Pharmacokinetic optimization of 3-amino-6-chloropyrazinone acetamide thrombin inhibitors. Implementation of P3 pyridine N-oxides to deliver an orally bioavailable series containing P1 N-benzylamides

Article abstract of DOI:10.1016/S0960-894X(03)00099-4

In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-b

Full text of DOI:10.1016/S0960-894X(03)00099-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1353–1357
Pharmacokinetic Optimization of 3-Amino-6-chloropyrazinone
Acetamide Thrombin Inhibitors. Implementation of P3 Pyridine
N-Oxides to Deliver an Orally Bioavailable Series Containing P1
N-Benzylamides
Christopher S. Burgey,a,* Kyle A. Robinson,a Terry A. Lyle,a Philippe G. Nantermet,a
a
a
HaroldG. Selnick, RichardC. A. Isaacs, S. Dale Lewis,b Bobby J. Lucas,b
Julie A. Krueger,b Rominder Singh,c Cynthia Miller-Stein,c Rebecca B. White,c
Bradley Wong,c Elizabeth A. Lyle,d Maria T. Stranieri,d Jacquelynn J. Cook,d
Daniel R. McMasters,e Janetta M. Pellicore,a Swati Pal,a Audrey A. Wallace,d
Franklin C. Clayton,d Dennis Bohn,d Denise C. Welsh,d Joseph J. Lynch, Jr.,d
Youwei Yan,f Zhongguo Chen,f Lawrence Kuo,f Stephen J. Gardell,b Jules A. Shaferb
a
andJoseph P. Vacca
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
dDepartment of Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA
eDepartment of Molecular Systems, Merck Research Laboratories, West Point, PA 19486, USA
fDepartment of Structural Biology, Merck Research Laboratories, West Point, PA 19486, USA
Received18 September 2002; accepted3 January 2003
Abstract—In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous
solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavail-
able P1 N-benzylamide thrombin inhibitors. An expedited investigation of the P1 SAR with respect to oral bioavailability, plasma
half-life, andhuman liver microsome stability revealed 5 as the best candidate for advanced evaluation.
# 2003 Elsevier Science Ltd. All rights reserved.
Deep vein thrombosis, pulmonary embolism, and
thromboembolic stroke represent a set of disorders that
can arise from the formation of an occlusive vascular
thrombus andare a major cause of mortality world-
wide.1 As a result of this medical need, an intensive
search continues for antithrombotics that are safer and,
at minimum, as efficacious as the current standard of
care for the treatment of venous thrombosis andthe
prevention of cardiogenic thromboembolism (i.e., low
molecular weight heparins andwarfarin). An equally
important requirement, particularly with regardto
chronic therapy, is the development of orally bioavail-
able drugs with a convenient dosing regimen (e.g., once-
daily dosing, no drug–drug interactions). Toward this
end, recent approaches to anticoagulant therapy have
been aimed at identifying direct small molecule inhibi-
tors of specific coagulation cascade enzymes which pos-
sess the pharmacokinetic properties requiredfor once
daily oral administration.2 Assuming predictable phar-
macokinetics, the direct mechanism of action of these
inhibitors shouldovercome the monitoring andsafety
liabilities associatedwith existing anticoagulation
therapies. A prominent target to emerge from this effort
is the trypsin-like serine protease thrombin (Factor IIa).
Thrombin occupies a central role in hemostasis:3 its
primary procoagulant actions are the activation of
*Corresponding author. Fax: +1-215-652-3971; e-mail: christopher_
urgey@merck.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00099-4

1354
C. S. Burgey et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1353–1357
platelets andcleavage of fibrinogen to fibrin, which
together constitute the primary components of a vas-
cular thrombus.
of the b-difluoro substitution renders the P3 pyridine
ring nitrogen of 1 relatively non-basic (pKa’s<2.1), and
in an effort to improve the low aqueous solubility of these
compounds this pyridine was converted to its N-oxide.
This modification not only improved the solubility (2:
44mg/mL @ pH 7.4), but provided an improvement in
enzyme binding potency (2: Ki=3.1 nM).11 This enhanced
solubility translates into improvedfunctional potency
(2ÂaPTT=0.54 mM) andexcellent performance in the rat
FeCl3 arterial thrombosis model (0/6 occlusions, final
plasma concentration=1079Æ69nM).12
The majority of previously disclosed thrombin inhibi-
tors retain a highly basic amidine that mimics the bind-
ing of the P1 Arg of natural peptide substrates to Asp-
189 at the base of the S1 pocket. Although this salt-
bridge interaction leads to a substantial amount of
binding energy,4 the incorporation of these extremely
basic moieties is generally associatedwith low levels of
5
oral bioavailability andhigh plasma clearance. In an
effort to produce orally bioavailable thrombin inhibi-
tors with acceptable pharmacokinetics, our laboratories
have focusedon establishing compounds that incorpo-
rate less basic P1 binding elements (e.g., 2-aminopyr-
idine).6 Toward this end, a recent report detailed the
metabolism directed optimization process which resul-
tedin the identification of a series of potent, selective
andorally bioavailable compounds containing weakly
basic pyridine P1 moieties.7 Although successful in pro-
ducing a thrombin inhibitor 1 (Fig. 1) which showed
oral bioavailability across three species andgoodstabi-
lity in the presence of human hepatocyte preparations,
an alternative approach to pharmacokinetic optimiza-
tion was desired. These compounds exhibited poor
solubility at physiologic pH (1: 11 mg/mL @ pH 7.4,
logP=1.50) and, most notably, the expedient produc-
tion of analogues was hindered by the multi-step syn-
theses of the substitutedP1 pyridines. In this paper, we
demonstrate that a modification principally directed
towardthe improvement of the aqueous solubility
additionally permitted a series of previously intractable
P1 N-benzylamides thrombin inhibitors to show useful
levels of oral bioavailability (Fig. 1). Due to the ready
availability of the P1 benzylamines, this result allowed
the rapidinvestigation of P1 SAR with respect to oral
bioavailability, plasma half-life, andhuman liver
microsome stability.
Upon oral dosing of 2 (1 mpk) to dogs, a 1.78 mM max-
imum plasma concentration (Cmax) anda 2.5 h plasma
half-life (t1/2) were achieved( Table 1). The substantial
decrease in plasma half-life observed upon the intro-
duction of the P3 N-oxide into 1 can be attributedto the
increasedplasma clearance ( Table 2). The excellent
microsome stability (2, Table 1) indicated that hepatic
clearance was low, therefore renal excretion was sus-
pectedto be the primary contributor to the higher
overall clearance; indeed collection of urine from the
dog pharmacokinetic experiment showed that 47% of 2
13
was excretedintact.
In an attempt to increase the extremely low plasma
protein binding (80% unbound), overall lipophilicity
(log P=0.19), andconcomitantly reudce the renal
clearance, the P1 3-fluoropyridine was replaced with a
3-chloropyridine (3, Table 1). This minimal substitution
increased protein binding (50% unbound; log P=0.75),
effectively reduced the plasma clearance (3.0 mL/min/
kg, Table 2) andaccordingly improvedthe observed
half-life (t1/2=6.0 h).
The incorporation of the P3 pyridine N-oxide success-
fully addressed the issue of low solubility, however the
multistep syntheses of the 2-aminomethylpyridine P1s
still prohibiteda rapidSAR investigation. It was envi-
sagedthat the enhancedpolarity acquiredupon P3 N-
oxide introduction could be exploited to improve the
poor oral bioavailability of thrombin inhibitors con-
taining benzylamine P1s;14 for example, 2-fluoro-
benzylamine thrombin inhibitor 4 exhibitedvery low
plasma levels in a dog oral pharmacokinetic experiment
(Fig. 2).15 Preparation of the N-oxide afforded 5, in
which the enzyme andanticoagulant potency were
improved( Table 1); more significantly, this compound
exhibitedhigh plasma levels ( Cmax=3.5 mM) anda good
half-life (t1/2=4.9 h) after oral dosing (F=40%). To
establish the general utility of this approach, the simple
N-benzylamide analogue 6 was preparedandalso
Figure 1. Modifications of thrombin inhibitor 1.
The inhibition constants (Ki) versus thrombin (IIa) and
the concentration needed to double the activated partial
thromboplastin time (2ÂaPTT) in human plasma were
determined for each compound (Table 1).8 As the prin-
cipal goal of this study was the optimization of phar-
macokinetics, compounds meeting our established
potency criteria9 were directly subjected to dog phar-
macokinetic andhuman liver microsome stability
experiments (Table 1).10 The electron withdrawing effect
Figure 2.

C. S. Burgey et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1353–1357
1355
Table 1. Effect of P1 substitution on thrombin inhibition (Ki), in vitro anticoagulant potency (2ÂaPTT), dog pharmacokinetic parameters, human
liver microsome stability, andlog P
1
CompdX
R
R2
Ki (nM)
2ÂaPTT (mM)
Cmax (mM)a
t1/2 (h)b
HLMe
Log P
2
3
N
N
F
Cl
H
H
3.1
2.9
0.54
0.47
1.78
1.09c
2.5
5.5
6.5
1.3
0.19
0.75
5
6
7
8
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
F
H
Cl
Me
H
H
H
H
H
H
H
H
H
H
2.3
7.0
2.5
6.3
3.6
3.0
5.2
3.0
2.4
1.8
0.40
0.94
0.54
0.77
0.93
0.56
0.87
0.69
0.81
0.58
3.50
3.20
0.70
1.76
1.43c
2.48c
0.77
2.03
1.67
2.29
4.9
6.3
2.9
3.6
3.7
3.8
1.2
2.8
3.0
2.5
1.1
1.3
0.15
0.29
0.05
0.44
0.08
0
1.39
1.23
1.89
1.74
2.24
1.36
1.91
1.63
2.34
1.93
9
CF3
OMe
OEt
OCHF2
OCF3
OCH2CF3
10
11
12
13
14
0
0
15
16
17
18
19
20
CH
CH
CH
CH
CH
CH
H
H
H
H
H
H
F
Cl
Br
Me
OMe
OCHF2
7.3
0.86
0.23
0.28
0.30
0.43
0.97
1.37d
1.91c
1.01
2.19
1.22
0.12
4.1
2.6
3.6
1.1
2.8
4.8
0.71
0
0.14
0.58
0.71
0.54
NA
NA
2.11
1.81
1.31
1.73
0.26
0.19
0.8
1.2
7.0
21
22
23
24
25
26
27
28
29
CH
CF
F
F
F
p-F
Cl
H
F
F
CF3
F
H
Cl
Cl
Cl
Cl
H
F
2.4
0.43
0.33
0.31
0.78
0.39
0.88
0.88
0.31
0.63
2.29
0.21
0.48
0.42
0.11
0.29
0.07c
1.51
0.99
3.9
6.2
2.1
2.4
2.1
2.5
2.4
3.5
2.8
0.05
3.4–0.61
0
NA
1.23
NA
1.98
2.61
2.14
1.89
1.42
1.99
0.85
0.16
3.6
0.087
2.0
1.3
CH
CH
CH
CCl
CCF3
CF
0
1.04
0.39
0
0.04
0
1.1
1.7
CH
F
aDose=1 mg/kg unless otherwise noted.
bpo, Half-life.
cDose=0.7 mg/kg.
dDose=0.5 mg/kg.
eHuman liver microsome stability versus an internal standard.10
Table 2. Comparison of dog pharmacokinetic parameters
(Table 1). The selection of P1s was biasedtowards simple,
1
substitutedbenzylamines (e.g., R , R2=halogen, fluor-
CompdPB (%free)
Dog Human
Clp (mL /min/kg) Vd (L/kg) iv t1/2 (h) F (%)
oalkylether, Table 1) which contain no obvious metabolic
‘soft-spots.’ Substitution at the ortho position exhibited
little effect on the thrombin inhibition with all prepared
analogues satisfying our establishedpotency criteria ( 5–14,
Table 1). This class of compounds exhibited a range of
Cmax (0.70–3.50mM), with a general trendtowardlower
log P compounds demonstrating higher plasma levels; the
oral half-lives within this group rangedfrom 1.2–6.3 h. As
anticipated,17 substitution at the meta position of the P1
phenyl ring hada more pronouncedeffect on binding,
with small lipophilic groups (e.g., Cl, Me) providing the
best potency (15–20, Table 1). These compounds also
exhibitedgoodplasma levels andhalf-lives after oral
dosing (Cmax=0.12–2.19 mM, t1/2=1.1–4.8 h). Disub-
stitutedP1s generally affordedcompounds with lower
plasma levels, possibly due to the higher log Ps observed
within this group (21–29, Table 1). Overall, there emerged
a trend, although not absolute, toward inhibitors with
lower log P exhibiting better human microsome stability.
1
2
3
33
80
50
14
70
41
2.2
4.91
3.03
0.62
1.13
1.17
4.5
2.9
6.0
66
83
80
shown to give high plasma levels anda goodhalf-life
upon oral administration (Cmax=3.2 mM, t1/2=6.3 h).
The better oral bioavailability is likely a result of
14
improvedoral absorption due to the enhanced solubi-
lity of the N-oxides versus the non-basic pyridines.16
Having demonstrated that the P3 pyridine N-oxide
confers bioavailability to inhibitors containing simple
P1 N-benzylamides, an investigation was undertaken to
explore the P1 SAR with respect to potency,
pharmacokinetics, andhuman microsome stability

1356
C. S. Burgey et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1353–1357
Basedupon thrombin andin vitro anticoagulant
potency, dog oral bioavailability and human liver
microsome stability, the best compoundto emerge from
this survey is 5. This inhibitor is extremely selective for
thrombin, exhibiting minimal (>49 mM) activity versus
trypsin, t-PA, plasmin, factor Xa, plasma kallikrein,
activatedprotein C, urokinase andchymotrypsin. Fur-
ther in vivo characterization (Table 3), revealedthat this
inhibitor exhibitedgoodbioavailability in ogd
(F=40%), rat (F=52%) andrhesus monkey ( F=21%).
Clearance is low in dogs (1.55 mL/min/kg) and, based
on in vitro metabolic scaling studies, is also predicted to
be low in man.13
Determination of the X-ray crystal structure of 5 com-
plexedto thrombin revealedthe same general binding
mode as observed with 1:7 the fluorobenzene ring occu-
pies the S1 specificity pocket, the chlorine of the pyr-
azinone fills the insertion loop, andthe P3 aryl group
binds in the distal hydrophobic pocket (Fig. 3). The
antiparallel b-sheet hydrogen bonding motif between
the aminopyrazinone andGly-216 is maintained
(d=2.63 and2.94 A ); similarly the Ser-214 H-bondto
the inhibitor amide is conserved (d=3.10 A). In S3, the
N-oxide projects toward solvent and appears not to be
involvedin any specific interaction; the improved
potency gainedupon incorporation of the p-deficient P3
pyridine N-oxide may be due to reinforcement of the
edge-to-face s–p interaction between the P3 aryl group
andthe p-rich Trp-215.18
Table 3. Pharmacokinetic parameters of 5 in dog, rat and rhesus
monkeya
Species Cmax (mM) Clp (mL/min/kg) Vd (L/kg) iv t1/2 (h) F (%)
Synthesis of the 2,2-difluoro-2-(2-pyridyl-N-oxide)ethyl-
amine 3319 commencedwith azide 31, preparedby our
previously disclosed route.7 Oxidation of the electron
deficient pyridine of 31 couldbe effectedby treatment
with MCPBA at elevatedtemperature in the presence of
Kishi’s radical inhibitor (Scheme 1).20 Staudinger
reduction afforded the 2,2-difluoro-2-(2-pyridyl-N-
oxide)ethylamine 33 as a low-melting solid.
Dog
Rat
Rhesus
3.28
1.94Æ0.79
1.05
1.55
43Æ7
19
0.49
0.93Æ0.12 0.43Æ0.05 52
1.5 1.1 21
6.3
40
aDose=1 mg/kg (dog), 10 mg/kg (rat), 5 mg/kg (rhesus).
This compound demonstrated dose-dependent efficacy
in the rat FeCl3 arterial thrombosis model upon iv dos-
ing (Table 4). At the maximum dose of 10 mg/kg/min
(solubility limit in vehicle) near complete efficacy (2/12
occlusions, 1RF) was observedat plasma levels that mini-
mally elevatedthe aPTT (ca. 1.5-foldelevation); a dose
dependent effect on the thrombin time (TT) was observed.
Scheme 1.
Coupling of the P3 amine 33 to 3-bromopyrazinone 34
requiredheating in a sealedtube at 120 ꢀC for 24 h
(Scheme 2). Chlorination of 35a with 1 equivalent of
NCS in DCE occurredwith complete regioselectivity to
affordthe 6-chloropyrazinone 35b. Hydrolysis of ethyl
ester afforded the corresponding acids, which under-
went EDC-mediated amide coupling with the P1 amines
to affordthe final products (e.g., 5).
In this paper, we have demonstrated that a modification
principally directed toward the improvement of the
aqueous solubility (i.e., introduction a P3 pyridine N-
oxide) of the previous lead compound afforded a new
series of potent orally bioavailable P1 N-benzylamide
Figure 3. X-ray crystal structure of 5 boundin the thrombin active site.
Table 4. Effects of intravenous administration of 5 in the rat model of FeCl3-induced carotid thrombosisa
Dose
Incidence of Occlusion
TTO
TT (s)
APTT (s)
Plasma level (mM)
b
—
19/22 (2RF)
2/12 (1RF)
3/6 (1RF)
5/6
15Æ1
26Æ5
15Æ1
19Æ1
14.4Æ0.2
64.7Æ5.4
59.3Æ6.5
31.6Æ1.9
17.2Æ0.4
25.4Æ0.9
22.6Æ0.9
24.1Æ1.1
—
10
5
788Æ51
402Æ30
219Æ20
3
aDose @ mg/kg/min infusion; TTO=time to occlusion; TT=thrombin time (ref 12).
bVehicle=0.3% DMSO.

C. S. Burgey et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1353–1357
1357
K.; Yan, Y.; Shafer, J. A.; Vacca, J. P. J. Med. Chem. 1998,
41, 4466.
7. Burgey, C. S.; Robinson, K. A.; Lyle, T. A.; Sanderson, P.
E. J.; Lewis, S. D.; Lucas, B. J.; Krueger, J. A.; Singh, R.;
Miller-Stein, C.; White, R. B.; Wong, B.; Lyle, E. A.; Wil-
liams, P. D.; Coburn, C. A.; Dorsey, B. D.; Barrow, J. C.;
Stranieri, M. T.; Holahan, M. A.; Sitko, G. R.; Cook, J. J.;
McMasters, D. R.; McDonough, C. M.; Sanders, W. M.;
Wallace, A. A.; Clayton, F. C.; Bohn, D.; Leonard, Y. M.;
Detwiler, Jr., T. J.; Lynch, Jr., J. J.; Yan, Y.; Chen, Z.; Kuo,
L.; Gardell, S. J.; Shafer, J. A.; Vacca, J. P. J. Med. Chem.
2003, 46, 461.
8. Lewis, S. D.; Ng, A. S.; Lyle, E. A.; Mellott, M. J.;
Appelby, S. D.; Brady, S. F.; Stauffer, K. S.; Sisko, J. T.; Mao,
S.-S.; Veber, D. F.; Nutt, R. F.; Lynch, J. J.; Cook, J. J.;
Gardell, S. J.; Shafer, J. A. Thromb. Haemost. 1995, 74, 1107.
9. For a discussion of the general criteria for thrombin inhib-
itor development see: Tucker, T. J.; Isaacs, R. C. A. Adv.
Amino Acid Mimetics Peptidomimetics 1999, 2, 53.
Scheme 2.
10. Human microsome stabilities are reportedas the ratio of
the percent remaining after a 1 h incubation versus the internal
standard thrombin inhibitor 3-(2-phenethylamino)-6-methyl-1-
(2 - amino - 6 - methyl - 5 - methylenecarboxamido - methylpyr-
idinyl)pyrazinone (ref 6).
thrombin inhibitors. This finding expedited an investi-
gation of the P1 SAR with respect to oral bioavail-
ability, plasma half-life, andhuman liver microsome
stability which revealed 5 as the best candidate for
advanced evaluation. Further studies are required to
evaluate the safety, tolerability, pharmacokinetics, and,
ultimately, the long-term utility of these thrombin inhi-
bitors as anticoagulant therapeutics.
11. This result is consistent with data from a related series:
Isaacs, R. C. A. Unpublishedresults.
12. Reportedas number of carotidartery vesssels ( n=6)
occluding after a 10 mg/kg/min 180-min infusion (arterial
injury is initiatedafter 120 min, followedby a 60-min obser-
vation period). Reflow (RF) indicates that the vessel occluded
then subsequently reopened: Kurz, M. D.; Main, B. W.; San-
dusky, G. E. Thromb. Res. 1990, 60, 269. See also ref 8.
13. Singh, R.; Wong, B. Unpublishedresults.
Acknowledgements
14. The beneficial effects of pyridine N-oxides in drug dis-
covery have been previously documented: Bell, I. M.; Erb,
J. M.; Freidinger, R. M.; Gallicchio, S. N.; Guare, J. P.; Gui-
dotti, M. T.; Halpin, R. A.; Hobbs, D. W.; Homnick, C. F.;
Kuo, M. S.; Lis, E. V.; Mathre, D. J.; Michelson, S. R.; Paw-
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We thank the analytical chemistry, mass spectroscopy,
andNMR analysis groups for their assistance, as well as
Carl Homnick for his skillful HPLC separations.
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¨
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compounds with low oral bioavailability.11
16. Compound 4 shows excellent stability to dog liver micro-
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