Journal of Chemistry

Volume 2013 (2013), Article ID 765768, 6 pages

http://dx.doi.org/10.1155/2013/765768

Research Article

Design, Synthesis, and In Vitro Antioxidant Activity of 1,3,5-Trisubstituted-2-pyrazolines Derivatives

P. C. Jagadish,¹ Neeraj Soni,² and Amita Verma³

¹Manipal College of Pharmaceutical Sciences, Manipal, India

²Novarstis Healthcare Private Limited, Hyderabad, Andhra Pradesh, India

³Department of Pharmaceutical Sciences, Sam Higginbottom Institute of Agriculture, Technology and Sciences Deemed to be University, Allahabad, Uttar Pradesh 211007, India

Received 31 May 2013; Revised 29 August 2013; Accepted 2 September 2013

Academic Editor: Mire Zloh

Copyright © 2013 P. C. Jagadish et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Mannich base of pyrazolines 3(a–e) under both conventional and microwave irradiation was synthesized. All the synthesised compounds were purified by recrystallisation, characterized on the basis of UV, IR, and NMR spectroscopy, and further supported by mass spectroscopy. The result obtained confirms superiority of microwave irradiation method over classical heating one. The molecular properties and Lipinski rule of five for compounds 3(a–e) were determined by Molinspiration. The synthesized compounds were subsequently evaluated for the antioxidant activity. All the compounds were found in compliance with Lipinski “Rule of Five”, and compound 3e having p-hydroxyl substitution showed best antioxidant activity as compared to ascorbic acid and rutin.

1. Introduction

Variously substituted pyrazolines and their derivatives are important biological agents, and a significant amount of research activity has been directed towards this class. In particular, they are used as antibacterial [1], antifungal [2], anti-inflammatory [3], central nervous system depressant [4], analgesic [5], mono amino oxidase inhibitor [6], anticancer [7], and antiviral agents [8]. Moreover, pyrazolines have played a crucial part in the development of theory in heterocyclic chemistry.

The advantages of microwave technology over conventional method have promoted us to synthesize pyrazolines from both conventional as well as microwave assisted method. There are several medical emergencies that result from free radical mediated pathology. The purpose of this study was to synthesize the pyrazoline derivatives in search for exploring the potential of this skeleton as antioxidant.

2. Materials and Methods

All the melting points reported were determined in open capillaries using Veego VMP-1 melting point apparatus expressed in °C and are uncorrected. The I. R. spectra of the compounds were recorded on Perkin-Elmer infrared 283 spectrometer in KBr disc and expressed in cm⁻¹. NMR spectra were recorded on Bruker DRX-300 spectrometer. Mass spectra were recorded on Jeol SX-102 (FAB) spectrometer. Synthesis and analytical studies of title compounds were carried out using laboratory grade reagents. Standard techniques like TLC were used to monitor reactions and to determine purity of the products. All the products were purified by recrystallization. The microwave-assisted syntheses were carried out in domestic oven, Midea PJ21B-A 400 W. All the compounds were subjected to molecular properties prediction and drug-likeness by Molinspiration software.

2.1. Procedure for Synthesis

2.1.1. Step  1: Synthesis of Chalcones (3-Furan-2-yl-1-aryl-prop-2-en-1-one) 1(a–e)

To a solution of ketone (0.01 mole) and furfuraldehyde (0.01 mole) in 30 mL methanol, catalytic quantity of solid sodium hydroxide pellets were added and stirred at room temperature. The solution was neutralized with diluted HCl. The precipitated product was filtered, washed with water, dried, and recrystallised from ethanol. The reaction was monitored by TLC using Toulene : Ethyl acetate (8 : 2) solvent system. By adopting this general procedure, five different chalcones were prepared using five different acetophenones [9].

2.1.2. Step  2: Synthesis of 5-Furan-2-yl-3-aryl-4, 5-Dihydro-1H-pyrazole 2(a–e)

Conventional Method. A mixture of chalcone 1(a–e) (0.01 mole) and hydrazine hydrate (0.02 mole) in 20 mL ethanol was refluxed for 4 to 6 hr; reaction was monitored by TLC and the resulting solution was left overnight in a refrigerator. Crystals separated were filtered and recrystalised from ethanol.

Microwave Irradiation Method. A mixture of chalcone 1(a–e) (0.01 mole) and hydrazine hydrate (0.02 mole) in 20 mL ethanol was subjected to microwave irradiation of 350 watt for a period of 2 to 3 minute. Reaction was monitored by TLC. Resulting solution was left overnight in refrigerator. Crystals separated were filtered and recrystalised from ethanol.

2.1.3. Step  3: Synthesis of 5-Furan-2-yl-3-aryl-1-[(3-chloro-phenyl) methyl amino]-2-pyrazoline 3(a–e)

A mixture of pyrazoline 2(a–e) (0.02 mole) and 3-chloro-aniline (0.02 mole), Paraformaldehyde (0.01 mole), and one drop of concentrated HCl in methanol (40 mL) was refluxed for 3-4 Hrs. Reaction was monitored by TLC. Excess solvent was removed by distillation under reduced pressure. After cooling the reaction mixture the product was separated out which was filtered and recrystallised from methanol.

2.2. Molecular Properties Prediction and Drug-Likeness by Molinspiration

2.2.1. Molecular Properties Prediction

Physicochemical parameters play a vital role in generation and determination of bioactivity of any compound. Molinspiration, web based software, was used to explore the various parameters such as miLogP, TPSA, MW, and drug likeness. MiLogP (octanol/water partition coefficient) was calculated by the method developed by Molinspiration as a sum of fragment based contributions and correction factors and used to predict the permeability of molecule across the cell membrane [10].

Topological Polar Surface Area (TPSA) is calculated based on the methodology published by Ertl et al. as the sum of fragment based contributions in which O- and N-centered polar fragments are to be considered and calculated by surface areas that are occupied by oxygen and nitrogen atoms and by hydrogen atoms attached to them [11]. TPSA has been used for characterizing drug absorption, including intestinal absorption, bioavailability, Caco-2 permeability, and blood brain barrier permeability. Method for calculation of molecular volume developed by molinspiration is based on group contributions.

Number of rotatable bonds (nrotb) is a simple topological parameter that measures molecular flexibility. It has been shown to be a very good descriptor of oral bioavailability of drug [12]. Rotatable bond is defined as any single nonring bond, bounded to nonterminal heavy (i.e., nonhydrogen) atom. Amide C–N bonds are not considered because they are having high rotational energy barrier.

2.2.2. Drug Likeness

Drug likeliness is a qualitative means of analysis to check whether the given molecule has drug-like properties, and it is defined as a complex balance of various molecular properties and structural features. These properties, mainly hydrophobicity, electronic distribution, hydrogen bonding characteristics, molecule size, and flexibility and presence of various pharmacophoric features, influence the behavior of a molecule in a living organism, including bioavailability, transport properties, affinity to proteins, reactivity, toxicity, and metabolic stability [10].

2.3. Antioxidant Activity

2.3.1. Scavenging of DPPH Radical [13]

Antioxidants react with stable free radical, DPPH, and convert it to 1,1-diphenyl-2-picryl hydrazine. The ability to scavenge the stable free radical DPPH was measured by decrease in absorbance at 517 nm. A standard drug ascorbic acid was also studied for comparison.

To the ethanolic solution of DPPH (0.5 mL of 0.0125 mM) an equal volume of the test compound dissolved in dimethyl sulfoxide [DMSO] was added at various concentrations; equal amount of DMSO was added to control solution. After 20 min at room temperature, absorbance at 517 nm was taken. The experiment was carried out in triplicate.

2.3.2. Scavenging of Nitric Oxide [14]

Sodium nitroprusside spontaneously generates nitric oxide in aqueous solutions. Nitric oxide, generated in this manner, was converted to nitric and nitrous acids on contact with dissolved oxygen and water. The liberated nitrous acid is estimated using Greiss reagent. In presence of test components, likely to be scavengers, the amount of nitrous acid will decrease. The degree of decrease will reflect the extent of scavenging.

Sodium nitroprusside (5 mM) in PBS was incubated with different concentrations of test compound in DMSO at 25°C for 5 hrs. Control experiments without test compounds but with equivalent amounts of DMSO were conducted in an identical manner. After 5 hrs, 0.5 mL of incubated solution was removed and 0.5 mL of Greiss reagent was added. The absorbance of the chromophore formed during diazotisation of nitrite with sulfanilamide and subsequent coupling with N-(1-naphthyl) ethelene diamine was read at 546 nm. Experiment was carried out in triplicate.

3. Results and Discussion

3.1. Chemistry

In this present work, the reaction of chalcones 1(a–e) with various ketones under microwave assisted technique as well as conventional heating method was studied (Figure 1). The result obtained confirms the superiority of microwave irradiation method over classical heating one (Table 1).

Table 1: Comparative statement of synthesis under conventional and microwave technique.

S. no.
Product code
Conventional
Microwave (350 watt)

Reaction time (hours)
Yield %
Irradiation time (minute)
Yield %

(1)
2a
4-5
50
2.5
56

(2)
2b
4-5
60
2.0
80

(3)
2c
4-5
60
2.0
80

(4)
2d
4-5
50
2.5
62

(5)
2e
4-5
50
2.5
52

Figure 1: Scheme for synthesis of compounds 3(a–e).

3.1.1. Spectral Data and Interpretation of the Compounds , , , and

In the IR spectrum of compound 2e, it showed absorption band at 3318 cm⁻¹ corresponding to the pyrazoline NH group and OH group. The absorption bands at 1603 and 1515 cm⁻¹ are attributed to the C=N stretching and N–N stretching, respectively. In the IR spectrum of compound 2b showed absorption band at 3349.25 cm⁻¹ corresponding to the pyrazoline NH group. The absorption bands at 1592 and 1493.88 cm⁻¹ are attributed to the C=N stretching and N–N stretching, respectively. In the IR spectrum of compound 3b absorption bands appear at 3416, 1585, and 1496 cm⁻¹ for NH, C=N, and N–N groups, respectively. The absence of carbonyl band clearly supported the formation of 2-pyrazoline system.

The ¹H NMR spectrum of compounds 2c, 2b, 2e, and 3b exhibits a peak in the region δ 8.25 characteristic of NH proton. The doublet of doublets in the region δ 3.28 (1H, d, d, , 10 Hz) and δ 3.38 (1H, d, d, , 11 Hz) were due to the methylene protons of pyrazoline nucleus. The splitting is due to vicinal and geminal coupling of the methylene protons. Another doublet of doublet at δ 5.00 region (1H, d, d, , 10 Hz) was due to the methine proton which was coupled with the magnetically nonequivalent methylene protons of pyrazoline ring. The doublets signals at δ 7.55 and 7.49 and the doublet of doublet at δ 6.30 region with a small coupling constant ( Hz) are due to the furan ring system. The pair of doublet in the δ 7.5 region ( Hz) are due to aromatic protons. The hydroxyl proton in compound 2e resonates at 4.95, and the aromatic proton signals in compound 3b resonate at δ 6.36 and 7.20 each integrating for two protons. All the signals in the compound 2e are in the upfield region when compared with the signals of compounds 2b, 2c, and 3b which may be due to the electron donating nature of the OH group.

In the compound 3b the presence of a singlet signal at δ 4.6 and the aromatic proton signals which resonate at δ 6.36 and 7.20 each integrating for two protons shows that the compound 2b has undergone Mannich reaction and yielded compound 3b.

In the mass spectrum of compounds 2b and 2e, they exhibit molecular ion peaks at 246/248 and 228, which also includes the isotope peak of halogen atom. The compounds exhibit peaks at 217 and 199 due to M⁺–N–NH characteristic of pyrazolines. The common peak at 93 and 107 present is due to furan nitrile and the furan nucleus, respectively. The p-chloro phenyl nitrile peak appears at 137 for compound 2b, and p-hydroxy phenyl nitrile peak appears at 119 for compound 2e as depicted in fragmentation Schemes 1 and 2.

Scheme 1: Fragmentation of Scheme 1.

Scheme 2: Fragmentation of Scheme 2.

In the mass spectrum of compound 3b M⁺ ion peak is observed at 386/388, which includes isotope peak of halogen atom. The other peaks 246, 217, and 138 are due to fragments shown in fragmentation of Scheme 3.

Scheme 3: Fragmentation of Scheme 3.

3.2. Molinspiration Calculation

Results of compounds 3(a–e) in terms of molecular properties (molecular weight, milogP and TPSA, nrotb, volume, etc.) are valued in Table 2. According to Lipinski rule of five, most “drug-like” molecules have log , number of hydrogen bond acceptors ≤10, molecular weight ≤500, and number of hydrogen bond donors ≤5. Molecules violating more than one of these rules may have problems with bioavailability [15]. On close inspection of Table 2, all molecules were found in compliance with Lipinski rule of five recommendations for new chemical entity to have good oral bioavailability with no violations (Nviolation). The milog valve of all compounds was found below five, suggesting that the molecules have good permeability across the cell membrane which in turn is needed for generation of bioactivity. All the compounds (3a–3e) are within the limit, that is, 160 Å in respect of TPSA, which showed that molecules are fulfilling the optimal requirement for drug absorption.

Table 2: Molinspiration calculation for synthesized compounds (3a–3e).

Compound
milogP
TPSA
natoms
MW
nON
nOHNH
Nviolation
Nrotb
Volume

3a
4.943
40.769
25
351.837
4
1
0
5
310.001

3b
5.621
40.769
26
386.282
4
1
1
5
323.536

3c
5.752
40.769
26
430.733
4
1
1
5
327.886

3d
4.883
60.997
26
367.836
5
2
0
5
318.018

3e
4.464
60.997
26
367.836
5
2
0
5
318.018

Ascorbic acid
−1.402
107.217
12
176.124
6
4
0
2
139.707

Rutin
−1.294
269.427
42
596.494
16
10
3
5
479.267

3.3. Antioxidant Activity

3.3.1. DPPH Radical Scavenging Activity

The effect of antioxidants on DPPH radical scavenging was thought to be due to their hydrogen donating ability. As shown in Tables 3 and 4, five compounds (2d, 2e, 3a, 3d, and 3e) showed radical scavenging activity. It is clear from the data that phenolic compounds are active (3e > 2e > 2d > 3d) and nonphenolic compounds are less or inactive. The scavenging activity order of the test compounds was 3e > 2e > 2d > 3a > 3d. The strong DPPH scavenging activity of can be attributed in part to the phenolic OH present in 3e, 2e, 2d, and 3d.

Table 3: Free radical scavenging activity of 5-furan-2-yl-3-aryl-4, 5-dihydro-1H-pyrazole 2(a–e).

Compound
Concentration in μg/mL
Percentage of scavenging ± SD

DPPH
Nitric oxide

2a
500

250

125

2b
500

250

2c
500

250

2d
500

250

125

62.5

2e
500

250

125

62.5

31.25

—

Ascorbic acid
50

—

Rutine
50

Table 4: Free radical scavenging activity of 5-furan-2-yl-3-aryl-1-[(3-chloro-phenyl) methyl amino]-2-pyrazoline 3(a–e).

Compound
Concentration in μg/mL
Percentage of scavenging ± SD

DPPH
Nitric oxide

3a
500

250

125

62.5

3b
500

250

3c
500

3d
500

250

125

3e
500

250

125

62.5

31.25

Ascorbic acid
50

—

Rutine
50

3.3.2. Nitric Oxide Radical Scavenging Activity

The effect of antioxidants on nitrogen centered nitric oxide radical scavenging. As shown in Tables 3 and 4, seven compounds (2d, 2e, 2a, 2b, 3a, 3d, and 3e) showed nitric oxide radical scavenging activity. The scavenging activity order of the test compounds was 3e > 3a > 2d > 2e >  3d > 2a > 2b.

4. Conclusion

As a result of our studies related to the development of synthetic protocols using both classical heating (conventional) method and microwave irradiation, we now report a novel and easy access to 1H-5(furan-2-yl) 3-aryl-2-pyrazolines using a one-pot procedure. This work demonstrates that all the synthesized compounds followed the Lipinski rule of five, that is, compounds may have the good oral bioavailability. By careful observation of the results of preliminary in vitro antioxidant activity of all the compounds, further screening on other in vitro, ex vivo and in vivo models will throw some light on the potential of compounds to be an antioxidant compound.

Acknowledgment

The authors are thankful to the Central Drug Research Institute, Lucknow, India, for scanning IR, NMR, and MS spectra of compounds.

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