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

Chemistry

Curcuminoids are the derivatives of curcumin. About 24 curcuminoids have been synthesized and investigated their cytotoxic properties against various cancer lines and thus established the structure–activity relationship for the future drugs development. In our experiments, we have synthesized three series of mono-carbonyl analogues of curcuminoids with cyclohexanone (1–10), acetone (11–17) and cyclopentanone (18–24). Three series were synthesized by Claisen–Schmidt condensation reaction by coupling the appropriate aromatic aldehydes with cyclohexanone, acetone and cyclopentanone by acid or base catalysed as previously stated by Wei [29]. In this project, β-diketone moiety of curcumin was modified with mono ketone and investigated their cytotoxic properties against Hele cell lines (human cervical cancer), K562 (Leukemia) cell lines, MCF-7 (an estrogen-dependent) and MDA-MB-231 (an estrogen-independent) cancer cell lines [30]. Additionally, we are going to report first time the data of (2E, 6E)-2,6-bis(2-methoxybenzylidene)cyclohexanone (1). Recently, we have reported the in vivo anti-tumour activity of 2,6-bis(4-hydroxy-3-methoxybenzylidene)cyclohexanone (5) on 4T1 breast cancer cells [31]. Previously, we have published another curcumin derivative DK1 and naturally occurring chalcone flavokawain B and its derivatives on various breast cancer cell lines [32–34].

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 ¹H NMR spectrum (600 MHz, CDCl₃) of compound 1 appeared at δ 1.75 as multiplet (2H) was assigned to the methylene proton (CH₂) 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 (OCH₃) 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 ¹³C 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 C₂₂H₂₂O₃ 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].

Structure–activity relationship

Most of curcuminoids are potent as compared to the curcumin with (IC₅₀ = 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, 40–42]. 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 Pna2₁.

Crystal data	4
CCDC	1548735
Chemical formula	C24H26O5
M r	394.45
Crystal system, space group	Orthorhombic, Pna21
Temperature (K)	296
a, b, c (Å)	8.529 (8), 25.65 (2), 9.430 (8)
α, β, γ (°)	90, 90, 90
V (Å3)	2063 (3)
Z	4
Radiation type	Mo Kα
µ (mm−1)	0.09
Crystal size (mm)	0.47 × 0.24 × 0.05
Data collection
Diffractometer	Bruker APEXII DUO CCD area-detector diffractometer
Absorption correction	Multi-scan (SADABS; Bruker, 2009)
Tmin, Tmax	0.8434, 0.9624
No. of measured, independent and observed \(\left[ {{\text{I}}\,{ > }\, 2\sigma \left( I \right)} \right]\) reflections	17,650, 3611, 1468
Rint	0.145
(sin θ/λ)max (Å−1)	0.594
Refinement
\(R\left[ {F^{ 2} > { 2}\sigma \left( {F^{ 2} } \right)} \right]\), wR(F2), S	0.071, 0.184, 1.00
No. of reflections	3611
No. of parameters	266
H-atom treatment	H-atom parameters constrained
Δρmax, Δρmin (e Å−3)	0.12, − 0.14

Chemistry

Synthetic procedures

Anticancer activity

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% CO₂ 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 × 10⁴ cells/well at 37 °C of CO₂ [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 (IC₅₀) values. Doxorubicin was used as a positive control.

Cell viability (%) = [OD sample at 570 nm/OD negative control at 570 nm] × 100%

IC₅₀ 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 [50–53]. The structures of compound 4 was solved by direct methods and refined by full-matrix least-squares techniques on F² 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).