Synthesis and In-Vitro Anti-Inflammatory Activity of New Pyrazoline Derivatives

 

K. Ishwar Bhat*, B. C. Revanasiddappa, M. Vijay Kumar, Banylla Felicity,

Ranee Kumari, Abhishek Kumar

Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences of NITTE

(Deemed to be University), Paneer, Deralakatte, Mangalore-575 018, Karnataka, India

 

ABSTRACT:

A new series of Chalcones (2a-i) were prepared by Claisen-Schmidth Condensation reaction involving substituted aromatic aldehydes and aromatic ketones in alcohol medium, in presence of 40% NaOH as base. The title compounds Pyrazolines (3a-i) were synthesized by reacting Chalcones (2a-i) and 2-thiophene carbohydrazide (1) in glacial acetic acid medium. The final synthesized compounds were screened for their In-vitro anti-inflammatory activity by egg albumin denaturation method and protein denaturation method. The structures of the newly synthesized compounds were assigned on the basis of IR, ¹H-NMR and Mass spectral data. The compounds 3b, 3c, 3d and 3f also showed good inhibition when compared to the standard diclofenac sodium

 

 

 

INTRODUCTION:

Pyrazolines and its derivatives belong to prominent class of nitrogen heterocycles. Formation of Pyrazolines has been reported by the action of nucleophiles like hydrazine hydrate/phenyl hydrazine/2,4-DNP on Chalcones. Many class of chemotherapeutic agents containing Pyrazoline nucleus are in clinical use such as antipyretic (Antipyrine, Oxyphenbutazone), analgesic (Novalgin), and Cox-II inhibitor (Celecoxib). Similarly the thiophene moiety is one of the important hetero atom and have been found to occur in fungi and higher plants. The thiophene ring is found in vitamin Biofin, Cephalosporins like Cephalothin, Cephaloridine, and antihistaminic Methapyrilene.

 

Chalcones are products of condensation of simple or substituted aromatic aldehydes with simple or substituted acetophenones in presence of alkali.

 

 

Chalcones constitute an important class of compounds in synthetic chemistry and some of them possess a wide range of biological activities such as antimicrobial¹, anticancer², anti-tubercular³, antiviral⁴ etc. The presence of reactive α, β-unsaturated keto group in Chalcones is found to be responsible for their biological activity. The chemical structure of Chalcones (1,3-diphenyl-2-propen-1-ones) consists of two aromatic rings joined by a three carbon α, β-unsaturated carbonyl system. The above prepared compounds are α, β-unsaturated ketones (Chalcones) containing the ketoethylenic group (–CO–CH=CH-).

 

Moreover, Chalcones are found to be useful as synthons in the synthesis of a large number of bioactive molecules such as Pyrazolines. Considerable interest has been focused on the Pyrazoline structure. Pyrazolines are the five membered heterocyclic compounds containing two nitrogen atoms. Pyrazolines are reported with wide variety of activities like anti-inflammatory^5-7, antimicrobial^8-10 etc.

 

Therefore, both the Chalcones and Pyrazolines possess worthy and imperative bioactivities which render them useful substances in drug research. In this study, based on the above findings, our aim was to synthesize a new series of Pyrazoline derivatives and to investigate their possible anti-inflammatory activity

 

MATERIALS AND METHODS:

The IR spectra are recorded by using Alpha Bruker IR Spectrometer using a thin film on KBr pellet technique and frequencies are expressed in cm^-1.The homogeneity of the compounds was checked by TLC using silica gel plates (Merck) using ethyl acetate: methanol as mobile phase. Melting points were determined by open capillary method and are uncorrected. The ¹H-NMR spectra were recorded on Bruker Avance II 400 MHz NMR Spectrometer. All the spectra were obtained in CDCl₃ and DMSO. Chemical shift values are reported as values in ppm relative to TMS (δ=0) as internal standard. Mass spectra were recorded on ESI.

 

 

 

Synthesis of Chalcones (2a-i):

A mixture of substituted aromatic aldehydes (0.01 mol) and substituted ketones (0.01 mol) in ethanol (20 ml) were stirred together for 24 hr, in presence of 40% NaOH (5 ml). The mixture was poured into crushed ice and acidified with HCl. The separated solid was filtered, washed with water and recrystalized from suitable solvents.

 

Synthesis of substituted Pyrazolines (3a-i):

Chalcones (0.01 mol) and 2-thiophene carbohydrazide (1) (0.01mol) were dissolved in glacial acetic acid (30ml) and the reaction mixture is refluxed for about 16-28 hrs. After the completion of the reaction, the reaction mixture is poured into 250 ml of ice cold water .The solid which is separated is filtered and washed with cold water. The separated compound is recrystalized by using alcohol. The physical data of Pyrazolines (3a-i) is given in table1.

 

Table-1: Physical data of Pyrazoline Derivatives (3a-i)

Comp.	Ar-CHO	Ar1-COCH3	Molecular weight	M.P (0C)	Yield (%)
3a	4-NO2	2-Cl	411	121-123	56
3b	3-NO2	4-OCH3	407	102-104	62
3c	4-NO2	4-F	395	110-112	64
3d	4-Br	4-OH	426	133-135	57
3e	4-Br	2-Cl	445	142-144	59
3f	4-OH	3,4-(OCH3)2	410	91-93	60
3g	2-OH	4-Br	426	97-99	61
3h	4-F	2-Cl	384	152-154	62
3i	4-Br	4-OCH3	440	160-162	62

 

(5-(4-fluorophenyl)-3-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(thiophen-2yl) methanone (3c):

IR (KBr)cm⁻¹: 2919 (CH), 1665 (C=O), 1591 (C=N), 1528 (C=C), 830 (C-F); ^1-H-NMR (CDCl₃, 400 MHz): δ 3.1-3.3 (dd, 1H, H_A) , 3.6-3.7(dd, 1H, H_B), 3.8-4.00 (dd, 1H, H_X) 7.008-8.583 (m, 11H, Ar), MS m/z: 395 (M+).

 

(5-(3,4-dimethoxyphenyl)-3-(3-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(thiophen-2-yl)methanone (3f):

IR (KBr)cm⁻¹: 3655 (OH), 3088 (CH), 1668 (C=O), 1510 (C=N), 11528 (C=C), ^1-H-NMR (CDCl₃, 400 MHz): δ 4.79 (OCH₃)₂, 6.89-6.91(dd, 1H, H_A), 6.93-6.95 (dd, 2H, H_B,, H_X7.18-8.88 (m, 11H, Ar), MS m/z: 410 (M+).

 

(3-(3-chlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl) (thiophen-2-yl) methanone (3h): IR (KBr)cm⁻¹: 2919 (CH), 1665 (C=O), 1591 (C=N), 1528 (C=C), 830 (C-F); ^1-H-NMR (CDCl₃, 400 MHz): δ 3.1-3.3 (dd, 1H, H_A) , 3.6-3.7(dd, 1H, H_B), 3.8-4.00 (dd, 1H, H_X) 7.008-8.583 (m, 11H, Ar), MS m/z: 395 (M+).

 

 

 

 

In-Vitro anti-inflammatory activity

Protein Denaturation by Bovine serum albumin method¹¹

Test solution (0.5ml) consists of 0.45ml of bovine serum albumin (5%w/v aqueous solution) and 0.05ml of test solution. Control solution (0.5ml) consists of 0.45ml of bovine serum albumin (5%w/v aqueous solution) and 0.05ml of distilled water. Product control (0.5ml) consists of 0.45ml of distilled water and 0.05ml of test solution. Standard solution (0.5ml) consists of 0.45ml of bovine serum albumin (5%w/v aqueous solution) and 0.05ml 0f diclofenac sodium. All of the above solutions were adjusted to pH 6.3 using a small amount of 1N HCl. The samples (10, 20, 30, 40, 50 µg/mL) were incubated at 37°C for 20 minutes and heated at 57°C for 3 minutes. After cooling, add 2.5ml of phosphate buffer to the above solutions. The absorbance of the solutions was measured using UV-Visible spectrophotometer at 416nm. The % percentage inhibition of protein denaturation by bovine serum albumin is given in table-2. Percentage inhibition of protein denaturation was calculated using the formula.

 

% Inhibition=100 × [V t/V C-1]

Where, V t=absorbance of the test sample, V c=absorbance of control

Table-2. Anti-inflammatory data of Pyrazolines (3a-f) by Bovine Serum Albumin Denaturation Method

Conc. ((𝜇g/mL)	Std.*	3a	3b	3c	3d	3e	3f
10	26	20	21	22	20	21	21.5
20	37	32	31	33	32	31	32
30	48	42	41	42	41	42	43.5
40	59	54	56	53	52	51.5	50
50	74	68	69	71.4	70.8	67	68

*Std. = Diclofenac sodium

 

Protein Denaturation by egg albumin method¹²:

The reaction mixture (5 mL) consisted of 0.2 mL of egg albumin (from fresh hen's egg), 2.8 mL of phosphate-buffered saline (PBS, pH 6.4) and 2 mL of varying concentrations (10, 20, 30, 40, 50 µg/mL) of test compounds. A similar volume of double-distilled water served as the control. Next, the mixtures were incubated at 37±2°C in a BOD incubator for 15 minutes and then heated at 70°C for five minutes. After cooling, their absorbance was measured at 660 nm by using the vehicle as a blank. Diclofenac sodium in the concentrations of 10, 20, 30, 40, 50 µg/mL was used as the reference drug and treated similarly for the determination of absorbance. The % percentage inhibition of protein denaturation by egg albumin is given in table-3. Percentage inhibition of protein denaturation was calculated by using the following formula

 

% Inhibition=100 × [V t/V C-1]

Where, V t=absorbance of the test sample, V c=absorbance of control

 

Table-3.Anti-inflammatory data of Pyrazolines (3a-f) by Egg Albumin Denaturation Method

Concentration ((𝜇g/mL)	Standard (Diclofenac sodium)	3a	3b	3c	3d	3e	3f
10	28	22	23	23	23.5	21	21
20	39	32	33	31	31	32	32
30	48	43	42	41	40	40.8	41
40	62	57	55	56	56	54	53
50	73	68	65	69	67	66	65

 

RESULTS AND DISCUSSION:

Chemistry:

The synthesized route to the target compounds is outlined in Scheme-01. A new series of Chalcones were synthesized as per the well known Claisen–Schmidt condensation reaction. The Chalcones undergoes selective cyclization with 2-thiophene carbohydrazide in glacial acetic acid medium to yield the title compounds Pyrazoline derivatives. The purity of the compounds was established by recrystallization technique. All the new compounds were established on the basis of spectral data, TLC and melting point.

 

 

 

The IR spectrum of compound 3c showed a characteristic absorption band at 2919 cm^-1 which was attributed to the CH group and a sharp absorption band appearing at 1665 cm^-1 was due to C=O group. ¹H-NMR spectra of compound 3c displayed characteristic signals for the aromatic protons in between 7.008-8.583 ppm. The signals belonging to protons in the Pyrazoline nucleus is represented in the form of H_A, H_B, H_X pattern. The protons were observed as doublets of doublets integrating for three protons in the region 3.1-3.3, 3.6-3.7, 3.9-4.00 respectively. Further evidence for the formation of Pyrazoline was obtained by recording its mass spectrum. Mass spectra of the compound 3c showed a [M⁺] peak, in line with their molecular formula.

 

Denaturation of proteins is a well documented cause of inflammation. As part of the investigation on the mechanism of the ant-inflammation activity, ability of the Pyrazolines to inhibit protein denaturation was studied. Some of the newly synthesized compounds were evaluated for In-Vitro anti-inflammatory activity.

 

In the bovine serum albumin denaturation method, all the tested compounds showed inhibition in the increasing order of concentration. None of the synthesized compounds was found to be effective when compared to the standard diclofenac sodium. Compounds 3c and 3d showed inhibition of 56%, 71.4% and 70.8 at 50 𝜇g/mL respectively. Compounds 3b and 3f also showed good inhibition close to the standard. The rest of the tested compounds showed moderate activity.

In the egg albumin denaturation method, compound 3d showed 23.5% inhibition at 10 𝜇g/mL, compound 3c showed 69% inhibition at 50 𝜇g/mL. All the tested compounds showed moderate activity when compared to the standard diclofenac sodium.

 

CONCLUSION:

In the present investigation a novel series of Pyrazoline derivatives were synthesized and evaluated for their In-Vitro anti-inflammatory activity. Some of the synthesized compounds showed good anti-inflammatory activity when compared to the standard diclofenac sodium.

 

ACKNOWLEDGEMENTS:

The author Dr. K. Ishwar Bhat thanks to Nitte University, Mangalore for providing the student research project and all the necessary facilities to carry out this research work.. The authors are thankful to SAIF, Vellore Institute of Technology, Vellore for providing the spectral data.

 

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Accepted on 12.07.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 3969-3972.