Design and synthesis of some new 1,3,4-thiadiazines with coumarin moieties and their antioxidative and antifungal activity

Article abstract of DOI:10.3390/molecules19011163

A series of newly disubstituted (compounds 4a,b) and trisubstituted 1,3,4-thiadiazines 5a-l with various substituents was prepared utilizing different thiosemicarbazides and 3-A-bromoacetylcoumarins as starting compounds. The structures of the synthesized

Full text of DOI:10.3390/molecules19011163

Molecules 2014, 19, 1163-1177; doi:10.3390/molecules19011163
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design and Synthesis of Some New 1,3,4-Thiadiazines with
Coumarin Moieties and Their Antioxidative and
Antifungal Activity
1
,
2
1
1
2
Milan Čačić *, Valentina Pavić , Maja Molnar , Bojan Šarkanj and Elizabeta Has-Schön
1
Department of Applied Chemistry and Ecology, Faculty of Food Technology, J. J. Strossmayer
University, Franje Kuhača 20, Osijek 31 000, Croatia; E-Mails: maja.molnar@ptfos.hr (M.M.);
bojan.sarkanj@ptfos.hr (B.S.)
2
Department of Biology, J. J. Strossmayer University, Cara Hadrijana 8/A, Osijek 31 000, Croatia;
E-Mails: valentina.pavic@biologija.unios.hr (V.P.); hasschon@biologija.unios.hr (E.H.-S.)
*
Author to whom correspondence should be addressed; E-Mail: mcacic@ptfos.hr
Tel.: +385-31-224-342; Fax: +385-31-207-117.
Received: 12 December 2013; in revised form: 13 January 2014 / Accepted: 14 January 2014 /
Published: 17 January 2014
Abstract: A series of newly disubstituted (compounds 4a,b) and trisubstituted
1
,3,4-thiadiazines 5a–l with various substituents was prepared utilizing different
thiosemicarbazides and 3-α-bromoacetylcoumarins as starting compounds. The structures
of the synthesized 1,3,4-thiadiazines are elucidated and confirmed utilizing the
corresponding analytical and spectroscopic data. All of the new thiadiazine derivatives
were tested for their antioxidant activity, employing different antioxidant assays (DPPH
scavenging activity, iron chelating activity, power reducing activity). Compounds 5b, 5f, 5j
and 4b were proven to be the best DPPH radical scavengers, while compounds 5h and 5j have
shown the best iron chelating activity. Thiadiazine derivatives were also tested on their
antifungal activity against four mycotoxicogenic fungi, Aspergillus flavus, A. ochraceus,
Fusarium graminearum and F. verticillioides. The best antifungal against A. flavus was proven
to be compound 5e, while compounds 4a and 5c were the best antifungals on A. ochraceus,
and compound 5g showed the best antifungal activity on F. verticillioides.
Keywords: coumarin; 1,3,4-thiadiazines; thiosemicarbazides; antifungal activity;
antioxidative activity

Molecules 2014, 19
. Introduction
Coumarin (2-oxo-2H-chromene) and its derivatives represent one of the most important classes of
1164
1
compounds possessing numerous biological activities [1–3]. Some of these compounds have proven to
be active as antibacterial [4–6], antifungal [7], anti-inflammatory [8], anticoagulant [9], anti-HIV [10]
and antitumor agents [11]. Coumarin derivatives are widely used as additives in food and cosmetics [12],
pharmaceuticals and optical brighteners [13] and laser dyes [14]. Coumarins have also proven to be an
excellent antioxidants as well as antifungal agents [15].
In recent years, interest in thiadiazines has increased due to the high biological activity and broad-
spectrum action of their derivatives [16]. Many thiadiazines have been discovered with possible
applications in medical practice as sedatives, antianxiety agents, antiasthmatic agents, anticonvulsants,
myorelaxants, coronary vasodilators, and spasmolytics. Synthesis of the 1,3,4-thiadiazine system
employing a reaction of α-bromoacetophenone with thiosemicarbazide was first reported by Bose [17].
This procedure is characterized by the formation of a few heterocyclic isomers. Which isomer is
+
formed depends on the H -ion concentration in the system, the polarity of the solvent, the reaction
temperature and on the substituents. Depending on whether condensation occurs at the N , N , or N of
1
2
4
the thiosemicarbazide, three different sulfur-containing heterocyclic rings are expected after
ring closure: the 6H-1,3,4-thiadiazines, the 2-substituted imino-2,3-dihydrothiazol-3-amines, and the
3
-substituted 2-hydrazono-2,3-dihydrothiazoles.
In addition, 1,3,4-thiadiazines may exist in three different tautomeric forms. Spectroscopic
investigations suggest that the 6H-form is preferred. The 4H-form represents a potentially anti-aromatic
π-system which can be transformed by valence isomerization into a thiahomopyrazole and by
8
subsequent extrusion of sulfur into a pyrazole [18]. In continuation of our research program on the
synthesis of novel heterocyclic compounds exhibiting antioxidative and biological activity, it was
thought to be interesting to synthesize compounds containing both coumarin and thiadiazine groups in
their structure.
2
. Results and Discussion
2
.1. Chemistry
In continuation of our ongoing research program to synthesize potentially biologically active 1,3,4-
thiadiazine derivatives, in a present report we describe a series of 6H-1,3,4-thiadizines (compounds 4a,
4
b) and 4H-1,3,4-thiadiazines (compounds 5a–l). The reaction sequence for the synthesis of 5-(2-oxo-
H-chromen-3-yl)-6H-1,3,4-thiadizin-2-aminium bromide (4a) and 3-(2-amino-6H-1,3,4-thiadiazin-5-
2
yl)-4-hydroxy-2H-chromen-2-one (4b), is outlined in Scheme 1. 3-(α-Bromoacetyl)coumarins 1a,b
were prepared according to the earlier literature methods [19,20]. Reaction of 1a with
thiosemicarbazide 2 in ethanol at room temperature as the first step, followed by refluxing of the
ethanolic solution of the obtained product in the presence of 48% HBr gave compound 4a. Compound
4
b was prepared from 1b and thiosemicarbazide in ethanol in a presence of small amount of HCl.
The structures of the products 4a and 4b were inferred from their analytical and spectral data. Thus,
their IR spectra showed characteristic absorption bands at 3426–3183 (NH; OH), 1714–1696 (lactone
−1
1
C=O), 1617–1600 (-C=N), 756–760 (CH-arom.) and (C-S-C) at 675 cm . The H-NMR spectra of

Molecules 2014, 19
1165
+
compounds 4a and 4b exhibited singlets at 3.91 (s, 2H, CH ), 4.80 (s, 2H, NH /-N H ) and 14.68 ppm
2
2
3
(s, 1H, -OH).
Scheme 1. The reaction sequence for the synthesis of 5-(2-oxo-2H-chromen-3-yl)-6H-
,3,4-thiadizin-2-aminium bromide (4a) and 3-(2-amino-6H-1,3,4-thiadiazin-5-yl)-4-
1
hydroxy-2H-chromen-2-one (4b).
1
,4-Thiosemicarbazide derivatives 3a–g were synthesized as previously described [21] by reacting
equimolar amounts of ethyl-, methyl- or phenylisothiocyanate with 4-hydroxybenzohydrazide,
-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetohydrazide and 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-
2
acetohydrazide.
The compounds 5a–l were prepared by heating 3-α-bromoacetylcoumarins 1a,b with the
corresponding 1,4-disubstituted thiosemicarbazides 3a–g in ethanol, with few drops of hydrochloric
acid for about 40 min (Scheme 2).
1
Newly synthesised compounds 5a–l were characterised on the basis of elemental analysis, IR, H
NMR and mass spectral data. The IR spectra of compounds 5a–l showed absorption bands in the range
−
1
from 3490–2920 cm due to –CH
2
- stretching, C-H arom. and NH. The strong band at 1740 and band
−1
−1
at 1690 cm is attributed to the C=O stretching vibration. The absorption band seen at a 1600–1560 cm
could be attributed to the C=N stretching. The weak absorption band showed in the range
−1
6
81–639 cm is attributed to the (C-S-C) stretching vibration.
1
The H-NMR spectra of 5a–l showed singlet in the range 5.56–5.67 ppm, which is characteristic for
S-CH=C- from 1,3,4-thiadiazine ring. The combined spectral data gave strong support to the proposed
structures of all the synthesized compounds.
Scheme 2. Synthetic path for compounds 5a–l.

Molecules 2014, 19
1166
Scheme 2. Cont.
Compound
R
1
R
2
R
3
5
a
H
CH
3
HO
O
O
5
5
5
b
c
H
H
H
CH
2
CH
3
ibid.
ibid.
C H
6
5
d
CH
3
O
O
O
5
5
e
f
H
OH
CH
CH
2
CH
3
ibid.
3
HO
O
O
5
5
5
g
h
i
OH
OH
OH
CH
2
CH
3
ibid.
ibid.
C H
6
5
CH
3
O
O
O
5
5
5
j
k
l
OH
OH
OH
CH
2
CH
5
3
ibid.
ibid.
C
C
H
H
6
6
5
HO
2
.2. DPPH-Scavenging Activity
The DPPH free radical exhibits a strong absorption maximum at 517 nm, which is purple in color.
Upon the addition of an antioxidant a reduction of DPPH radical to DPPH-H occurs and the color turns
from purple to yellow. The method used in this work determines the antioxidant capacity by measuring
the remaining amount of DPPH after 30 min of incubation. This DPPH-scavenging activity was
compared to ascorbic acid as a standard compound.
As it can be seen from Table 1, the thiadiazine derivatives are excellent antioxidants. The best ones
are compounds 5b, 5f, 5j and 4b, possessing DPPH scavenging activity in a range of ascorbic acid. All
of these compounds incorporate a -OH group in their structure—5f, 5j and 4b in position 4 of
coumarin core and 5b on the coumarin core linked to the 1,3,4-thaidiazine moiety of the compound.
Compounds 5g, 5e, 5h and 5l also exhibit very high DPPH scavenging activity, in a range 80%–90%,
but lower than that of the compounds mentioned above. From their structure it is evident that they all,
except for compound 5e, bear two hydroxyl groups in their structure. Compounds 5i and 5k exhibit
DPPH scavenging activity >70%, and the other compounds did not show significant activity. Since
these compounds are complex structures, most of them even containing two coumarin cores, their
activity is probably highly dependent on their stereochemistry.

Molecules 2014, 19
Table 1. DPPH scavenging activity and iron chelating activity of thiadiazine derivatives .
1167
a
%
DPPH scavenging
activity
% Chelating
Compound
activity
b
ascorbic acid
85.2 ± 6.1
-
-
c
EDTA
90.9 ± 7.22
4
4
5
5
5
a
b
a
b
c
17.2 ± 0.59
90.0 ± 1.24
36.0 ± 2.07
94.4 ± 1.73
17.7 ± 1.26
33.3 ± 0.27
80.7 ± 1.61
94.1 ± 1.35
82.3 ± 0.70
80.6 ± 0.98
79.7 ± 1.29
90.0 ± 1.08
77.2 ± 0.93
0.0
15.1 ± 4.27
-
0
0,0
5
d
-
5
e
0.0
5
f
0.0
5
5
g
h
0.0
62.5 ± 2.25
5.3 ± 0.56
53.5
5
5
i
j
5
k
5.18 ± 0.81
10.2 ± 0.42
5
l
80.2 ± 0.44
a
b
data are means ± standard deviation of three replicates; ascorbic acid was used as standard in DPPH
c
scavenging activity determination; EDTA was used as standard in iron chelating activity determination.
2
.3. Iron Chelating Activity
2
+
This assay is based upon the complex formation between ferrozine and Fe , which is red in color.
In the presence of chelating agents, the complex formation is disrupted and the red color of the
complex is decreased, which can be monitored by spectrophotometric measurement of the color
reduction at 562 nm.
Iron is known to be one of the most important lipid oxidation prooxidants due to its high reactivity
and may participate in hydroxyl radical generating Fenton type reactions. Thus effective ferrous ion
2+
chelators may be important in protection against oxidative damage by removing ferrous ion (Fe ) [22].
The most potent iron chelators amongst thiadiazine derivatives investigated in our research are shown
to be compounds 5h and 5j, while other compounds did not show significant activity.
2.4. Reducing Power
3
+
2+
This assay is based on the reduction of Fe from potassium ferricyanide to Fe (potassium
ferrocyanide), which then reacts with ferric chloride to form a ferric/ferrous complex with an
absorption maximum at 700 nm [23,24]. A higher absorbance indicates better reducing power of the
compound. Compounds possessing the highest reducing power in this assay were 4b, 5g, 5d, but their
activity was not comparable to that of ascorbic acid, which was used as standard compound (Figure 1).

Molecules 2014, 19
1168
Figure 1. Reducing power of tested thiadiazine derivatives.
2
.5. Antifungal Activity
The antifungal activity of thiadiazine derivatives strongly depends on the fungal species.
The best antifungal agent on A. flavus (Table 2) was proven to be compound 5e, while observing the
overall antifungal activity results, compared to other molds A. flavus was the most resistant to the
tested compounds.
Table 2. Minimal inhibitory concentration for 50% cell death for the tested
thiadiazine derivatives.
MIC50
Compound A. flavus A. ochraceus F. graminearum F. verticillioides
4
5
a
a
/
1
0.01
0.1
0.1
0.01
0.1
0.1
0.1
0.1
0.1
0.1
0.01
0.1
0.1
0.1
0.1
0.1
1
5
b
/
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
5
c
0.1
0.1
0.01
0.1
1
5
d
5
e
0.1
0.1
0.01
1
5
f
5
g
5
i
1
5
j
1
0.1
Compounds 4a and 5c were the best antifungals on A. ochraceus, and compound 5g showed the
best antifungal activity on F. verticillioides. F. graminearum was the most susceptible mold to the
tested compounds and most of the compounds showed high antifungal activity towards this mold. This
kind of behavior was also observed in our previous research [15], which could be important since this
mold was found to be a potential threat in production of malt barley and wheat in our region [25].
3. Experimental
3.1. General
Melting points were determined on a capillary melting point apparatus (Electrotermal, Rochford,
Great Britain) and are uncorrected. Thin-layer chromatography was performed with fluorescent silica

Molecules 2014, 19
1169
gel plates HF254 (Merck, Darmstadt, Germany), which were checked under UV (254 and 365 nm) light,
using benzene:acetone:acetic acid (8:1:1) as a solvent. The elemental analysis for C, H and N was done
−1
on a Analyzer 2440 (Perkin–Elmer, Boston, MA, USA). Infrared spectra (νmax/cm ) were recorded on
1
a FT-IR 3303 spectrometer (Beckman Instruments, Inc., Irvine, CA, USA), using KBr disks. H-NMR
spectra were recorded on a Bruker Avance 600 MHz NMR Spectrometer (Bruker Biospin GmbH,
Rheinstetten, Germany) at 293 K in DMSO-d6. The MS spectra were recorded on LC/MS/MS API
2
000 (Applied Biosystems/MDS SCIEX, Foster City, CA, USA). The absorbance was measured on a
UV visible spectrophotometer Heliosγ, (Thermo Spectronic, Cambridge, UK). Microplates were read
on a Sunrise absorbance reader (Tecan Group Ltd., Männedorf, Switzerland). Incubation was carried in
an Aqualytic AL 500-8 incubator (Aqualytic, Dortmund, Germany).
3.2. Synthesis
3
.2.1. General Procedure for the Preparation of 1,4-Disubstituted Thiosemicarbazides 3 [21]
To a solution of corresponding carbohydrazide (1 mmol) in ethanol (5–10 mL), alkyl/aryl
isothiocyanate (1 mmol) and sodium hydroxide (40 mg, 1 mmol, as a 2N solution) were added. The
mixture was refluxed for 2–4 h. The precipitate was filtered and crystallized from ethanol/water, to
give compounds 3a–g in 65%–85% yield.
3
.2.2. Synthesis of 5-(2-Oxo-2H-chromen-3-yl)-6H-1,3,4-thiadizin-2-aminium Bromide (4a)
A mixture of 3-α-bromoacetylcoumarin (1a, 0.01 mol) and thiosemicarbazide (0.01 mol) was
suspended in ethanol (35 mL) at 0 °C. The mixture was allowed to warm up to room temperature
overnight under stirring. The resulting slurry was cooled to −18 °C and the precipitate was collected by
filtration, washed with cold ethanol, and dried under vacuum. The yellow solid was again suspended in
ethanol (25 mL) which contained 48% aqueous hydrobromic acid (1 mL). The reaction mixture was heated
to reflux for 45 min. and then allowed to cool down overnight to room temperature. The precipitate was
filtered, recrystallized from ethanol, and dried over phosphorus pentoxide under high-vacuum at 40 °C.
Yellow crystals (70% yield); mp 240 °C, R
f
= 0.52; FT-IR νmax 3426, 3183, 1714, 1696, 1617, 1557,
−1
1
1
451, 1367, 1250, 1179, 965, 841, 756 and 675 cm ; H-NMR (DMSO-d
6
) δ (ppm): 3.91 (s, 2H,
+
CH ), 4.80 (s,2H, NH /-N H ), 7.02–7.27 (m, 4H, coum.), 7.45 (s, 1H, H-4 coum); MS m/z 339.0
2
2
3
+
3 2
[M-H] , (M = 340.2); Anal. Calcd. for C12H10BrN O S: C, 42.37; H, 2.96; N, 12.35%; Found: C,
4
2.39; H, 2.94; N, 12.37%.
3
.2.3. Synthesis of 3-(2-Amino-6H-1,3,4-thiadiazin-5-yl)-4-hydroxy-2H-chromen-2-one (4b)
Compound 4b was prepared by heating of 3-α-bromoacetylcoumarin (1b, 0.01 mol) with
thiosemicarbazide (0.01 mol) in ethanol (40 mL) with a few drops of hydrochloric acid for 20 min. The
reaction mixture was cooled and adjusted to pH 8-9 by adding a dilute solution of ammonia. A brown
colored precipitate was formed and filtered off, then recrystallized from 30% ethanol, and dried under
high-vacuum at 40 °C over phosphorus pentoxide. Pale brown crystals (75% yield); mp 210 °C;
−1
f
R = 0.66; FT-IR νmax 3416, 3143, 1687, 1656, 1571, 1557, 1410, 1257, 1110, 965, 891, 758 and 678 cm .
1
6 2 2
H-NMR (DMSO-d ) δ (ppm): 3.91 (s, 2H, CH ), 4.2 (s, 2H, NH ), 7.02–7.27 (m, 4H, coum.), 7.45 (s,

Molecules 2014, 19
1170
+
1
H, H-4 coum.), 14.68 (s, 1H, -OH); MS m/z 274.01[M–H] , (M = 275.04); Anal. Calcd. for
S: C, 52.36; H, 3.30; N, 15.26%; Found: C, 52.39; H, 3.29; N, 15.24%.
12 9 3 3
C H N O
3
.2.4. General Procedure for Preparation 2,4,5-Trisubstituted 4H-1,3,4- thiadiazines 5a–l
Compounds 5a–l were prepared by heating 3-α-bromoacetylcoumarin 1a,b (0.01 mol) with the
corresponding 1,4-disubstituted thiosemicarbazide 3a–g (0.01 mol) in ethanol (40 mL) with a few
drops of hydrochloric acid for 40 min. The reaction mixture was cooled and made alkaline with a diluted
solution of ammonia to adjust the pH to 8–9. The precipitate formed was filtered off, recrystallized from
30% water-ethanol, and dried under high-vacuum at 40 °C over phosphorus pentoxide.
3
-(4-(2-(7-Hydroxy-2-oxo-2H-chromen-4yl)acetyl)-2-(methylamino)-4H-1,3,4-thiadiazin-5-yl)-2H-
chromen-2-one (5a). Brown crystals (48% yield); mp 262 °C; R = 0.56; FT-IR ν 3227, 2979, 1710,
f
max
−1 1
1
682, 1607, 1560, 1478, 1395, 1330, 1209, 1139, 959, 843,759 and 639 cm ; H NMR (DMSO-d
6
) δ
(
ppm): 2.72 (d, 3H, CH ), 3.85 (s, 2H, CH ), 4.80 (q, 1H, NH), 5.56 (s, 1H, thiadiaz.), 6.04–7.37 (m,
3
2
+
9
6
H, coum. 10.86 (s, 1H, OH), MS m/z 475.1[M–H] , (M=475.5); Anal. Calcd. for C24
H
17
N O
3 6
S: C,
0.63; H, 3.60; N, 8.84; Found: C, 60.59; H, 3.60; N, 8.81%.
3
-(2-(Ethylamino)-4-(2-(7-hydroxy-2-oxo-2H-chromen-4yl)acetyl)-4H-1,3,4-thiadiazin-5-yl)-2H-
chromen-2-one (5b). Brown crystals (52% yield); mp 194 °C; R
f
= 0.54; FT-IR νmax 3492, 3134, 2978,
−1 1
1
6
708, 1673, 1607, 1566, 1552, 1478, 1392, 1210, 1062, 854, 759 and 639 cm ; H-NMR (DMSO-d )
δ (ppm): 2.42 (t, 3H, CH
3
), 2.69 (q, 2H, CH
2
), 2.85 (s, 2H, CH
2
), 4.90 (t, 1H, NH), 5.0 (s, 1H, OH), 5.60
+
(s, 1H, thiadiaz.), 6.04–7.37 (m, 9H, coum.), 11.20 (s, 1H, OH); MS m/z 489.1[M-H] , (M = 489.5);
19 3 6
Anal. Calcd. for C25H N O S: C, 61.34; H, 3.91; N, 8.58; Found: C, 61.32; H, 3.89; N, 8.53%.
3
-(4-(2-(7-Hydroxy-2-oxo-2H-chromen-4yl)acetyl)-2-(phenylamino)-4H-1,3,4-thiadiazin-5-yl)-2H-
chromen-2-one (5c). Pale grey crystals (62% yield); mp 252 °C; R = 0.51; FT-IR νmax 3350, 3231,
052, 1733, 1691, 1608, 1570, 1457, 1395, 1310, 1136, 1095, 996, 841, 759 and 693 cm ; H-NMR
DMSO-d ) δ (ppm): 2.85 (s, 2H, CH ), 4.6 (s, 1H, NH), 5.67 (s, 1H, thiadiaz.), 6.04–7.37 (m, 14H,
f
−1 1
3
(
6
2
+
coum. and arom.), 10.52 (s, 1H, OH); MS m/z 537.1[M-H] , (M = 537.5); Anal. Calcd. for
S: C, 64.80; H, 3.56; N, 7.82; Found: C, 64.79; H, 3.52; N, 7.80%.
29 19 3 6
C H N O
7
2
2
-(2-(2-(Methylamino)-5-(2-oxo-2H-chromen-2-oxoethoxy)-3-yl)-4H-1,3,4-thiadiazin-4-yl)-4-methyl-
H-chromen-2-one (5d). Pale grey crystals (72% yield); mp 205 °C; R = 0.51; FT-IR ν 3435, 3075,
f
max
970, 2830, 1715, 1677, 1610, 1558, 1449, 1391, 1284, 1213, 1183, 1080, 982, 852, 765 and 684
−1 1
cm ; H-NMR (DMSO-d
6
) δ (ppm): 1.71 (s, 3H, CH
3
), 2.74 (d, 3H, NCH
3
), 4.83 (s, 2H, OCH
2
), 5.1
+
(
q, 1H, NH), 5.64 (s, 1H, thiadiaz.), 5.90–7.28 (m, 8H, coum.); MS m/z 488.1[M-H] , (M = 489.5);
19 3 6
Anal. Calcd. for C25H N O S: C, 61.34; H, 3.91; N, 8.58; Found: C, 61.32; H, 3.90; N, 8.53%.
7
2
3
-(2-(2-(Ethylamino)-5-(2-oxo-2H-chromen-3-yl)-4H-1,3,4-thiadiazin-4-yl)-2-oxoethoxy)-4-methyl-
H-chromen-2-one (5e). Pale grey crystals (73% yield); mp 256 °C; R = 0.52; FT-IR νmax 3434, 3174,
029, 2734, 1765, 1715, 1607, 1556, 1449, 1391, 1277, 1153, 1072, 852, 759 and 681 cm ; H-NMR
) δ (ppm): 1.20 (t, 3H, CH ), 1.74 (s, 3H, CH ), 2.69 (q, 2H, CH ), 4.83 (s, 2H, OCH ),
f
−1 1
(DMSO-d
6
3
3
2
2

Molecules 2014, 19
1171
+
5
.21(q, 1H, NH), 5.60 (s, 1H, thiadiaz.), 5.90–7.28 (m, 9H, coum.); MS m/z 502.1[M-H] , (M =
03.5); Anal. Calcd. for C26 S: C, 62.02; H, 4.20; N, 8.35; Found: C, 61.92; H, 4.22; N, 8.30%.
5
21 3 6
H N O
4
5
3
-Hydroxy-3-(4-(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetyl)-2-(methylamino)-4H-1,3,4-thiadiazin-
-yl)-2H-chromen-2-one (5f). Pale yellow crystals (68% yield); mp 222 °C; R = 0.44; FT-IR ν
f
max
−1 1
449, 3337, 1707, 1692, 1613, 1560, 1448, 1395, 1269, 1139, 1032, 853, 759 and 693 cm ; H-NMR
) δ (ppm): 2.70 (d, 3H, CH ), 2.85 (s, 2H, CH ), 4.89 (q, 1H, NH), 5.65 (s, 1H, thiadi.),
(DMSO-d
6
3
2
6
.04–7.28 (m, 8H, coum.), 10.80 (s, 1H, OH-7 coum), 15.0 (s, 1H, OH-4 coum); MS m/z 490.1
+
[M-H] , (M = 491.5); Anal. Calcd. for C H N O S: C, 58.65; H, 3.49; N, 8.55; Found: C, 58.62; H,
2
4
17
3
7
3
.52; N, 8.53%.
4
-Hydroxy-3-(4-(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetyl)-2-(ethylamino)-4H-1,3,4-thiadiazin-5-
= 0.53; FT-IR νmax 3447,
yl)-2H-chromen-2-one (5g). Pale yellow crystals (72% yield); mp 261 °C; R
413, 2986, 1708, 1688, 1615, 1562, 1550, 1392, 1269, 1139, 1032, 829, 758 and 690 cm ; H-NMR
DMSO-d ) δ (ppm): 1.30 (t, 3H, CH ), 2.69 (q, 2H, CH ), 2.85 (s, 2H, CH ), 5.21 (t, 1H, NH), 5.63 (s,
f
−1 1
3
(
6
3
2
2
1
5
5
H, thiadiaz.), 6-04-7.27 (m, 8H, coum.), 10.86 (s, 1H, OH-7 coum), 15.1 (s,1H, OH-4 coum); MS m/z
+
04.1[M-H] , (M = 505.5); Anal. Calcd. for C25
9.42; H, 3.82; N, 8.34%.
19 3 7
H N O S: C, 59.40; H, 3.79; N, 8.31; Found: C,
4
5
3
-Hydroxy-3-(4-(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetyl)-2-(phenylamino)-4H-1,3,4-thiadiazin-
-yl)-2H-chromen-2-one (5h). Pale yellow crystals (85% yield); mp 289 °C; R = 0.65; FT-IR νmax
418, 3184, 1703, 1608, 1548, 1511, 1393, 1322, 1269, 1139, 1034, 852, 758 and 678 cm ; H-NMR
f
−1 1
(
DMSO-d
6
) δ (ppm): 2.85 (s, 2H, CH
2
), 5.62 (s, 1H, thiadiaz.), 6.04–7.27 (m, 13H, coum. and arom.),
+
8
.11 (s, 1H, NH), 15.0 (s, 1H, OH); MS m/z 552.1[M-H] , (M = 553.5); Anal. Calcd. for C29
H
19
N
3
O
7
S:
C, 62.92; H, 3.46; N, 7.59; Found: C, 62.89; H, 3.42; N, 7.53%.
4
-Hydroxy-3-(4-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)-2-(methylamino)-4H-1,3,4-thia-
f
diazin-5-yl)-2H-chromen-2-one (5i). Pale yellow crystals (68% yield); mp 222–223 °C; R = 0.67;
FT-IR νmax 3496, 2674, 1715, 1696, 1614, 1554, 1540, 1394, 1266, 1211, 1148, 867, 759 and 682
−1 1
cm ; H NMR (DMSO-d
6 3 3 2
) δ (ppm): 1.71 (s, 3H, CH ), 2.73 (d, 2H, CH -N), 4.83 (s, 2H, OCH ), 5.20
(
q, 1H, NH), 5.66 (s, 1H, thiadi.), 5.90–7.28 (m, 8H, coum.), 10.86 (s, 1H, OH-7 coum), 14.6 (s, 1H,
+
OH-4 coum), MS m/z 505.1[M-H] , (M = 505.5); Anal. Calcd. for C H N O S: C, 59.40; H, 3.79; N,
2
5
19
3
7
8
.31; Found: C, 59.32; H, 3.81; N, 8.30%.
4
5
3
6
-Hydroxy-3-(4-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)-2-(ethylamino)-4H-1,3,4-thiadiazin-
-yl)-2H-chromen-2-one (5j). Pale yellow crystals (78 % yield); mp 226 °C; R = 0.69; FT-IR νmax
433, 3169, 2707, 1723, 1676, 1611, 1553, 1492, 1391, 1267, 1210, 1143, 1070, 988, 841, 758 and
f
−1 1
81 cm ; H-NMR (DMSO-d
), 4.83 (s, 2H, OCH
21 3 7
5.0 (s, 1H, OH-4 coum); MS m/z 518.1[M-H] , (M = 519.5); Anal. Calcd. for C26H N O S: C, 60.11;
6
3 3
) δ (ppm): 1.24 (s, 3H, CH ), 1.74 (t, 3H, CH ), 2.0 (s, 1H, NH), 2.69
(q, 2H, CH
2
2
), 5.31 (s, 1H, NH), 5.67 (s, 1H, thiadi.), 5.90–7.28 (m, 8H, coum.),
+
1
H, 4.07; N, 8.09; Found: C, 60.12; H, 4.02; N, 8.10%.

Molecules 2014, 19
-Hydroxy-3-(4-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)-2-(phenylamino)-4H-1,3,4-thia-
1172
4
diazin-5-yl)-2H-chromen-2-one (5k). Pale yellow crystals (52% yield); mp 237–239 ° C; R = 0.45;
f
FT-IR νmax 3466, 3424, 3064, 2920, 1707, 1651, 1612, 1543, 1496, 1448, 1387, 1266, 1151, 1075, 842,
−1 1
7
55 and 697 cm ; H-NMR (DMSO-d ) δ (ppm): 1.71 (s, 3H, CH ), 4.83 (s, 2H, OCH ), 5.0 (q, 1H,
6 3 2
NH), 5.60 (s, 1H, thiadiaz.), 5.90–7.27 (m, 13H, coum. and arom.), 15.1 (s, 1H, OH); MS m/z
+
5
66.1[M-H] , (M = 567.6); Anal. Calcd. for C30
3.52; H, 3.62; N, 7.32%.
21 3 7
H N O S: C, 63.49; H, 3.73; N, 7.40; Found: C,
6
4
-Hydroxy-3-(4-(4-hydroxybenzoyl)-2-(phenylamino)-4H-1,3,4-thiadiazin-5-yl)-2H-chromen-2-one
3445, 3224, 1753, 1693,
(
5l). Ppale yellow crystals (72% yield); mp 285 °C; R = 0.69; FT-IR ν
f
max
−1 1
1612, 1560, 1532, 1438, 1258, 1103, 963, 845, 756 and 668 cm ; H-NMR (DMSO-d ) δ (ppm): 5.20
6
(
s, 1H, NH), 5.66 (s, 1H, thiadi.), 6.46–7.78 (m, 13H, coum. and arom.), 8.90 (s, 1H, OH), 15.0 (s, 1H,
+
OH-4 coum.); MS m/z 469.81[M-H] , (M = 471.5); Anal. Calcd. for C25
H
17
N
3
O
5
S: C, 63.69; H, 3.63;
N, 8.91; Found: C, 63.62; H, 3.42; N, 8.70%.
3
.3. DPPH-Scavenging Activity
Determination of antioxidant activity was performed according to the procedure described in the
literature [26,27] with some modifications described in our previous work [15]. Dimethyl sulfoxide
DMSO) was used as a solvent [28], due to the low solubility of synthesized compounds in ethanol and
(
methanol. A DMSO solution (750 µL) of the corresponding thiadiazine derivative (0.2 mM) was added
to a DMSO solution of DPPH radical (0.2 mM), so that the final concentration of DPPH radical and
the synthesized compound in a solution was 0.1 mM. The mixture was shaken and allowed to stand at
room temperature. After 30 min the absorbance at 517 nm was determined and the scavenging activity
was calculated according to the formula below:
ꢍ ꢏ ꢍ ꢑ ꢍ
ꢎ
ꢐ
ꢒ
%ꢀꢁꢂꢂꢃꢀꢄꢅꢆꢇꢈꢉꢊꢋꢉꢊ ꢌ
ꢓ 100
(
1)
ꢍꢎ
where A –absorbance of 0.1 mM DMSO solution of DPPH radical at 517 nm; A – absorbance of
b
s
0
.1 mM DMSO solution of test compound at 517 nm; A
m
– absorbance of DMSO mixture of test com-
pound and DPPH radical at 517 nm. Ascorbic acid (AA) was used as a reference compound.
3
.4. Iron Chelating Activity
2
+
The chelating activity of thiadiazine derivatives for ferrous ions Fe was measured according to the
literature method [29] with some modifications. FeCl (2 mM, 25 µL) were added to 2 mM
methanol/DMSO solution (1 mL, 4:1) of the compound investigated. After 30 s, ferrozine
5 mM, 50 µL) was added. Samples were incubated at room temperature for 10 min and the
2
(
2
+
absorbance of the complex formed between Fe and ferrozine was measured at 562 nm. Metal
chelating efficiency of samples was compared to the chelating activity of EDTA disodium salt. The
2
+
chelating activity of the extract for Fe was calculated as:
ꢍꢘ ꢑ ꢍꢙ
ꢍꢘ
%ꢀꢅꢔꢈꢕꢆꢖꢋꢉꢊ ꢗꢆꢖꢈ ꢌ
ꢓ 100
(
2)

Molecules 2014, 19
1173
A – absorbance of the control (blank, without samples) at 562 nm; A – absorbance in the presence of
0
1
the methanol/DMSO sample solution at 562 nm.
3.5. Reducing Power
The reducing power of thiadiazine derivatives was determined according to the method [30]. An
aliquot of the 0.2 mM thiadiazine derivatives in DMSO (250 µL), were mixed with 0.2 M phosphate
buffer (250 µL, pH 6.6) and potassium ferricyanide (K Fe(CN) , 250 µL, 1% w/v). The mixture was
3
6
incubated at 50 °C for 20 min, followed by the addition of trichloroacetic acid (TCA, 250 µL, 10%
w/v), centrifuged for 10 min at 2000 ×g. A portion of the upper layer of the solution (300 µL) was
mixed with distilled water (300 µL) and FeCl (0.5 mL, 0.1% w/v). The absorbance was measured at
3
7
3
3
00 nm. The higher absorbance indicates stronger reducing power of compound.
.6. Antifungal Activity
.6.1. General
Broth microdilution assays were performed in accordance with the guidelines detailed in CLSI
document M38-A [31].
3
.6.2. Tested Fungi
The fungi used in this experiment (Aspergillus flavus (NRRL 3251); Aspergillus ochraceus (CBS
89.68), Fusarium graminearum (CBS 110.250) and Fusarium verticillioides (CBS 119.825)) are the
5
major producers of mycotoxins and food contaminants [32].
3
.6.3. Preparation of Inoculum
The inocula were obtained from cultures that were grown on potato dextrose agar (PDA) slants.
Aspergillus strains were grown at 35 °C for 7 days, and Fusarium strains were grown at 35 °C for
days, followed by 7 days at 25 °C. Fungal spores were harvested by pouring 5 ml of sterile saline
2
onto the culture slants and scraping the surface with an inoculation loop. The number of spores in the
6
stock suspension was adjusted to 10 spores/mL, using a Bürker–Türk counting chamber
(Haemocytometer). Turbidity of each stock suspension was checked with a 0.5 McFarland standard
read at 530 nm. Working 2× spore suspension was prepared by diluting 200 µL of stock suspension
into 10 mL of RPMI 1640 medium [31].
3
.6.4. Medium
Antifungal susceptibility testing was performed, using RPMI 1640 medium buffered with 0.164 M
MOPS (34.53 g/L) and adjusted to pH 7.0 with (1 M) NaOH, as recommended by the National
Committee for Clinical Laboratory Standards. Medium was sterilized by filtration using a 0.45 µm filter.

Molecules 2014, 19
.6.5. Drug Dilutions
The drug dilutions were prepared following the additive two fold drug dilution scheme described in
1174
3
NCCLS M28-A method for water-insoluble compounds [31]. Briefly, stock drug solutions were first
diluted by 20× of the final concentrations in 100% dimethyl sulfoxide (DMSO), followed by further
dilutions to the final drug concentrations, 10, 1, 0.1 and 0.01 µg/mL. All drug dilutions were sterilized
by filtration using a 0.45 µm filter. Sterile polypropylene microtitre plates (96 U-shaped wells; Brand,
Wertheim, Germany) were used in the microdilution test. Rows 2–11 contained the series of drug
dilutions in 100 µL volumes and 100 µL of the 2× spore suspension. Row 1 contained 200 µL of
uninoculated, drug-free medium and served as the sterility control, while row 12 contained 100 µL of
drug-free medium and 100 µL of inoculum and served as growth control (the final volume in each well
was 200 µL).
3
.6.6. Incubation and MIC Determination
Following inoculation, all plates were incubated at 35 °C in an atmospheric incubator. After 48 h of
incubation, plates were read on a microplate reader at 450 nm. Minimal inhibitory concentration for
0% cell death (MIC 50) was defined as the lowest concentration reducing the optical density by 50%
at 450 nm compared with growth control [33,34].
5
4
. Conclusions
In this paper, and as a part of our ongoing research on biologically active heterocyclic compounds,
some thiadiazine derivatives were successfully synthesized and characterized by various spectral
methods. Compounds were subjected to antioxidant (DPPH scavenging, iron chelating and reducing
power) and antifungal investigation on four mycotoxicogenic fungi, A. flavus, A. ochraceus,
F. graminearum and F. verticillioides namely. The new thiadiazine derivatives were proven to possess
an excellent antioxidant activity, comparable to ascorbic acid, while not showing reducing power
activity at the same time. Also, most of the compounds were proven to possess potent antifungal
activity towards all the tested fungi. Compounds showing the best activity have been selected for
further investigation employing some in vivo models.
Conflicts of Interest
The authors declare no conflict of interest.
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©
2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(