Design, synthesis, and thrombin-inhibitory activity of pyridin-2-ones as P2/P3 core motifs

Article abstract of DOI:10.1016/j.bmcl.2008.01.122

Guided by available X-ray crystal structure data on the serine protease thrombin, a series of pyridin-2-one derivatives were designed and synthesized having diverse functionality at the P₁ and P₃ sites. Potent in vitro activity again

Full text of DOI:10.1016/j.bmcl.2008.01.122

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1972–1976
Design, synthesis, and thrombin-inhibitory activity
of pyridin-2-ones as P2/P3 core motifs
Stephen Hanessian,a,* Daniel Simard,a Malken Bayrakdarian,a
Eric Therrien,a Ingemar Nilssonb,* and Ola Fjellstro¨mb
aDepartment of Chemistry, Universite´ de Montre´al, C.P. 6128, Succ. Centre-Ville, Montre´al, Que., Canada H3C 3J7
bAstraZeneca R&D Mo¨lndal, Medicinal Chemistry Mo¨lndal, Sweden
Received 14 December 2007; revised 30 January 2008; accepted 30 January 2008
Available online 7 February 2008
Abstract—Guided by available X-ray crystal structure data on the serine protease thrombin, a series of pyridin-2-one derivatives
were designed and synthesized having diverse functionality at the P1 and P3 sites. Potent in vitro activity against thrombin, with
excellent selectivity over trypsin was found for selected analogues.
Ó 2008 Elsevier Ltd. All rights reserved.
Thromboembolic disorders are the major cause of mor-
tality and morbidity in the industrial world.1 Closely
linked to the clinical syndromes of thromboembolism is
an excessive stimulation of the coagulation cascade. The
serine protease thrombin plays a critical role in the blood
coagulation cascade. Predominantly, it is the key terminal
protease and holds a central role in both hemostasis and
thrombosis.1 Thrombin effectively catalyzes the proteo-
lytic cleavage of the soluble plasma-protein fibrinogen
to form insoluble fibrin leading to clot formation. Consid-
erable efforts have therefore been devoted to the discovery
of safe, orally active inhibitors of this enzyme in order to
replace those that are used nowadays.1
structure of indolizidinones with thrombin.5 Low nano-
molar inhibition of thrombin validated the initial design
concept of an indolizidinone motif with strategically
placed substituents as a constrained mimic of the natu-
ral substrate D-Phe-Pro-Arg.6 We have also reported
on related indolizidinones designed to target Factor
VIIa.7 Simpler indolizidinones and related sultams8 were
also synthesized as potential thrombin inhibitors. The
marine natural products Dysinosin A,9 Oscillarin,10
and Chlorodysinosin A11 were recently synthesized in
our laboratories to evaluate their inhibitory activity
against thrombin and Factor VIIa. Truncated analogues
of these natural products containing a 2-carboxy-octa-
hydroindole P2 core unit were also recently reported.12
X-ray crystal structures of thrombin-inhibitor com-
plexes have offered attractive options for the design
and synthesis of structure-based potential inhibitors.2
Indeed, several peptidomimetic-type drug candidates
known to interact with thrombin at specific sites are in
advanced stages of clinical studies.3 More recent efforts
have concentrated on non-peptidic inhibitors based on
heteroaromatic or heterocyclic core structures.4
In a previous publication, we reported on the design,
synthesis, and X-ray cocrystal structure of low nano-
molar phenolic thrombin inhibitors.13 We now report
on the design, synthesis and thrombin-inhibitory activ-
ity of a series of amines and sulfonamides appended
to a trisubstituted pyridin-2-one core structure repre-
sented by the generic expression 1 (Fig. 1). The requi-
site P1, P2, and P3 interactions with their respective
subsites in the enzyme as suggested by molecular mod-
eling augured well for anticipating in vitro activity as
well.
As part of our ongoing project in the discovery of new
potential thrombin inhibitors, we have previously re-
ported on the design, synthesis, and X-ray cocrystal
The pyridin-2-one core motif incorporating diverse
functionality in the P1/P3 positions was synthesized as
shown in Scheme 1. Thus, the 2-methoxy-4-methyl-pyr-
idine-3-carbaldehyde14 2 was converted into the acetoni-
trile analogue 3 in 57% overall yield using a three-step
Keywords: Enzyme inhibitors; Thrombin; 2-Pyridone.
*
Corresponding authors. Tel.: +1 514 343 6738; fax: +1 514 343 5728
(S.H.); tel.: +46 31 7761161; fax: +46 31 7763839 (I.N.); e-mail
addresses: stephen.hanessian@umontreal.ca; ingemar.nilsson@
astrazeneca.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.122

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1972–1976
1973
(Table 1, entries 1–3) showed excellent antithrombin
activity with IC50 below 100 nM. Thus, the best com-
pound in this series is the o-methyl phenethyl amine 8
with an IC50 of 23 nM against thrombin. Several substi-
tuted sulfonamides exhibited IC50 values in the range of
0.3–1.2 lM (Table 1, entries 4–9).
S2
P2
Me
O
N
P3
S3
N
N
H
H
O
P1
1
Unfortunately, no clear-cut SAR could be observed by
varying the position of aromatic substituents. The addi-
tion or deletion of a methyl group in the aromatic ring
led to a fourfold decrease of the in vitro activity (Table 1,
compare entries 1, 2, and 3). In addition, the nature of
the P3 amine seems important for binding as observed
by a 20-fold potency increase when the sulfonamide
functionality is replaced by an alkylamine (Table 1,
compare entries 2 and 11). Although the activities could
not be directly correlated with the nature or position of
aromatic substitution on the P3 part, we were intrigued
that analogue 8 showed good antithrombin activity with
a 42-fold selectivity over trypsin.
S1
Figure 1. Prototype of pyridin-2-one core with variations in the P1 and
P3 subunits.
procedure. Acidic hydrolysis of 3 furnished the expected
pyridone which was esterified with diazomethane to give
the methyl ester 4 in excellent overall yield. Then, the
N-amino functionality was introduced using a phos-
phine-based amino transfer reagent15 yielding the corre-
sponding N-amino pyridone 5. Arylsulfonylation or
alkylation via reductive amination of 5 with the desired
P3 appendage afforded 6. Hydrolysis of the ester followed
by amide formation, and subsequent deprotection yielded
the analogue 7. In order to provide sufficient diversity,
analogues featuring different aromatic P1 substituents
were prepared using the general protocol (Fig. 2).
Table 2 lists the IC50 values for the pyridin-2-one ana-
logues in the series B. Equipotent antithrombin activity
was observed for the substituted phenethyl amines
(Table 2, entries 1–2). However, aromatic or heterocy-
clic sulfonamides seemed detrimental to thrombin inhi-
bition (Table 2, entries 3–4). The same trend was
observed with the 2-amino-4-aminomethyl pyridine P1
subunit (series C, Table 3), as well as with the 2-amino-
methyl-3-fluoro-6-methyl pyridine P1 subunit (series D,
Table 4), although the phenethyl amine 28 did not show
any activity against thrombin in the series D.16
The sets of sulfonamides and amines harboring the pyri-
din-2-one core motif exhibited excellent purities by LC/
MS. The IC50 values of the synthesized analogues in the
4-amidinobenzyl P1 series (A) are listed in Table 1.
The pyridin-2-one analogues in this series exhibited IC50
values in the range of 0.02–2.5 lM with various selectiv-
ity over trypsin (Table 1). The amines 8, 9, and 10
Me
Me
Me
a) NaBH4, 85%
b) PPh3, NBS, 75%
a) HBr (37%)
b) CH2N2
CHO
OMe
CN
OMe
CO2Me
97% (two steps)
c) KCN, 90%
Me
N
N
N
O
H
2
3
4
a) R'SO2Cl,
pyridine
OR
O
P
Me
Ph
H2NO
Ph
CO2Me
W
N
CO2Me
R'
N
H
Cs2CO3, DMF
60%
b) R'CH2CHO,
NaBH3CN
N
O
O
NH2
5
6
Me
O
a) NaOH, THF/H2O
b) EDC, HOBt, R"NH2
W
N
N
R"
R'
N
H
H
c) H2, Pd/C, MeOH/HCl
OR HCl/MeOH
O
7
Scheme 1. General protocol for the synthesis of the pyridin-2-one analogues.
F
R" =
NH2
Me
N
NH2
N
N
NH2
NH
A
B
C
D
Figure 2. Different P1 substituents.

1974
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1972–1976
Table 1. IC50 values for the 4-amidinobenzyl P1 subunit (series A)
Me
R5
O
R4
R3
W
N
N
H
N
H
O
NH
R1
HCl
R2
NH2
IC50 (μM)
Entry
Compound
W
R1
R2
R3
R4
R5
IC50 (lM)
Ratioa
Thrombin
Trypsin
1
8
CH2CH2
CH2CH2
CH2CH2
SO2
Me
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.023
0.091
0.092
0.307
0.578
0.885
1.054
1.180
1.243
1.846
1.919
2.471
0.963
1.420
2.140
4.130
7.130
10.300
15.600
2.020
17.100
3.410
15.200
5.770
42
16
23
13
12
12
15
1.7
14
1.8
7.9
2.3
2
9
H
H
3
10
11
12
13
14
15
16
17
18
19
Me
OMe
Me
H
H
Me
H
4
Cl
H
5
SO2
SO2
Me
H
6
Cl
H
7
SO2
SO2
H
OMe
OMe
H
H
8
Me
H
H
9
SO2
SO2
CH@CHACH@CH
H
H
10
11
12
H
H
H
OMe
H
H
SO2CH2
SO2
H
H
H
H
H
a Ratio of (IC50 trypsin)/(IC50 thrombin).
Table 2. IC50 values for the 2-amino-5-aminomethyl pyridine P1 subunit (series B)
Me
R5
O
R4
R3
W
N
N
H
N
H
N
HCl
O
R1
NH2
R2
Entry
Compound
W
R1
R2
R3
R4
R5
IC50 (lM)
Ratioa
Thrombin
Trypsin
1
2
3
20
21
22
CH2CH
CH2CH
SO2
H
CF3
H
H
H
F
H
H
H
2.706
3.091
>44.400
> 44.400
> 44.400
> 4.400
>16
>14
—
Me
OMe
H
OMe
H
H
Me
4
23
SO2
>44.400
>44.400
—
S
Me
a Ratio of (IC50 trypsin)/(IC50 thrombin).
Table 3. IC50 values for the 2-amino-4-aminomethyl pyridine P1 subunit (series C)
Me
R5
O
R4
R3
W
N
NH2
N
N
H
H
HCl
O
N
R1
R2
Entry
Compound
W
R1
R2
R3
R4
R5
IC50 (lM)
Ratioa
Thrombin
Trypsin
1
2
24
25
SO2CH2
SO2
H
H
H
H
Cl
Et
H
H
H
H
>44.400
> 44.400
>44.400
>44.400
—
—
a Ratio of (IC50 trypsin)/(IC50 thrombin).

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1972–1976
1975
Table 4. IC50 values for the 2-aminomethyl-3-fluoro-4-methyl pyridine P1 subunit (series D)
Me
R5
O
F
R4
R3
W
N
Me
N
N
H
H
O
N
R1
R2
Entry
Compound
W
R1
R2
R3
R4
R5
IC50 (lM)
Thrombin
Ratioa
Trypsin
1
2
3
26
27
28
SO2
SO2
Me
SO2Me
F
H
H
H
H
H
H
H
H
Cl
Cl
H
H
>44.400
>44.400
>44.400
>44.400
>44.400
>44.400
—
—
—
CH2CH2
4
29
SO2
>44.400
>44.400
—
a Ratio of (IC50 trypsin)/(IC50 thrombin).
4. See for example, (a) Sanderson, P. E. J.; Cutrona, K. J.;
Dyer, D. L.; Krueger, J. A.; Kuo, L. C.; Lewis, S. D.;
Lucas, B. J.; Yan, Y. Bioorg. Med. Chem. Lett. 2003, 13,
161; (b) Sanderson, P. E.; Cutrona, K. J.; Savage, K. L.;
Naylor-Olsen, A. M.; Bickel, D. J.; Bohn, D. L.;
Clayton, F. C.; Krueger, J. A.; Lewis, S. D.; Lucas, B.
J.; Lyle, E. A.; Wallace, A. A.; Welsh, D. C.; Yan, Y.
Bioorg. Med. Chem. Lett. 2003, 13, 1441; (c) Soll, R. M.;
Lu, T.; Tomczuk, B.; Illig, C. R.; Fedde, C.; Eisennagel,
S.; Bone, R.; Murphy, L.; Spurlino, J.; Salemme, F. R.
Bioorg. Med. Chem. Lett. 2000, 10, 1; (d) Sanderson, P.
E. J.; Cutrona, K. J.; Dorsey, B. D.; Dyer, D. L.;
McDonough, C. M.; Naylor-Olsen, A. M.; Chen, I.-W.;
Chen, Z.; Cook, J. J.; Gardell, S. J.; Krueger, J. A.;
Lewis, S. D.; Lin, J. H.; Lucas, B. J., Jr.; Lyle, E. A.;
Lynch, J. J., Jr.; Stranieri, M. T.; Vastag, K.; Shafer, J.
A.; Vacca, J. P. Bioorg. Med. Chem. Lett. 1998, 8, 817;
(e) Tamura, S. Y.; Semple, J. E.; Reiner, J. E.; Goldman,
E. A.; Brunck, T. K.; Lim-Wilby, M. S.; Carpenter, S.
H.; Rote, W. E.; Oldeshulte, G. L.; Richard, B. M.;
Nutt, R. F.; Ripka, W. C. Bioorg. Med. Chem. Lett.
1997, 7, 1543; (f) Levy, O. E.; Semple, J. E.; Lim, M. L.;
Reiner, J.; Rote, W. E.; Dempsey, E.; Richard, B. M.;
Zhang, E.; Tulinsky, A.; Ripka, W. C.; Nutt, R. F. J.
Med. Chem. 1996, 39, 4527.
In conclusion, we have shown that an N-amino pyridin-
2-one can be used as a P2/P3 core motif for thrombin
inhibition. In general, the phenethyl amines obtained
by a reductive amination protocol were more active
against thrombin compared to the corresponding sul-
fonamides. In addition, the 4-amidinobenzyl substituent
was found to be the most effective amide P1 subunit (ser-
ies A). We also showed that the selectivity over trypsin
can be modulated according to the nature of the P3 sub-
unit. The simplicity of these structures and their relative
ease of synthesis should pave the way to a better under-
standing of the relative roles of the P2/P3 subunits, and
the subtle influences of aromatic substitutions on the
enzymatic activity.
Acknowledgments
We thank the NSERC of Canada, FQRNT (Quebec),
and AstraZeneca (Mo¨lndal, Sweden) for financial assis-
tance through the Medicinal Chemistry Chair program.
5. Hanessian, S.; Balaux, E.; Musil, D.; Olsson, L.-L.;
Nilsson, I. Bioorg. Med. Chem. Lett. 2000, 10, 243.
6. See for example, Danilewicz, J. C.; Abel, S. M.; Brown, A.
D.; Fish, P. V.; Hawkeswood, E.; Holland, S. J.; James,
K.; McElroy, A. B.; Overington, J.; Prowling, M. J.;
Rance, D. J. J. Med. Chem. 2002, 45, 2432, and references
cited therein.
7. Hanessian, S.; Therrien, E.; Granberg, K.; Nilsson, I.
Bioorg. Med. Chem. Lett. 2002, 12, 2907.
8. (a) Hanessian, S.; Sailes, H.; Munro, A.; Therrien, E.
J. Org. Chem. 2003, 68, 7219; (b) Hanessian, S.; Sailes, H.;
Therrien, E. Tetrahedron 2003, 59, 7047.
References and notes
1. (a) Colman, R. W.; Clowes, A. W.; George, J. N.;
Hirsch, J. L.; Marder, V. J. Overview of homeostasis.
In Hemostasis and Thrombosis. Basic Principles and
Clinical Practice; Colman, R. W., Hirsh, J. L., Marder,
V. J., Clowes, A. W., George, J. N., Eds., 4th ed.;
Lippincott Williams & Wilkins: Philadelphia, 2001; pp
3–16; (b) Dahlba¨ck, B. Lancet 2000, 355, 1627; (c)
Davie, E. W.; Fujikawa, K.; Kisiel, W. Biochemistry
1991, 30, 10363.
9. Hanessian, S.; Margarita, R.; Hall, A.; Johnstone, S.;
Tremblay, M.; Parlanti, L. J. Am. Chem. Soc. 2002, 124,
13342.
10. Hanessian, S.; Tremblay, M.; Petersen, J. F. W. J. Am.
Chem. Soc. 2004, 126, 6064.
11. Hanessian, S.; Del Valle, J. R.; Xue, Y.; Blomberg, N.
J. Am. Chem. Soc. 2006, 128, 10491; Hanessian, S.; Del
Valle, J. R.; Xue, Y.; Blomberg, N. J. Am. Chem. Soc.
2006, 128, 11727.
2. (a) Srivastava, S.; Goswami, L. N.; Dikshit, D. K. Med.
Res. Rev. 2005, 25, 66; (b) Gustafsson, D.; Bylund, R.;
Antonsson, T.; Nilsson, I.; Nystro¨m, J.-E.; Eriksson, U.;
Bredberg, U.; Teger-Nilsson, A.-C. Nat. Rev. Drug Disc.
2004, 3, 649; (c) Pfau, R. Curr. Opin. Drug Discov. Dev.
2003, 6, 437; (d) Kontigiorgis, C. A.; Hadjipavlou-
Litina, D. Curr. Med. Chem. 2003, 10, 525; (e) Sander-
son, P. E. J.; Naylor-Olsen, A. M. Curr. Med. Chem.
1998, 5, 289.
12. Hanessian, S.; Ersmark, K.; Wang, X.; Del Valle, J. R.;
Blomberg, N.; Xue, Y.; Fjellstro¨m, O. Bioorg. Med. Chem.
Lett. 2007, 17, 3480.
3. (a) Steinmetzer, T.; Sturzebecher, J. Curr. Med. Chem.
¨
2004, 11, 2297; (b) Steinmetzer, T.; Hauptmann, J.;
Sturzebecher, J. Exp. Opin. Invest. Drugs 2001, 10, 845.
¨

1976
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1972–1976
13. Hanessian, S.; Therrien, E.; van Otterlo, W. A. L.;
Bayrakdarian, M.; Nilsson, I.; Fjellstro¨m, O.; Xue, Y.
Bioorg. Med. Chem. Lett. 2006, 16, 1032.
14. (a) Comins, D. L.; LaMunyon, D. H. Tetrahedron Lett.
1988, 29, 773; (b) Comins, D. L.; Killpack, M. O. J. Org.
Chem. 1990, 55, 69.
Baldinger, H.; Karpitschka, E. M.; Knoflach, J. Synthe-
sis 1988, 592.
16. For examples of P1 substitution, see Rittle, K. E.;
Barrow, J. C.; Cutrona, K. J.; Glass, K. L.;
Krueger, J. A.; Kuo, L. C.; Lewis, S. D.; Lucas,
B. J.; Sanders, W. M.; Yan, Y.; Vacca, J. P.;
Selnick, H. G. Bioorg. Med. Chem. Lett. 2003, 13,
3477.
15. (a) Colvin, E. W.; Kirby, G. W.; Wilson, A. C.
Tetrahedron Lett. 1982, 23, 3835; (b) Klo¨tzer, W.;