Synthesis of optically pure norcantharidin analogue NCA-01, a highly selective protein phosphatase 2B inhibitor, and its derivatives

Article abstract of DOI:10.1002/asia.201200077

An efficient synthetic route to optically pure norcantharidin analogue NCA-01, a highly selective inhibitor of protein phosphatase 2B (PP2B; calcineurin), has been developed. The absolute stereochemistry of the enantiomers was determined by X-ray crystallographic analysis. Optically pure NCA derivatives that had various substituents at the C1 position were synthesized in a similar manner. The PP2B-inhibitory activities of NCA-01 and its derivatives were independent of the enantiomeric form. NCA-01 dimethyl ester potently inhibited IL-2 production in Jurkat cells.

Full text of DOI:10.1002/asia.201200077

DOI: 10.1002/asia.201200077
Synthesis of Optically Pure Norcantharidin Analogue NCA-01, a Highly
Selective Protein Phosphatase 2B Inhibitor, and its Derivatives
Tadashi Shimizu,[a, c] Masato Iizuka,[a] Hiroko Matsukura,[a] Daisuke Hashizume,[a] and
Mikiko Sodeoka*[a, b]
Abstract: An efficient synthetic route to optically pure norcantharidin analogue
NCA-01, a highly selective inhibitor of protein phosphatase 2B (PP2B; calcineur-
in), has been developed. The absolute stereochemistry of the enantiomers was de-
termined by X-ray crystallographic analysis. Optically pure NCA derivatives that
had various substituents at the C1 position were synthesized in a similar manner.
The PP2B-inhibitory activities of NCA-01 and its derivatives were independent of
the enantiomeric form. NCA-01 dimethyl ester potently inhibited IL-2 production
in Jurkat cells.
Keywords: biological activity
calcineurin · inhibitors · norcanthar-
idin · protein phosphatase
·
Introduction
learning and memory.[3] Immunosuppressant drugs cyclo-
sporine A (CsA) and FK506 inhibit PP2B and block activa-
tion of transcription factor NF-AT in T-lymphocytes.[4]
These drugs bind to immunophilins (cyclophilin and FKBP,
respectively) and the resulting complexes inhibit the phos-
phatase activity of PP2B. Remarkably, these immunosup-
pressants cannot directly inhibit PP2B, and the formation of
the immunophilin complex is essential. Because immunophi-
lins are involved in many cellular processes, such as the reg-
ulation of calcium ion channels, protein folding, and cell
death, FK506 and CsA may cause a variety of side-effects.
Thus, it is of interest to find direct and selective inhibitors of
PP2B that do not bind to immunophilins, both as biological
tools for studies of PP2B and as candidate therapeutic
agents.[5–11]
There are several reports on natural and synthetic prod-
ucts that inhibit PP2B without binding immunophilin, but
these compounds have potential problems. Polypeptide
PP2B inhibitors are potent and specific, but may have limit-
ed cell-permeability and short lifetimes.[5] Although pyreth-
roid insecticides, such as fenvalerate, cypermethrin, and per-
methrin, have been reported to be potent and specific PP2B
inhibitors,[6a] their effectiveness has been disputed by other
groups.[6b,c] Dibefurin,[7] PD144795,[8] and tyrphostin deriva-
tives[9] have also been reported to inhibit PP2B, but their se-
lectivity remains unknown. Gossypol is a potent inhibitor of
PP2B (IC50 =16 mm), but although it shows negligible inhibi-
tion of other protein phosphatases,[10a] it inhibits other en-
zymes, such as GgIP3K-A (IC50 =58 nm).[10c] Recently, ami-
noalkyl-substituted diarylheterocycles have been reported to
inhibit PP2B without inhibiting other protein phosphatases
and immunophilins and to be effective on the cell level,[11]
whereas there is still a need for direct and selective PP2B in-
hibitors.
Protein phosphatases are key enzymes in signal-transduction
pathways, by regulating entry into the cell cycle, mitosis, and
cell death. They can be categorized into three groups: pro-
tein serine/threonine-specific, tyrosine-specific, and dual-
specific phosphatases. Based on their biological properties,
substrate specificity, and sensitivity to specific inhibitors,
serine/threonine phosphatases have been further classified
into several subtypes, and, among them, protein phosphatase
1 (PP1), protein phosphatase 2A (PP2A), and protein phos-
phatase 2B (PP2B) have been well-studied.[1,2]
PP2B (also called calcineurin) is a calcium- and calmodu-
lin-dependent enzyme that is composed of calcineurin A
(catalytic subunit; 60 kDa), which shares high sequence ho-
mology with PP1, PP2A, and calcineurin B (calcium/calmo-
dulin-binding subunit; 15 kDa). PP2B is distributed widely
in the brain and lymphocytes and has established roles in T-
cell activation, neuronal cell death, vesicular trafficking, and
[a] Dr. T. Shimizu, M. Iizuka, H. Matsukura, Dr. D. Hashizume,
Prof. Dr. M. Sodeoka
RIKEN Advanced Science Institute
2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan)
Fax : (+81)48-462-4666
E-mail: sodeoka@riken.jp
[b] Prof. Dr. M. Sodeoka
Sodeoka Live Cell Chemistry Project, ERATO
Japan Science and Technology Agency (JST)
2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan)
[c] Dr. T. Shimizu
Present address:
Department of Pharmacy
Hyogo University of Health Sciences
1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan)
Cantharidin was isolated from the dried body of the Chi-
nese blister beetle (Mylabris phalerata or M. cichorii) in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201200077.
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FULL PAPERS
1810, and it has been used as a medicinal agent for over
2000 years. It is a moderately potent inhibitor of PP1 and
PP2A, but it inhibits PP2B only very weakly.[11] Although
numerous analogues of cantharidin have been reported as
potent and selective inhibitors of PP1 and PP2A in recent
years,[12] only a few were evaluated as PP2B inhibitors.[13]
We have previously reported that the racemic 1,5-disubsti-
tuted norcantharidin derivative 1a (NCA-01) is a highly se-
lective catalytic site-directed PP2B inhibitor that shows neg-
ligible inhibition of PP1 and PP2A (Scheme 1).[14] It is im-
evaluated all four stereoisomers.[15] Interestingly, isomers
(1S,10R)-2a and (1S,10S)-2b showed high selectivity for
PP2B and their inhibition of PP1 and PP2A was negligible.
On the other hand, isomers (1R,10S)-2a and (1R,10R)-2b
showed significant inhibition of all three PPs. These results
indicated that the absolute stereochemistry at the C1 posi-
tion was important for the selectivity. Furthermore, isomer
(1S,10S)-2b showed more-potent PP2B-inhibitory activity
than diastereomer (1S,10R)-2a and (1S)-benzoyloxymethyl
derivative 3, thus suggesting the importance of SAR study
for the C1 substituent. These facts encouraged us to synthe-
size optically pure compound 1a and its derivatives with var-
ious C1 substituents. Herein, we report a versatile synthetic
route to optically pure compound 1a and its derivatives 1b–
1j. The inhibitory activity of these compounds towards PP1,
PP2A, and PP2B was also evaluated.
Results and Discussion
Synthetic Route to Optically Pure NCA-01 and its
Derivatives
In addition to the difficulty of separation of the enantio-
mers, our original synthetic route (Scheme 2) had several
problems. The 2,4-dibenzoyloxymethylfuran (6) was synthe-
sized from 3-furanmethanol by benzoylation, Vilsmeier reac-
Scheme 1. Structures of cantharidin, norcantharidin, and cantharidin ana-
logues synthesized by ourselves.
portant to evaluate the activity
of each enantiomer to obtain
a better understanding of the
interaction between the inhibi-
tor and the enzyme. However,
our efforts to separate the
enantiomers of compound 1a
by chiral-phase HPLC were un-
successful, owing to the lability
of the acid anhydride moiety.
Therefore, we prepared two op-
tically active mono-substituted
norcantharidin analogues (2a
and 2b) starting from optically
Scheme 2. Problems in the original synthetic route to compound 1a.
pure 1-(2-furyl)ethanol, and we
tion, and reduction steps. However, the selectivity of the
Vilsmeier reaction was poor and the desired regioisomer
was only a minor product (16% yield over 3 steps). The key
Diels–Alder reaction and the subsequent hydrogenation re-
action were also troublesome. The yield of the desired prod-
uct (1a) was low, owing to the formation of stereoisomer 8
and hydrogenolysis product 9 in addition to the retro-Diels–
Alder product.
Abstract in Japanese:
To overcome these problems, we planned a versatile syn-
thetic route to the optically active 1,5-disubstituted norcan-
tharidin derivatives (1), as shown in Scheme 3. To allow for
the introduction of a variety of acyl groups at the C1 posi-
tion after separation of the enantiomers, we selected the
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Synthesis of Norcantharidin Analogue NCA-01
of compound 11 at high pres-
sure. The Diels–Alder reaction
of compound 11 with maleic an-
hydride in CH2Cl2 at 700 MPa
was complete within 24 hours,
Scheme 3. Synthetic route to optically active NCA derivatives.
thus affording compound 16 in
82% yield. However, for the
large-scale reaction, we pre-
stable diester 10 as a key intermediate. We expected that 4-
benzoyloxymethyl-2-tert-butyldimethylsilyloxymethylfuran
(11) would be obtainable from the commercially available 3-
bromofurfuryl aldehyde (12).
ferred to use the former conditions. The key hydrogenation
reaction of cycloadduct 16 was examined by using various
metal catalysts, such as Rh/Al2O3, Rh/C, PtO2, and
ꢀ
[Ir
G
G
(cod=1,5-cyclooctadiene). Un-
First, 3-bromofurfuryl aldehyde (12) was converted into
alcohol 13, which was protected with a tert-butyldimethylsil-
yl group (TBS) to give compound 14 (Scheme 4). Lithiation
fortunately, the yield of the desired product was not im-
proved. We also examined the diimide reduction of the
double bond, but the acid anhydride group was not compati-
ble with the hydrazine derivatives. Therefore, we investigat-
ed the conversion of compound 16 into the corresponding
benzyl ester (17). All of the reactions of compound 16 with
benzyl alcohol in the presence of various coupling reagents,
such as DCC (DCC=N,N’-dicyclohexylcarbodiimide),
DCC-HOBt (HOBt=1-hydroxy-1H-benzotriazole), EDC-
HOOBt (EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide,
HOOBt=3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzo-
triazine), and 2-chloropyridinium iodide, in the absence or
presence of base, such as DMAP, iPr2NEt, and Et3N, under
ambient conditions, were unsuccessful. Significant epimeri-
zation at the C2 or C3 positions was observed, in addition to
the retro-Diels–Alder reaction. Finally, we were pleased to
find that the reaction of compound 16 with benzyl alcohol
in the presence of TsCl (Ts=4-toluenesulfonyl) and N-
methylimidazole gave the desired dibenzyl ester (17) with-
out epimerization of the C2 and C3 positions.[17] Diimide re-
duction of compound 17 by using 2,4,6-triisopropylbenzene-
sulfonohydrazide proceeded smoothly to afford the desired
product (18) as a single diastereoisomer. The key intermedi-
ate (10) was obtained by deprotection of the TBS group of
compound 18 with boron trifluoride diethyl etherate. As ex-
pected, the separation of its
Scheme 4. Synthesis of 2,4-disubstituted furan derivative 11.
of compound 14, followed by the addition of N,N-dimethyl-
formamide (DMF), afforded aldehyde 15.[16] Reduction of
the formyl group and subsequent benzoylation of the alco-
hol furnished the desired 2,4-disubstituted furan derivative
(11).
The Diels–Alder reaction of compound 11 with maleic an-
hydride in toluene at 508C for 4 days produced cycloadduct
16 in 72% yield (Scheme 5). We also examined the reaction
enantiomers was achieved by
preparative HPLC by using
a
chiral stationary phase
(CHIRALPAKꢁ IA), and opti-
cally pure compounds (ꢀ)-10
and (+)-10 were obtained.
First, compound (ꢀ)-10 was
transformed into (ꢀ)-NCA-01
(1a) by a three-step sequence.
Benzoylation of the hydroxy
group with benzoyl chloride af-
forded compound (ꢀ)-19a. Fi-
nally, acid anhydride (ꢀ)-1a
was obtained by hydrogenolysis
of the benzyl ester of com-
pound (ꢀ)-19a, followed by
treatment with acetic anhy-
dride. Using the same synthetic
route, compound (+)-10 was
Scheme 5. Synthesis of common chiral intermediate 10.
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Mikiko Sodeoka et al.
FULL PAPERS
edly, the IC50 values of (1S,5R)-1a and (1R,5S)-1a were
equal (5.2 mm).
To understand this result, we carried out the molecular
docking of both enantiomers of compound 1a with the
PP2B active site based on the reported crystal structure
(PDB ID: 1TCO)[18] using DS Modeling 1.2 (Accelrys).
Figure 1 shows the binding modes of the dicarboxylate form
Scheme 6. Synthesis of optically active NCA derivatives.
converted into (+)-1a (Scheme 6). Furthermore, both enan-
tiomers of NCA derivatives 1b–1j, which had various acyl
groups at the C1 position, were also synthesized in a similar
manner.
The absolute stereochemistry of compounds (ꢀ)-10 and
(ꢀ)-1a were determined to be (1S,5R) by X-ray crystallogra-
phy of compound 20, which was derived from compound
(ꢀ)-1a by an imide-formation reaction with 4-bromoaniline
under the influence of triethylamine and 4-dimethylamino-
pyridine (DMAP; Scheme 7). The absolute stereochemistry
of all other derivatives was determined by correlation to
compound 10.
Figure 1. A,C) Binding model of (1S,5R)-1a with the PP2B active site.
B,D) Binding model of (1R,5S)-1a with the PP2B active site.
of isomers (1S,5R)-1a (A and C) and (1R,5S)-1a (B and D)
to PP2B.
In the X-ray structure of the PP2B–phosphate-anion com-
plex, the phosphate oxygen atoms coordinated to the metal
ions in the catalytic site (Zn, Fe). The conserved arginine
residues, Arg122 and Arg254, may play a role in substrate-
binding by forming hydrogen bonds with the oxygen atom
of the phosphate ester.[3b]
In our binding models of isomers (1S,5R)-1a and (1R,5S)-
1a, the carboxylate moieties were situated at positions in
which ionic interactions between the oxygen atoms of the
carboxylate groups and the metal ions were possible. How-
ever, the orientation of the bridging ether oxygen was differ-
ent for the two enantiomers. A hydrogen bond between the
bridging ether oxygen of the (1S,5R)-isomer and the Arg254
residue was observed in binding model A, whereas the
(1R,5S)-isomer formed a hydrogen bond with Arg122 in
binding model B. As a result, the C1 substituent of both
enantiomers of compound 1a fit well into the deep groove
of PP2B, and the C5 substituent interacted with the hydro-
philic region, which was characteristic of PP2B, but not of
PP1 or PP2A.
Recently, the crystal structure of the complex of protein
phosphatase 5 (PP5c) with norcantharidin was reported.[19]
As predicted, norcantharidin was bound as its dicarboxylate
form. Interestingly, multiple binding modes of the norcan-
tharidin dicarboxylate core structure were observed, thereby
suggesting flexible interactions between the core dicarboxy-
Scheme 7. Preparation of (1S,5R)-20 and an ORTEP of its crystal struc-
ture.[21]
Effect of the Absolute Stereochemistry on PP2B-Inhibitory
Activity
Initially, the PP2B-inhibitory activity of enantiomers of
NCA-01 (1a) were examined in terms of their ability to in-
hibit the dephosphorylation of an exogenous substrate (R-II
phosphopeptide) by human recombinant PP2B.[14] Unexpect-
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Synthesis of Norcantharidin Analogue NCA-01
late structure and the conserved catalytic-site structure of
the Ser/Thr protein phosphatases, which was composed of
two metal ions and basic amino acid residues. This result
seemed to be consistent with the finding that both enantio-
mers of compound 1a bound PP2B with similar affinity.
binding to PP1 and PP2A. However, the additional interac-
tion of the C1 acyl group with the hydrophobic amino acid
residues in the groove may be required for the high binding
affinity to PP2B.
Inhibition of IL-2 Production in Jurkat Cells by Optically
Active NCA-ME Derivatives
Effect of the C1 Substituent on the PP2B-Inhibition
Activity
It is known that the stimulation of Jurkat cells with mitogen
induces the production of IL-2 through the activation of
PP2B and NF-AT. Because PP2B inhibitors, such as cyclo-
sporin A, are known to suppress IL-2 production, we inves-
tigated the effect of NCA-01 at the cell level. However, the
inhibition of IL-2 production in Jurkat cells by NCA-01 was
very weak. We speculated that the acid anhydride
To examine the effect of the C1 substituent on the potency
and selectivity of PP2B inhibition, we evaluated various
NCA derivatives, 1b–1j. The percentage inhibition of both
enantiomers of these NCA derivatives are summarized in
Table 1. NCA-01 showed nearly 70% inhibition of PP2B ac-
group in NCA-01 would be easily hydrolyzed in
Table 1. Inhibitory activity of optically pure NCA derivatives towards PP2B, PP1, and
PP2A.
aqueous solution to give the dicarboxylic acid deriv-
ative, which could strongly bind PP2B, but was ex-
pected to have poor cell permeability. Therefore,
we prepared the more-chemically stable dimethyl
ester derivative of NCA-01, NCA-01-ME (21a), as
a pro-drug of NCA-01 dicarboxylate (Scheme 8).
Although neither of the enantiomers of compound
21a inhibited PP2B, we expected that the methyl
ester moieties would be hydrolyzed to dicarboxy-
late by endogenous esterases in cells.
Compound
R
PP2B Inhibition
PP1 Inhibition
PP2A Inhibition
[%] (10 mm)
[%] (100 mm)
[%] (100 mm)
A
E
N
U
G
ACHTUNGTRNE(NUNG 1R,5S)
1a
1b
1c
1d
1e
1 f
1g
1h
1i
C6H5
71ꢁ3.7 65ꢁ1.5
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
2-CH3-C6H4 72ꢁ7.2 61ꢁ3.7
3-CH3-C6H4 75ꢁ6.3 58ꢁ4.6
4-CH3-C6H4 63ꢁ8.2 60ꢁ2.7
c-C6H11
c-C5H9
c-C3H5
tert-C4H9
i-C3H7
CH3
71ꢁ4.1 61ꢁ5.4
68ꢁ1.1 54ꢁ6.2
48ꢁ9.0 25ꢁ6.8
62ꢁ1.3 50ꢁ4.4
62ꢁ6.5 45ꢁ7.1
17ꢁ10 15ꢁ14
Using a reported procedure,[20] we quantified IL-
2 production in the supernatant of Jurkat T lympho-
cytes that were stimulated with PHA (phytohemag-
1j
ni=no inhibition was observed.
tivity. The introduction of a methyl group at the ortho (1b),
meta (1c), or para (1d) position on the phenyl ring did not
greatly affect the PP2B-inhibition activity. The replacement
of the phenyl ring with a cyclohexyl (1e) or cyclopentyl ring
(1 f) that had similar hydrophobicity was also well-tolerated,
but its replacement with a cyclopropyl group (1g) reduced
the activity. Although no significant difference was generally
observed between the enantiomers, isomer (1R,5S)-1g was
less potent than the (1S,5R) isomer. NCA derivatives that
had branched alkyl substituents, such as tBu (1h) and iPr
(1i), showed effective PP2B inhibition, whereas the deriva-
tive that contained a small acetyl group (1j) showed a signifi-
cant loss of potency. These results suggested that the hydro-
phobicity and size of the substituents at the C1 ester group
were important for the PP2B-inhibition activity. Further-
more, almost all of the NCA derivatives had little or no in-
hibitory effect on PP1 and PP2A, even at 100 mm concentra-
tion, regardless of the size of the C1 substituent and the ab-
solute stereochemistry. We have previously reported that
the introduction of a hydrophobic substituent at the C5 posi-
tion of norcantharidin led to an increase in the PP2B-inhibi-
tion activity[13a] and binding to PP1 and PP2A was blocked
in the presence of a C1 benzoyloxymethyl group.[14] The
SAR results (Table 1) indicated that, in the presence of the
C5 benzoyloxymethyl group, a small substituent, such as an
acetyl group, at the C1 position was sufficient to block the
Scheme 8. Structures of NCA-01-ME derivatives (1S,5R)-21a and
(1R,5S)-21a.
glutinin) in the presence or absence of optically active
NCA-01-ME derivatives (30 mm). The cytotoxicity of these
compounds to Jurkat cells was also measured by an Ala-
marBlue assay.
As expected, the addition of 30 mm of NCA-ME deriva-
tive (1S,5R)- or (1R,5S)-21a suppressed the production of
PHA-stimulated IL-2 without affecting the cell viability.
These results indicated that the inhibitory effect of NCA-
ME derivatives on IL-2 production did not involve cytotox-
icity (Table 2).
Table 2. Inhibitory effects of NCA-ME derivatives on IL-2 production in
Jurkat cells.
Compound
Inhibition [%] (mm)
Cell viability [%] (mm)
DMSO
cyclosporine A
0
100ꢁ4.6
100ꢁ8.7 (1)
84ꢁ17 (30)
73ꢁ6.0 (30)
94ꢁ0.30 (1)
104ꢁ6.3 (30)
107ꢁ1.4 (30)
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
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4-Benzoyloxymethyl-2-tert-butyldimethylsiloxymethylfuran (11)
Conclusions
After azeotropic evaporation of compound 14 (1.85 g, 6.34 mmol) with
toluene, to a solution of 14 in dry Et2O (11 mL) was slowly added a solu-
tion of nBuLi (4.40 mL, 6.98 mmol, 1.59m in n-hexane) at ꢀ708C under
a nitrogen atmosphere. The reaction mixture was stirred for 1 h at
ꢀ408C, then DMF (540 mL, 6.98 mmol) was added at ꢀ708C. Stirring
was continued for 1 h at ꢀ408C and for 2 h at RT, then a saturated aque-
ous solution of NH4Cl was added. The aqueous layer was extracted with
EtOAc and the combined organic layers were dried over anhydrous
MgSO4 and concentrated under reduced pressure. The crude product
(15)[16] was dissolved in MeOH (30 mL), and NaBH4 (350 mg, 9.40 mmol)
was added at 08C. The reaction mixture was stirred for 12 h at RT, then
a saturated aqueous solution of NH4Cl was added. The aqueous layer
was extracted with EtOAc and the combined organic layers were dried
over anhydrous MgSO4 and concentrated under reduced pressure. The
crude product, Et3N (1.8 mL, 13 mmol), and DMAP (79 mg, 0.65 mmol)
were dissolved in dry CH2Cl2 (15 mL), and benzoyl chloride (900 mL,
7.2 mmol) was added to the solution at 08C. The reaction mixture was
stirred at RT for 3 h. The reaction was quenched with saturated sodium
hydrogen carbonate, and the mixture was extracted with EtOAc. The or-
ganic layer was washed with water and brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The crude product was purified by
column chromatography on silica gel (n-hexane/EtOAc, 8:1) to give com-
An improved synthetic route to optically active NCA deriv-
atives was developed and the absolute stereochemistry of
the products was determined by X-ray crystallographic anal-
ysis. The PP2B-inhibitory activity of the enantiomers of
NCA-01 was independent of the enantiomeric form. Molec-
ular modeling suggested that the two enantiomers bound to
PP2B in different modes. Furthermore, we synthesized
a series of 5-benzoyloxymethylnorcantharidin derivatives
with various substituents at the C1 position and evaluated
their inhibitory activities toward PP1, PP2A, and PP2B. The
NCA derivatives that had hydrophobic substituents at the
C1 position inhibited PP2B but showed negligible inhibition
of PP1 and PP2A, whilst the dimethyl ester NCA-01-ME
derivative (21a) significantly inhibited IL-2 production in
Jurkat cells. These compounds are the first step in the devel-
opment of PP2B inhibitors that are effective on the cellular
level and are expected to be useful tools for future studies
on the function of PP2B.
pound 11 as
a
colorless oil (1.90 g, 86% over 3 steps). 1H NMR
(400 MHz, CDCl3): d=0.09 (s, 6H), 0.90 (s, 9H), 4.61 (s, 2H), 5.19 (s,
2H), 6.35 (s, 1H), 7.45 (dd, J=7.1 and 7.8 Hz, 2H), 7.48 (s, 1H), 7.55 (t,
J=7.8 Hz, 1H), 8.04 ppm (d, J=7.1 Hz, 2H); 13C NMR (100 MHz,
CDCl3): d=ꢀ5.1 (2C), 18.5, 26.0 (3C), 58.2, 58.4, 108.1, 121.0, 128.2
(2C), 129.5 (2C), 130.0, 132.9, 140.9, 155.1, 166.3 ppm; MS (FAB, posi-
tive-ion mode, m-nitro benzyl alcohol): calcd for C19H27O4Si: 347
[M+H]+; found: 347.
Experimental Section
General Methods
1H NMR Spectra were recorded on JEOL JNM-AL-300 and AL-400
spectrometers, which operated at 300 and 400 MHz, respectively.
13C NMR Spectra were recorded on a AL-400 spectrometer, which oper-
ated at 100 MHz. 1H and 13C NMR shifts are reported downfield from
CHCl3 (d=7.26 and 77.0 ppm). MS (FAB) was performed on a JEOL
JMA-HM110 mass spectrometer by using m-nitrobenzyl alcohol
(mNBA). Column chromatography was performed with silica gel 60 (40–
50 mm and 40–100 mm) that was purchased from Kanto Chemical Co.
Preparative HPLC was performed on a JMM LC-6 A. Analytical HPLC
was done on a Waters HPLC system or on a TOSOH HPLC system.
Preparative TLC was performed with Silica gel 60 F254 (layer: 1 mm and
0.5 mm) that was purchased from Merck Ltd. All reactions were moni-
tored by TLC on 0.25 mm E. Merck silica gel plates 60 F254 by using UV
light or 10% ethanolic phosphomolybdic acid solution and heat as devel-
oping agents.
(1S*,2R*,3S*,4R*)-5-Benzoyloxymethyl-1-tert-butyldimethylsiloxymethyl-
7-oxabicycloACTHNUGRTNEUNG[2.2.1]hept-5-ene-2,3-dicarboxylic anhydride (16)
Ambient conditions: A mixture of compound 11 (2.6 g, 7.5 mmol) and
maleic anhydride (2.2 g, 23 mmol) in toluene (0.5 mL) was stirred at
508C for 4 days. The reaction mixture was cooled to RT and the crude
product was purified by column chromatography on silica gel (n-hexane/
EtOAc, 4:1) to give compound 16 as a white solid (2.4 g, 72%).
High-pressure conditions:
1.01 mmol) and maleic anhydride (120 mg, 1.21 mmol) was placed in
Teflon reaction vessel with CH2Cl2 (1.0 mL) and pressurized to
A mixture of compound 11 (350 mg,
a
700 MPa at RT for 5 h. After release of the pressure, the reaction mix-
ture was concentrated in vacuo, and purified by column chromatography
on silica gel (n-hexane/EtOAc, 4:1) to give compound 16 as a white solid
(370 mg, 82%). M.p.: 85–868C; 1H NMR (400 MHz, CDCl3): d=0.10 (s,
3H), 0.13 (s, 3H), 0.91 (s, 9H), 3.25 (d, J=6.6 Hz, 1H), 3.46 (d, J=
6.6 Hz, 1H), 4.07 (d, J=11.0 Hz, 1H), 4.25 (d, J=11.0 Hz, 1H), 5.02 (d,
J=1.5 Hz, 2H), 5.4 (s, 1H), 6.47 (t, J=1.5 Hz, 1H), 7.48 (dd, J=7.8 and
7.8 Hz, 2H), 7.61 (dt, J=1.4 and 7.8 Hz, 1H), 8.04 ppm (dd, J=1.4 and
7.8 Hz, 2H); MS (MALDI-TOF, positive-ion mode, a-cyano-4-hydroxy-
cinnamic acid): calcd for C23H28NaO7Si 467.15 [M+Na]+; found: 467.23.
4-Bromo-2-tert-butyldimethylsilyloxymethylfuran (14)[16]
To a solution of 4-bromo-2-furaldehyde (12, 3.0 g, 17.2 mmol) in MeOH
(90 mL) was added NaBH4 (980 mg, 25.8 mmol) at 08C. The reaction
mixture was stirred for 10 h at RT, then a saturated aqueous solution of
NH4Cl was added. The aqueous layers were extracted with EtOAc, and
the combined organic layers were dried over anhydrous MgSO4 and con-
centrated under reduced pressure. The crude product was dissolved in
DMF (18 mL) under
a nitrogen atmosphere, and imidazole (3.6 g,
(1S*,2R*,3S*,4R*,5R*)-5-Benzoyloxymethyl-1-tert-
52.6 mmol) and TBDMSCl (3.2 g, 21 mmol) were added at 08C. The re-
action mixture was stirred for 2 h at RT, then brine was added. The aque-
ous layer was extracted with EtOAc, and the combined organic layer was
dried over anhydrous MgSO4 and concentrated under reduced pressure.
The crude product was purified by column chromatography on silica gel
(n-hexane/EtOAc, 4:1) to give compound 14 as a colorless oil (4.8 g,
99% over 2 steps). 1H NMR (400 MHz, CDCl3): d=0.09 (s, 6H), 0.91 (s,
9H), 4.60 (s, 2H), 6.29 (d, J=0.7 Hz, 1H), 7.36 ppm (d, J=0.7 Hz, 1H);
13C NMR (100 MHz, CDCl3): d=ꢀ5.1 (2C), 18.5, 25.9 (3C), 58.1, 99.9,
110.4, 140.0, 155.2 ppm; MS (FAB, positive-ion mode, m-nitrobenzyl al-
cohol): calcd for C11H20BrO2Si: 291 [M+H]+; found: 291.
butyldimethylsiloxymethyl-7-oxa-bicycloACHTNUGTRNEUNG[2.2.1]heptane-2,3-dicarboxylic
acid dibenzyl ester (18)
To a solution of compound 16 (2.81 g, 6.30 mmol) and N-methylimidazole
(3.0 mL, 37.8 mmol) in CH3CN (12 mL) was added TsCl (2.65 g,
13.9 mmol) at 08C under a nitrogen atmosphere. The reaction mixture
was stirred for 30 min and
a solution of benzyl alcohol (2.87 mL,
27.7 mmol) in CH3CN (12 mL) was added at 08C. The reaction mixture
was stirred for 24 h at RT and then water was added. The aqueous layer
was extracted with EtOAc and the combined organic layers were washed
with water and brine, dried over anhydrous MgSO4, and concentrated in
vacuo.
To a solution of crude compound 17 in dry 1,2-dimethoxyethane (DME,
50 mL) was added 2,4,6-triisopropyl benzenesulfonyl hydrazide (3.3 g,
11 mmol) at RT and the reaction mixture was stirred at 508C for 5 h. The
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Chem. Asian J. 0000, 00, 0 – 0
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Synthesis of Norcantharidin Analogue NCA-01
reaction was quenched with saturated sodium hydrogen carbonate, and
the mixture was extracted with EtOAc. The organic layer was washed
with water and brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The crude product was purified by column chromatography on
silica gel (n-hexane/EtOAc, 6:1) to give compound 18 as a white solid
(1.66 g, 41% over 2 steps). 1H NMR (400 MHz, CDCl3): d=ꢀ0.04 (s,
3H), 0.00 (s, 3H), 0.83 (s, 9H), 1.26 (dd, J=6.3 and 12.3 Hz, 1H), 2.16
(dd, J=12.0 and 12.3 Hz, 1H), 2.74 (ddddd, J=4.9, 6.0, 6.3, 10.0, and
12.0 Hz, 1H), 3.25 (d, J=9.8 Hz, 1H), 3.44 (d, J=9.8 Hz, 1H), 3.79 (d,
J=10.4 Hz, 1H), 3.92 (d, J=10.4 Hz, 1H), 4.16 (dd, J=10.0 and 11.3 Hz,
1H), 4.52 (dd, J=6.0 and 11.3 Hz, 1H), 4.82 (d, J=12.1 Hz, 1H), 4.88 (d,
J=12.3 Hz, 1H), 4.90 (d, J=12.1 Hz, 1H), 5.06 (d, J=12.3 Hz, 1H), 5.16
(d, J=4.9 Hz, 1H), 7.22–7.31 (m, 10H), 7.42 (dd, J=7.8 and 7.8 Hz, 2H),
7.56 (dt, J=1.4 and 7.8 Hz, 1H), 7.98 ppm (dd, J=1.4 and 7.8 Hz, 2H);
13C NMR (100 MHz, CDCl3): d=ꢀ5.6, ꢀ5.3, 18.2, 25.7 (3C), 36.1, 40.5,
46.8, 54.2, 62.7, 64.6, 66.5, 66.8, 79.2, 89.3, 128.19 (2C), 128.21 (2C),
128.30 (2C), 128.44 (4C), 128.5 (2C), 129.58 (2C), 129.62 (2C), 133.2,
135.4, 166.2, 170.0, 170.4 ppm; MS (FAB, positive-ion mode, m-nitroben-
zyl alcohol) calcd for C37H45O8Si: 645 [M+H]+; found: 645.
(1S,2R,3S,4R,5R)-1,5-Bisbenzoyloxymethyl-7-oxa-bicycloACTHNGUTERNNU[G 2.2.1]heptane-
2,3-dicarboxylic acid dibenzyl ester ((1S,5R)-19a)
Yield: 42%; ½aꢂ2D8 =ꢀ47.1 (c=1.6, CHCl3); 1H NMR (400 MHz, CDCl3):
d=1.37 (dd, J=6.0 and 12.5 Hz, 1H), 2.12 (t, J=12.1 and 12.5 Hz, 1H),
2.81 (ddddd, J=4.8, 6.0, 6.5, 9.9, and 12.1 Hz, 1H), 3.34 (d, J=9.4 Hz,
1H), 3.47 (d, J=9.4 Hz, 1H), 4.14 (dd, J=9.9 and 11.5 Hz, 1H), 4.46 (dd,
J=6.5 and 11.5 Hz, 1H), 4.56 (d, J=11.5 Hz, 1H), 4.61 (d, J=11.5 Hz,
1H), 4.72 (d, J=12.0 Hz, 1H), 4.84 (d, J=12.0 Hz, 1H), 4.92 (d, J=
12.1 Hz, 1H), 5.05 (d, J=12.1 Hz, 1H), 5.22 (d, J=4.8 Hz, 1H), 7.03–7.22
(m, 10H), 7.34 (dd, J=7.2 and 7.2 Hz, 1H), 7.39 (dd, J=7.2 and 7.2 Hz,
2H), 7.51 (t, J=7.2 Hz, 2H), 7.53 (t, J=7.2 Hz, 1H), 7.92 (d, J=7.2 Hz,
2H), 7.94 ppm (d, J=7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=
36.98, 40.65, 46.75, 54.50, 63.89, 64.28, 67.00 (2C), 79.48, 87.04, 128.27
(2C), 128.33 (2C), 128.39 (2C), 128.49 (2C), 128.41 (4C), 128.49 (2C),
128.55 (4C), 129.47, 129.58, 129.70, 129.74, 133.14, 133.27, 165.63, 166.19,
169.65, 170.11 ppm.
(1R,2S,3R,4S,5S)-1,5-Bisbenzoyloxymethyl-7-oxa-bicycloACTHNUTRGNE[UNG 2.2.1]heptane-
2,3-dicarboxylic acid dibenzyl ester ((1R,5S)-19a)
(1S*,2R*,3S*,4R*,5R*)-5-Benzoyloxymethyl-1-hydroxymethyl-7-oxa-
bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic acid dibenzyl ester (10)
Yield: 72%; ½aꢂ2D8 =+49.6 (c=1.6, CHCl3).
Typical Procedure for the Synthesis of Compounds 1a–1j
To a solution of compound 18 (1.66 g, 2.57 mmol) in dry CH3CN (50 mL)
was added BF3·OEt2 (650 mL, 5.15 mmol) at 08C and the reaction mix-
ture was stirred at the same temperature for 1 h. The reaction was
quenched with saturated aqueous sodium hydrogen carbonate and the
mixture was extracted with EtOAc. The organic layer was washed with
water and brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The crude product was purified by column chromatography on
silica gel (n-hexane/EtOAc, 1:1) to give compound 10 as a white solid
(1.28 g, 94%). 1H NMR (400 MHz, CDCl3): d=1.24 (dd, J=6.3 and
12.0 Hz, 1H), 1.97–2.04 (brs, 1H), 2.19 (dd, J=12.0 and 12.0 Hz, 1H),
2.76 (ddddd, J=4.8, 6.0, 6.3, 10.0 and 12.0 Hz, 1H), 3.23 (d, J=9.5 Hz,
1H), 3.43 (d, J=9.5 Hz, 1H), 3.78 (brd, J=12.2 Hz, 1H), 3.86 (brd, J=
12.2 Hz, 1H), 4.19 (dd, J=10.0 and 11.4 Hz, 1H), 4.50 (dd, J=6.0 and
11.4 Hz, 1H), 4.86 (d, J=12.0 Hz, 1H), 4.95 (d, J=12.3 Hz, 1H), 5.00 (d,
J=12.0 Hz, 1H), 5.07 (d, J=12.3 Hz, 1H), 5.17 (d, J=4.8 Hz, 1H), 7.22–
7.38 (m, 10H), 7.43 (dd, J=7.2 and 7.2 Hz, 2H), 7.57 (dt, J=0.70 and
7.2 Hz, 1H), 7.98 ppm (dd, J=0.70 and 7.2 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d=35.2, 41.0, 46.5, 53.9, 62.2, 64.3, 66.9, 67.0, 89.4,
128.20 (2C), 128.37 (2C), 128.44 (2C), 128.46 (2C), 128.54 (2C), 128.64
(2C), 129.51, 129.56 (2C), 133.22, 135.2, 135.4, 166.2, 169.9, 170.5 ppm;
MS (FAB, positive-ion mode, m-nitrobenzyl alcohol): calcd for C30H31O8:
531 [M+H]+; found: 531.
To a solution of (1S,5R)- or (1R,5S)-19a–19j in EtOAc (0.1m) was added
10 wt.% Pd/C (Degussa type, about 10 mg) and the mixture was stirred
for 1 h at RT under a hydrogen atmosphere. The mixture was filtered
through a membrane filter and the filtrate was concentrated in vacuo.
The crude product was dissolved in acetic anhydride (0.1m) and the reac-
tion mixture was stirred for 4 h at 508C, concentrated in vacuo, and the
residue was recrystallized from Et2O, CH2Cl2, and n-hexane to give
(1S,5R)- or (1R,5S)-1a–1j.
(1S,2R,3S,4R,5R)-1,5-Dibenzoyloxymethyl-7-oxabicycloACTHUNRTGNEUNG[2.2.1]heptane-2,3-
dicarboxylic anhydride ((1S,5R)-1a)
Yield: 70%; ½aꢂ2D8 =ꢀ29.3 (c=2.6, CH3CN); H NMR (400 MHz, CDCl3):
d=1.45 (dd, J=5.9 and 12.3 Hz, 1H), 2.26 (dd, J=12.3 and 12.3 Hz,
1H), 2.98 (ddddd, J=4.9, 5.9, 6.0, 9.3, and 12.3 Hz, 1H), 3.33 (d, J=
7.5 Hz, 1H), 3.78 (d, J=7.5 Hz, 1H), 4.26 (dd, J=9.3 and 12.0 Hz, 1H),
4.54 (dd, J=6.0 and 12.0 Hz, 1H), 4.77 (d, J=12.4 Hz, 1H), 4.89 (d, J=
12.4 Hz, 1H), 5.10 (d, J=4.9 Hz, 1H), 7.46 (dd, J=7.5 and 7.5 Hz, 2H),
7.48 (dd, J=7.5 and 7.5 Hz, 2H), 7.59 (t, J=7.5 Hz, 1H), 7.62 (t, J=
7.5 Hz, 2H), 8.01 (d, J=7.5 Hz, 1H), 8.04 ppm (d, J=7.5 Hz, 2H);
13C NMR (100 MHz, CDCl3): d=34.69, 40.75, 47.63, 51.67, 62.19, 63.54,
81.80, 88.18, 128.41 (2C), 128.55 (2C), 129.05, 129.11, 129.48 (2C), 129.66
(2C), 133.31, 133.48, 165.50, 165.96, 168.49, 170.86 ppm; MS (FAB, posi-
tive-ion mode, m-nitrobenzyl alcohol): calcd for C24H21O8: 437 [M+H]+;
found: 437; elemental analysis calcd (%) for C24H20O8: C 66.05, H 4.62;
found: C 65.68, H 4.64.
1
Separation of Enantiomers (ꢀ)-(1S,5R)-10 and (+)-(1R,5S)-10
Racemate 10: (360 mg, 0.678 mmol) was separated by HPLC (CHIRAL-
PAK IA, 250 mmꢂ20 mm, 5 mm); flow rate: 6.0 mLminꢀ1, mobile phase,
n-hexane/CHCl3/2-propanol, 65:32:3) to give isomers (1R,5S)-10 (140 mg,
0.320 mmol) and (1S,5R)-10 (140 mg, 0.320 mmol).
(1R,2S,3R,4S,5S)-1,5-Dibenzoyloxymethyl-7-oxabicycloACTHUNRTGNEUNG[2.2.1]heptane-2,3-
dicarboxylic anhydride ((1R,5S)-1a)
A
(CHIRALPAK IA, 250 mmꢂ4.6 mm, 5 mm; flow rate: 1.0 mLminꢀ1, n-
Yield: 17%; ½aꢂ2D8 =+33.5 (c=0.54, CH3CN); elemental analysis calcd
hexane/CHCl3, 2:1).
(%) for C24H20O8: C 66.05, H 4.62; found: C 65.84, H 4.66.
A
(CHIRALPAK IA, 250 mmꢂ4.6 mm, 5 mm; flow rate: 1.0 mLminꢀ1, n-
(1S,2R,3S,4R,5R)-5-Benzoyloxymethyl-1-(2-toluoyloxymethyl)-7-oxa-
hexane/CHCl3, 2:1).
bicycloACHTUNTRGNEU[GN 2.2.1]heptane-2,3-dicarboxylic anhydride ((1S,5R)-1b)
Yield: 73%; ½aꢂ2D8 =ꢀ29.0 (c=2.6, CH3CN); H NMR (400 MHz, CDCl3):
d=1.45 (dd, J=6.0 and 12.4 Hz, 1H), 2.28 (dd, J=12.4 and 12.4 Hz,
1H), 2.61 (s, 3H), 2.93 (ddddd, J=4.8, 6.0, 6.4, 9.7, and 12.4 Hz, 1H),
3.34 (d, J=7.5 Hz, 1H), 3.77 (d, J=7.5 Hz, 1H), 4.26 (dd, J=9.7 and
12.0 Hz, 1H), 4.53 (dd, J=6.4 and 12.0, Hz, 1H), 4.72 (d, J=12.4 Hz,
1H), 4.82 (d, J=12.4 Hz, 1H), 5.09 (d, J=4.8 Hz, 1H), 7.20–7.35 (m,
2H), 7.43 (dd, J=7.6 and 7.6 Hz, 1H), 7.47 (dd, J=6.8 and 7.5 Hz, 2H),
7.61 (dd, J=7.6 and 7.6 Hz, 1H), 7.92 (d, J=7.6 Hz, 1H), 8.02 ppm (d,
J=6.8 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=21.89, 34.81, 40.80,
47.50, 51.78, 62.13, 63.53, 81.88, 88.10, 125.75, 128.46, 128.56 (2C), 129.07,
129.49 (2C), 130.58, 131.71, 132.35, 133.48, 140.49, 165.96, 166.24, 168.40,
170.85 ppm; MS (FAB, positive-ion mode, m-nitrobenzyl alcohol): calcd
1
Typical Procedure for the Synthesis of Compounds 19a–19j
To a solution of (1S,5R)-10 or (1R,5S)-10, iPr2NEt (2.0 equiv), and N-
methylimidazole (3.0 equiv) in CH2Cl2 (0.1m), was added an acid chloride
RCOCl (1.5 equiv) at 08C, and the mixture was stirred for 4 h at RT. The
reaction was quenched with saturated sodium hydrogen carbonate and
the mixture was extracted with EtOAc. The organic layer was washed
with water and brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The crude product was purified by column chromatography on
silica gel to give (1S,5R)- or (1R,5S)-19a–19j.
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Mikiko Sodeoka et al.
FULL PAPERS
for C25H23O8: 451 [M+H]+; found: 451; elemental analysis calcd (%) for
(1R,2S,3R,4S,5S)-5-Benzoyloxymethyl-1-cyclohexanecarbonyloxymethyl-7-
C25H22O8: C 66.66, H 4.92; found: C 66.49, H 4.94.
oxa-bicycloACTHNGUTRENNU[G 2.2.1]heptane-2,3-dicarboxylic anhydride ((1R,5S)-1e)
Yield: 80%; ½aꢂ2D8 =+26.9 (c=2.0, CH3CN); elemental analysis calcd (%)
(1R,2S,3R,4S,5S)-5-Benzoyloxymethyl-1-(2-toluoyloxymethyl)-7-oxa-
bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic anhydride ((1R,5S)-1b)
for C24H26O8: C 65.15, H 5.92; found: C 64.93, H 5.93.
Yield: 67%; ½aꢂ2D8 =+30.4 (c=2.8, CH3CN); elemental analysis calcd (%)
(1S,2R,3S,4R,5R)-5-Benzoyloxymethyl-1-cyclopentanecarbonyloxymethyl-
7-oxa-bicycloACTHNUTRGNEUG[N 2.2.1]heptane-2,3-dicarboxylic anhydride ((1S,5R)-1 f)
for C25H22O8: C 66.66, H 4.92; found: C 66.71, H 4.96.
Yield: 76%; ½aꢂ2D8 =ꢀ29.4 (c=2.2, CH3CN); H NMR (400 MHz, CDCl3):
d=1.38 (dd, J=6.0 and 12.4 Hz, 1H), 1.57–1.90 (m, 8H), 2.15 (dd, J=
12.4 and 12.4 Hz, 1H), 2.89 (tt, J=8.0 and 8.0 Hz, 1H), 2.90 (ddddd, J=
4.8, 6.0, 6.0, 9.5, and 12.4 Hz, 1H), 3.25 (d, J=7.6 Hz, 1H), 3.74 (d, J=
7.6 Hz, 1H), 4.24 (dd, J=9.5 and 11.5 Hz, 1H), 4.48 (d, J=12.7 Hz, 1H),
4.55 (dd, J=6.0 and 11.5 Hz, 1H), 4.58 (d, J=12.7 Hz, 1H), 5.06 (d, J=
4.8 Hz, 1H), 7.48 (dd, J=7.6 and 8.4 Hz„ 2H), 7.61 (t, J=7.6 Hz, 1H),
8.01 ppm (d, J=8.4 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=25.83
(2C), 29.98, 30.08, 39.88, 40.29, 43.98, 48.30, 51.98, 61.79, 63.67, 80.89,
88.04, 128.38 (2C), 129.04, 129.52 (2C), 133.65, 165.95, 168.39, 170.91,
175.61 ppm; MS (FAB, positive-ion mode, m-nitrobenzyl alcohol) calcd
for C23H25O8: 429 [M+H]+; found: 429; elemental analysis calcd (%) for
C23H24O8: C 64.48, H 5.62; found: C 64.54, H 5.68.
1
(1S,2R,3S,4R,5R)-5-Benzoyloxymethyl-1-(3-toluoyloxymethyl)-7-oxa-
bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic anhydride ((1S,5R)-1c)
Yield: 26%; ½aꢂ2D8 =ꢀ29.1 (c=0.9, CH3CN); H NMR (400 MHz, CDCl3):
d=1.45 (dd, J=6.0 and 12.4 Hz, 1H), 2.28 (dd, J=12.4 and 12.4 Hz,
1H), 2.41 (s, 3H), 2.93 (ddddd, J=4.8, 6.0, 6.0, 9.7, and 12.4 Hz, 1H),
3.34 (d, J=7.5 Hz, 1H), 3.78 (d, J=7.5 Hz, 1H), 4.26 (dd, J=9.7 and
12.0 Hz, 1H), 4.53 (dd, J=6.0 and 12.0, Hz, 1H), 4.73 (d, J=12.4 Hz,
1H), 4.88 (d, J=12.4 Hz, 1H), 5.10 (d, J=4.8 Hz, 1H), 7.20–7.40 (m,
1H), 7.43 (dd, J=7.6 and 7.6 Hz, 1H), 7.47 (dd, J=6.8 and 7.5 Hz, 1H),
7.61 (dd, J=7.6 and 7.6 Hz, 1H), 7.92 (d, J=7.6 Hz, 1H), 7.83 (d, J=
7.9 Hz, 1H), 7.85 (s, 1H), 8.01 ppm (d, J=7.9 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d=21.37, 34.65, 40.74, 47.60, 51.69, 62.12, 63.55,
81.81, 88.22, 126.81, 128.30, 128.56 (2C), 129.03, 129.06, 129.49 (2C),
130.18, 133.49, 134.10, 138.23, 165.71, 165.96, 168.45, 170.86 ppm; MS
(FAB, positive-ion mode, m-nitro benzyl alcohol) calcd for C25H23O8: 451
[M+H]+; found: 451; elemental analysis calcd (%) for C25H22O8: C 66.66,
H 4.92; found: C 66.42, H 4.94.
1
(1R,2S,3R,4S,5S)-5-Benzoyloxymethyl-1-cyclopentanecarbonyloxymethyl-
7-oxa-bicycloACTHNUTRGNEUG[N 2.2.1]heptane-2,3-dicarboxylic anhydride ((1R,5S)-1 f)
Yield: 64%; ½aꢂ2D8 =+27.1 (c=2.1, CH3CN); elemental analysis calcd (%)
for C23H24O8: C 64.48, H 5.62; found: C 64.41, H 5.66.
(1R,2S,3R,4S,5S)-5-Benzoyloxymethyl-1-(3-toluoyloxymethyl)-7-oxa-
bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic anhydride ((1R,5S)-1c)
(1S,2R,3S,4R,5R)-5-Benzoyloxymethyl-1-cyclopropanecarbonyloxymethyl-
7-oxa-bicycloACTHNUTRGNEUG[N 2.2.1]heptane-2,3-dicarboxylic anhydride ((1S,5R)-1g)
Yield: 64%; ½aꢂ2D8 =+28.4 (c=2.5, CH3CN); elemental analysis calcd (%)
for C25H22O8·0.2H2O: C 66.13, H 4.97; found: C 66.05, H 4.89.
Yield: 95%; ½aꢂ2D8 =ꢀ30.9 (c=2.3, CH3CN); H NMR (400 MHz, CDCl3):
d=0.89–0.94 (m, 2H), 1.03–1.06 (m, 2H), 1.38 (dd, J=6.0 and 12.2 Hz,
1H), 1.66 (dddd, J=4.3, 4.3, 4.3, and 4.3 Hz, 1H), 2.19 (dd, J=12.2 and
12.2 Hz, 1H), 2.90 (ddddd, J=4.8, 6.0, 6.0, 9.2, and 12.2 Hz, 1H), 3.25 (d,
J=7.6 Hz, 1H), 3.74 (d, J=7.6 Hz, 1H), 4.24 (dd, J=9.2 and 11.5 Hz,
1H), 4.49 (d, J=12.3 Hz, 1H), 4.52 (dd, J=6.0 and 11.5 Hz, 1H), 4.58 (d,
J=12.3 Hz, 1H), 5.06 (d, J=4.8 Hz, 1H), 7.48 (dd, J=7.6 and 7.8 Hz„
2H), 7.61 (t, J=7.6 Hz, 1H), 8.01 ppm (d, J=7.8 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d=9.01 (2C), 12.73, 34.44, 40.68, 47.57, 51.58, 61.80,
63.53, 81.76, 88.04, 128.55 (2C), 129.06, 129.47 (2C), 133.48, 165.95,
168.41, 170.88, 173.86 ppm; MS (FAB, positive-ion mode, m-nitrobenzyl
alcohol): calcd for C21H21O8: 401 [M+H]+; found: 401; elemental analysis
calcd (%) for C21H20O8: C 63.00, H 5.03; found: C 63.03, H 5.08.
1
(1S,2R,3S,4R,5R)-5-Benzoyloxymethyl-1-(4-toluoyloxymethyl)-7-oxa-
bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic anhydride ((1S,5R)-1d)
1
Yield: 50%; ½aꢂ2D8 =ꢀ28.3 (c=1.6, CH3CN); H NMR (400 MHz, CDCl3):
d=1.44 (dd, J=6.0 and 12.0 Hz, 1H), 2.26 (dd, J=12.0 and 12.0 Hz,
1H), 2.42 (s, 3H), 2.92 (ddddd, J=4.8, 6.0, 6.0, 9.1, and 12.0 Hz, 1H),
3.32 (d, J=7.6 Hz, 1H), 3.77 (d, J=7.6 Hz, 1H), 4.25 (dd, J=9.1 and
12.0 Hz, 1H), 4.53 (dd, J=6.0 and 12.0 Hz, 1H), 4.74 (d, J=12.4 Hz,
1H), 4.87 (d, J=12.4 Hz, 1H), 5.09 (d, J=4.8 Hz, 1H), 7.25 (d, J=
8.0 Hz„ 2H), 7.48 (dd, J=7.6 and 7.6 Hz, 2H), 7.61 (t, J=7.6 Hz, 1H),
7.93 (d, J=8.0 Hz, 2H), 8.02 ppm (d, J=7.6 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d=21.80, 34.72, 40.75, 47.61, 51.68, 62.00, 63.55,
81.81, 88.28, 126.37, 128.57 (2C), 129.07, 129.13 (2C), 129.50 (2C), 129.72
(2C), 133.49, 144.12, 165.58, 165.96, 168.41, 170.84 ppm; MS (FAB, posi-
tive-ion mode, m-nitrobenzyl alcohol) calcd for C25H23O8: 451 [M+H]+;
found: 451; elemental analysis calcd (%) for C25H22O8·0.2H2O: C 66.13,
H 4.97; found: C 66.15, H 5.04.
(1R,2S,3R,4S,5S)-5-Benzoyloxymethyl-1-cyclopropanecarbonyloxymethyl-
7-oxa-bicycloACTHNUTRGNEUG[N 2.2.1]heptane-2,3-dicarboxylic anhydride ((1R,5S)-1g)
Yield: 94%; ½aꢂ2D8 =+31.5 (c=2.6, CH3CN); elemental analysis calcd (%)
for C21H20O8: C 63.00, H 5.03; found: C 62.72, H 5.04.
(1S,2R,3S,4R,5R)-5-Benzoyloxymethyl-1-(2,2-dimethyl-
propionyloxymethyl)-7-oxa-bicycloACTHNUGRTNEUNG[2.2.1]heptane-2,3-dicarboxylic
anhydride ((1S,5R)-1h)
(1R,2S,3R,4S,5S)-5-Benzoyloxymethyl-1-(4-toluoyloxymethyl)-7-oxa-
bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic anhydride ((1R,5S)-1d)
Yield: 41%; ½aꢂ2D8 =+27.8 (c=1.2, CH3CN); elemental analysis calcd (%)
Yield: 32%; ½aꢂ2D8 =ꢀ28.0 (c=0.7, CH3CN); H NMR (400 MHz, CDCl3):
d=1.24 (s, 9H), 1.38 (dd, J=5.5 and 12.2 Hz, 1H), 2.15 (dd, J=12.2 and
12.2 Hz, 1H), 2.90 (ddddd, J=4.8, 5.5, 5.5, 9.5, and 12.2 Hz, 1H), 3.23 (d,
J=7.6 Hz, 1H), 3.74 (d, J=7.6 Hz, 1H), 4.23 (dd, J=9.5 and 11.9 Hz,
1H), 4.49 (d, J=12.8 Hz, 1H), 4.54 (dd, J=5.5 and 11.9 Hz, 1H), 4.58 (d,
J=12.0 Hz, 1H), 5.07 (d, J=4.8 Hz, 1H), 7.49 (dd, J=7.2 and 7.6 Hz,
2H), 7.62 (t, J=7.6 Hz, 1H), 8.02 ppm (d, J=7.2 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d=27.18 (3C), 34.93, 38.97, 40.73, 47.55, 51.57, 61.91,
63.53, 81.74, 88.16, 128.57 (2C), 129.06, 129.50 (2C), 133.52, 165.96
168.25, 170.81, 177.30 ppm; MS (FAB, positive-ion mode, m-nitrobenzyl
alcohol): calcd for C22H25O8: 417 [M+H]+; found: 417; elemental analysis
calcd (%) for C22H24O8·0.2H2O: C 62.91, H 5.86; found: C 62.79, H 5.96.
1
for C25H22O8: C 66.66, H 4.92; found: C 66.62, H 4.96.
(1S,2R,3S,4R,5R)-5-Benzoyloxymethyl-1-cyclohexanecarbonyloxymethyl-
7-oxa-bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic anhydride ((1S,5R)-1e)
Yield: 67%; ½aꢂ2D8 =ꢀ27.1 (c=1.5, CH3CN); H NMR (400 MHz, CDCl3):
d=1.21–1.50 (m, 6H), 1.51–1.80 (m, 3H), 1.83–1.98 (m, 2H), 2.15 (dd,
J=12.2 and 12.2 Hz, 1H), 2.37 (tt, J=3.6 and 11.1 Hz, 1H), 2.89 (ddddd,
J=4.8, 6.0, 6.3, 9.5, and 12.2 Hz, 1H), 3.25 (d, J=7.6 Hz, 1H), 3.74 (d,
J=7.6 Hz, 1H), 4.23 (dd, J=9.5 and 11.9 Hz, 1H), 4.48 (d, J=12.7 Hz,
1H), 4.54 (dd, J=6.0 and 11.9 Hz, 1H), 4.58 (d, J=12.7 Hz, 1H), 5.06 (d,
J=4.8 Hz, 1H), 7.48 (dd, J=7.6 and 8.4 Hz„ 2H), 7.61 (t, J=7.6 Hz,
1H), 8.00 ppm (d, J=8.4 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=
25.41, 25.44, 25.74, 28.93, 29.05, 34.69, 40.71, 42.95, 47.54, 51.58, 61.60,
63.53, 81.77, 88.08, 128.57 (2C), 129.07, 129.49 (2C), 133.49, 165.96,
168.35, 170.86, 174.86 ppm; MS (FAB, positive-ion mode, m-nitrobenzyl
alcohol) calcd for C24H27O8: 443 [M+H]+; found: 443; elemental analysis
calcd (%) for C24H26O8: C 65.15, H 5.92; found: C 65.22, H 5.93.
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Synthesis of Norcantharidin Analogue NCA-01
(1R,2S,3R,4S,5S)-5-Benzoyloxymethyl-1-(2,2-dimethyl-
(1S,2R,3S,4R,5R)-1,5-Bisbenzoyloxymethyl-7-oxa-bicycloACTHNGUTERNNU[G 2.2.1]heptane-
propionyloxymethyl)-7-oxa-bicyclo
G
2,3-dicarboxylic acid dimethyl ester ((1S,5R)-21a)
anhydride ((1R,5S)-1h)
To a solution of compound (1S,5R)-1a (13.9 mg, 0.032 mmol) in THF
(0.5 mL) and MeOH (0.5 mL) was added a solution of trimethylsilyldia-
zomethane (48 mL, 0.096 mmol, 2.0m in Et2O) at RT. The mixture was
stirred for 3 h and then concentrated in vacuo. The crude product was pu-
rified by preparative thin layer chromatography (n-hexane/EtOAc, 4:1)
to give the title compound as an amorphous white solid (10.9 mg, 71%).
½aꢂ2D8 =ꢀ34.4 (c=1.1, CHCl3); 1H NMR (400 MHz, CDCl3): d=1.42 (dd,
J=6.0 and 12.4 Hz, 1H), 2.20 (t, J=12.4 and 12.4 Hz, 1H), 2.86 (ddddd,
J=4.8, 6.0, 6.5 9.9, and 12.4 Hz, 1H), 3.33 (d, J=9.6 Hz, 1H), 3.46 (d, J=
9.6 Hz, 1H), 3.64 (s, 3H), 3.67 (s, 3H), 4.23 (dd, J=9.9 and 11.0 Hz, 1H),
4.51 (dd, J=6.2 and 11.0 Hz, 1H), 4.61 (d, J=11.5 Hz, 1H), 4.71 (d, J=
11.5 Hz, 1H), 5.20 (d, J=4.8 Hz, 1H), 7.34–7.62 (m, 7H), 8.01 (d, J=
7.2 Hz, 1H), 8.04 ppm (d, J=7.2 Hz, 2H); HRMS (FAB, positive-ion
mode, m-nitrobenzyl alcohol): calcd for C26H26NaO9: 505.1475 [M+Na]+;
found: 505.1475; purity>98% (HPLC).
Yield: 28%; ½aꢂ2D8 =+27.4 (c=0.7, CH3CN); elemental analysis calcd (%)
for C22H24O8: C 63.45, H 5.81; found: C 63.29, H 5.85.
(1S,2R,3S,4R,5R)-5-Benzoyloxymethyl-1-isobutyryloxymethyl-7-oxa-
bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic anhydride ((1S,5R)-1i)
Yield: 64%; ½aꢂ2D8 =ꢀ27.8 (c=2.2, CH3CN); H NMR (400 MHz, CDCl3):
d=1.19 (d, J=7.0 Hz, 3H), 1.20 (d, J=7.0 Hz, 3H), 1.39 (dd, J=6.0 and
13.1 Hz, 1H), 2.15 (dd, J=13.1 and 13.1 Hz, 1H), 2.62 (qq, J=7.0 and
7.0 Hz, 1H), 2.87–2.93 (m, 1H), 3.25 (d, J=7.6 Hz, 1H), 3.75 (d, J=
7.6 Hz, 1H), 4.24 (dd, J=9.1 and 11.4 Hz, 1H), 4.47 (d, J=12.3 Hz, 1H),
4.52 (dd, J=6.0 and 11.4 Hz, 1H), 4.58 (d, J=12.3 Hz, 1H), 5.06 (d, J=
5.2 Hz, 1H), 7.48 (dd, J=7.6 and 8.4 Hz„ 2H), 7.61 (t, J=7.6 Hz, 1H),
8.02 ppm (d, J=8.4 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=18.91,
19.02, 33.88, 34.72, 40.71, 47.60, 51.55, 61.75, 63.53, 81.73, 88.07, 128.56
(2C), 129.06, 129.47 (2C), 133.49, 165.95, 168.38, 170.87, 175.92 ppm; MS
(FAB, positive-ion mode, m-nitrobenzyl alcohol): calcd for C21H23O8 403:
[M+H]+; found: 403; elemental analysis calcd (%) for C21H22O8: C 62.68,
H 5.51; found: C 62.73, H 5.53.
1
(1R,2S,3R,4S,5S)-1,5-Bisbenzoyloxymethyl-7-oxa-bicycloACTHNUTRGNE[UNG 2.2.1]heptane-
2,3-dicarboxylic acid dimethyl ester ((1R,5S)-21a)
Yield: 93%; ½aꢂ2D8 =+30.7 (c=1.3, CHCl3); HRMS (FAB, positive-ion
mode, m-nitrobenzyl alcohol): calcd for C26H26NaO9: 505.1475 [M+Na]+;
found: 505.1451; purity>99% (HPLC).
(1R,2S,3R,4S,5S)-5-Benzoyloxymethyl-1-isobutyryloxymethyl-7-oxa-
bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic anhydride ((1R,5S)-1i)
Yield: 79%; ½aꢂ2D8 =+28.9 (c=1.7, CH3CN); elemental analysis calcd (%)
Construction of the Binding Model
for C21H22O8: C 62.68, H 5.51; found: C 62.59, H 5.52.
To construct the binding model of NCA-01 carboxylate and the catalytic
site of PP2B, a computational docking study was performed based on the
reported PP2B-FKBP-FK506 complex structure (pdb code, 1TCO). The
preliminary binding models were constructed in the Affinity module of
DS 1.2 (Accelrys). The structures were then subjected to energy-minimi-
zation based on molecular dynamics.
(1S,2R,3S,4R,5R)-1-Acetoxymethyl-5-benzoyloxymethyl-7-oxa-
bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic anhydride ((1S,5R)-1j)
1
Yield: 65%; ½aꢂ2D8 =ꢀ13.1 (c=0.82, CHCl3); H NMR (400 MHz, CDCl3):
d=1.38 (dd, J=5.8 and 12.8 Hz, 1H), 2.13 (s, 3H), 2.17 (dd, J=12.5 and
12.5 Hz, 1H), 2.81 (ddddd, J=4.9, 6.0, 6.3, 10.8, and 12.6 Hz, 1H), 3.24
(d, J=7.5 Hz, 1H), 3.76 (d, J=7.5 Hz, 1H), 4.24 (dd, J=9.4 and 11.8 Hz,
1H), 4.51 (d, J=12.3 Hz, 1H), 4.52 (dd, J=6.0 and 11.8 Hz, 1H), 4.58 (d,
J=12.3 Hz, 1H), 5.07 (d, J=4.9 Hz, 1H), 7.49 (dd, J=7.5 and 7.5 Hz,
2H), 7.62 (t, J=7.5 Hz, 1H), 8.01 ppm (d, J=7.5 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d=20.70, 34.64, 40.71, 47.60, 51.53, 61.92, 63.48,
81.80, 87.95, 128.59 (2C), 129.03, 129.50 (2C), 133.54, 165.96, 168.33,
169.91, 170.72 ppm; elemental analysis calcd (%) for C19H18O8·0.2H2O:
C 60.38, H 4.91; found: C 60.42, H 4.85.
Protein Phosphatase Assay
Phosphatases PP1 (rabbit skeletal muscle) and PP2A (rabbit skeletal
muscle, composed of a, b, and catalytic subunits) were purchased from
UBI (Upstate Biotechnology Inc.). Calmodulin (human brain) and PP2B
(human, recombinant) were purchased from CALBIOCHEM. Protein
phosphatase assays were carried out according to the UBI procedure in
the presence or absence of an appropriate concentration of compound.
For PP1 and PP2A assays, the Ser/Thr Phosphatase Assay Kit 1 (UBI)
was used, in which free phosphate ions that were released from the sub-
strate phosphopeptide (KRpTIRR) were quantified by colorimetric anal-
ysis (630 nm) by using the Malachite Green Method (enzyme concentra-
tions: 4 unitsmLꢀ1 PP1; 1.48 unitsmLꢀ1 PP2A). For the PP2B inhibition
assays, RII phosphopeptide (DLDVPIPGRFDRRVpSVAAE, BIOMOL)
was used as the substrate. Briefly, the phosphopeptide (90 mm) was incu-
(1R,2S,3R,4S,5S)-1-Acetoxymethyl-5-benzoyloxymethyl-7-oxa-
bicycloACHTUNGTRENNUNG[2.2.1]heptane-2,3-dicarboxylic anhydride ((1R,5S)-1j)
Yield: 60%; ½aꢂ2D7 =+14.7 (c=0.38, CHCl3); purity>97% (HPLC).
(1S,2R,3S,4R,5R)-1,5-Dibenzoyloxymethyl-7-oxabicycloACTHNUTRGNEU[GN 2.2.1]heptane-2,
3-N-4-bromophenyldicarboximide ((1S,5R)-20)
bated with PP2B (1.6 UmLꢀ1
) at 308C (in 40 mm Tris-HCl, pH 7.5,
100 mm KCl, 6 mm MgCl2, 0.1 mm CaCl2, 0.05 mm DTT, 0.1 mgmLꢀ1
BSA, 70 nm calmodulin) in the absence or presence of inhibitors.
To a solution of compound (1S,5R)-1a (48 mg, 0.11 mmol), Et3N (31 mL,
0.22 mmol), and DMAP (1.2 mg, 0.01 mmol) in toluene (1 mL) was
added 4-bromoaniline (29 mg, 0.17 mmol) and the mixture was stirred for
24 h at 1308C. The mixture was concentrated in vacuo. The crude product
IL-2 Production Assay
was purified by column chromatography on silica gel (n-hexane/EtOAc,
Jurkat cells (6ꢂ106 cellsmLꢀ1
) were placed in a 96-well microplate
28
2:1) to give the title compound as a white solid (48 mg, 66%). ½aꢂD
=
(145 mL/well) and incubated for 2 h. Test compound (6 mm in DMSO,
0.75 mL) and PHA (Sigma, 7.5 mg/well) were added to each well. The
plate was incubated in a CO2 incubator for 36 h, then the supernatant
was collected by centrifugation (400 g, 238C for 5 min). The concentra-
tion of IL-2 in each supernatant (50 mL) was measured by using the
human IL-2 ELISA system (Easy ELISA IL-2 human, GE Healthcare).
OD values at 450 nm were measured with a microplate reader (Spectra-
Max M2, Molecular Devices).
+33.5 (c=0.54, CH3CN); 1H NMR (400 MHz, CDCl3): d=1.49 (dd, J=
5.6 and 12.3 Hz, 1H), 2.33 (dd, J=12.3 and 12.3 Hz, 1H), 2.96 (ddddd,
J=5.1, 5.4, 5.6, 9.0, and 12.3 Hz, 1H), 3.21 (d, J=7.3 Hz, 1H), 3.58 (d,
J=7.3 Hz, 1H), 4.31 (dd, J=9.0 and 11.7 Hz, 1H), 4.58 (dd, J=5.4 and
11.7 Hz, 1H), 4.76 (d, J=12.7 Hz, 1H), 4.89 (d, J=12.7 Hz, 1H), 5.07 (d,
J=5.1 Hz, 1H), 7.15–7.17 (m, 2H), 7.36–7.62 (m, 8H), 8.00–8.05 ppm (m,
4H); 13C NMR (100 MHz, CDCl3): d=35.11, 40.88, 46.45, 51.19, 62.50,
63.87, 81.23, 87.54, 122.53, 127.74 (2C), 128.30 (2C), 128.50 (2C), 129.23,
129.32, 129.50 (2C), 129.63 (2C), 130.43, 132.16 (2C), 133.16, 133.36,
165.63, 166.01, 173.19, 175.41 ppm; MS (MALDI-TOF, positive ion, a-
cyano-4-hydroxycinnamic acid): calcd for C30H25BrNO7: 590.08 [M+H]+;
found: 590.24.
Cell Viability
Jurkat cells were incubated in the presence of a test compound and PHA
under the same conditions as that used for the IL-2-production assay, and
the cell viability was measured by using AlamarBlue reagent (Biosource
International; Ex. 560 nm, Em. 590 nm; SpectraMax M2, Molecular De-
vices).
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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Mikiko Sodeoka et al.
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Acknowledgements
We thank Prof. Dr. Takeshi Shimizu for helpful discussions about the re-
actions at high pressure. We thank Dr. Go Hirai for helpful discussions
about the chemical synthesis and molecular modeling. We also thank Dr.
Kosuke Dodo and Dr. Ayako Tsuchiya for helpful discussions about bio-
logical assays. This work was supported by a Grant-in-Aid for the “Crea-
tion of Biologically Functional Molecules” Scientific Research on Priority
Area from the Ministry of Education, Culture, Sports, Science and Tech-
nology. T.S. was supported by a Research Fellowship from the Japan So-
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Received: January 21, 2012
Revised: February 24, 2012
Published online: && &&, 0000
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www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 0000, 00, 0 – 0
ÝÝ These are not the final page numbers!

FULL PAPERS
Protein phosphatase 2B inhibitor:
Optically pure norcantharidin ana-
logue NCA-01, a highly selective
inhibitor of protein phosphatase 2B
(PP2B), has been synthesized. The
PP2B-inhibitory activity of NCA-01
and its derivatives were independent
of the enantiomeric form.
Protein Inhibitors
Tadashi Shimizu, Masato Iizuka,
Hiroko Matsukura,
Daisuke Hashizume,
Mikiko Sodeoka*
&&&& — &&&&
Synthesis of Optically Pure Norcan-
tharidin Analogue NCA-01, a Highly
Selective Protein Phosphatase 2B
Inhibitor, and its Derivatives
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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