FORM -2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See Section 10; rule 13)
THE FOLLOWING SPECIFICATION DESCRIBES THE NATURE OF THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
TITLE OF INVENTION:
2, 4- THIAZOLIDINEDIONE DERIVATIVES FOR THE
TREATMENT AND MANAGEMENT OF DIABETES MELLITUS
APPLICANTS
(a) Name:
1. AGRAWAL YOGESH PURUSHOTTAM
2. AGRAWAL MONA YOGESH
3. GUPTA ARUN KUMAR
4. SHAMKUWAR PRASHANT BABARAO

(b) Nationality: 1. INDIAN 2. INDIAN, 3. INDIAN, 4. INDIAN
(c) Address: GOVERNMENT COLLEGE OF PHARMACY, THIBA PALACE, RATNAGIRI, PIN NO. 415612, MAHARASHTRA, INDIA.

TITLE OF INVENTION:
2, 4- THIAZOLIDINEDIONE DERIVATIVES FOR THE TREATMENT AND MANAGEMENT OF DIABETES MELLITUS
FIELD OF THE INVENTION:
The present invention relates to the field of Pharmacy. Particularly, this invention relates to antidiabetic activity of synthesized novel Thiazolidinedione derivatives or solvates in vivo, which are utilized in the field of medicine.
BACKGROUND OF THE INVENTION:
Diabetes is associated with a variety of metabolic abnormalities, principle among them being hyperglycemia. This syndrome includes hypertriglyceridemia, reduced HDL, cholesterol and abnormal postprandial lipidemia (Diabetes, 1988, 37, 1597-1607).
Type 1 diabetes or insulin dependent diabetes mellitus (IDDM) is a complex, multifactorial disease involving severe destruction of pancreatic p cells. Type 2 diabetes or non-insulin dependent diabetes mellitus (NIDDM) typically accure at older age and obesity adds greater risk in its development (J. Clin. Endocrinol, metab. 1999, 84, 2329-2335). Although glycemic control, insulin treatment and other chemical therapies can control many aspects of diabetes, while risk of developing clinical complications due to microvascular or macrovascular diseases that includes nephropathy, retinopathy and neuropathy in diabetic patients.
A variety of biguanide, Sulphonylureas, DPP-4 Inhibitors and glinide derivatives have been used clinically as antidiabetic agents. However, DPP-4 Inhibitors and Glinide derivatives are now scarcely used as they cause weight gain and hypoglycemia, while biguanide like metformin tends to cause gastrointestinal disturbances and lactic acidosis, and use of sulfonylureas, though they have strong hypoglycemic activities, requires sufficient precaution, because they frequently cause serious hypoglycemia and weight gain. Therefore, a new type of antidiabetic agents free from these defects has been desired.

On the other hand, variety of therapeutic agents containing thiazolidinediones nucleus as a pharmacophore moiety (Rosiglitazone, Ciglitazone, Pioglitazone) were reported as both dual alpha and gamma PPAR receptor agonist lowering blood glucose level (JMC, 2001, 44, 2675-2678; JMC 2003, 46, 1306-1317) and as aldose reductase inhibitor (Sorbinil, Epalrestat, Fidarestat) for the management of secondary complication in DM (Bratisl Lek Listy, 2000,101(10) 541-551), Rosiglitazone and Pioglitazone Induce osteocalactogenesis while other agents like Muraglitazar and Tesaglitazar discontinued after phase III clinical trials due to adverse effect while Aleglitazar is currently in phase III clinical trials. Sorbinil causes hypersensitivity reaction in early weeks of therapy; Clinical trial of Zopolrestat was halted due to neuropathy while Fidarestat completed Phase III, Currently Epalrestat is only a drug, available in market.
In the present invention, inventors synthesized various compounds which can be practically used as Antidiabetic agents having a broad safety margin and not concretely described in any publication of unexamined patent applications and have made studies on them to find the compounds exhibiting potent pharmacological effects with lower toxicity.
OBJECTS OF THE INVENTION:
The main object of the present invention related to: 1. A compound of the formula(l):

and their pharmacologically acceptable salts 2. A novel compound of the formula (1), which exhibit an excellent Aldose Reductase Inhibiting activity for the management of Diabetes Mellitus.

3. An Antidiabetic agent contain compound of the formula (1) and their pharmacologically acceptable salts as an dual Paroxysmal Proliferated activating receptor (PPAR) alpha and gamma receptor agonist as an blood glucose and lipid lowering agent for the treatment of Diabetes Mellitus.
4. A method of synthesizing compound of the formula (1), and their pharmacologically acceptable salts
The compounds re-presentable by the above formula (1) include specific ones.
(E)-4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl 2-chlorobenzoate (1)
(E)-4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl4-chlorobenzoate(la)
(E)-4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl 2,4-dichlorobenzoate (lb)
The invented compound (1) contains acidic nitrogen in its molecule, and it can be converted to a pharmacologically desirable salt, by reacting with suitable acids. Such acid salts are exemplified as mineral salts (e.g. Hydrochloride, sulphate etc.), organic acid salts (e.g. succinate, fumarate, malate, tartrate etc.) and sulphonates.
The compound (1) of this invention and their pharmacologically acceptable salts exhibit an excellent blood glucose and lipid lowering action with broad safety margins, without toxicity or unfavorable side reaction and may be safely administered, orally or parentally as a valuable therapeutic agent for the treatment and management of Diabetes in mammals including man.

SUMMARY OF THE INVENTION:
The compound represented by the above mentioned formula (1) and their pharmacologically acceptable salts can be prepared as illustrated in scheme

In accordance with this invention the process of preparation of thiazolidinediones derivatives and their pharmacologically acceptable sails comprise
1. Conversion of Chloro substituted benzoic acid (6) into its acyl chloride derivatives by reacting with thionyl chloride in suitable organic solvent.

2. Reacting adduct thus obtain of Chloro substituted acyi chloride (5) with p-hydroxy benzaldehyde (4) in presence of suitable organic solvent and base like triethanolamine, triethylamine, ammonia, ammonium hydroxide etc.
3. The Chloro substituted benzoate (3) obtains is allowed to. react with thiazolidinediones (2) to give the compound represented by formula (1).
4. In this reaction, as the reaction proceeds, hydrogen chloride is produced as a by-product, and for capturing this by-product, the reaction may be conducted in the presence of suitable acetate like sodium acetate, potassium acetate etc. in an amount of usually 1-1.5 mole relative to 1 mole of compound 3.
5. The compound (1) and their pharmacologically acceptable salts produced as mentioned above can be isolated and purified by conventional means such as recrystallization, chromatography etc.
DETAILED DESCRIPTION OF THE INVENTION:
The present invention is illustrated by means of following non-limiting examples. Example 1:- Preparation of 4-formylphenyl 2-chlorobenzoate
Accurately weighted 15.6 gm of 2-Chloro Benzoic acid (O.lmol) was refluxed with 10 ml thionyl chloride for 3-4 hr. On completion of the reaction, excess of thionyl. chloride was removed under vacuum and the residue as an ar-yl chloride thus obtained was dissolved in RBF containing 40 ml of dichloromethane and cooled to 0°C 42 ml of triethylamine (0.3mol) was added slowly with constant stirring, followed by 12.2 gm of p-hydroxy benzaldehyde (O.lmol). The reaction mixture was stirred at 0°C for another 2 hr. and washed the resulting compound with saturated solution of sodium bicarbonate, brine solution and water in ethyl acetate. Organic phase was separated and pass through anhydrous Na9SO4. Solvent was removed under vacuum and recrystalized in ethanol give the pale yellow crystalline intermediate.
Example 2:- Preparation of 4-formylphenyl 4-chIorobenzoate
Synthesis was carried out using p-Chloro benzoic acid as the starting compound as per the procedure described in example 1.

Example 3:- Preparation of 4-formylphenyl 2,4-dichlorobenzoate
Synthesis was carried out using 19.1 gm. (0.1 mol) 2, 4-dichloro benzoic acid as the starting compound as per the procedure described in example 1.
Example 4:- Synthesis of 2,4-Thiazolidinedione:-
In a 250 ml three necked round-bottomed flask Chioroacetic acid (56.4 gm, 0.6 mol) in 60 ml of water was taken and solution of thiourea (45.6 gm, 0.6 mol) in 60 ml of water was added with stirring. Mixture was stirred for 15 min to form a white precipitate, accompanied by considerable cooling. To the contents of the flask 60 ml of concentrated hydrochloric acid was added slowly from a dropping funnel, the flask was then connected with a reflux condenser and gentle heat was applied to affect complete solution, after which the reaction mixture was stirred and refluxed for 8-10 h at 100-110°C. On cooling the contents of the flask solidified to a cluster of white needles. The product was filtered and washed with water to remove traces of hydrochloric acid and dried. Purified and recrystalized from ethyl alcohol.
Example 5:- Preparation of 4-((2,4-dioxothiazolidin-5-ylidene)methyi)phenyl 2-
chlorobenzoate derivatives (1).
Knoevenagel Condensation was carried out by mixing equimolar concentration of 4-
formylphenyl 2-chlorobenzoate (0.1 mol) and 11.7 gm. of 2,4-thiazolidinedione (0.1 mol)
in round-bottomed flask containing 40 ml of glacial acetic acid. To this a catalytic
amount (12.3 gm.) of sodium acetate (0.15 mol) was added and stirred the reaction
mixture at 100-105°C for 10-12 hr. After completion of reaction, crystalline the product
by keeping aside for overnight at RT. filtered, washed with cold acetic acid and purified
using column chromatography.
IR (cm-1):- 3033 (C-H), 1685 (C=C), 1159 (C-O), 1748 (C=0), 3648 (N-H), 736 (C-Cl)
1H NMR (DMSO) (δ):- 9.65 (s, 1H, N-H), 7.9-8.1 (d, 1H, arom. CH), 7.55-7.75 (m, 5H,
arom. CH), 7.45-7.55 (m, 2H: arom. CH), 7.38-7.45 (d, 1H, ethylene CH) MS (ESI+):360 [M+H]+

Example 6:- Preparation of 4-((2,4-dioxothiazoiidin-5-yiidene)methyI)phenyl 4-
chlorobenzoate derivatives (la).
Synthesis was carried out using 4-formyiphenyl 4-chlorobenzoate as the starting
compound as per the procedure described in example 5.
IR (cm-1):- 2984 (C-H), 1676 (C=C), 1267 (C-O), 1732 (C=0), 1590 (N-H), 759 (C-Cl)
1H NMR (DMSO) (δ):- 9.85 (s, 1H, N-H), 7.95-8.25 (m, 2H, arom. CH), 7.6-7.75 (m,
4H, arom. CH), 7.3-7.5 (m, 2H; arom. CH), 7.15-7.25(d, 1H, ethylene CH) MS (ESI+): 360 [M+H]+
Example 7:- 4-((2,4-dioxothiazolidin-5-ylidene)methyI)phenyl 2, 4-dichlorobenzoate derivatives (lb).
Synthesis was carried out using 4-formyJphenyl 2, 4-dichlorobenzoate as the starting compound as per the procedure described in example 5.
IR (cm-1):- 3084 (C-H), 1546 (G=C), 1252 (C-O), 3712 (C=0), 1610 (N-H), 785 (C-Cl) 1H NMR (DMSO) (δ):- 10.0 (s, 1H, N-H), 7.8-8.1 (d, 1H, atom. CH), 7.5-7.7 (m, 4H,
arom. CH), 7.35-7.45 (m, 2H; arom. CH), 7.05-7.3 (d, 1H, ethylene CH) MS(ESI+):371[M+H] +
Example 8:- In-Vitro Evaluation as Aldose Reductase Inhibitors
Inhibiting potential of compound (1) against purified Aldose reductase (ALR2) enzyme was extract and prepared from goat lenses. ALR2 activity was monitored at 30° C for ability of compound (1) to inhibit the Jn-vifro reduction of D,L-glyceraldehydes as compared to Sorbinil as an reference drug for 5min. Log dose inhibition curve was generated from absorbance at 280 nm using spectrophotometer at six dose level causing an inhibition between 10 to 90 % and IC50 values were determined by non linear regression analysis as shown in table 01.
Example 9:- In-Vitro evaluation as dual a/y-PPAR Agonist
The In-Vitro specific transcription factor DNA binding activity of proposed compound (1) was determined using commercial Cayman's PPAR α ,γ, δ assay kit and cell lysates

activity was determined in ELISA reader at 450 nm. Percent of maximal efficacy (Emax) of compound (1) for PPAR a and γ was calculated at three dose levels as compared to reference Wy-14643 and Rosiglitazone normalized to 100% respectively. The agonist study was performed by using 10 μM concentration of each drug and compound exhibited statistically significant PPAR a & y agonist activity as shown in table 01. Table 01:- In-Vitro evaluation of invented compounds

Compounds In-Vitro Evaluation

Aldose Reductase Inhibition (IC50) μM* α-PPAR Agonist
(% Emax)# γ-PPAR Agonist
(% Emax)#
Compound 1 1.82(1.13-1.49) 84.5 + 0.5 89.5 ± 2.8
Compound lb 5.89(5.63-6.29) 58.8 ±0.8 67.2+ 3.6
Compound lc 4.50 (4.36-4.89) 74.1 ±0.9 71.4 ±1.7
Sorbinil 1.32(1.12-1.65)

Wyl4643 100

Rosiglitazone 100

Note: - *IC50 (μM) (95% C.L.); "mean ± S.E.M. (n=3)
Example 10:- In-Vivo Antidiabetic activity of compounds in diabetic rats Animals
Male albino rats weighing about 125-170 gm. were maintained under controlled room temperature (24 ± 2°C) and humidity (55 ± 5%) with 12 hr. light and dark cycle. All the animals were housed in polypropylene cages in groups of 6 per cage and provided with standard pellet diet and water ad libitum. All the animals were acclimatized for one week prior to the experiment which was carried out in accordance with the guidelines of Institutional Animal Ethics Committee (IAEC).
Induction of diabetes in rats
The acclimatized albino rats were rendered diabetic by administering intraperitoneal injection of Alloxan Monohydrate (120 mg/Kg) in normal saline. A 5% dextrose solution

was given in feeding bottle for a day to overcome the early hypoglycemic phase. The blood glucose was monitored regularly after alloxination by withdrawing a drop of blood from the tail vein by Tail tipping method using digital blood glucometer. Those animals with fasting (3hr.) blood glucose level of >280 mg/dl were considered diabetic and included in the study.
Dosing, blood collection and measurement
The diabetic rats were randomly divided in different groups consisting each of 6 rats as negative control group, treated groups and positive control groups. The negative control, given vehicle 2% acacia solution (5ml/kg body weight) while the positive control group given standard Antihyperglycemic agent Roziglitazone (4 mg/kg body weight) orally in 2% acacia. The treated groups were given the synthesized compounds (15 mg/kg body weight) and sample was collected by withdrawing a drop of blood from the tail vein by mai1 tipping meethod. The blood glucose level was monitored at diffrent times 0, 1, 3, and 6h respectively. The decrease in blood glucose level against each compounds are shown in table 02.
Example 11:- In-Vivo Body Weight Index lowering activity in rats Animals
In-Vivo evaluation of Body Weight Index was carried out on alloxan induce albino rat diabetic model as describe in Example 10 which are kept on HF-HC-sucrose (High fat, high cholesterol and sucrose) diet for 21 days.
Dosing and measurement
The diabetic rats were grouped and dosed as describe in Example 10 where the positive control group given standard PPAR-a agonist W 14643 (4 mg/kg b. wt) orally in 2% acacia. The body weight index was monitored at regular interval of 1St 2nd. and 3rd Week as shown in table 02.

Table 02:- In-Vivo evaluation of invented compounds

Compounds In-Vivo Evaluation

Decrease in blood glucose (mg/dl) Body Weight ndex

lhra 3hra 6hra 1st Week 2nd Week 3rd Week
Compound 1 217.23+2.78*** 186.03+3.31*** 148.89+1.40*** 1 1.016 1.028
Compound lb 224.20+4.08*** 187.14±6.39*** 183.91±1.66*** 1.01 1.03 1.07
Compound lc 208.50+3.15** 171.82±15.27** 157.98+3.26*** 1.003 1.018 1.055
Wyl4643 1.004 1.02 1.031

Rosiglitazone 221.69±21.08* 197.72+27.88* 152.80±16.72
Note:- amean ± S.E.M.( n=6); *** P<0.001; **P<0.01; *P<0.05
Example 12:-Two-week Toxicity studies in rats Animals
Toxicity study was carried out on a group of albino rats as describe in Example 10.
Dosing and Evaluation
A group of albino rats (6 rats/ Group) was selected and compounds were administered orally once daily in 5% acacia for two weeks. The dose was 100 mg/kg b.wt./day for every group of test compound. The animals were sacrificed in about 20 hr. of fasting after termination of two week administration using ether anesthesia. Liver and heart were removed and weighed. Hematology analysis was carried out using an automatic cell counter as shown in table 03. Table represents % variation in reference to the control.

Table No. 03:- Two-week Toxicity Studies of invented compounds

Compounds Two-week Toxicity Studies (% variation)

Liver Weight Heart Weight No. of Erythrocyte
Compound 1 -0.85 -1.14 0.95
Compound lb 11.21 -0.67 2.13
Compound 1c 9.25 2.45 2.18
Sorbinil 0.52 -0.35 1.36
Wyl4643 1.45 0.9 0.47
Rosiglitazone -0.35 -0.83 -0.15
The compound (1) as represented in table 01, 02 and 03 is a compound under the coverage of the present invention while compounds (la) and (lb) are cited for comparison, since they are similar to compound (1) of this invention in their chemical structures. Sorbinil, Wy-14643 and Rosiglitazone are standard compound used as reference. As is apparent from the experimental results given in table, compound (1) of this invention is superior to the structural analog compounds (la) and (lb), referred standard Sorbinil and Wy-14643 while comparable to the Rosiglitazone only in hypoglycemic activity with better hypolipidemic activity, while showing extremely low toxicity as compared to prepared structural analog and referred standards. Such an effect as above caused by the introduction of a Chloro group at ortho position to the phenyl is quite expected. Thus compound (1) of the present invention exhibits excellent hypoglycemic and hypolipidemic effect with little toxicity to internal organs and blood even by continuous administration for a long period of time. Therefore, compound (1) is of valuable therapeutic agent for the treatment and management of type II Diabetes in mammals including man.

TECHNICAL ADVANCEMENT AND ECONOMICAL SIGNIFICANCE OF THE PRESENT INVENTION:
The present investigation concerned with the field of pharmaceutical sciences for the development and evaluation of a compound of the formula 1 and their pharmacologically acceptable salts, which exhibit a dual action for the treatment and management of Diabetes Mellitus. The Aldose Reductase inhibition potentiate the decrease in the secondary complications due to hyperglycemia while activation of Paroxysmal Proliferated activating receptor (PPAR) gamma receptors reduces the blood glucose level by increasing its catabolism. The toxicity of Thiazolidinedione associated with gaining weight is also regulated by activating alpha receptors of Paroxysmal Proliferated activating receptor (PPAR).
Formulation of a compound having a formula 1 and their pharmacologically acceptable salts will be the regimen in future as a valuable therapeutic agent for the treatment and management of Diabetes Mellitus.


WE CLAIM,
1. A compound of the formula(l):
and their pharmacologically acceptable salts
2. A compound as claimed in claim 1, wherein the compound is (E)-4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl 2-chlorobenzote
3. A compound as claimed in claim 1, wherein the compound is an hydrochloride salt of (E)-4-((2,4-dioxothiazolidin-5-elidene)methyl)phenyl)-2-chlorobenzoate
4. A method of synthesizing compound of the formula (1), and their pharmacologically acceptable salts.
5. A novel compound of the formula (1), which exhibit an excellent Aldose Reductase Inhibiting activity for the management of Diabetes Mellitus.
6. An Antidiabetic agent contain compound of the formula (1) and their pharmacologically acceptable salts as an dual Panoxysmal Proliferated activating receptor (PPAR) alpha and gamma receptor agonist as an blood glucose and lipid lowering agent for the treatment of Diabetes Mellitus.