Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Heterocyclic Communications

Editor-in-Chief: Henary, Maged


IMPACT FACTOR 2018: 0.810

CiteScore 2018: 0.77

SCImago Journal Rank (SJR) 2018: 0.208
Source Normalized Impact per Paper (SNIP) 2018: 0.264

Open Access
Online
ISSN
2191-0197
See all formats and pricing
More options …
Volume 23, Issue 1

Issues

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

Maja Molnar / Valentina PavićORCID iD: http://orcid.org/0000-0001-5369-1755 / Bojan Šarkanj / Milan Čačić / Dubravka Vuković / Jelena Klenkar
Published Online: 2017-01-18 | DOI: https://doi.org/10.1515/hc-2016-0078

Abstract

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.

Keywords: antifungal; antioxidant activity; dipicolinic acid; oxadiazole; thiadiazole; thiazolidinone; triazole

Introduction

Dipicolinic acid (DPA) is a unique constituent of endospores of Bacillus and Clostridium genuses [1] and is also produced and secreted by certain Penicillium strains and by several entomopathogenic fungi [2]. DPA and its derivatives show various biological activities including significant antimicrobial [3] and antioxidant properties [4]. As a strong complexing agent DPA is a potent metal-chelator functioning as a multidentate ligand [5] that inhibits lipid peroxidation and protects glutathione reductase from the copper-dependent inactivation [6]. A structural combination of DPA core with other heterocyclic 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], [10], [11], [12], [13], [14], triazoles [11], [13], [14], [15], thiazolidinones [16], [17], [18], [19], [20], [21], and oxadiazoles [22] have been documented. Only a slight change in structural characteristics can have a great effect on antifungal and antioxidant activity [4], [17]. To date, derivatives of dipicolinic acid were described by Milway in 2003 [23] and in our previous work on Schiff bases [4].

Results and discussion

The synthesis of the target compounds was carried out as outlined in Schemes 13. All products were purified and characterized by TLC, MS, 1H NMR and elemental analysis. Preparation of starting compounds 6-methoxycarbonyl-2-pyridinecarboxylic acid hydrazide (1) and 2,6-pyridinedicarboxylic acid bis-hydrazide (2) was described in our previous work [4]. Thiosemicarbazides were prepared by refluxing mono-hydrazide or bis-hydrazide with the corresponding isothiocyanate in 1:1 ratio for mono derivatives and 1:2 ratio for bis derivatives in ethanol (Scheme 1).

Synthetic route to compounds 1–4.
Scheme 1

Synthetic route to compounds 1–4.

Synthetic route to compounds 6a and 8a–c.
Scheme 2

Synthetic route to compounds 6a and 8a–c.

Synthetic route to compounds 9–12.
Scheme 3

Synthetic route to compounds 9–12.

Thiosemicarbazides exhibit characteristic 1H 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 only, since no expected products for mono derivatives were obtained, due to hydrolysis of the ester group by a strong acid. Bis derivatives of these compounds were published by Foroughifar and co-workers in 2014 [24]. Upon treatment of thiosemicarbazides with I2/KI system in NaOH solution, oxadiazoles were formed showing NH signals in the 1H NMR spectra, in addition to signals for groups characteristic for each substituent depending on isothiocyanate used in thiosemicarbazide synthesis. Oxadiazole synthesis using I2/KI system was successful for only two derivatives, monosubstituted with the methyl and disubstituted with phenyl groups. Treatment of thiosemicarbazides with NaOH resulted in cyclization to triazoles. The synthesized triazoles show characteristic peaks for NH group (triazole ring) at δ 14.15–14.27, indicating that they exist mainly in a thione form rather than with a thiol function. Only disubstituted derivatives of triazoles were synthesized [24], since the mono derivatives underwent hydrolysis of the methoxycarbonyl group. Refluxing monosubstituted and disubstituted thiosemicarbazides with ethyl bromoacetate in ethanol in the presence of sodium acetate yielded corresponding thiazolidinones. These compounds show a characteristic signal for the CH2 group of the thiazolidinone, which is not visible in the spectra of the corresponding thiosemicarbazides.

The synthesized compounds were assayed for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (Figure 1). As can be seen, compound 3c is 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 different substituents is obvious, since in almost all compounds the phenyl substitution results in a better DPPH radical scavenging activity in comparison to the alkyl substitution. 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.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of dipicolinic acid derivatives. AA is ascorbic acid taken as a reference.
Figure 1

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of dipicolinic acid derivatives.

AA is ascorbic acid taken as a reference.

Antibacterial properties of the synthesized compounds were studied against four bacteria, namely two Gram-positive (Bacillus subtilis and Staphylococcus aureus) and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) species. None of the compounds shows noticable antimicrobial activity comparable to antibiotic amikacin, an aminoglycoside that is highly active against most Gram-negative bacteria including many gentamicin-resistant strains [26]. Amikacin is markedly active against antibiotic-resistant clinical isolates [27].

Antifungal activity of the compounds was assayed against four fungal strains, namely Aspergillus flavus, Aspergillus ochraceus, Fusarium graminearum and Fusarium verticilioides. As in our previous work [17], F. graminearum was found to be highly susceptible to the tested compounds but most of the compounds exhibit impressive antifungal activity against all tested fungi (Table 1).

Table 1

Antifungal activity of dipicolinic acid derivatives expressed as MIC50.

In general, Fusarium strains are less resistant than Aspergilus strains (not shown). Aspergillus flavus is the most resistant to tested compounds and the best antifungal agent against this mold is compound 10b. Aspergillus ochraceus is the most affected by 3b, 11b, 12b, all of them incorporating different ring systems and possessing the ethyl group in their structure. For Fusarium strains almost all compounds exhibit significant antifungal activity at given concentrations. The difference between sensitivity of Aspergillus and Fusarium strains may be due to their differences in cell structure. Since Aspergillus spp. is mainly human pathogen [28] and according to phylogenetic tree is more closely related to Penicilium spp. [29], [30] that can produce DPA [2], it is expected to have higher resistance to DPA derivatives (as confirmed). On the other hand, Fusarium spp. is mainly a plant pathogen/saprophyte [31] and, therefore, more susceptible to DPA. Aspergillus spp. is also charaterized by a great number of cell wall associated enzymes that can degrade (hydrolyze) synthesized compounds due to their unspecific activity [32], [33]. On the other hand, Fusarium spp. possess a cell wall associated unspecific hydrolases [34] that are mainly intended to increase their virulence toward plants [31], [34]. This is a great indicator for design and synthesis of some future antifungal agents, especially against Aspergillus strains, 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 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 [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 peptidoglycan, the essential component of the bacterial cell wall. In contrast, most antifungal compounds target either the formation or the function of ergosterol, an important component of the fungal cell membrane. Bacteria employ an extensive repertoire of plasmids, transposons, and bacteriophages to facilitate the exchange of resistance and virulence determinants among and between species. Conversely, antifungal resistance generally involves the emergence of naturally resistant species [36].

Conclusions

Dipicolinic acid derivatives were synthesized and investigated for antimicrobial and antioxidant activity. Some compounds show excellent antioxidant activity, the thiosemicarbazide 3c, in particular. Thiosemicarbazide 3b possess good antioxidant activity, but it is also an excellent antifungal agent against all four investigated fungi strains. Data collected in this research indicate that thiosemicarbazides could be used as a unique antifungal and antioxidant agents.

Experimental

Melting points were determined on an Electrothermal capillary apparatus. The elemental analysis for C, H and N was done on a Perkin-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.

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 (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].

Methyl 6-(4-methylthiosemicarbazide-1-yl-carbonyl)pyridin-2-carboxylate (3a)

This compound was obtained from 1 and methyl isothiocyanate; yield 90%; mp 210°C; 1H NMR: δ 2.85 (m, 3H, CH3), 3.93 (s, 3H, OCH3), 8.23–8.25 (m, 3H, py-H); 8.01 (d, 1H, J=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-(4-ethylthiosemicarbazide-1-yl-carbonyl)pyridin-2-carboxylate (3b)

This compound was obtained from 1 and ethyl isothiocyanate; yield 92%; mp 208°C; 1H NMR: δ 1.07 (m, 3H, CH3), 3.46 (m, 2H, CH2), 3.94 (s, 3H, OCH3), 8.08 (t, 1H, J=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], (M=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.

Methyl 6-(4-phenylthiosemicarbazide-1-yl-carbonyl)pyridin-2-carboxylate (3c)

This compound was obtained from 1 and phenyl isothiocyanate; yield 80%; mp 162–164°C; 1H NMR: δ 3.94 (m, 3H, OCH3), 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], (M=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.

2,6-Bis(4-methylthiosemicarbazide-1-yl-carbonyl)pyridine (4a)

This compound was obtained from 2 and methyl isothiocyanate; yield 74%; mp 202–207°C; 1H NMR: δ 2.70 (d, 6H, J=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], (M=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-ethylthiosemicarbazide-1-yl-carbonyl)pyridine (4b)

This compound was obtained from 2 and ethyl isothiocyanate; yield 78%; mp 219–220°C; 1H 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], (M=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-phenylthiosemicarbazide-1-yl-carbonyl)pyridine (4c)

This compound was obtained from 2 and phenyl isothiocyanate; yield 68%; mp 234°C (lit mp 245–247°C [8]); 1H NMR: δ 7.13–7.47 (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], (M=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.

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 from N,N-dimethylformamide (DMF) [37]: yield 38%; mp 287°C; 1H NMR: δ 1.25 (s, 6H, CH3), 3.39–3.46 (m, 4H, CH2), 7.85 (brs, H, NH), 7.86–8.11 (m, 2H, py-H); MS: m/z 334.2 [M+H]+, (M=333.4).

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 reaction 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-(5-methylamino-1,3,4-oxadiazol-2-yl)pyridine-2-carboxylate (6a)

Yield 76%; mp >300°C; 1H NMR: δ 3.83 (s, 3H, CH3), 4.07 (s, 3H, OCH3), 7.92–7.98 (m, 3H, py-H); 8.61 (m, 1H, NH); MS: m/z 234.90 [M–H], (M=234.2).

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]+, (M=397.40).

Preparations of triazoles 11a–c from 4a–c

A solution of 4a–c (10 mmol) in aqueous NaOH (2N, 10 mL) was heated 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-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], (M=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.

2,6-Bis(4-ethyl-4H-5-sulfanyl-1,2,4-triazol-3-yl)pyridine (11b)

Yield 72%; mp >300°C; 1H NMR: δ 1.15–1.20 (t, 6H, J=7.0 Hz, 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], (M=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.

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 7.03–7.45 (m, 10H, arom), 7.74–8.02 (3H, py-H); 14.17 (br.s, 2H, NH); MS: m/z 428.00 [M–H], (M=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.

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 bromoacetate (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.

Methyl 6-[(2-methylimino-4-oxothiazolidin-3-yl)carbamoyl]pyridine-2-carboxylate (8a)

Yield 82%; mp 212°C; 1H NMR: δ 3.17–3.30 (s, 3H, CH3), 3.95 (s, 3H, OCH3), 4.12 (s, 2H, CH2), 8.23–8.26 (m, 3H, py-H); 10.57 (s, H, NH); MS: m/z 307.10 [M–H], (M=308.3). Anal. Calcd for C12H12N4O4S (308.06): 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)carbamoyl]pyridine-2-carboxylate (8b)

Yield 70%; mp >300°C; 1H NMR: δ 1.16–1.34 (t, 3H, J=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], (M=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)carbamoyl]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], (M=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]pyridine (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], (M=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.

2,6-Bis[(2-ethylimino-4-oxothiazolidin-3-yl)carbamoyl]pyridine (12b)

Yield 70%, mp 214–217°C; 1H NMR: δ 1.20–1.24 (t, 6H, J=7.0 Hz, 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+], (M=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]pyridine (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], (M=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.

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 incubation the absorbance was read at 517 nm and the scavenging activity was calculated as described previously [17].

Antibacterial activity

The antibacterial activities of DPA derivatives were evaluated against four test bacteria strains. Two Gram-positive Bacillus subtilis and 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 bacteria, respectively. Staphylococcus aureus and Pseudomonas aeruginosa 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 suspension of colonies from a 24 h Mueller-Hinton Agar (MHA) plate culture of the microorganisms. Each well was inoculated with 300×103 bacteria (density of the bacterial suspension at 0.5 McFarland scale, which is 150×106 CFU/mL. Amikacin sulfate (AMCK) was co-assayed as positive 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 concentration of compound at which there was no visual turbidity due to microbial growth. All assays were performed in duplicate.

Antifungal activity

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), Aspergillus 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].

Acknowledgments

The authors are grateful to the Microbiology Service of the Public Health Institute of Osijek-Baranja County, Osijek, Croatia, for providing test bacteria strains.

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. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [5]

    Büyükkιdan, N.; Yenikaya, C.; İlkimen, H.; Karahan, C.; Darcan, C.; Şahin, E. Synthesis, characterization, and antimicrobial activity of a novel proton salt and its Cu(II) complex. Russ. J. Coord. Chem. 2012, 39, 96–103. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [11]

    Zamani, K.; Faghihi, K.; Tofighi, T.; Shariatzadeh, M. R. Synthesis and antimicrobial activity of some pyridyl and naphthyl substituted 1,2,4-triazole and 1,3,4-thiadiazole derivatives. Turk. J. Chem. 2004, 28, 95–100. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [14]

    Bano, Q.; Tiwari, N.; Giri, S.; Nizamuddin, S. Synthesis and fungicidal activities of 3-aryloxymethyl-6-substituted-1,2,4-triazole [3,4-b]-1,3,4-thiadiazole. Indian J. Chem. 1992, 31B, 714–718. Google Scholar

  • [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. Google Scholar

  • [16]

    Molnar, M.; Šarkanj, B.; Cačić, M.; Gille, L.; Strelec, I. Antioxidant properties and growth-inhibitory activity of coumarin schiff bases against common foodborne fungi. Pharma Chem. 2014, 6, 313–320. Google Scholar

  • [17]

    Šarkanj, B.; Molnar, M.; Cačić, M.; Gille, L. 4-Methyl-7-Hydroxycoumarin antifungal and antioxidant activity enhancement by substitution with thiosemicarbazide and thiazolidinone moieties. Food Chem. 2013, 139, 488–495. Google Scholar

  • [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. Google Scholar

  • [19]

    RamaGanesh, C. K.; Bodke, Y. D.; Venkatesh, K. B. Synthesis and biological evaluation of some innovative coumarin derivatives containing thiazolidin-4-one ring. Indian J. Chem. 2010, 49B, 1151–1154. Google Scholar

  • [20]

    Salimon, J.; Salih, N.; Hameed, A.; Ibraheem, H.; Yousif, E. Synthesis and Antibacterial activity of some new 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives. J. Appl. Sci. Res. 2010, 6, 866–870. Google Scholar

  • [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 oxadiazole substituted coumarins. J. Heterocycl. Chem. 2014, 51, 132–137. Google Scholar

  • [22]

    Mayekar, A. N.; Yathirajan, H. S.; Narayana, B.; Sarojini, B. K.; Suchetha Kumari, N. Synthesis and antimicrobial studies on new substituted 1,3,4-oxadiazole derivatives bearing 6-bromonaphthalene moiety. Int. J. Chem. 2010, 2, 38–54. Google Scholar

  • [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. CrossrefGoogle Scholar

  • [24]

    Foroughifar, N.; Mobinikhaledi, A.; Rafiee, A. Efficient synthesis 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. Google Scholar

  • [25]

    Kulp, A.; Kuehn, M. J. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu. Rev. Microbiol. 2010, 64, 163–184. Google Scholar

  • [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. Google Scholar

  • [27]

    Price, K. E.; DeFuria, M. D.; Pursiano, T. A. Amikacin, an aminoglycoside with marked activity against antibiotic-resistant clinical isolates. J. Infect. Dis. 1976, 134, S249–S261. Google Scholar

  • [28]

    Klich, M. A. Aspergillus flavus: The major producer of aflatoxin. Mol. Plant Pathol. 2007, 8, 713–722. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [32]

    Bernard, M.; Latgé, J.-P. Aspergillus fumigatus Cell wall: composition and biosynthesis. Med. Mycol. 2001, 39, 9–17. Google Scholar

  • [33]

    de Vries, R. P.; Visser, J. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiol. Mol. Biol. Rev. 2001, 65, 497–522. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [37]

    Narwade, S. K.; Halnor, V. B.; Dalvi, N. R.; Gill, C. H.; Karale, B. K. Conventional and ultrasound mediated synthesis of some thiadiazoles, triazoles and oxadiazoles. Indian J. Chem. 2006, 45B, 2776–2780. Google Scholar

  • [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. Google Scholar

  • [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. Google Scholar

  • [40]

    Hussein, H. S.; Brasel, J. M. Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology 2001, 167, 101–134. CrossrefGoogle Scholar

About the article

Received: 2016-05-21

Accepted: 2016-10-11

Published Online: 2017-01-18

Published in Print: 2017-02-01


Citation Information: Heterocyclic Communications, Volume 23, Issue 1, Pages 35–42, ISSN (Online) 2191-0197, ISSN (Print) 0793-0283, DOI: https://doi.org/10.1515/hc-2016-0078.

Export Citation

©2017 Walter de Gruyter GmbH, Berlin/Boston.Get Permission

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

[1]
Pratibha Kumari, Shagufi Naz Ansari, Ravi Kumar, Anoop Kumar Saini, and Shaikh M. Mobin
Chemistry & Biodiversity, 2019
[2]
Anita Blagus Garin, Dunja Rakarić, Elvira Kovač Andrić, Martina Medvidović Kosanović, Tomislav Balić, and Franc Perdih
Polyhedron, 2019, Volume 166, Page 226
[3]
Maja Molnar, Ivana Periš, and Mario Komar
European Journal of Organic Chemistry, 2019, Volume 2019, Number 15, Page 2688
[6]
M. Molnar, M. Tomić, and V. Pavić
Pharmaceutical Chemistry Journal, 2018
[7]
Harshad Brahmbhatt, Maja Molnar, and Valentina Pavić
Karbala International Journal of Modern Science, 2018

Comments (0)

Please log in or register to comment.
Log in