A small library of 4-(alkylamino)-3-nitrocoumarin derivatives with potent antimicrobial activity against gastrointestinal pathogens

Article abstract of DOI:10.2298/JSC140619085R

Abstract: Due to a confirmed antimicrobial activity of both natural and synthetic coumarin derivatives, the present study was envisaged to provide a further insight into the antimicrobial potential of coumarins through a screening of a designed library of nine 4-(alkylamino)-3-nitrocoumarins against a panel of 24 laboratory strains and resistant (isolates) bacterial and fungal pathogens. All compounds showed some degree of strain-selective activity, that was, in some cases, very pronounced, reaching the value of 0.04 nmol/ml (i.e. 12 ng/ml) for the minimal inhibitory concentration against Candida albicans. The observed activity was higher against Gram-negative strains, among which the most susceptible strain, among both ATCC strains and clinical isolates, was Salmonella enteritidis. These results point out to a high potential of these coumarins as antimicrobials for the treatment of gastrointestinal and other infections caused by highly resistant microbial strains. Finally, a multivariate statistical analysis of the herein obtained and previous results on the antimicrobial activity of related selected coumarins was performed to allow an easier structure-activity discussion.

Full text of DOI:10.2298/JSC140619085R

J. Serb. Chem. Soc. 80 (3) 315–327 (2015)
UDC *Salmonella enterica:547.587.51+
JSCS–4718
547.21.024:579.86:616.34:615.282
Original scientific paper
A small library of 4-(alkylamino)-3-nitrocoumarin derivatives
with potent antimicrobial activity against gastrointestinal
pathogens
1
#
2
NIKO S. RADULOVIĆ * , ZORICA STOJANOVIĆ-RADIĆ ,
3
,4
3
PREDRAG STOJANOVIĆ , NIKOLA STOJANOVIĆ ,
5
#
5#
VIDOSLAV DEKIĆ and BILJANA DEKIĆ
1
Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Više-
2
gradska 33, 18000 Niš, Serbia, Department of Biology and Ecology, Faculty of Science and
Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia, Faculty of Medicine,
University of Niš, Zorana Đinđića 81, 18000 Niš, Serbia, Institute For Public Health Niš,
Center of Microbiology, 18000 Niš, Serbia and Department of Chemistry, Faculty of Science
3
4
5
and Mathematics, University of Priština, Lole Ribara 29, 38220 Kosovska Mitrovica, Serbia
(Received 19 June, revised 22 August, accepted 29 August 2014)
Abstract: Due to the confirmed antimicrobial activity of both natural and syn-
thetic coumarin derivatives, the present study was envisaged to provide further
insight into the antimicrobial potential of coumarins through the screening of a
designed library of nine 4-(alkylamino)-3-nitrocoumarins against a panel of 24
laboratory strains and resistant (isolates) bacterial and fungal pathogens. All
compounds showed some degree of strain-selective activity that in some cases
was very pronounced, reaching the value of 0.04 nmol mL- (i.e., 12 ng mL )
for the minimal inhibitory concentration against Candida albicans. The obs-
erved activities were higher against Gram-negative strains, among which the
most susceptible strain, among both ATCC strains and clinical isolates, was
Salmonella enterica subsp. enterica serovar Enteritidis. These results indicate
to a high potential of these coumarins as antimicrobials for the treatment of
gastrointestinal and other infections caused by highly resistant microbial
strains. Finally, a multivariate statistical analysis of the herein obtained and
previous results on the antimicrobial activity of related selected coumarins was
performed to allow an easier structure–activity discussion.
1
-1
Keywords: 4-(alkylamino)coumarins; antimicrobial activity; Candida albicans;
multivariate statistical analysis; Salmonella enterica.
*
Corresponding author. E-mail: nikoradulovic@yahoo.com
Serbian Chemical Society member.
#
doi: 10.2298/JSC140619085R
3
15
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3
16
RADULOVIĆ et al.
INTRODUCTION
The gastrointestinal (GI) tract represents an ideal place for the development
of various microorganisms that can affect the (human) organism in both bene-
ficial and adverse aspects, such as protection against infection, physiological
functions, activation of the immune system, carcinogenesis, aging and alteration
1
of the effect a therapeutic drug should have. GI bacterial infections, which can
range from mild (with few symptoms) to severe life-threatening disorders (e.g.,
hemorrhagic colitis, intestine perforation and kidney failure) are one of the most
frequent readons for patients to turn to physicians (one third of the people living
2
in industrialized countries per year). GI infections are mostly food-borne and
result from inadequate cooking and inadequate storage of food, while some com-
mensal bacteria can cause infections in immunocompromised patients (AIDS,
3
cancer, etc.). Hence, the search for new antimicrobial agents, renewed by the
rapid and alarming development of multiresistant bacterial and fungal pathogens,
has always been one of the most significant tasks of many medicinal chemists.
Coumarins are a widespread group of natural compounds present in the
seeds, roots and leaves of many plant species. Their function in the plant orga-
nism is far from clear, although some authors believe that they have growth
4
regulatory functions, or that they act as fungistatic and bacteriostatic agents.
This is substantiated by the extensive use of coumarin compound-containing her-
bal remedies in the traditional medicine of many nations due to their broad spec-
5–7
trum of pharmacological activities, including antibacterial and antifungal effects.
Significant antimicrobial activity of a series of 4-aryl-substituted 3-nitrocou-
8
marins was recently demonstrated by Debeljak and co-workers. In connection
with this, several arylamino- and (heteroarylamino)-3-nitrocoumarin derivatives
9
–14
were synthesized and their antimicrobial activity determined.
A number of
these compounds showed strong activity in reducing the microbial growth com-
9
,13,14
parable or even better than the activity of standard antibiotics.
-(1-naphthylamino)-3-nitrocoumarin possessed the strongest antimicrobial
potential as demonstrated by its action against Salmonella enterica in a low dose
Among them
4
1
5
of 9 µg per disc.
Additional fine-tuning of the antimicrobial activity of these coumarin deri-
vatives could be accomplished by the introduction of an alkyl chain instead of the
aromatic substituent on the nitrogen atom in position 4. In this way, the nucleo-
philicity and basicity would be increased that could lead to a stronger binding
with potential biological target molecules. The previous statements and the fact
that hitherto only the disc diffusion method was used to evaluate the activity of
the synthesized coumarin derivatives, it was decided to synthesize a small library
of coumarin derivatives possessing a 4-(alkylamino)-substituent on the 3-nitro-
coumarin parent structure and screen the compounds for their in vitro antimic-
robial activity against twenty four bacterial and fungal strains (both ATCC and
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NITROCOUMARIN DERIVATIVES WITH POTENT ANTIMICROBIAL ACTIVITY
317
clinical isolates, including three important GI pathogens Yersinia enterocolitica,
Salmonella enterica subsp. enterica serovar Enteritidis and Shigella flexneri)
using a microdilution method. Finally, another task of this work was to compare
previously published results concerning the antimicrobial activity of related cou-
marin derivatives and the herein obtained results using multivariate statistical
analysis with the aim of finding possible structure–activity relationships.
EXPERIMENTAL
General
The melting points were determined on a Kofler hot-plate apparatus and are uncorrected.
The HRMS(EI) spectra were recorded on a JEOL Mstation JMS 700 instrument (JEOL,
Germany). The IR measurements (ATR – attenuated total reflectance) were performed using a
Thermo Nicolet model 6700 FTIR instrument. The NMR spectra were recorded on a Varian
1
13
Gemini 200 spectrometer ( H-NMR at 200 MHz and C-NMR at 50 MHz), using DMSO-d6
as the solvent. The chemical shifts are expressed in δ (ppm) using TMS (Me Si) as the
4
internal standard. Microanalyses of carbon, hydrogen and nitrogen were performed with a
Carlo Erba 1106 microanalyser; the results agreed favorably with the calculated values. For
TLC, silica gel plates (Kieselgel 60 F254, Merck, Germany) were used. Visualization was
affected by spraying the plates with 1:1 (V/V) aqueous solution of sulfuric acid and then
heating. All the reagents and solvents were obtained from commercial sources (Aldrich, USA;
Merck, Germany; Fluka, Germany) and used as received, except for the solvents that were
purified by distillation.
Mass spectrometry
EI-MS and HRMS analyses were performed using a JEOL MStation JMS-700 mass
spectrometer with an ionization energy of 70 eV, an ionization trap current of 300 μA and a
source temperature of 230 °C. Perfluorokerosene (Sigma–Aldrich, USA) was used as the
internal mass reference in the HRMS studies. The conversion of the mass reference list to a
calibration was performed by the data system of the mass spectrometer. The corresponding
range of mass measurements was set to include two standard peaks that encompassed the
sample peak of interest. The mass resolution and scan speed used were 30,000 (10 % valley)
-
1
and 60 s decade , respectively. The accurate mass was calculated as the average of the values
+
•
measured in 5–10 scans, determined from the mass centroids of M and the other peaks. The
error for each elemental composition data is given in units of mmu as calculated using the
program installed in the data system.
Synthesis of 4-chloro-3-nitrocoumarin (3)
4
-Hydroxycoumarin (1) was nitrated using 72 % HNO in glacial AcOH to afford 4-hyd-
3
1
6
roxy-3-nitrocoumarin (2). The starting compound 4-chloro-3-nitrocoumarin (3) was pre-
pared from 2 following the method of Kaljaj et al. The preparation was carried out in the
following manner: N,N-dimethylformamide (DMF, 2 mL, 26 mmol) was cooled to 10 °C in an
17
ice bath. Under stirring, POCl (4.0 g, 26 mmol) was added dropwise, and the obtained
3
mixture was stirred for an additional 15 min. Then, the ice bath was removed and the reaction
was left to proceed at room temperature for a further 15 min. Finally, a solution of 2 (5.4 g; 26
mmol) in DMF (12.5 mL) was added dropwise. After 15 min of stirring, the reaction was
stopped by the addition of cold water (15 mL). The precipitated solid was collected by fil-
tration and washed with saturated sodium bicarbonate solution and water. Recrystallization
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3
18
RADULOVIĆ et al.
from a mixture of benzene–hexane (1:1,V/V) yielded yellow crystals of 3 (5.1 g; 22.6 mmol)
in 87 % yield, m.p. 162–163 °C. The procedure was repeated twice.
General procedure for the synthesis of 4-(alkylamino)-3-nitrocoumarins 5a–i
The solution of 4-chloro-3-nitrocoumarin (3, 1.0 g, 4.4 mmol) and the appropriate amine
nucleophile 4a–i (4.4 mmol) in ethyl acetate (10 ml) was refluxed in the presence of triethyl-
amine (1 ml, 7.2 mmol) for 1–3 h. After cooling, the precipitated solid was filtered off and
washed with ethyl acetate and water. Purity of the synthesized compounds was checked by
TLC.
Antimicrobial activity
The synthesized compounds were tested against a panel of microorganisms including
three Gram-positive (Bacillus cereus (isolate from food), Bacillus subtilis ATCC 6633 and
Staphylococcus aureus ATCC 6538), nineteen Gram-negative (Escherichia coli ATCC 8739,
E. coli (isolate from food), Klebsiella pneumoniae ATCC 10031, Salmonella enterica subsp.
enterica serovar Enteritidis ATCC 13076, S. enterica (5 clinical isolates), Shigella flexneri (5
clinical isolates) and Yersinia enterocolitica (5 clinical isolates), one yeast (Candida albicans
ATCC 10231) and one mold (Aspergillus brasiliensis ATCC 16404) strains.
Clinical isolates of Y. enterocolitica, S. enterica subsp. enterica serovar Enteritidis and S.
flexneri (5 isolates per strain), together with the food isolates (B. cereus and E. coli) were
obtained from the Institute of Public Health, Niš, Serbia and are stored in a private
microbiological collection. The isolation of the mentioned strains was performed from stool
(fecal) samples of patients with a diarrheal syndrome.
Testing of antimicrobial activity
The antimicrobial activity was evaluated using the broth microdilution method.18 Mini-
mum inhibitory concentration (MIC) determinations were performed by a serial dilution
method in 96-well microtitre plates. Bacterial species were cultured at 37 °C in Müller–Hinton
agar (MHA) and Sabouraud dextrose agar (SDA) was the culture medium for the yeast (30
°
C). After 18 h of cultivation, bacterial suspensions were made in Mueller Hinton broth and
their turbidity was standardized to 0.5 McFarland scale using a densitometer (DEN-1 McFar-
land densitometer, Biosan). The final density of the bacterial and the yeast inoculums corres-
5
ponded to 5×10 CFU (colony forming units). A suspension of the mould (A. niger) was made
in Sabouraud dextrose broth (SDB) and its turbidity was confirmed by viable counting in a
4
Thoma chamber with 1×10 CFU as the final size of the inoculum. The inoculums were added
to all wells and the plates were cultivated at 37 °C during 24 h (bacteria) or at 30 °C for 48 h
(
fungi). Tetracycline, chloramphenicol and nystatin served as positive controls, while the
solvent (ethanol) was used as a negative control. One inoculated well was included, to allow
control of the adequacy of the broth for organism growth. One non-inoculated well, free of
antimicrobial agent, was also included to ensure medium sterility.
The bacterial growth was determined by adding 20 μL of a 0.5 % aqueous triphenyl-
1
9
tetrazolium chloride (TTC) solution. The MIC was defined as the lowest concentration of
the tested compound that inhibited visible growth (red colored pellet on the bottom of the
wells after the addition of TTC). The experiments were performed in triplicate and mean
values are presented.
Statistical analyses
Agglomerative hierarchical clustering (AHC) was performed using the Excel program
20
plug-in XLSTAT version 2011.3.02. The method was applied utilizing the MIC values (in
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NITROCOUMARIN DERIVATIVES WITH POTENT ANTIMICROBIAL ACTIVITY
319
-
1
µmol mL ) as original variables without any recalculation. AHC was determined using Pear-
son dissimilarity, where the aggregation criterion were simple linkage, unweighted pair-group
average and complete linkage, the Euclidean distance, where the aggregation criterion were
weighted pair-group average, unweighted pair-group average, and the Ward method.
RESULTS AND DISCUSSION
Chemistry
In the first reaction step, 4-hydroxycoumarin (1) was nitrated in glacial
AcOH with HNO to afford 4-hydroxy-3-nitrocoumarin (2). The starting 4-chlo-
3
ro-3-nitrocoumarin (3) was prepared from 2 in a reaction with phosphorus
oxychloride and N,N-dimethylformamide.
The target compounds 5a–i (three new – 5e–g, three previously known – 5a,
5
c and 5i, and three commercially available but with no mention what so ever in
the literature – 5b, 5d and 5h), were prepared in the reaction of 3 and an alkyl-
amine 4a–i, respectively (1:1 mole ratio of starting materials) in ethyl acetate in
the presence of two equivalents of triethylamine. The compounds were obtained
in good yields (72–92 %). The synthesis scheme is presented in Fig. 1.`
Fig. 1. Synthesis of 4-(alkylamino)-3-nitrocoumarin derivatives 5a–i. (4a: butan-1-amine, 4b:
butan-2-amine, 4c: 2-methylpropan-2-amine, 4d: 3-aminopropan-1-ol, 4e: hexan-1-amine, 4f:
octan-1-amine, 4g: hexadecan-1-amine, 4h: furan-2-ylmethanamine and 4i: phenyl-
methanamine; R = 5a: CH (CH ) CH −; 5b: CH CH (CH )CH−; 5c: (CH ) C−;
3
2 2
2
3
2
3
3 3
5
d: HO(CH ) CH −; 5e: CH (CH ) CH −; 5f: CH (CH ) CH −; 5g: CH (CH ) CH −;
2
2
2
5
3
2 4
2
3
2 6
2
3
2 14
2
h: C H OCH − (furan-2-ylmethyl); 5i: C H CH −.
4 3 2 6 5 2
The structures of the synthesized compounds were confirmed by analytical
1
13
and spectral means (HRMS(EI), IR, H- and C-NMR). The physical, analytical
and spectral data for compounds 5a–i are given in the Supplementary material to
this paper.
IR spectra of the synthesized compounds contained characteristic vibrations
–
1
at 3030 to 3393 cm corresponding to absorptions of the Ar–H and N–H bonds,
and strong bands at 1677–1698 cm–1 corresponding to the C=O groups. The IR
absorptions due to the presence of the 3-NO group appeared at 1317–1364 and
2
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3
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RADULOVIĆ et al.
–
1
1
–
511–1556 cm , and the C–H bonds showed characteristic vibrations at 2925–
–
1
2960 cm .
Protons from the coumarin moiety showed very similar chemical shifts in the
1
H-NMR spectra of compounds 5a–i. Protons in the position 5 of the coumarin
core were more downfield and showed higher chemical shifts (doublet of doub-
lets at 8.35–8.52 ppm) than the other protons attached to that ring. Significant
differences in the chemical shifts of the mentioned protons existed for com-
pounds 5b, 5c and 5f where the signals for H-5 appear at 7.89–8.00 ppm. All
other protons on the coumarin moiety displayed much less variation in their che-
mical shifts. H-7 protons appeared as a doublet of triplets or only triplets at 7.69–
–
7.75 ppm. H-6 and H-8, the signals of which overlapped at 7.28–7.50 ppm,
were more shielded. The aromatic protons of the substituent groups in com-
pounds 5h and 5i showed similar values of chemical shifts and appeared as a
doublet of doublets at 7.64 ppm and a multiplet at 6.34–6.45 ppm, as well as a
multiplet at 7.26–7.34 ppm, respectively. The remaining protons, from the alkyl
groups bonded to the coumarin moiety, were as expected more upfield.
Antimicrobial activity
The created library was first evaluated for in vitro antimicrobial activity
against a wide range of bacteria (four Gram-negative and three Gram-positive) as
well as against a yeast and a mold species. The values of their minimal inhibitory
–
1
concentrations (MIC, nmol mL ), determined in a microdilution assay, revealed
the diverse effect of these compounds on the growth of microorganisms (all
strains were susceptible to the presence of these compounds in their nutritive
medium with an exception of E. coli (isolate) and A. brasiliensis (Table I)). The
-
1
TABLE I. Minimal inhibitory concentrations (MIC / nmol mL ) of the coumarins 5а–i; NT –
not tested, / – not sensitive in the range of the tested concentrations; AB – reference antibiotic
Compound
Bacterial strain
a
b
5
a
5b 5c 5d 5e 5f 5g 5h 5i AB AB
c
Gram-negative
E. coli
ATCC 8739 3.0 12
–
–
–
11 2.45 1.8
0.91
–
–
5.3 3.5 NT
5.3 0.88 NT
K. pneumoniae ATCC 10031 6.0 1.5 6.0
–
–
S. enterica
ATCC 13076 6.0 95 191 189 5.4 4.9 3.6 1.4 5.3 0.44 NT
Gram-positive
B. cereus
B. subtilis
S. aureus
Isolate from food
ATCC 6633
ATCC 6538
–
–
–
–
–
–
–
–
–
–
–
232 44
–
/
0.44 NT
0.22 NT
4.9 29
–
6.0 6.0 6.0 189 1.3 1.2 3.6 5.4 21 0.22 NT
d
Fungal strain
C. albicans ATCC 10231
0.34 0.05 3.0 11.8 0.04 0.08 0.44 1.4 0.64 NT 6.8
a
b
c
Tetracycline; nystatin; the food isolate of E. coli was resistant to all compounds in the tested range of con-
d
centrations; the mold A. brasiliensis (ATCC 16404) was resistant to all compounds in the tested range of con-
centrations
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NITROCOUMARIN DERIVATIVES WITH POTENT ANTIMICROBIAL ACTIVITY
321
–
1
active inhibitory concentrations ranged from 0.04 to 232 nmol mL , with 5g
being the most non-selective in its action. The best MIC value was observed for
compound 5e against C. albicans. The remaining compounds displayed antimic-
robial activity with no special selectivity towards either Gram-positive or Gram-
negative bacterial strains.
Among the Gram-positive strains, B. cereus was the most resistant strain,
susceptible to only two out of the nine tested compounds, but at relatively high
concentrations (5h and 5g, Table I). S. aureus was the most sensitive bacterial
strain inhibited by all tested compounds in a concentration range 1.2–189 nmol
–
1
mL . The activity of the compounds against Gram-negative bacteria was
characterized by two extreme cases – E. coli (a food isolate which showed com-
plete resistance to all the tested compounds, as well to the standard antibiotic)
and S. enterica (an ATCC reference strain (13076) showing the highest suscep-
tibility to the tested compounds with MIC values in the range (1.4 to 191 nmol
–
1
mL ) similar to the ones for Gram-positive bacteria). This strong resistance of
E. coli could be explained by its origin (food isolate, a probable cause of food-
borne infections) and this was corroborated by the higher registered MIC values
for an ATCC strain of E. coli (Table I). All compounds were also proven to pos-
–
1
sess good anticandidal activity (0.04–12 nmol mL ) but had no effect at the
highest tested concentration on the growth of the other tested fungal organism
(
the mold A. brasiliensis) (see Table I).
Prompted by the results of this initial screening of antimicrobial activity, a
selection of three compounds (based on their structural diversity, 5b having a
short N-alkyl substituent; 5g having the longest N-alkyl substituent and among
the most potent ones thus far and 5i possessing a N-benzyl group) were further
tested against fifteen bacterial strains representing gastrointestinal pathogenic
bacteria isolated from human clinical samples (Table II). Table II also contains
clinically relevant data on the patient diagnosed condition caused by the parti-
cular bacterial isolate.
Human sample isolates (five per strain) of Y. enterocolitica, S. enterica and
Sh. flexneri showed themselves to be highly resistant to the influence of the
tested coumarinic derivatives (most of which having MIC values higher than 1.9
–
1
nmol mL ). The differing effect on (clinical/food) isolates and ATCC strains
Tables I and II) was evident from the high resistance of the isolates to the con-
(
trol antibiotic used in this study as well. In the light of these facts, compound 5b
looks highly promising, since it was the only compound among the three tested,
which showed inhibitory action against S. enterica with MIC values of 0.48 and
–
1
0
.95 µmol mL . Although this does not appear significant at first, this activity is
more than relevant if one considers the fact that this strain was completely res-
istant to the highest tested concentration of the reference antibiotic (0.11 µmol
–
1
mL ).
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3
22
RADULOVIĆ et al.
TABLE II. Effect of the selected coumarin derivatives (5b, 5g and 5i) on growth of three
gastrointestinal pathogenic bacterial strains (isolates)
MIC / µmol mL-1
Bacteria
Isolate No.
Diagnosis
Chloram-
phenicol
5
b
5g
5i
Yersinia enterocolitica
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Acute enterocolitis >1.9
>1.2
>1.2
0.58
0.29
>1.2
>1.2
>1.2
>1.2
>1.2
>1.2
0.87
0.29
0.87
>1.2
0.87
>1.7
>1.7
0.85
>1.7
>1.7
>1.7
>1.7
>1.7
>1.7
>1.7
>1.7
1.3
0.11
0.11
0.027
0.054
0.11
>0.11
>0.11
>0.11
>0.11
>0.11
0.11
>1.9
>1.9
>1.9
>1.9
Salmonella enterica
Shigella flexneri
Gastroenterocolitis 0.95
0.95
0.48
>1.9
0.95
Shigellosis
>1.9
0.95
>1.9
>1.9
0.95
0.11
0.11
0.11
0.11
>1.7
>1.7
>1.7
Hitherto, a substantial amount of data on the antimicrobial potential of a num-
1
3–15,21
ber of N-substituted 4-amino-3-nitrocoumarins has been accumulated.
Using multivariate statistical analyses (MVA), in an attempt to determine their
similarities and selectivity against certain pathogenic strains, the relations
between the MIC values of the herein tested coumarin derivatives and those pre-
1
3–15,21
viously published were analyzed.
The compounds taken into consider-
ation (Fig. 2) for the statistical analysis were those that were previously tested
Fig. 2. Structures of coumarin derivatives (6–33) included in the multivariate
statistical analyses.
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NITROCOUMARIN DERIVATIVES WITH POTENT ANTIMICROBIAL ACTIVITY
323
against the same panel of microorganisms as the compounds tested in this work.
Agglomerative hierarchical analysis using MIC values against seven bacterial
and two fungal strains as variables resulted in the dendrogram shown in Fig. 3
with three clearly separated groups/classes of compounds (C1–C3). Group cen-
troids (the average representative of a group) were also plotted against microorg-
anism strains tested (Fig. 4) to give a better picture of the distinctions existing
between the groups.
Fig. 3. Dendrogram (AHC analysis) representing the dissimilarity in the antimicrobial activity
(
MIC values of selected coumarin derivatives used as variables against five bacterial and two
fungal strains) relationships of thirty-seven compounds obtained by Euclidean distance
dissimilarity, using the aggregation criterion-the Ward method. Three groups of compounds
were found: C1–C3.
Fig. 4. Graphical representation of
the scores (the lowest activity, i.e.,
the highest value of MIC, was
given the score 100 and the best
activity value 0) of the centroids
(
classes C1–C3) from the per-
formed AHC analysis (for details
see Fig. 3).
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3
24
RADULOVIĆ et al.
Group C1 (Figs. 3 and 4) consisted of compounds that had rather non-
selective activity with the lowest MIC values towards E. coli, K. pneumoniae, S.
enterica, B. subtilis, C. albicans and A. brasiliensis. Among these microorg-
anisms, K. pneumoniae and A. brasiliensis were the ones more resistant to the
action of compounds from group C1. Compounds 5f and 5g from the present
paper belong to this group. The second group, designated as C2, contained all
other compounds prepared in this work (5a–e, 5h and 5i). Group C2 compounds
demonstrated a greater degree of selectivity, highly affecting the growth of S.
aureus and C. albicans, while remaining relatively active against the other strains
in comparison to group C1. All the other statistically compared compounds were
grouped into C3 according to their overall low impact on bacterial growth (i.e.,
high MIC values) – the only exception being their action against B. subtilis (Fig. 4).
From all of the above mentioned, it could be conclude that group 1 (C1)
substances were the most potent (in terms of both their MIC values and broad
spectrum of bacteria that the compounds affect) and group 2 substances are the
most selective ones (even though they have lower MIC values).
Chemically speaking, compounds 5f and 5g differed from the rest of the
compounds prepared in this work by having the longest alkyl chains attached to
the nitrogen in position 4. This structural feature, a long lipophilic moiety (octyl
and hexadecyl groups in 5f and 5g, respectively), made them less selective in
their antimicrobial action, suggesting that lipophilicity, i.e., an interaction with
the cell membrane, might play a crucial role in their activity. It would be natural
then to expect, as observed, that such compounds have an effect on more differ-
ent strains (be less selective) by acting on/disrupting their membranes. Along
with these N-alkyl derivatives (5f and 5g), other members of C1 were almost
invariantly N-heteroaryl derivatives (20–30). These could be regarded as possible
hybrids of the coumarin core with other pharmacophoric groups (i.e., those hete-
rocycles that are known to possess an antimicrobial action). This hybridization
appears to have resulted in the onset of the strongest observed activity.
Group C2 compounds demonstrated that the presence of shorter alkyl sub-
stituents on N-4 lowers the activity but also that the nucleophilicity/basic nature
of this nitrogen could be relevant for the overall noted activity. The weakly nuc-
leophilic/basic anilino-type nitrogen, present in all compounds from group C3,
might be the reason why these compounds showed much lower potency in inhi-
biting the growth of microorganisms when compared to the activity of the much
more basic/nucleophilic (at N-4) compounds from C2. Not even the presence of a
phenol group in compounds 6 and 8 (known to be the carrier of antimicrobial act-
ivity of many natural compounds, thymol, for example) had a significant impact
on the activity of the N-aryl coumarin derivatives grouped in C3. Moreover, the
size of the N-substituent seems not to be of importance in the appearance of anti-
microbial activity, as exemplified by the low activity of compounds 12 and 18.
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NITROCOUMARIN DERIVATIVES WITH POTENT ANTIMICROBIAL ACTIVITY
325
Bearing all of this in mind, it could be concluded that the nature of the
N-substituent at position 4 profoundly affects the antimicrobial potency of 3-nit-
rocoumarins. The respective activity decreases in the following order N-long
alkyl chains ≈ N-heteroaryl > N-short alkyl chains >> N-aryl derivatives.
CONCLUSION
In the present paper, a wide range of activities, ranging from completely
inactive to active against antibiotic-resistant strains, was registered for the stu-
died 4-(alkylamino)-substituted 3-nitrocoumarins. The most important infor-
mation, relevant for any possible future applications of these compounds, is the
fact that the tested coumarins exhibited activity against strains isolated from fecal
samples of the patients with acute diarrheal syndrome. Bacterial infections of the
digestive tract are considered the major cause of morbidity and mortality, espe-
cially in children, the elderly and immunosuppressed patients, both in industrial-
2
2
ized and developing countries. These results demonstrate the substantial anti-
microbial potential of these coumarins in the therapy of GI and other infections
caused by highly resistant strains. Furthermore, the activity observed against S.
aureus was quite significant, knowing that this bacterial strain can be multidrug
2
3
resistant. A multivariate treatment of the results of the antimicrobial assays
obtained in this work and those previously attained enabled the tested compounds
to be classified according to their overall activity, selectivity and structural
requirements for the observed activities to be discussed. A general conclusion
could be reached that both a greater lipophilicity of the N-substituent, and nuc-
leophilicity and basicity of this nitrogen have beneficial effects on the antimic-
robial activity of these compounds. Such results and conclusions allow and direct
further studies focused on an even larger enhancement of the here obtained
activity by additional chemical modification of these coumarin antimicrobials.
SUPPLEMENTARY MATERIAL
Physical, analytic and spectral data for the prepared derivatives are available electro-
nically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request.
Acknowledgements. This work was supported by the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Project Nos. 172061 and 45022).
Available on line at www.shd.org.rs/JSCS/
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3
26
RADULOVIĆ et al.
И З В О Д
МАЛА СИНТЕТСКА БИБЛИОТЕКА 4-(АЛКИЛАМИНО)-3-НИТРОКУМАРИНА СА ЈАКОМ
АНТИМИКРОБНОМ АКТИВНОШЋУ ПРОТИВ ГАСТРОИНТЕСТИНАЛНИХ ПАТОГЕНА
1
2
3,4
3
НИКО С. РАДУЛОВИЋ , ЗОРИЦА СТОЈАНОВИЋ-РАДИЋ , ПРЕДРАГ СТОЈАНОВИЋ , НИКОЛА СТОЈАНОВИЋ ,
5
5
ВИДОСЛАВ ДЕКИЋ и БИЉАНА ДЕКИЋ
1
Департман за хемију, Природно–математички факултет, Универзитет у Нишу, Вишеградска 33,
2
18000 Ниш, Департман за биологију и екологију, Природно–математички факултет, Универзитет
3
у Нишу, Вишеградска 33, 18000 Ниш, Медицински факултет, Универзитет у Нишу, Зорана Ђинђића
8
4
5
1, 18000 Ниш, Институт за јавно здравље, Центар за микробиологију, 18000 Ниш, и Природно–
математички факултет, Универзитет у Приштини, Лоле Рибара 29, 38220 Косовска Митровица
Инфекције изазване микроорганизмима са развијеном резистентношћу према
антибиотицима тренутно представљају главни узрок озбиљних здравствених компли-
кација код пацијената. Стога су истраживања која за циљ имају откриће нових и/или
активнијих антимикробних агенаса веома важна. Имајући у виду потврђену антимик-
робну активност природних и синтетичких деривата кумарина, овај рад треба да пружи
додатни увид у антимикробни потенцијал кумаринских једињења испитивањем актив-
ности библиотеке од девет нових 4-(алкиламино)-3-нитрокумарина против 24 соја мик-
роорганизама, који представљају лабораторијске сојеве и резистентне (изолати) бакте-
ријске и фунгалне патогене. Сва једињења су показала одређени степен селективне
активности, која је у неким случајевима била нарочито изражена достижући минималну
инхибициону концентрацију од 0,04 nmol mL- (12 ng mL ) против гљивице Candida
albicans. Утврђена активност је већа према Грам-негативним сојевима, а најосетљивија,
и међу ATCC сојевима и клиничким изолатима, је била Salmonella enterica subsp. enterica
serovar Enteritidis. Ови резултати указују на потенцијал ових кумарина као антимик-
робних агенаса (већина је активна у концентрацијама реда nmol/ml) у третману гастро-
интестиналних и других инфекција изазваних високо резистентним сојевима микроор-
ганизама. Коначно, ради лакшег повезивања структуре и активности урађена је мулти-
варијантна статистичка анализа података добијених у овом раду и оних из претходних
студија антимикробне активности структурно сличних кумарина.
1
-1
(
Примљено 19. јуна, ревидирано 22. августа, прихваћено 29. августа 2014)
REFERENCES
1. D. J. Hentges, in Human Intestinal Microflora in Health and Disease, D. J. Hentges, Ed.,
Academic Press, New York, 1983, p. 311
2
. D. Gilliss, A. Cronquist, M. Carter, M, Tobin-D'Angelo, D. Blythe, K. Smith, S. Lathrop,
G. Birkhead, P. Cieslak, J. Dunn, K. G Holt, J. J. Guzewich, O. L. Henao, P. Griffin, S.
M. Crim, O. L. Henao, Vital Signs: Incidence and Trends of Infection with Pathogens
Transmitted Commonly Through Food – Foodborne Diseases Active Surveillance Net-
work, 10 U.S. Sites, 1996–2010, Morbidity and mortality weekly report 60, Centers for
Disease Control and Prevention, Atlanta, GA, 2011, p. 749
3
4
. H. Rotterdam, P. Tsang, Hum. Pathol. 25 (1994) 1123
. R. D. H. Murray, J. Mendez, S. A. Brown, The Natural Coumarins: Occurrence,
Chemistry and Biochemistry, Wiley, New York, 1982
5. R. O’Kennedy, R. D. Thornes, Coumarins - Biology, Applications and Mode of Action,
Wiley, Chichester, 1997
6. I. Kostova, Curr. Med. Chem.: Anti-Cancer Agents 5 (2005) 29
Available on line at www.shd.org.rs/JSCS/
_________________________________________________________________________________________________________________________
(CC) 2015 SCS. All rights reserved.

NITROCOUMARIN DERIVATIVES WITH POTENT ANTIMICROBIAL ACTIVITY
327
7
8
9
. F. Borges, F. Roleira, N. Milhazes, L. Santana, E. Uriarte, Curr. Med. Chem. 12 (2005)
87
. Z. Debeljak, A. Škrbo, I. Jasprica, A. Mornar, V. Plečko, M. Banjanac, M. Medić-Šarić,
J. Chem. Inf. Model. 47 (2007) 918
8
. N. Radulović, G. Stojanović, R. Vukićević, V. Dekić, B. Dekić, R. Palić, Monatsh Chem.
1
37 (2006) 1477
1
1
0. S. V. Dekić, V. S. Dekić, B. R. Dekić, M. S. Dekić, Chem. Pap. 61 (2007) 233
1. S. V. Dekić, V. S. Dekić, B. Vučić, B. R. Dekić, M. S. Dekić, Facta Univ., Ser.: Phys.,
Chem. Technol. 5 (2007) 85
12. M. S. Dekić, B. R. Dekić, V. S. Dekić, S. V. Dekić, J. Heterocycl. Chem. 45 (2008) 295
13. B. Dekić, V. Dekić, N. Radulović, R. Vukićević, R. Palić, Chem. Pap. 64 (2010) 354
14. B. Dekić, N. Radulović, V. Dekić, R. Vukićević, R. Palić, Molecules 15 (2010) 2246
15. N. Radulović, G. Bogdanović, P. Blagojević, V. Dekić, R. Vukićević, J. Chem. Crys-
tallogr. 41 (2011) 545
16. V. Savel’ev, O. Artamonova, V. Troitskaya, V. Vinokurov, V. Zagorevskii, Khim.
Geterotsikl. Soedin. 7 (1973) 885
1
1
7. V. Kaljaj, M. Trkovnik, L. Stefanović-Kaljaj, J. Serb. Chem. Soc. 52 (1987) 183
8. Clinical and Laboratory Standards Institute, Performance standards for antimicrobial
susceptibility testing. Eleventh informational supplement. Document M 100-S11,
NCCLS, Wayne, PA, 2003.
19. N. Radulović, M. Dekić, Z. Stojanović-Radić, R. Palić, Turk. J. Chem. 35 (2011) 499
20. N. S. Radulović, M. S. Dekić, Chem. Biodivers. 10 (2013) 2042
21. V. Dekić, N. Radulović, R. Vukićević, B. Dekić, Z. Stojanović-Radić, R. Palić, Afr. J.
Pharm. Pharmacol. 5 (2011) 371
2
2
2. B. Miljković-Selimović, T. Babić, P. Stojanović, Sci. J. Faculty of Med. Niš 27 (2010) 55
3. N. Radulović, P. Blagojević, Z. Stojanović-Radić, N. Stojanović, Curr. Med. Chem. 20
(
2013) 932.
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