*2.8. Anti-Proliferative Activity*

Following the synthesis of the arctigenin derivatives, their anti-proliferative activities were evaluated against colorectal cancer HCT-116 and triple negative breast cancer MDA-MB-231 cell lines (Table 1). Arctigenin has previously been shown to affect the growth of MDA-MB-231 [32] whilst the HCT-116 has been used for the anti-proliferative assessment of polyphenolic natural products [33,34]. All of the compounds showed improved activity in the HCT-116 cell line over MDA-MB-231 cells. Five of the fifteen compounds had better activity than the previously prepared hydroxylmethylene derivative **16** in the MDA-MB-231 cell line, and 10 out of the 15 compounds had better activity than that reported for arctigenin itself [32]. It was found that compounds with aromatic benzylic ethers tended to outperform their phenol counterparts, and *trans*,*trans* stereochemistry between C-8, C-8' and C-9 was favourable over a *trans*,*cis* relationship. The most potent four compounds, based upon their ability to inhibit cell growth at 10 μM, were the same across both cell lines; **24**, **29**, **32**, and **33**, and their IC50 values were determined. All of the tested compounds, **24**, **29**, **32** and **33** produced similar inhibition–with mean IC50 values ranging between 5.79–7.45 μM (MDA-MB-231) and 3.27–6.10 μM (HCT-116) (Table 2).

**Table 1.** Anti-proliferative activities when cells treated with compound (10 μM). Values are given as the average ± standard error of three experimental replicates. Most active compounds, selected for IC50 determination, in highlighted rows.


\* = determined using MTT assay, after 24 h [32].

**Table 2.** IC50 values. Values are given as the average ± standard error of three experimental replicates. Most active compounds in highlighted rows.


#### **3. Materials and Methods**

#### *3.1. Synthesis*

General experimental details: All reactions were carried out under an inert atmosphere using distilled anhydrous solvents unless otherwise specified. Triethylamine and diisopropylethylamine were each distilled and stored over activated 4 Å Molecular Sieves. All NMR spectra were recorded on a Bruker Avance DRX 400 MHZ spectrometer at ambient temperature. Chemical shifts are reported relative to the solvent peak of CDCl3 (<sup>δ</sup> 7.26 for 1H and <sup>δ</sup> 77.16 for 13C) or DMSO (<sup>δ</sup> 2.50 for 1H and <sup>δ</sup> 39.52 for 13C). 1H NMR data are reported as position (δ), relative integral, multiplicity (s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; dt, doublet of triplets; dq, doublet of quartets; t, triplet; td, triplet of doublets; q, quartet; m, multiplet), coupling constant (*J*, Hz), and the assignment of the atom. Proton-decoupled 13C NMR data are reported as position (δ) and assignment of the atom. NMR assignments were performed using HMBC, COSY and HSQC, experiments. 1H and 13C NMR spectra for all precursor and final compounds (Figures S1–S45) are found in the supplementary material. The numbering of arctigenin analogues was done according to lignan nomenclature, with the two C-6-C-3 units numbered 1–9 and 1'–9' [18]. All melting points for solid compounds are given in degrees Celsius (◦C), were measured using a Reicher-Kofler block, and are uncorrected. A Perkin-Elmer Spectrum 1000 series Fourier Transform Infrared ATR spectrometer was used to record infrared spectra. Absorption maxima are expressed in wavenumbers (cm<sup>−</sup>1). High-resolution mass spectroscopy (HRMS) was carried out by electrospray ionisation (ESI+) on a MicroTOF-Q II mass spectrometer. Fétizon's reagent was prepared following a literature procedure [35]. Unless noted, chemical reagents were used as purchased. General procedures, synthetic experimental methods, and full characterisation data (including copies of NMR spectra for all synthesised final compounds) can be found in the Supplementary Materials.
