Potent, small-molecule inhibitors of human mast cell tryptase. Antiasthmatic action of a dipeptide-based transition-state analogue containing a benzothiazole ketone

Article abstract of DOI:10.1021/jm030050p

Inhibitors of human mast cell tryptase (EC 3.4.21.59) have therapeutic potential for treating allergic or inflammatory disorders. We have investigated transition-state mimetics possessing a heterocycle-activated ketone group and identified in particular benzothiazole ketone (2S)-6 (RWJ-56423) as a potent, reversible, low-molecular-weight tryptase inhibitor with a K_i value of 10 nM. A single-crystal X-ray analysis of the sulfate salt of (2S)-6 confirmed the stereochemistry. Analogues 12 and 15-17 are also potent tryptase inhibitors. Although RWJ-56423 potently inhibits trypsin (K_i = 8.1 nM), it is selective vs other serine proteases, such as kallikrein, plasmin, and thrombin. We obtained an X-ray structure of (2S)-6 complexed with bovine trypsin (1.9-? resolution), which depicts inter alia a hemiketal involving Ser-189, and hydrogen bonds with His-57 and Gln-192. Aerosol administration of 6 (2R,2S; RWJ-58643) to allergic sheep effectively antagonized antigen-induced asthmatic responses, with 70-75% blockade of the early response and complete ablation of the late response and airway hyperresponsiveness.

Full text of DOI:10.1021/jm030050p

J . Med. Chem. 2003, 46, 3865-3876
3865
P oten t, Sm a ll-Molecu le In h ibitor s of Hu m a n Ma st Cell Tr yp ta se. An tia sth m a tic
Action of a Dip ep tid e-Ba sed Tr a n sition -Sta te An a logu e Con ta in in g a
Ben zoth ia zole Keton e
Michael J . Costanzo,† Stephen C. Yabut,† Harold R. Almond, J r.,† Patricia Andrade-Gordon,†
Thomas W. Corcoran,† Lawrence de Garavilla,† J ack A. Kauffman,† William M. Abraham,‡ Rosario Recacha,§
Debashish Chattopadhyay,§ and Bruce E. Maryanoff*,†
Drug Discovery, J ohnson & J ohnson Pharmaceutical Research & Development, Spring House, Pennsylvania 19477-0776,
Division of Pulmonary Disease, University of Miami at Mount Sinai Medical Center, Miami Beach, Florida 33140, and
Centre for Macromolecular Studies, University of Alabama at Birmingham, Birmingham, Alabama 35294
Received J anuary 30, 2003
Inhibitors of human mast cell tryptase (EC 3.4.21.59) have therapeutic potential for treating
allergic or inflammatory disorders. We have investigated transition-state mimetics possessing
a heterocycle-activated ketone group and identified in particular benzothiazole ketone (2S)-6
(RWJ -56423) as a potent, reversible, low-molecular-weight tryptase inhibitor with a Ki value
of 10 nM. A single-crystal X-ray analysis of the sulfate salt of (2S)-6 confirmed the
stereochemistry. Analogues 12 and 15-17 are also potent tryptase inhibitors. Although RWJ -
56423 potently inhibits trypsin (Ki ) 8.1 nM), it is selective vs other serine proteases, such as
kallikrein, plasmin, and thrombin. We obtained an X-ray structure of (2S)-6 complexed with
bovine trypsin (1.9-Å resolution), which depicts inter alia a hemiketal involving Ser-189, and
hydrogen bonds with His-57 and Gln-192. Aerosol administration of 6 (2R,2S; RWJ -58643) to
allergic sheep effectively antagonized antigen-induced asthmatic responses, with 70-75%
blockade of the early response and complete ablation of the late response and airway
hyperresponsiveness.
Activated mast cells secrete numerous proinflamma-
tory mediators including histamine, arachidonate me-
tabolites, and proteases. An important mediator is the
serine protease tryptase (EC 3.4.21.59), which is a
heparin-associated, homotetrameric trypsin-like enzyme
that constitutes 20-25% of the total protein of human
mast cells.1-3 Since tryptase is stored in a catalytically
active form (rather than as a zymogen) within the
secretory granules and released on stimulation, this
enzyme is thought to be highly relevant to mast cell-
dependent inflammatory conditions.1-3 Indeed, tryptase
has been directly implicated in the pathology of asthma.4
Consequently, inhibitors of human mast cell tryptase
have therapeutic potential for treating allergic or in-
flammatory disorders such as asthma, vascular injury
(e.g., restenosis and atherosclerosis), inflammatory bowel
disease, and psoriasis.5 In this respect, tryptase inhibi-
tors have attracted therapeutic interest, and several
types of compounds have been reported,5,6 such as APC-
366 (1), which advanced into phase 2 clinical trials but
was discontinued,5a,b Babim (2),6a and bifunctional
ligand 3 (Chart 1).6b
particularly detrimental because there are no endog-
enous protease inhibitors for it. Interestingly, tryptase
is one of the few proteases (trypsin being the other) that
can effectively activate protease-activated receptor-2
(PAR-2), a tethered-ligand G-protein-coupled receptor,
by cleavage of its elongated, extracellular N-terminus.7
PAR-2, which has been implicated in various inflam-
matory processes, may be responsible for some of the
physiological and pathophysiological effects of tryptase.
Since no receptor antagonists for PAR-2 have yet been
identified, inhibitors of tryptase offer one of the few
means for interrupting PAR-2 activity.
Our interest in tryptase emerged from the confluence
of research on thrombin inhibitors8 and PAR-2.9 In
studying tripeptide heterocycle-activated ketones that
potently inhibit thrombin, such as RWJ -50353 (4; Ki )
0.19 nM),8a,b we probed the minimally acceptable phar-
macophore by eliminating different segments of the
molecule. We prepared 5 (RWJ -51084), which is only
comprised of a single amino acid residue and found it
to be a weak thrombin inhibitor (Ki ) 12 300 nM), but
a reasonably potent inhibitor of trypsin (Ki ) 30 nM).10
Subsequently, we learned that 5 also effectively inhibits
tryptase, with a Ki value of 88 nM (Table 1), while being
quite selective over other serine proteases such as
kallikrein, factor Xa, and chymotrypsin. This level of
inhibitory potency is rather impressive given the rela-
tively low molecular weight (MW ) 387.5) and simple
structure of 5. Our reported X-ray structure determi-
nation on a cocrystal of 5 (actually the 2S enantiomer)
with trypsin10 shows the inhibitor occupying the active
site with its guanidine group in the S1 specificity pocket
The biological mechanisms by which tryptase con-
tributes to cellular inflammatory responses are not yet
completely understood. Although tryptase cleaves and
inactivates certain proteins, its range of activity is
somewhat limited. On the other hand, tryptase can be
* Address correspondence to this author. E-mail: bmaryano@
prdus.jnj.com. Fax: 215-628-4985.
† J ohnson & J ohnson Pharmaceutical Research & Development
(formerly The R. W. J ohnson Pharmaceutical Research Institute).
‡ University of Miami at Mount Sinai Medical Center.
§ University of Alabama at Birmingham.
10.1021/jm030050p CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/05/2003

3866 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Ta ble 1. Chemical Properties and Enzyme Inhibition Dataa
Costanzo et al.
Resu lts a n d Discu ssion
compd
salt
2S/2Rb
tryptase Ki, nMc
trypsin Ki, nMc
Dr u g Design . To explore structure vs activity sur-
rounding 5, and to enhance potency and selectivity, we
considered introducing some additional sites for interac-
tion with groups in the active site of tryptase, particu-
larly around the S2 region. Since thrombin inhibitor 4
is also a potent inhibitor of tryptase, with a single-digit
nanomolar Ki value (Table 1), we considered converting
the cyclopentyl ring of 5 into an L-proline and attaching
simple N-acyl groups. An N-acyl-L-proline could intro-
duce a hydrogen bond between the acyl carbonyl and
the backbone NH of Gly-216 in the active-site â-sheet.3
Secondarily, we thought that a polar group, such as a
hydroxyl, on the proline ring might provide an ad-
ditional hydrogen bond with the Gln-98 amide side
chain of tryptase. Compounds with these and related
modifications were synthesized and bioassayed, thereby
achieving a potency improvement of approximately 10-
fold with the best analogues, such as (2S)-6.
4
5
TFA
TFA
HNO3
HNO3
HCl
49:1
6.5 ( 3.0 (3)
88 ( 13 (4)
10 ( 3 (7)f
380 ( 120 (6)
22 ( 5 (13)
19 ( 7 (8)
3.1 ( 1.7 (3)d
30 ( 6 (6)
8.1 ( 1.8 (4)f
170 ( 100 (6)
6.0 ( 1.0 (9)
4.0 ( 1.0 (4)
960 ( 250 (4)h
9.9 ( 1.3 (3)
9.1 ( 3.8 (3)
53 ( 28 (5)h
7 (1)
1.7:1e
32:1
(2S)-6
(2R)-6
6
1:39
1.2:1
1.0:1g
1:2.5g
8.4:1
1.5:1
2.5:1i
1.2:1
ND
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2k
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
HCl
14 000 ( 600 (2)h
73 ( 22 (8)
38 ( 14 (5)
20 ( 5 (5)
41 ( 14 (6)
470 ( 90 (3)
33 ( 9 (3)
250 ( 120 (5)
180 ( 60 (3)
90 ( 25 (7)
NA (2)
NA (1)
NA (3)
140 ( 20 (16)
260 ( 110 (2)h
38 (1)h
2.1:1
2.8:1g
2.5:1g
1.1:1
160 ( 90 (8)h
81 ( 40 (3)
16 ( 3 (3)
NA (2)
j
-
ND
-
NA (1)
NA (2)
100 ( 15 (3)
-
a
All compounds were isolated and purified by reverse-phase
HPLC (typically water/MeCN/CF3CO2H, 70:30:0.1) and lyophilized
as trifluoroacetate (TFA) hydrates, except where noted. All
compounds were characterized by 300-MHz 1H NMR and electro-
spray MS; also, they were judged to be at least 95% pure by
analytical HPLC. The compounds are represented by standard
molecular formulas with the following adducts: 2 (3.0 HCl, 4.3
water), 4 (2.3 TFA, 0.8 water), 5 (1.25 TFA, 0.75 water), (2S)-6
(1.0 HNO3), (2R)-6 (1.0 HNO3, 0.3 TFA, 1.4 water), 6 (2.5 HCl, 2.2
water), 12 (1.3 TFA, 1.4 water), 13 (1.1 TFA, 1.1 water), 14 (1.2
TFA, 1.0 water), 15 (1.2 TFA, 0.8 water), 16 (1.2 TFA, 1.0 water),
17 (1.2 TFA, 0.5 water), 18 (1.28 TFA, 0.3 water), 19 (1.25 TFA,
1.15 water), 20 (1.3 TFA, 0.75 water), 21 (1.2 TFA, 1.0 water), 22
(1.0 TFA, 1.6 water), 23 (1.2 TFA, 1.0 water), 24 (1.0 water), 25
(1.2 TFA, 1.0 water). Microanalytical data (C, H, N; and water)
were within the accepted range, unless noted otherwise. Informa-
tion on the composition, microanalytical data, and enzymatic
assays is given in the Supporting Information. The composition
of 12, 14, 16, 18, 21, and 23-25 was presumed to be 1.2 TFA and
1.0 water for biological testing purposes because there was an
insufficient quantity of material to conduct combustion mi-
Syn th etic Ch em istr y. Representative chemistry is
given for target compound (2S)-6, which was synthe-
sized according to the route outlined in Scheme 1.
Commercially available methyl 4-benzyloxy-L-prolinate,
7, was converted by N-acetylation and ester saponifica-
tion to 813 in 41% yield. Arginine Weinreb amide 914
was reacted with excess 2-lithiobenzothiazole at low
temperature, and the intermediate ketone was purified
by chromatography on silica gel (EtOAc/hexane, 3:2) and
then reduced with NaBH4 to a diastereomeric mixture
of alcohols (ca. 1:1), which was deprotected to give 10.
The amino group was acylated with 8 to furnish 11,
which was purified by chromatography on silica gel
(CH2Cl2/MeOH, 19:1), and the alcohol was oxidized with
the Dess-Martin periodinane to the ketone, which was
largely comprised of the L-arginine isomer.15 Treatment
with anhydrous HF removed both the p-tosyl and benzyl
moieties, and the resultant (2S)-6 was purified by
reverse-phase HPLC (water/MeCN/CF3CO2H, 90:10:0.2
to 70:30:0.2; Bondapak C-18) and then converted into
an HCl salt. Most of the other analogues discussed
herein, 12-20 (Chart 2; Table 1), were prepared in a
similar fashion. The syntheses of 6 and 8-25 are
described in the Experimental Section.
As noted above,15 (2S)-6 can easily suffer base-induced
epimerization because of its labile proton R to the keto
group. Stability studies on closely related 4 in rat and
rabbit plasma showed that the arginine R-proton is
completely equilibrated in 2 h under physiological
conditions (pH 7.4, 37 °C). Consequently, we also
prepared a mixture of epimers, 6, by equilibrating (2S)-6
prior to HPLC purification and HCl salt formation (1.2:1
ratio of epimers). This epimeric mixture was used for
antiasthma studies in conscious allergic sheep.16
b
croanalysis. The 2S/2R epimeric ratio was determined by ana-
lytical HPLC, except where otherwise noted. ND ) not determined.
c Ki values except where noted otherwise. NA ) not active (<10%
inhibition at 100 µM). The number of experiments (N) is indicated
d
in parentheses; standard errors are given for N > 1. Value taken
from ref 8a. e Determined by 13C NMR via the integrals for the
peaks at 122.32 and 122.16 ppm, respectively, in the presence of
4.3 equiv of (R)-(-)-2,2,2-trifluoro-1-(9-anthryl)ethanol in CDCl3
at 40 °C. f IC50 values: tryptase, 29 ( 12 (N ) 5); trypsin, 22 (
g
12 (N ) 5). Determined by 1H NMR via the ratio of 2S and 2R
h
i
methine proton integrations. IC50 value. Determined by 1H
j
NMR via the ratio of methyl proton integrations. Mixture of four
unassigned stereoisomers; ratio of 5:4.7:3:1 as determined by 1H
k
NMR via the HC-OH methine proton integrations. Reference
tryptase inhibitor.
complexed to Asp-189, its ketone involved in a hemiketal
bond with the Oγ of Ser-195, and its benzothiazole
nitrogen hydrogen bonded to Nꢀ of His-57.8a,11 A com-
parison of this structural information with the active
site of human â-tryptase,3 by using computer graphics,
revealed an opportunity to manipulate 5 to enhance
potency as a tryptase inhibitor. In this manner, we
discovered the potent, reversible, low-molecular-weight
tryptase inhibitor RWJ-56423, (2S)-6. Herein, we present
the tryptase inhibitory activity for (2S)-6 and various
analogues, an X-ray crystal structure of (2S)-6 com-
plexed with bovine trypsin, and in vivo bioactivity for 6
(2R,2S mixture; RWJ -58643) in allergen-sensitized
sheep.12
En zym e In h ibition . Our compounds, along with
reference inhibitor 2, were tested for inhibition of
human â-tryptase and bovine trypsin (Table 1). The
data illustrate the importance of the native 2S absolute
stereochemistry in both the arginine and proline moi-
eties for high affinity: cf. (2S)-6 with (2R)-6; 12 with
13. As expected, a basic side chain substituent is
essential for potent inhibition: cf. 24 with 6 and 22.
Although an activated ketone is required for strong
inhibition, contributions from P2 are equally impor-
tant: cf. 6 with 23 and 25. In addition, hydrophobic

Inhibitors of Human Mast Cell Tryptase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3867
Ch a r t 1
Sch em e 1. Synthesis of (2S)-6
interactions of the P2 substituent are critical for good
affinity: cf. 17 with 18. A benzothiazole group provided
a more potent inhibitor than a thiazole group by a factor
of ca. 8: cf. 6 with 21. Acetyl substitution on proline
(12) led to affinity increases of 1.7-fold relative to mesyl
(19) and 13-fold relative to benzoyl (20). The X-ray
crystal structure of (2S)-6‚trypsin (vide infra) suggests
that this difference is associated with adverse steric
interactions between inhibitor molecule 20 and Gly-216/
Trp-215 of the â-sheet of tryptase. Proline and azetidine-
2-carboxylic acid substitution at the P2 position (S2
subsite) were equally effective, cf. 12 with 16; however,
sp2-hybridized proline ring carbons decreased affinity:
cf. 12 with 15 and (2S)-6 with 14. Substitution of proline
with a trans-4-hydroxyproline gave equivalent po-
tency: cf. 6 with 12.
Selectivity data for (2S)-6 relative to five other serine
proteases are presented in Table 2, along with data for
reference inhibitor 2. For comparison, published results
for inhibitor 1 are also offered. These enzyme assays
were conducted in the absence of any added zinc,
conditions which can dramatically increase the serine
protease affinity of 2 and its analogues.17 It is notewor-
thy that (2S)-6 is a considerably more potent tryptase
inhibitor than 2 (Ki value of 10 vs 140 nM), which may
be related to (2S)-6 having a better fit in the enzyme
active site or forming a covalent hemiacetal with the
Ser-195 hydroxyl of tryptase (see X-ray section). Neither
(2S)-6 nor 2 was selective for tryptase over trypsin.
Compound (2S)-6 was much more selective over throm-
bin than 2 (31 000 vs 32), and it was also significantly
more selective than 2 vs plasma kallikrein (32 vs 0.7),

3868 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Ch a r t 2. Analogues of (2S)-6
Costanzo et al.
Ta ble 2. Inhibition of Tryptase Relative to Other Serine Proteases
Ki (nM)a
selectivity (Ki other/Ki tryptase)b
compd
tryptase
trypsin
kallikrein
uPA
220
-
7.5
plasmin
thrombin
factor Xa
5700
(2S)-6c
10 ( 3 (7)
330
140 ( 20 (16)
0.8
0.5
1.3
32
>3000
0.7
810
>3000
20g
31 000
1.3
APC-366 (1)d
Babim (2)f
-
10
e
e
32g
a
b
Ki values (mean ( standard error) are given for tryptase inhibition; the number of experiments (N) is given in parentheses. Selectivity
is defined as the ratio of the Ki value of the serine protease over the Ki value for tryptase; uPA ) urokinase-type plasminogen activator.
For the original Ki data, see the paragraph at the end of this paper regarding Supporting Information. All Ki data were obtained without
the addition of exogenous zinc (refs 5b and 17). c Determined for the trifluoroacetate hydrate (1.2 TFA, 1.4 water). The tryptase Ki value
d
and the selectivities are derived from ref 5a. e Not reported. f Reference tryptase inhibitor, the tryptase Ki of which decreased to 0.48 (
g
0.17 nM (N ) 3) in the presence of 80 µM ZnCl2 (refs 5b and 17). Selectivity was calculated from the published plasmin and thrombin
Ki data (ref 6c).
urokinase plasminogen activator (220 vs 7.5), and
plasmin (810 vs 20).
The overall structure of (2S)-6‚trypsin (Figures 1 and
2) is analogous to that of (2S)-5‚trypsin, although there
are some significant differences. As expected, both
inhibitors form a hemiketal adduct between the ketone
carbonyl and the Ser-195 hydroxyl, a hydrogen bond
between the benzothiazole nitrogen and Nꢀ of His-57,
and a hydrogen bond between the P1 amide carbonyl
and the nitrogen of Gln-192. In the (2S)-6‚trypsin
complex, these bond lengths are 1.45, 3.16, and 2.71 Å,
respectively. By contrast to (2S)-5‚trypsin, the inhibitor
in (2S)-6‚trypsin extends more deeply into the S2
domain presumably because of additional hydrogen
bonding involving the acetyl group of (2S)-6. Even
though the hydroxyl group of (2S)-6 is a participant in
X-r a y Cr ysta l Str u ctu r e of (2S)-6‚Tr yp sin . Since
our attempts to obtain diffracting crystals of (2S)-6‚
tryptase were unsuccessful, the X-ray crystal structure
of a complex between (2S)-6 and bovine pancreatic
â-trypsin was determined (Figure 1).18,19 Crystals of the
(2S)-6‚trypsin complex, trigonal space group P3121, were
grown by the hanging-drop technique under the same
conditions as reported for (2S)-5.10 Diffraction data were
collected at -170 °C and processed to a resolution of
1.9 Å.19a The completeness of the data at this resolution
was 98.6% and the final refinement had convergence
at R ) 0.175 and Rfree ) 0.206 with 225 water molecules.

Inhibitors of Human Mast Cell Tryptase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3869
caused the expected airway resistance responses under
control conditions (no drug treatment). Immediately
after challenge there was a mean 300% increase over
baseline in specific lung resistance (SRL; early response).
SRL returned to baseline between 2 and 6 h post
challenge and then increased again between 6 and 8 h
post challenge to 100% over baseline (late response).
When these same sheep were treated with 6, however,
the early response was inhibited by 70-75% and the
late response was completely blocked. Panel B of Figure
3 depicts the post antigen-induced changes in airway
sensitivity. Prior to antigen challenge (BSL), the dose
of carbachol that elicited a 400% increase in SRL (PC400
)
was the same for both the control experiments and the
experiments with 6. Twenty-four hours after antigen
challenge, the sheep in the control group developed
airway hyper-responsiveness as evidenced by the 2-fold
decrease in the PC400. When these sheep were treated
with 6, this airway hyper-responsiveness was com-
pletely blocked, i.e., the post-challenge PC400 value was
not different from the preantigen value.
Con clu sion
We identified a series of the potent, reversible, low-
molecular-weight tryptase inhibitors, represented by 5
and (2S)-6. The compounds of interest are transition-
state mimetics that feature a heterocycle-activated
ketone group, which can form a hemiketal with the
γ-oxygen of Ser-195 and a hydrogen bond with the
ꢀ-nitrogen of His-57. An X-ray crystal structure of (2S)-6
bound to bovine trypsin at 1.9-Å resolution depicted
these key interactions. In comparing the structure of
(2S)-6‚trypsin with (2S)-5‚trypsin,10 (2S)-6 extends more
deeply into the S2 domain of the enzyme, possibly
because of the hydrogen-bonding interaction between
the acetyl group of (2S)-6 and Gly-216 of trypsin. Since
there is a high degree of homology between the active
site cavities of tryptase and trypsin, a similar hydrogen-
bonding interaction could occur in the (2S)-6‚tryptase
complex. Given that 6 is ca. four times more potent than
5 as a tryptase inhibitor, the introduction of this
planned interaction is an example of effective structure-
based drug design. The incorporation of a proline
hydroxyl into 12, to obtain 6, did not lead to increased
tryptase affinity, nor to better selectivity vs trypsin.
Hence, the intended hydrogen bond interaction between
the hydroxyl of 6 and the amide side chain of Gln-98 of
tryptase is probably not formed.
In our thrombin inhibitor studies,8a the 2-benzothia-
zole derivative was nearly 20 times more potent than
the corresponding 2-thiazole derivative. In that case, we
noted an aromatic stacking interaction between the
benzene ring of the benzothiazole and Trp-60D. For
tryptase inhibition, we found that benzothiazole 6 is
eight times more potent than thiazole 21. Here, the
benzothiazole group may be more effective than the
thiazole because of increased ketone electrophilicity and/
or increased electron density on the ring nitrogen in the
former.21 Additionally, the benzene ring of the ben-
zothiazole may participate in a hydrophobic interaction
with the Cys-42/Cys-58 disulfide bond of tryptase (cf.
Figure 2).
F igu r e 1. View of (2S)-6 (a generally white stick model with
the heteroatoms represented by standard colors) bound in the
active site of trypsin (orange ribbon bearing specific amino acid
side chains).
a hydrogen bond network with several water molecules,
this aspect does not lead to a significant increase in
either trypsin or tryptase affinity (Table 1; cf. 6 with
12). Specifically, the acetyl carbonyl of (2S)-6 forms a
hydrogen bond with the nitrogen of Gly-216 (length )
3.01 Å), and the hydroxyl group of (2S)-6 forms a
hydrogen bond network that links two water molecules
to the carbonyl group of Ser-96 (lengths ) 2.82, 2.64,
and 2.74 Å). Consequently, the minimum interatomic
distance between the benzothiazole phenyl ring and the
Cys-42/Cys-58 disulfide bond is increased by 1.07 Å,
indicating a significant decrease in van der Waals
interactions in (2S)-6‚trypsin relative to the (2S)-5‚
trypsin (5.37 Å vs 4.30 Å). In addition, the plane of the
proline ring of (2S)-6 is essentially parallel to the Ser-
213 through Ser-217 â-strand, whereas the cyclopentyl
ring of (2S)-5 is angled about 60° from the â-strand. This
change in ring orientation results in a decrease in van
der Waals interactions, which is reflected by a 1.05 Å
increase in the minimum interatomic distance between
the Leu-99 methyl groups and the proline ring of (2S)-6
relative to the cyclopentyl ring of (2S)-5 (4.32 Å vs 3.27
Å, respectively).
An tia sth m a tic Activity of 6. We evaluated 6 (2R,2S
mixture, RWJ -58643, HCl salt), for in vivo efficacy in
conscious, antigen-sensitized allergic sheep, which is a
well characterized asthma model.20 The inhibitor was
administered by aerosol as a total dose of 9 mg, twice a
day, for three consecutive days, and as a single, 9-mg
dose on day 4, 2 h prior to antigen challenge (Figure 3).
As depicted in panel A of Figure 3, inhaled antigen
In the well-characterized sheep model of asthma,
aerosol administration of 6 strongly blunted the early

3870 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Costanzo et al.
F igu r e 2. Schematic representing the interactions of (2S)-6 with trypsin.
N-[4-[(Am in oim in om eth yl)a m in o]-1-(2-ben zoth ia zolyl-
ca r bon yl)bu tyl]cyclop en ta n eca r boxa m id e (5). Compound
10 (0.676 g, 1.0 mmol) and cyclopentanecarboxylic acid (0.285
g, 2.5 mmol) were coupled, oxidized, and deprotected to give 5
according to methodology described for 6. Crude 5 was purified
by reverse phase HPLC (water/MeCN/CF3CO2H, 30:20:0.1).
The fractions containing the desired product were combined
and lyophilized to afford 5 as a white solid (0.336 g, 62%) with
a HPLC purity of 99%. The ratio of 2S/2R arginine epimers
was determined to be 1.7:1 by 13C NMR via the ratio of the
peak integrals at 122.32 and 122.16 ppm, respectively, in the
presence of 4.3 mol equiv of (R)-(-)-2,2,2-trifluoro-1-(9-an-
response (70-75% blockade) and completely abolished
the late response and airway hyper-responsiveness.
These noteworthy results reflect on the potential of this
compound for the treatment of asthma in humans, and
6 has advanced into clinical trials.
Exp er im en ta l Section
1
Gen er a l In for m a tion . H NMR spectra were acquired at
300.14 MHz on a Bruker Avance-300 spectrometer in CD3OD
unless indicated otherwise, using Me4Si as an internal stan-
dard. NMR abbreviations used: s, singlet; d, doublet; dd,
doublet of doublets; t, triplet; m, multiplet; br, broad; ov,
overlapping. 13C NMR spectra were acquired 125.76 MHz at
40 °C on a Bruker Avance-500 spectrometer. HPLC analyses
were performed on a Hewlett-Packard Series 1100 HPLC
instrument eluting with a gradient of water/MeCN/CF3CO2H
(10:90:0.2 to 90:10:0.2) over 4 min with a flow rate of 0.75 mL/
min on a Kromasil C18 column (50 × 2.0 mm; 3.5 µm particle
size) or on a Supelcosil ABZ+Plus column (50 × 2.1 mm; 3.0
µm particle size) at 32 °C. Signals were recorded simulta-
neously at 220, 254, and 305 nm with a diode array detector;
purity is reported for the 220-nm signal. Normal-phase
preparative chromatography was performed on an Isco Com-
biflash Separation System Sg 100c equipped with a Biotage
FLASH Si 40M silica gel cartridge (KP-Sil Silica, 32-63 µm,
60 Å; 4 × 15 cm) eluting at 35 mL/min with detection at 254
nm. Reverse-phase preparative chromatography was per-
formed on a Waters Delta-Prep 3000 HPLC equipped with 3
PrepPak reverse-phase cartridges connected in series (Bonda-
pak C-18; 40 × 300 mm; 15-20 µm, 125 Å) eluting at 40 mL/
min with detection at 220 nm unless noted otherwise. Melting
points were determined on a Thomas-Hoover apparatus cali-
brated with a set of melting point standards. Optical rotations
were measured on a Perkin-Elmer 241 polarimeter. Electro-
spray (ES) mass spectra were obtained on a Micromass
Platform LC single quadrupole mass spectrometer in the
positive mode. Accurate mass determination was performed
on an Autospec E high-resolution magnetic sector mass
spectrometer tuned to a resolution of 6K; the ions were
produced in a fast atom bombardment source at 8 kV. Linear
voltage scans were collected to include the sample ion and two
poly(ethylene glycol) ions, which were used as internal refer-
ence standards. Elemental analysis and Karl Fischer water
analysis were determined by Robertson Microlit Laboratories,
Inc., Madison, NJ .
thryl)ethanol in CDCl3 at 40 °C; mp 60-70 °C; [R]20 +5.1° (c
D
0.68, MeOH); 1H NMR (CDCl3) δ 1.40-2.10 (ov m, 12H), 2.10-
2.30 (br m, 1H), 2.55-2.80 (br m, 1H), 2.80-3.15 (br m, 1H),
3.15-3.40 (br m, 1H), 3.60-3.90 (br m, 1H), 5.70-5.90 (m,
1H), 6.80-7.30 (br s, 6H), 6.92 (d, 1H, J ) 6.9 Hz), 7.50-7.70
(m, 2H), 7.98 (d, 1H, J ) 7.3 Hz), 8.19 (d, 1H, J ) 6.9 Hz),
8.20-8.30 (br s, 1H); 13C NMR (CDCl3) δ 25.10, 25.85, 25.90,
30.45, 30.63, 30.97, 40.64, 45.66, 54.15, 122.36, 125.87, 127.44,
128.35, 137.26, 153.42, 157.59, 163.21, 178.18, 192.68; MS (ES)
m/z 388 (MH)+. Anal. (C19H25N5O2S‚1.25CF3CO2H‚0.75H2O)
C, H, N, F, H2O.
(2S,4R)-1-Acet yl-N-[4-[(a m in oim in om et h yl)a m in o]-1-
(2-b en zot h ia zolylca r b on yl)b u t yl]-4-h yd r oxy-2-p yr r oli-
d in eca r boxa m id e (6). Acetyl chloride (3.3 mL, 46 mmol) was
added dropwise to a solution of O-benzyl-L-4-trans-hydroxy-
proline methyl ester hydrochloride (7; 12.5 g, 46 mmol) and
triethylamine (6.4 mL, 46 mmol) in pyridine (150 mL) at 0 °C
while stirring under argon (Scheme 1). The reaction mixture
was stirred for 30 min at 0 °C then slowly warmed to room
temperature over 16 h. The reaction mixture was concentrated
in vacuo, diluted with CH2Cl2, washed with 1 N HCl (3×), 10%
aqueous Na2CO3, saturated aqueous NaHCO3, and brine, dried
(MgSO4), and concentrated in vacuo. The residue was purified
by chromatography on silica gel (CH2Cl2/MeOH, 49:1) to give
7.12 g (55%) of Ac-Hyp(OBzl)-OMe as an oil. This oil (5.38 g,
19.4 mmol) was dissolved in tetrahydrofuran (260 mL), cooled
to 0 °C, treated dropwise with 0.15 M LiOH (260 mL, 39
mmol), and stirred for 30 min. The reaction mixture was
concentrated in vacuo, acidified with 1 N HCl, and extracted
three times with ethyl acetate. The combined organic extracts
were dried (Na2SO4) and concentrated in vacuo to give 3.79 g
(73%) of trans-1-acetyl-4-benzyloxy-L-proline (8) as a white
solid.

Inhibitors of Human Mast Cell Tryptase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3871
were combined and lyophilized to give 6 (2S/2R ) 1.1:1) as
the trifluoroacetate (TFA) salt, which was converted to the HCl
salt by dissolving the TFA salt into 0.1 N HCl and concentrat-
ing in vacuo three times in succession. The resulting glass was
dissolved in water and lyophilized twice to afford 7.9 g (63%)
of the HCl salt of 6 as a light yellow solid with 95% purity by
HPLC and an L/D-arginine epimeric ratio of 1.2:1 by HPLC;
1H NMR δ 1.50-2.40 (ov m, 9H), 3.10-3.90 (ov m, 3H), 4.22-
4.90 (ov m, 3H), 5.52-5.63 (m, 0.4H), 5.63-5.74 (m, 0.6H),
7.50-7.80 (m, 2H), 8.00-8.28 (m, 2H); MS (ES) m/z 447 (MH)+.
Anal. (C20H26N6O4S‚2.50HCl‚2.2H2O) C, H, N, S, H2O.
(2S,4R)-1-Acetyl-N-[(1R)-4-[(am in oim in om eth yl)am in o]-
1-(2-b en zot h ia zolylca r b on yl)b u t yl]-4-h yd r oxy-2-p yr r o-
lid in eca r boxa m id e [(2R)-6]. The protocol for the synthesis
of 6 was repeated, and the product was purified by reverse
phase HPLC, eluting with a gradient of water/MeCN/CF3CO2H
(90:10:0.2 to 70:30:0.2) over 60 min. The fractions containing
the earlier eluting diastereomer were combined and lyophilized
to give (2R)-6 as a TFA salt. This material (160 mg, 0.258
mmol) was dissolved into 3.8 mL of warm MeCN/MeOH (3.8:
1) and treated with a solution of HNO3 (23 mg, 0.258 mmol)
in MeCN. The clear solution was concentrated under a stream
of nitrogen to yield an oil, which was dissolved in water and
lyophilized to give (2R)-6 as a white hygroscopic solid. 1H NMR
δ 1.60-2.00 (ov m, 4H), 2.08 (s, 3H), 2.10-2.30 (m, 2H), 3.45-
3.60 (m, 1H), 3.74 (dd, 1H, J ) 4.4, 11.1 Hz), 4.35-4.40 (m,
1H), 4.53 (t, 1H, J ) 8.2 Hz), 5.59 (dd, 1H, J ) 3.7, 9.2 Hz),
7.55-7.70 (m, 2H), 8.08 (d, 1H, J ) 7.4 Hz), 8.18 (d, 1H, J )
6.8); MS (ES) m/z 447 (MH)+. Anal. (C20H26N6O4S‚HNO3‚0.3CF3-
CO2H‚1.3H2O) C, H, N, F, S, H2O.
(2S,4R)-1-Acetyl-N-[(1S)-4-[(am in oim in om eth yl)am in o]-
1-(2-b en zot h ia zolylca r b on yl)b u t yl]-4-h yd r oxy-2-p yr r o-
lid in eca r boxa m id e [(2S)-6]. The protocol for 6 was repeated,
except the Dess-Martin oxidation was processed immediately
after quenching with 20% Na2S2O3 (w/w) in saturated aqueous
NaHCO3 to minimize epimerization. The product was purified
by reverse-phase HPLC (water/MeCN/CF3CO2H, 90:10:0.2 to
70:30:0.2) over 60 min, and the fractions containing the slower-
eluting diastereomer were combined and lyophilized to give
1.5 g of (2S)-6 as a TFA salt. The purified salt (1.5 g, 2.42
mmol) was dissolved into warm MeCN with a small amount
of MeOH and treated with a solution of HNO3 (0.21 g, 2.42
mmol) in MeCN. The white crystalline solid that formed on
cooling was washed with MeCN and dried in vacuo to give
F igu r e 3. Administration of 6 to conscious allergic sheep by
aerosol in a multiple-dosing protocol (see text). Panel A:
Increase in specific lung resistance (SRL); (O) control animals;
(9) animals treated with 6 (RWJ -58643). Panel B: Change in
airway responsiveness at baseline (BSL) and 24 h following
antigen challenge (post antigen); open bar, control experi-
ments; filled bar, treatment with 6 (RWJ -58643). Values are
given as the mean ( standard error for N ) 4.
1
(2S)-6 (1.04 g, 84%) as a nitrate salt, mp 174.5-176.5 °C; H
NMR δ 1.50-2.08 (ov m, 4H), 2.10 (s, 3H), 2.12-2.30 (m, 2H),
3.54 (d, 1H, J ) 11.1 Hz), 3.76 (dd, 1H, J ) 4.1, 11.1 Hz), 4.40-
4.49 (m, 1H), 4.57 (t, 1H, J ) 8.1 Hz), 5.70-5.82 (m, 1H), 7.55-
7.70 (m, 2H), 8.08 (d, 1H, J ) 7.4 Hz), 8.18 (d, 1H, J ) 6.8);
MS (ES) m/z 447 (MH)+. Anal. (C20H26N6O4S‚HNO3‚0.04H2O)
C, H, N, S, H2O.
Compound 10 (12.12 g, 0.027 mol), 8 (7.13 g, 0.027 mol),
and 1-hydroxybenzotriazole hydrate (HOBT; 9.16 g, 0.068 mol)
were combined in N,N-dimethylformamide (DMF, 270 mL) and
treated with 1,3-dicyclohexylcarbodiimide (DCC; 13.99 g, 0.068
mol). The reaction was stirred under argon at room temper-
ature for 18 h and filtered. The filtrate was diluted with water
(ca. 800 mL), extracted with ethyl acetate (3×), washed with
water, dried (Na2SO4), and concentrated in vacuo. The residue
was purified by chromatography on silica gel (CH2Cl2/MeOH,
19:1) to afford 15.0 g (80%) of 11 as a white solid.
The Dess-Martin periodinane (18.7 g, 0.044 mol) was added
to a solution of 11 (14.9 g, 0.022 mol) in CH2Cl2 (220 mL) under
argon at room temperature and stirred for 1 h. The reaction
mixture was quenched with a solution containing 20% Na2S2O3
(w/w) in saturated aqueous NaHCO3, and the mixture was
allowed to epimerize by stirring at 23 °C for 2 h. The organic
layer was separated, washed with brine, dried (Na2SO4), and
concentrated in vacuo to furnish a white solid. This solid was
dissolved in anhydrous anisole (ca. 12 mL) in a Teflon reaction
vessel, placed on a HF apparatus, and cooled to -78 °C.
Anhydrous HF (ca. 38 mL) was condensed into the reaction
vessel, and the reaction was warmed to 0 °C. The reaction was
stirred at 0 °C for 6 h, concentrated in vacuo, and triturated
with ethyl ether (3×) to furnish a white solid. This solid was
purified by reverse-phase HPLC eluting with a gradient of
water/acetonitrile/trifluoroacetic acid (90:10:0.2 to 70:30:0.2)
on six PrepPak cartridges connected in series (Bondapak C-18;
40 × 300 mm; 15-20 µm, 125 Å) eluting at 40 mL/min over
60 min. The fractions containing the both diastereomers of 6
(4S)-N-[[[4-Am in o-5-(2-ben zoth ia zolyl)-5-h yd r oxyp en -
t yl]a m in o]im in om et h yl]-4-m et h ylb en zen esu lfon a m id e
(10). Butyllithium in hexanes (2.5 M) (68 mL, 170 mmol) was
added dropwise to a stirred solution of distilled benzothiazole
(56.8 g, 420 mmol) in dry tetrahydrofuran (600 mL) at 78 °C
under argon at a rate that kept the reaction temperature below
-64 °C. Upon completion of addition, the reaction mixture was
stirred for 30 min at -70 °C, and a solution of 913 (20.0 g, 42.4
mmol) in dry tetrahydrofuran (500 mL) was added at a rate
to maintain the reaction temperature below -70 °C. The
resulting mixture was slowly warmed to room temperature
over 2 h and quenched with saturated aqueous NH4Cl (250

3872 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Costanzo et al.
mL). The resulting organic layer was separated, washed with
brine, dried (Na2SO4), and concentrated in vacuo. The residue
was triturated thrice with hexane and purified by chromatog-
raphy on silica gel (ethyl acetate/hexane, 3:2) to furnish ketone
26 as an amber solid.
NaBH4 (3.7 g, 0.097 mol) was added portionwise to a stirred
solution of 26 (17.8 g, 0.033 mmol) in dry methanol (165 mL)
under argon at -30 °C. After 3 h, the reaction was quenched
with acetone (30 mL) and concentrated in vacuo. The residue
was dissolved in CH2Cl2, washed sequentially with 10%
aqueous citric acid, water, and brine, dried (Na2SO4), and
concentrated in vacuo to give alcohol 27 as a yellow solid.
The thick syrupy residue was triturated with hexanes (3×)
and purified by chromatography on silica gel (CH2Cl2/ethyl
acetate, 7:3) to afford 29 as a light yellow solid.
NaBH4 (4.9 g, 129 mmol) was added portionwise to a stirring
solution of 29 (15.0 g, 27.4 mmol) in dry methanol (200 mL)
under argon at 0 °C. The reaction mixture was slowly warmed
to 23 °C over 1 h, quenched with acetone (30 mL), and
concentrated in vacuo. The residue was dissolved in ethyl
acetate, washed with water (2×) and brine, dried (Na2SO4),
and concentrated in vacuo to give 30 as a yellow solid.
p-Toluenesulfonic acid monohydrate (TsOH‚H2O) was added
at 23 °C to solution of 30 (1.0 g, 1.8 mmol) in CH2Cl2 (20 mL)
until the solution was saturated. The reaction was stirred at
23 °C for 6 h, diluted with ethyl acetate, extracted twice with
a 1:1 mixture (v/v) of brine and 10% aqueous Na2CO3, dried
(Na2SO4), and concentrated in vacuo to give 31 as a yellow
solid.
Compound 31 and N-acetyl-L-proline was coupled with DCC
and oxidized with the Dess-Martin periodinane according to
methodology described for 6. The resulting ketone (0.040 g,
0.062 mmol) was dissolved in trifluoroacetic acid (6 mL),
stirred at 23 °C for 6 h, and concentrated in vacuo to give crude
12, which was purified by reverse-phase HPLC with water/
MeCN/CF3CO2H (90:10:0.2 to 60:40:0.2) over 60 min to give
12 (0.010 g, 27%) as a white solid with 96% purity by HPLC
and an L/D-arginine ratio of 1.0:1 by 1H NMR; 1H NMR δ 1.8-
2.0 (ov m, 6H), 2.11 (s, 3H), 2.12-2.40 (br m, 2H), 3.20-3.75
(ov m, 4H), 4.35-4.50 (m, 1H), 5.50-5.65 (m, 0.5H), 5.65-
5.80 (m, 0.5H), 7.50-7.70 (m, 2H), 8.10 (d, 1H, J ) 7.2 Hz),
8.21 (d, 1H, J ) 6.9 Hz); MS (ES) m/z 431 (MH)+. Anal.
(C20H26N6O3S‚1.30CF3CO2H‚1.40H2O) C, H, N, F, H2O.
Compound 27 (1.0 g, 1.8 mmol) was treated with trifluoro-
acetic acid/CH2Cl2 (1:1 v/v; 50 mL) and stirred at 23 °C for 2
h. The reaction mixture was concentrated in vacuo, dissolved
in ethyl acetate, and extracted with a 1:1 mixture (v/v) of brine
and 10% aqueous Na2CO3. The organic layer was extracted
once with brine, dried (Na2SO4), and concentrated in vacuo to
1
furnish 10 as a yellow solid with a HPLC purity of 97%; H
NMR δ 1.10-1.80 (ov m, 4H), 2.36 (s, 3H), 2.90-3.40 (ov m,
3H), 4.83 (d, 0.5 H, J ) 4.4 Hz), 4.91 (d, 0.5 H, J ) 4.6 Hz),
7.25 (d, 2H, J ) 7.3 Hz), 7.33-7.55 (ov m, 2H), 7.60-7.70 (m,
2H), 7.94 (d, 1H, J ) 8.4 Hz), 7.98 (d, 1H, J ) 7.8 Hz); MS
(ES) m/z 448 (MH)+.
(2R)-1-Acet yl-N-[4-[(a m in oim in om et h yl)a m in o]-1-(2-
ben zoth ia zolylca r bon yl)bu tyl]-2-p yr r olid in eca r boxa m -
id e (13). Compound 10 (1.00 g, 1.83 mmol) and acetyl-D-
proline (0.426 g, 2.7 mmol) were converted crude 13 according
to methodology described for 6. The crude material was
purified by reverse-phase HPLC eluting with a gradient of
water/MeCN/CF3CO2H (90:10:0.2 to 60:40:0.2) over 60 min to
give 13 (37 mg) as a white solid with a HPLC purity of 95%
and an L/D-arginine epimeric ratio of 1:2.5 by 1H NMR; 1H
NMR δ 1.60-2.05 (ov m, 6H), 2.08 (s, 3H), 2.12-2.30 (br m,
2H), 3.10-3.3 (ov m, 2H), 3.46-3.72 (ov m, 2H), 4.40-4.45
(m, 0.7H), 4.45-4.55 (m, 0.3H), 5.58-5.65 (m, 0.7H), 5.65-
5.76 (m, 0.3H), 7.55-7.70 (m, 2H), 8.10 (d, 1H, J ) 7.2 Hz),
8.21 (d, 1H, J ) 6.9 Hz); MS (ES) m/z 431 (MH)+. Anal.
(C20H26N6O3S‚1.07CF3CO2H‚1.13H2O) C, H, N, F, H2O.
(2S)-1-Acet yl-N-[4-[(a m in oim in om et h yl)a m in o]-1-(2-
ben zoth ia zolylca r bon yl)bu tyl]-2-p yr r olid in eca r boxa m -
id e (12). Benzotriazol-1-yloxytris(dimethylamino)phospho-
nium hexafluorophosphate (BOP reagent; 25.0 g, 56 mmol) was
added in one portion to a stirring solution of N-R-t-Boc-NG-(4-
methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine (Boc-Arg-
(Mtr)-OH; 24.96 g, 51.3 mmol), N,O-dimethylhydroxylamine
hydrochloride (7.6 g, 56 mmol), and triethylamine (22 mL, 154
mmol) in dry DMF (100 mL) under argon at 0 °C. The reaction
mixture was allowed to slowly warm to room temperature over
2 h, filtered through diatomaceous earth, and concentrated in
vacuo. The residue was dissolved in ethyl acetate, washed
sequentially with H2O (three times), 1 M KHSO4, saturated
aqueous NaHCO3, and brine, dried (Na2SO4), and concentrated
in vacuo. The residue was purified by chromatography on silica
gel (ethyl acetate/CH2Cl2, 3:1) to give 2814d as a white solid.
Butyllithium in hexanes(2.5 M) (164 mL, 410 mmol) was
added dropwise at -78 °C under argon to a stirred solution of
benzothiazole (69.2 g, 512 mmol) in dry tetrahydrofuran (1 L)
at a rate that kept the reaction temperature below -64 °C.
On completion of addition, the reaction mixture was stirred
for 30 min at -70 °C, and a solution of 28 (27.11 g, 51.2 mmol)
in dry tetrahydrofuran (200 mL) was added at a rate that
maintained the temperature below -70 °C. The reaction was
stirred for 15 min, quenched with saturated aqueous NH4Cl
(500 mL), and stirred for 16 h at 23 °C. The resulting organic
layer was separated, diluted with ethyl acetate, washed with
water and brine, dried (Na2SO4), and concentrated in vacuo.
(2S)-1-Acet yl-N-[4-[(a m in oim in om et h yl)a m in o]-1-(2-
b en zot h ia zolylca r b on yl)b u t yl]-4-oxo-2-p yr r olid in eca r -
boxa m id e (14). Compound 31 (0.67 g, 1.32 mmol) and (4R)-
1-acetyl-4-hydroxy-L-proline (0.23 g, 1.32 mmol) were converted
to crude 14 according to methodology described for 12 with
the exception that 4 mol equiv of the Dess-Martin periodinane
was used instead of 2 mol equiv. The crude material was
purified by reverse-phase HPLC eluting with a gradient of
water/MeCN/CF3CO2H (90:10:0.2 to 70:40:0.2) over 60 min to
give 14 (8 mg) as a white solid with 99% purity and an L/D-
arginine epimeric ratio of 8.4:1 by HPLC; 1H NMR δ 1.70-
1.90 (br m, 3H), 2.09 (s, 0.3H), 2.11 (s, 2.7H), 2.12-2.30 (b m,
1H), 2.53 (dd, 1 H, J ) 3.4, 18.7 Hz), 3.00 (dd 1H, J ) 10.2,
18.7 Hz), 3.10-3.30 (m, 2H), 4.10 (dd, 2H, J ) 7.8, 16.7 Hz),
4.80-5.05 (m, 1H), 5.65-5.80 (b m, 1H), 7.50-7.70 (m, 2H),
8.17 (d, 1H, J ) 7.2 Hz), 8.21 (d, 1H, J ) 6.6 Hz); MS (ES) m/z
445 (MH)+. HRMS (FAB) m/z 445.1662 (445.1658 calcd for
C
20H24N6O4S + H+).
(2S)-1-Acet yl-N-[4-[(a m in oim in om et h yl)a m in o]-1-(2-
ben zoth ia zolylca r bon yl)bu tyl]-2,5-d ih yd r o-1H-p yr r ole-
2-ca r boxa m id e (15). 3,4-Dehydro-L-proline methyl ester
hydrochloride (0.50 g, 3.06 mmol, triethylamine (1.0 mL, 7.2
mmol), and acetyl chloride (0.22 mL, 3.1 mmol) were added to
CH2Cl2 (50 mL) with stirring under argon at 5 °C. The reaction
mixture was slowly warmed to 23 °C, stirred for 18 h, and
concentrated in vacuo. The residue was dissolve in 10 mL of
tetrahydrofuran/water (1:1), treated with LiOH‚H2O (0.13 g,
3.06 mmol), and stirred for 18 h. The mixture was concentrated

Inhibitors of Human Mast Cell Tryptase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3873
in vacuo and lyophilized. The resulting residue and 26 (1.47
g, 2.91 mmol) were converted into crude 15 according to
methodology described for 12 with the exception that 4 mol
equiv of the Dess-Martin periodinane was used instead of 2
mol equiv. The crude material was purified by reverse-phase
HPLC with water/MeCN/CF3CO2H (90:10:0.2 to 70:40:0.2) over
60 min to give 15 (125 mg) as a white solid with 99% purity
and an L/D-arginine epimeric ratio of 1.5:1 by HPLC; 1H NMR
δ 1.70-1.93 (br m, 3H), 2.08 (s, 1.2H), 2.11 (s, 1.8H), 2.12-
2.33 (m, 1H), 3.21-3.33 (m, 2H), 4.20-4.45 (m, 2H), 5.14-
5.30 (m, 1H), 5.60-5.90 (m, 2H), 5.95-6.10 (m, 1H), 7.59-
7.72 (m, 2H), 8.10 (d, 1H, J ) 7.4 Hz), 8.20 (d, 1H, 6.8 Hz);
MS (ES) m/z 429 (MH)+. Anal. (C20H24N6O3S‚1.22CF3CO2H‚
0.84H2O) C, H, N, F, H2O.
Compound 31 (1.38 g, 2.7 mmol) and 1-(methylsulfonyl)-L-
proline (0.40 g, 2.6 mmol) were converted to crude 19 according
to methodology described for 12. The crude material was
purified by reverse-phase HPLC with water/MeCN/CF3CO2H
(90:10:0.2 to 70:40:0.2) over 60 min to give 19 (70 mg) as a
white solid with 95.0% purity and an L/D-arginine epimeric
1
ratio of 2.1:1 by HPLC; H NMR δ 1.75-2.13 (m, 6H), 2.14-
2.35 (m, 2H), 2.95 (s, 2H), 2.99 (s, 1H), 3.22-3.33 (m, 2H),
3.35-3.49 (m, 1H), 3.50-3.61 (m, 1H), 4.23-4.36 (m, 1H),
5.59-5.67 (m, 1H), 7.55-7.70 (m, 2H), 8.12 (d, 1H, J ) 7.5
Hz), 8.20-8.29(m, 1H); MS (ES) m/z 467 (MH+). HRMS (FAB)
m/z 467.1533 (467.1535 calcd for C19H26N6O4S2 + H+). Anal.
(C19H26N6O4S2‚1.25CF3CO2H‚1.15H2O) C, H, N, F, H2O.
(2S)-N-[(1S)-4-[(Am in oim in om et h yl)a m in o]-1-(2-b en -
zoth ia zolylca r bon yl)bu tyl]-1-ben zoyl-2-p yr r olid in eca r -
boxa m id e (20). Compound 20 was prepared from L-proline
tert-butyl ester and benzoyl chloride in a manner analogous
to that described for 19. The crude material was purified by
reverse-phase HPLC with water/MeCN/CF3CO2H (90:10:0.2 to
60:40:0.2) over 60 min to give 20 (118 mg) as a white solid
with 98.3% purity by HPLC and an L/D-arginine epimeric ratio
(2S)-1-Acet yl-N-[4-[(a m in oim in om et h yl)a m in o]-1-(2-
b e n zot h ia zolylca r b on yl)b u t yl]-2-a ze t id in e ca r b oxa m -
id e (16). Compound 31 (0.55 g, 1.1 mmol) and (S)-2-
azetidinecarboxylic (0.26 g, 1.8 mmol) were converted to crude
16 according to methodology described for 12. The crude
material was purified by reverse-phase HPLC with water/
MeCN/CF3CO2H (90:10:0.2 to 70:40:0.2) over 60 min to give
16 (125 mg) as a white solid with 99.9% purity by HPLC and
1
1
of 2.8:1 by H NMR; H NMR δ 1.60-2.50 (br m, 6H), 2.17-
2.40 (m, 2H) 3.18-3.31 (m, 2H), 3.40-3.65 (m, 2H), 4.55-4.65
(m, 1H), 5.46-5.57 (m, 0.26H), 5.57-5.70 (m, 0.74 H), 7.37-
7.7 (ov m, 7H), 8.05-8.25 (m, 2H); MS (ES) m/z 493 (MH+).
Anal. (C25H28N6O3S‚1.30CF3CO2H‚0.75H2O) C, H, N, F, H2O.
(2S,4R)-1-Acet yl-N-[4-[(a m in oim in om et h yl)a m in o]-1-
(2-th ia zolylca r bon yl)bu tyl]-4-h yd r oxy-2-p yr r olid in eca r -
boxa m id e (21). Compound 21 was prepared from trans-1-
acetyl-4-benzyloxy-L-proline by methods analogous to those
described for 6 except that thiazole was used instead of
benzothiazole. The crude material was purified by reverse-
phase HPLC eluting with a gradient of water/acetonitrile/
trifluoroacetic acid (90:10:0.2 to 60:40:0.2) over 60 min to give
21 as a white solid (25 mg) with a HPLC purity of 95.0% and
1
an L/D-arginine epimeric ratio of 2.5:1 by H NMR; 1H NMR δ
1.70-1.89 (m, 3H), 1.90 (s, 2.1H), 1.94 (s, 0.9H), 2.10-2.36
(m, 2H), 2.40-2.70 (m, 1H), 3.22-3.35 (m, 2H), 4.10-4.20 (m,
2H), 4.95-5.05 (m, 1H), 5.60-5.82 (m, 1H), 7.55-7.70 (m, 2H),
8.13 (d, 1H, J ) 7.5 Hz), 8.23 (d, 1H, J ) 6.8 Hz); MS (ES) m/z
417 (MH)+. HRMS (FAB): m/z 417.1710 (417.1709 calcd for
C
19H24N6O3S + H+). Anal. (C19H24N6O3S‚1.22 CF3CO2H‚
0.84H2O) C, H, N, F, H2O.
(2S)-2-(Acetylam in o)-N-[4-[(am in oim in om eth yl)am in o]-
1-(2-ben zoth ia zolylca r bon yl)bu tyl]-4-m eth ylp en ta n a m -
id e (17). Compound 31 (1.95 g, 3.8 mmol) and 1-acetyl-L-
leucine (0.67 g, 3.8 mmol) were converted to crude 17 according
to methodology described for 12. The crude material was
purified by reverse-phase HPLC with water/MeCN/CF3CO2H
(90:10:0.2 to 70:40:0.2) over 60 min to give 17 (217 mg) as a
white solid with a HPLC purity of 99.9% and an L/D-arginine
epimeric ratio of 1.2:1 by HPLC; 1H NMR δ 0.86 (d, 2.7H),
0.95 (d, 3.3H,), 1.45-1.95 (ov m, 6H), 1.99 (s, 3H), 2.120-2.30
(br m, 1H), 3.21-3.35 (m, 2H), 4.35-4.49 (m, 1H), 5.50-5.6
(m, 0.4H), 5.65-5.73 (m, 0.6H), 7.55-7.70 (m, 2H), 8.13 (d,
1H, J ) 7.5 Hz), 8.21 (d, 1H, 6.8 Hz); MS (ES) m/z 447 (MH)+.
Anal. (C21H30N6O3S‚1.19CF3CO2H‚0.5H2O) C, H, N, F, H2O.
1
an L/D-arginine epimeric ratio of 2.5:1 by H NMR; 1H NMR δ
1.65-1.88 (br m, 3H), 1.95-2.05 (m, 1H), 2.09 (s, 3H), 2.10-
2.30 (m, 3.20-3.30, 2H), 3.53 (d, 1H, J ) 11.8 Hz), 3.75 (dd,
1H, J ) 3.3, 11.8 Hz), 4.4-4.50 (m, 1H), 4.50-4.60 (m, 1H),
5.45-5.60 (br m, 0.29H), 5.60-5.70 (br m, 0.71H), 8.00-8.06
(m, 1H), 8.07-8.20 (m, 1H); MS (ES) m/z 397 (MH+). HRMS
(FAB) m/z 397.1663 (397.1658 calcd for C16H24N6O4S + H+).
(2S,4R)-1-Acetyl-N-[5-a m in o-1-(2-ben zoth ia zolylca r bo-
n yl)p en tyl]-4-h yd r oxy-2-p yr r olid in eca r boxa m id e (22). A
solution of N,2-[(1,1-dimethylethoxy)carbonyl]-L-lysine methyl
ester hydrochloride (N-R-Boc-LysOMe; 5.0 g, 0.0168 mol) in
CH2Cl2 (80 mL) was treated with 4-methoxy-2,3,6-trimethyl-
benzenesulfonyl chloride (4.19 g, 0.0168 mol) at 0 °C while
stirring under an argon atmosphere. The reaction mixture was
allowed to slowly warm to 23 °C over 18 h. The reaction
mixture was extracted twice with aqueous 1 M KHSO4, twice
with saturated aqueous NaHCO3, and once with brine, dried
over anhydrous MgSO4, filtered, and concentrated in vacuo to
give N-R-Boc-Lys(Mtr)OMe (7.9 g; 99%). This material was
dissolved in methanol (150 mL) and treated with 50 mL of 2
M KOH with stirring at 23 °C. After 30 min, the mixture was
concentrated in vacuo, and the residue was dissolved in water,
acidified to pH 3 with concentrated HCl, and extracted three
times with ethyl acetate. The combined extracts were washed
twice with brine, dried (Na2SO4), and concentrated to yield 32
(Boc-Lys(Mtr)OH (7.7 g, 100%) as a yellow foam.
2-(Acet yla m in o)-N-[4-[(a m in oim in om et h yl)a m in o]-1-
(2-ben zoth ia zolylca r bon yl)bu tyl]a ceta m id e (18). Com-
pound 31 (0.501 g, 0.99 mmol) and N-acetylglycine (0.175 g,
1.49 mmol) were converted to crude 18 according to methodol-
ogy described for 12. The crude material was purified by
reverse-phase HPLC with water/MeCN/CF3CO2H (90:10:0.2 to
70:40:0.2) over 60 min to give 18 (27 mg) as a white solid with
1
a HPLC purity of 98.0%; H NMR δ 1.70-1.95 (m, 3H), 2.01
(s, 3H), 2.10-2.28 (m, 1H), 3.21-3.35 (m, 2H), 3.78, 4.04 (ABq,
2H, J ) 16.6 Hz), 5.70-5.85 (m, 1H), 7.60-7.70 (m, 2H), 8.14
(d, 1H, J ) 7.7 Hz), 8.22 (d, 1H, J ) 6.8 Hz); MS (ES) m/z 391
(MH+). HRMS (FAB) m/z ) 391.1557 (391.1552 calcd for
C17H22N6O3S + H+). Anal. (C17H22N6O3S‚1.28CF3CO2H‚0.3H2O)
C, H, N, F, H2O.
(2S)-N-[4-[(Am in oim in om eth yl)a m in o]-1-(2-ben zoth ia -
zolylca r bon yl)bu tyl]-1-(m eth yl-su lfon yl)-2-p yr r olid in e-
ca r boxa m id e (19). A solution of L-proline tert-butyl ester
(2.11 g, 12.3 mmol) and triethylamine (3.4 mL, 24.4 mmol) in
CH2Cl2 (20 mL) was cooled to 5 °C under an argon atmosphere
and treated dropwise with methanesulfonyl chloride (0.953
mL, 12.3 mmol). The reaction mixture was filtered through
filter agent and extracted sequentially with 10% aqueous citric
acid, saturated aqueous NaHCO3, and brine. The organic
extract was dried over anhydrous MgSO4 and concentrated in
vacuo to give 1-(methylsulfonyl)-L-proline tert-butyl ester (2.67
g, 87%). This material was dissolved in 20 mL of trifluoroacetic
acid/CH2Cl2 (1:1) and stirred at 23 °C for 30 min. The mixture
was concentrated in vacuo, triturated with hexane, and placed
under 2 Torr of vacuum to give 1-(methylsulfonyl)-L-proline
(0.40 g, 22%).
Compound 32 (0.92 g, 2.0 mmol) was converted to 22 by
methods analogous to those described for 12, except that the
final deprotection was performed in 9:1:1 trifluoroacetic acid/
anisole/dimethyl sulfide in the presence of 0.3 M methane-

3874 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Costanzo et al.
sulfonic acid over 1 h at 23 °C. The crude material was purified
by reverse-phase HPLC with water/MeCN/CF3CO2H (90:10:
0.2 to 60:40:0.2) over 60 min to give 22 as a white solid (106
mg) with 95.0% purity and an L/D-lysine epimeric ratio of 1.1:1
°C under argon at a rate that kept the reaction temperature
below -64 °C. Upon completion of addition, the reaction
mixture was stirred for 30 min at -70 °C, and a solution of
33 (0.34 g, 0.84 mmol) in dry tetrahydrofuran (25 mL) was
added at a rate that maintained the reaction temperature
below -70 °C. The resulting mixture was allowed to slowly
warm to 23 °C over 2 h and then quenched with saturated
aqueous NH4Cl (13 mL). The organic layer was separated,
washed with brine, dried (Na2SO4), and concentrated in vacuo.
The residue was triturated three times with hexane and
purified by chromatography on silica gel eluting with gradient
of 9:1 to 4:1 ethyl acetate/hexane over 30 min to furnish ketone
34 as an amber solid.
1
by HPLC; H NMR δ 1.50-1.92 (m, 6H), 1.93-2.07 (m, 1H),
2.10 (s, 3H), 2.11-2.30 (m, 1H), 2.90-3.10 (m, 2H), 3.48-3.58
(m, 1H), 3.70-3.80 (m, 1H), 4.30-4.68 (ov m, 2H), 5.55-5.67
(m, 0.5H), 5.71-5.80 (m, 0.5H), 7.55-7.70 (m, 2H), 8.08 (d,
1H, J ) 7.4 Hz), 8.18 (d, 1H, J ) 6.8 Hz); MS (ES) m/z 419
(MH+). Anal. (C20H26N4O4S‚0.95CF3CO2H‚0.05 CH3SO3H‚
1.55H2O) C, H, N, F, S, H2O.
(2S)-1-Acetyl-N-[1-(2-ben zoth ia zolylh yd r oxym eth yl)-4-
[[im in o[a m in o]m et h yl]a m in o]b u t yl]-2-p yr r olid in eca r -
boxa m id e (23). Compound 23 was prepared from 31 (2.48 g,
4.9 mmol)) by methods analogous to those described for 12
except that the Dess-Martin oxidation was not performed. The
crude material was purified by reverse-phase HPLC (water/
MeCN/CF3CO2H, 90:10:0.2 to 60:40:0.2) over 60 min to give
Compound 34 (0.14 g, 0.29 mmol) was dissolved in 6 mL of
trifluoroacetic acid/CH2Cl2 (1:1) and stirred at 23 °C. After 2
h, the reaction mixture was concentrated in vacuo, and the
residue was purified by reverse-phase HPLC with water/
MeCN/CF3CO2H (90:10:0.2) to furnish 25 as a white solid with
1
1
22 as a white solid (106 mg) with a HPLC purity of 99%; H
a HPLC purity of 99%. H NMR δ 1.60-1.75 (m, 2H), 1.76-
NMR δ 1.50-2.20 (ov m, 11H), 3.03-3.25 (m, 2H), 3.35-3.57
(m, 2H), 4.23-4.40 (m, 1H), 4.45-4.80 (ov m, 1H), 5.50-5.80
(ov m, 1H), 7.32-7.55 (m, 2H), 7.85-8.01 (m, 2H); MS (ES)
m/z 433 (MH+). HRMS (FAB) m/z 433.2012 (433.2024 calcd
for C20H28N6O3S + H+).
1.95 (m, 2H), 3.14-3.25 (m, 4H), 7.55-5.7 (m, 2H), 8.10 (d,
1H, J ) 7.4 Hz), 8.20 (d, 1H, J ) 6.8 Hz); MS (ES) m/z 277
(MH+). HRMS (FAB) m/z 277.1126 (277.1123 calcd for C13H16N4-
OS + H+).
â-Tr yp ta se En zym e Assa y. The â-tryptase assay was
conducted with human lung â-tryptase (Cortex Biochem
#CP3033) in an aqueous buffer (10 mM Tris, 10 mM Hepes,
150 mM NaCl, 0.1% PEG 8000, pH 7.4) using the chromogenic
substrate H-D-HHT-Ala-Arg-pNa (American Diagnostica #238)/
Km 580 µM) and a microplate reader (Molecular Devices). IC50
experiments were conducted by fixing the enzyme and sub-
strate concentrations (5 nM [E]/715 µM [S]) and varying the
inhibitor concentration. Changes in absorbance at 405 nm were
monitored using the software program Softmax (Molecular
Devices), upon addition of enzyme, with and without inhibitor
at 37 °C for 30 min. Percent inhibition was calculated by
comparing the initial reaction slopes of samples without
inhibitor to those with inhibitor. IC50 determination was made
using a four-parameter fit logistics model. Inhibition constants
(Ki values) were determined under conditions for the analysis
of Michaelis-Menten kinetics (1 nM [E]/30-700 µM [S]).
Changes in absorbance at 405 nm were monitored (37 °C for
30 min) on addition of enzyme with and without inhibitor
present using the software program Softmax (Molecular
Devices). Initial reaction slopes of samples were analyzed using
the program K-Cat (BioMetallics).
(2S,4R)-1-Acetyl-N-[1-(2-ben zoth ia zolylca r bon yl)p en -
tyl]-4-h yd r oxy-2-p yr r olid in eca r boxa m id e (24). Compound
24 was prepared from N-tert-butoxycarbonyl-L-norvaline (Boc-
Nva-OH; 2.5 g, 10.5 mmol) by methods analogous to those
described for 6. The crude material was purified by reverse-
phase HPLC with water/MeCN/CF3CO2H (90:10:0.2 to 60:40:
0.2) over 60 min to give 24 as a white solid (106 mg) with a
HPLC purity of 95%. 1H NMR δ 0.90-1.10 (m, 4H), 1.37-1.70
(m, 3H), 1.70-1.90 (m, 1H), 2.05 (s, 3H), 2.10-2.30 (m, 1H),
3.45-3.55 (m, 1H), 3.60-3.80 (m, 1H), 4.30-4.49 (m, 1H),
4.50-4.80 (m, 1H), 5.55-5.75 (m, 1H), 7.55-5.7 (m, 2H), 8.10
(d, 1H, J ) 7.4 Hz), 8.20 (d, 1H, J ) 6.8 Hz); MS (ES) m/z 390
(MH+). HRMS (FAB) m/z 390.1480 (390.1488 calcd for
C
19H23N3O4S + H+).
Oth er En zym e Assa ys. Enzyme-catalyzed hydrolysis rates
and IC50 and Ki values were determined, as described above
for â-tryptase, for the following enzymes: trypsin, kallikrein,
urokinase plasminogen activator (uPa), plasmin, and throm-
bin. The enzyme sources, substrates/Km, aqueous buffer, IC50
assay concentrations [E]/[S], and Ki assay concentrations
[E]/[S] were as follows: for bovine trypsin (Sigma #T8003),
Cbo-Gly-D-Ala-Arg-pNa (American Diagnostica # 216)/Km 770
µM, buffer A (10 mM Tris, 10 mM Hepes, 150 mM NaCl, 0.1%
PEG 8000, pH 7.4), 13.7 nM [E]/1000 µM [S], 13.7 nM [E]/100-
3000 µM [S]; for human plasma kallikrein (Sigma #K1004),
H-D-Pro-HHT-Arg-pNa (American Diagnostica # 302)/Km 100
µM, buffer A, 0.025 IU/mL [E]/1000 µM [S], 0.025 IU/mL
[E]/30-1000 µM [S]; for human urine uPa (American Diag-
nostica #124), Cbo-L-(γ)Glu(R-t-BuO)-Gly-Arg-pNa (American
Diagnostica #244)/Km 110 µM, buffer A, 150 U/mL [E]/1000
µM [S], 150 IU/mL [E]/30-1000 µM [S]; for human plasmin
(American Diagnostica #421), H-D-Nle-HHT-Lys-pNa (Ameri-
can Diagnostica #251)/Km 42 µM, buffer A, 1.4 µg/mL [E]/200
µM [S], 1.4 µg/mL [E]/20-500 µM [S]; for human R-thrombin
(American Diagnostica #470HT), H-D-HHT-Ala-Arg-pNa (Ameri-
can Diagnostica #238)/Km 24 µM, buffer A, 0.9 nM [E]/50 µM
[S], 0.9 nM [E]/5-100 µM [S].
1-(2-Ben zoth ia zolyl)-5-[(a m in oim in om eth yl)a m in o]-1-
p en ta n on e (25). A solution of 5-aminopentanoic acid (0.220
g, 1.88 mmol) and Na2CO3 (0.238 g, 2.24 mmol) in 3 mL of
water was added dropwise to a solution of 1H-pyrazole-1-N,N′-
bis(tert-butoxycarbonyl)carboxamidine (0.584 g, 1.88 mmol) in
10 mL of methanol at 23 °C. After 2 h, the reaction mixture
was concentrated in vacuo, diluted with water, and extracted
twice with ethyl acetate. The aqueous layer was acidified to
pH 3 with 1 M KHSO4 and extracted twice with 25 mL of ethyl
acetate. The combined extracts were washed with brine, dried
(MgSO4), and concentrated. The residue was crystallized from
ethyl acetate/hexanes to give 5-[bis(tert-butoxycarbonyl)guani-
dino]pentanoic acid (0.49 g, 72%). This material was combined
with N,O-dimethylhydroxylamine hydrochloride (0.146 g, 1.5
mmol) and triethylamine (0.63 mL, 4.5 mmol), dissolved in
DMF (10 mL), and treated with BOP reagent (0.654 g, 1.48
mmol) while stirring under argon at 0 °C. After 30 min, the
reaction was quenched with water (1 mL), diluted with ethyl
acetate (50 mL), and washed sequentially with water (3×), 10%
aqueous citric acid, saturated aqueous NaHCO3, and brine.
The organic layer was dried (MgSO4) and concentrated in
vacuo. The oily residue was purified by chromatography on
silica gel (hexane/EtOAc, 1.5:1) to give 33 (340 mg, 62%).
Butyllithium in hexanes (2.5 M) (2.7 mL, 6.7 mmol) was
added dropwise to a stirred solution of distilled benzothiazole
(1.1 g, 8.4 mmol) in anhydrous tetrahydrofuran (25 mL) at 78
X-r a y Cr ysta llogr a p h y of th e Su lfa te Sa lt of (2S)-6.22
The X-ray crystal structure of (2S)-6‚H2SO4 was determined
by Crystalytics Company (Lincoln, NE). Single yellowish-
orange crystals of (2S)-6‚H2SO4 were obtained as thin plates
from H2O/2-propanol (dimensions: 0.09 mm × 0.33 mm × 0.46
mm) and are, at -80 ( 2 °C, monoclinic, space group P21
-

Inhibitors of Human Mast Cell Tryptase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3875
2
C2 (No. 4) with a ) 10.653 (1) Å, b ) 10.425 (1) Å, c ) 11.448
performed with the CNS software suite (version 1.0),19b
converged at R ) 0.175 and Rfree ) 0.206 with 225 water
molecules.
(1) Å, â ) 108.897 (2)°, V ) 1202.8 (2) Å3 and Z ) 2 {dcalcd
)
1.504 g cm-3; µa (Mo KR) ) 0.281 mm-1}. A full hemisphere of
diffracted intensities (ω-scan width of 0.30°) was measured by
using graphite-monochromated Mo KR radiation (from
normal-focus sealed X-ray tube operated at 50 kV and 40 mA)
on Bruker Single-Crystal SMART CCD Area Detector
a
Ack n ow led gm en t. We thank Rekha Shah and
Kristina Klescz for HPLC assay development, Diane
Gauthier for NMR analyses, and Fan Zhang-Pasket for
the preparation of (2R)-6 and (2S)-6.
a
Diffraction System. Lattice constants were determined with
the Bruker SAINT software package using peak centers for
3134 reflections. A total of 7805 integrated reflection intensi-
ties having 2θ(Mo KR) < 57.47° were produced using the
Bruker program SAINT. Of these, 5101 reflections were unique
and gave Rint ) 0.036. The Bruker SHELXTL-PC software
package was used to solve the structure using “direct methods”
techniques. All stages of weighted full-matrix lest-squares
Su p p or tin g In for m a tion Ava ila ble: Ki values associated
with Table 2 and X-ray crystallographic data for the sulfate
salt of (2S)-6. This material is available free of charge via the
Internet at http://pubs.acs.org.
2
refinement were conducted using Fo data with the SHELTX-
Refer en ces
PC software package. Final agreement factors at convergence
are: R1 (unweighted, based on F) ) 0.051 for 3661 independent
reflections have 2θ(Mo KR) < 57.47° and I > 2σ(I); R1
(unweighted, based on F) ) 0.082 and wR2 (weighted, based
on F2) ) 0.120 for all 5101 independent reflections having 2θ-
(Mo KR) < 57.47°.
(1) (a) Caughey, G. H. Mast Cell Proteases in Immunology and
Biology; Marcel Dekker: New York, 1995. (b) Caughey, G. H.
Of mites and men: trypsin-like proteases in the lungs. Am. J .
Respir. Cell Mol. Biol. 1997, 16, 621-628. (c) Schwartz, L. B.
Mast cell tryptase: properties and roles in human allergic
responses. Clin. Allergy Immunol. 1995, 6, 9-23.
(2) (a) Schwartz, L. B. Tryptase:
a mast cell serine protease.
The structural model incorporated anisotropic thermal
parameters for all nonhydrogen atoms and isotropic thermal
parameters for all hydrogen atoms. Hydrogen atoms bonded
to oxygen and nitrogen were located from a difference Fourier
synthesis and included in the structural model as independent
isotropic atoms. The methyl group was refined as a rigid rotor
(using idealized sp3-hybridized geometry and a C-H bond
length of 0.96 Å) with three rotational parameters in least-
squares cycles. The final refined values of these three rota-
tional parameters gave C-C-H angles that ranged from 103°
to 119°. The remaining hydrogen atoms were included in the
structural model as fixed atoms (using idealized sp2- or sp3-
hybridized geometry and C-H bond lengths of 0.95-1.00 Å)
“riding” on their respective carbons. The isotropic thermal
parameters for hydrogen atoms were fixed at values 1.2
(nonmethyl) or 1.5 (methyl) times the equivalent isotropic
thermal parameters of the carbon atoms to which they are
covalently bonded.
X-r a y Cr ysta llogr a p h y of th e (2S)-6‚Tr yp sin Com p lex.
Bovine pancreatic trypsin (Worthington Biochemical) was
dissolved in 50 mM ammonium acetate buffer (pH 5.6)
containing 0.1 w/v% of CaCl2. A solution of (2S)-6 in DMSO
was added in a 1.5-fold molar excess. Crystals of (2S)-6‚trypsin
were grown at 23 °C by using the hanging-drop, vapor-
diffusion technique (drops of 2 mL of protein solution mixed
with 2 mL of reservoir solution were equilibrated against a
reservoir solution of 0.1 M Tris HCl buffer at pH 7.75
containing 1.2 M MgSO4).10 The crystals belong to the trigonal
space group P3121, with the unit cell parameters: a ) b )
54.67 Å, c ) 107.87 Å; R ) â ) 90°, γ ) 120°. Diffraction data
were collected at -170 °C using 25% glycerol as a cryopro-
tectant on an R-AXIS IV imaging plate (Rmerge ) 0.08; I/σ )
11.5) and processed with DENZO to a resolution of 1.9 Å.19a
The completeness of the data at this resolution is 98.6%.
The structure is isomorphous with a previously reported
bovine â-trypsin structure (PDB entry 3PTN).19c This model,
stripped of solvent molecules and calcium ion and with uniform
thermal and occupancy parameters, was used for the initial
refinement using XPLOR.19d Rigid body minimization, followed
by simulated annealing with slow cooling,19e and conjugate
gradient positional refinement were carried out. The molecular
model of the inhibitor (2S)-6 was constructed and built into
electron density by using CHEMNOTE of the QUANTA
program suite. The ideal parameters for the inhibitor were
derived from small-molecule structure information from the
Cambridge Crystal Data Base.19f Manual corrections of the
model complex were done during the refinement with the
QUANTA graphics program. The refinement data were gradu-
ally extended to 1.9 Å resolution during the refinement.
Solvent molecules were inserted at reasonable positions where
the difference electron density exceeded 2.5 σ. Finally, the
individual atomic B-values were refined. The final refinement,
Methods Enzymol. 1994, 244, 88-100. (b) Schwartz, L. B.; Lewis,
R. A.; Austen, K. F. Tryptase from human pulmonary mast cells.
Purification and characterization. J . Biol. Chem. 1981, 256,
11939-11943.
(3) Pereira, P. J . B.; Bergner, A.; Macedo-Ribeiro, S.; Huber, R.;
Matschiner, G.; Fritz, H.; Sommerhoff, C. P.; Bode, W. Human
â-tryptase is a ring-like tetramer with active sites facing a
central pore. Nature (London) 1998, 392, 306-311.
(4) (a) Molinari, J . F.; Scuri, M.; Moore, W. R.; Clark, J .; Tanaka,
R.; Abraham, W. M. Inhaled tryptase causes bronchoconstriction
in sheep via histamine release. Am. J . Respir. Crit. Care Med.
1996, 154, 649-653. (b) Clark, J .; Abraham, W. M.; Fishman,
C. E.; Forteza, R.; Ahmed, A.; Cortes, A.; Warne, R. L.; Moore,
W. R.; Tanaka, R. D. Tryptase inhibitors block allergen-induced
airway and inflammatory responses in allergic sheep. Am. J .
Respir. Crit. Care Med. 1995, 152, 2076-2083.
(5) (a) Clark, J . M.; Moore, W. R.; Tanaka, R. D. Tryptase inhibi-
tors: a new class of antiinflammatory drugs. Drugs Future 1996,
21, 811-816. (Update: Anon. APC-366. Drugs Future 1998, 23,
903.) (b) Rice, K. D.; Tanaka, R. D.; Katz, B. A.; Numerof, R. P.;
Moore, W. R. Inhibitors of tryptase for the treatment of mast
cell-mediated diseases. Curr. Pharm. Design 1998, 4, 381-396.
(c) Burgess, L. E. Mast cell tryptase as a target for drug design.
Drug News Perspect. 2000, 13, 147-156.
(6) (a) Caughey, G. H.; Raymond, W. W.; Bacci, E.; Lombardy, R.
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(22) See the paragraph at the end of this paper regarding Supporting
Information.
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