Lysyl 4-aminobenzoic acid derivatives as potent small molecule mimetics of plasminogen kringle 5

Article abstract of DOI:10.1016/j.bmcl.2003.12.034

Kringle 5, a proteolytic fragment of human plasminogen has been shown to potently inhibit angiogenesis. The tetrapeptide KLYD derived from kringle 5 has been shown to capture many activities of kringle 5 in vitro. Further simplification has been achieved

Full text of DOI:10.1016/j.bmcl.2003.12.034

Bioorganic & Medicinal ChemistryLetters 14 (2004) 965–966
Lysyl 4-aminobenzoic acid derivatives as potent small molecule
mimetics of plasminogen kringle 5
George S. Sheppard,* Megumi Kawai, Richard A. Craig, Donald J. Davidson,
Sandra M. Majest, RandyL. Bell and Jack Henkin
Abbott Laboratories, Global Pharmaceutical Research and Development, 100 Abbott Park Road, Abbott Park, IL 60064, USA
Received 8 October 2003; accepted 1 December 2003
Abstract—Kringle 5, a proteolytic fragment of human plasminogen has been shown to potently inhibit angiogenesis. The tetra-
peptide KLYD derived from kringle 5 has been shown to capture manyactivities of kringle 5 in vitro. Further simplification has
been achieved byreplacement of the two central amino acids with a 4-aminobenzoic acid spacer group. Molecules displaying the
required recognition groups on this core show similar in vitro properties to kringle 5, and are able to displace radiolabeled protein
from a high affinitybinding site on endothelial cells.
# 2003 Elsevier Ltd. All rights reserved.
Angiogenesis, the process of formation of new capil-
laries from existing blood vessels, is a highlyregulated
physiological process. In normal vasculature, the endo-
thelial cells remain in a quiescent state, and must be
activated and induced to undergo a number of processes
to create a new, stable structure. This complex process is
affected bya number of naturallyoccurring promoters
and inhibitors.1
with a suitablyprotected lysine derivative activated with
di-tert-butyl dicarbonate,5 followed byconversion to
the Na-acetyl derivative. Carbodiimide coupling with
aspartic acid di-tert-butyl ester and deprotection pro-
vided 2. Related compounds were prepared bya similar
route and evaluated in an endothelial cell chemotaxis
assayto provide an in vitro assessment of anti-angio-
genic activity.6 As indicated in Table 1, 2 was able to
inhibit the VEGF-stimulated migration of HMVECs
(human microvascular endothelial cells) with a similar
potencyto K5. Related compounds 3 and 4 with the
Kringle 5 of plasminogen (K5) has been reported to be a
potent inhibitor of angiogenesis and tumor growth.2
Studies of peptides representing portions of the K5
sequence indicated that the tetrapeptide KLYD found
on an inner loop of the kringle structure possessed sev-
3
eral keyin vitro properties of the protein.
In this
communication, we report that further structural sim-
plification can be achieved byreplacement of the amino
acids attached to lysine with appropriately functionalized
4-aminobenzoic acid derivatives.
Studies in search of Ras farnesyltransferase inhibitors
had indicated that the activityof CAAX tetrapeptides
could be mimicked byreplacing the central two hydro-
phobic amino acids with an aminobenzoic acid spacer.4
Applying a similar strategy to the KLYD peptide 1,
compound 2 was prepared bythe route shown in
Scheme 1. tert-Butyl 4-aminobenzoate was acylated
Scheme 1. Reagents: (a) BOC-Lys(CBZ)-OH, BOC2O, pyridine,
EtOAc; (b) TFA; (c) Ac2O, 2,6-lutidine, CH2Cl2, THF; (d) H-
Asp(OtBu)-OtBu, EDCI, HOBT, NMM, DMF; (e) H2, Pd/C, MeOH;
(f) HCl, dioxane.
* Corresponding author. Tel.: +1-847-937-8572; fax: +1-847-935-
5165; e-mail: george.sheppard@abbott.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.034

966
G. S. Sheppard et al. / Bioorg. Med. Chem. Lett. 14 (2004) 965–966
Table 1. Inhibition of VEGF-stimulated migration byK5 and com-
pounds 1–4
Scheme 2. Reagents: (a) 3-(BnO)phenylboronic acid, CsF, Pd(PPh3)4,
DME; (b) SnCl2, MeOH, CH2Cl2; (c) BOC-Lys(CBZ)-OH, BOC2O,
pyridine, EtOAc; (d) H2, Pd/C, MeOH.
Compd
R=
—
IC50, nMa
To confirm that the simplified structures were exerting
their effect on HMVEC chemotaxis through the same
pathwayas K5, a competitive binding studywas carried
out. K5 has been reported to displace I125-labeled K5
from a high affinitybinding site on HMVECs with a Kd
of 0.5 nM.7 Similar studies showed an IC50 of 0.7 nM
for 5, supporting the view that it acts as a mimetic of the
protein.
K5
1
2
3
4
0.015
1.2
0.01
—
(CH2)4NH2
H
(CH2)4NHAc
>100
>100
a Values are means of three experiments.
Table 2. Inhibition of VEGF-stimulated migration bycompounds 5–
8
In summary, we have demonstrated that small-molecule
mimetics of K5 derived from 4-aminobenzoic acid can
inhibit VEGF-induced HMVEC chemotaxis, and bind
to a high affinitybinding site on the target cell with
comparable affinityto the protein itself. Further studies
will be reported in due course.
Compd
R1=
R2=
IC50, nMa
5
6
7
8
NHBOC
H
NHBOC
NHBOC
(CH2)4NH2
(CH2)4NH2
(CH2)3CH3
(CH2)3NH2
0.020
0.06
>100
1.1
References and notes
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Haskell, C.; Wang, J.; Henkin, J. 215th ACS National
Meeting, April 1998. Abstract MEDI-207.
a Values are means of three experiments.
lysine sidechain removed or acetylated lost activity in
this assay.
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While examining the effects of substitution, it was dis-
covered that the aspartic acid residue could be replaced
as well. As indicated in Table 2, 5 is a potent inhibitor
of HMVEC chemotaxis. As in the aspartate-containing
series, the lysine side-chain plays a key role in the activ-
ity. Compound 6 lacking Na retains activity, while 7
lacking Ne does not. Compound 8 in which ornithine
replaces lysine maintains reasonable activity. Similar
results were obtained with other 2-substituted 4-amino-
benzoic acids, suggesting that the keyinteractions are
an ammonium and a carboxylate held in the proper
geometry. Scheme 2 shows the synthesis of compound 5;
related compounds were prepared in a similar manner.
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escentlylabeled with calcein AM (50-100 nM), plated into
a migration chamber (2.9Â104 /well) (Neuroprobe) and
stimulated to migrate with VEGF (10 ng/mL). After 4 h,
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Egan, D.; Wang, J.; Henkin, J. Proc. Am. Assoc. Cancer
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