P2 pyridine N-oxide thrombin inhibitors: A novel peptidomimetic scaffold

Article abstract of DOI:10.1016/j.bmcl.2005.03.110

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P ₁ benzylamide delivered the potent thrombin inhibitor 21 (K _i = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C_max = 2.6 μM, t _1/2 = 4.5 h).

Full text of DOI:10.1016/j.bmcl.2005.03.110

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2771–2775
P2 pyridine N-oxide thrombin inhibitors: a novel
peptidomimetic scaffold
a,
a
Philippe G. Nantermet,a,* Christopher S. Burgey, Kyle A. Robinson,
*
Janetta M. Pellicore,a Christina L. Newton,a James Z. Deng,a Harold G. Selnick,a
S. Dale Lewis,b Bobby J. Lucas,b Julie A. Krueger,b Cynthia Miller-Stein,c
Rebecca B. White,c Bradley Wong,c Daniel R. McMasters,d Audrey A. Wallace,e
Joseph J. Lynch, Jr.,e Youwei Yan,f Zhongguo Chen,f Lawrence Kuo,f Stephen J. Gardell,b
Jules A. Shafer,b Joseph P. Vaccaa and Terry A. Lylea
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
bDepartment of Biological Chemistry, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
dDepartment of Molecular Systems, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
eDepartment of Pharmacology, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
fDepartment of Structural Biology, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
Received 6 January 2005; revised 28 March 2005; accepted 29 March 2005
Available online 29 April 2005
Abstract—In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone
thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward
oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P1
benzylamide delivered the potent thrombin inhibitor 21 (Ki = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels
and half-life after oral dosing in the dog (Cmax = 2.6 lM, t1/2 = 4.5 h).
Ó 2005 Elsevier Ltd. All rights reserved.
Thrombosis-related disorders such as deep vein throm-
bosis, pulmonary embolism, and thromboembolic
stroke remain a major cause of morbidity worldwide.1
The limitations associated with current therapies2 have
driven the search for small-molecule direct inhibitors
of specific enzymes involved in the coagulation cascade.3
In this regard, inhibitors of both thrombin and factor
Xa have attracted considerable recent attention.4 In
our laboratories, the search for potent and orally bio-
available direct thrombin inhibitors has led to the
evaluation of pyrazinone based small molecule pepti-
domimetics (1, Fig. 1).5 Detailed metabolic studies indi-
cated a significant degree of oxidative metabolism
around the pyrazinone core.6 As a result, we sought to
Keywords: Thrombin; Peptidomimetic.
*
Corresponding authors. Tel.: +1 215 652 0945; fax: +1 215 652 3971;
e-mail addresses: philippe_nantermet@merck.com; christopher_burgey@
merck.com
Figure 1. From pyrazinone to pyridine N-oxide.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.110

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P. G. Nantermet et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2771–2775
Table 1. P1 SAR
identify alternative P2 peptidomimetic cores that would
retain the classical H-bond network with glycine-216 of
the enzyme (Fig. 1); one such alternative envisioned was
a pyridine N-oxide scaffold.7
Preparation of pyridine N-oxide derivative 2, the direct
analog of pyrazinone 1, afforded a 6 nM thrombin
inhibitor and provided support for this strategy. A cyano
group at the 5-position provided a further potency
enhancement (Fig. 1, 3). As anticipated, the correspond-
ing neutral pyridine derivatives are significantly less po-
tent against thrombin than their parent compounds
(pyridine of 2: Ki = 620 nM; pyridine of 3: Ki = 103 nM;
structures not shown), consistent with the N-oxide oxy-
gen serving as an H-bond acceptor. Additionally, X-ray
crystallographic analysis of a related inhibitor 4 bound
in the active site of thrombin (Fig. 2, PDB Code:
1Z71) clearly demonstrates the H-bond network with
glycine-216.
Compd
X
P1
Ki
(nM)a
2xaPTT
(lM)
5
CN
150
—
6
Cl
2.60
0.46
7
8
Cl
CN
2.50
0.23
0.40
0.15
An initial study focused on incorporating the optimized
P1 groups recently discovered in our laboratories into
the P2 pyridine N-oxide template (Table 1).8 Com-
pounds were evaluated for their thrombin inhibitory po-
tency and their functional ability to double the activated
partial thromboplastin time (2xaPTT) in human plas-
ma.9 The unsubstituted P1 benzyl amide 5 (Ki = 150 nM)
functions as the benchmark compound. Installation of
the N-linked triazole at the ortho-position of the P1 benz-
yl or pyridyl group8a yields inhibitors (6–8) with both
good intrinsic and functional potency. Introduction of
a 2-aminomethyl substituent8b also results in a signifi-
cant potency improvement, as illustrated by compounds
9 and 10. Incorporation of a meta-chloro substituent
affords a significant improvement in intrinsic potency
9
10
Cl
CN
0.40
<0.01
0.13
0.09
11
12
Cl
CN
0.42
0.08
0.73
0.20
13
14
Cl
H
0.05
1.50
0.23
0.43
15
16
H
CN
0.14
0.002
0.20
0.10
17
18
19
Cl
<0.01
0.001
0.04
0.15
0.07
0.10
CN
H
a Ki values are the average of at least two determinations, standard
error of the mean <10%.
(e.g., 12 vs 5), but, as noted in earlier studies, the
concomitant increase in lipophilicity is detrimental to
functional activity.10 The combination of the ortho-azole
and the meta-chloro substituents affords exquisitely po-
tent thrombin inhibitors (13–16); for example, com-
pound 16 displays a 2 pM Ki against thrombin, with a
2xaPTT of 100 nM. Similarly, the potency-enhancing
aminomethyl and chlorine substituents can be merged
Figure 2. X-ray crystal structure of 4 bound in the thrombin active
site.

P. G. Nantermet et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2771–2775
2773
in an additive fashion to yield extremely potent throm-
bin inhibitors (17–19). The most potent compound pro-
duced from this study is 18 (Ki = 1 pM), displaying a
2xaPTT of 70 nM.
potency increase (vs 21) attained with the hydroxy-
methyl analog 24.
The high levels of potency demonstrated by the ortho-
aminomethyl and tetrazole analogs (Table 1, 15–19) of-
fered the possibility that significant truncation of these
molecules could still deliver potent thrombin inhibitors.
Accordingly, complete excision of the P3 binding element
afforded inhibitors in the low to mid nanomolar range
(Table 3, 25–28); most notable is compound 28 with
a Ki = 10 nM and a 2xaPTT = 560 nM (MW = 346).
Unfortunately, removal of the P3 group afforded no sig-
nificant advantage regarding pharmacokinetic profile.
Having completed an investigation of the P1 SAR, we
turned our attention to the evaluation of the pharmaco-
kinetic profile of members of this series (Table 2). Upon
oral dosing of aminomethyl derivative 9 (0.65 mpk) to
dogs, a 0.82 lM maximum plasma concentration (Cmax
)
and a 1.5 h plasma half-life (t1/2) were achieved. The
meta-chloro analog 17 similarly displayed a promising
pharmacokinetic profile (Cmax = 0.70 lM, t1/2, = 1.3 h).
As the tri- and tetrazole analogs (13–16) had inferior
pharmacokinetic profiles, our efforts focused on the
optimization of the P1 aminomethyl series.
The synthesis of these P2 pyridine N-oxide inhibitors is
outlined in Scheme 1.11 Protection of 2-amino-6-methyl-
Metabolism studies involving human and dog micro-
somal incubations revealed that the desired objective
of minimizing the extent of P2 metabolism had
been achieved; however, P1 benzylic site oxidation and
subsequent N-dealkylation now arose as the primary
metabolic pathway. In an attempt to attenuate this
metabolism, a study to substitute the benzylic sites
was initiated (Table 2). Methylation of the benzylic po-
sition alpha to the P1 amide group (Table 2, Y = Me)
and resolution gave access to inhibitors 20 and 21.
The more active enantiomer 21 displays an 8-fold loss
in binding potency (vs 9) yet maintains good anticoagu-
lant activity (Ki = 3.2 nM, 2xaPTT = 360 nM); notably,
this modification resulted in a significant improvement
in the pharmacokinetic profile (21, Cmax = 2.6 lM,
t1/2 = 4.5 h). Methylation at the other benzylic site was
also tolerated, but did not lead to improved dog phar-
macokinetics (22–23). Inspection of the X-ray crystal
structure of the related inhibitor 4 bound in the active
site of thrombin (Fig. 2) suggested that strategic place-
ment of a hydrogen bonding group at the P1 benzylic
position could potentially gain access to an additional
interaction with residues contained within the active site
(e.g., Ser-195). This analysis is supported by the 5-fold
Table 3. Des-P3 analogs
Compd
X
P1
Ki (nM)a 2xaPTT (lM)b
25
Cl
140
18
—
26
CN
1.6
27
28
Cl
26
10
—
CN
0.56
a Ki values are the average of at least two determinations, standard
error of the mean <10%.
Table 2. P1 benzylic substitution
Compd
Y
Z
Ki (nM)a
2xaPTT
(lM)
Cmax
(lM)
t1/2 (h)
9
H
H
0.40
260
3.2
0.13
—
0.82d
—
1.5
—
20
21
22
23
24
Meb
Meb
H
H
H
0.36
5.9
2.6e
4.5
—
Meb
Meb
H
20
—
H
0.45
0.48
0.27
0.23
0.1f
0.34g
2.0
1.5
CH2OHc
a Ki values are the average of at least two determinations, standard error of the mean <10%.
b 20/21 and 22/23 are enantiomeric pairs.
c Single enantiomer, absolute configuration not established.
d po dose = 0.65 mpk.
e po dose = 0.95 mpk.
f po dose = 0.7 mpk.
g po dose = 0.85 mpk.

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P. G. Nantermet et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2771–2775
Scheme 1. Reagents and conditions: (a) Boc2O; (b) NCS, DCE; (c) LDA, diethyl carbonate, THF; (d) NaH, DMF, 32;5 (e) 1 N LiOH, THF;
(f) P1-NH2, EDC, HOAt, DMF; (g) TFA, DCM; (h) mCPBA, DCE.
pyridine with di-tert-butyl dicarbonate, followed by
regioselective chlorination affords 30. The pyridylacetate
31 is produced via benzylic metalation of 30 with LDA
and subsequent quenching with diethyl carbonate.
Alkylation of 31 with the P3 2,2-difluoro-2-(2-pyr-
idyl)ethyl-trifluoromethanesulfonate 325 completes the
assembly of the key P3–P2 subunit 33. Ester hydrolysis
and P1 amide coupling are followed by deprotection
and then pyridine oxidation12 with mCPBA to afford
the final products 34. The cyano pyridines are prepared
via an analogous route starting from 2-amino-5-cyano-
6-methylpyridine.
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In conclusion, we have demonstrated that the critical
hydrogen bonding motif of the established 3-aminopyr-
azinone thrombin inhibitors can be effectively mimicked
by a 2-aminopyridine N-oxide. As this peptidomimetic
core is more resistant toward oxidative metabolism, it
also overcomes the metabolic liability associated with
the pyrazinones. An optimization study of the P1 benzyl-
amide delivered the potent thrombin inhibitor, 21 which
exhibited good plasma levels and half-life after oral dos-
ing in the dog. Studies to explore the generality of 2-
aminopyridine N-oxides as peptidomimetics are
underway.
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Acknowledgments
We thank the analytical chemistry, mass spectroscopy,
and NMR analysis groups for their assistance.

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12. Oxidation was P2 regiospecific in the case of the chloro
derivatives but in the case of the cyano derivatives
careful monitoring of oxidant stoichiometry and reac-
tion time was necessary to avoid over-N-oxidation at
the P3 pyridyl.