Practical synthesis of Kainoids: A new chemical probe precursor and a fluorescent probe

Article abstract of DOI:10.1021/ol403434e

A practical total synthesis of kainoid MFPA (5) was achieved in only six steps, via a novel Ni-catalyst-mediated asymmetric conjugate addition reaction. Furthermore, a fluorescein-based fluorescent ionotropic glutamate receptor probe 28 was efficiently sy

Full text of DOI:10.1021/ol403434e

Letter
pubs.acs.org/OrgLett
Practical Synthesis of Kainoids: A New Chemical Probe Precursor and
a Fluorescent Probe
Shingo Sasaki,† Hiroto Suzuki,† Hitoshi Ouchi,† Tomohiro Asakawa,† Makoto Inai,† Ryuichi Sakai,‡
Keiko Shimamoto,§ Yoshitaka Hamashima,*,† and Toshiyuki Kan*,†
†School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan
‡Faculty of Fisheries and Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, 041-8611, Japan
§Suntory Foundation for Life Sciences, Bioorganic Research Institute, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka,
618-8503, Japan
S
* Supporting Information
ABSTRACT: A practical total synthesis of kainoid MFPA (5)
was achieved in only six steps, via a novel Ni-catalyst-mediated
asymmetric conjugate addition reaction. Furthermore, a
fluorescein-based fluorescent ionotropic glutamate receptor
probe 28 was efficiently synthesized from a precursor derived
from a synthetic intermediate of 5.
ainoids, as represented by kainic acid (1), have been
studied for more than 40 years due to their selective
K
binding to ionotropic glutamate receptors (iGluRs), which are
central neurotransmitters in the brain.1 Because 1 is a selective
ligand for kainate receptors (KARs), a subset of iGluRs, it has
been widely used as a standard tool in neuropharmacological
research. Since KARs are involved in the transmission of pain,
especially neuropathic pain, compounds that interact with
KARs are of particular interest as research tools or even
potential therapeutic agents. Natural (1,2 2,3 3−44) and
synthetic (5,5 6−96) kainoids have been used to investigate
neuropathic pain transmission, and it was reported that
allodynia induced by acromelic acids (3 and 4) was attenuated
by 3-carboxymethyl-4-(4-methylphenylthio)pyrrolidine-2-car-
boxylic acid (PSPA, 7) (Figure 1).6 Therefore, we wished to
develop a practical synthetic route to prepare various kainoids
in a small number of steps. We first validated our strategy by
preparing a potent kainoid (MFPA, 5)5a bearing an acromelic
acid motif in six steps from nitrostyrene derivative 15.7b We
then turned to the preparation of fluorescein-conjugated
kainoid 28. Such a fluorescent probe molecule with a high
affinity for KA receptors could be useful for characterizing the
behavior of synaptic KAR in live cells. Herein, we report a
practical total synthesis of MFPA (5). We also describe its
biological activity, as well as adaption of our synthesis to obtain
a fluorescent iGluR probe molecule, 28.
Figure 1. Structure of kainoids 1−9.
C2 position at a later stage. We envisioned that starting from α-
ketoester 14, which possesses two carboxylic acid moieties,
would allow a shorter synthetic route with retention of the
oxidation state. Furthermore, construction of the pyrrolidine
ring from α-ketoester 14 could be achieved by reduction of the
nitro group and subsequent intramolecular reductive amination
in a single step.
We recently reported an efficient organometal-catalyzed
construction of three consecutive chiral centers in a pyrrolidine
ring, leading to the total synthesis of MFPA (5).7a,b However, a
shorter, more practical synthesis of 5 was needed for efficient
preparation of iGluR probes. Thus, we set out to improve our
synthetic procedures.7b
The heart of our synthetic plan is illustrated in Scheme 1.
Our reported synthesis of 5 utilized nitroalkene 15 and α-
ketoester 13 to allow for the stepwise oxidation sequence at the
Received: November 27, 2013
© XXXX American Chemical Society
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Letter
derivative 22 was isolated in 90% yield from 18 in two steps.
According to our reported procedure, epimerization of the C-2
position could be accomplished by treatment with a
combination of t-BuOK and t-BuOH/benzene to give 23.
Finally, simultaneous cleavage of the Boc group and the t-Bu
esters of 23 was carried out by treatment with TFA in the
presence of anisole to afford the desired MFPA (5). Its spectral
data (1H NMR, 13C NMR, IR, and HRMS) were in full
agreement with reported values.3a,b
Scheme 1. Retrosynthetic Analysis of 5
With the enantioselective total synthesis of 5 having been
accomplished in six steps from o-anisaldehyde (overall yield:
62%), we next assessed the affinity of 5 for ionotropic
glutamate receptor sites and its excitatory toxicity in mice. As
described previously,5c−e 5 significantly inhibited [3H]KA
binding (Kd 1.8
0.3 nM), while its displacement of
80 nM).
[3H]AMPA binding was weaker (Kd 322
Intracerebroventricular (i.c.v.) injection in mice resulted in
the dose-dependent toxicity typically observed in excitatory
amino acids including generalized convulsion at higher doses
and catalepsy, stereotyped behaviors, and Straub tail response
at lower doses. The ED50 value was determined to be 0.046
nmol/mouse. Thus, 5 is more potent than kainic acid (ED50
0.28 nmol/mouse).
The two chiral centers of 12 would be constructed by Ni-
catalyzed diastereo- and enantioselective conjugate addition of
α-ketoesters 14 to nitroalkene 15, as developed by one of the
authors (Y. Hamashima).7b
As shown in Scheme 2, our total synthesis of 5 was achieved
in six steps, starting from nitrostyrene derivative 15.7b Reaction
We next turned to design a useful kainoid-based precursor
molecule that allows incorporation of various probes or
attachment to affinity media. Because, in our previous
study,10,11 terminal amine or azide groups extending from the
phenolic ring proved useful in incorporating probe function-
ality, we designed a precursor 26 bearing a linker at the para-
position of the methoxy group of 5, as a versatile platform for
chemical biological work on the kainoids. Though a bulky
fluorophore might interfere with binding of the ligand to the
relatively small binding cavity of the kainite type glutamate
receptors,12 we thought that the fluorescence probe 28 might
still provide useful data. As shown in Scheme 3, our synthesis of
Scheme 2. Synthesis of MFPA (5)
Scheme 3. Synthesis of Key Intermediate 26
of α-ketoester 168 and 159 in the presence of 1 mol % of our Ni
catalyst 17 resulted in a smooth asymmetric conjugate addition
reaction to afford 18 exclusively in 97% yield and 89% ee.
Sequential construction of the pyrrolidine ring of 21 was
accomplished by treatment of 18 with hydrogen (800 psi) and
Raney Ni. Tandem reduction of the nitro group, intramolecular
imine formation of 19, and reductive amination of 20
proceeded smoothly to provide the pyrrolidine 21. In the
reduction step, hydride attack occurred from the less hindered
β-face of the pyrrolidine ring of 20 to provide a single
diastereomer with α-stereochemistry. After protection of the
secondary amine of 21 with a Boc group, the pyrrolidine
the probe precursor 26, possessing an amino group, was
commenced with protected MFPA 23. For incorporation of the
linker unit, we employed the Sonogashira reaction.13
Regioselective iodination of the para-position of 23 was
accomplished by treatment with I2 and AgNO3 to afford
coupling precursor 24 in 88% yield. The reaction of iodide 24
and propargyl alcohol in the presence of catalytic quantities of
PdCl2(PPh3)2 and CuI in MeCN proceeded smoothly to give
B
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the desired cross-coupling product 25 in high yield.13 The
propargyl alcohol moiety of 25 was converted to propyl amine
26 in three steps via mesylation, displacement with an azido
group, and a simultaneous hydrogenation reaction of the azido
and alkyne groups.
With the desired probe precursor 26 in hand, we finally
focused on incorporation of a fluorescent moiety to obtain the
desired probe molecule 28. For this purpose, we selected a
reliable photophore, Tokyo-Green (TG, 27).14 As shown in
Scheme 4, condensation of the probe precursor 26 and TG 27
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the Uehara Memorial
Foundation (Y.H.), MEXT/JSPS KAKENHI Grant Numbers
23390007, 22380114 (R.S.), and a Grant-in-Aid for Scientific
Research on Priority Areas 12045232 and 24105530 from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy (MEXT) of Japan and, Platform for Drug Discovery,
Informatics, and Structural Life Science from the Ministry of
Education, Culture, Sports, Science and Technology. We also
thank Prof. Mikiko Sodeoka of RIKEN for the generous
support.
Scheme 4. Synthesis of Fluorescent Probe 28
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In conclusion, a practical total synthesis of 5 and the design
and efficient synthesis of the kainoid probe precursor 26 have
been accomplished by applying our Ni-catalyst-mediated
asymmetric conjugate addition reaction, together with the
efficient construction of the pyrrolidine ring by a tandem
reduction reaction. We have also prepared 28, a new
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linker groups, and is expected to afford a range of probe
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ASSOCIATED CONTENT
* Supporting Information
Experimental details and spectroscopic data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: kant@u-shizuoka-ken.ac.jp.
*E-mail: hamashima@u-shizuoka-ken.ac.jp.
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