Gastroprotective activity of synthetic coumarins: Role of endogenous prostaglandins, nitric oxide, non-protein sulfhydryls and vanilloid receptors

Article abstract of DOI:10.1016/j.bmcl.2016.10.056

Natural or synthetic coumarins showed gastroprotective and antiulcer activity in animal models. In this study, we have synthetized twenty coumarins using classic methods to evaluate their gastroprotective effects on the ethanol/HCl-induced gastric lesion model in mice at 20?mg/kg. Among the coumarins synthetized, compounds 6 and 10 showed the greatest gastroprotective activity being as active as lansoprazole at 20?mg/kg and reducing gastric lesions by 75 and 76%, respectively. Then, in a second experiment, compounds 6 and 10 were re-evaluated in order to understand the possible mode of gastroprotective activity. Regarding coumarin 6, the protective effect was reduced by pre-treatment of the mice with N-ethylmaleimide and l-NAME suggesting that sulfhydryl compounds and endogenous nitric oxide are involved in its gastroprotective activity. While for coumarin 10 the effect was reduced by pre-treatment with indomethacin suggesting that prostaglandins are positively involved in its gastroprotective activity.

Full text of DOI:10.1016/j.bmcl.2016.10.056

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Gastroprotective activity of synthetic coumarins: Role of endogenous
prostaglandins, nitric oxide, non-protein sulfhydryls and vanilloid
receptors
Beatriz Sepulveda a, Cristina Quispe b, Mario Simirgiotis c, Alfredo Torres-Benítez d, Johanna Reyes-Ortíz d,
Carlos Areche e, , Olimpo García-Beltrán d,
⇑
⇑
a Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andres Bello, Quillota 980, Viña del Mar, Chile
b Facultad de Ciencias de la Salud, Instituto de Etnofarmacología (IDEA), Universidad Arturo Prat. Casilla 121, Iquique, Chile
c Instituto de Farmacia, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia 5090000, Chile
d Facultad de Ciencias Naturales y Matemáticas, Universidad de Ibagué, Carrera 22 Calle 67, Ibagué, Colombia
e Departamento de Química, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural or synthetic coumarins showed gastroprotective and antiulcer activity in animal models. In this
study, we have synthetized twenty coumarins using classic methods to evaluate their gastroprotective
effects on the ethanol/HCl-induced gastric lesion model in mice at 20 mg/kg. Among the coumarins syn-
thetized, compounds 6 and 10 showed the greatest gastroprotective activity being as active as lansopra-
zole at 20 mg/kg and reducing gastric lesions by 75 and 76%, respectively. Then, in a second experiment,
compounds 6 and 10 were re-evaluated in order to understand the possible mode of gastroprotective
activity. Regarding coumarin 6, the protective effect was reduced by pre-treatment of the mice with
N-ethylmaleimide and l-NAME suggesting that sulfhydryl compounds and endogenous nitric oxide are
involved in its gastroprotective activity. While for coumarin 10 the effect was reduced by pre-treatment
with indomethacin suggesting that prostaglandins are positively involved in its gastroprotective activity.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 15 August 2016
Revised 18 October 2016
Accepted 19 October 2016
Available online xxxx
Keywords:
Coumarins
Gastroprotective
Ulcer
Heterocycles
Peptic ulcer develops when an imbalance occurs between the
defensive and aggressive factors.1 Defensive factors include mucus,
bicarbonate, prostaglandins, mucosal blood flow, nitric oxide, sulf-
hydryls and growth factors, while aggressive factors include
hydrochloride acid, pepsins, bile acids, hypoxia, smoking and alco-
hol. Up to date, around one in five persons suffer from ulcers asso-
ciated to stress, diet and certain types of drugs. Medicines used in
the treatment of gastric ulcers are mainly antiacids, H2-receptor
antagonists and proton pump inhibitors and when the gastric ulcer
is produced by Helicobacter pylori, antibiotics are included as well.
However, side effects produced by occidental medicine shows the
need for looking new antiulcer agents.1–4
Coumarins are a small group of molecules whose structures
contain the 2H-chromen-2-one or 1-benzopyran-2-one cores.
Many coumarins have numerous pharmacological applications as
anticoagulants, anti-inflammatory, antipyretics, antibacterials,
antihelminthics, and also as photoprotectors.5 The first coumarin
was isolated in 1820, and synthesized for the first time in 1868.6
Several synthetic routes have been published, being the most rep-
resentative those developed by Perkin, Pechmann, Knoevenagel,
Reformatsky, Wittig and Heck.7
In the course of our studies on gastroprotective drugs, we report
here the synthesis of twenty coumarins (1–20) and their gastro-
protective effect in mice. In addition, we discuss the mode of gas-
troprotective action of 6 and 16, including the involvement of
prostaglandins (PGs), nitric oxide (NO), sulfhydryl compounds
(SHs) and vanilloid receptors (VR).
Some twenty coumarins (Schemes 1–3) were synthetized to
disclosure structure activity relationship (SAR) based on the gas-
troprotective effect on the model of HCl/EtOH-induced gastric
lesions in mice (Table 1).8,9 All coumarins have been reported pre-
viously and were prepared through known synthetic routes. The
compounds 1–11 were synthetized through Pechmann condensa-
tion with modifications7b,10 (Schemes
1 and 2), while the
compounds 12–20 were synthetized through Knoevenagel
condensation (Scheme 3).7d,11 The synthesis and characterization
of the coumarins can be found in Supporting information.
⇑
Corresponding authors. Tel.: +56 2 29787218; fax: +56 2 22713888.
E-mail addresses: areche@uchile.cl (C. Areche), jose.garcia@unibague.edu.co
(O. García-Beltrán).
http://dx.doi.org/10.1016/j.bmcl.2016.10.056
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sepulveda, B.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.10.056

2
B. Sepulveda et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Scheme 1. Reagents and conditions: (a) H2SO4, respective ethylacetoacetate, rt, 6 h; (b) POCl3, ACN, or the corresponding benzaldehyde; (c) respective ethylacetoacetate or
malonate, piperidine, EtOH, reflux, 4 h.
Indeed, catechol moiety increases the gastroprotective effects for
both compounds. There were no differences (compounds 8 and
9) if a chlorine or methyl group is placed at C-4.
In the case of the coumarins 13–17, bearing a carbonyl group at
C-3, the highest gastroprotective activity was observed for com-
pound 16 (66%). The ester moiety decreases the gastroprotective
Scheme 2. Reagents and conditions: (a) ZnCl2, MW, 10 min, 400 W.
effects in compounds 13–15, while the effect of a carboxyl group
was lower than compound 16 (41%).
Finally, for the 7-hydroxycoumarins 18–20 with substitutions
at C-3 the activity collapsed when an acetyl group is present.
Table 1 shows the effect of the synthetic coumarins 1–20 at
20 mg/kg. The greatest gastroprotective activity was displayed by
compounds 6, and 10, which resulted as active as lansoprazole at
Regarding the carboxyl (60%) and ester (63%) groups, the gastro-
20 mg/kg and reduced gastric lesions by 75% and 76%, respectively.
protective activity was lower than lansoprazole (74%).
The gastroprotective activity of the coumarins 3, 11, 14 and 15 did
not differ statistically from the control. As for the rest of coumarins,
The best gastroprotective compounds were 6 and 10, so we
selected these compounds for further experiments. The possible
the gastroprotective activity were found over the range 15–68%.
mode of gastroprotective action by 6 and 10 on the gastric lesions
In the case of the 7-hydroxycoumarins 1–7, series where exists
induced by HCl/EtOH in mice pretreated with Indometacin9,12
substitution at C-4, a significant increase in the gastroprotective
(10 mg/kg, s.c.), N-ethylmaleimide9,12 (NEM, 10 mg/kg, s.c.), NG-
activity was observed for compound 6 bearing a morpholine moi-
nitro-L (L-NAME, 70 mg/kg, i.p.) or ruthe-
-arginine methyl ester9,12
ety (77%). The effect of a piperazine group in 7 (65%) was similar to
that of the compound 5 (68%) but less active than lansoprazole
(74%). The presence of an OH group (compound 3) at C-4, produced
a significant decrease in the gastroprotective effect (16%).
Regarding dihydroxycoumarins 8–12, the highest gastroprotec-
tive activity was observed for compounds 10 (76%) and 12 (61%).
nium red9,12 (RR, 3.5 mg/kg, s.c.) at an oral dose of 20 mg/Kg is
shown in Table 2.
Endogenous PGs are involved in the mechanism of gastropro-
tection induced by mild irritants, and necrotizing agents. In this
sense, PGs inhibit the gastric acid secretion, stimulate release of
Scheme 3. Reagents and conditions: (a) AlCl3, CH2Cl2, rt, 24 h; (b) malonic acid, phenylamine, pyridine, rt, 24 h; (c) pyridine/ethylene glycol (1:1.1); (d) K2CO3, acetone,
reflux, 2 h.
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B. Sepulveda et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 1
is related to the participation of endogenous SHs. Furthermore, the
mode of gastroprotective action of compound 10 is not involving
endogenous SHs because pretreatment with NEM did not reduced
the gastroprotective effect of 10.
Gastroprotective effect of synthetic coumarins 1–20 at 20 mg/kg on HCl/EtOH-
induced gastric lesions in mice
Compound
n
Lesion index (mm)
% lesion reduction
NO in the gastrointestinal tract play a role in the health, defense
and repair of the gastric mucosa.3,15 Furthermore, it has been
demonstrated that NO participates in gastric defense by regulating
the gastric mucosal blood flow, angiogenesis and gastric mucus
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
28.4 1.3⁄
17.6 1.1⁄
34.6 1.3
21.1 1.4⁄
13.4 1.1⁄
10.3 1.2⁄
14.6 1.1⁄
22.9 1.1⁄
24.1 1.6⁄
10.0 1.0⁄
34.3 1.5
16.1 1.1⁄
26.9 1.2⁄
35.3 1.9
43.4 1.2
14.0 0.8⁄
24.6 1.2⁄
16.6 1.1⁄
15.3 1.0⁄
35.3 1.5
10.8 1.3⁄
41.4 2.8
31⁄⁄
57⁄⁄
16⁄⁄
49⁄⁄
68⁄⁄
75
secretion. In this study, pretreatment with L-NAME (an inhibitor
65⁄⁄
45⁄⁄
42⁄⁄
76⁄⁄
17⁄⁄
61
of NO synthase) attenuated the gastroprotective activity of 6. This
finding suggests that endogenous NO have participation in the pro-
tective effect of this coumarin. The gastroprotective effect of 10
with L-NAME was similar to 10 without L-NAME. This fact suggests
that endogenous NO have null participation in the protective effect
of 10.
35⁄⁄
15⁄⁄
0⁄⁄
Capsaicin-sensitive sensory neurons via VR on the gastrointesti-
nal tract participate in gastric defense mechanisms by regulating
the gastric motility, acid secretion and gastric blood flow. This
action is promoted by calcitonin gene-related peptide (CGRP) and
stimulation of gastric mucus and bicarbonate.4,16 In this study, pre-
treatment with ruthenium red (a vanilloid receptor antagonist),
did not reduce the lesion index suggesting that the mechanism
of gastroprotection of 6 and 10 have no relationship with cap-
saicin-sensitive sensory neurons via VR.
In the last decades, many coumarins have been isolated from
natural sources and synthetized from simple precursors.17 Cou-
marins showed numerous biological activities such as anti-inflam-
matory, antiplatelet, anticancer, antibacterial, anti-obesity,
antiviral, antifungal, antioxidants, analgesic, anticonvulsivant,
antihyperlipidemic, gastroprotective, antiulcerogenic, antiParkin-
son and neuroprotective activities.17–19
66⁄⁄
41⁄⁄
60⁄⁄
63⁄⁄
15⁄⁄
74
17
18
19
20
Lansoprazole
Control
—
The results are expressed as mean SEM *P <0.01; significantly different compared
with the control and **P <0.01 significantly different compared with lansoprazole
(ANOVA followed by Dunnett’s test). n = number of mice.
Table 2
Effect of the coumarins (6 and 10) on the appearance of gastric lesions induced by
HCl/EtOH (po) in indomethacin-, NEM-, L-NAME- and RR-pretreated mice
Treatment
Dose (mg/kg)
Lesion index (mm)
Several coumarins were reported to be protective for the
induced lesions of gastric mucosa in different animal models.
Among them, esculin20 at doses of 25 and 50 mg/kg protected
the gastric mucosa against ethanol and indomethacin, while its
gastroprotective mechanism include stimulation of prostaglandins,
nitric oxide synthesis, opening of KATP channels, reduction of free
radicals, and modulation of antioxidant enzyme systems. In 2015,
Choi et al.21 reported the gastroprotective activity of scoparone
derivatives and showed that 5,6,7-trimethoxycoumarin and 6,7,8-
trimethoxycoumarin had a greater protection than rebamipide (a
standart drug), and suggested that the presence of methyl group
at position C-5 or C-8 of scoparone improves the gastroprotective
effects. Carvallo et al.22 reported an antiulcerogenic study of a cou-
marin (2H-1-benzopyran-2-one) isolate from Mikania laevigata and
suggested that this coumarin had anti-secretory activity mediated
by the parasympathetic system. Furthermore, Reyes-Chilpa et al.23
isolated and tested two coumarins known as mammea A/BA and
mammea C/OC and suggested that their gastroprotective proper-
ties are in part related to the inhibition of H+, K+-ATPase gastric
enzyme (proton pumps).
Control
IND
NEM
—
38.8 1.6
41.1 1.5
40.1 2.1
35.7 1.1
30
10
70
L
-NAME
RR
6
IND + 6
NEM + 6
3.5
20
30 + 20
10 + 20
70 + 20
39.8 1.8
10.3 1.2*
13.6 1.0*
28.8 1.1
34.7 1.5
14.4 1.8*
10.0 1.0*
42.0 1.2
12.4 1.0*
11.0 0.8*
L
-NAME + 6
RR + 6
10
IND + 10
NEM + 10
3.5 + 20
20
30 + 20
10 + 20
70 + 20
L
-NAME + 10
RR + 10
Carbenoxolone
3.5 + 20
100
12.6 1.2*
13.1 2.9*
Results are expressed as mean SEM, n = 7. Analysis of variance followed by Dun-
nett’s test.
*
P <0.01 compared with the respective control.
mucus and bicarbonate and increase blood flow on gastric
mucosal.13 In the present study, PGs are not involved in the gastro-
protective action of 6, because the activity of this compound was
not reduced by pretreatment with IND (an inhibitor of the PG syn-
thesis). While for compound 10, PGs seem to be involved in the
gastroprotective effect of 10, because IND reduced the activity of
this compound.
The carbonate dehydratases or carbonic anhydrases (CA) are a
family of very important zinc-containing metalloenzymes useful
in the maintenance of several physiological processes including
homeostasis, carbon dioxide and bicarbonate transportation, and
electrolytic balance.24 The enzymes interconvert carbon dioxide
and bicarbonate to maintain base–acid balance in blood and other
liquids and tissues. There are at least five distinct CA families (
a, b,
Endogenous sulfhydryls such as glutathione play an important
role in the protection of the gastric mucosa. Glutathione is known
to protect the integrity and permeability of the cell membrane and
may act as antioxidants, scavengers of free radicals, maintenance
of immune function, regulation of protein synthesis and degrada-
tion, and the maintenance protein structure.14 In this study, a pre-
treatment with NEM (an SH blocker) reduced the gastroprotective
activity of 6, suggesting that the protective effect of this coumarin
c
, d and ). The cytosolic isozymes CA II and CA VI are important
e
agents in the production of bicarbonate in the saliva and neutral-
ization of stomach acid.24 Indeed, several CA inhibitors have been
reported25–27 for the treatment of several ailments including epi-
lepsy, glaucoma, Alzheimer’s disease, obesity, microbial infection,
cáncer and osteoporosis.24,28 Therefore, CA inhibitors might be
used for generating new candidates for treatment of gastric and
duodenal ulcers.
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4
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seems to be related to stimulation of the defensive factors rather
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here the gastroprotective activity of twenty synthetic coumarins.
Compounds 6 and 10 exerted the best gastroprotective activity.
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Acknowledgments
Financial support came from FONDECYT INICIACION N°
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.10.
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