The composition and sequence specificity of Pro-Ala-Lys-OH for the thrombolytic activities of P6A and related oligopeptides

Article abstract of DOI:10.1016/j.bmc.2004.02.011

The in vitro and in vivo thrombolytic activities of Ala-Arg-Pro-Ala-Lys-OH, its analogs and the related peptides were assayed. The results indicate that when ⁵Lys of Ala-Arg-Pro-Ala-Lys-OH is changed into ⁵Arg, and ³Lys of

Full text of DOI:10.1016/j.bmc.2004.02.011

Bioorganic & Medicinal Chemistry 12 (2004) 2275–2286
The composition and sequence specificity of Pro-Ala-Lys-OH for
the thrombolytic activities of P6A and related oligopeptides
Ming Zhao,a Chao Wang,a Jiangyuan Liu,a Kexiang Zhouc and Shiqi Pengb,*
aCollege of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
bCollege of Pharmaceutical Sciences, Capital University of Medicinal Sciences, Beijing 100054, PR China
cGuangzhou Baiyunshan Pharmaceutical Co. Ltd., Guangzhou 510515, PR China
Received 9 December 2003; accepted 6 February 2004
Abstract—The in vitro and in vivo thrombolytic activities of Ala-Arg-Pro-Ala-Lys-OH, its analogs and the related peptides were
5
5
3
assayed. The results indicate that when Lys of Ala-Arg-Pro-Ala-Lys-OH is changed into Arg, and Lys of Pro-Ala-Lys-OH is
changed into 3Arg the thrombolytic activities are collapsed; when Pro-Ala-Lys-OH is changed into Ala-Pro-Lys-OH, and Ala-Arg-
5
Pro-Ala-Lys-OH is changed into Ala-Arg-Ala-Pro-Lys-OH the thrombolytic activities are also collapsed; when Lys of Ala-Arg-
Pro-Ala-Lys-OH is changed into 5nLeu the thrombolytic activities are again collapsed. All of the results indicate that for the
thrombolytic activities of Ala-Arg-Pro-Ala-Lys-OH and the related peptides Pro-Ala-Lys-OH exhibits either amino acid compo-
sition specificity or sequence specificity. The composition and sequence specificity of Pro-Ala-Lys-OH reflects its rule as the
pharmacophore of P6A and the related peptides.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
degradation products themselves may cause significant
thrombolysis, the inherent advantages of oligopeptides,
such as no hemorrhagenic tendency and immunogenic
reactions, and the correlations of P6A and thrombus,
P6A was used as the lead compound of thrombolytic
drug. In the rigidization and cyclization based modifi-
cations of P6A Gly-P6A and Gln-P6A,8;9 in the Arg-
Gly-Asp based hybridization of P6A 1Gly-P6A and
1Gln-P6A,10 particularly in the in vivo metabolism
identification the importance of Pro-Ala-Lys-OH
floated. For instance in the metabolism experiments we
found that in vivo P6A degraded into four metabolites
via two pathways (Scheme 1). The in vitro and in vivo
thrombolytic assays showed that the metabolites, Arg-
Pro-Ala-Lys-OH, and Pro-Ala-Lys-OH (12), exhibited
thrombolytic activity. The thrombolytic potency of Arg-
Pro-Ala-Lys-OH is comparable to that of P6A (1), and
It is known that the therapeutic use of thrombolytic
agents usually associates with the release of the fibrin-
ogen degradation products.1 In certain cases some
fibrinogen degradation products themselves may cause
significant vasodilation. Among the fibrinogen degra-
dation products a special sequence Ala-Arg-Pro-Ala-
Lys (P6A, 1) was firstly revealed as a permeability-
increasing factor in 1989.2 Afterwards the studies related
to P6A were mainly focused on the reestablishment
coronary blood flow in dogs,3;4 the synergic effect with
1
1
tPA or L
-arginine,5–7 and thus to thrombolytic effect.
Thrombus results in thromboembolic complications of
arteriosclerosis, heart attacks, strokes, and peripheral
vascular disease. The common and serious side effect
such as hemorrhagenic tendency and immunogenic
reactions, the inherent effect of macromolecular proteins
for the clinical thrombolytic drugs, tPA, UK, and SK,
attract a lot of attention to search of thrombolytic oli-
gopeptides. Considering in certain cases some fibrinogen
Ala-Arg-Pro-Ala-Lys-OH
Ala-Arg-Pro-Ala-OH
Arg-Pro-Ala-Lys-OH
Ala-Arg-Pro-OH
Pro-Ala-Lys-OH
Scheme 1. In vivo Ala-Arg-Pro-Ala-Lys-OH underwent stepwise
metabolites and degraded from both of its C-terminal and N-terminal
giving four metabolites Ala-Arg-Pro-Ala-OH, Ala-Arg-Pro-OH, Arg-
Pro-Ala-Lys-OH, and Pro-Ala-Lys-OH.
Keywords: SAR; P6A; Pro-Ala-Lys-OH; Thrombolysis; Pharmaco-
phore.
* Corresponding author. Tel.: +86-10-8280-2482; fax: +86-10-8280-
2311; e-mail: sqpeng@mail.bjmu.edu.cn
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.011

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M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
of Pro-Ala-Lys-OH (12) is higher than that of P6A.11
This observation suggests that even from such a five
amino acid residue containing peptide it is still possible
to have bioactive fragment. Based on the results drew
from the studies on Ala-Arg-Pro-Ala-Lys-OH (1) itself
it is conceivable that the studies on Pro-Ala-Lys-OH
(12) will perhaps provide more interesting results.
Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl, Boc-Gln-Arg-
(NO2)-Pro-Ala-Lys(Z)-OBzl, Boc-Lys(Z)-Arg-(NO2)-
Pro-Ala-Lys(Z)-OBzl, Boc-Arg(NO2)-Arg(NO2)-Pro-
Ala-Lys(Z)-OBzl or Boc-Gly-Arg(NO2)-Pro-Ala-Lys-
(Z)-OBzl were obtained in 85–90% yield. In the presence
of Pd/C (5%), formic acid in methanol (4.4%), and
hydrogen they were converted into corresponding Ala-
Arg-Pro-Ala-Lys-OH (1), Gln-Arg-Pro-Ala-Lys-OH
(2), Gly-Arg-Pro-Ala-Lys-OH (3), Lys-Arg-Pro-Ala-
Lys-OH (4), and Arg-Arg-Pro-Ala-Lys-OH (5) in 88–
90% yield.
In order to explore the necessity of the amino acid
composition, the exclusiveness of the sequence and the
importance of the positively charged side chain of Pro-
Ala-Lys-OH (12) for the thrombolytic Ala-Arg-Pro-
Ala-Lys-OH (1) and related peptides, in the present
paper the 5Lys of Ala-Arg-Pro-Ala-Lys-OH (1) was
substituted by 5nLeu, Pro-Ala-Lys-OH (12) sequence
was alternated to Ala-Pro-Lys-OH (7), and the posi-
tively charged side chain was introduced into the N-
terminal of Ala-Arg-Pro-Ala-Lys-OH (1). We hope that
the differences of thrombolytic activities resulted from
the structure modifications may reveal the possible
pharmacophore of P6A and the related peptides.
According to Scheme 3 the protective intermediate Boc-
Ala-Pro-Lys(Z)-OBzl, which was prepared with the
procedure in the literature11 was converted into
HClÆAla-Pro-Lys(Z)-OBzl. From Boc-Ala-OH and
HClÆArg(NO2)-OBzl
the
intermediate
Boc-Ala-
Arg(NO2)-OBzl was obtained in 86% total yield. After
the condensation of Boc-Ala-Arg(NO2)-OH and
HClÆAla-Pro-Lys(Z)-OBzl the protective intermediate
Boc-Ala-Arg(NO2)-Ala-Pro-Lys(Z)-OBzl was provided
in 92% yield. In the presence of Pd/C (5%), formic acid
in methanol (4.4%), and hydrogen Boc-Ala-Arg(NO2)-
Ala-Pro-Lys(Z)-OBzl and Boc-Ala-Pro-Lys(Z)-OBzl
were converted into Ala-Arg-Ala-Pro-Lys-OH (6) and
Ala-Pro-Lys-OH (7) in 92% and 90% yield, respectively.
2. Results
2.1. Peptide synthesis
According to Scheme 4 the protective intermediate Boc-
Ala-Arg(NO2)-Pro-Ala-OBzl, which was prepared with
the procedure in the literature10 was converted into Boc-
Ala-Arg(NO2)-Pro-Ala-OH in 95% yield. This was
coupled with HClÆnLeu-OBzl or HClÆArg(NO2)-OBzl to
provide the protective intermediates Boc-Ala-Arg(NO2)-
Pro-Ala-nLeu-OBzl and Boc-Ala-Arg(NO2)-Pro-Ala-
According to Scheme 2 the protective intermediate
Arg(NO2)-Pro-Ala-Lys(Z)-OBzl was prepared via the
solution method and stepwise synthesis (from C-termi-
nal to N-terminal) with Lys(Z)-OBzl as the starting
material in 62% total yield.9 Coupling HClÆArg(NO2)-
Pro-Ala-Lys(Z)-OBzl and Boc-Ala-OH, Boc-Gln-OH,
Boc-Lys(Z)-OH, Boc-Arg(NO2)-OH or Boc-Gly-OH,
AA-Arg-Pro-Ala-Lys-OH
Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
Lys(Z)-OBzl
1-5
Scheme 2. Preparation of AA-Arg-Pro-Ala-Lys-OH. (1) General procedure for removal of benzyl group of the N-terminal component: At 0 ꢁC the
mixture of 0.80 mmol of the benzyl ester in 10 mL of methanol and 5 mL of the solution of NaOH in methanol (2 mol/L) was stirred for 2 h then
neutralized to pH 7 and evaporated at room temperature to remove methanol. The residue was acidified to pH 1–2 with hydrochloric acid (2 mol/L).
(2) General procedure for coupling of C-terminal and N-terminal components: At 0 ꢁC the mixture of 0.20 mmol of the N-terminal component in
5 mL of anhydrous THF, 0.20 mmol of HOBt, and 0.25 mmol of DCC was stirred at 0 ꢁC for 24 h. To the solution 0.20 mmol of C-terminal
component and 0.26 mmol of N-methylmorpholine were added and stirred at room temperature for 24 h. (3) General procedure for removal of NO2,
Z, and Bzl: A suspension of 0.20 mmol of NO2, Z, and Bzl protected peptides, 5 mg of Pd/C (5%), and 8 mL of formic acid in methanol (4.4%) was
agitated with hydrogen at room temperature for 24 h. Wherein AA ¼ Ala (1), Gln (2), Gly (3), Lys (4), Arg (5).
Ala-Pro-Lys-OH
7
HCl.Ala-Pro-Lys(Z)-OBzl
Ala-Arg-Ala-Pro-Lys-OH
Boc-Ala-Pro-Lys(Z)-OBzl
Lys(Z)-OBzl
Boc-Ala-Arg(NO2)-OBzl
L-Arg(NO2)-OBzl
6
Scheme 3. Preparation of Ala-Pro-Lys-OH and Ala-Arg-Ala-Pro-Lys-OH. (1) General procedure for removal of benzyl group of the N-terminal
component: At 0 ꢁC the mixture of 0.80 mmol of the benzyl ester in 10 mL of methanol and 5 mL of the solution of NaOH in methanol (2 mol/L) was
stirred for 2 h then neutralized to pH 7 and evaporated at room temperature to remove methanol. The residue was acidified to pH 1–2 with
hydrochloric acid (2 mol/L). (2) General procedure for coupling of C-terminal and N-terminal components: At 0 ꢁC the mixture of 0.20 mmol of the
N-terminal component in 5 mL of anhydrous THF, 0.20 mmol of HOBt, and 0.25 mmol of DCC was stirred at 0 ꢁC for 24 h. To the solution
0.20 mmol of C-terminal component and 0.26 mmol of N-methylmorpholine were added and stirred at room temperature for 24 h. (3) General
procedure for removal of NO2, Z, and Bzl: A suspension of 0.20 mmol of NO2, Z, and Bzl protected peptides, 5 mg of Pd/C (5%), and 8 mL of formic
acid in methanol (4.4%) was agitated with hydrogen at room temperature for 24 h.

M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
2277
Boc-Ala-Arg(NO2)-Pro-Ala-OH
Boc-Ala-Arg(NO2)-Pro-Ala-OBzl
Ala-OBzl
L-AA-OBzl
Boc-Ala-Arg(NO2)-Pro-Ala-AA-OBzl
Ala-Arg-Pro-Ala-AA'-OH
8,9
Scheme 4. Preparation of Ala-Arg-Pro-Ala-nLeu-OH and Ala-Arg-Pro-Ala-Arg-OH. (1) General procedure for removal of benzyl group of the N-
terminal component: At 0 ꢁC the mixture of 0.80 mmol of the benzyl ester in 10 mL of methanol and 5 mL of the solution of NaOH in methanol
(2 mol/L) was stirred for 2 h then neutralized to pH 7 and evaporated at room temperature to remove methanol. The residue was acidified to pH 1–2
with hydrochloric acid (2 mol/L). (2) General procedure for coupling of C-terminal and N-terminal components: At 0 ꢁC the mixture of 0.20 mmol of
the N-terminal component in 5 mL of anhydrous THF, 0.20 mmol of HOBt, and 0.25 mmol of DCC was stirred at 0 ꢁC for 24 h. To the solution
0.20 mmol of C-terminal component and 0.26 mmol of N-methylmorpholine were added and stirred at room temperature for 24 h. (3) General
procedure for removal of NO2, Z, and Bzl: A suspension of 0.20 mmol of NO2, Z, and Bzl protected peptides, 5 mg of Pd/C (5%), and 8 mL of formic
acid in methanol (4.4%) was agitated with hydrogen at room temperature for 24 h. Wherein AA ¼ nLeu or Arg(NO2), AA0 ¼ nLeu (8) or Arg (9).
Arg(NO2)-OBzl in 90% and 87% yield, respectively. In
the presence of Pd/C (5%), formic acid in methanol
(4.4%), and hydrogen they were converted into Ala-Arg-
Pro-Ala-nLeu-OH (8) and Ala-Arg-Pro-Ala-Arg-OH (9)
in 91% and 88% yield, respectively.
Ala-Lys-OH (4), Arg-Arg-Pro-Ala-Lys-OH (5), Ala-
Arg-Ala-Pro-Lys-OH (6), Ala-Arg-Pro-Ala-nLeu-OH
(8), and Ala-Arg-Pro-Ala-Arg-OH (9) are significantly
different from that for Ala-Arg-Pro-Ala-Lys-OH (1),
Gln-Arg-Pro-Ala-Lys-OH (2), and Gly-Arg-Pro-Ala-
Lys-OH (3). The tested lysis times for Ala-Pro-Lys-OH
(7), Pro-Ala-nLeu-OH (10), and Pro-Ala-Arg-OH (11)
are within 190.45 15.24 to 208.16 18.01 min, for Pro-
Ala-Lys-OH (12) is 91.11 14.57 min, for Lys-Arg-Pro-
Ala-Lys-OH (4), Arg-Arg-Pro-Ala-Lys-OH (5), Ala-
Arg-Ala-Pro-Lys-OH (6), Ala-Arg-Pro-Ala-nLeu-OH
(8), and Ala-Arg-Pro-Ala-Arg-OH (9) are within
178.56 17.34 to 210.01 18.42 min, for Ala-Arg-Pro-
Ala-Lys-OH (1), Gln-Arg-Pro-Ala-Lys-OH (2), and
Gly-Arg-Pro-Ala-Lys-OH (3) are within 90.54 15.32 to
95.01 15.34 min, for NS is 202.32 14.35 min, and for
UK is 123.88 23.86 min, respectively. The tested lysis
time for Pro-Ala-Lys-OH (12) is significantly shorter
than that for Pro-Ala-nLeu-OH, Ala-Pro-Lys-OH, Pro-
Ala-Arg-OH, NS (p < 0:001), and UK (p < 0:05). The
tested lysis times for Ala-Arg-Pro-Ala-Lys-OH (1), Gln-
Arg-Pro-Ala-Lys-OH (2), and Gly-Arg-Pro-Ala-Lys-
OH (3) are significantly shorter than that for Lys-Arg-
Pro-Ala-Lys-OH (4), Arg-Arg-Pro-Ala-Lys-OH (5),
Ala-Arg-Ala-Pro-Lys-OH (6), Ala-Arg-Pro-Ala-nLeu-
OH (8), Ala-Arg-Pro-Ala-Arg-OH (9), NS (p < 0:001),
and UK (p < 0:05).
According to Scheme
5
using HClÆnLeu-OBzl,
HClÆArg(NO2)-OBzl, and HClÆLys(Z)-OBzl as the
starting materials the protective intermediates Boc-Pro-
Ala-nLeu-OBzl, Boc-Pro-Ala-Arg(NO2)-OBzl, and Boc-
Pro-Ala-Lys(Z)-OBzl, were obtained in 85%, 86%, and
75% total yield, respectively. In the presence of Pd/C
(5%), formic acid in methanol (4.4%), and hydrogen
they were converted into Pro-Ala-nLeu-OH (10), Pro-
Ala-Arg-OH (11), and Pro-Ala-Lys-OH (12) in 94%,
87%, and 90% yield, respectively.
2.2. In vitro euglobulin clot lysis time of the peptides
The rabbit euglobulin clots were prepared by use of the
method described in experimental section. To the rabbit
euglobulin clots P6A or related peptide was added and
the lysis time was determined and recorded using the
standard procedure. The tested lysis times for Ala-Pro-
Lys-OH (7), Pro-Ala-nLeu-OH (10), and Pro-Ala-Arg-
OH (11) are significantly different from that for Pro-Ala-
Lys-OH (12). The tested lysis times for Lys-Arg-Pro-
2.3. In vitro fibrinolytic lysis of the peptides
Pro-Ala-AA-OH
AA'-OBzl
Boc-Pro-Ala-AA'-OBzl
10-12
The fibrinogen–agarose mixture was coagulated in the
plastic dishes by thrombin according to the method
described in experimental section. The fibrinolytic assay
was carried out using the standard procedure. The
fibrinolytic activity of P6A and related peptides were
recorded and represented by the lysis area of the coag-
ulated fibrinogen–agarose mixture in the plastic dishes.
The tested lysis areas for Ala-Pro-Lys-OH (7), Pro-Ala-
nLeu-OH (10), and Pro-Ala-Arg-OH (11) are signifi-
cantly different from that for Pro-Ala-Lys-OH (12). The
tested lysis areas for Lys-Arg-Pro-Ala-Lys-OH (4), Arg-
Arg-Pro-Ala-Lys-OH (5), Ala-Arg-Ala-Pro-Lys-OH (6),
Ala-Arg-Pro-Ala-nLeu-OH (8), and Ala-Arg-Pro-Ala-
Arg-OH (9) are significantly different from that for Ala-
Arg-Pro-Ala-Lys-OH (1), Gln-Arg-Pro-Ala-Lys-OH
(2), and Gly-Arg-Pro-Ala-Lys-OH (3). The tested lysis
areas for Ala-Pro-Lys-OH (7), Pro-Ala-nLeu-OH (10),
and Pro-Ala-Arg-OH (11) are within 20.55 9.26 to
Scheme 5. Preparation of Pro-Ala-nLeu-OH (10), Pro-Ala-Arg-OH
(11), and Pro-Ala-Lys-OH (12). (1) General procedure for removal of
benzyl group of the N-terminal component: At 0 ꢁC the mixture of
0.80 mmol of the benzyl ester in 10 mL of methanol and 5 mL of the
solution of NaOH in methanol (2 mol/L) was stirred for 2 h then
neutralized to pH 7 and evaporated at room temperature to remove
methanol. The residue was acidified to pH 1–2 with hydrochloric acid
(2 mol/L). (2) General procedure for coupling of C-terminal and
N-terminal components: At 0 ꢁC the mixture of 0.20 mmol of the N-
terminal component in 5 mL of anhydrous THF, 0.20 mmol of HOBt,
and 0.25 mmol of DCC was stirred at 0 ꢁC for 24 h. To the solution
0.20 mmol of C-terminal component and 0.26 mmol of N-methyl-
morpholine were added and stirred at room temperature for 24 h. (3)
General procedure for removal of NO2, Z, and Bzl: A suspension of
0.20 mmol of NO2, Z, and Bzl protected peptides, 5 mg of Pd/C (5%),
and 8 mL of formic acid in methanol (4.4%) was agitated with
hydrogen at room temperature for 24 h. Wherein AA ¼ nLeu,
Arg(NO2) or Lys(Z), AA0 ¼ nLeu (10), Arg (11) or Lys (12).

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M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
27.64 8.44 mm2, for Pro-Ala-Lys-OH (12) is
236.11 10.01 mm2, for Lys-Arg-Pro-Ala-Lys-OH (4),
Arg-Arg-Pro-Ala-Lys-OH (5), Ala-Arg-Ala-Pro-Lys-
OH (6), Ala-Arg-Pro-Ala-nLeu-OH (8), and Ala-Arg-
Pro-Ala-Arg-OH (9) are within 20.38 9.44 to
29.81 10.52 mm2, for Ala-Arg-Pro-Ala-Lys-OH (1),
Gln-Arg-Pro-Ala-Lys-OH (2), and Gly-Arg-Pro-Ala-
Arg-Pro-Ala-Lys-OH (1), Gln-Arg-Pro-Ala-Lys-OH
(2), and Gly-Arg-Pro-Ala-Lys-OH (3) are significantly
larger than that for Lys-Arg-Pro-Ala-Lys-OH (4), Arg-
Arg-Pro-Ala-Lys-OH (5), Ala-Arg-Ala-Pro-Lys-OH (6),
Ala-Arg-Pro-Ala-nLeu-OH (8), Ala-Arg-Pro-Ala-Arg-
OH (9), and NS (p < 0:001), and substantially similar to
that for UK (p > 0:05).
Lys-OH
(3)
are
within
213.47 10.16
to
231.04 9.86 mm2, for NS is 21.01 9.26 mm2, and for
UK is 222.99 10.99 mm2, respectively. The tested lysis
area for Pro-Ala-Lys-OH (12) is significantly larger than
that for Ala-Pro-Lys-OH (7), Pro-Ala-nLeu-OH (10),
Pro-Ala-Arg-OH (11), and NS (p < 0:001), and sub-
stantially similar to that for UK (p > 0:05). The tested
lysis are as for Ala-Arg-Pro-Ala-Lys-OH (1), Gln-Arg-
Pro-Ala-Lys-OH (2), and Gly-Arg-Pro-Ala-Lys-OH (3)
are significantly larger than that for Lys-Arg-Pro-Ala-
Lys-OH (4), Arg-Arg-Pro-Ala-Lys-OH (5), Ala-Arg-
Ala-Pro-Lys-OH (6), Ala-Arg-Pro-Ala-nLeu-OH (8),
Ala-Arg-Pro-Ala-Arg-OH (9), and NS (p < 0:001), and
substantially similar to that for UK (p > 0:05).
3. Discussion
3.1. Amino acid composition specificity of Pro-Ala-Lys-
OH for thrombolytic activities of Ala-Arg-Pro-Ala-Lys-
OH and its analogs
The in vitro and in vivo thrombolytic experiments dem-
5
onstrated that when Lys of Ala-Arg-Pro-Ala-Lys-OH
(1) is changed into 5Arg, and when 3Lys of Pro-Ala-Lys-
3
3
OH (12) is changed into Arg and nLeu, the thrombo-
lytic activities are collapsed. This phenomenon suggests
that the amino acid composition of Pro-Ala-Lys-OH is
highly specific and this fragment is highly conservative.
Additionally in our previous paper10 we demonstrated
that comparing to Ala-Arg-Pro-Ala-Lys-OH (1) its in
vivo metabolites Ala-Arg-Pro-Ala-OH and Ala-Arg-
Pro-OH exhibited no any activity, Arg-Pro-Ala-Lys-OH
exhibited comparable thrombolytic activities, Pro-Ala-
Lys-OH (12) exhibited enhanced thrombolytic activities.
Pro-Ala-Lys-OH (12) is likely the active fragment of Ala-
Arg-Pro-Ala-Lys-OH (1). The differences of the throm-
bolysis activities of Ala-Arg-Pro-Ala-Lys-OH (1) and
Ala-Arg-Pro-Ala-Arg-OH (9), of Pro-Ala-Lys-OH (12)
and Pro-Ala-Arg-OH (11) give also the evidence that
Pro-Ala-Lys-OH (12) is likely the active fragment of Ala-
Arg-Pro-Ala-Lys-OH (1) and its analogs.
2.4. Thrombolytic activity in vivo
The thrombus prepared with artery blood of rat was
fixed on a special helix according to the literature.10
After the blood circulation outside the rat body was
established the helix with thrombus was inserted into the
polyethylene tube to stimulate the in vivo thrombus.
The in vivo thrombolytic assay was carried out
according to the mentioned method and the thrombo-
lytic activities of peptides were tested using the standard
procedure. The thrombolytic potencies of P6A (1) and
related peptides were recorded and represented by the
reduction of thrombus weight. The tested reduction of
thrombus weight of the rat for Ala-Pro-Lys-OH (7),
Pro-Ala-nLeu-OH (10), and Pro-Ala-Arg-OH (11) are
significantly different from that for Pro-Ala-Lys-OH.
The tested reduction of thrombus weight for Lys-Arg-
Pro-Ala-Lys-OH (4), Arg-Arg-Pro-Ala-Lys-OH (5),
Ala-Arg-Ala-Pro-Lys-OH (6), Ala-Arg-Pro-Ala-nLeu-
OH (8), and Ala-Arg-Pro-Ala-Arg-OH (9) are signifi-
cantly different from that for Ala-Arg-Pro-Ala-Lys-OH
(1), Gln-Arg-Pro-Ala-Lys-OH (2), and Gly-Arg-Pro-
Ala-Lys-OH (3). The tested reductions of thrombus
weight of the rat for Ala-Pro-Lys-OH (7), Pro-Ala-
nLeu-OH (10), and Pro-Ala-Arg-OH (11) are within
12.99 2.87 to 16.03 3.46 mg, for Pro-Ala-Lys-OH
(12) is 26.98 2.54 mg, for Lys-Arg-Pro-Ala-Lys-OH
(4), Arg-Arg-Pro-Ala-Lys-OH (5), Ala-Arg-Ala-Pro-
Lys-OH (6), Ala-Arg-Pro-Ala-nLeu-OH (8), and Ala-
Arg-Pro-Ala-Arg-OH (9) are within 13.81 2.50 to
17.11 3.40 mg, for Ala-Arg-Pro-Ala-Lys-OH (1), Gln-
Arg-Pro-Ala-Lys-OH (2), and Gly-Arg-Pro-Ala-Lys-
OH (3) are within 18.84 3.18 to 25.90 2.05 mg, for
NS is 15.31 3.57 mg, and for UK is 24.10 3.54 mg,
respectively. The tested reduction of thrombus weight of
the rat for Pro-Ala-Lys-OH (12) is significantly larger
than that for Ala-Pro-Lys-OH (7), Pro-Ala-nLeu-OH
(10), Pro-Ala-Arg-OH (11), and NS (p < 0:001), and
substantially similar to that for UK (p > 0:05). The
tested reductions of thrombus weight of the rat for Ala-
3.2. Sequence specificity of Pro-Ala-Lys-OH for throm-
bolytic activities of Ala-Arg-Pro-Ala-Lys-OH and its
analogs
The in vitro and in vivo thrombolytic experiments
demonstrated that when the sequence of Pro-Ala-Lys-
OH (12) is changed, for instance when Pro-Ala-Lys-OH
(12) is changed into Ala-Pro-Lys-OH (7), and when Ala-
Arg-Pro-Ala-Lys-OH (1) is changed into Ala-Arg-Ala-
Pro-Lys-OH (6) the thrombolytic activities are col-
lapsed. All the results mean that Pro-Ala-Lys (12) se-
quence exhibits high specificity for the thrombolytic
activities of P6A (1) and its analogs.
3.3. The importance of the positively charged side chain of
Pro-Ala-Lys-OH for the thrombolytic activities of Ala-
Arg-Pro-Ala-Lys-OH and its analogs
In the interactions of peptides or proteins with receptors
the positively charged side chain of Lys residue is gen-
erally considered as the most important group. In the
present paper it is found that removal of the positively
charged side chain of Pro-Ala-Lys-OH (12) will have
negative influence upon the thrombolytic activities, for
5
instance the substitution of Lys of Ala-Arg-Pro-Ala-

M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
2279
5
Lys-OH (1) with nLeu results in the collapse of the
anhydrous THF 0.20 mmol of C-terminal component
and 0.26 mmol of N-methylmorpholine were added. The
reaction mixture was stirred at room temperature for
24 h. On evaporation the residue was dissolved in 50 mL
of ethyl acetate. The solution was washed successively
with 5% sodium bicarbonate, 5% citric acid, and satu-
rated sodium chloride and the organic phase was sepa-
rated and dried over anhydrous sodium sulfate. After
filtration and evaporation under reduced pressure, and
purification by chromatography (CHCl3/CH3OH, 30:1)
to provide the protective intermediates.
thrombolytic activities. In the interactions of peptides or
proteins with receptors the positively charged side chain
of Lys residue is generally considered as an important
factor. In the present paper it is found that the intro-
duction of additional positively charged side chain into
the N-terminal of the peptides will also have negative
influence upon the thrombolytic activities, for instance
the substitution of 1Ala of Ala-Arg-Pro-Ala-Lys-OH (1)
with 1Arg or 1Lys results in also the collapse of the
thrombolytic activities. The observed phenomena imply
that the positively charged side chain of Pro-Ala-Lys-
OH (12), P6A and its analogs has critical influence upon
the thrombolytic activities.
4.3. Boc-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
Using the general procedure for removal of Boc of the
C-terminal component Boc-Pro-Ala-Lys(Z)-OBzl was
converted into HClÆPro-Ala-Lys(Z)-OBzl, which was
coupled with Boc-Arg(NO2)-OH according to the gen-
eral procedure for coupling of C-terminal and N-
terminal components to provide the title compound as a
3.4. Is Pro-Ala-Lys-OH the pharmacophore
of Ala-Arg-Pro-Ala-Lys-OH and its analogs?
It is very clear that to retain the thrombolytic activities of
Ala-Arg-Pro-Ala-Lys-OH (1) and its analogs the Lys
residue in the C-terminal of Ala-Arg-Pro-Ala-Lys-OH
(1) and Pro-Ala-Lys-OH (12) is not changeable, the Pro-
Ala-Lys-OH (12) sequence in Ala-Arg-Pro-Ala-Lys-OH
(1) is not alternatable, and the positively charged side
chain of Lys residue in Ala-Arg-Pro-Ala-Lys-OH (1) is
not removable. Since all of these requirements relate to
retain the integrity of Pro-Ala-Lys-OH (12) perhaps it
may be considered as the pharmacophore of Ala-Arg-
Pro-Ala-Lys-OH (1), its analogs and the related peptides.
colorless powder in 77% yield (129 mg), mp 70–72 ꢁC;
20
FAB-MS (m=e): 840 [M+H]þ; ½aꢀ )7.5 (c 0.2, CHCl3);
D
IR (KBr): 3360, 3350, 3344, 3033, 3011, 1765, 1692,
1600,1590, 1504, 1462, 1395, 1385, 1365, 760, 705 cmꢁ1
;
1H NMR (DMSO-d6): d ¼ 9.450 (1H), 8.250 (1H), 8.126
(1H), 8.120 (1H), 8.009 (1H), 7.985 (1H), 7.905 (1H),
7.365 (2H), 7.360 (2H), 7.328 (1H), 7.324 (1H), 7.205
(2H), 7.200 (2H), 5.151 (2H), 5.150 (2H), 4.315 (1H),
4.190 (1H), 4.013 (1H), 4.101 (1H), 3.390 (2H), 3.280
(2H), 3.040 (2H), 2.750 (2H), 2.050 (2H), 2.012 (2H),
1.766 (2H), 1.682 (2H), 1.535 (2H), 1.528 (2H), 1.440
(9H), 1.264 (3H). Amino acid analysis: calcd, Arg/Pro/
Ala/Lys ¼ 1.0:1.0:1.0:1.0; found, Arg/Pro/Ala/Lys ¼
0.98:1.00:1.02:0.99. Anal. Calcd for C40H57N9O11: C,
57.20; H, 6.84; N, 15.01. Found: C, 57.41; H, 6.67; N,
15.08.
4. Experimental. Synthesis of the peptides
4.1. General
The protected amino acids were of
purity of the intermediates and the products was con-
firmed by TLC (Merck silica gel plates of type 60 F254
L-configuration. The
,
0.25 mm layer thickness) and HPLC (waters, C18 column
3.9 · 150 mm). Melting points were measured on a XT5
hot stage microscope (Beijing keyi electro-optic factory),
and are uncorrected. Infrared spectra were recorded
with a Perkin–Elmer 983 instrument. FAB-MS was
determined by a VG-ZAB-MS and a HPES-5989x
instrument; 1H NMR spectra was determined by a
Varian INOVA-500 MHz spectrometer. Optical rota-
tions were determined at 20 ꢁC on a Schmidt + Haensch
Polartronic D instrument. The amino acid analysis was
performed by a Hitachi 835-50 instrument.
4.4. HClꢂArg(NO2)-Pro-Ala-Lys(Z)-OBzl
The solution of 168 mg (0.20 mmol) of Boc-Arg(NO2)-
Pro-Ala-Lys(Z)-OBzl, which were prepared according to
the literature,10 in 2 mL of hydrochloride in ethyl acetate
(6 mol/L) was stirred at room temperature for 3 h. The
reaction mixture was evaporated to remove the solvent.
The residue was dissolved in 10 mL of ethyl acetate and
the solution was evaporated to dry. The resulted solid
was used for coupling reaction directly.
4.5. Boc-Ala-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
4.2. General procedure for coupling C-terminal and
N-terminal components
Using the general procedure for coupling C-terminal
and N-terminal components from Boc-Ala-OH and
HClꢂArg(NO2)-Pro-Ala-Lys(Z)-OBzl the title com-
To a solution of 0.20 mmol of the N-terminal compo-
nent in 5 mL of anhydrous THF at 0 ꢁC 0.20 mmol of
HOBt and 0.25 mmol of DCC were added. The reaction
mixture was stirred at 0 ꢁC for 24 h. Precipitated DCU
was removed by filtration. The filtrate was evaporated
under reduced pressure and the residue was titrated with
petroleum ether to provide the corresponding active
ester. To the solution of the active ester in 10 mL of
pound was obtained as a colorless powder in 91% yield,
20
D
mp 93–95 ꢁC; FAB-MS (m=e): 911 [M+H]þ; ½aꢀ )9.5 (c
0.2, CHCl3); IR (KBr): 3358, 3349, 3340, 3035, 3010,
1760, 1690, 1605, 1588, 1500, 1460, 1397, 1383, 1360,
1
764, 701 cmꢁ1; H NMR (DMSO-d6): d ¼ 9.454 (1H),
8.200 (1H), 8.123 (1H), 8.120 (1H), 8.012 (1H), 7.989
(1H), 7.980 (1H), 7.366 (2H), 7.360 (2H), 7.329 (1H),

2280
M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
7.320 (1H), 7.205 (2H), 7.200 (2H), 5.153 (2H), 5.149
(2H), 4.324 (1H), 4.315 (1H), 4.311 (1H), 4.304 (1H),
3.988 (2H), 3.984 (1H), 3.552 (2H), 3.387 (2H), 3.031
(2H), 2.739 (2H), 2.045 (2H), 1.769 (2H), 1.701 (2H),
1.686 (2H), 1.530 (2H), 1.524 (2H), 1.442 (9H), 1.267
(3H), 1.254 (3H). Amino acid analysis: calcd, Ala/Arg/
Pro/Lys ¼ 2.00:1.00:1.00:1.00; found, Ala/Arg/Pro/Lys ¼
2.03:0.99:1.00:0.97. Anal. Calcd for C43H62N10O12: C,
56.69; H, 6.86; N, 15.37. Found: C, 56.41; H, 6.99; N,
15.19.
and HClꢂArg(NO2)-Pro-Ala-Lys(Z)-OBzl the title com-
pound was obtained as a colorless powder in 85% yield,
20
D
mp 90–92 ꢁC; FAB-MS (m=e): 1041 [M+H]þ; ½aꢀ )17.5
(c 0.2, CHCl3); IR (KBr): 3362, 3347, 3340, 3038, 3018,
1760, 1690, 1607, 1595, 1500, 1460, 1392, 1382, 1360,
1
764, 700 cmꢁ1; H NMR (DMSO-d6): d ¼ 9.454 (1H),
9.450 (1H), 8.207 (1H), 8.200 (1H), 8.134 (1H), 8.129
(1H), 8.124 (1H), 8.013 (1H), 7.980 (1H), 7.985 (1H),
7.712 (1H), 7.360 (2H), 7.357 (2H), 7.323 (1H), 7.320
(1H), 7.209 (2H), 7.201 (2H), 5.152 (2H), 5.151 (2H),
4.311 (1H), 4.306 (1H), 4.192 (1H), 3.986 (1H), 3.981
(1H), 3.977 (2H), 3.968 (2H), 3.392 (1H), 3.389 (2H),
2.755 (2H), 2.046 (2H), 2.033 (2H), 1.768 (2H), 1.684
(2H), 1.682 (2H), 1.677 (2H), 1.532 (2H), 1.525 (2H),
1.438 (9H), 1.260 (3H). Amino acid analysis: calcd, Arg/
Pro/Ala/Lys ¼ 2.00:1.00:1.00:1.00; found, Arg/Pro/Ala/
4.6. Boc-Gln-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
Using the general procedure for coupling C-terminal
and N-terminal components from Boc-Gln-OH and
HClꢂArg(NO2)-Pro-Ala-Lys(Z)-OBzl the title com-
Lys ¼ 1.96:1.01:1.03:0.98.
Anal.
Calcd
for
pound was obtained as a colorless powder in 86% yield,
20
D
mp 78–80 ꢁC; FAB-MS (m=e): 968 [M+H]þ; ½aꢀ )8.9 (c
C46H68N14O14: C, 53.07; H, 6.58; N, 18.83. Found: C,
52.98; H, 6.79; N, 18.67.
0.2, CHCl3); IR (KBr): 3358, 3352, 3341, 3035, 3014,
1761, 1689, 1662, 1602, 1588, 1500, 1464, 1392, 1387,
1
1360, 761, 700 cmꢁ1; H NMR (DMSO-d6): d ¼ 9.452
4.9. Boc-Lys(Z)-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
(1H), 8.207 (1H), 8.200 (1H), 8.126 (1H), 8.123 (1H),
8.012 (1H), 7.980 (1H), 7.912 (1H), 7.362 (2H), 7.358
(2H), 7.324 (1H), 7.321 (1H), 7.202 (2H), 7.199 (2H),
6.455 (2H), 5.153 (2H), 5.151 (2H), 4.312 (1H), 4.305
(2H), 4.191 (1H), 4.188 (1H), 3.986 (1H), 3.982 (1H),
3.393 (2H), 3.035 (2H), 2.753 (2H), 2.240 (2H), 2.053
(2H), 1.763 (2H), 1.687 (2H), 1.684 (2H), 1.665 (2H),
1.530 (2H), 1.526 (2H), 1.443 (9H), 1.265 (3H). Amino
acid analysis: calcd, Gln/Arg/Pro/Ala/Lys ¼ 1.00:
1.00:1.00:1.00:1.00; found, Glu/Arg/Pro/Ala/Lys ¼ 1.03:
0.98:1.00:1.03:0.99. Anal. Calcd for C45H65N11O13: C,
55.83; H, 6.77; N, 15.92. Found: C, 55.66; H, 6.99; N,
15.87.
Using the general procedure for coupling C-terminal
and N-terminal components from Boc-Lys(Z)-OH and
HClꢂArg(NO2)-Pro-Ala-Lys(Z)-OBzl the title com-
pound was obtained as a colorless powder in 81% yield,
mp 85–88 ꢁC; FAB-MS (m=e): 1102 [M+H]þ, 1124
20
[M+Na]þ; ½aꢀ )12.5 (c 0.2, CHCl3); IR (KBr): 3362,
D
3355, 3341, 3038, 3015, 1760, 1695, 1601, 1593, 1500,
1468, 1397, 1380, 1360, 765, 710 cmꢁ1
;
1H NMR
(DMSO-d6): d ¼ 9.456 (1H), 8.211 (1H), 8.199 (1H),
8.128 (1H), 8.122 (1H), 8.018 (1H), 8.016 (1H), 7.988
(1H), 7.711 (1H), 7.369 (2H), 7.364 (2H), 7.361 (2H),
7.348 (1H), 7.331 (1H), 7.327 (1H), 7.216 (2H), 7.210
(2H), 7.204 (2H), 5.153 (2H), 5.152 (2H), 5.150 (2H),
4.318 (1H), 4.309 (1H), 4.192 (1H), 4.173 (1H), 4.073
(1H), 3.986 (2H), 3.978 (2H), 3.975 (2H), 3.350 (2H),
2.756 (2H), 2.055 (2H), 1.768 (2H), 1.686 (2H), 1.683
(2H), 1.664 (2H), 1.538 (2H), 1.536 (2H), 1.524 (2H),
1.520 (2H), 1.446 (9H), 1.268 (3H). Amino acid analysis:
calcd, Lys/Arg/Pro/Ala ¼ 2.00:1.00:1.00:1.00; found,
Lys/Arg/Pro/Ala ¼ 1.97:0.96:1.02:1.02. Anal. Calcd for
C54H75N11O14: C, 58.84; H, 6.86; N, 13.98. Found: C,
58.71; H, 6.70; N, 13.69.
4.7. Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
Using the general procedure for coupling C-terminal
and N-terminal components from Boc-Gly-OH and
HClꢂArg(NO2)-Pro-Ala-Lys(Z)-OBzl the title com-
pound was obtained as a colorless powder in 93% yield,
20
D
mp 80–82 ꢁC; FAB-MS (m=e) 897 [M+H]þ; ½aꢀ )9.3 (c
0.2, CHCl3); IR (KBr): 3365, 3352, 3340, 3036, 3010,
1767, 1690, 1604, 1591, 1500, 1460, 1396, 1383, 1360,
1
761, 707 cmꢁ1; H NMR (DMSO-d6): d ¼ 9.452 (1H),
8.206 (1H), 8.124 (1H), 8.125 (1H), 8.102 (1H), 8.011
(1H), 7.980 (1H), 7.700 (1H), 7.368 (2H), 7.363 (2H),
7.324 (1H), 7.320 (1H), 7.205 (2H), 7.202 (2H), 5.152
(2H), 5.150 (2H), 4.315 (1H), 4.311 (1H), 4.305 (1H),
4.300 (2H), 4.192 (1H), 3.992 (2H), 3.388 (2H), 3.082
(2H), 2.742 (2H), 2.038 (2H), 1.864 (2H), 1.760 (2H),
1.677 (2H), 1.675 (2H), 1.530 (2H), 1.444 (9H), 1.260
(3H). Amino acid analysis: calcd, Gly/Arg/Pro/Ala/
Lys ¼ 1.00:1.00:1.00:1.00:1.00; found, Gly/Arg/Pro/Ala/
4.10. Boc-Ala-Arg(NO2)-OBzl
Using the general procedure for coupling C-terminal
and N-terminal components from 378 mg (2 mmol) of
Boc-Ala-OH and 618 mg (2 mmol) of Arg(NO2)OBzl
866 mg (90%) of the title compound were obtained as a
colorless powder, mp 85–87 ꢁC; FAB-MS (m=e): 481
20
[M+H]þ; ½aꢀ )12.0 (c 0.2, CHCl3); IR (KBr): 3350,
D
3344, 3021, 3018, 1748, 1672, 1601, 1570, 1502, 1470,
Lys ¼ 1.03:0.97:1.02:1.03:0.98.
Anal.
Calcd
for
1
1393, 1382, 1364, 760, 701 cmꢁ1; H NMR (DMSO-d6):
C42H60N10O12: C, 56.24; H, 6.74; N, 15.62. Found: C,
56.11; H, 6.89; N, 15.49.
d ¼ 9.350 (1H), 8.216 (1H), 8.122 (1H), 8.110 (1H), 8.105
(1H), 7.394 (2H), 7.310 (1H), 7.212 (2H), 5.153 (2H),
4.516 (1H), 4.405 (1H), 3.378 (2H), 2.104 (2H), 1.678
(2H), 1.421 (9H), 1.263 (3H). Amino acid analysis:
calcd, Ala/Arg ¼ 1.0:1.0; found, Ala/Arg ¼ 1.02:0.97.
Anal. Calcd for C21H32N6O7: C, 52.49; H, 6.71; N,
17.49. Found: C, 52.11; H, 6.93; N, 17.28.
4.8. Boc-Arg(NO2)-Arg(NO2)-Pro-Ala-Lys(Z)-OBzl
Using the general procedure for coupling C-terminal
and N-terminal components from Boc-Arg(NO2)-OH

M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
2281
4.11. Boc-Ala-Arg(NO2)-OH
4.14. Boc-Ala-Pro-Lys(Z)-OBzl
At 0 ꢁC to the solution of 481 mg (1.0 mmol) of Boc-Ala-
Arg(NO2)-OBzl, which were prepared according to the
literature,10 in 10 mL of methanol 5 mL of the solution
of NaOH in methanol (2 mol/L) were added. The reac-
tion mixture was stirred at 0 ꢁC for 2 h. The reaction
mixture was neutralized to pH 7 and evaporated at
room temperature to remove methanol. The residue
was acidified to pH 1–2 with hydrochloric acid
(2 mol/L) to provide the 375 mg (96%) of title com-
Using the general procedure for removal of Boc 567 mg
(1.00 mmol) of Boc-Pro-Lys(Z)-OBzl was converted into
HClÆPro-Lys(Z)-OBzl, which was coupled with 189 mg
(1.00 mmol) Boc-Ala-OH according to the general pro-
cedure for coupling of C-terminal and N-terminal
components to provide 581 mg (91%) of the title com-
pound as a colorless powder, mp 90–92 ꢁC; FAB-MS
20
(m=e) 639 [M+H]þ; ½aꢀ )7.9 (c 0.2, CHCl3); IR (KBr):
D
3361, 3345, 3021, 3010, 1763, 1694, 1600, 1577, 1504,
pound as colorless powder, mp 145–147 ꢁC; FAB-
1460, 1388, 1379, 1360, 761, 704 cmꢁ1 1H NMR
;
20
MS (m=e): 391 [M+H]þ; ½aꢀ )10.5 (c 0.2, CHCl3);
(DMSO-d6): d ¼ 8.311 (1H), 8.185 (1H), 7.976 (1H),
7.364 (2H), 7.358 (2H), 7.337 (1H), 7.322 (1H), 7.243
(2H), 7.237 (2H), 5.154 (2H), 5.152 (2H), 4.315 (1H),
4.228 (1H), 4.054 (1H), 3.325 (2H), 3.111 (2H), 2.727
(2H), 2.032 (2H), 1.753 (2H), 1.544 (2H), 1.531 (2H),
1.438 (9H), 1.263 (3H). Amino acid analysis: calcd,
Ala/Pro/Lys ¼ 1.0:1.0:1.0; found, Ala/Pro/Lys ¼ 1.00:
1.03:0.96. Anal. Calcd for C34H46N4O8: C, 63.93; H,
7.26; N, 8.77. Found: C, 63.77; H, 7.24; N, 8.67. Anal.
Calcd for C34H46N4O8: C, 63.93; H, 7.26; N, 8.77.
Found: C, 63.80; H, 7.35; N, 8.66.
D
IR (KBr): 3430, 3360, 3332, 3265, 3070, 2969, 1668,
1553, 1395, 1388, 1362 cmꢁ1 1H NMR (DMSO-d6):
;
d ¼ 10.991 (1H), 9.372 (1H), 8.213 (1H), 8.122 (1H),
8.114 (1H), 8.135 (1H), 4.119 (1H), 4.111 (1H), 3.378
(2H), 2.151 (2H), 1.678 (2H), 1.426 (9H), 1.265 (3H).
Amino acid analysis: calcd, Ala/Arg ¼ 1.0:1.0; found,
Ala/Arg ¼ 1.00:0.98. Anal. Calcd for C14H26N6O7:
C, 43.07; H, 6.71; N, 21.53. Found: C, 42.95; H, 6.90;
N, 21.37.
4.12. Boc-Pro-Ala-Arg(NO2)-OBzl
4.15. Boc-Ala-nLeu-OBzl
Using the general procedure for coupling C-terminal
and N-terminal components from 430 mg (2.00 mmol) of
Boc-Pro-OH and 654 mg (2.00 mmol) of HClꢂAla-Arg
(NO2)OBzl 833 mg (92%) of the title compound were
Using the general procedure for coupling C-terminal
and N-terminal components from 378 mg (2.00 mmol) of
Boc-Ala-OH and 515 mg (2.00 mmol) of HClꢂnLeu-OBzl
to provide 721 mg (92%) of the title compound as a
colorless powder, mp 79–81 ꢁC; FAB-MS (m=e) 393
obtained as a colorless powder, mp 105–107 ꢁC; FAB-
20
MS (m=e): 578 [M+H]þ; ½aꢀ )11.2 (c 0.2, CHCl3); IR
D
(KBr): 3359, 3340, 3025, 3010, 1750, 1666, 1604, 1567,
[M+H]þ; ½aꢀ20 )8.8 (c 0.2, CHCl3); IR (KBr): 3347, 3342,
D
3032, 3021, 1752, 1675, 1605, 1574, 1500, 1474, 1380,
1
1507, 1472, 1395, 1380, 1365, 762, 706 cmꢁ1; H NMR
1
(DMSO-d6): d ¼ 9.454 (1H), 8.216 (1H), 8.125 (1H),
8.012 (1H), 8.008 (1H), 7.390 (2H), 7.314 (1H), 7.210
(2H), 5.151 (2H), 4.306 (1H), 4.012 (1H), 4.115 (1H),
3.405 (2H), 3.064 (2H), 2.782 (2H), 2.061 (2H), 1.783
(2H), 1.689 (2H), 1.439 (9H), 1.268 (3H). Amino acid
analysis: calcd, Pro/Ala/Arg ¼ 1.0:1.0:1.0; found, Pro/
Ala/Arg ¼ 0.96:1.00:0.98. Anal. Calcd for C26H39N7O8:
C, 54.06; H, 6.81; N, 16.97. Found: C, 53.92; H, 6.91; N,
16.88.
765, 704 cmꢁ1; H NMR (DMSO-d6): d ¼ 8.120 (1H),
8.114 (1H), 7.358 (2H), 7.286 (1H), 7.203 (2H), 5.152
(2H), 4.304 (1H), 4.217 (1H), 1.686 (2H), 1.549 (2H),
1.547 (2H), 1.429 (9H), 1.274 (3H), 1.201 (3H). Amino
acid analysis: calcd, Ala/nLeu ¼ 1.0:1.0; found, Ala/
nLeu ¼ 1.01:1.00. Anal. Calcd for C21H32N2O5: C, 64.26;
H, 8.22; N, 7.14. Found: C, 64.18; H, 8.31; N, 8.09.
4.16. Boc-Pro-Ala-nLeu-OBzl
4.13. Boc-Pro-Ala-Lys(Z)-OBzl
Using the general procedure for removal of Boc of the
C-terminal component Boc-Ala-nLeu-OBzl was con-
verted into HClÆAla-nLeu-OBzl, which was coupled with
Boc-Pro-OH according to the general procedure for
coupling of C-terminal and N-terminal components to
Using the general procedure in the literature10 from
Lys(Z)-OBzl, Boc-Ala-OH, and Boc-Pro-OH the title
compound was obtained as a colorless powder in 75%
total yield (96 mg), mp 85–87 ꢁC; FAB-MS (m=e)
provide the title compound as a colorless powder in 92%
yield, mp 83–85 ꢁC; FAB-MS (m=e): 490 [M+H]þ; ½aꢀ
20
639 [M+H]þ; ½aꢀ )8.6 (c 0.2, CHCl3); IR (KBr): 3366,
20
D
D
3350, 3026, 3005, 1760, 1696, 1604, 1582, 1500, 1455,
)11.4 (c 0.2, CHCl3); IR (KBr): 3355, 3341, 3338, 3030,
3014, 1756, 1670, 1602, 1570, 1504, 1470, 1380, 763,
1
1392, 1384, 1363, 769, 702 cmꢁ1; H NMR (DMSO-d6):
1
d ¼ 8.118 (1H), 8.105 (1H), 7.990 (1H), 7.370 (2H), 7.364
(2H), 7.341 (1H), 7.301 (1H), 7.210 (2H), 7.208 (2H),
5.153 (2H), 5.152 (2H), 4.309 (1H), 4.209 (1H), 4.011
(1H), 3.969 (2H), 3.048 (2H), 2.746 (2H), 1.768 (2H),
1.672 (2H), 1.532 (2H), 1.526 (2H), 1.442 (9H), 1.269
(3H). Amino acid analysis: calcd, Pro/Ala/
Lys ¼ 1.0:1.0:1.0; found, Pro/Ala/Lys ¼ 1.00:1.03:0.96.
Anal. Calcd for C34H46N4O8: C, 63.93; H, 7.26; N, 8.77.
Found: C, 63.82; H, 7.37; N, 8.69.
702 cmꢁ1; H NMR (DMSO-d6): d ¼ 7.980 (1H), 7.975
(1H), 7.392 (2H), 7.320 (1H), 7.214 (2H), 5.150 (2H),
4.331 (1H), 4.215 (1H), 4.021 (1H), 3.059 (2H), 2.818
(2H), 1.890 (2H), 1.785 (2H), 1.554 (2H), 1.550 (2H),
1.418 (9H), 1.269 (3H), 1.221 (3H). Amino acid analysis:
calcd, Pro/Ala/nLeu ¼ 1.0:1.0:1.0; found, Pro/Ala/
nLeu ¼ 0.99:1.01:1.00. Anal. Calcd for C26H39N3O6: C,
63.78; H, 8.03; N, 8.58. Found: C, 63.69; H, 8.15; N,
8.50.

2282
M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
4.17. Boc-Ala-Arg(NO2)-Ala-Pro-Lys(Z)-OBzl
(KBr): 3424, 3364, 3336, 3260, 3070, 2960, 1660, 1550,
1390, 1382, 1360 cmꢁ1; 1H NMR (DMSO-d6): d ¼ 10.254
(1H), 9.454 (1H), 8.212 (1H), 8.120 (1H), 8.110 (1H),
8.102 (1H), 7.994 (1H), 4.301 (1H), 4.295 (1H), 4.198
(1H), 4.101 (1H), 3.541 (2H), 3.059 (2H), 2.396 (2H),
2.044 (2H), 1.789 (2H), 1.685 (2H), 1.439 (9H), 1.278
(3H), 1.265 (3H). Amino acid analysis: calcd, Ala/Arg/
Pro ¼ 2.0:1.0:1.0; found, Ala/Arg/Pro ¼ 1.97:0.98:1.01.
Anal. Calcd for C22H38N8O9: C, 47.30; H, 6.86; N,
20.06. Found: C, 47.18; H, 7.00; N, 19.95.
Using the general procedure for coupling 287 mg
(0.5 mmol) of C-terminal component HClÆAla-Pro-
Lys(Z)-OBzl and 195 mg (0.5 mmol) of N-terminal
component Boc-Ala-Arg(NO2)-OH 427 mg (93.8%) of
the title compound was obtained as a colorless powder,
20
D
mp 135–137 ꢁC; FAB-MS (m=e): 911 [M+H]þ; ½aꢀ )9.7
(c 0.2, CHCl3); IR (KBr): 3365, 3354, 3340, 3037, 3015,
1760, 1690, 1608, 1595, 1500, 1460, 1390, 1380, 1368,
1
768, 703 cmꢁ1; H NMR (DMSO-d6): d ¼ 9.447 (1H),
8.201 (1H), 8.169 (1H), 8.129 (1H), 8.127 (1H), 8.013
(1H), 7.980 (1H), 7.908 (1H), 7.361 (2H), 7.354 (2H),
7.332 (1H), 7.327 (1H), 7.201 (2H), 7.189 (2H), 5.152
(2H), 5.150 (2H), 4.377 (1H), 4.317 (1H), 4.310 (1H),
4.188 (1H), 4.032 (1H), 3.970 (2H), 3.758 (2H), 3.035
(2H), 2.747 (2H), 2.058 (2H), 2.012 (2H), 1.762 (2H),
1.685 (2H), 1.531 (2H), 1.526 (2H), 1.444 (9H), 1.267
(3H), 1.261 (3H). Amino acid analysis: calcd, Ala/Arg/
Pro/Lys ¼ 2.00:1.00:1.00:1.00; found, Ala/Arg/Pro/
4.20. Boc-Ala-Arg(NO2)-Pro-Ala-nLeu-OBzl
Using the general procedure from 112 mg of Boc-Ala-
Arg(NO2)-Pro-Ala-OH and 44 mg of nLeu-OBzl 137 mg
(90%) of the title compound was obtained as a colorless
20
D
powder, mp 105–107 ꢁC, ½aꢀ )32.1 (c 0.2, CHCl3);
FAB-MS (m=e): 762 [M+H]þ; IR (KBr): 3370, 3346,
3025, 3011, 1758, 1692, 1605, 1584, 1509, 1460, 1392,
Lys ¼ 2.01:0.99:1.01:0.98.
Anal.
Calcd
for
1380, 1365, 769, 708 cmꢁ1 1H NMR (DMSO-d):
;
C43H62N10O12: C, 56.69; H, 6.86; N, 15.37. Found: C,
56.58; H, 6.99; N, 15.25.
d ¼ 9.450 (1H), 8.214 (1H), 8.125 (1H), 8.113 (1H), 8.107
(1H), 8.015 (1H), 8.105 (1H), 7.384 (2H), 7.311 (1H),
7.211 (2H), 5.152 (2H), 4.305 (1H), 4.292 (1H), 4.256
(1H), 4.189 (1H), 3.393 (2H), 3.055 (2H), 2.747 (2H),
2.041 (2H), 2.010 (2H), 1.783 (2H), 1.686 (2H), 1.532
(2H), 1.530 (2H), 1.436 (9H), 1.273 (3H), 1.267 (3H),
1.263 (3H). Amino acid analysis: calcd, Ala/Arg/Pro/
nLeu ¼ 2.00:1.00:1.00:1.00; found, Ala/Arg/Pro/nLeu ¼
2.02:0.98:0.99:1.01. Anal. Calcd for C35H55N9O10: C,
55.18; H, 7.28; N, 16.55. Found: C, 55.06; H, 7.41; N,
16.42.
4.18. Boc-Ala-Arg(NO2)-Pro-Ala-OBzl
Using the general procedure for removal of benzyl
group of the N-terminal component Boc-Ala-Arg(NO2)-
Pro-OBzl was converted into Boc-Ala-Arg(NO2)-Pro-
OH, which was coupled with L-Ala-OBzl according to
the general procedure for coupling of C-terminal and N-
terminal components to provide the title compound as a
colorless powder in 80% yield (104 mg), mp 86–88 ꢁC;
20
FAB-MS (m=e): 649 [M+H]þ; ½aꢀ )9.6 (c 0.2, CHCl3);
4.21. Boc-Ala-Arg(NO2)-Pro-Ala-Arg(NO2)-OBzl
D
IR (KBr): 3364, 3336, 3031, 3009, 1760, 1695, 1600,
1
1580, 1500, 1466, 1390, 1382, 1360, 766, 700 cmꢁ1; H
Using the general procedure from 112 mg of Boc-Ala-
Arg(NO2)-Pro-Ala-OH and 62 mg of Arg(NO2)-OBzl
148 mg (87%) of the title compound was obtained as a
NMR (DMSO-d6): d ¼ 9.454 (1H), 8.212 (1H), 8.120
(1H), 8.110 (1H), 8.102 (1H), 7.994 (1H), 7.388 (2H),
7.308 (1H), 7.201 (2H), 5.151 (2H), 4.301 (1H), 4.295
(1H), 4.159 (1H), 4.145 (1H), 3.396 (2H), 3.059 (2H),
2.750 (2H), 2.044 (2H), 1.789 (2H), 1.679 (2H), 1.439
(9H), 1.278 (3H), 1.265 (3H). Amino acid analysis:
calcd, Ala/Arg/Pro ¼ 2.0:1.0:1.0; found, Ala/Arg/
Pro ¼ 1.98:0.97:1.00. Anal. Calcd for C29H44N8O9: C,
53.69; H, 6.84; N, 17.27. Found: C, 53.69496; H, 6.67;
N, 17.55. Anal. Calcd for C29H44N8O9:C, 53.69; H, 6.84;
N, 17.27. Found: C, 53.60; H, 6.93; N, 17.19.
20
colorless powder, mp 126–128 ꢁC, ½aꢀ )42.1 (c 0.2,
D
CHCl3); FAB-MS (m=e): 850 [M+H]þ; IR (KBr): 3366,
3341, 3032, 3016, 1762, 1688, 1602, 1590, 1515, 1464,
1
1390, 1382, 1364, 764, 702 cmꢁ1; H NMR (DMSO-d6):
d ¼ 9.452 (1H), 9.450 (1H), 8.215 (1H), 8.215 (1H), 8.214
(1H), 8.124 (1H), 8.114 (1H), 8.105 (1H), 7.995 (1H),
7.967 (1H), 7.389 (2H), 7.310 (1H), 7.206 (2H), 5.152
(2H), 4.307 (1H), 4.298 (1H), 4.195 (1H), 4.167 (1H),
4.298 (1H), 3.398 (2H), 3.394 (2H), 3.060 (2H), 2.050
(2H), 2.048 (2H), 2.046 (2H), 1.686 (2H), 1.786 (2H),
1.684 (2H), 1.441 (9H), 1.280 (3H), 1.270 (3H). Amino
acid analysis: calcd, Ala/Arg/Pro ¼ 2.00:2.00:1.00;
found, Ala/Arg/Pro ¼ 2.02:1.97:0.99. Anal. Calcd for
C35H55N13O12: C, 49.46; H, 6.52; N, 21.42. Found: C,
49.37; H, 6.60; N, 21.31.
4.19. Boc-Ala-Arg(NO2)-Pro-Ala-OH
At 0 ꢁC to the solution of 518 mg (0.80 mmol) of Boc-
Ala-Arg(NO2)-Pro-Ala-OBzl, which were prepared
according to the literature,10 in 10 mL of methanol 5 mL
of the solution of NaOH in methanol (2 mol/L) were
added. The reaction mixture was stirred at 0 ꢁC for 2 h.
The reaction mixture was neutralized to pH 7 and
evaporated at room temperature to remove methanol.
The residue was acidified to pH 1–2 with hydrochloric
acid (2 mol/L) to provide the 424 mg (95%) of title
4.22. General procedure for removal of NO2, Z, and Bzl
A suspension of 0.20 mmol of NO2, Z, and Bzl protected
peptides, 5 mg of Pd/C (5%) and 8 mL of formic acid in
methanol (4.4%) was agitated with hydrogen at room
temperature for 24 h. The reaction mixture was filtrated.
The filtrate was evaporated and the residue was titrated
compound as colorless powder, mp 146–148 ꢁC; FAB-
20
MS (m=e): 559 [M+H]þ; ½aꢀ )30.1 (c 0.2, CHCl3); IR
D

M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
2283
with ether and the resulted solid was purified on the
Sephadex G-10 column. The collected fractions were
lyophilized to provide the corresponding peptide.
Lys ¼ 1.00:0.99:1.02:1.03:0.99.
Anal.
Calcd
for
C22H41N9O6: C, 50.08; H, 7.83; N, 23.89. Found: C,
49.99; H, 7.95; N, 23.77.
4.26. Lys-Arg-Pro-Ala-Lys-OH (4)
4.23. Ala-Arg-Pro-Ala-Lys-OH (1)
Using general procedure for removal of NO2, Z, and Bzl
from 225 mg (0.2 mmol) of Boc-Lys(Z)-Arg(NO2)-Pro-
Ala-Lys(Z)-OBzl 96 mg (80%) of the title compound was
Using general procedure for removal of NO2, Z, and Bzl
from 182 mg (0.2 mmol) of Boc-Ala-Arg(NO2)-Pro-Ala-
Lys(Z)-OBzl 98 mg (90%) of the title compound was
obtained as colorless crystals, mp 168–170 ꢁC, FAB-
20
MS (m=e): 599 [M+H]þ, ½aꢀ )15.0 (c 1.87, H2O). IR
obtained as colorless crystals, mp 224–226 ꢁC; FAB-MS
20
(m=e): 542 [M+H]þ, ½aꢀ )40.0 (c ¼ 1.95, H2O); IR
D
(KBr): 3429, 3372, 3261, 3073, 2962, 1665, 1554,
D
(KBr): 3433, 3370, 3243, 3085, 2966, 1666, 1549,
1
1380 cmꢁ1; H NMR (DMSO-d6): d ¼ 9.885 (1H), 8.445
1
1384 cmꢁ1; H NMR (DMSO-d6): d ¼ 9.987 (1H), 8.432
(2H), 8.283 (1H), 8.214 (1H), 8.105 (1H), 7.565 (1H),
7.159 (1H), 4.269 (1H), 4.267 (1H), 4.224 (1H), 4.015
(1H), 4.174 (1H), 3.539 (2H), 3.360 (2H), 3.081 (2H),
3.078 (2H), 2.736 (2H), 2.295 (2H), 2.241 (2H), 2.229
(2H), 2.030 (2H), 2.011 (2H), 2.005 (2H), 1.868 (2H),
1.544 (2H), 1.541 (2H), 1.534 (2H), 1.530 (2H), 1.515
(2H), 1.258 (3H). Amino acid analysis: calcd, Lys/Arg/
Pro/Ala ¼ 2.00:1.00:1.00:1.00; found, Lys/Arg/Pro/
Ala ¼ 2.02:1.00:1.03:1.01. Anal. Calcd for C26H50N10O6:
C, 52.16; H, 8.42; N, 23.39. Found: C, 52.03; H, 8.55; N,
23.28.
(2H), 8.280 (1H), 8.220 (1H), 8.007 (1H), 7.598 (1H),
7.152 (1H), 4.266 (1H), 4.205 (1H), 4.219 (1H), 4.015
(1H), 3.673 (1H), 3.535 (2H), 3.353 (2H), 3.076 (2H),
2.730 (2H), 2.248 (2H), 2.242 (2H), 2.023 (2H), 2.006
(2H), 1.863 (2H), 1.676 (2H), 1.532 (2H), 1.531 (2H),
1.280 (3H), 1.267 (3H). Amino acid analysis: calcd, Ala/
Arg/Pro/Lys ¼ 2.00:1.00:1.00:1.00; found, Ala/Arg/Pro/
Lys ¼ 2.02:0.98:1.00:0.97. Anal. Calcd for C23H43N9O6:
C, 51.00; H, 8.00; N, 23.27. Found: C, 50.92; H, 8.11; N,
23.19.
4.24. Gln-Arg-Pro-Ala-Lys-OH (2)
4.27. Arg-Arg-Pro-Ala-Lys-OH (5)
Using general procedure for removal of NO2, Z, and Bzl
from 193 mg (0.2 mmol) of Boc-Gln-Arg(NO2)-Pro-Ala-
Lys(Z)-OBzl 134 mg (95%) of the title compound was
Using general procedure for removal of NO2, Z, and Bzl
from 208 mg (0.2 mmol) of Boc-Arg(NO2)-Arg(NO2)-
Pro-Ala-Lys(Z)-OBzl 106 mg (85%) of the title com-
obtained as colorless crystals, mp 180–182 ꢁC; FAB-MS
20
(m=e): 599 [M+H]þ, ½aꢀ )35.0 (c 2.01, H2O); IR (KBr):
pound was obtained as colorless crystals, mp 168–
D
20
D
H2O). IR (KBr): 3433, 3365, 3252, 3068, 2952, 1663,
170 ꢁC; FAB-MS (m=e): 627 [M+H]þ, ½aꢀ )13.3 (c 1.92,
3434, 3368, 3252, 3075, 2962, 1664, 1556, 1382 cmꢁ1; 1H
NMR (DMSO-d6): d ¼ 9.989 (1H), 8.446 (2H), 8.285
(1H), 8.211 (1H), 8.155 (1H), 7.578 (1H), 7.162 (1H),
6.405 (2H), 4.281 (1H), 4.272 (1H), 4.216 (1H), 4.106
(1H), 4.239 (1H), 3.541 (2H), 3.361 (2H), 3.075 (2H),
2.740 (2H), 2.395 (2H), 2.196 (2H), 2.034 (2H), 1.908
(2H), 1.768 (2H), 1.674 (2H), 1.532 (2H), 1.529 (2H),
2.290 (2H), 2.231 (2H), 1.265 (3H). Amino acid analysis:
calcd, Gln/Arg/Pro/Ala/Lys ¼ 1.00:1.00:1.00:1.00:1.00;
found, Gln/Arg/Pro/Ala/Lys ¼ 0.99:1.01:1.02:1.02:1.00.
Anal. Calcd for C25H46N10O7: C, 50.15; H, 7.74; N,
23.40. Found: C, 50.03; H, 7.82; N, 23.29.
1
1556, 1381 cmꢁ1; H NMR (DMSO-d6): d ¼ 9.899 (1H),
8.437 (2H), 8.435 (2H), 8.282 (1H), 8.280 (1H), 8.210
(1H), 7.602 (1H), 7.599 (1H), 7.212 (1H), 7.167 (1H),
4.277 (1H), 4.214 (1H), 4.210 (1H), 4.109 (1H), 4.096
(1H), 3.549 (2H), 3.537 (2H), 3.356 (2H), 3.078 (2H),
2.620 (2H), 2.256 (2H), 2.239 (2H), 2.136 (2H), 2.069
(2H), 2.025 (2H), 1.867 (2H), 1.670 (2H), 1.668 (2H),
1.536 (2H), 1.534 (2H), 1.265 (3H). Amino acid analysis:
calcd, Arg/Pro/Ala/Lys ¼ 2.00:1.00:1.00:1.00; found,
Arg/Pro/Ala/Lys ¼ 1.98:1.00:1.02:1.02. Anal. Calcd for
C26H50N12O6: C, 49.82; H, 8.04; N, 26.82. Found: C,
49.70; H, 8.17; N, 26.70.
4.25. Gly-Arg-Pro-Ala-Lys-OH (3)
Using general procedure for removal of NO2, Z, and Bzl
from 179 mg (0.2 mmol) of Boc-Gly-Arg(NO2)-Pro-Ala-
Lys(Z)-OBzl 99 mg (94%) of the title compound was
4.28. Ala-Arg-Ala-Pro-Lys-OH (6)
Using general procedure for removal of NO2, Z, and Bzl
from 182 mg (0.2 mmol) of Boc-Ala-Arg(NO2)-Ala-Pro-
Lys(Z)-OBzl 100 mg (92%) of the title compound was
obtained as colorless crystals, mp 168–170 ꢁC; FAB-MS
20
(m=e): 528 [M+H]þ, ½aꢀ )20.0 (c 1.98, H2O); IR (KBr):
D
3442, 3371, 3259, 3074, 2948, 1664, 1556, 1380 cmꢁ1; 1H
NMR (DMSO-d6): d ¼ 9.996 (1H), 8.563 (2H), 8.106
(1H), 7.987 (1H), 7.971 (1H), 7.629 (1H), 7.076 (1H),
4.624 (1H), 4.528 (1H), 4.254 (2H), 4.226 (1H), 4.108
(1H), 3.215 (2H), 3.388 (2H), 3.068 (2H), 2.739 (2H),
2.531 (2H), 2.530 (2H), 2.049 (2H), 1.988 (2H), 1.868
(2H), 1.677 (2H), 1.531 (2H), 1.530 (2H), 1.245 (3H).
Amino acid analysis: calcd, Gly/Arg/Pro/Ala/
Lys ¼ 1.00:1.00:1.00:1.00:1.00; found, Gly/Arg/Pro/Ala/
obtained as colorless crystals, mp 215–217 ꢁC; FAB-
20
MS (m=e): 542 [M+H]þ, ½aꢀ )30.0 (c 1.99, H2O). IR
D
(KBr): 3439, 3373, 3249, 3088, 2957, 1664, 1552,
1382 cmꢁ1; 1H NMR (DMSO-d6): d ¼ 10.050 (1H), 8.214
(1H), 8.184 (2H), 8.183 (1H), 8.101 (1H), 7.535 (1H),
7.106 (1H), 4.492 (1H), 4.381 (1H), 4.339 (1H), 4.125
(1H), 4.010 (1H), 3.356 (2H), 3.324 (2H), 3.059 (2H),
2.750 (2H), 2.051 (2H), 1.866 (2H), 1.668 (2H), 2.493
(2H), 2.491 (2H), 1.988 (2H), 1.535 (2H), 1.533 (2H),

2284
M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
1.315 (3H), 1.278 (3H). Amino acid analysis: calcd, Ala/
Arg/Pro/Lys ¼ 2.00:1.00:1.00:1.00; found, Ala/Arg/Pro/
Lys ¼ 2.03:0.99:1.00:0.98. Anal. Calcd for C23H43N9O6:
C, 51.00; H, 8.00; N, 23.27. Found: C, 50.89; H, 8.13; N,
23.15.
4.32. Pro-Ala-nLeu-OH (10)
Using general procedure for removal of NO2, Z, and Bzl
from Boc-Pro-Ala-nLeu-OBzl the title compound was
obtained as colorless crystals in 94% yield, mp 186–
20
188 ꢁC; FAB-MS (m=e): 300 [M+H]þ, ½aꢀ )25.7 (c 1.95,
D
H2O). IR (KBr): 3431, 3355, 3266, 3065, 2954, 1662,
1
4.29. Ala-Pro-Lys-OH (7)
1557 cmꢁ1; H NMR (DMSO-d6): d ¼ 9.986 (1H), 8.265
(1H), 8.161 (1H), 4.601 (1H), 4.329 (1H), 4.023 (1H),
3.044 (1H), 2.719 (2H), 2.276 (1H), 2.011 (2H), 1.762
(2H), 1.556 (2H), 1.552 (2H), 1.296 (3H), 1.210 (3H).
Amino acid analysis: calcd, Pro/Ala/nLeu ¼ 1.00:1.00:
1.00; found, Pro/Ala/nLeu ¼ 1.03:1.01:0.97. Anal. Calcd
for C14H25N3O4: C, 56.17; H, 8.42; N, 14.04. Found: C,
56.05; H, 8.61; N, 13.96.
Using general procedure for removal of NO2, Z, and Bzl
from 128 mg (0.2 mmol) of Boc-Ala-Pro-Lys(Z)-OBzl
57 mg (90%) of the title compound was obtained as col-
orless crystals, mp 165–167 ꢁC; FAB-MS (m=e): 315
[M+H]þ, ½aꢀ20 )30.3 (c 1.95, H2O). IR (KBr): 3430, 3262,
D
3060, 2954, 1663, 1550 cmꢁ1
;
1H NMR (DMSO-d6):
d ¼ 10.056 (1H), 8.214 (1H), 4.498 (1H), 4.127 (1H), 4.015
(1H), 3.345 (2H), 3.062 (2H), 2.782 (2H), 2.324 (2H),
2.331 (2H), 1.864 (2H), 2.015 (2H), 1.498 (2H), 1.491
(2H), 1.224 (3H). Amino acid analysis: calcd, Ala/Pro/
Lys ¼ 1.00:1.00:1.00; found, Ala/Pro/Lys ¼ 1.03:1.01:1.00.
Anal. Calcd for C14H26N4O4: C, 53.49; H, 8.34; N, 17.82.
Found: C, 53.38; H, 8.46; N, 17.70.
4.33. Pro-Ala-Arg-OH (11)
Using general procedure for removal of NO2, Z, and Bzl
from 115 mg (0.2 mmol) of Boc-Pro-Ala-Arg(NO2)-OBzl
60 mg (87%) of the title compound was obtained as col-
orless crystals, mp 195–197 ꢁC; FAB-MS (m=e): 343
20
[M+H]þ, ½aꢀ )31 (c 1.95, H2O). IR (KBr): 3437, 3346,
D
4.30. Ala-Arg-Pro-Ala-nLeu-OH (8)
3262, 3060, 2951, 1665, 1550, 1380 cmꢁ1
;
1H NMR
(DMSO-d6): d ¼ 9.986 (1H), 8.610 (2H), 8.071 (1H),
8.051 (1H), 7.323 (1H), 7.110 (1H), 4.310 (1H), 4.252
(1H), 4.035 (1H), 3.350 (2H), 3.070 (2H), 2.818 (2H),
2.511 (1H), 2.036 (2H), 1.764 (2H), 1.669 (2H), 1.265
(3H). Amino acid analysis: calcd, Pro/Ala/Arg ¼ 1.00:
1.00:1.00; found, Pro/Ala/Arg ¼ 1.00:1.02:0.97. Anal.
Calcd for C14H26N6O4: C, 49.11; H, 7.65; N, 24.54.
Found: C, 49.02; H, 7.81; N, 24.33.
Using general procedure for removal of NO2, Z, and Bzl
from 152 mg (0.2 mmol) of Boc-Ala-Arg(NO2)-Pro-Ala-
nLeu-OBzl 96 mg (91%) of the title compound was ob-
tained as colorless crystals, mp 201–203 ꢁC; FAB-MS
20
(m=e): 527 [M+H]þ, ½aꢀ )22.0 (c 1.95, H2O). IR (KBr):
D
3433, 3368, 3258, 3069, 2957, 1662, 1556, 1382 cmꢁ1; 1H
NMR (DMSO-d6): d ¼ 9.988 (1H), 8.601 (2H), 8.196
(1H), 8.064 (1H), 8.049 (1H), 7.501 (1H), 7.209 (1H),
4.527 (1H), 4.335 (1H), 4.261 (1H), 4.190 (1H), 4.086
(1H), 3.599 (2H), 3.098 (2H), 2.769 (2H), 2.684 (2H),
2.034 (2H), 1.982 (2H), 1.854 (2H), 1.684 (2H), 1.342
(2H), 1.335 (2H), 1.248 (3H), 1.204 (3H), 1.132 (3H).
Amino acid analysis: calcd, Ala/Arg/Pro/nLeu ¼ 2.00:
1.00:1.00:1.00; found, Ala/Arg/Pro/nLeu ¼ 2.03:0.99:
0.97:1.00. Anal. Calcd for C23H42N8O6: C, 52.46; H,
8.04; N, 21.28. Found: C, 53.38; H, 8.46; N, 17.70.
4.34. Pro-Ala-Lys-OH (12)
Using the general procedure in the literature10 after re-
moval of NO2, Z, and Bzl from 128 mg (0.2 mmol) of
Boc-Pro-Ala-Lys(Z)-OBzl 60 mg (95%) of the title
compound was obtained as colorless crystals in 95%
yield (60 mg), mp 150–152 ꢁC; FAB-MS (m=e): 315
20
[M+H]þ; ½aꢀ )35.6 (c 2.02, H2O); IR (KBr): 3421,
D
1
3259, 3063, 2950, 1660, 1554 cmꢁ1; H NMR (DMSO-
4.31. Ala-Arg-Pro-Ala-Arg-OH (9)
d6): d ¼ 9.987 (1H), 8.461 (1H), 8.132 (1H), 4.350 (1H),
4.086 (1H), 3.847 (1H), 3.335 (2H), 3.003 (2H), 2.745
(2H), 2.650 (2H), 2.509 (1H), 2.108 (1H), 1.710 (2H),
1.509 (2H), 1.307 (2H), 1.220 (3H). Amino acid analysis:
calcd, Pro/Ala/Lys ¼ 1.0:1.0:1.0; found, Pro/Ala/Lys ¼
1.00:1.01:0.96. Anal. Calcd for C14H26N4O4: C, 53.49;
H, 8.34; N, 17.82. Found: C, 53.36; H, 8.43; N, 17.73.
Using general procedure for removal of NO2, Z, and Bzl
from 170 mg (0.2 mmol) of Boc-Ala-Arg(NO2)-Pro-Ala-
Arg(NO2)-OBzl 100 mg (88%) of the title compound was
obtained as colorless crystals, mp 214–216 ꢁC; FAB-MS
20
(m=e): 570 [M+H]þ, ½aꢀ )34.3 (c 1.95, H2O). IR (KBr):
D
3440, 3365, 3254, 3064, 2955, 1660, 1553, 1380 cmꢁ1; 1H
NMR (DMSO-d6): d ¼ 9.968 (1H), 8.440 (2H), 8.436
(2H), 8.286 (1H), 8.284 (1H), 8.010 (1H), 7.562 (1H),
7.441 (1H), 7.260 (1H), 7.118 (1H), 4.270 (1H), 4.207
(1H), 4.115 (1H), 4.103 (1H), 4.025 (1H), 3.556 (2H),
3.540 (2H), 3.062 (2H), 2.741 (2H), 2.250 (2H), 2.034
(2H), 2.026 (2H), 1.865 (2H), 1.687 (2H), 1.681 (2H),
1.278 (3H), 1.269 (3H). Amino acid analysis: calcd, Ala/
Arg/Pro/nLeu ¼ 2.00:2.00:1.00; found, Ala/Arg/Pro/
nLeu ¼ 2.00:1.96:0.97. Anal. Calcd for C23H43N11O6: C,
48.49; H, 7.61; N, 27.05. Found: C, 48.38; H, 7.75; N,
26.96.
5. Bioassay of the peptides
5.1. In vitro euglobulin clot lysis time of the peptides
The rabbit euglobulin fraction was prepared according
to the literature.12 Plasma diluted 1:20 in distilled water
was precipitated at pH 5.7 with acetic acid (0.25%).
After 30 min at 4 ꢁC the suspension was centrifuged at
2000g for 15 min and the precipitate was resuspended to
the initial plasma volume with 50 mM barbital buffer

M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
Table 1. Euglobulin clot lysis time of the peptides Table 2. Effect of the peptides on fibrin plate
2285
Compound
Dosage (lg)
X
SD (min)
Compound
Dosage (lg)
X
SD (mm2)
NS
UK
––
202.32 14.35
123.88 23.86a
95.01 15.34a;b
92.22 13.19a;b
90.54 15.32a;b
178.56 17.34
201.22 17.60
189.99 16.66
196.77 15.66
191.61 14.52
210.01 18.42
190.45 15.24
208.16 18.01
91.11 14.57a;b
NS
UK
––
23.21 9.77
224.05 10.17a
213.47 10.16a
224.78 9.79a
231.04 9.86a
22.44 9.16
27.38 9.76
20.41 9.44
23.23 9.57
29.81 10.52
20.38 9.44
27.64 8.44
20.55 9.26
236.11 10.01a
5 IU
1.4
1.5
1.3
1.5
1.6
1.4
0.8
0.9
1.4
0.7
0.8
0.8
5 IU
1.4
1.5
1.3
1.5
1.6
1.4
0.8
Ala-Arg-Pro-Ala-Lys-OH (1)
Gln-Arg-Pro-Ala-Lys-OH (2)
Gly-Arg-Pro-Ala-Lys-OH (3)
Lys-Arg-Pro-Ala-Lys-OH (4)
Arg-Arg-Pro-Ala-Lys-OH (5)
Ala-Arg-Ala-Pro-Lys-OH (6)
Ala-Pro-Lys-OH (7)
Ala-Arg-Pro-Ala-Lys-OH (1)
Gln-Arg-Pro-Ala-Lys-OH (2)
Gly-Arg-Pro-Ala-Lys-OH (3)
Lys-Arg-Pro-Ala-Lys-OH (4)
Arg-Arg-Pro-Ala-Lys-OH (5)
Ala-Arg-Ala-Pro-Lys-OH (6)
Ala-Pro-Lys-OH (7)
Ala-Arg-Pro-Ala-nLeu-OH (8)
Ala-Arg-Pro-Ala-Arg-OH (9)
Pro-Ala-nLeu-OH (10)
Pro-Ala-Arg-OH (11)
Pro-Ala-Lys-OH (12)
Ala-Arg-Pro-Ala-nLeu-OH (8) 0.9
Ala-Arg-Pro-Ala-Arg-OH (9)
Pro-Ala-nLeu-OH (10)
Pro-Ala-Arg-OH (11)
1.4
0.7
0.8
0.8
Pro-Ala-Lys-OH (12)
n ¼ 6.
n ¼ 6.
a Compare to NS and peptides 4–11 p < 0:001.
a Compare to NS, and peptides 4–11 p < 0:001. The dosage of the
b Compare to UK p < 0:05. The dosage of the peptides and UK were
chosen based on the literature.11
peptides and UK were chosen based on the literature.11
Table 3. The reduction of thrombolytic mass
(pH 7.8, contained 1.66 mM of CaCl2, 0.68 mM of
MgCl2, and 93.96 mM of NaCl). To the rabbit euglob-
ulin clots the peptide was added and the euglobulin clot
lysis time (ECLT) was determined by the method in a 96
well microtiter plate.12 The data are listed in Table 1 and
the statistical analysis of the date was carried out by use
of ANOVA test, p < 0:05 is considered significant.
Compound
Dosage (lg)
X
SD (mg)
NS
UK
3 mL
20,000 IU
5.4
15.31 3.57
24.10 3.54a
18.84 3.18a
21.28 3.46a
25.90 2.05a
17.11 3.40
14.87 3.09
15.08 3.22
16.03 3.46
13.81 2.50
15.00 3.11
14.22 3.00
12.99 2.87
26.98 2.54a
Ala-Arg-Pro-Ala-Lys-OH (1)
Gln-Arg-Pro-Ala-Lys-OH (2)
Gly-Arg-Pro-Ala-Lys-OH (3)
Lys-Arg-Pro-Ala-Lys-OH (4)
Arg-Arg-Pro-Ala-Lys-OH (5)
Ala-Arg-Ala-Pro-Lys-OH (6)
Ala-Pro-Lys-OH (7)
6.0
5.3
6.0
6.3
5.4
3.1
5.2. In vitro fibrinolytic lysis of the peptides13
Ala-Arg-Pro-Ala-nLeu-OH (8)
Ala-Arg-Pro-Ala-Arg-OH (9)
Pro-Ala-nLeu-OH (10)
Pro-Ala-Arg-OH (11)
Pro-Ala-Lys-OH (12)
5.3
5.7
The plates were prepared by mixing equal volumes of
0.3% rabbit fibrinogen and 0.95% agarose solutions,
both dissolved in 50 mM of barbital buffer (pH 7.8,
contained 1.66 mM of CaCl2, 0.68 mM of MgCl2, and
93.96 mM of NaCl). The fibrinogen–agarose mixture
was coagulated with 100 mL thrombin (100 IU: mL) in
the plastic dishes (its diameter is 90 mm and the depth of
the fibrin plate is 1 mm). After 30 min at 4 ꢁC an ade-
quate number of wells, 5 mm in diameter, were perfo-
rated. To determine fibrinolytic activity, 30 lL aliquots
of the peptide to be tested were added to each well, the
plate was incubated, and areas of lysis were quantified as
described for the regular fibrin plates. The data are listed
in Table 2 and the statistical analysis of the date was
carried out by use of ANOVA test, p < 0:05 is consid-
ered significant.
3.0
3.4
3.1
n ¼ 10.
a Compare to NS, and peptides 4–11 p < 0:001. The dosage of the
peptides and UK were chosen based on the literature.11
tube. The polyethylene tube was fully injected with
heparin. Both ends of the tube were connected with
another polyethylene tube, which end was pulled to
change thin to inserted into artery and vein. The blood
and the tested peptide was flowed from the right carotid
artery to the left jugular vein via the polyethylene tube
for 90 min, after which the helix was taken out and
weighted accurately. The difference of the helix weight,
namely the reduction of thrombolytic mass, was re-
corded. The date are listed in Table 3 and the statistical
analysis of the date was carried out by use of ANOVA
test, p < 0:05 is considered significant.
5.3. Thrombolytic activity in vivo14
Male wistar rats weighing 200–300 g (purchased from
Animal Center of Perking University) were anesthetized
with pentobarbital sodium (80.0 mg/kg, ip), and the
right carotid artery and left vein jugular were separated.
Artery blood (1.0 mL) from the right carotid artery was
rapidly injected into the glass tube, then a stainless steel
filament helix (15 circles; L, 15 mm; D, 1.0 mm) was put
into the glass tube immediately. The helix with throm-
bus was carefully pulled out in 15 min later and weighted
exactly. This helix was put into the middle polyethylene
Acknowledgements
The author, Shiqi Peng, wishes to thank the pre-explore
of key Basic Research Project (2003CCA04400) of
China and National Natural Scientific Foundation of
China (20232020) and Guangzhou for financial support.

2286
M. Zhao et al. / Bioorg. Med. Chem. 12 (2004) 2275–2286
7. Saldeen, K.; Lawson, D. L.; Mehta, J. L. Thromb. Res.
1991, 61, 81.
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