Synthetic 4,4'-{(Arylmethylene)}bis(1H-pyrazol-5-ols): Efficient Radical Scavengers

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Byline: Qurat-ul-ain, Shahida Perveen, Munira Taj Muhammad, Sahar Yousuf, Khalid Mohammed Khan and M. Iqbal Choudhary

Summary: Functionalized 4,4'-{(arylmethylene)}bis(1H-pyrazol-5-ols) 1-22 were screened for their antioxidant activity. Antioxidant activity was performed by measuring their radical scavenging activity. These compounds demonstrated diverse in vitro DPPH radical scavenging activities with IC50 values ranging between 55.2 +- 1.2-149.6 +- 1.7 uM, as compared to standard BHT (butylated hydroxytoluene) (IC50 = 128.8 +- 2.1 uM).

Compounds 4,4'-((4-bromo-2,6-dimethoxyphenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol 18) IC50 = 55.2 +- 1.2 uM, 4,4'- (naphthalen-1-ylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol 20) (IC50 = 58.3 +- 0.9 uM), 4,4'- ((3-bromo-4-methoxyphenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol 19) (IC50 = 59.6 +- 0.1 uM), 4,4'-((4-methoxyphenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol 6) (IC50 = 60.01 +- 0.7 uM), 4,4'-((4-(methylthio)phenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol 10) (IC50 = 61.5 +- 0.6 uM), 4,4'-((2,4-dimethylphenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol) (1)

(IC50 = 68.3 +- 0.2 uM), 4,4'-((2-ethoxyphenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol) (8) (IC50 = 80.5 +- 1.5 uM), 4,4'-((3-nitrophenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol) (12) (IC50 = 70.8 +- 1.4 uM), 4,4'-((3-bromophenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol) (17) (IC50 = 77.9 +- 2.06 uM), and 4,4'-((2-fluorophenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol) (22) (IC50 80.0 +- 2.3 uM) showed better antioxidant activity than the standard BHT.

Varying substituents at aryl part (aldehydic part) have been responsible for diversified activity.

Keywords: Antioxidants, Radical Scavengers, DPPH, Bis-pyrazolone.

Introduction

Compounds with potential either to slow down or scavenge the process of oxidation can provide protection against reactive oxygen species (ROS) generated and are termed as antioxidants [1] Free radicals produce by the process of oxidation, further involve in chain reaction. Formation of these frees radicals in the cell resulting many diseases such as cancer, heart disease, diabetes, ageing, dementia, and eventually lead to death. Antioxidants, convert free radicals into harmless molecules. Antioxidant prevents the cell from oxidative stress and damage. Nature also maintain system of antioxidant in our human body e.g. vitamin E, N-acetylcysteine and ascorbic acid etc. [2]

In human body various ROS are generated as a result of metabolic and other functions [2, 3]. ROS leads to decreased membrane fluidity, loss of enzyme receptor activity and serious damage to membrane protein. Free radicals induced complications in human body include pre-mature aging, rheumatoid arthritis, Parkinson's disease, epilepsy, brain stroke, diabetes etc [4]. When ROS are circulating in excess in body, they cause serious damages to biological system. To encounter antioxidants [5] such as enzymes glutathione peroxidase, superoxide dismutase, catalase and non-enzymatic agents such as metallothionines, uric acid, albumin and bilirubin function. Low molecular weight antioxidants (LMWAs) can penetrate very easily into the regions of oxidative stress [6, 7].

In pathalogical conditions these endogenous antioxidants are not enough to fight these ROS and need to take some exogenous antioxidants arise [8]. Our group is perusing search of effective radical scavengers which can help in preventing diseases.

Our research group has reported, synthesis of pyrazolone and their antioxidant activity [9] and also structural similarity between pyrazolone and bis-pyrazolone, we therefore decided to synthesize and evaluate bis-pyrazolones for their antioxidant activity (Figure-1). The synthesis of these compounds has already been reported [10-13]. Screening of synthetic compound was performed in ethanolic solution which showed that compounds have polar or hydrophilic nature but as they are organic compounds so not completely soluble in aqueous solution.

We used DPPH assay method which is the best procedures for finding free radicals due to its free electron delocalization for a stable free radical. Free radical form of DPPH have deep violet color having I>>max value of 515 nm, when this solution is mixed with substrate, a stabilized non-radical form of DPPH showed a yellow color.

Experimental

Material and Methods

All the reagents used were purchased from Merck (Germany) and used as purchased. Thin layer chromatography was performed on pre-coated silica gel GF254 aluminum plates (Kieselgel, 60, 254, E-Merck, Germany). The 1H-NMR spectra were recorded on an Avance Bruker AM 300 MHz spectrometer. FAB-MS spectra were obtained on Finnigan MAT-113 spectrometer (Germany).

In Vitro DPPH Radical Scavenging Assay

The DPPH assay was typically carried out by the procedure by 5 uL of 500 uM solution of test compound which was mixed with solution of DPPH (95 uL, 300 uM) in ethanol. At 37 AoC, the reaction was allowed to progress for 30 minutes. Absorbance was recorded by multiple reader (Spectra Max 340, Molecular Devices, CA, USA) at 517 nm. Upon reduction, the color of the solution fades away (Violet to pale yellow). Activity is determined by comparing with a DMSO containing control. IC50 value is defined as the concentration of the test compound that decreases the concentration of DPPH up to 50%. The IC50 values of compounds were calculated by using the EZ-Fit Enzyme kinetics software program (Perrella Scientific Inc., Amherst, MA, USA). N-Acetylcysteine, ascorbic acid, and BHA are used as the reference compounds [14-15].

Results and Discussion

Chemistry

In present study, we synthesized twenty two (22) derivatives of bispyrazolone 1-22 from commercially available 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one by condensing them with different aldehydes. The reaction was performed in ethanol in the presence of CsF as catalyst. In CsF-catalyzed tandem Knoevenagel-Michael reaction, CsF plays two important roles. Firstly, it increases the positive charge on the carbonyl carbon of the aldehyde, and secondly the fluoride ion works as a base producing the enolate of pyrazolone. Consequently, the formation of carbon-carbon double bond by condensation with the aldehydes forms an intermediate 1,4-Michael type substrate serving for a 1,4-attack by the second fluoride-generated enolate of the pyrazolone resulting in the formation of the target bis-pyrazoles. Scheme-1 shows the general synthetic scheme.

Benzaldehydes with electron-donating and - withdrawing groups were reacted by using this method. It was found that the electron-withdrawing benzaldehydes provided an easy access and excellent yields to functionalized 4,4'-(arylmethylene)bis(1H-pyrazol-5-ols in a short reaction time, under mild conditions and simple work up. All synthetic compounds were characterized by 1H-NMR, and FAB-MS. The 1H-NMR spectra DMSO-d6 showed a broad signal for OH present in enol form of pyrazolone. A characteristic proton signal for the bis-pyrazolones resonated as singlet at I' 2 for 6H of two methyls of pyrazolone.

The reaction was performed by stirring both the reactants for 2-3 h. All the compounds were obtained as solid precipitates. In a typical experiment, 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one was reacted with various commercially available aldehydes in equimolar amount in ethanol as solvent for 2-3 h, the progress of the reacion was monitored by TLC. After completion of the reaction, precipetates were filtered and washed with dichloromethane to obtained pure products in good to excellent yields. Their structures were determined by 1H-NMR, and FAB mass spectrometry have been already published by our research group [10].

DPPH Radical Scavenging Activities

On the basis of previous knowledge that pyrazolone exhibited good anti-oxidant activity, we decided to evaluate compounds 1-22 for their antioxidant activity. Out of all the tested analogs only two of them were found to be inactive, and rest of the compounds except compound 3 were found to be more active than standard BHT (Table-1). Out of all the screened compounds, 18 (IC50 = 55.2 +- 1.2 uM) was found to be the most potent if compared with standard butylated hydroxytoluene (BHT) (IC50 = 128.8 +- 2.1 uM). Compounds 20 (IC50 = 58.3 +- 0.9 uM), 7 (IC50 = 62.8 +- 2.4 uM), 21 (IC50 = 65.3 +- 3.3 uM), 13 (IC50 = 59.80 +- 1.1 uM), 19 (IC50 = 59.6 +- 0.1 uM), 6 (IC50 = 60.01 +- 0.7 uM), 10 (IC50 = 61.5 +- 0.6 uM), and 14 (IC50 = 66.60 +- 1.1 uM) showed significant radical scavenging activity.

Compounds 12 (IC50 = 70.8 +- 1.4 uM), 22 (IC50 = 80. 0 +- 2.3 uM), 4 (IC50 = 90. 9 +- 2.1 uM), 1 (IC50 = 68.3 +- 0.2 uM), 9 (IC50 = 84. 1 +- 2.9 uM), 8 (IC50 = 80.5 +- 1.5 uM), 17 (IC50 = 77.9 +- 2.06 uM), 5 (IC50 = 90.8 +- 1.5 uM), and 2 (IC50 = 108.00 +- 1.5 uM) were found to have superior activity than BHT. Whereas, compound 3 (IC50 = 149.6 +- 1.7 uM) was moderate radical scavenger. Only two compounds of the series 15 and 11 have shown less than 50% inhibition and were not evaluated for their IC50 values.

Among all the synthesized alkoxy substituted analogs, compound 6 (IC50 = 60.01 +- 0.7 uM) was the most active one with 4-OMe group attached as substituent, comparing with compound 10 (IC50 = 61.5 +- 0.6 uM) with 4-SMe found to be slightly less active than its OMe counterpart. Comparison of compound 6 (IC50 = 60.01 +- 0.7 uM) with other alkoxy analogs such as compound 5 (IC50 = 90.8 +- 1.5 uM) which has 3-OMe residue indicated that the change in the position of the OMe group resulted in decreased activity. Similarly, comparing the compounds with di and tri-substituted analogs such as 4 (IC50 = 90.9 +- 2.1 uM) having 3,4-di-OMe, and 9 (IC50 = 84.1 +- 2.9 uM) having 2,3,4-tri-OMe residues a declined activity was observed.

Compound 7 (IC50 = 62.8 +- 2.4 uM) having 4-OEt substituent, was found to be good active than compound 8 (IC50 = 80.5 +- 1.5 uM) having 2-OEt, and 3 (IC50 = 149.6 +- 1.7 uM) with 3-OMe. This activity difference showed that the position of substituent greatly influences radical scavenging potential. There are few analogs in which OMe along with a halogen substituent such as 19 (IC50 = 59.6 +- 0.1 uM) with 3-Br and 4-OMe, showed good activity. In case of compound 18 (IC50 = 55.2 +- 1.2 uM) with 4-Br and 2,6-di-OMe, the highest radical scavenging activity was observed. This behavior indicates that if alkoxy and halogen substitutions are present in this class of compounds, a maximum radical scavenging activity may be achieved.

Among alkyl substituted compounds, 1 (IC50 = 68.3 +- 0.2 uM) and 2 (IC50 = 108.00 +- 1.5 uM) with 2,4 di-Me and 4-Me substitutions, respectively, were found to be more active than standard BHT.

Several mono nitro substituted analogs were synthesized including compound 11 (4-NO2), 12 (3-NO2), and 13 (2-NO2). Among various nitro derivatives, compound 13 (IC50 = 59.80 +- 1.1 uM) was more active than its meta analog 12 (IC50 = 70.8 +- 1.4 uM). However, compound 11 was found to be inactive with less than 50% inhibition. This difference in activity indicates that the position of substituent definitely effects the radical scavenging activity in bis-pyrazolone derivatives. Among single halogen substituted analogs, 4-chloro compound 14 demonstrated good activity (IC50 = 66.60 +- 1.1 uM) as compared to 2-chloro compound 15 which was inactive.

A compound with 2,4-dichloro substitution 16 (IC50 = 109.3 +- 0.9 uM) is less active than single halogen substituted analog 14.

Table-1: DPPH Radical scavenging activity of compounds 1-22.

Compounds###Structures###IC50 +- S.E.Ma (uM)

###1###68.3 +- 0.2

###2###108.00 +- 1.5

###3###149.6 +- 1.7

###4###90.9 +- 2.1

###5###90.8 +- 1.5

###6###60.01 +- 0.7

###7###62.8 +- 2.4

###8###80.5 +- 1.5

###9###84.1 +- 2.9

###10###61.5 +- 0.6

###11###NAb

###12###70.8 +- 1.4

###13###59.80 +- 1.1

###14###66.60 +- 1.1

###15###NAb

###16###109.3 +- 0.9

###17###77.9 +- 2.06

###18###55.2 +- 1.20

###19###59.6 +- 0.1

###20###58.3 +- 0.9

###21###65.3 +- 3.3

###22###80.0 +- 2.3

###BHT (st)###128.8 +- 2.1

Conclusion

The radical scavenging activity of bis-pyrazolones 1-22 is apparently based on the nature and position of substituents. Compounds 1, 2, 4-10, 12-14, and 16-22 exhibited superior activity than the standard BHT (IC50 = 128.8 +- 2.1 uM). Further chemical modifications and researchwork on these molecules may result in clinically useful antioxidants.

Acknowledgement

The authors acknowledge the financial support of the Higher Education Commission (HEC) Pakistan, under National Research Program for Universities (Project No. 20-1910).

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Gale Document Number: GALE|A546265682