Substituted benzocyloheptenes as potent and selective alpha(v) integrin antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00696-0

A novel series of potent and specific alpha(v) integrin antagonists has been obtained by aminoalkyl substitutions on benzocyloheptene acetic acids as a rigid GD bioisostere. The preferred compounds 1-2, 1-3 and 1-8, showed nano- to subnanomolar IC(50) values on alpha(v)beta(3) and alpha(v)beta(5) integrins, with favorable pharmacokinetics.

Full text of DOI:10.1016/S0960-894X(02)00696-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3291–3296
Substituted Benzocyloheptenes as Potent and Selective
ꢀv Integrin Antagonists
Francoise Perron-Sierra,a,* Dominique Saint Dizier,a Marc Bertrand,b
Annie Genton,a Gordon C. Tuckera and Patrick Casaraa,*
aInstitut de Recherches Servier, 125 chemin de Ronde, 78290 Croissy sur Seine, France
b
´
Technologie Servier, 25–27 rue E. Vignat, 45007 Orleans, France
Received 21 June 2002; revised 12 August 2002; accepted 19 August 2002
Abstract—A novel series of potent and specific av integrin antagonists has been obtained by aminoalkyl substitutions on benzo-
cyloheptene acetic acids as a rigid GD bioisostere. The preferred compounds 1–2, 1–3 and 1–8, showed nano- to subnanomolar IC50
values on avb3 and avb5 integrins, with favorable pharmacokinetics.
# 2002 Elsevier Science Ltd. All rights reserved.
Integrins belong to a large class of ubiquitous hetero-
dimeric cell surface glycoproteins involved in cell–cell
and cell–matrix interactions that are for the most part
mediated by the recognition of a tripeptidic RGD-
sequence present in the extracellular ligands.1
point to constrained orientation of the glycine moiety,
may be by intramolecular hydrogen bond, forming a
g-turn a (Fig. 1).10 This twisted conformation of the
RGD recognition sequence is an attractive hypothesis
for the design of avb3 and avb5 antagonists selective
versus the aIIbb3 integrin (Fig. 1). Compounds 1 are
proposed as constrained RGD bioisosteres, potentially
specific for av integrins. The benzocyloheptene acetic
acid replaces the GD g-turn a, ensuring conformational
rigidity, while various aminoalkyl side chains replace
the arginine (Fig. 1).
Upregulation of specific av-integrins such as avb3 and
avb5 (vitronectin receptors) has been associated with
various pathologies including arthritis2 and metastatic
cancer.3 Therefore regulation of these integrin receptors
has been acknowledged as a valuable therapeutic
approach.
Although numerous publications and patents have been
issued in this field,4 few integrin antagonists, among
them the cyclopeptide Cilengitide5 for cancer and the
peptidomimetic SB2730056 for osteoporosis, are in
clinical development.
Chemistry
A retrosynthetic analysis correlates 1 to the GD biomi-
metic 2, using classical coupling reactions. This amine 2
can derive from aldehyde 3, produced via one carbon
homologation of ketone 4. The racemic form of com-
pound 4 was generated from mono Michael addition on
the symmetrical benzocycloheptadienone 5, readily
obtained by a described procedure starting from
dibromo-o-xylene (Fig. 2).11
Because some integrins are also implicated in essential
phenomena, such as blood coagulation via the aIIbb3
receptor,7 selectivity of av integrin antagonists is essen-
tial for their development as drugs. For this purpose,
several cyclic and acyclic peptidomimetic derivatives
have been investigated.8
The monoalkylation of ketone 5, was controlled by
combination with a TBS-ketene acetal, at low tempera-
ture, in the presence of catalytic mercury iodide, to give
the corresponding TBS-enol ether 6.12 This intermediate
was then carefully hydrolysed with etheral hydrochloric
acid, to give ketone 4 in high yield. The one carbon
homologation of ketone 4 was achieved by 1,2-addition
Crystal structures of the truncated avb3 integrin9 and
the conformational analyses of cyclopeptide antagonists
*Corresponding author. Fax: +33-1-5572-2470; e-mail: patrick.
casara@fr.netgrs.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00696-0

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F. Perron-Sierra et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3291–3296
Figure 1. Integrin ligand and g-turn mimic 1.
Figure 2. Retrosynthesis.
of chloromethyl-TMS anion to form an epoxide inter-
mediate 7, which was quantitatively isomerized into
aldehyde 3, on silica gel.13 Various conditions of reduc-
tive aminations of aldehyde 3 led to partially isomerized
conjugated amine.14 Thus the stepwise procedure, com-
prising reduction of 3 in allylic alcohol,15 bromina-
tion,16 substitution with potassium phthalimide
followed by hydrazinolysis produced the amine 2,17 in
good overall yield, without double bond isomerisation
(Scheme 1).
non-substituted pyridylaminoalkyl acid species, the
corresponding triBOC derivatives 8–1 to 8–3 were used
and coupling with 2 could then be performed without
catalysis.20
Thiazole and benzimidazole containing derivatives 8–4
and 8–5 were produced in fair yield via Mitsunobu
coupling reaction of the appropriate activated N-BOC
heterocycles with g-hydroxybutyric amide,21 and sub-
sequent amide protection. The N-methylpyridylamino
butyric acid derivative 8–6, obtained directly from
2-fluropyridine, can not form a lactam and was used
without further protection (Scheme 2).
In the design of bioavailable vitronectin receptors
antagonists, it has been shown that the guanidine moi-
ety could be replaced advantageously by much less basic
amino heterocycle surrogates.18
The pyridylmethylamino derivative, 8–7, was synthe-
sised via Michael addition of the requisite methylami-
nopyridine onto acrylamide and subsequent protection
(Scheme 3).
In order to validate our conformational hypothesis, we
envisaged to couple synthon 2 with o-aminoalkyl-
carboxylic acid containing these various guanidinomi-
metics. Pyridylaminoalkyl side chains were readily
obtained from 2-chloropyridine-N-oxide and appro-
priate commercial b, g or d-aminoalkylcarboxylic acids
to give 8–1 to 8–319 (n=2–4, respectively) (Scheme 2).
To avoid any intramolecular lactam formation in the
The corresponding tetrahydronaphtyridyl derivative
8–8, also used as guanidine substitute,22 was produced
as described, via a base-catalysed ring closure of 2-ami-
nonicotaldehyde23 with methyl ketobutyric acid and
partial hydrogenation.24
Scheme 1. Synthesis of synthon 2: (i) CH2¼C(OtBu)(OTBS), HgI2, CH2Cl2, ꢀ78 ꢁC; (ii) 1N HCl, rt, 6 h (4, 95%); (iii) secBuLi, ClCH2TMS,
TMEDA, THF, ꢀ78 ꢁC; (iv) SiO2 (3, 95%); (v) (a) NaBH4, MeOH, CH2Cl2, 0 ꢁC, 1.5 h, (100%); (b) PPh3, CBr4, CH2Cl2, 0 ꢁC–rt, 2 h (50%); (c)
PhtNK, DMF, 40 ꢁC, 4 h, (60%); (d) NH2NH2, MeOH, CH2Cl2, rt, 48 h (2, 90%).

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3293
Scheme 2. Pyridylamino, thiazol and benzimidazol alkylcarboxylic derivatives: (i) (a) NaHCO3, H2O, 100 ꢁC; (b) AcCl, MeOH, 80 ꢁC, 2 h; (c) NH3,
MeOH, CH2Cl2, 90 ꢁC, 72 h; (d) BOC2O, tBuOH; (e) H2, Pd/C, rt, 72 h; (f) BOC2O, DMAP, rt, 72 h (n=2, 8–1, 12%; n=3, 8–2, 27%; n=4, 8–3,
8%); (ii) (a) DEAD, PPH3, DMF, rt, 24 h; (b) BOC2O, DMAP, CH3CN, rt, 48 h (n=3, 8–4, 37%; n=4, 8–5, 22%); (iii) NaHCO3, pyridine, H2O,
120 ꢁC, 120 h (8–6, 30%).
Biological and Pharmacological Data
The evaluation of the binding activities of these com-
pounds was measured on the human receptors avb3,
avb5 (vitronectin displacement)28,29a and aIIbb3 receptor
Scheme 3. Pyridylmethylamino derivative: (i) (a) acrylamide, 60 ꢁC, 24
h; (b) BOC2O, tBuOH, rt, 24 h; (c) BOC2O, DMAP (8–7, 21%).
(fibrinogen displacement),30 with comparison to the
clinical candidate Cilengitide (Table 1). Cellular adhe-
sion inhibition was also evaluated with the IGROV-1
cell line,29b,31 which interacts with vitronectin via av
integrin receptors. In vitro pharmacokinetic parameters
were determined using hepatic microsomes (rat/human)
to predict first pass metabolism and metabolic bioavail-
The various side chains 8–1 to 8–8 were combined with
amine 2 in the appropriate manner to afford, after
hydrolysis, the corresponding racemic acids 1–1 to 1–8
(Scheme 4); The cyclic amidines 1–9 and 1–10 were
synthesised via treatment with the appropriate hetero-
cyclic isothiouronium salt with the free amine 9.25 The
elaboration of this g-aminobutyryl side chain was per-
formed via combination of 2 with 4-bromobutanoyl
chloride, and subsequently, under classical conditions
via azide substitution and reduction,26 leading to the
corresponding amine 9 (Scheme 4, Table 1).27
ability (MF%),32a,b and Caco2 cell monolayers to pre-
dict oral absorption (A%) from permeability data
(Table 1).32c
In the pyridylamino derivatives, the best affinities were
obtained for the propyl and the butyl side chain, 1–2
and 1–3 respectively, with a preference for the propyl,
considering its better selectivity over aIIbb3 receptor and
activity in the cellular assay. Moreover, all these com-
pounds were found to be stable in the microsomal
assay, with good absorption (Table 1).
Scheme 4. Synthesis of compounds 1–1 to 1–10: (i) (a) CH2Cl2, rt, 24 h, [Y=N(BOC)2] or HOBT, DIEA, CH2Cl2, rt, 24 h, (Y=OH); (b) HCl, Et2O
(1–1 to 1–8, yield Table 1); (ii) (a) ClCO(CH2)3Br, Et3N, THF, 0 ꢁC, 3 h; (b) NaN3, DMF, 40 ꢁC, 12 h; (c) PPh3, THF, H2O, 12 h (9, 88%); (iii) (a)
Et3N, DMF, 50 ꢁC, 12 h (m=1, 2); (b) Et2O, HCl (m=1, 1–9; m=2, 1–10, yield Table 1).

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F. Perron-Sierra et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3291–3296
Table 1. Structures and biological data of compounds 1–1 to 1–10
No.
n
X
Yield
(%)
Binding28,29a,30 IC50, nM
Cell adhesion29b,31 IC50, nM
Stability MF
(%) rat/human
Absorption A
(%)
avb3
2.3
0.4
584
avb5
34
0.8
22
aIIbb3
860
110
Cilengitide5ꢀ10
Found
1–1
36
338
100/100
94/75
14
93
2
3
4
3
3
3
2
3
3
3
53
79
47
72
60
82
10
44
20
28
1700
1–2
1–3
1–4
1–5
1–6
1–7
1–8
1–9
1–10
1.9
2.7
0.8
4360
1200
>104
1020
667
2.0
12
83/77
77/65
95
96
0.9
45
20
40
100/100
87/94
96
0.6
0.5
1.8
<20
94
68
40
2034
2700
6.9
75/88
842
119
552
98/100
72/72
23
0.3
0.3
1495
110
92
0.3
0.2
0.2
0.1
18
86/100
92/92
<20
<20
330
2.7
Therefore, the replacement of the pyridylamine moiety
by various guanidine surrogates was performed on 1–2
preferentially.
density at the guanidinomimetic position of the RGD
sequence.18
Conversely, replacement of pyridylamino moiety in 1–2
by tetrahydronaphtyridine to give 1–8, increased recep-
tor affinities and maintained selectivity over aIIbb3 and
pharmacokinetics properties.
Introduction of a thiazole ring to give 1–4, entailed a
great loss of affinities on both av receptors. Interest-
ingly, a gain of one order of magnitude in av affinities
was observed for the benzimidazole, imidazoline and
tetrahydropyrimidine analogues 1–5, 1–9 and 1–10
respectively. However, despite a good cellular activity,
those compounds were not further examined due to an
absorption prediction below 20%.
The pharmacokinetic parameters were validated in vivo
in mice for two representative compounds, 1–2 and 1–5.
With a bioavailability of 71 and 16%, respectively, they
correlate nicely with the in vitro predictions, pointing
out the poor absorption of 1–5.
More specific modifications of the pyridylamine of 1–2,
such as N-methylation or nitrogen/carbon inversion to
give 1–6 or 1–7, decreased av affinities by more than one
or two orders of magnitude respectively. These data
confirmed the prevalence of steric effect over charge
For most compounds, adhesion and binding potencies
correlated well. However, for presently unexplained
reasons, several products were less efficient in cellular
assays with up to a 100-fold difference. Whether this

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3295
reflects a differential accessibility of these compounds to
the integrin binding site on the purified integrin versus
its native conformation at the cell surface needs to be
investigated.
6. Badger, A. M.; Blake, S.; Kapadia, R.; Sarkar, S.; Levin, J.;
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Cellular adhesion data revealed a potent inhibitory
activity for the pyridylamino compounds, 1–2 and 1–3,
and for the naphtyridine derivative 1–8 (Table 1) and
could favorably be compared with Cilengitide and have
subsequently been chosen for further in vivo evaluation.
Their asymmetric synthesis is in progress to obtain their
corresponding active enantiomer, as expected from ste-
reospecific receptor recognition for the (S)-aspartic acid
in the RGD sequence.33
9. (a) Xiong, J. P.; Stehle, T.; Diefenbach, B.; Zhang, R.;
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Conclusion
The design and synthesis of RGD peptidomimetic ben-
zocycloheptene derivatives 1 as potent vitronectin recep-
tor antagonists were described. A convergent synthetic
route allowed incorporation of various arginomimetics,
via coupling with the key synthon 2. Structure–activity
study revealed an optimal three to four carbon atom
spacer between the ‘GD’ scaffold and the guanidine
bioisosteres. The preferred compounds, namely pyr-
idylamino 1–2, 1–3 and tetrahydronaphtyridyl sub-
stituted derivative 1–8, showed nano- to subnanomolar
IC50 values on avb3 and avb5 integrins, with good selec-
tivity over the aIIbb3 fibrinogen receptor. Moreover the
favorable pharmacokinetics of these compounds justify
further pharmacological studies.
19. Tortorella, V.; Bettoni, G. Gazz. Chim. Ital. 1966, 97, 85.
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Acknowledgements
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The authors wish to thank Sophie Sciberras, Christine
Fouache, Sandra Haumont and Delphine Peyroulan-
Thorel for their skilful technical assistance and Solange
Huet for the preparation of the manuscript. The con-
tinuous support of Pr. John Hickman is also greatly
acknowledged.
22. Meissner, R. S.; Perkins, J. J.; Duong, L. T.; Hartman,
G. D.; Hoffman, W. F.; Huff, J. R.; Ihle, N. C.; Leu, C.-T.;
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3296
F. Perron-Sierra et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3291–3296
adhere exclusively through avb5. The cells were preincubated
with the compounds, and adhesion was evaluated by image
analysis after washing, fixation and crystal violet staining.
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after fitting the dose–response sets of at least two experiments
per product (with comparable results) with a sigmoid curve
with variable slope. For clarity, 95% confidence intervals are
not shown.
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