Anti-Helicobacter pylori activities of selected N-substituted cinnamamide derivatives evaluated on reference and clinical bacterial strains

Article abstract of DOI:10.1038/s41429-018-0027-1

In this study, thirty-five N-substituted derivatives of cinnamic acid amide (cinnamamide) were evaluated for anti-Helicobacter pylori activity using an agar disc-diffusion method. Qualitative screening was performed on a reference H. pylori strain (ATCC 43504), resulting in the identification of the three most active compounds, 8 (R,S-(2E)-3-(4-chlorophenyl)-N-(2-hydroxypropyl)prop-2-enamide, minimal inhibitory concentration, MIC = 7.5 μg/mL), 23 ((2E)-3-(4-chlorophenyl)-N-(2-hydroxycyclohexyl)prop-2-enamide, MIC = 10 μg/mL), and 28 ((2E)-3-(4-chlorophenyl)-N-(4-oxocyclohexyl)prop-2-enamide, MIC = 10 μg/mL). These compounds were further tested on twelve well-characterized clinical strains, yielding MIC values that ranged from 10 to 1000 μg/mL. Preliminary safety assessments of the compounds were made using the MTT viability test for cytotoxicity and Ames test for mutagenicity, which showed them to be generally safe, although compounds 8 and 28 showed mutagenic activity at some of the tested concentrations. The results of this study showed the anti-H. pylori potential of cinnamamide derivatives.

Full text of DOI:10.1038/s41429-018-0027-1

The Journal of Antibiotics
https://doi.org/10.1038/s41429-018-0027-1
BRIEF COMMUNICATION
Anti-Helicobacter pylori activities of selected N-substituted
cinnamamide derivatives evaluated on reference and clinical
bacterial strains
Karolina Klesiewicz1 Elżbieta Karczewska1 Paweł Nowak1 Iwona Skiba-Kurek1 Edward Sito2
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Katarzyna Pańczyk3 Paulina Koczurkiewicz4 Dorota Żelaszczyk3 Elżbieta Pękala4 Anna M. Waszkielewicz3
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Alicja Budak1 Henryk Marona3 Agnieszka Gunia-Krzyżak3
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Received: 24 August 2017 / Revised: 19 December 2017 / Accepted: 28 December 2017
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2018
Abstract
In this study, thirty-five N-substituted derivatives of cinnamic acid amide (cinnamamide) were evaluated for anti-
Helicobacter pylori activity using an agar disc-diffusion method. Qualitative screening was performed on a reference H.
pylori strain (ATCC 43504), resulting in the identification of the three most active compounds, 8 (R,S-(2E)-3-(4-
chlorophenyl)-N-(2-hydroxypropyl)prop-2-enamide, minimal inhibitory concentration, MIC = 7.5 µg/mL), 23 ((2E)-3-(4-
chlorophenyl)-N-(2-hydroxycyclohexyl)prop-2-enamide, MIC = 10 µg/mL), and 28 ((2E)-3-(4-chlorophenyl)-N-(4-oxocy-
clohexyl)prop-2-enamide, MIC = 10 µg/mL). These compounds were further tested on twelve well-characterized clinical
strains, yielding MIC values that ranged from 10 to 1000 µg/mL. Preliminary safety assessments of the compounds were
made using the MTT viability test for cytotoxicity and Ames test for mutagenicity, which showed them to be generally safe,
although compounds 8 and 28 showed mutagenic activity at some of the tested concentrations. The results of this study
showed the anti-H. pylori potential of cinnamamide derivatives.
Helicobacter pylori is a gram-negative, spiral shaped bac-
terium that inhabits the human gastric mucosa. H. pylori is
widespread in humans, with an estimated 50% of the
world’s population infected [1]. H. pylori is an etiological
agent of type B gastritis and is one of the pathogenic factors
that causes a predisposition to the development peptic ulcer
disease [2]. Moreover, according to The International
Agency for Research on Cancer of the World Health
Organization (IARC/WHO), since 1994, H. pylori has been
classified as a first class carcinogen, promoting the devel-
opment of gastric cancer and mucosa associated lymphoid
tissue (MALT) lymphoma [3].
Eradication of H. pylori infections plays an essential role
in overcoming gastric diseases. The recommendations for
the treatment of H. pylori infection, which were issued in
2016, include the use of three types of drugs, including
antisecretory (proton pump inhibitors (PPI)), cytoprotectant
(bismuth salts) and antibacterial drugs (clarithromycin,
amoxicillin, metronidazole, tetracycline, levofloxacin, and
rifabutin). Treatment regimens include triple therapy, con-
sisting of PPI and two antibiotics/chemotherapeutics men-
tioned above, or quadruple therapy, consisting of PPI,
metronidazole, tetracycline, and bismuth salt. Quadruple
therapy is especially recommended in regions with high
clarithromycin resistance [4].
* Agnieszka Gunia-Krzyżak
agnieszka.gunia@uj.edu.pl
1
Department of Pharmaceutical Microbiology, Faculty of
Pharmacy, Jagiellonian University Medical College, Medyczna 9,
Kraków, Poland
2
Falck Medycyna Outpatient Clinic of Gastroenterology,
Mazowiecka 4-6, Kraków, Poland
H. pylori eradication therapy failure is caused by resis-
tance of H. pylori to antibiotics and chemotherapeutics used
in empirical treatment, especially to clarithromycin, which
is conferred by the presence of point mutations in domain V
of the 23S rRNA gene, primarily A2143G, A2142G,
A2142C [5]. Thus, clarithromycin is not recommended if
3
Department od Bioorganic Chemistry, Chair of Organic
Chemistry, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9, Kraków, Poland
4
Department of Pharmaceutical Biochemistry, Faculty of
Pharmacy, Jagiellonian University Medical College, Medyczna 9,
Kraków, Poland

K. Klesiewicz et al.
the resistance level is above 15%, and its use is only
allowed after the exclusion of resistance using micro-
biological or genetic diagnostic methods [4]. For example,
in Poland, where the H. pylori clarithromycin resistance is
higher than 20%, this drug should not be used in empirical
treatments [6]. The ‘Kyoto global consensus report on
Helicobacter pylori gastritis’ recommended eradication
therapy in cases of confirmed H. pylori infection (especially
cagA positive strains), despite the absence of inflammatory
changes in the gastric mucosa to avoid the development of
inflammatory diseases and gastric cancer [7].
Currently, H. pylori eradications have diminished
because of the increasing resistance of H. pylori strains to
currently available antibiotics. The identification of novel
compounds that are active against H. pylori can lead to the
development of new alternative drugs to currently used
antibiotics and chemotherapeutics. Therefore, the search for
new anti-H. pylori agents is crucial to be able to conduct
effective therapy in the future.
Cinnamic acid and its derivatives, such as cinnamamides
and cinnamates, have been previously identified as potent
antimicrobial agents. Multiple studies have reported on their
antibacterial, antifungal, and antimycobacterial activities
[8]. However, there are only few published studies on the
anti-H. pylori activity of this group of compounds, and
moreover, the results primarily concerned plant extracts or
cinnamic acid itself rather than its synthetic derivatives [9].
Knowing their antimicrobial potential, we synthesized and
evaluated the anti-H. pylori activities of a series of N-sub-
stituted amide derivatives of cinnamic acid (cinnamamides).
The chemical structures of the tested compounds are
shown in Table 1. The compounds were synthesized by N-
acylation of the appropriate amine using (E)-cinnamoyl
chloride or (E)-4-chlorocinnamic acid chloride. The reac-
tions were carried out in a two-phase system (toluene/water/
K2CO3) or reagents were refluxed in toluene with anh.
K2CO3. Some of the obtained derivatives were subjected to
further oxidation of the hydroxyl group in N-substituent
using Dess-Martin periodinane. The purity and chemical
structures of the final compounds were confirmed by means
of high-performance liquid chromatography, elemental
analysis, and spectral methods (LC-MS, 1H NMR). All
synthesized compounds had the E configuration of the
double bond. The physicochemical properties of com-
pounds 1-22, 24-26, 29, and 31 were published previously
[10–13], while new compounds (23, 27, 28, 30, 32–35)
were characterized for the purpose of current study.
involved using the stock solution (1%) against the reference
H. pylori strain ATCC 43504. An inoculum of 3.0
McFarland standards in a sterile 0.85% NaCl solution was
prepared from a pure bacterial culture grown for 72 h. The
inoculum was spread onto Schaedler agar with 5% sheep
blood and then disc that was impregnated with 15 µL of the
stock solution of the compound being tested was placed
onto the plate. After 72 h of incubation under a micro-
aerophilic atmosphere at 37 °C, the diameter of the zone of
growth inhibition was measured. If the zone of growth
inhibition was ≤8 mm, the compound was recorded as
having no antibacterial activity. If the zone was >8 mm, the
compound was recorded as possessing antibacterial activ-
ity (Table 1).
In the second step, nine compounds with zones of growth
inhibition of >27 mm were observed as the most active
compounds against the reference H. pylori strain ATCC
43504 and were further quantitatively evaluated. To obtain
MIC values, the disc diffusion method was performed using
12 discs that were impregnated with decreasing concentra-
tions of a given compound. The MIC value was determined
as the lowest concentration of the tested compound that
inhibited bacterial growth for zones of inhibition that were
>8 mm (Table 1).
To ensure quality control of the disc diffusion method,
we conducted a quantitative assay to obtain the MIC value
for metronidazole against an H. pylori reference strain.
Additionally, the MIC value for metronidazole was assayed
by a reference method using strips impregnated with an
antibiotic gradient (E-test).
The investigated group of cinnamamide derivatives
possessed various N-substituents, including hydroxyalkyl,
hydroxycycloalkyl, carboxyalkyl, metoxycarbonylalkyl,
oxocycloalkyl, acetylhydroxyalkyl or arylalkyl moieties.
Moreover, in some compounds, the nitrogen atom of the
amide group was incorporated into a ring (piperidine or
piperazine). Another assayed modification was a 4-chloro
substitution in the phenyl ring.
The most active compounds identified were R,S-(2E)-3-
(4-chlorophenyl)-N-(2-hydroxypropyl)prop-2-enamide (8,
MIC = 7.5 µg/mL), (2E)-3-(4-chlorophenyl)-N-(2-hydro-
xycyclohexyl)prop-2-enamide (23, MIC = 10 µg/mL), and
(2E)-3-(4-chlorophenyl)-N-(4-oxocyclohexyl)prop-2-enam-
ide (28, MIC = 10 µg/mL), which significantly differed in
their N-substituent but all possessed a chlorine atom in their
phenyl rings. Indeed, for the majority of the compounds
tested, the introduction of a 4-chloro substituent in the
phenyl ring increased the anti-H. pylori activity, although
not in every case. Thus, the relationship between the 4-
chloro substitution and activity is unclear, as the obtained
results were surprising and a structure-activity relationship
could not be identified. Meaningful zones of bacterial
growth inhibition were observed for various derivatives,
The efficacy of the new potential antibacterial com-
pounds was assessed by determining minimal inhibitory
concentration (MIC) values using the disc-diffusion method
[14]. Tested compound were diluted in DMSO to obtain a
stock solution (1000 mg/L, 1%), from which 12 dilutions
were prepared in water. The first step of the screening

Anti-H. pylori activities of cinnamamide derivatives
Table 1 Chemical structures of the evaluated compounds and the
results of their anti-H. pylori activity screening using the reference
strain ATCC 43504
including those possessing in N-substituent moieties such as
hydroxyalkyl (compounds 2, 5, 8, 10, 14, 19), hydro-
xycycloalkyl (compound 23), and oxocycloalkyl (com-
pound 28), as well as compounds with the nitrogen atom of
the amide group incorporated into a ring (32, 35). One of
the three O-acetylated derivative (24) was more active than
the parent compound (22), while the remaining two were
not active (3 and 6), similar to their parental compounds (1
and 4, respectively). Interestingly, some similar compounds
significantly differed in their activities, e.g., 12 vs. other
hydroxyalkyl derivatives, 26 vs. 28, and 33 vs. 35. To
summarize, the described screening procedure is a useful
method to identify anti-Helicobacter pylori compounds of
various chemical compositions.
Compound R1
R2
Configuration Diameter
MIC
of growth value
inhibition (μg/mL)
(mm)
12
1
H
R,S
2
3
Cl
H
R,S
R,S
27
13
1000
200
4
H
R,S
R,S
R,S
12
54
11
5
6
Cl
H
Among the assayed compounds, the three with the lowest
MIC values (8, 23, and 28) were chosen for further eva-
luation using two additional H. pylori reference strains and
twelve clinical strains isolated from infected patients who
underwent gastroscopies at the Falck Outpatient Clinic in
Krakow, Poland. This study was approved by the Bioethical
Commission of the Jagiellonian University (Krakow,
Poland) (Approval no. 122.6120.273.2015), and each
patient signed an informed consent document. The clinical
strains were characterized by phenotypic and genotypic
studies. The presence of H. pylori in the collected samples
was confirmed by a positive test for urease, catalase, and
oxidase and the presence of spiral-shaped bacteria in gram-
stain slides. Antimicrobial susceptibility testing was con-
ducted according to the EUCAST recommendation for
clarithromycin, metronidazole, levofloxacin, tetracycline,
and amoxicillin using strips impregnated with the antibiotic/
chemotherapeutic gradient to obtain MIC values [15].
Quality control was ensured using the H. pylori reference
strain ATCC 43504. For the genotypic studies, isolation of
genomic DNA was performed using a Sherlock AX isola-
tion kit according to the manufacturer’s protocol. In the 16 S
rRNA-based identification, PCR reaction contained 2 μL of
bacterial DNA, 2 μL of each primer (HP-1 and HP-2, 5 μL
of GoTaq® Flexi Buffer, 1.5 μL of MgCl2, 0.5 μL of PCR
Nucleotide Mix, 0.125 μL of GoTaq® Flexi DNA Poly-
merase, and 25 μL of Nuclease-Free Water [16]. Genomic
DNA extracted from H. pylori strains was used for PCR-
based genotyping of the ureB, cagA, and vacA genes [17],
using HPU 50 and HPU 25 primers, D008 and R008 pri-
mers, and VAC3624F and VAC3853R primers, respec-
tively. Finally, to detect the most frequently occurring point
mutations that account for H. pylori clarithromycin resis-
tance (A2143G and A2142G), PCR followed by an RFLP
analysis was performed. The 425-bp fragment of the pep-
tidyl transferase region of the 23 S rRNA was amplified
with the primers K1 and K2 [18].
7
H
R,S
R,S
R,S
R,S
-
11
27
13
37
13
17
13
8
Cl
H
7.5
75
9
10
11
12
13
Cl
H
Cl
H
-
R,S
14
15
Cl
H
R,S
R,S
31
12
16
H
H
R,S
-
13
17
13
18
19
20
H
-
15
26
13
Cl
H
-
R,S
21
Cl
R,S
13
22
23
24
H
trans, D,L
trans, D,L
trans, D,L
19
34
38
Cl
H
10
200
25
26
H
trans
trans
13
16
Cl
27
H
-
13
28
29
30
31
32
33
Cl
H
-
27
14
25
11
43
18
10
-
Cl
H
-
R,S
R,S
-
Cl
H
150
500
34
35
Cl
H
-
-
8
44
The results of the phenotypic and genotypic identifica-
tion of clinical strains are presented in Table 2, together
The anti-H. pylori activity results are expressed as the diameter of the
zone of bacterial growth inhibition and MIC values

K. Klesiewicz et al.

Anti-H. pylori activities of cinnamamide derivatives
with the anti-H. pylori activity results for the investigated
compounds. The MIC values obtained for the clinical
strains ranged from 10 to 1000 µg/mL. To compare the
activities of the tested compounds, we calculated MIC50 and
MIC90 values, which are the minimal inhibitory con-
centrations observed for 50 and 90% of the tested strains,
respectively. For compound 8, the MIC50 = 150 µg/mL and
Acknowledgements This work was supported by Jagiellonian Uni-
versity Medical College in Kraków (Grants K/DSC/004292, K/ZDS/
005487, K/DSC/003549), the Polish National Science Center, decision
on grant no. DEC-2013/11/B/NZ7/04834, and by a research grant
funded by the Polish Government in the years 2011-2013
(NN404547640). Salmonella typhimurium strains TA98 and TA102
were kindly provided by Dr T Nohmi (Division of Genetics and
Mutagenesis, National Institute of Health Sciences, Tokyo, Japan).
the MIC90 = 200 µg/mL. For compound 23, the MIC50
=
Compliance with ethical standards
150 µg/mL and the MIC90 = 500 µg/mL. Lastly, for com-
pound 28, the MIC50 = 200 µg/mL and the MIC90 = 1000
µg/mL. These values suggest that compound 8 possesses the
highest anti-H. pylori potential among the tested
derivatives.
Conflict of interest The authors declare that they have no conflict of
interest.
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For compounds 8, 23, and 28, which were identified
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