Novel, potent, selective, and orally bioavailable human βII-tryptase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.04.088

The synthesis of novel [1,2,4]oxadiazoles and their structure-activity relationship (SAR) for the inhibition of tryptase and related serine proteases is presented. Elaboration of the P′-side afforded potent, selective, and orally bioavailable tryptase inhibitors.

Full text of DOI:10.1016/j.bmcl.2006.04.088

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4085–4089
Novel, potent, selective, and orally bioavailable human
bII-tryptase inhibitors
David Sperandio,* Vincent W.-F. Tai, Julia Lohman, Bernie Hirschbein,
Rohan Mendonca, Chang-Sun Lee, Jeffrey R. Spencer, James Janc, Margaret Nguyen,
Jerlyn Beltman, Paul Sprengeler, Heleen Scheerens, Tong Lin, Liang Liu, Ashwini Gadre,
Alisha Kellogg, Michael J. Green and Mary E. McGrath
Celera Genomics, 180 Kimball Way, South San Francisco, CA 94080, USA
Received 2 March 2006; revised 28 April 2006; accepted 28 April 2006
Available online 24 May 2006
Abstract—The synthesis of novel [1,2,4]oxadiazoles and their structure–activity relationship (SAR) for the inhibition of tryptase and
related serine proteases is presented. Elaboration of the P0-side afforded potent, selective, and orally bioavailable tryptase inhibitors.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Tryptase, a tetrameric serine protease, has been impli-
cated in many of the physiological responses observed
in asthma. For example, tryptase activates kininogens,
producing the potent bronchoconstrictor bradykinin,
and may also amplify its signal by stimulating further
mast cell degranulation. Tryptase also processes vasoac-
tive intestinal peptide, down-regulating this bronchodi-
lating agent, and it behaves as a potent mitogen for
smooth muscle cells, endothelial cells, and fibroblasts.
The mitogenic effects on smooth muscle cells may con-
tribute to the long term airway remodeling, a defining
characteristic of chronic asthma that is of therapeutic
concern in disease progression.1–14 Efficacy of the tryp-
tase inhibitor APC-366, in a sheep model of allergic
asthma15 and in clinical trials,6,12,16 prompted several
groups over the last decade to develop additional small
molecule inhibitors of tryptase. The successful crystalli-
zation of tryptase in 1998 by Pereira et al., revealed a
homotetrameric structure that was amenable to struc-
ture-guided design.17 Here we report that P0-side elabo-
ration of an a-ketoheterocycle affords potent and
selective tryptase inhibitors.
A screen of our in-house compound collection afforded
compound 1 (see Fig. 1) an a-ketoheterocycle with a
potency against tryptase of 2.7 lM. Compound 1 is
structurally related to RWJ-56423, which entered clini-
cal trials as an inhaled agent for asthma.18–20 Key inter-
actions of RWJ-56423, which has a potency against
tryptase of 10 nM, are a covalent reversible bond be-
tween the active site Ser195 and the ketone activated
by the benzthiazole moiety, a charge–charge interaction
of the guanidine moiety with Asp189 at S1, and several
H-bonds of the hydroxyproline moiety at S2.19
Most of the interactions at S2 are also likely to be
present when RWJ-56423 is bound to trypsin, which
explains the potency against trypsin of 8 nM.18 The
S2-sites of tryptase and trypsin are both open, hydro-
phobic patches on the enzymes which initially did not
seem to offer promise for developing tryptase-selective
HO
O
O
H
H
N
O
N
N
S
N
O
N
O
O
N
O
Keywords: a-Keto-[1,2,4]oxadiazoles; Structure–activity relationship;
Tryptase inhibitors; Serine proteases; P0-side; Asthma.
H2N
HN
*
Corresponding author. At present address: Gilead Sciences, Inc., 333
Lakeside Drive, Foster City, CA 94404, USA.Tel.: + 1 650 522 1641;
fax: +1 650 522 5899; e-mail: david.sperandio@gilead.com
Present address: Gilead Sciences, Inc., 333 Lakeside Drive, Foster
City, CA 94404, USA.
HN
NH2
1
RWJ-56423
Figure 1. Compound 1 and related a-ketobenzthiazole RWJ-56423.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.088

4086
D. Sperandio et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4085–4089
inhibitors. In contrast, the X-ray structure of tryptase
shows a hydrophobic pocket on the P0-side of the active
site, which is not present in trypsin.17 This pocket is
afforded the diastereomeric cyanohydrins 5a,b that were
converted to N-hydroxy-amidines 6a,b in the presence of
50% aqueous hydroxylamine in ethanol at 50 ꢁC. Phenyl
ethers 7 were prepared by a Mitsunobu-type coupling
reaction of 4-hydroxyphenylacetic acid methyl ester 9
and diversity alcohols 8, followed by saponification of
the ester and acidification. Acylation of the N-hy-
droxy-amidines 6a,b in the presence of phenylacetic
acids 9 and carbodiimide followed by thermal intramo-
lecular cyclization (pyridine, 120 ꢁC) afforded the
[1,2,4]oxadiazoles 11a,b. Diastereomeric alcohols 11a,b
were oxidized using Dess–Martin periodinane in dichlo-
romethane. Final deprotection of the Boc group (TFA–
dichloromethane) and conversion to the HCl salt of the
N-e-amino group provided the series of inhibitors 12.
˚
located about 12–14 A from Ser195, and was previously
explored with chelating inhibitors8,21 as well as with
mechanism-based azetidinone inhibitors.22–28 Modeling
studies of 1 docked into the X-ray structure of tryptase
indicate that elaboration of the [1,2,4]oxadiazole of 1 at
C-3 with a benzyl moiety affords a vector that points to-
ward the hydrophobic pocket on the P0-side. Based on
these observations, we elaborated the P0-side of 1 to im-
prove potency and selectivity.
The synthesis of [1,2,4]oxadiazole containing tryptase-
inhibitors is illustrated in Scheme 1. Orthogonally
protected N-a-benzyloxycarbonyl-N-e-tert-butyloxycar-
bonyl-L-lysine 2 was converted to aldehyde 4 via the
Weinreb-amide 3. Acetone cyanohydrin treatment of 4
We focused the diversity of the initial set of compounds
on the trajectory and length of the linker, and plan to
O
O
O
CbzHN
CbzHN
O
CbzHN
ii
i
OH
N
H
2
NHBoc
3
NHBoc
4
NHBoc
MeO
O
iii
OH
9
v
R
HO
8
OH
OH
HO
iv
CbzHN
NH2
CbzHN
CN
O
Ph
O
N
OH
7
6a, b
5a, b
NHBoc
NHBoc
vi
OH
OH
NH2
CbzHN
O
Ph
vii
O
CbzHN
N
N
O
O
N
Ph
O
10a, b
11a, b
NHBoc
NHBoc
viii
O
CbzHN
N
O
N
Ph
O
12
NH2
Scheme 1. Reagents and conditions: (i) CH3NHOCH3, EDC, HOBt, Hunig’s base, DMF, rt; (ii) LiAlH4, THF, 0 ꢁC; (iii) acetone cyanohydrin,
NEt3, CH2Cl2, rt; (iv) NH2OH 50 wt % in H2O, EtOH, 50 ꢁC; (v) a—PPh3, DEAD, CH2Cl2; b—NaOH, THF/H2O; (vi) EDC, HOBt, Hunig’s base,
DMF, rt; (vii) pyridine, 120 ꢁC; (viii) a—Dess–Martin periodinane, CH2Cl2, rt; b—TFA, CH2Cl2, rt, preparative RP-HPLC (H2O–acetonitrile, 0.1%
HCl).

D. Sperandio et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4085–4089
4087
explore the phenyl ‘end-cap’ SAR in a subsequent series
with an optimized linker moiety.
potent compounds 12d and 12e. Also, the longer linker
in 12e is more potent than the shorter in 12d, reversing
the SAR trend seen in the analogs 12b and 12c. These
observations suggest that the para trajectory points
more directly toward the hydrophobic binding pocket
than the meta trajectory (Table 1, Fig. 2).
Examples 12a and 12b did indeed show a significant in-
crease in potency against tryptase of 15· and 225·,
respectively. Extending the end-cap phenyl moiety out
by one more methylene group (12c) did not further im-
prove the potency. Changing the trajectory from the
para position to the meta position afforded the less
The distinctive SAR of compounds 12a–e for tryptase is
sharply contrasted by the flat SAR for trypsin. The
potency of all compounds against trypsin ranges from
24 to 92 nM. Thus, with 12b, we have improved the
potency and selectivity by a factor of 225· and 97·,
respectively, over the lead compound 1. This is strong
evidence that the phenethyloxy group is extending and
interacting with the P0-side hydrophobic binding
pocket30.
Table 1. Enzyme inhibition data for compounds 1 and 12a–e29
O
H
O
N
N
R
O
N
O
NH2
Table 2. Enzyme inhibition data for compounds 12f–q29
O
H
Compound R
bII-Tryptase Trypsin Selectivity
O
N
N
Ki(app)
[lM]
Ki(app)
[lM]
O
N
O
O
R
1
2.7
0.068
0.024
0.035
0.041
0.082
0.092
0.03
0.13
2.9
NH2
12a
12b
12c
12d
12e
0.180
0.012
0.025
0.360
0.120
Compound
R
bII-Tryptase Trypsin Selectivity
Ki(app)
[lM]
O
O
Ph
Ki(app)
[lM]
Ph
12f
0.019
0.041
0.024
0.078
0.027
1.10
0.034
0.059
0.011
0.095
0.140
1.0
1.8
OMe
1.6
OMe
O
O
Ph
Ph
12g
12h
12i
1.4
Ph
0.23
0.76
OMe
OMe
0.46
1.2
O
12k
121
12m
12n
12o
12p
12q
5.2
0.91
Cl
F
0.013
0.016
0.014
0.010
0.096
0.190
0.110
0.270
0.280
0.410
14.6
6.9
19.3
28.0
4.3
Cl
Cl
Cl
Figure 2. Docking studies of 12b in tryptase showing the P0-side
interaction of the phenyl end-cap.
Cl
Cl

4088
D. Sperandio et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4085–4089
We next explored the SAR of the end-cap phenyl moiety
using the optimized linker in compound 12b. Elabora-
tion of the end-cap phenyl moiety with electron-donat-
ing groups such as OMe (12f–h) did not improve
tryptase potency or positively affect the selectivity
against trypsin. Increasing steric bulk (12i,k) also had
little effect, with the exception of 12l, which lost
significant potency against both tryptase and trypsin.
However, electron-withdrawing groups deselected tryp-
sin, while maintaining the tryptase potency, resulting
in increased selectivity (12m–q). 3,4-Disubstitution with
chlorine afforded compound 12p with a selectivity
against trypsin of 28· and a potency of 10 nM (Table 2).
the potency against tryptase of 5.4 nM. Selectivity
against several related trypsin-like serine proteases is
striking (see Table 3). Modification of 13 to further im-
prove the selectivity against trypsin is reported in the
subsequent letter. Compound 13 has a modest oral
absorption of 18% and an oral bioavailability of 8.4%
in rat (data not shown).
Compound 13 was studied in a mast cell dependent
mouse model of allergic asthma and was demonstrated
to be efficacious in inhibiting ovalbumin-induced airway
hyperresponsiveness and airway inflammation dose
dependently.31
We speculated that the Cbz group might be responsible
for the low trypsin selectivity exhibited by the lead
compound 1. The very good density and low tempera-
ture factors in the X-ray structures of Cbz-containing
molecules in trypsin suggested that this group was well
suited for the S2 pocket of trypsin, while the crystal
structures of related compounds with Cbz P2 elements
in tryptase gave poor density and high temperature
factors suggesting that it does not bind tightly to the
P2-pocket in tryptase (data not shown). Deletion of
the hydrophobic contact of the P2 Cbz phenyl moiety
by replacement with an ethyl carbamate to give 13, in-
deed, significantly reduced potency against trypsin to
190 nM (compared to 35 nM for 12b), while preserving
In summary, a novel series of a-keto[1,2,4]oxadiazoles
was prepared using structure-guided design. These com-
pounds were demonstrated to be potent inhibitors of
tryptase when tested in biochemical assays and to show
biologic activity in a mast cell dependent model of aller-
gic asthma. In addition, when tested against a number of
other related enzymes, these compounds exhibited
exquisite selectivity for tryptase. Furthermore, this series
of analogs will serve to guide the design of more refined
therapeutic compounds with potential for the treatment
of asthma.
References and notes
1. Cairns, J. A. Pulm. Pharmacol. Ther. 2005, 18, 55.
2. Payne, V.; Kam, P. C. A. Anaesthesia 2004, 59, 695.
3. Fiorucci, L.; Ascoli, F. Cell. Mol. Life Sci. 2004, 61, 1278.
4. Caughey, G. H. Handbook of Proteolytic Enzymes, 2nd ed.
2004; Vol. 2, p 1535.
5. Bradding, P. Curr. Opin. Allergy Clin. Immunol. 2003, 3,
45.
6. Newhouse, B. J. IDrugs 2002, 5, 682.
Table 3. Enzyme inhibition and pharmacokinetics data for compound
1329
O
H
O
N
N
O
N
O
O
7. Abraham, W. M. Am. J. Physiol. 2002, 282, L193.
8. Gangloff, A. R. Curr. Opin. Investig. Drugs 2000, 1, 79.
9. Rice, K. D.; Sprengeler, P. A. Curr. Opin. Drug Discovery
Dev. 1999, 2, 463.
10. Rice, K.; Spencer, J. Exp. Opin. Ther. Patents 1999, 9,
1537.
11. Elrod, K. C.; Numerof, R. P. Emerg. Ther. Targets 1999,
3, 203.
12. Rice, K. D.; Tanaka, R. D.; Katz, B. A.; Numerof, R. P.;
Moore, W. R. Curr. Pharm. Des. 1998, 4, 381.
13. Clark, J. M.; Moore, W. R.; Tanaka, R. D. Drugs Future
1996, 21, 811.
14. Rice, K. D.; Moore, W. R. In High Throughput Screening
for Novel Anti-Inflammatories; Kahn, M., Ed.; Birkhaeus-
er: Basel, 2000; pp 101–121.
15. Clark, J. M.; Abraham, W. M.; Fishman, C. E.; Forteza,
R.; Ahmed, A.; Cortes, A.; Warne, R. L.; Moore, W. R.;
Tanaka, R. D. Am. J. Respir. Crit. Care Med. 1995, 152,
2076.
13
NH2
Potency [Ki (lM)]
Human bII-tryptase
Mouse tryptase (m-MCP-6)
Rat tryptase (r-MCP6)
Dog tryptase
Monkey tryptase
Trypsin
0.0054
0.0435
0.0055
0.380
0.0465
0.190
Thrombin
Plasmin
64
0.43
43
Kallikrein
APC
>150
>150
>150
>150
>150
>150
Chymotrypsin
Elastase
Chymase
Urokinase
Granzyme K
PK (iv–po, rat)
Cmax (lM)
CL (ml/min/kg)
0.59
16. Burgess, L. E. Drug News Perspect. 2000, 13, 147.
17. Pereira, P. J. B.; Bergner, A.; Macedo-Ribeiro, S.; Huber,
R.; Matschiner, G.; Fritz, H.; Sommerhoff, C. P.; Bode,
W. Nature 1998, 392, 306.
18. Costanzo, M. J.; Yabut, S. C.; Almond, H. R., Jr.;
Andrade-Gordon, P.; Corcoran, T. W.; De Garavilla, L.;
Kauffman, J. A.; Abraham, W. M.; Recacha, R.; Chatto-
padhyay, D.; Maryanoff, B. E. J. Med. Chem. 2003, 46,
3865.
93
910
7000
75
Vc (ml/kg)
Vss (ml/kg)
MRT (min)
AUC (lM min)
Absorption (%)
Bioavailability (%)
11
18
8.4
PK (ip, mouse)
Bioavailability (%)
68
19. Maryanoff, B. E. J. Med. Chem. 2004, 47, 769.

D. Sperandio et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4085–4089
4089
20. Breslav, M.; Harris, B.; Kenney, B.; Maryanoff, C. A.;
Villani, F. J.; Zhang-Plasket, F.; Zhong, H. M. Abstracts
of Papers, 229th ACS National Meeting, San Diego, CA,
USA, March 13–17, 2005, ORGN.
21. Katz, B. A.; Clark, J. M.; Finer-Moore, J. S.; Jenkins, T.
E.; Johnson, C. R.; Ross, M. J.; Luong, C.; Moore, W. R.;
Stroud, R. M. Nature 1998, 391, 608.
22. 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, 1057.
23. Sutton, J. C.; Bolton, S. A.; Davis, M. E.; Hartl, K. S.;
Jacobson, B.; Mathur, A.; Ogletree, M. L.; Slusarchyk, W.
A.; Zahler, R.; Seiler, S. M.; Bisacchi, G. S. Bioorg. Med.
Chem. Lett. 2004, 14, 2233.
24. Bisacchi, G. S.; Slusarchyk, W. A.; Bolton, S. A.; Hartl,
K. S.; Jacobs, G.; Mathur, A.; Meng, W.; Ogletree, M. L.;
Pi, Z.; Sutton, J. C.; Treuner, U.; Zahler, R.; Zhao, G.;
Seiler, S. M. Bioorg. Med. Chem. Lett. 2004, 14, 2227.
25. Sutton, J. C.; Bolton, S. A.; Hartl, K. S.; Huang, M.-H.;
Jacobs, G.; Meng, W.; Ogletree, M. L.; Pi, Z.; Schum-
acher, W. A.; Seiler, S. M.; Slusarchyk, W. A.; Treuner,
U.; Zahler, R.; Zhao, G.; Bisacchi, G. S. Bioorg. Med.
Chem. Lett. 2002, 12, 3229.
26. 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.
values were determined by a non-linear least squares
regression fit of the experimentally derived data to the
Morrison equation for tight-binding inhibitors as
described (Kuzmic, P., et al. Anal. Biochem. 2000, 281,
62). Example conditions for the tryptase and trypsin
assays are provided below, other enzyme assays for
which selectivity data are provided were performed in a
similar manner using enzyme from human origin.
Monkey tryptase: The enzyme was cloned from cyno-
molgus monkey, the cDNA used to generate recombi-
nant monkey tryptase was isolated from CD34+ mast
cell progenitors derived from cyno monkey blood.
Example enzyme assay conditions: Tryptase–tryptase
(recombinant human b-1 from Promega) was incubated
at 2 nM with variable concentrations of inhibitor in
50 mM Tris (pH 7.4), 150 mM NaCl, 0.02% Tween 20,
1 mM EDTA, 50 lg/ml heparin, and 10% DMSO. The
reaction was initiated with substrate, Tosyl-Gly-Pro-Lys-
pNA (Centerchem), supplied at the Km (400 lM). The
change in absorbance as
a function of time was
monitored at 405 nm. Trypsin–trypsin (Athens Research
Institute) was incubated at 10 nM with variable concen-
trations of inhibitor in 50 mM Tris (pH 7.4), 150 mM
NaCl, 1.5 mM EDTA, 0.05% Tween 20 and 10%
DMSO. The reaction was initiated with substrate,
Tosyl-Gly-Pro-Lys-pNA (Centerchem), supplied at the
Km (25 lM). The change in absorbance as a function of
time was monitored at 405 nm.
27. Sutton, J.; Bisacchi, G. S.; Bolton, S. A.; Hartl, K. S.;
Huang, M.-H.; Jacobs, G.; Ogletree, M. L.; Pi, Z.;
Schumacher, W. A.; Slusarchyk, W. A.; Treuner, U.;
Zhao, G.; Zahler, R.; Seiler, S. M. Abstracts of Papers,
221st ACS National Meeting, San Diego, CA, USA, April
1–5, 2001, MEDI-249.
28. Slusarchyk, W. A.; Bisacchi, G. S.; Hartl, K. S.; Huang,
M.-H.; Jacobs, G.; Ogletree, M. L.; Sutton, J.; Treuner,
U.; Zhao, G.; Zahler, R.; Seiler, S. M. Abstracts of Papers,
221st ACS National Meeting, San Diego, CA, USA, April
1–5, 2001, MEDI-250.
30. This flat SAR can be rationalized by rotamers about
the methylene unit bridging the oxadiazole heterocycle
and the end-cap linker moiety which afford confor-
mations that have weak, non-specific surface interac-
tions with the prime side of trypsin. This hypothesis
is supported by the X-ray analysis of related com-
pounds in trypsin: Attempts to soak these molecules
into trypsin afforded good density only for the P1-Lys
and the P2-Cbz moieties; no density was observed for
the atoms after the heterocyclic ring (data not
shown).
29. The velocity of the enzyme-catalyzed reaction was
determined from the linear portion of the progress
curve, typically the first five minutes of the reaction
after initiation with the addition of substrate. The Ki
31. Scheerens, H.; Tan, M. D.; Sweeney, T. V.; Perea, T. V.;
Baruch, A.; Beltman, J.; Graupe, D.; Liu, L.; Tai, V.; Lee,
S.; Mendonca, R.; Sperandio, D.; Dalrymple, S. A. Proc.
Am. Thorac. Soc. 2005, 2, A804.