Design, synthesis and biological activity of novel peptidyl benzyl ketone FVIIa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.05.025

Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formulations of rFVIIa, which is a serine protease used for the treatment of hemophilia A and B inhibitor patients. A library of compounds was synthesized with different tripeptide sequences, N-terminals and d-amino acids in the P3 position. Cbz-d-Phe-Phe-Arg-bk (33) was found to be the best candidate with a potency of K_i = 8 μM and no substantial inhibition of related blood coagulation factors (thrombin and FXa). Computational studies revealed that 33 has a very stable binding conformation due to intramolecular hydrogen bonds, which cannot be formed with l-Phe in the P3 position. Nonpolar amino acids were found to be superior, probably due to a minimization of the cost of desolvation upon binding to FVIIa.

Full text of DOI:10.1016/j.bmcl.2011.05.025

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3918–3922
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological activity of novel peptidyl benzyl ketone
FVIIa inhibitors
Morten Storgaard a,b, , Signe T. Henriksen a, , Florencio Zaragoza b,à, Bernd Peschke b, David Tanner a,
⇑
a Technical University of Denmark, Department of Chemistry, Kemitorvet, Building 201, DK-2800 Kgs. Lyngby, Denmark
b Novo Nordisk A/S, Biopharm Chemistry, Novo Nordisk Park, DK-2760 Måløv, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl
ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formula-
tions of rFVIIa, which is a serine protease used for the treatment of hemophilia A and B inhibitor patients.
Received 13 April 2011
Revised 9 May 2011
Accepted 9 May 2011
Available online 18 May 2011
A library of compounds was synthesized with different tripeptide sequences, N-terminals and
acids in the P3 position. Cbz- -Phe–Phe–Arg–bk (33) was found to be the best candidate with a potency
of Ki = 8 M and no substantial inhibition of related blood coagulation factors (thrombin and FXa).
Computational studies revealed that 33 has a very stable binding conformation due to intramolecular
hydrogen bonds, which cannot be formed with -Phe in the P3 position. Nonpolar amino acids were found
D-amino
D
l
Keywords:
Activated factor VII
Serine protease inhibitor
Peptidyl benzyl ketone
C-Terminal modified peptide
Stabilization agent
L
to be superior, probably due to a minimization of the cost of desolvation upon binding to FVIIa.
Ó 2011 Elsevier Ltd. All rights reserved.
NovoSevenÒ (rFVIIa)# is a serine protease used for the treat-
ment of hemophilia A and B inhibitor patients.1 In our efforts to
synthesize stabilization agents of liquid formulations of rFVIIa,
we were seeking to develop a series of FVIIa inhibitors with a dis-
tinct set of properties not accounted for in previously reported
FVIIa inhibitors.2 An ideal inhibitor in our respect would be revers-
H
S2
P2
HN
N
Pg2
P1'
NH
O
O
H
N
H
N
S4
R
N
H
S1'
P3
S3
O
P1
S1
Pg1
N
H
O
ible and only medium potent (preferably low lM Ki) and with no
substantial inhibition of related blood coagulation factors (e.g.,
FXa and thrombin). Potent FVIIa inhibitors would, on the other
hand, be valuable for development of novel anticoagulants target-
ing the initial stage of the coagulation cascade. This would avoid
prolonged bleedings as observed with traditional late-stage antico-
agulants (thrombin inhibitors).3 FVIIa inhibitors as stabilization
agents should be nontoxic, exhibit high clearance in vivo, have a
molecular weight of less than 700 g/mol to avoid an immune
response and have a sufficient aqueous solubility. Ideally the
Serine trap
Projected FVIIa inhibitors
Arginyl benzyl ketone
building block
Figure 1. The projected FVIIa inhibitors consist of a tripeptide sequence with a
benzyl ketone (bk) serine trap in the P1–P1’ position. With arginine in the P1
position, the inhibitors can be synthesized from the building block shown to the
right.
synthetic route should be short and efficient and allow easy
alternation of functional groups for a practical and fast structure-
activity relationship analysis. Stabilization of protease formula-
tions is not only important for FVIIa, but also in other applications
such as for the development of liquid detergents.4
To meet our preferred inhibitor properties, we elected to
explore a novel series of C-terminal modified peptides containing
a benzyl ketone (bk) in the P1–P1’ position ( Fig. 1). Molecules
containing a neutral electrophilic group (serine trap) in this posi-
tion are potential competitive and covalently-bound inhibitors of
serine proteases due to reaction with Ser195 in the active site.5
Abbreviations: bk, benzyl ketone; DIPEA, N,N-diisopropylethylamine; FXa,
activated Factor X; HOAt, 1-hydroxy-7-azabenzotrizole; HPLC-CLND, high perfor-
mance liquid chromatography–chemiluminescent nitrogen detection; MD, molec-
ular dynamics; Pmc, 2,2,5,7,8-pentamethyl-chroman-6-ylsulfonyl; PS-SCL,
positional-scanning synthetic combinatorial libraries; PyBOP, benzotriazol-1-yl-
oxytripyrrolidinophos-phonium hexafluorophosphate; rFVIIa, recombinant acti-
vated Factor VII; thr, thrombin; UPLC, ultra performance liquid chromatography.
⇑
Corresponding author. Tel.: +45 45252188; fax: +45 45933968.
E-mail address: dt@kemi.dtu.dk (D. Tanner).
Present address: Faculty of Pharmaceutical Sciences, Department of Medicinal
Chemistry, Jagtvej 162, DK-2100 Copenhagen Ø, Denmark.
à
Present address: Lonza AG, Rottenstraße 6, CH-3930 Visp, Switzerland.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.025

M. Storgaard et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3918–3922
3919
Highly electrophilic groups such as boronic acids, trifluoromethyl
sufficient space for the serine trap (Fig. 2). Interestingly, a cat-
ion– -interaction was observed with Lys192, which is a noncon-
ketones, aldehydes,
a
-keto carboxylates or
a
-keto heterocycles
p
are known to afford very potent serine protease inhibitor.6
served amino acid among the coagulation proteases.11
Initially, the arginyl benzyl ketone building block 3 was synthe-
sized in good overall yield with no racemization from commer-
cially available Boc–Arg(Pmc)-OH (1) via the Weinreb amide 2
(Scheme 1).7,12 More functionalized benzyl groups would not be
compatible with Grignard reagents, and the use of milder reactions
would be required. Although development of such reactions has
been part of our research,13 we decided to limit the number of
compounds by only considering an unsubstituted benzyl ketone
serine trap.
The benzyl ketone serine trap, which is less electrophilic, has
only been reported once in connection with one particular throm-
bin inhibitor,7 and therefore represents an unexplored class of po-
tential FVIIa inhibitors. This group can be introduced directly7
without the need of additional reduction/oxidation protection
steps8 or anchoring to a solid support,9 which is required with
more electrophilic serine traps. The benzyl group also gives the
unique possibility to probe the S1’ binding pocket by introduction
of a variety of substituents—this is not possible or very limited
with most of the other serine traps (e.g., aldehydes or
heterocycles).
a-keto
Three different dipeptides (4–6) were synthesized from the
building block
3 using solution-phase EDC/HOAt-mediated
A peptide-based inhibitor was chosen because such compounds
are easily degraded in vivo into nontoxic amino acids and because
a peptide much better resembles the natural FVIIa substrate than a
small molecule. Selection of the peptide sequences was based on a
specificity profile (PS-SCL) for FVIIa,10 suggesting that the opti-
mal substrates were Tyr–Thr–Arg > Tyr–Leu–Arg > Phe–Leu–Arg >
Phe–Phe–Arg. All of them have arginine at the P1 position
because, that is, the only acceptable amino acid (besides of Lys)
synthesis followed by synthesis of seven tripeptides (7–13) by an
analogous procedure (Scheme 2 and Table 1). Two peptides (11
and 13) have a D-Phe in the P3 position and two peptides (12
and 13) have an N-terminal Cbz-group. The Cbz-group was chosen
as a directly incorporated N-terminal, which resembles an N-ben-
zylsulfonyl group (BnSO2À), that is, known from the literature for
capping amino acids,14 as well as used for N-terminals of serine
protease inhibitors.8
in that position. Introduction of
D
-amino acids in the P3 position
The N-acetylated peptides Ac–Tyr(Bn)–Thr(Bn)–Arg(Pmc)–bk
(14) and Ac–Phe–Phe–Arg(Pmc)–bk (15) were synthesized by
selective N-terminal deprotection (20% TFA, DCM) followed by
reaction with Ac2O. Reaction with BnSO2Cl afforded the N-benzyl-
sulfonylated peptides 16–20 representing all five sequences
(Scheme 2 and Table 1).
is known from the literature to be beneficial for the potency.8 How-
ever, the exact reason for this has not previously been investigated
and we therefore decided to explore it further by introducing
D
-amino acids in the P3 position in some of the projected peptides.
It is not certain whether there is sufficient space in the active
site of FVIIa to accommodate the benzyl ketone group. Therefore,
we inspected visually the energy minimized H–Tyr–Thr–Arg–bk
complexed with human FVIIa (PDB ID: 2PUQ). The study revealed
O-Debenzylation of Boc–Tyr(Bn)–Thr(Bn)–Arg(Pmc)–bk (7) and
Ac–Tyr(Bn)–Thr(Bn)–Arg(Pmc)–bk (14) were successfully achieved
with H2 (80 bar) in MeOH + 10% AcOH (rt, 24 h) affording Boc–Tyr–
Thr–Arg(Pmc)–bk (21) and Ac–Tyr–Thr–Arg(Pmc)–bk (22), respec-
tively, in conversions >95%. Subjecting the N-benzylsulfonylated
peptides BnSO2–Tyr(Bn)–Thr(Bn)–Arg(Pmc)–bk (16) and BnSO2–
Tyr(Bn)–Leu–Arg(Pmc)–bk (17) to the same reaction conditions
unfortunately did not afford any traces of the desired products,
BnSO2–Tyr–Thr–Arg–bk (23) and BnSO2–Tyr–Leu–Arg–bk (24),
respectively. The reaction time was increased to several days, but
only a mixture of the starting material and the tyrosine mono-O-
debenzylated compounds were observed. Apparently, the N-ben-
zylsulfonyl group causes catalyst poisoning. Further optimization
was not pursued, and the synthesis of 23 and 24 was shelved.
Final Boc/Pmc-deprotection (95% TFA, DCM) followed by pre-
parative HPLC purification and lyophilization afforded the final
peptidyl benzyl ketones (25–33) in modest yield but in high purity
(Table 2). Two of the final peptides, H–Tyr–Thr–Arg–bk (25) and
H–Phe–Phe–Arg–bk (28) were not modified at the N-terminal.
The synthesized peptides were subjected to three competitive
binding assays for screening of inhibitory activity against FVIIa,
thrombin (thr) and FXa, respectively (Table 3). The solubility of
the peptides in the assay buffer varied considerably. The tyrosine-
and threonine-containing peptides 25 and 26 were highly soluble
Figure 2. Energy minimization of H–Tyr–Thr–Arg–bk complexed with human FVIIa
(2PUQ) revealed sufficient space for the benzyl ketone group and an interesting
cation–
p-interaction with the unique Lys192.
H
N
H
N
H
N
HN
HN
HN
Pmc
Pmc
Pmc
MgCl
(6 equiv)
H
NH
NH
NH
MeO
H
N
Cl
Me
Me
N
PyBOP, Et3N
DCM, rt, 1h
THF
oC, 10 min,
Boc
OH
Boc
Boc
N
H
N
H
OMe
N
H
0
O
O
O
then rt, 2h
1
2
3
85−93% yield
70−75% yield
99% ee
Scheme 1. Synthesis of the arginyl benzyl ketone building block 3.

3920
M. Storgaard et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3918–3922
H
H
HN
N
HN
N
Pmc
Pmc
NH
NH
Peptide
coupling (i)
Peptide
coupling (ii)
O
H
N
Boc
N
H
Boc
N
H
O
P2
O
3
4 6
-
H
N
HN
NH2
NH
HN
Pmc
NH
P3
O
1. N-Terminal modification (iii)
2. O-Debenzylation (iv)
P3
O
H
H
P4
P4
N
N
N
H
N
H
N
H
N
H
3. Deprotection (v)
O
P2
O
O
P2
11
O
7
25 33
-
−
(P4 = Boc)
12 − 13 (P4 = Cbz)
Scheme 2. Synthesis of peptidyl benzyl ketones. Reagents and conditions: (i) 3 to 4–6: 20% TFA, DCM, rt, 30 min, then Boc-P2-OH, EDC-HCl, HOAt, DIPEA, DCM, rt, 1 h; (ii) 4–6
to 7–13: 20% TFA, DCM, 0 °C, 4 h, then Boc/Cbz-P3-OH, EDC–HCl, HOAt, DIPEA, DCM, 0 °C, 1 h; (iii) 7, 10 to 14, 15: 20% TFA, DCM, rt, 30 min, then Ac2o, DIPEA, DCM, rt, 30 min,
7–11 to 16–20: 20% TFA, DCM, rt, 30 min, then BnSO2Cl, DIPEA, DCM, rt; (iv) 7, 14 to 21, 22: Pd/C (10 wt %), MeOH, AcOH, (10%), H2, (80 bar), rt, 24 h; (v) 10, 12, 13, 15, 18–33:
95% TFA, DCM, rt, 30 min.
Table 1
Table 2
Synthesis of protected peptidyl benzyl ketones, P1 = Arg
Synthesis of final peptidyl benzyl ketones, P1 = Arg
Entry
Peptide
P4a
P3a
P2a
Yieldb (%)
Entry
Peptide
P4a
P3a
P2a
Yieldb (%)
Purityc (%)
1
2
3
4
5
6
7
8
4
5
6
7
8
9
10
11
—
—
—
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Tyr(Bn)
Tyr(Bn)
Phe
Thr(Bn)
Leu
Phe
Thr(Bn)
Leu
Leu
67
62
60
62
39
29
54
27
1
2
3
4
5
6
7
25
26
27
28
29
30
31
H
Ac
BnSO2
H
Ac
Tyr
Tyr
Phe
Phe
Phe
Phe
Thr
Thr
Leu
Phe
Phe
Phe
Phe
35d
14d
30
93
89
97
95
96
95
95
49
57
BnSO2
BnSO2
24d
29d
Phe
Phe
Phe
D-Phe
D-Phe
8
9
32
33
Cbz
Cbz
Phe
Phe
Phe
21
22
97
99
9
10
12
13
Cbz
Cbz
Phe
Phe
Phe
32
27
D-Phe
D-Phe
a
b
c
Notation corresponds to the respective amino acid side-chain
Yield determined by HPLC–CLND and based on two steps.
Determined by UPLC analysis (5 ? 95% MeCN in H2O, 0.05% TFA).
11
12
13
14
15
16
17
14
15
16
17
18
19
20
Ac
Ac
Tyr(Bn)
Phe
Tyr(Bn)
Tyr(Bn)
Phe
Thr(Bn)
Phe
Thr(Bn)
Leu
Leu
Phe
67
70
BnSO2
BnSO2
BnSO2
BnSO2
BnSO2
23
d
>95c
74
Yield based on three steps, because N-terminal modification and/or deprotec-
tion step was used directly without purification.
Phe
32
d
Phe
—
D-Phe
18
19
20
21
21
22
23
24
Boc
Ac
BnSO2
BnSO2
Tyr
Tyr
Tyr
Tyr
Thr
Thr
Thr
Leu
>95c
>95c
Table 3
d
Inhibition of blood coagulation serine proteases
—
—
d
a
Entry
Peptide
IC50
(
lM)
Kib
(lM)
a
b
c
Notation corresponds to the respective amino acid side-chain.
Purified yield.
Conversion according to LC–MS analysis.
No product was formed.
FVIIa
Thr
FXa
FVIIa
1
2
3
4
5
6
7
8
9
25
26
27
28
29
30
31
32
33
>1340
>1340
>580
>1480
>400
26
640
109
16
>1340
>1340
>580
>1480
>400
>590
>640
>650
560
>1340
1078
>580
>1480
>400
>590
>640
508
—
—
—
–
d
in the buffer, whereas the N-benzylsulfonylated peptides 27, 30
and 31 and in particular the Cbz-modified peptides 32 and 33 were
less soluble. However concentrations of at least 400 lM were ob-
tained for all compounds, which do not restrict them to be used
as formulation aids.
—
43 1.9
—
89 2.8
440
8
2.4
a
IC50-value determined by a competitive binding assay against FVIIa, thrombin
The most potent FVIIa inhibitor was found to be Cbz-
D-Phe–
(thr) or FXa, respectively.
Phe–Arg–bk (33) with an IC50 = 16 and Ki = 8 M (Table 3). The
inhibitor has a 35- and 28-fold selectivity over thrombin and
l
b
Determined by linear fitting of (v0/vn) – 1 versus [I], where Ki = 1/slope.
FXa, respectively. Two less potent inhibitors were found to be
BnSO2–Phe–Phe–Arg–bk (30) with Ki = 43
lM and Cbz–Phe–Phe–
Arg–bk (32) with Ki = 89 M. Inhibitor 30 possesses at least a
l
and the two most potent inhibitors; Cbz-
D-Phe–Phe–Arg–bk (33,
23-fold selectivity against both thrombin and FXa, whereas 32 is
almost equally potent against all three coagulation factors. The se-
quence was not reported as being the most active by the FVIIa PS-
SCL,10 but it is in accordance with results obtained by Parlow and
South.8
Ki = 8 M) and the weaker -diastereomer 32 (Ki = 89 l
l
L
M), which
could possibly explain the importance of the P3 stereo configura-
tion in this case. MD cannot describe the bond formation between
Ser195 and the inhibitor, but it is assumed that the interactions
with the rest of the binding pocket will be approximately the same
in the pre-reactive complex as in the covalent complex. The simu-
lations of 33 and 32 in the active site of FVIIa were started (t = 0 ns)
Molecular dynamics (MD) simulations using GROMACS ver.
3.3.3 were employed to analyze the interaction between FVIIa

M. Storgaard et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3918–3922
3921
Figure 3. Conformations of peptides in the FVIIa active site during MD simulations. (a) Cbz-D-Phe–Phe–Arg–bk (33) at t = 0; (b) 33 at t = 18 ns; (c) Cbz–Phe–Phe–Arg–bk (32)
at t = 0 ns. d) 32 at t = 18 ns. Intermolecular interactions: H-bond (red), salt-bridge (blue),
p-stacking (gray). Intramolecular interactions (orange).
from their minimized docking poses (Fig. 3a and c). In the initial
complexes both inhibitors form the well-known salt-bridge to
Asp189, and they also both form hydrogen bonds to backbone
tion, where the cost of desolvation is minimized, since there are
fewer interactions with water that need to be replaced upon
binding. It will also reduce the loss in degrees of freedom and, cor-
respondingly, the loss in entropy if these intramolecular interac-
tions are already present in the unbound state of the peptide.
In conclusion, a novel class of medium potent FVIIa peptidyl
Gly216 and Gly218. Inhibitor 33 has a
p-stacking interaction with
Trp215, whereas 32 is -stacking with His57 and has two addi-
p
tional hydrogen bonds to Lys192 and Ser190.
During the simulations (t = 18 ns) an additional hydrogen
bond was formed to the unique Lys192 along with some very
favorable intramolecular interactions (Fig. 3b and d). An intramo-
lecular hydrogen bond between the Cbz oxygen and the P2
inhibitors was developed with Cbz-
D-Phe–Phe–Arg–bk (33) being
themostactive (Ki = 8 M). Thispeptiderepresentsa goodcandidate
l
for the future development of stabilization agents for liquid formu-
lations of rFVIIa. The usage of an N-terminal Cbz-group as a struc-
tural equivalent to BnSO2À has not previously been reported, and
is synthetically superior due to its easy introduction. The influence
amide N-H and an intramoleular
p-stacking interaction between
the phenyl groups of P2 and P3 were found. These conforma-
tional changes result in bringing the benzyl ketone group in
closer proximity to Ser195 (from 10.05 in the beginning to
7.11 Å after 18 ns, c.f. Fig. 3a and b), thus enabling the reaction
between these two groups. In contrast, the diastereomeric pep-
tide 32 is unable to form the same intramolecular interactions
due to steric restrictions. During the MD simulation, 32 lost all
its interactions with FVIIa, except for the salt-bridge to Asp189,
and the benzyl ketone group moves further away from Ser195
of the
to contribute to a stable binding conformation of Cbz-
Arg–bk (33) due to formation of intramolecular hydrogen bonds.
However, a -aminoacid in theP3 position is not tantamount to a po-
D
-configuration at the P3 position was investigated and found
D-Phe–Phe–
D
tent inhibitor since the overall intramolecular interactions have to
be taken into account, in particular the N-terminal group. A mini-
mized cost of desolvation upon binding to FVIIa might also explain
the activity of the inhibitor compared to the polar sequences.
(from 6.22 to 8.90 Å, Fig. 3c and d). In this particular case, a D-
configuration in the P3 position was found to be important.
However, it does not represent a general trend since BnSO2–
Phe–Phe–Arg–bk (30) was found to be a medium potent FVIIa
Acknowledgments
M.S. thanks Novo Nordisk A/S, Corporate Research Affairs, the
Danish Ministry of Science, Technology and Innovation, Oticon
Fonden, Augustinus Fonden and Ingeniør Alexandre Haynman
and Hustru Nina Haynmans Fond for financial support.
inhibitor (Ki = 43 lM), whereas BnSO2-D-Phe–Phe–Arg–bk (31)
was inactive. Since the Cbz oxygen was found to participate in
the favorable intramolecular interactions in 33, the N-terminal
group must have a significant influence on which P3 stereoiso-
mer is the most favorable.
Supplementary data
The fact that all active inhibitors have a large, hydrophobic N-
terminal, and furthermore are composed of nonpolar amino acids,
suggests that desolvation is also an important factor for binding.
The polar peptides, for example, H–Tyr–Thr–Arg–bk (25), will nat-
urally have to pay a higher cost of desolvation upon binding to
FVIIa, which might explain why 25 is an inactive compound.
Supplementary data (experimental procedures, characteriza-
tion of compounds 2–21 and 25–33 data from the competitive
binding assays and the computational studies) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.05.025.
Cbz-D-Phe–Phe–Arg–bk (33) has a very stable binding conforma-

3922
M. Storgaard et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3918–3922
5. (a) Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157; (b)
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