Synthesis and Na+/H+ exchanger inhibitory activity of benzoylguanidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.06.011

Twenty-two compounds of substituted benzoylguanidine derivatives were designed and synthesized as potent NHE1 inhibitors. Twelve compounds showed more potent NHE1 inhibitory activity than cariporide. The activities of compounds 7e, 7h and 7j (IC₅₀

Full text of DOI:10.1016/j.ejmech.2011.06.011

European Journal of Medicinal Chemistry 46 (2011) 4107e4116
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and Naþ/Hþ exchanger inhibitory activity
of benzoylguanidine derivatives
,
b
a,
*
Lu Jin a, Xiang-Min Jiang a, Ping Du a, Jing Xu b, Guo-qing Gong b , Qiu-juan Wang , Yun-gen Xu
**
a Department of Medicinal Chemistry, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China
b Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-two compounds of substituted benzoylguanidine derivatives were designed and synthesized as
potent NHE1 inhibitors. Twelve compounds showed more potent NHE1 inhibitory activity than car-
iporide. The activities of compounds 7e, 7h and 7j (IC50 ¼ 0.073 ꢀ 0.021, 0.084 ꢀ 0.012 and
0.068 ꢀ 0.021 nmol/L, respectively) were two orders of magnitude higher than that of cariporide
(30.7 ꢀ 2.5 nmol/L). Myocardial cells in vitro screening showed 7j had highlighted protective effect on
cardiomyocytes subjected to hypoxia/reoxygenation. Thus it is valuable for further investigation.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 1 April 2011
Received in revised form
7 June 2011
Accepted 8 June 2011
Available online 17 June 2011
Keywords:
Naþ/Hþ exchanger inhibitors
Myocardial ischemicereperfusion injury
Benzoylguanidine derivatives
1. Introduction
function and reduce Naþ and Ca2þ influx, and hence abolish the
post-ischemia Ca2þ overload in myocardial cells and lower the risk
The Naþ/Hþ exchanger (NHE), which regulates intracellular pH
by removing a proton in exchange for an extracellular sodium ion, is
an integral membraneprotein ubiquitouslyexpressed in mammalian
cells. To date, nine isoforms of the exchanger have been identified in
various organs in the human body and are designated NHE1eNHE9
[1]. NHE1 was the first isoform discovered and is ubiquitously
expressed in the plasma membrane of mammalian cells [2,3]. Other
isoforms have more restricted tissue distributions and some have
predominantly intracellular localization [4,5]. In mammals, NHE1
is the predominant isoform in heart and is involved in numerous
physiological processes, including regulation of intracellular pH,
cell-volume control, cytoskeletal organization, heart disease and
cancer [6,7]. The activity of NHE1 is elevated in animal models of
myocardial infarcts and in left ventricular hypertrophy [8]. During
ischemia and reperfusion of the myocardium, NHE activity catalyzes
increased uptake of intracellular Naþ. This in turn is exchanged for
extracellular calcium by the Naþ/Ca2þ exchanger resulting in calcium
overload and damage to the myocardium, such as myocardial
infarction activation, stunning and tissue necrosis [9,10].
of cell dysfunction and injury.
Aroylguanidine, a subunit typically present in most of the known
NHE1 inhibitors, such as cariporide and sabiporide (Fig. 1) [11e14], is
well-considered as the possible pharmacophore among the reported
NHE1 inhibitors [15]. TG-6 (Fig. 1) was synthesized and evaluated in
our laboratory before, which showed potent NHE1 inhibitory activity
and could excellently improve the cardiac function and reduce infarct
size against ischemia-reperfusion injury [16]. We replaced the
methylene which connected the piperazine nitrogen with the para-
position of benzoylguanidine with ethylene-1-oxy to afford another
compound TG-7(IC50 ¼ 1.06 ꢀ 0.18 nM) which exhibited more potent
NHE1 inhibitory potency than TG-6 (IC50 ¼ 1.82 ꢀ 0.36 nM). We
selected TG-7 as lead compound and introduced a series of
substituted amide groups to replace the nitro group at the meta-
positionof the aroylguanidinegroupbound tothe benzeneringofTG-
7, and then synthesized our target compounds 7ae7v (Table 1).
2. Results and discussion
NHE1 inhibitors, in their cation form, combine with NHE1 at the
2.1. Synthesis
extracellular Naþ binding site to competitively inhibit NHE1
The syntheticroute for target compounds is depicted in Scheme 1.
Intermediates 2a and 2b, obtained by condensation of 4-hydroxy-3-
nitro-benzoic acid ethyl ester and 1,2-dibromo ethane or 1,3-
dibromo propane, were coupled with 1-(2,3,4-trimethoxybenzyl)
* Corresponding author. Tel.: þ86 25 83322067; fax: þ86 25 83271487.
** Corresponding author.
E-mail address: xu_yungen@hotmail.com (Y.-g. Xu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.011

4108
L. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4107e4116
O
NH2
NH2
CH3
F3C
N
N
H3C
H3CO2S
N
NH2
NH2
N
O
N
H
O
Sabiporide
Cariporide
O
NH
NH2
OCH3
OCH3
O
NH
NH2
O2N
H3CO
H3CO
H3CO
H3CO
O2N
O
N
H
N
N
N
H
N
N
TG-6
TG-7
Fig. 1. Structures of cariporide, sabiporide, TG-6 and TG-7.
piperazine (trimetazidine) to give intermediates 3a and 3b. Treat-
ment of 3a and 3b with Fe/HCl afforded 4a and 4b. Compounds 4a
and 4b were coupled with acyl chloride to afford intermediates
5ae5v. Compounds 6ae6v, which are zwitterionic compounds
and can be separated from the reaction mixture simply by adjusting
their isoelectric points by 2% dilute hydrochloric acid, were
synthesized by hydrolysis of corresponding benzoates 5ae5v with
K2CO3 in MeOH/H2O (v/v ¼ 2:3). Compounds 7ae7v can be obtained
by reaction of the acids 6ae6v with 4 equiv of guanidine in the
NHE1 inhibitory activity screening. The experiment procedure was
similar as in the literature [18], with only minor modifications. The
IC50 of the tested compounds was obtained from the linear part of
the relationship between the log concentration and NHE activity
using linear regression analysis. The PSA results showed that most
of the tested compounds did inhibit rat platelet NHE1 in
a concentration-dependent manner. Twelve compounds showed
more potent NHE1 inhibitory activity than cariporide. The activities
of compounds 7e, 7h and 7j (IC50 ¼ 0.073 ꢀ 0.021, 0.084 ꢀ 0.012
and 0.068 ꢀ 0.021 nmol/L, respectively) were two orders of
magnitude higher than that of cariporide (30.7 ꢀ 2.5 nmol/L).
Compounds 7e, 7h and 7j, which showed a good NHE1 inhibi-
tory activity, together with TG-6, were evaluated in vitro for their
effect on cardiomyocytes viability subjected to hypoxia/reoxyge-
nation injury induced by Na2S2O4 (Tables 3 and 4). The protective
effect of these compounds against hypoxia/reoxygenation injury
induced by Na2S2O4 was determined in primary rat cardiomyocytes
hypoxia/reoxygenation model. After 2 h of hypoxia led by Na2S2O4,
presence
of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCI) and 1-hydroxybenzotrizole (HOBt) [17], and
purified by silica gel column chromatography (EtOAc/CH3OH/
Et3N ¼ 15:1:0.1) one time to be free from by-products and guanidine.
2.2. Biological activity
All synthesized compounds, along with reference compound
cariporide, were evaluated in rat platelet swelling assay (PSA) for
Table 1
Structures and characterization table of target compounds 7ae7v.
O
NH
NH2
OCH3
RHN
X
N
H
H3CO
N
O
N
H3CO
Compd. no.
X
R
Mol. formula
M.p. (ꢁC)
Analysis (%)
Found (calculated)
C
H
N
O
O
H3C
7a
7b
7c
(CH2)2
(CH2)2
(CH2)2
C32H40N6O6
141e145
180e185
184e189
63.27 (63.56)
63.97 (64.06)
60.35 (60.17)
7.06 (6.66)
13.79 (13.89)
C33H42N6O6
6.98 (6.84)
6.72 (6.62)
13.74 (13.58)
13.02 (13.15)
C2H5
O
C32H40N6O7$1H2O
H3CO
O
7d
7e
(CH2)2
(CH2)2
C32H37N7O6$0.5H2O
C31H37N7O8$0.5H2O
149e152
192e195
61.04 (61.52)
58.24 (57.75)
6.04 (6.13)
6.20 (5.94)
15.69 (15.69)
14.81 (15.21)
NC
O
NO2

L. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4107e4116
4109
Table 1 (continued )
Compd. no.
X
R
Mol. formula
M.p. (ꢁC)
Analysis (%)
Found (calculated)
C
H
N
O
S
O
7f
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
C31H40N6O7S
202e205
162e166
146e148
160e162
120e125
167e170
185e187
172e176
194e196
138e142
198e202
148e152
140e145
54e58
57.83 (58.11)
57.90 (57.89)
55.35 (54.95)
56.51 (56.66)
57.27 (57.30)
57.74 (57.50)
62.68 (62.63)
64.26 (64.54)
60.66 (60.72)
58.85 (59.15)
57.84 (57.90)
57.47 (57.38)
56.77 (57.06)
57.21 (57.06)
56.91 (57.31)
58.82 (58.75)
6.45 (6.29)
12.76 (13.11)
12.84 (13.06)
11.89 (12.40)
13.38 (13.67)
13.06 (13.36)
13.52 (13.87)
13.89 (13.69)
12.92 (13.28)
12.88 (12.87)
15.12 (15.09)
12.31 (12.66)
11.80 (12.16)
12.58 (12.47)
12.38 (12.47)
13.17 (13.36)
12.94 (13.26)
H3C
Cl
O
O
7g
7h
7i
C31H37N6O6Cl$1H2O
C31H36N6O6Cl2$1H2O
C29H36N6O6S$1H2O
C30H38N6O6S$1H2O
C29H36N6O6S$0.5H2O
C32H40N6O6.0.5H2O
C34H44N6O6
5.94 (6.11)
5.43 (5.65)
6.11 (6.23)
6.30 (6.41)
6.69 (6.15)
6.87 (6.73)
6.85 (7.00)
6.59 (6.79)
6.30 (6.05)
6.72 (6.53)
6.13 (5.98)
5.71 (5.68)
5.64 (5.68)
6.32 (6.41)
6.81 (6.52)
Cl
Cl
S
O
O
7j
S
O
7k
7l
S
O
O
O
7m
7n
7o
7p
7q
7r
7s
7t
C2H5
C33H42N6O7$1H2O
H3CO
NO2
O
C32H39N7O8
O
S
O
C32H42N6O7S.0.5H2O
C33H39N6O6F3$1H2O
C32H38N6O6Cl2
H3C
O
F3C
Cl
O
Cl
Cl
O
C32H38N6O6Cl2
Cl
S
C30H38N6O6S$1H2O
C31H40N6O6S$0.5H2O
174e178
183e186
O
O
7u
S
S
O
7v
(CH2)3
C30H38N6O6S$0.5H2O
183e185
58.14 (58.14)
6.34 (6.34)
13.18 (13.56)

4110
L. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4107e4116
O
O
O
OCH3
O2N
X
a
b
O2N
HO
OC2H5
O2N
H3CO
H3CO
OC2H5
OCH3
OC2H5
N
X
O
N
Br
O
3a-b
2a-b
O
O
OCH3
RHN
X
H2N
OC2H5
OC2H5
H3CO
H3CO
H3CO
H3CO
c
d
N
N
X
O
O
N
N
4a-b
5a-v
O
NH
NH2
O
OCH3
OCH3
RHN
RHN
N
H
H3CO
H3CO
OH
H3CO
H3CO
e
f
N
N
X
X
O
N
O
N
6a-v
7a-v
Scheme 1. Reagents and conditions: (a) 1,2-dibromo ethane/1,3-dibromo propane, K2CO3, CH3CN, reflux; (b) trimetazidine, K2CO3, CH3CN, 20e80 ꢁC; (c) Fe/HCl/50% (v/v) ethanol,
reflux; (d) CH2Cl2/Et3N, 0e5 ꢁC; (e) methanol/K2CO3, reflux; (f) guanidine/HOBt/EDCI/DMF, room temperature.
the cardiomyocytes were reoxygenated for 4 h. TG-6, 7e, 7h and 7j
were given 1 h before reoxygenation. The evaluation of car-
diomyocyte survival in a dose-dependent manner was measured as
an index of the ratio of treatment group OD to normal OD.
Compared with the model group, TG-6 and 7j could significantly
improve the survival at 10ꢂ4 mol/L (P < 0.01). TG-6 and 7j at
10ꢂ5 mol/L, 7e and 7h at 10ꢂ4 mol/L could improve the survival
(P < 0.05). There was no significant difference among the
compounds in the myocardial survival at 10ꢂ6 mol/L.
0.31 ꢀ0.07 nM of 7o, 0.084 ꢀ 0.012 nM of 7h vs 55.7 ꢀ 7.1 nM of 7s,
0.091 ꢀ0.017 nM of 7i vs 1.50 ꢀ 0.26 nM of 7t, 0.068 ꢀ 0.021 nM of
7j vs 0.92 ꢀ 0.08 nM of 7u).
4. Experiment protocol
Melting points were determined on a RDCSY-I capillary appa-
ratus and were uncorrected. The IR spectra (in KBr pellets) were
recorded on a Nicolet Impact 410 spectrophotometer. The 1H NMR
and 13C NMR spectra were recorded on a Brucker AV-300 or AV-500
NMR spectrometer using TMS as an internal standard and chemical
shifts were given in parts per million with tetramethylsilane (TMS).
Mass spectra were recorded on an Agilent 1100 series LC/MSD Tarp
(SL). All solvents were purchased from commercial sources and
used as received unless otherwise stated.
3. Conclusion
In summary, a series of novel substituted benzoylguanidine
derivatives were designed, synthesized and evaluated for their
NHE1 inhibitions. Most compounds showed more potent NHE1
inhibitory activity than cariporide, and 7e, 7h and 7j were more
potent than others. Further testing showed that, 7j could excel-
lently improve the cardiomyocytes survival rate against hypoxia/
reoxygenation injury induced by Na2S2O4.
4.1. General procedure for synthesis of compounds 2
To
a solution of ethyl 4-hydroxy-3-nitrobenzoate (5.1 g,
24 mmol) in anhydrous acetonitrile (70 mL), comminuted potas-
sium carbonate powder (10.4 g, 75 mmol) was added with stirring.
To the resulting mixture was then added 1,2-dibromo ethane (1,3-
dibromopropane) (48 mmol) and heated to reflux for 4 h. After
filtering, the filtrate was concentrated to dryness under reduced
pressure to give crude 2a and 2b, which were used for the next step
without further purification.
Preliminary structureeactivity relationship analysis indicated
that, introduction of electron-withdrawing group like nitro, chloro
or methylsulfonyl group to benzene ring of the amide group caused
better NHE1 inhibitory potency. Moreover, compounds whose
amide group has thiophene ring as core structure also exhibited
a good NHE1 inhibitory activity. On the contrary, the lengthening of
the connection chain between piperazine nitrogen and para-
position of benzoylguanidine from two carbons to three carbons
was likely to lead to decreased activity (0.073 ꢀ 0.021 nM of 7e vs
4.1.1. Ethyl 4-(2-bromoethoxy)-3-nitrobenzoate (2a)
Crude yield 40.6%, light yellow solid, m.p. 49e50 ꢁC.
Table 2
4.1.2. Ethyl 4-(3-bromopropoxy)-3-nitrobenzoate (2b)
Activities of target compounds 7ae7v.
Crude yield 60.0%, yellow oil.
a
Compd.
IC50a (nM)
Compd.
IC50 (nM)
Cariporide
30.7 ꢀ 2.5
1.06 ꢀ 0.18
154 ꢀ 9.5
TG-6
7l
1.82 ꢀ 0.36
139 ꢀ 11.8
49.3 ꢀ 11.0
225 ꢀ 13.2
0.31 ꢀ 0.07
0.56 ꢀ 0.07
1.10 ꢀ 0.36
1530 ꢀ 55.7
55.7 ꢀ 7.1
TG-7
7a
7b
7c
7d
7e
7f
7g
7h
7i
Table 3
7m
7n
7o
7p
7q
7r
7s
7t
7u
7v
Effect of TG-6, 7e, 7h and 7j on cardiomyocytes viability subjected to hypoxia/
reoxygenation injury induced by Na2S2O4 ðX ꢀ s; n ¼ 6Þ.
93.5 ꢀ 7.5
3020 ꢀ 245.8
142 ꢀ 12.5
0.073 ꢀ 0.021
0.11 ꢀ 0.04
125 ꢀ 6.2
Compd.
Concn.
10ꢂ4 mol/L
10ꢂ5 mol/L
10ꢂ6 mol/L
Normal (A490
Model (%)
TG-6 (%)
7e (%)
7h (%)
7j (%)
)
0.768 ꢀ 0.141
0.334 ꢀ 0.045##
0.532 ꢀ 0.025**
0.434 ꢀ 0.077*
0.488 ꢀ 0.084*
0.556 ꢀ 0.077**
0.736 ꢀ 0.095
0.364 ꢀ 0.040##
0.440 ꢀ 0.063*
0.379 ꢀ 0.058
0.409 ꢀ 0.080
0.454 ꢀ 0.061*
0.735 ꢀ 0.063
0.346 ꢀ 0.059##
0.370 ꢀ 0.035
0.353 ꢀ 0.054
0.362 ꢀ 0.067
0.391 ꢀ 0.085
0.084 ꢀ 0.012
0.091 ꢀ 0.017
0.068 ꢀ 0.021
0.53 ꢀ 0.09
1.50 ꢀ 0.26
0.92 ꢀ 0.08
0.67 ꢀ 0.16
7j
7k
a
Drug concentration to achieve half-maximal inhibition of acid-induced swelling
in rat platelets.
##P < 0.01 vs normal; *P < 0.05, **P < 0.01 vs model.

L. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4107e4116
4111
Table 4
agitation for 1 h. The reaction mixture was filtered under this
temperature, and the filtrate was concentrated to dryness under
reduced pressure to give compounds 4a and 4b.
Effect of TG-6, 7e, 7h and 7j on cardiomyocytes survival rate subjected to hypoxia/
reoxygenation injury induced by Na2S2O4 ðX ꢀ s; n ¼ 6Þ.
Compd.
Concn.
10ꢂ4 mol/L
10ꢂ5 mol/L
10ꢂ6 mol/L
4.3.1. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-amino benzoate (4a)
Model (%)
TG-6 (%)
7e (%)
7h (%)
7j (%)
43.43 ꢀ 1.45
49.46 ꢀ 6.20
59.78 ꢀ 4.74**
51.45 ꢀ 3.86
55.52 ꢀ 5.70
61.63 ꢀ 6.16**
47.10 ꢀ 4.56
50.41 ꢀ 5.29
48.05 ꢀ 4.85
49.27 ꢀ 4.21
53.22 ꢀ 7.58
Yield 98%, light yellow solid, m.p. 124e126 ꢁC. 1H NMR (CDCl3,
69.24 ꢀ 4.28**
56.48 ꢀ 3.60**
58.33 ꢀ 4.46**
72.41 ꢀ 6.94**
300 MHz)
d
(ppm): 1.36 (t, 3H, J ¼ 7.2, eCH3), 2.56e2.62 (m, 8H,
), 2.83 (t, 2H, J ¼ 5.1 Hz, eCH2), 3.51 (s, 2H, eCH2), 3.85 (s, 3H,
N
N
eOCH3), 3.87 (s, 3H, eOCH3), 3.88 (s, 3H, eOCH3), 4.16 (t, 2H, J ¼ 5.1 Hz,
eCH2), 4.31 (q, 2H, J ¼ 7.2, eCH2), 6.63 (d, 1H, J ¼ 8.7 Hz, ArH), 6.77 (d,
1H, J ¼ 8.4, ArH), 6.90 (d, 1H, J ¼ 8.4, ArH), 7.38e7.45 (m, 2H, ArH).
**P < 0.01 vs model.
4.2. General procedure for synthesis of compounds 3
4.3.2. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)
propoxy)-3-amino benzoate (4b)
Preparation of free trimetazidine: Trimetazidine hydrochloride
(3 g, 8.8 mmol) was dissolved in water (10 mL). An aqueous solu-
tion of potassium hydroxide (40 g in 100 mL of water) was then
added, the addition of alkali was continued till the pH of the
solution was raised to ca. 11. The basified solution was extracted
with ethyl acetate (3ꢃ10 mL). The combined organic phases were
washed with saturated sodium chloride solution, dried over
anhydrous sodium sulfate and concentrated under vacuum to give
free trimetazidine (1.88 g, 6.7 mmol).
Yield 98%, light yellow solid, m.p. 180e185 ꢁC. 1H NMR (CDCl3,
300 MHz)
J ¼ 6.3 Hz, eCH2), 2.50e2.55 (m, 10H,
d
(ppm): 1.36 (t, 3H, J ¼ 7.1 Hz, eCH3), 2.01 (q, 2H,
, eCH2), 3.49 (s, 2H,
N
N
eCH2), 3.84 (s, 3H, eOCH3), 3.87 (s, 3H, eOCH3), 3.88 (s, 3H,
eOCH3), 4.08 (t, 2H, J ¼ 6.3 Hz, eCH2), 4.31 (q, 2H, J ¼ 7.1 Hz, eCH2),
6.62 (d, 1H, J ¼ 8.55 Hz, ArH), 6.78 (d, 1H, J ¼ 8.55 Hz, ArH), 6.98 (d,
1H, J ¼ 8.4 Hz, ArH), 7.38 (1H, d, J ¼ 2.05 Hz, ArH), 7.44 (dd, 1H,
J1 ¼ 2.05 Hz, J2 ¼ 8.4 Hz, ArH).
Free trimetazidine (1.88 g, 6.7 mmol) was dissolved in anhy-
drous acetonitrile (10 mL). To this solution was added a solution of
ethyl 4-(2(3)-bromoethoxy)-3-nitrobenzoate (2) (4 mmol) in
anhydrous acetonitrile (20 mL), followed by anhydrous potassium
carbonate (3.1 g, 22 mmol) and the reaction mixture was refluxed
for 4 h. After filtering, the filtrate was concentrated to dryness
under reduced pressure. The residue was dissolved in ethyl acetate,
and the resulting solution was washed with water to remove the
excess free trimetazidine. The organic layer was dried over anhy-
drous sodium sulfate and concentrated under vacuum. The residue
was purified by silica gel column chromatography (petroleum/
acetone ¼ 8:1) to give 3a and 3b.
4.4. General procedure for synthesis of compounds 5
A mixture of ethyl 4-(2(3)-(4-(2,3,4-trimethoxybenzyl)piper-
azin-1-yl)ethoxy(propoxy))-3-amino benzoate (4) (2 mmol) and
triethylamine (0.4 mL) in dichloromethane (20 mL) was cooled to
0e5 ꢁC. A solution of appropriate acyl chloride (3 mmol) in
dichloromethane (1 mL) was then added dropwise. After stirring at
room temperature for 2 h, the reaction solution was washed three
times with saturated sodium bicarbonate solution and once with
saturated sodium chloride solution. The organic phase was dried
over anhydrous sodium sulfate and concentrated under vacuum.
The residue was purified by silica gel column chromatography
(petroleum/acetone/Et3N ¼ 8:1:0.1) to give compounds 5ae5v.
4.2.1. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-nitro benzoate (3a)
4.4.1. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(3-methylbenzamino)benzoate (5a)
Yield 85.0%, light yellow solid, m.p. 105e109 ꢁC. 1H NMR (CDCl3,
300 MHz)
d
(ppm): 1.36 (t, 3H, J ¼ 7.2 Hz, eCH3), 2.52e2.62 (m, 8H,
Yield 63.2%, yellow oil.
), 2.87 (t, 2H, J ¼ 5.7 Hz, eCH2), 3.49 (s, 2H, eCH2), 3.84 (s, 3H,
N
N
eOCH3), 3.86 (s, 3H, eOCH3), 3.87 (s, 3H, eOCH3), 4.29 (t, 2H,
J ¼ 5.7 Hz, eCH2), 4.33 (q, 2H, J ¼ 7.2 Hz, eCH2), 6.62 (d,1H, J ¼ 8.7 Hz,
ArH), 6.98(d,1H, J ¼ 8.4 Hz, ArH), 7.09 (d,1H, J ¼ 9.0 Hz, ArH), 8.18 (dd,
1H, J1 ¼ 2.4 Hz, J2 ¼ 9 Hz, ArH), 8.47 (d, 1H, J ¼ 2.1 Hz, ArH).
4.4.2. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(4-ethylbenzamino)benzoate (5b)
Yield 49.0%, white solid, m.p. 71e77 ꢁC.
4.4.3. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(4-methoxybenzamino)benzoate (5c)
4.2.2. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)propoxy)-
3-nitro benzoate (3b)
Yield 27%, yellow oil.
Yield 83.4%, yellow solid, m.p. 87e92 ꢁC. 1H NMR (CDCl3,
300 MHz)
J ¼ 6.7 Hz, eCH2), 2.49e2.56 (m, 10H,
d
(ppm): 1.39 (t, 3H, J ¼ 7.1 Hz, eCH3), 2.02 (q, 2H,
4.4.4. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(4-cyanobenzamino)benzoate (5d)
, eCH2), 3.48 (s, 2H,
N
N
eCH2), 3.85 (s, 3H, eOCH3), 3.87 (s, 3H, eOCH3), 3.88 (s, 3H,
eOCH3), 4.23 (t, 2H, J ¼ 6.2 Hz, eCH2), 4.38 (q, 2H, J ¼ 7.2 Hz, eCH2),
6.62 (d, 1H, J ¼ 8.5 Hz, ArH), 7.00 (d, 1H, J ¼ 8.5 Hz, ArH), 7.13 (d, 1H,
J ¼ 8.9 Hz, ArH), 8.18 (dd, 1H, J1 ¼ 2.2 Hz, J2 ¼ 8.8 Hz, ArH), 8.47 (d,
1H, J ¼ 2.1 Hz, ArH).
Yield 53.9%, white solid, m.p. 100e105 ꢁC.
4.4.5. N-(2-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)ethoxy)-5-
(guanidinocarbonyl)benzyl)-2-nitrobenzamide (5e)
Yield 36%, yellow solid.
4.3. General procedure for synthesis of compounds 4
4.4.6. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(4-methylphenylsulfonamino)benzoate (5f)
Yield 37.9%, yellow oil.
A mixture of ethyl 4-(2(3)-(4-(2,3,4-trimethoxybenzyl)piper-
azin-1-yl)ethoxy(propoxy))-3-nitro benzoate (3) (15 mmol), 50%
(v/v) ethanol (105 mL), reduced iron (6.7 g, 0.12 mol) and acid
alcohol (prepared by adding 0.1 mL concentrated hydrochloric acid
to 1 mL of 50% (v/v) ethanol) (1 mL) was refluxed with mechanical
4.4.7. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(4-chlorobenzamino)benzoate (5g)
Yield 51.8%, yellow oil.

4112
L. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4107e4116
Table 5
IR, MS and 1H NMR data of compounds 7ae7v.
NO.
IR (cmꢂ1
)
MS (m/z)a
1H NMR (
c,d2.40 (s, 3H, eCH3), 2.39e2.54 (m, 8H,
d)
7a
3396, 2938, 2822, 1652,
1533, 1495, 1344, 1268,
1096, 1014, 789.
605.3 [M þ H]þ
), 2.78
N
N
(t, 2H, J ¼ 5.1 Hz, eCH2), 3.38 (s, 2H, eCH2), 3.84 (s, 3H, eOCH3),
3.85 (s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 4.25 (t, 2H, J ¼ 5.1 Hz, eCH2),
6.61 (d, 1H, J ¼ 8.4 Hz, ArH), 6.92 (d, 1H, J ¼ 8.4 Hz, ArH), 6.99 (d, 1H,
J ¼ 8.7 Hz, ArH), 7.36e7.38 (m, 2H, ArH), 7.66e7.70 (m, 2H, ArH), 7.98
(dd, 1H, J1 ¼ 1.8 Hz, J2 ¼ 8.55 Hz, ArH), 8.78 (d, 1H, J ¼ 1.8 Hz, ArH),
8.85 (s, 1H, NHCO)
7b
3440, 3385, 2935, 2821,
1679, 1535, 1493, 1346,
1270, 1097, 1011, 788
619.4 [M þ H]þ
c,d1.28 (t, 3H, J ¼ 7.5 Hz, eCH3), 2.41e2.54 (m, 8H,
),
N
N
2.74 (q, 2H, J ¼ 7.5 Hz, eCH2), 2.76 (t, 2H, J ¼ 5.4 Hz, eCH2),
3.40 (s, 2H, eCH2), 3.84 (s, 3H, eOCH3), 3.85 (s, 3H, eOCH3),
3.86 (s, 3H, eOCH3), 4.25 (t, 2H, J ¼ 5.4 Hz, eCH2), 6.62 (d, 1H,
J ¼ 8.7 Hz, ArH), 6.93 (d, 1H, J ¼ 8.7 Hz, ArH), 6.99 (d, 1H,
J ¼ 8.7 Hz, ArH), 7.34 (d, 2H, J ¼ 8.1 Hz, ArH), 7.83 (d, 2H,
J ¼ 8.1 Hz, ArH), 7.97 (d, 1H, J ¼ 8.4 Hz, ArH), 8.71 (s, 1H,
ArH), 8.82 (s, 1H, NHCO)
7c
7d
7e
3326, 2934, 2817, 1650,
1607, 575, 1534, 1511,
1493, 1347, 1252, 1093,
1011, 788
621.3 [M ꢂ H]þ
616.2 [M þ H]þ
636.3 [M þ H]þ
c,d2.43e2.54 (m, 8H,
), 2.77 (t, 2H, J ¼ 5.3 Hz, eCH2),
N
N
3.40 (s, 2H, eCH2), 3.84 (s, 3H, eOCH3), 3.85 (s, 3H, eOCH3),
3.87 (s, 3H, eOCH3), 3.89 (s, 3H, eOCH3), 4.22 (t, 2H, J ¼ 5.3 Hz, eCH2),
6.61 (d, 1H, J ¼ 8.6 Hz, ArH), 6.93 (d, 1H, J ¼ 8.6 Hz, ArH), 6.97e7.00
(m, 3H, ArH), 7.85e7.89 (m, 2H, ArH), 7.94 (dd, 1H, J1 ¼ 2.0 Hz,
J2 ¼ 8.5 Hz, ArH), 8.7 (d, 1H, J ¼ 2.0 Hz, ArH), 8.79 (s, 1H, NHCO)
b,d2.27e2.50 (m, 8H,
), 2.72 (t, 2H, J ¼ 5.1 Hz, eCH2), 3.29
N
3339, 2938, 2823, 1689,
1535, 1494, 1344, 1269,
1095, 1010, 787
N
(s, 2H, eCH2), 3.73 (s, 3H, eOCH3), 3.74 (s, 3H, eOCH3), 3.78
(s, 3H, eOCH3), 4.19 (t, 2H, J ¼ 5.1 Hz, eCH2), 6.76 (d, 1H, J ¼ 8.4 Hz,
ArH), 6.89 (d, 1H, J ¼ 8.4 Hz, ArH), 7.13 (d, 1H, J ¼ 8.4 Hz, ArH), 7.93
(dd, 1H, J1 ¼ 1.8 Hz, J2 ¼ 8.4 Hz, ArH), 8.03 (d, 2H, J ¼ 8.1 Hz, ArH),
8.08 (d, 2H, J ¼ 8.1 Hz, ArH), 8.36 (s, 1H, ArH), 9.78 (d, 1H, NHCO)
c,d2.72e2.91 (m, 8H,
), 3.06 (t, 2H, eCH2, J ¼ 5.1 Hz), 3.68
N
3386, 2936, 2822, 1668,
1528, 1494, 1344, 1269,
1098, 1010, 783
N
(s, 2H, eCH2), 3.85 (s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 3.87
(s, 3H, eOCH3), 4.21 (t, 2H, J ¼ 5.1 Hz, eCH2), 6.61 (d, 1H,
J ¼ 8.7 Hz, ArH), 6.88 (d, 1H, J ¼ 8.4 Hz,
ArH), 7.03 (d, 1H, J ¼ 8.7 Hz, ArH), 7.64e7.75 (m, 3H, ArH), 7.98
(d, 1H, J ¼ 8.7 Hz,
ArH), 8.11 (d, 1H, J ¼ 7.8 Hz, ArH), 8.60 (s, 1H, ArH), 9.51 (s, 1H, NHCO)
7f
3348, 2928, 1695, 1499,
1466, 1320, 1269, 1095,
1012, 664
641.3 [M þ H]þ
c,d2.31 (s, 3H, eCH3), 2.44e2.49 (m, 8H,
(t, 2H, J ¼ 5.2 Hz, eCH2),
), 2.50
N
N
3.42 (s, 2H, eCH2), 3.76 (s, 3H, eOCH3), 3.77 (s, 3H, eOCH3), 3.78
(s, 3H, eOCH3),
3.87 (t, 2H, J ¼ 5.2 Hz, eCH2), 6.76 (d, 1H, J ¼ 8.5 Hz, ArH), 6.93
(d, 1H, J ¼ 8.5 Hz, ArH),
6.98 (d, 1H, J ¼ 8.5 Hz, ArH), 7.27 (d, 2H, J ¼ 8.0 Hz, ArH), 7.52
(d, 2H, J ¼ 8.0 Hz,
ArH), 7.83 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 8.5 Hz, ArH), 8.04
(d, 1H, J ¼ 1.9 Hz, ArH)
7g
3433, 2934, 2825, 1687,
1536, 1495, 1353, 1270,
1096, 1012, 787
625.2 [M þ H]þ
c,d2.40e2.53 (m, 8H,
3.40 (s, 2H, eCH2),
), 2.76 (t, 2H, J ¼ 5.1 Hz, eCH2),
N
N
3.84 (s, 3H, eOCH3), 3.85 (s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 4.24
(t, 2H, eCH2, J ¼ 5.1 Hz),
6.63 (d, 1H, J ¼ 8.4 Hz, ArH), 6.93 (d, 1H, J ¼ 8.4 Hz, ArH), 6.98
(d, 1H, J ¼ 8.7 Hz, ArH),
7.49 (d, 2H, J ¼ 8.7 Hz, ArH), 7.87 (d, 2H, J ¼ 8.4 Hz, ArH), 7.95
(dd, 1H, J1 ¼ 1.8 Hz, J2 ¼ 8.7 Hz,
ArH), 8.67 (d, 1H, J ¼ 1.8 Hz, ArH), 8.98 (s, 1H, NHCO).
7h
3372, 2938, 2822, 1671,
1602, 1582, 1532, 1494,
1347, 1270, 1097, 1009,
786
659.3 [M þ H]þ
c,e2.22e2.45 (m, 8H,
(s, 2H, eCH2), 3.84
), 2.67 (t, 2H, J ¼ 5.4 Hz, eCH2), 3.33
N
N
(s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 3.87 (s, 3H, eOCH3), 4.21
(t, 2H, J ¼ 5.4 Hz, eCH2),
6.62 (d, 1H, J ¼ 8.5 Hz, ArH), 6.91 (d, 1H, J ¼ 8.5 Hz, ArH), 7.00
(d, 1H, J ¼ 8.6 Hz, ArH),
7.37 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 8.2 Hz, ArH), 7.50 (d, 1H, J ¼ 1.9 Hz, ArH), 7.71
(d, 1H, J ¼ 8.2 Hz, ArH), 7.98 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 8.6 Hz, ArH), 8.77
(d, 1H, J ¼ 1.8 Hz, ArH), 9.33 (s, 1H, NHCO)

L. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4107e4116
4113
Table 5 (continued )
NO.
IR (cmꢂ1
)
MS (m/z)a
1H NMR (
d)
7i
3453, 3376, 2942, 1706,
1537, 1462, 1344, 1264,
1097, 1051, 799
597.3 [M þ H]þ
c,d2.47e2.58 (m, 8H,
), 2.82 (t, 2H, J ¼ 5.1 Hz, eCH2), 3.44 (s, 2H, eCH2),
N
N
3.85 (s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 3.87 (s, 3H, eOCH3), 4.24
(t, 2H, J ¼ 5.1 Hz, eCH2), 6.62 (d, 1H, J ¼ 8.4 Hz, ArH), 6.93e6.98 (m, 2H, ArH),
7.12e7.14 (m, 1H, ArH), 7.56e7.58 (m, 1H, ArH), 7.69e7.70 (m, 1H, ArH), 7.96
(dd, 1H, J1 ¼ 1.8 Hz, J2 ¼ 8.4 Hz, ArH), 8.71 (s, 1H, ArH), 8.76 (s, 1H, NHCO)
7j
3427, 3341, 2936, 1702,
1535, 1462, 1350, 1271,
1099, 1036, 750
611.3 [M þ H]þ
597.3 [M þ H]þ
605.3 [M þ H]þ
c,e2.41e2.53 (m, 8H,
), 2.62 (s, 3H, eCH3), 2.78 (t, 2H, J ¼ 5.5 Hz, eCH2),
N
N
3.41 (s, 2H, eCH2), 3.85 (s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 3.87 (s, 3H, eOCH3),
4.23 (t, 2H, J ¼ 5.5 Hz, eCH2), 6.62 (d, 1H, J ¼ 8.5 Hz, ArH), 6.93e6.97 (m, 3H, ArH),
7.37 (d, 1H, J ¼ 5.0 Hz, ArH), 7.95 (dd, 1H, J1 ¼ 2.0 Hz, J2 ¼ 8.5 Hz, ArH), 8.49 (s, 1H,
ArH), 8.73 (d, 1H, J ¼ 2.0 Hz, NHCO)
c,d2.46e2.56 (m, 8H,
), 2.80 (t, 2H, J ¼ 5.1 Hz, eCH2), 3.44 (s, 2H, eCH2),
N
7k
7l
3380, 2938, 1678, 1531,
1496, 1358, 1272, 1095,
1011, 790
N
3.84 (s, 3H, eOCH3), 3.85 (s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 4.23 (t, 2H, J ¼ 5.1
Hz, eCH2), 6.62 (d, 1H, J ¼ 8.5 Hz, ArH), 6.93e6.98 (m, 2H, ArH), 7.37e7.39 (m, 1H,
ArH), 7.53 (dd, 1H, J1 ¼ 1.2 Hz, J2 ¼ 5.0 Hz, ArH), 7.96 (dd, 1H, J1 ¼ 2.0 Hz, J2 ¼ 8.5 Hz,
ArH), 8.05e8.06 (m, 1H, ArH), 8.75 (s, 1H, ArH), 8.77 (d, 1H, J ¼ 2.0 Hz, NHCO)
c,d2.02 (m, 2H, eCH2), 2.50e2.55 (m, 10H,
, eCH2), 3.49 (s, 2H, eCH2), 3.86
N
3366, 2933, 2848, 1673,
1648, 1531, 1465, 1356,
1267, 1095, 1010, 790
N
(s, 3H, eOCH3), 3.87 (s, 3H, eOCH3), 3.88 (s, 3H, eOCH3), 4.16
(t, 2H, J ¼ 6.3 Hz, eCH2),
6.63 (d, 1H, J ¼ 8.7 Hz, ArH), 6.97e7.00 (m, 2H, ArH), 7.49e7.61 (m, 3H, ArH),
7.87e7.89 (m, 2H, ArH), 7.98 (dd, 1H, J1 ¼ 2.1 Hz, J2 ¼ 8.5 Hz, ArH), 8.45
(s, 1H, ArH), 8.82
(s, 1H, NHCO)
7m
3425, 2935, 2822, 1673,
1602, 1532, 1494, 1350,
1269, 1095, 1011, 788
633.4 [M þ H]þ
c,d1.28 (m, 3H, J ¼ 7.5 Hz, eCH3), 2.04 (m, 2H, eCH2), 2.50e2.54 (m, 10H,
,
N
N
eCH2), 2.73 (q, 2H, J ¼ 7.5 Hz, eCH2), 3.49
(s, 2H, eCH2), 3.85 (s, 3H, eOCH3), 3.87
(s, 3H, eOCH3), 3.88 (s, 3H, eOCH3), 4.17 (t, 2H, J ¼ 6.3 Hz, eCH2), 6.62
(d, 1H, J ¼ 8.7 Hz, ArH), 6.96e6.99 (m, 2H, ArH),
7.33 (d, 2H, J ¼ 8.4 Hz, ArH), 7.79 (d, 2H, J ¼ 8.4 Hz, ArH), 7.96 (dd, 1H,
J1 ¼ 1.8 Hz, J2 ¼ 8.55 Hz, ArH),
8.42 (s, 1H, ArH), 8.76 (d, 1H, J ¼ 1.8 Hz, NHCO)
c,e2.05 (m, 2H, eCH2), 2.51e2.54 (m, 10H,
, eCH2), 3.49 (s, 2H, eCH2),
N
7n
7o
3440, 2927, 2850, 1691,
1608, 1539, 1463, 1384,
1269, 1095, 1036, 743
635.4 [M þ H]þ
N
3.86 (s, 3H, eOCH3), 3.88 (s, 3H, eOCH3), 3.89 (s, 3H, eOCH3), 3.90 (s, 3H, eOCH3),
4.18 (t, 2H, J ¼ 6.2 Hz, eCH2), 6.64 (d, 1H, J ¼ 8.5 Hz, ArH), 6.98e6.99 (m, 2H, ArH),
7.01 (d, 2H, J ¼ 8.8 Hz, ArH), 7.86 (d, 2H, J ¼ 8.8 Hz, ArH), 7.95 (d, 1H, J ¼ 8.62 Hz, ArH),
8.35 (s, 1H, ArH), 8.7 (s, 1H, NHCO)
c,e1.98 (m, 2H, eCH2), 2.47e2.50 (m, 10H,
, eCH2), 3.47 (s, 2H, eCH2),
N
3376, 2939, 2821, 1673,
1601, 1528, 1494, 1346,
1267, 1096, 1010, 786
650.4 [M þ H]þ
N
3.84 (s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 3.87 (s, 3H, eOCH3), 4.13 (t, 2H, J ¼ 6.9
Hz, eCH2), 6.63 (d, 1H, J ¼ 8.5 Hz, ArH),
6.94e6.97 (m, 2H, ArH), 7.61e7.74 (m, 3H, ArH), 7.95 (d, 1H, J ¼ 8.5 Hz, ArH),
8.04 (d, 1H, J ¼ 8.2 Hz,
ArH), 8.73 (d, 1H, J ¼ 1.5 Hz, ArH)
c,e1.74 (m, 2H, eCH2), 2.31ee2.34 (m, 5H, eCH2, eCH3), 2.45e2.51
7p
3353, 2944, 2824, 1735,
1688, 1600, 1495, 1301,
1269, 1093, 1012, 767
655.3 [M þ H]þ
(m, 8H,
), 3.49 (s, 2H, eCH2), 3.79 (t, 2H, J ¼ 6.4 Hz, eCH2),
N
N
3.85 (s, 3H, eOCH3), 3.87 (s, 3H, eOCH3), 3.88 (s, 3H, eOCH3), 6.63
(d, 1H, J ¼ 8.5 Hz, ArH), 6.74 (d, 1H, J ¼ 8.5 Hz, ArH),
6.99 (d, 1H, J ¼ 8.5 Hz, ArH), 7.15 (d, 2H, J ¼ 8.0 Hz, ArH), 7.56
(d, 2H, J ¼ 8.0 Hz, ArH), 7.89
(dd, 1H, J1 ¼ 2.0 Hz, J2 ¼ 8.6 Hz,
ArH), 8.22 (d, 1H, J ¼ 2.0 Hz, ArH)
7q
3420, 2935, 2825, 1704,
1605, 1537, 1464, 1326,
1267, 1127, 1095, 1013, 758
673.4 [M þ H]þ
c,d2.04 (m, 2H, eCH2), 2.48e2.53 (m, 10H,
(s, 2H, eCH2), 3.85 (s, 3H, eOCH3),
, eCH2), 3.49
N
N
3.87 (s, 3H, eOCH3), 3.88 (s, 3H, eOCH3), 4.18 (t, 2H, J ¼ 6.3
Hz, eCH2), 6.63 (d, 1H, J ¼ 8.7 Hz, ArH), 6.96e7.01 (m, 2H, ArH),
7.78 (d, 2H, J ¼ 8.4 Hz, ArH), 7.97e8.01
(m, 3H, ArH), 8.46 (s, 1H, ArH), 8.81
(d, 1H, J ¼ 1.8 Hz, NHCO)
7r
3435, 2937, 2817, 1667,
1601, 1527, 1496, 1362,
1273, 1096, 1014, 796
673.3 [M þ H]þ
c,d1.97 (m, 2H, eCH2), 2.45e2.51 (m, 10H,
, eCH2), 3.47
N
N
(s, 2H, eCH2), 3.85 (s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 3.87
(s, 3H, eOCH3), 4.14 (t, 2H, J ¼ 6.3 Hz, eCH2), 6.63 (d, 1H, J ¼ 8.4 Hz, ArH),
6.96e7.00 (m, 2H, ArH), 7.30e7.41 (m, 3H, ArH), 7.95 (s, 1H, ArH),
8.01 (dd, 1H, J1 ¼ 1.5 Hz, J2 ¼ 8.8 Hz, ArH), 8.7
(d, 1H, J ¼ 1.5 Hz, NHCO)
(continued on next page)

4114
L. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4107e4116
Table 5 (continued )
NO.
IR (cmꢂ1
)
MS (m/z)a
1H NMR (
d)
c,e1.99 (m, 2H, eCH2), 2.47e2.50 (m, 10H,
, eCH2), 3.48
N
7s
3396, 2937, 2822, 1656,
1601, 1533, 1464, 1348,
1266, 1097, 1009, 786
673.3 [M þ H]þ
N
(s, 2H, eCH2), 3.85 (s, 3H, eOCH3), 3.87 (s, 3H, eOCH3), 3.88 (s, 3H, eOCH3),
4.15 (t, 2H, J ¼ 6.8 Hz, eCH2), 6.63 (d, 1H, J ¼ 8.5 Hz, ArH),
6.96e6.99 (m, 2H, ArH), 7.39 (dd, 1H, J1 ¼ 8.3 Hz, J2 ¼ 1.7 Hz, ArH), 7.49
(d, 1H, J ¼ 1.7 Hz, ArH), 7.77
(d, 1H, J ¼ 8.3 Hz, ArH), 7.99 (d, 1H, J ¼ 8.6 Hz, ArH), 8.57 (s, 1H, ArH), 8.87
(s, 1H, NHCO)
7t
3445, 2940, 2816, 1664,
1609, 1541, 1495, 1355,
1271, 1097, 1010, 746
611.3 [M þ H]þ
c,d2.05 (m, 2H, eCH2), 2.52e2.83 (m, 10H,
(s, 2H, eCH2), 3.85 (s, 3H, eOCH3),
3.87 (s, 3H, eOCH3), 3.88 (s, 3H, eOCH3), 4.17 (t, 2H, J ¼ 6.3 Hz, eCH2),
6.63 (d, 1H, J ¼ 8.7 Hz, ArH), 6.95e6.99 (m, 2H, ArH), 7.13e7.16
(m, 1H, ArH), 7.57e7.63 (m, 2H,
, eCH2), 3.49
N
N
ArH), 7.95 (d, 1H, J ¼ 8.4 Hz,
ArH), 8.34 (s, 1H, ArH), 8.68 (s, 1H, NHCO)
7u
3424, 2954, 2815, 1664,
1630, 1519, 1492, 1349,
1267, 1095, 1010, 784
625.3 [M þ H]þ
c,e2.03 (m, 2H, eCH2), 2.49e2.54 (m, 10H,
, eCH2), 2.62
N
N
(s, 3H, eCH3), 3.49 (s, 2H, eCH2), 3.85 (s, 3H, eOCH3), 3.87
(s, 3H, eOCH3), 3.88 (s, 3H, eOCH3), 4.17 (t, 2H, J ¼ 6.4 Hz, eCH2), 6.63
(d, 1H, J ¼ 8.5 Hz, ArH), 6.95e6.99 (m, 3H, ArH), 7.37 (d, 1H, J ¼ 5.0 Hz,
ArH), 7.96 (dd, 1H, J1 ¼ 2.0 Hz,
J2 ¼ 8.6 Hz, ArH), 8.23 (s, 1H, ArH), 8.77 (d, 1H, J ¼ 2.0 Hz, NHCO)
7v
3397, 2937, 2849, 1672,
1604, 1535, 1494, 1358,
1267, 1095, 1036, 797
611.4 [M þ H]þ
c,d2.05 (m, 2H, eCH2), 2.50e2.54 (m, 10H,
(s, 2H, eCH2), 3.85 (s, 3H, eOCH3),
, eCH2), 3.49
N
N
3.87 (s, 3H, eOCH3), 3.88 (s, 3H, eOCH3), 4.17 (t, 2H, J ¼ 6.3 Hz, eCH2),
6.63 (d, 1H, J ¼ 8.4 Hz, ArH),
6.96e6.99 (m, 2H, ArH), 7.41e7.48 (m, 2H, ArH), 7.93e7.98
(m, 2H, ArH), 8.26 (s, 1H, ArH),
8.7 (s, 1H, NHCO)
a
ESI(þ), 70 V.
b
c
DMSO-d6.
CDCl3.
d
e
300 MHz.
500 MHz.
4.4.8. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(2,4-dichlorobenzamino)benzoate (5h)
Yield 63.9%, yellow oil.
4.4.16. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(4-methylphenylsulfonamino)benzoate (5p)
Yield 61.6%, white solid, m.p. 125e127 ꢁC.
4.4.17. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(4-(trifluoromethyl)benzamino)benzoate (5q)
Yield 51.2%, yellow oil.
4.4.9. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(thiophene-2-carboxamino)benzoate (5i)
Yield 41.9%, yellow oil.
4.4.18. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(2,6-dichlorobenzamino)benzoate (5r)
Yield 44.4%, yellow oil.
4.4.10. Ethyl4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(3-methylthiophene-2-carboxamino)benzoate (5j)
Yield 34.1%, white solid, m.p. 121e125 ꢁC.
4.4.19. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(2,4-dichlorobenzamino)benzoate (5s)
Yield 35.5%, yellow oil.
4.4.11. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(thiophene-3-carboxamino)benzoate (5k)
Yield 31.5%, yellow oil.
4.4.20. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(thiophene-2-carboxamino)benzoate (5t)
Yield 38.5%, yellow oil.
4.4.12. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(benzamino)benzoate (5l)
Yield 61.9%, yellow oil.
4.4.21. Ethyl4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)
propoxy)-3-(3-methylthiophene-2-carboxamino)benzoate (5u)
Yield 39.1%, yellow oil.
4.4.13. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(4-ethylbenzamino)benzoate (5m)
Yield 69.4%, white solid, m.p. 97e102 ꢁC.
4.4.22. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)
propoxy)-3-(thiophene-3-carboxamino)benzoate (5v)
Yield 30.6%, yellow oil.
4.4.14. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(4-methoxybenzamino)benzoate (5n)
Yield 36.1%, white solid, m.p. 82e87 ꢁC.
4.5. General procedure for synthesis of compounds 6
4.4.15. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(2-nitrobenzamino)benzoate (5o)
Yield 33.8%, yellow oil.
The appropriate 3-amino substituted ethyl 4-(2(3)-(4-(2,3,4-
trimethoxybenzyl)piperazin1-yl)ethoxy(propoxy))benzoate
(5)

L. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4107e4116
4115
(1.5 mmol) was dissolved in a mixture of methanol (16 mL) and
4.5.14. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(4-methoxybenzamino)benzoic acid (6n)
Yield 77.1%, white solid, m.p. 67e72 ꢁC.
water (24 mL). Potassium carbonate (0.62 g, 4.5 mmol) was then
added and the reaction mixture was refluxed under stirring for 2 h.
After filtering, the methanol in the filtrate was removed under
vacuum. To the residue was dropped 2% dilute hydrochloric acid to
adjust the pH to 7 (isoelectric point), then the resulting solution
was extracted with ethyl acetate (3 ꢃ15 mL). The organic layer was
washed with saturated sodium chloride solution, dried over
anhydrous sodium sulfate and concentrated under vacuum to give
compound 6. If the product could be precipitated under isoelectric
point directly, it was filtered off and dried to afford compound 6.
4.5.15. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(2-nitrobenzamino)benzoic acid (6o)
Yield 67.5%, white solid, m.p. 83e87 ꢁC.
4.5.16. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(4-methylphenylsulfonamino)benzoic acid (6p)
Yield 79.0%, yellow oil.
4.5.1. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(3-methylbenzamino)benzoic acid (6a)
4.5.17. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(4-(trifluoromethyl)benzamino)benzoic acid (6q)
Yield 86.5%, white solid, m.p. 61e65 ꢁC.
Yield 87.5%, white solid, m.p. 78e83 ꢁC.
4.5.2. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(4-ethylbenzamino)benzoic acid (6b)
4.5.18. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(2,6-dichlorobenzamino)benzoic acid (6r)
Yield 88.4%%, white solid, m.p. 50e55 ꢁC.
Yield 56.0%, white solid, m.p. 79e84 ꢁC.
4.5.19. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(2,4-dichlorobenzamino)benzoic acid (6s)
Yield 67.5%, yellow oil.
4.5.3. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(4-methoxybenzamino)benzoic acid (6c)
Yield 90%, yellow oil.
4.5.20. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(thiophene-2-carboxamino)benzoic acid (6t)
Yield 89.9%, white solid, m.p. 121e125 ꢁC.
4.5.4. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(4-cyanobenzamino)benzoic acid (6d)
Yield 87.5%, white solid, m.p. 132e137 ꢁC.
4.5.21. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(3-methylthiophene-2-carboxamino)benzoic acid (6u)
Yield 69.0%, white solid, m.p. 75e80 ꢁC.
4.5.5. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(2-nitrobenzamino)benzoic acid (6e)
Yield 89.7%, white solid, m.p. 99e103 ꢁC.
4.5.22. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(thiophene-3-carboxamino)benzoic acid (6v)
Yield 79.9%, white solid, m.p. 96e101 ꢁC.
4.5.6. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(4-methylphenylsulfonamino)benzoicacid (6f)
Yield 87.0%, white solid, m.p. 50e55 ꢁC.
4.6. General procedure for synthesis of compounds 7
4.5.7. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(4-chlorobenzamino)benzoic acid (6g)
To a solution of the appropriate 3-amino substituted ethyl 4-(2(3)-
(4-(2,3,4-trimethoxybenzyl)piperazin1-yl)ethoxy(propoxy))benzoic
acid (6) (0.69 mmol), 1-hydroxybenzotriazole (HOBT) (0.09 g,
0.69 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCI) (0.13 g, 0.69 mmol) in anhydrous DMF (2 mL)
was added free guanidine (0.16 g, 2.79 mmol), followed by water
(2 mL). After stirring at room temperature for 2 h, the reaction
mixture was poured onto ethyl acetate/water (v/v ¼ 3:2) and
extracted with ethyl acetate. The combined organic layers were
washed with saturated sodium chloride solution, dried over anhy-
drous sodium sulfate and concentrated under vacuum. The residue
was purified by silica gel column chromatography (EtOAC/CH3OH/
Et3N ¼ 15:1:0.1) to give compounds 7ae7v.
Yield 75.5%, white solid, m.p. 89e94 ꢁC.
4.5.8. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(2,4-dichlorobenzamino)benzoic acid (6h)
Yield 69%, white solid, m.p. 91e93 ꢁC.
4.5.9. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(thiophene-2-carboxamino)benzoic acid (6i)
Yield 67.0%, yellow oil.
4.5.10. Ethyl4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(3-methylthiophene-2-carboxamino)benzoic acid (6j)
Yield 89.0%, white solid, m.p. 70e75 ꢁC.
For the physicochemical characters and spectral data of
compounds 7ae7v, see Tables 2 and 5.
4.5.11. Ethyl 4-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)ethoxy)-
3-(thiophene-3-carboxamino)benzoic acid (6k)
Yield 77.5%, yellow oil.
4.7. Rat platelet swelling assay
SpragueeDawley rats (380e420 g) were anesthetized with
ethyl ether and blood was collected from their eyeholes with 25%
(v/v) acidecitrateedextrose (ACD; sodium citrate 2.77 g, citric acid
1.07 g and glucose 3.07 g in 125 mL distilled H2O). Platelet-rich
plasma (PRP) was obtained by centrifugation of whole blood at
1300 g/min for 10 min at room temperature. The upper 2/3 of the
supernatants were used for the further measurements and stored
at r.t. until used. All measurements were performed within 4e5 h.
4.5.12. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-benzaminobenzoic acid (6l)
Yield 89.5%, white solid, m.p. 142e143 ꢁC.
4.5.13. Ethyl 4-(3-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)pro-
poxy)-3-(4-ethylbenzamino)benzoic acid (6m)
Yield 89.0%, white solid, m.p. 160e165 ꢁC.

4116
L. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4107e4116
All compounds were dissolved in DMSO and diluted with
Acknowledgments
propionate medium (pH 7.4). A solution of the tested compound
(25 mL) was added to 175 mL of propionate buffer (sodium propi-
onate, 140 mmol/L; HEPES, 20 qs; glucose, 10 mmol/L; KCl, 5 mmol/
L; MgCl2, 1 mmol/L; CaCl2, 1 mmol/L; pH 6.7) contained in a spec-
trophotometer cuvette. Then, 50 mL of PRP prewarmed to 37 ꢁC
was added. The suspension was stirred, and the change in optical
density (OD) was recorded each 7.5 s for 2 min at 550 nm (Ther-
moMultiscan Spectrum). The IC50 value was calculated according to
the regression analysis. For each molecule, the measurements were
performed in triplicate.
Financial support from the National Natural Science Foun-
dation of China (No. 30672512) is gratefully acknowledged. We
also thank Center for Instrumental Analysis at China Pharma-
ceutical University for their contribution in the structural
confirmation.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2011.06.011.
4.8. Screening of primary culture of myocardial cell in vitro in
neonatal rat
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