Novel 2-substituted nitronyl nitroxides as free radical scavengers: Synthesis, biological evaluation and structure-activity relationship

Article abstract of DOI:10.1016/j.bmc.2006.04.016

To develop more potent small molecules with enhanced free radical scavenger properties, we designed and synthesized a series of nitronyl nitroxide derivatives 4a-h. A lead compound 4f was discovered based on Ach-induced vascorelaxation assay. Further chemical modification based on this scaffold provided a new series of 2-substituted phenylnitronyl nitroxide derivatives 6a-s. The newly synthesized compounds 6a-s possess improved radical scavenger's activity based on PC12 cell survival assay. Compounds 6g,n,o, and s are some of the most potent compounds in terms of NO, H₂O₂, and OH scavenging ability. 2-Substitued phenylnitronyl nitroxides had a higher radical scavenging activity with the electron-donating group (EDG). In contrast, the introduction of electron-withdrawing group (EWG) to the aromatic ring led to a dramatic decrease in its radical scavenging activity. These results suggest that the electron-donating group (EDG) of the aromatic ring may be an important factor influencing the radical scavenging behavior of these compounds, and the potency of free radical scavenging activity largely depended on the position and electronic properties of the phenyl ring substituents. The enhanced radical scavenging capacities of the novel 2-substituted nitronyl nitroxides may be potential drug leads against the deleterious action of ROS (reactive oxygen species)/RNS (reactive nitrogen species).

Full text of DOI:10.1016/j.bmc.2006.04.016

Bioorganic & Medicinal Chemistry 14 (2006) 5711–5720
Novel 2-substituted nitronyl nitroxides as free radical
scavengers: Synthesis, biological evaluation
and structure–activity relationship
Yihui Wu,b Lanrong Bi,a Wei Bi,c Zeng Li,b Ming Zhao,b,* Chao Wang,b
Jingfang Jud,* and Shiqi Penga,*
aCollege of Pharmaceutical Sciences, Capital University of Medical Sciences, Beijing 100054, PR China
bCollege of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
cDepartment of Surgery, Second Affiliated Hospital of Hebei Medical University, Shijiazhuang 050000, PR China
dMitchell Cancer Institute, USA, 307 N. University Blvd, Mobile, AL 36688-0002, USA
Received 22 March 2006; revised 30 March 2006; accepted 6 April 2006
Available online 2 May 2006
Abstract—To develop more potent small molecules with enhanced free radical scavenger properties, we designed and synthesized
a series of nitronyl nitroxide derivatives 4a–h. A lead compound 4f was discovered based on Ach-induced vascorelaxation assay.
Further chemical modification based on this scaffold provided a new series of 2-substituted phenylnitronyl nitroxide derivatives
6a–s. The newly synthesized compounds 6a–s possess improved radical scavenger’s activity based on PC12 cell survival assay.
Compounds 6g,n,o, and s are some of the most potent compounds in terms of NO, H2O2, and OH scavenging ability. 2-Substit-
ued phenylnitronyl nitroxides had a higher radical scavenging activity with the electron-donating group (EDG). In contrast, the
introduction of electron-withdrawing group (EWG) to the aromatic ring led to a dramatic decrease in its radical scavenging activ-
ity. These results suggest that the electron-donating group (EDG) of the aromatic ring may be an important factor influencing the
radical scavenging behavior of these compounds, and the potency of free radical scavenging activity largely depended on the posi-
tion and electronic properties of the phenyl ring substituents. The enhanced radical scavenging capacities of the novel 2-substi-
tuted nitronyl nitroxides may be potential drug leads against the deleterious action of ROS (reactive oxygen species)/RNS
(reactive nitrogen species).
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
tis,7 asthma,8 diabetes,9 and degenerative eye disease.10
Therefore, it is important to find effective scavengers
of free radicals for prevention and treatment of such
disorders.
Free radicals play important roles in many physiological
and pathological conditions.1 In general, the generation
and scavenging of oxygen free radicals is balanced and
any imbalance or excessive amounts of active radicals
may contribute to disease development. It has been
found that free radical reactions can produce deleterious
modifications in membranes, proteins, enzymes, and
DNA,2 increasing the risk of diseases such as cancer,3
Alzheimer’s,4 Parkinson’s,5 angiocardiopathy,6 arthri-
Nitronyl nitroxides, synthesized more than 30 years
ago,11 have received considerable attention recently be-
cause of their capability to trap NO.12,13 More recently,
nitronyl nitroxides were found to react with free radicals
such as OH, H2O2, and O2, protecting endothelial cells
from the attack of free radicals.14,15
To develop small molecules with enhanced free radical
scavenger properties, a series of new nitronyl nitroxide
derivatives were designed and synthesized. In this study,
free radical scavenger properties of the newly synthe-
sized compounds were characterized to elucidate the
relationship among their activity, chemical structure,
and physicochemical parameters.
Keywords: Nitronyl nitroxide derivatives; Free radical scavenger;
Structure–activity relationship.
*
Corresponding authors. Tel.: +1 251 460 7393; fax: +1 251 460 6994
(J.J.); tel.: +86 10 8280 2482; fax: +86 10 8280 2482 (M.Z.); tel.: +86
10 8391 1528; fax: +86 10 8391 1528 (S.P.); e-mail addresses:
jju@usouthal.edu; sqpeng@mail.bjum.edu.cn
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.016

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Y. Wu et al. / Bioorg. Med. Chem. 14 (2006) 5711–5720
2. Results and discussion
2.2. NO scavenging activity determined using rat aortic
strip
2.1. Synthesis of 2-substituted nitronyl nitroxides
A variety of 2-substituted nitronyl nitroxides 4a–h were
prepared and their NO scavenging activities were evalu-
ated in the acetylcholine (Ach)-evoked, endothelium-
mediated relaxation assay.19
The nitronyl nitroxide derivatives were synthesized
according to Ullman’s procedure with minor modifica-
tion as shown in Scheme 1.16 Synthesis was initiated
with dinitro compound 1, followed by the reduction
with zinc in an ammonium chloride (Zn/NH4Cl) buf-
fered solution to yield the key intermediate bis(hydroxy-
amine) compound 2, which was subsequently subjected
to condensation with various aldehydes to generate the
corresponding tetramethylimidazoline derivatives 3a–h
and 5a–p with 51–85% yield, respectively. The final oxi-
dation by PdO2 gave the desired nitronyl nitroxide
derivatives 4a–h and 6a–p with 38–90% yield, respective-
ly. While the synthesis of 6s and q was slightly different
from the above-mentioned procedure. Treatment of 4g
with BrCH2COOC2H5 followed by hydrolysis in the
presence of NaOH (2 mol/L) provides 6q in 89% yield.
Similarly, the reaction of 4g and BrCH2CH2Br afforded
6s in 64% yields.
The endothelium controls the tone of the underlying
vascular smooth muscle through the production of vaso-
dilator mediators.20–22 In this experimental model, Ach
acts on the endothelium to release nitric oxide (NO), a
potent vasodilating mediator. The decreased relaxation
response in rat aortic strip could be attributed to a
reduction in NO (synthesis by the endothelium), there-
fore, the present study was undertaken to assess the
NO scavenging capability of these compounds.
The parental nitronyl nitroxide 4a was found to be a
weak inhibitor of Ach-induced vasorelaxation. Conse-
quently, nitronyl nitroxide ring was modified with a
variety of substituents. The effect of different substitu-
tions was then examined in Ach-induced vasorelaxtion
assay as shown in Table 1. Introduction of the aromatic
ring at the 2-position led to a significantly improved NO
scavenging activity, suggesting that in the 2-substituted
phenylnitronyl nitroxides series, the scaffold was opti-
mized compared to other substitutions at the 2-position
of nitronyl nitroxides.
The synthesis of nitronyl nitroxides relies almost exclu-
sively on the condensation of 2,3-bis(hydroxyamino)-
2,3-dimethylbutane with an aldehyde and oxidation of
the condensation product.16 The key intermediate 2
was prepared using the classical reduction process with
zinc in ammonium chloride buffered solution of the dini-
tro analogue. Utilizing THF instead of ethanol accord-
ing to the modified Lamchem–Mittag procedure can
avoid the formation of undesired nitroxides and favor
the reduction.17 In addition, the best ratio of Zn and
NH4Cl was 1:8 to yield best results. This was perhaps
due to the use of 8 equiv NH4Cl resulting in a well-de-
fined Zn complex, [Zn(NH3)2Cl2], which favors the
reduction proceed smoothly and in good yield. The for-
mation of the corresponding nitroxides requires only
mild oxidants to introduce the free radical-generating
moiety. Thus, PdO2 was utilized to insure completion
of the last oxidation step (Scheme 2) .
Among these compounds tested, Ach-induced relaxa-
tion was significantly reversed by compounds 4f–h. At
the concentration of 10 lmol, compound 4f showed
good inhibition ability with 62.7 5.5%, whereas com-
pounds 4g and h displayed even higher inhibition activ-
ity than 4f in this assay, and their inhibition percentage
of Ach-induced vasorelaxation was 73.1 5.9% and
71.3 8.8%, respectively. Clearly, compounds 4g and h
were more potent based on the inhibition assay. We
speculate that the NO scavenging activity of compounds
4f–h led to attenuation of the NO concentration in vitro,
iii
iv
N
O
O
O N
N OH
HO N
R
R
NO2
4a-h
3a-h
ii
i
CH
H3C
CH3
O2N
NO2
HO HN NH OH
1
2
N
R
N OH
O N
HO N
iv
iii
R
5a-p
6a-p
Scheme 1. Reagents and conditions: (i) Br2, NaOH (6 mol/L); (ii) Zn, NH4Cl; (iii) RCHO/MeOH; (iv) PdO2, MeOH. In 3a and 4a R = H, 3b and 4b
R = –CH2Cl, 3c and 4c R = –COOH, 3d and 4d R = fur-2-yl, 3e and 4e R = pyrid-3-yl, 3f and 4f R = phenyl, 3g and 4g R = 40-hydroxylphenyl, 3h
and 4h R = 4-nitrophenyl; in 5a and 6a R = 40-Br, 5b and 6b R = 40-Cl, 5c and 6c R = 40-CH3, 5d and 6d R = 40-OCH3, 5e and 6e R = 30-NO2, 5f and
6f R = 30-OH, 5g and 6g R = 30,40-dimethoxyl, 5h and 6h R = 40-OH–30-OCH3, 5i and 6i R = 20,40-dichloro, 5j and 6j R = 30,40-methylenedioxy, 5k
and 6k R = 20-F, 5l and 6l R = 20-NO2, 5m and 6m R = 30-OH-40-OCH3, 5n and 6n R = 40-N(CH3)2, 5o and 6o R = 20,40-dimethoxyl, 5p and 6p
R = 30-OH.

Y. Wu et al. / Bioorg. Med. Chem. 14 (2006) 5711–5720
5713
N
O
O N
N
O
N
O N
O
O N
N
O
O N
viii
vi
vii
OCH2CH2Br
6s
OH
4g
OCH2CO2C2H5
OCH2CO2H
6q
Scheme 2. Reagents and conditions: (vi) BrCH2COOC2H5 and NaOC2H5; (vii) NaOH/MeOH; (viii) BrCH2CH2Br, NaOC2H5.
Table 1. Inhibition of ACh-induced vasorelaxation of 4a–h
Compound
Inhibition percentage (X ꢁ SD%)
100 lmolb
10 lmolc
1 lmol
NS
4a
4b
4c
4d
4e
4f
1.63 1.45
8.6 3.7a
31.2 2.9a
1.7 1.5
1.6 1.5
1.7 1.6
1.6 1.5
1.7 1.7
9.8 7.8
26.1 7.0a
14.5 14.5
15.0 2.4a
7.7 9.8
14.3 6.5a
35.0 17.8a
20.2 4.2a
16.6 2.8a
62.7 5.5a,c,d
73.1 5.9a,c,d
71.3 8.8a,c,d
44.4 5.9a,b
48.6 2.9a,b
81.9 8.9a,c,d
99.2 1.4a,c,d
98.0 2.7a,c,d
Scheme 3. ESR spectrum of 6g at 10ꢀ5 mol/L in phosphate buffer (pH
7.4).
4g
4h
n = 6; NS, vehicle.
there was no difference among the compounds tested.
The ESR spectrum of 6g in phosphate buffer (pH 7.4)
is presented as an example in Scheme 3. The character-
istic spectra resulting from the interaction of a free elec-
tron are the two nitrogens in the nitronyl nitroxides.
a Compare to NS, P < 0.001.
b Compare to 4a–c, P < 0.01.
c Compare to 4a–c, P < 0.001.
d Compare to 4d and e, P < 0.001.
in return, the effect was also attributed to the inhibition
of Ach-induced vasorelaxation.
2.5. Scavenging activities determined by cell survival
assay in PC12 cells
2.3. Stability of compounds 4a–h in the aqueous solution
The chemical modification of lead compound 4f, focus-
ing on the benzene ring substituents, was carried out to
further improve the free scavenging ability. A series of
new 2-substituted phenyl nitronyl nitroxides derivatives
were synthesized. The free radical scavenging properties
of these 4f derivatives were evaluated to elucidate struc-
ture–activity relationships.
To test the stability of the compounds 4a–h at room
temperature, their pH value of aqueous solution
(10ꢀ5 mol/L) was adjusted to 2.0, 7.4 and 11.5. TLC
(CHCl3/CH3OH, 20:1) analysis indicated that, com-
pounds 4a–c started to decompose after 2 h, compounds
4d and e started to decompose after 15 h, whereas no
decomposition was observed for compounds 4f–h after
200 h. Based on TLC analysis, ESR spectra, and the re-
sults of the rat aortic strip assay, compound 4f was
selected as the lead compound for further chemical
modifications.
Various free radicals are formed during normal cell
metabolism. Therefore, the newly synthesized com-
pounds can be analyzed for their functional scavenging
capacity against the different active free radicals. The
free radical scavenging activities of these compounds
against NO, H2O2 , and ÅOH were evaluated by cell sur-
Å
2.4. Electron spin resonance (ESR) spectra of 4f–h and
6a–s
vival assay in PC12 cells using a published method with
minor modifications.23 The EC50 (lM) values are sum-
marized in Table 2.
ESR spectrometry is a widely accepted method for free
radical detections and has been successfully used to
study radical scavenging capacities of antioxidants.18
The unpaired electron in a free radical results in a spe-
cial ESR absorbance. This absorbance disappears when
the free radical obtains an electron from antioxidants.
The ESR spectra of 4f–h and 6a–s were obtained at
two different concentrations of 10ꢀ7 and 10ꢀ5 mol/L,
in water and phosphate buffer (pH 7.4), respectively.
They all exhibited free radical characteristics with spec-
tra showing a five-line pattern at 1:2:3:2:1 ratio, and
The derivatives exhibited varying degrees of scavenging
capacity on different active radicals. The scavenging
capacity (EC50) among all of the tested compounds ran-
ged from 8.32 0.25 to 114.82 4.35 lM for NO, from
25.29 0.72 to 107.86 4.37 lM for H2O2, from
27.73 1.32 to 167.06 7.91 lM for ÅOH. Among them,
compounds 6g,n,o, and s exhibited the highest scaveng-
ing capacity of active radical species. For example, com-
pound 6g was the most potent compound in inhibiting
NO free radical activity with EC50 8.32 0.25 lM,

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Y. Wu et al. / Bioorg. Med. Chem. 14 (2006) 5711–5720
Å
Table 2. The EC50 of 4f–h, 6a–s as NO, H2O2, and OH scavenger
Compound:
4f
4g
4h
6a
6b
6c
33.11 1.53
6d
EC50/NO
EC50/H2O2
EC50/ÅOH
67.61 4.22
51.63 2.41
66.51 2.33
39.81 1.52
48.18 2.44
56.03 2.47
114.82 4.35
107.86 4.37
167.06 7.91
46.77 2.38
55.32 2.05
60.66 2.97
63.10 3.57
58.88 2.73
74.62 2.56
30.90 1.28
42.94 1.78
59.28 2.54
38.27 1.91
64.99 3.15
Compound:
6e
6f
6g
6h
6i
6j
6k
EC50/NO
EC50/H2O2
EC50/ÅOH
87.10 2.81
62.07 2.95
63.52 3.02
61.66 2.42
57.93 2.86
69.64 2.88
8.32 0.25
30.40 1.42
29.71 1.24
25.70 1.43
32.57 1.63
42.94 2.03
42.76 1.97
64.99 3.15
78.14 3.85
19.50 2.24
29.71 1.46
47.08 2.11
44.67 1.75
76.36 3.82
85.68 3.24
Compound:
6l
6m
6n
6o
6p
6q
6s
EC50/NO
EC50/H2O2
EC50/ÅOH
47.86 2.10
60.66 3.03
74.62 3.52
27.54 1.17
40.08 2.05
54.06 2.48
9.12 0.14
29.03 1.48
31.11 1.47
9.33 0.31
25.29 0.72
27.73 1.32
44.67 0.82
52.83 3.26
55.32 2.77
91.20 2.23
74.62 4.12
100.67 4.56
9.55 0.24
27.73 2.75
39.16 1.85
Å
and the scavenging capacity of H2O2 and OH was also
quite effective with EC50 30.40 1.42, 29.71 1.24 lM,
respectively.
resulted in 2- to 3-fold enhancement of radical scavenging
activity (EC50/NO: 33.11 1.53; EC50/H2O2: 38.27
1.91; and EC50/ÅOH: 64.99 3.15).
We initially examined the effect of halogen of the phenyl
ring on the radical scavenging activity. Introduction of
different halogen at the 2- or 4-position of the phenyl
ring resulted in improved potency compared to unmod-
ified 4f. For example, 4-bromine derivative 6a (EC50/
NO: 46.77 2.38 lM) or 20-fluorine derivative 6k
(EC50/NO: 44.67 1.75 lM) exhibited better NO
scavenging activity than that of 4f (EC50/NO:
67.61 4.22 lM). It appeared that either a bromine or
fluorine atom at the 2- or 4-position on the phenyl ring
was beneficial for the enhanced NO scavenging activity.
In the case of substitution with a chlorine atom, that is, 40-
Cl derivative 6b (EC50/NO: 63.10 3.57 lM) showed less
improved activity compared to 40-Br 6a or 20-F derivative
6k. Interestingly, the effect of dichloro substitution on the
radical scavenging activity was quite different from that of
single-chlorine derivative. 20,40-Dichloro derivative 6i
(EC50/NO: 42.76 1.97 lM) exhibited higher NO
scavenging potency than that of 40-Cl derivative 6b.
Furthermore, it was noticed that the more distant
bromine (OCH2CH2Br) group on the phenyl ring furnish-
ing compound 6s yield remarkable NO scavenging
activity (EC50/NO: 9.55 0.24 lM). The enhanced NO
radical scavenging activity of bromoethoxy functionality
in 6s on the phenyl ring remained to be determined.
Interestingly, although introduction of a nitro group at
the 4-position of the phenyl ring resulted in a notable de-
crease in NO free radical scavenging potency, when a ni-
tro group was introduced at the 2-position of the phenyl
ring in 6l, the NO scavenging activity was greatly im-
proved (EC50/NO: 47.86 2.10 lM). Based on this obser-
vation, we speculated that the position of the nitro group
in the phenyl ring might be crucial for its NO inhibitory
activity. Furthermore, we assumed that, the nonplanar
torsional conformation of the nitro group in relation to
the phenyl ring had an effect on radical stabilization.
Finally, we focused on elucidating the impact of the elec-
tron-donating group of the phenyl ring. Replacement of
the methyl group in compound 6c with a methoxy group
led to compound 6d (EC50/NO: 30.90 1.28; EC50/H2O2:
42.94 1.78; and EC50/ÅOH: 59.28 2.54 lM), and its
free radical scavenging potency was effective and compa-
rable to that of 6c. Substitution with hydroxyl group at
the 3-position of the phenyl ring led to 6f (EC50/NO:
61.66 2.42; EC50/H2O2: 57.93 2.86; and EC50/ÅOH:
69.64 2.88 lM) with moderate radical scavenging activ-
ity. While the electron-donating hydroxyl group was
introduced at the 2-position of the phenyl ring in 6p
(EC50/NO: 44.67 0.82; EC50/H2O2: 52.83 3.26; and
EC50/ÅOH: 55.32 2.77 lM) leading to a higher radical
scavenging activity compared to 30-OH derivative 6f, sug-
gesting the hydroxyl group is favorable for radical scav-
enging activity specially at the 2-position of the phenyl
ring. This may be due to the ortho-hydroxyl group of
the phenyl ring increasing the stability of the radical form
and participating in electron delocalization. In addition,
other electron-donating group such as –N(CH3)2 also
plays an important role on the enhanced radical scaveng-
ing activity of nitronyl nitroxide derivative as shown in
compound 6n with highly potent radical scavenging activ-
ity (EC50/NO: 9.12 0.14; EC50/H2O2: 29.03 1.48; and
EC50/ÅOH: 31.11 1.47 lM).
Next, we continued to investigate the effect of the substit-
uents of the phenyl ring on radical scavenging capability
in P12 cell viability assay. As shown in Table 2, lead com-
pound 4f showed moderate radical inhibitory potency
(EC50/NO: 67.61 4.22; EC50/H2O2: 51.63 2.41; and
EC50/ÅOH: 66.51 2.33 lM). When an electron-with-
drawing nitro group was introduced at the 4-position of
the phenyl ring, 40-nitro derivative, 4h, a notable decrease
of activity was observed (EC50/NO: 114.82 4.35; EC50/
H2O2: 107.86 4.37; and EC50/ÅOH: 167.06 7.91 lM).
Similarly, when an electron-withdrawing carboxymethyl
group was introduced at the 4-position of the phenyl ring
of 4-carboxymethyl derivative, 6q, a marked decrease of
potency was observed (EC50/NO: 91.20 2.23; EC50/
H2O2: 74.62 4.12; and EC50/ÅOH: 100.67 4.56). In
contrast, introduction of a small, slightly electron-donat-
ing methyl group at the 4-position of the phenyl ring in 6c
Furthermore, it was revealed that the combined pres-
ence of hydroxyl and methoxy group on the phenyl ring
produced substances with higher activity as shown in 30-
OCH3–40-OH derivative 6h, 30-OH–40-OCH3 derivative

Y. Wu et al. / Bioorg. Med. Chem. 14 (2006) 5711–5720
5715
6m with good radical scavenging profiles. (6h: EC50/NO:
25.70 1.43; EC50/H2O2: 32.57 1.63; and EC50/ÅOH:
42.94 2.03 lM; 6m: EC50/NO: 27.54 1.17; EC50/
H2O2: 40.08 2.05; and EC50/ÅOH: 54.06 2.48 lM).
This increased activity due to the combined existence of
the OCH3 and OH most likely could be attributed to its
positive effect on the stabilization of the phenoxy free rad-
ical and hence increases the free radical scavenging effect.
the number of the methoxy groups and also with the
presence of other electron-donating groups in the phenyl
ring. Compounds 6g,n,o, and s are the most active com-
pounds in the PC12 radical scavenging assay. More gen-
erally the results suggest that radical scavenging ability
from 2-substituted phenylnitronyl nitroxide should be
strongly dependent on the nature and the position of
the substituents at the aromatic ring. The results sug-
gested that 2-substituted phenylnitronyl nitroxides were
potential useful free radical scavenger agents and further
in vivo evaluations will be necessary.
More importantly, compounds 6g and o have an identical
number of the methoxy groups, with 30,40- and 20,40-
dimethoxyl groups, respectively, on the phenyl ring.
Compared to lead compound 4f, they exhibited most po-
tent radical scavenging capacity with 8-fold increase in
NO scavenging activity (6g EC50/NO: 8.32 0.25; EC50/
H2O2: 30.40 1.42; and EC50/ÅOH: 29.71 1.24 lM; 6o
EC50/NO: 9.33 0.31; EC50/H2O2: 25.29 0.72; and
EC50/ÅOH: 27.73 1.32 lM). It was important to notice
that the dimethoxy groups on the phenyl ring significantly
enhanced the free radical scavenging activity of the nitro-
nyl nitroxide derivatives.
4. Experimental
The purity of all the compounds was confirmed by TLC
(Merck silica gel plates of type 60 F254, 0.25 mm layer
thickness) and HPLC (waters, C18 column
3.9 · 150 mm). Melting points were measured on a XT5
hot stage microscope (Beijing keyi electro-optic factory).
The infrared spectra were recorded with a Perkin-Elmer
983 instrument. The EI-MS was determined by Trace
MS System instrument (American Thermo Finnigan
company). The 1H NMR was determined by Varian INO-
VA-300 MHz spectrometer. The ESR spectra were ob-
tained from 10ꢀ5 mol/L phosphate-buffered saline, using
a BRUKER 300-E spectrometer. The conditions of mea-
surements are as follows: center field: 3440 G, sweep
width: 100 G, sweep time: 60 s, modulation amplitude:
1.1 G, time constant: 8.2 · 10ꢀ2 s, modulation frequency:
100 kHz, microwave frequency: 9.68 GHz, and micro-
wave power: 20 MW.
The radical scavenging capacity of the 2-substituted
phenylnitronyl nitroxide derivatives depends on several
factors such as the electron-donating and electron-with-
drawing substituents on the phenyl ring, the number of
methoxy groups or the chemical stability. It seemed that
the structural feature responsible for the highest free rad-
ical scavenging activity is the dimethoxy moiety on the
phenyl ring. In addition, the involvement of other elec-
tron-donating groups (–N(CH3)2 and –OCH2CH2Br)
may also play crucial roles on the radical scavenging
activity.
4.1. 2,3-Dimethyl-2,3-dinitrobutane (1)
Clearly, the presence of electron-donating hydroxyl or
methoxy groups attached to the aromatic ring is favorable
for the free radical scavenging activity. This observation
was consistent with the literature reported results.24,25
Currently, it is well recognized that phenolic compounds
in foods possess several interesting biological and chemi-
cal properties such as antioxidant activity and the ability
to scavenge reactive oxygen species. As a consequence,
they may prevent various diseases associated with oxida-
tive stress, such as cancers, cardiovascular diseases, and
inflammation.25,26 The newly synthesized compounds
containing phenolic substituents displayed good radical
scavenger’s activities and that might be of further signifi-
cance for the development of synthetic antioxidants. Fur-
ther in vivo studies will be needed to confirm these
findings.
At ꢀ5 ꢁC, 10 mL (0.19 mol) Br2 were added dropwise
within 1 h to the solution of 34.5 g (0.39 mol) of 2-nitro-
propane in 65 mL (6.0 mol/L) aqueous NaOH. Ethanol
(128 mL) was added into the solution via stirring. The
reaction mixture was stirred at 84 ꢁC for 3 h. The hot reac-
tion mixture was transferred into 400 mL of ice water. The
formed colorless crystals were collected by filtration to
yield 25 g (73%) of the title compound, mp 110–112 ꢁC.
4.2. 2,3-Bis(hydroxylamino)-2,3-dimethylbutane (2)
2,3-Dimethyl-2,3-dinitrobutane (17.6 g, 0.1 mol) was dis-
solved in a mixture of THF (300 mL) and water (50 mL).
Zn powder (27 g) was added all at once to this solution
cooled to 8–10 ꢁC in an ice bath. A solution of NH4Cl
(43 g, 08 mol) in H2O (150 mL) was added dropwise to
this slurry within 2 h, with continued stirring for 1 h at
10 ꢁC, and the flask was stored in a fridge (4–6 ꢁC) for
16 h. The slurry was filtered, and the precipitate was care-
fully washed with THF (4· 100 mL). The precipitate was
then dried by three washings with diethyl ether and care-
fully collected (59 g). The solution was evaporated under
vacuum until THF ceased to distill off. Then the solution
was protected from air, and sodium carbonate (50 g) and
sodium chloride (30 g) were added with cooling. Contin-
uous extraction with chloroform (400 mL) was performed
over 18 h. A white powder was obtained (9.4 g, 63%
yield), mp 182 ꢁC.
3. Conclusion
In this study, screening for new small molecule inhibi-
tors of Ach-induced vascorelaxation led to the discovery
of the lead compound 4f. Chemical modification on this
scaffold resulted in a series of 2-substituted phenylnitro-
nyl nitroxide derivatives 6a–s. Our results indicated the
newly synthesized compounds 6a–s exhibit good to
moderate radical scavenger’s activity as shown in
PC12 cell survival assay. The free radical scavenging
activity of the compounds increased significantly with

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Y. Wu et al. / Bioorg. Med. Chem. 14 (2006) 5711–5720
4.3. General procedure for the preparation of 3a–h
7.85 (s, 1H); ESI-MS (m/e) = 252 [M]+; Anal. Calcd for
C13H20N2O3: C, 61.88; H, 7.99; N, 11.10. Found: C,
61.92; H, 8.04; N, 11.17.
A solution of 296 mg (2 mmol) of 2,3-bis(hydroxylami-
no)-2,3-dimethylbutane and 2 mmol of various aldehyde
in 6 mL of methanol was stirred at room temperature
for 1 h. Compounds 3a–h were obtained by filtration.
The filtrate was evaporated under reduced pressure to
obtain the second crop of the title compound. Com-
pounds 3a–h were directly used for the next reaction
without further purification.
4.3.8. 1,3-Dihydroxy-2-(40-nitro)phenyl-4,4,5,5-tetramethyl-
imidazolidine (3h). Yield: 63%. Mp: 154–156 ꢁC. IR (KBr)
3425, 1625, 1520, 1384, 1350, 835 cmꢀ1 1H NMR
;
(DMSO) d = 1.09 (s, 12H), 4.71 (s, 1H), 7.70 (d,
J = 7.2 Hz, 2H), 8.21 (d, J = 7.2 Hz, 2H), 8.40 (s, 2H);
ESI-MS (m/e) = 281 [M]+; Anal. Calcd for C13H19N3O4:
C, 55.51; H, 6.81; N, 14.94. Found: C, 55.64; H, 6.93;
N, 14.89.
4.3.1. 1,3-Dihydroxy-4,4,5,5-tetramethylimidazolidine (3a).
Yield: 53%. IR (KBr): 3338, 1600, 1480, 1384 cmꢀ1; H
1
NMR (DMSO-d6) d = 0.966 (s, 12H), 3.873 (s, 2H),
7.644 (s, 2H); ESI-MS (m/e) = 160 [M]+; Anal. Calcd for
C7H16N2O2: C, 52.48; H, 10.07; N, 17.48. Found: C,
52.29; H, 10.22; N, 17.68.
4.4. General procedure for the preparation of 4a–h
At room temperature the suspension of 1 mmol of 3a–h
and 0.7 g of lead dioxide in 10 mL methanol was stirred
for 3 h. After filtration, the filtrate was evaporated un-
der reduced pressure, compounds 4a–h were obtained
as dark blue crystals.
4.3.2. 1,3-Dihydroxy-2-chloromethyl-4,4,5,5-tetramethyl-
imidazolidine (3b). Yield: 69%. IR (KBr): 3460, 1460,
1381 cmꢀ1 1H NMR (DMSO-d6) d = 0.951–0.985 (s,
;
12H), 3.82 (s, 1H), 7.998 (s, 2H), 3.609 (s, 2H); ESI-
MS (m/e) = 208 [M]+; Anal. Calcd for C8H17N2O2Cl:
C, 46.04; H, 8.21; N, 13.42. Found: C, 46.29; H, 8.25;
N, 13.38.
4.4.1. 4,4,5,5-Tetramethylimidazoline-3-oxide-1-oxyl (4a).
Yield: 90%. Mp: 92–93 ꢁC. IR (KBr) 1533, 1384,
889 cmꢀ1; ESI-MS (m/e) = 157 [M]+; Anal. Calcd for
C7H16N2O2: C, 53.49; H, 8.34; N, 17.82. Found: C,
53.29; H, 8.42; N, 17.68.
4.3.3. 1,3-Dihydroxy-2-carboxyl-4,4,5,5-tetramethylimi-
dazolidine (3c). Yield: 83%. Mp: 103–105 ꢁC. IR
4.4.2. 2-Chloromethyl-4,4,5,5-tetramethylimidazoline-3-
oxide-1-oxyl (4b). Yield: 38%. Mp: 73–75 ꢁC. IR (KBr)
1510, 1482, 1380 cmꢀ1; ESI-MS (m/e) = 205 [M]+; Anal.
Calcd for C8H14N2O2Cl: C, 46.72; H, 6.86; N, 13.62.
Found: C, 46.89; H, 6.42; N, 13.48.
(KBr): 3340, 2701–2402, 1460, 1381 cmꢀ1 1H NMR
;
(DMSO-d6) d = 0.99 (s, 12H), 4.064 (s, 1H), 8.05 (s,
2H), 12.248 (s, 1H); ESI-MS (m/e) = 204 [M]+; Anal.
Calcd for C8H16N2O4: C, 47.05; H, 7.90; N, 13.72.
Found: C, 47.19; H, 7.85; N, 13.72.
4.4.3. 2-Carboxyl-4,4,5,5-tetramethylimidazolidine-3-oxide-
1-oxyl (4c). Yield: 88%. Mp: 84–86 ꢁC. IR (KBr) 2705–
2400, 1709, 1500, 1462, 1380 cmꢀ1; ESI-MS (m/e) = 201
[M]+; Anal. Calcd for C8H13N2O4: C, 47.76; H, 6.51; N,
13.92. Found: C, 47.69; H, 6.33; N, 13.80.
4.3.4. 1,3-Dihydroxy-2-(fur-2-yl)-4,4,5,5-tetramethylimidaz-
olidine (3d). Yield: 73%. Mp: 93–95 ꢁC. IR (KBr): 3440,
1601, 1503, 1450, 1386 cmꢀ1; NMR (DMSO): d = 1.04
(s, 12H), 4.59 (s, 1H), 6.37 (d, J = 6.5 Hz, 1H), 6.44 (d,
J = 6.3 Hz, 1H), 7.58 (t, J = 6.6 Hz, 1H); ESI-MS (m/
e) = 226 [M]+; Anal. Calcd for C11H18N2O3: C, 58.39; H,
8.02; N, 12.38. Found: C, 58.35; H, 7.96; N, 12.37.
4.4.4. 2-(Fur-2-yl)-4,4,5,5-tetramethylimidazolidine-3-ox-
ide-1-oxyl (4d). Yield: 83%. Mp: 80–82 ꢁC. IR (KBr)
1619, 1505, 1450, 1382 cmꢀ1; ESI-MS (m/e) = 223
[M]+; Anal. Calcd for C11H15N2O3: C, 59.18; H, 6.77;
N, 12.55. Found: C, 59.30; H, 6.55; N, 12.68.
4.3.5. 1,3-Dihydroxy-2-(pyrid-3-yl)-4,4,5,5-tetramethylimi-
dazolidine (3e). Yield: 69%. Mp: 90-92 ꢁC. IR (KBr):
3440, 1601, 1505, 1450, 1383 cmꢀ1;NMR (DMSO):
d = 1.04 (s, 12H), 4.55 (s, 1H), 7.35 (s, 1H), 7.81 (s, 1H)
8.46 (s, 1H) 8.62 (s, 1H); ESI-MS (m/e) = 237 [M]+; Anal.
Calcd for C12H19N3O2: C, 60.74; H, 8.07; N, 17.71.
Found: C, 60.49; H, 7.99; N, 17.65.
4.4.5. 2-(Pyrid-3-yl)-4,4,5,5-tetramethylimidazolidine-3-
oxide-1-oxyl (4e). Yield: 79%. Mp: 81–83 ꢁC. IR (KBr)
1609, 1500, 1452, 1380 cmꢀ1; ESI-MS (m/e) = 234
[M]+; Anal. Calcd for C12H16N3O2: C, 61.52; H, 6.88;
N, 17.94. Found: C, 61.33; H, 6.69; N, 18.08.
4.3.6. 1,3-Dihydroxy-2-phenyl-4,4,5,5-tetramethylimidazoli-
dine (3f). Yield: 85%. Mp: 168–169 ꢁC. IR (KBr): 3340,
4.4.6. 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-
oxyl (4f). Yield: 80%. Mp: 84–85 ꢁC. IR (KBr) 1610,
1450, 1381, 1365 cmꢀ1; ESI-MS (m/e) = 233 [M]+; Anal.
Calcd for C13H17N2O2: C, 66.93; H, 7.34; N, 12.01.
Found: C, 67.02; H, 7.41; N, 12.09.
1
1606, 1500, 1450, 1381 cmꢀ1; H NMR (CDCl3) d = 1.14
(s, 12H), 4.78 (s, 1H), 7.31–7.51 (m, J = 6.9 Hz, 5H),
7.71 (s, 2H); ESI-MS (m/e) = 236.3 [M]+; Anal. Calcd for
C13H20N2O2: C, 66.07; H, 8.53; N, 11.85. Found: C,
66.12; H, 8.61; N, 11.93.
4.4.7. 2-(40-Hydroxyl)phenyl-4,4,5,5-tetramethylimidazo-
line-3-oxide-1-oxyl (4g). Yield: 52% Mp: 134–135 ꢁC. IR
(KBr) 3250, 1500, 1490, 1355, 840 cmꢀ1; ESI-MS
(m/e) = 249 [M]+; Anal. Calcd for C13H17N2O3: C,
62.64; H, 6.87; N, 11.24. Found: C, 62.75; H, 6.93; N,
11.21.
4.3.7. 1,3-Dihydroxy-2-(40-hydroxyl)phenyl-4,4,5,5-tetrame-
thylimidazolidine (3g). Yield: 51%. Mp: 161–162 ꢁC. IR
1
(KBr) 3455, 1610, 1506 1450, 1382, 830 cmꢀ1; H NMR
(DMSO) d = 1.03 (s, 12H), 4.39 (s, 1H), 6.70 (d,
J = 7.0 Hz, 2H), 7.23 (d, J = 7.0 Hz, 2H), 7.63 (s, 1H),

Y. Wu et al. / Bioorg. Med. Chem. 14 (2006) 5711–5720
5717
4.4.8. 2-(40-Nitrophenyl)-4,4,5,5-tetramethylimidazoline-
3-oxide-1-oxyl (4h). Yield: 73%. Mp: 175-176 ꢁC. IR
(KBr) 1360, 1600, 1450, 830 cmꢀ1; ESI-MS (m/e) = 278
[M]+; Anal. Calcd for C13H16N3O4: C, 56.11; H, 5.79;
N, 15.10. Found: C, 56.15; H, 5.82; N, 15.18.
4.5.6. 1,3-Dihydroxy-2-(30-hydroxyphen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (5f). Mp: 244–246 ꢁC. Rf = 0.56
(CHCl3/CH3OH, 6:1). EI-MS (m/z) 252 [M]+. IR (KBr)
3320, 1500, 880, 795, 695 cmꢀ1 1H NMR (DMSO-d6)
.
d = 1.03 (s, 6H), 1.05 (s, 6H), 4.51 (s, 1H), 6.89 (dd,
J = 8.4 Hz, J = 8.0 Hz, 1H), 6.92 (d, J = 6.2 Hz, 1H), 7.01
(d, J = 7.3 Hz, 1H), 7.71 (s, 1H), 7.86 (s, 1H), 7.95 (s,
2H). Anal. Calcd for C13H20N2O3: C, 61.88; H, 7.99; N,
11.10. Found: C, 61.85; H, 8.05; N, 11.18.
4.5. General procedure for the preparation of 5a–p
A solution of 296 mg (2 mmol) of 2,3-bis(hydroxyla-
mino)-2,3-dimethylbutane and 2 mmol of various
aldehydes in 3 mL of methanol was stirred at room
temperature for 16 h. The title compound was col-
lected by filtration. The filtrate was evaporated un-
der reduced pressure to obtain the second crop of
the title compound. The title compound was directly
used for the next reaction without further
purification.
4.5.7. 1,3-Dihydroxy-2-(30,40-dimethoxylphen-10-yl)-4,4,5,5-
tetramethylimidazolidine (5g). Mp: 147–149 ꢁC.Rf = 0.52
(CHCl3/CH3OH, 6:1). EI-MS (m/z) 296 [M]+. IR (KBr)
1
3310, 2825, 1610, 865, 815 cmꢀ1. H NMR (DMSO-d6)
d = 1.05 (s, 12H), 3.74 (s, 6H), 4.53 (s, 1H), 6.85 (s, 1H),
6.87 (d, J = 6.6 Hz, 1H), 6.92 (d, J = 6.4 Hz, 1H), 7.85
(s, 2H). Anal. Calcd for C15H24N2O4: C, 60.79; H, 8.16;
N, 9.45. Found: C, 60.82; H, 8.23; N, 9.54.
4.5.1. 1,3-Dihydroxy-2-(40-bromophen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (5a). Mp: 205–207 ꢁC. Rf = 0.69
(CHCl3/CH3OH, 10:1). EI-MS (m/z) 314 [M]+. IR (KBr)
4.5.8. 1,3-Dihydroxyl-2-(30-methoxyl-40-hydroxyphen-10-
yl)-4,4,5,5-tetramethylimidazolidine (5h). Mp: 235–
237 ꢁC, Rf = 0.47 (CHCl3/CH3OH, 6:1), EI-MS (m/z)
3310, 1590, 1450, 1075, 830 cmꢀ1 1H NMR (CDCl3)
.
d = 1.14 (s, 12H), 4.77 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H),
7.47 (d, J = 7.8 Hz, 2H), 7.73 (s, 2H). Anal. Calcd for
C13H19N2O2Br: C, 49.54; H, 6.08; N, 8.89. Found: C,
49.62; H, 6.14; N, 8.93.
282 [M]+, IR (KBr) 3310, 2840, 1600, 870, 815 cmꢀ1
.
1H NMR (DMSO-d6) d = 1.00 (s, 6H), 1.03 (s, 6H),
3.74 (s, 3H), 4.41 (s, 1H), 6.70 (d, J = 7.8 Hz, 1H),
6.85 (d, J = 8.1 Hz, 1H), 7.02 (s, 1H), 7.68 (s, 1H),
8.05 (s, 2H). Anal. Calcd for C14H22N2O4: C, 59.56;
H, 7.85; N, 9.92. Found: C, 59.61; H, 7.92; N, 9.85.
4.5.2. 1,3-Dihydroxy-2-(40-chlorophen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (5b). Mp: 213–215 ꢁC. Rf = 0.73
(CHCl3/CH3OH, 10:1). EI-MS (m/z) 270 [M]+. IR (KBr)
4.5.9. 1,3-Dihydroxyl-2-(20,40-dichlorophenyl)-4,4,5,5-tetra-
methylimidazolidine (5i). Mp: 201–203 ꢁC. Rf = 0.69
(CHCl3/CH3OH, 10:1). EI-MS (m/z) 305 [M]+. IR (KBr)
3325, 1600, 1500, 1085, 825 cmꢀ1 1H NMR (CDCl3)
.
d = 1.03 (s, 6H), 1.07 (s, 6H), 4.50 (s, 1H), 7.38 (d,
J = 8.7 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.80 (s, 2H).
Anal. Calcd for C13H16N2O2Cl: C, 57.67; H, 7.07; N,
10.35. Found: C, 57.72; H, 7.12; N, 10.43.
1
3315, 1590, 1450, 995, 820, 865 cmꢀ1. H NMR (DMSO-
d6) d = 1.11 (s, 12H), 5.01 (s, 1H), 7.50 (d, J = 7.6 Hz,
1H), 7.52 (d, J = 8.1 Hz, 1H), 7.58 (s, 1H), 7.79 (s, 2H).
Anal. Calcd for C13H18N2O2Cl2: C, 51.16; H, 5.94; N,
9.18. Found: C, 51.24; H, 5.99; N, 9.23.
4.5.3. 1,3-Dihydroxy-2-(40-methylphen-10-yl)-4,4,5,5-tetrame-
thylimidazolidine (5c). Mp: 199–201 ꢁC. Rf = 0.61 (CHCl3/
CH3OH, 10:1). EI-MS (m/z) 250 [M]+. IR (KBr) 3335,
4.5.10. 1,3-Dihydroxyl-2-(30,40-methylendioxyphen-10-yl)-
4,4,5,5-tetramethylimidazolidine (5j). Mp: 188–190 ꢁC,
Rf = 0.47 (CHCl3/CH3OH, 10:1), EI-MS (m/z) 280 [M]+,
1
2985, 1600, 815 cmꢀ1. H NMR (CDCl3) d = 1.14 (s, 6H),
1.19 (s, 6H), 1.37 (s, 3H), 4.90 (s, 1H), 7.68 (d,
J = 9.0 Hz, 2H), 8.22 (d, J = 8.4 Hz, 2H), 8.35 (s, 2H).
Anal. Calcd for C14H22N2O2: C, 67.17; H, 8.86; N, 11.19.
Found: C, 67.23; H, 8.81; N, 11.25.
1
IR (KBr) 3315, 1600, 915, 1235, 1105 cmꢀ1. H NMR
(DMSO-d6) d = 1.05 (s, 12H), 3.74 (s, 6H), 4.53 (s, 1H),
6.82 (d, J = 7.7 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.87
(s, 1H) 7.85 (s, 2H). Anal. Calcd for C14H20N2O4: C,
59.99; H, 7.19; N, 9.99. Found: C, 60.04; H, 7.25; N, 10.09.
4.5.4. 1,3-Dihydroxy-2-(40-methoxylphen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (5d). Mp: 179–181 ꢁC. Rf = 0.52
(CHCl3/CH3OH, 10:1). EI-MS (m/z) 266 [M]+. IR (KBr)
4.5.11. 1,3-Dihydroxy-2-(20-fluorophen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (5k). Mp: 172–173 ꢁC, Rf = 0.69
(CHCl3/CH3OH, 6:1), EI-MS (m/z) 254 [M]+, IR (KBr)
.
3340, 2835, 1500, 825 cmꢀ1 1H NMR (DMSO-d6)
d = 0.99 (s, 6H), 1.03 (s, 6H), 3.73 (s, 3H), 4.56 (s, 1H),
6.88 (d, J = 4.2 Hz, 2H), 7.38 (d, J = 5.7 Hz, 2H), 7.77 (s,
2H). Anal. Calcd for C14H22N2O3: C, 63.14; H, 8.33; N,
10.52. Found: C, 63.19; H, 8.38; N, 10.49.
1
3310, 1600, 1130, 1235, 775 cmꢀ1. H NMR (DMSO-d6)
d = 1.07 (s, 12H), 4.95 (s, 1H), 7.18 (t, J = 8.4 Hz, 1H),
7.30 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 8.7 Hz, 2H), 8.14 (s,
2H). Anal. Calcd for C13H19N2O4F: C, 61.40; H, 7.53; N,
11.02. Found: C, 61.49; H, 7.61; N, 11.09.
4.5.5. 1,3-Dihydroxy-2-(30-nitrophen-10-yl)-4,4,5,5-tetrame-
thylimidazolidine (5e). Mp: 179–181 ꢁC. Rf = 0.51 (CHCl3/
CH3OH, 10:1). EI-MS (m/z) 281 [M]+. IR (KBr) 3315,
4.5.12. 1,3-Dihydroxy-2-(20-nitrophen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (5l). Mp: 189–190 ꢁC. Rf = 0.54
(CHCl3/CH3OH, 10:1). EI-MS (m/z) 281 [M]+. IR (KBr)
.
1530, 1360, 1600, 875, 790, 685 cmꢀ1 1H NMR
(DMSO-d6) d = 0.52 (s, 12H), 4.19 (s, 1H), 7.04 (dd,
J = 8.7 Hz, J = 8.1 Hz, 1H), 7.34 (d, J = 6.0 Hz, 1H),
7.57 (d, J = 7.5 Hz, 1H), 7.77 (s, 1H), 8.03 (s, 2H). Anal.
Calcd for C13H19N3O4: C, 55.51; H, 6.81; N, 14.94.
Found: C, 55.61; H, 6.90; N, 14.87.
1
3325, 1600, 1535, 1365, 750 cmꢀ1. H NMR (DMSO-d6)
d = 1.00 (s, 6H), 1.04 (s, 6H), 5.37 (s, 1H), 7.52 (d,
J = 6.6 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.92 (t,
J = 7.6 Hz, 1H), 8.05 (d, J = 7.2 Hz, 1H), 8.23 (s, 2H).

5718
Y. Wu et al. / Bioorg. Med. Chem. 14 (2006) 5711–5720
Anal. Calcd for C13H19N3O4: C, 55.51; H, 6.81; N, 14.94.
Found: C, 55.46; H, 6.90; N, 14.86.
(KBr) 1590, 1500, 1365, 1090, 825 cmꢀ1. Anal. Calcd
for C13H13N2O2Cl: C, 58.32; H, 6.02; N, 10.46. Found:
C, 58.41; H, 6.14; N, 10.54. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.12 G, g = 2.00997.
4.5.13. 1,3-Dihydroxyl-2-(30-hydroxyl-40-methoxyl)phen-
yl-4,4,5,5-tetramethylimidazolidine (5m). Mp: 245–
247 ꢁC. Rf = 0.52 (CHCl3/CH3OH, 6:1). EI-MS (m/z)
4.6.3. 2-(40-Methylphen-10-yl)-4,4,5,5-tetramethylimidaz-
oline-3-oxide-1-oxyl (6c). Mp: 86–88 ꢁC. Rf = 0.88
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 247 [M]+. IR
(KBr) 2980, 1610, 1500, 1365, 810 cmꢀ1. Anal. Calcd
for C14H19N2O2: C, 67.99; H, 7.74; N, 11.33. Found:
C, 67.92; H, 7.83; N, 11.38. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.22 G, g = 2.00993.
282 [M]+. IR (KBr) 3340, 2825, 1500, 1450, 825 cmꢀ1
.
1H NMR (DMSO-d6) d = 1.03 (s, 6H), 1.04 (s, 6H)
3.72 (s, 3H), 4.53 (s, 1H), 6.82 (s, 1H), 6.89 (d,
J = 6.6 Hz, 1H), 6.94 (d, J = 6.4 Hz, 1H), 7.65 (s, 1H),
8.04 (s, 2H). Anal. Calcd for C14H22N2O4: C, 59.56;
H, 7.85; N, 9.92. Found: C, 59.65; H, 7.93; N, 9.89.
4.5.14. 1,3-Dihydroxyl-2-(40-N,N-dimethylphen-10-yl)-4,4,5,5-
tetramethylimidazolidine (5n). Mp: 168–169 ꢁC. Rf = 0.53
(CHCl3/CH3OH, 6:1). EI-MS (m/z) 279 [M]+. IR (KBr)
4.6.4. 2-(40-Methoxylphen-10-yl)-4,4,5,5-tetramethylimi-
dazoline-3-oxide-1-oxyl (6d). Mp: 89–92 ꢁC. Rf = 0.88
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 263 [M]+. IR
(KBr) 2830, 1600, 1355, 835 cmꢀ1. Anal. Calcd for
C14H19N2O3: C, 63.86; H, 7.27; N, 10.64. Found: C,
63.92; H, 7.35; N, 10.59. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.28 G, g = 2.00991.
1
3345, 2840, 1595, 1450, 840 cmꢀ1. H NMR (DMSO-d6)
d = 1.03 (s, 6H), 1.05 (s, 6H), 2.85 (s, 6H), 4.01 (s, 1H),
6.68 (d, J = 8.7 Hz, 2H), 7.25 (d, J = 8.7 Hz, 2H), 7.60 (s,
2H). Anal. Calcd for C15H25N3O2: C, 64.49; H, 9.02; N,
15.04. Found: C, 64.58; H, 9.12; N, 15.17.
4.6.5. 2-(30-Nitrophen-10-yl)-4,4,5,5-tetramethylimidazoline-
3-oxide-1-oxyl (6e). Mp: 161–162 ꢁC. Rf = 0.69 (CHCl3/
CH3OH, 20:1). EI-MS (m/z) 278 [M]+. IR (KBr) 1530,
1350, 1600, 1450, 880, 790, 680 cmꢀ1. Anal. Calcd for
C13H16N3O4: C, 56.11; H, 5.79; N, 15.10. Found: C,
56.19; H, 5.84; N, 15.16. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.10 G, g = 2.00998.
4.5.15. 1,3-Dihydroxyl-2-(20,40-dimethoxylphenyl)-4,4,5,5-
tetramethylimidazolidine (5o). Mp: 140–141 ꢁC. Rf = 0.49
(CHCl3/CH3OH, 10:1). EI-MS (m/z) 296 [M]+. IR (KBr)
1
3295, 2825, 1500, 865, 810 cmꢀ1. H NMR (DMSO-d6)
d = 1.03 (s, 12H), 3.72 (s, 6H), 4.99 (s, 1H), 7.02 (s, 1H),
7.42 (d, J = 6.7 Hz, 1H), 7.45 (d, J = 6.5 Hz, 1H), 7.79
(s, 2H). Anal. Calcd for C15H24N2O4: C, 60.79; H, 8.16;
N, 9.45. Found: C, 60.82; H, 8.21; N, 9.51.
4.6.6. 2-(30-Hydroxyphen-10-yl)-4,4,5,5-tetramethylimidazo-
line-3-oxide-1-oxyl (6f). Mp: 137–139 ꢁC. Rf = 0.33 (CHCl3/
CH3OH, 20:1). EI-MS (m/z) 249 [M]+. IR (KBr) 3340 (OH);
1590, 1450, 1380, 880, 800, 690 cmꢀ1. Anal. Calcd for
C13H17N2O3: C, 62.64; H, 6.87; N, 11.24. Found: C, 62.71;
H, 6.94; N, 11.18. ESR: a five-line pattern with intensity ra-
tios of 1:2:3:2:1, aN = 8.18 G, g = 2.00994.
4.5.16. 1,3-Dihydroxy-2-(20-hydroxylphen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (5p). Mp: 139–141 ꢁC. Rf = 0.73
(CHCl3/CH3OH, 6:1). EI-MS (m/z) 252 [M]+. IR (KBr)
1
3325, 1600, 1500, 765 cmꢀ1. H NMR (DMSO-d6) d = 1.08
(s, 12H), 4.63 (s, 1H), 6.70 (d, J = 6.3 Hz, 1H), 6.74 (t,
J = 6.5 Hz, 1H), 7.14 (t, J = 6.6 Hz, 1H), 7.20 (d,
J = 6.4 Hz, 1H), 8.12 (s, 1H), 8.35 (s, 2H). Anal. Calcd for
C13H20N2O3: C, 61.88; H, 7.99; N, 11.10. Found: C, 61.93;
H, 8.05; N, 11.16.
4.6.7. 2-(30,40-Dimethoxylphen-10-yl)-4,4,5,5-tetramethyl-
imidazoline-3-oxide-1-oxyl (6g). Mp: 95–97 ꢁC. Rf = 0.34
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 293 [M]+. IR
(KBr) 2830, 1595, 1355, 870, 820 cmꢀ1. Anal. Calcd for
C15H21N2O4: C, 61.42; H, 7.22; N, 9.55. Found: C,
61.56; H, 7.29; N, 9.61. ESR: a five-line pattern with inten-
sity ratios of 1:2:3:2:1, aN = 8.28 G, g = 2.00991.
4.6. General procedure for the preparation of compounds
6a–p
The mixture of 250 mg (0.8 mmol) of 5a–p and 0.5 g of
lead dioxide in 80 mL methanol was stirred at room
temperature for 20 min and TLC (CHCl3/CH3OH,
20:1) indicated the complete disappearance of 5a–p.
The reaction mixture was filtrated and the filtrate was
evaporated under vacuum to provide 230 mg (93%) of
the title compound as dark blue crystals.
4.6.8. 2-(30-Methoxyl-40-hydroxyphen-10-yl)-4,4,5,5-tetra-
methylimidazoline-3-oxide-1-oxyl (6h). Mp: 83–85 ꢁC.
Rf = 0.38 (CHCl3/CH3OH, 20:1). EI-MS (m/z) 279 [M]+.
IR (KBr) 3340, 2830, 1590, 1345, 875, 820 cmꢀ1. Anal.
Calcd for C14H19N2O4: C, 60.20; H, 6.86; N, 10.03. Found:
C, 60.23; H, 6.91; N, 10.08. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.29 G, g = 2.00990.
4.6.1. 2-(Phen-10-yl)-4,4,5,5-tetramethylimidazoline-3-oxide-
1-oxyl (6a). Mp: 89–91 ꢁC. Rf = 0.85 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 311 [M]+. IR (KBr) 1600, 1450, 1357,
1070, 825 cmꢀ1. Anal. Calcd for C13H16N2O2Br: C, 50.02;
H, 5.17; N, 8.97. Found: C, 50.12; H, 5.23; N, 9.05. ESR:
a five-line pattern with intensity ratios of 1:2:3:2:1,
aN = 8.16 G, g = 2.00997.
4.6.9. 2-(20,40-Dichlorophen-10-yl)-4,4,5,5-tetramethylim-
idazoline-3-oxide-1-oxyl (6i). Mp: 123–125 ꢁC. Rf = 0.54
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 302 [M]+. IR (KBr)
1590, 1450, 1365, 1000, 825, 870 cmꢀ1. Anal. Calcd for
C13H15N2O2Cl2: C, 51.67; H, 5.00; N, 9.27. Found: C,
51.73; H, 5.08, N 9.35. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.15 G, g = 2.00997.
4.6.2. 2-(40-Chlorophen-10-yl)-4,4,5,5-tetramethylimidaz-
oline-3-oxide-1-oxyl (6b). Mp: 103–105 ꢁC. Rf = 0.67
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 267 [M]+. IR
4.6.10. 2-(30,40-Methylendioxyphen-10-yl)-4,4,5,5-tetrame-
thylimidazoline-3-oxide-1-oxyl (6j). Mp: 97–99 ꢁC.

Y. Wu et al. / Bioorg. Med. Chem. 14 (2006) 5711–5720
5719
Rf = 0.75 (CHCl3/CH3OH, 20:1). EI-MS (m/z) 277 [M]+.
IR (KBr) 1600, 1365, 910, 1240, 1100 cmꢀ1. Anal. Calcd
for C14H17N2O4: C, 60.64; H, 6.18; N, 10.10. Found: C,
60.72; H, 6.24; N, 10.15. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.28 G, g = 2.00990.
vacuum and the residue was purified on silica gel chro-
matograph (CHCl3) to give 300 mg (90%) of 2-(40-ethoxy-
carbonylmethoxyphen-10-yl)-4,4,5,5-tetramethylimidazoli-
dine-3-oxide-1-oxyl. To the solution of 33 mg (1.0 mmol)
of 2-(40-ethoxycarbonylmethoxyphen-10-yl)-4,4,5,5-tetram-
ethylimidazolidine-3-oxide-1-oxyl 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 and TLC (CHCl3/CH3OH, 10:1) indicated the
complete disappearance of the materials. The reaction
mixture was evaporated under vacuum and the residue
was diluted with 2 mL of saturated NaCl aqueous solu-
tion. The solution was adjusted to pH 5–6 with HCl
(2 mol/L) and extracted with CHCl3 (3· 3 mL). The
organic phase was dried with anhydrous MgSO4 and fil-
tered. The filtrate was evaporated in vacuum to give
30 mg (89%) of title compound as blue acicular crystals.
Mp: 155–157 ꢁC. EI-MS (m/z) 307 [M]+. IR (KBr) 1760;
1605, 1490, 1450, 1260, 830 cmꢀ1. Anal. Calcd for
C15H19N2O5: C, 68.62; H, 6.23; N, 9.12. Found: C,
68.69; H, 6.31; N, 9.08. ESR: a five-line pattern with inten-
sity ratios of 1:2:3:2:1, aN = 8.15 G, g = 2.00998.
4.6.11. 2-(20-Fluorophen-10-yl)-4,4,5,5-tetramethylimidaz-
oline-3-oxide-1-oxyl (6k). Mp: 112–114 ꢁC. Rf = 0.52
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 251 [M]+. IR
(KBr) 1610, 1450, 1370, 1135, 770 cmꢀ1. Anal. Calcd
for C13H16N2O4: C, 62.14; H, 6.42; N, 11.15. Found:
C, 62.23; H, 6.58; N, 11.09. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.15 G, g = 2.00996.
4.6.12. 2-(20-Nitrophen-10-yl)-4,4,5,5-tetramethylimidazo-
line-3-oxide-1-oxyl (6l). Mp: 145–147 ꢁC. Rf = 0.49
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 278 [M]+. IR
(KBr) 1530, 1360, 1450, 740 cmꢀ1. Anal. Calcd for
C13H16N3O4: C, 56.11; H, 5.79; N, 15.10. Found: C,
56.21; H, 5.85; N, 15.04. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.14 G, g = 2.00997.
4.6.13. 2-(30-Hydroxyl-40-methoxylphen-10-yl)-4,4,5,5-tetrame-
thylimidazoline-3-oxide-1-oxyl (6m). Mp: 117–119 ꢁC.
Rf = 0.34 (CHCl3/CH3OH, 20:1). EI-MS (m/z) 279 [M]+.
IR (KBr) 3320, 2830, 1595, 1500, 1350, 820 cmꢀ1. Anal.
Calcd for C14H19N2O4: C, 60.20; H, 6.86; N, 10.03. Found:
C, 60.31; H, 6.79; N, 10.15. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.25 G, g = 2.00991.
4.7.2. 2-(40-Bromoethoxyphen-10-yl)-4,4,5,5-tetramethy-
limidazolidine-3-oxide-1-oxyl (6s). The mixture of
250 mg (1.0 mmol) of 4g, 0.5 mL of 1,2-dibromoethane,
and 150 mg of sodium ethylate in 5mL of anhydrous tet-
rahydrofuran was stirred at 60 ꢁC for 8 h and TLC
(CHCl3/CH3OH, 10:1) indicated the complete disaper-
ance of 5f. The reaction mixture was filtered and the fil-
trate was evaporated in vacuum. The residue was
purified with silica gel chromatograph (CHCl3/metha-
nol, 50:1) to give 162 mg (64%) of 6s as blue acicular
crystals. Mp: 117–118 ꢁC. EI-MS (m/z) 356 [M]+. IR
(KBr) 1600, 1500, 1450, 1250, 840 cmꢀ1. Anal. Calcd
for C15H20N2O2Br: C, 50.57; H, 5.66; N, 7.86. Found:
C, 50.52; H, 5.71; N, 7.93 ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.30 G, g = 2.00990.
4.6.14. 2-(40-N,N-dimethylphen-10-yl)-4,4,5,5-tetramethyl-
imidazoline-3-oxide-1-oxyl
(6n).
Mp:
151–154 ꢁC.
Rf = 0.51 (CHCl3/CH3OH, 20:1). EI-MS (m/z) 276 [M]+.
IR (KBr) 2850, 1595, 1500, 1360, 845 cmꢀ1. Anal. Calcd
for C15H22N3O2: C, 65.19; H, 8.02; N, 15.21. Found: C,
65.25; H, 8.13; N, 15.16. ESR: a five-line pattern with
intensity ratios of 1:2:3:2:1, aN = 8.27 G, g = 2.00992.
4.6.15. 2-(20,40-Dimethoxylphen-10-yl)-4,4,5,5-tetramethyl-
imidazoline-3-oxide-1-oxyl (6o). Mp: 75–77 ꢁC. Rf = 0.37
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 293 [M]+. IR (KBr)
2830, 1610, 1365, 805, 870 cmꢀ1. Anal. Calcd for
C15H21N2O4: C, 61.42; H, 7.22; N, 9.55. Found: C,
61.49; H, 7.31; N, 9.62. ESR: a five-line pattern with inten-
sity ratios of 1:2:3:2:1, aN = 8.32 G, g = 2.00989.
4.8. Determination of ESR
A center field of 3480 G, sweep width of 100 G, sweep time
of 100 s, modulation amplitude of 1.0 · 10ꢀ1 G, time con-
stant of 1.6 · 10ꢀ1 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 6a–s at two different concentrations, 10ꢀ7
and 10ꢀ5 mol/L, and in desecrated water and phosphate
buffer (pH 7.4), respectively, were recorded.
4.7. 2-(20-Hydroxylphen-10-yl)-4,4,5,5-tetramethylimi-
dazoline-3-oxide-1-oxyl (6p)
Mp: 83–85 ꢁC. Rf = 0.75 (CHCl3/CH3OH, 20:1). EI-MS
(m/z) 249 [M]+. IR (KBr) 3345, 1610, 1340, 760 cmꢀ1
.
4.9. NO scavenging activity determined using rat aortic
strip19
Anal. Calcd for C13H17N2O3: C, 62.64; H, 6.87; N,
11.24. Found: C, 62.58; H, 6.93; N, 11.35. ESR: a five-line
pattern with intensity ratios of 1:2:3:2:1, aN = 8.21 G,
g = 2.00993.
TheNO scavengingactivityof4a–hin the rat aortic stripwas
examined according to a published method.19 In brief,
immediately after decapitation rat aortic strips were ob-
tained 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 transduc-
ers and the relaxation–contraction curves were recorded.
Noradrenaline (NE, final concentration 10ꢀ9 mol/L) solu-
tion was added to induce contraction. When the hypertonic
contraction reached to the maximum level, NE was washed
4.7.1. 2-(40-Oxymethylcarboxylphen-10-yl)-4,4,5,5-tetrame-
thylimidazolidine-3-oxide-1-oxyl (6q). The mixture of
250 mg(1.0 mmol) of 4g, 0.32 mL of ethyl bromoacetate,
and 100 mg of sodium ethylate in 5 mL of anhydrous tet-
rahydrofuran was stirred at 60 ꢁC for 5 h and TLC
(CHCl3/CH3OH, 20:1) indicated the complete disappear-
ance of 4g. The reaction mixture was evaporated under

5720
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renewal of the solution, NE (final concentration 10ꢀ9 mol/
L) was added. When the hypertonic contraction value of
aortic strips reached the peak, 15 L of NS or the solution
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Å
Å
H2O2 , and OH in PC12 cells23,27,28
Free radical scavenging activities were evaluated in
PC12 cells using a method of Dawson with minor mod-
ifications. 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 bo-
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mL penicillin, and 100 g/mL streptomycin at 37 ꢁC, in
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Acknowledgments
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This work was supported by Beijing area major labora-
tory of peptide and small molecular drugs, National
Natural Scientific Foundation of China (30472071,
20232020), and National Natural Scientific Foundation
of Beijing (7052010).
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