A method for the synthesis of an oseltamivir PET tracer

Article abstract of DOI:10.1016/j.bmcl.2007.11.079

A protocol applicable for the synthesis of an oseltamivir positron emission tomography (PET) tracer was developed. Acetylation of amine 3 with CH₃COCl, followed by deprotection and aqueous workup, produced oseltamivir 4 from 3 within 10 min. The obtained 4 was sufficiently pure for PET studies. This method can be extended to PET tracer synthesis using CH₃¹¹COCl.

Full text of DOI:10.1016/j.bmcl.2007.11.079

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 600–602
A method for the synthesis of an oseltamivir PET tracer
Masataka Morita,a Toshihiko Sone,a Kenzo Yamatsugu,a Yoshihiro Sohtome,a
Shigeki Matsunaga,a Motomu Kanai,a,* Yasuyoshi Watanabeb and Masakatsu Shibasakia,*
aGraduate School of Pharmaceutical Sciences, The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bMolecular Imaging Research Program (MIRP), RIKEN, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
Received 29 October 2007; revised 19 November 2007; accepted 19 November 2007
Available online 28 November 2007
Abstract—A protocol applicable for the synthesis of an oseltamivir positron emission tomography (PET) tracer was developed.
Acetylation of amine 3 with CH3COCl, followed by deprotection and aqueous workup, produced oseltamivir 4 from 3 within
10 min. The obtained 4 was sufficiently pure for PET studies. This method can be extended to PET tracer synthesis using
CH311COCl.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Oseltamivir phosphate (Tamifluꢁ, 1: Fig. 1), invented by
the scientists of Gilead Science1 and licensed to Roche in
O
O
1996, is the first orally active antiviral drug for the treat-
ment of influenza types A and B. This drug potently
inhibits the influenza virus neuraminidase by binding
to the active site of the enzyme as a transition state mi-
mic of sialic acid hydrolysis, a critical molecular level
event for the influenza life cycle. As a result, release
and spread of the virus from infected cells are prevented.
Tamiflu is approved in many countries worldwide for
the treatment and prevention of influenza. Because the
active site of neuraminidase is highly conserved in all
virus subtypes, Tamiflu is thought to be consistently
effective for treating otherwise fatal avian influenza
(H5N1 type virus), a possible cause of a potential influ-
enza pandemic.2 According to the World Health Orga-
nization, stockpiling of Tamiflu is currently the only
way to guard against this possible pandemic. Some
countries are stockpiling or intending to stockpile en-
ough Tamiflu to treat 20–40% of their population.
NHAc
NHAc
NH2
EtO2C
NH2 H3PO4
HO2C
1
2
Figure 1.
cient synthetic route is under intensive investigation.
To date, five groups, including ours, have independently
reported alternative synthetic routes.4
The other concern is the possible side effects of Tamiflu
on the nervous system. Tamiflu is considered to be a safe
drug without severe side effects. Recently in Japan, how-
ever, where 80% of the world supply of Tamiflu is con-
sumed, it was reported that Tamiflu might cause
abnormal behavior (such as hallucinations and impul-
sive behavior). According to the Ministry of Health, La-
bor, and Welfare of Japan, 211 cases of abnormal
behavior that are believed to be associated with the
use of Tamiflu have been reported, of which eight re-
sulted in deaths. It is noteworthy that in most cases
the patients were adolescents or teenagers. More com-
prehensive statistical investigations are now underway
to clarify the relationship between the abnormal behav-
ior and Tamiflu administration. Meanwhile, very recent
findings of neuroexcitatory effects in animal models,
such as insects and mammals,5 have demonstrated that
Tamiflu and its carboxylate metabolite (Ro64-0802: 2)
There are two main concerns related to Tamiflu. One is
its manufacturing process. The current process utilizes
naturally occurring shikimic acid as the starting mate-
rial,3 which is not ideal to cover the worldwide demand
of Tamiflu. Therefore, the development of a more effi-
Keywords: Oseltamivir; Positron emission tomography (PET); Influ-
enza; Neuroexcitatory effects; Acetylation.
*
Corresponding authors. Tel.: +81 3 5841 4830; fax: +81 3 5684 5206
(M.S.); e-mail: mshibasa@mol.f.u-tokyo.ac.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.079

M. Morita et al. / Bioorg. Med. Chem. Lett. 18 (2008) 600–602
601
could indeed affect nervous function. Tamiflu (oseltam-
ivir) passes through the blood brain barrier in rats, but
its penetration into the brain is limited by an active ef-
flux transporter, P-glycoprotein, at the blood–brain bar-
rier, thereby attenuating the neuroexcitatory effects of
Tamiflu under normal conditions.6 Therefore, clarifica-
tion of the pharmacologic effects of Tamiflu on the hu-
man nervous system is an urgent task. We
hypothesized that PET studies might be an effective
way to more precisely examine the distribution of osel-
tamivir in the brains of higher animals. In this commu-
nication, we describe a method applicable for the
synthesis of an oseltamivir PET tracer.
We hypothesized that labeling the acetamide part of 1
(Tamiflu) or 4 (oseltamivir) would fulfill these require-
ments (Scheme 1). Based on our previous synthesis,4c
the acetyl group can be introduced in high yield to the late
stage-intermediate 3. In addition, there are several reports
11
on the synthesis and use of CH3 COCl in PET studies.8 A
possible side product, CH311CO2H, can be easily ex-
tracted or evaporated out from the target tracer molecule.
Because the half-life of 11C is 20.3 min, conditions should
be optimized to complete the synthesis and purification of
the tracer 4 from 3 within approximately 10 min. The pre-
cursor 3 would be synthesized by modifying our third-
generation synthetic route of Tamiflu.4e
PET imaging techniques allow for visualization of the dis-
tribution of a radioactive probe molecule under physio-
logic conditions in a living whole body system. A PET
tracer containing short-lived positron-emitting radio-
nuclides (such as 11C) spontaneously decomposes
through b+-decay, producing a positron and a stable
nucleus containing the same atomic mass (e.g., 11B from
11C). The generated positron then collides with an elec-
tron of a nearby molecule resulting in a pair of annihi-
lation c-ray emissions. PET imaging is created by
detecting this annihilation c-ray.7 The following three
main factors should be considered in designing a PET tra-
cer: (1) the availability of the labeling precursor with
short-lived positron-emitting radio-nuclides; (2) the tim-
ing of the introduction of short-lived positron-emitting
radio-nuclides to the tracer molecule; (3) the ease of puri-
fication of the final compound to eliminate contaminating
radioactive side products. For a high signal-to-noise ratio
in PET studies, the short-lived positron-emitting radio-
nuclides should be introduced to the target compound
through a clean conversion at a late stage of synthesis.
The key intermediate 3 was synthesized as shown in
Scheme 2. The known racemic carbamate 5, which
was rapidly synthesized through Diels–Alder reaction
and the Curtius rearrangement as key steps,4e was con-
verted to 6 via two straightforward steps. After oxidiz-
ing 6 to enone 7 under the modified Swern conditions,
enantiomers were separated using chiral preparative
HPLC [Daicel, Chiralpak AD-H, 2-propanol/hexane
1:20, flow 1.0 mL/min: tR 15.2 min (undesired) and
17.0 min (desired)].4c A Ni-catalyzed 1,4-addition of
TMSCN followed by bromination and elimination of
HBr produced b-cyano enone 8 in 66% yield in three
steps. Stereoselective reduction of 8 followed by Mits-
unobu reaction produced aziridine 9, to which the
3-pentyloxy group was introduced in the presence of
BF3ÆOEt2, producing di-Boc protected 10. Ethanolysis
of the nitrile under acidic conditions proceeded with
concomitant Boc cleavage, and the subsequent regiose-
lective mono-Boc protection of the less hindered ami-
no group produced the key intermediate 3 in high
yield.
Having obtained intermediate 3, the conditions were
then optimized for the conversion of 3 to 4 to fulfill
the requirements of PET tracer synthesis (Scheme 3).9
Amine 3 was treated with acetyl chloride (1.2 equiv)
and triethylamine (3 equiv) in hot (55 ꢂC) THF for
2 min. This reaction hardly proceeded at ambient tem-
perature, whereas N,N0-diacetylation proceeded in the
presence of an excess (5 equiv) amount of acetyl chlo-
ride. After completion of the acetylation, 4 M HCl in
ethyl acetate was added to the same reaction vessel to
O
O
H
N
within 10 min
CH3
NH2
*
C
O
EtO2C
4: C
NH2
EtO2C
NHBoc
*
=
11C in PET tracer
3
Scheme 1. Design of Tamiflu PET tracer and its synthetic plan.
O
1) Ni(COD)2 (20 mol %)
TMSCN
2) NBS; Et3N
OH
O
O
O
1) Boc2O
2) Cs2CO3, MeOH
1) isobutyric anhydride
DMSO, 97%
NH
NHBoc
NHBoc
NHBoc
NHBoc
NHBoc
NHBoc
2) resolution
84% (2 steps)
66% (3 steps)
NHBoc
NC
( )-5
( )-6
7
8
1) LiAlH(OtBu)3
85%
3-pentanol
BF3 OEt2, 60%
O
O
1) HCl, EtOH;
NH4OH, 89%
NBoc
NHBoc
NHBoc
NH2
2) Ph3P, DEAD
60%
2) Boc2O, Et3N
80%
NC
NHBoc
NC
EtO2C
NHBoc
9
10
3
Scheme 2. Synthesis of key intermediate 3.

602
M. Morita et al. / Bioorg. Med. Chem. Lett. 18 (2008) 600–602
Yamatsugu, K.; Kamijyo, S.; Suto, Y.; Kanai, M.;
1) AcCl (1.2equiv)
Et3N (3 equiv)
Shibasaki, M. Tetrahedron Lett. 2007, 48, 1403; (f) Sato,
N.; Akiba, T.; Yokoshima, S.; Fukuyama, T. Angew.
Chem., Int. Ed. 2007, 46, 5734; (g) Bromfield, K. M.;
Graden, H.; Hagberg, D. P.; Olsson, T.; Kann, N. Chem.
Commun. 2007, 3183; (h) Shie, J.; Fang, J.; Wang, S.; Tsai,
K.; Cheng, Y.; Yang, A.; Hsiao, S.; Su, C.; Wong, C. J.
Am. Chem. Soc. 2007, 129, 11892.
THF,55 oC, 2 min
O
O
2) 4 M HCl / EtOAc, 3 min
NH2
NHAc
NH2
3) evaporation, 2 min
EtO2C
NHBoc
EtO2C
4) CH2Cl2/satd. Na2CO3 aq.
partition;
evaporation, 3 min
3
4
5. (a) Izumi, Y.; Tokuda, K.; O’Dell, K. A.; Zorumski, C. F.;
Narahashi, T. Neurosci. Lett. 2007, 426, 54; (b) Ishii, K.;
Hamamoto, H.; Sasaki, T.; Ikegaya, Y.; Yamatsugu, K.;
Kanai, M.; Shibasaki, M.; Sekimizu, K. Drug Discov.
Ther., in press; (c) According to the Ministry of Health,
Labor, and Welfare of Japan, however, based on animal
experiments performed by the Chugai Pharmaceutical
Company, which sells Tamiflu in Japan, there is no
relationship between Tamiflu administration and abnor-
mal behavior (stated as a tentative conclusion on October
24, 2007).
6. (a) Morimoto, K.; Nakakariya, M.; Shirasaka, Y.; Kak-
inuma, C.; Fujita, T.; Tamai, I.; Ogihara, T. Drug Metab.
Dispos. 2007, 35. doi:10.1124/dmd.107.017699; (b) Ose,
A.; Kusuhara, H.; Yamatsugu, K.; Kanai, M.; Shibasaki,
M.; Fujita, T.; Yamamoto, A.; Sugiyama, Y. Drug Metab.
Dispos. 2007, 35. doi:10.1124/dmd.107.018556.
Total 10 min
Scheme 3. Rapid synthesis of oseltamivir.
cleave the Boc group. This deprotection was completed
in 3 min. Evaporation of the solvents (2 min), partition
of the residue in CH2Cl2/saturated Na2CO3 aq biphasic
system, phase separation, and evaporation of CH2Cl2
(3 min) produced 4 in an analytically pure form. There-
fore, sufficiently pure 4 for PET studies was obtained
from 3 in 10 min (20-mg scale).10 Yield of the two-step
conversion was 82% after silica gel column
chromatography.
7. For recent reviews, see: (a) Wood, K. A.; Hoskin, P. J.;
Saunders, M. I. Clin. Oncol. 2007, 19, 237; (b) Welsh, J. S.
Am. J. Clin. Oncol. 2007, 30, 437.
In summary, we developed a procedure that can be ap-
plied to the synthesis of a Tamiflu PET tracer. The selec-
tion of an appropriate precursor (3) for introduction of
an unstable nucleus and careful optimization of the
reaction conditions were key. Synthesis of the PET tra-
cer using this method and studies of its application to
PET imaging of [11C]Tamiflu in higher animals are cur-
rently ongoing in our group.
8. For examples of using CH311COCl in PET studies, see: (a)
Le Bars, D.; Luthra, S. K.; Pike, V. W.; Duc, C. Luu.
Appl. Rad. Isotop. 1987, 38, 1073; (b) Courtyn, J.;
Cornelissen, B.; Oltenfreiter, R.; Vandecapelle, M.; Sleg-
ers, G.; Strijckmans, K. Nucl. Med. Biol. 2004, 31, 649; (c)
Chen, J. J.; Fiehn-Schulze, B.; Brough, P. A.; Snieckus, V.;
Firnau, G. Appl. Rad. Isotop. 1998, 49, 1573; (d) Sasaki,
T.; Furukata, K.; Ishii, S.; Iimori, T.; Ikegami, S.; Nozaki,
T.; Senda, M. J. Labelled Compd. Radiopharm. 1996, 38,
337; (e) Bonnot-Lours, S.; Crouzel, C.; Prenant, C.;
Hinnen, F. J. Labelled Compd. Radiopharm. 1993, 33,
277; (f) Oberdorfer, F.; Siegel, T.; Guhlmann, A.; Keppler,
D.; Maier-Borst, W. J. Labelled Compd. Radiopharm.
1992, 31, 903; (g) Banks, W. R.; Tewson, T. J.; Digenis, G.
A. Appl. Rad. Isotop. 1990, 41, 719.
Acknowledgments
Financial support to this work was provided by a Grant-
in-Aid for Specially Promoted Research of MEXT.
K.Y. thanks JSPS for the research fellowships.
9. Unlabeled CH3COCl was used in this study.
10. Experimental
References and notes
details:
Acetyl
chloride
(4.6 lL,
0.0645 mmol, 1.2 equiv) was added to a solution of 3
(19.9 mg, 0.0537 mmol) and triethylamine (22.5 lL,
0.161 mmol, 3.0 equiv) in THF (0.4 mL) at 55 ꢂC. After
2 min, the reaction vessel was removed from the heating
bath, and 4 M hydrogen chloride in ethyl acetate (1.5 mL)
was added in one portion. After 3 min, the reaction
mixture was concentrated under vacuum. A saturated
aqueous solution of sodium hydrogen carbonate (1 mL)
was then added to the residue. The aqueous layer was
extracted with CH2Cl2 (2· 2 mL) using a Chem Elute
extraction column (available from Varian). The solvent
was evaporated under reduced pressure, producing ana-
lytically pure 4. The isolation of 4 was completed in 3 min.
The residue was passed through silica gel column chro-
matograph (CH2Cl2/MeOH = 7:1) to afford 4 (13.8 mg,
82%) as a colorless oil.
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