Novel 3-carboxamide-coumarins as potent and selective FXIIa inhibitors

Article abstract of DOI:10.1021/jm8002697

Recently, FXIIa was highlighted as an original attractive target for the development of new anticoagulant drugs with low rates of therapy-related hemorrhages. In this work, we describe the development of a new series of 3-carboxamide-coumarins that are the first potent and selective nonpeptidic inhibitors of FXIIa.

Full text of DOI:10.1021/jm8002697

J. Med. Chem. 2008, 51, 3077–3080
3077
Scheme 1. Synthesis of the Amide 19-27 and 28-33
Derivativesa
Novel 3-Carboxamide-coumarins as Potent
and Selective FXIIa Inhibitors
Se´verine Robert,† Carine Bertolla,† Bernard Masereel,
Jean-Michel Dogne´, and Lionel Pochet*
Department of Pharmacy, Drug Design and DiscoVery Center,
FUNDP, UniVersity of Namur, 61, Rue de Bruxelles,
B-5000 Namur, Belgium
ReceiVed March 12, 2008
Abstract: Recently, FXIIa was highlighted as an original attractive
target for the development of new anticoagulant drugs with low rates
of therapy-related hemorrhages. In this work, we describe the develop-
ment of a new series of 3-carboxamide-coumarins that are the first
potent and selective nonpeptidic inhibitors of FXIIa.
a Reagents: (a) diethyl malonate, piperidine, MW ; (b) 10% w/v NaOH;
(c) 6 N HCl; (d) SOCl2; (e) R-NH2; (f) EDC, HOBt, DMF; (g) R-NH2.
The coagulation cascade takes place through a series of
proteolytic reactions involving trypsin-like serine proteases in
order to generate sufficient amount of thrombin (THRa) to form
a fibrin clot. Initiation of fibrin formation through the “extrinsic
pathway” occurs when plasmatic activated factor VII (FVIIa)
forms a complex with the transmembrane protein tissue factor
(TF), which is exposed following a lesion of endothelium.
Alternatively, the coagulation cascade may be initiated through
the “intrinsic pathway” when factor XII (FXII) is activated on
a negatively charged surface by a process called “contact
activation”. Activation of FXII is followed sequentially by
activation of factor XI and factor IX. The intrinsic and extrinsic
pathways converge at the level of factor X (FX) activation.
Activated factor X (FXa) activates prothrombin to THR in the
presence of the cofactor activated factor V, and THR subse-
quently converts fibrinogen to fibrin.1
FXII has long been considered to be unnecessary for normal
hemostasis because humans with hereditary deficiencies of FXII
do not suffer from spontaneous or injury-related abnormal
bleedings.2,3 However, recent studies have highlighted the
potential role of FXII in pathological thrombogenesis.1,4,5 So
FXII-deficient mice were found to be protected against arterial
thrombosis, collagen- and epinephrine-induced thromboembo-
lism,6 and ischemic brain stroke.7 In all these in vivo models,
the protection was abolished by infusion of human FXII into
FXII-null mice. Wild type mice treated by D-Pro-Phe-Arg
chloromethyl ketone (PCK), which is known to inhibit the
amidolytic activity of activated factor XII (FXIIa),8 were also
found to be protected from ischemic brain stroke.7 Moreover,
as their human counterparts, the FXII-null mice do not suffer
from impaired hemostasis.9 FXIIa is thus an attractive target
for inhibitors designed to treat or prevent thromboembolic
disorders. Indeed, FXIIa inhibitors would likely have only
limited effects on hemostasis. Consequently, it is anticipated
that their use in clinic would be associated with relatively low
rates of therapy-related hemorrhages,10 a well-known adverse
drug reaction of the majority of anticoagulant agents.
Scheme 2. Synthesis of the Amide 34-48 Derivativesa
a Reagents: (a) diethyl malonate, piperidine, MW; (b) R-NH2, BMimPF6
ethanol, MW.
Natural anticoagulant proteins displaying FXIIa inhibitory
potency were reported from leguminous plants,11–17 hematopha-
gous insects,18,19 helminth parasites,20 and bacteria.21 However,
despite their efficacy, all these proteins were generally not
selective over blood coagulation proteases.
In our previous studies, we demonstrated the efficacy of 3,6-
disubstituted coumarins for inhibition of THR and FXa, two
enzymes implicated in the common pathway of the coagulation
cascade.22 Within this series, we highlighted two important
structural features to inhibit both enzymes: the ester link between
the coumarin ring and the side chain and the chloromethyl
function in the 6-position (which has been demonstrated to play
a key role in the inhibition process). The removal of these
structural features led to a drastic loss of inhibitory potency
toward THR and FXa. Taking into account the similarities
between the trypsin-like serine proteases THR, FXa, and FXIIa,
we decided to design coumarins as potent and selective inhibitors
of FXIIa. In order to afford selectivity toward THR and FXa,
we synthesized novel 3-carboxamide-coumarins deprived of a
chloromethyl moiety in the 6-position.
These novel coumarins have been synthesized according to
Schemes 1 and 2. In the first step, the commercially available
salicylaldehydes were converted into the corresponding 2-oxo-
2H-1-benzopyran-3-carboxylic acid ethyl esters via a Knoev-
enagel reaction under microwave irradiation (Schemes 1 and
2).23 This procedure allowed not only drastical reduction of the
reaction time (from 6-24 h to 2-30 min) but also improvement
in the yields (from 14-21% to 45-64%).
The ester hydrolysis was then easily realized by alkaline
hydrolysis followed by acidification to afford the intermediate
17 (Scheme 1). Several routes were investigated to obtain the
amide derivatives. For derivatives 19-27, the suitable amine
was reacted with the acyl chloride obtained by treatment of 17
with thionyl chloride. Alternatively, derivatives 28-33 were
* To whom correspondence should be addressed. Phone: +32 (0)81 72
42 90. Fax: +32(0)81 72 42 99. E-mail: lionel.pochet@fundp.ac.be.
†
These authors equally contributed to this work.
a Abbreviations: FVIIa, activated factor VII; FX, factor X; FXa, activated
factor X; FXII, factor XII; FXIIa, activated FXII; Kall, kallikrein; PCK,
D-Pro-Phe-Arg chloromethyl ketone; TF, tissue factor; THR, thrombin.
10.1021/jm8002697 CCC: $40.75
2008 American Chemical Society
Published on Web 05/07/2008

3078 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Letters
Table 1. Inhibitory Potency of N-Substituted
6-Methyl-2-oxo-2H-1-benzopyran-3-carboxamides toward FXIIa, THR,
FXa, TF/FVIIa, and Kallikrein
Finally, we investigated the influence of the nature and the
position of substituents on the phenyl side chain in the 3-position
(Table 3). These substituents strongly affect the inhibitory
potency. Indeed, in the 6-methyl series, a methyl group was
more favorable than a chloro group especially when this methyl
is in the 3′-position (28, 29, 30 vs 31, 32, 33). The introduction
of a second methyl group in 5′-position led to 34, which is as
active as the monosubstituted one (29). In the 6,8-dichloro series,
a single methyl group did not improve potency (42 vs 41)
whereas the introduction of the second methyl led to a 4-fold
activity increase (43 vs 41). Surprisingly, the introduction of
methyl substituents in the 8-bromo-6-chloro series led to a
decrease of inhibitory potency (45, 46 vs 44). The best
compounds among the newly designed 3-carboxamide-cou-
marins were 43 and 44 with IC50 values for inhibition of FXIIa
of 4.3 and 4.4 µM, respectively.
IC50 (µM)a
TF/
compd
R
FXIIa
THR FXa FVIIa Kall
19
21
22
23
20
24
25
26
27
isopropyl
NIb
NIb
NIb NIb >50 >50
NIb NIb NIb NIb
NIb NIb NIb NIb
NIb NIb NIb NIb
>50 NIb NIb >50
NIb NIb NIb NIb
NIb NIb NIb NIb
>50 NIb NIb NIb
cyclohexyl
cyclododecyl
adamantyl
phenyl
benzyl
ethylphenyl
33 (27-39)
29 (24-34)
23 (20-28)
>50
>50
naphthalen-1-ylmethyl 15 (11-20)
quinol-3-yl
8.3 (5.9-11.5) NIb NIb NIb NIb
Furthermore, the newly designed 3-carboxamide-coumarins
were selective for structurally related enzymes such as THR,
FXa, TF/FVIIa, and kallikrein (Kall) (Tables 1–3). The selectiv-
ity of these compounds toward THR could be explained by the
absence of an ester link between the coumarin and the side
chain in the 3-position but also by the lack of a chloromethyl
function in the 6-position. Indeed, it has been shown that the
chloromethyl group participates in the mechanism-based be-
havior of 3,6-disubstituted coumarins toward THR.22 With
this latter series, it has been postulated that the lactone carbonyl
would be first attacked by the catalytic nucleophilic serine 195.
The subsequent lactone ring opening would result in an increase
of the leaving properties of the chlorine atom and formation of
an electrophilic quinone methide. This latter compound would
form a covalent bond with a nucleophilic residue within the
THR active site leading to the formation of an alkyl enzyme.
a Calculated by nonlinear regression from dose-response curves.
Values in parentheses are 95% confidence intervals. b NI, no inhibition at
50 µM.
prepared by reaction between the suitable amine and the
carboxylic acid activated by EDC/HOBt.24
The 3-carboxamide-coumarins 34-48 were obtained in one
step by aminolysis of the corresponding 2-oxo-2H-1-benzopy-
ran-3-carboxylic acid ethyl ester under microwave irradiation
(Scheme 2). This latter route gave high yields (ranging from
50% to 81%) with phenylamine derivatives and reaction times
from 20 to 60 min.
The inhibitory potency of the newly 3-carboxamide-cou-
marins was investigated by measuring the IC50 value for
inhibition of FXIIa. First, we tested a series of N-substituted-
6-methyl-2-oxo-2H-1-benzopyran-3-carboxamides (Table 1).
Among this series, the compounds bearing a small aliphatic side
chain such as isopropyl (19) or cyclohexyl (21) were inactive
toward FXIIa. However, the presence of a bulky aliphatic group
such as cyclododecyl (22) or adamantyl (23) as well as a bulky
aromatic group such as naphthalen-1-ylmethyl (26) or quinol-
3-yl (27) led to active compounds. Phenylamide derivative (20)
was also found to be potent, but the introduction of a spacer
between this phenyl side chain and the carboxamide in the
3-position led to the lost of the inhibitory potency (24, 25 vs
20).
Second, we introduced various modifications on the aromatic
part of the coumarin ring keeping a 3-phenylcarboxamide side
chain (Table 2). We observed that the presence of a methyl
group in the 6-position was favorable to the inhibitory potency
on FXIIa (20 vs 18). Moreover, the replacement of this methyl
group in the 6-position by a halogen or a nitro moiety in the
same position did not significantly improve the inhibitory
potency (4, 35, 36 vs 20). However, a disubstitution in the 6
and 8 positions by halogens improved the inhibitory potency
(41, 44 vs 20). In contrast, the presence of a methoxy group in
the 8-position alone (40) or in combination with a bromo in
the 6-position (47) was unfavorable. The substitution in the 5,6-
positions by a benzo group (48) or in the 7-position by a
hydroxyl (37), a methoxy (38), or a diethylamino (39) always
led to inactive compounds.
For comparison purposes, we also tested compounds bearing
a 3-phenyl ester side chain. These derivatives presented no (1,
2) or lesser (3) inhibitory potency on FXIIa compared to their
amide counterparts (18, 20, 4). In conclusion, the best compound
in this series was the phenylamide of 2-oxo-2H-1-benzopyran-
3-carboxylic acid substituted by a 8-bromo-6-chloro group (44)
with IC50 for inhibition of FXIIa of 4.4 µM.
However, with the 3-carboxamide-coumarins presented in this
paper, the most potent FXIIa inhibitors were devoid of a latent
alkylating function, suggesting that this new series was not likely
acting through the same mechanism. We thus investigated the
inhibition profile of 44 toward FXIIa. Time dependent inhibition
was first observed, indicating a slow binding of 44 to FXIIa
(see Supporting Information). Moreover, this inhibition was
persistent even after elimination of the excess of inhibitor by
ultracentrifugation. These data demonstrate the irreversible
nature of the inhibition. The alkylation of FXIIa being impos-
sible, the inactivation is probably due to the formation of a stable
acyl enzyme. This acyl enzyme could be formed by the reaction
of the FXIIa active serine with the carbonyl group of the lactone
ring or with the exocyclic amide function. An acylation of the
lactone group has been already observed for the inhibition of
R-chymotrypsin by the ester of 3-chlorophenyl-6-methylcou-
marin 3-carboxylic acid.27 Therefore, these newly designed
3-carboxamide-coumarins would act as mechanism-based in-
hibitors of FXIIa through the formation of an acyl enzyme
instead of an alkyl enzyme as observed with thrombin and
6-chloromethyl ester coumarins.
For comparison purposes, we also tested the inhibitory
potency of the peptidic compound PCK. It had an IC50 for
inhibition of FXIIa of 0.18 µM. However, it was nonselective
toward THR, FXa, TF/FVIIa, and kallikrein with IC50 values
of 2.5, 1.9, 1.5, and 0.031 µM, respectively. Moreover, the
peptidic structure and the alkylating behavior of the chlorom-
ethyl function makes PCK unsuited for oral clinical use as
anticoagulant agent.
In this work, we successfully developed original 3-carboxa-
mide-coumarins that selectively inhibit FXIIa, a recent interest-

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3079
Table 2. Inhibitory Potency for Phenyl Esters or Amides of Substituted 2-Oxo-2H-1-benzopyran-3-carboxylic Acid toward FXIIa, THR, FXa, TF/FVIIa,
and Kallikrein
IC50 (µM)a
TF/
compd
X, Y
6-CH3
6-CH2Cl
6-NO2
FXIIa
THR
FXa
FVIIa
Kall
NIb
18
20
4c
42 (27-66)
25 (21-31)
17 (12-23)
22 (16-30)
24 (18-31)
19 (12-30)
4.4 (3.2-6.2)
>50
NIb
>50
>50
NIb
NIb
NIb
NIb
>50
NIb
>50
>50
>50
NIb
NIb
NIb
NIb
NIb
>50
NIb
NIb
NIb
NIb
NIb
>50
>50
>50
>50
NIb
NIb
NIb
>50
NIb
NIb
NIb
NIb
NIb
NIb
NIb
>50
NIb
>50
NIb
NIb
>50
NIb
NIb
NIb
>50
NIb
35
36
41
44
47
40
48
37
38
39
1d
2c
NIb
6-Br
NIb
6,8-diCl
6-Cl, 8-Br
6-Br, 8-OMe
8-OMe
5,6-benzo
7-OH
>50
NIb
NIb
NIb
>50
>50
>50
>50
>50
NIb
NIb
NIb
7-OMe
7-NH(C2H5)2
NIb
NIb
NIb
6-CH3
6-CH2Cl
NIb
NIb
3c
>50
7.0 (3.7-13.5)
NIb
a Calculated by nonlinear regression from dose-response curves. Values in parentheses are 95% confidence intervals. b NI, no inhibition at 50 µM.
c From ref 25. d From ref 26.
Table 3. Inhibitory Potency for Phenyl Amides of Substituted 2-Oxo-2H-1-benzopyran-3-carboxylic Acid toward FXIIa, THR, FXa, TF/FVIIa, and
Kallikrein
IC50 (µM)a
compd
X, Y
X′, Y′
2′-Cl
3′-Cl
4′-Cl
2′-CH3
3′-CH3
4′-CH3
3′,5′-diCH3
FXIIa
THR
FXa
TF/FVIIa
Kall
20
31
32
33
28
29
30
34
41
42
43
44
45
46
PCK
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6,8-diCl
6,8-diCl
6,8-diCl
23 (20-28)
30 (21-44)
31 (23-42)
34 (25-45)
32 (21-47)
10 (7.2-15)
20 (14-27)
9.8 (5.8-16.6)
19 (12-30)
25 (22-30)
4.3 (2.5-7.6)
4.4 (3.2-6.2)
10 (8.6-12)
32 (23-44)
0.18 (0.14-0.23)
>50
>50
NIb
>50
>50
>50
>50
>50
NIb
NIb
NIb
NIb
NIb
NIb
NIb
NIb
NIb
NIb
NIb
>50
NIb
NIb
>50
NIb
>50
NIb
NIb
NIb
NIb
NIb
NIb
NIb
>50
NIb
>50
NIb
NIb
>50
NIb
NIb
NIb
NIb
NIb
>50
NIb
NIb
3′-CH3
3′,5′-diCH3
NIb
NIb
>50
>50
6-Cl, 8-Br
6-Cl, 8-Br
6-Cl, 8-Br
NIb
NIb
3′-CH3
3′,5′-diCH3
>50
>50
2.5 (2.1-3.0)
>50
>50
1.5 (1.2-1.9)
D-Pro-Phe-Arg-CH2Cl
1.9 (1.4-2.5)
0.031 (0.024-0.039)
a Calculated by nonlinear regression from dose-response curves. Values in parentheses are 95% confidence intervals. b NI, no inhibition at 50 µM.
ing new target for the prevention and the treatment of
thromboembolic diseases. Despite a lower inhibitory potency
on FXIIa than PCK, these derivatives were selective toward
THR, FXa, TF/FVIIa, and kallikrein, contrary to PCK. To our
knowledge, these compounds are the first potent and selective
nonpeptidic inhibitors of FXIIa described in the literature to
date. Their uses as anticoagulant agents are currently evaluated.
They also constitute very interesting pharmacological tools to
investigate the role of FXIIa in thrombosis and hemostasis.
Further pharmacomodulations of the compounds should be
investigated in order to increase the inhibitory potency on FXIIa
while keeping the selectivity toward the other blood coagulation
proteases.
Acknowledgment. This work was partly supported by the
Fonds de la Recherche Scientifique Medicale Belge (Grant No.
1.5.066.07).
Supporting Information Available: Experimental procedures
and analytical data. This material is available free of charge via
the Internet at http://pubs.acs.org.
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JM8002697