Front. Chem. Frontiers in Chemistry Front. Chem. 2296-2646 Frontiers Media S.A. 1342784 10.3389/fchem.2024.1342784 Chemistry Original Research Choline chloride/urea as a green and efficient deep eutectic solvent in three-component and four-component synthesis of novel pyrazole and pyrano[2,3-c] pyrazole derivatives with antibacterial and antifungal activity Habeeb Naser et al. 10.3389/fchem.2024.1342784 Habeeb Naser Israa 1 Thoulfikar A. Alamir Hassan 2 Al-Shukarji Ali Hisham 3 Ahmed Batool Ali 4 Qassem Talal Aziz 5 Kamal Maher 6 Almeleebia Tahani M. 7 Alwaily Enas R. 8 Hasan Kadhum Eftikhaar 9 Alawadi Ahmed 10 11 12 * Alsalamy Ali 13 1 Medical Laboratories Techniques Department, Al-Mustaqbal University, Hillah, Iraq 2 Faculty of Pharmacy, Department of Pharmaceutics, University of Al-Ameed, Karbala, Iraq 3 Department of Medical Laboratories Technology, Al-Manara College for Medical Sciences, Maysan, Iraq 4 Department of Medical Engineering, Al-Nisour University College, Baghdad, Iraq 5 Department of Medical Laboratory Technics, Al-Noor University College, Nineveh, Iraq 6 Department of Dentistry, Al-Hadi University College, Baghdad, Iraq 7 Department of Clinical Pharmacy, College of Pharmacy, King Khalid University, Abha, Saudi Arabia 8 Microbiology Research Group, College of Pharmacy, Al-Ayen University, Thi-Qar, Iraq 9 National University of Science and Technology, Dhi Qar, Iraq 10 College of Technical Engineering, The Islamic University, Najaf, Iraq 11 College of Technical Engineering, The Islamic University of Al Diwaniyah, Al Diwaniyah, Iraq 12 College of Technical Engineering, The Islamic University of Babylon, Babylon, Iraq 13 College of Technical Engineering, Imam Ja’afar Al‐Sadiq University, Al‐Muthanna, Iraq

Edited by: Mohamed Saleh Abdelfattah, Helwan University, Egypt

Reviewed by: Ajay Kumar Chinnam, University of Idaho, United States

Gourav Kumar, Oregon Health and Science University, United States

 *Correspondence: Ahmed Alawadi, ahmedalawadi85@gmail.com 16 02 2024 2024 12 1342784 22 11 2023 15 01 2024 Copyright © 2024 Habeeb Naser, Thoulfikar A. Alamir, Al-Shukarji, Ahmed, Qassem, Kamal, Almeleebia, Alwaily, Hasan Kadhum, Alawadi and Alsalamy. 2024 Habeeb Naser, Thoulfikar A. Alamir, Al-Shukarji, Ahmed, Qassem, Kamal, Almeleebia, Alwaily, Hasan Kadhum, Alawadi and Alsalamy

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In this study, choline chloride/urea was used as a green deep eutectic solvent in the three-component reaction of hydrazine/phenylhydrazine, malononitrile, and aromatic aldehydes for synthesizing pyrazole derivatives, and in the four-component reaction of methyl/ethyl acetoacetate, hydrazine/phenylhydrazine, malononitrile, and aromatic aldehydes for synthesizing pyrano[2,3-c]pyrazole derivatives. Elemental analysis, 1H, and 13C NMR spectroscopy were used to confirm the structure of the synthesized pyrazole and pyrano[2,3-c] pyrazole derivatives. The antimicrobial effects of the synthesized pyrazole and pyrano[2,3-c] pyrazole derivatives were investigated. In antimicrobial tests, instructions from clinical and laboratory standards institutes were used. Antimicrobial study was done on pathogenic gram-positive and gram-negative species, and specialized aquatic strains and fungal species. Using choline chloride/urea, novel pyrazole derivatives and pyrano[2,3-c]pyrazole derivatives were synthesized, and other derivatives were synthesized with higher efficiency in less time than some previously reported methods. MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) obtained for derivatives were higher than some antibiotic drugs. Synthesis and reports of new derivatives of pyrazole and pyrano[2,3-c]pyrazole, and investigation and reports of their antimicrobial properties on gram-positive, gram-negative, and specialized aquatic and fungal species are among the novel and important findings of this study.

 choline chloride/urea deep eutectic solvent pyrazole pyrano[2,3-c]pyrazole biological evaluation antimicrobial agent section-at-acceptance Organic Chemistry 1 Introduction

The use of green processes in synthesizing organic and heterocyclic compounds is noteworthy (Nishanth Rao et al., 2021). Green methods in organic synthesis are essential because they are environmentally friendly (Kumar et al., 2020). Among the green techniques in organic chemistry are the synthesis of organic and heterocyclic compounds during multicomponent reactions, the use of recyclable catalysts, and the use of deep eutectic solvents (Calvo-Flores and Mingorance-Sánchez, 2021; Javahershenas, 2023). Multi-component reactions, in which several reactants lead to the synthesis of the desired product in one pot and one step, in addition to being green, are important in the synthesis of organic and heterocyclic compounds due to their high efficiency, cost-effectiveness, and time-saving characteristics (Bhaskaruni et al., 2020; John et al., 2021). There have been several reports of the synthesis of five-membered heterocycles, such as pyrazoles, or two-ring heterocyclic compounds, such as pyranopyrazoles, using multicomponent reactions (Mamaghani and Hossein Nia, 2021; Becerra et al., 2022; Khazaal and Ibraheam, 2022). Pyrazole is a useful heterocyclic compound due to its agricultural and drug-discovery applications (Kumar et al., 2013). There have been reports of this heterocyclic compound in nature, for example, isolation from fungi such as Colletotrichum gloeosporioides (Cui et al., 2023). The biological properties of this heterocyclic compound that can be mentioned are its anxiolytic, antidepressant, anti-tubercular, analgesic properties, and anti-malarial activities, etc. (Ramadan et al., 2021). As there is a pyrazole structure in the structure of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1h-pyrazol-1-yl]benzene-sulfonamide, which is a drug with the brand name Celecoxib, this heterocyclic compound can be used as a medicinal and bioactive compound (Küçükgüzel and Şenkardeş, 2015). Pyrano[2,3-c]pyrazole derivatives are one of the pyrazole derivatives that contain two heterocyclic compounds of pyrazole and pyran, which are connected by one facet. Derivatives of pyrano[2,3-c] pyrazole can maintain the biological properties of both heterocyclic compounds of pyran and pyrazole. Biological properties, such as cytotoxic, mutagenicity, cancer therapy, and antiviral properties, have been reported for the derivatives of the 6-membered heterocyclic compound of pyran with one oxygen in its structure (Yahiaoui et al., 2022). Biological properties, such as anti-tubercular, anti-malarial, anti-microbial, and anti-cancer properties, have been reported for the derivatives of pyrano[2,3-c]pyrazole (Mokariya et al., 2022; Parikh et al., 2022). In addition to the green method of multicomponent reaction, by using other green techniques such as the use of deep eutectic solvents, pyrazole derivatives and pyrano[2,3-c]pyrazole derivatives can be synthesized, and reports have been made in this field (Nguyen et al., 2021; Sikandar and Zahoor, 2021). As mentioned, the use of the deep eutectic solvent is one of the important and green methods in synthesizing organic and heterocyclic compounds. With this method, there is no need to use dangerous organic solvents or catalysts in the synthesis of organic and heterocyclic compounds (Rushell et al., 2019). A deep eutectic solvent plays the role of solvent and catalyst in the synthesizing of heterocyclic and organic compounds (Fekri et al., 2020). Deep eutectic solvents can be used during multicomponent reactions (Calvo-Flores and Mingorance-Sánchez, 2021). In this regard, we can refer to reports such as the use of glycerol: potassium carbonate in the four-component reaction of malononitrile, carbon disulfide, carbonyl, and methylene compounds, which led to the synthesis of [1,3]dithiine derivatives (Moghaddam-Manesh and Hosseinzadegan, 2021). Another deep eutectic solvent system that has been reported so far for the synthesis of pyrazolopyridines derivatives (Vanegas et al., 2019); tetrahydrobenzo[b]pyran and pyrano[2, 3-d]pyrimidinone (thione) (Biglari et al., 2020); and pyrazolines, pyrimidines, and naphthyridines (Mohamed et al., 2022) is the use of choline chloride/urea. According to reports, using choline chloride/urea reduces the synthesis time of the desired derivatives and has led to higher efficiency to synthesis of new derivatives. Therefore, choline chloride/urea was used as a deep eutectic solvent to synthesize pyrazole derivatives and pyrano[2,3-c]pyrazole derivatives. The synthesis of new derivatives and the experiments of antimicrobial and antifungal properties of the synthesized derivatives were among the other investigations carried out in this study.

 2 Materials and methods 2.1 Equipment and materials

The Brookfield DV-II + Pro EXTRA viscometer was used to measure solvent viscosity, and the G LAB melting point apparatus was used to measure the melting point of solvent and derivatives. The 1H, and 13C NMR spectra, CHNS/O elemental analyzer, and mass analysis of compounds were prepared using the Varian Inova 500MHz, the EMA 502, and the Agilent technologies 5975C. The Thermo biomate 5 Spectrophotometer was used to prepare the suspension of bacteria. The suspension of bacteria was obtained from the American Type Culture Collection (ATCC). All the reagent materials used for the synthesis of deep eutectic solvent and the synthesis of pyrazole derivatives and pyrano[2,3-c] pyrazole derivatives such as choline chloride, urea, malononitrile, aldehyde derivatives, and hydrazine derivatives were obtained from Merck and Sigma.

 2.2 Solvent preparation

To prepare the choline chloride/urea deep eutectic solvent, choline chloride and urea were weighed in different ratios, 1:1, 1:2, 1:3, and 1:4, and stirred at 80°C until a colorless homogeneous mixture was observed (Yadav and Pandey, 2014). The prepared mixture was cooled and used for other steps.

 2.3 Synthesis of pyrazole derivatives

To 1 g choline chloride/urea (1:2) deep eutectic solvent, 1 mmol malononitrile and 1 mmol aldehyde derivatives were added and stirred at 80°C for 5 min. Then 1 mmol hydrazine derivatives was added and stirred at 80°C until the reaction was complete. The completion of the reaction was checked by TLC. Finally, after the completion of the reaction, the temperature was brought to the ambient temperature, and 5 mL of distilled water was added and stirred for 30 min. The sediments, which were the desired product, were separated with filter paper. The desired synthesized product was purified by recrystallization in a mixture of distilled water and ethanol (1:1).

 2.4 Synthesis pyrano[2,3-c] pyrazole derivatives

To 1 g choline chloride/urea deep eutectic solvent, 1 mmol malononitrile and 1 mmol aldehyde derivatives were added and stirred at 70°C for 5 min. To 1 g choline chloride/urea deep eutectic solvent, 1 mmol ethyl acetoacetate and 1 mmol hydrazine derivatives were added and stirred at 80°C for 5 min. The mixtures were added together and stirred at 80°C until the reaction was complete. The completion of the reaction was checked by TLC. Finally, after the completion of the reaction, the temperature was brought to the ambient temperature, and 5 mL of distilled water was added and stirred for 30 min. The sediments, which were the desired product, were separated with filter paper. The desired synthesized product was purified by recrystallization in a mixture of distilled water and ethanol (1:1).

 2.5 Antimicrobial and antifungal activity pyrazole derivatives and pyrano[2,3-c]pyrazole derivatives

Based on previous studies reported according to relevant standards, the antimicrobial activity of synthetic derivatives on Streptomyces fradiae (10745), Staphylococcus aureus (29213), Streptococcus pyogenes (19615), Streptococcus agalactiae (12386), Yersinia enterocolitica (9610), Shigella dysenteriae (13313), Pseudomonas aeruginosa (15442), Acinetobacter baumannii (19606), Streptococcus iniae (29178), Loctococcus garvieae (43921), Yersinia ruckeri (29473), Edwardsiella tarda (15947), Candida albicans (10231), Cryptococcus neoformans (32045), Fusarium oxysporum (7601), and Aspergillus fumigatus Fresenius (1022) was investigated and evaluated. For this purpose, first, the half-McFarland concentration of the strains and the initial concentrations of 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, and 2048 μg/mL of the derivatives were prepared separately. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum fungicidal concentration (MFC), and inhibition zone diameter (IZD) were evaluated (Igei et al., 2016; Suksatan et al., 2022).

 3 Results and discussion

In this study, by using choline chloride/urea as a green and environmentally friendly deep eutectic solvent, in the three-component reaction of malononitrile, aldehyde derivatives, and hydrazine derivatives, novel pyrazole derivatives, and in the four-component reaction of malononitrile, aldehyde derivatives, ethyl acetoacetate, and hydrazine derivatives, novel pyrano[2,3-c]pyrazole derivatives were synthesized (Scheme 1).

Using choline chloride/urea as green method for synthesis of pyrazole derivatives and pyrano[2,3-c]pyrazole derivatives.

In general, the greenness of the reported method, high efficiency, and shorter synthesis time were the advantages of using choline chloride/urea in synthesizing derivatives. Additional information about the synthesis of derivatives is given below.

3.1 Three-component synthesis of pyrazole derivatives using choline chloride/urea

The three-component synthesis of pyrazole derivatives used choline chloride/urea, malononitrile, aldehyde derivatives, and hydrazine derivatives were used as raw materials.

For this purpose, according to the method presented in Section 2.2, different ratios of choline chloride and urea were prepared. For the synthesis of 4a, they were tested according to Table 1.

Optimization of molar ratio of choline chloride:urea and reaction temperature in synthesis of pyrazole.

Entry Product Molar ratio of choline chloride:urea Reaction temperature (°C) Tim (min) Yield (%)
1 4a 0: 1 80 60 N.R
2 4a 1: 0 80 60 N.R
3 4a 1: 1 80 60 67
4 4a 1: 2 80 20 97
5 4a 1: 3 80 30 82
6 4a 1: 4 80 30 55
7 4a 1: 2 50 30 52
8 4a 1: 2 60 30 76
9 4a 1: 2 70 30 88
10 4a 1: 2 90 30 95
11 4a 1: 2 100 30 90

N.R, no reaction.

In obtaining the optimal conditions (Table 1), in addition to using different ratios of choline chloride and urea, temperatures of 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C degrees Celsius were also tested. It was found that the ratio of 1:2 (choline chloride:urea) at 80°C had the highest efficiency in a shorter time for the synthesis.

Physical parameters such as viscosity (according to ASTM D445 testing method) and melting point for 1:2 choline chloride:urea mixture were obtained according to Table 2.

Physical parameters of a 1:2 ratio of choline chloride:urea deep eutectic solvent.

Viscosity (25°C, cPa) Melting point temperature (°C)
17.50 20

The above optimal conditions were used for synthesizing other derivatives, according to Table 3. Three of the derivatives reported in Table 3 are novel.

Information on synthesized pyrazole derivatives using choline chloride/urea as green method.

Product code R1 R2 Product structure Reported Mp (°C) Found Mp (°C) Synthesis time (min) Yield (%)
4a Ph Ph 158-159 (Poonam and Singh, 2019) 159-160 20 97
4b Ph 2,4-NO2-Ph 240-142 (Bakhtiarian and Khodaei, 2022) 240-243 95
4c 4-Me-Ph Ph 119-121 (Nemati et al., 2021) 120-121 97
4d 4-Me-Ph 2,4-NO2-Ph New 245-247 85
4e 4-OMe-Ph Ph 107-109 (Abshirini et al., 2020) 108-110 98
4f 4-OMe-Ph 2,4-NO2-Ph New 264-267 86
4g 4-NO2-Ph Ph 165-168 (Nemati et al., 2021) 166-168 97
4 h 4-NO2-Ph 2,4-NO2-Ph New 266-268 84
4i 4-Cl-Ph Ph 128-130 (Amirnejat et al., 2020) 129-131 98
4j 4-Cl-Ph 2,4-NO2-Ph 234-236 (Aryan et al., 2017) 235-236 93

Choline chloride:urea (1: 2); 80°C.

The results of 1H NMR, 13C NMR, and CHNS/O elemental analyzer of the newly synthesized derivatives that confirm their structure are given in the Supplementary Material.

As the results show, in general, electron-donating substituents attached to hydrazine, such as phenyl, and electron-withdrawing groups attached to the aldehyde, such as nitro, increased the efficiency of the products. Therefore, the following mechanism (Scheme 2) can be proposed for this 3-component reaction, which corresponds to the difference in the results of the product’s efficiency.

Mechanism for using choline chloride/urea as green method for synthesis of pyrazole derivatives (4a-j).

So far, many studies have been conducted on the three-component reaction of malononitrile, aldehyde derivatives, and hydrazine derivatives, which led to the synthesis of pyrazole derivatives. The conditions of some recent studies (from 2020 to 2023) compared to the conditions used in this study are given in Table 4.

The previously reported conditions and methods in the synthesis of pyrazole compared to the method presented in this study.

Reported year Product Condition (catalyst) Reaction temperature (°C) Time Yield (%) Reference
2023 4a Sulfonic acid modified 5-aminotetrazole-1,3,5-triazine-(3-aminopropyl)silylated carbon quantum dots-coated Fe3O4 magnetic nanoparticles 70 Not reported 95 Ghorbani et al. (2023)
2022 4a 2,6-diamino-1-(4-sulfobutyl)pyridin-1-ium hydrogen sulfate modified pectin nanoparticles 50 30 min 96 Bakhtiarian and Khodaei (2022)
2021 4a Sulfonic acid-functionalized polyvinyl alcohol 90 3 h 89 Patki et al. (2021)
2020 4a 1,3-disulfoimidazolium trifluoroacetate 80 50 min 94 Abshirini et al. (2020)
This work 4a Choline chloride:urea 80 20 min 97 -

Ease of reaction conditions and no need for catalyst and solvent, reporting of newly synthesized derivatives, being green, and having high efficiency and shorter synthesis time can be mentioned as advantages of using choline chloride/urea to synthesize derivatives compared to recently reported methods.

 3.2 Four-component synthesis of pyrano[2,3-c]pyrazole derivatives using choline chloride/urea

In the continuation of this study, choline chloride/urea was also used in the 4-component reaction of malononitrile, aldehyde derivatives, hydrazine derivatives, and ethyl acetoacetate for the synthesis of pyrano[2,3-c]pyrazole derivatives.

The steps for synthesizing derivatives here were the first optimization according to Section 3.1, the results of which are given in Table 5.

Optimization of molar ratio of choline chloride:urea and reaction temperature in synthesis of pyrano[2,3-c] pyrazoles.

Entry Product Molar ratio of choline chloride:urea Reaction temperature (°C) Tim (min) Yield (%)
1 6a 0: 1 70 60 N.R
2 6a 1: 0 70 60 N.R
3 6a 1: 1 70 60 42
4 6a 1: 2 70 17 95
5 6a 1: 3 70 30 66
6 6a 1: 4 70 30 54
7 6a 1: 2 50 30 62
8 6a 1: 2 60 30 79
9 6a 1: 2 80 30 90
10 6a 1: 2 90 30 88
11 6a 1: 2 100 30 88

N.R, no reaction.

Here, the optimal conditions for synthesizing pyrano[2,3-c]pyrazole derivatives with high efficiency and shorter time, were the ratio of 1:2 of choline chloride:urea and the reaction temperature of 70°C. With the obtained optimal conditions, other derivatives, including 15 derivatives, of which two were novel, were synthesized (Table 6).

Information on synthesized pyrano[2,3-c]pyrazole derivatives using choline chloride/urea as green method.

Product code R1 R2 Product structure Reported Mp (°C) Found Mp (°C) Synthesis time (min) Yield (%)
6a Ph H 241-243 (Yekke-Ghasemi et al., 2022) 240-242 17 95
6b Ph Ph 169-171 (Mathavan and Yamajala, 2023) 170-172 15 95
6c Ph 2,4-NO2-Ph 240-243 (Vasava et al., 2019) 241-242 30 90
6d 4-Me-Ph H 175-177 (El Mejdoubi et al., 2019) 176-178 25 90
6e 4-Me-Ph Ph 177-179 (Mohamadpour, 2020) 177-180 25 95
6f 4-Me-Ph 2,4-NO2-Ph New 270-273 35 85
6g 4-OMe-Ph H 209-211 (Yekke-Ghasemi et al., 2022) 209-212 20 92
6 h 4-OMe-Ph Ph 170 (Suryakumari and Sudhakar, 2023) 169-171 18 95
6i 4-OMe-Ph 2,4-NO2-Ph New 288-290 36 85
6j 4-NO2-Ph H 249-251 (Moghaddam et al., 2023) 249-251 20 95
6k 4-NO2-Ph Ph 195-197 (Kondabanthini et al., 2022) 194-196 19 95
6L 4-NO2-Ph 2,4-NO2-Ph 283-285 (Vasava et al., 2019) 284-285 34 88
6m 4-Cl-Ph H 234-236 (Yekke-Ghasemi et al., 2022) 235-236 20 94
6n 4-Cl-Ph Ph 175-177 (Kondabanthini et al., 2022) 175-177 17 97
6o 4-Cl-Ph 2,4-NO2-Ph 260-265 (Vasava et al., 2019) 263-266 35 80

Choline Chloride: Urea (1: 2); 70°C

The results of 1H NMR, 13C NMR, and CHNS/O elemental analyzer of the newly synthesized derivatives that confirm their structure are given in the Supplementary Material.

Comparing the results of the efficiency with the structure of the raw materials here was also the same as above, and it was observed that electron-donating substituents attached to hydrazine, such as phenyl, and electron-withdrawing groups attached to the aldehyde, such as nitro, increased the efficiency of the products. Therefore, the Scheme 3 mechanism is proposed for this reaction.

Mechanism for using choline chloride/urea as green method for synthesis of pyrazole pyrano[2,3-c]pyrazole derivatives (6a-o).

The result of the comparison of the method presented in this study to the recent studies in the synthesis of derivatives, as shown in Table 7, is similar to the conclusion for the synthesis of pyrazole derivatives. It can be stated here that ease of reaction conditions, catalyst and solvent not being necessary, newly synthesized derivatives being reported, being green, and having high efficiency and shorter synthesis time can be mentioned as advantages of using choline chloride/urea to synthesize derivatives compared to recently reported methods.

The previously reported conditions and methods in the synthesis of pyrano[2,3-c]pyrazole compared to the method presented in this study.

Reported year Product Condition (catalyst) Reaction temperature (°C) Time Yield (%) Reference
2018 6a Bael fruit ash 20 15 min 92 Shinde et al. (2018)
2018 6a Sodium L-ascorbate Reflux of water 10 min 88 Kiyani and Bamdad (2018)
This work 6a Choline Chloride: Urea 70 17 min 95 -
2023 6b Copper(II) oxide incorporated onto montmorilonite-K10 functionalized with tetraethyl orthosilicate, epichlorohydrine, melamine, and 5-sulphosalisilic acid 20 Not reported 90 Karrabi et al. (2023)
2023 6b Potassium dihydrogenphosphate 100 2 h 95 Mathavan and Yamajala (2023)
2022 6b Phenylazophenylenediamine-based lanthanum complex supported on silica-coated magnetic nano-Fe3O4 core-shell nanocatalyst 110 12 90 Ghorbani et al. (2022)
2021 6b Sugarcane bagasse ash-based silica-supported boric acid 80 35 min 86 Pandey et al. (2021)
2022 6b Tungstic acid immobilized on zirconium-L-aspartate amino acid metal-organic framework-grafted L-(+)-tartaric acid stabilized magnetic Fe3O4 nanoparticles 60 45 min 92 Khademi et al. (2021)
This work 6b Choline chloride:urea 70 15 min 95 -
3.3 The antimicrobial activity results

In the investigation of antimicrobial and antifungal activity of derivatives, gram-positive, gram-negative, and specialized aquatic strains were used. First, the MIC with a concentration in the range of 1–2048 μg/mL of the derivatives was checked. After obtaining this parameter, its MBC and MFC were acquired. Finally, using the MIC, the IZD parameter of the derivatives on the used strains was obtained.

For further comparison of pyrazole derivatives and pyrano[2,3-c]pyrazole derivatives, the antibacterial properties of gram-positive, gram-negative, and specialized aquatic and fungal strains are shown separately in Tables 8–11.

Antibacterial activity of pyrazole derivatives (4a-j) and pyrazole pyrano[2,3-c]pyrazole derivatives (6a-o) against Gram-positive strains.

Strains Pyrazole derivatives Drugs Pyrano[2,3-c]pyrazole derivatives
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j A B 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o
10745 (I) 2048 128 - 256 1024 128 512 128 64 64 - 16 512 512 128 1024 256 64 256 128 32 64 64 32 16 16 8
(II) 4096 256 - 512 2048 128 1024 128 128 128 - 32 1024 512 128 1024 512 128 256 256 64 64 64 64 16 32 16
(III) 12.6 16.8 - 16.2 12.8 17.3 13.7 15.9 18.2 17.3 - 17.3 14.5 14.9 14.3 15.3 14.8 18.9 17.6 18.5 19.1 18.2 21.2 20.3 22.5 19.9 22.4
29213 (I) - - - - - - - - - - 16 16 - - 2048 - - 512 - - 256 1024 1024 128 - - 64
(II) - - - - - - - - - - 32 16 - - 4096 - - 1024 - - 512 2048 1024 256 - - 128
(III) - - - - - - - - - - 15.1 16.9 - - 12.2 - - 13.8 - - 13.6 12.3 12.9 14.2 - - 14.1
19615 (I) 4096 4096 2048 1024 2048 2048 2048 2048 1024 1024 2 8 512 512 16 512 512 8 256 128 4 64 32 4 2 2 2
(II) 4096 4096 2048 4096 2048 2048 4096 2048 2048 2048 4 16 1024 1024 16 1024 1024 16 512 128 8 128 64 4 4 2 2
(III) 10.4 10.9 10.7 10.2 10.5 11.3 10.4 10.6 11.8 11.5 23.2 18.4 16.4 18.3 20.8 15.9 19.9 19.7 18.8 19.5 23.1 19.4 21.6 22.5 23.1 22.7 22.9
12386 (I) - - - - - - - - 1024 1024 - 4 - - 64 - - 32 256 128 16 128 128 8 8 4 2
(II) - - - - - - - - 4096 2048 - 8 - - 128 - - 64 512 256 16 128 128 16 8 8 4
(III) - - - - - - - - 18.2 17.5 - 19.1 - - 21.6 - - 21.0 17.3 16.6 22.1 16.2 19.4 22.4 21.5 21.7 22.3

(I): Minimum Inhibitory Concentration (μg/mL); (II): Minimum Bactericidal Concentration (μg/mL); (III): Inhibition Zone Diameter (mm);

A, penicillin; B, cefazolin.

Antibacterial activity of pyrazole derivatives (4a-j) and pyrazole pyrano[2,3-c]pyrazole derivatives (6a-o) against gram-negative strains.

Strains Pyrazole derivatives Drugs Pyrano[2,3-c]pyrazole derivatives
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j A B 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o
9610 (I) - - - - - - - - - - - 64 - - - - - - - - - - - - - - -
(II) - - - - - - - - - - - 128 - - - - - - - - - - - - - - -
(III) - - - - - - - - - - - 12.0 - - - - - - - - - - - - - - -
13313 (I) - - - - - - - - 1024 1024 - - - - - - - - - - - - - - 1024 512 512
(II) - - - - - - - - 4096 2048 - - - - - - - - - - - - - - 1024 1024 512
(III) - - - - - - - - 10.7 12.5 - - - - - - - - - - - - - - 13.6 12.4 12.3
15442 (I) - - - - - - - - - - - - - - 2048 - - 2048 4096 2048 1024 1024 1024 512 512 256 128
(II) - - - - - - - - - - - - - - 4096 - - 2048 4096 4096 1024 2048 2048 1024 512 256 256
(III) - - - - - - - - - - - - - - 11.8 - - 11.2 10.1 11.4 12.3 11.9 11.5 12.4 13.6 13.8 13.5
19606 (I) - - - - - - - - - 1024 - - - - - - - - - - - - - - - - 256
(II) - - - - - - - - - 2048 - - - - - - - - - - - - - - - - 512
(III) - - - - - - - - - 11.5 - - - - - - - - - - - - - - - - 12.3

(I): Minimum Inhibitory Concentration (μg/mL); (II): Minimum Bactericidal Concentration (μg/mL); (III): Inhibition Zone Diameter (mm);

A, penicillin; B, cefazolin.

Antibacterial activity of pyrazole derivatives (4a-j) and pyrazole pyrano[2,3-c]pyrazole derivatives (6a-o) against specialized aquatic strains.

Strains Pyrazole derivatives Drugs Pyrano[2,3-c]pyrazole derivatives
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j A B 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o
Gram- positive 29178 (I) 2048 256 1024 512 1024 512 1024 128 32 32 32 16 4096 - 512 2048 - 256 2048 1024 512 1024 512 128 32 32 32
(II) 4096 512 2048 1024 2048 512 1024 256 64 64 64 64 4096 - 1024 4096 - 512 4096 2048 512 1024 1024 256 64 64 64
(III) 11.7 13.8 12.5 11.9 13.0 13.4 11.8 14.2 15.9 16.7 16.6 14.2 10.5 - 1024 11.2 - 13.8 12.1 15.1 14.2 14.4 15.7 13.6 15.7 15.3 15.9
43921 (I) - 512 - 1024 2048 512 512 256 256 128 2 - - - 128 2048 - 64 512 256 32 256 256 16 4 2 2
(II) - 512 - 2048 4096 1024 1024 256 512 256 4 - - - 256 4096 - 128 1024 512 128 256 256 32 16 4 2
(III) - 12.9 - 11.5 11.9 13.6 14.2 15.4 16.5 17.7 19.7 - - - 21.8 10.2 - 20.7 17.6 19.3 21.5 19.8 17.5 22.9 22.6 23.5 23.1
Gram- negative 29473 (I) - - - - - - - - 2048 2048 - 32 - - 1024 - - 1024 4096 4096 1024 4096 2048 512 256 64 64
(II) - - - - - - - - 4096 4096 - 64 - - 1024 - - 2048 4096 4096 1024 4096 4096 512 512 256 128
(III) - - - - - - - - 11.6 11.1 - 15.9 - - 12.8 - - 11.3 12.2 10.3 12.6 10.7 10.9 12.4 13.7 14.5 15.1
15947 (I) - - - - - - - - - - 8 - - - 256 - - 128 2048 1024 64 512 512 16 16 8 4
(II) - - - - - - - - - - 16 - - - 512 - - 128 2048 2048 128 1024 1024 64 16 16 8
(III) - - - - - - - - - - 17.5 - - - 17.3 - - 16.4 15.5 14.9 18.7 17.7 16.2 17.8 19.1 19.5 19.6

(I): Minimum Inhibitory Concentration (μg/mL); (II): Minimum Bactericidal Concentration (μg/mL); (III): Inhibition Zone Diameter (mm);

A, penicillin; B, cefazolin.

Antifungal activity of pyrazole derivatives (4a-j) and pyrazole pyrano[2,3-c]pyrazole derivatives (6a-o).

Strains Pyrazole derivatives Drugs Pyrano[2,3-c]pyrazole derivatives
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j A B 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o
10231 (I) - 1024 - 4096 2048 1024 2048 1024 512 512 - 32 - 4096 512 - 1024 512 2048 1024 512 1024 512 256 128 128 64
(II) - 1024 - 4096 4096 2048 2048 2048 1024 1024 - 64 - 4096 1024 - 2048 1024 2048 2048 512 2048 1024 512 256 256 128
(III) - 12.6 - 11.4 11.7 12.1 11.8 13.0 15.9 15.5 - 21.6 - 11.7 16.6 - 12.3 16.9 13.5 14.1 16.3 15.9 16.9 17.8 17.6 17.3 18.4
32045 (I) - - - - - - - - - - - 64 - - - - - - - - - - - - - - -
(II) - - - - - - - - - - - 128 - - - - - - - - - - - - - - -
(III) - - - - - - - - - - - 17.2 - - - - - - - - - - - - - - -
7601 (I) - - - - - - - - 512 256 - 64 - - - - - - - - - - - - 256 256 128
(II) - - - - - - - - 1024 512 - 64 - - - - - - - - - - - - 512 256 256
(III) - - - - - - - - 12.7 12.8 - 20.8 - - - - - - - - - - - - 13.0 14.6 14.1
1022 (I) - 512 4096 2048 2048 1024 1024 512 256 256 - 32 2048 - 128 2048 - 64 1024 512 64 512 256 64 16 16 16
(II) - 1024 4096 4096 2048 1024 2048 1024 512 256 - 64 4096 - 256 4096 - 128 1024 1024 128 512 512 128 32 32 32
(III) - 12.2 14.6 14.8 13.1 13.7 13.8 12.8 15.9 15.5 - 20.3 10.8 - 17.9 11.8 - 18.6 1024 16.3 17.4 17.7 1024 18.2 19.5 19.8 19.3

(I): Minimum Inhibitory Concentration (μg/mL); (II): Minimum Fungicidal Concentration (μg/mL); (III): Inhibition Zone Diameter (mm);

A, tolnaftate; B, terbinafine.

Table 8 shows the results of investigating the antibacterial properties of the derivatives on the evaluated gram-positive strains.

Table 9 shows the results of investigating the antibacterial properties of the derivatives on the evaluated gram-negative strains.

Table 10 shows the results of investigating the antibacterial properties of the derivatives on the evaluated specialized aquatic strains.

Table 11 shows the results of investigating the antibacterial properties of the derivatives on the evaluated fungal strains.

The highest effectiveness in antibacterial activity between pyrazole derivatives and pyrano[2,3-c]pyrazole derivatives was related to 6o.

The IZD value results of some pyrano[2,3-c]pyrazole derivatives compared to Yersinia ruckeri are given in Figure 1.

The IZD value results of some pyrano[2,3-c]pyrazole derivatives against Yersinia ruckeri.

In general, the order of effectiveness of pyrazole derivatives was as follows: 4 j   >   4 i   >   4 h   >   4 f   >   4 d ≈ 4 b   >   4 g   >   4 e   >   4 b ≈ 4 a

In general, the order of effectiveness of pyrano[2,3-c]pyrazole derivatives was as follows: 6 o   >   6 n ≈ 6 m   >   6 l   >   6 i   >   6 f ≈ 6 c   >   6 j ≈ 6 k   >   6 h ≈ 6 g   >   6 e ≈ 6 d ≈ 6 b ≈ 6 a

By comparing the results of Tables 8–11, the pyrano[2,3-c]pyrazole derivatives, in general, are more effective than pyrazole derivatives because pyrano[2,3-c] pyrazoles contain two heterocyclic rings (pyran and pyrazole). Another general conclusion that can be drawn is that the order of effectiveness of derivatives is as described for compounds containing chlorine, nitrogen, and methoxy. These results can be justified based on previous studies on the effectiveness and antimicrobial properties of chlorine and nitrogen. The important point in determining antimicrobial activities is to compare them with commercial drugs, such as penicillin, cefazolin, Tolnaftate, and Terbinafine. As the results of Tables 8–11 show, some derivatives, such as 6o in many studied strains, are much more effective than tested drugs, which shows the value of synthetic compounds.

 4 Conclusion

Choline chloride/urea was used as a deep eutectic solvent in the one-pot method and during the multicomponent reaction in synthesizing pyrazole derivatives and pyrano[2,3-c]pyrazole derivatives. The greenness of the synthesis method, the synthesis of new derivatives, and the characteristics of high efficiency and less time were among the advantages and novel findings of this study compared to previously reported methods. The effectiveness on gram-positive, gram-negative, and specialized aquatic strains and fungal species is one of the novel findings of this project. In the investigation of antimicrobial activity, the highest effectiveness was observed in derivative 6o, which contained chlorine and nitrogen, where MIC and MBC values between 2 and 512 μg/mL were observed. In addition, 6o showed higher effectiveness in some strains than drugs in the market, such as penicillin, cefazolin, Tolnaftate, and Terbinafine. Finally, the relationship between the antimicrobial properties of the derivatives and their structure were observed and reported.

 Data availability statement

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

 Author contributions

IH: Project administration, Writing–review and editing. HT: Conceptualization, Writing–review and editing. AHA-S: Methodology, Writing–original draft. BA: Validation, Writing–original draft. TQ: Formal Analysis, Writing–original draft. MK: Resources, Writing–review and editing. TA: Data curation, Writing–original draft. EA: Methodology, Writing–original draft. EH: Investigation, Writing–original draft. AhA: Supervision, Writing–original draft. AlA: Funding acquisition, Writing–original draft.

 Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through the large research group program under grant number R.G.P.02/535/44.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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