Optimization of the β-aminoester class of factor Xa inhibitors. Part 1: P4 and side-chain modifications for improved in vitro potency

Article abstract of DOI:10.1016/S0960-894X(02)00212-3

A systematic modification of the C₃ side-chain of the β-aminoester class of factor Xa inhibitors and a survey of P₄ variations is described. These changes have resulted in the identification of sub-nanomolar inhibitors with improved selectivity versus related proteases. Coagulation parameters (i.e., APTT doubling concentrations) are also improved.

Full text of DOI:10.1016/S0960-894X(02)00212-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1667–1670
Optimization of the ꢀ-Aminoester Class of Factor Xa Inhibitors.
Part 1: P4 and Side-Chain Modifications for Improved In Vitro
Potency
Mark Czekaj,* Scott I. Klein, Kevin R. Guertin,*,y Charles J. Gardner, Allison L. Zulli,
Henry W. Pauls, Alfred P. Spada, Daniel L. Cheney, Karen D. Brown, Dennis J. Colussi,
Valeria Chu, Robert J. Leadley and Christopher T. Dunwiddie
Drug Innovation and Approval, Aventis Pharmaceuticals, Route 202-206, Bridgewater, NJ 08807, USA
Received 11 January 2002; accepted 28 February 2002
Abstract—A systematic modification of the C3 side-chain of the b-aminoester class of factor Xa inhibitors and a survey of P4 var-
iations is described. These changes have resulted in the identification of sub-nanomolar inhibitors with improved selectivity versus
related proteases. Coagulation parameters (i.e., APTT doubling concentrations) are also improved. # 2002 Elsevier Science Ltd.
All rights reserved.
The inhibition of the serine protease component of the
prothrombinase complex, factor Xa (fXa), is generally
recognized as a viable strategy for the development of
novel antithrombotic agents.1 The unique position of
fXa at the convergence of the intrinsic and extrinsic
pathways, its singular role in thrombin activation and in
the amplification of the coagulation cascade combine to
make it an attractive intervention point.2 In response, a
growing number of publications have appeared describing
small molecule fXa inhibitors and several comprehensive
reviews have been published.3
methoxycarbonyl group is positioned near the catalytic
triad, however, our biochemical studies did not support
this view.4 Neither were we able to elucidate the exact
role and importance of the C3 styryl side-chain. Sub-
sequent X-ray analysis indicated that the methoxy-
carbonyl group occupies a shallow groove remote from
the catalytic triad.5,6
In a previous publication,4 we reported the discovery
and initial SAR of a novel series of b-aminoester
derived fXa inhibitors 1 and 2. Therein, we established
the requirement for the ester moiety, the optimal ste-
reochemistry at the C2 and C3 positions (R,R) and in a
preliminary way, examined the SAR of the biaryl P4
moiety.
In this communication, we describe the systematic var-
iation of the C3 b-aminoester side-chain using a com-
mon synthetic approach. In addition, the SAR of the P4
group was further elucidated. This work resulted in the
identification of nanomolar inhibitors of factor Xa with
selectivity against related trypsin-like serine proteases.7
The subsequent article expands this theme to identify
the b-aminoester FXV673 as a development candidate
for acute thrombosis.6
A modeling study of 2 in the active site of des1-45 factor
Xa predicted that the benzamidine moiety binds in the
S1 pocket while the biphenyl group fills the aromatic
pocket (S4). The model also suggested that the C1
Chemistry
*Corresponding authors. E-mail: mark.czekaj@aventis.com (M. Cze-
kaj); kevin.guertin@roche.com (K. R. Guertin).
yCurrent address: Roche Research Center, 340 Kingsland St., Nutley,
NJ 07110, USA.
The b-aminoesters 4 bearing the requisite R-configur-
ation at C3 are prepared by Arndt–Eistert homologa-
tion of the corresponding Boc-d-amino acid derivatives
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00212-3

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M. Czekaj et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1667–1670
and proceed via the intermediary diazomethyl ketones
3.8 As expected, the homologated esters 4 are obtained
with retention of configuration at the asymmetric center
(Scheme 1).9 Subsequent deprotonation of 4 and alkyl-
ation with 3-cyanobenzyl bromide affords the adducts 5
The racemic inhibitor 10 was prepared as outlined in
Scheme 3. Acylation of commercially available b-ala-
nine methyl ester hydrochloride with 4-biphenylcarbonyl
chloride followed by alkylation with 3-cyanobenzyl
bromide afforded intermediate nitrile; which is
transformed to amidine 10 using standard conditions.
1
as single diastereomers (>95% de as determined by H
NMR). The Boc protecting group is then removed
under standard conditions providing the alkylated b-
aminoesters 6, which are then coupled to appropriate 4-
biphenylcarboxylic acids. Finally, conversion of the
nitrile moiety of intermediates 7 to their corresponding
benzamidines is carried out using Pinner/ammonolysis
chemistry delivering the target b-aminoester inhibitors
8, 9a, and 9c–g in good yield.
Discussion
Compound 1 is representative of the b-aminoester class
of fXa inhibitors originally discovered in our labora-
tories.4 Further optimization of this series began with a
systematic examination of the C3 substituent R (Table 1).
Initial efforts were focused on compounds bearing a
biphenyl moiety in the putative P4 position. Compounds
were assayed against human factor Xa, thrombin and
trypsin as previously described.4 In comparing inhibi-
tors 1, 2, 8, 9a, and 10, it is evident that large hydro-
phobic substituents are not particularly favored at C3.
Complete removal (R=H) resulted in only a modest
reduction in fXa potency for the racemate 10.
The suitably protected aminomethylbiphenyl carboxylic
acid 18 required for compounds 9b and 13–16 is pre-
pared via palladium catalyzed cross coupling of 3-bro-
mobenzonitrile and ethyl-4-iodobenzoate as outlined in
Scheme 2. Subsequent reduction affords 17 whose amine
function is Boc protected. Base-catalyzed hydrolysis of
the ester provides the requisite carboxylic acid 18, for
coupling to amines 6 (Scheme 1). The nitrile moiety of
coupled product 11 is converted in a three-step process,
to amidines 12; final Boc deprotection yields the desired
inhibitors 9b and 13–16.
Concurrent work had established the desirability of
appending substituents to the distal ring of the biphenyl
group;5 Table 2 summarizes the data. The marked
Scheme 1. Reagents and conditions: (a) i-BuCOCl, THF, À30 ꢀC; (b) CH2N2, ether, 0 ꢀC; (c) PhCO2Ag, NEt3, MeOH; (d) (i) LHMDS, THF, À78
to À25 ꢀC; (ii) 3-cyanobenzyl bromide, THF, À78ꢀC to rt; (e) TFA, CH2Cl2, 0 ꢀC; (f) Ar–Ph-4–CO2H, TBTU, iPr2NEt, DMF, rt; (g) (i) H2S, pyri-
dine, NEt3; (ii) MeI, acetone; (iii) NH4OAc; (h) HCl, MeOH, rt; (ii) NH3, MeOH, reflux; (i) 18, TBTU, iPr2NEt, DMF, rt.
Scheme 2. Reagents and conditions: (a) nBuLi, THF, À78 ꢀC; ZnCl2;
Scheme 3. Reagents and conditions: (a) 4-biphenylcarbonyl chloride,
Et3N, CH2Cl2, rt; (b) LHMDS, THF, À78 ꢀC then 3-cyanobenzyl
3-bromo-benzonitrile, Pd(Ph3P)2Cl2; (b) H2, Rh on alumina, NH3,
MeOH; (c) Boc2O, DMAP, DMF, rt; (d) aqueous NaOH.
bromide, À78 ꢀC to rt; (c) (i) HCl, MeOH, rt; (ii) NH3, MeOH, reflux.

M. Czekaj et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1667–1670
1669
increase in potency observed for 9b was attributed to
the charged nature of the benzyl amine, since a number
of dibasic inhibitors make use of the S4 cation hole of
factor Xa for productive interaction.3b Although this
interpretation was supported by entries 9c and 9d, the
result for 9e clearly indicates that a positive charge is
not a prerequisite for good activity and H-bonding
interactions of the carboxamide with the S4 pocket may
be involved. It appears that a number of different fac-
tors are important for binding and a variety of strategies
can be employed to optimize interaction with the S4
pocket.
isobutyl substituent (14) was the most effective while
steric crowding closer to the C3 branching point (15)
resulted in decreased fXa inhibitory activity. Although
the C3 substituent is required for stereocontrol at C2
during synthesis of b-aminoesters (Scheme 1), the
potency advantage for larger alkyls over methyl is mar-
ginal. Given the simplicity and ready availability of the
b-aminobutyric acid precursor 4 (i.e., R=Me), com-
pounds with a methyl group at the C3 position (e.g., 9a–g)
became the focus of further efforts.
The relative activity of several inhibitors verses related
trysin-like serine proteases is shown in Table 3.
Although very good selectivities have been achieved
against thrombin (fIIa), APC and tPA, selectivity
against trypsin and plasmin is modest. In comparing
compounds 9a, 9b, and 2, the C3 methyl side-chain
derivatives are no less selective than the styryl. The
incorporation of the basic aminomethyl substitutent in
P4 appears to impart better potency as well as across the
board selectivity (cf., 9a and 9b).
Variations at C3, which employ the aminomethyl-
biphenyl P4 group, allowed us to explore the SAR
around this position in more detail (13–16 and 9b). The
Table 1. SAR of C3 side-chain
Table 2. SAR of biaryl P4 ligands
Compd
R
R0
Ki10 (nM)
fXa
fIIa
Tryp.
67
1a
21
810
954
Compd
Ar
Ki10 (nM)
2a
9.4
221
75
fXa
fIIa
Tryp.
8
11
709
9a
9b
9c
9d
9e
9f
5.3
ꢁ3250
2920
69
9a
10b
13
14
15
5.3
3250
>3950
650
69
0.9
1.0
69
95
20
230
36
2660
4.0
>1200
>4000
>4000
>4000
>4000
>2900
90
0.40
8.0
990
38
0.5
2.0
>3950
300
16
9b
0.70
0.9
2100
63
69
42
ꢁ2900
9g
69
ND
aSynthesis described previously.4
bRacemic.
Table 3. In vitro10 comparisons of selected b-aminoester fXa inhibitors
Compd
fXa Ki (nM)
Ratio: Ki enz/Ki fXa
2 X APTT (mM)
fIIa
Tryp.
APC
Plasmin
tPA
Human plasma
Rat plasma
2
9a
9b
9.4
5.3
0.9
100
610
3250
24
13
77
>2050
>3500
650
80
87
230
>965
>1600
>9650
ND
3.9
0.33
ND
5.05
1.7

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M. Czekaj et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1667–1670
Anticoagulant activity in plasma from human and rat
was measured for select compounds. Inhibitors 9a and
9b were effective anticoagulants in vitro and reflected
the trend established against free factor Xa (Table 3).
Subtle species differences were observed perhaps due to
structural variations in enzyme.
Dunwiddie, C. T.; Perrone, M. H.; Chu, V. J. Med. Chem.
1998, 41, 437.
5. Maignan, S.; Guilloteau, J.-P.; Pouzieux, S.; Choi-Sledeski,
Y. M.; Becker, M. R.; Klein, S. I.; Ewing, W. R.; Pauls, H. W.;
Spada, A. P.; Mikol, V. J. Med. Chem. 2000, 43, 3226.
6. Guertin, K. R.; Gardner, C. J.; Klein, S. I.; Zulli, A. L.;
Czekaj, M.; Gong, Y.; Spada, A. P.; Cheney, D. L.; Maignan,
S.; Guilloteau, J. P.; Brown, K.; Colussi, D.; Chu, V.; Heran,
C. L.; Morgan, S. R.; Bentley, R. G.; Rebello, S.; Leadley,
R. J.; Dunwiddie, C. T.; Pauls, H. W. Bioorg. Med. Chem.
Lett. 2002, 12, 1671.
Conclusion
A systematic study on the effects of the C3 branching sub-
stituent on the potency and selectivity of the b-aminoester
series of factor Xa inhibitors has been carried out. The
results reveal that small alkyl substituents are preferred
at this position and the C3 substituent could be com-
pletely removed with only a modest reduction in fXa
inhibitory potency. Significant increases in potency
could also be achieved by substitutions on the distal P4
phenyl ring.
7. Parts of this work were communicated in poster form:
Czekaj, M.; Klein, S. I.; Gardner, C. J.; Guertin, K. R.; Zulli,
A. L.; Pauls, H.; Spada, A. P.; Chu, V.; Brown, K.; Colussi,
D.; Leadley, R. J.; Dunwiddie, C. T.; Morgan, S. R.; Heran,
C. L.; Perrone, M. H.; Maignan, S.; Guilloteau, J. P.; Liang,
G. Abstracts of Papers, 218th National Meeting of the Amer-
ican Chemical Society, New Orleans, LA; American Chemical
Society: Washington, DC, 1999; MEDI-032.
8. Alternatively, derivative 4 (R=Me) may be readily pre-
pared on a large scale via conjugate addition of Li (R)-N-(a-
methylbenzyl)benzylamide to methyl crotonate followed by
debenzylation using Pearlman’s catalyst. See: Davies, S. G.;
Ichihara, O. Tetrahedron: Asymm. 1991, 2, 183. Final Boc
protection (BOC2O, Et3N, CH2Cl2) then provides b-aminoe-
ster 4 (R=Me) in good yield.
9. Satisfactory spectral data were obtained for all compounds.
Final products were purified by C18 reverse-phase HPLC,
eluting with a 0.1% TFA H2O/CH3CN gradient, lyophilized
and isolated as powders.
10. In vitro enzyme assays and activated partial thrombo-
plastin times (APTT) were performed as described in: Choi-
Sledeski, Y. M.; McGarry, D. G.; Green, D. M.; Mason, H. J.;
Becker, M. R.; Davis, R. S.; Ewing, W. R.; Dankulich, W. P.;
Manetta, V. E.; Morris, R. L.; Spada, A. P.; Cheney, D. L.;
Brown, K. D.; Colussi, D. J.; Chu, V.; Heran, C. L.; Morgan,
S. R.; Bentley, R. G.; Leadley, R. J.; Maignan, S.; Guilloteau,
J. P.; Dunwiddie, C. T.; Pauls, H. W. J. Med. Chem. 1999, 42,
3572.
References and Notes
1. Kaiser, B.; Hauptmann, J. Cardiovasc. Drug Rev. 1994, 12,
225.
2. Davie, E. W.; Fujikawa, K.; Kisiel, W. Biochemistry 1991,
30, 10363.
3. (a) Pauls, H. W.; Ewing, W. R. Curr. Top. Med. Chem.
2001, 1, 83. (b) Ewing, W. R.; Pauls, H. W.; Spada, A. P.
Drugs Future 1999, 24, 771. (c) Zhu, B.-Y.; Scarborough,
R. M. Curr. Opin. Cardiov. Pulmon. Ren., Investig. Drugs
1999, 1, 63. (d) Al-Obeidi, F.; Ostrem, J. A. Exp. Opin. Ther.
Pat. 1999, 9, 931. (e) Betz, A. Exp. Opin. Ther. Pat 2001, 11,
1007.
4. Klein, S. I.; Czekaj, M.; Gardner, C. J.; Guertin, K. R.;
Cheney, D. L.; Spada, A. P.; Bolton, S. A.; Brown, K.;
Colussi, D.; Heran, C. L.; Morgan, S. R.; Leadley, R. J.;