Academic Editor: Ping-Chung Kuo

Received: 13 January 2020; Accepted: 22 January 2020; Published: 22 January 2020

**Abstract:** Compound **1** is a curcumin di-*O*-2,2-bis(hydroxymethyl)propionate that shows significant in vitro and in vivo inhibitory activity against MDA-MB-231 cells with eight to ten-fold higher potency than curcumin. Here, we modified the α-position (C-4 position) of the central 1,3-diketone moiety of **1** with polar or nonpolar functional groups to afford a series of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)propionate derivatives and evaluated their anticancer activities. A clear structure–activity relationship of compound **1** derivatives focusing on the functional groups at the C-4 position was established based on their anti-proliferative effects against the MDA-MB-231 and HCT-116 cell lines. Compounds **2**–**6** are 4,4-dimethylated, 4,4-diethylated, 4,4-dibenzylated, 4,4-dipropargylated and 4,4-diallylated compound **1**, respectively. Compounds **2m**–**6m**, the ester hydrolysis products of compounds **2**–**6**, respectively, were synthesized and assessed for anticancer activity. Among all compound **1** derivatives, compound **2** emerged as a potential chemotherapeutic agent for colon cancer due to the promising in vivo anti-proliferative activities of **2** (IC50 = 3.10 ± 0.29 μM) and its ester hydrolysis product **2m** (IC50 = 2.17 ± 0.16 μM) against HCT-116. The preliminary pharmacokinetic evaluation of **2** implied that **2** and **2m** are main contributors to the in vivo efficacy. Compound **2** was further evaluated in an animal study using HCT-116 colon tumor xenograft bearing nude mice. The results revealed a dose-dependent efficacy that led to tumor volume reductions of 27%, 45%, and 60% at 50, 100, and 150 mg/kg doses, respectively. The established structure–activity relationship and pharmacokinetic outcomes of **2** is the guidance for future development of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)- propionate derivatives as anticancer drug candidates.

**Keywords:** curcuminoid derivatives; prodrug; colon cancer; breast cancer; active metabolites

#### **1. Introduction**

Curcumin [(1*E*,6*E*)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione, the structure of which is shown in Scheme 1], the naturally occurring phytochemical from the rhizome of *Curcuma longa* L., is a polyphenol with a symmetrical structure composed of two *ortho*-methoxyphenol rings connected to each other through a flexible conjugated hydrocarbon chain. It is a versatile therapeutic agent against cancer [1] and exhibits diverse pharmacological effects, including antidiabetic [2], antiviral [3], analgesic [4], nephroprotective [5], and cardioprotective effects [6], via regulating the gene expression [7] and protein binding [8]. Similar to some phenolic natural products, curcumin is a multi-target anticancer agent [9] with the ability to interfere with several signaling pathways associated with tumor progression, metastasis, apoptosis, and angiogenesis [10].

**Scheme 1.** PLE mediated hydrolysis of **1**.

While curcumin shows promising therapeutic properties, its low solubility and poor chemical and metabolic stability hinder further drug development [11]. The low chemical stability of curcumin is demonstrated by its rapid decomposition upon exposure to light or high temperatures (>70 ◦C) [12]. The compound decomposes into several degradation products, including a bicyclopentadione derivative, vanillin, feruloylmethane, and ferulic acid. The degradation of curcumin presumably results from its autoxidation [13], wherein the starting radical species is formed through hydrogen atom removal from the phenolic moiety. The phenolic group with an easily abstractable proton and the keto-enol system display significant hydrogen-donating abilities [14] that could initiate the autoxidative degradation of curcumin. The poor metabolic stability of curcumin can be ascribed to the presence of two phenolic hydroxyl groups that are prone to the formation of glucuronide and sulfate conjugates through the phase II metabolic process [15]. Moreover, the enone moiety of curcumin is accessible to nicotinamide adenine dinucleotide phosphate (NADHP) cytochrome P450 reductase or alcohol dehydrogenases, allowing curcumin to be readily reduced to the hydrogenated metabolites dihydrocurcumin, tetrahydrocurcumin, hexahydrocurcumin, and hexahydrocurcuminol [16].

Medicinal chemists have performed structural optimizations of curcumin to improve its stability and activity. Most of these attempts were focused on modifying the phenolic hydroxyl groups, keto-enol system, and enone moiety of curcumin [17–20]. However, the first two abovementioned structural features are required for radical scavenging activity, which is regarded as vital for curcumin chemopreventive and anti-inflammatory activities [21]. In addition, the enone moiety of curcumin, serving as the electrophilic Michael acceptor, can interact with cysteine or selenocysteine residues in proteins to mediate biological activities [22]. Thus, finding a balance between structural stability and biological potency can become challenging. According to the FDA [23], about 30% of the curcumin clinical trials stalled at stage II, mostly due to discrepancies between in vitro activity and in vivo response. Accordingly, to achieve success in clinical trials, obtaining a more definite picture of signaling pathways and ADME profiling for curcumin is recommended.

We previously developed a series of curcuminoid prodrugs by modifying the phenolic hydroxyl groups of curcumin into a 2,2-bis(hydroxymethyl)propionate group [24]. Among the derivatives, the curcumin 2,2-bis(hydroxymethyl)propionate **1** exhibited stability and solubility superior to those of curcumin. Particularly, **1** showed comparable anticancer activity against MDA-MB-231 cells in vitro and in vivo and had 8 to 10 times more potency than curcumin. Subsequently, the α-position in the central 1,3-diketone moiety (C-4 position) of **1** were alkylated with methyl, ethyl, benzyl, and propargyl groups to give compounds **2**, **3**, **4**, and **5**, respectively. Among them, **2**, **3**, and **5** showed more potent

in vitro anti-proliferative activity than **1** against HCT-116 cells; therefore, were recommended them as potential therapeutic agents for colon cancer [24]. In our recent in vitro studies on the enzymatic hydrolysis of **1**, we demonstrated that treating **1** with porcine liver esterase (PLE) cleaves its two ester groups gradually to form curcumin (see supporting information). Compounds **2**-**5** are ester-based derivatives and structurally correlated to **1**. Therefore, we predicted that a high concentration of esterase, as particularly found in the intestinal tract and mucosa [25], would hydrolyze **2**, **3**, **4**, and **5** into their metabolites **2m**, **3m**, **4m**, and **5m**, respectively (Scheme 2). However, the anticancer activity of **2m**–**5m** should be evaluated prior to the further in vivo investigations. Compounds **2**–**5** were substituted with two nonpolar alkyl chains at the C-4 position with the loss of keto-enol tautomerism. The anticancer activity of compound **1** derivatives, which were substituted with two hydrophilic units or two polar side chains at the C-4 position, still remains unclear.

**Scheme 2.** In vivo enzymatic hydrolysis of **2**–**5**.

Herein, we report the successful replacement of the acidic α-hydrogens at the C-4 position of compound **1** with polar or nonpolar functional groups to produce compounds **6**, **9**, **10**, **11**, **12**, and **14**. Compounds **2m**–**6m** were prepared through the chemical hydrolysis of corresponding parent compounds **2**–**6**. The anticancer activities of **2**–**6**, **9**–**12**, **14** and **2m**–**6m** were evaluated in vitro. A selected compound was submitted for preliminary pharmacokinetic study and antitumor study. The detailed descriptions of the synthetic design, in vitro screening, and in vivo study of the abovementioned compounds are presented as follows.
