Conformationally restricted analogs of the direct thrombin inhibitor FM 19

Article abstract of DOI:10.1016/j.bmc.2011.10.045

The serine protease thrombin plays several key roles in the clotting cascade within the hemostatic system, such as in fibrin formation and platelet activation. Thus, development of an inhibitor that binds to the enzyme's active site (a direct thrombin inhibitor) offers an approach for the treatment of thrombus-associated diseases. Previous structure-activity relationship studies originally based on the bradykinin breakdown product Arg-Pro-Pro-Gly-Phe (RPPGF) led to the development of lead compound FM 19 (d-Arg-Oic-Pro-d-Ala-Phe(p-Me)- NH₂). The recently determined X-ray structure of FM 19 in the active site of thrombin has revealed sites of modification to potentially improve inhibition. In this study, we report the synthesis and biological characterization of nine peptides that replace only the d-Arg residue of the FM 19 sequence, investigating ways to add conformational restriction, modification of the basic moiety at the end of the side chain, and removal of the charge from the N-terminus. Two of these peptides, 6 and 7 (IC₅₀ values of 0.51 and 0.45 μM, respectively), show similar potency to the best compounds in the FM 19 series reported thus far.

Full text of DOI:10.1016/j.bmc.2011.10.045

Bioorganic & Medicinal Chemistry 19 (2011) 7425–7434
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Conformationally restricted analogs of the direct thrombin inhibitor FM 19
⇑
Elizabeth A. Girnys, Vanessa R. Porter, Henry I. Mosberg
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109-1065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The serine protease thrombin plays several key roles in the clotting cascade within the hemostatic sys-
tem, such as in fibrin formation and platelet activation. Thus, development of an inhibitor that binds
to the enzyme’s active site (a direct thrombin inhibitor) offers an approach for the treatment of throm-
bus-associated diseases. Previous structure–activity relationship studies originally based on the bradyki-
nin breakdown product Arg-Pro-Pro-Gly-Phe (RPPGF) led to the development of lead compound FM 19
Received 9 August 2011
Revised 7 October 2011
Accepted 15 October 2011
Available online 20 October 2011
(
D
-Arg-Oic-Pro-
site of thrombin has revealed sites of modification to potentially improve inhibition. In this study, we
report the synthesis and biological characterization of nine peptides that replace only the -Arg residue
D-Ala-Phe(p-Me)-NH2). The recently determined X-ray structure of FM 19 in the active
Keywords:
Thrombin inhibitor
Structure-based design
Peptide synthesis
D
of the FM 19 sequence, investigating ways to add conformational restriction, modification of the basic
moiety at the end of the side chain, and removal of the charge from the N-terminus. Two of these pep-
tides, 6 and 7 (IC50 values of 0.51 and 0.45
in the FM 19 series reported thus far.
l
M, respectively), show similar potency to the best compounds
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
covery, many compounds have been developed as thrombin inhib-
itors. Some work as bivalent inhibitors, similar to hirudin,4,6,7
others work through allosteric inhibition by binding to another
auxillary site, exosite II,8,9 and still others work as univalent DTIs,
and bind only to the enzyme’s active site.4–6
The serine protease thrombin plays a central role in the hemo-
static system and coagulation.1 One main function of thrombin is
the conversion of soluble fibrinogen into insoluble fibrin, the
protein that cross-links with platelets to form a thrombus.2 In
addition, thrombin is an effective platelet activator, via the prote-
ase-activated receptors (PARs) 1 and 4 present on the platelet cell
surface.3 The PARs, which are G protein-coupled receptors, can
send downstream signals resulting in platelet activation and aggre-
gation.3 Thus, thrombin is an attractive target for modulation of
the hemostatic system and the prevention of thrombus formation.
Due to its attractiveness as a target for anticoagulation therapy,
research focused on thrombin inhibition has been pursued by sev-
eral groups for at least the last 25 years.4,5 The 65 amino acid coag-
ulation protein originally isolated from leeches, hirudin, was one of
the early examples of the therapeutic potential of thrombin inhibi-
tion. Hirudin is a well-studied, bivalent direct thrombin inhibitor
(DTI), which binds to both the enzyme’s active site as well as an
auxiliary site, exosite I, with a sub-picomolar Ki.6,7 Since this dis-
Dabigatran, marketed as the prodrug dabigatran etexilate,10 has
recently been approved by the FDA as the first orally available uni-
valent DTI. When compared to warfarin and related compounds,
the only orally available agents indicated for anticoagulation treat-
ment,11 dabigatran was shown to be at least as effective at pre-
venting stroke in patients with atrial fibrillation, while also
showing a similar or decreased risk of major hemmorage.10 In
addition, since dabigatran shows fewer interactions with food
and other drugs and does not require frequent monitoring and
dose adjustment, dabigatran finally offers an alternative to warfa-
rin treatment10 and shows that anticoagulation can be achieved by
orally administered direct thrombin inhibitors.
Our development of a novel direct thrombin inhibitor was ini-
tially based upon the endogenous bradykinin breakdown product
Arg-Pro-Pro-Gly-Phe (RPPGF) (Fig. 1), which was shown to inhibit
thrombin substrate cleavage.12 Structure–activity relationship
(SAR) studies led to the development of the lead compound FM
Abbreviations: DTI, direct thrombin inhibitor; HOBt, hydroxybenzotriazole;
HBTU, O-benzotriazole-N,N,N0,N0-tetramethyl-uroniumhexafluorophosphate; DIEA,
N,N-diisopropylethylamine; Oic, (2S,3aS,7aS)-octahydroindole-2-carboxylic acid;
Phe(p-Me), p-methyl phenylalanine; NMP, N-methyl-2-pyrrolidone; TFA,
trifluoroacetic acid; THF, tetrahydrofuran; PAR, protease-activated receptor; SAR,
structure–activity relationships; Boc, tert-butoxycarbonyl; Fmoc, fluorenylmethyl-
oxycarbonyl.
19, with the sequence
D-Arg-Oic-Pro-D
-Ala-Phe(p-Me)-NH2,13,14
where Oic is (2S,3aS,7aS)-octahydroindole-2-carboxylic acid
(Fig. 1). FM 19 has been shown to be effective at prolonging throm-
bin clotting times and delaying carotid artery thrombosis following
oral administration in mice and thus represents a promising alter-
native lead toward the development of DTIs. Although the potency
of FM 19 is rather low, the X-ray structure of FM 19 in the active
⇑
Corresponding author. Tel.: +1 (734) 764 8117; fax: +1 (734) 763 5595.
E-mail address: him@umich.edu (H.I. Mosberg).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.045

7426
E. A. Girnys et al. / Bioorg. Med. Chem. 19 (2011) 7425–7434
N
N
N
N
NH
O
O
NH
O
NH
O
O
N
N
O
O
O
O
O
HN
H2N
HN
HN
HN
HN
O
O
NH2
OH
NH2
N
H
H2N
N
H
N
H
HN
O
H2N
NH2
NH2
O
O
Figure 1. Structures of pentapeptides RPPGF (left), FM 19 (middle) and compound 1 (right).
the two positively charged groups of FM 19, the N-terminal amine
and guanidino group on the side chain of -Arg, are in close prox-
Gly 218
D
Asp 189
Ala 190
imity, creating an unfavorable electrostatic interaction. This can
best be alleviated by removal of the N-terminal amine, since the
guanidino group makes key interactions with active site thrombin
residues Asp 189, Ala 190 and Gly 218, but the N-terminal amine
does not participate directly in binding to thrombin.15 Thus, all
replacements except 10 lack the N-terminal amine. Additionally,
the D-Arg side chain of FM 19 assumes a high energy, eclipsed con-
formation when bound to thrombin. To reduce this energetic pen-
alty, we have designed modifications to stabilize this eclipsed side
chain orientation, such as in compounds 1,16 2 and 3. We also re-
placed the guanidino with another basic group, an amidino moiety,
to explore another way to split the positive charge between more
than one atom at the end of the side chain. This splitting of the po-
sitive charge at the end of the amino acid side chain allows for im-
proved potency over a simple point charge, due to the side chain’s
ability to better interact with the catalytic Asp 189.17 An amidino
moiety has been shown to be effectively incorporated in the non-
peptide direct thrombin inhibitor argatroban.17 Here we used this
functional group while also adding conformational restriction, as
seen in compounds 4–7. Finally, we investigated replacing the N-
terminal amine with another group of comparable size, a methyl
group (compound 8), while still maintaining the same stereochem-
ical conformation, to see how removal of only the positive charge
impacts potency. For comparison, we have also made the methyl
and amino containing replacements with the opposite stereochem-
istry (compounds 9 and 10, respectively). Analytical data for all
compounds can be found in Table 2.
Figure 2. Structure of FM 19 in the active site of thrombin (PDB 3BV9, 1.8 Å). The D-
Arg residue is highlighted in yellow, with key interacting residues of thrombin
shown in green.
site of thrombin15 (Fig. 2), has guided the SAR for improved inhib-
itors. Previously, we have reported replacements to the D-Arg res-
idue of the FM 19 sequence, and two of these peptides show a
further increase in potency over FM 19. One of these pentapep-
tides, compound 1 (Fig. 1), also adds conformational restriction
to this side chain.16 In this study, we investigate additional replace-
ments of this crucial residue, to better understand the structural
features important for thrombin inhibition.
2. Results and discussion
Nine new replacements for the D-Arg residue in the FM 19 se-
quence were synthesized and are reported here. These replace-
ments were guided by insights gained from the X-ray structure
of FM 19 in the active site of thrombin15 (Fig. 2), and are summa-
rized in Figure 3. From the crystal structure, we can first see that
2.1. Chemistry
First, two saturated cyclic analogs of 1 were synthesized using
cyclohexanoic acids, with the guanidino group and acid moieties
in either the cis or trans-configurations, compounds 2 and 3,
respectively (Scheme 1). The starting material for the cis analog
was purchased as 11a, and the amino group was protected with
a tert-butoxycarbonyl (Boc) group as 12a. The starting material
for the trans analog was purchased with the amine already pro-
tected, as 12b. These acids could then be coupled to the Oic-Pro-
HN
HN
HN
H2N
H2N
O
O
H2N
O
NH
NH
NH
1
2
3
D-Ala-Phe(p-Me) tetrapeptide on the solid phase resin to yield pen-
tapeptides 13a and 13b, followed by cleavage from the resin and
simultaneous deprotection of the cyclohexanoic acid residue ami-
no group, resulting in 14a and 14b. The amine was converted to a
di-Boc protected guanidino group18 producing 15a and 15b, which
not only installed the desired functional group into the final pep-
tide, but also allowed for easier purification from the amine-con-
taining peptides. The Boc protecting groups were then removed,
giving pentapeptides 2 and 3, ready for testing.
Next, we synthesized four replacements containing an unsatu-
rated ring as in 1, but replacing the guanidino group with an ami-
dino group. This was done with both benzoic acid (4 and 6) and
phenylacetic acid (5 and 7) scaffolds, substituted in the meta (4
and 5) and para (6 and 7) positions. The synthesis of all four
replacements began with the commercially available correspond-
ing cyano acids 16a, 16b, 20a and 20b (Scheme 2). The nitrile
was first converted to the hydroxyamidino using hydroxylamine
O
O
H2N
HN
O
H2N
O
NH
NH
H2N
H2N
6
NH
4
5
7
NH
HN
NH
HN
NH
HN
H2N
H2N
H2N
H2N
O
H3C
O
H3C
O
9
8
10
Figure 3. Structures of
D
-Arg replacements (X) in FM 19 sequence (X-Oic-Pro-
Ala-Phe(p-Me)-NH2) for compounds 1–10.
D
-

E. A. Girnys et al. / Bioorg. Med. Chem. 19 (2011) 7425–7434
7427
O
TP
O
OH
O
TP-resin
O
OH
O
TP
O
TP
a
b
c
c
d
HN
N
O
HN
O
HN
O
O
NH2
11a
HN
NH
NH2
14a
O
NH
O
O
cis
NH2
cis
14b trans
12a
12b trans
cis
13a
13b trans
cis
O
2
cis
15a
15b trans
3 trans
cis
Scheme 1. Reagents and conditions: (a) di-tert-butyldicarbonate, NaOH, 1,4-dioxane–H2O 2:1, 0 °C to room temperature, 3 h. (b) HOBt, HBTU, DIEA, Oic-Pro-
D
-Ala-Phe(p-
Me)-NH2 tetrapeptide resin (designated TP-resin), NMP, room temperature, 1.5 h. (c) 95:5 TFA–H2O, room temperature, 2 h. (d) N,N0-di-Boc-N0-trifylguanidine, triethylamine,
1,4-dioxane, room temperature, 24 h.13
O
O
O
O
O
( )n
TP-resin
( )n
( )n OH
OH
( )n TP
( )n OH
d
a
b
c
NH
NH
NH
NHOH
NH
NH2
CN
NH2
NH2
16a n = 0
16b n = 1
17a n = 0
17b n = 1
19a n = 0
19b n = 1
4 n = 0
5 n = 1
18a n = 0
18b n = 1
O
O
O
O
O
( )n
OH
( )n
OH
( )n
( )n
TP
( )n
TP-resin
TP
a
c
d
e
CN
HOHN
NH
HOHN
NH
HOHN
NH
H2N
NH
20a n = 0
20b n = 1
21a n = 0
21b n = 1
22a n = 0
22b n = 1
6 n = 0
7 n = 1
23a n = 0
23b n = 1
Scheme 2. Reagents and conditions: (a) hydroxylamine hydrochloride, KOH, ethanol, reflux, 16–18.5 h.14 (b) H2, 10% Pd/C, H2O, 50 °C, 18–20 h. (c) HOBt, HBTU, DIEA, Oic-Pro-
D
-Ala-Phe(p-Me)-NH2 tetrapeptide resin (designated TP-resin), NMP, room temperature, 1–4 h. (d) 95:5 TFA–H2O, room temperature, 2 h. (e) acetic anhydride, H2, 10% Pd/C,
glacial acetic acid, room temperature, 21.5–23.5 h.14
O
O
O
O
O
O
NH
O
N
O
N
O
a
c
b
d
CN
HO
NC
27a
R
27b S
26a
25a
S,R
24a
S
S
26b R,S
25b R
O
O
O
O
O
f
g
e
HO
NH2
HO
N
H
O
TP-resin
N
H
O
29a
30a
R
30b S
R
28a
R
29b S
28b S
O
N
O
O
O
O
NH
NH2
h
g
TP
NH2
TP
N
H
NH
TP
N
H
O
O
31a
R
8
R
31b S
32a
R
32b S
9 S
Scheme 3. Reagents and conditions: (a) propionyl chloride, n-butyllithium, THF, ꢀ78 °C to room temperature, 14 h.16 (b) acrylonitrile, TiCl3(OiPr), DIEA, dichloromethane,
0 °C, 4 h.16 (c) LiOH, THF–H2O 2:1, 0 °C to room temperature, 1–15 h.16 (d) H2, PtO2, H2O–methanol 3:7, room temperature, 14–15 h.16 (e) di-tert-butyldicarbonate, NaOH, 1,4-
dioxane–H2O 2:1, 0 °C to room temperature, 4–4.5 h. (f) HOBt, HBTU, DIEA, Oic-Pro-
D-Ala-Phe(p-Me)-NH2 tetrapeptide resin (designated TP-resin), NMP, room temperature,
4.5–13 h. (g) 95:5 TFA–H2O, room temperature, 2 h. (h) N,N0-di-Boc-N0-trifylguanidine, triethylamine, 1,4-dioxane, room temperature, 15 h–6 days.13

7428
E. A. Girnys et al. / Bioorg. Med. Chem. 19 (2011) 7425–7434
hydrochloride and base19 (compounds 17a, 17b, 21a and 21b). The
meta-substituted amidino compounds 18a and 18b were synthe-
sized via hydrogenation and collected as their hydrochloride salts.
Compounds 25a and 25b were then functionalized with acryloni-
trile,21 to yield 26a and 26b, with the desired stereochemistry.
The chiral auxiliary was then hydrolyzed21 to give the cyanoacids
27a and 27b. Compounds 27a and 27b were then hydrogenated
using PtO2,21 resulting in amino acids 28a and 28b. The amino
group on 28a or 28b was protected with a Boc group to give 29a
and 29b, and the protected amino acids were then coupled to the
Compounds 18a and 18b were then coupled to the Oic-Pro-D-Ala-
Phe(p-Me) tetrapeptide on the resin to yield 19a and 19b, followed
by cleavage from the resin to give the final desired pentapeptides 4
and 5.
The synthesis of the para-substituted replacements followed a
slightly less direct route. Conversion of 4-hydroxyamidinobenzoic
acid (21a) to 4-amidinobenzoic acid was achieved through the
same procedure as described above for the meta-substituted
replacements, however the resulting compound was only soluble
in strong acids, and therefore not suitable for continued peptide
synthesis. As a result, 21a and 21b were first coupled to the Oic-
Oic-Pro-D-Ala-Phe(p-Me) tetrapeptide on the resin to yield 30a
and 30b. Compounds 30a and 30b were then cleaved from the re-
sin while simultaneously removing the Boc protecting group, to
give amino-containing pentapeptides 31a and 31b. The amine
was converted to a di-Boc protected guanidino group18 producing
32a and 32b, again, to simultaneously install the desired guanidino
group into the final peptide, as well as allow for easier purification
from the amine-containing peptides. The Boc protecting groups
were then removed, giving pentapeptides 8 and 9, ready for
testing.
Pro-D-Ala-Phe(p-Me) tetrapeptide on the resin to yield 22a and
22b, followed by cleavage from the resin to give pentapeptides
23a and 23b. The hydroxyamidino moiety in 23a and 23b was then
hydrogenated to the desired amidino through the use of acetic
anhydride as an acylating agent in acetic acid,19 to give desired
pentapeptides 6 and 7.
2.2. Inhibition of a-thrombin
Two compounds, 8 and 9, were synthesized that replaced the N-
terminal amine of FM 19 with a methyl group (Scheme 3). The
replacement in 8 has the R-configuration, which corresponds to
Peptides RPPGF and 2–10 were tested for their ability to inhibit
human -thrombin according to the general assay procedure. The
results of these assays are summarized in Table 1, along with re-
sults for 116 and FM 19.16 A representative inhibition profile for
compound 7 is shown in Figure 4. As seen in Figure 4, both FM
a
the same stereochemistry as the D-Arg residue in FM 19, while
the replacement in 9 has the opposite, S stereochemistry. Synthe-
ses of enantiopure replacements were achieved through the use
of Evans oxazolidinone chiral auxiliaries.20 The commercially avail-
able oxazolidinone 24a was functionalized using propionyl chlo-
ride and n-butyllithium,21 generating 25a. The other enantiomer
of this compound, 25b, was commercially available and purchased.
19 and compound 7 were able to completely inhibit
a-thrombin.
This was also observed for all compounds reported here.
When comparing the guanidino-containing saturated ring
replacements 2 and 3, we can see that the trans-substitution (3)
is more potent than the cis-substitution (2). Assuming the cyclo-
hexanoic acid residue in 3 adopts the expected, energetically
favored chair conformation with the trans para substituents ar-
ranged equatorially, the residue 1 side chain of 3 adopts a
Table 1
Inhibitory activities of RPPGF, FM 19 and compounds 1–10 against human
a-
thrombin
0.15
Compound
IC50 (lM)
D
-Arg replacement (X) in FM 19 sequence
Compound
FM 19
No inhibitor control
No enzyme control
7
(X-Oic-Pro-D-Ala-Phe(p-Me)-NH2)
RPPGF
FM 19
—
601 21
4.4 1.316
0.10
0.05
0.00
D-Arg
1
2
3
p-Guanidinobenzoic acid
0.57 0.1216
19.5 5.4
2.5 0.5
cis-4-Guanidino-1-cyclohexanecarboxylic acid
trans-4-Guanidino-1-cyclohexanecarboxylic
acid
3-Amidinobenzoic acid
3-Amidinophenylacetic acid
4-Amidinobenzoic acid
4-Amidinophenylacetic acid
(R)-5-Guanidino-2-methylpentanoic acid
(S)-5-Guanidino-2-methylpentanoic acid
4
5
6
7
8
9
10
1.03 0.25
26.2 2.3
0.51 0.19
0.45 0.08
98 27
-10
-5
0
9.8 1.0
73 36
log [inhibitor]
L
-Arg
Figure 4. Representative inhibition profile of 7 and FM 19.
Table 2
Analytical data for RPPGF, FM 19 and compounds 1–10
Compound
Expected [M+H] Found [M+H] Analytical HPLC retention time (min)
D-Arg replacement (X) in FM 19 sequence (X-Oic-Pro-D-Ala-Phe(p-Me)-NH2)
RPPGF
FM 19
—
573.3
573.1
17.4
654.416
654.416
26.516
D-Arg
1
2
3
4
5
6
7
8
9
p-Guanidinobenzoic acid
659.416
665.4
665.4
644.4
658.4
644.4
658.4
653.4
653.4
654.4
659.316
665.4
665.4
644.5
658.5
644.5
658.5
653.2
653.2
654.2
31.616
34.2
32.1
31.4
33.4
30.5
33.2
31.2
35.1
26.7
cis-4-Guanidino-1-cyclohexanecarboxylic acid
trans-4-Guanidino-1-cyclohexanecarboxylic acid
3-Amidinobenzoic acid
3-Amidinophenylacetic acid
4-Amidinobenzoic acid
4-Amidinophenylacetic acid
(R)-5-Guanidino-2-methylpentanoic acid
(S)-5-Guanidino-2-methylpentanoic acid
10
L-Arg

E. A. Girnys et al. / Bioorg. Med. Chem. 19 (2011) 7425–7434
7429
quasi-planar shape similar to that of 1. In a similar chair conforma-
tion of the saturated ring of 2, such a quasi-planar orientation is
not possible since one of the trans-substituents will be equatorial,
the other axial, thus making key interactions more difficult to
achieve. Since the three-dimensional shape of 3 more closely
resembles 1, it would be expected that 3 is more potent than 2.
However, 3 is still approximately four times less potent than 1,
and thus it appears as if the rigidity and planarity of the unsatu-
rated ring is preferred.
Three of the four amidino-containing peptides from Table 1 are
more potent than lead compound FM 19. Positioning of the ami-
dino substitution relative to the rest of the peptide appears to be
the key consideration. In 4 and 6 the connection of the aromatic
ring to remainder of the molecule is via a single rotatable bond,
which minimizes the positional variation of the amidino group
and maintains it in an orientation suitable for interaction with
thrombin. In 7 two rotatable bonds link the aromatic moiety to
the peptide, but conformational space accessible to the amidino re-
mains limited since rotation about the aryl–alkyl bond does not af-
fect its position. By contrast rotation about either the aryl–alkyl or
alkyl–carbonyl bond in 5 significantly alters the position of the
aminido group relative to the rest of the molecule, leading to a re-
duced population of the binding orientation and hence reduced
potency.
owe their improved potency to the incorporation of conforma-
tional restriction in the side chain to overcome the entropic pent-
alty of the flexible D-Arg side chain of FM 19. We have shown that
replacing the N-terminal residue with a saturated ring substituted
at the 4 position with a guanidine group can yield a potent com-
pound, and this guanidino group is preferred in the trans-orienta-
tion (3), over the cis-orientation (2). This is consistent with the
previously synthesized compound 1, as the guanidino group in 3
more closely resembles the positioning of this same group in 1.
Also, the guanidino group can be replaced with an amidino moiety
on an aromatic ring, and para-substitution (6 and 7) is preferred
over meta-substitution (4 and 5). Furthermore, although the N-ter-
minal amine in FM 19 makes an unfavorable electrostatic interac-
tion with the guanidino group on the side chain, this amine can
form a water-mediated hydrogen bond with thrombin’s active
site.15 Replacement of this amine with a methyl group (8) greatly
reduces potency, unless the stereochemistry around this chiral
center is changed to make the methyl group less solvent-exposed
(9). Additionally, burying an amine in the less-solvent exposed
area of the active site also results in decreased inhibition (10). Fi-
nally, previous work demonstrates that delocalization of the posi-
tive charge on the side chain of the N-terminal residue in this
peptide sequence is important for potency,16 as the splitting of this
charge has been shown to improve potency of direct thrombin
inhibitors by increasing interactions with catalytic Asp 189.17
The current work demonstrates that this delocalization can be suc-
cessfully achieved by the use of either a guanidino or amidino
moiety.
The methyl-substituted peptides 8 and 9 produce an interesting
result. Compound 8 has the same stereochemistry as the N-termi-
nal residue in FM 19, while 9 has the opposite stereochemistry. The
only difference between FM 19 and 8 is the elimination of the
charge due to replacement of the amino group by a methyl group,
since the sterics and stereochemistry of this part of the molecule
are maintained. Previous studies have suggested that the R-config-
4. Experimental section
uration around this stereocenter is preferred (
L
D-Arg is favored over
4.1. Chemistry general procedures
-Arg),13 and thus we would expect 8 to be more potent than 9, but
in fact 9 shows an order of magnitude improvement in potency
over 8. Compound 8 has the methyl group in the same position
as the amine of FM 19, which is more solvent-exposed, while 9
positions the methyl group in a more hydrophobic environment,
pointed further into the active site and directed more toward the
aliphatic side chain of Oic. Thus, it would be expected that there
would be more of a penalty for the methyl group to be solvent-ex-
posed as in 8, suggesting a reason for the increase in potency seen
in 9.
All reagents were purchased from commercial sources and used
without further purification. Purity of synthesized compounds was
determined on a Waters alliance 2690 Analytical HPLC (Waters
Corporation, Milford, MA, USA) and a Vydac Protein and Peptide
C18 reverse phase column, using a linear gradient of 0% Solvent B
(0.1% trifluoroacetic acid (TFA) in acetonitrile) in Solvent A (0.1%
TFA in water) to 70% Solvent B in Solvent A in 70 min, and UV
absorbance at 230 nm. ESI-MS was performed on either a Finnigan
LCQ mass spectrometer (ThermoFinnigan, San Jose, CA, USA) in po-
sitive mode or an Agilent Technologies LC/MS system using a 1200
Series LC and 6130 Quadrupole LC/MS (Agilent Technologies, Santa
Finally, due to the unexpected results of 8 and 9, compound 10
was synthesized to verify that
favored over -Arg. This has been confirmed, as we see that 10 is
approximately 16-fold less potent than FM 19. Although the
D-Arg as the N-terminal residue is
L
Clara, CA, USA) in positive mode with 10–100 lL injection volume
D
-
and a linear gradient of 0% Solvent D (0.02% TFA and 0.1% acetic
acid in acetonitrile) in Solvent C (0.02% TFA and 0.1% acetic acid
in water) to 60% Solvent D in Solvent C in 15 min. NMR data were
obtained on either a 300 or 500 MHz Bruker spectrometer. All col-
umn chromatography was performed using a Flash+ System (Bio-
tage Corporation, Sweden) and pre-packed silica Flash 40+M
Arg in FM 19 places the N-terminal amine in close proximity to
the guanidino group creating an unfavorable interaction, this
amine can make a water-mediated hydrogen bond to the backbone
carbonyl of Gly 216 in thrombin’s active site.15 The amine of 10 is
instead pointed in a different direction, deeper into the active site
and more toward the side chain of the Oic residue. In this orienta-
tion, the amine of 10 is unable to participate in hydrogen bonding
to the enzyme and is in an unfavorable hydrophobic environment,
hence its observed lower potency. These results are consistent with
cartridges (40 ꢁ 150 mm) and Flash 40+ samplets (40–63
lm par-
ticle distribution).
4.2. General procedure for solid phase peptide synthesis
findings from other research groups who have also utilized a
D-Arg
residue for thrombin inhibition.22,23
Peptides were synthesized using standard fluorenylmethyloxy-
carbonyl (Fmoc) chemistry, on a CS Bio CS336X Peptide Synthe-
sizer (CS Bio Company, Menlo Park, CA, USA), using previously
described protocols.24 Fmoc-Phe-Wang resin (NovaBiochem, EMD
Chemicals, Gibbstown, NJ, USA) was used to produce peptide
RPPGF as a C-terminal acid; Rink amide resin (Advanced Chem
Tech, Louisville, KY, USA) was used to produce peptides 2–10 as
C-terminal amides. A solution of 20% piperidine in N-methyl-2-
pyrrolidone (NMP) was used to remove the Fmoc protecting group
3. Conclusions
This study reports nine new replacements of the D-Arg residue
in the FM 19 sequence. Four of these compounds (3, 4, 6 and 7) are
more potent than lead compound FM 19, while two (6 and 7) show
equal, if not slightly improved, potency when compared to the best
previously reported compounds in this series.16 Like 1, 6 and 7

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E. A. Girnys et al. / Bioorg. Med. Chem. 19 (2011) 7425–7434
before beginning synthesis, and again to remove the Fmoc protect-
ing group after each coupling cycle. Coupling was performed using
three to four-fold excess of amino acid and a solution of 0.4 M 1-
hydroxybenzotriazole (HOBt; Advanced Chem Tech, Louisville,
KY, USA) and O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-
hexafluorophosphate (HBTU; Advanced Chem Tech, Louisville,
KY, USA) in dimethylformamide, in the presence of N,N-diisopro-
pylethylamine (DIEA). After the synthesis was complete, the resin
was washed with NMP followed by a wash with dichloromethane,
and dried.
fluoroacetate (white solid, 3.7 mg, 2.3% yield) which was >95% pure
by HPLC.
4.3.5. Synthesis of 3-amidinophenylacetic acid-Oic-Pro-D-Ala-
Phe(p-Me)-NH2 hydrotrifluoroacetate (5)
First, compound 18b (53 mg, 0.25 mmol) was dissolved in a
solution of 0.4 M HOBt/HBTU in NMP (3 mL) and then a 1 M DIEA
in NMP solution was added (2 mL). This solution was transferred to
a
vial containing Oic-Pro-D-Ala-Phe(p-Me)-resin (377 mg,
0.65 mmol/g substitution; synthesized according to the general so-
lid phase peptide synthesis procedure) in NMP (2 mL). The solution
was diluted with NMP (5 mL) and placed on a shaker at room tem-
perature for 1 h. The resin was collected, washed with dichloro-
methane and dried, to yield the resin-bound pentapeptide 19b
(373 mg). Compound 19b (304 mg) was stirred for 2 h at room
temperature with 95:5 TFA–H2O (10 mL). After filtration, the solu-
tion was concentrated in vacuo, followed by precipitation with
cold diethyl ether. After refrigeration overnight, 5 hydrotrifluoro-
acetate was collected (off-white solid, 86 mg) and purified by HPLC
to obtain 5 hydrotrifluoroacetate (white solid, 4.2 mg, 2.6% yield)
which was >95% pure by HPLC.
4.3. General peptide purification procedure
Crude peptides were purified using a Waters semipreparative
HPLC (Waters Corporation, Milford, MA, USA) with a Vydac or
Waters Protein and Peptide C18 column, using a linear gradient of
0–10% Solvent B in Solvent A to 50–60% Solvent B in Solvent A,
at a rate of 1% per minute.
4.3.1. Synthesis of RPPGF dihydrotrifluoroacetate
RPPGF was synthesized according to the general procedure for
solid phase peptide synthesis, using Fmoc-Phe-Wang resin
(520 mg, 0.39 mmol/g substitution). Once synthesis was complete,
the dried resin (627 mg) was stirred for 2 h at room temperature
with 95:5 TFA–H2O (10 mL). After filtration, the solution was con-
centrated in vacuo, followed by precipitation with cold diethyl
ether. After refrigeration overnight, RPPGF dihydrotrifluoroacetate
was collected (off-white solid, 120 mg) and purified by HPLC to ob-
tain RPPGF dihydrotrifluoroacetate (white solid, 98 mg, 60% yield)
which was >95% pure by HPLC.
4.3.6. Synthesis of 4-amidinobenzoic acid-Oic-Pro-
Me)-NH2 hydrotrifluoroacetate (6)
D-Ala-Phe(p-
The synthesis of 6 was completed using a modification of a pre-
viously reported procedure.19 In a reaction vessel for a Parr shaker,
compound 23a (15 mg, 0.02 mmol) and acetic anhydride (5 lL,
0.05 mmol) were dissolved in glacial acetic acid (40 mL) and de-
gassed. A catalytic amount of 10% Pd/C was added, and the solution
was placed on the shaker under 50 psi H2 at room temperature for
23.5 h. The solution was filtered through celite and the filtrate was
concentrated in vacuo to give 6 as a hydroacetate salt. The oil was
purified by HPLC to obtain 6 hydrotrifluoroacetate (white solid,
7.6 mg, 3.4% yield) which was >95% pure by HPLC.
4.3.2. Synthesis of cis-4-guanidino-1-cyclohexanecarboxylic
acid-Oic-Pro-D-Ala-Phe(p-Me)-NH2 hydrotrifluoroacetate (2)
Compound 15a (97 mg) was stirred for 2 h at room temperature
with 95:5 TFA–H2O (10 mL). The solution was concentrated in va-
cuo, followed by precipitation with cold diethyl ether. After refrig-
eration overnight, 2 hydrotrifluoroacetate was collected (off-white
solid, 56 mg) and purified by HPLC to obtain 2 hydrotrifluoroace-
tate (white solid, 42 mg, 39% yield) which was >95% pure by HPLC.
4.3.7. Synthesis of 4-amidinophenylacetic acid-Oic-Pro-D-Ala-
Phe(p-Me)-NH2 hydrotrifluoroacetate (7)
The synthesis of 7 was completed using a modification of a pre-
viously reported procedure.19 In a reaction vessel for a Parr shaker,
compound 23b (8.2 mg, 0.01 mmol) and acetic anhydride (5 lL,
0.05 mmol) were dissolved in glacial acetic acid (40 mL) and de-
gassed. A catalytic amount of 10% Pd/C was added, and the solution
was placed on the shaker under 50 psi H2 at room temperature for
21.5 h. The solution was filtered through celite and the filtrate was
concentrated in vacuo to give 7 as a hydroacetate salt. The oil was
purified by HPLC to obtain 7 hydrotrifluoroacetate (white solid,
2.1 mg, 0.9% yield) which was >95% pure by HPLC.
4.3.3. Synthesis of trans-4-guanidino-1-cyclohexanecarboxylic
acid-Oic-Pro-D-Ala-Phe(p-Me)-NH2 hydrotrifluoroacetate (3)
Compound 15b was stirred for 2 h at room temperature with
95:5 TFA–H2O (10 mL). The solution was concentrated in vacuo,
followed by precipitation with cold diethyl ether. After refrigera-
tion overnight, 3 hydrotrifluoroacetate was collected (off-white so-
lid, 42 mg) and purified by HPLC to obtain 3 hydrotrifluoroacetate
(white solid, 13 mg, 13% yield) which was 94% pure by HPLC.
4.3.8. Synthesis of (R)-5-guanidino-2-methylpentanoic acid-
Oic-Pro-D-Ala-Phe(p-Me)-NH2 hydrotrifluoroacetate (8)
4.3.4. Synthesis of 3-amidinobenzoic acid-Oic-Pro-
D-Ala-Phe(p-
Compound 32a was stirred for 2 h at room temperature with
95:5 TFA–H2O (10 mL). After filtration, the solution was concen-
trated in vacuo, followed by precipitation with cold diethyl ether.
After refrigeration overnight, 8 hydrotrifluoroacetate salt was col-
lected (off-white solid, 40 mg) and purified by HPLC to obtain 8
hydrotrifluoroacetate (white solid, 35 mg, 57% yield) which was
>95% pure by HPLC.
Me)-NH2 hydrotrifluoroacetate (4)
First, compound 18a (42 mg, 0.2 mmol) was dissolved in a solu-
tion of 0.4 M HOBt/HBTU in NMP (3 mL) and then a 1 M DIEA in
NMP solution was added (2 mL). This solution was transferred to
a
vial containing Oic-Pro-D-Ala-Phe(p-Me)-resin (321 mg,
0.65 mmol/g substitution; synthesized according to the general so-
lid phase peptide synthesis procedure) in NMP (2 mL). The solution
was diluted with NMP (5 mL) and placed on a shaker at room tem-
perature for 1 h. The resin was collected, washed with dichloro-
methane and dried, to yield the resin-bound pentapeptide 19a.
Compound 19a then was stirred for 2 h at room temperature with
95:5 TFA–H2O (10 mL). After filtration, the solution was concen-
trated in vacuo, followed by precipitation with cold diethyl ether.
After refrigeration overnight, 4 hydrotrifluoroacetate was collected
(off-white solid, 94 mg) and purified by HPLC to obtain 4 hydrotri-
4.3.9. Synthesis of (S)-5-guanidino-2-methylpentanoic acid-Oic-
Pro-D-Ala-Phe(p-Me)-NH2 hydrotrifluoroacetate (9)
Compound 32b was stirred for 2 h at room temperature with
95:5 TFA–H2O (10 mL). After filtration, the solution was concen-
trated in vacuo, to yield an oil containing 9 (39 mg). The oil was
purified by HPLC to obtain 9 hydrotrifluoroacetate (white solid,
22 mg, 57% yield) which was >95% pure by HPLC.

E. A. Girnys et al. / Bioorg. Med. Chem. 19 (2011) 7425–7434
7431
4.3.10. Synthesis of
dihydrotrifluoroacetate (10)
L
-Arg-Oic-Pro-
D
-Ala-Phe(p-Me)-NH2
was 75% pure by HPLC and carried on without further purification
or characterization.
Compound 10 was synthesized according to the general solid
phase peptide synthesis procedure, using Rink amide resin
(340 mg, 0.59 mmol/g substitution). Once synthesis was complete,
the dried resin (468 mg) was stirred for 2 h at room temperature
with 95:5 TFA–H2O (10 mL). After filtration, the solution was con-
centrated in vacuo, followed by precipitation with cold diethyl
ether. After refrigeration overnight, 10 dihydrotrifluoroacetate
was collected (off-white solid, 125 mg) and purified by HPLC to ob-
tain 10 dihydrotrifluoroacetate (white solid, 55 mg, 31% yield)
which was >95% pure by HPLC.
4.3.15. Synthesis of trans-4-(di-Boc)guanidino-1-
cyclohexanecarboxylic acid-Oic-Pro-D-Ala-Phe(p-Me)-NH2 (15b)
The synthesis of 15b was completed using a modification of a
previously reported procedure.18 To a stirred solution of N,N0-di-
Boc-N0-triflylguanidine (50 mg, 0.13 mmol) and triethylamine
(88 lL, 0.63 mmol) in 1,4-dioxane (2 mL) was added a solution of
14b (93 mg, 0.13 mmol) in 1,4-dioxane (13 mL). The solution stir-
red at room temperature for 24 h, and was then concentrated in
vacuo to yield a yellow-orange oil. The oil was dissolved in ethyl
acetate (20 mL), washed with 2 M KHSO4 (2 ꢁ 5 mL) followed by
saturated NaHCO3 (2 ꢁ 5 mL), dried over MgSO4 and concentrated
in vacuo to give an oily residue containing 15b. The solid was car-
ried on without further purification or characterization.
4.3.11. Synthesis of Boc-cis-4-amino-1-cyclohexanecarboxylic
acid (12a)
To a stirred solution of the commercially available 11a (286 mg,
2.0 mmol) in 2:1 1,4-dioxane–H2O (6 mL) at 0 °C was added 1 M
NaOH (3 mL), followed by di-tert-butyldicarbonate (480 mg,
2.2 mmol) dissolved in a minimal amount of 1,4-dioxane. The solu-
tion was stirred for 3 h and slowly warmed to room temperature,
and was then concentrated in vacuo to obtain a white solid. The so-
lid was dissolved in H2O (4 mL) and ethyl acetate (7 mL) and acid-
ified to pH 1.5 using 1 M HCl, prompting precipitation of 12a.
Compound 12a was collected by filtration (white solid, 388 mg,
73% yield) and was >95% pure by HPLC. 1H NMR (500 MHz, DMSO):
d 12.07 (s, 1H, br), 6.75 (s, 1H, br), 2.36 (m, 1H), 1.86 (m, 2H), 1.50–
1.37 (m, 7H), 1.37 (s, 9H).
4.3.16. Synthesis of 3-hydroxyamidiobenzoic acid19 (17a)
To
a suspension of the commercially available 16a (1 g,
6.8 mmol) in ethanol (100 mL) and H2O (5 mL) was added KOH
(954 mg, 17 mmol) and hydroxylamine hydrochloride (709 mg,
10.2 mmol), and the solution was held at reflux for 18.5 h. After
cooling to room temperature, the solid KCl was filtered away from
the solution, and the filtrate was concentrated in vacuo to give a
white solid. The solid was purified using the Flash+ system and a
mobile phase of 3:2 dicholoromethane–methanol (Rf = 0.33), to
yield 17a (white solid, 566 mg, 46% yield), which was >95% pure
by HPLC. 1H NMR (500 MHz, DMSO): d 9.71 (s, 1H, br), 8.28 (s,
1H), 7.94 (d, J = 7.7 Hz, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.46 (dd,
J1 = 7.7 Hz, J2 = 0.5 Hz, 1H), 5.89 (s, 1H)
4.3.12. Synthesis of cis-4-amino-1-cyclohexanecarboxylic acid-
Oic-Pro-D-Ala-Phe(p-Me)-NH2 hydrotrifluoroacetate (14a)
First, compound 13a was synthesized according to the general
procedure for solid phase peptide synthesis, using Rink amide resin
(270 mg, 0.75 mmol/g substitution), giving the resin-bound penta-
peptide (375 mg). Compound 13a was then stirred for 2 h at room
temperature with 95:5 TFA–H2O (10 mL). After filtration, the solu-
tion was concentrated in vacuo, followed by precipitation with
cold diethyl ether. After refrigeration overnight, 14a hydrotrifluo-
roacetate was collected (off-white solid, 108 mg, 73% yield) and
was >95% pure by HPLC. ESI-MS calculated [M+H]+, 623.4, found,
623.3.
4.3.17. Synthesis of 3-hydroxyamidinophenylacetic acid19 (17b)
To a solution of commercially available 16b (1 g, 6.25 mmol) in
ethanol (100 mL) was added KOH (877 mg, 15.6 mmol) and
hydroxylamine hydrochloride (651 mg, 9.4 mmol), and the solu-
tion was held at reflux for 16 h. After cooling to room temperature,
the solid KCl was filtered away from the solution, and the filtrate
was concentrated in vacuo to give a white solid. The solid was puri-
fied using the Flash+ system and a mobile phase of 3:2 dicholorom-
ethane–methanol (Rf = 0.27), to yield 17b (white solid, 608 mg, 50%
yield). 1H NMR (500 MHz, DMSO): d 12.49 (s, 1H, br), 9.60 (s, 1H),
7.57 (s, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.29 (dd, J1 = 7.6 Hz, J2 = 0.5 Hz,
1H), 7.25 (d, J = 6.8 Hz, 1 H), 5.77 (s, 2H), 3.54 (s, 2H).
4.3.13. Synthesis of trans-4-amino-1-cyclohexanecarboxylic
acid-Oic-Pro-D-Ala-Phe(p-Me)-NH2 hydrotrifluoroacetate (14b)
First, Compound 13b was synthesized using the commercially
available 12b according to the general procedure for solid phase
peptide synthesis, using Rink amide resin (310 mg, 0.70 mmol/g
substitution), giving the resin-bound pentapeptide (358 mg). Com-
pound 13b was then stirred for 2 h at room temperature with 95:5
TFA–H2O (10 mL). After filtration, the solution was concentrated in
vacuo, followed by precipitation with cold diethyl ether. After
refrigeration overnight, 14b hydrotrifluoroacetate was collected
(off-white solid, 93 mg, 58% yield) and was 67% pure by HPLC
and carried on without further purification or characterization.
4.3.18. Synthesis of 3-amidiobenzoic acid hydrochloride (18a)
In a reaction vessel for a Parr shaker, compound 17a (566 mg,
3.1 mmol) was suspended in H2O (50 mL) and degassed. A catalytic
amount of 10% Pd/C was added, and the solution was placed on the
shaker under 50 psi H2 at 50 °C for 20 h. No longer a suspension,
the solution was filtered through celite, and 1 M HCl (3.15 mL)
was added to the filtrate which was concentrated in vacuo to yield
18a hydrochloride (white solid, 518 mg, 82% yield). 1H NMR
(500 MHz, DMSO): d 9.61 (s, 2H), 9.44 (s, 2H), 8.25 (s, 1H), 8.20
(d, J = 7.3 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.72 (dd, J1 = 7.5 Hz,
J2 = 0.5 Hz, 1H).
4.3.14. Synthesis of cis-4-(di-Boc)guanidino-1-
cyclohexanecarboxylic acid-Oic-Pro-D-Ala-Phe(p-Me)-NH2 (15a)
The synthesis of 15a was completed using a modification of a
previously reported procedure.18 To a stirred solution of N,N0-di-
Boc-N0-triflylguanidine (48 mg, 0.12 mmol) and triethylamine
4.3.19. Synthesis of 3-amidinophenylacetic acid hydrochloride
(18b)
In a reaction vessel for a Parr shaker, compound 17b (608 mg,
3.1 mmol) was suspended in H2O (50 mL) and degassed. A catalytic
amount of 10% Pd/C was added, and the solution was placed on the
shaker under 50 psi H2 at 50 °C for 18 h. No longer a suspension,
the solution was filtered through celite, and 1 M HCl (3.13 mL)
was added to the filtrate which was concentrated in vacuo to yield
18b hydrochloride (white solid, 604 mg, 90% yield). 1H NMR
(500 MHz, DMSO): d 12.50 (s, 1H, br), 9.40 (s, 2H), 9.20 (s, 2H),
(96 lL, 0.69 mmol) in 1,4-dioxane (2 mL) was added a solution of
14a (101 mg, 0.14 mmol) in 1,4-dioxane (5 mL). The solution stir-
red at room temperature for 24.5 h, and was then concentrated
in vacuo to yield a yellow oil. The oil was dissolved in ethyl acetate
(20 mL), washed with 2 M KHSO4 (2 ꢁ 5 mL) followed by saturated
NaHCO3 (2 ꢁ 5 mL), dried over MgSO4 and concentrated in vacuo
to give a white solid containing 15a (97 mg, 60% yield). The solid

7432
E. A. Girnys et al. / Bioorg. Med. Chem. 19 (2011) 7425–7434
7.72 (s, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.57 (dd,
J1 = 7.9 Hz, J2 = 0.5 Hz, 1H), 3.63 (s, 2H). ESI-MS calculated [M+H]+,
179.1, found, 179.2.
was >70% pure by HPLC. ESI-MS calculated [M+H]+, 674.4, found,
674.5.
4.3.24. Synthesis of (S)-4-benzyl-3-propionyloxazolidin-2-one21
(25a)
4.3.20. Synthesis of 4-hydroxyamidiobenzoic acid19 (21a)
To a suspension of commercially available 20a (1 g, 6.8 mmol)
in ethanol (100 mL) and H2O (5 mL) was added KOH (954 mg,
17 mmol) and hydroxylamine hydrochloride (709 mg, 10.2 mmol),
and the solution was held at reflux for 18.5 h. After cooling to room
temperature, the solid KCl was filtered away from the solution, and
the filtrate was concentrated in vacuo to give a white solid. The so-
lid was purified using the Flash+ system and a mobile phase of 3:2
dicholoromethane–methanol (Rf = 0.35), to yield 21a (white solid,
616 mg, 50% yield). 1H NMR (500 MHz, DMSO): d 9.76 (s, 1H, br),
7.85 (d, J = 8.1 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 5.80 (s, 2H).
Commercially available compound 24a (800 mg, 4.5 mmol) was
dissolved in anhydrous THF (30 mL) in dry glassware under N2, and
cooled to ꢀ78 °C in a dry ice/acetone bath. A solution of 1.6 M n-
butyllithium in hexanes (5.0 mmol, 3.1 mL) was syringed in over
3 min. After mixing for 30 min, propionyl chloride (5.0 mmol,
0.43 mL) was syringed in over 3 min. The solution was allowed
to slowly warm to room temperature over 14 h and quenched by
the addition of saturated NH4Cl (10 mL) and H2O (30 mL). The final
aqueous layer was extracted with ethyl acetate (3 ꢁ 40 mL) and
the combined organic extracts were dried over MgSO4 and concen-
trated in vacuo to yield 25a (oil, 1.0 g, 96% yield) which was >95%
pure by HPLC. 1H NMR (500 MHz, CDCl3): d 7.33 (dd, J1 = 7.4 Hz,
J2 = 7.6 Hz, 2H), 7.29 (d, J = 6.9 Hz, 1H), 7.21 (d, J = 7.4 Hz, 2H),
4.67 (m, 1H), 4.18 (m, 2H), 3.31 (dd, J1 = 13.3 Hz, J2 = 3.2 Hz, 1H),
2.96 (m, 2H), 2.77 (dd, J1 = 13.3 Hz, J2 = 3.7 Hz, 1H), 1.21 (t,
J = 7.4 Hz, 3H).
4.3.21. Synthesis of 4-hydroxyamidinophenylacetic acid19 (21b)
To
a suspension of commercially available 20b (882 mg,
5.5 mmol) in ethanol (100 mL) was added KOH (767 mg,
13.7 mmol) and hydroxylamine hydrochloride (570 mg, 8.2 mmol),
and the solution was held at reflux for 15 h. After cooling to room
temperature, the solid KCl was filtered away from the solution, and
the filtrate was concentrated in vacuo to give a white solid. The so-
lid was purified using the Flash+ system and a mobile phase of 3:2
dicholoromethane–methanol (Rf = 0.27), to yield 21b (white solid,
286 mg, 27% yield). 1H NMR (300 MHz, DMSO): d 12.50 (s, 1H,
br), 9.59 (s, 1H, br), 7.59 (d, J = 7.3 Hz, 2H), 7.24 (d, J = 7.6 Hz,
2H), 5.78 (s, 2H), 3.53 (s, 2H).
4.3.25. Synthesis of (R)-5-((S)-4-benzyl-2-oxooxazolidin-3-yl)-4-
methyl-5-oxopentanenitrile21 (26a)
TiCl3(OiPr) was first prepared using dry glassware under N2. A
solution of 1 M TiCl4 in dichloromethane (3.4 mmol, 3.4 mL) was
cooled to 0 °C, and Ti(OiPr)4 (1.1 mmol, 0.34 mL) was added drop-
wise over 5 min. The solution was diluted with anhydrous dichlo-
romethane (3.5 mL) and stirred at 0 °C for 15 min. Meanwhile,
compound 25a (1.0 g, 4.3 mmol) was dissolved in anhydrous
dichloromethane (14 mL) in dry glassware under N2, and DIEA
(0.79 mL) was added dropwise over 5 min, with stirring, at room
temperature and then cooled to 0 °C. The TiCl3(OiPr) solution was
added to the 25a solution dropwise over 25 min. The TiCl3(OiPr)
flask was rinsed with anhydrous dichloromethane (2 mL), and
the rinse added to the combined flask. The solution was stirred
at 0 °C for 30 min, and then acrylonitrile (0.43 mL, 6.5 mmol) was
added dropwise over 10 min, and this solution stirred at 0 °C for
4 h. The reaction was quenched with the addition of saturated
NH4Cl (25 mL) and H2O (15 mL), and the aqueous layer was ex-
tracted with diethyl ether (3 ꢁ 40 mL). The organic extracts were
combined and washed with saturated NaHCO3 (55 mL) followed
by brine (55 mL), dried over MgSO4 and concentrated in vacuo to
yield a yellow oil. The oil was purified using the Flash+ system
and a mobile phase of 3:7 ethyl acetate–hexanes (Rf = 0.30), to
yield 26a as a very slightly yellow oil (890 mg, 72% yield). 1H
NMR (500 MHz, CDCl3): d 7.35 (dd, J1 = 7.6 Hz, J2 = 7.1 Hz, 2H),
7.30 (d, J = 7.3 Hz, 1H), 7.21 (d, J = 7.1 Hz, 2H), 4.69 (m, 1H), 4.20
(m, 2H), 3.83 (sextet, J = 6.9 Hz, 1H), 3.31 (dd, J1 = 13.4 Hz,
J2 = 3.2 Hz, 1H), 2.78 (dd, J1 = 13.3 Hz, J2 = 3.6 Hz, 1H), 2.41 (dt,
J1 = 7.7 Hz, J2 = 1.2 Hz, 2H), 2.19 (m, 1H), 1.81 (m, 1H), 1.24 (d,
J = 6.9 Hz, 3H).
4.3.22. Synthesis of 4-hydroxyamidinobenzoic acid-Oic-Pro-D-
Ala-Phe(p-Me)-NH2 hydrotrifluoroacetate (23a)
First, compound 21a (71 mg, 0.38 mmol) was dissolved in a
solution of 0.4 M HOBt/HBTU in NMP (2.5 mL) and then a 1 M DIEA
in NMP solution was added (1 mL). This solution was transferred to
a
vial containing Oic-Pro-D-Ala-Phe(p-Me)-resin (360 mg,
0.7 mmol/g substitution; synthesized according to the general so-
lid phase peptide synthesis procedure) in NMP (2 mL). The solution
was diluted with NMP (5 mL) and placed on a shaker at room tem-
perature for 4 h. The resin was collected, washed with dichloro-
methane and dried, to yield the resin-bound pentapeptide 22a
(417 mg). Compound 22a was then stirred for 2 h at room temper-
ature with 95:5 TFA–H2O (10 mL). After filtration, the solution was
concentrated in vacuo, followed by precipitation with cold diethyl
ether. After refrigeration overnight, 23a hydrotrifluoroacetate was
collected (off-white solid, 105 mg) and purified by HPLC to obtain
23a hydrotrifluoroacetate (white solid, 15 mg, 6.2% yield) which
was >80% pure by HPLC. ESI-MS calculated [M+H]+, 660.4, found,
660.3.
4.3.23. Synthesis of 4-hydroxyamidinophenylacetic acid-Oic-
Pro-D-Ala-Phe(p-Me)-NH2 hydrotrifluoroacetate (23b)
First, compound 21b (101 mg, 0.41 mmol) was dissolved in a
solution of 0.4 M HOBt/HBTU in NMP (2.5 mL) and then a 1 M DIEA
in NMP solution was added (1 mL). This solution was transferred to
4.3.26. Synthesis of (S)-5-((R)-4-benzyl-2-oxooxazolidin-3-yl)-4-
methyl-5-oxopentanenitrile21 (26b)
a
vial containing Oic-Pro-D-Ala-Phe(p-Me)-resin (371 mg,
0.7 mmol/g substitution; synthesized according to the general so-
lid phase peptide synthesis procedure) in NMP (2 mL). The solution
was diluted with NMP (5 mL) and placed on a shaker at room tem-
perature for 4 h. The resin was collected, washed with dichloro-
methane and dried, to yield the resin-bound pentapeptide 22b
(443 mg). Compound 22b was then stirred for 2 h at room temper-
ature with 95:5 TFA–H2O (10 mL). After filtration, the solution was
concentrated in vacuo, followed by precipitation with cold diethyl
ether. After refrigeration overnight, 23b hydrotrifluoroacetate was
collected (off-white solid, 100 mg) and purified by HPLC to obtain
23b hydrotrifluoroacetate (white solid, 14 mg, 5.1% yield) which
TiCl3(OiPr) was first prepared using dry glassware under N2. A
solution of 1 M TiCl4 in dichloromethane (3.6 mmol, 3.6 mL) was
cooled to 0 °C, and Ti(OiPr)4 (1.2 mmol, 0.35 mL) was added drop-
wise over 5 min. The solution was diluted with anhydrous dichlo-
romethane (3.5 mL) and stirred at 0 °C for 15 min. Meanwhile, the
commercially available compound 25b (1.05 g, 4.5 mmol) was dis-
solved in anhydrous dichloromethane (14 mL) in dry glassware un-
der N2, and DIEA (0.75 mL) was added dropwise over 5 min, with
stirring, at room temperature and then cooled to 0 °C. The Ti-
Cl3(OiPr) solution was added to the 25b solution dropwise over
25 min. The TiCl3(OiPr) flask was rinsed with anhydrous dichloro-

E. A. Girnys et al. / Bioorg. Med. Chem. 19 (2011) 7425–7434
7433
methane (2 mL), and the rinse added to the combined flask. The
solution stirred at 0 °C for 30 min, and then acrylonitrile
(0.45 mL, 6.8 mmol) was added dropwise over 10 min, and this
solution stirred at 0 °C for 4 h. The reaction was quenched with
the addition of saturated NH4Cl (25 mL) and H2O (15 mL), and
the aqueous layer was extracted with diethyl ether (3 ꢁ 40 mL).
The organic extracts were combined and washed with saturated
NaHCO3 (55 mL) followed by brine (55 mL), dried over MgSO4
and concentrated in vacuo to yield a yellow oil. The oil was purified
using the Flash+ system and a mobile phase of 3:7 ethyl acetate–
hexanes (Rf = 0.27), to yield 26b as a very slightly yellow oil
(919 mg, 71% yield). 1H NMR (500 MHz, CDCl3): d 7.35 (dd,
J1 = 7.5 Hz, J2 = 7.2 Hz, 2H), 7.30 (d, J = 7.4 Hz, 1H), 7.21 (d,
J = 7.2 Hz, 2H), 4.69 (m, 1H), 4.19 (m, 2H), 3.82 (sextet, J = 6.7 Hz,
1H), 3.31 (dd, J1 = 13.5 Hz, J2 = 3.3 Hz, 1H), 2.78 (dd, J1 = 13.4 Hz,
J2 = 3.6 Hz, 1H), 2.41 (dt, J1 = 8.2 Hz, J2 = 1.9 Hz, 2H), 2.18 (m, 1H),
1.81 (m, 1H), 1.23 (d, J = 7.1 Hz, 3H).
(s, 3H, br), 2.74 (sextet, J = 7.7 Hz, 2H), 2.34 (sextet, J = 6.1 Hz, 1H),
1.56 (m, 3H), 1.39 (m, 1H), 1.05 (d, J = 6.8 Hz, 3H).
4.3.31. Synthesis of Boc-(R)-5-amino-2-methylpentanoic acid
(29a)
To a stirred solution of 28a (237 mg, 1.4 mmol) in 2:1 1,4-diox-
ane–H2O (9 mL) at 0 °C was added 1 M NaOH (4 mL), followed by
di-tert-butyldicarbonate (340 mg, 1.6 mmol) dissolved in a mini-
mal amount of 1,4-dioxane. The solution stirred for 4 h and slowly
warmed to room temperature, and the solution was concentrated
in vacuo to obtain a white solid. The solid was dissolved in H2O
(10 mL) and acidified to pH 2 using 1 M HCl. The aqueous layer
was extracted with ethyl acetate (5 ꢁ 20 mL), and the combined
organic extracts were washed with brine (2 ꢁ 40 mL) followed by
H2O (2 ꢁ 40 mL), and dried over MgSO4. The organic layer was con-
centrated in vacuo to yield 29a (colorless oil, 248 mg, 76% yield).
1H NMR (500 MHz, DMSO): d 12.06 (s, 1H, br), 6.81 (t, J = 5.4 Hz,
1H), 2.88 (q, J = 6.3 Hz, 2H), 2.29 (sextet, J = 6.7 Hz, 1H), 1.50 (pen-
tet, J = 6.5 Hz, 2H), 1.37 (s, 9H), 1.32 (m, 2H), 1.02 (d, J = 7.0 Hz, 3H).
4.3.27. Synthesis of (R)-4-cyano-2-methylbutanoic acid21 (27a)
To a stirred solution of 26a (729 mg, 2.55 mmol) in THF (18 mL)
at 0 °C were added 1 M LiOH solution (5.95 mL) and H2O (50
l
L).
4.3.32. Synthesis of Boc-(S)-5-amino-2-methylpentanoic acid
(29b)
The solution slowly warmed to room temperature over 1 h. The
solution was diluted with H2O (20 mL) and extracted with ethyl
acetate (3 ꢁ 30 mL). The aqueous layer was acidified to pH 1.5 with
1 M HCl, and extracted with ethyl acetate (3 ꢁ 30 mL). These last
organic fractions were combined, dried over MgSO4, and concen-
trated in vacuo to yield 27a as a colorless oil (191 mg, 51% yield).
1H NMR (500 MHz, DMSO): d 12.38 (s, 1H, br), 2.51 (m, 2H), 2.41
(sextet, J = 7.1 Hz, 1H), 1.85 (m, 1H), 1.63 (m, 1H), 1.09 (d,
J = 7.1 Hz, 3H).
To a stirred solution of 28b (293 mg, 1.7 mmol) in 2:1 1,4-diox-
ane–H2O (9 mL) at 0 °C was added 1 M NaOH (4.5 mL), followed by
di-tert-butyldicarbonate (540 mg, 1.9 mmol) dissolved in a mini-
mal amount of 1,4-dioxane. The solution stirred for 4.5 h and
slowly warmed to room temperature, and the solution was con-
centrated in vacuo to obtain a white solid. The solid was dissolved
in H2O (10 mL) and acidified to pH 2 using 1 M HCl. The aqueous
layer was extracted with ethyl acetate (5 ꢁ 20 mL), and the com-
bined organic extracts were washed with brine (2 ꢁ 40 mL) fol-
lowed by H2O (2 ꢁ 40 mL), and dried over MgSO4. The organic
layer was concentrated in vacuo to yield 29b (colorless oil,
340 mg, 84% yield). 1H NMR (500 MHz, DMSO): d 12.06 (s, 1H,
br), 6.81 (t, J = 5.3 Hz, 1H), 2.87 (q, J = 6.3 Hz, 2H), 2.29 (sextet,
J = 6.8 Hz, 1H), 1.49 (pentet, J = 6.8 Hz, 2H), 1.37 (s, 9H), 1.33 (m,
2H), 1.02 (d, J = 6.9 Hz, 3H).
4.3.28. Synthesis of (S)-4-cyano-2-methylbutanoic acid21 (27b)
To a stirred solution of 26b (919 mg, 3.2 mmol) in THF (21 mL)
at 0 °C were added 1 M LiOH solution (6.42 mL) and H2O (580 lL).
The solution slowly warmed to room temperature over 15 h. The
solution was diluted with H2O (20 mL) and extracted with ethyl
acetate (3 ꢁ 30 mL). The aqueous layer was acidified to pH 1.5 with
1 M HCl, and extracted with ethyl acetate (3 ꢁ 30 mL). These last
organic fractions were combined, dried over MgSO4, and concen-
trated in vacuo to yield 27b as a colorless oil (233 mg, 57% yield).
1H NMR (500 MHz, DMSO): d 12.35 (s, 1H, br), 2.52 (m, 2H), 2.40
(sextet, J = 7.0 Hz, 1H), 1.89 (m, 1H), 1.62 (m, 1H), 1.09 (d,
J = 7.1 Hz, 3H).
4.3.33. Synthesis of (R)-5-amino-2-methylpentanoic acid-Oic-
Pro-D-Ala-Phe(p-Me)-NH2 hydrotrifluoroacetate (31a)
First, compound 29a (248 mg, 1.07 mmol) was dissolved in a
solution of 0.4 M HOBt/HBTU in NMP (2.75 mL) and then a 1 M
DIEA in NMP solution was added (1.1 mL). This solution was trans-
ferred to a vial containing Oic-Pro-D-Ala-Phe(p-Me)-resin (388 mg,
4.3.29. Synthesis of (R)-5-amino-2-methylpentanoic acid
hydrochloride21 (28a)
0.7 mmol/g substitution; synthesized according to the general so-
lid phase peptide synthesis procedure) in NMP (3 mL). The solution
was diluted with NMP (6 mL) and placed on a shaker at room tem-
perature for 4.5 h. The resin was collected, washed with dichloro-
methane and dried, to yield the resin-bound pentapeptide 30a
(437 mg). Compound 30a was then stirred for 2 h at room temper-
ature with 95:5 TFA–H2O (10 mL). After filtration, the solution was
concentrated in vacuo, followed by precipitation with cold diethyl
ether. After refrigeration overnight, 31a hydrotrifluoroacetate was
collected (off-white solid, 101 mg) and purified by HPLC to obtain
31a hydrotrifluoroacetate (white solid, 67 mg, 54% yield) which
was >95% pure by HPLC. ESI-MS calculated [M+H]+, 611.4, found,
611.2.
In a reaction vessel for the Parr shaker, 27a (191 mg, 1.5 mmol)
was dissolved in 3:7 H2O–methanol (40 mL) and degassed. A cata-
lytic amount of PtO2 was added, and the solution was placed on the
shaker at room temperature under 50 psi H2 for 15 h. The solution
was filtered through celite, and 1 M HCl (2 mL) was added to the
filtrate. The solution was concentrated in vacuo to yield 28a hydro-
chloride (oil, 237 mg, 94% yield). 1H NMR (500 MHz, DMSO): d 7.96
(s, 3H, br), 2.74 (sextet, J = 7.7 Hz, 2H), 2.33 (sextet, J = 6.9 Hz, 1H),
1.55 (m, 3H), 1.39 (m, 1H), 1.05 (d, J = 7.0 Hz, 3H).
4.3.30. Synthesis of (S)-5-amino-2-methylpentanoic acid
hydrochloride21 (28b)
In a reaction vessel for the Parr shaker, 27b (233 mg, 1.8 mmol)
was dissolved in 3:7 H2O–methanol (40 mL) and degassed. A cata-
lytic amount of PtO2 was added, and the solution was placed on the
shaker at room temperature under 50 psi H2 for 14 h. The solution
was filtered through celite, and 1 M HCl (2 mL) was added to the
filtrate. The solution was concentrated in vacuo to yield 28b hydro-
chloride (oil, 293 mg, 95% yield). 1H NMR (500 MHz, DMSO): d 7.97
4.3.34. Synthesis of (S)-5-amino-2-methylpentanoic acid-Oic-
Pro-D-Ala-Phe(p-Me)-NH2 (31b)
First, compound 29b (97 mg, 0.42 mmol) was dissolved in a
solution of 0.4 M HOBt/HBTU in NMP (2.5 mL) and then a 1 M DIEA
in NMP solution was added (1 mL). This solution was transferred to
a
vial containing Oic-Pro-D-Ala-Phe(p-Me)-resin (357 mg,
0.59 mmol/g substitution; synthesized according to the general

7434
E. A. Girnys et al. / Bioorg. Med. Chem. 19 (2011) 7425–7434
solid phase peptide synthesis procedure) in NMP (2 mL). The solu-
tion was diluted with NMP (6 mL) and placed on a shaker at room
temperature for 13 h. The resin was collected, washed with dichlo-
romethane and dried, to yield the resin-bound pentapeptide 30b
(397 mg). Compound 30b was then stirred for 2 h at room temper-
ature with 95:5 TFA–H2O (10 mL). After filtration, the solution was
concentrated in vacuo, followed by precipitation with cold diethyl
ether. After refrigeration overnight, 31b hydrotrifluoroacetate was
collected (off-white solid, 84 mg) and purified by HPLC to obtain
31b hydrotrifluoroacetate (white solid, 37 mg, 40% yield) which
was >95% pure by HPLC. ESI-MS calculated [M+H]+, 611.4, found,
611.2.
of substrate. The final concentration of enzyme in the assay was
2 nM and the final substrate concentration was 200 M. After
l
10 min, the OD405 (average and standard deviation of the three
wells for each concentration) was plotted as a function of the neg-
ative log of inhibitor concentration to generate a dose–response
curve using GraphPad Prism (version 5, GraphPad Software, Inc.,
La Jolla, CA, USA). From these data, an IC50 value was determined.
Each compound was tested in at least three independent experi-
ments, and the average and standard deviation are reported. A
sample inhibition profile, producing a single result for compound
7 can be found in Figure 4.
Acknowledgments
4.3.35. Synthesis of (R)-5-(di-Boc)guanidino-2-methylpentanoic
acid-Oic-Pro-
D
-Ala-Phe(p-Me)-NH2 (32a)
This work was supported by an American Foundation for Phar-
maceutical Education Pre-Doctoral Fellowship and the Fred M.
Lyons Fellowship, College of Pharmacy, University of Michigan to
E.A.G. This work was also supported by Rackham Merit Fellow-
ships, Rackham Graduate School, University of Michigan to E.A.G.
and V.R.P. The authors would also like to thank Michael R. Kuszpit
for his experimental assistance and helpful discussions.
The synthesis of 32a was completed using a modification of a
previously reported procedure.18 To a stirred solution of N,N0-di-
Boc-N0-triflylguanidine (32 mg, 0.08 mmol) and triethylamine
(57 lL, 0.41 mmol) in 1,4-dioxane (2 mL) was added a solution of
31a (56 mg, 0.08 mmol) in 1,4-dioxane (18 mL). The solution stir-
red at room temperature for 15 h, and was then concentrated in
vacuo to yield a yellow oil. The oil was dissolved in ethyl acetate
(20 mL), and washed with 2 M KHSO4 (2 ꢁ 10 mL) followed by sat-
urated NaHCO3 (2 ꢁ 10 mL), dried over MgSO4 and concentrated in
vacuo to give a white solid containing 32a. The solid was carried on
without further purification or characterization.
References and notes
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6. Straub, A.; Roehrig, S.; Hillisch, A. Angew. Chem. 2011, 50, 4574.
7. Alban, S. Curr. Pharm. Des. 2008, 14, 1152.
4.3.36. Synthesis of (S)-5-(di-Boc)guanidino-2-methylpentanoic
acid-Oic-Pro-D-Ala-Phe(p-Me)-NH2 (32b)
The synthesis of 32b was completed using a modification of a
previously reported procedure.18 To a stirred solution of N,N0-di-
Boc-N0-triflylguanidine (20 mg, 0.05 mmol) and triethylamine
8. Sidhu, P. S.; Liang, A.; Mehta, A. Y.; Aziz, M. H. A.; Zhou, Q.; Desai, U. R. J. Med.
Chem. 2011, 54, 5522.
9. Henry, B. L.; Monien, B. H.; Bock, P. E.; Desai, U. R. J. Biol. Chem. 2007, 282,
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Arterioscler. Thromb. Vasc. Biol. 2010, 30, 1885.
11. Gustafsson, D.; Bylund, R.; Antonsson, T.; Nilsson, I.; Nyström, Jan-Erik;
Eriksson, U.; Bredberg, U.; Teger-Nilsson, A.-C. Nat. Rev. Drug Disc. 2004, 3, 649.
12. Hasan, A. A. K.; Warnock, M.; Nieman, M.; Srikanth, S.; Mahdi, F.; Krishnan, R.;
Tulinsky, A.; Schmaier, A. H. Am. J. Physiol. Heart Circ. Physiol. 2003, 285, H183.
13. Nieman, M. T.; Warnock, M.; Hasan, A. A. K.; Mahdi, F.; Lucchesi, B. R.; Brown,
N. J.; Murphey, L. J.; Schmaier, A. H. J. Pharmacol. Exp. Ther. 2004, 311, 492.
14. Burke, F. M.; Warnock, M.; Schmaier, A. H.; Mosberg, H. I. Chem. Biol. Drug Des.
2006, 68, 235.
(36 lL, 0.26 mmol) in 1,4-dioxane (2 mL) was added a solution of
31b (37 mg, 0.05 mmol) in 1,4-dioxane (13 mL). The solution stir-
red at room temperature for 6 days, and was then concentrated
in vacuo to yield a yellow oil. The oil was dissolved in ethyl acetate
(20 mL), and washed with 2 M KHSO4 (2 ꢁ 10 mL) followed by sat-
urated NaHCO3 (2 ꢁ 10 mL), dried over MgSO4 and concentrated in
vacuo to give a white solid containing 32b. The solid was carried on
without further purification or characterization.
15. Nieman, M. T.; Burke, F.; Warnock, M.; Zhou, Y.; Sweigart, J.; Chen, A.; Ricketts,
D.; Lucchesi, B. R.; Chen, Z.; Cera, E. D.; Hilfinger, J.; Kim, J. S.; Mosberg, H. I.;
Schmaier, A. H. J. Thromb. Haemost. 2008, 6, 837.
4.4. Inhibition of human a-thrombin assay protocol
16. Girnys, E. A.; Sobczyk-Kojiro, K.; Mosberg, H. I. Chem. Biol. Drug Des. 2010, 75,
35.
17. Masic, L. P. Curr. Med. Chem. 2006, 13, 3627.
18. Feichtinger, K.; Sings, H. L.; Baker, T. J.; Matthews, K.; Goodman, M. J. Org. Chem.
1998, 63, 8432.
19. Judkins, B. D.; Allen, D. G.; Cook, T. A.; Evans, B.; Sardharwala, T. E. Synth.
Commun. 1996, 26, 4351.
20. Evans, D. A.; Bilodeau, M. T.; Somers, T. C.; Clardy, J.; Cherry, D.; Kato, Y. J. Org.
Chem. 1991, 56, 5750.
21. Attwood, M. R.; Conway, E. A.; Dunsdon, R. M.; Greening, J. R.; Handa, B. K.;
Jones, P. S.; Jordan, S. C.; Keech, E.; Wilson, F. X. Bioorg. Med. Chem. Lett. 1997, 7,
429.
22. Hellstern, P.; Sturzebecher, U.; Wuchold, B.; Haubelt, H.; Seyfert, U. T.; Bauer,
M.; Vogt, A.; Sturzebecher, J. J. Thromb. Haemost. 2007, 5, 2119.
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611, 579.
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Assays to measure the ability of the new synthetic peptides to
inhibit human -thrombin (>95% pure; specific activity = 5070 U/
a
mg, Haematologic Technologies, Essex Junction, VT, USA) cleavage
of the chromogenic substrate tetrapeptide Sarcosine-Pro-Arg-p-
nitroanilide (Bachem, Torrance, CA, USA) were completed as previ-
ously described.16 Before use in each assay,
a-thrombin solutions
were stored on ice in assay buffer (10 mM tris(hydroxy-
methyl)aminomethane (Tris), 150 mM NaCl, pH 7.6), with long-
term storage in a 50/50 glycerol/water solution at ꢀ20 °C.
Inhibition experiments were done in 96-well plates, using nine
different concentrations of synthetic peptides, each in triplicate.
Controls included no enzyme, no inhibitor and lead compound
FM 19. Enzyme was preincubated with peptide in assay buffer at
37 °C for 5 min, and the experiment was initiated with the addition