Design, synthesis and antibacterial study of new potent and selective coumarin-chalcone derivatives for the treatment of tenacibaculosis

Article abstract of DOI:10.1016/j.bmc.2015.09.028

With the aim of finding new chemical entities selective for fish pathogens to avoid drug resistance in humans, a series of coumarin-chalcone hybrid compounds with different patterns of substitution were designed and synthesized. Their antibacterial activi

Full text of DOI:10.1016/j.bmc.2015.09.028

Accepted Manuscript
Design, synthesis and antibacterial study of new potent and selective coumarin-
chalcone derivatives for the treatment of tenacibaculosis
Saleta Vazquez-Rodriguez, Raquel Lama López, Maria João Matos, Gabriel
Armesto-Quintas, Silvia Serra, Eugenio Uriarte, Lourdes Santana, Fernanda
Borges, Angeles Muñoz Crego, Ysabel Santos
PII:
S0968-0896(15)30053-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.09.028
BMC 12575
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
28 July 2015
14 September 2015
17 September 2015
Please cite this article as: Vazquez-Rodriguez, S., López, R.L., Matos, M.J., Armesto-Quintas, G., Serra, S., Uriarte,
E., Santana, L., Borges, F., Crego, A.M., Santos, Y., Design, synthesis and antibacterial study of new potent and
selective coumarin-chalcone derivatives for the treatment of tenacibaculosis, Bioorganic & Medicinal Chemistry
(
2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.09.028
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Design, synthesis and antibacterial study of
new potent and selective coumarin-chalcone
derivatives for the treatment of
tenacibaculosis.
Leave this area blank for abstract info.
a,
b
a
b
Saleta Vazquez-Rodriguez, * Raquel Lama López, Maria João Matos, Gabriel Armesto-Quintas, Silvia
c
c
c
a
b
b
Serra, Eugenio Uriarte, Lourdes Santana, Fernanda Borges, , Angeles Muñoz Crego, Ysabel Santos.
a
CIQUP/Department of Chemistry and Biochemistry, Faculty of Science, University of Porto, Rua Campo
Alegre 687, 4169-007, Porto, Portugal.
b
Department of Microbiology and Parasitology, CIBUS-Faculty of Biology, University of Santiago de
Compostela, 15782 Santiago de Compostela, Spain.
c
Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, Campus Vida
s/n, 15782, Santiago de Compostela, A Coruña, Spain.
O
Human pathogenic bacteria
O
O
Marine pathogen T. maritimum
coum arin-chalcone hybrid

Bioorganic & Medicinal Chemistry
Design, synthesis and antibacterial study of new potent and selective coumarin-
chalcone derivatives for the treatment of tenacibaculosis.
a,
b
a
b
c
Saleta Vazquez-Rodriguez, * Raquel Lama López, Maria João Matos, Gabriel Armesto-Quintas, Silvia Serra,
c
c
a
b
b
Eugenio Uriarte, Lourdes Santana, Fernanda Borges, , Angeles Muñoz Crego, Ysabel Santos.
a
CIQUP/Department of Chemistry and Biochemistry, Faculty of Science, University of Porto, Rua Campo Alegre 687, 4169-007, Porto, Portugal.
Department of Microbiology and Parasitology, CIBUS-Faculty of Biology, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
b
c
Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782, Santiago de Compostela, A Coruña, Spain
Corresponding author. Tel.: +34981594912; fax: +34981528070; e-mail: svre77@gmail.com
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
With the aim of finding new chemical entities selective for fish pathogens to avoid drug
resistance in humans, a series of coumarin-chalcone hybrid compounds with different patterns of
substitution were designed and synthesized. Their antibacterial activity was evaluated against
important types of human bacteria strains (E. coli, S. aureus and P. aeruginosa) and against a
fourteen strains of the marine pathogen Tenacibaculum maritimum, responsible for
tenacibaculosis in fish, which is an important disease that causes great economical loss in the
aquaculture industry. All the amino derivatives 5-12 presented high activity against different
strains of T. maritimum, no activity against any of the three human pathogenic bacteria strains
and no toxicity. Compounds 6, 7 and 11 were the most promising molecules. The most sensitive
strains to these compounds were LL01 8.3.8 and LL01 8.3.1, being compound 11 up to 20 times
more active than enrofloxacin. Therefore these scaffolds are good candidates for aquaculture
treatments, avoiding possible drug resistance problems in humans.
Available online
Keywords:
coumarin
chalcone
antibacterial
Tenacibaculum matitimum
Aquaculture
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
Even though tenacibaculosis has long been recognized as a
disease of fish in Japan, the infection was not considered of
Aquaculture is an emerging industrial sector that requires
economical importance until serious mortalities affecting Dover
continued research with scientific and technical developments
and innovation and contributes in 40.1% to the world total fish
production, and almost all the seaweeds production. World
aquaculture production continues to grow, albeit at a slowing
rate. According to the latest available statistics collected globally
by FAO, world aquaculture production attained another all-time
high of 90.4 million tones (live weight equivalent) in 2012
3,8
sole Solea solea in Scotland were reported.
To date,
tenacibaculosis is one of the most threatening bacterial infections
limiting the culture of many species of commercial value in
9
distinct geographical areas of the world. Until recently, no
vaccines were available to prevent the disease, but
a
flexibacteriosis vaccine (FM 95) has been patented by one of our
collaborating groups at the University of Santiago (Spain) and is
the only vaccine currently in the market to prevent mortalities
(
(
(
US$144.4 billion), including 66.6 million tones of food fish
US$137.7 billion) and 23.8 million tones of aquatic algae
mostly seaweeds, US$6.4 billion). Nowadays, the estimated
10
caused by T. maritimum . Therefore there is an urgent need to
1
develop new agents that can be used in aquaculture to treat this
fish disease not only in turbot but also in other fish species.
value of farmed food fish is USD 130 billion.
Tenacibaculum maritimum (T. maritimum), formerly known
as Flexibacter maritimus, and other members of the genus
Despite the significance of T. maritimum an other
Tenacibaculum spp. in the aquaculture industry, relatively little is
known about the pathogenicity of this bacteria. In vitro studies on
the susceptibility of T. maritimum to various chemotherapeutic
agents indicate that bacterial strains isolated from different host
species and geographical regions exhibit a similar pattern, with
respect to susceptibility to nitrofurans, penicillins, erythromycin,
tetracyclines, chloramphenicol, trimethoprim, potentiated
sulfonamides and fluoroquinolones, and resistance to colistin,
2
Tenacibaculum (T. ovolyticum, T. soleae, T. discolorand T.
gallaicum) are Gram-negative and filamentous bacterium,
responsible for tenacibaculosis in marine fish. The pathology of
the disease caused by this marine organism has mainly been
associated with characteristic gross lesions on the body surface of
fish such as ulcers, necrosis, eroded mouth, frayed fins and tail
3
,4,5,6,7
rots, and sometimes necrosis on the gills and eyes.

kanamicyn, neomicyn and the quinolones, oxolinic acid and
flumequine.
On the other hand, chalcones are prominent secondary
metabolites precursors of flavonoids and isoflavonoids in plants.
Chemically, they consist of two aromatic rings linked by a three-
carbon ,-unsatured systems that are known to exhibit an
1
1
Among the drugs used in the past few years in turbot and sole
cultures (tetracycline, enrofloxacin, flumequine and potentiated
sulfonamides), enrofloxacin proved to be the most useful
compound for controlling T. maritimum outbreaks, although the
rapid appearance of resistant strains has already been described
30
impressive array of biological properties. Among them, it is
31,32
33,34
remarkable to mention their antibacterial,
antimalarial,
properties.
35
36,37
38,39
antifungal, antiviral and anti-inflammatory
1
2
(
Figure 1). However, during the last few years, enrofloxacin, a
In humans, antibiotic resistance is a worldwide problem and
adds considerable and avoidable costs to the healthcare system.
The use of antibiotics is the single most important factor leading
to antibiotic resistance around the world. One of the major factors
in the growth of antibiotic resistance is spread of the resistant
strains of bacteria from person to person, or from the non-human
sources in the environment, including food.
member of the 4-quinolone group with broad spectrum activity,
has received growing attention for its potential in fish therapy
1
3,14
against infections caused by bacterial pathogens.
Based on our experience on 3-substituted coumarin
4
0,41,42,43
derivatives
, in previous results of our group with
44,45
aminocoumarins as antibacterial agents
and taking into
account the economic importance of the marine fish diseases in
the aquaculture industry, we have designed and synthesized
novel coumarin-chalcone hybrid compounds. These series of
compounds were tested in vitro for their antibacterial activity
against three human pathogenic bacteria (E. coli, S. aureus and P.
aeruginosa) and different species of marine pathogens of the
Tenacibaculum genus. The increasing need of finding new drug
candidates for the treatment of tenacibaculosis and the antibiotic-
resistance problems in humans makes this topic an interesting
research area.
Figure 1. Quinolone derivatives as antibacterial drugs.
Benzopyrone derivatives could be considered as structurally
similar quinolone drugs, widely used in clinic and with a broad
antimicrobial spectrum, including many encountered pathogens.
There are important natural occurring antibiotics containing the
benzopyrone moiety, such as novobiocin and coumermycin A1
1
5
(
Figure 2). This fact makes the study of quinolone-like
1
. Materials and Methods
compounds an interesting topic on this research area.
1
.1. Chemistry
Melting points were determined using
a
Reichert
Koflerthermopan or in capillary tubes on a Büchi 510 apparatus
1
13
and are uncorrected. H and C NMR spectra were recorded on a
Bruker AMX spectrometer at 300 and 75.47 MHz, respectively,
using TMS as internal standard (chemical shifts in δ values, J in
Hz). Mass spectra were obtained using a Hewlett-Packard 5988A
spectrometer. Elemental analyses were performed using a Perkin-
Elmer 240B microanalyser and were within ±0.4% of calculated
values in all cases. Silica gel (Merck 60, 230–00 mesh) was used
for flash chromatography (FC). Analytical thin layer
chromatography (TLC) was performed on plates precoated with
silica gel (Merck 60 F254, 0.25 mm).
1
.1.2. General procedure for the synthesis of 3-
benzoylcoumarins 1-4: To a solution of the appropriate -
ketoester (1 mmol) and the corresponding salicylaldehyde (1
mmol) in ethanol (3 mL) was added piperidine in catalytic
amount. The mixture was refluxed for 2–5 h and after completion
(followed by TLC), the reaction was cooled and the precipitated
was filtered and washed with cold ethanol and ether to afford the
desired compound. Compounds were further recrystallized from
MeOH/CH Cl .
Figure 2. Benzopyrone derivatives currently used as potent
antibacterial agents.
Coumarins are a large and well-known family of compounds
of natural and synthetic origin with great structural variety
2
2
1
6,17
46
47
responsible for a wide range of pharmacological activities.
Recent studies pay special attention to their anti-
Compounds 1 and 2 were previously described.
1
8,19
20,21
22
3-Benzoyl-8-ethoxycoumarin (3): Yield (60%), mp 141-143
inflammatory,
antitumor,
antioxidative,
cardioprotective,
1
2
3,24
25,26
27,28
ºC; H NMR (300 MHz, CDCl
) δ ppm 7.89 (s, 1H, H-4), 7.71 (d,
3
antimicrobial
and enzymatic inhibition
J = 8.3 Hz, 2H, H-2’, H-6’), 7.44 (t, J = 8.1 Hz, 1H, H-4’), 7.36-
.23 (m, 2H, H-3’, H-5’), 7.14-6.91 (m, 3H, H-5, H-6, H-7), 4.05
properties. Special attention can be paid to aminocoumarins,
which represents a class of antimicrobial agents that act by the
inhibition of the DNA gyrase enzyme involved in the cell
division in bacteria by binding tightly to its B subunit, thereby
7
13
(q, J = 7.0 Hz, 2H, CH ), 1.35 (t, J = 7.0 Hz, 3H, CH ). C NMR
2
3
(
75 MHz, CDCl ) δ ppm 192.15, 158.47, 147.00, 146.06, 145.06,
3
2
9
136.57, 134.16, 130.00, 128.93, 127.61, 125.18, 120.68, 119.27,
inhibiting this essential enzyme.
+
1
(
16.82, 65.50, 15.12. MS EI m/z (%): 295 ([M+1] , 28), 294
+
[M] , 78), 266 (43), 237 (35), 105 (100), 77 (77). Anal. Calcd.
for C H O : C, 73.46; H, 4.79; Found: C, 73.06; H, 4.45.
1
8
14
4

6
-Bromo-3-benzoylcoumarin (4): Yield (58%), mp 173-175
155.07, 152.92, 141.29, 135.13, 132.73, 131.28, 128.88, 123.03,
1
+
ºC; H-NMR (300 MHz, CDCl ) δ ppm 8.01 (s, 1H, H-4), 7.90
d, J = 7.5 Hz, 2H, H-2’, H-6), 7.81 – 7.72 (m, 2H, H-5, H-7),
120.48, 118.69, 116.35, 112.76. MS EI m/z (%): 345 ([M+2] ,
3
+
(
44), 343 ([M] , 44), 121 (14), 120 (100), 92 (28), 65 (20). Anal.
7
5
1
1
.67 (t, J = 7.5 Hz, 1H, H-4’), 7.52 (t, J = 7.5 Hz, 2H, H-3’, H-
Calcd. for C H BrNO : C, 55.84; H, 2.93; Found: C, 55.87; H,
2.88.
1
6
10
3
1
3
’), 7.33 (d, J = 9.5 Hz, 1H, H-8). C NMR (75 MHz, CDCl ) δ
3
91.48, 158.09, 153.91, 144.10, 136.64, 136.27, 134.43, 131.64,
29.98, 129.07, 128.51, 121.67, 120.03, 119.04, 117.91, 108.26.
3
-(3’-Aminobenzoyl)coumarin (9): Yield (94%), mp 147-148
1
+
+
+
ºC; H NMR (300 MHz, CDCl
.50 (m, 2H, H-5, H-7), 7.45-7.27 (m, 2H, H-6, H-8), 7.27-7.12
m, 3H, H-2’, H-5’, H-6’), 6.97-6.83 (m, 1H, H-4’), 3.85 (bs, 2H,
3
) δ ppm 8.00 (s, 1H, H-4), 7.72-
MS EI m/z (%): 331 ([M+2] , 26), 330 ([M+1] , 6), 329 ([M] ,
2
3
7
(
7), 105 (100), 77 (87). Anal. Calcd. for C H BrO : C, 56.85; H,
16 9 3
.09; Found: C, 56.51; H, 3.02.
13
NH2). C NMR (75 MHz, CDCl ) δ ppm 191.64, 158.23, 154.50,
3
1
1
46.77, 144.75, 137.05, 133.37, 129.32, 129.01, 126.97, 124.81,
20.35, 120.06, 117.98, 116.77, 114.97. MS EI m/z (%): 266
1
.1.3. General procedure for the synthesis of amino-3-
+
+
([M+1] , 30), 265 ([M] , 100), 236 (28), 172 (22), 120 (84), 89
benzoylcoumarins 5-12: Following the same procedure described
for the synthesis of derivatives 1-4, the nitro-3-benzoylcoumarin
derivatives were prepared and were used without further
purification. To a solution of the corresponding nitro-3-
benzoylcoumarin (1 mmol) in pure ethanol (5 mL) was treated
with SnCl .2H O and the resulting mixture was left under reflux
(15), 65 (44). Anal. Calcd. for C H NO : C, 72.45; H, 4.18;
1
6
11
3
Found: C, 72.31; H, 4.12.
3-(3’-Aminobenzoyl)-8-methoxycoumarin (10): Yield (74%),
1
mp 138-141 ºC; H NMR (300 MHz, DMSO-d ) δ ppm 8.30 (s,
1H, H-4), 7.47-7.27 (m, 3H, 3xAr-H), 7.16 (t, J = 7.8 Hz, 1H,
Ar-H), 7.12-6.96 (m, 2H, 2xAr-H), 6.85 (dd, J = 7.8, 2.3 Hz, 1H,
6
2
2
for 3-7 h. After completion of reaction monitored by TLC, water
was poured over the reaction mixture (15 mL), and the solution
1
3
Ar-H), 5.38 (bs, 2H, NH ), 3.93 (s, 3H, OCH ). C NMR (75
2
3
was further neutralized with a 5% NaHCO solution. Extraction
MHz, DMSO-d ) δ ppm 192.13, 157.77, 149.29, 146.57, 144.45,
3
6
was carried out with EtOAc (3x 20 mL), the combined organic
layers were dried over anhydrous Na SO and solvent was
completely removed. The crude was purified by flash
chromatography (7:3 Hexane/EtOAc) to afford the desired amino
derivatives (5-12).
143.33, 136.69, 129.40, 127.12, 125.04, 120.75, 119.43, 118.81,
+
117.22, 115.60, 114.21, 56.34. MS EI m/z (%): 296 ([M+1] , 31),
2
4
+
295 ([M] , 100), 266 (16), 120 (82). 92 (58), 65 (26). Anal.
Calcd. for C H NO : C, 69.15; H, 4.44; Found: C, 69.01; H,
1
7
13
4
4.45.
3
-(4’-Aminobenzoyl)coumarin (5): Yield (67%), mp 201-203
3-(3’-Aminobenzoyl)-8-ethoxycoumarin (11): Yield (69%), mp
1
1
ºC; H NMR (300 MHz, DMSO-d ) δ ppm 8.16 (s, 1H, H-4),
229-230 ºC; H NMR (300 MHz, DMSO-d ) δ ppm 8.26 (s, 1H,
6
6
7
7
5
1
.78 (d, J = 7.5 Hz, 1H, H-5), 7.71-7.56 (m, 3H, H-7, H2’, H-6’),
.49-7.32 (m, 2H, H-6, H-8), 6.59 (d, J = 8.2 Hz, 2H, H-3’, H-
H-4), 7.32-7.18 (m, 3H, 3xAr-H), 7.09 (t, J = 7.8 Hz, 1H, Ar-H),
7.01-6.84 (m, 2H, 2x Ar-H), 6.75 (dd, J = 7.8, 2.3 Hz, 1H, Ar-H),
5.32 (bs, 2H, NH ), 4.05 (q, J = 7.0 Hz, 2H, CH ), 1.35 (t, J = 7.0
1
3
’), 6.27 (bs, 2H, -NH2). C NMR (75 MHz, DMSO-d ) δ ppm
6
2
2
1
3
88.39, 158.41, 154.93, 153.83, 142.73, 132.95, 132.65, 129.39,
Hz, 3H, CH3). C NMR (75 MHz, DMSO-d ) δ ppm 192.15,
6
1
2
1
27.91, 124.93, 123.27, 118.51, 116.39, 112.79. MS EI m/z (%):
158.26, 148.52, 146.27, 144.45, 143.33, 136.59, 129.44, 127.77,
125.14, 120.67, 119.33, 118.81, 117.22, 115.60, 114.21, 65.50,
15.12. MS EI m/z (%): 310 ([M+1] , 20), 309 ([M] , 62), 120
+
+
66 ([M+1] , 40), 265 ([M] , 89), 237 (18), 236 (17), 121 (31),
20 (100), 89 (16), 65 (63), 63 (18). Anal. Calcd. for C H NO
3:
+
+
1
6
11
C, 72.45; H, 4.18; Found: C, 72.27; H, 4.02.
(100), 92 (28), 65 (18). Anal. Calcd. for C H NO : C, 69.89; H,
1
8
15
4
4
.89; Found: C, 69.75; H, 4.81.
3
-(4’-Aminobenzoyl)-8-methoxycoumarin (6): Yield (82%),
1
mp 165-167 ºC; H NMR (300 MHz, DMSO-d ) δ ppm 8.16 (s,
H, H-4), 7.62 (dd, J = 8.8, 2.3 Hz, 2H, H-2’, H-6’), 7.43-7.19
3-(3’-Aminobenzoyl)-6-bromocoumarin (12): Yield (61%), mp
6
1
1
205-207 ºC; H NMR (300 MHz, DMSO-d ) δ ppm 8.26 (s, 1H,
6
(
6
m, 3H, H-5, H-6, H-7), 6.56 (d, J = 8.8 Hz, 2H, H-3’, H-5’),
.33 (bs, 2H, NH ), 3.92 (s, 3H, OCH ). C NMR (75 MHz,
2 3
H-4), 8.07 (d, J = 2.9 Hz, 1H, H-5), 7.85 (dd, J = 8.9, 2.9 Hz, 1H,
H-7), 7.45 (d, J = 8.9 Hz, 1H, H-8), 7.25-6.96 (m, 3H, H-2’, H-
5’, H-6’), 6.86 (d, J = 7.3 Hz, 1H, H-4’), 5.38 (s, 2H, NH2). C
1
3
13
DMSO-d ) δ ppm 188.38, 154.93, 146.55, 142.94, 132.63,
6
1
28.10, 124.84, 123.25, 120.44, 119.07, 115.07, 112.76, 56.30.
NMR (75 MHz, DMSO-d ) δ ppm 191.74, 157.59, 153.11,
149.34, 142.86, 136.51, 135.73, 131.65, 129.44, 127.85, 120.19,
6
+
+
MS EI m/z (%): 296 ([M+1] , 23), 295 ([M] , 69), 121 (15), 120
(
4
100), 92 (28), 65 (17). Anal. Calcd. for C H NO C, 69.15; H,
.44; Found: C, 69.12; H, 4.43.
119.63, 118.79, 117.51, 116.54, 114.05. MS EI m/z (%): 345
1
7
13
4
+
+
([M+2] , 70), 343 ([M] , 70), 120 (100), 92 (85), 65 (52). Anal.
Calcd. for C H BrNO : C, 55.84; H, 2.93; Found: C, 55.77; H,
1
6
10
3
3
-(4’-Aminobenzoyl)-8-ethoxycoumarin (7): Yield (62%), mp
1
2.89.
2
21-222 ºC; H NMR (300 MHz, DMSO-d ) δ ppm 8.16 (s, 1H,
6
H-4), 7.61 (d, J = 8.7 Hz, 2H, H-2’, H-6’), 7.42-7.22 (m, 3H, H-
, H-6, H-7), 6.56 (d, J = 8.7 Hz, 2H, H-3’, H-5’), 6.33 (bs, 2H,
NH ), 4.18 (q, J = 6.9 Hz, 2H, CH ), 1.40 (t, J = 6.9 Hz, 3H,
5
2
.2. Antibacterial study
2
2
1
3
CH3). C NMR (75 MHz, DMSO-d ) δ ppm 188.38, 158.22,
6
2.2.1 Bacterial strains
1
1
54.92, 145.78, 143.02, 132.62, 128.11, 124.84, 123.26, 120.39,
On the present study a total of seventeen bacterial strains
19.16, 115.92, 112.74, 64.58, 14.76. MS EI m/z (%): 310
+
+
isolated from diseased fish belonging to the species
Tenacibaculum maritimum, Tenacibaculum discolor,
([M+1] , 24), 309 ([M] , 67), 281 (12), 280 (11), 121 (18), 120
(
100), 92 (29), 65 (20). Anal. Calcd. for C H NO : C, 69.89; H,
1
8
15
4
Tenacibaculum gallaicum, Tenacibaculum soleae and
Tenacibaculum ovolyticumwere used. Reference strains of
Tenacibaculum spp., Escherichia coli, Staphylococcus aureus
and Pseudomonas aeruginosa were included with comparative
purpose.
4
.89; Found: C, 69.68; H, 4.75.
3
-(4’-Aminobenzoyl)-6-bromocoumarin (8): Yield (42%), mp
1
2
43-245 ºC; H NMR (300 MHz, DMSO-d ) δ ppm 8.11 (s, 1H,
6
H-4), 8.03 (d, J = 2.5 Hz, 1H, H-5), 7.81 (dd, J = 8.9, 2.5 Hz, 1H,
H-7), 7.63 (d, J = 8.6 Hz, 2H, H-2’, H-6’), 7.43 (d, J = 8.9 Hz,
1
The Tenacibaculum spp. strains were cultured on Flexibacter
H, H-8), 6.57 (d, J = 8.3 Hz, 2H, H-3’, H-5’), 6.32 (bs, 2H,
4
9
13
maritimus agar medium (FMM agar), at 25 ºC for 48 h.
NH2). C NMR (75 MHz, DMSO-d ) δ ppm 187.93, 157.92,
6

Staphylococcus aureus, Escherichia coli, Pseudomona
aeruginosa and Aeromona salmonicida were cultured on Mueller
Hinton Agar, supplemented with 1 % NaCl (MHA-1) for 24-48 h
at 37 ºC and 18 ºC, respectively. Antimicrobial susceptibility
tests minimum inhibitory concentration (MIC) and minimum
bactericidal (MCB) concentration were carried out using 24 h
cultures of all strains. All strains were kept frozen at -30 ºC in the
completely inhibit the growth of the microorganism. The MIC
was established by comparing the amount of growth in the wells
containing coumarin-chalcone hybrids with the amount of growth
in the growth-control wells (no antimicrobial agent) as detected
by the unaided eye or reading (OD620) the microdilution tests
with a spectrophotometer (Microplate Reader Model 680,
BioRad).
TM
commercial medium Microbank (Pro-Lab Diagnostics) until
use.
2
.2.2.3. Minimum Bactericidal Concentration (MBC) assay.
The MBC was estimated by spectrophotometric and culture
methods. Eight strains of T. maritimum were used in this assay.
For culture method, the initial inoculum, the dilution representing
the MIC and at least two of the more concentrated test product
dilutions were plated and enumerated to determine viable
CFU/mL by using the plate dilution method. The cultures were
incubated at 25 ºC for 48 to 72 h and the antimicrobial
concentration that cause a reduction of the 99.9% of the original
inoculum was considered the MBC. The reduction of number of
viable cells was also measured by a colorimetric assay based on
the reduction of a tetrazolium salt (MTT method). Briefly, viable
bacteria present in the wells were quantified by the addition of
2
.2.2. Antibacterial activity
2
.2.2.1. Disk diffusion method assay
The antibacterial activity of the compounds was assayed by
the disk diffusion method, following the procedures of the
Clinical and Laboratory Standards Institute (CLSI, 2006). The
inoculum was prepared in sterile saline from a 24 h Mueller
Hinton Agar (MHA) (Cultimed, Spain) plate culture of reference
strains of E. coli, P. aeruginosa and S. aureus and from a 48 h
4
8
4
9
Flexibacter maritimum medium (FMM) plate culture of
Tenacibaculum spp. strains, containing approximately 1-2x10
9
colony forming units (CFU/mL) (OD620 of 0.08 to 0.13). Colony
counts on inoculum suspension were verified by the plate
dilution method using MHA and FMM plates and counting the
bacterial colonies produced. The entire surface of the MHA plate
was inoculated by streaking with the swab containing the
inoculum. Antimicrobial solution (concentration of 10000
µg/mL) was prepared by using DMSO as solvent. Sterile disks of
1
0µL of [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] (MTT, 5mg/mL), which is reduced in proportion to the
amount of viable bacteria present. The optical density was read at
6
20 nm 15 min later on a spectrophotometer (Microplate Reader
Model 680, BioRad). Experiments were carried out in triplicate.
2.2.3. Cytotoxicity test.
6
mm diameter (Liofilchem, Italy) embedded in the drug at a
Coumarin-chalcone hybrids 1-12 were assayed for
cytotoxicity in Epithelioma papillosum Cyprini (EPC) fish cell
line. The EPC cells were grown in 24 well flat bottom microtitre
plate (Costar) in Leibovitz' médium (L-15, Gibco BRL)
supplemented with 2% inactivated (56 ºC, 30 min) foetalcalf
serum (iFCS), 1 % penicillin-streptomycin (P/S, Gibco) at 20 ºC
for 48 h. Cell monolayers were inoculated with 0.1 mL of
coumarin-chalcone hybrid samples and incubated at 20 ºC. Total
or partial destruction of monolayers within a 3-day period was
scored as a positive cytotoxic effect. Cell control (cell
monolayers inoculated with sterile saline solution with DMSO
added and non-inoculated cells) was maintained throughout the
experiment.
final concentration of 100 µg/disk were kept on agar surface.
Sterile disks embedded with DMSO were used as a negative
control. The diameter of the zone of inhibition was determined
after incubation at 25 ºC (Tenacibaculum spp.) and at 37 ºC (S.
aureus, P. aeruginosa and E. coli) up to 72 h. Since in previous
assays the quality control strain Aeromonas salmonicida subsp.
salmonicida ATCC 33658, recommended by the Clinical
Laboratory Standards Institute (CLSI) document M42-A and
M49-A, was not sensitive to coumarins-chalcone hybrids tested,
the strain of T. maritimum NCIMB 2154, which did show
sensitivity to these compounds in the initial screening, was used
as positive control in all the assays.
Assays were carried out by triplicate and inhibition zones
were expressed in millimetres (average Sd).
2
.3. Theoretical evaluation of ADME properties
The theoretical drug-like properties of compounds 1-12 were
5
0
calculated using the Molinspiration property program.
2
.2.2.2. Minimum inhibitory concentration (MIC) assay.
2
. Results
The MIC for the most active coumarin-chalcone hybrids was
evaluated using the broth microdilution method, according to the
procedures of the Clinical and Laboratory Standards Institute
2
.1. Chemistry
The synthesis of the studied compounds 1-12 was performed
(CLSI, 2006) as above described. Coumarin-chalcone hybrids
following the synthetic route outlined in Scheme 1.
were serially diluted from 250 to 0.061035 µg/mL in FMM broth,
reaching a final volume of 100 µL in each well. Bacterial
Compounds 1-4 were efficiently synthesized with 58-70%
yield via Knoevenagel reaction treating the appropriate
salicylaldehyde and the corresponding ethyl substituted
benzoylacetate with piperidine in ethanol at reflux for 2–5 h. For
the preparation of the amino substituted derivatives 5-12, the
same strategy was employed to achieve the nitro substituted 3-
benzoylcoumarins precursors, which were treated without further
purification with SnCl .2H O in ethanol at reflux for 3-7 h to
9
suspensions, adjusted to contain 1-2x10 CFU/mL, were
inoculated to each well in a 1:10 proportion. FMM broth with
and without inoculum was used as positive and negative control
of growth, respectively. FMM broth with coumarin-chalcone
hybrids was used as blank. The plates were incubated at 25 ºC for
4
8 h. The strain NCIMB 2154 of T. maritimum was used as
quality control in every run. Eight strains of T. maritimum were
used to perform the MIC assay. The assays were carried out by
triplicate. Minimum Inhibitory Concentration (MIC) was defined
as the lowest concentration of antimicrobial agents able to
2
2
achieve the final amino substituted compounds 5-12 in 42-94%
overall yield after purification by flash chromatography in 7:3
Hexane/AcOEt.

ovolyticum tested. The experimental results of this in vitro
antibacterial evaluation for the T. maritimum strains are
summarized in Tables 1a and 1b. All T. marirtimum strains were
highly sensitives to compounds 6, 7, 10 and 11, with inhibition
zones similar to those obtained using enrofloxacin and ampicillin
commercial discs. All the strains were resistant to the compounds
1
and 4. For the remaining compounds the sensitivity varied
depending on the T. maritimum strains.
For the most active compounds (6, 7, 10 and 11) of the series
the MIC and MBC were evaluated in eight of the T. maritimum
strains, and the results are presented in Table 2. All the
compounds assayed showed bactericidal activity against T.
maritimum. Fifty percent of the strains tested were highly
sensitives to the compounds 6 and 11 with CMI and CMB values
in the range of 0.1-1.9 µg/mL, and showed intermediate
sensitivity for the remaining compounds tested (CMI and CMB
values in the range of 3.9-15.6 µg/mL). Thirty eight percent of
the strains were resistant to all the compounds tested with CMI
and CMB values of 62,5 to 125 µg/mL while the strain JIP31/99
showed intermediate sensitivity (CMI and CMB values in the
range of 7.8 to 15.6 µg/mL) for compounds 6, 7, 10 and 11.
Scheme 1. Reagents and conditions: i) EtOH, piperidine,
reflux, 2-5 h; ii) SnCl .2H O, EtOH, reflux, 3-7 h.
2
2
3
.2. Antibacterial study
In order to test the antibacterial activity of the target
compounds, the disk diffusion test was employed. The
antimicrobial drugs, oxolinic acid, enrofloxacin (quinolone
antibiotic, Figure 1) and ampicillin (-lactamic antibiotic), were
used as positive controls. The antibacterial activity of compounds
1
-12 was evaluated in vitro by measuring the inhibition zone (in
millimeters) for growth inhibitory effect over one Gram-positive
bacterium (S. aureus) and two Gram-negative bacteria (E. coli
and P. aeruginosa) and seventeen different strains of Gram-
negative marine bacteria of the Tenacibaculum genus. The
antibacterial activities were tested at concentrations of 100
3.3. Theoretical evaluation of ADME properties
In order to explore the drug-like properties of compounds 1-
2, the lipophilicity, expressed as the octanol/water partition
1
coefficient and herein called logP, as well as other theoretical
calculations such as the TPSA, the number of hydrogen acceptors
and the number of hydrogen bond donors were calculated.
Theoretical prediction of ADME properties of all compounds is
summarized in Table 3.

g/disk in DMSO. The bacterial cultures were inoculated and
incubated according to the described protocol of the experimental
section. The cytotoxicity of compounds 1-12 was also studied in
EPC (epithelioma papillosum Cyprini ) fish cell line.
Compounds 1-12 presented no significant inhibition zones
neither for the human pathogenic bacteria species nor the fish
bacteria strains T. discolor, T. gallaicum, T. soleae, and T.

a
Table 1a. In vitro antibacterial activity of the synthesized compounds 1-12 against the T. maritimum strains NCIMB 2154, Lyl-1,
b
LL01 8.3.8, LL01 8.3.1, LC 12.10.1.2, JIP 10/97 and JIP 31/99 expressed as inhibition zones (mm).
Cmpound
NCIMB 2154
Lyl-1
LL01 8.3.8
LL01 8.3.1.
LC 12.10.1.2
JIP 10/97
JIP 31/99
1
2
3
4
5
6
7
8
9
NA
NA
NA
NA
NA
NA
NA
NA
NA
12 
12 
NA
NA
NA
12.3  0.2
NA
NA
11.8  0.3
NA
12.1  0.3
NA
12.2  0.4
NA
NA
NA
NA
NA
NA
14.4  0.5
34.1  0.5
31.5  0.6
NA
15.8  0.6
37.7  0.3
32.1  0.2
9.4  0.2
15.8  0.5
27.4  0.5
34.2  0.3
14.9  0.3
17.4  0.3
30.2  0.2
38.3  0.2
14.5  0.6
41.4  0.2
36.5  0.4
11.2  0.6
23.8  0.4
32.3  0.1
37.4  0.3
16.2  0.2
15.1  0.3
25.2  0.1
33.1  0.3
14.5  0.6
41.3  0.2
36.3  0.2
11.4  0.5
23.7  0.4
32.5  0.3
37.2  0.2
16.3  0.3
17.5  0.3
27.1  0.2
34.2  0.1
NA
11.5  0.2
34.4  0.3
31.3  0.3
NA
12.3  0.2
34.5  0.3
28.4  0.2
NA
35.3  0.2
35.1  0.3
NA
NA
NA
10.8  0.3
24.1  0.2
31.2  0.2
10.3  0.6
15.3  0.2
30.3  0.1
40.4  0.3
11.7  0.3
16.1  0.4
28.2  0.4
9.3  0.2
14.9  0.2
30.3  0.1
30.3  0.2
1
1
1
0
1
2
23.9  0.6
33.2  0.4
13.4  0.3
15.1  0.4
27.5  0.2
32.2  0.1
30.4  0.4
35.4  0.3
13.9  0.2
16.2  0.3
26.1  0.1
35.2  0.2
Oxolinic A.
Enrofloxacin
Ampicillin
a
These results are average results of three experiments.
The compounds were used at the concentration of 100 μg/disk and the inhibition zones are stated in mm; NA = not active; diameter of inhibition zone ≤ 5 mm.
b
a
b
Table 1b. In vitro antibacterial activity and cytotoxicity of the synthesized compounds 1-12 against the T. maritimum strains LC07
c
1
0.1.2, DBa4a, 3.35, Tm Chile, RQ 07 10.1.2, LL 05 6.1.2 and LP2R expressed as inhibition zones (mm).
Cmpound.
LC07 10.1.2.
DBa4a
3.35
Tm Chile
RQ 07 10.1.2.
LL 05 6.1.2.
LP2R
Cytotoxicity
1
2
3
4
5
6
7
8
9
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
–
–
–
–
–
–
–
–
+
–
–
+
–
–
–
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
11.5  0.4
30.2  0.2
25.4  0.1
NA
10.5  0.4
30.2  0.3
31.1  0.2
NA
11.4  0.6
30.3  0.2
23.2  0.3
NA
17.3  0.5
41.2  0.4
NA
12.3  0.5
32.3  0.4
26.2  0.4
NA
NA
28.3  0.2
25.2  0.2
NA
29.3  0.2
24.4  0.3
NA
NA
22.4  0.3
18.1  0.5
28.2  0.3
NA
NA
NA
23.3  0.3
22.1  0.4
27.2  0.3
10.2  0.4
17.4  0.3
30.1  0.3
32.3  0.2
15.9  0.3
NA
NA
NA
1
1
1
0
1
2
17.3  0.3
28.4  0.1
11.3  0.1
20.8  0.2
30.4  0.1
31.8  0.2
15.2  0.3
30.4  0.2
10.1 0.3
22.3  0.4
35.9  0.2
40.1  0.2
19.2  0.2
28.3  0.3
15.4  0.3
18.4  0.2
30.1  0.2
31.3  0.2
NA
41.3  0.2
22.2  0.5
20.3  0.2
28.1  0.1
30.3  0.2
24.2  0.3
10.5 0.4
15.9  0.3
31.4  0.1
37.3  0.2
Oxolinic A.
Enrofloxacin
Ampicillin
18.4  0.3
28.1  0.2
34.3  0.2
a
These results are average results of three experiments.
No cytotoxic (–); cytotoxic (+)
b
c
The compounds were used at the concentration of 100 μg/disk and the inhibition zones are stated in mm; NA = not active; diameter of inhibition zone ≤ 5 mm.

Table 2. MICs and MBCs expressed in M of the most active compounds against eight strains of T. maritimum spp.
Compound 6
Compound 7
MIC MBC
Compound 10
Compound 11
Enrofloxacin
MIC MBC
Bacterial strain
NCIMB 2154
MIC
MBC
62.5
MIC
MBC
62.5
MIC
MBC
62.5
62.5
62.5
62.5
7.8
62.5
62.5
62.5
7.8
62.5
62.5
62.5
7.8
62.5
62.5
125
15.6
3.9
62.5
62.5
62.5
15.6
0.1
2
2
Tm Chile
LP2R
62.5
62.5
7.8
62.5
125
31.3
3.9
62.5
125
15.6
0.1
0.5
1
0.5
1
JIP 31/99
LL01 8.3.8.
LL01 8.3.1
DBa4a
2
2
0.5
0.5
0.2
0.2
2
2
0.5
0.5
0.2
0.2
3.9
3.9
0.1
0.1
2
2
0.5
1.0
15.6
7.8
15.6
3.9
7.8
15.6
7.8
0.5
1.9
0.5
1
0.5
1
3
.35
1.9
1.9
7.8
1.0
1.9
a
Table 3. Theoretical structural properties of the coumarin-chalconehybrids.
n-OH
n-OHNH
Cmpd.
logP
M.W. (g/mol)
TPSA
Volume
acceptors
donors
1
2
3
4
5
6
7
8
9
3.397
3.406
3.782
4.182
2.473
2.482
2.858
3.258
2.449
2.458
2.834
3.234
250.25
280.28
294.31
329.15
265.27
295.30
309.32
344.16
265.27
295.30
309.32
344.16
47.282
56.516
56.516
47.282
73.305
82.539
82.539
73.305
73.305
82.539
82.539
73.305
3
4
4
3
4
5
5
4
4
5
5
4
0
0
0
0
2
2
2
2
2
2
2
2
218.979
244.525
261.326
236.864
230.267
255.813
272.615
248.153
230.267
255.813
272.615
248.153
1
1
0
1
2
1
a
TPSA, topological polar surface area; n-OH, number of hydrogen acceptors; n-OHNH, number of hydrogen bond donors. The data was determined with
Molinspiration calculation software.
Regarding the 4’-amino substituted compounds (5-8),
compounds 6 and 7 were the most potent of this series,
presenting antibacterial activities higher or in the same range than
the enrofloxacin or ampicillin and twice more potent than
oxolinic acid for almost all the studied T. maritimum strains. The
same tendency was observed when studying the 3’-amino
substituted compounds 9-12, presenting inhibition zones with the
same pattern than the 4’amino substituted compounds, but
showing a slightly decrease in their potency. It is worth to
mention that for the 4’ and 3’-amino substituted compounds 5-
3
. Discussion
The obtained results revealed that all tested compounds 1-12
were inactive against the human pathogenic bacteria strains of E.
coli, S. aureus and P. aeruginosa. However most of the
synthesized compounds (5-12) presented antibacterial activity
against the T. maritimum marine bacteria strains, showing
inhibitions zones ranging between 10 to 41 mm, but were not
active against the other Tenacibaculum species (T. discolor, T.
gallaicum, T. soleae, and T. ovolyticum). Compounds with no
amino substituents (1-4) were inactive against the T. maritimum
strains, with exception of compound 3, which resulted to be
slightly sensitive to four of the T. maritimum strains (NCIMB
1
2, the most active molecules bear either a methoxy or ethoxy
group at the position 8 of the coumarin moiety (compounds 6 and
, and 10 and 11 respectively). Thus, amino 8-substituted
7
coumarin-chalcone derivatives (6, 7, 10 and 11) presented
antibacterial activity witn inhibition zones ranging from 15 to 41
mm for the fourteen T. maritimum strains, reaching inhibition
zone values almost twice higher than enrofloxacin, the currently
used drug for the treatment of tenacibaculosis. Therefore, when a
methoxy or ethoxy substituent at position 8 of the coumarin core
was introduced, the activity of these series notably increased
respect to the simplest derivatives of the series, compounds 5 and
2
154, LL01 8.3.8, LL01 8.3.1, and LC 12.10.1.2), presenting
aninhibition zones of 12 mm for all of them. However, when
introducing an amino substituent on the 3-benzoyl moiety in
either 3’ or 4’ positions (compounds 5-12), these compounds
improved their antimicrobial activity with inhibition zones from
good to excellent against almost all the T. maritimum strains.
9
, which have no substituents on the coumarin core. However,

when a substituent was introduced at position 6 of the coumarin
moiety (compounds and 12), the activity decreased
significantly or it was even lost.
In conclusion, the non toxicity of the compounds and the high
activity and selectivity only against the marine bacteria can avoid
drug resistance problems in humans, making these compounds
good candidates for the treatment of tenacibaculosis.
8
For the most promising molecules (compounds 6, 7, 10 and
1
1), the MICs and the MCBs were calculated for eight T.
maritimum strains. The data show that compounds 6 and 11 are
as active as enrofloxacin for DBa4a and 3.35 T. maritimum
strains. However, the highest activity was against the T.
maritimum strains LL01 8.3.8 and LL01 8.3.1 for compounds 6,
Acknowledgments
The authors thank the Foundation for Science and Technology
(FCT) of Portugal (PEst-C/QUI/UI0081/2013 project) and the
University of Santiago. S.V.R. thanks to the Xunta de Galicia for
the postdoctoral grant ED481B 2014/027-0. M.J.M. thanks to the
Fundação para a Ciência e Tecnologia (FCT), POPH (Programa
Operacional Potencial Humano) and QREN (Quadro de
Referência Estratégica Nacional) for the postdoctoral grant
7
and 11, presenting MIC values up to 20 times higher than
enrofloxacin.
The cytotoxicity studies of the compounds also revealed that
the molecules were non toxic when studied in EPC (epithelioma
papillosum of carp) fish cell line, with exception of compounds 9
and 12 that presented a weak sign of cytotoxicity at the tested
concentration, but these molecules did not present significant
activity.
(SFRH/BPD/95345/2013).
Reference and notes
On the other hand, analyzing the obtained theoretical results
of the ADME properties, it can be observed that no violations of
Lipinski’s rule (molecular weight, logP, number of hydrogen
donors and acceptors) were found for the described compounds
1
2
Food and Agriculture Organization of the United Nations. FAO
Fisheries and Aquaculture Department has published the Global
Aquaculture Production Statistics for the year 2011.
ftp://ftp.fao.org/fi/news/GlobalAquacultureProductionStatistics201
1
-12. Also, TPSA, described to be a predictive indicator of
1
.pdf
membrane penetration, was found to be positive for these
antibacterial drug candidates.
Suzuki, M.; Nakagawa, Y.; Harayama, S.; Yamamoto, S.
Phylogenetic analysis and taxonomic study of marine Cytophaga-
like bacteria: proposal for Tenacibaculum gen. nov.with
Tenacibaculum maritimum comb. nov.and Tenacibaculum
ovolyticum comb. nov., and description of Tenacibaculum
mesophilum sp. nov.and Tenacibaculum amylolyticum sp. nov.Int.
J. Syst. Evol. Microbiol. 2001, 51, 1639–1652.
McVicar, A. H.; White, P. G. Fin and skin necrosis of cultivated
Dover sole, Solea solea. J. Fish Dis. 1979, 2, 557–562.
Alsina, M.; Blanch, A. R. First isolation of Flexibacter maritimus
from cultivated turbot (Scophthalmus maximus). Bull.Eur. Assoc.
Fish Pathol. 1993, 13, 157–160.
It is worth to mention that the synthesized series of
compounds not only present high activity and no toxicity, but
also present high selectivity against the marine fish pathogen
Tenacibaculum maritimum. The fact that these compounds do not
present any activity on the human pathogenic bacteria it can be
seen as an advantage since it could avoid possible drug resistance
problems in humans if used as a treatment for tenacibaculosis in
aquaculture.
3
4
5
Ostland, V. E.; LaTrace, C.; Morrison, D.; Ferguson, H. W.
Flexibacter maritimus associated with a bacterial stomatitis in
Atlantic salmon smolts reared in net-pens in British Columbia. J.
Aquat. Anim. Health 1999, 11, 35–44.
Concluding Remarks
A series of coumarin-chalcone hybrid compounds have been
designed and synthesized and their antibacterial activity against
human pathogenic bacteria (E. coli, S. aureus and P. aeruginosa)
and different species of the genus Tenacibaculum were tested.
Compounds were non-active neither against any human
pathogenic bacteria nor against the Tenacibaculum species T.
discolor, T. gallaicum, T. soleae and T. ovolyticum. However,
compounds were highly selective and presented high antibacterial
activity against fourteen T. maritimum strains. Compounds that
did not bear an amino group were non active, while those having
an amino group at positions 3’ or 4’ showed antibacterial activity
against T. maritimum strains. Therefore, the presence of an amino
group in the benzoyl moiety (compounds 5-12) resulted to be a
key substitution for the activity of compounds against T.
maritimum. Additional substitutions on the coumarin core at
position 8 or 6 also played a key role on their activity against T.
maritimum, indicating that substitutions at position 8 (compounds
6
7
8
9
Cepeda, C.; Santos, Y. First isolation of Flexibacter maritimus
from farmed Senegalese sole (Solea senegalensis, Kaup) in Spain.
Bull. Eur. Assoc. Fish Pathol. 2002, 22, 388–392.
Baxa, D. V.; Kawai, K.; Kusuda, R. Characteristics of gliging
bacteria isolated from diseases cultured flounder Paralichthys
olivaceous. Fish Pathol. 1986, 21, 251–258.
McVicar, A. H.; White, P. G. The prevention and cure of an
infectious disease in cultivated juvenile Dover sole, Soleasolea
(
L.). Aquaculture 1982, 26, 213–222.
Toranzo, A. E.; Magariños, B.; Romalde, J. L. A review of the
main bacterial fish diseases in mariculture systems. Aquaculture.
2
005, 246, 37–61.
10 Santos, Y.; Pazos, F.; Barja, J. L. Flexibacter maritimus, causal
agent of flexibacteriosis in marine fish. In: Olivier, G. (Ed.), ICES
Identification Leaflets for Diseases and Parasites of Fish and
Shellfish. No. 55. International Council for the Exploration of the
Sea. Copenhagen, Denmark, 1999 pp. 1–6.
1
1
Baxa, D. V.; Kawai, K.; Kusuda, R. Chemotherapy of Flexibacter
maritimus infection. Rep. USA Mar. Biol. Inst. Kochi Univ. 1988,
1
0, 9–14.
1
2
Avendaño-Herrera, R. Avances en el conocimiento del patógeno
de peces Tenacibaculum maritimum: implicaciones en el
diagnóstico y prevención de la enfermedad. Ph. D. Thesis,
Universidad de Santiago de Compostela, 2005.
6
, 7, 10 and 11, the most active of the series) increases their
activity, whereas substitution at position 6 (compounds 8 and 12)
decreases T. maritimum antibacterial activity. These compounds
showed inhibition zones in the same range or higher than the
currently drug used for the treatment of tenacibaculosis,
enrofloxacin, which is not selective against T. maritimum since it
is also highly active against the tested human pathogenic
bacteria. The most active molecule was compound 11, presenting
MIC values 20 more active than enrofloxacin against the T.
maritimum strains LL01 8.3.8 and LL01 8.3.1. In addition, the
tested compounds were non cytotoxic in EPC and presented
appropriate ADME properties.
13 Williams, P. J.; Courtenay, S. C.; Vardy, C. Use of enrofloxacin to
control atypical Aeromonas salmonicida in atlantic tom cod. J.
Aquat. Anim. Health 1997, 9, 216–222.
1
4
Hsu, H. M.; Wooster, G. A.; Bowser, P. R. Efficacy of
Enrofloxacin for the Treatment of Salmonids with Bacterial
Kidney Disease, Caused by Renibacterium salmoninarum. J.
Aquat. Anim. Health 1994, 6, 220–223.
15 Bradbury, B. J.; Pucci, M. J. Recent advances in bacterial
topoisomerase inhibitors. Curr. Opin. Pharmacol. 2008, 8, 574–
5
81.

1
6
Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte,E.
Simple coumarins and analogues in medicinal chemistry:
occurrence, synthesis and biological activity. Curr. Med. Chem.
study using various yeast strains. Eur. J. Med. Chem. 2008, 43,
2220–2228.
36 Cheenpracha, S.; Karalai, C.; Ponglimanont, C.; Subhadhirasakul,
S.; Tewtrakul, S. Anti-HIV-1 protease activity of compounds from
Boesenbergiapandurata. Bioorg. Med. Chem. 2006, 14, 1710–
1714.
37 Wu, J. H.; Wang, X. H.; Yi, Y. H.; Lee, K. H. Anti-AIDS agents
54. A potent anti-HIV chalcone and flavonoids from genus
Desmos. Bioorg. Med. Chem. Lett. 2003, 13, 1813–1815.
38 Rojas, J.; Payá, M.; Domínguez, J. N.; Ferrándiz, M. L. ttCH, a
selective inhibitor of inducible nitric oxide synthase expression
with antiarthritic properties. Eur. J. Pharmacol. 2003, 465, 183–
189.
2
005, 12, 887–916.
1
1
7
8
Borges, F.; Roleira, F.; Milhazes, N.; Uriarte, E.; Santana, L.
Simple coumarins: Privileged scaffolds in medicinal chemistry.
Front. Med. Chem. 2009, 4, 23–85.
Melagraki, G.; Afantitis, A.; Igglessi-Markopoulou, O.; Detsi, A.;
Koufaki, M.; Kontogiorgis, C.; Hadjipavlou-Litina, D. J. Synthesis
and evaluation of the antioxidant and anti-inflammatory activity of
novel coumarin-3-aminoamides and their alpha-lipoic acid
adducts. Eur. J. Med. Chem. 2009, 44, 3020–3026.
1
9
Symeonidis, T.; Fylaktakidou, K. C.; Hadjipavlou-Litina, D. J.;
Litinas, K. E. Synthesis and anti-inflammatory evaluation of novel
angularly or linearly fused coumarins. Eur. J. Med. Chem. 2009,
39 Nowakowska, Z. A review of anti-infective and anti-inflammatory
chalcones. Eur. J. Med. Chem. 2007, 42, 125–137.
4
4, 5012–5017.
40 Vazquez-Rodriguez, S.; Matos, M. J.; Santana, L.; Uriarte, E.;
Borges, F.; Kachler, S.; Klotz, K. N. Chalcone-based derivatives
as new scaffolds for hA3 adenosine receptor antagonists. J.
Pharm. Parmacol. 2013, 65, 697–703.
2
2
0
1
Kostova, I.; Bhatia, S.; Grigorov, P.; Balkansky, S.; Parmar, V. S.;
Prasad, A. K.; Saso, L. Coumarins as antioxidants. Curr. Med.
Chem. 2011, 18, 3929–3951.
Rodriguez, S. A.; Nazareno, M. A.; Baumgartner, M. T. Effect of
different C3-aryl substituents on the antioxidant activity of 4-
hydroxycoumarin derivatives. Bioorg. Med. Chem. 2011, 19,
41 Matos, M. J.; Vazquez-Rodriguez, S.; Borges, F.; Santana, L.;
Uriarte, E. Synthesis of 3-arylcoumarins via Suzuki-cross-
coupling reactions of 3-chlorocoumarin.Tetrahedron Lett.2011,
72, 1225–1227.
6
233–6238.
2
2
2
2
2
3
4
5
Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yanez, M.; Fraiz,
N.; Alcaide, C.; Cano, E.; Orallo, F. Design, synthesis, and
vasorelaxant and platelet antiaggregatory activities of coumarin-
resveratrol hybrids. Bioorg. Med. Chem. Lett. 2006, 16, 257–261.
Belluti, F.; Fontana, G.; Bo, L.; Carenini, N.; Giommarelli, C.;
Zunino, F. Design, synthesis and anticancer activities of stilbene-
coumarin hybrid compounds: Identification of novel proapoptotic
agents. Bioorg. Med. Chem. 2010, 18, 3543–3550.
Riveiro, M. E.; Moglioni, A.; Vazquez, R.; Gomez, N.; Facorro,
G.; Piehl, L.; de Celis, E. R.; Shayo, C.; Davio, C. Structural
insights into hydroxycoumarin-induced apoptosis in U-937 cells.
Bioorg. Med. Chem. 2008, 16, 2665–2675.
42 Vazquez-Rodriguez, S.; Figueroa-Guíñez, R.; Matos, M. J.;
Santana, L.; Uriarte, E.; Lapier, M.; Maya, J. D.; Olea-Azar, C.
Synthesis of coumarin–chalcone hybrids and evaluation of their
antioxidant and trypanocidal properties. Med. Chem. Commun.
2013, 4, 993–1000.
43 Pérez-Cruz, F.; Vazquez-Rodriguez, S.; Matos, M. J.; Herrera-
Morales, A. Villamena, F. A.; Das, A.; Gopalakrishnan, B.; Olea-
Azar, C.; Santana, L.; Uriarte, E. Synthesis and electrochemical
and biological studies of novel coumarin-chalcone hybrid
compounds. J. Med. Chem. 2013, 56, 6136–6145.
44 Matos, M. J.; Vazquez-Rodriguez, S.; Santana, L.; Uriarte, E.;
Fuentes-Edfuf, C.; Santos, Y.; Munoz-Crego, A. Looking for new
targets: simple coumarins as antibacterial agents. Med.
Chem.2012, 8, 1140–1145.
45 Matos M. J.; Vazquez-Rodriguez, S.; Santana, L.; Uriarte, E.;
Fuentes-Edfuf, C.; Santos, Y.; Muñoz Crego, A. Synthesis and
structure-activity relationships of novel amino/nitro substituted 3-
arylcoumarins as antibacterial agents. Molecules. 2013, 18,1394–
404.
46 Rao. H. S. P.; Sivakumar, S. Condensation of -Aroylketene
Dithioacetals and 2-Hydroxyarylaldehydes Results in Facile
Synthesis of a Combinatorial Library of 3-Aroylcoumarins. J.
Org. Chem. 2006, 71, 8715–8723.
47 Tang, X.; Blake, A. J.; Lewis W, Woodward S. Asymmetric
conjugate additions to 1,1′-diactivated cyclic enones—a
comparative study. Tetrahedron: Asymmetry. 2009;20(16):1881–
1891.
48 Clinical and Laboratory Standards Institute (CLSI). Methods for
Antimicrobial Disk Susceptibility Testing of Bacteria Isolated
From Aquatic Animals; Approved Guideline. CLSI document
M42-A (ISBN 1-56238-611-5). Clinical and Laboratory Standards
Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania
19087-1898 USA, 2006.
Chimenti, F.; Bizzarri, B.; Bolasco, A.; Secci, D.; Chimenti, P.;
Granese, A.; Carradori, S.; Rivanera, D.; Zicari, A.; Scaltrito, M.
M.; Sisto, F. Synthesis, selective anti-Helicobacter pylori activity,
and cytotoxicity of novel N-substituted-2-oxo-2H-1-benzopyran-
3
-carboxamides.Bioorg. Med. Chem. Lett. 2010, 20, 4922–4926.
2
2
6
7
Shi, Y.; Zhou, C. Synthesis and evaluation of a class of new
coumarintriazole derivatives as potential antimicrobial
agents.Bioorg. Med. Chem. Lett.2011, 21, 956–960.
Matos, M. J.; Viña, D.; Picciau, C.; Orallo, F.; Santana, L.;
Uriarte, E. Synthesis and evaluation of 6-methyl-3-
phenylcoumarins as potent and selective MAO-B inhibitors.
Bioorg. Med. Chem. Lett.2009, 19, 5053-5055.
Matos, M. J.; Vazquez-Rodriguez, S.; Uriarte, E.; Santana, L.;
Viña, D. MAO inhibitory activity modulation: 3-Phenylcoumarins
versus 3-benzoylcoumarins.Bioorg. Med. Chem. Lett.2011, 21,
2
2
8
9
4
224–4227.
Galm, U.; Heller, S.; Shapiro, S.; Page, M.; Li, S. M.; Heide, L.
Antimicrobial and DNA gyrase-inhibitory activities of novel
clorobiocin derivatives produced by mutasynthesis.Agents
Chemother.2004, 48, 1307–1312.
Sahu, N. K.; Balbhadra, S. S.; Choudhary, J.; Kohli, D. V.
Exploring Pharmacological Significance of Chalcone Scaffold: A
Review. Curr. Med. Chem.2012, 19, 209–225.
Liaras, K.; Geronikaki, A.; Glamoclija, J.; Ciric, A.; Sokovic, M.
Thiazole-based chalcones as potent antimicrobial agents.
Synthesis and biological evaluation.Bioorg. Med. Chem.
Lett.2011, 19, 3135–3140.
3
3
0
1
49 Pazos, F.; Santos, Y.; Macías, A. R.; Núñez, S.; Toranzo, A. E.
Evaluation of media for the successful culture of Flexibacter
maritimum. J. Fish Dis. 1996, 19, 193-197.
50
.
M
cheminformatics,
Bratislava,
Slovak
Republic,
http://www.molinspiration.com/services/properties.html 2009.
3
2
Batovska, D.; Parushev, S.; Stamboliyska, B.; Tsvetkova, I.;
Ninova, M.; Najdenski, H. Examination of growth inhibitory
properties of synthetic chalcones for which antibacterial activity
was predicted.Eur. J. Med. Chem.2009, 44, 2211–2218.
Larsen, M.; Kromann, H.; Kharazmi, A.; Nielsen, S. F.
Conformationally restricted anti-plasmodialchalcones.Bioorg.
Med. Chem. Lett.2005, 15, 4858–4861.
Kumar, R.; Mohanakrishnan, D.; Sharma, A.; Kaushik, N. K.;
Kalia, K.; Sinha, A. K.; Sahal, D. Reinvestigation of structure-
activity relationship of methoxylatedchalcones as antimalarials:
synthesis and evaluation of 2,4,5-trimethoxy substituted patterns
as lead candidates derived from abundantly available natural β-
asarone. Eur. J. Med. Chem. 2010, 45, 5292–5301.
3
3
3
4
3
5
Lahtchev, K. L.; Batovska, D. I.; Parushev, S. P.; Ubiyvovk, V.
M.; Sibirny, A. A. Antifungal activity of chalcones: a mechanistic