Mono- and bis-dipicolinic acid heterocyclic derivatives - Thiosemicarbazides, triazoles, oxadiazoles and thiazolidinones as antifungal and antioxidant agents

Article abstract of DOI:10.1515/hc-2016-0078

A series of dipicolinic acid derivatives was synthesized and investigated for antimicrobial and antioxidant activity. Mono and bis derivatives of ethyl dipicolinate were utilized as starting materials for synthesis of mono- and bis-hydrazides. Thiosemicarbazides were obtained by reaction of hydrazides with isothiocyanates and cyclized into triazoles, thiadiazoles, oxadiazoles and thiazolidinones. Some of these products, especially those incorporating a thiazolidinone moiety in their structure, are excellent antioxidants, DPPH scavengers and antifungal agents.

Full text of DOI:10.1515/hc-2016-0078

Heterocycl. Commun. 2017; aop
Maja Molnar, Valentina Pavić*, Bojan Šarkanj, Milan Čačić, Dubravka Vuković
aMndoJenlenoa-Klaennkadr bis-dipicolinic acid heterocyclic
derivatives – thiosemicarbazides, triazoles,
oxadiazoles and thiazolidinones as antifungal
and antioxidant agents
DOI 10.1515/hc-2016-0078
reductase from the copper-dependent inactivation [6].
A structural combination of DPA core with other hetero-
cyclic compounds has already proven to be an excellent
tool for gaining antimicrobial and antioxidant activity [7,
8]. In this work, thiadiazole, triazole, thiazolidinone and
oxadiazole moieties were combined with the DPA core in
order to achieve the expected potent antimicrobial and/or
antioxidant activity. Diverse biological and/or antioxidant
properties of 1,3,4-thiadiazoles [9–14], triazoles [11, 13–15],
thiazolidinones [16–21], and oxadiazoles [22] have been
documented. Only a slight change in structural charac-
teristics can have a great effect on antifungal and antioxi-
dant activity [4, 17]. To date, derivatives of dipicolinic acid
were described by Milway in 2003 [23] and in our previous
Received May 21, 2016; accepted October 11, 2016
Abstract: A series of dipicolinic acid derivatives was
synthesized and investigated for antimicrobial and anti-
oxidant activity. Mono and bis derivatives of ethyl dipi-
colinate were utilized as starting materials for synthesis
of mono- and bis-hydrazides. Thiosemicarbazides were
obtained by reaction of hydrazides with isothiocyanates
and cyclized into triazoles, thiadiazoles, oxadiazoles and
thiazolidinones. Some of these products, especially those
incorporating a thiazolidinone moiety in their structure,
are excellent antioxidants, DPPH scavengers and antifun-
gal agents.
Keywords: antifungal; antioxidant activity; dipicolinic work on Schiff bases [4].
acid; oxadiazole; thiadiazole; thiazolidinone; triazole.
Results and discussion
The synthesis of the target compounds was carried out as
Introduction
Dipicolinic acid (DPA) is a unique constituent of outlined in Schemes 1–3. All products were purified and
endospores of Bacillus and Clostridium genuses [1] and is characterized by TLC, MS, 1H NMR and elemental analysis.
also produced and secreted by certain Penicillium strains Preparation of starting compounds 6-methoxycarbonyl-
and by several entomopathogenic fungi [2]. DPA and its 2-pyridinecarboxylic acid hydrazide (1) and 2,6-pyridin-
derivatives show various biological activities includ- edicarboxylic acid bis-hydrazide (2) was described in our
ing significant antimicrobial [3] and antioxidant prop- previous work [4]. Thiosemicarbazides were prepared by
erties [4]. As a strong complexing agent DPA is a potent refluxing mono-hydrazide or bis-hydrazide with the corre-
metal-chelator functioning as a multidentate ligand [5] sponding isothiocyanate in 1ꢀ:ꢀ1 ratio for mono derivatives
that inhibits lipid peroxidation and protects glutathione and 1ꢀ:ꢀ2 ratio for bis derivatives in ethanol (Scheme 1).
1
Thiosemicarbazides exhibit characteristic H NMR
peaks for NH groups and the difference between mono
and bis derivatives is clearly visible in the presence of
the characteristic peak at δ 3.93 corresponding to –OCH3
group of mono derivatives. Conventional cyclization of
thiosemicarbazides yields heterocyclic compounds such
as thiadiazole, oxadiazole, triazole and thiazolidinone.
Thus, cyclization of thiosemicarbazides in the presence of
an excess of sulfuric acid gave thiadiazoles. This reaction
was succesfully performed for bis-thiosemicarbazides
*Corresponding author: Valentina Pavić, Josip Juraj Strossmayer
University of Osijek, Department of Biology, Croatia,
e-mail: vpavic@biologija.unios.hr.
http://orcid.org/0000-0001-5369-1755
Maja Molnar, Bojan Šarkanj, Milan Čačić and Jelena Klenkar: Josip
Juraj Strossmayer University of Osijek, Faculty of Food Technology
Osijek, Croatia
Dubravka Vuković: Microbiology Service of the Public Health,
Institute of Osijek-Baranja County, Croatia
Brought to you by | University of Sussex Library
Authenticated
Download Date | 1/18/17 9:42 PM

ꢁꢁꢁꢂ
ꢀM. Molnar et al.: Mono- and bis-dipicolinic acid heterocyclic derivatives
2ꢀ
H3CO
OCH3
N
O
O
H2NNH2, EtOH, rt,18 h
H2NNH2, EtOH, rt, 18 h
H
H
N
H
N
N
H3CO
H2N
N
NH2
N
NH2
O
O
2
O
O
1
∆
∆
R-NCS, EtOH, , 3 h
R-NCS, EtOH, , 3 h
S
H
S
S
H
N
H
N
a: R = Me
H3CO
N
R
R
R
b
: R = Et
c: R = Ph
N
N
H
N
H
N
N
N
N
H
N
H
H
H
O
O
O
O
3a–c
4a–c
Scheme 1ꢁSynthetic route to compounds 1–4.
only, since no expected products for mono derivatives indicating that they exist mainly in a thione form rather
were obtained, due to hydrolysis of the ester group by than with a thiol function. Only disubstituted derivatives
a strong acid. Bis derivatives of these compounds were of triazoles were synthesized [24], since the mono deriva-
published by Foroughifar and co-workers in 2014 [24]. tives underwent hydrolysis of the methoxycarbonyl
Upon treatment of thiosemicarbazides with I2/KI system group. Refluxing monosubstituted and disubstituted
in NaOH solution, oxadiazoles were formed showing NH thiosemicarbazides with ethyl bromoacetate in ethanol
1
signals in the H NMR spectra, in addition to signals for in the presence of sodium acetate yielded correspond-
groups characteristic for each substituent depending ing thiazolidinones. These compounds show a charac-
on isothiocyanate used in thiosemicarbazide synthesis. teristic signal for the CH2 group of the thiazolidinone,
Oxadiazole synthesis using I2/KI system was success- which is not visible in the spectra of the corresponding
ful for only two derivatives, monosubstituted with the thiosemicarbazides.
methyl and disubstituted with phenyl groups. Treatment
The synthesized compounds were assayed for
of thiosemicarbazides with NaOH resulted in cyclization 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scaveng-
to triazoles. The synthesized triazoles show character- ing activity (Figure 1). As can be seen, compound 3c is
istic peaks for NH group (triazole ring) at δ 14.15–14.27, an excellent scavenger, with activity comparable to that
of ascorbic acid. The overall data show a predominant
antioxidant activity of thiosemicarbazides compared to
other synthesized derivatives of DPA. Also the influence of
H3CO
N
N
different substituents is obvious, since in almost all com-
pounds the phenyl substitution results in a better DPPH
radical scavenging activity in comparison to the alkyl sub-
stitution. The same trend has also been observed in our
previous work [4, 17, 25]. Triazoles 11a–c show moderate
DPPH scavenging activity that are better than activities of
thiazolidinones, oxadiazoles and thiadiazoles.
N
Conc. H2SO4, rt, 2.5 h
O
O
S
5a–c
HN
R
R
H3CO
N
N
N
∆
I2/KI, NaOH, , 4 h
O
6a
HN
3a–c
2N NaOH, ∆, 1.5 h, HCl
Antibacterial properties of the synthesized com-
pounds were studied against four bacteria, namely two
Gram-positive (Bacillus subtilis and Staphylococcus
aureus) and two Gram-negative (Escherichia coli and Pseu-
domonas aeruginosa) species. None of the compounds
shows noticable antimicrobial activity comparable to anti-
biotic amikacin, an aminoglycoside that is highly active
against most Gram-negative bacteria including many
H3CO
N
N
N
O
N
R
O
7a–c
SH
R
N
BrCH2COOEt, CH3COONa
H
a: R = Me
b: R = Et
c: R = Ph
H3CO
N
N
N
EtOH, ∆, 7 h
S
O
8a–c
O
Scheme 2ꢁSynthetic route to compounds 6a and 8a–c.
Brought to you by | University of Sussex Library
Authenticated
Download Date | 1/18/17 9:42 PM

ꢂꢁꢁꢁ
M. Molnar et al.: Mono- and bis-dipicolinic acid heterocyclic derivativesꢀ
ꢀ3
N
N
Conc.H2SO4, rt, 2.5 h
N
N
N
S
S
9b
NH
HN
R
R
R
R
N
N
N
I2/KI, NaOH, ∆, 4 h
N
N
O
N
O
N
10c
4a–c
NH
HN
N
N
N
N
N
N
N
2N NaOH, ∆, 1.5 h, HCl
N
HN
S
N
H
N
N
R
R
R
R
S
HS
SH
11a–c
R
R
N
N
S
BrCH2COOEt, CH3COONa
H
N
a: R = Me
b: R = Et
c: R = Ph
N
N
∆
N
EtOH, , 7 h
S
O
O
O
12a–c
O
Scheme 3ꢁSynthetic route to compounds 9–12.
100
90
80
70
60
50
40
30
20
10
0
AA 3a 3b 3c 4a 4b 4c 6a 8a 8b 8c 9b 10c 11a 11b 11c 12a 12b 12c
Compound
Figure 1ꢁ2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of dipicolinic acid derivatives.
AA is ascorbic acid taken as a reference.
gentamicin-resistant strains [26]. Amikacin is markedly agent against this mold is compound 10b. Aspergillus
active against antibiotic-resistant clinical isolates [27].
ochraceus is the most affected by 3b, 11b, 12b, all of them
Antifungal activity of the compounds was assayed incorporating different ring systems and possessing the
against four fungal strains, namely Aspergillus flavus, ethyl group in their structure. For Fusarium strains almost
Aspergillus ochraceus, Fusarium graminearum and Fusar- all compounds exhibit significant antifungal activity at
ium verticilioides. As in our previous work [17], F. gramine- given concentrations. The difference between sensitivity
arum was found to be highly susceptible to the tested of Aspergillus and Fusarium strains may be due to their dif-
compounds but most of the compounds exhibit impres- ferences in cell structure. Since Aspergillus spp. is mainly
sive antifungal activity against all tested fungi (Table 1).
human pathogen [28] and according to phylogenetic tree
In general, Fusarium strains are less resistant than is more closely related to Penicilium spp. [29, 30] that can
Aspergilus strains (not shown). Aspergillus flavus is the produce DPA [2], it is expected to have higher resistance
most resistant to tested compounds and the best antifungal to DPA derivatives (as confirmed). On the other hand,
Brought to you by | University of Sussex Library
Authenticated
Download Date | 1/18/17 9:42 PM

ꢁꢁꢁꢂ
ꢀM. Molnar et al.: Mono- and bis-dipicolinic acid heterocyclic derivatives
4ꢀ
Table 1ꢁAntifungal activity of dipicolinic acid derivatives expressed as MIC50.
Test compound ꢁ
ꢁ
MIC50 (μg/mL)
A. flavusꢁ
A. ochraceusꢁ
F. graminearumꢁ
F. verticilioides
3a
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
1
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
0.1
<ꢂ0.1
0.1
0.1
NA
0.1
0.1
0.1
10
0.1
NA
1
<ꢂ0.1
10
10
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
<ꢂ0.1ꢃ
<ꢂ0.1ꢃ
0.1ꢃ
<ꢂ0.1ꢃ
0.1ꢃ
<ꢂ0.1
<ꢂ0.1
0.1
<ꢂ0.1
0.1
0.1
0.1
0.01
10
0.1
0.1
<ꢂ0.1
<ꢂ0.1
0.1
0.1
<ꢂ0.1
<ꢂ0.1
3b
0.1
1
3c
4a
10
4b
NA
0.1
100
0.1
10
4c
<ꢂ0.1ꢃ
<ꢂ0.1ꢃ
0.1ꢃ
6a
8a
8b
0.1ꢃ
8c
0.1
10
0.1
<ꢂ0.1
10
0.1
0.1
1
0.1ꢃ
9b
0.1ꢃ
11a
11b
11c
12a
12b
12c
<ꢂ0.1ꢃ
<ꢂ0.1ꢃ
0.1ꢃ
0.1ꢃ
<ꢂ0.1ꢃ
0.1ꢃ
<ꢂ0.1
0.1
Fusarium spp. is mainly a plant pathogen/saprophyte [31] Conclusions
and, therefore, more susceptible to DPA. Aspergillus spp.
is also charaterized by a great number of cell wall asso- Dipicolinic acid derivatives were synthesized and inves-
ciated enzymes that can degrade (hydrolyze) synthesized tigated for antimicrobial and antioxidant activity. Some
compounds due to their unspecific activity [32, 33]. On compounds show excellent antioxidant activity, the thiosem-
the other hand, Fusarium spp. possess a cell wall associ- icarbazide 3c, in particular. Thiosemicarbazide 3b possess
ated unspecific hydrolases [34] that are mainly intended good antioxidant activity, but it is also an excellent antifun-
to increase their virulence toward plants [31, 34]. This is gal agent against all four investigated fungi strains. Data
a great indicator for design and synthesis of some future collected in this research indicate that thiosemicarbazides
antifungal agents, especially against Aspergillus strains, could be used as a unique antifungal and antioxidant agents.
since the triazole moiety substituted with the ethyl group
has a significant impact on this activity against both
A. flavus and A. ochraceus. Triazole derivatives may be
Experimental
Melting points were determined on an Electrothermal capillary appa-
ratus. The elemental analysis for C, H and N was done on a Perkin-
regarded as a new class of antifungal agents which can
inhibit fungi by blocking the biosynthesis of certain
fungal lipids, especially, ergosterol in cell membranes
Elmer CHNS/O analyzer 2400 Series II. 1H NMR spectra were recorded
in DMSO-d6 at 293 K on a Bruker Avance 600 MHz spectrometer. The
MS spectra were recorded on an LC-MS/MS API 2000 instrument
using electrospray ionization (ESI). The spectra were scanned in both
positive and negative ion modes. The absorbance was measured on
a UV visible spectrophotometer Helios γ. Microplates were read on a
Sunrise absorbance reader. Incubation was carried in an Aqualytic
AL 500-8 incubator.
[35]. The differences between antifungal and antibacterial
activities are probably caused by different mechanisms of
action. The structures of fungi and bacteria differ in very
significant ways. For example, many antibacterial agents
inhibit steps important for the formation of peptidogly-
can, the essential component of the bacterial cell wall.
In contrast, most antifungal compounds target either the
formation or the function of ergosterol, an important com-
ponent of the fungal cell membrane. Bacteria employ an
extensive repertoire of plasmids, transposons, and bac-
teriophages to facilitate the exchange of resistance and
virulence determinants among and between species. Con-
versely, antifungal resistance generally involves the emer-
gence of naturally resistant species [36].
Preparation of thiosemicarbazides 3a–c and 4a–c
A mixture of methyl 2-carbazoylpyridin-6-carboxylate (1, 10 mmol) or
2,6-bis-carbazoylpyridine (2, 10 mmol) and methyl, ethyl or phenyl
isothiocyanate (10 mmol for 1 and 20 mmol for 2) in absolute ethanol
Brought to you by | University of Sussex Library
Authenticated
Download Date | 1/18/17 9:42 PM

ꢂꢁꢁꢁ
M. Molnar et al.: Mono- and bis-dipicolinic acid heterocyclic derivativesꢀ
ꢀ5
(50 mL) was heated under reflux for 3 h. Progress of the reaction was
monitored by thin layer chromatography (TLC). Crude product, 3a–c
or 4a–c, was filtered and crystallized from ethanol [17].
Preparation of oxadiazoles 6a–c from 3a–c and 10c from 4c
A solution of compound 3a–c or 4c in EtOH (20 mL) was treated with I2
(500 mg), KI (640 mg in 20 mL of water) and NaOH (4N, 2 mL) and the
mixture was heated under mild reflux for 4.5 h [37]. Progress of the reac-
tion was monitored by TLC. Concentration to half a volume resulted in
precipitation 6a–c or 10c. The product was filtered and crystallized from
EtOH. The representative data for 6a and 10c are given below.
Methyl
6-(4-methylthiosemicarbazide-1-yl-carbonyl)pyridin-
2-carboxylate (3a)ꢁThis compound was obtained from 1 and methyl
1
isothiocyanate; yield 90%; mp 210°C; H NMR: δ 2.85 (m, 3H, CH3),
3.93 (s, 3H, OCH3), 8.23–8.25 (m, 3H, py-H); 8.01 (d, 1H, Jꢀ=ꢀ4.1 Hz, NH),
9.39 (brs, 1H, NH), 10.36 (brs, 1H, NH). Anal. Calcd for C10H12N4O3S
(268.06): C, 44.77; H, 4.51; N, 20.88. Found: C, 44.92; H, 4.68; N, 19.97.
Methyl 6-(5-methylamino-1,3,4-oxadiazol-2-yl)pyridine-2-car-
1
boxylate (6a)ꢁYield 76%; mp >ꢀ300°C; H NMR: δ 3.83 (s, 3H, CH3),
Methyl
6-(4-ethylthiosemicarbazide-1-yl-carbonyl)pyridin-
4.07 (s, 3H, OCH3), 7.92–7.98 (m, 3H, py-H); 8.61 (m, 1H, NH); MS: m/z
2-carboxylate (3b)ꢁThis compound was obtained from 1 and ethyl
234.90 [M–H]−, (Mꢀ=ꢀ234.2).
1
isothiocyanate; yield 92%; mp 208°C; H NMR: δ 1.07 (m, 3H, CH3),
3.46 (m, 2H, CH2), 3.94 (s, 3H, OCH3), 8.08 (t, 1H, Jꢀ=ꢀ5.5 Hz, NH), 8.24–
8.26 (3H, py-H), 9.36 (brs, 1H, NH), 10.36 (brs, 1H, NH); MS: m/z 281
[M–H]−, (Mꢀ=ꢀ282.3). Anal. Calcd for C11H14N4O3S (282.08): C, 46.80; H,
5.00; N, 19.85. Found: C, 46.02; H, 5.26; N, 19.38.
5,5′-Bis(phenylamino-1,3,4-oxadiazol-2-yl)pyridine (10c)ꢁYield
48%; mp 298°C (lit mp 303–305°C, [8]); 1H NMR: δ 7.36–7.67 (10H, arom),
+
8.22 (3H, py-H), 10.93 (s, 2H, NH); MS: m/z 396.10 [M–H] , (Mꢀ=ꢀ397.40).
Methyl 6-(4-phenylthiosemicarbazide-1-yl-carbonyl)pyridin-2-car- Preparations of triazoles 11a–c from 4a–c
boxylate (3c)ꢁThis compound was obtained from 1 and phenyl iso-
1
thiocyanate; yield 80%; mp 162–164°C; H NMR: δ 3.94 (m, 3H, OCH3),
A solution of 4a–c (10 mmol) in aqueous NaOH (2N, 10 mL) was heated
7.14–7.49 (5H, ArH), 8.24–8.26 (3H, py-H), 9.78 (brs, 2H, 2NH), 10.57 (s,
1H, NH); MS: m/z 328.9 [M–H]−, (Mꢀ=ꢀ330.3). Anal. Calcd for C15H14N4O3S
(330.08): C, 54.54; H, 4.27; N, 16.96. Found: C, 53.1; H, 4.32; N, 16.54.
under mild reflux for 1.5 h, then cooled, poured over ice water and the
mixture was acidified with diluted hydrochloric acid [37]. Product 11a–
c was separated by filtration and crystallized from DMF/water.
2,6-Bis(4-methylthiosemicarbazide-1-yl-carbonyl)pyridine
2,6-Bis(4-methyl-4H-5-sulfanyl-1,2,4-triazol-3-yl)pyridine
(11a)ꢁYield 75%; mp >ꢀ300°C; 1H NMR: δ 3.82 (s, 6H, CH3), 8.13–8.28
(m, 3H, py-H); 14.15 (br.s, 2H, NH); MS: m/z 304.20 [M–H]−, (Mꢀ=ꢀ305.4).
Anal. Calcd for C11H11N7S2 (305.05): C, 43.26; H, 3.63; N, 32.11. Found: C,
43.48; H, 3.91; N, 32.00.
(4a)ꢁThis compound was obtained from 2 and methyl isothiocy-
1
anate; yield 74%; mp 202–207°C; H NMR: δ 2.70 (d, 6H, Jꢀ=ꢀ4.1 Hz,
CH3), 8.14 (m, 2H, NH), 8.16 (3H, py-H), 9.53 (s, 2H, NH), 11.00 (s, 2H,
NH); MS: m/z 339.8 [M–H]−, (Mꢀ=ꢀ341.4). Anal. Calcd for C11H15N7O2S2
(341.07): C, 38.70; H, 4.43; N, 28.72. Found: C, 38.85; H, 4.95; N, 28.62.
2,6-Bis(4-ethyl-4H-5-sulfanyl-1,2,4-triazol-3-yl)pyridine
1
(11b)ꢁYield 72%; mp >ꢀ300°C; H NMR: δ 1.15–1.20 (t, 6H, Jꢀ=ꢀ7.0 Hz,
2,6-Bis(4-ethylthiosemicarbazide-1-yl-carbonyl)pyridine
CH3), 4.41–4.48 (m, 4H, CH2), 8.12–8.24 (m, 3H, py-H); 14.24 (br.s, 2H,
NH); MS: m/z 332.10 [M–H]−, (Mꢀ=ꢀ333.4). Anal. Calcd for C13H15N7S2
(333.08): C, 46.83; H, 4.53; N, 29.41. Found: C, 46,34; H, 4.51; N, 29.15.
(4b)ꢁThis compound was obtained from 2 and ethyl isothiocyanate;
1
yield 78%; mp 219–220°C; H NMR: δ 1.05 (s, 6H, CH3), 3.43–3.48 (m,
4H, CH2), 8.03–8.07 (m, 2H, NH), 8.22–8.25 (m, 3H, py-H), 9.33 (brs,
2H, NH), 10.32 (m, 2H, NH); MS: m/z 368.1 [M–H]−, (Mꢀ=ꢀ369.4). Anal.
Calcd for C13H19N7O2S2 (369.10): C, 42.26; H, 5.18; N, 26.54. Found: C,
42.75; H, 5.28; N, 26.75.
2,6-Bis(4-phenyl-4H-5-sulfanyl-1,2,4-triazole-3-yl)pyridine
(11c)ꢁYield 70%; mp >ꢀ300°C (lit mp 338–340°C, [8]); 1H NMR: δ 7.0 3 –
7.45 (m, 10H, arom), 7.74–8.02 (3H, py-H); 14.17 (br.s, 2H, NH); MS:
m/z 428.00 [M–H]−, (Mꢀ=ꢀ429.5). Anal. Calcd for C21H15N7S2 (429.08): C,
58.72; H, 3.52; N, 22.83. Found: C, 58.95; H, 3.71; N, 21.57.
2,6-Bis(4-phenylthiosemicarbazide-1-yl-carbonyl)pyridine
(4c)ꢁThis compound was obtained from 2 and phenyl isothiocy-
anate; yield 68%; mp 234°C (lit mp 245–247°C [8]); 1H NMR: δ 7.1 3 –7.47
(m, 10H, arom), 8.23–8.25 (m, 3H, py-H), 9.85–9.91 (4H, NH), 11.25
(brs, 2H, NH); MS: m/z 464.00 [M–H]−, (Mꢀ=ꢀ465.5). Anal. Calcd for
C21H19N7O2S2 (465.10): C, 54.18; H, 4.11; N, 21.06. Found: C, 53.65; H,
4.23; N, 20.27.
Preparations of thiazolidinones 8a–c from 3a–c and
12a–c from 4a–c
A mixture of thiosemicarbazide 3a–c or 4a–c (10 mmol), ethyl bromoac-
etate (11 mmol for 3a–c or 22 mmol for 4a–c) and anhydrous sodium
acetate (40 mmol) in absolute ethanol (30 mL) was heated under reflux
for 7–10 h [17]. Then the mixture was cooled, poured onto ice and water
and the resultant solid was filtered, dried and crystallized from EtOH.
2,6-Bis(5-ethylamino-1,3,4-thiadiazol-2-yl)
pyridine (9b)
A solution of compound 4b (5 mmol) in concentrated sulfuric acid
(5 mL) was kept at room temperature for 2.5 h and then poured over
ice water. The precipitated product 9b was filtered and crystalized
Methyl 6-[(2-methylimino-4-oxothiazolidin-3-yl)carba-
moyl]pyridine-2-carboxylate (8a)
1
from N,N-dimethylformamide (DMF) [37]: yield 38%; mp 287°C; H
1
NMR: δ 1.25 (s, 6H, CH3), 3.39–3.46 (m, 4H, CH2), 7.85 (brs, H, NH), Yield 82%; mp 212°C; H NMR: δ 3.17–3.30 (s, 3H, CH3), 3.95 (s, 3H,
+
7.86–8.11 (m, 2H, py-H); MS: m/z 334.2 [Mꢀ+ꢀH] , (Mꢀ=ꢀ333.4).
OCH3), 4.12 (s, 2H, CH2), 8.23–8.26 (m, 3H, py-H); 10.57 (s, H, NH); MS:
Brought to you by | University of Sussex Library
Authenticated
Download Date | 1/18/17 9:42 PM

ꢁꢁꢁꢂ
ꢀM. Molnar et al.: Mono- and bis-dipicolinic acid heterocyclic derivatives
6ꢀ
m/z 307.10 [M–H]−, (Mꢀ=ꢀ308.3). Anal. Calcd for C12H12N4O4S (308.06):
Staphylococcus aureus, and two Gram-negative Escherichia coli and
Pseudomonas aeruginosa were used. The four bacteria were isolates
from various clinical specimens obtained from Microbiology Service of
the Public Health Institute of Osijek-Baranja County, Croatia. Bacillus
subtilis and Escherichia coli were selected as two popular laboratory
model organisms representing Gram-positive and Gram-negative bac-
teria, respectively. Staphylococcus aureus and Pseudomonas aerugi-
nosa were selected as human pathogens representing Gram-positive
and Gram-negative bacteria, respectively. The antibacterial activity
was assessed in terms of minimum inhibitory concentrations (MICs) by
a modified broth microdilution method [38]. Broth microdilution tests
were performed with 96 sterile flat-bottom microtiter plates (Ratiolab,
Dreieich, Germany). Compounds dissolved in DMSO and one hundred
microliters of Mueller-Hinton Broth (MHB) (Cultimed) were prepared
in 96 well micro-trays. Antimicrobials were serially diluted (512 to 1 μg/
mL) in MHB. The inoculum was prepared by making a MHB suspen-
sion of colonies from a 24 h Mueller-Hinton Agar (MHA) plate culture of
the microorganisms. Each well was inoculated with 300ꢀ×ꢀ103 bacteria
(density of the bacterial suspension at 0.5 McFarland scale, which is
150ꢀ×ꢀ106 CFU/mL. Amikacin sulfate (AMCK) was co-assayed as posi-
tive control, and DMSO was used as negative control. Control samples
(positive and negative) were incubated under the same conditions.
After incubation at 37°C for 24 h with 5% CO2 and 50% humidity, the
trays were examined for the growth of the test microorganisms with
the unaided eye. The MIC value was defined as the lowest concentra-
tion of compound at which there was no visual turbidity due to micro-
bial growth. All assays were performed in duplicate.
C, 46.75; H, 3.92; N, 18.17. Found: C, 46.73; H, 3.85; N, 17.9.
Methyl 6-[(2-ethylimino-4-oxothiazolidin-3-yl)carba-
moyl]pyridine-2-carboxylate (8b)
1
Yield 70%; M.p. >ꢀ300°C; H NMR: δ 1.16–1.34 (t, 3H, Jꢀ=ꢀ7.0 Hz, CH3),
3.74–3.79 (m, 2H, CH2), 3.93 (s, 3H, OCH3), 4.03 (s, 2H, CH2), 8.15–8.28
(m, 3H, py-H); 10.64 (s, H, NH); MS: m/z 321.20 [M–H], (Mꢀ=ꢀ322.34).
Anal. Calcd for C13H14N4O4S (322.07): C, 48.44; H, 4.38; N, 17.38. Found:
C, 47.90; H, 4.43; N, 17.81.
Methyl 6-[(2-phenylimino-4-oxothiazolidin-3-yl)carba-
moyl]pyridine-2-carboxylate (8c)
Yield 88%; mp 235°C; 1H NMR: δ 3.94 (s, 3H, OCH3), 4.27 (m, 2H, CH2),
7.39–7.56 (5H, arom), 8.19–8.24 (m, 3H, py-H); 10.54 (br.s, H, NH); MS:
m/z 369.10 [M–H]−, (Mꢀ=ꢀ370.1); Anal. Calcd for C17H14N4O4S (370.07): C,
55.13; H, 3.81; N, 15.13. Found: C, 55.19; H, 3.71; N, 15.21.
2,6-Bis[(2-methylimino-4-oxothiazolidin-3-yl)carbamoyl]pyri-
dine (12a)ꢁYield 77%; mp >ꢀ300°C; 1H NMR: δ 3.21 (s, 6H, CH3), 4.09
(s, 4H, CH2), 8.21 (m, 3H, py-H); 11.60 (brs, 2H, NH); MS: m/z 420.0
[M–H]−, (Mꢀ=ꢀ421.4). Anal. Calcd for C15H15N7O4S2 (421.1): C, 42.75; H,
3.59; N, 23.26. Found: C, 42.49; H, 3.56; N, 22.66.
Antifungal activity
2,6-Bis[(2-ethylimino-4-oxothiazolidin-3-yl)carbamoyl]pyridine
1
(12b)ꢁYield 70%, mp 214–217°C; H NMR: δ 1.20–1.24 (t, 6H, Jꢀ=ꢀ7.0 Hz,
The method was previously described in detail [17] and was performed
in accordance with the guidelines detailed in [39]. Fungi strains used
in these experiments were Aspergillus flavus (NRRL 3251), Aspergil-
lus ochraceus (CBS 589.68), Fusarium graminearum (CBS 110.250) and
Fusarium verticillioides (CBS 119.825). These species were selected as
major producers of mycotoxins and food contaminants [40].
CH3), 3.77–3.83 (m, 4H, CH2), 4.08 (s, 4H, CH2), 8.21 (3H, py-H); 11.61 (s, 2H,
+
NH); MS: m/z 448.20 [M–H ]−, (Mꢀ=ꢀ449.50). Anal. Calcd for C17H19N7O4S2
(449.09): C, 45.42; H, 4.26; N, 21.81. Found: C, 44.92; H, 4.26; N, 22.38.
2,6-Bis[(2-phenylimino-4-oxothiazolidin-3-yl)carbamoyl]pyri-
dine (12c)ꢁYield 58%; mp 202°C; 1H NMR: δ 4.24–4.34 (m, 4H, CH2),
6.88–7.56 (10H, arom), 8.28–8.38 (3H, py-H); 11.88 (s, 2H, NH); MS:
m/z 544.0 [M–H]−, (Mꢀ=ꢀ545.6). Anal. Calcd for C25H19N7O4S2 (545.09):
C, 55.04; H, 3.51; N, 17.97. Found: C, 55.04; H, 3.51; N, 17.63.
Acknowledgments: The authors are grateful to the Microbi-
ology Service of the Public Health Institute of Osijek-Baranja
County, Osijek, Croatia, for providing test bacteria strains.
DPPH scavenging activity
Determination of antioxidant activity was performed according to
the procedure described in our previous work [17]. Briefly, 750 μL of
0.2 mm dimethyl sulfoxide (DMSO) solution of the compound was
added to 0.2 mm DMSO solution of 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical, resulting in 0.1 mm solution of tested compound.
Ascorbic acid (AA) was used as a reference. After 30 min of incuba-
tion the absorbance was read at 517 nm and the scavenging activity
was calculated as described previously [17].
References
[1] Navarro, A. K.; Peña, A.; Pérez-Guevara, F. Endospore dipicolinic
acid detection during Bacillus thuringiensis culture. Lett. Appl.
Microbiol. 2008, 46, 166–170.
[2] Assaf, A.; Cerda-García-Rojas, C.; de la Torre, M. Isolation of
dipicolinic acid as an insecticidal toxin from Paecilomyces
fumosoroseus. Appl. Microbiol. Biotechnol. 2005, 68, 542–547.
[3] Sugano, M. Soy in Health and Disease Prevention. In Nutrition
and Disease Prevention. Sugano M., Ed. CRC Press, Taylor &
Francis Group: Boca Raton, FL, USA, 2005; Vol. 20051651.
[4] Molnar, M.; Cacic, M.; Zec Zrinusic, S. Synthesis and antioxidant
evaluation of schiff bases derived from 2,6-pyridinedicarboxylic
acid. Lett. Org. Chem. 2012, 9, 401–410.
Antibacterial activity
The antibacterial activities of DPA derivatives were evaluated against
four test bacteria strains. Two Gram-positive Bacillus subtilis and
Brought to you by | University of Sussex Library
Authenticated
Download Date | 1/18/17 9:42 PM

ꢂꢁꢁꢁ
M. Molnar et al.: Mono- and bis-dipicolinic acid heterocyclic derivativesꢀ
ꢀ7
zole and 1,3,4-thiadiazole derivatives. J. Appl. Sci. Res. 2010, 6,
866–870.
[21] Reddy, K. R.; Mamatha, R.; Babu, M. S. S.; Kumar, K.
S.; Jayaveera, K. N.; Narayanaswamy, G. Synthesis and
antimicrobial activities of some triazole, thiadiazole, and oxa-
diazole substituted coumarins. J. Heterocycl. Chem. 2014, 51,
132–137.
[22] Mayekar, A. N.; Yathirajan, H. S.; Narayana, B.; Sarojini, B.
K.; Suchetha Kumari, N. Synthesis and antimicrobial stud-
ies on new substituted 1,3,4-oxadiazole derivatives bearing
6-bromonaphthalene moiety. Int. J. Chem. 2010, 2, 38–54.
[23] Milway, V. A.; Zhao, L.; Abedin, T. S. M.; Thompson, L. K.; Xu, Z.
Trinuclear complexes of a series of ‘tritopic’ hydrazide ligands
– structural and magnetic properties. Polyhedron 2003, 22,
1271–1279.
[24] Foroughifar, N.; Mobinikhaledi, A.; Rafiee, A. Efficient synthe-
sis of some novel symmetrical and unsymmetrical pyridine
bis-1,2,4-triazoles and bis-1,3,4-thiadiazoles. J. Chem. Res.
2014, 38, 111–114.
[5] Büyükkιdan, N.; Yenikaya, C.; İlkimen, H.; Karahan, C.;
Darcan, C.; Şahin, E. Synthesis, characterization, and antimi-
crobial activity of a novel proton salt and its Cu(II) complex.
Russ. J. Coord. Chem. 2012, 39, 96–103.
[6] Murakami, T.; Morita, Y. Morphology and distribution of the
projection neurons in the cerebellum in a teleost, Sebastiscus
marmoratus. J. Comp. Neurol. 1987, 256, 607–623.
[7] Aanandhi, M. V.; Mansoori, M. H.; Shanmugapriya, S.;
George, S.; Shanmugasundaram, P. Synthesis and in vitro
Antioxidant activity of substituted pyridinyl-1,3,4-oxadiazole
derivatives. Research J. Pharm. Biol. Chem. Sci. 2010, 1,
1083–1090.
[8] Ghozlan, S. A. S.; Mohamed, S. F.; Amr, A. E.-G.E.; Mustafa,
E.-S. E.; El-Wahab, A. A. A. Synthesis and reactions of some
new 2,6-bis-substituted pyridine derivatives as antimicrobial
agents. World J. Chem. 2009, 4, 83–88.
[9] Sancak, K.; Ünever, Y.; Er, M. Synthesis of 2-acylamino,
2-aroylamino and ethoxycarbonylimino-1,3,4-thiadiazoles as
antitumor agents. Turk. J. Chem. 2007, 31, 125–134.
[10] Yar, M. S.; Akhter, M. W. Synthesis and anticonvulsant activity
of substituted oxadiazole and thiadiazole derivatives. Acta Pol.
Pharm. – Drug Res. 2009, 66, 393–397.
[25] Kulp, A.; Kuehn, M. J. Biological functions and biogenesis of
secreted bacterial outer membrane vesicles. Annu. Rev. Micro-
biol. 2010, 64, 163–184.
[26] Pien, F. D.; Ho, P. W. Antimicrobial spectrum, pharmacology,
adverse effects, and therapeutic use of amikacin sulfate. Am. J.
Hosp. Pharm. 1981, 38, 981–989.
[27] Price, K. E.; DeFuria, M. D.; Pursiano, T. A. Amikacin, an amino-
glycoside with marked activity against antibiotic-resistant clini-
cal isolates. J. Infect. Dis. 1976, 134, S249–S261.
[28] Klich, M. A. Aspergillus flavus: The major producer of aflatoxin.
Mol. Plant Pathol. 2007, 8, 713–722.
[29] Samson, R. A.; Visagie, C. M.; Houbraken, J.; Hong, S.-B.;
Hubka, V.; Klaassen, C. H. W.; Perrone, G.; Seifert, K. A.; Susca,
A.; Tanney, J. B. Phylogeny, identification and nomenclature of
the genus Aspergillus. Stud. Mycol. 2014, 78, 141–173.
[30] Wang, H.; Xu, Z.; Gao, L.; Hao, B. A Fungal phylogeny based on
82 complete genomes using the composition vector method.
BMC Evol. Biol. 2009, 9, 195.
[31] Roncero, M. I. G.; Hera, C.; Ruiz-Rubio, M.; Maceira, F. I. G.;
Madrid, M. P.; Caracuel, Z.; Calero, F.; Delgado-Jarana, J.;
Roldán-Rodrí, R.; Martí, A. L. Fusarium as a Model for studying
virulence in soilborne plant pathogens. Physiol. Mol. Plant
Pathol. 2003, 62, 87–98.
[32] Bernard, M.; Latgé, J.-P. Aspergillus fumigatus Cell wall: com-
position and biosynthesis. Med. Mycol. 2001, 39, 9–17.
[33] de Vries, R. P.; Visser, J. Aspergillus enzymes involved in degra-
dation of plant cell wall polysaccharides. Microbiol. Mol. Biol.
Rev. 2001, 65, 497–522.
[34] Kikot, G. E.; Hours, R. A.; Alconada, T. M. Contribution of
cell wall degrading enzymes to pathogenesis of Fusarium
graminearum: a review. J. Basic Microbiol. 2009, 49,
231–241.
[35] Turan-Zitouni, G.; Kaplancıklı, Z. A.; Yıldız, M. T.; Chevallet,
P.; Kaya, D. Synthesis and antimicrobial activity of 4-phenyl/
cyclohexyl-5-(1-phenoxyethyl)-3-[n-(2-thiazolyl)acetamido]
thio-4h-1,2,4-triazole derivatives. Eur. J. Med. Chem. 2005, 40,
607–613.
[36] Ghannoum, M. A.; Rice, L. B. Antifungal agents: mode of
action, mechanisms of resistance, and correlation of these
mechanisms with bacterial resistance. Clin. Microbiol. Rev.
1999, 12, 501–517.
[11] Zamani, K.; Faghihi, K.; Tofighi, T.; Shariatzadeh, M. R.
Synthesis and antimicrobial activity of some pyridyl and naph-
thyl substituted 1,2,4-triazole and 1,3,4-thiadiazole deriva-
tives. Turk. J. Chem. 2004, 28, 95–100.
[12] Sankangoud, R. M.; Chatni, N. B.; Goudanavar, P. S. Synthesis
and evaluation of antibacterial activity of some new N,N′-(5-
(6-(4-substituted phenyl)imidazo[2, 1-b][1,3,4]thiadiazol-2-yl)
pyrimidine-2, 4-diyl)diacetamide derivatives. Pharma Chem.
2010, 2, 347–353.
[13] Dubey, A. K.; Sangwan, N. K. Synthesis and antifungal activity
of 5-(3,5-diphenylpyrazol-4-yl-methyl)-2-(4-oxo-2-substituted
phenyl-3-thiazolidinyl)-oxadiazol/thiadiazoles and related
compounds of potential pesticidal activity. Indian J. Chem.
1994, 33B, 1043–1047.
[14] Bano, Q.; Tiwari, N.; Giri, S.; Nizamuddin, S. Synthesis and fun-
gicidal activities of 3-aryloxymethyl-6-substituted-1,2,4-triazole
[3,4-b]-1,3,4-thiadiazole. Indian J. Chem. 1992, 31B, 714–718.
[15] Sharma, V.; Shrivastava, B.; Bhatia, R.; Bachwani, M.;
Khandelwal, R.; Ameta, J. Exploring potential of 1,2,4-triazole:
a brief review. Pharmacol. Online 2011, 1, 1192–1222.
[16] Molnar, M.; Šarkanj, B.; Cačić, M.; Gille, L.; Strelec, I. Anti-
oxidant properties and growth-inhibitory activity of coumarin
schiff bases against common foodborne fungi. Pharma Chem.
2014, 6, 313–320.
[17] Šarkanj, B.; Molnar, M.; Cačić, M.; Gille, L. 4-Methyl-7-Hydrox-
ycoumarin antifungal and antioxidant activity enhancement by
substitution with thiosemicarbazide and thiazolidinone moie-
ties. Food Chem. 2013, 139, 488–495.
[18] Chawla, R.; Arora, A.; Parameswaran, M. K.; Chan, P.;
Sharma, D.; Michael, S.; Ravi, T. K. Synthesis of novel
1,3,4-oxadiazole derivatives as potential antimicrobial agents.
Acta Pol. Pharm. – Drug Res. 2010, 67, 247–253.
[19] RamaGanesh, C. K.; Bodke, Y. D.; Venkatesh, K. B. Synthe-
sis and biological evaluation of some innovative coumarin
derivatives containing thiazolidin-4-one ring. Indian J. Chem.
2010, 49B, 1151–1154.
[20] Salimon, J.; Salih, N.; Hameed, A.; Ibraheem, H.; Yousif, E.
Synthesis and Antibacterial activity of some new 1,3,4-oxadia-
Brought to you by | University of Sussex Library
Authenticated
Download Date | 1/18/17 9:42 PM

ꢁꢁꢁꢂ
ꢀM. Molnar et al.: Mono- and bis-dipicolinic acid heterocyclic derivatives
8ꢀ
[37] Narwade, S. K.; Halnor, V. B.; Dalvi, N. R.; Gill, C. H.;
Karale, B. K. Conventional and ultrasound mediated synthe-
sis of some thiadiazoles, triazoles and oxadiazoles. Indian J.
Chem. 2006, 45B, 2776–2780.
[38] Gu, W.; Wang, S. Synthesis and antimicrobial activities of novel
1H-Dibenzo[a,c]carbazoles from dehydroabietic acid. Eur. J.
Med. Chem. 2010, 45, 4692–4696.
[39] National Committee for Clinical Laboratory Standards.
Reference Method for Broth Dilution Antifungal Susceptibility
Testing of Filamentous Fungi. Approved Standard; Second Edition;
NCCLS document M38-A, Wayne, PA, 2002; Vol. 22.
[40] Hussein, H. S.; Brasel, J. M. Toxicity, metabolism, and impact
of mycotoxins on humans and animals. Toxicology 2001, 167,
101–134.
Brought to you by | University of Sussex Library
Authenticated
Download Date | 1/18/17 9:42 PM