Synthesis of a New Class of β-Carbonyl Selenides Functionalized with Ester Groups with Antioxidant and Anticancer Properties—Part II

A series of phenyl β-carbonyl selenides with o-ester functionality substituted on the oxygen atom with chiral and achiral alkyl groups was synthesized. All compounds are the first examples of this type of organoselenium derivatives with an ester substituent in the ortho position. The obtained derivatives were tested as antioxidants and anticancer agents to see the influence of an ester functionality on the bioactivity of β-carbonyl selenides by replacing the o-amide group with an o-ester group. The best results as an antioxidant agent were observed for O-((1R,2S,5R)-(−)-2-isopropyl-5-methylcyclohexyl)-2-((2-oxopropyl)selanyl)benzoate. The most cytotoxic derivative against breast cancer MCF-7 cell lines was O-(methyl)-2-((2-oxopropyl)selanyl)benzoate and against human promyelocytic leukemia HL-60 was O-(2-pentyl)-2-((2-oxopropyl)selanyl)benzoate.


Introduction
Organic molecules, both of a synthetic and natural origin, are utilized in almost every field of industry, depending on their applicability, determined mainly by the particular functional groups incorporated into their structure.Among those, esters are one of the most exploited compounds, commonly known for their characteristic fragrance properties and widely used in the perfume industry [1].In addition to the natural presence in essential oils and pheromones, the ester bond is also a dominating functionality in many primary (e.g., lipids and carbohydrates [2]) and secondary (e.g., lactones, terpenoids and steroids [3]) metabolites [4].The ester linkage is essential for several biochemical pathways due to its ability and facile cleavage by esterases, hydrolase-type enzymes that split the ester into a carboxylic acid and alcohol [5][6][7].In the context of drug development, the introduction of an ester moiety bears several advantages.The transformation of bioactive compounds like polar carboxyl or hydroxyl groups into an ester prodrug improves its solubility and bioavailability [8].Increased membrane permeability facilitates the delivery and installation inside the cells, where the molecule can be hydrolyzed to its water-soluble active form [9,10].Using esterification in drug modification solves the bioavailability problem of many known bioactive agents.Examples of drugs applied in their ester form are presented in Figure 1 [11][12][13].Organoselenium compounds like selenides, diselenides and selenols are well known to possess a multitude of possible biological activities due to the ability to mimic the activity of several selenoenzymes, including the antioxidant enzyme glutathione peroxidase (GPx), and they are involved in thyroid physiology, thioredoxin reductase (TRx) and iodothyronine deiodinase (ID) [14][15][16].A catalytic reduction in peroxides by GPx is presented in Scheme 1 [17].L-selenocysteine 1 builds up the active side of the enzyme and catalyzes the ROOH reduction to ROH [18].The Se atom of 2 is maintained in its active ionized form due to the specific amino acid environment-glutamine and tryptophan-creating the catalytic triad with L-Sec [19].The formed seleninic acid 3 is reduced to the starting RSe − in a two-step process by two glutathione (GHS) molecules.Se-derivatives acting as artificial L-Sec can exhibit various bioactivities [20].Until now, we have synthesized a diversified series of GPx mimetics, including benzisoselenazolones 5, corresponding diselenides 6 [21] and phenyl selenides 7 [22].An evaluation of their antiproliferative activity revealed that installing a -SePh functionality significantly decreases the reactivity.To address this issue, we created a new type of selenide-type catalyst possessing a 2-(2-oxopropyl)selanyl group 8 and showed that this modification significantly enhanced the reactivity of the tested compounds [23].
Herein, we wanted to examine the influence of an ester functionality on the bioactivity of β-carbonyl selenides by replacing the o-amide group with an o-ester substituent.For this purpose, we synthesized the first β-carbonyl phenyl selenides possessing an o-ester group and evaluated if this modification changes their antioxidant and anticancer properties.Organoselenium compounds like selenides, diselenides and selenols are well known to possess a multitude of possible biological activities due to the ability to mimic the activity of several selenoenzymes, including the antioxidant enzyme glutathione peroxidase (GPx), and they are involved in thyroid physiology, thioredoxin reductase (TRx) and iodothyronine deiodinase (ID) [14][15][16].A catalytic reduction in peroxides by GPx is presented in Scheme 1 [17].L-selenocysteine 1 builds up the active side of the enzyme and catalyzes the ROOH reduction to ROH [18].The Se atom of 2 is maintained in its active ionized form due to the specific amino acid environment-glutamine and tryptophan-creating the catalytic triad with L-Sec [19].The formed seleninic acid 3 is reduced to the starting RSe − in a two-step process by two glutathione (GHS) molecules.Se-derivatives acting as artificial L-Sec can exhibit various bioactivities [20].Until now, we have synthesized a diversified series of GPx mimetics, including benzisoselenazolones 5, corresponding diselenides 6 [21] and phenyl selenides 7 [22].An evaluation of their antiproliferative activity revealed that installing a -SePh functionality significantly decreases the reactivity.To address this issue, we created a new type of selenide-type catalyst possessing a 2-(2-oxopropyl)selanyl group 8 and showed that this modification significantly enhanced the reactivity of the tested compounds [23].

Results and Discussion
The Herein, we wanted to examine the influence of an ester functionality on the bioactivity of β-carbonyl selenides by replacing the o-amide group with an o-ester substituent.For this purpose, we synthesized the first β-carbonyl phenyl selenides possessing an o-ester group and evaluated if this modification changes their antioxidant and anticancer properties.

Results and Discussion
The first step in the research involved synthesizing a new class of compounds-Osubstituted 2-((2-oxopropyl)selanyl)-benzoate derivatives.The procedure was based on the reaction of acetylated selenide 11, formed in situ from 2-(chloroseleno)-benzoyl chloride A and acetone, with commercially available alcohols.Initially, the reaction of acetone with sodium bicarbonate yielded a carbanion which was used to acylate the selenium atom.A subsequent reaction with alcohol B furnished the final product 10 with only a 16% yield (entry 1, Table 1).In the case of a reaction with several other alcohols, the product was not formed.In the next attempt, NaHCO 3 was replaced with triethylamine.In this way, the reaction efficiency increased to 54% (entry 2, Table 1).The reaction's key step was mixing the ketone with triethylamine to form the carbanion and remove the hydrogen chloride produced in the reaction.The selected conditions (entry 2, Table 1) were applied in further reactions with commercially available alkyl achiral and chiral alcohols, including monocyclic terpene alcohol.A series of β-carbonyl selenides, seven alkyl derivatives 12-18 and seven optically active compounds, including O-terpenyl selenide 19 and three pairs of enantiomers 10 and 20-24, were obtained with good yields (Scheme 2).
All derivatives were evaluated as antioxidants by two assays.Firstly, the NMR-based method was used, as presented by Iwaoka and co-workers [24].The ability to reduce hydrogen peroxide was measured by the rate of dithiothreitol oxidation to disulphide (DTTred to DTTox).The active Se-catalyst is formed through the oxidation of selenide 25 to selenoxide 26 by hydrogen peroxide.Dithiothreitol (DTTred) reduces compound 26 to the starting selenide 25.The substrate (DTTred) disappearance rate was estimated by recording changes in the 1H NMR spectrum in specific time intervals.The reaction equation and antioxidant test results (mean value ± standard deviation) are shown below (Table 2), and all obtained results are presented in the Supplementary Materials (Tables S1 and S2).The best result was observed for O-(propyl)-2-((2-oxopropyl)selanyl)benzoate 14.However, the conversion obtained for all derivatives was less efficient than for ebselen and the previously obtained derivatives with the o-amide group.As presented in Figure 2, it can be observed that the presence of an o-ester substituent instead of an o-amide group in the structure of β-carbonyl selenides significantly lowers the ability to reduce H 2 O 2 .In the case of enantiomers 20/21 and their counterparts with the o-amide group, these properties are reduced by as much as 9 times.
Ebselen 75 64 58 52 The best result was observed for O-(propyl)-2-((2-oxopropyl)selanyl)benzoate 14.However, the conversion obtained for all derivatives was less efficient than for ebselen and the previously obtained derivatives with the o-amide group.As presented in Figure 2, it can be observed that the presence of an o-ester substituent instead of an o-amide group in the structure of β-carbonyl selenides significantly lowers the ability to reduce H2O2.In the case of enantiomers 20/21 and their counterparts with the o-amide group, these properties are reduced by as much as 9 times.During our previous research, we also obtained a series of benzisoselenazolones and diselenides N-functionalized with long carbon chains terminated with ester groups.The obtained results indicate high reactivity of compounds possessing a Se-N bond along with the ester functionality.This suggests that the isoselenazolone core is essential for the elevated antioxidant potential of the ester-type Se-derivatives [25].
Because organoselenium compounds are well known for their ability to act as antioxidants and/or pro-oxidants, the compounds obtained were tested for their capacities to scavenge free radicals.The results obtained for the DPPH assay are presented in Figure 3 and listed in Table S1 (Supplementary Materials).During our previous research, we also obtained a series of benzisoselenazolones and diselenides N-functionalized with long carbon chains terminated with ester groups.The obtained results indicate high reactivity of compounds possessing a Se-N bond along with the ester functionality.This suggests that the isoselenazolone core is essential for the elevated antioxidant potential of the ester-type Se-derivatives [25].
Because organoselenium compounds are well known for their ability to act as antioxidants and/or pro-oxidants, the compounds obtained were tested for their capacities to scavenge free radicals.The results obtained for the DPPH assay are presented in Figure 3 and listed in Table S1 (Supplementary Materials).
In this study, the selected β-carbonyl selenides with the o-ester group were tested, and the loss of DPPH radical absorbance was detected in the presence of all.As observed, compound 19 exhibited the best free-radical scavenging capabilities.The calculated IC 50 value was 0.0933 mM at 15 min post-reaction initiation.Given the IC 50 value obtained for Trolox (0.0744 mM) and the remaining tested compounds, the free-radical scavenging capacity of compound 19 can be considered noticeable.
All tested organoselenium derivatives contained the same 2-((2-oxopropyl)selenyl)benzoate core but differed in the attached esterification functional substituents.O-((1R,2S,5R)-(−)-2-isopropyl-5-methylcyclohexyl)-2-((2-oxopropyl)selanyl)benzoate 19 differed from the other studied derivatives by the presence of a terpene moiety in its structure.It surprised us because monoterpenes without conjugated π bonds, such as menthol, did not have satisfactory free-radical scavenging properties in the DPPH assay [26] and antioxidant activity in the ABTS test [27].In our previous research, we obtained a new group of chiral benzisoselenazol-3(2H)-ones substituted on the nitrogen atom with monoterpene moieties, among others, p-menthane [28].The antioxidant activities of synthesized benzisoselenazolones (such as N-menthyl-1,2-benzisoselenazol-3(2H)-one) were evaluated based on the Iwaoka test [24].The pinene derivatives were the most effective, whereas the substrate conversion observed for the menthane derivative after 60 min was only 67%.However, this compound was characterized by the best anticancer activity against MCF-7 cells [28].The latter, the literature data and the values of IC 50 obtained in this study suggest that the presence of terpene moiety [26,27] in the structure may allow compounds with interesting antioxidant and anticancer properties to be obtained.In this study, the selected β-carbonyl selenides with the o-ester group were tested, and the loss of DPPH radical absorbance was detected in the presence of all.As observed, compound 19 exhibited the best free-radical scavenging capabilities.The calculated IC50 value was 0.0933 mM at 15 min post-reaction initiation.Given the IC50 value obtained for Trolox (0.0744 mM) and the remaining tested compounds, the free-radical scavenging capacity of compound 19 can be considered noticeable.
All tested organoselenium derivatives contained the same 2-((2oxopropyl)selenyl)benzoate core but differed in the attached esterification functional substituents.

O-((1R,2S,5R)-(−)-2-isopropyl-5-methylcyclohexyl)-2-((2oxopropyl)selanyl
)benzoate 19 differed from the other studied derivatives by the presence of a terpene moiety in its structure.It surprised us because monoterpenes without conjugated π bonds, such as menthol, did not have satisfactory free-radical scavenging properties in the DPPH assay [26] and antioxidant activity in the ABTS test [27].In our previous research, we obtained a new group of chiral benzisoselenazol-3(2H)-ones substituted on the nitrogen atom with monoterpene moieties, among others, p-menthane [28].The antioxidant activities of synthesized benzisoselenazolones (such as N-menthyl-1,2-benzisoselenazol-3(2H)-one) were evaluated based on the Iwaoka test [24].The pinene derivatives were the most effective, whereas the substrate conversion observed for the menthane derivative after 60 min was only 67%.However, this compound was characterized by the best anticancer activity against MCF-7 cells [28].The latter, the In the case of the remaining tested compounds, the organoselenium derivatives of chiral alcohols (20/21, 10/22 and 23/24) exhibited better free-radical scavenging properties compared to those derived from aliphatic alcohols (12)(13)(14)(15)(16)(17) or benzyl alcohol (18).Moreover, the capacity for free-radical inhibition among this group of compounds was the highest for butyl esters and the lowest for products incorporating methylbenzyl in their structure.
The inferior properties of inhibiting free radicals for organoselenium derivatives of aliphatic alcohols may stem from the absence of π bonds in the functional moiety.However, compound 17, containing a double bond, was not characterized by better free-radical quenching properties.The lowest TEAC value (0.0166) was determined for compound 18, which may be due to the appearance of an aromatic ring in the structure.A similar situation was observed by Wojtunik et al. [26] for p-cymene.It indicates that further research on these compounds should concentrate on elucidating their mechanisms of action as free-radical inhibitors.
The literature extensively discusses the relationship between the structures of wellknown antioxidants (such as polyphenols) and their antioxidant activity.However, for the discussed compounds, the literature data are very scarce.For this reason, the IC 50 values for the selected and obtained derivatives were compared with the results for analogous organoselenium compounds described previously [23] (Figure 4). of action as free-radical inhibitors.
The literature extensively discusses the relationship between the structures of wellknown antioxidants (such as polyphenols) and their antioxidant activity.However, for the discussed compounds, the literature data are very scarce.For this reason, the IC50 values for the selected and obtained derivatives were compared with the results for analogous organoselenium compounds described previously [23] (Figure 4).Given the obtained results, it is difficult to estimate how the replacement of functional groups allowed for organoselenium derivatives with better properties to inhibit the action of free radicals.For example, in the case of compounds 23/24, these properties increased modestly, while for compounds with a sec-butyl group (20/21), they increased significantly.On the other hand, we did not notice a decrease in the IC50 value for derivatives with an α-methyl benzyl group 10/22.Generally, one of the compounds with the highest reactivity for derivatives from the o-amide group was observed for N-(trans)-indanyl selenide, for which the IC50 value was 0.96 mM.Similar compounds synthesized in this work with an indanyl moiety 23/24 but without a hydroxyl group showed an even higher ability to scavenge free radicals.Given the obtained results, it is difficult to estimate how the replacement of functional groups allowed for organoselenium derivatives with better properties to inhibit the action of free radicals.For example, in the case of compounds 23/24, these properties increased modestly, while for compounds with a sec-butyl group (20/21), they increased significantly.On the other hand, we did not notice a decrease in the IC 50 value for derivatives with an α-methyl benzyl group 10/22.Generally, one of the compounds with the highest reactivity for derivatives from the o-amide group was observed for N-(trans)-indanyl selenide, for which the IC 50 value was 0.96 mM.Similar compounds synthesized in this work with an indanyl moiety 23/24 but without a hydroxyl group showed an even higher ability to scavenge free radicals.
Finally, the cytotoxic activity of the obtained compounds 10 and 12-24 against breast cancer MCF-7 and human promyelocytic leukemia HL-60 cell lines were assessed using the cell viability assay MTT [29].The highest cytotoxic potential was observed for the methyl β-carbonyl selenoesters 12 for the MCF-7 cell line and sec-amyl β-carbonyl selenoesters 16 for the HL-60 cell line.All results are shown in Table 3. Previously, we determined the IC 50 values for selenium derivatives like benzisoselenazolones, diselenides, phenyl selenides and β-carbonyl selenides with the o-amide group substituted with different achiral and chiral moieties, also in the form of enantiomeric and diastereomeric pairs.Through the analysis of these results, we can see the differentiation of values between individual groups of compounds and between enantiomers and diastereoisomers.As a representative example, a comparison of the results obtained for enantiomers 10/22 and corresponding Se-derivatives is presented (Table 4).[30,31] 94.2 ± 0.7 0.8 [32] Previously, we determined the IC50 values for selenium derivatives benzisoselenazolones, diselenides, phenyl selenides and β-carbonyl selenides with amide group substituted with different achiral and chiral moieties, also in the fo enantiomeric and diastereomeric pairs.Through the analysis of these results, we c the differentiation of values between individual groups of compounds and be enantiomers and diastereoisomers.As a representative example, a comparison results obtained for enantiomers 10/22 and corresponding Se-derivatives is pres (Table 4).Comparing the IC 50 values obtained for the same α-methylbenzyl derivatives, we can notice that for both molecules with an o-ester and an o-amide group, the best activity was achieved for the S configuration for the HL-60 cell line.Additionally, a greater sensitivity of the HL-60 cell line compared to the MCF-7 cell line to the obtained derivatives with an α-methylbenzyl group can be noticed.We can also see an increase in the anticancer properties of o-ester selenides compared to phenyl selenides.In general, we can observe that the exchange of the o-amide to o-ester moiety enhances the cytotoxic potential of the obtained β-carbonyl selenides.
An important scaffold in our research, which is particularly interesting due to the chirality of compounds and the change in biological activity for individual enantiomers and diastereoisomers, was the hydroxyindanyl moiety.In this work, for β-carbonyl selenide with the o-ester group, we obtained derivatives with an indanyl substituent without an additional hydroxyl group; therefore, the values can not be compared in a direct way.Looking at the results for the other groups of compounds, we can notice lower sensitivity of cancer cells to compounds 23 and 24, but it is difficult to determine whether this is the effect of replacing the amide group with an ester group in the ortho position or the lack of an additional hydroxyl group.
Looking ahead, our primary goals will involve advancing our understanding of apoptosis and DNA damage pathways, which are fundamental to the cytotoxicity mechanisms.By unravelling the molecular mechanisms governing these pathways, we will aim to elucidate the underlying basis for the anticancer effects of our compounds.Understanding apoptosis and DNA damage pathways is crucial because they regulate cell death and survival, especially in cancer.
For column chromatography, Merck 40-63D 60Å silica gel (Merck, Darmstadt, Germany) was used.Alcohols were commercially available from Merck (Merck, Darmstadt, Germany).Their chemical purities were above 98%, and optical purities were above 97%.The MCF-7 (human breast adenocarcinoma) cell line was obtained from the European Collection of Cell Cultures (ECACC, Nice, France), and the HL-60 cell line (human leukemia) was obtained from the European Collection of Authenticated Cell Cultures (ECACC Nice, France).

Conclusions
We developed an efficient method for synthesizing a new group of organoselenium compounds, β-carbonyl phenyl selenides, possessing an ester group in the ortho position.The key step in the synthesis was using triethylamine during the acylation of selenide.The first derivatives with alkyl achiral and chiral scaffolds were obtained.The obtained derivatives were tested for antioxidant and cytotoxic activity.The antioxidant activity was tested in two ways: the reduction in peroxide to water (Iwaoka test) and the quenching of free radicals (DPPH test).As a result of these studies, we observed that the exchange of the amide group to an ester group significantly lowers the H 2 O 2 reduction properties.However, it was observed that the obtained ester derivatives are better free-radical scavengers.The best results were obtained for the compound O-((1R,2S,5R)-(−)-2-isopropyl-5methylcyclohexyl)-2-((2-oxopropyl)selanyl)benzoate, for which the IC 50 value was close to the value for Trolox.Very good results of the DPPH test were also obtained for the sec-butyl and indanyl derivatives.In the case of cytotoxic activity, which was tested on two cell lines, MCF-7 (breast cancer) and HL-60 (promyelocytic leukemia), we did not notice a relevant improvement in these properties.

Figure 2 .
Figure 2. Results of the antioxidant activity measurements of 20/21, 10/22 and corresponding Se-derivatives with o-amide group.

Table 1 .
Optimization of reaction conditions.
Molecules 2024, 29, x FOR PEER REVIEW

.
Optimization of reaction conditions.

Table 2 .
Results of the antioxidant activity measurement.

Entry Solvent (Solubilization of A) Base Conditions and Order of Addition Yield (%)
Scheme 2. Synthetized β-carbonyl selenides with o-ester groups

Entry Solvent (Solubilization of A) Base Conditions and Order of Addition Yield (%)
Scheme 2. Synthetized β-carbonyl selenides with o-ester groups

Table 3 .
The antiproliferative activity of compounds