Synthetic Derivatives of Natural ent-Kaurane Atractyligenin Disclose Anticancer Properties in Colon Cancer Cells, Triggering Apoptotic Cell Demise

The antitumor activity of different ent-kaurane diterpenes has been extensively studied. Several investigations have demonstrated the excellent antitumor activity of synthetic derivatives of the diterpene atractyligenin. In this research, a series of new synthetic amides and their 15,19-di-oxo analogues obtained from atractyligenin by modifying the C-2, C-15, and C-19 positions were designed in order to dispose of a set of derivatives with different substitutions at the amidic nitrogen. Using different concentrations of the obtained compounds (10–300 μM) a reduction in cell viability of HCT116 colon cancer cells was observed at 48 h of treatment. All the di-oxidized compounds were more effective than their alcoholic precursors. The di-oxidized compounds had already reduced the viability of two colon cancer cells (HCT116 and Caco-2) at 24 h when used at low doses (2.5–15 μM), while they turned out to be poorly effective in differentiated Caco-2 cells, a model of polarized enterocytes. The data reported here provide evidence that di-oxidized compounds induced apoptotic cell death, as demonstrated by the appearance of condensed and fragmented DNA in treated cells, as well as the activation of caspase-3 and fragmentation of its target PARP-1.


Introduction
Cancer is a leading cause of death worldwide, with nearly 10 million deaths estimated in 2020 [1].Globally, nearly one in five deaths is due to cancer.At the beginning of 2023, the World Health Organization highlighted that approximately 70% of cancer deaths occur in low-and middle-income countries [2].Among all types, colorectal cancer is the fourth most common cancer in the world [3].The onset and progression of colorectal cancer is caused by a combination of multiple factors, with age, gender (it has a higher incidence in women than in men), family history, region, and personal history being the major risk factors [4].
Conventional treatments for colorectal cancer involve the use of surgery and/or chemotherapy.Drugs used for treatment can lead to cancer cell death by causing DNA damage or triggering sudden signaling pathways, including cell cycle arrest, global translation inhibition, DNA repair, etc. [5].However, it has been shown in many studies, including molecular pathological epidemiology studies, that the use of chemotherapy in patients with colorectal cancer varies according to the severity of the disease.The effects of cytotoxicity, drug resistance, and adverse reactions are the main problems associated with chemotherapy [6].
Natural products isolated from plants have led to the design and synthesis of a great number of biologically active compounds.They have shown excellent bioactivities, such as anti-inflammatory, antiviral [7], and antibacterial [8] properties.
In the last decade, metabolites of the genus Isodon (Benth.)Schrad.ex Spach, plants of the Lamiaceae family, have been biologically investigated, and they have proven to be promising phytopharmaceuticals due to their wide range of physiological effects, such as the inhibition of hepatitis viral replication [9] and bacterial infections of the lung or intestine [10], as well as antimalarial [11], anti-inflammatory [12], and anticancer properties [13].One of the most renowned ent-kauranic compounds is kaurenoic acid.Various biological assays have been carried out on this metabolite, attributing important properties to it.It is able to attenuate inflammatory mechanisms through various processes, such as the activation of the nuclear factor E2-related factor-2 (Nrf2) [14][15][16], inducing regulations in T-helper 2 (Th2) and nuclear factor-κB (NF-κB) pathways linked to specific cytokines [17,18].This particular acid is also able to dose-dependently inhibit the release of prostaglandin E2, influencing the production of nitric oxide (NO) through inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 [19][20][21][22].The ability to inhibit appropriate cytokines makes it an excellent analgesic product.This is because it is responsible for the activation of the NO cyclic GMP kinase G-ATP protein-sensitive potassium channel signaling pathway [23].
At the anticancer level, dynamics such as genotoxicity and mutagenicity must be seriously taken into consideration.Indeed, kaurenoic acid is considered a mutagenic compound according specific studies carried out in vitro and in vivo [24,25].In fact, clearly genotoxic and mutagenic effects have been found in human blood leukocytes and on various mouse and hamster cellular structures [24,25].It is assumed, from these studies, that the effect of this acid is mainly linked to the ability to break the filamentary structures of DNA [25].Structure-activity relationship studies have demonstrated that the double bond on C-16 is responsible for its genotoxicity [25].Considering these aspects, it was also highlighted that this compound shows important cytotoxic effects on several cell lines, such as HeLa, A-549, HEp-2, PC-3, and MCF-7, acting in a dose-dependent manner [26], and many of its derivatives have shown antitumor activity against several cancer cell lines, like MCF-7 [27], 4T1 [28], and U87 [29], as well as other tumor lines [30].
So far, more than 700 ent-kauranic-type diterpenoids with compact polycyclic ring systems have been isolated from the genus Isodon, especially the oridonin derivatives [31].A common structural unit present in these diterpene compounds is the α,β-unsaturated ketone in the D ring.Unfortunately, however, the use of these natural compounds as anticancer agents has been hampered by their moderate potency [31].Therefore, it is necessary to find new compounds with simple structures and more robust activity.Among the diterpenes with an ent-kaurenic structure, atractyligenin (1) (Figure 1), the aglycone of the highly toxic molecules atractyloside (2) and carboxyatractyloside (3) extractable in good amounts from Chamaeleon gummifer (L.) Cass., has undergone numerous chemical modifications in order to explore the biological properties of its derivatives.These investigations concerned the photoinduced functionalization of its C-20 methyl group, enzyme-catalysed transformations of its alcohol groups [32], and the synthesis of several oxidative derivatives with promising cytotoxic activity against some human cancer cell lines, including A549 (lung), PC-3 (prostate), 1A9 (ovary), MCF-7 (breast), KB These investigations concerned the photoinduced functionalization of its C-20 methyl group, enzyme-catalysed transformations of its alcohol groups [32], and the synthesis of several oxidative derivatives with promising cytotoxic activity against some human cancer cell lines, including A549 (lung), PC-3 (prostate), 1A9 (ovary), MCF-7 (breast), KB (nasopharynx), and KB-VIN (stresses multidrug-resistant KB) [33,34].One of them, 15-ketoatractyligenin methyl ester (4) (Figure 1), has shown potent tumour cell growth inhibition activity, the mechanism of action of which has been widely elucidated [34,35].According to the literature, the presence of the α,β-unsaturated carbonyl moiety seems to be important for the antiproliferative activity [33,36].Previous studies on the thioredoxin system (TrxR) have also demonstrated that the catalytic thiols of TrxR and/or the exposed selenocysteine [37,38] are good Michael donors for the α,β-unsaturated carbonyl group present in 4. The reactivity of the electrophilic group towards the Cys/Sec system is also supported by recent results on the Trx-inhibitory activity of oridonine [39].Furthermore, the evaluation of the antibiotic properties of 1, 2, and 4 against Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus [40]; their anti-leishmania activity [41]; and the inhibition of skin photoaging by atractyligenin (1) [42] have recently been reported.
In 2022 it was demonstrated that synthetic modifications (bromination, reduction, elimination, and oxidation) on the A ring of 1 modulated its antitumor activity, significantly increasing the antiproliferative efficacy in some cases [43].
Herein, we report the synthesis of modified derivatives on C-2 and C-19 of the A ring and on C-15 of the D ring of 1 using a semi-synthetic approach that involves the amidation of the carboxyl moiety and the oxidation of the alcoholic functionalities.Finally, the antiproliferative activity of all the designed compounds was evaluated at different concentrations and at different times on two lines of colon cancer cells, namely HCT116 and Caco-2, in addition to investigation of the effects on DNA and the mechanism of action.
After the standard purification procedures, all the synthesized compounds were characterized with extensive spectroscopic and spectrometric analyses, including HRES-IMS and 1 H-NMR, as well as 13 C-NMR spectral analysis, with the support of homotopic and heterotopic correlations such as COSY, HSQC, HMBC, and NOESY techniques.All NMR and mass spectra of compounds 5-18 are included as Supplementary Material file (Figures S1-S47).The amides of 5-18 have similar spectroscopic data.By way of example, the diagnostic analysis of compound 5 is discussed below.In the 13 C-NMR spectrum of 5 (Figure S2), the diagnostic signal at 174.29 ppm can be attributed to a quaternary sp 2 amide carbon (C-19), while the signal assignable to the carbon sp 3 of the methylene group (in α, to the nitrogenous function) was observed at 41.30 ppm (C-1 ′ ).To confirm the successful amidation, the signal at δ 3.13 (m, CH 2 -1 ′ ) in the 1 H-NMR spectrum (Figure S1), characteristic of the CH 2 -1 ′ group, showed an HSQC correlation with the C1 ′ carbon at 41.30 ppm (Figure S4) and an HMBC correlation (Figure S6) with the C-19 carbonilic carbon (174.29 ppm).These signals, together with those expected for the diterpenoid moiety and for the aliphatic portion of the amide pendant, confirmed the formation of atractyligenin N-propylamide 5.The assignments were also corroborated by DEPT (Figure S3), COSY (Figure S5), HSQC, and HMBC, in particular from the correlations between H 2 -3, H-4, H-5, H 2 -1 ′ , and C-19-especially NOESY correlations (Figure S7) which were absent between the H-2, the methylene protons CH 3 -20, and the proton in position 4 in this case-reconfirming an α orientation for the amide function at position 19.The other amides (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) showed NMR data, including 2D-NMR correlations, very similar to those observed for 5, thus allowing their structures to be unambiguously determined and all the 1 H and 13 C-NMR signals to be fully assigned, as reported in the Section 3.
In order to evaluate the importance of the oxidation states of C-2 and C-15 in terms of biological activity, it was planned to transform the two hydroxyl groups into the corresponding ketones (Scheme 2).All NMR and mass spectra of di-oxo-atractyligenin amides (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32) are included in the Supplementary Materials (Figures S48-S89).Also for the case of oxidations, only the model characterization of the modified amide 5 is reported.All the previously obtained amides (5-18) were reacted with DMP, a hypervalent iodine compound that is suitable for a mild oxidation of secondary alcohols to ketones.After one hour, the starting reagent disappeared on TLC, and a single product (19) was obtained Scheme 1.General procedure for the synthesis of atractyligenin amides 5-18.
In order to evaluate the importance of the oxidation states of C-2 and C-15 in terms of biological activity, it was planned to transform the two hydroxyl groups into the corresponding ketones (Scheme 2).All NMR and mass spectra of di-oxo-atractyligenin amides (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32) are included in the Supplementary Materials(Figures S48-S89).Also for the case of oxidations, only the model characterization of the modified amide 5 is reported.All the previously obtained amides (5-18) were reacted with DMP, a hypervalent iodine compound that is suitable for a mild oxidation of secondary alcohols to ketones.After one hour, the starting reagent disappeared on TLC, and a single product (19) was obtained from amide 5. Its proton and carbon spectra (Figures S48 and S49, respectively) showed a clear shift at the low field in the 1 H-NMR of exocyclic protons (δ H 5.96, H-17a; δ H 5.28, H-17b), the presence of two ketones function at δ C 2 × 209.78 (C-2 and C-15), and the absence of alcohol signals in the proton spectrum.Consequently, compound 19 is consistent with the structure of the di-oxo derivative of atractyligenin amide 5.The other di-oxo amides (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32) obtained by the same oxidative treatment showed NMR data very similar to those observed for 19, allowing us to determine their structures and, therefore, to completely assign all the proton and carbon signals, as reported in the Section 3.
As shown in Figure 3, except for compounds 20, 27, and 32, almost all other di-oxidates were capable of reducing the cell viability at a 10 µM dose (Figure 3).These data show that di-ketones are endowed with a remarkable efficacy in comparison to diols 5-18, indicating that the functional-group interconversion from alcohol to ketone on both C-2 and C-15 is fundamental for increasing the antiproliferative power of atractyligenin derivatives.
In light of the obtained results, the subsequent experiments were performed by incubating colon cancer cells with lower concentrations of the most active di-oxidates (range: 2.5-15 µM).Interestingly, as can be observed in the bar charts reported in Figure 4, except for 28 and 29, which showed scarce effects at low doses, all other tested compounds displayed clear cytotoxic effects in a dose-dependent fashion on HCT116 cells.Such an effect was already clearly visible at 24 h of incubation (Figure 4).
The insertion of additional nonpolar pharmacophores on the A ring of atractyligenin, as well as the introduction of the two ketone functionalities, led to significant results, contributing to the increase its antiproliferative efficacy in different ways.
Indeed, the targeting capacity of α,β-unsaturated carbonyl systems, such as those present in derivatives 19-32, has been demonstrated [44].This functionality can inhibit the activity of the thioredoxin (TrxR) system, comprising the coenzymes nicotinamide adenine dinucleotide phosphate (NADPH), thioredoxin (Trx), and Trx reductase (TrxR), in both cell-free systems and in Jurkat cells.Trx controls nuclear translocation and/or the activity of several transcription factors and those that prevent apoptosis.Trx, among other things, protects mitochondria by the opening of the permeability transition pore (PTP) [44] and the release of cytochrome c, in addition to inhibiting the activation of apoptosis signaling kinase 1 (ASK1) [45].The presence of functional thiol systems in Trx and TrxR [46] therefore makes these proteins suitable targets for the α,β-unsaturated carbonyl functional group [37,38].This aspect was also confirmed on synthetic derivatives of oridonin, a natural compound with a basic structure very similar to that of atractyligenin.All oridonin A-ring-modified compounds containing the α,β-unsaturated ketone presented IC 50 values ranging between 1.22 and 11.13 µM on four different human cell lines after 72 h of incubation [31].Derivatives 19-32 were also tested for 48 h for their effects on colon cancer HCT116 cells.It is interesting to note that the introduction of two ketone functionalities at the C-2 and C-15 positions caused an increase in antiproliferative effectiveness by 8-10 times compared to the corresponding derived amides (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).
As shown in Figure 3, except for compounds 20, 27, and 32, almost all other di-oxi-  In light of the obtained results, the subsequent experiments were performed by incubating colon cancer cells with lower concentrations of the most active di-oxidates (range: 2.5-15 μM).Interestingly, as can be observed in the bar charts reported in Figure 4, except for 28 and 29, which showed scarce effects at low doses, all other tested compounds displayed clear cytotoxic effects in a dose-dependent fashion on HCT116 cells.Such an effect was already clearly visible at 24 h of incubation (Figure 4).Table 1 presents reported IC 50 values of some di-oxidated derivates tested on HCT116 cancer cells after 24 h of treatment.Among the compounds tested, except for compounds 28 and 29, all derivative analogues were significantly more potent than 1 and the corresponding amides (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).The insertion of additional nonpolar pharmacophores on the A ring of atractyligenin, as well as the introduction of the two ketone functionalities, led to significant results, contributing to the increase its antiproliferative efficacy in different ways.
Indeed, the targeting capacity of α,β-unsaturated carbonyl systems, such as those present in derivatives 19-32, has been demonstrated [44].This functionality can inhibit the activity of the thioredoxin (TrxR) system, comprising the coenzymes nicotinamide adenine dinucleotide phosphate (NADPH), thioredoxin (Trx), and Trx reductase (TrxR), in both cell-free systems and in Jurkat cells.Trx controls nuclear translocation and/or the activity of several transcription factors and those that prevent apoptosis.Trx, among other things, protects mitochondria by the opening of the permeability transition pore (PTP) [44] and the release of cytochrome c, in addition to inhibiting the activation of apoptosis signaling kinase 1 (ASK1) [45].The presence of functional thiol systems in Trx and TrxR [46] therefore makes these proteins suitable targets for the α,β-unsaturated carbonyl functional group [37,38].This aspect was also confirmed on synthetic derivatives of oridonin, a natural compound with a basic structure very similar to that of atractyligenin.All oridonin A-ring-modified compounds containing the α,β-unsaturated ketone presented Compound 24 was the most active of the series, with an IC 50 value of 5.35 µM-very similar to the activity shown by compound 25 (IC 50 = 5.50 µM).Both compounds have a very similar structure, the first bearing an octyl linear chain on the amidic nitrogen, while the second is endowed with a decyl chain.The activity tended to worsen slightly when using short (compound 19) or medium (compounds 21-23) chains as N substituents or chains with small branches (compound 26).Finally, the introduction of an aromatic pendent (compound 31), compared to a totally aliphatic cycle (presented in 30), led to a clear improvement in the antiproliferative activity (IC 50 = 6.37 µM for 31 and 9.27 µM for 30).
The effect of the most active compounds was also evaluated on Caco-2 cells, another colon cancer cell line, as well as on differentiated Caco-2 cells, a model of polarized colon cells grown in culture according to the procedure reported by Natoli et al. [46] (Figure 5).It is noteworthy that cytotoxic effects also appeared in Caco-2 tumor cells, while no cytotoxicity was observed in differentiated Caco-2.The absence of any cytotoxic effect in this enterocyte-like model [47,48] suggests that the tested compounds exert a selective efficacy on tumor cells.However, we aim to better explore this aspect in our future research.To ascertain whether the effects exerted by active derivatives of atractyligenin on colon HCT116 cancer cells could be ascribed to the induction of apoptosis, it was evaluated whether chromatin condensation and its fragmentation (two typical changes of apoptotic cell death) occurred in treated cells.As shown in Figure 6, light microscopy analyses revealed that all tested compounds caused clear morphological changes at 24 h represented by cell detachment and shrinkage, as well as a consistent reduction in cellular density in comparison to untreated samples (upper panel in Figure 6).Interestingly, vital Hoechst staining (lower panel) highlighted that compounds 19, 21-26, and 28-31 caused the condensation of the nuclear material, as well as the formation of apoptotic bodies.To ascertain whether the effects exerted by active derivatives of atractyligenin on colon HCT116 cancer cells could be ascribed to the induction of apoptosis, it was evaluated whether chromatin condensation and its fragmentation (two typical changes of apoptotic cell death) occurred in treated cells.As shown in Figure 6, light microscopy analyses revealed that all tested compounds caused clear morphological changes at 24 h represented by cell detachment and shrinkage, as well as a consistent reduction in cellular density in comparison to untreated samples (upper panel in Figure 6).Interestingly, vital Hoechst staining (lower panel) highlighted that compounds 19, 21-26, and 28-31 caused the condensation of the nuclear material, as well as the formation of apoptotic bodies.In addition, Western blotting analyses were performed to elucidate the possible involvement of caspase-3 and PARP-1, typical apoptotic players [49,50].In particular, it was determined that all active compounds caused a downregulation of pro-enzymatic caspase-3 and the appearance of the fragmented and active forms with molecular weights of 19 and 17 kDa, respectively.Compounds 28 and 29, which were not found to be In addition, Western blotting analyses were performed to elucidate the possible involvement of caspase-3 and PARP-1, typical apoptotic players [49,50].In particular, it was determined that all active compounds caused a downregulation of pro-enzymatic caspase-3 and the appearance of the fragmented and active forms with molecular weights of 19 and 17 kDa, respectively.Compounds 28 and 29, which were not found to be significantly cytotoxic, did not show an appreciable effect on pro-caspase 3, confirming the relevant role of this protein in determining apoptosis in this cell line.On the other hand, the cytotoxicity of 19 does not seem to be related to an effect on the pro-caspase 3 pathway.A similar trend can be observed with the fragmentation of PARP-1, a typical caspase-3 target (Figure 7) commonly cleaved in apoptotic cell death programs.All active compounds, except 19, possess the ability to promote the fragmentation of both pro-caspase 3 and PARP-1.Also in this case, non-cytotoxic 28 and 29 do not exert any influence on PARP-1.The anomaly observed in the behaviour of compound 19 may suggest the involvement of other pathways in the cytotoxicity of this compound.Further investigations are on the way.Upon observing the chemical structures of the molecules discussed above, it comes into evidence that the more active compounds 24 and 25 possess a long, linear aliphatic chain (8C and 10C, respectively) linked to amide-nitrogen.On the contrary, molecules containing highly branched side chains, that is, 28 and 29, are not active.The bioactivity of the remaining set of tested molecules lies in between those extremes.This mere hypothesis, although quite reasonable, must be confirmed by docking investigations.significantly cytotoxic, did not show an appreciable effect on pro-caspase 3, confirming the relevant role of this protein in determining apoptosis in this cell line.On the other hand, the cytotoxicity of 19 does not seem to be related to an effect on the pro-caspase 3 pathway.A similar trend can be observed with the fragmentation of PARP-1, a typical caspase-3 target (Figure 7) commonly cleaved in apoptotic cell death programs.All active compounds, except 19, possess the ability to promote the fragmentation of both procaspase 3 and PARP-1.Also in this case, non-cytotoxic 28 and 29 do not exert any influence on PARP-1.The anomaly observed in the behaviour of compound 19 may suggest the involvement of other pathways in the cytotoxicity of this compound.Further investigations are on the way.Upon observing the chemical structures of the molecules discussed above, it comes into evidence that the more active compounds 24 and 25 possess a long, linear aliphatic chain (8C and 10C, respectively) linked to amide-nitrogen.On the contrary, molecules containing highly branched side chains, that is, 28 and 29, are not active.
The bioactivity of the remaining set of tested molecules lies in between those extremes.This mere hypothesis, although quite reasonable, must be confirmed by docking investigations.

Experimental Section
Optical rotations were measured in a CH 3 OH solution on a JASCO P-1010 digital polarimeter (Lecco, Italy) at 25 • C and at 589 nm.The NMR spectra were run on a Bruker Avance II (Milan, Italy) instrument operating at 600 MHz for 1 H-NMR and at 125 MHz for 13 C-NMR.Chemical shifts (δ) were indirectly referred to tetramethylsilane using residual solvent signals.Deuterated solvents such as CDCl 3 -d 1 , DMSO-d 6 , and CD 3 OD-d 4 were used for the solubilization of the various synthesized compounds.Residual solvent signals of δ = 7.27 ppm in 1 H and δ = 77.00ppm in 13 C for CDCl 3 -d 1 , δ = 2.50 ppm in 1 H and δ = 39.51 ppm in 13 C for DMSO-d 6 , and δ = 3.31 ppm in 1 H and δ = 49.15ppm in 13 C for CD 3 OD-d 4 were used as references in NMR spectra.DEPT, 1 H-1 H-COSY, HMBC, HSQC, and NOESY experiments were performed using Bruker microprograms.Mass spectra were obtained using an HPLC-ESI-QTOF HRMS apparatus (Agilent, Milan, Italy).The HPLC system was an Agilent 1260 Infinity.A reversed-phase C 18 column (ZORBAX Extended-C 18 2.1 × 50 mm, 1.8 µm) with a Phenomenex C 18 security guard column (4 × 3 mm) was used.The flow rate was 0.4 mL/min, and the column temperature was set to 30 • C. The mass spectra was recorded using an Agilent 6540 UHD accurate-mass Q-TOF spectrometer equipped with a Dual AJS ESI source working both in negative and positive modes.Merck Si gel (70-230 mesh) deactivated with 15% H 2 O was used for column chromatography.TLCs were performed on silica gel (Merck, Kieselgel 60 F 254 , 0.25 and 0.50 mm) plates.The spots were visualized by spraying with 5% 4-anisaldehyde in EtOH acid.All chemicals, such as dried DMF, DMAP, EDCI, HOBt, and Dess-Martin reagent, were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further purification; H 2 O, CH 3 CN, and HCOOH for HPLC-UV were of special grade (Carlo Erba, Milan, Italy).

General Procedure for the Synthesis of Atractyligenin Amides (5-18)
A general procedure previously reported for the synthesis of rosmarinic acid amides by Cardullo et al. [51] was followed for the synthesis of amides 5-18 of 1 (Figure 8).Atractyligenin (1.0 equiv.) was dissolved in dry DMF (2 mL) in a three-necked flask previously filled with N 2 , and DMAP (1.3 equiv.) was added.The mixture was stirred at 0 • C for 10 min; then, EDCI (1.3 equiv.)and HOBt (1.3 equiv.)were added dropwise to the solution with a syringe.The mixture was stirred at 0 • C for 30 min under an N 2 atmosphere.Finally, the suitable amine (1.3 equiv.) was added to the mixture, stirring for 3 h at r.t.The crude reaction was evaporated under vacuum to remove the solvent, and the residue was partitioned between EtOAc (20 mL) and 1 N HCl (3 × 20 mL); then, the organic layer was partitioned with saturated NaHCO 3 solution (3 × 20 mL).The organic phase was washed with water, dried over anhydrous Na 2 SO 4 , filtered, and taken to dryness.Each residue was purified by classic liquid column chromatography.

Experimental Section
Optical rotations were measured in a CH3OH solution on a JASCO P-1010 digital polarimeter (Lecco, Italy) at 25 °C and at 589 nm.The NMR spectra were run on a Bruker Avance II (Milan, Italy) instrument operating at 600 MHz for 1 H-NMR and at 125 MHz for 13 C-NMR.Chemical shifts (δ) were indirectly referred to tetramethylsilane using residual solvent signals.Deuterated solvents such as CDCl3-d1, DMSO-d6, and CD3OD-d4 were used for the solubilization of the various synthesized compounds.Residual solvent signals of δ = 7.27 ppm in 1 H and δ = 77.00ppm in 13 C for CDCl3-d1, δ = 2.50 ppm in 1 H and δ = 39.51 ppm in 13 C for DMSO-d6, and δ = 3.31 ppm in 1 H and δ = 49.15ppm in 13 C for CD3OD-d4 were used as references in NMR spectra.DEPT, 1 H-1 H-COSY, HMBC, HSQC, and NOESY experiments were performed using Bruker microprograms.Mass spectra were obtained using an HPLC-ESI-QTOF HRMS apparatus (Agilent, Milan, Italy).The HPLC system was an Agilent 1260 Infinity.A reversed-phase C18 column (ZORBAX Extended-C18 2.1 × 50 mm, 1.8 μm) with a Phenomenex C18 security guard column (4 × 3 mm) was used.The flow rate was 0.4 mL/min, and the column temperature was set to 30 °C.The mass spectra was recorded using an Agilent 6540 UHD accurate-mass Q-TOF spectrometer equipped with a Dual AJS ESI source working both in negative and positive modes.Merck Si gel (70-230 mesh) deactivated with 15% H2O was used for column chromatography.TLCs were performed on silica gel (Merck, Kieselgel 60 F254, 0.25 and 0.50 mm) plates.The spots were visualized by spraying with 5% 4-anisaldehyde in EtOH acid.All chemicals, such as dried DMF, DMAP, EDCI, HOBt, and Dess-Martin reagent, were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further purification; H2O, CH3CN, and HCOOH for HPLC-UV were of special grade (Carlo Erba, Milan, Italy).

General Procedure for the Synthesis of Atractyligenin Amides (5-18)
A general procedure previously reported for the synthesis of rosmarinic acid amides by Cardullo et al. [51] was followed for the synthesis of amides 5-18 of 1 (Figure 8).Atractyligenin (1.0 equiv.) was dissolved in dry DMF (2 mL) in a three-necked flask previously filled with N2, and DMAP (1.3 equiv.) was added.The mixture was stirred at 0 °C for 10 min; then, EDCI (1.3 equiv.)and HOBt (1.3 equiv.)were added dropwise to the solution with a syringe.The mixture was stirred at 0 °C for 30 min under an N2 atmosphere.Finally, the suitable amine (1.3 equiv.) was added to the mixture, stirring for 3 h at r.t.The crude reaction was evaporated under vacuum to remove the solvent, and the residue was partitioned between EtOAc (20 mL) and 1 N HCl (3 × 20 mL); then, the organic layer was partitioned with saturated NaHCO3 solution (3 × 20 mL).The organic phase was washed with water, dried over anhydrous Na2SO4, filtered, and taken to dryness.Each residue was purified by classic liquid column chromatography.
The crude mixture was purified on a silica gel column eluting with CH 2 Cl 2 -MeOH (98:2) to afford a fraction containing compound 5 (21.5
The crude mixture was purified on a silica gel column eluting with CH 2 Cl 2 -MeOH (98:2) to afford a fraction containing compound 8 (
The crude mixture was purified on a silica gel column eluting with CH
The crude mixture was purified on a silica gel column eluting with CH 2 Cl 2 -MeOH (98:2) to afford a fraction containing compound 11 (
The crude mixture was purified on a silica gel column eluting with CH 2 Cl 2 -MeOH (98:2) to afford a fraction containing compound 13 (21.6
The crude mixture was purified on a silica gel column eluting with CH 2 Cl 2 -MeOH (98:2) to afford a fraction containing compound 14 (25.4
The crude mixture was purified on a silica gel column eluting with CH 2 Cl 2 -MeOH (98:2) to afford a fraction containing compound 15 (24.5
The crude mixture was purified on a silica gel column eluting with CH 2 Cl 2 -MeOH (97:3) to afford a fraction containing compound 17 (21.7
The crude mixture was purified on a silica gel column eluting with CH

Cell Culture Conditions and Reagents
Colon cancer cells (HCT116 and Caco-2) were purchased from the Interlab Cell Line Collection (ICLC, Genoa, Italy) and cultured as monolayers in DMEM supplemented with 10% (v/v) heat-inactivated FCS and 2 mM glutamine in the presence of a 1% penicillin/streptomycin solution [52].Differentiated Caco-2 cells, used as an enterocyte-like model [53,54], were prepared as previously reported following the Natoli procedure [46].
For the reported experiments, cells were seeded in 96-well microplates for MTT assay or 6-well plates for the other experimental conditions.Then, cells were incubated at 37 • C in a humidified atmosphere containing 5% CO 2 for 24 h before proceeding with the treatment with the compounds or vehicle only.Stock solutions of amides or their di-oxidated derivatives were prepared in DMSO and stored at −20 • C until use.All working solutions were prepared in DMEM, never exceeding 0.01% (v/v) DMSO.The vehicle condition reported in each experiment as control was represented by untreated cells incubated in the presence of the corresponding DMSO volume.All cell culture media and culture reagents were provided by Euroclone SpA (Pero, Italy).All other reagents and chemicals, except where differently indicated, were purchased from Millipore Sigma (Milan, Italy).

Assessment of Cell Viability
The cytotoxic effects of analysed compounds were assessed by the colorimetric 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.MTT was purchased from Millipore Sigma (Milan, Italy).For this assay, 8 × 10 3 cells/well were plated in a 96-well plate and incubated with compounds for periods reported in the Results section.Then, 10 µL of MTT solution (5 mg/mL in PBS) of added to the cell medium, and the incubation was protracted for 2 h at 37 • C in the dark.MTT is metabolised to formazan salt in viable cells [55].Afterwards, the media were taken out, and cells were lysed in lysis buffer (20% SDS and 10% dimethylformamide).The cell viability was determined by analysing the absorbance of the formazan read at 490 nm with 630 nm as a reference wavelength using an automatic ELISA plate reader (OPSYS MR, Dynex Technologies, Chantilly, VA, USA).IC 50 values were determined by Graphpad Prism 7.0 software (San Diego, CA, USA).

Analysis of Apoptotic Cell Death by Vital Hoechst Staining
Apoptotic cell death was assessed by vital Hoechst 33342 staining [56] (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to vendor's specifications.Morphological analysis of condensed or fragmented chromatin was estimated by a fluorescence microscope and pictures were taken with Leica Q Fluoro software (Leica Microsystems, Wetzlar, Germany; https://www.leica-microsystems.com/it, (accessed on 21 March 2024)).

Western Blotting Analysis
Whole-cell extracts prepared in ice-cold lysis RIPA buffer (1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS in PBS, pH 7.4) and supplemented with a protease inhibitor cocktail were subjected to SDS-PAGE and consequent immunoblot.PARP-1 antibody (cat.no.sc-53643) was provided by Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), while anti-caspase-3 was from Cell Signaling Technology (cat.no.#9662; Cell Signaling Technology Inc., Beverly, MA, USA).In all performed experiments, the analysed proteins were normalised with γ-tubulin (cat.no.T3559; Sigma-Aldrich, Milan, Italy) used as loading control.In all analyses, protein bands were detected with an ECL™ Prime Western Blotting System (Cytiva, Merck KGaA, Milan, Italy) using a ChemiDoc XRS

Figure 2 .
Figure 2. Effects of atractyligenin and its amide derivatives on cell viability of HCT116 colon cancer cells.Cells were incubated in the presence of compounds for 48 h; then, cell viability was assessed by MTT assay as reported in Section 3.Each value reported in the histogram represents the mean of three independent experiments ± SD.Significant differences compared with the untreated sample are represented by (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 calculated by Student's t-test and oneway analysis of variance.

Figure 2 .
Figure 2. Effects of atractyligenin and its amide derivatives on cell viability of HCT116 colon cancer cells.Cells were incubated in the presence of compounds for 48 h; then, cell viability was assessed by MTT assay as reported in Section 3.Each value reported in the histogram represents the mean of three independent experiments ± SD.Significant differences compared with the untreated sample are represented by (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 calculated by Student's t-test and one-way analysis of variance.

Figure 3 .
Figure 3. Effects of high doses of 19-32 on HCT116 cell viability after 48 h of treatment.Values reported in the histogram represent the mean of three independent experiments ± SD.Significant differences compared with the untreated sample are represented by (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 calculated by Student's t-test and one-way analysis of variance.

Figure 3 .
Figure 3. Effects of high doses of 19-32 on HCT116 cell viability after 48 h of treatment.Values reported in the histogram represent the mean of three independent experiments ± SD.Significant differences compared with the untreated sample are represented by (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 calculated by Student's t-test and one-way analysis of variance.

Figure 4 .
Figure 4. Effects of low doses of some di-oxidated derivates of atractyligenin amides on HCT116 cell viability at 24 h.Values reported in the histogram represent the mean of three independent experiments ± SD.Significant differences compared with the untreated sample are represented by (*) p < 0.05, and (**) p < 0.01, (***) p < 0.001 calculated by Student's t-test and one-way analysis of variance.

Figure 4 .
Figure 4. Effects of low doses of some di-oxidated derivates of atractyligenin amides on HCT116 cell viability at 24 h.Values reported in the histogram represent the mean of three independent experiments ± SD.Significant differences compared with the untreated sample are represented by (*) p < 0.05, and (**) p < 0.01, (***) p < 0.001 calculated by Student's t-test and one-way analysis of variance.

Figure 5 .
Figure 5. Effects of low doses of some di-oxidated derivates of atractyligenin amides on colon cancer Caco-2 cells and differentiated Caco-2 cells at 24 h of treatment.Values reported in the histogram represent the mean of three independent experiments ± SD.Significant differences compared with the untreated sample are represented by (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 calculated by Student's t-test and one-way analysis of variance.

Figure 5 .
Figure 5. Effects of low doses of some di-oxidated derivates of atractyligenin amides on colon cancer Caco-2 cells and differentiated Caco-2 cells at 24 h of treatment.Values reported in the histogram represent the mean of three independent experiments ± SD.Significant differences compared with the untreated sample are represented by (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 calculated by Student's t-test and one-way analysis of variance.Indeed, a comparative analysis of the IC 50 values in both colon cancer cells, HCT116 and Caco-2, after 24 h of treatment (Table2) unveiled 23 and 24 among the most efficacious compounds in reducing viability in Caco-2 cells, while compounds 24 and 25 were the most active for the HCT116 cell line.The activity of compounds 19 and 25 worsened considerably compared to that shown on the HCT116 cell line, while the activity of compounds 21 and 26 improved slightly on the Caco-2 cell line.

26 Figure 6 .
Figure 6.Effects of some di-oxidated derivates of atractyligenin amides on chromatin condensation and fragmentation visualized by Hoechst staining.Cells were treated with a 10 μM dose for 24 h; then, morphological analysis of HCT116 cells was performed under light microscopy (upper panel).After Hoechst 33342 staining, condensed or fragmented chromatin (lower panel) was visualized using a DAPI filter in a Leica inverted fluorescence microscope.The condensation of the nuclear material (arrowheads), followed by the formation of apoptotic bodies (arrows), is indicated in the figure.White squares correspond to a magnification of stained nuclei.Pictures were taken by Leica Q Fluoro Software (version 2.0, Leica Microsystems S.r.l, Wetzlar, Germany, original magnification of 200×).

Figure 6 .
Figure 6.Effects of some di-oxidated derivates of atractyligenin amides on chromatin condensation and fragmentation visualized by Hoechst staining.Cells were treated with a 10 µM dose for 24 h; then, morphological analysis of HCT116 cells was performed under light microscopy (upper panel).After Hoechst 33342 staining, condensed or fragmented chromatin (lower panel) was visualized using a DAPI filter in a Leica inverted fluorescence microscope.The condensation of the nuclear material (arrowheads), followed by the formation of apoptotic bodies (arrows), is indicated in the figure.White squares correspond to a magnification of stained nuclei.Pictures were taken by Leica Q Fluoro Software (version 2.0, Leica Microsystems S.r.l, Wetzlar, Germany, original magnification of 200×).

Figure 7 .
Figure 7.Western blotting analysis of the apoptotic markers caspase-3 and PARP-1 in HCT116 cells incubated in the presence of a 10 μM dose of di-oxidated derivates of atractyligenin amides for 24 h.Each band was determined by densitometric analysis and normalized with γ-tubulin used as a loading control.Bar charts of densitometric analyses report the mean values of three independent

Figure 7 .
Figure 7.Western blotting analysis of the apoptotic markers caspase-3 and PARP-1 in HCT116 cells incubated in the presence of a 10 µM dose of di-oxidated derivates of atractyligenin amides for 24 h.Each band was determined by densitometric analysis and normalized with γ-tubulin used as a loading control.Bar charts of densitometric analyses report the mean values of three independent experiments.Significant differences compared with the untreated sample are represented by (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 calculated by Student's t-test and one-way analysis of variance.

Table 1 .
IC 50 values of di-oxidated derivates of atractyligenin amides on HCT116 colon cancer cells after 24 h of treatment.

Table 2 .
Comparative analysis of IC 50 values of some di-oxidated derivates of atractyligenin amides tested on HCT116 and Caco-2 colon cancer cells after 24 h of treatment.