Identification, synthesis and bioassay for the metabolites of P6A

Article abstract of DOI:10.1016/j.bmc.2003.09.021

The metabolites Ala-Arg-Pro-Ala-OH, Ala-Arg-Pro-OH, Arg-Pro-Ala-Lys-OH and Pro-Ala-Lys-OH were identified by HPLC/ESI/MS from the in vivo blood of Ala-Arg-Pro-Ala-Lys-OH (P6A) received mice. The in vitro incubation of P6A in the blood of mice the same met

Full text of DOI:10.1016/j.bmc.2003.09.021

Bioorganic & Medicinal Chemistry 11 (2003) 4913–4920
Identification, Synthesis and Bioassay for the Metabolites of P6A
Ming Zhao, Chao Wang, Jian Yang, Jiangyuan Liu, Youxuan Xu, Yanfen Wu
and Shiqi Peng*
College of Pharmaceutical Sciences, Peking University, Beijing 100083, China
Received 6 July 2003; accepted 12 September 2003
Abstract—The metabolites Ala-Arg-Pro-Ala-OH, Ala-Arg-Pro-OH, Arg-Pro-Ala-Lys-OH and Pro-Ala-Lys-OH were identified by
HPLC/ESI/MS from the in vivo blood of Ala-Arg-Pro-Ala-Lys-OH (P6A) received mice. The in vitro incubation of P6A in the
blood of mice the same metabolites were also found by use of the Prep LC System. The protective intermediates of these meta-
bolites were prepared via the solution method using the stepwise synthesis in 77.4, 90, 88, and 80% yield, respectively. After
deprotection with catalytic hydrogenation the intermediates were converted into the corresponding sequences Arg-Pro-Ala-Lys-
OH, Pro-Ala-Lys-OH, Ala-Arg-Pro-Ala-OH, and Ala-Arg-Pro-OH in 90, 95, 85% and 86% yield, respectively. In the thrombolysis
in vivo assay the synthetic Ala-Arg-Pro-Ala-OH, and Ala-Arg-Pro-OH exhibited no activity. On the other hand the thrombolytic
activity of Arg-Pro-Ala-Lys-OH was comparable to P6A, and an enhanced thrombolytic activity was observed for Pro-Ala-Lys-
OH. In the in vitro fibrinolytic lysis tests the approximate results were obtained and an enhanced activity was also observed for Pro-
Ala-Lys-OH. In the euglobulin clot lysis time tests P6A, Arg-Pro-Ala-Lys-OH and Pro-Ala-Lys-OH gave significantly shorter time
than that given by UK, demonstrating their fast action.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
saline (NS) was injected into the tail vein of the mice
(body weight, 40g). Using the proper procedure the
metabolites containing sample extracted from the
plasma was injected on to the HPLC column of the
HPLC/ESI/MS. From the plasma of 0.4 and 4.0 mg of
P6A received mice a set of the same ions were mon-
itored, based on which the same metabolites Ala-Arg-
Pro-Ala-OH, Ala-Arg-Pro-OH, Arg-Pro-Ala-Lys-OH
and Pro-Ala-Lys-OH were identified (Table 1, Scheme
2). If the whole blood from 0.3 mL of NS received mice
(vehicle control) was treated by use of the same proce-
dure not any mentioned sequence was observed.
P6A, one of the products obtained from the degradation
of fibrinogen, was used as the lead compound in our
previous study on thrombolytic agents.1ꢀ3 Though a
number of biological researches related to P6A were
reported4ꢀ6 only few was focused on its metabolism and
no lead compound was obtained from the correspond-
ing metabolite. It was known that the incubation of
P6A with angiotension converting enzyme (ACE) in
vitro gave metabolites Ala-Arg-Pro-OH and Ala-Lys-
OH (Scheme 1).7 Considering the importance of the
metabolite for our investigations in the present paper
the in vitro and in vivo metabolism of P6A was
observed, the corresponding metabolites were identified
and synthesized, and their related bioactivities were tested
in vitro and in vivo.
In the identification of the in vitro metabolites of P6A
the solution of 40.0, 4.0, or 0.12 mg of P6A in the mice
blood was incubated. Using the proper procedure the
metabolites containing sample extracted from the
plasma was injected on to the Nova-Park HR C18
reverse phase column for preparation purpose or on to
the HPLC column of the HPLC/ESI/MS for analytic
purpose. For preparation the obtained fractions corres-
ponding to the interested peaks (retention time, 4.95,
5.16, 5.78 and 5.99 min) gave four crystals in 4.2–0.24,
2.9–0.13, 3.3–0.17 and 5.0–0.31 mg yield, respectively.
They were then tested by FAB/MS for sequence analy-
sis. Based on the corresponding analytic ion and the
resulted fragments the sequences of the four crystals
Results
In the identification of the in vivo metabolites of P6A a
solution of 0.4 or 4.0 mg of P6A in 0.3 mL of normal
*Corresponding author. Tel.: +86-10-8280-2482; fax: +86-10-8280-
2311; e-mail: sqpeng@mail.bjmu.edu.cn
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.09.021

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M. Zhao et al. / Bioorg. Med. Chem. 11 (2003) 4913–4920
Scheme 1. P6A metabolized in the presence of ACE in vitro.
Table 1. The trapped ions and the corresponding sequences from in vivo metabolism of P6A
m/z
414
343
471
315
Sequence
Peak area
Ala-Arg-Pro-Ala-OH
4.02ꢂ107
Ala-Arg-Pro-OH
9.54ꢂ106
Arg-Pro-Ala-Lys-OH
6.92ꢂ106
Pro-Ala-Lys-OH
2.88ꢂ106
were assigned to Arg-Pro-Ala-Lys-OH, Pro-Ala-Lys-
OH, Ala-Arg-Pro-OH and Ala-Arg-Pro-Ala-OH,
respectively. For analysis the sequences of the metabo-
lites were determined by retention time and analytic ion
of Ala-Arg-Pro-Ala-OH, Ala-Arg- Pro-OH, Arg-Pro-
Ala-Lys-OH and Pro-Ala-Lys-OH.
According to Scheme 4 via the solution method and
stepwise synthesis (from C-terminal to N-terminal) with
l-Pro-OBzl as the starting material the protective inter-
mediates Boc-Ala-Arg(NO2)-Pro-OBzl was prepared in
88.0% yield. After saponification of Boc-Ala-
Arg(NO2)-Pro-OBzl the resulting Boc-Ala-Arg(NO2)-
Pro-OH was coupled with l-Ala-OBzl to provide Boc-
Ala-Arg(NO2)-Pro-Ala-OBzl in 80% yield. Removing
the Boc group with hydrogen chloride in ethyl acetate,
removing NO2, Z, and Bzl with catalytic hydrogenation
the protective intermediates were converted into the
desirable products Ala-Arg-Pro-OH and Ala-Arg-Pro-
Ala-OH in 86.0and 85.0% yield, respectively.
In the examination of the stability of P6A the solution of
0.04 mg of P6A in 1.0 mL of water, or in 1.0 mL of buffer
(0.1 mol/L of phosphate, pH 7.4), or in 1.0 mL of pro-
tein free plasma, or in 1.0mL of plasma containing NaF
(final concentration 2 mol/L), or in 1.0mL of plasma
containing EDTA (final concentration 1.5%), or in 1.0
mL of buffered (NaHCO3–K2CO3, 3:2, pH 9.4) plasma
was incubated at 37 ꢁC. After the proper treatment the
HPLC/ESI/MS analysis of all the obtained samples
gave no degradation product of P6A.
According to the literature8 the in vitro fibrinolytic lysis
test was performed and the quantified lysis areas of the
regular fibrin plates for Ala-Arg-Pro-Ala-OH and Ala-
Arg-Pro-OH were significantly smaller than that for
Arg-Pro-Ala-Lys-OH and Pro-Ala-Lys-OH (Table 2).
According to Scheme 3 the protective intermediates of
the in vivo metabolites of P6A, Boc-Arg (NO2)-Pro-
Ala-Lys(Z)-OBzl and Boc-Pro-Ala-Lys(Z)-OBzl, were
prepared via the solution method and stepwise synthesis
(from C-terminal to N-terminal) with l-Lys(Z)-OBzl as
the starting material in 77.4 and 90.0% yield, respec-
tively. After the removal of Boc group with hydrogen
chloride in ethyl acetate, the removal of NO2, Z, and Bzl
with catalytic hydrogenation the protective inter-
mediates were converted into the desirable products
Arg-Pro-Ala-Lys-OH and Pro-Ala-Lys-OH in 90and
95% yield, respectively.
According to the literature8ꢀ10 the in vitro euglobulin
clot lysis test was performed and the resulting euglobu-
lin clot lysis time for Arg-Pro-Ala-Lys-OH and Pro-
Ala-Lys-OH were significantly shorter than that for
Ala-Arg-Pro-Ala-OH and Ala-Arg-Pro-OH (Table 3).
According to the literature8ꢀ10 the in vivo thrombolytic
activity was tested and no difference between the reduc-
tion of thrombolytic mass for Ala-Arg-Pro-Ala-OH or
Ala-Arg-Pro-OH and NS was observed, and a sig-
Scheme 2. The metabolism ways of P6A in vivo.
Scheme 3. Preparation of Arg-Pro-Ala-Lys-OH and Pro-Ala-Lys-OH. (1) General procedure for removal of Boc of the C-terminal component: The
solution of 0.20 mmol of Boc protected compound in 2 mL of hydrogen chloride in ethyl acetate (4 mol/L) was stirred at room temperature for 3 h.
(2) General procedure for coupling of C-terminal and N-terminal components: To a solution of 0.20 mmol of the N-terminal component 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. To the solution
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.

M. Zhao et al. / Bioorg. Med. Chem. 11 (2003) 4913–4920
4915
Scheme 4. Preparation of Ala-Arg-Pro-OH and Ala-Arg-Pro-Ala-OH. (1) General procedure for removal of benzyl group of the N-terminal compo-
nent: At 0 ꢁC to the solution of 0.80 mmol of protected peptide benzyl ester 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 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: To a
solution of 0.20 mmol of the N-terminal component 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. To the solution 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.
Table 2. Effect of synthetic metabolites of P6A on fibrin plate
Table 4. The reduction of thrombolytic mass
-
2
ꢀ
Compound
Dosage (mg)
XꢃSD (mm )
Compound
Dosage (mg)
XꢃSD (mg)
NS
UK
P6A
—
5 IU
1.4
1.1
0.9
21.01ꢃ9.26
NS
UK
P6A
3 mL
20 000 IU
5.4 mg
4.2 mg
3.4 mg
4.7 mg
3.1 mg
12.31ꢃ2.57
222.99ꢃ10.99a,b
210.84ꢃ12.08a,b
31.01ꢃ9.26
22.10ꢃ2.54a,c,d
17.84ꢃ2.18a,b
14.01ꢃ2.26
Ala-Arg-Pro-Ala-OH
Ala-Arg-Pro-OH
Arg-Pro-Ala-Lys-OH
Pro-Ala-Lys-OH
Ala-Arg-Pro-Ala-OH
Ala-Arg-Pro-OH
Arg-Pro-Ala-Lys-OH
Pro-Ala-Lys-OH
29.81ꢃ10.52
13.81ꢃ2.50
1.2
0.8
211.12ꢃ10.41a,b
234.39ꢃ9.39a,b,c
18.22ꢃ2.40a,c
23.68ꢃ2.34a,c,d
n=6.
aCompare to NS P<0.001.
bCompare to Ala-Arg-Pro-Ala-OH and Ala-Arg-Pro-OH P<0.001.
cCompare to P6A and Arg-Pro-Ala-Lys-OH P<0.01.
n=10.
aCompare to NS p<0.001.
bCompare to Ala-Arg-Pro-Ala-OH and Ala-Arg-Pro-OH P<0.01.
cCompare to Ala-Arg-Pro-Ala-OH and Ala-Arg-Pro-OH P<0.001.
dCompare to P6A P<0.001.
Table 3. Euglobulin clot lysis time of synthetic metabolites of P6A
area indicated that the metabolism pathways of P6A
either from C-terminal or from N-terminal by cleavage
of the corresponding peptide bond successively had
approximately equal probability. The observation that
the vehicle control gave no related sequences indicated
that the metabolites did result from the degradation of
P6A. The fact that when the mixture of P6A and the
blood of mice was incubated and treated according to
the procedure used for in vivo experiments as the
metabolites Ala-Arg-Pro-Ala-OH, Ala-Arg-Pro-OH,
Arg-Pro-Ala- Lys-OH and Pro-Ala-Lys-OH were also
isolated by use of Waters Prep LC System indicated that
the in vivo and in vitro metabolism took the same
pathways. When the incubation of P6A was carried out
in the solution without plasma or in the plasma con-
taining enzyme inhibitor, for instance when the incu-
bation of 0.04 mg of P6A was carried out in 1.0 mL of
water, or in 1.0mL of buffer (0.1 mol/L of phosphate,
pH 7.4), or in 1.0mL of plasma containing EDTA (final
concentration 1.5%), or in 1.0mL of buffered
(NaHCO3–K2CO3, 3:2, pH 9.4) plasma at the same
condition and treated with the same procedure as men-
tioned above no any degradation of P6A was occured.
The result suggested that the observed metabolism of
P6A was plasma enzyme dependent.
ꢀ
Compound
Dosage (mg)
XꢃSD (min)
NS
UK
P6A
—
5 IU
1.4
1.1
0.9
200.01ꢃ15.26
122.19ꢃ24.90a
94.84ꢃ16.28a,b
186.54ꢃ19.16
191.61ꢃ14.52
92.02ꢃ16.21a,b
90.98ꢃ15.69a,b
Ala-Arg-Pro-Ala-OH
Ala-Arg-Pro-OH
Arg-Pro-Ala-Lys-OH
Pro-Ala-Lys-OH
1.2
0.8
n=6.
aCompare to NS, Ala-Arg-Pro-Ala-OH and Ala-Arg-Pro-OH
P<0.001.
bCompare to UK P<0.05.
nificant difference between the reduction of thromboly-
tic mass for Arg-Pro-Ala-Lys-OH or Pro-Ala-Lys-OH
and NS was observed (Table 4).
Discussion
At the dose of 0.4 or 4.0 mg/kg the metabolites (Ala-
Arg-Pro-Ala-OH, Ala-Arg-Pro-OH, Arg- Pro-Ala-Lys-
OH and Pro-Ala-Lys-OH) of P6A in mice may be
clearly identified by HPLC/ESI/MS. The peak area of
the metabolites Ala-Arg-Pro-Ala-OH, Ala-Arg-Pro-
OH, Arg-Pro-Ala-Lys-OH and Pro-Ala-Lys-OH identi-
fied by HPLC/MS directly from the plasma of P6A
received mice was 4.02ꢂ107, 9.54ꢂ106, 6.92ꢂ106 and
2.88ꢂ106, respectively. The approximately equal peak
The fact that the synthetic metabolites and the in vitro
metabolites gave substantially equal specific rotation indi-
cated that in the metabolism of P6A no any configuration

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M. Zhao et al. / Bioorg. Med. Chem. 11 (2003) 4913–4920
change was occurred. Since in both of the HPLC/ESI/
MS analysis and the HPLC preparation no metabolite
Ala-Lys-OH was found the situation of ACE promoted
in vitro metabolism of P6A (Scheme 1) was obviously
different from the case of P6A in the blood of mice. The
in vivo thrombolytic assay demonstrated that there was
no significant difference between the thrombolytic
activities of Ala-Arg-Pro-Ala-OH (the reduction of
thrombolytic mass, 14.01ꢃ2.26 mg), Ala-Arg-Pro-OH
(the reduction of thrombolytic mass, 13.81ꢃ2.50mg )
and NS (the reduction of thrombolytic mass,
12.31ꢃ2.57 mg). On the other hand the thrombolytic
activity of Arg-Pro-Ala-Lys-OH (the reduction of
thrombolytic mass, 18.22ꢃ2.40mg) is comparable to
that of P6A (the reduction of thrombolytic mass,
17.84ꢃ2.18 mg), and an enhanced thrombolytic activity
was observed for Pro-Ala-Lys-OH (the reduction of
thrombolytic mass, 23.68ꢃ2.34 mg). Combining the
data of metabolism and bioassay one may consider Pro-
Ala-Lys-OH as the pharmacophore of P6A and its
analogues. Considering two of four metabolites of P6A
(Ala-Arg-Pro-Ala-OH and Ala-Arg-Pro-OH) exhibited
no thrombolytic effect the result that the thrombolytic
activity of Pro-Ala-Lys-OH was higher than that of
P6A became understandable. The quantified lysis area
of the regular fibrin plate for Ala-Arg-Pro-Ala-OH,
Ala-Arg-Pro-OH, Arg-Pro-Ala-Lys-OH and Pro-Ala-
Lys-OH was 31.01ꢃ9.26, 29.81ꢃ10.52, 211.12ꢃ10.41
and 234.39ꢃ9.39 mm2, respectively. Comparing to Arg-
Pro-Ala-Lys-OH and P6A (the quantified lysis area,
210.84ꢃ12.08 mm2) Pro-Ala-Lys-OH exhibited an
enhanced activity. The euglobulin clot lysis time of Ala-
Arg-Pro-Ala-OH, Ala-Arg-Pro-OH, Arg-Pro-Ala-Lys-
OH and Pro-Ala-Lys-OH was 186.54ꢃ19.16,
191.61ꢃ14.52, 92.02ꢃ16.21 and 90.98ꢃ15.69 min,
respectively, which exhibited significant difference
(P<0.001). All of the fibrinolytic lysis tests of the
metabolites suggested that the in vitro and in vivo
results were parallel. These results demonstrate that the
investigated oligopeptides may exhibite fast action as
well.
(a) Identification of the metabolites from the plasma of
high dose of P6A received mice. The solution of 4.0mg
of P6A in 0.3 mL of normal saline was injected into the
tail vein of the mice (body weight 38–40g, n=4). After
20min the whole blood of the mice was centrifuged at
0 ꢁC and 3500 g for 5 min to isolate the plasma. To 100
mL of the plasma 100 mL of perchloric acid (1 mol/L)
were added in order to free the residual P6A and all its
metabolites from the plasma proteins. The plasma mix-
ture was centrifuged at 0 ꢁC and 3500 g for 15 min. The
separated upper layer from the plasma of 4.0mg/kg of
P6A received mice, as a clean solution, was used as the
test sample and injected into the HPLC column of the
HPLC/ESI/MS directly. According to the trapped ions
the corresponding sequences were Ala-Arg-Pro-Ala-OH
(m/z, 414), Ala-Arg-Pro-OH (m/z, 343), Arg-Pro-Ala-
Lys-OH (m/z, 471), and Pro-Ala-Lys-OH (m/z, 315).
(b) Identification of the metabolites from the plasma of
low dose of P6A received mice. Using the same proce-
dure as that used in (a) with the solution of 0.4 mg of
P6A in 0.3 mL of normal saline instead of the solution
4.0mg of P6A in 0.3 mL of normal saline to inject into
the tail vein of the mice (body weight 38–40g, n=4).
After the plasma was concentrated to 1/10volume the
separated upper layer, as a clean solution, was used as
the test sample and injected on to the HPLC column of
the HPLC/ESI/MS. According to the trapped ions the
corresponding sequences were Ala-Arg-Pro-Ala-OH (m/
z, 414), Ala-Arg-Pro-OH (m/z, 343), Arg-Pro-Ala-Lys-
OH (m/z, 471), and Pro-Ala-Lys-OH (m/z, 315).
(c) Identification of the metabolites from the plasma of
normal saline received mice. Using the same procedure
as that used in (a) with 0.3 mL of normal saline instead
of the solution 4.0mg of P6A in 0.3 mL of normal sal-
ine to inject into the tail vein of the mice (body weight
38–40g, n=4). According to the trapped ion only P6A
was identified and no any metabolite was found.
Isolation and identification of the in vitro metabolite of
P6A
General. The separation was proceeded on the Nova-
Park HR C18 reverse phase column (40ꢂ100 mm, 6 mm)
with the support of Waters PrepLe System. The LC flow
rate was 18 mL/min. The mobile phase consisted of
acetic acid (A, 1%) and acetonitrile (B). The grant pro-
gram was that in 15 min 0% B was increased to 20% B
and in another 15 min increased to 70% B.
Experimental
Identification of the in vivo metabolites of P6A
General. HP5989B MS engine mass spectrometer was
used with analysis of Brandford LC/ESI/MS interfaces.
A Hewlett-Packard Model 1090 system was used for
HPLC separation of the metabolites of P6A and deliv-
ery of the eluant to the mass spectrometer. The HPLC
column used was Agilent Zorbax SB-C18 reverse phase
column (2.1ꢂ150mm, 5 mm). The LC flow rate was 0.25
mL/min. The mobile phase consisted of acetic acid (A,
1%) and acetonitrile (B). The gradient program was
that in 10 min 0% B was increased to 20% B and in
another 10min increased to 70% B. The temperature of
the drying gas was 400 ꢁC. The flow rate of the drying
gas was 3.3l/min. The flow rate of the spray gas was
2.0l/min. The spray voltage was 4500 V. All the possible
ions of the metabolites from P6A in vivo were
monitored by ESI/MS.
Isolation of the metabolites from the incubation of P6A
in mouse blood. To 10mL of the mouse blood 40.0or
4.0mg of P6A were added. The solution was incubated
at 37 ꢁC for 20min and then was centrifuged at ꢁC and
3500 g for 5 min to isolate the plasma. To the obtained
plasma 2 mL of perchloric acid (1 mol/L) were added to
free the residual P6A and all its metabolites from the
plasma proteins. The plasma was centrifuged at 0 ꢁC
and 3500 g for 15 min and the separated upper layer, as
a clean solution, was frozen and lyophilized. The resul-
ted crystals were dissolved in 1 mL of water and the
obtained solution was injected on to the column for

M. Zhao et al. / Bioorg. Med. Chem. 11 (2003) 4913–4920
4917
separation. The fractions corresponding to the inter-
ested peaks were collected, frozen and lyophilized. The
resulted 4 crystals were analyzed with FAB/MS. The m/
at 0 ꢁC and 3500 g for 15 min and the separated upper
layer, as a clean solution, was frozen. The resulted resi-
due was dissolved in 0.01 mL of water and the resulted
solution was injected on to the column of HPLC/MS
for analysis. According to the trapped ion the corres-
ponding sequence was only Ala-Arg-Pro-Ala-Lys-OH
(m/z, 541) no metabolite was found.
24
z, retention time, sequence and ½aꢄD of the metabolites
was 471 [M+H]+, 4.95 min, Arg-Pro-Ala-Lys-OH,
ꢀ46.5ꢁ (c=0.31, H2O); 315 [M+H]+, 5.16 min, Pro-
Ala-Lys-OH, ꢀ34.6ꢁ (c=0.22, H2O); 343 [M+H]+,
5.78 min, Ala-Arg-Pro-OH, ꢀ43.0ꢁ (c=0.24, H2O) and
414 [M+H]+, 5.99 min, Ala-Arg-Pro-Ala-OH ꢀ54.7ꢁ
(c=0.35, H2O), respectively.
(c) Stability of P6A in NaHCO3–K2CO3 (3:2, pH 9.4)
buffer. To 5 mL of saturated solution of NaHCO3–
K2CO3 (3:2, pH 9.4) in water 0.4 mg of P6A were
added. The solution was incubated at 37 ꢁC for 20min
and then was centrifuged at 0 ꢁC and 3500 g for 5 min.
To the solution 0.5 mL of perchloric acid (0.25 mol/L)
were added. The solution was centrifuged at 0ꢁ and 3500
g for 15 min and the separated upper layer, as a clean
solution, was frozen. The resulted residue was dissolved
in 0.01 mL of water and the resulted solution was
injected on to the column of HPLC/MS for analysis.
According to the trapped ion the corresponding
sequence was only Ala-Arg-Pro-Ala-Lys-OH (m/z, 541)
no any metabolite was found.
The incubation of 40.0 mg of P6A in 10 mL of mouse
blood yielded 4.2 mg of Arg-Pro-Ala-Lys-OH, 2.9 mg of
Pro-Ala-Lys-OH, 3.3 mg of Ala-Arg-Pro-OH and 5.0
mg of Ala-Arg-Pro-Ala-OH.
The incubation of 4.0mg of P6A in 10mL of mouse
blood yielded 0.24 mg of Arg-Pro-Ala-Lys-OH, 0.13 mg
of Pro-Ala-Lys-OH, 0.17 mg of Ala-Arg-Pro-OH and
0.31 mg of Ala-Arg-Pro-Ala-OH.
Identification of the metabolites from the incubation of
P6A in mouse blood. To 2 mL of the mouse blood 0.4
mg of P6A were added. The solution was incubated at
37 ꢁC for 20min and then was centrifuged at 0 ꢁC and
3500 g for 5 min to isolate the plasma. To 1 mL of the
plasma 0.5 mL of perchloric acid (0.25 mol/L) were
added to free the residual P6A and all its metabolites
from the plasma proteins. The plasma mixture was cen-
trifuged at 0 ꢁC and 3500 g for 15 min and the separated
upper layer, as a clean solution, was frozen and
lyophilized to provide 20 mg of crystals. The resulted
crystals were dissolved in 0.01 mL of water and the
obtained solution was injected on to the column of
HPLC/MS for analysis. According to the trapped ions
and LC the corresponding sequences were also Ala-Arg-
Pro-Ala-OH (m/z, 414), Ala-Arg-Pro-OH (m/z, 343),
Arg-Pro-Ala-Lys-OH (m/z, 471), and Pro-Ala-Lys-OH
(m/z, 315) and the relative proportion for them was
approximately 2.0:1.0:1.2:2.2.
Synthesis of the in vivo metabolite of P6A
General. The protected amino acids were of l-configur-
ation. The purity of the intermediates and the products
was confirmed by TLC (Merck silica gel plates of type
60F 254, 0.25 mm layer thickness) and HPLC (waters,
C18 column 3.9ꢂ150mm). Melting points were mea-
sured on a XT5 hot stage microscope (Beijing keyi
electro-optic factory), and are uncorrected. Infrared
spectra were recorded with a Perkin–Elmer 983 instru-
ment. FAB-MS was determined by a VG-ZAB-MS and
1
a HPES-5989x instrument. H NMR spectra was deter-
mined by a Varian INOVA-500 MHz spectrometer.
Optical rotations were determined at 20 ꢁC on a
Schmidt+Haensch Polartronic
D instrument. The
amino acid analysis was performed by a Hitachi 835-50
instrument.
General procedure for removal of Boc of the C-terminal
component. The solution of 0.20 mmol of Boc pro-
tected compound in 2 mL of hydrogen chloride in ethyl
acetate (4 mol/L) was stirred at room temperature for 3
h. The reaction mixture was evaporated to remove the
solvent. The residue was dissolved in 10mL of ethyl
acetate and the solution was evaporated to dry. The
resulted solid was used for coupling reaction directly.
Stability of P6A in solution
(a) Stability of P6A in phosphate buffer (pH=7.4). To 2
mL of phosphate buffer (pH=7.4) 0.4 mg of P6A were
added. The solution was incubated at 37 ꢁC for 20min
ꢁ
and then was centrifuged at 0 C and 3500 g for 5 min.
To the solution 0.5 mL of perchloric acid (0.25 mol/L)
were added. The solution was centrifuged at 0 ꢁC and
3500 g for 15 min and the separated upper layer, as a
clean solution, was frozen. The resulting residue was
dissolved in 0.01 mL of water and the resulting solution
was injected on to the column of HPLC/MS for analy-
sis. According to the trapped ion the corresponding
sequence was only Ala-Arg-Pro-Ala-Lys-OH (m/z, 541)
no metabolite was found.
General procedure for coupling of C-terminal and
N-terminal components. To a solution of 0.20 mmol of
the N-terminal component 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 triturated with petroleum ether to provide
the corresponding active ester. To the solution of the
active ester in 10mL of anhydrous THF 0.20mmol of
C-terminal component and 0.26 mmol of N-methyl-
morpholine were added. The reaction mixture was stir-
red at room temperature for 24 h. On evaporation the
(b) Stability of P6A in enzyme inhibitor containing solu-
tion. To 2 mL of water 0.4 mg of P6A and 150 mg of
EDTA were added. The solution was incubated at 37 ꢁC
for 20min and then was centrifuged at 0 ꢁC and 3500 g
for 5 min. To the solution 0.5 mL of perchloric acid
(0.25 mol/L) were added. The solution was centrifuged

4918
M. Zhao et al. / Bioorg. Med. Chem. 11 (2003) 4913–4920
residue was dissolved in 50mL of ethyl acetate. The
solution was washed successively with 5% sodium bicar-
bonate, 5% citric acid, and saturated sodium chloride
and the organic phase was 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.
Pro=1.0:1.0:1.0; found, Ala:Arg:Pro=1.02:0.97:1.00.
Anal. calcd for C26H39N7O8: C, 54.06; H, 6.81; N,
16.97. Found: C, 54.30; H, 6.55; N, 16.70.
Boc-Ala-Arg(NO2)-Pro-Ala-OBzl. Using the general
procedure for removal of benzyl group of the N-term-
inal component Boc-Ala-Arg(NO2)-Pro-OBzl was con-
verted into Boc-Ala-Arg(NO2)-Pro-OH which was
coupled with l-Ala-OBzl according to the general pro-
cedure for coupling of C-terminal and N-terminal
components to provide the title compound as a color-
less powder in 80% yield (104 mg). Mp 86–88 ꢁC;
FAB-MS (m/e): 649 [M+H]+; [a]D20=ꢀ9.6ꢁ (c=0.2,
CHCl3), IR (KBr): 3364, 3336, 3031, 3009, 1760, 1695,
1600, 1580, 1500, 1466, 1390, 1382, 1360, 766, 700
General procedure for removal of benzyl group of the
N-terminal component. At 0 ꢁC to the solution of 0.80
mmol of protected peptide benzyl ester in 10mL 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 protected
peptide carboxylic acid.
1
cmꢀ1, H NMR (CDCl3): d=9.454 (1H), 8.212 (1H),
8.120(1H), 8.110(1H), 8.102 (1H), 7.664 (1H), 7.388
(2H), 7.308 (1H), 7.201 (2H), 4.301 (1H), 4.295 (1H),
3.884 (1H), 3.778 (1H), 3.541 (2H), 3.396 (2H), 3.059
(2H), 2.750(2H), 24.04 (2H), 1.789 (2H), 1.439
(9H), 1.278 (3H), 1.265 (3H). Amino acid analysis:
calcd Ala:Arg: Pro=2.0:1.0:1.0; found, Ala:Arg:
Pro=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.
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 he residue
was triturated with ether and the resulted solid was
purified on the Sephadex G-10column. The collected
fractions were lyophilized to provide the corresponding
peptide.
Boc-Ala-Lys(Z)-OBzl. Using the general procedure for
coupling of C-terminal and N-terminal components
from Boc-l-Ala-OH and l-Lys(Z)-OBzl the title com-
pound was obtained as a colorless powder in 90% yield
(98 mg). Mp 88–90 ꢁC; FAB-M (m/e) 542 [M+H]+;
[a]2D0=ꢀ6.6 (c=0.2, CHCl3), IR (KBr): 3366, 3030,
3015, 1765, 1690, 1610, 1590, 1506, 1460, 1395, 1385,
1366, 760, 710 cmꢀ1, 1H NMR (CDCl3): d=8.123 (1H),
8.102 (1H), 8.089 (1H), 7.365 (2H), 7.360 (2H), 7.310
(1H), 7.295 (1H), 7.208 (2H), 7.201 (2H), 4.315 (1H),
4.211 (1H), 3.988 (2H), 3.565 (2H), 3.550(2H), 1.655
(2H), 1.529 (2H), 1.520(2H), 1.450(9H), 1.269 (3H).
Amino acid analysis: calcd Ala:Lys=1.0:1.0; found,
Ala:Lys=1.00:0.97. Anal. calcd for C29H39N3O7: C,
64.31; H, 7.26; N, 7.76. Found: C, 64.10; H, 7.08; N,
7.54.
Boc-Arg(NO2)-Pro-OBzl. Using the general procedure
for coupling of C-terminal and N-terminal components
from Boc-l-Arg(NO2)-OH and l-Pro-OBzl the title
compound was obtained as a colorless powder in 86%
yield (87 mg), Mp 70–72 ꢁC FAB-MS (m/e): 507
[M+H]+; [a]D20=ꢀ7.8ꢁ (c=0.2, CHCl3); IR (KBr):
3340, 3030, 3010, 1750, 1692, 1600, 1580, 1500, 1463,
1
1394, 1382, 1364, 760, 700 cmꢀ1; H NMR (CDCl3):
d=9.465 (1H), 8.225 (1H), 8.115 (1H), 8.111 (1H), 7.401
(2H), 7.332 (1H), 7.201 (2H), 4.301 (1H), 4.124 (1H),
3.905 (1H), 3.561 (2H), 3.402 (2H), 2.746 (2H), 1.886
(2H), 1.465 (9H), amino acid analysis: calcd Arg:
Pro=1.0:1.0; found, Arg:Pro=0.97:1.00. Anal. calcd
for C23H34N6O7: C, 54.53; H, 6.77; N, 16.59. Found: C,
54.25; H, 6.44; N, 16.35.
Boc-Pro-Ala-Lys(Z)-OBzl. Using the general procedure
for removal of Boc of the C-terminal component Boc-
.
Ala-Lys(Z)-OBzl was converted into HCl Ala-Lys(Z)-
OBzl which was coupled with Boc-l-Pro-OH according
to the general procedure for coupling of C-terminal
and N-terminal components to provide the title com-
pound as a colorless powder in 90% yield (115 mg),
mp 85–87 ꢁC, FAB-M (m/e) 639 [M+H]+;
[a]2D0=ꢀ8.6ꢁ (c=0.2, CHCl3), IR (KBr): 3366, 3350,
3026, 3005, 1760, 1696, 1604, 1582, 1500, 1455, 1392,
Boc-Ala-Arg(NO2)-Pro-OBzl. Using the general proce-
dure for removal of Boc of the C-terminal component
Boc-Arg(NO2)-Pro-OBzl
was
converted
into
.
HCl Arg(NO2)-Pro-OBzl which was coupled with Boc-
l-Ala-OH according to the general procedure for cou-
pling of C-terminal and N-terminal components to
provide the title compound as a colorless powder in
87% yield (100 mg). Mp 82–84 ꢁC; FAB-MS (m/e) 578
[M+H]+; [a]D20=ꢀ7.0ꢁ (c=0.2, CHCl3), IR (KBr):
3355, 3348, 3025, 3015, 1758, 1677, 1605, 1575, 1509,
1384, 1363, 769, 702 cmꢀ1
,
1H NMR (CDCl3):
d=8.118 (1H), 8.150 (1H), 7.690(1H), 7.370(2H),
7.364 (2H), 7.341 (1H), 7.301 (1H), 7.210 (2H), 7.208
(2H), 7.201 (2H), 4.309 (1H), 4.209 (1H), 3.969 (2H),
3.546 (2H), 3.540(2H), 3.048 (2H), 2.746 (2H), 1.768
(2H), 1.672 (2H), 1.532 (2H), 1.442 (9H), 1.269 (3H).
Amino acid analysis: calcd, Pro:Ala:Lys=1.0:1.0:1.0;
found, Pro:Ala:Lys=1.00:1.03:0.96. Anal. calcd for
C34H46N4O8: C, 63.93; H, 7.26; N, 8.77. Found: C,
63.68; H, 7.20; N, 8.54.
1472, 1396, 1387, 1369, 765, 704 cmꢀ1 1H NMR
,
(CDCl3): d=9.354 (1H), 8.218 (1H), 8.125 (1H), 8.114
(1H), 8.109 (1H), 7.399 (2H), 7.315 (1H), 7.214 (2H),
3.884 (1H), 3.778 (1H), 3.533 (2H), 3.382 (2H), 3.100
(2H), 2.698 (2H), 2.102 (2H), 1.764 (2H), 1.426 (9H),
1.269 (3H). Amino acid analysis: calcd Ala:Arg:

M. Zhao et al. / Bioorg. Med. Chem. 11 (2003) 4913–4920
4919
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
calcd for C14H26N4O4: C, 53.49; H, 8.34; N, 17.82.
Found: C, 53.65; H, 8.54; N, 17.61.
.
into HCl Pro-Ala-Lys(Z)-OBzl which was coupled with
H-Arg-Pro-Ala-Lys-OH. Using the general procedure
for removal of NO2, Z and Bzl from Boc-Arg(NO2)-
Pro-Ala-Lys(Z)-OBzl the title compound was obtained
as a colorless crystals in 90% yield (85 mg), mp 158–
160 ꢁC, FAB-MS (m/e) 471 [M+H]+, [a]2D0=ꢀ47.5ꢁ
(c=1.95, H2O), IR(KBr): 3425, 3377, 3256, 3070, 2955,
1668, 1552 cmꢀ1, 1H NMR (DMSO-d6): d=8.560(3H),
8.172 (1H), 7.955 (1H), 7.600 (1H), 7.231 (2H), 6.898
(1H), 4.433 (1H), 4.253 (1H), 4.155 (4H), 3.489 (2H),
3.097 (2H), 2.744 (2H), 1.837 (2H), 1.817 (2H), 1.736
(2H), 1.530(2H), 1.304 (2H), 1.255 (3H), amino acid
analysis: calcd Arg:Pro:Ala: Lys=1.0:1.0:1.0:1.0; found,
Arg:Pro:Ala:Lys=0.96:1.01:1.00:0.97. Anal. calcd for
C20H38N8O5: C, 51.05; H, 8.14; N, 23.81. Found: C,
51.28; H, 8.30; N, 23.57.
Boc-Arg(NO2)-OH according to the general procedure
for coupling of C-terminal and N-terminal components
to provide the title compound as a colorless powder in
77% yield (129 mg), Mp 70–72 ꢁC; FAB-MS (m/e): 840
[M+H]+; [a]D20=ꢀ7.5ꢁ (c=0.2, CHCl3), IR (KBr):
3360, 3350, 3344, 3033, 3011, 1765, 1692, 1600, 1590,
1
1504, 1462, 1395, 1385, 1365, 760, 705 cmꢀ1, H NMR
(CDCl3): d=8.450(1H), 8.205 (1H), 8.126 (1H), 8.120
(1H), 8.009 (1H), 7.985 (1H), 7.705 (1H), 7.365 (2H),
7.360 (2H), 7.328 (1H), 7.324 (1H), 7.205 (2H), 7.200
(2H), 4.315 (1H), 4.301 (1H), 4.190 (1H), 3.989 (1H),
3.980(2H), 3.548 (2H), 3.544 (2H), 3.390(2H), 3.040
(2H), 2.750(2H), 2.050(2H), 1.766 (2H), 1.681 (2H),
1.535 (2H), 1.528 (2H), 1.440(9H), 1.264 (3H), amino
acid analysis: calcd, Arg:Pro:Ala:Lys=1.0:1.0:1.0:1.0;
found, Arg:Pro:Ala:Lys=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.
Bioassay of the synthetic metabolites of P6A
In vitro euglobulin clot lysis time of the synthetic meta-
bolites of P6A. The rabbit euglobulin fraction was pre-
pared according to the literature.9 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 2000 g for 15 min and the precipitate was
re-suspended to the initial plasma volume with 50mM
barbital buffer (pH 7.8, contained 1.66 mM of CaCl2,
0.68 mM of MgCl2 and 93.96 mM of NaCl). Euglobulin
clot lysis time (ECLT) was measured using a 96 well
microtiter plate.8,10
H-Ala-Arg-Pro-OH. Using the general procedure for
removal of NO2, Z and Bzl from Boc-Ala-Arg(NO2)-
Pro-OBzl the title compound was obtained as colorless
crystals in 86% yield (59 mg), mp 150–152 ꢁC, FAB-MS
(m/e) 343 [M+H]+, [a]D20=ꢀ43.7ꢁ (c=2.04, H2O), IR
(KBr): 3424, 3260, 3070, 2960, 1660, 1550 cmꢀ1 1H
,
NMR (DMSO-d6): d=8.825 (3H), 8.415 (2H), 8.200
(1H), 8.195 (1H), 8.065 (1H), 4.023 (1H), 4.016 (1H),
4.001 (1H), 3.405 (2H), 2.745 (2H), 1.796 (2H), 1.365
(3H). Amino acid analysis: calcd Ala:Arg:
Pro=1.0:1.0:1.0; found, Pro:Ala:Lys=1.00:0.97:1.01.
Anal. calcd for C14H26N6O4: C, 49.11; H, 7.65; N,
24.54. Found: C, 49.28; H, 7.42; N, 24.33.
In vitro fibrinolytic lysis of the synthetic metabolites of
P6A. The plates were prepared by mixing equal
volumes of 0.3% rabbit fibrinogen and 0.95% agarose
solutions, both dissolved in 50mM of barbital buffer
(pH 7.8, contained 1.66 mM of CaCl2, 0.68 mM of
MgCl2 and 93.96 mM of NaCl).8 The fibrinogen–agar-
ose mixture was coagulated with 100 mL thrombin (100
IU: mL) in the plastic dishes (its diameter is 90mm and
the depth of the fibrin plate is 1 mm). After 30min at
4 ꢁC an adequate number of wells, 5 mm in diameter,
were perforated. To determine fibrinolytic activity,
30 mL aliquots of the metabolites 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.
H-Ala-Arg-Pro-Ala-OH. Using the general procedure
for removal of NO2, Z and Bzl from Boc-Ala-Arg
(NO2)-Pro-Ala-OBzl the title compound was obtained
as colorless crystals in 85% yield (70mg), mp 157–ꢁ
159 ꢁC, FAB-MS (m/e) 414 [M+H]+, [a]2D0=ꢀ55.5
(c=1.75, H2O), IR (KBr): 3450, 3266, 3240, 3069, 2966,
1
1655, 1558 cmꢀ1, H NMR (DMSO-d6): d=8.621 (3H),
8.401 (2H), 8.211 (1H), 8.185 (1H), 8.010 (1H), 7.605
(1H), 4.206 (1H), 3.901 (1H), 3.820 (1H), 3.816 (1H),
3.360(2H), 2.740(2H), 1.855 (2H), 1.276 (3H), 1.275
(3H), amino acid analysis: calcd, Ala:Arg:
Pro=2.0:1.0:1.0; found, Pro:Ala:Lys=1.98:0.98:1.00.
Anal. calcd for C17H31N7O5: C, 49.38; H, 7.56; N,
23.71. Found: C, 49.16; H, 7.80; N, 23.49.
In vivo thrombolytic activities of the synthetic metabo-
lites of P6A.2 Male Wistar rats weighing 200–1300 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 (1.0mL) from the right carotid artery of
the animal a stainless steel filament helix (15 circles; L,
15 mm; D, 1.0mm) was put immediately. After 15 min
the helix with thrombus was carefully taken out and
weighted exactly, which was put into the middle poly-
ethylene tube. The polyethylene tube was full with
heparin sodium (50IU/mL of NS) and one end was
inserted into the left jugular vein. Heparin sodium was
H-Pro-Ala-Lys-OH. Using the general procedure for
removal of NO2, Z and Bzl from Boc-Pro-Ala-Lys(Z)-
OBzl the title compound was obtained as colorless crystals
in 95% yield (60mg). Mp 150–152 ꢁC; FAB-MS (m/e) 315
[M+H]+; [a]D20=ꢀ35.6ꢁ (c=2.02, H2O), IR (KBr): 3421,
3259, 3063, 2950, 1660, 1554 cmꢀ1, 1H NMR (DMSO-d6):
d=8.650(3H), 8.461 (1H), 8.132 (1H), 4.350(1H), 4.086
(1H), 3.847 (1H), 3.335 (2H), 3.003 (2H), 2.745 (2H),
2.108 (1H), 1.710 (4H), 1.509 (2H), 1.307 (2H), 1.220
(3H). Amino acid analysis: calcd Pro:Ala:Lys=
1.0:1.0:1.0; found, Pro:Ala:Lys=1.00:1.01:0.96. Anal.

4920
M. Zhao et al. / Bioorg. Med. Chem. 11 (2003) 4913–4920
injected via the other end of the polyethylene tube as the
anticoagulant, following which the tested compound
was injected. The blood was circulated through the
polyethylene tube for 90min, after which the helix was
taken out and weighted accurately. The reduction of
thrombolytic mass was recorded. The data are listed
in Table 4. The statistical analysis of the date was
carried out by use of ANOVA test, P<0.05 is con-
sidered significant.
References and Notes
1. Zhao, M.; Peng, S. J. Prakt. Chem. 1999, 341, 668.
2. Lin, N.; Zhao, M.; Wang, C.; Peng, S. Bioorg. Med. Chem.
Lett. 2002, 12, 585.
3. Wu, Y..; Zhao, M.; Wang, C.; Peng, S. Bioorg. Med. Chem.
Lett. 2002, 12, 2331.
4. Mehta, J. L.; Nichols, W. W.; Saldeen, T. G. P. J. Cardio-
vasc. Pharmacol. 1989, 13, 803.
5. Saldeen, K.; Lawson, D. L.; Mehta, J. L. Thrombosis
Research 1991, 61, 81.
6. Saldeen, T. G. P.; Nichols, W. W.; Saldeen, K.; Wallin, R.;
Mehta, J. L. J. Cardiovasc. Pharmacol. 1994, 23, 103.
7. Saldeen, T.; James, W.; Berryer, R.; Berryer, P. Thrombosis
Research 1981, 23, 465.
Acknowledgements
8. Kluft, C. Thromb. Haemost. 1979, 41, 356.
9. Urano, T.; Sakakibara, K.; Rydzewski, A.; Urano, S.;
Takada, Y.; Takada, A. Thromb. Haemost. 1990, 63, 82.
10. Lin, N.; Yang, J.; Wang, C.; Zhao, M.; Peng, S.; Song, D.;
Wong, J. J. Beijing Mwed. Univ. 2000, 32, 283.
The author Shiqi Peng wishes to thank the key Basic
Research Project (G1998051111) of China and 985 of
Peking University for financial support.