Derivatives of 7-amino-1,2,3,4-tetrahydroisoquinoline and isophthalic acids as novel fibrinogen receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.07.090

The novel fibrinogen receptor antagonists containing fragments of 7-amino-1,2,3,4-tetrahydroisoquinoline and isophthalic acids were synthesized and successfully tested for their ability to inhibit platelet aggregation in vitro and to block FITC-Fg binding

Full text of DOI:10.1016/j.bmcl.2006.07.090

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5294–5297
Derivatives of 7-amino-1,2,3,4-tetrahydroisoquinoline
and isophthalic acids as novel fibrinogen receptor antagonists
Olga L. Malovichko, Anna S. Petrus, Andrei A. Krysko,* Tatyana A. Kabanova,
Sergei A. Andronati, 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 11 July 2006; revised 31 July 2006; accepted 31 July 2006
Available online 21 August 2006
Abstract—The novel fibrinogen receptor antagonists containing fragments of 7-amino-1,2,3,4-tetrahydroisoquinoline and isophtha-
lic acids were synthesized and successfully tested for their ability to inhibit platelet aggregation in vitro and to block FITC-Fg bind-
ing to aIIbb3 on washed human platelets.
ꢀ 2006 Elsevier Ltd. All rights reserved.
One of the main causes of myocardial infarction, stroke,
peripheral ischemia, and paralysis is the process of
thrombus formation.1 Anticoagulants, fibrinolytics,
and antiaggregants are used for the therapeutic treat-
ment of thrombotic events. Fibrinogen receptor antago-
nists are the most interesting objects as antiaggregants.2
Formation of the supramolecular complexes of fibrino-
gen with its receptor (aIIbb3 integrin) leads to platelet
aggregative properties of RGDF mimetics.4 The com-
pounds containing 2H-1,4-benzoxazine-3(4H)-one
scaffold were synthesized by the authors5,6 and found
to be potent aIIbb3 antagonists. The distance between
basic moiety (guanidino-, amidino-, amino-, etc.) and
carboxylic function in molecules of potent RGDF
4,7
˚
mimetics is of 10–15 A. The structure of peptidomi-
metic 1 developed by the authors7 through modification
of cyclopeptide 2 (Fig. 1) corresponds to all these
requirements. Insertion of the residue of m-aminobenzo-
ic acid makes a mimetic molecule fairly rigid and im-
proves its activity. The IC50 value of antiaggregative
activity for the compound 1 is 200 nM.7
aggregation.
Tripeptide
fragment—RGD
(Arg-
Gly-Asp) is responsible for this process.2 Binding of
fibrinogen to aIIbb3 may be blocked by various RGD
containing peptides. Consequently, RGD sequence has
become a base for the design of new aIIbb3 antagonists,
platelet aggregation inhibitors. Antagonists of aIIbb3
receptor are represented by monoclonal antibodies,
RGDF and RGDS containing peptides, RGD
mimetics.3 RGDF mimetics receive a great interest from
investigators as promising synthetic aIIbb3 antagonists
which may be obtained relatively easily.
(Aminobenzamidino)succinyl was proposed as Arg-Gly
surrogate for obtaining RGDF mimetics (ABAS series).8
The linear mimetics 3 and 4 based on the residues of
4-(isoindoline-5-yl)amino-4-oxobutyric9 and 4-(1,2,3,4-
tetrahydroisoquinoline-7-yl-amino)-4-oxobutyric10 acids
as Arg-Gly fragment surrogates were synthesized by us
earlier (Fig. 2). Asp-Phe motif was replaced by the resi-
dues of b-alanine and D,L-b-phenyl-b-alanine. Obtained
mimetics have demonstrated a high in vitro antiaggrega-
tive activity and a high affinity for aIIbb3 in suspension of
washed human platelets.9,10
It is considered4 that RGDF mimetics should incorpo-
rate both basic and acidic binding centers for the effec-
tive interaction with aIIbb3 receptor. The presence of
hydrophobic fragment in the linker which connects these
basic and acidic centers positively influences on the anti-
Study in this paper concerns the possibility to use the
residue of 3-[(1,2,3,4-tetrahydroisoquinoline-7-yl-ami-
no)carbonyl]benzoic acid as Arg-Gly surrogate for
RGDF mimetic creation. b-Alanines containing various
substituents in b-position are successfully used for the
Keywords: Fibrinogen receptor antagonists; aIIbb3; RGD mimetics;
1,2,3,4-Tetrahydroisoquinoline; Platelet aggregation.
*
Corresponding author. Tel.: +38 0482 66 30 41; fax: +38 0482 65 20
12; e-mail: peptides@paco.net
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.090

O. L. Malovichko et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5294–5297
5295
S
S
O
H
HN
O
N
OH
O
N
N
O
O
H
H
HN
O
O
N
N
H
OH
1
H
N
NH2
O
HN
2
NH2
Figure 1. Structures of mimetic 1 and cyclopeptide 2 (SK&F 106760).
O
H
N
HCl*HN
HCl*HN
OH
OH
N
H
3a R = H, 3b R = C6H5
4a R = H, 4b R = C6H5
O
O
R
R
O
O
H
N
N
H
O
Figure 2. Structures of mimetics 3 and 4.
synthesis of RGDF mimetics, fibrinogen receptor block-
ers. The residue of b-substituted b-alanine is considered
to mimic Asp-Phe motif.4,11 Aromatic substituent gives
a possibility for additional binding of a ligand to the
aIIbb3, and carboxylic group of b-alanine imitates the
side chain of aspartic acid residue.
ethyl esters of isophthalic or 5-nitroisophthalic acids
has given derivatives 6a and 6b, and further saponifi-
cation of their ester groups has afforded corresponding
acids 7a and 7b. The compounds 8 were obtained by
condensation of the acids 7a and 7b with b-alanines’
sodium salts conducted by DCC method with SuOH
addition. Removal of Boc-groups of intermediate
compounds 8 afforded target RGDF mimetics 9. All
b-substituted b-alanines used in syntheses represented
racemic mixtures.
7-Amino-2-Boc-1,2,3,4-tetrahydroisoquinoline10
was used as a initial compound (Scheme 1). Acylation
of the compound 5 by chloroanhydrides of monom-
(5)
O
O
a
O
N
O
N
N
O
NH2
O
70 - 75 %
H
O
O
5
R1
6a R = H, 6b R = NO2
b, c
92 - 96 %
d, e, c
O
O
N
N
OH
O
H
O
61 - 77 %
R1
O
O
R2
O
7a R = H, 7b R = NO2
N
N
N
OH
H
H
O
f
8a R1 = H, R2 = H;
R 1
92 - 99 %
8b R1 = H, R2 = C6H5;
8c R1 = H, R2 = pCH3OC6H4;
8d R1 = H, R2 = m,p(CH3O)2C6H3;
8e R1 = NO2, R2 = H;
8f R1 = NO2, R2 = C6H5;
8g R1 = NO2, R2 = pCH3OC6H4;
8h R1 = NO2, R2 = m,p(CH3O)2C6H3.
O
O
R2
O
HCl*HN
N
N
OH
H
H
9a R1 = H, R2 = H;
R1
9b R1 = H, R2 = C6H5;
9c R1 = H, R2 = pCH3OC6H4;
9d R1 = H, R2 = m,p(CH3O)2C6H3;
9e R1 = NO2, R2 = H;
9f R1 = NO2, R2 = C6H5;
9g R1 = NO2, R2 = pCH3OC6H4;
9h R1 = NO2, R2 = m,p(CH3O)2C6H3.
Scheme 1. Reagents and conditions: (a) chloroanhydrides of monomethyl esters of isophthalic or 5-nitroisophthalic acids, Et3N, chloroform, 0 ꢁC;
(b) 1 M NaOH, MeOH/H2O, 20 ꢁC; (c) 1 M HCl, H2O, 0 ꢁC; (d) DCC, SuOH, THF, 0 ꢁC; (e) b-amino acids, NaHCO3, H2O, 0 ꢁC; (f) 4 M HCl in
dioxane, 0 ꢁC.

5296
O. L. Malovichko et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5294–5297
Table 1. Biological properties of RGDF mimetics 3a, 3b, 4a, 4b and 9a–9h
Compound
Antiaggregative activity,
in vitro assays on human PRP, IC50 (nM)
Inhibition of FITC-Fg binding to aIIbb3
on the surface of human activated platelets, IC50 (nM)
a
a
3a
3b
4a
4b
9a
9b
9c
9d
9e
9f
2750.0 ( 370.0)9
860.0 ( 120.0)9
30.0 ( 1.6)10
13.0 ( 1.0)10
78.0 ( 6.5)
14.0 ( 2.1)9
8.3 ( 1.4)9
1.2 ( 0.14)10
1.0 ( 0.12)10
9.0 ( 0.56)
1.0 ( 0.41)
0.8 ( 0.07)
0.7 ( 0.07)
32.0 ( 2.2)
8.0 ( 0.6)
25.0 ( 1.7)
17.0 ( 1.6)
11.6 ( 1.3)
2300.0 ( 160.0)
710.0 ( 39.0)
450.0 ( 41.0)
340.0 ( 23.0)
9g
9h
5.0 ( 0.4)
3.7 ( 0.4)
a Values are means of three experiments, standard deviation is given in parentheses.
The compounds 9 have demonstrated a high in vitro
antiaggregative activity in bioassays on a human
platelet-rich plasma (PRP) (Table 1) by Born’s meth-
od12,13 in blood obtained from at least three donors.
Platelet aggregation was induced by ADP. In order
to reveal the molecular mechanism of antiaggregatory
action of RGDF mimetics 9, their influence on spe-
cific binding of fluoresceinisothiocyanate-labeled
fibrinogen (FITC-Fg) to aIIbb3 (in a suspension of
human washed platelets) was examined by the proce-
dure.14,15 FITC-Fg obtained by the method16 specifi-
cally bound to platelet receptors with a dissociation
constant (Kd) of 1.02 lM. Experimental data (Table
1) evidently show high affinities of the compounds
9 for aIIbb3.
possible to note negative influence of a nitro group
on inhibition of FITC-Fg binding to aIIbb3 and anti-
aggregative activity. RGDF mimetics 9c and 9d have
demonstrated maximum affinity for aIIbb3 receptors
on the surface of human washed platelets.
Experimental data obtained on antiaggregative activity
and inhibition of FITC-Fg binding to fibrinogen recep-
tor allow to consider the novel RGDF mimetics based
on
3-[(1,2,3,4-tetrahydroisoquinoline-7-yl-amino)car-
bonyl]benzoic acid and b-substituted b-alanines as po-
tent platelet aggregation inhibitors and aIIbb3 receptor
antagonists.
References and notes
Earlier, it was demonstrated by us that incorporation
of phenyl in the b-position of b-alanine residue in the
compound 3a brought a 3-fold increase in antiaggre-
gative activity for the compound 3b and a 2-fold
increase in its affinity.9 For the derivatives of 4-
(1,2,3,4-tetrahydroisoquinoline-7-yl-amino)-4-oxobutyr-
ic acid, this structure modification, practically,
produced no effect on the affinity of mimetic 4b for
aIIbb3, while its antiaggregative activity had enhanced
almost twice corresponding to the compound 4a.10
Similar tendency could be traced also for the peers
mimetics 9a and 9b, 9e and 9f. In general, biological
activity of the compounds containing fragment of
isophthalic acid (mimetics 9a and 9b) was lower than
that of their analogs 4a and 4b. The presence of meth-
oxy substituents in para position of the phenyl groups
situated in b-alanine moieties of RGDF mimetics 9c
and 9g afforded a minor enhancement of indexes for
inhibition of FITC-Fg binding to aIIbb3 and antiag-
gregative activity of these compounds relative to their
analogs 9b and 9f, respectively. Introduction of second
methoxy group in the meta position of benzene ring
also insignificantly improved values of IC50 both for
binding to aIIbb3 and for platelet aggregation inhibi-
tion in in vitro assays for the compounds 9d and
9h, compared to their analogs 9c and 9g. Comparison
of biological properties of the mimetics 9a–9d without
nitro group in isophthalic acid fragment with those of
their analogs 9e–9h containing nitro groups makes it
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15. Fibrinogen (Sigma) was labeled with fluoresceinisothi-
ocyanate (ACROS). Fresh platelet concentrate was
centrifuged (900g, 15 min), and the platelet pellet was
washed at pH 5.6 in the presence of PGE1 and finally
resuspended carefully in Tyrode’s buffer, containing
BSA, pH 7.4. Platelet suspension was incubated for
30 min with peptidomimetics at concentrations in the
range from 0.001 nM to 1 lM at 37 ꢁC. FITC-Fg was
added after addition of agonist (ADP, 2 lM). Follow-
ing 60-min incubation at room temperature (under
protection from the light), the reaction mixtures were
layered onto a 20% sucrose cushion and centrifuged at
6000 rpm for 15 min. The tips of each tube were
clipped off, and the platelet pellet, containing the
bound fibrinogen, solubilized with a 3% SDS solution.
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13. PRP was prepared by centrifugation of whole blood at
150 g for 10 min, and the platelet count was adjusted to
1 · 108 platelets/mL with time matched platelet-poor
plasma (PPP). PRP (250 ll) was preincubated with 50 ll
of various concentrations of compounds to be tested, or
saline, for 2 min at 37 ꢁC prior to the addition of ADP
(10 ll). Platelet aggregation was measured by the change
of light transmittance (PPP represents 100%) under
stirring conditions (1000 rpm) on a ‘THROMLITE-1006
A’ aggregometer. The ability of the compounds to inhibit
platelet aggregation was measured and the IC50 was
determined as the concentration of the compound
required to produce 50% inhibition of the response to
ADP.
The
spectrofluorimetrically.
16. Hantgan, R. Biochim. Biophys. Acta 1987, 927, 55.
bound
FITC-Fg
was
quantitated