Isolation and Characterization of a Process Impurity in Tizanidine Hydrochloride

Indian Journal of Pharmaceutical Sciences 360 May June 2010 S, Gopalakrishan S, et al. Microwave assisted synthesis of 3,6-disubstituted-5,6-dihyro-s-triazolo (3,4-b) (1,3,4)-thiadiazoles and their biological activities. Indian J Heterocycl Chem 2008;17:263-64. 2. Kumar MP, Ravi TK, Gopalakrishnan S, Bhat AR, Chawla R, George S, et al. Synthesis of certain 5-substituted4-arylideneamino-3-mercapto-(4H)-1,2,4-triazoles and 1-substituted-4-arylidene-3-methyl-5-pyrazolones and their antimicrobial and antioxidant activities. Indian Drugs 2008;45:626-30. 3. Bishnoi A, Saxena R. Synthesis of hexahydro-1,3,5-tri (8-arylaminomethyl-7-hydroxy-4-methyl quinolinyl)-s-triazine as possible antiviral agents. Indian J Heterocycl Chem 2001;11:47-50. 4. Shastri AL, Ghate DM, Kulkarni VM. Dual fl uorescence and biological evaluation of paracetamol ethers from 4-bromomethylcoumarin. Indian J Chem 2004;43B:2416-22. 5. Ghate M, Manohar D, Kulkarni V, Shoba R. Synthesis of vanillin ethers from 4-(bromomethyl) coumarins as anti-infl ammatory agents. Eur J Med Chem 2003;38:297-302. 6. Gupta R, Sudan S, Kachroo PL. Reaction of 3-substituted-4-amino-smercapto-1,2,4-triazoles with substituted cinnamic acid. Indian J Chem 1996;35B:718-20. 7. Udupi RH, Setty SR, Srinivasulu N. Synthesis of condensed triazole system and their biological evaluation. Indian Drugs 2002;39:318-21. 8. Gupta R, Paul S, Gupta AK, Kachroo PL. Improved synthesis of some substituted 5,6-dihydro-s-triazolo (3,4-b) (1,3,4)-thiadiazoles in a microwave oven. Indian J Chem 1994; 33B:888-91. 9. Kumar MP, Ravi TK, Lakshmimadhuri M, Chawla R, Sonia G, Gopalakrishnan S. Microwave assisted synthesis of 3,5-dimethyl-4(arylazo)pyrazoles and isoxazoles over conventional method and their therapeutic activities. Indian Drugs 2009;46:384-90. 10. Bauer AN, Kirby WNM, Sherries JC, Truck M. Antibiotic susceptibility testing by a standardised single disc method. Amer J Clin Path 1996;45:493-96.

antioxidant studies, compounds 2d (IC 50 -5.7 μg/ ml) (4-dimethylaminophenyl derivative) and 2h (IC 50 -5.6μg/ml) (3,4-dimethoxyphenyl derivative) were identified to be more potent than rest of the compounds.From the results of biological screening we could conclude that 3,4-dimethoxyphenyl substituent and 4-N,N-dimethylphenyl substituent at position 6 of triazolothiadiazole are vital in improving the scavenging capacity of free radicals as evident from the antioxidant activity of compounds 2h and 2d, respectively which was comparable to that of standard ascorbic acid.Similarly the results of antimicrobial screening showed that substitution of 3-nitrophenyl substituent at position 6 of triazolothiadiazole confers both antibacterial and antifungal activity as seen with compound 2b.The compounds 2d and 2h can be chosen as lead moieties for antimicrobial and antioxidant studies and compounds 2b, 2j and 2f for antimicrobial studies.

Sample of tizanidine hydrochloride (I) was synthesized and characterized in Mylan India
Pvt. Ltd (Formerly Merck Development Centre Pvt.Ltd.), India.Sodium dihydrogen phosphate, methanol (HPLC grade) and phosphoric acid were procured from Merck India Ltd., Mumbai, India.The chromatographic purifi cation was performed on a Nova Prep 200 (Merck Hitachi) preparative HPLC system consisting of L-7400 UV detector and HSM software and a built-in autosampler for fraction collection.The purity of the fractions was checked on a Merck Hitachi HPLC system consisting of L-7100 pump, L-7300 Column oven, L-7200 Autosampler, L-7420 detector and HSM data acquisition software.The mass spectra were obtained on a Applied Biosystems API 4000 triple quadrupole spectrometer using electrospray ionization in positive mode.HR-MS spectrum was obtained on a Micromass Q-TOF micro spectrometer using electrospray ionization in positive mode.NMR spectra were recorded on a Bruker AV 300 spectrometer.
The mobile phase A was a mixture of 0.02 M aqueous sodium dihydrogen phosphate, pH 3.0 with dilute orthophosphoric acid-methanol (90:10 v/v), while mobile phase B was 0.02 M aqueous sodium dihydrogen phosphate, pH 3.0 with dilute orthophosphoric acid-methanol (20:80 v/v).The gradient program used was min/%B: 10/0, 30/50, 31/0 and 40/0.The preparative HPLC column used was Waters C18 Symmetry (19×150 mm), 7 .The monitoring wavelength was 225 nm and the flow rate was 24 ml/min.A stock solution of 3% w/v tizanidine hydrochloride (I) was prepared in water for the isolation of process impurity (II) and 4 ml was injected per run.The fraction containing the enriched impurity (II) was re-chromatographed using a C18 Symmetry (7.8×150 mm), 7 . at a fl ow rate of 4.0 ml/min with an injection volume of 50 l.Analytical HPLC was performed using the same conditions except that the column used was Symmetry C18 (150×3.9mm), 5  with a fl ow rate of 1 ml/min for checking the purity.
The preparative HPLC fraction containing impurity was evaporated to dryness on a rotavapor at 30 o under vacuum.The residue was suspended in minimum quantity of dry methanol, sonicated for 2 min and kept overnight in refrigerator.The suspension was filtered immediately under vacuum to remove the undissolved phosphates.The fi ltrate was concentrated on a rotavapor at 30 o under vacuum to dryness to get impurity (II) as a yellow powder.
The unknown process impurity (II) was obtained as a yellow powder having a purity of 94% by HPLC.The ESI-MS gave a molecular ion peak at m/z 280.3 (M+H) + .A comparison of NMR spectra (Table 1) of process impurity (II) with those of tizanidine hydrochloride (I) indicated the presence of additional signals [ H : 3.18 (q, 2H) and 1.28 (t, J=7.3 Hz, 3H);  C : 25.9 (CH 2 ) and 14.6 (CH 3 )] in II, which were attributed to an ethyl group.The relatively downfi eld shift of methylene was due to its attachment to a hetero atom.The presence of only two aromatic methines [ H : 8.09 (d, J=9.3 Hz, 1H) and 7.86 (d, J=9.3 Hz, 1H);  C : 129.3 and 121.3] together with the absence of isotopic pattern for chlorine in the mass spectrum suggested that S-ethyl group was present in place of chlorine.During the conversion of tizanidine to the corresponding HCl salt, II was also expected to form a HCl salt, which was confirmed by silver nitrate test.Thus, the structure of impurity (II) was established as 5-S-ethyl-N-(4,5-dihydro-1H-imidazol-2-yl)-2,1,3-benzothiadiazol-4-amine hydrochloride (fi g. 1).The structure was further confi rmed by comparison with synthetic sample prepared subsequently.The possible route for the formation of II was due to the displacement of chlorine atom by ethanethiol liberated during the conversion of precursor viz.S-ethyl-N-(5chloro-2,1,3-benzothiadiazole-4-yl) isothiouronium bromide (III) to tizanidine hydrochloride (I) using ethylenediamine/p-toluenesulfonic acid in toluene/ water [7] .