Synthesis of Thienopyrimidines and their Antipsychotic Activity

A series of thienopyrimidines and related heterocycles were synthesized by refluxing related imidoyl chloride with primary and secondary amines under microwave irradiation and classical heating. The imidoyl chlorides were synthesized from corresponding cyclic imides with phosphorus oxychlorides under microwave irradiation and classical heating. The structures of the compounds were confirmed by FT-IR, NMR. The synthesized compounds were screened for anti psychotic activity.


Introduction
Activation of central dopaminergic systems is generally considered to be the most important factor in the etiology of schizophrenia 1,2 .Several dopamine and serotonin antagonists (atypical antipsychotics) such as clozapine, olanzapine, risperidone, quietapine, sertindole, seroquel have been found to exhibit effective atypical antipsychotic activity.These compounds still are associated with some extrapyramidal side effects 3 .Literature reports that the compounds synthesized by removal of one of the benzene ring of clozapine were having same affinity on dopaminergic receptors to that of clozapine with minimization of its extrapyramidal side effects 4 .Clozapine, olanzapine, seroquel and other atypical antipsychotics have greater affinity for serotonin receptor than dopamine receptors 5,6 .An attempt has been made in direction of synthesizing olanzapine analogues by removal of one of the benzene rings of it and replacement of its seven membered diazepine ring by six membered pyrimidine ring for the sake of improving its dopamine selectivity and minimization of extrapyramidal side effects.

Experimental
Melting points were determined in open capillary on Veego (model: VMP-D) electronic apparatus and were uncorrected.Purity of the compounds were verified by running TLC, using precoated plates.The IR spectra of the compounds were recorded on Shimadzu 8400-S FT-IR Spectrophotometer using KBr (cm -1 ). 1 H NMR Spectra was recorded in CDCl 3 using NMR Varian-Mercury 300 MHZ spectrometer using TMS as internal standard (chemical shifts in δ ppm).

Synthetic of 2-amino-5-substituted-thiophene-3-carboxylate (2a-c)
The compounds (2a-c) were synthesized by Gewald reaction 7,8 .The eqimolar quantities of ethylcyanoacetate, aldehyde / ketone (propionaldehyde for 2a, butyraldehyde for 2b, cyclohexanone for 2c, sulphur and TEA for 2a/b) or DEA (for 2c), in DMF (for 2a,b) ethanol (for 2c) were taken.The reaction mixture was kept for 24 h (24 h for 2a, 20 h. for 2b, 1 h.for 2c) with constant stirring.Then it was refluxed in a microwave for 20 min.at power level 4. The resulting solution of 2a, 2b was added in equal volume of ice-cold water, and extracted with methylene chloride.Organic layer was washed 2-3 times with 0.1 M HCl and then with brine solution.Organic layer was separated and dried over anhydrous Na 2 SO 4 .For 2c the solid obtained was filtered and washed with water and recrystalized by using CH 3 OH.

Synthesis of thienopyrimidines
The compound 4a and n-phenylpiperazine (for 5a 1 ) or morpholine (for 5a 2 ) in proportion of 1: 1.5 were dissolved in dioxane and reaction mixture 10 was refluxed for 3.5 h.After cooling, equal quantity of water was added to this reaction mixture and extracted with CHCl 3 , purification was done by using column chromatography (CHCl 3 : CH 3 OH; 98.2: 0.2) for 5a 1 and CHCl 3 for 5a 2 .

Synthesis of 5b 1 , 5b 2
The compound 4b and n-methylpiperazine (for 5b 1 ) or morpholine (for 5b 2 ) in proportion of 1: 1.5 were dissolved in dioxane and reaction mixture was refluxed for 3.5 h.After cooling, equal quantity of water was added to this reaction mixture and extracted with CHCl 3 .Purification was done by using column chromatography (CHCl 3 : CH 3 OH; 98.2: 0.2) for 5b 1 and 5b 2 .

Antipsychotic activity
The Antipsychotic activity of the synthesized compounds was evaluated by using models like spontaneous motor activity using Actophotometer, behavioral effect in mice, catalepsy test in mice.
Control group was treated with 0.1% acacia solution p.o. and remaining groups were treated with test compounds (10 mg/kg and 20 mg/kg p.o. by suspending test compounds in 0.1% acacia solution) and reference compound (Olanzapine 2.5 mg/kg and 5 mg/kg p.o. by suspending reference compounds in 0.1% acacia solution) respectively.Immediately after drug administration the animals were closely observed for their spontaneous locomotor activity.It was recorded by using Actophotometer.Where in interruption of beam of light generated a pulse which was recorded on digital counter.The locomotor count for each animal was recorded for 5 min at 60 min interval for 1 h.

Catalepsy test 16
Control group was treated with 0.1% acacia solution p.o. and remaining groups were treated with test compounds (5 mg/kg and 10 mg/kg p.o. by suspending test compounds in 0.1% acacia solution) and reference compound (Olanzapine 2.5 mg/kg and 5 mg/kg p.o. by suspending reference compound in 0.1% acacia solution) respectively.The animals were placed individually in clear acrylic cages and allowed a minimum 30 min to acclimatize to the new environment.Catalepsy was assessed by positioning mice with their hind paws on the floor and their forelimbs rested on an elevated bar (set at 2.5 cm.).The time that the paws remained on the bar was determined at 60 min.interval for 2 h.

Behavioral effects
The behavioral effect of test drug (20 mg/kg p.o.) was assessed by the method described by Irwin et al. 17 .Briefly, the effects of test compound on different behavioral paradigms in animals were scored with the use of nine degrees, that is, with a scale of 0-8.The base score for normal signs or effects is 4, scores below 4 are subnormal responses, those above 4, for supernormal.The base score for abnormal signs is 0 and the maximal score is 8.In the items mentioned below, the base score is given in parentheses.The animals were observed for 2 h after treatment for alertness (4), stereotypy (0), and reactivity to touch response (4), body position (4), righting reflex (0) and lacrimation (0).

Results and Discussion
Gewald products 2 were synthesized by stirring reaction mixture or by refluxing ethyl cyano acetate, aldehyde/ ketone, sulphur, solvent (DMF/Ethanol), base (TEA/DEA) in microwave.The compound 2 were refluxed with excess of formamide to synthesize thienopyrimidines 3 which on refluxing with phosphorus oxychloride give 4-chloro-5,6disubstitutedthieno [2,3-d]pyrimidine (4).Target compounds were prepared by refluxing 4 with different primary and secondary amines in the presence of DMF/dioxane in microwave (Scheme 1).
Compound 2 showed a characteristic primary amine group peak in the range 3300-3400 cm -1 and sharp carbonyl stretching vibration for ester in the range of 1640 -1680.0cm -1 .The 1 H NMR spectrum showed singlet of allylic proton of thiophene ring at 6.5 ppm and amine protons showed singlet at 5.5 ppm.Methylene and methyl protons of ester gave quartet and triplet at 4.2 and 1.3 ppm respectively.Methyl protons attached to thiophene ring gave singlet at 2.2 ppm.
Compound 3 showed characteristic stretching of secondary amine with broadning of peak in the range of 3200-3300 cm -1 and sharp carbonyl stretching vibration for the cyclic imide at 1640-1690 cm -1 .In 1 H NMR spectrum characteristics pyrimidine proton showed signal at 8.0 ppm as a singlet.Proton of thiophene ring showed singlet at 7.1 ppm.Due to keto-enol tautomerization in the structure, singlet of hydroxyl group was found at 11.65 ppm.Methyl protons attached to the thiophene ring showed singlet at 2.5 ppm.
Compound 5 showed characteristics pyrimidine proton singlet at 8.0 ppm.and singlet of proton of thiophene ring at around 7 ppm.All aromatic protons came in the region of 6.9-7.6.The protons of piperazine and morpholine ring showed signals between 3-4.0 ppm.Secondary amine proton comes in the region of 6 -7 ppm.

Scheme 1.
Synthesized compounds showed significant decrease in locomotor activity was at dose 20 mg/kg (Figure 1, 2, 3) and less cataleptic behavior than standard compound at 10 mg/kg (Figure 4, 5, 6).The compounds were found to be safe after oral administration the dose of 20 mg/kg.No mortality was observed at this dose up to 24 h.There was decrease in alertness, and reactivity to touch stimuli.The animals did not show loss of righting reflex, and body position was normal and no stereotypy and lacrimation was observed.