Non-amidine-containing 1,2-dibenzamidobenzene inhibitors of human factor Xa with potent anticoagulant and antithrombotic activity [2]

Full text of DOI:10.1021/jm990625b

J . Med. Chem. 2000, 43, 2087-2092
2087
Non -Am id in e-Con ta in in g
1,2-Diben za m id oben zen e In h ibitor s of
Hu m a n F a ctor Xa w ith P oten t
An ticoa gu la n t a n d An tith r om botic
Activity
J ohn J . Masters,* J effry B. Franciskovich,
J ennifer M. Tinsley, Charles Campbell,
J ack B. Campbell, Trelia J . Craft, Larry L. Froelich,
Donetta S. Gifford-Moore, Lynne A. Hay,
David K. Herron, Valentine J . Klimkowski,
Kenneth D. Kurz, J ames T. Metz, Andrew M. Ratz,
Robert T. Shuman, Gerald F. Smith, Tommy Smith,
Richard D. Towner, Michael R. Wiley,
Alex Wilson, and Ying K. Yee
Lilly Research Laboratories,
A Division of Eli Lilly and Company,
Lilly Corporate Center, Indianapolis, Indiana 46285
Received December 23, 1999
In tr od u ction . Human factor Xa (fXa) is a trypsin-
like serine protease which serves a critical role in blood
coagulation events.1 Competitive inhibitors of fXa have
demonstrated potent anticoagulant activity in vitro and
antithrombotic efficacy in preclinical models in vivo.2
Within the past decade, research efforts to identify small
molecule inhibitors of this coagulation enzyme as novel
therapies for thromboembolic disorders have increased.3
We recently disclosed a series of 1,2-dibenzamidoben-
zene derivatives that are selective, sub-micromolar
inhibitors of fXa (1, Figure 1).4 In contrast to most
inhibitors of fXa reported which contain amidine func-
tionality,5 these compounds use selected hydrophobic
groups for occupying both the S1 and S4 binding
domains of the enzyme.6 In the case of 1, the 4-meth-
oxyphenyl and 4-tert-butylphenyl groups reside in the
S1-site and “aryl binding” S4-site of fXa, respectively.7
Incorporation of an S1 binding amidine into these
constructs yielded 2.8 Amidine 2 was significantly more
potent than 1 in both binding affinity for fXa and
prothrombin time (PT) activity (Table 1).9 Moreover, the
potency observed with 2 in vitro translated into potent
antithrombotic efficacy in vivo. There is precedent that
amidine-containing molecules, in general, display un-
desired pharmacokinetic properties after oral adminis-
tration.10 We describe here our efforts to identify fXa
inhibitors that have potency similar to that of amidine
2 but lack this undesirable S1 binding functionality
altogether.
F igu r e 1. 1,2-Benzamidobenzene inhibitors of human factor
Xa 1-4.
Ta ble 1. Human fXa Binding and Prothrombin Time Activity
of 1,2-Benzamidobenzene Inhibitors 1-4
a
compd
Kass (× 106 L/mol)
PTb (µM)
1
2
3
4
6.6
470
9.2
>50
0.83
9.5
2.3
9.7
a
Kass represents the apparent association constant as measured
by the methods of Smith, G.F.; et al.8
PT is defined as the
b
concentration of compound (µM) required to double the time to
clot formation in the prothrombin time assay as described in ref
7.
potent anticoagulant activity.11 This was further ex-
tended to the 1-(4-pyridyl)piperidine-containing deriva-
tive 4.12 Since the 1-(4-pyridyl)piperidine compound 4
(pKa ∼ 9) is significantly more basic than the 4-phe-
nylpyridine-containing 3 (pKa ∼ 5), compound 4 should
carry a positive charge at plasma pH, further reducing
undesirable interactions with plasma proteins. Consis-
tent with this hypothesis, both 3 and 4 had similar PT
activities even though 3 had ∼4-fold higher binding
affinity for fXa. Derivatives of 4 with enhanced binding
affinity for fXa would be expected to be even more potent
anticoagulants.
In this communication, we describe the SAR of
altering the linkage of the 1-(4-pyridyl)piperidine to the
central ring of 4 in order to optimize the placement of
this group in the S4-site of fXa. Toward this end, we
prepared various urethane and urea derivatives and
measured their fXa binding affinity. Most of these
compounds were found to have significantly higher
binding affinity for fXa than 4. Further, these data
provided opportunity to examine the translation of fXa
binding affinity into anticoagulant potency in this
structurally related series. The most potent anticoagu-
Previous reported structure-activity relationship
(SAR) studies of 1 revealed that 4-arylbenzamides are
acceptable S4 binding elements.4a Derivative 3 was of
particular note since it had a binding affinity for fXa
similar to that of 1; however, 3 was at least 5-fold more
potent in PT assays (Table 1). This suggested the
increased hydrophilicity of 3 significantly reduced in-
teractions with plasma proteins, therefore allowing the
fXa binding affinity to more effectively translate into
* To whom correspondence should be addressed. Phone: 317-277-
7969. Fax: 317-433-0715. E-mail: jjm@lilly.com.
10.1021/jm990625b CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/12/2000

2088 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11
Sch em e 1. Preparation of 3 and 4a
Communications to the Editor
a
Reagents: (a) 4-(4-pyridyl)benzoic acid, thionyl chloride; then, 5, pyridine, CHCl3; (b) 4-chloropyridine hydrochloride, triethylamine,
EtOH, 120 °C; (c) oxalyl chloride; then, 5, pyridine, CHCl3.
Sch em e 2. Preparation of 17-25a
a
Reagents: (a) phthalimide, triphenylphosphine, diethylazodicarboxylate, THF; then, hydrazine monohydrate, EtOH, 70 °C; (b) 7-11,
CHCl3/THF; (c) H2, 10% Pd/C, EtOH; (d) p-anisoyl chloride, pyridine, CHCl3; (e) aryl acid chloride, pyridine, CHCl3.
lant identified in these studies was examined in a rabbit
arterio-venous shunt model of thrombosis in vivo, and
these results are compared to amidine 2 of the related
1,2-dibenzamidobenzene series. Finally, with represen-
tative compounds, selectivity for inhibition of fXa versus
other proteases is also discussed.
Ch em istr y. The synthesis of 3 and 4 is outlined in
Scheme 1.13 Treatment of N1-(4-methoxybenzoyl)-1,2-
benzenediamine (5)4a with 4-(4-pyridyl)benzoyl chlo-
ride14 afforded 3. The amide lead 4 was obtained from
5 and the acid chloride of 6.12
Synthesis of the urethane and urea derivatives 17-
25 is described in Scheme 2.13 Preparation of 17-21
required the 1-(4-pyridyl)piperidine-containing inter-
mediates 7-11. Using a procedure similar to that
described for the synthesis of acid 6, 4-hydroxypiperi-
dine and 4-chloropyridine afforded 7. Conversion of this
alcohol to amine 8 was accomplished using a two-step
sequence involving a Mitsunobu reaction with phthal-
imide followed by a hydrazine deprotection sequence.
Primary carbinol 9 was obtained by lithium aluminum
hydride mediated reduction of the corresponding ethyl
ester of acid 6. Amine 10 was prepared using the same
procedure described for 8. Finally, alcohol 11 was
obtained using the same protocol developed for con-
structs 6 and 7. Reaction of 7-11 with 2-nitrophenyl
isocyanate followed by reduction of the nitro functional-
ity afforded the anilines 12-16. The final compounds
17-21 were completed by treatment of 12-16 with
4-anisoyl chloride, respectively. The ureas 22-25 were
prepared by reaction of aniline 15 with the correspond-
ing aryl acid chlorides.
Resu lts a n d Discu ssion . Illustrated in Table 2 are
the results obtained with 17-25 in fXa binding affinity
and PT assays in vitro. The constructs 17-21 have two
characteristics worth mentioning. First, they maintain

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11 2089
Ta ble 2. Human fXa Binding and Prothrombin Time Activity
of 21-29
a
compd X )
Y )
Kass (× 106 L/mol) PTb (µM)
17
18
19
20
21
22
23
24
25
OMe NH(CO)O
OMe NH(CO)NH
OMe NH(CO)OCH2
OMe NH(CO)NHCH2
OMe NH(CO)O(CH2)2
17
12
20
62
2.6
2.4
1.9
0.96
2.5
5.9
100
15
0.04
>15
H
Cl
NH(CO)NHCH2
NH(CO)NHCH2
6.2
0.58
2.6
OEt NH(CO)NHCH2
tBu NH(CO)NHCH2
a
Kass represents the apparent association constant as measured
by the methods of Smith, G.F.; et al.8
PT is defined as the
b
concentration of compound (µM) required to double the time to
clot formation in the prothrombin time assay as described in ref
7. Kass and PT values are the average of at least three separate
experiments with a standard deviation of less than 20%.
the 1,2-diamide-like functionality of lead compounds
1-4. Second, these derivatives sequentially extend the
1-(4-pyridyl)piperidine group from the central core of
4, allowing for alternate placement of this group in the
S4-site of fXa. Published SAR studies in the 1,2-
dibenzamidobenzene series revealed the benefits of the
central amides to high fXa binding affinity.4 Further-
more, molecular modeling studies of this series consis-
tently indicated H-bonding interactions between the
carbonyl of the amide linking the S4 binding group and
Gly218 of fXa.7 Modeling studies with 4 also displayed
these particular interactions (Figure 2).15 These studies
with 4 further revealed that the 1-(4-pyridyl)piperidinyl
functionality could reside “deeper” in the S4 binding site
of fXa, which could further enhance binding affinity to
the enzyme.
All of the derivatives 17-21 had higher binding
affinity for fXa relative to lead compound 4. Extension
of the amide linkage of 4 by one atom, as in urethane
17 and urea 18, afforded ∼7-fold and ∼5-fold more
potent analogues, respectively. Even more active com-
pounds resulted when the amide linker was extended
by two atoms. In this case, the urea 20 was ∼27-fold
more potent than 4 and had >3-fold greater binding
affinity for fXa than carbamate 19. Extension of the
amide linkage of 4 by three atoms, as in 21, provided
comparable fXa binding activity. Overall, these data
suggest that various positioning of the 4-(1-piperidinyl)-
pyridine group within the S4-site of fXa is tolerated and
high binding affinity for fXa can result for each.16
Having identified several linkers that provided en-
hanced fXa binding affinity, we focus on SAR of the
4-methoxyphenyl group. Assuming that this substituent
serves as the S1 binding element, SAR trends consistent
with those observed earlier in the 1,2-dibenzamidoben-
zene series were expected.4a,8 The phenyl analogue 22
indicates the contribution of the 4-methoxy substituent
to fXa binding affinity. The ∼10-fold decrease in activity
of 22 compared to 20 is consistent with previously
reported results. Similarly, 4-chlorophenyl also serves
as a suitable S1 binding functionality as exemplified by
compound 23. Compounds 24 and 25 confirm the
F igu r e 2. Proposed energy-minimized binding model of the
compound 4 complexed with the active site of factor Xa. The
orientation presented is looking directly into the S1 and S4
binding pockets. Intermolecular hydrogen bonds are shown as
dashed lines, and the molecular surface for the S4-S4′ region
is highlighted by white dots.
sensitivity of the S1-site to the size of the 4-substituent
of the phenyl ring. Overall, these results strongly
suggest that the 4-substituted benzamide in this series
resides in the S1-site of fXa.
The range of the fXa binding affinity of compounds
17-25 enables an assessment of how this activity
translates into anticoagulant potency for this series of
fXa inhibitors. Among all these structurally related
compounds, a clear relationship between higher binding
affinity for fXa and an increase in anticoagulant potency
was observed. Even though this correlation exists within
this series of inhibitors, it does not hold when directly
comparing these with other inhibitors of fXa that
display different physiochemical properties.17 For ex-
ample, compound 23 was ∼2-fold more potent than
amidine 2 as an anticoagulant, even though it was 4.5-
fold less active in fXa binding assays. Additionally,
compound 20 was ∼7-fold less potent in fXa binding
assays, but had similar activity in the PT assay. These
data illustrate the importance of considering both the
fXa binding affinity and anticoagulant activity in vitro
when attempting to compare the relative antithrombotic
potential of fXa inhibitors that arise from distinct
structural classes.
The antithrombotic activity for 23, a non-amidine
inhibitor of fXa with comparable activity to potent
amidines in vitro, was evaluated in vivo. To choose an
appropriate animal model, we measured the PT activity
of 23 in plasma from various animal species.18 The
similarity between the rabbit and human plasma dose-
response curves prompted us to evaluate 23 in a rabbit
arterio-venous shunt model. As illustrated in Figure 3,
23 displayed dose-dependent antithrombotic efficacy
after intravenous administration. An ED50 was obtained
at a dose of 1.8 mg/kg/h. Initial evaluation of these data

2090 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11
Communications to the Editor
Ta ble 3. Binding Affinity of Compounds 20 and 23 for Various
Enzymes
Kass for 20a
Kass for 23a
enzyme
(× 106 L/mol)
(× 106 L/mol)
fXa
62
100
IIa
fXIa
fXIIa
activated protein C
plasmin
t-PA
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.06
<0.01
<0.01
<0.01
0.09
0.14
<0.01
<0.01
urokinase
trypsin (bovine)
a
Kass represents the apparent association constant as measured
by the methods of Smith, G.F.; et al.8
Con clu sion s
Variation of the linkage which connects the central
ring of 4 to the S4 binding 1-(4-pyridyl)piperidine group
produced several potent nonamidine inhibitors of hu-
man fXa. Similar to amidine-containing inhibitors of
fXa, this series shows efficient translation of enzyme
binding affinity into anticoagulant potency. The most
potent compound, 23, was 43-fold more active than lead
molecule 4 in fXa binding assays and ∼10-fold more
potent in prothrombin clotting time assays. Dose-
dependent antithrombotic efficacy was observed with 23
when evaluated intravenously in a rabbit arterio-venous
shunt model in vivo. In this thrombosis model, similar
PT ex vivo and antithrombotic effects in vivo were noted
with 23 and the potent amidine 2 of the 1,2-dibenza-
midobenzene series. Compound 23 was highly selective
for binding to fXa. These potent and selective nonami-
dine containing inhibitors of fXa not only represent
novel anticoagulants, they are also useful tools to
further our preclinical understanding of competitive fXa
inhibition.
F igu r e 3. Antithrombotic dose response for compounds 2 and
23 in an AV shunt model of thrombosis in the rabbit during
continuous infusion of compound for 30 min. Thrombosis was
induced during the final 15 min of the infusion period.
Ackn owledgm en t. J.J.M. thanks Matthew J. Fisher,
Thomas E. J ackson, and J effrey A. Dodge for helpful
discussions and review of the manuscript.
F igu r e 4. Relationship between prothrombin time ex vivo and
antithrombotic response in the AV shunt model of thrombosis
in the rabbit. Extension of prothrombin time reflects the
average of samples collected from each animal before and after
the period of thrombosis.
Su p p or tin g In for m a tion Ava ila ble: Mean and standard
deviations of measurements in vitro, elemental analyses, and
1H NMR and MS spectral data for 3, 4, and 17-25. This
material is available free of charge via the Internet at http://
pubs.acs.org.
suggests amidine 2 was significantly more antithrom-
botic in this model, requiring only a dose of 0.06 mg/
kg/mL to achieve an ED50 response. In contrast, inspec-
tion of the relationship between PT ex vivo and clot size
suggests they have similar antithrombotic activity in
vivo (Figure 4). Since 2 and 23 have similar dose-
response curves in rabbit PT measurements, similar
antithrombotic effects result at comparable plasma
concentrations of these two inhibitors.18 Individual
pharmacokinetic properties of 2 and 23 likely explain
potency differences observed in dose.17
Since many trypsin-like proteases have essential
physiological functions,19 an important development
consideration for fXa inhibitors is the ability of the
molecule to bind selectively. Toward this end, both 20
and 23 were evaluated in several enzyme binding assays
(Table 3). Both compounds displayed >500-fold selectiv-
ity for fXa versus several coagulation (IIa, fXIa, fXIIa,
and activated protein C) and fibrinolytic enzymes (plas-
min, t-PA, urokinase). Further, these compounds had
no significant binding to bovine trypsin.
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Franciskovich, J . B.; Sawyer, J . S.; Beight, D. W.; Ratz, A. M.;
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2092 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11
Communications to the Editor
(13) All new compounds gave spectral (1H NMR, MS) and elemental
analysis data consistent with their structure. See Supporting
Information for details.
(14) Ali, N. M.; McKillop, A.; Mitchell, M. B.; Rebelo, R. A.; Wallbank,
P. J . Palladium-Catalysed Cross-Coupling Reactions of Arylbo-
ronic Acids with π-Deficient Heteroaryl Chlorides. Tetrahedron
1992, 48 (37), 8117-8126.
containing 4-aminopyridine fXa inhibitor FX-2212a: Kamata,
K.; Kawamoto, H.; Honma, T.; Iwama, T.; Kim, S. H. Structural
Basis for Chemical Inhibition of Human Blood Coagulation
Factor Xa. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6630-6635.
(16) In addition to interactions with the “aryl binding” region of fXa,
this basic functionality could further interact with the S4′-site
of fXa:3a
a series of exposed carbonyl groups consisting of
(15) The molecular modeling was carried out using the QUANTA/
CHARMm software (Molecular Simulations Inc., San Diego, CA)
and the human des(1-45) fXa X-ray crystal structure (1HCG):
Padmanabhan, K.; Padmanabhan, K. P.; Tulinsky, A.; Park, C.
H.; Bode, W.; Huber, R.; Blankenship, D. T.; Cardin, A. D.; Kisiel,
W. Structure of Human Des(1-45) Factor Xa at 2.2 Å Resolution.
J . Mol. Biol. 1993, 232, 947-966. The approach used for 4 was
the same as previously described.4,7 The charges assigned to each
ligand were smoothed to give a total sum of 1.0. The initial
position of the water complexed to the ligand in the S4′ region
was taken from the X-ray crystallographic water 518 of the
1HCG fXa coordinate set. Key binding interactions of 4 with fXa
include: (a) hydrophobic interactions of methoxyphenyl group
with S1-site, (b) H-bonding of the 1,2-bisamide with Gly218, (c)
the pyridine ring of 4 with the “aryl binding” site of S4 composed
of residues Tyr99, Phe174, and Trp215, and (d) H-bonding with
protonated pyridine of 4 and a buried water molecule in S4-
S4′-site which is further hydrogen bonded to carbonyl oxygens
of Thr98 and Ile175 and the hydroxyl of Thr98 in fXa. Although
positioning of water within the context of a molecular force field
is uncertain, a similar interaction was observed in the X-ray
structure determination of des[1-44]fXa with the amidine-
residues Glu97, Thr98, Ile175, and Thr176 of fXa (Figure 2).
(17) The pKa values for compounds 2, 4, and 23 were 11.0 ( 0.2 (3.9
( 0.1-acid), 8.7 ( 0.02, and 8.9 ( 0.1, respectively. The LogD
values measured at pH ) 7.4 for compounds 2, 4, and 23 were
-1.0, 1.1, and 1.9, respectively.
(18) The importance of using species anticoagulant selectivity profiles
to guide the selection of the appropriate animal model was
revealed in our earlier reported studies.8 The dose response of
prothrombin clotting times in various species plasma for 2 and
23 was determined as described in ref 8. The PT concentrations
of 2 and 23 in rabbit plasma were 0.84 ( 0.05 µM and 0.78 (
0.03 µM, respectively. The PT for 23 was >20 µM in both rat
and dog plasma.
(19) For a discussion on the importance of enzyme binding selectivity,
see: Wiley, M. R.; Chirgadze, N. Y.; Clawson, D. K.; Gifford-
Moore, D. S.; J ones, N. D.; Olkowski, J . L.; Schacht, A. L.; Weir,
L. C.; Smith, G. F. Serine Protease Selectivity of the Thrombin
Inhibitor D-Phe-Pro-Agmatine and its Homologues. Bioorg. Med.
Chem. Lett. 1995, 5 (23), 2835-2840 and references therein.
J M990625B