Synthesis and in-Vitro Anti-Inflammatory Activity of Novel Pyrazoline Derivatives

 

Jennifer Fernandes*, B. C. Revanasiddappa, K. Ishwarbhat, M. Vijay Kumar, Lidwin D’Souza,

Shanal Smitha Alva

Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical, Sciences of Nitte University, Paneer, Deralakatte, Mangalore-575 018, Karnataka, India

 

ABSTRACT:

A new series of Chalcones (2a-j) were prepared by reacting 2-acetyl Pyrrole and substituted ketones in alcohol medium in presence of NaOH. The Chalcones undergoes selective cyclization with phenoxy acetic acid hydrazide (1) in glacial acetic acid medium to yield the title compounds 1,3,5-trisubstituted Pyrazolines (3a-j). The newly synthesized compounds were evaluated for their In-vitro anti-inflammatory activity by egg albumin denaturation and protein denaturation method. All the new compounds were established on the basis of ¹H-NMR, IR and Mass spectral data. Some of the tested compounds 3a, 3f showed good anti-inflammatory activity when compared to the standard drug diclofenac sodium.

 

 

 

 

INTRODUCTION:

Chalcones are the simple condensation products of substituted aromatic aldehydes with substituted acetophenones in presence of alkali in alcohol medium. Chalcones constitute an important class of compounds, specially in the synthetic chemistry. They are reported with a wide range of activities such as antimicrobial¹, anticancer², antitubercular³, antiviral⁴ etc. The presence of reactive α, β- unsaturated keto group in Chalcones is found to be responsible for the various biological activites. 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-).Basically Chalcones are used as one of the key intermediates in the synthesis of  large number of five/six/seven membered heterocyclic compounds.. Nitrogen heterocycles are found be as one of the important class of compounds in heterocyclic chemistry. Pyrazolines are the five membered heterocyclic compounds containing two nitrogen atoms. Pyrazolines are reported to possess a wide variety of activities such as such as antinflammatory^5-7, antimicrobial^8-10 etc. The reaction sequence for the title compounds is given in Scheme-01. Therefore, both the Chalcones and Pyrazolines possess wide rage of biological and pharmacological activities which render them useful substances in drug research. In the present 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:

Melting points were determined by open capillary method and are uncorrected. The homogeneity of the compounds was checked by TLC using silica gel plates (Merck) using ethyl acetate: methanol as mobile phase 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. Mass spectra were recorded on ESI. 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.

 

Synthesis of Chalcones (2a-j):

A mixture of substituted aromatic aldehydes (0.01 mol) and 2-acetyl pyrolle (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-j):

Chalcones (2a-j) [0.01 mol] and phenoxy acetic acid hydrazide (1) [0.01mol] were dissolved in glacial acetic acid [25ml], and the reaction mixture is refluxed for about 22-34 hrs. After the completion of the reaction, the reaction mixture is poured into ice cold water. The solid which is separated, is filtered and washed with cold water and recrystalized by using alcohol. The physical data of Pyrazolines (3a-j) is given in table-1.

 

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

Comp.	Ar-CHO	Molecular weight	M.P (°C)	Yield (%)
3a	C6H5	345	112-114	62
3b	4-Cl	379	125-127	63
3c	4-OCH3	375	134-136	66
3d	2,5-(OCH3)2	405	102-104	60
3e	4-OH	361	92-94	61
3f	4-NO2	390	165-167	62
3g	Furfural	335	144-46	61
3h	4F	363	139-141	62
3i	4-Br	423	120-122	63
3j	Vanillin	391	177-79	65

3b:IR (KBr)cm⁻¹: 3605(NH), 3239 (CH), 1649 (C=O), 1582 (C=N), 1470 (C=C), 783 (C-F); ¹H-NMR (CDCl₃, 400 MHz): δ  6.36(dd, 1H, H_A) , 7.09-7.15(dd, 2H, H_B, H_X), 7.25(s, 2H, OCH₂),  7.25-8.24 (m, 11H, Ar),  10.10 (s, 1H, NH)  MS: 345 (M+).

 

3c:IR (KBr)cm⁻¹: 3712(NH), 3257 (CH), 1645 (C=O), 1569 (C=N), 1510 (C=C);  ¹H-NMR (CDCl₃, 400 MHz): δ  3.84 (s, 3H, OCH₃)  6.34 (dd, 1H, H_A) , 6.92-6.94 (dd, 2H, H_B, H_X),   7.06 (s, 2H, OCH₂),  7.11-7.82 (m, 11H, Ar),  10.10 (s, 1H, NH)  MS: 375(M+).

 

3d:IR (KBr)cm⁻¹: 3650(NH), 3241 (CH), 1643 (C=O), 1588 (C=N), 1516 (C=C);  ¹H-NMR (CDCl₃, 400 MHz): δ  3.93 (s, 6H, 2XOCH₃)  6.34-6.35 (dd, 1H, H_A) , 6.89-6.91 (dd, 2H, H_B, H_X),   7.09 (s, 2H, OCH₂),  7.15-7.81 (m, 11H, Ar),  9.90 (s, 1H, NH)  MS:405 (M+).

 

3g: ¹H-NMR (CDCl₃, 400 MHz): δ 6.27 (dd, 1H, H_A), 6.43 (dd, 1H, H_B),  6.60 (dd, 1H,  H_X),   7.00 (s, 2H, OCH₂),  7.08-7.52 (m, 11H, Ar),  9.50 (s, 1H, NH)  MS: 335(M+).

 

In-Vitro anti-inflammatory activity:

Protein Denaturation by Bovine serum albumin method [11]:

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) Bovine Serum Albumin Denaturation Method

Concentration (𝜇g/mL)	Standard (Diclofenac sodium)	3a	3b	3c	3d	3e	3f
10	27	22	21.22	23	21	23.5	22.5
20	36	33	30.24	31.5	30.5	29	29.5
30	47	44	38.5	42.5	42.5	42	43.5
40	59	55	50.5	52.5	56.5	51.5	51.5
50	70	66	61.5	62	63.5	60.5	67

 

 

 

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

Concentration(𝜇g/mL)	Standard (Diclofenac sodium)	3a	3b	3c	3d	3e	3f
10	30	24	22	23	22.5	21.5	22
20	37	33	30	29	28	31.5	32
30	48	41	42	43	43.5	44	44.5
40	58	52	51	52	51	51.5	52
50	68	61	60	63	62.5	63	63.5

 

 

Protein Denaturation by egg albumin method [12]:

The reaction mixture (5mL) consisted of 0.2mL of egg albumin (from fresh hen's egg), 2.8mL of phosphate-buffered saline (PBS, pH 6.4) and 2mL of varying concentrations (10, 20, 30, 40, 50µg/mL) of test compounds. A similar volume of double-distilled water served as the control. The mixtures were incubated at 37°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, 50µg/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

RESULTS AND DISCUSSIONS:

Denaturation of proteins is a well documented cause of inflammation. As part of the investigation on the mechanism of the anti 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 (table-2) all the tested compounds showed inhibition in the increasing order of concentration. None of the synthesized compounds was found to be effective when compared with the standard diclofenac sodium. Compounds 3a and 3f showed inhibition of 66% and 67% at 50𝜇g/mL respectively. The rest of the tested compounds showed moderate activity. In the egg albumin denaturation method (table-3), compound 3a showed 33% inhibition at 20𝜇g/mL, compound 3f showed 44.5% inhibition at 30𝜇g/mL. All the other tested compounds showed moderate activity when compared to the standard diclofenac sodium.

 

CONCLUSION:

In the present investigation a novel series of Pyrazoline derivatives were prepared and evaluated for their In-Vitro anti-inflammatory activity. Some of the synthesized compounds exhibited good anti-inflammatory activity. The study further requires in the structural modification for the development of novel potent anti-inflammatory agents.

 

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Accepted on 27.04.2017        © RJPT All right reserved