Novel fibrinogen receptor antagonists. RGDF mimetics, derivatives of 4-(isoindoline-5-yl)amino-4-oxobutyric acid

Article abstract of DOI:10.1016/j.bmcl.2004.09.002

The novel RGDF mimetics 9a and 9b were synthesized with the use of 4-(isoindoline-5-yl)amino-4-oxobutyric acid as a surrogate of Arg-Gly motif. The synthesized compounds have demonstrated a high potency to inhibit platelet aggregation in vitro and to bloc

Full text of DOI:10.1016/j.bmcl.2004.09.002

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5533–5535
Novel fibrinogen receptor antagonists. RGDF mimetics,
derivatives of 4-(isoindoline-5-yl)amino-4-oxobutyric acid
Andrei A. Krysko,* Boris M. Chugunov, Olga L. Malovichko, Sergei A. Andronati,
Tatyana A. Kabanova, Tamara L. Karaseva and Anna V. Kiriyak
Department of Medicinal Chemistry, A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine,
86 Lustdorfskaya doroga, 65080 Odessa, Ukraine
Received 5 May 2004; accepted 2 September 2004
Available online 21 September 2004
Abstract—The novel RGDF mimetics 9a and 9b were synthesized with the use of 4-(isoindoline-5-yl)amino-4-oxobutyric acid as a
surrogate of Arg-Gly motif. The synthesized compounds have demonstrated a high potency to inhibit platelet aggregation in vitro
and to block FITC-Fg binding to aIIbb3 on washed human platelets.
Ó 2004 Elsevier Ltd. All rights reserved.
Last decade, interest of medicinal chemists essentially
focuses on the design, synthesis and development of
compounds, which prevent a thrombosis through the
inhibition of platelet aggregation. A fundamental phys-
iological role of a platelet, as a blood element, is the
interaction with fibrinogen, which leads to the platelet
aggregation and thereby initiates the formation of
hemostatic clot, preventing bleeding at the site of vascu-
lar injury. It is established that a platelet membrane-
bound glycoprotein GP IIb/IIIa (or aIIbb3 integrin) is
highly responsible for this aggregation process. This gly-
coprotein, which serves as a receptor for plasma protein
fibrinogen is a member of superfamily of cell adhesion
proteins, called integrins, which bind Arg-Gly-Asp
(RGD) sequences occurring in fibrinogen. Various
RGD containing peptides are able to block the native
ligand from binding to its receptor. Therefore, the
RGD sequence is a lead structure for the design of novel
platelet aggregation inhibitors.1
RGDF mimetics.2 Named fragments may be referred
as bioisosters of the side function of arginine residue.
The synthesis of the series of RGDF mimetics—(amino-
benzamidino)succinyl derivatives, has been reported.3
The compounds of this series have demonstrated a high
affinity for platelet fibrinogen receptor and effectively
inhibited platelet aggregation. The (aminobenzami-
dino)succinyl (1) residue has been recognized as a
mimetic of Arg-Gly moiety of RGDF sequence (Fig. 1).
In the present study, we have proposed the residue
of 4-(isoindoline-5-yl)amino-4-oxobutyric acid (2) as
Arg-Gly surrogate.
b-Alanines with different substituents in b position are
successfully applied in the synthesis of RGDF mimet-
ics—fibrinogen receptor blockers.2,4 The residue of
b-substituted b-alanine is considered as a surrogate of
Asp-Phe motif. Carboxylic group of b-alanine mimics
the side chain of aspartic acid residue. b-Substituted b-
alanines have been used by us as starting blocks for
preparation of novel RGDF mimetics.
The fragment containing the residues of p-benzamidine,
piperidine, p-benzguanidine, p-benzmethyleneamine,
etc. are known to be useful as surrogates of arginine d-
guanidine group in the design of active and selective
The aim of the present study was to ascertain the possi-
bility of use of 4-(isoindoline-5-yl)amino-4-oxobutyric
acid (2) for obtaining of novel RGDF mimetics, aIIbb3
antagonists, as well as, to offer the application of Arg-
Gly surrogate for the further design of potent aIIbb3
antagonists. The synthesis of this Arg-Gly surrogate is
rather simple and short staged.
Keywords: Fibrinogen receptor antagonists; GP IIb/IIIa; RGD mimet-
ics; Isoindoline; Platelet aggregation.
*
Corresponding author. Tel.: +38 0482 663041; fax: +38 0482
652012; e-mail: peptides@paco.net
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.002

5534
A. A. Krysko et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5533–5535
NH2
O
HN
O
HN
N
H
N
H
2
1
O
O
Figure 1. Structures of Arg-Gly mimetics: (aminobenzamidino)succinyl (1) and fragment of 4-(isoindoline-5-yl)amino-4-oxobutyric acid (2).
To obtain RGDF mimetics on the base of 4-(isoindo-
line-5-yl)amino-4-oxobutyric acid derivatives, we have
used 5-nitroisoindoline (3). Blocking of 5-nitroisoindo-
line amino group, then reduction of nitro group in the
obtained N-Boc-5-nitroisoindoline (4) followed by acyl-
ation of the amine 5 with succinic anhydride, afforded
4-(N-Boc-isoindoline-5-yl)amino-4-oxobutyric acid (6)
(Scheme 1).5
The synthesized RGDF mimetics, derivatives of 4-(iso-
indoline-5-yl)amino-4-oxobutyric acid, have showed a
high antiaggregative in vitro activity in human platelet
rich plasma. The IC50 values are of 2.75lM (for 9a)
and 0.86lM (for 9b). It should be noted that an IC50
value for RGDF peptide is of 21lM. In bioassays on
the suspension of human washed platelets IC50 values
are of 2.09lM (for 9a) and 0.27lM (for 9b). The exper-
iments have been carried out by Bornꢀs method in blood
samples of at least three different donors.8 At both ver-
sions of the experiment, ADP in a final concentration of
10lM was used as an inductor of the platelet
aggregation.
The method of mixed anhydrides selected for condensa-
tion of acid 6 with b-alanines esters has brought us to
acceptable results (Scheme 2).6 The condensation of b-
alanines esters with the compound 6 gave the protected
mimetics 7a and 7b. Subsequent saponification of their
ester groups and elimination of Boc-protective groups
yielded the target mimetics 9a and 9b. The use of given
schemes of synthesis makes it possible to obtain the
products of all stages with high yields and of a high
purity, without HPLC processing. RGDF mimetics
obtained are chemically stable substances, both in dried
form and in water solution at normal conditions. The
compounds 9a and 9b are highly water soluble. This fact
is substantial for in vitro bioassays.7
To define the mechanism of antiaggregatory action of
RGDF mimetics 9a and 9b, their influence on fluoresce-
inisothiocyanate labeled fibrinogen (FITC-Fg) binding
to its receptor on the suspension of washed human
platelets was assessed by the procedure of Xia et al.9
FITC-Fg obtained by method described by Hantgan,
R.10 specifically binded to its platelet receptor with a
value Kd (dissociation constant) of 1.02lM. It was
established that compounds 9a and 9b had inhibited
O
a
N
HBr*HN
O
4
NO2
3
NO2
b
O
O
O
O
O
c
N
N
OH
N
H
NH2
6
O
5
Scheme 1. Reagents and conditions: (a) Boc2O; (b) N2H4ÆH2O, 3% Pd in carbon; (c) succinic anhydride.
6
a, b
O
O
H
N
N
O
O
N
H
O
R
O
R = H, 7a; R = C6 H5 , 7b.
c, d
O
O
H
N
N
OH
R = H , 8a; R = C6 H5 , 8b.
O
O
N
H
O
R
e
O
H
N
HCl*HN
OH
N
H
R = H , 9a; R = C6 H5 , 9b.
O
R
O
Scheme 2. Reagents and conditions: (a) Et3N, isobutyl chloroformate; (b) b-amino acid methyl ester, Et3N; (c) 1M NaOH, H2O; (d) 1M HCl, H2O;
(e) 4M HCl in dioxane.

A. A. Krysko et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5533–5535
5535
Tjoeng, F. S.; Lindmark, R. J.; Toth, M. V.; Zupec, M. E.;
McMackins, D. E.; Adams, S. P.; Miyano, M.; Markos,
C. S.; Milton, M. N.; Paulson, S.; Herin, M.; Jacqmin, P.;
Nicholson, N. S.; Panzerknodle, S. G.; Haas, N. F.; Page,
J. D.; Szalony, J. A.; Taite, B. B.; Salyers, A. K.; King, L.
W.; Campion, J. G.; Feigen, L. P. J. Med. Chem. 1995, 38,
2378–2394.
FITC-Fg binding to its receptor in suspension of washed
human platelets with IC50 values of 0.014lM (for 9a)
and 0.0083lM (for 9b). At that, Ki values were of
0.0071lM and 0.0042lM for compounds 9a and 9b,
respectively.
The introduction of phenyl in b position of b-alanine
residue has led to 10-fold enhancement in the antiaggre-
gatory activity and affinity for aIIbb3 of the compound
9b. Similar tendency can be observed, also, for ABAS
series (10): at R=H, an IC50 value is of 1.09lM
(Collagen-induced platelet aggregation, dog platelet-rich
plasma), and at R=Ph, IC50 = 0.294lM (the similar
experimental conditions).3
4. Andronati, S. A.; Krysko, A. A.; Kabanov, V. M.;
Karaseva, T. L.; Kabanova, T. A. Acta Polonia Pharma-
ceut.—Drug Res. 2000, 57(Suppl.), 15–21.
1
5. For 4: mp 159–162°C. H NMR (300MHz, DMSO-d6) d
1.47 (s, 9H), 4.67 (s, 4H), 7.59 (dd, J = 2.5Hz, J = 8.4Hz,
1H), 8.18 (dd, J = 1.9Hz, J = 8.4Hz, 1H), 8.23 (s, 1H). MS
(FAB): 265 (M+H). For 6: mp 205–207°C. 1H NMR
(300MHz, DMFA-d7): d 1.49 (s, 9H), 2.62–2.70 (m, 4H),
4.58 (dd, J = 5.9Hz, J = 10.0Hz, 4H), 7.26 (dd J = 4.8Hz,
J = 9.1Hz, 1H), 7.53 (d, J = 9.1Hz, 1H), 7.75 (d,
J = 9.1Hz, 1H), 10.12 (s, 1H). MS (FAB): 335 (M+H).
6. For 7a: mp 145–148°C. 1H NMR (300MHz, DMFA-d7) d
1.49 (s, 9H), 2.50–2.56 (m, 4H), 2.66 (t, J = 6.8Hz, 2H),
3.41 (q, J = 6.8Hz, 2H), 3.63 (s, 3H), 4.58 (dd, J = 6.3Hz,
J = 9.8Hz, 4H), 7.26 (dd J = 4.8Hz, J = 8.7Hz, 1H), 7.53
(d, J = 8.7Hz, 1H), 7.75 (d, J = 8.7Hz, 1H), 8.00 (t,
J = 5.6Hz, 1H), 10.08 (s, 1H). MS (FAB): 420 (M+H).
For 7b: The oil. 1H NMR (300MHz, DMSO-d6) d 1.45 (s,
9H), 2.41–2.57 (m, 4H), 2.73–2.77 (m, 2H), 3.55 (s, 3H),
4.53 (t, J = 7.3Hz, 4H), 5.22 (q, J = 7.8Hz, 1H), 7.19–7.33
(m, 6H), 7.40 (dd, J = 1.4Hz, J = 8.4Hz, 1H), 7.61 (d,
J = 19.6Hz, 1H), 8.47 (d, J = 8.4Hz, 1H), 9.97 (s, 1H). MS
(FAB): 496 (M+H). For 8a: mp 142–148°C. 1H NMR
(300MHz, DMFA-d7) d 1.49 (s, 9H), 2.47–2.56 (m, 4H),
2.67 (t, J = 6.8Hz, 2H), 3.40 (q, J = 6.8Hz, 2H), 4.58 (dd,
J = 6.7Hz, J = 9.4Hz, 4H), 7.26 (m, 1H), 7.53 (d,
J = 8.1Hz, 1H), 7.75 (d, J = 8.1Hz, 1H), 7.97 (t,
J = 5.6Hz, 1H), 10.10 (s, 1H). MS (FAB): 406 (M+H),
428 (M+Na). For 8b: mp 138–141°C. 1H NMR (300MHz,
DMFA-d7) d 1.49 (s, 9H), 2.57–2.69 (m, 4H), 2.83 (m, 2H),
4.58 (t, J = 6.1Hz, 4H), 5.39 (q, J = 7.5Hz, 1H), 7.21–7.45
(m, 6H), 7.53 (d, J = 8.1Hz, 1H), 7.72 (d, J = 8.1Hz, 1H),
8.46 (d, J = 8.1Hz, 1H), 10.07 (s, 1H). MS (FAB): 482
NH
2
HN
O
H
N
OH
N
H
10
O
R
O
The values for antiaggregative activity and affinity for
aIIbb3 of RGDF mimetics synthesized by us are compa-
rable with the values obtained for well-known RGDF
mimetics containing p-benzamidine group as argrinine
side function bioisoster. Furthermore, it should be men-
tioned that further synthesis of the analogs in the series
of 4-(isoindoline-5-yl)amino-4-oxobutyric acid (2) deriv-
atives is promising for the purposes of SAR studies.
The experimental data obtained for antiaggregatory
activity and inhibition of FITC-Fg binding to fibrinogen
receptor by the novel RGDF mimetics on the base of 4-
(isoindoline-5-yl)amino-4-oxobutyric acid give the pos-
sibility to consider them as potent platelet aggregation
inhibitors and aIIbb3 antagonists.
1
(M+H), 504 (M+Na). For 9a: mp 197–200°C. H NMR
(300MHz, DMSO-d6) d 2.35–2.41 (m, 4H), 2.53–2.61 (m,
2H), 3.23 (q, J = 6.2Hz, 2H), 4.40–4.48 (m, 4H), 7.29 (d,
J = 8.2Hz, 1H), 7.48 (d, J = 8.2Hz, 1H), 7.75 (s, 1H), 8.02
(t, J = 4.7Hz, 1H), 10.06 (s, 2H), 10.22 (s, 1H). MS (FAB):
306 (M+H), 328 (M+Na). For 9b: mp 150–153°C. 1H
NMR (300MHz, DMSO-d6) d 2.42–2.56 (m, 4H), 2.6–2.69
(m, 2H), 4.40–4.47 (m, 4H), 5.39 (q, J = 7.3Hz, 1H), 7.19–
7.34 (m, 6H), 7.47 (d, J = 8.1Hz, 1H), 7.74 (s, 1H), 8.51 (d,
J = 8.1Hz, 1H), 10.05 (t, J = 5.4Hz, 2H), 10.20 (s, 1H).
MS (FAB): 382 (M+H), 404 (M+Na).
Acknowledgements
´
´
We thank Prof. J. Gonzalez-Rodrıguez (Instituto de
´
´
Quımica Fısica, CSIC, Madrid, Spain) for his help in
apply method of binding FITC-Fg and RGDF mimetics
to aIIbb3 on washed human platelets.
7. Eldred, C. D.; Evans, B.; Hindley, S.; Judkins, B. D.;
Kelly, H. A.; Kitchin, J.; Luley, P.; Porter, B.; Ross, B. C.;
Smith, K. J.; Taylor, N. R.; Wheatcroft, J. R. J. Med.
Chem. 1994, 37, 3882.
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