Monocharged inhibitors of mast cell tryptase derived from potent and selective dibasic inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00444-9

Truncation of potent and selective dibasic inhibitors afforded monocharged inhibitors of human mast-cell tryptase. Using two classes of analogues as lead structures, several monocharged derivatives were identified with K_i values ranging from 0.

Full text of DOI:10.1016/S0960-894X(01)00444-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2325–2330
Monocharged Inhibitors of Mast Cell Tryptase Derived from
Potent and Selective Dibasic Inhibitors
Jeffrey M. Dener,* Vivian R. Wang, Kenneth D. Rice,y Anthony R. Gangloff,
Elaine Y.-L. Kuo, WilliamS. Newcomb, Daun Putnamand Martin Wong
Departments of Medicinal Chemistry, Biochemistry, and Enzymology, Axys Pharmaceuticals, Inc. 180 Kimball Way,
South San Francisco, CA 94080, USA
Received 29 April 2001; accepted 14 June 2001
Abstract—Truncation of potent and selective dibasic inhibitors afforded monocharged inhibitors of human mast-cell tryptase.
Using two classes of analogues as lead structures, several monocharged derivatives were identified with Ki values ranging from
0.084 to 0.21 mM against the enzyme. # 2001 Elsevier Science Ltd. All rights reserved.
Human mast-cell tryptase is a trypsin-like serine pro-
tease that comprises up to 23% of the protein in the
mast cell.1 The enzyme is believed to play a central role
in mediating both allergic and inflammatory
responses2À4 and has recently been shown in the clinic to
be an important therapeutic target for treatment of
asthma.5 Structurally, the active formof human b-tryp-
tase is a glycosylated tetramer, requiring heparin to sta-
bilize this tetrameric structure.6,7 Several in vitro and in
vivo mechanisms of tryptase inactivation have appeared
in the literature. Both neutrophil myeloperoxidase
(MPO)8 and lactoferrin9 have been shown to inactivate
the tetramer by interfering with the heparin-mediated
stabilization. In addition, secretory leukocyte protease
inhibitor (SLPI) may regulate tryptase activity in vivo
through inhibition of the tryptase-dependent degradation
of vasoactive intestinal peptide (VIP).10
Recently, examples of synthetic inhibitors of this unu-
sual tetrameric protease have appeared in the litera-
ture,11À17 including APC-366, which was the first
tryptase inhibitor to enter clinical trials for asthma.5,12
Furthermore, a series of potent and selective symme-
trical and unsymmetrical dibasic inhibitors of human
b-tryptase has been reported.16,17 As part of a program
to identify tryptase inhibitors with optimum therapeutic
utility, including oral bioavailability, we describe our
efforts to develop monocharged analogues based on
dibasic inhibitors 1 and 3, which are potent and selective
for tryptase (Scheme 1).18À20 Our initial synthetic efforts
Scheme 1. General approach to monocharged tryptase inhibitors.
*Corresponding author at present address: ChemRx Advanced Technologies, 385 Oyster Point Blvd., Suite 1, South San Francisco, CA 94080,
USA. Tel.: +1-650-829-1030; fax: +1-650-829-1123; e-mail: dener@chemrx.com
yPresent address: Exeliscis Pharmaceuticals, Inc., 170 Harbor Way, PO Box 511, South San Francisco, CA 94080, USA.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00444-9

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J. M. Dener et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2325–2330
Scheme 2. Synthesis of 4-(guanidinophenyl)urea derivatives (2). Reagents and conditions: (a) MeOH, CH2Cl2, DIPEA; (b) TFA; (c) R1–N¼C¼O
(8), DIPEA, DMF; (d) R1R2NCOCl (9), DIPEA, DMF.
Scheme 3. Preparation of 4-(guanidinophenyl)acetylpiperazine derivatives. Reagents and conditions: (a) SOCl2, reflux; (b) tert-butyl 1-piper-
azinecarboxylate, DIPEA, THF, À78 ꢀC; (c) 10% Pd/C, H2, MeOH; (d) NH2CN, 4 N HCl in 1,4-dioxane, 60 ꢀC; (e) TFA; (f)
Ph2CH(CH2)2N¼C¼O (12), DIPEA, DMF; (g) NaH, t-butyl bromoacetate, 0 ꢀC; (h) Ph(CH2)2NH2, EDC, HOBt, DMF.
began with replacement of the central core and the sec-
ond charged group of both 1 and 3 with small molecular
fragments to give inhibitors of general structure 2, con-
taining the piperazine scaffold, and 4, which possesses a
4-(aminomethyl)piperidine moiety.
ality, employing conditions similar to those described for
13.
Compounds described in Table 3 were prepared from
amidines 17 and 18,20 which were then acylated with
either 12 or 4-nitrophenylcarbamate 19,23 to give the
urea derivatives 20 and 21, as shown in Scheme 4.
Scheme 2 outlines the chemistry employed to prepare
the piperazine-containing inhibitors of general structure
2, varying substitution on the urea nitrogen, whose
activity is shown in Table 1. Commercially available
4-aminobenzylamine was converted to 4-guanidino-
benzylamine dihydrochloride 5 according to the litera-
Scheme 5 outlines the preparation of homologues of the
4-(aminomethyl)piperidine unit of 4, reported in Table 4.
4-Amino-1-benzylpiperidine (22) was protected as the
phthalimide under standard conditions and the benzyl
group was removed by hydrogenolysis. The 3,3-diphenyl-
propyl-1-aminocarbonyl moiety was introduced with
either 12 or 19 as described previously, then the phthal-
imide group removed with hydrazine in ethanol.
Finally, acylation of 23 with 4-cyanobenzoyl chloride
and conversion of the nitrile to the amidine moiety
afforded inhibitor 25. The two-carbon homologue of 25,
inhibitor 29, was prepared from4-(2-aminoethyl)pyr-
ture procedure.21 Formation of the urea
7 was
accomplished by reaction of 5 with carbamoyl chloride
622 under basic conditions, followed by BOC deprotec-
tion. (4-Guanidinophenyl)piperazine 7 was reacted with
either isocyanates (8) to give trisubstituted ureas of 2
(R2=H), or carbamoyl chlorides (9) to afford tetra-
substituted urea derivatives of 2, where R2 is alkyl.
Scheme 3 outlines representative chemical routes used
to prepare the inhibitors shown in Table 2, which
contain the 4-(guanidinophenyl)acetylpiperazine motif.
4-Nitrophenylacetic acid (10) was converted to the acid
chloride and reacted with tert-butylpiperazine 1-car-
boxylate to afford piperazine amide 11. This material
was then converted to 13 by reduction of the nitro
functionality, followed by guanidine formation, BOC-
deprotection and urea formation with 3,3-diphenyl-
propylisocyanate (12). Substituted derivative 16 was
prepared from 11 by alkylation with t-butyl bromoacetate
using sodiumhydride as base to give amino acid 14 after
treatment with TFA. The monosubstituted piperazine
14 was acylated with isocyanate 12 then the carboxylic
acid functionality was converted to N-phenethyl amide
15 under standard conditions. The synthesis of 16 was
completed by introduction of the guanidine function-
Scheme 4. Representative syntheses of tryptase inhibitors containing
the 4-(aminomethyl)piperidine scaffold. Reagents and conditions: (a)
12 or Ph2CH(CH2)2NHCO2C6H4NO2 (19), DIPEA, DMF.

J. M. Dener et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2325–2330
2327
Scheme 6. Preparation of the 3-aminopiperidine analogue of inhibitor
20. Reagents and conditions: (a) (Boc)2O, MeOH; (b) Lawesson’s
reagent, THF, reflux; (c) NiCl2ꢂ 6H2O, NaBH4, THF/MeOH, 0 ꢀC; (d)
4-cyanobenzoyl chloride, DIPEA, CH2Cl2; (e) H2NOHꢂ HCl, DIPEA,
EtOH, reflux; (f) H2, 10% Pd–C, EtOAc/EtOH (1:1); (g) 4 N HCl in
1,4-dioxane; (h) 12, DIPEA, DMF.
Scheme 5. Preparation of inhibitors containing homologues of 4-
(aminomethyl)piperidine. Reagents and conditions: (a) N-Car-
boethoxyphthalimide, Na2CO3, CH2Cl2; (b) (Boc)2O, H2, Pd–C,
EtOH; (c) TFA, CH2Cl2; (d) 12, DIPEA, CH2Cl2; (e) H2NNH2,
EtOH, reflux; (f) 4-cyanobenzoyl chloride, DIPEA, CH2Cl2; (g)
(Boc)2O, MeOH; (h) H2, PtO2, MeOH/AcOH (7.5:1); then HCl; (i) 12
or 19, DIPEA, CH2Cl2; (j) H2NOHꢂ HCl, DIPEA, EtOH, reflux; (k)
H2, 10% Pd–C, EtOAc/EtOH (1:1).
activity. Restricting rotation of the two phenyl groups
of 2k, as found in fluorenyl derivative 2n reduced activ-
ity 13-fold. In general, tetrasubstituted urea derivatives
2g and 2h were slightly less active (1.7–2ꢁ) than their
trisubstituted analogues (e.g., 2b and 2d).
idine (26), first by protection of the free amine followed
by reduction of the pyridine ring. Acylation of the
resulting secondary amine with isocyanate 12 afforded
the urea derivative, which was deprotected to give (2-
aminoethyl)urea 27. Acylation of 27 with 4-cyano-
benzoyl chloride gave 28, which was converted to the
amidine 29 under identical conditions employed for 24.
The data presented in Table 1 shows the 3,3-diphenyl-
propyl motif to be the optimal binding fragment
attached to the urea functionality found in 2. Based on
these results, a series of derivatives containing the [(3,3-
diphenylpropylamino)carbonyl]piperazine motif was
synthesized, varying the 4-guanidinophenyl charged
group (arginine mimetic) found in 2 and compounds
2a–n. These variations included amide and a-substituted
carboxamide analogues of the guanidinophenyl moiety
Lastly, Scheme 6 shows the preparation of the 3-isomer
of 20. Nipecotamide (30) was converted to the Boc-
protected thioamide 31 first by Boc-protection followed
by reaction of the amide with Lawesson’s reagent.24
Reduction of the thioamide to the amine was performed
using nickel boride25 and the crude amine was acylated
with 4-cyanobenzoyl chloride in the presence of
DIPEA to give 4-cyanobenzamide 32. Conversion of 32
to the inhibitor 34 was accomplished using reaction
conditions developed for benzamidines 25 and 29
(Scheme 5).
Table 1. Tryptase inhibition data for (4-guanidinophenyl)urea deri-
vatives 2
Compd
R1
R2
Tryptase Selectivityb
(Ki, mM)a
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
CH2CH3
CH2Ph
(CH2)2Ph
(CH2)3Ph
(CH2)4Ph
CH2Ph
CH2Ph
(CH2)3Ph
H
H
H
H
H
CH3
CH2Ph
CH2Ph
H
H
H
370
16
42
0.38
12
3.6
21
10
0.80
2.5
4.2
170
14
360
>150
170
48
Our investigation into monocharged derivatives of 1
began by synthesis of urea derivatives of general struc-
ture 2, which were prepared according to the chemical
routes described above.
7.3
11
80
29
12
1.3
7.4
0.87
6.9
0.89
6.9
Apparent inhibition constants (Ki values)18 and selec-
tivity over trypsin26 for these inhibitors, using the gua-
nidine as the charged group, are reported in Table 1.
The data given in Table 1 exhibit some important
trends. First, urea derivatives containing hydrophobic
residues based on the gem-diphenyl motif (compounds
2k and 2m) were the most potent in the series, posses-
sing Ki values around 0.8–0.9 mM. In addition, the
2-naphthylmethyl derivative 2i also exhibited modest
2-Naphthymethyl
2-(1-Naphthyl)ethyl
(CH2)2CHPh2
(CH2)2CPh3
H
H
H
(4,4-Diphenyl)-1-cyclohexyl
2-(9-Fluorenyl)ethyl
aData reported is for a single determination. See ref 18 for assay
protocols.
bRatio of Ki(trypsin)/Ki(tryptase).

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J. M. Dener et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2325–2330
Table 2. Tryptase inhibition data for various charged group deriva-
tives of 2k
The most potent compounds included benzamidine 35
(0.40 mM), guanidinophenylsuccinic diamide 16 (0.21 mM)
and carboxymethyl derivative 36 (0.16 mM). Further-
more, the latter two compounds possessed excellent
selectivity over trypsin. However, kinetic analysis of 36
suggested that this compound was a substrate and not
an inhibitor of tryptase.
Compd
R
Tryptase (Ki, mM)
Selectivity
With the optimal urea substituent identified, we then
prepared charged group variants using the 4-(amino-
methyl)piperidine unit found in dibasic inhibitor 3.
These derivatives are shown in Table 3.
13
7.0
>140
The inhibition data for monocharged inhibitors derived
from 3 indicates that arylamidines are preferred over
arylguanidines as the arginine mimetic, with para-sub-
stitution on the benzamide being optimal. This pref-
erence for the benzamidine moiety is more dramatic in
the (aminomethyl)piperidine series by a factor of 110, as
seen by comparison of benzamidine 20 to guanidine 37.
In contrast, benzamidine 35 was only twice as potent as
guanidine 2k in the piperazine series (Tables 1 and 2).
Interestingly 4-(amidinobenzene)sulfonamide analogue
21 was considerably less potent (ca. 100-fold) than the
corresponding 4-amidinobenzamide 20.
16
35
0.21
0.40
>4800
200
36
0.16
>6300
of 2k, as well as the corresponding benzamidine ana-
logue of 2k. The structure–activity relationships for
these compounds are shown in Table 2.
Table 4 shows some additional variations in the charged
moiety (arginine mimetic) and (4-aminomethyl)piper-
idine motif of 20. These results confirmthat the 4-(ami-
nomethyl)piperidine is required for optimum activity.
Simple N-methylation of the benzamide nitrogen of 20
(compound 41) reduced activity almost 600-fold. Fur-
thermore, variation of the chain length of the amino-
methyl fragment, or incorporation of the 3-
Generally only the 4-isomer of phenylguanidine pos-
sessed significant activity against tryptase (Ki values less
than 1 mM). In addition, several alkyl guanidine deriva-
tives of 2k were also prepared and were less potent (Ki
values=14–29 mM; structures not shown) than the 4-
phenylguanidine and benzamidine derivatives reported
in Table 3.
Table 4. Arginine mimetic and scaffold analogues of benzamidine 20
Table 3. Tryptase inhibition data for benzamidine and phenylguani-
dine derivatives of 4 containing the 3,3-diphenylpropylurea moiety
from 2k
Compound
R
Tryptase (Ki, mM) Selectivity
2.6 44
25
Compd
20
R
X
Tryptase
(Ki, mM)
Selectivity
29
34
39
40
41
11
6.8
CO
SO2
CO
CO
0.084
9.7
950
3.5
13
0.7
21
0.30
490
37
9.8
>100
6
0.34
270
16
38
24
50

J. M. Dener et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2325–2330
2329
aminomethyl isomer of piperidine was not tolerated.
These variations, represented by compounds 25, 29, and
34, afforded inhibitors with inhibition constants in the
low micromolar range. The best compounds in this
series, the ‘retro-inverso’ amide 39 and the 4-amino-
methylbenzamide derivative 40, were 3.5–4.0 times less
active than 20.
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tors 1 and 3 can be used as lead structures to design
monocharged inhibitors of tryptase. From these deriva-
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the identification of the 3,3-diphenylpropyl fragment as
a potent binding motif. Optimization of the charged
binding fragment afforded compounds with Ki values
ranging from0.084 to 0.21 mM, including guanidino-
phenylsuccinamide 16 and benzamidine 20. Being the
most potent mast cell tryptase inhibitor in this series,
compound 20 was selected for pharmacokinetic analysis
employing a rat model. Unfortunately 20 exhibited low
bioavailability (<1%) after id administration at 6 mg/kg.
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17. Ono, S.; Kuwahara, S.; Takeuchi, M; Sakashita, H.; Naito,
Y.; Kondo, T. Bioorg. Med. Chem. Lett. 1999, 9, 3285 In addi-
tion, inhibition constants for several monocharged derivatives
of the dimeric inhibitors are described in this publication.
18. Rice, K. D.; Gangloff, A. R.; Kuo, E. Y.-L.; Dener, J. M.;
Wang, V. R.; Lum, R.; Newcomb, W. S.; Havel, C.; Putnam,
D.; Cregar, L.; Wong, M.; Warne, R. L. Bioorg. Med. Chem.
Lett. 2000, 10, 2357 This reference should be consulted for
experimental details concerning measurement of apparent
inhibition constants (Ki values) reported in Tables 1–4. Data
reported in this work is for a single determination. All
enzymes employed were human form and obtained commer-
cially with the exception of mast cell tryptase, which was
obtained according to ref 11.
Acknowledgements
The authors would like to thank Mark Dreyer and Lil-
ing Fang for chemical analyses, and Mike Venuti and
Heinz Gschwend for their guidance with this project.
Bayer AG is gratefully acknowledged for financial sup-
port of the tryptase research programat Axys Pharma-
ceuticals, Inc. Lastly we would like to thank Elizabeth
Daniels for proofreading the manuscript.
19. Rice, K. D.; Wang, V. R.; Gangloff, A. R.; Kuo, E. Y.-L.;
Dener, J. M.; Newcomb, W. S.; Young, W. B.; Putnam, D.;
Cregar, L.; Wong, M.; Simpson, P. J. Bioorg. Med. Chem.
Lett. 2000, 10, 2361.
20. Dener, J. M.; Rice, K. D.; Newcomb, W. S.; Wang, V. R.;
Young, W. B.; Gangloff, A. R.; Kuo, E. Y.-L.; Cregar, L.; Put-
nam, D.; Wong, M. Bioorg. Med. Chem. Lett. 2001, 11, 1629.
21. Safir, S. R.; Kushner, D.; Brancone, L. M.; Subbarow, Y.
J. Org. Chem. 1948, 13, 924.
22. Prepared from commercially available tert-butyl-1-piper-
azinecarboxylate (Aldrich) and triphosgene.
23. Prepared according to: Izdebski, J.; Pawlak, D. Synthesis
1989, 423.
References and Notes
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2. Molinari, J. F.; Scuri, M.; Moore, W. R. Am. J. Respir.
Crit. Care Med. 1996, 154, 649.
3. Molinari, J. F.; Moore, W. R.; Clark, J. J. Appl. Phys.
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4. Elrod, K. C.; Numerof, R. P. Emerging Ther. Targets 1999,
3, 203.

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J. M. Dener et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2325–2330
26. In general, most monocharged inhibitors were the least
selective for trypsin relative to the other serine proteases
examined (thrombin and plasmin). Furthermore, similarities
in the active sites of the tryptase monomer and trypsin support
the lack of observed selectivity; see: Sommerhoff, C. P.; Bode,
W.; Matschiner, G.; Bergner, A.; Fritz, H. Biochim. Biophys.
Acta 2000, 1477, 75.