d-Phenylglycinol-derived non-covalent factor Xa inhibitors: Effect of non-peptidic S4 linkage elements on affinity and anticoagulant activity

Article abstract of DOI:10.1016/j.bmcl.2007.08.051

Analogs to a series of d-phenylglycinamide-derived factor Xa inhibitors were discovered. It was found that the S4 amide linkage can be replaced with an ether linkage to reduce the peptide character of the molecules and that this substitution leads to an i

Full text of DOI:10.1016/j.bmcl.2007.08.051

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5801–5805
D-Phenylglycinol-derived non-covalent factor Xa inhibitors:
Effect of non-peptidic S4 linkage elements on affinity
and anticoagulant activity
Valentine J. Klimkowski,a Brian M. Watson,a Michael R. Wiley,a John Liebeschuetz,b,ꢀ
Jeffry B. Franciskovich,a Jothirajah Marimuthu,a Jolie A. Bastian,a Daniel J. Sall,a
Jeffrey K. Smallwood,a Nikolay Y. Chirgadze,a Gerald F. Smith,a Ronald S. Foster,a
Trelia Craft,a Philip Sipes,a Marcia Chastaina and Scott M. Sheehana,*
aLilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN 46285, USA
bTularik Ltd, Amgen, Aleutian House, Tytherington Business Park, Macclesfield, Cheshire, UK
Received 2 July 2007; revised 22 August 2007; accepted 23 August 2007
Available online 28 August 2007
Abstract—Analogs to a series of D-phenylglycinamide-derived factor Xa inhibitors were discovered. It was found that the S4 amide
linkage can be replaced with an ether linkage to reduce the peptide character of the molecules and that this substitution leads to an
increase in binding affinity that is not predicted based on modeling. Inhibitors which incorporate ether, amino, or alkyl S4 linkage
motifs exhibit similar levels of binding affinity and also demonstrate potent in vitro functional activity, however, binding affinity in
this series is strongly dependent on the nature of the S1 binding element.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The need for a new oral therapy for the prevention of
thrombosis has driven more than a decade of research
into inhibition of the trypsin-like serine proteases in-
volved in the coagulation cascade.1 Reports of phase
III clinical trial efficacy of ximelegatran, an oral direct
thrombin inhibitor, for prevention of venous thrombo-
embolism highlight the potential value of this overall ap-
proach even though ximelegatran was ultimately
withdrawn due to adverse liver effects.2 Amplification
during the coagulation cascade suggests that a strategy
of inhibition of coagulation factors that operate earlier
in the cascade sequence may result in lower concentra-
tions of inhibitor necessary to achieve efficacy and
may thus lead to therapeutic agents with potential for
fewer non-target related side effects. Factor Xa, a serine
protease centrally located at the junction of the intrinsic
and extrinsic pathways, forms a prothrombinase com-
plex which is directly responsible for the activation of
prothrombin to thrombin. It has been reported that
one molecule of factor Xa in the prothrombinase com-
plex can activate 138 molecules of thrombin per min-
ute.3 As such, inhibition of factor Xa has been
specifically targeted by many industrial and academic
laboratories.4
As part of our research program aimed at the discovery
of selective non-covalent factor Xa inhibitors, we evalu-
ated a series of D-phenylglycinamide-derived inhibitors
having a secondary amide linkage connecting the S4
binding element to the core of the scaffold represented
by structure 1 (Fig. 1).5
O
N
H
N
H
N
Keywords: Factor Xa; Serine protease inhibitors; Non-covalent inhib-
itors; Coagulation cascade.
N
H
*
Corresponding author. Tel.: +1 317 277 8633; e-mail: sheehan_scott
@lilly.com
O
1
Cl
Figure 1. A D-phenylglycinamide-derived inhibitor of factor Xa.
ꢀ
Present address: Cambridge Crystallographic Database Centre, 12
Union Road, Cambridge CB2 1EZ, UK.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.051

5802
V. J. Klimkowski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5801–5805
Compounds were discovered that inform the model with
respect to the role of the proposed amide hydrogen bond
between the S4 amide linker and the protein backbone.
The corresponding ether analog 2 can be prepared via
alkylation of D-phenyl glycinol 3 with N-boc-4-metha-
nesulfonyloxymethylpiperidine 4 to provide 5 in 52%
yield. Amide coupling with 3-chloroindole-6-carboxyl-
ate 6 provides intermediate 7 which can be carried on
without purification. Standard deprotection of the tert-
butoxycarbonyl protecting group followed by reductive
amination with acetone provides 2 in straightforward
fashion (Scheme 1).7
D189
S195
Q192
S1
S3
G218
H57
W215
Surprisingly, 2 demonstrates an improvement in binding
affinity for factor Xa relative to compound 1, and has an
apparent association constant (Kass) of 1700 · 106 L/mol
compared with a Kass of 460 · 106 L/mol for 1.8 Re-
moval of the potential hydrogen bonding interaction
predicted by modeling does not diminish activity and
an X-ray co-crystal structure of 2 bound in the active
site of factor Xa helps to explain the key interactions
(Fig. 3).
Y99
S4
F174
Figure 2. Computationally derived binding model of factor Xa
inhibitor 1 having a secondary amide S4 linker.
Analysis of the factor Xa co-crystal structure with 2
shows the inhibitor binding in a very similar fashion
as the model for 1 (Fig. 2). Without an amide S4 linkage,
no hydrogen bond exists between the inhibitor and
backbone amide nitrogen of Gly218. However, in its
place, a crystallographically resolved water molecule ap-
pears to have the capability of creating a hydrogen
bonding network between the ether linkage and the pro-
tein backbone amide nitrogen of Gly213. Structurally it
is unclear why 2 is more active than 1, but this result
suggests that there may be differences in the binding
mode of 1 that the model has not adequately captured.
It is possible that there is more strain energy associated
with the binding mode derived for 1 compared to that
observed in the crystal structure for 2. Another explana-
tion may be that the unassociated 1 may exist in solution
in a peptide-like C7 conformation having a well-defined
intramolecular hydrogen bond. This internal interaction
would need to be broken before 1 could adopt the
Computational modeling based on X-ray co-crystal
structures of related molecules can be used to develop
a binding mode for structure 1 in the factor Xa active
site (Fig. 2).6 Using this approach, the chloroindolyl
moiety fits into the S1 pocket such that the indole nitro-
gen lies between a carboxyl oxygen of Asp189 and the
carbonyl oxygen of Gly218 to which it forms a hydrogen
bond. This arrangement positions the chlorine near the
face of Tyr228 which is located in the back of the pock-
et. Projecting out of the S1 pocket is the phenyl glycine-
derived central portion of the inhibitor which places the
edge of the phenyl ring within proximity to interact with
the disulfide bridge formed by Cys191 and Cys220. The
cationic box of the S4 pocket, defined by Tyr99, Trp215,
and Phe174, is then filled by the N-isopropyl piperidinyl
tail section of the inhibitor. Interestingly, the amide car-
bonyl which serves as the link to the S4 binder is mod-
eled to be within hydrogen bonding distance of Gly218.
Boc
N
MsO
Boc
N
4
O
OH
H2N
a
H2N
5
3
O
H
N
OH
b
Cl
6
Boc
O
N
O
N
c,d
H
H
O
O
N
N
N
N
H
H
2
Cl
7
Cl
Scheme 1. General synthesis of S4-ether linked factor Xa inhibitors illustrated by the preparation of 2. Reagents and condition: (a) 4, NaH,
THF:CH3CN (5:1), 65 ꢁC, 52%; (b) 6, EDCl, HOAt, DMF; (c) 4 N HCl in dioxane; (d) acetone, NaCNBH3, HOAc, MeOH, 54% (three steps).

V. J. Klimkowski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5801–5805
5803
(Table 1). Additional analogs containing an amino func-
tionality in the linker can be prepared in straightforward
fashion from D-phenyl glycine to provide additional
information. Secondary amine containing 12 also dem-
onstrates similar affinity to 2 and 11, but the N-methyl-
ated analog 13 is much less active in this assay.
D189
Q192
S195
G218
Additional inhibitors containing S4 ether linkers show
that the 3-chloroindolyl S1 binder and the centrally lo-
cated phenyl ring are key to maintaining strong affinity
for the enzyme (Table 2). Interestingly, ortho-chloro
substitution on the central phenyl ring does not provide
an increase in affinity as has been reported in related sys-
tems containing a secondary amide S4 linkage.5 Isopro-
pyl and n-butyl replacements for the phenyl ring afford
inhibitors 19 and 20 with markedly decreased affinity
for factor Xa further highlighting the importance of
the phenyl’s interaction with the disulfide bridge.
Bridging
Water
H57
W215
Y99
F174
S4 Water
Variably sized substituents on the piperidine S4 binder
demonstrate interesting SAR trends (Table 3). While
Figure 3. X-ray co-crystal structure of inhibitor 2 bound in factor Xa
˚
(1.9 A resolution).
Table 1. Effect of linker on binding to factor Xa
bound conformation modeled in Figure. 2. No such
hydrogen bond is possible for 2, so there may be less
of an energy penalty to assuming the observed binding
mode (Fig. 3).
O
N
H
Y
N
N
X
H
The preparation of analog 11, which replaces the ether
in 2 with an alkyl linkage, is shown below (Scheme 2).
Readily available 8 can be subjected to sequential oxime
formation, reduction, and boc-protection, to provide 9
in good overall yield. Reduction of the pyridine ring
using platinum oxide followed by reductive amination
with acetone and sodium cyanoborohydride provides
intermediate amine 10. Boc-deprotection of 10 followed
by coupling with 6 provides the racemic material 11 in
20% yield (unoptimized over four steps).7
Cl
Kass (106 L/mol)
a
Compound
X
Y
2
CH2
CH2
CH2
CH2
C@O
O
1700
700b
1200
60
11
12
13
1
CH2
NH
NMe
NH
460
a Kass is the apparent association constant reported in units of 106 L/mol
and is approximately equal to 1/Ki. Kass values were obtained by the
method of Smith et al.9 and are the result of a single experiment
(performed in triplicate at each of four to eight inhibitor
concentrations).10
The all carbon linker, which is not able to engage in a
bridged hydrogen bond with water, demonstrates bind-
ing affinity similar to that found for ether linked 2
b Compound 11 was tested as a racemate.11
a,
b
,
c
N
N
Boc
N
O
H
9
8
d, e
f, g
O
N
N
H
Boc
N
N
N
H
H
10
11
Cl
Scheme 2. Preparation of carbon-linked analog of 2. Reagents and condition: (a) HONH2 Æ HCl, NaOAc, EtOH, 80 ꢁC, 89%; (b) Pd/C, H2 (60 psi),
87%; (c) Boc2O, Et3N, THF, 82%; (d) PtO2, AcOH, H2 (60 psi);(e) acetone, NaCNBH3, AcOH, MeOH; (f) 4 N HCl, dioxane; (g) 6, EDCl, HOAt,
DMF, 20% (four steps).

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V. J. Klimkowski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5801–5805
Table 2. Effect of substitution of R1 and R2 on binding to factor Xa
Xa sequence differs between species in a loop that resides
near the S4 pocket.12
O
R2
N
In conclusion, replacement of the secondary amide
linkage in specific D-phenylglycinamide derived factor
Xa inhibitors with an alkyl ether linkage provides
inhibitors with improved measured binding affinity
for factor Xa. X-ray co-crystallography confirms the
binding hypothesis around this series and can serve
to refine future computational docking models for this
series.
O
R1
R1
N
H
Compound
R2
Kass (106 L/mol)
2
3-Cl-6-indole
3-Me-6-indole
6-Indole
Ph
Ph
Ph
Ph
1700
160
35
14
15
16
17
18
19
20
4-MeO-Ph
3
3-Cl-6-indole
3-Cl-6-indole
3-Cl-6-indole
3-Cl-6-indole
2-Cl-Ph
2-F-Ph
i-Pr
850a
650a
14
Acknowledgments
n-Bu
41
a Compound was tested as a racemate.11
S.M.S. thanks John M. Schaus for helpful discussions
regarding the research. The authors thank Timothy I.
Richardson and Thomas E. Jackson for helpful com-
ments on the manuscript.
Table 3. Effect of piperidine substitution on binding to factor Xa
R
O
Cl
O
N
References and notes
H
N
N
H
1. (a) Agnelli, G.; Sonaglia, F. Haematologica 2002, 87, 757–
770; (b) Zhu, B.-Y.; Scarborough, R. M. Ann. Rep. Med.
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1998, 82, 11L–14L; (d) Wiley, M. R.; Fisher, M. J. Expert
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2. Schulman, S.; Wahlander, K.; Lundstrom, T.; Clason, S.
B.; Eriksson, M. D. N. Eng. J. Med. 2003, 18, 349, 1713.
3. Zhu, B.-Y.; Scarborough, R. M. Curr. Opin. Cardiovasc.
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C.; Liebeschuetz, J. W.; Jones, S. D.; Murray, C. W.;
Franciskovich, J. B.; Engel, D. B.; Weber, W. W.;
Marimuthu, J.; Kyle, J. A.; Smallwood, J. K.; Farmen,
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Webber, W. W. III; Marimuthu, J.; Kyle, J.; Sheehan, S.;
Watson, B.; Smallwood, J. K.; Foster, R.; Craft, T. J.;
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6. The coordinate set for the protein structure used for
the molecular modeling was the human factor Xa
X-ray structure of Brandstetter et al.14 (RCSB PDB
file 1fax.pdb). The initial structure of inhibitor 1 was a
model built starting from the related phenylglycine
ligand contained in the trypsin X-ray structure
1eb2.pdb.15 Using residue identity, the trypsin structure
complex was superimposed onto the factor Xa protein
structure, and then the ligand coordinates of the
former were extracted and modified. The initial struc-
Cl
Compound
R
Kass (106 L/mol)
17
21
22
23
i-Pr
Et
850a
150a
65a
Me
4-Pyridyl
1300a
a Compound was tested as a racemate.11
cyclic alkyl groups maintain useful affinity for the en-
zyme, isopropyl provides the greatest amount of bind-
ing affinity. It is interesting that the S4 pocket can
accommodate a 4-pyridyl substituent and maintain
high affinity. Modeling suggests that the pyridine ring
may be able to interact with or displace a water mole-
cule that is located at the bottom of the S4 pocket
(Fig. 3).
The potential of phenylglycinol-derived inhibitors as
therapeutic agents is further demonstrated by the
in vitro anticoagulant profile of 2 (Table 4). Anticoagu-
lant activity as measured by prolongation of prothrom-
bin time shows that both ether and amide linkages
provide inhibitors with similar activity when measured
in human plasma. Interestingly, 2 provides improved
anticoagulant activity in rat and dog plasma relative
to its amide comparator 1. The reason for the marked
difference in preclinical species anticoagulant profile is
unclear, however, it has been reported that the factor
Table 4. Effect of linker on species in vitro anticoagulant profile13
ture of
structure. Molecular modeling then proceeded in
similar fashion as detailed previously in Ref. 9a. All
molecular modeling was performed with QUANTA
and CHARMm, version 98 (Accelrys Inc., San Diego,
CA 92121).
1 was then inserted into the factor Xa
a
Compound
Human
1.5XPT
(lM)
Rat
1.5XPT
(lM)
Dog
1.5XPT
(lM)
Monkey
1.5XPT
(lM)
2
1
0.4
0.5
0.6
2.6
0.8
3.2
0.4
0.6

V. J. Klimkowski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5801–5805
5805
7. For detailed experimental procedures: Liebeschuetz, J. W.;
Sheehan, S. M.; Watson, B. M. PCT Int. Appl.
WO2004060872.
experiment have been determined to have a minimum
significant ratio of 2.13; (b) Bland, J. M.; Altman, D. G.
Lancet 1986, 1, 307.
8. Compound 2 has >1000· selectivity with respect to the
following enzymes: Thrombin, Plasmin, Bovine Trypsin,
Urokinase, aPC, Kallikrein, and tPA.
11. We have found that the majority of Xa binding affinity
results from one enantiomer (unpublished results), and
test inhibitors as racemates when it is synthetically
expedient to do so.
12. Rai, R.; Sprengeler, P. A.; Elrod, K. C.; Young, W. B.
Curr. Med. Chem. 2001, 8, 101.
13. The minimum significant ratio for this assay has been
determined to be 2.26.
14. Brandstetter, H.; Kuhne, A.; Bode, W.; Huber, R.; von
der Saal, W.; Wirthersohn, K.; Engh, R. A. J. Biol. Chem.
1996, 271, 29988.
15. Liebeschuetz, J. W.; Jones, S. D.; Morgan, P. J.; Murray,
C. W.; Rimmer, A. D.; Roscoe, J. M. E.; Waszkowycs, B.;
Welsh, P. M.; Wylie, W. A.; Young, S. C.; Martin, H.;
Mahler, J.; Brady, L.; Wilkinson, K. J. Med. Chem. 2002,
45, 1221.
9. (a) Herron, D. K.; Goodson, T., Jr.; Wiley, M. R.; Weir, L.
C.; Kyle, J. A.; Yee, Y. K.; Tebbe, A. L.; Tinsley, J. M.;
Mendel, D.; Masters, J. J.; Franciskovich, J. B.; Sawyer, J.
S.; Beight, D. W.; Ratz, A. M.; Milot, G.; Hall, S. E.;
Klimkowski, V. J.; Wikel, J. H.; Eastwood, B. J.; Towner,
R. D.; Gifford-Moore, D. S.; Craft, T. J.; Smith, G. F.
J. Med. Chem. 2000, 43, 859; (b) Smith, G. F.; Gifford-Moore,
D. S.; Craft, T. J.; Chirgadze, N. K.; Ruterbories, K. J.;
Lindstrom, T. D.; Satterwhite, J. H. In New Anticoagulants
for the Cardiovascular Patient; Piffare, R., Ed.; Hanley &
Belfus Publishers: Philadelphia, 1997; pp 265–300.
10. (a) Utilizing the statistical method of Bland and Altman,
apparent Kass data generated in this fashion from a single