N-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)thiosemicarbazones of 6-alkoxy-2-oxo-2H-chromene-4-carbaldehydes: synthesis, evaluation of their antibacterial, anti-MRSA, antifungal activity, and docking study

Article abstract of DOI:10.1007/s00044-020-02688-0

Reaction of 6-alkoxy-2-oxo-2H-chromen-4-carbaldehydes with N-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)thiosemicarbazide yielded corresponding thiosemicarbazones having 2H-chromen-2-one ring. In vitro evaluations showed that these 2H-chromen-2-one compounds exhibited remarkable antibacterial and antifungal activities against some typical bacteria and fungi. Representative compounds with MIC values of 0.78 ? 1.56 μg/mL were 6c, 6g (against S. aureus), 6a, 6f (against S. epidermidis) (Gram-positive bacterial strains), 6e, 6g (against E. coli), 6b, 6e (against K. pneumoniae), and 6d–f (against S. typhimurium) (Gram-negative bacterial strains). Almost all thiosemicarbazones 6a–g had no activity against Gram-positive bacterial strain B. subtilis at these MIC values. Some compounds had strong inhibitory activity against several bacteria, such as 6b (for K. pneumoniae and S. typhimurium), 6d, 6e (for E. coli, K. pneumoniae, and S. typhimurium), 6f (for S. aureus, E. coli, and S. typhimurium), and 6g (for B. subtilis, S. aureus, E. coli, and K. pneumoniae). Some compounds had remarkable inhibitory activity against three clinical MRSA isolates with MIC values of 0.78–6.25 μg/mL. Docking study showed that compound 6g is compatible with the active site of S. aureus DNA gyrase 2XCT, which suggested that the tested compounds inhibited the synthesis of this enzyme. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-020-02688-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:743–759
https://doi.org/10.1007/s00044-020-02688-0
ORIGINAL RESEARCH
N-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)thiosemicarbazones of 6-
alkoxy-2-oxo-2H-chromene-4-carbaldehydes: synthesis, evaluation of
their antibacterial, anti-MRSA, antifungal activity, and docking study
2
Vu Ngoc Toan1,2 Nguyen Dinh Thanh
Vu Hong Khuyen2 Luu Thi Cam Tu2 Nguyen Minh Tri1,2
●
●
●
●
●
Nguyen Thi Thu Huong2
Received: 16 September 2020 / Accepted: 7 December 2020 / Published online: 13 January 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Reaction of 6-alkoxy-2-oxo-2H-chromen-4-carbaldehydes with N-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)thiosemicarbazide
yielded corresponding thiosemicarbazones having 2H-chromen-2-one ring. In vitro evaluations showed that these 2H-chromen-2-
one compounds exhibited remarkable antibacterial and antifungal activities against some typical bacteria and fungi. Representative
compounds with MIC values of 0.78 − 1.56 μg/mL were 6c, 6g (against S. aureus), 6a, 6f (against S. epidermidis) (Gram-positive
bacterial strains), 6e, 6g (against E. coli), 6b, 6e (against K. pneumoniae), and 6d–f (against S. typhimurium) (Gram-negative
bacterial strains). Almost all thiosemicarbazones 6a–g had no activity against Gram-positive bacterial strain B. subtilis at these MIC
values. Some compounds had strong inhibitory activity against several bacteria, such as 6b (for K. pneumoniae and
S. typhimurium), 6d, 6e (for E. coli, K. pneumoniae, and S. typhimurium), 6f (for S. aureus, E. coli, and S. typhimurium), and 6g
(for B. subtilis, S. aureus, E. coli, and K. pneumoniae). Some compounds had remarkable inhibitory activity against three clinical
MRSA isolates with MIC values of 0.78–6.25 μg/mL. Docking study showed that compound 6g is compatible with the active site
of S. aureus DNA gyrase 2XCT, which suggested that the tested compounds inhibited the synthesis of this enzyme.
Graphical Abstract
* Nguyen Dinh Thanh
2
Faculty of Chemistry, VNU University of Science, 19 Le Thanh
Tong, Hoan Kiem, Ha Noi, Vietnam
nguyendinhthanh@hus.edu.vn
1
Department of Toxicological Chemistry and Radiation, Institute
for Advanced Technology, Vietnam Academy of Military Science
and Technology, 17 Hoang Sam, Cau Giay, Ha Noi, Vietnam

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Keywords 6-Alkoxy-2-oxo-2H-chromene-4-carbaldehydes Antibacterial Antifungal Anti-MRSA Thiosemicarbazones
Molecular docking
Introduction
active coumarin compounds against Staphylococcus aur-
eus tyrosyl-tRNA synthetase and topoisomerase II DNA
gyrase [32]. Maxwell found that three glycoside com-
pounds derived from different Streptomyces species with
coumarin structure, including novobiocin (D, Fig. 1, also
known as albamycin or cathomycin, an aminocoumarin
antibiotic), clorobiocin (E), and coumermycin A1, dis-
played antibiotic activity as potent inhibitors of DNA
topoisomerase type II (DNA gyrase) [33]. Kayser and
Kolodziej found that both lipophilic characteristics as well
as a planar structure were necessary for high antibacterial
effects [34]. Sardari et al. indicated that 6- and 7-hydroxyl
groups in coumarin ring played an important role for
antifungal and antibacterial activity [35]. In fact, cou-
marins with 6-, 7-, or 8-hydroxyl/alkoxyl moiety had
antibacterial effect against a broad spectrum of bacteria
[18, 19, 36].
Thiosemicarbazone derivatives containing mono-
saccharide moiety had synthesized due to their remark-
able anti-microorganisms and antioxidant activity both
in vivo and in vitro [1, 3, 4, 6, 37, 38], including anti-
bacterial [2, 37], antifungal [37], antioxidant [6], anti-
dyslipidemic [3], antituberculosis [1], and anticancer
[38] activities. Figure 2 displayed some examples of
bioactive thiosemicarbazones-bearing sugar moiety.
Sugar-based thiosemicarbazone of peracetylated gluco-
side (compound F) was discovered to be a potent Rho-
desian (major cysteine protease from Trypanosoma
brucei) inhibitor (IC50 = 1.2 μg/mL) [39]. Thiosemi-
Formyl functional group that presented in 2H-chromen-2-
one ring (coumarin ring) could contribute to extend the
reactivity of this ring. Various compounds that carry this
ring could be formed based on the properties of formyl
functional group, such as thiosemicarbazones [1–8],
2H-chromen-2-one substituted benzothiazole derivatives
[9], Schiff’s bases of 2H-chromen-2-one [7, 10], etc. The
formyl group could be attached to benzene ring or to 2H-
pyran-2-one ring at any position of those moieties of
coumarin ring [11–16]. Coumarin (2H-chromen-2-one), a
compound of cinnamon, was a family of benzopyrone
(1,2-benzopyrone or 2H-1-benzopyran-2-ones) that
widely distributed in the nature in many plants [17, 18].
Coumarin itself and its derivatives possessed various
pharmacological activities, such as antibacterial [19],
antioxidant [20], anti-inflammatory [21], anticancer [22],
antifungal [23] activity, etc. Nowadays these compounds
are still of interest to the scientific due to these biological
effects [24, 25]. For instances, compound A (Fig. 1)
exhibited potent anticancer activity with IC50 value of
5.18 mM against KB cell lines [26]. Compound B pre-
sented the highest oxygen radical absorbance capacity
value of 14.1, good scavenging capacity, low cytotoxi-
city, and high cytoprotecting values (almost 100% cell
viability at 20 μg/mL) [27]. Hydroxycoumarin C was
used as selective MAO-B inhibitor with IC50 = 2.79
0.19 μg/mL [28].
Some study results demonstrated that the action of the
coumarin derivative was against the structure of the fun-
gal cell wall [29, 30]. Coumarin derivatives inhibited
cytochrome P450 lanosterol 14α-demethylase (CYP51)
[31]. Molecular docking studies showed that some most
carbazones
G
containing simultaneously mono-
saccharide and isatin moieties exhibited in vitro
antibacterial and in vivo antioxidant activities. When
R′=Br, R=H the compound showed selective cytotoxic
effects against some cancer (LU-1, HepG2, MCF7, P338,
Fig. 1 Some bioactive and
typical clinically well-known
drugs or naturally occurring
compounds containing
coumarin ring

Medicinal Chemistry Research (2021) 30:743–759
745
SW480, KB) cell lines and normal fibroblast cell line
NIH/3T3. This compound had good inhibitory activity
with minimum inhibitory concentration (MIC) of
1.56 μg/mL against Bacillus subtilis, Staphylococcus
aureus, and Staphylococcus epidermidis [37].
Based on above-mentioned literatures, it is possible to
see that the hybridization between coumarin ring and
monosaccharide moiety could bring new activity or
enhance the inherent activity of coumarin rings. In this
article we reported the design and the synthesis of some 6-
alkoxy-2-oxo-2H-chromene-4-carbaldehydes
thiosemicarbazones-bearing D-glucose (Schemes 1 and 2).
The inhibitory activity of these compounds against some
typical bacterial and fungal strains was evaluated and
docking studies were performed on the most potent com-
pounds to investigate their binding mode with the active
site of the representative enzyme.
Results and discussion
Chemistry
We have provided the synthetic pathway of 6-alkoxy-2H-
chromen-2-ones (3a–g) that prepared from hydroquinone 2
(Scheme 1). These hydroxy derivatives were readily
obtained from corresponding dihydroxy benzene (hydro-
quinone and resorcinol); the formers were transformed into
ether by using Williamson ether synthesis, followed by
oxidation reaction when using selenium dioxide as an oxi-
dant. Almost all obtained 6-alkoxy-2-oxo-2H-chromen-2-
ones in this study are new compounds. Yields of
these ethers were 75–85%. The IR spectra of compounds
3a–g displayed characteristic absorption bands at ν =
1737–1708 cm−1 (C=O lactone), 1293–1242 cm−1 and
1170–1142 cm−1 (C–O–C group in lactone and ether
1
functional groups). The H NMR spectra showed chemical
shifts at δ = 7.66–6.17 ppm (protons on 2H-chromen-2-one
ring), 4.03–0.88 ppm (protons in the alkoxy group).
Methylene group attached to oxygen atom had signal at
about δ = 4.07–4.00 ppm. The 13C NMR spectra displayed
resonance signals of carbon atoms in molecules, for
examples, at δ = 160.6 − 101.1 ppm (carbon atoms in the
2H-chromen-2-one component, except C=O lactone atom),
162.2–159.9 ppm (carbon atom in C=O lactone group), and
30.7–10.7 ppm (carbon atoms in the alkoxy group, except
Fig. 2 Some bioactive thiosemicarbazones-bearing sugar moiety
Scheme 1 Synthetic path for
6-alkoxy-2H-chromene-4-
carbaldehydes from 6-hydroxy-
2H-chromen-2-ones. Reagents
and conditions: (i) 1.70%
H2SO4, 10 °C (for 20 min) to
25 °C (for 24 h); 2.5% NaOH
solution to pH 9, then 20%
H2SO4 solution to pH 5 [44]; (ii)
R−Br and KI or R−I, K2CO3,
dried acetone, or DMF, 12
−16 h, under reflux (for chloride
and bromide derivatives: KI
(1 mol%) was added); (iii)
activated SeO2, xylene, for 24 h
under reflux

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Scheme 2 Synthetic path for
substituted 2-oxo-2H-chromene-
4-carbaldehyde N-(tetra-O-
acetyl-β-D-glucopyranosyl)
thiosemicarbazones. Reaction
conditions: glacial acetic acid
(cat.), MeOH (solv.) under MW-
assisted conditions at microwave
power of 600 W
methylene carbon atom attached to oxygen) depending on
their positions in carbon alkyl chain, and δ = 74.3–64.2
ppm (methylene carbon atom attached to oxygen).
9–13 min at microwave power of 600 W. We found that
strong inorganic acids, such as sulfuric or perchloric acids,
did not apply to catalyze this reaction, although these acids
usually were used for this reaction type [41]. Weak organic
acids, such as acetic acid, even trichloroacetic acid were
sufficient to activate the carbonyl group without protonation
of amin group of thiosemicarbazide.
Oxidation of 4-methyl group on 2H-chromen-2-one ring
of compounds 3a–g performed by using activated selenium
dioxide as selective oxidant in xylene (Riley’s oxidation)
according to modified literature procedure (Scheme 1) [40].
Yields of corresponding 4-formyl derivatives 4a–g achieved
41–67%. The IR spectra of these aldehydes displayed the
characteristic absorption bands for functional groups that
are present in the molecule. Intense absorption bands
characterizing stretching vibration for functional groups, for
examples, in region at ν = 1737–1712 cm−1 (νC=O lactone),
1710–1700 cm−1 (νC=O aldehyde), 1270–1243 cm−1, and
1151–1145 cm−1 (νCOC lactone and ether). Benzene ring of
2H-chromen-2-one had some characteristic absorption
bands for aromatic C=C bonds in region at ν =
The IR spectra of 6-alkoxy-2-oxo-2H-chromene-4-carbal-
dehyde N-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)thiose-
micarbazones (6a–g) showed characteristic absorptions in the
range of ν = 3354–3447 and 3328–3234 cm−1 belonging to
stretching vibrations of N–H groups of thiosemicarbazone
linkage group (–NHCSNHN=C<); this linkage group had
chemical shift at δ = 8.53–8.44 ppm. Chemical shifts
appeared at δ = 12.21–12.20 (singlet, proton NH-2) and δ =
9.27–9.16 ppm (doublet, proton NH-4) confirmed the pre-
sence of N–H groups in molecular structure of thiosemi-
carbazone 6a–g. Proton NH-4 had coupling interaction to
proton H-1 on pyranose ring with coupling constant J =
9.5–9.0 Hz. β-Anomeric configuration of these thiosemi-
carbazones was confirmed by the values of coupling constants
1540–1480 cm−1 1H NMR spectra exhibited chemical
.
shifts in accordance with the structure of compounds 4a–g.
Protons of 2H-chromen-2-one ring had chemical shifts in
region at δ = 8.00–6.33 ppm with the splitting pattern fac-
tions conforming with the substitution on benzene ring of
2H-chromen-2-one. Proton of formyl group gave resonance
signal at δ = 10.16–10.08 ppm. Alkyl protons had chemical
shifts in region at δ = 4.10–0.84 ppm. The 13C NMR
spectra displayed resonance signals of carbon atoms that are
present in the molecule. The carbon atoms in the aldehyde
group have a resonance signal of nearly δ = 194.1–193.6
ppm, the carbon atoms of 2H-chromen-2-one ring are in
region at δ = 161.9–100.9 ppm, and C-alkyl atoms had
signals at δ = 69.6–10.2 ppm.
Synthesis of target molecules, 6-alkoxy-2-oxo-2H-chro-
mene-4-carbaldehyde N-(2,3,4,6-tetra-O-acetyl-β-D-gluco-
pyranosyl)-thiosemicarbazones, performed as following.
N-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl thiosemicarba-
zide (5) reacted with above-synthesized 6-alkoxy-2-oxo-2H-
chromene-4-carbaldehydes (4a–g) to yield corresponding
thiosemicarbazones 6a–g. The condensation reaction was
carried out in absolute methanol in the presence of glacial
acetic acid as catalyst using microwave-assisted heating
method (Scheme 2). Reaction mixture was irradiated for
1
of J = 10.0–9.0 Hz between protons H-1 and H-2 in the H
NMR spectra. An absorption bands with medium intensities
lying in region at ν = 1373–1327 cm‒1 belonged to the
stretching vibration of C=S group of thiosemicarbazone
linkage. Resonance of carbon atom C-1 on pyranose ring was
easily recognized, at δ = 82.2–82.0 ppm, which is the signal
lying in the weakest field in resonance region of pyranose’s
carbon. Chemical shift at δ = 5.98–5.94 ppm was assigned to
proton H-1. The 13C NMR spectra of these thiosemicarba-
zones showed resonance signals at δ = 179.1–178.9 ppm (for
carbon atom in C=S group) and 170.0–169.3 ppm (for carbon
atoms in C=O bond of acetyl groups). Carbon atoms in 2H-
chromen-2-one ring had chemical shifts at δ = 155.5–107.4
ppm, whereas resonance signals at δ = 82.2–61.3 ppm
belonged carbon atoms on D-glucopyranose ring and chemical
shifts at δ = 20.5–20.3 ppm belonged methyl carbons in
acetyl groups, except for the carbon attached to oxygen atom,
which had chemical shift at δ = 74.3–63.8 ppm. Carbon atom
in lactone carbonyl group on 2H-chromen-2-one ring had
chemical shift at δ = 160.3–160.0 ppm.

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Table 1 Antibacterial activity of thiosemicarbazones 6a–g
Entry
Alkoxy groups
Microorganisms/MIC (μg/mL)
Gram-positive bacteria
Gram-negative bacteria
B. subtilis
S. aureus
S. epidermidis
E. coli
K. pneumoniae
P. aeruginosa
S. typhimurium
6a
6b
6c
6d
6e
6f
OCH2CH3
400
200
400
25
100
0.78
25
12.5
25
50
100
100
200
25
12.5
3.125
6.25
1.56
1.56
0.78
6.25
1.56
–
OCH2CH2CH3
OCH(CH3)2
400
0.78
400
3.125
1.56
25
12.5
400
25
25
OCH2(CH2)2CH3
OCH2CH(CH3)2
OCH2(CH2)3CH3
OCH2CH2CH(CH3)2
Ciprofloxacin
Vancomycin
12.5
50
3.125
1.56
0.78
3.125
1.56
–
25
1.56
3.125
0.78
3.125
3.125
400
400
50
50
1.56
50
6g
3.125
3.125
1.56
400
3.125
1.56
–
50
3.125
0.78
400
1.56
–
Methicillin
400
400
400
400
Pharmacology
0.78 μg/mL) against S. aureus; 6a (MIC = 0.78 μg/mL), 6f
(MIC = 1.56 μg/mL) against S. epidermidis; 6d, 6g (MIC =
3.125 μg/mL), 6e (MIC = 1.56 μg/mL), and 6f (MIC =
0.78 μg/mL) against E. coli; 6d, 6g (MIC = 3.125 μg/mL),
6e (MIC = 1.56 μg/mL), and 6b (MIC = 0.78 μg/mL)
against K. pneumoniae), 6c, 6g (MIC = 6.25 μg/mL), 6b
(MIC = 3.125 μg/mL), 6d, 6e (MIC = 1.56 μg/mL), and 6f
(MIC = 0.78 μg/mL) against S. typhimurium. Almost all thio-
semicarbazones 6a–g had no activity against strain B. subtilis
(except compound 6g with 6-isopentoxy, MIC = 3.125 μg/mL)
and P. aeruginosa (MIC values were 25–400 μg/mL). Thus,
the synthesized thiosemicarbazone 2H-chromen-2-one−D-glu-
cose conjugates were more inhibitory active against Gram-
negative bacteria than Gram-positive ones. Gram-positive
bacterium B. subtilis inhibited only by thiosemicarbazone 6g
with MIC value of 3.125 μg/mL.
Some compounds had strong inhibitory activity against
several bacteria simultaneously in MIC ranges of 0.78–6.25 μg/
mL. Compound 6b had activity against K. pneumoniae and
S. typhimurium with MIC values of 0.78 and 3.125 μg/mL,
respectively. Compound 6d exhibited inhibitory activity
against Gram-negative bacterial strains, including E. coli, K.
pneumoniae, and S. typhimurium with MIC values of 3.125,
3.125, and 1.56 μg/mL, respectively. Compound 6e had
stronger activity against S. aureus, E. coli, K. pneumoniae, and
S. typhimurium with all MIC values of 1.56 μg/mL. Compound
6f had also stronger inhibition against S. aureus, S. epidermidis,
E. coli, and S. typhimurium with MIC values of 3.125, 1.56,
0.78, and 0.78 μg/mL. Especially, compound 6g expressed
remarkable inhibitory activity against five bacterial strains,
including B. subtilis, S. aureus, E. coli, K. pneumoniae, and S.
typhimurium, with MIC values of 3.125, 0.78, 3.125, 3.125,
and 6.25 μg/mL, respectively. The remaining thiosemicarba-
zones had MIC values of 25–400 μg/mL. Several thiosemi-
carbazones had inhibitory activity against each Gram-negative
Antibacterial assays
The evaluations of biological activity of compounds 6a–g
against some above-mentioned typical bacteria were repre-
sented in Table 1. In general, almost all compounds had
remarkable biological activities against tested microorgan-
isms. The results in Table 1 showed that almost all novel
molecule exhibited antibacterial activity against the tested
bacteria at low and high concentrations. In general, it has been
observed that almost all tested compounds showed mild-to-
moderate activity against the tested bacteria in comparison
with the MIC values of the references compound. MIC values
of these reference drugs are as follows: Ciprofloxacin,
3.12 μg/mL (for Gram-positive bacteria), 1.56 μg/mL (for
Gram-negative bacteria); vancomycin, 0.78–3.12 μg/mL (for
Gram-positive bacteria). Methicillin had the least inhibitory
activity for all tested bacteria (all MICs = 400 μg/mL). In
ranges of MIC below 6.25 μg/mL, these thiosemicarbazones
exhibited strong inhibitory activity against bacteria S. aureus,
E. coli, K. pneumoniae, and S. typhimurium, medium activity
against S. epidermidis, weak activity against B. subtilis, and
had no activity against P. aeruginosa.
In general, almost all synthesized thiosemicarbazones were
similar or more inhibitory active than ciprofloxacin but less
than vancomycin. In vitro evaluations in Table 1 showed that
these 2H-chromen-2-one compounds exhibited remarkable
antibacterial and antifungal activities against some typical
bacteria and fungi. Some thiosemicarbazones had higher ability
to inhibit to Gram-positive bacteria (B. subtilis, S. aureus,
and S. epidermidis) with MIC values of 1.56–6.25 μg/mL.
Amongst these thiosemicarbazones were compound 6g
(MIC = 3.125 μg/mL) against B. subtilis; compounds 6e
(MIC = 1.56 μg/mL), 6f (MIC = 3.125 μg/mL), 6g (MIC =

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Table 2 MRSA antibacterial activity of thiosemicarbazones 6a–g
Table 3 Inhibitory assessment (IC50 in μM) of compound 6g on S.
aureus DNA gyrase, Topoisomerase IV, and Dihydrofolate reductase
enzymes
Entry Alkoxy groups
MRSA198-1 MRSA198-2 MRSA198-3
6a
6b
6c
6d
6e
6f
OCH2CH3
400
12.5
50
100
200
6.25
50
6.25
6.25
12.5
100
25
Compound
IC50 (μM)
OCH2CH2CH3
OCH(CH3)2
S. aureus
DNA gyrase
Topoisomerase IV Dihydrofolate
reductase
OCH2(CH2)2CH3 200
OCH2CH(CH3)2 25
OCH2(CH2)3CH3 12.5
6g
1.82 0.11
79.52 1.20
26.12 1.01
−
0.12 0.05
−
400
25
Ciprofloxacin 1.65 0.13
Methotrexate
1.56
0.78
−
0.34 0.07
6g
OCH2CH2CH
(CH3)2
12.5
1.56
Ciprofloxacin
Vancomycin
Methicillin
400
400
1.56
NE
400
1.56
NE
1.56
NE[a]
IV enzymes. The obtained results as IC50 were presented in
Table 3. The suitable positive controls were used, including
ciprofloxacin and methotrexate.
NE not evaluated
Table 3 showed that compound 6g exhibited an equipotent
inhibitory activity toward DNA gyrase (IC50 = 1.82 0.11 μM
vs. IC50 = 1.65 0.13 μM for ciprofloxacin) in comparison
with the reference ciprofloxacin, and weak activity against
Topoisomerase IV (IC50 = 79.52 1.20 μM vs. IC50 = 26.12
1.01 μM for ciprofloxacin). In addition, compound 6g dis-
played twofolds increase in the suppression effect toward
dihydrofolate reductase comparing with methotrexate (IC50 =
0.08 1.15 μM and 0.14 1.07 μM, respectively).
bacterium similar or stronger than ciprofloxacin, with MIC
value of 0.78 or 1.56 μg/mL, such as 6e (MIC = 1.56 μg/mL)
and 6f (MIC = 0.78 μg/mL) against E. coli, 6b (MIC =
0.78 μg/mL) and 6e (MIC = 1.56 μg/mL) against K. pneumo-
niae, 6d, 6e (MIC = 1.56 μg/mL), 6f (MIC = 0.78 μg/mL)
against S. typhimurium. Thus, the compounds mentioned
herein were more active than vancomycin (MIC = 3.12 μg/mL)
and methicillin (MIC = 400 μg/mL) for all tested Gram-
negative bacteria. The presence 6-alkoxy substituents on cou-
marin ring also caused the similar changes on antibacterial
activity ò compounds bearing coumarin ring [18, 19, 36].
We have evaluated the antibacterial activities of these
thiosemicarbazones against a series of MRSA strains,
signed MRSA198-1, MRSA198-2, and MRSA198-3
from clinical sources [42]. Among the tested compounds
(Table 2), 6g exerted excellent anti-MRSA activity against
all tested MRSA strains with MIC values of 1.56, 1.56, and
0.78 μg/mL, respectively. Compound 6f expressed good
inhibitory activity against MRAS198-3 with MIC value of
1.56 μg/mL. In addition, the inhibitory effects of thiosemi-
carbazones 6a and 6b were obviously observed by treating
it against strain MRSA198-3 with MIC values of 6.25 μg/
mL. Thiosemicarbazone 6c had better inhibition activity
against strain MRAS198-2 with MIC value of 6.25 μg/mL.
Compounds that showed mild anti-MRSA effect with MIC
values of 12.5–25 μg/mL included 6b, 6e, 6f (against
MRSA198-1), 6f (against MRSA198-2), and 6c, 6e (against
MRSA198-3). Other compounds did not show any sig-
nificance in anti-MRSA activity (MIC = 50–100 μg/mL).
Antifungal assay
We evaluated the antifungal activities of the above thiose-
micarbazones 6a–g against some fungi, such as Aspergillus
niger (ATCC 439), Aspergillus flavus (ATCC 204304),
Candida albicans (ATCC 7754), and Saccharomyces cer-
evisiae (SH 20). Miconazole and fluconazole were used as
references. Miconazole is mainly used externally for the
treatment and prophylactic treatment of Candida infection
in the oral cavity and the digestive tract. Fluconazole is a
first-generation triazole antifungal medication. MIC values
of miconazole were 1.56, 1.56, 3.12, and 3.12 μg/mL for
each fungus, respectively, and of fluconazole were 1.56,
0.78, 0.78, and 0.78 μg/mL for each fungus, respectively.
The obtained results were given in Table 4.
Results in Table 4 showed that in ranges of MIC values of
0.78–6.25 μg/mL almost all of the tested thiosemicarbazones
6a–g were stronger active against fungi A. niger and A. fla-
vus, but medium active against S. cerevisiae, and weak active
against C. albicans. Thiosemicarbazones 6b, 6f, and 6g were
more resistant to A. niger and A. flavus than miconazole and
fluconazole against A. niger. Almost all of the compounds
were less active than miconazole and fluconazole against
C. albicans and S. cerevisiae, except compound 6a (MIC =
1.56 μg/mL) that was more active than miconazole and less
active than fluconazole (against C. albicans), and compounds
6c, 6d (MIC = 3.125 and 6.25 μg/mL, respectively) were less
active than fluconazole. Notable active compounds included
In vitro kinase assessment
To order, understand the preliminary mechanism of the com-
pound 6g with potent antibacterial activity and to confirm the
results of docking study, an enzyme inhibitory assay was
performed toward S. aureus DNA gyrase and Topoisomerase

Medicinal Chemistry Research (2021) 30:743–759
749
Table 4 Antifungal activity of
Entry
Alkoxy groups
Fungi/ MIC (μg/mL)
thiosemicarbazones 6a–g
A. niger
A. flavus
C. albicans
S. cerevisiae
6a
6b
6c
6d
6e
6f
OCH2CH3
200
3.125
0.78
50
200
100
0.78
6.25
25
1.56
50
400
100
3.125
6.25
50
OCH2CH2CH3
OCH(CH3)2
25
OCH2(CH2)2CH3
OCH2CH(CH3)2
OCH2(CH2)3CH3
OCH2CH2CH(CH3)2
Miconazole
50
25
50
6.25
6.25
1.56
1.56
3.125
25
25
200
25
6g
100
3.125
0.78
1.56
0.78
3.125
0.78
Fluconazole
6b (MIC = 3.125 μg/mL) and 6c (MIC = 0.78 μg/mL)
against A. niger, 6c (MIC = 0.78 μg/mL) and 6f (MIC =
3.125 μg/mL) against A. flavus, 6a (MIC = 1.56 μg/mL)
against C. albicans, 6c (MIC = 3.125 μg/mL) against S.
cerevisiae. Remarkably, compound 6c was more active
against A. niger, C. albicans, and S. cerevisiae with MIC
values of 0.78, 0.78, and 3.125 μg/mL, respectively. Two
other compounds that had notable activity included 6d
(against A. flavus and S. cerevisiae) and 6f (against A. niger
and A. flavus) with MIC values of 3.125–6.25 μg/mL. These
such influents of 6-alkoxy substituents on antifungal activity
also observed in some compounds having coumarin ring [35].
base guanine (DG9 on chain G). There was two π–π
stacking interactions of 2H-pyran ring of coumarin core
with DNA base thymine (DT8 on chain E) of ribonucleo-
tide 2-deoxythymidine-5-monophosphate and with
DNA base guanine (DG9 on chain G) of ribonucleotide
2-deoxyguanosine-5-monophosphate. As a result of dock-
ing study, it can be declared that compound 6g is compa-
tible with the active site of S. aureus DNA gyrase.
Conclusions
Various 6-alkoxy-4-methyl-2H-chromen-2-ones were pre-
pared using Williamson ether synthesis starting from 6-
hydroxy-2H-chromen-2-ones. These ethers were converted
into corresponding 4-carbaldehydes using Riley’s oxidation
procedure. Microwave-assisted heating method was effi-
cient and convenient method for synthesis of thiosemi-
carbazones of 6-alkoxy-4-methyl-2-oxo-2H-chromene-4-
carbaldehydes with N-(2,3,4,6-tetra-O-acetyl-β-D-glucopyr-
anosyl)thiosemicarbazide. These compounds displayed
significant inhibition in vitro against bacteria S. aureus,
E. coli, K. pneumoniae, and S. typhimurium and fungi A.
niger, A. flavus. The compound 6g exhibited good inhibi-
tory activity against five tested bacterial strains with MIC
values of 0.78–6.25 μg/mL. Compounds 6a–c, 6f, and 6g
were active against three clinical MRSA isolates with MIC
values of 0.78–6.25 mM, especially 6g exerted excellent
anti-MRSA activity against all tested MRSA strains with
MIC values of 0.78–1.56 μg/mL. Molecular docking study
was performed to observe binding efficiency and steric
interactions of the lead compound 6g with isopentoxy
substituent at C-6 position of 2H-chromene moiety. Dock-
ing results showed that compound 6g is compatible with the
active site of S. aureus DNA gyrase 2XCT with some
hydrogen-bonding interaction and two π–π stacking inter-
action, which suggested that the tested compounds inhibited
the synthesis of this enzyme in S. aureus.
Molecular docking
As above discussion, the newly synthesized compounds
have exhibited good antibacterial activity, in particular
against Gram-positive bacterium S. aureus. To understand
the mechanism of antibacterial activity of newly synthe-
sized compounds, molecular docking study was performed
on X-ray crystal structure of S. aureus DNA gyrase with its
bound inhibitor ciprofloxacin and the protein-ligand com-
plex was constructed based on the X-ray structure of this
enzyme [43]. The most active compound 6g was docked
into the empty binding site of this enzyme. The docking
pose of 6g in the active site of 2XCT is presented in Figs 3
and 4. The figures revealed the disposition of protein side-
chains and DNA that formed the drug binding pockets. Our
docking study showed that compound 6g bonds to DNA
cleavage as similar as ciprofloxacin did [43]. As shown in
Fig. 3, this compound formed some hydrogen-bonding
interactions at the active site of enzyme 2XCT. Oxygen
atom of acetate ester in position 4 of pyranose ring formed
hydrogen-bonding interaction with amino-hydrogen of
DNA base cytosine (DC11 on chain H). Other hydrogen-
bonding interactions included the ones of amino acid
residue arginine ARG1122 (on chain D) with carbonyl-
oxygen of coumarin ring and of amino-hydrogen of DNA

750
Medicinal Chemistry Research (2021) 30:743–759
Fig. 3 Interacting mode of
compound 6g (colored elements)
without and with helix form in
the active region surrounded in
DNA gyrase active site. The
numbers in violet colors were
distances for hydrogen bonding
and π–π stacking interactions.
DNA helices were in magenta
colors. Residues that had active
interactions were in thin-tube
representations. Ligand 6g was
in thick tube in color by
elements
Experimental
Chemistry
was visualized with UV light. Chemical reagents in high
purity were purchased from the Merck Chemical Company
(in Viet Nam). All materials were of reagent grade for
organic synthesis. 4-Methyl-6-hydroxy-2H-chromen-2-one
(2) and was prepared by reaction of hydroquinone (1) with
ethyl acetoacetate in the presence of 70% sulfuric acid
according to Russell’s procedure (see Scheme 1) [44].
N-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)thiosemicarba-
zide (5) was prepared from D-glucose via corresponding
bromide and isocyanate derivatives according to the literature
procedure (see Scheme 2) [37, 45].
Instruments
Melting points were determined by open capillary method
on STUART SMP3 instrument (BIBBY STERILIN, UK)
and are uncorrected. IR spectra (KBr disc) were recorded on
1
an Impact 410 FT-IR Spectrometer (Nicolet, USA). H and
13C NMR spectra were recorded on Bruker Avance Spec-
trometer AV500 (Bruker, Germany) at 500 and 125 MHz,
respectively, using DMSO-d6 as solvent and TMS as an
internal standard; ESI-EI-HRMS and ESI/HR mass spectra
were recorded on EI-MS LTQ Orbitrap XL or Thermo
Scientific Exactive Plus Orbitrap spectrometers (Thermo-
Scientific, USA) in methanol using the ESI method. The
analytical thin-layer chromatography was performed on
silica gel 60WFS254 aluminum sheets (Merck, Germany) and
General procedure for the synthesis of 6-alkoxy-4-methyl-
2H-chromen-2-ones (3a–g)
To the suspension of 4-methyl-6-hydroxy-2H-chromen-2-
one (2a, 0.02 mol) in dry acetone (20 mL) was added
anhydrous potassium carbonate (0.04 mol, 5.5 g). The
mixture was heated under reflux for 10 min with stirring.

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751
Fig. 4 Two-dimensional
diagram (top) in ligand
interaction of compound 6g in
DNA gyrase active site showed
active ligand-receptor
interactions. The pocket on this
enzyme was shown by
electrostatic potential surface
(bottom). Ligand 6g was in thick
tube in color by elements
(carbon was in green color,
oxygen in red, nitrogen in blue,
sulfur in yellow)
Appropriate alkyl halide (bromide or iodide) was added. In
cases of bromide derivatives, KI (0.001 mol, 0.17 g) was
supplemented. Reaction mixture continued to heat under
reflux for 20 h. Solvent was removed completely under
reduced pressure. Water (20 mL) was added to residue in
order to dissolve inorganic salts. The precipitate was fil-
tered, washed with water until pH 7, and crystallized from
96% ethanol to yield the title compounds 3a–g.
d6, ppm), δ (ppm): 7.27 (d, J = 9.0 Hz, 1H, H-8), 7.16
(dd, J = 9.0, 2.5 Hz, 1H, H-7), 7.11 (d, J = 2.5 Hz, 1H,
H-5), 6.34 (s, 1H, H-3), 4.07 (q, J = 7.0 Hz, 2H, 6-
OCH2CH3), 2.39 (s, 3H, 4-CH3), 1.35 (t, J = 7.0 Hz, 3H,
6-OCH2CH3); 13C NMR (125 MHz, DMSO-d6), δ (ppm):
160.3 (C=O lactone, C-2), 155.3 (C-6), 153.4 (C-8a),
147.6 (C-4), 120.5 (C-5), 119.7 (C-7), 117.8 (C-8), 115.1
(C-4a), 109.2 (C-3), 64.2 (6-OCH2CH3), 18.6 (4-CH3),
15.0 (6-OCH2CH3); ESI-MS: C12H12O3, calc. for M =
204.1 Da, found: m/z 204.2 ([M]+).
4-Methyl-6-ethoxy-2H-chromen-2-one
(3a) From
2a
(0.02 mol, 3.52 g), ethyl iodide (0.024 mol, 1.93 mL).
Reaction time: 16 h. Yield: 3.47 g (85%) of 3a as pale-
yellow crystals. M.p.: 116–117 °C (from 96% ethanol),
ref. [46], 116–118 °C (from 96% ethanol). IR (KBr),
ν (cm−1) 1715 (νC=O lactone), 1572, 1485 (νC=C arene),
4-Methyl-6-propoxy-2H-chromen-2-one (3b) From 2a
(0.02 mol, 3.52 g), propyl iodide (0.022, 2.4 mL). Reaction
time: 16 h. Yield: 3.27 g (75%) of 3b as pale-yellow crys-
tals. M.p.: 119 − 121.5 °C (from 96% ethanol). IR (KBr),
ν cm−1: 1713 (νC=O lactone), 1576, 1497 (νC=C arene),
1
1165, 1067 (νCOC lactone); H NMR (500 MHz, DMSO-

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1
1173, 1029 (νCOC lactone); H NMR (500 MHz, CDCl3,
ppm), δ (ppm): 7.32 (d, J = 8.5 Hz, 1H, H-8), 7.21 (d, J =
8.5 Hz, 1H, H-7), 7.18 (s, 1H, H-5), 6.38 (s, 1H, H-3), 4.02
(t, J = 7.0 Hz, 2H, 6-OCH2CH2CH3), 2.43 (s, 3H, 4-CH3),
1.76 (sextet, J = 6.5 Hz, 2H, 6-OCH2CH2CH3), 1.00 (t, J =
7.25.0 Hz, 3H, 6-OCH2CH2CH3); 13C NMR (125 MHz,
DMSO-d6), δ (ppm): 160.4 (C=O lactone, C-2), 155.5 (C-
6), 153.5 (C-8a), 147.6 (C-4), 120.6 (C-5), 119.9 (C-7),
117.9 (C-8), 115.1 (C-4a), 109.4 (C-3), 70.2 (6-
OCH2CH2CH3), 22.5 (4-CH3), 18.6 (6-OCH2CH2CH3),
10.9 (6-OCH2CH2CH3); ESI-MS: C13H14O3, calc. for M +
H = 219.10 Da, M + Na = 241.08 Da, found: m/z 219.16
([M + H]+), 241.09 ([M + Na]+).
4-Methyl-6-isobutoxy-2H-chromen-2-one (3e) From 2a
(0.02 mol, 3.52 g), isobutyl bromide (0.022 mol, 2.59 mL), and
KI (0.1 mmol, 0.17 g). Reaction time: 12 h. Yield: 3.49 g (75%)
of 3f as white crystals. M.p.: 103–105 °C (from 96% ethanol).
IR (KBr), ν (cm−1): 1718 (νC=O lactone), 1520, 1500, 1480
1
(νC=C arene), 1248, 1167 (νCOC lactone); H NMR (500 MHz,
DMSO-d6), δ (ppm): 7.60 (d, J = 8.75 Hz, 1H, H-7), 6.92 −
6.87 (m, 2H, H-5, and H-8), 6.16 (s, 1H, H-3), 4.04 [d, J =
8.0 Hz, 2H, 6-OCH2CH(CH3)2], 2.36 (s, 3H, 4-CH3),
1.73–1.67 [m, 1H, 6-OCH2CH(CH3)2], 0.93 [d, J = 7.0 Hz, 6H,
6-OCH2CH(CH3)2]; 13C NMR (125 MHz, DMSO-d6), δ
(ppm): 162.2 (C=O lactone, C-2), 160.6 (C-6), 155.2 (C-4),
153.8 (C-8a), 126.8 (C-4a), 113.4 (C-8), 112.8 (C-7), 111.5
(C-3), 101.5 (C-5), 68.4 [6-OCH2CH(CH3)2], 31.0 [6-
OCH2CH(CH3)2], 19.1 [6-OCH2CH(CH3)2], 18.5 (4-CH3);
ESI-MS: C14H16O3, calcd. for M + H = 233.12 Da, M + Na
= 255.10 Da, found: m/z 233.19 ([M + H]+), 255.08 ([M +
Na]+); ESI-HRMS: calcd. for M + H = 233.1178 Da, found:
233.1185 ([M + H]+, 100%); elemental anal. for C14H16O3,
calcd.: C, 72.39; H, 6.94%; found: C, 72.57; H, 6.61%.
4-Methyl-6-isopropoxy-2H-chromen-2-one (3c) From 2a
(0.02 mol, 3.52 g), isopropyl iodide (0.022, 2.4 mL).
Reaction time: 16 h. Yield: 2.62 g (60%) of 3d as pale-
yellow crystals. M.p.: 120–122 °C (from 96% ethanol). IR
(KBr), ν (cm−1): 1709 (νC=O lactone), 1573, 1490 (νC=C
1
arene), 1170, 1020 (νCOC lactone); H NMR (500 MHz,
DMSO-d6, ppm), δ (ppm): 7.68 (d, J = 3.0 Hz, 1H, H-5),
6.93–6.88 (m, 2H, H-7, and H-8), 6.48 (s, 1H, H-3),
4.23–4.14 [m, 1H, 6-OCH(CH3)2], 2.43 (s, 3H, 4-CH3),
1.02–0.98 [m, 6H, 6-OCH(CH3)2]; 13C NMR (125 MHz,
DMSO-d6), δ (ppm): 162.2 (C=O lactone, C-2), 160.6 (C-
6), 155.2 (C-4), 153.8 (C-8a), 126.8 (C-4a), 113.4 (C-8),
112.8 (C-7), 111.5 (C-3), 101.5 (C-5), 68.2 [6-OCH(CH3)
2], 22.3 [6-OCH(CH3)2], 19.5 (4-CH3); ESI-MS: C13H14O3,
calcd. for M + H = 219.10 Da, M + Na = 241.08 Da,
found: m/z 219.15 ([M + H]+), 241.15 ([M + Na]+); Ele-
mental anal. for C13H14O3, calcd.: C, 71.54; H, 6.47%;
found: C, 71.32; H, 6.24%.
4-Methyl-6-pentoxy-2H-chromen-2-one (3f) From 2a
(0.02 mol, 3.52 g), pentyl bromide (0.022 mol, 3.12 mL), and
KI (0.001 mol, 0.17 g). Reaction time: 12 h. Yield: 4.88 g
(84%) of 3d as white crystals. M.p.: 90–92 °C (from 96%
ethanol). IR (KBr), ν cm−1: 1719 (νC=O lactone), 1568, 1492
(νC=C arene), 1248, 1170 (νCOC lactone); 1H NMR (500 MHz,
DMSO-d6), δ (ppm): 7.28 (d, J = 9.0 Hz, 1H, H-7), 7.18 (dd,
J = 8.75, 2.3 Hz, 1H, H-8), 7.15 (d, J = 3.0 Hz, 1H, H-3), 6.45
(d, J = 0.5 Hz, 1H, H-3), 4.00 (t, J = 6.5 Hz, 2H, 6-
OCH2CH2CH2CH2CH3), 2.40 (s, 3H, 4-CH3), 1.72 (quintet,
J = 6.5 Hz, 2H, 6-OCH2CH2CH2CH2CH3), 1.37–1.35 (m,
4H, 6-OCH2CH2CH2CH2CH3), 0.88 (t, J = 7.0 Hz, 3H, 6-
OCH2CH2CH2CH2CH3); 13C NMR (125 MHz, DMSO-d6), δ
(ppm): 159.9 (C=O lactone, C-2), 155.0 (C-6), 153.0 (C-8a),
147.1 (C-4), 120.1 (C-5), 119.3 (C-7), 117.4 (C-8), 114.6 (C-
4a), 108.8 (C-3), 68.1 (6-OCH2CH2CH2CH2CH3], 28.3 (6-
OCH2CH2CH2CH2CH3), 27.7 (6-OCH2CH2CH2CH2CH3),
21.8 (6-OCH2CH2CH2CH2CH3), 18.1 (4-CH3), 13.9 (6-
OCH2CH2CH2CH2CH3]; ESI-MS: C15H18O3, calc. for (M +
H) = 247.1 Da, found: m/z 247.2 ([M + H]+).
4-Methyl-6-butoxy-2H-chromen-2-one
(3d) From
2a
(0.02 mol, 3.52 g), butyl bromide (0.022 mol, 2.58 mL), and
KI (0.001 mol, 0.17 g). Reaction time: 12 h. Yield: 4.65 g
(85%) of 3c as white crystals. M.p.: 107–109 °C (from 96%
ethanol). IR (KBr), ν cm−1: 1721 (νC=O lactone), 1569, 1428
(νC=C arene), 1242, 1166 (νCOC lactone); 1H NMR
(500 MHz, DMSO-d6), δ (ppm): 7.28 (d, J = 9.0 Hz, 1H, H-
7), 7.18 (dd, J = 9.0, 3.0 Hz, 1H, H-8), 7.15 (d, J = 2.3 Hz,
1H, H-3), 6.35 (d, J = 1.0 Hz, 1H, H-5), 4.01 (t, J = 6.5 Hz,
2H, 6-OCH2CH2CH2CH3), 2.40 (s, 4-CH3), 1.70 (quintet,
J = 6.5 Hz, 2H, 6-OCH2CH2CH2CH3), 1.44 (sextet, J =
7.0 Hz, 2H, 6-OCH2CH2CH2CH3), 0.93 (t, J = 7.0 Hz,
3H, 6-OCH2CH2CH2CH3); 13C NMR (125 MHz, DMSO-
d6), δ (ppm): 159.8 (C=O lactone, C-2), 155.0 (C-6), 152.9
(C-8a), 147.0 (C-4), 120.0 (C-5), 119.3 (C-7), 117.3 (C-8),
114.6 (C-4a), 108.7 (C-3), 67.8 (6-OCH2CH2CH2CH3),
30.7 (6-OCH2CH2CH2CH3), 18.7 (4-CH3), 18.1 (6-
OCH2CH2CH2CH3), 13.6 (6-OCH2CH2CH2CH3); ESI-MS:
C14H16O3, calc. for (M + 2H) = 234.2 Da, found: m/z 234.1
([M + 2H]+).
4-Methyl-6-isopentoxy-2H-chromen-2-one (3g) From 2a
(0.02 mol, 3.52 g), isopentyl bromide (0.024 mol, 2.88 mL),
and KI (0.1 mmol, 0.17 g). Reaction time: 12 h. Yield:
3.85 g (79%) of 3h as white crystals. M.p.: 99–101 °C (from
96% ethanol). IR (KBr), ν (cm−1): 1708 (νC=O lactone),
1
1571 (νC=C arene), 1243, 1169 (νCOC lactone); H NMR
(500 MHz, DMSO-d6), δ (ppm): 7.24 (t, J = 6.0 Hz, 1H, H-
7), 7.14 (dd, J = 8.75, 3.0 Hz, 1H, H-8), 7.10 (s, 1H, H-3),
6.31 (d, J = 1.0 Hz, 1H, H-5), 4.00 [t, J = 6.5 Hz, 2H, 6-
OCH2CH2CH(CH3)2], 2.37 (s, 3H, 4-CH3), 1.76–1.74 [m,
1H, 6-OCH2CH2CH(CH3)2], 1.59 [q, J = 13.5 Hz, 2H,

Medicinal Chemistry Research (2021) 30:743–759
753
6-OCH2CH2CH(CH3)2], 0.91 [t, J = 7.0 Hz, 6H, 6-
OCH2CH2CH(CH3)2]; 13C NMR (125 MHz, DMSO-d6), δ
(ppm): 159.8 (C=O lactone, C-2), 154.9 (C-6), 152.9 (C-4),
147.0 (C-8a), 120.0 (C-4a), 119.3 (C-8), 117.3 (C-7), 114.6
(C-3), 108.8 (C-5), 66.5 [6-OCH2CH2CH(CH3)2], 37.6 [6-
OCH2CH2CH(CH3)2], 24.5 [6-OCH2CH2CH(CH3)2], 22.3
[6-OCH2CH2CH(CH3)2], 18.1 (4-CH3); ESI-MS: C15H18O3,
calcd. for M + H = 247.1 Da, found: m/z 247.3 ([M + H]+);
ESI-HRMS: calcd. for M + H = 247.1334 Da, found: m/z
247.1341 ([M + H]+, 100%); elemental anal. for C15H18O3,
calcd.: C, 73.15; H, 7.37%; found: 73.42; H, 7.57%.
C-2), 155.8 (C-6), 148.7 (C-4), 143.6 (C-8a), 125.9 (C-4a),
120.5 (C-7), 118.2 (C-8), 115.9 (C-3), 109.7 (C-5), 70.2 (6-
OCH2CH2CH3), 22.4 (6-OCH2CH2CH3), 10.8 (6-
OCH2CH2CH3); ESI-MS: C13H12O4, calc. for M + H =
233.08 Da, M + Na = 255.06 Da, found: m/z 233.12 ([M +
H]+), 255.17 ([M + Na]+).
6-Isopropoxy-2-oxo-2H-chromene-4-carbaldehyde
(4c)
From 3c (0.2 mol, 4.4 g). Reaction time: 24 h. Yield: 2.32 g
(50%) of 4c as yellow crystals. M.p.: 134–135 °C (from
toluene). IR (KBr), ν (cm−1): 1734 (νC=O lactone), 1704
(νC=O aldehyde), 1525, 1432 (νC=C arene), 1246, 1054
1
General procedure for synthesis of 6-alkoxy-2-oxo-2H-
chromene-4-carbaldehydes (4a–g)
(νCOC lactone); H NMR (500 MHz, DMSO-d6), δ (ppm):
10.12 (s, 4-CHO), 8.54 (d, J = 9.0 Hz, 1H, H-5), 6.92 (m,
1H, H-3), 6.96–6.94 (m, 1H, H-8), 6.35–6.34 (d, J =
8.0 Hz, 1H, H-6), 4.05–4.03 [m, 1H, 6-OCH(CH3)2],
0.99–0.96 [m, 6H, 6-OCH(CH3)2]; 13C NMR (125 MHz,
DMSO-d6), δ (ppm): 194.2 (4-CHO), 161.5 (C-4), 161.3
(C-7), 156.4 (C=O lactone, C-2), 143.4 (C-8a), 121.5 (C-5),
112.6 (C-3), 110.9 (C-4a), 108.1 (C-6), 101.4 (C-8), 70.1
[6-OCH(CH3)2], 10.6 [6-OCH(CH3)2]; ESI-MS: C13H12O4,
calcd. for M + H = 233.08 Da, M + Na = 255.06 Da,
found: m/z 233.15 ([M + H]+), 255.19 ([M + Na]+); ESI-
HRMS: calcd. for M + H = 233.0814 Da, found: m/z
233.0819 ([M + H]+, 100%); elemental anal. for C13H12O4,
calcd.: C, 67.23; H, 5.21%; found: C, 67.41; H, 5.09%.
To the solution of corresponding 6-alkoxy-4-methyl-2-oxo-
2H-chromen-2-ones (3a–g, 0.02 mol) in xylene (50 mL)
was added activated selenium dioxide (0.027 mol, 3.8 g).
The reaction mixture was boiled under reflux for 24 h with
gently stirring. The hot reaction mixture then was filtered,
the filtrate was cooled in ice bath, and separated product
was filtered, crystallized from toluene to afford the title
aldehydes 4a–g.
6-Ethoxy-2-oxo-2H-chromene-4-carbaldehyde (4a) From
3a (0.2 mol, 4.08 g). Reaction time: 24 h. Yield: 2.83 g
(65%) of 4a as yellow crystals. M.p.: 163–168 °C (from
toluene). IR (KBr), ν cm−1: 1736 (νC=O lactone), 1708
(νC=O aldehyde), 1565, 1430 (νC=C arene), 1243, 1054
6-Butoxy-2-oxo-2H-chromene-4-carbaldehyde (4d) From
3c (0.2 mol, 4.64 g). Reaction time: 24 h. Yield: 3.31 g (67%)
of 4d as yellow crystals. M.p.: 110–112 °C (from toluene). IR
(KBr), ν cm−1: 1735 (νC=O lactone), 1700 (νC=O aldehyde),
1565, 1435 (νC=C arene), 1244, 1150 (νCOC lactone); 1H NMR
(500 MHz, DMSO-d6), δ (ppm): 10.16 (s, 4-CHO), 7.99 (d,
J = 3.0 Hz, 1H, H-5), 7.46 (d, J = 9.0 Hz, 1H, H-8), 7.32 (dd,
J = 9.5, 2.5 Hz, 1H, H-7), 7.17 (s, H, 1H, H-3), 4.04 −
4.01 (m, 2H, 6-OCH2CH2CH2CH3), 1.76–1.70 (m,
2H, 6-OCH2CH2CH2CH3), 1.50–1.42 (m, 2H, 6-
OCH2CH2CH2CH3), 0.95 (t, J = 7.0 Hz, 3H, 6-
OCH2CH2CH2CH3); 13C NMR (125 MHz, DMSO-d6), δ
(ppm): 194.1 (4-CHO), 160.6 (C=O lactone, C-2), 155.8 (C-
6), 148.7 (C-4), 143.5 (C-8a), 125.8 (C-4a), 120.5 (C-7), 119.5
(C-8), 118.2 (C-3), 112.0 (C-5), 68.4 (6-OCH2CH2CH2CH3),
31.1 (6-OCH2CH2CH2CH3), 19.2 (6-OCH2CH2CH2CH3),
14.1 (6-OCH2CH2CH2CH3); ESI-MS: C14H14O4, calc. for M
+ H = 247.10 Da, M + Na = 269.08 Da, found: m/z 247.12
([M + H]+), 269.15 ([M + Na]+).
1
(νCOC lactone); H NMR (500 MHz, DMSO-d6), δ (ppm):
10.15 (s, 4-CHO), 7.96 (d, J = 3.0 Hz, 1H, H-5), 7.40 (d,
J = 9.0 Hz, 1H, H-8), 7.27 (dd, J = 9.5, 3.0 Hz, 1H, H-7),
7.16 (s, H, 1H, H-3), 4.09 (q, J = 7.0 Hz, 2H, 6-OCH2CH3),
1.37 (t, J = 7.0 Hz, 3H, 6-OCH2CH3); 13C NMR (125 MHz,
DMSO-d6), δ (ppm): 193.6 (4-CHO), 160.1 (C=O lactone,
C-2), 155.0 (C-6), 148.2 (C-4), 143.0 (C-8a), 125.3 (C-4a),
119.9 (C-7), 117.3 (C-8), 109.1 (C-3), 63.7 (6-OCH2CH3),
14.5 (6-OCH2CH3); ESI-MS: C12H10O4, calc. for M + H =
219.07 Da, M + Na = 241.05 Da, found: m/z 219.15 ([M +
H]+), 219.11 ([M + Na]+).
6-Propoxy-2-oxo-2H-chromene-4-carbaldehyde (4b) From
3b (0.2 mol, 4.4 g). Reaction time: 24 h. Yield: 3.1 g (67%)
of 4b as yellow crystals. M.p.: 143–145 °C (from toluene).
IR (KBr), ν cm−1: 1723 (νC=O lactone), 1703 (νC=O alde-
hyde), 1542, 1434 (νC=C arene), 1244, 1045 (νCOC lactone);
1H NMR (500 MHz, DMSO-d6), δ (ppm): 10.16 (s, 4-
CHO), 7.99 (d, J = 3.0 Hz, 1H, H-5), 7.43 (d, J = 9.0 Hz,
1H, H-8), 7.30 (dd, J = 9.5, 2.5 Hz, 1H, H-7), 7.19 (s, H,
1H, H-3), 3.98 (t, J = 7.0 Hz, 2H, 6-OCH2CH2CH3), 1.77
(sextet, J = 7.0 Hz, 2H, 6-OCH2CH2CH3), 1.01 (t, J =
7.0 Hz, 2H, 6-OCH2CH2CH3); 13C NMR (125 MHz,
DMSO-d6), δ (ppm): 194.1 (4-CHO), 160.6 (C=O lactone,
6-Isobutoxy-2-oxo-2H-chromene-4-carbaldehyde
(4e)
From 3e (0.2 mol, 4.64 g). Reaction time: 24 h. Yield:
3.38 g (65%) of 5e as yellow crystals. M.p.: 123–125 °C
(from toluene). IR (KBr), ν (cm−1): 1733 (νC=O lactone),
1694 (νC=O aldehyde), 1515, 1425 (νC=C arene), 1250, 1154
1
(νCOC lactone); H NMR (500 MHz, DMSO-d6), δ (ppm):

754
Medicinal Chemistry Research (2021) 30:743–759
10.16 (s, 4-CHO), 7.99 (d, J = 3.5 Hz, 1H, H-5), 7.43 (d,
J = 9.0 Hz, 1H, H-8), 7.29 (dd, J = 9.0, 3.0 Hz, 1H, H-7),
7.19 (s, H, 1H, H-3), 3.83 [d, J = 6.5 Hz, 2H, 6-OCH2CH
(CH3)2], 2.03 [septet, J = 6.5 Hz, 1H, 6-OCH2CH(CH3)2],
0.98 [d, J = 7.0 Hz, 6H, 6-OCH2CH(CH3)2]; 13C NMR
(125 MHz, DMSO-d6), δ (ppm): 194.1 (4-CHO), 160.6 (C-
4), 155.8 (C-6), 148.7 (C=O lactone, C-2), 143.6 (C-8a),
125.9 (C-4a), 120.5 (C-3), 118.2 (C-8), 115.9 (C-7), 109.7
(C-5), 74.3 [6-OCH2CH(CH3)2], 27.5 [6-OCH2CH(CH3)2],
18.9 [6-OCH2CH(CH3)2]; ESI-MS: C14H14O4, calcd. for
M + H = 247.10 Da, M + Na = 269.08 Da, found: m/z
247.15 ([M + H]+), 269.17 ([M + Na]+); ESI-HRMS:
calcd. for M + H = 247.0970 Da, found: m/z 247.0978
([M + H]+, 100%); Elemental anal. for C14H14O4, calcd.: C,
68.28; H, 5.73%; found: C, 68.47; H, 5.51%.
M + Na = 283.09 Da, found: m/z 261.18 ([M + H]+), 283.21
([M + Na]+); ESI-HRMS: calcd. for M + H = 261.1127 Da,
found: 261.1121 ([M + H]+, 100%); elemental anal. for
C15H16O4, calcd.: C, 69.22; H, 6.20%; found: C, 69.39;
H, 6.04%.
General procedure for synthesis of 6-alkoxy-2-oxo-2H-
chromene-4-carbaldehyde N-(2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosyl)thiosemicarbazones (6a–g)
To a slurry of N-(1,2,3,4-tetra-O-acetyl-β-D-glucopyranosyl)
thiosemicarbazide 5 (2 mmol) in absolute ethanol (10 mL)
was added appropriate 6-alkoxy-2-oxo-2H-chromene-4-
carbaldehydes (4a–g, 2 mmol). Glacial acetic acid (5 mol%)
as catalyst was added dropwise with stirring. The obtained
mixture was then irradiated in microwave oven at power of
600 W for 9–13 min, cooled to room temperature, and the
separated precipitate was filtered and recrystallized from
96% ethanol to yield the compounds 6a–g.
6-Pentoxy-2-oxo-2H-chromene-4-carbaldehyde (4f) From
3f (0.2 mol, 4.92 g). Reaction time: 24 h. Yield: 3.22 g (61%) of
4f as yellow crystals. M.p.: 112–114 °C. IR (KBr), ν (cm−1):
1738 (νC=O lactone), 1703 (νC=O aldehyde), 1567, 1437 (νC=C
arene), 1243, 1156 (νCOC lactone); 1H NMR (500 MHz, DMSO-
d6), δ (ppm): 10.14 (s, 4-CHO), 7.94 (d, J = 3.5 Hz, 1H,
H-5), 7.39 (d, J = 9.0 Hz, 1H, H-8), 7.32 (dd, J = 9.0, 3.5 Hz,
1H, H-7), 7.16 (s, H, 1H, H-3), 3.98 (t, 2H, J = 7.0 Hz,
6-OCH2CH2CH2CH2CH3), 1.74 (quintet, 2H, J = 7.0 Hz,
6-Ethoxy-2-oxo-2H-chromene-4-carbaldehyde
N-(2,3,4,6-
tetra-O-acetyl-β-D-glucopyranosyl)thiosemicarbazone
(6a) From 4a (2 mmol, 218 mg) and 5 (2.2 mmol, 421 mg).
Reaction time: 9 min. Yield: 397 mg (64%) of 6a as yellow
crystals. M.p.: 189–191 °C (from 96% ethanol), ½αŠ25 + 76.1
D
6-OCH2CH2CH2CH2CH3),
1.43–1.33
(m,
4H,
6-
(c = 0.21, CHCl3). IR (KBr), ν (cm−1): 3330 and 3250 (νNH),
1748 (νC=O ester), 1730 (νC=O lactone), 1518 (νC=C arene),
OCH2CH2CH2CH2CH3), 0.91 (t, J = 7.0 Hz, 3H, 6-
OCH2CH2CH2CH2CH3); 13C NMR (125 MHz, DMSO-d6),
δ (ppm): 194.1 (4-CHO), 160.6 (C=O lactone, C-2), 155.7 (C-
6), 148.7 (C-4), 143.5 (C-8a), 125.8 (C-4a), 120.4 (C-7),
118.2 (C-8), 115.8 (C-3), 109.6 (C-5), 68.6 (6-
OCH2CH2CH2CH2CH3), 28.7 (6-OCH2CH2CH2CH2CH3),
28.1 (6-OCH2CH2CH2CH2CH3), 22.4 (6-OCH2CH2CH2
CH2CH3), 14.4 (6-OCH2CH2CH2CH2CH3); ESI-MS:
C15H16O4, calc. for M = 260.1 Da, found: m/z 260.8 ([M]+).
1
1235, 1042 (νCOC ester), 1090 (νC=S); H NMR (500 MHz,
DMSO-d6), δ (ppm): 12.06 (s, 1H, NH-2), 9.24 (d, J = 8.5 Hz,
1H, NH-4), 8.53 (s, 1H, CH=N), 7.40 (d, J = 9.0 Hz, 1H, H-
8″), 7.37 (s br, 1H, H-3″), 7.30 (s br, 1H, H-5″), 7.26 (dd, J =
9.25, 2.75 Hz, 1H, H-7″), 5.98 (t, J = 9.0 Hz, H-1′), 5.38 (dd,
J = 10.0, 3.5 Hz, 1H, H-3′), 5.35 (d, J = 9.0 Hz, 1H, H-2′),
5.33 (d, J = 3.5 Hz, H-4′), 4.35 (t, J = 6.5 Hz, 1H, H-5′), 4.11
(q, 2H, J = 7.0 Hz, 6-OCH2CH3), 4.05 (d, 2H, J = 6.5 Hz, 1H,
H-6′a, 1H, H-6′b), 2.14–1.94 (s, 4 × 3H, 4 × CH3CO), 1.37 (t,
3H, J = 7.0 Hz, 6-OCH2CH3); 13C NMR (125 MHz, DMSO-
d6), δ (ppm): 178.9 (C=S), 170.0–169.4 (C=O ester, 4 ×
CH3CO), 160.0 (C=O lactone, C-2″), 154.8 (C-6″), 147.8 (C-
8″a), 144.0 (C-4″), 136.9 (CH=N), 120.0 (C-7″), 118.1 (C-8″),
117.5 (C-4″a), 112.4 (C-3″), 107.5 (C-5″), 82.1 (C-1′), 71.8 (C-
5′), 70.7 (C-3′), 68.7 (C-2′), 67.5 (C-4′), 63.8 (6-OCH2CH3),
61.3 (C-6′), 20.5–20.3 (4 × CH3CO), 14.5 (6-OCH2CH3); ESI-
MS: C27H31N3O12S, calc. for M–H=620.2 Da, found: m/z
620.9 ([M–H]−); ESI-HRMS(+): calc. for. M + H = 622.1707
Da, M + Na = 644.1526 Da, found: m/z 622.1713 [M + H]+,
644.1531 [M + Na]+.
6-Isopentoxy-2-oxo-2H-chromene-4-carbaldehyde
(4g)
From 3g (0.2 mol, 4.92 g). Reaction time: 24 h. Yield: 2,60 g
(50%) of 5g as yellow crystals. M.p.: 114–116 °C (from
toluene). IR (KBr), ν (cm−1): 1738 (νC=O lactone), 1700 (νC=O
aldehyde), 1562, 1438 (νC=C arene), 1273, 1156 (νCOC lactone);
1H NMR (500 MHz, DMSO-d6), δ (ppm): 10.16 (s, 4-CHO),
7.97 (d, J = 3.0 Hz, 1H, H-5), 7.45–7.39 (m, 1H, H-8),
7.33–7.27 (m, 1H, H-7), 7.18 (s, 1H, H-3), 4.03 [t, J = 6.75 Hz,
2H, 6-OCH2CH2CH(CH3)2], 1.84–1.76 [m, 1H, 6-
OCH2CH2CH(CH3)2], 1.66–1.61 [m, 2H, 6-OCH2CH2CH
(CH3)2], 0.94 [d, 6H, J = 6.5 Hz, 6-OCH2CH2CH(CH3)2]; 13
C
NMR (125 MHz, DMSO-d6), δ (ppm): 193.6 (4-CHO), 160.1
(C-4), 154.5 (C-6), 148.2 (C=O lactone, C-2), 143.1 (C-8a),
125.2 (C-4a), 119.9 (C-3), 117.7 (C-8), 117.3 (C-7), 109.3
(C-5), 66.6 [6-OCH2CH2CH(CH3)2], 37.3 [6-OCH2CH2CH
(CH3)2], 24.6 [6-OCH2CH2CH(CH3)2], 22.4 [6-OCH2CH2CH
(CH3)2]; ESI-MS: C15H16O4, calcd. for M + H = 261.11 Da,
6-Propoxy-2-oxo-2H-chromene-4-carbaldehyde N-(2,3,4,6-
tetra-O-acetyl-β-D-glucopyranosyl)-thiosemicarbazone
(6b) From 4b (2 mmol, 232 mg) and 5 (2.2 mmol, 421 mg).
Reaction time: 11 min. Yield: 470 mg (74%) of 6b as yellow
crystals. M.p.: 121–122 °C (from 96% ethanol), ½αŠ25 + 83.5
D

Medicinal Chemistry Research (2021) 30:743–759
755
(c = 0.25, CHCl3). IR (KBr), ν (cm−1): 3539 and 3277 (νNH),
1748 (νC=O ester), 1737 (νC=O lactone), 1516 (νC = C arene),
1252 and 1049 (νCOC ester), 1080 (νC=S); 1H NMR (500 MHz,
DMSO-d6), δ (ppm): 12.05 (s, 1H, NH-2), 9.26 (d, J = 8.5 Hz,
1H, NH-4), 8.51(s, 1H, CH=N), 7.34 (d, J = 9.5 Hz, 1H, H-
8″), 7.33 (s, 1H, H-3″), 7.26 (dd, J = 8.7, 1.75 Hz, 1H, H-7″),
7.22 (s, 1H, H-5″), 5.98 (t, J = 9.0 Hz, H-1′), 5.39 (dd, J =
10.2, 3.75 Hz, 1H, H-3′), 5.37 (d, J = 9.0 Hz, 1H, H-2′), 5.33
(d, J = 2.3 Hz, H-4′), 4.35 (t, J = 6.5 Hz, 1H, H-5′), 4.06 (d,
2H, J = 6.5 Hz, 1H, H-6′a, 1H, H-6′b), 3.96 (t, 2H, J =
7.0 Hz, 6-OCH2CH2CH3), 2.15–1.94 (s, 4 × 3H, 4 × CH3CO),
1.76 (sextet, 2H, J = 7.0 Hz, 6-OCH2CH2CH3), 0.99 (t, 3H,
J = 7.0 Hz, 6-OCH2CH2CH3); 13C NMR (125 MHz,
DMSO-d6), δ (ppm): 178.9 (C=S), 170.0–169.4 (C=O
ester, 4 × CH3CO), 160.1 (C=O lactone, C-2″), 154.8 (C-
6″), 147.8 (C-8″a), 143.9 (C-4″), 136.7 (CH=N), 119.9
(C-7″), 118.1 (C-8″), 118.0 (C-4″a), 111.9 (C-3), 107.4
(C-5″), 82.2 (C-1′), 71.8 (C-5′), 70.8 (C-3′), 69.6
(OCH2CH2CH3), 68.7 (C-2′), 67.5 (C-4′), 61.3 (C-6′),
21,9 (6-OCH2CH2CH3), 20.5–20.3 (4 × CH3CO), 10.3 (6-
OCH2CH2CH3); ESI-MS: C28H33N3O12S, calc. for M =
635.2 Da, found: m/z 635.4 ([M]+); ESI-HRMS(+): calc.
for. M + H = 636.1863 Da, M + Na = 658.1683 Da,
found: m/z 636.1868 [M + H]+, 658.1689 [M + Na]+.
6-Butoxy-2-oxo-2H-chromene-4-carbaldehyde N-(2,3,4,6-
tetra-O-acetyl-β-D-glucopyranosyl)-thiosemicarbazone
(6d) From 4d (2 mmol, 246 mg) and 5 (2.2 mmol,
421 mg). Reaction time: 13 min. Yield: 402 mg (62%) of 6d
as yellow crystals. M.p.: 119–121 °C (from 96% ethanol),
½αŠ25 + 81.7 (c = 0.24, CHCl3). IR (KBr), ν (cm−1): 3286
D
and 3251 (νNH), 1753 (νC=O ester), 1718 (νC=O lactone),
1530, 1500 (νC = C arene), 1241 and 1042 (νCOC ester), 1089
1
(νC=S); H NMR (500 MHz, DMSO-d6), δ (ppm): 12.04 (s,
1H, NH-2), 9.27 (d, J = 8.0 Hz, 1H, NH-4), 8.52 (s, 1H,
CH = N), 7.36 (d, J = 9.5 Hz, 1H, H-8″), 7.34 (s, 1H, H-
3″), 7.23 (d, J = 7.0 Hz, 1H, H-7″), 7.22 (s, 1H, H-5″), 5.98
(t, J = 9.25 Hz, H-1′), 5.39 (dd, J = 10.0, 3.5 Hz, 1H, H-3′),
5.35 (d, J = 10.0 Hz, 1H, H-2′), 5.33 (d, J = 2.3 Hz, H-4′),
4.35 (t, J = 6.25 Hz, 1H, H-5′), 4.06 (d, 2H, J = 6.5 Hz,
1H, H-6′a, 1H, H-6′b), 4.01 (t, 2H, J = 7.5 Hz, 6-
OCH2CH2CH2CH3), 2.15–1.94 (s, 4 × 3H, 4 × CH3CO),
1.72 (quintet, 2H, J = 7.5 Hz, 6-OCH2CH2CH2CH3), 1.44
(sextet, 2H, J = 7.5 Hz, 6-OCH2CH2CH2CH3), 0.94 (t, 3H,
J = 7.5 Hz, 6-OCH2CH2CH2CH3); 13C NMR (125 MHz,
DMSO-d6), δ (ppm): 178.9 (C=S), 170.0–169.3 (C=O
ester, 4 × CH3CO), 160.0 (C=O lactone, C-2″), 155.0 (C-
6″), 147.8 (C-8″a), 143.9 (C-4″), 136.7 (CH=N), 119.9 (C-
7″), 118.0 (C-8″), 117.5 (C-4″a), 111.9 (C-3″), 107.4 (C-
5″), 82.1 (C-1′), 71.8 (C-5′), 70.7 (C-3′), 68.7 (C-2′), 67.5
(C-4′), 67.8 (6-OCH2CH2CH2CH3), 61.3 (C-6′), 30.7 (6-
OCH2CH2CH2CH3), 20.5–20.3 (4 × CH3CO), 18.7 (6-
OCH2CH2CH2CH3), 13.6 (6-OCH2CH2CH2CH3); ESI-MS:
C29H35N3O12S, calc. for M − H = 648.2 Da, found: m/z
648.8 ([M − H]−); ESI-HRMS(+): calc. for. M + H =
650.2020 Da, M + Na = 672.1839 Da, found: m/z 650.2025
[M + H]+, 672.1834 [M + Na]+.
6-Isopropoxy-2-oxo-2H-chromene-4-carbaldehyde N-(2,3,4,
6-tetra-O-acetyl-β-D-glucopyranosyl)-thiosemicarbazone
(6c) From 4c (2 mmol, 232 mg) and 5 (2.2 mmol, 421 mg).
Reaction time: 12 min. Yield: 432 g (68%) of 6c as yellow
25
D
crystals. M.p.: 191–193 °C (from 96% ethanol), ½αŠ
+
84.5 (c = 0.25, CHCl3). IR (KBr), ν (cm−1): 3540 and 3266
(νNH), 1751 (νC=O ester), 1715 (νC=O lactone), 1600, 1522,
and 1490 (νC=C arene), 1242 and 1043 (νCOC ester), 1090
(νC=S); 1H NMR (DMSO-d6) δ (ppm): 12.09 (s, 1H, NH-2),
9.17 (d, 1H, J = 9.0 Hz, NH-4), 8.54 (s, 1H, CH=N), 8.45
(s, 1H, H-3″), 7.83–7.45 (m, 3H, H-5″, H-7″, H-8″), 6.03 (t,
1H, J = 9.25 Hz, H-1′), 5.43 (t, 1H, J = 9.25 Hz, H-3′), 5.29
(t, 1H, J = 9.25 Hz, H-2′), 4.97 (t, 1H, J = 9.75 Hz, H-4′),
4.22 (dd, 1H, J = 12.25, 4.75 Hz, H-6′a), 4.12 (ddd, 1H,
J = 9.88, 5.13, 2.63 Hz, H-5′), 4.08 [sextet, 2H, J = 6.5 Hz,
6-OCH(CH3)2], 3.99 (dd, 1H, J = 10.5, 2.0 Hz, H-6′b),
2.00–1.92 (s, 4 × 3H, CH3CO), 0.95 [d, 6H, J = 6.5 Hz, 6-
OCH(CH3)2]; 13C NMR (DMSO-d6) δ (ppm): 179.0 (C=S),
170.0–169.4 (C=O ester, 4 × CH3CO), 160.1 (C=O
lactone, C-2″), 155.0 (C-8″), 147.0 (C-8″a), 144.0 (C-4″),
137.4 (CH=N), 120.0 (C-5″), 118.1 (C-7″. C-6″),
117.6 (C–4″a), 107.6 (C-3″), 81.7 (C-1′), 72.7 (C-5′), 72.4
(C-3′), 71.0 (C-2′), 67.8 (C-4′), 61.8 (C-6′), 66.6 [6-OCH
(CH3)2], 22.4 [6-OCH(CH3)2], 20.5–20.3 (4 × CH3CO);
ESI-MS: C28H33N3O12S, calc. for M = 635.2 Da, found: m/z
635.5 ([M]+); ESI-HRMS(+): calc. for. M + H =
636.1863 Da, M + Na = 658.1683 Da, found: m/z 636.1869
[M + H]+, 658.1691 [M + Na]+.
6-Isobutoxy-2-oxo-2H-chromene-4-carbaldehyde N-(2,3,4,6-
tetra-O-acetyl-β-D-glucopyranosyl)-thiosemicarbazone
(6e) From 4e (2 mmol, 246 mg) and 5 (2.2 mmol, 421 mg).
Reaction time: 13 min. Yield: 480 mg (74%) of 6e as yellow
crystals. M.p.: 120–122 °C (from 96% ethanol), ½αŠ25 + 81.7
D
(c = 0.24, CHCl3). IR (KBr), ν (cm−1): 3263 (νNH), 1752 (νC=O
ester), 1715 (νC=O lactone), 1600, 1529 (νC=C arene), 1237 and
1035 (νCOC ester), 1090 (νC=S); 1H NMR (DMSO-d6) δ (ppm):
12.22 (s, 1H, NH-2), 9.11 (d, 1H, J = 9.0 Hz, NH-4), 8.45 (s,
1H, CH = N), 7.84 (d, 1H, J = 8.5 Hz, H-5″), 7.07–7.04 (m,
2H, H-6″, H-8″), 7.04 (s, 1H, H-3″), 5.95 (t, 1H, J = 9.25 Hz,
H-1′), 5.43 (t, 1H, J = 9.5 Hz, H-3′), 5.36 (t, 1H, J = 9.0 Hz,
H-2′), 4.95 (t, 1H, J = 9.75 Hz, H-4′), 4.26 (dd, 1H, J = 12.3,
5.0 Hz, H-6′a), 4.12 (ddd, 1H, J = 10.0, 4.5, 2.25 Hz, H-5′),
3.99 (dd, 1H, J = 12.25, 1.75 Hz, H-6′b), 3.85 [d, 2H, J =
6.5 Hz, 6-OCH2CH(CH3)2]; 2.05 [sextet, 1H, J = 6.5 Hz, 6-
OCH2CH(CH3)2], 2.00–1.93 (s, 12H, 4 × CH3CO); 0.99 [d,
6H, J = 6.5 Hz, 6-OCH2CH(CH3)2]; 13C NMR (DMSO-d6), δ
(ppm): 179.0 (C=S), 170.1–169.3 (C=O ester, 4 × CH3CO),
161.9 (C=O lactone, C-2″), 160.3 (C-7″), 155.5 (C-8″a), 144.5

756
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(C-4″), 137.4 (CH=N), 125.7 (C-5″), 113.0 (C-8″), 110.3 (C-
4″a), 109.2 (C-3″), 101.6 (C-6″), 81.6 (C-1′), 72.7 (C-5′), 72.3
(C-3′), 70.8 (C-2′), 67.8 (C-4′), 61.7 (C-6′), 67.8 (OCH2), 27.6
(OCH2CH(CH3)2), 20.5-20.3 (4 × CH3CO), 18.9 (OCH2CH
(CH3)2); ESI-MS: C29H35N3O12S, calc. for M − H = 648 Da,
found: m/z 648 ([M−H]−); ESI-HRMS(+): calc. for. M +
H = 650.2020 Da, M + Na = 672.1839 Da, found: m/z
650.2026 [M + H]+, 672.1844 [M + Na]+.
(s, 1H, H-3″), 7.83–7.45 (m, 3H, H-5″, H-7″, H-8″), 6.03 (t,
1H, J = 9.25 Hz, H-1′), 5.43 (t, 1H, J = 9.25 Hz, H-3′), 5.29
(t, 1H, J = 9.25 Hz, H-2′), 4.97 (t, 1H, J = 9.75 Hz, H-4′),
4.22 (dd, 1H, J = 12.25, 4.75 Hz, H-6′a), 4.12 (ddd, 1H,
J = 9.88, 5.13, 2.63 Hz, H-5′), 4.08 [t, 2H, J = 6.5 Hz, 6-
OCH2CH2CH(CH3)2], 3.99 (dd, 1H, J = 10.5, 2.0 Hz, H-6′
b), 2.00–1.92 (s, 4 × 3H, CH3CO); 1.81 [sextet, 1H, J =
6.5 Hz, 6-OCH2CH2CH(CH3)2], 1.65 [q, 2H, J = 6.5 Hz, 6-
OCH2CH2CH(CH3)2], 0.95 [d, 6H, J = 6.5 Hz, 6-
OCH2CH2CH(CH3)2]; 13C NMR (DMSO-d6) δ (ppm):
179.0 (C=S), 170.0–169.4 (C=O ester, 4 × CH3CO), 160.1
(C=O lactone, C-2″), 155.0 (C-8″), 147.0 (C-8″a), 144.0
(C-4″), 137.4 (CH=N), 120.0 (C-5″), 118.1 (C-7″. C-6″),
117.6 (C-4″a), 107.6 (C-3″), 81.7 (C-1′), 72.7 (C-5′), 72.4
(C-3′), 71.0 (C-2′), 67.8 (C-4′), 61.8 (C-6′), 66.6 (OCH2),
37.4 [6-OCH2CH2CH(CH3)2], 24.6 [6-OCH2CH2CH(CH3)
2], 22.4 [6-OCH2CH2CH(CH3)2], 20.5–20.3 (4 × CH3CO);
ESI-MS: C30H37N3O12S, calc. for [M + H]+ = 664 Da,
found: m/z 664; ESI-HRMS(+): calc. for. M + H =
664.2176 Da, M + Na = 686.1996 Da, found: m/z 664.2182
[M + H]+, 686.1990 [M + Na]+.
6-Pentoxy-2-oxo-2H-chromen-2-one-4-carbaldehyde N-(2,3,
4,6-tetra-O-acetyl-β-D-glucopyranosyl)thiosemicarbazone
(6f) From 4f (2 mmol, 260 mg) and 5 (2.2 mmol, 421 mg).
Reaction time: 13 min. Yield: 464 mg (70%) of 6f as yellow
25
D
crystals. M.p.: 166–168 °C (from 96% ethanol), ½αŠ
+
80.3 (c = 0.26, CHCl3). IR (KBr), ν (cm−1): 3203 and 3263
(νNH), 1756 (νC=O ester), 1715 (νC=O lactone), 1571, 1527
1
(νC=C arene), 1224 and 1038 (νCOC ester), 1089 (νC=S); H
NMR (500 MHz, DMSO-d6), δ (ppm): 12.04 (s, 1H, NH-2),
9.28 (d, J = 8.0 Hz, 1H, NH-4), 8.53 (s, 1H, CH=N), 7.37
(d, J = 9.0 Hz, 1H, H-8″), 7.36 (s, 1H, H-6″), 7.25 (s, 1H,
H-5″), 7.24 (d, J = 7.5 Hz, 1H, H-7″), 5.98 (t, J = 8.75 Hz,
H-1′), 5.39 (dd, J = 10.0, 4.5 Hz, 1H, H-3′), 5.35 (d, J =
9.0 Hz, 1H, H-2′), 5.33 (d, J = 4.5 Hz, H-4′), 4.35 (t, J =
6.25 Hz, 1H, H-5′), 4.05 (d, 2H, J = 6.5 Hz, 1H, H-6′a, 1H,
H-6′b), 4.01 [t, 2H, J = 7.0 Hz, 6-OCH2CH2(CH2)2CH3],
2.15–1.94 (s, 4 × 3H, CH3CO), 1.74 (quintet, 2H, J =
7.0 Hz, 6-OCH2CH2CH2CH2CH3), 1.42–1.32 (m, 4H,
Biological assays
In vitro antimicrobial activity
All the synthesized thiosemicarbazone 6a–g were evaluated
for in vitro antibacterial and antifungal activities against
Gram-positive and Gram-negative bacteria organisms.
Gram-positive were chosen Bacillus subtilis (ATCC
11774), Staphylococcus aureus (ATCC 11632), Staphylo-
coccus epidermidis (ATCC 12228); Gram-negative bacteria
were Escherichia coli (ATCC 25922), Klebsiella pneumo-
niae (ATCC 4352), Pseudomonas aeruginosa (ATCC
25923), and Salmonella typhimurium (ATCC 1402). Three
methicillin-resistant S. aureus (MRSA198-1, MRSA198-2,
and MRSA198-3 were also chosen. The evaluations were
performed by using MIC, as described in our previous
article [42]. The microbroth dilutions technique was applied
using Mueller-Hinton broth [47]. The drug references were
ciprofloxacin, methicillin, and vancomycin (Table 1). The
solutions that had the concentrations of 400, 200, 100, 50,
25, 12.5, 6.25, 3.12, 1.56, and 0.78 μg/mL were prepared by
further diluting the test compounds and standard drugs
prepared above. The inoculum was prepared using a 4–6-h
broth adjusted to a turbidity equivalent of a 0.5 McFarland
standard, diluted in broth media to give a final concentration
of 5 × 105 CFU/mL in the test tray. The plates were incu-
bated at 35 °C for 18–20 h. The MIC was defined as the
lowest concentration of compound giving complete inhibi-
tion of visible growth. All the experiments were performed
three times. The MIC values for all tested compounds and
reference drug are listed in Tables 1 and 2.
(6-OCH2CH2CH2CH2CH3),
0.87
(t,
3H,
6-
OCH2CH2CH2CH2CH3); 13C NMR (125 MHz, DMSO-
d6), δ (ppm): 179.0 (C=S), 170.0–169.3 (C=O ester, 4 ×
CH3CO), 160.1 (C=O lactone, C-2″), 155.0 (C-6″), 147.8
(C-8″a), 144.0 (C-4″), 136.6 (CH=N), 119.9 (C-7″), 118.0
(C-8″), 117.6 (C-4″a), 112.0 (C-3″), 107.4 (C-5″), 82.2 (C-
1′), 71.8 (C-5′), 70.7 (C-3′), 68.7 (C-2′), 68.0 (6-
OCH2CH2CH2CH2CH3), 67.5 (C-4′), 61.3 (C-6′), 28.3 (6-
OCH2CH2CH2CH2CH3), 27.6 (6-OCH2CH2CH2CH2CH3),
21.8 (6-OCH2CH2CH2CH2CH3), 20.5–20.3 (4 × CH3CO),
13.8 (6-OCH2CH2CH2CH2CH3); ESI-MS: C30H37N3O12S,
calc. for M = 663.2 Da, found: m/z 663.2 ([M]+); ESI-
HRMS(+): calc. for. M + H = 664.2176 Da, M + Na =
686.1996 Da, found: m/z 664.2181 [M + H]+, 686.1991
[M + Na]+.
6-Isopentoxy-2-oxo-2H-chromen-2-one-4-carbaldehyde N-
(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)thiosemicarba-
zone (6g) From 4g (2 mmol, 260 mg) and 5 (2.2 mmol,
421 mg). Reaction time: 13 min. Yield: 464 mg (70%) of 6g
as yellow crystals. M.p.: 179–181 °C (from 96% ethanol),
½αŠ25 + 80.5 (c = 0.26, CHCl3). IR (KBr), ν (cm−1): 3570
D
and 3270 (νNH), 1753 (νC=O ester), 1719 (νC=O lactone),
1600, 1530 (νC=C arene), 1241 and 1043 (νC−O ester), 1090
(νC=S); 1H NMR (DMSO-d6) δ (ppm): 12.09 (s, 1H, NH-2),
9.17 (d, 1H, J = 9.0 Hz, NH-4), 8.54 (s, 1H, CH=N), 8.45

Medicinal Chemistry Research (2021) 30:743–759
757
In vitro antifungal activity
docking was accomplished and analyzed via the Glide v.
7.8 docking tool. The receptor grid was located in the center
based on the active site of the protein, using the receptor
grid generation tool. The ligands were flexibly docked in
grid box using the Monte Carlo-based simulation algorithm
and a high-throughput virtual screening (HTVS) method
without any constraints was employed that generated
binding poses based on energy.
The compounds 6a–g were evaluated for their in vitro
antifungal activity against three fungi, including Aspergillus
niger (439), Aspergillus flavus (ATCC 204304), Sacchar-
omyces cerevisiae (SH 20), and Candida albicans (ATCC
7754), using the agar dilution method with Saburoud’s
dextrose agar (Hi-Media), as described in our previous
article [42]. Miconazole and fluconazole were used as drug
references for antifungal activity. The solutions, which had
the concentrations of 400, 200, 100, 50, 25, 12.5, 6.25,
3.12, 1.56, and 0.78 μg/mL of each tested compound and
standard drugs, were prepared. Suspensions of each
microorganisms were prepared to contain 10 CFU/mL and
applied to agar plates, which had been serially diluted with
compounds to be tested. The plates were incubated at 35 °C.
After 72 h, the MICs were determined [47]. Minimal inhi-
bitory concentrations for each compound were investigated
against standard fungal strains. All the experiments were
performed three times. The MIC values for all tested
compounds and reference drug are shown in Table 4.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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