Synthesis of potent and highly selective inhibitors of human tryptase

Article abstract of DOI:10.1016/S0960-894X(02)00689-3

The serine protease tryptase has been implicated in allergic and inflammatory diseases and associated with asthma. The synthesis and SAR of a series of N1-activated-4-carboxy azetidinones are described, resulting in identification of BMS-363131 (2) as a potent inhibitor of human tryptase (IC₅₀<1.7 nM) with high selectivity (>3000-fold) for tryptase versus related serine proteases including trypsin.

Full text of DOI:10.1016/S0960-894X(02)00689-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3235–3238
Synthesis of Potent and Highly Selective Inhibitors of
Human Tryptase
William A. Slusarchyk,* Scott A. Bolton, Karen S. Hartl, Ming-Hsing Huang,
Glenn Jacobs, Wei Meng, Martin L. Ogletree, Zulan Pi, William A. Schumacher,
Steven M. Seiler, James C. Sutton, Uwe Treuner, Robert Zahler,
Guohua Zhao and Gregory S. Bisacchi
The Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 4 January 2002; accepted 24 May 2002
Abstract—The serine protease tryptase has been implicated in allergic and inflammatory diseases and associated with asthma. The
synthesis and SAR of a series of N1-activated-4-carboxy azetidinones are described, resulting in identification of BMS-363131 (2) as
a potent inhibitor of human tryptase (IC50<1.7 nM) with high selectivity (>3000-fold) for tryptase versus related serine proteases
including trypsin.
# 2002 Elsevier Science Ltd. All rights reserved.
Tryptase is a serine-like protease that is the major pro-
tein component found in mast cells. Upon activation,
mast cells degranulate, releasing tryptase, histamine,
chymase, and other mediators of allergic and inflam-
matory conditions. Tryptase has been implicated in
allergic and inflammatory diseases as well as auto-
immune diseases and is believed to play an important
role in inflammatory, bronchoconstrictive, and remo-
deling processes associated with asthma.1ꢀ3 It is there-
fore believed that inhibition of tryptase may offer a
therapeutic benefit for the treatment of asthma. Our
preceding report4 described the identification of BMS-
262084 (1) as a potent inhibitor of tryptase (IC50=4
nM) with moderate to good selectivity against related
serine proteases but not for trypsin.
efforts to improve upon BMS-262084 by exploring the
effects of conformationally constrained guanidino groups
at C-3 and new substituents at N-1 of the azetidinone
nucleus. These investigations led to the discovery of
BMS-363131 (2) as a potent inhibitor of tryptase having
excellent selectivity versus other serine proteases includ-
ing trypsin and having improved hydrolytic stability.
In ovalbumin-sensitized guinea pig models, intra-
tracheally dosed BMS-262084 demonstrated efficacy in
preventing allergen-induced bronchoconstriction and in
protecting against inflammatory cell infiltration into the
lung. It was recognized that a significant portion of any
inhaled drug would be swallowed; consequently, good
selectivity against the gastric protease trypsin was con-
sidered highly desirable. In this Letter, we report on our
The general Scheme for preparation of compounds 9b–f
in Table 1 is outlined in Scheme 1. Alkylation of (S)-
azetidinone 45 with iodides 3 gave compounds 5, which
were converted in two steps to 6. Removal of the Boc
group and then guanylation gave the bis-Cbz protected
guanidine compounds 7. Subsequent acylation with
chlorocarbonylpiperazine 8a and hydrogenation pro-
vided (3R,4S) compounds 9b–f.
*Corresponding author. Tel.: +1-609-818-5295; fax: +1-609-818-
3331; e-mail: william.slusarchyk@bms.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00689-3

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W. A. Slusarchyk et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3235–3238
Table 1. Tryptase inhibition, selectivities and chemical stability for compounds 1 and 9
Compd
n
Tryptase
Selectivity ratio
uPA
Stability, t1/2 at 37 ꢁC (h)
IC50 (nM)
Plasmin
Thrombin
tPA
Trypsin
pH 7
pH 9
1
4
4
12
9
71
63
204
430
364
394
2625
>9170
>2845
135
843
862
>8250
9170
2845
18
5
7
>48
>48
>48
8.0
7.3
14.4
9a
9b
9ca
9da
9eb
9fb
0
1
2
2
2
4
>465
>465
>162
>465
>465
>162
>465
>465
>162
>465
>465
>162
258
364
>162
>48
>48
aMixture of diastereomers at C-6.
bSingle diastereomer at C-6, compounds 9e and 9f are the isolated diastereomers of 9d.
Scheme 1. (a) LDA, THF; (b) TBAF, THF; (c) BnBr, NaHCO3, DMF; (d) TFA, CH2Cl2; (e) 1-[C(NCbz)(NHCbz)]pyrazole, TEA, DMF or
MeSC(NCbz)(NHCbz), Hg(OAc)2, TEA, DMF; (f) TEA, DMAP, DMF; (g) Pd/C, H2, dioxane, aq 1 N HCl.
The iodo compounds 3a–c employed in Scheme 1 were
prepared from the intermediate Boc protected amino
alcohols 11, 13, and 15 (Scheme 2), which could be
obtained from the commercially available amino acid,
amino alcohol, or amino ester. Chlorocarbonyl-piper-
azines (8a–g) used in either Scheme 1 or Scheme 4 were
prepared as shown in Scheme 3. Compounds 8a–e were
prepared from Boc-piperazine (16) by acylation,
removal of the Boc group, and finally treatment with
phosgene. The synthesis of compounds 8f–g involved
the formation of the carbamoylchloride of Boc-piper-
azine (16) followed by sequential treatment with an
amine, removal of the Boc group, and treatment with
phosgene.
The synthesis of diastereomeric mixtures 18a–f and the
single diastereomer BMS-363131 (2), which are listed in
Table 2, are outlined in Scheme 4. The mixtures 18a–f
were prepared by acylation of diastereomeric mixtures
7a with chlorocarbonylpiperazines 8b–g and deprotec-
tion. BMS-363131 (2), however, was prepared from the
single diastereomer 21 by acylation with 8b and depro-
tection. Compound 21 was derived from (R)-ethyl
Scheme 3. (a) HO2C-R, EDAC, DIEA, CH2Cl2; (b) TFA, CH2Cl2; (c)
Cl2CO. CH2Cl2, TEA or NaHCO3; (d) ClOC-R, TEA, DMF; (e)
H2NR1(CH2)4Ph R1=H or Me, NaHCO3 or TEA, CH2Cl2.
Scheme 2. (a) Procedures described in ref 7; (b) I2, PPh3, imidazole,
CH2Cl2; (c) Pd/C, H2, MeOH or iPrOH; (d) Boc2O, THF; (e) LAH,
THF.

W. A. Slusarchyk et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3235–3238
3237
Scheme 4. (a) TEA, cat. DMAP, DMF, 82%; (b) Pd/C, H2, dioxane, aq 1 N HCl, (c) polyvinylpyridine resin, H2O, 92% 2 steps; (d) Boc2O , CHCl2,
2
87%; (e) LAH, THF, 87%; (f) I2, PPh3, imidazole, CH2Cl2, 77%; (g) 4, LDA, THF, ꢀ70 ! ꢀ20 ꢁC; (h) TBAF, THF; (i) BnBr, NaHCO3, DMF,
76% 3 steps; (j) TFA, CH2Cl2; (k) 1-[C(NCbz)(NHCbz)]pyrazole, TEA, DMF, 73% 2 steps. Yields are for the synthesis of BMS-363131 (2).
nipecotate (19), which along with the (S)-enantiomer
was obtained by resolution of racemic ethyl nipecotate.6
Boc protection of 19, followed by reduction and iodi-
nation gave compound 20. Alkylation of (S)-azetidi-
none 4 with 20 and sequential desilylation, benzylation,
removal of the Boc group, and guanylation provided
intermediate 21. BMS-363130 (22), which has the (S)-
configuration at C-6 in contrast to BMS-363131 (2)
which has the (R)-configuration at C-6, was prepared in
the same manner as BMS-363131, however, starting
from (S)-ethyl nipecotate in place of 19.
BMS-262084 (1), which differ in their N-1 substituent,
were equally potent against tryptase (IC50=4 nM) and
exhibited similar hydrolytic stability profiles. However,
the overall selectivity profile of 9a offered an improved
template to investigate constrained guanidine analogues.
The amidinoazetidinyl analogue 9b showed potent
activity against tryptase (IC50=12 nM) but no sig-
nificant improvement in selectivity against plasmin,
thrombin, urokinase (uPA), tissue plasminogen acti-
vator (tPA), or trypsin. The amidinopyrrolidinyl ana-
logue 9c, although potent against tryptase, similarly
showed poor selectivity against trypsin. Activity against
tryptase was substantially reduced for the unsymme-
trical amidinopiperidinyl diastereomeric mixture 9d
(diastereomeric at the piperidinylmethyl juncture) and
the single diastereomers 9e and 9f. However, 9d dis-
played greatly improved selectivity for tryptase over
trypsin (258-fold), as well as enhanced aqueous stability
(t1/2>48 h at pH 9). The diastereomers 9e and 9f, com-
prising the mixture 9d, were similarly selective.
Guanidinylpropyl compounds 9a (Table 1) and 23
(Table 2) were prepared using chlorocarbonylpiper-
azines 8a and 8b, respectively, following methodology
described in the previous Letter to prepare BMS-
262084.
The tryptase inhibition, selectivities, and chemical sta-
bility for the new conformationally constrained guani-
dino compounds are shown in Tables 1 and 2. Table 1
displays several constrained guanidino analogues of
guanidinylpropyl compound 9a. Compound 9a and
SAR in the amidinopiperidinyl series was studied fur-
ther by examining substituents on N-4 of the piperazine
Table 2. Tryptase inhibition, selectivities and chemical stability for compounds 2, 18, 22, and 23
Compd
R
Tryptase
Selectivity ratio
uPA
Stability, t1/2 at 37 ꢁC (h)
IC50 (nM)
Plasmin
Thrombin
tPA
Trypsin
pH 7
pH 9
23
<1.7
<1.7
<1.7
6
1.9
4.6
<1.7
<1.7
<1.7
>26
>16,280
>16,070
3621
8421
>7147
>92,476
>12,900
>19,410
>16,500
>19,410
>19,410
>5500
>17,370
>7147
>19,410
>19,410
>19,410
>226
>17,480
>8785
>5500
12,100
5000
>19,410
>11,820
>19,410
>16,500
>19,410
>19,410
>5500
>17,370
>7147
>19,410
>19,410
>19,410
>8
>1941
>1364
2166
1684
739
>1676
>3389
>3279
18a
18b
18c
18d
18e
18f
2
–(CH2)5–Ph
>48
>48
>48
>17.5
12
23.9
–NH–(CH2)4–Ph
–NMe–(CH2)4–Ph
–(CH2)5–cyclohexyl
–(CH2)5–2-pyridyl
–O–(CH2)3–O–Ph
>48
>48
>17.5
>17.5
22

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W. A. Slusarchyk et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3235–3238
in 9d. This led to a series of diastereomeric mixtures
(18a–f) of highly potent inhibitors of tryptase with
excellent selectivity against related serine proteases
including trypsin as shown in Table 2. The diaster-
eomeric mixture 18a was comparable to the guanidinyl-
propyl analogue 3 in potency versus tryptase but
exceedingly more selective against plasmin, uPA, and
trypsin. Additionally, 18a exhibited improved hydro-
lytic stability at pH 9 (t1/2>17.5 h) compared to BMS-
262084 (1) in Table 1 (t1/2=8.0 h). The other amidino-
piperidinyl diastereomeric mixtures in Table 2 displayed
potent activity versus tryptase and high selectivity
against other serine proteases including trypsin (most
>1000-fold).
shown that conformationally constrained guanidinyl
groups at C-3 and extended substituents appended to
the piperazine at N-1 provide potent inhibitors of typ-
tase with highly improved selectivity versus other serine
proteases including trypsin. One of the most potent and
selective compounds, BMS-363131 (2), was shown to be
efficacious in a guinea pig model of asthma.
References and Notes
1. Elrod, K. C.; Numerof, R. P. Emerging Ther. Targets 1999,
3, 203 and references therein.
2. Gangloff, A. R. Curr. Opin. Investig. Drugs 2000, 1, 79 and
references therein.
3. Zhang, M.-Q.; Timmerman, H. Mediators Inflammation
1997, 6, 311 and references therein.
4. Sutton, J. C.; Bolton, S. A.; Hartl, K. S.; Huang, M.-H.;
Jacobs, G.; Meng, W.; Ogletree, M. L.; Pi, Z.; Schumacher,
W. A.; Seiler, S. M.; Slusarchyk, W. A.; Treuner, U.; Zahler,
R.; Zhao, G.; Bisacchi, G. S. Bioog. Med. Chem. Lett. 2002,
12, 3229.
5. Baldwin, J. E.; Adlington, R. M.; Gollins, D. W.; Schofield,
C. J. Tetrahedron 1990, 46, 4733.
6. (a) Zheng, X.; Gollamudi, R.; Barnes, C. L. Med. Chem.
Res. 1994, 4, 597. (b) Zheng, X.; Day, C.; Gollamudi, R.
Chirality 1995, 7, 90.
7. (a) Anderson, A. G.; Lok, R. J. Org. Chem. 1972, 37, 3953.
(b) Murata, M.; Chiba, T.; Tsutsumi, H.; Yamada, A.; Hat-
tori, K. PCT Patent Application WO9202521, 1992; Chem.
Abstr. 1992, 117, 48211.
Potency versus tryptase and selectivity for the two single
diastereomers BMS-363131 (2) and BMS-363130 (22)
that comprise the mixture 18a are also shown in Table
2. Each of the diastereomers had an IC50 <1.7 nM
against tryptase with selectivity >3,000-fold versus
trypsin and >10,000-fold versus plasmin, thrombin,
uPA, and tPA. BMS-363131 was selected over BMS-
363130 for further evaluation following comparisons of
other biological properties of the two compounds. In
the ovalbumin-sensitized guinea pig model of lung
inflammation,4 intratracheal dosing of BMS-363131
effected a reduction in inflammatory cell count in the
lungs compared to control.
In summary our investigation of the SAR at N-1 and C-
3 of the azetidinone nucleus of BMS-262084 (1) has