Synthesis of enantiomerically pure αvβ3 integrin ligands based on a 5,6-dihydropyridin-2-one scaffold

Article abstract of DOI:10.1016/j.tetasy.2005.12.006

The fundamental importance of α_vβ₃ integrin in a diverse range of biological processes, makes the search for new ligands of this receptors a significant therapeutic goal. We herein report our initial results on the synthesis of 5,6-d

Full text of DOI:10.1016/j.tetasy.2005.12.006

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 17 (2006) 167–170
Synthesis of enantiomerically pure avb3 integrin ligands
based on a 5,6-dihydropyridin-2-one scaffold
Fides Benfatti,a Giuliana Cardillo,a,* Serena Fabbroni,a Luca Gentilucci,a
Rossana Perciaccante,a Alessandra Tolomelli,a,* Monica Baiulab and Santi Spampinatob,*
a
`
Dipartimento di Chimica ‘G. Ciamician’, Universita di Bologna, Via Selmi 2, 40126 Bologna, Italy
b
`
Dipartimento di Farmacologia, Universita di Bologna, Via Irnerio 48, 40126 Bologna, Italy
Received 25 October 2005; revised 7 December 2005; accepted 8 December 2005
Available online 23 January 2006
Abstract—The fundamental importance of avb3 integrin in a diverse range of biological processes, makes the search for new ligands
of this receptors a significant therapeutic goal. We herein report our initial results on the synthesis of 5,6-dihydropyridin-2-one based
ligands, containing a rigid heterocyclic core and two appendages mimicking arginine and aspartic acid moieties. In particular, we
explored the influence of the scaffold stereochemistry on bioactivity, performing SK-MEL-24 cell-fibronectin adhesion tests on dia-
stereoisomers, that differ in the configuration at C6.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
migration along the bone surface.6 For these reasons,
the reduction of osteoclast activity by antagonism of
integrin avb3, offers a potential therapy for the preven-
tion and treatment of osteoporosis.7
Many functions in multicellular organisms involve com-
plex interactions between cells and the extracellular
matrix (ECM).1 Integrin-mediated cell adhesion is there-
fore of fundamental importance in a diverse range of
biological processes, including cell differentiation,
embryonic cell migration, maintenance of tissue integ-
rity and blood coagulation.2
Linear and cyclic peptides,8 containing the RGD
sequence, have been reported as excellent ligands of inte-
grin avb3 and have significant therapeutic potential but
serious limitations, especially for oral dosing. The need
for antagonists with higher bioavailability and lower
molecular weight has prompted several research groups
to develop small constrained non-peptidic molecules
mimicking the RGD motif, which would be more prom-
ising for drug development.9 Most of the structures pro-
posed so far, share a common pattern, consisting of a
polyfunctionalized rigid core, linked to appendages cor-
responding to arginine and aspartic acid side chains.
The basicity and length of the arginine-mimicking group
was found to play a central role.10 Moreover, the pres-
ence of a carboxylic function, mimicking the aspartic
acid residue in the original binding motif, is a fundamen-
tal feature to create a ionic interaction with the metal
cation in the receptor active site.11
In the family of integrin, the avb3 is one of the more
versatile receptors since it binds through the tripeptide
sequence RGD (Arg-Gly-Asp) to a number of ECM
components such as fibronectin, fibrinogen, vitronectin
and osteopontin.3 This integrin is expressed on several
malignant tumour infiltrating endothelial cells and rep-
resents an attractive investigational target for therapeu-
tic intervention.4 In addition, the preferential expression
of avb3 integrin on new forming blood vessels enhances
its potential application as an antitumour target, since
tumour proliferation and metastasis are highly depen-
dant on neovascularization.5 The avb3 receptor is also
highly expressed in osteoclasts, the cells responsible for
bone resorption, and it is thought to be involved both
in cellular adhesion of osteoclasts and in regulating their
We envisaged 3-bromo-5,6-dihydropyridin-2-one12 as a
scaffold that could be converted into a potential avb3
integrin ligand introducing a carboxylic acid and a
guanidinic appendage (Fig. 1). Based on these premises,
we embarked on synthetic efforts to explore whether this
*
Corresponding authors. Tel.: +39 051 2099570; fax: +39 051 2099456
(G.C.); e-mail: giuliana.cardillo@unibo.it
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.12.006

168
F. Benfatti et al. / Tetrahedron: Asymmetry 17 (2006) 167–170
Figure 1. Racemic peptidomimetic 1, containing 5,6-dihydropyridin-2-
one as a rigid core.
scaffold could adopt a rigid conformation, amenable for
the interaction with the integrin binding site and we syn-
thesized racemic compound 1.13
2. Results and discussion
Scheme 1. Synthesis of 3-bromo-4-methyl-5,6-dihydropyridin-2-ones
4a–4b.
The activity and selectivity of 1 on RGD recognizing
integrins in a cellular environment was evaluated per-
forming cell adhesion assays both on melanoma cell
with H2 on Pd/C allowed compound 7a to be obtained.
This compound, which has an amino function on the
aromatic ring, was easily transformed into the Boc-pro-
tected guanidine derivative 8a by treatment with carb-
oxyamidine. Finally, hydrolysis of the methyl ester
with LiOH, followed by treatment with trifluoroacetic
acid to remove the Boc-protecting groups, afforded 9a,
which was further purified on a cationic exchange resin
(Scheme 2).
line SK-MEL-24 (avbPOS 14 and human erythroleukemic
)
3
cell line K562 (a5bPOS).15 The results obtained are mod-
1
erate (this compound inhibited SK-MEL-24 cell
adhesion to FN-coated wells showing an IC50 of
1.2 · 10ꢀ4 M; it inhibited only 27% K562 cell adhesion
to FN-coated wells at 10ꢀ5 M concentration), neverthe-
less suggested that the substrate bound to the receptor
and could be used as a model to evaluate the influence
of the scaffold stereochemistry on the interaction with
integrins.
The same reaction sequence was performed on com-
pound 4b to obtain the diastereoisomer 9b.
In order to gain further insight into the nature of ligand–
receptor interactions, we focused our attention on
the preparation of enantiomerically pure 3-bromo-4-
methyl-5,6-dihydropyridin-2-ones to be exploited as
rigid cores for novel RGD mimetics and studied the
influence of the stereochemistry at the C6 of the scaffold
on the bioactivity.
To evaluate the activity of these optically active novel
compounds in a cellular environment, we tested their
ability to hinder initial cell attachment mediated by
avb3 integrin, by using a cell adhesion assay. The inte-
grin ligand fibronectin was immobilized on tissue culture
plates and the adhesion of human melanoma cell line
SK-MEL-24 (avbPOS
)
analyzed in the presence or
14
3
Ketenes have long been used in the synthesis of various
heterocyclic compounds.16 We have recently reported a
good approach to the synthesis of 3-bromo-4-methyl-
5,6-dihydropyridin-2-ones by treatment of 2-bromo-3-
methyl-2-butenoyl chloride 2 and TEA in the presence
of an appropriate Schiff base 3.12 Starting from the
chiral imine 3, deriving from the condensation of benz-
aldehyde and (S)-p-nitro-phenylethylamine,17 dihydro-
pyridin-2-ones (10S,6R)-4a and (10S,6S)-4b were
obtained in 88% yield and 63:37 d.r. (Scheme 1). The
diastereomeric mixture was easily separated by flash
chromatography.
absence of the peptidomimetics. The results are summa-
rized in Table 1. The inhibitory activity of the well-
known avb3 integrin antagonist H3528 was measured
as a positive control.18
The data reported in the table show that no significant
difference was found between the diastereomeric (6R)-
and the (6S)-derivatives. Nevertheless compound 9a
with the (6R)-configuration seems to be slightly more
active. This behaviour suggests that the configuration
at C6 is not crucial for activity.
A likely interaction of compound 9a with the avb3 inte-
grin binding site is reported in Figure 2. The appendages
of dihydropyridinone 9a have been arranged by super-
imposition to the corresponding side chains of
c(RGDf-N[Me]V) in the binding region of avb3 integrin
extracellular domain.19,20 The dihydropyridinone inserts
into a crevice between the a and b subunits, disposing
the aromatic ring linked to C6 towards the external
space.
The presence of the bromide at C3 allows easy introduc-
tion of an appendage mimicking the aspartic acid func-
tion, while the nitro group on the aromatic ring is a
useful precursor of the arginine mimic (Fig. 1). Com-
pound 5a was obtained by reaction of 4a with allylamine
at rt.12 Treatment of 5a with malonyl chloride in CH2Cl2
in the presence of TEA, afforded, in good yield, malonyl
derivative 6a (Scheme 2). Reduction of the nitro group

F. Benfatti et al. / Tetrahedron: Asymmetry 17 (2006) 167–170
169
Scheme 2. Synthesis of compound 8a. Reagents and conditions: (a) allylamine, 48 h, reflux; (b) TEA (1.5 equiv), malonyl chloride (1 equiv), CH2Cl2,
0 ꢁC to rt; (c) Pd/C (30 mg/mmol), H2 (1 atm), MeOH, rt; (d) N-Boc-1H-pyrazol-1-carboxyamidine (1.2 equiv), DMF, rt; (e) LiOHÆH2O (3 equiv),
THF/MeOH/H2O (3.6:1:1); (f) TFA, rt; (g) Dowex, MeOH, NH4OH.
Table 1. SK-MEL-24 cell-fibronectin (avb3 mediated) adhesion inhi-
bition results for compounds 9a and 9b
a cellular environment, showed a small effect of the scaf-
fold stereochemistry on the bioactivity. Further modifi-
cations will be addressed to the nature and length of the
arginine and aspartate mimicking branches.
Compounds
Adhesion inhibitiona IC50
9a
9b
(8.1 1.1) 10ꢀ5 M
(1.1 0.3) 10ꢀ4 M
(1.5 0.2) 10ꢀ10 M
H3528
Acknowledgements
a Concentration necessary to inhibit cell attachment to extracellular
matrix coated with fibronectin to 50% of the control. Each value is
the mean of at least three separate experiments carried out in
quadruplicate.
We thank MIUR (PRIN 2004), CNR-ISOF and Uni-
versity of Bologna (Funds for selected topics) for finan-
cial support. Mr. Andrea Garelli and Mr. Alessandro
Mignogna are gratefully acknowledged.
References
1. (a) Curley, G. P.; Blum, H.; Humphries, M. J. Cell. Mol.
Life Sci. 1999, 56, 427–441; (b) Gottschalk, K.-E.; Kessler,
H. Angew. Chem., Int. Ed. 2002, 41, 3767–3773.
2. (a) Marchisio, P. C. Adv. Neur. 1991, 1, 214–234; (b)
Coutre, S.; Leung, L. Ann. Rev. Med. 1995, 46, 257–265;
(c) Georges-Labousse, E.; Messaddeq, N.; Yehia, G.;
Cadalbert, L.; Dierich, A.; LeMeur, M. Nature Gen. 1996,
13, 370–373.
3. (a) Anderson, D. H.; Johnson, L. V.; Hageman, G. S. J.
Comp. Neurol. 1995, 360, 1–16; (b) Horton, M. A. Int. J.
Biochem. Cell Biol. 1997, 29, 721–725; (c) Henry, C.;
Moitissier, N.; Chapleur, Y. Mini Rev. Med. Chem. 2002,
2, 531–542.
4. Buckley, C. D.; Simmons, D. L. Mol. Med. Today 1997, 3,
449–456.
5. Haubner, R.; Finsinger, D.; Kessler, H. Angew. Chem.,
Int. Ed. 1997, 36, 1374–1389.
Figure 2. Possible interactions of 5,6-dihydropyridin-2-one 9a with key
residues in avb3 integrin receptor. Blue labels correspond to residues in
the av subunit and red ones to residues in the b3 chain.
6. (a) Duong, L. T.; Rodan, G. A. Front. Biosci. 1998, 3,
d757–d768; (b) Coleman, P. J.; Brashear, K. M.; Askew,
B. C.; Hutchinson, J. H.; McVean, C. A.; Duong, L. T.;
Feuston, B. P.; Fernandez-Metzler, C.; Gentile, M. A.;
Hartman, G. D.; Kimmel, D. B.; Leu, C.-T.; Lipfert, L.;
Merkle, K.; Pennypacker, B.; Prueksaritanont, T.; Rodan,
G. A.; Wesolowski, G. A.; Rodan, S. B.; Duggan, M. E. J.
Med. Chem. 2004, 47, 4829–4837.
3. Conclusion
In conclusion, although the activity values are not satis-
factory, these compounds show a promising interaction
with the receptor, confirming that 5,6-dihydropyridin-2-
one could represent a useful scaffold in the preparation
of a novel class of avb3 integrin. Attachment assays in

170
F. Benfatti et al. / Tetrahedron: Asymmetry 17 (2006) 167–170
7. (a) Lark, M. W.; Stroup, G. B.; Hwang, S. M.; James, I.
10. (a) Nicolaou, K. C.; Trujillo, J. I.; Jandeleit, B.; Chibale,
K.; Rosenfeld, M.; Diefenbach, B.; Cheresh, D. A.;
Goodman, S. L. Bioorg. Med. Chem. 1998, 6, 1185–
1208; (b) Pitts, W. J.; Wityak, J.; Smallheer, J. M.; Tobin,
A. E.; Jetter, J. W.; Buynitsky, J. W.; Harlow, P. P.;
Solomon, K. A.; Corjay, M. A.; Mousa, S. A.; Wexler, R.
R.; Jadhav, P. K. J. Med. Chem. 2000, 43, 27–40.
11. Neustad, B. R.; Smith, E. M.; Lindo, N.; Nechuta, T.;
Bronnenkant, A.; Wu, A.; Armstrong, L.; Kumar, C.
Bioorg. Med. Chem. Lett. 1998, 8, 2395–2398.
12. Cardillo, G.; Fabbroni, S.; Gentilucci, L.; Perciaccante,
R.; Piccinelli, F.; Tolomelli, A. Tetrahedron 2004, 60,
5031–5040.
13. Compound 1 was prepared starting from 2-bromo-3-
methyl-2-butenoyl chloride and N-benzyl imine following
the procedure reported later for 9a–9b.
E.; Rieman, D. J.; Drake, F. H.; Bradbeer, J. N.; Mathur,
A.; Erhard, K. F.; Newlander, K. A.; Ross, S. T.; Salyers,
K. L.; Smith, B. R.; Miller, W. H.; Huffman, W. F.;
Gowen, M. J. Pharmacol. Exp. Ther. 1999, 291, 612–617;
(b) Miller, W. H.; Alberts, D. P.; Bhatnagar, P. K.;
Bondinell, W. E.; Callahan, J. F.; Calvo, R. R.; Cousins,
R. D.; Erhard, K. F.; Heerding, D. A.; Keenan, R. M.;
Kwon, C.; Manley, P. J.; Newlander, K. A.; Ross, S. T.;
Samanen, J. M.; Uzinskas, I. N.; Venslavsky, J. W.; Yuan,
C. C.-K.; Haltiwanger, R. C.; Gowen, M.; Hwang, S.-W.;
James, I. E.; Lark, M. W.; Rieman, D. J.; Stroup, G. B.;
Azzarano, L. M.; Salyers, K. L.; Smith, B. R.; Ward, K.
W.; Johanson, K. O.; Huffman, W. F. J. Med. Chem. 2000,
43, 22–26.
8. (a) Muller, G.; Gurrath, M.; Kessler, H. Angew. Chem.,
Int. Ed. Engl. 1992, 31, 326–328; (b) Belvisi, L.; Bernardi,
A.; Checchia, A.; Manzoni, L.; Potenza, D.; Scolastico,
C.; Castorina, M.; Capelli, A.; Giannini, G.; Carminati,
P.; Pisano, C. Org. Lett. 2001, 3, 1001–1004.
14. Caltabiano, S.; Hum, W. T.; Attwell, G. J.; Gralnick, D.
N.; Budman, L. J.; Cannistraci, A. M.; Bex, F. J. Biochem.
Pharmacol. 1999, 58, 1567–1578.
15. Lundell, B. I.; McCarthy, J. B.; Kovach, N. L.; Verfaille,
C. M. Blood 1996, 87, 2450–2458.
9. (a) Fisher, M. J.; Gunn, B.; Harms, C. S.; Kline, A. D.;
Mullaney, J. T.; Nunes, A.; Scarborough, R. M.; Arfsten,
A. E.; Skelton, M. A.; Um, S. L.; Uttenback, B. G.;
Jakubowski, J. A. J. Med. Chem. 1997, 40, 2085–2101; (b)
Breslin, M. J.; Duggan, M. E.; Halczenko, W.; Fernandez-
Metzler, C.; Hunt, C. A.; Leu, C.-T.; Merkle, K. M.;
Naylor-Olsen, A. M.; Prueksaritanont, T.; Stump, G.;
Wallace, A.; Rodan, S. B.; Hutchinson, J. H. Bioorg. Med.
Chem. Lett. 2003, 13, 1809–1812; (c) Bourguet, E.;
Banares, J.-L.; Parello, J.; Lusinghi, X.; Girare, J.-P.;
Vidal, J.-P. Bioorg. Med. Chem. Lett. 2003, 13, 1561–1564;
(d) Perkins, J. J.; Duong, L. T.; Fernandez-Metzler, C.;
Hartman, G. D.; Kimmel, D. B.; Leu, C.-T.; Lynch, J. J.;
Prueksaritanont, T.; Rodan, G. A.; Rodan, S. B.; Duggan,
M. E.; Meissner, R. S. Bioorg. Med. Chem. Lett. 2003, 13,
4285–4288; (e) Biltresse, S.; Attolini, M.; Dive, G.; Cordi,
A.; Tucker, G. C.; Marchand-Brynaert, J. Bioorg. Med.
Chem. 2004, 12, 5379–5393; (f) Zartman, A. E.; Duong, L.
T.; Fernandez-Metzler, C.; Hartman, G. D.; Leu, C.-T.;
Prueksaritanont, T.; Rodan, G. A.; Rodan, S. B.; Duggan,
M. E.; Meissner, R. S. Bioorg. Med. Chem. Lett. 2005, 15,
1647–1650.
16. (a) Tidwell, T. T. Acc. Chem. Res. 1990, 23, 273–279; (b)
Hyatt, J. A.; Raynolds, P. W. In Organic Reactions;
Paquette, L. A., Ed.; John Wiley: New York, 1994; Vol.
45, pp 159–646; (c) Orr, R. K.; Calter, M. A. Tetrahedron
2003, 59, 3545–3565.
17. Ollis, D. W.; Rey, M.; Sutherland, I. O. J. Chem. Soc.,
Perkin Trans. 1 1983, 1009–1027.
18. Fujii, H.; Komazawa, H.; Mori, H.; Kojima, M.; Itoh, I.;
Murata, J.; Azuma, I.; Saiki, I. Biol. Pharm. Bull. 1995, 18,
1681–1688.
19. The three-dimensional model of compound 9a was gen-
erated by HYPERCHEM 7.0 package, using AMBER
force field. The model of the inhibitor was manually
docked into the active site on the basis of docking studies
for the binding of RGD active ligands.
20. (a) Xiong, J.-P.; Stehle, T.; Zhang, R.; Joachimiak, A.;
Frach, M.; Goodman, S. L.; Arnaout, M. A. Science 2002,
296, 151–155, PDB file deposited with code 1L5G; (b)
Marinelli, L.; Lavecchia, A.; Gottschalk, K.-E.; Novel-
lino, E.; Kessler, H. J. Med. Chem. 2003, 46, 4393–
4404.