Design and synthesis of bicyclic pyrazinone and pyrimidinone amides as potent TF-FVIIa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.01.094

Bicyclic pyrazinone and pyrimidinone amides were designed and synthesized as potent TF-FVIIa inhibitors. SAR demonstrated that the S2 and S3 pockets of FVIIa prefer to bind small, lipophilic groups. An X-ray crystal structure of optimized compound 9b boun

Full text of DOI:10.1016/j.bmcl.2013.01.094

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1604–1607
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of bicyclic pyrazinone and pyrimidinone
amides as potent TF–FVIIa inhibitors
⇑
Xiaojun Zhang , Peter W. Glunz, Wen Jiang, Aaron Schmitt, Makenzie Newman, Frank A. Barbera,
Jeffery M. Bozarth, Alan R. Rendina, Anzhi Wei, Xiao Wen, Karen A. Rossi, Joseph M. Luettgen,
Pancras C. Wong, Robert M. Knabb, Ruth R. Wexler, E. Scott Priestley
Bristol-Myers Squibb R&D, 311 Pennington-Rocky Hill Rd, Pennington, NJ 08534, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Bicyclic pyrazinone and pyrimidinone amides were designed and synthesized as potent TF–FVIIa inhib-
itors. SAR demonstrated that the S2 and S3 pockets of FVIIa prefer to bind small, lipophilic groups. An X-
ray crystal structure of optimized compound 9b bound in the active site of FVIIa showed that the bicyclic
scaffold provides 5 hydrogen bonding interactions in addition to projecting groups for interactions within
the S1, S2 and S3 pockets. Compound 9b showed excellent FVIIa potency, good selectivity against FIXa,
Xa, XIa and chymotrypsin, and good clotting activity.
Received 12 November 2012
Revised 14 January 2013
Accepted 22 January 2013
Available online 30 January 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
TF–FVIIa inhibitor
Bicyclic pyrazinone
Bicyclic pyrimidinone
Vascular injury or atherosclerotic plaque rupture results in
exposure of tissue factor (TF) to circulating factor VIIa (FVIIa) in
plasma. Binding of TF to FVIIa leads to a large enhancement of
the catalytic activity of FVIIa. The TF–FVIIa complex then initiates
the extrinsic coagulation pathway by activating factor IX to IXa
and factor X to Xa, which in turn activates prothrombin to throm-
bin. Thrombin cleaves fibrinogen to fibrin, which forms blood clots
with activated platelets.1,2 Inappropriate clot formation in blood
vessels causes cardiovascular disease such as stroke and myocar-
dial infarction. Recent preclinical studies have shown that selective
inhibition of TF–FVIIa is effective in anticoagulation with a low risk
of bleeding.3–6 Herein, we report our discovery of bicyclic pyrazi-
none and pyrimidinone amides as potent TF–FVIIa inhibitors.
Based on knowledge that serine proteases typically bind their
peptide substrate in an extended conformation and our previous
work on 3-aminobicyclic pyrazinone and pyrimidinone as b-sheet
mimetics,7–9 we reasoned that by attaching an aminomethyl ben-
zamidine to interact with Asp189 in the S1 pocket to the bicyclic
pyrazinone or pyrimidinone scaffold, a series of potent TF–FVIIa
inhibitors may be obtained (Fig. 1). Further supporting this pro-
posal is the report by South et al. that mono-cyclic pyrazinone
amides are potent TF–FVIIa inhibitors.10–12 Bicyclic pyrazinone
amide 1 was thus prepared and found to be a potent TF–FVIIa
inhibitor with Ki of 30 nM (Fig. 1). Although compound 1 showed
the same level activity against thrombin, it was highly selective
against FXa. We considered it to be a good lead that warranted fur-
ther investigation.13
The general synthesis of 3-amino bicyclic pyrazinone amides is
shown in Scheme 1. Boc-protected pyroglutamate 2 was treated
with lithium bis(trimethysilyl)amide and alkylated with an elec-
trophile to give a 4-substituted pyroglutamate. This alkylation pro-
cedure is stereoselective and can be carried out twice to give the
4,4-disubstituted derivatives 3.14 The second electrophile reacts
with the enolate preferentially trans to the 2-carboxylate, allowing
stereocontrol of R1 and R2 simply by changing the order of addition
of the electrophiles. Pyroglutamate 3 was reduced with lithium tri-
ethylborohydride to the aminal, which was converted to acetoxy
derivative 4. Intermediate 4 reacted with trimethylsilyl cyanide
catalyzed by BF3ÁOEt2 in methylene chloride to give 5-cyano pro-
line derivative 5. The Boc protecting group was removed to gener-
ate aminonitrile salt 6, which underwent cyclocondensation with
oxalyl chloride to give dichloropyrazinone 7.7 Heating a solution
of dichloropyrazinone 7 in ethyl acetate in the presence of excess
amine R3NH2 resulted in a selective nucleophilic displacement of
the C-3 chlorine to give intermediate 8. Saponification of com-
pound 8 resulted in the acid, which was coupled with benzyl (4-
(aminomethyl)phenyl)(imino)methylcarbamate. After removal of
the Cbz group, compound 9 was obtained for biological testing.
Scheme 2 describes the preparation of bicyclic pyrimidinones.
In this series, the N-terminal substituent was maintained as a pro-
pyl group since it was shown to be a preferred substituent in the
pyrazinone series. Pyrimidinone 108 was N-allylated and the
carboxylic acid was masked as a phenyl amide 11. Compound 11
⇑
Corresponding author.
E-mail address: xiaojun.zhang@bms.com (X. Zhang).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.094

X. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1604–1607
1605
Peptide substrate binding:
Bicyclic mimetics:
P2
Y
X
P3
P3/P4
N
O
H
N
N
O
P2
N
H
N
H
O
N
H
O
O
H
N
O
O
O
H
N
H
P1
H
N
P1
structural class
Pyrazinone
Pyrimidinone
X
N
CH
Y
CR
N
O
8
Scissile bond
Cl
1
N
3
N
N
H
NH
O
1
NH
O
FVIIa Ki (nM)
Thrombin Ki (nM) 35
FXa Ki (nM) > 15000
30
NH2
Figure 1. Design of bicyclic pyrazinone and pyrimidinone FVIIa inhibitors and initial lead 1.
R2
R2
N
R1
R2
N
R1
R1
AcO
a, b
e
c, d
f
O
CO2Me
NC
CO2Me
O
CO2Me
N
CO2Me
N
Boc
2
Boc
5
Boc
3
Boc
4
R1
Cl
Cl
R1
R2
Cl
R2
R1
R2
R2
R1
NC
N
i, j, k
N
N
R3
N
h
g
N
R3
N
N
H
CO2Me
N
H
N
Cl
NH2
NH
H2
-
O
N
H
CF3CO2
O
O
OMe
OMe
O
O
O
9
6
7
8
Scheme 1. Reagents and conditions: (a) LHMDS, THF, À78 °C, R1-X; (b) LHMDS, THF, À78 °C, R2-X, 30–90%, 2 steps; (c) LiBHEt3, THF, À78 °C; (d) Ac2O, À78 °C to rt, 50–90%, 2
steps; (e) TMSCN, BF3ÁOEt2, CH2Cl2, À78 °C, 50–70%; (f) TFA, CH2Cl2, >80%; (g) (COCl)2, toluene, 85 °C, 25–70%; (h) R3NH2, EtOAc, 80 °C, >65%; (i) LiOH, THF, H2O; (j) IBCF,
NMM, THF, À20 °C, benzyl ((4 (aminomethyl)phenyl)(imino)methyl)carbamate; (k) H2, Pd/C, MeOH, 50–90%.
R2
R1
N
N
O
N
c, d
a, b
N
N
N
CbzN
CbzN
CbzHN
CONHPh
CONHPh
O
CO2H
O
10
12
11
R1
R2
N
R2
R1
N
N
g, h
e, f
N
N
H
CbzN
NH2
NH
CO2H
O
N
H
O
O
14
13
Scheme 2. Reagents and conditions: (a) NaH, AllylBr; (b) PhNH2, EDC, HOAt, 58%, 2 steps; (c) LHMDS, R1-X, (d) LHMDS, R2-X, 40–95%, 2 steps, (e) Boc2O, DMAP; (f) LiOOH, 80–
91%, 2 steps; (g) EDC, HOAt, benzyl ((4-(aminomethyl)phenyl)(imino)methyl)carbamate; (h) H2, Pd/C, MeOH, 50–90%, 2 steps.
was doubly alkylated stereoselectively to give intermediate 12.
Compound 12 was activated to the imide with Boc2O and then
hydrolyzed to acid 13. Coupling of compound 13 with benzyl
(4-(aminomethyl)phenyl)(imino)methylcarbamate and hydrogen-
olysis to remove the Cbz group and reduce the allyl to propyl gave
final compound 14.
We expected the potency of compound 1 should be further im-
proved with an optimal R2 group engaging interactions in the S2
pocket. The SAR of compounds with varied R1 and R2 groups is sum-
marized in Table 1.
It is clear that a small hydrophobic group is preferred at R2. An
allyl group (9c) is optimal, followed by ethyl (9b). Both compound
9b and 9c showed improved binding for FVIIa compared to
compound 1. Selectivity of compound 9b against thrombin is also
improved compared to compound 1, and it remains more than
450-fold selective against FXa. Polar R2 groups are not well
We believed that the R2 group at 8-position of the bicyclic pyraz-
inone should occupy the S2 pocket in the FVIIa active site based on
modeling studies and analogy to binding of this chemotype to HCV
NS3 serine protease, another chymotrypsin-like serine protease.7

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X. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1604–1607
Table 1
Bicyclic pyrazinone-R1 and R2 SAR
Cl
O
R1
R2
N
N
N
H
N
O
H
NH2
NH
Cmpd
R1
R2
FVIIa Ki (nM)
Thrombin Ki (nM)
FXa Ki (nM)
1
H
Me
Et
Me
Me
n-Pr
n-Pr
H
Me
Et
Allyl
n-Pr
Me
n-Pr
30
23
7.3
5.8
23
21
28
99
72
35
200
210
220
36
110
170
78
>15,000
>15,000
3300
650
1000
2200
550
>15,000
>15,000
1100
9a
9b
9c
9d
9e
9f
9g
9h
9i
–CH2CH2CH2CH2–
Me
Me
Me
CH2OH
CONHMe
CONHBn
>15,000
>15,000
>15,000
2100
560
9j
3700
Ki’s for the indicated enzymes were determined by chromogenic substrate assays at 25 °C (n = 2).15
Table 2
Table 3
Bicyclic pyrazinone-R3 SAR
Bicyclic pyrimidinone-R1 and R2 SAR
R1
Cl
O
R2
Me
N
Me
N
N
N
R3
N
H
N
H
O
N
N
H
O
H
O
NH2
NH
NH2
NH
Compd R3
FVIIa Ki (nM) Thrombin Ki
FXa Ki (nM)
Compd
R1
R2
FVIIa Ki (nM)
Thrombin Ki (nM)
FXa Ki (nM)
(nM)
14a
14b
14c
14d
14e
H
Me
Et
Me
n-Pr
H
Me
Et
Et
n-Pr
186
290
75
150
620
150
>15,000
>15,000
12,000
>15,000
3600
1800
5200
2100
1200
9k
9l
Me
Et
i-Pr
c-Bu
c-Pr
i-Bu
c-Pentyl
t-Bu
24
19
17
23
37
43
60
68
230
260
130
200
360
220
160
220
>15,000
>15,000
>15,000
>15,000
>15,000
>15,000
>15,000
2800
9m
9a
9n
9o
9p
9q
Ki’s for the indicated enzymes were determined by chromogenic substrate assays at
25 °C (n = 2).15
et al.,11,12 small alkyls, such as Me (9k), Et (9l), i-Pr (9m), c-Bu
(9a) and c-Pr (9n) were among the best groups. FVIIa binding affin-
ity decreased as the size of R3 group increased (9o–w) beyond c-Bu
and i-Pr. The R3 group did not seem to affect the selectivity against
thrombin and FXa.
9r
9s
9t
70
76
84
380
220
ND
>15,000
>15,000
ND
OH
A number of bicyclic pyrimidinone amides have also been pre-
pared, varying the R1 and R2 groups. In general, the SAR follows the
trend observed in the pyrazinone series, with diethyl as the pre-
ferred P2 group (Table 3). Bicyclic pyrimidinone amides are less
potent than the pyrazinone amides, likely due to the absence of
the chlorine substituent on the ring. The des-chloro analog of com-
pound 1 in the pyrazinone amide series is 2-fold less potent than
the parent compound 1 (data not shown), suggesting the beneficial
effect of the chlorine on binding.
9u
9v
MeO
130
132
190
ND
32
ND
290
ND
ButO2CCH2-
O
H3C
9w
ND
Ki’s for the indicated enzymes were determined by chromogenic substrate assays at
25 °C (n = 2).15
Compound 9b has been further characterized because of its
excellent potency. It showed very good selectivity (>160-fold)
against FIXa, Xa, XIa and chymotrypsin and moderate selectivity
(30-fold) against thrombin. However, selectivity against trypsin
and plasma kallikrein is limited (<3-fold). Free fraction of compound
9b in human plasma is 6.8%. It is efficacious in a human plasma clot-
tolerated, as seen with hydroxy methyl (9h) and amides (9i and 9j).
A spiro compound 9g was synthesized to further constrain the
diethyl side chains, however it was found to be less active than
compound 9b.
SAR of substituents R3 on the amino terminus has been investi-
gated using dimethyl groups as the P2 side chain for synthetic sim-
plicity. The results are summarized in Table 2. Similar to the
observation in the monocyclic pyrazinones reported by South
ting assay with FVII deficient PT of 5.6 l
M.15 Attempts to replace the
highly basic benzamidine P1 in compound 9b (Fig. 2) with a less ba-
sic amine such as benzyl amine (15), 1-aminoisoquinonline (16) and

X. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1604–1607
1607
Cl
O
O
Et
N
Et
P1
NH
NH2
N
NH2
N
NH2
NH2
N
N
H
Cmpd
9b
15
16
360
17
1900
P1
N
H
O
FVIIa Ki (nM)
7.3
300
Figure 2. Replacement of bezamidine in compound 9b with weakly basic amines.
hydrogen bonding interactions in addition to projecting groups
for interactions within the S1, S2 and S3 pockets. Compound 9b
showed excellent FVIIa potency, good selectivity against FIXa, Xa,
XIa and chymotrypsin and good clotting activity.
Acknowledgement
We thank Ge Zhang and the Lead Evaluation department for
carrying out the in vitro enzyme assays. We would also like to
thank Steven Sheriff for helping to prepare the coordinates and
data for PDB deposition.
References and notes
1. Golino, P. Thromb. Res. 2002, 106, 257.
2. Houston, D. S. Expert Opin. Ther. Targets 2002, 6, 159.
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South, M. S.; Wood, R. S.; Nicholson, N. S. J. Pharmacol. Exp. Ther. 2003, 306,
1115.
5. Szalony, J. A.; Suleymanov, O. D.; Salyers, A. K.; Panzer-Knodle, S. G.; Blom, J. D.;
LaChance, R. M.; Case, B. L.; Parlow, J. J.; South, M. S.; Wood, R. S.; Nicholson, N.
S. Thromb. Res. 2004, 112, 167.
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Thromb. Thrombolysis 2002, 14, 113.
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Chem. Lett. 2003, 13, 1157.
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10. Parlow, J. J.; Case, B. L.; Dice, T. A.; Fenton, R. L.; Hayes, M. J.; Jones, D. E.;
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Clare, M.; Stegeman, R. A.; Stevens, A. M.; Stallings, W. C.; Kurumbail, R. G.;
South, M. S. J. Med. Chem. 2003, 46, 4050.
Figure 3. X-ray crystal structure of compound 9b bound in the active site of FVIIa.
11. South, M. S.; Case, B. L.; Wood, R. S.; Jones, D. E.; Hayes, M. J.; Girard, T. J.;
Lachance, R. M.; Nicholson, N. S.; Clare, M.; Stevens, A. M.; Stegeman, R. A.;
Stallings, W. C.; Kurumbail, R. G.; Parlow, J. J. Bioorg. Med. Chem. Lett. 2003, 13,
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15. For full experimental details of the biological assays; see: Wong, P. C.; Luettgen,
J. M.; Rendina, A. R.; Kettner, C. A.; Xian, B.; Knabb, R. M.; Wexler, R. R.;
Priestley, E. R. Thromb. Haemost. 2010, 104, 261.
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3-aminobenzoisoxazole (17) all resulted in significant loss (40–260-
fold) of potency. One of the major challenges in the development of
drugs inhibiting TF–VIIa is attaining oral bioavalability. Only limited
reports of non-benzamidine TF–FVIIa inhibitors have recently ap-
peared in literature.16–18
An X-ray crystal structure of compound 9b bound to FVIIa was
solved (Fig. 3) to better understand how these bicyclic pyrazinone
amides bind in the active site of the enzyme.20 As we anticipated,
besides the key salt bridge interactions of the benzamidine with
Asp189 in the bottom of the S1 pocket, the pyrazinone scaffold
provided three additional key hydrogen bond interactions with
the enzyme backbone: a pair from the pyrazinone carbonyl and
terminal NH to interact with Gly216, and the amide NH to interact
with Ser214 hydroxyl. The R2-ethyl group engages in a hydropho-
bic interaction with the side chain methyl of Thr99 in the S2 pocket
while R1 group is solvent exposed. This R2-ethyl group also dis-
places a structural water molecule from the S2 pocket that is often
seen in other TF–FVIIa/inhibitor crystal structures.19 The cyclobu-
tyl group attached to the terminal nitrogen makes hydrophobic
interactions with Pro170I.
In summary, we have designed and synthesized bicyclic pyraz-
inone and pyrimidinone amides as potent TF–FVIIa inhibitors. SAR
shows that the S2 and S3 pocket of the enzyme prefer to bind
small, lipophilic groups. The bicyclic scaffolds provide three
20. PDB deposition number is 4ISI.