Microwave Assisted Synthesis and Antibacterial Evaluation of 1, 3, 4-Thiadiazole Derivatives
1College of Pharmaceutical Sciences, Government Medical College, Thiruvananthapuram, Kerala, India.
2Nirmala College of Pharmacy, Muvattupuzha, Kerala, India.
*Corresponding Author E-mail: Pmjayalakshmi94@gmail.com
1,3,4-Thiadiazole is an important heterocyclic moiety, forms an integral core structural component of different categories of drugs such as antimicrobial, antitubercular, anti-inflammatory, antiepileptic, antiviral, antineoplastics, and analgesic agents. It is a key moiety in current discovery and designing of new drugs. The compounds were synthesised by both conventional method and microwave method. The targeted derivatives can be synthesised in a shorter time under microwave condition than under conventional reaction condition. Their structures were confirmed by FT-IR and NMR Spectroscopy. Antibacterial property of two synthesised analogs were evaluated by Agar well diffusion method against Escherichia coli and Staphylococcus aureus. The results of antibacterial activity showed that both the compounds were active against Staphylococcus aureus and inactive against Escherichia coli. Results of invitro studies showed that modifications in SB-2-PHB and SB-8-PHB will make it as a promising lead molecule for further research.
KEYWORDS: 1, 3, 4-thiadiazole; Microwave method; Antibacterial activity; Agar well diffusion.
Multi-drug-resistant pathogens are a challenge for existing therapeutic options, and their increasing occurrence mandates the discovery and development of novel treatment strategies.1 Heterocyclic compounds are one of the most preferred compounds in antimicrobial studies and many involve 1,3,4-thiadiazole and their derivatives2. Thiadiazole nucleus is a core structural component of different categories of drugs3 such as antimicrobial4, antitubercular5, anti-convulsant6, anti- inflammatory7, antiviral8, antineoplastic9 and analgesic agents10.
For example, Acetazolamide, megazol, methazolamide, cefopram etc11. It also possess antioxidant12 and antidiabetic activity13. Hence, synthesis and characterization of such bioactive compounds containing 1, 3, 4-thiadiazole are being studied intensely14. Conventional synthesis reactions suffered from drawbacks such as the use of high boiling solvents, long reaction time and lower yields. MW irradiation is currently used to carry out a wide range of reactions.15,16 Compared with the traditional heating reactions, the microwave (MW) reaction technique is often rapid, more convenient and has environmental, and economic advantages17,18.
The purpose of this study is to synthesise various 1, 3, 4- thiadiazole derivatives by using both conventional and microwave method and perform the evaluation of antibacterial activity of synthesised molecules.
All the chemicals and reagents used were of analytical or synthetic grades.
Fig. No. 2.1: General scheme for synthesis
Step 1: Synthesis of 2-amino-5-aryl-1, 3, 4-thiadiazole derivatives (Conventional Method):
Thiosemicarbazide (0.05M) and appropriate aromatic carboxylic acid (0.05M) were taken into an RB flask and dissolved in alcohol (25ml) by shaking. To this solution concentrated sulphuric acid (10 drops) was added while shaking and the reaction mixture was heated under reflux for 1-2h, on a hot water bath. After completion of the reaction (monitored by TLC) alcohol was removed to a possible extent by distillation and the residue was cooled and triturated with crushed ice. The product was filtered, washed with small portion of cold water and dried. It was purified by recrystallization from hot alcohol TLC was carried out using ethanol: chloroform (0.5:4.5)
Thiosemicarbazide (0.05M) and appropriate aliphatic or aromatic carboxylic acid (0.05M) were taken into a beaker and dissolved in minor quantity of dimethyl formamide (10ml). To this solution concentrated sulphuric acid (10 drops) was added while stirring. A funnel was hanged in the beaker and covered with a watch glass. The reaction mixture was subjected to the microwave irradiation at 480w for 3-6 min, with a pulse rate of 30sec, each in a domestic LG little chef microwave oven. The solvent was removed by distillation and residue was cooled and triturated with crushed ice. The resultant product was filtered, washed with small portions of cold water and dried. It was purified by recrystallization from hot alcohol.19
TLC was carried out using ethanol: chloroform (0.5:4.5)
A mixture of compound 1 (0.01mole) and a suitable aromatic aldehyde (0.01mole) was refluxed in absolute ethanol (25ml) in presence of a few drops of glacial acetic acid for 4-6hours. The reaction mixture was cooled and the precipitate was filtered and recrystallized20.
TLC was carried out using ethylacetate: hexane (3:2)
A mixture of 2-amino-5-aryl-1,3,4-thiadiazole derivatives (0.01mole) and Suitable aromatic aldehyde (0.014mole) and a few drops of glacial acetic acid in toluene was stirred and irradiated in a WF4000M microwave fast reaction system under 300W for a few minutes at 110oC.
After cooling and filtering, crude compound was obtained. The pure compound was obtained after recrystallization from ethanol.21
TLC was carried out using ethylacetate: hexane (3:2)
Table No. 3.1: Physical characterisation of synthesised compounds-step1
|
Compound |
Molecular formula |
Molecular weight |
Colour |
Rf value |
Melting Point (ͦC) |
Percentage yield (%) |
|
|
Conventional |
Microwave |
||||||
|
ACT-02 |
C8H7N3OS |
364.39 |
Off white |
0.49 |
130 |
75 |
89 |
|
ACT-04 |
C9H9N3O2S |
394.42 |
Pale brown |
0.42 |
142 |
60 |
85 |
|
ACT-07 |
C8H6N4O2S |
393.39 |
Yellowish white |
0.39 |
150 |
70 |
90 |
|
ACT-08 |
C8H6BrN3S |
427.29 |
Off white |
0.46 |
165 |
56 |
80 |
Table No. 3.2: Physical characterisation of synthesised compounds-step2
|
Compound |
Molecular formula |
Molecular weight |
Colour |
Rf value |
Melting Point (ͦC) |
Percentage yield (%) |
|
|
Conventional |
Microwave |
||||||
|
SB-8-4NB |
C15H9BrN4O2S |
389.23 |
Brown |
0.52 |
180 |
67 |
85 |
|
SB-2-4NB |
C15H10N4O3S |
326.34 |
White |
0.73 |
146 |
72 |
82 |
|
SB-4-4NB |
C16H12N4O4S |
356.36 |
Yellow |
0.51 |
180 |
59 |
75 |
|
SB-8-PHB |
C15H10BrN3OS |
360.24 |
Brown |
0.64 |
195 |
62 |
75 |
|
SB-7-4CLB |
C15H9ClN4O2S |
344.78 |
Yellow |
0.48 |
165 |
71 |
89 |
|
SB-2-PHB |
C15H11N3O2S |
297.84 |
White |
0.62 |
140 |
75 |
86 |
|
SB-4-PHB |
C16H13N3O3S |
327.37 |
Brown |
0.5 |
167 |
68 |
78 |
In vitro antimicrobial screening of selected synthesised compounds was carried out using cultures of bacterial strains. It includes Gram positive bacteria, Staphylococcus aureus (NCIM 2127) and Gram negative bacteria Escherichia coli (NCIM 2065). Streptomycin was used as standard antimicrobial agent to evaluate the potency of the tested compounds under the same conditions.
The microorganism inoculums were uniformly spread using sterile cotton swabs on a sterile Petri dish nutrient agar. Each sample (250, 500 and 1000μL) was added to each well (6 mm diameter holes cut in the agar gel, 20 mm apart from one another). The systems were incubated for 24–48 h at 37 °C. After incubation, microorganism growth was observed. Inhibition of the bacterial growth were measured in mm. Tests were performed two times.22,23,24
3.1.1. Physical characterization:
The physical characterisation of synthesised compounds are shown in the table 3.1and 3.2.
Spectral characterisation of synthesised compounds are as follows
ACT-02: IR(KBr): 3064.89 cm-1, 1654.92 cm-1, 3387
cm-1 , 759.95 cm-1, 3421.72 cm-1, 1249.87 cm-1, 1296.16 cm-1,1386.82 cm-1,1614.42 cm-1
ACT-04: IR(KBr): 3080.32 cm-1, 1541.12 cm-1,
3485.37 cm-1, 3421.72 cm-1, 2845 cm-1, 1240.23 cm-1,
1028.06 cm-1, 881.47 cm-1, 1280.73 cm-1, 1182.36 cm-1,
1114.86 cm-1, 1386.82 cm-1, 1598.99 cm-1
ACT-07: IR (KBr): 3093.82 cm-1, 1618.28 cm-1, 1276.88 cm-1, 705.95 cm-1, 3373.5 cm-1, 1533.41 cm-1,
1348.24 cm-1, 1705.07 cm-1
ACT-08: IR (KBr):2873.94 cm-1, 1313.52 cm-1, 1261.45 cm-1, 702.09 cm-1, 3066.82 cm-1, 669.3 cm-1,
1172.72 cm-1, 1683.86 cm-1
SB-8-4NB: IR (KBr): 3046.81 cm-1, 1591.80 cm-1, 701.48 cm-1, 2802 cm-1, 1677.01 cm-1, 1066.50 cm-
1,1564.94 cm-1, 665.17 cm-1 1H NMR (400MHz, CDCl3) δ= 7.261(s, H), 7.349-7.388(m, 2H), 7.736-
7.764(m, 2H), 8.037-8.090(m, 2H), 8.253-8.261(m, 2H)
ppm.
SB-2-4NB: IR(KBr): 3106.02 cm-1, 1605.27 cm-1,
677.45 cm-1, 2850.16 cm-1, 1677.91 cm-1, 1194.69 cm-1,
1521.83 cm-1, 3491.44 cm-1, 1243.12 cm-1, 1341.83 cm-1
SB-4-4NB: IR(KBr): 3105.28 cm-1, 1596.00 cm-1,
676.56 cm-1, 2980.49 cm-1,1673.91 cm-1, 1285.88 cm-1,
1520.22 cm-1, 3480.93 cm-1, 1285.88 cm-1, 1343.47 cm-1,
2849.19 cm-1, 1232.89 cm-1, 1027.20 cm-1
SB-8-PHB: IR(KBr): 2918.57 cm-1, 1565.50 cm-1,
701.10 cm-1, 2855 cm-1, 1678.30 cm-1, 1066 cm-1,
3518.24 cm-1, 1258.40 cm-1, 1406.50 cm-1, 547.26 cm-1
1H NMR (400MHz, CDCl3) δ= 7.261(she), 7.349-
7.388(m, 2H), 7.737-7.764(m, 2H), 8.038-8.064(m, 2H),
8.253-8.262(m, 2H) ppm
SB-7-4CLB: IR(KBr): 3084.19 cm-1, 1615.97 cm-1, C-
S-C stretching:700.66 cm-1, 2823 cm-1, 1697.06 cm-1, 1149.34 cm-1, 1525.22 cm-1, 614.23 cm-1
SB-2-PHB: IR(KBr): 3064.98 cm-1, 1580.52 cm-1,
690.20 cm-1, 2850.67 cm-1, 1651.95 cm-1,CH1206.90
cm-1, OH stretching:3228.90 cm-1, 1243.16 cm-1, 1206.90 cm-1 1H NMR (400MHz, CDCl3) δ= 6.926-
6.966(m, 2H), 7.008-7.031(m, 2H), 7.259(she), 7.512-
7.556(m, 2H), 7.927-7.951(m, 2H), 10.372(s, 2H) ppm SB-4-PHB: IR(KBr):3097.38 cm-1, 1595.26 cm-1, 636.45 cm-1, 2953.40 cm-1, 1670.86 cm-1, 1109.56 cm-1 ,
3480.33 cm-1, 1201.87 cm-1, 1376.67 cm-1, 2848.86 cm-1,
1236.85 cm-1, 1027.31cm-1
Table No. 3.3: Antibacterial activity of selected 1, 3, 4-thiadiazole derivatives
|
Compound |
Parameters |
Test result |
Test method |
|||||
|
Zone of Inhibition in diameter (mm) |
||||||||
|
1000 μg/ml |
500 μg/ml |
250 μg/ml |
||||||
|
SB-2-PHB |
Staphylococcus aureus (NCIM 2127) |
I |
II |
I |
II |
I |
II |
Agar well diffusion method |
|
10mm |
11mm |
10mm |
11mm |
No Zone |
No Zone |
|||
|
Escherichia coli (NCIM 2065) |
No Zone |
No Zone |
No Zone |
No Zone |
No Zone |
No Zone |
||
|
SB-8-PHB |
Staphylococcus aureus (NCIM 2127) |
10mm |
10mm |
No Zone |
No Zone |
No Zone |
No Zone |
Agar well diffusion method |
|
Escherichia coli (NCIM 2065) |
No Zone |
No Zone |
No Zone |
No Zone |
No Zone |
No Zone |
||
The synthesised compounds were subjected to invitro Antibacterial screening against Staphylococccus aureus (NCIM 2127) and Escherichia coli (NCIM 2065). Streptomycin was used as standard for both gram positive (Staphylococcus aureus NCIM 2127) and gram negative organism (Escherichia coli NCIM 2065) (Table 3.3).
Compounds were synthesised by microwave and conventional methods where percentage yield of compound obtained is higher for microwave method than conventional method. Physical characterisation and spectral characterisation of synthesised compounds were performed. Among these synthesised compounds, two compounds, SB-2-PHB and SB-8-PHB were selected for evaluation of antibacterial activity. Both the compounds were inactive against E coli and shows moderate to good activity against Staphylococcus aureus. The entire work focused on the development of better antibacterial agents and these compounds can be considered as a promising lead molecule for further research.
Our heartful thanks to the Principal, College of Pharmaceutical sciences, government medical college, Thiruvananthapuram, for granting permission to utilize the facilities to carry out this work. I express my sincere gratitude to Care Keralam, Koratty for doing pharmacological screening of synthesised compounds.
The authors have no conflicts of interest to declare that they are directly relevant to the content of this manuscript
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Received on 06.09.2020 Modified on 08.11.2020
Accepted on 17.12.2020 © RJPT All right reserved
Research J. Pharm. and Tech 2021; 14(10):5293-5296.
DOI: 10.52711/0974-360X.2021.00923