Application of stereocontrolled stepwise [3+2] cycloadditions to the preparation of inhibitors of α4β1-integrin- mediated hepatic melanoma metastasis

Article abstract of DOI:10.1002/anie.200462497

(Chemical Equation Presented) Inhibitors of the interaction between protein VLA-4 and its natural ligand VCAM-1 have been designed, even though the structure of the protein remains unresolved. The rational design relied on the simulation of the steric and electronic properties of the active loop of VCAM-1, whose structure is known (see picture), and the inhibitors were readily prepared by stereoselective stepwise [3+2] cycloadditions.

Full text of DOI:10.1002/anie.200462497

Angewandte
Chemie
Synthetic Inhibitors
Application of Stereocontrolled Stepwise [3+2]
Cycloadditions to the Preparation of Inhibitors of
a4b1-Integrin-Mediated Hepatic Melanoma
Metastasis**
Aizpea Zubia, Lorea Mendoza, Silvia Vivanco,
Eneko Aldaba, Teresa Carrascal, Begoꢀa Lecea,
Ana Arrieta, Tahl Zimmerman, Fernando Vidal-Vana-
clocha, and Fernando P. Cossꢁo*
Dedicated to Professor Cecilia Sarasola
Intravascular circulation of cancer cells represents a frequent
event along neoplasic progression that seriously increases the
probability of occurrence of metastasis. However, the adhe-
sion of blood-borne cancer cells to the microvascular wall is a
prerequisite for metastatic cell implantation at target organs.
Similar to capillary homing of leukocytes, this process is
proinflammatory cytokine-inducible and the mechanism
involves specific binding between cancer and endothelial
cell adhesion molecules.[1] Previously, we and others reported
that integrin “very late antigen 4” (VLA-4, a4b1) accounts for
melanoma cell adhesion to endothelium in metastasized
organs.[2] As vascular adhesion cell molecule 1 (VCAM-1) is
the natural ligand of VLA-4, the VLA-4–VCAM-1 interac-
[*] Dr. A. Zubia, Dr. S. Vivanco, E. Aldaba, Prof. B. Lecea, Prof. A. Arrieta,
Prof. F. P. Cossꢀo
Facultad de Quꢀmica-Kimika Fakultatea
Universidad del Paꢀs Vasco-Euskal Herriko Unibertsitatea
P. O. Box 1072, 20080 San Sebastiꢁn-Donostia (Spain)
Fax: (+34)943-212-236
E-mail: fpcossio@sq.ehu.es
Dr. L. Mendoza, Dr. T. Carrascal
Dominion Pharmakine, Ltd.
Zamudio Technology Park
48170 Zamudio (Spain)
Prof. F. Vidal-Vanaclocha
Facultad de Medicina y Odontologꢀa-Medikuntza eta Odontologia
Fakultatea
Universidad del Paꢀs Vasco-Euskal Herriko Unibertsitatea
48940 Lejona-Leioa (Spain)
T. Zimmerman
Centro Nacional de Investigaciones Oncolꢂgicas (CNIO)
Structural and Computational Biology Programme
Melchor Fernꢁndez Almagro 3, 28029 Madrid (Spain)
[**] This work was supported by the Universidad del Paꢀs Vasco-Euskal
Herriko Unibertsitatea, Gobierno Vasco-Eusko Jaurlaritza (grants 9/
UPV00170.215-13548/2001 and -13641/2001), Dominion Pharma-
kine Ltd., and by the Spanish Ministerio de Educaciꢂn y Ciencia
(grants BQU2001-0904, BIO2003-02246, and SAF99-0042). A.Z. and
E.A. are recipients of fellowships from the GV-EJ. We thank Dr.
Francisco J. Blanco and Dr. Pascal Garcꢀa (CNIO, Spain) for their
advice on the 15N–1H HSQC experiments. We also thank Dr. Antonio
Llamas (Unidade de Raios X, Universidade de Santiago de
Compostela, Spain) for the X-ray diffraction analysis of compound
11d.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2005, 44, 2903 –2907
DOI: 10.1002/anie.200462497
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2903

Communications
tion may, therefore, constitute an interesting molecular target
inhibitors.[4] The crystal structure of domains 1 and 2 of
VCAM-1 has been resolved by X-ray diffraction analysis.[5]
Additionally, directed mutagenesis studies have determined
that the CD loop in domain 1 of VCAM-1 is crucial for
binding to VLA-4[5] (Figure 1 a). In the solvated molecule, the
carboxymethyl group of Asp40 has considerable conforma-
tional freedom and is surrounded by a relatively hydrophobic
environment (Figure 1b). We reasoned that the compounds
shown in Table 1 could mimic the structural and electronic
for innovative therapy against melanoma dissemination.
However, no results on the antimetastatic effects of synthetic
VLA-4/VCAM-1 inhibitors have been reported to date,
although interesting studies on synthetic inhibitors of the
VLA-4/VCAM-1 interaction have been recently reported.[3]
Herein, we demonstrate that small synthetic molecules which
are designed to mimic the minimal requirements for binding
between the natural macromolecules completely abrogate
1) melanoma cell adhesion to cytokine-activated endothelial
cells and 2) melanoma cell responses to soluble and immo-
bilized VCAM-1, and 3) efficiently inhibit metastatic devel-
opment in vivo, without significantly affecting their cell
viability, proliferation rate, or major metabolic activities.
Structural and electronic features of VCAM-1 were first
examined in order to reproduce these features in the synthetic
Table 1: General structure and retrosynthetic analysis of the novel
inhibitors of the VLA-4/VCAM-1 interaction.
11
R1
R2
R3[a]
R4
a
b
c
d
e
f
g
h
i
Me
Me
Me
Et
Me
Et
Et
Et
Et
Et
Ph
Ph
Ph
tBu
Ph
Ph
Ph
cHex
Ph
cPr
Ph
H
H
H
H
H
H
H
H
H
Me
2-FC6H4
Ph
2,6-F2C6H3
Ph
3,5-F2C6H3
Ph
2,3-F2C6H3
Ph
j
Ph
k
Et
Ph
H
[a] cHex=cyclohexyl, cPr=cyclopropyl, Bn=benzyl.
features of the Ile39-Asp40-Ser41-Pro42 sequence of
domain 1 of VCAM-1 (Figure 1c). Thus, the carboxymethyl-
amido chain of these novel compounds reproduces the
binding ability of Asp40, whereas the remaining groups
provide the environment required for simulating the struc-
tural and electrostatic features of the remaining residues.
Furthermore, the pyrrolidine ring confers the necessary
restriction of conformational freedom to the molecule to
mimic the energetically available conformations of the CD
loop in the b barrel of domain 1 of VCAM-1 (Figure 1b and
c). Therefore, these unnatural highly substituted pyrrolidine
rings should bind to VLA-4 to disrupt VLA-4/VCAM-1-
interaction-dependent mechanisms.
The new compounds 11 were synthesized in 12 prepara-
tive steps according to the design depicted in Table 1 and
Scheme 1 (for more details, see Supporting Information). The
starting materials were readily accessible and inexpensive
chemicals such as l-a-amino acids (glycine, alanine, valine,
and leucine), halides 3, and aldehydes 7. The key step in the
synthetic route shown in Scheme 1 is the formal [3+2]
cycloaddition between E-nitroalkenes 6 and imines 9 to
yield pyrrolidines 10. This reaction takes place with complete
Figure 1. a) X-ray crystal structure (taken from ref. [5]) of the active
loop of VCAM-1 which is involved in binding with integrin VLA-4.
b), c) Electrostatic potential projected onto the electron density of the
Ile39-Asp40-Ser41-Pro42 tetramer (b) and inhibitor 11d (c); energies
range from À138.2 (red) to +100.2 kcalmolÀ1 (blue); asterisks ( )
*
indicate the hydrophobic regions in both structures.
2904
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2005, 44, 2903 –2907

Angewandte
Chemie
mechanism, to give the [3+2] cycloadduct. The first step
comprises a Michael-type nucleophilic attack of the a carbon
atom of the azomethine ylide A, which forms in situ, on the
b carbon atom of the nitroalkene (Scheme 2). The cyclization
step takes place by means of an intramolecular Henry-type
reaction between the intermediate nitronate moiety and the
iminic fragment of the zwitterionic intermediate denoted as B
in Scheme 2.
Scheme 1. Synthesis of compounds 11. a) 1 (1.0 equiv in 1n H2SO4),
NaNO2 (1.5 equiv in H2O), 08C, 24 h, 83–90%; b) DMP (1.0 equiv),
TsOH·H2O (0.007 equiv), MeOH, 458C, 24 h, 88–96%; c) 2 (1.0 equiv),
NaH (1.0 equiv), anhydrous THF/DMF (2:0.6), 08C, 10 min, then
3 (1.2 equiv), RT, 24 h, 45–64%; d) LiAlH4 (1.0 equiv), Et2O, 08C, then
4 (1.0 equiv), Et2O, RT, 3 h, 80–90%; e) ClCOCOCl (1.5 equiv),
DMSO (2.0 equiv), TEA (4.0 equiv), CH2Cl2, À678C, 5 h, 79–90%;
f) 5 (1.0 equiv), CH3NO2 (5.0 equiv), TEA (0.14 equiv), RT, overnight,
87–96%; g) MsCl (1.2 equiv), DIPEA (2.5 equiv), CH2Cl2, À788C, 2 h,
85–95%; h) 8 (1.15 equiv), TEA (1.25 equiv), MgSO4, CH2Cl2, RT, 1 h,
then 7 (1.0 equiv), RT, overnight, 75–95%; i) 6 (1.0 equiv),
Scheme 2. Mechanism of formation of cycloadducts 10 from the
*
reaction between imines 9 and nitroalkenes 6. Xc =chiral group.
To understand the origins of the stereocontrol of the
reaction, we computed the two possible diastereomeric
transition-state structures TS1 and TS1’ for the model
system depicted in Figure 2.[7] As shown in Figure 2 and
Scheme 2, part of the stereocontrol of the reaction stems from
the retention of configuration of the E-nitroalkene and the
9 (1.0 equiv), AgOAc (0.1 equiv), TEA (1 equiv), CH3CN, RT, 5 h, 60–
92%; j) 1n DME/LiOH (5:3, aq), 08C, 1–4 h, 82–95%;
k) 10 (1.0 equiv), MeGly·HCl (1 equiv), DECP (1.2 equiv), DMF, 08C,
then TEA (2.0 equiv), RT, overnight, 64–95%; l) 1n DME/LiOH (5:3,
aq), 08C, 1–4 h, 85–97%. DMP=2,2-dimethoxypropane, Ts=p-
toluenesulfonyl, DMF=N,N-dimethylformamide, DMSO=dimethyl-
sulfoxide, DMP=2,2-dimethoxypropane, TEA=triethylamine,
DIPEA=diisopropylethylamine, DME=1,2-dimethoxyethane, MeGly=
glycine methyl ester, DECP=diethylcyanophosphonate.
stereocontrol when R3 is a phenyl group, and R1 = Me or Et
(compounds 10a,c–e,g,i,j). When R3 is an alkyl group, the
stereocontrol is variable and ranges from greater than 99:1
when R3 = tBu (10b) to 91:9 when R3 = cHex (10 f) or cPr
(10h). In the case of pyrrolidine 10k, which has a chiral group
at R3, three stereoisomers were obtained in a ratio of 77:15:8.
Previous studies[6] have shown that the reaction between
p-deficient alkenes and metalated azomethine ylides takes
place through a stepwise mechanism, not a concerted
Figure 2. Ball-and-stick representations of the fully optimized
structures of transition-state structures TS1 and TS1’ (C gray, O red,
N dark blue, Ag light blue); green lines represent the new sigma bonds
that form; see ref. [7] for further details.
Angew. Chem. Int. Ed. 2005, 44, 2903 –2907
www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2905

Communications
chelated nature of the metalated azomethine ylide. The
diastereoselectivity can be explained in terms of a two-
electron interaction between the localized s* orbital of the
À
C C bond that forms and the localized s orbital of the
À
antiperiplanar C CH3 bond, with the alkoxy group occupying
the inside position with respect to the pyrrolidine ring that
forms, as seen in Figure 2. The alternative diastereomeric
À
transition-state structure TS1’, in which the C H bond is anti
À
with respect to the new C C bond that forms, is more than
1 kcalmolÀ1 higher in energy than TS1 and corresponds to
high stereocontrol in favor of the (2S,3R,4S,5S) cycloadduct,
provided that groups which are bulkier than methyl are used.
The absolute configuration of the new stereogenic centers was
verified by X-ray diffraction analysis on compound 11d and
confirmed the validity of our computational results.[8]
Next, the potential VLA-4 antagonism of compounds 11
was tested through a previously established model[2b] on
VLA-4/VCAM-1-interaction-dependent B16M cell adhesion
to primary cultured hepatic sinusoidal endothelial (HSE) cells
in vitro. As shown in Figure 3, both B16M cell adhesion to
TNF-a (tumor necrosis factor alpha)-treated HSE—which
increases VCAM-1 expression on endothelial cells—and
H2O2-treated B16M cell adhesion to unstimulated HSE
cells—which increases VLA-4 activation in cancer cells—
significantly increased relative to their respective untreated
control cells (P < 0.01, where P is the statistical probability
value).[2b] Preincubation of B16M cells with compounds 11
and measurement of adhesion to HSE resulted in variable
responses as shown in Figure 3. Interestingly, compounds 11b
and 11h with tBu and cPr groups at R3, respectively, did not
show any significant antiadhesive activity. Similarly, com-
pound 11e, with a 2,6-difluorophenyl group at R2 and a
methyl group at R1 was also almost inactive. In contrast, the
presence of a quaternary atom at the a-position of the
pyrrolidine ring (R4 = Me), such as in 11j, did not result in a
significant loss of activity.
To assess if interactions other than VLA-4/VCAM-1 were
involved in the inhibitory activity of compounds 11, we used
the chemical shift perturbation method[9] on the I domain of
the a2-subunit of a2b1-VLA-2 integrin which is also relevant in
melanoma metastasis and binds different ligands such as
collagen or laminin.[10] 15N–1H heteronuclear single-quantum
coherence (HSQC) NMR spectra[9] of uniformly 15N-labeled
I domain were recorded in the presence and the absence of
inhibitors 11a–j. In these spectra each amino acid of the
I domain appeared as a cross-peak that corresponds to the
NH group of its amide backbone. Analysis of the spectra at
different pH values (pH 6.5 and 7.4) and at different temper-
atures (T= 283 and 298 K, see Supporting Information) did
not lead to any significant shift of the cross-signals upon
addition of the inhibitors, which indicates the lack of binding
and, therefore, the high selectivity of these compounds with
respect to integrin VLA-2.
Figure 3. Inhibition of adhesion between HSE and B16M cells by com-
pounds 11. The degree of inhibition was measured on a) TNF-a-acti-
vated cultured HSE cells and b) H2O2-pretreated melanoma B16M
cells. Asterisks ( ) indicate the inhibition measured on unstimulated
*
HSE. The concentration of compounds 11 was 50 mg per 1ꢃ106 B16M
cells. The lyophilized compounds were dissolved in DMSO and diluted
to a final working stock solution of 5 mgmLÀ1 in serum-free medium.
1ꢃ106 B16M cells resuspended in 300 mL of serum-free medium were
preincubated with 11 (10 mL of stock solution, 50 mg) for 15 min at
378C before the adhesion assay. m(VLA-4)Ab denotes rat anti-mouse
CD49d monoclonal antibody (Serotec Ltd, Oxford, UK) against VLA-4.
In this case, B16M cells were incubated with 10 mgmLÀ1 of m(VLA-
4)Ab before the adhesion assay. The results are the mean standard
deviation of three independent experiments, each performed in sextu-
plicate (n=18).
addition of 11d compound to IL-1b (interleukin-1b)-treated
melanoma cells completely abrogated their adhesion to
immobilized VCAM-1 and prevented vascular endothelial
growth factor (VEGF) production induced by soluble-
VCAM-1-stimulated cells (see Supporting Information),
which confirms the potent VLA-4 antagonism of compound
11d. Finally, metastasis density and volume significantly
decreased (P < 0.01) by 60% and 95%, respectively, in mice
that were intrasplenically injected with 11d-pretreated B16M
cells relative to those that received untreated cells (Figure 4).
Parallel measurements on cell viability and cytotoxicity,
intracellular oxidative metabolism, and cell-proliferation
rates of 11d-treated melanoma cells excluded that antimeta-
static effects of 11d compound may have been caused by
Although the best activity in vitro was shown by
compound 11 f, which has a cyclohexyl group at R3, prelimi-
nary assays in vivo were carried out with 11d (R3 = Ph)
because of its higher solubility in water. Additionally, 11d
displayed comparable antiadhesive activity to monoclonal
antibodies against murine VLA-4 (Figure 3b). Furthermore,
2906 ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2005, 44, 2903 –2907

Angewandte
Chemie
Reznikov, S.-H. Kim, Novick, M. Rubinstein, C. A. Dinarello,
Proc. Natl. Acad. Sci. USA 2000, 97, 734.
[3] For example, see: a) J. Boer, D. Gottschling, A. Schuster, B.
Holzmann, H. Kessler, Angew. Chem. 2001, 113, 3988; Angew.
Chem. Int. Ed. 2001, 40, 3870; b) S. Wattanasin, B. Weidmann, D.
Roche, S. Myers, A. Xing, Q. Guo, M. Sabio, P. v. Matt, R. Hugo,
S. Maida, P. Lake, M. Weetall, Bioorg. Med. Chem. Lett. 2001, 11,
2955; c) L. Chen, J. Tilley, R. v. Trilles, W. Yun, D. Fry, C. Cook,
K. Rowan, V. Schwinge, R. Campbell, Bioorg. Med. Chem. Lett.
2002, 12, 137; d) G. A. Doherty. T. Kamenecka, E. McCauley,
G. V. Riper, R. A. Mumford, S. Tong, W. K. Hagmann, Bioorg.
Med. Chem. Lett. 2002, 12, 729; e) I. E. Kopka, D. N. Young, L. S.
Lin, R. A. Mumford, P. A. Magriotis, M. MacCoss, S. G. Mills,
G. V. Riper, E. McCauley, L. E. Egger, U. Kidambi, J. A.
Schmidt, K. Lyons, R. Stearns, S. Vincent, A. Coletti, Z. Wang,
S. Tong, J. Wang, S. Zheng, K. Owens, D. Levorse, W. K.
Hagmann, Bioorg. Med. Chem. Lett. 2002, 12, 637; f) D. R.
Leone, K. Giza, A. Gill, B. M. Dolinski, W. Yang, S. Perper,
D. M. Scott, W.-C. Lee, M. Cornebise, K. Wortham, C. Nick-
erson-Nutter, L. L. Chen, D. Lepage, J. C. Spell, E. T. Whalley,
R. C. Petter, S. P. Adams, R. R. Lobb, R. B. Pepinski, J.
Pharmacol. Exp. Ther. 2003, 305, 1150.
Figure 4. Antimetastatic activity of compound 11d on male C57BL/6J
mice (6 to 8 weeks old). At least 30 mice were used per experiment,
and each experiment was carried out 3 times. The bars on the left cor-
respond to the control group. The test group was treated with 11d-pre-
incubated B16M viable cells (50 mg per 1ꢃ106 B16M cells). Livers were
removed on day 12 after intrasplenical injection of B16M cells and
photographed. Photographs on the left and on the right correspond to
livers obtained from the control and test groups, respectively. Metasta-
ses can be identified as black melanotic nodules. Metastasis density
(dm) was measured as the number of foci per 100 mm3 of liver, and
metastasis volume (Vm) was measured as the percentage fraction of
liver volume occupied by metastases.
[4] For a related computational analysis of VCAM-1, see: A.
Macchiarulo, G. Constantino, M. Meniconi, K. Pleban, G. Ecker,
D. Bellocchi, R. Pellicciari, J. Chem. Inf. Comput. Sci. 2004, 44,
1829.
[5] E. Y. Jones, K. Harlos, M. J. Bottomley, R. C. Robinson, P. C.
Driscoll, R. M. Edwards, J. M. Clements, T. J. Dudgeon, D. I.
Stuart, Nature 1995, 373, 539.
[6] S. Vivanco, B. Lecea, A. Arrieta, P. Prieto, I. Morao, A. Linden,
F. P. Cossꢀo, J. Am. Chem. Soc. 2000, 122, 6078.
[7] These calculations were performed at the B3LYP level of theory;
see: a) A. D. Becke, J. Chem. Phys. 1993, 98, 5648; b) A. D.
Becke, Phys. Rev. A 1988, 38, 3098; c) C. Lee, W. Yang, R. G.
Parr, Phys. Rev. B 1980, 37, 785; d) S. H. Vosko, L. Wilk, M.
Nusair, Can. J. Phys. 1980, 58, 1200. Carbon, nitrogen, oxygen,
and hydrogen atoms were described by means of the 6-31G(d)
basis set, whereas silver atoms were described by the Hay – Wadt
effective core potential; see: P. J. Hay, W. R. Wadt, J. Chem.
Phys. 1985, 82, 299. Harmonic analyses on TS1 and TS1’ showed
that both saddle points have only one imaginary frequency
associated with nuclear motion along the reaction coordinate
indirect effects on cell functions other than those specifically
operated through a VLA-4-dependent mechanism.[11]
In summary, we have described a new family of inhibitors
of the VLA-4/VCAM-1 interaction that 1) block the in vitro
adhesion of melanoma cells to microvascular endothelium
induced by proinflammatory cytokines and oxidative stress,
2) prevent melanoma cell production of VEGF induced by
VCAM-1 in vitro, and 3) exhibit a potent antimetastatic
activity in vivo. These small synthetic molecules fulfill the
bioavailability requirements proposed by Lipinski et al.[12]
Finally, the synthetic route developed for these inhibitors
was convergent, completely regio- and stereoselective, versa-
tile, and did not require sophisticated experimental devices or
purifications. Therefore, it is well-suited to extensive studies
of structural variation to refine the biological properties of the
most promising lead compounds.
À
associated with formation of a new C C bond. Differences in
energy include zero-point vibration energy corrections.
[8] CCDC 252339 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from
the Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/data_request/cif.
[9] a) A. Bax, M. Ikura, L. E. Kay, D. A. Tochia, R. Tschudin, J.
Magn. Reson. 1990, 86, 304; b) G. Bodenhausen, D. J. Buben,
Chem. Phys. Lett. 1980, 69, 185.
[10] a) R. Mortarini, A. Anichini, G. Parmiani, Int. J. Cancer 1991, 47,
551; b) D. Baronas-Lovell, J. L. Lauer-Fields, J. A. Borgia, G. F.
Sferrazza, M. Al-Ghoul, D. Minond, G. B. Fields, J. Biol. Chem.
2004, 279, 43503.
[11] F. Vidal-Vanaclocha, L. Mendoza, N. Gallot, N. Telleria, F. P.
Cossio, A. Zubia, E. Aldaba, unpublished results.
[12] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Freeney, Adv.
Drug Delivery Rev. 1997, 23, 3.
Received: November 2, 2004
Revised: February 14, 2005
Published online: April 13, 2005
Keywords: cell adhesion · cycloaddition · drug design ·
.
inhibitors · proteins
[1] a) I. J. Fidler, Nat. Rev. Cancer 2003, 3, 1; b) J. D. Hood, D. A.
Cheresh, Nat. Rev. Cancer 2002, 2, 91.
[2] a) G. E. Rice, M. P. Bevilacqua, Science 1989, 246, 1303; b) F.
Vidal-Vanaclocha, G. Fantuzzi, L. Mendoza, A. M. Fuentes,
M. J. Anasagasti, J. Martꢀn, T. Carrascal, P. Walsh, L. L.
Angew. Chem. Int. Ed. 2005, 44, 2903 –2907
www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2907