Discovery of potent & selective inhibitors of activated thrombin-activatable fibrinolysis inhibitor for the treatment of thrombosis

Article abstract of DOI:10.1021/jm0702433

Thrombin-activatable fibrinolysis inhibitor (TAFI) has emerged as a key link between the coagulation and fibrinolysis cascades and represents a promising new target for the treatment of thrombosis. A novel series of imidazolepropionic acids has been designed that exhibit high potency against activated TAFI (TAFIa) and excellent selectivity over plasma carboxypeptidase N (CPN). Structure activity relationships suggest that the imidazole moiety plays a key role in binding to the catalytic zinc of TAFIa, and this has been supported by crystallographic studies using porcine pancreatic carboxypeptidase B as a surrogate for TAFIa. The SAR program led to the identification of 21 (TAFIa Ki = 10 nM, selectivity TAFIa/CPN > 1000) as a candidate for clinical development. Compound 21 exhibited antithrombotic efficacy in a rabbit model of venous thrombosis, yet had no effect on surgical bleeding in the rabbit. In addition, 21 exhibited an excellent preclinical and clinical pharmacokinetic profile, characterized by paracellular absorption, low clearance, and a low volume of distribution, fully consistent with its physicochemical properties of low molecular weight (MW = 239) and high hydrophilicity (log D = -2.8). These data indicate 21 (UK-396,082) has potential as a novel TAFIa inhibitor for the treatment of thrombosis and other fibrin-dependent diseases in humans.

Full text of DOI:10.1021/jm0702433

J. Med. Chem. 2007, 50, 6095-6103
6095
Discovery of Potent & Selective Inhibitors of Activated Thrombin-Activatable Fibrinolysis
Inhibitor for the Treatment of Thrombosis
Mark E. Bunnage,*,† Julian Blagg,† John Steele,† Dafydd R. Owen,† Charlotte Allerton,† Andrew B. McElroy,† Duncan Miller,†
Tracy Ringer,† Ken Butcher,† Kevin Beaumont,‡ Karen Evans,‡ Andrew J. Gray,§ Stephen J. Holland,§ Neil Feeder,#
Robert S. Moore, and David G. Brown
Pfizer Global Research and DeVelopment, Sandwich Laboratories, Ramsgate Road, Kent CT13 9NJ, U.K.
ReceiVed March 4, 2007
Thrombin-activatable fibrinolysis inhibitor (TAFI) has emerged as a key link between the coagulation and
fibrinolysis cascades and represents a promising new target for the treatment of thrombosis. A novel series
of imidazolepropionic acids has been designed that exhibit high potency against activated TAFI (TAFIa)
and excellent selectivity over plasma carboxypeptidase N (CPN). Structure activity relationships suggest
that the imidazole moiety plays a key role in binding to the catalytic zinc of TAFIa, and this has been
supported by crystallographic studies using porcine pancreatic carboxypeptidase B as a surrogate for TAFIa.
The SAR program led to the identification of 21 (TAFIa Ki ) 10 nM, selectivity TAFIa/CPN > 1000) as
a candidate for clinical development. Compound 21 exhibited antithrombotic efficacy in a rabbit model of
venous thrombosis, yet had no effect on surgical bleeding in the rabbit. In addition, 21 exhibited an excellent
preclinical and clinical pharmacokinetic profile, characterized by paracellular absorption, low clearance,
and a low volume of distribution, fully consistent with its physicochemical properties of low molecular
weight (MW ) 239) and high hydrophilicity (log D ) -2.8). These data indicate 21 (UK-396,082) has
potential as a novel TAFIa inhibitor for the treatment of thrombosis and other fibrin-dependent diseases in
humans.
Introduction
of potential new antithrombotic agents that act on the coagula-
tion cascade, particularly inhibitors of thrombin and factor Xa.
Until recently, very little attention has been devoted to agents
that can enhance fibrinolysis.
Thrombotic diseases are among the more common causes of
mortality and morbidity in the developed world. A number of
agents are available for the treatment and prevention of
thrombotic disease, but these are generally anti-coagulants with
significant drawbacks, including varying degrees of adverse
hemorrhagic side-effects. Heparin and low-molecular-weight
heparins are administered parenterally and are thus generally
used as short-term treatments only. Chronic oral antithrombotic
therapy is centered on the use of warfarin, which requires regular
patient monitoring because of its significant safety liabilities,
including severe adverse bleeding events and significant drug-
drug interactions. Overall, there remains a clear medical need
for new antithrombotic agents that are safer than established
therapies and are suitable for chronic oral administration.
Activation of the coagulation cascade leads ultimately to
thrombus formation through the generation of thrombin and the
subsequent conversion of soluble fibrinogen to fibrin. Fibrin
polymerizes to form an insoluble matrix that acts as an essential
scaffold for the developing thrombus. Fibrin is regulated by the
balance between its synthesis via the coagulation cascade and
its clearance via the fibrinolysis cascade. Fibrinolysis results
from the generation of plasmin, a serine protease that breaks
down fibrin to soluble fibrin degradation products (FDPs),a
ultimately leading to dissolution of the thrombus. The pharma-
ceutical industry has devoted considerable effort to the discovery
Thrombin-activatable fibrinolysis inhibitor (TAFI)1,2 is a 60-
kDa glycoprotein zymogen found in human plasma.3 Thrombin
has been shown to convert TAFI from the inactive zymogen to
its activated form (TAFIa), a proteolytic cleavage reaction that
is significantly augmented by thrombomodulin. TAFIa (also
known as plasma carboxypeptidase B4 and carboxypeptidase
U5) is an unstable basic carboxypeptidase that has been found
to significantly inhibit endogenous fibrinolysis, and TAFI has
thus emerged as the molecular link between the coagulation
and fibrinolysis cascades.3,6 It is believed that the inhibition of
fibrinolysis by TAFIa is due to its ability to down-regulate the
formation of plasmin. Plasmin is generated from the zymogen
plasminogen by the action of tissue-type plasminogen activator
(t-PA). Both t-PA and plasminogen contain kringle domains
that recognize key C-terminal lysine residues on fibrin and aid
the formation of a ternary complex between t-PA, plasminogen,
and fibrin that is believed to be critical to the generation of
plasmin at the site of a thrombus. In addition, cleavage of fibrin
to the FDPs by plasmin results in exposure of new C-terminal
lysine residues, thereby amplifying the generation of plasmin
and further enhancing clot lysis. TAFIa is implicated in the
removal of the C-terminal lysine residues from both fibrin and
FDPs, thereby inhibiting plasmin generation and reducing
fibrinolysis. Consequently, inhibition of TAFIa has the potential
to enhance endogenous fibrinolysis and thus represents an
* To whom correspondence should be addressed. Address: Pfizer Global
Research and Development, Sandwich Laboratories, ipc 432, Ramsgate
Road, Kent CT13 9NJ, U.K. Tel: +44 1304 648974. Fax: +44 1304
651987. E-mail: mark.bunnage@pfizer.com.
a Abbreviations: FDPs, fibrin degradation products; TAFI, thrombin-
activatable fibrinolysis inhibitor; TAFIa, activated thrombin-activatable
fibrinolysis inhibitor; t-PA, tissue-type plasminogen activator; CPA, bovine
pancreatic carboxypeptidase A; ACE, angiotensin-converting enzyme;
pCPB, pancreatic carboxypeptidase B; CPN, human plasma carboxypep-
tidase N; GFR, glomerular filtration rate.
† Department of Discovery Chemistry.
‡ Department of Pharmacokinetics, Dynamics & Metabolism.
§ Department of Discovery Biology.
# Department of Pharmaceutical Sciences.
Department of Structural Biology.
10.1021/jm0702433 CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/09/2007

6096 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Bunnage et al.
Scheme 1a
Scheme 3a
a Reagents: (a) NaOAc, tert-butyl N-(2-oxoethyl)carbamate, NaCNBH3,
4 Å molecular sieves (b) i, TFA, MeOH, H2O; ii, aq. NaOH; iii, Dowex
50WX8-200.
Scheme 2a
a Reagents: (a) i, NaH, THF; ii, N-(3-bromopropyl)-N-tritylamine 37,
18-crown-6; (b) i, NaH, THF; ii, aldehyde 39 or 40; (c) Pd/C, H2, EtOH (R
) SEM) or NaBH4, CuCl, MeOH (R ) n-Pr); (d) i, aq. NaOH, dioxane; ii,
aq. HCl; iii, Dowex 50WX8-200.
concentrated aqueous hydrochloric acid, in line with the
literature precedent. For certain substrates, however, we found
that these conditions also led to the generation of an impurity
that corresponded to the regioisomeric (1,5)-substituted imida-
zole. We reasoned that this apparent loss of regiospecificity
could stem from a chloride-ion-mediated dealkylation of the
salt occurring in competition with the hydrolytic ring-opening
reaction. Subsequent ring hydrolysis of this dealkylated inter-
mediate would then provide an unsubstituted imidazole that
could react in a non-regioselective manner with the alkyl
chloride that had been generated in situ. On the basis of this
analysis, the hydrolysis step was examined using concentrated
aqueous sulfuric acid in an attempt to avoid such in situ
dealkylation (the sulfate counterion being much less nucleophilic
than chloride). Notably, under these conditions, the hydrolysis
step proceeded without complication to afford the desired
analogues as single pure regioisomers.
The racemic targets 6 and 20 were prepared as illustrated in
Scheme 3. Deprotonation of triethyl phosphonoacetate 36 with
sodium hydride followed by alkylation with the known11
bromide 37 provided intermediate 38. Wadsworth-Emmons
coupling of 38 with the appropriate imidazole carboxaldehydes
39 or 40 afforded the corresponding alkenes 41 and 42 as
mixtures of E and Z stereoisomers. The imidazolecarboxaldeydes
39 and 40 were readily prepared under standard conditions12
via alkylation of imidazole 4-carboxaldehyde9 with 2-(trimeth-
ylsilyl)ethoxymethyl chloride (SEMCl) and n-propyl bromide,
respectively. Reduction of the alkene 41 was achieved by
catalytic hydrogenation to afford the protected intermediate 43
as a racemate. In some cases, this catalytic hydrogenation
reaction was found to be somewhat capricious because of
competitive detritylation and lactam formation. Consequently,
a NaBH4/CuCl-mediated conjugate reduction strategy13 was
a Reagents: (a) carbonyldiimidazole, DMF; (b) RX (X ) Br or I),
CH3CN; (c) i, concd HCl or H2SO4; ii, Dowex 50WX8-200.
attractive new antithrombotic approach that does not rely on
the direct inhibition of coagulation. As such, an inhibitor of
TAFIa could potentially exhibit antithrombotic efficacy with
greatly reduced potential for hemorrhagic side effects compared
to an anticoagulant. In this paper we describe the discovery of
a series of novel, potent, and selective small molecule inhibitors
of TAFIa and the identification of 21 (UK-396,082) as a
candidate for clinical evaluation in the treatment of thrombosis
and related diseases.7
Chemistry
Synthesis of the thiol-containing compounds 4 and 5 (Chart
1) has been reported in the literature.8 The imidazole-based
targets described in this study were prepared as illustrated in
Schemes 1-4. Compound 8 was prepared starting from L-
histidine methyl ester 229 (Scheme 1). Reductive alkylation of
22 with tert-butyl N-(2-oxoethyl)carbamate9 gave the protected
intermediate 23. Hydrolysis of 23 using TFA in methanol/water
was followed by saponification and ion exchange chromatog-
raphy to afford 8. The (1,4)-substituted alkyl analogues of 8
(compounds 9-19) were prepared (Scheme 2) using a variation
of the regiospecific histidine alkylation methodology reported
by Jain and Cohen.10 Thus, intermediate 23 was condensed with
carbonyldiimidazole to afford the cyclic intermediate 24.
Alkylation of 24 with the appropriate alkyl bromide or iodide
then provided the salt intermediates 25-35, which were
subjected to acidic hydrolysis to afford the targets 9-19.
Initially, this latter acid hydrolysis step was conducted using

TAFIa Inhibitors for the Treatment of Thrombosis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6097
Scheme 4a
a Reagents: (a) i, LHMDS, THF; ii, aldehyde 40; (b) i, Pd black, H2,
EtOH; ii, chiral HPLC resolution; (c) i, aq. LiOH, THF; ii, aq. HCl; iii,
Dowex 50WX8-20.
Figure 1. Single-crystal X-ray structure of 49 confirming the (2S)
stereochemistry of 21.
employed to reduce 42 to the corresponding racemic intermedi-
ate 44. Global deprotection of both 43 and 44 could be achieved
via sequential saponification and acid hydrolysis to afford, after
ion exchange chromatography, the racemic targets 6 and 20.
Compound 21 was prepared as a single enantiomer using a
lactam-based synthetic strategy (Scheme 4), commencing with
the known Boc-substituted piperidinone 45.14 Compound 45 was
deprotonated with lithium hexamethyldisilazide (LHMDS), and
the resultant enolate was treated with carboxaldehyde 40. Upon
workup, the aldol addition product readily dehydrated to afford
46 as a single stereoisomer following chromatography. Catalytic
hydrogenation of 46 over palladium-black, and chiral high-
performance liquid chromatography (HPLC) resolution of the
racemic product provided the intermediate 47 as a single
enantiomer (g99% e.e.). Ring opening of the Boc-substituted
lactam moiety in 47 was effected under the mild lithium
hydroxide conditions described by Grieco and co-workers,14 and
the product was subjected to acid hydrolysis and ion exchange
chromatography to provide 21 (g95% e.e.). The (2S) absolute
stereochemistry of 21 (and thus 47) was evident from an X-ray
structural analysis of 21 cocrystallized in porcine pancreatic
carboxypeptidase B (pCPB) (Vide infra). This (2S) stereochem-
istry was subsequently confirmed through single-crystal X-ray
crystallography of the heavy-atom containing derivative 49
(Figure 1), which could be readily secured from the known15
quinidine salt 48 (Scheme 5). An efficient large-scale route to
21 has also recently been reported.15
Scheme 5a
a Reagents: (a) EDCI, HOBt, N-methyl morpholine, 4-bromobenzy-
lamine, CH2Cl2.
Figure 2. Schematic model of the binding site of CPA illustrating the
proposed binding mode of the inhibitor and peptide substrate.
Results and Discussion
At the outset of our program, there had been no reports of
potent, nonpeptidic inhibitors of TAFIa.16,17 However, although
carboxypeptidases had attracted little interest as potential drug
targets, pioneering historical work on small-molecule inhibitors
of pancreatic carboxypeptidases suggested to us that a TAFIa
inhibitor could potentially be identified by de novo design.
Indeed, the rational design of carboxypeptidase inhibitors in the
1970s proved key landmarks in modern medicinal chemistry
and the basis for the discovery of angiotensin-converting enzyme
(ACE) inhibitors. For example, in 1973, Byers and Wolfenden18
demonstrated the “byproduct analogue concept” in the design
of benzylsuccinic acid (1) as a potent, selective, and competitive
inhibitor of bovine pancreatic carboxypeptidase A (CPA). CPA
recognizes C-terminal hydrophobic amino acids, and it was
reasoned19 that 1 binds to CPA as illustrated schematically in
Figure 2, with the R-benzyl group in the S1′ specificity pocket,
the â-acid moiety binding to the catalytic zinc (mimicking the
scissile amide bond in the substrate), and the R-acid mimicking
the C-terminal peptide acid. Cushman and Ondetti20,21 extended
the binding model of 1 in CPA to the design of inhibitors for
the related zinc-dependent dipeptidase ACE, leading to the
discovery of the antihypertensive agent 2 (captopril, (1-[(2S)-
3-mercapto-2-methylpropionyl]-L-proline) (Chart 1), in which
a thiol moiety was utilized as a high affinity ligand for zinc. In
addition, Cushman and Ondetti also demonstrated19 that a thiol
moiety could be used as a zinc ligand in carboxypeptidases and
illustrated how specificity could be achieved through judicious
choice of S1′ substitution. Thus, 3 (SQ-14,603) was found to

6098 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Bunnage et al.
Chart 1
Chart 2
Figure 3. Schematic model of the binding site of TAFIa illustrating
the possible binding modes of compound 8.
the thiol moiety in 4. En route to 4, the simple amino analogue
5 was also prepared8 and found to exhibit moderate activity
against TAFIa (Ki ) 300 nM), and compound 6 was therefore
identified (Chart 2) as a key target to examine the potential for
imidazole-based inhibitors of TAFIa. In addition, while the
chemistry to access 6 was in development, compounds 7 and 8
(Chart 2) were identified as analogues of 6 that could be readily
assembled from D- or L-histidine, and these compounds were
thus prepared for examination as potential TAFIa inhibitors.
Compound 8 proved to be a significant new lead. The
compound was a moderately potent, non-thiol inhibitor of TAFIa
(Ki ) 344 nM) and, in dramatic contrast to the original thiol
leads, was inactive against CPN (Ki >10 µM). By comparison,
the antipode 7 exhibited only very weak inhibition of TAFIa
(Ki ) 17 µM). The observation that 8 was significantly more
active than 7 at first sight appeared highly surprising: if the
imidazole moiety was binding to the catalytic zinc in TAFIa as
predicted (Figure 3, Mode A), then the sense of chirality of 8
would be opposite that found in the endogenous peptide
substrate. This raised the possibility that 8 could potentially bind
to TAFIa in an unexpected orientation in which the imidazole
moiety resided in the S1′ specificity pocket and the diamine
fragment coordinated zinc (Figure 3, Mode B). Efforts to resolve
this binding mode dichotomy through direct cocrystallization
and structural elucidation of 8 complexed with TAFIa were
unsuccessful. Moreover, attempts to cocrystallize 8 with porcine
pCPB as a potential surrogate for TAFIa (Vide infra) also failed.
In the absence of structural information, we embarked on an
SAR analysis of 8 with a view towards the optimization of
potency against TAFIa.
exhibit excellent activity against CPA (Ki ) 11 nM) and high
selectivity (>10 000-fold) over porcine pancreatic carboxypep-
tidase B (porcine pCPB), a closely related carboxypeptidase with
specificity for C-terminal basic residues (i.e., Arg and Lys).
Conversely, replacing the Phe mimetic in 3 with an Arg mimetic
gave 4 (SQ-24,798), which exhibited excellent potency for
porcine pCPB (Ki ) 0.42 nM) and high selectivity over CPA
(>10 000-fold). Since porcine pCPB and TAFIa are both basic
carboxypeptidases, we decided to prepare 4 and determine its
activity against TAFIa. Gratifyingly, 4 proved a highly potent
TAFIa inhibitor (Ki ) 4 nM) and became the starting point for
our SAR program.
Although 4 exhibited excellent activity against TAFIa, further
profiling highlighted a number of significant issues with the
compound. In particular, attempts to establish the pharmaco-
kinetic properties of 4 were complicated by the instability of
the compound in plasma (due to the free thiol group undergoing
rapid disulfide conjugation reactions). In addition, we also found
4 to exhibit high potency (Ki ) 2 nM) against human plasma
carboxypeptidase N (CPN), a result consistent with that obtained
previously by Plummer and Ryan.22 Achieving high selectivity
for TAFIa over CPN was a key objective since CPN is known
to play a critical role in the clearance of anaphylatoxins such
as C5a.23,24 In light of the inverse selectivity of 4 and the
complexity associated the free thiol moiety, attention was turned
to the identification of a new template that utilized an alternative
to a thiol moiety as the ligand for zinc.
Potency optimization was sought through the incorporation
of hydrophobic binding, and the template was examined for
toleration of a methyl substituent probe. In this regard, analysis
of imidazole substitution provided promise since the methyl-
substituted imidazole analogue 9 exhibited encouraging activity
against TAFIa (Table 1). This led us to undertake a full analysis
of alkyl-substitution SAR at this position, and data for selected
analogues are collected in Table 1. Notably, alkyl chain
extension resulted in the optimization of activity against TAFIa,
but this reached a plateau between n-propyl and n-butyl, and
further increases in alkyl chain length were detrimental. In
Despite the prevalence of histidine as an endogenous ligand
for zinc in proteins, the use of an imidazole moiety as the zinc
ligand in metalloprotease inhibitors has been underexplored.
Notably, however, Kim and co-workers25 described a key study
on the potential for imidazole to ligate zinc in a series of CPA
inhibitors. In light of this work, we decided to investigate
whether an imidazole moiety could serve as a replacement for

TAFIa Inhibitors for the Treatment of Thrombosis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6099
Table 1. TAFIa Inhibition Data for Compounds 8-19
Table 2. Active Site Sequence Differences within 5.0 Å of the Shell of
21 for Pancreatic Carboxypeptidasesa
203 207 243 247 251 254 255
pCPB, boVine
bpCPB L... S... G... I...
pCPB, porcine ppCPB L... S... G... I...
S...
A...
S
S
G
D
D
D
TAFIa V... S... G... L... P...
a All other residues within 5.0 Å are identical in each species.
compound
R
Ki (nM)
8
9
H
Me
Et
n-Pr
n-Bu
n-pentyl
i-Pr
i-Bu
Bn
phenethyl
cyclobutylmethyl
3-hydroxypropyl
344
235
250
84
10
11
12
13
14
15
16
17
18
19
70
160
430
100
269
140
124
111
Chart 3
Figure 4. Structure of 21 in porcine pCPB (1.4 Å).
addition, although large lipophilic groups could be well-tolerated
(e.g., 17), groups with larger cone angles (e.g., 16) were
somewhat less favored. These data suggested to us that the
N-substituent resided in a lipophilic channel that projected out
of the active site into solvent. In this context, it was notable
that substitution of the alkyl chain with polar functionality was
also well-tolerated (e.g., 19).
The SAR in Table 1 was of particular note in the context of
the potential binding modes discussed above (Figure 3). Indeed,
if Mode B binding was in operation, such alkyl substitution
would serve to localize the imidazole sp2 lone pair onto the
opposite nitrogen to that invoked as having potential to bridge
to the specificity Asp at the base of the S1′ pocket. This analysis
suggested that the binding mode in which imidazole ligated zinc
(Mode A) remained the most likely, despite having the opposite
sense of chirality to that of the endogenous peptide. Notably,
in this binding mode, the secondary amino functionality in
compounds 8-19 would potentially be redundant.
On the basis of the above SAR analysis, examination of the
original carbon-chain series, exemplified by 6, continued to
represent a key objective. Indeed, a route to this series was
secured, and racemic 6 (Ki ) 140 nM) was found to be a
significantly more potent TAFIa inhibitor than (enantiopure)
8, further supporting the Mode A binding orientation. In
addition, the imidazole-substitution SAR established for 8 was
found to track directly into this new template, with the racemic
n-propyl analogue 20 (Chart 3) exhibiting excellent potency
against TAFIa (Ki ) 46 nM). Resolution confirmed that the
binding interaction was also highly stereospecific in this
template, and 21 was identified as the preferred enantiomer (Ki
) 10 nM, cf. ent-21, Ki > 700 nM). Enzyme kinetic studies
with 21 confirmed the compound as a competitive reversible
inhibitor of TAFIa (with a derived Ki of 40 nM by Lineweaver-
Burke analysis). Importantly, 21 also exhibited excellent
selectivity for TAFIa over CPN (>1000-fold).
Figure 5. Structure of 21 in porcine pCPB showing key interactions
and distances (Å). The catalytic zinc in this representation is displayed
as a purple sphere.
cocrystallization of 21 with porcine pCPB as a surrogate for
TAFIa. Porcine pCPB was considered a suitable TAFIa sur-
rogate due to its high degree of sequence identity (80%) in the
active site region (Table 2). Additionally, homology models of
TAFIa based on the available carboxypeptidase structures
predicted that none of the residues that differed between porcine
pCPB and TAFIa would be significantly involved in inhibitor
binding. Attempts to cocrystallize 21 with porcine pCPB were
successful, and the X-ray structure of the complex was
determined to high resolution (1.4 Å) (Figure 4). Examination
of the active site of this structure (Figure 5) confirmed our SAR
analysis that the imidazole moiety in 21 was indeed involved
in ligation of the catalytic zinc (Mode A). This crystallographic
data further highlights the utility of an imidazole moiety as a
suitable zinc ligand in zinc-metalloprotease inhibitors. The
Compound 21 was found to exhibit moderate potency against
porcine pCPB (Ki ) 206 nM), and we decided to attempt the

6100 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Bunnage et al.
remaining key interactions were also as predicted (Figure 5):26
the primary amine in 21 bridged to the key Asp255 at the base
of the S1′ specificity pocket, the acid moiety formed a salt bridge
with Arg145, mimicking the C-terminal acid of the substrate,
and the imidazole alkyl substituent was found to reside in a
lipophilic channel, which projected out of the active site into
solvent, adjacent to Tyr 199. Finally, this high-resolution
structure indicated that the absolute configuration of 21 was
(2S) (Figure 5), in line with the stereochemistry of 8. The
observation that 21 has an opposite stereochemical orientation
relative to the endogenous peptide substrate is very interesting
and may reflect the geometric constraints associated with a
highly directional imidazole-zinc interaction. Further, the ability
of TAFIa to accommodate a ligand with this stereochemistry
may also provide a rationale for the high CPN selectivity of 21
relative to the original thiol-based lead 4.
Conclusion
In this paper, we have described a novel series of inhibitors
of TAFIa that utilize imidazole as a zinc ligand. These studies
led to the identification of 21 as a potent (Ki ) 10 nM), highly
selective (sel. TAFIa/CPN > 1000) competitive inhibitor of
TAFIa. Compound 21 exhibited antithrombotic efficacy in a
rabbit model of venous thrombosis, yet had no effect on surgical
bleeding in the rabbit. In addition, 21 exhibited excellent
preclinical and clinical pharmacokinetics characterized by good
paracellular absorption, low clearance, and a low volume of
distribution, fully consistent with its physicochemical properties
of low molecular weight (MW ) 239) and high hydrophilicity
(log D ) -2.8). These data indicate 21 has potential as a novel
TAFIa inhibitor for the treatment of thrombosis and related
fibrin-dependent diseases in humans.
Experimental Section
The antithrombotic potential of TAFIa inhibition was estab-
lished through exploring the efficacy of 21 in rabbit models of
venous thrombosis and surgical bleeding. Compound 21 dem-
onstrated efficacy in the thrombosis model with a maximal
inhibition of thrombus formation of approximately 35%, yet
caused no increase in bleeding in the surgical blood loss model
when compared to vehicle-treated animals. In contrast, admin-
istration of t-PA caused a 66% inhibition in thrombus generation,
but this was associated with a dramatic (14-fold) increase in
blood loss. These data support the conclusion that TAFIa
inhibitors have potential as novel, stand-alone antithrombotic
agents with significantly enhanced safety margins over estab-
lished agents.27
(A) Biology. (1) Assay for Inhibition of TAFIa. (i) TAFI
Activation. Human TAFI (recombinant or purified) was activated
by incubating 20 µL of stock solution (360 µg/mL) with 10 µL of
human thrombin (10 NIH units/mL), 10 µL of rabbit thrombo-
modulin (30 µg/mL), 6 µL of calcium chloride (50 mM) in 50 µL
of 20 mM N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]
(HEPES) buffer containing 150 mM sodium chloride and 0.01%
TWEEN 80 (polyoxyethylene-sorbitan monooleate), pH 7.6, for
20 min at 22 °C. At the end of the incubation period, thrombin
was neutralized by the addition of 10 µL of PPACK (D-Phe-Pro-
Arg chloromethyl ketone) (100 nM). TAFIa solution was stored
on ice for 5 min and finally diluted with 175 µL of HEPES buffer.
(ii) Ki Determination (TAFIa). A number of different dilutions
of the test compound in water were made up. To 20 µL of each
dilution was added 150 µL of HEPES buffer and 10 µL of TAFIa,
which was then preincubated for 15 min at 24 °C. To each dilution
was then added 20 µL furylacryloyl-alanyl-lysine at a standard
concentration. Substrate turn over was measured by reading the
absorbance of the reaction mixture at 330 nm every 15 s for 30
min. The reaction was performed at 24 °C, and samples were mixed
for 3 s prior to each absorbance reading. A graph of percent
inhibition against test compound concentration was then plotted,
from which the IC50 value was calculated. The Ki value was then
calculated using the Cheng-Prusoff equation.
(iii) Effects of Compound 21 in Rabbit Models of Venous
Thrombosis and Surgical Bleeding. Thrombus formation in the
anesthetized rabbit (New Zealand White, Harlan, male, 2.0-3.0
kg) was induced by the insertion of a collagen-coated thread into
both jugular veins for 1 h, after which the thrombi were removed
and weighed. Intravenously administered 21 had no effect on
thrombus weight at projected free plasma concentrations of below
80 nM (n ) 3; estimated to be approximately 8 nM), but at
approximately 80 nM (n ) 6) it caused a significant 37% inhibition
in thrombus weight compared to that in vehicle-treated animals (n
) 10). Increasing the free plasma concentrations of 21 had no
greater inhibitory effect. In comparison, t-PA (10 µg/kg/min; n )
6), at a dose that caused a significant 45% depletion in circulating
fibrinogen, caused a significant 66% inhibition in thrombus
generation compared to that in vehicle-treated animals (n ) 13).
To confirm the antithrombotic activity and maximal effect of 21,
a repeat study was carried out. 21 (at approximately 800 nM; n )
6) caused a significant 36% inhibition of thrombus formation
compared to vehicle-treated animals (n ) 5). In an additional group
of anesthetized rabbits, 21, at a dose (100 µg/kg/min pretreated for
30 min and infused throughout; n ) 3) that is estimated to have
achieved a free plasma concentration of approximately 80 µM, had
no effect on surgical blood loss when compared to that of vehicle-
treated animals (n ) 4). In contrast, t-PA, infused at a dose (10
µg/kg/min; n ) 4) that caused a 66% inhibition in thrombus
generation in the efficacy study, caused an approximately 14-fold
increase in surgical bleeding (mean increase from 1 to 14 g total
blood loss). All the above in vivo studies involved terminal
The pharmacokinetics of 21 were found to be fully consistent
with its physicochemical properties of low molecular weight
(MW ) 239) and high hydrophilicity (log D ) -2.8). Following
intravenous (iv) bolus administration in a dog (0.6 mg/kg), 21
had pharmacokinetics characterized by low total plasma clear-
ance (Clp ) 3.1 mL/min/Kg), commensurate with passive renal
elimination (glomerular filtration rate (GFR) in dogs ≈ 3 mL/
min/Kg), and a very low volume of distribution (0.35 L/Kg).
Renal elimination was confirmed by urinary excretion studies
in a dog in which 96% of an iv dose of 21 was recovered in the
urine as unchanged drug. The terminal elimination half-life of
21 in a dog was 1.4 h and, on the basis of oral dog
pharmacokinetic studies (1 mg/kg), oral bioavailability was
found to be 42%. Since 21 displayed exclusively non-hepatic
clearance in the dog, this moderate bioavailability likely reflects
incomplete absorption of 21 via the paracellular route. Indeed,
21 was found to have negligible flux across Caco-2 monolayers,
confirming that transcellular absorption was unlikely.28 Since
a rat is considered a more reliable predictor of paracellular
absorption potential in man,29 the absorption of 21 in this species
was also assessed. Gratifyingly, 21 was found to exhibit ∼20%
absorption in the rat, suggesting that the compound had
excellent potential for bioavailability in humans. On the basis
of the above studies, compound 21 was selected for clinical
development.
In Phase I clinical studies, compound 21 was very well-
tolerated and exhibited an excellent pharmacokinetic profile,
fully consistent with the preclinical predictions. Human half-
life following iv dosing was found to be 4 h, characterized by
a low total clearance (Clp ) 1.5-2 mL/min/Kg), in line with
human GFR, and a low volume of distribution (0.5 L/Kg). Oral-
dosing of 21 confirmed good oral bioavailability (Foral ) 23%),
a Tmax of 4-6 h, and dose-linear pharmacokinetics. Notably,
bioavailability and Tmax were not effected by co-administration
of 21 with food.

TAFIa Inhibitors for the Treatment of Thrombosis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6101
anaesthesia of the rabbits, were conducted in compliance with
national legislation, and were ethically reviewed.
mmol) and water (28 mL) were added to a solution of the lactam
47 (3 g, 9.33 mmol) in tetrahydrofuran (THF; 45 mL), and the
reaction was stirred at room temperature for 18 h. The solution
was neutralized using aqueous hydrochloric acid (6 N), then more
acid (15 mL, 6 N) was added, and the solution was stirred at room
temperature for 4 h. The mixture was purified directly by ion
exchange chromatography (DOWEX 50WX8-200), eluting with a
solvent gradient of deionized water/0.88 ammonia (100:0 to 97:3),
(B) Chemistry. Melting points were determined on a Gallenkamp
melting point apparatus using glass capillary tubes and were
uncorrected. Unless otherwise indicated, all reactions were carried
out under a nitrogen atmosphere, using commercially available
anhydrous solvents. The term“0.88 Ammonia” refers to a com-
mercially available aqueous ammonia solution of about 0.88 specific
gravity. Thin-layer chromatography was performed on glass-backed
precoated Merck silica gel (60 F254) plates, and compounds were
visualized using UV light, 5% aqueous potassium permanganate,
or chloroplatinic acid/potassium iodide solution. Silica gel column
chromatography was carried out using 40-63 µm silica gel (Merck
silica gel 60). Ion exchange chromatography was performed using
DOWEX 50WX8-200 ion-exchange resin that had been prewashed
with deionized water. Proton NMR spectra were measured on a
Varian Inova 300, Varian Inova 400, or Varian Mercury 400
spectrometer in the solvents specified. In the NMR spectra, only
exchangeable protons that appeared distinct from the solvent peaks
are reported. Low-resolution mass spectra (LRMS) were recorded
on either a Fisons Trio 1000, using thermospray positive ionization,
or a Finnigan Navigator, using electrospray positive or negative
ionization. High-resolution mass spectra (HRMS) were recorded
on a Bruker Apex II FT-MS using electrospray positive ionization.
Combustion analyses were conducted by Exeter Analytical U.K.
Ltd., Uxbridge, Middlesex, U.K. Optical rotations were determined
at 25 °C using a Perkin-Elmer 341 polarimeter using the solvents
and concentrations specified.
1
to give the title compound as a white solid, 2.1 g, 94% yield. H
NMR (D2O, 400 MHz) δ: 0.60 (t, 3H), 1.30 (m, 2H), 1.40 (m,
2H), 1.55 (m, 2H), 2.26-2.40 (m, 2H), 2.57 (dd, 1H), 2.76 (m,
2H), 3.68 (t, 2H), 6.66 (s, 1H), 7.36 (s, 1H). HRMS: m/z 240.1699
(MH+), calcd 240.1706. [R]D ) +2.80 (c 0.14, deionized water).
[R]D ) -4.9 (c 0.16, methanol). [R]D ) -5.0 (c 0.10, ethanol),
g95% e.e. by capillary electrophoresis analysis. Anal. (C12H21N3O2‚
0.3H2O) C, H, N.
(5) Methyl (2S)-2-({2-[(tert-Butoxycarbonyl)amino]ethyl}-
amino)-3-(1H-imidazol-4-yl)propanoate (23). l-Histidine methyl
ester 22 (7.93 g, 32.8 mmol) and sodium acetate (10.75 g, 131
mmol) were added to a stirred solution of tert-butyl N-(2-oxoethyl)-
carbamate (5.22 g, 32.8 mmol) in methanol (100 mL). Molecular
sieves (4 Å) and sodium cyanoborohydride (4.12 g, 65.6mmol) were
added, and the mixture was stirred at room temperature for 17 h.
Aqueous hydrochloric acid (2N, 4 mL) was added, and the mixture
was then basified with saturated aqueous sodium carbonate solution
to pH ) 10. The mixture was filtered to remove solid, which was
washed with methanol. Methanol was removed by evaporation
under reduced pressure, and the residual aqueous solution was
extracted with ethyl acetate (2 × 300 mL). The combined organic
extracts were then dried (MgSO4), filtered, and concentrated under
reduced pressure. The resultant residue was purified by column
chromatography on silica gel, eluting with a solvent gradient of
dichloromethane/methanol (96:4 to 92:8), to afford the title
compound as a colorless oil, 8.07 g, 79% yield. 1H NMR (CDCl3,
300 MHz) δ: 1.42 (s, 9H), 2.65 (m, 1H), 2.90 (m, 2H), 3.07 (m,
1H), 3.19 (m, 1H), 3.30 (m, 1H), 3.58 (m, 1H), 3.73 (s, 3H), 5.22
(br s, 1H), 6.97 (s, 1H), 7.02 (br s, 2H), 7.91 (s, 1H). LRMS: m/z
313.1 (MH+).
(1) (2S)-2-[(2-Aminoethyl)amino]-3-(1H-imidazol-4-yl)pro-
panoic Acid (8). Trifluoroacetic acid (17 mL) was added dropwise
to a stirred solution of 23 (2.58 g, 8.2 mmol) in methanol/water
(27:14 mL). The reaction was slightly exothermic with evolution
of carbon dioxide gas. The mixture was stirred at room temperature
for 4 h, and the solvent was removed by evaporation under reduced
pressure to give a colorless oil, which was dried in vacuo overnight.
The resultant oil was treated with aqueous sodium hydroxide
solution (1 N) until the solution was at pH ) 8. A further portion
of aqueous sodium hydroxide solution (1 N, 30 mL) was added,
and the solution was stirred at room temperature for 72 h. The
solution was concentrated under reduced pressure to 10 mL and
purified by ion exchange chromatography (DOWEX 50WX8-200),
eluting with a solvent gradient of deionized water/0.88 ammonia
solution (100:0 to 97:3). The solvent was removed by evaporation
under reduced pressure to afford a yellow oil, which was dissolved
in deionized water (15 mL) and freeze-dried overnight to afford a
foam. This material was dissolved in deionized water/methanol (95:
5) and further purified using MCI gel (55 g) chromatography,
eluting with a solvent gradient of deionized water/methanol (95:5)
(6) Methyl (7S)-6-{2-[(tert-Butoxycarbonyl)amino]ethyl}-5-
oxo-5,6,7,8-tetrahydroimidazo[1,5-c]pyrimidine-7-carboxylate
(24). Carbonyldiimidazole (156 mg, 0.959 mmol) was added to a
stirred solution of 23 (300 mg, 0.959 mmol) in N,N-dimethylfor-
mamide (5 mL), and the mixture was heated at 60-70 °C for 17
h. The solvent was removed by evaporation under reduced pressure,
and the residue was dissolved in saturated aqueous sodium hydrogen
carbonate solution and extracted with dichloromethane. The
combined organic extracts were dried (MgSO4), filtered, and then
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel, eluting with dichloromethane/
methanol (95:5), to afford the title compound as a colorless oil,
1
to afford the title compound, 1.13 g, 69% yield. H NMR (D2O,
300 MHz) δ: 2.61-2.87 (m, 4H), 2.92 (m, 2H), 3.25 (t, 1H), 6.81
(s, 1H), 7.59 (s, 1H). LRMS: m/z 199.2 (MH+). [R]D ) +1.74 (c
0.12, deionized water). Anal. (C8H14N4O2‚1.3H2O) C, H, N.
1
210 mg, 67% yield. H NMR (D2O, 300 MHz) δ: 1.40 (s, 9H),
3.20-3.60 (m, 5H), 3.70 (s, 3H), 4.08 (m, 1H), 4.33 (m, 1H), 4.82
(br m, 1H), 6.80 (s, 1H), 8.13 (s, 1H). LRMS: m/z 339 (MH+).
[R]D ) +39.2 (c 0.12, dichloromethane).
(2) General Procedure for Hydrolysis of Salts 25-35 (Method
B). Either concentrated hydrochloric acid (6 N, 5 mL) or concen-
trated sulfuric acid (2-4 N, 5 mL) was added to a stirred solution
of the requisite salt 25-35 (0.32 mmol) in water (5 mL), and the
mixture was heated at reflux for 17 h. The mixture was allowed to
cool to room temperature, and the solvent was removed by
evaporation under reduced pressure. The residue was purified by
ion exchange chromatography (DOWEX 50WX8-200), eluting with
deionized water/0.88 ammonia (97:3).
(3) (2S)-2-[(2-Aminoethyl)amino]-3-(1-methyl-1H-imidazol-
4-yl)propanoic Acid (9). Prepared from the product of 25 according
to Method B (using concd H2SO4) and isolated in 52% yield: 1H
NMR (D2O, 400 MHz) δ: 2.60-2.80 (m, 4H), 2.90 (m, 2H), 3.24
(t, 1H), 3.53 (s, 3H), 6.77 (s, 1H), 7.45 (s, 1H). LRMS: m/z 211
(M+). [R]D ) -5.8 (c 0.12, methanol). Anal. (C9H16N4O2‚1.45H2O)
C, H, N.
(7) General Procedure for Alkylation of 24 (Method A). The
appropriate alkyl bromide or iodide (2-5 equiv) was added to a
stirred solution of 24 (200 mg, 0.592 mmol) in acetonitrile (5 mL),
and the mixture was heated at reflux for 17 h under a nitrogen
atmosphere. The mixture was allowed to cool to room temperature,
and the solvent was removed by evaporation under reduced pressure.
The residue was purified by column chromatography on silica gel,
eluting with dichloromethane/methanol (90:10), to afford the
product salts 25-35.
(8) (7S)-6-{2-[(tert-Butoxycarbonyl)amino]ethyl}-7-(methoxy-
carbonyl)-2-methyl-5-oxo-5,6,7,8-tetrahydroimidazo[1,5-c]pyri-
midin-2-ium Iodide (25). Prepared according to Method A using
methyl iodide (3 equiv) and isolated in 75% yield. 1H NMR
(DMSO, 400 MHz) δ: 1.32 (s, 9H), 3.09-3.23 (m, 3H), 3.45 (m,
2H), 3.64 (s, 3H), 3.86 (s, 3H), 3.95 (m, 1H), 4.86 (m, 1H), 6.91
(s, 1H), 7.60 (s, 1H), 9.80 (s, 1H). [R]D ) +36.2 (c 0.11, methanol).
Anal. (C16H15N4O5I‚1.25H2O) C, H, N.
(4) (2S)-5-Amino-2-[(1-n-propyl-1H-imidazol-4-yl)methyl]-
pentanoic Acid (21). Lithium hydroxide monohydrate (1.1 g, 28

6102 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Bunnage et al.
(9) tert-Butyl (3E)-2-Oxo-3-[(1-n-propyl-1H-imidazol-4-yl)-
methylene]-1-piperidinecarboxylate (46). A solution of lithium
bis(trimethylsilyl)amide in THF (43.5 mL, 1 M, 43.5 mmol) was
added dropwise to a cooled (-78 °C) solution of tert-butyl 2-oxo-
1-piperidinecarboxylate 4513 (8.7 g, 43.5 mmol) in THF (120 mL),
and, once the addition was complete, the solution was allowed to
warm to 0 °C and then stir for an hour. The solution was recooled
to -78 °C, a solution of the aldehyde 40 (4 g, 28.9 mmol) in THF
(40 mL) was added, and the reaction was then allowed to warm to
room temperature. The reaction mixture was stirred for 18 h and
then partitioned between water and ethyl acetate. The phases were
separated, and the organic phase was dried (MgSO4), filtered, and
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel, eluting with dichloromethane/
complex with 21 were grown by vapor diffusion (2 µL protein and
2 µL reservoir buffer) using the hanging drop method. The reservoir
was 100 mM NaKPO4, pH 6.8; 200 mM zinc acetate; 10%
PEG8000. Precipitate, which formed upon mixing of the reservoir
solution, was removed by centrifugation. Crystals grew over a
period of several weeks and were of tetragonal form, space group
P41212, with cell dimensions a ) 82.248, b ) 82.248, and c )
93.761 Å and R ) â ) γ ) 90.0. Crystals were cryoprotected
using 25% glycerol, and data were collected to 2.0 Å at 100 °K on
a Rigaku RU-H3R and R-axis IV image plate. Additional data, on
the same sample, was collected to 1.4 Å at the ESRF on station
ID14 EH2. All data were processed using DENZO.30 The data were
combined for refinement with an overall R merge of 9% for all
data and completeness of 98.5% in the final resolution shell between
1.45 and 1.4 Å.
1
methanol (95:5), to give the title compound, 4 g, 43% yield. H
NMR (CDCl3, 300 MHz) δ: 0.89 (t, 3H), 1.50 (s, 9H), 1.78 (m,
2H), 1.86 (m, 2H), 3.00 (m, 2H), 3.70 (t, 2H), 3.85 (t, 2H), 7.07
(s, 1H), 7.46 (s, 1H), 7.62 (s, 1H). LRMS: m/z 320.3 (MH+).
(10) tert-Butyl (3S)-2-Oxo-3-[(1-propyl-1H-imidazol-4-yl)-
methyl]-1-piperidinecarboxylate (47). A mixture of 46 (6.6 g, 20.6
mmol) and palladium black (700 mg) in ethanol (120 mL) was
hydrogenated at 4 atm and 60 °C for 18 h. The cooled mixture
was filtered through Arbocel, washing through with ethyl acetate,
and the filtrate concentrated under reduced pressure. The crude
product was purified by column chromatography on silica gel,
eluting with dichloromethane/methanol (97:3), to afford the race-
mate of the title compounds as a yellow oil, 4.3 g, 65% yield. This
racemic compound was resolved by HPLC using a Chiralcel OG
250 column (20 mm) and hexane/isopropanol (70:30) as eluant, at
a rate of 10 mL/minute, to give the title compound (1.56 g, retention
time 15.23 min, 98.9% e.e.) and its antipode (1.56 g, retention time
10.10 min, 99.5% e.e.). Data for 47: 1H NMR (CDCl3, 300 MHz)
δ: 0.92 (t, 3H), 1.54 (s, 9H), 1.80 (m, 4H), 2.00 (m, 2H), 2.63-
2.85 (m, 2H), 3.19 (m, 1H), 3.58 (m, 1H), 3.90-3.98 (m, 3H),
6.72 (s, 1H), 7.37 (s, 1H). LRMS: m/z 322.3 (MH+). [R]D ) +27.7
(c 0.22, dichloromethane).
All data reduction and subsequent refinement were performed
using the CCP4 program suite.31 The structure was solved by
molecular replacement methods using the program AMORE.32 The
deposited coordinates pdb1nsa.ent were used to build a search model
where the pro-domain residues were removed. Analysis clearly
identified the presence of the inhibitor 21 in the active site of the
protein. Additionally the high-resolution structure indicated se-
quence anomalies with the deposited sequence in the PDB entry
1NSA. A patent publication US 5672496-A reveals a corrected
sequence (PID gi2731308) for porcine pancreatic carboxypeptidase
B, with which this high-resolution structure is in agreement (as
used in more recent PDB structural deposition 1Z5R). Sequence
changes and modeling of the inhibitor were performed using the
graphics program QUANTA.
The structure was refined using REFMAC31 with the inclusion
of two zinc atoms and 305 solvents molecules with a final R factor
33
of 19% and an Rfree of 21%. The Ramachandran conformational
parameters generated by PROCHECK34 indicate that the structure
is in accordance with expected values.
Coordinates for this complex have been deposited with the
Protein Data Bank (PDB code2JEW).
(D) X-ray Data Collection and Structure Solution for
Compound 49 (Figure 1). X-ray diffraction data were recorded at
room temperature using a Bruker AXS SMART-APEX CCD area-
detector diffractometer (Mo KR radiation). Intensities were inte-
grated35 from several series of exposures. Each exposure covered
0.3° in ω, with an exposure time of 60 s, and the total data set was
more than a sphere. Data were corrected for absorption using the
multiscans method.36 The crystal structure was solved by direct
methods using SHELXS-9737 and refined by the method of least-
squares using SHELXL-97.38
There were two molecules in the asymmetric unit, each with
the same chirality (ORTEP drawings of both molecules are included
in Supporting Information, Figure 6). Refinement of the structure
proceeded normally, although the propyl groups of both molecules
were found to be disordered, and each were modeled over two
overlapping conformations. The occupancy of the two conforma-
tions was determined from a refinement job where the thermal
parameters of the atoms in question were fixed at some reasonable
value while their occupancies were allowed to refine; atoms in the
same group were assigned a common occupancy, while occupancies
of the two conformations were constrained to sum to 1. In this
way, the two propyl orientations in both molecules were found to
have a 0.5 occupancy. The occupancies were fixed at these values
for all subsequent jobs. Mild restraints were placed on these
disordered groups to give them a reasonable geometry.
All heavy atoms were refined with anisotropic temperature
factors, except those in the disordered propyl groups which were
refined with isotropic temperature factors. All hydrogen atoms were
placed in calculated positions and allowed to refine with a riding
model and isotropic temperature factors.
(11) (2S-N-(4-Bromobenzyl)-5-[(tert-butoxycarbonyl)amino]-
2-[(1-n-propyl-1H-imidazol-4-yl)methyl]pentanamide (49). 1-(3-
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
(2.9 g, 1.5mmol) was added to a stirred mixture of known quinidine
salt 4815 (1 g, 1.5 mmol), 1-hydroxybenzotriazole hydrate (HOBt)
(0.205 g, 1.5 mmol), N-methyl morpholine (0.33 mL, 3 mmol) and
4-bromo benzylamine (2.8 g, 1.5 mmol) in dry CH2Cl2 at 0-5 °C.
The mixture was allowed to warm to room temperature and was
stirred for 72 h. The resultant solution was then concentrated under
reduced pressure, and the residue was suspended in saturated sodium
carbonate solution (20 mL) and then extracted with ethyl acetate
(3 × 20 mL). The ethyl acetate solution was then dried (Na2SO4),
filtered, and concentrated under reduced pressure. The crude product
was purified by column chromatography on silica gel, using an
elution gradient of dichloromethane/methanol (100:0 to 97.5:2.5),
to give the title compound as a glass following suspension and
evaporation from ether, 0.6 g, 80% yield. The glass was recrystal-
lized from ethyl acetate/hexane to give colorless crystals, which
were filtered and dried and confirmed to be g99.5% e.e. by chiral
HPLC (chiral column OG250 × 4.6 mm; retention time 5.92). 1H
NMR (CDCl3, 400 MHz) δ: 0.90 (t, 3H), 1.43 (s, 9H), 1.49-1.53
(m, 3H), 1.69-1.78 (m, 3H), 2.66-2.85 (m, 2H), 3.76 (t, 2H), 4.17
(dd, 1H), 4.40 (dd, 1H), 4.65 (s, 1H), 6.58 (s, 1H), 6.73 (s, 1H),
7.00 (d, 2H), 7.28 (s, 1H), 7.37 (d, 2H). LRMS: m/z 506.5, 508.5
(MH+). [R]D ) -10.7 (c 0.103, methanol). MPt 102-103 °C. Anal.
(C24H35BrN4O3) C, H, N. Recrystallization of a small (50 mg)
sample of the crystalline product from ethyl acetate/hexane gave
single crystals of 49 suitable for X-ray crystallography studies
(confirming absolute configuration as S).
(C) Crystallization, X-ray Data Collection and Structure
Solution for Compound 21 in Porcine pCPB (Figure 4). Porcine
pCPB was purchased from Sigma (C6158) in the activated form.
The protein was dissolved in Milli-Q water at a concentration of
10 mg/mL. Inhibitor complex was preformed with a 60-fold molar
excess of inhibitor dissolved in Milli-Q water. Crystals of the
Pertinent crystal, data collection, and structure refinement details
are summarized in the Supporting Information (Table 3). Atomic
coordinates, bond lengths, bond angles, torsion angles, and tem-
perature factors are also included in the Supporting Information
(Tables 4-8).

TAFIa Inhibitors for the Treatment of Thrombosis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6103
(17) Over recent years, a number of nonpeptidic TAFIa inhibitors have
been disclosed, including a related series of imidazole-based inhibitors
from Merck; see: (a) Barrow, J. C.; Nantermet, P. G.; Stauffer, S.
R.; Ngo, P. L.; Steinbeiser, M. A.; Mao, S.-S.; Carroll, S. S.; Bailey,
C.; Colussi, D.; Bosserman, M.; Burlein, C.; Cook, J. J.; Sitko, G.;
Tiller, P. R.; Miller-Stein, C. M.; Rose, M.; McMasters, D. R.; Vacca,
J. P.; Selnick, H. G. Synthesis and evaluation of imidazole acetic
acid inhibitors of activated thrombin-activatable fibrinolysis inhibitor
as novel antithrombotics. J. Med. Chem. 2003, 46, 5294-5297. (b)
Nantermet, P. G.; Barrow, J. C.; Lindsley, S. R.; Young, M.; Mao,
S.-S.; Carroll, S.; Bailey, C.; Bosserman, M.; Colussi, D.; McMasters,
D. R.; Vacca, J. P.; Selnick, H. G. Imidazole acetic acid TAFIa
inhibitors: SAR studies centered around the basic P1′ group, Bioorg.
Med. Chem. Lett. 2004, 14, 2141-2145.
The absolute configuration for this structure was determined by
the method of Flack.39 Both molecules in the asymmetric unit were
found to have an ‘S’ configuration at the chiral center, for a final
refined Flack parameter of -0.016 (7).
Acknowledgment. We would like to thank Ed Hawkeswood,
Keith Holmes, Christine Bell, and Audrey Sequira for their
contributions to the biology studies, and Edel Evrard, Keith
Reeves, Graham Smith, Morgan Sproates, Ross Strang, and Nick
Summerhill for assistance with the chemistry program. Thanks
also to Gorkhn Sharma-Singh and Sylvie Gelebart for perform-
ing chiral HPLC resolutions and analytical studies. Valerie
Horne is also thanked for her contributions to the structural
studies.
(18) Byers, L. D. Wolfenden, R. Binding of the by-product analog
benzylsuccinic acid by carboxypeptidase A. Biochemistry 1973, 12,
2070-2078.
(19) Ondetti, M. A.; Condon, M. E.; Reid, J.; Sabo, E. F.; Cheung, H. S.;
Cushman, D. W. Design of potent and specific inhibitors of
carboxypeptidases A and B. Biochemistry 1979, 18, 1427-1430.
(20) Ondetti, M. A.; Rubin, B.; Cushman, D. W. Design of specific
inhibitors of angiotensin-converting enzyme: New class of orally
active antihypertensive agents. Science 1977, 196, 441-444.
(21) Cushman, D. W.; Ondetti, M. A. Inhibitors of angiotensin-converting
enzyme. Prog. Med. Chem. 1980, 17, 41-104.
Supporting Information Available: ORTEP plots and crystal
structure data for compound 49. Experimental details for compounds
6, 10-20, 26-35, and 38-44. Analytical data for target compounds
6 and 8-21 and selected intermediates. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(22) Plummer, T. H., Jr.; Ryan, T. J. A potent mercapto bi-product analog
inhibitor for human carboxypeptidase N. Biochem. Biophys. Res.
Commun. 1981, 98, 448-454.
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