Semi-synthesis of phenolic-amides and their cytotoxicity against THP-1, HeLa, HepG2 and MCF-7 cell lines

Abstract In the present study, we derivatized several hydroxycinnamic and hydroxybenzoic acids to phenolic amides (PAMs) via one step BOP mediated amide coupling reactions. Fifteen PAMs were synthesized in >40% yields and were screened for their cytotoxic activities against four cancer cell lines: THP-1 (leukaemia), HeLa (cervical), HepG2 (liver), and MCF-7 (breast), in comparison to 5-flurouracil (5-FU). Four amides showed IC50 ranging from 5 to 55 µM against all four cell lines. In contrast, tetradecyl-gallic-amide (13) affected only THP-1 leukaemia cells with IC50 of 3.08 µM. The activities of these compounds support the promise of phenolic amides as anticancer agents. Graphical Abstract


Introduction
About 8000 phenolic compounds are identified and various bioactivities and health benefits are attributed to them (Kumar and Pandey 2013;Ahmed et al. 2016).The bran of grains like rice and wheat contains several bioactive secondary metabolites, including antioxidant phenolic acids, flavonoids, anthocyanins, glycoconjugates, and γ-oryzanols (Brodniewicz and Grynkiewicz 2012;Ciulu et al. 2018).Among the phenolic compounds found in bran and other plant components, phenolic acids are of interest to this study.
Phenolic acids (PAs) consist of hydroxylated derivatives of benzoic acid and cinnamic acid.PAs serve as free radical scavengers and this property protects the cell from damage caused by oxidative stress.Several hydroxybenzoic acids and cinnamic acids from different plant sources and their anti-oxidant, anti-inflammatory, anti-pyretic, anti-fungal, anti-bacterial, insecticidal, and cytotoxic properties have been well reviewed (Khadem and Marles 2010;Saibabu et al. 2015;Kumar and Goel 2019;Kiokias et al. 2020).Their health benefits make them attractive to the food and nutraceutical industries to produce and market in the form of nutritional supplements.Although many beneficial biological properties have been attributed to PAs, their bioavailability is limited by their being easily excreted from the body after sulfation and glucuronidation, (Wu et al. 2011) which diminishes their utility in drug discovery.
Only a few branded US Federal Drug Administration (FDA)-approved polyphenol drugs are in the market.Polyphenon E (a green tea polyphenol mixture), which was approved in 2006 for treating genital warts, is one (Blumenthal 2007).In 2012, another macromolecular polyphenol drug Mytesi/Crofelemer (oligomeric proanthocyanidin) was marketed for managing the side effects of certain HIV drugs (Hoffman and Kishter 2013).
To improve the bioactivity and bioavailability of natural products, many have been semi-synthetically modified.Most of the new drugs that have been approved by the FDA have been extended by doing semi-synthetic modifications on the original natural products (Newman and Cragg 2016).Ultimately, natural products are playing a pivotal role as drug leads in the pharmaceutical industry.For example, the well-known bioactive compound curcumin and its reduced form tetrahydro-curcumin have been semi-synthetically modified and these derivatives were reported to have cytotoxic and antiproliferative activities toward cancer cells (Mahal et al. 2017;2019;Duan et al. 2022).In a similar fashion, naturally occurring PAs have been semi-synthetically modified to improve their bioactivity and bioavailability (Cos et al. 2002;Serafim et al. 2011;Li et al. 2012;Paiva et al. 2013;Khatkar et al. 2017;Lan et al. 2020).
Despite these many studies, there are not many reports in the literature with respect to the PAMs' anti-cancer activity against THP-1 leukaemia cancer cells.Herein, we report the one-step functionalization of the readily available free carboxylic group on PAs with alkyl, aryl, and phenyloxy primary amines via amide coupling reactions, resulting in a panel of fifteen novel PAMs.The PAMs were then screened for their cytotoxic efficacy against four cancer cell lines THP-1 (leukemia) HeLa (cervical), HepG2 (liver), and MCF-7 (breast).

Chemistry
We have chosen trans/E isomers of unsaturated p-coumaric acid, caffeic acid, sinapic acid, and saturated syringic acid, vanillic acid, and gallic acid as the PAs for the synthesis of alkyl and aryl amide derivatives by coupling reactions.Our initial attempts to react the PAs with different amines under EDC coupling reaction conditions using base and DMAP in DMF resulted in no reaction.Changing the amide coupling agent and reacting in the presence of HATU and DIPEA in DMF overnight resulted in a new non-polar compound (Vrettos et al. 2017).However, the coupling of histamine hydrochloride with p-coumaric acid under the above conditions resulted in no reaction.Unfortunately, impurities were observed in the reactions that were carried out under HATU coupling reaction conditions, so we switched to BOP coupling reactions (Shi et al. 2014).p-Coumaric acid was coupled with 3-isopropoxypropylamine (a) under BOP coupling reaction conditions using Et 3 N as base in DMF:DCM (1:1 v/v) (Shi et al. 2014) (Method A, S3) to yield compound 1 in 56% yield.Caffeic acid was reacted with a, 4-phenoxy aniline (b), and 4-isopropoxyaniline (c) by method A, afforded caffeic-amide derivatives 2, 3, and 4 in over 40% yields (Figure 1).
Sinapic acid was reacted with a, b, c, and dodecyl amine (d), by method A (see S3), giving rise to sinapic-amide derivatives 5, 6, 7 and 8 in good yields (Figure 1).Syringic acid was reacted with b, and 3-alkynyl aniline (f) under the optimized method A (see S3), which afforded syringic-amide derivatives 9 and 10 in 51% and 49% yields.Vanillic acid was reacted with b and d by method A, resulting in vanillic amides 11 and 12 in 42% and 44% yield, respectively.Surprisingly, no reaction was observed with propanol diamine under the same conditions.In the case of gallic acid, a benzyl protection strategy was applied, resulting in a per benzylated gallate (PBGA) (Belin et al. 2003).PBGA was then conjugated with tetradecyl amine (e) and c by method A to generate the per-benzylated-amides PBGTDA and PBGPA, which were debenzylated by hydrogenolysis using 10% Pd/C (method B, S4) to yield the gallic-amide derivatives 13 and 14 in 35% and 41% yield over two steps (Figure 1).Peracetylated-glucosamine (g) was coupled with caffeic acid under method A and afforded 15 in 51% yield.The E/Z configuration in the unsaturated phenolic amides (1, 2, 3, 4, 5, 6, 7, 8, and 15) was determined to be the E isomer, which was confirmed by the olefinic protons coupling constant J = >15 Hz (See SI) with multiplicity of a doublet.The newly formed amide bond carbonyl carbon peaks for all the fifteen compounds were observed around 165-168ppm in 13 C NMR.All the semi-synthetic PAMs (1-15) were characterized by 1 H and 13 C NMR, FT-IR, and HR-ESI-MS.

Cytotoxicity assay of PAMs along with standard drug 5-flurouracil
Each of the pure PAMs was assessed for its cytotoxicity against the leukaemia monocyte THP-1 cell line by the slightly modified standard MTT assay protocol, as described in the cell culture methods section (see S8).We found PAMs 2, 3, 5, 6, 9, and 13 exhibited cytotoxicity and the other PAMs (1, 4, 7, 8, 10, 11, 12, 14, and 15) were found to be non-cytotoxic at up to 150 µM concentration in 24 h.Later, we chose PAMs that showed cytotoxicity at concentrations below 150 µM and continued assays for 72 h of incubation.The IC 50 values of 2, 3, 5, 6, 9, and 13 were found to be 11.6, 11.4, 19.3, 54.9, 49.3, 4.17 and 3.08 µM, respectively (Figure S18 and Table S1, see S65).Compound 13 was slightly more potent than the standard anti-cancer drug 5-FU against THP-1 cells (Figure S22A).To confirm that cell death may be due to apoptosis, we carried out the apoptotic DNA fragmentation assay with 13 (see S9).The agarose gel showed the DNA fragmentation bands increased with the concentration of 13 from 0 to 4 µM, as shown in Figure S22B.In contrast, gallic acid has no cytotoxic effect against THP-1 at 10 µM (Figure S22C).
The parent PAs were also tested against THP-1 cells for 72 h and the results showed that caffeic acid, sinapic acid, and syringic acid (Figure S18) were not cytotoxic up to 50 µM.These results suggest that the parent PAs are relatively non cytotoxic, but the PAMs exhibited significant toxicity at low concentrations.Our initial cytotoxicity assay screening of PAMs on THP-1 cells directed us to explore the cytotoxicity on other cancer cell lines, i.e.HeLa, HepG2, and MCF-7.
Morphological changes were observed in all four cell lines after 72 h with compound 5. Significant cell losses were seen at 50 µM compound 5 and complete loss of cell morphology was observed at 100 µM concentration (Figure S23).

Conclusions
In summary, fifteen PAMs were synthesized via a one-step amide coupling reactions.All the derivatives were screened for cytotoxic efficacy against four cancer cell lines.Caffeic and sinapic amides (2, 3, 5, and 6) were found to be cytotoxic and exhibited IC 50 between 5 and 50 µM in 72 h incubations with THP-1, HeLa, HepG2, and MCF-7 cells.Compounds 3 and 13 were found to be the most cytotoxic to HeLa and THP-1 cells, respectively, but 13 was not active against the cell lines other than THP-1.In THP-1 cells, 13 was confirmed to cause apoptosis by DNA fragmentation.This suggests that compound 13 may be a good therapeutic compound to target leukaemia.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Financial support from the Thailand Research Fund (IRN62W0004) and National Research Council of Thailand (NRCT) and Suranaree University of Technology (SUT), Thailand Science Research and Innovation (TSRI), and the National Science Research and Innovation Fund (NSRF), Grant No. 90464 are gratefully acknowledged.

Figure 1 .
Figure 1.scheme of the reaction, structures of phenolic-amides and primary amines.