A new class of analgesic agents toward prostacyclin receptor inhibition: Synthesis, biological studies and QSAR analysis of 1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazolines

Article abstract of DOI:10.1016/j.ejmech.2007.07.007

By studying the structural similarity of analgesic imidazolines and 2-phenylnitronyl nitroxides, 20 1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazolines (2a-t) were newly synthesized as selective antagonists of prostacyclin receptor (IP receptor). In the in vivo tail-flick assay, 2a-t (dose, 0.13 mmol/kg) receiving mice showed increased pain thresholds ranging from 20.52 ± 7.25% to 90.94 ± 11.97%, which were significantly higher than that ranged from 12.27 ± 9.56% to 17.71 ± 7.00% shown by normal saline (NS) receiving mice. In the in vivo tail bleeding assay, 2a-t (dose, 1.30 mmol/kg) receiving mice gave a bleeding time ranging from 116.3 ± 8.0 s to 119.6 ± 7.1 s, and NS receiving mice gave a bleeding time ranging from 116.7 ± 7.5 s to 119.1 ± 8.7 s, which were at a substantially equal level. These observations imply that no bleeding risk occurred even when 10-fold dose of analgesic assay was used. In the in vitro vasorelaxation assay, it was found that when the aortic strip contracted by noradrenaline (NE, final concentration, 10^-7 M) was treated with the solution of 2a-t in NS (final concentration, 5 × 10^-3 M) only lower percentage inhibitions ranged from 6.63 ± 2.72% to 46.28 ± 2.63% were recorded. Relatively higher concentration of 2a-t (5 × 10^-3 M) and relatively lower percentage inhibitions (13 of 20 less than 23.27 ± 3.47%) suggest that 2a-t exhibit few vasodilation activity. To shed some light on the potential analgesic mechanisms of 2a-t, moreover, a QSAR analysis was carried out by using the multiple linear regression method. Taken altogether, the current study confirms that as selective antagonist of IP receptor 1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazoline may be a promising lead compound of analgesic agent without cardiovascular and bleeding side effects.

Full text of DOI:10.1016/j.ejmech.2007.07.007

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European Journal of Medicinal Chemistry 43 (2008) 1048e1058
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Original article
A new class of analgesic agents toward prostacyclin receptor inhibition:
Synthesis, biological studies and QSAR analysis of
1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazolines
Ming Zhao a, Zheng Li b, Li Peng a, Yu-Rong Tang c, Chao Wang b,
Ziding Zhang c, , Shiqi Peng
*
a,**
a College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
b College of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
c College of Biological Sciences, China Agricultural University, Beijing 100094, PR China
Received 22 March 2007; received in revised form 1 June 2007; accepted 9 July 2007
Available online 27 July 2007
Abstract
By studying the structural similarity of analgesic imidazolines and 2-phenylnitronyl nitroxides, 20 1-hydroxyl-2-substituted phenyl-4,4,5,5-
tetramethylimidazolines (2aet) were newly synthesized as selective antagonists of prostacyclin receptor (IP receptor). In the in vivo tail-flick
assay, 2aet (dose, 0.13 mmol/kg) receiving mice showed increased pain thresholds ranging from 20.52 ꢀ 7.25% to 90.94 ꢀ 11.97%, which were
significantly higher than that ranged from 12.27 ꢀ 9.56% to 17.71 ꢀ 7.00% shown by normal saline (NS) receiving mice. In the in vivo tail
bleeding assay, 2aet (dose, 1.30 mmol/kg) receiving mice gave a bleeding time ranging from 116.3 ꢀ 8.0 s to 119.6 ꢀ 7.1 s, and NS receiving
mice gave a bleeding time ranging from 116.7 ꢀ 7.5 s to 119.1 ꢀ 8.7 s, which were at a substantially equal level. These observations imply that
no bleeding risk occurred even when 10-fold dose of analgesic assay was used. In the in vitro vasorelaxation assay, it was found that when the
aortic strip contracted by noradrenaline (NE, final concentration, 10ꢁ7 M) was treated with the solution of 2aet in NS (final concentration,
5 ꢂ 10ꢁ3 M) only lower percentage inhibitions ranged from 6.63 ꢀ 2.72% to 46.28 ꢀ 2.63% were recorded. Relatively higher concentration
of 2aet (5 ꢂ 10ꢁ3 M) and relatively lower percentage inhibitions (13 of 20 less than 23.27 ꢀ 3.47%) suggest that 2aet exhibit few vasodilation
activity. To shed some light on the potential analgesic mechanisms of 2aet, moreover, a QSAR analysis was carried out by using the multiple
linear regression method. Taken altogether, the current study confirms that as selective antagonist of IP receptor 1-hydroxyl-2-substituted phe-
nyl-4,4,5,5-tetramethylimidazoline may be a promising lead compound of analgesic agent without cardiovascular and bleeding side effects.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Tetramethylimidazoline; Analgesic; Vasorelaxation; QSAR
1. Introduction
[9], aimed at developing chemotherapy for the diseases medi-
ated by those receptors, including inflammatory, glaucoma,
stroke, hypertension, rheumatoid arthritis, Alzheimer’s
disease, nasal decongestion, hypotension, hyperglycemia,
depression, liver cell proliferation, hyperaggregability of
platelets, benign prostatic hyperplasia, erectile dysfunction
and dermal necrosis. In order to find selective antagonists of
individual receptor and finally selective chemotherapy for in-
dividual disease, a lot of interest was attracted by the structural
diversity of imidazolines, and the intense efforts were mounted
to prepare analgesic imidazolines.
During the past years a number of imidazolines were pre-
pared to discover the antagonists for human adenosine recep-
tors (A3 Ars) [1e3], a-adrenergic receptor subtypes [4e7],
P2X7 receptor [8] and prostacyclin receptor (IP receptor)
* Corresponding author. Tel.: þ86 10 6273 4376; fax: þ86 10 6273 1332.
** Corresponding author. Tel.: þ86 10 8391 1528; fax: þ86 10 8391 1528.
E-mail addresses: zidingzhang@cau.edu.cn (Z. Zhang), sqpeng@mail.
bjum.edu.cn (S. Peng).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.07.007

M. Zhao et al. / European Journal of Medicinal Chemistry 43 (2008) 1048e1058
1049
It was observed that as a major mediator of inflammation
and pain prostacyclin, one of several prostanoids released
upon tissue damage, may alter the pain and inflammatory re-
sponses of transgenic mice lacking IP receptor. Thus it was
further hypothesized that an IP receptor antagonist could be
an analgesic/anti-inflammatory agent without cardiovascular
and thrombotic side effects [10]. Based on this understanding
2-phenylaminoimidazoline was used as selective antagonist of
IP receptor and thus as selective blocker of inflammatory pain.
With the assistance of the radioligand binding technology,
the IP receptor affinities of 460 2-phenylaminoimidazolines
were determined and [(4,5-dihydro-1H-imidazol-2-yl)-[4-(4-
iso-propoxybenzyl)phenyl]amine] was found to have analgesic
activity in the rat [11,12]. In addition, 4,4,5,5-tetramethylimi-
dazoline was particularly used as a building block to construct
2-substituted nitronyl nitroxides such as 2-substituted phenyl-
nitronyl nitroxides [13,14].
By comparing the structures of analgesic imidazolines
(without cardiovascular and thrombotic side effects) and 2-
substituted phenyl-4,4,5,5-tetramethylimidazolines (general
structure of nitronyl nitroxides), we hypothesized that 1-hy-
droxyl-2-phenyl-4,4,5,5-tetra-methylimidazoline could have
analgesic potential via selectively inhibiting IP receptor acti-
vation. In the present paper, 20 1-hydroxyl-2-substituted phe-
nyl-4,4,5,5-tetramethylimidazolines were prepared, their in
vivo analgesic potential was evaluated by the tail-flick test,
the lack of cardiovascular and thrombotic side effects was con-
firmed by the in vitro vasorelaxation assay and the in vivo tail
bleeding time assay, respectively. By selecting appropriate
molecular descriptors, the QSAR equation of 20 1-hydroxyl-
2-substituted phenyl-4,4,5,5-tetra-methylimidazolines was
constructed using the multiple linear regression method.
The condensation of 2,3-bis(hydroxylamino)-2,3-dimethylbu-
tane and substituted phenyl aldehydes provided 1,3-dihy-
droxyl-2-substituted-phenyl-4,4,5,5-tetramethylimidazolidines
(1aet) and the yield significantly depended on the lipophilic-
ity and space hindrance of the substituents at the phenyl.
Hydrophilic and multiple substitutions usually led to lower
yields (31e85%). With anhydrous magnesium sulfate as the
dehydration agent one of the two hydroxyl groups of 1aet
was removed via water elimination, and 1-hydroxyl-2-
(substituted phen-10-yl)-4,4,5,5-tetramethylimidazoline (2aet)
were obtained. The yields of the eliminations depended on the
electrical properties of the substituents at the phenyl group,
when the phenyl group were substituted by electron releasing
groups such as methyl, methoxy and methylenedioxy the
yields ranged from 85% to 87%, and when the phenyl group
were substituted by electron withdrawing groups such as
NO2 the yields ranged from 72% to 74%. Considering that
water elimination depends on the leaving ability of the
hydroxyl group and the electron releasing substituent at the
phenyl assists the hydroxyl group to leave, the mentioned
electrical effects should be understandable.
2.2. As antagonists of IP receptor 1-hydroxyl-2-
substituted phenyl-4,4,5,5-tetramethylimidazolines
significantly increasing pain threshold of the treated mice
As antagonist of IP receptor the in vivo analgesic action of
2aet was first evaluated with a tail-flick assay [17,18]. The ba-
sic pain threshold (BPT) and the pain threshold after a single
intraperitoneal (i.p.) injection (PTAD) of 2aet (dose,
0.13 mmol/kg) receiving mice were measured at 30 min inter-
vals totally for 150 min. Using BPT, PTAD and AAPT values
(PTAD minus BPT), and via the equation PTV ¼AAPT/BPT
the pain threshold variation (PTV) was calculated. All the
PTV values for each animal were averaged and constituted
one sample. The statistical analysis was carried out by use
of ANOVA test, and p < 0.05 is considered significant.
2. Results and discussion
2.1. Preparing 1-Hydroxyl-2-substituted phenyl-4,4,5,5-
tetramethylimidazolines via simple procedures
It was found that nearly all 2aet receiving mice gave an in-
creased PTV value during the experimental 150 min, suggest-
ing that 2aet exhibited obvious analgesic activity at the
dosage of 0.13 mmol/kg (Table 1). On the other hand, how-
ever, after 30 min of a single i.p. injection the pain thresholds
in mice were increased by 2aet, but the maximal pain thresh-
olds occurred at different time points. The maximal pain
thresholds for 2a,g,h,p receiving mice was measured after
To prepare the desired 1-hydroxyl-2-substituted
phenyl-4,4,5,5-tetramethylimidazolines (2aet) in acceptable
yield, the synthetic route shown in Scheme 1 was chosen
[15,16]. The bromination of 2-nitropropane gave 2,3-
dimethyl-2,3-dinitrobutane in 73% yield. Upon reduction
2,3-dimethyl-2,3-dinitrobutane was smoothly converted into
2,3-bis(hydroxyl-amino)-2,3-dimethylbutane (63% yield).
N
N
HO
N
OH
HO
N
NO2
CH
iv
i
H3C
CH3
HO
O2N
NO2
OH
NHHN
R
2a-t
R
1a-t
Scheme 1. Synthetic route of 1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazolines. (i) Br2 and NaOH (6 mol/l), (ii) Zn and NH4Cl, (iii) ReC6H4CHO,
(iv) anhydrous magnesium sulfate. In (1,2)a R ¼ H, (1,2)b R ¼ 2-OH, (1,2)c R ¼ 3-OH, (1,2)d R ¼ 4-OH, (1,2)e R ¼ 4-CH3, (1,2)f R ¼ 4-OCH3, (1,2)g R ¼ 2,4-
dimethoxyl, (1,2)h R ¼ 3,4-dimethoxyl, (1,2)i R ¼ 3,4-methylenedioxy, (1,2)j R ¼ 3-OH-4-OCH3, (1,2)k R ¼ 3-OCH3-4-OH, (1,2)l R ¼ 4-N(CH3)2, (1,2)m R ¼ 2-F,
(1,2)n R ¼ 4-Cl, (1,2)o R ¼ 4-Br, (1,2)p R ¼ 2,4-di-Cl, (1,2)q R ¼ 3,4- di-Cl, (1,2)r R ¼ 2-NO2, (1,2)s R ¼ 3-NO2, (1,2)t R ¼ 4-NO2.

1050
M. Zhao et al. / European Journal of Medicinal Chemistry 43 (2008) 1048e1058
Table 1
The effect of 2aet on the pain threshold in mice
Compd.
Pain threshold variation ðx ꢀ SD%Þ
30 min
60 min
90 min
120 min
150 min
NS
2a
2b
2c
2d
2e
2f
12.45 ꢀ 7.21
13.40 ꢀ 8.47
17.71 ꢀ 7.00
12.27 ꢀ 9.56
17.43 ꢀ 9.18
43.67 ꢀ 12.29a
50.76 ꢀ 9.58a
50.69 ꢀ 11.74a
57.60 ꢀ 10.64a
42.06 ꢀ 10.02a
64.55 ꢀ 10.06a
52.93 ꢀ 11.36a
50.90 ꢀ 10.06a
58.82 ꢀ 11.00a
44.33 ꢀ 9.14a
47.57 ꢀ 7.24a
34.30 ꢀ 10.19a
66.15 ꢀ 10.15a
63.85 ꢀ 11.45a
29.02 ꢀ 7.40a
46.97 ꢀ 9.96a
20.52 ꢀ 7.25b
28.95 ꢀ 7.72a
52.76 ꢀ 11.61a
44.15 ꢀ 10.91a
37.68 ꢀ 10.65a
49.93 ꢀ 10.48a
59.38 ꢀ 11.70a
74.74 ꢀ 12.54a
55.16 ꢀ 10.35a
76.13 ꢀ 11.76a
44.85 ꢀ 8.62a
50.12 ꢀ 10.68a
68.46 ꢀ 10.79a
59.85 ꢀ 10.64a
61.52 ꢀ 11.6a
39.44 ꢀ 9.37a
72.72 ꢀ 11.16a
70.21 ꢀ 11.6a
89.78 ꢀ 12.43a
46.31 ꢀ 9.56a
21.43 ꢀ 7.42b
45.14 ꢀ 9.2a
37.07 ꢀ 10.00a
68.27 ꢀ 11.64a
59.94 ꢀ 11.71a
79.80 ꢀ 12.68a
48.82 ꢀ 10.60a
66.49 ꢀ 10.85a
37.41 ꢀ 9.33a
73.13 ꢀ 11.90a
77.78 ꢀ 11.00a
53.45 ꢀ 11.44a
39.96 ꢀ 9.07a
65.23 ꢀ 11.70a
38.34 ꢀ 7.48a
38.43 ꢀ 9.55a
59.77 ꢀ 10.48a
50.77 ꢀ 11.76a
52.21 ꢀ 10.62a
37.34 ꢀ 9.66a
75.97 ꢀ 10.14a
74.74 ꢀ 10.40a
75.77 ꢀ 11.72a
46.12 ꢀ 8.89a
40.17 ꢀ 7.87a
50.91 ꢀ 10.25a
60.26 ꢀ 12.18a
35.97 ꢀ 8.90a
35.48 ꢀ 11.12a
67.30 ꢀ 10.15a
72.88 ꢀ 10.40a
27.62 ꢀ 7.54b
45.41 ꢀ 9.24a
46.74 ꢀ 10.77a
37.89 ꢀ 8.76a
35.37 ꢀ 8.41a
60.22 ꢀ 11.07a
41.62 ꢀ 9.32a
a
2g
2h
2i
44.96 ꢀ 9.96
54.54 ꢀ 10.38a
67.15 ꢀ 11.33a
60.71 ꢀ 11.50a
60.65 ꢀ 11.33a
40.17 ꢀ 11.05a
73.25 ꢀ 11.61a
75.61 ꢀ 11.22a
75.18 ꢀ 11.36a
44.10 ꢀ 9.32a
41.90 ꢀ 8.24a
45.93 ꢀ 9.88a
63.46 ꢀ 10.03a
36.31 ꢀ 7.44a
2j
a
2k
2l
40.56 ꢀ 9.57
31.27 ꢀ 9.00a
90.94 ꢀ 11.97a
88.42 ꢀ 10.70a
70.19 ꢀ 12.06a
45.99 ꢀ 9.83a
50.62 ꢀ 10.10a
40.04 ꢀ 9.84a
70.83 ꢀ 12.26a
47.81 ꢀ 8.51a
2m
2n
2o
2p
2q
2r
2s
62.07 ꢀ 11.20a
2t
38.32 ꢀ 9.69a
n ¼ 10; dose ¼ 0.13 mmol/kg; NS (normal saline) ¼ vehicle.
a
Compared to NS, p < 0.01.
b
Compared to NS, p < 0.05.
30 min of their i.p. injection, for 2e,f,iek receiving mice was
measured after 60 min of their i.p. injection, for 2d receiving
mice was measured after 90 min of its i.p. injection, for 2b,c,r
receiving mice was measured after 120 min of their i.p. injec-
tion, and for 2m,n,q,s,t receiving mice was measured after
150 min of their i.p. injection. During the measurements,
2a,g,h,p,t steadily exerted their moderate analgesic action.
Among all 2aet, the analgesic potentials of 2meo were sig-
nificantly higher than those of the others. Based on these ob-
servations, we recognized that the effect of the substituents
at the phenyl on the analgesic potential can be simply de-
duced. Comparing to phenyl itself, the substitution of Me,
OMe, di-OMe, di-Cl, N(Me)2, and NO2 did not enhance the
analgesic activity of the imidazolines, the substitution of OH
moderately enhanced the analgesic activity of the imidazo-
lines, and the substitution of F, Cl and Br usually significantly
enhanced the analgesic activity of the imidazolines.
i.p. injection of a mixed solvent of 0.2 ml of NS and 0.01 ml
of DMSO and 0.02 ml of Tween-80, or a solution of 2aet
(dose, 1.30 mmol/kg) in the mixed solvent, the mouse was re-
placed in the tube holder with its tail protruding. After 5, 15,
30 and 60 min of the i.p. injection 1 mm cut was also made on
the tail. Flowing blood until it stopped was gently wiped away
with a filter paper every 30 s and BT was recorded. All the BT
values for each animal were averaged and constituted one
sample. The statistical analysis was carried out by use of
ANOVA test, and p < 0.05 is considered significant.
It was found that the BT values before i.p. injection or after 5,
15, 30 and 60 min of the i.p. injection for the mice receiving the
solvent only and for the mice receiving the solution of 2aet in
the solvent fell into a range of 116.7 ꢀ 7.5 s to 119.1 ꢀ 8.7 s
and 116.3 ꢀ 8.0 s to 119.6 ꢀ 7.1 s, respectively (Table 2),
2aet exhibiting no thrombotic action. It should be mentioned
that the dose used in bleeding assay is ten-fold higher than
that used in analgesic assay. Based on these experimental
observations and the dose comparison one may conclude
that as IP targeting analgesic 1-hydroxyl-2-substituted
phenyl-4,4,5,5-tetramethylimidazolines have no thrombotic
risk.
2.3. As antagonists of IP receptors 1-hydroxyl-2-
substituted phenyl-4,4,5,5-tetramethyl-imidazolines
having no thrombotic risk
According to the hypothesis that an IP receptor antagonist
could be an analgesic agent without thrombotic side effects
[10], such as bleeding action, the tail bleeding assays of
2aet were consequently performed using a method reported
in the literature [18]. In brief, the mouse was placed in
a tube holder with its tail protruding, and a 1 mm cut was
made on the tail. Flowing blood until it stopped was gently
wiped away with a filter paper every 30 s and the bleeding
time (BT) was recorded. Then the mouse was given a single
2.4. As antagonists of IP receptor 1-hydroxyl-2-
substituted phenyl-4,4,5,5-tetramethylimidazolines
exhibiting few vasorelaxation activities
According to another hypothesis that an IP receptor antag-
onist could be an analgesic agent without cardiovascular side
effects [10], and 2aet share a structural similarity with the va-
sodilator imidazolines [15,19], they were examined to see if

M. Zhao et al. / European Journal of Medicinal Chemistry 43 (2008) 1048e1058
1051
Table 2
Effect of 2aet on the tail bleeding time ðx ꢀ SDsÞ of mice
Compd.
Before administration
After administration
5 min
15 min
30 min
60 min
NS
2a
2b
2c
2d
2e
2f
117.1 ꢀ 8.4
119.5 ꢀ 8.3
118.6 ꢀ 8.4
116.6 ꢀ 9.2
118.3 ꢀ 9.3
117.6 ꢀ 8.5
119.6 ꢀ 8.2
118.2 ꢀ 9.1
117.5 ꢀ 8.4
118.3 ꢀ 8.5
116.3 ꢀ 8.0
119.5 ꢀ 8.3
117.7 ꢀ 9.2
116.6 ꢀ 8.4
118.6 ꢀ 9.0
118.1 ꢀ 8.5
116.4 ꢀ 9.1
117.5 ꢀ 9.2
119.5 ꢀ 8.7
116.6 ꢀ 9.1
118.1 ꢀ 9.2
119.1 ꢀ 8.7
116.7 ꢀ 7.3
118.2 ꢀ 9.2
116.6 ꢀ 8.5
116.3 ꢀ 8.3
119.1 ꢀ 8.7
119.3 ꢀ 8.3
117.5 ꢀ 8.5
116.7 ꢀ 8.1
119.6 ꢀ 7.3
119.6 ꢀ 7.1
119.2 ꢀ 9.2
117.4 ꢀ 9.5
117.1 ꢀ 8.3
117.2 ꢀ 8.4
118.4 ꢀ 8.1
116.7 ꢀ 8.7
118.4 ꢀ 8.3
119.0 ꢀ 8.1
116.8 ꢀ 8.2
117.6 ꢀ 8.5
116.7 ꢀ 7.5
119.0 ꢀ 7.2
116.8 ꢀ 7.3
119.1 ꢀ 8.2
118.3 ꢀ 7.4
118.5 ꢀ 8.3
118.1 ꢀ 7.2
118.2 ꢀ 8.0
116.9 ꢀ 9.3
118.6 ꢀ 8.5
118.1 ꢀ 9.4
118.2 ꢀ 9.3
116.9 ꢀ 8.6
116.8 ꢀ 8.1
119.0 ꢀ 9.0
119.1 ꢀ 8.3
116.7 ꢀ 8.5
117.5 ꢀ 9.0
119.1 ꢀ 8.4
118.2 ꢀ 8.2
117.0 ꢀ 8.6
116.9 ꢀ 8.0
117.7 ꢀ 8.2
119.2 ꢀ 7.4
117.7 ꢀ 9.0
119.0 ꢀ 7.3
116.9 ꢀ 9.2
117.8 ꢀ 8.4
119.1 ꢀ 7.5
117.8 ꢀ 9.0
119.2 ꢀ 9.1
116.7 ꢀ 7.4
119.2 ꢀ 8.2
116.9 ꢀ 8.5
119.0 ꢀ 8.2
116.9 ꢀ 8.1
118.1 ꢀ 8.0
119.3 ꢀ 9.4
117.5 ꢀ 8.6
119.4 ꢀ 8.2
119.1 ꢀ 8.3
116.9 ꢀ 8.5
118.1 ꢀ 8.0
116.9 ꢀ 7.4
118.3 ꢀ 8.3
119.2 ꢀ 8.2
119.4 ꢀ 8.0
119.1 ꢀ 7.5
117.5 ꢀ 9.2
116.9 ꢀ 8.1
118.2 ꢀ 9.2
118.1 ꢀ 7.7
119.0 ꢀ 7.8
116.8 ꢀ 8.1
119.2 ꢀ 9.4
119.1 ꢀ 8.7
119.4 ꢀ 8.2
119.5 ꢀ 8.0
119.2 ꢀ 9.0
116.9 ꢀ 9.2
118.2 ꢀ 8.5
116.8 ꢀ 9.2
116.9 ꢀ 8.3
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
n ¼ 10, dose ¼ 1.3 mmol/kg; NS (normal saline) ¼ vehicle.
they can convert the vasoconstriction induced by noradrena-
line (NE) using the rat aortic strip model. When the aortic strip
contracted by NE (final concentration 10ꢁ7 M) was treated
with the solution of 2aet in NS (final concentration
5 ꢂ 10ꢁ3 M), only lower percentage inhibitions were observed
(Table 3). The relatively higher final concentration and the
lower vasorelaxation potency ranging from 6.63 ꢀ 2.72% to
46.28 ꢀ 2.63% suggest that 2aet exhibit few vasodilation
activities and thus without cardiovascular side effects.
profile (Table 4). Statistical analysis was carried out using
one way ANOVA test with p < 0.05 as significant cut-off. Af-
ter i.p. injection of the solution of 0.13 mmol/kg, 13 mmol/kg
or 1.3 mmol/kg of 2m in NS, the observed pain thresholds
were 66.15 ꢀ 10.15, 45.44 ꢀ 9.33 and 27.64 ꢀ 9.35, respec-
tively; of 2n were 63.85 ꢀ 11.45, 39.87 ꢀ 9.48 and
24.97 ꢀ 7.37, respectively; of 2o were 29.02 ꢀ 7.40,
19.33 ꢀ 8.25 and 11.32 ꢀ 7.32, respectively (Table 4). These
data demonstrate that the mice receiving different doses
of 1-hydroxyl-2-substituted-phenyl-4,4,5,5-tetramethylimida-
zolines may give different pain response.
2.5. As antagonists of IP receptor 1-hydroxyl-2-
substituted phenyl-4,4,5,5-tetramethyl- imidazolines
(2meo) dose-dependent increasing pain threshold of the
treated mice
2.6. QSAR of 2aet
Selecting the molecular descriptors generated from the
e-dragon server [20], the multiple linear regression method
was employed to derive the QSAR equation of 2aet. In the
present study, four descriptors (IDET, Mor07e, Mor13e and
MATS6v) can result in the following regression model with
the highest predictive accuracy.
To reveal the dose-dependent analgesic action, the com-
pounds with significant activity (2meo) were administrated
using three different dosages (0.13 mmol/kg, 13 mmol/kg and
1.3 mmol/kg) to obtain a detailed pharmacological activity
Table 3
Conversion of 2aet during vasoconstriction induced by NE
PTV90 ¼ ꢁ1424:11ð ꢀ 240:56Þ þ 795:06ð ꢀ 126:04Þ ꢂ IDET
ꢁ 20:92ð ꢀ 3:71Þ ꢂ Mor07e þ 20:49ð ꢀ 5:13Þ ꢂ JGI1
ꢁ 10:26ð ꢀ 2:65Þ ꢂ MATS6v
Compd.
x ꢀ SD%
Compd.
x ꢀ SD%
2a
2b
2c
2d
2e
2f
22.93 ꢀ 4.15
22.85 ꢀ 1.20
46.28 ꢀ 2.63
13.60 ꢀ 1.83
16.73 ꢀ 3.06
19.69 ꢀ 1.05
23.27 ꢀ 3.47
17.92 ꢀ 2.37
21.95 ꢀ 1.13
35.11 ꢀ 1.92
2k
2l
20.63 ꢀ 3.25
6.63 ꢀ 2.72
22.70 ꢀ 3.83
23.78 ꢀ 2.19
18.40 ꢀ 3.46
15.93 ꢀ 1.13
22.78 ꢀ 2.73
30.20 ꢀ 3.72
31.80 ꢀ 3.21
32.70 ꢀ 3.36
2m
2n
2o
2p
2q
2r
2s
N ¼ 20; R ¼ 0:94; S ¼ 4:47; e ¼ 3:84;
RLOO ¼ 0:88; SLOO ¼ 6:06; eLOO ¼ 5:22;
F ¼ 27:75; p < 10ꢁ5; AIC ¼ 44:40; FIT ¼ 3:05 ð1Þ
2g
2h
2i
where PTV90 (i.e. the PTV value measured at 90 min) is con-
sidered as the analgesic activity of a compound. The regression
coefficient R is 0.94. The resubstitution analysis gives an
2j
2t
n ¼ 6; the final concentration of 2aet was 5 ꢂ 10ꢁ3 M.

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M. Zhao et al. / European Journal of Medicinal Chemistry 43 (2008) 1048e1058
Table 4
Effect of 2meo on the pain threshold in mice at different dosages
Compd., dose (mmol/kg)
Pain threshold variation ðx ꢀ SD%Þ
30 min
60 min
90 min
120 min
150 min
Control
12.45 ꢀ 7.21
66.15 ꢀ 10.15a
45.44 ꢀ 9.33b
27.64 ꢀ 9.35c
63.85 ꢀ 11.45a
39.87 ꢀ 9.48b
24.97 ꢀ 7.37
29.02 ꢀ 7.40a
19.33 ꢀ 8.25b
11.32 ꢀ 7.32
13.40 ꢀ 8.47
72.72 ꢀ 11.16a
53.11 ꢀ 9.72b
36.02 ꢀ 9.41c
70.21 ꢀ 11.6a
47.25 ꢀ 9.15b
30.02 ꢀ 9.06c
89.78 ꢀ 12.43a
68.89 ꢀ 9.12b
43.90 ꢀ 9.30c
17.71 ꢀ 7.00
73.25 ꢀ 11.61a
54.00 ꢀ 9.65b
36.96 ꢀ 9.33c
75.61 ꢀ 11.22a
50.88 ꢀ 9.19b
32.13 ꢀ 8.98c
75.18 ꢀ 11.36a
53.75 ꢀ 9.29b
34.49 ꢀ 9.48c
12.27 ꢀ 9.56
75.97 ꢀ 10.14a
56.97 ꢀ 10.00b
37.80 ꢀ 9.14c
74.74 ꢀ 10.40a
50.00 ꢀ 9.22b
31.55 ꢀ 9.50c
75.77 ꢀ 11.72a
52.98 ꢀ 9.02b
31.87 ꢀ 9.33c
17.43 ꢀ 9.18
90.94 ꢀ 11.97a
69.89 ꢀ 9.06b
47.03 ꢀ 9.14c
88.42 ꢀ 10.70a
62.76 ꢀ 9.09b
42.74 ꢀ 8.99c
70.19 ꢀ 12.06a
47.70 ꢀ 9.33b
26.57 ꢀ 9.30c
2m, 0.1300
2m, 0.0130
2m, 0.0013
2n, 0.1300
2n, 0.0130
2n, 0.0013
2o, 0.1300
2o, 0.0130
2o, 0.0013
n ¼ 10.
a
Compared to group of 0.0130 mmol/kg dose, p < 0.01.
Compared to group of 0.0013 mmol/kg dose, p < 0.01.
Compared to control p < 0.01.
b
c
absolute average error ðeÞ of 3.84, whereas the absolute aver-
age error in the Leave-One-Out (LOO) test (i.e. eLOO) is 5.22.
More details about the statistical parameters in Eq. (1) are
available in Section 3. The IDET is one of the descriptors of
information indices [21]. The Mor07e, Mor13e belong to the
3D-MoRSE descriptors, which are based on the idea of obtain-
ing information from the 3D atomic coordinates by the trans-
form used in electron diffraction studies for preparing
theoretical scattering curves [22e24]. The Mor07e and
Mor13e correspond to signal 7 and 13, weighted by atomic
Sanderson electronegativities [23]. Grouped into the 2D auto-
correlation descriptors, the MATS6v is related to the atomic
van der Waals volumes of a molecule [25]. A pairwise corre-
lation analysis of the above four variables was carried out
and all the pairwise correlation coefficients between different
variables are in the range from ꢁ0.39 to 0.22 (Table 5), imply-
ing that there is no significant correlation existing between any
two variables.
be their electrostatic and steric effects. It can be envisioned
that 2aet are involved in the interaction with the same recep-
tor, therefore a more elegant 3D-QSAR analysis (e.g. CoMFA
[30]) may provide new insight into the molecular mechanisms
of their analgesic effects.
3. Experimental section
3.1. General
The purity of all the compounds were confirmed by TLC
(Merck silica gel plates of type 60 F254, 0.25 mm layer thick-
ness) and HPLC (waters, C18 column 3.9 ꢂ 150 mm). Melting
points were measured on a XT5 hot stage microscope (Beijing
keyi electro-optic factory). The IR spectra were recorded with
a PerkineElmer 983 instrument. The EI-MS was determined
by Trace MS System instrument (American Thermo Finnigan
1
Company). The H NMR was determined by Varian INOVA-
As intuitively shown in Fig. 1, the current QSAR model of
2aet is of good predictive capability, implying that it may be
promising to forecast the analgesic activities of new 1-
hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazoline
derivatives. The QSAR studies on the analgesic activities of
different types of compounds have been widely reported in
the literature [26e29]. In addition to obtaining the predictable
QSAR models, the molecular mechanisms of analgesic effects
were also derived to some extent. In Eq. (1), two descriptors
(Mor07e and Mor13e) mainly reflect the general 3D structure
and electrostatic effect of a molecule and the other two de-
scriptors (IDET and MATS6v) are more involved in describing
the compounds’ topological and steric effects. Therefore, the
major determinants for the analgesic activities of 2aet would
100
90
80
70
60
50
40
30
20
Table 5
Correlation matrix of the variables in Eq. (1)
20
30
40
50
60
70
80
90
100
IDET
1.00
Mor07e
Mor13e
MATS6v
Tested PTV
IDET
ꢁ0.27
ꢁ0.15
0.22
1.00
ꢁ0.20
ꢁ0.19
ꢁ0.39
1.00
Fig. 1. Comparison of the predicted analgesic activities of 2aet in the LOO
test with the tested activities. The data points situated at the zone between
the two diagonal-dash lines correspond to the compounds, in which the abso-
lute prediction errors are less than 20.0.
Mor07e
Mor13e
MATS6v
1.00

M. Zhao et al. / European Journal of Medicinal Chemistry 43 (2008) 1048e1058
1053
300 MHz spectrometer. The ESR spectra were obtained from
10ꢁ5 mol/L phosphate buffer saline, using a BRUKER 300-E
spectrometer. The measurement conditions: center field,
3440 Gauss; sweep width, 100 Gauss; sweep time, 60 s; mod-
ulation amplitude, 1.1 Gauss; time constant, 8.2 ꢂ 10ꢁ2 s;
modulation frequency, 100 kHz; microwave frequency,
9.68 GHz; microwave power, 20 MW.
mentioned above, 286 mg (57%) of the title compound was
obtained. Mp 139 e 141 ꢃC. Rf ¼ 0.73 (CHCl3/CH3OH, 6:1).
EI-MS (m/z) 252 [M]þ. IR (KBr) 3325, 1600, 1500,
1
765 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.08 (s, 12H), 4.63
(s, 1H), 6.70 (d, J ¼ 6.3 Hz, 1H), 6.74 (t, J ¼ 6.5 Hz, 1H),
7.14 (t, J ¼ 6.6 Hz, 1H), 7.20 (d, J ¼ 6.4 Hz, 1H), 8.12 (s,
1H), 8.35 (s, 2H).
3.2. Synthesis of the compounds
3.2.3.3. 1,3-Dihydroxyl-2-(30-hydroxyphen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (1c). Using the general procedure men-
tioned above, 304 mg (60%) of the title compound was
obtained. Mp 244e246 ꢃC. Rf ¼ 0.56 (CHCl3/CH3OH, 6:1).
EI-MS (m/z) 252 [M]þ. IR (KBr) 3320, 1500, 880, 795,
3.2.1. 2,3-Dimethyl-2,3-dinitrobutane
At ꢁ5 ꢃC, to the solution of 34.5 g (0.39 mol) of 2-nitro-
propane in 65 ml (6.0 mol/l) of aqueous NaOH 10 ml
(0.19 mol) of Br2 were added dropwise within 1 h. With
stirring 128 ml of ethanol were added into the solution. The re-
action mixture was stirred at 84 ꢃC for 3 h. The hot reaction
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 110e112 ꢃC.
1
695 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.03 (s, 6H), 1.05
(s, 6H), 4.51 (s, 1H), 6.89 (dd, J ¼ 8.4 Hz, J ¼ 8.0 Hz, 1H),
6.92 (d, J ¼ 6.2 Hz, 1H), 7.01 (d, J ¼ 7.3 Hz, 1H), 7.71
(s, 1H), 7.86 (s, 1H), 7.95 (s, 2H).
3.2.3.4. 1,3-Dihydroxyl-2-(40-hydroxyphen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (1d). Using the general procedure men-
tioned above, 275 mg (51%) of the title compound was
obtained. Mp 240e242 ꢃC. Rf ¼ 0.67 (CHCl3/CH3OH, 6:1).
EI-MS (m/z) 252 [M]þ. IR (KBr) 3310, 1610, 1500, 1450,
3.2.2. 2,3-Bis(hydroxylamino)-2,3-dimethylbutane
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 8e10 ꢃC in an ice bath. A solution of NH4Cl (43 g,
0.8 mol) in H2O (150 ml) was added dropwise to this slurry
within 2 h, with continued stirring for 1 h at 10 ꢃC, and the
flask was stored in a fridge (4e6 ꢃC) for 16 h. The slurry
was filtered, and the precipitate was carefully washed with
100 ml of THF four times. The collected precipitates were
dried by three washings with diethyl ether and carefully col-
lected (59 g). The solution was evaporated under vacuum until
THF ceased to distil off. Then the solution was protected from
air, and sodium carbonate (50 g) and sodium chloride (30 g)
were added with cooling. Continuous extraction with chloro-
form (400 ml) was performed over 18 h. A white powder
was obtained (9.4 g, 63% yield). Mp 182 ꢃC.
1
794, 693 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.03 (s, 6H),
1.05 (s, 6H), 4.39 (s, 1H), 6.70 (d, J ¼ 6.9 Hz, 2H), 7.23 (d,
J ¼ 6.9 Hz, 2H), 7.63 (s, 1H), 7.85 (s, 2H).
3.2.3.5. 1,3-Dihydroxyl-2-(40-methylphen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (1e). Using the general procedure men-
tioned above, 350 mg (70%) of the title compound was
obtained. Mp 199e201 ꢃC. Rf ¼ 0.61 (CHCl3/CH3OH, 10:1).
EI-MS (m/z) 250 [M]þ. IR (KBr) 3335, 2985, 1600,
1
815 cmꢁ1. H NMR (CDCl3) d/ppm ¼ 1.14 (s, 6H), 1.19 (s,
6H), 1.37 (s, 3H), 4.90 (s, 1H), 7.68 (d, J ¼ 9.0 Hz, 2H), 8.22
(d, J ¼ 8.4 Hz, 2H), 8.35 (s, 2H).
3.2.3.6. 1,3-Dihydroxyl-2-(40-methoxyphen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (1f). Using the general procedure men-
tioned above, 372 mg (70%) of the title compound was
obtained. Mp 179e181 ꢃC. Rf ¼ 0.52 (CHCl3/CH3OH, 10:1).
EI-MS (m/z) 266 [M]þ. IR (KBr) 3340, 2835, 1500,
3.2.3. General procedure for preparing 1,3-dihydroxyl-2-
substituted- phenyl-4,4,5,5-tetramethylimidazolidine (1aet)
A solution of 296 mg (2 mmol) of 2,3-bis(hydroxylamino)-
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 collected by filtration. The filtration was evap-
orated under reduced pressure to obtain the second crop of the
title compound. The title compounds were directly used for the
next reaction without further purification.
1
825 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 0.99 (s, 6H), 1.03
(s, 6H), 3.73 (s, 3H), 4.56 (s, 1H), 6.88 (d, J ¼ 4.2 Hz, 2H),
7.38 (d, J ¼ 5.7 Hz, 2H), 7.77 (s, 2H).
3.2.3.7. 1,3-Dihydroxyl-2-(20,40-dimethoxylphenyl)-4,4,5,5-tet-
ramethylimidazolidine (1g). Using the general procedure men-
tioned above, 349 mg (59%) of the title compound was
obtained. Mp 140e141 ꢃC. Rf ¼ 0.49 (CHCl3/CH3OH, 10:1).
EI-MS (m/z) 296 [M]þ. IR (KBr) 3295, 2825, 1500, 865,
3.2.3.1. 1,3-Dihydroxyl-2-phenyl-4,4,5,5-tetramethylimidazoli-
dine (1a). Using the general procedure mentioned above,
401 mg (85%) of the title compound was obtained. Mp 168e
169 ꢃC. IR (KBr): 3340, 1606, 1500, 1450, 1381 cmꢁ1; H
1
810 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.03 (s, 12H), 3.72
1
(s, 6H), 4.99 (s, 1H), 7.02 (s, 1H), 7.42 (d, J ¼ 6.7 Hz, 1H),
7.45 (d, J ¼ 6.5 Hz, 1 H), 7.79 (s, 2H).
NMR (CDCl3) d/ppm ¼ 1.14 (s, 12H), 4.78 (s, 1H), 7.31e7.51
(m, J ¼ 6.9 Hz, 5H), 7.71 (s, 2H); ESI-MS (m/e) ¼ 236 [M]þ.
3.2.3.8. 1,3-Dihydroxyl-2-(30,40-dimethoxyphen-10-yl)-4,4,5,5-
tetramethylimidazolidine (1h). Using the general procedure
mentioned above, 390 mg (66%) of the title compound was
3.2.3.2. 1,3-Dihydroxyl-2-(20-hydroxylphen-10-yl)-4,4,5,5-tet-
ramethylimidazolidine (1b). Using the general procedure

1054
M. Zhao et al. / European Journal of Medicinal Chemistry 43 (2008) 1048e1058
1
obtained. Mp 147e149 ꢃC. Rf ¼ 0.52 (CHCl3/CH3OH, 6:1).
EI-MS (m/z) 296 [M]þ. IR (KBr) 3310, 2825, 1610, 865,
825 cmꢁ1. H NMR (CDCl3) d/ppm ¼ 1.03 (s, 6H), 1.07 (s,
6H), 4.50 (s, 1H), 7.38 (d, J ¼ 8.7 Hz, 2H), 7.48 (d,
J ¼ 8.4 Hz, 2H), 7.80 (s, 2H).
1
815 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.05 (s, 12H), 3.74
(s, 6H), 4.53 (s, 1H), 6.85 (s, 1H), 6.87 (d, J ¼ 6.6 Hz, 1H),
6.92 (d, J ¼ 6.4 Hz, 1H), 7.85 (s, 2H).
3.2.3.15. 1,3-Dihydroxyl-2-(40-bromophen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (1o). Using the general procedure men-
tioned above, 320 mg (51%) of the title compound was
obtained. Mp 205e207 ꢃC. Rf ¼ 0.69 (CHCl3/CH3OH, 10:1).
EI-MS (m/z) 314 [M]þ. IR (KBr) 3310, 1590, 1450, 1075,
3.2.3.9. 1,3-Dihydroxyl-2-(30,40-methylendioxyphen-10-yl)-4,4,
5,5-tetramethylimidazolidine (1i). Using the general procedure
mentioned above, 240 mg (43%) of the title compound was
obtained. Mp 188e190 ꢃC, Rf ¼ 0.47 (CHCl3/CH3OH, 10:1),
EI-MS (m/z) 280 [M]þ, IR (KBr) 3315, 1600, 915, 1235,
1
830 cmꢁ1. H NMR (CDCl3) d/ppm ¼ 1.14 (s, 12H), 4.77 (s,
1H), 7.37 (d, J ¼ 8.4 Hz, 2H), 7.47 (d, J ¼ 7.8 Hz, 2H), 7.73
1
1105 cmꢁ1. H NMR(DMSO-d6) d/ppm ¼ 1.05 (s, 12H), 3.74
(s, 2H).
(s, 6H), 4.53 (s, 1H), 6.82 (d, J ¼ 7.7 Hz, 1H), 6.84
(d, J ¼ 8.0 Hz, 1H), 6.87 (s, 1H) 7.85 (s, 2H).
3.2.3.16. 1,3-Dihydroxyl-2-(20,40-dichlorophenyl)-4,4,5,5-tet-
ramethylimidazolidine (1p). Using the general procedure men-
tioned above, 427 mg (70%) of the title compound was
obtained. Mp 201e203 ꢃC. Rf ¼ 0.69 (CHCl3/CH3OH, 10:1).
EI-MS (m/z) 305 [M]þ. IR (KBr) 3315, 1590, 1450, 995,
3.2.3.10. 1,3-Dihydroxyl-2-(30-hydroxyl-40-methoxyl) phenyl-
4,4,5,5-tetramethylimidazolidine (1j). Using the general pro-
cedure mentioned above, 276 mg (49%) of the title compound
was obtained. Mp 245e247 ꢃC. Rf ¼ 0.52 (CHCl3/CH3OH,
6:1). EI-MS (m/z) 282 [M]þ. IR (KBr) 3340, 2825, 1500,
820, 865 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.11 (s, 12H),
1
5.01 (s, 1H), 7.50 (d, J ¼ 7.6 Hz, 1H), 7.52 (d, J ¼ 8.1 Hz,
1
1450, 825 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.03 (s, 6H),
1H), 7.58 (s, 1H), 7.79 (s, 2H).
1.04 (s, 6H) 3.72 (s, 3H), 4.53 (s, 1H), 6.82 (s, 1H), 6.89 (d,
J ¼ 6.6 Hz, 1H), 6.94 (d, J ¼ 6.4 Hz, 1H), 7.65 (s, 1H), 8.04
(s, 2H).
3.2.3.17. 1,3-Dihydroxyl-2-(30,40-dichlorophenyl)-4,4,5,5-tet-
ramethylimidazolidine (1q). Using the general procedure men-
tioned above, 427 mg (70%) of the title compound was
obtained. Mp 212e214 ꢃC. Rf ¼ 0.69 (CHCl3/CH3OH, 10:1).
EI-MS (m/z) 305 [M]þ. IR (KBr) 3313, 1592, 1453, 992,
3.2.3.11. 1,3-Dihydroxyl-2-(30-methoxyl-40-hydroxyphen-10-yl)-
4,4,5,5-tetramethylimidazolidine (1k). Using the general pro-
cedure mentioned above, 186 mg (33%) of the title compound
was obtained. Mp 235e237 ꢃC, Rf ¼ 0.47 (CHCl3/CH3OH,
6:1), EI-MS (m/z) 282 [M]þ, IR (KBr) 3310, 2840, 1600, 870,
863, 822 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.06 (s, 12H),
1
4.99 (s, 1H), 7.66 (m, 3H), 7.92 (s, 2H).
1
815 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.00 (s, 6H), 1.03
3.2.3.18. 1,3-Dihydroxyl-2-(20-nitrophen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (1r). Using the general procedure men-
tioned above 410 mg (73%) of the title compound was
obtained. Mp 189e190 ꢃC. Rf ¼ 0.54 (CHCl3/CH3OH, 10:1).
EI-MS (m/z) 281 [M]þ. IR (KBr) 3325, 1600, 1535, 1365,
(s, 6H), 3.74 (s, 3H), 4.41 (s, 1H), 6.70 (d, J ¼ 7.8 Hz, 1H),
6.85 (d, J ¼ 8.1 Hz, 1H), 7.02 (s, 1H), 7.68 (s, 1H), 8.05 (s, 2H).
3.2.3.12. 1,3-Dihydroxyl-2-(40-N,N-dimethylphen-10-yl)-4,4,5,
5-tetramethylimidazolidine (1l). Using the general procedure
mentioned above, 173 mg (31%) of the title compound was
obtained. Mp 168e169 ꢃC. Rf ¼ 0.53 (CHCl3/CH3OH, 6:1).
EI-MS (m/z) 279 [M]þ. IR (KBr) 3345, 2840, 1595, 1450,
1
750 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.00 (s, 6H), 1.04
(s, 6H), 5.37 (s, 1H), 7.52 (d, J ¼ 6.6 Hz, 1H), 7.60
(t, J ¼ 7.8 Hz, 1H), 7.92 (t, J ¼ 7.6 Hz, 1H), 8.05 (d, J ¼ 7.2 Hz,
1H), 8.23 (s, 2H).
1
840 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.03 (s, 6H), 1.05
(s, 6H), 2.85 (s, 6H), 4.01 (s, 1H), 6.68 (d, J ¼ 8.7 Hz, 2H),
7.25 (d, J ¼ 8.7 Hz, 2H), 7.60 (s, 2H).
3.2.3.19. 1,3-Dihydroxyl-2-(30-nitrophen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (1s). Using the general procedure men-
tioned above, 421 mg (75%) of the title compound was
obtained. Mp 179e181 ꢃC. Rf ¼ 0.51 (CHCl3/CH3OH, 10:1).
EI-MS (m/z) 281 [M]þ. IR (KBr) 3315, 1530, 1360, 1600,
3.2.3.13. 1,3-Dihydroxyl-2-(20-fluorophen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (1m). Using the general procedure men-
tioned above, 366 mg (72%) of the title compound was
obtained. Mp 172e173 ꢃC, Rf ¼ 0.69 (CHCl3/CH3OH, 6:1),
EI-MS (m/z) 254 [M]þ, IR (KBr) 3310, 1600, 1130, 1235,
875, 790, 685 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 0.52 (s,
1
12H), 4.19 (s, 1H), 7.04 (dd, J ¼ 8.7 Hz, J ¼ 8.1 Hz, 1H),
7.34 (d, J ¼ 6.0 Hz, 1H), 7.57 (d, J ¼ 7.5 Hz, 1H), 7.77 (s,
1H), 8.03 (s, 2H).
1
775 cmꢁ1. H NMR (DMSO-d6) d/ppm ¼ 1.07 (s, 12H), 4.95
(s, 1H), 7.18 (t, J ¼ 8.4 Hz, 1H), 7.30 (d, J ¼ 8.1 Hz, 1H),
7.68 (d, J ¼ 8.7 Hz, 2H), 8.14 (s, 2H).
3.2.3.20. 1,3-Dihydroxyl-2-(40-nitrophen-10-yl)-4,4,5,5-tetrame-
thylimidazolidine (1t). Using the general procedure mentioned
above, 335 mg (63%) of the title compound was obtained. Mp
172e174 ꢃC. Rf ¼ 0.59 (CHCl3/CH3OH, 10:1). EI-MS (m/z)
281 [M]þ. IR (KBr) 3325, 1532, 1590, 1500, 1450, 1365,
3.2.3.14. 1,3-Dihydroxyl-2-(40-chlorophen-10-yl)-4,4,5,5-tetra-
methylimidazolidine (1n). Using the general procedure men-
tioned above, 330 mg (61%) of the title compound was
obtained. Mp 213e215 ꢃC. Rf ¼ 0.73 (CHCl3/CH3OH, 10:1).
EI-MS (m/z) 270 [M]þ. IR (KBr) 3325, 1600, 1500, 1085,
835, 790, 685 cmꢁ1
.
1H NMR (DMSO-d6) d/ppm ¼ 1.00

M. Zhao et al. / European Journal of Medicinal Chemistry 43 (2008) 1048e1058
1055
(s, 6H), 1.05 (s, 6H), 4.71 (s, 1H), 7.70 (d, J ¼ 9.0 Hz, 2H), 8.21
(d, J ¼ 7.2 Hz, 2H), 8.40 (s, 2H).
(s, 6H). Anal. Calcd for C13H18N2O2: C, 66.64; H, 7.74; N,
11.96. Found: C, 66.53; H, 7.61; N, 11.82.
3.2.4. General procedure for preparing 1,3-dihydroxyl-2-
(substituted phen-10-yl)-4,4,5,5-tetramethyl-imidazolidines
(2aet)
3.2.4.5. 1-Hydroxyl-2-(40-methylphen-10-yl)-4,4,5,5-tetrame-
thylimidazoline (2e). Using the general procedure mentioned
above, 162 mg (70%) of the title compound was obtained as
yellow powder. Mp 79e81 ꢃC. Rf ¼ 0.93 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 232 [M]þ. IR (KBr) 3348, 2980, 1603,
A suspension of 1 mmol of 1,3-dihydroxyl-2-(substituted
phen-10-yl)-4,4,5,5-tetramethylimidazolidine (1aet) and
360 mg of anhydrous MgSO4 in 40 ml of anhydrous THF
was stirred at 80 ꢃC for 24 h and TLC (CHCl3/CH3OH,
20:1) indicated the complete disappearance of 1aet. The reac-
tion mixture was cooled to room temperature. After removal of
MgSO4 by filtration the filtrate was evaporated to dryness. The
residue was purified on chromatography column of silica gel
(CHCl3/CH3OH, 30:1) to give the title compound.
1
1501, 1369, 800 cmꢁ1. H NMR (CDCl3-d ) d/ppm ¼ 7.526
(d, J ¼ 7.50 Hz, 2H), 7.124 (d, J ¼ 7.50 Hz, 2H), 2.644 (s,
1H), 1.377 (s, 3H), 1.159 (s, 6H), 1.137 (s, 6H). Anal. Calcd
for C14H20N2O: C, 72.38; H, 8.68; N, 12.06. Found: C,
72.53; H, 8.52; N, 12.12.
3.2.4.6. 1-Hydroxyl-2-(40-methoxylphen-10-yl)-4,4,5,5-tetrame-
thylimidazoline (2f). Using the general procedure mentioned
above, 206 mg (83%) of the title compound was obtained as
yellow powder. Mp 81e83 ꢃC. Rf ¼ 0.15 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 248 [M]þ. IR (KBr) 3346, 2830, 1610,
3.2.4.1. 1-Hydroxyl-2-(phen-10-yl)-4,4,5,5-tetramethylimidazo-
line (2a). Using the general procedure mentioned above,
152 mg (69%) of the title compound was obtained as yellow
powder. Mp 173e175 ꢃC. Rf ¼ 0.15 (CHCl3/CH3OH, 20:1).
EI-MS (m/z) 218 [M]þ. IR (KBr) 3342, 3031, 1600, 1505,
1500, 1456, 1372, 840 cmꢁ1
.
1H NMR (CDCl3-d ) d/
ppm ¼ 7.496 (d, J ¼ 7.50 Hz, 2H), 6.945 (d, J ¼ 7.50 Hz,
2H), 3.734 (s, 3H), 2.640 (s, 1H), 1.159 (s, 6H), 1.137
(s, 6H). Anal. Calcd for C14H20N2O2: C, 67.71; H, 8.12; N,
11.28. Found: C, 67.56; H, 8.00; N, 11.14.
1452, 1359, 1070, 811 cmꢁ1
.
1H NMR (CDCl3-d ) d/
ppm ¼ 7.603 (d, J ¼ 7.50 Hz, 2H), 7.304 (t, J ¼ 7.50 Hz,
1H), 7.118 (t, J ¼ 7.38 Hz, 2H), 2.643 (s, 1H), 1.154
(s, 12H). Anal. Calcd for C13H18N2O: C, 71.53; H, 8.31; N,
12.83. Found: C, 71.74; H, 8.50; N, 12.68.
3.2.4.7. 1-Hydroxyl-2-(20,40-dimethoxylphen-10-yl)-4,4,5,5-tet-
ramethylimidazoline (2g). Using the general procedure men-
tioned above, 175 mg (63%) of the title compound was
obtained as yellow powder. Mp 97e99 ꢃC. Rf ¼ 0.12
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 278 [M]þ. IR (KBr)
3.2.4.2. 1-Hydroxyl-2-(20-hydroxylphen-10-yl)-4,4,5,5-tetrame-
thylimidazoline (2b). Using the general procedure mentioned
above, 115 mg (49%) of the title compound was obtained as
yellow powder. Mp 150e151 ꢃC. Rf ¼ 0.11 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 234 [M]þ. IR (KBr) 3341, 1611, 1504,
3342, 2853, 1607, 1598, 1502, 833, 800 cmꢁ1 1H NMR
.
(CDCl3-d ) d/ppm ¼ 7.488 (d, J ¼ 7.50 Hz, 1H), 6.940
(d, J ¼ 7.50 Hz, 1H), 6.442 (s, 1H), 3.730 (s, 3H), 3.720
(s, 3H), 2.637 (s, 1H), 1.161 (s, 6H), 1.139 (s, 6H). Anal.
Calcd for C15H22N2O3: C, 64.73; H, 7.97; N, 10.06. Found:
C, 64.88; H, 7.82; N, 10.20.
1
1453, 760 cmꢁ1. H NMR (CDCl3-d ) d/ppm ¼ 8.155(s, 1H),
7.445 (d, J ¼ 7.51 Hz, 1H), 7.353 (t, J ¼ 7.51 Hz, 1H), 7.278
(t, J ¼ 7.50 Hz, 1H), 7.244 (t, J ¼ 7.50 Hz, 1H), 2.653
(s, 1H), 1.136 (s, 12H). Anal. Calcd for C13H18N2O2: C,
66.64; H, 7.74; N, 11.96. Found: C, 66.53; H, 7.57; N, 12.12.
3.2.4.8. 1-Hydroxyl-2-(30,40-dimethoxylphen-10-yl)-4,4,5,5-tet-
ramethylimidazoline (2h). Using the general procedure men-
tioned above, 111 mg (40%) of the title compound was
obtained as yellow powder. Mp 89e90 ꢃC. Rf ¼ 0.13
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 278 [M]þ. IR (KBr)
3.2.4.3. 1-Hydroxyl-2-(30-hydroxyphen-10-yl)-4,4,5,5-tetrame-
thylimidazoline (2c). Using the general procedure mentioned
above, 129 mg (55%) of the title compound was obtained as
yellow powder. Mp 139e141 ꢃC. Rf ¼ 0.10 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 234 [M]þ. IR (KBr) 3350, 1610, 1503,
3345, 2836, 1604, 1502, 1461, 1378, 870, 820 cmꢁ1 1H
.
1
1450, 760 cmꢁ1. H NMR (CDCl3-d ) d/ppm ¼ 7.814(s, 1H),
NMR (CDCl3-d ) d/ppm ¼ 7.208 (d, J ¼ 7.51 Hz, 1H), 7.149
(s, 1H), 6.848 (d, J ¼ 7.51 Hz, 1H), 3.750 (s, 3H), 3.747
(s, 3H), 2.639 (s, 1H), 1.162 (s, 6H), 1.135 (s, 6H). Anal.
Calcd for C15H22N2O3: C, 64.73; H, 7.97; N, 10.06. Found:
C, 64.60; H, 7.79; N, 10.22.
7.423 (d, J ¼ 7.50 Hz, 1H), 7.304 (t, J ¼ 7.50 Hz, 1H), 7.108
(s, 1H), 6.889 (d, J ¼ 7.50 Hz, 1H), 2.643 (s, 1H), 1.334
(s, 6H), 1.154 (s, 6H). Anal. Calcd for C13H18N2O2: C,
66.64; H, 7.74; N, 11.96. Found: C, 66.79; H, 7.91; N, 12.13.
3.2.4.4. 1-Hydroxyl-2-(40-Hydroxyphen-10-yl)-4,4,5,5-tetrame-
thylimidazoline (2d). Using the general procedure mentioned
above, 138 mg (59%) of the title compound was obtained as
yellow powder. Mp 197e198 ꢃC. Rf ¼ 0.10 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 234 [M]þ. IR (KBr) 3359, 1605, 1462,
3.2.4.9. 1-Hydroxyl-2-(30,40-methylendioxyphen-10-yl)-4,4,5,5-
tetramethylimidazoline (2i). Using the general procedure
mentioned above, 189 mg (72%) of the title compound was
obtained as yellow powder. Mp 109e110 ꢃC. Rf ¼ 0.12
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 262 [M]þ. IR (KBr)
1380, 885, 840, 693 cmꢁ1
.
1H NMR (CDCl3-d ) d/
3425, 1602, 1504, 1453, 1371, 1262, 930 cmꢁ1 1H NMR
.
ppm ¼ 7.734 (s, 1H), 7.463 (d, J ¼ 7.50 Hz, 2H), 6.764
(CDCl3-d ) d/ppm ¼ 7.202 (d, J ¼ 7.51 Hz, 1H), 7.145
(d, J ¼ 7.50 Hz, 2H), 2.643 (s, 1H), 1.158 (s, 6H), 1.132
(s, 1H), 6.855 (d, J ¼ 7.51 Hz, 1H), 5.99 (s, 2H), 2.642

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M. Zhao et al. / European Journal of Medicinal Chemistry 43 (2008) 1048e1058
1
(s, 1H), 1.157 (s, 6H), 1.133 (s, 6H). Anal. Calcd for
C14H18N2O3: C, 64.10; H, 6.92; N, 10.68. Found: C, 64.29;
H, 6.80; N, 10.86. d/ppm ¼ 1.15 (s, 12H, eCH3), 5.99
(s, 2H, eCH2), 7.02 (m, 3H, AreH)
1369, 1092, 821 cmꢁ1. H NMR (CDCl3-d ) d/ppm ¼ 7.590
(d, J ¼ 7.51 Hz, 2H), 7.392 (t, J ¼ 7.51 Hz, 2H), 2.648
(s, 1H), 1.151 (s, 6H), 1.135 (s, 6H). Anal. Calcd for
C13H17N2OCl: C, 61.78; H, 6.78; N, 14.03. Found: C, 61.62;
H, 6.71; N 14.20.
3.2.4.10. 1-Hydroxyl-2-(30-hydroxyl-40-methoxylphen-10-yl)-4,
4,5,5-tetramethylimidazoline (2j). Using the general procedure
mentioned above, 106 mg (40%) of the title compound was
obtained as yellow powder. Mp 156e157 ꢃC. Rf ¼ 0.19
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 264 [M]þ. IR (KBr)
3.2.4.15. 1-Hydroxyl-2-(40-bromophen-10-yl)-4,4,5,5-tetrame-
thylimidazoline (2o). Using the general procedure mentioned
above, 241 mg (81%) of the title compound was obtained as
yellow powder. Mp 73e75 ꢃC. Rf ¼ 0.13 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 297 [M]þ. IR (KBr) 3351, 1599, 1452,
1
3345, 2836, 1602, 1501, 1376, 810 cmꢁ1. H NMR (CDCl3-
d ) d/ppm ¼ 7.597 (s, 1H), 7.200 (d, J ¼ 7.51 Hz, 1H), 7.005
(s, 1H), 6.859 (d, J ¼ 7.51 Hz, 1H), 3.732 (s, 2H), 2.646
(s, 1H), 1.155 (s, 6H), 1.131 (s, 6H). Anal. Calcd for
C14H20N2O3: C, 63.62; H, 7.63; N, 10.60. Found: C, 63.78;
H, 7.75; N, 10.41.
1401, 1375, 872, 823 cmꢁ1 1H NMR (CDCl3-d ) d/
.
ppm ¼ 7.514 (d, J ¼ 7.71 Hz, 2H), 7.465 (t, J ¼ 7.71 Hz,
2H), 2.647 (s, 1H), 1.155 (s, 6H), 1.137 (s, 6H). Anal. Calcd
for C13H17N2OBr: C, 52.54; H, 5.77; N, 9.43. Found: C,
52.41; H, 5.11; N, 9.62.
3.2.4.11. 1-Hydroxyl-2-(30-methoxyl-40-hydroxyphen-10-yl)-4,4,
5,5-tetramethylimidazoline (2k). Using the general procedure
mentioned above, 85 mg (32%) of the title compound was ob-
tained as yellow syrup. Mp 113e115 ꢃC. Rf ¼ 0.11 (CHCl3/
CH3OH, 20:1). EI-MS (m/z) 264 [M]þ. IR (KBr) 3341,
3.2.4.16. 1-Hydroxyl-2-(20,40-dichlorophen-10-yl)-4,4,5,5-tetra-
methylimidazoline (2p). Using the general procedure men-
tioned above, 227 mg (79%) of the title compound was
obtained as yellow powder. Mp 86e88 ꢃC. Rf ¼ 0.15
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 287 [M]þ. IR (KBr)
1
2838, 1599, 1448, 1374, 875, 811 cmꢁ1. H NMR (CDCl3-
3355, 1598, 1459, 1401, 1377, 870, 820 cmꢁ1 1H NMR
.
d ) d/ppm ¼ 7.625(s, 1H), 7.018 (d, J ¼ 7.51 Hz, 1H), 7.001
(s, 1H), 6.855 (d, J ¼ 7.51 Hz, 1H), 3.736 (s, 3H), 2.646
(s, 1H), 1.158 (s, 6H), 1.134 (s, 6H). Anal. Calcd for
C14H20N2O3: C, 63.62; H, 7.63; N, 10.60. Found: C, 63.50;
H 7.58; N, 10.78.
(CDCl3-d ) d/ppm ¼ 7.574 (d, J ¼ 7.51 Hz, 1H), 7.320
(s, 1H), 7.195 (t, J ¼ 7.51 Hz, 1H), 2.643 (s, 1H), 1.153
(s, 6H), 1.138 (s, 6H). Anal. Calcd for C13H16N2OCl2: C,
54.37; H, 5.62; N, 9.75. Found: C, 54.49; H, 5.77; N, 9.60.
3.2.4.17. 1-Hydroxyl-2-(30,40-dichlorophen-10-yl)-4,4,5,5-tetra-
methylimidazoline (2q). Using the general procedure men-
tioned above, 198 mg (69%) of the title compound was
obtained as yellow powder. Mp 83e85 ꢃC. Rf ¼ 0.19
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 287 [M]þ. IR (KBr)
3.2.4.12.
1-Hydroxyl-2-(40-N,N-dimethylaminophen-10-yl)-
4,4,5,5-tetramethylimidazoline (2l). Using the general proce-
dure mentioned above, 117 mg (45%) of the title compound
was obtained as yellow powder. Mp 65e67 ꢃC. Rf ¼ 0.11
(CHCl3/CH3OH, 20:1). EI-MS (m/z) 261 [M]þ. IR (KBr)
3343, 1604, 1502, 1453, 1372, 1005, 870, 817 cmꢁ1 1H
.
3345, 2857, 1602, 1500, 1455, 1371, 831 cmꢁ1
.
1H NMR
NMR (CDCl3-d ) d/ppm ¼ 7.570(s, 1H), 7.481 (d, J ¼ 7.51 Hz,
1H), 7.245 (d, J ¼ 7.51 Hz, 1H), 2.649 (s, 1H), 1.151 (s, 6H),
1.135 (s, 6H). Anal. Calcd for C13H16N2OCl2: C, 54.37; H,
5.62; N, 9.75. Found: C, 54.22; H, 5.54; N, 9.90.
(CDCl3-d ) d/ppm ¼ 7.515 (d, J ¼ 7.51 Hz, 2H), 6.712
(d, J ¼ 7.51 Hz, 2H), 2.886 (s, 6H), 2.651 (s, 1H), 1.150
(s, 6H), 1.131 (s, 6H). Anal. Calcd for C15H23N3O: C,
68.93; H, 8.87; N, 16.08. Found: C, 69.10; H, 8.99; N, 16.00.
3.2.4.18. 1-Hydroxyl-2-(20-nitrophen-10-yl)-4,4,5,5-tetramethy-
limidazoline (2r). Using the general procedure mentioned
above, 189 mg (71%) of the title compound was obtained as
yellow powder. Mp 113e115 ꢃC. Rf ¼ 0.13 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 263 [M]þ. IR (KBr) 3345, 1600, 1502,
3.2.4.13. 1-Hydroxyl-2-(20-fluorophen-10-yl)-4,4,5,5-tetrame-
thylimidazoline (2m). Using the general procedure mentioned
above, 180 mg (76%) of the title compound was obtained as
yellow powder. Mp 111e112 ꢃC. Rf ¼ 0.12 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 236 [M]þ. IR (KBr) 3343, 2852, 1604,
1
1450, 1371, 742 cmꢁ1. H NMR (CDCl3-d ) d/ppm ¼ 8.220
1452, 1377, 1132, 770 cmꢁ1
.
1H NMR (CDCl3-d ) d/
(d, J ¼ 7.20 Hz, 1H), 7.881 (d, J ¼ 7.21 Hz, 1H), 7.685 (t,
J ¼ 7.21 Hz, 1H), 7.554 (t, J ¼ 7.21 Hz, 1H), 2.643 (s, 1H),
1.152 (s, 6H), 1.133 (s, 6H). Anal. Calcd for C13H17N3O3:
C, 59.30; H, 6.51; N, 15.96. Found: C, 59.14; H, 6.38; N,
16.13.
ppm ¼ 7.664 (d, J ¼ 7.51 Hz, 1H), 7.412 (t, J ¼ 7.51 Hz,
1H), 7.123 (t, J ¼ 7.51 Hz, 1H), 7.059 (d, J ¼ 7.51 Hz, 1H),
2.650 (s, 1H), 1.152 (s, 6H), 1.133 (s, 6H). Anal. Calcd for
C13H17N2OF: C, 66.08; H, 7.25; N, 11.86. Found: C, 66.23;
H, 7.39; N 11.70.
3.2.4.19. 1-Hydroxyl-2-(30-nitrophen-10-yl)-4,4,5,5-tetramethy-
limidazoline (2s). Using the general procedure mentioned
above, 174 mg (66%) of the title compound was obtained as
yellow powder. Mp 97e99 ꢃC. Rf ¼ 0.13 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 263 [M]þ. IR (KBr) 3345, 1602, 1513,
3.2.4.14. 1-Hydroxyl-2-(40-chlorophen-10-yl)-4,4,5,5-tetrame-
thylimidazoline (2n). Using the general procedure mentioned
above, 185 mg (73%) of the title compound was obtained as
yellow powder. Mp 94e95 ꢃC. Rf ¼ 0.14 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 252 [M]þ. IR (KBr) 3350, 1598, 1501,
1455, 1370, 811, 790, 680 cmꢁ1 1H NMR (CDCl3-d )
.

M. Zhao et al. / European Journal of Medicinal Chemistry 43 (2008) 1048e1058
1057
d/ppm ¼ 8.549 (s, 1H), 8.219 (d, J ¼ 7.20 Hz, 1H), 7.989 (d,
J ¼ 7.21 Hz, 1H), 7.545 (t, J ¼ 7.21 Hz, 1H), 2.646 (s, 1H),
1.153 (s, 6H), 1.131 (s, 6H). Anal. Calcd for C13H17N3O3: C,
59.30; H, 6.51; N, 15.96. Found: C, 59.44; H, 6.64; N, 16.11.
immediately after decapitation rat aortic strips were obtained
and put in a perfusion bath with 5 ml of warmed (37 ꢃC), ox-
ygenated (95% O2, 5% CO2) Krebs solution (pH 7.4). The aor-
tic strips were mounted to the tension transducers and the
relaxationecontraction curves were recorded. Noradrenaline
(NE, final concentration 10ꢁ9 M) solution was added to induce
contraction. When the hypertonic contraction reached to the
maximum level, NE was washed and the vessel strips were sta-
bilized for 30 min. After the renewal of the solution, NE (final
concentration 10ꢁ9 M) was added. When the hypertonic con-
traction value of aortic strips reached the peak, 15 ml of alco-
hol (vehicle), or the solution of 2aet in 15 ml of alcohol (final
concentration 5 ꢂ 10ꢁ3 M) were added, respectively, to ob-
serve the relaxant response of the aortic strip to them. The per-
centage inhibitions of NE-induced vasoconstriction by 2aet
were recorded.
3.2.4.20. 1-Hydroxyl-2-(40-nitrophen-10-yl)-4,4,5,5-tetramethy-
limidazoline (2t). Using the general procedure mentioned
above, 166 mg (63%) of the title compound was obtained as
yellow powder. Mp 131e132 ꢃC. Rf ¼ 0.13 (CHCl3/CH3OH,
20:1). EI-MS (m/z) 263 [M]þ. IR (KBr) 3344, 1601, 1510,
1454, 862, 791, 680 cmꢁ1
.
1H NMR (CDCl3-d ) d/
ppm ¼ 8.219(d, J ¼ 7.20 Hz, 2H), 7.788 (d, J ¼ 7.21 Hz,
2H), 2.641 (s, 1H), 1.151 (s, 6H), 1.128 (s, 6H). Anal. Calcd
for C13H16N3O3: C, 59.53; H, 6.15; N, 16.02. Found: C,
59.66; H, 6.28; N, 15.89.
3.3. Pain threshold in vivo assay
3.6. QSAR analysis
Male ICR mice weighing 25 ꢀ 2 g, were housed in a 12/12
light/dark cycle at a room temperature of 21 ꢀ 2 ꢃC for one
day before being used. Each of them in drug receiving groups
was given a single intraperitoneal (i.p.) injection of one of
2aet at a level of 0.13 mmol/kg in 0.2 ml of saline mixed
with 0.01 ml of DMSO and 0.02 ml of Tween-80. The mouse
in control group was given the same mixed solvent as the drug
receiving groups received. Analgesic effects of 2aet were
evaluated by the tail-flick test. The basic pain threshold value
of each mouse was measured four times.
After 2aet were administered, the pain thresholds were
measured at 30 min intervals. This measurement was carried
out for 150 min. The potency of analgesia was indicated by
the pain threshold variation. The values were calculated ac-
cording to equation PTV ¼AAPT/BPT wherein PTV ¼ pain
threshold variation, BPT ¼ basic pain threshold and
AAPT ¼ pain threshold after administration-basic pain thresh-
old. All values of the pain threshold variation for each animal
were averaged and constituted one sample.
To perform a QSAR analysis, firstly the SMILE formats of
compounds 2aet were generated using JME server (http://
www.molinspiration.com/cgi-bin/properties). Moreover, the
obtained SMILE files were submitted to e-dragon server
(http://www.vcclab.org/lab/edragon/) to calculate molecular
descriptors. To calculate 3D descriptors, it should be men-
tioned that e-dragon requires 3D optimized structures as input.
In the current study, the 3D structures were automatically gen-
erated using CORINA (http://www2.chemie.uni-erlangen.de/
software/corina/), which has been implemented in e-dragon
server as an option. Finally, the 1664 different molecular
parameters for each compound were obtained.
The multiple linear regression method was employed to de-
rive the QSAR equations of 2aet. Concerning the analgesic
effect of a molecule, the PTV value measured at 90 min (i.e.
PTV90) was considered in the QSAR analysis. As the number
of compounds in each case (2aet) is only 20, the optimum
number of molecular descriptors used in the QSAR analysis
is around 4. To achieve the best regression equation, two-
step descriptor selection procedure was applied. The first
step consisted in the elimination of the descriptors that are
not able to provide useful statistical information. For instance,
the absolute value of the correlation coefficient between the
selected descriptor ðXiÞ and the activity (PTV90) under inves-
tigation ðjcorðXi; PTV90ÞjÞ should be larger than 0.4. At the
second step, the descriptor subset was optimized by means
of a genetic algorithm (GA). Here, a GA program previously
developed in our laboratory was used with the following con-
figuration: (1) genetic operators: selection process of stochas-
tic sampling with replacement [31], simple crossover and
random mutation [32]; (2) fitness function: regression coeffi-
cient (R); (3) number of variables: 4; (4) population size:
500; (5) generation: 5000; (6) crossover probability: 80%;
(7) mutation probability: 10%. Finally, the optimal QSAR
equation in describing the analgesic effects of 2aet was
determined.
3.4. Tail bleeding time in vivo assay
Male ICR mice weighing 25 ꢀ 2 g, were housed in a 12/12
light/dark cycle at a room temperature of 21 ꢀ 2 ꢃC for one
day before being used. Each of them in drug receiving groups
was given a single intraperitoneal (i.p.) injection of one of
2aet at a level of 1.30 mmol/kg in 0.2 ml of saline mixed
with 0.01 ml of DMSO and 0.02 ml of Tween-80. The mouse
in control group was given the same mixed solvent as the drug
receiving groups received. At 5, 15, 30 and 60 min, a mouse
was placed in a tube holder with its tail protruding, and
a 2 mm cut was made on the tail. Flowing blood until it
stopped was gently wiped away with a filter paper every
30 s, yielding the bleeding time (BT).
3.5. Vasorelaxation in vitro assay
The conversion of 2aet during vasoconstriction induced by
NE was examined using the rat aortic strip. In brief,
To systematically assess a QSAR model, the tests of resub-
stitution and LOO were carried out. In the resubstitution test,

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M. Zhao et al. / European Journal of Medicinal Chemistry 43 (2008) 1048e1058
three statistical parameters (R, S and e) were used to assess the
performance. The regression coefficient R is defined as
follows:
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Acknowledgements
[31] J.H. Holland, Adaptation in Natural and Artificial Systems, second ed.
MIT Press, Cambridge, USA, 1992.
This work was supported by Beijing area major laboratory
of peptide and small molecular drugs, the 973 Project of China
(2006CB708501) and National Natural Scientific Foundation
of China (30472071) and Natural Scientific Foundation of
Beijing (7052010). Z.Z. thanks Dr. Paolo F. Mazzatorta
(Nestle Research Center, Switzerland) and Dr. Laurence
Miguet (CERMAV,CNRS, France) for a number of discussions
concerning QSAR.
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