The Effect of Different Ester Chain Modifications of Two Guaianolides for Inhibition of Colorectal Cancer Cell Growth

Several sesquiterpene lactones (STLs) have been tested as lead drugs in cancer clinical trials. Salograviolide-A (Sal-A) and salograviolide-B (Sal-B) are two STLs that have been isolated from Centaurea ainetensis, an indigenous medicinal plant of the Middle Eastern region. The parent compounds Sal-A and Sal-B were modified and successfully prepared into eight novel guaianolide-type STLs (compounds 1–8) bearing ester groups of different geometries. Sal-A, Sal-B, and compounds 1–8 were tested against a human colorectal cancer cell line model with differing p53 status; HCT116 with wild-type p53 and HCT116 p53−/− null for p53, and the normal-like human colon mucosa cells with wild-type p53, NCM460. IC50 values indicated that derivatization of Sal-A and Sal-B resulted in potentiation of HCT116 cell growth inhibition by 97% and 66%, respectively. The effects of the different molecules on cancer cell growth were independent of p53 status. Interestingly, the derivatization of Sal-A and Sal-B molecules enhanced their anti-growth properties versus 5-Fluorouracil (5-FU), which is the drug of choice in colorectal cancer. Structure-activity analysis revealed that the enhanced molecule potencies were mainly attributed to the position and number of the hydroxy groups, the lipophilicity, and the superiority of ester groups over hydroxy substituents in terms of their branching and chain lengths. The favorable cytotoxicity and selectivity of the potent molecules, to cancer cells versus their normal counterparts, pointed them out as promising leads for anti-cancer drug design.


Introduction
Terpenes have attracted considerable attention of medicinal chemists due to their numerous biological activities [1,2]. Sesquiterpene lactones (STLs) are a group of compounds commonly isolated from various genera of the Asteraceae family and are known to possess diverse biological activities [3]. In addition, several STLs have been tested as lead drugs in cancer clinical trials [4]. Therefore, the cytotoxicity and structure-activity relationship of STLs are the subject of intense interest. The biological activity of these complex plant secondary metabolites appears to be associated with their ability to act as alkylating agents due to a Michael addition reaction by biological nucleophiles to the α-methylene part of the lactone moiety [5,6]. α-methylene-γ-lactone was shown to react rapidly with cysteines to form stable adducts, whereas the endocyclic α, β-unsaturated-γ-lactones reacts slowly with cysteines to form unstable adducts [7]. It was also postulated that the reactions between words, the optimum Sal-A or Sal-B derivatives would exhibit a maximized anti-growth activity while maintaining their cytotoxicity at a minimum. Once absorbed into the cell, such acyl groups might be hydrolyzed in the cell by some esterases back to the original Sal-A or Sal-B [40].
The ester series of acetate, isopropylate, isovalerate, and isobutyrate acyl groups of varying chain length and branching were used to esterify the hydroxy groups at positions C-8 and C-9 in Sal-A and at C-8 in Sal-B. Eight novel compounds (1-4 from Sal-A and 5-8 from Sal-B) were synthesized ( Figure 1, and supplementary material), purified, isolated, and tested. The standard method includes the treatment of Sal-A and Sal-B with the relative carboxylic acid anhydride (RCOOCOR), tri-ethylamine (Et 3 N), and 4dimethylaminopyridine (DMAP) in chloroform produced the respective esters 1-4 and 5-8. The structures of all synthesized salograviolide derivatives were confirmed by 1 H-NMR, 13 C-NMR, and MS spectra. The purity of all compounds was higher than 95%, verified by HPLC before evaluation of their biological efficacies (please see supplementary material for more details). search for the adequate chain length or the suitable molecular geometry to increase lipophilicity without significantly increasing cytotoxicity becomes important. In other words, the optimum Sal-A or Sal-B derivatives would exhibit a maximized anti-growth activity while maintaining their cytotoxicity at a minimum. Once absorbed into the cell, such acyl groups might be hydrolyzed in the cell by some esterases back to the original Sal-A or Sal-B [40]. The ester series of acetate, isopropylate, isovalerate, and isobutyrate acyl groups of varying chain length and branching were used to esterify the hydroxy groups at positions C-8 and C-9 in Sal-A and at C-8 in Sal-B. Eight novel compounds (1-4 from Sal-A and 5-8 from Sal-B) were synthesized ( Figure 1, and supplementary material), purified, isolated, and tested. The standard method includes the treatment of Sal-A and Sal-B with the relative carboxylic acid anhydride (RCOOCOR), tri-ethylamine (Et3N), and 4-dimethylaminopyridine (DMAP) in chloroform produced the respective esters 1-4 and 5-8. The structures of all synthesized salograviolide derivatives were confirmed by 1 H-NMR, 13 C-NMR, and MS spectra. The purity of all compounds was higher than 95%, verified by HPLC before evaluation of their biological efficacies (please see supplementary material for more details).

Anti-Growth Effects of Sal-A, Sal-B, and Derivatives against Colorectal Cancer In Vitro Model and Structure-Activity Analysis
The anti-growth effects of Sal-A, Sal-B, and the eight synthesized derivatives (1)(2)(3)(4)(5)(6)(7)(8) were evaluated in an in vitro model of human colorectal cancer using the 3-(4,5-dimethylthiazol-2-yl-),5-diphenyl tetrazolium bromide (MTT) dye-reduction assay as previously described [41]. This cell line model consists of the HCT116 wild type for p53 and HCT116 p53 −/− null for p53 cells and the normal-like human colon mucosa cells, NCM460, with wild-type p53. The concentrations that induce 50% cell growth inhibition (IC50) are reported in Table 1. The tested derivatives displayed more potent anti-growth effects compared with the parent molecules (Sal-A and Sal-B). Compounds 2, 4, 6, and 8 were found to be the most potent, while 3 and 7 were the least effective among the derivatives but more than the parent molecules (Figures 2 and 3). These active derivatives showed minor anti-growth activity against the normal-like NCM460 cells, indicating differential properties in cancer versus normal cells (Figure 4). The potency of these compounds was then compared to 5-FU on both HCT116 and HCT116 p53 −/− cells ( Figure 5). Remarkably, all the tested compounds, except the parental compound Sal-A, displayed a higher anticancer effect at lower concentrations then 5-FU (approximate IC50 = 19.2 µM at 48 h of treatment). The properties and biological potencies against HCT116 and HCT116 p53 −/− of Sal-A, Sal-B, and their derivatives were analyzed based on the bearing molecular functional groups and their chain geometry.

Anti-Growth Effects of Sal-A, Sal-B, and Derivatives against Colorectal Cancer In Vitro Model and Structure-Activity Analysis
The anti-growth effects of Sal-A, Sal-B, and the eight synthesized derivatives (1)(2)(3)(4)(5)(6)(7)(8) were evaluated in an in vitro model of human colorectal cancer using the 3-(4,5-dimethylthiazol-2yl-),5-diphenyl tetrazolium bromide (MTT) dye-reduction assay as previously described [41]. This cell line model consists of the HCT116 wild type for p53 and HCT116 p53 −/− null for p53 cells and the normal-like human colon mucosa cells, NCM460, with wild-type p53. The concentrations that induce 50% cell growth inhibition (IC 50 ) are reported in Table 1. The tested derivatives displayed more potent anti-growth effects compared with the parent molecules (Sal-A and Sal-B). Compounds 2, 4, 6, and 8 were found to be the most potent, while 3 and 7 were the least effective among the derivatives but more than the parent molecules (Figures 2 and 3). These active derivatives showed minor anti-growth activity against the normal-like NCM460 cells, indicating differential properties in cancer versus normal cells (Figure 4). The potency of these compounds was then compared to 5-FU on both HCT116 and HCT116 p53 −/− cells ( Figure 5). Remarkably, all the tested compounds, except the parental compound Sal-A, displayed a higher anticancer effect at lower concentrations then 5-FU (approximate IC 50 = 19.2 µM at 48 h of treatment). The properties and biological potencies against HCT116 and HCT116 p53 −/− of Sal-A, Sal-B, and their derivatives were analyzed based on the bearing molecular functional groups and their chain geometry.

The Potency of Sal-A, Sal-B, and Their Derivatives on Colorectal Cancer Cells Is Not Due to Cell Toxicity
The cytotoxicity of the tested compounds on HCT116 cells was quantitatively measured at six hours post-treatment. It was assessed by the measurement of the activity of the stable cytosolic lactate dehydrogenase (LDH) enzyme released to the outer milieu after lysis of damaged cells. When quantified at early time points, this assay allows the detection of the acute effect of the drug as highlighted by cell bursting and LDH release. Cells were treated with the previously selected concentrations. Sal-A, Sal-B, and compound 3 were non-cytotoxic up to 100 µg/mL, while a significant cytotoxic effect was noted starting  The cytotoxicity of the tested compounds on HCT116 cells was quantitatively measured at six hours post-treatment. It was assessed by the measurement of the activity of the stable cytosolic lactate dehydrogenase (LDH) enzyme released to the outer milieu after lysis of damaged cells. When quantified at early time points, this assay allows the detection of the acute effect of the drug as highlighted by cell bursting and LDH release. Cells were treated with the previously selected concentrations. Sal-A, Sal-B, and compound 3 were non-cytotoxic up to 100 µg/mL, while a significant cytotoxic effect was noted starting at concentrations of 10-15 µg/mL for the other derivatives (Figures 6-8). However, these concentrations are at least two folds higher than the previously calculated IC 50 values. at concentrations of 10-15 µg/mL for the other derivatives (Figures 6-8). However, these concentrations are at least two folds higher than the previously calculated IC50 values.    at concentrations of 10-15 µg/mL for the other derivatives (Figures 6-8). However, these concentrations are at least two folds higher than the previously calculated IC50 values.   dehydrogenase assay. Results are expressed as percentage of control and represent the average of three independent experiments ± SEM. Statistical significance is reported by one-way ANOVA post hoc Tukey test indicating differences between treatment concentrations and control (**, p < 0.01; ***, p < 0.001).

Comparing Potencies between Sal-A and Sal-B: Effect of Hydroxy (OH)-Group at the C-8 Position on Colorectal Cancer Cell Growth
The presence of an OH group at the C-8 position next to the α-methylene-γ-lactone moiety in both Sal-A and Sal-B influenced the potency of both molecules as previously reported [8,[42][43][44]. Neighboring OH groups to the molecule's main alkylating center have been shown to enhance the rate of cysteine addition by presumably facilitating the incorporation of the sulfur anion RS− or the proton transfer at an intermediate stage in the Michael-type addition reaction [8]. Sal-B showed enhanced potency over Sal-A (IC50 of 4.4 vs. 31.9 for HCT116, and 5.1 versus 69.5 for HCT116 p53 −/− ) a property attributed to a lower number of OH groups. An increasing number of free OH groups, as in the case of Sal-A, seems to decrease the growth inhibitory activity. A lower number of OH groups correlates with higher lipophilicity followed by enhanced penetration through the cell membranes. Figure 8. Cytotoxic effect of Sal-B derivatives on HCT116 cells. Cells were treated with 1% DMSO (control) or the indicated concentrations of the compounds for six hours. The cytotoxic activity was determined in triplicate wells by the lactate dehydrogenase assay. Results are expressed as percentage of control and represent the average of three independent experiments ± SEM. Statistical significance is reported by one-way ANOVA post hoc Tukey test indicating differences between treatment concentrations and control (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Comparing Potencies between Sal-A and Sal-B: Effect of Hydroxy (OH)-Group at the C-8 Position on Colorectal Cancer Cell Growth
The presence of an OH group at the C-8 position next to the α-methylene-γ-lactone moiety in both Sal-A and Sal-B influenced the potency of both molecules as previously reported [8,[42][43][44]. Neighboring OH groups to the molecule's main alkylating center have been shown to enhance the rate of cysteine addition by presumably facilitating the incorporation of the sulfur anion RS− or the proton transfer at an intermediate stage in the Michael-type addition reaction [8]. Sal-B showed enhanced potency over Sal-A (IC 50 of 4.4 vs. 31.9 for HCT116, and 5.1 versus 69.5 for HCT116 p53 −/− ) a property attributed to a lower number of OH groups. An increasing number of free OH groups, as in the case of Sal-A, seems to decrease the growth inhibitory activity. A lower number of OH groups correlates with higher lipophilicity followed by enhanced penetration through the cell membranes. Sal-B is more hydrophobic than Sal-A as predicted by the calculated log P values (log P of 10.35 mg/mL and 1.06 mg/mL for Sal-A and Sal-B, respectively). This is in agreement with previous reports suggesting the existence of an optimal number of OH groups for STL activity [43].

Neighboring O-Acyl Group Affects Cysteine Addition Rate
The substitution of OH groups by ester functions positioned at C-8 and C-9 in Sal-A and at C-8 in Sal-B showed an increase in the potencies of compounds 1-4 in comparison with Sal-A and compounds 5-8 when contrasted to Sal-B. Relative to Sal-A, the IC 50 of compounds 1, 2, 3, and 4 decreased by 90%, 92%, 78%, and 94%, respectively. Derivatives of Sal-B were also more potent with IC 50 decreasing by 50% for compound 5, 59% for 6, 14% for 7, and 50% for 8. These findings are in agreement with Kupchan et al. [8] who found that the neighboring O-acyl group produced a marked enhancement in the rate of cysteine addition. Therefore, the existence of such number of ester groups in one molecule seems to be an important feature for the growth inhibitory activity.

Steric Hindrance Effects: Lipophilicity and Tertiary Alpha Carbon
The carbon branching of the alkyl chain in the different types of ester derivative seems to have a unique effect on the anti-growth properties. An increase, by one carbon, in the carbon branch from compounds 1 to 2 and 5 to 6 correlates with enhanced potency of the sought derivatives by up to 30%. However, when an additional aliphatic carbon was added to the branch, as it is the case in 3 and 7, potencies lower by approximately 60% than the ones calculated for compounds 2 and 6 were observed. This behavior is attributed to a steric hindrance caused by the isovaleryl group. The bulkiness of the molecule is mostly shown in the least active compound 7 where two isovaleryl groups at C-8 and C-9 are present. In line with previous literature findings, the lipophilic enhancement obtained using larger ester groups is size-limited by steric hindrance effects on the exocyclic methylene group, preventing it from approaching its target [45]. For example, in the bifunctional helenalin and mexicanin I analogs, the addition of a lipophilic character enhanced cytotoxicity against Ehrlich ascites carcinoma in vitro and in vivo; however, there was a size optimum of lipophilic ester groups beyond which STL toxicity decreased [44,45].
Interestingly, the pronounced anti-growth effect of the isopropyl and isobutyl esters (2, 4, 6, and 8) to HCT116 and HCT116 p53 −/− cell lines might be attributed to the presence of a tertiary alpha-carbon in the ester group. The same structure seems to also enhance the cytotoxicity by up to 75% than the primary (1 and 5) and secondary (3 and 7) alpha-carbon.

Conclusions
In the current work, a series of eight new guaianolide-type STLs derivatives were synthesized with ester group(s) modification at either C8-OH, C9-OH, or both, positions. All of the derivatives examined for their inhibition of colorectal cancer cell growth showed that compounds with isopropyl and isobutyl chains were active against HCT116 and HCT116 p53 −/− cells. The difference in anti-growth potency between the synthesized derivatives (compounds 1-8) and their parent compounds (Sal-A and -B) suggests a positive contribution of the ester chain at C-8 and C-9 and confirms the ability of STLs to inhibit colorectal cancer cell growth independently of p53 status. Unspecific cell lysis, as demonstrated by LDH release into the medium, is observed at concentrations about 3-4-times the IC 50 against the normal control cells and at concentrations about 3-6 times against the cancer cells. This finding indicates that the new guaianolide-type STLs derivatives have a specific cell death effect on colorectal cancer cells. Colorectal cancer is one of the most common and deadliest cancers worldwide and there is an urgent need for novel therapies [46,47] It would be interesting to test the effect of the most active derivatives on colorectal cancer cell lines with mutant p53 as the majority of observed mutations in human cancers are missense-type, some of them conferring oncogenic "gain-of-function" mechanisms [28].
Although the target enzymes/proteins affected by the STLs in the tested cell lines used in this study are not yet known, our findings demonstrate that the position of the hydroxy group at C-8, the lipophilicity, the nature of substituent group (OH versus ester), the carbon chain size, and the presence of tertiary alpha-carbon in the ester groups, are all relevant factors to be considered in the interpretation of STL biological activity. These findings highlight the fact that potent ester derivatives adjacent to the α-methylene-δ-lactone in STLs at the C-8 and C-9 positions need to be lipophilic, of an optimal size, and have a tertiary alpha-carbon in the ester group. Interestingly, the derivatization of Sal-A and Sal-B molecules enhanced their anti-growth properties versus 5-FU which is the drug of choice in colorectal cancer therapy for the last six decades.
Taken together, the introduction of ester groups into Sal-A and Sal-B enhanced the growth suppressive activity and improved the selectivity compared with the corresponding parent compounds and increased their potency versus 5-FU. Importantly, this study provides insight into developing novel anti-cancer compounds with high degree of selectivity for cancer versus normal cells. Based on these structure-activity relationships, further optimization-in terms of engineering other ester-based derivatives with tertiary α-carbons and even quaternary ones-and evaluating their biological activity and cell death mechanism of action as anti-colorectal cancer growth inhibitors are ongoing in our laboratory.

Plant Material
The plant material of C. ainetensis was collected from the Bcharre Cedars area in Northern Lebanon at an altitude of 1650 m during the flowering stage in June 2018 (GPS 34 • 11 56.74" N, 36 • 5 14.60" E). Voucher specimens were deposited in the herbarium of the Faculty of Agriculture and Food Sciences at the American University of Beirut (Beirut, Lebanon). The aerial parts were dried by leaving the plant samples in the shade for two weeks before grinding into approximately 10 mm pieces using a blender.

Derivatization
Triethylamine (Et 3 N) (0.5 mmol) was added to a mixture of Sal-A and Sal-B (0.1 mmol each) and DMAP (5 mg) in dichloromethane (CH 2 Cl 2 ) (5 mL). The reaction mixture was cooled to 0 • C and treated with acetic anhydride (0.5 mmol) to prepare compounds 1 and 5, with isobutyric anhydride (0.5 mmol) to prepare compounds 2 and 6, with isovaleric anhydride (0.5 mmol) to prepare compounds 3 and 7, and with (S)-(+)-2-methylbutyric anhydride (0.5 mmol) to prepare compounds 4 and 8. The resulting solution was warmed to room temperature for 12 h. After completion, the reaction mixture was diluted with CH 2 Cl 2 (10 mL), washed with water, and brine. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated in vacuo. The residues were purified via preparative HPLC Maria Galmarini, PharmaMar, Madrid, Spain. The NCM460 normal-like cell line was purchased from INCELL Corporation, LLC San Antonio, Texas. The human colorectal cell line HCT116 was cultured in RPMI medium supplemented with 10% fetal bovine serum, 1% sodium pyruvate, and 1% penicillin-streptomycin. HCT116 p53 −/− cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% sodium pyruvate, 1% nonessential amino acids, and 1% penicillin-streptomycin. The NCM460 normal-like cell line was cultured in M3: Base medium (INCELL Corporation, LLC) supplemented with 10% fetal bovine serum. All cells were maintained in a humidified incubator at 37 • C, 95% air, and 5% CO 2 .
5-FU (Sigma) was dissolved in DMSO at 0.5 mM concentration and stored at −20 • C. Cells in logarithmic phase were seeded in 96-well plates. When 40-50% confluent, cells were treated with vehicle solvent (DMSO) as control or with increasing concentrations of the tested compounds (1, 2.5, 5, 10, 15, 25, and 100 µg/mL) up to 48 h. The use of high concentrations of the tested compounds was mandatory for the calculation of the IC 50 values. The highest used concentrations for the solvent controls did not exceed 1% DMSO; this concentration did not affect the viability of the tested cell lines.

Cytotoxicity and Growth Assays
The cytotoxicity of the tested compounds was quantitatively assessed by the Cytotoxicity Detection Kit PLUS (LDH) (Sigma-Aldrich, Taufkirchen, Germany) at 6 h post-treatment. This kit measures the activity of the stable cytosolic lactate dehydrogenase (LDH) enzyme released to the outer milieu after lysis of damaged cells. When quantified at short time points, it allows the detection of the acute effect of the drug highlighted by cell bursting and LDH release. Briefly, cells were seeded in 96-well plates and treated with increasing concentrations of the tested compounds. Six hours post-treatment, 50 µL of the medium and 50 µL of the reaction mix were incubated for 30 min and then stopped by the addition of the stop solution. The absorbance was detected at 490 nm with the use of an ELISA microplate reader. The results are representative of three independent experiments done in triplicate wells expressed as percentage of control cells and plotted as the mean ± SEM.
Cell growth was assessed by the use of the thiazolyl blue tetrazolium bromide (MTT) assay (Sigma M5655). It quantifies the metabolic activity of the mitochondria converting tetrazolium salt into a blue formazan crystal. Briefly, exponentially growing cells were seeded into 96-well plates and treated with the indicated concentrations of the compounds up to 48 h. At each time point, the resulting formazan crystals were dissolved in a solubilization solution (6 M HCl, 10% SDS, and 5% isobutanol). The absorbance was recorded at 595 nm using an ELISA microplate reader. Cell growth was assayed in triplicates. The results are expressed as percentage of control and represent the average of three independent experiments ± SEM. Cell viability was confirmed by the trypan blue dye exclusion assay.

Statistical Analysis
Data are expressed as mean ± standard error of the mean (SEM) of three independent experiments. Results were analyzed by analysis of variance ANOVA and Bonferroni as post hoc using GraphPad Prism 7. The exact IC 50 values at 48 h were calculated using GraphPad Prism 7 software ± standard error. Differences were considered significant if p < 0.05. Approximate IC 50 values for Sal-A were calculated as 100% cell death was not achieved at 100 µg/mL. Higher Sal-A concentrations than 100 µg/mL were not possible due to solubility limitations.
Supplementary Materials: The following are available online. Figure S1: Effect of Sal-A and Sal-B on the growth of HCT116 colorectal cancer cells. Figure S2: Effect of Sal-A and Sal-B on the growth of HCT116 p53-/-colorectal cancer cells. Figure S3: Effect of the different Sal-A derivatives on the growth of HCT116 cells. Figure S4: Effect of the different Sal-B derivatives on the growth of HCT116 cells. Figure S5: Effect of the different Sal-A derivatives on the growth of HCT116 p53-/-cells. Figure S6: Effect of the different Sal-B derivatives on the growth of HCT116 p53-/-cells. Figure S7: Effect of Sal-A and its most potent derivatives on the growth of NCM460 cells. Figure S8: Effect of