The discovery of fluoropyridine-based inhibitors of the Factor VIIa/TF complex

Article abstract of DOI:10.1016/j.bmcl.2005.07.059

The activated Factor VII/tissue factor complex (FVIIa/TF) plays a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. An X-ray crystal structure of a fluoropyridine-based FVIIa/TF inhibitor bound in the active site of the enzyme complex suggested that incorporation of substitution at the 5-position of the hydroxybenzoic acid side chain could lead to the formation of more potent inhibitors through interactions with the S1′/S2′ pocket.

Full text of DOI:10.1016/j.bmcl.2005.07.059

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4752–4756
The discovery of fluoropyridine-based inhibitors of the Factor
VIIa/TF complex
Jeffrey T. Kohrt,a,* Kevin J. Filipski,a Wayne L. Cody,a Cuiman Cai,a Danette A. Dudley,a
Chad A. Van Huis,a J. Adam Willardsen,a Stephen T. Rapundalo,a Kamlai Saiya-Cork,a
Robert J. Leadley,a Lakshmi Narasimhan,a Erli Zhang,a Marc Whitlow,b Marc Adler,b
Kirk McLean,b Yuo-Ling Chou,b Cecile McKnight,b Damian O. Arnaiz,b
Kenneth J. Shaw,b David R. Lightb and Jeremy J. Edmundsa
aPfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
bBerlex Biosciences, 2600 Hilltop Drive, PO Box 4099, Richmond, CA 94804, USA
Received 2 June 2005; revised 25 July 2005; accepted 25 July 2005
Available online 24 August 2005
Abstract—The activated Factor VII/tissue factor complex (FVIIa/TF) plays a key role in the formation of blood clots. Inhibition of
this complex may lead to new antithrombotic drugs. An X-ray crystal structure of a fluoropyridine-based FVIIa/TF inhibitor bound
in the active site of the enzyme complex suggested that incorporation of substitution at the 5-position of the hydroxybenzoic acid
side chain could lead to the formation of more potent inhibitors through interactions with the S10/S20 pocket.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The activated Factor VII/tissue factor complex (FVIIa/
TF) is known to play a key role in the formation of
blood clots.1 Theoretically, inhibition of either the
FVIIa/TF complex formation or its active site responsi-
ble for the cleavage of Factor X (FX) to activated Fac-
tor X (FXa) will block the key extrinsic coagulation
pathway, but keeping intact the intrinsic pathway to sus-
tain normal hemostasis. This may lead to enhanced anti-
thrombotic drugs with decreased bleeding side effects.2
There are several strategies for disruption of the
FVIIa/TF complex formation and function,3 but work
still seems to be incomplete with regard to the develop-
ment of small molecule, FVIIa/TF active-site inhibi-
tors.4 In this communication, we describe the
exploration of the structure/activity relationships of a
series of fluoropyridine-derived, small molecule inhibi-
tors that appeared to utilize the S10/S20 binding pocket
of the FVIIa/TF active site.
no measurable inhibition of FXa (IC50 > 10 lM,
Fig. 1). Synthesis of a subsequent analog substituted
at the 5-position of the hydroxybenzoic acid side chain
of
1 led to a more potent 5-dimethylamino 2
(IC50 = 190 nM). This result suggested that substitution
at the 5-position could be optimized further to enhance
the binding affinity of a new class of FVIIa/TF inhibi-
tors. Also in this series, a limited optimization of the
4-position of the central pyridine ring led to the potent
FVIIa/TF inhibitor 3 (IC50 = 86 nM).
Ph
NH
F
F
F
F
O
N
O
O
N
O
CO2H
CO2H
From a focused screening of compounds previously syn-
thesized as potential FXa inhibitors,5 we identified 1
(IC50 = 1200 nM) as a modest FVIIa/TF inhibitor with
H2N
H2N
NH
R
NH
R
5-position
1 R = H
IC50 = 1200 nM
3 R = H IC50 = 86 nM
2 R = NMe2 IC50 = 190 nM
*
Corresponding author. Tel.: +1 734 622 1371; fax: +1 734 622
3909; e-mail: jeffrey.kohrt@pfizer.com
Figure 1. Lead inhibitors of FVIIa/TF.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.059

J. T. Kohrt et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4752–4756
4753
Analysis of the X-ray crystallography data of a complex
of 3 (R = H) with TF/FVIIa showed that the R-methyl
enantiomer 4-R (coordinates deposited in the RCSB
database as PDB ID code 2AEI) crystallized with the
phenylamidine pharmacophore hydrogen bonding to
the Asp189, Ser190, and Gly219 in the S1 pocket of
the FVIIa active site, while the benzoic acid moiety
interacted with Ser195, Gly193, and His57 in the oxy-
anion hole (Fig. 2). The 1-methyl-3-phenyl-propylamine
side chain was shown to be filling the S2 and S3 binding
pockets through hydrophobic interactions with the
(R)-methyl and phenyl groups, respectively.
S1’/S2’
S1
Oxy-Anion
Hole
S3
The crystal structure also revealed a potential binding
pocket close to the carboxylate group bound in the
oxy-anion hole. This area of the active site is made up
of both the S10 and S20 pockets (Fig. 3). Therefore, ana-
logs that could access this S10/S20 pocket via incorpora-
tion of a substituent at the 5-position on the benzoic
acid side chain should result in more potent FVIIa/TF
inhibitors.
S2
Figure 3. Connolly surface depicting the binding pockets of the FVIIa/
TF active site with respect to 4-R. The enzyme is colored by residue
electrostatic charge: red, negatively charged residue; blue, positively
charged residue; white, residue of negligible charge.
The synthesis of these analogs started with the 5-substi-
tuted salicylates 6 and 8 (Scheme 1). Formation of
5-alkyl substituted 6 resulted from heating a carbon tet-
rachloride solution of 4-alkyl-phenol 5 (R1 = alkyl
group) in the presence of copper and base, followed by
acid-catalyzed esterification.6 5-N,N-Dialkylamino
substituted 8a was formed by reductive amination of
OH
OH
CO2Me
a, b
R1 = Alkyl
OH
R1
R1
5
6
OH
CO2Me
c or d or e
CO2Me
R3
R
3 = N(Alkyl)2 = 8a
OAlkyl = 8b
Ph = 8c
R2
S3
Ph
S2
8
7
Scheme 1. Reagents and conditions: (a) R1 = alkyl group; CCl4, Cu,
KOH, H2O, reflux; (b) H2SO4, MeOH, reflux; (c) R2 = NH2; R3CHO,
HOAc, NaBH(OAc)3, DCE, RT; (d) R2 = OH; Alkyl-I, K2CO3,
acetone, reflux; (e) R2 = I; PhB(OH)2, K3PO4, Pd(PPh3)4, DMF,
110 ꢁC.
S1'/S2'
S1
HN
Ser190
HO
F
F
NH2
O
N
O
O
H
NH2
N
O
O
H2
N
O
Asp189
O
Gly193
OH
His57
Gly219
Ser195
HN
an alkylaldehyde, and 7 (R2 = NH2), while 5-alkoxy
Oxy-anion
Hole
4-R
substituted 8b was synthesized by heating
7
(R2 = OH), an alkylhalide, and dimethylformamide in
the presence of sodium hydride.7 Finally, formation of
the 5-phenyl analog of 8c was dependent on the palladi-
um-catalyzed Suzuki coupling of 7 (R2 = I) and phen-
ylboronic acid.8
( )-1-Methyl-3-phenyl-propylamine 9 was added to
pentafluoropyridine in the presence of triethylamine to
provide a 4-amino-substituted pyridine (Scheme 2).
Addition of the salicylate side chains was effected in a
two-step procedure by first adding the trifluoromethy-
loxadiazole-phenol 104c in the presence of potassium
carbonate to give the aryl ether 11. Subsequent addition
of a 5-substituted salicylate 6 or 8 in the presence of cesi-
um carbonate produced the trisubstituted pyridine 12.
Hydrogenation of the oxadiazole over Raney nickel
revealed the amidine.4c Final hydrolysis of the methyl
ester with lithium hydroxide produced analogs 15–29.
All inhibitors were isolated by reverse-phase preparative
HPLC as TFA salts.
Figure 2. Crystal structure of 4-R in the active site of the FVIIa/TF
complex.

4754
J. T. Kohrt et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4752–4756
Ph
Ph
Ph
O
N
PMB
F
PMB
F
F3C
OH
NH
N
N
N
N
N
N
F
F
F
F
F
10
a, b
a, b
Ph
NH2
O
O
O
O
O
(+/-) 9
CO2Me
CO2Me
11
N
N
N
O
OH
O
O
N
CO2Me
N
I
N
13
14
O
R4
c
F3C
F3C
F3C
6 or 8
R1,3
c - e
Ph
Ph
Ph
NH
NH
NH
N
F
F
F
F
d, e
F
F
O
O
N
O
O
N
O
O
CO2H
CO2Me
CO2H
H2N
TFA
N
H2N
TFA
O
R1,3
R1,3
NH
N
NH
15 - 29
12
F3C
O
R4
30 - 41
Scheme 2. Reagents and conditions: (a) pentafluoropyridine, Et3N,
CH2Cl2, RT; (b) 10, K2CO3, DMSO, 70 ꢁC; (c) 6 or 8, Cs2CO3,
DMSO, 80 ꢁC; (d) Raney nickel, H2, Et3N, MeOH, RT; (e) LiOH,
water, dioxane, RT.
Scheme 3. Reagents and conditions: (a) CO, Pd(OAc)2, K2CO3, DMF/
H2O (4:1), 100 ꢁC; (b) amine, HOAt, HATU, Et3N, DMF, RT; (c) H2,
Raney nickel, MeOH, Et3N, RT; (d) LiOH, MeCN, H2O, RT; (e)
TFA, CH2Cl2, RT.
Synthesis of the isophthalamic acid derivatives 30–41
was achieved via a Pd(0)-catalyzed carboxylation of
5-iodo 13 to yield carboxylic acid 14 (R4 = OH).9 Using
p-methoxybenzyl (PMB) protected 9, formed via reduc-
tive amination with 4-methoxy-benzaldehyde and sodi-
um triacetoxyborohydride, the 5-iodo 13 was prepared
in a similar manner to 12. The use of this protecting
group was necessary to obtain good yield in the subse-
quent carboxylation step. The amides were formed with
amines via a HOAt/HATU mediated coupling reac-
tion.4c Raney nickel reduction of the oxadiazole to the
amidine and LiOH hydrolysis of the ester were followed
by TFA-mediated deprotection of the PMB protecting
group to reveal the analogs 30–41 (Scheme 3).
Table 1. 5-Substituted FVIIa/TF inhibitorsa
FVIIa/TF R1,3
inhibitors
FVIIa/TF
11
IC50 (lM) (lM)
2· PT FXa
12
IC50 (lM)
3
H
0.086
0.045
0.015
0.020
0.021
0.079
0.013
0.016
0.017
0.036
0.056
0.017
0.019
0.029
0.039
35
29
55
51
4-R
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
H
iPr
28
4.8
29
CH3
t-Bu
NH2
NMe2
NEt2
NHMe
NBu2
OH
ND
24
3.3
11
ND
49
6.8
9.6
8.0
ND
11
83
41
ND
ND
52
OiPr
OPr
OEt
OMe
12
26
To enhance the binding affinity of this series of inhibi-
tors, investigation of analogs with substitution at the
5-position of the hydroxybenzoic acid side chain was
undertaken (Table 1). Substitution of the 5-hydrogen
with a 5-methyl group on the hydroxybenzoic acid ring
resulted in 16 with a 4-fold increase in binding
(IC50 = 20 nM) from our lead 3. Compared to 16, addi-
tion of a larger tert-butyl group 17 (21 nM), phenyl
group 29 (23 nM), or isopropyl group 15 (15 nM) did
not substantially increase the binding. Unsubstituted
hydrogen-bond-donating groups, such as the 5-amino
analog 18 and 5-hydroxy analog 23, decreased the bind-
ing from 16 by 3.5- and 2.5-fold respectively, but alkyl-
ation of the respective nitrogen or oxygen atom regained
potency. For example, mono- or dialkylation of 5-amino
18 produced analogs 19–22 with an increased potency of
approximately 2- to 5-fold over 18. Similarly, alkylation
of 5-hydroxy 23 produced alkoxy analogs 24–27 with in-
creased potency. Finally, incorporation of a 4-substitu-
ent on the hydroxybenzoic acid ring did not increase
the binding. For example, analog 28 (15 nM) substituted
by a 4-methyl substituent showed potency similar to the
95
94
17
11
ND
16
NMe2 4-Me 0.015
Ph 0.023
4.5
17
112
a All compounds tested as racemic mixtures except where noted.
4-H analog 19 (13 nM). In all cases, these inhibitors
maintained selectivity for the inhibition of FVIIa/TF
over that of FXa.
Molecular modeling did not provide clues as to addi-
tional interactions that led to higher binding affinities
observed for the 5-substituted inhibitors, but it did
indicate that the S10/S20 pocket was potentially a deep-
er binding site than was assumed initially. Additional
attempts to fully exploit this binding pocket to gain
more potent FVIIa/TF inhibitors were explored
through the synthesis of analogs containing an amide
at the 5-position of the hydroxybenzoic acid side chain
(Table 2).4c,10

J. T. Kohrt et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4752–4756
4755
Table 2. 5-Benzamide FVIIa/TF inhibitors
the 4-methyl substituted 28 (16 lM) had a significant
lowering of the 2· PT value from the parent 3.
FVIIa/TF
inhibitors
R4
FVIIa/TF 2· PT FXa
IC50 (lM) (lM) IC50 (lM)
While it appeared that the amide series of inhibitors fa-
vored branched alkyl and benzyl-amides for a modest
increase in binding affinity, a more significant relation-
ship was apparent in regard to the lowering of 2· PT
potency (Table 2). Branched alkyl-amides had a superi-
or doubling of PT than the benzyl-amides. While both
the isobutyl amide 33 and 3-methoxybenzyl-amide 30
had similar binding affinities, the former analog was
2.5-fold more potent for the doubling of PT. Another
2-fold improvement in 2· PT potency was gained in
the alkyl-hydroxyalkyl-amide series. For example,
weaker binding inhibitors, such as diol 39
(IC50 = 46 nM), offered a better 2· PT profile (9.0 lM)
than the stronger binding 3-methoxybenzyl-amide 30
(IC50 = 8 nM, 2· PT = 53 lM). While we are uncertain
as to the reasons for the variations in the observed
2· PT data, it has been suggested that the changes in
pharmacological parameters (clogP, solubility, perme-
ability, protein binding, etc.) resulting from the hydroxyl
groups are accounting for some of the differences.14
OMe
OH
HN
30
31
32
33
34
35
36
37
38
39
40
41
0.008
0.039
0.140
0.010
0.014
0.067
0.071
0.016
0.023
0.046
0.200
0.730
53
9.5
4.1
ND
9.6
4.3
20
HN
49
OH
OH
HN
ND
22
HN
HN
15
OH
16
HN
HN
12
ND
5.9
5.7
13
OH
OH
11
HN
HN
Examination of 5-substituted hydroxybenzoic acid side
chains of a fluoropyridine-based series of FVIIa/TF
inhibitors showed a modest improvement in binding
affinity for this enzyme complex. Enhancement of the
interactions needed for increased binding affinity that
can lead to inhibitors that also display a more potent
2· PT profile has not yet been fully optimized. Further
investigations into the synthesis of analogs that continue
to probe the S10/S20 pocket are in progress.
10
OH
OH
9.0
ND
ND
OH
HN
OH
HO
ND
ND
O
OH
HN
Acknowledgment
The authors thank Yun-Wen Peng for her work on the
PT assay.
A substantial increase in FVIIa/TF binding affinity was
obtained by the formation of 3-methoxybenzyl-amide 30
(8 nM). All other substituted benzyl-amides showed de-
creased binding affinity from 30. Interestingly, the isobu-
tyl-amide 33 (10 nM) was of similar potency to analog
30, while the trimethylpropyl-amide 34 (14 nM) showed
only a small loss in activity. Reduced binding was also
observed in the hydroxyalkyl-amides 35 and 36, but
the alkyl-hydroxyalkyl-amides 37–39 helped to regain
some binding activity. The serine-based amide 41
(730 nM) was an exception displaying significantly re-
duced activity potentially as a result of the presence of
the free carboxylic acid. Likewise, the incorporation of
a carboxylic acid 40 (200 nM) directly at the 5-position
also significantly decreased binding.
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