Inhibitors of plasmin that extend into both the S and S′ binding sites: Cooperative interactions between S1 and S2

Article abstract of DOI:10.1021/jo0160569

A new procedure for the synthesis of cyclohexanone-based inhibitors of serine proteases is reported. In this procedure the reactive ketone functionality is carried through the synthesis in masked form as a TBDMS-protected alcohol. Deprotection followed by oxidation of the alcohol generates the final form of the inhibitor. Two new inhibitors, which interact with the S1-S3 and S2′ subsites of plasmin, are synthesized using this procedure. Inhibitors 1 and 2 have IC₅₀ values against plasmin of 20 and 24 μM, respectively. The inhibition studies show that cooperative binding of inhibitors in the S1 and S2 subsites of plasmin is important for determining inhibitor selectivity.

Full text of DOI:10.1021/jo0160569

1184
J . Org. Chem. 2002, 67, 1184-1191
In h ibitor s of P la sm in th a t Exten d in to Both th e S a n d S′ Bin d in g
Sites: Coop er a tive In ter a ction s betw een S1 a n d S2
Paul Abato, Courtney M. Yuen, J eanne Y. Cubanski, and Christopher T. Seto*
Department of Chemistry, Brown University, 324 Brook Street, Box H, Providence, Rhode Island 02912
Christopher_Seto@brown.edu
Received August 24, 2001
A new procedure for the synthesis of cyclohexanone-based inhibitors of serine proteases is reported.
In this procedure the reactive ketone functionality is carried through the synthesis in masked form
as a TBDMS-protected alcohol. Deprotection followed by oxidation of the alcohol generates the
final form of the inhibitor. Two new inhibitors, which interact with the S1-S3 and S2′ subsites of
plasmin, are synthesized using this procedure. Inhibitors 1 and 2 have IC50 values against plasmin
of 20 and 24 µM, respectively. The inhibition studies show that cooperative binding of inhibitors in
the S1 and S2 subsites of plasmin is important for determining inhibitor selectivity.
In tr od u ction
the diverse roles that are played by this enzyme in both
normal and pathological processes.6 In this paper we
report on two new inhibitors (compounds 1 and 2) that
are designed to make a variety of specific contacts with
the active site of plasmin. The aminohexyl, R1, and Cbz
groups are designed to bind in the S1-S3 subsites,
respectively, while the C-terminal Trp residue will bind
in the S2′ subsite. These multiple noncovalent interac-
tions position the ketone moiety of the inhibitors so that
it can react with the active site serine nucleophile. The
inhibitors show reasonable activity and moderate speci-
ficity for plasmin when compared to other related serine
proteases including trypsin, thrombin, and kallikrein.
Plasmin is a serine protease that is central to a number
of normal physiological processes such as lysis of fibrin
clots, tissue remodeling, and cell migration.1 There is now
mounting evidence that also implicates plasmin in the
processes of angiogenesis and metastasis during the
progression of cancer.2 Plasmin acts by hydrolyzing
components of the basement membrane including fibrin,
type IV collagen, fibronectin, and laminin. It also has an
indirect mode of action through hydrolytic activation of
matrix metalloproteases.2 During angiogenesis, degrada-
tion of the basement membrane allows epithelial cells to
migrate into the extracellular matrix and form new blood
vessels. Lesions in the basement membrane also promote
metastasis by allowing cancer cells to penetrate into the
underlying tissues and form secondary tumors.3 Thus,
plasmin is a potential target for the development of new
chemotherapeutic agents that could act by inhibiting
degradation of the basement membrane, and as a result,
inhibiting angiogenesis and metastasis.2
Design of In h ibitor s. Over the past several years we
have been investigating 4-heterocyclohexanone deriva-
tives as inhibitors for serine and cysteine proteases.7
These compounds are reversible inhibitors of the pro-
teases and are designed to give a reversibly formed
covalent bond between the enzyme active site nucleophile
and the electrophilic ketone functionality of the inhibitor.8
In previous studies we have synthesized inhibitors 3-5
(Table 1) that interact with the S1-S3 subsites of
plasmin.9 The aminohexyl side chains in compounds 3
and 4 are designed to form an electrostatic interaction
Most of the current pharmaceutical agents that inhibit
plasmin, such as ꢀ-aminocaproic acid and trans-4-amino-
methylcyclohexanecarboxylic acid, are targeted to the
lysine binding site.4 This binding site anchors plasmino-
gen, which is the inactive precursor to plasmin, to fibrin.5
Therefore, the compounds are good inhibitors of fibrin-
olysis. However, they do not affect the active site of the
protease, which is separate from the lysine binding site.
Inhibitors that are targeted directly to the active site of
plasmin may be useful as potential anticancer agents.
In addition, they may lead to a better understanding of
(1) (a) Dano, K.; Andreasen, P. A.; Grondahl-Hansen, J .; Kristensen,
P. L.; Nielsen, L. S.; Skriver, L. Adv. Cancer Res. 1985, 44, 139. (b)
Tryggvason, K.; Hoyhtya, M.; Salo, T. Biochim. Biophys. Acta 1987,
907, 191.
(2) (a) Pepper, J . S.; Montesano, R.; Mandriots, S. J .; Orci, L.;
Vassalli, J . Enzyme Protein 1996, 49, 138. (b) Kobayashi, H.; Shino-
hara, H.; Takeuchi, K.; Itoh, M.; Fujie, M.; Saitoh, M.; Terao, T. Cancer
Res. 1994, 54, 844.
(6) 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. Thrombosis 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 4.
(3) Liotta, L. A. Sci. Am. 1992, Feb., 54.
(4) Okada, Y.; Tsuda, Y.; Teno, N.; Wanaka, K.; Bohgaki, M.;
Hijikata-Okunomiya, A.; Naito, T.; Okamoto, S. Chem. Pharm. Bull.
1988, 36, 1289.
(7) Conroy, J . L.; Sanders, T. C.; Seto, C. T. J . Am. Chem. Soc. 1997,
119, 4285.
(5) Sherry, S. Fibrinolysis, Thrombosis, and Hemostasis; Lea &
Febiger: Philadelphia, 1992; chapter 1.
(8) Conroy, J . L.; Seto, C. T. J . Org. Chem. 1998, 63, 2367.
(9) Sanders, C. T.; Seto, C. T. J . Med. Chem. 1999, 42, 2969.
10.1021/jo0160569 CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/22/2002

Inhibitors of Plasmin
J . Org. Chem., Vol. 67, No. 4, 2002 1185
Sch em e 1a
Ta ble 1. In h ibition of P la sm in by Cycloh exa n on e-Ba sed
In h ibitor sa
compd Xaa
R
Yaa Zaa
Ki (µM)b
3c
4c
5c
6d
7d
8d
9d
Phe (CH2)6NH2
Trp (CH2)6NH2
50 ( 5, 130 ( 10
240, >1000
Phe
H
9000 ( 1000, 16000 ( 1300
Trp Trp 5 ( 0.6, 10 ( 2
Phe Trp 420 ( 40e
Trp Phe 100 ( 20, 150 ( 25
Phe Phe 3000 ( 1000e
a
b
One of two diastereomers of compounds 6-9 are shown. The
two values correspond to two separate diastereomers. c Data taken
from refs 9 and 11. Data take from ref 12. e Mixture of two
d
diastereomers.
with the aspartic acid residue at the base of the S1
subsite. This side chain is attached to the inhibitor via
the exocyclic amide nitrogen atom in a “peptoid-like”
arrangement;10 an arrangement that avoids formation of
a quaternary center R to the ketone functionality. The
length of the side chain was chosen based upon molecular
modeling studies. A comparison of the inhibition con-
stants for compounds 3 and 5 shows that binding of this
side chain into the S1 subsite is critical for good affinity
in this series of inhibitors. The Xaa and the D-isoleucine
side chains of the inhibitors are designed to bind in S2
and S3, respectively. Comparison of compounds 3 and 4
shows that, in this series, Phe is moderately preferred
over Trp at the P2 position. The difference in inhibition
constants between these two compounds is a factor of
approximately five.9,11
Compounds 6-9 represent a second series of inhibitors
that were synthesized using combinatorial methods.12
The Yaa and Zaa groups in these inhibitors are designed
to bind in the P2 and P2′ subsites of plasmin. Comparison
of compound 6 with 7, and 8 with 9, shows that, in
contrast to what we observed for inhibitors 3-5, in this
series Trp is strongly preferred over Phe at the P2 (Yaa)
position. It is important to note that, unlike inhibitors 3
and 4, compounds 6-9 do not incorporate a functional
group that interacts with the S1 subsite of the enzyme.
To further explore the specificity of the S2 subsite of
plasmin in the context of cyclohexanone-based inhibitors,
we have synthesized compounds 1 and 2. These inhibitors
are similar in structure to compounds 6 and 7, but they
also incorporate an aminohexyl group that can interact
with the S1 subsite of the protease. This work has three
major objectives. First, we have developed a revised
strategy for synthesizing the inhibitors. This new protocol
avoids one of the shortcomings of our previous synthetic
route (see below). Second, we have synthesized inhibitors
that are capable of forming multiple interactions with
the active site of serine proteases in an attempt to
increase their potency and specificity for plasmin over
other related proteases. Finally, we have explored the
possible role that cooperative binding with the S1 and
a
Reagents: (a) NaBH4, 60%; (b) TBDMSCl, 80%; (c) NaOH,
MeOH, 68%; (d) (C6H5O)2PON3; (e) BnOH, n-BuLi; (f) KMnO4,
NaIO4, 75% (three steps); (g) H2NTrp(Boc)OMe, HBTU, 84%; (h)
H2, Pd/C, 62%; (i) BocNH(CH2)5CHO, AcOH; NaBH4, 30%.
S2 subsites of plasmin plays in determining the relative
binding affinity and selectivity of the inhibitors.
In this work we do not address the specific question of
whether the ketones in inhibitors 1 and 2 react with the
active site serine of plasmin to give a reversibly formed
hemiketal adduct. However, in other related systems we
have unambiguously demonstrated the formation of this
type of adduct.7-9 As a result, we expect that the
inhibitors reported herein will react in an analogous
fashion.
Resu lts a n d Discu ssion
Syn th esis of In h ibitor s. In the syntheses of cyclo-
hexanone-based inhibitors that we have reported previ-
ously, the ketone moiety has been carried through the
syntheses as a protected cyclic acetal or thioacetal.7,9,12,13
The acetal is then removed at the end of the synthesis
to unmask the ketone using aqueous trifluoroacetic acid,
while the thioacetal can be deprotected using aqueous
N-bromosuccinimide. However, we have found that when
the inhibitors incorporate a tertiary amide functional
group as do compounds 1 and 2, this final deprotection
proceeds in poor yield and is accompanied by significant
hydrolysis of the acid labile tertiary amide. To avoid this
problem we have developed a modified synthetic strategy
in which the ketone is first reduced to the corresponding
alcohol, and then it is carried through the synthesis in
protected form as the TBDMS ether. Near the end of the
synthesis the TBDMS protecting group is removed and
the alcohol is subsequently reoxidized to give back the
ketone. This strategy is outlined in Schemes 1 and 2.
The synthesis began with reduction of â-ketoester 10
to give the corresponding alcohol 11 as a mixture of
(10) Simon, R. J .; Kania, R. S.; Zuchermann, R. N.; Huebner, V. D.;
J ewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 9367.
(11) Sanders, T. C. Ph.D. Dissertation, Brown University, Provi-
dence, RI, 2000.
(12) Abato, P.; Conroy, J . L.; Seto, C. T. J . Med. Chem. 1999, 42,
4001.
(13) Conroy, J . L.; Abato, P.; Ghosh, M.; Austermuhle, M. I.; Kiefer,
M. R.; Seto, C. T. Tetrahedron Lett. 1998, 39, 8253.

1186 J . Org. Chem., Vol. 67, No. 4, 2002
Abato et al.
Sch em e 2a
Ta ble 2. In h ibition of F ou r Ser in e P r otea ses by
In h ibitor s 1 a n d 2
IC50 (µM)a
serine
protease
1
2
plasmin
trypsin
thrombin
kallikrein
20 ( 7
160 ( 40
80 ( 60
36 ( 6
24 ( 1
140 ( 30
40 ( 10
130 ( 30
a
Mixture of two diastereomers.
with pyridinium chlorochromate to give the correspond-
ing ketones 23 and 24. Finally, the Boc protecting groups
on the tryptophan and aminohexyl side chains were
removed with trifluoroacetic acid. We were unable to
separate the two diastereomers of each of the inhibitors,
which were present in approximately a 1:1 mixture.
In h ibition Stu d ies. Inhibitors 1 and 2 were assayed
against four related serine proteases: plasmin, trypsin,
thrombin, and kallikrein (Table 2). All four of these
proteases have a specificity for substrates that incorpo-
rate a positively charged amino acid (either Lys or Arg)
at the P1 position. The proteases were assayed using UV
spectroscopy to monitor the hydrolysis of the p-nitro-
anilide substrates D-Val-Leu-Lys-pNA, D-Phe-Pip-Arg-
pNA, D-Phe-Pip-Arg-pNA, and D-Pro-Phe-Arg-pNA, re-
spectively. The assay mixtures contained 50 mM sodium
phosphate buffer at pH 7.4, and 10% DMSO to maintain
solubility of the inhibitors. Under these conditions, the
KM values for the four substrates were measured to be
180, 120, 30, and 60 µM, respectively.
a
Reagents: (a) CbzTrp(Boc)OH, HBTU, 69%; (b) CbzPheOH,
HBTU, 81%; (c) Zn(BF4)2 21: 79%, 22: 35%; (d) pyridinium
chlorochromate 23: 78%, 24: 45%; (e) TFA, 1: 67%, 2: 40%. One
of two diastereomers of compounds 1 and 2 are shown.
Compounds 1 and 2 are both reasonable inhibitors of
plasmin with IC50 values of 20 and 24 µM. These values
are the same within experimental error. Examination of
the inhibition constants of compounds 1-4 shows that
plasmin does not exhibit a strong bias for Phe over Trp
at the P2 position. For compounds 3 and 4 the preference
is approximately 5-fold for Phe, while for compounds 1
and 2 there is no preference. These observations are
consistent with data from the recent literature concerning
the substrate specificity of plasmin. Using combinatorial
methods, Ellman, Craik, and co-workers have constructed
libraries of peptide-aminomethylcoumarin (AMC) sub-
strates and screened them against a variety of pro-
teases.16 They found that plasmin prefers Lys and Arg
at P1, the aromatic amino acids Phe, Trp, and Tyr at P2,
and shows broad substrate specificity at P3. Substrates
containing Phe, Trp, and Tyr at the P2 position all had
similar activity, and there was no strong preference
among the three amino acids. In complementary studies,
Corey, Madison, and co-workers have examined the
substrate specificity of plasmin using phage display.17
They also found that the P2 position is specific for
aromatic amino acids, with no strong preference among
Phe, Trp, and Tyr. Thus, the results from studies with
synthetic inhibitors, AMC-based substrates, and the
purely peptide-based substrates are all in agreement and
show that plasmin prefers aromatic amino acids in
general at P2, but it does not discriminate among Phe,
Trp, and Tyr. All of these inhibitors and substrates have
one structural feature in common: they all possess Lys-
diastereomers, followed by protection of the alcohol with
TBDMSCl to give ester 12 (Scheme 1). The synthesis of
compound 10 has been reported previously.13 Ester 12
was saponified with NaOH to give carboxylic acid 13,
which was then treated with diphenylphosphoryl azide
in refluxing benzene to induce the Curtius rearrange-
ment. The resulting isocyanate was trapped with the
anion of benzyl alcohol to generate the Cbz-protected
amine 14. The alkene in compound 14 was then oxida-
tively cleaved with potassium permanganate and sodium
periodate to give carboxylic acid 15, which was coupled
with the methyl ester of Trp using HBTU to give the
corresponding amide 16. The indole nitrogen atom of Trp
was protected as the tert-butyl carbamate. Catalytic
hydrogenation of the Cbz protecting group gave amine
17, which was subjected to a two-step reductive alkyla-
tion procedure. First, the imine was formed between
compound 17 and N-Boc-6-aminohexanal using acetic
acid as a catalyst.14 Second, this imine was reduced with
sodium borohydride to give secondary amine 18 in modest
yield. Other reductive alkylation procedures using so-
dium cyanoborohydride or sodium triacetoxyborohydride
gave lower yields of the desired secondary amine.
The secondary amine 18 was next coupled with Cbz-
Trp(Boc)OH or CbzPheOH using HBTU to give com-
pounds 19 and 20 (Scheme 2). These two compounds
were subjected to the same three-step procedure to give
inhibitors 1 and 2. First, the TBDMS protecting group
was removed using zinc tetrafluoroborate to give alcohols
21 and 22.15 Second, the resulting alcohol was oxidized
(16) (a) Backes, B. J .; Harris, J . L.; Leonetti, F.; Craik, C. S.; Ellman,
J . A. Nat. Biotechnol. 2000, 18, 187. (b) Harris, J . L.; Backes, B. J .;
Leonetti, F.; Mahrus, S.; Ellman, J . A.; Craik, C. S. Proc. Natl. Acad.
Sci, USA 2000, 97, 7754.
(14) The synthesis of the requisite BocNH(CH2)5CHO has been
reported previously. See ref 9.
(15) Ranu, B. C.; J ana, U.; Majee, A. Tetrahedron Lett. 1999, 40,
1985.
(17) Hervio, L. S.; Coombs, G. S.; Bergstrom, R. C.; Trivedi, K.;
Corey, D. R.; Madison, E. L. Chem. Biol. 2000, 7, 443.

Inhibitors of Plasmin
J . Org. Chem., Vol. 67, No. 4, 2002 1187
or Arg-type side chains that extend into the S1 subsite
of the protease.
3 may be caused by the D-Ile residue with the free
N-terminus in compound 3. This residue may impart
significant selectivity for plasmin compared to the other
serine proteases. Replacing this residue with the simple
Cbz group that is present in compound 2 results in lower
specificity.
In summary, we have developed a new procedure for
the synthesis of cyclohexanone-based inhibitors of serine
proteases. This procedure avoids the removal of a ketone
protecting group during the late stages of the synthesis
under conditions that are not compatible with other
functional groups in the molecule. Inhibitors 1 and 2,
which result from this synthesis, are reasonable inhibi-
tors of plasmin and show modest selectivity when com-
pared to other related serine proteases. Comparison of
the IC50 values for these inhibitors with results from
previous studies highlight the fact that inhibitor selectiv-
ity can be dependent on cooperative binding interactions
between the S1 and S2 subsites.
The results summarized above can be contrasted with
what we observe for inhibitors 6-9. These inhibitors do
not incorporate a functional group that interacts with the
S1 subsite. Comparison of the inhibition constants for
compounds 6 and 7 shows an approximate 80-fold
selectivity for Trp over Phe at the P2 (Yaa) position.
Comparison of compounds 8 and 9 demonstrates a
similar high preference (approximately 30-fold) for Trp
at P2. What accounts for the apparent change in selectiv-
ity of the S2 subsite in the different studies?
One reasonable explanation for these observations is
the presence of cooperative binding between the S1 and
S2 sites. Inhibitors 1-5, the AMC-based substrates, and
the purely peptide substrates from phage display all
incorporate a positively charged side chain that binds in
the S1 subsite. This binding interaction could position
the molecules in the active site so that Phe, Trp, or Tyr
at the P2 position are all bound similarly in the S2
subsite. Thus, there is little discrimination among the
three aromatic amino acids. For compounds 6-9 that do
not interact with S1, the inhibitors may bind in a more
relaxed conformation that allows Trp to form more
favorable van der Waals or hydrogen bonding contacts
with residues in the S2 subsite, when compared to Phe.
This would explain the high selectivity for Trp at the P2
position that is observed for inhibitors 6-9. Since binding
in the S1 and S2 subsites is linked, the substrate
specificity of S2 can be altered depending on whether the
S1 subsite is occupied or not.
Inhibitors 6-9 and 1 and 2 are different in two
respects. First, compounds 1 and 2 incorporate an amino-
hexyl side chain at the P1 position. This group is not
present in 6-9, and we have attributed the differences
in specificity at the P2 position that we observe among
the different inhibitors to this structural feature. Second,
compounds 6-9 are C-terminal carboxylic acids, while 1
and 2 are methyl esters. If this second difference plays
an important role in determining inhibitor specificity, we
would expect inhibitor 1 to have a significantly different
inhibition constant than its corresponding carboxylic acid.
However, 1 and its free acid derivative have IC50 values
against plasmin that are the same within experimental
error.18 Thus, the difference between the C-terminal
carboxylic acid and methyl ester groups does not play an
important role in determining inhibitor selectivity.
Plasmin, trypsin, thrombin, and kallikrein all prefer
to cleave substrates that incorporate Lys or Arg at P1.
Thus, it is reasonable to expect that inhibitors 1 and 2
would have similar activity against all four proteases.
The inhibition data shown in Table 1 demonstrates that
we have been at least partly successful in our goal of
developing inhibitors that are specific for plasmin. Com-
pound 1 has 8- and 4-fold selectivity for plasmin when
compared to trypsin and thrombin, respectively. How-
ever, the selectivity between plasmin and kallikrein is
less than a factor of 2. Inhibitor 2 has reasonable
selectivity for plasmin when compared to trypsin and
kallikrein, but its selectivity against thrombin is less
than a factor of 2. By contrast, compound 3 has a greater
than 10-fold selectivity for plasmin when compared to
the other three serine proteases.9 The differences in
specificity that are observed between compounds 2 and
Exp er im en ta l Section
Gen er a l Meth od s. NMR spectra were recorded on Bruker
Avance-300 or Avance-400 instruments. Spectra were cali-
brated using TMS (δ ) 0.00 ppm) for 1H NMR and CDCl3 (δ
) 77.0 ppm) or CD3OD (δ ) 49.0 ppm) for 13C NMR. IR spectra
were recorded on a Perkin-Elmer 1700 series FT-IR spectrom-
eter. Mass spectra were recorded on a Kratos MS 80 under
electron impact (EI), chemical ionization (CI), or fast-atom
bombardment (FAB) conditions. HPLC analyses were per-
formed on a Rainin HPLC system with Rainin Microsorb silica
or C18 columns, and UV detection. Semipreparative HPLC
was performed on the same system using a semipreparative
column (21.4 × 250 mm).
Reactions were conducted under an atmosphere of dry
nitrogen in oven-dried glassware. Anhydrous procedures were
conducted using standard syringe and cannula transfer tech-
niques. THF was distilled from sodium and benzophenone.
Other solvents were of reagent grade and were stored over 4
Å molecular sieves. All reagents were used as received. Organic
solutions were dried over MgSO4 unless otherwise noted.
Solvent removal was performed by rotary evaporation at water
aspirator pressure.
Alcoh ol 11. Ketoester 10 (12.9 g, 57.6 mmol) was dissolved
in 20 mL of MeOH and cooled in an ice bath. To this solution
was added NaBH4 (4.40 g, 115.2 mmol) that was also dissolved
in 20 mL of ice cold MeOH. After 1 h the reaction was
quenched with 300 mL of 1 N HCl, and the methanol was
removed by rotary evaporation. The resulting material was
dissolved in 1 L of EtOAc, and the organic layer was washed
with 600 mL of 1 N HCl, 600 mL of saturated aqueous
NaHCO3, 500 mL of brine, and dried over MgSO4. The
resulting solution was concentrated by rotary evaporation
yielding 9.87 g of a clear oil. The crude material was purified
by flash chromatography (7-9% EtOAc in hexanes) to yield
11 (6.13 g, 27.1 mmol, 47%): 1H NMR (300 MHz, DMSO-d6) δ
1.19 (t, J ) 7.1 Hz, 5H), 1.24-1.33 (m, 3H), 1.37-1.46 (m, 1H),
1.48-1.60 (m, 1H), 1.59-1.74 (m, 2H), 1.94-2.19 (m, 2H), 2.29
(ddd, J ) 12.2, 4.1, 2.3 Hz, 1H), 3.97-4.12 (m, 3H), 4.25-4.61
(br s, 1H), 4.94 (dddd, J ) 10.2, 2.3, 1.1, 1.1 Hz, 1H), 5.01 (ddd,
J ) 17.1, 3.6, 1.5 Hz, 2H), 5.80 (dddd, J ) 16.9, 10.2, 6.6, 6.5
Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ 175.0, 140.0, 115.3,
68.6, 60.3, 48.9, 41.9, 32.5, 31.4, 25.6, 22.2, 15.0; HRMS-FAB
(M + Na+) calculated for C13H22NaO3 249.1467, found 249.1466.
TBDMS Eth er 12. To the alcohol 11 (4.64 g, 20.5 mmol)
was added 10 mL of DMF followed by DIEA (2.54 mL, 1.88 g,
24.6 mmol), imidazole (2.09 g, 31.0 mmol), and TBDMSCl (7.74
g, 51.3 mmol). The reaction was stirred at 50 °C for 12 h. The
mixture was diluted with 600 mL of EtOAc, and the organic
layer was washed with 2 × 500 mL of 1 N HCl, 500 mL of
saturated aqueous NaHCO3, and 500 mL of brine. The organic
layer was dried over MgSO4, and the solvent was removed by
rotary evaporation. The crude oil was purified by flash
(18) Abato, P.; Seto, C. Unpublished results.

1188 J . Org. Chem., Vol. 67, No. 4, 2002
Abato et al.
chromatography (3-9% EtOAc in hexanes) to give 12 (6.05 g,
17.8 mmol, 87%): 1H NMR (300 MHz, DMSO-d6) δ -0.10 (d,
J ) 14.8 Hz, 6H), 0.82 (s, 9H), 1.14 (t, J ) 6.2 Hz, 3H), 1.25-
1.35 (m, 2H), 1.35-1.58 (m, 4H), 1.61-1.77 (m, 3H), 1.94-
2.15 (m, 2H), 2.48-2.58 (m, 1H), 3.89-4.11 (m, 3H), 4.94-
5.06 (m, 2H), 5.80 (dddd, J ) 16.9, 10.2, 6.6, 6.5 Hz, 1H); 13C
NMR (75 MHz, DMSO-d6) δ 174.1, 139.5, 115.8, 73.1, 60.5,
44.3, 39.9, 32.0, 29.7, 26.4, 24.7, 23.3, 20.0, 18.5, 14.9, -3.6,
-4.4; HRMS-FAB (M + H+) calculated for C19H37O3Si
341.2512, found 341.2517.
Alk en e Acid 13. To compound 12 (5.50 g, 16.2 mmol) were
added 300 mL of MeOH and 300 mL of 66.7 mM aqueous
NaOH. The reaction was stirred at 85 °C for 72 h. The
methanol was removed by rotary evaporation, and the aqueous
layer was acidified with concentrated HCl to pH 2. The volume
of the aqueous layer was increased to 500 mL with 1 N HCl
and extracted with 3 × 400 mL of EtOAc. The organic layers
were combined, washed with 500 mL of brine, and dried over
MgSO4, and the solvent was removed by rotary evaporation.
The crude material was purified by flash chromatography (2-
50% EtOAc in hexanes) to yield 13 (3.53 g, 11.3 mmol, 70%):
1H NMR (300 MHz, DMSO-d6) δ -0.01(d, J ) 5.0 Hz, 6H),
0.83 (s, 9H), 1.13-1.27 (m, 2H), 1.26-1.56 (m, 4H), 1.56-1.72
(m, 4H), 1.95-2.19 (m, 2H), 2.33-2.46 (m, 1H), 3.95-4.06 (m,
1H), 4.89-5.09 (m, 2H), 5.78 (dddd, J ) 16.9, 10.2, 6.6, 6.5
Hz, 1H), 11.0-12.5 (br s, 1H); 13C NMR (75 MHz, MeOH-d4)
δ 177.1, 138.8, 114.2, 73.0, 44.0, 40.3, 31.9, 29.8, 25.4, 24.1,
22.7, 19.7, 17.9, -5.2, -5.8; HRMS-FAB (M + H+) calculated
for C17H33O3Si 313.2199, found 313.2194.
Ca r ba m a te 14. To a solution of 13 (3.00 g, 9.62 mmol) in
15 mL of toluene were added DIEA (2.01 mL, 1.49 g, 11.5
mmol) and diphenylphosphoryl azide (1.81 mL, 2.32 g, 11.5
mmol). The reaction was heated to 50 °C under nitrogen for 5
h. The formation of the isocyanate was monitored by the
increase in IR intensity at 2256 cm-1. The alkoxide from benzyl
alcohol was prepared in a separate flask as follows. Benzyl
alcohol (5.00 mL, 5.23 g, 48.1 mmol) was added to 30 mL of
THF at 0 °C under nitrogen, followed by dropwise addition of
n-butyllithium (11.6 mL of a 2.5 M solution in hexanes, 28.9
mmol) over a period of 10 min. The isocyanate solution was
then 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 quenched
by slow addition of 100 mL of 1 N HCl followed by removal of
the THF by rotary evaporation. The remaining solution was
diluted with 300 mL of EtOAc and washed with 300 mL of 1
N HCl, 300 mL of saturated aqueous Na2CO3, and 300 mL of
brine solution. The organic layer was dried over MgSO4, and
the solvents were removed by rotary evaporation. The crude
material was purified by flash chromatography (1:19 EtOAc/
hexanes) to yield 14 (2.21 g, 5.27 mmol, 55%): 1H NMR (300
MHz, DMSO-d6) δ 0.00 (s, 6H), 0.85 (s, 9H), 0.98-1.10 (m, 1H),
1.13-1.31 (m, 1H), 1.33-1.44 (m, 3H), 1.50-1.74 (m, 4H),
3.55-3.69 (m, 2H), 4.91-5.06 (m, 4H), 5.70-5.88 (m, 1H), 6.81
(d, J ) 7.4 Hz, 1H), 7.27-7.38 (m, 5H); 13C NMR (75 MHz,
CDCl3) δ 156.2, 138.9, 128.9, 128.4, 115.1, 74.0, 66.8, 50.7, 40.3,
32.0, 30.0, 28.2, 26.2, 25.1, 20.1, 18.5, 11.8, -4.3, -4.4; HRMS-
FAB (M + H+) calculated for C24H40NO3Si 418.2778, found
418.2797.
Ca r boxylic Acid 15. To compound 14 (2.20 g, 5.28 mmol)
was added 200 mL of acetone followed by 66 mL of an aqueous
solution containing the following: NaIO4 (5.64 g, 26.4 mmol),
KMnO4 (0.835 g, 5.28 mmol), and NaHCO3 (0.488 g, 5.81
mmol). The solution was stirred for 18 h at room temperature.
The acetone was removed by rotary evaporation, and the
remaining material was diluted with 600 mL of EtOAc and
washed with 3 × 500 mL of 1 N HCl and 400 mL of brine. The
organic layer was dried over MgSO4, and the solvent was
removed by rotary evaporation. The crude material was
purified by flash chromatography (10-50% EtOAc in hexanes)
to yield 15 (1.61 g, 3.95 mmol, 75%): 1H NMR (300 MHz,
DMSO-d6) δ 0.01 (s, 6H), 0.85 (s, 9H), 0.98-1.10 (m, 1H), 1.30-
1.45 (m, 4H), 1.55-1.80 (m, 4H), 2.10-2.30 (m, 2H), 3.56-
3.70 (m, 2H), 4.99 (d, J ) 12.7 Hz, 2H), 5.04 (d, J ) 12.4 Hz,
1H), 7.35-7.40 (m, 5H), 12.00 (br s, 1H); 13C NMR (75 MHz
CDCl3) δ 179.3, 156.4, 137.0, 128.9, 128.7, 128.5, 73.8, 67.0,
50.7, 40.5, 23.5, 29.1, 28.1, 26.2, 20.0, 18.4, -4.4; HRMS-FAB
(M + Na+) calculated for C23H37NaNO5Si 458.2339, found
458.2327.
F m oc-Tr p (Boc)-OMe. To commercially available Fmoc-
Trp(Boc)-OH (3.00 g, 5.69 mmol) were added 15 mL of
methanol, DMAP (69.5 mg, 0.569 mmol), and EDC (2.18 g,
11.4 mmol). The reaction was allowed to stir at room temper-
ature for 1 h. The reaction was diluted with 500 mL of EtOAc
and washed with 500 mL of 1 N HCl, 500 mL of saturated
aqueous Na2CO3, and 500 mL of brine. The organic layer was
dried over MgSO4, and the solvent was removed by rotary
evaporation. The resulting material was purified by flash
chromatography (3:7 EtOAc/hexanes) to yield compound Fmoc-
Trp(Boc)-OMe (2.56 g, 4.74 mmol, 83%): 1H NMR (300 MHz,
CDCl3) δ 1.69 (s, 9H), 3.27-3.33 (m, 2H), 3.74 (s, 3H), 4.24 (t,
J ) 7.1 Hz, 1H), 4.35-4.47 (m, 2H), 4.76-4.83 (m, 1H), 5.45
(d, J ) 7.4 Hz, 1H), 7.23-7.35 (m, 4H), 7.37-7.45 (m, 3H),
7.56 (dd, J ) 15.1, 7.6, Hz, 3H), 7.79 (d, J ) 8.5 Hz, 2H), 8.15
(d, J ) 8.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 172.5, 156.2,
150.0, 144.2, 141.7, 135.8, 130.9, 128.1, 127.5, 125.6, 125.0,
124.6, 123.1, 120.4, 119.2, 115.8, 115.3, 84.2, 67.6, 54.6, 52.9,
47.6, 28.6, 28.3; HRMS-FAB (M + Na+) calculated for C32H32
NaN2O6 563.2158, found 563.2153.
-
H2N-Tr p (Boc)-OMe. To compound Fmoc-Trp(Boc)-OMe
(2.50 g, 4.62 mmol) was added 15 mL of a 1:1 solution of
piperidine:DMF. The reaction was stirred at room temperature
for 30 min, after which time it was diluted with 500 mL of
EtOAc. The reaction was washed with 2 × 500 mL of saturated
aqueous Na2CO3 and 500 mL of brine. The organic layer was
dried over MgSO4, and the solvent was removed by rotary
evaporation. The crude material was purified by flash chro-
matography (1:1 EtOAc/hexanes) to yield H2N-Trp(Boc)-OMe
(0.995 g, 3.13 mmol, 67%): 1H NMR (300 MHz, MeOH-d4) δ
1.67 (s, 9H), 3.06 (ddd, J ) 14.4, 6.7, 0.8 Hz, 1H), 3.16 (ddd, J
) 14.3, 6.0, 0.9 Hz, 1H), 3.67 (s, 3H), 3.85 (t, J ) 6.2 Hz, 1H),
7.22-7.34 (m, 2H), 7.50 (s, 1H), 7.56-7.58 (m, 1H), 8.12 (d, J
) 8.1 Hz, 1H); 13C NMR (75 MHz, MeOH-d4) δ 175.4, 149.9,
136.0, 130.7, 124.5, 124.3, 122.7, 119.0, 116.2, 115.2, 83.8, 54.3,
51.6, 30.1, 27.5; HRMS-FAB (M + H+) calculated for
C
17H22N2O4 319.1658, found 319.1666.
Am id e 16. To compound 15 (0.921 g, 2.12 mmol) were added
H2N-Trp(Boc)-OMe (0.875 g, 2.73 mmol), HBTU (1.61 g, 4.24
mmol), DIEA (1.48 mL, 1.10 g, 8.47 mmol), and 4 mL of DMF.
The reaction was stirred for 2.5 h at room temperature and
diluted with 500 mL of EtOAc. The organic layer was washed
with 2 × 400 mL of 1 N HCl, 400 mL of saturated aqueous
Na2CO3, and 400 mL of brine. The organic layer was dried
over MgSO4, and the solvent was removed by rotary evapora-
tion. The crude material was purified by flash chromatography
(1:2 EtOAc/hexanes) to give 16 (1.31 g, 1.78 mmol, 84%): 1H
NMR (300 MHz, CDCl3) δ -0.12 to -0.05 (m, 6H), 0.86-0.93
(m, 9H), 1.04-1.16 (m, 1H), 1.35-1.54 (m, 6H), 1.72 (s, 9H),
1.81-1.90 (m, 3H), 2.10-2.23 (m, 2H), 3.21-3.38 (m, 2H)
3.80-3.91 (m, 4H), 4.85-5.00 (m, 2H), 5.09 (s, 2H), 6.01 (t, J
) 8.9 Hz, 1H), 7.15-7.45 (m, 8H), 7.49 (d, J ) 7.5 Hz, 1H)
8.12 (d, J ) 7.9 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ 170.2,
153.9, 147.5, 134.8, 133.3, 128.6, 126.5, 126.0, 122.6, 120.0,
116.8, 113.3, 113.1, 81.7, 71.6. 64.4, 50.5, 48.4, 38.2, 38.0, 32.3,
26.7, 26.2, 25.8, 24.2, 17.6, 16.0, 12.2, -6.7, -6.8; HRMS-
FAB (M + H+) calculated for C40H58N3O8Si 736.3993, found
736.3992.
Am in e 17. To compound 16 (2.73 g, 3.71 mmol) were added
20 mL of MeOH and 10% Pd on carbon (1.0 g). The reaction
flask was then purged with and kept under 1 atm of hydrogen
gas while stirring for 3 h at room temperature. The reaction
was then filtered through a pad of Celite, and the solvent was
removed by rotary evaporation. The crude material was
purified by flash chromatography (5-10% MeOH in CH2Cl2)
to give 17 (2.84 g, 3.07 mmol, 83%): 1H NMR (300 MHz,
MeOH-d4) δ -0.04-0.13 (m, 6H), 0.87-0.94 (m, 9H), 0.99-
1.12 (m, 1H), 1.20-1.36 (m, 2H), 1.36-1.49 (m, 3H), 1.51-
1.66 (m, 3H), 1.69 (s, 9H), 1.73-1.94 (m, 2H), 2.06-2.34 (m,
2H), 2.80-2.90 (m, 1H), 3.05-3.17 (m, 1H), 3.26 (d, J ) 5.3
Hz, 1H), 3.43-3.48 (m, 1H), 3.72 (s, 3H), 4.76-4.88 (m, 1H),

Inhibitors of Plasmin
J . Org. Chem., Vol. 67, No. 4, 2002 1189
7.23-7.35 (m, 2H), 7.50 (s, 1H), 7.59 (d, J ) 7.7 Hz, 1H), 8.11
(d, J ) 8.1 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ 170.7,
170.3, 147.0, 133.3, 128.6, 122.6, 122.0, 120.6, 116.8, 113.3,
81.7, 50.5, 50.4, 48.6, 36.7, 32.3, 28.8, 26.2, 25.4, 25.1, 24.0,
17.4, 12.2, -6.4, -6.6; HRMS-FAB (M + H+) calculated for
9H), 1.64 (s, 9H), 1.73-1.87 (m, 2H), 2.00 (dt, J ) 11.7, 3.1
Hz, 1H), 2.54 (dt, J ) 13.3, 4.3 Hz, 1H), 2.72-2.85 (m, 1H),
2.89-3.05 (m, 1H), 3.05-3.15 (m, 3H), 3.18-3.40 (m, 3H), 3.55
(s, 2H), 3.72 (s, 3H), 4.58 (br s, 1H), 4.76-4.94 (m, 3H), 5.12
(dd, J ) 14.1, 6.6 Hz, 1H), 5.31 (s, 1H), 5.97 (d, J ) 8.9 Hz,
1H), 7.15-7.26 (m, 5H), 7.31-7.41 (m, 3H), 7.49 (s, 1H), 7.59
(d, J ) 7.0 Hz, 1H), 7.79 (d, J ) 7.6 Hz, 1H), 7.94 (d, J ) 7.8
C
32H52N3O6Si 602.3625, found 602.3648.
Secon d a r y Am in e 18. To compound 17 (1.39 g, 2.32 mmol)
were added 15 mL of CDCl3, N-Boc-6-aminohexaldehyde14
(2.50 g, 11.6 mmol), AcOH (155 µL, 163 mg, 2.58 mmol) and
1.0 g of 4 Å molecular sieves, and the reaction was heated to
Hz, 1H), 8.05 (d, J ) 7.7 Hz, 1H), 8.14 (d, J ) 8.1 Hz, 1H); 13
C
NMR (75 MHz, CDCl3) δ 173.3, 172.9, 171.3, 156.4, 156.2,
150.0, 149.7, 136.4, 135.8, 135.7, 131.0, 130.3, 128.8, 128.5,
128.3, 125.6, 124.9, 124.2, 124.0, 122.9, 119.4, 119.3, 116.2,
115.8, 115.7, 115.1, 84.0, 83.9, 79.4, 73.9, 67.3, 54.9, 53.8, 52.9,
52.7, 50.9, 45.9, 45.5, 41.0, 35.1, 32.3, 31.2, 30.1, 30.0, 29.8,
29.6, 28.9, 28.6, 27.9, 27.5, 26.8, 26.2, 25.7, 25.7, 25.1, 23.1,
20.3, 18.1, -4.4, -4.7; HRMS-FAB (M + Na+) calculated for
1
50 °C. Formation of the imine was monitored using H NMR
spectroscopy by following the appearance of a resonance for
the imine at 9.36 ppm and disappearance of a resonance for
the aldehyde at 9.77 ppm. After formation of the imine was
complete, the reaction was poured into a solution of NaBH4
(5.00 g, 135 mmol) in 200 mL of methanol at 0 °C, and the
solution was allowed to warm to room temperature over 30
min. The volume of the solution was then reduced to 50 mL
by rotary evaporation. This solution was diluted with 500 mL
of EtOAc, washed with 500 mL of saturated Na2CO3 and 500
mL of brine. The organic layer was dried over MgSO4 and the
solvents were removed by rotary evaporation. The resulting
material was purified by flash chromatography (1:1 EtOAc/
hexanes to elute the N-Boc-6-aminohexanol, followed by a
gradient of 0.1:0.9:99 to 1:9:90 concentrated aqueous NH4OH:
MeOH:CH2Cl2) to give compound 18 (0.574 g, 0.835 mmol,
36%): 1H NMR (300 MHz, DMSO-d6) δ -0.1-0.5 (m, 6H),
0.81-0.84 (m, 9H), 0.84-0.87 (m, 1H), 1.10-1.30 (m, 7H),
1.30-1.35 (m, 1H), 1.37 (s, 9H), 1.39-1.60 (m, 4H), 1.62 (s,
9), 1.90-2.15 (m, 3H), 2.25-2.38 (m, 1H), 2.50-2.61 (m, 0.5H),
2.75-2.81 (m, 0.5H), 2.85-2.91 (m, 2H), 2.95-3.04 (m, 1H),
3.13 (dd, J ) 14.9, 5.0 Hz, 1H), 3.36-3.46 (m, 1H), 3.61 (s,
3H), 4.50-4.60 (m, 1H), 6.65-6.80 (m, 1H), 7.29 (ddd, J ) 15.2,
7.2, 7.3 Hz, 2H), 7.51 (s, 1H), 7.58 (d, J ) 7.5 Hz, 1H), 8.03 (d,
J ) 8.1 Hz, 1H), 8.27 (dd, J ) 7.7, 3.1 Hz, 1H); 13C NMR (75
MHz, DMSO-d6) δ 173.1, 172.6, 156.4, 149.9, 135.7, 131.0,
125.0, 124.4, 123.0, 119.2, 115.7, 115.4, 84.1, 79.4, 77.9, 77.7,
77.5, 77.0, 58.0, 52.9, 47.6, 41.0, 39.6, 34.9, 34.8, 30.9, 39.5,
30.1, 29.6, 28.8, 28.1, 27.8, 27.2, 26.9, 26.2, 19.8, 18.4, -3.0,
-4.0, -4.2; HRMS-FAB (M + H+) calculated for C43H73N4O8-
Si 801.5198, found 801.5225.
Cbz-Tr p (Boc)-OH. To commercially available H2N-Trp-
(Boc)-OH (1.48 g, 5.14 mmol) were added 25 mL of DMF, DIEA
(1.5 mL, 1.13 g, 11.3 mmol) and 1-benzyloxycarbonyl-benzo-
triazole (1.95 g, 7.71 mmol). The reaction was allowed to stir
for 3 h at room temperature. The reaction was then diluted
with 1 L of EtOAc and washed with 3 × 600 mL of 1 N HCl
and 400 mL of brine. The organic layer was dried over MgSO4
and the solvent removed by rotary evaporation. The crude
material was purified by flash chromatography (1-5% MeOH
in CH2Cl2) to yield compound Cbz-Trp(Boc)-OH (2.02 g, 4.61
mmol, 90%): 1H NMR (300 MHz, DMSO-d6) δ 1.67 (s, 9H),
3.27 (dd, J ) 14.9, 6.2 Hz, 1H), 3.37 (dd, J ) 14.9, 5.2 Hz,
1H), 4.80 (dd, J ) 13.2, 6.0 Hz, 1H), 5.12 (d, J ) 3.8 Hz, 2H),
5.50 (d, J ) 7.9 Hz, 1H), 7.20 (t, J ) 7.3 Hz, 1H), 7.29-7.33
(m, 2H), 7.35-7.37 (m, 4H), 7.50 (s, 1H), 7.56 (d, J ) 7.8 Hz,
1H), 8.12 (d, J ) 8.1 Hz, 1H), 9.37 (br s, 1H); 13C NMR (75
MHz, CDCl3) δ 175.9, 156.6, 150.1, 136.5, 136.0, 131.0, 129.5,
129.0, 128.9, 125.0, 124.8, 123.1, 119.4, 115.7, 115.4, 84.3, 67.6,
61.1, 54.3, 28.6, 28.0, 21.5, 14.6; HRMS-FAB (M + H+)
calculated for C24H27NaN2O6 461.1689, found 461.1702.
Am id e 19. To compound 18 (132 mg, 165 µmol) were added
410 µL of DMF, DIEA (147 µL, 107 mg, 825 µmol), Cbz-Trp-
(Boc)-OH (223 mg, 494 µmol), and HBTU (250 mg, 660 µmol).
The reaction was stirred at 40 °C for 6 h followed by the
addition of 100 mL of methylene chloride. The organic layer
was washed with 50 mL of 0.25 N HCl, 100 mL of saturated
aqueous NaHCO3, and 100 mL of brine. The organic layer was
then dried over MgSO4. After the solvents were removed by
rotary evaporation, the crude material was purified by flash
chromatography (2% MeOH in methylene chloride) to give
compound 19 as a mixture of diastereomers (140 mg, 114 µmol,
69%): 1H NMR (300 MHz, CDCl3) δ -0.19 (s, 3H), -0.12 (s,
3H), -0.01-0.16 (m, 1H), 0.82 (s, 9H), 0.87-1.01 (m, 2H),
1.03-1.21 (m, 3H), 1.22-1.37 (m, 5H), 1.46 (s, 13H), 1.63 (s,
C
67H95NaN6O13Si 1243.6703, found 1243.6693.
Alcoh ol 21. To compound 19 (35 mg, 29 µmol) was added
1.3 mL of MeCN followed by 1.56 mL of a 92 mM solution of
Zn(BF4)2 in 1:9 water:MeCN. The reaction was allowed to stir
at room temperature for 7 h. The solvents were then removed
by rotary evaporation, and the crude material was diluted with
15 mL of EtOAc and washed with 10 mL of saturated aqueous
Na2CO3 and 10 mL of brine. The organic layer was dried over
MgSO4, and the solvents were removed by rotary evaporation.
The resulting material was purified by flash chromatography
(1.5% MeOH in methylene chloride) to give compound 21 as a
mixture of diastereomers (25 mg, 23 µmol, 79%): 1H NMR (300
MHz, MeOH-d4) δ 0.10-0.30 (m, 1H), 0.73-0.83 (m, 1H),
0.85-1.07 (m, 4H), 1.13 (br s, 1H), 1.20-1.39 (m, 9H), 1.48-
1.52 (m, 2H), 1.53-1.58 (m, 2H), 1.63 (s, 10H), 1.66-1.71 (m,
13H), 2.02-2.23 (m, 1H), 2.17-2.34 (m, 1H), 2.35-2.60 (m,
1H), 2.85-2.97 (m, 1H), 3.03 (t, J ) 6.9 Hz, 3H), 3.06-3.14
(m, 2H), 3.15-3.24 (m, 2H), 3.24-3.31 (m, 1H), 3.41-3.59 (m,
1H), 3.74 (s, 3H), 3.76-3.83 (m, 1H), 4.79 (s, 2H), 4.94-4.99
(m, 1H), 5.00-5.19 (m, 1H), 7.10-7.21 (m, 3H), 7.23-7.40 (m,
9H), 7.45 (s, 1H), 7.51 (s, 1H), 7.54-7.81 (m, 1H), 7.67 (d, J )
7.7 Hz, 1H), 8.01 (d, J ) 7.8 Hz, 1H), 8.13 (d, J ) 8.3 Hz, 1H);
13C NMR (75 MHz, CDCl3) δ 172.9, 172.3, 171.9, 156.4, 156.1,
150.6, 150.0, 149.7, 136.6, 136.3, 135.9, 135.6, 135.2, 131.0,
130.3, 128.9, 128.7, 128.6, 128.3, 128.2, 128.9, 125.7, 125.2,
125.0, 124.8, 124.7, 124.4, 123.7, 123.2, 122.9, 119.3, 119.2,
119.1, 115.9, 115.7, 115.5, 115.3, 84.9, 84.2, 84.0, 79.4, 77.7,
73.2, 72.0, 67.4, 67.2, 60.8, 54.7, 53.8, 53.0, 52.9, 52.3, 51.9,
51.8, 45.2, 41.8, 40.9, 40.7, 35.2, 35.0, 33.7, 32.3, 31.0, 30.2,
29.5, 28.9, 28.5, 28.1, 27.8, 27.4, 26.4, 26.2, 25.4, 25.0, 24.5,
24.1, 23.1, 21.5, 20.2, 14.6; HRMS-FAB (M + Na+) calculated
for C61H82NaN6O13 1129.5838, found 1129.5867.
Keton e 23. Compound 21 (25 mg, 23 µmol) was treated with
a solution of PCC (15 mg, 70 µmol) and Al2O3 (90 mg, 883 µmol)
in 2.3 mL of methylene chloride. The reaction was allowed to
stir for 5 h at room temperature. The solvent was then
removed by rotary evaporation, and the crude material was
diluted with 15 mL of EtOAc, washed with 10 mL of saturated
aqueous Na2CO3, and 10 mL of brine. The organic layer was
dried over MgSO4 and the solvent removed by rotary evapora-
tion. The resulting material was purified by flash chromatog-
raphy (gradient of 1-2% MeOH in methylene chloride) to give
compound 23 as a mixture of two diastereomers (20 mg, 18
µmol, 78%): 1H NMR (300 MHz, MeOH-d4) δ 0.80-0.97 (m,
2H), 1.07 (s, 3H), 1.20-1.36 (m, 6H), 1.45 (s, 12H), 1.62 (s,
4H), 1.63 (s, 3H), 1.65 (s, 9H), 1.67 (s, 1H), 1.91-2.12 (m, 2H),
2.41-2.63 (m, 1H), 2.85-3.08 (m, 4H), 3.08-3.20 (m, 2H), 3.25
(d, J ) 5.2 Hz, 1H), 3.27-3.40 (m, 2H), 3.68-3.73 (m, 3H),
4.20-4.35 (m, 1H), 4.77 (s, 1H), 5.04-5.15 (m, 2H), 6.35-6.56
(m, 1H), 7.69 (d, J ) 7.5 Hz, 0.5H), 7.75 (d, J ) 7.9 Hz, 0.5H),
7.95-8.04 (m, 0.5H), 7.43 (s, 1H), 7.46-7.51 (m, 2H), 7.53-
7.58 (m, 1H), 7.64 (d, J ) 8.5 Hz, 0.5H), 7.14-7.38 (m, 10H),
8.04-8.15 (m, 1H); 13C NMR (75 MHz, CD2Cl2) δ 209.2, 208.9,
172.9, 172.5, 172.3, 172.2, 156.4, 156.1, 156.0, 155.9, 150.1,
149.9, 149.7, 136.7, 136.2, 136.0, 135.7, 131.0, 130.3, 128.8,
128.6, 128.5, 128.2, 126.0, 125.1, 124.9, 124.5, 123.0, 119.3,
119.2, 116.2, 116.0, 115.8, 115.7, 115.4, 84.3, 84.1, 84.0, 79.4,
77.7, 67.4, 67.2, 62.7, 52.8, 52.7, 51.4, 51.1, 50.7, 50.5, 48.8,
48.4, 47.0, 44.7, 40.7, 34.2, 32.3, 31.6, 30.6, 30.3, 30.0, 29.5,
28.8, 28.6, 28.2, 27.9, 27.6, 27.1, 26.4, 20.3, 19.8; HRMS-FAB

1190 J . Org. Chem., Vol. 67, No. 4, 2002
Abato et al.
(M + Na+) calculated for C61H80NaN6O13 1127.5681, found
1127.6714.
1.06-1.18 (m, 2H), 1.28 (br s, 12H), 1.44 (s, 10H), 1.51-1.59
(m, 3H), 1.68 (s, 9H), 1.91-2.02 (m, 1H), 2.10-2.28 (m, 2H),
2.45-2.68 (m, 1H), 2.85-2.96 (m, 1H), 2.97-2.16 (m, 3H),
3.16-3.35 (m, 2H), 3.60-3.75 (m, 3H), 4.49-4.67 (m, 1H),
4.87-4.99 (m, 2H), 5.01-5.12 (m, 2H), 5.45-5.57 (m, 1H),
6.40-6.55 (m, 1H), 7.10-7.37 (m, 10H), 7.38-7.44 (m, 1H),
7.50 (d, J ) 7.3 Hz, 1H), 8.00-8.16 (m, 1H); 13C NMR (75 MHz,
CDCl3) δ 173.0, 172.7, 171.8, 171.5, 156.6, 156.4, 150.0, 136.5,
136.2, 135.7, 131.0, 130.0, 129.8, 129.6, 129.2, 129.1, 129.0,
128.9, 128.6, 128.5, 128.3, 127.6, 127.5, 124.9, 124.5, 123.0,
119.2, 115.7, 115.5, 84.1, 79.4, 77.7, 67.5, 60.8, 55.7, 55.1, 53.0,
52.8, 47.1, 40.9, 38.1, 36.6, 34.3, 32.3, 30.4, 30.1, 28.8, 28.6,
27.8, 27.3, 27.2, 27.0, 26.9, 23.0, 21.5, 19.5, 14.6, 14.5; HRMS-
ESI (M + H+) calculated for C54H74N5O11 968.5385, found
968.5408.
In h ibitor 1. To compound 23 (20 mg, 18 µmol) was added
1 mL of a 1:1 solution of TFA:methylene chloride. The reaction
was allowed to stir at room temperature for 20 min, the solvent
was removed by rotary evaporation, and the residue was dried
under vacuum. The crude material was diluted with 15 mL of
EtOAc and washed with 10 mL of saturated aqueous Na2CO3
and 10 mL of brine. The organic layer was then dried over
MgSO4 and the solvent removed by rotary evaporation. The
resulting material was purified by flash chromatography (5%
MeOH in methylene chloride) to give inhibitor 1 as a mixture
of two diastereomers (10 mg, 12 µmol, 67%): 1H NMR (300
MHz, MeOH-d4) δ 1.00-1.15 (m, 2H), 1.19-1.35 (m, 3H),
1.61-1.82 (m, 18H), 1.82-1.95 (m, 1H), 1.96-2.20 (m, 1H),
2.25-2.39 (m, 2H), 2.56-2.78 (m, 1H), 2.82-3.19 (m, 3H),
3.19-3.23 (m, 1H), 3.25 (d, J ) 5.2 Hz, 1H), 3.48-3.59 (m,
1H), 3.67-3.74 (m, 3H), 4.20-4.35 (m, 1H), 4.63-4.73 (m, 1H),
4.78 (s, 1H), 4.92-4.96 (m, 1H), 5.08 (d, J ) 4.0 Hz, 1H), 7.10-
7.20 (m, 1H), 7.22-7.33 (m, 8H), 7.35-7.38 (m, 1H), 7.41-
7.43 (m, 1H), 7.48-7.53 (m, 1H), 7.53-7.58 (m, 1H), 7.69 (d,
J ) 7.4 Hz, 0.5H), 7.75 (d, J ) 7.7 Hz, 0.5H), 8.00 (d, J ) 7.7
Hz, 0.5H), 8.13 (q, J ) 8.1 Hz, 1H), 8.20 (d, J ) 8.2 Hz, 0.5H);
13C NMR (75 MHz, CD2Cl2) δ 209.7, 209.4, 173.2, 173.0, 172.4,
172.1, 156.0, 137.1, 136.9, 136.6, 128.8, 128.7, 128.3, 128.2,
127.9, 124.3, 124.1, 124.0, 123.7, 123.0, 122.2, 120.7, 119.6,
118.8, 118.7, 112.1, 111.7, 110.7, 110.3, 110.2, 67.1, 63.0, 52.6,
52.5, 51.8, 50.1, 46.1, 44.7, 41.9, 34.2, 33.8, 33.6, 32.9, 32.6,
32.1, 31.0, 30.4, 30.1, 29.4, 29.0, 28.3, 28.0, 27.1, 26.6, 26.5,
25.6, 24.4, 20.3, 19.7; HRMS-FAB (M + Na+) calculated for
Keton e 24. Alcohol 22 (125 mg, 129 µmol) was subject to a
solution containing PCC (109 mg, 505 µmol) and Al2O3 (375
mg, 3.68 mmol) in 6.25 mL of methylene chloride. The reaction
was allowed to stir for 8 h at room temperature. The solvent
was removed by rotary evaporation, and the crude material
was diluted with 150 mL of EtOAc and washed with 100 mL
of saturated aqueous Na2CO3 and 100 mL of brine. The organic
layer was dried over MgSO4 and the solvent removed by rotary
evaporation. The resulting material was purified by flash
chromatography (gradient of 1-2% MeOH in methylene
chloride) to give compound ketone 24 as a mixture of two
diastereomers (56 mg, 58 µmol, 45%): 1H NMR (300 MHz,
CDCl3) δ 0.80-1.01 (m, 1H), 1.05-1.35 (m, 6H), 1.45 (s, 9H),
1.54-1.59 (m, 2H), 1.60-1.63 (m, 10H), 1.75-1.94 (m, 5H),
1.96-2.12 (m, 3H), 2.18-2.29 (m, 1H), 2.33-2.65 (m, 2H),
2.76-2.94 (m, 1H), 2.95-3.16 (m, 4H), 3.16-3.31 (m, 2H),
3.60-3.77 (m, 3H), 4.36-4.54 (m, 1H), 4.64-4.83 (m, 1H),
4.86-4.99 (m, 1H), 5.00-5.15 (m, 2H), 6.17 (t, J ) 8.0 Hz, 1H),
6.50-6.67 (m, 1H), 7.10-7.36 (m, 13H), 7.40-7.60 (m, 2H),
7.95-8.16 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 210.4, 210.0,
209.2, 208.6, 173.0, 172.7, 172.6, 172.7, 172.4, 172.2, 172.0,
171.8, 171.7, 171.5, 170.9, 170.7, 156.4, 156.1, 156.0, 150.0,
137.1, 136.8, 136.6, 135.7, 131.0, 130.0, 129.7, 129.5, 129.1,
129.0, 128.8, 128.5, 128.4, 128.2, 128.1, 127.5, 127.4, 127.2,
125.0, 124.9, 124.6, 123.0, 119.3, 119.2, 116.3, 115.7, 115.4,
84.2, 84.1, 84.0, 79.4, 77.6, 67.4, 67.2, 67.1, 62.5, 60.8, 56.5,
55.6, 53.0, 52.9, 52.7, 50.6, 49.5, 49.4, 48.3, 40.8, 40.4, 40.3,
39.2, 35.0, 34.7, 34.4, 34.1, 32.8, 32.5, 30.3, 30.2, 29.7, 29.3,
28.8, 28.6, 28.2, 27.8, 27.1, 27.0, 26.7, 26.7, 26.6, 21.4, 20.5,
20.3, 20.0, 19.8, 14.6; HRMS-FAB (M + Na+) calculated for
C
46H56NaN6O7 827.4109, found 827.4099.
Am id e 20. To the compound 18 (464 mg, 579 µmol) were
added 410 µL of DMF, DIEA (515 µL, 374 mg, 2.90 mmol),
commercially available Cbz-Phe-OH (520 mg, 1.74 mmol), and
HBTU (878 mg, 2.32 mmol). The reaction was stirred at 40
°C for 6 h, and then 150 mL of methylene chloride was added.
The organic layer was washed with 150 mL of 0.25 N HCl,
150 mL of saturated aqueous NaHCO3, and 150 mL of brine
and then dried over MgSO4. After the solvents were removed
by rotary evaporation, the crude material was purified by flash
chromatography (2% MeOH in methylene chloride) to give
compound 20 as a mixture of diastereomers (507 mg, 469 µmol,
81%): 1H NMR (300 MHz, DMSO-d6) δ -0.40-0.10 (m, 6H),
0.74-0.87 (m, 9H), 0.91-1.04 (m, 1H), 1.07-1.27 (m, 6H), 1.37
(br s, 10H), 1.43-1.53 (m, 4H), 1.61 (s, 5H), 1.62 (s, 4H), 2.10-
2.25 (m, 2H), 2.69 (s, 3H), 2.74 (s, 1H), 2.80-2.95 (m, 5H),
2.98-3.07 (m, 2H), 3.09-3.21 (m, 2H), 3.57-3.70 (m, 3H),
4.47-4.61 (m, 2H), 4.86-4.95 (m, 2H), 4.95-5.08 (m, 1H),
6.67-6.80 (m, 1H), 7.15-7.35 (m, 12H), 7.49-7.70 (m, 2H),
C
54H71NaN5O11 988.5048, found 988.5077.
In h ibitor 2. To ketone 24 (24 mg, 25 µmol) was added 1
mL of a 1:1 solution of TFA:methylene chloride. The reaction
was allowed to stir at room temperature for 20 min, the solvent
was removed by rotary evaporation, and the residue was dried
under vacuum. The resulting crude material was dissolved in
15 mL of EtOAc and washed with 10 mL of saturated aqueous
Na2CO3 and 10 mL of brine. The organic layer was dried over
MgSO4 and the solvent removed by rotary evaporation. The
resulting material was purified by flash chromatography (5%
MeOH in methylene chloride) to give inhibitor 2 as a mixture
of two diastereomers (6 mg, 8 µmol, 25%): 1H NMR (300 MHz,
CDCl3) δ 0.61-1.05 (m, 3H), 1.10-1.42 (m, 8H), 1.43-1.92 (m,
8H), 1.93-2.10 (m, 3H), 2.14-2.25 (m, 1H), 2.26-2.70 (m, 3H),
2.80-3.29 (m, 2H), 3.25-3.55 (m, 1H), 3.61-3.85 (m, 3H),
4.25-4.56 (m, 2H), 4.80-5.00 (m, 1H), 5.01-5.20 (m, 2H),
5.21-5.50 (m, 1), 5.90-6.25 (m, 1H), 6.45-6.85 (m, 1H), 7.05-
7.45 (m, 13H), 7.46-7.75 (m, 1H), 8.30-6.65 (m, 1H); 13C NMR
(75 MHz, CDCl3) δ 213.5, 210.5, 210.2, 208.6, 208.4, 173.1,
172.8, 172.7, 172.5, 171.6, 171.4, 171.0, 170.9, 170.7, 156.4,
136.7, 136.5, 129.7, 129.2, 129.0, 128.6, 128.4, 128.0, 127.7,
127.6, 123.7, 123.5, 123.3, 123.1, 122.7, 122.6, 120.1, 120.0,
118.9, 111.9, 111.7, 110.2, 110.1, 77.6, 67.6, 60.8, 58.5, 58.3,
56.5, 55.5, 53.0, 52.8, 52.7, 49.5, 49.3, 49.0, 39.1, 39.0, 36.2,
36.0, 35.5, 35.1, 34.8, 34.3, 34.1, 34.0, 33.9, 32.5, 32.3, 30.1,
29.8, 28.2, 28.0, 27.8, 27.3, 26.9, 25.4, 25.3, 23.8, 23.7, 23.1,
7.75-7.89 (m, 1H), 7.98-8.05 (m, 1H), 8.28-8.40 (m, 1H); 13
C
NMR (75 MHz, CDCl3) δ 173.6, 173.5, 173.2, 172.8, 172.7,
172.6, 171.2, 170.4, 163.0, 156.4, 156.1, 156.0, 155.9, 150.0,
137.5, 136.8, 136.7, 136.5, 136.4, 135.7, 131.0, 130.0, 129.8,
129.7, 129.6, 129.3, 129.1, 129.0, 128.9, 128.8, 128.5, 128.4,
128.2, 128.1, 127.7, 127.5, 127.4, 125.0, 124.5, 124.4, 124.1,
123.0, 122.9, 119.3, 119.2, 116.2, 115.7, 84.1, 84.0, 79.4, 77.7,
73.7, 72.4, 67.4, 67.2, 60.8, 54.9, 53.0, 52.9, 52.8, 52.7, 52.6,
45.9, 45.7, 42.5, 42.1, 41.0, 40.9, 40.6, 40.5, 39.0, 36.9, 35.3,
34.7, 34.4, 32.1, 31.8, 30.4, 30.1, 29.5, 28.8, 28.6, 28.3, 28.2,
27.8, 27.4, 26.9, 26.8, 26.5, 26.3, 26.2, 26.1, 26.0, 24.8, 24.3,
24.1, 21.0, 20.8, 19.8, 18.3, 18.1, 14.6, -4.2, -4.3, -4.5, -4.7,
-4.9; HRMS-FAB (M + Na+) calculated for C60H87NaN5O11
Si 1104.6069, found 1104.6048.
-
Alcoh ol 22. To compound 20 (428 mg, 396 µmol) was added
20.1 mL of MeCN followed by 21.4 mL of a 92 mM solution of
Zn(BF4)2 in 1:9 water:MeCN. The reaction was allowed to stir
at room temperature for 7 h. The solvents were removed by
rotary evaporation, and the residue was dissolved in 250 mL
of EtOAc and washed with 250 mL of saturated aqueous Na2-
CO3 and 250 mL of brine. The organic layer was then dried
over MgSO4 and the solvent removed by rotary evaporation.
The resulting material was purified by flash chromatography
(1.5% MeOH in methylene chloride) to give compound 22 as a
mixture of diastereomers (135 mg, 140 µmol, 35%): 1H NMR
(300 MHz,CDCl3) δ 0.67-0.79 (m, 1H), 0.80-0.96 (m, 3H),
21.5, 19.8, 14.6; HRMS-FAB (M + Na+) calculated for C44H55
NaN5O7 788.4000, found 788.3964.
-
En zym e Assa ys. IC50 values for inhibitors 1 and 2 were
determined for plasmin, kallikrein, thrombin, and trypsin

Inhibitors of Plasmin
J . Org. Chem., Vol. 67, No. 4, 2002 1191
using the chromogenic substrates D-Val-Leu-Lys-pNA, d-Pro-
Phe-Arg-pNA, D-Phe-Pip-Arg-pNA, and D-Phe-Pip-Arg-pNA,
respectively. Enzymes and substrates were purchased from
Sigma or Chromogenix (distributor: DiaPharma Group, Inc.).
Initial rates were determined by monitoring the formation of
p-nitroaniline at 405 nm using a Fluostar Galaxy UV/
fluorescence plate reader from BMG Labtechnologies, Inc. in
a 384-well format (plate purchased from Nalge Nunc Inter-
national). Rate data was collected from 3 to 15 min after
preparation of the assay mixtures. In all cases the amount of
enzyme used was adjusted to give rates of approximately 3 ×
10-4 au/s, and in all cases conversion was kept to less than
10% of substrate. All assay mixtures contained 50 mM sodium
phosphate buffer adjusted to pH 7.4. DMSO (10%) was needed
in the assay mixtures to keep the inhibitors soluble. During
the determinations of IC50 values the substrates were kept at
a concentration of KM. The KM values for the respective
substrates of plasmin, kallikrein, thrombin, and trypsin were
measured to be 180, 60, 30, and 120 µM. The absorbance data
was analyzed using Microsoft Excel and IC50 values were
determined using a Dixon analysis with the commercial
graphing package Grafit (Erithacus Software Ltd.).
Ack n ow led gm en t. This research was supported by
the NIH (Grant GM57327), and the U.S. Army Medical
Research and Materiel Command-Breast Cancer Re-
search Initiative (Grant DAMD17-96-1-6161, Career
Development Award to C.T.S.). P.A. was supported by
a GAANN Fellowship from the Department of Educa-
tion.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra for all new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O0160569