Polymer-Assisted Solution-Phase (PASP) parallel synthesis of an α-ketothiazole library as tissue factor VIIa inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00398-6

A solution-phase synthesis of an α-ketothiazole library of the general form D-Phe-L-AA-L-Arg-α-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification protocols, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multi-step synthesis affords the desired α-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of the tissue factor (TF) VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10e bound to the TF/VIIa complex was obtained that explains the observed selectivity. The α-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue factor VIIa, with some analogues demonstrating selectivity versus thrombin.

Full text of DOI:10.1016/S0960-894X(03)00398-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2363–2367
Polymer-Assisted Solution-Phase (PASP) Parallel Synthesis of an
ꢀ-Ketothiazole Library as Tissue Factor VIIa Inhibitors
Michael S. South,a,* Thomas A. Dice,a Thomas J. Girard,b Rhonda M. Lachance,b
Anna M. Stevens,c Roderick A. Stegeman,c William C. Stallings,c
Ravi G. Kurumbailc and John J. Parlowa
aDepartment of Medicinal and Combinatorial Chemistry, Pharmacia Corporation,
800 North Lindbergh Boulevard, St.Louis, MO 63167, USA
bDepartment of Cardiovascular and Metabolic Disease, Pharmacia Corporation,
800 North Lindbergh Boulevard, St.Louis, MO 63167, USA
cStructure and Computational Chemistry, Pharmacia Corporation, 700 Chesterfield Village Pkwy.,
St.Louis, MO 63198, USA
Received 31 January 2003; revised 15 April 2003; accepted 16 April 2003
Abstract—A solution-phase synthesis of an a-ketothiazole library of the general form d-Phe-l-AA-l-Arg-a-ketothiazole is descri-
bed. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase pur-
ification protocols, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multi-step synthesis
affords the desired a-ketothiazole products in excellent purities and yields. A variety of l-amino acid inputs were used to probe the
S2 pocket of the tissue factor (TF) VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound
10e bound to the TF/VIIa complex was obtained that explains the observed selectivity. The a-ketothiazoles were found to be
potent, reversible-covalent inhibitors of tissue factor VIIa, with some analogues demonstrating selectivity versus thrombin.
# 2003 Elsevier Science Ltd. All rights reserved.
Cardiovascular disease is the most common cause of
mortality in the western world. The disease is charac-
terized by the acute coronary syndromes unstable
angina and myocardial infarction, which often lead to
sudden death.1 This inappropriate thrombus formation
is initiated via the extrinsic coagulation cascade by a
plaque rupture, which exposes tissue factor to the serine
protease VIIa in circulating blood forming the TF/VIIa
complex. This complex then further activates the serine
proteases IX to IXa and X to Xa, which in turn activate
prothrombin to thrombin and fibrinogen to fibrin.
Upon combination with activated platelets, a fibrin clot
may result in a life threatening thrombus formation.2
There is evidence from our own laboratories as well as
others that selective inhibition of the TF/VIIa complex
may provide effective anticoagulation while lessening
the risk of bleeding side effects when compared to other
antithrombotic mechanisms such as IIb/IIIa antagonists
and inhibitors of Xa and thrombin.3
An approach to the design of serine protease inhibitors
has been the replacement of the scissile amide bond by
an electron-deficient carbonyl group.4 A series of alde-
hydes,5 a-fluoroketones,6 a-keto esters/amides,7 and
a-ketothiazoles8 have been incorporated into peptidyl
protease inhibitors. Since the report of the X-ray crystal
structure of d-Phe-l-Phe-l-Arg chloromethyl ketone
(DFFRCMK) bound to the active site of tissue factor
VIIa,9 comparisons of the various pockets with other
closely related proteases such as thrombin and Xa have
been possible. The most notable difference is the S2
pocket of VIIa. The S2 pocket of TF/VIIa is larger than
that of thrombin and Xa and differs by a key residue,
Asp 60, contained only in TF/VIIa. We initiated a pro-
gram to identify a selective tissue factor VIIa inhibitor.
At the outset of the program, we sought to utilize tri-
peptide transition state analogues closely related to
DFFRCMK. The design was of the general form
d-Phe-l-AA-l-Arg-a-ketothiazole,
utilizing
the
DFFRCMK backbone with a-ketothiazole in place of
the chloromethyl ketone to provide a covalent-rever-
sible inhibitor. The l-amino acid allows for variation at
the P2 position, which directly overlaps the S2 pocket of
*Corresponding author. Tel.:+1-314-274-5104; fax:+1-314-274-1621;
e-mail: michael.s.south@pharmacia.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00398-6

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M.S.South et al./ Bioorg.Med.Chem.Lett.13 (2003) 2363–2367
Scheme 1. PASP Synthesis of an a-ketothiazole library.
the enzyme and is expected to influence both potency
and selectivity. In an effort to rapidly prepare libraries
of a-ketothiazole peptidyl protease inhibitors in a par-
allel format, a solution-phase library synthesis was
developed utilizing polymer-assisted solution-phase
(PASP) technology.10 Herein, we report a five-step
PASP synthesis and biological activity of a-ketothia-
zoles with the structural motif d-Phe-l-AA-lArg-a-
ketothiazole.
compared to thrombin and Xa, a variety of l-amino
acids were used to probe the S2 pocket with the hope of
providing potency and selectivity.
The first step of the synthesis in Scheme 1 involved an
amide coupling using the starting scaffold 3 and a vari-
ety of BOCprotected l-amino acids 1, which are shown
in Scheme 2. The polymer-bound carbodiimide resin
was used as the coupling agent with hydroxy-
benzotriazole. Upon completion of the reaction, a
A summary description of the library synthesis is
depicted in Scheme 1. Each of the steps in the synthesis
underwent independent validation in order to optimize
conditions such that each transformation could be per-
formed in a high-yielding, parallel format. The synthesis
was accomplished using a combination of polymeric
reagents and polymer-assisted solution-phase purifi-
cation protocols, including reactant-sequestering resins,
reagent-sequestering resins, and tagged reagents. Com-
pound 3 was selected as the starting scaffold in its
reduced form as the alcohol to avoid side reactions
during the ensuing steps. As a result, the synthesis was
devised such that oxidation of the alcohol to the ketone
was conducted near the end. The synthesis is general
and allows for variation of either amino acid. The initial
library was designed with d-Phe as the terminal amino
acid capped as the benzylsulfonamide. The benzylsulfon-
amide moiety, expected to occupy the S3 pocket, was
selected based on the structure activity relationship
(SAR) from the thrombin literature and the similarity
of the S3 pockets of factor VIIa and thrombin. Con-
sidering the larger size of the S2 pocket of TF/VIIa
Scheme 2. Amino acid inputs.

M.S.South et al./ Bioorg.Med.Chem.Lett.13 (2003) 2363–2367
2365
mixed-resin bed of the polyamine and carbonate resins
were added to sequester the remaining reactants. Simple
filtration and evaporation of the solvents left highly
purified products 4.
of p-nitroaniline when the substrate is cleaved by the
protease. Inhibition of the enzyme reduces the change in
absorbance with the data reported as IC50 values. All of
the a-ketothiazoles 10 prepared were active against TF/
VIIa with IC50 values ranging from 0.042 to 1.45 mM.
The most potent compound of the library was 10d with
the l-amino acid as phenylalanine. The TF/VIIa activ-
ity was fairly flat across the set of compounds with the
substituted phenylalanine derivatives exhibiting con-
sistent activity with IC50’s below 1 mM. A key require-
ment for the program was to demonstrate that selectivity
of TF/VIIa versus thrombin could be achieved. Several
compounds, 10e, f, h, m have selectivity ratios (VIIa/IIa)
of greater than 100, with 10e having a selectivity factor
of 500. The data in Table 1 confirmed that a potent TF/
VIIa inhibitor with selectivity versus thrombin could
indeed be developed.
Deprotection of the BOCgroup was accomplished by
treatment with 4N hydrochloric acid in dioxane to
afford the amine hydrochloride salts 5. The next step of
the synthesis involved an amide coupling using the
amine intermediate 5 and N-(benzylsulfonyl)-d-phenyl-
alanine 6. The same reaction conditions were applied
from step 1 except that the sequestration process
involved using only the polyamine resin. The carbonate
resin was omitted in this step since the strongly basic
carbonate resin sequesters the desired products 7 due to
the acidic proton on the sulfonamide. The oxidation of
the alcohol to the ketone utilized a PASP purification
protocol.11 The alcohol 7 was oxidized with an excess of
the periodinane reagent 8. Upon completion of the
reaction, the thiosulfate resin was added to the product
mixture, which reduced the excess Dess–Martin reagent
8 and the IIII by-product species to the sequesterable
2-iodobenzoic acid. In addition, the thiosulfate resin
sequestered the majority of the 2-iodobenzoic acid.
Amberlyst A-21 was added to sequester the remaining
acid. Simple filtration and rinsing with dichloromethane
yielded a filtrate, whereupon evaporation of the solvents
left purified products 9 from each parallel reaction. The
last step of the synthesis involved deprotection of the
2,3,6-trimethyl-4-methoxybenzenesulfonyl (Mtr) pro-
tecting group using thioanisole in TFA to afford the
unprotected guanidine. Upon completion of the reac-
tion, the solvent was evaporated and the residue was
triturated to afford desired product 10 from each reac-
tion chamber. The HPLCpurities for the desired
a-ketothiazoles 10 ranged from 70 to 99% with an
average purity level of 88%. The overall yields ranged
from 12 to 37% based on mass recovery.12
The X-ray crystal structure of compound 10e bound in
the active site of TF/VIIa complex is shown in Figure 1.
The ketothiazole inhibitor 10e is bound well in the
active site of TF/VIIa, forming several hydrogen bond-
ing interactions with the protein atoms. The arginine
side chain at P1 forms four strong hydrogen bonds at
the bottom of the S1 pocket with the side chains of Asp
189 and Ser 190 as well as to the backbone carbonyl
oxygen of Gly 219. The active site serine, Ser 195, forms
The compounds 10a–m were screened for potency on
TF/VIIa and for other enzymes affecting coagulation,
such as Xa and Thrombin (IIa), to determine specificity,
Table 1. Each enzyme assay consisted of the specific
enzyme and chromogenic substrate for that enzyme.
Enzyme activity was determined by monitoring the
increase in absorbance at 405 nm caused by the release
Table 1. IC50 Values of a-ketothiazoles 10
Compd
10
IC50 (mM)
Xa
2.4
VIIa
1.45
IIa
VIIa/IIa
a
b
c
d
e
f
g
h
i
100
69
64
0.30
1.03
0.042
0.20
0.09
0.20
0.80
0.16
0.24
0.17
0.23
0.34
19.3
60
4.0
0.38
2.1
58
95
500
>333
75
106
70
90
71
0.027
0.29
0.21
0.25
1.0
Figure 1. Crystal structure of ketothiazole inhibitor 10e bound in the
active site of TF/VIIa complex. Some of the key side chains of factor
VIIa are displayed (C: green, N: dark blue, O: red, S: yellow and H:
orange). The carbon atoms of the inhibitor are shown in gold color.
The hydrogen bonds formed by the inhibitor and a bound water
molecule in the S2 site are shown in dotted white lines. The close
interaction between the bound solvent and the pyridyl nitrogen of the
inhibitor is shown in dotted green line. The active site serine, Ser 195,
forms a covalent bond (thin solid line) with the activated carbon of the
inhibitor.
100
>30
15
84.9
11.2
21.7
12.0
14.6
50
0.21
0.33
0.16
0.27
0.90
j
k
l
63
147
m

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M.S.South et al./ Bioorg.Med.Chem.Lett.13 (2003) 2363–2367
enzymes affecting coagulation to determine specificity,
Table 1. All of the compounds exhibited activity on tis-
sue factor VIIa with IC50’s ranging from 0.042 to 1.45
mM. Several compounds had excellent selectivity for
VIIa versus thrombin, such as compound 10e with a
selectivity ratio (IIa/VIIa) of 500. This demonstrated
that selectivity for TF/VIIa versus thrombin was pos-
sible. The structural information from the X-ray crystal
structure of compound 10e from this study, combined
with the crystal structure of other related serine pro-
teases such as thrombin and Xa, facilitated the devel-
opment of a noncovalent-reversible non-peptidic tissue
factor VIIa inhibitor which will be the topic of a future
publication.
Acknowledgements
Figure 2. Expanded view of the bound solvent molecule in the S2
pocket. Shown in yellow is the |Fo|-|Fc| electron density contoured at
3.0 s. The atoms are colored as in Figure 1. The hydrogen bonds
formed by the solvent are shown in dotted white line while the close
interaction between the water molecule and the pyridyl nitrogen is
shown in dotted green line.
The authors thank Dr. Huey Shieh for some of the early
crystallographic refinements of the TF/VIIa structure.
Diffraction data for the TF/VIIa complex with the
ketothiazole inhibitor were collected at beamline 17-ID
in the facilities of the Industrial Macromolecular Crys-
tallography Association Collaborative Access Team
(IMCA-CAT) at the Advanced Photon Source. IMCA-
CAT facilities are supported by the corporate members
of the IMCA and through a contract with Illinois Insti-
tute of Technology (IIT), executed through the IIT’s
Center for Synchrotron Radiation Research and
Instrumentation. Use of the Advanced Photon Source
was supported by the US Department of Energy, Basic
Energy Sciences, Office of Science, under Contract No.
W-31-109-Eng-38.
a covalent bond to the activated carbon of the inhibitor
as expected. This results in the formation of a transition
state analogue. The resulting hydroxyl group binds in
the oxyanion hole, forming two hydrogen bonds with
the amide nitrogen of Gly 193 and Ser 195. The thiazole
ring stacks parallel to the side chain of active site histi-
dine, His 57, with a hydrogen bond between the nitrogen
of the thiazole and one of the nitrogens of the histidine
side chain. The inhibitor forms two other hydrogen
bonds with peptide nitrogens of Gly 216 and Gly 219.
The side chain pyridyl ring traps a water molecule at the
S2 site of the enzyme active site, Figure 2. The bound
solvent molecule forms hydrogen bonding interactions
with the side chains of Asp 60 and Tyr 94. In addition,
two other hydrogen bonds are formed by the solvent
molecule with the backbone atoms of Gly 97 and Thr
98. Moreover, the pyridyl nitrogen of the inhibitor is 3.0
A away from the water molecule although the geometry
is not appropriate for a direct hydrogen bond. The pyr-
idyl ring of the inhibitor is almost orthogonal to the
plane of the His 57 side chain. The S2 pocket of factor
VIIa is relatively open and has a negative potential due
to the presence of Asp 60. The pyridyl group of the
inhibitor takes advantages of these structural features to
form strong interactions in the S2 pocket of VIIa.
Among the coagulation proteases, only factor VIIa has
a negatively charged residue at position 60. In contrast,
factor Xa has a tyrosine at position 60. Moreover, the
S2 pocket of factor Xa is occluded by the side chain of
Tyr 99. Thrombin has a large insertion in the S2 pocket
with a number of aromatic amino acid residues. These
structural differences would account for the enhanced
selectivity of compound 10e for TF/VIIa versus thrombin.
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