Structure-based design of a highly selective catalytic site-directed inhibitor of Ser/Thr protein phosphatase 2B (calcineurin)

Article abstract of DOI:10.1021/ja034694y

Protein serine/threonine phosphatases (PP1, PP2A and PP2B) play important roles in intracellular signal transductions. The immunosuppressant drugs FK506 and cyclosporin A (CsA) bind to immunophilins, and these complexes selectively inhibit PP2B (calcineurin), leading to the suppression of T-cell proliferation. Both FK506 and CsA must, however, form complexes with immunophilins to exert their inhibitory action on PP2B. Thus, it is of interest to find a direct and selective inhibitor of PP2B that does not involve the immunophilins as a biological tool for studies of PP2B and also as a candidate therapeutic agent. We selected the simple natural product cantharidin, a known PP2A-selective inhibitor, as a lead compound for this project. Primary SAR indicated that norcantharidin (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride) inhibits not only PP1 and PP2A but also PP2B, and a binding model of norcantharidin carboxylate to the PP2B catalytic site was computationally constructed. Based on this binding model, we designed and synthesized several cantharidin derivatives. Among these compounds, 1,5-dibenzoyloxymethyl-substituted norcantharidin was found to inhibit PP2B without inhibiting PP1 or PP2A. To our knowledge, this is the first highly selective catalytic site-directed inhibitor of PP2B.

Full text of DOI:10.1021/ja034694y

Published on Web 07/22/2003
Structure-Based Design of a Highly Selective Catalytic
Site-Directed Inhibitor of Ser/Thr Protein Phosphatase 2B
(Calcineurin)
Yoshiyasu Baba,†,‡,§ Nozomu Hirukawa,†,‡ Naoto Tanohira,‡ and Mikiko Sodeoka*,†,‡
Contribution from the Institute of Multidisciplinary Research for AdVanced Materials
(IMRAM), Tohoku UniVersity, 2-1-1 Katahira, Aoba, Sendai, Miyagi 980-8577, Japan, and
Sagami Chemical Research Center, Nishi-Ohnuma, Sagamihara Kanagawa 229-0012, Japan
Received February 17, 2003; E-mail: sodeoka@tagen.tohoku.ac.jp
Abstract: Protein serine/threonine phosphatases (PP1, PP2A and PP2B) play important roles in intracellular
signal transductions. The immunosuppressant drugs FK506 and cyclosporin A (CsA) bind to immunophilins,
and these complexes selectively inhibit PP2B (calcineurin), leading to the suppression of T-cell proliferation.
Both FK506 and CsA must, however, form complexes with immunophilins to exert their inhibitory action on
PP2B. Thus, it is of interest to find a direct and selective inhibitor of PP2B that does not involve the
immunophilins as a biological tool for studies of PP2B and also as a candidate therapeutic agent. We
selected the simple natural product cantharidin, a known PP2A-selective inhibitor, as a lead compound for
this project. Primary SAR indicated that norcantharidin (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic
anhydride) inhibits not only PP1 and PP2A but also PP2B, and a binding model of norcantharidin carboxylate
to the PP2B catalytic site was computationally constructed. Based on this binding model, we designed and
synthesized several cantharidin derivatives. Among these compounds, 1,5-dibenzoyloxymethyl-substituted
norcantharidin was found to inhibit PP2B without inhibiting PP1 or PP2A. To our knowledge, this is the first
highly selective catalytic site-directed inhibitor of PP2B.
Introduction
of PP2B is known to cause suppression of T-lymphocyte
activation. The therapeutic immunosuppressants FK506 and
The reversible phosphorylation of proteins catalyzed by
protein kinase and phosphatase plays an important role in
intracellular signal transductions, such as regulation of cell
proliferation or differentiation.1 Protein phosphatases can be
divided into two classes according to the substrate specificity,
that is, protein serine/threonine phosphatase (PP) and protein
tyrosine phosphatase (PTP). Furthermore, PP is categorized into
four major subtypes, PP1, PP2A, PP2B, and PP2C.1d PP2B
(calcineurin) is a calcium and calmodulin-regulated phosphatase
which is a heterodimer composed of a catalytic subunit (CnA,
59 kDa) and a calcium/calmodulin-binding subunit (CnB, 19
kDa).2 The catalytic subunit of CnA shares a high degree of
amino acid identity with PP1 and PP2A. PP1 and PP2A
inhibitors, such as okadaic acid, tautomycin, and microcystin-
LR, are well-known as tumor promoters.3 In contrast, inhibition
cyclosporin A form complexes with their binding proteins
(FKBP and cyclophilin, respectively), and these complexes bind
to CnB and inhibit its activity, leading to the suppression of
T-cell proliferation.4 It is important to note that neither FK506
nor cyclosporin A alone can inhibit PP2B, and formation of
the immunophilin complex is required for the PP2B inhibition.
Recently, immunophilins have been shown to regulate the
intracellular calcium ion channel or to be involved in various
other biological processes.5 Some of the side effects of the above
immunosuppressants may be caused by inhibition of the
immunophilins. However, a direct and selective inhibitor of
PP2B has not been reported so far. We started a project aiming
at the development of a novel immunosuppressant, which binds
the CnA catalytic site directly, without requiring immunophilins,
to inhibit PP2B. In this paper, we describe the design and
synthesis of highly selective catalytic site-directed inhibitors of
PP2B.
† Tohoku University.
‡ Sagami Chemical Research Center. This facility has moved and is
currently at 2743-1 Hayakawa, Ayase, Kanagawa 252-1193, Japan.
§ Present address: International Innovation Center, Kyoto University,
Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
The catalytic site structure of PPs is highly conserved
(conserved amino acid sequence, GDXHG(X)nGDXVDRG-
(X)nRGNHE) but is distinct from that of PTPs, which is
characterized by the highly conserved loop structure, C(X)5R.
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J. AM. CHEM. SOC. 2003, 125, 9740-9749
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Inhibitor of Ser/Thr Protein Phosphatase 2B (Calcineurin)
A R T I C L E S
Figure 2. Crystal structures of PP1 and PP2B catalytic sites. (A) PP1-
microcystin covalent complex (conserved residues are shown as sticks,
unique residues are shown as ball-and-stick structures, and microcystin-
LR is shown as a line). (B) PP2B-phosphate ion complex (conserved
residues and phosphate anion are shown as sticks, and unique residues are
shown as ball-and-stick structures). (C) PP1-microcystin covalent complex
(solvent contact surface is shown). (D) PP2B-phosphate ion complex
(solvent contact surface is shown).
Figure 1. Strategy for the development of catalytic site-directed protein
phosphatase inhibitors.
The conserved catalytic site interacts with phosphate oxygen
atoms of the substrate, and each enzyme has a unique site
surrounding the catalytic site that recognizes its specific
substrate. Based on these general structural features of these
enzymes, we designed a strategy for the development of
selective protein phosphatase inhibitors. Briefly, (i) an enzyme
in the same family as the target enzyme, which has a known
tertiary structure, is selected, and a known inhibitor of the former
enzyme is picked up as the lead compound for the target
enzyme. (ii) Primary structure-activity relationships (SARs)
between this inhibitor and the target enzyme are clarified, and
a “core” structure that interacts with the highly conserved
catalytic site is identified and optimized. (iii) A binding model
of the “core” structure and the catalytic site of the enzyme is
constructed by using computational docking techniques. (iv)
Substituents which can interact with the unique regions of the
target enzyme are introduced on to the “core” structure, based
on the binding model.6 According to this strategy, we have
identified a selective “core” structure which binds strongly to
PTPs but not PPs. We found moderately selective inhibitors of
several PTPs from a library of compounds having this “PTP
core” structure.7 For the development of a highly selective
inhibitor of PP2B, we decided to take a rational structure-based
approach instead of the library approach (Figure 1).
reported. As shown in Figure 2, the catalytic sites are highly
conserved. All amino acid residues act as metal ligands (D90,
H92, D118, N150, H199, H281, PP2B numbering), and basic amino
acid residues which interact with the phosphate group (R122
,
H151, R254, PP2B numbering) are completely conserved. There
are possible recognition sites near the catalytic site. A hydro-
phobic amino acid cluster is located near the upper ridge of the
catalytic site in PP2B (L312, V314, Y315). In contrast, there is a
hydrophilic amino acid in the corresponding region of PP1 (C273
,
G274, E275) and also PP2A (C262, Y263, R264). The molecular
topologies of these regions are also very different (Figure 2C
and D).
Known PP1/PP2A inhibitors, including okadaic acid, micro-
cystin-LR, tautomycin, calyculin A and cantharidin, have a
variety of structures (Figure 3).3,10,11 These inhibitors contain a
carboxylic acid, acid anhydride, or phosphate moiety, and it is
likely that these functional groups mimic a phosphate group of
the substrate phosphopeptide. The reported crystal structure of
the PP1-microcystin complex reveals interaction between the
carboxylic acids of microcystin and the basic amino acid residue
in the catalytic site, in addition to the covalent bond with C273
(Figure 2A). It is a substantial challenge to identify the “PP-
selective core” structure in these PP1/PP2A-selective inhibitors
and to create a PP2B-selective inhibitor by rational design based
on the structural difference between PP1/PP2A and PP2B.
Among these PP1/PP2A-selective inhibitors, we chose canthari-
The catalytic site structure is highly conserved among the
major PPs (PP1, PP2A and PP2B), and two metal ions and
coordinating and/or basic amino acid residues play a key role
in the catalysis. The crystal structures of the PP1-microcystin
complex8 and PP2B-FKBP-FK506 complex9 have been
(6) Approaches for increasing affinity to a target protein by tethering two
individual fragments have already been successfully demonstrated. See:
(a) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science
1996, 274, 1531-1534. (b) Mammen, M.; Choi, S.-K.; Whitesides, G. M.
Angew. Chem., Int. Ed. 1998, 37, 2754-2794 and references therein.
(7) (a) Sodeoka, M.; Sampe, R.; Kojima, S.; Baba, Y.; Usui, T.; Ueda, K.;
Osada, H. J. Med. Chem. 2001, 44, 3216-3222. (b) Usui, T.; Kojima, S.;
Kidokoro, S.; Ueda, K.; Osada, H.; Sodeoka, M. Chem. Biol. 2001, 8,
1209-1220. (c) Sodeoka, M.; Baba, Y. Yuki Gosei Kagaku Kyokaishi 2001,
59, 1095-1102.
(8) Goldberg, J.; Huang, H. B.; Kwon, Y. G.; Greengard, P.; Nairn, A. C.;
Kuriyan, J. Nature 1995, 376, 745-753. Recently, the crystal structure of
the PP1-calyculin complex was also reported. See: Kita, A.; Matsunaga,
S.; Takai, A.; Kataiwa, H.; Wakimoto, T.; Fusetani, N.; Isobe, M.; Miki,
K. Structure 2002, 10, 715-724.
(9) (a) Kissinger, C. R.; Perge, H. E.; Knighton, D. R.; Lewis, C. T.; Pelletier,
L. A.; Tempczyk, A.; Kalish, V. J.; Tucker, K. D.; Showalter, R. E.;
Moomaw, E. W.; Gastinel, L. N.; Habuka, N.; Chen, X.; Maldonado, F.;
Barker, J. E.; Bacquet, R.; Villafranca, J. E. Nature 1995, 378, 641. (b)
Griffith, J. P.; Kim, J. L.; Kim, E. E.; Sintchak, M. D.; Thomson, J. A.;
Fitzgibbon, M. J.; Fleming, M. A.; Caron, P. R.; Hsiao, K.; Navia, M. A.
Cell 1995, 82, 507-522.
(10) Sheppeck, J. E.; Gauss, C.-M.; Chamberlin, A. R. Bioorg. Med. Chem.
1997, 5, 1739-1750 and references therein.
(11) (a) Li, Y.-M.; Casida, J. E. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11867-
11870. (b) Li, Y.-M.; MacKintosh, C.; Casida, J. E. Biochem. Pharmacol.
1993, 46, 1435-1443. (c) Honkanen, R. E. FEBS Lett. 1993, 330, 283-
286 and references therein.
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A R T I C L E S
Baba et al.
Figure 4. Basic structure-activity relationships of cantharidin derivatives
1-8. Black, red, and blue bars represent the inhibition of PP1, PP2A, and
PP2B, respectively. Solid bars and striped bars indicate inhibition at 1 mM
and 100 µM drug concentrations, respectively.
examined the basic structure-activity relationship (SAR) of
cantharidin analogues for PP1, PP2A, and PP2B (Figure 4).13
Several cantharidin analogues were tested for their inhibition
of PP1, PP2A, and PP2B at concentrations of 1 mM and 100
µM. As reported previously,11c cantharidin (1) strongly inhibited
PP1 and PP2A, but only a very weak inhibition of PP2B was
observed. Norcantharidin (2), the analogue without the two
methyl substituents at the C2 and C3 positions of cantharidin,
showed a stronger inhibition of PP2B. Norcantharidic acid
sodium salt (3) (endothal-sodium, a herbicide14) also inhibited
all three PPs.15 It is likely that the dicarboxylic acid (salt) formed
from the acid anhydride by hydrolysis under aqueous assay
conditions is an active form, as observed in the case of
tautomycin.16 We were pleased to find that 3 did not inhibit
PTP-1B, a representative tyrosine phosphatase, even at >1 mM
concentrations, indicating that this rigid norcantharidin dicar-
boxylate structure can be considered as the “PP-selective core”.
Introduction of methyl groups at both the C1 and C4 positions
(compound 4) almost abolished the inhibitory activity toward
all phosphatases. We further synthesized the acid anhydride
derivatives, because of the ease of synthesizing and handling
these compounds, and examined their SAR. Compounds 5 and
6 having only one substituent at the bridgehead position also
showed decreased inhibition of all PPs, but some inhibitory
activity remained. The C1 substituent was more unfavorable in
relation to PP1/PP2A than PP2B. A comparison of compounds
7 and 8 having a substituent at the C5-endo position was
interesting. Compound 7, having an acetoxymethyl group,
Figure 3. Naturally occurring Ser/Thr phosphatase inhibitors. Reported
IC50 values for PPs are as follows:10,11c (okadaic acid) PP1, 3 nM, PP2A,
0.2-1 nM, PP2B, >10 mM; (microcystin-LR) PP1, 0.1 nM, PP2A, 0.1
nM, PP2B, not determined; (tautomycin) PP1, 0.7 nM, PP2A, 0.7 nM, PP2B,
∼70 mM; (calyclin A) PP1, 0.3-0.7 nM, PP2A, 0.2-1 nM, PP2B, >10
mM; (cantharidin) PP1, 1.7 µM, PP2A, 0.16 µM, PP2B, >1 mM.
din (1) as a lead compound. Cantharidin, an active constituent
of the dried body of a Chinese blister beetle (Mylabris phalerata
or M. cichorii), is a chemical defense material. The inhibitory
activities of cantharidin on PP1 and PP2A (IC50 of sub µM
order) are much weaker11 than those of other okadaic acid
class compounds such as microcystin, tautomycin, and so forth
(IC50s of sub nM order),10 but we considered that the simple
and rigid structure with an acid anhydride moiety was well suited
as a lead for finding a good PP core. In fact, cantharidin was
reported to inhibit PP2B very weakly (IC50 ) 1 mM), although
inhibition of PP2B by other okadaic acid class compounds was
negligible.11c
Results and Discussion
Basic SAR of Cantharidin Derivatives. Several reports on
the SAR of cantharidin analogues as PP1/PP2A inhibitors have
already been published,11,12 but there has been no systematic
study comparing the inhibition of all three enzymes. We first
(12) (a) McCluskey, A.; Taylor, C.; Quinn, R. J. Bioorg. Med. Chem. Lett. 1996,
6, 1025-1028. (b) Enz, A.; Zenke, G.; Pombo-Villar, E. Bioorg. Med.
Chem. Lett. 1997, 7, 2513-2518. (c) McCluskey, A.; Keane, M. A.;
Mudgee, L.-M.; Sim, A. T. R.; Sakoff, J.; Quinn, R. J. Eur. J. Med. Chem.
2000, 35, 957-964. (d) McCluskey, A.; Bowyer, M. C.; Collins, E.; Sim,
A. T. R.; Sakoff, J. A.; Baldwin, M. L. Bioorg. Med. Chem. Lett. 2000,
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S. P.; Gardiner, E.; Sakoff, J. A. Bioorg. Med. Chem. Lett. 2001, 11, 2941-
2946. (f) McCluskey, A.; Keane, M. A.; Walkom, C. C.; Bowyer, M. C.;
Sim, A. T. R.; Young, D. J.; Sakoff, J. A. Bioorg. Med. Chem. Lett. 2002,
12, 391-393,
(13) Preliminary SAR results have already been reported; see: Sodeoka, M.;
Baba, Y.; Kobayashi, S.; Hirukawa, N. Bioorg. Med. Chem. Lett. 1997, 7,
1833-1836.
(14) Matsuzawa, M.; Graziano, M. J.; Casida, J. E. J. Agric. Food Chem. 1987,
35, 823-829.
(15) Increased inhibition of PP2B by 1 and 2 has also been reported; see ref
11c.
(16) Sugiyama, Y.; Ohtani, I. I.; Isobe, M.; Takai, A.; Ubukata, M.; Isono, K.
Bioorg. Med. Chem. Lett. 1996, 6, 3-8.
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Inhibitor of Ser/Thr Protein Phosphatase 2B (Calcineurin)
A R T I C L E S
Scheme 1. Preparation of Disubstituted Derivativesa
Figure 5. Proposed binding model of norcantharidin carboxylate to PP2B.
a (a) BzCl, Et3N, DMAP, CH2Cl2; (b) Ph(CH2)3COOH, DCC, DMAP,
THF; (c) DMF, POCl3, then sat. NaHCO3; (d) NaBH3CN, THF-MeOH-
AcOH; (e) BzCl, Et3N, DMAP, CH2Cl2; (f) toluene, 23 °C; (g) H2, 5%
Rh/Al2O3, THF-Et2O.
showed a slightly weakened inhibition of PP2B, whereas
compound 8, having a benzoyloxymethyl group, showed an
increased inhibition of PP2B.17 These facts indicated that a
hydrophobic group at this position interacts favorably with
PP2B.
Computational Docking Model of PP2B and the “Core”.
We next constructed a binding model of the core structure to
the PP2B catalytic site. To construct a binding model of
norcantharidin carboxylate and the catalytic site of PP2B, a
computational docking study was performed based on the
reported PP2B-FKBP-FK506 complex structure. The pre-
liminary binding model was constructed using the Affinity
module of the Insight II molecular modeling program. The
structures were then subjected to energy minimization. Figure
5 shows a possible energy-minimized docking model of
norcantharidin dicarboxylate and PP2B. Interactions between
likely that several binding modes other than this model exist
for this simple core structure, but the C1 substituent is expected
to restrict the binding orientation in any of them. As a result,
even a small group such as methyl at the C4 position might
effectively prevent binding of 4 to all the enzymes. The
observation that 8 showed stronger inhibition than 7 suggested
an attractive interaction between the benzoyl group of 8 and
hydrophobic residues of PP2B. All of the experimental results
can be well explained by this model. To develop a PP2B-
selective inhibitor, we next planned to synthesize 1,5- or 1,6-
disubstituted derivatives based on this model. We expected that
the C1 substituent would fix the binding orientation, and a
hydrophobic substituent at the C5 or C6 position should have
an attractive interaction with PP2B but a repulsive interaction
with PP1/PP2A.
phosphate oxygens, two metals (Fe, Zn), and amino acids (R254
,
R122, H151) were observed in the crystal structure of the PP2B-
phosphate complex (Figure 2B). In this model, the carboxylate
moieties interact with this region as a phosphate mimic. The
SAR indicated that introduction of two methyl groups at the
C1 and C4 positions (4) completely abolished inhibitory activity
toward all PPs. In contrast, monosubstituted compounds at this
position (5 and 6) still retained some inhibitory activity. As
shown in Figure 2C and D, a narrow and deep groove exists in
the catalytic site of PP1 and PP2B, which is supposed to be the
substrate recognition site of each enzyme. This groove ends at
the phosphate-binding pocket, and there is no room around the
dicarboxylate except in this direction. According to this model,
the C1 substituent of 5 and 6 can be fitted into the groove. It is
Disubstituted Cantharidin Derivatives. According to Scheme
1, we synthesized the disubstituted derivatives 9-12. Disub-
stituted furan derivatives 19-22 were prepared by Vilsmeier
reaction of 3-acyloxymethylfuran 13 and 14. The mixture of
formylated products was reduced with NaBH3CN under an
acidic condition (THF-acetic acid) to yield 2-hydroxymethyl-
3-acyloxymethylfuran and 2-hydroxymethyl-4-acyloxymethyl-
furan without acyl transfer. The alcohols were treated with
benzoyl chloride to give 2-benzoyloxymethyl-4-acyloxymeth-
ylfurans 19 and 21 and 2-benzoyloxymethyl-3-acyloxymethyl-
furans 20 and 22. Diels-Alder reaction of the disubstituted furan
derivatives with maleic anhydride proceeded at 23 °C in toluene
to yield the corresponding adducts in a moderate yield.
Hydrogenation of the resulting adducts by using a Rh/Al2O3
complex as a catalyst gave the desired 1,6- and 1,5-disubstituted
(17) Tatlock et al. also reported that substitution of the 5-endo-hydrogen of
norcantharidin by hydrophobic residues increased inhibition of PP2B; see:
Tatlock, J. H.; Linton, M. A.; Hou, X. J.; Kissinger, C. R.; Pelletier, L. A.;
Showalter, R. E.; Tempczyk, A.; Villafranca, J. E. Bioorg. Med. Chem.
Lett. 1997, 7, 1007-1012.
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Baba et al.
Figure 6. Inhibition of PP1, PP2A, and PP2B by disubstituted derivatives
9-12. Black, red, and blue bars represent the inhibition of PP1, PP2A, and
PP2B, respectively. Solid bars and striped bars indicate inhibition at 1 mM
and 100 µM drug concentrations, respectively.
exo,exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride
derivatives 9-12.
The inhibitory activities for all PPs were examined. As
expected, these compounds showed selective inhibition of PP2B.
In particular, the 1,5-dibenzoyloxymethyl deriVatiVe 9 showed
greatly increased inhibition of PP2B (IC50 ) 50 µM) compared
to 6 (IC50 ) >1 mM) and no inhibition of PP1 or PP2A (Figure
6). It is likely that introduction of a large hydrophobic substituent
at the C5 position not only increases the attractive interaction
between 9 and hydrophobic residues of PP2B (AA312-314) as
expected but also effectively prevents the binding of 9 to PP1
and PP2A, probably through an unfavorable interaction between
the rigid and hydrophobic residue at the C5 position and a
hydrophilic amino acid residue (E275 of PP1 and R264 of PP2A).
A more flexible 5-endo-phenylbutyroyloxymethyl derivative 11
(IC50 ) 45 µM) and 6-substituted derivatives 10 (IC50 ) 170
µM) and 12 (IC50 ) 60 µM) were less selective, suggesting
that a substituent at the C6 position and a flexible substituent
at the C5 position are less effective to block binding to PP1
and PP2A.
Figure 7 shows dose-response curves of phosphatase inhibi-
tion by compound 9 and known inhibitors. No inhibition of PP1
by 9 was observed over the range of concentration tested (5
µM-1 mM), and PP2A inhibition by 9 was also negligible.
Okadaic acid and cantharidin strongly inhibited these enzymes
under the same assay conditions (Figure 7A and B). The
cyclosporin A-cyclophilin complex and FK506-FKBP com-
plex are known to be ineffective if p-nitrophenyl phosphate
(pNPP) was used as a substrate.4a,18 In contrast to these
regulatory site-directed inhibitors, 9 was effective even if this
small substrate was used (Figure 6). To compare the inhibitory
activity of 9 with that of a cyclosporin A-cyclophilin A
complex, we next carried out PP2B inhibition assays using RII
phosphopeptide18 as a substrate (Figure 7C). Compound 9 was
effective for the inhibition of the PP2B-catalyzed phosphopep-
tide hydrolysis (IC50 ) 7 µM).
Figure 7. Inhibition of PP1, PP2A, and PP2B by okadaic acid, cantharidin,
cyclosporin-cyclophilin complex, and 9. (A) Inhibition of PP1 by okadaic
acid (blue), cantharidin (pink), and 9 (green, not shown on the graph). (B)
Inhibition of PP2A by okadaic acid (blue), cantharidin (pink), and 9 (green).
(C) Inhibition of PP2B by cyclophilin A-cyclosporin A complex (orange),
cantharidin (pink), and 9 (green).
Conclusion
In conclusion, we have succeeded in developing a highly
selective catalytic site-directed inhibitor of PP2B using a rational
approach. Norcantaridin dicarboxylate was found to be a good
“PP-selective core” structure which could interact with the
highly conserved catalytic site of PPs but not that of PTPs. The
basic SAR was clarified, and a binding model of the “core”
and PP2B was constructed. Finally, the highly selective PP2B
inhibitor 9 was designed and synthesized based on the binding
model and the structural difference between PP2B and PP1. To
our knowledge, this is the first example of a specific inhibitor
of PP2B which directly binds to the catalytic site. Work to obtain
a more potent PP2B inhibitor by fine-tuning of the structure is
continuing. The successful example of a complete change of
subtype specificity described here indicates the potency of our
approach. The basic strategy shown in Figure 1 should be
applicable not only for protein phosphatases but also for other
families of proteins.
Experimental Section
General Methods. Infrared (IR) spectra were measured on a JASCO
FT/IR-5300 spectrometer. 1H and 13C NMR spectra were recorded on
a Bruker AM-400 or AC-200P, AVANCE 500 NMR, or JEOL JNM-
LA-400. Chemical shifts are reported downfield from tetramethylsilane
1
()0) for H NMR. For 13C NMR, chemical shifts are reported in the
(18) Swanson, S. K.-H.; Born, T.; Zydowsky, L. D.; Cho, H.; Chang, H. Y.;
Walsh, C.; Rusnak, F. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 3741-3745.
scale relative to the solvent used as an internal reference. Mass spectra
9
9744 J. AM. CHEM. SOC. VOL. 125, NO. 32, 2003

Inhibitor of Ser/Thr Protein Phosphatase 2B (Calcineurin)
A R T I C L E S
(MS) were obtained with a Hitachi M-80B or JEOL JMS-AX500 using
an electron-impact or chemical ionization method or with a JEOL JMS-
GCmateII using a fast atom bombardment method. Column chroma-
tography was performed with silica gel 60 (40-100 µm) purchased
from Kanto Chemical Co. In general, reactions were carried out under
anhydrous conditions in dry solvents under an argon atmosphere. Purity
of the compounds obtained was determined to be more than 95% based
in dry pyridine (10 mL) was added benzoyl chloride (2.15 g, 15.3 mmol)
at room temperature. The mixture was stirred for 3 h and then acidified
with 1 N HCl (5 mL). After neutralization with saturated aqueous
sodium bicarbonate, the mixture was extracted with EtOAc, and the
organic layer was washed with water and brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The crude product was purified
by silica gel column chromatography (20:1 hexane/EtOAc) to give
2-benzoyloxymethylfuran (647 mg, 31%) as a colorless oil. IR (neat,
cm-1) 1720, 1600, 1580, 1500, 1370, 1310, 1280; 1H NMR (400 MHz,
CDCl3) δ 5.24 (s, 2H), 6.51 (dd, J ) 1.7, 1.7 Hz, 1H), 7.41-7.46 (m,
3H), 7.53-7.59 (m, 2H), 8.02-8.08 (m, 2H); 13C NMR (100 MHz,
CDCl3) δ 58.2, 110.6, 120.5, 128.3, 129.6, 130.1, 133.0, 141.6, 143.4,
166.4; LRMS (EI, m/z) 202 (M+), 105, 81. HRMS (FAB, m/z) calcd
for C12H10O3, 202.0630; found, 202.0631.
1
on the H NMR spectral analysis.
Synthesis of Cantharidin Derivatives. Compounds 1 and 4 were
commercial products, and other compounds were synthesized as follows.
Norcantharidin (2).12c To a solution of exo-3,6-epoxy-1,2,3,6-
tetrahydrophthalic anhydride (392 mg, 2.36 mmol) in THF (20 mL)
was added 10% Pd/C (100 mg), and the mixture was stirred at room
temperature under a hydrogen atmosphere for 4 h. The reaction mixture
was filtered through Celite and concentrated in vacuo to give 2 (425
A solution of 2-benzoyloxymethylfuran (647 mg, 3.12 mmol) and
maleic anhydride (314 mg, 3.12 mmol) in toluene (5 mL) was stirred
at 80 °C for 456 h. The reaction mixture was cooled to room
temperature, and the crude product was collected by filtration and
washed with hexane to afford 1-benzoyloxymethyl-7-oxabicyclo[2.2.1]-
hept-5-ene-2,3-dicarboxylic anhydride (437 mg, 46%) as colorless
crystals. Mp 152-154 °C; IR (KBr, cm-1) 3020, 1860, 1790, 1720,
1600, 1450; 1H NMR (250 MHz, CDCl3) δ 3.30 (d, J ) 6.9 Hz, 1H),
3.37 (d, J ) 6.9 Hz, 1H), 4.81 (d, J ) 13.0 Hz, 1H), 5.20 (d, J ) 13.0
Hz, 1H), 5.48 (d, J ) 1.5 Hz, 1H), 6.53-6.65 (m, 2H), 7.40-7.63 (m,
3H), 8.02-8.07 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 49.3, 50.9,
60.6, 81.8, 90.5, 128.0, 128.8, 129.4, 133.0, 137.1, 137.6, 165.4, 167.4,
168.9; LRMS (EI, m/z) 301 (M++1), 283, 203.; Anal. Calcd for
C16H12O6: C, 64.00; H, 4.03. Found: C, 63.92; H, 4.01.
mg, quant.) as colorless crystals. Mp 116-119 °C; IR (KBr, cm-1
1870, 1790, 1720, 1600, 1450; H NMR (250 MHz, CDCl3) δ 1.61
(dt, J ) 14.1, 3.4 Hz, 2H), 1.87-1.90 (m, 2H), 3.15 (s, 2H), 5.02 (dd,
J ) 3.4, 2.2 Hz, 2H).
)
1
Norcantharidin Disodiumsalt (3).12c To a solution of 2 (4.80 mg,
0.029 mmol) in THF (0.5 mL) was added 0.1 N sodium bicarbonate in
water (57.1 µL, 0.058 mmol). The mixture was stirred at room
temperature for 1 h and concentrated in vacuo to give 3 (6.67 mg,
1
quant.) as a white solid. H NMR (400 MHz, D2O) δ 1.37 (m, 2H),
1.49 (m, 2H), 2.69 (s, 2H), 4.55 (brs, 2H).
1-Acetoxymethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic An-
hydride (5). To a solution of furfuryl alcohol (4.91 g, 50.1 mmol) and
Et3N (20.9 mL, 150 mmol) in dichloromethane (25 mL) was added
acetic anhydride (10.2 g, 100 mmol), and the mixture was stirred at
room temperature for 19 h. The reaction was quenched with saturated
ammonium chloride, and the mixture was extracted with dichloro-
methane. The organic layer was washed with water and brine, dried
over anhydrous Na2SO4, and concentrated in vacuo. The crude product
was purified by silica gel column chromatography (10:1 hexane/EtOAc)
To a solution of 1-benzoyloxymethyl-7-oxabicyclo[2.2.1]hept-5-ene-
2,3-dicarboxylic anhydride (96.3 mg, 0.32 mmol) in THF (5 mL) was
added 10% Pd/C (20 mg), and the mixture was stirred at room
temperature under a hydrogen atmosphere for 18 h. The reaction mixture
was filtered through Celite and concentrated in vacuo. The crude product
was purified by silica gel column chromatography (EtOAc) and
recrystallized with 2-propanol to give 6 (51.0 mg, 53%) as colorless
crystals. Mp 109-110 °C; IR (KBr, cm-1) 1870, 1790, 1720, 1600,
1
to give 2-acetoxymethylfuran (6.40 g, 91%) as a yellow oil. H NMR
(200 MHz, CDCl3) δ 2.07 (s, 3H), 5.05 (s, 2H), 6.36 (dd, J ) 3.2, 1.8
Hz, 1H), 6.41 (brd, J ) 3.2 Hz, 1H), 7.42 (dd, J ) 1.8, 0.8 Hz, 1H);
13C NMR (100 MHz, CDCl3) δ 20.9, 58.0, 110.5, 110.6, 143.3, 149.4,
170.6. HRMS (FAB, m/z) calcd for C7H8O3, 140.0473; found, 140.0474.
A solution of 2-acetoxymethylfuran (700 mg, 5.00 mmol) and maleic
anhydride (491 mg, 5.00 mmol) in toluene (5 mL) was stirred at room
temperature for 97 h. The reaction mixture was filtered, washed with
hexane-EtOAc (1:1 v/v), and dried in vacuo to give 1-acetoxymethyl-
7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic anhydride (880 mg,
74%) as colorless crystals. Mp 113-115 °C; IR (KBr, cm-1) 1740,
1
1450; H NMR (250 MHz, CDCl3) δ 1.68-1.83 (m, 2H), 1.92-2.20
(m, 2H), 3.32 (d, J ) 7.5 Hz, 1H), 3.36 (d, J ) 7.5 Hz. 1H), 4.79 (d,
J ) 12.5 Hz, 1H), 4.94 (d, J ) 12.5 Hz, 1H), 5.08 (d, J ) 5.0 Hz,
1H), 7.39-7.60 (3H, m), 8.03-8.07 (m, 2H); 13C NMR (125 MHz,
CDCl3) δ 29.2, 31.2, 51.5, 51.9, 62.3, 80.1, 87.6, 128.5, 129.4, 129.8,
133.3, 165.7, 169.0, 170.8; LRMS (CI, m/z) 303 (M+ + 1), 181, 105.
Anal. Calcd for C16H14O6: C, 63.57; H, 4.67. Found: C, 63.37; H,
4.65.
5-Acetoxymethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic An-
hydride (7). To a solution of 3-furanmethanol (1.02 g, 3.72 mmol)
and Et3N (1.04 mL, 7.44 mmol) in dichloromethane (30 mL) was added
acetic anhydride (573 mL, 4.09 mmol), and the mixture was stirred at
room temperature for 30 h. The reaction was quenched with saturated
ammonium chloride, and the mixture was extracted with dichlo-
romethane. The organic layer was washed with water and brine, dried
over anhydrous Na2SO4, and concentrated in vacuo. The crude product
was purified by silica gel column chromatography (dichloromethane)
1
1660, 1380, 1040, 1090; H NMR (400 MHz, CDCl3) δ 2.13 (s, 3H),
3.23 (d, J ) 6.8 Hz, 1H), 3.35 (d, J ) 6.8 Hz, 1H), 4.55 (d, J ) 12.8
Hz, 1H), 4.92 (d, J ) 12.8 Hz, 1H), 5.46 (d, J ) 1.6 Hz, 1H), 6.48 (d,
J ) 5.7 Hz, 1H), 6.64 (dd, J ) 5.7, 1.6 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 20.6, 49.6, 51.3, 60.7, 82.2, 90.7, 137.4, 138.0, 167.8, 169.3,
170.3. LRMS (EI, m/z) 239 (M+ + 1).
To a solution of 1-acetoxymethyl-7-oxabicyclo[2.2.1]hept-5-ene-2,3-
dicarboxylic anhydride (28.3 mg, 0.12 mmol) in THF (2 mL) was added
10% Pd/C (5 mg), and the mixture was stirred at room temperature
under a hydrogen atmosphere for 4 h. The reaction mixture was filtered
through Celite and concentrated in vacuo to give 5 (28.5 mg, quant.)
as colorless crystals. Mp 125-129 °C; IR (KBr, cm-1) 2960, 1740,
1370; 1H NMR (400 MHz, CDCl3) δ 1.64-1.78 (m, 2H), 1.87 (ddd, J
) 12.5, 11.8, 3.4 Hz, 1H), 2.02-2.10 (m, 1H), 2.12 (s, 3H), 3.24 (d,
J ) 7.5 Hz, 1H), 3.34 (d, J ) 7.5 Hz, 1H), 4.54 (d, J ) 12.5 Hz, 1H),
4.62 (d, J ) 12.5 Hz, 1H), 5.05 (d, J ) 5.3 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ 20.6, 29.2, 31.0, 51.3, 51.8, 61.9, 80.1, 87.3, 168.9,
170.2, 170.7. HRMS (FAB, m/z) calcd for C11H12O6, 240.0634; found,
240.0623.
1
to give 3-furylmethyl acetate (711 mg, quant.) as a colorless oil. H
NMR (200 MHz, CDCl3) δ 2.07 (s, 3H), 4.98 (s, 2H), 6.43 (d, J ) 1.3
Hz, 1H), 7.40 (d, J ) 1.3 Hz, 1H), 7.47 (brs, 1H); 13C NMR (100
MHz, CDCl3) δ 21.0, 57.7, 110.6, 120.3, 141.6, 143.4, 170.9; LRMS
(EI, m/z) 140 (M+). HRMS (FAB, m/z) calcd for C7H8O3, 140.0473;
found, 140.0468.
A solution of 3-furylmethyl acetate (695 mg, 4.96 mmol) and maleic
anhydride (486 mg, 4.96 mmol) in toluene (3 mL) was stirred at room
temperature for 117 h. The reaction mixture was filtered and dried in
vacuo to give 5-acetoxymethyl-7-oxabicyclo[2.2.1]hept-5-ene-2,3-di-
carboxylic anhydride (961 mg, 81%) as colorless crystals. Mp 112-
116 °C; IR (KBr, cm-1) 2960, 1780, 1740, 1650, 1370, 1040, 1090;
1H NMR (400 MHz, CDCl3) δ 2.12 (s, 3H), 3.23 (d, J ) 6.9 Hz, 1H),
1-Benzoyloxymethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxyl-
ic Anhydride (6). To a solution of furfuryl alcohol (1.00 g, 10.2 mmol)
9
J. AM. CHEM. SOC. VOL. 125, NO. 32, 2003 9745

A R T I C L E S
Baba et al.
3.29 (d, J ) 6.9 Hz, 1H), 4.75 (dd, J ) 14.5, 1.7 Hz, 1H), 4.80 (dd, J
) 14.5, 1.4 Hz, 1H), 5.36 (s, 1H), 5.45 (d, J ) 1.4 Hz, 1H), 6.38 (ddd,
J ) 1.7, 1.4, 1.4 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 20.7, 48.4,
49.6, 59.0, 82.8, 82.9, 132.6, 147.0, 169.6, 169.8, 170.5.
stirred for 27 h. The reaction mixture was filtered through Celite, and
1 N HCl (5 mL) was added. The mixture was diluted with EtOAc (10
mL), washed with saturated aqueous sodium bicarbonate, water, and
brine, dried over anhydrous MgSO4, and concentrated in vacuo. The
crude product was purified by silica gel column chromatography (3:1
hexane/EtOAc) to give 14 (3.48 g, 69%) as a colorless oil. IR (neat,
cm-1) 2926, 2853, 1740, 1600, 1500, 1480, 1430, 1140, 1040; 1H NMR
(250 MHz, CDCl3) δ 1.96 (tt, J ) 7.5, 7.5 Hz, 2H), 2.34 (t, J ) 7.5
Hz, 2H), 2.64 (t, J ) 7.5 Hz, 2H), 4.98 (s, 2H), 6.42 (d, J ) 1.3 Hz,
1H), 7.17-7.47 (m, 7H); 13C NMR (125 MHz, CDCl3) δ 26.5, 33.6,
35.1, 57.6, 110.5, 120.5, 126.0, 128.4, 128.5, 141.3, 141.5, 143.4, 173.3;
LRMS (EI, m/z) 244 (M+), 163, 117, 81. HRMS (FAB, m/z) calcd for
C15H16O3, 244.1100; found, 244.1093.
To a solution of 5-acetoxymethyl-7-oxabicyclo[2.2.1]hept-5-ene-2,3-
dicarboxylic anhydride (119 mg, 0.50 mmol) in THF (5 mL) was added
10% Pd/C (25 mg). The mixture was stirred at room temperature under
a hydrogen atmosphere for 3 h, filtered through Celite, and concentrated
in vacuo to give 7 (105 mg, 88%) as colorless crystals. Mp 94-97 °C;
1
IR (KBr, cm-1) 2960, 1760, 1690, 1370, 1280, 1240; H NMR (400
MHz, CDCl3) δ 1.21 (dd, J ) 12.7 Hz, 5.4 Hz, 1H), 2.11 (s, 3H), 2.17
(dd, J ) 12.7, 5.6 Hz, 1H), 2.64 (m, 1H), 3.14 (d, J ) 7.4 Hz, 1H),
3.50 (d, J ) 7.4 Hz. 1H), 3.96 (dd, J ) 11.7 Hz, 9.8 Hz, 1H), 4.26
(dd, J ) 11.7 Hz, 5.6 Hz, 1H), 4.98 (d, 5.6 Hz, 1H), 5.01 (d, 4.9 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 20.8, 31.7, 39.4, 46.0, 50.5, 63.3,
80.6, 82.1, 170.8, 170.9, 171.6. HRMS (FAB, m/z) calcd for C11H12O6
+ H+, 241.0712; found, 241.0717.
4-Benzoyloxymethyl-2-hydroxymethylfuran (15) and 3-Benzoyl-
oxymethyl-2-hydroxymethylfuran (16). A mixture of DMF (3.00 g,
41.1 mmol) and phosphorus oxychloride (6.31 g, 41.1 mmol) was stirred
at 0 °C for 1 h to give a Vilsmeier reagent. To the Vilsmeier reagent
was slowly added 13 (5.55 g, 27.4 mmol) at 0 °C, and stirring was
continued for 1.5 h. After the resulting mixture was warmed to room
temperature over 20 h, it was poured into saturated aqueous sodium
bicarbonate (100 mL) and stirring was continued for 2.5 h. The reaction
mixture was extracted with Et2O, and the organic layer was washed
with water and brine, dried over anhydrous MgSO4, and concentrated
in vacuo. The crude product was purified by silica gel column
chromatography (8:1 hexane/EtOAc) to give a 1:3 mixture of 4-benzo-
yloxymethyl-2-formylfuran and 3-benzoyloxymethyl-2-formylfuran
(5.61 g, 89%) as a colorless oil.
3-Furylmethyl Benzoate (13). To a solution of 3-furanmethanol
(1.09 g, 11.1 mmol) in dry pyridine (10 mL) was added benzoyl chloride
(2.35 g, 16.7 mmol) at room temperature. The reaction mixture was
stirred for 3 h, acidified with 1 N HCl (5 mL), neutralized with saturated
aqueous sodium bicarbonate, and extracted with Et2O. The organic layer
was washed with water and brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The crude product was purified by silica gel
column chromatography (20:1 hexane/EtOAc) to give 13 (2.22 g, 97%)
as a colorless oil. IR (neat, cm-1) 1720, 1600, 1580, 1500, 1370, 1310,
1
1280; H NMR (400 MHz, CDCl3) δ 5.24 (s, 2H), 6.51 (dd, J ) 1.7,
1.7 Hz, 1H), 7.41-7.46 (m, 3H), 7.53-7.59 (m, 2H), 8.02-8.08 (m,
2H); 13C NMR (100 MHz, CDCl3) δ 58.2, 110.6, 120.5, 128.3, 129.6,
130.1, 133.0, 141.6, 143.4, 166.4; LRMS (EI, m/z) 202 (M+), 105, 81.
HRMS (FAB, m/z) calcd for C12H10O3, 202.0630; found, 202.0618.
5-Benzoyloxymethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarbox-
ylic Anhydride (8). A solution of 13 (1.05 g, 5.25 mmol) and maleic
anhydride (477 mg, 4.87 mmol) in toluene (5 mL) was stirred at 80 °C
for 14 h. The reaction mixture was cooled at room temperature, and
then the product was collected by filtration, washed with benzene, and
dried in vacuo to give 5-benzoyloxymethyl-7-oxabicyclo[2.2.1]hept-
5-ene-2,3-dicarboxylic anhydride (1.29 g, 89%) as a white solid. Mp,
134-137 °C; IR (KBr, cm-1) 1860, 1790, 1720, 1600, 1580, 1490,
1450, 1310, 1270; 1H NMR (400 MHz, CDCl3) δ 3.25 (d, J ) 6.9 Hz,
1H), 3.37 (d, J ) 6.9 Hz, 1H), 5.04 (d, J ) 1.5 Hz, 2H), 5.45 (s, 1H),
5.47 (d, J ) 1.3 Hz, 1H), 6.45 (m, 1H), 7.45-7.51 (m, 3H), 8.02-
8.06 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 48.7, 50.0, 59.9, 81.9,
82.3, 128.7, 129.2, 132.7, 133.5, 146.1, 165.3, 171.2, 171.3; LRMS
(EI, m/z) 301 (M+ + 1), 202, 105. Anal. Calcd for C16H12O6: C, 64.00;
H, 4.03. Found: C, 63.86; H, 3.84.
To a solution of 5-benzoyloxymethyl-7-oxabicyclo[2.2.1]hept-5-ene-
2,3-dicarboxylic anhydride (199 mg, 0.66 mmol) in THF (5 mL) was
added 10% Pd/C (20 mg), and the mixture was stirred at room
temperature under a hydrogen atmosphere for 3 h. The reaction mixture
was filtered through Celite and concentrated in vacuo. The crude product
was recrystallized with 2-propanol to give 8 (133 mg, 67%) as colorless
crystals. Mp 145-149 °C; IR (KBr, cm-1) 1860, 1840, 1780, 1710,
1600, 1590, 1470, 1450, 1390, 1320, 1290; 1H NMR (200 MHz, CDCl3)
δ 1.32 (dd, J ) 12.7, 5.4 Hz, 1H), 2.22 (ddd, J ) 12.7, 12.7, 5.7 Hz,
1H), 2.72-2.87 (m, 1H), 3.20 (d, J ) 7.5 Hz, 1H), 3.61 (d, J ) 7.5
Hz, 1H), 4.21 (dd, J ) 11.8, 9.5 Hz, 1H), 4.55 (dd, J ) 11.8, 5.7 Hz,
1H), 5.06 (d, J ) 5.5 Hz, 1H), 5.08 (d, J ) 4.7 Hz, 1H), 7.43-7.67
(m, 3H), 8.00-8.07 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 31.8,
39.7, 46.1, 50.6, 63.7, 128.7, 129.3, 129.6, 133.6, 166.2, 170.8, 171.4;
LRMS (CI, m/z) 303 (M+ + 1), 180, 105. Anal. Calcd for C16H14O6:
C, 63.57; H, 4.67. Found: C, 63.50; H, 4.53.
To a solution of 4-benzoyloxymethyl-2-formylfuran and 3-benzoyl-
oxymethyl-2-formylfuran (5.61 g, 24.4 mmol) in a mixture of methanol
(55 mL), dry THF (55 mL), and acetic acid (2.7 mL) was added NaBH3-
CN (2.30 g, 36.6 mmol) at room temperature. The mixture was stirred
for 2 h and then diluted with EtOAc (170 mL), washed with saturated
aqueous sodium bicarbonate, water, and brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The crude product was purified
by silica gel column chromatography (4:1 hexane/EtOAc) to give 15
(0.98 g, 19%) as a yellow oil and 16 (2.84 g, 57%) as a colorless solid.
15: IR (neat, cm-1) 3370, 2950, 1720, 1610, 1460, 1280, 1280, 1110;
1H NMR (500 MHz, CDCl3) δ 1.94 (s, 1H), 4.59 (s, 2H), 5.19 (s, 2H),
6.42 (s, 1H), 7.42-7.45 (m, 2H), 7.51 (s, 1H), 7.54-7.57 (m, 1H),
8.03-8.05 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 57.5, 58.2, 108.8,
121.3, 128.3, 129.6, 130.0, 133.0, 141.5, 155.0, 166.5; Mass (EI, m/z)
232 (M+), 110, 105. HRMS (EI, m/z) calcd for C20H16O5, 232.0736;
found, 232.0758. 16: mp, 66-67 °C; IR (KBr, cm-1) 3370, 2950, 1720,
1610, 1460, 1280, 1280, 1110; 1H NMR (250 MHz, CDCl3) δ 2.75 (s,
1H), 4.75 (s, 2H), 5.27 (s, 2H), 6.46 (d, J ) 1.8 Hz, 1H), 7.36 (d, J )
1.8 Hz, 1H), 7.41-7.44 (m, 2H), 7.54-7.57 (m, 1H), 8.01-8.03 (m,
2H); 13C NMR (125 MHz, CDCl3) δ 55.5, 58.1, 111.7, 117.0, 128.4,
129.7, 130.0, 133.2, 142.2, 152.9, 167.0; LRMS (EI, m/z) 232 (M+),
110, 105. HRMS (EI, m/z) calcd for C20H16O5, 232.0736; found,
232.0750.
2-Hydroxymethyl-4-(4-phenylbutanoyloxymethyl)furan (17) and
2-Hydroxymethyl-3-(4-phenylbutanoyloxymethyl)furan (18). A mix-
ture of DMF (1.14 mL, 41.7 mmol) and phosphorus oxychloride (1.48
g, 14.7 mmol) was stirred at 0 °C for 30 min to give a Vilsmeier reagent.
To the Vilsmeier reagent was slowly added 14 (1.80 g, 7.37 mmol) at
0 °C, and the reaction mixture was stirred for 30 min. After the reaction
mixture was warmed to room temperature, the reaction mixture was
further stirred for 70 h and then poured into saturated aqueous sodium
bicarbonate (30 mL) at 0 °C, and stirring was continued for 4 h. The
reaction mixture was extracted with Et2O, and the organic layer was
washed with water and brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The crude product was purified by silica gel
column chromatography (7:1 hexane/EtOAc) to give 2-formyl-3-(4-
phenylbutanoyloxymethyl)furan (1.48 g, 74%) and 2-formyl-4-(4-
phenylbutanoyloxymethyl)furan (503 mg, 25%), each as a colorless
3-Furylmethyl-4-phenylbutanoate (14). To a solution of 3-furan-
methanol (2.04 g, 20.7 mmol) in dry THF (20 mL) was added
4-phenylbutyric acid (3.24 g, 19.7 mmol), DCC (8.56 g, 41.5 mg), and
DMAP (1.25 g, 10.4 mmol) at room temperature, and the mixture was
oil. 2-Formyl-3-(4-phenylbutanoyloxymethyl)furan: IR (neat, cm-1
)
9
9746 J. AM. CHEM. SOC. VOL. 125, NO. 32, 2003

Inhibitor of Ser/Thr Protein Phosphatase 2B (Calcineurin)
A R T I C L E S
2926, 2853, 1740, 1670, 1600, 1480, 1450, 1430, 1270, 1140; 1H NMR
(250 MHz, CDCl3) δ 1.98 (tt, J ) 7.5, 7.5 Hz, 2H), 2.39 (t, J ) 7.5
Hz, 2H), 2.66 (t, J ) 7.5 Hz, 2H), 5.33 (s, 2H), 6.61 (d, J ) 1.5 Hz,
1H), 7.17-7.28 (m, 5H), 7.60 (d, J ) 1.5 Hz, 1H), 9.85 (s, 1H). 13C
NMR (125 MHz, CDCl3) δ 26.3, 33.3, 35.0, 56.9, 113.4, 126.0, 128.4,
128.4, 130.9, 141.1, 147.2, 148.5, 172.9, 178.7; LRMS (CI, m/z) 273
(M+ + 1), 255, 163, 109, 91. 2-Formyl-4-(4-phenylbutanoyloxymethyl)-
furan: IR (neat, cm-1) 2820, 1740, 1670, 1600, 1520, 1450, 1270, 1140;
1H NMR (250 MHz, CDCl3) δ 1.93 (tt, J ) 7.5, 7.5 Hz, 2H), 2.35 (t,
J ) 7.5 Hz, 2H), 2.64 (t, J ) 7.5 Hz, 2H), 5.00 (s, 2H), 7.14-7.31 (m,
6H), 7.70 (s, 1H), 9.63 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 26.3,
33.4, 35.0, 56.7, 120.9, 123.5, 126.0, 128.4, 128.4, 141.1, 146.7, 153.2,
173.1, 177.8; LRMS (EI, m/z) 272 (M+), 255, 163, 109, 91.
room temperature for 30 min. The solution was acidified with 1 N
HCl (2 mL) and neutralized with saturated aqueous sodium bicarbonate.
The resulting mixture was extracted with dichloromethane. The organic
layer was washed with water and brine, dried over anhydrous MgSO4,
and concentrated in vacuo. The crude product was purified by silica
gel column chromatography (2:1 hexane/EtOAc) to give 20 (4.36 g,
92%) as a pale yellow solid. Mp 56-58 °C; IR (KBr, cm-1) 2950,
1720, 1610, 1460, 1280, 1100; 1H NMR (250 MHz, CDCl3) δ 5.36 (s,
2H), 5.44 (s, 2H), 6.53 (d, J ) 1.8 Hz, 1H), 7.38 (d, J ) 1.8 Hz, 1H),
7.39-7.53 (m, 4H), 7.56-7.68 (m, 2H), 8.00-8.18 (m, 4H); 13C NMR
(125 MHz, CDCl3) δ 56.7, 57.8, 112.0, 120.3, 128.9, 129.7, 129.8,
130.0, 130.6, 133.0, 133.1, 143.0, 147.9, 162.4, 166.2, 166.4; LRMS
(EI, m/z) 336 (M+), 105; HRMS (EI, m/z) calcd for C20H16O5, 336.0993;
found, 336.0992.
To a solution of 2-formyl-4-(4-phenylbutanoyloxymethyl)furan (390
mg, 1.43 mmol) in methanol-THF-acetic acid (17.5 mL, 20:20:1 v/v)
was added NaBH3CN (225 mg, 3.58 mmol) at 0 °C. The mixture was
stirred for 23 h at room temperature, then poured into saturated aqueous
sodium bicarbonate (3 mL) at 0 °C, and 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 silica gel column chromatography (3:1 hexane/EtOAc) to give 17
(287 mg, 73%) as a colorless oil. IR (neat, cm-1) 3450, 2940, 2853,
2-Benzoyloxymethyl-4-(4-phenylbutanoyloxymethyl)furan (21).
To a solution of 17 (280 mg, 1.02 mmol), Et3N (284 mL, 2.04 mmol),
and DMAP (10.3 mg, 0.10 mmol) in dichloromethane (8 mL) was added
benzoyl chloride (189 µL, 1.34 mmol) at 0 °C, and the mixture was
stirred for 57 h at room temperature. The reaction was quenched with
1 N HCl (1 mL) at 0 °C and extracted with dichloromethane. The
organic layer was washed with saturated aqueous sodium bicarbonate,
water, and brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The crude product was purified by silica gel column chroma-
tography (3:1 hexane/EtOAc) to give 21 (361 mg, 94%) as a colorless
oil. IR (neat, cm-1) 2820, 1720, 1600, 1500, 1450, 1270, 710; 1H NMR
(250 MHz, CDCl3) δ 1.96 (tt, J ) 7.5, 7.5 Hz, 2H), 2.35 (t, J ) 7.5
Hz, 2H), 2.64 (t, J ) 7.5 Hz, 2H), 4.96 (s, 2H), 5.27 (s, 2H), 6.51 (s,
1H), 7.14-7.47 (m, 9H), 8.03-8.06 (m, 2H); 13C NMR (125 MHz,
CDCl3) δ 26.4, 33.5, 35.1, 57.5, 58.4, 111.6, 121.5, 126.0, 128.4, 128.4,
128.5, 129.6, 129.8, 133.1, 141.3, 142.1, 150.4, 166.2, 173.2; LRMS
(CI, m/z) 379 (M+ + 1), 163, 105. HRMS (EI, m/z) calcd for C23H22O5,
378.1467; found, 378.1463.
1
1740, 1500, 1460, 1260, 1200, 1120, 1023, 754, 692; H NMR (250
MHz, CDCl3) δ 1.96 (tt, J ) 7.5, 7.5 Hz, 2H), 2.34 (t, J ) 7.5 Hz,
2H), 2.64 (t, J ) 7.5 Hz, 2H), 4.58 (s, 2H), 4.94 (s, 2H), 6.33 (s, 1H),
7.14-7.31 (m, 5H), 7.43 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 26.4,
33.6, 35.1, 57.5, 57.6, 108.7, 121.3, 126.0, 128.4, 128.5, 141.3, 141.5,
154.9, 173.3; LRMS (EI, m/z) 274 (M+), 257, 163; HRMS (EI, m/z)
calcd for C16H18O4, 274.1205; found, 274.1199.
To a solution of 2-formyl-3-(4-phenylbutanoyloxymethyl)furan (1.46
g, 5.35 mmol) in methanol-THF-acetic acid (30.8 mL, 20:20:1 v/v)
was added NaBH3CN (672 mg, 10.7 mmol) at 0 °C. The mixture was
stirred for 18 h at room temperature, then poured into saturated aqueous
sodium bicarbonate (10 mL) at 0 °C, and 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 silica gel column chromatography (3:1 hexane/EtOAc) to give 18
(1.21 g, 83%) as a colorless oil. IR (neat, cm-1) 3450, 2940, 2853,
2-Benzoyloxymethyl-3-(4-phenylbutanoyloxymethyl)furan (22).
To a solution of 2-hydroxymethyl-3-(4-phenylbutanoyloxymethyl)furan
18 (1.02 g, 3.72 mmol), Et3N (1.04 mL, 7.44 mmol), and DMAP (45.0
mg, 0.34 mmol) in dichloromethane (30 mL) was added benzoyl
chloride (573 µL, 4.09 mmol) at 0 °C, and the mixture was stirred for
23 h at room temperature. The reaction was quenched with 1 N HCl
(5 mL) at 0 °C and extracted with dichloromethane. The organic layer
was washed with saturated aqueous sodium bicarbonate, water, and
brine, dried over anhydrous MgSO4, and concentrated in vacuo. The
crude product was purified by silica gel column chromatography (4:1
hexane/EtOAc) to give 22 (1.19 g, 95%) as a colorless oil. IR (neat,
cm-1) 2940, 2853, 1740, 1600, 1500, 1460, 1260, 1140, 1100, 940,
1
1740, 1600, 1500, 1450, 1260, 1200, 1120, 1023, 754, 692; H NMR
(250 MHz, CDCl3) δ 1.84 (tt, J ) 7.5, 7.5 Hz, 2H), 2.23 (t, J ) 7.5
Hz, 2H), 2.52 (t, J ) 7.5 Hz, 2H), 4.59 (s, 2H), 4.93 (s, 2H), 6.30 (d,
J ) 1.8 Hz, 1H), 7.04-7.26 (m, 5H), 7.27 (d, J ) 1.8 Hz, 1H); 13C
NMR (125 MHz, CDCl3) δ 26.3, 33.6, 35.0, 55.5, 57.5, 111.6, 117.0,
126.0, 128.4, 141.2, 142.2, 152.7, 174.0; LRMS (EI, m/z) 274 (M+),
257, 163, 111. HRMS (EI, m/z) calcd for C16H18O4, 274.1205; found,
274.1213.
1
754, 792; H NMR (250 MHz, CDCl3) δ 1.93 (tt, J ) 7.5, 7.5 Hz,
2H), 2.32 (t, J ) 7.5 Hz, 2H), 2.61 (t, J ) 7.5 Hz, 2H), 5.10 (s, 2H),
5.38 (s, 2H), 6.43 (d, J ) 1.8 Hz, 1H), 7.12-7.55 (m, 9H), 8.01-8.05
(m, 2H); 13C NMR (125 MHz, CDCl3) δ 26.4, 33.5, 35.0, 56.5, 57.1,
111.8, 120.1, 126.0, 128.3, 128.3, 128.4, 129.7, 129.8, 133.1, 141.3,
143.0, 147.7, 166.1, 173.2. LRMS (CI, m/z) 379 (M+ + 1), 257. HRMS
(EI, m/z) calcd for C23H22O5, 378.1467; found, 378.1423.
2,4-Di(benzoyloxymethyl)furan (19). To a solution of 15 (273 mg,
1.18 mmol), Et3N (328 µL, 2.36 mmol), and DMAP (14.2 mg, 0.12
mmol) in dry dichloromethane (6.8 mL) was added benzoyl chloride
(181 µL, 1.29 mmol) at 0 °C, and the reaction mixture was stirred at
room temperature for 1.5 h. After addition of 1 N HCl (2 mL), the
resulting mixture was extracted with dichloromethane. The organic layer
was washed with water and brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The crude product was purified by silica gel
column chromatography (2:1 hexane/EtOAc) to give 19 (375 mg, 95%)
as a pale yellow oil. IR (neat, cm-1) 2950, 1720, 1610, 1460, 1280,
1,5-Dibenzoyloxymethyl-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicar-
boxylic Anhydride (23). A solution of 19 (355 mg, 1.06 mmol) and
maleic anhydride (310 mg, 3.18 mmol) in toluene (2 mL) was stirred
at room temperature for 12 h. After addition of further maleic anhydride
(87.5 mg, 0.89 mmol), the reaction mixture was stirred at room
temperature for 183 h. The solvent was evaporated under reduced
pressure, and the crude compound was purified by silica gel column
chromatography (4:1 hexane/EtOAc) to give 23 (297 mg, 64%) as a
colorless oil. IR (neat, cm-1) 3100, 2975, 1865, 1830, 1785, 1720, 1700,
1600, 1580, 1450, 1270, 1120, 710, 680; 1H NMR (400 MHz, CDCl3)
δ 3.36 (d, J ) 6.8 Hz, 1H), 3.57 (d, J ) 6.8 Hz, 1H), 4.79 (d, J ) 13.0
Hz, 1H), 5.03 (d, J ) 1.7 Hz, 2H), 5.16 (d, J ) 13.0 Hz, 1H), 5.47 (s,
1H), 6.42 (d, J ) 1.7 Hz, 1H), 7.43-7.49 (m, 4H), 7.59-7.61 (m,
2H), 8.01-8.07 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 50.7, 51.1,
1
1100; H NMR (250 MHz, CDCl3) δ 5.14 (s, 2H), 5.21 (s, 2H), 6.53
(s, 1H), 7.32 (s, 1H), 7.33-7.45 (m, 4H), 7.48-7.61 (m, 2H), 7.92-
7.96 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 58.1, 58.4, 111.7, 121.5,
128.9, 129.7, 129.8, 129.8, 130.1, 130.6, 133.1, 133.1, 150.4, 162.3,
166.2, 166.4; LRMS (EI, m/z) 336 (M+), 105. HRMS (EI, m/z) calcd
for C20H16O5, 336.0998; found, 336.1023.
2,3-Di(benzoyloxymethyl)furan (20). To a mixture of 16 (3.27 g,
14.1 mmol), Et3N (3.92 mL, 28.2 mmol), and DMAP (172 mg, 1.41
mmol) in dry dichloromethane (30 mL) was added benzoyl chloride
(2.45 mL, 21.2 mmol) at 0 °C, and the reaction mixture was stirred at
9
J. AM. CHEM. SOC. VOL. 125, NO. 32, 2003 9747

A R T I C L E S
Baba et al.
1
59.3, 61.0, 82.8, 91.8, 128.5, 128.6, 129.0, 129.2, 129.7, 129.8, 133.1,
133.4, 133.6, 148.2, 165.9, 166.0, 167.8, 169.3; LRMS (EI, m/z) 337,
307, 105.
3100, 2950, 1865, 1780, 1730, 1600, 1500, 1450, 1270, 920, 710; H
NMR (250 MHz, CDCl3) δ 1.97 (tt, J ) 7.5, 7.5 Hz, 2H), 2.37 (t, J )
7.5 Hz, 2H), 2.66 (t, J ) 7.5 Hz, 2H), 3.32 (d, J ) 7.0 Hz, 1H), 3.46
(d, J ) 7.0 Hz, 1H), 4.75 (s, 2H), 4.77 (d, J ) 12.8 Hz, 1H), 5.16 (d,
J ) 12.8 Hz, 1H), 5.36 (s, 1H), 6.33 (s, 1H), 7.15-7.59 (m, 8H), 8.02-
8.06 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 26.2, 33.2, 35.0, 50.7,
51.0, 58.8, 61.0, 82.7, 91.7, 126.0, 128.4, 128.5, 129.2, 129.8, 132.9,
133.5, 140.9, 165.8, 167.7, 169.3, 172.9; LRMS (EI, m/z) 378, 163,
105.
1,5-Dibenzoyloxymethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicar-
boxylic Anhydride (9). To a solution of 23 (83.2 mg, 1.91 mmol) in
Et2O (2 mL) and THF (1 mL) was added 5% Rh/Al2O3 (15 mg). The
reaction mixture was stirred at room temperature under a hydrogen
atmosphere, filtered through Celite, and concentrated in vacuo. The
oily crude product was taken up in hexanes-EtOAc (2 mL 5:1 v/v) to
afford a precipitate of a white solid. This product was purified by gel
permeation chromatography to give 9 (21.8 mg, 26%) as colorless
crystals. Mp 149-152 °C; IR (KBr, cm-1) 2970, 1865, 1830, 1785,
1720, 1600, 1580, 1450, 1270, 1120, 710; 1H NMR (500 MHz, CDCl3)
δ 1.47 (dd, J ) 12.4, 5.5 Hz, 1H), 2.28 (ddd, J ) 12.4, 12.4, 5.5 Hz,
1H), 2.93-2.96 (m, 1H), 3.33 (d, J ) 7.5 Hz, 1H), 3.78 (d, J ) 7.5
Hz, 1H), 4.26 (dd, J ) 11.8, 9.4 Hz, 1H), 4.52 (dd, J ) 11.8, 5.8 Hz,
1H), 4.89 (d, J ) 12.8 Hz, 1H), 4.95 (d, J ) 12.8 Hz, 1H), 5.10 (d, J
) 4.9 Hz, 1H), 7.45-7.50 (m, 4H), 7.60-7.62 (m, 2H), 8.02-8.06
(m, 4H); 13C NMR (125 MHz, CDCl3) δ 33.6, 39.7, 46.5, 50.6, 61.2,
62.5, 80.8, 87.2, 127.5, 127.7, 128.2, 128.2, 128.6, 128.9, 132.4, 132.6,
164.7, 165.2, 167.7, 170.1; LRMS (CI, m/z) 437 (M+ + 1), 315, 123,
105. Anal. Calcd for C24H20O8: C, 66.05; H, 4.62. Found: C, 65.82;
H, 4.69.
1-Benzoyloxymethyl-5-(4-phenylbutanoyloxymethyl)-7-oxabicyclo-
[2.2.1]heptane-2,3-dicarboxylic Anhydride (11). To a solution of 25
(200 mg, 0.42 mmol) in Et2O (2 mL) and THF (2 mL) was added 5%
Rh/Al2O3 (15 mg). The reaction mixture was stirred at room temperature
under a hydrogen atmosphere, filtered through Celite, and concentrated
in vacuo to give 11 (125 mg, 62%) as a white solid. Mp 100-103 °C;
IR (KBr, cm-1) 2950, 1865, 1780, 1730, 1600, 1500, 1450, 1270, 1100,
980, 920, 760, 710; 1H NMR (250 MHz, CDCl3) δ 1.34 (dd, J ) 12.5,
5 Hz, 1H), 1.92 (tt, J ) 7.5, 7.5 Hz, 2H), 2.24-2.37 (m, 3H), 2.63 (t,
J ) 7.5 Hz, 2H), 2.70-2.79 (m, 1H), 3.30 (1H, d, J ) 7.5 Hz), 3.54
(d, J ) 7.5 Hz, 1H), 3.97 (dd, J ) 12.5, 7.5 Hz, 1H), 4.34 (dd, J )
12.5, 7.5 Hz, 1H), 4.77 (d, J ) 12.0 Hz, 1H), 4.95 (d, J ) 12.0 Hz,
1H), 5.00 (d, J ) 5.0 Hz, 1H), 7.12-7.30 (m, 5H), 7.42-7.48 (m,
2H), 7.55-7.58 (m, 1H), 8.00-8.03 (m, 2H); 13C NMR (125 MHz,
CDCl3) δ 26.1, 32.8, 33.3, 34.9, 40.3, 47.1, 51.8, 61.9, 62.8, 79.7, 89.1,
126.1, 128.4, 128.5, 129.3, 129.7, 133.4, 141.0, 146.8, 165.5, 169.4,
170.4, 172.9; LRMS (EI, m/z) 478, 315, 147, 105. HRMS (EI, m/z)
calcd for C27H26O8, 478.1628; found, 478.1641.
1,6-Dibenzoyloxymethyl-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicar-
boxylic Anhydride (24). A solution of 20 (370 mg, 1.10 mmol) and
maleic anhydride (324 mg, 3.30 mmol) in toluene (2 mL) was stirred
at room temperature for 68 h. After addition of further maleic anhydride
(108 mg, 1.10 mmol), the reaction mixture was stirred at room
temperature for 216 h. The solvent was evaporated under reduced
pressure, and the crude compound was purified by silica gel column
chromatography (4:1 hexane/EtOAc) to give 24 (305 mg, 63%) as a
colorless oil. IR (neat, cm-1) 2820, 1865, 1830, 1720, 1705, 1600, 1580,
1-Benzoyloxymethyl-6-(4-phenylbutanoyloxymethyl)-7-oxabicyclo-
[2.2.1]hept-5-ene-2,3-dicarboxylic Anhydride (26). A solution of 22
(487 mg, 1.29 mmol) and maleic anhydride (379 mg, 3.86 mmol) in
toluene (2 mL) was stirred at room temperature for 136 h. The solvent
was evaporated under reduced pressure, and the crude compound was
purified by silica gel column chromatography (7:2 hexane/EtOAc) to
give 26 (314 mg, 51%) as a colorless oil. IR (neat, cm-1) 2950, 1865,
1
1450, 1270, 1120, 710; H NMR (250 MHz, CDCl3) δ 3.41 (d, J )
6.9 Hz, 1H), 3.55 (d, J ) 6.9 Hz, 1H), 5.05 (m, 4H), 5.48 (d, J ) 2.0
Hz, 1H), 6.54 (d, J ) 2.0 Hz, 1H), 7.40-7.48 (m, 4H), 7.55-7.63 (m,
2H), 7.96-8.03 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 49.7, 52.3,
59.1, 60.7, 82.1, 91.0, 128.6, 128.7, 129.0, 129.1, 129.6, 129.8, 133.5,
133.7, 134.5, 146.9, 165.5, 165.9, 167.8, 169.1; LRMS (EI, m/z) 337,
307.
1
1780, 1730, 1600, 1500, 1450, 1270, 1100, 920, 710; H NMR (250
MHz, CDCl3) δ 1.92 (tt, J ) 7.5, 7.5 Hz, 2H), 2.32 (t, J ) 7.5 Hz,
2H), 2.62 (t, J ) 7.5 Hz, 2H), 3.37 (d, J ) 7.0 Hz, 1H), 3.43 (d, J )
7.0 Hz, 1H), 4.79 (s, 2H), 4.98 (s, 2H), 5.45 (d, J ) 1.75 Hz, 1H),
6.45 (d, J ) 1.75 Hz, 1H), 7.13-7.59 (m, 8H), 7.98-8.02 (m, 2H);
13C NMR (125 MHz, CDCl3) δ 33.2, 35.0, 49.6, 52.3, 58.6, 60.6, 82.0,
90.9, 126.2, 128.4, 128.5, 128.6, 129.1, 129.7, 133.5, 134.5, 140.9,
146.8, 165.4, 167.8, 169.1, 172.8; LRMS (EI, m/z) 379, 105.
1,6-Dibenzoyloxymethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicar-
boxylic Anhydride (10). To a solution of 24 (94.4 mg, 220 mmol) in
THF (1.5 mL) was added 5% Rh/Al2O3 (10 mg), and the mixture was
stirred at room temperature under a hydrogen atmosphere. The reaction
mixture was filtered through Celite and concentrated in vacuo. The
oily crude product was taken up in 2 mL of hexane/EtOAc (4:1) to
afford a precipitate of 10 (78.3 mg, 83%) as a colorless solid. Mp 75-
78 °C; IR (KBr, cm-1) 2820, 1865, 1830, 1785, 1720, 1600, 1580,
1450, 1270, 1120, 755, 710, 680; 1H NMR (500 MHz, CDCl3) δ 1.47
(dd, J ) 12.4, 5.5 Hz, 1H), 2.89 (ddd, J ) 12.4, 12.4, 5.5 Hz, 1H),
2.95-2.99 (m, 1H), 3.39 (d, J ) 7.5 Hz, 1H), 3.67 (1H, d, J ) 7.5 Hz,
1H), 4.29 (dd, J ) 12.0, 9.3 Hz, 1H), 4.61 (dd, J ) 12.0, 5.5 Hz, 1H),
4.87 (d, J ) 13.0 Hz, 1H), 4.95 (d, J ) 13.0 Hz, 1H), 5.04 (d, J ) 5.5
Hz, 1H), 7.40-7.47 (m, 4H), 7.54-7.61 (m, 2H), 7.95-8.02 (m, 4H);
13C NMR (125 MHz, CDCl3) δ 32.8, 40.6, 47.2, 51.9, 62.0, 63.5, 79.8,
89.5, 128.5, 128.7, 129.0, 129.2, 129.5, 129.7, 133.3, 133.6, 165.6,
166.1, 169.4, 170.5; LRMS (EI, m/z) 436 (M+) 315, 122, 105. HRMS
(EI, m/z) calcd for C24H20O8, 436.1158; found, 436.1140.
1-Benzoyloxymethyl-6-(4-phenylbutanoyloxymethyl)-7-oxabicyclo-
[2.2.1]heptane-2,3-dicarboxylic Anhydride (12). To a solution of 26
(249 mg, 0.52 mmol) in Et2O (3 mL) and THF (3 mL) was added 5%
Rh/Al2O3 (15 mg). The reaction mixture was stirred at room temperature
under a hydrogen atmosphere, filtered through Celite, and concentrated
in vacuo to give 12 (250 mg, quant.) as colorless crystals. Mp 91-93
°C; IR (KBr, cm-1) 2950, 1865, 1780, 1730, 1600, 1500, 1450, 1270,
1
1100, 980, 920, 760, 710; H NMR (250 MHz, CDCl3) δ 1.34 (dd, J
) 12.5, 5.0 Hz, 1H), 1.92 (tt, J ) 7.5, 7.5 Hz, 2H), 2.24-2.37 (m,
3H), 2.63 (t, J ) 7.5 Hz, 2H), 2.70-2.79 (m, 1H), 3.30 (d, J ) 7.5
Hz, 1H), 3.54 (d, J ) 7.5 Hz, 1H), 3.97 (dd, J ) 12.5, 7.5 Hz, 1H),
4.34 (dd, J ) 12.5, 7.5 Hz, 1H), 4.77 (d, J ) 12 Hz, 1H), 4.95 (d, J
) 12 Hz, 1H), 5.00 (d, J ) 5.0 Hz, 1H), 7.12-7.30 (m, 5H), 7.42-
7.48 (m, 2H), 7.55-7.58 (m, 1H), 8.00-8.03 (m, 2H); 13C NMR (125
MHz, CDCl3) δ 26.1, 32.8, 33.3, 34.9, 40.3, 47.1, 51.8, 61.9, 62.8,
79.7, 89.1, 126.1, 128.4, 128.5, 129.3, 129.7, 133.4, 141.0, 146.8, 165.5,
169.4, 170.4, 172.9; LRMS (EI, m/z) 478, 315, 147, 105. HRMS (EI,
m/z) calcd for C27H26O8, 478.1628; found, 478.1641.
1-Benzoyloxymethyl-5-(4-phenylbutanoyloxymethyl)-7-oxabicyclo-
[2.2.1]hept-5-ene-2,3-dicarboxylic Anhydride (25). A solution of 21
(350 mg, 0.93 mmol) and maleic anhydride (272 mg, 2.78 mmol) in
toluene (0.5 mL) was stirred at room temperature for 17 h. After further
addition of maleic anhydride (90.7 mg, 0.93 mmol) and toluene (0.5
mL), the reaction mixture was stirred at room temperature for 72 h.
The solvent was evaporated under reduced pressure, and the crude
product was purified by silica gel column chromatography (3:1 hexane/
Construction of the Binding Model. To construct a binding model
of norcantharidin 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 model was constructed in the Affinity module of
the Insight II molecular modeling program developed by MSI (now
EtOAc) to give 25 (297.2 mg, 68%) as a colorless oil. IR (neat, cm-1
)
9
9748 J. AM. CHEM. SOC. VOL. 125, NO. 32, 2003

Inhibitor of Ser/Thr Protein Phosphatase 2B (Calcineurin)
A R T I C L E S
succeeded by Accelrys, San Diego). Affinity is a program for
automatically docking a ligand to a receptor using a combination of
Monte Carlo type and simulated annealing procedures. The molecular
mechanics program employed in Affinity is the Discover 3 module in
conjunction with the force fields supported by that program. In the
first orientation, we constructed a “grid” which is partitioned into bulk
(nonflexible) and movable atoms at the ligand/receptor system; interac-
tions among bulk atoms are approximated by using the accurate and
efficient molecular mechanical/grid (MM/Grid) method, while interac-
tions among movable atoms are treated using a full force field
representation. The grid was defined by the amino acid residues within
6 Å around the phosphate ion, including in the catalytic site of PP2B.
The initial placements of norcantharidin within the PP2B catalytic site
were made using a Monte Carlo type procedure to search both
conformational and Cartesian space. Second, a simulated annealing
phase optimized each ligand placement. The structures were then
subjected to energy minimization based on molecular dynamics.
Protein Phosphatase Inhibition Assays. Phosphatases PP1 (rabbit
skeletal muscle), PP2A (rabbit skeletal muscle, composed of R, â and
catalytic subunit), and PP2B (bovine brain) were purchased from UBI
(Upstate Biotechnology Inc.). PTP1B was purchased from BIOMOL
(BIOMOL Research Laboratories Inc). Cyclophilin A (calf thymus)
and cyclosporin A were purchased from SIGMA and CALBIOCHEM,
respectively. Protein phosphatase assays were carried out according to
the UBI protocol in the presence or absence of the appropriate
concentration of a cantharidin derivative. For PP1 and PP2A assays,
the Ser/Thr Phosphatase Assay Kit 1 (UBI) was used, in which free
phosphate ion released from a substrate phosphopeptide (KRpTIRR)
is quantified by colorimetric analysis (630 nm) using the Malachite
Green method (enzyme concentrations: 4 units/mL PP1; 56 nM PP2A).
For PP2B inhibition assays shown in the Figure 7C, RII phosphopeptide
(DLDVPIPGRFDRRVpSVAAE, BIOMOL)18 was used as a substrate.
Briefly, the phosphopeptide (90 µM) was incubated with PP2B (230
nM) at 37 °C (in 40 mM Tris‚HCl buffer pH 7.5, 0.1 M KCl, 6 mM
MgCl2, 0.1 mM CaCl2, 0.1 mg/mL BSA, 0.05 mM DTT, 0.25 µM
calmodulin) in the absence or presence of inhibitors. For evaluation of
the cyclosporin A-cyclophilin A complex, concentration of cyclophilin
A was varied in the presence of 10 µM cyclosporin A. p-Nitrophenyl
phosphate (pNPP) was used as a substrate for the other preliminary
PP2B assays (in 50 mM Tris‚HCl buffer pH 7.0, 0.1 mM CaCl2, 2.5
mM NiCl2, 0.3 mg/mL BSA, 0.25 µM calmodulin, 2.4 mM pNPP, 57
nM PP2B) and PTP1B assays (in 50 mM Hepes buffer pH 7.2, 50
mM NaCl, 1.0 mM EDTA, 1.0 mM DTT, 1.0 mg/mL BSA, 8.0 nM
PTP1B). Hydrolysis of the substrate was followed in terms of the
absorption of the hydrolysis product, p-nitrophenol (415 nm).
Acknowledgment. This work was supported in part by grants
from The Naito Foundation, Suzuken Memorial Foundation, and
Hoh-ansha Foundation and by a Grant-in-Aid for Creative
Scientific Research (No. 13NP0401) and a Grant-in-Aid for
Scientific Research from the Japan Society for the Promotion
of Science, a Grant-in-Aid for Scientific Research on Priority
Areas (A), and a Grant-in-Aid for the COE project, Giant
Molecules and Complex Systems, from The Ministry of
Education, Culture, Sports, Science and Technology, as well
as the Chugai Pharmaceutical Award in Synthetic Organic
Chemistry.
JA034694Y
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J. AM. CHEM. SOC. VOL. 125, NO. 32, 2003 9749