Identification, Synthesis, Isolation and Spectral Characterization of Multidrug-Resistant Tuberculosis (MDR-TB) Related Substances

Several related substances were detected at trace level in (2R)-2,3-dihydro-2methyl-6-nitro-2-[[4-[4-[4-(trifluoromethoxy)phenoxy]-1-piperidinyl] phenoxy] methyl]imidazo[2,1-b]oxazole drug substance by a newly developed high-performance liquid chromatography method. All related substances were characterized rapidly but some impurities were found to be intermediates. Proposed structures were further confirmed by characterization using NMR, FT-IR, and HRMS techniques. Based on the spectroscopic data; unknown related substances were characterized as 1-(Methylsulfonyl)-4-[4-(trifluoromethoxy) phenoxy]piperidine; 4-{4-[4-(Tri-fluoromethoxy)-phenoxy]piperidin-1-yl}phenol and 4-{4-[4-(trifluoromethoxy)phenoxy]piperidin-1-yl}phenyl methane sulfonate; 4-Bromophenyl methane sulfonate, Ethyl 3,6-dihydro-1(2H)-pyridine carboxylate, (2S)-3-(4-Bromophenoxy)-2-hydroxy-2-methylpropyl methane sulfonate, (2S)-3-(4-Bromophenoxy)-2-methylpropane-1,2-diyldimethane-sulfonate, (2S)-2-Methyl-3-(4-{4-[4-(trifluoromethoxy) phenoxy]-piperidin-1-yl} phenoxy)-propane-1,2-diyldimethane sulfonate, (S)-3-(4-Bromophenoxy)-2methyl-propane-1,2-diol and corresponding Enantiomer, (2R)-2-[(4-Bromophenoxy)methyl]-2-methyloxirane and (2R)-2-[(4-bromophenoxy)methyl]-2methyl-6-nitro-2,3-dihydroimidazo[2,1-b][1,3]oxazole. A possible mechanism for the formation of these related substances is also proposed.


Introduction
Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis. Among acid-fast bacteria, human Mycobacterium tuberculosis has been widely known. It is said that one-third of the human population is infected with this bacterium. Tuberculosis, an airborne lung infection, still remains as a major public health problem worldwide. It is estimated that about 32% of the world population is infected with Tuberculosis bacillus and of those, approximately 8.9 million people develop active Tuberculosis and 1.7 million die annually as a result, as per 2004 figures [1]. Human immunodeficiency virus (HIV) infection has been a major contributing factor in the current resurgence of Tuberculosis [2] [3]. In addition to the human Mycobacterium tuberculosis, Mycobacterium africanus and Mycobacterium bovis have also been known to belong to the Mycobacterium tuberculosis group. The recent emergence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis that are resistant to two major effective drugs, isonicotinic acid hydrazide (INH) [4] and rifampicin (RFP) [5], have further complicated the world situation [6].
Tuberculosis is positioned as the second leading cause of death from a single infectious agent worldwide, with an incidence rate of almost 9 million cases and a fatality rate of 1.4 million in 2011. The World Health Organization (WHO) has estimated that if the present conditions remain unchanged, more than 30 million lives will be claimed by TB, between 2000 and 2020 [7].
The current therapy consists of an intensive phase with four drugs, INH, RFP, pyrazinamide (PZA), 8 and streptomycin (SM) [8] or ethambutol (EB) [9], administered for 2 months followed by a continuous phase with INH and RFP for 4 months [10]. These bacteria known as Mycobacteria, have a strong pathogenicity to humans. Hepatotoxicity, which is induced by rifampicin, isoniazid, and ethambutol, out of the 5 agents used in combination of first-line treatment, is known as an adverse reaction that is developed most frequently. At the same time, Mycobacterium tuberculosis resistant to antitubercular agents, multi-drug-resistant Mycobacterium tuberculosis, and the likes have been increasing of Mycobacterium tuberculosis, making the treatment more difficult. Notwithstanding the successful implementation of directly observed treatment short course (DOTS) in many countries, high prevalence of multi-drug-resistant (MDR) and extensively drug resistant shorten the current duration of therapy [11].

(2R)-2,3-Dihydro-2-methyl-6-nitro-2-[[4-[4-[4-(trifluoromethoxy)phenoxy]-1-piperidinyl] phenoxy] methyl]imidazo[2,1-b]oxazole
The presence of its related compounds in a drug substance, can have a significant impact on the quality and safety of the drug product. Therefore, it is necessary to study the impurity profile of any active pharmaceutical ingredient (API) and control it during the manufacturing of a drug product. As per the general guidelines recommended by ICH [4] to qualify the drug substance, the amount of acceptable level for a known and unknown related compound should be less than 0.15% and 0.10%, respectively. To meet the stringent regulatory requirements, the impurities present in the drug substance must be identified and characterized thoroughly.

Ethyl 4-[4-(trifluoromethoxy)phenoxy]piperidine-1-carboxylate
Under inert atmosphere, to a mixture of N-Carbethoxy-4-Hydroxy piperidine (194 g; 1.120 mol.) and methane sulfonyl chloride (141 g, 1.231 mol.) in toluene (500 ml), Triethylamine (148 g, 1.462 mol.) was added at <20˚C and stirred at 20˚C ± 3˚C. After completion of reaction, reaction mass was quenched with water and organic layer was separated. 4-(Trifluoromethoxy)phenol (100 g, 0.561 mol.), aqueous Tetra butyl ammonium bromide solution (62 g in water 62 ml, 0.191 mol.) and aqueous sodium hydroxide solution (87.3 g in 350 ml of water) was added sequentially to the obtained organic layer and temperature was raised to 86˚C ± 3˚C slowly. After completion of reaction, reaction mass was cooled to 20˚C ± 3˚C, organic layer was separated and washed with water. To the obtained organic layer, ethanol (200 ml) and potassium hydroxide (283 g, 5.035 mol.) were added sequentially. Thereafter, reaction mass temperature was raised to 86˚C ± 3˚C slowly. After completion of reaction, reaction mass was concentrated and product was extracted/washed using toluene/water. Thus, obtained organic layer was concentrated and product was crystallized with isopropyl alcohol/water mixture (100 ml/1000ml) and aqueous sodium hydroxide solution (12.5 g in 50 ml water) to provide 140 g of 4-(4-Trifluoromethoxyphenoxy) piperidine. Synthesis Synthetic Scheme-4:
After completion of reaction, reaction mass was quenched with water and organic layer was separated. The organic layer was washed with aq. sodium bicarbonate solution. The organic layer was concentrated to afford 11 g of 4-Bromophenyl methane sulfonate.  Ethyl 3,6-dihydro-1(2H)-pyridine carboxylate
The reaction mixture was then stirred for 30 minutes and subjected to filtration.
The toluene layer was washed with water (500 ml), and then the toluene and cumyl alcohol were distilled off under reduced pressure. Toluene was added to the concentrated residue followed by heating to 70˚C to dissolve the concen- acetate (20 ml) and raise the temperature to 98˚C ± 3˚C and stirred the contents for 1 hr. After reaction completion, mass was cooled to 27˚C -30˚C and diluted with methanol (140 ml). Further, reaction temperature was lowered to 2˚C ± 3˚C and added aqueous sodium hydroxide solution slowly (15.57 g). Again, temperature was raised to 27˚C -30˚C and stirred. After reaction completion, reaction mass was diluted with water (30 ml) and with ethyl acetate (30 ml) and temperature raised to 50˚C ± 5˚C. After 1hr. of stirring contents were cooed to 23˚C ± 3˚C, stirred for 1 hr. and filtered and washed with methanol and water and dried the product under vacuum to yield 14.5 g of (2R)-2-[(4-bromophenoxy) methyl]-2-methyl-6-nitro-2,3-dihydroimidazo[2,1-b] [1,3]oxazole .

Conclusion
In summary, related substances plausible to be contemporary in (2R)-2,3-dihydro- and extensively characterized. Also, plausible origin for these related substances has been provided to have a better control for these and make the product free from these.