Synthesis and biological activity of nitronyl nitroxide containing peptides

Article abstract of DOI:10.1021/jm050050k

[1-(1′,3′-Dioxyl-4′,4′,5′, 5′-tetramethyldihydroimidazol-2-yl)-phenyl-4-yl]oxyacetic acid (4), a nitronyl nitroxide, and its peptide derivatives, N-[1-(1′,3′-dioxyl- 4′,4′,5′,5′-tetramethyldihydroimidazol-2-yl) -phenyl-4-yl]oxyacetyl-ARPAK (9a), -GRPAK (9b), and -QRPAK (9c), were synthesized and characterized. Judging from the results of electron spin resonance analysis, the newly synthesized nitronyl nitroxide containing peptides, 9a, 9b, and 9c, demonstrated the characteristics of free radicals. The free radical scavenging activities of 9a, 9b, and 9c were assessed using in vitro free radical scavenging tests. The thrombolysis effect of 9a, 9b, and 9c was evaluated using an euglobulin clot lysis test, a fibrinolytic lysis test, and in vivo thrombolysis tests. Results indicated that these nitronyl nitroxide containing peptides possessed both free radical scavenging activity and thrombolytic activity.

Full text of DOI:10.1021/jm050050k

J. Med. Chem. 2005, 48, 4285-4292
4285
Synthesis and Biological Activity of Nitronyl Nitroxide Containing Peptides
Ming Zhao,‡ Junling Liu,† Chao Wang,‡ Lili Wang,*,§ Hu Liu,§ and Shiqi Peng*,†
College of Pharmaceutical Sciences, Capital University of Medical Sciences, Beijing 100054, PR China,
College of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China, and School of Pharmacy,
Memorial University of Newfoundland, St. John’s, NL A1B 3V6, Canada
Received January 18, 2005
[1-(1′,3′-Dioxyl-4′,4′,5′,5′-tetramethyldihydroimidazol-2-yl)-phenyl-4-yl]oxyacetic acid (4), a nit-
ronyl nitroxide, and its peptide derivatives, N-[1-(1′,3′-dioxyl-4′,4′,5′,5′-tetramethyldihydroimi-
dazol-2-yl)-phenyl-4-yl]oxyacetyl-ARPAK (9a), -GRPAK (9b), and -QRPAK (9c), were synthe-
sized and characterized. Judging from the results of electron spin resonance analysis, the newly
synthesized nitronyl nitroxide containing peptides, 9a, 9b, and 9c, demonstrated the
characteristics of free radicals. The free radical scavenging activities of 9a, 9b, and 9c were
assessed using in vitro free radical scavenging tests. The thrombolysis effect of 9a, 9b, and 9c
was evaluated using an euglobulin clot lysis test, a fibrinolytic lysis test, and in vivo
thrombolysis tests. Results indicated that these nitronyl nitroxide containing peptides possessed
both free radical scavenging activity and thrombolytic activity.
Introduction
brinogen â chain and identified as the active fraction
responsible for increased microvascular permeability in
rat and human skin.16,17 We have since searched for
other potential peptides of low molecular weight pos-
sessing the same biological effect, thrombolytic activity.
Gly-Arg-Pro-Ala-Lys (GRPAK) and Gln-Arg-Pro-Ala-Lys
(QRPAK) have been reported by our research group.18-22
Considering the NO scavenging activity exhibited by
nitronyl nitroxides and the thrombolytic activity dem-
onstrated by ARPAK, GRPAK, and QRPAK, the present
study attempted to link a nitronyl nitroxide, [1-(1′,3′-
dioxyl-4′,4′,5′,5′-tetramethyldihydroimidazol-2-yl)-phen-
yl-4-yl]oxyacetic acid (4), to the respective thrombolytic
oligopeptides hoping that the resulting nitronyl nitrox-
ide containing peptides would exhibit both NO scaveng-
ing activity and thrombolytic activity and thus be
beneficial for the prevention of ischemia/reperfusion
brain injuries.
A number of previous studies suggested that the
impact of nitric oxide (NO) on an ischemic brain injury
was dependent on the stage of tissue damage.1,2 In the
early stages following cerebral ischemia (<2 h), NO was
found to be beneficial by promoting collateral circulation
and microvascular flow. However, a glutamate-NO
pathway may occur in ischemic penumbra, the risky
region for infarction, by which large amounts of NO are
produced by neural nitric oxide synthase (nNOS) and
might contribute to the metabolic deterioration of the
penumbra leading to a larger infarction.1 At later times
(>6 h), the expression of inducible nitric oxide synthase
(iNOS) is triggered and the large amounts of NO thus
produced by this enzyme accelerates the progression of
the tissue damage.2 Considerable evidence suggests that
NO is also involved in ischemia/reperfusion brain
injury.3 Therapeutic strategies have attempted to reduce
the damage either by intervening in the formation
process of NO or by scavenging NO already formed.
Nitronyl nitroxides, which were first synthesized more
than 30 years ago,4 have recently received considerable
attention because of their capability to trap NO.5,6
Among other biological activities, nitronyl nitroxides
were found to be effective against various viral infec-
tions and endotoxin shock.7-13 In addition, nitronyl
nitroxides were found to react with free radicals such
Results
Synthesis of [1-(1′,3′-Dioxyl-4′,4′-5′,5′-tetrameth-
yldihydroimidazol-2-yl)-phenyl-4-yl]- oxyacetic Acid
(4). The compound 2,3-bis-(hydroxylamino)- 2,3-di-
methylbutane was condensed with salicylaldehyde (c)
according to the synthetic route depicted in Scheme 1
to provide 1,3-dihydroxy-2-(4′-hydroxy)-phenyl-4,4,5,5-
tetramethylimidazolidine (1), and the chemical yield
was 51%. Oxidation of 1 by lead dioxide resulted in 2
with a yield of 52%. The reaction of 2 and BrCH2-
COOC2H5 provided [1-(1′,3′-dioxyl-4′,4′,5′,5′-tetrameth-
yldihydroimidazol- 2-yl)phenyl-4-yl]oxyacetic ethyl ester
(3), and the yield was 90%. In the presence of NaOH (2
mol/L), 3 was converted to its corresponding acid 4.
Synthesis of [(1′,3′-Dioxyl-4′,4′,5′,5′-tetramethyl-
dihydroimidazol-2-yl)-phenyl-4-yl]- oxyacetic Acid
Containing ARPAK, GRPAK, and QRPAK (9a, 9b,
and 9c). Protected peptide intermediates, 6a, 6b, and
6c, respectively, were prepared as shown in Scheme 2
and the chemical yields were found to be in the range
of 83-96%. Preparation of the intermediates started
as •OH, H2O2 , and O2 , protecting endothelial cells from
•
•
the attack of free radicals.14,15
In addition to NO, thrombogenesis and thrombolysis
are also known to play an important role in ischemic
brain injuries.1-4
A pentapeptide, Ala-Arg-Pro-Ala-Lys (ARPAK), was
isolated from products degraded by plasmin from fi-
* To whom correspondence should be addressed. Dr. Lili Wang: tel
(709)-777-7053; fax (709)-777-7044; e-mail lwang@mun.ca. Dr. Shiqi
Peng: tel86-10-62092482;fax86-10-62092482;e-mailsqpeng@mail.bjmu.edu.cn.
† College of Pharmaceutical Sciences, Capital University of Medical
Sciences.
‡ College of Pharmaceutical Sciences, Peking University.
§ School of Pharmacy, Memorial University of Newfoundland.
10.1021/jm050050k CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/03/2005

4286 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Zhao et al.
Scheme 1. Preparation of [1-(1′,3′-Dioxyl-4′,4′,5′,5′-
tetramethyldihydroimidazol-2-yl)-phenyl-4-yl]oxyacetic
Acid (4)a
again. It was found that the spectra changed to a seven-
line pattern, which suggests that the nitronyl nitroxide
had been converted into its corresponding imino nitrox-
ides. The ESR spectra of 9a in water and phosphate
buffer (pH 7.4), prior to and after NO gas was intro-
duced, are provided in Scheme 3 as an example.
Scavenging Activities Determined in PC12 Cells.
The free radical scavenging activities of 9a, 9b, and 9c
•
•
against NO, H2O2 , and OH were evaluated in PC12
cells and compared with that of 4 using a published
method23 with minor modifications. The results are
expressed as EC50 (µM) values. As shown in Table 1,
the EC50 values of 9a, 9b, and 9c were found to be
similar to that of 4, suggesting that they were effective
•
•
scavengers of NO, H2O2 , and OH as 4.
NO Scavenging Activity Determined Using Rat
Aortic Strip. The NO scavenging activity of 4, 7a-c,
and 9a-c was also determined using rat aortic strip
according to a published method.25 The results ex-
pressed as the percentage inhibition of acetylcholine
(ACh)-induced vasorelaxation by test compounds are
summarized in Table 2. The results indicated that 9a,
9b, and 9c significantly inhibited ACh-induced vaso-
relaxation as 4, while 7a, 7b, and 7c had little effect.
Determination of Euglobulin Clot Lysis Time
(ECLT). The ECLT of 9a-c was determined using
rabbit euglobulin clots prepared according to the litera-
ture26,27 and was compared with that of 7a-c. Normal
saline (NS) and 4 were tested as the controls. As shown
in Table 3, the results indicated that like 7a-c, 9a-c
significantly shortened the ECLT in comparison with
NS or 4.
Fibrinolytic Activity. The fibrinolytic activities of
9a-c were determined according to a published proce-
dure28 and expressed by lysis area. The results were
compared with those of 7a-c and urokinase (UK). NS
and 4 were used as the controls. Data provided in Table
4 indicated that like UK and 7a-c, 9a-c significantly
enlarged fibrinolytic lysis areas in comparison with NS
and 4.
Thrombolytic Activity. The thrombolytic activity
of 9a-c determined according to literature19 was ex-
pressed as the reduction of thrombus mass and com-
pared with that of 7a-c. The results are summarized
in Table 5. It was found that like 7a-c, 9a-c signifi-
cantly reduced thrombus mass and the effect is dose
dependent.
a Reagents: (a) Br2, NaOH (6 mol/L); (b) Zn, NH4Cl; (c) HO-
C6H4CHO; (d) PbO2; (e) BrCH2COOC2H5, NaOEt, THF; (f) NaOH
(2 mol/L).
from the C-terminal, and L-Lys(Z)OBzl was the starting
material. In the presence of hydrogen fluoride (HF),
compounds 6a, 6b, and 6c were converted to 7a, 7b,
and 7c, and chemical yields were 90%, 94%, and 94%,
respectively. After removal of the Boc protecting group,
6a, 6b, and 6c were coupled with compound 4 to give
compounds 8a, 8b, and 8c, and chemical yields were
92%, 90%, and 90%, respectively. The protective group
on the side chain of the amino acid residue was removed
with anhydrous HF. The resulting products were ini-
tially colorless. However, if left in air, they may turn
into blue after a couple of hours. After purification by
chromatography using Sephadex G-10, [N-(1′,3′-dioxyl-
4′,4′,5′,5′- tetramethyldihydroimidazol-2-yl)-phenyl-4-yl]-
oxyacyl-ARPAK (9a), -GRPAK (9b), and -QRPAK (9c)
were obtained and chemical yields were 84%,75%, and
80%, respectively.
Electron Spin Resonance (ESR) Spectra of 4,
8a-c, and 9a-c and Their Reactivity with NO. The
ESR spectra of 4, 8a-c, and 9a-c were obtained at two
different concentrations, 10-7 and 10-5 mol/L, in water
and phosphate buffer (pH 7.4), respectively. They all
exhibited free radical characteristics with spectra show-
ing a five-line pattern at a 1:2:3:2:1 ratio, and there was
no difference observed among 4, 8a-c, and 9a-c. The
characteristic spectra resulted from the interaction of
a free electron with the two nitrogens in the nitronyl
nitroxides. The preparations of 4 and 9a-c were then
exposed to NO gas, and ESR spectra were recorded
Scheme 2. Preparation of 7a-c, 8a-c, and 9a-ca
a AA ) Ala in 7a, 8a, and 9a; AA ) Gly in 7b, 8b, and 9b; AA ) Gln in 7c, 8c, and 9c.

Nitronyl Nitroxide Containing Peptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4287
Table 4. Determination of Lysis Areas
Scheme 3. The ESR Spectra of 9a at 10-5 mol/L in
Water (a) and in Phosphate Buffer (pH 7.4) (b), Prior to
(Panel A) and After (Panel B) the Introduction of NO
compd
dosage (µg)
Xh ( SD (mm2)
NS
UK
4
24.05 ( 9.98
5 IU
0.8
1.4
1.3
1.5
0.8
0.4
0.2
0.7
0.4
0.2
0.8
0.4
0.2
224.05 ( 10.17a
23.96 ( 9.33
7a
7b
7c
9a
9a
9a
9b
9b
9b
9c
9c
9c
212.78 ( 10.54a
230.54 ( 9.95a
223.99 ( 9.86a
215.23 ( 9.65a,b
194.54 ( 8.98a,c
172.33 ( 10.22a
217.35 ( 9.57a,b
196.87 ( 10.34a,c
175.04 ( 10.11a
230.15 ( 9.68a,b
215.12 ( 8.99a,c
195.38 ( 10.14a
aIn comparison with NS and 4, p < 0.001, n ) 6. b In comparison
with 0.4 and 0.2 µg of the same compound, p < 0.05. c In
comparison with 0.2 µg of the same compound, p < 0.05.
Table 5. Reduction of Thrombus Mass
Table 1. The Free Radical Scavenging Activities of 4 and
9a-c
compd
dosage
Xh ( SD (mg)
EC50 (µM) values ((SD)a
NS
UK
4
(3 mL/kg)
15.11 ( 3.70
25.40 ( 2.50a
15.89 ( 2.53
18.54 ( 3.28c
25.82 ( 2.15a
21.17 ( 3.06b
26.88 ( 1.99a,f
19.91 ( 3.59c,g
16.27 ( 1.98
26.64 ( 1.85a,f
19.70 ( 3.76c,g
16.34 ( 1.96
28.85 ( 3.80a,d,e,g
20.43 ( 1.84b,h
16.40 ( 3.40
20 000 IU/kg
10.0 (µmol/kg)
10.0 (µmol/kg)
10.0 (µmol/kg)
10.0 (µmol/kg)
15.0 (µmol/kg)
10.0 (µmol/kg)
5.0 (µmol/kg)
15.0 (µmol/kg)
10.0 (µmol/kg)
5.0 (µmol/kg)
10 (µmol/kg)
5 (µmol/kg)
•
compd
NO
H2O2
•OH
4
91.20 ( 2.23
89.09 ( 2.02
92.34 ( 2.11
91.76 ( 2.00
30.18 ( 2.12
28.05 ( 1.99
25.39 ( 2.04
74.62 ( 1.97
99.87 ( 2.35
98.08 ( 2.41
95.38 ( 1.86
94.17 ( 2.53
7a
7b
7c
9a
9a
9a
9b
9b
9b
9c
9c
9c
9a
9b
9c
a SD ) standard deviation, n ) 6.
Table 2. Inhibition of ACh-Induced Vasorelaxation
compd
% Xh ( SD
compd
% Xh ( SD
NS
1.6 ( 1.4
99.2 ( 1.4a
9.5 ( 7.2
9.2 ( 8.5
8.9 ( 8.3
9a (10-8 mol/L) 66.8 ( 2.7a
9b (10-6 mol/L) 96.0 ( 1.4a,b
9b (10-7 mol/L) 80.9 ( 1.9a,c
9b (10-8 mol/L) 69.3 ( 2.2a
9c (10-6 mol/L) 95.6 ( 0.8a,b
9c (10-7 mol/L) 80.1 ( 1.9a,c
9c (10-8 mol/L) 68.3 ( 2.1a
4 (10-6 mol/L)
7a (10-6 mol/L)
7b (10-6 mol/L)
7c (10-6 mol/L)
1 (µmol/kg)
a In comparison with NS, p < 0.001, n ) 10. b In comparison
with NS, p < 0.01. c In comparison with NS, p < 0.05. d In
comparison with 7a and 7c, p < 0.001. e In comparison with UK
and 7b, p < 0.05. f In comparison with 10 and 5 µmol/kg of the
same compound, p < 0.001. g In comparison with 5 and 1 µmol/kg
of 9c, p < 0.001. h In comparison with 1 µmol/kg of 9c, p < 0.001.
9a (10-6 mol/L) 92.9 ( 3.2a,b
9a (10-7 mol/L) 78.6 ( 3.0a,c
a In comparison with normal saline (NS), p < 0.001, n ) 6. b In
comparison with 10-7 and 10-8 mol/L of the same compound, p <
0.001. c In comparison with 10-8 mol/L of the same compound, p
< 0.001.
(7b), and QRPAK (7c), resulting in 9a, 9b, and 9c,
respectively. The ESR spectrometry revealed the exist-
ence of unpaired electrons, suggesting that 9a, 9b, and
9c were of the same free radical characteristics as 4. In
addition, it was shown that they are reactive with NO
as demonstrated by the ESR results (Scheme 3). It is
important to note that 9a, 9b, and 9c retained the free
Table 3. Euglobulin Clot Lysis Time
compd
dosage (µg)
Xh ( SD (min)
NS
UK
4
204.03 ( 14.55
125.65 ( 16.76a
218.56 ( 15.34
96.38 ( 14.84a,b
91.09 ( 14.98a,b
93.01 ( 13.97a,b
104.12 ( 14.22a-c
123.32 ( 12.25a
145.61 ( 11.98a,d
105.01 ( 14.40a-c
126.51 ( 11.87a,d
147.22 ( 12.37a
100.09 ( 14.35a-c
117.96 ( 13.01a,d
136.00 ( 12.44a
5 IU
0.8
1.4
1.3
1.5
2.2
1.1
0.6
2.1
1.1
0.6
2.3
1.1
0.6
7a
7b
7c
9a
9a
9a
9b
9b
9b
9c
9c
9c
•
radical scavenging activity of 4 against NO, H2O2 , and
•OH and the thrombolytic activity of 7a, 7b, and 7c.
Compounds 9a-c demonstrated similar free radical
scavenging activity as 4 as demonstrated by the EC50
values. The euglobulin lysis test, fibrinolytic lysis test,
ACh-induced vasorelaxation test, and thrombolysis tests
indicated that 9a, 9b, and 9c also possess thrombolytic
activity similar to that of 7a, 7b, and 7c.
The results showed that the coupling of a nitronyl
nitroxide of free radical scavenging property with a
peptide of thrombolytic activity provided a means of
synthesizing compounds with dual properties, free radi-
cal scavenging property and thrombolytic activity.
a In comparison with NS and 4, p < 0.001, n ) 6. b In
comparison with urokinase (UK), p < 0.05. c In comparison with
1.1 and 0.6 µg of the same compound, p < 0.05. d In comparison
with 0.6 µg of the same compound, p < 0.05.
Discussion
It is known that both NO and thrombogenesis are
important risk factors in ischemic brain injuries. The
compounds synthesized in this study may be beneficial
for the prevention of ischemia/reperfusion brain injuries.
[(1′,3′-Dioxyl-4′,4′,5′,5′-tetramethyldihydroimidazol-2-
yl)-phenyl-4-yl]oxyacetic acid (4) was successfully in-
troduced into the N-terminal of ARPAK (7a), GRPAK

4288 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Zhao et al.
slowly added which took about 3 h. The reaction mixture was
stirred at room temperature for another 3 h and TLC (ethyl
acetate/petroleum, 2:1) indicated the complete disappearance
of 2,3-dimethyl-2,3-dinitrobutane. The reaction mixture was
filtrated under reduced pressure, and the collected solids were
washed with aqueous ethanol solution repeatedly. The com-
bined filtrate was adjusted to pH 2 with concentrated hydro-
chloric acid and was subjected to evaporation under reduced
pressure. The residue was mixed with 5 g of potassium
carbonate and extracted with chloroform. The extracts were
evaporated under reduced pressure to give 2.60 g (44%) of 2,3-
bis(hydroxylamino)-2,3-dimethylbutane which was a colorless
powder with a mp of 157-159 °C.
1,3-Dihydroxy-2-(4′-hydroxylphenyl)-4,4,5,5-tetra-
methylimidazolidine (1). The solution of 296 mg (2 mmol)
of 2,3-bis(hydroxylamino)-2,3-dimethylbutane and 244 mg (2
mmol) of 4-hydroxylbenzaldehyde in 3 mL of methanol was
stirred at room temperature for 8 h, and TLC (CHCl3/CH3-
OH, 6:1) indicated the complete disapperance of 4-hydroxyl-
benzaldehyde. The precipitate was collected, and the filtrate
was evaporated under vaccum to dryness. The residue and the
precipitate were combined to give 257 mg (51%) of the product
which was directly used for the next reaction without purifica-
tion. Rf value: 0.67 (CHCl3/CH3OH, 6:1). MS (EI) m/z: 252
[M]+. IR (KBr): 3310, 1610, 1450, 830 cm-1. 1H NMR (DMSO-
d6): δ 1.03 (s, 6H), 1.05 (s, 6H), 4.39 (s, 1H), 6.70 (d, J ) 6.9
Hz, 2H), 7.23 (d, J ) 6.9 Hz, 2H), 7.63 (s, 1H), 7.85 (s, 2H).
Experimental Section
General. The protected peptides were of the L-configuration.
The purity of all of the products was checked by thin-layer
chromatography (TLC) (Merck silica gel plates of type 60 F254
,
0.25 mm layer thickness) and high-performance liquid chro-
matography (HPLC) (Waters, C18 column, 3.9 mm × 150 mm).
Melting points (mp) were measured using a XT5 hot stage
microscope (Beijing keyi electrooptic factory), which was not
calibrated. Infrared (IR) spectra were recorded with a Perkin-
Elmer 983 instrument. Fast atom bombardment mass spec-
trometry (FAB-MS) was obtained by a VG-ZAB-MS and an
HPES-5989x instrument. 1H NMR spectra were obtained using
a Varian INOVA-500 MHz spectrometer. Optical rotations
were determined at 20 °C on a Schmidt + Haensch Polartronic
D instrument. Amino acid analyses were performed using a
Hitachi 835-50 instrument. Statistical analysis was carried out
using an ANOVA test.
General Procedure for Coupling 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 dicyclohexylcarbodiimide
were added. The reaction mixture was stirred at 0 °C for 24
h. Precipitated dicyclohexyl urea was removed by filtration.
The filtrate was evaporated under reduced pressure, and the
residue was triturated with petroleum ether to provide the
corresponding ester. To the solution of the ester in 10 mL of
anhydrous THF, 0.20 mmol of C-terminal component and 0.26
mmol of N-methylmorpholine were added. The reaction mix-
ture was stirred at room temperature for 24 h. Upon evapora-
tion, the residue was dissolved in 50 mL of ethyl acetate. The
solution was washed with 5% sodium bicarbonate, followed
by 5% citric acid and saturated sodium chloride, and the
organic phase was separated and dried over anhydrous sodium
sulfate. After filtration and evaporation under reduced pres-
sure, purification by chromatography (CHCl3/CH3OH, 30:1)
provided the desired product.
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. 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 dryness. The resulting solid was
used for the coupling reaction directly without further puri-
fication.
General Procedure for Removal of Side Chain Pro-
tective Groups of the Peptides. The mixture of 0.2 mmol
of the protected intermediate of the peptide, 1 mL of phenyl
methyl ether, 1 mL of dimethyl sulfide, and 4 mL of HF were
stirred at 0 °C for 2 h. The reaction mixture was subjected to
evaporation. The residue was mixed with 1 mL of phenyl
methyl ether, 1 mL of dimethyl sulfide and 8 mL of HF and
stirred at 0 °C for 2 h. The reaction mixture was evaporated
in a vacuum and the residue was triturated with ether. For
the preparation of 7a-c, the triturated residue was directly
purified on Sephadex G-10 and HPLC to provide the desired
peptide. For the preparation of 9a-c, the triturated residue
was left in air for 2 h prior to purification on Sephadex G-10
and HPLC to provide the desired product.
2-(4′-Hydroxylphenyl)-4,4,5,5-tetramethylimidazoline-
3-oxide-1-oxyl (2). One hundred and twenty six milligrams
(0.5 mmol) of 1 and 500 mg of lead dioxide in 30 mL of
methanol were mixed and stirred at room temperature for 40
min, and TLC (CHCl3/CH3OH, 20:1) indicated the complete
disapperance of 1. The reaction mixture was filtrated, and the
filtrate was evaporated under vacuum to provide 65 mg (52%)
of 2 as dark blue crystals. mp: 134-135 °C. Rf value: 0.13
(CHCl3/CH3OH, 20:1). EI-MS (m/z): 249 [M]+. IR (KBr) 3250,
1500, 1490, 1355, 840 cm-1
.
[1-(1′,3′-dioxyl-4′,4′,5′,5′-tetramethyldihydroimidazol-
2-yl)-phenyl-4-yl]oxyacetic Acid (4). The mixture of 250 mg
(1.0 mmol) of 2, 0.32 mL of ethyl bromoacetate, and 100 mg of
sodium ethylate in 5 mL of anhydrous tetrahydrofuran was
stirred at 60 °C for 5 h, and TLC (CHCl3/CH3OH, 20:1)
indicated the complete disapperance of 2. The reaction mixture
was evaporated under vaccum, and the residue was purified
on a silica gel chromatograph (CHCl3 was the eluent, giving
300 mg (90%) of 3). To the solution of 33 mg (1.0 mmol) of 3
in 3 mL of methanol, 0.5 mL of NaOH aqueous solution (2
mol/L) was added, and the mixture was stirred at room
temperature for 30 min. TLC (CHCl3/CH3OH, 10:1) indicated
the complete disapperance of 3. The reaction mixture was
evaporated under vaccum, and the residure was diluted with
2 mL of saturated NaCl aqueous solution. The solution was
adjusted to pH 5-6 with HCl (2 mol/L) and extraced with
CHCl3 (3 × 3 mL). The organic phase was dried with
anhydrous MgSO4 and filtered. The filtrate was evaporated
in vaccum to give 30 mg (100%) of the product as blue acicular
crystals. mp: 155-157 °C. EI-MS: 307 [M]+. IR (KBr): 1760,
1605, 1490, 1450; 830, 1260 cm-1
.
Boc-Lys(Z)OBzl. To the solution of 73 mg (0.20 mmol) of
Boc-Lys(Z)-OH in 5 mL of anhydrous ethanol the solution of
33 mg (0.10 mmol) of Cs2CO3 in 2 mL of water was added.
The mixed solution was stirred at room temperature for 1 h
and then evaporated under reduced pressure to remove the
solvent. The residue was kept in a desiccator overnight. The
dried product was dissolved in 2 mL of anhydrous DMF. To
the solution, 34 mg (0.2 mmol) of benzyl bromide was added.
The reaction mixture was stirred at 50 °C for 16 h and then
filtered to remove the resulting precipitate of CsBr. The filtrate
was evaporated under reduced pressure to remove the solvent.
The residue was dissolved in 30 mL of ethyl acetate and
washed successively with 5% sodium bicarbonate, 5% citric
acid, and saturated sodium chloride, and the organic phase
was separated and dried over anhydrous sodium sulfate. After
2,3-Dimethyl-2,3-dinitrobutane. At -5 °C to the solution
of 34.5 g (0.39 mol) of 2-nitropropane and 15.6 g (0.39 mol) of
NaOH in 65 mL of water, 10 mL (0.19 mol) of Br2 were added
dropwise, which took about 1 h. Ethanol (128 mL) was added
while stirring. The reaction mixture was stirred at 84 °C for
3 h and TLC (ethyl acetate/petroleum, 2:1) indicated the
complete disappearance of 2-nitropropane. The reaction mix-
ture was poured into 400 mL of icy water. Twenty five grams
of 2,3-dimethyl-2,3-dinitrobutane (mp 110-112 °C) was col-
lected by filtration as colorless crystals, and the chemical yield
was 73%.
2,3-Bis(hydroxylamino)-2,3-dimethylbutane. The sus-
pension of 7.0 g (40 mmol) of 2,3-dimethyl-2,3-dinitrobutane,
4.0 g of NH4Cl, 40 mL of ethanol, and 40 mL of water was
stirred at 0 °C. To the suspension, 16.0 g of zinc powder were

Nitronyl Nitroxide Containing Peptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4289
filtration and evaporation under reduced pressure, 93 mg
J ) 6.78 Hz, J ) 4.65 Hz, 2H), 2.382 (s, 3H), 2.254 (t, J )
5.64 Hz, 2H), 2.182 (t, J ) 6.78 Hz, 1H), 2.15 (t, J ) 6.76 Hz,
1H), 1.975 (m, J ) 5.66 Hz, 2H), 1.922 (dt, J ) 6.69 Hz, J )
4.88 Hz, 2H), 1.792 (m, J ) 4.72 Hz, 2H), 1.612 (m, J ) 4.72
Hz, 2H), 1.553 (m, J ) 4.69 Hz, 2H), 1.511 (d, J ) 6.69 Hz,
3H), 1.445 (9H), 1.301 (m, J ) 4.72 Hz, 2H).
HCl‚Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. A solution of 195 mg
(0.20 mmol) of Boc-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl prepared
according to literature10 in 2 mL of hydrochloride in ethyl
acetate (6 mol/L) was stirred at room temperature for 3 h. The
mixture was subjected to evaporation to remove the solvent.
The residue was dissolved in 10 mL of ethyl acetate, and the
solution was evaporated to dryness. The resulting solid was
used directly for the following coupling reaction.
(99%) of the Boc-Lys(Z)OBzl were obtained.
HCl‚Lys(Z)-OBzl. By use of the general procedure de-
scribed above for the removal of the Boc of the C-terminal
component, 91 mg (0.2 mmol) of Boc-Lys(Z)-OBzl was con-
verted into HCl‚Lys(Z)-OBzl. The resulting solid was used for
the coupling reaction directly.
Boc-Ala-Lys(Z)-OBzl. By use of the general procedure
described above for the coupling of C-terminal and N-terminal
components from 38 mg (0.2 mmol) of Boc-Ala-OH and 81 mg
(0.2 mmol) of HCl‚Lys(Z)-OBzl, 98 mg of the title compound
was obtained as a colorless powder (90% yield). mp: 88-90
°C. FAB-MS (m/e): 542 [M + H]+. [R]20D -6.6 (c ) 0.2, CHCl3).
IR (KBr): 3366, 3030, 3015, 1765, 1690, 1610, 1590, 1506,
1
1460, 1395, 1385, 1366, 760, 710 cm-1. H NMR (CDCl3): δ
Boc-Ala-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. By use of the
general procedure for the coupling C-terminal and N-terminal
components, Boc-Ala-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl was ob-
tained as a colorless powder. Boc-Ala-OH and HCl‚Arg(Tos)-
Pro-Ala-Lys(Z)-OBzl were obtained in 91% yield. mp: 98-101
8.123 (d, J ) 6.69 Hz, 1H), 8.102 (d, J ) 6.70 Hz, 1H), 8.089
(t, J ) 4.59 Hz, 1H), 7.365 (d, J ) 7.86 Hz, 2H), 7.360 (d, J )
7.79 Hz, 2H), 7.310 (t, J ) 7.86 Hz, 1H), 7.295 (t, J ) 7.79 Hz,
1H), 7.208 (t, J ) 7.86 Hz, 2H), 7.201 (t, J ) 7.79 Hz, 2H),
4.315 (m, J ) 6.69 Hz, 1H), 5.332 (s, 2H), 5.315 (s, 2H), 4.211
(dt, J ) 6.70 Hz, J ) 4.64 Hz, 1H), 2.950 (dt, J ) 4.72 Hz, J
) 4.59 Hz, 2H), 1.952 (dt, J ) 4.77 Hz, J ) 4.70 Hz, 2H), 1.529
(m, J ) 4.74 Hz, 2H), 1.318 (m, J ) 4.69 Hz, 2H), 1.469 (s,
3H), 1.450 (s, 9H).
°C. FAB-MS (m/e) 1020 [M + H]+. [R]20 ) -8.8° (c ) 0.2,
D
CHCl3). IR (KBr): 3363, 3351, 3339, 3031, 3012, 1754, 1685,
1
1608, 1592, 1507, 1462, 1408, 1385, 762, 707 cm-1. H NMR
(DMSO-d6): δ 9.362 (s, 1H), 8.318 (d, J ) 6.70 Hz, 1H), 8.286
(d, J ) 6.69 Hz, 1H), 8.130 (d, J ) 6.68 Hz, 1H), 8.123 (d, J )
6.67 Hz, 1H), 8.012 (t, J ) 5.89 Hz, 1H), 7.861 (d, J ) 7.75
Hz, 2H), 7.391 (d, J ) 7.77 Hz, 2H), 7.332 (t, J ) 7.56 Hz,
1H), 7.321 (t, J ) 7.54 Hz, 1H), 7.228 (d, J ) 7.64 Hz, 2H),
7.218 (d, J ) 7.61 Hz, 2H), 7.211 (t, J ) 7.49 Hz, 2H), 7.199
(t, J ) 7.64 Hz, 2H), 5.345 (s, 2H), 5.335 (s, 2H), 4.748 (dt, J
) 6.70 Hz, J ) 4.83 Hz, 1H), 4.694 (dt, J ) 6.74 Hz, J ) 4.67
Hz, 1H), 4.664 (m, J ) 6.68 Hz, 1H), 4.672 (m, J ) 6.65 Hz,
1H), 4.430 (t, J ) 5.65 Hz, 1H), 3.461 (t, J ) 5.57 Hz, 2H),
2.957 (dt, J ) 5.96 Hz, J ) 4.87 Hz, 2H), 2.661 (dt, J ) 6.76
Hz, J ) 4.67 Hz, 2H), 2.390 (s, 3H), 2.262 (t, J ) 5.62 Hz,
2H), 2.203 (t, J ) 6.78 Hz, 1H), 2.157(t, J ) 6.74 Hz, 1H),
1.977 (m, J ) 5.68 Hz, 2H), 1.924 (dt, J ) 6.66 Hz, J ) 4.85
Hz, 2H), 1.794(m, J ) 4.70 Hz, 2H), 1.614 (m, J ) 4.70 Hz,
2H), 1.551 (m, J ) 4.71 Hz, 2H), 1.508 (d, J ) 6.67 Hz, 3H),
1.489 (d, J ) 6.67 Hz, 3H), 1.447 (s, 9H), 1.312 (m, J ) 4.70
Hz, 2H).
HCl‚Ala-Lys(Z)-OBzl. By use of the general procedure
described above for the removal of the Boc of the C-terminal
component, 108 mg (0.2 mmol) of Boc-Ala-Lys(Z)-OBzl was
converted into HCl‚Ala-Lys(Z)-OBzl, which was used directly
for the coupling reaction.
Boc-Pro-Ala-Lys(Z)-OBzl. By use of the general procedure
described above for the coupling of C-terminal and N-terminal
components, 43 mg (0.2 mmol) of Boc-Pro-OH and 96 mg (0.2
mmol) of HCl‚Ala-Lys(Z)-OBzl resulted in 115 mg of Boc-Pro-
Ala-Lys(Z)-OBzl as a colorless powder and the chemical yield
was 90%. mp: 85-87 °C. FAB-MS (m/e): 639 [M + H]+. [R]20
D
-8.6 (c ) 0.2, CHCl3). IR (KBr): 3366, 3350, 3026, 3005, 1760,
1696, 1604, 1582, 1500, 1455, 1392, 1384, 1363, 769, 702 cm-1
.
1H NMR (CDCl3): δ 8.125 (d, J ) 6.67 Hz, 1H), 8.106 (d, J )
6.71 Hz, 1H), 8.086 (t, J ) 4.62 Hz, 1H), 7.367 (d, J ) 7.88
Hz, 2H), 7.362 (d, J ) 7.76 Hz, 2H), 7.312 (t, J ) 7.87 Hz,
1H), 7.294 (t, J ) 7.78 Hz, 1H), 7.215 (t, J ) 7.84 Hz, 2H),
7.210 (t, J ) 7.76 Hz, 2H), 4.321 (m, J ) 6.67 Hz, 1H), 5.334
(s, 2H), 5.321 (s, 2H), 4.221 (dt, J ) 6.72 Hz, J ) 4.66 Hz,
1H), 3.442 (t, J ) 4.78 Hz, 2H), 2.954 (dt, J ) 4.75 Hz, J )
4.58 Hz, 2H), 2.228 (t, J ) 4.69 Hz, 2H), 1.974 (m, J ) 4.77
Hz, 2H), 1.953 (dt, J ) 4.79 Hz, J ) 4.72 Hz, 2H), 1.531 (m, J
) 4.76 Hz, 2H), 1.321 (m, J ) 4.71 Hz, 2H), 1.471 (s, 3H), 1.462
(s, 9H).
HCl‚Ala-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. By use of the
general procedure for the removal of the Boc of the C-terminal
component, 204 mg (0.2 mmol) of Boc-Ala-Arg(Tos)-Pro-Ala-
Lys(Z)-OBzl was converted to HCl‚Ala-Arg(Tos)-Pro-Ala-Lys-
(Z)-OBzl. The resulting solid was used for the coupling reaction
directly without further purification.
Ala-Arg-Pro-Ala-Lys-OH (7a). By use of the general
procedure for the removal of side chain protective groups of
the peptides from 204 mg (0.2 mmol) of Boc-Ala-Arg(Tos)-Pro-
Ala-Lys(Z)-OBzl, 98 mg (90%) of (6a) was obtained as colorless
HCl‚Pro-Ala-Lys(Z)-OBzl. By use of the general procedure
described above for the removal of the Boc of the C-terminal
component, 128 mg (0.2 mmol) of Boc-Pro-Ala-Lys(Z)-OBzl was
converted into HCl‚Pro-Ala-Lys(Z)-OBzl, which was used
directly for the following coupling reaction.
crystals. mp: 224-226 °C. FAB-MS (m/e): 542 [M + H]+. [R]20
D
) -40.4° (c ) 2, H2O). IR (KBr): 3434, 3368, 3245, 3083, 2965,
1
1667, 1550, 1386 cm-1. H NMR (DMSO-d6): δ 9.982 (s, 1H),
8.432 (s, 2H), 8.282(d, J ) 6.68 Hz, 1H), 8.224 (d, J ) 6.72
Hz, 1H), 8.011 (d, J ) 6.70 Hz, 1H), 7.587 (d, J ) 6.65 Hz,
1H), 7.160 (d, J ) 6.68 Hz, 1H), 4.266 (1H), 4.215 (dt, J )
6.69 Hz, J ) 4.84 Hz, 1H), 4.223 (dt, J ) 6.72 Hz, J ) 4.65
Hz, 1H), 4.022 (m, J ) 6.67 Hz, 1H), 3.671 (m, J ) 6.67 Hz,
1H), 3.535 (t, J ) 6.74 Hz, 2H), 3.349 (t, J ) 5.55 Hz, 2H),
3.076 (t, J ) 5.93 Hz, 2H), 2.732 (t, J ) 5.64 Hz, 2H), 2.249 (t,
J ) 6.75 Hz, 1H), 2.242 (m, J ) 5.66 Hz, 2H), 2.021 (dt, J )
6.64 Hz, J ) 4.83 Hz, 2H), 2.006 (m, J ) 4.72 Hz, 2H), 1.861
(m, J ) 4.72 Hz, 2H), 1.674 (m, J ) 4.73 Hz, 2H), 1.534 (m, J
) 4.73 Hz, 2H), 1.533 (m, J ) 6.64 Hz, 2H), 1.282 (d, J ) 6.65
Hz, 3H), 1.268 (d, J ) 6.64 Hz, 3H).
Boc-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. By use of the general
procedure described above for the removal of the Boc of the
C-terminal component, Boc-Pro-Ala-Lys(Z)-OBzl was con-
verted into HCl‚Pro-Ala-Lys(Z)-OBzl, which was then coupled
with Boc-Arg(Tos)-OH according to the general procedure for
the coupling of C-terminal and N-terminal components to
provide Boc-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl as a colorless pow-
der in 79% yield. mp: 76-78 °C. FAB-MS (m/e): 977 [M +
H]+. [R]20 ) -9.4° (c ) 0.2, CHCl3). IR (KBr): 3360, 3350,
D
3344, 3033, 3011, 1744, 1681, 1605, 1592, 1502, 1464, 1405,
1
1390, 1380, 762, 708 cm-1. H NMR (DMSO-d6): δ 9.356 (s,
1H), 8.230 (d, J ) 6.72 Hz, 1H), 8.126 (d, J ) 6.69 Hz, 1H),
8.120 (d, J ) 6.68 Hz, 1H), 8.009 (t, J ) 5.98 Hz, 1H), 7.855
(d, J ) 7.81 Hz, 2H), 7.385 (d, J ) 7.81 Hz, 2H), 7.325 (t, J )
7.54 Hz, 1H), 7.318 (t, J ) 7.49 Hz, 1H), 7.228 (d, J ) 7.62
Hz, 2H), 7.224 (d, J ) 7.63 Hz, 2H), 7.205 (t, J ) 7.55 Hz,
2H), 7.200 (t, J ) 7.61 Hz, 2H), 5.342 (s, 2H), 5.331 (s, 2H),
4.785 (d, J ) 6.76 Hz, 2H), 4.745 (dt, J ) 6.72 Hz, J ) 4.84
Hz, 1H), 4.691 (dt, J ) 6.72 Hz, J ) 4.68 Hz, 1H), 4.674 (m, J
) 6.69 Hz, 1H), 4.421 (t, J ) 5.64 Hz, 1H), 3.459 (t, J ) 5.561
Hz, 2H), 2.954 (dt, J ) 5.98 Hz, J ) 4.84 Hz, 2H), 2.657 (dt,
Boc-Gln-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. By use of the
general procedure for the coupling C-terminal and N-terminal
components from Boc-Gln-OH and HCl‚Arg(Tos)-Pro-Ala-Lys-
(Z)-OBzl, Boc-Gln-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl was obtained
as a colorless powder in 86% yield. mp: 90-92 °C. FAB-MS
(m/e): 1077 [M + H]+. [R]20 ) -8.9° (c ) 0.2, CHCl3). IR
D
(KBr): 3361, 3355, 3345, 3033, 3010, 1758, 1682, 1660, 1600,
1
1590, 1506, 1460, 1394, 1385, 1361, 760, 702 cm-1. H NMR
(DMSO-d6): δ 9.401 (s, 1H), 8.332(d, J ) 6.72 Hz, 1H), 8.314

4290 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Zhao et al.
(d, J ) 6.64 Hz, 1H), 8.138 (d, J ) 6.66 Hz, 1H), 8.126 (d, J )
6.65 Hz, 1H), 8.025 (t, J ) 5.92 Hz, 1H), 7.859 (d, J ) 7.73
Hz, 2H), 7.396 (d, J ) 7.75 Hz, 2H), 7.341 (t, J ) 7.60 Hz,
1H), 7.325 (t, J ) 7.60 Hz, 1H), 7.230 (d, J ) 7.60 Hz, 2H),
7.221 (d, J ) 7.60 Hz, 2H), 7.215 (t, J ) 7.50 Hz, 2H), 7.196
(t, J ) 7.62 Hz, 2H), 6.458 (t, J ) 6.69 Hz, 2H), 5.346 (s, 2H),
5.338 (s, 2H), 4.744 (dt, J ) 6.71 Hz, J ) 4.81 Hz, 1H), 4.692
(dt, J ) 6.72 Hz, J ) 4.66 Hz, 1H), 4.666 (m, J ) 6.66 Hz,
1H), 4.670 (m, J ) 6.67 Hz, 1H), 4.432 (t, J ) 5.66 Hz, 1H),
3.463 (t, J ) 5.60 Hz, 2H), 2.961 (dt, J ) 5.98 Hz, J ) 4.85
Hz, 2H), 2.664 (dt, J ) 6.74 Hz, J ) 4.70 Hz, 2H), 2.392 (s,
3H), 2.264 (t, J ) 5.64 Hz, 2H), 2.205 (t, J ) 6.76 Hz, 1H),
2.191 (t, J ) 6.69 Hz, 1H), 2.155 (t, J ) 6.72 Hz, 1H), 2.075 (t,
J ) 6.67 Hz, 1H), 1.978 (m, J ) 5.66 Hz, 2H), 1.926 (dt, J )
6.67 Hz, J ) 4.85 Hz, 2H), 1.930 (dt, J ) 6.65 Hz, J ) 4.83
Hz, 2H), 1.796 (m, J ) 4.74 Hz, 2H), 1.621 (m, J ) 4.72 Hz,
2H), 1.556 (m, J ) 4.70 Hz, 2H), 1.511 (d, J ) 6.66 Hz, 3H),
1.445 (s, 9H), 1.309 (m, J ) 4.72 Hz, 2H).
product obtained was used for the coupling reaction directly
without further purification.
Gly-Arg-Pro-Ala-Lys-OH (7b). By use of the general
procedure for the removal of side chain protective groups of
the peptides from 201 mg (0.2 mmol) of Boc-Gly-Arg(Tos)-Pro-
Ala-Lys(Z)-OBzl, 99 mg (94%) of the title compound was
obtained as colorless crystals. mp: 168-170 °C. FAB-MS (m/
e): 528 [M + H]+. [R]20 ) -20.0° (c ) 1.98, H2O). IR (KBr):
D
3440, 3373, 3256, 3071, 2946, 1667, 1558, 1384 cm-1. 1H NMR
(DMSO-d6): δ 9.994 (s, 1H), 8.563 (s, 2H), 8.104 (d, J ) 6.67
Hz, 1H), 7.985 (d, J ) 6.72 Hz, 1H), 7.973 (d, J ) 6.71 Hz,
1H), 7.627 (d, J ) 6.67 Hz, 1H), 7.077 (t, J ) 5.86 Hz, 1H),
4.625 (dt, J ) 6.70 Hz, J ) 4.83 Hz, 1H), 4.525 (dt, J ) 6.75
Hz, J ) 4.64 Hz, 1H), 4.256 (d, J ) 6.67 Hz, 2H), 4.228 (m, J
) 6.65 Hz, 1H), 4.106 (t, J ) 5.65 Hz, 1H), 3.217 (t, J ) 5.62
Hz, 2H), 3.385 (dt, J ) 5.96 Hz, J ) 4.82 Hz, 2H), 3.068 (t, J
) 5.48 Hz, 2H), 2.736 (dt, J ) 6.71 Hz, J ) 4.63 Hz, 2H), 2.534
(t, J ) 5.65 Hz, 2H), 2.531 (t, J ) 5.66 Hz, 2H), 2.046 (m, J )
5.67 Hz, 2H), 1.985 (dt, J ) 6.65 Hz, J ) 4.84 Hz, 2H), 1.865
(m, J ) 4.74 Hz, 2H), 1.675 (m, J ) 4.74 Hz, 2H), 1.531 (m, J
) 4.75 Hz, 2H), 1.532 (m, J ) 4.71 Hz, 2H), 1.243 (d, J ) 6.61
Hz, 3H).
HCl‚Gln-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. By use of the
general procedure for the removal of the Boc of the C-terminal
component, 216 mg (0.2 mmol) of Boc-Gln-Arg(Tos)-Pro-Ala-
Lys(Z)-OBzl was converted to HCl‚Gln-Arg(Tos)-Pro-Ala-Lys-
(Z)-OBzl. The resulting solid was used directly for the coupling
reaction without further purification.
N-[1-(1′,3′-Dioxyl-4′,4′,5′,5′-tetramethyldihydroimidazol-
2-yl)phenyl-4-yl]oxyacetyl-Ala-Arg(Tos)-Pro-Ala-Lys(Z)-
OBzl (8a). By use of the general procedure for coupling of
C-terminal and N-terminal components from 61 mg (0.2 mmol)
of 4 and 191 mg (0.2 mmol) of HCl‚Ala-Arg(Tos)-Pro-Ala-Lys-
(ClZ)-OBzl 222.3 mg (92%) of the title compound was obtained
as a colorless powder. mp: 92-94 °C. FAB-MS (m/e): 1209
Gln-Arg-Pro-Ala-Lys-OH (7c). By use of the general
procedure for the removal of the side chain protective groups
of the peptides, 215 mg (0.2 mmol) of Boc-Gln-Arg(Tos)-Pro-
Ala- Lys(Z)-OBzl was converted to 134 mg (95% of yield) of 6c
as colorless crystals. mp: 180-182 °C. FAB-MS(m/e): 599 [M
+ H]+. [R]20D ) -35.0° (c ) 2.01, H2O). IR (KBr): 3436, 3367,
3254, 3073, 2964, 1662, 1558, 1385 cm-1.1H NMR (DMSO-
d6): δ 9.989 (s, 1H), 8.446 (s, 2H), 8.283 (d, J ) 6.74 Hz, 1H),
8.214 (d, J ) 6.66 Hz, 1H), 8.157 (d, J ) 6.68 Hz, 1H), 7.575
(d, J ) 6.66 Hz, 1H), 7.164 (t, J ) 5.94 Hz, 1H), 6.401 (s, 2H),
4.283 (dt, J ) 6.73 Hz, J ) 4.84 Hz, 1H), 4.273 (dt, J ) 6.70
Hz, J ) 4.68 Hz, 1H), 4.218 (m, J ) 6.68 Hz, 1H), 4.112 (m, J
) 6.65 Hz, 1H), 4.241 (t, J ) 5.67 Hz, 1H), 3.541 (dt, J ) 5.96
Hz, J ) 4.87 Hz, 2H), 3.359 (t, J ) 5.62 Hz, 2H), 3.072 (t, J )
5.64 Hz, 2H), 2.742 (t, J ) 5.62 Hz, 2H), 2.393 (t, J ) 6.67 Hz,
2H), 2.291 (t, J ) 6.65 Hz, 2H), 2.233 (t, J ) 6.64 Hz, 2H),
2.191 (t, J ) 6.70 Hz, 2H), 2.031 (m, J ) 5.68 Hz, 2H),1.912
(dt, J ) 6.65 Hz, J ) 4.83 Hz, 2H), 1.765 (dt, J ) 6.67 Hz, J
) 4.81 Hz, 2H), 1.672 (m, J ) 4.76 Hz, 2H), 1.535 (m, J )
4.74 Hz, 2H), 1.527 (m, J ) 4.72 Hz, 2H), 1.267 (d, J ) 6.64
Hz, 3H).
[M + H]+. [R]20 ) 24.0 (c ) 2, CHCl3).
D
N-[1-(1′,3′-Dioxyl-4′,4′,5′,5′-tetramethyldihydroimidazol-
2-yl)phenyl-4-yl]oxyacetyl-Gly-Arg(Tos)-Pro-Ala-Lys(Z)-
OBzl (8b). By use of the general procedure for the coupling
of C-terminal and N-terminal components from 61 mg (0.2
mmol) of 4 and 188 mg (0.2 mmol) of HCl‚Gly-Arg(Tos)-Pro-
Ala-Lys(ClZ)OBzl, 219.7 mg (92%) of 8b was obtained as a
colorless powder. mp: 90-92 °C. FAB-M (m/e): 1195 [M +
H]+. [R]20 ) 13.0 (c ) 2, CHCl3).
D
N-[1-(1′,3′-Dioxyl-4′,4′,5′,5′-tetramethyldihydroimidazol-
2-yl)phenyl-4-yl]oxyacetyl-Gln-Arg(Tos)-Pro-Ala-Lys(Z)-
OBzl (8c). By use of the general procedure for the coupling of
C-terminal and N-terminal components from 61 mg (0.2 mmol)
of 4 and 203 mg (0.2 mmol) of HCl‚Gln-Arg(Tos)-Pro-Ala-Lys-
(ClZ)OBzl, 225.4 mg (89%) of compound 8c was obtained as a
colorless powder. mp: 96-98 °C. FAB-MS (m/e) 1267 [M +
Boc-Gly-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. By use of the
general procedure for the coupling C-terminal and N-terminal
components, Boc-Gly-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl was gener-
ated from Boc-Gly-OH and HCl‚Arg(Tos)-Pro-Ala-Lys(Z)-OBzl
as a colorless powder, and the chemical yield was 93%. mp:
H]+. [R]20 ) 29.0 (c ) 2, CHCl3).
D
N-[1-(1′,3′-Dioxyl-4′,4′,5′,5′-tetramethyldihydroimidazol-
2-yl)phenyl-4-yl]oxyacetyl-ARPAK (9a). By use of the
general procedure for the removal of side chain protective
groups, 136 mg (0.1 mmol) of 8a was converted to 70 mg of
compound 9a and was obtained as a blue powder. The chemical
93-95 °C. FAB-MS (m/e): 1006 [M + H]+. [R]20 ) -10.6° (c
D
) 0.2, CHCl3). IR (KBr): 3368, 3350, 3346, 3032, 3008, 1765,
yield was 84%. mp: 170 °C(dec). ESI-MS (m/e): 831 [M]+. [R]20
) 35.0 (c ) 1, H2O).
D
1688, 1602, 1561, 1501, 1462, 1401, 1387, 1361, 765, 701 cm-1
.
1H NMR (DMSO-d6): δ 9.355 (s, 1H), 8.320 (d, J ) 6.69 Hz,
1H), 8.279 (d, J ) 6.70 Hz, 1H), 8.141 (d, J ) 6.70 Hz, 1H),
8.128 (d, J ) 6.66 Hz, 1H), 8.015 (t, J ) 5.87 Hz, 1H), 7.864
(d, J ) 7.74 Hz, 2H), 7.394 (d, J ) 7.79 Hz, 2H), 7.335 (t, J )
7.58 Hz, 1H), 7.324 (t, J ) 7.52 Hz, 1H), 7.232 (d, J ) 7.65
Hz, 2H), 7.220 (d, J ) 7.58 Hz, 2H), 7.214 (t, J ) 7.47 Hz,
2H), 7.196 (t, J ) 7.66 Hz, 2H), 5.346 (s, 2H), 5.336 (s, 2H),
4.746 (dt, J ) 6.72 Hz, J ) 4.81 Hz, 1H), 4.692 (dt, J ) 6.76
Hz, J ) 4.65 Hz, 1H), 4.667 (d, J ) 6.69 Hz, 2H), 4.659 (m, J
) 6.67 Hz, 1H), 4.432 (t, J ) 5.67 Hz, 1H), 3.458 (t, J ) 5.60
Hz, 2H), 2.956 (dt, J ) 5.94 Hz, J ) 4.85 Hz, 2H), 2.663 (dt,
J ) 6.74 Hz, J ) 4.65 Hz, 2H), 2.392 (s, 3H), 2.260 (t, J )
5.64 Hz, 2H), 2.205 (t, J ) 6.76 Hz, 1H), 2.155 (t, J ) 6.72 Hz,
1H), 1.974 (m, J ) 5.69 Hz, 2H), 1.921 (dt, J ) 6.67 Hz, J )
4.86 Hz, 2H), 1.793 (m, J ) 4.71 Hz, 2H), 1.616 (m, J ) 4.72
Hz, 2H), 1.554 (m, J ) 4.74 Hz, 2H), 1.487 (d, J ) 6.64 Hz,
3H), 1.445 (s, 9H), 1.321 (m, J ) 4.72 Hz, 2H).
N-[1-(1′,3′-Dioxyl-4′,4′,5′,5′-tetramethyldihydroimidazol-
2-yl)phenyl-4-yl]oxyacetyl-GRPAK (9b). By use of the
general procedure for the removal of side chain protective
groups and from 130 mg (0.1 mmol) of 8b, 60 mg of 9b was
obtained as a blue powder and the chemical yield was 75%.
mp: 174 °C(dec). ESI-MS (m/e): 817[M]+. [R]20D ) 21.0 (c ) 1,
H2O).
N-[1-(1′,3′-Dioxyl-4′,4′,5′,5′-tetramethyldihydroimidazol-
2-yl)phenyl-4-yl]oxyacetyl-QRPAK (9c). By use of the
general procedure for the coupling of C-terminal and N-
terminal components and from 136 mg (0.1 mmol) of 8c, 70
mg of 9c were obtained as a blue powder and the chemical
yield was 80%. mp: 178 °C(dec). ESI-MS (m/e): 889 [M + 2
H]+. [R]20 ) -27.0 (c ) 1, H2O).
D
Determination of ESR. A center field of 3480 G, sweep
width of 100 G, sweep time of 100 s, modulation amplitude of
1.0 × 10-1 G, time constant of 1.6 × 10-1 S, modulation
frequency of 100 kHz, microwave frequency of 9.72 GHz, and
microwave power of 10 MW were all used for the ESR
measurement. The ESR spectra of 4, 8a-c, and 9a-c at two
HCl‚Gly-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. By use of the
general procedure for the removal of the Boc of the C-terminal
component, 202 mg (0.2 mmol) of Boc-Gly-Arg(Tos)-Pro-Ala-
Lys(Z)-OBzl was converted into the title compound. The

Nitronyl Nitroxide Containing Peptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4291
different concentrations, 10-7 and 10-5 mol/L, and in deaerated
water and phosphate buffer (pH 7.4), respectively, were
recorded.
To study the reactivity of respective compounds with NO,
the deaerated water or phosphate buffer containing 4, 8a-c,
and 9a-c (10-5 mol/L) were bubbled with NO gas for 30 s,
following which the ESR spectra were then recorded.
ylene tube outside male Wistar rat according to the standard
procedure.19 After administration of NS, UK, 4, 7a-c, or 9a-
c, the blood was circulated through the polyethylene tube for
90 min and the helix was taken out and weighed accurately.
The reduction in thrombus mass was recorded. The results
are summarized in Table 5. The effect of three different
dosages of 9a-c was compared, and the results are also listed
in Table 5. Statistical analysis of the results was carried out
using an ANOVA test. The results indicated that 9a-c
significantly reduced thrombus mass, and the effect appeared
to be dose dependent.
Male Wistar rats weighing 200-300 g (purchased from the
Animal Center of Peking University) were anesthetized with
pentobarbital sodium (80.0 mg/kg, intraperitoneally). The right
carotid artery and left jugular vein of the animals were
separated. To the glass tube containing 1.0 mL of blood
obtained from the right carotid artery of the animal, a stainless
steel filament helix (15 circles; 15 mm × 1.0 mm) was added
immediately. Fifteen minutes later, the helix with thrombus
was carefully taken out and weighed. It was then put into a
polyethylene tube which was filled with heparin sodium (50
IU/mL of NS), and one end was inserted into the left jugular
vein. Heparin sodium was injected via the other end of the
polyethylene tube as the anticoagulant, following which the
test compound was injected. The blood was circulated through
the polyethylene tube for 90 min, after which the helix was
taken out and weighed. The reduction of the thrombolytic mass
was recorded.
Evaluation of 9a-c as Scavengers of NO, H2O2‚, and
‚OH in PC12 Cells. Free radical scavenging activities were
evaluated in PC12 cells using a method of Dawson23 with
minor modifications. In brief, PC12 cells were grown in
Dulbecco’s modified eagle’s medium supplemented with 10%
of heat inactivated horse serum (Hyclone), 5% of fetal bovine
serum (GIBCO), 1.0 mM sodium pyruvate, 100 U/mL penicil-
lin, and 100 µg/mL streptomycin at 37 °C in 5% CO2 atmo-
sphere. PC12 cells were seeded in 96-well plates coated with
poly-L-lysine at a density of 20 000 cells per well during the
exponential phase of growth. After 24 h, attachment period
fresh media containing 12.5 , 25 , 50 , 100 , or 200 µM of 4,
9a, 9b, or 9c, respectively, were added to each well and were
incubated for 1 h. NO damage was then induced by adding 2
mM of sodium nitroprusside followed by 2 h of incubation. The
media were replaced with fresh media and cells were incubated
for 14 h, following which cell survival was measured by a
colorimetric assay with MTT according to the method of
Mosmann.24 Similarly, H2O2 damage was induced by 1 mM
•
•
H2O2 followed by 1 h of incubation, while OH damage was
induced by 1 mM H2O2/30 µM Fe(II) followed by 1 h of
incubation.
Acknowledgment. The authors wish to thank the
Pre-exploration Program of the Key Basic Research
Project (2003CCA04400) of China, the National Natural
Scientific Foundation of China (30472071), and the
Scientific Foundation of Guangzhou for their financial
support.
NO Scavenging Activity Determined Using Rat Aortic
Strip. The NO scavenging activity of 4, 7a-c, and 9a-c in
the rat aortic strip was examined according to a published
method.25 In brief, immediately after decapitation rat aortic
strips were obtained 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 mounted to the tension
transducers and the relaxation-contraction curves were re-
corded. Noradrenaline (NE, final concentration 10-9 mol/L)
solution was added to induce contraction. When the hypertonic
contraction reached to the maximum level, NE was washed
and the vessel strips were stabilized for 30 min. After the
renewal of the solution, NE (final concentration 10-9 mol/L)
was added. When the hypertonic contraction value of aortic
strips reached the peak, 15 µL of NS, or the solution of 4, 7a-
c, and 9a-c in 15 µL of water (final concentration 10-6 mol/
L) were added, respectively. Upon stabilization, 1.5 µL of ACh
(final concentration 10-6 mol/L) were added and the percentage
inhibition of ACh-induced vasorelaxation by test compounds
was determined.
Determination of ECLT. The rabbit euglobulin clots were
prepared according to a published method.26,27 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
centrifuged at 2000g for 15 min and the precipitate was
resuspended to the initial plasma volume with 50 mM sodium
barbiturate buffer (pH 7.8, containing 1.66 mM CaCl2, 0.68
mM MgCl2, and 93.96 mM NaCl). To the rabbit euglobulin
clots, NS, UK, 4, 7a-c, or 9a-c was added, and the ECLT or
time to clot lysis was determined in a 96-well microtiter plate.
Determination of Fibrinolytic Activity. The fibrinogen-
agarose mixture was prepared and coagulated with thrombin
in plastic dishes according to a published procedure.28 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 x
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 µL of NS, UK, 4, 7a-c, or 9a-c was
added to each well. The plate was incubated and areas of lysis
were quantified by the lysis area.
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