INTRODUCTION:

Pain is the local swelling compresses sensory nerve ending. The tissue injury causes the release of chemical mediators which activates nociceptor. The prostaglandins, leucotriens and thromboxanes are release on the site of injury as chemical mediators. Cyclooxygenase is the key enzyme which catalyses the conversion of arachidonic acid to prostaglandins and thromboxanes. There are two types of cyclooxygenase enzymes, COX-1 and COX-2. COX-1 is constitutive enzyme and COX-2 is inducible which produce inflammation.¹ Non steroidal anti-inflammatory drugs (NSAIDs) widely used in the treatment of pain and inflammation. These compounds non-selectively inhibit the two isoform of cyclooxygenase enzymes and thus prevent the metabolism of arachidonic acid and upgration of prostaglandin production.²

Aspirin is used as a standard drug because it inhibits the COX enzymes irreversibly by the process of acetylation.³

NSAIDs inhibit COX enzymes reduces the basal production of cytoprotective PGE₂ and PGI₂causes ulceration but does not show any inhibitory action on 5-lipoxygenase causes tissue damage. The benzothiazole suppressed the production of leukotriens and thramboxane which mediate inflammation but does not positional suppress the production of PGE₂ which protect tissue mucosa.¹

The 2-aminobenzothiazole derivatives shows various biological activity that includes anti-alzheimers activity ⁴, antitumor activity,⁵ anticonvulsant activity,⁶ antimicrobial activity,⁷anti-HIV-1,⁸

On the basis of above observation the present research work was carried out to synthesize some 2-amino benzothiazole derivatives and evaluated for analgesic activity with the hope to potentiate the biological activity with lesser or limited amount of toxicity.

MATERIAL AND METHODS:

The melting points of compounds were determined by open capillary tubes on a Veego melting point apparatus and are uncorrected. The purity of the compounds was routinely checked by thin layer chromatography (TLC) using precoated silica gel-G (Merck) with solvent system toluene: ethyl acetate: formic acid (5:4:1). Iodine chamber was used for the visualization of TLC spot. The spectroscopic data were taken by the following instruments: UV spectra were taken on the Shimadzu 1700s at VNS Institute of Pharmacy, Bhopal. IR spectra were taken on the VEEGO 8400 S with KBr pellets B. R. Nahata College of pharmacy, mandsaur. ¹H-NMR spectra were taken on a JEOL GSX 270 MHz NMR spectrophotometer (chemical shift in δ ppm) using solution CDCl₃ (TMS as internal reference) from IIT Mumbai.

EXPERIMENTAL:

Synthesis of benzothiazol-2-ylamine (1a):

To aniline (25ml), concentrated hydrochloric acid (25ml) was added and the solution was warmed. A saturated solution of ammonium thiocyanate in water (50%) was

Figure 1: Synthetic scheme

added slowly in above solution, the mixture was boiled until the solution got turbid. The turbid solution was poured in cold water. The separated precipitated of phenyl thiourea was filtered and crystallized from aqueous ethanol (80%) so as to obtain pure compound.

The phenyl thiourea (15g, 0.098 mol) in chloroform (75ml) was brominated by using bromine solution in chloroform (5%) till the orange-yellow colour appeared. The slurry was kept overnight. The precipitate obtained was filtered and wash with chloroform until the colour disappeared. The precipitate, as hydro bromide, was dissolved in rectified spirit (100ml) and basified with liquid ammonia solution. The ppt was filtered, washed with water, dried and recrystallized using aqueous ethanol (80%). ⁹ (yield= 80%, m.p 156-157 ^oC).

Synthesis of substituted benzoylisothiocyanates (2a-d):

Substituted benzoic acid (0.1mol) and thionyl chloride (0.1mol) were refluxed in benzene (50ml) for one hour. The reaction mixture upon filtration yielded substituted benzoyl chloride as a plumy liquid. Ammonium thiocyanate (0.1mol) was added to substituted benzoyl chloride (0.1mol) and refluxed for 30 min in benzene. The resultant mixture was filtered and substituted benzoylisothiocyanates were obtained in the form of liquid and ammonium chloride as a residue. Ammonium chloride was removed by filtration as a residue from the mixture.

Figure 2: Effect of different synthesized compounds in analgesic activity

Synthesis of 1-Benzothiazol-2yl-3-substituted-benzoyl-thiourea (PG-1 to PG-4):

The final compound were obtained when benzothiazol-2-ylamine (0.02 mol) and substituted benzoylisothiocyanates (0.02mol) were refluxed in benzene for 5 hours, solid material obtained was filtered and recrystallized from benzene.¹⁰ The synthetic scheme is given in figure 1.

Synthesis of 1-Benzothiazol-2yl-3-benzoyl-thiourea (PG -1)

Benzoic acid was used as substituted benzoic acid in procedure described in synthesis of 2a-d.

PG-1. R₁=H, R₂=H, Yield 32.06%, m.p.144°C, R_f0.90, λmax 250.5 nm, FTIR: (KBr) ν_max(cm^-1) 3308.08 (NH), 1666.30 (C=O), 1614(C=N), 1002.03 (C=S), 821.62 (Ar-H),¹H-NMR (δ ppm), (CDCl₃), 7.26-7.92 (m, 8H, Ar-H), 9.10 (s, 1H, NHC=O), 12.59 (s, 1H, NHC=S).

Synthesis of 1-(4-amino-benzoyl)-3-benzothiazol-2-yl-thiourea (PG-2)

4-amino benzoic acid was used as substituted benzoic acid in procedure described in synthesis of 2a-d.

PG-2. R₁=NH₂, R₂=H, Yield 38.00%, m.p.138°C, R_f 0.51, λmax 258.5nm, FTIR: (KBr) ν_max(cm^-1) 3425.61 (NH), 1633.31 (C=O), 1226.64 (C=N), 1006.02 (C=S), 748.33 (Ar-CH),¹H-NMR (δ ppm), (CDCl₃): 7.22-7.88 (m, 7H, Ar-H), 7.88 (s, 1H, NHC=O), 8.17 (s, 1H, NHC=S).

Synthesis of 1-Benzothiazol-2-yl-3-(4-nitro-nenzoyl)-thiourea (PG -3)

4-nitro benzoic acid was used as substituted benzoic acid in procedure described in synthesis of 2a-d.

PG-3. R₁-NO₂, R₂=H, Yield 70.00%, m.p.154°C, R_f 0.58, λmax 266.0 nm, FTIR: (KBr) ν_max(cm^-1) 3419.11 (NH), 1652.88 (C=O), 1614.20 (C=N), 1064.63 (C=S), 746.40 (Ar-CH)¹H-NMR (δ ppm), (CDCl₃): 7.23-7.47 (m, 7H, Ar-H) 7.90 (s, 1H, NHC=O), 8.09 (s, 1H, NHC=S).

Synthesis of 1-Benzothiazol-2-yl-3-(2-hydroxy-benzoyl)-thiourea (PG -4)

2-hydroxy benzoic acid was used as substituted benzoic acid in procedure described in synthesis of 2a-d.

PG-4. R₁-H, R₂=OH, Yield 75.00%, m.p.148°C, R_f 0.63, λmax 268.5 nm, FTIR: (KBr) ν_max(cm^-1) 3263.33 (NH), 1758.96 (C=O), 1604.20 (C=N), 1060.78 (C=S), 850.55 (Ar-CH)¹H-NMR (δ ppm), (Acetone): 7.12-7.79 (m, 7H, Ar-H), 9.85 (s, 1H, NHC=O), 12.10 (s, 1H, NHC=S).

Table 1: The reaction time of synthesized compounds at different latency time interval in hot plate method

Group

Treatment

Dose

Reaction time (sec.)

0 min

30 min

60 min

90 min

120 min

Control

Standard

PG-1

PG-2

PG-3

PG-4

100 mg/kg

30 mg/kg

4.83 ± 0.40

5.50 ± 0.42

5.00 ± 0.36

5.16 ± 0.40

4.66 ± 0.33

6.16 ± 0.30

5.00±0.44

5.00±0.36

5.50±0.50

4.83±0.30

5.33±0.55

5.83±0.47

5.33±0.47

7.33±0.49

6.00±0.44

7.16±0.30*

5.55±0.42

7.02±0.25*

5.16±0.40

8.66±0.42

6.66±0.33

8.16±0.30**

6.00±0.25

7.83±0.30**

5.16±0.40

9.00±0.51

6.33±0.42

8.33±0.33**

6.33±0.21

8.33±0.33**

The results was expressed as the mean ± SEM of N=6 animals per group. (*) P<0.05, (**) P<0.01 compared with control group

Animals:

The experiment was performed on experimental animals consisting Swiss albino rats (100-150g) of either sex were chosen for analgesic activity. They were kept in cages in an animal house with a 12 h light-dark cycle. They were fed on pellets and tap water ad libitum. The test was carried out in a quiet laboratory setting with ambient illumination and temperature close to those of the animal house. Rats were allowed to acclimatize to the laboratory for 1 h before the experiments begin. Animals study was performed in department of pharmacology, VNS institute of Pharmacy, Bhopal. The Institutional Animal Ethics Committee approved the experimental protocol (Registration No. 778/03/c/CPCSEA).

Analgesic activity:

Thirty six rats divided in to six groups of six each. Group I- Control, receiving only 0.5% CMC suspension. Group II- Standard, receiving aspirin (dose: 100mg/kg body weight)¹¹ in 0.5% CMC suspension and all test compounds PG-1 to PG-4 receiving (dose: 30mg/kg body weight) in 0.5% CMC suspension. The compounds were administered orally. After that rat was placed on a hot plate and paw licking or

jumping response was observed on eddy’s hot plate apparatus. The percent increase in reaction time at time interval of 0, 30, 60, 90 and 120 min was calculated.¹²

Statistical analysis:

The responses measured were expressed as mean ± S.E.M. Statistical significance for analgesic activity was calculated using the Dunnet t-test and P < 0.05 was considered significant.

RESULTS AND DISCUSSION:

The analgesic effect of synthesized benzothiazole derivatives PG-1 to PG-4 was investigated on hot plate apparatus. The results of hot plate method (figure 2) showed that the test compounds significantly increased the latency to jumping response without affecting the animal’s ability to detect pain threshold (licking response) of thermal origin.

The compound PG-2 and PG-4 exhibited more analgesic activity as shown in table 2. Compared with control group, the potency of the tested compounds was found to be as follows: PG2>PG4>PG3>PG-1 at 120 min. According to the structure–activity relationship (SAR) it can be concluded that synthesized compound PG-2 and PG-4 show significant analgesic activity after 30 min later from administration and it may be due to presence of electron releasing group -NH₂ and -OH on para and ortho position of heterocyclic ring respectively. The lowest activity of PG-1 and PG-3 was assumed due to electron withdrawing group at para position of heterocyclic ring.

ACKNOWLEDGEMENT:

The authors are thankful to VNS Institute of Pharmacy, Bhopal for providing the laboratory facilities and analgesic activity.

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Received on 10.09.2009 Modified on 09.11.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 47-50