Synthesis and structure-activity relationships of novel amino/nitro substituted 3-arylcoumarins as antibacterial agents

Article abstract of DOI:10.3390/molecules18021394

A new series of amino/nitro-substituted 3-arylcoumarins were synthesized and their antibacterial activity against clinical isolates of Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) was evaluated. Some of these molecules exhibited antibacterial activity against S. aureus comparable to the standards used (oxolinic acid and ampicillin). The preliminary structure-activity relationship (SAR) study showed that the antibacterial activity against S. aureus depends on the position of the 3-arylcoumarin substitution pattern. With the aim of finding the structural features for the antibacterial activity and selectivity, in the present manuscript different positions of nitro, methyl, methoxy, amino and bromo substituents on the 3-arylcoumarin scaffold were reported.

Full text of DOI:10.3390/molecules18021394

Molecules 2013, 18, 1394-1404; doi:10.3390/molecules18021394
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Structure-Activity Relationships of
Novel Amino/Nitro Substituted 3-Arylcoumarins as
Antibacterial Agents
Maria J. Matos 1,*, Saleta Vazquez-Rodriguez 1, Lourdes Santana 1, Eugenio Uriarte 1,
Cristina Fuentes-Edfuf 2, Ysabel Santos 2 and Angeles Muñoz-Crego 2
1
Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela,
15782 Santiago de Compostela, Spain; E-Mails: svre77@gmail.com (S.V.-R.);
lourdes.santana@usc.es (L.S.); eugenio.uriarte@usc.es (E.U.)
2
Departamento de Microbiología y Parasitología, Facultad de Biología,
Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain;
E-Mails: cristina.fuentesedfuf@gmail.com (C.F.-E.); ysabel.santos@usc.es (Y.S.);
a.munoz.crego@usc.es (A.M.-C.)
* Author to whom correspondence should be addressed; E-Mail: mariacmatos@gmail.com;
Tel.: +34-981-528-070; Fax: +34-981-594-912.
Received: 24 December 2012; in revised form: 15 January 2013 / Accepted: 17 January 2013 /
Published: 24 January 2013
Abstract: A new series of amino/nitro-substituted 3-arylcoumarins were synthesized and
their antibacterial activity against clinical isolates of Staphylococcus aureus (Gram-positive)
and Escherichia coli (Gram-negative) was evaluated. Some of these molecules exhibited
antibacterial activity against S. aureus comparable to the standards used (oxolinic acid and
ampicillin). The preliminary structure-activity relationship (SAR) study showed that the
antibacterial activity against S. aureus depends on the position of the 3-arylcoumarin
substitution pattern. With the aim of finding the structural features for the antibacterial
activity and selectivity, in the present manuscript different positions of nitro, methyl,
methoxy, amino and bromo substituents on the 3-arylcoumarin scaffold were reported.
Keywords: amino/nitro substituted 3-arylcoumarins; Perkin reaction; antibacterial assays;
S. aureus; E. coli

Molecules 2013, 18
1395
1. Introduction
Drug discovery is considered a complex and slow activity. However, with the intention of
discovering new chemical entities in a new and more efficient way, medicinal chemistry researchers
have developed new approaches and methods. Some of the new products are based on diversity
molecules found and extracted from natural sources. Coumarins are a large family of compounds of
natural or synthetic origin, associated with remarkable pharmacological activities [1,2]. They occur
naturally in plants and microorganisms and approximately 1,000 coumarin derivatives have been
isolated from over 800 species [2]. The fused heterocyclic framework of coumarins has been used as a
prototype scaffold for the synthesis of a wide variety of analogues in order to study and improve their
biological properties. Its structural variety is responsible for the important place they occupy in the
realm of natural products and synthetic organic chemistry [2,3]. Some coumarins have been studied for
their antimicrobial [4–7], cardioprotective [8], anticancer [9,10], antioxidative [11] and enzymatic
inhibition properties [12–17]. Goth described the antibacterial properties of coumarins for the first time
in 1945 when he studied the dicoumarol (Figure 1) [18]. Some other coumarin derivatives have proven
to display antibacterial and antifungal activities [19–22]. A study of coumarin derivatives substituted
on the pyrone ring indicated that 3-carboxyl derivatives show significant activities [23]. Different
4-substituted coumarins, such as 4-chlorocoumarins, exhibit also an interesting antimicrobial
profile [24,25]. Novobiocin (Figure 1), chlorobiocin and coumermycin A1 are important natural
occurring antibiotics in which the coumarin nucleus is present in their skeleton [26].
Figure 1. Chemical structures of dicoumarol and novobiocin.
The most active of them is novobiocin, isolated from Streptomyces niveus and which is mainly
active against Gram-positive bacteria. Although they antagonize the B subunit of the essential E. coli
DNA gyrase supertwisting activity in vitro and the bacterial multiplication, they are not used in the
clinic due to their relatively weak activity against Gram-negative bacteria, side effects and poor water
solubility [4,26]. On the other hand, these three examples are antibiotics, which present activity
against methicillin-resistant S. aureus (MRSA) [26]. The number of multidrug-resistant (MDR)
bacteria is increasing and the Gram-positive MRSA are of particular importance. The ability of

Molecules 2013, 18
1396
S. aureus species to develop resistance to virtually all antibiotics is a major concern and the discovery
of new antimicrobial agents is essential to combat this problem [27]. MRSA are often an important
problem for severe infections in patients, especially if they are immunosuppressed, during long stays in
hospitals [27]. The dramatic worldwide increase of dangerous infections by resistant and multi-resistant
microbes makes, therefore, the search of new molecules and new chemical entities an important topic
in medicinal chemistry [28,29]. As the ideal drug candidate has not been attained, an intensive search
for new and innovative antimicrobials is still needed. Unfortunately, due to economic reasons,
pharmaceutical companies have considerably decreased this effort in recent years [30].
2. Results and Discussion
Based on previous findings in the area [24,25], and in our experience with substituted
3-arylcoumarins [15,31,32], in the present work we proposed the synthesis and antibacterial evaluation
of a series of different substituted amino/nitro 3-arylcoumarins (Scheme 1). With the aim of finding
the structural features for the antibacterial activity and selectivity, we decided to explore the
importance of the nature and position of small groups (methoxy, bromo, nitro, amino and methyl
substituents) into both coumarin nucleus and 3-aryl ring (Scheme 1).
Scheme 1. Synthesis of 3-arylcoumarins (1–11).
Reagents and conditions: (a) NaH, acetic anhydride, r.t., 3 h; (b) H2, Pd/C, ethanol, r.t., 3 h.
New derivatives 2, 4–7 and 9–11, as well as the already described compounds 1, 3 and 8 [33–36],
were efficiently synthesized according to the synthetic protocol outlined in Scheme 1.
3-Arylcoumarins 1–10 were synthesized in a dry Schlenk tube, in presence of sodium hydride and with
acetic anhydride as solvent, at room temperature for three hours. The reaction mixture was purified by
flash chromatography, using hexane/ethyl acetate as eluent in a proportion of 9:1. Starting from
different commercially available substituted salicylaldehydes and the respectively substituted
phenylacetic acids, we obtained ten derivates in good yields (60–75%). The derivative 11 was obtained

Molecules 2013, 18
1397
from 4, in ethanol, with palladium/carbon as catalyst and under hydrogen atmosphere for three hours.
The reaction mixture was purified by flash chromatography, using hexane/ethyl acetate as eluent, in a
proportion of 9:1. We obtained the desired derivate in good yield (80%). Then, two different
methodologies were employed for the antibacterial evaluation of these compounds: disk diffusion test
and microdilution method. Clinical antimicrobial drugs–oxolinic acid and ampicillin–were used as
positive controls. The inhibition zones for growth inhibitory effects of the new compounds and
controls were measured in millimetres (Table 1, method A). The minimum inhibitory concentrations
(MICs) of the new compounds and controls were measured in microgram/millilitre (Table 1, method B).
a
Table 1. In vitro antibacterial activity of amino/nitro substituted 3-arylcoumarins 1–11
(A) expressed as inhibition zones (mm) b and (B) expressed as MICs (µg/mL).
Method A (mm)
S. aureus E. coli
Method B (µg/mL)
S. aureus E. coli
128
Compounds
1
19
25
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
31
-
2
64
32
-
-
3
22
4
22
32
-
5
25
64
-
6
32
8
-
7
14
256
>512
>512
>512
128
2
-
8
NA
NA
NA
18
-
9
10
-
-
11
-
Oxolinic acid
Ampicillin
26
<1
8
32
23
2
a
b
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.
The obtained inhibition zones revealed that all the 6-nitro derivatives (compounds 1–7) presented
antibacterial activity against S. aureus, showing inhibition zones ranging between 14 and 32 mm. The
amino derivative 11 presented activity against the same bacterial strain. The 3-arylcoumarins nitro
substituted only in the 3-aryl ring (compounds 8–10) were inactive against S. aureus in the in vitro
disk diffusion test assays, independently of the relative position of this nitro group (para or meta
positions). From the data, it is shown that compound 6 and ampicillin presented the same inhibition
zone against S. aureus (32 mm) and a higher inhibition zone than the oxolinic acid (26 mm). Hence,
the preliminary experimental data revealed that some of the tested compounds showed a very
interesting activity profile against S. aureus when compared with the standards ampicillin and oxolinic
acid. In addition, the different nature and position of the substituent in the coumarin scaffold seems to
influence the antibacterial activity.
In order to deeply study the antibacterial activities and quantify the previously mentioned
information, MICs of compounds 1–11 against S. aureus were also determined. This second
methodology (method B) is used to clarify and refine the previously results obtained by method A,
which could be influenced by differences in the diffusion of the compounds. This study was performed

Molecules 2013, 18
1398
against the same bacteria strain, by a microdilution assay with serial dilutions (from 512 to 1 μg/mL)
of the synthesized and the reference compounds. MICs of compounds 1–11 against E. coli were not
evaluated, taking into account the lack of inhibition zones in the previous disk diffusion test
(inhibition zones ≤ 5 mm). Analysing the MICs results for S. aureus, it is clear that the presence of a
nitro substituent at the six position of coumarin moiety (compounds 1–7) is more beneficial than its
presence only at meta or para positions of the 3-aryl ring (compounds 8–10).
Compounds with nitro substituents either in the coumarin moiety and in the aryl group (compounds
1 and 4) are not better than compounds where the nitro in the aryl ring is substituted for a methyl or a
methoxy group (compounds 2 and 3 compared to compound 1, and compounds 5 and 6 compared to
compound 4). However, compound 4 (MIC = 32 µg/mL), with the nitro substituent in meta position of
the 3-aryl ring, is better than compound 1 (MIC = 128 µg/mL), with the same substituent in para
position. The presence of a methyl group in the meta position is the best substitution when one nitro
substituent is present at six position of the coumarin moiety (compound 6, MIC = 8 µg/mL). In
general, meta substitutions are more favourable than para, being the meta-methyl substituent the most
active, followed by meta-nitro and finally meta-methoxy. There is one exception to this conclusion.
The introduction of a bromo atom at the same position was the worst substitution of the studied
3-aryl-6-nitrocoumarins (compound 7, MIC = 256 µg/mL). A para-methyl substituent in the 3-aryl
ring (compound 3, MIC = 32 µg/mL) is the best substitution in this para position. Therefore, the
para-methyl substitution is better than the para-nitro and para-methoxy substitutions. Comparing the
two positions in the 3-aryl ring, the substitutions by methyl groups are more favourable for the desired
activity. The substitution of nitro by amino groups significantly decreases the activity of the described
compounds (comparing compounds 4 and 11).
In order to evaluate if there was a correlation between the activity and the lipophilicity, and to better
understand the overall properties of the described compounds, logP (octanol/water partition
coefficient) values were calculated using the Molinspiration property calculation program [37]. The
results are presented in Table 2.
Table 2. LogP (octanol/water partition coefficient) values calculated for the amino/nitro
substituted 3-arylcoumarins 1–11 a.
Compounds
LogP
3,631
3,729
4,120
3,607
3,705
4,096
4,457
3,729
3,705
4,120
1,841
1
2
3
4
5
6
7
8
9
10
11
a The data was determined with Molinspiration calculation software [37].

Molecules 2013, 18
1399
Analysing the obtained data, it can be concluded that there are no linear correlations between the
lipophilicity and the activity of the compounds. Compounds 5 and 6 are two of the best compounds
against S. aureus, presenting intermediate, but different, logP values (3.705 and 4.096 respectively).
The compounds with the lowest (compound 11, 1.841) and the highest (compound 7, 4.457) logP
presented activity. Compound 9, with the same logP as compound 5, resulted to be inactive. Also,
compound 10, with the same lipophilicity profile as compound 3 (logP = 4.12), resulted to be inactive.
Therefore, there are no clear correlation between activity and logP values. It is important to highlight
that the calculated logP values for all the described compounds are lower than 5.
3. Experimental
3.1. Chemistry
Merck supplied the entire chemicals used in the synthesis. Melting points (mp) are uncorrected
were determined using a Reichert Kofler thermopan or in capillary tubes on a Büchi 510 apparatus and
are uncorrected. 1H-NMR (300 MHz) and 13C-NMR (75.4 MHz) spectra were recorded with a Bruker
AMX spectrometer. Chemical shifts (δ) are expressed in parts per million (ppm) using TMS as an
internal standard. Spin multiplicities are given as s (singlet), d (doublet), dd (doublet of doublets) and
m (multiplet). Mass spectrometry was carried out with a Kratos MS-50 or a Varian MAT-711
spectrometer. Elemental analyses were performed by a Perkin-Elmer 240B microanalyzer and were
within ±0.4% of calculated values in all cases. Flash Chromatography (FC) was performed on
silica gel (Merck 60, 230–400 mesh); analytical TLC was performed on precoated silica gel plates
(Merck 60 F254).
General Procedure for the preparation of 3-arylcoumarins 1–10. In a 20 mL dry Schlenk tube, to a
solution of the conveniently substituted salicylaldehyde (2.46 mmol) and the arylacetic acid (2.46 mmol),
in acetic anhydride (6 mL), NaH (2.46 mmol) was added in small portions, and the reaction mixture
was stirred for 3 hours, at room temperature. The obtained crude was filtered and washed with diethyl
ether. The solid was then purified by flash chromatography (hexane/ethyl acetate 9:1) to give the
desired coumarins 1–10.
1
3-(4′-Methoxyphenyl)-6-nitrocoumarin (2). Pale yellow solid, 75% yield. Mp: 62–63 °C. H-NMR
(CDCl3) δ (ppm): 3.80 (s, 3H, OCH3), 7.05 (d, 2H, H-3′, H-5′, J = 9.45 Hz), 7.62–7.69 (m, 3H, H-2′, H-6′,
H-8), 8.36 (s, 1H, H-4), 8.73 (d, 2H, H-5, H-7, J = 2.93 Hz). 13C-NMR (CDCl3) δ (ppm): 55.9, 116.4,
119.7, 127.9, 128.4, 128.6, 129.2, 132.2, 132.5, 134.3, 139.1, 139.4, 151.5, 155.8, 161.2. ESI-MS m/z:
297 (M+, 100). Anal. Calcd. for C16H11NO5: C, 64.65; H, 3.73; Found: C, 64.67; H, 3.76.
3-(3′-Nitrophenyl)-6-nitrocoumarin (4). Pale yellow solid, 69% yield. Mp: 87–88 °C. 1H-NMR (CDCl3) δ
(ppm): 7.57–7.63 (m, 2H, H-8, H-6′), 7.71–7.77 (m, 1H, H-5′), 8.15 (s, 1H, H-4), 8.42-8.63 (m, 4H,
H-4′, H-2, H-5, H-7). 13C-NMR (CDCl3) δ (ppm): 118.1, 120.2, 123.1, 123.3, 124.3. 124.5, 124.7,
129.4, 133.6, 135.1, 141.2, 144.6, 147.9, 159.2, 160.5. ESI-MS m/z: 312 (M+, 100). Anal. Calcd. for
C15H8N2O6: C, 50.70; H, 2.58. Found: C, 50.72; H, 2.60.

Molecules 2013, 18
1400
1
3-(3′-Methoxyphenyl)-6-nitrocoumarin (5). Pale yellow solid, 71% yield. Mp: 53–54 °C. H-NMR
(CDCl3) δ (ppm): 3.71 (s, 3H, OCH3), 6.80 (s, 1H, H-2′), 7.17–7.22 (m, 3H, H-4′, H-5′, H-6′), 7.57 (d,
13
1H, H-8, J = 6.5 Hz), 7.77 (s, 1H, H-5), 8.19 (d, 1H, H-7, J = 6.5 Hz), 8.39 (s, 1H, H-4). C-NMR
(CDCl3) δ (ppm): 55.4, 84.5, 112.4, 115.5, 122.0, 123.5, 125.8, 126.8, 129.5, 129.7, 136.9, 145.7,
153.2, 159.6, 168.7, 173.0. ESI-MS m/z: 297 (M+, 100). Anal. Calcd. for C16H11NO5: C, 64.65; H,
3.73. Found: C, 64.63; H, 3.70.
1
3-(3′-Methylphenyl)-6-nitrocoumarin (6). Pale yellow solid, 69% yield. Mp: 55–56 °C. H-NMR
(CDCl3) δ (ppm): 2.25 (s, 3H, CH3), 6.90–7.30 (m, 5H, H-2′, H-4′, H-5′, H-6′, H-8), 7.75 (s, 1H, H-4),
8.44 (d, 1H, H-7, J = 2.8 Hz), 8.62 (d, 1H, H-5, J = 2.8 Hz). 13C-NMR (CDCl3) δ (ppm): 21.5, 118.4,
123.3, 124.5, 124.6, 124.8, 125.9, 126.6, 128.3, 128.5, 132.7, 138.4, 141.1, 144.6, 159.3, 160.9.
ESI-MS m/z: 281 (M+, 100). Anal. Calcd. for C16H11NO4: C, 68.32; H, 3.94. Found: C, 68.31; H, 3.92.
1
3-(3′-Bromophenyl)-6-nitrocoumarin (7). Pale yellow solid, 60% yield. Mp: 240–241 °C. H-NMR
(CDCl3) δ (ppm): 7.12–7.26 (m, 2H, H-4′, H-5′), 7.46–7.71 (m, 5H, H-2′, H-6′, H-5, H-7, H-8), 7.78
(s, 1H, H-4). 13C-NMR (CDCl3) δ (ppm): 117.9, 122.7, 123.1, 124.5, 124.6, 124.8, 127.9, 129.5, 129.7,
130.8, 134.6, 140.7, 144.6, 159.1, 161.1. ESI-MS m/z: 345 (M+, 100). Anal. Calcd. for C15H8BrNO4:
C, 52.05; H, 2.33. Found: C, 52.01; H, 2.30.
1
6-Methoxy-3-(3′-nitrophenyl)coumarin (9). Pale yellow solid, 73% yield. Mp: 84–85 °C. H-NMR
(CDCl3) δ (ppm): 3.77 (s, 3H, OCH3), 7.01–7.10 (m, 2H, H-5, H-7), 7.54–7.59 (m, 2H, H-5′, H-6′),
13
7.71 (d, 1H, H-8, J = 7.6 Hz), 8.06–8.09 (m, 2H, H-2′, H-4′), 8.15 (s, 1H, H-4). C-NMR (CDCl3) δ
(ppm): 56.7, 115.0, 116.8, 122.3, 124.9, 128.7, 130.2, 134.2, 135.5, 137.2, 138.0, 143.3, 148.3, 156.8,
167.7, 172.7. ESI-MS m/z: 297 (M+, 100). Anal. Calcd. for C16H11NO5: C, 64.65; H, 3.73. Found: C,
64.63; H, 3.70.
1
6-Methyl-3-(4′-nitrophenyl)coumarin (10). Pale yellow solid, 75% yield. Mp: 100–101 °C. H-NMR
(CDCl3) δ (ppm): 2.36 (s, 3H, CH3), 7.21 (d, 2H, H-7, H-8, J = 8.2 Hz), 7.38 (s, 1H, H-5), 7.63–7.70
(m, 2H, H-2′, H-6′), 7.99–8.20 (m, 2H, H-3′, H-5′), 8.38 (s, 1H, H-4). 13C-NMR (CDCl3) δ (ppm):
20.6, 122.6, 123.9, 125.1, 127.9, 130.2, 131.4, 136.4, 136.6, 136.8, 137.2, 148.1, 149.3, 170.2, 190.6.
ESI-MS m/z: 281 (M+, 98). Anal. Calcd. for C16H11NO4: C, 68.32; H, 3.94. Found: C, 68.38; H, 3.99.
Procedure for the Preparation of 3-(3′-Aminophenyl)-6-Aminocoumarin (11)
The previously prepared 3-(3′-nitrophenyl)-6-nitrocoumarin (4, 2.46 mmol) was dissolved in
ethanol (5 mL) and a catalytic amount of Pd/C was added to the mixture. The solution was stirred, at
room temperature, under a H2 atmosphere, for 3 h. After completion of the reaction, the mixture was
filtered to eliminate the catalyst. The obtained crude was then purified by flash chromatography
(hexane/ethyl acetate 9:1) to give the desired coumarin 11 as a white solid in 80% yield. Mp:
105–106 °C. 1H-NMR (CDCl3) δ (ppm): 4.02 (s, 2H, NH2), 6.50–6.55 (m, 1H, H-4′) 6.56 (s, 1H, H-2′),
6.69–6.73 (m, 1H, H-6′) 6.80 (dd, 2H, H-5, H-7, J = 7.8, J = 2.4 Hz), 7.30–7.38 (m, 2H, H-5′, H-8),
13
8.4 (s, 1H, H-4). C-NMR (CDCl3) δ (ppm): 114.9, 115.7, 116.7, 120.9, 123.6, 125.4, 127.2, 128.6,

Molecules 2013, 18
1401
133.7, 135.8, 136.2, 143.3, 145.5, 149.7, 161.9. ESI-MS m/z: 252 (M+, 100). Anal. Calcd. for
C15H12N2O2: C, 71.42; H, 4.79. Found: C, 71.45; H, 4.82.
3.2. Biological Assays
Disk Diffusion Test: The antimicrobial 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 as a saline suspension of colonies from a 24 h Mueller Hinton Agar (MHA)
(Cultimed, Barcelona, Spain) plate culture of the microorganisms, containing approximately 1 × 108
colony forming units (CFU)/mL (OD620 of 0,09). Colony counts on inoculum suspension were verified
by the plate dilution method using MHA 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 using DMSO as solvent. Sterile
disks of 6 mm diameter (Liofilchem, Roseto degli Abruzzi, Italy) embedded in the drug at a final
concentration of 100 µg/disk were kept on agar surface. Sterile disks embedded with DMSO were used
as a negative control. The plates were incubated at 37 °C for 24 h. Zones of inhibition were measured
in millimeter.
Disk Minimum Inhibitory Concentrations (MICs): The MICs were evaluated using the broth
microdilution method, following the procedures of the Clinical and Laboratory Standards Institute
(CLSI, 2006). Broth microdilution tests were performed with 96 sterile flat-bottom microtiter plates
(Becton Dickinson Labware Europe, Madrid, Spain). Antimicrobials were serially diluted (512 to
1 μg/mL) in Mueller-Hinton Broth (MHB) (Cultimed) and then one hundred microliters of each
dilution were deposited into each well. The inoculum was prepared by making a MHB suspension of
colonies from a 24 h MHA (Cultimed) plate culture of the microorganisms, containing approximately
1 × 108 colony forming units (CFU)/mL (OD620 of 0,09). The inoculum was diluted 1:100 in MHB to
yield 1 × 106 CFU/mL. Ten microliters of this suspension was inoculated into the each well. Colony
counts on inoculum suspension were verified by the plate dilution method using MHA plates and
counting the bacterial colonies produced. MHB with and without inoculum was used as growth-control
and negative control, respectively. The plates were incubated at 37 °C for 24 h. The MIC was
established comparing the amount of growth in the wells containing the antimicrobial agents with the
amount of growth in the growth-control wells both with the unaided eye and by using a photometric
device (OD620).
4. Conclusions
In conclusion, in the present study it was shown that eight out of the eleven synthesized
amino/nitro-substituted 3-arylcoumarins have inhibitory activity against S. aureus. MIC determinations
proved that the tested compounds presented different profiles against S. aureus due to their
substituents, being 3-(3′-methylphenyl)-6-nitrocoumarin (6) the best one. A nitro substituent at the
6-position of the coumarin moiety seems to be essential for the antibacterial activity of this kind of
compounds. The introduction of an amino substituent seems to decrease the described activity. The
pharmacological potential of these amino/nitro-substituted 3-arylcoumarins confirms that this scaffold
can be effectively optimized into a candidate for the treatment of some bacterial infectious diseases.

Molecules 2013, 18
1402
Acknowledgments
Thanks are due to the Ministerio Español (PS09/00501), Xunta da Galicia
(PGIDIT09CSA030203PR and PGIDT08MMA011200PR) and IN845B-2010/089 for the financial
support. M.J.M. thanks to Fundação para a Ciência e Tecnologia for a PhD grant
(SFRH/BD/61262/2009). S.V.R. thanks the Ministerio de Educación y Ciencia for a PhD grant
(AP2008-04263).
References
1. 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. 2005, 12,
887–916.
2. Borges, F.; Roleira, F.; Milhazes, N.; Uriarte, E.; Santana, L. Simple coumarins: Privileged
scaffolds in medicinal chemistry. Front. Med. Chem. 2009, 4, 23–85.
3. Chilin, A.; Battistutta, R.; Bortolato, A.; Cozza, G.; Zanatta, S.; Poletto, G.; Mazzorana, M.;
Zagotto, G.; Uriarte, E.; Guiotto, A.; et al. Coumarin as Attractive Casein Kinase 2 (CK2)
Inhibitor scaffold: An integrate approach to elucidate the putative binding motif and explain
structure–activity relationships. J. Med. Chem. 2008, 51, 752–759.
4. Hooper, D.C.; Wilfson, J.S.; McHugh, G.L.; Winters, M.B.; Swartz, M.N. In vitro activity of
DNA gyrase inhibitors, singly and in combination, against Mycobacterium avium complex.
Antimicrob. Agents Chemother. 1982, 22, 662–671.
5. Ostrov, D.A.; Hernández Prada, J.A.; Corsino, P.E.; Finton, K.A.; Le, N.; Rowe, T.C. Discovery of
novel DNA gyrase inhibitors by high-throughput virtual screening. Antimicrob. Agents Chemother.
2007, 51, 3688–3698.
6. Chimenti, F.; Bizzarri, B.; Bolasco, A.; Secci, D.; Chimenti, P.; Granese, A.; Carradori, S.;
Rivanera, D.; Zicari, A.; Scaltrito, M.M.; et al. 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.
7. Matos, M.J.; Vazquez-Rodriguez, S.; Santana, L.; Uriarte, E.; Fuentes-Edfuf, C.; Santos, Y.;
Muñoz-Crego, A. Looking for new targets: Simple coumarins as antibacterial agents. Med. Chem.
2012, 8, 1140–1145.
8. 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.
9. 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.
10. 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.

Molecules 2013, 18
1403
11. Roussaki, M.; Kontogiorgis, C.; Hadjipavlou-Litina, D.J.; Hamilakis, S. A novel synthesis of
3-aryl coumarins and evaluation of their antioxidant and lipoxygenase inhibitory activity.
Bioorg. Med. Chem. Lett. 2010, 20, 3889–3892.
12. Fais, A.; Corda, M.; Era, B.; Fadda, M.B.; Matos, M.J.; Quezada, E.; Santana, L.; Picciau, C.;
Podda, G.; Delogu, G. Tyrosinase inhibitor activity of coumarin-resveratrol hybrids. Molecules
2009, 14, 2514–2520.
13. Matos, M.J.; Viña, D.; Quezada, E.; Picciau, C.; Delogu, G.; Orallo, F.; Santana, L.; Uriarte, E.
A New series of 3-phenylcoumarins as potent and selective MAO-B inhibitors. Bioorg. Med.
Chem. Lett. 2009, 19, 3268–3270.
14. Matos, M.J.; Viña, D.; Janeiro, P.; Borges, F.; Santana, L.; Uriarte, E. New halogenated
3-phenylcoumarins as potent and selective MAO-B inhibitors. Bioorg. Med. Chem. Lett. 2010,
20, 5157–5160.
15. Matos, M.J.; Terán, C.; Pérez-Castillo, Y.; Uriarte, E.; Santana, L.; Viña, D. Synthesis and study
of a series of 3-arylcoumarins as potent and selective monoamine oxidase B inhibitors.
J. Med. Chem. 2011, 54, 7127–7137.
16. 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.
17. Delogu, G.; Picciau, C.; Ferino, G.; Quezada, E.; Podda, G.; Uriarte, E.; Viña, D. Synthesis,
Human monoamine oxidase inhibitory activity and molecular docking studies of
3-heteroarylcoumarin derivatives. Eur. J. Med. Chem. 2011, 49, 1147–1152.
18. Goth, A. The antibacterial properties of dicumarol. Science 1945, 101, 383.
19. Hoettecke, N.; Rotzoll, S.; Albrecht, U.; Lalk, M.; Fischer, C.; Langer, P. Synthesis
and antimicrobial activity of 2-alkenylchroman-4-ones, 2-alkenylthiochroman-4-ones and
2-alkenylquinol-4-ones. Bioorg. Med. Chem. 2008, 16, 10319–10325.
20. Shi, Y.; Zhou, C. Synthesis and evaluation of a class of new coumarin triazole derivatives as
potential antimicrobial agents. Bioorg. Med. Chem. Lett. 2011, 21, 956–960.
21. Patil, S.A.; Unki, S.N.; Kulkarni, A.D.; Naik, V.H.; Badami, P.S. Synthesis, Characterization,
In vitro antimicrobial and DNA cleavage studies of Co(II), Ni(II) and Cu(II) complexes with
ONOO donor coumarin Schiff bases. J. Mol. Struct. 2011, 985, 330–338.
22. Sabry, N.M.; Mohamed, H.M.; Khattab, E.S.A.E.H.; Motlaq, S.S.; El-Agrody, A.M. Synthesis of
4H-chromene, Coumarin, 12H-chromeno[2,3-d]pyrimidine derivatives and some of their
antimicrobial and cytotoxicity activities. Eur. J. Med. Chem. 2011, 46, 765–772.
23. Kawase, M.; Varu, B.; Shah, A.; Motohashi, M.; Tani, S.; Saito, S. Antimicrobial activity of new
coumarin derivatives. Arzneimittelforschung 2001, 51, 67–71.
24. Patel, D.; Kumari, P.; Patel, N. Synthesis, characterization and biological evaluation of some
thiazolidinone derivatives as antimicrobial agents. J. Chem. Pharm. Res. 2010, 2, 84–91.
25. Patel, D.; Kumari, P.; Patel, N. Synthesis and characterization of some new thiazolidinones
containing coumarin moiety and their antimicrobial study. Arch. Appl. Sci. Res. 2010, 2, 68–75.
26. Laurin, P.; Ferroud, D.; Klich, M.; Dupuis-Hamelin, C.; Mauvais, P.; Lassaigne, P.; Bonnefoy, A.;
Musicki, B. Synthesis and in vitro evaluation of novel highly potent coumarin inhibitors of gyrase
B. Bioorg. Med. Chem. Lett. 1999, 9, 2079–2084.

Molecules 2013, 18
1404
27. Nathan, C. Antibiotics at the crossroads. Nature 2004, 431, 899–902.
28. Rachakonda, S.; Cartee, L. Challenges in antimicrobial drug discovery and the potential of
nucleoside antibiotics. Curr. Med. Chem. 2004, 11, 775–793.
29. Walsh, C. Antibiotics, Actions, Origins, Resistance; ASM: Washington, DC, USA, 2003.
30. Von Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.; Häbich, D. Antibacterial natural
products in medicinal chemistry - exodus or revival? Angew. Chem. Int. Ed. 2006, 45, 5072–5129.
31. Matos, M.J.; Delogu, G.; Podda, G.; Santana, L.; Uriarte, E. Regioselective synthesis of
bromo-substituted 3-arylcoumarins. Synthesis 2010, 16, 2763–2766.
32. 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.
33. Krishnaswamy, N.R.; Seshadri, T.R.; Sharma, B.R. Nitro derivatives of 3-phenylcoumarins.
Ind. J. Chem. 1969, 7, 49–55.
34. Rao, D.V.; Sayigh, A.R.; Ulrich, H. Aminophenyl alkoxy coumarins. US 3676436 A, 11 July 1972.
35. Kremky, E. Chromatographic method for the separation and identification of some coumarin
derivatives. Organika 1979, 159–163.
36. Piazzi, L.; Cavalli, A.; Belluti, F.; Bisi, A.; Gobbi, S.; Rizzo, S.; Bartolini, M.; Andrisano, V.;
Recanatini, M.; Rampa, A. Extensive SAR and computational studies of
3-{4-[(benzylmethylamino)methyl]phenyl}-6,7-dimethoxy-2H-2-chromenone
Derivatives. J. Med. Chem. 2007, 50, 4250–4254.
(AP2238)
37. M
cheminformatics,
Bratislava,
Slovak
Republic.
Available
online:
http://www.molinspiration.com/services/properties.html (accessed on 14 December 2009).
Sample Availability: Samples of the compounds are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).