Synthesis and evaluation of in?vivo antioxidant, in?vitro antibacterial, MRSA and antifungal activity of novel substituted isatin N-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)thiosemicarbazones

Article abstract of DOI:10.1016/j.ejmech.2016.07.074

Some new isatin N-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)thiosemicarbazones 4a-t with different substituents at 1-, 5- and 7-positions of isatin ring have been synthesized by reaction of N-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)thiosemicarbazide 2 with corresponding isatins 3a-t. Compounds 4a-t were evaluated in?vivo for antioxidant activity and in?vitro for anti-microorganism activities. The MIC values were found for Gram positive bacteria (MIC?=?1.56–6.25?μM), for Gram negative bacteria (MIC?=?12.5?μM), and for fungi Aspergillus niger (MIC?=?3.12–12.5?μM), Fusarium oxysporum (MIC?=?6.25–12.5?μM) and Saccharomyces cerevisiae (MIC?=?6.25–12.5?μM). Regarding the antioxidant activity, the SOD, GHS-Px and catalase activities of 4c-i and 4m-r were MIC?=?10.57–10.85, 0.27–0.93 and 345.45–399.75 unit/mg protein, respectively. Compounds 4e-h had MIC values of 0.78, 1.56, and 3.12?μM for three clinical MRSA isolates. Compound 4e showed the selective cytotoxic effects against some cancer (LU-1, HepG2, MCF7, P338, SW480, KB) cell lines and normal fibroblast cell line NIH/3T3.

Full text of DOI:10.1016/j.ejmech.2016.07.074

European Journal of Medicinal Chemistry 123 (2016) 532e543
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and evaluation of in vivo antioxidant, in vitro antibacterial,
MRSA and antifungal activity of novel substituted isatin
N-(2,3,4,6-tetra-O-acetyl-
b- -glucopyranosyl)thiosemicarbazones
D
,
Nguyen Dinh Thanh a *, Nguyen Thi Kim Giang b, Tran Ha Quyen a, Doan Thi Huong a,
Vu Ngoc Toan c
a Faculty of Chemistry, VNU University of Science (Vietnam National University, Ha Noi), 19 Le Thanh Tong, Hoan Kiem, Ha Noi, Viet Nam
b Lab of Profession Chemistry, Institute of Biochemical Technology and Profession Documents, General Department 4 (Ministry of Public Security), 80 Tran
Quoc Hoan, Cau Giay, Ha Noi, Viet Nam
c Institute for Chemistry and Materials, Military Institute of Science and Technology, 17 Hoang Sam, Cau Giay, Ha Noi, Viet Nam
a r t i c l e i n f o
a b s t r a c t
Article history:
Some new isatin N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)thiosemicarbazones 4a-t with different
Received 5 May 2016
Received in revised form
28 July 2016
Accepted 31 July 2016
Available online 2 August 2016
substituents at 1-, 5- and 7-positions of isatin ring have been synthesized by reaction of N-(2,3,4,6-tetra-
O-acetyl- -glucopyranosyl)thiosemicarbazide 2 with corresponding isatins 3a-t. Compounds 4a-t were
evaluated in vivo for antioxidant activity and in vitro for anti-microorganism activities. The MIC values
were found for Gram positive bacteria (MIC 1.56e6.25 M), for Gram negative bacteria
(MIC ¼ 12.5 M), and for fungi Aspergillus niger (MIC ¼ 3.12e12.5 M), Fusarium oxysporum (MIC ¼ 6.25
e12.5 M). Regarding the antioxidant activity, the
M) and Saccharomyces cerevisiae (MIC ¼ 6.25e12.5
SOD, GHS-Px and catalase activities of 4c-i and 4m-r were MIC ¼ 10.57e10.85, 0.27e0.93 and 345.45
e399.75 unit/mg protein, respectively. Compounds 4e-h had MIC values of 0.78, 1.56, and 3.12 M for
b-D
¼
m
m
m
m
m
Keywords:
Antibacterial
Antifungal
m
three clinical MRSA isolates. Compound 4e showed the selective cytotoxic effects against some cancer
(LU-1, HepG2, MCF7, P338, SW480, KB) cell lines and normal fibroblast cell line NIH/3T3.
© 2016 Elsevier Masson SAS. All rights reserved.
Antioxidant activity
Cytotoxic effects
D-glucose
Isatins
Microwave-assisted synthesis
Methicillin-resistant Staphylococcus aureus
Thiosemicarbazones
1. Introduction
one of the first clinically used synthetic antiviral agent for treat-
ment of smallpox [6], also of other groups of viruses, such as
The ring of isatin, 1H-indole-2,3-dione, is a heterocyclic com-
pound of significant importance in organic chemistry and also in
medicinal chemistry. Thiosemicarbazone molecules bearing isatin
moiety have diverse types of biological activity, including antiviral,
antibacterial, anticancer, anticonvulsant and antidepressant activ-
adenovirus, herpesvirus, picornavirus, reovirus, arbovirus, myxo-
and paramyxovirus, and retrovirus [7]. Some isatin derivatives also
have antifungal and anti-mycotoxin activities [8].
It is known that methicillin-resistant Staphylococcus aureus
(MRSA) causes serious public health problems. MRSA is defined as
difficult-to-treat strain of Staphylococcus aureus which resists to
almost all antibiotics [9]. However, infections caused by MTSA have
been a major threat to public health in hospitals and the commu-
ity [1,2]. On the other hand, isatin-
b-thiosemicarbazone and N-
methylisatin- -thiosemicarbazone (methisazone, marboran) are
b
extensively studied thiosemicarbazones that demonstrate an
inhibitory effect against the replication of pox-viruses [3,4].
Methisazone has been described as being used in prophylaxis since
at least 1965 [5]. Methisazone (See Fig. 1), which works by inhib-
iting mRNA and protein synthesis, especially in pox viruses, was
nity during the past decade. Some substituted isatin-b-thio-
semicarbazones (IBTs) of substituted benzaldehydes have been
synthesized and shown the inhibit the growth of methicillin-
resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
Enterococcus (VRE) [10].
On the other hand, the chemistry of monosaccharide thio-
semicarbazides is interesting because these derivatives could be
* Corresponding author.
E-mail address: nguyendinhthanh@hus.edu.vn (N.D. Thanh).
http://dx.doi.org/10.1016/j.ejmech.2016.07.074
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

N.D. Thanh et al. / European Journal of Medicinal Chemistry 123 (2016) 532e543
533
used as versatile intermediates for preparing various compounds
(e.g., heterocycles [11]) as well as be used for making complex
formation of several metallic ions [12]. Thiosemicarbazones exhibit
various biological activities, such as antituberculosis [11], antimi-
crobial [13], antibacterial [14], anticancer [15], anticonvulsant [16],
antidiabetic [17], antifungal [18], antitumor [19], cytotoxic activities
[20], and some of them were used as antioxidant [22e24], anti-
dyslipidemic [24] agents.
thiosemicarbazide formed in reaction is well dissolved in
dichloromethane, then the solvent must be removed completely
and 96% ethanol must be added to isolate the product. The
advantage of using absolute ethanol as a reaction solvent is that the
thiosemicarbazide product is precipitated when formed, isolating
the product more easily, just by the filtering the precipitate. The
high yield of 95% is achieved.
The condensation reaction of substituted isatins 3a-l and N-
Thiosemicarbazone derivatives containing monosaccharide
moiety have shown the remarkable anti-microorganisms and
antioxidant activity both in vivo and in vitro [23,24]. Some articles in
the past reported about the synthesis of substituted aromatic
alkylisatins 3m-t with N-(tetra-O-acetyl-b-D-glucopyranosyl)thio-
semicarbazide was carried out in absolute ethanol in the presence
of glacial acetic acid as catalyst under microwave irradiation
(Scheme 1) [22,26]. Reaction solvent is absolute ethanol, the sol-
vent 5 volume is usually from 5 mL to 10 mL depending on solu-
bility of substituted isatins or N-alkylisatins, for example, ethanol
volume is 5 mL for isatin 3a-l, while for N-alkylisatins 3m-t is 10 mL
due to lower solubility of the latter. The reaction mixture in sus-
pension type became a clear solution after irradiating for several
minutes, and at the end of reaction process, the precipitate
appeared. Reaction products usually separated as yellow or orange
solids after cooling to room temperature. The products are soluble
in almost common organic solvents, such as toluene, DMF, acetone,
and ethyl acetate. The reaction yields were 70e88% for substituted
isatin series 4a-l and 74e89% for N-alkylisatin series 4m-t. The
synthesized products were characterized by elemental analysis, FT-
IR, 1H NMR, 13C NMR and mass spectral data.
aldehyde/ketone
semicarbazones [11,23,24e27]. These compounds have been syn-
thesized by reaction of N-(per-O-acetyl- -glycosyl)
N-(per-O-acetylated
glucopyranosyl)thio-
b-D
thiosemicarbazides with the corresponding carbonyl compounds,
but the thiosemicarbazones containing simultaneously mono-
saccharide and isatin moieties have not been reported yet.
Continuing our previous studies on synthesis and reactivity of N-
(per-O-acetyl-D-glycopyranosyl)thiosemicarbazides [22,26], in the
present paper we have reported a study for the synthesis and
spectral characterization of a series of substituted isatin N-(tetra-O-
acetyl-b-D-glucopyranosyl)thiosemicarbazones using microwave-
assisted method. The biological activities of synthesized com-
pounds have been estimated.
2.2. The in vitro antimicrobial activity evaluation
2. Results and discussion
The antimicrobial activities in vitro against Staphylococcus
aureus (ATCC 11632), Escherichia coli (ATCC 25922), Pseudomonas
aeruginosa (ATCC 25923), Klebsiella pneumoniae (ATCC 4352),
Staphylococcus epidermidis (ATCC 12228), Bacillus subtilis (ATCC
11774), and Enterobacter aerogenes (ATCC 13048) were screened. A
series of methicillin resistant Staphylococcus aureus (MRSA) strains
was isolated from 198th hospital of Ministry of Public Security (in
Ha Noi), numbered MRSA198-1, MRSA198-2 and MRSA198-3, was
also included in these tests. The compounds 4a-t were also evalu-
ated for their antifungal activity in vitro against Aspergillus niger
(439), Candida albicans (ATCC 7754), Fusarium oxysporum (M42),
and Saccharomyces cerevisiae (SH20).
The results of antibacterial activity study for thio-
semicarbazones 4a-t showed that the new molecule exhibited
antibacterial activity against the studied bacteria at low and high
concentrations (Table 1). It has been observed that all the com-
pounds tested showed mild to moderate activity against tested
bacteria in comparison with the MIC values of the reference com-
2.1. Chemistry
Unsubstituted
N-(tetra-O-acetyl-b-D-glucopyranosyl)thio-
semicarbazide, in particular, and other thiosemicarbazides linked
to carbohydrate (N-glycosylthiosemicarbazides), to the best of our
knowledge, could only be prepared from corresponding isothio-
cyanate. It's known that the conversion of N-(tetra-O-acetyl-
glucopyranosyl)isothiocyanate into corresponding thio-
semicarbazide usually could take place in aprotic solvent (such as
dichloromethane [24], dioxane [27]) due to the higher reactivity
towards water of peracetated glucopyranosyl isothiocyanate in
comparing with aromatic isocyanate [21,27,28]. We found that a
protic solvent, such as absolute ethanol, could be used for this re-
action, but the reaction must be performed at a low temperature
(<10 ꢀC) to prevent the decomposition of isothiocyanate derivative
[27]. The use of absolute ethanol as solvent allowed to avoid the
reaction of this isocyanate to water, while 96% ethanol isn't a good
solvent for that reaction as above mentioned. Reaction outcome
was improved by using an 85% solution of hydrazine solution [22]
instead of 100% hydrazine hydrate [24] by dropping into the solu-
b-D-
pound (ciprofloxacin, MIC ¼ 3.12 and 1.56
mM, respectively). For
Gram positive bacteria, B. subtilis, Staphylococcus aureus and
S. epidermidis, thiosemicarbazones 4c-i had the higher ability to
tion of this isocyanate. In this way, N-(tetra-O-acetyl-b-D-gluco-
pyranosyl)thiosemicarbazide was synthesized from corresponding
isothiocyanate derivative by reaction with 85% hydrazine hydrate
inhibit to these bacteria with MIC values of 1.56e6.25 mM, respec-
tively. The order of inhibitory activity against these bacteria was 4e
> 4c,4d,4f > 4g,4h,4i > 4j,4k > 4l > 4a,4b,4m,4n,4o,4p,4q,4s >
4r,4t. The compound 4e with two bromine atoms at 5th and 7th
positions is most active amongst these thiosemicarbazones. The
compounds 4j-l bearing alkyl group at 5th or 7th positions had
(Scheme 1) by similar method for synthesis of N-(tetra-O-acetyl-
b-
D-galactopyranosyl)thiosemicarbazide using 85% hydrazine hy-
drate in dichloromethane [22]. In case of the latter,
medium activity against these bacteria (MIC ¼ 12.5e25
compounds 4a,b,m-t bearing alkyl groups at 1st position had
M). For
mM). The
lowest activity against these bacteria (MIC ¼ 100e200
m
Gram negative bacteria, Enterobacter spp., E. coli, P. aeruginosa and
K. pneumoniae, thiosemicarbazones 4m-q having alkyl substituents
at 1st position exhibited higher activity at all (MIC ¼ 12.5
whereas compounds 4c,4d,4j,4k bearing alkyl group at 5th or 7th
M). The substitutions of
mM),
positions were less active (MIC ¼ 25
m
allylic, benzylic or phenethylic groups at 1st position in compounds
Fig. 1. Methisazone.
4r-t decreased the inhibitory ability of these derivatives; in these

534
N.D. Thanh et al. / European Journal of Medicinal Chemistry 123 (2016) 532e543
Scheme 1. The synthesis route for preparation of the title compounds 4a-t.
Table 1
Antibacterial activities of the compounds 4a-t.
Entry
R
Micro-organisms/MIC (
mM)
Gram positive
Gram negative
MRSA
B.s.a
S.a.a
S.e.a
En.a
E.c.a
K.p.a
P.a.a
MRSA198-1
MRSA198-2
MRSA198-3
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
H
100
100
3.12
3.12
1.56
3.12
6.25
6.25
6.25
25
200
200
6.25
6.25
1.56
3.12
6.25
6.25
6.25
12.5
12.5
12.5
100
100
100
100
100
200
200
200
3.12
1.21
NE
100
100
6.25
6.25
1.56
3.12
6.25
6.25
6.25
12.5
12.5
12.5
100
100
100
100
100
200
200
200
3.12
2.10
NE
50
50
25
25
50
50
50
50
50
25
25
50
12.5
12.5
12.5
12.5
12.5
100
100
100
1.56
NE
50
50
25
25
50
50
50
50
50
25
25
50
12.5
12.5
12.5
12.5
12.5
100
100
100
1.56
NE
50
50
25
25
50
50
50
50
25
25
25
25
12.5
12.5
12.5
12.5
12.5
100
100
100
1.56
NE
50
50
25
25
50
50
50
50
25
25
25
25
200
200
6.25
6.25
0.78
1.56
0.78
1.56
6.25
12.5
12.5
12.5
100
100
100
100
100
200
200
200
NE
200
200
6.25
6.25
0.78
3.12
0.78
1.56
6.25
12.5
12.5
12.5
100
100
100
100
100
200
200
200
NE
200
200
6.25
6.25
0.78
1.56
0.78
3.12
6.25
12.5
12.5
12.5
100
100
100
100
100
200
200
200
NE
5-NO2
5-Cl-7-NO2
5-Br-7-NO2
5,7-diBr
5-F
5-Cl
5-Br
5-I
5-Me
7-Me
5-iPr
1-Me
1-Et
1-Pr
1-Bu
1-iBu
25
25
100
100
100
100
100
200
200
200
3.12
0.83
NEb
25
12.5
12.5
12.5
12.5
100
100
100
1.56
NE
NE
4q
4r
4s
4t
1-Allyl
1-Benzyl
1-Phenethyl
(Reference)
(Reference)
(Reference)
Ciprofloxacin
Vancomycin
Methicillin
1.65
>200
1.65
>200
1.65
>200
NE
NE
NE
a
B.s.: Bacillus subtilis, S.a.: Staphylococcus aureus, S.e.: Staphylococcus epidermidis, En.: Enterobacter spp., E.c.: Escherchia coli, K.p.: Klebsiella pneumoniae, P.a.: Pseudomonas
aeruginosa.
b
NE ¼ not evaluated.
cases, the MIC values were 200
(for Gram negative).
m
M (for Gram positive) and 100
m
M
showed weak anti-MRSA effect against three MRSA strains. Other
compounds did not show any statistical significance in anti-MRSA
activity (Table 1).
Next, we evaluated the antifungal of these thiosemicarbazones
against some fungi, such as Aspergillus niger, Candida albicans,
Fusarium oxysporum, Saccharomyces cerevisiae. Notably, almost all
tested thiosemicarbazones 4a-t had more remarkable activities
against fungi C. albicans, F. oxysporum, and S. cerevisiae but were
more resistant to A. niger than miconazole (the reference com-
Next, we further evaluated the antibacterial activities of these
compounds against series of MRSA strains (MRSA198-1,
a
MRSA198-2 and MRSA198-3) from clinical sources. Among the
isolated compounds, compounds 4c-i exerted anti-MRSA activities
against all the strains tested with MIC values ranging from 0.78 to
6.25
obviously observed by treating it against these three strains (with
IC50 of 0.78, 1.56, and 3.12 M, respectively). Compounds 4c,4d,4j-l
mM. In addition, the inhibitory effects of compounds 4e-h were
m
pound with MIC ¼ 1.56, 3.12, 3.12 and 3.12
mM, respectively). The

N.D. Thanh et al. / European Journal of Medicinal Chemistry 123 (2016) 532e543
535
data in Table 2 indicated that in case of fungus Aspergillus niger, all
compounds are less active than miconazole (MIC ¼ 7.50 M). The
compounds 4c-i had the remarkable ability to inhibit to this fungus
with MIC values of 3.12e12.5 M. The remaining compounds
exhibited worst activity (MIC ¼ 25e200 M), especially, for com-
pounds 4n,4o,4s,4t bearing long-chain alkylic, allylic, benzylic or
M). It is observed
that the activity was increased due to the presence of halogen atom
(F, Cl, Br, I), and the order of inhibitory activity against A. niger was
4e > 4i,4h > 4c,4d,4f,4g. The compound 4e with two bromine
atoms at 5th and 7th positions is most active amongst these thio-
one catalase molecule can convert millions of molecules of
hydrogen peroxide to water and oxygen each second [35]. Anti-
oxidative enzyme glutathione peroxidase (GSH-Px, EC 1.11.1.9) is
the general name of an enzyme family with peroxidase activity
whose main biological role is to protect the organism from oxida-
tive damage. The biochemical function of glutathione peroxidase is
to reduce lipid hydrogen peroxides to their corresponding alcohols
and to reduce free hydrogen peroxide to water [36].
The SOD, GHS-Px and catalase activities of thiosemicarbazones
4a-t were shown in Table 3. Some of the compounds 4a-t caused
significant elevation of SOD activity than the one of referent drug
(Resveratrol). Thiosemicarbazones 4c-i and 4m-t caused significant
elevation of SOD activity, especially in cases of compounds 4c-e,g-i.
This activity is extremely considerable. The SOD activity of these
thiosemicarbazone is higher than the one of compounds 4m-t. It
seems that the presence of halogens, excepting fluorine, contrib-
uted to enhance the SOD activity. Thiosemicarbazones with
different R substituents had the MIC values below: R ¼ 5-Cl-7-NO2
m
m
m
phenethylic groups at 1st position (MIC ¼ 400
m
semicarbazones (MIC ¼ 3.12
compounds are less active than miconazole (MIC ¼ 12.5e400
vs. 3.12 M of reference compound). This one also was observed in
mM). In case of C. albicans, almost all
mM
m
cases of fungi F. oxysporum and S. cerevisiae. Again, these thio-
semicarbazones having halogen atoms exhibited higher activity at
all (MIC ¼ 3.12e25
mM). The compounds bearing alkyl groups had
worst activities against tested fungi.
(4c), MIC ¼ 10.57
0.31 unit/mg protein, 5-Br-7-NO2 (4d),
0.33 unit/mg protein, 5,7-diBr (4e),
MIC 10.76
¼
2.3. Study on in vivo antioxidant activity
MIC ¼ 10.89 0.25 unit/mg protein, 5-Cl (4g), MIC ¼ 10.20 0.29
unit/mg protein, 5-Br (4h), MIC ¼ 10.47 0.19 unit/mg protein and
5-I (4i), MIC ¼ 10.85 0.32 unit/mg protein, whereas compound
It's known that the superoxide dismutase (SOD), glutathione
peroxidase (GSH-Px) and catalase (CAT) are important ones of
antioxidant defense systems. Compounds 4a-t were evaluated
in vivo for their antioxidant activity as the similar previous methods
that was described in Ref. [22] below and the results are shown in
Table 3. The referent drug is resveratrol. These thiosemicarbazones
and resveratrol, when administered i.p., with a dry weight equiv-
alent dosage of 100 mg/kg/day of total extract for seven consecutive
days in the CCl4-intoxicated rats, was shown to cause a significant
elevation of free radical scavenging enzyme activities such as su-
peroxide dismutases, catalase, and glutathione peroxidase. Super-
oxide dismutase (SOD, EC 1.15.1.1) is enzyme that catalyze the
dismutation of superoxide into oxygen and hydrogen peroxide [34].
Thus, it is an important antioxidant defense in nearly all cells
exposed to oxygen. Catalase (CAT, EC 1.11.1.6) is a common enzyme
found in nearly all living organisms exposed to oxygen and cata-
lyzes the decomposition of hydrogen peroxide to water and oxygen.
Catalase has one of the highest turnover numbers of all enzymes;
with R ¼ 5-F (4f) has MIC ¼ 9.84
0.35 unit/mg protein. The
substitution of alkyl groups at position 1 of isatin moiety made the
SOD activity to decrease little. The MIC values of thio-
a
semicarbazones 4m-t were from 8.45 unit/mg protein to 9.89 unit/
mg protein. The order of SOD activity in this case was
4t < 4s < 4m < 4n < 4o < 4p < 4q < 4r. It seems that allylic group
with alkene bond made the van der Waals interactions to receptor
to be better than other groups at position 1 of isatin moiety,
whereas the presence of benzene ring in 4s and 4t made these ones
to become lesser due to their steric effects. The MIC values of 4s and
4t were 8.60 0.41 and 8.45 0.43 unit/mg protein, respectively,
while these ones of thiosemicarbazones 4m-r are 9.52e9.91 unit/
mg protein.
The similar situation also appeared in cases of GHS-Px and
catalase activity. The GSH-Px activity of these compounds had some
little picture: some compounds expressed negligible GHS-Px ac-
tivity, such as 4a, 4b, 4j-l, 4s, 4t, with the MIC values were in range
of 1.69e1.89 unit/mg protein and the increasing order of GHS-Px
activity is 4b < 4a <4l < 4t < 4j < 4k < 4s, whereas the remained
compounds exhibited the better GHS-Px activity with the
decreasing order 4r > 4e,4g > 4h > 4c,4i > 4d > 4f > 4m > 4n > 4o >
4q > 4p in MIC ranges of 0.27e0.93 unit/mg protein. The MIC values
of resveratrol and control were 0.33 0.02 and 0.28 0.01 unit/mg
protein, respectively. Again, the halogens made the thio-
semicarbazones being more GHS-Px active, on the other hand, the
substitution of alkyl groups in the nitrogen atom of isatin moiety
also triggered this activity significantly (MIC ¼ 0.31e0.39 unit/mg
protein). The compound 4r exhibited the best GHS-Px activity
(MIC ¼ 0.27 0.01 unit/mg protein).
Table 2
Antifungal activities of MIC for compounds 4a-t.
Entry
R
Micro-organisms/MIC (mM)
A.n.a
C.a.a
F.o.a
S.c.a
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
H
100
100
12.5
12.5
3.12
12.5
12.5
6.25
6.25
100
100
100
25
200
100
6.25
12.5
6.25
6.25
12.5
12.5
6.25
100
100
100
100
400
400
100
100
100
400
400
3.12
100
100
25
100
100
25
12.5
6.25
6.25
12.5
12.5
6.25
25
5-NO2
5-Cl-7-NO2
5-Br-7-NO2
5,7-diBr
5-F
5-Cl
5-Br
5-I
5-Me
7-Me
5-iPr
1-Me
1-Et
1-Pr
1-Bu
1-iBu
6.25
12.5
12.5
12.5
12.5
12.5
100
25
Compounds 4c-i,m-r had higher catalase activity with the MIC
values of 345.45e399.75 unit/mg protein. But as showed in Table 3,
the catalase activity of 4m,4n,4o,4r exhibited the lower activity
(MIC values of 345.45e367.63 unit/mg protein). It may be
explained that the compounds 4m,4n,4o,4r could protect the CCl4
in toxicated rats from oxidant injury but not caused significant
elevation of catalase activity. The increasing order of high catalase
activity was 4i < 4e < 4h < 4g < 4c < 4d < 4f < 4p <
4q < 4o < 4n < 4r < 4m. The MIC values of halogen-bearing thio-
semicarbazones 4c-i were in range of 379.56e399.75 unit/mg
protein, and the ones of N-alkylisatin thiosemicarbazones 4m-t
were MIC ¼ 345.45e377.61 unit/mg protein. The highest catalase
activity found in compound 4i (R ¼ I, MIC ¼ 399.75 12.12 unit/mg
25
50
25
200
200
100
100
100
200
100
200
3.12
100
400
400
100
100
100
400
400
3.12
25
50
100
100
200
100
200
1.56
4q
4r
4s
4t
1-Allyl
1-Benzyl
1-Phenethyl
Miconazole
a
A.n.: Aspergillus niger, C.a.: Candida albicans, F.o.: Fusarium oxysporum, S.c.:
Saccharomyces cerevisiae.

536
N.D. Thanh et al. / European Journal of Medicinal Chemistry 123 (2016) 532e543
Table 3
Effect of compounds 4a-t on the liver cytosolic sod, the liver cytosolic GSH-Px, the liver cytosolic catalase activities and the hepatic MDA production.
Entry
R
SOD (unit/mg protein)
GHS-Px (unit/mg protein)
Catalase (unit/mg protein)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
H
5.81 0.33
6.45 0.37
10.57 0.31
10.76 0.33
10.89 0.25
9.84 0.35
10.20 0.29
10.47 0.19
10.85 0.32
5.91 0.30
4.60 0.31
5.82 0.33
9.52 0.32
9.68 0.42
9.77 0.34
9.89 0.43
9.88 0.32
9.91 0.29
8.60 0.41
8.45 0.43
7.31 0.42
5.27 0.22
1.77 0.02
1.89 0.02
0.35 0.04
0.37 0.03
0.31 0.01
0.39 0.02
0.31 0.01
0.32 0.02
0.35 0.01
1.70 0.01
1.69 0.01
1.75 0.01
0.67 0.02
0.75 0.01
0.84 0.01
0.93 0.01
0.91 0.02
0.27 0.01
1.69 0.01
1.73 0.01
0.33 0.02
0.28 0.01
295.32 10.32
283.53 12.43
389.53 13.34
381.67 11.71
397.53 11.47
379.56 10.53
390.63 12.62
394.43 11.43
399.75 12.12
258.47 12.33
250.63 12.13
260.61 11.73
350 12.25
358.47 12.33
367.63 12.13
377.61 11.73
375.45 12.25
357.47 12.33
250.63 12.13
260.61 11.73
282.12 10.26
220.15 11.43
5-NO2
5-Cl-7-NO2
5-Br-7-NO2
5,7-diBr
5-F
5-Cl
5-Br
5-I
5-Me
7-Me
5-iPr
1-Me
1-Et
1-Pr
1-Bu
1-iBu
4q
4r
4s
4t
1-Allyl
1-Benzyl
1-Phenethyl
Resveratrol
Control
protein). These obtained results showed the important role of
halogen in order to enhance the biological activity.
4. Experimental section
Since compound 4e had best activity for antibacterial, antifungal
and antioxidant activity, then it was screened the cell cytotoxicity on
cells of NIH/3T3 (normal fibroblast cell line), LU-1 (Lung carcinoma),
HepG2 (Lepatocellular carcinoma), MCF7 (Breast adenocarcinoma),
P338 (Leukemia), SW480 (Colon adenocarcinoma) and KB (Epider-
moid carcinoma). The results are listed in Table 4. The IC50 values of
cell cytotoxicity of 4e againts these cell lines were 1.53 0.11 (NIH/
3T3), 5.53 0.16 (for LU-1), 8.41 0.28 (for HepG2), 2.75 0.65 (for
4.1. Chemistry
All solvents, chemicals, and reagents were obtained commer-
cially and used without purification. Melting points were deter-
mined by open capillary method on STUART SMP3 instrument
(BIBBY STERILIN, UK) and are uncorrected. FT-IR spectra (KBr disc)
were recorded on an Impact 410 FT-FT-IR Spectrometer (Nicolet,
USA). 1H and 13C NMR spectra were recorded on Bruker Avance
Spectrometer AV500 (Bruker, Germany) at 500.13 MHz and
125.77 MHz, respectively, using DMSO-d6 as solvent and TMS as an
internal standard. Mass spectra were recorded on mass spectrom-
eter LC-MS LTQ Orbitrap XL (ThermoScientific, USA) in methanol,
using ESI method, or AutoSpec Premier (Water, USA) using EI
method. Thin-layer chromatography was performed on silica gel
plate 60F254 No. 5715 (Merck, Germany) with ethyl acetate and light
petroleum (bp 60e90 ꢀC) or toluene, and spots were visualized
with UV light or iodine vapor. All substituted isatins 3, except isatin
3a, were prepared in our lab. Required 5-, 7-substituted and 5,7-
disubstituted isatins 3b-l were synthesized according to refer-
ences [3,29,30]. N-Alkylisatins 3m-t were prepared by using
alkylation reaction of unsubstituted isatin 3a with corresponding
alkyl bromides in the presence of potassium carbonate as base in
DMF under microwave-assisted heating method [31,32]. Tetra-O-
MCF7), 18.41
12.75 0.19
the ones of drug reference, ellipticine. The corresponding IC50 values
of this drug were 0.32 0.05 (NIH/3T3), 1.39 0.12 (for LU-1),
0.31 (for P338), 4.75
0.27 (for SW480) and
mM (for KB), respectively. These values were higher than
1.95 0.30 (for HepG2), 0.53 0.23 (for MCF-7), 1.27 0.32 (for
P338), 0.75 0.14 (for SW480), and 0.52 0.17 (for KB).
3. Conclusion
In conclusion, new substituted isatin N-(2,3,4,6-tetra-O-acetyl-
b-
D-glucopyranosyl)thiosemicarbazones have been synthesized
from N-(2,3,4,6-tetra-O-acetyl- -glucopyranosyl)thio-
b-D
semicarbazide and substituted isatin or N-alkylisatin using con-
ventional heating and microwave-assisted heating method. Almost
all obtained thiosemicarbazones had in vivo antioxidant and in vitro
antibacterial, MRSA and antifungal activities. These obtained re-
sults allow to direct the synthetic design of new thiosemicarbazone
molecules bearing both monosaccharide and isatin moieties having
interesting biological activities.
acetyl-
the reaction of tetra-O-acetyl-
prepared from -glucose) [38] with dry ammonium thiocyanate in
b
-D
-glucopyranosyl isothiocyanate [37] were prepared by
b-D
-glucopyranosyl bromide (which
D
absolute acetonitrile using tetrabutylammonium bromide as
transfer catalyst (modified the Tashpulatov's method [27]).
4.1.1. Synthesis of N-(2,3,4,6-tetra-O-acetyl-
thiosemicarbazide (2) in absolute ethanol as solvent
To a solution of 2,3,4,6-tetra-O-acetyl- -glucopyranosyl iso-
b-D-glucopyranosyl)
Table 4
Cell cytotoxicity data (IC50
,
m
M) of compound 4e.
b-D
Cells
4e
Ellipticine
thiocyanate 1 (3.89 g, 10 mmol) in 25 mL of absolute ethanol, a
solution of 85% hydrazine hydrate (10 mmol, 1.2 mL) in 10 mL of
absolute ethanol was added dropwise slowly with stirring in
30 min so that the reaction temperature is below 10 ꢀC. The white
precipitate appeared immediately when several drops of hydrazine
were added due to low solubility of this thiosemicarbazide in
ethanol. The temperature of solution was maintained between 10
Normal fibroblast cell line NIH/3T3
Lung carcinoma LU-1
Hepatocellular carcinoma HepG2
Breast adenocarcinoma MCF-7
Leukemia P338
1.53 0.11
5.53 0.16
8.41 0.28
2.75 0.65
18.41 0.31
4.75 0.27
12.75 0.19
0.32 0.05
1.39 0.12
1.95 0.30
0.53 0.23
1.27 0.32
0.75 0.14
0.52 0.17
Colon adenocarcinoma SW480
Epidermoid carcinoma KB

N.D. Thanh et al. / European Journal of Medicinal Chemistry 123 (2016) 532e543
537
and 12 ꢀC. The mixture was continuously stirred at 20 ꢀC for 30 min.
The solid product then was isolated by filtering with suction. The
crude product was crystallized from 96% ethanol to yield 3.75 g of
white product 2. Yield 85%, m.p. 156e158 ꢀC; ref. [27]: 169e171 ꢀC.
1H, NHb), 11.83 (s, 1H, NH isatin), 9.90 (d, J ¼ 9.0 Hz, 1H, NHa), 8.63
(d, J ¼ 2.5 Hz, 1H, H-40), 8.30 (dd, J ¼ 8.75, 2.5 Hz, 1H, H-60), 7.13 (d,
J ¼ 8.75 Hz,1H, H-70), 6.06 (t, J ¼ 9.25 Hz,1H, H-1), 5.47 (t, J ¼ 9.5 Hz,
1H, H-3), 5.35 (t, J ¼ 9.25 Hz, 1H, H-2), 5.01 (t, J ¼ 9.75 Hz, 1H, H-4),
4.24 (dd, J ¼ 12.5, 5.0 Hz, 1H, H-6a), 4.19 (dq, J ¼ 10.0, 2.0 Hz, 1H, H-
5), 4.01 (dd, J ¼ 12.25, 1.75 Hz, H-6b), 2.02e1.93 (s, 12H, 4 ꢂ CH3CO);
FT-IR (KBr, cmꢁ1):
1242, 1043 (nCOC ester); 1H NMR (DMSO-d6)
n
3322, 3129 (nNH),1752 (nC]O ester),1355 (nC]S),
d
(ppm): 12.77 (s, 1H,
NHb), 9.23 (s,1H, NH), 8.17 (s,1H, NH), 4.58 (s, 2H, NH2), 5.80 (m,1H,
H-1), 5.07 (t, J ¼ 9.5 Hz, 1H, H-2), 5.34 (t, J ¼ 9.75 Hz, 1H, H-3), 4.91
(t, J ¼ 9.75 Hz, 1H, H-4), 4.14 (dd, J ¼ 12.25, 4.75 Hz, 1H, H-6a),
13C NMR (DMSO-d6)
d
(ppm): 178.0 (C]S), 169.9e169.1
(4 ꢂ COCH3), 162.9 (C]O lactam), 147.7 (C-7a0), 142.8 (C-50), 131.5
(C-30), 127.3 (C-60), 120.7 (C-4a0), 116.7 (C-40), 111.3 (C-70), 82.0 (C-1),
72.7 (C-3), 72.5 (C-5), 70.9 (C-2), 67.6 (C-4), 61.7 (C-6), 20.5e20.3
(4 ꢂ CH3CO); MS (þESI): m/z (%) 593.90 (100) [MeH]þ; calcd. for
3.98e3.93 (m, 2H, H-5 & H-6b), 1.98e1.94 (s, 12H, 4 ꢂ CH3CO); 13
C
NMR (DMSO-d6)
d
(ppm): 182.1 (C]S), 169.9e169.2 (4 ꢂ COCH3),
81.0 (C-1), 70.5 (C-2), 72.5 (C-3), 68.1 (C-4), 72.1 (C-5), 61.8 (C-6),
20.4e20.2 (4 ꢂ CH3CO); MS (þESI): m/z (%) ¼ 422.42 (45) [MþH]þ,
462.28 (100) [MþK]þ; calcd. for C15H23N3O9S ¼ 421.12 Da.
C
23H25N5O12S ¼ 595.12. Anal. calcd for C23H25N5O12S (595.54): C,
46.39; H, 4.23; N, 11.76%. Found: C, 46.34; H, 4.27; N, 11.70%.
4.1.2.3. 50-Chloro-70-nitroisatin N-(2,3,4,6-tetra-O-acetyl-
b-D
-gluco-
4.1.2. General methods for synthesis of substituted isatin N-(2,3,4,6-
pyranosyl)thiosemicarbazone (4c). Starting from 5-chloro-7-
nitroisatin 3c (0.58 g) and thiosemicarbazide 2 (0.84 g), in 5 mL
of absolute ethanol, irradiation time: 15 min, the title compound 4c
was obtained as yellow solid, recrystallization from mixture of
toluen-96% ethanol (2:1 v/v); yield: 0.98 g (71%), m.p. 206e207 ꢀC;
tetra-O-acetyl-
b-D-glucopyranosyl)thiosemicarbazones (4a-l)
A suspension mixture of N-(2,3,4,6-tetra-O-acetyl-
b-D-gluco-
pyranosyl)thiosemicarbazide 2 (2 mmol, 0.84 g) and appropriate
substituted isatin 3a-l (2 mmol) and glacial acetic acid (0.02 mL) in
absolute ethanol was microwave irradiated for 5e15 min
(depending on substituents on benzene ring of isatins). After irra-
diating for 1e3 min, the suspension mixture became a clear solu-
tion. The irradiation was continued in a given time. At the end of
reaction process, the precipitate appeared. Upon completion,
monitored by TLC, the reaction mixture was cooled to room tem-
perature; the color precipitate was filtered with suction. The crude
product was recrystallized from 96% ethanol or mixture of toluene-
96% ethanol (2:1 in volume) to afford the title compounds of
½
a 2D5
ꢃ
þ45.0 (c ¼ 0.22, CHCl3); FT-IR (KBr, cmꢁ1):
n
3365, 3101 (nNH),
C]N), 1375 ( C]S),
d (ppm): 12.44 (s, 1H,
1742 (nC
]
ester), 1710 (nC
]
lactam), 1633 (
n
n
O
O
1243, 1041 (nCOC ester); 1H NMR (DMSO-d6)
NHb), 11.99 (s, 1H, NH isatin), 9.85 (d, J ¼ 9.0 Hz, 1H, NHa), 8.23 (d,
J ¼ 2.25 Hz, 1H, H-60), 8.16 (d, J ¼ 2.25 Hz, 1H, H-40), 6.05 (t,
J ¼ 9.25 Hz, 1H, H-1), 5.48 (t, J ¼ 9.5 Hz, 1H, H-3), 5.31 (t, J ¼ 9.25 Hz,
1H, H-2), 5.00 (t, J ¼ 9.75 Hz, 1H, H-4), 4.25 (dd, J ¼ 12.25, 4.75 Hz,
1H, H-6a), 4.20 (dq, J ¼ 10.0, 2.0 Hz, 1H, H-5), 4.02 (d, J ¼ 11.0 Hz, H-
6b), 2.01e1.93 (s, 12H, 4 ꢂ CH3CO); 13C NMR (DMSO-d6)
d (ppm):
substituted isatin N-(2,3,4,6-tetra-O-acetyl-
b
-
D
-glucopyranosyl)
178.96 (C]S), 169.90e169.11 (4 ꢂ COCH3), 162.60 (C]O lactam),
136.60 (C-30), 132.0 (C-70), 129.4 (C-40), 126.1 (C-60), 126.1 (C-7a0),
125.4 (C-4a0), 113.1 (C-50), 81.9 (C-1), 72.6 (C-3), 72.5 (C-5), 71.0 (C-
2), 67.6 (C-4), 61.6 (C-6), 20.7e20.3 (4 ꢂ CH3CO); MS (þESI): m/z
(%) ¼ 652.00/653.96 (100/42) [Mþ35Cl]þ/[Mþ37Cl]þ; calcd. for
thiosemicarbazones 4a-l. The spectral (FT-IR, 1H NMR, 13C NMR and
ESI-MS or EI-HRMS) data are in good agreement with their
structures.
35
37
4.1.2.1. Isatin
N-(2,3,4,6-tetra-O-acetyl-
b
-D-glucopyranosyl)thio-
C
H
ClN5O12S/C23
H
ClN5O12S ¼ 629.08/631.08 Da. Anal. calcd
23 24
24
semicarbazone (4a). Starting from isatin 3a (0.29 g) and thio-
semicarbazide 2 (0.84 g) in 5 mL of absolute ethanol, irradiation
time: 15 min, the title compound 4a was obtained as yellow solid,
recrystallization from 96% ethanol; yield: 0.81 g (74%), m.p.
for C23H24ClN5O12S (629.98): C, 43.85; H, 3.84; N, 11.12%. Found: C,
43.89; H, 3.79; N, 11.17%.
4.1.2.4. 50-Bromo-70-nitroisatin N-(2,3,4,6-tetra-O-acetyl-
b-D-gluco-
251e252 ꢀC; ½a 2D5
ꢃ
þ34.0 (c ¼ 0.23, CHCl3); FT-IR (KBr, cmꢁ1):
n
3335,
pyranosyl)thiosemicarbazone (4d). Starting from 5-bromo-7-
nitroisatin 3d (0.66 g) and thiosemicarbazide 2 (0.84 g), in 5 mL
of absolute ethanol, irradiation time: 8 min, the title compound 4d
was obtained as yellow solid, recrystallization from mixture of
toluen-96% ethanol (2:1 v/v); yield: 1.09 g (75%), m.p. 239e240 ꢀC;
3265 (nNH), 1750 (nC
]
O ester), 1731 (nC
]
O lactam), 1425 (
n
C]N), 1373
(n d (ppm): 12.77 (s,
C]S),1225,1037 (nCOC ester); 1H NMR (DMSO-d6)
1H, NHb), 11.21 (s, 1H, NH isatin), 9.52 (d, J ¼ 9.0 Hz, 1H, NHa), 7.72
(d, J ¼ 7.5 Hz, 1H, H-40), 7.39 (td, J ¼ 7.5, 0.75 Hz, 1H, H-60), 7.12 (td,
7.5, 0.75 Hz, 1H, H-50), 6.94 (d, J ¼ 8.0 Hz, 1H, H-70), 5.99 (t,
J ¼ 9.0 Hz, 1H, H-1), 5.44 (t, J ¼ 9.5 Hz, 1H, H-3), 5.33 (t, J ¼ 9.25 Hz,
1H, H-2), 4.98 (t, J ¼ 9.75 Hz,1H, H-4), 4.14 (dq, J ¼ 9.5, 2.5 Hz,1H, H-
5), 4.23 (dd, J ¼ 12.5, 4.5 Hz, 1H, H-6a), 4.01 (d, J ¼ 12.5, 1.65 Hz, 1H,
½
a 2D5
1744 (nC
ꢃ
þ45.0 (c ¼ 0.22, CHCl3); FT-IR (KBr, cmꢁ1):
n
3358, 3093 (nNH),
C]N), 1373 ( C]S),
d (ppm): 12.45 (s, 1H,
]
O
ester), 1720 (nC
]
lactam), 1627 (
n
n
O
1229, 1040 (nCOC ester); 1H NMR (DMSO-d6)
NHb), 11.99 (s, 1H, NH isatin), 9.86 (d, J ¼ 9.5 Hz, 1H, NHa), 8.27 (s,
1H, H-40), 8.34 (d, J ¼ 1.5 Hz, 1H, H-60), 6.05 (t, J ¼ 9.25 Hz, 1H, H-1),
5.48 (t, J ¼ 9.5 Hz, 1H, H-3), 5.32 (t, J ¼ 9.25 Hz, 1H, H-2), 5.00 (t,
J ¼ 9.5 Hz, 1H, H-4), 4.25 (dd, J ¼ 12.25, 4.75 Hz, 1H, H-6a), 4.20 (dq,
J ¼ 10.0, 2.0 Hz, 1H, H-5), 4.01 (dd, J ¼ 11.0, 1.5 Hz, H-6b), 2.02e1.93
H-6b), 2.01e1.93 (s, 12H, 4 ꢂ CH3CO); 13C NMR (DMSO-d6)
d (ppm):
179.0 (C]S), 169.8e169.2 (4 ꢂ COCH3), 162.4 (C]O lactam), 142.7
(C-7a0), 133.3 (C-30), 131.7 (C-70), 122.3 (C-60), 121.2 (C-40), 119.6 (C-
4a0),111.1 (C-50), 81.8 (C-1), 72.7 (C-3), 72.4 (C-5), 70.9 (C-2), 67.8 (C-
4), 61.1 (C-6), 20.4e20.2 (4 ꢂ CH3CO); MS (þESI): m/z (%) ¼ 551.21
(s, 12H, 4 ꢂ CH3CO); 13C NMR (DMSO-d6)
d (ppm): 179.0 (C]S),
(20)
[MþH]þ,
573.16
(100)
[MþNa]þ;
calcd.
for
169.9e169.1 (4 ꢂ COCH3), 162.5 (C]O lactam), 137.0 (C-30), 132.3
(C-70), 129.3 (C-40), 128.7 (C-7a0), 127.0 (C-60), 125.6 (C-4a0), 113.1 (C-
50), 81.9 (C-1), 72.7 (C-3), 72.5 (C-5), 71.0 (C-2), 67.6 (C-4), 61.6 (C-6),
C
23H26N4O10S ¼ 550.14 Da. Anal. calcd for C23H26N4O10S (550.54):
C, 50.18; H, 4.76; N, 10.18%. Found: C, 50.25; H, 4.72; N, 10.13%.
20.5e20.3 (4 ꢂ CH3CO); MS (þESI): m/z (%) ¼ 695.91/697.94 (100/
79
4.1.2.2. 50-Nitroisatin N-(2,3,4,6-tetra-O-acetyl-
b
-D
-glucopyranosyl)
98)
[Mþ79Br]þ/[Mþ81Br]þ;
calcd.
for
C23H BrN5O12S/
24
81
thiosemicarbazone (4b). Starting from 5-nitroisatin 3b (0.39 g) and
thiosemicarbazide 2 (0.84 g), in 5 mL of absolute ethanol, irradia-
tion time: 15 min, the title compound 4b was obtained as yellow
solid, recrystallization from 96% ethanol; yield: 0.84 g (70%), m.p.
C
C
H
23 24
BrN5O12
S
¼
673.03/675.03 Da. Anal. calcd for
23H24BrN5O12S (674.43): C, 40.96; H, 3.59; N, 10.38%. Found: C,
40.92; H, 3.63; N, 10.43%.
242e243 ꢀC; ½a 2D5
ꢃ
þ45.0 (c ¼ 0.22, CHCl3); FT-IR (KBr, cmꢁ1):
n
3632,
4.1.2.5. 50,70-Dibromoisatin N-(2,3,4,6-tetra-O-acetyl-
b-D-glucopyr-
anosyl)thiosemicarbazone (4e). Starting from 5,7-dibromoisatin 3e
(0.62 g) and thiosemicarbazide 2 (0.84 g), in 5 mL of absolute
3102 (nNH), 1751 (nC
]
O ester), 1710 (nC
]
O lactam), 1626 (
nC]N), 1347
(n d (ppm): 12.59 (s,
C]S), 1212, 1049 (nCOC ester); 1H NMR (DMSO-d6)

538
N.D. Thanh et al. / European Journal of Medicinal Chemistry 123 (2016) 532e543
37
ethanol, irradiation time: 10 min, the title compound 4e was ob-
C
H
23 25
ClN4O10S ¼ 584.10/586.09 Da. Anal. calcd for C23H25ClN4O10
S
tained as orange solid, recrystallization from mixture of toluen-96%
(584.98): C, 47.22; H, 4.31; N, 9.58%. Found: C, 47.26; H, 4.36; N,
9.54%.
ethanol (2:1 v/v); yield: 1.14 g (79%), m.p. 243e245 ꢀC; ½a D25
ꢃ þ38.0
(c ¼ 0.20, CHCl3); FT-IR (KBr, cmꢁ1):
n 3359, 3167 (nNH), 1748 (nC]
O
ester), 1688 (nC
]
O lactam), 1616 (
n
C]N), 1372 (
nC]S), 1224, 1040 (nCOC
4.1.2.8. 50-Bromoisatin
N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyr-
ester); 1H NMR (DMSO-d6)
d
(ppm): 12.56 (s, NHb), 11.67 (s, NH
anosyl)thiosemicarbazone (4h). Starting from 5-bromoisatin 3h
(0.45 g) and thiosemicarbazide 2 (0.84 g), in 5 mL of absolute
ethanol, irradiation time: 10 min, the title compound 4h was ob-
tained as orange solid, recrystallization from 96% ethanol; yield:
isatin), 9.75 (d, J ¼ 9.0 Hz, NHa), 7.96 (s, H-40), 7.82 (dd, J ¼ 8.5,
1.5 Hz, H-60), 6.03 (t, J ¼ 9.25 Hz, H-1), 5.46 (t, J ¼ 9.25 Hz, H-3), 5.32
(t, J ¼ 9.25 Hz, H-2), 4.99 (t, J ¼ 9.75 Hz, H-4), 4.24 (dd, J ¼ 12.5,
4.5 Hz, H-6a), 4.19 (d, J ¼ 9.5 Hz, H-5), 4.01 (d, J ¼ 11.5 Hz, H-6b),
1.15 g (79%), m.p. 197e198 ꢀC; ½a D25
ꢃ
þ36.0 (c ¼ 0.22, CHCl3); FT-IR
O ester), 1709 (nC
C]S), 1216, 1050 (nCOC ester); 1H NMR
2.01e1.93 (4 ꢂ CH3CO); 13C NMR (DMSO-d6)
d
(ppm): 179.0 (C]S),
(KBr, cmꢁ1):
n
3343, 3215 (nNH), 1746 (nC
]
]
O lac-
170.0e169.2 (4 ꢂ COCH3), 162.3 (C]O lactam), 141.1 (C-7a0), 135.1
(C-30), 131.7 (C-40), 123.5 (C-70), 122.7 (C-60), 114.5 (C-4a0), 104.3 (C-
50), 81.9 (C-1), 72.7 (C-3), 72.5 (C-5), 71.0 (C-2), 67.7 (C-4), 61.7 (C-6),
20.5e20.3 (4 ꢂ CH3CO); MS (þESI): m/z (%) ¼ 728.86/730.83/732.85
(52/100/54) [Mþ79Brþ79Br]þ/[Mþ79Brþ81Br]þ/[Mþ81Brþ81Br]þ;
tam), 1624 (
(DMSO-d6)
n
C]N), 1372 (
n
d
(ppm): 12.64 (s, 1H, NHb), 11.33 (s, 1H, NH isatin), 9.77
(d, J ¼ 9.0 Hz, 1H, NHa), 7.99 (d, J ¼ 1.5 Hz, 1H, H-40), 7.54 (dd, J ¼ 8.0,
2.0 Hz, 1H, H-60), 6.91 (d, J ¼ 8.0 Hz, 1H, H-70), 5.98 (t, J ¼ 9.0 Hz, 1H,
H-1), 5.42e5.35 (m, 1H, H-3), 5.33e5.34 (m, 1H, H-2), 4.40 (t,
J ¼ 9.5 Hz, 1H, H-4), 4.35 (d, J ¼ 9.5 Hz, 1H, H-5), 4.09e4.07 (m, 2H,
H-6a & H-6b), 2.01e1.93 (s, 12H, 4 ꢂ CH3CO); 13C NMR (DMSO-d6)
79
79
calcd.
for
Br2N5O12
C H Br2N4O10S/C23H
Br81BrN4O10S/
23 24 24
81
C
C
H
23 24
S
¼
705.96/705.96/709.95 Da. Anal. calcd for
23H24Br2N5O12S (708.33): C, 39.00; H, 3.42; N, 7.91%. Found: C,
d
(ppm): 179.1 (C]S), 170.0e169.4 (4 ꢂ COCH3), 162.2 (C]O lac-
39.03; H, 3.47; N, 7.95%.
tam), 141.7 (C-7a0), 133.7 (C-30), 131.9 (C-60), 123.8 (C-40), 122.1 (C-
4a0), 114.2 (C-50), 113.1 (C-70), 82.4 (C-1), 72.0 (C-3), 70.7 (C-5), 68.6
(C-2), 67.5 (C-4), 61.3 (C-6), 20.5e18.5 (4 ꢂ CH3CO); MS (þESI): m/z
(%) ¼ 650.97/652.94 (100/98) [Mþ79Br]þ/[Mþ81Br]þ; calcd. for
4.1.2.6. 50-Fluoroisatin N-(2,3,4,6-tetra-O-acetyl-
b-D-glucopyranosyl)
thiosemicarbazone (4f). Starting from 5-fluoroisatin 3f (0.33 g) and
thiosemicarbazide 2 (0.84 g), in 5 mL of absolute ethanol, irradia-
tion time: 10 min, the title compound 4f was obtained as yellow
solid, recrystallization from 96% ethanol; yield: 0.98 g (86%), m.p.
79
81
C
H
BrN4O10S/C23
H
BrN4O10S ¼ 628.05/630.05 Da. Anal. calcd
23 25
25
for C23H25BrN4O10S (628.43): C, 43.89; H, 4.00; N, 8.90%. Found: C,
43.85; H, 4.04; N, 8.95%.
187e189 ꢀC; ½a 2D5
ꢃ
þ45.0 (c ¼ 0.25, CHCl3); FT-IR (KBr, cmꢁ1):
n 3183
C]N), 1375
(
nNH), 1748 (nC
]
ester), 1697 (nC
]
lactam), 1610 (
n
4.1.2.9. 50-Iodoisatin N-(2,3,4,6-tetra-O-acetyl-
b-D-glucopyranosyl)
O
O
(n
C]S), 1243, 1045 (nCOC ester); 1H NMR (DMSO-d6)
d
(ppm): 12.65
thiosemicarbazone (4i). Starting from 5-iodoisatin 3i (0.55 g) and
thiosemicarbazide 2 (0.84 g), in 5 mL of absolute ethanol, irradia-
tion time: 10 min, the title compound 4i was obtained as orange
solid, recrystallization from 96% ethanol; yield: 1.17 g (87%), m.p.
(s,1H, NHb),11.24 (s,1H, NH isatin), 9.64 (d, J ¼ 9.5 Hz,1H, NHa), 7.57
(s, H-40), 7.22 (dd, J ¼ 9.0, 2.3 Hz, 1H, H-60), 6.93 (dd, J ¼ 8.5, 4.0 Hz,
1H, H-70), 6.02 (t, J ¼ 9.0 Hz, 1H, H-1), 5.46 (t, J ¼ 9.5 Hz, 1H, H-3),
5.32 (t, J ¼ 9.25 Hz, 1H, H-2), 4.98 (t, J ¼ 9.75 Hz, 1H, H-4), 4.24 (dd,
J ¼ 12.25, 4.75 Hz,1H, H-6a), 4.18 (ddd, J ¼ 10.0, 4.5, 2.0 Hz,1H, H-5),
4.00 (dd, J ¼ 12.5, 1.5 Hz, 1H, H-6b), 2.01e1.93 (s, 12H, 4 ꢂ CH3CO);
171e173 ꢀC; ½a 2D5
ꢃ
þ46.0 (c ¼ 0.27, CHCl3); FT-IR (KBr, cmꢁ1):
n 3256
C]N), 1372
(
nNH), 1753 (nC
]
O
ester), 1702 (nC
]
lactam), 1617 (
n
O
(n
C]S), 1246, 1036 (nCOC ester); 1H NMR (DMSO-d6)
d (ppm): 12.64
13C NMR (DMSO-d6)
d
(ppm): 179.0 (C]S), 170.0e169.2
(s, 1H, NHb), 11.32 (s, 1H, NH isatin), 9.70 (d, J ¼ 9.5 Hz, 1H, NHa),
8.07 (d, J ¼ 1.0 Hz, 1H, H-40), 7.69 (dd, J ¼ 8.25, 1.5 Hz, 1H, H-60), 6.78
(d, J ¼ 8.5 Hz, 1H, H-70), 6.04 (t, J ¼ 9.25 Hz, 1H, H-1), 5.33 (t,
J ¼ 9.25 Hz, 1H, H-2), 4.99 (t, J ¼ 9.75 Hz, 1H, H-4), 5.46 (t, J ¼ 9.5 Hz,
1H, H-3), 4.24 (dd, J ¼ 12.25, 4.75 Hz, 1H, H-6a), 4.18 (ddd, J ¼ 10.0,
4.75, 1.5 Hz, 1H, H-5), 4.01 (d, J ¼ 11.5 Hz, 1H, H-6b), 2.01e1.92 (s,
(4 ꢂ COCH3), 162.6 (C]O lactam), 157.2 (C-70), 139.0 (C-4a0), 133.7
(C-30), 121.1 (C-50), 117.2 (C-7a0), 112.3 (C-60), 108.1 (C-40), 81.8 (C-1),
72.7 (C-3), 72.4 (C-5), 71.0 (C-2), 67.7 (C-4), 61.4 (C-6), 20.5e20.3
(4 ꢂ CH3CO); MS (þESI): m/z (%) ¼ 591.55 (37, [MþNa]þ), 588.69
(100, [M]þ, 551.55, 464.81, 337.72, 303.65, 241.70, 225.68, 128.58;
calcd. for C23H25FN4O10S ¼ 568.13 Da. Anal. calcd for C23H25FN4O10
S
12H, 4 ꢂ CH3CO); 13C NMR (DMSO-d6)
d (ppm): 178.9 (C]S),
(568.53): C, 48.59; H, 4.43; N, 9.85%. Found: C, 48.55; H, 4.47; N,
9.81%.
170.0e169.2 (4 ꢂ COCH3), 161.9 (C]O lactam), 142.2 (C-60), 129.2
(C-30), 139.5 (C-40), 131.9 (C-4a0), 122.2 (C-50), 113.5 (C-7a0), 91.7 (C-
70), 81.9 (C-1), 72.7 (C-3), 72.4 (C-5), 71.0 (C-2), 67.7 (C-4), 61.6 (C-6),
20.5e20.3 (4 ꢂ CH3CO); MS (eESI): m/z (%) ¼ 676.5 (100%, [M]þ);
4.1.2.7. 50-Chloroisatin
N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyr-
anosyl)thiosemicarbazone (4g). Starting from 5-chloroisatin 3g
(0.36 g) and thiosemicarbazide 2 (0.84 g), in 5 mL of absolute
ethanol, irradiation time: 10 min, the title compound 4g was ob-
tained as yellow solid, recrystallization from 96% ethanol; yield:
calcd. for C23H25IN4O10S ¼ 676.03 Da. Anal. calcd for C23H25IN4O10
S
(676.43): C, 40.84; H, 3.73; N, 8.28%. Found: C, 40.89; H, 3.70; N,
8.35%.
0.87 g (73%), m.p. 202e203 ꢀC; ½a D25
ꢃ
þ40.0 (c ¼ 0.24, CHCl3); FT-IR
O ester), 1709 (nC
C]S), 1227, 1043 (nCOC ester); 1H NMR
4.1.2.10. 50-Methylisatin
N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyr-
(KBr, cmꢁ1):
n
3558, 3259 (nNH), 1748 (nC
]
]
O lac-
anosyl)thiosemicarbazone (4j). Starting from 5-methylisatin 3j
(0.32 g) and thiosemicarbazide 2 (0.84 g), in 5 mL of absolute
ethanol, irradiation time: 5 min, the title compound 4j was ob-
tained as dark yellow solid, recrystallization from 96% ethanol;
tam), 1619 (
n
C]N), 1375 (
n
(DMSO-d6)
d
(ppm): 12.61 (s, 1H, NHb), 11.32 (s, 1H, NH isatin), 9.67
(d, J ¼ 9.5 Hz, 1H, NHa), 7.82 (s, H-40), 7.42 (dd, J ¼ 8.5, 1.5 Hz, 1H, H-
60), 6.95 (d, J ¼ 8.0 Hz, 1H, H-70), 6.03 (t, J ¼ 9.0 Hz, 1H, H-1), 5.46 (t,
J ¼ 9.0 Hz, 1H, H-3), 5.32 (t, J ¼ 9.25 Hz, 1H, H-2), 4.99 (t, J ¼ 9.5 Hz,
1H, H-4), 4.24 (dd, J ¼ 12.5, 4.5 Hz,1H, H-6a), 4.19 (d, J ¼ 10.0 Hz,1H,
H-5), 4.01 (d, J ¼ 11.5 Hz, 1H, H-6b), 2.01e1.93 (s, 12H, 4 ꢂ CH3CO);
yield: 0.95 g (84%), m.p. 153e155 ꢀC; ½a D25
ꢃ
þ54.0 (c ¼ 0.27, CHCl3);
FT-IR (KBr, cmꢁ1):
n
3331 (nNH), 1751 (nC
]
O ester), 1700 (nC O lactam),
]
1620 (
d6) (ppm): 12.78 (s, 1H, NHb), 11.31 (s, 1H, NH isatin), 9.56 (d,
nC]N), 1375 (n
C]S), 1235, 1058 (nCOC ester); 1H NMR (DMSO-
d
13C NMR (DMSO-d6)
d
(ppm): 179.00 (C]S), 170.0e169.2
J ¼ 9.0 Hz, 1H, NHa), 7.53 (s, 1H, H-40), 7.18 (d, J ¼ 7.5 Hz, 1H, H-70),
6.81 (d, J ¼ 8.0 Hz, 1H, H-60), 6.03 (t, J ¼ 9.25 Hz, 1H, H-1), 5.45 (t,
J ¼ 9.5 Hz, 1H, H-3), 5.36 (t, J ¼ 9.25 Hz, 1H, H-2), 4.99 (t, J ¼ 9.75 Hz,
1H, H-4), 4.25 (dd, J ¼ 12.5, 4.5 Hz, 1H, H-6a), 4.17 (ddd, J ¼ 9.75, 4.5,
2.5 Hz, 1H, H-5), 4.01 (d, J ¼ 11.5 Hz, 1H, H-6b), 2.31 (s, 3H, 50-CH3)
(4 ꢂ COCH3), 162.3 (C]O lactam), 141.4 (C-7a0), 132.1 (C-30), 131.0
(C-60),126.6 (C-50),121.6 (C-40),120.9 (C-70),112.7 (C-4a0), 81.9 (C-1),
72.7 (C-3), 72.5 (C-5), 71.0 (C-2), 67.7 (C-4), 61.7 (C-6), 20.5e20.3
(4 ꢂ CH3CO); MS (þESI): m/z (%) ¼ 607.03/609.05 (100/40)
[Mþ35Cl]þ/[Mþ37Cl]þ;
calcd.
for
C23
H
ClN4O10S/
2.01e1.93 (s, 12H, 4 ꢂ CH3CO); 13C NMR (DMSO-d6)
d (ppm): 178.9
35
25

N.D. Thanh et al. / European Journal of Medicinal Chemistry 123 (2016) 532e543
539
(C]S), 170.0e169.3 (4 ꢂ COCH3), 162.6 (C]O lactam), 140.5 (C-70),
133.4 (C-4a0), 132.2 (C-30), 131.4 (C-60), 121.6 (C-40), 119.7 (C-50),
110.9 (C-7a0), 81.8 (C-1), 72.7 (C-3), 72.4 (C-5), 71.0 (C-2), 67.7 (C-4),
61.7 (C-6), 21.5 (50-CH3), 20.5e20.3 (4 ꢂ CH3CO); calcd. for
(96% ethanol), ref [39]: 198 ꢀC; N-butylisatin (3p), 4 min, 63%,
34e36 ꢀC (diethyl ether), ref [6]: 36 ꢀC; N-allylisatin (3r), 2 min,
72%, 90e91 ꢀC (96% ethanol), ref [39]: 88e89 ꢀC; N-benzylisatin
(3s), 3 min, 88%, 129e130 ꢀC (96% ethanol), ref [39]: 131e132 ꢀC.
Compounds 3q and 3t were prepared similarly and used for syn-
thesis of corresponding thiosemicarbazones.
C
24H28N4O10S ¼ 564.15 Da. Anal. calcd for C24H28N4O10S (564.56):
C, 51.06; H, 5.00; N, 9.92%. Found: C, 51.10; H, 5.03; N, 9.97%.
N-Isobutylisatin (3q), 5 min, 74%, 88e89 ꢀC (from 96% ethanol);
4.1.2.11. 70-Methylisatin
N-(2,3,4,6-tetra-O-acetyl-
b
-D-glucopyr-
1H NMR (DMSO-d6)
d
(ppm): 7.67 (t, 1H, J ¼ 8.0 Hz, H-6), 7.55 (d, 1H,
anosyl)thiosemicarbazone (4k). Starting from 7-methylisatin 3k
(0.32 g) and thiosemicarbazide 2 (0.84 g), in 5 mL of absolute
ethanol, irradiation time: 5 min, the title compound 4k was ob-
tained as dark yellow solid, recrystallization from 96% ethanol;
J ¼ 8.5 Hz, H-4), 7.23 (t, 1H, J ¼ 8.0 Hz, H-7), 7.15 (t, 1H, J ¼ 8.5 Hz, H-
5), 4.68 (d, 2H, J ¼ 7.0 Hz, CH2), 2.78 (septet, 1H, J ¼ 7.0 Hz, CH), 0.91
(d, 6H, J ¼ 7.0 Hz, 2 ꢂ CH3); 13C NMR (DMSO-d6)
d (ppm): 160.5 (C-
2), 179.8 (C-3), 148.6 (C-7a), 134.4 (C-6), 129.8 (C-5), 125.5 (C-4),
117.7 (C-4a), 116.2 (C-7), 53.9 (CH2), 26.2 (CH), 20.7 (2 ꢂ CH3).
N-Phenethylisatin (3t), 4 min, 90%, 110e112 ꢀC (from 96%
yield: 0.99 g (88%), m.p. 225e227 ꢀC; ½a D25
ꢃ
þ57.0 (c ¼ 0.27, CHCl3);
FT-IR (KBr, cmꢁ1):
n
3183 (nNH), 1743 (nC
]
O ester), 1706 (nC O lactam),
]
1624 (nC]N), 1375 (
n
C]S), 1225, 1033 (nCOC ester); 1H NMR (DMSO-
ethanol); 1H NMR (DMSO-d6)
d (ppm): 7.92 (m, 2H, H-6 & H-7), 7.56
d6) (ppm): 12.81 (s, 1H, NHb), 11.30 (s, 1H, NH isatin), 9.55 (d,
d
(d, 1H, J ¼ 8.0 Hz, H-4), 7.33e7.28 (m, 5H, C6H5, N-phenethyl), 7.15
J ¼ 9.5 Hz, 1H, NHa), 7.55 (d, J ¼ 7.5 Hz, 1H, H-40), 7.20 (d, J ¼ 8.0 Hz,
1H, H-60), 7.03 (t, J ¼ 7.5 Hz, 1H, H-50), 6.01 (t, J ¼ 9.0 Hz, 1H, H-1),
5.45 (t, J ¼ 9.5 Hz, 1H, H-3), 5.35 (t, J ¼ 9.25 Hz, 1H, H-2), 4.98 (t,
J ¼ 9.75 Hz, 1H, H-4), 4.23 (dd, J ¼ 12.5, 4.5 Hz, 1H, H-6a), 4.16 (ddd,
J ¼ 9.75, 4.5, 2.5 Hz, 1H, H-5), 3.99 (dd, J ¼ 12.5, 1.5 Hz, 1H, H-6b),
2.22 (s, 3H, 70-CH3) 2.01e1.93 (s, 12H, 4 ꢂ CH3CO); 13C NMR (DMSO-
(m, 1H, H-5), 4.65 (s, 2H, CH2, N-phenethyl); 13C NMR (DMSO-d6)
d
(ppm): 179.4 (C-3), 160.7 (C-2), 148.3 (C-7a), 136.4 (C-10), 135.1 (C-
6), 129.3 (C-5), 128.1 (C-30 & C-50), 126.6 (C-40), 126.2 (C-20 & C-60),
124.9 (C-4), 117.2 (C-4a), 116.8 (C-7), 47.4 (CH2).
4.1.4. General procedure for synthesis of N-alkylisatin N-(2,3,4,6-
d6)
d
(ppm): 179.0 (C]S), 170.0e169.3 (4 ꢂ COCH3), 163.0 (C]O
tetra-O-acetyl-
b-
D-glucopyranosyl)thiosemicarbazones (4m-t)
lactam), 141.3 (C-4a0), 133.7 (C-50), 133.0 (C-60), 122.3 (C-30), 120.6
(C-40), 119.4 (C-70), 118.8 (C-7a0), 81.8 (C-1), 72.7 (C-3), 72.4 (C-5),
70.9 (C-2), 67.7 (C-4), 61.7 (C-6), 20.5e20.3 (4 ꢂ CH3CO), 15.8 (70-
To a solution of N-(2,3,4,6-tetra-O-acetyl-
b-D-glucopyranosyl)
thiosemicarbazide 2 (2 mmol, 0.84 g) in absolute ethanol, the
corresponding N-alkylisatins 3m-t (2 mmol) was added. Glacial
acetic acid (0.02 mL) as catalyst was added dropwise with stirring.
The obtained suspension mixture was then microwave irradiated
for 5e20 min (depending on alkyl substituents on nitrogen atom of
isatin ring). After irradiating for 3e5 min, the suspension mixture
became a clear solution. The irradiation was continued in a given
time. At the end of reaction process, the precipitate appeared. Upon
completion, monitored by TLC, the reaction mixture was cooled to
room temperature; the color precipitate was filtered with suction.
The crude product was recrystallized from mixture of toluene-96%
ethanol (1:1 in volume) to afford the title compounds of N-(2,3,4,6-
CH3);calcd. for C24H28N4O10
S
¼
564.15 Da. Anal. calcd for
C
24H28N4O10S (564.56): C, 51.06; H, 5.00; N, 9.92%. Found: C, 51.02;
H, 5.03; N, 9.88%.
4.1.2.12. 50-Isopropylisatin N-(2,3,4,6-tetra-O-acetyl-
b-D-glucopyr-
anosyl)thiosemicarbazone (4l). Starting from 5-isopropylisatin 3l
(0.38 g) and thiosemicarbazide 2 (0.84 g), in 5 mL of absolute
ethanol, irradiation time: 5 min, the title compound 4l was ob-
tained as deep yellow solid, recrystallization from 96% ethanol;
yield: 0.95 g (80%), m.p. 150e152 ꢀC; ½a D25
ꢃ
þ59.0 (c ¼ 0.26, CHCl3);
FT-IR (KBr, cmꢁ1):
n
3348 (nNH),1750 (nC
]
O ester),1704 (nC O lactam),
]
tetra-O-acetyl-b-D-glucopyranosyl)-2-(1-alkyl-2-oxoindolin-3-
1628 (
d6) (ppm): 12.83 (s, 1H, NHb), 11.20 (s, 1H, NH isatin), 9.52 (d,
nC]N), 1375 (n
C]S), 1240, 1047 (nCOC ester); 1H NMR (DMSO-
ylidene)hydrazinecarbothioamide 4m-t. The spectral (FT-IR, 1H
NMR, 13C NMR and ESI-MS or EI-HRMS) data are in good agreement
with their structures.
d
J ¼ 9.5 Hz, 1H, NHa), 7.58 (s, 1H, H-40), 7.27 (dd, J ¼ 8.0, 1.5 Hz, 1H, H-
70), 6.86 (d, J ¼ 8.0 Hz, 1H, H-60), 6.01 (t, J ¼ 9.0 Hz, 1H, H-1), 5.46 (t,
J ¼ 9.5 Hz, 1H, H-3), 5.36 (t, J ¼ 9.25 Hz,1H, H-2), 5.00 (t, J ¼ 9.75 Hz,
1H, H-4), 4.24 (dd, J ¼ 12.5, 5.0 Hz, 1H, H-6a), 4.17 (ddd, J ¼ 10.0, 4.5,
2.0 Hz, 1H, H-5), 4.00 (dd, J ¼ 12.0, 1.5 Hz, 1H, H-6b), 2.90 [septet,
J ¼ 7.25 Hz, 1H, 50-CH (CH3)2], 2.01e1.90 (s, 12H, 4 ꢂ CH3CO), 1.23 [d,
4.1.4.1. N-Methylisatin N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)
thiosemicarbazone (4m). Starting from N-methylisatin 3m (0.33 g)
and thiosemicarbazide 2 (0.84 g), in 10 mL of absolute ethanol,
irradiation time: 5 min, the title compound 4m was obtained as
yellow solid, recrystallization from mixture of toluene-96% ethanol
J ¼ 7.25 Hz, 1H, 50-CH (CH3)2]; 13C NMR (DMSO-d6)
d (ppm): 178.9
(C]S), 170.0e169.3 (4 ꢂ COCH3), 162.7 (C]O lactam), 142.8 (C-4a0),
140.8 (C-50), 133.7 (C-60), 129.8 (C-30), 119.9 (C-70), 119.6 (C-40), 111.0
(C-7a0), 81.9 (C-1), 72.7 (C-3), 72.4 (C-5), 71.0 (C-2), 67.7 (C-4), 61.7
(C-6), 33.2 (50-CH (CH3)2], 24.0 (50-CH (CH3)2], 20.5e20.3
(4 ꢂ CH3CO); calcd. for C26H32N4O10S ¼ 592.18 Da. Anal. calcd for
(1:1 v/v); yield: (0.92 g, 76%), m.p. 208e209 ꢀC; ½a D25
ꢃ þ69.0
(c ¼ 0.25, CHCl3); FT-IR (KBr, cmꢁ1):
n 3320 (nNH), 1755 (nC]
O ester
and nC]O lactam), 1610 (nC]N), 1370 (nC]S), 1228, 1040 (nCOC ester);
1H NMR (DMSO-d6)
d (ppm): 12.71 (s, 1H, NHb), 9.61 (d, 1H,
C
26H32N4O10S (615.61): C, 52.70; H, 5.44; N, 9.45%. Found: C, 52.75;
J ¼ 9.0 Hz, NHa), 7.75 (d, 1H, J ¼ 8.0 Hz, H-70), 7.48 (t, 1H, J ¼ 7.75 Hz,
H-50), 7.19 (t, 1H, J ¼ 7.5 Hz, H-60), 7.16 (d, 1H, J ¼ 7.5 Hz, H-40), 6.01
(t, 1H, J ¼ 9.0 Hz, H-1), 5.46 (t, 1H, J ¼ 9.5 Hz, H-3), 5.34 (t, 1H,
J ¼ 9.0 Hz, H-2), 4.99 (t, 1H, J ¼ 9.75 Hz, H-4), 4.24 (t, 1H, J ¼ 12.5,
5.0 Hz, H-6a), 4.18e4.17 (m, 1H, H-5), 4.00 (dd, 1H, J ¼ 11.0 Hz, H-
H, 5.40; N, 9.42%.
4.1.3. General procedure for synthesis of N-alkylisatins (3m-t)
To a solution of isatin 3a (0.03 mol) in DMF (7 mL), the corre-
sponding alkyl halides (0.03 mol) and K2CO3 (0.042 mol, 6.20 g)
were added. Reaction mixture was microwave irradiated for an
appropriate time and then cooled to room temperature and mixed
thoroughly with ice-water. The product recrystallized from the
suitable solvent. Reaction time, yield, melting point and recrystal-
lization solvents for these N-alkylisatins are following: N-methyl-
isatin (3m), 2 min, 84%, 130e131 ꢀC (96% ethanol), ref [39]:
130e133 ꢀC; N-ethylisatin (3n), 3 min, 71%, 86e87 ꢀC (96% ethanol),
ref [39]: 88e89 ꢀC; N-propylisatin (3o), 3 min, 75%, 196e197 ꢀC
6b), 3.21 (s, 3H, CH3, N-methyl), 2.01e1.93 (s, 12H, 4 ꢂ CH3CO); 13
C
NMR (DMSO-d6) d (ppm): 179.0 (C]S), 169.3e169.9 (COCH3), 160.7
(C]O isatin), 144.0 (C-30), 132.5 (C-40), 131.6 (C-40a), 122.9 (C-50),
121.0 (C-60),119.0 (C-70),109.9 (C-70a), 81.9 (C-1), 72.7 (C-3), 72.4 (C-
5), 70.9 (C-2), 67.7 (C-4), 61.7 (C-6), 25.7 (CH3, N-methyl), 20.5e20.3
(4
ꢂ
CH3CO);
EI-HRMS:
C24H28N4O10S,
calc.
for
[M]þ
¼
564.1526 Da, found: m/z 564.1533. Anal. calcd for
ꢄ
C24H28N4O10S (564.56): C, 51.06; H, 5.00; N, 9.92%. Found: C, 51.02;
H, 5.05; N, 9.95%.

540
N.D. Thanh et al. / European Journal of Medicinal Chemistry 123 (2016) 532e543
4.1.4.2. N-Ethylisatin N-(2,3,4,6-tetra-O-acetyl-
b
-
D
-glucopyranosyl)
J ¼ 6.5 Hz, CH2, N-butyl), 2.07 (m, 1H, CH, N-butyl); 2.01e1.93 (s,
12H, 4 ꢂ CH3CO), 1.61 (m, 2H, CH2, N-butyl), 1.61 (m, 2H, CH2, N-
butyl), 0.90 (t, 3H, J ¼ 7.0 Hz, CH3, N-butyl); 13C NMR (DMSO-d6)
thiosemicarbazone (4n). Starting from N-ethylisatin 3n (0.29 g) and
thiosemicarbazide 2, in 10 mL of absolute ethanol, irradiation time:
6 min, the title compound 4n was obtained as yellow solid,
recrystallization from mixture of toluene-96% ethanol (1:1 v/v);
yield: 1.16 g (89%), m.p. 152e153 ꢀC (from 96% ethanol), yield 81%;
d
(ppm): 179.1 (C]S), 169.9e169.3 (4 ꢂ COCH3), 160.7 (C]O isatin),
143.2 (C-70a), 132.4 (C-30), 131.6 (C-60), 122.8 (C-50), 121.3 (C-70),
119.1 (C-40a), 110.1 (C-40), 82.3 (C-1), 70.7 (C-3), 71.9 (C-5), 68.6 (C-
2), 67.5 (C-4), 61.3 (C-6), 40.0 (CH2CH2CH2CH3, N-butyl), 29.0
½
a 2D5
ꢃ
þ76.2 (c ¼ 0.25, CHCl3); FT-IR (KBr, cmꢁ1):
n
3350 (nNH), 1751
C]S), 1232, 1039
(nC
]
and nC
]
O lactam), 1614 (
n
C]N), 1367 (
n
(CH2CH2CH2CH3, N-butyl), 20.5e20.3 (4
ꢂ
CH3CO), 19.4
O ester
(
nCOC ester); 1H NMR (DMSO-d6)
d
(ppm): 12.72 (s, 1H, NHb), 9.61 (d,
(CH2CH2CH2CH3, N-butyl), 13.5 (CH2CH2CH2CH3, N-butyl); EI-
1H, J ¼ 9.5 Hz, NHa), 7.76 (d, 1H, J ¼ 7.5 Hz, H-70), 7.72 (d, 1H,
J ¼ 8.0 Hz, H-40), 7.46 (t, 1H, J ¼ 7.75 Hz, H-60), 7.19 (t, 1H, J ¼ 7.75 Hz,
H-50), 6.01 (t,1H, J ¼ 9.0 Hz, H-1), 5.46 (t,1H, J ¼ 9.5 Hz, H-3), 5.34 (t,
1H, J ¼ 9.25 Hz, H-2), 4.98 (t, 1H, J ¼ 9.75 Hz, H-4), 4.24 (dd, 1H,
J ¼ 12.5, 4.5 Hz, H-6a), 4.17 (ddd, 1H, J ¼ 9.5, 4.5, 2.0 Hz, H-5), 4.00
(dd, 1H, J ¼ 12.0, 1.5 Hz, H-6b), 3.77 (t, 2H, CH2, N-ethyl; 2.01e1.93
(s, 12H, 4 ꢂ CH3CO), 1.20 (t, 3H, CH3, N-ethyl); 13C NMR (DMSO-d6)
HRMS: C27H34N4O10S, calc. for [M]þ ¼ 606.1996 Da, found: m/z
ꢄ
606.2008. Anal. calcd for C27H34N4O10S (606.64): C, 53.46; H, 5.65;
N, 9.24%. Found: C, 53.49; H, 5.61; N, 9.29%.
4.1.4.5. N-Isobutylisatin
N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyr-
anosyl)thiosemicarbazone (4q). Starting from N-isobutylisatin 3q
(0.29 g) and thiosemicarbazide 2, in 10 mL of absolute ethanol,
irradiation time: 17 min, the title compound 4q was obtained as
yellow solid, recrystallization from mixture of toluene-96% ethanol
d
(ppm): 179.0 (C]S), 169.9e169.3 (4 ꢂ COCH3), 160.3 (C]O isatin),
142.9 (C-30), 132.6 (C-40), 131.7 (C-40a), 122.8 (C-50), 121.3 (C-60),
119.2 (C-70), 110.0 (C-70a), 81.9 (C-1), 72.7 (C-3), 72.4 (C-5), 70.9 (C-
2), 67.7 (C-4), 61.7 (C-6), 34.1 (CH2CH3, N-ethyl), 20.5e20.3
(4 ꢂ CH3CO), 12.5 (CH2CH3, N-ethyl); EI-HRMS: C25H30N4O10S, calc.
(1:1 v/v); yield: 1.04 g (86%), m.p. 190e193 ꢀC; ½a D25
ꢃ
þ83.0 (c ¼ 0.25,
CHCl3); FT-IR (KBr, cmꢁ1):
n
3287 (nNH), 1747 (nC
]
O ester and nC
]
O
lactam), 1617 (
(DMSO-d6)
nC]N), 1370 (n
C]S), 1230, 1040 (nCOC ester); 1H NMR
(ppm): 12.73 (s, 1H, NHb), 9.63 (d, 1H, J ¼ 9.0 Hz, NHa),
for [M]þ ¼ 578.1682 Da, found: m/z 578.1692. Anal. calcd for
ꢄ
d
C
25H30N4O10S (578.59): C, 51.90; H, 5.23; N, 9.68%. Found: C, 51.95;
7.77 (d,1H, J ¼ 7.5 Hz, H-70), 7.45 (t,1H, J ¼ 7.75 Hz, H-60), 7.20 (d,1H,
J ¼ 7.5 Hz, H-40), 7.19 (t, 1H, J ¼ 7.75 Hz, H-50), 6.01 (t, 1H, J ¼ 9.0 Hz,
H-1), 5.46 (t, 1H, J ¼ 9.25 Hz, H-3), 5.34 (t, 1H, J ¼ 9.25 Hz, H-2), 4.98
(t, 1H, J ¼ 9.75 Hz, H-4), 4.24 (dd, 1H, J ¼ 12.5, 4.5 Hz, H-6a), 4.17
(ddd, 1H, J ¼ 10.0, 4.75, 2.0 Hz, H-5), 3.98 (dd, 1H, J ¼ 11.25, 1.5 Hz,
H-6b), 3.55 (d, 2H, CH2, N-isobutyl), 2.07 (m, 1H, CH, N-isobutyl);
H, 5.20; N, 9.65%.
4.1.4.3. N-Propylisatin N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)
thiosemicarbazone (4o). Starting from N-propylisatin 3o (0.29 g)
and thiosemicarbazide 2, in 10 mL of absolute ethanol, irradiation
time: 13 min, the title compound 4o was obtained as yellow solid,
recrystallization from mixture of toluene-96% ethanol (1:1 v/v);
2.01e1.93 (s, 12H, 4 ꢂ CH3CO), 0.91 [d, 6H, (CH3)2, N-isobutyl]; 13
C
NMR (DMSO-d6)
d
(ppm): 179.0 (C]S), 170.0e169.3 (4 ꢂ COCH3),
yield: 0.89 g (75%), m.p. 187e189 ꢀC; ½a D25
ꢃ
þ59.5 (c ¼ 0.2, CHCl3); FT-
161.0 (C]O isatin), 143.6 (C-30), 132.4 (C-40), 131.6 (C-40a), 122.8 (C-
50), 121.1 (C-60), 119.0 (C-70), 110.4 (C-70a), 81.8 (C-1), 72.7 (C-3), 72.4
(C-5), 70.9 (C-2), 67.7 (C-4), 61.7 (C-6), 46.6 [CH2CH (CH3)2, N-iso-
butyl], 26.8 [CH2CH (CH3)2, N-isobutyl), 20.5e18.3 (4 ꢂ CH3CO), 19.8
IR (KBr, cmꢁ1):
n
3294 (nNH), 1743 (nC
]
O ester and nC O lactam), 1615
]
(nC]N), 1367 (n
C]S), 1235, 1035 (nCOC ester); 1H NMR (DMSO-d6)
d
(ppm): 12.73 (s, 1H, NHb), 9.62 (d, 1H, J ¼ 9.0 Hz, NHa), 7.77 (d, 1H,
J ¼ 7.5 Hz, H-70), 7.46 (t, 1H, J ¼ 7.75 Hz, H-60), 7.21 (t, 1H, J ¼ 8.0 Hz,
H-40), 7.19 (t, 1H, J ¼ 7.75 Hz, H-50), 6.01 (t, 1H, J ¼ 9.0 Hz, H-1), 5.46
(t, 1H, J ¼ 9.5 Hz, H-3), 5.34 (t, 1H, J ¼ 9.0 Hz, H-2), 4.98 (t, 1H,
J ¼ 9.75 Hz, H-4), 4.24 (dd, 1H, J ¼ 12.25, 4.75 Hz, H-6a), 4.17 (ddd,
1H, J ¼ 9.5, 4.5, 2.0 Hz, H-5), 4.00 (d, 1H, J ¼ 12.0, H-6b), 3.71 (t, 2H,
J ¼ 7.25 Hz, NCH2, N-propyl; 2.01e1.93 (s, 12H, 4xCH3CO), 1.65
(sextet, 2H, J ¼ 7.25 Hz, CH2, N-propyl), 0.89 (t, 3H, J ¼ 7.25 Hz, CH3,
[CH2CH (CH3)2, N-isobutyl]; EI-HRMS: C27H34N4O10S, calc. for
[M]þ
¼
606.1996 Da, found: m/z 606.2011. Anal. calcd for
ꢄ
C27H34N4O10S (606.64): C, 53.46; H, 5.65; N, 9.24%. Found: C, 53.42;
H, 5.69; N, 9.21%.
4.1.4.6. N-Allylisatin N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)
N-propyl); 13C NMR (DMSO-d6)
d
(ppm): 179.1 (C]S), 169.9e169.3
thiosemicarbazone (4r). Starting from N-allylisatin 3r (0.29 g) and
thiosemicarbazide 2, in 10 mL of absolute ethanol, irradiation time:
20 min, the title compound 4r was obtained as yellow solid,
recrystallization from mixture of toluene-96% ethanol (1:1 v/v);
(4 ꢂ COCH3), 160.7 (C]O isatin), 143.3 (C-70a), 132.4 (C-30), 131.6 (C-
60), 122.8 (C-50), 121.3 (C-70), 119.1 (C-40a), 110.1 (C-40), 82.3 (C-1),
71.9 (C-3), 70.7 (C-5), 68.6 (C-2), 67.5 (C-4), 61.3 (C-6), 20.5e20.3
(4 ꢂ CH3CO), 40.8 (CH2CH2CH3, N-propyl), 20.4 (CH2CH2CH3, N-
propyl), 11.1 (CH2CH2CH3, N-propyl); EI-HRMS: C26H32N4O10S, calc.
yield: 1.04 g (74%), m.p. 150e151 ꢀC; ½a D25
ꢃ
þ91.0 (c ¼ 0.25, CHCl3);
FT-IR (KBr, cmꢁ1):
n
3303 (nNH), 1747 (nC
]
O ester
and nC]O lactam),
for [M]þ ¼ 592.1839 Da, found: m/z 592.1852. Anal. calcd for
1611 (
d6)
nC]N), 1373 (n
C]S), 1229, 1037 (nCOC ester); 1H NMR (DMSO-
(ppm): 12.68 (s, 1H, NHb), 9.64 (d, 1H, J ¼ 9.5 Hz, NHa), 7.78 (d,
ꢄ
C
26H32N4O10S (592.62): C, 52.70; H, 5.44; N, 9.45%. Found: C, 52.67;
d
H, 5.47; N, 9.41%.
1H, J ¼ 2.5 Hz, H-70), 7.44 (t, 1H, J ¼ 7.75 Hz, H-60), 7.19 (t, 1H,
J ¼ 7.5 Hz, H-50), 7.09 (d, 1H, J ¼ 8.0 Hz, H-40), 6.02 (t, 1H, J ¼ 9.0;
9.0 Hz, H-1), 5.88e5.87 (m, 1H, NeCH2CH]CH2), 5.46 (t, 1H,
J ¼ 9.5 Hz, H-3), 5.35 (t, 1H, J ¼ 9.25 Hz, H-2), 5.21 (m, 2H,
NeCH2CH]CH2), 4.99 (t, 1H, J ¼ 9.75 Hz, H-4), 4.37e4.36 (m, 2H,
NeCH2CH]CH2), 4.24 (dd, 1H, J ¼ 12.5, 5.0 Hz, H-6a), 4.17 (ddd, 1H,
J ¼ 10.0, 4.5, 2.0 Hz, H-5), 4.00 (dd, 1H, J ¼ 11.75, 1.5 Hz, H-6b),
4.1.4.4. N-Butylisatin N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)
thiosemicarbazone (4p). Starting from N-butylisatin 3p (0.29 g) and
thiosemicarbazide 2, in 10 mL of absolute ethanol, irradiation time:
15 min, the title compound 4p was obtained as yellow solid,
recrystallization from mixture of toluene-96% ethanol (1:1 v/v);
yield: 1.05 g (87%), m.p. 186e189 ꢀC; ½a D25
ꢃ
þ76.1 (c ¼ 0.22, CHCl3);
2.01e1.93 (s, 12H, 4 ꢂ CH3CO); 13C NMR (DMSO-d6)
d (ppm): 179.00
FT-IR (KBr, cmꢁ1):
n
3346 (nNH), 1749 (nC
]
and nC]O lactam),
(C]S), 169.3e167.0 (4 ꢂ COCH3), 160.4 (C]O isatin), 143.0 (C-30),
132.3 (C-40), 131.5 (C-40a), 131.3 (NeCH2CH]CH2), 122.9 (C-50),
121.2 (C-60), 119.1 (C-70), 117.4 (NeCH2CH]CH2), 110.4 (C-70a), 81.9
(C-1), 70.9 (C-2), 72.7 (C-3), 67.7 (C-4), 72.4 (C-5), 61.7 (C-6), 41.3
O ester
1612 (
d6)
nC]N), 1376 (n
C]S), 1227, 1043 (nCOC ester); 1H NMR (DMSO-
(ppm): 12.73 (s, 1H, NHb), 9.61 (d, 1H, J ¼ 9.0 Hz, NHa), 7.77 (d,
d
1H, J ¼ 7.5 Hz, H-70), 7.46 (t, 1H, J ¼ 7.5 Hz, H-60), 7.20 (d, 1H,
J ¼ 7.5 Hz, H-40), 7.19 (t,1H, J ¼ 7.75 Hz, H-50), 6.01 (t,1H, J ¼ 9.25 Hz,
H-1), 5.46 (t, 1H, J ¼ 9.5 Hz, H-3), 5.34 (t, 1H, J ¼ 9.25 Hz, H-2), 4.98
(t, 1H, J ¼ 9.75 Hz, H-4), 4.24 (dd, 1H, J ¼ 12.5, 4.5 Hz, H-6a), 4.17 (dt,
1H, J ¼ 7.5, 2.0 Hz, H-5), 4.00 (d, 1H, J ¼ 12.0 Hz, H-6b), 3.74 (t, 2H,
(NeCH2eCH]CH2), 20.5e20.3 (4
C
Anal. calcd for C26H30N4O10S (590.60): C, 52.88; H, 5.12; N, 9.49%.
Found: C, 52.86; H, 5.16; N, 9.46%.
ꢂ
CH3CO); EI-HRMS:
26H30N4O10S, calc. for [M]þ ¼ 590.1683 Da, found: m/z 590.1692.
ꢄ

N.D. Thanh et al. / European Journal of Medicinal Chemistry 123 (2016) 532e543
541
4.1.4.7. N-Benzylisatin N-(2,3,4,6-tetra-O-acetyl-
b
-
D
-glucopyranosyl)
diphenyltetrazolium bromide were purchased from Sigma-Aldrich
Chemical Co. (Viet Nam). All other chemicals and reagents were
analytical grade.
thiosemicarbazone (4s). Starting from N-benzylisatin 3s (0.29 g)
and thiosemicarbazide 2, in 10 mL of absolute ethanol, irradiation
time: 15 min, the title compound 4s was obtained as yellow solid,
recrystallization from mixture of toluene-96% ethanol (1:1 v/v);
4.2.2. In vitro antimicrobial activity
yield: 1.21 g (94%), m.p. 195e196 ꢀC; ½a D25
ꢃ
þ90.1 (c ¼ 0.22, CHCl3);
The in vitro antibacterial and antifungal activities were screened
by the twofold serial dilution technique using the principles of
Clinical and Laboratory Standards Institute [33]. Antimicrobial ac-
tivity in vitro against S. aureus (ATCC 11632), E. coli (ATCC 25922),
P. aeruginosa (ATCC 25923), K. pneumoniae (ATCC 4352),
S. epidermidis (ATCC 12228), B. subtilis (ATCC 11774), E. aerogenes
(ATCC 13048), and a series of methicillin resistant S. aureus
(MRSA198-1, MRSA198-2 and MRSA198-3 were investigated by
minimum inhibitory concentration (MIC) using microbroth di-
lutions technique using MuellereHinton broth [33]. Test com-
FT-IR (KBr, cmꢁ1):
n
3341 (nNH), 1750 (nC
]
and nC]O lactam),
O ester
1608 (
d6)
nC]N), 1370 (n
C]S), 1229, 1064 (nCOC ester); 1H NMR (DMSO-
(ppm): 12.72 (s, 1H, NHb), 9.67 (d, 1H, J ¼ 9.5 Hz, NHa), 7.79 (d,
d
1H, J ¼ 7.5 Hz, H-70), 7.39e7.34 (5H, C6H5, N-benzyl), 7.33 (t, 1H,
J ¼ 7.25 Hz, H-60), 7.17 (t, 1H, J ¼ 7.5 Hz, H-50), 7.04 (d, 1H, J ¼ 8.0 Hz,
H-40), 6.04 (t,1H, J ¼ 9.0 Hz, H-1), 5.47 (t,1H, J ¼ 9.5 Hz, H-3), 5.36 (t,
1H, J ¼ 9.25 Hz, H-2), 5.00 (t, 1H, J ¼ 9.75 Hz, H-4), 4.98 (s, 2H, CH2,
N-benzyl), 4.24 (dd, 1H, J ¼ 12.5, 5.0 Hz, H-6a), 4.19 (ddd, 1H, J ¼ 9.5,
4.75, 1.5 Hz, H-5), 4.01 (dd, 1H, J ¼ 11.0 Hz, H-6b), 2.01e1.93 (s, 12H,
4 ꢂ CH3CO); 13C NMR (DMSO-d6)
d
(ppm): 13C NMR (DMSO-d6)
pounds were dissolved in dimethyl sulfoxide (DMSO) at a
d
(ppm): 179.0 (C]S), 169.9e169.3 (4 ꢂ COCH3), 160.7 (C]O (isa-
concentration of 1 mg/mL. Ciprofloxacin, methicillin and vanco-
mycin was used as references for antibacterial activity (Table 1).
Further dilutions of the test compounds and standard drugs were
prepared test medium to provide concentrations are in which 400,
tin), 142.9 (C-30), 135.6 (C-100, N-benzyl), 132.2 (C-40), 131.5 (C-400, N-
benzyl), 128.8 (C-40a), 128.6 (C-300 & C-500, N-benzyl), 127.4 (C-200
&
C-600, N-benzyl), 123.0 (C-50), 121.2 (C-60), 119.2 (C-70), 110.4 (C-70a),
81.9 (C-1), 72.7 (C-3), 72.4 (C-5), 70.9 (C-2), 67.7 (C-4), 61.8 (C-6),
200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 mM. The inoculum was
42.6 (CH2, N-benzyl), 21.0e20.8 (4
C
Anal. calcd for C30H32N4O10S (640.66): C, 56.24; H, 5.03; N, 8.75%.
Found: C, 56.29; H, 5.06; N, 8.71%.
ꢂ
CH3CO); EI-HRMS:
prepared using a 4e6-h broth adjusted to a turbidity equivalent of
an 0.5 McFarland standard, diluted in broth media to give a final
concentration of 5 ꢂ 105 CFU/mL in the test tray; they were covered
and placed in plastic bags to prevent evaporation. The plates were
incubated at 35 ꢀC for 18e20 h. The MIC was defined as the lowest
concentration of compound giving complete inhibition of visible
growth. All the experiments were performed three times. The MIC
values for all tested compounds and reference drug are listed in
Table 1.
The compounds 4a-t were also evaluated for their in vitro
antifungal activity against A. niger (439), C. albicans (ATCC 7754),
F. oxysporum (M42), (and) S. cerevisiae (SH20) using agar dilution
method with Saburoud's dextrose agar (Hi-Media). Miconazole was
used as reference for antifungal activity. The tested compounds and
standard drug were prepared test medium to provide concentra-
30H32N4O10S, calc. for [M]þ ¼ 640.1839 Da, found: m/z 640.1850.
ꢄ
4.1.4.8. N-Phenethylisatin
N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyr-
anosyl)thiosemicarbazone (4t). Starting from N-phenethylisatin 3t
(0.29 g) and thiosemicarbazide 2, in 10 mL of absolute ethanol,
irradiation time: 9 min, the title compound 4t was obtained as
yellow solid, recrystallization from mixture of toluene-96% ethanol
(1:1 v/v); yield: 1.34 g (96%), m.p. 200e203 ꢀC; ½a D25
ꢃ
þ96.0 (c ¼ 0.25,
CHCl3); FT-IR (KBr, cmꢁ1):
n
3290 (nNH), 1735 (nC
]
O ester and nC
]
O
lactam), 1614 (
(DMSO-d6)
nC]N), 1367 (n
C]S), 1234, 1040 (nCOC ester); 1H NMR
d
(ppm): 12.66 (s, 1H, NHb), 9.62 (d, 1H, J ¼ 9.0 Hz, NHa),
7.75 (d, 1H, J ¼ 7.0 Hz, H-70), 7.42 (t, 1H, J ¼ 7.5 Hz, H-60), 7.28e7.21
(5H, C6H5, N-phenethyl), 7.19e7.16 (m, 2H, H-40 & H-50), 6.00 (t, 1H,
J ¼ 9.0 Hz, H-1), 5.46 (t, 1H, J ¼ 9.5 Hz, H-3), 5.33 (t, 1H, J ¼ 9.25 Hz,
H-2), 4.98 (t, 1H, J ¼ 9.75 Hz, H-4), 4.34 (t, 2H, J ¼ 5.0 Hz, CH2, N-
phenethyl), 4.24 (dd, 1H, J ¼ 12.25, 4.75 Hz, H-6a), 4.17 (ddd, 1H,
J ¼ 6.5, 4.5, 1.5 Hz, H-5), 4.02 (d, 1H, J ¼ 9.0 Hz, H-6b), 3.46e3.41 (m,
2H, CH2, N-phenethyl), 2.08e1.93 (s, 12H, 4 ꢂ CH3CO); 13C NMR
tions are in which 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 mM.
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 during 72 h and MIC's were determined [33]. Minimal
inhibitory concentrations for each compound were investigated
against standard fungal strains. All the experiments were per-
formed three times. The MIC values for all tested compounds and
reference drug are shown in Table 2.
(DMSO-d6)
d
(ppm): 179.1 (C]S), 169.9e169.3 (4 ꢂ COCH3), 160.5
(C]O isatin), 143.0 (C-70a), 138.1 (C-100, N-phenethyl), 132.2 (C-30),
131.6 (C-60), 128.8 (C-300 & C-500, N-phenethyl), 128.4 (C-200 & C-600,
N-phenethyl), 126.5 (C-400, N-phenethyl), 122.8 (C-50), 119.0 (C-40a),
110.2 (C-40),121.3 (C-70), 82.3 (C-1), 71.9 (C-3), 70.7 (C-5), 68.6 (C-2),
67.5 (C-4), 61.3 (C-6), 40.8 (NeCH2CH2C6H5, N-phenethyl), 33.5
4.2.3. In vivo antioxidant activity
4.2.3.1. Anti-oxidant assay. Albino rats of Wistar strain, weighing
100e150 g were used in all experiments. Rats were maintained on
12 h light/dark cycle at approximately 22 ꢀC and allowed food and
water ad libitum, then were injected i.p., with a mixture of CCl4 in
olive oil (1:1) at a dose of 0.6 mL/kg to induce hepatotoxicity. These
animals were divided randomly into four groups and seven rats
each. Control animals were given the vehicle alone. Rats were
(NeCH2CH2C6H5, N-phenethyl), 20.5e20.3 (4 ꢂ CH3CO); EI-HRMS:
ꢄ
C
31H34N4O10S, calc. for [M]þ ¼ 654.1996 Da, found: m/z 654.2007.
Anal. calcd for C31H34N4O10S (654.69): C, 56.87; H, 5.23; N, 8.56%.
Found: C, 56.92; H, 5.20; N, 8.59%.
4.2. Screening for biological activity
pretreated once with DL-a-tocopherol acetate (a dose of 400 mg/
4.2.1. Chemicals
kg) and test samples were given i.p. at a dose of 100 mg/kg/day for
seven consecutive days prior to the administration of CCl4. Animals
were sacrificed 24 h after CCl4 dosing and blood was collected by
decapitation for the determination of serum transaminases. He-
patic tissues were carefully excised and homogenized in cold 1.15%
KClꢁ10 mM phosphate buffer with EDTA (pH 7.4) and centrifuged
at 12000 rpm for 8 min. The supernatant was further centrifuged at
45000 rpm for 50 min to obtain cytosolic extract for the mea-
surement of liver cytosolic SOD, catalase and GSH-Px activities. The
protein contents were measured by the method of Lowry et al. [40]
Chrysin, dicyclohexylcarbodiimide (DCC) and diethylphosphoryl
cyanide (DEPC), sodium azide, ethylenediamine tetraacetic acid
(EDTA),
form (NADPH), cumene hydroperoxide, glutathione reductase, DL-
-tocopherol acetate, carbon tetrachloride (CCl4), xanthine, potas-
b-nicotinamide adenine dinucleotide phosphate, reduced
a
sium cyanide (KCN), sodium dodecylsulfate, trichloroacetic acid
(TCA), cytochrome C, thiobarbituric acid, n-butanol, pyridine,
HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid],
glutamine, sodium pyruvate, and 3-(4,5-dimethylthiazol-2-yl)-2,5-
L-

542
N.D. Thanh et al. / European Journal of Medicinal Chemistry 123 (2016) 532e543
with bovine serum albumin as a standard. The MICs were tested
twice in triplicate and the mean absorbance values were calculated.
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