dd, J=12.9, 3.0 Hz, H-30), 2.84 (1H,
m, H-10), 2.93 (1H, dd, J=12.9, 5.1 Hz, H-30), 3.20 (1H, m,
H-40), 3.39 (2H, CH2NHCO, overlapped with CH3OD), 3.95
(1H, dt-like, J=ca. 11, 6 Hz, H-12), 4.10 (1H, dt-like, J=ca.
11, 5 Hz, H-12), 4.33 (1H, ddd, J=7.8, 5.1, 3.0 Hz, H-20), 4.52
(1H, dd, J=7.8, 4.7 Hz, H-10), 5.10 (1H, ddd, J=6.4, 4.0, 1.1
Hz, H-5), 5.21 (1H, d, J=9.4 Hz, H-9), 5.76 (1H, dd, J=15.8,
6.4 Hz, H-6), 6.04 (1H, dd, J=9.7, 1.1 Hz, H-2), 6.73 (1H, d,
J=15.8 Hz, H-7), 7.11 (1H, dd, J=9.7, 5.6 Hz, H-3). FAB-
MS m/z: 660 (M+H)+. HR FAB-MS m/z: calcd for
C36H58N3O6S: 660.4046, found: 660.4050.
In summary, we have elucidated a new Rev-transport
inhibitor from the nucleus to cytoplasm with appro-
priate lipophilicity as medicinal leads, valtrate (1), from
Valerianae Radix according to bioassay-guided separation
using fission yeast expressing the fusion proteins of
GST-NLS-GFP-RevNES. Furthermore, the biotinylated
probe 13 was synthesized in order to compare the
modes of action between 1 and 3. The analysis of the
binding protein to 1 using 13 and the reactant 15 of 1
with N-acetyl-cystein methyl ester (14) demonstrated
that both 1 and 3 inhibit Rev-transport in the same
fashion. Exploration for synthetic leads having more
potent anti-HIV activity than 1 is in progress in our
laboratory.
14. KB 3–1 cells (8.0ꢂ105 cells) in 10 mL of RPMI medium
1640 containing with 10% fetal bovine serum were cultured in
the presence of biotinylated probe 13 at 37 ꢀC for 3 h. Valtrate
(1) and callystatin A (3) were injected 1 h prior to addition of
13, respectively. All compounds were inoculated as 10 mL
EtOH solutions and the final concentrations of 1, 3, and 13
were 2.0, 5.4, and 10 mM, respectively. After harvesting the
cells, 1.6 mL of 0.1% Nonidet P40–Tris buffer saline (TBS, pH
7.4) was added and the mixture was stirred with a vortex mixer
for 10 min at 4 ꢀC. The whole was centrifuged at 15,000 rpm
Acknowledgements
This work was supported in part by Grants-in-Aid for
Scientific Research on Priority Areas (Grant No.
13015212, N.M.) from the Ministry of Education,
Science, Culture and Sports. The authors are grateful to
the Naito Foundation, the Takeda Science Foundation,
the Senri Life Science Foundation, and the San-Ei Gen

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N. Murakami et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2807–2810
for 30 min. This procedure to collect solubilized protein was
conducted three times. The combined supernatant was treated
with 150 mL of 50% (v/v) immobilized streptoavidin (Sigma)
in TBS under rotation for 15 h, then the mixture was cen-
trifuged at 10,000 rpm for 5 min. After removing the super-
natant and washing the pellets, the bound proteins were eluted
from the pellets by SDS-PAGE sample buffer (20 mL) under
boiling at 95 ꢀC for 5 min. Each eluate was applied to poly-
acrylamide gel (Ready Gels J, Bio-Rad), then the gel was
stained with Silver Stain II Kit (Wako).
15. Yellow oil, [a]D+102.3ꢀ (c 0.20, CHCl3, 25 ꢀC). IR nmax
(KBr): 3304, 1740, 1659, 1244. 1H NMR (500 MHz, CDCl3) d:
1.01, 1.00, 0.93, 0.92 (3H each, all d, J=6.7 Hz,
CH(CH3)2ꢂ2), 2.04, 2.01 (3H each, both s, Acꢂ2), 2.06 (1H,
m, CH(CH3)2), 2.14 (2H, d, J=6.1 Hz, COCH2), 2.15 (1H, m,
CH(CH3)2), 2.29 (1H, dd, J=15.0, 6.7 Hz, COCH2), 2.31 (1H,
dd, J=15.0, 8.0 Hz, COCH2), 2.76 (1H, d, J=14.0 Hz, H-10),
2.82 (1H, dd, J=14.6, 8.5 Hz, SCH2), 2.87 (1H, dd, J=9.4, 2.4
Hz, H-9), 2.94 (1H, dd, J=14.6, 3.3 Hz, SCH2), 3.36 (1H, d,
J=14.0 Hz, H-10), 3.75 (3H, s, OCH3), 4.66, 4.60 (2H, ABq,
J=12.2 Hz, H2-11), 4.88 (1H, ddd, J=10.4, 8.5, 3.3 Hz,
CHNHAc), 5.31 (1H, d, J=3.1 Hz, H-7), 5.76 (1H, dd,
J=3.1, 2.4 Hz, H-6), 6.23 (1H, d, J=10.4 Hz, NH), 6.25 (1H,
d, J=9.4 Hz, H-1), 6.63 (1H, s, H-3). 13C NMR (125 MHz,
CD3OD)
dc:
174.6,
173.4
(2C),
173.4,
173.1
(COCH2CH(CH3)2ꢂ2, Acꢂ2), 150.1 (C-3), 142.0 (C-5), 119.2
(C-6), 111.3 (C-4), 95.2 (C-1), 86.2 (C-7), 83.3 (C-8), 62.8 (C-11),
55.0 (OCH3), 53.7 (CHNAc), 52.7 (C-9), 45.2, 44.8 (COCH2ꢂ2),
41.9 (C-10), 38.3 (SCH2), 27.7, 27.6 (CH(CH3)2ꢂ2), 23.6, 23.5,
23.4, 23.4, 23.2 (CH(CH3)2ꢂ2, NHAc), 21.6 (OAc). FAB-MS
m/z: 622 (M+Na), FAB-HRMS m/z: calcd for C28H40NO11SNa:
622.2298, found: 622.2275.
16. Gagnaire, D.; Vincendon, M. Chem. Commun. 1977, 509.
17. MT-4 cells were suspended at a density of 5ꢂ105 cells/mL
of RPMI medium 1640 supplemented with 10% fetal bovine
serum. The cells were infected with HIV-1 strain NL43 for 2 h
at 37 ꢀC and washed twice, then 100 mL of infected cell sus-
pension (4ꢂ105 cells/mL) was added to each well of microtiter
trays containing 100 mL of appropriate dilution of the test
compound. After incubation for 3 days at 37ꢀC, the production
of HIV-p24 antigen in the supernatants was evaluated by using a
commercially available HIV-antigen kit (ZeptoMetrix).