Design of novel, potent, and selective human β-tryptase inhibitors based on α-keto-[1,2,4]-oxadiazoles

Article abstract of DOI:10.1016/j.bmcl.2006.05.009

A series of novel α-keto-[1,2,4]-oxadiazoles has been synthesized as human tryptase inhibitors for evaluation as a new class of anti-asthmatic agent. The inhibitor design is focused on using a prime-side hydrophobic pocket and the S2 pocket of β-tryptase

Full text of DOI:10.1016/j.bmcl.2006.05.009

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4036–4040
Design of novel, potent, and selective human b-tryptase inhibitors
based on a-keto-[1,2,4]-oxadiazoles
Chang-Sun Lee,* Weili Liu, Paul A. Sprengeler, John R. Somoza, James W. Janc,
David Sperandio, Jeffrey R. Spencer, Michael J. Green and Mary E. McGrath
Departments of Chemistry and Biology, Celera, 180 Kimball Way, South San Francisco, CA 94080, USA
Received 23 March 2006; revised 2 May 2006; accepted 2 May 2006
Available online 22 May 2006
Abstract—A series of novel a-keto-[1,2,4]-oxadiazoles has been synthesized as human tryptase inhibitors for evaluation as a new
class of anti-asthmatic agent. The inhibitor design is focused on using a prime-side hydrophobic pocket and the S2 pocket of
b-tryptase to achieve inhibition potency and selectivity over other serine proteases.
Ó 2006 Elsevier Ltd. All rights reserved.
Interest in tryptase as a therapeutic target for asthma
has increased following the proof of principle studies
with APC-366 in sheep and humans.1 Tryptase is a
homotetrameric serine protease with trypsin-like speci-
ficity that is stored in mast cell granules and released
upon IgE stimulation.2 The proteolytic activity of tryp-
tase is believed to be responsible for immediate- and
long-term effects that contribute to the physiological
effects experienced by asthma sufferers.3 Therefore,
inhibiting tryptase with potent, selective, and preferably
oral small molecule drugs could have important
therapeutic value.4 Several groups have identified small
Figure 1. Tryptase inhibitors containing an activated ketone as a
warhead.
molecule inhibitors of tryptase as potential drugs for
asthma.5 However, designing compounds that
selectively inhibit tryptase over antitargets such as
trypsin has proven difficult.
containing a piperazine linker, which should have higher
solubility relative to 1b.
Although we achieved good tryptase inhibition potency
with 1b containing a phenyl ether linkage, compounds in
the series had at least three problems: poor trypsin selec-
tivity, low kinetic solubility, and high hERG K+ channel
binding affinity (Fig. 1).6 We therefore turned our atten-
tion to other linkages to improve not only anti-target
selectivity but also physicochemical properties such as
solubility and log P.7 We first designed compounds 6
The synthesis of the inhibitors 6 is outlined in Scheme 1.
Protected (L)-Lys 2 was converted to 3 by reduction of
Weinreb amide and hydrocyanation reaction with
acetone cyanohydrin. Compound 3 was transformed
to the corresponding hydroxyamidine,8 which was
coupled with 2-chloroacetylchloride. Thermal intramo-
lecular cyclization of the coupling product provided
the [1,2,4]-oxadiazole ring 4.9 Compound 4 was convert-
ed to 5 by N-alkylation with an excess amount of piper-
azine followed by the second N-alkylation/N-acylation
with alkyl/acyl halides. The alcohols 5 were oxidized
under Swern conditions,10 and final deprotection of
the BOC group from the lysine side chain provided the
series of compounds 6.
Keywords: Tryptase inhibitor; Asthma; Activated carbonyl;
Oxadiazole.
*
Corresponding author. Tel.: +1 925 242 0501; e-mail: csleesammy@
gmail.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.009

C.-S. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4036–4040
4037
Scheme 1. Reagents and conditions: (a) MeNHOMe, HATU, DMF;
(b) LiAlH4, THF, 0 °C; (c) acetone cyanohydrin, CH2Cl2; (d) NH2OH,
EtOH; (e) ClCH2COCl, CHCl3; (f) 100 °C, DMF; (g) piperazine,
K2CO3, CH3CN; (h) R2-Cl, K2CO3, CH3CN; (i) (COCl)2, DMSO,
TEA, CH2Cl2; (j) HCl; (g) CH3CN.
Table 1 shows the tryptase inhibition potency for
compounds 6a–e. Both 6a and 6b have similar activities
as 1a, suggesting that the linker groups may not be long
enough to reach to the prime-side binding pocket.
Increasing the chain length by one methylene, as in 6c,
increases potency by 7-fold. Replacing the carbonyl
group of 6b by a methylene 6d or an ethylene 6e
increases potency further by 21- and 40-fold,
respectively, indicating that the piperazine modification
has successfully allowed the phenyl group to reach the
distal pocket of the prime side.
Figure 2. X-ray crystal structure of 6c bound to human b-tryptase.
The synthesis of inhibitors 10 containing a benzamide
group is outlined in Scheme 2. Compound 3 was trans-
formed to 7 as shown in Scheme 2. Saponification of the
ester 7 was followed by amide coupling reaction with
water-soluble carbodiimide reagent (EDCI, HOBt,
As expected, kinetic solubility of most compounds was
high. Compared with 1b, the piperazine linkage increas-
es solubility of 6c–e by at least 15-fold. The X-ray
co-crystal structure of tryptase with 6c (Fig. 2) clearly
shows the phenyl end cap group occupying the prime-
side hydrophobic binding pocket.13 To our delight, 6c
also had significantly reduced hERG K+ channel bind-
ing affinity (2.3% at 10 lM concentration) relative to
ether-containing compound 1b, suggesting that the
amide might be a focal point for further optimization.
In order to improve poor trypsin selectivity and micro-
somal stability of 6c, we then adopted the benzamide
linkage as shown in the series of compounds 10 for the
inhibitor design.
Scheme 2. Reagents and conditions: (a) NH2OH, EtOH; (b) 4-
(CO2Me)–PhCH2CO2H (4). HATU, DMF; (c) 100 °C, DMF; (d)
LiOH, aq THF; (e) R3NH2, EDCI, HOBt, DMF; (f) Dess–Martin
periodinane, CH2Cl2; (g) HCl, CH3CN.
Table 1. Structure–activity relationship of tryptase inhibitors 6 containing the piperazine linkage
Compounds
R2
b-Tryptase Ki (nM)
Trypsin Ki (nM)
Solubility (lM) at pH 7.4
Microsome stabilitya (%)
ND
6a
470
42
ND
6b
6c
6d
230
34
33
22
11
12
ND
>500
329
ND
2.0
ND
22
11
6e
5.8
466
ND, not detected.
a Microsome stability is measured as percent of parent compound remaining after 1 h incubation with human liver microsomes as determined by
HPLC.

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C.-S. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4036–4040
DMF) to provide the series of compounds 9. Alcohols 9
were oxidized with Dess–Martin periodinane in dichlo-
romethane,11 which significantly reduced racemization
at the a-position of the ketone compared to other oxida-
tion conditions. Final deprotection of the BOC group
(HCl(g), CH3CN) from the lysine side chain provided
the series of compounds 10.
Table 2 shows the structure–activity relationship of
inhibitors 10 which have a P1 (L)-Lys and a P2 Cbz
group. The initial compound 10a showed 20-fold im-
proved tryptase potency compared with compound
1a, but this was not further improved in analogs
10b, 10c or 10d. Increasing the chain length between
the amide and the phenyl-capping group such as
Table 2. Structure–activity relationship of the benzamide inhibitors 10 in the prime-side distal-pocket binding region
Compounds
R3NH2
b-Tryptase Ki (nM)
Trypsin Ki (nM)
Selectivity
0.62
Solubility (lM) at pH 7.4
10a
37
23
459
10b
10c
39
11
34
15
0.87
1.4
245
100
10d
13
26
2.0
77.2
10e
10f
3.2
3.0
20
19
6.3
6.3
440
370
10g
10h
60
14
0.23
188
472
140
3.7
0.026
10i
3.6
220
8.2
18
27
30
18
31
23
5.0
0.12
3.7
5.1
1.9
13
226
227
284
333
ND
109
10j
10k
10l
3.5
10m
10n
16
1.8
10o
10p
2.9
2.4
11
13
3.8
5.4
299
484
10q
10r
9.9
3.7
29
21
2.9
5.7
364
206

C.-S. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4036–4040
4039
compounds 10e–g, 10e and 10f resulted in 10-fold
increase in tryptase potency from 10a. We then chose
10e for further optimization to achieve not only better
tryptase potency but also selectivity against other
antitargets. 10n with an m-chlorophenyl group showed
Ki below 2 nM of tryptase inhibition and 13-fold
selectivity over trypsin. Compound 10q demonstrates
that aliphatic end cap groups can also bind in the
distal pocket. We were pleased to find that most of
compounds 10 in the benzamide series showed high
kinetic solubility at pH 7.4 and very low affinity for
the hERG K+ channel.12
carbamate (EOC) group of 11a (Table 3). Substituted
benzamides and aliphatic amides also provided im-
proved selectivity against trypsin, ranging from 130- to
270-fold. Among them, difluorobenzamides 11d and
11e had over 250-fold selectivity. We speculate that the
smaller P2 residues may reduce trypsin affinity due to
the weaker hydrophobic interaction at S2 pocket of
trypsin. In addition, the less flexible P2 residue may fur-
ther increase selectivity possibly by steric interactions
with Leu99 in trypsin, but not the smaller Ala97 in
tryptase.
Further optimization led to 12b in Table 4, which shows
very good selectivity over all of the serine proteases.
Replacement of the benzyl group 12a with the ethyl
group 12b improved not only trypsin selectivity more
than 10-fold, but also kinetic solubility to over 500 lM
at pH 7.4. Further evaluation of 12b also revealed in-
creased microsomal stability compared to 6c.
However, selectivity versus trypsin was still very low.
This may not be unexpected since compounds 10 are
very flexible and have many different conformers due
to the rotational freedom between the activated car-
bonyl and the oxadiazole ring. In addition, we ob-
served there was poorly defined electron density
around the P2 in the crystal structures of 6c and
10e, and speculated that the P2 residue might not
be making a good interaction with the S2 pocket of
tryptase. We decided to synthesize a series of P2
analogs 11 to improve selectivity without reducing
tryptase binding affinity. Synthesis of 11a–h was
achieved by using a modification of the sequence in
Scheme 2.
While further optimization of P2 is needed, we were able
to obtain an X-ray co-crystal structure of 12b with
human b-tryptase (Fig. 3).13 The activated ketone is
covalently linked with the hydroxyl group of Ser195.
The nitrogen of the oxadiazole ring forms hydrogen-
bonding interaction with His57. The e-amino group of
lysine residue of 12b interacts with Asp189 at S1 pocket
through a salt-bridge. The benzamide NH– of 12b is well
positioned for hydrogen bonding with the carbonyl
group of Cys 58.
The selectivity against trypsin is significantly improved
by replacing the Cbz group of 10n with the shorter ethyl
Table 3. Structure–selectivity relationship of 11 in the P2 side
Compounds
R4
b-Tryptase Ki (nM)
Trysin Ki (nM)
Selectivity
95
Solubility (lM) at pH 7.4
11a
1.9
180
471
11b
11c
11d
11e
11f
11g
1.8
1.7
1.7
1.5
1.9
2.1
2.0
240
280
440
410
330
330
470
133
165
265
273
174
157
235
390
422
349
313
ND
337
30
11h
ND, not detected.

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C.-S. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4036–4040
Table 4. Serine proteases selectivity and stability data for 12a and 12b
Compounds b-Tryptase Ki (nM) Trypsin Ki (nM) Thrombin Ki (nM) FVIIA Ki (nM) FXA Ki (nM) Microsome stability (%)
12a
12b
3.7
2.8
21
170
48,000
60,000
140,000
>150,000
100,000
140,000
ND
64
ND, not detected.
at the Advanced Light Source (ALS) in the Lawrence
Berkeley Laboratory.
References and notes
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Figure 3. X-ray crystal structure of 12b bound to human b-tryptase.
5. (a) Hopkins, C. R.; Czekaj, M.; Kaye, S. S.; Gao, Z.;
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The phenyl-capping group of 12b is able to have van der
Waals interaction with a unique hydrophobic pocket
consisting of a loop (Val59–Leu64) and two b-sheets
(Leu33–Gly37 and Tyr37B–Phe41) of b-tryptase. In
comparison, trypsin has no specific pocket in this
area since hydrogen bonding between the backbones
of Phe41 and Lys60 prevents the formation of a
well-defined pocket.
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In summary, we have achieved highly potent and selec-
tive inhibitors of human tryptase with good water solu-
bility. Significant improvements in microsomal stability
and hERG channel binding were also demonstrated in
analogs from the benzamide series relative to previously
described analogs. X-ray co-crystal structures of two
amide-containing analogs typifying the series confirmed
that the molecules are capable of occupying a distal
binding pocket in tryptase that is thought to be impor-
tant for potency and selectivity.
11. Dess, D. B.; Martin, J. J. Org. Chem 1983, 48, 4155.
12. Compounds 10p and 10q show 7.2% at 10 lM and 0%
hERG channel binding at 1 lM substrate concentration,
respectively.
Acknowledgment
13. The tryptase crystal structures have been deposited in the
Protein Data Bank (PDB) under Accession codes 2GDD
and 2FS9.
The authors thank Joseph D. Ho for his contribution
to X-ray crystallography work, which was conducted