A class of novel nitronyl nitroxide labeling basic and acidic amino acids: Synthesis, application for preparing ESR optionally labeling peptides, and bioactivity investigations

Article abstract of DOI:10.1016/j.bmc.2008.01.022

Aimed at optional ESR label 2-(4′-hydroxyl)phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl was introduced into the guanido of l-Arg-OH, the ω-amino group of l-Lys-OH with methylcarboxyl as a linker, and into the β-carboxyl of l-Asp-OH and the γ-carboxyl of l-Glu-OH with ethylamino as a linker. It was explored that the synthetic 30 novel ESR labeling amino acid derivatives were stable enough to the reaction conditions of peptide synthesis. Their incorporation led to 12 novel ESR optionally labeling PAK, RGDS, RGDV, and ECG. A series of NO related chemical tests, the in vitro and in vivo assays of these peptides confirmed that this strategy was practical.

Full text of DOI:10.1016/j.bmc.2008.01.022

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4019–4028
A class of novel nitronyl nitroxide labeling basic and acidic
amino acids: Synthesis, application for preparing ESR
optionally labeling peptides, and bioactivity investigations
Jianwei Zhang, Ming Zhao,* Guohui Cui and Shiqi Peng*
College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
Received 10 December 2007; revised 14 January 2008; accepted 14 January 2008
Available online 19 January 2008
Abstract—Aimed at optional ESR label 2-(40-hydroxyl)phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl was introduced into
the guanido of L-Arg-OH, the x-amino group of L-Lys-OH with methylcarboxyl as a linker, and into the b-carboxyl of L-Asp-
OH and the c-carboxyl of L-Glu-OH with ethylamino as a linker. It was explored that the synthetic 30 novel ESR labeling amino
acid derivatives were stable enough to the reaction conditions of peptide synthesis. Their incorporation led to 12 novel ESR option-
ally labeling PAK, RGDS, RGDV, and ECG. A series of NO related chemical tests, the in vitro and in vivo assays of these peptides
confirmed that this strategy was practical.
ꢀ 2008 Published by Elsevier Ltd.
1. Introduction
decomposed to lose the electronic spin resonance signal
and can not be recovered in basic solution. On the other
hand even in the presence of HF the N–O of the piperidine
nitroxide moiety was protonated though it can be reverted
upon treatment with 0.02 M ammonium acetate at pH 9.0
for 3 h.8 In the literature another effort was given to
prepare ESR labeling peptides directly using (1,3-dioxyl-
4,4,5,5-tetramethyldihydroimidazol-2-yl)-L-alanine. This
effort was also not successful because (1,3-dioxyl-
4,4,5,5-tetramethyldihydroimidazol-2-yl)-L-alanine can
be decomposed not only in strong acid media but also in
the presence of anhydrous trifluoroacetic acid.7 Thus
there has been a long-standing interest to identify novel,
more stable, and credible peptide spin labels especially
ESR labeling amino acids to be directly used for the syn-
thesis of peptide. The present paper describes fifteen novel
nitronyl nitroxide modified L-Lys, L-Arg, L-Asp, and L-
Glu derivatives, their synthesis, applications for preparing
ESR labeling peptides, seven optionally ESR labeling
peptides, and biological activities.
Electronic spin resonance (ESR), as one of the most useful
biological techniques, is able to provide information
about conformation properties of peptides and their inter-
action with macromolecules and membrane of biological
interest by the use of stable free radical spin labels.1–4 So
far most of the reports related to the use of ESR for
labeling proteins and peptides involving chemically intro-
ducing the piperidine nitroxides to the side chains of their
amino acid residues, their N- or C-terminal, or to any po-
sition of their sequences.5,6 Unfortunately the synthesis of
peptides with piperidine nitroxides as spin labels was of
limited success because of the ability of the piperidine nitr-
oxide moiety, which was decomposed during the depro-
tection steps required to remove the protecting groups
of a-amino groups and side chains of each coupled amino
acid residue, and to remove the protecting groups of a-
carboxyl groups or cleave the peptides from the resin.7
For instance in the presence of reagent K, the piperidine
nitroxide moiety of 9-fluorenylmethyoxy-carbonyl-
2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carbox-
ylic acid which was usually used to label peptides was
2. Results and discussion
2.1. Preparing nitronyl nitroxides 2g and 3g capable of
labeling
Keywords: Nitronyl nitroxide; Label; Basic and acidic amino acids;
Incorporating; Peptide.
*
Corresponding authors. Tel./fax: +86 10 8391 1535 (M.Z.); tel./fax:
+86 10 8391 1528 (S.P.); e-mail addresses: maozhao@126.com;
sqpeng@mail.bjum.edu.cn
To find a suitable nitronyl nitroxide capable of modify-
ing L-Lys, L-Arg, L-Asp, and L-Glu a series of 2-alkyl,
0968-0896/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.01.022

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J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 4019–4028
carboxyl, heteroaryl, and aryl substituted nitronyl nitr-
oxides were prepared according to the synthetic route
depicted in Scheme 1,9 their NO free radical characteris-
tic, their NO scavenging activity, and their chemical sta-
bility were tested and compared.
bath with 5 ml of warmed (37 ꢁC) and oxygenated (95%
O2, 5% CO2) Krebs solution (pH 7.4) according to a
standard procedure. The data are expressed by inhibi-
tion percentage of ACh-induced vasorelaxation which
was recorded by the tension transducers and are listed
in Table 1. The results indicated that though 100 lM
2a–h consistently inhibited ACh-induced vasorelaxa-
tion, only 10 lM 2f–h significantly inhibited ACh-in-
duced vasorelaxation. Among nine compounds only 2g
and 3g gave significant and dose-dependent inhibition
for ACh-induced vasorelaxation.
In the presence of Br2 and NaOH 2-nitropropane was
smoothly converted into 2,3-dimethyl-2,3-dinitrobutane
which was reduced by zinc powder and NH4Cl to give
2,3-dimethyl-2,3-dihydroxylaminobutane in 40% total
yield. The condensation of hydroxylamine and eight
substituted aldehydes, including methyl aldehyde, chlo-
romethyl aldehyde, aldehydic acid, a-furaldehyde, b-
pyridyaldehyde, benzaldehyde, 4-hydroxylbenzaldehyde,
and 4-nitrobenzaldehyde, provided eight 2-substituted
phenyl-1,3-dioxyl-4,4,5,5-tetramethylimidazolindines 1a–
h in 53%, 69%, 83%, 73%, 69%, 85%, 51%, and 63% yield,
respectively. With PbO2 as oxidation agent 1a–h were
converted into the corresponding nitronyl nitroxides
2a–h in 99%, 38%, 88%, 83%, 79%, 80%, 52%, and 73%
yield, respectively (Scheme 1).
It is well known that in preparation of ESR labeling
peptides nitronyl nitroxide-amino acids will undergo a
series of treatments of acidic and basic aqueous solution
and may be reduced to imino nitroxide-amino acids. To
observe the stability of 2a–h in acidic and basic aqueous
solution they were dissolved in the aqueous solutions of
pH 2.0, 7.4, and 11.5 (final concentration 10ꢀ2 mM),
kept at room temperature for up to 200 h, during which
TLC (CHCl3/CH3OH, 20:1) monitor was conducted.
The results are listed in Table 2. TLC monitor indicated
that at the mentioned conditions 2a–c were decomposed
between 2 and 18 h, 2d–e were decomposed between 15
and 30 h, and 2f–h and 3g did not decompose up to
200 h. According to the results of the ESR spectral, aor-
tic strip assay and TLC analysis 2-(40-hydroxylphe-
nyl)nitronyl nitroxide (2g) should be the most suitable
In the determination of NO free radical characteristic
the ESR spectra of 2a–h were recorded on ESR spec-
trometer. It was found that 2a–h consistently gave iden-
tical ESR spectrum as shown in Figure 1, which consists
of a five-line pattern with 1:2:3:2:1 intensity ratios. This
kind of spectrum obviously resulted from an electron
interacting with two equivalent nitrogens in the nitronyl
nitroxides. The spectra imply that 2a–h are desirable
nitronyl nitroxides with correct electronic structure.
Table 1. Inhibition of ACh-induced vasorelaxation of 2a–h and 3ga
Inhibition potency
In the determination of NO scavenging capacity the rat
aortic strip assays of 2a–h were performed in a perfusion
100 lM
10 lM
1 lM
1.7 1.5
NS
2a
2b
2c
2d
2e
2f
1.63 1.45
8.6 3.7b
31.2 2.9b
25.0 2.4b
34.3 6.5b
44.4 5.9b
48.6 2.9b
81.9 8.9c
99.2 1.4c
98.0 2.7c
99.0 1.8c
7.7 9.8
1.6 1.5
1.7 1.6
25.0 17.8b
20.2 4.2b
16.6 2.8b
62.7 5.5c
73.1 5.9c
71.3 8.8c
72.4 5.2c
NO2
i
ii
iii
HO NH NH OH
1.6 1.5
1.7 1.7
H3C CH CH3
HO
1a-h
N
N
OH
O
O2N
NO2
R
9.8 7.8
iv
2g
2h
3g
26.1 7.0b
ꢀ14.5 14.5
25.3 6.2b
v
vi
C6H5-[OCH2CO2H]-p
O
N
N
O
O
O
N
3g
N
O
N
N
2a-h
R
C6H5-[OCH2CO2CH3]-p
a Inhibition is expressed by X ꢁ SD%; NS (normal saline) = vehicle;
n = 6.
b Compared to NS p < 0.001.
Scheme 1. Route for preparing nitronyl nitroxides. Reagents: (i) Br2,
NaOH (6 M); (ii) Zn, NH4Cl; (iii) Methyl aldehyde, chloromethyl
aldehyde, aldehydic acid, a-furaldehyde, b-pyridyaldehyde, benzalde-
hyde, 4-hydroxylbenzaldehyde or 4-nitrobenzaldehyde; (iv) PbO2; (v)
BrCH2CO2C2H5, NaOEt, THF; (vi) NaOH (2 M) in 1a and 2a R = H,
1b and 2b R = –CH2Cl, 1c and 2c R = –COOH, 1d and 2d R = fur-2-
yl, 1e and 2e R = pyrid-3-yl, 1f and 2f R = phenyl, 1g and 2g R = 40-
hydroxylphenyl, 1h and 2h R = 4-nitrophenyl.
c Compared to NS p < 0.001 and to 2a–e, p < 0.01.
Table 2. Stability of 2a–h and 3g in pH 2.0, 7.4, and 11.5 aqueous
solution
Compound
Decomposition time (h)
pH 7.4
pH 2.0
pH 11.5
2a
2b
2c
2d
2e
2f
2g
2
2
18
18
2.5
2.5
2
2
15
18
30
2.5
18
15
30
18
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
Figure 1. Typical ESR spectrum given by 2g, 3g, the modified amino
acids, and the peptides.
3g

J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 4019–4028
4021
labeling block. The O-alkylation of 2g and BrCH2CO2H
provided [1-(10,30-dioxyl-40,40,50,50-tetramethyldihydro-
imidazol-2-yl)phenyl-4-yl]oxyacetic acid (3g) in theoret-
ical yield. The stability evaluation indicated that 3g
should be suitable like 2g as a labeling block.
were tested. The testing samples were obtained by dis-
solving them in water or phosphate buffer (pH 7.4, final
concentration 10 lM). The tests demonstrated that all of
them gave similar ESR spectrum to that of 2g and 3g.
The spectrum (Fig. 1) consists of a five-line pattern with
1:2:3:2:1 intensity ratios. This spectrum obviously re-
sulted from an electron interacting with two equivalent
nitrogens in the nitronyl nitroxides. Standard ESR spec-
trum of nitronyl nitroxide was recorded for 2g and 3g
modified amino acids suggested that after a series of pre-
parative reactions and treatments they were still desir-
able nitronyl nitroxide derivatives. This was also true
for the peptides containing 2g and 3g modified amino
acids. The standard ESR spectrum of nitronyl nitroxides
gained from all of the labeling compounds themselves
prepared here not only establishes a base for their struc-
tural determination but also provides a sensitive signal
for their use as a probe. These are obviously impossible
for the previous unstable ESR labeling compounds.
2.2. Introducing 2g or 3g into L-Lys, L-Arg, L-Asp, and L-
Glu
Considering the high appearance frequency and avail-
ability of labeling L-Lys-OH, L-Arg-OH, L-Asp-OH,
and L-Glu-OH in peptides, 2g or 3g was introduced
either into the side chain or into the a-amino group of
these by use of the routes depicted in Schemes 2–7 and
L-Na-3g-Lys(Z)-OBzl (4, 65% yield), L-Na-3g-Lys(Z)-
OH (5, 91% yield), L-Na-3g-Lys-OH (6, 90% yield), L-
Nx-3g-Lys-OCH3 (7, 83% yield), L-Nx-3g-Lys-OH (8,
93% yield), L-Na-Boc-Nx-3g-Lys-OH (9, 91% yield), L-
Na-3g-Arg-OMe (10, 87% yield), L-Na-3g-Arg-OH (11,
73% yield), L-Na-Boc-NG-3g-Arg-OMe (12, 87% yield),
L-Na-Boc-NG-3g-Arg-OH (13, 73% yield), L-NG-3g-
Arg-OMe (14, 98% yield), L-Na-Boc-b-2g-Asp-OH (17,
39% yield), and L-Na-Boc-b-2g-Asp-OCH3 (18, 46%
yield), L-b-2g-Asp-OH (19, 94% yield) and L-b-2g-
Asp-OCH3 (20, 94% yield), L-Na-Boc-c-2g-Glu-OH
(23, 48% yield), and L-Na-Boc-c-2g-Glu-OCH3 (24,
48% yield) were obtained. Their detailed data were
provided as supporting information. The structures
of the labeling amino acids 4–14, 17–20, 23, and 24
are shown in Table 3. In contrast to the lack of
ESR labeling amino acid previously, these derivatives
of Table 1 not only form a family of ESR labeling
amino acids but also provide structural diversity to
meet the necessary selection in the preparation of
optionally labeling peptides.
2.5. NO trapping activity of 2g and 3g modified amino
acids and peptides
To examine the NO trapping activity of the 2g and 3g
modified amino acids and peptides the NO trapping as-
say was performed. The testing samples were obtained
by dissolving them in a deaerated phosphate buffer
(pH 7.4, final concentration 10 lM), NO gas was bub-
bled and the ESR spectra were recorded. The tests ex-
plored that all 2g and 3g modified amino acids and
peptides gave similar ESR spectrum of imino nitroxide
(Fig. 2), which consists of a seven-line pattern attribut-
ing to an electron interacting with two equivalent nitrog-
enes. The formed imino nitroxide products obviously
resulted from the oxidation–reduction reaction of nitro-
nyl nitroxide and NO, suggesting that 2g and 3g modi-
fied amino acids and peptides are desirable NO
scavengers. A desirable NO scavenging property was re-
corded for 2g and 3g modified amino acids, suggesting
that after a series of preparative reactions and treat-
ments they were still desirable free radicals. A desirable
NO scavenging property was recorded for the peptides
containing 2g and 3g modified amino acids, suggesting
that after a series of preparative reactions and treat-
ments they were also still free radicals. The results imply
that these labeling methods are able to provide ESR
optionally labeling peptides with typical NO scavenging
property. The standard ESR spectrum of imino nitrox-
ides gained from the deductive products of all the label-
ing compounds prepared here not only establishes a
base for determining their capacity removing NO free
radical but also provides a sensitive signal for their use
as a probe. These are also obviously impossible for the
previous unstable ESR labeling compounds.
2.3. Incorporating 2g or 3g modified L-Lys, L-Arg, L-Asp
or L-Glu into bioactive peptides
In order to clarify the application of the mentioned 2g or
3g modified L-Lys, L-Arg, L-Asp or L-Glu in the prepa-
ration of ESR label peptides they were incorporated into
some bioactive peptides such as Pro-Ala-Lys (PAK),
Glu-Cys-Gly (ECG), and Arg-Gly-Asp (RGD) peptides.
The preparations were carried out according to the
routes depicted in Schemes 8–13 and H-Pro-Ala-(Nx-
3g-Lys)-OH (31, 78% total yield), (NG-3g-Arg)-Gly-
Asp-Val-OH (36, 54% total yield), H-Arg-Gly-(b-2g-
Asp)-Ser-OH (43, 25% total yield), and H-(c-2g-Glu)-
Cys-Gly-OH (48, 35% total yield) were obtained. Their
detailed data were provided as supporting information.
The structures of the labeling peptides 31, 36, 43, and
48 are also shown in Table 3. In contrast to dull N-ter-
minal labeling peptides previously, these derivatives of
Table 3 not only form a family of ESR labeling peptides
but also provide a strategy preparing optionally labeling
peptides.
2.6. Compounds 2g and 3g modified amino acids and
peptides preventing PC12 cells from free radical damage
2.4. Typical ESR spectra of 2g and 3g modified amino
acids and peptides
To examine the prevention function of 2g and 3g mod-
ified amino acids and peptides the PC12 cell damage as-
say was performed. In the experiment a method of
Dawson with minor modifications was used,10 NO,
H2O2Æ, and ÆOH were used as the damage radicals. The
To examine the free radical characteristics of the 2g and
3g modified amino acids and peptides their ESR spectra

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J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 4019–4028
Table 3. Structures of 2g and 3g modified amino acids and peptides
Compound
Chemical structure
Compound
Chemical structure
O
O
O
O
N
N
N
O
N
N
2g
3g
C6H5-(OCH2CO2H)-p
C6H5-OH-p
O
O
O
N
N
N
5
7
9
6
C6H4-[O-CH2CONH-CH-CO2H]-p
Z-NH-(CH2)3 CH2
C6H4-[O-CH2CONH-CH-CO2H]-p
NH2-(CH2)3 CH2
O
N
O
O
N
N
O
O
N
8
C6H4-[O-CH2CONH-(CH2)3CH2
H2N CH CO2CH3]-p
C6H4-[O-CH2CONH-(CH2)3CH2
H2N CH CO2H]-p
O
N
N
O
O
O
N
N
10
C6H4-[O-CH2CONH
CH CO2CH3]-p
C6H4-[O-CH2CONH-(CH2)3CH2
Boc-HN CH CO2H]-p
CH2(CH2)2-NH-C-NH2
NH
O
N
N
O
O
O
N
N
NH
C6H4-[O-CH2CONH-C-NH-(CH2)2CH2
11
13
17
19
23
12
14
18
20
24
C6H4-[O-CH2CONH
CH CO2H]-p
CH2(CH2)2-NH-C-NH2
NH
Boc-HN CH CO2CH3]-p
O
O
N
N
N
N
NH
C6H4-[O-CH2CONH-C-NH-(CH2)2CH2
NH
C6H4-[O-CH2CONH-C-NH-(CH2)2CH2
Boc-HN CH CO2H]-p
H2N CH CO2CH3]-p
O
O
N
N
O
O
O
N
N
O
C6H4-[O-(CH2)2-NHCO-CH2-CH-CO2H]-p
Boc-NH
C6H4-[O-(CH2)2-NHCO-CH2-CH-CO2CH3]-p
Boc-NH
N
N
O
N
N
O
C6H4-[O-(CH2)2-NHCO-CH2-CH-CO2H]-p
NH2
C6H4-[O-(CH2)2-NHCO-CH2-CH-CO2CH3]-p
NH2
O
O
N
N
O
O
N
N
O
O
C6H4-[O-(CH2)2-NHCO-(CH2)2-CH-CO2H]-p
Boc-NH
C6H4-[O-(CH2)2-NHCO-(CH2)2-CH-CO2CH3]-p
Boc-NH
N
N
O
N
N
O
25
26
C6H4-[O-(CH2)2-NHCO-(CH2)2-CH-CO2H]-p
NH2
C6H4-[O-(CH2)2-NHCO-(CH2)2-CH-CO2CH3]-p
NH2

J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 4019–4028
4023
Table 3 (continued)
Compound
Chemical structure
Compound
Chemical structure
29
30
O
O
O
O
N
N
N
O
O
N
N
N
C6H4-[O-CH2CONH-(CH2)3CH2
Boc-Pro-Ala-HN CH CO2CH3]-p
C6H4-[O-CH2CONH-(CH2)3CH2
Boc-Pro-Ala-HN CH CO2H]-p
O
N
N
O
31
42
34
35
36
41
43
C6H4-[O-CH2CONH-(CH2)3CH2
H-Pro-Ala-HN CH CO2H]-p
C6H4-[O-(CH2)2-NHCO-CH2-CH-CO-Ser-OCH3]-p
BocArg-Gly-HN
O
N
N
O
N
N
O
O
C6H4-[O-(CH2)2-NHCO-CH2-CH-CO-Ser-OH]-p
BocArg-Gly-HN
C6H4-[O-(CH2)2-NHCO-CH2-CH-CO-Ser-OH]-p
H-Arg-Gly-HN
O
N
N
NH
C6H4-[O-CH2CONH-C-NH-(CH2)2CH2
Boc-HN CH CO-Gly-Asp(OBzl)-Val-OCH3]-p
O
O
N
N
NH
C6H4-[O-CH2CONH-C-NH-(CH2)2CH2
Boc-HN CH CO-Gly-Asp(OBzl)-Val-OH]-p
O
O
N
N
NH
C6H4-[O-CH2CONH-C-NH-(CH2)2CH2
H2N CH CO-Gly-Asp-Val-OH]-p
O
N
N
O
46
47
C6H4-[O-(CH2)2-NHCO-(CH2)2-CH-CO-Cys-Gly-OCH3]-p
Boc-HN
O
O
N
N
O
C6H4-[O-(CH2)2-NHCO-(CH2)2-CH-CO-Cys-Gly-OH]-p
Boc-HN
N
N
O
48
C6H4-[O-(CH2)2-NHCO-(CH2)2-CH-CO-Cys-Gly-OH]-p
H2N
potency is expressed by EC50(lM) and the data are
listed in Table 4. The observation gave substantially
similar EC50 values for 2g and 3g modified amino acids
and peptides. The recorded capacity of 2g and 3g mod-
ified amino acids preventing PC12 cell from free radical
damage suggested that after a series of preparative reac-
tions and treatments they still had desirable free radical
scavenging activity. The recorded ability of the peptides
containing 2g and 3g modified amino acids preventing
PC12 cell from free radical damage suggested that after
a series of preparative reactions and treatments they still
had desirable free radical scavenging activity. The
capacity of all the labeling compounds themselves pre-
venting PC12 cell from free radical damage not only
establishes a base for directly using them in cell preven-
tion but also provides a cell model for screening free
radical scavengers with each the present compound as
positive control. These are obviously impossible for
the previous unstable ESR labeling compounds with
bioactivity loss.

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J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 4019–4028
Table 5. Inhibition of 2g and 3g modified compounds to ACh-induced
vasorelaxationa
Inhibition
100 lM
10 lM
1 lM
NS
PAK
ECG
RGDS
RGDV
2g
1.6 1.4
9.5 7.2b
9.2 8.5b
8.9 8.3b
10.5 8.0b
98.3 4.8c
99.2 4.5c
98.9 4.9c
98.6 4.8c
97.9 4.2c
15.9 5.3b
95.0 5.5c
96.1 4.9c
97.5 4.7c
99.1 5.0c
96.9 5.2c
97.1 4.9c
99.1 5.6c
96.6 5.4c
95.9 4.6c
97.3 5.1c
6.1 5.4b
5.2 6.5b
4.5 4.8b
3.2 4.1b
2.9 3.3b
5.3 4.1b
27.8 5.6
26.7 5.2
28.7 5.4
25.4 5.1
28.0 5.5
7.8 5.6b
24.1 5.3
25.7 5.6
26.7 5.2
29.2 5.3
26.0 5.1
26.2 5.2
26.6 5.5
26.2 5.6
24.0 4.9
25.9 5.0
4.3 5.0b
7.6 6.2b
74.5 5.2d
73.1 5.1d
74.0 5.7d
72.3 5.0d
72.7 4.9d
10.7 5.5b
71.2 5.0d
72.4 5.3d
72.8 5.0d
76.0 5.1d
72.4 4.9d
72.4 5.0d
73.9 5.4d
72.8 5.3d
71.3 5.0d
72.2 4.9d
3g
6
Figure 2. Typical ESR spectrum recorded from the reactions of 2g, 3g,
the modified amino acids and the peptides with NO gas.
8
9
11
Table 4. PC12 cell prevention activity of 2g and 3g modified
compoundsa
13
14
19
20
Compound
EC50 (lM)
H2O2Æ
NO
ÆOH
56.90 2.39
23
25
2g
3g
6
40.02 1.98
92.30 2.05
90.60 2.79
90.56 2.64
89.90 2.78
92.88 2.95
93.00 2.90
93.61 3.00
42.31 3.04
38.99 2.78
42.66 2.18
41.77 2.55
88.99 3.22
92.86 2.92
93.01 3.00
37.84 3.06
49.05 2.75
31.64 2.20
30.07 2.88
29.85 2.66
29.33 2.85
32.07 2.69
32.41 2.94
33.36 2.93
48.62 3.31
47.82 2.93
49.89 2.98
49.62 2.80
28.83 3.11
32.83 2.87
32.04 3.19
46.98 2.91
31
36
100.08 2.17
99.11 3.09
98.77 3.06
97.72 3.22
100.92 3.12
101.76 2.99
102.85 2.92
58.22 3.11
55.63 3.00
59.00 2.99
58.06 2.62
99.00 3.03
101.78 3.20
101.95 3.24
54.90 3.04
43
48
8
9
a Inhibition is expressed by X ꢁ SD%; n = 6.
11
13
14
19
20
23
25
31
36
43
48
b Except 11, compared to all 2g, and 3g modified amino acids and
peptides p < 0.01.
c Compared to 10 lM group p < 0.01.
d Compared to 1 lM group p < 0.01.
antagonists with each the present compound as positive
control. These are obviously impossible for the previous
unstable ESR labeling compounds with bioactivity loss.
a EC50 is expressed by X ꢁ SDlM; n = 6.
2.8. Compound 2g modified RGDS and 3g modified
RGDV inhibiting in vitro platelet aggregation
To examine the in vitro anti-platelet aggregation activity
of 2g modified RGDS and 3g modified RGDV the anti-
platelet aggregation effect of 36 and 43 was examined
with normal rabbit blood according to a published
method,12 platelet-activating factor (PAF, final concen-
tration 10ꢀ7 M) and adenosine diphosphate (ADP, final
concentration 10ꢀ5 M) were used as the activator. The
maximal rate of platelet aggregation (Am%) was repre-
sented by the peak height of aggregation curve and the
data are listed in Table 6. It was found that the
in vitro anti-platelet aggregation activity of 10ꢀ6 M 43
equaled to the in vitro anti-platelet aggregation activity
of 10ꢀ6 M RGDS and significantly higher than that of
2g, while the in vitro anti-platelet aggregation activity
of 10ꢀ6 M 2g equaled to that of NS. This comparison
suggested that 2g contributed nothing to the in vitro
anti-platelet aggregation activity of 43. It was also found
that the in vitro anti-platelet aggregation activity of
10ꢀ6 M 36 equaled to the in vitro anti-platelet aggrega-
tion activity of 10ꢀ6 M RGDV and significantly higher
than that of 3g, while the in vitro anti-platelet aggrega-
tion activity of 10ꢀ6 M 3g equaled to that of NS. This
comparison suggested that 3g contributed nothing to
the in vitro anti-platelet aggregation activity of 36.
Without anti-platelet aggregation activity loss for
2.7. Compounds 2g and 3g modified amino acids and
peptides inhibiting ACh-induced vasorelaxation
To examine the in vitro NO scavenging ability of 2g and
3g modified amino acids and peptides the rat aortic strip
assay was performed according to a published method.11
The data are expressed by the percentage inhibition of
ACh-induced vasorelaxation and listed in Table 5. The
data demonstrated that except 11, all 2g and 3g modified
amino acids effectively inhibited ACh inducing vasore-
laxation, suggesting that after a series of preparative
reactions and treatments they had desirable in vitro
NO scavenging activity. For L-Na-3g-Arg-OH (11) only
very weak vitro NO scavenging activity was tested. This
observation should be understandable considering that
the interaction of the guanido of 11 with the NOS in
the rat aortic strip may generate NO to counteract the
NO scavenging activity of 11 and thus the ACh-induced
vasorelaxation may be retained. Compared to PAK,
RGDV, RGDS, and ECG 3g modified peptides 31, 36,
43, and 48 significantly inhibited ACh-induced vasore-
laxation. The capacity of all the labeling compounds
themselves inhibiting ACh-induced vasorelaxation not
only establishes a base for directly using them to inhibit
ACh but also provides a tissue model for screening ACh

J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 4019–4028
Table 6. Inhibition of 36 and 43 to ADP and PAF-induced platelet aggregationa
4025
Inhibition to ADP
10ꢀ6 M
Inhibition to PAF
10ꢀ6 M
10ꢀ7 M
10ꢀ5 M
10ꢀ7 M
10ꢀ5 M
NS
2g
50.40 5.23
50.39 3.22
39.62 3.00b
39.43 3.14b
50.65 4.00
33.41 2.83b
39.60 3.03b
56.70 4.11
52.45 3.01
36.47 3.00b
34.33 2.22b
51.40 3.11
34.84 2.51b
32.80 2.70b
49.28 2.63
51.50 2.43
50.00 3.11
51.98 2.45
49.78 3.20
50.20 3.13
49.01 3.08
16.60 2.61c
16.11 2.51c
49.33 4.02
12.65 1.84c
16.38 2.50c
54.00 3.25
53.69 3.10
51.59 3.11
53.80 3.01
52.07 3.27
50.26 3.22
53.04 2.19
22.03 2.31c
21.52 2.19c
52.45 2.09
20.01 2.12c
19.88 2.21c
RGDS
43
3g
RGDV
36
a Inhibition is expressed by X ꢁ SD%, n = 8, NS = vehicle.
b Compared to NS and 10ꢀ7M group p < 0.01.
c Compared to NS and 10ꢀ6M group p < 0.01.
Table 7. In vivo anti-thrombotic activity of 36 and 43a
to that of NS. This comparison suggested that 3g con-
tributed nothing to the in vivo anti-thrombotic activity
of 36 and introducing 3g into RGDV resulted in no de-
crease of its in vivo anti-thrombotic activity. Without
anti-thrombotic activity loss for RGDS with ESR on
its R guanido and RGDV with ESR on its D b-carboxyl
not only establishes a base for directly using them to in-
hibit thrombosis but also provides an in vitro model for
screening ESR containing anti-thrombotic agents with
each the present compound as positive control. Though
these were also true for previously reported RGD pep-
tides with ESR on their N-termini,9 this optional ESR
labeling strategy provides a general means for structural
diversity design.
Compound
Dose
Wet thrombus
weight
Dry thrombus
weight
NS
Asprin
2g
3 ml/kg
39.89 3.60
24.16 2.90b
38.90 3.10
27.90 2.18b
25.01 2.12b
38.76 3.18
28.60 3.20b
26.11 2.94b
6.95 1.01
4.04 1.03b
6.80 0.88
4.22 1.10b
4.08 1.11b
6.67 0.79
4.43 1.06b
4.18 1.12b
20 mg/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
RGDS
43
3g
RGDV
36
a Thrombus weight is expressed by X ꢁ SDmg, n = 10, NS=vehicle.
b Compared to NS, 2g and 3g p < 0.01.
RGDS with ESR on its R guanido and RGDV with
ESR on its D b-carboxyl not only establishes a base
for directly using them to inhibit platelet aggregation
but also provides an in vitro model for screening ESR
containing anti-platelet aggregators with each the pres-
ent compound as positive control. Though these were
also true for previously reported RGD peptides with
ESR on their N-termini,9 this optional ESR labeling
strategy provides a general means for structural diver-
sity design.
2.10. Compound 3g modified PAK prolong euglobulin clot
lysis time
To examine the in vitro thrombolytic activity of 3g mod-
ified PAK the euglobulin clot lysis time (ECLT) assay
was performed. In the determination the rabbit euglobu-
lin clots were prepared in a 96-well microtiter plate
according to a published method.14,15 To the rabbit
euglobulin clots, NS, UK, 3g, PAK, and 31 were added
and the ECLT was recorded. The data are listed in Table
8. It was found that the ECLT of 4.5 lM 31 equaled to
the ECLT of 4.5 lM PAK and significantly shorter than
that of 4.5 lM 3g, while the ECLT of 4.5 lM 3g equaled
to that of NS. This comparison suggested that 3g con-
tributed nothing to the in vitro thrombolytic activity of
31 and introducing 3g into PAK resulted in no decrease
of its in vitro thrombolytic activity. The ECLTs of 31 at
2.9. Compound 2g modified RGDS and 3g modified
RGDV possessing in vivo anti-thrombotic activity
To examine the in vivo anti-thrombotic activity of 2g
modified RGDS and 3g modified RGDV 43 and 36 were
evaluated with a rat model. In the evaluation a pub-
lished method was followed,13 and the thrombus weight
was used to express the activity, which is listed in Table
7. It was found that the in vivo anti-thrombotic activity
of 5 lmol/kg 43 equaled to the in vivo anti-thrombotic
activity of 5 lmol/kg RGDS and significantly higher
than that of 3g, while the in vivo anti-thrombotic activ-
ity of 5 lmol/kg 3g equaled to that of NS. This compar-
ison suggested that 3g contributed nothing to the in vivo
anti-thrombotic activity of 43 and introducing 3g into
PAK resulted in no decrease of its in vivo anti-throm-
botic activity. It was also found that the in vivo anti-
thrombotic activity of 5 lmol/kg 36 equaled to the
in vivo anti-thrombotic activity of 5 lmol/kg RGDV
and significantly higher than that of 3g, while the
in vivo anti-thrombotic activity of 5 lmol/kg 3g equaled
Table 8. Euglobulin clot lysis time of 31a
Compound
Concentration
ECLT
NS
—
5 IU
210.22 15.31
130.34 15.35b
223.11 16.00
101.60 15.41c
102.95 14.82d
128.80 12.77e
149.00 13.19b
UK
3g
4.5 lM
4.5 lM
4.5 lM
2.2 lM
1.1 lM
PAK
31
a ECLT is expressed by X ꢁ SDmin; NS = vehicle; n = 6.
b Compared to NS and 3g p < 0.01.
c Compared to NS p < 0.01, to UK p < 0.05.
d Compared to NS p < 0.01, to 2.2 lM 31 p < 0.05.
e Compared to 1.1 lM 31 p < 0.05.

4026
J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 4019–4028
Table 10. Thrombolytic activity of 31a
different concentration indicated that its in vitro throm-
bolytic activity depended on the dose. Without euglobu-
lin clot lysis activity loss for PAK with ESR on its K x-
amino group not only establishes a base for directly using
it as euglobulin clot lysis agent but also provides an
in vitro model for screening ESR containing thrombo-
lytic agents with it as positive control, and provides a
general means for structural diversity design as well.
Compound
Dosage
Reduce thrombus weight
NS
5 ml/kg
12.02 2.35
23.62 2.45b
12.11 2.85
24.11 2.31b
24.20 2.40c
15.38 2.25d
11.06 2.30
UK
3g
20,000 IU/kg
10.0 lmol/kg
10.0 lmol/kg
10.0 lmol/kg
5.0 lmol/kg
2.5 lmol/kg
PAK
31
a Reduced thrombus weight is expressed by X ꢁ SDmg; NS = vehicle;
2.11. Compound 3g modified PAK enlarging fibrinogen–
agarose lysis area
n = 10.
b Compared to NS and 3g p < 0.01.
c Compared to NS and 3g p < 0.01, to 2.2 lM 31 p < 0.05.
d Compared to NS and 3g p < 0.05, to 1.1 lM 31 p < 0.01.
To further examine the in vitro thrombolytic activity of
3g modified PAK the fibrinogen lysis area assay was also
performed according to a published procedure.16–18 In
the determination the rabbit fibrinogen–agarose mixture
was prepared and coagulated with thrombin in plastic
dishes. To observe the fibrinolytic activity, 30 lL of
NS and the solution of UK, PAK or 31 in NS were
added to each well. The plate was incubated and areas
of lysis were quantified. The data are listed in Table 9.
It was found that the lysis area of 2.2 lM 31 equaled
to the lysis area of 4.5 lM PAK and significantly larger
than that of 4.5 lM 3g, while the lysis area of 4.5 lM 3g
equaled to that of NS. This comparison suggested that
3g contributed nothing to the in vitro thrombolytic
activity of 31 and introducing 3g into PAK resulted in
no decrease of its in vitro thrombolytic activity. The ly-
sis areas of 31 at different concentration indicated that
its in vitro thrombolytic activity depended on the dose.
Without fibrinogen–agarose lysis activity loss for PAK
with ESR on its K x-amino group not only establishes
a base for directly using it as fibrinogen–agarose lysis
agent but also provides an in vitro model for screening
ESR containing thrombolytic agents with it as positive
control, and provides a general means for structural
diversity design as well.
ethylene tube is used to represent the thrombolytic activ-
ity. The data are listed in Table 10. It was found that the
reduced thrombus weight of 10.0 lmol/kg 31 equaled to
the reduced thrombus weight of 10.0 lmol/kg PAK and
significantly higher than that of 10.0 lmol/kg 3g, while
the reduced thrombus weight of 10.0 lmol/kg 3g
equaled to that of 3 ml/kg NS. This comparison sug-
gested that 3g contributed nothing to the in vivo throm-
bolytic activity of 31 and introducing 3g into PAK
resulted in no decrease of its in vivo thrombolytic activ-
ity. The in vivo thrombolytic activities of 31 at different
dose indicated that its in vivo thrombolytic activity de-
pended on the dose. Without thrombolytic activity loss
for PAK with ESR on its K x-amino group not only
establishes a base for directly using it as thrombolytic
agent but also provides an in vivo rat model for screen-
ing ESR containing thrombolytic agents with it as posi-
tive control, and provides a general means for structural
diversity design as well.
3. Conclusion
2.12. Compound 3g modified PAK reducing thrombus
weight in rat model
Using 2-(40-hydroxyl)phenyl-4,4,5,5-tetramethylimidaz-
oline-3-oxide-1-oxyl (2g) and [1-(10,30-dioxyl-40,40,50,50-
tetramethyldihydroimidazol-2-yl)-phenyl-4-yl]oxyacetic
acid (3g) as ESR labeling block L-Asp-OH, L-Glu-OH,
L-Lys-OH, L-Arg-OH, RGDS, RGDV, and ECG were
successively modified and 30 related compounds with
free radical property were obtained (Table 3). A series
of NO free radical related chemical, cell and rat aortic
strip tests confirmed that all these 2g and 3g labeling
18 amino acid derivatives were stable enough to the
reaction conditions of peptide synthesis. A series of
NO free radical related chemical, cell and rat aortic strip
tests, as well as the in vitro and in vivo assays of 12
labeling peptides confirmed that the strategy of labeling
bioactive peptides via incorporating 2g and 3g labeling
amino acid was practical.
To examine the in vivo thrombolytic activity of 3g mod-
ified PAK the thromboclasis assay was performed
according to a published procedure.12 In the assay the
in vivo thrombolytic activity of 31 was tested on the
pentobarbital sodium anesthetized male Wistar rats.
The reduced weight of the thrombus in an inserted poly-
Table 9. Fibrinogen–agarose lysis areas of 31a
Compound
Concentration
Lysis area
NS
—
5 IU
25.09 9.96
225.33 11.02b
26.60 9.85
214.11 10.20b
233.50 10.00c
216.23 9.24d
197.02 10.03b
UK
3g
4.5 lM
4.5 lM
4.5 lM
2.2 lM
1.1 lM
PAK
31
4. Experimental
4.1. General
a Fibrinogen–agarose lysis area is expressed byX ꢁ SDmm2;
NS = vehicle; n = 6.
b Compared to NS and 3g p < 0.01.
c Compared to NS and 3g p < 0.01, to 2.2 lM 31 p < 0.05.
Unless otherwise stated, all reactions were under a nitro-
gen atmosphere (1 bar). Melting points are uncorrected.
d Compared to NS and 3g p < 0.01, to 1.1 lM 31 p < 0.05.

J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 4019–4028
4027
The agents used in this work were purchased from Sig-
ma Chemical Co (USA). Chromatography was per-
formed on Qingdao silica gel H (Qingdao of China).
The purities of the intermediates and the products were
confirmed by TLC (Merck silica gel plates of type
60 F254, 0.25 mm layer thickness, Germany) and HPLC
(Waters, C18 column 4.6 · 150 mm, USA). NMR spec-
tra were recorded at 300 MHz on a VXR-300 instru-
ment or at 500 MHz on a Bruker Am-500 instrument
in CDCl3 with tetramethylsilane as internal standard.
EI-MS was determined by Trace MS System (Thermo
Finnigan, USA). Optical rotations were determined with
a Schmidt + Haensch Polartromic D instrument (Ger-
many). The statistical analysis of all the biological data
was carried out by use of ANOVA test, p < 0.05 is con-
sidered significant.
colorimetric assay with MTT according to the method
of Mosmann. Similarly, H2O2Æ damage was induced by
1 mM of H2O2 followed by 1 h of incubation, while
ÆOH damage was induced by 1 mM of H2O2/30 lM Fe
(II) followed by 1 h of incubation.
4.5. Rat aortic strip relaxation assay
Immediately after decapitation rat aortic strips were pre-
pared and put in a perfusion bath with 5 ml of warmed
(37 ꢁC), oxygenated (95% O2, 5% CO2) Krebs solution
(pH 7.4). The aortic strips were connected to the tension
transducers and their relaxation–contraction curves were
recorded. The noradrenaline (NE) solution (final concen-
tration 10ꢀ9 mol/L) was added for inducing the contrac-
tion of the strips, and when the hypertonic contraction
reached the maximal level NE was washed and the vessel
strips were stabilized for 30 min. After renewal of the
solution NE (final concentration 10ꢀ9 M) was added.
When the hypertonic contraction value of aortic strips
reached the peak 15 ll of water (vehicle), or the solution
of nitroxide modified L-Lys-OH, L-Arg-OH, L-Asp-OH,
L-Glu-OH, ECG, PAK, RGDS, and RGDV in 15 ll of
water (final concentration 10ꢀ6 M), was added, respec-
tively. After stabilization 1.5 ll of Ach (final concentra-
tion 10ꢀ6 M) was added and the NO scavenging
activities of the compounds are expressed with the inhi-
bition percentage of Ach-induced vasorelaxation.
4.2. Preparing compounds 1a–h, 2a–h, 3g, and 4–48
Compounds 1a–h, 2a–h, 3g, and 4–48 were prepared
according to the procedures described in supporting
information which also provides their physical chemical
data.
4.3. Determination of ESR spactrum
Center field 3480 G, sweep width 100 G, sweep time
100 s, modulation amplitude 1.0 · 10ꢀ1 G, time constant
1.6 · 10ꢀ1 s, modulation frequency 100 kHz, microwave
frequency 9.72 GHz, and microwave power 10 MW
were used for ESR measurement. The ESR spectra of
nitronyl nitroxide modified L-Lys, L-Arg-OH, L-Asp-
OH, L-Glu-OH, ECG, PAK, RGDS, RGDV peptides
and the related intermediates at two different concentra-
tions, 10ꢀ7 M and 10ꢀ5 M, in deaerated water and phos-
phate buffer (pH 7.4), were recorded. The deaerated
phosphate buffer containing nitronyl nitroxide modified
L-Lys-OH, L-Arg-OH, L-Asp-OH, L-Glu-OH, ECG,
PAK, RGDS, RGDV and the related intermediates
(10ꢀ5 M) were bubbled with NO gas for 30 s and the
ESR spectra of reaction products were recorded.
4.6. In vitro platelet aggregation assay
Platelet-rich plasma was prepared by centrifugation of
normal rabbit blood anti-coagulation with sodium cit-
rate at a final concentration of 3.8%. The platelet counts
were adjusted to 2 · 105/ll by addition of autologous
plasma. Platelet aggregation tests were conducted in
an aggregometer using the standard turbidimetric tech-
nique. The agonists used were platelet-activating factor
(PAF, final concentration 10ꢀ5–10ꢀ7 M) and adenosine
diphosphate (ADP, final concentration 10ꢀ5–10ꢀ7 M).
The effects of 36 and 43 on PAF or ADP induced plate-
let aggregation were observed. The maximal rate of
platelet aggregation (Am%) was represented by the peak
height of aggregation curve.
4.4. NO, H2O2Æ and ÆOH induced PC12 cell damage assay
Free radical scavenging activities were evaluated in
PC12 cells using a method of Dawson with minor mod-
ifications. PC12 cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% heat
inactivated horse serum (Hyclone), 5% fetal bovine ser-
um (GIBCO), 1.0 mM sodium pyruvate, 100 U/mL pen-
icillin, and 100 lg/mL streptomycin at 37 ꢁC in 5% CO2
atmosphere. During the exponential phase of growth,
PC12 cells were seeded in 96-well plates coated with
poly-L-lysine at a density of 20,000 cells per well. After
24 h attachment period fresh media containing
12.5 lM, 25 lM, 50 lM, 100 lM, 200 lM of nitronyl
nitroxide modified L-Lys, L-Arg-OH, L-Asp-OH, L-
Glu-OH, ECG, PAK, RGDS, and RGDV were added
to each well and were incubated for 1 h. NO damage
was then induced by adding 2 mM of sodium nitroprus-
side (SNP) followed by 2 h of incubation. Media were
replaced with fresh media and cells were incubated for
14 h, following which cell survival was measured by a
4.7. ECLT assay
The rabbit euglobulin clots were prepared according to
a published method. Plasma diluted at 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 centri-
fuged at 2000g for 15 min and the precipitate was resus-
pended to the initial plasma volume with 50 mM sodium
barbiturate buffer (pH 7.8, containing 1.66 mM of
CaCl2, 0.68 mM of MgCl2 and 93.96 mM of NaCl).
To the rabbit euglobulin clots, NS, UK, PAK or 31
was added and the ECLT or time to clot lysis was deter-
mined in a 96-well microtiter plate.
4.8. Fibrinogen–agarose lysis area assay
The fibrinogen–agarose mixture was prepared and coag-
ulated with thrombin in plastic dishes according to a

4028
J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 4019–4028
published procedure. The fibrinogen–agarose mixture
was prepared by mixing equal volumes of 0.3% rabbit
fibrinogen and 0.95% agarose solutions, both dissolved
in 50 mM sodium barbiturate buffer (pH 7.8). The
fibrinogen–agarose mixture was coagulated with
100 ml of thrombin (100 IU/ mL) in plastic dishes
(90 mm diameter · 1 mm depth). After 30 min at 4 ꢁC
an adequate number of wells, 5 mm in diameter, were
perforated. To determine fibrinolytic activity, 30 ll of
NS, UK, PAK, and 31 was added to each well. The plate
was incubated and areas of lysis were quantified by lysis
area. The statistical analysis of the data was carried out
by use of ANOVA test, p < 0.05 is considered
significant.
carried out by use of ANOVA test, p < 0.05 is consid-
ered significant.
Acknowledgments
This work supported by Beijing area major laboratory
of peptide and small molecular drugs, the 973 Project
of China (2006CB708501), National Natural Scientific
Foundation of China (30672513), and Natural Scientific
Foundation of Beijing (7052010).
Supplementary data
4.9. In vivo anti-thrombosis assay
Synthetic routes, preparing procedures, physical, analyt-
ical, and spectrometric data of all compounds. This
material is available free of charge via the Internet at
http://www.siencedirect.com/science/journal/09680896.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2008.01.022.
Male Wistar rats weighing 250–300 g (purchased from
Animal Center of Peking University) were used. The
tested compounds were dissolved in NS just before use
and kept in an ice bath. The rats were anesthetized with
pentobarbital sodium (80.0 mg/kg, ip), and the right car-
otid artery and left jugular vein were separated. A 6 cm
thread with exact weight was put into the middle of the
polyethylene tube. The polyethylene tube was full with
heparin sodium (50 IU/ml of NS) and one end was in-
serted into the left jugular vein. From the other end of
the polyethylene tube heparin sodium was injected as
anti-coagulant, then the tested compounds were in-
jected, which was inserted into the right carotid artery.
In the case the tube was full of NS or tested compound
dissolved in NS. The blood was flowed from the right
carotid artery to the left jugular vein via the polyethyl-
ene tube for 15 min. The thread was taken out, weighed
to record the weight of the wet thrombus was recorded,
kept in a desiccator for 2 weeks, and weighed to record
the weight of the dry thrombus. The statistical analysis
of the data was carried out by use of ANOVA test,
p < 0.05 is considered significant.
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Male Wistar rats weighing 200–300 g (purchased from
Animal Center of Peking University) were anesthetized
with pentobarbital sodium (80.0 mg/kg, ip). The right
carotid artery and left vein jugular of the animals were
separated. To the glass tube filled with artery blood
(1.0 ml) from the right carotid artery of the animal a
stainless steel filament helix (15 circles; L, 15 mm; D,
1.0 mm) was put immediately. After 15 min the helix
with thrombus was carefully taken out and weighted ex-
actly, which was put into the middle polyethylene tube.
The polyethylene tube was full with heparin sodium
(50 IU/ml of NS) and one end was inserted into the left
jugular vein. Heparin sodium was injected via the other
end of the polyethylene tube as the anti-coagulant, fol-
lowing which the tested compound was injected. The
blood was circulated through the polyethylene tube for
90 min, after which the helix was taken out and
weighted accurately. The reduction of thrombus mass
was recorded. The statistical analysis of the data was