Synthesis and antiaggregative activity of a new RGDF mimetic

Article abstract of DOI:10.1023/B:RUBI.0000049769.62906.0d

m-[4-Oxo-4-(piperazin-1-yl)butyrylamino)benzoyl-D,L-β-(3, 4-methylenedioxyphenyl)-β-alanine, a new mimetic of the peptide RGDF, was synthesized. This compound inhibited ADP-induced platelet aggregation in human blood plasma enriched with platelets with IC

Full text of DOI:10.1023/B:RUBI.0000049769.62906.0d

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 6, 2004, pp. 534–538. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 6, 2004, pp. 594–598.
Original Russian Text Copyright © 2004 by Krysko, Malovichko, Kabanova, Mazepa.
Synthesis and Antiaggregative Activity
of a New RGDF Mimetic
1
A. A. Krysko , O. L. Malovichko, T. A. Kabanova, and A. V. Mazepa
Bogatsky Physicochemical Institute, National Academy of Sciences of Ukraine,
Lustdorfskaya doroga 86, Odessa, 65080 Ukraine
Received October 15, 2003; in final form, February 1, 2004
Abstract—m-[4-Oxo-4-(piperazin-1-yl)butyrylamino)benzoyl-D,L-β-(3,4-methylenedioxyphenyl)-β-alanine,
a new mimetic of the peptide RGDF, was synthesized. This compound inhibited ADP-induced platelet aggre-
gation in human blood plasma enriched with platelets with IC50 = 3.5 × 10–8 M.
Key words: GP IIb/IIIa, peptide synthesis, platelet aggregation, RGDF mimetic
1
INTRODUCTION
Previously, we synthesized a number of linear
RGDF peptidomimetics (III) and (IV) on the basis of
4-oxo-4-(piperazin-1-yl)butyric acid mimicking the
Arg residue. The Asp-Phe sequence was replaced by
the residues of β-substituted β-alanines [3–5]. These
compounds exhibited sufficiently high antiaggregative
activities (IC50 varied from 10–6 to 10–8 M). Affinities of
the most active compounds to the fibrinogen receptors
was studied by the method of fluorescent analysis
[4, 5].
A search for new antithrombotic and antiaggrega-
tive agents with high efficiency and low toxicity is one
of the most important problems of bioorganic and
2
medicinal chemistry.
In the last ten years, sedulous attention has been
paid to a group of antiaggregative agents that are antag-
onists of the GP IIb/IIIa fibrinogen receptors. The tri-
peptide sequence RGD (Arg-Gly-Asp) is responsible
for the binding of fibrinogen to its receptors. Small pro-
teins and peptides containing the RGD sequence are
antagonists of the fibrinogen receptors, and the peptides
involving this sequence can be used as inhibitors of the
platelet aggregation. The molecular design of potential
antagonists of the fibrinogen receptors is aimed at an
increase in their selectivity and affinity and involves the
modeling of structures containing this sequence or
fragments mimicking it.
RESULTS AND DISCUSSION
The goal of this study was the synthesis of a new
RGDF peptidomimetic (XII) that contains the residue
of 4-oxo-(4-piperazin-1-yl)butyric acid instead of Arg,
a fragment of m-aminobenzoic acid, and the residue of
D,L-β-(3,4-methylenedioxyphenyl)-β-alanine.
The
C-terminal hydrophobic radical is necessary for the
effective binding of the mimetic to the receptor [6].
The molecules of highly active RGDF mimetics
should have a certain distance (10–15 Å) between two
binding centers: the terminal basic group (guanidino,
amidino, amino, or another group) and carboxyl func-
tion [1]. The structure of peptidomimetic (II), which
was prepared by Alig et al. [2] by a modification of
cyclopeptide (I) (Scheme 1), meets these requirements.
Introduction of the residue of m-aminobenzoic acid
provides for the substantial rigidity of the mimetic mol-
ecule and exerts a positive effect on its activity. The IC50
The synthesis of pseudopeptide (XII) is presented in
scheme 1. 4-Oxo-4-(4-Boc-piperazin-1-yl)butyric acid
(V) was used as a starting compound [3]. The conden-
sation of m-aminobenzoic acid and Boc-derivative (V)
was carried out by three methods. The first method con-
sisted in the use of mixed anhydride (VIII), which was
prepared from the triethylammonium salt of acid (V)
and isobutyl chloroformate. Succinimide ester (VI) and
pentafluorophenyl ester (VII) were used in the second
and in the third methods, respectively. The esters (VI)
value for the antiaggregative activity of (II) is 200 nM and (VII) were synthesized using DCC and N-hydrox-
[2].
ysuccinimide or pentafluorophenol, respectively. The
amino group of m-aminobenzoic acid was acylated
with derivatives (VI)–(VIII). The best result was
achieved with pentafluorophenyl ester (VII); an aver-
age yield of Boc-derivative (IX) was approximately
80%. The next stage was an activation of carboxyl
group in (IX) by DCC in the presence of HONSu. The
1
Corresponding author; phone: +38-482-66-3041; e-mail: pep-
tides@paco.net
2
Abbreviations: β-Ala, β-alanine, DCC, N,N'-dicyclohexylcarbo-
diimide; FAB, fast atom bombardment; HONSu, N-hydroxysuc-
cinimide; PfpOH, pentafluorophenol; Piz, piperazinyl; and Suc,
succinyl.
1068-1620/04/3006-0534 © 2004 MAIK “Nauka /Interperiodica”

SYNTHESIS AND ANTIAGGREGATIVE ACTIVITY OF A NEW RGDF MIMETIC
535
Template
S
S
O
H
HN
O
N
OH
O
N
N
O
O
H
H
N
HN
O
O
N
OH
(II)
H
N
NH2
H
HN
O
(I)
NH2
Scheme 1. The conversion of cyclopeptide (I) into the RGDF mimetic (II).
corresponding succinimide ester (X) resulted. It was was used as an internal standard. The mass spectrum of
reacted with the sodium salt of D,L-β-(3,4-methylene- (VI) was recorded on an MX-1321 mass spectrometer
dioxyphenyl)-β-alanine [7], which resulted in Boc- (NPO Nauchnyi Pribor, Russia) at the ionization energy
derivative (XI). The target (XII) was prepared by the of 70 eV and at the temperature of 200°ë in the ioniza-
acidolytic removal of Boc-group from (XI).
tion chamber. The FAB mass spectra were recorded on
a VG 7070 spectrometer (VG, UK) using a glycerol
matrix with the ionization by a beam of Xe atoms with
the energy of 8 kV. The purity of compounds was exam-
ined by HPLC on a DuPont 8800 chromatograph
(DuPont Instruments, United States) equipped with a
Zorbax C8 analytical column (DuPont Instruments,
Structures of the synthesized compounds were con-
1
firmed by mass spectrometry and H NMR spectros-
copy. All the characteristic signals from protons with
the corresponding integral intensities were present in
the 1H NMR spectra of the synthesized compounds.
The antiaggregative activity of the prepared RGDF United States) eluted with a 4 : 1 acetonitrile–water
mimetic (XII) was studied on human blood plasma rich mixture. TLC was carried out on Silufol sheets (Kava-
with platelets according to the Born method [8]. Com- lier, Czech Republic) and Kieselgel 60 precoated plates
pound (XII) inhibited the ADP-induced aggregation of (Merck, Germany) in the following chromatographic
platelets with IC50 of 3.5 × 10–8 M. We should note that
systems: (A) 100 : 50 : 1 benzene–acetone–acetic acid,
the IC50 value for the RGDF peptide was 1.9 × 10–6 M.
(B) 4 : 1 methanol–ammonia, (C) 9 : 3 : 2 chloroform–
ethyl acetate–methanol, and (D) 9 : 3 : 2 : 1 chloro-
form–ethyl acetate–methanol–acetic acid. The sub-
stances were detected by ninhydrin or by chlorine–tolu-
idine reagent.
Thus, the RGDF mimetic (XII) exhibited a high antiag-
gregative activity.
EXPERIMENTAL
The following designations are used in the descrip-
tion of 1H NMR spectra: Ar-βAla, aromatic protons of
1
The H NMR spectra were recorded on a Varian
the β-(3,4-methylenedioxyphenyl)-β-alanine residue;
αëç2-β-Ala, α-protons of the β-aryl-substituted β-ala-
WXP-300 spectrometer (Varian, Germany) with a
working frequency of 299.95 MHz in DMSO-d6
(99.9%) at the temperature of 25°ë. Tetramethylsilane nine residue; βëç-β-Ala, β-proton of the residue of the
O
OH
H
N
O
O
O
O
O
H
N
N
N
H
OH
HCl · HN
O
O
O
(III)
R
O
H
N
N
N
H
OH
(IV), R = H, Ar, Alk.
HCl · HN
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 6 2004

536
KRYSKO et al.
O
O
N
O
N
OH
O
(V)
DCC, HONSu
DCC, PfpOH
Cl
O
O
O
O
NEt3
O
N
O
O
N
O
N
N
OSu
OPfp
O
O
(VI)
(VII)
O
O
N
O
N
O
O
O
O
(VIII)
O
(VI) or (VII) or (VIII)
OH
N
O
N
O
H2N
N
O
O
N
H
O
O
OH
DCC, HONSu
(IX)
O
N
O
OSu
(X)
H2N
OH
N
H
1) NaHCO3
2) H+
O
O
O
O
O
O
O
N
O
H
N
N
OH
N
H
O
O
O
HCl · HN
O
HCl in dioxane
H
N
N
OH
O
N
(XI)
O
H
O
O
O
(XII)
O
O
Scheme 2. The synthesis of m-[4-oxo-4-(piperazin-1-yl)butyrylamino]benzoyl-DL-β-(3,4-methylenedioxyphenyl)-β-alanine
(XII).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 6 2004

SYNTHESIS AND ANTIAGGREGATIVE ACTIVITY OF A NEW RGDF MIMETIC
537
aryl-substituted β-alanine; CH2-NSu, protons of the carried out as described in the method B. The yield
87%; Rf 0.39 (C), 0.45 (D); mp 155°ë; FAB MS, m/z:
succinyl residue; CH2-Piz, protons of the piperazinyl
1
residue; and NH-β-Ala, amide proton of the residue of 406 [M + H]+; H NMR (δ, ppm): 1.41 (9 H, s,
β-substituted β-alanine.
CH3-Boc), 2.63 (4 H, dd, CH2-Suc), 3.28–3.36 (4 H, m,
CH2-Piz), 3.42–3.46 (4 H, m, CH2-Piz), 7.41 (1 H, t,
Succinimidyl
4-oxo-4-(4-Boc-piperazin-1-
yl)butyrate (VI). HONSu (2.414 g, 20.9 mmol) and Ar), 7.59 (1 H, d, Ar), 7.80 (1 H, d, Ar), 8.24 (1 H, s,
DCC (4.329 g, 20.9 mmol) were added to a solution of Ar), 10.14 (1 H, s, NH), and 12.92 (1 H, br. s, COOH).
(V) (5 g, 17.5 mmol) [3] in anhydrous THF (40 ml).
Succinimidyl
m-[4-oxo-4-(4-Boc-piperazin-1-
The reaction mixture was cooled to 0°ë and stirred at
this temperature for 4 h. When the reaction was com-
pleted, the precipitated N,N'-dicyclohexylurea was fil-
tered off, and the filtrate was evaporated in a vacuum at
the temperature no higher than 35°ë. The residue was
recrystallized from isopropanol, washed with cool iso-
propanol and ether, and dried in desiccator over CaCl2
to give (VI); yield 5.825 g (87%); Rf 0.41 (A), 0.56 (D);
mp 155–159°ë; mass spectrum, m/z (I, %): 383 (5.1),
327 (6.4), 310 (12.7), 269 (22.4), 213 (40.6), 212
(14.4), 211 (5.2), 168 (6.0), 167 (5.8), 115 (9.3), 100
(11.5), 97 (12.5), 86 (5.0), 84 (20.9), 83 (6.8), 69 (16.4),
yl)butyrylamino]benzoate (X). HONSu (2.414 g,
20.9 mmol) and DCC (4.329 g, 20.9 mmol) were added
to a solution of (IX) (7.087 g, 17.5 mmol) in anhydrous
THF (30 ml). The reaction mixture was cooled to 0°ë
and stirred at this temperature for 4 h. The precipitated
N,N'-dicyclohexylurea was filtered off, and the filtrate
was evaporated at the temperature no higher than 35°ë.
The residue was recrystallized from isopropanol. The
precipitated product was washed with cool isopropanol
and ether and dried in a desiccator over CaCl2 to give
(X); yield 7.028 g (80%); Rf 0.52 (C); mp 185–189°ë;
FAB MS, m/z: 503 [M + H]+; 1H NMR (δ, ppm): 1.41
(9 H, s, CH3-Boc), 2.63 (4 H, dd, CH2-Suc), 2.79 (4 H,
s, CH2-NSu), 3.29–3.36 (4 H, m, CH2-Piz), 3.42–3.46
(4 H, m, CH2-Piz), 7.58 (1 H, t, Ar), 7.74 (1 H, d, Ar),
7.91 (1 H, d, Ar), 8.49 (1 H, s, Ar), 10.33 (1 H, s, NH).
1
58 (6.1), 57 (100), 56 (33.8), 55 (45.1); H NMR (δ,
ppm): 1.41 (9 H, s, CH3-Boc), 2.70 (2 H, t, CH2-NSuc),
2.79 (4 H, s, CH2-NSu) 2.87 (2 H, t, CH2-Suc), 3.28–
3.34 (4 H, m, CH2-Piz), and 3.42–3.4 (4 H, m, CH2-
Piz).
m-[4-Oxo-4-(4-Boc-piperazin-1-yl)butyrylami-
no]benzoyl-DL-b-(3,4-methylenedioxyphenyl)-b-ala-
nine (XI). A solution of β-(3,4-methylenedioxyphe-
nyl)-β-alanine (1.421 g, 8.2 mmol) [7] and NaHCO3
(0.687 g, 8.2 mmol) in water (20 ml) was added to a
solution of (X) (3.413 g, 6.8 mmol) in a freshly distilled
THF (20 ml). When the reaction was over, the reaction
mixture was evaporated in a vacuum to the half of its
initial volume, acidified to pH 4 with 3 N HCl, and
extracted with chloroform (3 × 50 ml). The organic
layer was washed with 1 N HCl (10 ml) and water
(10 ml), dried with anhydrous Na2SO4, and evaporated
in a vacuum. The oily residue was triturated with hot
ether for the further purification, cooled, and filtered to
give (XI); yield 3.85 g (95%); Rf 0.47 (C) and 0.58 (D);
m-[4-Oxo-4-(4-Boc-piperazin-1-yl)butyrylamino]-
benzoic acid (IX). Method A. Triethylamine (0.58 ml,
4.2 mmol) was added to a solution of (V) (1 g,
3.5 mmol) in chloroform (15 ml). The solution was
cooled to –5°ë, and isobutyl chloroformate (0.5 ml,
3.8 mmol) was added. The reaction mixture was kept
for 1 h at –5°ë, and m-aminobenzoic acid (0.578 g,
4.2 mmol) was added. The reaction mixture was
refluxed for 3 h, washed with 1 M solution of HCl (2 ×
10 ml), water (2 × 10 ml), and a saturated solution of
NaCl (10 ml), and dried with anhydrous sodium sulfate.
Sodium sulfate was filtered off, chloroform was evapo-
rated, and the residue was dried in a vacuum. Ether was
added to the oily residue, and the mixture was triturated
to crystallization of the product. The crystals were fil-
tered, washed with ether, and dried. The yield 0.76 g
(35%).
Method B. m-Aminobenzoic acid (4.29 g, 31 mmol)
was added to a solution of (VI) (10 g, 26mmol) in THF
(40 ml). The reaction mixture was refluxed for 3 h and
evaporated under a reduced pressure. Water (50 ml)
was added to the residue, and the mixture was acidified
to pH 3–4 with concentrated HCl and extracted with
chloroform. The chloroform extract was washed with
1 M HCl (2 × 50 ml), water (2 × 50 ml), and a saturated
solution of NaCl (50 ml); dried with anhydrous sodium
sulfate; and evaporated. The residue was dried in a vac-
uum, mixed with ether, and triturated to the crystalliza-
tion of oily residue. The crystals were filtered, washed
with ether, and dried. The yield was 6.66 g (63%).
mp 160°ë; FAB MS, m/z: 619 [M + Na]+, 641 [M – H +
2Na]+; 1H NMR (δ, ppm): 1.41 (9 H, s, CH3-Boc), 2.60
(4 H, dd, CH2-Suc), 2.80 (2 H, ddd, αëç2-βAla), 3.29–
3.45 (8 H, m, CH2-Piz), 5.34 (1 H, q, βëç-βAla), 5.97
(2 H, s, OCH2O), 6.82–6.88 (2 H, m, Ar-βAla), 7.00
(1 H, s, Ar-βAla), 7.36 (1 H, t, Ar), 7.47 (1 H, d, Ar),
7.77 (1 H, d, Ar), 7.95 (1 H, s, Ar), 8.74 (1 H, d, NH-
βAla), 10.06 (1 H, s, NH-Ar), and 12.21 (1 H, br. s,
COOH).
m-[4-Oxo-4-(piperazin-1-yl)butyrylamino]benzoyl-
DL-b-(3,4-methylenedioxyphenyl)-b-alanine hydro-
chloride (XII). A suspension of Boc-derivative (XI)
(0.5 g, 1 mmol) in 4 M solution of HCl in dioxane
(10 ml) was stirred for 1 h at room temperature and
evaporated. The solid residue was dried for 5 h at 40°C
and at the pressure of 3 mmHg. The substance (XII) is
hygroscopic; yield 0.536 g (quantitative); Rf 0.6 (B);
Method C. This method is based on the reaction of
pentafluorofenyl ester (VII) [3] with m-aminobenzoic
acid. Preparation of the product and its isolation were
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 6 2004

538
KRYSKO et al.
FAB MS, m/z: 497 [m/z: 497 [M + H]+, 519 [M + Na]+,
541 [M – H + 2Na]+; 1H NMR (δ, ppm): 2.62 (4 H, dd,
CH2-Suc), 2.70–2.90 (2 H, m, αëç2-βAla), 3.49 (4 H,
REFERENCES
1. Zablocki, J.A., Rico, J.G., Garland, R.B., Rogers, T.E.,
Williams, K., Schretzman, L.A., Rao, S.A., Bovy, P.R.,
Tjoeng, F.S., Lindmark, R.J., Toth, M.V., Zupec, M.E.,
McMackins, D.E., Adams, S.P., Miyano, M., Mar-
kos, C.S., Milton, M.N., Paulson, S., Herin, M., Jac-
qmin, 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., and Feigen, L.P., J. Med.
Chem., 1995, vol. 38, pp. 2378–2394.
2. Alig, L., Edenhofer, A., Hadvary, P., Hurzeler, M.,
Knopp, D., Muller, M., Steiner, B., Trzeciak, A., and
Weller, T., J. Med. Chem., 1992, vol. 35, pp. 4393–4407.
3. Krys’ko, A.A., Kabanov, V.M., Polishchuk, P.G., Chugu-
nov, B.M., Pavlovskii, V.I., Andronati, S.A., Mazepa, A.V.,
Kabanova, T.A., and Karaseva, T.L., Zh. Obshch. Khim.,
2000, vol. 70, pp. 1013–1017.
4. Andronati, S.A., Krysko, A.A., Kabanov, V.M.,
Karaseva, T.L., and Kabanova, T.A., Acta Poloniae Phar-
maceutica, Drug Research, 2000, vol. 57, pp. 15–21.
5. Andronati, S.A., Krysko, A.A., Kabanov, V.M., Kaba-
nova, T.A., Karaseva, T.L., Chugunov, B.M., Mesh-
kova, S.B., and Topilova, Z.M., Acta Poloniae Pharma-
ceutica, Drug Research, 2004, vol. 60, pp. 375–381.
6. Sugihara, H., Fukushi, H., Miyawaki, T., Imai, Y.,
Terashita, Z., Kawamura, M., Fujisawa, Y., and Kita, S.,
J. Med. Chem., 1998, vol. 41, pp. 489–502.
d, CH2-Piz), 3.71 (4 H, d, CH2-Piz), 5.33 (1 H, q, βëç-
βAla), 5.98 (2 H, s, OCH2O), 6.85 (2 H, s, Ar-βAla),
7.01 (1 H, s, Ar-βAla), 7.37 (1 H, t, Ar), 7.49 (1 H, d,
Ar), 7.76 (1 H, d, Ar), 7.97 (1 H, s, Ar), 8.79 (1 H, d,
NH-βAla), 9.25 (2 H, s, H2N+), 10.16 (1 H, s, NH-Ar),
and 12.41 (1 H, br. s, COOH).
Pharmacological part. The platelet aggregation
was studied on the platelet rich plasma (PRP) [8] pre-
pared by centrifugation of citrate human blood at 200 g
for 15 min. The aggregation of platelets was measured
according to increase in the light transparency on a
Thromlite 1006A aggregometer (Biokhimmak, Mos-
cow, Russia) connected with a recording device. PRPs
(250 µl) were incubated with the studied compound
(XII) taken in various concentrations at 37°C for 1 min
before the addition of ADP (the final concentration was
10 µM). The platelet aggregation was measured for
2 min (maximum aggregation). The biological activity
of RGDF mimetic (XII) was quantitatively character-
ized by the IC50 value, the substance concentration at
which the maximum amplitude of the platelet aggrega-
tion is 50% from the starting level.
7. Rodionov, V.M., Izbrannye trudy (Selected Works),
Moscow: Akad. Nauk SSSR, 1958.
8. Born, G.V.R., Nature, 1962, vol. 194, pp. 927–929.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 6 2004