Dimerization of β-tryptase inhibitors, does it work for both basic and neutral P1 groups?

Article abstract of DOI:10.1016/j.bmcl.2012.01.021

The tetrameric folding of β-tryptase and the pair-wise distribution of its substrate binding sites offer a unique opportunity for development of inhibitors that span two adjacent binding sites. A series of dimeric inhibitors with two basic P1 moieties was

Full text of DOI:10.1016/j.bmcl.2012.01.021

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3370–3376
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dimerization of b-tryptase inhibitors, does it work for both basic and neutral
P1 groups?
Guyan Liang a, , Yong Mi Choi-Sledeski a, Xin Chen a, Yong Gong a, , Eric W. MacMillan a, Joseph Tsay b,
⇑
Keith Sides b, Jennifer Cairns c, Berndt Kulitzscher d, David J. Aldous a, Isabelle Morize a,
Henry W. Pauls a,à
a Molecular Innovative Therapeutics, Sanofi Pharmaceuticals, United States
b Fibrosis and Wound Repair, Sanofi Pharmaceuticals, United States
c Immunology and Inflammation, Sanofi Pharmaceuticals, United States
d Chemical Development, Sanofi Pharmaceuticals, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The tetrameric folding of b-tryptase and the pair-wise distribution of its substrate binding sites offer a
unique opportunity for development of inhibitors that span two adjacent binding sites. A series of
dimeric inhibitors with two basic P1 moieties was discovered using this design strategy and exhibited
tight-binder characteristics. Using the same strategy, an attempt was made to design and synthesize
dimeric inhibitors with two neutral-P1 groups in hope to exploit the dimeric binding mode to achieve
a starting point for further optimization. The unsuccessful attempt, however, demonstrated the impor-
tant role played by Ala190 in neutral-P1 binding and casted further doubt on the possibility of developing
neutral-P1 inhibitors for b-tryptase.
Received 10 December 2011
Revised 4 January 2012
Accepted 9 January 2012
Available online 14 January 2012
Keywords:
Serine protease
Asthma
Ó 2012 Elsevier Ltd. All rights reserved.
COPD
Tryptase
Inhibitor
Molecular modeling
Structure-based drug design
X-ray
Benzylamine
Human b-tryptase (EC 3.4.21.59), a mast cell-specific trypsin-
like serine protease, has been implicated in inflammatory and
allergic disorders, especially asthma and chronic obstructive pul-
monary disease (COPD).1–6 Among all forms of human tryptase en-
chymotrypsin.19–24 Aiming at development of orally bioavailable
drugs, most publications on the topic of b-tryptase inhibitors have
focused on small organic molecules with various core scaffolds,
such as amidinobenzofuran,25 azetidinone,26–29 azepanone,30 calix-
arene,31 pyrazinone,32 piperidinylamide,33–36 oxadiazoles,37–39
naphthalenesulphoamide,40 and phenylphthalimide derived from
thalidomide.41 The present Letter describes the discovery of di-
meric inhibitors with two basic-P1 moieties and our effort to lever-
age this approach to design a dimeric inhibitor with two neutral-P1
groups.
zymes identified so far (a-, b-, c-, d-, and e-tryptases), b-tryptase is
the most abundant form in mast cells.7–13 Stabilized by negatively
charged heparin, human b-tryptase is secreted as an active enzyme
and stored in the secretory granules.14–17 Following mast cell acti-
vation upon allergen stimulation, b-tryptase is released, which
consequently triggers allergic and inflammatory responses.
The therapeutic potential of modulating b-tryptase has at-
tracted widespread attention with a variety of inhibitors reported
in scientific journals and patent publications.2,18 One of the earliest
published inhibitors is a 46-amino acid polypeptide, leech derived
tryptase inhibitor form C (LDTI-C), which inhibits both b-tryptase
and some other trypsin-like serine proteases, such as trypsin and
Three major categories of small organic inhibitors have been
published for trypsin-like serine proteases. The first category con-
sists of mechanism-based inhibitors, where the inhibitor forms a
covalent bond with the catalytic serine of the enzyme (Ser195
for trypsin-based numbering system).29,42–46 The covalent bond
between the inhibitor and the enzyme significantly enhances the
inhibitory potency. This enhancement, however, is mainly attrib-
uted to the catalytic triad, which is conserved among all serine pro-
teases. Therefore, it may potentially decrease selectivity against
other enzymes that have a similar reactive serine residue, and thus
lead to undesirable side-effects.
⇑
Corresponding author. Tel.: +1 908 231 4573.
E-mail address: guyan.liang@verizon.net (G. Liang).
Presently with Johnson & Johnson.
Presently with Campbell Family Institute for Breast Cancer Research.
à
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.021

G. Liang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3370–3376
3371
The second major category of trypsin-like serine protease inhib-
itors includes molecules with a basic-P1 moiety that can form an
H-bonding interaction with Asp189 in the S1 pocket. The presence
of Asp189 in the S1 binding pocket is the most conserved feature of
trypsin-like serine proteases besides the catalytic triad. Most tryp-
sin-like serine proteases with known competitive non-mechanism-
based inhibitors have inhibitors of this kind. While the H-bonding
interaction between inhibitors and Asp189 in the S1 pocket of the
enzyme contributes significantly to the binding affinity, this inter-
action prefers a strong basic group, which often leads to inhibitors
with undesirable pharmacokinetic properties, especially poor oral
bioavailability.47–49
The third category covers inhibitors with a non-basic-P1 group,
or more precisely, competitive inhibitors without either a reactive
‘warhead’ to form a covalent bond with Ser195 or a basic group to
form an H-bonding interaction with Asp189. Inhibitors in this
category generally have better selectivity and pharmacokinetic
profiles, which is best exemplified by the successful development
of ApixabanÒ,50 EdoxabanÒ,51 and RivaroxabanÒ.52 While this cat-
egory of inhibitors has been identified for trypsin-like serine prote-
ases with Ala190, such as factor Xa (fXa) and thrombin, it has not
been reported for those with Ser190, such as b-tryptase.50–58
It is debatable what the nature of the interaction for neutral-P1
binders is and whether Ser190 is an insurmountable obstacle for a
neutral-P1 moiety to bind in the S1 pocket.59 Numerous in-house
efforts to develop possible neutral binders for b-tryptase have been
unsuccessful. All designed molecules with a neutral-P1 moiety
showed binding affinity too weak to be detected through either
spanning two adjacent substrate-binding sites. By doubling the
binding enthalpy, this type of dimeric inhibitor should have a
better chance than monomeric inhibitors to achieve a synergetic
binding affinity detectable by physical methods, such as X-ray
crystallography, thus offering a platform to explore the nature of
the neutral P1 interaction.
The enhanced inhibition for dimeric b-tryptase inhibitors span-
ning two adjacent substrate-binding sites have been evidenced by
compounds shown in Figure 2. The monomeric inhibitor, com-
pound 1, with a benzylamine P1, a piperidine-amide scaffold, and
a simple phenyl P4 group showed a Ki of 1900 nM against b-tryp-
tase in a chromogenic assay. Based on in silico models, compound
1 exhibits a typical binding mode of category 2 inhibitors discussed
above with the NH2 of benzylamine H-bonding with the side chain
of Asp189 and the backbone oxygen of Gly219. The carbonyl oxy-
gen of the piperidine-amide scaffold interacts with the backbone
NH of Gly219 through another conserved H-bonding interaction.
The P4 phenyl ring has a
p-stacking interaction with the amide
side chain of Gln98 in the S4 pocket. Because the two S4 pockets
of the adjacent substrate-binding sites are next to each other, the
two P4 phenyl rings in the adjacent binding sites are facing each
0
other with approximately 6.5 ÅA space in-between, which can be
bridged with various linker groups to form dimeric inhibitors, such
as compounds 2 and 3 in Figure 2.
The synthesis of dimeric b-tryptase inhibitors 2 and 3 is shown
in Scheme 1. Acetylene intermediate 8 was prepared in 83% yield
from TBTU activated coupling of di-Boc protected 3-piperidin-4-
ylbenzylamine 7 with 3-ethynyl benzoic acid 6. Under Sonogashira
conditions, acetylene 8 reacted with 1-fluoro-2-iodobenzene and
formed arylation product 10 as the major product (58%) and
dimeric compound 9 as a minor product (38%). Removal of Boc
groups from compound 9 in TFA-dichloromethane gave a clean di-
mer 2. Further hydrogenation of dimer 2 with H2 in the presence of
10% Pd/C produced the saturated dimer 3 quantitatively.
enzymatic assay (>30 lM) or by X-ray crystallography.
Compared to other trypsin-like serine proteases, the most un-
ique feature of b-tryptase is its tetrameric folding, which was first
discovered by Bode and co-workers.60–62 As shown in Figure 1, the
bioactive form of b-tryptase is a donut-shaped tetramer with four
substrate binding sites facing toward the central pore of the tetra-
mer in a pair-wise distribution. Within each pair, the S4 pockets of
the two substrate-binding sites face each other and are located at
the interface between two adjacent monomers. Although each
monomer has its own independent catalytic center and S1 pocket,
the S4 pockets of two adjacent substrate-binding sites are
connected to each other without any physical barrier. This unique
tetrameric folding offers an opportunity to design an inhibitor
Inhibition of b-tryptase activity was measured by means of a
chromogenic assay using human recombinant b-tryptase and a
chromogenic substrate L-pyroglutamyl-L-prolyl-L-arginine-p-nitro-
aniline hydrochloride, S-2366 (DiaPharmaÒ). Eight inhibitor
O
NH2
N
1
Ki = 1900 nM
O
N
NH2
N
H2N
O
2
Ki = 0.34 nM
O
N
NH2
N
H2N
O
3
Ki = 2.0 nM
Figure 1. The tetrameric folding of b-tryptase. Two adjacent monomers are colored
in yellow and magenta, respectively, with only
a-carbon atoms shown. Inhibitors
Figure 2. Monomeric and dimeric b-tryptase inhibitors with a benzylamine P1
(compound 3) in the substrate-binding sites are colored in green.
group and a piperidine-amide scaffold.

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G. Liang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3370–3376
O
O
NH
NBoc2
a
b
N
NBoc2
HO
+
6
7
8
O
O
NBoc2
N
F
NBoc2
N
N
+
Boc2N
O
9
10
c
O
NH2
N
N
H2N
O
2
O
NH2
d
N
N
H2N
O
3
a)TBTU, NEt3, DMF, r.t., 1 h, 83%.
b) 1-fluoro-2-iodobenzene, Pd(PPh3)2Cl2, CuI, NEt3, DMF, r.t., 22 h, 38% 9, 58% 10.
c) 20% TFA/CH2Cl2, r.t., 2 h, 92%.
d) 10% Pd/C, H2 (1 atm), CH3OH, r.t., 2 h, quantitatively.
Scheme 1. The reaction scheme for synthesizing compounds 2 and 3.
concentrations, 0.0046, 0.0137, 0.04, 0.12, 0.37, 1.11, 3.3, and
10 M, were used to generate IC50 curves. Various b-tryptase con-
Neutral-P1 inhibitors, such as compound 4 in Figure 3, can bind
in the substrate-binding site of fXa and thrombin with good bind-
ing affinity. As shown in Figure 4, binding models of compounds 4
and 5 in fXa exhibited a binding mode different from conventional
basic-P1 inhibitors. Instead of an H-bonding interaction with
Asp189, the chlorothiophene group was situated above the cen-
troid of the Tyr228 side chain phenol ring. While the exact nature
of the interaction between the chlorothiophene and the Tyr228
side chain is debatable, an induced dipole–dipole interaction, in
addition to hydrophobic interaction, was believed to contribute
to the binding affinity. The oxygen and nitrogen of the isoxazole
ring face the direction of catalytic triad. Even if there is no direct
interaction between the isoxazole ring and catalytic triad, water-
mediated H-bonding interaction may still contribute to the overall
binding affinity. The oxygen atom of the ketopiperidine scaffold
maintained an H-bonding interaction with the NH of the Gly219
backbone, which further contributed to the binding affinity.
l
centration were explored during assay optimization. While the IC50
value of compound 1 was not dependent on the b-tryptase concen-
tration, those for compounds 2 and 3 changed significantly when
different enzyme concentrations were used. This phenomenon
suggests that compounds 2 and 3 are tight-binding inhibitors for
b-tryptase. Copeland et al. demonstrated that calculating Ki values
of tight binders based on the classical method under the assump-
tion of [It] > >[Et] was invalid.63 They suggested to derive the true
Ki value for a tight binder, where It ꢀ Et, using Henderson’s linear
plot of It/(1ꢁvi/v0) versus v0/vi. Using this approach, Ki values of
compounds 2 and 3 were calculated to be 0.34 and 2.0 nM, respec-
tively, which are 1000-fold more potent than compound 1. A sim-
ilar enhancement in potency was observed for other dimeric
inhibitors with either a benzylamine or a benzamidine P1 group,
as shown in Table 1.

G. Liang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3370–3376
3373
Table 1
for chlorothiophene. Molecular Dynamics (MD) simulation indi-
cated that the S1 binding pocket of b-tryptase was still capable
to accommodate the binding of the chlorothiophene group if
surrounding residues were allowed to relax their conformations
slightly. The relaxation of those residues, however, increased the
free energy of the system, which may potentially decrease the
binding affinity of the compound.
In vitro potency for b-tryptase
O
X
N
R
N
X
O
Compd#
X
R
b-tryptase (Ki, nM)
The second difference affecting the binding of compound 4 in
the S1 pocket of b-tryptase is the electrostatic nature of the local
binding area. The interaction between chlorothiophene and the
S1 pocket of fXa is largely hydrophobic in nature. For b-tryptase,
whereas the hydrophobic interaction with Tyr228 is conserved,
the mutation from Ala190 to Ser190 decreases the hydrophobic
interaction between the chlorothiophene group and the side chain
of Ser190. This analysis is consistent with our previous observation
that numerous compounds designed to incorporate a neutral moi-
ety into the S1 pocket of b-tryptase failed to achieve a detectable
NH2
2
3
0.34
2.0
NH2
NH2
–(CH2)4–
H
N
H
N
18
3.0
NH2
NH2
19
20
0.29
0.3
HN
HN
inhibition (<30 lM).
Comparing to monomeric inhibitors, a dimeric inhibitor occu-
pies two adjacent substrate-binding sites, which doubles the bind-
ing enthalpy and decreases the binding entropy. The synergetic
effect should result in higher potency for dimeric inhibitors, which
is evidenced by the 1000-fold enhancement in Ki values for dimeric
basic-P1 inhibitors discussed above. Although the enhancement is
not directly transferable from dimerization of basic-P1 inhibitors
to neutral-P1 inhibitors, a similar amplification of binding free en-
ergy is expected for dimeric inhibitors with two neutral-P1 groups.
With this amplification of binding free energy, it was envisioned
that one should have a better chance to detect the neutral-P1 bind-
ing in the S1 pocket of b-tryptase with a dimeric inhibitor than
with its monomeric counterpart.
Following this strategy, a homodimeric inhibitor with two neu-
tral-P1 groups, identical to the one for compound 4, was designed
to span two adjacent b-tryptase binding sites to preserve all its S1-
binding features in fXa, such as the chloro-aromatic interaction
with Tyr228 and the H-bonding interaction with the backbone
NH of Gly219. Additionally, the P4 exiting vector of this neutral-
P1 dimer (shown in white-carbon in Fig. 5) was highly similar to
the one for the basic-P1 dimer (shown in green-carbon in Fig. 5)
discussed above. Therefore, a similar aliphatic bridge linker was
–(CH2)4–
The S1 binding pocket of b-tryptase is identical to the one of fXa
or thrombin except that b-tryptase has serine at residue 190 posi-
tion and fXa and thrombin have alanine. As shown in Figure 4, all
residues lining the surface of the S1 pocket of b-tryptase (with yel-
low-colored carbon atoms) and fXa (with white-colored carbon
atoms) are superimposed well on each other, which allows the
neutral-P1 moiety of fXa inhibitors to maintain the same binding
mode in the S1 pocket of b-tryptase. The side chain phenol ring
of Tyr228 in b-tryptase is situated at the same location to receive
the chlorothiophene group. The backbone NH of Gly219 presents
the same H-bonding accommodation for the oxygen of the scaffold
ketopiperidine moiety, while the water-mediated H-bonding inter-
action with catalytic triad also remains unchanged.
One difference in the S1 pocket between fXa and b-tryptase is
that the side chain of Ser190 for b-tryptase intrudes more into
the S1 pocket than Ala190 for fXa. Based on the in silico model
of compound 4 in the substrate-binding site of b-tryptase, mutat-
ing alanine to serine at the 190 position shrinks the binding space
O
N
N
N
S
Cl
O
N
N
N
4
O
N
N
N
S
O
Cl
Cl
O
S
N
N
O
N
5
Figure 3. Molecules with neutral-P1 groups. Compound 4 is a known fXa inhibitor with a neutral-P1 moiety. Compound 5 is a dimeric inhibitor with the same neutral-P1
moiety designed to span the two adjacent substrate-binding sites in b-tryptase.

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G. Liang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3370–3376
Figure 4. A stereo view of superimposed S1 pockets of b-tryptase and factor Xa. Carbon atoms of b-tryptase are colored in yellow and those of fXa are colored in white.
Compound 4 (green carbon) was docked into fXa using GlideÒ from SchroedingerÒ with the binding mode confirmed by X-ray structures. Compound 5 (magenta carbon) was
docked into b-tryptase using GlideÒ from SchroedingerÒ with a constraint to mimic the neutral-P1 binding mode of compound 4 in fXa.
Figure 5. A stereo view of dimeric inhibitors spanning two adjacent substrate-binding sites of b-tryptase. The top figure shows the solvent accessible protein surface while
the bottom figure shows residues around the two adjacent binding sites. Two monomers of b-tryptase are colored in yellow and magenta, respectively. Carbon atoms of the
basic-P1 inhibitor (compound 3) are colored in green whereas those of the neutral-P1 inhibitor (compound 5) are colored in white.
used to construct the neutral-P1 dimer without changing the over-
all binding mode, especially interactions in the S1 pocket. As
shown in Figure 5, the bridge linkers for the neutral-P1 dimer
and the basic-P1 dimer bound at the same position, with similar
binding vectors, and interacted with b-tryptase in a similar fashion.
As shown in Scheme 2, the Claisen condensation of 2-acetyl-5-
chloro-thiophene 11 with ethyl oxalate 12 proceeded in the pres-
ence of hydroxylamine hydrochloride to give directly isoxazole
13. Upon reduction of 13 with sodium borohydride, the resulting
alcohol 14 was converted to the mesylate 15, which was further
coupled with ketopiperazine to form compound 16, a monomer
with a neutral-P1 group. Compound 16 was further coupled with
1,10-dichloro-decane under room temperature in DMF, producing
a mixture of monomer 17 and dimer 5, from which the dimeric
compound 5 was separated and purified.
With the same chromogenic assay, no inhibition of b-tryptase,
however, was detected for the neutral-P1 dimer 5. This result,
while disappointing, is consistent with the previous observation

G. Liang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3370–3376
3375
O
O
HONH2HCl
S
Cl
O
+
Cl
S
O
O
O
O
N
96%
O
11
12
13
O
S
S
O
O
O
NaBH4
88%
O
O
O
Cl
Cl
S
S
S
O
O
89%
O
N
O N
14
15
N
N
O
Cl(CH2)10Cl
NaH, DMF
Cl
S
N
O
O
N
89%
N
16
O
N
O
N
N
N
(CH2)10Cl
N
S
+
O
Cl
Cl
Cl
O
N
S
N
Cl
O
N
O
N
5
17
Scheme 2. Reaction scheme for synthesizing compound 5, the dimeric inhibitor with two neutral-P1 groups.
that monomeric compounds with various known neutral-P1 moi-
eties, including the one in the present design, did not exhibit any
meaningful inhibition for b-tryptase. The negative result of
previous monomeric neutral-P1 inhibitors indicates that a neu-
tral-P1 moiety may not be able to offer adequate binding affinity
to a monomeric inhibitor to render its b-tryptase inhibition
detectable. However, what we did not know was how significant
the inadequacy was and whether it was possible to supplement
the inadequacy with the optimization of the rest of the molecule.
The negative result of the present neutral-P1 dimer 5 suggests
that even doubling the binding enthalpy the neutral-P1 moiety
still does not bind with b-tryptase effectively, at least up to the
enforces our hypothesis that alanine at the 190 position is critically
important for the binding of a neutral-P1 inhibitor. With a serine at
the same position, the probability of effective binding of the same
neutral-P1 inhibitor is significantly decreased, if not impossible.
In conclusion, the tetrameric fold of b-trytpase offers an oppor-
tunity for a dimeric inhibitor to span two adjacent substrate-bind-
ing sites. The synergetic binding effect can significantly improve
binding affinity of dimeric inhibitors, which is evidenced by more
than 1000-fold enhancement in Ki values. A group of dimeric inhib-
itors with either benzylamine or benzamidine P1 groups exhibited
sub-nano molar Ki for b-tryptase and tight-binder kinetics. A di-
meric inhibitor designed using the same strategy with two identi-
cal neutral-P1 moieties, however, did not show any detectable
binding, which further demonstrated the important role played
by Ala190 in neutral-P1 binding and casted further doubt on the
possibility of developing neutral-P1 inhibitors for b-tryptase.
detectable level of 30
lM. Although it is theoretically impossible
to rule out the possibility of neutral-P1 binding solely based on
this negative result, the probability of success is significantly
decreased.
This conclusion has been further supported by our in-house
X-ray crystallographic studies. Compounds 1, 2, and 3 were soaked
into b-tryptase crystals with well-defined electron density map,
where compounds 2 and 3 spanning the two adjacent binding sites.
Similar crystallographic experiments were also carried out for both
Compounds 4 and 5 under the same condition, as well as enhanced
conditions generally used for weaker binders, such as increased
compound concentration, prolonged soaking time, and co-crystal-
lization. However, neither compounds 4 nor 5 was ever detected
in the binding pocket of b-tryptase. This negative result further
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