Synthesis of a bioprobe for elucidation of target molecule of spongean anti-malarial peroxides

Article abstract of DOI:10.1016/j.bmcl.2004.04.086

The reactants of an anti-malarial peroxide having a 6-carbomethoxymethyl-3- methoxy-1,2-dioxane moiety treated with FeSO₄ were analyzed. For mechanistic study of the anti-malarial peroxide, two biotinylated probes to elucidate the target molecu

Full text of DOI:10.1016/j.bmcl.2004.04.086

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3513–3516
Synthesis of a bioprobe for elucidation of target molecule of
spongean anti-malarial peroxides
Nobutoshi Murakami,a Motoyuki Kawanishi,a Sawako Itagaki,b Toshihiro Horiib and
Motomasa Kobayashia,*
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
bResearch Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Received 5 March 2004; accepted 19 April 2004
Available online
Abstract—The reactants of an anti-malarial peroxide having a 6-carbomethoxymethyl-3-methoxy-1,2-dioxane moiety treated with
FeSO4 were analyzed. For mechanistic study of the anti-malarial peroxide, two biotinylated probes to elucidate the target molecules
were designed and synthesized. The two synthesized probes showed potent anti-malarial activity, and one of them was proved to
form an irreversible binding with protein in a model experiment.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Since the discovery of artemisinin, many kinds of cyclic
OCH3
O
O
CH3O
peroxides with anti-malarial activity were reported from
synthetic and natural resources.1 In the course of our
continuous investigation in a search for new bioactive
principles from marine organisms, the two methyl esters
(1, 2) of cyclic peroxides2 from a marine sponge were
shown to exhibit potent anti-malarial activity. Further-
more, a facile construction of the pharmacophore
framework, 6-carbomethoxymethyl-3-methoxy-1,2-di-
oxane, was established.3 By application of this protocol,
readily available peroxides showing not only more po-
tent activity than 1 and 24 but also in vivo efficacy
against Plasmodium berghei infected mice5 were dis-
closed. For the purpose of rational and evidence-based
design of new anti-malarials utilizing 1 and 2 as scaf-
folds, elucidation of the target molecule was intensively
required. In this context, we have undertaken to explore
a molecular probe to elucidate the target protein of 1
and 2. This communication deals with a synthesis of
biotinylated probes with irreversible binding affinity
against protein (Fig. 1).
6
O
1 : 6α-H
2 : 6β-H
OCH3
O
O
CH3O
O
3
Figure 1.
Fe(II) of heme in the food vacuole, a characteristic
organelle in P. falciparum, to provide alkyl radicals.6
The resulting radical species were presumed to form
covalent adducts with proteins in the food vacuole to
inhibit the proliferation of malaria parasites.7 Previ-
ously, we disclosed that a synthetic peroxide 3 having a
6-carbomethoxymethyl-3-methoxy-1,2-dioxane moiety
with a pentyl residue at the C-3 position showed anti-
malarial activity with IC50 of 0.12 lM against P. falci-
parum.4 In order to design a bioprobe molecule for
studying the mode of action of 3, the genuine active
species of 3 were investigated from the reactants of 3
with Fe(II). Treatment of 3 with FeSO4 in H2O afforded
From a study of the mode of action of artemisinin and
its model compounds, the endoperoxide bridge was
proved to be degradated by one-electron reduction with
* Corresponding author. Tel.: +81-6-6879-8215; fax: +81-6-6879-8219;
e-mail: kobayasi@phs.osaka-u.ac.jp
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.086

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N. Murakami et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3513–3516
O
O
CH3O
O
O
OCH3
a
HN NH
CH3O
(CH2)4CH3
O
H
(CH2)4CH3
OH
O
O
O
S
4
O
i
7
8
O
b, c
O
O
CH3O
O
CH3O
HN NH
OCH3
O
a
H
N
O
O
iii
3
5
S
NH2
O
10
9
(CH2)4CH3
iv
O
O
O
CH3O
OCH3
HN NH
CH3O
O
O
OCH3
(CH2)4CH3
HN NH
O
O
OH
d
O
S
O
N
HO
6
H
S
ii
O
11
12
Figure 2. Plausible reaction pathway giving 4–6 in the FeSO4 treat-
ment of 3.
Scheme 1. Reagents and conditions: (a) 11, EDCIÆHCl, pyridine, 93%
for 8, 88% for 10; (b) MsCl, Et3N, toluene, then aq NaN3, Bu4NBr; (c)
n-Bu3P, CH3CN–H2O, 2 steps 43%; (d) DPPA, Et3N, CH3CN, 55 ꢁC
then 7, 84%.
4, 5, and 6 in 18%, 28%, and 43% yields, respectively. A
plausible reaction pathway from 3 to 4, 5, and 6 is de-
picted in Figure 2. Taking this product distribution into
account, a pentyl radical iv generated in the formation
of a major intermediate iii was presumed to be respon-
sible for the anti-malarial activity of 3. Based on this
finding, we planned to introduce biotin, which possesses
strong affinity to avidin, at the terminal of the pentyl
residue.
For effective labeling of the target protein by the probe
molecule and successive isolation of the labeled protein
by the tight affinity between biotin and avidin, the
linkage between the carbon radical and the biotin resi-
due should be stable during a few hours’ exposure in the
culture medium. Therefore, the stability of three kinds
of linkages (ester, amide, and carbamate) in the culture
medium was evaluated as candidate functionalities to
connect the carboxylic group in biotin with the C-3 alkyl
moiety. For this purpose, three model compounds
having 1-phenyl-1-yne chromophore were synthesized as
shown in Scheme 1. Condensation of 1-phenyl-1-pen-
tyn-5-ol (7) and biotin (11) by 3-(3-dimethylaminoprop-
yl)-1-ethylcarbodiimide hydrochloride (EDCIÆHCl) in
pyridine gave an ester 8 in 93% yield. The mesylate of
7 was subjected to azidation followed by reduction with
n-Bu3P to afford a primary amine 9 in 43% yield for two
steps. The amine 9 was coupled with biotin (11) under
the same conditions as for the preparation of 8 to pro-
vide an amide 10. The carbamate 12 was synthesized by
the coupling of 7 with an isocyanate, which was gener-
ated from biotin (11) through diphenylphosphoryl azide
(DPPA)-mediated Curtius rearrangement,8 in 84% yield.
Next, the stability in RPMI 1640 medium supplemented
with heat-inactivated 10% type A human serum for the
three model compounds was evaluated, and the time
course of the remains is depicted in Figure 3. As shown
in Figure 3, the ester 8 was readily metabolized and
Figure 3. Stability of 8, 10, and 12 in the human serum.
disappeared completely within 3 h. The amide 10
showed moderate stability in the human serum. On the
contrary, the carbamate 12 was appreciably stable and
almost all remained even after 6 h exposure. On the basis
of this behavior, the carbamate linkage was chosen for
the linkage with biotin.
Synthesis of the biotinylated probe 17 was executed as
shown in Scheme 2. Treatment of the Weinreb amide 134
with 1-pentylmagnesiumbromide, which was prepared
from 1-bromopentane, afforded a ketoalcohol. Swern
oxidation of the ketoalcohol followed by Wittig reaction
with (carbethoxymethylene)triphenylphosphorane pro-
vided a ketoester 14 in 70% yield for three steps. The
ketoester 14 was submitted to hydroboration with

N. Murakami et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3513–3516
3515
O
O
O
OMe
Me
CH3O
d, e
HO
a, b, c
CH3O
OTBDPS
O
N
O
O
13
15
14
HN
NH
OCH3
f, g, h
i
CH3O
OCH3
O O
O
CH3O
O O
OH
O
(CH2)5O
S
O
N
H
16
17
O
O
O
HN NH
HN NH
j
O
O
O
i
HO
OH
OCN
O
O
N
O
O
S
HO
O
N
H
S
N
H
H
18
v
19
O
HN NH
CH3O
OCH3
O
O
O O
H
N
O
O
N
H
O
O
S
O
N
H
O
20
Scheme 2. Reagents and conditions: (a) CH2@CHCH2CH2CH2MgBr, THF; (b) (COCl)2, DMSO, Et3N, CH2Cl2; (c) CH3OCOCH@PPh3, CH2Cl2,
3 steps 70%; (d) 9-BBN, THF, then H2O2, aq NaOH, 69%; (e) TBDPSCl, imidazol, CH2Cl2, quant.; (f) H2O2ÆH2NCONH2, Sc(OTf)3, MeOH, 52%;
(g) Et2NH, CF3CH2OH, 87%; (h) HF–pyridine, THF, quant.; (i) 11, DPPA, Et3N, CH3CN, 75% for 17, 71% for 19; (j) 1,4-diisocyanatobutane,
pyridine, then 16, 58%.
9-borabicyclo[3.3.1]nonane (9-BBN) and subsequent
oxidation with H2O2 to give a primary alcohol, the hy-
droxyl group of which was protected as a t-butyldi-
phenylsilyl ether to afford 15 in 69% yield. After
construction of the 6-carbomethoxymethyl-3-methoxy-
1,2-dioxane framework by the Sc(OTf)3 catalyzed per-
oxyhemiacetalization followed by intramolecular hetero
Michael addition, the protecting group was removed by
HF-pyridine treatment to provide a peroxide 16 in 45%
yield for three steps. Furthermore, biotin (11) was con-
nected with 16 through a carbamate linkage in the same
fashion as for the preparation of 12 to furnish the de-
sired biotinylated probe 179 in 75% yield.
they still preserved moderate anti-malarial potency de-
spite some reduction of activity in comparison with the
parent peroxide 3 (IC50 ¼ 0.12 lM), they were expected
to bind to the target protein of the spongean anti-
malarial peroxides (1, 2). As a preliminary experiment,
we examined the ability of 17 to generate a carbon
radical species and label proteins in the presence of
FeSO4 under the same acidic medium (acetate buffer,
pH 5.2) as the food vacuole in P. falciparum. After the
probe 17 was incubated with bovine albumin, the reac-
tion mixture was analyzed by avidin bound to horse-
radish peroxidase on the SDS-page.12 In only the case of
coexistence of 17 and FeSO4, the protein band due to
bovine albumin was stained.
On the other hand, the adjacent region to the biotin-
recognizing domain in avidin was known to consist of
hydrophilic amino acid residues.10 Therefore, introduc-
tion of a hydrophilic function was expected to bring
about an effective biotin–avidin binding. This pre-
sumption directed us to synthesize another biotinylated
probe 20 possessing a hydrophilic spacer as illustrated in
Scheme 2. The isocyanate derived from biotin (11) was
treated with diethylene glycol 18 to give a half-carba-
mate 19 in 71% yield. Coupling of 19 and 1,4-di-
isocyanatobutane in pyridine and successive treatment
of the resulting half-isocyanate v with the alcohol 16
in situ furnished the probe 20 in 58% yield.11
From this observation, the biotinylated probe 17 was
clarified to generate the expected carbon radical and
possess affinity against proteins. Labeling of target
proteins in the malaria parasites is currently under
investigation in our laboratory.
Acknowledgements
This study was financially supported by Grants-in-Aid
for Scientific Research from the Ministry of Education,
Science, Sports, and Culture of Japan. The authors are
also grateful to the Research Foundation for Pharma-
ceutical Sciences for financial support.
The anti-malarial activity of both biotinylated probes
(17, 20) was assessed. Both compounds inhibited the
proliferation of malaria parasite with IC50 values of
0.91 lM for 17 and 0.57 lM for 20, respectively.4 Since

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N. Murakami et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3513–3516
10. Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5076.
References and notes
11. 20: colorless powder. syn:anti ¼ 7:1. IR mmax (KBr) cmꢀ1
:
1. Jefford, C. W. Curr. Med. Chem. 2001, 8, 1803.
2. Kobayashi, M.; Kondo, K.; Kitagawa, I. Chem. Pharm.
Bull. 1993, 41, 1324.
3. Murakami, N.; Kawanishi, M.; Itagaki, S.; Horii, T.;
Kobayashi, M. Tetrahedron Lett. 2001, 42, 7281.
4. Murakami, N.; Kawanishi, M.; Itagaki, S.; Horii, T.;
Kobayashi, M. Bioorg. Med. Chem. Lett. 2002, 12, 69.
5. Murakami, N.; Kawanishi, M.; Mostaqul, H. M.; Li, J.;
Itagaki, S.; Horii, T.; Kobayashi, M. Bioorg. Med. Chem.
Lett. 2003, 13, 4081.
1734, 1702, 1260. 1H NMR (500 MHz, CDCl3) d: 1.10–
1.31, 1.35–1.71 (total 22H, m), 2.31 (1H, dd, J ¼ 16:0,
5.9 Hz, MeO2CCH2-a), 2.43 (1H, dd, J ¼ 16:0, 7.8 Hz,
MeO2CCH2-b), 2.68 (1H, d, J ¼ ca. 13 Hz, SCH2-a), 2.86
(1H, dd, J ¼ ca. 13, 4 Hz, SCH2-b), 3.06–3.16 (7H, m,
CHS, 3 · OCONHCH2), 3.19 (3H, s, 3-OCH3, syn), 3.21
(3H, s, 3-OCH3, anti), 3.55–3.68 (8H, m, 2 · OCH2CH2O),
3.63 (3H, s, CO2CH3), 3.97 (2H, t-like, J ¼ ca. 7 Hz,
CH2OCONH), 4.17–4.24 (1H, br s, NHCONHCH), 4.25–
4.48 (1H · 2, m, NHCONHCH, 6-H). FAB-MS: m=z 764
(M+H)þ. FAB-HRMS m=z calcd for C33H58N5O13S:
764.3752, found: 764.3750.
6. O’Neill, P. M.; Miller, A.; Bishop, L. P. D.; Hindley, S.;
Maggs, J. L.; Ward, S. A.; Roberts, S. M.; Scheinmann,
F.; Stachulski, A. V.; Posner, G. H.; Park, B. K. J. Med.
Chem. 2001, 44, 58.
12. A solution of albumin (2.5 lg) in acetate buffer (25 lL,
pH ¼ 5.2) was treated with a solution of 17 (0.1 mM) in
DMSO (5 lL) in the presence or absence of a solution of
FeSO4ÆH2O (0.5 nM) in acetate buffer (20 lL). After
standing at 37 ꢁC for 6 h, the whole mixture was treated
with SDS-PAGE sample buffer (20 lL, 100 mM Tris–HCl,
4% SDS, 12% mercaptoethanol, 20% glycerol, 0.005%
bromophenol blue), and boiled for 5 min. The mixture was
subjected to electrophoresis on SDS-polyacrylamide gel
(4–20%, gradient), then transferred to a PVDF membrane.
The membrane was blocked with PBS buffer containing
5% non-fat dry milk and 0.1% NP-40. After triplicate
washing with PBS buffer, the blot was incubated with
horseradish peroxidase-conjugated avidin (Bio-Rad) for
1 h. After additional rinses with PBS buffer, the immuno-
blots were developed using ECL detection kit (Amersham)
on Hyperfilm ECL film (Amersham).
7. Meshnick, S. R.; Jefford, C. W.; Posner, G. H.; Avery, M.
A.; Peters, W. Parasitol. Today 1996, 12, 79.
8. Ninomiya, K.; Shioiri, T.; Yamada, S. Tetrahedron 1974,
30, 2151.
9. 17: Colorless powder. syn:anti ¼ 6.7:1. IR mmax (KBr) cmꢀ1
:
1737, 1703, 1256. 1H NMR (500 MHz, CDCl3) d: 1.00–
2.10 (18H, m), 2.31 (1H, dd, J ¼ 15:9, 5.5 Hz,
MeO2CCH2-a), 2.44 (1H, dd, J ¼ 15:9, 7.9 Hz,
MeO2CCH2-b), 2.66 (1H, d, J ¼ 12:8 Hz, SCH2-a),
2.84 (1H, dd, J ¼ 12:8, 3.7 Hz, SCH2-b), 2.91–3.14 (3H,
br s, CHS, OCONHCH2), 3.19 (3H, s, 3-OCH3, syn),
3.21 (3H, s, 3-OCH3, anti), 3.63 (3H, s, CO2CH3), 3.97
(2H, t, J ¼ 6:1 Hz, CH2OCONH), 4.27 (1H, br s,
NHCONHCH), 4.38–4.48 (1H · 2, m, NHCONHCH, 6-
H). FAB-MS: m=z 518 (M+H)þ. FAB-HRMS m=z calcd
for C23H40N3O8S: 518.2536, found: 518.2533.