A conformationally constrained inhibitor with an enhanced potency for β-tryptase and stability against semicarbazide-sensitive amine oxidase (SSAO)

Article abstract of DOI:10.1016/j.bmcl.2010.08.141

A novel β-tryptase inhibitor with a basic benzylamine P1 group, a piperidine-amide linker, and a substituted indole P4 group was discovered. A substitution at 4-indole position was introduced to constrain the conformational flexibility of the inhibitor to the bioactive conformation exhibited by X-ray structures so that entropic penalty was decreased. More importantly, this constrained conformation limited the accessibility of this molecule to anti-targets, especially SSAO, so that an enhanced metabolic profile was achieved.

Full text of DOI:10.1016/j.bmcl.2010.08.141

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6721–6724
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A conformationally constrained inhibitor with an enhanced potency for
b-tryptase and stability against semicarbazide-sensitive amine oxidase (SSAO)
Guyan Liang a, , Yong Mi Choi-Sledeski b, Gregory Poli b, Xin Chen c, Patrick Shum b, Anne Minnich d,
⇑
Qingping Wang e, Joseph Tsay d, Keith Sides d, Jennifer Cairns d, Gregory Stoklosa b, Thaddeus Nieduzak b,
Zhicheng Zhao b, Jie Wang e, Roy J. Vaz a
a Drug Design, Chemical and Analytical Science, Sanofi-Aventis Pharmaceuticals, Route 202-206, Bridgewater, NJ 08807, USA
b Medicinal Chemistry, Internal Medicine, Sanofi-Aventis Pharmaceuticals, Route 202-206, Bridgewater, NJ 08807, USA
c Structural Biology, Chemical and Analytical Science, Sanofi-Aventis Pharmaceuticals, Route 202-206, Bridgewater, NJ 08807, USA
d In Vitro Pharmacology, Internal Medicine, Sanofi-Aventis Pharmaceuticals, Route 202-206, Bridgewater, NJ 08807, USA
e Drug Metabolism and Pharmacokinetics, Sanofi-Aventis Pharmaceuticals, Route 202-206, Bridgewater, NJ 08807, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel b-tryptase inhibitor with a basic benzylamine P1 group, a piperidine-amide linker, and a substituted
indole P4 group was discovered. A substitution at 4-indole position was introduced to constrain the confor-
mational flexibility of the inhibitor to the bioactive conformation exhibited by X-ray structures so that
entropic penalty was decreased. More importantly, this constrained conformation limited the accessibility
of this molecule to anti-targets, especially SSAO, so that an enhanced metabolic profile was achieved.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 12 July 2010
Revised 27 August 2010
Accepted 31 August 2010
Available online 6 September 2010
Keywords:
Tryptase
Inhibitor
Molecular modeling
Structure-based drug design
X-ray
Benzylamine
SSAO
Mast cell stimulation leads to secretion of various types of prote-
olytic mediators among which is b-tryptase, a trypsin-like serine
protease. Recent studies demonstrated a strong correlation between
mast cell b-tryptase activity and a broad range of inflammatory and
allergic reactions, which suggested that b-tryptase activity could be
a keyaspectof manyinflammatory andallergicdiseases, particularly
asthma.1 As a result, b-tryptase attracted wide-spread attention as a
promising therapeutic target since the early 1990s.2–5 Bode and co-
workerssuccessfullycrystallizedandresolvedb-tryptasestructures,
which explained many of its biochemical properties and provided a
strong foundation for structure-based drug design.6 In the last two
decades, a variety of b-tryptase inhibitors have been discovered
including amidino derivatives,2,3,7,8 di-basic inhibitors,9,10 inhibitors
with an azetidinone11,12 or azepanone linker,13 oxadiazole deriva-
tives,14,15 piperidine analogs,16–20 andinhibitorswitha benzylamine
P1 group.
Comparing amidino-containing compounds, inhibitors with a
benzylamine P1 group are preferable. While the best oral bioavail-
ability achieved for benzamidine-like inhibitors without using a
pro-drug strategy is ꢀ5%, the one for benzylamine-containing com-
pounds can reach 30% or higher. Benzylamine and benzamidine
inhibitors with the same scaffold have very similar, if not identical,
binding vectors to form the critical H-bonding interaction with
D189. Benzylamine is an ideal bioisosteric replacement for ben-
zamidine as a P1 group for inhibitors of trypsin-like serine prote-
ases. When benzamidine is replaced by benzylamine without
structural change to the rest of the inhibitor, the potency decreases
more significantly for some trypsin-like serine proteases than for
others. Fortunately, b-tryptase belongs to the later category and
we were able to identify some highly potent inhibitors with a ben-
zylamine P1 group. The liability of benzylamine, however, is that it
is a known substrate for semicarbazide-sensitive amine oxidase
(SSAO). Inhibitors with a benzylamine group are subject to oxida-
tion under physiological condition, which renders them inactive
towards b-tryptase. A major challenge was to develop benzyl-
amine-containing b-tryptase inhibitors with a reasonable potency
and metabolic profile, especially towards SSAO stability, which is
the focus of the present study.
⇑
Corresponding author. Address: 1041 route 202-206, Mail Stop JR1-203A,
Bridgewater, NJ 08807-0800, United States. Tel.: +1 908 231 4573; fax: +1 908 231
3605.
E-mail address: guyan.liang@sanofi-aventis.com (G. Liang).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.141

6722
G. Liang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6721–6724
As evidenced by both published X-ray structures6,21,22 and our
enhance the SSAO stability of b-tryptase inhibitors is to constrain
the conformation of the inhibitor in such a way that it is able to
bind b-tryptase but not to pass through the accessing channel to
the catalytic center of SSAO.
own in-house structural biology studies, the bioactive b-tryptase
is a ring-like tetramer with four substrate binding pockets facing
the central pore of the ring. Benzylamine inhibitors with a piperi-
dine-amide linker exhibit conventional competitive binding modes
in the b-tryptase substrate binding pocket. As shown in Figure 1,
the P1 amino hydrogen atoms form H-bonding interactions with
the backbone carbonyl oxygen of G219 and the side chain of
D189, which represents the most recognizable interaction for tryp-
sin-like serine proteases. The piperidine ring, meta-connected to
the aminomethyl group, bypasses the catalytic triad which is
formed by S195, D102, and H57, and projects the exiting binding
vector directly into the S4 pocket located above W215. The car-
bonyl oxygen atom of the piperidine-amide has a direct H-bonding
interaction with the backbone NH of G219 which is also a con-
served interaction within this scaffold. Various aromatic P4 groups
were explored and many of them exhibited promising activity
against b-tryptase. In the present study, our effort was focused
on various substituted indole rings as a P4 group which occupied
Various approaches to constrain the piperidine-amide confor-
mation were explored and the present study focused on the con-
formational effect of substitutions on the 4-indole position. Due
to conjugation, the amide of the piperidine-amide linker largely re-
mains planar, which is evidenced by both X-ray structures and in-
silico models. However, the torsion angle between the amide and
the indole ring is twisted by about 55° in its bound conformation
within the b-tryptase binding pocket. This twisted torsion angle
is not preferred by the conjugation between the amide and the in-
dole ring. Even if the steric repulsion between the piperidine and
the 4-position of the indole ring helps to stabilize this twisted con-
formation, significant conformational flexibility remains for this
part of the molecule. Another major area of the conformational
flexibility for the linker is the 4-position of the piperidine ring,
which can adopt either a chair or a boat conformation. Substitution
at the 4-indole position constrains the conformational flexibility of
the linker for both of these areas. It strictly locks the torsion angle
between the amide and the indole ring to the X-ray bound confor-
mation and further increases the energy difference between the
X-ray bound conformation (chair) and other possible conforma-
tions including the boat form.
The effect of the conformational constraint was further evi-
denced by a molecular dynamics (MD) simulation, as shown in
Figure 2. The compound with or without the 4-indole substitution
went through identical MD simulations, in which a 500 ps trajec-
tory was collected following a heating period and a 100 ps equili-
bration period. The simulation was carried out using the
CHARMm force field through Discovery Studio v2.1 from Accelrys.
The time step for the simulation was 1 fs under 300 K. The ‘Shake’
option was turned on to scale down the movement of hydrogen
atoms. Two exiting vectors of the piperidine-amide linker, the
one between piperidine ring and the benzylamine ring (C1–C2)
and the one between the amide and the indole ring (C3–C4), were
monitored and the torsion angle between them was analyzed to re-
flect the conformational flexibility of the piperidine-amide linker.
As shown in Figure 2, the molecule without the 4-indole substitu-
tion (the top trajectory) was much more flexible than the one with
the dimethylamide substitution at the 4-indole position (the bottom
the S4 pocket above the side chain of W215 and had a
interaction with the side chain of Q98.
p-stacking
Human SSAO is a dimeric membrane protein with a short N-ter-
minal cytoplasmic tail, a membrane-spanning domain, and an
extracellular catalytic domain.23,24 The catalytic center of SSAO is
featured by a cofactor, trihydroxyphenylalanine quinone (TPQ),
and a copper ion coordinated by three conserved histidine resi-
dues. The catalytic center is deeply buried within the enzyme
and is accessible only through a narrow channel with a diameter
of about 4.5 Å. This channel is gated by the side chain of L469
which, along with the copper–TPQ coordination, controls the cata-
lytic activity of SSAO. While specific interactions with residues lin-
ing the surface of the accessing channel are important for substrate
specificity, the flexibility of substrates also plays an important role.
As observed in molecular dynamics and induced docking studies,
residues that form the accessing channel have reasonable flexibil-
ity to be able to reform the channel to various sizes and shapes to
accommodate different substrates. However, such change in con-
formation increases the free energy of the system. The more rigid
the substrate, the more significant the conformational change
those residues must undergo to accommodate the substrate, which
in turn further increases the free energy and decreases the proba-
bility of the substrate oxidation. Therefore, one possible strategy to
Figure 1. Stereo view of b-tryptase catalytic pocket. Carbon atoms of the inhibitors and two adjacent monomer of b-tryptase are colored white, green, and magenta,
respectively. While the inhibitor in one substrate binding pocket is shown in whole, the one in the adjacent binding pocket is only shown in part to demonstrate the interface
between the copies of the inhibitors.

G. Liang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6721–6724
6723
Table 1
360
300
240
180
120
60
b-Tryptase activity and selective DMPK profiling data for of compound 7 where R is
N,N-dimethylamide
N
O
C3
C4
N
O
N
N
O
C1 C2
F
H
N
O
N
R
F
Where R is N,N-dimethylamide
0
b-Tryptase (Ki)
20.0 nM
100%
67%
69%
92%
0
100
200
time (ps)
300
400
500
Compound remaining (SSAO, 24 h)
Metabolic stability (human)
Metabolic stability (guinea pig)
Metabolic stability (rat)
360
300
240
180
120
60
hERG (percent inhibition at 10
CYP inhibition (3A4)
l
M)
16%
>30 lM
N
O
C3
C4
N
O
N
C1 C2
F
Where R is N,N-dimethylamide
R
the substituted molecule was 8.5° and the one for the unsubstituted
molecule was 10.1°. Based on this MD simulation, the amide substi-
tution at the 4-indole position properly constrained the conforma-
tion to the one exhibited in the X-ray structures and significantly
reduced the flexibility of the molecule.
Synthesis of the dimethylamide substituted indole, inhibitor 7, is
described in Scheme 1. 4-Dimethylcarbamoyl-1-(2-methoxy-
ethyl)-1H-indole-3-carboxylic acid 4 was prepared via N-alkylation
of 1H-indole-4-carboxylic acid methyl ester 1 with 2-methoxyethyl
bromide using powdered KOH followed by trifluoroacetylation to
provide fluoroform 2. Selective base hydrolysis of the ester 2 then
subsequent treatment with the coupling agent carbonyl diimidazole
and dimethylamine gave the corresponding amide 3. Hydrolysis of
fluoroform 3 under refluxing 6 N NaOH solution provided the key
carboxylic acid intermediate 4 in high yield. Acid 4 was coupled with
2,2,2-trifluoro-N-(4-fluoro-3-piperidin-4-yl-benzyl)-acetamide
hydrochloride 5 (see reference for synthesis) in the presence of EDCI
to afford the penultimate intermediate 6. Removal of the trifluoro-
acetyl protecting group of 6 with aqueous potassium carbonate fol-
lowed by 2 N HCl in Et2O provided the desired product 7.
0
0
100
200
time (ps)
300
400
500
Figure 2. Torsion angle between the two exiting vectors of the piperidine-amide
scaffold during a course of 500 picosecond (ps) molecular dynamics (MD) simula-
tion. The bottom figure represented the trajectory of a molecule with a dimeth-
ylamide substitution at 4-indole position and the top represented the one without
such substitution.
trajectory). The molecule without the 4-indole substitution experi-
enced nine major conformational transformations for the linker
within 500 ps while the molecule with a 4-indole substation stayed
at the X-ray bound conformation through the entire simulation.
Even during the timeframe in which neither molecule experienced
any major conformational change (from 150 ps to 350 ps), the aver-
age linker flexibility of the molecule without the 4-indole substitu-
tion was higher than the one with the substitution. The standard
deviation of the torsion angle between the two exiting vectors for
The inhibition of human b-tryptase was assayed with a chromo-
genic substrate S-2366 (Diapharma),
L-pyroglutamyl-L-prolyl-L-
arginine-p-nitroaniline hydrochloride. For determining IC50, eight
F
F
N
O
O
F
F
O
O
O
O
O
F
c. 1N NaOH
MeOH
a. i. KOH, DMSO
ii. 2- methoxyethyl bromide
25%
F
e. 6N NaOH
reflux
45 oC 2h, 91%
N
b. TFAA, DMF
0 oC, 55%
N
96%
N
d. dimethylamine,
carbonyl diimidazole
THF, 62%
H
1
3
2
O
O
O
NH2
HCl
N
O
O
F
N
N
O
O
OH
f. EDCI, Et3N,
N
O
H
F
O
N
F
N
F
CH2Cl2, RT
g. i. K2CO3, H2O/MeOH
N
F
N
50%
7
O
N
O
RT, 50%
ii. 2N HCl/Et2O
6
F
F
4
N
H
O
F
O
HCl HN
5
F
Scheme 1. The reaction scheme for synthesizing compound 7.

6724
G. Liang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6721–6724
Table 2
especially SSAO, which is critically important for further develop-
ment of this series of compounds as therapeutic agents.
b-Tryptase activity and SSAO stability of the reference compound
N
References and notes
O
N
O
N
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concentrations of the compound were used: 0.0046, 0.0137, 0.04,
0.12, 0.37, 1.11, 3.3, and 10 M. Briefly, the compound was mixed
with 50 ng/mL b-tryptase, followed by addition of 500 M substrate
l
l
S-2366. The reaction mixture was incubated at room temperature
for 30 min. The absorbance of released product, p-nitroaniline, was
measured at a wavelength of 405 nm with Wallac Victor2 V spectro-
photometer. The IC50 value was determined by fitting the dose–re-
sponse data into a sigmoid curve and the Ki value was calculated
from a competitive inhibition equation: Ki = IC50/(1 + [S]/Km). The
Ki of compound 7 was determined to be 20 nM, as shown in Table
1, which represented a reasonable enhancement as compared to
the reference compound without the dimethylamide substitution
at the 4-indole position, as shown in Table 2.
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For stability determination in the presence of SSAO, 0.5
lM of
compound was incubated with 56 g/mL of recombinant human
l
full-length SSAO over-expressed in CHO cells for 0 and 24 h. The
mixture was then extracted with acetonitrile containing an inter-
nal standard. After centrifugation, 50 ll of the acetonitrile fraction
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Dupuy, A.; Davidson, J.; Harrison, T.; Morley, A.; Watson, S.; Fenton, G.;
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was subjected to LC/MS analysis with a PE Sciex API 3000 mass
spectrometer. The percentage remaining of the compound was
determined by the ratio of the integrated area at 24 h to the inte-
grated area at 0 h. While significant metabolism was observed for
many similar compounds without the dimethylamide substitution,
compound 7 was found to be 100% stable after 24-h incubation
with SSAO, which represented a significant improvement over
the reference compound, as shown in Table 2.
An in vitro liver microsome stability assay with appropriate
co-factors was used for further metabolic stability evaluation. Com-
pound 7 was incubated with liver microsomes for 20 min, before the
reaction was stopped and the amount of compound remaining was
measured. As shown in Table 1, the metabolic stability of compound
7 for human, guinea pig, and rat are 67%, 69%, and 92%, respectively.
The inhibition against hERG and CYP (3A4) was also measured and
found to be satisfactory, as shown in Table 1.
In conclusion, a substitution of N,N-dimethylamide at the 4-in-
dole position significantly decreases the flexibility of the piperi-
dine-amide linker, which constrained the conformation of the
inhibitor to the X-ray bound conformation. Such a constraint de-
creases the entropic penalty of binding, which leads to enhanced
potency against b-tryptase. More importantly, the reduced flexibil-
ity decreases the chance for the inhibitor to bind with anti-targets,
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