New fibrinolytic agents: Benzothiophene derivatives as inhibitors of the t-PA-PAI-1 complex formation

Article abstract of DOI:10.1016/S0960-894X(03)00233-6

The synthesis and activity of novel benzothiophene derivatives are described. In the t-Pa-induced fibrin clot lysis assay, several compounds inhibit the formation of the tPa-PAI-1 complex with submicromolar IC₅₀. This class of compounds potentially represents a new generation of antithrombotic-fibrinolytic agents.

Full text of DOI:10.1016/S0960-894X(03)00233-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1705–1708
New Fibrinolytic Agents: Benzothiophene Derivatives as
Inhibitors of the t-PA–PAI-1 Complex Formation
Guillaume De Nanteuil,a,* Christine Lila-Ambroise,a,y
Alain Rupin,b Marie-Odile Vallezb and Tony J. Verbeurenb
aDivision D of Medicinal Chemistry, Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France
bDivision of Angeiologie, Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France
Received 2 December 2002; revised 27 January 2003; accepted 5 March 2003
Abstract—The synthesis and activity of novel benzothiophene derivatives are described. In the t-Pa-induced fibrin clot lysis assay,
several compounds inhibit the formation of the tPa–PAI-1 complex with submicromolar IC50. This class of compounds potentially
represents a new generation of antithrombotic-fibrinolytic agents.
# 2003 Elsevier Science Ltd. All rights reserved.
The conversion of plasminogen to plasmin through the
proteolytic cleavage of the Plasminogen Activators
(Tissue type Plasminogen Activator, t-PA, and Urinary
Plasminogen Activator, or urokinase, u-PA) is the
main event occurring in the fibrinolytic pathway, ulti-
mately leading to the dissolution of the clot by plas-
min. The proteolytic activity of t-PA is regulated by
Plasminogen Activator Inhibitor-1 (PAI-1). The mole-
cular mechanism of inhibition of t-PA by PAI-1 is
common to all the members of the serpin superfamily,
and is characterised by the formation of a stable 1:1
stoechiometric complex.1
Numerous animal studies have highlighted the central
role of PAI-1 in the development of thrombotic events,
among which those obtained using transgenic mice:
animals overexpressing human PAI-1 displayed venous
occlusions, while deficient mice are virtually protected
against venous thrombosis.3 In human, elevated PAI-1
levels have been shown to be correlated with several
thrombotic disease states, including venous throm-
boembolism, coronary artery disease, acute myocardial
infarction, obesity, diabetes, sepsis, surgery and
trauma.4
Beside this important role in the fibrinolytic pathway,
PAI-1 has also been implicated in several other crucial
biological processes including tumor invasion, neo-
vascularization, inflammation and wound healing.5
When released from endothelial cells into the circula-
tion, a fraction of PAI-1 is spontaneously inactivated
into a latent form; a second fraction of circulating PAI-
1 gives a 1:1 complex with vitronectin. This stabilized
complex allows PAI-1 to act as a pseudo-substrate for
t-PA by mimicking plasminogen sequence, using a ‘bait’
dipeptidic residue Arg 346-Met 347, located on the
reactive center loop. Cleavage of this peptidic bond by
Ser 478 of t-PA leads to the formation of the t-PA/PAI-
1 1:1 complex. Deactivation into the latent form as well
as cleavage of the bait residue give rise to the inter-
nalization of the reactive center loop into b-sheet A.2
Several hypothesis have been put forward in order to
decrease PAI-1 activity and to restore fibrinolysis: for
example, it has been shown that the inhibition of the
formation of the stoechiometric t-PA/PAI-1 complex by
anti-PAI-1 monoclonal antibodies such as MAI-126
could be of value in order to decrease prothrombotic
PAI-1 activity. More recently, a tetradecapeptide based
upon the peptidic sequence of the reactive center loop
was shown to decrease PAI-1 activity in an in vitro clot
lysis model.7 In 1996, Xenova reported the first low
molecular weight inhibitor of the t-PA/PAI-1 interac-
tion: XR 334 1 was found to enhance fibrinolysis ex vivo
and reduce the formation of the thrombus in the rat
electrically stimulated carotid artery model.8
*Corresponding author. Tel.: +33-1-5572-2234; e-mail: guillaume.
denanteuil@fr.netgrs.com
yPresent address: Isochem,
France.
9 rue Lavoisier, 91710 Vert-le-Petit,
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00233-6

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G. De Nanteuil et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1705–1708
Optimization of this very insoluble lead gave rise to
compounds with improved activity and solubility, such
as XR 5082 2 and XR 5118 3.9 Meanwhile, the optimi-
zation of a family of 2,3-disubstituted benzothiophenes
selected from our corporate library giving sub-
micromolar activity in the t-Pa-induced fibrin clot lysis
assay has been reported, yielding compounds exempli-
fied here by 4.10 More recently, Xenova has disclosed
the structures of novel inhibitory templates.11
IC50 of 59.4Æ5.7, 51Æ3 and 106.7Æ1.6 mM, respec-
tively. In our previous report, we had already shown
that 4 was significantly more active with an IC50 of
0.47Æ0.15 mM (Table 1).10
Variously substituted biphenyl systems were prepared:
the biphenyl-4-yl methyloxy moiety (7) was preferred to
its 2- (5) and 3-yl (6) counterparts. Activity was main-
tained with a 40-methoxy substitution in 8. Finally,
introduction of a spacer between the two phenyl rings in
9–12 proved to be advantageous for activity, culminat-
ing with 11 which came out as the most active tPa–PAI-
1 complex formation inhibitor of this chemical series
with an IC50 of 39 nM. Having established the biphenyl-
4-yl methyloxy moiety as one of the optimal substitu-
tions at positions 5 and 6, modifications were effected at
position 3 (Table 2): the non-substituted derivative 13
proved to be as potent as starting compound 7, while
introduction of a fluorine atom at position 3 on the 4-
chloro substituted phenyl ring gave a sharp drop in
activity (14). One methyl group at position 3 was
acceptable (15), while 3,5-dimethyl substitution found in
16 was 2 times less potent; 3-fluoro (17) and 3,4-difluoro
(18) substitutions restored interesting sub-micromolar
activities. Finally, introducing a pyridine ring (19–21) as
well as substitution with larger polycyclic ring systems
such as in 22–24, led to an overall decrease in activity.
The compounds described herein were prepared accord-
ing to Scheme 1. Starting from 3,4-methylenedioxy-cin-
namic acid i, Higa-type cyclization12 gave the acid
chloride of 3-chloro-5,6-methylenedioxy-benzo[b]thio-
phene-2-carboxylic acid ii with 75% yield. Methanolic
hydrolysis and removal of the methylenedioxy group
afforded the key dihydroxy intermediate iv. Alkylation
with the appropriate halide yielded the corresponding
5,6-disubstituted benzothiophene derivative v. Reduction
of the ester function gave alcohol vi, which was further
oxidized to aldehyde vii. Functionalisation at position 3
on the benzothiophene ring was then realized by sub-
stitution of the chlorine atom with a properly substituted
phenol; finally, Knoevenagel reaction followed by sapo-
nification gave the final carboxylic acid derivatives 5–24.
Biological evaluation of this new series of compounds
was realized by measuring the inhibitory potency of the
new compounds against the formation of the t-PA–PAI-1
complex in a t-PA-induced fibrin clot lysis assay,
according to our previously published protocol.13,14
In summary, optimization of a family of benzothiophene
derivatives as inhibitors of the t-Pa/PAI-1 complex for-
mation led to novel structures with sub-micromolar IC50
values. Further structural modification as well as in
vivo evaluation of the new inhibitors will be reported
in due course.
Xenova compounds 1, 2 and 3 proved to be potent
inhibitors of the t-PA–PAI-1 complex formation giving
Scheme 1. Reagents: (a) SOCl2, pyridine, chlorobenzene; (b) methanol, toluene; (c) BBr3, methylene chloride; (d) R1-Cl, K2CO3, DMF; (e) LiAlH4,
THF; (f) MnO2, toluene; (g) R1-substituted phenol, NaH, DMF; (h) ethyl aryl acetate, Ac2O; (i) NaOH, EtOH.

G. De Nanteuil et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1705–1708
Table 1. tPa/PAI-1 complex formation inhibition data for compounds 5–12
1707
Compd
R1
IC50 (mM)
10.8Æ0.1
Compd
R1
IC50 (mM)
0.27Æ0.01
5
9
6
7
8
0.22Æ0.05
0.13Æ0.06
0.14Æ0.04
10
11
12
0.10Æ0.08
0.039Æ0.01
0.77Æ0.1
Table 2. tPa/PAI-1 complex formation inhibition data for compounds 13–24
Compd
R2
IC50 (mM)
Compd
R2
IC50 (mM)
13
14
15
0.10Æ0.04
2.28Æ0.72
0.48Æ0.15
19
20
21
2.40Æ0.87
2.48
2.07
16
17
18
1.09Æ0.29
0.24Æ0.04
0.59Æ0.04
22
23
24
6.06
2.91Æ1.5
10.7Æ2

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G. De Nanteuil et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1705–1708
9. Charlton, P.; Faint, R.; Barnes, C.; Bent, F.; Folkes, A.;
Acknowledgements
Templeton, D.; Mackie, I.; Machin, S.; Bevan, P. Fibrinolysis
Proteolysis 1997, 11, 51.
10. De Nanteuil, G.; Lila, C.; Rupin, A.; Verbeuren, T. J.
16th International Symposium on Medicinal Chemistry,
Bologne, Sept. 18–22, 2000, Abst. PA 112.
The authors wish to thank Daniele Heno and Edith
Bonhomme for their skillful technical assistance.
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