Novel pyrazinone inhibitors of mast cell tryptase: Synthesis and SAR evaluation

Article abstract of DOI:10.1016/j.bmcl.2004.07.051

In this manuscript, the synthesis and SAR evaluation of a novel pyrazinone class of tryptase inhibitors is described. Chemical optimization of the P1 and P4 groups led to the identification of 7p (K_i = 93 nM) as a potent inhibitor of mast cell tryptase.

Full text of DOI:10.1016/j.bmcl.2004.07.051

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4819–4823
Novel pyrazinone inhibitors of mast cell tryptase:
synthesis and SAR evaluation
a
a
a
Corey R. Hopkins,a, Kent Neuenschwander, Anthony Scotese, Sharon Jackson,
Thaddeus Nieduzak,a Henry Pauls,a Guyan Liang,b Keith Sides,c Dona Cramer,c
Jennifer Cairns,c Sebastien Maignand and Magali Mathieud
*
aDepartment of Medicinal Chemistry, Drug Innovation and Approval, Aventis Pharmaceuticals,
Route 202-206, Bridgewater, NJ 08807, USA
bDepartment of Medicinal Chemistry/Molecular Modeling, Drug Innovation and Approval, Aventis Pharmaceuticals,
Route 202-206, Bridgewater, NJ 08807, USA
cDepartment of Molecular Validation and Cell Biology, Drug Innovation and Approval, Aventis Pharmaceuticals,
Route 202-206, Bridgewater, NJ 08807, USA
dDepartment of Structural Biology, Aventis Pharma, Vitry 94403, France
Received 7 June 2004; revised 22 July 2004; accepted 23 July 2004
Available online 13 August 2004
Abstract—In this manuscript, the synthesis and SAR evaluation of a novel pyrazinone class of tryptase inhibitors is described.
Chemical optimization of the P1 and P4 groups led to the identification of 7p (Ki = 93nM) as a potent inhibitor of mast cell tryptase.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Asthma is a disease of ever increasing proportions, one
that is expected to nearly double over the next decade.1
Even though asthma has been afflicting the world popu-
lation for many years, there still does not exist a general
therapy for this indication. The most common therapy
includes the use of bronchodilators and anti-inflamma-
tory agents and there has been only one new major ther-
apy approved over the past few years, Singulairꢁ.2
However, even with this new class of therapy, a
great number of asthmatics still rely on a combination
therapy of inhaled drugs. Thus, discovery of a novel
medication for asthma, preferably an orally acting
drug, is the major concentration of a number of research
departments.
arin-bound tetramer5 and is released upon mast cell
stimulation. Once released, tryptase has been shown to
cleave and degrade numerous substrates all of which
play important roles as bronchodilators or vasodilators
by causing smooth muscle relaxation.4,6 In addition to
these effects, tryptase is also a known mitogen of both
dog tracheal7 and human8 smooth muscle cells as well
as human lung and dermal fibroblasts.9 These effects
are known as airway remodeling10 and thus tryptase
inhibitors would be a novel therapy for this condition.
There have been a number of reports of small molecule
inhibitors of tryptase,11 and this report details efforts
from our laboratories to discover a novel inhibitor of
tryptase.
One such possible target that has been receiving atten-
tion from a number of laboratories is tryptase inhibition
due to the fact that tryptase has been directly linked to
the pathology of asthma.3 It is a trypsin-like serine-class
protease that is found almost exclusively in mast cells.4
Tryptase is stored in mast cell granules as a stable, hep-
Cl
N
O
N
NH2
H2N
N
H
N
H
.
TFA
O
P1
P4
7p
Ki (tryptase) = 93 nM
Keywords: Mast cell tryptase; SAR; Pyrazinone.
*
Pyrazinone compounds of similar structure to 7 are
reported serine protease inhibitors.12 It was at this
Corresponding author. Tel./fax: +1-908-231-3351; e-mail: corey.
hopkins@aventis.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.051

4820
C. R. Hopkins et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4819–4823
Cl
O
ClCOCOCl
N
N
O
dichlorobenzene, 100 o
C
CH2Cl2
O
O
O
.
N
HCl
+
+
Si
N
H2N
Cl
OBn
HN
OBn
OBn
.
HCl
2
1
Cl
O
Cl
O
LiOH,
R-NH2, EtOAc,
reflux
CH3CN, Et3N, TBTU
H2N
THF:MeOH:H2O
N
O
N
O
R
N
H
N
R
N
OH
N
H
OBn
NHBoc
5
3a-p
4a-p
Cl
N
Cl
TFA, CH2Cl2
O
N
O
R
N
NHBoc
R
N
NH2
N
H
N
H
N
H
N
H
.
TFA
O
O
6a-p
7a-p
Scheme 1. Synthesis of the pyrazinones 7.
juncture that we started our program looking at these
compounds as possible mast cell tryptase inhibitors.
The synthesis of the pyrazinone core structure 2 is out-
lined in Scheme 1.13 Glycine benzyl ester hydrochloride,
trimethylsilylcyanide, and acetaldehyde were reacted to-
gether under modified Strecker conditions to yield the
nitrile amino ester 1. Alternatively, the ethyl ester of 1
could be prepared by a literature method.14 Next, the
pyrazinone 2 was formed by cyclization of 1 with oxalyl
chloride in dichlorobenzene15 at 100ꢂC. The dichloro-
pyrazinone 2 was treated with a variety of amines in
refluxing ethyl acetate to give the amino pyrazinones
3c–p after chloride displacement.12a Chloride displace-
ment with anilines could be accomplished by heating
the ethyl ester of 2 with neat aniline in the microwave
(variable 300W max, 12min, 120ꢂC). Saponification
(LiOH, THF/MeOH/H2O) to acids 4a–p followed by
coupling with aniline 5 (CH3CN, Et3N, TBTU)16 deliv-
ered the amides 6a–p. The final targets 7 were realized,
after Boc deprotection (TFA, CH2Cl2), in the salt form.
Table 1 (continued)
Compd
R-NH
Tryptase Ki, nMa
NH
7f
1550
875
615
580
518
426
390
290
245
138
O
NH
NH2
7g
7h
7i
NH
H2N
H2N
NH
N
NH
7j
N
H
N
7k
7l
HN
NH
NH
H2N
Compound 7c was our starting point for the pyrazinone
series (Table 1). The compound showed moderate acti-
vity (1.5lM) and we were interested in improving this
HN
7m
7n
7o
NH
H2N
H2N
Table 1. Tryptase inhibition of P4 group modifications for compounds
7a–p17
NH
Compd
R-NH
Tryptase Ki, nMa
NH
NH
7a
NAb
F
NH
H2N
7p
93
NH
Babimd
140
7b
7c
NAc
1550
a Values are means of three experiments.
b 24% inhibition @ 5lM.
Cl
c 11% inhibition @ 5lM.
NH
d Reference compound.18
7d
7e
NH
970
activity by making changes to the P4 group. The first
changes included the incorporation of hydrophobic por-
tions on to the amine; however, these compounds did
not improve the activity (7c–7f). Substituted anilines
(7a,b) exhibited a marked decrease in activity. It was
N
N
1238
F
N

C. R. Hopkins et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4819–4823
4821
Table 2. Tryptase inhibition of P1 group modifications for compounds
9a–g
Cl
BocNH
Cl
O
BocNH
N
O
NH2
N
O
N
Compd
R0-NH
Tryptase Ki, lM
N
N
H
OBn
Cl
OBn
EtOAc, reflux
O
O
HN
9a
18% @ 5lM
3o
2
NH2
Cl
1. LiOH, THF:MeOH:
H2O
BocNH
HN
N
O
9b
9c
9d
9e
31% @ 5lM
16% @ 5lM
29% @ 5lM
7% @ 5lM
N
R'
NH2
NH2
2. R'-NH2, CH3CN,
Et3N, TBTU
N
H
N
H
O
O
HN
HN
8a-g
Cl
.
H2N
TFA
TFA, CH2Cl2
N
O
R'
N
O
N
H
N
H
NH2
NH2
9a-g
HN
Scheme 2. Synthesis of 9a–g, P1 replacements.
HN
HN
N
9f
8% @ 5lM
2% @ 5lM
N
H
not until more polar groups were investigated that the
tryptase activity reached more acceptable levels. To this
end, the aryl amines and benzyl amines (7g–7i) showed
improvement in activity to sub-micromolar levels. The
P4 site also tolerated non-aromatic substituents. A ste-
rically bulky group delivered affinity for tryptase at a
more interesting level (7p, Ki = 93nM). It is of note that
this represents a 10-fold increase in tryptase activity by
modification of the P4 group.
9g
NH2
Concurrent with our efforts at improving the P4 group
we pursued optimization of the P1 group on the trans-
1,4-cyclohexylamine P4 derivative 7m, which was the
best compound at the time and due to the ease of syn-
thesis (Scheme 2). Thus, the mono-Boc protected
trans-1,4-cyclohexylamine was added to the dichloro-
pyrazinone 2 in refluxing EtOAc to yield 3o. Next, the
acid was saponified (LiOH, THF/MeOH/H2O) followed
by amide formation by coupling with the appropriate
amine (CH3CN, Et3N, TBTU) to give the intermediates
8a–g. Finally, the targets 9a–g were realized after Boc
deprotection (TFA, CH2Cl2).
The results from the primary assay for compounds 9a–g
can be seen in Table 2. The benzyl amine replacements
for 3m all gave inactive compounds. It is interesting to
note that even the para substituted derivative 9c, was
inactive. These results suggest that the meta substituted
benzyl amine is the favored P1 group.
Figure 1. X-ray structure of inhibitor 7o soaked into b-tryptase. The
carbonyl of the pyrazinone ring is engaged in both an external
hydrogen bond interaction with Glu192 and an internal hydrogen
bond with the amide NH of the benzylamine moiety, which is situated
in the active site pocket. The benzyl amine makes an interaction with
both Asp189 and the carbonyl of Gly219.
These results are supported by X-ray structure analysis
for inhibitor 7o (Fig. 1) obtained using similar condi-
tions as Bode and co-workers.5 The analysis shows a
number of key interactions for this compound. Namely,
the meta-benzylamine (P1 group) interacts with both the
Asp189 and the carbonyl of Gly219. This explains why
the P1 replacements did not show the same activity as
7o. When the benzyl amine is replaced with either an
amide (9a,9b), or aniline (9d,9e), these groups do not
have the same length nor geometry to retain this interac-
tion. Also, when the benzyl amine is moved to the para
position (9c) this too does not have the required geo-
metry to retain the interaction and hence activity.
The X-ray structure revealed an alternative binding
mode for the P4 group. As shown in Figure 2, instead
of binding in the S4 pocket, the ÔP4Õ groups actually
stayed in an area close to N146 and Q221, which is
not utilized by natural substrates for typical trypsin-like
serine proteases. While the S4 pocket in a typical tryp-
sin-like serine protease prefers an aromatic/hydrophobic
group, this alternative binding region is more hydrophi-
lic featuring several charged residues; including Q221 di-
rectly underneath, N146/D147 on one side, and E217/
D60 on another. This alternative binding mode explains
the SAR shown in Table 1. As the ÔP4Õ groups become

4822
C. R. Hopkins et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4819–4823
Figure 2. Stereoview of two tryptase inhibitors indicating the alternative binding mode (7n, blue; 7o, grey).
(b) Hartmann, T.; Ruoss, S. J.; Raymond, W. W.; Seuwen,
K.; Caughey, G. H. Am. J. Physiol. Lung Cell Mol.
Physiol. 1992, 262, L528–L534.
more polar and bulky, the activity increases. Molecular
modeling studies indicate that it is rather difficult to
achieve tight binding in this surface exposed shallow
pocket. As a result, the mobility of these ÔP4Õ groups
was high and the H-bonding interactions between inhib-
itors and enzyme could also be further diluted by water
molecules.
10. Bousquet, J.; Jeffrey, P. K.; Busse, W. W.; Johnson, M.;
Vignola, A. M. Am. J. Respir. Crit. Care Med. 2000, 161,
1720–1721.
11. (a) Clark, J. M.; Moore, W. R.; Tanaka, R. D. Drugs
Future 1996, 21, 811–816; (b) Burgess, L. E. Drug News
Perspect. 2000, 13, 147–156; (c) Sutton, J. C.; Bolton, S.
A.; Hartl, K. S.; Huang, M.-H.; Jacobs, G.; Meng, W.;
Ogletree, M. L.; Pi, Z.; Schumacher, W. A.; Seiler, S. M.;
Slusarchyk, W. A.; Treuner, U.; Zahler, R.; Zhao, G.;
Bisacchi, G. S. Bioorg. Med. Chem. Lett. 2002, 12,
3229–3233; (d) Slusarchyk, W. A.; Bolton, S. A.; Hartl,
K. S.; Huang, M.-H.; Jacobs, G.; Meng, W.; Ogletree, M.
L.; Pi, Z.; Schumacher, W. A.; Seiler, S. M.; Sutton, J. C.;
Treuner, U.; Zahler, R.; Zhao, G.; Bisacchi, G. S. Bioorg.
Med. Chem. Lett. 2002, 12, 3235–3238; (e) Zhao, G.;
Bolton, S. A.; Kwon, C.; Hartl, K. S.; Seiler, S. M.;
Slusarchyk, W. A.; Sutton, J. C.; Bisacchi, G. S. Bioorg.
Med. Chem. Lett. 2004, 14, 309–312; (f) Burgess, L. E.;
Newhouse, B. J.; Ibrahim, P.; Rizzi, J.; Kashem, M. A.;
Hartman, A.; Brandhuber, B. J.; Wright, C. D.; Thomson,
D. S.; Vigers, G. P. A.; Koch, K. Proc. Natl. Acad. Sci.
1999, 96, 8348–8352; (g) Costanzo, M. J.; Yabut, S. C.;
Almond, H. R.; Andrade-Gordon, P.; Corcoran, T. W.;
de Garavilla, L.; Kauffman, J. A.; Abraham, W. M.;
Recacha, R.; Chattopadhyay, D.; Maryanoff, B. E.
J. Med. Chem. 2003, 46, 3865–3876.
12. (a) Burgey, C. S.; Robinson, K. A.; Lyle, T. A.; Sander-
son, 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.; Williams, 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, T. J., Jr.; Lynch, J. J., Jr.; Yan,
Y.; Chen, Z.; Kuo, L.; Gardell, S. J.; Shafer, J. A.; Vacca,
J. P. J. Med. Chem. 2003, 46, 461–473; (b) 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, 4050–4062.
In conclusion, our investigation of the SAR at the P4
and P1 chain modifications of the pyrazinone nucleus
has identified a potent inhibitor of mast cell tryptase
(7p, Ki = 93nM). Chemical optimization has shown that
bulky, polar substituents for the P4 chain are desired.
Also, optimization of the P1 chain has revealed (along
with the X-ray structure) that a meta-substituted benzyl-
amino amide is the optimal substituent.
References and notes
1. (a) Barnes, P. J.; Chung, K. F.; Page, C. P. Pharmacol.
Rev. 1998, 50, 515–596; (b) Nimmaggadda, S. R.; Evans,
R. Pediatr. Rev. 1999, 20, 111–115.
2. Nayak, A. Expert Opin. Pharmacother. 2004, 5, 679–686.
3. Molinari, J. F.; Scuri, M.; Moore, W. R.; Clark, J.;
Tanaka, R.; Abraham, W. M. Am. J. Respir. Crit. Care.
Med. 1996, 154, 649–653.
4. Brown, J. K.; Jones, C. A.; Rooney, L. A.; Caughey, G.
H.; Hall, I. P. Am. J. Physiol. Lung Cell Mol. Physiol.
2002, 282, L197–L206.
5. Pereira, P. J. B.; Bergner, A.; Macedo-Ribeiro, S.; Huber,
R.; Matschiner, G.; Fritz, H.; Sommerhoff, C. P.; Bode,
W. Nature 1998, 392, 306–311. Resolution for reported
˚
structures 2.0–2.6A.
6. Abraham, W. M. Am. J. Physiol. Lung Cell Physiol. 2002,
282, L193–L196.
7. Brown, J. K.; Tyler, C. L.; Jones, C. A.; Ruoss, S. J.;
Hartmann, T.; Caughey, G. H. Am. J. Respir. Cell Mol.
Biol. 1995, 13, 227–236.
8. Berger, P.; Perng, D. W.; Thabrew, H.; Hollenberg, M. D.;
Sanjar, S.; Laurent, G. J.; McAnulty, R. J. J. Appl.
Physiol. 2001, 91, 1372–1379.
9. (a) Akers, I. A.; Parsons, M.; Hill, M. R.; Hollenberg, M.
D.; Sanjar, S.; Laurent, G. J.; McAnulty, R. J. Am. J.
Physiol. Lung Cell Mol. Physiol. 2000, 278, L193–L201;
13. All new compounds were characterized by high resolution
LCMS and 1H NMR and found to be in agreement
with their structures.
14. Araldi, G. L.; Semple, J. E. PCT Int. Appl. 2001, 236.

C. R. Hopkins et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4819–4823
4823
15. Vekemans, J.; Pollers-Wieers, C.; Hoornaert, G. J.
Heterocycl. Chem. 1983, 20, 919–923.
16. Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.;
Zhang, C.; Lee, Y.; Foxman, B. M.; Henklein, P.; Hanay,
C.; Mugge, C.; Wenschuh, H.; Klose, J.; Beyermann, M.;
¨
add compound in duplicates, final concentration of 20lM,
volume 20lL, add enzyme at a final concentration of
50ng/mL in a volume of 20lL. Total volume for each well
is 100lL. Agitate briefly to mix and incubate at room
temperature in the dark for 30min. Read absorbances at
405nM. Each plate has the following controls––Totals:
60lL of substrate, 20lL of buffer (with 0.2% final
concentration of DMSO), 20lL of enzyme. Nonspecific:
60lL of substrate, 40lL of buffer (with 0.2% DMSO).
Totals: 60lL of substrate, 20lL of buffer (no DMSO),
20lL of enzyme. Nonspecific: 60lL of substrate, 40lL of
buffer (no DMSO) Protocol (IC50 determination). The
protocol is essentially the same as above except that the
compound is added in duplicates at the following final
concentrations: 0.01, 0.03, 0.1, 0.3, 1, 3, 10lM (all
dilutions carried out manually). For every assay, whether
single point or IC50 determination, a standard compound
is used to derive IC50 for comparison; (b) McEuen, A. R.;
He, S.; Brander, M. L.; Walls, A. F. Biochem. Pharm.
1996, 52, 331–340.
Bienert, M. Angew. Chem., Int. Ed. 2002, 41, 441–
445.
17. (a) Tryptase inhibition activity is confirmed using rec-
ombinant human bII tryptase expressed in yeast cells. The
assay procedure employs a 96 well microplate (Costar
3590) using L-pyroglutamyl-L-prolyl-L-arginine-para-
nitroanilide (S2366: Quadratech) as substrate (essentially
as described by McEuen et al.17b). Assays are performed at
room temperature using 0.5mM substrate (2 · Km) and
the microplate is read on a microplate reader (Beckman
Biomek Plate reader) at 405nm wavelength. Materials and
methods for tryptase primary screen (Chromogenic assay):
assay buffer––50mM Tris (pH8.2), 100mM NaCl, 0.05%
Tween 20, 50lg/mL heparin; substrate-S2366 (stock
solutions of 2.5mM); purified recombinant bII tryptase
enzyme stocks of 310lg/mL. Protocol (single point
determination): add 60lL of diluted substrate (final
concentration of 500lM in assay buffer) to each well,
18. Caughey, G. H.; Raymond, W. W.; Bacci, E.; Lombardy,
R. J.; Tidwell, R. R. J. Pharm. Exp. Exp. Ther. 1993, 264,
676–682.