Design, synthesis, and biological evaluation of pyrazinones containing novel P1 needles as inhibitors of TF/VIIa

Article abstract of DOI:10.1016/j.bmcl.2007.05.090

Herein is described the design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of the TF/VIIa complex. These inhibitors were designed to explore replacement and variation of the P1 amidine described previously [J. Med. Chem. 2003, 46, 4050]. The P1 needle replacements were selected based upon their reduced basicity compared to the parent phenyl amidine (pKa ～ 12). A contributing factor towards the oral bioavailability of a compound is the ionization state of the compound in the intestinal tract. The desired outcome of the study was to identify an orally bioavailable TF-VIIa inhibitor.

Full text of DOI:10.1016/j.bmcl.2007.05.090

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4568–4574
Design, synthesis, and biological evaluation of
pyrazinones containing novel P1 needles as inhibitors of TF/VIIa
John I. Trujillo,* Horng-Chih Huang, William L. Neumann, Matthew W. Mahoney,
Scott Long, Wei Huang, Danny J. Garland, Carrie Kusturin, Zaheer Abbas,
Michael S. South and David B. Reitz
Department of Medicinal Chemistry, Pfizer Global Research and Development, Chesterfield, MO 63017, USA
Received 3 April 2007; revised 29 May 2007; accepted 30 May 2007
Available online 6 June 2007
Abstract—Herein is described the design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of
the TF/VIIa complex. These inhibitors were designed to explore replacement and variation of the P1 amidine described previously
[J. Med. Chem. 2003, 46, 4050]. The P1 needle replacements were selected based upon their reduced basicity compared to the
parent phenyl amidine (pKa ꢀ 12). A contributing factor towards the oral bioavailability of a compound is the ionization state
of the compound in the intestinal tract. The desired outcome of the study was to identify an orally bioavailable TF–VIIa
inhibitor.
Ó 2007 Elsevier Ltd. All rights reserved.
Cardiovascular disease is perhaps the most common
cause for mortality in the western world.1 The current
therapies of heparin or warfarin are limited due to
their slow onset of action and lack of selectivity,
which can lead to bleeding side effects, thus requiring
close patient monitoring.2 The goal of current research
in the area of antithrombotics is the development of
more efficacious drugs that possess an improved safety
profile and pharmacodynamics over the existing treat-
ments.3 Recent research in the area of anticoagulants
has targeted almost every step in the coagulation
pathway, with the most research being pursued
around the development of selective thrombin or
factor Xa inhibitors.4
on neointimal formation and restenosis after vascular
intervention, an advantage especially in the manage-
ment of atherosclerotic complications.
Many approaches have been described by investigators
for antagonizing the effect of the TF/VIIa complex.
For example, active site inhibition of factor VIIa
(VIIai), tissue factor mutants, antibodies directed to
TF or VIIa, naturally occurring protein-based inhibi-
tors, peptide-based exosite inhibitors, and small mole-
cule active site inhibitors have appeared in the
literature.5
Recent findings by our laboratories and others have
highlighted the advantages of inhibiting the TFVIIa
complex, in particular a lower risk of bleeding side ef-
fects versus other coagulation cascade targets.6 We
have detailed our work in a series of publications that
described the design and synthesis of compounds that
inhibit the TF/VIIa complex. The articles discuss the
optimization of the P1, P2, and P3 sites, with the sub-
sequent identification of a low nanomolar, pyrazinone-
based inhibitor (see Fig. 1).7
Of the enzymes in the coagulation cascade, TF/VIIa
has most recently drawn significant attention.5a The
inhibition of the coagulation cascade at this stage of
cascade has been shown to have significant advanta-
ges. For example the inhibitors act locally at the site
injury, thus lessening unwanted side effects. In addi-
tion, the inhibitors have been shown to have an effect
An inspection of the compound reveals a highly basic
amidine bound in the S1 pocket of TF/FVIIa in con-
tact with Asp 189.7b In general, compounds with
highly basic functionality, such as an amidine
Keywords: Factor VIIa; Anticoagulants; Thrombosis; Serine protease;
Amidine; TF/VIIa; Antithrombotics; Oral; Pyrazinones.
*
Corresponding author. Tel.: +1 636 244 1937; e-mail:
john.i.trujillo@pfizer.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.090

J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4568–4574
4569
The compounds were assayed against VIIa, Xa, IIa
(thrombin), and trypsin. Most of the compounds retained
their activity against VIIa with the exception of com-
pounds 5, 6, and 7, which lost >10-fold activity. Interest-
ingly, compound 3 retained TF–VIIa activity as well as
displaying selectivity over Xa and thrombin (>100-fold).
Compound 3 was dosed orally into rats at 10 mpk, but
disappointingly very little oral exposure was observed.
The syntheses of the P1 needles for compounds 1–7 are
described in Scheme 1. For compounds 2, 3, 5, and 7 the
P1 needle was coupled to the acid as previously repor-
ted,7c followed by reductive removal of the Cbz group.
For compounds 4 and 6 the P1 needle was coupled fol-
lowed by reduction of NAO bond to give the final prod-
uct (see Scheme 2).
Figure 1. Structure of TF–VIIa inhibitor.
(pKa ꢀ 12), have difficulty in being orally absorbed.8
In addition to the incorporation of substitutions into the
aryl ring of the amidine other variations were explored.
Previous work by others in the Xa area had demonstrated
success in the replacement of an amidine with a less basic
functionality.9 Given the success of the strategy, we
sought to apply a similar strategy toward the identifica-
tion of a replacement P1 needle for our TF–VIIa inhibitor
1. The compounds designed, synthesized, and assayed are
presented in Table 2. Unlike the F-substituted amidine
derivatives, all of the analogs failed to show any activity
less than 3 lM against TF–VIIa.
Indeed, when
1 was dosed orally into rats at
10 mpk, very little absorption into the systemic circu-
lation was observed. A common strategy used to en-
hance the absorption of such compounds is to
identify a substitute for the amidine moiety that is less
basic, while still being capable of making the requisite
interaction with the enzyme.8a,b In addition, to the
replacement of the amidine an alternative is the prep-
aration of pro-drugs which can be absorbed and then
converted in vivo to the active compound.9 In this
article, we wish to disclose our efforts toward the
identification of a suitable P1 amidine substitute.
These results suggest an essential requirement for the
amidine moiety for a compound to possess TF–VIIa
inhibitory activity. The syntheses of the P1 needles for
compounds 54, 55, and 57–59 are described in Scheme
3, for compound 56 the corresponding P1 needle was
obtained from commercial sources.
Our initial approach to the design of a suitable amidine
replacement was to lower the basicity of the amidine
(pKa) by incorporating substituents into the aryl ring.
The designed compounds all possess a reduced pKa rel-
ative to compound 1 (see Table 1).10
Table 1. Calculated pKa for amidine analogs 1–7
a
Compound
P1-substitution
pKa
IC50 (lM)
VIIa
Xa
IIa
Try
1
2
3
4
5
6
7
H
3-F
11.6
10.8
0.02
0.04
0.06
0.11
5.48
0.19
0.79
0.04
0.05
>30
10.6
12.8
>30
>42
>30
0.10
34.4
4.7
2-F
9.84
10.8
6.60
2-OH, 3,5,6-F
2,3,5,6-F
2-OH
>100
>30
>100
>100
>100
>30
—
10.6
>100
>30
7.55
0.23
3-CF3
a Values calculated using the ACD/Physchem Batch 5.0 Program.

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J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4568–4574
compounds 43 and 44 the fluoronitrile derivative was
treated with acetohydroxamic acid in the presence of
potassium tert-butoxide in DMF to provide the oxa-
zole derivative 43. On the other hand, the thiazole
derivative was prepared by treating the fluoronitrile
derivative with sodium sulfide in DMSO at 70 °C
followed by cooling and treatment with aqueous
ammonia and sodium hypochlorite to give the com-
pound 44.
The synthesis of compounds 47 and 48 required mod-
ification of the pendant acid in the core template. This
was accomplished by a four-step protocol to give the
primary amine 46, which served as the key intermedi-
ate. For 47, the amine was treated with 4-cyanoben-
zene-1-sulfonyl chloride followed by conversion of
the nitrile to the amidine with LiHMDS,11 which
was protected in situ as the Cbz derivative to facilitate
isolation. The Cbz and nitro group were then reduced
simultaneously to give the desired product. Compound
48 was prepared by coupling 4-(N-(benzyloxycarbonyl)
carbamimidoyl) benzoic acid12 to the amine under
standard conditions, followed by reduction and re-
moval of the nitro and Cbz group, respectively
(Scheme 4).
The syntheses of compounds 52 and 53 are described
in Scheme 5. The Cl-isoquinoline 51 was prepared
from 6-methylisoquinoline in five steps. The Cl-iso-
quinoline was then coupled to 44, followed by Cl dis-
placement and reduction of the nitro group to give the
final compound. Compound 53 was prepared through
the sequence described (Scheme 5, steps i–n) to give
the final product 53.
In conclusion, a series of analogs with various P1 nee-
dles were prepared to replace the benzamidine func-
tionality present in the TF–VIIa inhibitor 1.7 Despite
the synthesis and biological evaluation of a variety
of less basic P1 needles a suitable replacement was
not identified that possessed the desired enzymatic
activity against the panel of VIIa, Xa, IIa (thrombin)
and trypsin enzymes. Due to the lack of activity of the
compounds in the primary screen, none were further
evaluated in rat oral PK models to determine if indeed
an improvement in oral absorption was achieved. Cur-
rent research is being pursued around a pro-drug
strategy for the future development of a TF–VIIa
inhibitor and will be reported in due course.13
Scheme 1. Preparation of P1 needles for compounds 2–7. Reagents
and conditions: (a) NBS, CCl4, reflux; (b) NaH, THF, HNBoc2; (c)
NH2OHÆHCl, K2CO3, EtOH/H2O (2:1), reflux; (d) AcOH, Ac2O, Pd/
C, H2 (40 psi); (e) NaOH, Cbz–Cl, THF; (f) 4 N HCl in dioxane; (g)
Boc2O, 10% Pd/C, EtOH, H2 (70 psi); (h) tBuoK, THF
(CH3)2C@NOH; (i) EtOH:H2O, concd HCl, reflux; (j) 5 N HCl,
EtOH, 5% Pd/C, H2 (60 psi).
Following the synthesis of the P1 needles, the amines
were coupled to the acid 23 followed by removal of
the Cbz group via hydrogenation as shown in Scheme
2 to give the compounds 54–59. For the synthesis of
Scheme 2. Coupling of P1 needles to acid core.

J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4568–4574
Table 2. Structure and biological activity of compounds 44–60
4571
a
Compound
Substitution
pKa
IC50 (lM)
VIIa
12.5
Xa
IIa
Try
O
N
H
54
9.92
8.59
>30
>30
16
H
N
N
O
N
H
53
47
43
48
55
56
44
57
52
58
7.02
>100
>30
>30
>30
>30
>30
>30
>30
>30
>30
>100
>30
>30
>30
>30
>30
>30
>30
>30
>30
>3
NH
HN
NH2
O
S
N
O
H
10.9
30
9.1
NH
NH2
O
O
N
N
H
4.62
12.0
10.7
>30
3.00
26.8
>30
>30
>30
>30
2.53
>30
0.23
>30
>30
>30
NH2
O
N
H
10.8
NH
NH2
O
S
N
H
6.32
9.31
6.89
6.12
7.50
4.6
N
H2N
O
N
H
NH2
O
S
N
N
H
NH2
O
O
N
H
NH
N
NH2
O
N
H
N
NH2
O
N
H
N
N
NH2
(continued on next page)

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J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4568–4574
Table 2 (continued)
a
Compound
Substitution
pKa
IC50 (lM)
VIIa
>30
Xa
IIa
Try
>30
O
HN
N
59
7.16
>30
>30
N
H
NH2
N
O
HN
60
8.61
>30
>30
>30
6.20
N
H
a Values calculated using the ACD/Physchem Batch 5.0 Program.
Scheme 3. Preparation of P1 needles for compounds 54, 55, 57, 58, and 59. Reagents and conditions: (a) 5% Pd/C, EtOH:THF, H2 (40 psi); (b)
H2N(CH2)2NH2, MeOH, reflux; (c) 4 N HCl in dioxane; (d) Cbz–Cl, pyridine, CH2Cl2; (e) CuCN, CMF; (f) NBS, CCl4, benzoyl peroxide, reflux; (g)
NaH, HNBoc2, THF, 0 °C; (h) TFA, CH2Cl2, rt; (i) Boc2O, TEA, CH2Cl2, 0 °C; (j) HS(CH2)2NH2ÆHCl, KOH, nBuOH, 100 °C; (k) NaOH, CbzCl,
THF, 0 °C; (l) TFA, CH2Cl2, 0 °C; (m) Pd2(dba)3, dppf, Zn(CN)2, DMF, 120 °C; (n) H2NNH2, MeOH, 50 °C; (o) NaNO2, p-BrPhNH2, H2O, 0 °C;
(p) PtO2, H2 (35 psi), EtOH, rt; (q) BuLi, TMEDA, Br(CH2)2OTBS, THF, 0 °C; (r) Boc2O, TEA, DMAP, DCM, rt; (s) TBAF, THF, rt; (t) MsCl,
Et3N, PhMe, 0 °C; (u) NaN3, TBAl, PhMe:H2O, 60 °C; (v) 10% Pd/C, H2 (10 psi), EtOH.

J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4568–4574
4573
Scheme 4. Preparation of compounds 43, 44 and 47, 48. Reagents and conditions: (a) MeCONHOH, DMF, KOtBu, rt; (b) Na2S, DMSO, 70 °C then
NH3(aq), 5% NaOCL(aq), ꢁ5 °C ! 0 °C; (c) BH3–THF, 0 °C; (d) MsCl, Et3N, CH2Cl2, 0 °C; (e) NaN3, DMF, 60 °C; (f) PPh3, THF:H2O, 50 °C; (g)
ClSO2PhCN, Et3N, CH2Cl2, 0 °C; (h) LiHMDS, THF, 0 °C, then CbzCl, Na2CO3, THF:H2O; (i) 10% Pd/C, H2 (40 psi), EtOH, HCl; (j)
HO2CPh(C@NH)NHCbz, EDC, HOBt, Et3N, DMF, 0 °C.
Acknowledgment
We thank Rhonda LaChance for running the biological
assays.
References and notes
1. Braunwald, E.; Califf, R. M.; Cannon, C. P.; Fox, K. A.;
Fuster, V.; Gibler, W. B.; Harrington, R. A.; King, S. B.;
Kleiman, N. S.; Theroux, P.; Topol, E. J.; Van de Werf,
F.; White, H. D.; Willerson, J. T. Am. J. Med. 2000, 108,
41.
2. Gage, B. F.; Fihn, S. D.; White, R. H. Am. J. Med. 2000,
109, 481.
3. Hirsh, J. Thromb. Res. 2003, 109, S1.
4. Gresele, P.; Agnelli, G. Trends Pharmacol. Sci. 2002, 23,
25.
5. For reviews in the area of TF–VIIa inhibitors, see: (a)
Kranjc, A.; Kikelj, D.; Peterlin-Masic, L. Curr. Pharm.
Des. 2005, 11, 4207; (b) Lazarus, R. A.; Oliver, A. G.;
Eigenbrot, C.; Kirchofer, D. Curr. Med. Chem. 2004, 11,
Scheme 5. Preparation of compounds 51–53. Reagents and conditions:
2275; (c) Robinson, L. A.; Saiah, E. Ann. Rep. Med. Chem.
(a) mCPBA, CH2Cl2, rt then HCl/MeOH, 0 °C; (b) POCl3, 90 °C; (c)
2002, 12, 85.
6. (a) Szalony, J. A.; Taite, B. B.; Girard, T. J.; Nicholson,
(f) PS–DCC, HOBt, Et3N, CH2Cl2/DMF; (g) NH4OAc, DMF, reflux;
NBS, CCl4, Bz2O; (d) PhtNH, NaH, THF; (e) H2NNH2, MeOH, 0 °C;
N. S.; LaChance, R. M. J. Thromb. 2002, 14, 113; (b)
Zoldhelyi, P.; McNatt, J.; Shelat, H. S.; Yamamoto, Y.;
(h) 5% Pd/C, MeOH, H2 (25 psi); (i) 4-(aminomethyl)benzonitrile,
EDC, HOBt, Et3N, DMF, 0 °C; (j) Boc2O, Et3N, DMAP, THF; (k)
Chen, Z.-Q.; Willerson, J. T. Circulation 2000, 101, 289;
H2S, Et3N, pyridine, 0 °C; (l) Mel, acetone, rt; (m) H2NNH2, MeOH,
(c) Himber, J.; Kirchhofer, D.; Riedereer, M.; Tschopp, T.
rt; (n) TFA, CH2Cl2, rt.

4574
J. I. Trujillo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4568–4574
B.; Steiner, B.; Roux, S. P. Thromb. Haemost. 1997, 7,
1142; (d) Harker, L. A.; Hanson, S. R.; Wilcox, J. N.;
Pharmacol. 1994, 259, 165; (d) Hauptmann, J.; Mark-
wardt, F. Semin. Thromb. Hemost. 1992, 18, 200; For a
recent report of TF/FVIIa inhibitors with non-amidine P1
substituents, see: (e) Miura, M. et al. Bioorg. Med. Chem.
2006, 14, 7688; . Bioorg. Med. Chem. 2007, 15, 160.
9. (a) Eriksson, U.; Bredberg, U.; Hoffmann, K.; Thuresson,
A.; Gabrielsson, M.; Ericsson, H.; Ahnoff, M.; Gislen, K.;
Fager, G.; Gustafsson, D. Drug Metab. Dispos. 2003, 31,
29; (b) Clement, B. Drug Metab. Rev. 2002, 34, 565; (c)
Riggs, J. R.; Kolesnikov, A.; Hendrix, J.; Young, W. B.;
Shrader, W. D.; Vijaykumar, D.; Stephens, R.; Liu, L.;
Pan, L.; Mordenti, J.; Green, M. J.; Sukbuntherng, J.
Bioorg. Med. Chem. Lett. 2006, 16, 2224.
Kelly, A. B. Haemostasis 1996, 26, 76.
7. (a) Parlow, J. J.; South, M. S. Tetrahedron 2003, 59, 7695;
(b) 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, 2319; (c) South, M. S.; Dice, T.
A.; Girard, T. J.; Lachance, R. M.; Stevens, A. M.;
Stegeman, R. A.; Stallings, W. C.; Kurumbail, R. G.;
Parlow, J. J. Bioorg. Med. Chem. Lett. 2003, 13, 2363; (d)
Parlow, J. J.; Case, B. L.; Dice, T. A.; Fenton, R. L.;
Hayes, M. J.; Jones, D. E.; Neumann, W. L.; Wood, R. S.;
Lachance, R. M.; Girard, T. J.; Nicholson, N. S.; Clare,
M.; Stegeman, R. A.; Stevens, A. M.; Stallings, W. C.;
Kurumbail, R. G.; South, M. S. J. Med. Chem. 2003, 46,
4050; (e) Parlow, J. J.; Stegeman, R. A.; Stevens, A. M.;
Stallings, W. C.; Kurumbail, R. G.; South, M. S. J. Med.
Chem. 2003, 46, 4297; (f) Parlow, J. J.; Stegeman, R. A.;
Stevens, A. M.; Stallings, W. C.; Kurumbail, R. G.; South,
M. S. Bioorg. Med. Chem. Lett. 2003, 13, 3721; (g) Parlow,
J. J.; Dice, T. A.; Lachance, R. M.; Girard, T. J.; Stevens,
A. M.; Stegeman, R. A.; Stallings, W. C.; Kurumbail, R.
G.; South, M. S. J. Med. Chem. 2003, 46, 4043; (h) Parlow,
J. J.; Kurumbail, R. G.; Stegeman, R. A.; Stevens, A. M.;
Stallings, W. C.; South, M. S. J. Med. Chem. 2003, 46,
4696; (i) Parlow, J. J.; Stevens, A. M.; Stegeman, R.;
Stallings, W. C.; Kurumbail, R. G.; South, M. S. J. Med.
Chem. 2003, 46, 4297.
10. (a) pKa values calculated using the ACD/Physchem
Batch 5.0 Program; (b) South, M. S.; Parlow, J. J.;
Jones, D. E.; Case, B.; Dice, T.; Lindmark, R.; Hayes, M.
J.; Rueppel, M. L.; Fenton, R.; Franklin, G. W.; Huang,
H.; Huang, W.; Kusturin, C.; Long, S. A.; Neumann, W.
L.; Reitz, D.; Trujillo, J. I.; Wang, C.; Wood, R.; Zeng,
Q.; Mahoney, M. W. WO 2001087854 A1, 2001; (c)
South, M. S.; Parlow, J. J.; Jones, D. E.; Case, B.; Dice,
T.; Lindmark, R.; Hayes, M. J.; Rueppel, M. L.; Fenton,
R.; Franklin, G. W.; Huang, H.; Huang, W.; Kusturin,
C.; Long, S. A.; Neumann, W. L.; Reitz, D. B.; Trujillo,
J. I.; Wang, C.; Wood, R.; Zeng, Q. WO 2000069834 A1,
2001.
11. Boere, R. T.; Oakley, R. T.; Reed, R. W. J. Organomet.
Chem. 1987, 331, 161.
12. Bondinell, W. E.; Callahan, J. F.; Huffman, W. F.;
Keenan, R. M.; Ku, T. W. F.; Newlander, K. A. WO
9300095A2, 1993.
8. (a) Pinto, D. J. P.; Orwat, M. J.; Wang, S.; Fevig, J. M.;
Quan, M. L.; Amparo, E.; Cacciola, J.; Rossi, K. A.;
Alexander, R. S.; Smallwood, A. M.; Luettgen, J. M.;
Liang, L.; Aungst, B. J.; Wright, M. R.; Knabb, R. M.;
Wong, P. C.; Wexler, R. R.; Lam, P. Y. S. J. Med. Chem.
2001, 44, 566; (b) Pauls, H. W.; Ewing, W. R. Curr. Top.
Med. Chem. 2001, 1, 83; (c) Schumacher, W. A.; Bala-
subramanian, N.; St. Laurent, D. R.; Seiler, S. M. Eur. J.
13. (a) South, M. S.; Webber, R. K.; Huang, H. C.; Toth, M.
V.; Moorman, A. E.; Snyder, J. S.; Scholten, J. A.;
Garland, D. J.; Rueppel, M. L.; Neumann, W. L.; Long,
S.; Huang, W.; Trujillo, J.; Parlow, J. J.; Jones, D. E.;
Case, B.; Hayes, M. J.; Zeng, Q. WO 2003028729 A2,
2003; (b) Peterlin-Masic, L.; Cesar, J.; Zega, A. Curr.
Pharm. Des. 2006, 12, 73.