Amido-(propyl and allyl)-hydroxybenzamidines: Development of achiral inhibitors of factor Xa

Article abstract of DOI:10.1016/S0960-894X(99)00673-3

The design, synthesis and SAR of amido-(propyl and allyl)-hydroxybenzamidine coagulation factor Xa inhibitors is described. These achiral inhibitors are selective for fXa vis a vis structurally related serine proteases and are readily prepared in 6-7 linear steps. The most potent member 9j (fXa K(i) = 0.75 nM) is selective (>1000-fold) and an effective anticoagulant in mammalian plasma. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(99)00673-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 217±221
Amido-(Propyl and Allyl)-hydroxybenzamidines:
Development of Achiral Inhibitors of Factor Xa
Yong Gong, a Henry W. Pauls, a,* Alfred P. Spada, a Mark Czekaj, a
Guyan Liang, b Valeria Chu, c Dennis J. Colussi, c Karen D. Brown c and Jingbo Gao c
aDepartment of Medicinal Chemistry, Rhone-Poulenc Rorer, PO Box 1200, 500 Arcola Rd, Collegeville, PA 19426-0107, USA
bComputer Assisted Drug Design, Rhone-Poulenc Rorer, PO Box 1200, 500 Arcola Rd, Collegeville, PA 19426-0107, USA
cCardiovascular Biology, Rhone-Poulenc Rorer, PO Box 1200, 500 Arcola Rd, Collegeville, PA 19426-0107, USA
Received 25 October 1999; accepted 18 November 1999
AbstractÐThe design, synthesis and SAR of amido-(propyl and allyl)-hydroxybenzamidine coagulation factor Xa inhibitors is
described. These achiral inhibitors are selective for fXa vis a vis structurally related serine proteases and are readily prepared in 6±7
linear steps. The most potent member 9j (fXa Ki=0.75 nM) is selective (>1000-fold) and an e€ective anticoagulant in mammalian
plasma. # 2000 Elsevier Science Ltd. All rights reserved.
The serine protease component of the prothrombinase
complex, factor Xa (fXa), is the singular enzyme
responsible for the conversion of prothrombin to
thrombin (fIIa).1 Inhibitors of thrombin, the terminal
enzyme of the coagulation cascade,2 have been studied
for some time as potential antithrombotic agents.3 More
recently, inhibitors of fXa, the penultimate enzyme in
the cascade, have been pursued as anticoagulant thera-
pies.4,5 A variety of factor Xa inhibitors have now been
described and several comprehensive reviews have
appeared recently.4±9
isomer was the most active. A chiral synthesis was
described which requires a diazomethane mediated
homologation of an amino acid, a costly and potentially
hazardous process.
Given the perceived drawbacks of the chiral series and
the liabilities of the ester function, we sought to develop
simpler analogues in which the branched chain is
replaced by an achiral tether (e.g. 2a). Maintaining
potency appeared to be a daunting task since the simple
prototype 2a (fXa Ki ꢀ1 mM) was much less potent12,13
than the b-amidoester 1a (fXa Ki=5.0 nM). Several
groups,14±16 including ourselves,17,18 have described the
potency enhancing e€ects of incorporating hydroxyl
groups para to the amidine attachment point of the P-1
benzamidine. This strategy was applied to the amido-
propylbenzamidines as a way to increase anti-fXa
activity. The development of potent fXa inhibitors
based on compound 3a is described.
Compounds 1a and b are representative of the b-ami-
doester series of fXa inhibitors, discovered in our
laboratories.10,11 Previously, the optimization of the
biaryl group (P-4) and the structure activity relation-
ships for the branching substituents were described. The
ester function, a possible hydrolytic center, was found
to be necessary for optimal activity and the R,R stereo-
*Corresponding author. Tel.: +1-610-454-5636; fax: +1-610-454-
3311; e-mail: heinz.pauls@aventis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00673-3

218
Y. Gong et al. / Bioorg. Med. Chem. Lett. 10 (2000) 217±221
Scheme 1. Synthesis of amido-(propyl and allyl)-benzamidines 9 and 3. (i) NaH, MEMCl, THF; (ii) AllylNPth, 1% equiv. Pd(OAc)2, 2% equiv.
P(o-Tol)3, Et3N; (iii) N2H4, H2O, EtOH, re¯ux, 1 h; (iv) Et3N, TBTU, DMF; (v) EtOH±HCl; (vi) MeOH±NH3; HPLC; (vii) 10% Pd/C, H2.
Scheme 2. Synthesis of biaryl acids. (viii) Pd(PPh3)4, aq. Na2CO3, MeCN, 90 ꢁC; (ix) aq. NaClO2/NaH2PO4.
Chemistry19
ligand. For example, the most potent analogue 3b is
35-fold more e€ective against fXa than the imidazoly-
phenyl derivative 3f. Selectivity against thrombin and
trypsin varies 10±500-fold depending on the nature of
the P-4 ligand.
The synthesis begins with the commercially available
3-bromo-4-hydroxybenzonitrile which is protected as
the MEM ether 4 (Scheme 1). Heck reaction is followed
by removal of the phthaloyl group to yield the free
amine 6. At this stage a variety of carboxylic acids 7
can be coupled to give compounds 8 with a diverse set of
P-4 ligands. Pinner reaction followed by ammonolysis
gives the amidoallylhydroxybenzamidines 9. Subsequent
catalytic reduction yields the amidopropylhydroxy-
benzamidines 3.
A molecular modeling study of the amidopropyl
hydroxybenzamidines bound in the fXa active site was
initiated in an e€ort to enhance the activity of this series.27
The study suggested that incorporation of unsaturation
into the chain would yield conformationally restricted
analogues with potentially higher binding anities.
Compound 9a for example makes many of the same
interactions which have been shown to be important for
binding of the b-amidoesters to fXa10,11,28 (Fig. 1).
These contacts include a salt bridge (benzamidine/
D189), a hydrogen bond (amide carbonyl/G218) and
Van der Waals interactions (biaryl/S-4). In addition, the
hydroxyl group on the benzamidine is within H-bond-
ing distance of the side chain of S195.
The aryl carboxylic acids used in this paper were com-
mercially available (entries a and c) or prepared by
standard methods. The preparation of biaryl acids 7b, e,
f, g, h and i has been previously described.19 The acid
required for entry j is prepared from the methyl ester of
4-(1-oxypyridin-4-yl)benzoate19 by application of cyano-
trimethylsilane;20 subsequent acid hydrolysis21 (85%
H2SO4) gives the desired 7j. Alternatively we have found
the Suzuki reaction to be a general method for biaryl
acid synthesis22 (Scheme 2). For example, 4-carboxy-
benzene boronic acid is heated with the appropriate
halogenated aryl or heteroaryl in aqueous sodium car-
bonate and acetonitrile, in the presence of Pd(PPh3)4.
The desired acids 7k and l are obtained by precipitation
from the reaction medium and require no further puri-
®cation. Acids 7n23 and 7d24 were prepared according to
literature methods.
The allyl analogues were readily obtained (Scheme 1). A
modest increase in activity was observed for 9a versus
its saturated analogue 3a and this trend (up to 3.5-fold)
was observed across a range of P-4 moieties (Table 2).
Potency was markedly increased by meta substitution
on the distal phenyl ring with a carboxamide group (9b).
A similar enhancement was seen in the b-aminoester
series.10,11 This observation was extended to sulfona-
mide substituents; in this case the ortho derivative 9m
was found to be the most e€ective.29 Note also that by
judicious choice of the P-4 ligand (9j, fXa Ki=0.75 nM)
it was possible to achieve anti-fXa potency in the ami-
doallyl hydroxybenzamidine series comparable to the
b-aminoesters (1b, fXa Ki=0.5 nM).
Results and Discussion
Table 1 summarizes the results for a limited set of ami-
dopropyl hydroxybenzamidines. Note that the para-
hydroxybenzamidine (3a) is 11-fold more active than
the comparable unsubstituted benzamidine (2a). This
substitution, to some degree, compensates for the absent
ester function.26 The entries illustrate the dependence of
activity on the nature of the aryl group (Ar), i.e. the P-4
The most potent inhibitor 9j was tested against a panel
of trysin-like serine proteases and shown to be selective
for factor Xa (Table 3). In fact the achiral inhibitors
were in general more selective for fXa than the b-ami-
doesters. This is illustrated by the activities of 3b and

Y. Gong et al. / Bioorg. Med. Chem. Lett. 10 (2000) 217±221
219
Table 1. Coagulation factor Xa inhibition data25 for amidopropylhydroxybenzamidines 3
Compound
Ar
fXa
Ki (nM)
fIIa
Ki (nM)
Tryp.
Ki (nM)
Compound
Ar
fXa
Ki (nM)
fIIa
Ki (nM)
Tryp.
Ki (nM)
3a
88
>4000
>2900
3e
58
>4000
ꢀ2900
3b
7.0
>4000
>2900
3f
254
>4000
ꢀ2900
3c
3d
59
>4000
2200
3g
3h
131
38
>4000
>4000
>2900
1900
181
ꢀ4000
>2900
Figure 1. A stereoview of 9a bound in the active site of factor Xa.27
9j as compared to compound 1b against trypsin and
plasmin.
observed with other factor Xa inhibitors and may re¯ect
subtle structural di€erences in the factor Xa from dif-
ferent species.31
Inhibitor 9j was tested for its ability to prolong the
activated partial thromboplastin time (APTT) in plasma
from four species.30 This assay provides a measure of
the compound's ability to inhibit the prothrombinase
bound factor Xa i.e. the physiologically relevant form of
the enzyme. Some species di€erences were observed; the
concentration required to double the APTT for human,
dog, rabbit and rat was found to be 0.87, 0.99, 0.51
and 1.7 mM, respectively. This phenomenon has been
In summary, achiral factor Xa inhibitors have been
described whose synthesis is readily achieved in 6±7
linear steps. The amidoallyl hydroxybenzamidines are
nanomolar inhibitors of fXa with better than a 1000-
fold selectivity against related serine proteases. The
e€ectiveness of inhibitor 9j against the prothrombinase
complex is demonstrated by the APTT prolongation in
mammalian plasma.32

220
Y. Gong et al. / Bioorg. Med. Chem. Lett. 10 (2000) 217±221
Table 2. Coagulation factor Xa inhibition data25 for amidoallyhydroxybenzamidines 9
Compound
Ar
fXa
Ki (nM)
fIIa
Ki (nM)
Tryp.
Ki (nM)
Compound
Ar
fXa
Ki (nM)
fIIa
Ki (nM)
Tryp.
Ki (nM)
9a
51
>4000
>4000
>2900
>2900
9i
20
2900
1400
2200
9b
5.0
9j
0.75
>4000
9c
9e
61
3200
2300
1400
9k
9l
12
>4000
>4000
1600
240
128
>4000
9.0
9f
19
37
>4000
>4000
1800
1400
9m
9n
2.0
>4000
>4000
1500
1400
9g
184
Table 3. Selectivity of coagulation factor Xa inhibitors against related serine proteases25
Compound
fXa Ki (nM)
fIIa Ki (nM)
ꢀ4000
Tryp. Ki (nM)
APC Ki (nM)
Plasm. Ki (nM)
tPA KI (nM)
1b
3b
9j
0.50
90
>18,500
140
>8700
7.0
>4000
>4000
>2900
2200
15,000
3500
>8700
7700
0.75
ꢀ18,500
>7300
Acknowledgements
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sured with a MLA Electra 800 automatic coagulation timer
(Orthodiagnostics, NJ) as described in reference 17.
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32. Selected members from this series were found to be e€ec-
tive in animal models of thrombosis upon iv dosing; this data
will be published separately.