Dibasic inhibitors of human mast cell tryptase. Part 3: Identification of a series of potent and selective inhibitors containing the benzamidine functionality

Article abstract of DOI:10.1016/S0960-894X(01)00254-2

A survey of charged groups and linkers for a series of symmetrical and unsymmetrical dibasic inhibitors is described, leading to several classes of potent and selective inhibitors. In particular, the benzamidine functionality was identified as the most potent charged group investigated.

Full text of DOI:10.1016/S0960-894X(01)00254-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1629–1633
Dibasic Inhibitors of Human Mast Cell Tryptase. Part 3:
Identification of a Series of Potent and Selective Inhibitors
Containingthe Benzamidine Functionality
Jeffrey M. Dener,* Kenneth D. Rice,y William S. Newcomb, Vivian R. Wang,
Wendy B. Young, Anthony R. Gangloff, Elaine Y.-L. Kuo, Lynne Cregar,
Daun Putnam and Martin Wong
Departments of Medicinal Chemistry, Biochemistry, and Enzymology, Axys Pharmaceuticals, Inc.,
180 Kimball Way, South San Francisco, CA 94080, USA
Received 8 February 2001; accepted 27 March 2001
Abstract—A survey of charged groups and linkers for a series of symmetrical and unsymmetrical dibasic inhibitors is described,
leading to several classes of potent and selective inhibitors. In particular, the benzamidine functionality was identified as the most
potent charged group investigated. # 2001 Elsevier Science Ltd. All rights reserved.
The development of protease inhibitors as potential
human therapeutic agents is an active area of research
in the pharmaceutical industry.1 In particular, discovery
of inhibitors of serine proteases such as thrombin,2,3 fac-
tor Xa,3 and elastase4 has been vigorously investigated in
recent years. Another serine protease, mast cell tryptase,
has been implicated in certain allergic and inflammatory
disease states.5ꢀ8 Furthermore, an inhibitor of tryptase,
APC-366, has been validated in a clinical setting for the
modulation of the asthmatic response.8 In previous
reports, we described a series of dibasic inhibitors of
tryptase, leading to the identification of APC-1390 (1), a
potent and selective tryptase inhibitor (Ki=70 pM).9
Furthermore, variations in the cyclooctane core, charged
functionality (P1 recognition element), and unsymme-
trical, dibasic analogues of 1 were also reported.9,10
Syntheses of several representative side-chain fragments
are shown in Scheme 1. These were incorporated into
the target inhibitors according to conditions described
in Scheme 2. Typically the symmetrical inhibitors were
prepared by reaction of the P1–P2 fragments (200
mol%) under basic conditions with the bis-chloro-
formate of either 1,4-bis(hydroxymethyl)benzene or cis-
(1,4-dihydroxy)cyclooctane.9 Unsymmetrical inhibitor
23 was prepared by reaction of excess chloroformate
2110 with (N-BOC-piperidino)urea derivative 12 to give
the monochloroformate 22, which was then condensed
with the benzamidine moiety 16. Deprotection of the
BOC group afforded the desired unsymmetrical deriva-
tive 23. Alternatively, monochloroformate 2410 could be
converted to 25 after introduction of the benzamidine
moiety 7. Subsequent BOC deprotection of 25 and
A
representative potent and selective unsymmetrical deri-
vative of 1 is the primary amine 2 (Ki=500 pM). Herein
we would like to present additional SARs directed
toward the identification of potent and selective tryptase
inhibitors related to APC-1390. In particular, our
efforts focused on evaluation of different charged and
linker functionalities to accomplish these objectives.
*Corresponding author at present address: ChemRx Advanced Tech-
nologies, 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: Exelixis Pharmaceuticals, Inc., 280 E. Grand Ave-
nue, South San Francisco, CA 94080, USA.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00254-2

1630
J. M. Dener et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1629–1633
acylation of the distal piperazine with 4-(N-t-butylox-
ycarbonyl-4-piperidinyl)butyric acid (20) and BOC-
deprotection gave unsymmetrical inhibitor 26. Other
derivatives were prepared in analogous fashion
employing similar amine-, amidine-, and guanidine-
containing fragments. Finally, all inhibitors were iso-
lated as their HCl salts after preparative reverse-phase
HPLC.
aminobutane unit of 29 with the more rigid 4-
aminomethylpiperidine linker in inhibitor 30 resulted in
a 5-fold increase in potency. However, incubation of
primary amine 30 with plasma amine oxidase (PAO)
showed that rapid metabolism took place (Vmax
/
Km=1870), indicating that the primary amine of the 4-
aminomethylpiperidine unit was unsuitable as a charged
group for in vivo applications.
Tryptase inhibition data for a series of similar symme-
trical inhibitors containing various charged and linking
groups based on either a 1,4-benzenedimethanol or cis-
(1,4-cyclooctane)diol central scaffold is reported in
Table 1. Ki values for mast cell tryptase, thrombin,
plasmin, and trypsin were obtained according to meth-
ods described previously.9 Replacement of the 1,4-di-
Inhibitors 31 and 32, containing the more rigid P1–P2
units (R fragment), are clearly potent and selective for
tryptase (Table 1). Furthermore, a 60-fold preference
for the benzamidine 31 over the primary amine 30 was
noted. The benzamidine 32 possessed inhibitory potency
comparable to inhibitor 31, indicating that this charged
group/linker combination is critical for optimum
Scheme 1. Preparation of key charged fragment-linker and distal element-linker moieties. Reagents and conditions: (a) PhCHO, toluene, reflux; (b)
CBz-Cl, DIPEA; (c) KHSO4, H2O, then NaOH; (d) 4-cyanobenzoyl chloride or 4-cyanobenzenesulfonyl chloride, DIPEA, CH2Cl2; (e)
H2NOHꢁ HCl, DIPEA, EtOH, reflux; (f) H2, 10% Pd–C, EtOH–AcOH (1:1), then HCl; (g) H2, PtO2, AcOH–H2O; (h) NaOH, (BOC)2O, then
KHSO4; (i) DPPA, Et3N, benzene, 90 ꢂC, then N-CBz-piperazineꢁ HCl, Et3N; (j) H2, 10% Pd–C, EtOH; (k) NaN3, aqueous acetone, reflux; (l) Ph3P,
5% H2O in THF, N-CBz-benzotriazole; (m) H2S, Et3N, pyridine; (n) CH3I, acetone, reflux; (o) NH4OAc, MeOH, reflux, then NaOH; (p) 30% HBr
in AcOH; (q) 4-(tert-butyloxycarbonyl)piperazine-1-carbonyl chloride, DIPEA, DMF; (r) 4 N HCl in 1,4-dioxane; (s) triphenylphosphine, toluene;
(t) NaOH, H2O; (u) 4-N-BOC-piperidinone, toluene, 95–105 ꢂC; (v) H2, 10% Pd–C, EtOH; (w) NaOH, THF–H2O–MeOH (3:1:1), then KHSO4.
Scheme 2. Representative syntheses of unsymmetrical tryptase inhibitors. Reagents and conditions: (a) 12, DIPEA, CH2Cl2; (b) 16, DIPEA, DMF;
(c) HCl or TFA; (d) 7 (100 mol%), DIPEA, DMF, then NaOH; (e) 20, EDC, HOBt, DMF.

J. M. Dener et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1629–1633
1631
Table 1. Inhibition constants of tryptase and other serine proteases for symmetrical dibasic inhibitors derived from scaffold A or B
Compound
Scaffold
R
Ki (mM)
Tryptase
0.155
Thrombin
386
Plasmin
Trypsin
466
27
A
>1000
28
29
30
31
32
33
A
A
A
A
B
0.110
423
>1000
>1000
42.2
>1000
>1000
>1000
>1000
>1000
515
0.112
0.024
0.0004
0.00085
0.0087
0.006
4.4
20.8
62.2
4.67
25
B
59.5
134
activity. In the cyclooctane series, preference for the
amidinobenzamide/aminomethylpiperidinyl unit 7 over
amidinobenzyl/piperazinyl fragment 16 was observed,
as indicated by the 10-fold potency increase for 32
relative to benzamidine 33. Lastly, the alkyl amines and
guanidines represented in Table 1 were much less active
against tryptase than the corresponding benzamidine
derivatives. Next, additional SAR studies of unsymme-
trical inhibitors similar to compound 2 were under-
taken, employing the benzamidine motif incorporated
in inhibitors 31 and 32.
charged group in the asymmetric series (cf. 23 to 34,
Table 2), whereas fragment 7 provided the more potent
symmetric inhibitor 32 (Table 1). Table 2 also shows
further variations in the charged group and linker frag-
ments. Benzamidines 23 and 35 were the most potent
and selective tryptase inhibitors in this series. Further-
more 23 was about 10 times more potent than the par-
ent symmetrical inhibitor 33. Other variations reported
in this table include the retro-inverso amide analogue 37
and sulfonamide 36, both of which were less potent than
either 23 or 35. Lastly, the carbamate derivative of
amidine 23 (39) was prepared as a potential prodrug11,12
and possessed modest activity against tryptase. Studies
addressing the viability of 39 as a prodrug were not
pursued since this material, isolated as the bis-HCl salt,
proved to be unstable in aqueous solution. We believe
that hydrolysis of 39 to the corresponding acyl methyl-
carbamate 40 was taking place (Scheme 3), based on
LC–MS data and control experiments with the parent
N-(methoxycarbonyl)benzamidine.13,14
The piperidine moiety, the most potent terminal nitro-
gen base identified in our previous report,10 was used
for these studies since other charged groups incorpo-
rated at the distal site followed trends observed for
compounds reported in the phenylguanidine series.
Tryptase inhibition data for these benzamidine deriva-
tives are shown in Table 2. Replacement of one benz-
amidine moiety of symmetrical inhibitor 32 with
(piperidino)amide and (piperidino)urea fragments
afforded compounds 26 and 34 that were 19-fold less
active than the parent inhibitor 32. Furthermore, com-
pounds 26 and 34 demonstrate that the nature of the
linkage (amide or urea) at the distal site does not sig-
nificantly contribute to binding of the inhibitor to the
enzyme. In addition, fragment 16 was the preferred
In summary, we have identified potent and selective
dibasic inhibitors of mast cell tryptase, including 23, 31,
32, and 35, each of which features a benzamidine
charged group. Future reports will outline additional
modifications based on these inhibitors, including
monocharged derivatives.

1632
J. M. Dener et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1629–1633
Table 2. Inhibition constants of tryptase and other serine proteases for P1–P2 variations of unsymmetrical dibasic inhibitors
Compound
R
X
Ki (mM)
Tryptase
0.00091
Thrombin
111
Plasmin
287
Trypsin
47.2
23
NH
26
34
35
36
37
38
39
CH2
NH
CH2
NH
NH
NH
NH
0.0164
0.016
31.9
41.2
102
320
279
37.4
38.8
52.1
16.8
21.1
63.1
554
0.00112
0.028
310
>1000
52.9
94.4
561
>1000
293
0.0089
0.0067
0.0406
>1000
>1000
4. Edwards, P. D.; Berstein, P. R. Med. Res. Rev. 1994, 14,
127.
5. Molinari, J. F.; Scuri, M.; Moore, W. R. Am. J. Respir.
Crit. Care Med. 1996, 154, 649.
6. Molinari, J. F.; Moore, W. R.; Clark, J. J. Appl. Phys.
1995, 79, 1966.
7. Elrod, K. C.; Numerof, R. P. Emerging Ther. Targets 1999,
3, 203.
Scheme 3. Hydrolytic instability of methoxycarbonylbenzamidine
prodrug 39.
8. Krishna, M. T.; Chauhan, A. J.; Little, L.; Sampson, K.;
Mannt, T. G. K.; Hawksworth, R.; Djukanovic, R.; Lee, T. H.;
Holgate, S. T. Am. J. Respir. Crit. Care Med. 1998, 157, A456.
9. Part 1: 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.
10. Part 2: Rice, K. D.; Wang, V. R.; Gangloff, A. R.; Kuo,
E. Y.-L.; Dener, J. M.; Newcomb, W. S.; Young, W. B.; Put-
nam, D.; Cregar, L.; Wong, M.; Simpson, P. J. Bioorg. Med.
Chem. Lett. 2000, 10, 2361.
Acknowledgements
The authors would like to thank Mark Dreyer and Liling
Fang for analytical support, and Mike Venuti and Heinz
Gschwend for their guidance with this project. Bayer AG
is also acknowledged for financial support of the tryp-
tase research program at Axys Pharmaceuticals, Inc.
11. Weller, T.; Alig, L.; Beresini, M.; Blackburn, B.; Bunting,
S.; Hadvary, P.; Muller, M. H.; Knopp, D.; Levet-Trafit, B.;
Lipari, M. T.; Modi, N. B.; Muller, M.; Refino, C. J.; Schmitt,
M.; Schonholzer, P.; Weiss, S.; Steiner, B. J. Med. Chem.
1996, 39, 3139.
12. Himmelsbach, F.; Austel, V.; Guth, B.; Linz, G.; Muller,
T. H.; Pieper, H.; Seewaldt-Becker, E.; Weisenberger, H. Eur.
J. Med. Chem. 1995, 30, S243.
References and Notes
1. Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem.
2000, 43, 305.
2. Rewinkel, J. B. M.; Adang, A. E. P. Curr. Pharm. Des.
1999, 5, 1043.
3. Fevig, J. M.; Wexler, R. M. In Annual Reports in Medicinal
Chemistry; Doherty, A. M., Ed.; Academic: San Diego, 1999;
Vol. 34, pp 81–100.
13. Control experiments with N-(methoxycarbonyl)benzamidine
indicate that aqueous solutions containing this functionality at

J. M. Dener et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1629–1633
1633
low pH may lead to this hydrolysis product. Solutions of N-
carboxymethylphenylamidine in 50% aqueous acetonitrile
were stirred overnight at either pH 2.0 (with added hydro-
chloric acid) or pH 6.8 (no added acid). HPLC–MS analysis of
both solutions showed that hydrolysis to the corresponding
acyl carbamate had occurred only in the presence of acid.
14. After these studies were completed, a similar observa-
tion for an N-(ethoxycarbonyl)benzamidine prodrug was
reported: Shahrokh, Z.; Lee, E.; Olivero, A. G.; Mata-
moros, R. A.; Robarge, K. D.; Lee, A.; Weise, K. J.;
Blackburn, B. K.; Powell, M. F. Pharm. Res. 1998, 15,
434