INTRODUCTION:

Pain can be categorized according to several variables, including its duration such as acute, convalescent and chronic. Secondly depending upon the pathophysiologic mechanisms such as physiologic, nociceptive and neuropathic and thirdly on its clinical context like postsurgical, malignancy related, neuropathic and degenerative. Nociceptive pain has also been termed inflammatory because peripheral inflammation and inflammatory mediators play major roles in its initiation and development. In general, the intensity of nociceptive pain is proportional to the magnitude of tissue damage and release of inflammatory mediators¹. Traditionally, the analgesic action of Nonsteroidal anti-inflammatory drugs (NSAIDs) has been explained on the basis of their inhibition of the enzymes that synthesize prostaglandins. The treatment of pain requires analgesics including inflammatory drugs. Hence most of Non-steroidal antiinflammatory agents also have analgesic activity. Although many analgesic and anti-inflammatory agents are present in the market but modern drug therapy is associated with adverse side effects like gastrointestinal irritation, broncho spasm etc therefore, it is necessary to shift the focus on new drugs with no or less side effects.

Inflammation can be define as a defensive but exaggerated local tissue reaction in response to exogenous or endogenous insult. It is complex phenomenon, comprising of biochemical as well as immunological factors. It is recognised by the following symptoms: Calor (Heat), Rubor (Redness), Tumours (Swellings), Dolor (Pain). Tissue damage initiates or activates the local release of various chemotatic factors that provoke directly or indirectly the appearance of the mediators of pain and inflammation².A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process³. NSAIDs which include both selective and nonselective cyclooxygenase (COX) inhibitors are most widely used for the treatments of pain. All of these drugs are reported for their side effects and toxic effects⁴. The activity of NSAIDs in rheumatoid arthritis and other rheumatic diseases does not seem to be only due to the inhibition of the production of endogenous prostaglandins⁵, it also prevents the denaturation of proteins. When albumin is heated it undergoes denaturation, it expresses antigens associated to Type III hyper-sensitive reaction and which are related to diseases such as serum sickness, rheumatoid arthritis and systemic lupus erythematosus⁶. Denatured proteins disrupt cellular redox homeostasis and increase reactive oxygen species levels⁷ and ROS induces signalling cascades that trigger the production of proinflammatory cytokines and chemokines. Many reports claim that all the NSAIDs possess anti-denaturation activity. Thus the present study was framed to evaluate reduced albumin anti-denaturation property to assess its anti-inflammatory activity. Phthalimide analogues have been extensively used in medicinal chemistry owing to their wide range of applications as anticonvulsant⁸, antiinflammatory⁹, analgesic¹⁰, hypoglycemic¹¹ and immunomodulatory activities.

Most clinically important medicine belongs to steroidal or non-steroidal anti-inflammatory chemical therapeutics for treatment of inflammation related diseases. Though these have potent activity and long term administration is required for treatments of chronic diseases. Furthermore, these drugs have various and severe adverse effects. Thus there is need for newer and safer analgesic and antiinflammatory agents. Discovery of thalidomide as TNF-α inhibitor has led to an impetus on development of phthalimide derivatives as potential analgesic antiinflammatory agents.

The aim of present work was to attach the substituted 1,3,4-thiadiazole residue to 1,3-isoindolinedione in order to find new pharmacologically active molecule. Thus the synthesis of novel 1, 3-isoindolinedione derivatives has been achieved and evaluated for their analgesic and invitro anti-inflammatory activity.

MATERIAL AND METHODS:

General procedure:

Melting points were measured on an Veego electronic apparatus and and IR spectra were recorded on a JASCO FTIR 4100 spectrophotometer by using KBr. The ¹H NMR spectra of synthesized compounds were recorded at department of chemistry, University of Pune, Pune, on Varian NMR 300 MHz spectrophotometer using TMS as an internal standard and chloroform as a solvent. All the reagents were purchased from Fluka, Merck and Rankem and used without further purification.

Steps in synthesis of compounds:

Step I General procedure for synthesis of N-hydroxymethyl phthalimide (1): The mixture of phthalimide (0.1mole) and 37 % w/w formaldehyde in water was taken in 250 ml of round bottom flask and refluxed for 4 hours. Reaction was monitored by TLC using mobile phase chloroform: methanol (5:1) and iodine vapors as visualsing agent. After completion of reaction the hot solution was allowed to cool to room temperature. N-hydroxymethylphthalimide precipitated as white crystals, product was filtered off and purified by recrystallization from hot benzene and dried.

Step II: General procedure for synthesis of 5-(substituted phenyl)-2-amino-1,3,4 thiadiazole: (6a -6l)

A mixture of thiosemicarbazide (0.1 mole), aryl carboxylic acid (0.1 mole) and concentrated sulphuric acid (10 drops) was reflux for 2-3 hours on water bath. Reaction was monitored by TLC using mobile phase chloroform: methanol (5:1). After completion of reaction the reaction mixture was poured onto crushed ice. The solid separated out was filtered, washed with water and recrystallized from ethanol and dried.

Step III: General procedure for synthesis of 2-(5-substituted phenyl)-1,3,4-thiadiazole-2-yl)amino)methyl)-1H-isoindole-1,3(2H)-dione. (7a-7l)

An equimolar mixture of N-hydroxymethylphthalimide and 5-(substituted phenyl)-2-amino-1,3,4-thiadiazole) (6a-6l) in methanol along with few drops of triethylamine was refluxed on water bath for 3-4 hours. Reaction was monitored by TLC using benzene: ethyl acetate (4:2) as mobile phase. After completion of reaction mixture was allow to cool at room temperature the solid material was separates out after solvent evaporation. The solids separated out were filtered and recrystallized from ethanol and dried.

The compounds 7a-l were obtained in satisfactory yields and the synthetic pathways are described in following scheme

Step I:

Step II:

Step III:

3 6a-1 7a-1

Scheme: The synthetic route of 2-(5-substituted phenyl)-1,3,4-thiadiazole-2-yl)amino)methyl)-1H-isoindole-1,3(2H)-dione derivatives. (7a – l)

Table 1. Physical properties of synthesized compounds

Sr. No	Compound Code	R	Rf Value	Yield (%)
1	7a	H	0.89	66.00
2	7b	C₆H₅-COCH₃	0.79	88.00
3	7c	4-NH₂	0.84	62.50
4	7d	4-NO₂	0.36	84.76
5	7e	4-Cl	0.45	60.00
6	7f	2-OH,5-Cl	0.32	96.89
7	7g	2-OH,3-CH₃	0.44	79.00
8	7h	3,5-(NO₂)₂	0.38	83.33
9	7i	3,4-(OCH₃)₂	0.95	85.00
10	7j	3-OH	0.34	74.60
11	7k	2,4-(OH)₂	0.30	77.97
12	7l	2-Cl	0.92	68.42

Biological screening:

Analgesic activity:

The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC) and conducted according to the guidelines for the use and care of experimental animals. Analgesic activity was determined by acetic acid induced writhing in mice model of Koster et al.

Albino mice of either sex weighing 25-30 g were used. The test compounds 7a-l, and control were administered orally to mice and standard (diclofenac sodium 5 mg/kg body weight) was injected intraperitonally. 0.6% acetic acid solution (10 ml/kg) injected after the administration of synthesized compounds and standard. The number of writhings in each mouse was observed for 20 min period starting 10 min after injection of acetic acid. Analgesic activity was expressed as percentage of inhibition of number of writhings, when compared with the control group.

Percentage analgesic activity of compounds was calculated using following formula,% Analgesic activity = (n – n’/n) x 100

Where n = Mean number of writhes of control group and

n’ = Mean number of writhes of test group

The results of the analgesic activity are presented in table 2

Invitro antiinflammatory activity:

All the compounds were subjected to invitro antiinflammatory activity by protein denaturation technique in various concentration i.e. 10, 50,100, 150, 200, 250, 300, 350, 400, 450 and 500 μg/ml. The standard drug and synthesized compounds (7a-7l) were dissolved in minimum

quantity of dimethyl formamide (DMF) and diluted with phosphate buffer (0.2 M, pH 7.4). Final concentration of DMF in all solution was less than 2%. Test solution (1ml) containing different concentrations of drug was mixed with 1 ml of 1mM albumin solution in phosphate buffer and incubated at 27° + 1° C in BOD incubator for 15 min. Denaturation was induced by keeping the reaction mixture at 60° + 1° C in water bath for 10 min. After cooling, the turbidity was measured at 660 nm. Percentage of inhibition of denaturation was calculated from control where no drug was added. Each experiment was done in triplicate and average is taken. The diclofenac was used as standard drug. The percentage inhibition of denaturation was calculated by using following formula.% Of Inhibition = 100 X [Vt / Vc – 1]

Where,

Vt = Mean absorbence of test sample.

Vc = Mean absorbence of control

The results of the Invitro antiinflammatory activity are presented in table 3

Table 2: Analgesic activity of data 2-(5-substituted phenyl)-1,3,4-thiadiazole-2-yl amino)methyl)-1H-isoindole-1,3(2H)-dione.

Group	Dose	No. of writhes	Percentage protection
Control	0.1ml/10gm	48±0.954	-
Standard	5mg/kg	8.00±0.424**	83.33
7a	25mg/kg	43.66 ±0.881^NS	9.04
7b	25mg/kg	24.60±1.155**	48.75
7c	25mg/kg	27.60±1.000**	42.50
7d	25mg/kg	10.00±0.8819**	79.16
7e	25mg/kg	12.33±0.666**	74.31
7f	25mg/kg	14.60±1.155**	69.58
7g	25mg/kg	20.00±0.333**	58.33
7h	25mg/kg	16.00±0.577**	66.64
7i	25mg/kg	30.00±1.453**	37.50
7j	25mg/kg	17.00±1.000**	64.58
7k	25mg/kg	13.66±0.577**	71.54
7l	25mg/kg	26.80±0.577**	44.16

** p≤0.01 represent the significant difference when compared with control group.

NS= Non significant

Data expressed as Mean ± SEM. n=6

Data was analyzed by one-way ANNOVA followed by Dunnett’s test.

RESULTS AND DISCUSSION:

All the compounds obtained are solids melting at around the range of 210-265 ⁰C. The solid state IR (KBr,cm^-1) spectra of these compounds reveal a characteristic aromatic stretch between 3000-3100 cm^-1 and sharp imide carbonyl stretching vibration for phthalimide at 1747 cm^-1and 1775 cm^-1 . Only single peak was observed for secondary amino group at around 3342.75 cm^-1 indicating incorporation of primary amine group of substituted thiadiazole into the phthalimide ring. The ¹H NMR spectra were recorded in CDCl₃. The secondary amine 1-H revealed singlet at around 1.4 ppm, and CH₂revealed singlet at around 3.4 ppm. The aromatic protons appeared at 7.6 – 9.2 ppm as multiplet. Physical properties of the final derivatives (7a-7l) are given in table 1.

Table 3: The inhibitory effects of different concentration of test compounds and standard drug.

Concentration (µg/ml)	% Inhibition
	Std	7d	7e	7f	7g	7h	7i	7j	7k	7l
10	18	26	24	15	21	13	19	21	12	19
50	24	32	29	19	32	16	27	28	23	22
100	29	38	32	22	36	21	36	31	29	28
150	34	46	38	26	39	28	44	36	32	37
200	42	54	42	32	42	29	48	42	39	43
250	55	62	45	38	46	32	49	45	48	47
300	59	68	48	42	48	38	51	48	49	49
350	65	70	51	46	52	41	56	51	55	54
400	72	73	56	48	58	48	59	53	68	59
450	76	79	68	55	65	58	61	55	71	62
500	89	83	78	56	68	61	62	59	74	64
IC₅₀	242	189.6	285.6	406.4	295.5	403.5	289.4	348.4	287.5	309.8