Synthesis and thrombolytic activity of fibrinogen fragment related cyclopeptides

Article abstract of DOI:10.1016/S0960-894X(02)01072-7

In the modification of the fibrinogen fragment related sequences ARPAK, QRPAK GRPAK and KRPAK, the corresponding cyclo-ARPAK, cyclo-QRPAK, cyclo-GRPAK, and cyclo-KRPAK were prepared in the diluted solution. The bioassay in vivo indicated that the thrombolytic potencies of cyclo-ARPAK, cyclo-GRPAK, cyclo-QRPAK, and cyclo-KRPAK were significantly higher than that of ARPAK, QRPAK, GRPAK, and KRPAK. In water, the cyclopeptides were incubated with pepsin or trypsin at 37°C for 64 h. There was no degradation product observed, on the other hand, with the same condition, the peptides were completely hydrolyzed in 8 h. The relationships among the rigidity or the conformation and the thrombolytic activity in vivo and the stability to enzyme-induced hydrolysis in vitro of the cyclopeptides were discussed.

Full text of DOI:10.1016/S0960-894X(02)01072-7

Bioorganic & Medicinal Chemistry Letters 13(2003) 961–964
Synthesis and Thrombolytic Activity of Fibrinogen Fragment
Related Cyclopeptides
Ming Zhao, Na Lin, Chao Wang and Shiqi Peng*
College of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
Received 9 October 2002; accepted 3December 2002
Abstract—In the modification of the fibrinogen fragment related sequences ARPAK, QRPAK GRPAK and KRPAK, the corre-
sponding cyclo-ARPAK, cyclo-QRPAK, cyclo-GRPAK, and cyclo-KRPAK were prepared in the diluted solution. The bioassay in
vivo indicated that the thrombolytic potencies of cyclo-ARPAK, cyclo-GRPAK, cyclo-QRPAK, and cyclo-KRPAK were sig-
nificantly higher than that of ARPAK, QRPAK, GRPAK, and KRPAK. In water, the cyclopeptides were incubated with pepsin or
trypsin at 37 ꢀC for 64 h. There was no degradation product observed, on the other hand, with the same condition, the peptides
were completely hydrolyzed in 8 h. The relationships among the rigidity or the conformation and the thrombolytic activity in vivo
and the stability to enzyme-induced hydrolysis in vitro of the cyclopeptides were discussed.
# 2003Published by Elsevier Science Ltd.
In the metabolism studies in vitro, it was found that
when ARPAK, the fragment from fibrinogen, and its
derivatives QRPAK and GRPAK, were incubated with
pepsin or trypsin in water at 37 ꢀC for 8 h, complete
degradation was observed. Comparing to the unmodi-
fied peptide, the introduction of 3-(S)-1,2,3,4-tetra-
hydrocarboline- 3-carboxylic acid into their C-terminal
improved the thrombolytic potency and the stability to
pepsin or trypsin hydrolysis for N-ARPAK-,
N-GRPAK-, and N-QRPAK-3-(S)-1,2,3,4-tetra- hydro-
beta-carboline-3-carboxylic acid.1 The results from the
bioassay in vitro, in vivo, and the conformation analysis
with discover program particularly by Sybyl version 6.4
suggested that the increase of the conformation rigidity
of ARPAK, GRPAK, QRPAK and related sequences,
resulted from the introduction of 3-(S)-1,2,3,4-tetra-
hydro-beta-carboline-3-carboxylic acid, was probably
responsible for the enhanced potency in vitro and in
vivo as mentioned above.1ꢁ3 As the further modification
aimed at the increase of the conformation rigidity, the
corresponding cyclo-ARPAK, cyclo-GRPAK, cyclo-
QRPAK and cyclo-QRPAK were prepared and their
stability to enzyme-induced hydrolysis in vitro, the
thrombolytic potency in vivo and the conformation
were observed.
Chemistry
The protected intermediates (1a–d and 11a–d) were pre-
pared via the solution method mentioned in the litera-
ture.4 The stepwise synthesis (from C-terminal to
N-terminal, in 83–96% yield) was carried out starting
with l-Lys(Z)-OBzl and l-Ala-OBzl, respectively.
According to Scheme 1, by use of the suitable proce-
.
dures, 1a was converted into HCl Ala-Arg(Tos)-Pro-
.
Ala-Lys(Z)-N3 (9) via HCl Ala-Arg(Tos)-Pro-Ala-
.
Lys(Z)-NH2NH2 in 70% total yield, into HCl Ala-
Arg(Tos)-Pro-Ala-Lys(Z)-ONP (6) via Boc-Ala-Arg
(Tos)-Pro-Ala-Lys(Z)-OH in 76% total yield, and into
.
HCl Ala-Arg(Tos)-Pro-Ala-Lys(Z)-OH (3a) in 80%
yield. The cyclization of 9 in the presence of DIEA, of
3a in the presence of TBTU/HOBT/NMM, HBTU/
HOBt/NMM, BOP/HOBt/NMM, or BOP/DIEA/
NMM gave no desirable product, cyclo-Ala- Arg(Tos)-
Pro-Ala-Lys(Z) (4a). Cyclization of 3a in the presence
of DCC/NMM or 6 in the presence of NMM afforded
4a in 18 and 5% yield, respectively. Thus, the cycli-
zation of the diluted solution of the protective peptides
(3a–d, 10ꢁ3 mol/L) was carried out by use of the routes
described in Schemes 2 and 3. The yields of 4a–d were
*Corresponding author. Tel.:+86-10-6209-2482; fax:+86-10-6209-
2311; e-mail: sqpeng@mail.bjmu.edu.cn
0960-894X/03/$ - see front matter # 2003Published by Elsevier Science Ltd.
doi:10.1016/S0960-894X(02)01072-7

962
M. Zhao et al. / Bioorg. Med. Chem. Lett. 13 (2003) 961–964
Scheme 1. Selection of the reaction conditions for cyclization: (a) 2 mol/L of NaOH; (b) 4 mol/L of HCl in EtOAc; (c) TBTU/HOBt/NMM, HTBU/
.
HOBt/NMM, BOP/HOBt/NMM, BOP/DIEA/NMM, DCC/NMM; (d) p-NO2-phenol/DCC; (e) NMM; (f) NH2NH2 H2O; (g) HCl/NaNO2;
(h) DIEA.
Scheme 2. Preparation of cyclo-AA0-Arg-Pro-Ala-Lys from Boc-AA0-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl: (a) 2 mol/L of NaOH; (b) 4 mol/L of HCl in
EtOAc; (c) DCC/NMM; (d) HF; for 4a AA’=Ala, for 4b AA0=Gln, for 4c AA0=Gly, for 4d AA0=Lys, for 10a AA=Ala, for 10b AA=Gln, for
10c AA=Gly, for 10d AA=Lys.
Scheme 3. Preparation of cyclo-Pro-Arg-AA0-Lys-Ala from Boc-Pro-Arg(Tos)-AA0-Lys(Z)-Ala-OBzl: (a) 2 mol/L of NaOH; (b) 4 mol/L of HCl in
EtOAc; (c) DCC/NMM; (d) HF; for 4a AA0=Ala, for 4b AA0=Gln, for 4c AA0=Gly, for 4d AA0=Lys, for 10a AA=Ala,for 10b AA=Gln, for
10c AA=Gly, for 10d AA=Lys.

M. Zhao et al. / Bioorg. Med. Chem. Lett. 13 (2003) 961–964
963
listed in Table 1. In the presence of HF, 4a–d were con-
verted into the target cyclopeptides, 10a–d, in 53, 64, 82
and 80% yield, respectively.
Thrombolytic activities in vivo5
Male Wistar rats weighing 200–300 g (purchased from
Animal Center of Peking University) were anesthetized
with pentobarbital sodium (80.0 mg/kg, ip). The right
carotid artery and left vein jugular of the animals were
separated. To the glass tube filled with artery blood (1.0
mL) from the right carotid artery of the animal, a
stainless steel filament helix (15 circles; l: 15 mm; d: 1.0
mm) was put immediately. After 15 min, the helix with
thrombus was carefully taken out and weighted accu-
rately, which was put into the middle polyethylene tube.
The polyethylene tube was filled with heparin sodium
(50 IU/mL of NS) and one end was inserted into the left
jugular vein. Heparin sodium was injected via the other
end of the polyethylene tube as the anticoagulant, fol-
lowed by the injection of the tested compound. The
blood was circulated through the polyethylene tube for
90 min, after which the helix was taken out and weigh-
ted accurately. The reduction of thrombus mass was
recorded. The data are listed in Table 3. The statistical
analysis of the date was carried out by use of ANOVA
test, p<0.05 is considered significant.
Stability to pepsin or trypsin in vitro
10 mg of the tested compound were dissolved in 1 mL of
phosphate buffer (pH 8). To the solution 0.5 mg of
pepsin or trypsin were added. The reaction mixture was
kept at 37 ꢀC and the concentration of the tested com-
pound was monitored every 1 h by HPLC, the mobile
phase is 25% of CH3OH in water containing 0.1% of
CF3COOH. Results indicated that the depletion of
ARPAK (14), QRPAK (15), GRPAK (16), and
KRPAK (17) was observed after the enzyme promotion
hydrolysis was proceeded for 8 h. On the other hand,
however, the concentrations of 10a, 10b, 10c and 10d
were not changed at all even the enzyme promotion
hydrolysis was proceeded for 64 h. The related data are
listed in Table 2.
Table 1. Yield in cyclizationa
Material
Condensation agents
Product
Yield (%)
3a
3a
3a
3a
3a
6
9
3b
3c
3d
13a
13b
13c
13d
TBTU, HOBt, NMM
TBTU, HOBt, NMM
BOP, HOBt, NMM
BOP, DIEA, NMM
DCC, NMM
NMM
—
—
—
—
4a
4a
—
4b
4c
4d
4a
4b
4c
4d
—
—
—
—
18
5
—
15
31
10
9
Conformational Analysis
Based on the calculated results by Sybyl version 6.4, the
conformations of 10a–d were analyzed. The conform-
DIEA
DCC, NMM
DCC, NMM
DCC, NMM
DCC, NMM
DCC, NMM
DCC, NMM
DCC, NMM
Table 3. The thrombolytic activity of the peptides
Compd
Dosage (mg)
XÆSD mg
14
29
10
NS
UK
14
10a
15
10b
16
16
16
10c
10c
10c
17
3mL
20,000 IU
5.0
0.76 Æ2.40
12.81Æ3.15a
6.07Æ2.14a
5.0
5.0
5.0
5.0
10.0
20.0
5.0
10.0
20.0
5.0
10.62Æ3.10a,b
7.02Æ2.25a
a(1) Solvent=anhydrous DMF; (2) concentration of the material was
10ꢁ3 mol/L; (3) deprotection of 4a–d with HF gave 10a–d in 53, 61, 64
and 60% yield, respectively; (4) there is no significant difference of the
yield was observed between the cyclization 13a–d and 130a–d.
9.68Æ2.20a,c
9.31Æ2.14a
13.17Æ3.13a
16.81Æ3.40a,e
11.86Æ2.16a,c
17.31Æ2.39a,f
18.88Æ2.08a,g
0.28Æ2.13
Table 2. Stability of the peptides to enzyme promoting hydrolysis
Compd
Incubation time (h)
%Degradation
In pepsin
In trypsin
10d
5.0
6.13Æ2.31a,d
10a
10a
10b
10b
10c
10c
10d
10d
14
14
14
15
15
15
16
16
16
17
17
17
64
84
64
84
64
84
64
84
1
2
8
1
2
8
1
2
8
1
2
8
0
40
0
25
0
0
38
0
20
0
N=8, NS=vehicle
aComparing to NS, p<0.001.
bComparing to 14, p<0.01.
cComparing to 15 or 16, p<0.05.
dComparing to 17, p<0.001.
eComparing to UK, p<0.05.
fComparing to UK, p<0.01.
gComparing to UK, p<0.001.
10
0
20
0
30
10
50
98
45
78
92
56
75
93
50
80
95
35
79
95
99
20
72
95
25
77
Table 4. Key data for conformation analysis
Compd
Distance between
O and H (Arg)
The lowest energy
(kcal/mol)
Conformation
Beta II0 turn
Beta II0 turn
Beta II0 turn
Beta II0 turn
98
10a
10b
10c
10d
2.004
1.930
1.619
2.550
0.619
ꢁ2.610
ꢁ8.317
5.219
3
70
96
0

964
M. Zhao et al. / Bioorg. Med. Chem. Lett. 13 (2003) 961–964
ation analysis indicated that at the used conditions, the
lowest conformation energy for 10a–d was 0.619,
ꢁ2.610, ꢁ5.20, and 5.219 kcal/mol, respectively, under
which 10a–d exhibited beta II0 turn with intramolecular
hydrogen bond consisted of the alpha NH of Lys1 resi-
due and the C¼O of Arg4. The exact data were listed in
Table 4.
The bioassay of 14–17 and 10a–d in vivo suggested that
in general the thrombolytic activities of 14–17 were
enhanced. In accordance with the conformation analysis
using of Sybyl version 6.4, it may be clear that the flex-
ibility of the fibrinogen fragment related peptides were
significantly reduced after the cyclization. The beta II0
turn resulted from the intramolecular hydrogen bond
consisted of the alpha NH of Lys1 residue and the C¼O
of Arg4 resulted not only the increase of the conforma-
tion rigidity, but also the enhance of the stability of the
enzyme promotion hydrolysis and the thrombolytic
potency.
Discussion
The stepwise synthesis (from C-terminal to N-terminal)
and the related procedures starting from l-Lys(Z)-OBzl
or l-Ala-OBzl, may be provide all of the desirable pro-
tective intermediates in good yields. Using the dilute
Acknowledgements
solution (10ꢁ3 mmol/L) of HCl Ala-Arg(Tos)-Pro-Ala-
.
.
Lys(Z)-N3, HCl Ala-Arg(Tos)-Pro-Ala- Lys(Z)-ONP
The author Shiqi Peng wishes to thank the key Basic
Research Project (G1998051111) of China for financial
support.
.
and HCl Ala-Arg(Tos)-Pro-Ala- Lys(Z)-OH in DMF,
the cyclization resulted in different yield and only the
.
cyclization for HCl Ala-Arg(Tos)-Pro-Ala-Lys(Z)-OH
gave acceptable yield. On the other hand, however,
when the cyclization was carried out in the presence of
TBTU/HOBT/NMM, HBTU/HOBt/NMM, BOP/
HOBt/NMM, BOP/DIEA/NMM, or DCC/NMM, only
the latter gave acceptable yields for all of the protective
cyclopeptides. The solutions of cyclo-ARPAK, cyclo-
GRPAK, cyclo-QRPAK, or cyclo-KRPAK in phos-
phate buffer (pH 8) showed excellent enzymatic stabi-
lity. At 37 ꢀC in the presence of pepsin or trypsin, the
solutions were kept even for 64 h not any degradation
product was observed.
References and Notes
1. Wu, Y.; Zhao, M.; Wang, C.; Peng, S. Bioorg. Med. Chem.
Lett. 2002, 12, 2331.
2. Zhao, M.; Peng, S. J. Prakt. Chem 1999, 668.
3. Zhao, M.; Zhang, L.; Yu, T.; Peng, S. Chinese J. Med.
Chem. 6, 125; Chem. Abstr. 1999, 124, 290228y.
4. Lin, N.; Zhao, M.; Wang, C.; Peng, S. Bioorg. Med. Chem.
Lett. 2002, 12, 585.
5. Lin, N.; Yang, J.; Wang, C.; Zhao, M.; Peng, S.; Song, D.;
Wong, J. J. Beijing Med. Uni. 2000, 32, 283.