To an oven-dried, 100 mL round bottomed flask, fitted with magnetic stir-bar, 0.425 g (1.18 mmol) of Fenofibrate (1) was added and then dissolved in 16 mL of methanol. When partially dissolved, an ice water bath was placed under the reaction flask and 0.093 g (2.46 mmol) of NaBH₄ was added slowly in small portions. The ice water bath was removed and the solution was allowed to stir at room temperature (24˚C) for 3 hours. The reaction was monitored by TLC (7:3 ethyl acetate/hexane) and it resulted in two spots with corresponding Rf values of 0.73 and 0.77. The reaction mixture was placed in an ice water bath and 15 mL of chilled 5% HCl was added very slowly. The crude reaction mixture was poured onto 10 mL water, separated, extracted with ethyl acetate (2 × 15 mL) and dried over MgSO₄. Using a Whatman #1 filter pad, the reaction mixture was filtered and the solvent was removed in vacuo resulting in a clear oil with 66.7% yield. Fibratol (2a/2b) has the following spectroscopic properties: ¹H NMR: δ 1.21 (d, 6H, ³J= 5.76 Hz), 1.57 (s, 6H), 2.16 (s, 2H), 5.07 (septet, 1H, ³J = 6.31 Hz), 5.76 (s, 2H), 6.79 (pd (FF’ part of FF’HH’ pattern) ³J = 9.12 Hz, 2H), 7.19 (pd (GG’ part of GG’II’ pattern) ³J = 8.84 Hz, 2H), 7.29 (pd (HH’ part of HH’FF’ pattern and (pd (II’ part of II’GG’ pattern) ³J = 8.79 Hz, 4H); ¹³C NMR: δ 21.53, 25.39, 69.35, 79.47, 117.31, 128.55, 130.28, 131.16, 131.94, 136.47, 138.37, 159.77, 173.10, 194.25; ESI+ m/z (calculated by ACDLab2014): 362.8; m/z (experimental/found): 361.2 + 23 (385.2); IR spectrum (cm⁻¹): 3466, 2985, 2939, 2876, 1904, 1728, 1606, 1505, 1465, 1381, 1096, 1012, 972, 827, 718, 684, 618.

High Performance liquid chromatography (HPLC) analyses were performed on a Waters 1525 Binary HPLC pump coupled to a Waters 2487 dual λ absorbance detector using Breeze software. All solvents were degassed for 1 hr under helium. A Lux Amylose-2 chiral column (5 µm, 4.6 × 250 mm) was selected for the separation of chiral compounds that had a unique selector amylose tris(5-chloro- 2-methylphenylcarbamate). Reduced racemic fenofibrate was separated by chiral separations techniques. A 5 mg of reaction product was dissolved in 1.5 mL of HPLC grade methanol. A 20 µL of this solution was injected by a 250 µL syringe at a flow rate of 1 mL/min and analyzed using HPLC by monitoring the flow through at 275 nm.

Specific rotation [α], reflects the magnitude of the rotation of each enantiomer. In addition the optical rotation is dependent on the wavelength of light used, temperature, solvent, concentration and the length of the polarimeter tube/cell. When monochromatic light from a sodium lamp D-line at 589 nm is used as the source, α can be defined by (Equation (1)) as

[ α ] D 25 = { observedrotation ( degrees ) / lengthofsampletube ( d m ) × concentration ( g m l ) } (1)

where T (=25˚C) is the measurement temperature, λ is the wavelength of light employed, α is the observed rotation, l is the path length and c is the concentration in grams per mL (the density of the pure substances) or grams per 100 mL. Specific rotation may also be expressed as degrees per mole of the substance where the conditions of measurement (i.e. solvent used, light source and path length) are also specified [9] .

The reduction (Scheme 1) of Fenofibrate (1), to Fibratol (2a/2b) was monitored via TLC (7:3 ethyl acetate/hexane v:v) until complete conversion of (1) to the final product (2a/2b) occur. Completion of the reaction after 3 hours is supported by 1) the shift of the -OH signal at 5.76 ppm in ¹H NMR; 2) presence of an experimental m/z peak at 361.2, corresponding to the calculated m/z at 362.8 in MS; 3) appearance of an absorbance band at 3466 cm⁻¹ in the IR spectra and 4) the disappearance of the signal at 194.0 ppm and appearance of 74.9 ppm peak in ¹³C NMR and further indicate the absence of the ketone carbonyl and the presence of OH group in the product.

HPLC technique was selected for the enantiomeric separation of Fibratol (2a/b). The mobile phase 1 consisted of 500 mL of acetonitrile and 0.5 mL of diethylamine and mobile phase 2 comprised of 500 mL of isopropanol and 0.5 mL of diethylamine. For 20 µL sample size, the run time of 20 minutes at 275 nm with constant flow rate at 1 mL/min was used. Corresponding retention times for separation of each enantiomer are shown in Table 1 and Figure 1.

The difference in the retention time between peaks 2a and 2b increases from trial 1 to 4. Moreover trial 4 gave better resolved peaks for 2a and 2b compared to trial 1. Although there are minor differences in their retention times when varying the solvent systems, 60% of mobile phase 1 and 40% of mobile phase 2 yielded the best separation of the peaks.

Scheme 1. Formation of Fibratol (2a/b) from Fenofibrate (1).

The angle of rotation, α, of the plane of polarized light by an optically active forms of Fibratol (2a/2b) were measured by polarimetry technique. A 100 mg of Fibratol (2a) and Fibratol (2b) that were separated by chiral column were dissolved individually in 2 mL of methanol. The optical activity of Fibratol (2a) and Fibratol (2b) were −5.2˚ reflecting counterclockwise (levo) and 8.0˚ suggesting clockwise (dextro) rotation of plane polarized light, respectively. The trend demonstrated in resolving the chiral isomers of fibratol under otherwise identical condition (method, column, rate, pressure) reflects the amount of mobile phase 1 and 2 used in trials.