*2.6. LC-MS*/*MS*

In order to quantify HSG4112(S) and HSG4112(R), the Acquity UPLC-MS/MS system (Waters, Milford, MA, USA) was used with an electrospray ionization source. Mass detection was performed in the negative ion mode, and the column temperature was maintained at 40 ◦C using a thermostatically controlled column oven. The column used for the separation was a CHIRALPAK® IC-U (1.6 µm, 3.0 × 100 mm; DAICEL, New York, NY, USA). The mobile phases consisted of D.W (solvent A) and ACN (solvent B). For quantification of the analytes, isocratic elution was performed at a flow rate of 0.5 mL/min. Solvent B was maintained at 60%. For multiple reaction monitoring (MRM) analyses, the target ions used were *m*/*z* 353.3→137.1 for HSG4112(S) and HSG4112(R) and *m*/*z* 358.3→142.0 for HSG4112-d5. The capillary voltage was 3 kV and cone voltage was 50 V. Collision energy was 23 V. Nitrogen was used as the desolvation gas at a flow rate of 650 L/h and at 450 ◦C. The representative chromatograms of HSG4112(S) and HSG4112(R) in rat and dog plasma are provided in Supplementary data (Figure S1). The validation for the quantitation method was conducted and the resulting data were satisfactory (Supplementary data). For metabolite profiling, an ACQUITY UPLC BEH C18 column (2.1 × 150 mm, 1.7 µm; Waters, Milford, MA, USA) was used and a gradient elution program was used

as follows: 30%B to 75% B at 10 min, to 30% B at 10.1 min, and held at 30% B for 3 min. The metabolite levels were measured using selected ion monitoring based on the *m*/*z* values of the deprotonated ions of metabolites. Other mass spectrometer conditions were the same as above.

#### *2.7. LC-QTOF*/*MS for Metabolite Analysis*

The high performance liquid chromatography quadruple time of the flight mass spectrometer (LC-QTOF/MS) system consisted of an Agilent 1260 series binary pump HPLC system and an Agilent 6530 Q-TOF/MS/MS equipped with an electrospray ionization source (Agilent Technologies, Palo Alto, CA, USA). The column used for the separation was a Thermo Hypersil Gold column (2.1 × 150 mm, 3 µm; Thermo Fisher Scientific Inc., Waltham, MA, USA). Column temperature was maintained at 40 ◦C using a thermostatically controlled column oven. The HPLC mobile phases consisted of 0.1% formic acid in distilled water (A) and 90% acetonitrile in 0.1% formic acid (B). A gradient program was used for the HPLC separation with a flow rate of 0.2 mL/min. The initial composition of the mobile phase was 30% B and it was changed to 90% B over 13 min and followed by a 7 min re-equilibration to the initial condition. The entire column eluent was directly introduced into the mass spectrometer. Nitrogen was used both as the nebulizing gas at 20 psi and as the drying gas at a flow rate of 10 L/min at 300 ◦C. The mass spectrometer was operated in the negative ion mode in *m*/*z* 50–400.

#### *2.8. Pharmacokinetic and Data Analysis*

All data were expressed as mean ± SD. Pharmacokinetic parameters were calculated by non-compartmental analysis using Pheonix WinNonlin (Ver. 6.2, Pharsight-A Certara Company, USA). The area under the concentration–time curve from time zero to the last measurable concentration (AUClast), area under the plasma concentration–time curve to the infinite time (AUCinf), maximum plasma concentration (*C*max), time to reach *C*max (*T*max), terminal elimination half-life (*t*1/2), total body clearance (*C*<sup>l</sup> ), apparent volume of distribution (*V*z), apparent volume of distribution at steady state (*V*ss), and equation for the mean residence time (MRT) were estimated by non-compartmental analysis of the plasma concentration versus time. The significance of the pharmacokinetic parameters was assessed using the paired Student's *t*-test. The analysis was performed on log-transformed data. When *p* value was less than 0.05, it was judged to be significant.

#### **3. Results**

#### *3.1. Analysis of HSG4112(S) and HSG4112(R) in Rat Plasma*

The time–plasma concentration plots of HSG4112(S) and HSG4112(R) are presented in Figure 2A, and the pharmacokinetic parameters are described in Table 1. The plasma concentration levels of HSG4112(S) were significantly higher than those of HSG4112(R) throughout all time points. The *C*max of HSG4112(S) and HSG4112(R) were 2904.9 ng·h/mL and 984.6 ng/mL, respectively. The area under the curve (AUC) of HSG4112(S) and HSG4112(R) were 45,733.3 and 12,190.6 ng·h/mL, respectively. The pharmacokinetics of HSG4112(S) and HSG4112(R) after IV administration of HSG4112 were also investigated. The plasma concentration profiles of HSG4112(S) and HSG4112(R) after IV injection of HSG4112 at a dose of 10 mg/kg are presented in Figure 2B, and the pharmacokinetic parameters are described in Table 2. The stereoselective differences in plasma concentration were also observed in the IV administration. The AUC of HSG4112(S) and HSG4112(R) were 3806.7 ± 894.6 ng·h/mL and 1390.4 ± 92.7 ng·h/mL, respectively. Validation Data are presented in Tables S2–S5.

**Figure 2.** Plot for time-plasma concentration of HSG4112 after (**A**) oral (100 mg/kg/day, 28th day, *n* = 3) and (**B**) iv (10 mg/kg, single dose, *n* = 3) administration in rats.

**Table 1.** Pharmacokinetic parameters of HSG4112(S) and HSG4112(R) after PO administration at a repeated dose of 100 mg/kg/day to rats.


∙ <sup>a</sup> The blood samples were collected by a sparse sampling method from six rats (Table S1). Accordingly, the pharmacokinetic parameters were calculated from the mean plasma concentration data.

**Table 2.** Pharmacokinetic parameters of HSG4112(S) and HSG4112(R) after IV administration at a single dose of 10 mg/kg to rats.


∙ \*: *p* < 0.05, \*\*: *p* < 0.01, \*\*\*: *p* < 0.005 versus HSG4112(S).

#### *3.2. Analysis of HSG4112(S) and HSG4112(R) in Dog Plasma*

The time–plasma concentration plots of HSG4112(S) and HSG4112(R) are presented in Figure 3A, and the pharmacokinetic parameters are described in Table 3. The *C*max of HSG4112(S) and HSG4112(R) were 102.6 ± 69.6 and 707.6 ± 442.7, respectively. The AUC of HSG4112(S) and HSG4112(R) were 1408.4 ± 1418.0 and 12,324.8 ± 9715.7, respectively. The plasma concentration profiles of HSG4112(S) and HSG4112(R) after IV injection of HSG4112 at a dose of 2 mg/kg are presented in Figure 3B, and the pharmacokinetic parameters are described in Table 4. After IV injection of 2 mg/kg to beagle dogs,

the AUC of HSG4112(S) and HSG4112(R) were 1002.6 ± 163.6 and 1837.4 ± 20.4 ng·h/mL, respectively. Validation Data are presented in Tables S6–S9. ∙

**Figure 3.** Plot for time–plasma concentration of HSG4112 after (**A**) oral (100 mg/kg/day, 28th day, *n* = 3) and (**B**) iv (2 mg/kg, single dose, *n* = 2) administration in dogs.

**Table 3.** Pharmacokinetic parameters of HSG4112(S) and HSG4112(R) after PO administration at a repeated dose of 100 mg/kg/day to dogs.


\*: *p* < 0.05, \*\*: *p* < 0.01, \*\*\*: *p* < 0.005 versus HSG4112(S). NA: not available.

∙

**Table 4.** Pharmacokinetic parameters of HSG4112(S) and HSG4112(R) after IV administration at a single dose of 2 mg/kg to dogs.

