Polymer-assisted solution-phase library synthesis and crystal structure of α-ketothiazoles as tissue factor VIIa inhibitors

Article abstract of DOI:10.1021/jm030130t

A solution-phase synthesis of an α-ketothiazole library of the general form D-Phe-L-AA-Arg-α-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification concepts, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multistep synthesis affords desired α-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S₂ pocket of tissue Factor VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10k 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 over thrombin.

Full text of DOI:10.1021/jm030130t

J . Med. Chem. 2003, 46, 4043-4049
4043
P olym er -Assisted Solu tion -P h a se Libr a r y Syn th esis a n d Cr ysta l Str u ctu r e of
r-Ketoth ia zoles a s Tissu e F a ctor VIIa In h ibitor s
J ohn J . Parlow,*,† Thomas A. Dice,† Rhonda M. Lachance,‡ Thomas J . Girard,‡ Anna M. Stevens,§
Roderick A. Stegeman,§ William C. Stallings,§ Ravi G. Kurumbail,§ and Michael S. South†
Department of Medicinal and Combinatorial Chemistry, Pharmacia Corporation, 800 North Lindbergh Boulevard,
St. Louis, Missouri 63167, Department of Cardiovascular and Metabolic Disease, Pharmacia Corporation,
800 North Lindbergh Boulevard, St. Louis, Missouri 63167, and Structure and Computational Chemistry,
Pharmacia Corporation, 700 Chesterfield Village Parkway, St. Louis, Missouri 63198
Received March 20, 2003
A solution-phase synthesis of an R-ketothiazole library of the general form D-Phe-L-AA-Arg-
R-ketothiazole is described. The five-step synthesis is accomplished using a combination of
polymeric reagents and polymer-assisted solution-phase purification concepts, including
reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multistep
synthesis affords desired R-ketothiazole products in excellent purities and yields. A variety of
L-amino acid inputs were used to probe the S2 pocket of tissue Factor VIIa enzyme to influence
both potency and selectivity. An X-ray crystal structure of compound 10k bound to the TF/
VIIa complex was obtained that explains the observed selectivity. The R-ketothiazoles were
found to be potent, reversible-covalent inhibitors of tissue Factor VIIa, with some analogues
demonstrating selectivity over thrombin.
Tissue Factor (TF) VIIa is a serine protease and a key
enzyme in the blood coagulation cascade.1-4 Tissue
factor is the essential cofactor for the coagulation
protease Factor VIIa, initiating the coagulation cascade.
The role of tissue factor in thrombotic diseases is
becoming increasingly evident. Recent findings suggest
that inhibition of TF/VIIa activity could be important
in the prevention of clinical sequelae associated with
plaque rupture or vessel damage that exposes tissue
factor to blood. Furthermore, selective inhibitors of
tissue Factor VIIa may be associated with less bleeding
risk than other antithrombotic agents.5-8
R-ketothiazole in place of the chloromethyl ketone to
provide a covalent-reversible inhibitor. The L-amino acid
allows for variation at that position, to probe the S2
pocket of the enzyme, which is expected to influence
both potency and selectivity. In an effort to rapidly
prepare libraries of R-ketothiazole peptidyl protease
inhibitors in a parallel format, a solution-phase library
synthesis was developed utilizing polymer-assisted solu-
tion-phase (PASP) technology.27-29 Herein, we report a
five-step PASP synthesis and biological activity of
R-ketothiazoles with the structural motif D-Phe-L-AA-
Arg-R-ketothiazole.30
An approach to the design of serine protease inhibi-
tors has been the replacement of the scissile amide bond
by an electron-deficient carbonyl group.9,10 Series
of aldehydes,11 R-fluoroketones,12-14 R-keto esters/
amides,15-18 and R-ketothiazoles19-23 have been incor-
porated into peptidyl protease inhibitors. Since the
report of the X-ray crystal structure of D-Phe-L-Phe-Arg
chloromethyl ketone (DFFRCMK) bound to the active
site of tissue Factor VIIa,24-26 comparisons of the
various pockets with other closely related proteases such
as thrombin and Factor Xa have been possible. The most
notable difference is the S2 pocket of TF/VIIa. The S2
pocket of TF/VIIa is larger than that of thrombin and
Factor Xa and differs by a key residue, Asp 60, con-
tained only in TF/VIIa. We initiated a program to
identify a selective tissue Factor VIIa inhibitor. At the
outset of the program, we sought to utilize tripeptide
transition state analogues closely related to DFFRCMK.
The design was of the general form D-Phe-L-AA-Arg-R-
ketothiazole, utilizing the DFFRCMK backbone with
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 identify
and optimize conditions such that each transformation
could be performed in a high-yielding, parallel format.
The synthesis was accomplished using a combination
of polymeric reagents and polymer-assisted solution-
phase purification concepts, including reactant-seques-
tering resins, reagent-sequestering resins, and tagged
reagents. Compound 3 was selected as the starting
scaffold in its unoxidized 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 ketone was conducted near the end. The synthesis is
general such that variation of either amino acid is easily
allowed. The initial library was designed with D-Phe as
the terminal amino acid capped as the benzylsulfon-
amide. The benzylsulfonamide moiety, expected to oc-
cupy 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. Considering the larger size of the S2 pocket
of TF/VIIa compared to thrombin and Factor Xa, a
variety of L-amino acids was used to probe the S2 pocket
with the hope of providing potency and selectivity.
* To whom correspondence should be addressed. Phone: 314-274-
3494. Fax 314-274-1621. E-mail: john.j.parlow@pfizer.com.
† Department of Medicinal and Combinatorial Chemistry.
‡ Department of Cardiovascular and Metabolic Disease.
§ Department of Crystallography and Molecular Modeling.
10.1021/jm030130t CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/09/2003

4044 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Sch em e 1. PASP Synthesis of R-Ketothiazole Library
Parlow et al.
Sch em e 2. Synthetic Step 1 of PASP Ketothiazole Synthesis
The first step of the synthesis (Scheme 2) involved
an amide coupling using the starting scaffold31 3 and a
variety of BOC protected L-amino acids 1. The amino
acids 1 were incubated with the polymer-bound carbo-
diimide 11, hydroxybenzotriazole 12, and a catalytic
amount of Hunig’s base 13 to afford the activated esters
2. Diisopropylaminomethyl resin 14 and a limiting
amount of the amine hydrochloride salt 3 were added
to each reaction vessel. An excess of the polymer-bound
base, diisopropylaminomethyl resin 14, was used to
generate the free amine of scaffold 3 since it existed as
a dihydrochloride salt. After total consumption of the
scaffold 3 was observed, the reaction mixture was
diluted with dimethylformamide to accommodate the
addition of sequestering resins. A mixed-resin bed of the
polyamine resin 16 and carbonate resin 15 were added
to sequester the hydroxybenzotriazole 12, activated
ester 2, and any remaining amino acid 1. Acylation of
the hydroxy group was observed as a minor impurity
(<8%) in some of the product mixtures when using a
small amino acid input. As a result, the combination of
both basic resins 15 and 16 was used and incubated for
a period of 20 h, resulting in hydrolysis of the ester.
Simple filtration and rinsing with dimethylformamide
yielded a filtrate, whereupon evaporation of the solvents
left highly purified product 4 from each parallel reac-

R-Ketothiazoles as Tissue Factor VIIa Inhibitors
Sch em e 3. Amino Acid Inputs
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4045
Sch em e 4. Oxidation of Alcohol to Ketone
tion. The products were characterized by LC/MS and
carried on to the next step of the synthesis. A repre-
sentative set of BOC protected amino acids used for the
amide coupling is depicted in Scheme 3.
The oxidation of the alcohol 7 to ketone 9 using a
PASP purification protocol is shown in Scheme 4.32
A
mild oxidizing agent was required that would tolerate
the functionalities present within the library members.
Several oxidizing conditions were attempted and the
periodinane Dess-Martin reagent 8 proved superior.
The alcohol 7 was oxidized with an excess of the
periodinane reagent 8 to drive the reaction to comple-
tion, affording a product mixture containing the desired
ketone 9, excess oxidizing agent 8, and a reactant
byproduct 17. The thiosulfate resin 18 was added to the
product mixture, which reduced the excess Dess-
Martin reagent 8 and the IIII byproduct species 17 to
the sequesterable 2-iodobenzoic acid 19 (II species). In
addition, the thiosulfate resin 18 sequestered the major-
ity of the 2-iodobenzoic acid 19. Amberlyst A-21 20 was
The next step of the synthesis involved a straightfor-
ward deprotection of the BOC group using 4 N hydro-
chloric acid in dioxane to afford the amine hydrochloride
salts 5 (Scheme 1). The third step of the synthesis was
an amide coupling using the amine intermediate 5 and
N-(benzylsulfonyl)-D-phenylalanine 6 (Scheme 1). The
same reaction conditions were applied from step 1
(Scheme 2) except that the sequestration process in-
volved using only the polyamine resin 16. The carbonate
resin 15 was omitted in this step as the desired products
7, possessing an acidic proton from the sulfonamide, are
sequestered by the strongly basic carbonate resin 15.

4046 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Parlow et al.
Ta ble 1. IC50 Values of R-Ketothiazoles 10
IC50 (uM)
compd 10
VIIa
IIa
100
0.04
19.3
9.20
0.44
60
12.9
15.4
24.2
4.0
100
3.19
>30
15
3.15
84.9
11.2
6.82
-
21.7
12.0
7.48
28.1
14.6
3.42
6.3
50
>30
4.59
Xa
VIIa/IIa
a
1.45
0.11
0.30
0.82
0.70
1.03
2.4
69
0.4
64
11
0.6
58
1
b
0.24
0.38
2.12
1.03
2.1
c
d
e
f
g
h
i
j
k
l
13.7
2.0
1.7
2.82
3.30
0.042
0.20
0.76
0.09
0.20
0.17
0.80
0.16
0.19
0.20
0.24
0.17
1.29
0.70
0.23
0.68
1.56
0.34
5.88
0.57
9.97
1.95
5.6
5
7
2.70
0.027
0.29
0.38
0.21
0.25
0.185
1.0
0.21
0.18
0.25
0.33
0.16
0.57
2.77
0.27
0.61
0.50
0.90
2.18
0.30
2.4
95
500
4
>333
75
19
106
70
36
-
m
n
o
p
q
r
s
t
90
71
6
u
v
w
x
40
63
5
y
z
4
a a
bb
cc
d d
ee
ff
gg
h h
ii
jj
k k
ll
147
>5
8
9.92
2.4
1
1
0.77
1.8
>30
15.4
9.89
11.2
1.68
10.4
4.99
>5
F igu r e 1. Crystal structure of ketothiazole inhibitor 10K
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, 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 the dotted green line. The active site serine, Ser 195,
forms a covalent bond (thin solid line) with the activated
carbon of the inhibitor.
0.50
1.53
17.8
1.83
0.90
3.18
0.15
0.31
3.14
1.07
0.54
1.25
3
6
1
1
12
2
added to sequester the remaining acid 19. Strongly basic
resins, such as polymer-bound 1,3,5-triazabicylcodec-5-
ene (TBD), were avoided at this step due to sequestra-
tion of the product. Simple filtration and rinsing with
dichloromethane yielded a filtrate, whereupon evapora-
tion of the solvents left purified products 9 from each
parallel reaction. The products were characterized by
LC/MS and carried on to the next step of the synthesis.
The last step of the synthesis involved deprotection
of the 2,3,6-trimethyl-4-methoxybenzenesulfonyl (Mtr)
protecting group to afford the unprotected guanidine.
Thioanisole was added to the ketones 9 in trifluoroacetic
acid. Upon completion of the reaction, the solvent was
evaporated and the residue was triturated to afford
desired product 10 from each reaction chamber. The
HPLC purities and overall yields for the desired R-
ketothiazoles 10 are listed in Table 2 in Supporting
Information. Purity levels ranged from 70 to 99% with
an average purity level of 82%. The yields ranged from
9 to 45% based on mass recovery. Considering that this
is a five-step synthesis, with a portion of the sample
taken for characterization of each intermediate and the
physical manipulation of the reaction mixtures to and
from the reaction block, these are quite acceptable
overall yields (i.e., 80% yield for each reaction of a five
step synthesis affords an overall 33% yield).
enzyme. Enzyme activity was determined by monitoring
the increase in absorbance at 405 nm caused by the
release of p-nitroaniline when the substrate is hydro-
lyzed. Inhibition of the enzyme reduces the change in
absorbance with the data reported as IC50 values.
Several of the intermediate alcohols 7 were not oxidized
to the ketone and tested as R-hydroxythiazoles. As
anticipated, the unoxidized compounds showed no bio-
logical activity. All of the R-ketothiazoles 10 prepared
were active against TF/VIIa with IC50 values ranging
from 0.042 to 17.8 uM. The most potent compound of
the library was 10j with the L-amino acid as phenyla-
lanine. The TF/VIIa activity was fairly flat across the
set of compounds with the substituted phenylalanine
derivatives exhibiting consistent activity with IC50’s
below 1 uM. A key requirement for the program was to
demonstrate that selectivity of TF/VIIa over thrombin
could be achieved. Several compounds, 10k ,m ,p ,a a ,
have selectivity ratios (VIIa/IIa) of greater than 100,
with 10k having a selectivity factor of 500. These
selectivity factors confirmed that a potent TF/VIIa
inhibitor with selectivity versus thrombin could be
attained.
The compounds 10a -ll were screened for potency on
TF/VIIa and for other enzymes affecting coagulation,
such as Factor Xa and Thrombin (IIa), to determine
specificity (Table 1). Each enzyme assay consisted of the
specific enzyme and chromogenic substrate for that
An X-ray crystal structure of compound 10k bound
in the active site of TF/VIIa complex was obtained to
gain a better understanding of the structural differences
responsible for selectivity (Figure 1). The ketothiazole

R-Ketothiazoles as Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4047
F igu r e 2. Expanded view of the bound solvent molecule in the S2 pocket. Shown in yellow is the |Fo| - |Fc| electron density
contoured at 3.0σ. The atoms are colored as in Figure 1. The hydrogen bonds formed by the solvent are shown in dotted white
lines while the close interaction between the water molecule and the pyridyl nitrogen is shown in the dotted green line.
inhibitor 10k is bound well in the active site of TF/VIIa,
forming several hydrogen bonding 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 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 histidine, 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.
Con clu sion
A series of R-ketothiazoles with the structure BzSO2-
D-Phe-L-AA-Arg- R-ketothiazole was synthesized using
combinations of polymer-assisted solution-phase puri-
fication concepts. The compounds were screened for
potency on TF/VIIa and for other enzymes affecting
coagulation to determine specificity (Table 1). All of the
compounds exhibited activity on tissue Factor VIIa with
IC50’s ranging from 0.042 to 17 uM. Several compounds
have excellent selectivity for TF/VIIa over thrombin,
such as compound 10k with a selectivity ratio (IIa/VIIa)
of 500, demonstrating that selectivity of TF/VIIa over
thrombin can be achieved. The structural information
from the X-ray crystal structure of compound 10k from
this study, combined with the crystal structure of other
related serine proteases such as thrombin and Factor
Xa, facilitated the development of a noncovalent-revers-
ible nonpeptidic tissue Factor VIIa inhibitor which will
be the topic of a future publication.
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 inter-
actions 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 Å away from the water molecule although the
geometry is not appropriate for a direct hydrogen bond.
The pyridyl 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 struc-
tural 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. 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 10k for TF/VIIa over thrombin.
Exp er im en ta l Section
Gen er a l. Solvents and chemicals were reagent grade or
better and were obtained from commercial sources. All BOC
protected amino acids were obtained from commercial sources.
PS-carbodiimide resin, PS-DIEA, PS-polyamine resin, and MP-
carbonate resin were purchased from Argonaut Technologies.
Amberlyst A-21 was purchased from Sigma-Alrich Chemical
Company. 1H and 13C NMR spectra were recorded using a 300
or 400 MHz NMR spectrometer. Sample purities were deter-
mined by HPLC analysis equipped with a mass spectrometer
detector using a C18 3.5 µm 30 × 2.1 mm column, eluting with
a gradient system of 5/95 to 95/5 acetonitrile/H2O with a buffer
consisting of 0.1% TFA over 4.5 min at 1 mL/min and detected
by ELS. Reported yields are not optimized, with emphasis on
purity of products rather than quantity. Recombinant soluble
TF, consisting of amino acids 1-219 of the mature protein
sequence was expressed in E. coli and purified using a Mono
Q Sepharose FPLC. Recombinant human VIIa was purchased
from American Diagnostica, Greenwich CT. Chromogenic
substrate N-methylsulfonyl-D-phe-gly arg-p-nitroaniline was

4048 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Parlow et al.
prepared by American Peptide Company, Inc., Sunnyvale, CA.
Factor Xa was obtained from Enzyme Research Laboratories,
South Bend IN, thrombin from Calbiochem, La J olla, CA, and
trypsin and L-BAPNA from Sigma, St. Louis, MO. The chro-
mogenic substrates S-2765 and S-2238 were purchased from
Chromogenix, Sweden.
under high vacuum for 24 h to afford the crude product 10.
The product was purified by trituration with ether and then
ethyl acetate followed by filtration to afford the pure product
10. The products were characterized by LC/MS and HRMS
with yields based on mass recovery as shown in Table 2 in
Supporting Information.
Assa ys for Biologica l Activity. TF -VIIa Assa y. Recom-
binant soluble tissue factor (100 nM) and recombinant human
Factor VIIa (2 nM) were added to a 96-well assay plate
containing 0.4 mM of the substrate, N-methylsulfonyl-D-phe-
gly arg-p-nitroaniline, and either inhibitor or buffer (5 mM
CaCl2,50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1% BSA). The
reaction, in a final volume of 100 µL, was measured im-
mediately at 405 nm to determine background absorbance. The
plate was incubated at room temperature for 60 min, at which
time the rate of hydrolysis of the substrate was measured by
monitoring the reaction at 405 nm for the release of p-
nitroaniline. All compounds were assayed in duplicate at seven
concentrations. Percent inhibition at each concentration was
Gen er a l P r oced u r e A. Cou p lin g of Am in o Acid s 1 w ith
Am in e Sca ffold 3 to Affor d P r od u ct 4. The amino acid 1
(0.60 mmol) was added to each reaction vessel containing
hydroxybenzotriazole 12 (0.65 M in dimethylformamide) (1.0
mL, 0.65 mmol), N,N-diisopropylethylamine (0.20 M in di-
methylformamide) (0.25 mL, 0.05 mmol), and PS-carbodiimide
resin 11 (1.91 mequiv/g) (0.34 g, 0.658 mmol). Dichloromethane
(2.0 mL) was added to each well to ensure effective mixing.
The vials were agitated by orbital shaking for 15 min. The
amine scaffold 3 (0.30 M in dimethylformamide) (1.0 mL, 0.30
mmol) was added along with PS-DIEA resin 14 (3.86 mequiv/
g) (0.35 g, 1.35 mmol), and the vials were agitated by orbital
shaking for 3.5 h. Additional dimethylformamide (2.0 mL) was
added along with PS-polyamine resin 16 (4.78 mequiv/g) (0.50
g, 2.4 mmol) and MP-carbonate resin 15 (2.81 mequiv/g) (0.50,
1.4 mmol), and the vials were agitated by orbital shaking
overnight (approx 20 h). The reaction mixtures were filtered
and rinsed four times with 2.0 mL of dimethylformamide. The
combined filtrate and washings were evaporated to afford pure
product 4.
calculated from OD405
value from the experimental and
nm
control sample. IC50 values were calculated from a four-
parameter logistic regression equation. For each compound the
individual IC50 values were within 10% of each other. The
reported IC50 represents an average of the duplicates.
Xa Assa y. Human Factor Xa (0.3 nM) and N-R-benzyloxy-
carbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroaniline dihydro-
chloride (S-2765) (0.15 mM) were added to a 96-well assay
plate containing either inhibitor or buffer (50 mM Tris-HCl,
pH 8.0, 100 mM NaCl, 0.1% BSA). The reaction, in a final
volume of 100 uL, was measured immediately at 405 nm to
determine background absorbance. The plate was incubated
at room temperature for 60 min, at which time the rate of
hydrolysis of the substrate is measured by monitoring the
reaction at 405 nm for the release of p-nitroaniline. All
compounds were assayed in duplicate at seven concentrations.
Percent inhibition at each concentration was calculated from
OD405 nm value from the experimental and control sample. IC50
values were calculated from a four-parameter logistic regres-
sion equation. For each compound the individual IC50 values
were within 10% of each other. The reported IC50 represents
an average of the duplicates.
Th r om bin Assa y. Human thrombin (0.28 nM) and H-D-
phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline dihydrochlo-
ride (0.06 mM) were added to a 96-well assay plate containing
either inhibitor or buffer (50 mM Tris-HCl, pH 8.0, 100 mM
NaCl, 0.1% BSA). The reaction, in a final volume of 100 µL,
was measured immediately at 405 nm to determine back-
ground absorbance. The plate was incubated at room temper-
ature for 60 min, at which time the rate of hydrolysis of the
substrate was measured by monitoring the reaction at 405 nm
for the release of p-nitroaniline. All compounds were assayed
in duplicate at seven concentrations. Percent inhibition at each
concentration was calculated from OD405nm value from the
experimental and control sample. IC50 values were calculated
from a four-parameter logistic regression equation. For each
compound the individual IC50 values were within 10% of each
other. The reported IC50 represents an average of the dupli-
cates.
Gen er a l P r oced u r e B. Dep r otection of th e BOC Gr ou p
of Com p ou n d s 2 t o Affor d t h e Am in e Hyd r och lor id e
Sa lts 5. Hydrochloric acid in 1,4-dioxane (4.0 N, 3 mL) was
added to the BOC protected amine 4 (∼0.3 mmol) in a reaction
vessel, and the solution was agitated on an orbital shaker at
room temperature for 3 h. Evaporation of the solvents afforded
the product 5.
Gen er a l P r oced u r e C. Cou p lin g of N-(Ben zylsu lfon yl)-
D-p h en yla la n in e 6 w it h Am in e 5 t o Affor d P r od u ct 7.
The N-(benzylsulfonyl)-D-phenylalanine 6 (0.60 mmol) was
added to each reaction vessel containing hydroxybenzotriazole
12 (0.60 M in 1/1 dimethylformamide/dichloromethane) (1.0
mL, 0.60 mmol), N,N-diisopropylethylamine (0.20 M in dim-
ethylformamide) (0.25 mL, 0.05 mmol), and PS-carbodiimide
resin 11 (1.91 mequiv/g) (0.34 g, 0.658 mmol). Dichloromethane
(2.0 mL) was added to each well to ensure effective mixing.
The vials were agitated by orbital shaking for 15 min. The
amine 5 (0.20 M in dimethylformamide) (1.5 mL, ∼0.3 mmol)
was added along with PS-DIEA resin 14 (3.86 mequiv/g) (0.35
g, 1.35 mmol), and the vials were agitated by orbital shaking
for 6 h. Additional dimethylformamide (3.0 mL) was added
along with PS-polyamine resin 16 (4.78 mequiv/g) (1.28 g, 6.1
mmol), and the vials were agitated by orbital shaking over-
night (approx 17 h). The reaction mixtures were filtered and
rinsed four times with 2.0 mL of dimethylformamide. The
combined filtrate and washings were evaporated to afford pure
product 7.
Gen er a l P r oced u r e D. Oxid a t ion of Alcoh ol 7 t o
Affor d Ket on e P r od u ct 9. The Dess-Martin periodinane
reagent 8 (0.30 M in dichloromethane) (1.0 mL, 0.30 mmol)
was added to the alcohol 7 (∼0.30 mmol) in 2.0 mL of
dichloromethane, and the vials were agitated by orbital
shaking for 2 h. Upon completion of the reaction, the thiosul-
fate resin 18 (1.73 mmol/g) (1.0 g, 1.7 mmol) was added
followed by 3.0 mL of dichloromethane, and the vials were
agitated by orbital shaking for 3 h. The Amberlyst A-21 resin
20 (4.81 mmol/g) (0.40 g, 1.9 mmol) was added followed by 3.0
mL of dichloromethane, and the vials were agitated by orbital
shaking for 3 h. The reaction mixtures were filtered and rinsed
four times with 2.0 mL of dimethylformamide. The combined
filtrate and washings were evaporated to afford pure product
9.
Cr ysta l Str u ctu r e. Crystals of TF/VIIa complex were
obtained by slight modification of the procedure described by
Banner et al. (ref 20). Diffraction data were measured at the
Advanced Photon Source to 2.6 Å resolution, and the structure
has been refined with good agreement between the data and
the model (Rfree of 28.6% and Rwork: 22.2%).
Ack n ow led gm en t. 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 Crystallography Association
Collaborative Access Team (IMCA-CAT) at the Ad-
vanced Photon Source. IMCA-CAT facilities are sup-
Gen er a l P r oced u r e E. Dep r otection of th e Mtr Gr ou p
to Affor d Desir ed r-Ketoth ia zole 10. Thioanisole (100 uL,
0.85 mmol) was added to the ketone (∼0.3 mmol) in trifluo-
roacetic acid (2.0 mL), and the vials were agitated by orbital
shaking for 5 h. Upon completion of the reaction, the product
mixture was evaporated by a stream of nitrogen and placed

R-Ketothiazoles as Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4049
(15) Angelastro, M. R.; Mehdi, S.; Burkhart, J . P.; Peet, N. P.; Bey,
P. R-Diketone and R-keto ester derivatives of N-protected amino
acids and peptides as novel inhibitors of cysteine and serine
proteinases. J . Med. Chem. 1990, 33, 11-13.
ported by the corporate members of the IMCA and
through a contract with Illinois Institute 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 U.S.
Department of Energy, Basic Energy Sciences, Office
of Science, under Contract No. W-31-109-Eng-38.
(16) Ocain, T. D.; Rich, D. H. R-Keto amide inhibitors of aminopep-
tidases. J . Med. Chem. 1992, 35, 451-456.
(17) Yuan, W.; Wong, C.-H.; Haeggstrom, J . Z.; Wetterholm, A.;
Samuelsson, B. Novel tight-binding inhibitors of leukotriene A4
hydrolase. J . Am. Chem. Soc. 1992, 114, 6552-6553.
(18) Semple, J . E.; Rowley, D. C.; Brunck, T. K.; Ripka, W. C.
Synthesis and biological activity of P2-P4 azapeptidomimetic P1-
argininal and P1-ketoargininamide derivatives: A novel class
of serine protease inhibitors. Bioorg. Med. Chem. Lett. 1997, 7,
315-320.
(19) Saito, M.; Hashimoto, M.; Kawaguchi, N.; Fukami, H.; Tanaka,
T.; Higuchi, N.Synthesis and inhibitory activity of acyl-peptidyl-
prolinal derivatives toward post-proline cleaving enzyme as
nootropic agents. J . Enzymol. Inhib. 1990, 3, 163-178.
(20) Tsutsumi, S.; Okonogi, T.; Shibahara, S.; Patchett, A. A.;
Christensen, B. G. R-Ketothiazole inhibitors of prolyl endopep-
tidase. Bioorg. Med. Chem. Lett. 1994, 4, 831-834.
(21) Tao, M.; Bihovsky, R.; Kauer, J . C. Inhibition of calpain by
peptidyl heterocycles. Bioorg. Med. Chem. Lett. 1996, 6, 3009-
3012.
(22) Akiyama, Y.; Tsutsumi, S.; Hatsushiba, E.; Ohuchi, S.; Okonogi,
T. Peptidyl R-keto thiazole as potent thrombin inhibitors. Bioorg.
Med. Chem. Lett. 1997, 7, 533-538.
(23) Subramanyam, C.; Chang, S. P. Solid-phase synthesis of peptidyl
R-keto heterocycles. Tetrahedron Lett. 2002, 43, 6313-6315.
(24) Banner, D. W.; D’Arcy, A.; Chene, C.; Winkler, F. K.; Guha, A.;
Konigsberg, W. H.; Nemerson, Y.; Kirchhofer, D. The crystal
structure of the complex of blood coagulation factor VIIa with
soluble tissue factor. Nature 1996, 380, 41-46. Publications after
completion of the R-ketothiazole project:
(25) Pike, A. C.; Brzozowski, A. M.; Roberts, S. M.; Olsen, O. H.;
Persson, E. Structure of human factor VIIa and its implications
for the triggering of blood coagulation. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 8925-8930.
(26) Kemball-Cook, G.; J ohnson, D. J .; Tuddenham, E. G.; Harlos,
K. Crystal structure of active site-inhibited human coagulation
factor VIIa (des-Gla). J . Struct. Biol. 1999, 127, 213-223.
(27) Flynn, D. L.; Devraj, R. V., Parlow, J . J . Polymer-Assisted
Solution-Phase Methods for Chemical Library Synthesis; Bur-
gess, K., Ed.; Solid-Phase Organic Synthesis; J ohn Wiley &
Sons: New York, 2000; Chapter 5.
Su p p or tin g In for m a tion Ava ila ble: A table of yields and
HPLC purities of R-ketothiazole products 10. This material is
available free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Houston, D. S. Tissue factor - a therapeutic target for throm-
botic disorders. Expert Opin. Ther. Targets 2002, 6, 159-174.
(2) Girard, T. J .; Nicholson, N. S. The role of tissue factor/factor
VIIa in the pathophysiology of acute thrombotic formation. Curr.
Opin. Pharmacol. 2001, 1, 159-163.
(3) Bajaj, M. S.; Birktoft, J . J .; Steer, S. A.; Bajaj, S. P. Structure
and biology of tissue factor pathway inhibitor. Thromb. Haemost.
2001, 86, 959-972.
(4) Girard, T. J . Tissue Factor Pathway Inhibitor, In New Thera-
peutic Agents in Thrombosis and Thrombolysis; Sasahara, A. A.;
Loscalzo, J ., Eds.; Marcel Dekker: New York, 1998.
(5) Szalony, J . A.; Taite, B. B.; Girard, T. J .; Nicholson, N. S.;
Lachance, R. M. Pharmacological intervention at disparate sites
in the coagulation cascade: Comparison of anti-thrombotic
efficacy vs bleeding propensity in a rat model of acute arterial
thrombosis. J . Thromb. Thrombolysis 2003, 14, 113-121.
(6) Zoldhelyi, P.; McNatt, J .; Shelat, H. S.; Yamamoto, Y.; Chen,
Z.-Q.; and Willerson, J . T. Thromboresistance of balloon-injured
porcine carotid arteries after local gene transfer of human tissue
factor pathway inhibitor. Circulation 2000, 101, 289-95.
(7) Himber, J .; Kirchhofer, D.; Riedereer, M.; Tschopp, T. B.; Steiner
B.; Roux, S. P. Dissociation of antithrombotic effect and bleeding
time prolongation in rabbits by inhibiting tissue factor function.
Thromb. Haemost. 1997, 7, 1142-1149.
(8) Harker, L. A.; Hanson, S. R.; Wilcox, J . N.; Kelly, A. B.
Antithrombotic and antilesion benefits without hemorrhagic
risks by inhibiting tissue factor pathway. Haemostasis 1996, 26
(suppl 1), 76-82.
(9) Wiley: R. A.; Rich, D. H. Peptidomimetics derived from natural
products. Med. Res. Rev. 1993, 13, 327-384 and references
therein.
(28) Parlow, J . J .; Devraj, R. V., South, M. S. Solution-phase chemical
library synthesis using polymer-assisted purification techniques.
Curr. Opin. Chem. Biol. 1999, 3, 320-336.
(29) Flynn, D. L.; Devraj, R. V., Naing, W., Parlow, J . J .; Weidner,
J . J ., Yang, S. Polymer-assisted solution phase (PASP) chemical
library synthesis. Med. Chem. Res. 1998, 8, 219-243.
(30) South, M. S.; Dice, T. A.; Lachance, R. M.; Girard, T. J .; Stevens,
A. M., Stegeman, R. A.; Stallings, W. C.; Kurumbail, R. G.;
Parlow, J . J . Polymer-assisted solution-phase (PASP) parallel
synthesis of an R-ketothiazole library as tissue factor VIIa
inhibitors. Bioorg. Med. Chem. Lett. 2003, 13, 2363-2367.
(31) Boatman, P. D.; Ogbu, C. O.; Eguchi, M.; Kim, H.-O.; Nakanishi,
H.; Cao, B.; Shea, J . P.; Kahn, M.. Secondary structure peptide
mimetics: Design, synthesis, and evaluation of â-strand mimetic
thrombin inhibitors. J . Med. Chem. 1999, 42, 1367-1375.
(32) Parlow, J . J .; Case, B. L.; South, M. S. High-throughput
purification of solution-phase periodinane mediated oxidation
reactions utilizing a novel thiosulfate resin. Tetrahedron 1999,
55, 6785-6796.
(10) Mehdi, S. Synthetic and naturally occurring protease inhibitors
containing an electrophilic carbonyl group. Biorg. Chem. 1993,
21, 249-259 and references therein.
(11) Andersson, L.; Isley, T. C.; Wolfenden, R. R-Aminoaldehydes:
transition state analogue inhibitors of leucine aminopeptidase.
Biochemistry 1982, 21, 4177-4180.
(12) Angliker, H.; Anagli, J .; Shaw, E. Inactivation of calpain by
peptidyl fluoromethyl ketones. J . Med. Chem. 1992, 35, 216-
220.
(13) Damon, D. B.; Hoover, D. J . Synthesis of the ketodifluorometh-
ylene dipeptide isostere. J . Am. Chem. Soc. 1990, 112, 6439-
6442.
(14) Stein, R. L.; Strimpler, A. M.; Edwards, P. D.; Lewis, J . J .;
Mauger, R. C.; Schwartz, J . A.; Stein, M. M.; Trainor, P. A.;
Wildonger, R. A.; Zottola, M. A. Mechanism of slow-binding
inhibition of human leukocyte elastase by trifluoromethyl ke-
tones. Biochemistry 1987, 26, 2682-2689.
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