Synthesis of a bis-azido analogue of acromelic acid for radioisotope-free photoaffinity labeling and biochemical studies

Article abstract of DOI:10.1016/j.bmcl.2006.01.083

A novel acromelic acid analogue containing a phenyl group possessing two different types of azido functional groups, of which one is the aromatic N ₃ acting as a photoaffinity group to bind to a target protein by photoirradiation and the other

Full text of DOI:10.1016/j.bmcl.2006.01.083

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2433–2436
Synthesis of a bis-azido analogue of acromelic acid
for radioisotope-free photoaffinity labeling and biochemical studies
Pi Sun, Guang Xing Wang,* Kyoji Furuta and Masaaki Suzuki
Regeneration and Advanced Medical Science, Graduate School of Medicine, Gifu University, Gifu 501-1193, Japan
Received 14 December 2005; revised 12 January 2006; accepted 24 January 2006
Available online 15 February 2006
Abstract—A novel acromelic acid analogue containing a phenyl group possessing two different types of azido functional groups, of
which one is the aromatic N3 acting as a photoaffinity group to bind to a target protein by photoirradiation and the other is alkyl N3
group which survives photolysis acting as a detecting group through the Staudinger–Bertozzi reaction to identify the ligated prod-
uct, was designed and synthesized as a radioisotope-free biochemical probe potentially for studies on kainoid receptors.
Ó 2006 Elsevier Ltd. All rights reserved.
Acromelic acid (1) isolated from a toadstool, citocybe
acromelalga,1 is a potent neuroexcitatory amino acid
which belongs to a class of so-called kainoids bearing
a pyrrolidine dicarboxylic acid structure represented
by kainic acid (2).2 These compounds possess a structure
similar to that of glutamic acid, a major excitatory neu-
rotransmitter in the human central nervous system.
Therefore, they can be looked as conformationally con-
strained glutamic acid analogues and are believed to ex-
ert their biological activities through glutamate
receptors3 that are classified as ionotropic and metabo-
tropic receptors comprising of three and eight subtypes,
respectively.4 By binding and acting at the subclass of
kainate receptor and AMPA (a-amino-3-hydroxy-5-
methylisoxazole-4-propionic acid) receptor, the kainoids
have been shown to display powerful neuroexcitatory
activity in the mammalian central nervous system.3 Like
kainic acid (2), acromelic acid (1) can also strongly
depolarize the neurons, but its in vivo behavioral and
pathological effects are reportedly different from those
of kainic acid,5 suggesting the existence of distinct types
of kainoid receptors. Therefore, the actual receptor for
acromelic acid and its signaling pathway are yet to be
determined. During our efforts to elucidate the molecular
mechanism behind the neuro-toxicity of acromelic acid
and associated receptor functions, we have designed and
synthesized an acromelic acid analogue (GIF-0448, 3)
possessing an azido group as photoaffinity group and
125I as a radioactive detecting group as a bifunctional
probe for the object.6 The compound is successfully
utilized as a substitute of acromelic acid in the biochem-
istry study of receptor signaling analysis, but the radio
property of this compound is obviously inconvenient
and unhealthy for the treatment and preparation.
Recently, we have developed a novel method for radio-
isotope-free photoaffinity labeling, in which a bifunc-
tional ligand is connected to a target protein by
activation of a photoreactive group, such as an aromatic
azido group, and identification of the ligated product is
achieved by anchoring of a detectable tag through the
Staudinger–Bertozzi reaction with an alkyl azido moiety
that survives photolysis. The chemical ground of this
method was also confirmed using model compounds
with the bifunctional group under photoirradiation in
the presence of trapping agents for reactive intermedi-
ates and the method was demonstrated by specific label-
ing of the catalytic portion of human HMG-CoA
reductase.7 In this letter, we wish to adopt this idea
and report the design and synthesis of an acromelic acid
analogue (4) related to compound (3) possessing a
phenyl group functionalized with two different azido
groups as the radioisotopic-free probe for the acromelic
acid receptor signaling analysis (see Fig. 1).
Retrosynthetically, compound 4 could be obtained by
deprotection of N-Boc and mild hydrolysis of bis-methyl
ester of compound 5 under mild condition without
destroying the azido group.6 The alkyl N3-group could
be introduced by the substitution of the corresponding
mesylate 6, which could be prepared from the alcohol
Keywords: Acromelic acid; Radioisotopic free; Molecular probe;
Bis-azodo analogue; Staudinger–Bertozzi reaction; Photoaffinity.
*
Corresponding author. Tel.: +86 13524617726; e-mail: wgxwys@126.
com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.083

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P. Sun et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2433–2436
HOOC
H
dure for preparing compound 7 starting from the begin-
N
O
ning was envisaged (Scheme 3). TBS protection of the
primary OH of 2-hydroxymethyl-4-nitrophenol afforded
compound 12 smoothly. Coupling 12 with compound 9
under Mitsunobu condition gave compound 13 in 69%
yield. The stereochemistry of compound 13 was deduced
by the general mechanism of Mitsunobu reaction, which
usually reverses the stereochemistry of hydroxy group.
COOH
COOH
COOH
COOH
N
H
N
H
2 Kainic Acid A
1 Acromelic Acid A
I
N3
1
Also the H NMR spectrum of 13 represented the char-
N3
N3
O
O
acteristic pattern of 3,4-cis-configuration as judged by
accumulated data of analogs.6,9 The nitro group in com-
pound 13 was then converted to the amino group by
hydrogenation catalyzed by 10% Pd/C to give com-
pound 14. Removal of TBS group in 14 using TBAF
in THF afforded compound 15 in high yield. With both
amino and hydroxy groups in the body, compound 15 is
much soluble in the 3 N HOAc which is beneficial to the
next azidation step. Thus, dissolving compound 15 in
3 N HOAc and treatment with NaNO2 and then NaN3
provided the compound 7 in 86% combined yield.10
Mesylation of the hydroxy group using MsCl in the
presence of Et3N gave almost quantitative yield of the
mesylate 6, which was displaced by another azido group
using NaN3 in DMSO at 40 °C to provide the bis-azido
compound 5. Removal of the N-Boc group of 5 with tri-
fluoroacetic acid and hydrolysis of methyl esters by
treatment with lithium hydroxide in methanol–water
afforded the desired compound 411 as a pale yellow
amorphous solid in 94% yield, after ion-exchange chro-
matography and lyophilization (Scheme 3).
COOH
COOH
COOH
N
COOH
N
H
H
3 GIF-0448
4
Figure 1.
7. Like the step in the preparation of compound 3, the
compound 7 could be prepared by incorporating the
azidophenol 8, prepared from 2-hydroxymethyl-4-nitro-
phenol, to the pyrrolidine derivatives 9 by Mitsunobu
condition (see Scheme 1).
The pyrrolidine derivative 9 was synthesized from com-
mercially available trans-4-hydroxyproline following the
reported procedure.6,8 The azido phenol 8 was prepared
from 2-hydroxymethyl-4-nitrophenol as follows. Hydro-
genation of nitro group in 2-hydroxymethyl-4-nitrophe-
nol under the atmosphere of hydrogen catalyzed by 10%
Pd/C afforded the aminophenol 10. Without purifica-
tion, compound 10 was treated with NaNO2 in 2 N
HCl at 0 °C and then exchanged with NaN3 to give
the azido phenol 11 in 50% yield over 3 steps. Selective
protection of the primary hydroxy group as TBS ether
was achieved by using TBSCl and Et3N in DMF
(Scheme 2).
The complete preservation the biological property of
acromelic acid A (1) by GIF-0448 (3) is quite intriguing
OH
OH
OH
OH
b
a
H2N
O2N
With both intermediates in hand, the synthesis of com-
pound 5 was started. Though phenol is a well-known
substrate for the Mitsunobu reaction, coupling azido-
containing phenol seemed to be uneasy task because
the azido group could be reduced with Ph3P. We per-
formed this reaction by changing the addition sequence
of reagents in order to avoid this reduction, but the reac-
tion still gave a complex mixture and after removal of
TBS ether using TBAF compound 7 could be obtained
but only in 13% yield. Therefore, an improved proce-
10
OTBS
OH
OH
OH
c
N3
N3
8
11
Scheme 2. Reagents and conditions: (a) H2, Pd/C, EtOAc, 6 h: (b) 1—
NaNO2, 2 N HCl, 0 °C, 5 min; 2—NaN3, 1 h, 50% for 3 steps; (c)
TBSCI, DMF, Et3N, rt, 5 h, 55%.
OMs
N3
N3
O
CO2Me
N3
O
CO2H
CO2Me
CO2Me
N3
O
N3
CO2Me
N
CO2H
Boc
N
H
N
Boc
4
6
5
OH
O
OTBS
OH
CO2Me
CO2Me
HO
CO2Me
+
N3
N3
N
CO2Me
Boc
N
Boc
9
8
7
Scheme 1. Retrosynthetic analysis.

P. Sun et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2433–2436
2435
OH
O
1)TPP
DIAD
THF
CO2Me
CO2Me
OTBS
OH
HO
CO2Me
CO2Me
N3
+
N3
2) TBAF
13%
N
Boc
N
Boc
8
9
OTBS
O
CO2Me
OTBS
OH
HO
a
CO2Me
O2N
+
O2N
CO2Me
N
CO2Me
Boc
N
Boc
13
12
9
OH
O
OTBS
O
c
CO2Me
b
H2N
CO2Me
H2N
CO2Me
N
CO2Me
N
Boc
Boc
15
14
OH
O
OMs
O
e
d
CO2Me
CO2Me
CO2Me
N3
N3
N
CO2Me
N
Boc
6
Boc
16
N3
O
N3
O
f
g
CO2H
CO2H
CO2Me
N3
N3
CO2Me
N
H
N
Boc
5
4
Scheme 3. Reagents and conditions: (a) TPP, DIAD, THF, rt overnight, 69%; (b) H2, Pd/C, EtOH, rt, 90%; (c) TBAF, THF, rt, 86%; (d) NaNO2,
3 N HOAc, 0 °C, 5 min, then NaN3, 1 h, 86%; (e) MsCl, Et3N, CH2Cl2, 0 °C, 3 h, 99%; (f) NaN3, DMSO, 50 °C, overnight, 76%; (g) 1—LiOH,
MeOH/H2O, rt, 6 h; 2—TFA, CH2Cl2, rt, 3 h; 3—ion-exchange resin, 94%.
because they have the substantial differences in struc-
ture.6 Particularly interesting, lots of analogues of
GIF-0448 (3) with different substitute patterns in the
phenyl ring have shown almost the same biological
activity as GIF-0448 (3).12 It seems reasonable to as-
sume that the phenyloxy substitute at C-4 of the pyrrol-
idine rings plays a crucial role for binding and biological
activity. As one of the close and similar analogues of
GIF-0448 (3), compound 4 should exhibit the same
biological activity as GIF-0448 (3).13
Culture, Sports, Science and Technology, Japan.
G.X.W. thanks the JSPS Postdoctoral Fellowship for
Foreign Researchers.
References and notes
1. (a) Konno, K.; Shirahama, H.; Matsumoto, T. Tetrahe-
dron Lett. 1983, 24, 939; (b) Konno, K.; Hashimoto, K.;
Ohfune, Y.; Shirahama, H.; Matsumoto, T. J. Am. Chem.
Soc. 1988, 110, 4807.
2. For reviews, see: (a) Shinozaki, H. In Kainic Acid as a Tool
in Neurobiology; McGeer, E. G., Olney, J. W., McGeer, P.
L., Eds.; Raven: New York, 1978; pp 17–35; (b) Hashim-
oto, K.; Shirahama, H. Trends Org. Chem. 1991, 2, 1; (c)
Parsons, A. F. Tetrahedron 1996, 52, 4149; (d) Bleakman,
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3. (a) Tsai, C.; Schneider, J. A.; Lehmann, J. Neurosci. Lett.
1988, 92, 298; (b) Hashimoto, K.; Ohfune, Y.; Shirahama,
H. Tetrahedron Lett. 1995, 36, 6235; (c) Cantrell, B. E.;
Zimmerman, D. M.; Monn, J. A.; Kamboj, R. K.; Hoo,
K. H.; Tizzano, J. P.; Pullar, I. A.; Farrell, L. N.;
Bleakman, D. J. Med. Chem. 1996, 39, 3617; (d) Holl-
mann, M.; Heinemann, S. Annu. Rev. Neurosci. 1994, 17,
31; (e) Chevliakov, M. V.; Montgomery, J. J. Am. Chem.
Soc. 1999, 121, 11139.
In conclusion, we elaborated a simple acromelic acid
analogue 4 bearing a 4-phenyloxy group possessing
two different types of azido groups as a photoisotopic-
free biochemical probe for acromelic acid. The com-
pound is more stable and safer and would be usable
not only as a photoaffinity labeling probe, but also as
a biochemical tool for acromelic acid receptor signaling
analysis.13,14 The simplicity of the synthesis would allow
diverse structural modification for further investigations
on kainoid activities. Designs along this way are per-
formed in this laboratory and will be reported in due
course.
4. For recent reviews, see: (a) Conn, P. J.; Pin, J. P. Annu.
Rev. Pharmacol. Toxicol. 1997, 37, 205; (b) Moloney, M.
G. Nat. Prod. Rep. 1998, 15, 205; (c) Dingledine, R.;
Borges, K.; Bowie, D.; Traynelis, S. F. Pharmacol. Rev.
1999, 51, 7; (d) Brauner-Osborne, H.; Egebjerg, J.;
Nielsen, E. O.; Madsen, U.; Krogsgaard-Larsen, P.
J. Med. Chem. 2000, 43, 2609; (e) Schoepp, D. D.
J. Pharmacol. Exp. Ther. 2001, 299, 12.
Acknowledgments
This work was supported in part by a Grant-in-Aid for
Creative Scientific Research ‘In vivo Molecular Science
for Discovery of New Biofunctions and Pharmaceutical
Drugs’ No. 13NP0401, from the Ministry of Education,

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P. Sun et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2433–2436
5. (a) Shinozaki, H.; Ishida, M.; Gotoh, Y.; Kwak, S. Brain
J = 8.8 Hz, 1H), 4.94(t, J = 3.6 Hz, 1H), 4.60(s, 2H),
4.11(d, J = 9.2 Hz, 1H), 3.83(d, J = 12.8 Hz, 1H), 3.79(s,
3H), 3.68 (d, J = 12.8 Hz, 1H), 3.66(s, 3H), 2.85–2.92(m,
2H), 2.69(dd, 18.9/3.4 Hz, 1H), 1.41(s, 9H). Compound 6:
1HNMR (CDCl3, 400 MHz, d ppm): 7.05(d, J = 2.4 Hz,
1H), 7.01(dd, J = 8.8/2.4 Hz, 1H), 6.83(d, J = 8.8 Hz, 1H),
5.18(s, 2H), 5.03(t, J = 3.2 Hz, 1H), 4.18(d, J = 9.6 Hz,
1H), 3.82(d, J = 12.8 Hz, 1H), 3.78(s, 3H), 3.69–3.72(m,
1H), 3.64(s, 3H), 2.96–3.01(m, 2H), 2.93(s, 3H), 2.69(dd,
Res. 1989, 503, 330; (b) Kwak, S.; Aizawa, H.; Ishida, M.;
Shinozaki, H. Life Sci. 1991, 49, PL91; (c) Tsuji, K.;
Nakamura, Y.; Ogata, T.; Mitani, A.; Kataoka, K.;
Shibata, T.; Ishida, M.; Shinozaki, H. Neuroscience 1995,
68, 585.
6. Furuta, K.; Wang, G. X.; Minami, T.; Nishizawa, M.; Ito,
S.; Suzuki, M. Tetrahedron Lett. 2004, 45, 3933.
7. Hosoya, T.; Hiramatsu, T.; Ikemoto, T.; Nakanishi, M.;
Aoyama, H.; Hosoya, A.; Iwata, T.; Maruyama, K.;
Endo, M.; Suzuki, M. Org. Biomol. Chem. 2004, 2, 637.
8. Baldwin, J. E.; Bamford, S. J.; Fryer, A. M.; Rudolph, M.
P. W.; Wood, M. E. Tetrahedron 1997, 53, 5233.
9. Baldwin, J. E.; Fryer, A. M.; Pritchard, G. J. J. Org.
Chem. 2001, 66, 2597.
10. Azidation using compound 14 as starting material affor-
ded the corresponding product in low yield because
compound 14 is insoluble in the reaction media.
1
J = 18.9/3.4 Hz, 1H), 1.41(s, 9H). Compound 5: HNMR
(CDCl3, 400 MHz, dppm): 6.97(d, J = 8.8 Hz, 1H), 6.95(d,
J = 2.4 Hz, 1H), 6.82(d, J = 8.8 Hz, 1H), 5.00(t,
J = 3.2 Hz, 1H), 4.26(s, 2H), 4.13(d, J = 9.2 Hz, 1H),
3.82(d, J = 12.8 Hz, 1H), 3.78(s, 3H), 3.67–3.69(m, 1H),
3.65(s, 3H), 2.88–2.96(m, 2H), 2.67(dd, J = 18.6/3.4 Hz,
1H), 1.4(s, 9H). Compound 4: pale yellow amorphous
1
powder. H NMR (D2O, 400 MHz, d ppm): 7.13(s, 1H),
7.09(d, J = 8.8 Hz, 1H), 7.03(d,J = 8.8 Hz, 1H), 5.09(t,
J = 3.4 Hz, 1H), 4.61(d, J = 14.0 Hz, 1H), 4.39(d,
J = 14.0 Hz, 1H), 3.54(dd, J = 12.6/4.8 Hz, 1H), 3.44(d,
J = 10.4 Hz, 1H), 2.96(d, J = 13.2 Hz, 1H), 2.6–2.67(m,
2H), 2.52–2.58(m, 1H). MALDI-TOF-MS (m/z) [M+Na]+
calcd for C14H15NaN7O5 384.1032. Found: 384.1029.
12. The analogues of GIF-0448 with non-substituted phenyl-
oxy group at 4-position also showed almost the same
biological activity as acromelic acid A, unpublished
results.
13. Incorporation of a fluorescence detector and biological
assays, such as the measurements of electrophysiological
responses and intracellular Ca2+ increase in neurons, is
currently underway and detailed descriptions will be
reported separately.
11. Physical data: compound 13: 1H NMR (CDCl3, 400 MHz,
dppm): 8.24(d, J = 2.0 Hz, 1H), 7.98(dd, J = 8.8/2.0 Hz,
1H), 6.67(d, J = 8.8 Hz, 1H), 4.98(t, J = 3.6 Hz, 1H),
4.55(s, 2H), 3.95(d, J = 10.0 Hz, 1H), 2.75 ꢀ 2.81(m, 1H),
2.65–2.69(m, 1H), 2.5–2.6(m, 1H), 1.41(s, 9H), 0.9(s, 9H),
0.1(s, 6H). Compound 14: 1H NMR (CDCl3, 400 MHz,
dppm): 6.85(d, J = 8.8 Hz, 1H), 6.54(d, J = 8.8 Hz, 1H),
6.52(d, J = 8.8 Hz, 1H), 4.81(t, J = 3.6 Hz, 1H), 4.64(d,
J = 14 Hz, 1H), 4.55(d, J = 14 Hz, 1H), 4.10(d, J = 9.2 Hz,
1H), 3.76(s, 3H), 3.76(s, 3H), 3.64(s, 3H), 3.56–3.61(m,
2H), 2.86–2.89(m, 2H), 2.59–2.66(m, 1H), 1.41(s, 9H).
Compound 15: 1HNMR (CDCl3, 400 MHz, d ppm):
6.72(s, 1H), 6.58(d, J = 8.8 Hz, 1H), 6.25(d, J = 8.8 Hz,
1H), 4.76(t, J = 3.2 Hz, 1H), 4.45(s, 2H), 4.07(d,
J = 9.6 Hz, 1H), 3.73(s, 3H), 3.63(s, 3H), 3.38(br s, 2H),
2.80–2.90(m, 2H), 2.68(d, J = 12.6 Hz, 1H), 1.38(s, 9H).
Compound 16: 1HNMR (CDCl3, 400 MHz, d ppm):
7.26(d, J = 0.8 Hz, 1H), 6.88(d, J = 8.8 Hz, 1H), 6.78(d,
14. The compound can be handled for routine purpose of
biochemical experiments without special care under lab-
oratory conditions and kept for months unchanged in the
refrigerator unless exposed to UV light.