Design of bivalent ligands using hydrogen bond linkers: Synthesis and evaluation of inhibitors for human β-tryptase

Article abstract of DOI:10.1016/j.bmcl.2004.09.065

Alternative to a covalent linker, hydrogen bonds can be used to link ligands for maintaining simultaneous interactions with multiple binding sites. This is exemplified by the synthesis and evaluation of amide derivatives as potent inhibitors of human β-tryptase. We exploit the concept of using hydrogen bonds to link multiple ligands for maintaining simultaneous interactions with polyvalent binding sites. This approach is demonstrated by the syntheses and evaluation of pseudo-bivalent ligands as potent inhibitors of human β-tryptase.

Full text of DOI:10.1016/j.bmcl.2004.09.065

Bioorganic & Medicinal Chemistry Letters 14 (2004) 6053–6056
Design of bivalent ligands using hydrogen bond linkers:
synthesis and evaluation of inhibitors for human b-tryptase
Roy J. Vaz,a Zhongli Gao,a James Pribish,a Xin Chen,a Julian Levell,a Larry Davis,a
Eva Albert,b Maurice Brollo,b Antonio Ugolini,b Dona M. Cramer,a Jennifer Cairns,a
Keith Sides,a Feng Liu,a Jennifer Kwong,a Jiesheng Kang,a Sam Rebello,a Michael Elliot,a
HengKeang Lim,a Vinolia Chellaraj,a Robert W. Singletona and Yi Lia,*
aAventis Pharmaceuticals, 1041 Route 202/206 N, Bridgewater, NJ 08807, USA
bAventis Pharma, 13 quai Jules Guesde, 94403 Vitry-sur-Seine Cedex, France
Received 9 September 2004; revised 23 September 2004; accepted 23 September 2004
Abstract—We exploit the concept of using hydrogen bonds to link multiple ligands for maintaining simultaneous interactions with
polyvalent binding sites. This approach is demonstrated by the syntheses and evaluation of pseudo-bivalent ligands as potent inhibi-
tors of human b-tryptase.
Ó 2004 Elsevier Ltd. All rights reserved.
Symmetrical molecular assembly of identical subunits is
essential for many biological functions such as ion chan-
nel conductance,1 signal transduction,2 gene expres-
sion,3 and enzyme catalysis.4 There are often symmetry
related multiple binding sites in these assemblies that
are targeted by drug or modulator molecules. Thus,
chemically symmetrical drugs involving identical func-
tional groups separated by a spacer may be of signifi-
cance in order to provide optimal activity.5
illustrated in Scheme 1. In comparison to a covalent
linkage, a hydrogen bond linker may offer greater toler-
ance in terms of both distance and vector of bonding. It
remains the relative size of individual monomers, which
aggregates ideally under the cooperative template effect
only upon binding with the biological molecule.7 We
report here an example of this approach from the syn-
thesis and evaluation of amide derivatives as potent
inhibitors of human b-tryptase.
As an alternative to the common approach of covalently
linking identical functional groups in a drug molecule,6
van der Waals interactions such as hydrogen bonds
may be invoked in constructing a molecular cluster that
is optimal for the corresponding biological molecule as
Human b-tryptase is a trypsin-like serine protease spe-
cific to mast cells and is implicated in the pathogenesis
of allergic and inflammatory diseases.8 The X-ray analy-
sis of its crystal structure revealed a tetrameric architec-
ture consisting of four quasi-identical monomers.9 The
A
H D
A
D H
Monomeric ligand binding
dimeric ligand binding
pseudo-dimeric ligand binding
Scheme 1.
Keywords: Hydrogen bonds; Tryptase; Enzyme inhibitors; Structure-based design.
*
Corresponding author. Tel.: +1 908 231 5862; fax: +1 908 541 5548; e-mail: yi.li@aventis.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.065

6054
R. J. Vaz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6053–6056
OH
O
OH
O
O
HO
NH2
OH
O
O
O
HO
OH
O
S
H
N
H
O
O
O
H
N
O
H
N
HO
H2N
S
NH
HN
N
O
H
N
O O
H
O
H2N
NH
N
OH
HO
O
O
O
O
O
O
O
OH
HO
HO
N
N
H
NH
H
O
HO
O
N
OH
O
N
H
NH2
OH
O
OH
HO
O
O
O
HO
1 (APC-2059)
2
Scheme 2.
tetramer is believed to be the enzymatically active form
of human b-tryptase, although its monomeric form has
recently been reported to be catalytically active.10
NH2
R
~ 23 Å
Many symmetrical inhibitors with dibasic units cova-
lently linked by various bridging skeletons have recently
been reported among potent inhibitors of human mast
cell tryptase.11–13 For example, compound 1, APC-
2059, had reached the stage of clinical development,11b
whereas compound 2 incorporated a rigid cyclodextrin
template as a spacer (Scheme 2).12a
N
O
X
H
O
X
R'
O
R'
O
H
N
Our approach is based on the structural information
obtained from the crystal structure of tetrameric b-tryp-
tase (Fig. 1).9,13h It revealed close contact between two
identical monomeric inhibitors. We envisioned that
incorporation of a primary amide group at the specific
position would provide an almost perfect bridge to link
two inhibitors at the binding sites by forming bifurcated
hydrogen bonds (Scheme 3).
R
X = NH, O
H2N
Scheme 3. Dimeric assembly via hydrogen bond. The distance between
˚
the centroids of benzylamine aromatic rings is about 23A.
We first made compound 3a (Scheme 4). To our sur-
prise, it was a weaker inhibitor of b-tryptase than the
similar compound 3b where the bifurcating primary
amide is replaced by a chloro atom.13d We speculate that
intermolecular hydrogen bonding is insufficient to com-
pensate for the desolvation energy that is required for
compound 3a binding to the enzyme.
Subsequently, compounds 4a–e (Table 1) were synthe-
sized according to Scheme 5. Thus, the acid 5 was con-
densed with thiophene N-hydroxy-acetamidine 6,
prepared by refluxing thiophene acetonitrile with hydr-
oxylamine in methanol, to produce oxadiazole 7. After
hydrolysis of the ester, the acid 8 was obtained. This
acid set the stage for the condensation reaction with
amine template 9 to give compound 10. Removal of
the protection group yielded the desired compound
4a–e.
X
Y
N
O
3a (X = CONH2, Y = H)
H2N
Figure 1. Ribbon rendered tetrameric structures of b-tryptase com-
plexed with inhibitors shown in space-filled spheres (PDB entry:
1AOL).
3b (X, Y = Cl)
Scheme 4. Structures of b-tryptase inhibitors.

R. J. Vaz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6053–6056
Table 1. Tryptase inhibitors binding potency
6055
Compound
R1
R2
Ki (nM) Tryptase
IC50 (lM)
hERG
CYP2C9
CYP2C19
2.19
ND
4a
4b
4c
4d
4e
F
F
F
H
F
H
4.3
59
0.8
NDa
76.8
>30
0.48
ND
>50
CO2Et
CO2H
CONH2
CONH2
88
>50
ND
1.5
1.3
ND
17.1
5.38
9.69
a ND = not determined.
O
O
R2
O
R2
O
HO
R2
HO
HN
NH
O
b
+
a
N
O
S
O
N
S
78%
85%
O
N
HO
N
S
5
6
7
8
NH
R1
c
O
75%
O
N
H
9
O
R1
O
R1
R2
R2
N
N
d
O
H2N
O
O
N
88%
N
N
O
N
N
H
S
S
4
10
Scheme 5. Syntheses of the pseudo-dimer compounds. Reagents and conditions: (a) (1) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCÆHCl), (2) toluene, 120°C; (b) (1) LiOH, dioxane, H2O, rt, 2h, (2) NH4Cl, DMF, O-[(ethoxycarbonyl)cyanomethylenamino]-
N,N,N0,N0-tetramethyluronium tetrafluoroborate (TOTU), Pr2NEt, 0°C to rt, overnight, (3) LiOH, dioxane, H2O, rt, 2h; (c) DMF, TOTU, Pr2NEt,
0°C to rt, overnight; (d) HCl, dioxane, rt.
Compounds 4a–e were tested in a tryptase assay14 and
the results are summarized in Table 1. From Table 1,
it is apparent that the compounds with the hydrogen
bond formation capability showed increased potency
(compare 4a–b vs 4d–e). Since carboxylic groups are sel-
dom found to be in proximity in protein structures,
except for those involving special functions as in the
active site of an aspartyl protease, a favorable inter-
action between two ionized carboxylic acids is unlikely.
Thus, it is not surprising that 4c is less potent than the
corresponding amide 4e. Preliminary X-ray crystallo-
graphic study of the complex between b-tryptase enzyme
and 4e revealed indeed the assembly of the expected
dimer via hydrogen bonds between the two inhibitors,
which contributed to the increased potency.
proved profile against the hERG channel blockage. In-
deed, the experimental measurements16 indicated these
compounds, as compared to compound lacking the
bifurcating amide/acid substituent, are less potent inhib-
itors against the hERG channel, CYP 2C9 and 2C19
isozymes.
In conclusion, we have demonstrated that hydrogen
bonds, instead of a covalent bond, may be invoked in
constructing a molecular cluster that is optimal for the
corresponding biological molecule. As compared to
dimers linked by a covalent bond, the pseudo-dimer
approach via hydrogen bond may offer some advantages
in terms of lower molecule weight, preferable solubility
and pharmacokinetic property.
To assess the potential likelihood of drug-induced QT
prolongation and drug–drug interactions, compounds
4a–e were profiled in hERG channel and CYP inhibition
assays. According to the predictions of our QSAR
CoMSiA model,15 the amides were expected to have im-
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