Design, synthesis, and biological activity of Schiff bases bearing salicyl and 7-hydroxycoumarinyl moieties

Article abstract of DOI:10.1007/s00706-018-2325-5

Abstract: 18 (11 novel) Schiff bases, derivatives of salicylaldehyde, 2-hydroxyacetophenone, and 6-acetyl-, 8-acetyl-, and 8-formyl-7-hydroxy-4-methylcoumarin were synthesized and characterized by their spectral studies. 6-Acetyl-7-hydroxy-4-methylcoumarin was prepared by novel method under microwave assistance. These Schiff bases were evaluated for antibacterial activities against 12 bacterial and six fungi strains in vitro. N-(3,5-Dichloro-2-hydroxybenzylidene)-4-aminobenzenesulfonic acid sodium salt proved to be the most active against Staphylococcus aureus and MRSA strains (MIC 0.0194?μmol/cm³). The substitution pattern, two chlorine atoms in the salicylidene ring and the SO₃Na group, is probably the most beneficial for the activity against Gram-(+) bacteria strains. All Schiff bases were evaluated for cancer efficacy against CFPAC-1 and HeLa cell cultures originating from human pancreas cancer or human cervical cancer. Schiff bases derived from salicylaldehyde are highly effective in pancreas and cervical cancer cells; however, they demonstrate also substantial toxicity towards NIH3T3 cells. Derivatives of coumarin contain three highly selective compounds: 7-hydroxy-8-[(4-methoxyphenylimino)methyl]-4-methyl-2H-chromen-2-one, N-[(7-hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)methylene]-4-aminobenzenesulfonic acid sodium salt, and 7-hydroxy-8-[1-(4-hydroxyphenylimino)ethyl]-4-methyl-2H-chromen-2-one suggesting more promising potential of the second group of substances.

Full text of DOI:10.1007/s00706-018-2325-5

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-018-2325-5
ORIGINAL PAPER
Design, synthesis, and biological activity of Schif bases bearing salicyl
and 7‑hydroxycoumarinyl moieties
Elżbieta Hejchman1 · Hanna Kruszewska2 · Dorota Maciejewska1 · Barbara Sowirka‑Taciak1 ·
Magdalena Tomczyk1 · Alicja Sztokfsz‑Ignasiak3 · Jan Jankowski3 · Izabela Młynarczuk‑Biały3
Received: 24 July 2018 / Accepted: 15 October 2018
© The Author(s) 2019
Abstract
18 (11 novel) Schif bases, derivatives of salicylaldehyde, 2-hydroxyacetophenone, and 6-acetyl-, 8-acetyl-, and 8-formyl-
7-hydroxy-4-methylcoumarin were synthesized and characterized by their spectral studies. 6-Acetyl-7-hydroxy-4-methyl-
coumarin was prepared by novel method under microwave assistance. These Schif bases were evaluated for antibacterial
activities against 12 bacterial and six fungi strains in vitro. N-(3,5-Dichloro-2-hydroxybenzylidene)-4-aminobenzenesulfonic
acid sodium salt proved to be the most active against Staphylococcus aureus and MRSA strains (MIC 0.0194 µmol/cm3). The
substitution pattern, two chlorine atoms in the salicylidene ring and the SO3Na group, is probably the most benefcial for the
activity against Gram-(+) bacteria strains. All Schif bases were evaluated for cancer efcacy against CFPAC-1 and HeLa
cell cultures originating from human pancreas cancer or human cervical cancer. Schif bases derived from salicylaldehyde are
highly efective in pancreas and cervical cancer cells; however, they demonstrate also substantial toxicity towards NIH3T3
cells. Derivatives of coumarin contain three highly selective compounds: 7-hydroxy-8-[(4-methoxyphenylimino)methyl]-
4-methyl-2H-chromen-2-one, N-[(7-hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)methylene]-4-aminobenzenesulfonic acid
sodium salt, and 7-hydroxy-8-[1-(4-hydroxyphenylimino)ethyl]-4-methyl-2H-chromen-2-one suggesting more promising
potential of the second group of substances.
Graphical abstract
Keywords Antitumor agents · Drug research · Aldehydes · Coumarins · Antimicrobial activity
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-018-2325-5) contains
supplementary material, which is available to authorized users.
3
* Elżbieta Hejchman
Department of Histology and Embryology, Center
of Biostructure Research, Medical University of Warsaw, 5
Chałubińskiego Str, 02 004 Warsaw, Poland
ehejchman@wum.edu.pl; ehejchman@gmail.com
1
Department of Organic Chemistry, Faculty of Pharmacy,
Medical University of Warsaw, 1 Banacha Str,
02 097 Warsaw, Poland
2
Department of Antibiotics and Microbiology, National
Medicines Institute, 30/34 Chełmska Str, 00 725 Warsaw,
Poland
Vol.:(0123456789)
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E. Hejchman et al.
A series of 26 Schif bases were synthesized by react-
ing 5-chloroaldehyde and primary amines. The compounds
were assayed for antibacterial (Bacillus subtilis, Escheri-
chia coli, Pseudomonas fuorescens, and Staphylococ-
cus aureus) and antifungal (Aspergillus niger, Candida
albicans, and Trichophyton rubrum) activities by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) method. Among the compounds tested, (E)-
4-chloro-2-[(4-fluorobenzylimino)methyl]phenol (2b)
showed the most favorable antimicrobial activity with
MICs of 45.2, 1.6, 2.8, 3.4, and 47.5 µg/cm3 against B.
subtilis, E. coli, P. fuorescens, S. aureus, and A. niger,
respectively. Compound 2a showed signifcant activity
against E. coli (3.1 μg/cm3, kanamycin: 3.9 µg/cm3). Com-
pound 2c showed signifcant activity against P. fuorescens
(3.1 µg/cm3, kanamycin 3.9 µg/cm3) and S. aureus (1.8 µg/
cm3, kanamycin 1 µg/cm3, penicillin 2 μg/cm3).
Introduction
Schif bases, formed in the reaction of aromatic or het-
eroaromatic substances’ primary amine with aldehyde
or ketone, are also classifed as imines or azomethines.
They are a signifcant class of compounds in medicinal
and pharmaceutical chemistry having a variety of biologi-
cal applications that include antibacterial, antifungal, and
antitumor activities. The imine group present in such com-
pounds (of natural as well as synthetic origin) has been
shown to be critical to their biological activities [1–4].
The increase in the mortality rate associated with infec-
tious diseases is directly related to antimicrobial resistance
to antibiotics [5]. New drugs to combat this problem are,
therefore, in great demand [6].
Schif bases have been shown to be interesting moieties
for the design of antimicrobial agents [1–4]. Especially,
several Schif bases having dichloro or bromophenyl moi-
eties have been proved to be biologically important [4].
The presence of halogen atom in the molecule increases
the lipophilicity of the molecule and, therefore, afects
the partitioning of a molecule into membranes and also
facilitates hydrophobic interactions of the molecule with
specifc binding sites on either receptor or enzymes [7].
Salicylidene (2-hydroxybenzaldehyde-derived) Schiff
bases (Fig. 1) are reported as promising antimicrobial
drugs [4, 8, 9].
The authors claimed that increased hydrophilicity and
aromaticity, as well as the presence of heteroatoms resulted
in an increase in the antimicrobial activity [9].
Hybrid Schif bases containing salicylidene and sulfona-
mide moieties show promising antimicrobial activity [10].
4-[(2-Hydroxybenzylidene)amino]-N-(pyrimidin-2-yl)ben-
zenesulfonamides (3) (Fig. 2) were active against S. aureus,
including a methicillin-resistant strain (MRSA) (MIC values
within the range of 0.125–0.250 µmol/cm3 [10]. In general,
dihalogenation of the salicylic moiety improved the antibac-
terial and antifungal activity but also increased the cytotox-
icity, particularly with an increasing atomic mass.
De Souza reports the determination of the minimal
inhibitory concentration (MIC) in vitro for M. tubercu-
losis H37Rv of a diverse group of compounds, including
N-(salicylidene)-derived Schif bases. The most efective
compound among tested samples was N-(salicylidene)-
2-hydroxyaniline (1) with a MIC of 8 µmol/dm3. Com-
pound 1 showed a weak cytotoxicity to J774 macrophages
and at 1000 µmol/dm3 80% of cells were viable indicating
a higher selectivity of the drug to the pathogen than to
the mammalian cells. The results for this compound are
indicative of the promising antimycobacterial activity of
this molecule [8].
In the recent few years, the organic compounds contain-
ing coumarin Schif bases as integral part of their structures,
in particular, the transition metal complexes, have gained
much attention not only because of their antimicrobial [11,
12] but also cytotoxic activity [13]. A variety of coumarin-
derived Schif bases have been identifed or developed for
treatment of diferent types of cancers [14, 15]. For example,
they have been recently shown to be inhibitors of carbonic
anhydrases (CA). Wang and coworkers have designed and
synthesized a series of imine-linked sulfonamide–coumarin
derivatives [16]. The leading compounds 4a and 4b (Fig. 3)
Fig. 1 Salicylidene Schif bases:
N-salicylidene-2-hydroxyaniline
(1) and derivatives of 5-chloro-
salicylaldehyde (2)
1 3

Design, synthesis, and biological activity of Schif bases bearing salicyl and…
Fig. 4 Design of the novel Schif bases—a series of compounds hav-
ing salicylic moiety (5a) and coumarin moiety (5b). R=H, CH3; X
and Y are substituents with various electronic efects
The aim of this study is evaluation of an impact of diverse
moieties and substituents in the series of Schif bases on
antimicrobial and cytotoxic activity in vitro and fnding pre-
liminary conclusions about the structure–activity relation-
ship. Furthermore, the objective of our work is to prepare a
pool of compounds that can be used as ligands in the copper
or silver complexes with increased biological activity [1].
Fig. 2 4-[(2-Hydroxybenzylidene)amino]-N-(pyrimidin-2-yl)benzene
sulfonamides 3
Results and discussion
Chemistry
The classical synthesis of Schif base involves the condensa-
tion of a carbonyl compound with primary amine. Various
in situ methods for water elimination (for example, using
dehydrating solvents or molecular sieves or carrying the
reaction under azeotropic distillation) were developed. It is
known, that efciency of this synthesis is dependent on the
use of highly electrophilic carbonyl components and strongly
nucleophilic aromatic amines. If necessary, Brönsted–Lowry
or Lewis acids were proposed as catalysts, to activate the
carbonyl group of aldehydes or ketones and facilitate the
nucleophilic attack by amines, then to eliminate a mol-
ecule of water. Recently, several new techniques have been
reported. Among these innovations, microwave irradiation
has been extensively used due to its operational simplicity,
enhanced reaction rates, and great selectivity [4].
Fig. 3 4-Chloro-3-coumarinyl derived Schif bases 4a and 4b
were active in vitro against mouse melanoma B16-F10 and
MCF7 cells, to obtain the IC50 values≤0.19 μM. Both com-
pounds inhibited the hCAs II (cytosolic, oftarget isoform)—
the values of IC50 were 23 nM; and hCA IX (transmembrane,
tumor associated enzyme)—24 nM, respectively.
Compounds 8–13, derivatives of salicylaldehyde (Fig. 4,
structure 5a), were obtained under conditions described in
earlier reports [17–19]. Aldehydes 6a–6d were stirred with
equimolar amount of appropriate aniline or sodium salt of
sulfanilic acid (7a, 7e, 7f) at ambient temperature. Com-
pound 14 was prepared by heating 2-hydroxyacetophenone
and 4-methoxyaniline with the catalyst—lemon juice [20] in
xylene at 100 °C (Scheme 1). The structures of Schif bases
8–14 are listed in the Table 1.
To the best of our knowledge, there are no data concern-
ing the biological activity of Schif bases having benzene-
sulfonic acid salt moiety combined with halogenated sali-
cylidene or 7-hydroxycoumarin scafolds. To fll this gap,
in the present study, we report the synthesis, characteriza-
tion, and in vitro cytotoxic and antimicrobial evaluation of
a series of hybrid Schif bases, derived from salicylaldehyde
(structure 5a) or 7-hydroxycoumarin scafold (structure 5b)
and para-substituted aniline, with a view to explore their
potency as better chemotherapeutic agents (Fig. 4).
8-Formyl-7-hydroxy-4-methylcoumarin (15) and
8-acetyl-7-hydroxy-4-methylcoumarin (17) were prepared
according to earlier reports [21, 22]. As the yield of syn-
thesis of 6-acetyl-7-hydroxy-4-methylcoumarin (16c) is
1 3

E. Hejchman et al.
Scheme 1
Table 1 The structures of the reported salicylaldehyde-derived Schif
bases 8–14
base 23. Heating of the coumarin 17 with the correspond-
ing aniline 7a–7g under catalytic amount of lemon juice in
xylene resulted in the Schif bases 24–28. The structures of
Schif bases 15–28 are listed in Table 2.
Compounds
R
X1
X2
Y
8
H
H
H
H
H
H
CH3
H
H
Cl
Cl
Cl
OCH3
H
H
H
Cl
Cl
Cl
Br
H
OCH3
SO3Na
SO3Na
OH
OCH3
SO3Na
OCH3
9
Microbiology
10
11
12
13
14
Compounds 8–28 were screened in vitro for their antimicro-
bial activity. The panel of pathogens involved Gram-(+) bac-
teria strains: Micrococcus luteus, Bacillus subtilis, Staphy-
lococcus aureus, the MRSA strains and Enterococcus hirae;
Gram-(−) bacteria strains: Escherichia coli, Pseudomonas
aeruginosa; fungi: Candida albicans, Candida parapsi-
losis, Saccharomyces cerevisiae, Aspergillus brasiliensis.
The MIC values of the active compounds are summarized
in Table 3.
low via Fries migration, we have synthesized it by novel
method under microwave assistance in about 40%. Heating
7-hydroxy-4-methylcoumarin (16a) with acetic anhydride
and anhydrous alumina in methanesulfonic acid resulted in
the mixture of 7-acetoxy-4-methylcoumarin (16b) and the
only product of Fries rearrangement: 6-acetyl-7-hydroxy-
4-methylcoumarin (16c). Isolation by column chromatog-
raphy on silica gel yielded the required compound 16c
(Scheme 2). The synthesis of Schif bases, derivatives of
coumarin 18–28 is depicted in Scheme 3.
Salicylidene-derived Schiff bases (compounds 8, 12,
14), coumarins 15, 16a–16c, and 17 as well as coumarin-
derived Schif bases (18–23, 25–28) showed no signifcant
antimicrobial activity. N-(2-hydroxybenzylidene)-4-amin-
obenzenesulfonic acid sodium salt (9) caused only some
antifungal activity. N-(3,5-dichloro-2-hydroxybenzylidene)-
4-aminobenzenesulfonic acid sodium salt (10) proved to be
the most active against S. aureus and MRSA strains (MIC
0.0194 µmol/cm3). According to the Table 2C from Clinical
and Laboratory Standards Institute [23], the susceptibility
of Staphylococcus sp. to linezolid, an antibiotic used for the
treatment of infections caused by Gram-positive bacteria,
is defned as<0.012–0.024 µmol/cm3. Compared with lin-
ezolid, the novel compound 10 is a promising structure for
Schif bases 18–22, derivatives of coumarin 15, were
prepared by traditional but efcient method refuxing the
ethanolic mixture of compound 15, substituted aniline 7a,
7b, 7d, 7e, and 7f, and catalytic amount of glacial acetic
acid [4]. Coumarin 16 was more reactive, compared to the
isomeric compound 17. The reaction of compound 16 with
4-methoxyaniline (7f) in xylene at 100 °C yielded Schif
Scheme 2
1 3

Design, synthesis, and biological activity of Schif bases bearing salicyl and…
Scheme 3
further research towards potential medicines active against
S. aureus strains, including MRSA.
Table 2 The structures of the reported coumarins 15–17 and cou-
marin-derived Schif bases 18–28
Two other halogenated Schif bases 11 and 12 did not
possess signifcant activity against these bacterial strains—
replacement of the sulfonic acid salt substituent present in
the compound 10 by hydroxy (compound 11) or methoxy
(compound 12) groups results in reducing the antibacterial
activity. The 3-methoxy-5-bromo substitution pattern and
presence of the sulfonic substituent in compound 13 does not
provide a benefcial efect on its activity against S. aureus
and MRSA strains. We conclude that the substitution pattern,
two chlorine atoms in the salicylidene ring and the SO3Na
group, is probably the most benefcial for the activity against
Gram-(+) bacteria strains.
Compounds
R
X1
X2
Y
15
16c
17
18
19
20
21
22
23
24
25
26
27
28
–
–
–
–
–
–
–
–
–
–
–
–
–
–
H
HC=O
H
CH3C=O
–
–
–
Br
CH3C=O
H
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
OH
OCH3
CH3
SO3Na
OCH3
OH
OCH3
OC2H5
CH3
C2H5
Compounds 10 and 13 show antifungal activity against
C. albicans strains (MIC: 0.0776 and 0.0704 µmol/cm3).
Compared with the susceptibility of Candida sp. to fucona-
zole, an antifungal medication used for a number of fungal
infections which are defned as<0.026–0.11 µmol/cm3 [24],
1 3

E. Hejchman et al.
Table 3 The antibacterial and
antifungal activity of Schif
bases
Minimal inhibitory concentration (MIC)/µmol cm−3
Compounds
9
10
11
13
22
24
Bacteria
Micrococcus luteus ATCC 10240
Bacillus subtilis ATCC 6633
Bacillus cereus ATCC 11178
Staphylococcus epidermidis ATCC 12228
Staphylococcus aureus ATCC 6538
Staphylococcus aureus ATCC 6538P
MRSA A854
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.0388
0.0388
0.0388
NA
0.0194
0.0194
0.0194
0.0194
0.0776
NA
0.440
0.440
0.440
0.440
0.440
0.440
0.440
0.440
0.440
NA
0.2933
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
0.404
NA
NA
NA
NA
0.404
NA
NA
NA
NA
MRSA A876
Enterococcus hirae ATCC 10541
Escherichia coli ATCC 8739
Pseudomonas aeruginosa ATCC 9027
Pseudomonas aeruginosa ATCC 15442
Fungi
NA
NA
NA
NA
Candida albicans ATCC 10231
Candida albicans ATCC 2091
Candida parapsilosis ATCC 22019
Saccharomyces cerevisiae ATCC 9763
Aspergillus brasiliensis ATCC 16404
NA
0.390
NA
NA
NA
0.0776
0.0776
0.1618
0.1618
0.1618
NA
0.440
NA
NA
0.440
0.0704
0.0704
0.1466
0.2933
0.2933
NA
0.313
NA
NA
NA
NA
NA
NA
NA
NA
NA not active in concentration below 0.5 µmol/cm3
the MICs of compounds 10 and 13 look promising. Unfortu-
nately, the use of coumarin scafold instead of salicylidene
for the synthesis of Schif bases 22 and 24 causes a signif-
cant reduction in antimicrobial activity.
showed moderate activity against tumor cells and can be
categorized into the “little efective group” defned above.
In particular, for CFPAC-1 cells compounds: 10–13, 18, 19,
21, 22, and 24 were little efective, whilst in HeLa cells
compounds 10, 11, 13, 18–21, and 23–27 were little efec-
tive. Compound 20 for CFPAC-1 cells turned out highly
efective. Compounds 8 and 9 in used concentrations were
not efective for both tumor cell lines. HeLa cells turned
out to be resistant against compound 22, whereas CFPAC-1
cells were little sensitive to 22 (Table 4). However, the IC50
value was relatively high for all tested compounds showing
little cytotoxicity compared to known and well-established
chemotherapeutics working in vitro in nanomolar concen-
trations, such as bortezomib. For instance, published so far
data with most active imine-linked sulfonamide—coumarin
derivatives—demonstrated the IC50 values≤0.19 μM [16].
In our experiments except for only compound 14 against
NIH3T3 cells (IC50 = 4 μM) and compound 20 against
CFPAC-1 cells (IC50 =10 μM), all other substances were
less cytotoxic.
Cytotoxicity
Compounds 8–14 and 18–28 were examined in tumor and
non-tumor cells to estimate theirs antitumor activity and
selectivity. In experiments, we used following cell lines:
CFPAC-1 and HeLa originating from human pancreas can-
cer or human cervical cancer, respectively, as well as mouse
NIH-3T3 fbroblasts as non-tumor cells.
Within cytotoxicity assay, inhibitory concentrations
required to inhibit 50% or 90% of control cell growth were
calculated for each cell line and are displayed in the Table 4.
According to this, the examined compounds are considered
as not efective (IC50 values in the range 499 µM–100 µM),
little effective (11 µM < IC50 < 99 µM) and effective
IC50 ≤10 µM. All experiments were done three times inde-
pendently. SD value was in range 2–15 for all substances
and was omitted for more descriptive results. For better ori-
entation in this table, values that are lower than those for
non-tumor fbroblasts are indicated bold.
The ideal antitumor drug candidate should selectively tar-
get tumor cells and be relatively harmless for normal non-
tumor cells. For assessment of tumor cell selectivity, selec-
tivity index (SI) was calculated and is displayed in Table 5.
According to the defnition, selective agents have the SI
higher than one [16]. Among novel Schif bases selective
Coumarins 15, 16a, 16b, 16c, and 17 showed no signif-
cant cytotoxic activity. Most of synthesized Schif bases
1 3

Design, synthesis, and biological activity of Schif bases bearing salicyl and…
Table 4 Compilation of
Compounds
Inhibitory concentration (IC50 or IC90)/μM
cytostatic/cytotoxic efects
by CVDE assay as inhibitory
concentrations, IC50 and IC90,
respectively, after incubation of
given compound for 48 h with
indicated cell line: CFPAC,
HeLa or NIH3T3
Cell lines
CFPAC-1
IC50
HeLa
IC50
NIH3T3
IC50
IC90
IC90
IC90
99
380
101
8
9
>500
>500
32
>500
>500
104
110
107
107
350
120
115
120
122
142
190
100
350
350
150
132
>500
320
64
38
150
34
111
91
72
92
68
>500
>500
116
48
144
35
10
11
12
13
14
18
19
20
21
22
23
24
25
26
27
28
54
51
27
123
>500
110
55
42
30
119
106
105
207
36
332
170
4
62
160
170
32
126
160
43
96
124
175
10
47
148
>500
310
71
380
72
158
>500
126
80
>500
68
135
76
37
73
73
91
89
144
>500
38
>500
123
207
207
117
104
144
300
150
91
>500
170
110
332
68
148
IC50 and IC90 is an inhibitory concentration of compound required to inhibit cell proliferation by 50% or
90%, respectively, in comparison with untreated controls
Table 5 Tumor cell selectivity of novel compounds
for CFPAC-1 cell line are agents 10, 11, 13, 18–22, and 24,
whereas selective for HeLa cells are compounds 11, 20,
21, 23, 24, and 26. It is worth to emphasize that three com-
pounds turned out to be highly selective: 20, 22, and 24.
The most selective compound 24 has SI yielding approxi-
mately 6.5 for CFPAC-1, and 13.5 for HeLa cell line. It
has to be admitted that this good selectivity of compound
24 is only resulted from insensitivity of NIH3T3 cells
to those substances, in this context relatively high IC50
76 μM and 37 μM (in CFPAC1 and HeLa cells, respec-
tively), in denominator, gave such a good selectivity index.
Compound 24 is little cytotoxic for tested tumor cell lines
and this should be improved to fnd out it as a potential
antitumor drug.
Compounds
NIH3T3/CFPAC-1
NIH3T3/HeLa
8
9
<1
<1
<1
<1
10
11
12
13
14
18
19
20
21
22
23
24
25
26
27
28
1.09
1.02
0.82
1.11
0.02
1.72
1.34
9.60
1.51
4.75
0.53
6.58
0.18
0.72
0.78
0.65
0.55
1.45
0.28
0.88
0.04
0.68
0.60
1.04
1.04
<1
1.06
13.51
0.52
2.05
1.00
0.62
It seems that benzene ring substituted with OH group in
para position added to the coumarin moiety (17) gives the
best tumor cell selectivity; however, IC50 for compound 24
should drop to nanomolar concentrations to talk about the
antitumor activity of 24.
When we compare two chemically diferent group of
compounds, namely derivatives of salicylaldehyde (10–13)
and coumarin derivatives (18–28) in context of their anti-
cancer activity, we can take some general conclusions. Schif
bases derived from salicylaldehyde are efective in pancreas
and cervical cancer cells; however, they demonstrate also
Numbers displayed in the table represent the ratio of the IC50 values
obtained for non-tumor NIH3T3 vs. IC50 for tumor-derived CFPAC,
or HeLa cells, respectively. Bold are numbers demonstrating relative
selectivity of the given compound
1 3

E. Hejchman et al.
substantial toxicity towards NIH3T3 cells. Derivatives of
coumarin represented by compounds 18–28 contain three
highly selective compounds (20–22, 24) suggesting more
promising potential of the second group of substances.
High-resolution accurate mass determinations (HRMS)
were recorded in the Faculty of Chemistry the University of
Warsaw, on MAS Quattro LCT (TOF) mass spectrometer
equipped with electrospray ionization (ESI). Melting points
were determined on a ElectroThermal 9001 Digital Melting
Point Melting Point apparatus. Nuclear magnetic resonance
(1H NMR and 13C NMR, HMBC and HSQC) spectra were
recorded on a Varian NMRS-300 NMR spectrometer with
TMS as an internal standard. Atoms’ numbering is presented
in Scheme 3. The chemical shifts were reported in parts per
million (ppm); the coupling constants (J) were expressed
in hertz (Hz). IR spectra (KBr or Nujol) were recorded on
Shimadzu FTIR 8300 spectrophotometer in the Department
of Technology of Drugs and Pharmaceutical Technology,
Medical University of Warsaw or FTIR Nicolet iS5 in The
Institute of Physics PAN in Warsaw. Elemental analysis (C,
H, N, S) was conducted using the Elemental Analyser Vario
EL III (Elementar) in the University of Wrocław, and their
results were found to be in good agreement ( 0.3%) with
the calculated values.
Conclusion
The present work comprises the new, microwave-assisted
method of synthesis of 6-acetyl-7-hydroxy-4-methylcou-
marin as well as the preparation of 18 (11 novel) Schif bases,
derivatives of salicylaldehyde, 2-hydroxyacetophenone, and
6-acetyl-, 8-acetyl-, and 8-formyl- 7-hydroxy-4-methylcou-
marin. All these compounds were assayed for antimicrobial
and cytotoxic activity. Several interesting structure–activity
relationships in some Schif bases were found. The novel
compound N-(3,5-dichloro-2-hydroxybenzylidene)-4-am-
inobenzenesulfonic acid sodium salt (10) shows the highest
antibacterial activity. It is noteworthy that compound 10 is
soluble in water what potentially makes possible transport
of the compound to the interior of the cell or interferences
in the structure of the microbial cell membrane.
2‑[(4‑Methoxyphenylimino)methyl]phenol (8) Equimolar
amounts of salicylaldehyde and 4-methoxyaniline dissolved
in ethanol were heated to refux for 1 h. Then the precipitate
was fltered of and dried. Light green solid; yield 62%; m.p.:
81.5–82.6 °C (lit. 81 °C [17]).
All but only three compounds (8, 9, 22 on HeLa cells)
were active against cancer cells in micromolar concentra-
tions ranging from ca 300 to 10 µM. Coumarin derivatives
turned out to be more selective against cancer cells than
derivatives of salicylaldehyde. The most selective com-
pounds are 20 and 24. Compound 20 is the Schif base
derived from 8-formyl-7-hydroxy-4-methylcoumarin and
4-methoxyaniline; compound 24 is derived from 8-acetyl-
7-hydroxy-4-methylcoumarin and 4-aminophenol. Both
compounds are structural isomers difering in the position-
ing of the methyl group in the molecule. For these mol-
ecules, the desirable action is separated from an unwanted
toxicity, thus constituting a promising hit for further struc-
ture optimization and development of potential anticancer
agents. Especially, they are intended to be used as ligands
in the copper or silver complexes with potentially increased
biological activity.
N‑(2‑Hydroxybenzylidene)‑4‑aminobenzenesulfonic acid
sodium salt (9) Sodium salt of compound 9 was prepared
according to [18]. Sulfanilic acid (2.5 g, 14.3 mmol) was
suspended in 25 cm3 methanol and heated to refux with
0.58 g powdered sodium hydroxide (14.4 mmol) for 1 h.
Then the reaction mixture (white suspension) was left for
10 min at ambient temperature and salicylaldehyde was
added dropwise with stirring. Change of color to yellow and
precipitation of yellow solid were observed. Stirring was
continued for 2 h. The precipitate was fltered of and washed
with chloroform. Yellow solid; yield 98%; m.p.:>300 °C;
NMR spectra were found to be identical with the ones
described in Ref. [18].
Experimental
N‑(3,5‑Dichloro‑2‑hydroxybenzylidene)‑4‑aminobenzene‑
sulfonic acid sodium salt (10, C13H9Cl2NO4S) This product
was prepared according to the procedure given for com-
pound 9. Orange solid; yield 74%; m.p.:>300 °C; 1H NMR
(300 MHz, DMSO-d6): δ=7.44 (d, J=8.4 Hz 2H, H-9,13),
7.70 (d, J = 8.4 Hz, 2H, H-10,12), 7.74 (s, 2H, H-4,6),
9.05 (s, 1H, H-7) ppm; 13C NMR (75 MHz, DMSO-d6):
δ = 120.44 (C-1), 120.84 (C-9,13), 121.67 (C-3), 121.94
(C-5), 126.86 (C-10,12), 130.71 (C-6), 132.26 (C-4),
145.89 (C-11), 147.85 (C-8), 156.02 (C-7), 162.42 (C-2)
ppm; IR (KBr): ̄ꢀ = 3455 (OH), 1620 (C=N), 1320, 1045
(sulfone), 1175 (sulfonate salt), 850 (substitution para di)
All reagents were obtained from Sigma-Aldrich and sol-
vents from CHEMPUR S.A. or Polskie Odczynniki Chem-
iczne S.A. and were used without purifcation. 100% lemon
juice LEMONDOR from Giancarlo Polenghi was used
as a catalyst. All reaction mixtures were monitored using
thin-layer chromatography (TLC) on 60F254 silica gel TLC
plates (Merck) (developing system; chloroform–ethyl ace-
tate 20:1). Column chromatography was carried out with
silica gel (230–400 mesh) from Merck. Visualization was
made with the use of UV laboratory lamp (λ=254, 365 nm).
1 3

Design, synthesis, and biological activity of Schif bases bearing salicyl and…
cm−1; HR-MS (TOF, ESI-): m/z calcd for C13H8Cl2NO4S
([M−H]−) 343.9551, found 343.9546.
6‑Acetyl‑7‑hydrox y‑4‑methyl‑2H‑ chromen‑2‑ one
(16c) 7-Hydroxy-4-methylcoumarin (16ª, 8.80 g, 50 mmol)
was dissolved in 35 cm3 methanesulfonic acid (540 mmol).
Then 35 cm3 acetic anhydride (370 mmol) and 40 g anhy-
drous alumina (390 mmol) were added. The reaction mixture
was heated in microwave oven; conditions: two cycles of
heating, time: 4 min (refux), power: 500 W. Then the mix-
ture was allowed to reach ambient temperature and fltered
through silica gel under reduced pressure. The fltrate was
poured to 60 g ice water and extracted with dichloromethane
(3×60 cm3). The organic layer was washed with saturated
aqueous solution of NaHCO3. Solvent was evaporated and
the product 2 was isolated by column chromatography on
silica gel 230–400 mesh (chloroform–ethyl acetate). Color-
less solid; yield 42%; m.p.: 207–208 °C (lit. 210–211 °C
[26]); 1H and 13C NMR spectra were found to be identical
with the ones described in Ref. [26].
4,6‑Dichloro‑2‑[(4‑hydroxyphenylimino)methyl]phe‑
nol (11) 3,5-Dichloro-2-hydroxybenzaldehyde (0.955 g,
5 mmol) and 0.546 g 4-aminophenol (5 mmol) were dis-
solved in 23 cm3 methanol. The mixture was heated to refux
for 1 h and concentrated; then it was left at ambient tempera-
ture. The precipitate was fltered of and purifed by crystalli-
zation (ethanol). Red solid; yield 76%; m.p.: 192.9–193.5 °C
(lit. 224 °C [19]).
4,6‑Dichloro‑2‑[(4‑methoxyphenylimino)methyl]phe‑
nol (12) 3,5-Dichloro-2-hydroxybenzaldehyde (0.907 g,
4.75 mmol) and 0.585 g 4-methoxyaniline (4.75 mmol)
were dissolved in 13 cm3 methanol. The mixture was left at
ambient temperature for 0.5 h. The precipitate was purifed
by crystallization (ethanol). Orange solid; yield 55%; m.p.:
114–115 °C (lit. 116 °C [19]).
8‑Acetyl‑7‑hydrox y‑4‑methyl‑2H‑ chromen‑2‑ one
(17) Colorless solid; yield 80%; m.p.: 174–175 °C (lit.
174–175 °C [26]); 1H and 13C NMR spectra were found to
be identical with the ones described in Ref. [26].
N‑(5‑Bromo‑2‑hydroxy‑3‑methoxybenzylidene)‑4‑amin‑
obenzenesulfonic acid sodium salt (13, C14H12BrNO5S) This
product was prepared according to the procedure given
for compound 9. Red solid; yield 50%; m.p.:>300 °C; 1H
NMR (300 MHz, DMSO-d6): δ=3.85 (s, 3H, OCH3), 7.26
(d, J=2.4 Hz, H-4), 7.35 (d, J=8.4 Hz, 2H, H-9,13), 7.47
(d, J=2.4 Hz, H-6), 7.67 (d, J=8.4 Hz, 2H, H-10,12), 8.95
(s, 1H, H-7) ppm; 13C NMR (75 MHz, DMSO-d6): δ=55.88
(OCH3), 117.74 (C-2), 120.30 (C-4), 120.67 (C-9,13),
125.13 (C-6), 126.80 (C-1), 126.86 (C-10,12), 147.14 (C-8),
149.14 (C-3), 150.07 (C-7) ppm; IR (KBr): ꢀ̄ =3700-3400
(OH), 1612 (C=N), 1320, 1045 (sulfone), 1182 (sulfonate
salt), 851 (substitution para di) cm−1; HR-MS (TOF, ESI-
): m/z calcd for C14H11BrNO5S ([M−H]−) 383.9541, found
383.9534.
8‑Formyl‑7‑hydroxy‑4‑methyl‑2H‑chromen‑2‑one (15)
derived Schiff bases—general procedure Compound 15
(0.408 g, 2 mmol), appropriate aniline or the sodium salt
of sulfanilic acid (2 mmol) were dissolved in 5 cm3 ethanol,
one drop of glacial acetic acid was added, and the mixture
was magnetically stirred and heated to refux in the oil bath
(1–4 h). The progress of reaction was monitored by TLC
(chloroform–ethyl acetate 20:1). When the reaction was
complete, the mixture was left at ambient temperature over-
night. The precipitate was fltered of under reduced pres-
sure, and purifed by crystallization (ethanol).
7‑Hydroxy‑8‑[(4‑bromophenylimino)methyl]‑4‑me‑
thyl‑2H‑chromen‑2‑one (18, C17H12BrNO3) This product
was prepared according to the general procedure. Orange
2‑[1‑(4‑Methoxyphenylimino)ethyl]phenol (14) 2-Hydroxy-
acetophenone (1.365 g, 10 mmol) and 1.60 g 4-methoxyani-
line (13 mmol) were dissolved in 5 cm3 xylene. Then the cat-
alyst 1.4 cm3 lemon juice was added. The reaction mixture
was stirred magnetically and heated in the oil bath at 100 °C.
The progress of reaction was monitored by TLC (chloro-
form–ethyl acetate 20:1). After 8 h the mixture was allowed
to reach ambient temperature, then it was left in refrigerator
for 24 h. Chloroform (10 cm3) and anhydrous MgSO4 were
added to remove water. Then the fltrate was concentrated
and the resulting precipitate was fltered of. The crude com-
pound was purifed by crystallization (methanol). Yellow
solid; yield 23%; m.p.: 101–103 °C (lit. 98–99 °C [25]).
1
solid; yield 68%; m.p.: 222–223.5 °C; Rf= 0.68; H NMR
(300 MHz, CDCl3): δ=2.43 (d, J=1.2 Hz, 3H, H-9), 6.16
(bq, J=1.2 Hz, 1H, H-3), 6.95 (d, J=9 Hz, 1H, H-6), 7.24–
7.29 (m, 2H, H-12,16), 7.55–7.60 (m, 2H, H-13,15), 7.61
(d, J=9 Hz, 1H, H-5), 9.36 (s, 1H, H-10) ppm; 13C NMR
(75 MHz, CDCl3): δ = 19.18 (C-9), 107.18 (C-8), 111.44
(C-3), 111.63 (C-4a), 114.87 (C-6), 121.52 (C-14), 123.25
(C-12,16), 129.68 (C-5), 132.88 (C-13,15), 153.41 (C-4),
157.28 (C-8a,10), 160.36 (C-2), 166.06 (C-7) ppm; IR
(Nujol): ̄ꢀ =1710 (C=O), 1620 (C=N) cm−1; HR-MS (TOF,
ESI-): m/z calcd for C17H11NO3Br ([M−H]−) 355.9922,
found 355.9915.
8‑Formyl‑7‑hydroxy‑4‑methyl‑2H‑chromen‑2‑one (15) Of-
white solid; yield 11%; m.p.: 182–184 °C (lit. 174–176 °C
[21]).
7‑Hydroxy‑8‑[(4‑hydroxyphenylimino)methyl]‑4‑me‑
thyl‑2H‑chromen‑2‑one (19, C17H13NO4) This product was
1 3

E. Hejchman et al.
prepared according to the general procedure. Orange solid;
7‑Hydroxy‑6‑[1‑(4‑methoxyphenylimino)ethyl]‑4‑me‑
thyl‑2H‑chromen‑2‑one (23, C19H17NO4) Coumarin 16c
(10 mmol) and 1.60 g 4-methoxyaniline (13 mmol) were
dissolved in 5 cm3 xylene. The reaction mixture was stirred
magnetically and heated in the oil bath at 100 °C. The pro-
gress of reaction was monitored by TLC (chloroform–ethyl
acetate 20:1). After 6 h the mixture was allowed to reach
ambient temperature, then it was left in refrigerator for 24 h.
Chloroform (10 cm3) and anhydrous MgSO4 were added to
remove water. Then the fltrate was concentrated and the
resulting precipitate was fltered of. The crude compound
was purifed by crystallization (methanol). Yellow solid;
yield 83%; m.p.: 239–241 °C; Rf =0.57; 1H NMR (300 MHz,
acetone-d6): δ=2.53 (d, 3H, J=3.0 Hz, H-9), 2.77–2.80 (m,
3H, C10-CH3), 3.84 (s, 3H, C14-OCH3), 6.12 (bs, 1H, H-3),
6.75 (d, 1H, J=4.2 Hz, H-8), 7.03 (m, 4H, H-12,13,15,16),
8.13 (s, 1H, H-5) ppm; 13C NMR (75.4 MHz, acetone-d6):
δ=18.60 (C-9), 55.88 (C14-OCH3), 105.02 (C-8), 112.20
(C-3), 112.32 (C-4a), 115.36 (C-13,15), 123.89 (C-6),
123.97 (12,16), 128.22 (C-5), 139.77 (C-11), 153.95 (C-4),
158.66 (C-8a), 160.57 (C-2), 167.14 (C-7), 172.81 (C-10)
ppm; IR (KBr): ̄ꢀ =2841, 1723 (C=O), 1612 (C=N), 1500
(C=C), 858 (substitution para di) cm−1; HR-MS (TOF,
ESI+): m/z calcd for C19H17NNaO4 ([M+Na]+) 346.1055,
found 346.1049.
1
yield 60%; m.p.: 253 °C (decomposition); Rf = 0.65; H
NMR (300 MHz, DMSO-d6): δ = 2.42 (d, J = 0.9 Hz, 3H,
H-9), 6.24 (bq, J = 0.9 Hz, 1H, H-3), 6.85–6.90 (m, 2H,
H-13,15), 6.91 (d, J = 9 Hz, 1H, H-6), 7.39–7.44 (m, 2H,
H-12,16), 7.77 (d, J=9 Hz, 1H, H-5), 9.19 (s, 1H, H-10),
9.81 (s, 1H, H-10) ppm; 13C NMR (75 MHz, DMSO-d6):
δ=18.40 (C-9), 106.22 (C-8), 110.18 (C-3), 110.69 (C-4a),
114.31 (C-6), 116.16 (C-13,15), 122.75 (C-12.16), 129.81
(C-5), 137.34 (C-11), 153.17 (C-10), 153.81 (C-8a), 154.06
(C-4), 157.54 (C-14), 159.25 (C-2), 165.67 (C-7) ppm; IR
(Nujol): ̄ꢀ =3250 (O–H), 1696 (C=O), 1622 (C=N) cm−1.
7‑Hydroxy‑8‑[(4‑methoxyphenylimino)methyl]‑4‑me‑
thyl‑2H‑chromen‑2‑one (20, C18H15NO4) This product
was prepared according to the general procedure. Orange
solid; yield 69%; m.p.: 203.7–205.6 °C; Rf =0.46; 1H NMR
(300 MHz, CDCl3): δ=2.42 (d, J=0.9 Hz, 3H, H-9), 3.86
(s, 3H, C14-OCH3), 6.14 (bq, J=0.9 Hz, 1H, H-3), 6.96 (d,
J=9 Hz, 1H, H-6) 6.95–7.00 (m, 2H, H-13,15), 7.40–7.56
(m, 2H, H-12,16), 7.58 (d, J=9 Hz, 1H, H-5), 9.33 (s, 1H,
H-10) ppm; 13C NMR (75 MHz, CDCl3): δ=19.18 (C-9),
55.81 (C14-OCH3), 107.16 (C-8), 111.06 (C-3), 111.18
(C-4a), 114.99 (C-13,15), 115.21 (C-6), 122.75 (C-12.16),
129.09 (C-5), 139.45 (C-11), 153.54 (C-8a), 154.28 (C-4),
154.57 (C-10), 159.68 (C-2), 160.61 (C-14), 166.4 (C-7)
ppm; IR (Nujol): ̄ꢀ = 1721 (C=O), 1602 (C=N), 1379
(C-O-Casym) cm−1; HR-MS (TOF, ESI-): m/z calcd for
C18H14NO4 ([M−H]−) 308.0923, found 308.0924.
8‑Acetyl‑7‑hydroxy‑4‑methyl‑2H‑chromen‑2‑one (17)
derived Schiff bases—general procedure Coumarin 17
(10 mmol) and appropriate aniline (13 mmol) were dis-
solved in 5 cm3 xylene. Then the catalyst—1.4 cm3 lemon
juice—was added. The reaction mixture was stirred magneti-
cally and heated in the oil bath at 100 °C. The progress of
reaction was monitored by TLC (chloroform–ethyl acetate
20:1). After 8 h the mixture was allowed to reach ambient
temperature, then it was left in refrigerator for 24 h. Chloro-
form (10 cm3) and anhydrous MgSO4 were added to remove
water. Then the fltrate was concentrated and the resulting
precipitate was fltered of. The crude compound was puri-
fed by crystallization (methanol).
7‑Hydroxy‑8‑[(4‑methylphenylimino)methyl]‑4‑me‑
thyl‑2H‑chromen‑2‑one (21) This product was prepared
according to the general procedure. Orange solid; yield 57%;
m.p.: 196.5–199.0 °C (lit.188–189 °C [27]).
N‑[(7‑Hydroxy‑4‑methyl‑2‑oxo‑2H‑chromen‑8‑yl)
methylene]‑4‑aminobenzenesulfonic acid sodium salt (22,
C17H13NO6S) This product was prepared according to the
general procedure. Orange solid; yield 90%; m.p.:>300 °C;
1H NMR (300 MHz, DMSO-d6): δ=2.43 (s, 3H, H-9), 6.26
(d, J=0.9 Hz, 1H, H-3), 6.95 (d, J=9 Hz, 1H, H-6,) 7.48 (d,
J=8.4 Hz, 2H, H-12,16), 7.71 (d, J=8.4 Hz, 2H, H-13,15),
7‑Hydroxy‑8‑[1‑(4‑hydroxyphenylimino)ethyl]‑4‑me‑
thyl‑2H‑chromen‑2‑one (24) This product was prepared
according to the general procedure. Yellow solid; yield 85%;
m.p.: 256–257 °C (lit. 217 °C [28]); 1H NMR spectrum was
found to be diferent from the one described in Ref. [28].
7.82 (d, J=9.0 Hz, 1H, H-5), 9.28 (s, 1H, H-10) ppm; 13
C
NMR (75 MHz, DMSO-d6): δ=18.37 (C-9), 106.20 (C-8),
110.38 (C-3), 110.88 (C-4a), 114.29 (C-6), 120.72 (C-12,16),
126.96 (C-13,15), 130.63 (C-5), 145.94 (C-14), 147.62
(C-11), 154.00 (C-8a), 154.12 (C-4), 156.89 (C-10), 159.11
(C-2), 165.62 (C-7) ppm; IR (Nujol): ̄ꢀ =3260 (OH), 1728
(C=O), 1610 (C=N), 1319, 1050 (sulfone), 1180 (sulfonate
salt), 849 (substitution para di) cm−1; HR-MS (TOF, ESI-
): m/z calcd for C17H12NO6S ([M−H]−) 358.0385, found
358.0380.
7‑Hydroxy‑8‑[1‑(4‑methoxyphenylimino)ethyl]‑4‑me‑
thyl‑2H‑chromen‑2‑one (25, C19H17NO4) This product
was prepared according to the general procedure. Yellow
1
solid; yield 87%; m.p.: 198–200 °C; Rf= 0.48; H NMR
(300 MHz, acetone-d6): δ = 2.47 (d, 3H, J = 1.2 Hz, H-9),
2.68 (m, 3H, C10-CH3), 3.85 (s, 3H, C14-OCH3), 6.11 (s,
1H, H-3), 6.89 (dd, 1H, J = 9.0, 3.6 Hz, H-6), 7.03–7.12
1 3

Design, synthesis, and biological activity of Schif bases bearing salicyl and…
(m, 4H, H-12,13,15,16), 7.75 (d, 1H, J=9.0 Hz, H-5) ppm;
13C NMR (75.4 MHz, acetone-d6): δ = 19.15 (C-9), 33.99
(C10-CH3), 55.79 (C14-OCH3), 110.41 (C-4a), 111.68
(C-3), 114.96 (C-13,15), 115.37 (C-6), 116.34 (C-8),
121.43 (C-12,16), 129.81 (C-5), 133.01 (C-7), 146.12
(C-11), 154.56 (C-8a), 154. 89 (C-4), 159.55 (C-2), 160.13
(C-10,14) ppm; IR (KBr): ꢀ̄ = 3001, 2964, 1709 (C=O),
1597 (C=N), 1502 (C=C), 827 (substitution para di) cm−1;
HR-MS (TOF, ESI-): m/z calcd for C19H16NO4 ([M−H]−)
322.1079, found 322.1073.
(d, 3H, J=0.9 Hz, H-9), 2.64–2.80 (m, 5H, C10-CH2CH3),
6.12 (br.s, 1H, H-3), 6.93 (d, 1H, J=9.0 Hz, H-6), 7.04 (d,
2H, J=8.1 Hz, H-13,15), 7.32 (d, 2H, J=8.1 Hz, H-14,16),
7.76 (d, 1H, J=8.7 Hz, H-5), 13.40 (s, 1H, C7-OH) ppm;
13C NMR (75 MHz, acetone-d6) δ=18.18 (C14-CH3), 33.01
(C-11, C14-CH2-CH3), 110.70 (C-8), 114.28 (C-3), 114.43
(C-4a), 119.31 (C-6), 119.42 (C-12,16), 128.13 (C-5), 132.04
(C-13,15), 134.00 (C-14), 146.62 (C-11), 151.41 (C-4), 156.46
(C-8a), 155.81 (C-10), 161.59 (C-2), 160.39 (C-7) ppm; IR
(KBr): ̄ꢀ =2962, 2930, 1667 (C=O), 1600 (C=N), 1514
(C=C), 835 (substitution para di) cm−1; HR-MS (TOF, ESI-):
m/z calcd for C20H18NO3 ([M−H]−) 320.1289, found 320.1292.
7‑Hydroxy‑8‑[1‑(4‑ethoxyphenylimino)ethyl]‑4‑me‑
thyl‑2H‑chromen‑2‑one (26, C20H19NO4) This product was
prepared according to the general procedure. Yellow solid;
yield 76%; m.p.: 167–170 °C; Rf =0.39; 1H NMR (300 MHz,
acetone-d6): δ = 1.40 (t, 3H, J = 6.9 Hz, H-19), 2.47 (d,
3H, J=0.9 Hz, H-9), 2.67, 2.69 (m, 3H, C14-OCH2CH3),
4.10 (q, 2H, J = 6.9 Hz, C14-OCH2CH3), 6.11 (s, 1H,
H-3), 6.88 (d, 1H, J = 8.7 Hz, H-6), 7.02–7.11 (m, 4H,
In vitro antibacterial activity assay
Strains
The following microbial strains were chosen from American
Type Culture Collection (ATCC): Gram-(+) bacteria strains:
Micrococcus luteus 10240, Bacillus subtilis 6633, Staphy-
lococcus aureus (6538, 6538P), Enterococcus hirae 10541;
Gram-(−) bacteria strains: Escherichia coli 8739, Pseu-
domonas aeruginosa (9027, 15442); fungi (yeast strains):
Candida albicans (10231, 2091), Candida parapsilosis 22019,
Saccharomyces cerevisiae 9763; fungi (mold strain): Aspergil-
lus brasiliensis 16404. The MRSA hospital strains (methicil-
lin-resistant S. aureus) from Clinical Hospital Prof. Orlowski
in Warsaw collection: A854 (isolated from throat mucosa,
30.03.2011) resistance: CIP, E, DA, AK and A876 (isolated
from lower leg injury, 19.02.2012) resistance: E, DA, CIP, AK.
H-12,13,14,15,16), 7.75 (d, 1H, J=9.0 Hz, H-5) ppm; 13
C
NMR (75.4 MHz, acetone-d6): δ=15.25 (C14-OCH2CH3),
19.19 (C-9), 64.14 (C-11), 64.40 (C14-OCH2CH3), 110.40
(C-4a), 111.70 (C-3), 115.27 (C-13,15), 116.00 (C-6),
121.40 (12,16), 124.35 (C-11), 129.78 (C-5), 133.03 (C7),
154.53 (C-8a), 154.79 (C-4), 160.09 (C-2), 168.61 (C-14),
173.12 (C-10) ppm; IR (KBr): ̄ꢀ =2984, 1722 (C=O), 1590
(C=N), 1507 (C=C), 832 (substitution para di) cm−1
;
HR-MS (TOF, ESI-): m/z calcd for C20H18NO4 ([M−H]−)
336.1236, found 336.1234.
7‑Hydroxy‑8‑[1‑(4‑methylphenylimino)ethyl]‑4‑me‑
thyl‑2H‑chromen‑2‑one (27, C19H17NO3) This product
was prepared according to the general procedure. Yellow
Antimicrobial activity: preliminary test
1
solid; yield 81%; m.p.: 149–150 °C; Rf= 0.54; H NMR
In the preliminary tests, antimicrobial activity was deter-
mined by a modifed cylinder–plate method. Stainless steel
cylinders of 6 mm diameter were put on a Mueller–Hinton
two agar (for bacteria strains) or Sabouraud agar (for yeast
strains) plate inoculated with one of the tested strains. An
aliquot of 100 mm3 of each compound in concentration of
10 mg/cm3 (dissolved in 10% DMSO, in a 0.08 M phosphate
bufer, pH 7.0) was placed into the cylinder. As a negative
control 10% DMSO (in 0.08 M phosphate bufer, pH 7.0)
without any compound was used. The plates with bacterial
strains were incubated at 37°C for 24 h, whereas plates with
yeast strains at 30°C for 48 h. The inhibition of bacterial
growth was observed as a halo around the cylinder contain-
ing the tested compound. Size of inhibition zone refected
an antimicrobial activity of the compound.
(300 MHz, acetone-d6): δ=2.37 (s, 3H, C14-CH3), 2.47 (d,
3H, J = 1.2 Hz, H-9), 2.65–2.80 (m, 3H, C10-CH3), 6.12
(br.s, 1H, H-3), 6.89 (d, 1H, J=9.3 Hz, H-6), 7.15 (d, 2H,
J=9.0 Hz, H-13,15), 7.30 (d, 2H, J=7.8 Hz, H-14,16), 7.76
(d, 1H, J=9.0 Hz, H-5) ppm; 13C NMR (75 MHz, acetone-
d6): δ=19.15 (C-9), 21.02 (C14-CH3), 34.00 (C-11), 111.67
(C-8), 113.10 (C-4a), 115.24 (C-6), 115.39 (C-3), 120.26
(C-12,16), 129.85 (C-5), 130.78 (C-13,15), 133.00 (C-14),
146.82 (C-11), 150.41 (C-4), 154.56 (C-8a), 154.80 (C-10),
159.60 (C-2), 160.09 (C-7) ppm; IR (KBr): ꢀ̄ =3023 (OH),
2985, 1722 (C=O), 1603 (C=N), 1505 (C=C), 839 (substi-
tution para di) cm−1; HR-MS (TOF, ESI+): m/z calcd for
C19H17NNaO3 ([M+Na]+) 307.1208 found 307.1186.
7‑Hydroxy‑8‑[1‑(4‑ethylphenylimino)ethyl]‑4‑me‑
thyl‑2H‑chromen‑2‑one (28, C20H19NO3) This product was
prepared according to the general procedure. Yellow solid;
yield 61%; m.p.: 122–123 °C; Rf=0.53; 1H NMR (300 MHz,
acetone-d6): δ=1.26 (t, 3H, J=7.5 Hz, C14-CH2CH3), 2.47
Minimum inhibitory concentration
For the compounds, which showed some activity against any
of the tested strains, a minimum inhibitory concentration
1 3

E. Hejchman et al.
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(MIC) was determined based on M7-A9 method [23]. The
stock solution was prepared by dissolving each compound
in DMSO. A series of gradient solutions in the range from
1 to 0.0156 mg/cm3 was obtained by the double dilution in
0.08 M phosphate bufer (pH 7.0). The 250 mm3 aliquot of
the successive dilution of tested compound was mixed with
4.75 cm3 of a liquefed Mueller–Hinton two or Sabouraud
agar, precooled to 45°C. The suspension of the particular
strain of density 0.5 unit (McFarland scale) was diluted ten
times and aliquot of 2 mm3 was applied on agar plate sur-
face. The lowest concentration of tested compound, which
totally inhibited growth of the examined strain, was taken as
MIC value. As a negative control the solution of the highest
concentration of DMSO in 0.08 M phosphate bufer (pH 7.0)
was used. All tests were performed in triplicates. In control
samples, MIC values of ciprofoxacin ranging between 0.08
and 1.36×10−3 µmol/cm3 for bacterial strains.
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of Warsaw, Faculty of Pharmacy, FW24/NM1/17.
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