The design of phenylglycine containing benzamidine carboxamides as potent and selective inhibitors of factor Xa

Article abstract of DOI:10.1016/S0960-894X(01)00042-7

Factor Xa. a critical serine protease in the blood coagulation cascade, has become a target for inhibition as a strategy for the invention of novel anti-thrombotic agents. Here we describe the development of phenylglycine containing benzamidine carboxamides as novel, potent and selective inhibitors of factor Xa.

Full text of DOI:10.1016/S0960-894X(01)00042-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 733±736
The Design of Phenylglycine Containing Benzamidine
Carboxamides as Potent and Selective Inhibitors of Factor Xa
Stuart D. Jones,a,* John W. Liebeschuetz,a Phillip J. Morgan,a Christopher W. Murray,a
Andrew D. Rimmer,a Jonathan M. E. Roscoe,a,y Bohdan Waszkowycz,a
Pauline M. Welsh,a,{ William A. Wylie,a Stephen C. Young,a Harry Martin,a
Jacqui Mahler,a Leo Bradyb and Kay Wilkinsonb
aProtherics Molecular Design, Beech®eld House, Lyme Green Business Park, Maccles®eld, SK11 0JL, UK
bDepartment of Biochemistry, University of Bristol, Bristol BS8 1TD, UK
Received 28 April 2000; revised 29 November 2000; accepted 16 January 2001
AbstractÐFactor Xa, a critical serine protease in the blood coagulation cascade, has become a target for inhibition as a strategy for
the invention of novel anti-thrombotic agents. Here we describe the development of phenylglycine containing benzamidine car-
boxamides as novel, potent and selective inhibitors of factor Xa. # 2001 Elsevier Science Ltd. All rights reserved.
Serine protease factor Xa is central to the blood coagu-
lation cascade, responsible for the generation of throm-
bin by both intrinsic and extrinsic pathways. This is
achieved by combination of factor Xa, Ca2+ and phos-
pholipids, forming the prothrombinase complex, which
generates active thrombin by proteolysis of the zymogen
prothrombin. Thrombin is responsible for the activation
of platelets, the feedback activation of other coagula-
tion factors and the conversion of ®brinogen to ®brin
which crosslinks and stabilises the aggregating platelet
plug. Following the extensive investigation of inhibitors
of thrombin as potential antithrombotic agents,1 small
molecule inhibitors of factor Xa are increasingly being
reported as potential anti-thrombotic agents.2,3
result in an additional interaction with the lipophilic
`disulphide' pocket adjacent to S1, comprising residues
Gln192, Cys191, Cys220 and Gly218. Accordingly a
small library of compounds was generated and synthe-
sised using primarily lipophilic d-amino acids. Addi-
tional non-preferred l-amino acids were also included
as control molecules. Thus, bis-1,4-aminomethylcyclo-
hexane, immobilised on chlorotrityl resin, was con-
densed with Fmoc-amino acids using TBTU/DIPEA.6
Fmoc deprotection with piperidine and condensation
with benzamidine-3-carboxylic acid using HOBt/DIPCI6
gave a set of 32 compounds.7 Table 1 shows binding
data8 for some of these compounds (1±8) against factor
Xa and the structurally related enzyme trypsin (used as
a measure of selectivity). The results for compounds 2±7
show a marked preference for compounds derived from
d-amino acids as suggested by the model with, for the
most part, some small selectivity for factor Xa over
trypsin. Compound 8, a racemic mixture,9 showed the
greatest anity for factor Xa. A second library of 20
aryl glycines and analogues was designed and synthe-
sised as before, selected results are also shown in Table 1
compounds 9±15. In this second library for the most
part racemic starting amino acids were used, the exception
being d- and l-cyclohexylglycine.
Benzamidine-3-carboxamide 1, Ki 12 mM against factor
Xa, was selected from a small library of compounds
designed as potential factor Xa inhibitors using
PRO_SELECT, a module4 within our proprietary
modelling software suite PrometheusTM. Examination
of a model of 1 docked into the active site of factor Xa,5
suggested that further elaboration by replacement of the
central glycine with lipophilic d-amino acids might
*Corresponding author. Tel.: +44-1625-500555; fax: +44-1625-
500666; e-mail: stuart.jones@protherics.com
yPresent address: Tripos Receptor Research, Bude, Cornwall, EX23
8LY, UK.
{Present address: AstraZeneca, Alderley Park, Cheshire, SK10 4TF,
UK.
Compounds 14 and 15 derived from l- and d-cyclo-
hexylglycine, respectively, show the same preference for
d-amino acid derived compounds as previously
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00042-7

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S. D. Jones et al. / Bioorg. Med. Chem. Lett. 11 (2001) 733±736
Table 1. Replacement of glycine in compound 1
noted. Molecular modelling of the two phenylglycine
enantiomers of 8 in the active site of factor Xa shows
the benzamidine in the S1 pocket with a bidentate
interaction with Asp189 whilst the cyclohexyl-
aminomethyl moiety projects towards the S4 pocket
bordered by Phe174, Trp215 and Tyr99. The benzene
ring of d-phenylglycine is accommodated in the lipo-
philic `disulphide' pocket described above. This interac-
tion is not possible with the enantiomer derived from
l-phenylglycine. Despite the greater activity of com-
pound 13, derived from d,l-1-naphthylglycine, further
studies were carried out using the more accessible and
less lipophilic d-phenylglycine.
Amino acid AA
FXa Ki (mM)
Trypsin Ki (mM)
1
2
3
4
5
6
7
8
Gly
l-Val
12.0
38.0
6.6
10.5
2.3
33.9
25.7
0.3
0.3
2.5
23.0
12.6
16.2
7.9
10.0
12.6
17.4
4.5
3.9
6.0
5.1
1.1
d-Val
l-Met
d-Met
l-Phe
To explore the S4 region of factor Xa, several diverse
compound libraries were designed and synthesised using
coupling conditions which gave much reduced racemi-
sation of the d-phenylglycine10 (typically <5% racemi-
sation by HPLC examination of products derived from
chiral amines). Tables 2 and 3 show the activities of
some of these compounds (selected from a total of
approximately 200) assayed against factor Xa, trypsin
and thrombin (as a measure of selectivity against related
d-Phe
d,l-Phga
d,l-3-Cl-Phg
d,l-4-Cl-Phg
d,l-3-Cl-Phg
d,l-4-Ph-Phg
d,l-1-Nagb
l-Chgb
9
10
11
12
13
14
15
0.3
0.8
0.1
7.8
2.1
3.6
8.5
d-Chgb
0.5
aCompounds prepared from d- and l-phenyl glycine su€ered com-
plete racemisation using our initial coupling conditions in ref 9.
bThg, thienylglycine, Nag, naphthylglycine, Chg, cyclohexylglycine.
Table 3. Secondary amine±S4 library
Table 2. Primary amine±S4 library
RFXa
Ki
(mM)
Trypsin Ki
(mM)
Thrombin Ki
(mM)
RFXa
Ki
(mM)
Trypsin Ki
(mM)
Thrombin
6.5
22
0.030
0.65
1.35
8
0.275
5.6
23
24
25
26
27
28
29
0.013
0.010
0.120
0.282
0.016
0.015
0.013
0.32
0.54
2.20
0.46
0.34
1.26
0.56
1.26
1.18
7.94
7.94
1.74
3.80
17.80
16
17
18
19
20
21
0.500
0.910
0.850
0.145
0.042
0.028
4.1
5.4
3.3
2.2
4.9
5.5
26
8.5
3.1
NAa
1.9
2.3
aNA denotes data not available.

S. D. Jones et al. / Bioorg. Med. Chem. Lett. 11 (2001) 733±736
735
`disulphide' pocket which is also accessible in trypsin.
The ligand makes two other hydrogen bonds with tryp-
sin, the ®rst with Gly216 through the 3-benzamide NH
and the second with Gly218 through the phenylglycine
carbonyl. The small di€erences observed in the con-
formation of the benzoylpiperidine possibly re¯ect the
more open nature of the structure of trypsin in the S4
region.
Conclusions
Using PRO_SELECT, a number of highly focused
libraries of compounds have been designed and synthe-
sised giving rapid access to a series of potent and selec-
tive inhibitors of factor Xa. Key to the potency of these
compounds is the lipophilic interaction between the
phenylglycine residue and the lipophilic `disulphide'
pocket comprising Gln192, Cys191, Cys220 and
Gly218. This is the ®rst example reported of the use of
modelling of this interaction in the design of inhibitors
for factor Xa.
Figure 1. Compound 24 as modelled in the active site of fXa (blue)
compared to the X-ray structure of 24 crystallised in trypsin (green).
enzymes). It may be seen that replacement of the
cyclohexane ring of 8 with a benzene ring 16 gave only a
modest loss of activity against factor Xa (8 0.27 mM vs
16 0.5 mM) with a signi®cant gain in selectivity against
thrombin (6.5 vs 26.0 mM).
We are currently researching additional novel series of
orally available inhibitors of factor Xa design using
PRO_SELECT.
Deletion of the terminal methylene amino group, 17,
gave a loss in activity (0.91 mM) which was recovered by
a two carbon chain extension, 19 (0.145 mM), where the
benzene ring can make a closer lipophilic interaction in
S4. (Further chain extension was detrimental to activ-
ity.) Substitution in the 4-position of the benzene ring of
compound 17 gave 20 (0.042 mM) and 21 (0.028 mM)
showing a 21- and 32-fold increase in activity against
factor Xa compared to compound 17 with no increase
in activity against trypsin.
References
1. Wiley, M. R.; Fisher, M. J. Exp. Opin. Ther. Patents 1997,
7, 1265.
2. Sinha, U. Exp. Opin. Invest. Drugs 1999, 8, 567. Walenga,
J. M.; Jeske, W. P.; Hoppensteadt, D.; Kaiser, B. Current
Opinion in Cardiovascular, Pulmonary & Renal Investigational
Drugs 1999, 1, 13. Bing-Yan Scarborough, Z. R. M, Current
Opinion in Cardiovascular, Pulmonary & Renal Investigational
Drugs 1999, 1, 63.
Libraries designed using more constrained secondary
amines, for example, piperidines and piperazines, were
particularly e€ective, yielding compounds in the low
nanomolar range albeit with some increased activity
towards trypsin and thrombin. Thus, substituted piper-
idines 22, 23 and 24, more rigid examples of the exten-
ded alkyl amine 18 and 19, showed improved activity
towards factor Xa, the larger gains seen with a carbonyl
linkage between the piperidine and the benzene ring.
3. Herron, D.; Goodson, T.; Wiley, M.; Weir, L.; Yee, Y.;
Tinsley, J.; Mendel, D.; Masters, J.; Franciskovich, J.; Sawyer,
J.; Beight, D.; Ratz, A.; Milot, G.; Hall, S.; Klimkowski, V.;
Wikel, J.; Eastwood, B.; Towner, R.; Gi€ord-Moore, D.;
Craft, T.; Smith, G. J. Med. Chem. 2000, 43, 859. Yee, Y.;
Tebbe, A.; Linebarger, J.; Beight, D.; Craft, T.; Gi€ord-
Moore, D.; Goodson, T.; Herron, D. K.; Klimkowski, V.;
Kyle, J.; Sawyer, J.; Smith, G.; Tinsley, J.; Towner, R.; Weir,
L.; Wiley, M. J. Med. Chem. 2000, 43, 873. Wiley, M.; Weir,
L.; Briggs, S.; Bryan, N.; Buben, J.; Campbell, C.; Chirgadze,
N.; Conrad, R.; Craft, T.; Ficorilli, J.; Franciskovich, J.;
Froelich, L.; Gi€ord-Moore, D.; Goodson, T.; Herron, D.;
Klimkowski, V.; Kurz, K.; Kyle, J.; Masters, J.; Ratz, A.;
Milot, G.; Shuman, R.; Smith, T.; Smith, G.; Tebbe, A.;
Tinsley, J.; Towner, R.; Wilson, A.; Yee, Y. J. Med. Chem.
2000, 43, 883.
This was not seen in the piperazine series where the
resulting amide of compound 26 would favour a non-
optimal conformation for the benzene ring. Here, the
best activity against factor Xa was found with the ben-
zene ring directly attached to the piperazine nitrogen as
in compounds 27, 28 and 29.
4. Murray, C. W.; Clark, D. E.; Auton, T. R.; Firth, M. A.;
Li, J.; Sykes, R. A.; Waszkowycz, B.; Westhead, D. R.;
Young, S. C. J. Comput.-Aided Mol. Des. 1997, 11, 193. For
additional information on Protherics software suite see
www.protherics.com.
5. Initial work in this series of compounds was carried out
using the crystal structure as described by Padmanabhan,
K. P.; Tulinsky, A.; Park, C. H.; Bode, W.; Huber, R.; Blan-
kenship, D. T.; Cardin, A. D.; Kiesel, W. J. Mol. Biol. 1993
232, 947.
Support for our modelled binding mode was provided
by crystallographic studies using the surrogate enzyme
trypsin which is structurally similar to factor Xa. Figure 1
shows compound 23 as modelled in the active site of
factor Xa (blue) as compared to the X-ray structure of 23
crystallised in trypsin (green). The benzamidine is sited
in the S1 pocket forming a bidentate interaction with
Asp189 whilst the benzene ring of the d-phenylglycine
makes the proposed lipophilic interaction in the

736
S. D. Jones et al. / Bioorg. Med. Chem. Lett. 11 (2001) 733±736
6. The following abbreviations have been used. TBTU: 2-(1H-
benzotriazole-1-yl)-1,3,3-tetramethyluronium tetra¯uoroborate.
DIPEA: diisopropylethylamine. HOBt: 1-hydroxybenzotriazole.
DIPCI: diisopropylcarbodiimide. EDC: 1-ethyl-3-(30-dimethyl-
aminopropyl)-carbodiimide HCl. HOAt: 1-hydroxy-7-azabenzo-
triazole.
milder coupling conditions were developed for solution-phase
chemistry of later compounds.
10. General method of solution-phase synthesis of library
compounds: A solution of EDC (1.1 equiv) and HOAt (1.3
equiv) in 1:1 dichloromethane/DMF was stirred at ꢀ5 ꢁC for
10 min. Boc-phenylglycine (1.0 equiv) was added and stirring
continued for 10 min before adding to the amine (1.1 equiv).
After 2 h, during which time the mixture was allowed to warm
to room temperature, the solution was partitioned between
water and ethyl acetate. The product was isolated by eva-
poration of solvent and puri®ed as necessary using ¯ash chro-
matography. (HPLC examination of products derived from
chiral amines typically showed <5% of the diastereomeric
product.) Removal of the Boc protection was carried out using
1:1 dichloromethane/tri¯uoroacetic acid and the products iso-
lated as free bases by partitioning between ethyl acetate and
sodium bicarbonate solution. The ®nal coupling with 3-ami-
dino-benzoic acid was carried out as follows: HOBt (1 mmol,
136 mg) in DMF (4 mL) was stirred in an ice bath and treated
with DIPCI (1 mmol, 160 mL). After 10 min, 3-amidino-ben-
zoic acid TFA salt (1 mmol, 278 mg) was added and stirring
continued for a further 10 min at room temperature. This
mixture was then added to the d-phenylglycine `amide'
(1 mmol) and stirred overnight. The crude product was then
dissolved in water/acetonitrile (20 mL), ®ltered and puri®ed by
preparative HPLC.
.
7. Satisfactory spectral data was obtained for all compounds.
8. Ki values were determined (minimum n=2) according to
the method of Tapparelli. Compounds were dissolved in
Me2SO, and diluted into assay bu€er: 100 mM phosphate
bu€er, 0.1% bovine serum albumin, pH 7.4. Enzymes were
supplied by Calbiochem, San Diego, CA and substrates by
Pentapharm, Basel, Switzerland. The ®nal concentrations of
enzyme and substrate were: 100 pM human fXa/125 mM Pefa-
chrome-FXA (Km 72 mM); 1 nM human thrombin/125 mM
Pefachrome-TH (Km 5.4 mM); 1 nM human trypsin/125 mM
Pefachrome-TRY (Km 47 mM) (Tapparelli, C.; Metternich, R.;
Ehrhardt, C.; Zurini, M.; Claeson, G.; Scully, M. F.; Stone, S.
R. J. Biol. Chem. 1993, 268, 4734). Increasing optical density
at 405 nM caused by substrate hydrolysis was measured on a
Labsystems Ascent Microplate reader (Labsystems, Cam-
bridge, UK). Km and Ki were calculated using SAS PROC
NLIN (SAS Institute, Cary, NC, USA, Release 6.11).
9. Initial coupling conditions used on solid phase gave com-
plete racemisation of d- and l-phenylglycines, the results
obtained are reported as racemic d,l mixtures. Accordingly