Design, synthesis, and biological activity of potent and selective inhibitors of mast cell tryptase

Article abstract of DOI:10.1016/j.bmcl.2005.04.002

A new series of novel mast cell tryptase inhibitors is reported, which features the use of an indole structure as the hydrophobic substituent on a m-benzylaminepiperidine template. The best members of this series display good in vitro activity and excellent selectivity against other serine proteases.

Full text of DOI:10.1016/j.bmcl.2005.04.002

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2734–2737
Design, synthesis, and biological activity of potent and
selective inhibitors of mast cell tryptase
Corey R. Hopkins,a,* Mark Czekaj,a Steven S. Kaye,a Zhongli Gao,a James Pribish,a
Henry Pauls,a Guyan Liang,b Keith Sides,c Dona Cramer,c Jennifer Cairns,c Yongyi Luo,d
Heng-Keang Lim,d Roy Vaz,d Sam Rebello,d Sebastian Maignan,e Alain Dupuy,e
Magali Mathieue and Julian Levella,*
aDepartment of Medicinal Chemistry, Aventis Pharmaceuticals, Route 202-206, Bridgewater, NJ 08807, USA
bDepartment of Medicinal Chemistry/Molecular Modeling, Aventis Pharmaceuticals, Route 202–206, Bridgewater, NJ 08807, USA
cDepartment of Molecular Validation and Cell Biology, Aventis Pharmaceuticals, Route 202–206, Bridgewater, NJ 08807, USA
dDepartment of eADME/DMPK, Drug Innovation and Approval, Aventis Pharmaceuticals, Route 202–206,
Bridgewater, NJ 08807, USA
eDepartment of Structural Biology, Aventis Pharma, Vitry 94403, France
Received 7 February 2005; revised 31 March 2005; accepted 4 April 2005
Available online 4 May 2005
Abstract—A new series of novel mast cell tryptase inhibitors is reported, which features the use of an indole structure as the hydro-
phobic substituent on a m-benzylaminepiperidine template. The best members of this series display good in vitro activity and excel-
lent selectivity against other serine proteases.
Ó 2005 Elsevier Ltd. All rights reserved.
Asthma is a very complex disease that is mediated by a
number of factors. The number of cases is expected to
double in the next decade1 and thus there is a huge effort
in the pharmaceutical industry to find novel therapies to
treat this disease. One target that has garnered the atten-
tion of the industry is b-tryptase. It is a serine-like pro-
tease that is found almost exclusively in mast cells.2
Tryptase is stored in intracellular granules as the active
form as a heparin bound tetramer and is released upon
stimulation of mast cells. Since tryptase has been di-
rectly linked to the pathology of asthma,3 we4 and oth-
ers5 have been working toward finding a small-molecule
inhibitor.
the synthesis, SAR, and PK properties of this type of
inhibitor.
The synthesis of inhibitors 9 is shown in Scheme 1. The
appropriately substituted indole 1 was treated with tri-
fluoroacetic anhydride in DMF to yield the indole-3-tri-
fluoroacetate 2. The trifluoroacetate was hydrolyzed
(20% NaOH (aq)) and the acid was esterified giving
4. The indole nitrogen was alkylated (NaH, R1X, and
THF or Cs2CO3, R1X, and DMPU) giving 5, which
was then saponified (NaOH (aq), MeOH, and THF)
yielding the indole-3-carboxylic acid 6. Alternatively,
the trifluoroacetate 2 could be directly alkylated at the
indole nitrogen followed by subsequent hydrolysis to
yield acid 6. This acid was then coupled with the piper-
idine 74b (TBTU, DIEA, and DMF) followed by bis-Boc
cleavage (TFA and DCM) to realize the desired inhibi-
tors 9 as their TFA salts. Alternative deprotection with
HCl/dioxane resulted in the isolation of the HCl salt.
Having established a preferred P1 ligand,4b we next
explored alternative hydrophobic units to improve
potency and hopefully moderate PK and eADMET
properties. Based on in-house computer models, we felt
that a 3-substituted indole scaffold would lead to desir-
able inhibitors of mast cell tryptase. Herein we describe
Our first efforts concentrated on making unsubstituted
indole compounds. After making several compounds, it
was apparent (Table 1) that substitution in the 5- and
7-position (9b,d, and e) of the indole was tolerated while
substitution in the 4- and 6-positions gave less active
*
Corresponding authors. Tel./fax: +1 908 231 3351 (C.R.H.); tel./fax:
+1 908 231 4045 (J.L.); e-mail addresses: corey.hopkins@aventis.
com; julian.levell@aventis.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.002

C. R. Hopkins et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2734–2737
2735
Scheme 1. Synthesis of indole inhibitors 9. Reagents and conditions: (i) TFAA, DMF (70–85%); (ii) 20% NaOH (aq) (60–80%); (iii) H2SO4, MeOH;
(iv) TMSCHN2, Tol, MeOH; (v) NaH, R1X, THF; (vi) Cs2CO3, R1X, DMPU (50–90%, 2 steps); (vii) NaOH (aq), MeOH, THF (85–95%); (viii)
TBTU, DIEA, DMF (90% quant.); (ix) TFA, DCM (50–95%).
Table 1. Tryptase inhibition for indole substituents6
Compd
R
R1
Tryptase Ki, nM
Sol (lM)
CombiPK,7 F (%)
9a
9b
9c
4-Me
5-Me
6-Me
7-Me
7-F
H
H
>2000
101
>2000
61
27
40
40
26
40
21
23
13
32
5.9
7.4
40
36
29
27
38
10
30
9
ND
ND
ND
19
H
H
9d
9e
H
H
368
290
197
373
240
246
128
162
310
497
185
155
141
124
116
57
ND
22
9f
H
9g
9h
9i
H
3-Pyridyl
2-Thiazole
Acetyl
ND
ND
ND
ND
17
H
H
9j
H
Thiophene-2-carbonyl
Benzenesulfonyl
Methanesulfonyl
Isopropyl
Cyclohexylmethyl
Propyl
9k
9l
H
H
ND
ND
ND
ND
ND
ND
11
9m
9n
9o
9p
9q
9r
H
H
H
H
Methyl
H
Cyclopropylmethyl
Ethyl
Isobutyl
H
9s
H
ND
ND
9.3
9t
H
2-Methoxyethyl
Butyl
Hexyl
30
34
30
14
11
30
8.1
9u
9v
H
49
H
125
32
ND
ND
ND
26
9w
9x
9y
9z
7-Me
7-Me
7-Me
7-Me
2-Methoxyethyl
Propyl
Ethyl
41
41
Butyl
68
140
33
Babim8
compounds (9a and c). Also, substitution at the 2-posi-
tion (2-Me) led to a ꢀ10-fold loss of activity (not shown).
The 7-Me substituted compound gave very acceptable
potency, solubility, and displayed bioavailability that

2736
C. R. Hopkins et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2734–2737
was a reasonable starting point. Next, our efforts moved
toward indoles with varying N-substitution (entries 9f–
v). A number of groups were investigated (aryl, alkyl,
acyl, and sulfonyl). As can be seen in the table, no substi-
tution (9f) as well as aryl, acyl, and sulfonyl groups (9g–l)
were all inferior to the alkyl substituents. It appears that
an sp3 atom adjacent to the nitrogen is optimal for
potency. Also, as the chain length grows, the potency
increases (9t–v). However, there seems to be an optimal
chain length as the potency increases to a point (9u)
and then starts to diminish as the hexyl (9v) substituents
potency is less.
Next, we turned our attention to evaluating compounds
with variable R1 while retaining R = 7-Me (entries 9w–
z). There is no significant difference in potency between
these four compounds shown, each showing good po-
tency. The combination of the 2-methoxyethyl and butyl
N-alkyl groups with R = 7-Me substituent did not offer
significant improvement in the potency (9t vs w,
57 nM vs 32 nM) and (9u vs z, 49 nM vs 68 nM). How-
ever, the presence of the R = 7-Me improved the
potency with the shorter propyl and ethyl groups (9o vs
x, 185 nM vs 41 nM) and (9r vs y, 124 nM vs 41 nM).
Evaluation of oral bioavailability for selected com-
pounds showed the addition of R = 7-Me afforded
improvements in oral bioavailability (9r vs y, 11% vs
26%) and (9u vs z, 9.3% vs 33%).
Figure 1. Computer modeled (using InsightII and Discover from
Accelrys, and FlexX from BioSolveIT) binding mode of inhibitor 9z in
b-tryptase. The amide carbonyl makes a key hydrogen bond interac-
tion with the nitrogen of Gly219. The benzyl amine inserts into the S1
pocket, forms a salt bridge to Asp189 and hydrogen bonds to oxygens
of Gly219 and Ser190. The indole moiety is making good hydrophobic
contacts.
activity. This is likely due to the change in the torsion
angle of the inhibitor about the amide bond rather than
a steric clash with the protein. Second, substitution at
the C-6 position (9c) also led to a decrease in activity,
which may be explained by a steric clash between the
two inhibitors in adjacent active sites of the tryptase tet-
ramer. Substitution at either the C-5 or C-7 (9b,d) are
tolerated because they do not either change the torsion
angle, or cause steric interactions with the adjacent
inhibitor or protein backbone.
The SAR results are supported by computer modeling
analysis for inhibitor 9z (Fig. 1). The analysis shows a
number of key interactions for this compound. Namely,
the meta-benzylamine (P1 group) interacts with both the
Asp189 and the carbonyl of Gly219. The meta-benzyl-
amine has been shown in our laboratories to be a key
component for potent tryptase activity.4 Any modifica-
tion to this group drastically reduces the inhibitory
activity. The indole portion fills a hydrophobic void in
the enzyme.
Another key interaction is likely that of Tyrosine-95 and
the proximal N-indole group (Figs. 1 and 2). Thus, due
to the constraints of the system, flexible groups on the
N-indole lead to the most active compounds (9r–u).
Analysis of modifications of the phenyl portion of the
indole ring showed two distinct differences. First, substi-
tution at the C-4 position (9a) leads to a decrease in
Figure 2. Stereoview of tryptase inhibitor 9z.

C. R. Hopkins et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2734–2737
Table 2. Selectivity data for potent tryptase inhibitor, 9z
2737
Compd
IC50 (nM)
Factor Xa
>10,000
b-Tryptase
a-Tryptase
Chymase
>10,000
Thrombin
>10,000
Trypsin
>10,000
9z
68
3694
3. Molinari, J. F.; Scuri, M.; Moore, W. R.; Clark, J.; Tanaka,
R.; Abraham, W. M. Am. J. Respir. Crit. Care Med. 1996,
154, 649.
Groups such as acyl, sulfonyl, or branched alkanes are
too sterically hindered to rotate away from the Tyro-
sine-95. In addition, the bottom face of the indole group
is blocked and thus inhibitors that do not have an un-
branched sp3 center a (and preferably b) cannot rotate
above the indole group. However, as the chain length
grows, the activity seems to hit a peak at butyl and starts
to decrease with hexyl (9v). Combining the two substitu-
tions (7-Me and N-alkyl) offered an advantage in
potency in the N-ethyl and N-propyl derivatives (9r vs
y; 9o vs x), but did not offer any substantial advantage
using N-2-methoxyethyl (9w) or the N-butyl (9z).
4. (a) Hopkins, C. R.; Neuenschwander, K.; Scotese, A.;
Jackson, S.; Nieduzak, T.; Pauls, H.; Liang, G.; Sides, K.;
Cramer, D.; Cairns, J.; Maignan, S.; Mathieu, M. Bioorg.
Med. Chem. Lett. 2004, 14, 4819; (b) Levell, J.; Astles, P.;
Eastwood, P.; Cairns, J.; Houille, O.; Aldous, S.; Merri-
man, G.; Whiteley, B.; Pribish, J.; Czekaj, M.; Liang, G.;
Maignan, S.; Gouilloteau, J.-P.; Dupuy, A.; Davidson, J.;
Harrison, T.; Morley, A.; Watson, S.; Fenton, G.; McCar-
thy, C.; Romano, J.; Mathew, R.; Engers, D.; Sides, K.;
Kwong, J.; Tsay, J.; Rebello, S.; Shen, L.; Wang, J.; Luo,
Y.; Giardino, O.; Lim, H.-K.; Smith, K.; Pauls, H. Bioorg.
Med. Chem. 2005, 13, 2859.
5. (a) Clark, J. M.; Moore, W. R.; Tanaka, R. D. Drugs
Future 1996, 21, 811; (b) Burgess, L. E. Drug News
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Ogletree, M. L.; Pi, Z.; Schumacher, W. A.; Seiler, S. M.;
Slusarchyk, W. A.; Treuner, U.; Zahler, R.; Zhao, G.;
Bisacchi, G. S. Bioorg. Med. Chem. Lett. 2002, 12, 3229; (d)
Slusarchyk, W. A.; Bolton, S. A.; Hartl, K. S.; Huang,
M.-H.; Jacobs, G.; Meng, W.; Ogletree, M. L.; Pi, Z.;
Schumacher, W. A.; Seiler, S. M.; Sutton, J. C.; Treuner,
U.; Zahler, R.; Zhao, G.; Bisacchi, G. S. Bioorg. Med.
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Lastly, we turned our attention to selectivity. We
wanted to make sure that these compounds would be
selective against other, very similar, serine proteases.
As can be seen in Table 2, inhibitor 9z shows excellent
selectivity against factor Xa, thrombin, and trypsin
(ꢀ1000-fold). This compound also shows very good
selectivity against other tryptase homologues, for exam-
ple, a-tryptase (ꢀ90-fold selectivity).
In conclusion, we have reported the synthesis and SAR
evaluation of a novel class of small-molecule mast cell
tryptase inhibitors. The compounds are an extension
of our tryptase program and are very potent, orally bio-
available inhibitors. In addition, these compounds have
also been shown to be very selective against other simi-
lar serine proteases.
References and notes
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H.; Hall, I. P. Am. J. Physiol. Lung Cell Mol. Physiol. 2002,
282, L197; (b) Cairns, J. A. Pulm. Pharmacol. Ther. 2005,
18, 55.
6. For assay details, see Ref. 4.
7. CombiPK was performed in rats using IV and PO doses of
1 and 2 mg/kg, respectively.
8. Reference compound, see: Caughey, G. H.; Raymond, W.
W.; Bacci, E.; Lombardy, R. J.; Tidwell, R. R. J.
Pharmacol. Exp. Ther. 1993, 264, 676.