Potent 4-amino-5-azaindole factor VIIa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.06.016

The 4-amino-5-azaindole as an amidino-benzimidazole replacement is described. A series of potent and selective analogs were discovered and showed desirable ex vivo efficacy as measured by PT.

Full text of DOI:10.1016/j.bmcl.2006.06.016

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4567–4570
Potent 4-amino-5-azaindole factor VIIa inhibitors
Huiyong Hu,* Aleksandr Kolesnikov, Jennifer R. Riggs, Kieron E. Wesson,
Robin Stephens, Ellen M. Leahy, William D. Shrader, Paul A. Sprengeler,
Michael J. Green, Ellen Sanford, Margaret Nguyen, Erik Gjerstad,
Ronnel Cabuslay and Wendy B. Young
Celera Genomics, 180 Kimball Way, South San Francisco, CA 94080, USA
Received 19 April 2006; revised 3 June 2006; accepted 6 June 2006
Available online 21 June 2006
Abstract—The 4-amino-5-azaindole as an amidino-benzimidazole replacement is described. A series of potent and selective analogs
were discovered and showed desirable ex vivo efficacy as measured by PT.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The development of novel anticoagulants with improved
therapeutic indices is an attractive goal in the pharma-
ceutical industry. The activated factor VIIa-tissue factor
complex (fVIIaÆTF), which exists in the extrinsic path-
way of the coagulation cascade, has been an appealing
molecular target for the treatment of various thrombosis
related disorders.1–4
and >10-fold selectivity against the antitargets. Most
recently, we identified the 4-amino-5-azaindole (1H-pyr-
rolo[3,2-c]pyridin-4-ylamine) moiety as another viable
amidine replacement. Compound 3, which was made
as a direct comparison of 2, has further improved poten-
cy (fVIIa Ki = 0.081 lM) and selectivity (>400-fold
against antitargets). The 4-amino-5-azaindole analogs
retained desirable potency10 and ex vivo efficacy in hu-
man plasma as monitored by the coagulation assays
PT (prothrombin time) and aPTT (activated partial
thromboplastin time).11 In this communication, we dis-
close our discovery of analogs with good ex vivo efficacy
based on the 4-amino-5-azaindole.
We have previously reported on the discovery and devel-
opment of small molecule fVIIaÆTF complex inhibi-
tors.5–7 Many of these described inhibitors contain an
amidino moiety, which forms a salt bridge with Asp
189 in the S1 pocket of fVIIa providing increased bond-
ing and potency. The amidino compound 1 (Fig. 1) has a
0.013 lM inhibition Ki for fVIIaÆTF and good selectivity
(>200-fold) versus primary antitargets fXa, thrombin,
and trypsin. The strongly basic amidino group, howev-
er, is considered to be a major limitation to oral bio-
availability. We have studied the oral bioavailability of
various amidine and amidine prodrugs in our biaryl
scaffold.8 These compounds suffer from low oral bio-
availability due to low absorption or low conversion
of prodrug to parent amidine. In an effort to replace
the 5-amidino-benzimidazole, we explored less basic P1
elements including 5-azaindoles9,10 and 4-chloro-5-
azaindoles10 as well as others.9 The 5-azaindole com-
pound 2 (Fig. 1) has a potency of 0.22 lM for fVIIa
We have discovered in our 5-amidino-benzimidazole
analogs that substituents bearing an acid moiety at the
C50 position of the central aryl ring improved potency
against fVIIa by interacting with Lys 192 of the en-
zyme.5 Starting from 3, we installed various acid substi-
tutions at the C50 position in order to achieve more
desirable potency (compounds 4 and 5, Table 1). The
C50 benzoic acid 4 gave the best potency against fVIIa,
as well as ex vivo efficacy, as measured by the fVIIaÆTF
dependent clotting assay PT. However, compound 4 was
also quite active in the fVIIaÆTF independent clotting as-
say aPTT, possibly by inhibiting proteases along the
intrinsic coagulation pathway. Compound 5, although
less potent compared to 4, has moderately differentiated
the PT and aPTT. To target more specifically the
fVIIaÆTF complex, which is associated with the extrinsic
and common coagulation pathway, we choose the po-
tent compound 5 as a starting point to further optimize
our analogs for an improved PT profile.
Keywords: Factor VIIa; Tissue factor; Anticoagulant; Serine protease;
Amidine; Amidine replacement; PT; aPTT; 4-amino-5-azaindole; 1H-
pyrrolo[3,2-c]pyridin-4-ylamine.
*
Corresponding author. Tel.: +1 408 835 2435; e-mail:
huiyong.hu@yahoo.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.016

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H. Hu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4567–4570
OH
H2N
N
HN
NH OH
HO
NH OH
HO
N
N
NH OH
H2N
HN
HN
HN
H2N
O
O
O
1
2
3
Cmpd
fVIIa·TF Ki( M)
0.013
fXa Ki ( M)
Thrombin Ki ( M)
Trypsin Ki ( M)
1
2
3
2.7
30
54
90
1.0 X 102
99
3.6
12
38
0.22
0.081
Figure 1. Potency for fVIIaÆTF and selected antitargets in amidino and non-amidino scaffolds.
In our effort to improve the potency and selectivity of
the 5-azaindole series, we had identified substituted phe-
nylureas as the optimal S10 substitution.9 In the 5-azain-
dole series, the para-benzoic acid-substituted phenylurea
gave desirable potency and decreased lipophilicity,
which is believed to lower binding to serum albumin
and therefore improve the PT. The same SAR transfers
to the current series where the corresponding compound
6 (Table 2) retained potency against fVIIa and ex vivo
efficacy with a greater differentiation between PT and
aPTT. The 2,6-difluorophenylurea moiety was also
found to provide further improvement to fVIIa potency
and selectivity in 5-azaindole series.9 Accordingly, com-
pound 7 showed an increase in potency against fVIIa by
ꢀ10-fold as well as selectivity against other trypsin fam-
ily enzymes. The PT profile was further improved to
2.4 lM, while aPTT was greater than 20 lM. This 4-
amino-5-azaindole series marks an improvement in the
PT and the differentiation between the intrinsic and
extrinsic coagulation pathways when compared to the
5-azaindole series.
rile 8 followed by hydrolysis of the nitrile, deprotection of
the methyl ether, and finally esterification provided ester
9. Compound 9 was selectively ortho-formylated with
paraformaldehyde and MgCl2.13 The resulting salicylal-
dehyde was brominated with N-bromosuccinamide in
DMF, followed by benzyl protection of the phenol to af-
ford ether 10. Suzuki coupling between bromide 10 and
the boronic acid 11 led to biaryl compound 12. Subse-
quent treatment of aldehyde 12 with the diazophospho-
nate Ohira reagent14,15 generated alkyne 13. The
4-chloro-5-azaindole ring was established via a Sonagash-
ira reaction between 13 and mesylate 1416 to form a tran-
sient biaryl alkyne, which cyclized spontaneously to form
the N-mesyl indole. The indole was subsequently treated
with NaOH/MeOH to induce cleavage of the sulfonamide
and hydrolysis of the ester providing compound 15. The
chloro-substituted indole 15 was treated with NH4OAc
in meltingphenoltodisplace the4-chlorogroupproviding
the 4-amino-5-azaindole. The boc and benzyl protecting
groups were removed upon treatment with 6 N HCl(aq)
followed by hydrogenation withPearlman’s catalyst to af-
ford 16. The amine 16 was treated with 2,6-difluoroisocy-
anate 17 under basic conditions followed by purification
on reverse phase HPLC provided 7 as a mono HCl salt.
Synthesis of analog 7 is outlined in Scheme 1). Methyla-
tion of commercially available 4-methoxyphenylacetonit-
Table 1. SAR at the C50-position of central aryl ring12
NH2
R
N
O
N
H
HO
HN
OH
Compound
R
fVIIaÆTF Ki (lM)
2· PT (lM)
2· aPTT (lM)
Selectivity for fVIIa versus
Thrombina
fXab
Trypsinc
3
4
5
H
COOH
C(CH3)2CO2H
0.081
0.0013
0.015
12
22
3.7
17
1.2 · 103
4.0 · 104
6.7 · 102
5.0 · 103
2.8 · 103
4.7 · 102
3.5 · 103
3.2 · 103
4.5
6.4
>1.0 · 104
a Thrombin Ki (lM)/fVIIaÆTF Ki (lM) = fold selectivity.
b fXa Ki (lM)/fVIIaÆTF Ki (lM) = fold selectivity.
c Trypsin Ki (lM)/fVIIaÆTF Ki (lM) = fold selectivity.

H. Hu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4567–4570
4569
Table 2. SAR on distal aryl ring12
O
NH2
OH
N
O
N
H
HO
HN
R
Compound
R
fVIIaÆTF Ki (lM)
2· PT (lM)
2· aPTT (lM)
Selectivity for fVIIa versus
Thrombin
fXa
Trypsin
5
0.015
6.4
17
>1.0 · 104
2.8 · 103
3.2 · 103
OH
H
N
6
7
0.020
5.8
2.4
>20
>20
>7.5 · 103
>5.8 · 104
5.5 · 103
2.8 · 104
5.5 · 103
2.9 · 104
OH
O
F
H
N
0.0026
F
In conclusion, we have identified potent and selective
fVIIaÆTF inhibitors in a novel non-amidino scaffold
which differentiates between the intrinsic and extrinsic
coagulation pathways. This series was dropped in lieu
of pursuing a more attractive series and absorption data
were never obtained. We would like to suggest that from
a potency standpoint, the 4-amino-5-azaindole is a suit-
able surrogate for the benzamidine and that others con-
(HO)2B
MeO2C
NHBoc
MeO2C
NC
MeO2C
11
a-d
e,f,g
90%
h
H
O
H
Br
NHBoc
58%
95%
OBn
OMe
O
OBn
OH
9
10
12
8
Cl
I
N
HO2C
MeO2C
14
NHMs
i
l, m, n
15%
j, k
Cl
NHBoc
NHBoc
85%
63%
NH OBn
OBn
N
15
13
F
HO2C
OCN
F
17
H2N
N
o
NH2
7
NH OH
30%
16
Scheme 1. Reagents and conditions: (a) t-BuOK, MeI, THF, rt; (b) KOH, ethylene glycol/water (5:1), 150 ꢁC; (c) PyÆHCl, 180 ꢁC; (d) SOCl2, MeOH;
(e) MgCl2, anhydrous paraformaldehyde, Et3N, CH3CN, reflux; (f) NBS, DMF; (g) BnBr, DIEA, CH3CN; (h) 11, Pd(PPh3)4, K2CO3, DME, reflux;
(i) Ohira’s reagent, K2CO3, MeOH; (j) 14, PdCl2(PPh3)2, CuI, Et3N, CH3CN, 80 ꢁC; (k) 50% NaOH, MeOH, 60 ꢁC; (l) NH4OAc, PhOH, 105 ꢁC; (m)
6 N HCl, reflux; (n) 20% Pd(OH)2 on carbon (Pearlman’s catalyst)/H2, EtOH; (o) 17, Et3N, DMF.

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H. Hu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4567–4570
assays (pH 7.4) and forms water bridged hydrogen bonds
with Asp 189 of the enzyme to afford desired nanomolar
potency against fVIIa. The 4-chloro-5-azaindole P1, which
has a calculated pKa ꢀ5.7, is not protonated and therefore
has little interaction with Asp 189, which is consistent with
the loss of potency against fVIIa (micro-molar Ki for
fVIIa.TF). The 4-amino-5-azaindole P1, which has a
calculated pKa ꢀ9.9, is mostly protonated and forms a
stronger interaction with Asp 189 through water bridged
hydrogen bonds. In addition, the 4-amino group is also
thought to interact with G219 of the enzyme. Both
interactions are considered to contribute to the improved
potency.
sider such a moiety in their drug development programs,
especially in the challenging arena of developing fVIIa
inhibitors.
References and notes
1. Harker, L. A.; Hanson, S. R.; Wilcox, J. N.; Kelly, A. B.
Haemostasis 1996, 26, 76.
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12. Inhibition assays for factor Xa and thrombin were
performed as described (Cregar, L.; Elrod, K. C.;
Putnam, D.; Moore, W. R. Arch. Biochem. Biophys.
1999, 366, 125), with the pH adjusted to 7.4. The
trypsin and fVIIa assays were performed and analyzed
as in the above reference with the following additional
details. Factor VIIa (Enzyme Research) was incubated
at 7 nM and CH3SO2-D-CHA-But-Arg-pNA (Center-
chem) was used as the substrate. The buffer for the
factor VIIa assay was supplemented with 11 nM relip-
idated tissue factor and 5 mM CaCl2. Trypsin (Athens
Research Institute) was incubated at 10 nM with vari-
able 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 (Centerchem), supplied at the
Km (25 lM). Coagulation assays (PT and aPTT) were
carried out at 37 ꢁC in human plasma using a Beckman
Coulter ACL100 in accordance with the manufacturer’s
instructions.
4. Olivero, A. G.; Eigenbrot, C.; Goldsmith, R.; Robarge,
K.; Artis, D. R.; Flygare, J.; Rawson, T.; Sutherlin, D. P.;
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Katz, B.; Yu, C.; Cabuslay, R.; Sanford, E.; Janc, J.
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Shrader, W. D.; Stephens, S.; Liu, L.; Pan, L.; Mordenti,
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Wesson, K. E.; Shrader, W. D.; Vijaykumar, D.; Wahl, T.
A.; Tong, Z.; Sprengeler, P. A.; Green, M. J.; Yu, C.;
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Rai, R.; Kolesnikov, A.; Sprengeler, P. A.; Torkelson, S.;
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¨
Synthesis 2004, 59.
16. The synthesis of intermediate 14 is shown in Scheme 2.
Commercially available 4-amino-2-chloropyridine 18 was
iodinated to afford a mixture of iodopyridines 19 and 20
(ꢀ6:4). Compound 20 can be easily isolated by chroma-
tography on silica. Treatment of 20 with methanesulfonyl
chloride led to a mixture of monomesylate 14 and bis-N-
mesylated byproduct. Subsequent treatment with NaO-
H(aq)/THF converted the bismesylate to monomesylate
14.
10. The 5-azaindole, which has a calculated pKa ꢀ7.8 (ACD
Labs/pKa DB), is thought to be partially protonated in the
Cl
Cl
Cl
Cl
I
NHMs
I
NH2
N
b, c
N
a
N
N
+
NH2
NH2
I
18
20
14
19
Scheme 2. Reagents and conditions: (a) ICl, KOAc/HOAc, 60–70 ꢁC; (b) MsCl, Et3N, DCM, 0 ꢁC; (c) 10% NaOH, THF.