Novel 5-azaindole factor VIIa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.03.049

The discovery and development of 5-azaindole factor VIIa inhibitors will be described.

Full text of DOI:10.1016/j.bmcl.2006.03.049

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3197–3200
Novel 5-azaindole factor VIIa inhibitors
Jennifer R. Riggs,* Huiyong Hu, Aleksandr Kolesnikov, Ellen M. Leahy,
Kieron E. Wesson, William D. Shrader, Dange Vijaykumar, Troy A. Wahl,
Zhiwei Tong, Paul A. Sprengeler, Michael J. Green, Christine Yu, Brad A. Katz,
Ellen Sanford, Margaret Nguyen, Ronnel Cabuslay and Wendy B. Young
Celera Genomics, 180 Kimball Way, South San Francisco, CA 94080, USA
Received 1 February 2006; revised 15 March 2006; accepted 16 March 2006
Available online 18 April 2006
Abstract—The discovery and development of 5-azaindole factor VIIa inhibitors will be described.
Ó 2006 Elsevier Ltd. All rights reserved.
In an effort to develop novel anti-thrombotic agents, we
have been actively pursuing inhibition of the trypsin-like
serine protease, factor VIIa (fVIIa). Although inhibition
of related proteases, such as fIIa and fXa, has proven
efficacious, inhibition earlier in the coagulation cascade,
such as factor VIIa, may provide a safety advantage.1–5
Our previous reports focus on the discovery and optimi-
zation of potent and selective amidine-containing P1
fVIIa inhibitors.6,7
scaffold represented a promising lead with a less basic P1
and moderate potency and selectivity.
Toward our goal of a potent and selective fVIIa inhibi-
tor, we sought fVIIa potency <0.100 lM with selectivity
>250-fold against the three main anti-targets fXa,
thrombin, and trypsin. To gain potency and selectivity
in our 5-azaindole series, we began to optimize for the
S10 pocket. This region offers variety within the tryp-
sin-like serine proteases affording the opportunity for
selectivity and affinity.
Earlier, we reported on the development of compounds
such as 1 as an injectable agent for the treatment of
thromboembolic disorders (Fig. 1).7 These biaryl ami-
dine-containing compounds suffer from low oral bio-
availability.8 In an attempt to apply the knowledge we
have gained from our amidine fVIIa inhibitor parenteral
program to an oral program, we began with our 5-ami-
dinobenzimidazole (1) scaffold and replaced the amidine
with a 5-azaindole (1H-pyrrolo[3,2-c]pyridine, 2) in con-
junction with removal of the distal phenol to decrease
the overall polar surface area. The amidine-containing
analog has good potency for fVIIa (0.013 lM) and
>200-fold selectivity versus the primary anti-targets
fXa, fIIa (thrombin), and trypsin. The 5-azaindole
retains respectable potency at 0.80 lM and >50-fold
selectivity for the anti-targets. Overall, this non-amidino
To exploit the S10 pocket for potency and selectivity, a
number of analogs based on compound 2 were generat-
ed. Of these, 33 selected compounds are depicted in
Table 1. Of the 33 compounds generated, a range of
potencies (fVIIa Ki 0.028–130 lM) and selectivities
versus the anti-targets (1–5000) were achieved including
24 compounds superior to the initial urea lead 2.
Keywords: Factor VIIa; Anti-coagulant; Serine protease; Amidine;
Amidine replacement; Azaindole; Pyrrolopyridine.
*
Figure 1. Potency for fVIIa and selected anti-targets in an amidino and
non-amidino scaffold.9,10
Corresponding author. Tel.: +1 415 846 3717; e-mail: jennifer.
riggs@yahoo.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.049

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J. R. Riggs et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3197–3200
Table 1. Inhibition data for compounds 2–34
NH OH
N
NH
O
R
Compound
R
fVIIa Ki (lM)
Xa
156
IIa
89
Trypsin
2
3
NH2
Pentylamine
0.80
0.335
0.27
0.35
0.0615
0.057
0.13
0.11
0.033
0.10
0.33
0.83
0.057
0.047
0.10
0.088
0.028
0.06
0.19
0.058
0.16
0.13
2.7
58
181
96
388
417
429
1008
1930
923
382
2697
1700
455
181
2632
1723
1600
705
2643
2167
789
966
600
623
67
343
174
169
2114
2456
1154
1364
3939
1200
455
181
2632
3191
1500
1023
3179
2000
305
2241
938
769
56
4
Phenethylamine
2-Thiophen-2-yl-ethylamine
Phenylamine
5
120
2033
2632
1154
1364
2182
1500
455
181
2632
3191
1500
1591
5000
2167
495
1103
538
1154
56
6
7
2-Fluoro-phenylamine
3-Fluoro-phenylamine
4-Fluoro-phenylamine
2,6-Difluoro-phenylamine
2,4-Difluoro-phenylamine
3,4-Difluoro-phenylamine
3,5-Difluoro-phenylamine
2-Chloro-phenylamine
2-Methoxy-phenylamine
3-Methoxy-phenylamine
4-Methoxy-phenylamine
2,4-Dimethoxy-phenylamine
1-(3-Amino-phenyl)-ethanone
1-(4-Amino-phenyl)-ethanone
4-Amino-benzoic acid
Thiophen-2-ylamine
Thiophen-3-ylamine
Pyridin-2-ylamine
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Pyridin-3-ylamine
Pyrrolidine
0.155
17
635
3
968
5
935
9
1-Methylpiperazine
Toluene
130
1
46
1
24
1
26
2.0
9.5
1,3-Difluoro-2-methyl- benzene
2-Phenyl-ethanol
10
16
16
57
0.22
0.61
0.85
2.8
136
149
80
470
107
68
2-Pyridin-3-yl-ethanol
Phenyl-methanol
Propyl-benzene
34
57
61
653
28
750
10
375
N-Benzyl-hydroxylamine
0.072
Data shown are factor VIIa Ki and fold-selective ratios (anti-target Ki/fVIIa Ki) for coagulation factors Xa, IIa (thrombin), and trypsin.9,10
Incorporation of an alkyl moiety on the urea as in 3
yielded a moderate increase in potency and selectivity
versus 2. Remarkably, addition of a phenyl group on
the urea as in 6 offered a 10-fold increase in potency
Phenyl isosteres such as thiophenes and heterocycles
such as pyridines were not well tolerated (compounds
22–25). Finally, cyclic ureas such as compounds 26
and 27 lost potency for all the serine proteases
tested, emphasizing the importance of the distal urea
N–H.
and
a 6- to 35-fold increase in selectivity as
compared to 2. To further develop 6, substituted
phenylurea analogs (7–21) were produced. Interesting-
ly, ortho-substitution seems to be well tolerated often
increasing potency and selectivity, while meta- and
para-substitution often leads to decreased potency
and selectivity (e.g., compounds 7–9 and 15–17). Fol-
lowing this trend, 2,6-substituted fluoro compound 10
showed a 2-fold increase in potency and selectivity
over the phenyl analog 6. Substitution of the
terminal aryl ring with either electron-donating or
electron-withdrawing groups was tolerated, suggesting
that the advantage of substitution may be to disrupt
the planarity between the urea and the phenyl group.
To diversify away from ureas, a number of amides
were completed based on the same base scaffold
(Table 1). The amide analog 28 was found to be
30-fold less potent and >20-fold less selective than
the homologous urea counterpart 6. This trend
continues as the 2,6-difluoromethyl amide 29 is far less
potent and selective than the corresponding urea 10.
Substitution of the phenethyl chain with an a-hydrox-
yl group (30) via coupling of the amine with L-phen-
yllactic acid surprisingly retrieves some potency and
selectivity. As might be predicted, the N-hydroxy urea

J. R. Riggs et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3197–3200
3199
34 retains intermediate values of potency and
selectivity between urea 6 and the phenyllactic amide
30 through a possible H-bonding interaction similar
to the N–H of the urea.
Synthesis of the urea S10 analogs began with the
commercially available 2-bromophenol 35 (Scheme 1).
Selective ortho-formylation using paraformaldehyde
and MgCl2 followed by benzyl protection provided
salicylaldehyde 36.12 Palladium-catalyzed Suzuki cou-
pling between bromide 36 and 3-cyanophenylboronic
acid followed by conversion of the aldehyde to the al-
kyne via the diazophosphonate Ohira reagent gave
intermediate 37. Construction of the indole ring was
achieved using a Sonogashira coupling of the alkyne
37 and mesylate 38.13 Reduction of the nitrile and
benzyl ether of 39 was achieved via hydrogenation
with Pearlman’s catalyst followed by addition of
phenylisocyanate to provide the target urea 6. Urea
compounds 3–25 were constructed following the above
procedure. Amides 28–33 were completed using
standard amide coupling procedures with the
deprotected benzylamine of 39.
In an attempt to understand the binding of this
structural series, we obtained the crystal structure of
compound 2 in fVIIa (Fig. 2). An array of hydrogen
bonds to the catalytic residues are observed including
the short interactions between Ser195, the hydroxypy-
razole oxygen, and a water molecule in the oxyanion
hole. The residues from 215 to 220 which define one
side of the S1 pocket are significantly rearranged from
the position observed for the amidine-based inhibitors
such that the indole ring of Trp215 is relocated into
the position of the S2 pocket. This ring now provides
an edge to face contact with the phenol ring of the
inhibitor. The P1 pyridine ring does not interact
directly with the enzyme but is likely protonated and
binds through
a
water bridge to Asp189. The
The replacement of the amidino P1 group in trypsin-
family serine protease inhibitors has been a major fo-
cus of research in the pharmaceutical industry. Herein,
we have demonstrated that the 5-azaindole is a viable
replacement for the 5-amidinobenzimidazole for fVIIa
inhibition. Furthermore, the P10-phenylurea 6 provides
a substantial increase in potency and selectivity from
our original azaindole lead 2. A systematic SAR study
on phenylurea 6 revealed that ortho-substitution was
optimal with 2,6-difluoro compound 10 increasing
potency 25-fold and selectivity over 15-fold for all
anti-targets compared to the initial lead 2. Crystallo-
graphic data support the potency achieved by the
5-azaindole urea 2 and phenylurea 6 through a combi-
nation of hydrogen-bonding effects and phenyl edge to
face interactions. These potent and selective non-ami-
dino fVIIa inhibitors are undergoing pharmacokinetic
substituent on the distal ring of 2 does not extend into
the S10 pocket. The distal phenyl ring on the urea
likely binds in an edge to face interaction with the
rearranged Trp215.
evaluation and will be presented in
publication.
a
future
Acknowledgments
The authors thank Colin O’Bryan and Robert Yee for
the synthesis of key intermediates.
Figure 2. Crystal structure of 2 bound to fVIIa.11
I
a,b
c,d
N
N
H
+
Br
Br
NHMs
H
OBn
OH
35
O
OBn
36
37
38
O
N
g,h
e,f
N
N
H
H
NH OBn
NH OH
N
N
39
6
Scheme 1. Synthesis of 6. Reagents and conditions: (a) MgCl2, TEA, paraformaldehyde, 95%; (b) BnBr, K2CO3, 95%; (c) 3-benzonitrile boronic acid,
Pd(PPh3)4, K2CO3, DME, reflux, 80%; (d) (1-diazo-2-oxo-propyl)-phosphonic acid dimethyl ester (Ohira reagent), K2CO3, MeOH, 80%; (e)
PdCl2(PPh3)2, TEA, CuI; (f) NaOH, MeOH, reflux, 60% over 2 steps; (g) Pd(OH)2, H2, MeOH, trace of HCl, 95%; (h) phenylisocyanate, TEA,
DMF, 30–50%.

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J. R. Riggs et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3197–3200
human tissue factor (11 nM) with variable concentra-
References and notes
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NaCl, 5.0 mM CaCl2, 1.5 mM EDTA, 0.05% Tween
20, and 10% DMSO. The reaction was initiated with
the addition of substrate, CH3SO2-D-CHA-But-Arg-
pNA (Centerchem), supplied at the Km (500 lM). The
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9. General inhibition assays inhibitor potency measure-
ments were performed at room temperature using
either a Molecular Device’s SpectraMax 250 (absor-
bance assays) or fMax (fluorescence assays) 96-well
kinetic plate readers. Reaction velocities were moni-
tored at varying inhibitor concentrations by following
change in absorbance as
a function of time was
monitored at 405 nm. Factor IIa (thrombin): thrombin
(Calbiochem) was incubated at 12.7 nM with variable
concentrations of inhibitor in 50 mM Tris (pH 7.4),
150 mM NaCl, 5.0 mM CaCl2, 1 mM EDTA, 0.05%
Tween 20, and 10% DMSO. The reaction was
initiated with the addition of substrate, Tosyl-Gly-
Pro-Lys-pNA (Centerchem), supplied at the Km
(25 lM). The change in absorbance as a function of
time was monitored at 405 nm. Factor Xa: factor Xa
(Haematologic Technologies) was incubated at 2 nM
with variable concentrations of inhibitor in 50 mM
Tris (pH 7.4), 150 mM NaCl, 5.0 mM CaCl2, 1.5 mM
EDTA, 0.05% Tween 20, and 10% DMSO. The
reaction was initiated with the addition of substrate,
CH3OCO-D-Cha-Gly-Arg-pNA (Centerchem), supplied
at the Km (1.0 mM). The change in absorbance as a
function of time was monitored at 405 nm. Trypsin:
trypsin (Athens Research Institute) was incubated at
10 nM with variable concentrations of inhibitor in
50 mM Tris (pH 7.4), 150 mM NaCl, 1.5 mM EDTA,
0.05% Tween 20, and 10% DMSO. The reaction was
initiated with substrate, Tosyl-Gly-Pro-Lys-pNA (Cen-
terchem), supplied at the Km (25 lM). The change in
absorbance as a function of time was monitored at
405 nm.
10. The Ki apparent (K0i) values were determined by a non-
linear least-squares regression fit of the experimentally
derived data to the Morrison equation for tight-binding
inhibitors as described Kuzmic, P. et al. Anal. Biochem.
2000, 281, 62, Enzyme and inhibitor were incubated
30 min prior to initiation of reaction with the addition of
substrate.
the hydrolysis of either p-nitroanalide (A405nm
aminomethylcoumarin substrates (ex355 em460). All
substrates were added at concentrations equal to or
near their Km. All reactions were performed in
) or
11. The PDB access code for the X-ray coordinates is 2FLR.
12. Hofslokken, N. U.; Skattebol, L. Acta Chem. Scand. 1999,
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,
a
13. Choi-Sledeski, Y. M. et al. J. Med. Chem. 2003, 46, 681,
The mesylate was prepared from the amine by treatment
with methanesulfonyl chloride, triethylamine at 0 °C
followed by 10% NaOH in THF.
total volume of 100 lLs. Control reactions in the
absence of inhibitor were performed in parallel.
Factor VIIa: factor VII (Enzyme Research) was
incubated at 7 nM together with recombinant soluble