Bioisostere of valtrate, anti-HIV principle by inhibition for nuclear export of Rev

Article abstract of DOI:10.1016/j.bmcl.2010.02.038

Rational design by the MO calculation disclosed 5,6-dihydrovaltrate (2) as the bioisostere of valtrate (1), the Rev-export inhibitor with anti-HIV activity. The synthesis of 2 was accomplished by ingenious use of asymmetric Diels-Alder reaction and stereoselective epoxidation associated with the adjacent hydroxyl group. Because of similar biological potency to 1, the analog 2 should be recognized as a promising scaffold for new anti-HIV agents with an unprecedented mechanism of action, inhibition for nuclear export of Rev protein, in the conventional remedy.

Full text of DOI:10.1016/j.bmcl.2010.02.038

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2159–2162
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Bioisostere of valtrate, anti-HIV principle by inhibition for nuclear export of Rev
Satoru Tamura a, Nobuhiro Shimizu b, Katsuaki Fujiwara a, Masafumi Kaneko b, Tominori Kimura c,d
,
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
c College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji Higashi, Kusatsu, Shiga 525-8577, Japan
d Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8506, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Rational design by the MO calculation disclosed 5,6-dihydrovaltrate (2) as the bioisostere of valtrate (1),
the Rev-export inhibitor with anti-HIV activity. The synthesis of 2 was accomplished by ingenious use of
asymmetric Diels–Alder reaction and stereoselective epoxidation associated with the adjacent hydroxyl
group. Because of similar biological potency to 1, the analog 2 should be recognized as a promising scaf-
fold for new anti-HIV agents with an unprecedented mechanism of action, inhibition for nuclear export of
Rev protein, in the conventional remedy.
Received 26 December 2009
Revised 1 February 2010
Accepted 9 February 2010
Available online 13 February 2010
Keywords:
Valtrate
Ó 2010 Elsevier Ltd. All rights reserved.
Rev-export inhibitor
Anti-HIV
Iridoid
Bioisostere
The acquired immunodeficiency syndrome (AIDS) is a life-
threatening disease caused by HIV-1 and the HIV pandemic
remains one of the most serious threats to worldwide public
health.1 Rev protein of HIV-1 was clarified to play an essential role
in viral replication by constructing the structural proteins after
export of the viral mRNA from the nucleus to the cytoplasm with
the aid of the cargo protein CRM1.2,3 Inhibition for nuclear export
of Rev was, therefore, recognized as one of the attractive targets for
new anti-HIV drugs with an unprecedented mechanism of action.
In practice, leptomycin B, the first nuclear export inhibitor of
Rev, was reported to show anti-HIV activity.4,5 On the other hand,
we found out valtrate (1) as the inhibitor for nuclear export of Rev
from the medicinal plant Valerianae Radix (root of Valeriana fau-
riei) and revealed 1 to inhibit proliferation of HIV-1. Furthermore,
valtrate (1) was shown to be linked to the Cys-529 residue in
CRM1 by the covalent bond between the epoxy moiety in 1 and
the thiol group in Cys-529, this indicating that the epoxy portion
is the conclusive function to exert the bioactivity of 1 (Fig. 1).6
Despite the potential biological activity as well as the elucidated
mechanism of action, utilization of 1 as a scaffold toward anti-
HIV agents seemed infeasible because of both scarce supply from
natural resource (6.3 ꢀ 10ꢁ3% from crude drug) and strict limita-
tion of derivatization of 1; removal of the isovaleryl group at C-1
facilely brought about decomposition by way of dial with involving
elimination of the acetoxy group. In this context, search for the bio-
isosteres of 1 was intensively considered to be important for devel-
opment of new anti-HIV agents. This manuscript deals with the
* Corresponding author. Address: Kyoto Pharmaceutical University. Tel.: +81 75
595 4634; fax: +81 75 595 4768.
E-mail addresses: murakami@phs.osaka-u.ac.jp, murakami@poppy.kyoto-
phu.ac.jp (N. Murakami).
Figure 1. Stable conformations of valtrate (1) and 5,6-dihydrovaltrate (2) by MO
calculation.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.038

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S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2159–2162
Figure 2. Retrosynthetic analysis of 5,6-dihydrovaltrate (2).
synthesis and biological property of 5,6-dihydrovaltrate (2), the
bioisostere of valtrate (1), which will be anticipated to possess
fairly similar steric environment around the epoxy pharmacophore
to 1 through rational design by the molecular orbital calculation.
In the first instance, the most stable conformations of 1 and 2
were calculated by a semi-empirical molecular orbital method
PM3. As a result of the MO calculation, no serious difference in ste-
ric environment around the C-8 epoxy moiety appeared between 1
and 2, which intensively presented that 5,6-dihydroanalog (2)
exhibited nearly similar inhibitory activity for nuclear export of
Rev as compared with valtrate (1) (Fig. 1).
methyl acetal iii bearing the fully oxygen-functionalized iridoid
skeleton, we conducted retrosynthetic analysis of 2 as illustrated
in Figure 2. Namely, the labile 1,1-disubstituted epoxy moiety
would be constructed by taking advantage of the
a-oriented hy-
droxyl group at C-7 in the late stage. The isovaleryloxy group
would be introduced by Mitsunobu inversion for the hydroxyl
group at C-7 in the final stage. Exo-olefin ii as the precursor of
epoxide i would be afforded from methyl acetal iii. Moreover, the
acetal iii was planned to be yielded by intramolecular transacetal-
ization of enol iv, which would be obtained by condensation
between the optical active cyclopentane v and ethyl formate. The
cyclopentane v would be prepared by methanolysis of lactone vi,
which would be available from cyclic ether vii by oxidation
followed by Baeyer–Villiger rearrangement. The cyclic ether vii
was expected to be attained by stereoselective epoxidation and
intramolecular etherization from the asymmetric Deals–Alder
adduct viii between cyclopentadiene (3) and diethyl fumarate (4)
in high optical purity.
Since 5,6-dihydroanalog (2) was presumed to be synthesized
from the highly optical pure cyclopentane v by way of the bicyclic
The synthesis of the methyl acetal 12 with the optical active irid-
oid skeletonwas executed as shownin Scheme 1. AsymmetricDiels–
Alder reaction using Corey’s chiral ligand7 between cyclopentadiene
(3) and diethyl fumarate (4) afforded the adduct 5 quantitatively
with 95% ee. After reduction of the two ester functions in 5, treat-
ment of the resulting diol with mCPBA promoted not only epoxida-
tion but also intramolecular etherization to give tricyclic ether 6. The
primary hydroxyl group in 6 was selectively protected as TBDPS
ether and subsequent Dess–Martin oxidation8 provided cyclic ke-
tone 7. Reductive cleavage of the cyclic ether in 7 with Al–Hg fol-
lowed by oxidation of the resultant primary hydroxyl function
affordedaldehyde 8. Theformylgroupin 8 was subjectedtoselective
acetalization by treatment with methoxytrimethylsilane (MeOTMS)
in the presence of TMSOTf, then Baeyer–Villiger rearrangement by
mCPBA gave seven-membered lactone 9. Successive methanolysis
of 9 with NaOMe and protection of theresulting hydroxylgroupwith
the TBS residue provided cyclopentane 10, which was submitted to
installation of the formyl group mediated with ethyl formate and
lithium diisopropyl amide to provide enol 11. BF3 catalyzed trans-
acetalization of 11 smoothly proceeded concomitant with deprotec-
tion of the TBS function. In the last step, the partly obtained
hemiacetal was entirely converted to methyl acetal 12 possessing
thedesired iridoidskeletonbytreatmentwithCH(OMe)3 in thepres-
ence of p-TsOH.
Scheme 1. Reagents and conditions: (a) ligand, CH2Cl2, ꢁ35 °C, quant.; (b) LiAlH4,
THF, 0 °C, quant.; (c) mCPBA, CH2Cl2, 90%; (d) TBDPSCl, DBU, CH2Cl2, 95%; (e) Dess–
Martin periodinane, CH2Cl2, 88%; (f) Al–Hg, THF, EtOH, 92%; (g) Dess–Martin
periodinane, CH2Cl2, quant.; (h) MeOTMS, TMSOTf, CH2Cl2, 80%; (i) mCPBA, KH2PO4,
CH2Cl2, 81%; (j) NaOMe, MeOH, 90%; (k) TBSOTf, 2,6-lutidine, CH2Cl2, 80%; (l) LDA,
HCOOEt, THF, ꢁ78 °C; (m) BF3ꢂOEt2, CH2Cl2, 0 °C; (n) p-TsOH, CH(OMe)3, MeOH,
three steps 54%.
Subsequently, the synthesis of 5,6-dihydroanalog (2) was
accomplished from 12 by ingenious use of stereoselective epoxida-
tion associated by the adjacent hydroxyl group as displayed in
Scheme 2. The secondary hydroxyl group in 12 was protected as
MOM ether, then the TBDPS group was removed by treatment of

S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2159–2162
2161
by tert-butyl hydroperoxide (TBHP) and vanadium oxyacetylaceto-
nate [VO(acac)2] mediated by the adjacent hydroxyl group at C-79
gave the desired
and 10-Ha, 9-H and 10-Hb in the NOESY spectrum of 18 unequiv-
ocally established the desired -epoxy configuration. Introduction
a-epoxyalcohol 18. NOE correlations between 7-H
a
of the isovaleryloxy function to C-7 in 18 involving the steric inver-
sion of the hydroxyl function under Mitsunobu condition10 com-
pleted the synthesis of the target 5,6-dihydroanalog (2).11
Finally, the synthesized analog (2) was evaluated for inhibitory
activity for nuclear export of Rev protein.12 After transfection of
plasmid coding Rev tagged with human influenza haemagglutinin
(HA) into HeLa cells, localization of Rev protein was examined by
an indirect fluorescent antibody technique aiming at HA tag
(Fig. 3). Consequently, 5,6-dihydroanalog (2), presumed to possess
the similar steric environment around the epoxy pharmacophore
to 1 by molecular orbital calculation, inhibited export of Rev from
nucleus with IC50 of 4.4
most entirely disrupted nuclear export of Rev at the concentration
of 10 M. On the basis of these biological scores, 5,6-dihydroanalog
(2) was undoubtedly revealed to be the bioisostere of valtrate (1,
IC50: 2.5 M) as the promising scaffold of new anti-HIV agents.
lM in practice. In addition, the analog 2 al-
l
l
In conclusion, we synthesized and disclosed 5,6-dihydrovaltrate
(2) as the bioisostere of valtrate (1) with anti-HIV activity through
the rational design by the MO calculation. The analog should be
recognized as the promising scaffold for new anti-HIV agents with
the unprecedented mechanism of action, inhibition for nuclear
export of Rev protein, in the conventional remedy. Exploration
for more potent analogs modifying the three acyl groups in 2 is
currently under investigation in our laboratory.
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific
Research (Grant No. 19590100) from the Ministry of Education,
Science, Culture and Sports. The authors are grateful to the Shorai
Foundation for Science and Technology financial support.
Scheme 2. Reagents and conditions: (a) MOMCl, iPr2NEt, CH2Cl2; (b) TBAF, THF; (c)
I2, PPh3, imidazole, C6H6; (d) tBuOK, THF, 0 °C, four steps 40%; (e) DIBAL, CH2Cl2,
ꢁ78 °C; (f) Dess–Martin periodinane, CH2Cl2, two steps 80%; (g) 10% HCl–THF, 43%,
recovery of 15 42%; (h) isovaleric acid, Im2CO, DBU, CH2Cl2, 0 °C, 77%; (i) BCl3,
CH2Cl2, 0 °C, 65%; (j) NaBH4, CeCl3ꢂ7H2O, MeOH; (k) AcCl, iPr2NEt, CH2Cl2, 0 °C, two
steps 58%; (l) TBHP, VO(acac)2, C6H6, 80%; (m) isovaleric acid, DEAD, PPh3, C6H6,
85%.
References and notes
1. del Rio, C. Arch. Med. Res. 2005, 36, 682.
2. Zapp, M. L.; Stern, S.; Green, M. R. Cell 1993, 74, 969.
3. Werstuck, G.; Zapp, M. L.; Green, M. R. Chem. Biol. 1996, 3, 129.
4. Kudo, N.; Wolff, B.; Sekimoto, T.; Schreiner, E. P.; Yoneda, Y.; Yanagida, M.;
Horinouchi, S.; Yoshida, M. Exp. Cell Res. 1998, 242, 540.
5. Wolff, B.; Sanglier, J.-J.; Wang, Y. Chem. Biol. 1997, 4, 139.
6. Murakami, N.; Ye, Y.; Kawanishi, M.; Aoki, S.; Kudo, N.; Yoshida, M.; Nakayama,
E. E.; Shioda, T.; Kobayashi, M. Bioorg. Med. Chem. Lett. 2002, 12, 2807.
7. Ryu, D. H.; Lee, T. W.; Corey, E. J. J. Am. Chem. Soc. 2002, 124, 9992.
8. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
TBAF to afford 13. Iodination of the hydroxyl group in 13 and the
following dehydrohalogenation by tBuOK gave exo-olefin 14. After
reduction of 14 with DIBAL, Dess–Martin oxidation of the resulting
primary alcohol provided conjugated aldehyde 15. Selective hydro-
lysis of the methyl acetal in 15 in 10% HCl–THF gave hemiacetal,
which was coupled with isovaleric acid in the presence of DBU
and N,N0-carbonyldiimidazole (CDI) to afford isovaleryl ester 16.
Selective removal of the MOM protection by BCl3 followed by
reduction by NaBH4 in the presence of CeCl3 led 16 to the corre-
sponding diol, of which the primary hydroxyl group was selec-
tively acetylated to afford 17. Stereoselective epoxidation of 17
9. Sharpless, K. B.; Michaelson, R. C. J. Am. Chem. Soc. 1973, 95, 6136.
10. Mitsunobu, O. Synthesis 1981, 1.
11. Compound 2: colorless oil, ½a D20
ꢁ48.6 (c 2.1, MeOH), IR (KBr): 1761 (sh), 1738,
ꢃ
1670 cmꢁ1 1H NMR (500 MHz, CDCl3) d: 6.52 (1H, s, 3-H), 5.84 (1H, d,
,
J = 5.5 Hz, 1-H), 4.97 (1H, dd, J = 6.1, 6.1 Hz, 7-H), 4.65 (1H, d, J = 12.2 Hz, 11-
Ha), 4.46 (1H, d, J = 12.2 Hz, 11-Hb), 3.06 (1H, d, J = 5.5 Hz, 10-Ha), 2.95 (1H,
ddd, J = 8.5, 6.1, 6.1 Hz, 5-H), 2.82 (1H, d, J = 5.5 Hz, 10-Hb), 2.72 (1H, dd, J = 8.5,
5.5 Hz, 9-H), 2.28 (1H, J = 13.4, 6.1, 6.1 Hz, 6-Ha), 2.21 (2H, d, J = 7.3 Hz,
CO2CH2CH(CH3)2), 2.17 (2H, d, J = 7.3 Hz, CO2CH2CH(CH3)2), 2.08 (3H, s,
OCOCH3), 2.07 (1H, m, CO2CH2CH(CH3)2), 2.05 (1H, m, CO2CH2CH(CH3)2),
1.99 (1H, J = 13.4, 6.1, 6.1 Hz, 6-Hb), 0.99 (3H, d, J = 6.7 Hz, CO2CH2CH(CH3)2),
0.97 (3H, d, J = 6.7 Hz, CO2CH2CH(CH3)2), 0.96 (3H, d, J = 6.7 Hz,
CO2CH2CH(CH3)2), 0.94 (3H, d, J = 6.7 Hz, CO2CH2CH(CH3)2), FAB-MS m/z: 425
(M+H)+, FAB-HRMS m/z: Calcd for C22H32O8+H: 425.2097, Found: 425.2117.
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
HA-tagged Rev protein) and pCRRE/DRev (plasmid encoding Gag protein)
plasmids into HeLa cells were performed using PolyFectÒ 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 (ꢁ) twice and fixed with 4% formaldehyde/D-PBS (ꢁ) for 20 min. Then the
cells were defatted with MeOH under shaking for 10 min and washed with cold
Figure 3. Inhibitory activity for nuclear export of Rev by 2.

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S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2159–2162
D-PBS (ꢁ) thrice. After treatment with 10% FBS in Dulbecco’s MEM medium for
under a fluorescence microscope, then image analysis was conducted by Scion
image software (Scion) to determine Rev-export inhibitory activity. In the
depicted pictures, several cells free from transfection displayed disperse weak
fluorescence due to nonspecific binding of the antibodies.
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