Research paper
New bis(hydroxymethyl) alkanoate curcuminoid derivatives exhibit activity against triple-negative breast cancer in vitro and in vivo

https://doi.org/10.1016/j.ejmech.2017.03.006Get rights and content

Highlights

  • Bis(hydroxymethyl) alkanoate curcuminoid derivatives were designed and synthesized.

  • The new compounds showed more potent antiproliferative activity than curcumin.

  • Activity was shown against triple-negative breast cancer (TBNC) in vitro and in vivo.

  • Synergistic activity was found when 9a was used in combination with doxorubicin.

Abstract

Novel bis(hydroxymethyl) alkanoate curcuminoid derivatives were designed, synthesized and screened for in vitro antiproliferative and in vivo antitumor activity. Selected new compound 9a and curcumin were further evaluated for inhibitory activity against ER+/PR+ breast cancer (MCF-7, T47D), HER 2+ breast cancer (SKBR3, BT474, and MDA-MB-457) and triple negative breast cancer (TNBC) (HS-578T, MDA-MB-157, and MDA-MB-468) cell lines. In addition, compound 9a was evaluated in the MDA-MB-231 xenograft nude mice model. Compound 9a exhibited greater inhibitory activity than curcumin against TNBC cells and also demonstrated significant inhibitory activity against doxorubicin-resistant MDA-MB-231 cells, with ten-fold higher potency than curcumin. Furthermore, when evaluated against the MDA-MB-231 xenograft nude mice model, compound 9a alone was ten-fold more potent than curcumin. Moreover, synergistic activity was observed when 9a was used in combination with doxorubicin against MDA-MB-231 breast cancer cells.

Introduction

Breast cancer is the most prevalent form of cancer diagnosed in women worldwide. According to a recent 2015 report [1], the estimated number of new cases of female breast cancer in the United States was 2,310,840, accounting for 29.5% of total cancer cases. Between 1975 and 2011, the incidence rates of breast cancer remained the highest compared with other cancers [1].

Triple-negative breast cancer (TNBC) represents about 15–20% of all breast cancer cases. The designation TNBC denotes that this cancer cell type lacks estrogen receptors (ERs) and progesterone receptors (PRs), as well as has low expression of human epidermal growth factor receptor-2 (HER-2) [2]. TNBC is more common in young women and also is an aggressive cancer that generally has a poor prognosis and is very prone to exacerbation. Due to a lack of well-defined molecular target drugs, TNBC is normally treated with cytotoxic agents, such as doxorubicin and taxoids [3]. These agents are cytotoxic in nature, and TNBC cells readily develop drug resistance to them. Therefore, the development of an anti-TNBC drug with less toxicity and drug resistance remains an important research subject.

Natural product mimics are a continuous major source of drug leads and drug candidates [4]. According to a 2012 analysis of new marketed medicines approved by the US Food and Drug Administration (FDA) from 1981 to 2010, about 34% of new drugs are based on small molecules derived from natural products [5]. The plant sources of such natural products are generally part of folkloric medicine practice. For example, plants of the Zingiberaceae or ginger family, especially their rhizomes, have been used as spices, flavoring agents, food preservatives, coloring agents, and folk medicines in Asian countries for centuries. Curcuma longa, common name turmeric, is a rhizomatous herbaceous perennial plant of the ginger family. Its major component, curcumin [(E,E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], was isolated about two centuries ago [6], and its structure was subsequently elucidated in 1910 [7].

Curcumin has a highly conjugated structure containing a heptadiene-3,5-diketone linking two methoxylated phenols. Therefore, curcumin is a potent antioxidant with strong free radical trapping ability [8], [9], [10]. Extensive research has shown that curcumin modulates numerous targets [11] and exhibits many biological activities [12]. In addition, curcumin inhibits different stages of cancer cell progression [13]. Moreover, curcumin is proven safe, even at doses up to 8 g per day in one clinical study [14]. Therefore, curcumin seems to be an ideal drug candidate that affects multiple pathways, and more importantly, is pharmacologically safe.

The critical disadvantages of curcumin as a drug are its low bioavailability, caused by poor hydrophilicity, and its rapid in vivo metabolism [15]. To address its poor hydrophilicity and improve its bioavailability, several new dosage forms of curcumin, including nanoparticles [16], liposomes [17], cyclodextrin encapsulation [18], micelles [19], and phospholipid complexes [20], have been tested in recent years. So far, however, none of these dosage forms have passed clinical trials and new drug approval [14], [15]. The key to its second disadvantage of rapid in vivo metabolism is the presence of two phenolic OH groups on the curcumin structure which are easily metabolized by transferases, through a phase II transformation, into glucuronides and sulfates [21], [22], [23]. Since the resulting water soluble metabolites are easily eliminated, the half-life of curcumin is very short and the bioavailability is low. Several studies reported the derivatization of curcumin on its phenolic OH groups to ester prodrugs, leading to succinate-conjugated [24], amino acid-conjugated [25], [26], [27], [28], [29], and phosphorylated curcumin derivatives [24]. Nonetheless, the in vivo efficacy data of these curcumin prodrugs were not available in the literature. However, recent reports illustrate the continuing intense interest in curcumin and its analogs as anticancer agents, for instance, in glioblastoma or breast cancer prevention and treatment [30].

In this study, curcumin was selected as a lead compound and bis(hydroxymethyl) alkanoate curcuminoid analogs (9a18) were designed as target compounds. The design concept was based on modification of the lead curcumin to give ((1E,3Z,6E)-3-hydroxy-5-oxohepta-1,3,6-triene-1,7-diyl)bis (2-methoxy-4,1-phenylene)bis (3-hydroxy-2-hydroxymethyl)-2-methyl propanoate (9a). This representative target compound is described schematically as shown in Fig. 1.

In this target structure (9a), both of the phenolic OH groups on curcumin are esterified and bulky groups with aliphatic OH groups are present at the ester terminus. Reportedly, alcoholic OH groups undergo glucuronidation less readily than phenolic OH groups [31], presumably due to the lower pKa value of phenolic OH groups. However, the four aliphatic OH groups on 9a could cause potential metabolic problems.

The calculated logarithmic partition coefficient (log P) of 9a is 1.73, implying better hydrophilicity than curcumin (calculated log P 3.38); calculations performed with ChemBioDraw Ultra 14.0. In fact, the solubility of 9a in either water or alcohol is better than that of curcumin (Table S1, Supporting Information).

Based on the above description, the target compound 9a may improve upon the critical pharmacokinetic disadvantage of curcumin. As discussed below, the research results indicated that 9a is more potent than curcumin, as shown not only by in vivo, but also by in vitro antiproliferative assays, which were not predictable in advance. In this study, new 9a analogs were designed, synthesized, and evaluated for antitumor activity. Several potential drug candidates were identified and are reported herein.

Section snippets

Chemistry

The target compounds (9a18) were synthesized according to Scheme 1A–C. As shown in Scheme 1, three curcuminoids including curcumin, bisdemethoxycurcumin, and compound 3, were reacted with acid 1 or 2 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 1-hydroxybenzotriazole (HOBt) and N,N-dimethylaminopyridine (DMAP) in DMF to give diverse ester derivatives (4a-8b). The obtained esters then underwent a deprotection reaction in THF under acid catalysis at 45–50 °C to give

Results and discussion

Growth inhibitory activity of target compounds 9a18 against MDA-MB-231, HCT116, and PC-3 cancer cell lines.

The synthesized target compounds 9a18 and curcumin as the positive control were screened against MDA-MB-231 (TNBC), HCT-116 (colon cancer), and PC-3 (prostate cancer) cell lines, and the results are summarized in Table 1. Furthermore, the stability of 9a in the culture media was tested. After 72 h treatment, the media containing the representative target compound 9a was analyzed for the

Conclusion

Novel bis(hydroxymethyl) alkanoate analogs of curcuminoids were designed, synthesized, and screened for in vitro antiproliferative activity. The screening results indicated that these analogs exhibited more potent activity than curcumin. Among these new analogs, compound 9a was selected for further evaluation. In additional in vitro testing, compound 9a exhibited 3.4–7.4 times greater antiproliferative activity than curcumin against TNBC cells and 10 times more activity than curcumin toward

Experimental

The reactions were performed under an air atmosphere unless otherwise stated. All solvents and reagents were employed as received. Analytical thin layer chromatography was performed on SiO2 60 F-254 plates and flash column chromatography was carried out using SiO2 60 (particle size 0.040–0.055 mm, 230–400 mesh), both of which are available from E. Merck. Visualization was performed under UV irradiation at 254 nm followed by staining with aqueous potassium permanganate [KMnO4 (3 g) and K2CO3

Acknowledgment

We are grateful to China Medical University Hospital (DMR-104-107) and Chinese Medicine Research Center, China Medical University (the Ministry of Education, the Aim for the Top University Plan) for financial support.

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