Synthesis of some novel 2-mercapto-3-(substituted amino)- 5,6,7,8-tetrahydro-3 H -benzo[4,5]thieno[2,3-d ]pyrimidin-4-ones as analgesic and anti-inflammatory agents

A variety of novel 2-mercapto-3-(substituted amino)-5,6,7,8-tetrahydro-3 H -benzo[4,5] thieno[2,3-d ]pyrimidin-4-ones were synthesized by reacting 3-Amino-2-mercapto-5,6,7,8-tetrahydro-3 H -benzo[4,5]thieno[2,3-d ]pyrimidin-4-one with different aldehydes and ketones; the starting material 3-amino-2-mercapto-5,6,7,8-tetrahydro-3 H -benzo[4,5]thieno[2,3-d ]pyrimidin-4-one was synthesized from 2-amino-3-carbethoxy-4,5,6,7-tetrahydrobenzo thiophene by a novel innovative route. The title compounds were investigated for analgesic, anti-inflammatory and ulcerogenic index activities. While the test compounds exhibited significant activity, compounds AS1, AS2 and AS3 showed more potent analgesic activity. The compound AS3 showed more potent anti-inflammatory activity when compared to the reference standard diclofenac sodium. Interestingly the test compounds showed only mild ulcerogenic potential when compared to aspirin.

The title compounds 2-mercapto-3-(substituted amino)-5,6,7,8-tetrahydro-3H-benzo [4,5]  thieno [2,3-d]pyrimidin-4-ones AS1-AS10 were obtained by the condensation of amino group of 3-amino-2-mercapto-5,6,7,8-tetrahydro-3H-benzo [4,5]thieno [2,3-d]pyrimidin-4-one (3) with different aldehydes and ketones.The formation of title product is indicated by the disappearance of peak due to 3-NH 2 of the starting material in IR and NMR spectra of all the compounds AS1-AS10.The IR and NMR spectra of these compounds showed the presence of peaks due to (N=CR 1 R 2 ) carbonyl (C=O) and cyclohexyl ring.The molecular ion recorded in the mass spectra is in agreement with the molecular weight of the compounds.Elemental (C,H,N) analysis indicated that the calculated and observed values were within the acceptable limits (± 0.4%).Characterization data of the title compounds is represented in Table 1.

Molecular weight determination by mass spectral analysis
The biological screening results revealed that all the test compounds (AS1-AS10) showed significant analgesic activity (Table 2).Compound AS1 with N-3-aliphatic substituent showed good activity, when it was replaced by araliphatic group (compound AS2 and AS3) leads to retaining in activity.Placement of aryl group at N-3 (compounds AS4, AS5 and AS10) results in decreasing activity.Placement of electron withdrawing group at N-3 aryl ring (compounds AS6-AS9) results in further decrease in activity.Compound 3-sec-butylideneamino-2-mercapto-5,6,7,8-tetrahydro-3H-benzo [4,5]thieno [2,3-d]pyrimidin-4-one (AS1) emerged as the most active analgesic agent and it is more potent when compared to the reference standard diclofenac sodium.The anti-inflammatory activity data (Table 3) indicated that all the test compounds protected rats from carrageenan-induced inflammation.The compound 3-sec-butylideneamino-2mercapto-5,6,7,8-tetrahydro-3H-benzo [4,5]thieno [2,3-d]pyrimidin-4-one (AS1) showed more potent anti-inflammatory activity and compounds AS2 and AS3 showed equipotent antiinflammatory activity when compared to the reference standard diclofenac sodium.The ulcer index of the test compounds (Table 4) reveals that the compounds AS6-AS9 possessing electron-withdrawing groups exhibited higher ulcer index than the other test compounds.The high ulcer index score for these compounds may be due to the suppression of the prostaglandin synthesis.In our earlier studies [6][7][8][9] we observed that the presence of alkyl groups exhibited more analgesic and anti-inflammatory activities over aryl groups at the N-3 position.Hence in the the C-2 position also we made a substitution in such a way to increase lipophilicity of the molecule.The placement of such a group enhanced the analgesic and anti-inflammatory activities.To compare the increase in activity we have taken the average of all the readings of reaction time noted for each compound for each pharmacological activity.The most active compound of the C-2 phenyl series showed 43% analgesic and 36% anti-inflammatory activity (Fig 1, I) 6 , whereas the C-2 methyl series lead molecule showed 50% analgesic and 44% anti-inflammatory activity (Fig 1, II) 7 .Introduction of sulfur atom at C-2 position in the above series i.e. by placing methyl thio group at C-2 position showed 54% analgesic and 43% anti-inflammatory activity (Fig 1, III) 9 .The results of the analgesic and anti-inflammatory activities of the present series showed that enhancement of activity (56% analgesic and 46% anti-inflammatory activity).Interestingly these compounds showed negligible ulcer index unlike other Non Steroidal Anti-inflammatory Drugs (NSAID's), Hence this series could be developed as a novel class of analgesic and antiinflammatory agents.However further structural modification is planned to increase the analgesic and anti-inflammatory activities with the decreased ulcerogenic index.

Experimental Section
General Procedures.Melting points were determined in open capillary tubes on a Thomas Hoover apparatus and are uncorrected.IR spectra were recorded in KBr on a Shimadzu FT-IR, 8300 spectrometer (cm -1 ), mass spectra on a MASPEC msw 9629 mass spectrometer at 70 eV and 1 H NMR spectra on Varian 300 MHz spectrometer, using tetramethylsilane as internal standard.Elemental analyses were performed on Carlo Erba 1108.The 2-amino-3-carbethoxy-4,5,6,7-tetrahydrobenzothiophene 1, was prepared as per the procedure described by Gewald and Schinke 10 .

Pharmacology
The synthesized compounds were evaluated for analgesic, anti-inflammatory and ulcerogenic activities.Student t-test was performed to ascertain the significance of all the exhibited activities.
The test compounds and the standard drugs were administered in the form of a suspension (1% carboxyl methyl cellulose as vehicle) by the same route of administration.Each group consisted of six animals.The Institutional Animal Ethics Committee approved the protocol of the animal studies.
Animals.The animals were procured from the Tetrex Biological Center, Madurai, India, and were maintained in colony cages at 25 ±2°C, relative humidity of 45-55%, under a 12h light and dark cycle; they were fed standard animal feed.All the animals were acclimatized for a week before use.Analgesic activity.Test for analgesic activity was performed by tail-flick technique using Wistar albino mice (25 -35 gm) of either sex selected by random sampling technique. 11,12iclofenac sodium at a dose level of 10 mg/kg and 20 mg/kg was administered orally as reference drug for comparison.The test compounds at two dose levels (10, 20 mg/kg) were administered orally.The reaction time was recorded at 30min, 1, 2 and 3h after the treatment, and cut-off time was 10 sec.The percent analgesic activity (PAA) was calculated by the following formula, where T 1 is the reaction time (s) before treatment, and T 2 is the reaction time (s) after treatment.Anti-inflammatory activity.Anti-inflammatory activity was evaluated by carrageenan-induced paw oedema test in rats. 13Diclofenac sodium 10, 20 mg/kg was administered as a standard drug for comparison.The test compounds were administered at two dose levels (10 mg, 20 mg/kg).The paw volumes were measured using the mercury displacement technique with the help of a plethysmograph immediately before and 30 min, 1, 2 and 3h after carrageenan injection.The percent inhibition of paw oedema was calculated using the following formula Percent inhibition I = 100[1-(a-x)/ (b-y)] where x is the mean paw volume of rats before the administration of carrageenan and test compounds or reference compound (test group), a is the mean paw volume of rats after the administration of carrageenan in the test group (drug treated), b is the mean paw volume of rats after the administration of carrageenan in the control group, y is the mean paw volume of rats before the administration of carrageenan in the control group.Evaluation of ulcerogenicity index.Ulceration in rats was induced as described by Goyal et al. 14 Albino rats of wistar strain weighing 150-200 g of either sex were divided into various groups each of six animals.Control group of animals were administered only with 10% v/v Tween 80 suspension intraperitonially.One group was administered with Aspirin intraperitoneally at a dose of 20 mg/kg once daily for three days.The remaining group of