3.2.5. LC-MS Analysis Method

All samples obtained from in vivo pharmacokinetic studies were assayed using LC-MS for the analysis of **2** and the corresponding metabolites. The Waters ACQUITY UPLC I-Class system (Waters Corp., Milford, MA, USA) was utilized. System control and all of the mass spectrometry data were acquired and analyzed using UNIFI software (Waters Corp.). For sample separation, the LC was equipped with an Inertsil ph-3 (50 × 2.1 mm, 2 μm particle size) RPLC column (GL Sciences Inc. Tokyo, Japan) in which the column temperature was held at 35 ◦C, injection volume was 7.5 μL, and the flow rate of the mobile phase was 200 μL/min. The elution started from 50% mobile phase A (ultrapure water + 0.1% formic acid) and 50% mobile phase B (100% methanol + 0.1% formic acid), held at 50% B for 0.5 min, raised to 95% B in 5.5 min, held at 95% B for 1 min, and then lowered to 50% B in 1 min. Before sample injection, the column was equilibrated by pumping 50% B for 4 min. The analysis of target compounds for all samples was performed by using a Waters Vion IMS QTof mass spectrometer. Data were acquired in the electrospray ionization (ESI) positive ion MSE mode with range of *m*/*z* 100–1000 and 0.5 s scan time. Parameters were set as capillary voltage of 2.5 kV, source temperature of 100 ◦C, desolvation temperature at 250 ◦C, cone gas maintained at 10 L/h, desolvation gas maintained

at 600 L/h. The acquired m/z and isotope pattern were processed by UNIFI software to illustrate chromatogram, and the amounts of compound was calculated with the integrated peak area of signals.

#### **4. Conclusions**

Here, structural modifications at the C-4 position of compound **1**, a 2,2-bis(hydroxymethyl) propionate curcumin prodrug, with polar and nonpolar functional groups were performed to form a series of 4,4-disubstituted analogs of **1** and **DMC**. The in vitro anti-proliferative assay of these derivatives elucidated an obvious SAR in which the enhancement of polarity on C-4 resulted in the erosion of anticancer activity. The ester prodrug characteristics of **2**–**6** motivated us to synthesize their hydrolysis compounds **2m**–**6m**, **15**, and **16**, whose anticancer activities were highly dependent on the volume and length of the C-4 alkyl chains. The preliminary pharmacokinetic evaluations indicated that the esterase-mediated hydrolysis of **2** proceeded smoothly to produce **2m**, which in turn was reduced to **18** and **19** as major phase I metabolites. The phase II metabolic pathway was confirmed by the detection of glucuronide **22** and sulfate **23**. However, it is still possible that reduction precedes ester hydrolysis to provide the hydrogenated counterparts of **2**, which possess novel chemical skeletons with unknown biological activity.

Our preliminary pharmacokinetic results also shed light on future directions for the structural modification of the original compounds. Additional modifications have been scheduled to reduce the high propensity towards ester hydrolysis by converting the methyl of 2,2-bis(hydroxymethyl) propionate group into more bulky alkyl groups. Pharmacokinetic studies have been designed to identify, biologically evaluate, and quantify the possible metabolites. Apart from this, compound **2** and **2m** may behave like **1** which acts on multiple targets and signaling pathways. The differences between the mechanism of action of **2**, **2m** and **1** are not definitively understood. Currently, we are actively investigating the mechanism of action and pharmacokinetic parameters of **2** and **2m**, including T1/2, AUC, and Cmax, and the results will be reported in due course. The animal study showed that compound **2** inhibits tumor growth by 45% at a dose of 100 mg/kg in HCT-116 xenograft nude mice. Thus, twice-a-day (BID) dosing formulations for achieving higher therapeutic efficacy at lower doses should be considered in further development of title curcuminoid derivatives as anticancer drug candidates.

**Supplementary Materials:** The following are available online. **Scheme S1**: PLE mediated hydrolysis of curcumin 2,2-bis(hydroxymethyl)propionate **1**. **Figure S1**: The HPLC analysis of compound **18**, **19**, **20**, **21**, **22** and **23**. Copies of 1H and 13C NMR spectra of the new products.

**Author Contributions:** D.-Y.L. performed the preliminary pharmacokinetic analysis and wrote experimental section and supplementary material file. Y.-C.H. designed the preliminary pharmacokinetic study and integrated the experimental data. J.-S.Y. and H.-Y.L. performed the in vivo assay and in vivo animal study. K.-H.L. and T.-Y.C. reviewed and revised the manuscript. S.-C.K. designed the target compounds and interpreted the experimental data. M.-T.H. designed and synthesized targeted compounds. S.-C.K. and M.-T.H. are the responsible researchers, who wrote and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** We are grateful to Ministry of Science and Technology, Taiwan (MOST 107-2320-B-039-028) for financial support. Partial support was also provided by the "Chinese Medicine Research Center, China Medical University" from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan (CMRC-CHM-5) awarded to S. C. Kuo.

**Conflicts of Interest:** The authors declare no conflict of interest.
