4-heterocyclohexanone-based inhibitors of the serine protease plasmin

Article abstract of DOI:10.1021/jm990110k

Three inhibitors that are based upon a 4-heterocyclohexanone nucleus were synthesized and evaluated for activity against the serine protease plasmin. Inhibitors of plasmin have potential as cancer chemotherapeutic agents that act by blocking both angiogen

Full text of DOI:10.1021/jm990110k

J . Med. Chem. 1999, 42, 2969-2976
2969
4-Heter ocycloh exa n on e-Ba sed In h ibitor s of th e Ser in e P r otea se P la sm in
Tanya C. Sanders and Christopher T. Seto*
Department of Chemistry, Brown University, 324 Brook Street, Box H, Providence, Rhode Island 02912
Received March 11, 1999
Three inhibitors that are based upon a 4-heterocyclohexanone nucleus were synthesized and
evaluated for activity against the serine protease plasmin. Inhibitors of plasmin have potential
as cancer chemotherapeutic agents that act by blocking both angiogenesis and metastasis.
Inhibitor 1 has moderate activity against plasmin but shows good selectivity for this enzyme
compared to other serine proteases including trypsin, thrombin, and kallikrein. Inhibitor 2
shows both good activity and selectivity for plasmin. Inhibitor 3, which does not incorporate
an aminohexyl group that can interact with the S1 subsite, has poor activity. These results,
along with previous work, demonstrate that the 4-heterocyclohexanone nucleus can effectively
serve as the basis for designing inhibitors of both serine and cysteine proteases.
In tr od u ction
olysis inhibitors have no effect on the active site of the
enzyme, which retains its catalytic activity. Thus plas-
min that is already activated retains its catalytic
activity even after treatment with inhibitors that are
directed toward the lysine binding site. To overcome this
problem, we are interested in developing inhibitors that
are targeted to the active site of plasmin and are
designed to shut down catalytic activity. In this paper
we report the synthesis and evaluation of compounds
1-3 which are active site- directed inhibitors of plasmin.
Compound 2 has both good activity and specificity
against plasmin when compared to several other serine
proteases.8
Angiogenesis and metastasis are two processes that
are central to the progression of cancer. As such, they
have become important targets for the development of
chemotherapeutic agents. Several recent reports in the
literature have demonstrated that suppressing angio-
genesis is an effective method for limiting the growth
of primary tumors and producing dormancy in second-
ary metastases.1,2 Both angiogenesis and metastasis
require a proteolytic cascade that involves serine, cys-
teine, and metalloproteases. This proteolytic cascade
degrades the basement membrane which surrounds
blood vessels.3 During angiogenesis the resulting lesion
in the basement membrane allows epithelial cells to
extend into the neighboring tissues and form new blood
vessels. During metastasis cancer cells penetrate through
the degraded basement membrane and extracellular
matrix, become implanted in the underlying tissues, and
subsequently form secondary tumors.4 Compounds which
inhibit enzymes in the proteolytic cascade may be useful
for blocking these processes.
Plasmin is a serine protease that plays an important
role in the proteolytic cascade. This protease acts
directly by hydrolyzing components of the basement
membrane such as fibrin, type IV collagen, fibronectin,
and laminin and also acts indirectly by activating other
enzymes in the cascade such as matrix metallopro-
teases.3 Degradation of the basement membrane by
plasmin is a multistep process. For example, during the
first step in fibrin hydrolysis, plasminogen, which is the
inactive precursor to plasmin, binds to fibrin via a lysine
binding site. Next plasminogen is converted to active
plasmin in a reaction that is catalyzed by urokinase
plasminogen activator. Finally catalytic residues in the
active site of plasmin, which is separate from the lysine
binding site, hydrolyze fibrin via the mechanism that
is common to serine proteases.5 Most current pharma-
ceutical agents that are designed to inhibit plasmin are
targeted to the lysine binding site.6 These agents inhibit
fibrinolysis by blocking the binding of plasminogen to
fibrin, and thus halting production of new plasmin. R2-
Antiplasmin, a natural plasmin inhibitor, is also tar-
geted to the lysine binding site.7 However these fibrin-
Design of In h ibitor s
We have recently reported a new class of inhibitors
for cysteine proteases that are based upon a 4-hetero-
cyclohexanone pharmacophore.9 13C NMR studies using
a
13C-labeled inhibitor confirm that these molecules
react with the enzyme to give a reversibly formed
covalent hemithioketal adduct between the active site
cysteine residue and the ketone of the inhibitor.10 The
key design feature in these molecules is the through-
space electrostatic repulsion that occurs between the
heteroatom and ketone functionalities in the 4-hetero-
cyclohexanone pharmacophore. This repulsive interac-
tion controls the electrophilicity of the ketone, which in
turn controls the potency of the inhibitors.9
Because serine and cysteine proteases share a similar
mechanism for hydrolyzing amide bonds, we expect that
4-heterocyclohexanones should be good inhibitors of
both classes of enzymes. Reaction of the active site
nucleophile of a serine protease with a 4-heterocyclo-
hexanone-based inhibitor would give a reversibly formed
hemiketal adduct. However, several reversible protease
10.1021/jm990110k CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/07/1999

2970 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Sanders and Seto
Sch em e 1a
F igu r e 1. Shifting of the P1 side chain from the position R to
the ketone to the exocyclic nitrogen to avoid formation of the
quaternary center. R ) (CH2)6NH2.
inhibitors show activity against one class of enzyme and
not the other. For example, trifluoromethyl ketones and
boronic acids are good inhibitors of serine proteases11
but not cysteine proteases.12,13 Nitriles have the opposite
specificity, while aldehydes and R-dicarbonyl compounds
are good inhibitors of both classes of enzymes.13 Thus
one of our motivations for synthesizing compounds 1-3
was to determine if 4-heterocyclohexanones would prove
to have activity against serine proteases, in addition to
cysteine proteases as we have shown previously.9
Plasmin has a strong specificity for substrates with
positively charged side chains in the P1 position. To
accommodate this specificity we have included a lysine-
like side chain in the structure of compounds 1 and 2.
However, attachment of this side chain in its “natural”
peptide-like position would place it on the tetrahy-
drothiopyranone ring between the ketone and the exo-
cyclic nitrogen (Figure 1). This placement would create
a sterically demanding quaternary center R to the
reactive ketone. Space-filling molecular models suggest
that this quaternary center would sterically inhibit
addition of an active site nucleophile to the ketone and
thus decrease the potency of the inhibitor. To overcome
this difficulty we have attached the P1 side chain to the
amide nitrogen that is connected to the ring. This type
of modification is well-precedented in peptoids.14 To
ensure that the lysine-like side chain of the inhibitor
makes good contact with the aspartic acid at the base
of the S1 binding site, we have increased the length of
the aminoalkyl chain to six carbons. This chain length
is based upon molecular modeling studies of inhibitor
2 bound in the active site of trypsin. The X-ray crystal
structure of the active site of plasmin has not been
solved; however, the active sites of plasmin and trypsin
share significant homology.15
a
Reagents and conditions: (a) TFA, 85%; (b) 10, NaBH(OAc)3,
22%; (c) TFA, H2O, triisopropylsilane, thioanisole, 50%; (d) 6 N
HCl, 99%.
Sch em e 2a
a
Reagents and conditions: (a) (Boc)2O, 88%; (b) pyridinium
chlorochromate, 91%.
Sch em e 3a
Compound 1 contains three functionalities that are
designed to make specific contacts with the active site.
The ketone will react with the active site nucleophile
to give a hemiketal. In addition one of the aminoalkyl
chains will bind in the S1 subsite, while the second
aminoalkyl chain will extend along the main channel
of the active site to make contacts with the S3 subsite.
Okada and co-workers have shown that peptide-based
substrates and inhibitors that contain a free N-terminus
at the P3 position bind well to the enzyme.16 In
compound 2, one of the aminoalkyl chains has been
replaced by phenylalanine and D-isoleucine in order to
include additional functionality that will interact with
the S2 and S3 subsites.16 The sulfur atom was incor-
porated into the cyclohexanone rings of the three
inhibitors because the related tetrahydrothiopyranone-
based inhibitor of the cysteine protease papain had good
activity and its synthesis was relatively straightfor-
ward.9 Compound 3, which lacks an aminoalkyl func-
tionality, was synthesized in order to determine how
a
Reagents and conditions: (a) 10, NaBH(OAc)3, dichloroethane,
50%; (b) Fmoc-Phe-F, DIEA, 12 (75%); (c) Fmoc-Phe, EDC, HOBT,
13 (36%); (d) piperidine, DMF, 14A and 14B (67%), 15A and 15B
(81%); (e) Boc-D-Ile, EDC, HOBT, 16A (78%), 16B (63%), 17A
(88%), 17B (86%); (f) TFA, H2O, TIS, thioanisole, 2A (50%), 2B
(9%), 3A (34%), 3B (82%). A and B represent two different
diastereomers.
much the P1 side chain contributes to the affinity of the
inhibitors for plasmin. We have also synthesized com-
pound 22 (Scheme 4), which is similar in structure to 3
but lacks the electrophilic ketone functionality. This
molecule provides a useful control for probing the
mechanism of inhibition by inhibitors 1-3.

Inhibitors of the Serine Protease Plasmin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2971
Sch em e 4a
Ta ble 1. Inhibition of Serine Proteases by Inhibitors 1-3
and 22
Ki (µM)
compda
plasmin
trypsin
thrombin
kallikrein
>10000
1
400 ( 35
50 ( 5
130 ( 10
9000 ( 1000
16000 ( 1300
520 ( 30
1400 ( 110 >10000
1700 ( 1500 720 ( 550
2A
2B
3A
3B
22
630 ( 125
a
A and B represent two different diastereomers.
Control compound 22 was synthesized in a manner
similar to that of inhibitor 2 (Scheme 4). Reductive
alkylation of cyclohexylamine with aldehyde 10 gave
secondary amine 19. This material was coupled to Fmoc-
Phe-F followed by removal of the Fmoc protecting group
to give compound 20. After a second coupling reaction
with Boc-D-Ile, the Boc protecting groups were removed
to give 22.
a
Reagents and conditions: (a) 10, NaBH(OAc)3, dichloroethane,
25%; (b) Fmoc-Phe-F, DIEA; (c) piperidine, DMF, 66% (2 steps);
(d) Boc-D-Ile, EDC, HOBT, 91%; (e) TFA, triisopropylsilane,
ethanedithiol, 59%.
Resu lts a n d Discu ssion
Compound 1, which incorporates two simple amino-
hexyl side chains, was assayed against four different
serine proteases; plasmin, trypsin, thrombin, and kal-
likrein (Table 1). All of these proteases have a strong
specificity for positively charged side chains such as
lysine or arginine at the P1 position and thus provide a
reasonable test of the specificity of the inhibitors for
plasmin compared to other related enzymes. Compound
1 has modest activity against plasmin with an inhibition
constant of 400 µM. It has greater than 25-fold selectiv-
ity for this protease when compared to thrombin and
kallikrein and a 3-fold selectivity when compared to
trypsin. The similar affinity of this inhibitor for plasmin
and trypsin is reasonable based upon the sequence
homology between the two enzymes.15
Ch em istr y
The synthesis of inhibitor 1, which is outlined in
Scheme 1, began with deprotection of the Boc-protected
nitrogen in compound 4 with trifluoroacetic acid to give
amine 5. The synthesis of 4 has been reported previ-
ously.9 Dialkylation of 5 by reductive amination with 2
equiv of aldehyde 10 gave the tertiary amine 6. The Boc
protecting groups were removed with TFA to give 7, and
the ketal was hydrolyzed using aqueous HCl to give
inhibitor 1. Aldehyde 10 was synthesized starting from
6-amino-1-hexanol (8) (Scheme 2). The amino group in
8 was first protected using (Boc)2O to give alcohol 9,
followed by oxidation of the alcohol using pyridinium
chlorochromate.
To increase both the potency and specificity of the
inhibitors, we have replaced one of the aminohexyl
chains in compound 1 with a D-Ile-L-Phe dipeptide to
give inhibitors 2A and 2B. The free N-terminus of the
D-Ile residue in these compounds positions a positive
charge in the S3 enzyme subsite, which has been shown
to be beneficial for binding.16 In addition, the D-Ile and
Phe side chains provide hydrophobic contacts with the
S2 and S3 subsites. Compound 2A is a good inhibitor
of plasmin with an inhibition constant of 50 µM. By
comparison it has significantly lower affinity for trypsin,
thrombin, and kallikrein. The low activity of this
inhibitor against trypsin is somewhat surprising given
the reported similarity between the active sites of
plasmin and trypsin.16 Lineweaver-Burk analysis of 2A
against plasmin demonstrates that it is a reversible
competitive inhibitor. This observation is consistent
with a mechanism of inhibition that involves addition
of the active site serine residue to the tetrahydrothi-
opyranone carbonyl group of the inhibitor to give a
reversibly formed hemiketal. This mechanism has been
demonstrated for related inhibitors of the cysteine
protease papain.10
The synthesis of inhibitors 2 and 3 began with
reductive amination of 5 with 1 equiv of aldehyde 10
using sodium triacetoxyborohydride in dichloroethane
to give secondary amine 11 (Scheme 3).17 An alternate
strategy for the preparation of 11 involving monoalky-
lation of 5 with the appropriate alkyl bromide gave a
poor yield of the secondary amine. Amine 5 was coupled
to Fmoc-Phe using a standard peptide coupling proce-
dure that employed 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide (EDC) and 1-hydroxybenzotriazole
(HOBT). However, the more sterically hindered second-
ary amine 11 did not couple under these conditions and
required reaction with the acid fluoride of Fmoc-Phe
using methodology developed by Carpino.18 These reac-
tions gave compounds 12 and 13, each as a mixture of
two diastereomers. The Fmoc protecting groups in 12
and 13 were removed using piperidine in DMF to give
amines 14 and 15. The diastereomers of both com-
pounds were separated at this stage using flash chro-
matography, and each of the diastereomers was carried
separately through the remainder of the synthesis. The
amines were next coupled to Boc-D-Ile using EDC and
HOBT to give compounds 16 and 17. Final removal of
the Boc and ketal protecting groups in 16 and 17 was
accomplished by treatment with trifluoroacetic acid and
H2O to give inhibitors 2 and 3.
Compound 2B has an affinity for plasmin that is 2-3
times lower than that of its diastereomer 2A. In
contrast, there is a 100-fold difference in the binding of

2972 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Sanders and Seto
two diastereomers of analogous inhibitors for papain.9
Papain has a relatively deep and narrow binding cleft19
that discriminates strongly between two diastereomers
that differ in stereochemistry at the position R to the
reactive ketone. Based upon the similarities between
plasmin and trypsin,16 it is likely that plasmin has a
binding cleft that is much more open and shallow when
compared to papain. This sterically unrestrictive active
site can accommodate both diastereomers of inhibitor
2, and it is reasonable to expect that both diastereomers
can find a conformation in the active site that allows
reaction with the active site serine residue. Thus 2A
and 2B can bind to plasmin with similar affinities.
inhibitors using the amide nitrogen in a strategy that
is borrowed from peptoids.14 Our future work will focus
on extending the noncovalent interactions of these
inhibitors into the leaving group subsites of plasmin in
order to increase both their potency and specificity.
Exp er im en ta l Section
Gen er a l Meth od s. NMR spectra were recorded on
a
Bruker WM-250, Avance-300, or Avance-400 instrument.
1
Spectra were calibrated using TMS (δ ) 0.00 ppm) for H 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 spectrometer. Mass spectra were recorded on a Kratos
MS 80 spectrometer under electron impact (EI), chemical
ionization (CI), or fast-atom bombardment (FAB) conditions.
HPLC analyses were performed 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).
We have synthesized compounds 3A and 3B in order
to determine how much the aminohexyl side chain
contributes to the potency of the inhibitors. These
compounds are similar in structure to 2A and 2B but
are missing the side chain which interacts with the S1
pocket in the enzyme active site. Since plasmin is
specific for substrates that incorporate a lysine residue
at P1, we expected that removing the aminohexyl group
from the inhibitor should have a significant negative
impact on its ability to bind. Compounds 3A and 3B
have inhibition constants against plasmin of 9.0 and
16.0 mM, respectively. These values are 200-300 times
higher than the inhibition constant for 2A. This result
confirms that the aminohexyl side chain, which mimics
a lysine residue at the P1 position of the inhibitor, is
critical for good recognition and affinity for the protease.
Reactions were conducted under an atmosphere of dry
nitrogen in oven-dried glasswear. 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 other 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.
P r im a r y Am in e 5. A solution containing 9.5 mL of trif-
luoroacetic acid (TFA), 0.25 mL of triisopropylsilane (TIS), and
0.25 mL of thioanisole was added to the carbamate 49 (4.8 g,
17 mmol) dissolved in 2 mL of CH2Cl2. After stirring at room
temperature for 10 min the solvent was evaporated under
reduced pressure. The crude product was purified by column
chromatography (1:10:89 concentrated NH4OH/CH3OH/CH2-
Cl2) affording 2.8 g (15 mmol, 89%) of the primary amine 5:
1H NMR (300 MHz, MeOH-d4) δ 1.44 (dm, J ) 13.5 Hz, 1H),
1.65 (ddd, J ) 15.0, 11.6, 3.5 Hz, 1H), 1.94-2.11 (m, 1H), 2.50
(dm, J ) 13.8 Hz, 1H), 2.61 (ddd, J ) 13.1, 5.6, 1.8 Hz, 1H),
2.73 (ddd, J ) 13.9, 11.4, 2.6 Hz, 1H), 2.96 (dd, J ) 13.0, 11.2
Hz, 1H), 3.21 (dm, J ) 14.4 Hz, 1H), 3.30 (m, 1H), 3.89 (m,
2H), 4.10 (m, 2H); 13C NMR (75 MHz, MeOH-d4) δ 25.9, 26.4,
The design of these 4-heterocyclohexanone-based
inhibitors depends on the supposition that the ketone
of the inhibitors reacts in a reversible covalent fashion
with the active site nucleophile. This mechanism has
been confirmed for cysteine proteases10 but remains
unproven for serine proteases. Thus it is possible that
compounds 1-3 are inhibiting plasmin through simple
noncovalent interactions. To further explore the mech-
anism of inhibition, we have synthesized compound 22
(Scheme 4) which is missing the thioether and ketone
functionalities that are present in inhibitor 2. If the
4-heterocyclohexanones are interacting with the enzyme
solely through noncovalent interactions such as salt
bridges, hydrogen bonds, and hydrophobic interactions,
control compound 22 should be a good mimic of inhibitor
2, and the two molecules would be expected to have
similar affinities for plasmin. However, if the ketone of
inhibitor 2 is also reacting with the active site serine to
give a reversibly formed covalent adduct, we would
expect 22 to be a significantly weaker inhibitor. Table
1 shows that compound 22 has an inhibition constant
against plasmin of 520 µM; a value that is 10-fold higher
than that observed for inhibitor 2. Although this result
does not unambiguously prove the mechanism of inhibi-
tion, it is consistent with the reasonable mechanistic
hypothesis that inhibitors 1-3 react with the active site
serine to give a hemiketal adduct.
28.5, 31.1, 57.9, 60.9, 61.1, 96.5; HRMS-EI (M+) calcd for C8H15
NO2S 189.0824, found 189.0827.
-
Ter tia r y Am in e 6. Amine 5 (0.15 g, 0.79 mmol) was
dissolved in 5 mL of 1,2-dichloroethane (DCE) before the
aldehyde 10 (0.38 g, 1.7 mmol) and sodium triacetoxyborohy-
dride (0.23 g, 1.1 mmol) were added. After 6.5 h at room
temperature the reaction was partitioned between saturated
NaHCO3 solution and EtOAc. The organic layer was dried over
MgSO4 and concentrated. The crude product was purified by
flash chromatography (EtOAc) affording 0.10 g (0.18 mmol,
22%) of the tertiary amine 6: 1H NMR (400 MHz, MeOH-d4)
δ 1.24-1.35 (m, 37H), 1.96 (m, 1H), 2.28 (m, 1H), 2.51-2.63
(m, 3H), 2.78 (m, 3H), 2.93-3.00 (m, 5H), 3.23-3.27 (m, 2H),
3.75-4.02 (m, 4H); 13C NMR (100 MHz, MeOH-d4) δ 25.9, 26.5,
27.1, 27.9, 28.2, 28.8, 30.2, 31.1, 33.0, 41.4, 53.4, 59.6, 59.8,
68.2, 79.8, 100.8, 158.6; HRMS-FAB (M + Na+) calcd for
C
30H57N3NaO6S 610.3866, found 610.3882.
Keta l 7. Tertiary amine 6 (100 mg, 0.17 mmol) was
dissolved in 1 mL of a solution containing 92.5% TFA, 2.5%
TIS, 2.5% thioanisole, and 2.5% H2O. The reaction was stirred
at room temperature for 1 h before the TFA was removed
under reduced pressure. The resultant material was dissolved
in MeOH to which Et2O was added until the solution turned
cloudy. The ketal 7 (53 mg, 0.86 mmol, 50%) which settled
out of the solution as an oily liquid was used without further
purification: 1H NMR (250 MHz, MeOH-d4) δ1.46 (m, 9H),
1.66-1.85 (m, 10H), 2.07 (m, 1H), 2.49-2.54 (m, 1H), 2.79-
3.01 (m, 7H), 3.25 (m, 2H), 3.45 (m, 1H), 3.59-3.64 (dd, J )
12.0, 2.9 Hz, 1H), 3.93-4.28 (m, 5H); 13C NMR (75 MHz,
MeOH-d4) δ 23.8, 25.9, 26.0, 26.5, 26.6, 27.4, 27.5, 27.8, 28.7,
28.8, 32.6, 40.9, 54.6, 55.6, 61.2, 61.4, 69.9, 98.3, 118 (q), 163.2
(q).
In conclusion, this work has shown that the 4-het-
erocyclohexanone nucleus can serve as the basis for
designing good inhibitors of plasmin. In addition, our
experiments highlight the versatility of the 4-heterocy-
clohexanone nucleus because we have now confirmed
that it can be used to synthesize inhibitors of both serine
and cysteine proteases. We have also demonstrated the
feasibility of attaching P1 recognition elements to the

Inhibitors of the Serine Protease Plasmin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2973
Alcoh ol 9. 6-Amino-1-hexanol (8) (2.0 g, 17 mmol) was
dissolved in a 5:1 mixture of 1,4-dioxane/H2O and cooled to 0
°C. Di-tert-butyl dicarbonate (7.5 g, 34 mmol) was added, and
the reaction mixture was allowed to warm to room tempera-
ture and stirred for 12 h. The dioxane was evaporated under
reduced pressure, and the remaining material was partitioned
between EtOAc and saturated NaHCO3 solution. The organic
layer was washed with brine, dried over MgSO4, and concen-
trated under reduced pressure. The crude product was purified
by flash chromatography (4:1 EtOAc/hexanes) to afford alcohol
9 (3.2 g, 15 mmol, 88%): 1H NMR (300 MHz, CDCl3) δ 1.35-
1.76 (m, 18H), 3.11 (q, J ) 6.1 Hz, 2H), 3.64 (t, J ) 6.3 Hz,
2H), 4.60 (bs, 1H); 13C NMR (75 MHz, CDCl3) δ 25.4, 26.4,
26.5, 28.4, 30.0, 32.6, 40.0, 62.4, 79.1, 156.2; HRMS-CI (M +
H+) calcd for C11H24NO3 218.1756, found 218.1760.
Ald eh yd e 10. The alcohol 9 (7.2 g, 33 mmol) was added to
a CH2Cl2 solution (500 mL) containing 51 g of neutral alumina
and pyridinium chlorochromate (11 g, 50 mmol). The reaction
was allowed to stir at room temperature for 3 h and then was
loaded directly onto a flash chromatography column. The
product was eluted with 1:1 EtOAc/hexanes to afford 6.5 g (30
mmol, 91%) of the aldehyde 10: IR 1704 cm-1 (CO); 1H NMR
(250 MHz, CDCl3) δ 1.26-1.49 (m, 13H), 1.61 (pent, J ) 7.2
Hz, 2H), 2.40 (t, J ) 7.2 Hz, 2H), 3.08 (q, J ) 6.6 Hz, 2H),
4.59 (bs, 1H), 9.76 (t, J ) 1.7 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 22.5, 27.1, 29.2, 30.7, 41.4, 44.5, 79.8, 156.8, 203.0;
HRMS-CI (M + H+) calcd for C10H22NO2 216.1600, found
216.1600.
Secon d a r y Am in e 11. Aldehyde 10 (0.52 g, 2.4 mmol) was
dissolved in 2 mL of DCE and added to a solution of primary
amine 5 (0.51 g, 2.7 mmol) dissolved in 3 mL of DCE. After 10
min sodium triacetoxyborohydride (0.80 g, 3.8 mmol) was
added, and the reaction was allowed to stir for an additional
3 h at room temperature. The reaction was then quenched with
saturated NaHCO3 solution and extracted with EtOAc. The
organic layer was dried over MgSO4 and concentrated under
reduced pressure. The crude product was purified by flash
chromatography (2:1:7 EtOAc/MeOH/Et2O) providing the sec-
ondary amine 11 (0.53 g, 1.40 mmol, 50%): 1H NMR (300 MHz,
MeOH-d4) δ 1.36-1.72 (m, 21H), 2.02 (m, 1H), 2.50 (dm, J )
13.7 Hz, 1H), 2.69 (ddd, J ) 9.7, 9.7, 2.6 Hz, 1H), 2.76-2.89
(m, 4H), 3.00-3.07 (m, 4H), 3.87 (m, 2H), 4.02 (ddd, J ) 11.9,
9.3, 2.5 Hz, 1H), 4.12 (ddd, J ) 12.0, 12.0, 2.7 Hz, 4H); 13C
NMR (75 MHz, MeOH-d4) δ 24.7, 25.2, 25.5, 25.9, 26.7, 28.2,
30.2, 40.4, 46.0, 60.0, 60.1, 62.8, 79.3, 96.1, 158.0; HRMS-FAB
(M + Na+) calcd for C19H36N2NaO4S 411.2294, found 411.2306.
F m oc Keta l 12. Fmoc-Phe-F18 (0.34 g, 0.89 mmol) and
diisopropylethylamine (DIEA; 0.10 mL, 0.60 mmol) were added
to a solution of the secondary amine 11 (0.11 g, 0.30 mmol)
dissolved in 15 mL of CH2Cl2. The reaction was heated at
reflux for 5 h, then cooled, and washed with 10 mL of 1 N
NaOH, 15 mL of 1 N HCl, and 15 mL of saturated NaHCO3
solution. The organic layer was then dried over MgSO4 and
concentrated under reduced pressure. Flash chromatography
(2:3 EtOAc/hexanes) of the resultant material afforded a
mixture of two diastereomers of Fmoc ketal 12 (0.17 g, 0.22
mmol, 75%): 1H NMR (300 MHz, CDCl3) δ 1.02-2.07 (m, 21H),
2.28-2.45 (m, 1H), 2.56-5.09 (m, 18H), 5.39-5.90 (m, 1H),
7.20-7.83 (m, 13H); 13C NMR (75 MHz, CDCl3) δ 25.0, 26.5,
27.1, 27.3, 28.4, 28.7, 29.1, 30.1, 31.3, 40.5, 41.4, 44.9, 47.1,
47.3, 52.1, 52.4, 58.8, 59.0, 63.0, 66.6, 66.9, 97.1, 120.0, 125.1,
125.2, 126.5, 126.8, 127.0, 127.6, 128.3, 128.4, 128.5, 128.6,
129.4, 129.7, 136.4, 136.8, 141.3, 143.9, 144.0, 155.2, 156.0,
172.8; HRMS-FAB (M + Na+) calcd for C43H55N3NaO7S
780.3659, found 780.3663.
The organic layer was washed with 100 mL of H2O, 50 mL of
saturated KHSO4 solution, and 50 mL of saturated Na2CO3
solution, dried over MgSO4, and concentrated under reduced
pressure. Flash chromatography (1:1 EtOAc/hexanes) afforded
a mixture of two diastereomers of Fmoc ketal 13 (0.56 g, 1.0
mmol, 36%): 1H NMR (300 MHz, CDCl3) δ 1.62-1.81 (m, 3H),
2.28-3.19 (m, 7H), 3.72-3.93 (m, 4H), 4.10-4.46 (m, 5H), 5.44
(m, 1H), 6.24-6.48 (m, 1H), 7.24-7.79 (m, 13H); 13C NMR (75
MHz, CDCl3) δ 25.0, 25.3, 25.4, 30.6, 30.7, 32.0, 32.2, 39.2,
39.5, 47.6, 56.6, 56.9, 59.4, 59.5, 59.6, 67.5, 96.3, 96.4, 120.4,
125.47, 125.54, 127.3, 127.5, 128.1, 129.0, 129.1, 129.8, 129.9,
136.8, 137.0, 141.7, 144.2, 156.2, 170.4, 170.6; HRMS-FAB
(M + Na+) calcd for C32H34N2NaO5S 581.2086, found 581.2099.
Am in o Keta ls 14A a n d 14B. A DMF solution (35 mL) of
Fmoc ketal 12 (1.75 g, 2.3 mmol) and piperidine (1.4 mL, 14
mmol) was stirred at room temperature for 1 h. The solvent
was evaporated under reduced pressure, and the crude mate-
rial was purified by flash chromatography (98:2 CH2Cl2/MeOH)
to give the two separate diastereomers of the amino ketals 14A
(0.43 g, 0.50 mmol) and 14B (0.41 g, 0.76 mmol) with a
combined yield of 67%. 14A: 1H NMR (300 MHz, CDCl3) δ
1.08 (m, 1H), 1.09-1.34 (m, 7H), 1.36-1.52 (m, 14H), 1.68-
1.73 (m, 4H), 1.84-1.97 (m, 1H), 2.36 (dq, J ) 13.5, 1.8 Hz,
1H), 2.65-2.82 (m, 2H), 2.91-3.02 (m, 2H), 3.09-3.19 (m, 4H),
3.44-3.59 (m, 1H), 3.63 (dd, J ) 11.4, 3.3 Hz, 1H), 3.71-3.91
(m, 4H), 4.02 (dt, J ) 11.9, 2.4 Hz, 1H), 4.10 (dd, J ) 10.0, 5.7
Hz, 1H), 4.60 (bm, 1H), 7.19-7.39 (m, 5H); 13C NMR (75 MHz,
CDCl3) δ 27.6, 29.2, 29.8, 30.3, 31.2, 31.9, 32.8, 34.1, 45.4, 47.5,
55.5, 61.6, 62.0, 65.2, 100.1, 129.1, 131.2, 132.2, 140.6, 180.0;
HRMS-ESI (M + H+) calcd for C28H46N3O5S 536.3158, found
536.3163. 14B: 1H NMR (300 MHz, CDCl3) δ 1.25-1.67 (m,
27H), 1.96-2.02 (m, 2H), 2.43-2.51 (m, 2H), 2.67 (dd, J ) 13.6,
9.3 Hz, 1H), 2.79-2.83 (m, 1H), 3.11-3.30 (m, 6H), 3.36-3.52
(m, 2H), 3.69-4.05 (m, 6H), 4.27 (dd, J ) 11.2, 2.9 Hz, 1H),
4.61 (bs, 1H), 7.20-7.34 (m, 5H); 13C NMR (75 MHz, CDCl3) δ
24.0, 24.1, 24.3, 25.6, 25.8, 26.1, 26.4, 27.5, 27.7, 28.0, 28.3,
29.3, 30.2, 30.7, 30.9, 39.8, 41.5, 42.0, 43.6, 43.8, 52.6, 52.9,
57.9, 58.0, 58.1, 58.3, 62.3, 78.2, 78.3, 96.7, 97.2, 125.5, 125.9,
127.6, 127.7, 128.6, 128.7, 137.6, 138.6, 155.2, 155.5, 175.0,
175.8; HRMS-ESI (M + H+) calcd for C28H46N3O5S 536.3158,
found 536.3140.
Am in o Keta ls 15A a n d 15B. A solution of piperidine (0.6
mL, 6.0 mmol) and Fmoc ketal 13 (0.56 g, 1.0 mmol) in 5 mL
of DMF was allowed to stir at room temperature for 5 h. The
reaction mixture was then partitioned between 50 mL of
EtOAc and 50 mL of H2O. The organic layer was washed with
H2O, dried over MgSO4, and concentrated. The crude material
was purified by flash chromatography (2:98 MeOH/CH2Cl2) to
afford the two separate diastereomers of the amino ketals 15A
(0.17 g, 0.50 mmol) and 15B (0.11 g, 0.32 mmol) with a
combined yield of 81%. 15A: 1H NMR (300 MHz, CDCl3) δ
1.26 (s, 2H), 1.47 (m, 2H), 1.68 (m, 1H), 2.30 (bm, 1H), 2.50
(m, 2H), 2.73 (dd, J ) 13.7, 9.2 Hz, 5H), 2.93 (m, 1H), 3.26
(dd, J ) 13.7, 3.9 Hz, 1H), 3.67 (dd, J ) 9.2, 4.0 Hz, 1H), 3.81
(m, 1H), 3.93 (m, 2H), 4.14 (m, 1H), 4.66 (bm, 1H), 7.23-7.34
(m, 5H), 8.01 (d, J ) 8.6 Hz, 1H); 13C NMR (75 MHz, CDCl3)
δ 25.9, 26.7, 31.3, 32.7, 42.6, 57.6, 60.5, 60.8, 97.9, 128.2, 129.9,
131.0, 134.3, 139.2, 176.8; HRMS-FAB (M + H+) calcd for
C17H25N2O3S 337.1586, found 337.1579. 15B: 1H NMR (300
MHz, CDCl3) δ 1.53-1.72 (m, 4H), 2.29 (bs, 1H), 2.52 (m, 2H),
2.76 (dd, J ) 13.7, 9.1 Hz, 2H), 2.96 (dd, J ) 10.8, 2.1 Hz,
1H), 3.26 (dd, J ) 13.7, 4.5 Hz, 1H), 3.64 (dd, J ) 9.1, 4.6 Hz,
1H), 3.82 (m, 1H), 3.94 (m, 2H), 3.96 (m, 1H), 4.70 (bm, 1H),
7.22-7.35 (m, 5H), 7.90 (d, J ) 9.2 Hz, 1H); 13C NMR (75 MHz,
CDCl3) δ 25.9, 26.6, 31.2, 32.7, 42.8, 57.9, 60.5, 60.7, 97.8,
128.1, 130.0, 130.9, 139.4, 176.8; HRMS-FAB (M + H+) calcd
for C17H25N2O3S 337.1586, found 337.1592.
F m oc Keta l 13. A DMF solution (75 mL) containing
hydroxybenzotriazole (HOBT; 0.37 g, 2.8 mmol), N-(3-dim-
ethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC;
0.69 g, 3.6 mmol), and Fmoc-phenylalanine (1.1 g, 2.8 mmol)
was stirred at room temperature for 1 h. A solution of the
primary amine 5 (0.52 g, 2.8 mmol) and 4-methylmorpholine
(0.60 mL, 5.5 mmol) dissolved in 25 mL of DMF was then
added to the reaction mixture. After 2 h the reaction mixture
was partitioned between 100 mL of EtOAc and 100 mL of H2O.
Boc Keta l 16A. A DMF solution (10 mL) containing HOBT
(103 mg, 0.76 mmol), EDC (192 mg, 1.0 mmol), and Boc-D-Ile
(172 mg, 0.76 mmol) was stirred at room temperature for 20
h. A solution of the amino ketal 14A (0.41 g, 0.76 mmol) and
4-methylmorpholine (0.17 mL, 1.5 mmol) dissolved in 10 mL
of DMF was then added to the reaction mixture. After 4 h the
reaction mixture was partitioned between EtOAc and H2O. The

2974 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Sanders and Seto
organic layer was washed with H2O, dried over MgSO4, and
concentrated under reduced pressure. Flash chromatography
(4:1 EtOAc/hexanes) afforded the Boc ketal 16A (0.44 g, 0.59
mmol, 78%). This compound appears in the NMR spectra as a
mixture of two conformational isomers: 1H NMR (300 MHz,
CDCl3) δ 0.75-0.99 (m, 6H), 1.06-1.36 (m, 34H), 2.27-2.32
(m, 1H), 2.62 (t, J ) 13.1 Hz, 1H), 2.75 (t, J ) 12.3 Hz, 1H),
2.87 (t, J ) 11.5 Hz, 1H), 2.97-3.17 (m, 5H), 3.46-4.03 (m,
6H), 4.56 (m, 0.5H), 4.97-5.30 (m, 1.5H), 6.90 (d, J ) 7.1 Hz,
1H), 7.17-7.31 (m, 5H); 13C NMR (100 MHz, CDCl3) δ 11.9,
15.6, 15.77, 15.83, 25.0, 25.2, 25.3, 26.3, 26.8, 27.4, 27.5, 28.6,
28.7, 29.4, 30.4, 31.4, 37.8, 38.2, 40.4, 40.8, 41.2, 41.3, 45.3,
51.2, 51.3, 59.2, 63.3, 79.3, 79.9, 97.3, 98.3, 127.1, 128.7, 129.0,
129.7, 130.0, 130.1, 136.6, 137.0, 155.9, 170.3, 170.4, 173.0,
192.2, 201.5; HRMS-FAB (M + Na+) calcd for C39H64N4NaO8S
771.4166, found 771.4334 for a mixture of diastereomers 16A
and 16B.
Boc Keta l 16B. Compound 16B was prepared from 14B
(270 mg, 0.51 mmol), HOBT (68 mg, 0.51 mmol), EDC (130
mg, 0.67 mmol), Boc-D-Ile (120 mg, 0.51 mmol), and 4-meth-
ylmorpholine (0.11 mL, 1.0 mmol) in 20 mL of DMF using the
method described for the synthesis of 16A. The crude material
was purified by HPLC (1.5% MeOH/CH2Cl2 over 45 min) to
afford 16B (240 mg, 0.32 mmol, 63%). This compound appears
in the NMR spectra as a mixture of two conformational
isomers: 1H NMR (300 MHz, CDCl3) δ 0.67-1.07 (m, 6H),
1.32-1.48 (m, 26H), 1.62-1.79 (m, 2H), 1.91-2.03 (m, 2H),
2.44 (m, 1.5H), 2.76-3.49 (m, 8H), 3.66 (m, 0.5H), 3.80-4.05
(m, 4H), 4.57 (m, 1H), 4.92-5.29 (m, 3H), 6.48 (m, 0.5H), 6.65
(m, 0.5H), 7.18-7.30 (m, 5H); 13C NMR (100 MHz, CDCl3) δ
11.9, 12.1, 15.6, 15.8, 24.8, 24.9, 25.0, 25.36, 25.41, 26.5, 26.9,
27.4, 28.4, 28.7, 28.8, 28.9, 29.2, 30.1, 31.3, 31.6, 37.7, 38.2,
38.5, 40.6, 40.9, 44.8, 45.2, 50.7, 51.1, 53.8, 59.1, 59.2, 59.3,
59.4, 59.5, 63.0, 77. 7, 79.4, 80.0, 97.9, 98.2, 126.8, 127.4, 128.6,
128.9, 129.8, 129.9, 137.0, 138.0, 155.9, 171.1, 173.1; HRMS-
FAB (M + Na+) calcd for C39H64N4NaO8S 771.4166, found
771.4334 for a mixture of diastereomers 16A and 16B.
Boc Keta l 17A. Compound 17A was prepared from com-
pound 15A (110 mg, 0.32 mmol), HOBT (43 mg, 0.32 mmol),
EDC (80 mg, 0.42 mmol), Boc-D-Ile (74 mg, 0.32 mmol), and
4-methylmorpholine (0.070 mL, 0.64 mmol) in 15 mL of DMF
by the method described for the synthesis of 16A. The crude
material was purified by flash chromatography (4:1 EtOAc/
hexanes) to afford 17A (156 mg, 0.28 mmol, 88%): 1H NMR
(300 MHz, CDCl3) δ 0.80-0.95 (m, 7H), 1.24-1.44 (m, 10H),
1.60-1.81 (m, 6H), 2.46-2.59 (m, 3H), 2.82-3.13 (m, 3H),
3.76-4.01 (m, 5H), 4.44 (m, 1H), 4.73 (q, J ) 6.9 Hz, 1H), 5.01
(m, 1H), 6.58 (m, 2H), 7.19-7.32 (m, 5H); 13C NMR (100 MHz,
CDCl3) δ 11.9, 15.8, 24.9, 25.4, 28.7, 30.6, 31.9, 37.8, 38.6, 54.8,
59.6, 80.4, 96.3, 127.3, 129.0, 129.8, 136.8, 156.0, 170.4, 171.8;
HRMS-FAB (M + H+) calcd for C28H44N3O6S 550.2951, found
550.2961.
afford 53 mg (0.09 mmol, 99%) of inhibitor 1: 1H NMR (300
MHz, MeOH-d4) δ 1.31 (m, 10H), 1.67-1.83 (m, 10H), 2.92-
3.04 (m, 9H), 3.14 (m, 1H), 4.61 (dd, J ) 11.6, 5.3 Hz, 1H); 13
C
NMR (75 MHz, MeOH-d4) δ 24.9, 25.9, 26.06, 26.12, 26.3, 27.3,
28.1, 29.0, 39.5, 44.5, 53.0, 53.2, 69.9, 201.9.
In h ibitor 2A. The Boc ketal 16A (100 mg, 0.14 mmol) was
dissolved in 1 mL of a solution containing 92.5% TFA, 2.5%
TIS, 2.5% thioanisole, and 2.5% H2O. After 18 h the TFA was
removed under reduced pressure. The crude mixture was
purified by reverse-phase HPLC (0-50% MeCN/H2O over 45
min) affording 49 mg (0.068 mmol, 50%) of the inhibitor 2A:
1H NMR (300 MHz, MeOH-d4) δ 0.83-0.97 (m, 7H), 1.31-1.41
(m, 7H), 1.71-1.79 (m, 4H), 2.79-2.99 (m, 7H), 3.05-3.21 (m,
2H), 3.39-3.48 (m, 2H), 3.69 (d, J ) 5.5 Hz, 1H), 4.10 (dd, J
) 11.1, 5.9 Hz, 1H), 5.12 (dd, J ) 9.3, 5.8 Hz, 1H), 7.26-7.38
(m, 5H); 13C NMR (75 MHz, MeOH-d4) δ 12.0, 15.3, 25.4, 27.5,
27.6, 27.9, 28.0, 28.8, 28.9, 30.7, 32.3, 34.6, 38.1, 39.8, 41.0,
45.2, 50.8, 52.1, 52.8, 53.7, 54.2, 59.4, 61.3, 67.8, 128.7, 130.2,
130.3, 130.8, 131.1, 138.0, 138.2, 163.4, 169.5, 172.7, 190.3,
204.6; HRMS-FAB (M + H+) calcd for C26H43N4O3S 491.3056,
found 491.3067.
In h ibitor 2B. Inhibitor 2B was prepared from compound
16B (240 mg, 0.32 mmol) and 1 mL of the TFA solution
specified in the synthesis of 2A. The crude product was purified
by RPHPLC (0-50% MeCN/H2O over 45 min) to afford
inhibitor 2B (21 mg, 0.030 mmol, 9%): 1H NMR (300 MHz,
MeOH-d4) δ 0.52-0.82 (m, 7H), 1.06-1.12 (m, 1H), 1.19-1.36
(m, 5H), 1.49-1.64 (m, 5H), 2.64-2.85 (m, 6H), 2.91-2.98 (m,
1H), 3.08 (dd, J ) 14.5, 4.6 Hz, 1H), 4.93 (dd, J ) 10.2, 4.6
Hz, 1H), 7.00-7.24 (m, 5H); 13C NMR (75 MHz, MeOH-d4) δ
12.1, 15.2, 25.4, 27.6, 28.9, 31.0, 32.6, 38.2, 39.1, 41.0, 45.3,
50.8, 52.9, 59.4, 68.1, 128.6, 130.1, 130.5, 130.7, 138.5, 169.6,
173.1, 204.0; HRMS-ESI (M + H+) calcd for C26H43N4O3S
491.3056, found 491.3065.
In h ibitor 3A. Compound 17A (53 mg, 0.10 mmol) was
dissolved in 1 mL of a solution containing 92.5% TFA, 2.5%
TIS, 2.5% H2O, and 2.5% thioanisole. After 1 h the TFA was
removed under reduced pressure. The crude mixture was
purified by flash chromatography (10:89:1 MeOH/CH2Cl2/
concentrated NH4OH) before the final purification was per-
formed using RPHPLC (0-100% MeCN/H2O over 45 min)
affording the inhibitor 3A (17 mg, 0.03 mmol, 34%). In MeOH-
d4 solution, the inhibitor is visible as an approximate 1:1
mixture of hemiketal and ketone: 1H NMR (400 MHz, MeOH-
d4) δ 0.70-0.78 (m, 7H), 1.15 (m, 1H), 1.65 (m, 1H), 1.83 (m,
0.5H), 1.99 (m, 0.5H), 2.17 (m, 0.5H), 2.38 (m, 0.5H), 2.59-
3.00 (m, 5H), 3.13 (ddd, J ) 13.2, 5.6, 2.8 Hz, 0.5H), 3.28 (m,
0.5H), 3.67 (m, 1H), 4.13 (m, 0.5H), 4.72 (dd, J ) 11.6, 4.8 Hz,
0.5H), 4.82 (dd, J ) 11.2, 7.2 Hz, 0.5H), 7.23-7.35 (m, 5H);
13C NMR (100 MHz, MeOH-d4) δ 10.7, 13.6, 24.0, 24.7, 25.0,
25.1, 30.45, 30.49, 34.7, 35.2, 36.7, 37.8, 38.0, 44.4, 64.99, 55.02,
58.00, 58.03, 59.8, 95.5, 96.2, 115.5 (q, J ) 284 Hz), 160.3 (q,
J ) 34 Hz), 168.3, 168.4, 172.4, 172.68, 172.71, 204.4; HRMS-
FAB (M + Na+) calcd for C20H29N3NaO3S 414.1827, found
414.1823.
In h ibitor 3B. Inhibitor 3B was prepared from compound
17B (60 mg, 0.11 mmol) and 1 mL of the TFA solution specified
in the synthesis of 3A. The crude product was purified by
RPHPLC (0-100% MeCN/H2O over 45 min) to afford inhibitor
3B (45 mg, 0.090 mmol, 82%). In MeOH-d4 solution, the
inhibitor is visible as an approximate 1:1 mixture of hemiketal
and ketone: 1H NMR (400 MHz, MeOH-d4) δ 0.58-1.05 (m,
7H), 1.16-1.40 (m, 1H), 1.65-1.81 (m, 1H), 1.94-2.14 (m, 1H),
2.39-3.05 (m, 6H), 3.15-3.23 (m, 1H), 3.69 (d, J ) 5.2 Hz,
1H), 4.05-4.11 (m, 0.5H), 4.67 (dd, J ) 11.5, 5.3 Hz, 0.5H),
4.73 (dd, J ) 10.1, 5.8 Hz, 0.5H), 4.84 (dd, J ) 10.2, 5.2 Hz,
0.5H), 7.22-7.32 (m, 5H); 13C NMR (75 MHz, MeOH-d4) δ 14.2,
14.3, 27.7, 27.8, 28.5, 33.8, 34.0, 38.1, 38.8, 40.30, 40.33, 41.4,
41.7, 48.0, 58.5, 59.0, 61.5, 61.7, 63.6, 99.2, 130.6, 130.7, 132.2,
132.8, 140.6, 140.8, 172.0, 172.4, 175.6, 176.1, 207.9; HRMS-
FAB (M + Na+) calcd for C20H29N3NaO3S 414.1827, found
414.1834.
Boc Keta l 17B. Compound 17B was prepared from com-
pound 15B (106 mg, 0.32 mmol), HOBT (43 mg, 0.32 mmol),
EDC (78 mg, 0.41 mmol), Boc-D-Ile (73 mg, 0.32 mmol), and
4-methylmorpholine (0.070 mL, 0.64 mmol) in 15 mL of DMF
by the method described for the synthesis of 16A. The crude
material was purified by flash chromatography (4:1 EtOAc/
hexanes) to give compound 17B (148 mg, 0.27 mmol, 86%):
1H NMR (300 MHz, CDCl3) δ 0.81-1.02 (m, 7H), 1.23-1.80
(m, 16H), 2.27-2.74 (m, 4H), 2.95-3.18 (m, 2H), 3.70-3.99
(m, 5H), 4.38 (s, 1H), 4.67 (q, J ) 8.2 Hz, 1H), 5.11 (m, 1H),
6.41 (m, 1H), 6.81 (d, J ) 7.5 Hz, 1H), 7.20-7.31 (m, 5H); 13
C
NMR (100 MHz, CDCl3) δ 12.0, 15.8, 24.9, 25.3, 28.7, 30.5,
32.0, 38.1, 38.8, 55.2, 59.5, 59.6, 80.3, 96.3, 127.4, 129.1, 129.7,
137.0, 156.0, 170.1, 171.7; HRMS-FAB (M + H+) calcd for
C
28H44N3O6S 550.2951, found 550.2953.
In h ibitor 1. Ketal 7 (53 mg, 0.09 mmol) was dissolved in a
solution of 5 mL of MeOH and 10 mL of 6 N HCl. The reaction
was heated at reflux for 1 h before the solvent was removed
under reduced pressure. The crude material was dissolved in
a small amount of MeOH to which Et2O was added until the
solution turned cloudy. The Et2O was pipetted off and the oily
residue further purified by RPHPLC (H2O with 0.1% TFA) to
Secon d a r y Am in e 19. Compound 19 was prepared from
18 (3.8 mL, 33.5 mmol), compound 10 (6.6 g, 30.5 mmol), and

Inhibitors of the Serine Protease Plasmin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2975
sodium triacetoxyborohydride (3.7 g, 17.5 mmol) in 20 mL of
DCE using the method described for the synthesis of 11. The
crude material was purified by flash chromatography (2:1:7
EtOAc/MeOH/Et2O) to afford the secondary amine 19 (1.3 g,
4.4 mmol, 25%): 1H NMR (300 MHz, CDCl3) δ 0.96-1.47 (m,
23H), 1.57-1.73 (m, 3H), 1.84 (m, 2H), 2.32-2.42 (m, 1H), 2.58
on a Perkin-Elmer 8452A diode array UV-vis spectrometer.
All enzymes were assayed at 25 °C in 50 mM sodium
phosphate buffer (pH 7.4) with or without inhibitor. Due to
solubility, inhibitors 2 and 3 were assayed in a solution with
a final concentration of 10% DMSO. Initial rates were deter-
mined by monitoring the change in absorbance at 404 nm from
60 to 120 s after mixing. None of the inhibitors showed
evidence of slow binding behavior. Inhibitor 2A was subjected
to full kinetic analysis against plasmin. For each inhibitor
concentration examined (2A: 0, 8.6, 43, 86, 170, 260 µM) five
substrate concentrations were used (75, 150, 300, 600, 1200
µM) with at least two independent determinations at each
concentration. Ki values were determined by nonlinear fit to
the Michaelis-Menten equation for competitive inhibition
using simple weighing. Competitive inhibition was confirmed
by Lineweaver-Burk analysis using simple statistical weigh-
ing to the linear fit of 1/v vs 1/[S]. For the less potent
compounds (1, 2B, 3A, 3B) a substrate concentration of 300
µM was monitored with six different inhibitor concentrations
(1: 0, 110, 210, 430, 860, 1700 µM; 2B: 0, 110, 230, 460, 690,
920 µM; 3A: 0, 0.8, 1.6, 2.4, 3.1, 3.9 mM; 3B: 0, 0.9, 1.8, 2.7,
3.6, 4.5 mM). For inhibitor 2A assayed against thrombin (Thr),
kallikrein (Kal), and trypsin (Try), a single substrate concen-
tration (Thr: 50 µM; Kal: 100 µM; Try: 50 µM) was monitored
with six different inhibitor concentrations (Thr: 0, 26, 53, 79,
110, 160 µM; Kal: 0, 48, 96, 144, 190, 240 µM; Try: 0, 26, 53,
79, 110, 160 µM). Competitive inhibition was assumed, and
Ki values were calculated using a Dixon analysis. Data analysis
was performed with the commercial graphing package Grafit
(Erithacus Software Ltd.). Km values for the substrates were
determined both with and without 10% DMSO (without
DMSO: plasmin 220 µM, Thr 10 µM, Kal 117 µM, Try 42 µM;
with DMSO: plasmin 370 µM, Thr 22 µM, Kal 63 µM, Try 50
µM).
(t, J ) 7.1 Hz, 2H), 3.07 (q, J ) 6.5 Hz, 2H), 4.60 (bs, 1H); 13
C
NMR (75 MHz, CDCl3) δ 25.5, 26.6, 27.1, 27.5, 28.5, 30.4, 30.8,
34.0, 40.7, 47.3, 57.3, 79.3, 156.4; HRMS-FAB (M + Na+) calcd
for C17H34N2NaO2 321.2518, found 321.2522.
P r im a r y Am in e 20. A solution of Fmoc-Phe-F (5 g, 13.2
mmol), DIEA (2.3 mL, 13.2 mmol), and the secondary amine
19 (1.3 g, 4.4 mmol) in 100 mL of CH2Cl2 was heated at reflux
for 18 h. The mixture was then diluted with 100 mL of EtOAc
and washed with 100 mL each of 1 N NaOH, 1 N HCl, and
saturated NaHCO3 solution. The resultant organic layer was
dried over MgSO4 and concentrated. The crude material was
purified by flash chromatography (1:1 EtOAc/hexanes) which
provided a mixture of Fmoc-Phe and the expected coupling
product. The mixture was dissolved in 85 mL of DMF, and
piperidine (2.2 mL, 22 mmol) was added. After 15 min the
solution was partitioned between 150 mL of EtOAc and 150
mL of H2O. The organic layer was dried over MgSO4 and
concentrated. The crude material was purified by flash chro-
matography (2% MeOH/CH2Cl2) to afford the primary amine
20 as a mixture of two conformational isomers that intercon-
vert slowly on the NMR time scale (1.3 g, 2.9 mmol, 66%): 1H
NMR (300 MHz, CDCl3) δ 1.02-1.74 (m, 27H), 2.65-3.34 (m,
6H), 3.67 (t, J ) 7.0 Hz, 0.5H), 3.89 (t, J ) 7.0 Hz, 0.5H), 4.22
(m, 0.5H), 4.67 (m, 0.5H), 7.16-7.33 (m, 5H); 13C NMR (75
MHz, CDCl3) δ 25.6, 25.9, 26.3, 26.4, 26.7, 26.8, 27.4, 28.8,
29.7, 30.4, 30.9, 31.2, 31.7, 32.1, 32.6, 42.7, 43.9, 53.3, 54.2,
54.4, 57.1, 127.0, 128.8, 128.9, 129.1, 129.7, 138.3, 156.4, 174.5;
HRMS-FAB (M + Na+) calcd for C26H43N3NaO3 468.3202,
found 468.3216.
Boc Dip ep tid e 21. Compound 21 was prepared from 20
(1.3 g, 2.9 mmol), HOBT (0.39 g, 2.9 mmol), EDC (0.73 g, 3.8
mmol), Boc-D-Ile (0.68 g, 2.9 mmol), and 4-methylmorpholine
(0.66 g, 6 mmol) in 75 mL of DMF using the method described
for the synthesis of 16A. The crude material was purified by
flash chromatography (4:1 EtOAc/hexanes) to afford Boc
dipeptide 21 (1.7 g, 2.6 mmol, 91%): 1H NMR (300 MHz,
CDCl3) δ 0.76-0.82 (m, 7H), 0.94-1.77 (m, 36H), 2.60-3.29
(m, 6H), 4.00-4.12 (m, 2H), 4.69-5.53 (m, 3H), 7.07-7.28 (m,
5H); 13C NMR (75 MHz, CDCl3) δ 11.9, 15.8, 24.87, 24.94, 25.5,
26.0, 26.1, 26.8, 27.3, 28.7, 28.79, 28.81, 30.3, 31.0, 31.4, 40.4,
40.5, 42.7, 50.5, 54.6, 57.4, 59.5, 79.8, 127.2, 128.8, 129.8, 129.9,
136.8, 137.0, 155.9, 170.7, 171.0, 171.3; HRMS-FAB (M +
Na+) calcd for C37H62N4NaO6 681.4567, found 681.4550.
Ack n ow led gm en t. This research was supported by
the NIH (Grant 1 R01 GM57327-01), the Petroleum
Research Fund administered by the American Chemical
Society (Grant 30544-G4), 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.). T.C.S. was supported
by a Predoctoral Fellowship from the U.S. Army Medical
Research and Materiel Command-Breast Cancer Re-
search Initiative (Grant DAMD17-96-1-6037).
Refer en ces
Am in e 22. The Boc dipeptide 21 (180 mg, 0.27 mmol) was
dissolved in 1 mL of CH2Cl2 before 1 mL of a solution
containing 95% TFA, 2.5% TIS, and 2.5% ethanedithiol was
added. After 30 min the solvent was removed under vacuum.
The crude product was purified by flash chromatography (10:
89:1 MeOH/CH2Cl2/concentrated aqueous NH4OH) to afford
amine 22 (0.11 g, 0.16 mmol, 59%): 1H NMR (300 MHz, CDCl3)
δ 0.83-0.92 (m, 6H), 1.00-1.20 (m, 3H), 1.33-1.87 (m, 19H),
2.89-3.12 (m, 5H), 3.24-3.34 (m, 3H), 3.38 (m, 0.5H), 3.68
(dd, J ) 8.3, 5.7 Hz, 1H), 3.94-4.18 (m, 1H), 5.15 (t, J ) 7.7
Hz, 1H), 7.23-7.35 (m, 5H); 13C NMR (75 MHz, CDCl3) δ 10.67,
10.69, 13.9, 14.0, 24.1, 25.2, 25.5, 25.8, 25.9, 26.0, 26.1, 26.2,
26.4, 26.8, 27.4, 27.5, 29.1, 30.6, 31.0, 31.2, 31.5, 37.0, 38.4,
38.9, 39.58, 39.63, 42.5, 43.9, 51.3, 52.2, 55.5, 58.0, 116.0 (q, J
) 293 Hz), 127.1, 127.22, 127.24, 128.6, 128.7, 128.8, 129.4,
129.5, 129.6, 136.8, 137.0, 162.1 (q, J ) 34 Hz), 168.6, 168.7,
171.6, 171.9; HRMS-FAB (M + Na+) calcd for C27H46N4NaO2
481.3519, found 481.3523.
(1) Cao, Y.; O′Reilly, M. S.; Marshall, B.; Flynn, E.; J i, R.; Folkman,
J . Expression of Angiostatin cDNA in Murine Fibrosarcoma
Suppresses Primary Tumor Growth and Produces Long-Term
Dormancy of Metastases. J . Clin. Invest. 1998, 101, 1055.
(2) Wu, Z.; O′Reilly, M. S.; Folkman, J .; Shing, Y. Suppression of
Tumor Growth With Recombinant Murine Angiostatin. Biochem.
Biophys. Res. Commun. 1997, 236, 651.
(3) Pepper, J . S.; Montesano, R.; Mandriots, S. J .; Orci, L.; Vassalli,
J . Angiogenesis: A Paradigm for Balanced Extracellular Pro-
teolysis During Cell Migration and Morphogenesis. Enzyme
Protein 1996, 49, 138.
(4) Liotta, L. A. Cancer Cell Invasion and Metastasis. Sci. Am. 1992,
February, 54.
(5) Sherry, S. Fibrinolysis, Thrombosis, and Hemostasis; Lea &
Febiger: Philadelphia, PA, 1992; Chapter 1.
(6) Okada, Y.; Tsuda, Y.; Teno, N.; Wanaka, K.; Bohgaki, M.;
Hijikata-Okunomiya, A.; Naito, T.; Okamoto, S. Synthesis of
Active Center-Directed Peptide Inhibitors of Plasmin. Chem.
Pharm. Bull. 1988, 36, 1289.
(7) Ganu, V. S.; Shaw, E. Improved Synthetic Inactivators of
Plasmin. Thromb. Res. 1987, 45, 1.
En zym e Assa ys. The amidolytic activity of plasmin, throm-
bin, kallikrein, and trypsin was determined using chromogenic
substrates D-Val-Leu-Lys-pNA, H-D-Phe-Pip-Arg-pNA, H-D-
Pro-Phe-Arg-pNA, and H-D-Phe-Pip-Arg-pNA, respectively.20
Enzymes and substrates were used as received from Sigma-
Aldrich or Chromogenix (distributor: DiaPharma Group, Inc.)
without further purification. Reaction progress was monitored
(8) For other active site-directed inhibitors of plasmin, see: Teno,
N.; Wanaka, K.; Okada, Y.; Taguchi, H.; Okamoto, U.; Hijikata-
Okunomiya, A.; Okamoto, S. Development of Active Center-
Directed Plasmin and Plasma Kallikrein Inhibitors and Studies
on the Structure-Inhibitory Activity Relationship. Chem. Pharm.
Bull. 1993, 41, 1079. Wanaka, K.; Okamoto, S.; Horie, N.;
Hijikata-Okunomiya, A.; Okamoto, U.; Naito, T.; Ohno, N.;
Bohgaki, M.; Tsuda, Y.; Okada, Y. Use of an Active Center-

2976 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Sanders and Seto
Directed Plasmin Inhibitor Elucidates the Multiplicity of Plas-
min Actions. Thromb. Res. 1996, 82, 79. Tamura, S. Y.; Goldman,
E. A.; Brunck, T. K.; Ripka, W. C.; Semple, J . E. Rational Design,
Synthesis, and Serine Protease Inhibitory Activity of a Novel
P1-Argininal Derivative Featuring a Conformationally Con-
strained P2-P3 Bicyclic Lactam Moiety. Bioorg. Med. Chem. Lett.
1997, 7, 331; and refs 6 and 16.
(15) Matsuzaki, T.; Sasaki, C.; Umeyama, H. A Predicted Tertiary
Structure of a Thrombin Inhibitor-Trypsin Complex Explains
the Mechanisms of the Selective Inhibition of Thrombin, Factor
Xa, Plasmin, and Trypsin. J . Biochem. 1988, 103, 537.
(16) Teno, N.; Wanaka, K.; Okada, Y.; Tsuda, Y.; Okamoto, U.;
Hijikata-Okunomiya, A.; Naito, T.; Okamoto, S. Development
of Active Center-Directed Inhibitors Against Plasmin. Chem.
Pharm. Bull. 1991, 39, 2340.
(17) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C.
A.; Shah, R. D. Reductive Amination of Aldehydes and Ketones
with Sodium Triacetoxyborohydride. Studies in Direct and
Indirect Reductive Amination Procedures. J . Org. Chem. 1996,
61, 3849.
(18) Carpino, L. A.; Sadat-Aalace, D.; Chao, H. G.; DeSelms, R. H.
((9-Fluorenylmethyl)oxy)carbonyl (Fmoc) Amino Acid Fluorides.
Convenient New Peptide Coupling Reagents Applicable to the
Fmoc/tert-Butyl Strategy for Solution and Solid-Phase Synthesis.
J . Am. Chem. Soc. 1990, 112, 9651.
(19) Kamphuis, I. G.; Kalk, K. H.; Swarte, M. B. A.; Drenth, J .
Structure of Papain Refined at 1.65 Å Resolution. J . Mol. Biol.
1984, 179, 233.
(9) Conroy, J . L.; Sanders, T. C.; Seto, C. T. Using the Electrostatic
Field Effect to Design a New Class of Inhibitors for Cysteine
Proteases. J . Am. Chem. Soc. 1997, 119, 4285.
(10) Conroy, J . L.; Seto, C. T. Demonstration by 13C NMR Studies
that Tetrahydropyranone-Based Inhibitors Bind to Cysteine
Proteases by Reversible Formation of a Hemithioketal Adduct.
J . Org. Chem. 1998, 63, 2367.
(11) Brady, K.; Abeles, R. H. Inhibition of Chymotrypsin by Peptidyl
Trifluoromethyl Ketones: Determinants of Slow-Binding Kinet-
ics. Biochemistry 1990, 29, 7608.
(12) Smith, R. A.; Copp, L. J .; Donnelly, S. L.; Spencer, R. W.; Krantz,
A. Inhibition of Cathepsin B by Peptidyl Aldehydes and Ke-
tones: Slow-Binding Behavior of a Trifluoromethyl Ketone.
Biochemistry 1988, 27, 6568.
(13) Otto, H.-H.; Schirmeister, T. Cysteine Proteases and Their
Inhibitors. Chem. Rev. 1997, 97, 133.
(14) Simon, R. J .; Kania, R. S.; Zuchermann, R. N.; Huebner, V. D.;
J ewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.
Peptoids: A Modular Approach to Drug Discovery. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 9367.
(20) Hitomi, Y.; Ikari, N.; Fujii, S. Inhibitory Effect of a New
Synthetic Protease Inhibitor (FUT-175) on the Coagulation
System. Haemostasis 1985, 15, 164.
J M990110K