Synthesis and X-ray crystal structures of substituted fluorobenzene and benzoquinone inhibitors of the tissue factor VIIa complex

Article abstract of DOI:10.1016/j.bmcl.2003.08.002

Multistep syntheses of substituted benzenes and benzoquinone inhibitors of tissue Factor VIIa are reported. The benzene analogues were designed such that their substitution pattern would occupy and interact with the S₁, S₂, and S

Full text of DOI:10.1016/j.bmcl.2003.08.002

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3721–3725
Synthesis and X-ray Crystal Structures of Substituted
Fluorobenzene and Benzoquinone Inhibitors of the
Tissue Factor VIIa Complex
John J. Parlow,a,* Ravi G. Kurumbail,b Roderick A. Stegeman,b Anna M. Stevens,b
William C. Stallingsb and Michael S. Southa
aDepartment of Medicinal and Combinatorial Chemistry, Pharmacia Corporation, 800 North Lindbergh Boulevard,
St.Louis, MO 63167, USA
bStructure and Computational Chemistry, Pharmacia Corporation, 700 Chesterfield Village Parkway,
St.Louis, MO 63198, USA
Received 11 April 2003; revised 4 August 2003; accepted 5 August 2003
Abstract—Multistep syntheses of substituted benzenes and benzoquinone inhibitors of tissue Factor VIIa are reported. The benzene
analogues were designed such that their substitution pattern would occupy and interact with the S1, S2, and S3 pockets of the tissue
Factor VIIa (TF/VIIa) enzyme. The compounds exhibited modest potency on TF/VIIa with selectivity over Factor Xa and
thrombin. The X-ray crystal structures of the targeted fluorobenzene 12a and benzoquinone 14 inhibitors bound to TF/VIIa were
obtained and will be described.
# 2003 Elsevier Ltd. All rights reserved.
Cardiovascular disease is the most common cause of
mortality in the Western world.1 The disease is char-
acterized by the acute coronary syndromes (ACS)
unstable angina and myocardial infarction, which often
lead to sudden death. Often, the root cause for ACS is
due to deposition of a thrombus in coronary arteries.
This inappropriate thrombus formation is initiated via
the extrinsic coagulation cascade caused by a plaque
rupture, which exposes cell surface tissue factor (TF) to
the serine protease VIIa in circulating blood forming the
TF/VIIa complex. This cascade is critical in normal
hemostasis, but is also involved in the pathogenesis of
various thrombotic diseases. Under normal conditions,
TF expressed in the sub-endothelium of healthy blood
vessels is not exposed to blood. However, in a disease
state or during injury, TF comes in contact with Factor
VIIa and the ensuing TF/VIIa complex activates Fac-
tors X and IX to Xa and IXa, respectively. The complex
of Factor Xa and Factor Va on a membrane surface
converts prothrombin to thrombin, leading to fibrin
formation, deposition and subsequent thrombus forma-
tion.2 Effective and safe antithrombotics are needed to
combat cardiovascular diseases. Most research has
focused on thrombin and Factor Xa inhibitors as
potentially valuable therapeutic agents for these dis-
eases.3 More recently, small molecule inhibitors of tissue
Factor VIIa have been the point of much research effort
because of their potential to inhibit the coagulation
cascade while minimizing the risk of bleeding side
effects.4
We previously reported the preparation of pyrazinone
analogues as tissue Factor VIIa inhibitors.5 These pyr-
azinone compounds are active-site inhibitors for TF/
VIIa exhibiting potency at the nanomolar level with
excellent selectivity over thrombin (IIa) and Factor Xa.
In an effort to increase the potency and influence the
pharmocokinetic properties, other core ring systems
were evaluated. Depicted in Figure 1 is the general pyr-
azinone structure I. Focusing on the central ring, one of
the exercises was to replace the nitrogens with carbons
on the pyrazinone ring, resulting in benzene II and
benzoquinone as central ring systems.6,7 Based on the
X-ray crystal structures of TF/VIIa,5,8 several key
interactions were crucial for both potency for TF/VIIa
and selectivity over thrombin and Factor Xa. It was
*Corresponding author. Tel.: +1-314-274-3494; fax: +1-314-274-
1621; e-mail: john.j.parlow@pfizer.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.002

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J.J.Parlow et al./ Bioorg.Med.Chem.Lett.13 (2003) 3721–3725
Scheme 1. Synthesis of substituted benzene TF/VIIa inhibitors. Reagent and conditions: (i) triflic anhydride, Et3N, DCM, À10 ꢀC–rt; (ii) Pd(PPh3)4,
LiCl, dioxane, 85 ꢀC; (iii) Fe, MeCO2H, 80 ꢀC; (iv) Me2CO, NaHB(OAc)3, DCM/THF; (v) NaOH, MeOH, 65 ꢀC; (vi) HOBt, NMM, PS-CD, DCM/
DMF, then PS-aldehyde and PS-polyamine resins; (vii) H2, Pd/C, MeOH.
found in the pyrazinone series that the benzamidine
binds, as expected, in the S1 pocket mediated by an ion-
pair between the basic amidine moiety and the carboxyl-
ate of Asp 189. The substituted meta-aminophenyl ring
occupies the S2 pocket and the amino group at the 3-
position of the pyrazinone occupies and interacts with
the S3 pocket. In addition, the ketone at the 2-position
on the pyrazinone ring forms a hydrogen bond with the
peptide backbone of Gly 216. We explored the possibi-
lity of replacing the pyrazinone core with a simple ben-
zene ring system that could orient the various
substituents in the correct spatial arrangement to probe
the S1, S2, and S3 pockets. In addition, the benzene ring
could be substituted at the 2-position with various
hydrogen bond accepting groups to engage Gly 216.
Reported herein is the discovery, synthesis, biological
activity, and X-ray structures of novel substituted ben-
zene and benzoquinone analogues as tissue Factor VIIa
inhibitors.
butylstannyl)phenylcarbamate
5a
using
tetrakis
(triphenylphosphine)palladium(0) and lithium chloride
in dioxane at 85 ꢀCto afford the 6-phenyl derivative
6a.10 Iron reduction of the nitro group afforded the
desired amine 7a. Reductive amination of the amino
compound 7a with acetone using sodium triacetox-
yborohydride afforded the isopropylamine 8a. Hydro-
lysis of the methyl ester using sodium hydroxide
afforded the carboxylic acid 9a. The amide coupling was
accomplished by treating the acid 9a with polymer-
bound carbodiimide, hydroxybenzotriazole, and N-
methylmorpholine as base followed by addition of the
amine 10, benzyl amino[4-(aminomethyl)phenyl]-
methylcarbamate, to yield the Cbz protected product
11a. Hydrogenation deprotection of the Cbz groups was
used to afford the desired 2-fluorobenzene product 12a.
A benzoquinone core maintains the ketone in the same
position as the pyrazinone I for hydrogen bonding with
Gly 216. The precursor to prepare a benzoquinone ring
system was synthesized as described above and shown in
Scheme 1. 2-Methoxybenzene 2 was used as the starting
A key feature required in replacing the pyrazinone ring
with a benzene ring system was to have a hydrogen
bond accepting group at the 2-position. Thus, a sub-
stituted benzene ring with a fluorine in the 2-position
was designed with the fluorine acting as the hydrogen
bond acceptor to interact with Gly 216. The fluoro-
benzene target 12a was prepared as shown in Scheme 1.
Compound 1 was used as the starting material9 allowing
for substitution at the 6-position while maintaining the
fluorine at the 2-position. The fluoro compound 1 was
reacted with triflic anhydride to afford the triflate 3. The
triflate 3 underwent a Stille coupling with benzyl 3-(tri-
Figure 1. Pyrazinone and benzene core structures.

J.J.Parlow et al./ Bioorg.Med.Chem.Lett.13 (2003) 3721–3725
3723
comparable with the benzoquinone 14. However, these
analogues are less potent against TF/VIIa than their
corresponding pyrazinone analogues.5
The crystal structure of compound 12a bound to TF/
VIIa is shown in Figure 2. The bound conformation of
fluorobenzene inhibitor 12a in the active site of VIIa
resembles that of pyrazinone inhibitors that have been
reported previously.5 The benzamidine moiety engages
the carboxylate of Asp 189 in the S1 site, similar to the
interactions observed in the crystal structures of other
serine proteases. The peptide nitrogen of the acetate
linker forms a hydrogen bond (3.2 A) with the carbonyl
oxygen of Ser 214. The anilino nitrogen attached to the
central fluorobenzene core donates a hydrogen bond
(3.4 A) to the main chain oxygen of Gly 216. As
anticipated, the fluorine atom in the central ring accepts
a hydrogen bond (3.4 A) from the amide nitrogen of
Gly 216. Some of these hydrogen bonds are longer and
perhaps indicate non-optimized interactions of the inhi-
bitor in the active site. This might explain the relatively
weaker binding affinity of this class of inhibitors com-
pared to that of pyrazinone inhibitors.
Scheme 2. Benzoquinone synthesis. Reagent and conditions: (i) BBr3,
DCM, À10 ꢀC; (ii) (KSO3)2NO, H2O/THF.
material to afford the benzoquinone precursor 11b.
Compound 11b was designed with the isopropylamine
at the 3-position and the unsubstituted phenyl ring at
the 6-position. The less potent unsubstituted phenyl ring
was chosen over the aminophenyl ring at the 6-position
to avoid quinone formation of that phenyl ring in the
final step of the synthesis. The synthesis was carried out
as shown in Scheme 2. The methoxybenzene 11b was
treated with boron tribromide to afford the phenol 13.
Boron tribromide was used as the reagent to provide
concomitant deprotection of the methyl ether and Cbz
group affording the desired phenol 13 with the 2-
hydroxy serving as the hydrogen bond accepting group
to interact with Gly 216. Reacting the phenol 13 with
Fremy’s salt11 afforded the targeted benzoquinone 14.
The amino group attached to the P2 phenyl ring forms
tight interactions with Asp 60, Tyr 94, and the carbonyl
oxygen of Thr 98. Potential collision of the phenyl group
of the inhibitor at P2 with the side chain of Tyr 99 in
Factor Xa is probably why it does not inhibit Factor Xa.
The target compounds were screened for potency on
TF/VIIa and for other enzymes affecting coagulation to
determine specificity (Table 1). Each enzyme assay con-
sists 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 of p-nitroaniline when the substrate is
hydrolyzed. Inhibition of the enzyme reduces the change
in absorbance with the data reported as IC50 values.
Compound 12a was the most potent compound
(IC50=340 nM) on TF/VIIa, having the amino sub-
stitution on the P2 phenyl ring. The benzoquinone 14,
which maintains the ketone at the 2-position from the
pyrazinone core, also exhibited activity on TF/VIIa
with excellent selectivity over both Factor Xa and
thrombin. The 2-hydroxybenzene 13 and 2-methoxy-
benzene 12b analogues were both active against TF/
VIIa with the 2-hydroxy compound having potency
Table 1. IC50 values
Figure 2. Crystal structure of 12a (fluorobenzene) bound in the active
site of TF/VIIa complex. Crystals of TF/VIIa complex were obtained
by slight modification of the procedure described by Banner et al.8a
The structure has been refined to an Rfree of 29.1% at 2.4 A resolution
(Rcrystal: 23.7%). Some of the key side chains of Factor VIIa are dis-
played (C: green, N: dark blue, O: red, S: yellow and H: orange). The
inhibitor is represented with carbon, nitrogen, oxygen, and fluorine
atoms displayed in gold, blue, red, and purple, respectively. The
hydrogen bonds formed by the inhibitor are shown in dotted white
lines.
Compd
IC50 (uM)
Xa
VIIa
Thrombin
12a
12b
13
0.34
14.7
2.5
>30
>30
>30
>30
0.95
7.9
>30
>30
14
2.8

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J.J.Parlow et al./ Bioorg.Med.Chem.Lett.13 (2003) 3721–3725
benzene 12a and benzoquinone 14 inhibitors bound to
TF/VIIa were obtained. The crystal structure of the
fluorobenzene 12a analogue clearly shows that the
fluorine acts as the hydrogen bond acceptor and enga-
ges with Gly 216. Similarly, one of the ketones of the
benzoquinone 14 registers with the peptide backbone
of Gly 216 via a hydrogen bond. The progression of
the synthesis of substituted benzene analogues from
the discovery of the lead compound to the develop-
ment of potent analogues will be the topic of a future
publication.
Acknowledgements
The authors thank Rhonda M. Lachance for the biolo-
gical evaluation and Dr. Huey Shieh for some of the
early crystallographic refinements of the TF-VIIa struc-
ture. Diffraction data for the TF-VIIa complex with the
inhibitors were collected at beamline 17-ID in the facil-
ities of the Industrial Macromolecular Crystallography
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 Institute of Technology
(IIT), executed through the IIT’s Center for Synchro-
tron 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.
Figure 3. Crystal structure of 14 (benzoquinone) bound in the active
site of TF/VIIa. The structure was refined to an Rfree of 28.0% at
2.2 A resolution (Rcrystal: 22.5%). The atoms are colored as in Figure
2. Some of the key side chains of Factor VIIa are displayed. The
hydrogen bonds are shown as dotted lines (magenta). One of the qui-
none oxygen atoms accepts a hydrogen bond from the amide nitrogen
of Gly 216 as observed in the structures of pyrazinone inhibitors.
References and Notes
The crystal structure of compound 14 bound to TF/
VIIa is shown in Figure 3. The binding orientation of
the benzoquinone inhibitor 14 is similar to that of the
fluorobenzene 12a. The ion-pair that is formed by the
amidine moiety of the inhibitor with the carboxylate of
Asp 189 functions as the main anchor for the inhibitor
in the enzyme active site. In addition to this, the amidine
group also forms hydrogen bonds to the main chain
carbonyl of Gly 219 and the hydroxyl group of the side
chain of Ser 190. Three other hydrogen bonds are
formed by the inhibitor with the peptide backbone of
residues Ser 214-Gly 216 of VIIa. The amide nitrogen of
the acetate linker interacts with the main chain oxygen
of Ser 214 (3.2 A) while the secondary nitrogen
attached to the quinone scaffold donates a hydrogen
bond (3.0 A) to the carbonyl oxygen of Gly 216. One
of the quinone oxygen atoms accepts a hydrogen bond
(3.3 A) from the peptide nitrogen of Gly 216 as antici-
pated in our design. The other quinone oxygen forms
van der Waals interactions (3.5 A) with the carbonyl
oxygen of Gly 97.
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The substituted benzene analogues were successfully
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tized to prepare a benzoquinone 14. These compounds
exhibit modest potency for TF/VIIa and some of them
display excellent selectivity over Factor Xa and throm-
bin. The X-ray crystal structures of the targeted fluoro-

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