Design, synthesis and biological evaluation of novel substituted benzoylguanidine derivatives as potent Na+/H+ exchanger inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.04.079

A novel series of substituted benzoylguanidine derivatives were designed and synthesized as potent NHE1 inhibitors. Most compounds can significantly inhibit NHE1-mediated platelet swelling in a concentration-dependent manner, among which compound 5f (IC

Full text of DOI:10.1016/j.bmcl.2009.04.079

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3283–3287
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of novel substituted
benzoylguanidine derivatives as potent Na+/H+ exchanger inhibitors
b,
a
Wen-Ting Xu a, Ning Jin a, Jing Xu b, Yun-Gen Xu a, , Qiu-Juan Wang , Qi-Dong You
*
*
a Department of Medicinal Chemistry, China Pharmaceutical University, 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:
A novel series of substituted benzoylguanidine derivatives were designed and synthesized as potent
NHE1 inhibitors. Most compounds can significantly inhibit NHE1-mediated platelet swelling in a concen-
tration-dependent manner, among which compound 5f (IC50 = 3.60 nM) and 5l (IC50 = 4.48 nM) are 18
and 14 times respectively more potent than cariporide (IC50 = 65.0 nM). Furthermore, when tested
in vivo and in vitro, compound 5f showed superior cardioprotective effects against SD rat myocardial
ischemic-reperfusion injury over cariporide, representing a promising lead compound for further
exploration.
Received 14 December 2008
Revised 30 March 2009
Accepted 20 April 2009
Available online 23 April 2009
Keywords:
Na+/H+ exchanger inhibitors
Myocardial ischemic-reperfusion injury
Benzoylguanidine derivatives
Synthesis
Ó 2009 Elsevier Ltd. All rights reserved.
The Na+/H+ exchanger (NHE) is an integral membrane protein
ubiquitously expressed in mammalian cells. It regulates intracellu-
lar pH by removing a proton in exchange for an extracellular so-
dium ion. To date, nine isoforms of the exchanger have been
identified and are designated NHE1–NHE9. NHE1 was the first iso-
form discovered and is ubiquitously expressed in the plasma mem-
brane of mammalian cells. Other isoforms have more restricted
tissue distributions and some have predominantly intracellular
localization.1,2 In mammals, NHE1 is the predominant isoform in
heart and is involved in numerous physiological processes, includ-
ing regulation of intracellular pH, cell-volume control, cytoskeletal
organization, heart disease and cancer.3,4 The activity of NHE1 is
elevated in animal models of myocardial infarcts and in left ven-
tricular hypertrophy.5 During ischemia and reperfusion of the
myocardium, NHE activity catalyzes increased uptake of intracellu-
lar Na+. This in turn is exchanged for extracellular calcium by the
Na+/Ca2+ exchanger resulting in calcium overload and damage to
the myocardium, such as myocardial infarction activation, stun-
ning and tissue necrosis.6,7
Aroylguanidine, a subunit typically presented in most of the
known NHE1 inhibitors, such as cariporide and sabiporide
(Fig. 1),8 is well-considered as the possible pharmacophore among
the reported NHE1 inhibitors.9 Upon analyzing the structures of car-
iporide and sabiporide, we chose benzoylguanidine as core
structure, and introduced 4-(2,3,4-trimethoxy-benzyl)-piperazin-
1-ylmethyl group to the ortho, meta or para position of benzoylgua-
nidine, and then synthesized our target compounds 5a–l (Table 1).
The synthetic route for target compounds is depicted in Scheme
1. Substituted bromo-methyl benzoic acid ethyl esters 2a–l, ob-
tained by bromination of methyl (or ethyl)-substituted benzoic
acid ethyl esters 1a–l, were reacted with 1-(2,3,4-trimethoxy-ben-
zyl)-piperazine (3, trimetazidine) to give intermediate 4a–l. Treat-
ment of 4a–l with excessive guanidine in anhydrous THF or
isopropyl alcohol afforded the free base of the target compounds
5a–l. The crude bases were purified by silica gel column chroma-
tography (petroleum ether/acetone, 2–5:1), followed by salt for-
mation with saturated HCl (g) in anhydrous acetone.
NHE1 inhibitors, in their cation form, combine with NHE1 at the
extracellular Na+ binding site to competitively inhibit NHE1 func-
tion and reduce Na+ and Ca2+ influx, and hence abolish the post-
ischemia Ca2+ overload in myocardial cells and lower the risk of
cell dysfunction and injury.
NH
O
F
2
F
CH
3
F
N
NH
2
H C
3
N
NH
N
2
H CO S
3
2
N
O
NH
2
cariporide
N
H
* Corresponding authors. Tel.: +86 25 83322067; fax: +86 25 83271487 (Y.-G.X.);
tel.: +86 25 83271341; fax: +86 25 8330 1606 (Q.-J.W.).
E-mail addresses: xu_yungen@hotmail.com (Y.-G. Xu), qjwangnj@sina.com (Q.-J.
Wang).
O
sabiporide
Figure 1. Structures of cariporide and sabiporide.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.079

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W.-T. Xu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3283–3287
Table 1
leum ether/acetone, 3:1) one time to be free from by-products and
guanidine.
All synthesized compounds, along with reference compound
NHE1 inhibitory activity of compounds 5a–l
OCH
O
3
2
NH
NH
2
R
cariporide, were evaluated in rat platelet swelling assay (PSA) for
NHE1 inhibitory activity screening. The experiment procedure
was similar as in the literature,13 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 concen-
tration-dependent manner. The potential of 5f and 5l is superior to
that of cariporide. Preliminary structure–activity relationship anal-
ysis indicated that, compounds with electron-withdrawing group
possess better NHE1 inhibitory activity. The introduction of nitro
or methylsulfonyl group led to a significant increase in NHE1
inhibitory potency.
Next, compound 5f, which showed a good NHE1 inhibitory
activity, was evaluated for its cardioprotective efficacy against
ischemia-reperfusion injury in vivo (Figs. 2 and 3). The protective
effect was determined in rat heart coronary artery occlusion and
reperfusion model.14 After 45 min of occlusion, the coronary artery
was reperfused by loosing the ligature for 90 min. 5f (1 mg/kg,
0.5 mg/kg, 0.25 mg/kg), cariporide were intravenously given
5 min before reperfusion. The evaluation of cardioprotective effect
was measured as an index of the ratio of myocardial infarction size
to left ventricle area (IS/LV). The activity of creatine kinase (CK),
lactate dehydrogenase (LDH), superoxide dismutase (SOD) and
the content of malondialdehyde (MDA) in prepared serum were
also measured as the indicators of myocardial injury. Compared
with the model group, 5f could significantly diminish the myocar-
dial infarct size, decrease the release of CK and LDH, the content of
MDA and increase the activity of SOD in serum. These protective
effects were in a dose-dependent manner. There was no significant
difference between 5f (1 mg/kg) and cariporide (1 mg/kg) in the
myocardial protective effect.
H CO
3
.
N
N
HCl
N
N
2
H CO
3
1
R
R1
R2
Position of
IC50 (nM)
a
Compound
OCH
3
OCH
3
N
OCH
3
1
R
Cariporide
65.0
36.4
36.5
11.1
40.5
248
3.60
14.2
18.7
11.5
1150
15.0
4.48
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
H
H
CH3
H
H
H
CH3
H
H
H
H
H
H
4
3
2
4
4
4
3
4
4
4
4
4
3-Br
3-NO2
4-NO2
3-NO2
3-SO2NH2
3-SO2NHC3H7
3-SO2CH3
5j
5k
5l
H
CH3
3-SO2CH3
a
Drug concentration to achieve half-maximal inhibition of acid-induced swelling
in rat platelets.
According to the synthetic method of 5f described in Scheme 1,
1 equiv of 4f was needed to react with 20 equiv of guanidine in order
to obtain moderate yield of crude 5f.10 The crude product could only
be purified by silica gel column chromatography (petroleum ether/
acetone, 2–5:1) two times to be free from guanidine. The improved
synthetic route for compound 5f11 is depicted in Scheme 2. The
crude ester 4f was hydrolyzed with K2CO3 in MeOH/H2O (V/V, 1:1)
to give the corresponding acid 4f–1, which is a zwitterionic
compound and can be separated from the reaction mixture simply
by adjusting its isoelectric point using 0.1 mol/L HCl. Compound 5f
can be obtained by reaction of the acid 4f–1 with 3 equiv of
guanidine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotrizole
(HOBt),12 and purified by silica gel column chromatography (petro-
The protective effect of compound 5f in vitro was also deter-
mined in improved Langendorff system (Figs. 4 and 5).15 The iso-
lated rat hearts were experienced a 20 min equilibration period,
perfused at a low rate (0.5 ml/min) for 30 min, and then reper-
fused for 30 min. 5f (50 lM, 10 lM, 2 lM), cariporide(10 lM)
contained in perfusate were administered during the low rate
perfusion period. For the evaluation of their cardioprotective ef-
fect, the ratios of the coronary flow at the beginning and the
end of reperfusion to the end of equilibration value were mea-
sured. Additionally, the content of ATP, lactic acid (LD) and the
OCH3
H3CO
N
O
NH
O
R2
R1
Br
R2
H3CO
OC2H5
3
a
OC2H5
b
R1H2C
1a-l
2a-l
OCH3
O
OCH3
O
R2
R1
R2
R1
NH2
NH2
H3CO
H3CO
H3CO
H3CO
c
.
N
N
HCl
N
OC2H5
N
N
5a-c, e-g, i-k (R = H)
5d, 5h, 5l (R = CH3)
4a-l
Scheme 1. Reagents and conditions: (a) NBS, benzoyl peroxide, CCl4, reflux; (b) trimetazidine, (C2H5)3N, CH3CN, 20–80 °C; (c) (i) Guanidine, THF or isopropyl alcohol, reflux;
(ii) satd HCl (g) in anhydrous acetone.

W.-T. Xu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3283–3287
3285
OCH3
OCH3
H3CO
H3CO
COOC2H5
H3CO
H3CO
COOH
a
N
N
N
N
NO2
NO2
4f-1
4f
OCH3
O
NH2
b
H3CO
H3CO
.
HCl
N
N
N
NH2
NO2
5f
Scheme 2. Reagents and conditions: (a) K2CO3, MeOH/H2O, reflux, 92%; (b) (i) EDCI, HOBt, guanidine, DMF, rt; (ii) satd HCl (g) in anhydrous acetone, 60%.
Figure 2. Effect of compound 5f on myocardial infarct size in rats subjected to 45 min occlusion followed by 90 min reperfusion. Drugs were given by iv bolus at 5 min before
**
reperfusion. The myocardial infarct size was expressed as a percentage of the left ventricle area (IS/LV%) (n = 8). *P <0.05, P <0.01 versus sham group; 4P <0.05, 44P <0.01
versus model group.
Figure 3. Effect of compound 5f on LDH, CK, MDA and SOD included in the prepared serum of rats subjected to 45 min occlusion followed by 90 min reperfusion. Drugs were
**
given by iv bolus at 5 min before reperfusion (n = 8). *P <0.05, P <0.01 versus sham group; 4P <0.05, 44P <0.01 versus model group.

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W.-T. Xu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3283–3287
Figure 4. Effect of NHE1 inhibitor 5f on coronary flow of isolated rat hearts subjected to 30 min low rate perfusion followed by 30 min reperfusion. Drugs were contained in
the perfusate. The coronary flow at the beginning and the end of reperfusion was expressed as the ratio to the end of equilibration value (n = 10). *P <0.05, **P <0.01 versus
sham group; 4P <0.05, 44P <0.01 versus model group.
Figure 5. Effect of NHE1 inhibitor 5f on the activity of Na+–K+ATPase, the content of LD and ATP included in the tissue homogenate of isolated rat hearts subjected to 30 min
**
low rate perfusion followed by 30 min reperfusion (n = 10). *P <0.05, P <0.01 versus sham group; 4P <0.05, 44P <0.01 versus model group.
activity of Na+–K+ ATPase in prepared heart homogenate were
measured to determine the cardioprotective effect. The testing
results showed that, 5f could significantly increase the coronary
flow, the content of ATP, the activity of Na+–K+ ATPase and re-
duce the content of LD in tissue homogenates. These effects of
relaxing coronary artery and improving myocardial energy
metabolism were in a dose-dependent manner. In improving
NHE1 inhibitions. Most compounds show more potent NHE1
inhibitory activity than cariporide, and the most potent one was
5f. Further testing showed that, 5f could excellently improve
the cardiac function and reduce infarct size against ischemia-
reperfusion injury. Preliminary structure–activity relationship
showed that, the introduction of electron-withdrawing group
led to an increase in NHE1 inhibitory potency. Further biological
evaluation of these derivatives and extensive lead optimization
are ongoing in our laboratory and will be reported in due course.
myocardial energy metabolism, 5f (10
oride(10 M). In relaxing coronary artery, 5f (50
(10 M) showed better effect. But there was no significant differ-
ence between 5f (10 M) and cariporide (10 M).
l
M) was better than carip-
l
l
M) and 5f
l
l
l
Acknowledgments
All values are expressed as mean S.E.M. Data were analyzed by
one-way analysis of variance (ANOVA) and unpaired Student’s t-
test. In all comparison, the difference was considered to be statis-
tically significant at P <0.05 and extremely significant at P <0.01.
In summary, a series of novel substituted benzoylguanidine
derivatives were designed, synthesized and evaluated for their
Financial support from the National Natural Science Founda-
tion of China (No. 30672512) is gratefully acknowledged.
We also thank Center for Instrumental Analysis at China Phar-
maceutical University for their contribution in the structural
confirmation.

W.-T. Xu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3283–3287
3287
3:1). The product was dissolved in acetone, and followed by hydrochlorination
with gaseous HCl to give 5f as light yellow solid. Yield: 35%; mp: 230–233 °C;
IR (KBr, cmꢀ1): 3401, 2936, 1703(C@O), 1612, 1582, 1543(NO2), 1345(NO2),
References and notes
1106(OCH3); 1H NMR (500 MHz, CDCl3): 2.53–3.29 (8H, m,
); 3.77
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column chromatography (petroleum ether/acetone, 3:1). The resulting oil was
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