A comparison of cyclohexanone and tetrahydro-4H-thiopyran-4-one 1,1-dioxide as pharmacophores for the design of peptide-based inhibitors of the serine protease plasmin

Article abstract of DOI:10.1021/jo0508954

The plasminogen system is important in the proteolytic cascade that facilitates angiogenesis, a process that is essential for tumor growth and metastasis. The serine protease plasmin has a central role in the plasminogen system. This protease acts by degr

Full text of DOI:10.1021/jo0508954

A Comparison of Cyclohexanone and
Tetrahydro-4H-thiopyran-4-one 1,1-Dioxide as Pharmacophores for
the Design of Peptide-Based Inhibitors of the Serine
Protease Plasmin
Fengtian Xue and Christopher T. Seto*
Department of Chemistry, Brown University, 324 Brook Street, Box H, Providence, Rhode Island, 02912
christopher_Seto@brown.edu
Received May 4, 2005
The plasminogen system is important in the proteolytic cascade that facilitates angiogenesis, a
process that is essential for tumor growth and metastasis. The serine protease plasmin has a central
role in the plasminogen system. This protease acts by degrading several components of the basement
membrane and by activating other proteases. Therefore, inhibition of plasmin may be an effective
method for blocking angiogenesis and, as a result, inhibiting the growth of primary tumors and
secondary metastases. Three pairs of plasmin inhibitors were synthesized to compare the relative
potency of inhibitors that are based upon a cyclohexanone or a tetrahydro-4H-thiopyran-4-one 1,1-
dioxide nucleus. Compounds 1, 3, and 5 were cyclohexanone-based inhibitors, whereas compounds
2, 4, and 6 were tetrahydro-4H-thiopyran-4-one 1,1-dioxide-based inhibitors. Compounds 5 and 6
are reasonable inhibitors with IC50 values of 25 and 5.5 µM, respectively. Comparisons of the IC50
values of the three pairs show that the electron-withdrawing sulfone functional group is a beneficial
element for the design of plasmin inhibitors. The presence of the sulfone increases inhibitor potency
by a factor of 3-5 when compared to inhibitors that are based upon a simple cyclohexanone core.
Introduction
formation of secondary metastases.4-6 Angiogenesis is
mediated by a proteolytic cascade that involves a large
number of enzymes. Components of the plasminogen
activator (PA)-plasmin system and matrix metallopro-
teases (MMPs) play critical roles in this proteolytic
cascade.7 Hence, therapeutic strategies that are aimed
at inhibiting these enzymes may have significant poten-
tial for the treatment of cancer and other diseases that
are dependent on angiogenesis.
Angiogenesis, the growth of new blood vessels from pre-
existing ones, is critical for the development of cancer.1,2
Without a blood supply, tumors are usually limited in
size to 1-2 mm3.3 Growth of a tumor beyond this size is
angiogenesis-dependent, since vascularization of the
tumor is required to supply it with oxygen and nutrients
and to dispose of waste products. As a result, angiogen-
esis allows the tumor to grow rapidly beyond a micro-
scopic size and also provides cancer cells with a route to
spread to other parts of the body.
Plasmin is a serine protease that is a primary con-
stituent of the proteolytic cascade. This protease degrades
(4) Ingber, D.; Fujita, T.; Kishimoto, S.; Sudo, K.; Kanamaru, T.;
Brem, H.; Folkman, J. Nature 1990, 348, 555.
(5) Cao, Y.; O’Reilly, M. S.; Flynn, E.; Ji, R.; Folkman, J. J. Clin.
Invest. 1998, 101, 1055.
(6) Reisfeld, R. A.; Niethammer, A. G.; Luo, Y.; Xiang, R. Int. Arch.
Allergy Immunol. 2004, 133, 295.
(7) Rakic, J. M.; Maillard, C.; Jost, M.; Bajou, K.; Masson, V.; Devy,
L.; Lambert, V.; Foidart, J. M.; Noe¨l, A. Cell. Mol. Life Sci. 2003, 60,
463.
In recent decades, Folkman and several other research
groups have demonstrated that angiogenesis inhibitors
can impede both the growth of primary tumors and the
(1) Folkman, J. N. Engl. J. Med. 1971, 285, 1182.
(2) Carmeliet, P.; Jain, R. K. Nature 2000, 407, 249.
(3) Folkman, J. N. Engl. J. Med. 1995, 333, 1757.
10.1021/jo0508954 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/14/2005
J. Org. Chem. 2005, 70, 8309-8321
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Xue and Seto
a wide variety of substrates and components of the
extracellular matrix including fibrin, glycoproteins such
as laminin and fibronectin, and proteoglycans. It acti-
vates other proteases such as the pro-metalloproteinases
MMP-1, MMP-3, and MMP-9.8 Additionally, plasmin
activates or releases a number of growth factors from the
extracellular matrix including latent transforming growth
factor (TGF-â), basic fibroblast growth factor (bFGF), and
vascular endothelial growth factor (VEGF).8 Thus, plas-
min plays a vital role in the breakdown of the basement
membrane and facilitates angiogenesis and the migration
of cancer cells. Because of these features, plasmin is a
potential target for the development of chemotherapeutic
agents that could block degradation of the basement
membrane and the activation of other enzymes. Such
activity, in turn, could lead to the inhibition of angio-
genesis and metastasis.
Considerable advances have been made in the design
of inhibitors for plasmin and other related serine pro-
teases. Currently, there are two primary families of
inhibitors for plasmin. They are classified by the different
domains within the enzyme that are targeted by the
inhibitors. The first family targets the lysine binding site
of plasmin and includes inhibitors such as ꢀ-aminocaproic
acid, p-aminomethylbenzoic acid, and trans-4-aminocy-
clohexane carboxylic acid, several of which have been
used in clinical applications.9 The lysine binding site
mediates the interaction between plasmin and fibrin,
which is a key step in the processes of fibrinolysis and
the generation of active plasmin from its inactive precur-
sor plasminogen.10 This family of inhibitors effectively
inhibits these processes. However, these inhibitors cannot
block the activity of plasmin that has already been
activated by cleavage of plasminogen. The second family
of inhibitors, which are targeted to the active site of
plasmin, can overcome this limitation. They act by
blocking the catalytic activity of the serine residue in the
active site of the enzyme.11
FIGURE 1. Enhancement of the electrophilicity of the ketone
by introducing an electronegative sulfone group into the
cyclohexanone ring system. R ) H or (CH2)6NH2.
valent hemiketal bond with the active site serine residue
of the protease. The electrophilicity of the ketone, and
thus the potency of the inhibitors, can be enhanced by
incorporating an electronegative functional group in place
of the C-4 atom of the cyclohexanone ring. The dipole of
the electronegative group induces a through-space elec-
trostatic repulsion with the dipole of the ketone.14-16 This
unfavorable electrostatic interaction destabilizes the
ketone and makes it more susceptible to attack by
nucleophiles, including the serine hydroxyl group in the
active site of plasmin (Figure 1). In previous studies, we
demonstrated that cyclohexanone-based inhibitors react
with the active site cysteine residue of papain to give a
reversibly formed hemithioketal linkage.14b
Herein, we report the synthesis and evaluation of three
pairs of inhibitors for plasmin (compounds 1-6, Figure
2). The inhibitors in each pair have identical structures,
except that one inhibitor in each pair is based upon a
cyclohexanone nucleus, whereas in its partner the C-4
carbon of the cyclohexanone ring has been replaced by
an SO2 group. These pairs of inhibitors were designed to
test how effective the sulfone functional group is at
enhancing the electrophilicity of the ketone moiety via a
through-space electrostatic interaction and to gauge the
influence of the sulfone group on the potency of the
inhibitors. The cyclohexanone or tetrahydro-4H-thiopy-
ran-4-one 1,1-dioxide nucleus of the inhibitors is deriva-
tized with one or two peptide appendages. These peptides
target the inhibitors to the enzyme active site and are
designed to correctly align the electrophilic ketone moiety
of the inhibitor for reaction with the active site serine
nucleophile.
Design of the Inhibitors. Over the last several years,
we have been investigating active site-directed inhibitors
for plasmin that are based upon a 4-heterocyclohexanone
nucleus.12-14 These inhibitors contain an electrophilic
ketone moiety that is designed to form a reversible co-
Compounds 1 and 2 are the simplest of the three pairs
of inhibitors. They incorporate a single Trp residue to
bind in the S2 subsite of the enzyme.17 To increase the
number of contacts between the inhibitors and the
enzyme, we synthesized a second inhibitor pair, 3 and 4,
which employ two binding elements in their peptide-
based side chains. First, the aminohexyl group mimics
the side chain of positively charged amino acids such as
Lys and Arg. This group is targeted to the S1 subsite,
since it is know that trypsin-like serine proteases prefer
to bind substrates with positively charged amino acids
at the P1 position.10 Second, the dipeptide D-Ile-Phe was
chosen to interact with the S3 and S2 subsites of plasmin,
based upon work that has been published by Okada and
(8) Noe¨l, A.; Gilles, C.; Bajou, K.; Devy, L.; Kebers, F.; Lewalle, J.
M. Invasion Metastasis 1997, 17, 221.
(9) (a) Okada, Y.; Tsuda, Y.; Teno, N.; Wanaka, K.; Bohgaki, M.;
Hijikata-Okunomiya, A.; Naito, T.; Okamoto, S. Chem. Pharm. Bull.
1988, 36, 1289. (b) Okamoto, S. Keio J. Med. 1959, 8, 211. (c) Okamoto,
S.; Sato, S.; Tanaka, Y.; Okamoto, U. Keio J. Med. 1964, 13, 177.
(10) (a) Sherry, S. Fibrinolysis, Thrombosis, and Hemostasis; Lea
& Febiger: Philadelphia, 1992; Chapter 1. (b) Backes, B. J.; Harris,
J. L.; Leonetti, F.; Craik, C. S.; Ellman, J. A. Nat. Biotechnol. 2000,
18, 187. (c) Harris, J. L.; Backes, B. J.; Leonetti, F.; Nahrus, S.; Ellman,
J. A.; Craik, C. S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7754.
(11) For other active site-directed inhibitors of plasmin, see: (a)
Teno, N.; Wanaka, K.; Okada, Y.; Tsuda, Y.; Okamoto, U.; Hijikata-
Okunomiya, A.; Naito, T.; Okamoto, S. Chem. Pharm. Bull. 1991, 39,
2340. (b) Teno, N.; Wanaka, K.; Okada, Y.; Taguchi, H.; Okamoto, U.;
Hijikata-Okunomiya, A.; Okamoto, S. Chem. Pharm. Bull. 1993, 41,
1079. (c) Wanaka, K.; Okamoto, S.; Horie, N.; Hijikata-Okunomiya,
A.; Okamoto, U.; Naito, T.; Ohno, N.; Bohgaki, M.; Tsuda, Y.; Okada,
Y. Thromb. Res. 1996, 82, 79. (d) Tamura, S. Y.; Goldman, E. A.;
Brunck, T. K.; Ripka, W. C.; Semple, J. E. Bioorg. Med. Chem. Lett.
1997, 7, 331. See also ref 9.
(15) (a) Roberts, J. D.; Moreland, W. T., Jr. J. Am. Chem. Soc. 1953,
75, 2167. (b) Holtz, H. D.; Stock, L. M. J. Am. Chem. Soc. 1964, 86,
5188. (c) Geneste, P.; Durand, R.; Hugon, I.; Reminiac, C. J. Org. Chem.
1979, 44, 1971. (d) Das, G.; Thornton, E. R. J. Am. Chem. Soc. 1993,
115, 1302. (e) Burkey, T. J.; Fahey, R. C. J. Org. Chem. 1985, 50, 1304.
(16) Chen, C.-T.; Siegel, J. S. J. Am. Chem. Soc. 1994, 116, 5959.
(17) Abato, P.; Conroy, J. L.; Seto, C. T. J. Med. Chem. 1999, 42,
4001.
(12) Sander, T. C.; Seto, C. T. J. Med. Chem. 1999, 42, 2969.
(13) Abato, P.; Yuen, C. M.; Cubanski, J. Y.; Seto, C. T. J. Org. Chem.
2002, 67, 1184.
(14) (a) Conroy, J. L.; Sanders, T. C.; Seto, C. T. J. Am. Chem. Soc.
1997, 119, 4285. (b) Conroy, J. L.; Seto, C. T. J. Org. Chem. 1998, 63,
2367.
8310 J. Org. Chem., Vol. 70, No. 21, 2005

Design of Inhibitors of the Serine Protease Plasmin
FIGURE 2. Structures of inhibitors 1-6. One of two diastereomers is shown for each inhibitor.
SCHEME 1. Synthesis of Inhibitor 1a
Results and Discussion
Inhibitor 1. The synthesis of inhibitor 1 (Scheme 1)
began with coupling of primary amine 7 with Cbz-Trp-
OH employing a standard peptide coupling procedure
with O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU) and N,N-diisopropylethyl-
amine (DIEA) to give amide 8 as a mixture of two
diastereomers. The synthesis of compound 7 has been
reported previously.13 The ketal-protecting group in
compound 8 was removed with aqueous TFA to give
inhibitor 1.
Inhibitor 2. Inhibitor 2 was synthesized according to
the procedure shown in Scheme 2. â-Ketoester 9 was
reduced with NaBH4 to give a mixture of alcohols 10 and
11. The synthesis of compound 9 has been reported
previously.13 Compounds 10 and 11 were separated by
flash chromatography, and racemic 11 was carried
through the remainder of the synthesis. The hydroxyl
group in compound 11 was protected with TBDMSCl to
give TBDMS ether 12, which was subsequently saponi-
fied with aqueous NaOH in methanol to yield carboxylic
acid 13. Compound 13 was treated with diphenylphos-
phoryl azide in toluene at 60 °C to first generate the acyl
a Reagents: (a) Cbz-Trp-OH, HBTU, DIEA, 80%; (b) TFA, H2O,
55%. Only one of the two diastereomers is shown for compounds
8 and 1.
co-workers and work from our own laboratory.12 The final
pair of inhibitors, 5 and 6, have peptides attached on both
sides of the ketone group. One Trp reside is designed to
bind in the S2 subsite, similar to inhibitors 1 and 2. The
second Trp residue extends toward the leaving group
subsites and binds in the S2′ subsite.15 The results that
are presented in the next section comparing inhibition
constants among these three pairs of inhibitors demon-
strate that inhibitor potency is increased by a factor of
3-5 when the C-4 atom of a cyclohexanone-based inhibi-
tor is replaced by the more electronegative sulfone
functional group.
SCHEME 2. Synthesis of Inhibitor 2a
a Reagents: (a) NaBH4, 60%; (b) TBDMSCl, imidazole, 86%; (c) NaOH, MeOH, 70%; (d) (C6H5O)2PON3, DIEA; (e) BnOH, n-BuLi, 84%
(two steps); (f) NaIO4, KMnO4, NaHCO3, 74%; (g) H2, Pd/C, 100%; (h) Cbz-Trp(Boc)-OH, HBTU, DIEA, 86%; (i) TBAF, 89%; (j) Dess-
Martin periodinane; (k) TFA, 34% (two steps). Only one of the two diastereomers is shown for compounds 17, 18, and 2.
J. Org. Chem, Vol. 70, No. 21, 2005 8311

Xue and Seto
SCHEME 3. Synthesis of Inhibitor 3a
SCHEME 4. Synthesis of Inhibitor 4a
a Reagents: (a) BocNH(CH2)5CHO, Ti(OiPr)4, followed by NaBH4;
(b) Fmoc-Phe-OH, HATU, DIEA, 31% (two steps); (c) piperidine;
(d) Boc-D-Ile-OH, HBTU, DIEA, 78% (two steps); (e) TFA; (f) TFA,
H2O, 59% (two steps). Only one of the two diastereomers is shown
for compounds 20, 21, and 3.
a Reagents: (a) BocNH(CH2)5CHO; NaBH(OAc)3, 75%; (b) BSA;
(c) Fmoc-Phe-F, catalytic TBAF, 65%; (d) piperidine; (e) Boc-D-Ile-
OH, HBTU, DIEA, 84% (two steps); (f) TBAF; (g) Dess-Martin
periodinane, 69% (two steps); (h) TFA, 70%. Only one of the two
diastereomers is shown for compounds 24, 25, 26, and 4.
azide and then to induce the Curtius rearrangement. The
resulting isocyanate was trapped with the anion of benzyl
alcohol to generate Cbz-protected amine 14. Oxidation
of the thioether in compound 14 with NaIO4 and KMnO4
gave sulfone 15. An alternate method for this oxidation
employing NaBO3 also gave a reasonable yield of the
desired sulfone.18
Catalytic hydrogenation of the Cbz-protecting group
generated primary amine 16, which was coupled with
Cbz-Trp(Boc)-OH to give amide 17 as a mixture of two
diastereomers. The TBDMS-protecting group was re-
moved using tetrabutylammonium fluoride (TBAF) to
give alcohol 18, which was oxidized back to the ketone
with Dess-Martin periodinane. Finally, the Boc-protect-
ing group on the side chain of Trp was removed by TFA
to give inhibitor 2.
a two-step procedure. First, amide 22 was treated with
N,O-bis(trimethylsilyl)acetamide (BSA) to generate the
N-TMS derivative 23.20 This compound was reacted in
situ with Fmoc-Phe-F to furnish amide 24 as a mixture
of two diastereomers. The synthesis of Fmoc-Phe-F has
been reported previously.11 The coupling reaction be-
tween secondary amine 22 and Fmoc-Phe-OH could be
accomplished using HATU at 60 °C. However, this
method gave a lower yield when compared to the proce-
dure employing the acid fluoride. The Fmoc group in
compound 24 was removed, and the resulting amine was
coupled with Boc-D-Ile-OH to give dipeptide 25. The
TBDMS ether was cleaved with TBAF, and the resulting
alcohol was oxidized to ketone 26 with Dess-Martin
periodinane. Finally, the Boc-protecting groups were
removed with TFA to give inhibitor 4.
Inhibitor 5. Unlike inhibitors 1-4, inhibitors 5 and
6 incorporate functionality that extend toward the leav-
ing group subsites of the enzyme active site. This
extension was accomplished using the strategy outlined
in Scheme 5. Compound 27 was treated with 2 equiv of
LDA to generate the corresponding dienolate, which was
alkylated with 1 equiv of allyl bromide to give compound
28.21 The ketone group in compound 28 was converted
to ketal 29 using 1,3-propanediol in the presence of
TMSCl. Compound 29 was obtained as a racemic mixture
in which the ethoxycarbonyl and the allyl groups are
oriented cis to one another.22 Saponification of the ester
group in compound 29, followed by treatment of the
resulting carboxylic acid with diphenylphosphoryl azide,
induced the Curtius rearrangement to give the corre-
sponding isocyanate. This isocyanate was treated with
t-BuOK to generate the Boc-protected amine 30. Oxida-
tive cleavage of the alkene with KMnO4 and NaIO4
resulted in a carboxylic acid, which was coupled with
Inhibitor 3. Reductive alkylation of amine 7 was
accomplished using a two-step procedure (Scheme 3).
First, the imine was formed between the primary amine
and the aldehyde BocNH(CH2)5CHO. The synthesis of
this aldehyde has been reported previously.10 We exam-
ined several dehydrating reagents to promote formation
of the imine including Ti(OiPr)4, 4 Å molecular sieves,
and MgSO4. Ti(OiPr)4 gave the highest yield of imine as
1
determined by H NMR spectroscopy. In a second step,
the resulting imine was reduced with NaBH4 to give
secondary amine 19.19 Other reducing reagents including
NaCNBH3 and NaBH(OAc)3 gave lower yields of the
desired secondary amine.
Compound 19 was coupled with Fmoc-Phe-OH using
O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroni-
um hexafluorophosphate (HATU) at 60 °C to generate
amide 20 as a mixture of two diastereomers. Substitution
of HBTU for HATU in this coupling reaction gave low
yields of the desired amide. The Fmoc-protecting group
in compound 20 was removed with piperidine, and the
resulting amine was coupled with Boc-D-Ile-OH to gener-
ate compound 21. Finally, the two Boc-protecting groups
were removed with TFA, and the ketal was hydrolyzed
using aqueous TFA to give inhibitor 3.
Inhibitor 4. In contrast to the reductive alkylation of
amine 7 (Scheme 3), amine 16 underwent smooth reduc-
tive alkylation with BocNH(CH2)5CHO10 without the need
for a dehydrating agent. In this case, NaBH(OAc)3 was
used as the reducing agent (Scheme 4). The resulting
secondary amine 22 was coupled with Fmoc-Phe-F using
(18) McKillop, A.; Kemp, D. Tetrahedron 1989, 45, 3299.
(19) Bhattacharyya, S. Tetrahedron Lett. 1994, 35, 2401.
(20) Rajeswari, S.; Jones, R. J.; Cava, M. P. Tetrahedron Lett. 1987,
28, 5099.
(21) Huckin, S. N.; Weiler, L. J. Am. Chem. Soc. 1974, 96, 1082.
(22) Conroy, J. L.; Abato, P.; Ghosh, M.; Austermuhle, M. I.; Kiefer,
M. R.; Seto, C. T. Tetrahedron Lett. 1998, 39, 8253.
8312 J. Org. Chem., Vol. 70, No. 21, 2005

Design of Inhibitors of the Serine Protease Plasmin
SCHEME 5. Synthesis of Inhibitor 5a
a Reagents: (a) LDA (2 equiv), allyl bromide, 78%; (b) 1,3-propanediol, TMSCl, 84%; (c) NaOH, MeOH; (d) (PhO)2PON3, DIEA; (e)
t-BuOK, 65% (three steps); (f) NaIO4, KMnO4, NaHCO3; (g) H-Trp-OMe, HBTU, DIEA, 80%; (h) TFA; (i) Cbz-Trp-OH, HBTU, DIEA, 77%
(two steps); (j) TFA, H2O, 67%. Only one of the two diastereomers is shown for compounds 31, 32, and 5.
H-Trp-OMe to yield amide 31 as a mixture of two
diastereomers. The Boc-protecting group was removed
with TFA, and the resulting amine was coupled with Cbz-
Trp-OH to give compound 32. Finally, the ketal-protect-
ing group was removed using aqueous TFA to give
inhibitor 5.
Inhibitor 6. The synthetic strategy that we had
employed for the preparation of inhibitor 5 did not work
for the preparation of inhibitor 6. When â-ketoester 9
(Scheme 2) was treated with 2 equiv of LDA it decom-
posed rather than giving clean formation of the dienolate.
This decomposition may have been caused by â-elimina-
tion of the thioether, although such elimination reactions
are often slow in six-membered rings.23
Since our attempts to alkylate compound 9 were
unsuccessful, we chose an alternate strategy for the
preparation of inhibitor 6 that is shown in Scheme 6.
Ketone 33 was converted to its enol ether 34 using allyl
alcohol, 2,2-dimethoxypropane, and catalytic p-toluene-
sulfonic acid in benzene at 80 °C. Without isolating the
product, the reaction solvent was switched to toluene. It
was then heated at reflux to induce a Cope rearrange-
ment to give alkene 35.24 We also prepared compound
35 by treating ketone 33 with LDA, followed by alkyla-
tion of the enolate with allyl bromide. However, the
product of this reaction was contaminated with a signifi-
cant amount of the dialkylated ketone, and this material
was difficult to separate from the desired monoalkylated
ketone.
ate and methyl chloroformate were not as satisfactory.
Reduction of compound 36 with NaBH4 gave alcohol 37
as a mixture of several diastereomers. Protection of
alcohol 37 with TBDMSCl, followed by saponification of
the ester generated carboxylic acid 38. At this point, the
diastereomers with the carboxylic acid, OTBDMS, and
allyl substituents cis to one another were separated from
the other diastereomers by flash chromatography, and
these two diastereomers were carried on through the
remainder of the synthesis. The relative configuration of
the three substituents on the ring in compound 38 was
confirmed by 2D-COSY NMR. Compound 38 was con-
verted into Cbz-protected amine 39 using the procedure
that was described earlier for the synthesis of compound
14 (Scheme 2). Compound 39 was treated with an excess
of NaIO4 in the presence of catalytic KMnO4 to ac-
complish both oxidative cleavage of the alkene and
oxidation of the thioether to give sulfone 40.
We planned to measure the activity of the two separate
diastereomers of inhibitor 6 against plasmin. Thus, after
compound 40 was coupled with Trp(Boc)-OMe, the two
resulting diastereomers were separated by flash chro-
matography to give 41a and b, and these two diaster-
eomers were separately carried on through the synthesis.
Deprotection of the Cbz group and coupling with Cbz-
Trp(Boc)-OH gave compounds 42a and b. The silyl ether
was then removed to give alcohols 43a and b. When these
two alcohols were oxidized to the corresponding ketone
(44) with Dess-Martin periodinane, we found that the
two reactions gave identical products that were ap-
proximately a 1:1 mixture of two diastereomers, even
though the starting materials for both reactions were
single diastereomers. The arrangement of the sulfone and
the ketone in the six-membered ring makes the ketone
very prone to enolization. As a result, the two stereo-
centers R to the ketone racemized under the reaction
conditions. The synthesis was completed by removing the
two Boc groups in the 1:1 mixture of diastereomers of
compound 40 with TFA to yield the desired inhibitor.
To add the second substituent to the cyclohexanone
core, alkene 35 was deprotonated with LDA and the
resulting enolate was treated with methyl cyanoformate
to give â-ketoester 36 as predominantly the enol tau-
tomer.25 Other CO2 equivalents such as dimethyl carbon-
(23) (a) Tsuji, J.; Nagashima, H.; Hori, K. Tetrahedron Lett. 1982,
23, 2679. (b) Taguchi, H.; Ghoroku, K.; Tadaki, M.; Tsubouchi, A.;
Takeda, T. J. Org. Chem. 2002, 67, 8450.
(24) Matsuyama, H.; Miyazawa, Y.; Takei, Y.; Kobayashi, M. J. Org.
Chem. 1987, 52, 1703.
(25) Mander, L. S.; Sethi, S. P. Tetrahedron Lett. 1983, 24, 5425.
J. Org. Chem, Vol. 70, No. 21, 2005 8313

Xue and Seto
SCHEME 6. Synthesis of Inhibitor 6a
a Reagents: (a) allyl alcohol, 2,2-dimethoxypropane, p-toluenesulfonic acid, 93%; (b) LDA; MeO2CCN, 64%; (c) NaBH4, 78%; (d) TBDMSCl,
imidazole; (e) NaOH, MeOH, 42% (two steps); (f) (C6H5O)2PON3, DIEA; (g) BnOH, n-BuLi, 72% (two steps); (h) NaIO4, KMnO4, NaHCO3,
65%; (i) H-Trp(Boc)-OMe, HBTU, DIEA, 41a: 35%, 41b: 31%; (j) H2, Pd/C; (k) Cbz-Trp(Boc)-OH, HBTU, DIEA, 42a: 70%, 42b: 76%
(two steps); (l) TBAF, 43a: 81%, 43b: 80%; (m) Dess-Martin periodinane, 92%; (n) TFA, 76%. For compounds 41-43, a and b refer to
two different diastereomers. Only one of the two diastereomers is shown for compounds 41-44 and 6.
TABLE 1. Inhibition Constants of Compounds 1-6
Compounds 1 and 2 have similar structures. They both
incorporate a simple Trp side chain at the P2 position
that binds in the S2 subsite of the enzyme. Inhibitor 1
has low activity with an IC50 value of 800 µM. This result
is not surprising since the inhibitor has only a single
amino acid, in addition to the electrophilic ketone, to
make contacts with active site residues. In inhibitor 2,
the C-4 atom of the cyclohexanone ring has been replaced
with an SO2 group. This replacement induces a repulsive
electrostatic interaction between the sulfone and the
ketone, which increases the electrophilicity of the ketone
and enhances its interaction with the active site serine
nucleophile. As a result, there is a 4-fold increase in
potency when inhibitor 2 is compared with 1.
Inhibitors 3 and 4 incorporate a dipeptide side chain
and are expected to make a greater number of noncova-
lent contacts with the active site. The aminohexyl group
in these inhibitors mimics the side chain of Lys and is
designed to bind in the S1 subsite. The aromatic ring of
Phe fits into the hydrophobic S2 subsite, whereas D-Ile
is aligned with the S3 subsite. The free N-terminus of
the D-Ile residue is likely to be protonated at physiological
pH. This positions a positive charge at the base of the
S3 subsite, which has been reported to be beneficial for
binding.11 Comparison of inhibitors 1 and 3 shows that
replacement of the Cbz-Trp with D-Ile-L-Phe increases the
against Plasmin
inhibitor
IC50 (µM)
inhibitor
IC50 (µM)
1
3
5
800 ( 60
122 ( 5
25 ( 3
2
4
6
200 ( 16
45 ( 5
5.5 ( 0.5
The enolization of compounds 44 and 6 prevented us
from being able to prepare inhibitor 6 as two separate
diastereomers. This same behavior is likely to be seen
in the other sulfone-containing inhibitors 2 and 4.
Therefore, we elected to measure the activity of all of the
inhibitors as mixtures of two diastereomers. In previous
studies, we found that the diastereomers of related
inhibitors have similar activities; in most cases, the
diastereomers have IC50 values that are within a factor
of 2 of each other.12,17
Inhibition Studies. Inhibitors 1-6 were assayed
against plasmin using D-Val-Ile-Lys-pNA (pNA ) p-
nitroanilide) as the substrate (Table 1). Initial rates were
measured using UV spectroscopy to monitor formation
of p-nitroaniline. The assay mixture contained 50 mM
sodium phosphate buffer at pH 7.4, and 10% DMSO to
ensure solubility of the inhibitors. Under these condi-
tions, the KM value for this substrate was measured to
be 170 µM.
8314 J. Org. Chem., Vol. 70, No. 21, 2005

Design of Inhibitors of the Serine Protease Plasmin
Plasmin was assayed at 25 °C in a 50 mM sodium phosphate
buffer (pH 7.4) with or without inhibitors. A final concentration
of 10% DMSO was used in the assay mixtures to ensure
solubility of the inhibitors. Initial rates of the enzymatic
reactions were determined by monitoring the formation of
p-nitroaniline at 405 nm from 30 to 120 s after mixing on a
UV-vis spectrometer. When measuring the IC50 of inhibitors,
the substrate concentration was held constant at its KM value,
which was measured to be 170 µM. Data analysis was
performed with the commercial graphing package Grafit
(Erithacus Software Ltd.).
Amide 8. Amine 7 (200 mg, 1.18 mmol) was dissolved in
DMF (5 mL). To this solution, Cbz-Trp-OH (598 mg, 1.77
mmol), HBTU (671 mg, 1.77 mol), and DIEA (620 µL, 460 mg,
3.54 mmol) were added. After the reaction was stirred for 1 h
at room temperature, the reaction mixture was partitioned
between EtOAc (200 mL) and 1 N HCl (200 mL). The organic
layer was washed with 1 N HCl (150 mL), saturated NaHCO3
(150 mL), and brine (100 mL). It was then dried over MgSO4,
and the solvent was removed by rotary evaporation. The crude
oil was purified by flash chromatography (EtOAc/hexanes, 2:1)
to yield amide 8 as a mixture of two diastereomers (463 mg,
944 µmol, 80%): 1H NMR (300 MHz, CDCl3) δ 1.00-1.45 (m,
4H), 1.46-1.70 (m, 4H), 1.71-1.95 (m, 1H), 2.40-2.70 (m, 1H),
3.05-3.35 (m, 2H), 3.50-3.95 (m, 5H), 4.45-4.71 (br s, 1H),
5.05-5.25 (m, 2H), 5.40-5.80 (m, 1H), 5.81-6.35 (m, 1H),
6.95-7.25 (m, 3H), 7.29-7.45 (m, 6H), 7.58-7.85 (m, 1H),
8.10-8.30 (br s, 1H); 13C NMR (75 MHz, CDCl3) δ 14.6, 17.0,
21.8, 21.99, 22.05, 23.9, 24.1, 25.6, 25.7, 28.0, 29.0, 29.3, 29.6,
54.3, 55.6, 55.9, 59.5, 59.6, 60.8, 67.2, 69.7, 75.4, 77.7, 97.5,
97.7, 110.6, 111.5, 119.3, 119.5, 120.0, 120.1, 122.4, 122.6,
123.7, 123.8, 128.45, 128.50, 128.90, 128.93, 136.6, 136.7,
156.3, 170.7, 171.1; HRMS-FAB (M + H+) calcd for C28H34N3O5
492.2498, found 492.2503.
Inhibitor 1. To compound 8 (200 mg, 407 µmol), an aqueous
TFA solution (10 mL, 50%) was added at 0 °C. The reaction
was warmed to room temperature, stirred for an additional
12 h, and then concentrated by rotary evaporation. The
resulting residue was diluted with EtOAc (50 mL) and washed
with saturated aqueous Na2CO3 (50 mL) and brine (50 mL).
It was then dried over MgSO4, and the solvent was removed
by rotary evaporation. The crude oil was purified by flash
chromatography (EtOAc/hexanes, 2:1) to yield inhibitor 1 as
a mixture of two diastereomers (97 mg, 224 µmol, 55%): 1H
NMR (300 MHz, CDCl3) δ 1.05-1.25 (m, 1H), 1.40-1.92 (m,
5H), 2.10-2.70 (m, 2H), 2.85-3.35 (m, 3H), 3.90-4.60 (m, 1H),
5.00-5.25 (m, 3H), 5.60-6.80 (m, 1H), 6.92-7.21 (m, 4H),
7.28-7.90 (m, 6H), 8.31-8.80 (m, 1H); 13C NMR (75 MHz,
CDCl3) δ 17.0, 21.8, 22.9, 24.3, 24.5, 26.0, 26.6, 28.3, 30.0, 33.0,
33.8, 34.3, 35.5, 41.2, 41.3, 56.1, 56.7, 58.2, 58.5, 59.6, 60.4,
60.5, 67.4, 67.8, 68.0, 69.7, 77.7, 108.8, 110.3, 110.5, 111.4,
111.7, 118.8, 119.2, 119.9, 120.2, 122.5, 122.8, 123.8, 127.1,
127.8, 128.4, 128.5, 128.6, 128.8, 128.90, 128.96, 129.1, 135.2,
136.20, 136.23, 136.3, 136.7, 155.1, 156.3, 170.3, 171.4, 207.2;
HRMS-FAB (M + Na+) calcd for C25H27N3NaO4 456.1899,
found 456.1892.
Alcohol 10. Ketoester 9 (10 g, 57 mmol) was dissolved in
dry THF (150 mL) at 0 °C. To this solution, NaBH4 (4.25 g,
115 mmol) was slowly added. After 30 min, the reaction was
quenched with 1 N HCl (200 mL), and the THF was removed
by rotary evaporation. The resulting material was partitioned
between EtOAc (500 mL) and 1 N HCl (300 mL). The organic
layer was washed with 1 N HCl (300 mL), saturated aqueous
NaHCO3 (300 mL), and brine (500 mL). It was then dried over
MgSO4, and the resulting solution was concentrated by rotary
evaporation. The crude oil was purified by flash chromatog-
raphy (EtOAc/hexanes, gradient of 1:9 to 1:2) to yield com-
pounds 10 (1.52 g, 8.6 mmol, 15%) and 11 (6.07 g, 34.5 mmol,
60%). 10: 1H NMR (300 MHz, CDCl3) δ 1.55-1.75 (m, 1H),
2.15-2.32 (dm, J ) 13.5 Hz, 1H), 2.49-2.70 (m, 4H), 2.71-
2.90 (dm, J ) 12.3 Hz, 1H), 3.25 (s, 1H), 3.55-3.65 (m, 4H);
13C NMR (75 MHz, CDCl3) δ 27.8, 30.0, 35.6, 52.3, 52.5, 70.4,
potency of the cyclohexanone-based inhibitors by a factor
of approximately 6. Inhibitor 4 also incorporates the
sulfone functional group and shows a further increase
in potency to give an IC50 value of 45 µM.
Inhibitors 5 and 6 incorporate peptide side chains that
extend into both the nonprimed and the primed subsites.
Like inhibitors 1 and 2, the Cbz-Trp moiety is designed
to bind in the S2 subsite. The Trp-OMe group on the
C-terminal side of the inhibitors is aligned to bind in the
S2′ subsite. We chose Trp for P2′ position because our
earlier studies with a combinatorial library of inhibitors
demonstrated that Trp is the optimal amino acid for this
position of these inhibitors.17 Inhibitor 5 has an IC50 value
of 25 µM, which represents a 5-fold increase in potency
over inhibitor 3, and a 32-fold increase over inhibitor 1.
One plausible explanation for the good activity of inhibi-
tor 5 is that this molecule is anchored into the active site
on both the S and S′ sides. This set of interactions may
be more effective at properly aligning the electrophilic
ketone for reaction with the serine nucleophile, when
compared to the inhibitors 1-4 that anchor the inhibitors
only through interactions with the S subsites. Thus, a
combination of both favorable noncovalent and covalent
interactions between enzyme and inhibitor would lead
to better inhibition by compound 5.
Inhibitor 6, which is structurally similar to inhibitor
5 but incorporates the sulfone group, has an IC50 value
of 5.5 µM and is the best inhibitor out of the series. Across
all three pairs of inhibitors, we observe that incorporation
of the sulfone increases inhibitor potency by a factor of
3-5. This result is consistent with our proposed mech-
anism of inhibition, which involves reversible formation
of a hemiketal linkage between the active site serine
reside and the ketone group in the inhibitors. It is also
consistent with the trend that we observed in previous
studies between inhibitor potency and ketone elecrophi-
licity.14 An alternate explanation for the enhanced activ-
ity of the sulfone-containing inhibitors is that the sulfone
could be forming a hydrogen bond with nearby residues
in the active site. However, since we have observed
similar increases in potency for a number of inhibitors
of both serine and cysteine proteases, we believe that the
main role of the sulfone is to increase the electrophilicity
of the ketone.
Conclusions
In summary, we synthesized three pairs of compounds
(1-6) as inhibitors against the serine protease plasmin.
Compounds 1, 3, and 5 are based upon a cyclohexanone
pharmacophore, while compounds 2, 4, and 6 are based
upon the tetrahydro-4H-thiopyran-4-one 1,1-dioxide struc-
ture. The direct comparisons of the three pairs of inhibi-
tors show that the strongly electronegative sulfone group
increases inhibitor potency by a factor of 3-5 when
compared to the cyclohexanone analogues. In addition,
inhibitors that bind in both the S and S′ subsites have
higher activity when compared to inhibitors that contact
only half of the active site.
Experimental Section
Enzyme Assays. IC50 values for inhibitors 1-6 were
measured for plasmin using the chromogenic substrate D-Val-
Leu-Lys-pNA. Enzyme and substrate were used as received.
J. Org. Chem, Vol. 70, No. 21, 2005 8315

Xue and Seto
174.2; HRMS-FAB (M + H+) calcd for C7H12O3S 176.0507,
found 176.0510. 11: 1H NMR (300 MHz, CDCl3) δ 1.85-2.00
(tm, J ) 14.5 Hz, 1H), 2.03-2.25 (m, 1H), 2.30-2.40 (dm, J )
15.0 Hz, 1H), 2.55-2.65 (dm, J ) 13.5 Hz, 1H), 2.85-2.92 (dt,
J ) 10.5, 3.0 Hz, 1H), 2.95-3.12 (tm, J ) 13.8 Hz, 1H), 3.05-
3.25 (m, 2H), 3.78 (s, 3H) 4.15-4.25 (br s, 1H); 13C NMR (75
MHz, CDCl3) δ 23.2, 25.4, 33.7, 47.8, 52.5, 66.2, 174.6; HRMS-
FAB (M + H+) calcd for C7H12O3S 176.0507, found 176.0512.
TBDMS Ether 12. To a solution of alcohol 11 (6.07 g, 34.5
mmol) in DMF (12 mL), imidazole (3.52 g, 51.7 mmol) and
TBDMSCl (12.9 g, 86.3 mmol) were added. The reaction was
stirred at 50 °C for 12 h. The mixture was diluted with EtOAc
(1 L), and the organic layer was washed with 1 N HCl (2 ×
500 mL), saturated aqueous NaHCO3 (500 mL), and brine (500
mL). It was then dried over MgSO4, and the solvent was
removed by rotary evaporation. The crude oil was purified by
flash chromatography (EtOAc/hexanes, 1:10) to give compound
12 (8.60 g, 29.7 mmol, 86%): 1H NMR (300 MHz, CDCl3) δ
-0.04-0.04 (d, J ) 19.5 Hz, 6H), 0.85 (s, 9H), 1.83-1.90 (tq,
J ) 12.3, 1.8 Hz, 1H), 2.02-2.09 (dm, J ) 13.9 Hz, 1H), 2.18-
2.23 (dm, J ) 13.2 Hz, 1H), 2.47-2.52 (dm, J ) 13.5 Hz, 1H),
2.64-2.71 (dt, J ) 12.0, 3.0 Hz, 1H), 2.96-3.06 (td, J ) 13.1,
2.4 Hz, 1H), 3.17-3.26 (dd, J ) 13.3, 12.1 Hz, 1H), 3.66 (s,
3H), 4.51-4.54 (m, 1H); 13C NMR (75 MHz, CDCl3) δ -5.0,
-4.0, 18.3, 21.9, 23.1, 26.0, 35.4, 49.6, 52.0, 67.3, 173.2; HRMS-
FAB (M + H+) calcd for C13H27O3SSi 291.1450, found 291.1455.
Carboxylic Acid 13. To compound 12 (6.00 g, 20.7 mmol),
MeOH (120 mL) and 1 N aqueous NaOH (120 mL) were added.
The mixture was heated at reflux for 24 h. The MeOH was
removed by rotary evaporation, and to the aqueous layer were
added EtOAc (500 mL) and 1 N HCl (500 mL). The organic
layer was washed with brine (300 mL) and dried over MgSO4.
The solvent was removed by rotary evaporation. The crude oil
was purified by flash chromatography (EtOAc/hexanes, 1:2)
to yield carboxylic acid 13 (3.87 g, 14.5 mmol, 70%): 1H NMR
(300 MHz, CDCl3) δ 0.04-0.09 (d, J ) 15.0 Hz, 6H), 0.98 (s,
9H), 1.87-1.97 (tt, J ) 14.0, 3.4 Hz, 1H), 2.03-2.12 (dm, J )
13.7 Hz, 1H), 2.28-2.33 (dm, J ) 13.2 Hz, 1H), 2.52-2.58 (dm,
J ) 13.4 Hz, 1H), 2.74-2.81 (dt, J ) 11.1, 2.8 Hz, 1H), 2.96-
3.01 (m, 1H), 3.20-3.25 (m, 1H), 4.48-4.49 (dm, J ) 2.0 Hz,
1H); 13C NMR (75 MHz, CDCl3) δ -4.9, -4.0, 18.4, 22.6, 23.7,
26.1, 35.3, 49.0, 67.8, 177.8; HRMS-FAB (M + Na+) calcd for
C12H24NaO3SSi 299.1113, found 299.1112.
Carbamate 14. To a solution of 13 (3.8 g, 13.8 mmol) in
dry toluene (100 mL), DIEA (3.6 mL, 2.69 g, 20.7 mmol) and
(PhO)2PON3 (4.56 mL, 4.57 g, 16.6 mmol) were added. The
reaction was heated at 60 °C under nitrogen for 4 h. The
formation of the isocyanate was followed by IR spectroscopy
at 2257 cm-1. The alkoxide from benzyl alcohol was prepared
in a separate flask as follows: Benzyl alcohol (4.50 mL, 4.47
g, 41.4 mmol) was added to dry THF (60 mL) at 0 °C under
nitrogen, followed by dropwise addition of n-butyllithium (11.0
mL, 2.5 M solution in hexanes, 27.6 mmol) over a period of 5
min. The isocyanate solution was then cooled to room tem-
perature and added dropwise to the alkoxide solution at 0 °C.
After the addition was complete, the reaction was allowed to
warm to room temperature over 30 min. The reaction was then
quenched with 1 N HCl (100 mL), followed by the removal of
the THF by rotary evaporation. The remaining solution was
partitioned between EtOAc (500 mL) and 1 N HCl (300 mL)
and washed with saturated aqueous NaHCO3 (300 mL) and
brine (300 mL). It was then dried over MgSO4, and the solvents
were removed by rotary evaporation. The crude oil was purified
by flash chromatography (EtOAc/hexanes, 1:9) to yield com-
pound 14 (3.60 g, 9.49 mmol, 84%): 1H NMR (300 MHz, CDCl3)
δ 0.08 (s, 6H), 0.93 (s, 9H), 1.95-2.00 (tm, J ) 11.6 Hz, 1H),
2.06-2.12 (m, 1H), 2.21-2.26 (dm, J ) 13.6 Hz, 1H), 2.41-
2.46 (dd, J ) 10.0, 2.6 Hz, 1H), 2.84-2.93 (t, J ) 11.8 Hz,
2H), 3.87-4.00 (m, 2H), 5.01-5.05 (d, J ) 9.2 Hz, 1H), 5.11-
5.16 (m, 2H), 7.30-7.38 (m, 5H); 13C NMR (75 MHz, CDCl3) δ
-4.6, -4.1, 18.5, 22.1, 26.1, 26.2, 28.0, 35.4, 53.0, 67.0, 69.3,
128.4, 128.5, 128.9, 129.0, 137.0, 155.8; HRMS-FAB (M + Na+)
calcd for C19H31NNaO3SSi 404.1692, found 404.1680.
Sulfone 15. Compound 14 (2.0 g, 5.25 mmol) was dissolved
in acetone (200 mL) and H2O (100 mL). To this solution, NaIO4
(6.74 g, 31.5 mmol), KMnO4 (620 mg, 3.94 mmol), and NaHCO3
(450 mg, 5.25 mmol) were added. The solution was stirred for
6 h at room temperature. The acetone was removed by rotary
evaporation, and the remaining material was partitioned
between EtOAc (500 mL) and 1 N HCl (300 mL). The organic
layer was washed with 1 N HCl (2 × 300 mL) and brine (300
mL) and then dried over MgSO4. The solvent was removed by
rotary evaporation, and the crude oil was purified by flash
chromatography (EtOAc/hexanes, 1:2) to yield 15 (1.60 g, 3.88
mmol, 74%): 1H NMR (300 MHz, CDCl3) δ 0.10 (s, 6H), 0.92
(s, 9H), 2.06-2.15 (m, 1H), 2.15-2.25 (t, J ) 13.0 Hz, 1H),
2.83-2.90 (dm, J ) 14.0 Hz, 1H), 3.15-3.28 (m, 2H), 4.10-
4.15 (m, 1H), 4.28-4.35 (m, 1H), 4.90-4.93 (d, J ) 9.2 Hz,
1H), 5.13 (s, 2H), 7.27-7.40 (m, 5H); 13C NMR (75 MHz,
CDCl3) δ -4.6, -4.2, 18.4, 26.1, 29.2, 45.1, 51.4, 51.8, 67.1,
67.6, 128.6, 128.8, 129.0, 136.4, 155.3; HRMS-FAB (M + Na+)
calcd for C19H31NaNO5SSi 436.1590, found 436.1579.
Primary Amine 16. To compound 15 (2.0 g, 4.84 mmol),
dry MeOH (60 mL) and 10% Pd on carbon (1.0 g) were added.
The reaction flask was purged and kept under 1 atm of
hydrogen gas. The reaction was stirred at room temperature
for 5 h. The Pd/carbon was then removed by filtration. The
clear solution was concentrated by rotary evaporation, and the
resulting residue was dried under vacuum for 24 h to give
compound 16 (1.35 g, 4.84 mmol, 100%): 1H NMR (300 MHz,
CDCl3) δ 0.14-0.16 (d, J ) 9.0 Hz, 6H), 0.94 (s, 9H), 2.10-
2.20 (m, 2H), 2.78-2.86 (dq, J ) 13.8, 3.7 Hz, 1H), 2.93-3.01
(dt, J ) 13.2, 3.6 Hz, 1H), 3.10-3.26 (m, 2H), 3.33-3.40 (dq,
J ) 11.4, 2.1 Hz, 1H), 4.02-4.04 (t, J ) 2.0 Hz, 1H); 13C NMR
(75 MHz, CDCl3) δ 0.6, 0.8, 23.4, 31.1, 34.3, 49.8, 57.7, 58.9,
59.9, 74.3; HRMS-FAB (M + Na+) calcd for C11H25NNaO3SSi
302.1222, found 302.1228.
Amide 17. Compound 16 (200 mg, 0.72 mmol) was dissolved
in DMF (2 mL). To this solution, Cbz-Trp(Boc)-OH (346 mg,
0.80 mmol), HBTU (545 mg, 1.44 mmol), and DIEA (375 µL,
281 mg, 2.16 mmol) were added. After 2 h at room tempera-
ture, the reaction mixture was partitioned between EtOAc (100
mL) and 1 N HCl (100 mL). The organic layer was then washed
with 1 N HCl (50 mL), saturated NaHCO3 (50 mL), and brine
(50 mL). It was dried over MgSO4, and the solvent was
removed by rotary evaporation. The crude oil was purified by
flash chromatography (EtOAc/hexanes, 1:2) to yield compound
17 as a mixture of two diastereomers (433 mg, 0.62 mmol,
86%): 1H NMR (300 MHz, CDCl3) δ -0.4-0.0 (m, 6H), 0.70-
0.80 (d, J ) 10.0 Hz, 9H), 1.66 (s, 9H), 1.85-2.00 (m, 1H),
2.00-2.16 (m, 1H), 2.50-3.40 (m, 7H), 4.19-4.40 (m, 2H),
5.00-5.20 (m, 2H), 5.25-6.15 (m, 2H), 7.10-7.70 (m, 9H),
8.11-8.21 (s, 1H); 13C NMR (75 MHz, CDCl3) δ -5.1, -5.0,
-4.4, -4.3, 18.0, 18.2, 25.9, 26.0, 28.6, 29.1, 29.2, 29.3, 45.0,
45.1, 50.0, 50.4, 50.6, 50.8, 55.7, 66.7, 67.6, 67.7, 84.3, 84.6,
115.5, 115.8, 116.0, 119.3, 119.5, 123.2, 123.4, 124.8, 124.9,
125.2, 125.6, 128.6, 128.8, 129.0, 130.2, 130.7, 135.8, 135.9,
136.3, 136.4, 170.2, 170.6; HRMS-FAB (M + H+) calcd for
C35H49N3NaO8SSi 722.2907, found 722.2899.
Alcohol 18. To a solution of compound 17 (100 mg, 0.17
mmol) in dry THF (5 mL), a solution of TBAF (66 mg, 0.25
mmol) in dry THF (5 mL) was added. The reaction was stirred
at room temperature for 30 min. The solvents were then
removed by rotary evaporation, and the resulting material was
purified by flash chromatography (EtOAc/chloroform 2:1) to
give compound 18 as a mixture of two diastereomers (87 mg,
0.15 mmol, 89%): 1H NMR (400 MHz, acetone-d6) δ 0.68 (s,
9H), 2.00-2.35 (m, 2H), 2.75-3.00 (m, 2H), 3.11-3.35 (m, 4H),
3.89-4.11 (m, 1H), 4.30-4.70 (m, 3H), 4.96-5.12 (m, 2H),
6.10-6.75 (m, 1H), 7.15-7.43 (m, 6H), 7.45-7.80 (m, 3H),
8.11-8.21 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 32.8, 33.2,
50.1, 50.2, 55.3, 55.5, 55.7, 60.46, 60.51, 69.9, 70.0, 70.8, 71.4,
88.8, 88.9, 120.4, 121.58, 121.63, 124.6, 127.8, 127.9, 129.65,
8316 J. Org. Chem., Vol. 70, No. 21, 2005

Design of Inhibitors of the Serine Protease Plasmin
129.69, 129.8, 130.1, 133.1, 133.2, 133.8, 136.0, 136.1, 140.8,
142.5, 154.77, 154.81, 161.4, 161.5, 176.0, 176.1; HRMS-FAB
(M + Na+) calcd for C29H35N3NaO8S 608.2043, found 608.2047.
125.5, 125.6, 125.7, 126.8, 127.2, 127.4, 128.0, 128.6, 128.8,
129.9, 130.1, 137.2, 137.4, 138.3, 141.60, 141.62, 141.7, 144.2,
144.35, 144.41, 144.5, 155.6, 155.9, 156.1, 156.4, 171.1, 173.1,
173.3; HRMS-FAB (M + Na+) calcd for C44H57N3NaO7 762.4094,
found 762.4091.
Inhibitor 2. To a solution of alcohol 18 (50 mg, 85 µmol) in
dry CH2Cl2 (10 mL), Dess-Martin reagent (55 mg, 128 µmol)
was added. The reaction was stirred at room temperature for
24 h. The reaction mixture was then partitioned between
EtOAc (30 mL) and saturated Na2CO3 (30 mL), and the organic
layer was washed with saturated Na2CO3 (30 mL) and brine
(30 mL). It was then dried over MgSO4, and the solvent was
removed by rotary evaporation. The crude ketone was purified
by flash chromatography (EtOAc/hexanes, 2:1). The resulting
ketone was redissolved in CH2Cl2 (5 mL), and to this solution
TFA (2.5 mL) was added. The reaction was stirred at room
temperature for 1.5 h, the solvents were removed by rotary
evaporation, and the resulting material was partitioned
between EtOAc (25 mL) and saturated Na2CO3 (10 mL). The
organic layer was washed with saturated Na2CO3 (10 mL) and
brine (10 mL) and then dried over MgSO4. The solvent was
removed by rotary evaporation, and the crude material was
purified by flash chromatography (EtOAc/CH2Cl2, 2:1) to yield
inhibitor 2 as a mixture of two diastereomers (14 mg, 29 µmol,
34%): 1H NMR (300 MHz, acetone-d6) δ 2.69-2.89 (m, 1H),
3.11-3.79 (m, 8H), 4.43-4.62 (m, 1H), 4.99-5.15 (m, 3H),
Dipeptide 21. Compound 20 (200 mg, 271 µmol) was stirred
in a 1:1 mixture of piperidine and DMF (10 mL) at room
temperature for 1 h. The solvents were then removed by rotary
evaporation. The resulting residue was dried under vacuum
for 12 h to remove excess piperidine. The white solid was
redissolved in DMF (5 mL). To this solution, Boc-D-Ile-OH (126
mg, 542 µmol), HBTU (205 mg, 542 µmol), and DIEA (143 µL,
106 mg, 813 µmol) were added. The reaction was stirred for 2
h at room temperature, and the mixture was partitioned
between EtOAc (100 mL) and 1 N HCl (75 mL). The organic
layer was washed with 1 N HCl (75 mL), saturated aqueous
NaHCO3 (75 mL), and brine (75 mL). It was then dried over
MgSO4 and concentrated. The crude oil was purified by flash
chromatography (EtOAc/hexanes, 2:3) to yield compound 21
as a mixture of two diastereomers (155 mg, 211 µmol, 78%):
1H NMR (300 MHz, CDCl3) δ 0.52-1.03 (m, 8H), 1.04-1.35
(m, 8H), 1.36-1.68 (m, 22H), 1.69-2.08 (m, 6H), 2.48-3.00
(m, 3H), 3.01-3.17 (m, 3H), 3.28-3.66 (m, 2H), 3.67-4.10 (m,
4H), 4.11-4.65 (m, 1H), 4.58-5.30 (m, 2H), 6.30-7.00 (m, 1H),
7.00-7.27 (m, 5H), 7.30-7.82 (m, 1H); 13C NMR (75 MHz,
CDCl3) δ 12.0, 12.1, 15.57, 15.63, 15.9, 22.1, 22.3, 22.4, 24.6,
24.7, 24.9, 25.1, 25.3, 25.6, 25.7, 25.9, 26.4, 26.6, 26.7, 26.9,
27.5, 28.2, 28.3, 28.4, 28.7, 28.9, 29.0, 29.4, 30.4, 30.5, 37.9,
38.4, 38.8, 40.7, 40.9, 41.2, 44.5, 45.1, 45.2, 50.6, 51.9, 55.3,
58.0, 59.3, 59.4, 59.5, 62.0, 62.3, 77.7, 79.4, 79.9, 98.5, 99.1,
99.5, 99.9, 125.8, 126.7, 127.2, 128.6, 128.7, 128.8, 129.78,
129.84, 130.1, 137.3, 138.2, 155.8, 156.0, 156.4, 156.5, 170.3,
171.0, 173.0, 173.3; HRMS-FAB (M + Na+) calcd for C40H66N4-
NaO8 753.4778, found 753.4788.
Inhibitor 3. Compound 21 (50 mg, 68 µmol) was dissolved
in CH2Cl2 (2 mL). To this solution TFA (1 mL) was added. The
reaction was stirred at room temperature for 30 min. The
solvents were removed by rotary evaporation. To the resulting
residue, 50% aqueous TFA (3 mL) was added, and the reaction
was stirred at room temperature for an additional 12 h. The
solvents were removed by rotary evaporation. The crude
material was purified by flash chromatography (5-15% MeOH
in CH2Cl2) to yield inhibitor 3 as a mixture of two diastereo-
mers (19 mg, 40 µmol, 59%): 1H NMR (300 MHz, MeOH-d4) δ
0.62-1.09 (m, 7H), 1.18-1.54 (m, 6H), 1.57-1.85 (m, 6H),
1.92-2.23 (m, 4H), 2.36-2.63 (m, 3H), 2.81-3.11 (m, 4H),
3.14-3.49 (m, 2H), 3.57-3.87 (m, 2H), 4.27-4.46 (m, 1H),
4.47-4.59 (m, 1H), 4.70-5.19 (m, 2H), 7.00-7.25 (m, 5H),
7.30-8.00 (m, 1H); 13C NMR (75 MHz, MeOH-d4) δ 10.60,
10.65, 10.73, 13.6, 13.8, 21.4, 22.5, 23.9, 24.0, 24.4, 24.5, 24.6,
24.9, 25.2, 25.4, 25.5, 26.1, 26.3, 26.6, 27.5, 29.8, 30.1, 30.3,
31.0, 36.6, 36.7, 36.8, 39.6, 40.9, 43.8, 51.6, 51.8, 54.7, 57.7,
58.0, 59.0, 65.0, 65.1, 65.5, 66.3, 120.6, 124.9, 127.0, 127.2,
127.8, 128.67, 128.73, 129.1, 129.27, 129.33, 129.6, 129.7,
136.9, 137.0, 141.4, 142.7, 168.1, 171.8, 206.2, 206.3; HRMS-
FAB (M + Na+) calcd for C27H44N4NaO3 495.3311, found
495.3318.
6.96-7.15 (m, 2H), 7.20-7.51 (m, 8H), 7.51-7.72 (m, 1H); 13
C
NMR (75 MHz, acetone-d6) δ 14.0, 20.3, 32.7, 36.3, 49.9, 50.0,
54.08, 54.14, 54.2, 54.3, 56.0, 60.0, 66.3, 110.3, 110.5, 111.7,
118.8, 119.1, 121.7, 124.0, 128.0, 128.1, 128.7, 136.2, 136.9,
127.6, 156.3, 170.4, 170.6, 199.9, 200.0; HRMS-FAB (M + Na+)
calcd for C24H25N3NaO6S 506.1362, found 506.1345.
Amide 20. Primary amine 7 (200 mg, 1.18 mmol) was
dissolved in CDCl3 (7 mL). To this solution, a solution of
BocNH(CH)5CHO (216 mg, 1.00 mmol) in CDCl3 (3 mL) and
titanium(IV) isopropoxide (585 µL, 568 mg, 2.00 mmol) were
added. The reaction was stirred at room temperature, and the
formation of the imine was monitored using 1H NMR spec-
troscopy by following the disappearance of a resonance for the
aldehyde at 9.77 ppm. After the formation of the imine was
complete, the reaction was added dropwise to a solution of
NaBH4 (185 mg, 5.0 mmol) in MeOH (10 mL) at 0 °C. The
solution was warmed to room temperature and stirred for an
additional 1 h. Water (2 mL) was added with stirring, and the
resulting inorganic precipitate was filtrated and washed with
EtOAc (50 mL). The filtrate was then partitioned between
EtOAc (75 mL) and 1 N HCl (75 mL). The organic layer was
washed with 1 N HCl (50 mL), saturated aqueous Na2CO3 (50
mL), and brine (50 mL). It was then dried over MgSO4, and
the solvents were removed by rotary evaporation. The result-
ing oil was purified by flash chromatography (0-15% MeOH
in CH2Cl2) to give a mixture of starting material 7 and
secondary amine 19. To this mixture, Fmoc-Phe-OH (685 mg,
1.77 mmol), HATU (673 mg, 1.77 mmol), DIEA (620 µL, 460
mg, 3.54 mmol), and DMF (800 µL) were added. The reaction
was stirred at 60 °C for 3 h. The reaction mixture was cooled
to room temperature and partitioned between EtOAc (100 mL)
and 1 N HCl (100 mL). The organic layer was washed with 1
N HCl (75 mL), saturated aqueous NaHCO3 (75 mL), and brine
(75 mL). It was then dried over MgSO4, and the solvent was
removed by rotary evaporation. The crude material was
purified by flash chromatography (0-15% MeOH in CH2Cl2)
to give compound 20 (229 mg, 310 µmol, 31% for two steps) as
a mixture of two diastereomers: 1H NMR (300 MHz, CDCl3)
δ 0.70-1.00 (m, 1H), 1.11-1.56 (m, 19H), 1.57-2.04 (m, 4H),
2.63-3.22 (m, 6H), 3.43-3.60 (m, 1H), 3.62-3.96 (m, 3H),
3.97-4.29 (m, 3H), 4.31-4.48 (m, 1H), 4.49-4.61 (m, 1H),
4.62-5.15 (m, 2H), 5.34-6.03 (m, 1H), 6.89-7.26 (m, 4H),
Secondary Amine 22. The aldehyde BocNH(CH2)5CHO
(277 mg, 1.29 mmol) was dissolved in DCE (dichloroethane)
(5 mL). It was added to a solution of compound 16 (300 mg,
1.08 mmol) in DCE (5 mL). After 25 min, sodium triacetoxy-
borohydride (366 mg, 1.73 mmol) was added. The reaction was
stirred at room temperature for 5 h. The reaction was then
partitioned between saturated aqueous NaHCO3 (200 mL) and
EtOAc (200 mL). The organic layer was washed with brine
(200 mL) and then dried over MgSO4. It was concentrated by
rotary evaporation, and the crude oil was purified by flash
chromatography (EtOAc/hexanes, 1:2) to yield the secondary
amine 22 (387 mg, 0.81 mmol, 75%): 1H NMR (300 MHz,
CDCl3) δ 0.12-0.13 (d, J ) 3.0 Hz, 6H), 0.93 (s, 9H), 1.31-
1.34 (m, 5H), 1.33-1.47 (m, 13H), 2.07-2.20 (m, 2H), 2.55-
2.65 (m, 2H), 2.75-2.95 (m, 1H), 3.05-3.20 (m, 5H), 3.20-
7.28-7.47 (m, 5H), 7.47-7.69 (m, 2H), 7.70-7.89 (m, 2H); 13
C
NMR (75 MHz, CDCl3) δ 17.0, 21.7, 22.1, 22.3, 25.4, 25.7, 25.9,
26.0, 26.6, 26.87, 26.93, 27.1, 27.5, 28.2, 28.3, 28.4, 28.6, 28.8,
29.0, 29.4, 30.3, 30.5, 38.9, 41.0, 41.7, 45.1, 45.2, 47.4, 47.49,
47.54, 47.7, 52.4, 53.1, 53.3, 58.3, 59.3, 59.4, 59.56, 59.63, 62.4,
67.0, 67.3, 69.7, 75.4, 77.7, 79.4, 98.6, 99.1, 99.5, 100.0, 120.3,
J. Org. Chem, Vol. 70, No. 21, 2005 8317

Xue and Seto
3.31 (m, 1H), 4.10-4.18 (s, 1H), 4.43-4.60 (s, 1H); 13C NMR
(75 MHz, CDCl3) δ -4.4, -4.1, 18.4, 26.1, 27.0, 27.3, 28.1, 29.6,
30.2, 30.4, 30.7, 40.9, 45.4, 46.6, 51.9, 58.5, 66.7, 69.7, 77.6,
79.4, 102.7, 156.4; HRMS-FAB (M + Na+) calcd for C22H46N2-
NaO5SSi 501.2794, found 501.2782.
HRMS-FAB (M + Na+) calcd for C36H58N4NaO9S 745.3822,
found 745.3833.
Inhibitor 4. Compound 26 (50 mg, 69 µmol) was dissolved
in CH2Cl2 (2 mL). To this solution TFA (0.5 mL) was added.
The reaction was stirred at room temperature for 30 min. The
solvents were removed by rotary evaporation. The crude
material was purified by flash chromatography (5-15% MeOH
in CH2Cl2) to yield inhibitor 4 as a mixture of two diastereo-
mers (25 mg, 48 µmol, 70%): 1H NMR (300 MHz, MeOH-d4) δ
0.72-0.98 (m, 4H), 0.98-1.09 (m, 3H), 1.12-1.57 (m, 7H),
1.58-2.06 (m, 5H), 2.86-3.25 (m, 6H), 2.81-3.11 (m, 3H),
3.32-3.57 (m, 3H), 3.58-3.80 (m, 2H), 3.81-4.08 (m, 1H),
Amide 24. To a solution of compound 22 (200 mg, 418 µmol)
in acetonitrile (8 mL), BSA (203 µL, 170 mg, 209 µmol) was
added. The reaction was stirred at room temperature under
an atmosphere of nitrogen. After 2 h, Fmoc-Phe-F (364 mg,
936 µmol) and catalytic TBAF (5 mg) were added. The reaction
was stirred at 60 °C for an additional 4 h, then cooled to room
temperature. The reaction was diluted with CH2Cl2, and the
organic layer was washed with saturated aqueous NaHCO3
(150 mL), 1 N HCl (150 mL), and brine (150 mL). It was then
dried over MgSO4, and the solvents were removed by rotary
evaporation. The crude oil was purified by flash chromatog-
raphy (EtOAc/hexanes, 1:2) to give compound 24 as a mixture
of two diastereomers (230 mg, 272 µmol, 65%): 1H NMR (300
MHz, CDCl3) δ -0.12-0.08 (m, 6H), 0.72-0.92 (m, 9H), 1.00-
1.33 (m, 4H), 1.33-1.58 (m, 11H), 1.92-2.35 (m, 2H), 2.70-
3.73 (m, 9H), 4.00-4.48 (m, 5H), 4.49-5.17 (m, 3H), 5.42-
5.73 (m, 1H), 7.09-7.48 (m, 9H), 7.48-7.68 (m, 3H), 7.68-
7.86 (m, 3H); 13C NMR (75 MHz, CDCl3) δ -4.9, -4.6, -4.3,
-4.2, -3.9, 14.4, 18.2, 18.3, 26.2, 26.4, 26.79, 26.84, 27.1, 27.3,
28.9, 29.56, 29.62, 29.8, 29.9, 30.2, 30.4, 32.0, 32.1, 40.3, 40.5,
40.7, 40.8, 40.9, 45.3, 45.6, 45.8, 46.2, 47.4, 50.2, 50.4, 50.7,
51.6, 52.9, 53.5, 54.0, 56.3, 67.5, 67.65, 67.73, 69.0, 69.7, 77.7,
79.4, 120.4, 125.4, 125.5, 125.6, 127.46, 127.53, 127.7, 128.12,
128.16, 128.20, 129.1, 129.4, 129.6, 129.8, 130.0, 135.5, 136.6,
136.7, 141.67, 141.70, 143.95, 144.08, 144.1, 144.2, 155.9,
156.0, 156.36, 156.40, 171.9, 172.6, 173.6; HRMS-FAB (M +
Na+) calcd for C46H65NaN3O8SSi 870.4159, found 870.4129.
4.09-4.56 (m, 1H), 4.94-5.18 (m, 1H), 7.15-7.35 (m, 5H); 13
C
NMR (75 MHz, MeOH-d4) δ 10.7, 10.8, 13.8, 13.9, 14.1, 23.9,
24.1, 24.3, 26.1, 26.2, 27.5, 28.8, 29.1, 29.3, 36.6, 36.8, 37.2,
37.7, 38.4, 39.6, 49.6, 50.5, 51.2, 51.4, 51.5, 51.7, 52.1, 52.5,
57.7, 57.9, 62.1, 115.0, 118.9, 127.2, 127.4, 128.5, 128.77,
128.82, 129.1, 129.3, 129.4, 129.5, 129.6, 129.7, 136.7, 136.8,
161.2, 161.7, 168.1, 168.3, 171.9, 172.2, 197.4, 198.0; HRMS-
FAB (M + Na+) calcd for C26H42N4O5S 523.2954, found
523.2966.
Alkene 28. To a solution of diisopropylamine (8.42 mL, 6.06
g, 60.0 mmol) in THF (60 mL), n-butyllithium (23.5 mL, 58.8
mmol, 2.5 M in hexanes) was added at -78 °C under an
atmosphere of nitrogen. The temperature of the solution was
slowly increased to 0 °C and maintained at that temperature
for an additional 10 min. To this solution, compound 27 (5.0
g, 29.4 mmol) was slowly added. After 15 min, allyl bromide
(2.60 mL, 3.72 g, 31.0 mmol) was added dropwise. The reaction
was stirred at 0 °C for 30 min and then quenched with water.
The THF was removed by rotary evaporation, and the mixture
was partitioned between EtOAc (300 mL) and 1 N HCl (200
mL). The organic layer was washed with 1 N HCl (200 mL),
saturated NaHCO3 (200 mL), and brine (200 mL). It was then
dried over MgSO4, and the resulting solution was concentrated
by rotary evaporation. The crude oil was purified by flash
chromatography (EtOAc/hexanes, 1:18) to yield 28 as a
mixture of ketone and enol tautomers (4.63 g, 22 mmol, 75%):
1H NMR (300 MHz, CDCl3) δ 1.10-1.30 (m, 3.5H), 1.31-1.55
(m, 1.6H), 1.56-1.77 (m, 2.3H), 1.80-2.05 (m, 1.4H), 2.06-
2.28 (m, 2.7H), 2.29-2.45 (m, 1.1H), 2.46-2.66 (m, 1.2H),
3.20-3.40 (m, 0.4H), 4.00-4.35 (m, 2H), 4.80-5.10 (m, 2H),
5.50-5.80 (m, 1H), 14.42 (s, 0.5H); 13C NMR (75 MHz, CDCl3)
δ 14.06, 14.13, 14.2, 20.0, 21.6, 21.9, 22.4, 22.8, 24.0, 26.7, 29.0,
30.2, 30.8, 33.0, 33.4, 33.7, 36.3, 38.2, 48.9, 50.5, 56.2, 57.8,
60.0, 60.2, 60.8, 61.2, 98.0, 116.3, 116.6, 128.18, 128.28, 128.32,
135.9, 136.0, 136.4, 169.9, 172.9, 173.7; HRMS-FAB (M + H+)
calcd for C12H19O3 211.1334, found 211.1338.
Ketal 29. A solution of compound 28 (4.0 g, 19 mmol) in
THF (10 mL) was cooled in an ice bath. To this solution, 1,3-
propanediol (41 mL, 43.3 g, 570 mmol) and TMSCl (4.77 mL,
4.1 g, 38.0 mmol) were added. The reaction was stirred at room
temperature for 48 h and then partitioned between EtOAc (400
mL) and saturated NaHCO3 (400 mL). The organic layer was
washed with saturated NaHCO3 (400 mL) and brine (400 mL).
It was then dried over MgSO4, and the resulting solution was
concentrated by rotary evaporation. The crude oil was purified
by flash chromatography (EtOAc/hexanes, 1:18) to yield 29
(4.33 g, 16.2 mmol, 85%): 1H NMR (300 MHz, CDCl3) δ 1.05-
1.35 (m, 5H), 1.36-1.55 (m, 2H), 1.56-1.70 (m, 2H), 1.71-
1.79 (m, 4H), 2.05-2.32 (m, 1H), 2.40-2.60 (m, 1H), 3.70-
3.95 (m, 3H), 4.00-4.25 (m, 3H), 4.80-5.10 (m, 2H), 5.55-
5.85 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 14.2, 20.0, 25.2,
25.5, 26.1, 32.4, 59.1, 59.4, 60.0, 98.4, 115.3, 138.4, 172.4;
HRMS-FAB (M + Na+) calcd for C15H24NaO4 291.1572, found
291.1575.
Ketone 26. Compound 24 (200 mg, 236 µmol) was stirred
in a 1:1 mixture of piperidine and DMF (10 mL) at room
temperature for 1 h. The solvents were removed by rotary
evaporation. The resulting residue was dried under vacuum
for 12 h to remove excess piperidine. The white solid was then
redissolved in DMF (5 mL). To this solution, Boc-D-Ile-OH (110
mg, 472 µmol), HBTU (179 mg, 472 µmol), and DIEA (124 µL,
92 mg, 708 µmol) were added. The reaction was stirred at room
temperature for 2 h, and the mixture was partitioned between
EtOAc (100 mL) and 1 N HCl (75 mL). The organic layer was
washed with 1 N HCl (50 mL), saturated aqueous NaHCO3
(50 mL), and brine (50 mL). It was then dried over MgSO4
and concentrated. The crude oil was purified by flash chro-
matography (EtOAc/hexanes, 1:1) to yield compound 25. To a
solution of dipeptide 25 in dry THF (5 mL), a solution of TBAF
(92 mg, 354 µmol) in dry THF (5 mL) was added. The reaction
was stirred at room temperature for 30 min. The solvents were
removed by rotary evaporation, and the resulting material was
purified by flash chromatography (EtOAc/chloroform, 2:1) to
give the corresponding alcohol. The alcohol was redissolved
in CH2Cl2 (15 mL). To this solution, Dess-Martin reagent (150
mg, 354 µmol) was added. The reaction was stirred at room
temperature for 12 h and then partitioned between EtOAc (150
mL) and saturated Na2CO3 (150 mL). The organic layer was
washed with saturated Na2CO3 (100 mL) and brine (100 mL).
It was then dried over MgSO4, and the solvent was removed
by rotary evaporation. The crude material was then purified
by flash chromatography (EtOAc/hexanes, 2:1) to yield ketone
26 as a mixture of two diastereomers (98 mg, 136 µmol, 58%
for three steps): 1H NMR (300 MHz, CDCl3) δ 0.72-0.95 (m,
6H), 1.06-1.19 (m, 2H), 1.19-1.32 (m, 4H), 1.32-1.59 (m,
23H), 1.70-1.96 (m, 1H), 2.88-3.03 (m, 3H), 3.03-3.15 (m,
4H), 3.16-3.47 (m, 2H), 3.58-4.10 (m, 4H), 4.58-4.85 (m, 1H),
4.86-5.21 (m, 2H), 6.55-6.86 (m, 1H), 7.11-7.50 (m, 5H); 13
C
NMR (75 MHz, CDCl3) δ 11.8, 11.9, 12.0, 14.6, 15.8, 15.9, 16.0,
24.9, 25.0, 26.1, 26.4, 26.5, 26.6, 28.7, 28.8, 28.9, 29.4, 30.0,
30.2, 37.0, 37.2, 37.47, 37.53, 37.7, 39.0, 40.4, 40.8, 48.3, 48.4,
50.2, 50.4, 50.5, 51.2, 51.6, 59.5, 60.8, 62.6, 77.6, 79.5, 80.4,
127.6, 127.9, 129.1, 129.2, 129.8, 129.9, 136.0, 136.1, 156.0,
156.5, 171.3, 171.4, 171.6, 171.7, 171.8, 172.2, 195.6, 196.0;
Alkene 30. To a solution of compound 29 (5.0 g, 18.7 mmol)
in MeOH (50 mL), 2 N aqueous NaOH (50 mL) was added.
The reaction was heated at reflux for 48 h, then cooled to room
temperature. The MeOH was removed by rotary evaporation,
and the resulting solution was partitioned between EtOAc (400
mL) and 1 N HCl (400 mL). The organic layer was washed
8318 J. Org. Chem., Vol. 70, No. 21, 2005

Design of Inhibitors of the Serine Protease Plasmin
with brine (300 mL) and then dried over MgSO4. The solvent
was removed by rotary evaporation to yield the corresponding
carboxylic acid as a white solid. The carboxylic acid was
redissolved in toluene (50 mL). To this solution, DIEA (4.91
mL, 3.65 g, 28.0 mmol) and (PhO)2PON3 (4.87 mL, 6.2 g, 22.5
mmol) were added. The reaction was heated at 85 °C under
nitrogen for 16 h, then cooled to room temperature. To a
separated flask containing a solution of potassium tert-
butoxide (5.24 g, 46.8 mmol) in THF (150 mL) at 0 °C, the
isocyanate solution was added dropwise. The reaction was
allowed to warm to room temperature over 30 min, and then
it was quenched with water (30 mL). The THF was removed
by rotary evaporation, and the resulting material was parti-
tioned between EtOAc (400 mL) and 1 N HCl (400 mL). The
organic layer was washed with 1 N HCl (300 mL), saturated
NaHCO3 (300 mL), and brine (300 mL). It was then dried over
MgSO4, and the solvent was removed by rotary evaporation.
The crude oil was purified by flash chromatography (EtOAc/
hexanes, 1:9) to give compound 30 (4.54 g, 14.6 mmol, 78%):
1H NMR (300 MHz, CDCl3) δ 1.20-1.38 (m, 2H), 1.39-1.45
(m, 9H), 1.46-1.55 (m, 2H), 1.56-1.80 (m, 4H), 1.85-2.10 (m,
2H), 2.28-2.53 (m, 1H), 3.72-3.98 (m, 4H), 3.99-4.30 (m, 1H),
between EtOAc (150 mL) and 1 N HCl (150 mL). The organic
layer was washed with 1 N HCl (100 mL), saturated NaHCO3
(100 mL), and brine (100 mL). It was then dried over MgSO4,
and the solvent was removed by rotary evaporation. The crude
oil was purified by flash chromatography (EtOAc/hexanes, 2:1)
to yield 32 as a mixture of two diastereomers (296 mg, 327
µmol, 77%): 1H NMR (300 MHz, CDCl3) δ 1.00-1.68 (m, 9H),
1.77-1.97 (m, 1H), 2.83 (s, 2H), 2.99-3.21 (m, 1H), 3.22-3.49
(m, 3H), 3.60-3.90 (m, 7H), 4.42-4.73 (br s, 1H), 4.83-5.06
(m, 1H), 5.07-5.28 (m, 2H), 5.68-6.08 (m, 1H), 6.35-6.72 (m,
1H), 6.85-7.05 (m, 2H), 7.06-7.25 (m, 5H), 7.28-7.46 (m, 7H),
7.47-7.92 (m, 2H), 8.60-9.00 (m, 2H); 13C NMR (75 MHz,
CDCl3) δ 17.0, 19.3, 21.8, 25.0, 25.2, 28.0, 28.8, 39.1, 52.77,
52.82, 53.1, 55.9, 59.3, 59.6, 67.4, 69.7, 75.4, 77.7, 98.3, 110.1,
110.3, 111.8, 111.9, 118.8, 118.9, 119.1, 120.0, 122.47, 122.52,
123.4, 123.8, 127.9, 128.0, 128.5, 128.6, 129.0, 136.59, 136.64,
136.7, 136.9, 156.4, 156.5, 171.2, 171.5, 173.3; HRMS-FAB (M
+ Na+) calcd for C42H47N5NaO8 772.3322, found 772.3308.
Inhibitor 5. To compound 32 (120 mg, 160 µmol) was added
aqueous TFA (10 mL, 50%) at 0 °C. The reaction was warmed
to room temperature, stirred for 12 h, and then concentrated
by rotary evaporation. The resulting residue was diluted with
EtOAc (50 mL) and washed with saturated aqueous Na2CO3
(50 mL) and brine (50 mL). It was then dried over MgSO4, the
solvent was removed by rotary evaporation, and the crude oil
was purified by flash chromatography (EtOAc/hexanes, 2:1)
to yield 5 as a mixture of two diastereomers (74 mg, 107 µmol,
67%): 1H NMR (300 MHz, CDCl3) δ 1.32-1.80 (m, 4H), 1.87-
2.58 (m, 3H), 2.60-3.46 (m, 5H), 3.58-3.80 (m, 3H), 4.10-
4.96 (m, 2H), 5.00-5.87 (m, 3H), 6.27-7.05 (m, 2H), 7.05-
7.25 (m, 4H), 7.28-7.90 (m, 13H), 8.31-8.91 (m, 2H); 13C NMR
(75 MHz, CDCl3) δ 14.6, 19.3, 23.1, 23.6, 26.8, 27.6, 27.9, 28.6,
28.9, 29.7, 32.0, 34.8, 35.5, 35.7, 35.8, 36.1, 36.6, 39.6, 46.9,
47.2, 52.87, 52.93, 53.1, 53.3, 55.9, 56.2, 56.6, 58.1, 58.3, 64.7,
67.4, 67.6, 77.7, 108.8, 109.6, 110.1, 110.8, 111.7, 111.8, 111.9,
112.0, 118.81, 118.84, 118.9, 119.0, 119.7, 119.97, 120.05,
120.1, 120.2, 120.6, 122.4, 122.5, 122.6, 122.70, 122.72, 122.9,
123.1, 123.4, 123.7, 126.3, 127.5, 127.9, 128.4, 128.5, 128.6,
128.7, 128.9, 129.0, 132.9, 136.2, 136.5, 136.56, 136.61, 156.3,
156.4, 171.1, 171.2, 171.4, 171.8, 173.1, 173.3, 176.9, 207.3,
208.3; HRMS-FAB (M + Na+) calcd for C39H41N5NaO7 714.2904,
found 714.2930.
Alkene 35. To a solution of compound 33 (10.0 g, 86 mmol)
in benzene (60 mL), 2,2-dimethoxypropane (12.5 mL, 10.7 g,
103 mmol), allyl alcohol (7.83 mL, 11.0 g, 189 mmol), and
p-toluenesulfonic acid (35 mg, 0.09 mmol) were added. The
reaction was heated at 80-85 °C, and the byproducts (acetone
and MeOH) were collected in a Dean-Stark trap. After 4 h,
the temperature of the oil bath was increased to distill off most
of the benzene. Toluene (100 mL) was added to the solution,
and allyl alcohol was distilled out as an azeotropic mixture
with toluene. The resulting dark brown oil was purified by
distillation (bp 72-76 °C, 1.0-1.5 mmHg) to yield alkene 35
(12.5 g, 80 mmol, 93%): 1H NMR (300 MHz, CDCl3) δ 2.05-
2.22 (m, 1H), 2.46-2.80 (m, 5H), 2.85-3.09 (m, 3H), 4.95-
5.15 (m, 2H), 5.54-5.86 (m, 1H); 13C NMR (75 MHz, CDCl3) δ
31.3, 34.0, 35.6, 44.4, 117.8, 135.5, 209.9; HRMS-FAB (M +
Na+) calcd for C8H12NaOS 179.0507, found 170.0510.
Ketoester 36. To a solution of diisopropylamine (3.0 mL,
21.1 mmol) in dry diethyl ether (150 mL), n-butyllithium (8.5
mL, 21.1 mmol, 2.5 M in hexanes) was added at -78 °C under
an atmosphere of nitrogen. The temperature of the solution
was slowly increased to -20 °C and maintained there for an
additional 30 min. The mixture was then cooled to -100 °C,
and then compound 35 (3.0 g, 19.2 mmol) was added as a
solution in diethyl ether (20 mL). After 1 h, methylcyanofor-
mate (1.71 mL, 1.72 g, 20.2 mmol) was added dropwise. The
reaction was stirred at -100 °C for an additional 4 h and then
quenched with ice water. The organic layer was washed with
saturated Na2CO3 (150 mL) and brine (150 mL). It was then
dried over MgSO4, and the resulting solution was concentrated
by rotary evaporation. The crude oil was purified by flash
4.60-4.90 (m, 1H), 4.91-5.12 (m, 2H), 5.60-5.86 (m, 1H); 13
C
NMR (75 MHz, CDCl3) δ 18.9, 25.2, 28.4, 31.6, 58.9, 59.0, 79.0,
98.7, 115.7, 137.8, 155.9; HRMS-FAB (M + Na+) calcd for
C19H29NNaO4 334.1994, found 334.1988.
Amide 31. Alkene 30 (284 mg, 912 µmol) was dissolved in
a 2:1 mixture of acetone and water (40 mL). To this solution,
NaIO4 (976 mg, 4.56 mmol), KMnO4 (108 mg, 648 µmol), and
NaHCO3 (78 mg, 912 µmol) were added. The reaction was
stirred at room temperature for 2 h, and then the acetone was
removed by rotary evaporation. The remaining material was
partitioned between EtOAc (100 mL) and 1 N HCl (75 mL).
The organic layer was washed with 1 N HCl (3 × 75 mL) and
brine (75 mL) and dried over MgSO4. The solvent was removed
by rotary evaporation, and the crude oil was purified by flash
chromatography (EtOAc/hexanes, 1:2) to yield the correspond-
ing carboxylic acid. The carboxylic acid was redissolved in
DMF (5 mL). To this solution, H2N-Trp-OMe (300 mg, 1.37
mmol), HBTU (519 mg, 1.37 mmol), and DIEA (480 µL, 356
mg, 2.74 mmol) were added. The reaction was stirred at room
temperature for 2 h and then partitioned between EtOAc (200
mL) and 1 N HCl (200 mL). The organic layer was washed
with 1 N HCl (100 mL), saturated aqueous NaHCO3 (100 mL),
and brine (100 mL). It was then dried over MgSO4, and the
solvent was removed by rotary evaporation. The crude material
was purified by flash chromatography (EtOAc/hexanes, 2:1)
to give 31 as a mixture of two diastereomers (386 mg, 730
µmol, 80%): 1H NMR (300 MHz, CDCl3) δ 1.26-1.38 (m, 5H),
1.38-1.58 (m, 11H), 1.58-1.86 (m, 3H), 1.87-2.00 (m, 1H),
2.42-2.75 (m, 1H), 3.17-3.36 (m, 2H), 3.55-3.70 (s, 3H), 3.70-
3.94 (m, 4H), 4.76-5.10 (m, 2H), 6.05-6.36 (m, 1H), 6.95-
7.20 (m, 3H), 7.30-7.41 (m, 1H), 7.43-7.62 (m, 1H), 8.30-
8.55 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 14.8, 17.1, 19.7,
21.9, 25.4, 25.5, 28.2, 28.3, 29.0, 52.9, 53.0, 53.4, 59.5, 59.7,
59.8, 61.0, 69.8, 75.6, 77.8, 79.8, 98.7, 110.5, 111.9, 119.0, 119.1,
120.0, 120.1, 122.6, 122.7, 123.4, 128.1, 128.2, 136.7, 156.5,
173.0, 173.1; HRMS-FAB (M + Na+) calcd for C28H39N3NaO7
552.2686, found 552.2698.
Amide 32. Compound 31 (300 mg, 567 µmol) was dissolved
in CH2Cl2 (8 mL). To this solution, TFA (4 mL) was added.
The reaction was stirred at room temperature for 30 min. The
solvents were removed by rotary evaporation, and the residue
was diluted with EtOAc (150 mL). The organic layer was
washed with saturated aqueous Na2CO3 (100 mL) and brine
(100 mL) and then dried over Na2CO3. The solvent was
removed by rotary evaporation to give the corresponding
primary amine. The resulting amine was redissolved in DMF
(5 mL). To this solution, Cbz-Trp-OH (287 mg, 851 µmol),
HBTU (323 mg, 851 µmol), and DIEA (298 µL, 221 mg, 1.70
mmol) were added. The reaction was stirred at room temper-
ature for 1 h, and then the reaction mixture was partitioned
J. Org. Chem, Vol. 70, No. 21, 2005 8319

Xue and Seto
chromatography (EtOAc/hexanes, 1:18) to yield 36 as a
mixture of tautomers (2.63 g, 12.3 mmol, 64%): 1H NMR (300
MHz, CDCl3) δ 1.98-2.25 (m, 0.6H), 2.38-2.85 (m, 3.8H),
2.85-3.10 (m, 1.4H), 3.15-3.47 (m, 1.5H), 3.61-3.85 (m, 3.6H),
4.95-5.18 (m, 2H), 5.68-5.90 (m, 1H); 13C NMR (75 MHz,
CDCl3) δ 24.1, 29.1, 33.3, 33.5, 33.8, 34.0, 35.4, 36.4, 39.5, 51.3,
52.3, 53.0, 53.4, 57.8, 60.4, 77.7, 98.0, 118.1, 118.2, 135.0, 135.2,
136.0, 169.5, 169.6, 172.5, 174.8, 205.2, 205.4; HRMS-FAB (M
+ Na+) calcd for C10H14NaO3S 237.0561, found 237.0562.
Alcohol 37. Ketoester 36 (2.0 g, 9.4 mmol) was dissolved
in dry THF (150 mL) at 0 °C. To this solution, NaBH4 (1.04 g,
28.0 mmol) was added. After 30 min the reaction was quenched
with 1 N HCl (200 mL), and the THF was removed by rotary
evaporation. The resulting material was partitioned between
EtOAc (300 mL) and 1 N HCl (200 mL). The organic layer was
washed with 1 N HCl (200 mL), saturated aqueous NaHCO3
(200 mL), and brine (200 mL). It was then dried over MgSO4,
and the resulting solution was concentrated by rotary evapo-
ration. The crude oil was purified by flash chromatography
(EtOAc/hexanes, gradient of 1:9 to 1:4) to yield alcohol 37 as
a mixture of diastereomers (1.58 g, 7.3 mmol, 78%): 1H NMR
(300 MHz, CDCl3) δ 1.72-1.85 (m, 1H), 2.09-2.30 (m, 3H),
2.49-2.62 (d, J ) 9.8 Hz, 2H), 2.75-2.99 (m, 2H), 3.05-3.25
(t, J ) 9.6 Hz, 1H), 3.68-3.80 (s, 3H), 4.23 (s, 1H), 4.98-5.15
(m, 2H), 5.69-5.88 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 23.7,
26.5, 38.1, 43.3, 49.8, 52.5, 67.6, 117.5, 136.2, 174.7; HRMS-
FAB (M + Na+) calcd for C10H16NaO3S 239.0718, found
239.0721
Carboxylic Acid 38. To a solution of compound 37 (3.0 g,
13.9 mmol) in DMF (10 mL), imidazole (3.67 g, 20.9 mmol)
and TBDMSCl (5.21 g, 34.8 mmol) were added. The reaction
was stirred at 50 °C for 12 h and then diluted with EtOAc
(500 mL). The organic layer was washed with 1 N HCl (2 ×
300 mL), saturated aqueous NaHCO3 (300 mL), and brine (300
mL). It was then dried over MgSO4, and the solvent was
removed by rotary evaporation. The crude oil was purified by
flash chromatography (EtOAc/hexanes, 1:9) to give the corre-
sponding TBDMS ether. The TBDMS ether was dissolved in
MeOH (50 mL). To this solution, 1 N aqueous NaOH (50 mL)
was added. The mixture was heated at reflux for 24 h, and
the MeOH was removed by rotary evaporation. The aqueous
layer was partitioned between EtOAc (300 mL) and 1 N HCl
(300 mL). The organic layer was washed with brine (200 mL)
and then dried over MgSO4. The solvent was removed by
rotary evaporation, and the crude oil was purified by flash
chromatography (EtOAc/hexanes, gradient of 1:18 to 1:4) to
yield carboxylic acid 38 (2.75 g, 8.69 mmol, 42% for two
steps): 1H NMR (300 MHz, CDCl3) δ 0.06-0.08 (d, J ) 19.0
Hz, 6H), 0.89 (s, 9H), 1.94-1.99 (m, 1H), 2.12-2.25 (dm, J )
9.6 Hz, 1H), 2.25-2.39 (m, 1H), 2.40-2.59 (m, 2H), 2.80-3.00
(dm, J ) 11.7 Hz, 1H), 3.15-3.28 (t, J ) 13.0 Hz, 2H), 4.20-
4.25 (s, 1H), 4.96-5.16 (m, 2H), 5.62-5.88 (m, 1H), 9.35-11.43
(br, 1H); 13C NMR (75 MHz, CDCl3) δ -4.9, -4.1, 1.4, 16.9,
18.4, 21.2, 21.7, 23.0, 26.1, 26.4, 33.8, 40.8, 44.4, 59.5, 69.7,
71.1, 75.4, 117.5, 136.2, 177.9, 179.5; HRMS-FAB (M + Na+)
calcd for C15H28NaO3SSi 339.1426, found 339.1421.
Carbamate 39. To a solution of compound 38 (2.0 g, 6.33
mmol) in dry toluene (50 mL), DIEA (1.66 mL, 1.23 g, 9.49
mmol) and (PhO)2PON3 (2.05 mL, 2.61 g, 9.49 mmol) were
added. The reaction was heated at 80 °C under nitrogen for
16 h. The formation of the isocyanate was followed by IR
spectroscopy at 2256 cm-1. The alkoxide from benzyl alcohol
was prepared in a separate flask as follows: Benzyl alcohol
(2.05 mL, 2.05 g, 19.0 mmol) was added to dry THF (40 mL)
at 0 °C under nitrogen. To the same container, n-butyllithium
(5.06 mL, 2.5 M solution in hexanes, 12.7 mmol) was added
dropwise over a period of 5 min. The isocyanate solution was
then cooled to room temperature and added slowly to the
alkoxide solution at 0 °C. After the addition was complete, the
reaction was allowed to warm to room temperature over 30
min. The reaction was quenched with 1 N HCl (100 mL). The
THF was removed by rotary evaporation, and the remaining
solution was partitioned between EtOAc (300 mL) and 1 N
HCl (300 mL) and washed with saturated aqueous NaHCO3
(200 mL) and brine (200 mL). It was then dried over MgSO4,
and the solvents were removed by rotary evaporation. The
crude oil was purified by flash chromatography (EtOAc/
hexanes, 1:9) to yield carbamate 39 (1.92 g, 4.56 mmol, 72%):
1H NMR (300 MHz, CDCl3) δ 0.08 (s, 6H), 0.94 (s, 9H), 1.91-
2.03 (m, 1H), 2.10-2.20 (dd, J ) 9.9, 3.1 Hz, 1H), 2.30-2.55
(m, 3H), 2.78-2.92 (dd, J ) 9.7, 9.1 Hz, 1H), 3.65-3.75 (s,
1H), 3.95-4.10 (m, 1H), 4.95-5.25 (m, 5H), 5.66-5.85 (m, 1H),
7.30-7.45 (m, 5H); 13C NMR (75 MHz, CDCl3) δ -4.8, -4.6,
18.1, 25.8, 26.4, 28.2, 33.7, 41.7, 49.1, 66.7, 72.7, 117.3, 128.0,
128.1, 128.5, 135.9, 136.6, 155.5; HRMS-FAB (M + H+) calcd
for C22H35NNaO3SSi 444.2005, found 444.2003.
Carboxylic Acid 40. Compound 39 (1.00 g, 2.38 mmol) was
dissolved in a mixture of acetone (100 mL) and H2O (40 mL).
To this solution, NaIO4 (5.08 g, 23.8 mmol), KMnO4 (282 mg,
1.79 mmol), and NaHCO3 (205 mg, 2.38 mmol) were added.
The reaction was stirred at room temperature for 8 h. The
acetone was removed by rotary evaporation, and the remaining
material was partitioned between EtOAc (200 mL) and 1 N
HCl (200 mL). The organic layer was washed with 1 N HCl (2
× 200 mL) and brine (200 mL). It was then dried over MgSO4,
and the solvent was removed by rotary evaporation. The crude
oil was purified by flash chromatography (EtOAc/hexanes,
gradient of 1:2 to 2:1) to yield acid 40 (730 mg, 1.55 mmol,
65%): 1H NMR (300 MHz, CDCl3) δ 0.12-0.15 (m, 6H), 0.92
(s, 9H), 2.60-2.75 (m, 2H), 2.90-3.15 (m, 3H), 3.18-3.40 (m,
2H), 3.90-3.96 (s, 1H), 4.30-4.45 (m, 1H), 5.00-5.18 (m, 3H),
7.28-7.41 (m, 5H); 13C NMR (75 MHz, CDCl3) δ -4.6, -4.3,
14.6, 18.3, 23.1, 26.1, 32.0, 33.5, 36.4, 48.9, 51.4, 67.8, 70.7,
77.6, 128.7, 128.8, 129.0, 136.2, 155.5, 176.2; HRMS-FAB (M
+ Na+) calcd for C21H33NaNO7SSi 494.1645, found 494.1652.
Amides 41a and 41b. To compound 40 (400 mg, 849 µmol),
H2N-Trp(Boc)-OMe (406 mg, 1.27 mmol), HBTU (481 mg, 1.27
mmol), DIEA (445 µL, 330 mg, 2.54 mmol), and DMF (4 mL)
were added. The reaction was stirred at room temperature for
2 h and then partitioned between EtOAc (200 mL) and 1 N
HCl (200 mL). The organic layer was washed with 1 N HCl
(100 mL), saturated aqueous NaHCO3 (100 mL), and brine
(100 mL). It was then dried over MgSO4, and the solvent was
removed by rotary evaporation. The crude material was
purified by flash chromatography (EtOAc/hexanes, gradient
of 1:4 to 1:1) to give compound 41a (228 mg, 297 µmol, 35%):
1H NMR (300 MHz, CDCl3) δ 0.08-0.13 (d, J ) 6.8 Hz, 6H),
0.91 (s, 9H), 1.69 (s, 9H), 2.40-2.60 (m, 1H), 2.65-2.85 (m,
3H), 3.00-3.12 (dm, J ) 11.8 Hz, 1H), 3.15-3.35 (m, 4H), 3.70
(s, 3H), 3.90-3.95 (s, 1H), 4.30-4.45 (m, 1H), 4.80-5.05 (m,
2H), 5.10 (s, 2H), 6.35-6.45 (d, J ) 7.5 Hz, 1H), 7.18-7.27
(m, 2H), 7.29-7.45 (m, 5H), 7.46-7.60 (m, 2H), 8.05-8.20 (d,
J ) 7.3 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ -4.7, -4.3, 18.3,
26.1, 27.8, 28.6, 35.8, 37.1, 48.3, 48.7, 51.3, 52.9, 53.1, 67.6,
70.7, 84.2, 115.2, 115.8, 119.1, 123.1, 124.7, 125.0, 128.6, 128.8,
129.0, 130.6, 135.8, 136.3, 150.0, 155.4, 170.4, 172.2; HRMS-
FAB (M + Na+) calcd for C38H53N3NaO10SSi 794.3119, found
794.3128; and 41b (202 mg, 263 µmol, 31%): 1H NMR (300
MHz, CDCl3) δ 0.06-0.16 (d, J ) 13.5 Hz, 6H), 0.91 (s, 9H),
1.69 (s, 9H), 2.25-2.41 (m, 1H), 2.65-2.85 (m, 2H), 3.00-3.40
(m, 5H), 3.73 (s, 3H), 3.80-3.90 (s, 1H), 4.30-4.45 (m, 1H),
4.80-5.05 (m, 2H), 5.12 (s, 2H), 6.05-6.15 (d, J ) 6.8 Hz, 1H),
7.18-7.27 (m, 2H), 7.29-7.45 (m, 5H), 7.46-7.60 (m, 2H),
8.05-8.20 (d, J ) 7.3 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ
-4.6, -4.2, 18.3, 26.1, 27.7, 28.6, 35.8, 37.7, 47.1, 48.8, 51.4,
53.0, 53.1, 67.7, 71.0, 84.2, 115.3, 115.8, 119.1, 123.1, 124.5,
125.0, 128.6, 128.6, 128.8, 129.0, 130.6, 136.3, 149.9, 155.3,
170.6, 172.3; HRMS-FAB (M + Na+) calcd for C38H53N3NaO10-
SSi 794.3119, found 794.3134.
Amide 42a. To a solution of compound 41a (200 mg, 261
µmol) in dry MeOH (40 mL), 10% Pd on carbon (60 mg) was
added. The reaction flask was purged and kept under 1 atm
of hydrogen gas. The reaction was stirred at room temperature
for 2 h, and then the Pd/carbon was removed by filtration. The
8320 J. Org. Chem., Vol. 70, No. 21, 2005

Design of Inhibitors of the Serine Protease Plasmin
clear solution was concentrated by rotary evaporation, and the
residue was dried under vacuum for an additional 24 h to give
the corresponding primary amine. The resulting primary
amine was dissolved in DMF (3 mL). To this solution, Cbz-
Trp(Boc)-OH (320 mg, 467 µmol), HBTU (198 mg, 467 µmol),
and DIEA (137 µL, 101 mg, 784 µmol) were added. The
reaction was stirred at room temperature for 1 h and then
partitioned between EtOAc (60 mL) and 1 N HCl (60 mL). The
organic layer was washed with 1 N HCl (40 mL), saturated
NaHCO3 (40 mL), and brine (40 mL). It was then dried over
MgSO4, and the solvent was removed by rotary evaporation.
The crude oil was purified by flash chromatography (EtOAc/
hexanes, gradient of 1:4 to 1:2) to yield 42a (193 mg, 183 µmol,
70%): 1H NMR (300 MHz, CDCl3) δ -0.22 (s, 3H), -0.02 (s,
3H), 0.72 (s, 9H), 1.66 (s, 18H), 2.30-2.50 (m, 1H), 2.50-2.80
(m, 5H), 2.95-3.35 (m, 5H), 3.40-3.55 (s br, 1H), 3.67 (s, 3H),
4.22-4.42 (m, 1H), 4.43-4.67 (m, 1H), 4.80-4.95 (m, 1H),
5.00-5.25 (m, 2H), 5.40-5.65 (s br, 1H), 5.70-5.90 (s br, 1H),
6.50-6.60 (d, J ) 7.4 Hz, 1H), 7.15-7.26 (m, 2H), 7.28-7.42
(m, 7H), 7.41-7.56 (m, 3H), 7.57-7.75 (s br, 1H), 8.00-8.25
(s br, 2H); 13C NMR (75 MHz, CDCl3) δ -5.4, -4.8, 17.7, 25.5,
27.5, 28.1, 28.2, 28.7, 35.4, 36.7, 46.2, 47.7, 50.2, 52.5, 52.7,
55.4, 67.3, 70.0, 83.8, 84.2, 114.8, 115.1, 115.4, 115.6, 118.8,
119.1, 122.7, 123.0, 124.3, 124.6, 125.1, 128.3, 128.6, 129.8,
130.2, 135.5, 135.9, 149.4, 149.6, 155.7, 169.8, 170.0, 172.0;
HRMS-FAB (M + Na+) calcd for C54H71N5NaO13SSi 1080.4436,
found 1080.4405.
Amide 42b. Amide 42b was synthesized with a procedure
similar to that used for amide 42a (210 mg, 199 µmol, 76%):
1H NMR (300 MHz, CDCl3) δ -0.19 (s, 3H), -0.01 (s, 3H), 0.79
(s, 9H), 1.68 (s, 18H), 2.05-2.15 (m, 1H), 2.50-2.80 (m, 3H),
2.82-3.40 (m, 8H), 3.73 (s, 3H), 4.22-4.42 (m, 2H), 4.80-4.95
(m, 1H), 5.00-5.20 (m, 2H), 5.30-5.45 (d, J ) 6.1 Hz, 1H),
5.95-6.05 (d, J ) 6.0 Hz, 1H), 6.60-6.70 (d, J ) 7.4 Hz, 1H),
7.15-7.26 (m, 1H), 7.28-7.42 (m, 9H), 7.41-7.56 (m, 4H),
8.00-8.25 (m, 2H); 13C NMR (75 MHz, CDCl3) δ -4.8, -4.7,
14.2, 17.8, 21.1, 25.6, 27.3, 28.0, 28.3, 35.4, 37.4, 46.9, 50.4,
52.6, 52.8, 55.8, 60.4, 67.4, 70.3, 83.8, 84.1, 114.9, 115.4, 115.6,
118.7, 118.9, 119.1, 122.9, 124.1, 124.5, 124.9, 125.1, 128.3,
128.5, 128.7, 130.3, 135.3, 135.8, 149.4, 149.6, 155.9, 169.9,
170.3, 171.9; HRMS-FAB (M + Na+) calcd for C54H71N5NaO13-
SSi 1080.4436, found 1080.4415.
Alcohol 43a. To a solution of compound 42a (150 mg, 142
µmol) in dry THF (8 mL) was added a solution of TBAF (56
mg, 213 µmol) in dry THF (7 mL). The reaction was stirred at
room temperature for 30 min. The solvents were removed by
rotary evaporation, and the crude material was purified by
flash chromatography (EtOAc/hexanes, gradient of 2:1 to 5:1)
to give alcohol 43a (107 mg, 114 µmol, 81%): 1H NMR (300
MHz, CDCl3) δ 1.55-1.72 (s, 18H), 1.98 (s, 1H), 2.20-2.35 (m,
2H), 2.36-2.57 (m, 2H), 2.58-2.70 (dm, J ) 11.0 Hz, 1H),
2.71-2.82 (m, 1H), 3.00-3.22 (m, 4H), 3.23-3.31 (m, 1H),
3.32-3.45 (m, 1H), 3.60-3.75 (m, 4H), 4.28-4.41 (m, 1H),
4.41-4.55 (m, 1H), 4.85-4.95 (m, 1H), 5.00-5.20 (s, 2H), 5.75-
5.90 (d, J ) 8.1 Hz, 1H), 6.65-6.75 (d, J ) 8.1 Hz, 1H), 6.90-
7.00 (m, 1H), 7.16-7.26 (m, 2H), 7.28-7.41 (m, 7H), 7.42-
7.65 (m, 5H), 8.00-8.25 (s br, 2H); 13C NMR (75 MHz, CDCl3)
δ 1.1, 14.2, 27.4, 28.1, 28.2, 36.0, 47.5, 50.4, 52.4, 52.6, 55.7,
67.2, 84.1, 114.9, 115.1, 115.4, 115.6, 118.8, 122.7, 124.3, 124.5,
124.7, 124.8, 128.1, 128.3, 128.6, 130.3, 135.3, 136.0, 149.7,
156.1, 170.4, 172.1; HRMS-FAB (M + Na+) calcd for C48H57N5-
NaO13S 966.3571, found 966.3591.
3.05-3.20 (m, 4H), 3.21-3.33 (m, 2H), 3.34-3.60 (m, 2H), 3.72
(s, 3H), 4.30-4.65 (m, 2H), 4.80-4.95 (m, 1H), 5.00-5.20 (s,
2H), 5.55-5.65 (d, J ) 8.1 Hz, 1H), 6.60-6.70 (d, J ) 8.1 Hz,
1H), 6.80-7.10 (s br, 1H), 7.15-7.26 (m, 2H), 7.28-7.41 (m,
7H), 7.42-7.65 (m, 4H), 8.00-8.20 (m, 2H); 13C NMR (75 MHz,
CDCl3) δ 27.2, 27.9, 28.1, 28.2, 36.1, 47.4, 50.9, 52.3, 52.6, 55.2,
67.4, 76.7, 84.0, 84.2, 84.3, 114.9, 115.0, 115.4, 115.6, 118.7,
118.8, 119.1, 122.7, 122.9, 124.1, 124.3, 124.5, 124.8, 130.1,
130.3, 135.4, 135.9, 149.6, 149.7, 170.5, 172.9; HRMS-FAB (M
+ Na+) calcd for C48H57N5NaO13S 966.3571, found 966.3595.
Ketone 44. Alcohol 43a or 43b (50 mg, 53 µmol) was
dissolved in CH2Cl2 (5 mL). To this solution, Dess-Martin
reagent (33 mg, 79 µmol) was added. The reaction was stirred
at room temperature for 12 h and then partitioned between
EtOAc (25 mL) and saturated aqueous Na2CO3 (20 mL). The
organic layer was washed with saturated Na2CO3 (20 mL) and
brine (25 mL). It was then dried over MgSO4, and the solvent
was removed by rotary evaporation. The crude material was
purified by flash chromatography (EtOAc/hexanes, 5:1) to yield
ketone 44 as a mixture of two diastereomers (starting from
43a: 46 mg, 49 µmol, 92%; starting from 43b: 46 mg, 49 µmol,
92%): 1H NMR (300 MHz, CDCl3) δ 1.60-1.68 (m, 18H), 2.32-
2.78 (m, 3H), 3.00-3.38 (m, 6H), 3.40-3.57 (m, 1H), 3.58-
3.70 (m, 1H), 3.70-3.76 (m, 3H), 4.32-4.61 (m, 2H), 4.80-
4.94 (m, 1H), 5.00-5.21 (m, 2H), 5.50-5.70 (m, 1H), 6.20-
6.80 (m, 1H), 7.05-7.26 (m, 2H), 7.28-7.78 (m, 12H), 7.99-
8.22 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 14.6, 21.5, 27.6,
27.8, 28.6, 33.3, 34.3, 34.4, 36.1, 41.4, 42.1, 42.2, 50.3, 53.0,
53.1, 53.2, 53.3, 54.1, 54.2, 54.6, 54.9, 55.4, 55.6, 56.0, 60.8,
67.5, 68.1, 77.6, 84.4, 84.7, 95.1, 115.0, 115.1, 115.2, 115.4,
115.6, 115.8, 115.9, 119.1, 119.4, 119.5, 123.1, 123.2, 123.3,
124.5, 124.6, 124.8, 125.1, 125.2, 125.5, 128.38, 128.44, 128.5,
128.6, 128.7, 128.8, 128.92, 128.97, 129.03, 130.5, 130.8, 130.9,
131.0, 132.1, 133.6, 135.6, 135.8, 136.4, 142.1, 149.9, 150.0,
156.3, 168.7, 169.1, 169.4, 169.5, 171.3, 171.4, 171.6, 172.2,
172.4, 172.5, 201.0, 201.4, 201.7; HRMS-FAB (M + Na+) calcd
for C48H55N5NaO13S 964.3415, found 964.3405.
Inhibitor 6. Ketone 44 (50 mg, 47 µmol) was dissolved in
CH2Cl2 (2 mL). To this solution, TFA (0.5 mL) was added. The
reaction was stirred at room temperature for 1.5 h, and then
the solvents were removed by rotary evaporation. The crude
material was purified by flash chromatography (2-15% MeOH
in CH2Cl2) to yield inhibitor 6 as a mixture of two diastereo-
mers (26 mg, 36 µmol, 76%): 1H NMR (400 MHz, DMSO-d6) δ
2.05-2.45 (m, 2H), 2.60-2.79 (m, 1H), 2.80-3.22 (m, 5H),
3.39-3.48 (s, 2H), 3.49-3.62 (m, 3H), 3.63-3.81 (m, 1H), 4.15-
4.49 (m, 3H), 4.87-5.07 (m, 2H), 6.88-7.20 (m, 7H), 7.21-
7.39 (m, 7H), 7.40-7.70 (m, 3H), 8.30-8.59 (m, 1H), 10.70-
10.95 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 27.9, 28.0, 28.7,
31.3, 33.9, 42.2, 42.3, 52.7, 54.0, 54.1, 54.2, 54.3, 56.2, 66.1,
110.1, 110.3, 110.8, 110.9, 112.2, 112.3, 118.9, 119.1, 119.3,
121.7, 121.8, 124.6, 124.8, 127.9, 128.1, 128.3, 128.4, 128.6,
129.2, 136.9, 137.8, 138.2, 138.4, 139.7, 141.3, 156.7, 168.0,
170.55, 170.61, 170.7, 171.7, 172.2, 172.6, 172.8, 173.1, 173.2,
174.7, 201.4, 201.5; HRMS-FAB (M + Na+) calcd for C38H39N5-
NaO9S 764.2366, found 764.2379.
Acknowledgment. This research was supported by
the NIH NIGMS (Grant R01 GM057327).
Supporting Information Available: 1H and 13C NMR
spectra for all new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
Alcohol 43b. Alcohol 43b was synthesized with a procedure
similar to that used for amide 43a (106 mg, 113 µmol, 80%):
1H NMR (300 MHz, CDCl3) δ 1.60-1.80 (s, 18H), 1.90 (s, 1H),
2.06-2.20 (m, 1H), 2.22-2.65 (m, 3H), 2.70-3.05 (m, 2H),
JO0508954
J. Org. Chem, Vol. 70, No. 21, 2005 8321