Total synthesis and adjuvant activity of all stereoisomers of pinellic acid

Article abstract of DOI:10.1016/S0960-894X(02)01069-7

Pinellic acid is a novel and potentially useful oral adjuvant when used in conjunction with intranasal inoculation of influenza HA vaccines. All stereoisomers of pinellic acid have been synthesized via regioselective asymmetric dihydroxylation, regioselective inversion, and stereoselective reduction, and their adjuvant activities were characterized. Among this series of isomers, 9S, 12S, 13S compound has the most potent adjuvant activity. Structure-activity relationships are discussed.

Full text of DOI:10.1016/S0960-894X(02)01069-7

Bioorganic& Medicinal Chemistry Letters 13 (2003) 937–941
Total Synthesis and Adjuvant Activity of All Stereoisomers of
Pinellic Acid
Tatsuya Shirahata, Toshiaki Sunazuka, Kiminari Yoshida, Daisuke Yamamoto,
Yoshihiko Harigaya, Takayuki Nagai, Hiroaki Kiyohara, Haruki Yamada,
Isao Kuwajima and Satoshi Omura*
-
The Kitasato Institute for Life Science, and School of Pharmaceutical Science, Kitasato University, and The Kitasato Institute,
Shirokane, Minatoku, Tokyo 108-8641, Japan
Received 15 October 2002; accepted 6 December 2002
Abstract—Pinellic acid is a novel and potentially useful oral adjuvant when used in conjunction with intranasal inoculation of
influenza HA vaccines. All stereoisomers of pinellic acid have been synthesized via regioselective asymmetric dihydroxylation,
regioselective inversion, and stereoselective reduction, and their adjuvant activities were characterized. Among this series of iso-
mers, 9S, 12S, 13S compound has the most potent adjuvant activity. Structure–activity relationships are discussed.
# 2003 Elsevier Science Ltd. All rights reserved.
Influenza is a highly infectious acute respiratory disease
caused by specific influenza viruses leading to both
worldwide pandemics and local outbreaks.
When (À)-1 was found, the absolute configuration of
(À)-1 was unknown. Thus, we attempted the first total
synthesis of (À)-1 and determined the relative and
absolute configuration of (–)-1, which is 9S, 12S, 13S.
(Fig. 1).3
Influenza virus infection is sometimes critical for
patients with respiratory diseases such as bronchial
asthma, immunosuppressive syndrome such as AIDS,
or for aged persons with cardiopulmonary disease.1
This paper deals with the total synthesis of all stereo-
isomers of pinellic acid, 9, 12, 13-trihydroxy-10-octa-
deconoic acid, and their adjuvant activities to clarify the
structure–activity relationships.
Influenza vaccine is useful as prophylaxis of influenza
virus infection.1 Intranasal inoculation of influenza
vaccine has been tried to increase its safety and to pre-
vent the antigenicvariation of influenza viruses. None-
theless, it has generally been considered that intranasal
inoculation of the vaccine alone cannot readily induce
high levels of antibodies. Therefore, the development of
effective adjuvants for influenza vaccine administered
intranasally is necessary to enhance the potency of the
vaccine.
For the purpose of stereoselective syntheses of all the
stereoisomers of (À)-1, the following syntheticplan was
devised as shown in Figure 2. There are three key
points: (1) stereoselective reduction from dienone in order
to prepare the allylicalochol at C-9, (2) regioseletcive
asymmetricdihydoxylation of the dienone to give
12,13-syn-diol, (3) inversion of the C-13 hydroxy group
from C-12, 13-syn-diol for C-12, 13-anti-diol (vide infra).
Pinellicaicd (À)-1 isolated from an oriental medicine,
Pinelliae tuber, is a novel and potentially useful oral
adjuvant when used in conjunction with intranasal
inoculation of influenza HA vaccines.2 (À)-1 may prove to
be a useful and safe oral adjuvant for mucosal vaccines.
In our previous work,3 a syntheticroute of C-12, 13-
syn-diol was established the outline of which is shown in
Scheme 1. A syntheticproblem for C-12, 13- syn-diol
(À)-3 was solved by use of regioselective asymmetric
dihydroxylation4 of 2 and stereoselective reduction5 of
(+)-4. The 12, 13-anti-diol could be constructed via
regioselective protection of the C-12 hydroxy group in
12, 13-syn-diol followed by inversion of the C-13
hydroxy group.
*Corresponding author. Tel.:+81-3-5791-6101; fax:+81-3-3444-8360;
e-mail: omura-s@kitasato.or.jp
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01069-7

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T. Shirahata et al. / Bioorg. Med. Chem. Lett. 13 (2003) 937–941
(TIPSOTf, 2,6-lutidine) in good yield without formation
of the C-13-O-TIPS compound because of high reactiv-
ity of the allylicalcohol. Next, we attempted inversion
of the hydroxy group at C-13. While the Mitsunobu
inversion6 was not successful because of the hindered
O-TIPS group, Nakata’s method,7 using mono-
chloromethanesulfonyl (ClSO2CH2Cl, pyridine) group
followed by treatment with CsOAc, gave C-12, 13-anti-
diol in good yield. Deprotection of dithioacetal furn-
ished enone (+)-8. The stereoselective reduction of (+)-8
by (S)-BINAL-H gave 9S alcohol (+)-9, which was
easily separated as a single isomer by silica gel chroma-
tography (diastereoselectivity; 13:1). Finally deprotec-
tion of acetyl and tert-butyl groups by hydrolysis and
desilylation with TBAF gave (+)-10 (Scheme 2).
Figure 1. Structure of pinellic acid (À)-1.
The preparation of (+)-10 which has 9S, 12S, 13R con-
figuration is shown in Scheme 2.3 The selection of the
protecting group for this plan is important because only
the C12 hydroxy group should be protected. The chemo-
selective protection of the C-12 hydroxy group in (À)-33
was accomplished by the use of the TIPS group
Figure 2. Synthetic strategy of construction of three hydroxy groups.
Scheme 1. Total synthesis of (À)-1, 12, 13-syn-diol.
Scheme 2. Synthesis of (+)-10. Reagent and conditions: (a) TIPSOTf, 2, 6-lutidine, CH2Cl2, À78 ꢀC, 8 h, (79%), (b) (1) ClCH2SO2Cl, pyridine, 0 ꢀC,
1 h; (2) CsOAc, 18-crown-6, benzene, 80 ꢀC, 20 h, (75%); (c) Hg(ClO4)2, CaCO3, THF/H2O (5:1), rt, 5 min, (89%); (d) (S)-BINAL-H, THF, À78 ꢀC,
1 h, (99%, d.r.; 13:1), (e) (1) 1.0 N KOH in EtOH/H2O (4:1), rt, 5 day; (2) TBAF, THF, 45 h, (98%).

T. Shirahata et al. / Bioorg. Med. Chem. Lett. 13 (2003) 937–941
939
Synthesis of (+)-11 having 9S, 12R, 13S was also success-
ful according to the following synthetic route, utilizing
(+)-33 as the starting material (Scheme 3).
structures and the [a]D values of all isomers are shown
in Figure 3.
Oral administration of pinellicaicd analogues as an
adjuvant for the intranasal inoculation of influenza HA
vaccine was investigated.2 Mice were orally adminis-
tered pinellic acid analogues (1 microgram/mouse) with
intragastricgavage followed by intranasal inouclation
of HA vaccine (1 microgram/mouse), and 3 weeks later,
According to the synthetic routes for C-12, 13-syn- and
anti-diol as shown in Schemes 4 and 5, all the stereo
isomers of pinellicaicd [ (À)-10, (À)-11, (À)-12 and
(+)-1] could be prepared from the corresponding
intermediates [(À)-8, (+)-8, (+)-4 and (À)-4].8 The
Scheme 3. Synthesis of (+)-12.
Scheme 4. Synthesis of (À)-11 and (À)-13.
Scheme 5. Synthesis of (+)-1 and (+)-15.
Figure 3. The structures and the [a]D values of all stereoisomers of pinellicacid.

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T. Shirahata et al. / Bioorg. Med. Chem. Lett. 13 (2003) 937–941
Figure 4. Adjuvant activity of all stereoisomers.
the same procedure was repeated. The IgA and IgG
antibody responses against anti-influenza virus in the
nasal and serum in the vaccinated mice were examined 1
week after vaccination.
In summary, we have established syntheticroutes to
enable preparation of all the stereoisomers of 1 via
regioselective asymmetric dihydroxylation, stereo-
selective inversion, and stereoselective reduction. In this
series, 9S, 12S, 13S compound has the most potent
adjuvant activity. Studies on the mechanism of adjuvant
and protective effects of pinellic acid with nasal influ-
enza HA vaccine against influenza virus infection are
currently under way.
The results of adjuvant activity of all stereoisomers are
shown in Figure 4.
The antiviral IgA and IgG antibody responses, induced
in the nasal of mice given pinellic acid (À)-1 with vac-
cine, were enhanced 5.2- and 2-fold, respectively com-
pared with control mice given the vaccine and solvent
alone. Among the C9-isomers of pinellicacid, 9 S-com-
pounds showed much stronger activity compared with
9R-compounds. Thus, stereochemistry at the C-9
hydroxyl group is very important for adjuvant activity.
Among the 9S-derivatives, the adjuvant activities of
13S-compounds are stronger than that of 13R-com-
pounds. However, the stereochemistry of the C-12
hydroxyl group is not important for adjuvant activity.
Acknowledgements
This work was supported in part by a grant from the
Ministry of Education, Science and Culture of Japan
and Keirin Association.
References and Notes
1. Murphy, B. R.; Webster, R. G. Orthomyxoviruses; Fields,
B. N., Knipe, D. M., Eds.; In Virology, 2nd ed.; Raven: New
York, 1990; p 1091.
2. Nagai, T.; Kiyohara, H.; Munakata, K.; Shirahata, T.;
Sunazuka, T.; Harigaya, Y.; Yamada, H. Int. Immunopharm.
2002, 2, 1183.
Interestingly, the adjuvant activity of (+)-1, the enan-
tiomer of natural pinellicacid, was weaker than that of
the natural one.
In the data shown in Figure 4, adjuvant activity of
pinellicacid (À)-1 from a natural source was lower than
that of the syntheticone. The result is presumably due
to the chemical purity of the available sample.
3. Sunazuka, T.; Shirahta, T.; Yoshida, K.; Yamamoto, D.;
Harigaya, Y.; Nagai, T.; Kiyohara, H.; Yamada, H.; Omura,
S. Tetrahedron Lett. 2002, 43, 1265.
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T. Shirahata et al. / Bioorg. Med. Chem. Lett. 13 (2003) 937–941
941
4. Review, Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless,
K. B. Chem. Rev. 1994, 94, 2483.
5. Noyori, R.; Tomino, I.; Tanimoto, Y.; Nishizawa, M. J.
Am. Chem. Soc. 1984, 106, 6709.
6. Review: Mitsunobu, O. Synthesis 1981, 1.
7. Shimizu, T.; Hiranuma, S.; Nakata, T. Tetrahedron Lett.
1996, 37, 6145.
8. The non-selective reduction of (+)-4 (NaBH4, CeCl3)
afforded 1:1 mixture of 9,12-anti, and 9, 12-syn-triol. So, the
stereoselectivity on the reduction of (+)-4 depended upon the
chiral reagents [(R)-BINAL, diastereoselectivity; >20:1, (S)-
BINAL, diastereoselectivity, 1:>20]. On the other hand, the
non-selective reduction of (+)-8 (NaBH4, CeCl3) afforded 3:2
mixture of 9,12-anti, and 9, 12-syn-triol, because of the steric
hindrance of 12-OTIPS. The reduction of (+)-8 by (S)-BINAL
gave the lower diastereoselectivity; 13:1 (mis-matched), how-
ever, by (R)-BINAL gave the higher diastereoselectivity;
>20:1 (matched).