Synthesis, selective anti-Helicobacter pylori activity, and cytotoxicity of novel N-substituted-2-oxo-2H-1-benzopyran-3-carboxamides

Article abstract of DOI:10.1016/j.bmcl.2010.06.048

N-substituted-3-carboxamido-coumarin derivatives were prepared and evaluated for selective antibacterial activity against 20 isolates of Helicobacter pylori clinical strains, including five metronidazole resistant ones. Some of them possessed the best activity against H. pylori metronidazole resistant strains with MIC values lower than the drug reference (metronidazole). Furthermore, anti-inflammatory activity through the inhibition of the IL-8 production was investigated.

Full text of DOI:10.1016/j.bmcl.2010.06.048

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4922–4926
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, selective anti-Helicobacter pylori activity, and cytotoxicity
of novel N-substituted-2-oxo-2H-1-benzopyran-3-carboxamides
a
a
a
a
Franco Chimenti a, Bruna Bizzarri a, , Adriana Bolasco , Daniela Secci , Paola Chimenti , Arianna Granese ,
*
Simone Carradori a, Daniela Rivanera b, Alessandra Zicari c, M. Maddalena Scaltrito d, Francesca Sisto d
a Dipartimento di Chimica e Tecnologie del Farmaco, Università ‘La Sapienza’, P.le A. Moro 5, 00185 Rome, Italy
b Dipartimento di Scienze di Sanità Pubblica, Università ‘La Sapienza’, P.le A. Moro 5, 00185 Rome, Italy
c Dipartimento di Medicina Sperimentale, Università ‘La Sapienza’, V.le Regina Elena 324, 00161 Rome, Italy
d Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano, via Pascal 36, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
N-substituted-3-carboxamido-coumarin derivatives were prepared and evaluated for selective antibacte-
rial activity against 20 isolates of Helicobacter pylori clinical strains, including five metronidazole resistant
ones. Some of them possessed the best activity against H. pylori metronidazole resistant strains with MIC
values lower than the drug reference (metronidazole). Furthermore, anti-inflammatory activity through
the inhibition of the IL-8 production was investigated.
Received 13 May 2010
Revised 7 June 2010
Accepted 8 June 2010
Available online 19 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Coumarin
Antimicrobial activity
Helicobacter pylori
Cytotoxicity
Helicobacter pylori are spiral-shaped Gram-negative bacteria
with polar flagella that live near the surface of the human gastric
mucosa. They have evolved intricate mechanisms to avoid the bac-
tericidal acid environment in the gastric lumen and to survive near,
to attach to, and to communicate with the human gastric epithe-
lium and host immune system. This interaction sometimes results
in severe gastric pathology. H. pylori infection is the strongest
known risk factor for the development of gastroduodenal ulcers,
with infection being present in 60–80% of duodenal ulcers.1
Helicobacter pylori is also the first bacterium to be classified as a
definite carcinogen by the World Health Organization’s Interna-
tional Agency for Research on Cancer because of its epidemiologic
relationship to gastric adenocarcinoma and gastric mucosa-associ-
ated lymphoid tissue lymphoma.2 Most H. pylori transmission
occurs in childhood, and, in some countries, up to 90% of children
become infected by age 10 years, with reports of infection as early
as the first months of life.3
transiently buffering the acidic environment by the breakdown of
urea to generate ammonia and carbon dioxide. This enzymatic activ-
ityis highlyconserved in different H. pylori strains and therefore use-
ful for diagnostic purposes.4,5 To remain in the mucus layer, H. pylori
need to utilize their polar flagella for motility. Both the ability to
swim with flagellar motion and the ability to control the direction
ofmovementby chemotacticresponsesare essentialfor H. pylori col-
onization.6,7 H. pylori causes its damage by initiating chronic inflam-
mation in the gastric mucosa. This inflammation is mediated by an
array of pro- and anti-inflammatory cytokines. For this reason,
anti-inflammatoryactivitythroughtheinhibitionoftheIL-8produc-
tion was also investigated.
Helicobacter pylori infections are difficult to cure and successful
treatment generally requires the administration of several antibac-
terial agents simultaneously. Duration of therapy is also important
and depends upon whether resistance is present. Antibiotic resis-
tance has resulted in unsatisfactory eradication with dual and
now triple therapy in many countries. Newer antibiotics and
changes in dosing and duration of therapy may overcome resistant
strains but may only provide limited improvement in eradication
rates. Sequential therapy with amoxicillin and a proton pump
inhibitor given for 5 days followed by clarithromycin and tinida-
zole for 5 days is now a first-line therapy for H. pylori in some
countries. This work is aimed at trying to overcome these problems
as well as evaluating a series of N-substituted-2-oxo-2H-1-benzo-
pyran-3-carboxamides as a part of a screening program.
From the medical point of view, H. pylori is a formidable pathogen
responsible for much morbidity and mortality worldwide. However,
H. pylori infection occurs in approximately half of the world popula-
tion, with disease being the exception rather than the rule. To avoid
the bactericidal activity of acid, H. pylori generates large quantities of
cytosolic and cell surface-associated urease, an enzyme capable of
* Corresponding author. Tel.: +39 649913259; fax: +39 649913923.
E-mail address: bruna.bizzarri@uniroma1.it (B. Bizzarri).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.048

F. Chimenti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4922–4926
4923
In our previous works,8,9 we have pointed out that a carboxam-
ide function in the coumarin ring might play an important role
leading to activity. On the basis of the SAR studies and from the
analysis of the structure of natural coumarins reported as potent
anti-H. pylori agents,10–12 we have demonstrated that a halogen
in the position 6, no bulky substituents in the position 7 and an
acyl group on the p-position of the N-aromatic moiety seemed to
be crucial for the activity.
Moving from these indications, in this report we described the
synthesis and selective antimicrobial evaluation of a new series
of 20 derivatives substituted in the positions 3, 6, 7, and 8 of the
coumarin ring (Table 1).
We have synthesized 2-oxo-2H-chromene-3-carboxylic acid
ethylesterderivatives[coumarins(a)] byaKnoevenagelreactionbe-
tween the appropriate benzaldehyde and diethylmalonate in etha-
nol by using piperidine as catalyst. They were hydrolyzed with
NaOH 20% and HCl 3 N to obtain the corresponding carboxylic acid
and treated with thionyl chloride at reflux to give the reactive acyl
chlorides (b), differently substituted on the 6, 7, and 8 positions of
coumarin nucleus. Compounds (b) were reacted with 4-amino-ben-
zoic acid ethyl ester and with 4-aminopiperidine-1-carboxylic acid
ethyl ester in anhydrous ethyl ether to yield derivatives 1–9 and
19–20, respectively, which contain the benzamidic structure impor-
tant for anti-H. pylori activity.
Table 1
Chemical and physical data of derivatives 1–20
O
R2
R1
NH X
O
O
R
Compound
R
R1
R2
X
1
2
3
4
5
H
H
H
Cl
Br
OH
H
H
H
H
H
H
Cl
Br
OH
H
H
H
OH
H
H
H
H
H
H
OCH3
H
OH
H
H
H
H
H
H
OCH3
H
H
H
H
(C6H4)-40-COOEt
(C6H4)-40-COOEt
(C6H4)-40-COOEt
(C6H4)-40-COOEt
(C6H4)-40-COOEt
(C6H4)-40-COOEt
(C6H4)-40-COOEt
(C6H4)-40-COOEt
(C6H4)-40-COOEt
(C6H4)-40-COOH
(C6H4)-40-COOH
(C6H4)-40-COOH
(C6H4)-40-COOH
(C6H4)-40-COOH
(C6H4)-40-COOH
(C6H4)-40-COOH
(C6H4)-40-COOH
(C6H4)-40-COOH
(C5H9N)-10-COOEt
(C5H9N)-10-COOEt
NO2
CH3
Br
Br
H
OH
H
OCH3
H
NO2
CH3
Br
Br
H
OH
H
OCH3
H
6
7
8a
9
10
11
12
13
14
15
16
17a
18
19
20
H
H
Compounds 1–9 were submitted to mild alkaline hydrolysis with
LiOH in H2O/MeOH and treated with HCl 3 N to obtain derivatives
10–18 (Scheme 1). All synthesized compounds (which possessed
Br
a
Ref. 14.
O
R2
R1
CHO
OH
R2
COOEt
CH2
COOEt
EtOH
OEt
+
a
R1
O
O
piperidine
R
R
1)NaOH 20%, HCl 3N
2)SOCl2
O
O
R2
R1
Cl
b
O
R
H2N
COOEt
H2N
N COOEt
O
Et2O
anhydrous
O
O
O
N
OEt
R2
R1
R2
NH
COOEt
N
H
O
O
O
R2= H, Br
R
1-9
19-20
1) LiOH H2O/MeOH
2) HCl, 3N
O
R= H, OH, Br
R1= H, OH, OCH3
R2= H, NO2, CH3, Cl, Br, OH, OCH3
R2
R1
NH
COOH
O
O
R
10-18
Scheme 1. General synthesis of derivatives 1–20.

4924
F. Chimenti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4922–4926
Table 2
Minimal inhibitory concentration of tested compounds and metronidazole (M) against 20 clinical strains of Helicobacter pylori
Compound
MIC (lg/mL)
All strains
MIC50
Metronidazole resistant strains
Range
MIC90
NCTC 11637
1
8
18
20
1
2
3
2–8
ND
1–32
8
ND
4
8
ND
8
8
ND
4
8
ND
8
4
ND
8
8
ND
2
2
ND
2
4
5
6
7
4–32
8
16
64
>128
8
16
64
>128
8
8
4
8
8
32–128
8 to >128
2–8
64
>128
4
64
>128
4
64
>128
2
64
>128
8
64
>128
2
8
9
ND
2–16
ND
4
ND
8
ND
4
ND
8
ND
4
ND
4
ND
4
10
11
12
13
14
15
16
17
18
19
20
M
4–16
32–128
ND
2–32
2–32
4 to >128
4 to >128
>128
8
16
8
8
4
8
8
128
ND
8
16
>128
16
>128
4
128
ND
32
128
ND
32
16
4
16
>128
4
128
ND
16
16
>128
4
>128
8
128
32
64
64
ND
32
16
>128
16
>128
4
128
ND
16
16
>128
16
>128
4
64
ND
32
16
>128
4
>128
4
>128
32
128
32
>128
>128
>128
8
>128
64
2–16
128 to >128
32–64
0.5–128
>128
64
2
>128
64
128
>128
64
64
128
32
128
128
ND, not determined (slightly soluble in DMSO).
calculated log P values less than 5) were fully characterized by
means of analytical and spectral data.13
only for those compounds which were soluble enough in DMSO.
Compounds that have been demonstrated to achieve the strongest
anti-H. pylori activity (1, 3, 4, 7, 9, 10, and 18) were analyzed to esti-
mate their cytotoxicity on cultured cell lines in two steps. First, we
performed Trypan blue test on an immortalized hybrid cell line dis-
playing an endothelial phenotype, EAhy, derived from the fusion of
human umbilical vein endothelial cells (HUVEC) with lung carci-
noma cells. In a second moment, the MTT test was performed on a
human gastric epithelial cell line AGS, to better quantify the cyto-
toxic activity showed by Trypan blue test. The results were reported
in Table 3.16 IL-8 release by the same human gastric epithelial cell
line AGS was also analyzed.17
The synthesized compounds were first assayed against several
species of Gram-positive and Gram-negative bacteria and against
various strains of pathogenic fungi in order to identify those with lit-
tle or no activity as leading compounds. Ceftazidime and clotrima-
zole were used as reference compounds. The data obtained against
all the assayed species mostly showed a MIC value >128 lg/mL.
Based on these results, all synthesized derivatives could be submit-
ted to subsequent screening toward H. pylori.15 The activity of the
substances was compared with the reference compound metronida-
zole against 20 clinical strains of H. pylori including five metronida-
zole resistant ones (Table 2). We reported on the biological results
A series of new coumarin derivatives were prepared, character-
ized, and evaluated for selective antimicrobial activity. Most of the
assayed compounds showed interesting activity against H. pylori
clinical strains (MIC 1–32 lg/mL). The modifications on the posi-
Table 3
Cytotoxic effect of the most active compounds tested on EAhy 926 cells after 24 h of
incubation at 37 °C, using Trypan blue exclusion test (a), and MTT assay (b), expressed
as cell survival fraction (%)a
tion 6 of the coumarin nucleus (as just revealed by the introduction
of Br or Cl in our previous work) brought good inhibitory activity
toward H. pylori and, in particular, compounds 3, 7, and 9 (bearing
R2 as a methyl, hydroxy, and methoxy group) showed the lowest
MIC value, so they might be considered lead compounds for further
development in this field.
Compound (a)
Concentrationb
5
(lg/mL)
50
0.5
0.05
1
3
4
7
9
64 1.6
14 4.9
58 7.1
77 6.1
36 7.8
82 1.0
62 2.6
92 3.5
59 4.6
71 6.5
80 4.1
42 1.5
92 1.7
69 1.7
95 4.1
77 4.6
86 4.5
83 7.5
67 4.3
93 2.5
77 5.4
96 3.8
80 5.2
94 2.3
93 6.6
72 4.4
98 2.6
85 4.2
Furthermore, these results have stated that the disubstitution in
the 6 and 8 positions with halogens (Br and Cl) led to moderately
10
18
only benzocaine derivatives
Br, Cl >>
Compound (b)
Concentrationb
(lg/mL)
OH, CH3, OCH3
O
50
5
0.5
3
6
NH
COX
1
3
4
7
9
78 2.8
82 4.3
81 2.7
75 3.1
99 0.3
58 1.6
99 0.3
99 0.2
99 0.2
99 0.1
98 0.6
99 0.2
98 0.6
99 0.1
99 0.1
63 1.2
64 1.1
82 4.3
41 3.8
77 4.3
77 4.6
7
O
O
8
OEt
OH
bulky substituents,
OCH3
OH
10
18
H >> Cl, Br
OH
a
Cells incubated with culture medium alone represented the controls and the
cell viability was always greater than 99.80%.
b
Data represent arithmetic means SD of at least three independent
Figure 1. SAR studies of substituents on coumarin nucleus against Helicobacter
pylori.
experiments.

F. Chimenti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4922–4926
4925
226 °C, 79% yield; 1H NMR (400 MHz, DMSO-d6) d 1.41–1.44 (t, 3H, CH3),
4.21–4.25 (m, 2H, CH2), 7.68–7.72 (m, 4H, Ar), 8.35–8.39 (m, 3H, Ar), 8.80 (s,
1H, CH@), 10.49 (s, 1H, NH, D2O exch.).
active derivatives (but weaker than the monohalogenated ones),
and no bulky groups in the 7 position (as a polar hydroxy group
better than a methoxy one) of the coumarin ring affected positively
the biological activity. As regards the acyl function on the N-aro-
matic moiety, the ester derivatives showed better activity with re-
spect to the corresponding carboxylic acids. The substitution of
benzocaine linked to the C3 position with a bioisosteric group
led to inactive compounds, thus demonstrating the importance of
this nucleus to display a strong inhibition activity, as shown in
Figure 1 which is a resume of all the obtained results of our per-
formed studies on this scaffold (SAR studies).
All tested compounds possessed the best selectivity against the
metronidazole resistant strains with MIC values lower than the
drug reference. That could be useful in overcoming the increasing
emergence of drug resistance to the conventional therapy.
In general, most of the derivatives presented a discrete cyto-
Ethyl 4-(6-methyl-2-oxo-2H-chromene-3-carboxamido)benzoate (3). Mp 227–
228 °C, 70% yield; 1H NMR (400 MHz, DMSO-d6) d 1.40–1.42 (t, 3H, CH3), 2.47
(s, 3H, ArCH3), 4.40–4.42 (m, 2H, CH2), 7.26 (s, 1H, Ar), 7.51–7.53 (m, 2H, Ar),
7.83 (s, 2H, Ar), 8.04 (s, 1H, Ar), 8.99 (s, 1H, CH@), 11.09 (s, 1H, NH, D2O exch.).
Ethyl 4-(6-bromo-8-chloro-2-oxo-2H-chromene-3-carboxamido)benzoate (4).
Mp >290 °C, 79% yield; 1H NMR (400 MHz, DMSO-d6)
d 1.29–1.32 (t, 3H,
CH3), 4.29–4.30 (m, 2H, CH2), 7.82–7.83 (m, 2H, Ar), 7.97–7.99 (m, 2H, Ar), 8.14
(s, 1H, Ar), 8.19 (s, 1H, Ar), 8.77 (s, 1H, CH@), 10.78 (br s, 1H, NH, D2O exch.).
Ethyl 4-(6,8-dibromo-2-oxo-2H-chromene-3-carboxamido)benzoate (5). Mp 223–
224 °C, 70% yield; 1H NMR (400 MHz, DMSO-d6) d 1.23–1.28 (t, 3H, CH3), 4.16–
4.20 (m, 2H, CH2), 6.55–6.58 (m, 2H, Ar), 7.62–7.64 (m, 2H, Ar), 8.29 (s, 1H, Ar),
8.44 (s, 1H, Ar), 8.62 (s, 1H, CH@), 10.70 (br s, 1H, NH, D2O exch.).
Ethyl 4-(8-hydroxy-2-oxo-2H-chromene-3-carboxamido)benzoate (6). Mp
280–281 °C, 77% yield; 1H NMR (400 MHz, DMSO-d6) d 1.24–1.27 (t, 3H,
CH3), 4.16-4.22 (m, 2H, CH2), 6.62–6.64 (m, 2H, Ar), 7.20–7.21 (m, 2H, Ar),
7.31–7.32 (m, 1H, Ar), 7.65–7.67 (m, 2H, Ar), 8.70 (s, 1H, CH@), 10.50 (s, 1H,
NH, D2O exch.), 11.20 (br s, 1H, OH, D2O exch.).
Ethyl 4-(6-hydroxy-2-oxo-2H-chromene-3-carboxamido)benzoate (7). Mp
210–211 °C, 95% yield; 1H NMR (400 MHz, DMSO-d6)
d 1.30–1.31 (t, 3H,
toxic profile at concentration between 0.5 and 5 lg/mL, especially
CH3), 4.29–4.31 (m, 2H, CH2), 7.29–7.40 (m, 3H, Ar), 7.88–7.97 (m, 4H, Ar), 8.89
(s, 1H, CH@), 10.99 (s, 1H, NH, D2O exch.), 11.99 (br s, 1H, OH, D2O exch.).
Ethyl 4-(6-methoxy-2-oxo-2H-chromene-3-carboxamido)benzoate (9). Mp 209–
210 °C, 71% yield; 1H NMR (400 MHz, DMSO-d6) d 1.30–1.32 (t, 3H, CH3), 3.82
(s, 3H, OCH3), 4.27–4.29 (m, 2H, CH2), 7.39–7.49 (m, 4H, Ar), 7.80–7.92 (m, 3H,
Ar), 8.64 (s, 1H, CH@), 10.99 (s, 1H, NH, D2O exch.).
4-(7-Hydroxy-2-oxo-2H-chromene-3-carboxamido)benzoic acid (10). Mp 242–
243 °C, 76% yield; 1H NMR (400 MHz, DMSO-d6) d 6.73 (s, 2H, Ar), 6.82–6.85
(m, 2H, Ar), 7.72–7.74 (m, 3H, Ar), 8.65 (s, 1H, CH@), 10.80 (s, 1H, NH, D2O
exch.), 11.25 (br s, 1H, OH, D2O exch.), 13.01 (br s, 1H, COOH, D2O exch.).
4-(6-Nitro-2-oxo-2H-chromene-3-carboxamido)benzoic acid (11). Mp 224–
225 °C, 72% yield; 1H NMR (400 MHz, DMSO-d6) d 7.71–7.74 (m, 4H, Ar),
8.39–8.43 (m, 3H, Ar), 8.86 (s, 1H, CH@), 10.59 (s, 1H, NH, D2O exch.), 12.93 (br
s, 1H, COOH, D2O exch.).
in relation to their MIC values. In the current study, we also inves-
tigated a possible anti-inflammatory effect of new synthesized
compounds in H. pylori-induced gastric inflammation. None of
the compounds tested was able to significantly reduce IL-8 produc-
tion, thus demonstrating a different mechanism of action for this
scaffold of derivatives.
Acknowledgment
This work was supported by grants from FIRB RBI067F9E (Italy).
References and notes
4-(6-Methyl-2-oxo-2H-chromene-3-carboxamido)benzoic acid (12). Mp >290 °C,
77%; 1H NMR (400 MHz, DMSO-d6) d 2.50 (s, 3H, ArCH3), 7.36 (s, 1H, Ar), 7.56–
7.65 (m, 4H, Ar), 7.93 (s, 1H, Ar), 8.44 (s, 1H, Ar), 8.99 (s, 1H, CH@), 11.09 (s, 1H,
NH, D2O exch.), 12.96 (br s, 1H, COOH, D2O exch.).
1. Hunt, R. H. Scand. J. Gastroenterol. 1996, 220, 3.
4-(6-Bromo-8-chloro-2-oxo-2H-chromene-3-carboxamido)benzoic acid (13).
Mp >290 °C, 84% yield; 1H NMR (400 MHz, DMSO-d6) d 7.79–7.82 (m, 2H,
Ar), 7.95–7.98 (m, 2H, Ar), 8.29 (s, 1H, Ar), 8.40 (s, 1H, Ar), 8.67 (s, 1H, CH@),
10.81 (s, 1H, NH, D2O exch.), 13.02 (br s, 1H, COOH, D2O exch.).
4-(6,8-Dibromo-2-oxo-2H-chromene-3-carboxamido)benzoic acid (14). Mp 275–
276 °C, 82% yield; 1H NMR (400 MHz, DMSO-d6) d 7.80–7.82 (d, Jo = 8.0 Hz, 2H,
Ar), 7.94–7.96 (d, Jo = 8.0 Hz, 2H, Ar), 8.27 (s, 1H, Ar), 8.58 (s, 1H, Ar), 8.70 (s,
1H, CH@), 10.79 (s, 1H, NH, D2O exch.), 13.01 (br s, 1H, COOH, D2O exch.).
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207 °C, 86% yield; 1H NMR (400 MHz, DMSO-d6) d 7.15–7.20 (m, 4H, Ar), 7.30–
7.32 (m, 3H, Ar), 8.67 (s, 1H, CH@), 10.35 (s, 1H, NH, D2O exch.), 11.30 (br s, 1H,
OH, D2O exch.), 13.11 (br s, 1H, COOH, D2O exch.).
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10. Kawase, M.; Motohashi, N. Curr. Med. Chem.-Anti-Infective Agents 2004, 3, 89.
11. Takeda, K.; Utsunomiya, H.; Kakiuchi, S.; Okuno, Y.; Oda, K.; Inada, K. I.;
Tsutsumi, Y.; Tanaka, T.; Kakudo, K. J. Oleo Sci. 2007, 565, 253.
12. Lee, J. H.; Bang, H. B.; Han, S. Y.; Jun, J. G. Tetrahedron Lett. 2007, 48, 2889.
13. The chemicals, solvents for synthesis and spectral grade solvents were
purchased from Sigma–Aldrich (Italy) and used without further purification.
4-(6-Methoxy-2-oxo-2H-chromene-3-carboxamido)benzoic
acid
(18).
Mp >290 °C, 78% yield; 1H NMR (400 MHz, DMSO-d6) d 3.81 (s, 3H, OCH3),
7.37–7.48 (m, 4H, Ar), 7.84–7.96 (m, 3H, Ar), 8.69 (s, 1H, CH@), 10.97 (s, 1H,
NH, D2O exch.), 12.98 (br s, 1H, COOH, D2O exch.).
Ethyl 4-(2-oxo-2H-chromene-3-carboxamido)piperidine-1-carboxylate (19). Mp
220–221 °C, 85% yield; 1H NMR (400 MHz, DMSO-d6) d 1.42–1.44 (t, 3H, CH3),
1.52–1.55 (m, 4H, 2ꢀ CH2), 2.77–2.81 (m, 4H, 2ꢀ CH2), 2.92–2.95 (m, 1H, CH),
4.14–4.18 (m, 2H, CH2), 7.19–7.27 (m, 3H, Ar), 7.93–7.97 (m, 1H, Ar), 8.89 (s,
1H, CH@), 10.80 (s, 1H, NH, D2O exch.).
Ethyl 4-(6-bromo-2-oxo-2H-chromene-3-carboxamido)piperidine-1-carboxylate
(20). Mp 201–202 °C, 76% yield; 1H NMR (400 MHz, DMSO-d6) d 1.29–1.32 (t,
3H, CH3), 1.53–1.55 (m, 4H, 2ꢀ CH2), 2.78–2.82 (m, 4H, 2ꢀ CH2), 2.91–2.93 (m,
1H, CH), 4.29–4.30 (m, 2H, CH2), 7.82–7.83 (m, 2H, Ar), 8.19 (s, 1H, Ar), 8.77 (s,
1H, CH@), 10.78 (br s, 1H, NH, D2O exch.).
Melting points (uncorrected) were determined automatically on
a FP62
apparatus (Mettler–Toledo). 1H NMR spectra were recorded at 400 MHz on a
Bruker spectrometer using DMSO-d6 as solvent. Chemical shifts are expressed
as d units (parts per million) relative to the solvent peak. Coupling constants J
are valued in Hertz (Hz). Elemental analysis for C, H, and N were recorded on a
Perkin-Elmer 240
B
microanalyzer and the analytical results were
within 0.4% of the theoretical values for all compounds.
General procedure for the synthesis of derivatives 1–20. The appropriate
salicylaldehyde (5 g) in ethanol (50 mL) is refluxed under magnetic stirring
with diethyl malonate and catalytic amounts of piperidine for 24 h. After
cooling to room temperature, the suspension is filtered to give the desired
ethyl ester of 3-coumarin carboxylic acid (a). Hydrolysis with 10% NaOH
(100 mL) and addition of HCl 3 N led to the desired product. Following
filtration, the corresponding carboxylic acid was refluxed at 100 °C with
thionyl chloride for 2 h to obtain the reactive acyl chloride (b). Then, it was
condensed with the appropriate amine in anhydrous diethyl ether to give the
crude products in high yields (1–9 and 19–20). Subsequently, ester derivatives
were transformed into carboxylic acids by alkaline hydrolysis for 2 h at room
temperature with equimolar quantities of LiOH in water/methanol (1:4; v/v)
(10–18).
14. Shiping, X.; Xiaoguang, C.; Song, X.; Lanmin, L.; Longfei, X.; Hongyan, L.; Yan, L.;
Guifang, C. WO 2004/050082.
15. Antibacterial and antifungal activity. All synthesized derivatives were evaluated
for their antimicrobial and antifungal activity when dissolved in DMSO.
Organisms, from routine clinical Gram-positive (S. aureus, S. epidermidis, S.
hominis, S. a-hemolyticus, S. faecalis), Gram-negative (E. coli, K. pneumoniae, K.
oxytoca, Enterobacter spp., E. aerogenes, C. freundii, P. vulgaris), and Candida
strain isolates (C. albicans, C. sakè, C. krusei) from the respiratory tract, were
collected from specimens of patients at the Hospital ‘Azienda Policlinico
Umberto I°’ of Rome ‘La Sapienza’ University. The isolates were subcultured on
a qualified medium to ensure purity and an optimal growth. The isolates were
identified by conventional methodologies. The in vitro antibacterial activities
of the compounds were determined with the broth micro dilution method, as
recommended by the National Committee for Clinical Laboratory Standards18
with Mueller–Hinton II broth (BBL Microbiology Systems, Cockeysville, MD).
Microtiter plates containing serial dilutions of each compound ranging from
Ethyl 4-(7-hydroxy-2-oxo-2H-chromene-3-carboxamido)benzoate (1). Mp 282–
283 °C, 71% yield; 1H NMR (400 MHz, DMSO-d6) d 1.39–1.42 (t, 3H, CH3), 4.20–
4.21 (m, 2H, CH2), 6.70–6.77 (m, 3H, Ar), 7.78–7.91 (m, 4H, Ar), 8.88 (s, 1H,
CH@), 10.90 (s, 1H, NH, D2O exch.), 11.25 (br s, 1H, OH, D2O exch.).
Ethyl 4-(6-nitro-2-oxo-2H-chromene-3-carboxamido)benzoate (2). Mp 225–

4926
F. Chimenti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4922–4926
128 to 0.5
l
g/mL were inoculated with each organism to yield the appropriate
supplemented with 2 mM L-glutamine, HAT supplement and containing
density (105/mL) in
a
100 final volume; each plate included positive
l
L
antibiotic mixture. Experiments were performed in cells grown to 60–70%
confluency.21 The stock solutions of the investigated compounds were
prepared in sterile DMSO and successive dilutions were made in culture
medium; the DMSO percent present in culture medium never exceeded 0.5%.
EAhy cells in the exponential phase of growth (1 ꢀ 105/mL) were seeded into
24-well microplate and incubated for 24 h with four different concentrations
controls (bacteria without a compound), and a negative control (medium only).
The plates were incubated for 18–22 h at 35 °C. The Minimal Inhibitory
Concentration (MIC) for all isolates was defined as the lowest concentration of
antibacterial agent that completely inhibited the growth of the organism, as
detected by the unaided eye.
The in vitro antifungal activity of the compounds was determined with the
broth micro dilution method with Sabouraud dextrose broth (BBL
Microbiology Systems, Cockeysville, MD) as recommended by the NCCLS.19
Microtiter plates containing serial dilutions of each compound were inoculated
of the compounds (50–0.05 lg/mL). Some plates containing cells alone or cells
and DMSO represented the negative controls, whereas cells incubated with
1 mM natrium nitroprusside represented the positive one. After the incubation
period, cells were mechanically scraped off from the plates and an aliquot was
diluted (1:1) with a solution 0.4% Trypan blue stain. After few minutes at room
temperature, cells were counted under an optical microscope in a Thoma
hemocytometer chamber by two different operators. On the basis that Trypan
blue is a vital dye22 and can enter and interact with the cells unless the
plasmatic membrane is damaged, blue stained cells were considered as having
died. Values are expressed as % of viable cells. Cell viability in control samples
was always 97–98%.
with each organism to yield the appropriate density (103/mL) in a 100
lL final
volume; each plate included positive controls (fungi without a compound), and
a negative control (medium only). The plates were incubated for 24 h at 37 °C.
The MIC for all isolates was defined as the lowest concentration of antifungal
agent that completely inhibited the growth of the organism, as detected by the
unaided eye.
Anti-H. pylori activity. Twenty clinical strains of H. pylori isolated from patients
with duodenal ulcer or gastritis were tested. Four of these strains had known
metronidazole resistance. H. pylori ATCC 43504 was used as control in each
assay session.They were maintained at ꢁ80 °C in Wilkins Chalgren broth with
10% (v/v) horse serum (Seromed) and 20% (v/v) glycerol (Merck) until required
for the experiments. Before being used the bacteria were subcultured twice on
Columbia agar base (Difco Laboratories) supplemented with 10% horse serum
and 0.25% Bacto yeast extract (Difco). Plates were incubated for 72 h at 37 °C in
an atmosphere of 10% CO2 in a gas incubator.
Antimicrobial activity against H. pylori was determined by the agar dilution
standard method.20 The strains were inoculated onto Columbia agar base
(Difco) supplemented with 10% horse serum and 0.25% bacto yeast extract
(Difco), and were incubated for 72 h at 37 °C in an atmosphere of 10% CO2 in a
gas incubator. Colonies were suspended in Wilkins Chalgren broth to achieve a
turbidity equivalent to 0.5 Mc Farland. Columbia agar plates with 10% horse
serum were prepared by using twofold dilutions of the compounds (128–
MTT staining (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
was applied on AGS cell cultures. Cells were seeded in triplicate in 96-well
plates at the density of 5 ꢀ 104 cell/well in complete medium and incubated
with selected compounds at concentration of 50, 5, and 0.5 lg/mL for 24 h.
Then, 20 ll of a 5 mg/mL solution of MTT in phosphate-buffered saline was
added, and incubation continued for an additional 3 h at 37 °C. The plates were
then centrifuged, the supernatants were discarded, and the dark blue formazan
crystals were dissolved using 100 mL of lysing buffer consisting of a solution of
20% (w/v) sodium dodecyl sulfate and 40% N,N-dimethylformamide in H2O at
pH 4.7 adjusted with 80% acetic acid. The plates were then read on a microplate
reader (Molecular Devices Co., Menlo Park, CA) using a test wavelength of
550 nm and a reference wavelength of 650 nm. The optical density at 650 nm
(OD650) was subtracted from the OD550 to eliminate nonspecific background.
17. IL-8 assessment. AGS cells (1 ꢀ 105 mL) were seeded in 24-well culture plates
for 24 h and then preincubated for 1 h with selected compounds at
0.0039
l
g/mL), and metronidazole (256–0.0039
l
g/mL), as control. The
concentration of 5 and 0.5 lg/mL. After that, AGS cells were incubated with
inoculum was delivered to the surface of the agar plates with
a
Steer’s
H. pylori according to literature.23
replicator in order to obtain approximately 5 ꢀ 105 CFU per spot. Growth
control plates without antibiotics were inoculated in each series of tests. All
plates were incubated at 37 °C for 72 h under conditions (10% CO2 in a gas
incubator). The minimal inhibitory concentration was defined as the lowest
concentration of drug inhibiting visible bacterial growth.
18. National Committee for Clinical Laboratory Standards. Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically; approved
standard, 8th ed. M07-A8. National Committee for Clinical Laboratory
Standards, Wayne, PA, 2009.
19. National Committee for Clinical Laboratory Standards. Reference methods for
broth dilution antifungal susceptibility testing of yeasts; approved standard,
3rd ed. Supplement M27-A3. National Committee for Clinical Laboratory
Standards, Wayne, PA, 2006.
20. National Committee for Clinical Laboratory Standards. Methods for
antimicrobial susceptibility testing of anaerobic bacteria; approved standard
M11-A6, 6th ed. National Committee for Clinical Laboratory Standards,
Villanova, PA, 2004.
16. In vitro cytotoxicity. EAhy human cell line has been obtained from a hybridoma
between HUVEC cells and epithelial cells from a lung carcinoma, AGS cell line
(gastric adenocarcinoma, ATCC CRL 1739) was purchased from the American
Type Culture Collection (Rockville, MD, USA). Cells were grown in complete
medium, consisting of D-MEM for EAhy cells and F-12K medium for AGS ones,
supplemented with 10% fetal bovine serum, 1% glutamine, 100 U/mL penicillin
G and 100 lg/mL streptomycin. Cell cultures were maintained at 37 °C in a
humidified atmosphere of 95% air and 5% CO2. After attaining confluence the
cells were subcultured following trypsinization with 0.25% trypsin–EDTA
solution.
Trypan blue dye exclusion assay was applied on EAhy cell cultures. Cells were
maintained as adherent type cultures under humidified atmosphere in 5% CO2
at 37 °C, Dulbecco’s modified Eagle’s culture medium (high glucose)
21. Edgell, C. J.; McDonald, C. C.; Graham, J. B. Proc. Natl. Acad. Sci. U.S.A. 1983, 80,
3734.
22. Freshney, R. In Culture of Animal Cells: A Manual of Basic Technique, Liss, A. R.,
Inc.; New York, 1987.
23. Shih, Y. T.; Wu, D. C.; Liu, C. M.; Yuan-Chieh, Y.; Ing-Jun, C.; Yi-Ching, L. J.
Ethnopharmacol. 2007, 112, 537.