Synthesis and characterisation of CuO nanoparticle: its electrochemical paracetamol sensor activity and substituted-2-aminothiophene synthesis applications

Abstract

In the present work, the synthesised nanoparticle was examined by multiple characterisation techniques for physico-chemical, surface-morphological, elemental, and optical properties. These structural parameters of achieved nano-catalyst was studied by using P-XRD, SEM-EDAX, FT-IR and Energy band gap techniques. The electrochemical analysis of synthesised CuO nanoparticle modified with carbon paste was investigated using 0.1 M KCl under different scan rates of 0.01–0.05 V/s. The sensing activity of prepared material was performed for Paracetamol medicine at concentration 1–5 mM in the potential range of–1.0 V to + 1.0 V using cyclic voltametric analysis. Microwave accelerated synthesis of substituted-2-aminothiophene by a 3-component (acetophenone, malononitrile and elemental sulphur) one pot Gewald reaction using low-cost nano-catalyst (CuO nanoparticle) to perform for chemical reactions. The synthesised compounds were confirmed by FT-IR, ¹HNMR, ¹³C NMR and Mass spectral techniques. The antibacterial activity was examined using Bacillus cereus (gram positive) & Ciprofoxin (gram negative) organism and evaluated antifungal activities using Candida albicans (gram positive) & Itraconazole (gram negative) organism for prepared organic compounds tested under agar diffusion method. Molecular docking studies were discussed for both carboxylate and carbonitrile derivatives at the active site of the bacterial protein GlcN-6-P synthase. Green chemistry encourages the design of synthetic processes that minimise the use and generation of hazardous substances.

Keywords:

• Substituted-2-aminothiophene
• gewald reaction
• microwave accelerated organic synthesis
• nano-catalyst

1. Introduction

The thiophene derivatives were synthesised by Gewald reaction method, which is a highly versatile and significant reaction for the development of biological active compounds and intermediates containing a thiophene moiety (Abd-El-Aziz and Afifi, 2006; Abd-El-Aziz and Afifi, 2006; Anil Kumar et al., 2018; Anil Kumar et al., 2018; Basavaraju et al., 2021). The benzene derivative of thiophene compounds plays a vital role in bio-isosterism. However, the replacement of a benzene moiety by a thiophene ring in a pharmacologically active substance for the development of drug design activities (reaction-1) (Bhagyalakshmi and Surendra, 2022; Channagiri and Reddy, 2015). The necessity of synthesising different substituted-2-aminothiophenes has been revealed by the Pharmaceutical, Agriculture and Food industries due to their stable structure of thiophene derivatives. Therefore, the current investigation focused on designing and synthesis of substituted thiophene derivative carried by using specific nanoparticle under a microwave oven in order to improve yield and reduce the reaction time. The Gewald reaction for the synthesis of active substances containing a thiophene moiety is well known in research survey towards access of several 2-aminothiophenes in a simple method. The most elegant and simple one-pot route involves the reaction among ketones or 1,3-dicarbonyl species, activated nitriles and elemental sulfur using CuO nanoparticles.

Nowadays, the nanomaterials have got more attention due to their physico-chemical properties like low cost, more efficiency, easy separation, recyclability, and eco-friendly nature towards materials having size come close to the nanoscale (Chavan et al., 2012; Chinnam et al., 2012; Demir et al., 2020; Isildak et al., 2018; Jagdale and Pathade, 2019). The nanomaterials have been gaining the attention of researchers to enhancing new way for the synthesis of sustainable compounds and hence, which has been successfully applied as a heterogeneous catalyst for organic synthesis. Thus, the nanomaterials are makes them an exciting candidate for multiple applications such as supercapacitors, waste water treatment, gas sensors, electrochemical, sensors, and photocatalytic degradation (Jamballi Manjunatha, 2020; Jamballi et al., 2021). Therefore, the CuO nanoparticles are potential candidate for selectivity and catalytic studies towards substituted thiophene derivatives. Further, the achieved compounds were subjected to examine antibacterial and antifungal activities and their effects by interaction with living cells (Kubelka and Munk, 1931).

Nowadays, the extensive research has been focused on nanotechnology for demonstrating the importance of paying attention towards electrochemical analysis (Mahadeva Swamy et al., 2021; Department of Chemistry, FMKMC College, Mangalore University Constituent College Madikeri–571201, Karnataka, India, 2018). The copper-based nanoparticle research is drawn its attentions much potentially towards their excellent properties in multiple applications such as electrical resistivity, photocatalysis, antimicrobial applications, sensor activity for humidity and chemical detections. Presently, our research focused on the qualitative measurement of electrochemical reactions of modified CuO-carbon electrode paste in a 3-electrode system using 0.1 M KCl electrolytic solution by performing Cyclic Voltammetric (CV) and Electrochemical Impedance Spectroscopy (EIS) techniques. The CV studies reveals an potential activity towards measurement of thermodynamics of redox reaction and capacitance carried out by cycling the potential of modified CuO-carbon working electrode in the different scan rates of 0.01–0.05 V/s at RT. The antimicrobial assets are seriously investigated because of a vast increase in bacterial resistance against the unnecessary and frequent usage of antibiotics, which supports the development of new drug substances and challenging task (Manjunatha, 2013; Manjunatha, 2020; Manjunatha et al., 2011). Thus, we are focused for examination of antimicrobial studies of achieved organic compounds of by Gewald reaction process. The present work reported that the synthesis of substituted 2-aminothiophene using nano CuO catalyst in one pot Gewald reaction (Reaction 1) and its structural characterisations were investigated by well spectroscopic methods. Further, its application extended towards antimicrobial activities carried out by disc diffusion method.

2. Experimental section

2.1. Preparation of CuO NPs

The nano CuO catalyst was achieved by taking stoichiometric ratios of analytical grade chemical precursors such as Cu(NO₃)₂.3H₂O (S.D. Fine Chemicals Company Ltd.) and Urea as a fuel.This chemical mixture was placed into silica crucible and subjected to stirring in presence of less quantity distilled water for about 30 min to attaining homogeneous nature. Further, the prepared mixture was placed in to muffle furnace with retained temperature of 430 °C, which is instantly started to boiling and catches the fire inside and produced a blackish powder material within 3 min duration.

2.2. Typical procedure for the synthesis of (3D) 5-acetyl-2-amino-4-methyl thiophene-3-carbonitrile under microwave accelerated method

A mixture of Acetyl acetone (0.1 mole), Malononitrile (0.1 mole), Elemental Sulphur (0.05 mole) in Ethanol (15 ml) were charged to a 250 ml round bottom flask in presence of 0.001 mole of nano catalyst CuO. The above solution mixture was kept in microwave oven with maintained temperature of 70 °C for 8 minutes. The progress of the reaction was monitored by TLC. After the completion of reaction, filter the reaction mixture, wash with ethanol, to the filtrate check pH by using litmus paper then keep the filtrate in oven 5 minutes to remove excess ethanol, after attaining room temperature add ice cubes we get brown coloured precipitate, workup using cold water, quenched with NH₄Cl, extracted by diethyl ether and dried over anhydrous Na₂SO₄, recrystallized by hot ethanol and hot water, dried to afford corresponding substituted 2-aminothiophene, The achieved yield of the product has been reported in the Table 1. the schematic representation of the synthesised 5-acetyl-2-amino-4-methyl thiophene-3-carbonitrile under microwave method as shown in Figure 1.

Figure 1. Flow chart for the synthesis of 5-acetyl-2-amino-4-methyl thiophene-3-carbonitrile under microwave accelerated method.

Table 1. Chemical data of synthesised compounds 3D,NR9 & NR10.

S.N	Name of Compound	Molecular formula & Molecular mass	Structural formula	Melting Point(°C)	Yield %	Colour
1	5-acetyl-2-amino-4-methyl thiophene-3-carbonitrile	C8H8ON2S & 180.23 g/mole		253-255	45.52	Brown
2	2-amino-4-(4-nitrophenyl) thiophene-3-carbonitrile	C11H7N3O2S & 245.26 g/mole		99-102	49.66	Light brown
3	2-amino-4-(2-bromophenyl) thiophene-3-carbonitrile	C11H7BrN2S & 279.16 g/mole		323-325	25.19	Brown
4	Ethyl-5-amino-4-cyano-3- methylthiophene- 2-carboxylate	C9H10N2O2S & 211.17 g/mole		294-295	24.74	Black
5	2-amino-4-(3- hydroxyphenyl) thiophene-3- carbonitrile	C11H8N2OS & 217.26 g/mole		361-363	14.34	Light brown
6	2-amino-4- (4-flurophenyl) thiophene-3- carbonitrile	C11H7FN2S & 219.24 g/mole		265-266	2.126	White

2.3. Characterisations

The synthesised compounds were identified from their spectroscopic data. FTIR spectra were obtained on a Brucker infra-red spectrometer, ¹HNMR spectra were recorded in CDCl₃ on a Jeol 400 MHz spectrometer, ¹³C NMR spectra in CDCl₃/DMSO-d6 as a solvent was recorded on Bruker spectrometer with TMS as internal standard & Mass spectral data was obtained using thermo scientific corporation, DSQ II mass spectrometer.The phase identity and crystalline size of CuO Nps were characterised by Shimadzu Xray diffractometer (PXRD-7000) using Cu-Kα radiation of wavelength λ = 1.541 Ǻ.Morphological features were studied by using Hitachi-7000 Scanning Electron Microscopy (SEM).v

3. Results and discussion

3.1. X-ray diffraction examination

The structural formation and confirmation of CuO nanoparticle prepared by microwave irradiation process was demonstrated by XRD analysis as shown in Figure 2. In the XRD pattern, the diffraction lines of prepared nanoparticle are with 2θ standards of 34.2°, 36.95°, 39.28°, 46.92°, 50.58°, 54.2°, 56.4°, 60.2°, 61.7°, 64.8° and 67.1°, which are matching to the diffraction peaks of (11 0), (−1 1 1), (111), (−202), (020), (202), (−113), (−311), (220), (311), and (004) shows the monoclinic phase of CuO nanoparticle. Thus, the existence of diffraction peaks at $2\mathrm{\theta }$ values were well matchs with JCPDS Card No. 41-254 (Mohammad et al., 2020). The obtained XRD pattern of prepared CuO nanoparticle was confirms purity of the material by observing absence of additional peaks in the plot. The existence sharp and strong intensity of peaks designated as high crystallinity and its average particle size was found to be ∼ 21 nm calculated bythe Scherrer’s process (Equation 1(1) $$\mathrm{d}=\frac{\text{kλ}}{\text{β cosθ}}$$(1) ) (Mylarappa et al., 2022; N et al., 2021). (1) $$\mathrm{d}=\frac{\text{kλ}}{\text{β cosθ}}$$(1)

Figure 2. PXRD Patterns of CuO nanoparticle prepared by microwave method.

3.2 Scanning Electron Microscopy (SEM) studies

The SEM images of synthesised CuO nano-material are presented in Figure 3a&b. The morphological changes of the CuO sample is somewhat porous and well agglomerated, which is believed to be advantageous to enhance the properties. Generally, the formation of voids and pores are the characteristics of combustion method (PrinithJamballi and Manjunatha, 2020). Figure 3c shows the EDAX analysis of CuO nanomaterial, which is confirms the elemental compositions present in prepared CuO nanomaterial. The prepared CuO nanoparticles consists Cu and O elements without any additional elements (Inset Figure 3c), which corresponds to the purity of prepared sample.

Figure 3. (a&b) SEM Micrographs;and (c) EDAX analysis of prepared CuO nanoparticles.

3.3 FT-IR examination

The FT-IR spectrum of synthesised CuO nanoparticle from by chemical combustion method was observed in the range between 4000–400 cm⁻¹ as shown in the Figure 4. The broad band appear at 3347 cm⁻¹ indicating stretching vibrations of OH^- group in prepared CuO nanoparticle, which is due to existence of water content in the crystal lattice of material. Similarly, the appearance of bands at 1512 cm⁻¹shows stretching vibrations of unsaturated carbon-carbon (C=C) or carboxyl (C=O)functional groups. The bands appeared in low frequency region representing the bending vibrations of metal-oxygen (M-O) linkages observed in the range between 1000-500 cm⁻¹. The observed band at 632 cm⁻¹ in finger-print area representing a bending vibrations of co-ordination linkage of Cu-O group (Naresh et al., 2014; Nasihat Sheno and Morsali, 2012).

Figure 4. FT-IR spectral examination of CuO nanoparticle synthesised by combustion method.

3.4. Energy band gapinvestigation of CuO nanoparticle

The energy band gap of prepared CuO nanoparticle was examined using Diffuse Reflectance spectroscopy (DRS)in the range of 800–200 nm. Figure 5 shows band gap of CuO nanoparticle achieved by combustion process and its optical properties were calculated by using Kubelka-Munk method (Equation 2(2) $$F\left(R\right)=\frac{{(1-R)}^2}{2R}=\frac{K}{S}$$(2) ) with correlation between the diffuse reflectance of the CuO nanoparticle(R), the absorption coefficient (K) and the scattering coefficient (S) (Pompapathi et al., 2023); (2) $$F\left(R\right)=\frac{{(1-R)}^2}{2R}=\frac{K}{S}$$(2)

Figure 5. Diffuse Reflection spectra and wood and tauc’s(inset Fig) plot to find energy band gap CuO nanoparticle.

Tauc equation used for band-gap energy and linear absorption coefficient (α) of prepared CuO nanoparticle as expressed by Equations 3 & 4(3) $$\mathrm{⍺}=\frac{{C_1(h\nu -Eg)}^{\frac{1}{2}}}{h\nu }$$(3) ; (3) $$\mathrm{⍺}=\frac{{C_1(h\nu -Eg)}^{\frac{1}{2}}}{h\nu }$$(3) (4) $${\left[F\right(R\left)h\nu \right]}^2=C_2\left[h\nu -Eg\right]$$(4)

From the [F(R)hν]²versus hνplot, the value of Egwas obtained by extrapolating the linearly fitted regions to [F(R)hν]²=0 and the energy band gap of CuO nanoparticle was found to be 3.39 eV (inset Figure 5).

4. Synthesis of substituted-2-aminothiophene via Gewald reaction under microwave process

4.1 Synthesis of (3 C) ethyl-5-amino-4-cyano-3-methylthiophene-2-carboxylate

The 0.01 mole of ethylacetoacetate, 0.01 mole of malononitrile, 0.005 mole of sulphur are dissolved by heating with 5 ml of toluene in round bottom flask using 0.001 mole of nano catalyst CuO. Add 20 ml of ethanol maintain temperature of 70 °C for 8 minutes and place TLC for checking reaction completion, work-up using ethanol:methanol (1:2 ratio) solvent (Reaction 2 and its mechanism). The achieved yield of the product has been reported in the Table 1. Black precipitate, m.p.: 294-295 °C, MS: m/e = 211.17 (M + 1); ¹H NMR (400 MHz, DMSO-d₆) δ: 4.29(s, 2H, CH₂), 4.0(s, 2H, NH₂), 2.21(d, 3H, CH₃), 1.30 (d, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.6, 152.2, 147, 146, 115.3, 84.7, 60.9, 14.1, 4.0.

4.2 Compound 3D: 5-acetyl-2-amino-4-methyl thiophene-3-carbonitrile

A mixture of Acetyl acetone (0.01 mole), Malononitrile (0.01 mole), Elemental Sulphur (0.005 mole) in Ethanol (15 ml) were charged to a 250 ml round bottom flask in presence of 0.001 mole of nano catalyst CuO. The above solution mixture was kept in microwave oven with maintained temperature of 70 °C for 8 minutes. The progress of the reaction was monitored by TLC. After the completion of reaction, filter the reaction mixture, wash with ethanol, to the filtrate check pH by using litmus paper then keep the filtrate in oven 5 minutes to remove excess ethanol, after attaining room temperature add ice cubes we get brown coloured precipitate, workup using cold water, brown amorphous precipitate was formed filter and washed with cold water, dried to afford corresponding substituted 2-aminothiophene (Reaction 3 and its mechanism), The achieved yield of the product has been reported in the Table 1. Brown precipitate; m.p.: 253–255 °C; IR (KBr) (cm⁻¹): 3100 (C-H), 2607 (C≡N), 1600 (C-H), 1030 (C-S); ¹H NMR (400 MHz, DMSO) δ: 2.21(d, 3H, CH₃), 2.55 (d, 3H, CH₃), 4.0(s, 2H, NH2); ¹³C NMR (100 MHz, DMSO-d₆) δ: 190.5, 163.7, 146, 146, 115.3, 84.8, 28.4, 4.11.

4.3 Compound NR9: 2-amino-4-(4-nitrophenyl)thiophene-3-carbonitrile

A mixture of 4-nitroacetophenone (0.1 mole), Malononitrile (0.1 mole), Elemental Sulphur (0.05 mole), CuO nanocatalyst (0.001 mole), Ethanol (15 ml) were charged to a 250 ml Round bottom flask, kept in microwave oven maintain temperature 70 °C for 14 minutes. The reaction completion was monitered by TLC, after the completion filter the reaction mixture, check pH then keep the filtrate in oven for few minutes to remove excess of ethanol, remove filtrate from oven keep it for attaining room temperature add ice cubes we get light brown coloured precipitate, washed with cold water, quenched with NH₄Cl, extracted by diethyl ether and dried over anhydrous Na₂SO₄, recrystallised by hot ethanol and hot water, dried to afford corresponding substituted-2-aminothiophene. (Reaction 4 and its mechanism). The achieved yield of the product has been reported in the Table 1. Light brown precipitate, m.p.: 99–102 °C, MS: m/e = 246.11 (M + 1); ¹H NMR (400 MHz, DMSO) δ: 8.25(t, 1H, Ar-H), 7.74(t, 1H, Ar-H), 8.25 (t, 1H, Ar-H), 6.5(m,1H, Ar-H), 4.0(s, 2H, NH2); ¹³C NMR (100 MHz, DMSO-d₆) δ: 148.4, 145.0, 142.5, 140.0, 128.9, 128.4, 128.4, 121.6, 121.6, 115.8, 108.0.

4.4 Compound NR10: 2-amino-4-(2-bromophenyl)thiophene-3-carbonitrile

A mixture of 2-bromoacetophenone (0.1 mole), Malononitrile (0.1 mole), Elemental Sulphur (0.05 mole), CuO nanocatalyst (0.001 mole), Ethanol (15 ml) were charged to a 250 ml Round bottom flask, kept in microwave oven maintain temperature 70 °C for 14 minutes. The reaction completion was monitered by TLC, after the completion filter the reaction mixture, check pH then keep the filtrate in oven for few minutes to remove excess of ethanol, remove filtrate from oven keep it for attaining room temperature add ice cubes we get brown coloured precipitate, washed with cold water, quenched with NH₄Cl, extracted by diethyl ether and dried over anhydrous Na₂SO₄, recrystallised by hot ethanol and hot water, dried to afford corresponding substituted-2-aminothiophene. (Reaction 5 and its mechanism). The achieved yield of the product has been reported in the Table 1. brown precipitate, m.p.: 323-325 °C, MS: m/e = 281.17 (M + 2); ¹H NMR (400 MHz, DMSO-d₆) δ: 7.11(t, 1H, Ar-H), 7.26(t, 1H, Ar-H), 7.37 (t, 1H, Ar-H), 6.5 (m, 1H, Ar-H), 7.49 (t, 1H, Ar-H), 4.0 (s, 2H,NH2); ¹³C NMR (100 MHz, DMSO-d₆) δ: 145.0, 140.0, 136.2, 132.2, 131.0, 129.7, 128.9, 128.3, 120.3, 115.3, 108.

4.5 Compound NR11: 2-amino-4-(3-hydroxyphenyl)thiophene-3-carbonitrile

A mixture of 3-hydroxy acetophenone (0.01 mole), malononitrile (0.01 mole), sulphur (0.005 mole) were dissolved by heating with 5 ml of toluene in a round bottom flask using 0.001 mole of nano catalyst CuO. Add 20 ml of ethanol maintain temperature of 70 °C for 8 minutes and place TLC for checking reaction completion, work-up using ethanol:methanol (1:2 ratio) solvent (Reaction 6 and its mechanism). The achieved yield of the product has been reported in the Table 1. Light brown precipitate, m.p.: 361-363 °C, MS: m/e = 217.26 (M + 1); ¹³C NMR (100 MHz, DMSO-d₆) δ: 159.0, 145, 140, 137.8, 130.7, 128.9, 120.1, 115.9, 115.3, 112.9, 108.

4.6 Compound NR13: 2-amino-4-(4-fluorophenyl)thiophene-3-carbonitrile

Take 0.01 mole of 4-fluoro acetophenone, 0.01 mole of malononitrile, 0.005 mole of sulphur dissolved by heating with 5 ml of toluene, add dissolved sulphur to round bottom flask, add 20 ml of ethanol maintain temperature of 70 °C for 20 minutes, put TLC for checking reaction completion, work-up using ethanol:methanol = 1:2 ratio.kept ppt for dry. The achieved yield of the product has been reported in the Table 1. White precipitate, m.p.: 265–266 °C, MS: m/e = 219.24 (M + 1).

5. Biological activity

The antimicrobial activity of newly synthesised compounds 3 C, 3D, NR9, NR10, NR11 & NR13 were performed under disc diffusion method, as recommended by the National committee for clinical laboratory, the synthesised compound is used at the concentration of 50 µl, 150 µl and 200 µl. The antibacterial activity of 5-acetyl-2-amino-4-methylthiophene-3-carbonitrile, 2-amino-4-(4-nitrophenyl) thiophene-3-carbonitrile & 2-amino-4-(2-bromophenyl) thiophene-3-carbonitrile were screened against Bacillus cereus (gram positive) & Ciprofoxin (gram negative) organism, synthesised compound exhibit moderate antibacterial activity against both bacteria, antibacterial activity as shown in the Table 2.

Table 2. Antibacterial activity of synthesised.

Organism: Bacillus cereus (Gram + ve), Control : 11mm (Ciprofoxacin)
Compound	Conc.1 (50 µl)	Conc.2 (150 µl)	Conc.3 (200 µl)
3C	6mm	9mm	10mm
3D	4mm	6mm	9mm
NR9	6mm	9mm	14mm
NR10	7mm	10mm	15mm
NR11	6mm	8mm	10mm
NR13	–	–	–

The antibacterial agents are allowed to diffused out into the medium and interact in a plate freshly seeded with the test organisms. The resulting zones of inhibition will be uniformly circular as there will be a confluent lawn of growth. The diameter of zone of inhibition can be measured in millimetres. The disc diffusion method for antibiotic susceptibility testing is the Kirby Bauer method. The agar used is LB agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values. There is also a zone of intermediate resistance indicating that some inhibition occurs using this antimicrobial but it may not be sufficient inhibition to eradicate the organism from the body (Pradeepa Kumara et al., 2017; Sivakumar and Haranadha Reddy, 2011; Srivastava and Das, 2011; Surendra et al., 2022; Surendra et al., 2020).

Petriplates containing 25 ml MH agar were seeded using glass rod with 24 hr (old) culture of different bacterial strains separately. Spread plate method was followed. Wells were made using well-borer. Stock concentration 10 mg in 700 µl and standard drug (cifrofloxacin) 30 µl.The plates were then incubated at 37 °C for 24 hours. The antibacterial activity was confirmed by measuring the diameter of the inhibition zone formed around the well as shown in Figure 6.

Figure 6. (a) Control; inhibition zone of Bacillus cereus (gram positive) & Ciprofoxin (gram negative) organism for control; and (b-f) inhibition zone of Bacillus cereus (gram positive) organism for synthesised 3 C, 3D, NR9, NR10, and NR11 compounds respectively.

Antifungal activity of 5-acetyl-2-amino-4-methylthiophene-3-carbonitrile was screened against Candida albicans (gram positive), Itraconazole (gram negative) organism, synthesised NR10 compound exhibit excellent anti-fungal activity against both fungi, anti-fungal activity thanthose of 3 C, 3D, NR9, NR11 & NR13 as shown in the following Table 3. Petriplates containing 25 ml optimised media were seeded using glass rod with 24 hr (old) culture of Candida albicans strains separately. Spread plate method was followed. Wells were made using well-borer. Stock concentration 10 mg in 700 µl and standard drug (Itracanozole) 30 µl.The plates were then incubated at 37 °C for 24 hours. The antifungal activity was confirmed by measuring the diameter of the inhibition zone formed around the well as shown in Figure 7.

Figure 7. (a–e) Inhibition zone of Candida albicans (gram positive), Itraconazole (gram negative) organism for synthesised compound.

Table 3. Antifungal activity of synthesised.

Organism: Candida albicans, Control : 20mm (Itraconazole)
Compound	Conc.1 (50 µl)	Conc.2 (150 µl)	Conc.3 (200 µl)
3C	12mm	14mm	17mm
3D	–	3mm	9mm
NR9	15mm	20mm	24mm
NR10	12mm	13mm	13mm
NR11	–	–	–
NR13	–	–	–

6. Antidiabetic activity

The alpha amylase inhibitory activity was found at 250 µg/ml concentration as given in Table (4-6) and its plot as displayed in Figure (8–10). The IC50 value of NR9, NR10 and the standard drug was 181.86 µg/ml (Table 4), 125.21 µg/ml (Table 5) and 100.14 µg/ml (Table 6) respectively. These results indicate that the NR9 & NR10 derivative is more effective than the standard drug in inhibiting alpha amylase activity. Therefore, the NR9 compound can be used to treat diabetes.

Figure 8. The alpha amylase inhibitory activity at of 250 µg/ml concentration for standard Acarbose.

Figure 9. The alpha amylase inhibitory activity at of 250 µg/ml concentration for NR9.

Figure 10. The alpha amylase inhibitory activity at of 250 µg/ml concentration for NR10.

Table 4. Antidiabetic activity of standard α-amylase (Pancreatic) inhibition assay.

Concentrations µg/ml	1	2	3	Mean	Std. Dev	Std. error	% Std. error	% Inhibition
50	0.977	0.989	0.974	0.98	0.0079373	0.004582576	0.319640248	31.64380377
100	0.688	0.681	0.692	0.687	0.0055678	0.00321455	0.224218804	52.08091142
150	0.467	0.473	0.458	0.466	0.0075498	0.004358899	0.304038522	67.49593118
200	0.254	0.263	0.269	0.262	0.0075498	0.004358899	0.304038522	81.72518019
250	0.0846	0.0853	0.0862	0.08536667	0.0008021	0.000463081	0.047398308	94.0455708

Table 5. Antidiabetic activity of NR9 compound.

Concentrations µg/ml	1	2	3	Mean	Std Dev	Std error	% Std error	% Inhibition
50	1.318	1.327	1.314	1.31966667	0.0066583	0.003844188	0.268136773	7.951639154
100	1.135	1.139	1.143	1.139	0.004	0.002309401	0.161083544	20.55335968
150	0.892	0.885	0.881	0.886	0.0055678	0.00321455	0.224218804	38.20041851
200	0.577	0.581	0.573	0.577	0.004	0.002309401	0.161083544	59.75354569
250	0.412	0.424	0.416	0.41733333	0.0061101	0.003527668	0.267653142	70.89049058

Table 6. Antidiabetic activity of NR10 compound.

Concentrations µg/ml	1	2	3	Mean	Std Dev	Std error	% Std error	% Inhibition
50	1.008	0.991	0.988	0.99566667	0.0107858	0.006227181	0.434353446	30.55103464
100	0.842	0.852	0.841	0.845	0.0060828	0.003511885	0.244958237	41.06021855
150	0.614	0.617	0.606	0.61233333	0.0056862	0.003282953	0.228989951	57.28900256
200	0.433	0.429	0.427	0.42966667	0.0030551	0.001763834	0.123029589	70.03022553
250	0.197	0.211	0.216	0.208	0.0098489	0.005686241	0.564111181	85.49174611

7. Anti-inflammatory activity

The highest rate of inhibition of protein denaturation was observed at a concentration of 250 µg/ml as given in Table-(7-9) and its plot as displayed in Figure (11–13). The inhibition rates of NR9, NR10 were significantly higher than aspirin (p < 0.05). the IC50 value of NR9, NR10 and aspirin was found to be 152.59 µg/ml (Table 7), 180.78 µg/ml (Table 8), and 109.09 µg/ml respectively (Table 9). These findings demonstrate the potency of the NR9, NR10 molecules as an inflammatory drug.

Figure 11. The plots of anti-inflammatory activity study of standard protein.

Figure 12. The plots of anti-inflammatory activity study of NR9.

Figure 13. The plots of anti-inflammatory activity study of NR10.

Table 7. Anti-inflammatory activity study of standard protein denaturation method.

Concentrations ug/ml	1	2	3	Mean	Std Dev	Std error	% Std error	% Activity
50	0.2562	0.2371	0.2448	0.24603333	0.0096095	0.005548073	1.574665867	30.17029328
100	0.1988	0.1883	0.1678	0.18496667	0.0157665	0.009102808	2.583578393	47.50236518
150	0.1431	0.1235	0.1142	0.12693333	0.0147527	0.008517498	2.417454587	63.97350993
200	0.06413	0.08422	0.05419	0.06751333	0.0152982	0.00883243	2.506839029	80.83822138
250	0.02365	0.03372	0.04367	0.03368	0.0100101	0.005779311	1.640296348	90.44087039

Table 8. Anti-inflammatory activity study of NR9:.

Concentrations ug/ml	1	2	3	Mean	Std Dev	Std error	% Std error	% Activity
50	0.2929	0.2734	0.2616	0.27596667	0.0158071	0.009126214	2.590221505	21.67455061
100	0.2359	0.2167	0.2281	0.2269	0.0096561	0.005574944	1.582292511	35.60075686
150	0.1833	0.1642	0.1692	0.17223333	0.0099047	0.005718489	1.623033643	51.11636708
200	0.1411	0.1344	0.1223	0.1326	0.0095284	0.005501212	1.561365749	62.36518448
250	0.09651	0.08448	0.07539	0.08546	0.0105941	0.006116478	1.735991791	75.74456008

Table 9. Anti-inflammatory activity study of NR10:.

Concentrations ug/ml	1	2	3	Mean	Std Dev	Std error	% Std error	% Activity
50	0.3029	0.2819	0.2922	0.29233333	0.0105006	0.006062544	1.72068431	17.02932829
100	0.2533	0.2345	0.2448	0.2442	0.0094144	0.005435378	1.542680588	30.69063387
150	0.2178	0.1966	0.1871	0.2005	0.0157172	0.009074323	2.575493684	43.09366131
200	0.1768	0.1573	0.1566	0.16356667	0.0114657	0.006619752	1.87883205	53.57615894
250	0.1251	0.1139	0.1047	0.11456667	0.0102163	0.005898399	1.674096224	67.48344371

8. Molecular Docking study of NR9, NR10,NR11 and NR13 compounds

Molecular docking studies were performed for both carboxylate and carbonitrile derivatives at the active site of the bacterial protein GlcN-6-P synthase. AutoDock 4.0 software was used to find the different binding confirmations. The PRODRG server is used for the above derivatives along with the standard fluconazole molecule for 3D structure construction. The PreADMET server is used for minimising the energy and predicting the drug probability. The 3D structure file of the protein molecule (PDB ID: 1Jxa) was downloaded from www.rcsb.org/pdb and the structure was cleaned up by removing the heteroatoms and adding the C-terminal oxygen molecule. Docking calculations were applied to the ligands for further docking studies. CASTp server is used to find all active sites and the amino acids located there in GlcN-6-P synthase. Autodock 4.0 methods were used to minimise protein-drug interaction studies. The binding energy, minimum docking energy, and inhibition constant were documented as shown in table 10.

Table 10. Docking calculations of synthesised compounds.

Entry	Binding Energy (Kcal/mol)	Inhibition Constant (M)	RMSD
NR9	−7.43	3.55x10^-6	1.54
NR10	−12.13	1.66 x10^-9	0.76
NR11	−8.98	9.52x10^-7	1.12
NR13	−12.90	2.91 x10^-9	0.73

9. Electrochemical characterisation of CuO NPs

The electrochemical performance of modified nano-based electrode with graphite was measured using cyclic voltammetry (CV) and impedance spectroscopy analysis were performed. The CV examination of synthesised CuO NPs was performed using 3-electrode configuration at different scan rate in 0.1 M KCl electrolyte. Figure 14a shows the CV graphs synthesised CuO-graphite electrode in the potential range of +0.85 to -0.2 V with varying scan rates from 0.01 to 0.05 V/s. The increasing redox peaks in CV curves represents the excellent performance of CuO-graphite electrode with increasing in scan rate. The electrochemical impedance spectroscopy analysis was examined for synthesised CuO NPs from 1 Hz to 1000 kHz frequency range using 0.1 M KCl electrolyte as depicted in Figure 14b. The appearance of semi-circle arc indicating the pseudo-capacitance nature of CuO-graphite electrode. High-frequency regions of the prepared electrodes comprises of a semi-circle and the lower frequency region comprises of a straight line. Using the diameter of the samll semicircle, the charge transfer resistance (R_ct) values of electrode was found to be 156 Ω indicating the high capacitance and lesser the resistance and hence provides better catalytic and microbial activity (Surendra et al., 2021; Surendra and Veerabhadraswamy, 2017; Surendra et al., 2018; Uma et al., 2022).

Figure 14. (a) CV plots of CuO-graphite electrode at the scan rate of 0.01–0.05 V/s in 0.1 M KCl and (b) Nyquist plots of CuO-graphite electrode in 0.1 M KCl electrolyte.

The electrochemical sensor performance of synthesised CuO-graphite electrode was investigated using paracetamol medicinal compound in same electrolytic conditions. Figure 15a shows the CV analysis of prepared graphite CuO-graphite electrode for sensing paracetamol compound of concentration 1-5 mM in the potential range of +0.85 to -0.2 V. As represented in Figure 15b, the sensing of paracetamol compound was confirmed by the presence of oxidation and reduction potential peaks at + 0.68 V and -0.34 V respectively. The sensibility by prepared electrode towards paracetamol compound was confirmed by the appearance of redox peaks at varied potential position with respect to CV curve of bare electrolyte. Thus, the obtained data reveals that the prepared graphite electrode shows excellent sensing activity towards paracetamol and the synthesised NPs can be used as a better candidate for sensor applications (Uma et al., 2023; Lakshmi Ranganatha et al., 2020; Vasudha et al., 2021).

Figure 15. (a) CV plot of CuO-graphite electrode for sensing paracetamol at 1–5 mM in 0.1 M KCl solution. (b) presence of additional oxidation and reduction potential Peak for sensing paracetamol by CuO-graphite electrode.

10. Conclusion

The substituted-2-aminothiophenes were successfully synthesised by one pot Gewald reaction using low-cost CuO nano-catalyst under microwave technique. This nano-catalyst was prepared by solution combustion route and well characterised by spectral techniques. The XRD pattern confirms purity with crystallinity of CuO nanomaterial and its average particle size was found to be ∼ 21 nm. The smaller size of prepared material shows excellent catalytic activity towards organic synthesis and its energy band gap was found to be 3.39 eV recorded by UV-Visible absorption spectra. The increasing antifungal and antibacterial activities of synthesised substituted-2-aminothiophenes against Candida albicans (gram positive), Itraconazole (gram negative) organism and Bacillus cereus (gram positive) & Ciprofoxin (gram negative) organism respectively showed excellent performance, which is attributed to the presence of substituted-2-aminothiophene. The inhibition zone of Bacillus cereus (gram positive) organism for synthesised NR10 has showed excellent activity at 200 µl than those of 3 C, 3D, NR9, and NR11 compounds. The excellent inflammatory drug action of NR10 due to its highest rate of inhibition on protein denaturation was observed than those of NR9 and aspirin at concentration of 250 µg/ml. The excellent antidiabetic action of NR9 (181.86 µg/ml) was observed due to its more effective than those of standard drug in inhibiting alpha amylase activity at concentration of 250 µg/ml and it can be used to treat diabetes. The reported research provides better insights towards development of multifunctional applications of substituted-2-aminothiophenes using nano-catalyst and excellent multifaceted biomedical applications

Aknowledgement

Dr. Ravishankar shastri P R and Rathnamma D, thanks for financial support and providing necessary facilities and thankful to research guide Dr. Suresha Kumara T H, department of chemistry, UBDT college of engineering for kind support to carry out this research work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability materials

The data available on request from the authors.