Design and Enantiopure Synthesis of ( R )-2-( ( 2-Oxooxazolidin-5-yl ) methyl ) isoindoline-1 , 3-dione : A Key Precursor to Build 2-Oxazolidinone Class of Antibacterial Agents

A new synthetic method for the preparation of high enantiopure (R)-2-((2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione has been developed. The enantiopurity of the obtained (R)-2-((2-oxooxazolidin-5-yl)methyl) isoindoline-1,3-dione is established using chiral high performance liquid chromatography (HPLC) i.e. enantiomeric excess (ee) as 100%. One among the two proposed approaches, is succeeded in preparing enantiopure targeted chiral building block using (R)-2-(chloromethyl)oxirane ((R)-epichlorohydrin) as precursor. This heterocyclic 2-oxazolidinone moiety could be useful to prepare a series of antibacterial agents containing 2-oxazolidinone.


Introduction
The antimicrobial properties of 2-oxazolidinone were discovered by researchers at E.I. duPont de Nemours in the 1970s 1 .In 1978, duPont patented a series of oxazolidinone derivatives as being effective in the treatment of bacterial and fungal plant diseases and in 1984, another patent described their usefulness in treating bacterial infections in mammals 1,2 .
In 1987, duPont scientists presented a detailed description of the oxazolidinones as a new class of antibiotics with a novel mechanism of action 1,3 .Pharmacia & Upjohn (now part of Pfizer) started its own oxazolidinone research program in the 1990s to study structure-activity relationships (SAR) of substituted 2-oxazolidinones led to the development of several subclasses of oxazolidinone derivatives, with varying safety profiles and antimicrobial activity.Two compounds (Figure 1) were considered as drug candidates: eperezolid (PNU-100592) and linezolid (PNU-100766) 4,5 .In preclinical stages of development, they were similar in safety and antibacterial activity, so they were taken to phase I clinical trials to identify any difference in pharmacokinetics 6,7 .Linezolid was found to have a pharmacokinetic advantage requiring only twice-daily dosage, while eperezolid needed to be given thrice a day to achieve similar exposure and therefore only linezolid was preceded to further trials 4 .The U.S. Food and drug administration (FDA) approved linezolid on April 18, 2000 and as of 2009, linezolid is the only oxazolidinone antibiotic available in the market 8 .

Experimental
All reagents and solvents employed were of commercial grade and were used as such, unless otherwise specified.Reaction flasks were oven-dried at 200 °C, flame-dried and flushed with dry nitrogen prior to use.All moisture and air-sensitive reactions were carried out under an atmosphere of dry nitrogen.TLC was performed on Kieselgel 60 F254 silicacoated aluminium plates (Merck) and visualized by UV light (λ = 254 nm) or by spraying with a solution of KMnO 4 .Organic extracts were dried over anhydrous Na 2 SO 4 .Flash chromatography was performed using Kieselgel 60 brand silica gel (230-400 mesh).The melting points were determined in an open capillary tube using a Büchi B-540 melting point instrument and were uncorrected.The IR spectra were obtained on a Nicolet 380 FT-IR instrument (neat for liquids and as KBr pellets for solids).NMR spectra were recorded with a varian 300 MHz mercury plus spectrometer at 300 MHz ( 1 H) and at 75 MHz ( 13 C).Chemical shifts were given in ppm relative to trimethylsilane (TMS).Mass spectra were recorded on waters quattro premier XE triple quadrupole spectrometer using either electron spray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) technique.

Preparation of (R)-5-(chloromethyl)oxazolidin-2-one (5)
To a stirred solution of (R)-2-(Chloromethyl)oxirane 4 (5.0 g, 0.054 mol) in water (50 mL), potassium cyanate (8.76 g, 0.108 mol) and magnesium sulfate (13.0 g, 0.108 mol) were added at ambient temperature.The temperature of the reaction mixture was raised to 100 °C and maintained at the same temperature for 5 h.The reaction mixture was filtered to remove solids and the resulted filtrate was extracted with ethyl acetate (2×25 mL).The combined organic layer was washed with saturated sodium chloride solution (25 mL), dried over anhydrous sodium sulfate and the solvent was removed by evaporation under reduced pressure.The obtained solid was triturated with n-hexane and filtered to give (R)-5-(chloromethyl) oxazolidin-2-one (5) as a white solid (75% yield); mp 67-68 °C.

Results and Discussion
Herein we wish to report a new and efficient synthesis of (R)-2-((2-oxooxazolidin-5-yl)methyl) isoindoline-1,3-dione (1) using commercially available (R)-2-(chloromethyl)oxirane (4) (epichlorohydrin).The key challenge of the synthesis is achieving high enantiopurity of the targeted compound.Initially, we designed a two step synthesis to accomplish our goal.The first step involves the straight away transformation of the starting material, (R)-2-(chloromethyl) oxirane into (R)-5-(chloromethyl)oxazolidin-2-one (5) by following the reported procedure with minor modification which includes introduction of MgSO 4 in the reaction condition.The obtained (R)-5-(chloromethyl)oxazolidin-2-one was converted to the required compound 1 by reaction with potassium phthalimide in DMF under PTC condition (Scheme 1).
Figure 1.Structures of linezolid and eperezolidScientists utilize preferably convergent or linear approach to develop the synthesis of final drug molecules.In linear synthesis, the oxazolidinone skeleton will be constructed in the way of its preparation where as in convergent synthesis initially the oxazolidinone basic moiety will be prepared using readily available chemicals.These basic precursors further connected with other corresponding structural component(s) to build final drug molecules.As in the case of linezolid and also in other oxazolidinone class of antibacterial agents, it is evident that small chiral building blocks are used as key synthetic precursors[9][10][11][12] e.g.(R)-2-((2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione (1), (R)-5-azidomethyl-2-oxazolidinone (2), (S)-glyceraldehyde acetonide (3) etc.( Figure 2).

A
Scheme 1

Figure 3 .
Figure 3. Chiral HPLC chromatograms of racemic and (R)-2-((2-oxooxazolidin-5-yl) methyl)isoindoline-1,3-dione (chiral purity: 90.4%)Even though, the described synthetic approach (Scheme 1) is high yielding but unfortunately the prepared (R)-compound, 1 shows the low enantiopurity, 90.4% and we have not further carried out any research on this approach to determine how and where the racemization might happen.Immediately, we switched our attention on alternative routes.The (R)-2-(chloromethyl) oxirane is regarded as starting material even in the alternative route.(R)-Epichlorohydrin, 4 was stereo selectively ring-opened with NaN 3 in AcOH, whilst maintaining acidic condition,