Phenyltriazolinones as potent factor Xa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.011

We have discovered that phenyltriazolinone is a novel and potent P₁ moiety for coagulation factor Xa. X-ray structures of the inhibitors with a phenyltriazolinone in the P₁ position revealed that the side chain of Asp189 has reorient

Full text of DOI:10.1016/j.bmcl.2010.01.011

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1373–1377
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phenyltriazolinones as potent factor Xa inhibitors
*
Mimi L. Quan , Donald J. P. Pinto, Karen A. Rossi, Steven Sheriff, Richard S. Alexander, Eugene Amparo,
Kevin Kish, Robert M. Knabb, Joseph M. Luettgen, Paul Morin, Angela Smallwood, Francis J. Woerner,
Ruth R. Wexler
Research and Development, Bristol-Myers Squibb, PO Box 5400, Princeton, NJ 08543-5400, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We have discovered that phenyltriazolinone is a novel and potent P1 moiety for coagulation factor Xa. X-
ray structures of the inhibitors with a phenyltriazolinone in the P1 position revealed that the side chain of
Asp189 has reoriented resulting in a novel S1 binding pocket which is larger in size to accommodate the
phenyltriazolinone P1 substrate.
Received 17 November 2009
Revised 27 December 2009
Accepted 4 January 2010
Available online 11 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Factor Xa inhibitors
Coagulation
Cardiovascular diseases are the leading cause of death in devel-
oped countries, and most cardiovascular events are primarily due
to thrombosis.1 Despite the extensive efforts to discover and devel-
op new antithrombotic agents,2 warfarin remains the only
approved oral anticoagulant in the United States.3 Other agents
such as heparin and fondaparinux are available only by parenteral
administration. Limitations of these agents have prompted exten-
sive research for novel anticoagulants.3 Factor Xa (fXa) has been
a major focus of pharmaceutical intervention in the past decade
because of its central and unique position in the coagulation
cascade.4 Significant progress has been made in the discovery
and development of fXa inhibitors as anticoagulants. Several small
molecule fXa inhibitors have been in phase III clinical trials. Among
them the first oral fXa inhibitor, rivaroxaban, has recently been
approved in both Europe and Canada for the prophylaxis of venous
thromboembolism.5
Our efforts to discover and develop orally active inhibitors of
fXa led to clinical candidates DPC423 (1),6 razaxaban (2),7 and
apixaban (3)8 (Fig. 1). Razaxaban and apixaban were studied in
phase II clinical trials and shown to be efficacious in the treatment
of deep vein thrombosis.9 Apixaban, which has an overall better
pharmacokinetic profile, is currently in phase III clinical trials for
both venous and arterial indications.9b The discovery effort which
ultimately led to apixaban significantly advanced our understand-
ing of moieties that were sufficiently tolerated on the P1 phenyl
group.10,11 As part of our efforts to further diversify the P1 portfolio,
we discovered a novel phenyltriazolinone P1 moiety as shown in
structures 4b and 5. Herein we describe the synthesis and X-ray
crystal structures of these potent and selective fXa inhibitors
containing a phenyltriazolinone in the P1 position.
F
F3C
F
F3C
N
N
SO2Me
H
N
H
N
N
N
N
N
N
O
O
2, Razaxaban
1, DPC423
Xa Ki = 0.19 nM
Xa Ki = 0.15 nM
NH2
NH2
O
N
F3C
F
H2NOC
H
N
SO2Me
O
N
N
N
N
N
N
O
O
H
N
3, Apixaban
4b
Xa Ki = 0.08 nM
O
N
OMe
HN
R1
N
R2
N
N
O
H
N
5
O
N
HN
* Corresponding author. Tel.: +1 609 818 5301; fax: +1 609 818 3331.
E-mail address: mimi.quan@bms.com (M.L. Quan).
Figure 1. Structures of key compounds.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.011

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M. L. Quan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1373–1377
Table 1
P1 SAR in DPC423 series
The syntheses of compounds listed in Tables 1 and 2 are shown
in Schemes 1–3. The glycinamide 4a was prepared from DPC4236a
in a two step manner. DPC423 was coupled to NBocGlycine and
deprotected with TFA to afford compound 4a. Compounds 4b–d
were prepared from compound 76a (Scheme 1). The cyano group
in 7 was readily converted to the triazolinone derivative 4b by
treatment with gaseous HCl in methanol, followed by treatment
with semicarbazide/N-methylmorpholine in dioxane at reflux.
Using a similar but modified protocol, the aminothiadiazole 4c
and aminotriazole 4d were also prepared as shown in Scheme 1.
Triazolinones 5a–f were prepared in a similar fashion as shown
in Scheme 2. Compound 8 was synthesized according to the
procedures described in Ref. 10. The cyano group was converted
to triazolinone 9 as described above. Suzuki coupling of 9 with
2-formylphenylboronic acid (10), followed by reductive amination
with appropriate amines afforded compounds 5c–f in Table 2.
Suzuki coupling of 9 with 2-methylsulfonylphenylboronic acid
(11) provided compound 5a. Ullmann coupling of compound 8
with imidazole 12 gave compound 13, which was converted to
triazolinone 5b via methods described above.
F3C
F
H
SO2Me
N
N
N
O
4
R
Compd
R
fXa Ki (nM) IIa Ki (nM) Trpsin Ki (nM)
DPC423
0.15
9
6000
60
NH2
H
N
4a
4b
NH2
>21,000
1200
O
H
N
O
0.5
46
>21,000
>21,000
>2500
>2500
N
S
N
H
N
N
4c
Scheme 3 shows the synthesis for compound 5g wherein the
CF3 on the pyrazole was replaced with a carboxamide moiety.
Compounds 14 and 15 were prepared according to the procedures
described in Ref. 10. Reaction of 14 and 15 with triethylamine in
refluxing toluene gave bicycle 16. The ester in compound 16 was
converted to the carboxamide to yield 17. Formation of the
triazolinone followed by Suzuki reaction and reductive amination
as described previously produced compound 5g in Table 2.
NH2
N
N
4d
15
4900
160
HN
NH2
The Ki’s obtained from purified human enzymes and are averaged from multiple
determinations (n = 2).
Table 2
SAR of triazolinone P1 in the bicyclic series
R1
N
N
N
O
A
H
N
O
N
5
N
H
Compd
R1
A
fXa Ki (nM)
IIa Ki (nM)
PT EC2x (lM)
SO2Me
N
5a
CF3
2.2
>6000
>80
N
5b
5c
CF3
CF3
2.0
1.1
>6000
>6000
11
33
N
N
N
OH
5d
5e
CF3
CF3
0.84
0.40
>6000
>6000
59
39
N
N
N
5f
CH3
8.1
>6300
Not tested
5g
CONH2
0.25
>6000
4.4
Razaxaban
Apixaban
0.19
0.08
540
3100
2.1
3.8
The Ki’s obtained from purified human enzymes and are averaged from multiple determinations (n = 2). All compounds have Ki > 4200 nM for trypsin.

M. L. Quan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1373–1377
1375
The suboptimal trypsin selectivity profile of DPC423 prompted
us to investigate additional P1 moieties for potency and selectivity.
Glycinamide P1 (4a) showed low nanomolar binding affinity
against fXa which suggested that this P1 moiety sits deeper into
the S1 pocket of fXa. To further elaborate on this finding, a set of
constrained heterocyclic analogs were evaluated (4b–d). Although
most of these compounds were less potent, the triazolinone
compound 4b retained the fXa affinity with a fXa Ki of 0.5 nM
(Table 1). With the exception of 4d, these compounds show signif-
icantly weaker affinity for thrombin and trypsin compared with
DPC423.
Ar
F3C
F3C
F
H
H
N
N
Ar
N
N
SO2Me
N
N
a
O
O
H
45%
N
7
CN
O
N
4b
NH
c
b
35%
> 80%
F3C
F3C
H
H
N
Ar
N
N
Ar
N
We next evaluated the triazolinone P1 group appended to the
bicyclic pyrazole scaffold of apixaban. As shown in Table 2,
N
N
O
O
compounds 5a–e with
a trifluoromethyl substitution on the
N
N
N
bicyclic pyrazole core achieved low nanomolar to sub-nanomolar
affinity. However, weak in vitro anticoagulant activity was
observed with these compounds. This was attributed to higher
lipophilicity which contributes to higher protein binding (for a
discussion of the effect of plasma protein binding on translation
from fXa Ki to in vitro anticoagulant activity see Ref. 7a). To lower
the protein binding of compound 5e, the C-3 pyrazole trifluoro-
methyl group was replaced with C-3 methyl and C-3 carboxamide
moieties, which have previously been shown to impact higher free
fraction in related compounds.8a While the methyl analog 5f lost
fXa affinity, the carboxamide compound 5g had a fXa Ki of
0.25 nM and improved in vitro anticoagulant potency (PT
N
HN
4d
S
4c
NH2
NH2
Scheme 1. Reagents and conditions: (a) (1) HCl/MeOH; (2) NMM, semicarbazide,
dioxane, reflux; (b) thiosemicarbazide, TFA, reflux; (c) (1) HCl/MeOH; (2) cyano-
guanidine, K2CO3, AcOH, dioxane, 140 °C.
R
R
B(OH)2
CHO
N
N
N
N
I
I
N
N
a
10
O
O
5c-f
M).12 In comparison with razaxaban (fXa Ki = 0.19 nM
M), compound 5g was within twofold in terms
H
45%
EC2x = 4.4
and PT EC2x = 2.1
l
b, c
40-60%
N
O
CN
l
9
N
NH
8
of in vitro anticoagulant activity, and despite a threefold difference
in fXa affinity, was comparable in the PT assay to apixaban (fXa
Ki = 0.08 nM and PT EC2x = 3.8 lM).
N
N
B(OH)2
SO2Me
R = CF3, CH3
HN
11
12
d
b
Modeling of the phenyltriazolinone into the S1 pocket of the fXa
active site indicated that the phenyltriazolinone moiety was not
expected to fit in the S1 pocket. The increased size of the triazoli-
none moiety was predicted to result in steric clashes with
Asp189. To better understand the observed results, X-ray struc-
tures of compounds 4b, 5a, and 5d–f bound to human fXa were
determined.13 All of these compounds bind to fXa in a similar
mode as depicted in Figure 2, which shows the overlay of all five
compounds in the fXa active site. The X-ray structures show all
compounds maintain the general binding mode previously
observed with other pyrazole-based fXa inhibitors except in the
35%
50%
N
N
5a
F3C
N
N
a
N
N
5b
O
40%
13
CN
Scheme 2. Reagents and conditions: (a) (1) HCl/MeOH; (2) NMM, semicarbazide,
dioxane, reflux; (b) (1) Pd(PPh3)4, 2 M Na2CO3, toluene/EtOH or DME/H2O, reflux;
(c) amine, NaCNBH3, MeOH; (d) CuI/K2CO3, 1,10-phenantholine, DMSO, 130 °C,
12 h.
Cl
CO2Et
EtO2C
N
N
N
N
HN
N
I
N
+
O
a
O
O
I
16
15
96%
CN
CN
14
b
76%
H2NOC
N
N
H2NOC
I
N
N
O
N
e, f
45%
c,d
I
N
H
N
5g
O
O
36% for two steps
N
N
H
CONH2
18
17
Scheme 3. Reagents and conditions: (a) TEA, toluene, reflux; (b) formamide, NaOMe, DMF, 100 °C; (c) HCl/MeOH; (d) NMM, semicarbazide, dioxane, reflux; (e) 2-
formylphenylboronic acid, Pd(PPh3)4, 2 M Na2CO3, toluene/EtOH or DME/H2O, reflux; (f) amine, NaCNBH3, MeOH.

1376
M. L. Quan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1373–1377
Figure 2. Overlay of the crystal structures of 4b (orange), 5a (green), 5d (cyan), 5e
(magenta), and 5f (tan) in the fXa active site (Figure created with PyMol14).
Figure 5. Interactions of 18 in the S1 pocket.
Figure 6. Comparison of fXa (cyan) to thrombin (grey).
Figure 3. Crystal structure of 5d bound to human fXa: Initial 2Fo–Fc electron
density (magenta) contoured at 1r is shown with the final model of compound 5d.
Carbon atoms are shown in cyan for compound 5d and green for fXa. Selected water
shows that Asp189 has moved and the triazolinone has extended
much deeper into the S1 site (Fig. 4).
molecules are shown as spheres. Figure created with PyMol.14
Figure 5 shows the detailed interactions of the phenyltriazoli-
none in the S1 pocket. One of the nitrogen atoms of the triazolinone
forms a H-bond with the Gly218 carbonyl. The triazolinone
carbonyl forms two H-bonds, one directly with Ala221 nitrogen
and another with one of the carboxylic oxygens of Asp189 side
chain through a water molecule. Another nitrogen of the triazoli-
none ring forms a H-bond with the second oxygen of Asp189 side
chain which in turn forms a H-bond with Tyr223 OH through an
additional water molecule. This network of H-bonds enables the
triazolinone moiety to have strong interactions with the enzyme.
The selectivity of the triazolinones against trypsin can be ratio-
nalized by a change in residue in the S1 pocket. FXa has Ala190
adjacent to Asp189, whereas trypsin has a bulkier serine residue
in this position. These larger S1 substituents are unable to bind
when the serine is present, therefore yielding selectivity over tryp-
sin. Comparison of the S1 pockets of fXa and thrombin reveals a
possible reason for the selectivity of the triazolinones against
thrombin. The fXa structure shows the movement of Asp189;
however, thrombin would be unable to adopt this conformation
due to differences in residues near Asp189 that would create unfa-
vorable steric and electronic interactions with Asp189. The
triazolinone P1 moiety moves Asp189 deeper into the fXa S1 pock-
et, and puts it adjacent to Ala221. In thrombin, this residue is
Asp221, which partially occludes the pocket and provides an unfa-
vorable electrostatic environment for Asp189 to rotate towards,
resulting in improved selectivity against thrombin (Fig. 6).
Figure 4. Overlay of the phenyltriazolinone (Cyan) and the aminobenzisoxazole
(yellow) moieties in the S1 site.
S1 pocket. With all five compounds, the Asp189 side chain in the S1
binding site has rotated away from its normal location. This move-
ment creates a novel larger pocket to accommodate the phenyl-
triazolinone. Figure 3 shows the crystal structure of compound
5d with the initial electron density of the ligand. An overlay of
the phenyltriazolinone with the aminobenzisoxazole7a clearly

M. L. Quan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1373–1377
1377
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In summary, we have discovered the first examples of move-
ment of Asp189 upon binding to fXa inhibitors. A series of novel
and potent fXa inhibitors with a phenyltriazolinone P1 moiety were
identified. Although the general binding mode compared to our
previously-reported fXa inhibitors was maintained, a larger cavity
to accommodate this P1 moiety was created by the movement of
the Asp189 side chain. X-ray structures revealed an intricate
network of H-bonds between the triazolinone and the enzyme
including several structural water molecules.
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Acknowledgements
The authors thank Tracy Bozarth for clotting times, and Joanne
Smallheer for helpful discussions and suggestions.
References and notes
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12. PT values were measured according to Refs. 6b,7a.
13. The X-ray structures of 4b, 5a, and 5d–f in fXa were obtained using
conditions similar to those described in Ref. 8a. The coordinates for these
enzyme inhibitor structures have been deposited with the Protein Data Bank
with the accession codes of 4b (3KQB), 5a (3KQC), 5d (3FFG), 5e (3KQD),
and 5f (3KQE).
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