Design, synthesis, and biochemical evaluation of novel α vβ3 integrin ligands

Article abstract of DOI:10.1016/j.bmcl.2004.06.017

Studies on integrin α_vβ₃ have implicated this receptor in a number of pathologies. In this article we describe some of our initial efforts to design small molecules α_vβ ₃ ligands incorporating an indole core tem

Full text of DOI:10.1016/j.bmcl.2004.06.017

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4553–4555
Design, synthesis, and biochemical evaluation of novel
avb3 integrin ligands
Juan Jose Marugan,* Kristin D. Haslow and Carl Crysler
3-Dimensional Pharmaceuticals, Inc, Eagleview Corporate Center, 665 Stockton Drive, Suite 104, Exton, PA 19341, USA
Received 9 April 2004; revised 8 June 2004; accepted 8 June 2004
Available online 3 July 2004
Abstract—Studies on integrin avb3 have implicated this receptor in a number of pathologies. In this article we describe some of our
initial efforts to design small molecules avb3 ligands incorporating an indole core template and an oxyguanidine as basic ending.
Synthesis, biochemical activity and pharmacological properties are analyzed.
Ó 2004Elsevier Ltd. All rights reserved.
The integrins are a family of heterodimeric cell surface
receptors that regulate the attachment of cells to extra-
cellular matrix.1 The dimerization of a and b glyco-
proteins subunits give rise to the heterodimeric
receptors. Both integrin a and b subunits have a single
large extracellular domain, one transmembrane region,
and a short cytoplasmic tail without known enzymatic
activity. The exception to this is the b4subunit, which
has an extended cytoplasmic domain containing four
fibronectin (FN) type III-like domains. Integrins bind to
a variety of extracellular ligands including other trans-
membrane proteins such as IgG-domain proteins in
addition to the extracellular matrix (ECM). The binding
of ligands can modulate a number of intracellular pro-
cesses including activation of focal adhesion kinase
(FAK), integrin-linked kinase (ILK), Abl kinase and
reorganization of the actin cytoskeleton. The integrins
also mediate inside-out signaling whereby cytoplasmic
molecules such as protein kinase C can modulate the
affinity of integrins for their ligands. Because of their
ability to regulate cell mobility and cell–cell interaction
there has been considerable work focused on modula-
ting the activity of integrins over the last decade. Of
particular interest to our research group was the vitro-
nectin receptor avb3. The integrin has been implicated in
osteoclast adhesion and in mobility of endothelial cells
during vascular injury.2 Thus, avb3 emerged as a
potential therapeutic target for the treatment of osteo-
porosis, restenosis, ocular diseases, tumor-induced
angiogenesis, metastasis formation, and sickle cell ane-
mia disease.2;3 A number of research groups have
4
reported small molecule inhibitors of avb3 receptor. In
this paper we describe our initial efforts in the design
and synthesis of novel, nonpeptidic avb3 receptor
antagonists. The selectivity of compounds toward a5b1,
a1b5, and GP IIbIIIa was also evaluated.
Most of the potent avb3 inhibitors reported in the lit-
erature incorporate a RGD mimetic motif: (i) a basic
group at the N-terminus, (ii) a carboxylic acid in the
carboxy terminus, (iii) and a molecular core which can
organize the molecule in the proper spatial geometry.
Although this RGD motif is essential for activity,5 the
zwitterionic nature of the pharmacaphore often becomes
the ‘Achilles’ Heel’ of the inhibitor. The highly charged
compounds usually have high clearance rates and/or
poor oral bioavailability. One potential way to address
this problem is to choose a basic group with a relatively
low pKa (7–8). The oxyguanidine functional group
(pKa ¼ 7–7.5) was chosen as a possible replacement for
the more basic guanidine moiety as the means to a more
oral bioavailable drugs with improved pharmacokinetic
properties.6 A second possible strategy to potentate the
overall charge of the molecule is incorporate a-substi-
tuted amino acids at the acidic end.7 The a-substituent,
usually a sulfonamide or a carbamate, has been shown
to increase the potency while modulating the acidity of
these inhibitors. Based on these results, we designed and
Keywords: Integrin; avb3.
* Corresponding author. Tel.: +1-610-458-5264; fax: +1-610-458-8249;
e-mail: jmaruga1@prdus.jnj.com
0960-894X/$ - see front matter Ó 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.017

4554
J. J. Marugan et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4553–4555
HO
Table 1. Inhibition of avb3-vitronectin interaction
O
O
i, ii
O
O
O
iii, iv, v
N
H
OH
N
H
O
O
R1
O
N
2
N
H
1
OH
H
R2
H
N
H2N
O
O
BocHN
O
O
O
vi
O
a
O
Com-
pounds
R1
R2
avb3 IC50
(nM)
N
NBoc
O
N
H
O
H
3
O
4
NH
NH
H
N
S
O
O
1
3.4
BocHN
O
H N
2
N
H
O
vii, viii
ix, x
NBoc
N
H
HN
O
O
NH
O
HN
S
O
NH
5
S
O
O
O
O
O
O
2
9.0
O
H N
2
N
H
O
H
H2N
N
O
O
NH
NH
O
S
O
NH
3
6.1
N
H
HN
O
H N
2
N
H
Cl
Cl
NH
HN
S
OH
6
O
O
NH
O
S
O
4
10.5
H N
2
N
H
Scheme 1. Reagents and conditions: (i) (trimethylsilyl)diazomethane,
MeOH, 98%; (ii) 2-benzyloxyethanol, DEAD, triphenylphosphine,
THF, 99%; (iii) 10% Pd(C), MeOH, H2, 100%; (iv) N-hydroxyph-
thalamide DEAD, triphenylphosphine, THF, 45%; (v) methylamine,
THF, 96%; (vi) bis-Boc-pyrazole, THF, 97%; (vii) LiOH, MeOH–H2O,
95%; (viii) 3-amino-2-benzenesulfonylamino-propionic acid methyl
ester, DIEA, EDCI, 78%; (ix) LiOH, MeOH–H2O, 97%; (x) TFA,
CH2Cl2, 93%.
NH
O
O
S NH
5
0.4
H N
2
N
H
NH
6
NH2
>20,000
7.2
H N
2
N
H
NH
O
O
O
7
O
N
H
H N
N
H
2
synthesized molecules bearing both an oxyguanidine at
the basic ending and a-substitution at the carboxy ter-
minus.
O
O
8
>20,000
310
O
N
H
N
H
N
H
We looked at the 5-oxy-2-indole carboxylic amide as a
suitable template for the synthesis of avb3 ligands and
the synthesis of these molecules is shown in Scheme 1.
The commercially available 5-hydroxy-2-indole carbox-
ylic acid 1 was transformed to the corresponding methyl
ester using (trimethylsilyl)diazomethane in MeOH. The
methyl ester was coupled with 2-benzyloxyethanol via a
Mitsunobu reaction to yield compound 2. Deprotection
of the benzylic alcohol of 2 was achieved by hydroge-
nation, followed by a Mitsunobu coupling with N-hy-
droxyphthalamide and subsequent deprotection with
methylamine to give O-amine 3. The oxyguanidine
functional group was introduced by the reaction of
compound 3 with bis-Boc-pyrazole to give 4. This was
hydrolyzed using lithium hydroxide and the free car-
boxylic acid was coupled with the previously synthesized
3-amino-2-benzenesulfonylamino-propionic acid methyl
ester,7b using diisopropylethylamine as base and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDCI) as coupling reagent to give indole 5.
Cleavage of the ester, using lithium hydroxide as a base
and deprotection of the Boc group with trifluoroacetic
acid yielded the target compound 6.
NH
O
O
O
9
N
H
H N
2
N
H
NH
10
11
12
>20,000
130
H N
2
N
H
O
NH
NH
O
NH
S
O
O
O
N
H
N
H
O
N
S
O
O
100
N
H
N
H
a In vitro inhibition, as measured by ELISA, of avb3-vitronectin pro-
tein interactions.
Substitution a to the carboxy terminus was found to be
essential to maintain activity (entry 6). Aliphatic (entries
4 and 5) or aromatic (entries 1–3) sulfonamides or car-
bamates (entry 7) gave the most potent inhibitors.
Amide or benzyl substitution alpha to the acid resulted
in a loss of all activity (entries 9 and 10). With regard to
substitution at the basic end, the free O-guanidinium
provides better activity (entries 1 vs 12). Mono-Boc
protection and imidazolyl O-guanidinium analogues
A series of modifications to the two termini was car-
ried out as a means of generating a rapid SAR evalua-
tion. The results from these modifications are listed in
Table 1.

J. J. Marugan et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4553–4555
4555
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Cl
O
Cl
H
N
H2N
O
O
O
N
NH
N
H
H
OH
13
Figure 1. Antagonist with substitution b to the carboxy terminus.
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Table 2. Selectivity profile toward other integrins
IC50 (nM)a
Compounds
avb3
a5b1
1000
>1000
avb5
aIIbbIIIa
1
3.4ND
6.1
100
13
ND
>20,000
3
12
25
170
>20,000
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a In vitro inhibition, as measured by ELISA, of avb3-vitronectin, avb5-
vitronectin, a5b1-fibronectin, and aIIbbIIIa-fibrinogen protein interac-
tions.
retain some of the activity (entries 11 and 12), but
introduction of other functional groups such as the
benzyl urea (entry 8) results in the complete loss of
activity.
Although substitutions alpha to the carboxy terminus
are well tolerated, substitution b to the carboxy termi-
nus had a negative effect on activity (compound 13, avb3
IC50 >20 lM) (Fig. 1).
Several compounds were tested for their selectivity
against other integrins, shown in Table 2. The potencies
for the avb3 receptor are in the nM range, but the
selectivity for aIIbbIIIa is less than 10.
Permeability and pharmacokinetic proprieties of this
series was also determined. None of the compounds
showed good oral bioavailability or desirable PK
parameters. We attributed these negative results to the
high number of heteroatoms contained in the molecules.
In following papers, we will present another indole
series, which address and resolve this problem.
Acknowledgements
We would like to thanks to Jen Kirpatrick for her help
in the determination of some of the IC50 in Table 2, to
Dr. Kevin Moriarty and Dr. Bruce Tomczuk for their
help in the edition of this manuscript.