INTRODUCTION:

A number of thiazolidinone derivatives have been reported to possess diversified activities including hypoglycemic action.¹ Thiazolidinone ring is a main pharamacophoric group responsible for antidiabetic activity. Therefore, it was planned to choose thiazolidinone as a lead molecule for molecular modification to enhance the specificity and potency of action and to reduce the toxicity. Compounds carrying the thiazolidinone ring have been reported to demonstrate a wide range of pharmacological activities which include anticonvulsant², antimicrobial³, antiinflammatory⁴, antihistaminic⁵, anti-hypertensive⁶, and hypnotic⁷, antidiabetic^{8, 9} activities. Heterocyclic ring like pyridine ring also plays important role in antidiabetic activity of some drugs (pioglitazone, rosiglitazone). In general, pyridine ring and substituted thiazolidinone ring are essential for antidiabetic activity. The proposed work involves syntheses of some novel N’-[3-(aryl/alkyl substituted)-4-oxo-1, 3-thiazolidin-2-ylidene]- -(pyridine-2-yloxy) acetohydrazides with the aim of obtaining the new antidiabetic agents.

Ethyl (pyridine-2-yloxy) acetate (compound 2) was synthesized in an excellent yield by electrophillic substitution on 2-hydroxy pyridine using ethyl chloroacetate under the reflux condition. Compound 2 on amination with hydrazine hydride yield 2-(pyridine-2-yloxy) acetohydrazide 3. Reaction of 3 with alkyl/aryl isothiocynate in ethanol gives compounds 4-8. The cyclization reaction of 4-8 with chloroacetic acid in

boiling ethanol containing fused sodium acetate gives the corresponding N’-[3-(aryl/alkyl substituted)-4-oxo-1, 3-thiazolidin-2-ylidene]-2-(pyridine-2-yloxy) acetohydrazides (9-13). The synthetic route is depicted in Scheme 1.

Thus in the present investigation, five different derivatives of N’-[3-(aryl/alkyl substituted)-4-oxo-1, 3-thiazolidin-2-ylidene]-2-(pyridine-2-yloxy) acetohydrazides were synthesized and evaluated for their antidiabetic activity.

EXPERIMENTAL:

Melting points and boiling points were determined in open capillaries and were uncorrected. Purity of the compounds was ascertained by TLC plates using silica gel G coated glass plates using chloroform-methanol as irritant and iodine vapour as detecting agent. IR spectra were recorded using KBr pellets on FTIR 8101, Shimadzu, Japan, ¹H NMR spectra and Mass spectra (FAB-MS) were recorded on Varian 300 MHz instrument and 70eV on Jeol D-300 spectrometer (Jeol Ltd, Tokyo, Japan). All the solvents and chemicals used for the synthesis were of S. d. Fine–chemicals Limited, Mumbai. The starting materials were obtained from Lancaster Limited and Alkali Metals Limited, Hyderabad.

Synthesis of 2: To a mixture of triethylamine (5.32 g, 0.0525 mol) and 2-hydroxy pyridine (5 g, 0.0525 mol), a solution of ethyl chloroacetate (5.6ml, 0.0525 mol) in 1:4 Dioxane (50 ml) was added drop wise. The temperature was maintained at 90°C for 1 hr and then the reaction mixture was stirred for 7-8 hrs. The excess solvent was removed under reduced pressure. Then the reaction mixture was poured in ice-cold water and was

Table 1: Physical data of N-(substituted aryl/alkyl)-2-[(pyridine-2- yloxy) acetyl] carbothioamides (4-11).

Compound No.	Name of aliphatic / aromatic isothiocynate	Yield	R_f value	m.p.(⁰c) (Uncorrected)
4	Phenyl	95%	0.68	262-264
5	Ethyl	93%	0.65	116-118
6	p-chlorophenyl	89%	0.59	226-228
7	2, 4- dichlorophenyl	88%	0.60	215-216
8	Methyl	90%	0.64	136-137

Table 2: Physical and analytical data of compounds.

Compound No.	R	Molecular formula	m.p.(⁰C) uncorrected	Yield^a(%)	Mass[M+2]
9	Phenyl	C₁₆H₁₄N₄O₃S	84-86	65	342^b
10	Ethyl	C₁₂H₁₄N₄O₃S	116-118	62	294
11	p-chlorophenyl	C₁₆H₁₃N₄O₃S	130-132	55	376^b
12	2,4-dichlorophenyl	C₁₆H₁₂N₄O₃S	116-118	62	410^b
13	Methyl	C₁₁H₁₂N₄O₃S	118-120	59	279

^a All the compounds were recrystalized from ethanol. ^b Values represent [M+2] due to appearance of an isotopic peak.

Table 3: Intraday Effect of Different Aryl/ Alkyl Substituted Thiazolidinone Derivatives on Serum Glucose at 1^st day.

Compound No.	Average Serum Glucose Level (mg/dL) at (1^st day)
Compound No.	0 hr	1 hr	3 hr	5 hr	7 hr
Control	275.15±1.87	276.12±0.78	269.21±2.22	266.02±3.10	262.01±2.43
Alloxan	270.6±2.80	275.23±1.87	289.40±1.01	296.08±3.12	302.61±2.09
Standard	282.00±2.80	242.27±1.63	201.42±4.21	159.11±0.99	122.15±5.63
9	276.02±3.36	245.42±2.50	212.12±3.10	170.21±1.56	126.06±1.23
10	294.31±5.32	274.10±3.21	247.02±1.02	191.38±0.19	138.15±0.96
11	298.32±4.23	290.43±0.49	261.23±3.10	220.31±5.20	181.26±2.14
12	294.43±1.93	288.11±2.63	276.40±2.90	253.24±0.62	245.22±1.83
13	283.11±1.23	276.10±2.01	243.17±1.94	196.16±1.03	148.19±0.96

The values are presented as mean ± S. E. M. of six determinations p<0.01, Student’s t- test compared with diabetic control.

Table 4: Effect of Different Aryl/ Alkyl Substituted Thiazolidinone Derivatives on Serum Glucose at 1^st, 3^rd, 7^th day.

Compound No.	Average Serum Glucose Level (mg/dL)
Compound No.	1^st day	3^rd day	7^th day
Control	237.02±3.10	229.21±1.23	198.13±3.21
Alloxan	270.6±2.80	336.02±0.98	302.31±1.34
Standard	159.11±0.99	124.15±2.63	105.21±5.32
9	170.21±1.56	140.08±5.20	118.16±1.10
10	191.38±0.19	164.32±1.03	124.43±0.69
11	290.31±5.20	261.11±3.36	221.12±2.12
12	253.24±0.62	220.43±0.36	191.03±2.26
13	196.16±1.03	176.21±4.20	159.21±1.21

The values are presented as mean ± S. E. M. of six determinations p<0.01, Student’s t- test compared with diabetic control.

Scheme 1: General scheme for syntheses of compounds 9-13 (R= phenyl, ethyl, p-chlorophenyl, 2, 4- dichlorophenyl, methyl)

The yield and physical data are summarized in Table 1.

4: IR: 3382, 3355 cm^-1(NHNH₂), 1720 cm^-1(C=O), 1360 cm^-1(C=S); ¹H NMR (CDCl₃): δ 7.58 (m, 4H, Ar.), 4.82 (s, 2H, OCH₂), 7.78 (s, 1H, CONH), 6.34 (t, 1H, pyridine C-3), 7.72 (q, 1H, pyridine C-4), 6.79 (q, 1H, pyridine C-5), 7.76 (t, 1H, pyridine C-6).

5: IR: 3212, 3225 cm^-1(NH), 1730 cm^-1(C=O), 1365 cm^-1(C=S); ¹H NMR (CDCl₃): δ 1.28 (m, 3H, CH₃), 4.72 (s, 2H, OCH₂), 7.75 (s, 1H, CONH), 6.44 (t, 1H, pyridine C-3), 7.76 (q, 1H, pyridine C-4), 6.69 (q, 1H, pyridine C-5), 7.66 (t, 1H, pyridine C-6).

6: IR: 3217, 3232 cm^-1(NH), 1736 cm^-1(C=O), 1315 cm^-1(C=S); ¹H NMR (CDCl₃): δ 7.39-7.65 (m, 4H, ArH), 4.72 (s, 2H, OCH₂), 7.75 (s, 1H, CONH), 6.34 (t, 1H, pyridine C-3), 7.73 (q, 1H, pyridine C-4), 6.62 (q, 1H, pyridine C-5), 7.76 (t, 1H, pyridine C-6).

7: IR: 3219, 3222 cm^-1(NH), 1734 cm^-1(C=O), 1315 cm^-1(C=S); ¹H NMR (CDCl₃): δ 7.29-7.65 (m, 3H, ArH), 4.73 (s, 2H, OCH₂), 7.75 (s, 1H, CONH), 6.44 (t, 1H, pyridine C-3), 7.73 (q, 1H, pyridine C-4), 6.65 (q, 1H, pyridine C-5), 7.73 (t, 1H, pyridine C-6).

8: IR: 3216, 3215 cm^-1(NH), 1735 cm^-1(C=O), 1362 cm^-1(C=S); ¹H NMR (CDCl₃): δ 1.18(m, 2H, CH₂), 4.70 (s, 2H, OCH₂), 7.73 (s, 1H, CONH), 6.54 (t, 1H, pyridine C-3), 7.72 (q, 1H, pyridine C-4), 6.72 (q, 1H, pyridine C-5), 7.76 (t, 1H, pyridine C-6).

Syntheses of 9-13: A mixture of the N-(substituted aryl/alkyl)-N’’(-2-pyridine-2-yloxy) acetyl thiosemicarbazides (3.03g, 0.01 mol), chloroacetic acid (0.93g, 0.01 mol) and sodium acetate (0.81g, 0.01 mol) in ethanol (60 ml) was refluxed for 10 hrs. The mixture was cooled and diluted with enough water to develop turbidity and left overnight for complete separation of the product. Then the compounds were filtered and recrystallized from ethanol. The yield and physical data are summarized in Table 2.

9. IR: 3220 cm^-1 (N-H), 1720 cm^-1 (C=O), 1585 cm^-1 (C=N), 3010 cm^-1 (C-H); ¹H NMR :( CDCl₃) d 6.38, 7.75, 6.68, 7.79 (C-H, 2-pyridine), d 3.76, 4.83, 3.24 (CH₂), d 7.0 (-NH-), d 1.20(CH₃); FAB-MS: (m/z, 100%): 342 ([M⁺], 100%)

10. IR: 3325 cm^-1 (N-H), 1722 cm^-1 (C=O), 1583 cm^-1 (C=N), 3015 cm^-1 (C-H); ¹H NMR :( CDCl₃) d 6.38, 7.69, 6.68, 7.75 (C-H, 2-pyridine), d 3.76, 4.83 (CH₂), d 7.0 (-NH-), d 7.14, 7.06, 7.06, 7.14, 7.07 (Phenyl ring); FAB-MS: (m/z, 100%): 294 ([M⁺], 100%)

11. IR: 3400 cm^-1 (C-H str. pyridine), 1552 cm^-1 (N-H str.), 1730 cm^-1, 1650 cm^-1 (C=O), 1525 cm^-1 (C=N), 3035 cm^-1 (C-H, Aromatic str.), 1208 cm^-1 (C-O-C str.); ¹H NMR (CDCl₃) d 6.38, 7.69, 6.68, 7.75 (C-H, 2-pyridine), d 3.81, 4.83 (CH₂), d 7.0 (-NH-), d 7.58, 7.25, 7.25, 7.58, (Phenyl ring); FAB-MS: (m/z, 100%): 378 ([M⁺+2], 100%)).

12: IR: 3410 cm^-1 (C-H str. pyridine), 1552 cm^-1 (N-H str.), 1710 cm^-1, 1640 cm^-1 (C=O), 1580 cm^-1 (C=N), 3020 cm^-1 (C-H, Aromatic str.), 1260 cm^-1 (C-O-C str.); ¹H NMR (CDCl₃) d 6.38, 7.69, 6.68, 7.75 (C-H, 2-pyridine), d 3.81, 4.83 (CH₂), d 7.0 (-NH-), d 7.26, 7.13, 7.52 (Phenyl ring); FAB-MS: (m/z, 100%): 412 ([M⁺ +2], 100%.

13. IR: 3326 cm^-1 (N-H), 1729 cm^-1 (C=O), 1583 cm^-1 (C=N), 3025 cm^-1 (C-H); ¹H NMR :( CDCl₃) d 6.38, 7.69, 6.68, 7.75 (C-H, 2-pyridine), d 3.76, 4.83 (CH₂), d 7.0 (-NH-), d 7.14, 7.06, 7.06, 7.14, 7.07 (Phenyl ring); FAB-MS: (m/z, 100%): 294 ([M⁺], 100%)

PHARMACOLOGICAL EVALUATION:

Antidiabetic activity:

Animals:

Wistar albino rats of either sex weighing between 150 – 200 g were used for the study. The animals were housed in standard environmental conditions of temperature (25±2⁰C), humidity (55±10%) and light (12:12 hr light: dark cycle). Rats were supplied with standard laboratory diet and water ad libitum. Animals were deprived of food for at least 18 hrs but were allowed free access to drinking water.

Cut-Off Lethal Dose (LD₅₀):

All the compounds synthesized were tested for acute toxicity test. No toxicity was observed at the doses of 300, 1000, 2000 mg/kg of body weight but it was observed that more than 50% of animals were died at the dose of 2000 mg/kg of body weight. Thus for the screening of antidiabetic activity, the dose selected was 200 mg/kg of body weight (i.e., 1\10 of the 2000 mg/kg of body weight) as per the OECD guidelines.¹⁰

Drugs Used:

Metformin was given to rats at a dose of 5 mg/kg body weight, as a reference standard.

Induction of Diabetes:

A single dose (150 mg/kg, body weight) of Alloxan monohydrate (5%w/v in sterile water) was dissolved in normal saline used for the induction of diabetes and injected intraperitoneally to Wistar albino rats weighing 150-200 g. The induction of diabetes was confirmed by estimation of elevated fasting blood glucose level. The rats having blood glucose level above 200 mg/dl of blood were selected for the study.

Groups Design:

These rats were divided into various groups with 6 rats each. The rats in group I (control) were administered distilled water orally. Group II was treated as the diabetic control (Alloxan 150 mg/kg, i.p.). Group III was treated with metformin (5mg/kg, orally), while groups IV, V, VI, VII, VIII were treated with test compounds. Treatment with compounds was started on the 6^th day of Alloxan treatment (i.e. Day 1) and was continued for 8^th day (i.e. Day 3), 12^th day (i.e. Day 7) of Alloxan treatment. Before this treatment, intraday serum glucose estimation was also carried out (i.e. after 0hr, 1hr, 3hr, 5hr, and 7hr on the 6^th day of Alloxan treatment). All the drugs were given orally as a single dose. All the groups were subjected to serum glucose estimation by withdrawing 0.5 ml of blood from the retro orbital plexus under light ether anesthesia. The blood glucose concentration was estimated in spectrophotometer at 505 nm.

Sample Collection:

Blood was collected from retro orbital plexus of the eye under light ether anesthesia using capillary tube. Blood was collected in fresh vials containing sodium fluoride and sodium oxalate as anti coagulant.

All the compounds synthesized were tested for antidiabetic activity, the fasting serum glucose levels were determined according to GOD-POD method.¹¹

RESULTS AND DISCUSSION:

In the present investigation, different derivatives of N’-[3-(aryl/alkyl substituted)-4-oxo-1, 3-thiazolidin-2-ylidene]-2-(pyridine-2-yloxy) acetohydrazides (9-13) were synthesized and evaluated for their physical, analytical and spectral data (Table 2).

The structures of compounds 9-13 were confirmed on the basis of spectral data. IR spectrum showed absorption peaks at 1552 cm^-1 and 1650 cm^-1 for the N-H stretching and C=O stretching of amide groups respectively. The ¹H-NMR spectrum exhibited signals attributed to the proton atd 6.38, 7.69, 6.68, 7.75 indicating the presence of pyridine ring, while the signals atd 7.0 indicated the presence of acetohydrazide linkage.

The results of antidiabetic activity of test compounds were given in Table 3, 4. Compounds No. 9 and 10 were found to be most efficient i.e. 48% and 52% reduction of serum glucose level respectively at 200 mg/kg dose.

CONCLUSION:

A series of N’-[3-(4-alkyl/aryl substituted)-4-oxo-1, 3-thiazolidin-2 ylidene]-2-(pyridine-2-yloxy) acetohydrazides were synthesized using appropriate synthetic route and screened for antidiabetic activity. It can be concluded that, the number of compounds showed antidiabetic activity, out of which 9 and 10 showed appreciable antidiabetic activity. Thus research work was undertaken for substitution at 3 position of thiazolidinone ring. The encouraging results showed may lead to the development of novel antidiabetic drugs if explored further.

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Received on 06.04.2009 Modified on 21.05.2009

Asian J. Research Chem. 2(2): April.-June, 2009 page 157-161