Guanylpiperidine peptidomimetics: Potent and selective bis-cation inhibitors of factor Xa

Article abstract of DOI:10.1016/S0960-894X(00)00105-0

A novel series of rigid P₃-guanylpiperidine peptide mimics 3-14 was designed as potential factor Xa and prothrombinase inhibitors. Incorporation into a P₂-gly-P₁-argininal motif led to highly potent and selective inhibitors. The synthesis and biological activities of these derivatives are reported herein. (C) 2000 Published by Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00105-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 745±749
Guanylpiperidine Peptidomimetics: Potent and Selective
bis-Cation Inhibitors of Factor Xa
Susan Y. Tamura, Odile E. Levy, Theresa H. Uong, John E. Reiner,
Erick A. Goldman,y Jonathan Z. Ho, Cheryl R. Cohen,{ Peter W. Bergum,
Ruth F. Nutt,x Terence K. Brunckx and J. Edward Semple*
Corvas International, Inc., Department of Medicinal Chemistry, 3030 Science Park Road, San Diego, CA 92121, USA
Received 16 December 1999; accepted 2 February 2000
AbstractÐA novel series of rigid P3-guanylpiperidine peptide mimics 3±14 was designed as potential factor Xa and prothrombinase
inhibitors. Incorporation into a P2-gly-P1-argininal motif led to highly potent and selective inhibitors. The synthesis and biological
activities of these derivatives are reported herein. # 2000 Published by Elsevier Science Ltd. All rights reserved.
As a key member of the trypsin class of serine proteases
involved in blood coagulation, factor Xa catalyzes the
penultimate step in this biochemical cascade, the for-
mation of the serine protease thrombin. Factor Xa
mediated thrombin formation occurs following the
assembly of the prothrombinase complex, which is
composed of factor Xa and the non-enzymatic cofactor
factor Va assembled on an appropriate phospholipid
surface such as activated platelets.1,2 A variety of small
molecule synthetic sca€olds that selectively inhibit fac-
tor Xa as well as prothombinase activity are currently
receiving considerable attention and are emerging as
potential therapeutic agents for the prevention and
treatment of thrombotic vascular disease.3
The application of peptidomimetic inhibitors that con-
tain rigid heterocyclic motifs is currently an area of
active investigation. Conformationally constrained pep-
tidomimetics can contribute a favorable entropic com-
ponent to binding in the enzyme active site relative to
their highly ¯exible peptide counterparts. Recent studies
in our laboratories demonstrated that incorporation of
a rigidi®ed P1-3-guanylpiperidinylalaninal arginine sur-
rogate into the appropriate peptidyl or peptidomimetic
template led to a potent and highly selective series of
novel thrombin inhibitors.7 The S3 pocket in factor Xa
features the cylinder-like orientation of the aromatic
residues Trp215, Tyr99, and Phe174, constituting a site
that can accommodate critical cation-p interactions.4 In
this investigation, rigidi®ed arginine surrogates of the P3
d-arginine residue were utilized in the design of inhibi-
tors of factor Xa to enhance this cation-p site interac-
tion (Fig. 1). The synthesis and biological activity of this
family of P3 d-arginine mimics incorporating 3-gua-
nylpiperidinylalanine, 4-guanylpiperidinylalanine and 4-
guanylpiperidinylglycine moieties is described in this
letter.
The incorporation of bis-cation binding elements in the
design of new targets has recently emerged as an active
area of investigation,3 and is based upon the topo-
graphy of the factor Xa active site.4 Compounds 1 and
2, discovered during the initial stages of work in this
area,5,6 are potent inhibitors of factor Xa (IC50's of 35
and 0.9 nM, respectively); however, they su€er from
mediocre to poor selectivity pro®les, especially against
the key digestive enzyme trypsin.
Chemistry
Synthetic route to inhibitors 3a and 3b8
*Corresponding author. Tel.: +1-858-455-9800, ext. 1140; fax: +1-
858-455-5169; e-mail: ed_semple@corvas.com
yPresent address: COR Therapeutics Inc., 256 E. Grand Avenue, San
Francisco, CA 94080, USA.
N-a-Boc-d-3-pyridylalanine (Synthetech, Albany, OR)
was converted to the di-protected 3-guanylpiperidine 15
in two steps by hydrogenation followed by guanylation.
The rigid Arg mimic 15 was coupled to glycine ethyl
ester and deprotected with HCl to produce the dipeptide
{Present address: Allergan Pharmaceuticals, 2525 Dupont Drive,
Irvine, CA 92612, USA.
xPresent address: Bioreason, Inc., 150 Washington Avenue, Suite 303,
Santa Fe, NM 87501, USA.
0960-894X/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00105-0

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S. Y. Tamura et al. / Bioorg. Med. Chem. Lett. 10 (2000) 745±749
Figure 1. Strategy for the design of guanylpiperidine inhibitors 3±14.
16. Dipeptide 16 was capped with benzylsulfonyl chlo-
ride and then hydrolyzed to a€ord the free acid 17.
Intermediate 17 was coupled to nitroArg ethyl aminal9
using standard peptide coupling techniques to produce
the fully elaborated compound 18. Conversion of com-
pound 18 to targets 3a/3b was easily accomplished by
hydrogenolysis of the Cbz and nitro protecting groups
followed by mild acidic hydrolysis of the aminal. The
diastereomeric pair 3a and 3b was separated during RP
HPLC puri®cation (Scheme 1).
Synthetic route to inhibitors 6±13
4-Guanylpiperidine targets 6±13 were prepared via pro-
tected dipeptide 22 in a similar manner to 3-guanyl-
piperidine targets 3±5; however, the reaction sequence
was changed to optimize yields (Scheme 3). In this case,
compound 21 was prepared by coupling Boc-4-pyr-
idylalanine to glycine methyl ester, and subsequent
reduction to the piperidine and functionalization to the
bis-Cbz-guanylpiperidine. To remove the Boc protec-
tion, compound 21 was then treated with HCl to deliver
compound 22, which was used to prepare targets 6±13
in a route analogous to the preparation of targets 3±5.
Synthetic route to inhibitors 4 and 5
Intermediate 19 (Scheme 2) was prepared from com-
pound 16 in four steps substituting 2-carbomethoxy-
benzylsulfonyl chloride for benzylsulfonyl chloride
following the strategy used in Scheme 1 for inhibitors 3a
and 3b. The methyl ester of compound 19 was subjected
to lithium hydroxide hydrolysis to a€ord the free acid
20. Intermediates 19 and 20 were each hydrolyzed to
a€ord targets 4a/4b and 5a/5b, which were separated
during the ®nal RP HPLC puri®cation.
Synthetic route to inhibitor 14
The preparation of the guanylpiperidylglycine inter-
mediate 26 was accomplished in eight steps. The key
step in the synthesis featured a modi®ed Ugi reaction10
between 4-pyridinecarboxaldehyde, formic acid, ethyl
isocyanoacetate, and tetrapivaloyl-d-galactopyranosyl-
amine following the Kunz protocol11 and produced 23
Scheme 1. Reagents and conditions: (a) H2, PtO2, EtOH, AcOH, H2O, quant; (b) MeSC(NCbz)(NHCbz), DIEA, THF, Á, 41%; (c) H-Gly-OEt,
HCl salt, EDC, HOBt, DIEA, CH3CN, 53%; (d) HCl, EtOH; (e) BnSO2Cl, NMM, CH3CN, 16±26% for two steps; (f) 1.0 N LiOH, EtOH; (g)
EDC, HOBt, nitroargininal ethyl aminal.HCl, NMM, CH3CN, 43±88% for two steps; (h) H2, 10% Pd/C, EtOH, H2O, HOAc; (i) 6.0 N HCl;
preparative RP HPLC, 14% for 3a, 24% for 3b.

S. Y. Tamura et al. / Bioorg. Med. Chem. Lett. 10 (2000) 745±749
747
Scheme 2. Reagents and conditions: (a) 1.0 N LiOH, EtOH; (b) 6 N HCl; preparative RP HPLC puri®cation, 35±55%.
Scheme 3. Reagents and conditions: (a) H-Gly-OMe, HCl salt, EDC, HOBt, DIEA, CH3CN, 97%; (b) H2, PtO2, EtOH, AcOH, H2O, quant; (c)
MeSC(NCbz)(NHCbz), DIEA, THF, Á, 55%; (d) HCl, EtOAc, quant.
in excellent yield (Scheme 4). HPLC analysis of crude 23
indicated a 4:1 diastereomeric ratio at the newly created
pyridylglycine a-methine center, with the desired a-(R)-
stereochemistry predominating. Silica gel ¯ash chroma-
togaphic puri®cation readily e€ected diastereomer
separation and provided pure a-(R)-23. Simultaneous
hydrolysis of the N-formyl, ester and sugar moieties,
followed by re-esteri®cation and a-N-protection with
Boc2O a€orded compound 24. Platinum-catalyzed
hydrogenation of the pyridyl moiety proceeded smoothly
to provide the expected piperidine intermediate, which
in turn was converted to the bis-Cbz-guanylpiperidine
25. Acid-catalyzed cleavage of the a-N-Boc protecting
group delivered advanced intermediate 26, which was
elaborated to the ®nal target 14 via a protocol analogous
to that described for the preparation of targets 3a and
3b above.
Results and Discussion
Fifteen bis-cation argininals 3±14 were prepared and
evaluated for their ability to inhibit four relevant serine
proteases (Table 1). In accordance with our design
strategies, highly potent factor Xa inhibitors were dis-
covered that were considerably more trypsin-selective
than their corresponding non-rigid counterparts, vide
supra. They also displayed favorable selectivity pro®les
against the ®brinolytic protease plasmin. Within the 3-
guanylpiperidine class, the diastereomeric pairs 3, 4 and
Scheme 4. Reagents and conditions: (a) ZnCl2, Et2O, THF, 30ꢀ to 0 ꢀC, 96%, 81% a-(R)-isomer; (b) EtOH, HCl; H2O; (c) EtOH, HCl; 80% for
two steps; (d) Boc2O, Et3N, DMAP, dioxane, 23%; (e) H2, PtO2, EtOH, H2O, HOAc; (f) MeSC(NCbz)(NHCbz), THF, 41% for two steps; (g)
EtOH, HCl, quant.

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S. Y. Tamura et al. / Bioorg. Med. Chem. Lett. 10 (2000) 745±749
Table 1. In vitro IC50 values (nM) of inhibitors 3±14 against human factor Xa, thrombin, plasmin and trypsina
Compound
R
X
n
R0
Factor Xa
Thrombin
Plasmin
Trypsin
3a
3b
4a
4b
5a
5b
6
7
8
9
10
11
12
13
14
Bn
Bn
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
O(CO)
O(CO)
SO2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
3-Guanylpiperidine
3-Guanylpiperidine
3-Guanylpiperidine
3-Guanylpiperidine
3-Guanylpiperidine
3-Guanylpiperidine
4-Guanylpiperidine
4-Guanylpiperidine
4-Guanylpiperidine
4-Guanylpiperidine
4-Guanylpiperidine
4-Guanylpiperidine
4-Guanylpiperidine
4-Guanylpiperidine
4-Guanylpiperidine
1.2
0.92
1.08
1.37
2.1
>2500
>2500
>2500
1100
>2500
352
>2500
>2500
>2500
>2500
>2500
>2500
>2500
>2500
>2500
919
726
894
120
31
124
15
240
33
169
147
145
447
211
537
158
412
25
(20-CO2Me)Bn
(20-CO2Me)Bn
(20-CO2H)Bn
(20-CO2H)Bn
Bn
1950
2500
>2500
869
965
387
2500
231
4.2
0.83
0.94
2.2
5.7
4.3
(20-CO2Me)Bn
(30-CO2Me)Bn
(20-CO2H)Bn
(30-CO2H)Bn
(20-Tetrazole)Bn
(40-CO2Et)phenethyl
Me
2.6
872
16b
23b
1.1
1440
>2500
678
Bn
Reference standards
1
2
Bn
Bn
O(CO)
SO2
d-Arg
d-Arg
34.9
0.9
>2500
>2500
>2500
1190
115
75
aConcentration of compounds 1±14 necessary to inhibit human enzymes (factor Xa, factor IIa, plasmin and trypsin) by the cleavage of the chro-
mogenic substrates described in ref 13 by 50%.
bEstimated Ki for classical fast inhibition.14
5 demonstrated similar activities against factor Xa, but
displayed di€erent selectivity pro®les. Targets 3a, 4a
and 5a appear to have better trypsin selectivity. The in
vitro results for the 3-guanylpiperidinylalanine, 4-gua-
nylpiperidinylalanine and 4-guanylpiperidinylglycine
sca€olds indicate that high potency is retained in all
three spatial permutations, suggesting that they indeed
serve as ecient arginine surrogates. Furthermore, the
mode of presentation of the P3-cation to the S4 binding
pocket appears to have little e€ect on the relative in
vitro activity within this group of analogues. In general,
P4-sulfonamide-containing targets display an increased
potency against factor Xa when compared to the P4-
carbamate containing targets.
interact covalently with the catalytic triad when com-
plexed to free factor Xa, but does when complexed to
the prothrombinase active site.
In vivo pharmacokinetic studies on bis-cations indicated
substantial blood pressure/MAP e€ects during iv dosing
that are attenuated by the presence of P4-ester, P4-acid
or P4-tetrazole functions. The iv dosing (5 mg/kg) of
compound 6 produced a profound and prolonged MAP
e€ect. This e€ect was attenuated in the dosing of 7, and
non-existent in the dosing of 9.
Table 2. In vitro IC50 values (nM) of inhibitors against factor Xa and
the prothrombinase complex (Ptase)
The assembly of the prothrombinase complex results in
speci®c structural and kinetic changes in factor Xa,
resulting in a 100 000-fold improvement in the catalytic
eciency of prothrombin activation by factor Xa.12 The
potency of our inhibitors was measured against the
natural substrate prothrombin by the prothrombinase
complex (Table 2). In most of the analogues examined,
inhibitor potency against the prothrombinase complex
was measured by using IC50 values and was 2- to 4-fold
higher than the corresponding results using the amido-
lytic substrate with the uncomplexed factor Xa.
Signi®cantly, compounds 12 and 13, both containing
N-terminal carbamate caps, display di€erent kinetic
pro®les for factor Xa and prothrombinase. These com-
pounds display classical kinetics when inhibiting factor
Xa, and slow-tight binding kinetics when inhibiting
prothrombinase.15 It may be inferred that for com-
pounds 12 and 13 the aldehyde carbonyl does not
Compound
Factor Xaa
Ptaseb
Ratio Ptase/FXa
3a
3b
6
9
10
11
1.2
0.92
0.83
5.3
4.2
3.3
3.1
12
14
69
2.0
3.6
3.7
2.3
0.9
3.0
16c
23c
Reference standards
1a
1b
35
0.9
7.9
3.4
0.23
3.8
aConcentration of compounds necessary to inhibit the cleavage of the
chromogenic substrate by human enzyme factor Xa described in ref 13
by 50%.
bConcentration of compounds necessary to inhibit the activation of
human prothrombin in a preformed complex of factor Xa, factor Va
and phospholipid vesicles as described in ref 16 by 50%.
cEstimated Ki for classical fast inhibition.14

S. Y. Tamura et al. / Bioorg. Med. Chem. Lett. 10 (2000) 745±749
749
Conclusion
4. (a) Padmanabhan, K.; Padmanabhan, K. P.; Tulinsky, A.;
Park, C. H.; Bode, W.; Huber, R.; Blankenship, D. T.; Cardin,
A. D.; Kisiel, W. J. Mol. Biol. 1993, 232, 947. (b) Brandstetter,
H.; Kuhne, A.; Bode, W.; Huber, R.; von der Saal, W.;
Wirthensohn, K.; Engh, R. A. J. Biol. Chem. 1996, 271, 29988.
5. Abelman, M. M.; Miller, T. A.; Nutt, R. F. PCT Patent
Application WO 96/19493, Chem Abstr. 1996, 125, 196382.
6. Marlowe, C. K.; Scarborough, R. M.; Laibelman, A. M.;
Sinha, U.; Zhu, B.-Y. US Patent 5721214, 1998; Chem Abstr.
1998, 128, 205143.
7. Levy, O. E.; Reiner, J. R.; Semple, J. E.; Nutt, R. F.; Ripka,
W. C. J. Med. Chem. 1996, 39, 4527. (b) Krishnan, R.; Zhang,
E.; Hakansson, K.; Arni, R. V.; Tulinsky, A.; Lim-Wilby M.
S. L.; Levy, O. E.; Semple, J. E.; Brunck, T. K. Biochemistry
1998, 37, 12094.
A series of guanylpiperidine amino acids was designed
and appended onto a P1-argininal moiety resulting in
the discovery of a novel class of highly potent and
selective factor Xa inhibitors. From this work, com-
pounds 6, 7, 9 and 11 emerged as leading candidates
that combined both high in vitro potency versus factor
Xa with desirable selectivity pro®les towards other
trypsin-like serine proteases. These sca€olds may ®nd
utility in the design and synthesis of new classes of drug
targets, especially protease inhibitors which incorporate
arginine-like residues, and may lead to inhibitors with
improved levels of activity and/or selectivity.
8. All new compounds were characterized by full spectroscopic
(NMR, low resolution MS) data. Yields refer to spectro-
scopically and chromatographically homogeneous (=95% by
1H NMR, HPLC, TLC) materials.
Acknowledgements
9. Tamura, S. Y.; Semple, J. E.; Ardecky, R. A.; Leon, P.;
Carpenter, S.; Ge, Y.; Shamblin, B. M.; Weinhouse, M. I.;
Ripka, W. C.; Nutt, R. F. Tetrahedron Lett. 1996, 37, 4091.
10. (a) Kunz, H.; Pfrengle, W.; Sager, W. Tetrahedron Lett.
1989, 30, 4109. (b) Kunz, H.; Pfrengel, W.; Ruck, K.; Sager,
W. Synthesis 1991, 1039.
The authors wish to thank S. Anderson, L. Truong, and
E. Ganio for technical support in performing the in
vitro assays.
11. Kunz, H.; Sager, W. Angew. Chem., Int. Ed. Engl. 1987, 26,
557.
12. Mann, K. G.; Jenny, R. J.; Krishnaswamy, S. Annu. Rev.
Biochem. 1988, 57, 915.
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