Synthesis and Evaluation of Imidazole Acetic Acid Inhibitors of Activated Thrombin-Activatable Fibrinolysis Inhibitor as Novel Antithrombotics

Article abstract of DOI:10.1021/jm034141y

Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis, and inhibitors of this enzyme have potential use in antithrombotic and thrombolytic therapy. Appropriately substituted imidazole acetic acids such as 10j were fo

Full text of DOI:10.1021/jm034141y

5294
J . Med. Chem. 2003, 46, 5294-5297
Syn th esis a n d Eva lu a tion of Im id a zole
Acetic Acid In h ibitor s of Activa ted
Th r om bin -Activa ta ble F ibr in olysis
In h ibitor a s Novel An tith r om botics
J ames C. Barrow,*,† Philippe G. Nantermet,†
Shaun R. Stauffer,† Phung L. Ngo,†
Melissa A. Steinbeiser,† Shi-Shan Mao,|
Steven S. Carroll,| Carolyn Bailey,| Dennis Colussi,|
Michelle Bosserman,| Christine Burlein,|
J acquelynn J . Cook,# Gary Sitko,# Philip R. Tiller,‡
Cynthia M. Miller-Stein,‡ Mark Rose,‡
Daniel R. McMasters, J oseph P. Vacca,† and
Harold G. Selnick†
Departments of Medicinal Chemistry, Biological Chemistry,
Pharmacology, Drug Metabolism, and Molecular Systems,
Merck Research Laboratories, P.O. Box 4,
West Point, Pennsylvania 19486
Received J uly 8, 2003
Abstr a ct: Thrombin-activatable fibrinolysis inhibitor (TAFI)
is an important regulator of fibrinolysis, and inhibitors of this
enzyme have potential use in antithrombotic and thrombolytic
therapy. Appropriately substituted imidazole acetic acids such
as 10j were found to be potent inhibitors of activated TAFI
and selective versus the related carboxypeptidases CPA, CPN,
and CPM but not CPB. Further, 10j accelerated clot lysis in
vitro and was shown to be efficacious in a primate model of
thrombosis.
F igu r e 1. Homology model of the TAFI active site.
carboxypeptidase inhibitor or “PCI”), as well as TAFIa
knock-out mice,8 has allowed several groups to demon-
strate that inhibition of TAFIa does indeed enhance
fibrinolysis in vitro and in vivo.9 Importantly, complete
inhibition of TAFIa does not impart significant increases
in bleeding,8 suggesting that TAFIa inhibitors may have
a wider therapeutic index than traditional antithrom-
botics. TAFIa inhibitors have also shown promise in
animal models when used in combination with existing
thrombolytic agents such as tPA10 or with anticoagu-
lants such as thrombin inhibitors.11
TAFIa is a zinc metalloprotease with significant
amino acid sequence homology (approximately 50%) to
the digestive carboxypeptidases A and B.12 CPA recog-
nizes and cleaves C-terminal hydrophobic residues such
as phenylalanine, while CPB, like TAFIa, prefers C-
terminal basic residues (lysine and arginine). An inter-
esting difference between TAFIa and other carboxypep-
tidases is that it is conformationally unstable (thus the
alternative nomenclature2a,3 “CPU”), losing half of its
activity in about 10 min at 37 °C.13
Greatly assisting the inhibitor design process is the
presence of high-resolution X-ray crystallographic struc-
tures of CPA and CPB with and without inhibitors
bound.14 A TAFI homology model was created15 from
these structures, and the active site is depicted in Figure
1. The C-terminal carboxylate of the substrate or
inhibitor is bound by salt bridges from two arginine
residues (Arg235 and Arg217)16 as well as by hydrogen
bonds from Asn234 (not shown) and a mobile tyrosine
(Tyr341) that covers the active site after binding. The
catalytic zinc atom (green sphere) is coordinated by two
histidines, a glutamate, and a displaceable water mol-
ecule. CPA features an isoleucine residue located at the
bottom of the S1′ specificity pocket to help recognize
hydrophobic residues, while in CPB and TAFIa this
residue is an aspartic acid (Asp348) that binds to basic
side chains. The S1 region is bordered in part by the
Thrombin-activatable fibrinolysis inhibitor (TAFI, EC
3.4.17.20)1 was recently identified2 as an important
regulator of fibrinolysis.3 It is present in plasma as a
zymogen, which is activated by limited proteolysis
primarily by thrombin/thrombomodulin.4 The activated
form, designated TAFIa, is a carboxypeptidase that
serves to remove C-terminal arginine and lysine resi-
dues from protein and peptides. Initial degradation of
fibrin by plasmin exposes new C-terminal lysine and
arginine residues on the surface of fibrin. These then
serve as binding sites for tissue plasminogen activator
(tPA) and its substrate plasminogen, thereby bringing
them in proximity and accelerating the production of
plasmin.5 The action of TAFIa serves to stabilize clots
by removing these binding sites that allow for more
rapid plasmin generation. Therefore, a TAFIa inhibitor
should stimulate endogenous fibrinolysis and thereby
exert an antithrombotic effect.
In the search for treatments of thrombotic disorders,
much effort has been expended toward development of
inhibitors of the coagulation cascade.6 Since hemostasis
results from a balance of procoagulant and fibrinolytic
forces, an alternative approach would be agents that
result in the enhancement of fibrinolysis. The avail-
ability of a natural peptide inhibitor of carboxypepti-
dases A, B, and TAFIa from potato tubers7 (potato
* To whom correspondence should be addressed. Phone: 215-652-
4780. Fax: 215-652-3971. E-mail: james_barrow@merck.com.
† Department of Medicinal Chemistry.
| Department of Biological Chemistry.
#
Department of Pharmacology.
‡ Department of Drug Metabolism.
Department of Molecular Systems.
10.1021/jm034141y CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/01/2003

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5295
Ta ble 1. In Vitro Inhibition of Carboxypeptidasesa
compd config
PCI
R
TAFIab IC50 (µM) CPBb IC50 (µM)
CPAc IC50 (µM)
CPNc IC50 (µM) CPMc IC50 (µM)
CLT50 (µM)
0.0021 ( 0.0005
>50
0.001 ( 0.0002
1.6 ( 0.4
>100
0.0009 ( 0.00016 >50
0.4(0.1
1.7(0.3
>50
0.015 ( 0.005
1
(
(
(
(
+
-
(
(
(
(
(
(
-
>50
>50
>50
2
3
>100
>50
>50
>50
0.2 ( 0.02
0.08 ( 0.003
1.8 ( 0.15
0.04 ( 0.006
0.042 ( 0.008
0.048 ( 0.002
0.069 ( 0.006
0.008 ( 0.001
0.028 ( 0.0007
0.0082 ( 0.0009 >50
0.017 ( 0.004
>50
0.013 ( 0.003
>50
3.5
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
H
H
H
0.067 ( 0.002
1.0 ( 0.2
0.035 ( 0.01
0.05 ( 0.01
0.036 ( 0.01
0.052 ( 0.009
0.006 ( 0.002
0.021 ( 0.001
0.004 ( 0.0004
>50
0.97
>10
0.5
0.42
0.65
1.06
0.15
0.74
0.12
0.056 ( 0.021
>50
>50
>50
>50
>50
>50
Me
Et
iPr
Bu
Bn
NT
>50
>50
NT
6.4 ( 0.3
NT
NT
2.2 ( 0.3
>50
>50
>50
>50
>50
>50
>50
>50
iPent 0.005 ( 0.001
iPent 0.002 ( 0.0005
>50
>50
0.0027 ( 0.0006 1.4 ( 0.4
>50
>50
a
b
Values represent the average of two runs or the mean ( standard error of the mean. Tested in ECL format; see ref 21. c Tested in
spectrophotometric format; see ref 22.
protecting groups, hydrolyzed the esters, and induced
decarboxylation. Reesterification (HCl, EtOH) afforded
key intermediate 8, which could be selectively alkylated
on the least hindered imidazole nitrogen (3:1 ratio) when
treated with NaH and an appropriate alkylating agent.
Hydrolysis of the ester afforded the products shown in
Table 1. Enantioenriched 10b, 10c, and 10j were
produced by resolution (chiral preparative HPLC) of 8
or 9 followed by hydrolysis. This hydrolysis did not cause
F igu r e 2. Carboxypeptidase inhibitors.
any decarboxylation, and racemization was minimal
(e5%) with short reaction times.
Sch em e 1. Synthesis of Inhibitors
Compounds were assayed for carboxypeptidase activ-
ity using ECL21 or spectrophotometric22 assays and are
reported in Table 1 as IC50 values. As expected, conver-
sion from the lipophilic phenyl P1 group present in 2 to
the basic aminopyridine 10a decreased CPA activity and
improved TAFIa and CPB activity presumably by
interaction with Asp348 at the bottom of the S1′ pocket
(Figure 1). Although the absolute stereochemistry of the
resolved enantiomers 10b and 10c has not been estab-
lished, activity resides predominately with the (-)-
isomer 10c. This small compound (MW ) 232) is
remarkably potent (40 nM) and selective versus other
carboxypeptidases, save CPB, and serves as a core
structure that can be decorated with other potency and
selectivity enhancing features. The imidazole N1 posi-
tion proved to be a good site for further substitution and
was explored further. As seen in 10d , 10e, and 10g,
longer alkyl chains gradually improved potency; how-
a
(a) MeOH, HCl; (b) TsCl, Et3N, CH2Cl2; (c) LHMDS, MeO2CCN,
ever, bigger chains were not as beneficial as seen by the
THF; (d) NaH, 5-bromomethyl-2-(boc-amino)pyridine, DMF, 0 °C;
modest improvements of isopropyl (10f) and benzyl
(10h ). Branching further from the imidazole with an
isopentyl chain (10i) was beneficial, and the (-)-isomer
10j proved to be a low nanomolar inhibitor of TAFIa
suitable for further study. The inhibitory activity of
these compounds can be rationalized by examining 10j
in the TAFIa homology model (Figure 3). The imidazole
acetic acid anchors the inhibitor between the zinc and
the carboxylate-binding arginines because the aminopyr-
idine reaches down into S1′, forming a salt bridge with
Asp348. The N1 imidazole nitrogen then orients the
isopentyl group toward S1.
Importantly for in vivo studies, 10a -j were inactive
versus the critical regulatory enzymes23 CPN and CPM
in part because of the low sequence identity between
TAFIa and CPN & M (<15%). Unfortunately, no differ-
ences between TAFIa and CPB were revealed in the P1
area because all the compounds prepared have similar
(e) 6 N HCl, 100 °C; (f) EtOH, HCl; (g) NaH, R-Br, DMF; (h) 6 N
HCl, 90 °C, 2 h.
â-sheet at the “front” of Figure 1.17 At the initiation of
this work, several inhibitors of carboxypeptidases A and
B such as 1 (CPA),18 2 (CPA),19 and 3 (CPB)20 (Figure
2) were known and were attractive starting points
because of their small size and high water solubility.
Inhibitor 2 was chosen for further modification be-
cause imidazole was considered to be the most attractive
zinc ligand and offered several points for exploration of
S1. We began by converting the lipophilic phenyl of CPA
inhibitor 2 into the basic aminopyridine group as shown
in Scheme 1. Direct alkylation of 5 was somewhat
problematic; however, after conversion to malonate
derivative 6 (LHMDS, MeO2CCN), alkylation proceeded
smoothly to give adduct 7 in good yield. Heating in
aqueous 6 N HCl removed the tosyl and carbamate

5296 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Letters
F igu r e 4. 10j accelerates human plasma clot lysis in a dose-
dependent manner. Human plasma (125 µL) was mixed with
thrombin (25 nM), CaCl2 (25 mM), and tPA (0.065 µg/mL) in
100 µL of buffer and varying concentrations of 10j (µM): A, 0;
B, 0.008; C, 0.016; D, 0.063; E, 0.125; F, 0.250; G,0.500; H,
1.0; I, 2.0; J , PCI (1 µM) as a control for maximal acceleration
of clot lysis.
F igu r e 3. 10j docked in the TAFI homology model.
Ta ble 2. Pharmacokinetic Profile of 10j
however, the effect is saturable and lysis faster than
50 min does not occur under these conditions even at
extremely high doses of inhibitor. The CLT50 value
represents the concentration of inhibitor that gives 50%
of the maximum acceleration of lysis, and as seen in
Table 1, activity in this functional assay tracks well with
the IC50 for enzyme inhibition.
species
% F
T1/2 (h)
Cl (mL/(min‚kg))
Vdss (L/kg)
rata
47
38
12
1.1 ( 0.2
0.6
0.8
32 ( 5.7
6.1
6.1
1.36 ( 0.06
0.28
0.26
dogb
rhesusc
a
b
2 mg/kg iv (n ) 3); 14 mg/kg po (n ) 3) noncrossover. 0.55
mg/kg iv (n ) 2); 0.9 mg/kg po (n ) 2) crossover. c 1 mg/kg iv (n )
2); 2 mg/kg po (n ) 2) crossover.
While the saturable effect on clot lysis represents a
potential safety feature, whether a potent TAFIa inhibi-
tor will display useful antithrombotic efficacy is a more
difficult question to answer. Of concern is the report that
TAFI knock-out mice responded no differently than
wild-type to several acute models of thrombosis.8 It is
difficult to accurately model human disease, including
deep-vein thrombosis. However, a model of electrolytic
vascular injury in the African green monkey (AGM)25
wherein an occlusive thrombus forms gradually over
∼90 min is suitable to evaluate the antithrombotic effect
of a TAFIa inhibitor. This model has been validated with
novel and standard-of-care clinical agents such as
antiplatelet (aspirin and glycoprotein IIb/IIIa inhibitors)
and anticoagulant (direct and indirect thrombin inhibi-
tors) compounds. In preparation for this model, 10j was
assayed for activity versus AGM TAFIa (IC50 ) 18 nM)
and in AGM plasma (CLT50 ) 35 nM). Although
intrinsic potency was slightly diminished, functional
activity was improved possibly because of decreased
protein binding in AGM plasma (40% bound) versus
human (87% bound). As was seen for PCI,25 10j did
indeed prolong time-to-occlusion (TTO) in the jugular
vein in a dose-dependent manner without a physiologi-
cally meaningful difference in template bleeding time
(Table 3). Further, there was no effect on APTT, platelet
aggregation, hemoglobin, platelet count, or hemody-
namics at the initial high dose (0.3 mg/kg bolus +
infusion of 0.1 mg/(kg‚min)). The success of 10j in this
model suggests that TAFIa inhibitors should be consid-
ered as novel, stand-alone antithrombotic agents.
activity against the two enzymes. The significance of
inhibiting CPB (thought to only be present in the gut)
remains to be determined, but the presence of carboxy-
peptidase inhibitors in potatoes and tomatoes24 suggests
that some CPB activity might be manageable.
Compound 10j was extensively counterscreened ver-
sus other coagulation enzymes, Zn metalloenzymes, and
others (e.g., thrombin, factor Xa, plasmin, uPA, tPA,
trypsin, chymotrypsin, MMP-1, NEP, and ACE) and
found to be inactive (>50 µM). The potency and specific-
ity for TAFIa make 10j an excellent tool for further
studies and prompted further evaluation in vivo.
Compound 10j was first examined for ADMET prop-
erties and has an excellent profile. Even though 10j is
very polar (log P ) -0.9), it is bioavailable in rat and
dog (Table 2), although the terminal half-life is some-
what short. Microsomal incubations show no metabo-
lites, and 80% of an iv dose was recovered unchanged
in rats. Further, 10j was found not to be an inhibitor of
the cytochrome P450 enzyme isoforms CYP3A4, 2D6,
or 2C9, despite containing an imidazole moiety.
A clot lysis assay was developed to measure functional
activity of TAFIa inhibitors in pooled human plasma.
Clot formation and lysis were triggered by the addition
of thrombin, CaCl2, and tPA and were detected by
turbidity changes. As shown in Figure 4, clots form
rapidly (increased turbidity and thus absorbance at 405
nM) and lyse after 2 h with no TAFIa inhibitor present
(curve A). Increasing concentration of inhibitor 10j
(curves B-I) decreases the time required for clot lysis;

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5297
(8) Nagashima, M.; Yin, Z.-F.; Zhao, L.; White, K.; Zhu, Y.; Lasky,
N.; Halks-Miller, M.; Broze, G. J .; Fay, W. P.; Morser, J .
Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is
compatible with murine life. J . Clin. Invest. 2002, 109, 101.
(9) See ref 3.
Ta ble 3. AGM in Vivo Data for 10j
dosea
artery
bleeding
time
(min)
(mg/kg +
mg/(kg‚min))
vein TTO
(min)
TTO
(min)
concnb
(µM)
n
(10) Nagashima, M.; Werner, M.; Wang, M.; Zhao, L.; Light, D. L.;
Pagila, R.; Morser, J .; Verhallen, P. An Inhibitor of Activated
Thrombin-Activatable Fibrinolysis Inhibitor Potentiates Tissue-
Type Plasminogen Activator-Induced Thrombolysis in a Rabbit
J ugular Vein Thrombolysis Model. Thromb. Res. 2000, 98, 333-
342.
10 saline
96 ( 6
51 ( 6
2.0 ( 0.1
0
6
8
8
8
0.3 + 0.1
216 ( 22c 85 ( 14c 2.9 ( 0.2 26.1 ( 2
0.1 + 0.03
0.05 + 0.02
0.03 + 0.01
171 ( 24c 52 ( 7
147 ( 17d 62 ( 5
128 ( 18
2.3 ( 0.2 9.6 ( 1
2.2 ( 0.1 5.7 ( 0.7
2.0 ( 0.1 2.4 ( 0.1
61 ( 10
(11) Abrahamsson, T.; Nerme, V.; Polla, M.
A pharmaceutical
a
b
iv bolus followed by continuous infusion for 300 min. Steady-
formulation containing an inhibitor of carboxypeptidase U and
a thrombin inhibitor. PCT Int. Appl. WO 0066152A1, CAN 133:
344617, 2000.
state plasma concentration measured by LC/MS. c p < 0.01 vs
vehicle. p < 0.05 vs vehicle.
d
(12) Skidgel, R. A. Structure and function of mammalian zinc
carboxypeptidases. In Zinc Metalloproteases in Health and
Disease; Hooper, N. M., Ed.; Taylor & Francis, London, 1996;
pp 241-283.
These studies demonstrate that the imidazole acetic
acid framework 10 serves as a good scaffold for con-
structing potent TAFIa inhibitors with good selectivity
versus most other enzymes. Further, 10j accelerates clot
lysis in vitro and is efficacious in a primate model of
thrombosis. Studies aimed at improving CPB selectivity
and terminal half-life are ongoing and will be reported
in due course.
(13) Boffa, M. B.; Wang, W.; Bajzar, L.; Nesheim, M. E. Plasma and
Recombinant Thrombin-Activatable Fibrinolysis Inhibitor and
Activated TAFI Compared with Respect to Glycosylation, Throm-
bin/Thrombomodulin-Dependent Activation, Thermal Stability,
and Enzymatic Properties. J . Biol. Chem. 1998, 273, 2127-2135.
(14) (a) Aviles, F. X.; Vendrell, J .; Guasch, A.; Coll, M.; Huber, R.
Advances in metallo-procarboxypeptidases. Eur. J . Biochem.
1993, 211, 381-389. (b) Vendrell, J .; Querol, E.; Aviles, F. X.
Metallocarboxypeptidases and Their Protein Inhibitors Struc-
ture, Function, and Biomedical Properties. Biochim. Biophys.
Acta 2000, 1477, 284-298. (c) Christianson, D. W.; Lipscomb,
W. N. Carboxypeptidase A. Acc. Chem. Res. 1989, 22, 62-69.
(15) The homology model of TAFIa was created from the crystal
structure of CPA (PDB code 2CTC) using MOE software
(Chemical Computing Group, Inc., Montreal). Ten intermediate
models were created, and the best-scoring model was minimized
to a root-mean-squared gradient of 1 kcal/(mol‚Å). The positions
of the Zn, Zn-binding residues (His159, Glu162, and His288),
and Asp348 were altered to those found in the crystal structure
of procarboxypeptidase B (PDB code 1NSA). These residues,
along with the residues flanking them, were allowed to relax in
the context of the entire protein using the AMBER force field
and the GB/SA solvation model as implemented in BatchMin
(Schro¨dinger, Inc., Portland, OR) with the Zn-ligand distances
constrained to those found in 1NSA.
Ack n ow led gm en t. The authors thank C. Homnick
for chiral separations and ee determinations, B. Lucas
for counterscreening data, Ping Lu for P450 inhibition
data, M. Zrada, K. Hoffman, and K. Anderson for
analytical support, and R. Woodward for technical
assistance.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and compound characterization data. This material
is available free of charge via the Internet at http://pubs.
acs.org.
(16) The numbering system is based on full-length, unactivated TAFI.
(17) S1 has historically been filled with hydrophobic, L-configured
residues. See ref 14c and the following. Abramowitz, N.; Schechter,
I.; Berger, A. On the Size of the Active Site in Proteases II.
Carboxypeptidase A. Biochem. Biophys. Res. Commun. 1967, 29,
862-867.
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Information for details.
J M034141Y