Solid-phase synthesis of a small library of 3-phenylthio-3-nicotinyl propionic acid derivatives acting as antagonists of the integrin αVβ3

Article abstract of DOI:10.1016/j.bmcl.2003.11.045

We describe the synthesis of a series of low molecular weight inhibitors of the αvβ3 integrin obtained by modifying a high-throughput screening hit with micromolar activity. A solid phase synthesis to prepare 3-phenylthio-3-nicotinyl propionic acid derivatives, exemplified by 13c, was set up. Compounds with nanomolar activity in the biochemical assay and able to efficiently inhibit cell adhesion mediated by vitronectin have been obtained.

Full text of DOI:10.1016/j.bmcl.2003.11.045

Bioorganic & Medicinal Chemistry Letters 14 (2004) 657–661
Solid-phase synthesis of a small library of 3-phenylthio-3-nicotinyl
propionic acid derivatives acting as antagonists of the
integrin ꢀVꢁ3
Paola Vianello,a,* Paolo Cozzi,a Arturo Galvani,c Maurizio Meroni,a Mario Varasi,a
Daniele Volpib and Tiziano Bandieraa
aDepartment of Chemistry, Pharmacia Italia S.p.A–Gruppo Pfizer, Discovery Research Oncology, Viale Pasteur 10,
20014 Nerviano (MI), Italy
bDepartment of Biology, Pharmacia Italia S.p.A–Gruppo Pfizer, Discovery Research Oncology, Viale Pasteur 10,
20014 Nerviano (MI), Italy
cDepartment of Pharmacology, Pharmacia Italia S.p.A–Gruppo Pfizer, Discovery Research Oncology, Viale Pasteur 10,
20014 Nerviano (MI), Italy
Received 29 August 2003; revised 23 October 2003; accepted 18 November 2003
Abstract—We describe the synthesis of a series of low molecular weight inhibitors of the avb3 integrin obtained by modifying a
high-throughput screening hit with micromolar activity. A solid phase synthesis to prepare 3-phenylthio-3-nicotinyl propionic acid
derivatives, exemplified by 13c, was set up. Compounds with nanomolar activity in the biochemical assay and able to efficiently
inhibit cell adhesion mediated by vitronectin have been obtained.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
ligation with a specific anti-avb3 monoclonal antibody
(LM609) or the cyclic peptide c[RGDfV] was shown to
inhibit angiogenesis in the chick chorioallantoic mem-
brane (CAM) and rabbit cornea assays when variously
elicited by bFGF, TNF-a or tumor cells, whereas anti-
bodies against related integrins and a control peptide
had no effect.4 Similarly, a reduction of angiogenesis
and tumor cell growth was reported following adminis-
tration of LM609 to SCID mice transplanted with full
thickness human skin containing avb3-negative human
breast tumor cells.5 Immunohistochemical studies on
clinical specimens have shown that integrin avb3 is fre-
quently expressed on the endothelium of tumor-asso-
ciated blood vessels,6 and expression level on these
vessels is a negative prognostic factor in breast and
colon cancer patients.7,8
Integrins are a family of heterodimeric transmembrane
glycoproteins involved in cell–cell and cell–extracellular
matrix (ECM) interactions.1 Integrin-mediated adhe-
sion induces a variety of signal transduction events that
influence many aspects of cell behavior including survi-
val, proliferation, differentiation, gene expression, and
migration.2 Integrins are composed of two non-cova-
lently associated subunits, a and b, and each ab
combination may recognize a single or various ECM
proteins. Integrin avb3 is the most promiscuous member
of the family since it recognizes many extracellular
matrix proteins, including vitronectin, fibrinogen and
fibronectin, through the tripeptide RGD (Arg-Gly-Asp)
sequence. Despite its promiscuous behavior, integrin
avb3 is not expressed in many normal tissues but is sig-
nificantly upregulated on endothelial cells of angiogenic
blood vessels.3 In preclinical studies, disruption of avb3
In addition to its expression on vasculature, avb3 integ-
rin has also been found to be overexpressed on tumor
cells themselves in various cancers, including mela-
noma,9 breast,10 and ovarian,11 and a wide body of evi-
dence suggests various roles for this integrin in
promoting invasive and metastatic tumor growth inde-
pendently of angiogenesis, including enhancing tissue
arrest of blood-borne tumor cells,12 cooperating with
matrix metalloprotease activity in cell migration pro-
cesses,13 and mediating tumor cell tissue invasion.14
Keywords: Integrins; avb3; Cancer; Angiogenesis; Solid phase.
Abbreviations: DBU, 1,8-diazabicyclo(5.4.0)undec-7-ene; DIC, 1,3-di-
isopropylcarbodiimide; DIPEA, N,N-diisopropylethylamine; DMAP,
4-(dimethylamino)pyridine; DMF, N,N-dimethylformamide; TFA,
trifluoroacetic acid.
* Corresponding author. Tel.: +39-02483-83878; Fax: +39-02483-
83833; e-mail: paola.vianello@pharmacia.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.045

658
P. Vianello et al. / Bioorg. Med. Chem. Lett. 14 (2004) 657–661
A critical role for integrin avb3 has also been demon-
strated in the bone resorption process brought about by
osteoclasts. Anti-avb3 antibodies, peptide and small mole-
cule inhibitors of integrin avb3 all resulted in inhibition of
osteoclast activity.15 Therefore, antagonists of integrin
avb3 may represent a viable therapeutic strategy for the
treatment of diseases dependent on angiogenesis, including
tumor growth and metastasis, and of osteoporosis.
In the search for small molecule inhibitors of integrin
avb3, we screened our research compound collection and
identified compound 1a as an interesting hit with activ-
ity in the mM range. Compound 1a contains a b-aryl pro-
pionic acid function, a motif present in many avb3
inhibitors,16 and a b-aryl thioether group which replaces
the commonly encountered b-glycyl-amino moiety. The
aryl-thioether group simultaneously reduces the flexibility
Table 1.
a
b
c
Compd
R
Method
A
IC50 (mM) avb3
IC50 (mM) aIIbb3
IC50 (mM) avb3
IC50 (mM) aIIbb3
IC50 (mM) avb3
IC50 (mM) aIIbb3
1
4
45
2.9
30n.t.
1.5
2
3
4
A
A
A
25
n.t.
3.8
3030
4
>304.2
n.t.
4.2
21
>30
a
>302.6
n.t.
19
5
C
A
A
A
A
C
A
C
10
>100
0.8
31
0.2
81
6
9.6
n.t.
5.8
30n.t.
5.8
7
>3014.3
n.t.
15.2
n.t.
n.t.
8
29.6
n.t.
11.2
n.t.
10.6
n.t.
a
9
5.9
n.t.
4.5
30
10
11
12
5
100
0.3
10.4
0.2
87
9.2
n.t.
1.8
8.9
2.9
24
a
0.051
2.4
0.12
>30
13
B
B
4
16
0.068
1.4
0.016
9.8
14
9
>30
0.015
0.02
1.8
34
a Compound not obtained.

P. Vianello et al. / Bioorg. Med. Chem. Lett. 14 (2004) 657–661
659
Scheme 1. (a) Wang resin, DIC, DMAP CH2Cl2, 20h rt; (b) o-, m- or p-amiothiophenol, DBU cat. DMF, rt 48 h.
Scheme 2. Methods A and B: (a) RCOOH, DIC, CH2Cl2, rt, one week; (b) RCOOH, SOCl2, Pyr or DMF, rt, one week; (c) TFA, CH2Cl2,1:1, rt, 1 h.
Scheme 3. Method C: (a) DIC, CH2Cl2, rt 10days; (b) piperidine 4%, DMF, rt, 2 ꢀ1 min, 1ꢀ5 min; (c) CH2Cl2; (d) TFA, CH2Cl2.
and increases the lipophilicity of compounds originating
from the expansion of 1a compared to analogues con-
taining the b-glycyl-amino moiety. The hit isomers 1b
and 1c retained activity on avb3 integrin, thus confirm-
ing the potential interest of this class. Therefore, a small
library of derivatives was prepared to verify if potency
against avb3 and selectivity versus the related integrin
aIIbb3 could be improved. Most of the selected sub-
stituents (R, Table 1) carry a basic residue, since the tri-
peptide recognition element (RGD) of proteins binding
to avb3 integrin contains the basic arginine side chain.
The selection was made to study the effect of the chain
length on activity. In addition, since compound 1c showed
a ca. 1 mM IC50, we decided to include in the design of the
library a few substituents devoid of the basic center to
verify whether additional hydrophobic interactions were
beneficial for activity.
2. Chemistry
In the solid-phase approach, the commercially available
trans-3-(3-pyridyl)acrylic acid was loaded onto the
Wang resin by activation with DIC in methylene chlo-
ride and in the presence of DMAP to give 15 (Scheme 1).
Michael addition on compound 15 with o-, m- and p-
aminothiophenol with DBU as a catalyst in DMF at
room temperature gave the three intermediates 16a–c.
The amino derivatives 16a–c thus obtained (0.166
mmol) were condensed with the appropriate acids (1.66
mmol), pre-activated with DIC (2.49 mmol, method A)
or with thionyl chloride (3.2 mmol, method B), as illu-
strated in Scheme 2. After coupling, compounds were
cleaved from the resin by treatment with trifluoroacetic
acid in dichloromethane.
In this communication we describe the solid-phase
synthesis and the pharmacological evaluation of this
library. The synthesis of the most interesting compound
via homogeneous phase will also be presented. Within
this series, compounds with nanomolar activity in the
biochemical assay and sub-micromolar EC50 in inhibit-
ing cell–ECM interactions mediated by vitronectin have
been obtained.
A different procedure (method C, Scheme 3) was used to
obtain the derivatives bearing a guanidino-function on
an aliphatic chain (see Table 1, rows 5, 10, 12). The
synthesis was performed by condensing the FMOC-
protected amino acid (1.66 mmol) pre-activated with
DIC (2.49 mmol), with the aminothiophenols 16a–c
(0.166 mmol), deprotecting the amino function and
generating the guanidino moiety by reaction with N,N0-

660
P. Vianello et al. / Bioorg. Med. Chem. Lett. 14 (2004) 657–661
Scheme 4. (a) p-Methoxybenzylalcohol, DIC, DMAP, CH2Cl2, rt, overnight, 82%; (b) 4-aminothiophenol, DBU, DMF, rt, 8 h, 50%; (c) DIC,
DMAP, CH2Cl2, rt, 24 h, 51%; (d) TFA, CH2Cl2, 3 h, rt, 90%.
Table 2.
showed low nanomolar activity but only 13c displayed
good selectivity with respect to aIIbb3, with an aIIbb3/
Biochemical assays (IC50
avb3: 0.016ꢁ0.009 mM
aIIbb3: 9.8ꢁ4.6 mM
)
avb3 IC50 ratio of ca. 600. In cell adhesion assays,20
compound 13c confirmed as a specific inhibitor of the
avb3-mediated adhesion to extracellular matrix proteins
(Table 2). The compound did not show activity in
adhesion assays dependent on aIIbb3, a5b1 and a4b1
integrin, and did not inhibit in vitro growth of CEMD9
cells21 at 72 h after continuous treatment.
Cytotoxicity (ED50
)
CEMD9: >100 mM
Cell Adhesion (EC50
)
CEMD9-Vitronectin: 0.706ꢁ0.37 mM
CHOaIIbb3-Fibrinogen: >100 mM
K562-Fibronectin (a5b1): >100 mM
CEMD9-VCAM1 (a4b1): >100 mM
In conclusion, a nanomolar avb3 inhibitor with a good
selectivity versus the aIIbb3 integrin in both the bio-
chemical and cell-based assays, and devoid of cytotoxi-
city, has been obtained. This compound represented the
starting point for further expansion of this class.
di-Boc-N00-triflyl-guanidine.17 In the deprotection of the
amino group, a low concentration of piperidine and a
short reaction time were mandatory to avoid the retro-
Michael reaction.18 Subsequent cleavage with tri-
fluoroacetic acid in dichloromethane gave the desired
compounds with 80–90% purity.
References and notes
1. Hynes, R. O. Cell 1992, 69, 11.
The most interesting compound 13c was re-prepared in
homogeneous phase as reported in Scheme 4. trans-3-(3-
Pyridyl)acrylic acid was protected as p-methoxybenzyl
ester (20), treated with 4-aminothiophenol to give 21,
which in turn was reacted with compound 22, obtained
by reaction of m-amino benzoic acid with N,N0-di-Boc-
N00-triflyl-guanidine in presence of triethylamine, to
yield 23. Subsequent treatment with trifluoroacetic acid
in dichloromethane led to 13c.
2. Giancotti, F. G.; Ruoslahti, E. Science 1999, 285, 1028.
3. Brooks, P. C.; Clark, R. A.; Cheresh, D. A. Science 1994,
264, 569.
4. Brooks, P. C.; Montgomery, A. M. P.; Rosenfeld, M.;
Reisfeld, R. A.; Hu, T.; Klier, G.; Cheresh, D. A. Cell
1994, 79, 1157.
5. Brooks, P. C.; Stromblad, S.; Klemke, R.; Visscher, D.;
Sarkar, F. H.; Cheresh, D. A. J. Clin. Invest. 1995, 96, 1815.
6. Max, R.; Gerritsen, R. R.; Nooijen, P. T.; Goodman,
S. L.; Sutter, A.; Keilholz, U.; Ruiter, D. J.; De Waal,
R. M. Int. J. Cancer 1997, 71, 320.
7. Gasparini, G.; Brooks, P. C.; Biganzoli, E.; Vermeulen,
P. B.; Bonoldi, E.; Dirix, L. Y.; Ranieri, G.; Miceli, R.;
Cheresh, D. A. Clin. Cancer Res. 1998, 4, 2625.
8. Vonlaufen, A.; Wiedle, G.; Borisch, B.; Birrer, S.; Luder,
P.; Imhof, B. A. Mod. Pathol. 2001, 14, 1126.
9. Natali, P. G.; Hamby, C. V.; Felding-Habermann, B.;
Liang, B.; Nicotra, M. R.; Di Filippo, F.; Giannarelli, D.;
Temponi, M.; Ferrone, S. Cancer Res. 1997, 57, 1554.
10. Pignatelli, M.; Cardillo, M. R.; Hanby, A.; Stamp, G. W.
Hum. Pathol. 1992, 23, 1159.
11. Liapis, H.; Adler, L. M.; Wick, M. R.; Rader, J. S. Hum.
Pathol. 1997, 28, 443.
12. Pilch, J.; Habermann, R.; Felding-Habermann, B. J. Biol.
Chem. 2002, 277, 21930.
13. Rolli, M.; Fransvea, E.; Pilch, J.; Saven, A.; Felding-
Habermann, B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
9482.
14. Hsu, M. Y.; Shih, D. T.; Meier, F. E.; Van Belle, P.; Hsu,
J. Y.; Elder, D. E.; Buck, C. A.; Herlyn, M. Am. J. Pathol.
1998, 153, 1435.
3. Conclusions
An efficient method for the preparation of 3-phenylthio-
3-nicotinyl propionic acids on solid phase has been
developed. A preliminary exploration of this class of
compounds as avb3 antagonists has been carried out
preparing 39 derivatives (Table 1).
Binding studies19 demonstrated that compounds bear-
ing a guanidino residue have good affinity for the avb3
receptor, and para- and meta-aminothiophenol deriv-
atives showed submicromolar IC50s. Among those
compounds, replacement of the aliphatic chain with a
phenyl ring led to more potent derivatives. In the latter
series of compounds the position of the guanidino
group affects both the binding to avb3 and the selectiv-
ity with respect to the closely related aIIbb3 integrin. As
reported in Table 1, compounds 13b, 13c and 14b all

P. Vianello et al. / Bioorg. Med. Chem. Lett. 14 (2004) 657–661
661
15. Hartman, G. D.; Duggan, M. E. Exp. Opin. Invest. Drugs
2000, 9, 1281.
85, 683. Briefly, the integrin was coated onto a 96-well
plate (50mg/well) and 5 nM biotinylated vitronectin was
allowed to bind for 30min at 37 ꢂC, with or without
increasing concentrations of the competing molecules.
The plate was then rinsed and incubated 45 min with
streptavidin conjugated to peroxidase. The amount of
vitronectin bound was monitored after addition of Turbo-
TMB as peroxidase substrate, and the yellow color pro-
duced was read by a Packard ELISA reader, at 450nm
wavelength. Data were analyzed using the computerized
program ‘GraphPad Prism’.
16. (a) See for instance: Coleman, P. J.; Askew, B. C.;
Hutchinson, J. H.; Whitman, D. B.; Perkins, J. J.; Hart-
man, G. D.; Rodan, G. A.; Leu, C.; Prueksaritanont, T.;
Fernandez-Metzler, C.; Merkle, K. M.; Lynch, R.; Lynch,
J. J.; Rodan, S. B.; Duggan, M. E. Bioorg. Med. Chem.
Lett. 2002, 12, 2463 and references cited therein. (b)
Lange, U. E. W.; Backfisch, G.; Delzer, J.; Geneste, H.;
Graef, C.; Hornberger, W.; Kling, A.; Lauterbach, A.;
Subkowski, T.; Zechel, C. Bioorg. Med. Chem. Lett. 2002,
12, 1379. (c) Gabor, S. A. G.; Gibson, C.; Goodman,
S. L.; Holzemann, G.; Wiesner, M.; Kessler, H. J. Med.
Chem. 2001, 44, 1938. (d) Carron, C. P.; Meyer, D. M.;
Pegg, J. A.; Engleman, V. W.; Nickols, M. A.; Settle, S. L.;
Westlin, W. F.; Ruminski, P. G.; Nickols, G. A. Cancer
Res. 1998, 58, 1930.
20. Cells in log phase of growth were used. Cells were washed
counted, and diluted in adhesion medium (0.1% heat
denatured BSA in HBSS+1 mM MnCl2). Then, 10 mL of
adhesion medium containing inhibitors at suitable con-
centrations were added to test wells. 90 mL/well of cell
suspension was plated and incubated for 1 h at 37 ꢂC.
Following adhesion, plates were gently washed and cells
fixed with 50 mL TCA 50% (trichloroacetic acid). Eval-
uation of cell adhesion was performed by staining with
sulforhodamine B.
21. CEMD9 is a recombinant sub-line, derived from an acute
lymphoblastic leukemia (ATCC CCL-119), stably expres-
sing avb3 integrin obtained by co-transfecting cDNAs
encoding human av and b3 subunits. Cells were dispensed
in 96 wells and treatment was performed 2 h later. Eval-
uation of growing inhibition was done 72 h later.
17. Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J.
Org. Chem. 1998, 63, 3804.
18. Chemical stability was measured on the final compounds,
in the Tris buffer, pH 7.4, used for the biochemical assays.
19. (a) Binding assays were performed as described in the lit-
erature: Wong, A.; Hwang, S. M.; McDevitt, P.;
McNulty, D.; Stadel, J. M.; Johanson, K. Mol. Pharm.
1996, 50, 529. (b) Brooks, P. C.; Stromblad, S.; Sanders,
L. C.; von Schalscha, T. L.; Aimes, R. T.; Stetler-Ste-
venson, W. G.; Quigley, J. P.; Cheresh, D. A. Cell 1996,