Synthesis and Biological Evaluation of Some Novel Substituted Thiazolidine and Pyrazolone Derivatives

 

Jignesh B. Patel* and Dr. Dhrubo Jyoti Sen

Department of Pharmaceutical Chemistry, Shri Sarvajanik Pharmacy College, Gujarat Technological University, Arvind Baug, Mehsana-384001, Gujarat, India

ABSTRACT:

In the present investigation, our aim of synthesis is to find a molecule which resists the anti inflammatory and microbial infection. So, thiazolidine and its derivatives and pyrazolone and their condensed derivatives was prepared and tested the Anti inflammatory activities of synthesized compound by rat paw edema method using phenylbutazone as a standard and in-vitro antibacterial activity of synthesized compound against Gram positive and Gram negative microorganisms (Staphylococcus aureus MTCC 96 and Escherichia coli MTCC 521) tested by filter paper disk method using Ofloxacin as a standard drug. All the compounds were characterized by IR, Mass and NMR. All Compounds possess a molecular ion M+ and M+2 peaks due to presence of chlorine. In the 1H NMR spectra of  7a compound, signal for OCH₃ proton was observed with value 3.73 and CH₃ proton was observed with value 1.71. In anti inflammatory activities Compound 7b found to have better anti-inflammatory activity while Compounds 7a, 7e and 7f showed this activity but less potent than compound 7b and phenylbutazone and Compound 7c and 7d were found to be least potent among the series. In antibacterial screening all compounds found to have more activity against gram negative bacteria E.coli. Compound 7b, 7c and 7d found to have better antibacterial activity against gram negative bacteria E.coli and compounds 7a, 7e and 7f shown activity against E.coli, but less potent than compound 7b,7c, 7d and Ofloxacin.

 

 

 

INTRODUCTION:

It is evident from reported articles that bacterial infections often produce pain and inflammation. In normal practice, two groups of agents (chemotherapeutics and NSAIDs) are prescribed simultaneously. Unfortunately, none of the drugs possesses these activities in a single component. Therefore, our aim is to find a compound having dual effect both analgesic-anti-inflammatory and antimicrobial activities. (twin drug concept). In following our aim we find from the literature survey Thiazolidine ring and its derivatives was found to be having diverse activity like anti-inflammatory, antimicrobial, anti HIV and anti cancer etc.  From the literature survey Pyrazolone their condensed derivatives constitute compounds with anti-inflammatory activities. So we have planned to attach Thiazolidinone ring with Pyrazolone their condensed derivatives to screen for various biological activities like anti-inflammatory, antimicrobial, etc.

 

EXPERIMENTAL:

MATERIALS AND METHOD:

The entire chemicals were supplied by S. D. Fine Chem. (Mumbai), Finar Chem. Ltd (Ahmedabad) and Loba Chemie. Pvt. Ltd. (Mumbai). Melting points were determined by open tube capillary method and were uncorrected. Purity of compounds was checked by thin layer chromatography (TLC) on silica gel-G in solvent system chloroform-methanol (9:1) and the spots were located under iodine vapours and UV light. IR spectra of all compounds were recorded on FT-IR 8400S Shimadzu spectrophotometer using KBr. Mass spectra were obtained using 2010EV LCMS Shimadzu instrument. The ¹H-NMR was recorded on Bruker advanced –II NMR-500MHz instruments using CDCL₃ as solvent and TMS (Tetra methyl silane) as internal standard, chemical shifts were expressed as δ values (ppm).

 

Preparation of 2-(4-chlorophenyl)-3-(4-methoxyphenyl) thiazolidin-4-one^1-3

p-anisidine (0.1mol) was dissolved in toluene in 250 ml Round bottom flask and then p-chloro benzaldehyde (0.1mol) and thioglycollic acid (0.1mol) was added. Then reaction mixture was refluxed for 48-50 hr. After reflux the reaction mixture was distilled to remove excess solvent. Then after reaction mixture was treated with dil. HCl to remove unreacted p-anisidine. Solid was separated out and it was washed with cold water. Solid was recrystallized with ethanol. The yield was 79.65%; R_f: 0.90 (Chloroform  9.5 : methanol  0.5); MP=180-183^oC ; IR (υ, cm^-1) 1670.24(>N-C=O),  1126.35(C-O) , 1029.92 (C-Cl), 840.91,810.05 (p-sub) ,  1353.94 (CH₃); MASS:  319.9[M+], 321.0[M+2].    

 

 

             (4)

 

 

 

 

Synthetic Pathway:

 

 

General procedure for the synthesis of 1-(2-(4-chlorophenyl)-3-(4-methoxyphenyl) thiazolidin-4-ylidene)-2-substituted hydrazine

Substituted hydrazine salt (0.01mol) with added glacial acetic acid (act as a catalyst) was prepared.2-(4-chlorophenyl)-3-(4-methoxyphenyl) thiazolidin-4-one (0.01mol) was dissolved in ethanol to prepare solution.Ethanol solution was added in to  substituted hydrazine salt. The reaction mixture was heated on water bath for 6-7 hours. Then cooled to obtain solid product, it was filtered and then wash with water or rectified spirit and recrystallized with methanol.

 

(5a-5c)

 

 

General procedure for the synthesis of 1-(2-(4-chlorophenyl)-3-(4-methoxyphenyl) thiazolidin-4-yl)-2 substituted hydrazine

1-(2-(4-chlorophenyl)-3-(4-methoxyphenyl)thiazolidin-4-ylidene)-2-phenylhydrazine (0.01mol) was dissolved in  ethanol  to prepare solution. Zinc amalgam (0.01mol) in presence of concentrated hydrochloric acid was added. The reaction mixture was heated under reflux for 13-14 hours. Then cooled and basify it with solution of sodium hydroxide. The separated solid product was filtered and recrystallized with methanol.

 

 

(6a-6c)

 

General procedure for the synthesis of 1-(2-(4-chlorophenyl)-3-(4-methoxyphenyl) thiazolidin-4-yl)-5- substituted l,2-substituted-1,2-dihydropyrazol-3-one^4-8

1-(2-(4-chlorophenyl)-3-(4-methoxyphenyl) thiazolidin-4-yl)-2- substituted hydrazine (0.01mol) was dissolved in ethanol to prepare solution. Ethylacetoacetate (0.01mol) or Ethylcynoacetate (0.01mol) was added. The reaction mixture was refluxed on a water-bath for 10 hrs in presence of catalytic amount of glacial acetic acid (2-3 drops). Distilled ethanol from reaction mixture and wash reaction mixture with ether to remove excess Ethylacetoacetate or Ethylcynoacetate. The obtained product was dried and purified by recrystallized with ethanol.

(7a-7f)

Table: 1 Physical Characteristics of synthesized compounds:

Compound Code	R	R1	Molecular Formula	Molecular Weight (g/mol)	Melting Point (oC)	% Yield (%w/w )	Rf
7a	C6H5	CH3	C26H24Cl1N3O2S	477.41	193-195	27.55	0.86
7b	C6H5	NH2	C25H23Cl1N4O2S	478.99	200-202	39.46	0.70
7c	2,4 NO2C6H3	CH3	C26H22Cl1N5O6S	567.01	217-219	24.65	0.82
7d	2,4 NO2C6H3	NH2	C25H21Cl1N6O6S	568.20	244-246	29.54	0.60
7e	H	CH3	C20H20Cl1N3O2S	401.30	201-203	43.54	0.76
7f	H	NH2	C19H19Cl1N4O2S	402.09	223-225	52.22	0.63

 

Table: 2 Spectral data of synthesized compounds :

Compd Code	IR (cm-1 )	Mass (m/z)	NMR (δ, ppm)
7a	1573.81   (C=C), 1385.00   ( CH3) , 1657.10   (>NC=O)	478.2 [M+], 480.1 [M+2]	1.71 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 2.7 (d, 2H, CH2), 4.04 (t, 1H, NCHCH2), 4.95 (s, 1H,  CH),  5.42 (s, 1H, CH), 6.4-7.2 (m, 13H, ArH)
7b	1639.22    (C=C) 3417.63    (NH2) 1675.52    (>NC=O) 678.60      (O-Sub.)	479.4 [M+], 481.2 [M+2]	…….
7c	1661.10    (>NC=O)     690.47      (O-Sub.)  1531.11    (-N=0)  1373.73    (CH3)	567.1 [M+], 569.1 [M+2]	…….
7d	1677.52     (>NC=O)     1554.11     (-N=0) 3352.32     (NH2) 1647.22     (-C=C-)	568.0 [M+], 570.3 [M+2]	…….
7e	1675.41     (>NC=O)  1641.22     (-C=C)  1375.41     (CH3) 3101.32     (-NH-)	402.1 [M+], 403.9 [M+2]	…….
7f	1679.24     (>NC=O) 1639.22     (-C=C)  3388.65     (-NH2)	402.9 [M+], 404.3 [M+2]	…….

 

Biological Evaluation

1. Anti-Inflammatory Screening Method^9-11

Anti inflammatory activities were analyzed by rat paw edema method. Phenylbutazone was used as standard reference drug for Anti-inflammatory screening.

 

Anti-inflammatory activity:

The anti-inflammatory activity of newly synthesized substituted Thiazolidine and Pyrazolone derivatives was carried out using Carrageenan induced rat hind paw edema method.

·         Method: Inhibition of carrageenan induced inflammation in rat paw

·         Animals used: Swiss Albino rat

·         No. of animals used: 3 (in each group)

·         Dose of compound: 100mg/kg

·         Dose of std. drug: 100mg/kg (Phenylbutazone)

·         Route of administration: Oral (suspended in 1% tween-80 solution)

Rat was assigned into 6 groups of 3 animals each. They were marked with picric acid for individual animal identification. The animals were starved overnight with water ad libitum prior to the day of experiment. First 0.1ml of 1%w/v of carrageenan in normal saline was injected in to the sub planter region of the left hind paw of rat. Synthetic compounds and std. Compounds were administered after 1hr of the injection of carrageenan. Dose volume not exceeding 0.5ml/100gm orally administered. Group I: The solvent control received vehicle orally.

Group II: Positive control received phenylbutazone (100mg/kg). Group III: Received test compound 7a at a dose of 100mg/kg suspended in 1%w/v tween-80. Group IV: Received test compound -7b at a dose of 100mg/kg suspended in 1%w/v tween-80. Group V: Received test compound -7c at a dose of 100mg/kg suspended in 1%w/v tween-80. Group VI: Received test compound-7d at a dose of 100mg/kg suspended in 1%w/v tween-80. Group VII: Received test compound-7e at a dose of 100mg/kg suspended in 1%w/v tween-80. Group VIII: Received test compound-7f at a dose of 100mg/kg suspended in 1%w/v tween-80. Immediately after administered the test compounds and Std.compounds, the volume of its displacement was measured using plethysmometer. The reading was recorded at 0, ½, 1, 2, 3 hrs. The percentage inhibition calculates by the following equation.

 

% Inhibition =

 

Table 3: Anti-inflammatory activity:

Compound Code	Inhibition of Inflammation(mm)±SEM				% Inhibition
	0 hr	1hr	2hr	3hr	1hr	2hr	3hr
Control	0.75±0.03	0.74±0.03	0.74±0.04	0.74±0.03	-	-	-
7a	0.75±0.03	0.59±0.02	0.49±0.01	0.41±0.03	20.0	33.7	44.5
7b	0.74±0.03	0.55±0.03	0.43±0.03	0.33±0.03	26.0	42.6	56.0
7c	0.75±0.02	0.69±0.02	0.62±0.03	0.55±0.04	06.7	16.2	25.6
7d	0.74±0.03	0.66±0.02	0.55±0.03	0.51±0.03	12.0	26.6	32.0
7e	0.73±0.03	0.63±0.02	0.53±0.02	0.46±0.03	14.0	28.3	37.8
7f	0.75±0.02	0.6±0.03	0.51±0.03	0.42±0.03	17.0	30.1	42.4

 

 

 

Figure 1: Histogram of Anti-inflammatory activity

 

2. Antimicrobial activity^12-15

In our current study, evaluation of antimicrobial activity was carried out by using filter disk method and the response of microorganisms to the synthesized compounds has been measured with that of the standard drug Ofloxacin and microorganisms were selected for the study was Escherichia coli (Gram –ve), Bacillus subtilis (+ve) Staphylococcus aureus (+ve).

 

Antibacterial activity: All the petri dishes were sterilized in oven at 160°C for 1 hour, Agar media, absorbent paper and test solutions were sterilized in autoclave at 121°C at 15psi then molten sterile agar was poured in sterile petridishes aseptically. The agar was allowed to cool and the bacterial suspension was poured into the petridishes aseptically. Placing the absorbent paper was absorbed with solutions of the compound in the petridishes aseptically. Incubated the petridishes at 37°C for antimicrobial for 24 hrs and observed the Zone of inhibition.

 

Table 4: Anti microbial Activity:

Compound Code	Concentration (μg/ml)	Zone of Inhibition (mm)
		Gram +ve		Gram –ve
		S. aureus	B. subtilis	E. coli
Control	100	-	-	-
	200	-	-	-
	300	-	-	-
Ofloxacin	100	14	15	24
	200	14	16	26
	300	15	18	29
7a	100	04	08	13
	200	05	10	15
	300	05	11	19
7b	100	04	00	19
	200	04	02	21
	300	05	02	24
7c	100	09	00	21
	200	11	00	22
	300	12	02	26
7d	100	08	02	22
	200	07	03	22
	300	08	03	24
7e	100	04	04	14
	200	07	05	15
	300	08	05	17
7f	100	05	04	13
	200	06	06	16
	300	09	06	15

 

Figure 2: Histogram of Antibacterial  Activity

 

 

RESULT AND DISCUSSION:

Table: 1 represents the physical characteristics of the synthesized compounds and Table: 2 for spectral datas of the same. The pharmacological screening of the synthesized compounds showed anti-inflammatory activity ranging from 6.7 to 66.6% inhibition of rat paw oedema volume after 3hr, where as the standard drug Phenylbutazone showed 66.6% inhibition of rat paw oedema volume after 3hr. The compound 7b was found to be nearly equipotent to Phenylbutazone which is used as standard drug. Compounds 7a, 7e, and 7f shown this activity but less potent than compound 7b and Phenylbutazone. Compound 7c and 7d was found to be least potent among the series. Table: 3 shows the anti-inflammatory screening report of the synthesized compounds and histogram of the same in Figure: 1.

 

The Antimicrobial activity was carried out against three microorganisms, S.aureus, B.subtilis and E.coli, and it was found that synthesized Compounds showed more activity against gram negative bacteria E.coli. Among that synthesized Compounds 7b, 7c and 7d have shown highest zone of inhibition against E.coli, which is nearly equipotent to Ofloxacin, used as a standard drug. Compounds 7a, 7e, and 7f shown activity against E.coli, but less potent than compound 7b, 7c, 7d and Ofloxacin. All synthesized compound were less active against S.aureus and B.subtilis. Antibacterial screening report has been mentioned in Table: 4 and histogram in Figure: 2.

 

ACKNOWLEDGEMENT:

The author Jignesh B. Patel is thankful to the project guide Prof. Dr. D. J. Sen and thankful to the all staff members of Shri Sarvajanik Pharmacy College, Mehsana, Gujarat to fulfill the project successfully. He is also thankful to the Quality Assurance Department of Shri Sarvajanik Pharmacy College, Mehsana for IR spectral data, Oxygen Healthcare, Ahmadabad for Mass spectral data and NIPER, Panjab University, Mohali for NMR spectral Datas.

 

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Received on 01.06.2012        Modified on 08.06.2012

Accepted on 20.06.2012        © AJRC All right reserved