A novel class of selective anti-Helicobacter pylori agents 2-oxo-2H-chromene-3-carboxamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2007.03.050

A novel class of selective anti-Helicobacter pylori agents, 2-oxo-2H-chromene-3-carboxamide derivatives, were prepared and evaluated for their anti-bacterial activity. All synthesized compounds showed little or no activity against different species of Gram-positive and Gram-negative bacteria and against various strains of pathogenic fungi. Some of them exhibited a potent and specific inhibitory effect on the growth of H. pylori, including metronidazole-resistant strains, in the 0.0039-16 μg/mL MIC range. A cytotoxic screening by the Trypan blue dye exclusion assay was also carried out on the most active compounds as anti-H. pylori agents. Among the derivatives examined for their cytotoxic potential, a number of them induced low cytotoxic effects.

Full text of DOI:10.1016/j.bmcl.2007.03.050

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3065–3071
A novel class of selective anti-Helicobacter pylori agents
2-oxo-2H-chromene-3-carboxamide derivatives
Franco Chimenti,a Bruna Bizzarri,a,* Adriana Bolasco,a Daniela Secci,a Paola Chimenti,a
Simone Carradori,a Arianna Granese,a Daniela Rivanera,b Daniela Lilli,b
Alessandra Zicari,c M. Maddalena Scaltritod and Francesca Sistod
a
`
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita ‘‘La Sapienza’’,
P.le A. Moro 5, 00185 Rome, Italy
b
`
Dipartimento di Scienze di Sanita Pubblica, Universita ‘‘La Sapienza’’, P.le A. Moro 5, 00185 Rome, Italy
Dipartimento di Medicina Sperimentale, Universita ‘‘La Sapienza’’, V.le Regina Elena 324, 00161 Rome, Italy
`
c
`
d
`
Istituto di Microbiologia, Universita degli Studi di Milano, via Pascal 36, 20133 Milano, Italy
Received 16 February 2007; revised 14 March 2007; accepted 14 March 2007
Available online 19 March 2007
Abstract—A novel class of selective anti-Helicobacter pylori agents, 2-oxo-2H-chromene-3-carboxamide derivatives, were prepared
and evaluated for their anti-bacterial activity. All synthesized compounds showed little or no activity against different species of
Gram-positive and Gram-negative bacteria and against various strains of pathogenic fungi. Some of them exhibited a potent
and specific inhibitory effect on the growth of H. pylori, including metronidazole-resistant strains, in the 0.0039–16 lg/mL MIC
range. A cytotoxic screening by the Trypan blue dye exclusion assay was also carried out on the most active compounds as anti-
H. pylori agents. Among the derivatives examined for their cytotoxic potential, a number of them induced low cytotoxic effects.
ꢀ 2007 Elsevier Ltd. All rights reserved.
For more than 20 years, Helicobacter pylori, a spiral-
shaped microaerophilic Gram-negative bacterium, has
been known to colonize the human gastric mucosa.1
The persistence of H. pylori leads to the development
of a subset of gastric pathologies, including chronic ac-
tive gastritis, peptic ulceration, and the promotion of
malignant disorders.2 Hence since 1994, the World
Health Organization (WHO) has classified H. pylori as
a Class 1 carcinogen responsible for its leading role in
the development of gastric neoplasia in humans. Thus
the eradication of H. pylori can significantly reduce the
risk of ulcer relapse and may help prevent the lym-
phoma of gastric mucosa-associated lymphoid tissue
(MALT-lymphoma) and other gastric malignancies.3
a combination treatment with anti-secretory drugs, such
as proton-pump inhibitors (PPI) or ranitidine bismuth
citrate (RBC), and two antibiotics from among clari-
thromycin, amoxicillin, and metronidazole for 1–2
weeks.4
Furthermore, basic recommendations are given on the
choice treatment in the event of failure of a first-line
eradicating strategy, suggesting a quadruple therapy
based on tetracycline, metronidazole, bismuth salts,
and anti-secretory agents (PPI or ranitidine) for at least
7 days, but discordant results have been reported in tri-
als attempting to evaluate its efficacy, with remission
rates ranging from 57% to 95%.
The guidelines established by several Consensus Confer-
ences held recently in Europe, America, Pacific Asia,
Japan, and Canada have proposed, as a first-line choice
for the eradication of H. pylori, a triple therapy based on
Despite this careful approach, the treatment regimens
have not always been successful, achieving variable
eradication rates of about 70–85%. Multiple drugs, fre-
quent dosing, length of treatment, and occurrence of
harmful side effects often contribute to poor patient
compliance. In addition, an increasing emergence of
drug resistance, which is more frequent and more severe
in the case of quadruple therapy,5 can lead to treatment
failure.
Keywords: Anti-Helicobacter pylori activity; Coumarins.
*
Corresponding author. Tel.: +39 06 49913975; fax: +39 06
49913772; e-mail: bruna.bizzarri@uniroma1.it
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.050

3066
F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3065–3071
Since known anti-microbial agents may present a poten-
tial for the disruption of intestinal microbial flora, which
may cause side effects, the development of new alterna-
tive therapeutic agents with highly selective anti-bacte-
rial activity against H. pylori, but without the risk of
resistance or other untoward effects, has become
necessary.
Moreover, coumarin derivatives are well known for
their anti-microbial activity toward different microor-
ganisms. Some authors have reported a study on the
anti-microbial activity of some coumarin derivatives
with reference to anti-H. pylori activity.8 For all assayed
coumarins, no activity has been reported except for the
derivatives with carboxylic or hydroxyl groups. Based
on these results, the authors have pointed out that a car-
boxylic acid function in the coumarin ring might play an
important role leading to activity. In addition, either
coumarins derived directly from natural products or
synthetic coumarins, both bearing hydroxyl substitu-
ents, were identified as potent anti-H. pylori agents,
comparable to metronidazole.6
This work is aimed at trying to overcome these problems
as well as evaluating a series of 2-oxo-2H-chromene-3-
carboxamide derivatives as part of a screening program.
Naturally occurring coumarins, widely found in plants
belonging to the families Rutaceae, Umbelliferae, and
Compositae,6 are endowed with different types of biolog-
ical applications. It has been reported that coumarin
derivatives exhibit an ever-increasing variety of uses,
including platelet anti-aggregating activity, anti-inflam-
matory activity, and anti-tumor activity.7
Moving from these literature indications and pursuing
our research in the field,9,10 in this report we describe
the synthesis and anti-microbial evaluation of a new ser-
ies of coumarin derivatives against the most common
R1
COOEt
CH2
COOEt
CHO
OH
+
R= H, -OCH2C6H5, -OCH2-(4'-Cl-C6H4), -OCH2-(4'-F-C6H4)
R1= H, Cl, Br
R
ii
i
R1
COCl
O
O
R
A
v
iv
iii
O
O
O
R1
R
R1
R
O
O
HN
O
NH
R1
R
HN
s
NH
COY
O
O
O
N
X
O
O
N
O
OH
C17
C1 - C12
C13 - C16
R = -OCH2C6H5, -OCH2-(4'-F-C6H4)
R1 = H
X = -CH3, -CH2OCOCH3
R = H, -OCH2C6H5, -OCH2-(4'-Cl-C6H4)
R1 = H, Cl, Br
Y = OEt, OH, Cl
R= -OCH2C6H5
R1= H
O
NH
COCl
O
O
B
vi
vii
O
O
O
O
H
NH
CONHNH
HN
O
N
S
O
O
O
N
O
O
X
N
OH
C18-C19
C20
X= -CH3, -CH2OCOCH3
Scheme 1. Reagents: (i) EtOH, piperidine, D; (ii) a—NaOH 20%; b—SOCl2; (iii) a—benzocaine, Et2o; b—NaOH 20%; c—SOCl2; (iv) 7-ACA, 7-
ADCA, THF; (v) INH, CHCl3, Et3N; (vi) 7-ACA, 7-ADCA, THF; (vii) INH, CHCl3, Et3N.

F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3065–3071
3067
pathogens, both bacterial and fungal, and against
H. pylori.
ity studies.13 Starting from 4-[(2-oxo-2H-chromene-3-
carbonyl)-amino]-benzoyl chloride (B),10 we have also
synthesized compounds C18–C20, made up of couma-
rins with cephem derivatives as substituents such as
7-ACA, 7-ADCA, and also isoniazid (INH) (see
Scheme 1).
We have focused on the development of a number of
coumarin derivatives aimed at identifying features of
the structure that could be important for anti-H. pylori
activity.
All synthesized compounds were fully characterized by
means of analytical and spectral data as reported in
Tables 1 and 2.14
In order to establish the cytotoxic activity of this novel
series of coumarin 3-carboxamides, an immortalized
hybrid cell line displaying an endothelial phenotype,
EAhy, derived from the fusion of human umbilical vein
endothelial cells (HUVEC) with lung carcinoma cells
was incubated in the presence of compounds C7–C12
which demonstrated to possess the most potent activity
against H. pylori and the cell viability determined by the
Trypan blue dye exclusion assay.
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 little or no activity as
leading compounds. Ceftazidime and clotrimazole are
used as reference compounds.
The coumarin moiety of all derivatives is obtained fol-
lowing a Knoevenagel reaction between the appropriate
benzaldehydes and diethylmalonate.
The data obtained against all the assayed species mostly
show a MIC value >128 lg/mL. Based on these results,
all synthesized compounds can be submitted to subse-
quent screening toward H. pylori.15
In particular, on the basis of previous investigation,11
starting from the acyl chloride (A) we have synthesized
coumarins C1–C12 (C10 is commercially available12)
which contain the benzamidic structure important for
anti-H. pylori activity. In addition, we have synthesized
compounds C13–C16, which are made up of coumarins
with cephem derivatives as substituents such as 7-ami-
nocephalosporanic acid (7-ACA), 3-deacetyloxy-7-ami-
nocephalosporanic acid (7-ADCA), and also isoniazid
(INH) for compound C17, according to structure-activ-
The activity of the substances is compared with the ref-
erence compound metronidazole against 16 strains of
H. pylori, including the reference strain NCTC 11637
and two other metronidazole-resistant strains.
The MIC ranges and the MIC at which 50% (MIC50)
and 90% (MIC90) of the H. pylori tested strains
are inhibited by compounds C1–C20 are shown in
Table 3, together with the MIC values of the synthesized
Table 1. Chemical and physical data of derivatives C1–C20
O
R1
R
R2
O
O
Compound
R
R1
R2
mp (ꢁC)
Yield (%)
C1
C2
–OCH2C6H5
–OCH2C6H5
H
H
H
H
H
H
Cl
Cl
Cl
Br
Br
Br
H
H
H
H
H
H
H
H
–NH–(40-COOEt–C6H4)
–NH–(40-COOH–C6H4)
–NH–(40-COCl–C6H4)
–NH–(40-COOEt–C6H4)
–NH–(40-COOH–C6H4)
–NH–(40-COCl–C6H4)
–NH–(40-COOEt–C6H4)
–NH–(40-COOH–C6H4)
–NH–(40-COCl–C6H4)
–NH–(40-COOEt–C6H4)
–NH–(40-COOH–C6H4)
–NH–(40-COCl–C6H4)
7-ACAa
263–265
200–203
153–155
260–262
220–225
130–135
279–281
>290
83
32
45
87
95
82
94
56
67
96
54
70
30
70
73
82
30
34
69
30
C3
C4
–OCH2C6H5
–OCH2–(40-Cl–C6H4)
–OCH2–(40-Cl–C6H4)
–OCH2–(40-Cl–C6H4)
C5
C6
C7
C8
H
H
C9
H
>290
C1012
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
H
260–264
>280
>290
H
H
–OCH2–(40-F–C6H4)
135–138
162–166
156–160
210–213
260–263
240–242
229–232
186–200
–OCH2–(40-F–C6H4)
–OCH2C6H5
7-ADCAb
7-ACA
7-ADCA
INHc
–NHC6H4–40-CO–7-ACA
–NHC6H4–40-CO–7-ADCA
–NHC6H4–40-CO–INH
–OCH2C6H5
–OCH2C6H5
H
H
H
a 7-ACA, 7-aminocephalosporanic acid.
b 7-ADCA, 3-deacetiloxy-7-aminocephalosporanic acid.
c INH, isoniazid.

3068
F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3065–3071
Table 2. 1H NMR data of derivatives C1–C20
Compound
1H NMR d (ppm)
C1a
1.39–1.42 (t, 3H, CH3); 4.37–4.39 (q, 2H, CH2); 5.20 (s, 2H, CH2); 6.98–7.17 (m, 2H, Ar); 7.43–7.45 (m, 5H, Ar); 7.64–7.66 (d,
1H, Ar, J = 8.8 Hz); 7.81–7.83 (d, 2H, Ar, J = 8.6 Hz); 8.06–8.08 (d, 2H, Ar, J = 8.6 Hz); 8.95 (s, 1H, CH@); 11.00 (s, 1H, NH,
D2O exch)
C2a
5.20 (s, 2H, CH2); 7.00 (s, 2H, Ar); 7.08–7.10 (d, 2H, Ar, J = 8.6 Hz); 7.39–7.43 (m, 6H, Ar); 7.64–7.66 (d, 2H, Ar, J = 8.9 Hz);
8.86 (s, 1H, CH@); 11.50 (br s, 1H, NH, D2O exch)
C3b
5.25–5.26 (d, 2H, CH2, J = 3.4 Hz); 7.15–7.25 (m, 4H, Ar); 7.35–7.41 (m, 4H, Ar); 7.46–7.48 (m, 4H, Ar); 8.71–8.72 (d, 1H,
CH@, J = 3.3 Hz); 11.32 (br s, 1H, NH, D2O exch)
C4b
1.55–1.57 (t, 3H, CH3); 4.50–4.51 (q, 2H, CH2); 5.30 (s, 2H, CH2); 7.10–7.19 (m, 5H, Ar); 7.30–7.60 (m, 6H, Ar); 8.86 (s, 1H,
CH@); 10.88 (s, 1H, NH, D2O exch)
C5b
5.25 (s, 2H, CH2); 7.05–7.11 (m, 3H, Ar); 7.42–7.51 (m, 6H, Ar); 7.75–7.81 (m, 2H, Ar); 8.60 (s, 1H, CH@); 10.95 (s, 1H, NH,
D2O exch)
C6b
5.30 (s, 2H, CH2); 7.03–7.12 (m, 3H, Ar); 7.47–7.53 (m, 6H, Ar); 7.80–7.87 (m, 2H, Ar); 8.72 (s, 1H, CH@); 10.89 (s, 1H, NH,
D2O exch)
C7a
1.38–1.42 (t, 3H, CH3); 4.33–4.39 (q, 2H, CH2); 7.26–7.28 (d, 1H, Ar, J = 8.6 Hz); 7.72–7.81 (m, 4H, Ar); 8.03–8.05 (d, 2H, Ar,
J = 8.6 Hz); 9.05 (s, 1H, CH@); 10.85 (s, 1H, NH, D2O exch)
C8b
7.58–7.61 (dd, 1H, Ar, J = 8.7 Hz); 7.80–7.85 (m, 3H, Ar); 7.94–7.97 (m, 2H, Ar); 8.14–8.15 (t, 1H, Ar); 8.84–8.85 (s, 1H,
CH@); 10.88 (s, 1H, NH, D2O exch); 13.85 (s, 1H, COOH, D2O exch)
C9b
7.52–7.54 (m, 1H, Ar); 7.77–7.80 (m, 3H, Ar); 7.90–7.92 (m, 3H, Ar); 8.10–8.12 (m, 1H, Ar); 8.82 (s, 1H, CH@); 10.96 (s, 1H,
NH, D2O exch)
C11b
C12b
C13b
C14b
C15a
C16b
C17b
C18b
C19b
C20b
7.51–7.53 (d, 1H, Ar, J = 7.8 Hz); 7.82–7.84 (d, 2H, Ar, J = 7.9 Hz); 7.90–7.96 (m, 3H, Ar); 8.26 (s, 1H, Ar); 8.86 (s, 1H,
CH@); 10.90 (s, 1H, NH, D2O exch); 12.82 (s, 1H, COOH, D2O exch)
7.51–7.54 (m, 1H, Ar); 7.82–7.85 (m, 2H, Ar); 7.90–7.97 (m, 3H, Ar); 8.26–8.27 (m, 2H, Ar); 8.82–8.83 (s, 1H, CH@); 10.92–
10.93 (d, 1H, NH, D2O exch J = 2.6 Hz)
2.05 (s, 3H, CH3); 3.75 (m, 2H, CH2); 4.90 (m, 2H, CH2); 5.10 (m, 2H, CH2); 5.30 (d, 1H, CH, J = 4.3); 6.05 (m, 1H, CH);
7.20–8.00 (m, 7H, Ar); 9.00 (s, 1H, CH@); 9.30 (s, 1H, NH, D2O exch); 13.10 (s, 1H, COOH, D2O exch)
2.00 (s, 3H, CH3); 3.70 (s, 2H, CH2); 5.20 (s, 2H, CH2); 5.25 (d, 1H, CH, J = 4.3); 5.90 (m, 1H, CH); 7.30–8.00 (m, 7H, Ar);
8.90 (s, 1H, CH@); 9.30 (s, 1H, NH, D2O exch); 13.15 (s, 1H, COOH, D2O exch)
2.15 (s, 3H, CH3); 3.60 (s, 2H, CH2); 5.05 (s, 2H, CH2); 5.10 (s, 2H, CH2); 5.20 (d, 1H, CH, J = 4.3); 6.20 (m, 1H, CH); 7.00–
7.70 (m, 8H, Ar); 8.90 (s, 1H, CH@); 9.60 (s, 1H, NH, D2O exch); 13.10 (s, 1H, COOH, D2O exch)
2.05 (s, 3H, CH3); 3.55 (s, 2H, CH2); 5.25 (s, 2H, CH2); 5.30 (d, 1H, CH, J = 4.3); 6.00 (m, 1H, CH); 7.20–8.00 (m, 8H, Ar);
8.95 (s, 1H, CH@); 9.35 (s, 1H, NH, D2O exch); 13.20 (s, 1H, COOH, D2O exch)
5.30 (s, 2H, CH2); 7.10–7.95 (m, 8H, Ar and 2H, Pyr); 8.75 (m, 2H, Pyr); 8.95 (s, 1H, CH@); 10.55 (s, 1H, NH, D2O exch);
11.35 (s, 1H, NH, D2O exch)
2.00 (s, 3H, CH3); 3.70 (s, 2H, CH2); 4.80 (s, 2H, CH2); 5.20 (d, 1H, CH, J = 4.3); 5.90 (m, 1H, CH); 7.50–8.00 (m, 8H, Ar);
8.95 (s, 1H, CH@); 9.40 (s, 1H, NH, D2O exch); 10.95 (s, 1H, NH, D2O exch); 13.2 (s, 1H, COOH, D2O exch)
1.95 (s, 3H, CH3); 3.60 (s, 2H, CH2); 5.10 (d, 1H, CH, J = 4.3); 5.80 (m, 1H, CH); 7.45–8.00 (m, 8H, Ar); 8.90 (s, 1H, CH@);
9.30 (s, 1H, NH, D2O exch); 10.85 (s, 1H, NH, D2O exch); 13.2 (s, 1H, COOH, D2O exch)
7.45–8.00 (m, 8H, Ar and 2H, Pyr); 8.75 (m, 2H, Pyr); 8.95 (s, 1H, CH@); 10.60 (s, 1H, NH, D2O exch); 10.80 (s, 1H, NH,
D2O exch); 10.90 (s, 1H, NH, D2O exch)
a CDCl3.
b DMSO-d6.
compounds against the metronidazole-resistant strains
of H. pylori.
Compounds C1–C6, 7-benzyloxy substituted, show low-
er activity, except for C4 derivative whose MIC was of
0.031 lg/mL against the NCTC 11637 strain. Com-
pounds C13–C20 show no activity.
The best activity (MIC values ranging between 0.0039
and 8 lg/mL) was observed for derivatives C7–C12
bearing an halogen (chlorine or bromine) in the 6-posi-
tion, a 3-benzamidic group and hydrogen atom in the
7-position of the coumarin moiety. All these compounds
show good activities against all strains and very good
activities against the metronidazole-resistant strains.
These values are very much lower in comparison with
those of metronidazole.
From these results we can point out that anti-H. pylori
activity strongly depends on the presence of an acyl
function like acid, ester or acyl chloride in the 3-benz-
amidic group and of an halogen in the 6-position of
the coumarin ring. The choice of the acyl functions
and particularly of the potentially unstable acyl chloride
as substituents in the 3-position of the coumarin ring

F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3065–3071
3069
Table 3. MIC of compounds C1–C20 and metronidazole (M) against 16 H. pylori strains, including three metronidazole-resistant strains
Compound
Range
All strains
Metronidazole resistant
MIC50 (lg/mL)
MIC90 (lg/mL)
NCTC 11637
5
14
C1
C2
0.5–64
0.5–4
0.5–4
8
64
4
4
64
4
32
4
2
2
C3
C4
2
4
32
2
4
32
4
<0.0039–32
2–16
1–8
4
0.031
8
16
8
C5
C6
8
16
8
4
4
4
4
8
C7
C8
<0.0039–16
<0.0039–2
<0.0039–0.25
<0.0039–8
<0.0039–0.125
<0.0039–0.125
32–128
0.25
0.0039
0.0078
0.5
0.015
0.0039
64
8
<0.0039
<0.0039
<0.0039
0.031
<0.0039
<0.0039
64
8
<0.0039
0.015
0.015
0.5
0.062
0.062
4
0.062
0.0078
2
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
M
0.125
0.031
128
128
128
128
>128
64
0.062
0.015
128
128
128
128
>128
64
0.015
<0.0039
128
16–128
16–128
32–128
64
64
32
64
64
64
64
32
64
>128
32
32–>128
8–128
4–>128
128
32
>128
32
32
128
128
0.25
>128
>128
64
>128
>128
64
>128
>128
64
16–>128
0.125–64
>128
64
Table 4. Cytotoxic effect of compounds C7–C12 tested on EAhy 926 cells after 24 h of incubation at 37 ꢁC, using Trypan blue exclusion test,
expressed as cell survival fraction (%)a
Compound
Concentration (lg/mL)b
50
5
0.5
0.05
0.005
C7
C8
10 0.5
10 0,8
15 0.5
30 2,2
20 1.6
10 1.1
30 1.2
12 0,8
22 1.4
70 2.3
37 3.2
31 1.4
60 2.0
45 3.0
58 4.0
70 2.8
70 3.8
56 4.2
77 4.4
68 3.4
80 6.4
87 3.5
90 5.2
70 4.4
80 5.5
79 6.2
78 5.8
92 4.0
90 3.8
85 5.6
C9
C10
C11
C12
a Cells incubated with culture medium alone represented the controls and the cell viability was always greater than 97%.
b Data represent the arithmetic mean SD of at least three independent experiments.
was admissible on the basis of our previous studies done
on the stability of similar compounds in the assay con-
References and notes
1
ditions by H NMR and HPLC techniques.16,17
1. Marshall, B. J.; Warren, J. R. Lancet 1984, 8390, 1311.
2. Peek, R. M., Jr.; Crabtree, J. E. J. Pathol. 2006, 208,
233.
3. NIH Consensus Development Panel on Helicobacter
pylori in peptic ulcer disease. J. Am. Med. Assoc. 1994,
272, 65.
4. Candelli, M.; Nista, E. C.; Carloni, E.; Pignataro, G.;
Zocco, M. A.; Cazzato, A.; Di Campli, C.; Fini, L.;
Gasbarrini, G.; Gasbarrini, A. Curr. Med. Chem. 2005, 12,
375.
5. McLoughlin, R. M.; O’Morain, C. A.; O’Connor, H.
J. Fundam. Clin. Pharmacol. 2005, 19, 421.
6. Kawase, M.; Motohashi, N. Curr. Med. Chem.—Anti-
Infective Agents 2004, 3, 89.
If a benzyloxy group is present in the 7-position the
activity decreases. Finally, the presence of bulky substit-
uents like 7-ACA, 7-ADCA or INH leads to inactive
compounds.
The cytotoxic profile of the compounds C7–C12, the
most active agents against H. pylori, (Table 4) showed
that the derivatives C10 and C11 were less toxic at con-
centrations below 0.5 lg/mL with percentage of viable
cells remaining of 87 and 90, respectively.18
7. Finn, G. J.; Kenealy, E.; Creaven, B. S.; Egan, D. A.
Cancer Lett. 2002, 183, 61.
8. Kawase, M.; Varu, B.; Shah, A.; Motohashi, N.; Tani, S.;
Saito, S.; Debnath, S.; Mahapatra, S.; Dastidar, S. G.;
Chakrabarty, A. N. Arzneimittelforschung/Drug Res. 2001,
51, 67.
9. Chimenti, F.; Bizzarri, B.; Manna, F.; Bolasco, A.; Secci,
D.; Chimenti, P.; Granese, A.; Rivanera, D.; Lilli, D.;
Scaltrito, M. M.; Brenciaglia, M. I. Bioorg. Med. Chem.
Lett. 2005, 15, 603.
Acknowledgments
This work was supported by grants from MURST. We
also acknowledge Mr. Anton Gerada, a professional
translator, Fellow of the Institute of Translation and
Interpreting of London and Member of AIIC (Associa-
ˆ
tion Internationale des Interpretes de Conferences—
Geneva) for the revision of the manuscript.
´

3070
F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3065–3071
10. Chimenti, F.; Bizzarri, B.; Bolasco, A.; Secci, D.; Chim-
enti, P.; Carradori, S.; Granese, A.; Rivanera, D.; Lilli, D.;
Scaltrito, M. M.; Brenciaglia, M. I. Eur. J. Med. Chem.
2006, 41, 208.
11. Kobayashi, I.; Muraoka, H.; Hasegawa, M.; Saika, T.;
Nishida, M.; Kawamura, M.; Ando, R. J. Antimicrob.
Chemother. 2002, 50, 129.
12. Zhang, Y.; Chen, B.; Zheng, K.; Xu, M.; Zhang, L.; Lei,
X. Yaoxue Xuebao 1982, 17, 17.
13. Yoshida, Y.; Matsuda, K.; Sasaki, H.; Matsumoto, Y.;
Matsumoto, S.; Tawara, S.; Takasugi, H. Bioorg. Med.
Chem. Lett. 2004, 14, 2627.
14. General procedure for the preparation of 2-oxo-2H-chro-
mene-3-acyl chloride (A). The appropriate salicylaldehyde
(5 g) in EtOH (90 mL) is refluxed under magnetic stirring
with diethyl malonate and catalytic amounts of piperidine
for 24 h. After cooling to room temperature, the solution
is filtered to give the desired 2-oxo-2H-chromene-3-
carboxylic acid ethyl ester. After hydrolysis with NaOH
20% (100 mL) and addition of hydrochloric acid 4 N, the
solution is filtered to obtain the desired 2-oxo-2H-
chromene-3-carboxylic acid. The 2-oxo-2H-chromene-3-
carboxylic acid (0.05 mol) is refluxed for 3 h at 100 ꢁC
under magnetic stirring with thionyl chloride (0.5 mol).
After cooling to room temperature, the solution is filtered
to give the desired compound A.
defined as the lowest concentration of anti-bacterial agent
that completely inhibited the growth of the organism, as
detected by the unaided eye. The in vitro anti-fungal
activities of the compounds were determined with the
broth micro dilution method with Sabouraud dextrose
broth (BBL Microbiology Systems, Cockeysville, MD) as
recommended by the NCCLS.20 Microtiter plates con-
taining serial dilutions of each compound were inoculated
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 anti-fungal agent that com-
pletely inhibited growth of the organism, as detected by
the unaided eye.
Anti-H. pylori activity. Fifteen clinical H. pylori strain
isolates and the reference strain NCTC 11637 were used.
Three of these strains were metronidazole resistant. They
were maintained at ꢀ80 ꢁC in Wilkins Chalgren with 10%
(v/v) horse serum (Seromed) and 20% (v/v) glycerol
(Merck) until they could be used for the experiments.
The bacteria were grown on Columbia agar base (Difco
Laboratories) supplemented with 10% horse serum (Ser-
omed) and 0.25% Bacto yeast extract (Difco) incubated
for 72 h at 37 ꢁC under microaerobic conditions (10%
CO2) in a gas incubator (Haereus). Before use, the media
were always preincubated under the same microaerobic
conditions for a minimum 2 h to allow equilibration, and
none of the cultures were kept in the air for more than
15 min. The MICs were determined by the agar dilution
standard method21 incubating the bacteria at microaero-
bic conditions. By serial double dilutions, they were
diluted in agar medium to give concentrations ranging
from 128 to 0.0039 lg/mL. The plates of Columbia agar
with horse serum and yeast extract containing anti-
microbial agents were prepared on the day they were
used. The inoculum was prepared as follows: a 72 h
growth of each strain on agar plates was suspended in
Wilkins Chalgren broth (Difco) at a turbidity equivalent
to the 0.5 McFarland standard. The plates were inoculated
General procedure for the synthesis of 2-oxo-2H-chromene-
3-carboxamide derivatives C1–C20. A solution of the
appropriate amine derivatives (0.0072 mol) in the suitable
solvent (30 mL) is added dropwise to a solution of the
appropriate 2-oxo-2H-chromene-3-acyl chloride (A)
(0.0072 mol) or 4-[(2-oxo-2H-chromene-3-carbonyl)-ami-
no]-benzoyl chloride (B) (0.0072 mol) in the same solvent
(30 mL) and triethylamine (1 mL). The reaction mixture is
stirred for 96 h at reflux, then cooled to room temperature
and filtered off to remove triethylammonium salt. The
organic layer is concentrated under vacuum, cooled to
4 ꢁC to give the desired compounds. Chemical and
physical data of synthesized compounds and 1H NMR
spectra are reported in Tables 1 and 2.
15. Anti-bacterial and anti-fungal activity. All synthesized
derivatives C1–C20 were evaluated for their anti-microbial
and anti-fungal activity dissolved in dimethylsulfoxide
(DMSO). Organisms from routine clinical Gram-positive
(Staphylococcus aureus, Staphylococcus epidermidis, Staph-
ylococcus hominis, Staphylococcus warneri, Streptococcus
a-hemolyiticus, Streptococcus faecalis) and Gram-negative
isolates (Escherichia coli, Klebsiella pneumoniae, Klebsiella
oxytoca, Enterobacter spp., Enterobacter aerogenes, Citro-
bacter freundii, Proteus vulgaris) and four Candida strain
using
a multipoint inoculator (Denley A 400 PBI)
dispensing 5 lL and incubated at 37 ꢁC for 72 h under
microaerobic conditions (10% CO2 in a gas incubator).
The MIC was defined as the lowest concentration capable
of inhibiting any visible bacterial growth.
16. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.;
Granese, A.; Befani, O.; Turini, P.; Alcaro, S.; Ortuso,
F. Bioorg. Med. Chem. Lett. 2004, 14, 3697.
17. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.;
Granese, A.; Gagliardi, L.; De Orsi, D. Chromatographia
2005, 61, 519.
`
isolates (C. albicans, C. sake, C. krusei) from the respira-
tory tract were collected from specimens of patients at the
Hospital ‘Azienda Policlinico Umberto Iꢁ’ of Rome ‘La
Sapienza’ University. The isolates were subcultured on a
qualified medium to ensure purity. The isolates were
identified by conventional methodologies; all isolates were
subcultured to ensure optimal growth. The in vitro anti-
bacterial activities of the compounds were determined
with the broth micro dilution method, as recommended by
the National Committee for Clinical Laboratory Stan-
dards19 with Mueller–Hinton II broth (BBL Microbiology
Systems, Cockeysville, MD). Microtiter plates containing
serial dilutions of each compound ranging from 128 to
0.5 lg/mL were inoculated with each organism to yield the
appropriate density (105/mL) in a 100 lL final volume;
each plate included positive controls (bacteria without a
compound), and a negative control (medium only). The
plates were incubated for 18 to 22 h at 35 ꢁC. The Minimal
Inhibitory Concentration (MIC) for all isolates was
18. In vitro cytotoxicity. The cytotoxicity of the newly
synthesized compounds under investigation was tested
against EAhy, human cell line obtained from a hybridoma
between HUVEC cells and epithelial cells from a lung
carcinoma. The viability of cells exposed to test com-
pounds was estimated by the Trypan blue dye exclusion
assay. Cell lines were maintained as adherent type cultures
under humidified atmosphere in 5% CO2 at 37 ꢁC,
Dulbecco’s modified Eagle’s culture medium (high glu-
cose) supplemented with 2 mM L-Glutamine, HAT sup-
plement and containing antibiotic mixture. Experiments
were performed in cells grown to 60–70% confluency.22
The stock solutions of the investigated compounds were
prepared in sterile dimethylsulfoxide (DMSO) and the
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

F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3065–3071
3071
(1 · 105/mL) were seeded into 24-well microplate and
incubated for 24 h with five different concentrations of the
compounds (50–0.005 lg/mL). Some plates containing
cells alone or cells and DMSO represented the controls.
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 rt cells were counted under an optical micro-
scope in a Thoma hemocytometer chamber by two
different operators. On the basis that Trypan blue is a
vital dye23 and can enter and interact with the cells unless
the plasmatic membrane is damaged, blue stained cells
were considered as died. Values are expressed as % of
viable cells. Cell viability in control samples was always
97–98%.
bility tests for bacteria that grow aerobically. Approved
standard M7-A6. National Committee for Clinical Lab-
oratory Standards, Wayne, PA.
20. National Committee for Clinical Laboratory Standards
2003. Performance standards for antimicrobial suscepti-
bility testing. Supplement M100-S13. National Committee
for Clinical Laboratory Standards, Wayne, PA.
21. National Committee for Clinical Laboratory Standards
2004. Methods for Antimicrobial Susceptibility Testing of
Anaerobic bacteria. Approved standard M11-A6. 6th ed.
National Committee for Clinical Laboratory Standards,
Villanova, PA.
22. Edgell, C. J. S.; McDonald, C. C.; Graham, J. B. Proc.
Natl. Acad. Sci. U.S.A. 1983, 80, 3734.
23. Freshney, R. In Culture of animal cells: a manual of
basic technique; Alan R. Liss, Ed.; New York, 1987; p
117.
19. National Committee for Clinical Laboratory Standards
2002/2003. Methods for dilution antimicrobial suscepti-