Low molecular weight activated protein C inhibitors as a potential treatment for hemophilic disorders

Article abstract of DOI:10.1021/jm0606950

The synthesis and evaluation of inhibitors of activated protein C (aPC) are reported. This serine protease is partly responsible for the degradation of factor Villa, involved in the regulation of bleeding in hemophilia A. Benzamidine-containing derivatives were found to be potent aPC inhibitors, some of them showing selectivity against the procoagulant protease thrombin. Moreover, compound 1 significantly restored the generation of thrombin in hemophiliac plasma.

Full text of DOI:10.1021/jm0606950

J. Med. Chem. 2006, 49, 5047-5050
5047
Letters
circulating FVIII levels of 2-5%. This would also decrease the
amount of FVIII necessary to regulate the bleeding tendencies
and, as a consequence, the development of resistance mechanism
to FVIII.
Low Molecular Weight Activated Protein C
Inhibitors as a Potential Treatment for
Hemophilic Disorders
Activated protein C (aPC) is a trypsin-like serine protease
synthesized from protein C through activation by thrombo-
modulin-bound thrombin. aPC enzymatic activity requires the
presence of a cell surface, calcium ions, and a cofactor protein
S (PS), which potentiates the cleavage by aPC of its two main
substrates, FVa and FVIIIa.3
Guillaume De Nanteuil,*,§ Philippe Gloanec,§
Suzette Be´guin,# Peter L. A. Giesen,# H. Coenraad Hemker,#
Philippe Mennecier,† Alain Rupin,† and Tony J. Verbeuren†
DiVision D of Medicinal Chemistry and DiVision of Angiology,
Institut de Recherches SerVier, 11 Rue des Moulineaux,
92150 Suresnes, France, and Synapse b.V., CardioVascular
Research Institute Maastricht, UniVersity of Maastricht,
P.O. Box 616, 6200 MD Maastricht, The Netherlands
The concept of using aPC inhibitors as procoagulant agents
has been mentioned by Butenas in 199. In an experiment
mimicking hemophilia A, the authors showed that thrombin
generation was barely detectable, while in the presence of the
pseudopeptidic inhibitors Arg-ANSN-Lys(Z)-Lys(Z)-OBz (ANSN
) 6-aminonaphthalene-2-sulfonyl) and Lys-Lys-Thr-Arg-ANSN-
Lys(Z)-Lys(Z)-OBz, the absence of FVIII was fully compen-
sated. aPC inhibition was said to be responsible for an increased
generation of thrombin.4 Thus, potent inhibitors of aPC devoid
of inhibitory properties against procoagulant proteases such as
thrombin (Thr) represent structural targets able to improve
thrombin generation. Herein, we report for the first time the
discovery of a new class of benzamidine-containing aPC
inhibitors and show that some of these compounds are able to
increase the physiological levels of thrombin in human hemo-
philiac plasma.
ReceiVed June 9, 2006
Abstract: The synthesis and evaluation of inhibitors of activated
protein C (aPC) are reported. This serine protease is partly responsible
for the degradation of factor VIIIa, involved in the regulation of bleeding
in hemophilia A. Benzamidine-containing derivatives were found to
be potent aPC inhibitors, some of them showing selectivity against the
procoagulant protease thrombin. Moreover, compound 1 significantly
restored the generation of thrombin in hemophiliac plasma.
Hemophilia A is a severe inherited disorder characterized by
an increased bleeding tendency. The incidence of the disease
in humans is estimated at 1 in 5000. The major symptom of
hemophilia is hemarthrosis located at the major articulations,
most of the time degenerating to chronic arthropathy, which is
characterized by severe degeneration of the cartilagineous tissues
and subchondral bone. Other types of bleeding can also occur
in the muscles and the kidney and intracerebrally, this latter
situation being an important cause of mortality.1
In the course of our quest for novel anticoagulants, all the
compounds prepared were counterscreened against aPC.5 Com-
pound 1 was found to be a potent inhibitor of aPC, with an
IC50 of 823 nM. In addition, 1 showed promising selectivity
against thrombin with an IC50 around 47 µM.
Bleeding tendency in hemophilia A is correlated with a defect
in the levels or in the functionality of factor (F) VIII. Patients
with less than 1% of the normal level are considered severe
hemophiliacs. Those with 1-5% are considered moderate, and
those with 5-25% are considered mild.1 FVIII is cleaved by
thrombin to activated FVIII (FVIIIa). FVIIIa plays a major role
in the intrinsic pathway of the coagulation cascade where it is
responsible through formation of the tenase complex FVIIIa-
FIXa for the amplification of the initiation signal ascribed to
the extrinsic, tissue-factor-dependent pathway, ultimately leading
to the generation of procoagulant thrombin.
Current treatments for the hemophiliac patient are mainly
composed of FVIII concentrates; dramatic progress in the
purification and virus inactivation procedures has allowed the
preparation of human-derived FVIII and recombinant FVIII.
Although these two sources of FVIII have similar pharmaco-
kinetic and clinical properties, the major drawback in the
utilization of FVIII concentrates is the development of inhibitory
antibodies in up to 25% of hemophilia A patients, who develop
bleeding tendencies unresponsive to FVIII infusions.2
The synthesis of 1 is shown in Scheme 1 and is representative
of the preparation of all the compounds described here. It was
accomplished in six steps starting from the commercially
available amino ester 2, which was coupled with Boc-(R)-
cyclohexylglycine to give the dipeptidic intermediate 3 in 97%
yield. Saponification of the C-terminal ester with sodium
hydroxide in dioxane (95%) was followed by coupling with
protected aminomethylbenzamidine6 to give 4 in 84% yield.
Deprotection of the N-terminal amino group under acidic
conditions was realized in 95% yield and was followed by
alkylation with benzyl bromoacetate in a nonoptimized yield
of 54% to afford 5. Finally, a double deprotection was performed
by hydrogenolysis to give the desired compound in 66% yield.
To improve the activity and selectivity profiles of 1, structural
modifications at the different subsites were considered. We
started by replacing (R)-cyclohexylglycine at the P3 subsite by
other simple amino acids (named as A); the corresponding IC50
values for aPC and thrombin are given in Table 1.
It has been shown that the severity of the disease could be
dramatically decreased, even with a modest elevation of
* To whom correspondence should be addressed. Phone: +33 1 55 72
22 34. Fax: +33 1 55 72 24 30. E-mail: guillaume.denanteuil@fr.netgrs.com.
§ Division D of Medicinal Chemistry, Institut de Recherches Servier.
# University of Maastricht,.
The (R)-configuration for this amino acid appeared to be
critical for optimum potency because the (S)-cyclohexylglycine
† Division of Angiology, Institut de Recherches Servier.
10.1021/jm0606950 CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/25/2006

5048 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Scheme 1. Synthetic Route to Compound 1a
Letters
a Reaction conditions: (a) HOBT, TBTU, DIEA, DMF, 97%; (b) NaOH, dioxane, 95%; (c) protected aminomethylbenzamidine, DIEA-EDCI, DMF,
84%; (d) HCl, dioxane-CH2Cl2, 95%; (e) aqueous saturated NaHCO3-EtOAc, then BrCH2COOCH2Ph, K2CO3, CH3CN, 54%; (f) H2, Pd-C, CH3OH,
86%.
Table 1. aPC and Thrombin Inhibition Values for Compounds with
Modified Amino Acids at the P3 Subsite
Table 2. aPC and Thrombin Inhibition Values for Compounds with
Modified Amino Acids at the P2 Subsitea
containing analogue 6 was totally devoid of activity. The glycine
derivative 7 was also completely inactive. Conversion of
cyclohexylglycine to alanine or ethylglycine (8 or 9) led to a
loss of potency with IC50 greater than 1 µM. In contrast,
medium-sized amino acids such as valine (10) or leucine (12)
restored the inhibitory activity with IC50 against aPC of 0.80 or
0.75 µM, respectively. Interestingly, 10 was found to be at least
1 order of magnitude more selective than 1 or 12 with an IC50
against thrombin greater than 300 µM.
Further increases in the size of the amino acid side chain
with a tert-butylglycine (11) or an isoleucine (13) were again
deleterious for activity.
Replacement of cyclohexylglycine with cyclohexylalanine
gave 14, the most potent aPC inhibitor of this first series of
compounds with an IC50 of 0.10 µM; unfortunately, inhibition
of thrombin with 14 was also enhanced with an IC50 value in
the micromolar range. In contrast, the aromatic analogue 15
a The asterisk (/) represents monosubstitution on the R-carbon.
was much less active. Finally, introduction of 1-aminoisobutyric
acid in 16 or 1-aminocyclopentane carboxylic acid in 17 gave
inactive derivatives.
Taking these first results into account, we maintained (R)-
valine as the N-terminal amino acid to study the influence of
the central amino acid located at the P2 subsite (named as B in
Table 2). 2-Aminoindane-2-carboxylic acid used in 10 was
replaced by several structural analogues; dimethoxy substitution
on the phenyl ring of the indane nucleus (18) led to an important
drop in activity. Introduction of a nitrogen atom at position 4

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5049
Figure 1. Thrombin generation with increasing concentrations of 1
((-) 0 nM, (- - -) 10 nM, (‚‚‚) 20 nM) in PPP from a normal volunteer.
Figure 2. Thrombin generation with increasing concentrations of 1
((-) 0 nM, (- - -) 10 nM, (‚‚‚) 20 nM) in PPP from hemophiliac patients.
in the indane ring gave two diastereomers 19 and 20 that could
be separated. This was not the case with 21 with the nitrogen
atom at position 5 in the indane ring. These three compounds
gave micromolar values for aPC inhibition. Introduction of a
second aromatic ring in a cyclopenta[a]- or cyclopenta[b]-
naphthalene system gave 22 or 23, respectively. The former
was found to be a potent aPC inhibitor with an IC50 of 0.25
µM but was not selective vs thrombin, while the latter was much
less active with an IC50 around 10 µM.
Increasing the size of the central amino acid by using
2-amino-2,3-dihydro-1H-phenalene-2-carboxylic acid gave in-
hibitor 24, almost twice as potent as 10 and with a similar
impressive selectivity profile. The 6-amino-6,7-dihydro-5H-
dibenzo[a,c]cycloheptene-6-carboxylic acid containing 25 was
much less active with an IC50 greater than 10 µM. Potency was
also improved by extruding the indane ring from the amino acid
skeleton to give 26. This compound was also very selective
with an IC50 against thrombin greater than 33 µM. Using (S)-
phenylalanine in 27 proved to be an interesting variation with
an inhibition value for aPC similar to that of 1, albeit with a
decrease in selectivity. Introducing a second phenyl ring gave
diphenylalanine containing 28, which was completely devoid
of inhibitory activity.
As a summary of these preliminary investigations, it is worth
noting that the best compromise between inhibitory potency for
aPC and selectivity against thrombin was obtained by using a
medium-sized amino acid at P3 (e.g., valine). As far as P2 was
concerned, the simple indane ring remained the favored moiety
mostly because of its easy synthetic access either as a spiro
system such as in 10 or as a substituent on the R-carbon in 26.
Because of its improved selectivity vs thrombin, 24 containing
a central tricyclic system was also selected as an interesting
candidate for further studies.
(20 nM), it is suggested that the inhibitory efficacy of 1 on
aPC is higher in the presence of its physiological substrate than
with its synthetic chromogenic substrate.
In summary, we have reported here the preparation and
biochemical evaluation of potent low molecular weight inhibitors
of activated protein C, some of them showing a promising
selectivity against the procoagulant protease thrombin. In a
thrombin generation assay, inhibitor 1 was shown to be inactive
in normal human plasma while it significantly restored thrombin
generation in hemophilia A plasma. Since compounds described
here contain a hydrophilic benzamidine moiety, prodrugs similar
to those obtained from melagatran9 or dabigatran10 in the field
of thrombin inhibition will have to be prepared to obtain orally
available inhibitors. On the basis of these preliminary results,
it appears here for the first time that a low molecular weight
selective inhibitor of aPC could become a potential oral
treatment of great value for increasing FVIII levels in hemo-
philiacs, thus improving the quality of life of people enduring
this devastating genetic disease.
Acknowledgment. The authors acknowledge the skillful
technical assistance in medicinal chemistry of Camille Poindron,
Sophie Cornet, and Christian Me´riaux. They also thank Dr. Jean-
Paul Bouchet and his group for analytical and spectral deter-
minations.
Supporting Information Available: Synthetic procedures;
spectral and elemental analysis data for the tested compounds; and
experimental protocols for aPC, thrombin inhibition, and thrombin
generation. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
As a preliminary indication that an aPC inhibitor might be
of interest as a potential treatment for hemophilia disorders, 1
was studied on thrombin generation using the calibrated
automated thrombogram assay.7 In normal pooled human
platelet poor plasma (PPP),8 thrombin generation induced by
intrinsic coagulation pathway was observed with peak levels
around 400 nM. Under these conditions, inhibitor 1 barely
increased thrombin generation when incubated at increasing
concentrations of 10 and 20 nM (Figure 1). In contrast, in a
human moderate hemophilia A PPP (FVIII < 5%), where
thrombin generation was almost absent (Figure 2), inhibitor 1
used at 10 and 20 nM significantly restored the thrombin
generation to a maximum level of 160 nM.
Moreover, in human severe hemophilia A PPP spiked with
5% normal human PPP where the thrombin generation peak
was at 55 nM, inhibitor 1 (20 nM) increased thrombin generation
to a maximum peak level of 201 nM. Notably, since IC50 of 1
in the screening assay (0.82 µM) is far above the concentration
that gives a maximal effect in the thrombin generation assay
(1) Klinge, J.; Ananyeva, N. M.; Hauser, C. A. E.; Saenko, E. L.
Hemophilia A. From Basic Science to Clinical Practice. Semin.
Thromb. Hemostasis 2002, 28, 309-321.
(2) Bishop, P.; Lawson, J. Recombinant Biologics for Treatment of
Bleeding Disorders. Nat. ReV. Drug DiscoVery 2004, 3, 684-694.
(3) Kraus, M. The Anticoagulant Potential of the Protein C System in
Hereditary and Acquired Thrombophilia: Pathomechanisms and New
Tools for Assessing Its Clinical Relevance. Semin. Thromb. Hemo-
stasis 1998, 24, 337-354.
(4) Butenas, S.; Mann, K. Peptidomimetics Containing 6-Peptidylamino-
1-Naphthalenesulfonamide Moieties. World Patent WO 98/22125,
1998.
(5) Activity of the compounds was evaluated by determining the
concentrations that inhibit by 50% the proteasic action of aPC against
the chromogenic substrate Pyroglu-Pro-Arg-pNA (S-2366, Chro-
mogenix) (IC50 expressed in µM). Selectivity was evaluated by
comparing this IC50 value with that obtained against thrombin in the
presence of its natural substrate, fibrinogen (Enzyme Research
Laboratories).
(6) Lila, C.; Gloanec, P.; Cadet, L.; Herve´, Y.; Fournier, J.; Leborgne,
F.; Verbeuren, T. J.; De Nanteuil, G. Large Scale Preparation of
Protected 4-Aminomethylbenzamidine. Application to the Synthesis
of the Thrombin Inhibitor Melagatran. Synth. Commun. 1998, 28,
4419-4429.

5050 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Letters
(7) Hemker, H. C.; Giesen, P.; Al Dieri, R.; Regnault, V.; de Smedt, E.;
Wagenvoord, R.; Lecompte, T.; Be´guin, S. The Calibrated Automated
Thrombogram (CAT): A Universal Routine Test for Hyper and
Hypocoagulability. Pathophysiol. Haemostasis Thromb. 2002, 32,
249-253.
(8) Hemker, H. C.; Giesen, P.; Al Dieri, R.; Regnault, V.; de Smedt, E.;
Wagenvoord, R.; Lecompte, T.; Be´guin, S. Calibrated Automated
Thrombin Generation Measurement in Clotting Plasma. Pathophysiol.
Haemostasis Thromb. 2003, 33, 4-15.
(9) Gustafsson, D.; Nystro¨m, J.-E.; Carlsson, S.; Bredberg; U.; Eriksson,
U.; Gyzander, E.; Elg, M.; Antonsson, T.; Hoffmann, K.-J.; Ungell,
A.-L.; So¨rensen, H.; Någård, S.; Abrahamsson, A.; Bylund, R. The
Direct Thrombin Inhibitor Melagatran and Its Oral Prodrug H 376/
95: Intestinal Absorption Properties, Biochemical and Pharmaco-
dynamic Effects. Thromb. Res. 2001, 101, 171-181.
(10) Hauel, N. H.; Nar, H.; Priepke, H.; Ries, U.; Stassen, J.-M.; Wienen,
W. Structure-Based Design of Novel Potent Nonpeptide Thrombin
Inhibitors. J. Med. Chem. 2002, 45, 1757-1766.
JM0606950