Introduction

Pyrazole is considered to be a heterocyclic compound that is consisted of a five-membered ring with one double bond included. It contains three carbon atoms and two nitrogen atoms at positions 1 and 2 in a partially saturated non-aromatic ring with the formula C₃H₃N₂H [1]. Pyrazoline is dihydropyrazole and can be present in three isomeric forms (1-pyrazoline, 2- pyrazoline, and 3- pyrazoline) depending on the position of the double bond [2], among the three types of these compounds, 3-pyrazoline earned a wide pharmaceutical interest. Pyrazole synthesis has been reported through a cyclization reaction of chalcones using succinicdihydrazide [3] and phenylhydrazine [4].

Pyrazoline compounds can be converted to pyrazole through an oxidation reaction using (bromine or oxygen) after the one-pot condensation reaction step of an aldehyde with ketones and hydrazine monohydrochloride [5]. Pyrazole and its derivatives showed significant activity in the biological field, such as anti-diabetic [6], antimicrobial [7], anti-breast cancer [8], antiviral [9], and antioxidant [10].

The aim of this work is comprehensive research and to achieve a further step forward for a previously published work. This includes synthesizing a new series of bisdihydropyrazolylbuta-1,4-dione derivatives and evaluating their biological activity against some gram-positive and negative bacteria. Synthesis of novel organic compounds and studying their biological activity are academically significant science values.

Materials and Methods

Melting points for the prepared compounds have been measured by a (Stuart SMP II) device in the Northern Technical University - College of Technical in Kirkuk, Department of Engineering Technologies of Fuel and Energy. A Shimadzu FT-IR 8400S with a range of (4000-400) cm^-1 was used to identify the products. The FT-IR and KBr tablets are available in the Department of Chemistry-College of Education, the University of Tikrit. In addition, the ¹H-NMR and ¹³C-NMR spectra for some of the prepared compounds have been taken as the main tool to confirm the product’s structure. Dimethyl sulfoxide was used as a solvent to run the ¹H-NMR and ¹³C-NMR analyses.

2-(4-cinnamoylphenyl) isoindoline-1,3-dione (C₁-₁₀)

A serious of chalcone compounds (C_1-10) was synthesized according to the previously published work.

Preparation of 1,4-bis[5-(4-(isoindolin-2-yl-1,3-dione) phenyl)-3-phenyl-2,3-dihydro-1H-pyrazol-1-yl] butane-1,4-dione (P_1-10).

In a circular flask (100 mL), a mixture of (0.006 mol of one of the chalcone compounds (C₁-₁₀) and (0.003 mol, 0.438 g) of succinicdihydrazide was dissolved in (20 mL) of acetic acid. The mixture was refluxed for 4 hours, and the solution was cooled down at room temperature, poured on crushed ice, and left in the beaker until crystals formed. The precipitant was purified by filtered and recrystallized from ethanol [3]; some physical properties are given in Table 1.

Preparation of saturated disks of the bacterial suspension

The biological effect of final products has been evaluated against gram-positive bacteria like (staphylococcus aureus and staphylococcus epidermidis) and gram-negative bacteria like (escherichia coli and pseudomonas aeruginosa). The micro-organisms have been isolated and identified at Medical Laboratory Techniques Department/ Technical College in Kirkuk. The single protectorate was transferred to the test tube containing 5 mL of nutritious, and the broth brooded and kept at 37 °C for 24 hours. The bacterial suspension prepared and compared with tube number 0.5 of McFarland- standards giving a cell density of 1.5×108 cell/mL) [11].

An antiseptic cotton sweep was dunked into a bacterial suspension and wiped equally on the surface of a Muller-Hinton agar plate, and the plates were brooded at 37 °C for 30 minutes. The saturated disks have been prepared from Whatman number 1 and maintained for 24 hours with the compounds 0.1 mg/mL, applied on Mueller-Hinton agar using Kirby–Bauer disc spread method [12]. Forceps were pressed firmly to guarantee the connection with agar, and in the next step, the plates inverted and brooded at 37 °C for 14-18 hours.

Results and Discussion

The discussion details about chalcone compounds (C_1-10) are available in the previously published work. 1,4-bis[5-(4-(isoindolin-2-yl-1,3-dione) phenyl)-3-phenyl-2,3-dihydro-1H-pyrazol-1-yl] butane-1,4-dione (P_1-10) have been synthesized via cyclization of substituted chalcones with succinicdihydrazide. The structures of all prepared compounds were diagnosed by FT-IR, and some of them were diagnosed by ¹H-NMR and ¹³C-NMR techniques. Synthesis of 1,4-bis[5-(4-(isoindolin-2-yl-1,3-dione) phenyl)-3-substituted phenyl-2,3-dihydro-1H-pyrazol-1-yl] butane-1,4-dione (P₁-₁₀) will be discussed as shown in Scheme 1.

The suggested mechanism for the cyclization reaction of chalcone compounds (C₁-₁₀) with succinicdihydrazide in acid media is a nucleophilic substitution, as shown in Scheme 2.

Scheme 1: Preparation of bis-pyrazole compounds (P_1-10)

Scheme 2: Mechanism of bis-pyrazole compounds synthesis (P_1-10)

The FT-IR spectra of compounds (P₁-₁₀) showed bands for the secondary amine (-NH) in the pyrazoline ring at (3376-3265) cm^-1 with the disappearance of the olefinic bond band and no shifting in the carbonyl band compared to the starting materials [13]. This could be taken as evidence for achieving a change in the olefinic bond attached to the ketone group [14]. The remaining packages appeared in their expected locations, as shown in Table 2, Figure 1 and 2.

The ¹H-NMR spectrum of compound (P₁) shows a singlet at δ 2.91 for the four protons in the two methylene groups, with signal δ 5.74 for the protons in the fifth position for the pyrazole ring. Moreover, aromatic protons signals are seen in the range at δ 7.40-8.30 with a signal at δ 6.20 for the two olefinic protons in the pyrazole ring, as shown in Figure 3. The interesting signal is δ 5.14 for the N-H proton, which is strong evidence for transforming chalcones to pyrazole. Compound (P₂) gives multiple signals at δ 7.40-8.29 for aromatic protons and δ 6.60 for olefinic protons in the pyrazole ring. In addition, a singlet appears at δ 2.81 and δ 4.99 for the protons in methylene and the protons of the fifth carbon in the pyrazole ring, respectively, as shown in Figure 4. The N-H group signal is clearly seen at δ 5.74, ascribed to achieving the cyclization reaction for the olefinic bond in the chalcone. The ¹H-NMR spectra of (P₁) and (P₂) confirmed the achieving of cyclization to the unsaturated bond attached to the ketone group.

The ¹³C-NMR spectrum of compound (P₁) showed signals at δ 124-143 referring to the aromatic carbon group. Furthermore, signals appeared at δ 165 and δ 186 for the carbonyl in imide and ketone groups, respectively (Figure 5). The methylene carbon band clearly appeared at δ 24, which is evidence of a change in the unsaturated carbon bond bonded with the ketone group. Further confirmation of producing the interested compound is the appearance of signals at δ 65.0 and δ 93.4 for the fifth and fourth carbons in the pyrazole ring, respectively.

Compound (P₂) showed signals for the aromatic at the range δ 123-143, with signals appearing at δ 167 and δ 180 for the carbonyl in imide and ketone groups, as shown in Figure 6. In addition, signals appeared at δ 21, δ 68, and δ 113 for carbons in methylene, the fifth and the fourth carbons groups, respectively. The ¹³C-NMR spectra of compounds (P₁) and (P₂) confirmed the cyclization reaction for chalcones. The combined spectroscopy data confirms the transformation of chalcone to pyrazole. The heat of formation and steric energy of final compounds are shown in Table 3, and the 3D structure of the most stable formula for the compounds (P₃^-P₈) is shown in Figure 7.

Biological study

The antimicrobial activity of the synthesized compounds has been evaluated in vitro against several pathogenic representative microorganism's gram-positive bacteria [staphylococcus aureus and staphylococcus epidermidis] and gram-negative bacteria like [escherichia coli, pseudomonas aeruginosa], using agar well diffusion method [15]. Ciprofloxacin and norfloxacin were used as standard drugs for studying the potential activities of these compounds. The compounds under the test were injected using a loop onto plates containing nutrient agar (NA) media and brooded at 37 °C for 24 hours. The agar diffusion was carried out by preparing bacterial suspensions in distilled water. The results indicated that the prepared compounds did not have any effect on the two types of gram-negative (escherichia coli and pseudomonas aeruginosa), while showed a different effect on both types of gram-positive (Staphylococcus aureus and Staphylococcus epidermidis). This relates to the difference in the cell wall structure for the gram-negative compared to the gram-positive which the former contains an outer membrane increasing bacteria resistance towards the tested chemicals as antibiotics [16].

Figure 7: 3D-structure for some of synthesized compounds; (a): (P₃) and (P₄) and (b): (P₇) and (P₈)

In addition, it possesses some virulence factors such as capsule and biofilm compared to the gram negative, providing high resistivity against anti-chemical materials. This is described as the tested chemicals can be hindered for penetrating the cell wall causing to decrease in the inhibition effect [17]. Compounds (P₅-P₁₀) with the concentration of 0.01 mg/mL, showed a higher effect on bacteria (staphylococcus aureus) than the antibiotic norfloxacin and less than the antibiotic ciprofloxacin. However, at concentration of 0.001 mg/mL and 0.0001 mg/mL these compounds showed a similar effect less than both of the antibiotics. Similarly, to the case of (staphylococcus epidermidis) bacteria, compounds (P₃ and P₅-P₁₀) at a concentration of 0.01 mg/mL, and compounds (P₆ and P₇) at a concentration of 0.001 mg/mL showed a higher effect than the antibiotic norfloxacin. One of the reasons could be the presence of NO₂ and Cl withdrawal groups compared to the others, as this is reported in the literature [18]. The remaining compounds showed a similar effect less than the two antibiotics. The effect of the prepared compounds against all the tested bacteria is shown in Table 4, and the results are summarized in Figures 8 and 9. Some pictures of the biological activity disks are shown in Figures 10 and 11.

Figure 8: The inhibition efficiency of final products against Staphylococcus aureus

Figure 9: The inhibition efficiency of tested compounds on the growth of Staphylococcus aureus bacteria; (a): (P₁) and (P₂) and (b): (P₇) and (P₈)

Conclusion

A new series of substituted bisdihydropyrazolylbuta-1,4-dione compounds have been successfully synthesized through a cyclization reaction of new substituted chalcone compounds as starting materials with succinicdihydrazide. The results of characterization were in agreement with the structures of the prepared compounds. P₆ and P₇ compounds have a high biological effect on the gram-positive of both types (staphylococcus aureus and staphylococcus epidermidis) at a concentration of 0.01 mg/mL and 0.001 mg/mL. This may be related to the presence of withdrawal groups (NO₂ and Cl) compared to the others. The theoretical properties like the heat of formation (HF) and steric energy (SE) have been studied, the heat of formation and steric energy has a slight difference from one compound to another, and the highest value was to P_6, and P₇ compounds may have related to withdrawal group.

Acknowledgements

The authors are highly acknowledged everyone supporting this research to be carried out.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' contributions

All authors contributed to data analysis, drafting, and revising of the paper and agreed to be responsible for all the aspects of this work.

Conflict of Interest

We have no conflicts of interest to disclose.

ORCID:

Shakhawan Beebany

https://orcid.org/0000-0002-8231-5481

Saad Salem Jasim

https://orcid.org/0000-0003-3893-8769

Mohammad M.Al-Tufah

https://orcid.org/0000-0002-0797-4882

Sevgi Samih Hidayet Arslan

https://orcid.org/0000-0001-7386-4174

HOW TO CITE THIS ARTICLE

Shakhawan Beebany, Saad Salem Jasim, Mohammad M.Al-Tufah, Sevgi Samih Hidayet Arslan. Preparation and Identification of New 1,4-bis (5,3-substituted-2,3-dihydro-1H-pyrazole-1-yl) buta-1,4-dione Derivatives with Their Antibacterial Effect Evaluation. Chem. Methodol., 2023, 7(2) 123-136

https://doi.org/10.22034/CHEMM.2023.365060.1614

URL: http://www.chemmethod.com/article_160325.html