Synthesis, characterization and biological evaluation of

4-{1-aza-2-[(aryl) amino]}-3-methyl-2-pyrazolin-5-ones derivatives

 

Gurdeep Singh¹*, Ritesh Patel², Sudeep Mandal³, Deepak Kumar Basediya⁴

¹School of Pharmaceutical Science Lovely Professional University Phagwara, Punjab- 144001, India.

²Department of Pharmaceutical Chemistry, Indore Institute of Pharmacy, Indore-453331, M. P., India.

³Faculty of Pharmacy, Kalinga University, Raipur-492101, C.G., India.

⁴Department of Pharmacy, Technocrats Institute of Technology, Bhopal-462021, M. P., India.

 

ABSTRACT:

A series of substituted 4-{1-aza-2-[(aryl) amino)]}-3-methyl-2-pyrazolin-5-ones has been synthesized and evaluated for their biological activity. The title compounds (4a-l) were prepared by the diazotization of substituted anilines (1a-l) to form substituted phenyl hydrazine derivatives (2a-l) which synthesized substituted 4-{1-aza-2-[(aryl) amino)]}-3-methyl-2-pyrazolin-5-ones (4a-l) by Michael addition reaction, which is a nucleophilic addition of enolate anion to the carbon-carbon double bond of a α, β–unsaturated carboxylic acid derivatives. Twelve different pyrazolinone derivatives (4a to 4l) were synthesized. Structural assignments of these compounds have been made by elemental analysis, FTIR, ¹HNMR and Mass spectral data and the purity of the compounds was determined by TLC. The antimicrobial activity of the newly Synthesized heterocyclic compounds were evaluated against Gram-positive, Gram-negative bacteria and fungi. Most of the compounds showed a moderate degree of potent antimicrobial activity. While analgesic activities were tested via both hot plate and acetic acid induced writhing methods. The study concluded that the compound 4b and4f were found to exhibit significant analgesic activity when compared to Diclofenac as standard drug while other derivatives exhibit moderate to good analgesic activity.

 

 

1. INTRODUCTION:

Microorganisms that resist antimicrobial drugs are a complex problem affecting the health of people all over the world. More than one million people die from microbial infections every year, and the number of deaths is expected to increase as antimi­crobial drug resistance increases^[1]. Innovation must be strengthened in research activities related to effective antimicrobial and antifungal drugs^[2]. However, because conventional antibiotics are often abused/overused to treat microbial infections, some microorganisms have developed resistance to some of these antibiotics ^[3]. Analgesics are the primary need for patients to get rid of any kind of pain. Pain is one of the basic symptoms of all human ailments which is a sensorial modality and primary protective. Analgesics only relieve pain in a particular complaint without affecting its cause^[4]. A prolonged painful stimulation may generate increased blood flow and inflammation, and vice versa, inflammation may lead to pain^[5]. The effect of Nitricoxide (NO) on peripheral nociception and with its local vasodilation, spinal pain associated with herniated discs can be classified as peripheral nociception because the spine is in this case the site of inflammation^{[6, 7]}. It has been concluded that pain associated with Rheumatoid arthritis (RA) is due to synovial inflammation, and that pain in RA is associated with enhanced substance P and IL-2 in the synovial fluid. Both these substances induce production of Nitric oxide. It has been shown in murine models that NO enhances the sensitivity of peripheral nociceptors and that inhibitors of Nitric oxide synthase can act as analgesic agents at the cerebral, spinal and peripheral levels^{[8, 9]}. There is a direct-evidence that NO can induce pain locally.

 

 

On the other hand, the pyrazolinone ring that contains a five membered heterocyclic organic compound with two adjacent nitrogen atoms is a prominent heterocyclic scaffold in lots of bioactive molecules. They are important substances and have gained widespread attention in agrochemical, pharmaceutical and chemical industries^[10]. They possess a wide range of biological activities^{[11, 14]}, including antimicrobial^{[15, 16]}, antiviral^{[17, 18]}, anticancer^{[19, 20]}, anti-inflammatory ^{[21, 22]}, antihistaminic^[23], pesticidal^[24], antifungal^[25], rheumatoid arthritis^[26], anticonvulsant^[27], antidepressant^[28], antipyretic^[29], antibacterial^[30] agents, etc. and these bio-activities have inspired chemists to synthesize substituted pyrazolinone systems to explore the usefulness of this heterocyclic template. In view of these reports the present research deals with a novel synthesis of substituted 4-{1-aza-2-[(aryl) amino)]}-3-methyl-2-pyrazolin-5-ones and evaluated their anti-microbial and analgesic activity.

 

2. MATERIALS AND METHODS:

2.1 Experimental:

All the chemicals used in the synthesis of the intermediates and final products were of analytical grade. Melting points of all the compounds were recorded in Digital melting point apparatus and were uncorrected. The IR spectra were recorded on Perkin Elmer FTIR spectrometer using KBr. ¹HNMR spectra were recorded on Bruker Avance II 400MH_Z NMR. The chemical shifts were reported as parts per million downfield from tetra methyl silane as internal standard. Mass spectra were performed on LC-MSD-Tranp-SL2010A SHIMADZU using CDCl₃ as solvent. The purity of the compound was checked by TLC using precoated silica gel G plate method (R_f value given in Table 1) using ethyl acetate: petroleum ether: chloroform in the ratio of 0.6:0.8:8.6 and iodine vapors as visualizing agent.

 

Table - 1: Physico-Chemical Characteristics of Synthesized Pyrazolinone Derivatives

 

Scheme of Synthesis:

 

2.2 Procedure for the synthesis of substituted phenyl hydrazine from substituted anilines (2a-l)[31]:

Substituted aniline (0.03 mol) was dissolved in a mixture of 10.5 ml of concentrated HCl and an equal volume of water, cooled rapidly to 0°C in order to obtain the hydrochloride of the base in a fine state of division. Gradual addition of a solution of sodium nitrite (0.03 mol) in 6 ml of water was performed for diazotization. Stirring was continued for a few minutes, and the solution was filtered and added by using a separatory funnel to an ice-cold solution of sodium sulphite (96% Na₂SO₃⋅7H₂O) (0.15 mol) in 100 ml of water containing 4 g of NaOH. The solution was allowed to stand for 5 minutes, acidify with 35 ml of concentrated HCl, and heat on a water bath at 25⁰C for 3 minutes, when yellow needles commence separating. This solution was kept overnight, filtered off the crystals, heated with 10 ml of concentrated HCl on a water bath for 7 minutes, and allowed to cool. The precipitate was filtered off and dissolved in water and the solution was treated with a concentrated solution of sodium acetate. The free base separated out in an almost pure state. Recrystallized the product with methylated spirit.

 

2.3 Synthesis of Ethyl -2- acetyl-3-aza-3-{(substituted phenyl amino} prop-2-enolate (3a-l)

Substituted phenyl hydrazine (0.002 mol) was dissolved in minimum amount of cold water then ethanolic KOH was added. The solution was then refluxed for 40 min at 70°C in the presence of ethylacetoacetate. The precipitate was filtered, washed with water and dried. Recrystallized the product with methylated spirit.

 

2.4 Synthesis of 4-{1-aza[(substituted phenyl) amino)]}-3-methyl-2-pyrazolin-5-ones (4a-l)

Ethyl-2-acetyl-3-aza-3-{(substituted phenyl amino}prop-2-enolate (0.002 mol) was dissolved in glacial acetic acid (25ml) and hydrazine hydrate (0.002mol) in glacial acetic acid was added. The mixture was refluxed for 6 hr, cooled and allowed to stand overnight. The resulting solid was dried and recrystallized from ethanol. Similarly other members of 4a-l were prepared and their physical and analytical data were recorded.

 

2.5 4-(2-(2-chlorophenylhydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4a)

Molecular formula: C₁₀H₉ClN₄O, Molecular weight: 236.66Yield: 64.53%, M.P.: 166-168^oC, R_f value: 0.71, FT-IR (KBr, υ, cm-1): 3476.89 (N-H Str.), 3145.57 (=C-H str.), 2966.78 (C-H str.), 1706.67 (C=O str.), 1602.97 (C=C str.), 1225.67 (C-N str.), 743.35 (Ar C-H Bend.), 782.03 (C-Cl Bend). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 6.40-6.79 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 238.57 (M⁺²). Anal. Calcd.:C, 50.75; H, 3.83; Cl, 14.98; N, 23.67; O, 6.76. Found:C, 50.43; H, 3.91; Cl, 14.54; N, 23.02; O, 6.97.

 

2.6 4-(2-(3-chlorophenylhydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4b)

Molecular formula: C₁₀H₉ClN₄O, Molecular weight: 236.66Yield: 65.92%, M.P.: 176-179^oC, R_f value: 0.69, FT-IR (KBr, υ, cm-1): 3407.25 (N-H Str.), 3157.35 (=C-H str.), 2972.35 (C-H str.), 1714.97 (C=O str.), 1612.25 (C=C str.), 1234.73 (C-N str.), 739.29 (Ar C-H Bend.), 778.27 (C-Cl Bend). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 6.34-6.91 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 238.76 (M⁺²). Anal. Calcd.:C, 50.75; H, 3.83; Cl, 14.98; N, 23.67; O, 6.76. Found:C, 50.67; H, 4.02; Cl, 14.93; N, 22.98; O, 6.91.

 

2.7 4-(2-(4-chlorophenylhydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4c)

Molecular formula: C₁₀H₉ClN₄O, Molecular weight: 236.66Yield: 70.55%, M.P.: 181-183^oC, R_f value: 0.52, FT-IR (KBr, υ, cm-1): 3389.57 (N-H Str.), 3146.78 (=C-H str.), 2936.86 (C-H str.), 1712.35 (C=O str.), 1607.57 (C=C str.), 1238.54 (C-N str.), 749.78 (Ar C-H Bend.), 774.59 (C-Cl Bend). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 6.31-6.86 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 238.93 (M⁺²). Anal. Calcd.:C, 50.75; H, 3.83; Cl, 14.98; N, 23.67; O, 6.76. Found:C, 50.96; H, 3.67; Cl, 14.78; N, 23.06; O, 6.56.

 

2.8 4-(2-tolylhydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4d)

Molecular formula: C₁₁H₁₂N₄O, Molecular weight: 216.24Yield: 68.19 %, M.P.: 155-157^oC, R_fvalue: 0.67, FT-IR (KBr, υ, cm-1): 3423.57 (N-H Str.), 3178.70 (=C-H str.), 2956.56 (C-H str.), 1710.67 (C=O str.), 1603.68 (C=C str.), 1278.94 (C-N str.), 752.43 (Ar C-H Bend.). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 2.35 (s, 3H, CH₃) 6.34-6.82 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 217.67 (M⁺1). Anal. Calcd.:C, 61.10; H, 5.59; N, 25.91; O, 7.40. Found:C, 61.56; H, 5.76; N, 25.43; O, 7.50.

 

2.9 4-(3-tolylhydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4e)

Molecular formula: C₁₁H₁₂N₄O, Molecular weight: 216.24Yield: 69.22 %, M.P.: 152-154^oC, R_f value: 0.56, FT-IR (KBr, υ, cm-1): 3405.59 (N-H Str.), 3136.93 (=C-H str.), 2963.63 (C-H str.), 1707.73 (C=O str.), 1600.67 (C=C str.), 1209.94 (C-N str.), 743.93 (Ar C-H Bend.). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 2.35 (s, 3H, CH₃) 6.30-6.77 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 217.18 (M⁺¹). Anal. Calcd.:C, 61.10; H, 5.59; N, 25.91; O, 7.40. Found:C, 61.53; H, 5.98; N, 25.09; O, 7.73.

 

2.10 4-(4-tolylhydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4f)

Molecular formula: C₁₁H₁₂N₄O, Molecular weight: 216.24Yield: 66.93 %, M.P.: 159-161^oC, R_fvalue: 0.62, FT-IR (KBr, υ, cm-1): 3476.84 (N-H Str.), 3133.75 (=C-H str.), 2922.45 (C-H str.), 1711.44 (C=O str.), 1608.35 (C=C str.), 1256.77 (C-N str.), 758.67 (Ar C-H Bend.). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 2.35 (s, 3H, CH₃) 6.29-6.70 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 217.88 (M⁺¹). Anal. Calcd.:C, 61.10; H, 5.59; N, 25.91; O, 7.40. Found:C, 61.23; H, 5.87; N, 25.67; O, 7.69.

 

2.11 4-(2-(2-methoxyphenyl)hydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4g)

Molecular formula: C₁₁H₁₂N₄O₂, Molecular weight: 232.24Yield: 69.47 %, M.P.: 169-171^oC, R_f value: 0.88, FT-IR (KBr, υ, cm-1): 3411.24 (N-H Str.), 3108.67 (=C-H str.), 2975.66 (C-H str.), 1719.76 (C=O str.), 1618.54 (C=C str.), 1234.56 (C-N str.), 1145.32 (C-O str.), 767.76 (Ar C-H Bend.). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 3.73 (s, 3H, OCH₃) 6.35-6.57 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 233.45 (M⁺¹). Anal. Calcd.:C, 56.89; H, 5.21; N, 24.12; O, 13.78. Found:C, 57.01; H, 5.29; N, 24.53; O, 13.90.

 

2.12 4-(2-(4-methoxyphenyl)hydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4h)

Molecular formula: C₁₁H₁₂N₄O₂, Molecular weight: 232.24Yield: 63.74 %, M.P.: 176-178^oC, R_f value: 0.67, FT-IR (KBr, υ, cm-1): 3433.64 (N-H Str.), 3134.45 (=C-H str.), 2955.76 (C-H str.), 1709.67 (C=O str.), 1606.46 (C=C str.), 1245.87 (C-N str.), 1123.98 (C-O str.), 756.94 (Ar C-H Bend.). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 3.73 (s, 3H, OCH₃) 6.29-6.75 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 233.92 (M⁺¹). Anal. Calcd.:C, 56.89; H, 5.21; N, 24.12; O, 13.78. Found:C, 56.76; H, 5.87; N, 24.69; O, 13.56.

 

2.13 4-(2-(2-nitrophenyl)hydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4i)

Molecular formula: C₁₀H₉N₅O₃, Molecular weight: 247.21Yield: 67.87 %, M.P.: 201-203^oC, R_fvalue: 0.59, FT-IR (KBr, υ, cm-1): 3389.62 (N-H Str.), 3101.36 (=C-H str.), 2967.12 (C-H str.), 1713.89 (C=O str.), 1609.37 (C=C str.), 1273.98 (C-N str.), 1535.72 (N-O str.), 787.27 (Ar C-H Bend.). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 6.48-6.82 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 248.34 (M⁺¹). Anal. Calcd.:C, 48.58; H, 3.67; N, 28.33; O, 19.42. Found::C, 48.98; H, 3.88; N, 28.76; O, 19.27.

 

2.14 4-(2-(4-nitrophenyl)hydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4j)

Molecular formula: C₁₀H₉N₅O₃, Molecular weight: 247.21Yield: 52.03 %, M.P.: 207-209^oC, R_fvalue: 0.63, FT-IR (KBr, υ, cm-1): 3414.37 (N-H Str.), 3167.98 (=C-H str.), 2947.76 (C-H str.), 1711.56 (C=O str.), 1605.78 (C=C str.), 1276.48 (C-N str.), 1515.76 (N-O str.), 767.22 (Ar C-H Bend.). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 6.32-6.78 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 248.99 (M⁺¹). Anal. Calcd.:C, 48.58; H, 3.67; N, 28.33; O, 19.42. Found: C, 49.12; H, 3.56; N, 28.87; O, 19.67.

 

2.15 4-(2-(2-hydroxyphenyl)hydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4k)

Molecular formula: C₁₀H₁₀N₄O₂, Molecular weight: 218.21Yield: 49.85 %, M.P.: 187-189^oC, R_f value: 0.58, FT-IR (KBr, υ, cm-1): 3512.85 (O-H Str.), 3456.57 (N-H Str.), 3189.34 (=C-H str.), 2967.34(C-H str.), 1719.47 (C=O str.), 1600.17 (C=C str.), 1243.56 (C-N str.), 777.38 (Ar C-H Bend.). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 5.0 (s, 1H, OH) 6.29-6.57 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 219.56 (M⁺¹). Anal. Calcd.:C, 55.04; H, 4.62; N, 25.68; O, 14.66. Found:C, 55.67; H, 4.87; N, 25.48; O, 14.87.

 

2.16 4-(2-(4-hydroxyphenyl)hydrazono)-3-methyl-1H-pyrazol-5(4H)-one (4l)

Molecular formula: C₁₀H₁₀N₄O₂, Molecular weight: 218.21Yield: 52.03 %, M.P.: 196-198^oC, R_f value: 0.63, FT-IR (KBr, υ, cm-1): 3543.76 (O-H Str.), 3433.85 (N-H Str.), 3145.75 (=C-H str.), 2976.78(C-H str.), 1711.84 (C=O str.), 1610.67 (C=C str.), 1249.83 (C-N str.), 766.39 (Ar C-H Bend.). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.9 (s, 3H, CH₃), 5.0 (s, 1H, OH) 6.32-6.72 (m, 4H, Ar-H), 7.0 (s, 2H, NH). MS [m/z]: 219.89 (M⁺¹). Anal. Calcd.:C, 55.04; H, 4.62; N, 25.68; O, 14.66. Found:C, 56.23; H, 4.60; N, 25.87; O, 14.77.

 

2.17 Biological evaluation:

2.17.1 Antimicrobial activity:

All test compounds and standard drugs were dissolved in dimethyl formamide for screening the anti-microbial activity. All the synthesized compounds were screened for anti-bacterial and anti-fungal activities by the paper disk diffusion technique. The anti-bacterial activity of the compounds was evaluated against S. aureus, E. coli, S. typhi, P. aureus and B. subtilis using the nutrient agar medium (Hi-Media Laboratories, India). The anti-fungal activities of the synthesized compounds were evaluated against C. albicans, A. niger and A. fumigates using the Sabouraud dextrose agar medium (Hi-Media Laboratories, India). The minimum inhibitory concentrations (MIC) of the compounds were also determined by the agar streak dilution method.

 

2.17.2 Paper Disk Diffusion Technique:

The sterilized (autoclaved at120°C for 30 min) medium (40 – 50°C) was inoculated (1 ml/100 ml of medium) with the suspension (10⁵cfu/ml) of the microorganism and poured into a petridish to give a depth of 3 – 4 mm. The paper impregnated with the test compounds dissolved in dimethyl formamide (100 µg/disk) and was placed on the solidified medium. The plates were pre-incubated for one hour at room temperature and incubated at 37°C for 24 and 48 hours for anti-bacterial and anti-fungal activities, respectively. Streptomycin sulphate (100µg/disk) and Greseofulvin (100 µg/disk) were used as the standards for anti-bacterial and anti-fungal activities, respectively. The observed zone of inhibition is presented in Table 2.

 

Table 2: Antibacterial and antifungal activity of synthesized compounds

 

2.17.3 Minimum Inhibitory Concentration:

The Minimum inhibitory concentration (MIC) of the compound were determined by the agar streak dilution method. A stock solution of the synthesized compound (100 µg/ml), in dimethyl formamide, was prepared and graded quantities of the test compounds were incorporated in a specified quantity of molten sterile agar (nutrient agar for anti-bacterial activity and Sabouraud dextrose agar medium for anti-fungal activity). A specified quantity of the medium (40 – 50°C) containing the compound was poured into a petridish to give a depth of 3 – 4 mm, and allowed to solidify. A suspension of the microorganism were prepared, to contain approximately 10⁵ cfu/ml, and applied on the plates with serially diluted compounds in dimethyl formamide, to be tested, and was incubated at 37°C for 24 and 48 hours for bacteria and fungi, respectively. The MIC was considered to be the lowest concentration of the test substance, exhibiting no visible growth of bacteria or fungi on the plate. The observed MIC is presented in Table 2.

 

2.17.4 Evaluation of Analgesic Activity^[32-35]:

Animals:

Adult male albino mice (20-25 g) were used for studying the analgesic activity. The animals (five per cage) were maintained under standard laboratory conditions (light period of 12 hrs/day, temperature 27±2°C with relative humidity of 45-55%). They were fed with standard animal feed and water ad libitum. The experimental procedures were carried out in strict compliance with the Institutional Animal Ethics Committee. All experiments were performed in the morning according to the guidelines for the care of laboratory animals.

 

2.17.5 The hot-plate method:

Analgesic activity of the tested compounds was determined by the hot-plate method. A total number of 70 mice were divided into 14 groups of five animals each. The first group was administered DMSO orally (0.2 ml/mice) and kept as negative control. Diclofenac was given as standard drug (50 mg/kg) to the second group and the tested compounds 4a to 4l dissolved in DMSO were administered at a dose of 100 mg/kg body weight to the rest of the groups. Each animal was placed individually on a hot plate and maintained at 55°C. The time taken by the animals to lick the hind paw or jump out of the plate was taken as the reaction time, which was measured at 30 min., 1 hrs, 2 hrs and 3 hrs. A cut off period of 30 s was considered as maximal latency to avoid paw injury. The pain inhibition percentage (PIP) was calculated according to the following formula:

 

Pain Inhibition Percentage (PIP) = (T_t– T_c/T_c) × 100

Where T_c and T_t are the latency for the control and drug-treated animal groups.

 

2.17.6 The acetic acid-induced writhing test:

This test was conducted using the method described by Collier et al^[36]. Muscle contractions were induced in 14 groups of mice (five animals per group) by intraperitoneal injection of 0.6% solution of acetic acid (10 ml/kg). Thirty minutes before this administration, the animals in the first group were treated orally with DMSO (0.2 ml/mice) and they served as negative controls. Diclofenac as the reference standard (50 mg/kg) and the tested compounds 4a to 4l dissolved in DMSO were administered orally (100 mg/kg) to the animals of the rest of the groups. Immediately after administration of acetic acid the animals were placed in glass cages and the number of ‘stretching’ per animal was recorded during the course of the next 15 min. Writhing movement was accepted as contraction of the abdominal muscles accompanied by stretching of hind limbs. There was significant reduction in the number of writhes in the drug-treated animals as compared with vehicle-treated animals. This was considered a positive analgesic response and the percentage inhibition of writhing was calculated according to the following formula:

% Analgesic activity = [No. of writhings for control - No. of writhings for test compounds / No. of writhings for control] × 100

 

Table 3: Analgesic activity of the tested compounds in mice using hot-plate method.

Values are expressed as mean ± SEM of five animals in each group. **Statistically

significant (P<0.05). ***Statistically significant (P<0.01)

 

Table 4: % Inhibition of Analgesic activity of the tested compounds in mice using Hot-plate method.

Values are expressed as mean ± SEM of five animals in each group. **Statistically significant

  (P<0.05).***Statistically significant (P<0.01)

 

 

Table 5: Acetic acid induced writhing response of the tested compounds and % analgesic activity.

Values are expressed as mean ± SEM of five animals in each group. **Statistically significant

  (P<0.05).***Statistically significant (P<0.01)

 

Fig. 1: Zone of inhibition of Pyrazolone derivatives and Standard Drugs.

 

Fig. 2: Effect of various treatments on mean latency time by using hot plate method.

 

Fig. 3: % inhibition of mean latency time by using hot plate method.

 

 

Fig. 4: Acetic acid induced writhing test of compounds.

 

3. RESULTS  AND DISCUSSION:

The main focus of this research work was to synthesize novel series of pyrazolinone derivatives, purify, characterize and evaluate their anti-microbial and analgesic activity. The synthesized compounds were characterized by spectral data (¹HNMR, IR, Mass) and elemental analysis.

 

3.1 Antimicrobial activity:

The compounds were subjected to in –vitro antibacterial activity assays against S. aureus, E.coli, S. typhi, P. aureus, B. subtilis and antifungal activity assays against C. albicans, A. niger and A. fumigate. The results showed that the synthesized compounds possessed weak to good antibacterial and antifungal activities against the tested bacteria and fungi, with compounds 4d, 4h and 4j displaying good activity. Further studies are currently under way to establish a definite structure activity relationship.

 

3.2 Analgesic Activity:

The analgesic activity was assessed by using hot plate and acetic acid induced writhing methods using Diclofenac as the standard drug. The analgesic activity data by hot plate method was obtained as mean latency time at 30 min., 1 hrs, 2 hrs and 3 hrs intervals and expressed in % inhibition as shown in Table 3 and 4. Compounds 4b and 4f showed excellent analgesic activity as 83.88% and 81.90% inhibition respectively at 3^rd hrs, which were nearby 99.34 % inhibition of the standard Diclofenac drug used and also greater than the other Pyrazolone derivatives. Compounds 4b and 4f showed significant% of analgesic activity as 88.49% and 87.23% in acetic acid induced writhing method as shown in Table 5.

 

4. CONCLUSIONS:

The main focus of this research work was to synthesize novel series of pyrazolinone derivatives, purify, characterize and evaluate their anti-microbial and analgesic activity. The synthesized compounds were characterized by spectral data (¹HNMR, IR, Mass) and elemental analysis. The compounds were subjected to in-vitro antibacterial activity assays against S. aureus, E.coli, S. typhi, P. aureus, B. subtilis and antifungal activity assays against C. albicans, A. niger and A. fumigate. The results showed that the synthesized compounds possessed weak to good antibacterial and antifungal activities against the tested bacteria and fungi, with compounds 4d, 4h and 4j displaying good activity. On other hand, the synthesized compounds were subjected to analgesic activity by using hot plate and acetic acid induced writhing test methods. The results showed that among the derivatives, compound 4b and 4f displayed the significance analgesic activity as compared to other Pyrazolinone derivatives. Further studies are currently under way to establish a definite structure activity relationship.

 

5. ACKNOWLEDGEMENT:

The authors are thankful to Department of Pharmaceutical Chemistry, Oriental College of Pharmacy and Research, Oriental University, Indore for providing chemicals and facilities to conduct research work.

 

6. CONFLICT OF INTEREST:

The authors declared no conflict of interest.

 

7. REFERENCES:

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Received on 22.09.2020            Modified on 05.11.2020

Accepted on 10.12.2020           © RJPT All right reserved

Research J. Pharm.and Tech 2021; 14(12):6645-6652.