Synthesis and biological properties of tensyuic acids B, C, and E, and investigation of the optical purity of natural tensyuic acid B

Article abstract of DOI:10.1016/j.tet.2008.05.035

The first, concise total synthesis of (±)-tensyuic acids B, C, and E, using chemoselective formal S_N2′ type Grignard reactions and selective esterification, is described. In addition, the optical purity of natural (±)-tensyuic acid B was determined using Chirabite-AR. Synthetic tensyuic acids, together with their intermediate compounds, were found to possess useful bioactive properties, with some of them showing potent activity against Trypanosoma brucei brucei strain GUTat 3.1.

Full text of DOI:10.1016/j.tet.2008.05.035

Tetrahedron 64 (2008) 7369–7377
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and biological properties of tensyuic acids B, C, and E,
and investigation of the optical purity of natural tensyuic acid B
*
Takanori Matsumaru, Toshiaki Sunazuka , Tomoyasu Hirose, Aki Ishiyama, Miyuki Namatame,
ꢀ
*
Takashi Fukuda, Hiroshi Tomoda, Kazuhiko Otoguro, Satoshi Omura
Kitasato Institute for Life Sciences & Graduate School of Infection Control Sciences, Research Center for Tropical Diseases and School of Pharmaceutical Sciences,
Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
a r t i c l e i n f o
a b s t r a c t
The first, concise total synthesis of (ꢀ)-tensyuic acids B, C, and E, using chemoselective formal SN20 type
Grignard reactions and selective esterification, is described. In addition, the optical purity of natural (ꢀ)-
tensyuic acid B was determined using Chirabite-AR. Synthetic tensyuic acids, together with their in-
termediate compounds, were found to possess useful bioactive properties, with some of them showing
potent activity against Trypanosoma brucei brucei strain GUTat 3.1.
Article history:
Received 18 April 2008
Received in revised form 8 May 2008
Accepted 8 May 2008
Available online 13 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Tensyic acid
Total synthesis
Formal SN2 reaction
Selective esterification
Chirabite-AR
Anti-trypanosomal activity
1. Introduction
reaction3 and p53-HDM2 interaction,4 studies of the biological
properties of tensyuic acids have been hampered by insufficient
During the course of screening for new antibiotics, six new
compounds, designated tensyuic acids A–F (Fig. 1), were isolated
from a culture broth of Aspergillus niger FKI-2342.1 The structure
of tensyuic acids A–F is known to belong to the itaconic acid
family.2–4 In addition, tensyuic acids are novel itaconic acid de-
rivatives having the ester carboxyl moieties at the bottom of the
alkyl side chain. Although tensyuic acids and structurally related
quantities of products from the natural source. Consequently, the
supply of these natural products produced synthetically will allow
detailed investigation of their biological properties. Here, we de-
scribe the first total synthesis of (ꢀ)-tensyuic acids B (1), C (2), and E
(3), the optical purity of naturally occurring 1, and the biological
activities of them.
hexylitaconic acid3 have low [
a
]
values (tensyuic acid A: þ1.8, B:
D
2. Results and discussion
ꢁ1.7, C: ꢁ6.0, D: þ3.0, E: þ4.9, F: ꢁ3.1; hexylitaconic acid: ꢁ8) in
methanol,1,3 the absolute configuration of related itaconic acid
derivatives (i.e., hexylitaconic acid3 and ceriporic acid A4) has
never been reported. The property of biological activities for an-
timicrobial and anticancer actions has been traced to the novel
tensyuic acids, and tensyuic acid C (2) has been identified as being
antibiotic agent against Bacillus subtilis with moderate concen-
2.1. Retrosynthetic analysis of tensyuic acids B, C, and E
Tensyuic acids containing exo-type olefin are generally synthe-
sized using a Wittig reaction,5 coupling reactions involving re-
duction of carbon–carbon triple bond,6 and SN20 coupling reactions
of appropriate substrates and nucleophiles.7 In our synthetic plan,
we envisaged construction of the basic structure of the tensyuic
acids to permit a shorter synthetic route via chemoselective formal
SN20 coupling between Grignard reagents and dimethyl bromo-
methylfumarate (8) as shown in Scheme 1.
tration for MIC (inhibition zone: 10 mm at 50 m
g/disk).1 Although
other itaconic acid derivatives have been reported to exhibit
various activity, such as inhibitor of the glyoxylate cycle,2 the
production of a cellulolytic active oxygen species, the iron redox
The C–C linkage for this formal SN20 reaction will allow access to
all forms of tensyuic acids from 8 with suitable nucleophiles. Our
approach also has the feature of efficient selective esterification
from hydrazide, with appropriate side chains.
* Corresponding authors.
E-mail addresses: sunazuka@lisci.kitasato-u.ac.jp (T. Sunazuka), omuras@
ꢀ
insti.kitasato-u.ac.jp (S. Omura).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.05.035

7370
T. Matsumaru et al. / Tetrahedron 64 (2008) 7369–7377
O
O
O
O
HO
O
HO
O
HO
OH
OMe
OH
EtO
MeO
MeO
O
O
O
Tensyuic acid C (2)
O
Tensyuic acid B (1)
Tensyuic acid A
O
O
O
HO
O
HO
O
HO
OH
OMe
OH
EtO
MeO
MeO
O
O
O
Tensyuic acid F
Tensyuic acid D
Tensyuic acid E (3)
Figure 1. Structure of tensyuic acids A–F.
O
O
O
HO
Selective
OH Esterification
H2NHN
O
HO
OH
RO
n
n
O
O
( )-Tensyuic acid B (1) n=1, R=Me
C (2) n=1, R=Et
n=1 (4)
n=3 (5)
E (3) n=3, R=Me
Chemoselective
formal SN2'
Coupling Reaction
O
Br
O
MgBr
n
TBDPSO
MeO
n=1 (6)
n=3 (7)
OMe
8
OH
n
HO
n=1 (9)
n=3 (10)
O
11
O
O
Scheme 1. Retrosynthetic analysis of (ꢀ)-tensyuic acids B, C, and E.
2.2. Preparation of fragments and chemoselective formal SN20
coupling
Grignard reagent from unreactive halides by direct reaction of the
halides and magnesium. This difficultly was conquered by the use
of I2 to activate magnesium metal in freshly distilled Et2O, and the
long chain Grignard reagent (7) was obtained as a dark gray so-
lution in quantitative conversion. The Grignard reagent reacted in
a highly chemo- and regioselective fashion with 8 and the exclu-
sive Michael addition, followed by elimination of the allylic bro-
mine atom, gave the formal SN20 product in 66% yield. In
a previous report,8 Argade and Kar reported that HMPA as an
additive is very effective for this formal SN20 coupling reaction, but
we obtained a better yield in the absence of HMPA for our
substrate.
The total synthesis of (ꢀ)-tensyuic acid E (3), which has the
longest alkyl chain among the tensyuic acids, began with the
preparation of the 8C-Gignard reagent (7) and dimethyl bromo-
methylfumarate (8).8 The fumarate derivative (8), conveniently
available from citraconic anhydride (11), is a convenient starting
material via the Argade protocol8 (Scheme 2). The reaction of 11
with methanol/H2SO4 under reflux gave the desired diester (12) in
90% yield. Treatment of 12 with NBS/AIBN in refluxing carbon tet-
rachloride underwent allylic bromination and isomerization of the
carbon–carbon double bond to yield 8 in 89% yield.
The precursor of Grignard reagent (14)9 was prepared from 1,8-
octanediol (10) as a starting material by mono-bromination with
HBr under reflux in 99% yield and protection of the remaining hy-
droxyl group with TBDPS in 99% yield (Scheme 3).
Turning to the formal SN20 coupling tactic to construct the C3-
alkyl itaconic acid structure, the Grignard reagent (7) was derived
from 14 with activated magnesium metal to react with 8 (Scheme
4). However, it is difficult to prepare the high-molecular weight
O
O
Br
NBS
AIBN
H2SO4
MeO
MeO
MeO
OMe
CCl4
MeOH
reflux
90%
O
O
O
reflux
89%
O
12
O
11
8
Scheme 2. Preparation of dimethyl bromomethylfumarate (8).

T. Matsumaru et al. / Tetrahedron 64 (2008) 7369–7377
7371
TBDPSCl
imid.
HBr
TBDPSO
HO
HO
Br
Br
OH
CH2Cl2
99%
toluene
Δ
99%
10
13
14
Scheme 3. Preparation of 1-bromo-8-silyloxy octane (14).
O
Mg
I2
8
MeO
TBDPSO
14
MgBr
Et2O
Et2O
r.t.
OMe
TBDPSO
7
–78 °C
( )-15
O
66%
Scheme 4. Synthesis of (ꢀ)-3-alkyl itaconic acid derivative (15).
2.3. Completion of total synthesis of ( )-tensyuic acid E
hydrazide being treated with CAN11 as oxidant in methanol to
directly furnish (ꢀ)-3 in 58% yield in three steps from (ꢀ)-19
(Scheme 7).
Having the basic carbon frame of 3, we turned our attention to
complete the synthesis of (ꢀ)-3 via a selective esterification strat-
egy (Scheme 5). Deprotection of TBDPS from (ꢀ)-15 with TBAF in
THF gave the desired exo-olefin product (ꢀ)-16 in 47% yield with
the more thermodynamically stable endo-olefin as a by-product in
53% yield. To avoid this isomerization, we examined several
deprotection conditions, with 47% HF aq/pyridine (1/2.4, v/v) being
the most effective for suppression of the undesired isomerization,
affording 88% yield of exo-product (ꢀ)-16 and none of the endo-
product. Subsequently, primary alcohol (ꢀ)-16 was subjected to
Parikh–Doering oxidation and Pinnick oxidation to furnish the
desired carboxylic acid (ꢀ)-17 in 81% yield in two steps. Then,
(ꢀ)-17 was converted to acid chloride with (COCl)2, followed by
condensation with Boc-hydrazine to give the Boc-hydrazide (ꢀ)-18
in 99% yield in two steps. Because hydrazide (ꢀ)-18 is a key in-
termediate in this total synthesis, two benefits are bestowed.
Firstly, hydrazide can be easily converted to the desired ester under
simple oxidation conditions in the solution of suitable alcohol.
Secondly, hydrazide can be maintained under saponification con-
ditions. Consequently, dicarboxylic acid with Boc-hydrazide (ꢀ)-19
was easily prepared under basic condition, from (ꢀ)-18, with
(ꢀ)-19 then being subjected to the next reaction without silica gel
purification, due to its high polarity.
The mechanism of the reaction of hydrazide with CAN and es-
terification with alcohol is unclear.11,12 Although the reaction pro-
cess is not fully understood, a proposed mechanism for the
formation of the corresponding ester is depicted in Scheme 8.
Regarding the evolution of nitrogen, an acyldiazonium salt (c)
would be generated by the oxidation of hydrazide. Alcohol would
then attack c to give the ester (d).
2.4. Total synthesis of ( )-tensyuic acids B and C
After completion of the total synthesis of (ꢀ)-3, our focus turned
to the total synthesis of tensyuic acids B (1) and C (2). Accordingly,
the Grignard reagent (6) was prepared by the same reaction se-
quences as for the preparation of 7, using commercially available
1,6-hexanediol (9). Subsequently, 6 and 8 underwent the same
reaction sequence as (ꢀ)-15 for the synthesis of (ꢀ)-tensyuic acid E
(3) to give (ꢀ)-tensyuic acid B (1) (Scheme 9). Additionally,
(ꢀ)-tensyuic acid C (2) was prepared via the use of ethanol instead
of methanol as a reaction solvent for final reaction step from (ꢀ)-4
to selectively give the ethyl ester (Scheme 9).
The three specific synthetic natural products, (ꢀ)-tensyuic acids
B, C, and E, were found to be identical to the naturally occurring
tensyuic acid products in all respect.
After deprotection of the Boc group with TFA from the crude
(ꢀ)-19, azide (ꢀ)-5 was prepared using sodium nitrate and aqueous
HCl.10 The reaction was quenched with suitable alcohol (in this
case, methanol) to complete full construction of (ꢀ)-tensyuic acid E
(3) in 30% yield from (ꢀ)-19 in three steps (Scheme 6).
2.5. Application of Chirabite-AR to determine ee% of
natural-(1)
Utilization of HONO for this selective esterification, under sev-
eral conditions, was examined but (ꢀ)-tensyuic acid E (3) could not
be obtained in satisfactory yields. Esterification with another oxi-
dizing agents eventually proved successful, the deprotected
In possession of the racemic tensyuic acids, we next investigated
the optical purity of naturally occurring tensyuic acids using Chir-
abite-AR, a commercially available chiral shift reagent, newly de-
veloped by Ema et al.13 (Fig. 2).
1) SO3 Py
Et3N
O
O
MeO
CH2Cl2
O
MeO
( )-17
HF. aq
OMe
( )-15
OMe
O
2) NaClO2
NAH2PO4
t-BuOH/H2O
2 steps 81%
HO
Pyridine
88%
HO
O
( )-16
O
O
1) (COCl)2
Benezene
O
HO
O
MeO
LiOH
OH
OMe
2) BocNHNH2
Et3N
2 steps 99%
THF : H2O
BocHNHN
BocHNHN
( )-19
( )-18
O
O
Scheme 5. Synthesis of (ꢀ)-19.

7372
T. Matsumaru et al. / Tetrahedron 64 (2008) 7369–7377
O
O
1)TFA
2)NaNO2
HCl aq.
quenched
O
HO
O
HO
with MeOH
( )-19
OH
OH
MeO
N3
3 steps 30%
( )-5
O
O
( )-Tensyuic acid E (3)
Scheme 6. Completion of the synthesis of (ꢀ)-tensyuic acid E (3).
mL)22a (Fig. 5), which is still widely used as therapeutic agent in
humans. Currently, only four drugs are registered for the treatment
of Human African trypanosomiasis (HAT): pentamidine, suramin,
melarsoprol, and eflornithine.22a All four drugs used to treat HAT
are unsatisfactory, since they cannot be given orally and are all
hampered by severe toxicity and increasing resistance of the par-
asites. Consequently, there is an urgent need for new anti-trypa-
nosomal drugs, which have novel structures and mechanisms of
action and which are both safe and effective. Tensyuic acid C and
a synthetic intermediate (16) may develop to be novel anti-trypa-
nosomal drugs.
O
1) TFA
O
HO
2) CAN / MeOH
( )-19
OH
MeO
3 steps 58%
from ( )-18
( )-3
O
Scheme 7. Advanced condition for the selective esterification.
So far, various types of chiral shift reagents such as lanthanide
complexes,14 cyclodextrins,15 crown ethers,16 calixarenes,17 por-
phyrins,18 and others have been developed. However, few have
been commercialized except for lanthanide complexes, which often
cause signal broadening, particularly in a high magnetic field be-
cause of the paramagnetic metal. To demonstrate the practical
utility of Chirabite-AR, at first, synthetic (ꢀ) and naturally occurring
tensyuic acid B (1) was tested. Before comparison between syn-
thetic and natural tensyuic acid B with Chirabite-AR, we examined
the effect of differing amounts of Chirabite-AR regarding (ꢀ)-1, to
determine sufficient signal separations between (þ)- and (ꢁ)-1. As
shown in Figure 3, two signals of exo-olefin of (ꢀ)-1 were strongly
influenced by Chirabite-AR in CDCl3 at room temperature, the ex-
tent depending on increasing amounts of Chirabite-AR, together
with the signal broadening. Fortunately, the problem of signal
broadening was resolved by heating the mixture of (ꢀ)-1 and
Chirabite-AR in CDCl3 to 50 ꢂC.
3. Conclusion
In conclusion, we have achieved the first, a concise, total syn-
thesis of (ꢀ)-tensyuic acids B (1), C (2), and E (3) using chemo-
selective formal SN20 type Grignard reactions and selective
esterification. In addition, using Chirabite-AR we found that the
optical purity of natural 1 is 25% ee. We have also identified some
important bioactive properties for (ꢀ)-tensyuic acids and their
synthetic intermediates, and found potent activity against Trypa-
nosoma brucei brucei for compounds (ꢀ)-2 and 16. Further in vitro
and in vivo studies on tensyuic acid C and analogues are in progress.
4. Experimental
4.1. General
A mixture of (ꢀ)-tensyuic acid B with 70 mol % of Chirabite-AR
1
was measured sequentially by 400 MHz H NMR at 50 ꢂC in CDCl3
(Fig. 4-1), marked signal separations were observed for exo-olefin,
without notable line broadening, and good enantiomeric discrim-
ination was achieved for racemic 1.
NMR analysis of natural 1 under the same conditions as used to
obtain the results displayed in Figure 4-1 indicated that separated
signals exhibited 5/3 ratio in numerical integration value (Fig. 4-2).
Therefore, the optical purity of natural 1 was determined as 25% ee.
Dry tetrahydrofuran (THF), toluene, and CH2Cl2 were purchased
from Kanto Chemical Co., Inc. Dry diethyl ether (Et2O) was freshly
distilled from sodium/benzophenone under argon. Activated
magnesium turnings were purchased from Kanto Chemical Co., Inc.
Chirabite-AR was purchased from Tokyo Chemical Industry (TCI)
Co., Ltd. Pre-coated silica gel plates with a fluorescent indicator
(Merck 60 F254) were used for analytical and preparative thin layer
chromatography. Flash column chromatography was carried out
with Kanto Chemical silica gel (Kanto Chemical Co., Inc., Silica gel
60N, spherical neutral, 0.040–0.050 mm, Cat. No. 37563-84). 1H
NMR spectra were recorded at 270 MHz or 400 MHz and 13C NMR
spectra were recorded at 67.5 MHz or 100 MHz on JEOL JNM-EX270
(270 MHz) or Varian XL-400 (400 MHz) or Varian UNITY-400
(400 MHz). Chemical shifts are expressed in parts per million
downfield from internal solvent peaks CHCl3 (7.26 ppm, 1H NMR)
and J values are given in hertz. The coupling patterns are expressed
by s (singlet), d (doublet), t (triplet), and m (multiplet). The all in-
frared spectra were measured on a Horiba FT-210 spectrometer.
High- and low-resolution mass spectra were measured on a JEOL
2.6. Biological activity
The bioactivity of synthetic tensyuic acids, including all syn-
thetic intermediates, was examined, with respect to their anti-in-
fectious properties, notably those focused on Type III secretion
systems,19 influenza,20 methicillin-resistant Staphylococcus aureus
(MRSA),21 Trypanosoma,22 vancomycin-resistant Enterococcus
(VRE),21 and toll-like receptor (TLR).23
Both (ꢀ)-tensyuic acid C (2) and a synthetic intermediate (16)
show promising anti-trypanosomal properties in vitro, with IC50
values of 2.23
mg/mL and 1.95
m
g/mL, respectively (Table 1). The
g/
values of these IC50 are close to the IC50 value of suramin (1.58
m
O
O
O
H
N
O
[O]
R'OH
[O]
N
N
R
–
N2
OR'
R
N
H
R
N
H
R
N
H
a
c
b
d
Scheme 8. Proposed mechanism for esterification from hydrazide by CAN.

T. Matsumaru et al. / Tetrahedron 64 (2008) 7369–7377
7373
O
MeO
HF. aq
TBDPSO
MgBr
8
OMe
Et2O
63%
Pyridine
78%
TBDPSO
6
( )-20
O
O
O
1) SO3 Py
Et3N
1) (COCl)2
Benezene
O
MeO
MeO
CH2Cl2
OMe
OMe
O
HO
HO
2) NaClO2
NaH2PO4
t-BuOH/H2O
2 steps 85%
2) BocNHNH2
Et3N
2 steps 78%
( )-21
( )-22
O
O
O
1) LiOH
2) TFA
O
MeO
O
HO
OMe
OH
O
BocHNHN
H2NHN
( )-23
( )-4
O
CAN, EtOH
3 steps42% from 23
CAN, MeOH
3 steps 45% from 23
O
O
O
HO
O
HO
OH
OH
MeO
EtO
O
O
( )-Tensyuic acid C (2)
( )-Tensyuic acid B (1)
Scheme 9. Total synthesis of (ꢀ)-tensyuic acids B (1) and C (2).
JMS-DX300 and JEOL JMS-AX505 HA spectrometers. Liquid chro-
matographic preparation was conducted on a Jasco PU-980 with
Senshu Pak-PEGASIL ODS.
reaction mixture was stirred for further 19 h. The reaction was then
quenched by addition of NH4Cl aq solution (10 mL) and warmed to
room temperature. After stirring for 10 min, an additional Et2O
(10 mL) was added to the mixture and two layers were separated.
The aqueous layer was extracted with Et2O (3ꢃ30 mL) and the
combined organic layers were washed with brine (30 mL), dried
over Na2SO4, and concentrated in vacuo. Flash chromatography
(hexane/ethyl acetate¼70:1) afforded (ꢀ)-15 (730 mg, 66%) as
a colorless oil. IR nmax (NaCl): 1739, 1724 (C]O), 1199, 1141 (C–O),
4.2. Total synthesis of ( )-3
4.2.1. 1-(tert-Butyldiphenylsilyloxy)-8-octylmagnesium bromide (7)
and methyl 11-(tert-butyldiphenylsilyloxy)-2-methylene-3-
methoxycarbonylundecanate ((ꢀ)-15)
1101 (C–O–Si) cmꢁ1; 1H NMR (270 MHz, CDCl3)
d: 7.68 (4H, m, Ar–
A solution of Grignard reagent in Et2O was prepared as follows:
a catalytic amount of I2 (one crystal) was added to activated mag-
nesium turnings (310 mg, 12.9 mmol), which were soaked in dry
Et2O (0.5 mL) under argon at room temperature, and the mixture
was stirred for 10 min. To the mixture with gentle stirring was
added the solution of 14 (1.9 g, 4.26 mmol) in Et2O (0.5 mL), and the
solution was stirred for further 3 h to afford the desired Grignard
reagent in Et2O. Subsequently, to the solution of dimethyl bromo-
methylfumarate (8) (500 mg, 2.13 mmol) in ether (5 mL) was
slowly added the prepared Grignard reagent 7 at ꢁ78 ꢂC. The
H), 7.36 (6H, m, Ar–H), 6.36 (1H, s, 10-H), 5.75 (1H, s, 10-H), 3.76 (3H,
s, 1-OCH3), 3.67 (3H, s, 100-OCH3), 3.64 (2H, t, J¼6.4 Hz, 11-H), 3.50
(1H, t, J¼7.3 Hz, 3-H), 1.98–1.44 (4H, complex m, 4, 10-H2), 1.42–1.17
(10H, complex m, 5, 6, 7, 8, 9-H2), 1.04 (9H, s, SiC(CH3)3); 13C NMR
(67.5 MHz, CDCl3) d
: 173.7 (C-100), 166.6 (C-1), 138.3 (C-2), 135.5 (2C,
Arꢃ2), 134.1 (Ar), 129.4 (Ar), 127.5 (2C, Arꢃ2), 126.6 (C-10), 63.9 (C-
11), 52.0 (COOCH3), 51.9 (COOCH3), 46.5 (C-3), 32.5 (C-10), 31.2 (C-
4), 29.2 (3C, C-6, 7, 8), 27.4 (C-5), 26.8 (3C, SiC(CH3)3), 25.7 (C-9),
19.1 (SiC(CH3)3). HRMS (FAB, m-NBA) m/z: 547.2829 [MþNa]þ;
calcd for C31H44O5SiNa: 547.2856 [MþNa].
4.2.2. Methyl 11-hydroxy-2-methylene-3-methoxycarbonyl-
undecanate ((ꢀ)-16)
Compound (ꢀ)-15 (250 mg, 0.464 mmol) was dissolved into
a solution of 47% HF aq (1.4 mL) and pyridine (3.3 mL) at room
temperature. The resultant mixture was stirred for 30 min, and
then quenched by addition of saturated NaHCO3 aq (20 mL) and
extracted with CHCl3 (3ꢃ20 mL). The combined organic layers were
washed with saturated NaHCO3 aq (20 mL), dried over Na2SO4, and
concentrated in vacuo. Flash chromatography (hexane/ethyl
acetate¼4:1) afforded (ꢀ)-16 (117 mg, 88%) as a colorless oil. IR nmax
O
O
O
O
HN
NH
N
O
N
O
NH
HN
(NaCl): 3473 (OH), 1735, 1725 (C]O), 201, 1143 (C–O) cmꢁ1
;
1H
NMR (270 MHz, CDCl3)
d
: 6.36 (1H, s, 10-H), 5.75 (1H, s, 10-H), 3.77
(3H, s, 1-OCH3), 3.67 (3H, s, 100-OCH3), 3.63 (2H, t, J¼6.4 Hz, 11-H),
3.50 (1H, t, J¼7.3 Hz, 3-H), 1.98–1.40 (4H, complex m, 4, 10-H2),
1.40–1.13 (10H, complex m, 5, 6, 7, 8, 9-H2); 13C NMR (67.5 MHz,
NO2
Figure 2. Structure of Chirabite-AR.

7374
T. Matsumaru et al. / Tetrahedron 64 (2008) 7369–7377
O
H
a
H
O
HO
b
Chirabite-AR
mol%
OH
MeO
Hc, Hd
H
H
d
c
O
Ha
Hb
(
)-Tensyuic acid B (1 mg, 3.9 µmol)
70
(2.15 mg)
60
(1.83 mg)
50
(1.56 mg)
40
(1.27 mg)
30
(0.99 mg)
20
(0.65 mg)
10
(0.33 mg)
No additive
9
8
7
6
5
4
3
2
ppm
Figure 3. Superposition of 1H NMR (400 MHz) spectra in the presence of increasing amounts of Chirabite-AR (10–70 mol %, from bottom to top) in a CDCl3 solution of synthetic
(ꢀ)-1 (1 mg, 3.9 mol) at room temperature.
m
CDCl3)
d
: 173.8 (C-100), 166.7 (C-1), 138.3 (C-2), 126.7 (C-10), 64.0
(3.1 mL) at 0 ꢂC was added SO3$pyrdine (630 mg, 3.96 mmol). After
warming to room temperature, the reaction mixture was stirred for
1 h, and then quenched by addition H2O (5 mL) and extracted with
hexane/AcOEt (1/1) (3ꢃ5 mL). The combined organic layers were
dried over Na2SO4 and concentrated in vacuo to give crude aldehyde,
which was used in the next step without further purification. To the
solution of crude aldehyde in t-BuOH (15.7 mL) were added 2-
methyl-2-butene (3.3 mL, 31.4 mmol) and a solution of NaHPO4
(3.1 g, 20.4 mmol) and NaClO2 (1.4 g, 15.7 mmol) in H2O (3.4 mL) at
(C-11), 52.1 (COOCH3), 52.0 (COOCH3), 46.5 (C-3), 32.7 (C-10), 31.2
(C-4), 29.2 (3C, C-6, 7, 8), 27.4 (C-5), 25.6 (C-9). HRMS (FAB, m-NBA)
m/z: 309.1682 [MþNa]þ; calcd for C15H26O5Na: 309.1687 [MþNa]þ.
4.2.3. Methyl 10-carboxy-2-methylene-3-methoxycarbonyl-
decanate ((ꢀ)-17)
To a solution of (ꢀ)-16 (450 mg, 1.57 mmol), dimethyl sulfoxide
(1.1 mL,15.7 mmol) and triethyl amine (1.1 mL, 7.86 mmol) in CH2Cl2
( )- 1
with Chirabite-AR
(70mol%)
1 : 1
1 : 1
1
( )- 1
5 : 3
5 : 3
Natural 1
with Chirabite-AR
(70mol%)
2
Natural 1
6.6
6.4
6.2
6.0
5.8
5.6
5.4
5.2
5.0 ppm
Figure 4. Comparison of 1H NMR (400 MHz) spectra between (ꢀ)-1 and natural 1, in 70 mol % of Chirabite-AR in CDCl3 at 50 ꢂC.

T. Matsumaru et al. / Tetrahedron 64 (2008) 7369–7377
7375
Table 1
In vitro anti-trypanosomal activity against Trypanosoma brucei brucei strain GUTat 3.1 for (ꢀ)-tensyuic acids and synthetic intermediate
Compound
IC50
(
mg/mL)
Compound
IC50
(
mg/mL)
Compound
IC50
(
m
g/mL)
O
O
O
O
HO
O
HO
O
HO
>12.5
2.23
>12.5
>12.5
OH
OH
OH
MeO
EtO
MeO
O
O
( )-Tensyuic acid E
O
( )-Tensyuic acid C
( )-Tensyuic acid B
O
O
O
MeO
O
MeO
O
MeO
4.39
>12.5
OMe
OMe
OMe
BocHNHN
HO
HO
( )-23
( )-22
O
( )-21
O
O
O
O
O
O
MeO
O
MeO
( )-17
MeO
( )-18
1.95
>12.5
4.93
OMe
OMe
OMe
BocHNHN
HO
HO
O
O
O
( )-16
3-H), 2.21 (2H, t, J¼7.6 Hz, 2-H2), 1.95–1.49 (4H, complex m, 3,8-H2),
1.47(9H, s, C(CH3)3),1.35–1.15(8H, complexm, 4, 5, 6, 7-H2);13C NMR
O
O
H
N
(67.5 MHz, CDCl3)
(–NHNHCOOC(CH3)3), 138.2
d
: 173.7 (C-100), 172.6 (C-1), 166.7 (C-10), 155.7
HN
N
H
NaO3S
(C-10), 126.7
(C-11),
81.5
O
CH3
(–NHNHCOOC(CH3)3), 52.1 (2C, COOCH3ꢃ2), 46.4 (C-9), 33.9 (C-2),
31.0 (C-8), 28.9 (3C, C-4, 5, 6), 28.0 (3C, –NHNHCOOC(CH3)3), 27.2 (C-
7), 25.1 (C-3); HRMS (FAB, m-NBA) m/z: 415.2430 [MþH]þ; calcd for
C20H35N2O7: 415.2444 [MþH].
NaO3S
SO3Na
Figure 5. Structure of suramin.
2
4.2.5. 3-Carboxy-2-methylene-10-methoxycarbonyldecanic acid
[tensyuic acid E, (ꢀ)-3]
To a solution of LiOH (14.4 mg, 0.6 mmol) in H2O (450
m
L) was
room temperature. The reaction mixture was stirred for 1 h, and
then quenched by addition of saturated NH4Cl solution (20 mL). The
resultant mixture was extracted with CHCl3 (30 mL) and the aque-
ous layer was extracted with CHCl3 (3ꢃ60 mL). The combined or-
ganic layers were dried over Na2SO4 and concentrated in vacuo.
Flash chromatography (hexane/ethyl acetate¼30/1 to 1/2) afforded
(ꢀ)-17 (450 mg, 94%) as a light yellow oil. IR nmax (NaCl): 3151
added a solution of (ꢀ)-18 (300 mg, 1.0 mmol) in THF (150
mL) at
room temperature and stirred for 5 h. The reaction was quenched
by addition of 1 N HCl aq solution (0.3 mL) and extracted with
AcOEt (3ꢃ5 mL). The combined organic layers were dried over
Na2SO4 and concentrated in vacuo to furnish crude dicarboxylic
acid, which was used in the next step without purification. Crude
product was dissolved into TFA (0.2 mL) and the resultant mixture
was stirred for 1 h at room temperature. The reaction mixture was
then concentrated in vacuo to give crude product, which was
subjected to next reaction without further purification. To a solu-
tion of crude hydrazide in MeOH (1.0 mL) at room temperature was
added CAN (183 mg, 0.334 mmol) and stirred for 30 min. The re-
action was quenched by addition of 0.1 N HCl aq solution (0.5 mL).
The resultant two layers were separated and the aqueous layer was
extracted with AcOEt (3ꢃ3 mL). The combined organic layers were
dried over Na2SO4 and concentrated in vacuo. Preparative HPLC
(Senshu Pak-PEGASIL ODS 20Bꢃ250 mm, MeCN/H2O¼35/65,
8.0 mL/min, UV at 210 nm) afforded tensyuic acid E ((ꢀ)-3) (12 mg,
58%) as a colorless oil. IR nmax (NaCl): 3482 (COOH), 1704 (C]O),
(COOH), 1739, 1731, 1724 (C]O), 1199, 1141 (C–O) cmꢁ1 1H NMR
;
(270 MHz, CDCl3) d
: 6.35 (1H, s,10-H), 5.75 (1H, s,10-H), 3.77 (3H, s,1-
OCH3), 3.68 (3H, s, 100-OCH3), 3.50 (1H, t, J¼7.3 Hz, 3-H), 2.34 (2H, t,
J¼7.2 Hz, 10-H2), 1.98–1.50 (4H, complex m, 4, 9-H2), 1.38–1.19 (8H,
complex m, 5, 6, 7, 8-H2); 13C NMR (67.5 MHz, CDCl3)
d: 179.5 (C-1%),
173.8 (C-100), 166.7 (C-1), 138.3 (C-2), 126.7 (C-10), 52.1 (COOCH3),
52.0 (COOCH3), 46.5 (C-3), 32.7 (C-10), 31.2 (C-4), 29.2 (3C, C-6, 7, 8),
27.4 (C-5), 25.6 (C-9). HRMS (FAB, m-NBA) m/z: 323.1457 [MþNa]þ;
calcd for C15H24O6Na: 323.1471 [MþNa].
4.2.4. N0-tert-Butyl ester-9,10-dimethoxycarbonyl-
undecanohydrazide-10-ene ((ꢀ)-18)
To a solution of (ꢀ)-17 (300 mg, 1.0 mmol) in benzene (2.0 mL)
1205 (C–O) cmꢁ1 1H NMR (400 MHz, CDCl3) : 6.53 (1H, s, 10-H),
; d
was added oxalyl chloride (428 mL, 5.0 mmol) at room temperature.
5.82 (1H, s, 10-H), 3.66 (3H, s, 1%-OCH3), 3.50 (1H, t, J¼7.2 Hz, 3-H),
2.30 (2H, t, J¼7.5 Hz, 10-H2), 2.01–1.67 (2H, m, 4-H), 1.66–1.54 (2H,
m, 9-H2), 1.39–1.21 (8H, complex m, 5, 6, 7, 8-H2); 13C NMR
The mixture was then warmed to 80 ꢂC and stirred for 2 h. After
stirring, the reaction mixture was cooled to room temperature and
concentratedinvacuotoaffordcrudeacidchloride,whichwasusedin
thenextstepwithoutpurification. Tothesolutionofcrudeproductin
CH2Cl2 (15.7 mL) was added the solution of BocNHNH2 (145 mg,
(100.6 MHz, CDCl3) d
: 179.1 (C-100), 174.4 (C-1%), 171.3 (C-1), 137.2
(C-2), 129.8 (C-10), 51.4 (C1%–OCH3), 47.3 (C-3), 34.0 (C-10), 29.5 (C-
4), 29.0 (C-6, 7, 8), 27.2 (C-5), 24.9 (C-9); HRMS (FAB, m-NBA) m/z:
287.1505 [MþH]þ; calcd for C14H23O6: 287.1495 [MþH].
1.1 mmol) and Et3N (121 m
L,1.1 mmol) in CH2Cl2 (1.0 mL) at 0 ꢂC; the
resultant mixture being stirred for 30 min. The reaction was then
quenchedbyadditionofsaturatedNH4Clsolution(5 mL)followedby
extractionwith CHCl3 (3ꢃ10 mL). The combined organic layers were
dried overNa2SO4 and concentrated in vacuo. Flash chromatography
(hexane/ethylacetate¼1:1)afforded(ꢀ)-18 (315 mg,99%)asayellow
oil. IR nmax (NaCl): 3282 (C]O), 2360 (NH), 1704 (C]O), 1205 (C–
4.3. Total synthesis of tensyuic acids B and C
4.3.1. Methyl 9-(tert-butyldiphenylsilyloxy)-2-methylene-3-
methoxycarbonylnonanate ((ꢀ)-20)
O) cmꢁ1; 1H NMR (270 MHz, CDCl3)
d: 6.35 (1H, s, 11-H), 5.75 (1H, s,
11-H),3.77(3H,s,10-OCH3),3.68(3H,s,100-OCH3),3.50(1H,t,J¼7.3 Hz,
The Et2O solution of Grignard reagent 6, which was prepared
from the corresponding alkyl bromide24 (2.0 g, 4.78 mmol) by same

7376
T. Matsumaru et al. / Tetrahedron 64 (2008) 7369–7377
procedure for the preparation of 7, and 8 (561 mg, 2.38 mmol) was
converted to (ꢀ)-20 (750 mg, 63%) by same procedure as that de-
scribed above for the formation of (ꢀ)-15. IR nmax (NaCl): 1739, 1724
described above for the formation of (ꢀ)-tensyuic acid E. IR nmax
(NaCl): 3482 (COOH), 1737, 1698 (C]O), 1216 (C–O) cmꢁ1; 1H NMR
(400 MHz, CDCl3) d
: 6.53 (1H, s, 10-H), 5.83 (1H, s, 10-H), 3.66 (3H, s,
(C]O), 1199, 1141 (C–O), 1110 (C–O–Si) cmꢁ1
;
1H NMR (270 MHz,
1%-OCH3), 3.40 (2H, t, J¼8.8 Hz, 3-H2), 2.30 (2H, t, J¼9.2 Hz, 8-H2),
CDCl3)
d
: 7.68 (4H, m, Ar–H), 7.40 (6H, m, Ar–H), 6.36 (1H, s, 10-H),
2.02–1.54 (4H, complex m, 4, 7-H2), 1.44–1.28 (4H, complex m, 5, 6-
5.75 (1H, s, 10-H), 3.76 (3H, s, 1-OCH3), 3.68 (3H, s, 100-OCH3), 3.64
(2H, t, J¼6.4 Hz, 9-H), 3.50 (1H, t, J¼7.4 Hz, 3-H), 1.97–1.45 (4H,
complex m, 4, 8-H2), 1.42–1.18 (6H, complex m, 5, 6, 7-H2), 1.04 (9H,
H2); 13C NMR (100.6 MHz, CDCl3) : 179.4 (C-100), 174.3 (C-1%), 171.5
d
(C-1), 137.2 (C-2), 129.9 (C-100), 51.5 (9-OCH3), 47.0 (C-3), 33.9 (C-8),
29.5 (C-4), 28.7 (C-6), 27.0 (C-5), 24.6 (C-7). HRMS (FAB, m-NBA)
m/z: 259.1192 [MþH]þ; calcd for C12H19O6: 259.1182 [MþH].
s, SiC(CH3)3); 13C NMR (67.5 MHz, CDCl3) : 173.6 (C-100),166.5 (C-1),
d
138.3 (C-2), 135.4 (2C, Arꢃ2), 134.0 (Ar),129.4 (Ar),127.5 (2C, Arꢃ2),
126.6 (C-10), 63.9 (C-9), 52.2 (COOCH3), 51.8 (COOCH3), 46.4 (C-3),
32.3 (C-8), 31.1 (C-4), 28.9 (C-6), 27.3 (C-5), 26.8 (3C, SiC(CH3)3),
25.4 (C-9), 19.1 (SiC(CH3)3); HRMS (FAB, m-NBA) m/z: 519.2557
[MþNa]þ; calcd for C29H40O5SiNa: 519.2534 [MþNa].
4.3.6. 3-Carboxy-2-methylene-8-ethoxycarbonyloctanic acid
((ꢀ)-tensyuic acid C)
Boc-hydrazide (ꢀ)-23 (245 mg, 0.90 mmol) was converted to
(ꢀ)-tensyuic acid C (36 mg, 45%) with ethanol by the procedure as
that described above for the formation of (ꢀ)-tensyuic acid E. IR
4.3.2. Methyl 9-hydroxy-2-methylene-3-methoxycarbonyl-
nonanate ((ꢀ)-21)
nmax (NaCl): 3482 (COOH), 1707 (C]O), 1193 (C–O) cmꢁ1 1H NMR
;
(400 MHz, CDCl3) d
: 6.51 (1H, s, 10-H), 5.82 (1H, s, 10-H), 4.12 (2H, q,
TBDPS ether (ꢀ)-20 (700 mg, 1.41 mmol) was converted to
(ꢀ)-21 (270 mg, 78%) by the same procedure as that described
above for the formation of (ꢀ)-16. IR nmax (NaCl): 3473 (OH), 1737,
J¼7.2 Hz, 1%-OCH2CH3), 3.38 (2H, t, J¼8.2 Hz, 3-H2), 2.28 (2H, t,
J¼9.2 Hz, 8-H2), 2.0–1.67 (2H, m, 4-H), 1.67–1.54 (2H, m, 7-H2),
1.42–1.29 (4H, complex m, 5, 6-H2), 1.25 (3H, t, J¼10.0 Hz, 9-
1727 (C]O), 1143 (C–O) cmꢁ1
;
1H NMR (270 MHz, CDCl3)
d
: 6.34
OCH2CH3); 13C NMR (100.6 MHz, CDCl3) : 179.1 (C-100), 173.9 (C-
d
(1H, s, 10-H), 5.73 (1H, s, 10-H), 3.75 (3H, s, 1-OCH3), 3.66 (3H, s, 100-
OCH3), 3.60 (2H, t, J¼6.6 Hz, 9-H), 3.48 (1H, t, J¼7.3 Hz, 3-H), 1.98–
1.43 (4H, complex m, 4, 8-H2), 1.39–1.19 (6H, complex m, 5, 6, 7-H2);
1%), 171.2 (C-1), 137.3 (C-2), 129.6 (C-100), 60.3 (100-OOCH2CH3), 47.2
(C-3), 34.2 (C-8), 29.5 (C-4), 28.7 (C-6), 27.0 (C-5), 24.7 (C-7), 14.2
(100-OOCH2CH3). HRMS (FAB, m-NBA) m/z: 273.1339 [MþH]þ; calcd
for C13H21O6: 273.1338 [MþH].
13C NMR (67.5 MHz, CDCl3) : 173.8 (C-100), 166.7 (C-1), 138.3 (C-2),
d
126.7 (C-10), 62.8 (C-9), 52.1 (COOCH3), 52.0 (COOCH3), 46.5 (C-3),
32.6 (C-8), 31.1 (C-4), 29.0 (C-6), 27.3 (C-5), 25.4 (C-7). HRMS (FAB,
m-NBA) m/z: 259.1537[MþH]þ; calcd for C13H23O5: 259.1545
[MþH].
4.4. In vitro anti-trypanosomal assay
T. brucei brucei strain GUTat 3.1 was cultured in IMDM with
various supplements containing 10% heat-inactivated FBS at 37 ꢂC,
under 5% CO2/95% air. Ninety five microliters of the trypanosomal
suspension (2.0–2.5ꢃ104 trypanosomes/mL) of the bloodstream
4.3.3. Methyl 8-carboxy-2-methylene-3-methoxycarbonyloctanate
((ꢀ)-22)
Alcohol (ꢀ)-21 (270 mg, 1.05 mmol) was converted to (ꢀ)-22
(245 mg, 85%) by the same procedure as that described above for
the formation of (ꢀ)-17. IR nmax (NaCl): 3151 (COOH), 1737, 1731,
form was seeded in a 96-well microplate, and 5 mL of a test com-
pound solution (dissolved in 5% dimethyl sulfoxide, DMSO) was
added, followed by incubation for 72 h (long incubation–low in-
oculation test: LILIT). Ten microliters of the fluorescent dye Alamar
Blue was added to each well. After the incubation for 3–6 h, the
resulting solution was read at 528/20 nm excitation wavelength
and 590/35 nm emission wavelength by an FLꢃ800 fluorescent
plate reader (Bio-Tek Instrument, Inc., Vermont, USA). Data were
transferred into a spreadsheet program (Excel) and IC50 values
were determined using fluorescent plate reader software (KC-4,
Bio-Tek).
1712 (C]O), 1174, 1143 (C–O) cmꢁ1 1H NMR (270 MHz, CDCl3)
; d:
6.35 (1H, s, 10-H), 5.74 (1H, s, 10-H), 3.73 (3H, s, 1-OCH3), 3.66 (3H, s,
100-OCH3), 3.48 (1H, t, J¼7.4 Hz, 3-H), 2.32 (2H, t, J¼7.3 Hz, 8-H2),
1.99–1.53 (4H, complex m, 4, 8-H2), 1.43–1.20 (4H, complex m, 5, 6-
H2); 13C NMR (67.5 MHz, CDCl3) : 179.2 (C-100), 173.7 (C-1%), 166.6
d
(C-1), 138.2 (C-2), 126.8 (C-10), 52.2 (COOCH3), 52.1 (COOCH3), 46.5
(C-3), 33.8 (C-8), 31.0 (C-4), 28.6 (C-6), 27.1 (C-5), 24.4 (C-7). HRMS
(FAB, m-NBA) m/z: 273.1343 [MþH]þ; calcd for C13H21O6: 273.1338
[MþH].
Acknowledgements
4.3.4. N0-tert-Butyl ester-7,8-dimethoxycarbonylnonanohydrazide-
8-ene ((ꢀ)-23)
We wish to thank Ms. A. Nakagawa, Ms. C. Sakabe, and Ms. N.
Sato (all Kitasato University) for various instrumental analyses. We
wish to thank Prof. H. Yamada, Prof. K. Shiomi, Prof. H. Hanaki, and
Mr. M. Iwatsuki (all Kitasato University) for testing the biological
activities and valuable discussions. This work was supported in part
by funds from the Drugs for Neglected Diseases initiative (DNDi),
a grant for All Kitasato Project Study (AKPS), the 21st Century COE
Program, and Ministry of Education, Culture, Sports, Science and
Technology, Japan.
Carboxylic acid (ꢀ)-22 (245 mg, 0.90 mmol) was converted to
(ꢀ)-23 (270 mg, 78%) by the same procedure as that described
above for the formation of (ꢀ)-18. IR nmax (NaCl): 3288 (C]O), 1737,
1731, 1727 (C]O), 1438 (CH), 1201 (C–O) cmꢁ1; 1H NMR (270 MHz,
CDCl3) d
: 6.33 (1H, s, 9-H), 5.72 (1H, s, 9-H), 3.74 (3H, s, 10-OCH3),
3.65 (3H, s, 100-OCH3), 3.47 (1H, t, J¼7.4 Hz, 7-H), 2.18 (2H, t,
J¼7.5 Hz, 2-H2), 1.95–1.53 (2H, complex m, 5-H2), 1.43 (9H, s,
C(CH3)3), 1.40–1.19 (4H, complex m, 3, 4-H2); 13C NMR (67.5 MHz,
CDCl3)
d:
173.7 (C-100), 172.6 (C-1), 166.6 (C-10), 155.7
(NHNHCOC(CH3)3),138.2 (C-8),126.8 (C-9), 81.7 (NHNHCOC(CH3)3),
52.1 (2C, COOCH3ꢃ2), 46.4 (C-7), 33.8 (C-2), 30.9 (C-6), 28.0 (3C,
NHNHCOC(CH3)3), 28.7 (C-4), 27.0 (C-5), 24.9 (C-3). HRMS (FAB, m-
NBA) m/z: 409.1936 [MþNa]þ; calcd for C18H30N2O7Na: 409.1951
[MþNa].
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