Design, synthesis and cytotoxic effects of curcuminoids on HeLa, K562, MCF-7 and MDA-MB-231 cancer cell lines

Background Curcumin is one of the leading compound extracted from the dry powder of Curcuma longa (Zingiberaceae family), which possess several pharmacological properties. However, in vivo administration exhibited limited applications in cancer therapies. Results Twenty-four curcumin derivatives have synthesized, which comprises cyclohexanone 1–10, acetone 11–17 and cyclopentanone 18–24 series. All the curcuminoids were synthesized by the acid or base catalyzed Claisen Schmidt condenstion reactions, in which β-diketone moiety of curcumin was modified with mono-ketone. These curcuminoids 1–24 were screened against HeLa, K562, MCF-7 (an estrogen-dependent) and MDA-MB-231 (an estrogen-independent) cancer cell lines. Among them, acetone series 11–17 were found to be more selective and potential cytotoxic agents. The compound 14 was exhibited (IC50 = 3.02 ± 1.20 and 1.52 ± 0.60 µg/mL) against MCF-7 and MDA-MB-231 breast cancer cell lines. Among the cyclohexanone series, the compound 4 exhibited (IC50 = 11.04 ± 2.80, 6.50 ± 01.80, 8.70 ± 3.10 and 2.30 ± 1.60 µg/mL) potential cytotoxicity against four proposed cancer cell lines, respectively. All the curcucminoids were characterized with the detailed 1H NMR, IR, UV–Vis, and mass spectroscopic techniques. The structure of compound 4 was confirmed by using the single X-ray crystallography. Additionally, we are going to report the first time spectral data of (2E,6E)-2,6-bis(2-methoxybenzylidene)cyclohexanone (1). Structure–activity relationships revealed that the mono-carbonyl with 2,5-dimethoxy substituted curcuminoids could be an essential for the future drugs against cancer diseases. Conclusions Curcuminoids with diferuloyl(4-hydroxy-3-methoxycinnamoyl) moiety with mono carbonyl exhibiting potential cytotoxic properties. The compound 14 was exhibited (IC50 = 3.02 ± 1.20 and 1.52 ± 0.60 µg/mL) against MCF-7 and MDA-MB-231 breast cancer cell lines.


Introduction
Cancer is one of the leading causes of death worldwide, with approximately 14 million new cases in 2012 [1]. The number of new cases is expected to rise by about 70% over the next two decades. Cancer causes of death globally and was responsible for 8.8 million deaths in 2015. Globally, nearly 1 in 6 deaths is due to cancer [2]. In 2016, 1,685,210 new cancer cases and 595,690 cancer deaths are projected to occur in the United States [3]. Breast cancer was the commonest cancer in women amongst all races from the age of 20 years in Malaysia for 2003 to 2005. According to the National Cancer Institute, 232,340 female breast cancers and 2240 male breast cancers are reported in the USA. It accounts for 16% of all female cancers and 22.9% of invasive cancers in women [4][5][6]. Curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a natural diarylheptanoid extracted from the rhizome of Curcuma longa [7,8]. Curcumin is a fascinating symmetrical molecules possessing interesting skeleton of β-diketone with diferuloyl (4-hydroxy-3-methoxycinnamic acid) moieties [9]. It exhibited remarkable biological activities mainly anticancer [10][11][12], anti-inflammatory [13][14][15], antioxidant [16,17], anti-hepatotoxic [18], nephroprotective [19], thrombosis suppressing [20], and hypoglycemic activities [21]. Curcuminoids have been identified as a potent antibreast cancer agent available from natural food ingredients including turmeric. This effect maybe contributed through targeting the estrogen receptors [22]. Advance understanding of bioactive metabolites through chemical synthesis has further enhanced the potential of these natural products including curcumin as the anticancer agent. For example, 4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidone (PAC), which is the analogue of curcumin were reported with enhanced antitumor effect against breast cancer via targeting the estrogen receptor [23]. On the other hand, modification of cyclohexanone derivative of curcumin was reported to enhance cytotoxicity against estrogen receptor-negative breast cancer cells [24]. Although it is well known natural remedies for pain still have bioavailability problems such as absorption, distribution, metabolism etc. [25,26]. Due to its significant anti-cancer properties on the various cancers such as gastrointestinal, genitourinary, gynecological, hematological, pulmonary, breast, and bone diseases, curcumin becomes a promising lead compound to develop a novel drugs [27,28].
The compound was 1 purified as yellow liquid. The UV spectrum of compound 1 showed the absorption wavelength, λ max at 339 nm corresponding to the α,β conjugated carbonyl group (C=O) compound. The IR absorption bands at 1636 cm −1 corresponding to carbonyl (C=O) and 2942-3001 cm −1 referred to aromatic C-H stretching functional groups. The 1 H NMR spectrum (600 MHz, CDCl 3 ) of compound 1 appeared at δ 1.75 as multiplet (2H) was assigned to the methylene proton (CH 2 ) at C4. A methylene protons at 2.84 as a multiplet (4H) integrated was corresponding to the C3 and C5 atoms. A singlet appeared at 3.86 integrated by 6H was assigned to the methoxy protons (OCH 3 ) at C2′ and C2″ position. A multiplet appeared at 6.92 was assigned to the aromatic protons at C3′ and C3″ methine protons. Two protons (2H) integrated at 6.96 shown a multiplet were assigned to the C5′ and C5″ protons. Another multiplet appeared at 7.33-730 (4H) was assigned to the C4′, C4″, C6′ and C6″ as aromatic methine protons. A broad singlet appeared at 7.98 integrated by 2H was due to the olefinic protons (-C=C-H). The board band decoupled spectra 13 C NMR showed the presence seven quaternary carbons, three methylene and ten methine carbons atoms. The compound showed EI-MS molecular mass was at m/z 334. The molecular formula of compound 1 was supported by HREI-MS calculated C 22 H 22 O 3 334.1575, found for 334.1580, which supported the proposed structure of compound 1 (Fig. 1). Previously, the radical scavenger and enzyme inducer activity of compound 1 obtained from Aldrich was reported by Dinkova-Kostavo et al. [35]. Interestingly, the data of all the compounds were characterized precisely on 600 MHz Bruker and 500 MHz and assignments were made carefully. The data of known compounds were compared with the previously published by Wei, Hosoya and Du [29,36,37].
Most of curcuminoids are potent as compared to the curcumin with (IC 50 = 22.50 ± 5.50 and 26.50 ± 1.40 µg/ mL) against MCF-7 and MDA-MB-231 (Table 1). Several reports on curcuminoids with mono-carbonyl (acetone series) have been even better pharmacological properties than curcumin [22,38]. Due to enolization and chelating (hydrogen bonding with the diketone), curcumin exhibited slightly lower cytotoxic effect than the modified derivatives. This could be due to the weak binding with the receptors, thus cause the weak pharmacokinetic profiles [39]. All curcuminoids possessed bis-enone conjugated system, which is quite reasonable site to binding with the Michael receptor selectivity with target nucleophile [30,[40][41][42]. The curcuminoids with mono-carbonyl 1-10 could be potential analogues for the drug discovery against cancer. In this respect, curcumin derivatives bearing a mono-carbonyl and methoxy groups especially cyclohexanone (1-10) and acetone 11-17 series could be a remarkable approach for the improvement of bioavailability problems related to curcumin [43,44].

X-ray structure description
Crystal data of compound 4 was given in Table 2. One crystal structure was determined by using X-ray diffraction method. Figure 2 showed the molecular structure of compound 4. Compound 4 crystalized in orthorhombic crystal system, space group Pna2 1 .

Synthetic procedures Method A (acid-catalyzed)
A typical Claisen-Schmidt condensation reaction procedure was used to prepare all curuminoids. Appropriate mono ketone (cyclohexanone, acetone and cyclopentanone) 10 mol (1 equiv) was dissolved in absolute ethanol (15-20 mL). Substituted benzaldehydes 20 mol, (2 equiv) was added slowly. About 1-2 mL concentrated HCl was added drop wise over 5-10 min in a stirred mixture of ketone. The reaction mixture was stirred overnight (12-24 h). The product was monitored by comparing the Co-TLC with the starting material. The products were extracted with ethyl acetate by dissolving the compounds in distilled water (100 mL). Curcuminoids were purified by silica gel column chromatography (ethyl acetate/hexane) and re-crystallized with hot solution of ethyl acetate and ethanol.

Method B (base-catalyzed)
The general procedure Claisen-Schmidt condensation reaction was used to synthesize curcuminoids by using this method involved in addition of certain amount of mono ketone (cyclohexanone, acetone and cyclopentanone) to a solution of substituted aldehydes in MeOH or C 2 H 5 OH by adding KOH or NaOH. The reaction mixture is stirred at room temperature and monitored by TLC. The products are extracted and purified as described as in acid catalysed [43,44].

Anticancer activity Sample preparation
Stock samples at 1 mg/mL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) were prepared and keep at 4 °C.

MTT cell viability assay
Breast cancer MCF-7 and MDA-MB-231 cells, chronic myelogenous leukemia K562 cells, and cervical cancer HeLa cells lines were purchased from American Type Culture Collection (ATCC, USA) and cultured at 37 °C, 5% CO 2 and 90% humidity using RPMI-1640 medium (Sigma-Aldrich, USA) supplemented with 10% Foetal Bovine Serum (FBS) (Thermo Fisher Scientific, USA). For MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) cell viability assay [48], MCF-7, MDA-MB-231, K562 and HeLa cells were seeded overnight in 96-well plates at 8 × 10 4 cells/well at 37 °C of CO 2 [49]. Then, 100 µL of media was discarded for all well-plates and compounds were serially diluted into the seeded cells at the concentration ranging between 30-0.47 µg/mL with cells treated with 3% DMSO (Sigma-Aldrich, USA) as the negative control. All samples were tested for triplicates. After 72 h of incubation, all well was added with 20 µL of MTT solution (5 mg/mL) and further incubated for 3 h. At that point, 170 µL of solution were discarded and 100 µL of DMSO (Sigma-Aldrich, USA) was added to all wells. Finally, absorbance was recorded by ELISA plate reader (Biotek-Instruments, USA) at the wavelength of 570 nm. Percentage of cell viability was calculated using following formula [38,39]. The assay was performed in triplicate to calculate the half maximal inhibitory concentration (IC 50 ) values. Doxorubicin was used as a positive control.
Cell viability (%) = [OD sample at 570 nm/OD negative control at 570 nm] × 100% IC 50 value (concentration of compounds inhibited 50% of cell viability) was determined from the graph of cell viability vs absorbance.

X-ray crystallographic analysis
X-ray analysis for all these samples were performed using Bruker APEX II DUO CCD diffractometer, employing MoKα radiation (λ = 0.71073 Å) with φ and ω scans, at room temperature. Data reduction and absorption correction were performed using SAINT and SADABS programs [50][51][52][53]. The structures of compound 4 was solved by direct methods and refined by full-matrix least-squares techniques on F 2 using SHELXTL software package. Crystallographic data of the reported structures have been deposited at the Cambridge Crystallographic Data Centre with CCDC deposition numbers of 1548735. Copy of available material can be obtained free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-(0)1223-336033 or e-mail: deposit@ ccdc.cam.ac.uk).

Conclusions
In conclusion, we have examined three series of curcumin analogues against four types (HeLa, K562, MCF-7 and MDA-MB-231) cancer cell lines. Curcuminoids with diferuloyl (4-hydroxy-3-methoxycinnamoyl) moiety with mono carbonyl exhibiting potential cytotoxic properties. The compound 14 was exhibited (IC 50 = 3.02 ± 1.20 and 1.52 ± 0.60 µg/mL) against MCF-7 and MDA-MB-231 breast cancer cell lines. Structure activity relationship revealed that the role of methoxy groups are important. Curcumin derivatives, 4, 5, 9, 14, 11 and 17 exhibited significant cytotoxic activity (Table 1). Curcuminoids with acetone series such as 2,5-dimethoxy substituted with mono ketones were found to be more selective and potential cytotoxic agents, which could be the best templet for future drug discovery against selective cancer especially breast cancer lines.
Authors' contributions SNHZ, MNA and SZ carried the literature and designed synthetic schemes (synthesis and purification), SKY, NBA and YH contributed to study of cancer cell lines of curcuminoids, CKQ and WSL contributed to X-ray analysis of compound, SAAS record the NMR of all compounds. All authors read and approved the final manuscript.