Synthesis and bioactivity of substituted benzoylguanidine derivatives as potent Na+/H+ exchanger inhibitors

Article abstract of DOI:10.1002/cjoc.201180470

A novel series of substituted benzoylguanidine derivatives were designed and synthesized in order to evaluate their NHE1 inhibitory activity. Most of them were found to inhibit NHE1-mediated platelet swelling in a concentration-dependent manner, and eight compounds showed more potent NHE1 inhibitory activity than Cariporide. Compound 6f with an IC₅₀ value of 1.08 × 10^-10 molmiddot;L^-1, was 39 times more potent than lead compound CPU-X-050420 in vitro tests. A novel series of 5-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)acetyl)benzoylguanidine derivatives 6a-6i have been synthesized and screened for their NHE1 inhibitory activity. Copyright

Full text of DOI:10.1002/cjoc.201180470

FULL PAPER
DOI: 10.1002/cjoc.201180470
Synthesis and Bioactivity of Substituted Benzoylguanidine
Derivatives as Potent Na＋/H＋ Exchanger Inhibitors
Jin, Ninga(金宁)
Yang, Yuna(杨芸)
Xu, Wentinga(徐文婷)
Yang, Xiaozhia(杨小治)
Gong, Guoqingb(龚国清)
Xu, Yungen*,a(徐云根)
a Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
b Department of Pharmacology, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
A novel series of substituted benzoylguanidine derivatives were designed and synthesized in order to evaluate
their NHE1 inhibitory activity. Most of them were found to inhibit NHE1-mediated platelet swelling in a concentra-
tion-dependent manner, and eight compounds showed more potent NHE1 inhibitory activity than Cariporide. Com-
pound 6f with an IC50 value of 1.08×10－ mol•L－ 1, was 39 times more potent than lead compound
10
CPU-X-050420 in vitro tests.
Keywords NHE1 inhibitor, benzoylguanidine derivatives, ischemia-reperfusion injury
Introduction
050420 as lead compounds, we chose the benzoylgua-
nidine as core structure, using combination principles,
introduced [4-(2,3,4-trimethoxy-benzyl)-piperazin-1-yl]-
acetyl group to the 5 position and alkoxy group to the 2
position of benzoylguanidine, and then synthesized tar-
get compounds 6a—6i. NHE1 inhibitory activity of
these compounds was tested.
＋
Intracellular Ca2 overload and massive free radical
produced during ischemia and reperfusion are the major
pathophysiological factors.[1] It has been demonstrated
that activated Na＋/H＋ exchanger (NHE) led to the ex-
＋
cessive Ca2 influx during ischemia and reperfusion.[2]
NHE is a kind of integral protein membrane family ex-
pressed in various types of mammalian cells, which
regulates intracellular pH (pHi) by exchanging one in-
tracellular H＋ for an extracellular Na＋. From the data,
at least nine NHE isoforms have been identified and
characterized (NHE1—NHE9).[3] The most predominant
isoform is NHE-1 which is involved in intracellular pH,
cell-volume control, cytoskeletal organization, heart
disease and cancer.[4]
CH3
OCH3
H3CO
H3CO
H3C
H3CO2S
N
N
NH2
NH2
NH
O
Trimetazidine
Cariporide
O
N
NHE inhibitors have been shown to provide signifi-
＋
cant myocardial protection in reducing reperfusion Ca2
NH2
O2N
N
H2N
HCl
overload in myocardial cells, reducing the mitochon-
drial ultrastructural injury in cardiac cell and leakage of
enzymes, and reducing cell edema, cardiac stunned and
arrhythmia incidence, which contribute to reduced
post-ischemic recovery of mechanical function.[5] The
structure-activity relationship study on various types of
NHE1 inhibitors shows that the acylguanidine moiety is
the pharmacophore of NHE1 inhibitors, although there
are several exceptions. During the course of our efforts
to discover novel inhibitors of NHE1, our laboratory
found that CPU-X-050420 (Figure 1) showed a similar
potency against NHE1 activity compared to cariporide
on rat platelet swelling assay, and significantly im-
provement on myocardial ischemia and reperfusion in-
jury compared with trimetazidine.[6] With CPU-X-
H3CO H2C N
H3CO
H3CO
CPU-X-050420
Figure 1 The structures of cariporide, trimetazidine and CPU-
X-050420.
Experimental
Apparatuses and materials
Melting points were obtained with an RDCSY-I cap-
illary apparatus and were uncorrected. The IR spectra
*
E-mail: xyg64@126.com
Received March 21, 2011; accepted September 21, 2011.
Chin. J. Chem. 2012, 30, 333—340
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
333

Jin et al.
FULL PAPER
General procedure for the synthesis of compounds 2
(in KBr pellets) were recorded on a Nicolet Impact 410
spectrophotometer. The H NMR spectra were recorded
1
Methyl 5-acetyl-2-hydroxybenzoate (10 mmol) was
dissolved in anhydrous DMF (20 mL) with stirring, then
KI (1.8 mmol) and the powder of K2CO3 (20 mmol)
were added and the mixture was stirred for 1 h at room
temperature. After slowly dropping a mixture of alkyl
halide (10 mmol) in DMF (5 mL), the mixture was
heated to 50 ℃ for 5 h. After cooling, the resultant
mixture was poured into water (100 mL), then the
aqueous phase was extracted with acetic ether (50 mL×
3), and the combined organic layers were washed with
saturated sodium chloride solution and dried over anhy-
drous MgSO4. The filtrate was concentrated to dryness
under reduced pressure to give compounds 2.
on a Brucker AV-300 or AV-500 NMR spectrometer us-
ing TMS as an internal standard and chemical shifts
were given relative to tetramethylsilane (TMS). Mass
spectra were taken in an Agilent 1100 series LC/MSD
Tarp (SL).
Preparation and analysis data
The synthetic route for target compounds 6a—6i is
depicted in Scheme 1. The key intermediates 4a—4i
were obtained from methyl 5-acetyl-2-hydroxybenzoate
(1) by O-alkylation with alkyl halides to form 2a—2i
which were subjected to α-bromination with bromine,
followed by reaction with trimetazidine. Hydrolysis of
4a—4i with potassium carbonate in methanol-water
(6∶4, w/w) afforded 5a—5i. Treatment of 5a—5i with
excessive guanidine, 1-hydroxybenzotrizole (HOBt) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (EDCI) in anhydrous N,N-dimethylformamide
(DMF) afforded the target compounds 6a—6i which
were purified by silica gel column chromatography
(acetic ether/methanol/triethylamine ＝ 15 ∶ 1 ∶ 0.1,
V/V/V).
Methyl 5-acetyl-2-methoxy-benzoate (2a): Yield
97.0%, white solid, m.p. 95—97 ℃ (Lit.[7]: 95—96 ℃).
Methyl 5-acetyl-2-ethoxy-benzoate (2b): Yield
87.8%, white solid, m.p. 56—58 ℃ (Lit.[7]: 47—50 ℃).
Methyl 5-acetyl-2-propoxy-benzoate (2c): Yield
1
89.7%, white solid, m.p. 49—51 ℃; H NMR (CDCl3,
300 MHz) δ: 1.09 (t, J＝7.5 Hz, 3H, CH3), 1.85—1.92
(m, 2H, CH2), 2.58 (s, 3H, COCH3), 3.92 (s, 3H, OCH3),
4.08 (t, J＝6.4 Hz, 2H, OCH2), 7.00 (d, J＝8.8 Hz, 1H,
ArH), 8.07 (dd, J1＝8.8 Hz, J2＝2.4 Hz, 1H, ArH), 8.37
(d, J＝2.4 Hz, 1H, ArH).
Scheme 1 Syntheses of compounds 6a—6i
Methyl 5-acetyl-2-butoxy-benzoate (2d): Yield
1
O
O
O
O
92.0%, white solid, m.p. 53—56 ℃; H NMR (CDCl3,
(1)
300 MHz) δ: 0.99 (t, J＝7.4 Hz, 3H, CH3), 1.50—1.57
(m, 2H, CH2), 1.81—1.86 (m, 2H, CH2), 2.58 (s, 3H,
COCH3), 3.91 (s, 3H, OCH3), 4.11 (t, J＝6.4 Hz, 2H,
OCH2), 7.00 (d, J＝8.8 Hz, 1H, ArH), 8.08 (dd, J1＝8.8
Hz, J2＝2.4 Hz, 1H, ArH), 8.39 (d, J＝2.4 Hz, 1H,
ArH).
H3C
OCH3
OH
H3C
OCH3
OR
2a － 2i
(3)
1
O
O
(2)
Br
OCH3
OR
Methyl 5-acetyl-2-isobutoxy-benzoate (2e): Yield
1
3a － 3i
89.3%, pale yellow oil; H NMR (CDCl3, 300 MHz) δ:
OCH3
1.08 (d, J＝2.6 Hz, 6H, CH3), 2.12—2.21 (m, 1H, CH),
2.58 (s, 3H, COCH3), 3.87 (d, J＝6.4 Hz, 2H, OCH2),
3.92 (s, 3H, OCH3), 6.99 (d, J＝8.8 Hz, 1H, ArH), 8.08
(dd, J1＝8.8 Hz, J2＝2.4 Hz, 1H, ArH), 8.40 (d, J＝2.4
Hz, 1H, ArH).
H3CO
H3CO
N
O
O
(4)
N
OCH3
OR
4a － 4i
OCH3
Methyl 5-acetyl-2-pentyloxy-benzoate (2f): Yield
1
H3CO
H3CO
89.3%, white solid, m.p. 27—30 ℃; H NMR (CDCl3,
N
N
O
O
(5)
300 MHz) δ: 0.94 (t, J＝7.2 Hz, 3H, CH3), 1.36—1.52
(m, 4H, CH2CH2), 1.82—1.91 (m, 2H, CH2), 2.58 (s,
3H, COCH3), 3.91 (s, 3H, OCH3), 4.11 (t, J＝6.6 Hz,
2H, OCH2), 7.01 (d, J＝8.7 Hz, 1H, ArH), 8.08 (dd, J1
＝8.7 Hz, J2＝2.4 Hz, 1H, ArH), 8.39 (d, J＝2.4 Hz, 1H,
ArH).
OH
OR
5a － 5i
OCH3
H3CO
H3CO
N
N
O
O
NH
NH2
N
Methyl 5-acetyl-2-hexyloxy-benzoate (2g): Yield
H
1
95.9%, yellow oil; H NMR (CDCl3, 500 MHz) δ: 0.94
OR
6a － 6i
(t, J＝4.1 Hz, 3H, CH3), 1.37—1.41 (m, 4H, CH2CH2),
1.50—1.56 (m, 2H, CH2), 1.85—1.91 (m, 2H, CH2),
2.60 (s, 3H, COCH3), 3.93 (s, 3H, OCH3), 4.14 (t, J＝
3.9 Hz, 2H, OCH2), 7.03 (d, J＝8.8 Hz, 1H, ArH), 8.11
(dd, J1＝8.8 Hz, J2＝2.4 Hz, 1H, ArH), 8.41 (d, J＝2.4
Hz, 1H, ArH).
a R = CH3; b R = CH2CH3; c R = CH2CH2CH3; d R = (CH2)3CH3;
e R = CH2CH(CH3)2; f R = (CH2)4CH3; g R = (CH2)5CH3;
h R = (CH2)6CH3; i R = (CH2)7CH3
Reagents and conditions: (1) RX, K2CO3, KI, anhydrous DMF,
50 ℃; (2) Br2, CHCl3, room temperature; (3) trimetazidine,
K2CO3, anhydrous CH3CN, room temperature; (4) K2CO3, 60%
methanol, reflux; (5) a) HOBt, EDCI, DMF; b) guanidine, room
temperature.
Methyl 5-acetyl-2-heptyloxy-benzoate (2h): Yield
1
97.6%, white solid, m.p. 34—35 ℃; H NMR (CDCl3,
334
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 333—340

Synthesis and Bioactivity of Substituted Benzoylguanidine Derivatives
300 MHz) δ: 0.90 (t, J＝6.6 Hz, 3H, CH3), 1.26—1.41
(m, 6H, CH2CH2CH2), 1.44—1.51 (m, 2H, CH2), 1.81
—1.90 (m, 2H, CH2), 2.58 (s, 3H, COCH3), 3.91 (s, 3H,
OCH3), 4.10 (t, J＝6.6 Hz, 2H, OCH2), 7.00 (d, J＝8.7
Hz, 1H, ArH), 8.08 (dd, J1＝8.7 Hz, J2＝2.4 Hz, 1H,
ArH), 8.41 (d, J＝2.4 Hz, 1H, ArH).
Methyl 5-acetyl-2-octyloxy-benzoate (2i): Yield
93.5%, white solid, m.p. 35—36 ℃; 1H NMR (CDCl3,
300 MHz) δ: 0.89 (t, J＝6.9 Hz, 3H, CH3), 1.29—1.34
(m, 8H, CH2CH2CH2CH2), 1.47—1.52 (m, 2H, CH2),
1.81—1.88 (m, 2H, CH2), 2.58 (s, 3H, COCH3), 3.91 (s,
3H, OCH3), 4.11 (t, J＝6.6 Hz, 2H, OCH2), 7.00 (d, J＝
8.7 Hz, 1H, ArH), 8.08 (dd, J1＝8.7 Hz, J2＝2.4 Hz, 1H,
ArH), 8.39 (d, J＝2.4 Hz, 1H, ArH).
ArH), 8.12 (dd, J1＝9 Hz, J2＝2.4 Hz, 1H, ArH), 8.42
(d, J＝2.4 Hz, 1H, ArH).
Methyl
5-(2-bromo-acetyl)-2-hexyloxy-benzoate
1
(3g): Yield 64.4%, white crystal, m.p. 89—91 ℃; H
NMR (CDCl3, 500 MHz) δ: 0.94 (t, J＝6.7 Hz, 3H,
CH3), 1.36—1.38 (m, 4H, CH2CH2), 1.51—1.60 (m, 2H,
CH2), 1.85—1.91 (m, 2H, CH2), 3.94 (s, 3H, OCH3),
4.15 (t, J＝6.5 Hz, 2H, OCH2), 4.44 (s, 2H, COCH2),
7.05 (d, J＝8.8 Hz, 1H, ArH), 8.14 (dd, J1＝8.8 Hz, J2
＝2.1 Hz, 1H, ArH), 8.44 (d, J＝2.1 Hz, 1H, ArH).
Methyl
5-(2-bromo-acetyl)-2-heptyloxy-benzoic
acid methyl ester (3h): Yield 47.8%, white crystal, m.p.
1
96—98 ℃; H NMR (CDCl3, 300 MHz) δ: 0.90 (t, J＝
6.9 Hz, 3H, CH3), 1.25—1.39 (m, 6H, CH2CH2CH2),
1.45—1.52 (m, 2H, CH2), 1.82—1.91 (m, 2H, CH2),
3.92 (s, 3H, OCH3), 4.13 (t, J＝6.6 Hz, 2H, OCH2),
4.42 (s, 2H, COCH2), 7.03 (d, J＝9 Hz, 1H, ArH), 8.14
(dd, J1＝9 Hz, J2＝2.4 Hz, 1H, ArH), 8.44 (d, J＝2.4
Hz, 1H, ArH).
5-(2-Bromo-acetyl)-2-octyloxy-benzoate (3i): Yield
50.1%, white crystal, m.p. 87—88 ℃; 1H NMR (CDCl3,
300 MHz) δ: 0.89 (t, J＝6.9 Hz, 3H, CH3), 1.29—1.34
(m, 8H, CH2CH2CH2CH2), 1.45—1.52 (m, 2H, CH2),
1.81—1.91 (m, 2H, CH2), 3.92 (s, 3H, OCH3), 4.12 (t,
J＝6.3 Hz, 2H, OCH2), 4.42 (s, 2H, COCH2), 7.03 (d,
J＝9 Hz, 1H, ArH), 8.12 (dd, J1＝9 Hz, J2＝2.4 Hz, 1H,
ArH), 8.42 (d, J＝2.4 Hz, 1H, ArH).
General procedure for synthesis of compounds 3
2 (12.7 mmol) was dissolved in chloroform (20 mL)
with stirring, then added 2 drops of a mixture of bro-
mine (19.0 mmol) and chloroform (10 mL). Added the
remaining mixture dropwise after color fading, and fin-
ished in 4 h. The mixture was concentrated to dryness
under reduced pressure to give yellow solid. The crude
product was recrystallised from methanol to give com-
pounds 3.
Methyl 5-(2-bromo-acetyl)-2-methoxy-benzoate (3a):
Yield 72.2%, white crystal, m.p. 150—153 ℃ (Lit.[7]:
149—153 ℃).
Methyl 5-(2-bromo-acetyl)-2-ethoxy-benzoate (3b):
Yield 44.3%, white crystal, m.p. 148—150 ℃ (Lit.[7]:
147—149 ℃).
Methyl 5-(2-bromo-acetyl)-2-propoxy-benzoate (3c):
Yield 65.3%, white crystal, m.p. 102—105 ℃; 1H NMR
(CDCl3, 300 MHz) δ: 1.09 (t, J＝7.4 Hz, 3H, CH3), 1.86
—1.93 (m, 2H, CH2), 3.92 (s, 3H, OCH3), 4.10 (t, J＝
6.4 Hz, 2H, OCH2), 4.42 (s, 2H, COCH2), 7.03 (d, J＝
8.8 Hz, 1H, ArH), 8.11 (dd, J1＝8.8 Hz, J2＝2.4 Hz, 1H,
ArH), 8.42 (d, J＝2.4 Hz, 1H, ArH).
General procedure for synthesis of compounds 4
K2CO3 powder (17.5 mmol) was added to a solution
of 3 (3.5 mmol) in anhydrous acetonitrile (20 mL).
Trimetazidine (1-(2,3,4-trimethoxybenzyl)piperazine)
(3.5 mmol) in anhydrous acetonitrile (5 mL) was added
dropwise. TLC showed the completion of the reaction in
a flash, and then the mixture was filtered, concentrated,
and submitted to silica gel column chromatography (pe-
troleum/EtOAc＝4∶1, V/V).
Methyl 5-(2-bromo-acetyl)-2-butoxy-benzoate (3d):
Yield 52.1%, white crystal, m.p. 93—95 ℃; H NMR
Methyl 5-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-
1-yl)acetyl)-2-(methyl)benzoate (4a): Yield 74.5%, yel-
low oil; H NMR (CDCl3, 300 MHz) δ: 2.58 (s, 8H,
piperazine H), 3.52 (s, 2H, CH2), 3.76 (s, 2H, CH2),
3.85—3.92 (m, 12H, OCH3), 3.98 (s, 3H, OCH3), 6.64
(d, J＝8.4 Hz, 1H, ArH), 6.99 (d, J＝8.4 Hz, 1H, ArH),
7.03 (d, J＝9 Hz, 1H, ArH), 8.19 (dd, J1＝9 Hz, J2＝2.1
Hz, 1H, ArH), 8.49 (d, J＝2.1 Hz, 1H, ArH).
1
1
(CDCl3, 300 MHz) δ: 0.99 (t, J＝7.4 Hz, 3H, CH3), 1.51
—1.58 (m, 2H, CH2), 1.82—1.87 (m, 2H, CH2), 3.92 (s,
3H, OCH3), 4.14 (t, J＝6.4 Hz, 2H, OCH2), 4.42 (s, 2H,
COCH2), 7.03 (d, J＝8.8 Hz, 1H, ArH), 8.11 (dd, J1＝
8.8 Hz, J2＝2.4 Hz, 1H, ArH), 8.42 (d, J＝2.4 Hz, 1H,
ArH).
Methyl
5-(2-bromo-acetyl)-2-isobutoxy-benzoate
Methyl 5-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-
1-yl)acetyl)-2-(ethyl)benzoate (4b): Yield 77.8%, yel-
low oil; H NMR (CDCl3, 300 MHz) δ: 1.50 (t, J＝7.2
1
(3e): Yield 53.7%, white crystal, m.p. 98—100 ℃; H
NMR (CDCl3, 300 MHz) δ: 1.08 (d, J＝6.7 Hz, 6H,
CH3), 2.14—2.23 (m, 1H, CH), 3.90 (d, J＝6.4 Hz, 2H,
OCH2), 3.93 (s, 3H, OCH3), 4.42 (s, 2H, COCH2), 7.02
(d, J＝8.8 Hz, 1H, ArH), 8.12 (dd, J1＝8.8 Hz, J2＝2.4
Hz, 1H, ArH), 8.43 (d, J＝2.4 Hz, 1H, ArH).
1
Hz, 3H, CH3), 2.59 (s, 8H, piperazine H), 3.52 (s, 2H,
CH2), 3.77 (s, 2H, CH2), 3.85—3.92 (m, 12H, OCH3),
4.20 (q, J＝6.9 Hz, 2H, OCH2), 6.64 (d, J＝8.4 Hz, 1H,
ArH), 7.03 (d, J＝9 Hz, 2H, ArH), 8.15 (dd, J1＝9 Hz,
J2＝2.4 Hz, 1H, ArH), 8.46 (d, J＝2.4 Hz, 1H, ArH).
Methyl 5-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-
1-yl)acetyl)-2-(propoxy)benzoate (4c): Yield 63.0%,
yellow oil; 1H NMR (CDCl3, 300 MHz) δ: 1.8 (t, J＝7.4
Hz, 3H, CH3), 1.86—1.90 (m, 2H, CH2), 2.62 (s, 8H,
piperazine H), 3.55 (s, 2H, CH2), 3.76 (s, 2H, CH2),
Methyl
5-(2-bromo-acetyl)-2-pentyloxy-benzoate
1
(3f): Yield 67.2%, white crystal, m.p. 91—93 ℃; H
NMR (CDCl3, 300 MHz) δ: 0.94 (t, J＝7.2 Hz, 6H,
CH3), 1.36—1.52 (m, 4H, CH2CH2), 1.82—1.90 (m, 2H,
CH2), 3.92 (s, 3H, OCH3), 4.13 (t, J＝6.6 Hz, 2H,
OCH2), 4.42 (s, 2H, COCH2), 7.03 (d, J＝9 Hz, 1H,
Chin. J. Chem. 2012, 30, 333—340
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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335

Jin et al.
FULL PAPER
1
3.85—3.91 (m, 12H, OCH3), 4.07 (t, J＝6.4 Hz, 2H,
OCH2), 6.64 (d, J＝8.4 Hz, 1H, ArH), 6.98 (d, J＝8.4
Hz, 1H, ArH), 7.00 (d, J＝8.8 Hz, 1H, ArH), 8.14 (dd,
J1＝8.8 Hz, J2＝2.3 Hz, 1H, ArH), 8.46 (d, J＝2.3 Hz,
1H, ArH).
pale yellow oil; H NMR (CDCl3, 300 MHz) δ: 0.86 (t,
J＝6.9 Hz, 3H, CH3), 1.29—1.32 (m, 8H, CH2CH2-
CH2CH2), 1.47—1.51 (m, 2H, CH2), 1.83—1.87 (m, 2H,
CH2), 2.60 (s, 8H, piperazine H), 3.52 (s, 2H, CH2),
3.76 (s, 2H, CH2), 3.85—3.91 (m, 12H, OCH3), 4.10 (t,
J＝6.6 Hz, 2H, OCH2), 6.64 (d, J＝8.4 Hz, 1H, ArH),
6.99 (d, J＝8.7 Hz, 1H, ArH), 8.15 (dd, J1＝8.7 Hz,
J2＝2.1 Hz, 1H, ArH), 8.46 (d, J＝2.1 Hz, 1H, ArH).
Methyl 5-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-
1-yl)acetyl)-2-(butoxy)benzoate (4d): Yield 79.2%, pale
1
yellow oil; H NMR (CDCl3, 300 MHz) δ: 0.99 (t, J＝
7.4 Hz, 3H, CH3), 1.50—1.60 (m, 2H, CH2), 1.81—1.88
(m, 2H, CH2), 2.63 (s, 8H, piperazine H), 3.56 (s, 2H,
CH2), 3.76 (s, 2H, CH2), 3.85—3.92 (m, 12H, OCH3),
4.11 (t, J＝6.4 Hz, 2H, OCH2), 6.64 (d, J＝8.5 Hz, 1H,
ArH), 6.98 (d, J＝8.5 Hz, 1H, ArH), 7.01 (d, J＝8.8 Hz,
1H, ArH), 8.14 (dd, J1＝8.8 Hz, J2＝2.3 Hz, 1H, ArH),
8.45 (d, J＝2.3 Hz, 1H, ArH).
Methyl 5-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-
1-yl)acetyl)-2-(isobutoxy)benzoate (4e): Yield 59.6%,
pale yellow oil; 1H NMR (CDCl3, 500 MHz) δ: 1.07 (d,
J＝6.7 Hz, 6H, CH3), 2.13—2.19 (m, 1H, CH), 2.63 (s,
8H, piperazine H), 3.55 (s, 2H, CH2), 3.75 (s, 2H, CH2),
3.85—3.91 (m, 12H, OCH3), 3.92 (d, J＝12.5 Hz, 2H,
OCH2), 6.64 (d, J＝8.5 Hz, 1H, ArH), 6.97 (d, J＝8.8
Hz, 1H, ArH), 7.01 (d, J＝8.5 Hz, 1H, ArH), 8.14 (dd,
J1＝8.8 Hz, J2＝2.0 Hz, 1H, ArH), 8.46 (d, J＝2.0 Hz,
1H, ArH).
General procedure for synthesis of compounds 5a—
5g
K2CO3 (4.8 mmol) was added to a solution of 4 (1.6
mmol) in 60% methanol solution (35 mL), and the mix-
ture was stirred and refluxed for 1 h, then methanol was
removed under vacuum. Acidified to pH 7 with 2% HCl,
then extracted with dichlormethane (25 mL×3). The
combined organic layers were dried over anhydrous
MgSO4. Then, the filtrate was concentrated to dryness
under reduced pressure, and the resultant solid was used
without further purification.
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(methoxy)benzoic acid (5a): Yield 81.6%,
1
yellow solid, m.p. 110—114 ℃; H NMR (CDCl3, 300
MHz) δ: 2.89—3.24 (m, 8H, piperazine H), 3.48 (s, 2H,
CH2), 3.86—4.02 (m, 12H, OCH3), 4.22 (s, 2H, CH2),
6.70 (d, J＝8.7 Hz, 1H, ArH), 7.04 (d, J＝9 Hz, 1H,
ArH), 7.33 (d, J＝8.7 Hz, 1H, ArH), 8.07 (d, J＝8.1 Hz,
1H, ArH), 8.43 (s, 1H, ArH).
Methyl 5-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-
1-yl)acetyl)-2-(pentyloxy)benzoate (4f): Yield 73.9%,
1
pale yellow oil; H NMR (CDCl3, 300 MHz) δ: 0.94 (t,
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(ethoxy)benzoic acid (5b): Yield 77.3%, yel-
low solid, m.p. 78—80 ℃; 1H NMR (CDCl3, 300 MHz)
δ: 1.51 (t, J＝6.9 Hz, 3H, CH3), 2.85—3.15 (m, 8H,
piperazine H), 3.86 (s, 9H, OCH3), 3.95 (s, 2H, CH2),
4.14 (s, 2H, CH2), 4.26 (q, J＝6.9 Hz, 2H, OCH2), 6.68
(d, J＝8.4 Hz, 1H, ArH), 7.00 (d, J＝8.7 Hz, 1H, ArH),
7.25 (d, J＝8.4 Hz, 1H, ArH), 8.02 (dd, J1＝8.7 Hz,
J2＝2.1 Hz, 1H, ArH), 8.30 (d, J＝2.1 Hz, 1H, ArH).
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(propoxy)benzoic acid (5c): Yield 79.5%, pale
J＝6.9 Hz, 3H, CH3), 1.36—1.51 (m, 4H, CH2CH2),
1.84—1.88 (m, 2H, CH2), 2.60 (s, 8H, piperazine H),
3.52 (s, 2H, CH2), 3.76 (s, 2H, CH2), 3.85—3.91 (m,
12H, OCH3), 4.11 (t, J＝6.6 Hz, 2H, OCH2), 6.64 (d,
J＝8.4 Hz, 1H, ArH), 6.99 (d, J＝8.7 Hz, 2H, ArH),
8.15 (dd, J1＝8.7 Hz, J2＝2.1 Hz, 1H, ArH), 8.47 (d,
J＝2.1 Hz, 1H, ArH).
Methyl 5-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-
1-yl)acetyl)-2-(hexyloxy)benzoate (4g): Yield 83.6%,
1
pale yellow oil; H NMR (CDCl3, 300 MHz) δ: 0.91 (t,
J＝6.6 Hz, 3H, CH3), 1.34—1.36 (m, 4H, CH2CH2),
1.50—1.52 (m, 2H, CH2), 1.80—1.87 (m, 2H, CH2),
2.65 (s, 8H, piperazine H), 3.63 (s, 2H, CH2), 3.78 (s,
2H, CH2), 3.85—3.94 (m, 12H, OCH3), 4.10 (t, J＝6.3
Hz, 2H, OCH2), 6.64 (d, J＝8.4 Hz, 1H, ArH), 6.98 (d,
J＝8.4 Hz, 1H, ArH), 7.05 (d, J＝8.7 Hz, 1H, ArH),
8.13 (dd, J1＝8.7 Hz, J2＝2.1 Hz, 1H, ArH), 8.46 (d,
J＝2.1 Hz, 1H, ArH).
1
yellow solid, m.p. 74—78 ℃; H NMR (CDCl3, 300
MHz) δ: 1.06 (t, J＝7.3 Hz, 3H, CH3), 1.89—1.92 (m,
2H, CH2), 2.85—3.04 (m, 8H, piperazine H), 3.86 (s,
2H, CH2), 3.92 (s, 9H, OCH3), 4.05 (s, 2H, CH2), 4.13 (t,
J＝6.5 Hz, 2H, OCH2), 6.64 (d, J＝8.6 Hz, 1H, ArH),
7.00 (d, J＝8.8 Hz, 1H, ArH), 7.23 (d, J＝8.6 Hz, 1H,
ArH), 8.02 (d, J＝8.0 Hz, 1H, ArH), 8.31 (s, 1H, ArH).
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(butoxy)benzoic acid (5d): Yield 80.2%, pale
Methyl 5-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-
1-yl)acetyl)-2-(heptyloxy)benzoate (4h): Yield 87.3%,
1
1
yellow solid, m.p. 81—82 ℃; H NMR (CDCl3, 300
pale yellow oil; H NMR (CDCl3, 300 MHz) δ: 0.90 (t,
MHz) δ: 0.99 (t, J＝7.4 Hz, 3H, CH3), 1.49—1.56 (m,
2H, CH2), 1.87—1.92 (m, 2H, CH2), 3.00—3.15 (m, 8H,
piperazine H), 3.86—3.93 (m, 9H, OCH3), 3.94 (s, 2H,
CH2), 4.13 (s, 2H, CH2), 4.26 (t, J＝6.5 Hz, 2H, OCH2),
6.71 (d, J＝8.6 Hz, 1H, ArH), 7.07 (d, J＝8.8 Hz, 1H,
ArH), 7.35 (d, J＝8.6 Hz, 1H, ArH), 8.09 (dd, J1＝8.8
Hz, J2＝2.3 Hz, 1H, ArH), 8.55 (d, J＝2.3 Hz, 1H,
ArH).
J＝6.6 Hz, 3H, CH3), 1.26—1.52 (m, 8H, CH2CH2-
CH2CH2), 1.81—1.87 (m, 2H, CH2), 2.60 (s, 8H,
piperazine H), 3.52 (s, 2H, CH2), 3.76 (s, 2H, CH2),
3.85—3.91 (m, 12H, OCH3), 4.10 (t, J＝6.6 Hz, 2H,
OCH2), 6.64 (d, J＝8.4 Hz, 1H, ArH), 6.99 (d, J＝8.7
Hz, 1H, ArH), 8.15 (dd, J1＝8.7 Hz, J2＝2.1 Hz, 1H,
ArH), 8.46 (d, J＝2.1 Hz, 1H, ArH).
Methyl 5-(2-(4-(2,3,4-trimethoxybenzyl)piperazin-
1-yl)acetyl)-2-(octyloxy)benzoate (4i): Yield 62.5%,
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
336
www.cjc.wiley-vch.de
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 333—340

Synthesis and Bioactivity of Substituted Benzoylguanidine Derivatives
acetyl)-2-(isobutoxy)benzoic acid (5e): Yield 74.6%,
pale yellow solid, m.p. 88—91℃; H NMR (CDCl3,
4.2 Hz, 1H, ArH), 8.06 (d, J＝8.4 Hz, 1H, ArH), 8.41 (s,
1H, ArH).
1
500 MHz) δ: 1.06 (d, J＝6.7 Hz, 6H, CH3), 2.18—2.20
(m, 1H, CH), 2.94—3.10 (m, 8H, piperazine H), 3.85 (s,
2H, CH2), 3.92 (s, 9H, OCH3), 3.96 (d, J＝6.4 Hz, 2H,
OCH2), 4.07 (s, 2H, CH2), 6.68 (d, J＝8.6 Hz, 1H, ArH),
7.01 (d, J＝8.9 Hz, 1H, ArH), 7.30 (d, J＝8.6 Hz, 1H,
ArH), 8.04 (d, J＝8.5 Hz, 1H, ArH), 8.45 (s, 1H, ArH).
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(pentyloxy)benzoic acid (5f): Yield 83.7%,
General procedure for synthesis of compounds 6
1-Hydroxybenzotriazole (0.53 mmol) and 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide hydrochloride
(0.53 mmol) were added to a solution of compounds 5
(0.41 mmol) in DMF (3 mL), and the mixture was
stirred for 0.5 h at room temperature, and then gua-
nidine (1.65mmol) was quickly added. The resultant
mixture was stirred for 10 min at room temperature and
poured into 50 mL of EtOAc/H2O (3∶2, V/V), the
aqueous phase was extracted with ethyl acetate (30 mL
×3), and the combined organic layers were washed
with saturated sodium chloride solution (50 mL×3) and
dried over anhydrous MgSO4. The filtrate was concen-
trated to dryness under reduced pressure. The residue
was purified by silica gel column chromatography (ace-
tic ether/methanol/triethylamine＝15∶1∶0.1, V/V/V).
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(methoxyl)benzoyl guanidine (6a): Yield
30.5%, yellow solid, m.p. 69—71 ℃; 1H NMR (CDCl3,
300 MHz) δ: 2.57 (s, 8H, piperazine H), 3.49 (s, 2H,
CH2), 3.79 (s, 2H, CH2), 3.85 (s, 3H, OCH3), 3.87 (s,
3H, OCH3), 3.88 (s, 3H, OCH3), 3.99 (s, 3H, OCH3),
6.64 (d, J＝8.4 Hz, 1H, ArH), 6.97—7.00 (m, 2H, ArH),
8.08 (d, J＝9 Hz, 1H, ArH), 8.34 (d, J＝2.1 Hz, 1H,
ArH); IR (KBr) ν: 3405, 2925, 2849, 1688 (C＝O),
1601 (C＝O), 1495, 1463, 1355, 1264, 1095 (OCH3),
1
pale yellow solid, m.p. 81—84 ℃; H NMR (CDCl3,
300 MHz) δ: 0.93 (t, J＝6.9 Hz, 3H, CH3), 1.35—1.50
(m, 4H, CH2CH2), 1.87—1.94 (m, 2H, CH2), 3.03—
3.20 (m, 8H, piperazine H), 3.85—3.93 (m, 9H, OCH3),
3.96 (s, 2H, CH2), 4.17 (s, 2H, CH2), 4.25 (t, J＝6.6 Hz,
2H, OCH2), 6.72 (d, J＝8.4 Hz, 1H, ArH), 7.07 (d, J＝
8.7 Hz, 1H, ArH), 7.37 (d, J＝8.4 Hz, 1H, ArH), 8.09
(dd, J1＝8.7 Hz, J2＝1.8 Hz, 1H, ArH), 8.56 (d, J＝1.8
Hz, 1H, ArH).
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(hexyloxy)benzoic acid (5g): Yield 83.6%,
1
pale yellow solid, m.p. 59—62 ℃; H NMR (CDCl3,
300 MHz) δ: 0.88 (t, J＝6.9 Hz, 3H, CH3), 1.31—1.33
(m, 4H, CH2CH2), 1.46—1.48 (m, 2H, CH2), 1.85—
1.92 (m, 2H, CH2), 2.86—3.06 (m, 8H, piperazine H),
3.86 (s, 9H, OCH3), 3.92 (s, 2H, CH2), 4.07 (s, 2H,
CH2), 4.18 (t, J＝6.6 Hz, 2H, OCH2), 6.67 (d, J＝8.4
Hz, 1H, ArH), 7.01 (d, J＝8.7 Hz, 1H, ArH), 7.26 (d,
J＝8.4 Hz, 1H, ArH), 8.05 (dd, J1＝8.7 Hz, J2＝2.1 Hz,
1H, ArH), 8.38 (d, J＝2.1 Hz, 1H, ArH).
－
＋
1
1004, 818 cm ; MS (ESI(＋)70 V) m/z: 500.3 [M＋H] .
Anal. calcd for C25H33N5O6•1.8H2O: C 56.44, H 6.93, N
13.16; found C 56.74, H 7.32, N 12.71.
General procedure for synthesis of compounds 5h
and 5i
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(ethoxyl)benzoyl guanidine (6b): Yield 29.8%,
yellow solid, m.p. 146—148 ℃; 1H NMR (CDCl3, 500
MHz) δ: 1.46 (t, J＝7.0 Hz, 3H, CH3), 2.55—2.60 (m,
8H, piperazine H), 3.49 (s, 2H, CH2), 3.76 (s, 2H, CH2),
3.85 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.90 (s, 3H,
OCH3), 4.22 (q, J＝7.1 Hz, 2H, OCH2), 6.63 (d, J＝8.5
Hz, 1H, ArH), 6.93—7.00 (m, 2H, ArH), 8.04 (dd, J1＝
8.8 Hz, J2＝2.0 Hz, 1H, ArH), 8.33 (d, J＝2.0 Hz, 1H,
ArH); IR (KBr) ν: 3372, 2938, 2805, 1662 (C＝O),
1601 (C＝O), 1495, 1468, 1363, 1266, 1096 (OCH3),
K2CO3 (1.8 mmol) was added to a solution of com-
pounds 4 (0.58 mmol) in 60% methanol solution (15
mL), and the mixture was stirred and refluxed for 1 h,
then methanol was removed under vacuum. Acidified to
pH 7 with 2% HCl, then filtered, followed by drying
under IR lamp.
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(heptyloxy)benzoic acid (5h): Yield 77.3%,
1
white solid, m.p. 58—60 ℃; H NMR (CDCl3, 300
MHz) δ: 0.86 (t, J＝6.3 Hz, 3H, CH3), 1.29—1.48 (m,
8H, CH2CH2CH2CH2), 1.85—1.94 (m, 2H, CH2), 2.85
—3.07 (m, 8H, piperazine H), 3.86 (s, 9H, OCH3), 3.93
(s, 2H, CH2), 4.08 (s, 2H, CH2), 4.17 (t, J＝6.9 Hz, 2H,
OCH2), 6.67 (d, J＝8.4 Hz, 1H, ArH), 7.00 (d, J＝8.7
Hz, 1H, ArH), 7.04 (d, J＝8.4 Hz, 1H, ArH), 8.03 (d,
J＝8.7 Hz, 1H, ArH), 8.32 (s, 1H, ArH).
－
1
＋
1009, 698 cm ; MS (ESI(＋)70 V) m/z: 514.3 [M＋H] .
Anal. calcd for C26H35N5O6: C 60.80, H 6.87, N 13.64;
found C 60.59, H 7.08, N 13.65.
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(propoxy)benzoyl guanidine (6c): Yield 28.5%,
1
yellow solid, m.p. 83—85 ℃; H NMR (CDCl3, 300
MHz) δ: 1.02 (t, J＝7.4 Hz, 3H, CH3), 1.80—1.87 (m,
2H, CH2), 2.53—2.56 (m, 8H, piperazine H), 3.48 (s,
2H, CH2), 3.78 (s, 2H, CH2), 3.85 (s, 3H, OCH3), 3.87
(s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.04 (t, J＝6.7 Hz,
2H, OCH2), 6.63 (d, J＝8.6 Hz, 1H, ArH), 6.92 (d, J＝
8.8 Hz, 1H, ArH), 6.97 (d, J＝8.6 Hz, 1H, ArH), 7.97
(dd, J1＝8.8 Hz, J2＝2.3 Hz, 1H, ArH), 8.19 (d, J＝2.3
Hz, 1H, ArH); IR (KBr) ν: 3405, 2938, 2822, 1602 (C＝
O), 1527, 1495, 1465, 1353, 1265, 1166, 1095 (OCH3),
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(octyloxy) benzoic acid (5i): Yield 75.5%,
white solid, m.p. 71—74 ℃; H NMR (CDCl3, 300
MHz) δ: 0.86 (t, J＝6.9 Hz, 3H, CH3), 1.25—1.30 (m,
8H, CH2CH2CH2CH2), 1.45—1.47 (m, 2H, CH2), 1.85
—1.92 (m, 2H, CH2), 2.89—3.08 (m, 8H, piperazine H),
3.86 (s, 9H, OCH3), 3.92 (s, 2H, CH2), 4.08 (s, 2H,
CH2), 4.20 (t, J＝6.6 Hz, 2H, OCH2), 6.68 (d, J＝8.7
Hz, 1H, ArH), 7.02 (d, J＝9 Hz, 1H, ArH), 7.06 (d, J＝
1
Chin. J. Chem. 2012, 30, 333—340
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
337

Jin et al.
FULL PAPER
1003, 819 cm ; MS (ESI(＋)70 V) m/z: 527.3 [M＋H] .
Anal. calcd for C27H37N5O6: C 61.46, H 7.07, N 13.27;
found C 61.15, H 7.40, N 12.78.
－
1
＋
1.79 — 1.85 (m, 2H, CH2), 2.55 — 2.59 (m, 8H,
piperazine H), 3.48 (s, 2H, CH2), 3.77 (s, 2H, CH2),
3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 4.09 (t, J＝6.8 Hz, 2H, OCH2), 6.63 (d, J＝8.5
Hz, 1H, ArH), 6.93 (d, J＝8.8 Hz, 1H, ArH), 6.98 (d,
J＝8.5 Hz, 1H, ArH), 7.99 (dd, J1＝8.8 Hz, J2＝2.1 Hz,
1H, ArH), 8.46 (s, 1H, ArH); IR (KBr) ν: 3374, 2933,
2810, 1663 (C＝O), 1601 (C＝O), 1531, 1495, 1467,
1362, 1266, 1096 (OCH3), 1008, 698 cm － 1; MS
(ESI(＋)70 V) m/z: 570.4 [M＋H]＋. Anal. calcd for
C30H43N5O6: C 63.25, H 7.61, N 12.29; found C 63.52,
H 7.44, N 12.14.
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(heptyloxy)benzoyl guanidine (6h): Yield
31.4%, white solid, m.p. 160 —162 ℃ ; 1H NMR
(CDCl3, 500 MHz) δ: 0.86 (t, J＝6.7 Hz, 3H, CH3), 1.26
—1.34 (m, 6H, CH2CH2CH2), 1.40—1.45 (m, 2H, CH2),
1.79—1.83 (m, 2H, CH2), 2.57 (d, J＝20.4 Hz, 8H,
piperazine H), 3.48 (s, 2H, CH2), 3.77 (s, 2H, CH2),
3.84 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 4.08 (t, J＝6.8 Hz, 2H, OCH2), 6.63 (d, J＝8.5
Hz, 1H, ArH), 6.93 (d, J＝8.8 Hz, 1H, ArH), 6.97 (d,
J＝8.5 Hz, 1H, ArH), 7.98 (dd, J1＝8.8 Hz, J2＝2.1 Hz,
1H, ArH), 8.46 (s, 1H, ArH); IR (KBr) ν: 3376, 2930,
2854, 1662 (C＝O), 1602 (C＝O), 1529, 1495, 1466,
1363, 1266, 1096 (OCH3), 1006, 821 cm － 1; MS
(ESI(＋)70 V) m/z: 584.4 [M＋H]＋. Anal. calcd for
C31H45N5O6•0.5H2O: C 62.82, H 7.82, N 11.81; found C
62.71, H 7.91, N 11.81.
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(butoxy)benzoyl guanidine (6d): Yield 36.1%,
yellow solid, m.p. 88—92 ℃; 1H NMR (CDCl3, 300
MHz) δ: 0.94 (t, J＝7.4 Hz, 3H, CH3), 1.44—1.51 (m,
2H, CH2), 1.75—1.85 (m, 2H, CH2), 2.56 (s, 8H,
piperazine H), 3.48 (s, 2H, CH2), 3.78 (s, 2H, CH2),
3.85 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 4.09 (t, J＝6.6 Hz, 2H, OCH2), 6.63 (d, J＝8.5
Hz, 1H, ArH), 6.93 (d, J＝8.8 Hz, 1H, ArH), 6.97 (d,
J＝8.5 Hz, 1H, ArH), 7.98 (dd, J1＝8.8 Hz, J2＝2.1 Hz,
1H, ArH), 8.20 (s, 1H, ArH); IR (KBr) ν: 3406, 2936,
2821, 1604 (C＝O), 1527, 1495, 1464, 1352, 1271,
－
1
1166, 1095 (OCH3), 1004, 801 cm ; MS (ESI(＋)70 V)
m/z: 542.3 [M＋H]＋ . Anal. calcd for C28H39N5O6•
0.5H2O: C 61.07, H 7.32, N 12.71; found C 60.91, H
7.45, N 12.32.
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(isobutoxy)benzoyl guanidine (6e): Yield
37.6%, yellow solid, m.p. 140—144 ℃; 1H NMR
(CDCl3, 500 MHz) δ: 1.01 (d, J＝6.7 Hz, 6H, CH3),
2.11—2.14 (m, 1H, CH), 2.55—2.59 (m, 8H, piperazine
H), 3.47 (s, 2H, CH2), 3.77 (s, 2H, CH2), 3.83 (d, J＝6.6
Hz, 2H, OCH2), 3.84 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 6.63 (d, J＝8.5 Hz, 1H, ArH), 6.91
(d, J＝8.8 Hz, 1H, ArH), 6.97 (d, J＝8.5 Hz, 1H, ArH),
7.96 (dd, J1＝8.8 Hz, J2＝2.3 Hz, 1H, ArH), 8.16 (d,
J＝2.3 Hz, 1H, ArH); IR (KBr) ν: 3415, 2937, 2821,
1603 (C＝O), 1528, 1495, 1465, 1351, 1274, 1167,
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(octyloxy)benzoyl guanidine (6i): Yield 34.8%,
white solid, m.p. 135—138 ℃; H NMR (CDCl3, 300
－
1
1
1096 (OCH3), 1002, 819 cm ; MS (ESI(＋)70 V) m/z:
542.3 [M＋H]＋. Anal. calcd for C28H39N5O6 ·0.5H2O: C
61.07, H 7.32, N 12.71; found C 61.08, H 7.79, N 12.48.
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(pentyloxy)benzoyl guanidine (6f): Yield
26.4%, yellow solid, m.p. 80—83 ℃; 1H NMR (CDCl3,
300 MHz) δ: 0.90 (t, J＝6.3 Hz, 3H, CH3), 1.26—1.39
(m, 4H, CH2CH2), 1.82—1.84 (m, 2H, CH2), 2.57—
2.62 (m, 8H, piperazine H), 3.49 (s, 2H, CH2), 3.63 (s,
2H, CH2), 3.78 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.87
(s, 3H, OCH3), 4.11 (t, J＝6.3 Hz, 2H, OCH2), 6.63 (d,
J＝8.1 Hz, 1H, ArH), 6.94 (d, J＝8.4 Hz, 1H, ArH),
6.98 (d, J＝8.4 Hz, 1H, ArH), 8.02 (d, J＝8.1 Hz, 1H,
ArH), 8.27 (s, 1H, ArH); 13C NMR (CDCl3, 75 MHz) δ:
13.9, 22.3, 28.0, 28.6, 29.7, 52.7 (piperazine C), 53.7
(piperazine C), 56.0, 60.7, 61.2, 64.2, 69.2, 107.2, 112.4,
124.2, 125.1, 128.4, 129.4, 130.3, 131.3, 142.4, 152.6,
152.9, 160.7, 161.8, 177.4, 195.5 (C＝O); IR (KBr) ν:
3420, 2353, 2310, 1632 (C＝O), 1458, 1096 (OCH3),
MHz) δ: 0.86 (t, J＝6.9 Hz, 3H, CH3), 1.25—1.43 (m,
10H, CH2CH2CH2CH2CH2), 1.77—1.84 (m, 2H, CH2),
2.57 (s, 8H, piperazine H), 3.48 (s, 2H, CH2), 3.78 (s,
2H, CH2), 3.85 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.88
(s, 3H, OCH3), 4.08 (t, J＝6.6 Hz, 2H, OCH2), 6.63 (d,
J＝8.7 Hz, 1H, ArH), 6.92 (d, J＝9 Hz, 1H, ArH), 6.98
(d, J＝8.7 Hz, 1H, ArH), 7.98 (dd, J1＝9 Hz, J2＝1.8
Hz, 1H, ArH), 8.46 (d, J＝1.8 Hz, 1H, ArH); 13C NMR
(CDCl3, 75 MHz) δ: 14.1, 22.6, 25.8, 52.6 (piperazine
C), 53.7 (piperazine C), 56.0, 56.6, 60.8, 61.2, 64.1,
69.2, 107.0, 112.3, 124.1, 125.2, 128.2, 130.3, 131.3,
142.3, 152.6, 152.9, 160.7, 161.7, 172.5, 195.5 (C＝O);
IR (KBr) ν: 3373, 2929, 2855, 1663 (C＝O), 1601 (C＝
O), 1531, 1495, 1467, 1363, 1267, 1096 (OCH3), 1009,
－
＋
1
844 cm ; MS (ESI(＋)70 V) m/z: 598.4 [M＋H] .
Anal. calcd for C32H47N5O6: C 64.30, H 7.92, N 11.72;
found C 63.94, H 8.14, N 11.76.
－
＋
1
Rat platelet swelling assay
668 cm ; MS (ESI(＋)70 V) m/z: 556.3 [M＋H] .
Anal. calcd for C29H41N5O6: C 62.68, H 7.43, N 12.60;
found C 62.47, H 7.48, N 12.22.
All target compounds, along with the reference
compound cariporide and CPU-X-050420, were evalu-
ated in rat platelet swelling assay (PSA) for NHE1 in-
hibitory activity screening. The experimental procedure
was similar as in the literature,[8] with only minor modi-
fications. Collect 5 mL blood from the eyeholes of each
adult male and female Sprague-Dawley rat (160—250
5-(2-(4-(2,3,4-Trimethoxybenzyl)piperazin-1-yl)-
acetyl)-2-(hexyloxy)benzoyl guanidine (6g): Yield
38.2%, white solid, m.p. 153 —155 ℃ ; 1H NMR
(CDCl3, 500 MHz) δ: 0.87 (t, J＝7.1 Hz, 3H, CH3), 1.30
—1.33 (m, 4H, CH2CH2), 1.43—1.46 (m, 2H, CH2),
338
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 333—340

Synthesis and Bioactivity of Substituted Benzoylguanidine Derivatives
g), then mix acid-citrate-dextrose (ACD: citric acid 65
mmol/L, sodium citrate 85 mmol/L and 2% glucose) in
the test tubes. After centrifugation of whole blood at
1300 g/min for 10 min at r.t., the upper two-thirds of the
supernatants were used for the further measurements
and performed within 4 h.
Each compound was tested for six concentrations,
and the measurement of each concentration repeated
three times. All compounds were dissolved in DMSO,
and diluted to test concentration with standard media.
Add 25 μL of the solution of the tested compound and
175 μL of propionate buffer (sodium propionate 140
mmol/L, glucose 10 mmol/L, KCl 5 mmol/L, MgCl2 1
of 9 was increased when prolonging the reaction time.
For example, compound 9f was separated out during the
preparation of 6f, and its structure was identified by
spectral data and elemental analysis.
9f: 1H NMR (CDCl3, 300 MHz) δ: 0.88 (t, J＝7.2 Hz,
3H, CH3), 1.26—1.44 (m, 4H, CH2CH2), 1.75—1.84 (m,
2H, CH2), 2.46—2.58 (m, 4H, piperazine H), 3.37 (m,
2H, piperazine H), 3.51 (s, 2H, CH2), 3.75 (s, 2H,
piperazine H), 3.85 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 4.07 (t, J＝6.6 Hz, 2H, OCH2),
6.64 (d, J＝8.7 Hz, 1H, ArH), 7.00 (m, 2H, ArH), 7.75
(d, J＝2.1 Hz, 1H, ArH), 8.00 (s, 1H, ArH); 13C NMR
(CDCl3, 75 MHz) δ: 13.9, 22.3, 27.8, 27.9, 28.4, 52.2
(piperazine C), 52.7 (piperazine C), 55.9, 60.7, 61.1,
64.2, 69.1, 107.0, 112.9, 123.1, 124.5, 125.0, 130.7,
130.9, 131.6, 142.3, 152.6, 153.1, 161.8, 162.7, 166.1,
177.4, 189.5 (C＝O); MS (ESI(＋)70 V) m/z: 570.3
[M＋H]＋. Anal. calcd for C29H39N5O7•0.5H2O: C 60.19,
H 6.96, N 12.10; found C 59.84, H 6.91, N 11.71.
When compared with the 1H NMR spectrum of 6f, it
can clearly be seen that the peak of methylene between
piperazine nitrogen and carbonyl disappeared. In the 13C
NMR spectrum, the peak of δ 56.5 (saturated carbon) in
6f disappeared, but a new peak of δ 166.1 (unsaturated
carbon) appeared. Mass spectrum data showed that the
molecular weight of 9f was 14 times bigger than that of
6f. Based on the above spectral data, we speculated that
this methylene may be converted into carbonyl groups
(Scheme 3). The percentage of C, H and N obtained by
elemental analysis was consistent with the data
calculated on 9f.
mmol/L, CaCl2
1
mmol/L, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) 20 (quantum
sufficit, pH 6.7) to a spectrophotometer cuvette. After
pre-heated to 37 ℃, 50 μL of platelet-rich plasma (PRP)
was added. Each 7.5 s for 2 min recorded the change in
optical density (OD) due to the platelet swelling at 550
nm. 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. As the result, the average of IC50 of three sam-
ples was presented in this paper.
Results and Discussion
Key intermediates 4a—4i can readily be obtained
from 1 using the method described in Scheme 1, but this
route is time-consuming and low-efficient. In order to
improve the synthetic efficiency of 4a—4i, we designed
an alternative route (Scheme 2) to prepare 4a—4i using
7 as starting materials by α-bromination, condensation
with trimetazidine, O-deacylation, esterification and
O-alkylation. Compound 8 could be obtained in good
yield (overall yield 56%). However, a lot of impurities
were formed when converting 8 to 4. The isolated yield
of 4 after column chromatography was less than 10%.
In the synthesis of target compounds 6a—6i, we
isolated corresponding analogue 9 from the reaction
mixture of 5 and guanidine, and found that the content
Scheme 3
OCH3
H3CO
N
O
O
O
NH
NH2
N
H3CO
CH3
6f
OCH3
H3CO
H3CO
N
O
O
O
NH
NH2
N
Scheme 2
O
CH3
O
O
O
(1)
9f
H3C
OCH3
Ph
The β-position methylene of N,N-dialkyl-α-oxo-
β-phenylethylamine is susceptible to be oxided into
carbonyl in the presence of oxidants such as NaIO4,[9]
O2/FeCl3.[10] But such oxidation conversion under our
experimental conditions is not clear.
O
7
OCH3
H3CO
H3CO
NHE1 inhibitory activities of 6a—6i, along with the
reference compound cariporide and CPU-X-050420,
were determined in rat platelet swelling assay, and the
result (Table 1) showed that all the tested compounds
and positive controls inhibited rat platelet NHE1 in a
concentration-dependent manner, among which com-
pounds 6b, 6d, 6f, 6g and CPU-X-050420 were signifi-
cantly more effective than cariporide in NHE1 inhibi-
N
O
O
(2)
4a
4i
N
OH
OH
8
Reagents and conditions: (1) (a) Br2/CHCl3, r.t., 71.5%; (b)
trimetazidine/K2CO3/CH3CN, r.t., 86%; (c) K2CO3/60% methanol, 50
℃, 88.7%; (2) (a) SOCl2/CH3OH; (b) RX/K2CO3/CH3CN, reflux.
Chin. J. Chem. 2012, 30, 333—340
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
339

Jin et al.
FULL PAPER
Table 1 NHE1 inhibitory activities of compounds 6a—6i
Science Foundation of China (No. 30672512).
Compd.
IC50/(mol•L－
)
Compd. IC50/(mol•L－
)
1
1
6.50×10－
6e
6f
1.08×10－
8
7
References
Cariporide
CPU-X-050420
4.22×10－
1.08×10－
5.32×10－
5.86×10－
9.80×10－
9
10
[1] (a) Tani, M.; Weis, C. R.; Geisinger, C. Rev. Physiol. 1990, 52, 543;
(b) Opie, L. H. Cardiovasc. Drugs Ther. 1991, 5, 237.
6a
6b
6c
6d
9.59×10－
6g
6h
6i
8
9
8
8
6.58×10－
9
[2] (a) Tani, M.; Neely, J. R. Circ. Res. 1989, 65, 1045; (b) DuToit, E.;
Opie, L. Circ. Res. 1992, 70, 960.
3.90×10－
8
2.28×10－
9
[3] Fliegel, L. Int. J. Biochem. Cell Biol. 2005, 37, 33.
[4] (a) Schulze, C. J.; Wang, W.; Suarez-Pinzon, W. L.; Jolanta, S.;
Grzegorz, S.; Richard, S. Circulation 2003, 107, 2487; (b) Luo, Z. C.
Mod. Med. Health 2004, 20, 970.
tion, and compound 6f was 39 times more potent than
CPU-X-050420.
[5] (a) Yasutake, M.; Ibuki, C.; Hearse, D.; Avkiran, M. Am. J. Physiol.
1994, 267, 2430; (b) Scholz, W.; Albus, U.; Counillon, L.; Gogelein,
H.; Lang, H.; Linz, W.; Weichert, A.; Scholens, B. Cardiovasc. Res.
1995, 29, 260; (c) Fukuta, M.; Wakida, Y.; Uesugi, M.; Kobayashi, T.
Pacing Clin. Electrophysiol. 1996, 19, 2027; (d) Piper, H. M.; Balser,
C.; Ladilov, Y. V.; Schafer, M.; Siegmund, B.; Ruiz-Meana, M.;
Garcia-Dorado, D. Cardiology 1996, 91, 191; (e) Xue, Y. X.; Aye, N.
N.; Hashimoto, K. Eur. J. Pharmacol. 1996, 317, 309.
Preliminary structure-activity relationship showed
that the good inhibitory effects of target compounds on
NHE1 can be achieved when the number of carbon
atom in straight-chain alkoxy group is 4 to 6. Because
of the relatively less number of tested compounds, the
conclusion above need to be further confirmed.
Conclusions
[6] Xu, W. T.; Jin, N.; Xu, J.; Xu, Y. G.; Wang, Q. J.; You, Q. D. Bioorg.
Med. Chem. Lett. 2009, 19, 3283.
In summary, a series of novel substituted benzoyl-
guanidine derivatives on the basis of the structure of an
initial lead compound CPU-X-050420, were designed,
synthesized and evaluated for their NHE1 inhibitions.
Most compounds showed more potent NHE1 inhibitory
activities than cariporide, and the most potent one was
6f. Further pharmacological studies are in progress.
[7] Grisar, J. M.; Claxton, G. P.; Bare, T. M.; Dage, R. C.; Cheng, H. C.;
Woodward, J. K. J. Med. Chem. 1981, 24, 327.
[8] (a) Rosskopf, D.; Morgenstern, E.; Scholz, W.; Osswald, U.; Siffert,
W. J. Hypertens. 1991, 9, 231; (b) Laeckmann, D.; Rogister, F.;
Dejardin, J. V.; Prosperi-Meys, C.; Géczy, J.; Delarge, J.; Masereel,
B. Bioorg. Med. Chem. 2002, 10, 1793.
[9] Takagi, K.; Ohshima, T.; Hasegawa, N.; Katoh, C.; Kanaoka, A.;
Kanno, H. EP 0657421A1, 1994 [Chem. Abstr. 1995, 123, 169370].
[10] Murata, S.; Suzuki, K.; Miura, M.; Nomura, M. J. Chem. Soc.
Perkin Trans. 1990, 2, 361.
Acknowledgement
This work was supported by the National Natural
(E1103212 Cheng, B.)
340
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 333—340