Original Article

Antimicrobial Activities of Some Pyrazoline and Hydrazone Derivatives


  • Suna Sibel GÜRPINAR
  • Müjde ERYILMAZ

Received Date: 29.04.2019 Accepted Date: 22.08.2019 Turk J Pharm Sci 2020;17(5):500-505 PMID: 33177930


Resistance to antibiotics is recognized as one of the biggest threats to human health worldwide. Frequent and unnecessary use of antibiotics has caused infectious agents to adapt to antibiotics and thus drugs have become less effective. The resistance to many antibiotics necessitates the discovery of new antibiotics. In this study, two new and 23 previously reported 2-pyrazoline derivatives and one hydrazone derivative were evaluated for their in vitro antibacterial and antifungal activities.

Materials and Methods:

For the determination of the minimum inhibitory concentration (MIC) values of compounds, microbroth dilution was used.


The antimicrobial activities of the compounds were found in a wide range with MIC values of 32-512 μg/mL.


The synthesized compounds showed moderate antimicrobial activity compared with the standards. They can be used as lead molecules for the synthesis of more effective compounds.

Keywords: Synthesis, antimicrobial activity, pyrazoline derivatives, hydrazone derivatives


Antimicrobials are drugs that kill or inhibit the growth of microorganisms. Resistance to antimicrobials occurs when microorganisms change in a way that reduces the effectiveness of drugs. Antibiotic resistance has become a major clinical and public health problem worldwide today. Resistance rates are rising dangerously in the world. New resistance mechanisms are emerging, making it difficult to treat infectious diseases.1,2,3,4 In order to control this global problem, all government sectors and societies should take the necessary precautions and should support investigations on developing new antimicrobial drugs.

Hydrazones are formed as intermediates in the reaction of hydrazine and its derivatives with β-unsaturated carbonyl compounds but they are often not isolated due to their low stability and give pyrazolines with ring closure.5,6 These compounds have interesting biological properties, such as antimicrobial, antituberculous, antidepressant, analgesic, anticonvulsant, antitumor, antiviral, and antiinflammatory activities.7 Pyrazolines are five-membered and two neighboring nitrogen-containing heterocyclic compounds. They can be synthesized by the reaction of chalcones and hydrazines/hydrazides.8,9,10 Pyrazoline derivatives are electron-rich compounds that are thought to cause a wide variety of biological activities. Pyrazolines are important compounds because of their antimicrobial, analgesic, antiinflammatory, and antidepressant activities.11,12,13 According to the literature above, both pyrazoline and hydrazone compounds have antimicrobial activity. Therefore, we tested our compounds for their antimicrobial activity. In the present study, two new and 23 previously reported 2-pyrazoline derivatives and one hydrazone derivative were tested for their antibacterial and antifungal activity.


Antimicrobial activity tests

In the antibacterial activity tests, Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were used as test bacteria. For antifungal activity testing, Candida albicans ATCC 10231 was used. The cultures were prepared in Mueller Hinton Broth (Difco, Difco Laboratories, Detroit, MI, USA). For determination of minimum inhibitory concentration (MIC) values, microbroth dilution was used.14,15 Serial two-fold dilutions ranging from 1024 µg/mL to 8 µg/mL were made in the medium. The incubation conditions for the bacteria were 18-24 h at 35±1 °C and for the fungi were 48 h at 35±1 °C; the last well with no microbial growth was noted as the MIC value (mg/mL). Ampicillin, ofloxacin, and fluconazole were used as the positive control and 10% dimethyl sulfoxide (DMSO) was used as the negative control. All experiments were repeated three times. There was no statistical data analysis.


All compounds except compounds 20 and 24 have been reported earlier.10,13

Synthesis of chalcone derivatives (A, B)

2’-Hydroxy-4’-methoxy acetophenone/5’-chloro-2’-hydroxy acetophenone (4.99 mmol) and 4-bromobenzaldehyde/4-benzyloxybenzaldehyde (4.99 mmol) were reacted in ethanol (20 mL) using KOH solution (50% w/v) in water (5 mL) as catalyzer at room temperature overnight. Ice was added to the mixture and pH was set to 3-4 with 1 M HCl. Then the mixture was filtered and crystallized from ethanol.16,17,18

(E)-3-(4-bromophenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one (A): Yellow product. 61.14% yield. M.p. 141.0 °C. [lit. 138.0-140.0 °C].19 C16H13BrO3.

(E)-3-(4-(benzyloxy)phenyl)-1-(5-chloro-2-hydroxyphenyl)prop-2-en-1-one (B): Orange product. 93.70% yield. M.p. 138.0 °C. [lit. 100.0-102.0 °C].20 C22H17ClO3.

Synthesis of compounds 20 and 24

First, 1 equiv of compound A/compound B and 1 equiv of isoniazid were heated and stirred in ethanol (20 mL) for 4-25 h. Then the filtered products recrystallized from ethanol to give 20 and 24.21,22,23

(5-(4-(benzyloxy)phenyl)-3-(5-chloro-2-hydroxyphenyl)-4,5-dihydropyrazol-1-yl)(pyridin-4-yl)methanone (20): Beige product. Yield: 24.8%. M.p. 237.1 °C. IR (n, cm-1): 3167 (OH), 1641 (amide C=O), 1585 (C=N). 1H NMR (DMSO-d6, 400 MHz): 2.91 (dd, 1H, J1=16.4 Hz, J2=12.4 Hz, HA), 3.46 (dd, 1H, J1=3.2 Hz, J2=3.2 Hz HB), 5.14 (s, 2H, -OCH2Ph), 5.22 (dd, 1H, J1=2.4 Hz, J2=2.8 Hz, Hx), 7.02-8.74 (16H, aromatic-H), 11.11 (s, 1H, OH). MS (ESI): m/z=484 [M+H] (100%). C28H22ClN3O3 . 1.25 H2O: C 66.36, H 4.44, N 8.05; calcd. C 66.14, H 4.72, N 8.26.

(5-(4-bromophenyl)-3-(2-hydroxy-4-methoxyphenyl)-4,5-dihydropyrazol-1-yl) (pyridin-4-yl)methanone (24): Cream colored product. Yield 29.5%. M.p. 225.5 °C. IR (n, cm-1): 3174 (OH), 1641 (amide C=O), 1576 (C=N). 1H NMR (DMSO-d6, 400 MHz): 2.82 (dd, 1H, J1=12.4 Hz, J2=12.8 Hz, HA), 3.41 (dd, 1H, J1=2.8 Hz, J2=3.2 Hz, HB), 3.79 (s, 3H, -OCH3), 5.29 (dd, 1H, J1=2.8 Hz, J2=2.4 Hz, Hx), 6.59-8.73 (11H, aromatic-H), 11.02 (s, 1H, OH). MS (ESI): m/z=452 [M+H], 454 [M+H+2] (100%). C22H18BrN3O3 . 0.5 H2O: C 57.15, H 4.38, N 9.36; calcd. C 57.23, H 4.12, N 9.10.


A number of pyrazoline derivatives (compounds 2-26) and one hydrazone derivative (compound 1) were prepared. The structures of the target compounds are outlined in Figure 1.

Twenty-six compounds were tested for their antibacterial and antifungal activities. Antimicrobial activity was screened against two Gram-negative (E. coli ATCC 25922 and P. aeruginosa ATCC 27853) and three Gram-positive (S. aureus ATCC 29213, E. faecalis ATCC 29212, and B. subtilis ATCC 6633) bacteria and a fungus (C. albicans ATCC 10231) using ampicillin, ofloxacin, and fluconazole as the standard drugs. The results are given in Table 1.

Compound 1, the hydrazone, showed moderate activity against all the bacteria and the fungus. Pyrazoline derivatives were found to possess moderate activity against the bacteria and fungus. Whether the ring was open (hydrazone) or closed (pyrazolines) generally did not appear to make a large difference in antimicrobial effect. Compounds 5, 19, and 24 exhibited the highest antibacterial activity against S. aureus, with a MIC value of 64 µg/mL among the tested bacteria. Compounds 19 and 22 were found to have the best activity against P. aeruginosa. Compounds 22 and 26 showed the best activity against B. subtilis, with a MIC value of 64 µg/mL. Compounds 22 and 24 exhibited the highest antimicrobial activity against E. faecalis, with a MIC value of 32 µg/mL. Compound 5 was found the most active compound against C. albicans, with a MIC value of 64 µg/mL.

Karad et al.24 synthesized (2-morpholinoquinolin-3-yl)-4,5-dihydro-1H-pyrazol-1-yl) derivatives and studied their antibacterial activity. They found that the existence of -OCH3 substituent at position-4 in the phenyl ring at the C-3 position in the pyrazoline scaffold enhanced the antibacterial activity and antimalarial potency. For our compounds, a methoxy substituent in this position increased the antibacterial activity against S. aureus and E. faecalis, when it had bromo at the R7 position and pyridin-4-yl at the R8 position (compound 24).

Replacement of 4-methyl with 4-bromo substitution on the B ring in the pyrazoline nucleus enhanced the activity against S. aureus and E. faecalis (compounds 23 and 24).

According to Hamada and Abdo,9 the addition of pharmacophores such as chloro and bromo substituents with lipophilic properties increased the antimicrobial activity. For our compounds 7, 12, and 14, the substitution of chloro and bromo atoms at the 5-position of the A ring tended to increase the biological activity.

When the C ring had a phenyl scaffold, replacement of the 2-hydroxy-5-bromo phenyl (A ring) by 2-hydroxy-5-chloro phenyl increased the antibacterial activity against E. coli, S. aureus, and E. faecalis (compounds 7 and 12). When the compound carried a pyridine as the C ring, the substitution of 2-hydroxy-3,5-dichloro phenyl decreased the antimicrobial and antifungal activity. Replacement of this group by 2-hydroxy-5-bromo phenyl enhanced the antimicrobial activity against all bacteria and the fungus (compounds 4 and 8). Replacement of 2-hydroxy-3,5-dichloro phenyl by 2-hydroxy-5-chloro phenyl increased the activity against E. coli, P. aeruginosa, B. subtilis, and C. albicans (compounds 4 and 21).

The addition of the naphthyl group instead of phenyl on the A ring in compound 2 resulted in increased efficacy against E. coli. It reduced the activity against S. aureus, P. aeruginosa, E. faecalis, and B. subtilis. Compound 25 showed no antimicrobial activity against E. coli, S. aureus, or B. subtilis. Compound 26 showed no antimicrobial activity against E. coli.

Addition of phenyl instead of 2-furyl as the C ring increased the activity (compounds 6 and 9; compounds 10 and 11; compounds 13 and 17; compounds 7 and 16). The presence of phenyl instead of pyridine as the C ring increased the antimicrobial activity against E. coli. However, addition of pyridine instead of phenyl as the C ring enhanced the antibacterial activity against S. aureus, P. aeruginosa, E. faecalis, and B. subtilis (compounds 18 and 22). The substitution by a methoxy group at the fourth position on the B ring produced comparable antimicrobial activity against S. aureus and E. faecalis to the substitution by a methyl group (compounds 3 and 9).

Meta methoxy substitution on the B ring increased the activity against P. aeruginosa and S. aureus in the presence of pyridine as the C ring (compounds 19 and 21). Para methoxy substitution on the B ring enhanced the antimicrobial activity against P. aeruginosa, E. faecalis, and B. subtilis in the case of a pyridine substituent at the R8 position (compounds 8 and 22). Orto methoxy substitution on the B ring is not preferable, especially when the C ring is pyridine. According to Manna and Agrawal25 ortho substitution in the phenyl ring with a methoxy group at the 5th position of the pyrazoline ring caused less or inactive antibacterial activity against Gram-negative bacteria.

Replacement of 5-bromo with 5-methyl substitution on the A ring enhanced the activity against S. aureus and E. faecalis (compounds 3 and 18).

The presence of a methyl group at the fifth position of the A ring instead of a methoxy group at the fourth position of the A ring increased the antifungal activity and antimicrobial activity against E. coli, S. aureus, E. faecalis, P. aeruginosa, and C. albicans (compounds 6 and 15).


In this work, several pyrazoline derivatives and one hydrazone derivative were synthesized and screened for their antibacterial and antifungal activities. We noted that the pyridine ring as the C ring and methoxy and bromo substitutions on the B ring are preferable for a good antibacterial effect. 2-hydroxy-5-chloro substitution and 2-hydroxy-4-methoxy substitution substituents are favorable as the A ring. Further studies are necessary in order to understand the relation between the substitutions and activity, which could guide the design of more potent antimicrobial agents for therapeutic use.


I would like to thank to the staff of the Central Laboratory of the Pharmacy Faculty of Ankara University for the acquisition of the nuclear magnetic resonance mass spectrometer, and elemental analyzer of this work.

Conflicts of interest: No conflict of interest was declared by the authors. The authors alone are responsible for the content and writing of the paper.


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