Anticancer, Antimicrobial, and Antioxidant Activities of Organodiselenide-Tethered Methyl Anthranilates

Novel methyl anthranilate-based organodiselenide hybrids were synthesized, and their chemical structures were confirmed by state-of-the-art spectroscopic techniques. Their antimicrobial properties were assessed against Staphylococcus aureus, Escherichia coli, and Candida albicans microbial strains. Moreover, the antitumor potential was estimated against liver and breast carcinomas, as well as primary fibroblast cell lines. The Staphylococcus aureus and Candida albicans strains were more sensitive than Escherichia coli toward the OSe compounds. Interestingly, methyl 2-amino-5-(methylselanyl) benzoate (14) showed similar antifungal activity to the standard drug clotrimazole (IA% = 100%) and manifested promising antibacterial activity against E. coli (IA% = 91.3%) and S. aureus (IA% = 90.5%). Furthermore, the minimum inhibitory concentration experiments confirmed the antimicrobial activity of the OSe 14, which in turn was comparable to clotrimazole and ampicillin drugs. Interestingly, the anticancer properties were more pronounced in the HepG2 cells. The OSe 14 was the most cytotoxic (IC50 = 3.57 ± 0.1 µM), even more than the Adriamycin drug (IC50 = 4.50 ± 0.2 µM), and with therapeutic index (TI) 17 proposing its potential selectivity and safety. Additionally, OSe compounds 14 and dimethyl 5,5′-diselanediylbis(2-aminobenzoate) (5) exhibited promising antioxidants in the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) in vitro assays with 96%, 92%, 91%, and 86% radical scavenging activities compared to 95% by vitamin C in the DPPH and ABTS assays, respectively. These results point to promising antimicrobial, anticancer, and antioxidant activities of OSe 14 and 5 and warrant further studies.


Introduction
Organoselenium (OSe) compounds have raised much more concern than their inorganic selenium (Se) peers due to their numerous pharmaceutical applications as well as improved pharmacokinetics and bioavailability [1,2]. The non-metal Se element belongs to the oxygen (a.k.a. chalcogen) group. Se has a leading function in immune systems protection and cancer cell proliferation inhibition [3,4]. Various infectious and autoimmune illnesses are somehow linked with the deficiency of Se [4]. On the other hand, Se dietary supplementation was associated with improving different inflammatory disorders and chemoprevention of various types of cancer [5]. Within this context, OSe compounds have shown antiviral, antimicrobial, and antioxidant activities [6]. Due to their diverse pharmacological activities, organodiselenide (OSe 2 ) compounds are among the most evaluated OSe derivatives [7][8][9]. Therefore, researchers have recently been directed to disclose new OSe 2 for biological testing. For instance, diphenyl diselenide (I) is an antidepressant and an antioxidant agent [10]. Moreover, compounds 1,2-bis(chloropyridazinyl) diselenide (II) and bis(4-amino-3-carboxyphenyl) diselenide (III) were able to inhibit the growth of breast cancer cells and manifest efficient anti-leishmanial properties, respectively [5]. Accordingly, combining the pharmacologically active anthranilate scaffolds and OSe2 would promote overall biological activities. Therefore, we aim to design and synthesize new OSe2-tethered anthranilate hybrids and evaluate their respective antimicrobial, anticancer, and antioxidant properties.

Material and Methods
All reagents and solvents used in this study were purchased from Sigma and used without purification. Melting points (MP) were measured on the Gallenkamp apparatus in degrees centigrade. The IR spectra (KBr, λmax.cm −1 ) were recorded on a Mattson spectrophotometer (5000 FTIR) at King Faisal University. The 1 HNMR and 13 CNMR spectra were measured using Varian Spectrophotometer (400 MHz), employing DMSO-d6 as the solvent and TMS internal standard at Mansoura University. The chemical shifts (δ, ppm) were recorded regarding the solvent residual peaks. GC-MS-QP-100 EX Shimadzu apparatus was used for mass measurements at Cairo University. All biological tests were carried out at the Faculty of Pharmacy, Mansoura University. All cell lines and microorganisms were purchased from the VACSERA Company (ATCC), Cairo, Egypt. DPPH and ABTS probes were obtained from Sigma. Compound 2 was synthesized following our literature method [13]. Copies of 1 H & 13 CNMR spectra IR and MS can be found in the supplementary materials Section S2.

Chemistry
Procedure I: The preparation of selenocyanate 4 and diselenide 5. Methyl 2-amino-5-selenocyanatobenzoate (4) was synthesized from the reaction of methyl 2-aminobenzoate with triselenium dicyanide prepared in situ from malononitrile and selenium dioxide in 96% yields. Briefly, selenium dioxide (30 mmol, 3300 mg) was Accordingly, combining the pharmacologically active anthranilate scaffolds and OSe 2 would promote overall biological activities. Therefore, we aim to design and synthesize new OSe 2 -tethered anthranilate hybrids and evaluate their respective antimicrobial, anticancer, and antioxidant properties.

Material and Methods
All reagents and solvents used in this study were purchased from Sigma and used without purification. Melting points (MP) were measured on the Gallenkamp apparatus in degrees centigrade. The IR spectra (KBr, λ max .cm −1 ) were recorded on a Mattson spectrophotometer (5000 FTIR) at King Faisal University. The 1 HNMR and 13 CNMR spectra were measured using Varian Spectrophotometer (400 MHz), employing DMSO-d6 as the solvent and TMS internal standard at Mansoura University. The chemical shifts (δ, ppm) were recorded regarding the solvent residual peaks. GC-MS-QP-100 EX Shimadzu apparatus was used for mass measurements at Cairo University. All biological tests were carried out at the Faculty of Pharmacy, Mansoura University. All cell lines and microorganisms were purchased from the VACSERA Company (ATCC), Cairo, Egypt. DPPH and ABTS probes were obtained from Sigma. Compound 2 was synthesized following our literature method [13]. Copies of 1 H & 13 CNMR spectra IR and MS can be found in the Supplementary Materials Section S2.

Chemistry
Procedure I: The preparation of selenocyanate 4 and diselenide 5. Methyl 2-amino-5-selenocyanatobenzoate (4) was synthesized from the reaction of methyl 2-aminobenzoate with triselenium dicyanide prepared in situ from malononitrile and selenium dioxide in 96% yields. Briefly, selenium dioxide (30 mmol, 3300 mg) was added to malononitrile (15 mmol, 1000 mg) in 10 mL DMSO, and the mixture was stirred for 20 min at room temperature. Next, the mixture was filtered off to get rid of any formed black selenium, and methyl 2-aminobenzoate (12.5 mmol, 1800 mg) was then added, and the reaction mixture was stirred for a further 2 hr at room temperature. Finally, adding 10 g of ice terminated the reaction, and the formed precipitate was filtered, washed several times with H 2 O and sodium carbonate solution, dried, and recrystallized from petroleum ether.
Compound dimethyl 5,5-diselanediylbis(2-aminobenzoate) (5) was synthesized from the reaction of 4 and sodium hydroxide in 92% yields. Briefly, compound 4 (4 mmol, 1000 mg) was dissolved in EtOH (20 mL), and then sodium hydroxide (4 mmol, 160 mg) was added. The reaction mixture was stirred for 2 h at room temperature, and the resulting precipitate was filtered, washed several times with H 2 O, and recrystallized from chloroform.
Procedure II: The preparation of the OSe azo dyes 6. Dimethyl 5,5 -diselanediylbis(2-aminobenzoate) (5) (2 mmol,916.4 mg) was dissolved in aqueous HCl (4 mL) and cooled to 0-5 • C. Sodium nitrite (4.4 mmol, 331 mg in 10 mL H 2 O) was then added to the previously prepared solution while maintaining the temperature at 0-5 • C. The freshly prepared diazonium salt solution was then added dropwise to a cooled and stirred solution of methylene compounds, e.g., ethyl cyanoacetate (4.4 mmol) and sodium acetate (2000 mg) dissolved in (10 mL) of H 2 O. After stirring the reaction mixture for 3 h at 0-5 • C, the resulting precipitate was filtered, washed with H 2 O, and recrystallized from EtOH.
Procedure III: The preparation of OSe amide-acids 11 and 13. Dimethyl 5,5 -diselanediylbis(2-aminobenzoate) (5) (2 mmol) was added to a stirred solution of maleic or succinic anhydride (4.4 mmol) in dry toluene (15 mL). The mixture was vigorously stirred, and the formed precipitate was separated by filtration. The residue was washed with hot toluene, dried under reduced pressure, and recrystallized from EtOH.
Procedure IV: The preparation of cyclic imide 12.
A mixture containing amide acids (11) (0.5 mmol) and sodium acetate (250 mg) in acetic anhydride (5 mL) was heated at 60-65 • C for three h. The reaction mixture was poured into ice H 2 O and neutralized with sodium carbonate. The precipitate formed was collected by filtration, washed 3x with sodium carbonate solution, and recrystallized from EtOH.

The Biological Assays
The OSe candidates were prepared in DMSO stock solution (10 mM) and kept at −20 • C for further use.

The Anticancer Activity
The anticancer activity of the OSe was performed using the MTT assay against liver (HepG2) and breast (MCF-7) carcinoma cells, as well as normal WI-38 cells following the reported method [14][15][16]. Experimental details can be found in the Supplementary Materials Section S1.

The Antimicrobial Activity
According to the reported method, the OSe agents' antimicrobial properties were estimated against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Candida albicans (C. albicans) microbial strains using the agar well diffusion assay [16,17]. In addition, the MICs (in M) were also recorded via the microdilution method following the reported procedure. Experimental details can be found in the Supplementary Materials Section S1 [16,18].

The Antioxidant Activity
The DPPH and ABTS in vitro bioassays were used to assess the OSe antioxidant activities following the reported method [16,[19][20][21]. Experimental details can be found in the Supplementary Materials Section S1.

Synthesis
Recently, OSe 2 compounds have drawn much attention in pharmaceutical chemistry owing to their broad medicinal applications (e.g., antioxidant, chemopreventive, and anticancer activities) [22][23][24][25]. Unluckily, the synthesis of OSe 2 compounds is limited by using toxic, expensive, and air-sensitive reagents (e.g., potassium selenocyanate) and commercially available selenium precursors. Furthermore, poor functional-group tolerance and sophisticated reaction conditions (e.g., under inert gas) are significant drawbacks of synthesizing the OSe 2 compounds [15,26]. Accordingly, synthesizing new and stable OSe 2 synthons is required to develop potential libraries for biological testing. Moreover, anthranilic acid derivatives manifested diverse pharmacological applications and were used to prepare Biomolecules 2022, 12, 1765 7 of 14 different marketed drugs [11,12,27]. Therefore, combining the diselenide functionality into the backbone of anthranilic acid will enable access to unprecedented organic candidates designed to interfere with biotargets. The compound 2-amino-5-selenocyanatobenzoic acid (3) [13] drew our attention and was employed as a start to our synthetic approach. Compound 3 was synthesized by the selenocyanation of 2-aminobenzoic acid (1) [13]. However, our attempts to synthesize the diselenide functionality via hydrolysis or reduction of the selenocyanate group were unsuccessful, and we could not isolate any products. This is attributed to the zwitterionic nature of compound 3, which arises from the presence of the carboxylic group in the ortho position to the amino group, which in turn causes difficulties in product isolation.
Furthermore, compound 3 is characterized by its low solubility in most organic solvents and high polarity limited its synthetic applications. Therefore, our synthetic strategy was oriented to mask the carboxylic or the amino group to overcome these difficulties. Accordingly, the alcoholic esterification of compound 3 furnished the respective methyl 2-amino-5-selenocyanatobenzoate (4); however, in low yield (25%). Therefore, we used an alternative synthetic approach to increase the yield starting from methyl 2-aminobenzoate (2) instead of 2-aminobenzoic acid (1). In this case, selenocyanation proceeded smoothly, and the yield was improved to 96% (Scheme 1). As a result, the methyl 2-amino-5selenocyanatobenzoate (4) is new and features good solubility in most organic solvents and lower polarity compared to the corresponding 2-amino-5-selenocyanatobenzoic acid (3). In addition, the hydrolysis of 4 using NaOH in EtOH afforded the corresponding diselenide 5 in 92% yield, thus, giving access to the symmetrical diselenide scaffolds known for their hepatoprotective activities (Scheme 1). The spectral data were used to identify the chemical structure of the diselenide 5. Compound 5 exhibited a characteristic absorption band at 3455 cm −1 and 3344 cm −1 for NH 2 . The 1 HNMR spectrum of compound 5 showed two singlet signals at δ7.00 ppm and δ3.74 ppm related to the proton of NH 2 and OCH 3 , respectively. The MS spectrum of compound 5 showed molecular ion peaks at 460.15(M+H, 20.76) and the base peak at m/z 91. Similarly, bisdiazotization of the diselenide 5 and subsequent coupling with two equivalents of ethyl cyanoacetate afforded the corresponding disdiazo-based diselenide 6 in 81% yield (Scheme 2). Furthermore, the reaction of diselenide 5 with acetic formic anhydride, acetic anhydride, chloroacetyl chloride, and phenoxy acetyl chloride afforded the corresponding diselenide-based formamide 7, acetanilide 8, chloroacetamide 9, and phenoxy acetamide 10 were obtained in 51%, 85%, 93%, and 55% yields, respectively (Scheme 2). Based on its spectrum data, the structure of compound 7 was established. The IR showed distinctive peaks at 3255 cm −1 for the NH and 1681 cm −1 , and 1573 cm −1 for carbonyl groups. The 1 HNMR spectrum of compound 8 showed three singlet signals at δ10.42 ppm for NH, at δ8.39 ppm related to the proton of the formyl group, and at δ3.75 ppm for OCH 3 . Its MS showed a molecular ion peak at 516.10(M, 25.78) and a base peak at m/z 198. Moreover, the reaction of the diselenide 5 with different succinic and maleic anhydrides afforded the corresponding diselenide-based succinanilic 11 and mealanilic acids 13 in 75% and 82% yields, respectively (Scheme 3). Additionally, warming the diselenidebased succinanilic 11 with acetic anhydride afforded the corresponding cyclic succinimide 12 via dehydration and subsequent cyclization, however, in low yield (39%) (Scheme 3). Unfortunately, our attempts to perform the same reaction with the diselenide-based Moreover, the reaction of the diselenide 5 with different succinic and maleic anhydrides afforded the corresponding diselenide-based succinanilic 11 and mealanilic acids 13 in 69% and 75% yields, respectively (Scheme 3). Additionally, warming the diselenide-based succinanilic 11 with acetic anhydride afforded the corresponding cyclic succinimide 12 via dehydration and subsequent cyclization, however, in low yield (39%) (Scheme 3). Unfortunately, our attempts to perform the same reaction with the diselenide-based mealanilic acid 13 were unsuccessful, and the starting material decomposition was often observed (Scheme 3).
Eventually, the one-pot alkaline reduction of the diselenide 5 began employing a mixture of NaBH 4 and NaOH (1:1) in MeOH and subsequent reaction with alkyl halides, namely methyl iodide, benzyl chloride, and 2-chloro-N-phenylacetamide afforded the corresponding organoselenides 14, 15, and 16 in 82%, 93%, and 96% yields, respectively (Scheme 3). The IR spectra of compound 14 demonstrated characteristic absorption bands of NH 2 at 3474 cm −1 , 3363 cm −1 , and 1686 cm −1 for the C=O. The 1 HNMR spectrum of compound 14 exhibited singlet signals at δ6.75 ppm for NH 2 , singlet signal at δ3.62 ppm for OCH 3, and singlet signal at δ2.2 ppm for the SeCH 3 . The mass spectrum coincided with the predicted molecular mass for the proposed structure at m/z 245.10. Eventually, the one-pot alkaline reduction of the diselenide 5 began employing a mixture of NaBH4 and NaOH (1:1) in MeOH and subsequent reaction with alkyl halides, namely methyl iodide, benzyl chloride, and 2-chloro-N-phenylacetamide afforded the corresponding organoselenides 14, 15, and 16 in 82%, 93%, and 96% yields, respectively (Scheme 3). The IR spectra of compound 14 demonstrated characteristic absorption bands of NH2 at 3474 cm −1 , 3363 cm −1, and 1686 cm −1 for the C=O. The 1 HNMR spectrum of compound 14 exhibited singlet signals at δ6.75 ppm for NH2, singlet signal at δ3.62 ppm for OCH3, and singlet signal at δ2.2 ppm for the SeCH3. The mass spectrum coincided with the predicted molecular mass for the proposed structure at m/z 245.10.
Accordingly, the anticancer properties of the OSe hybrids were assessed against two cancer cell lines, i.e., HepG2 and MCF-7 cells. Furthermore, their corresponding cytotoxicity was also estimated against the immortalized lung WI-38 fibroblasts employing the MTT assay. The Adriamycin cancer drug was used as the positive control. The concentration inhibition, 50%, needed to kill half of the cells (IC 50 ) was estimated from (concentrationresponse plots) and tabulated in Table 1. Furthermore, the safety and selectivity of drugs are assessed from their respective therapeutic indices (TI), defined as the ratio between the IC 50 exhibited by the compound against WI38 cells to the compound's respective IC 50 against cancer cells, i.e., HepG2 and MCF-7 cells (Table 1).
Drugs with high TI values kill cancer cells, leaving normal cells unaffected. Therefore, these drugs are highly preferable in chemotherapy, and the high TI values point to the potential selectivity and safety of a specific drug candidate [18,[33][34][35][36]. Within this context, better TI values were noticed in the case of HepG2 cells compared to the MCF-7 cells. The best selective cytotoxicity patterns for HepG2 cells were observed in the case of OSe compounds 14 and 5 with TI values of 17 and 5. On the other hand, OSe compounds 14, 5, and 6 showed modest selective cytotoxicity patterns for MCF-7 cells with TI values of 11, 3.6, and 2.7 (Table 1). Eventually, such interesting selective anticancer activity merits more investigations using a broader panel of normal and cancerous cells and in vivo experiments.

Evaluation of the Antimicrobial Activities of the OSe Compounds
The promising anticancer activities manifested by the OSe compounds encourage us to further evaluate their respective antimicrobial activity against a panel of gram-negative bacteria (e.g., E. coli) and the gram-positive bacteria (e.g., S. aureus) as well as a fungal strain (C. albicans) using the method of agar diffusion. The clotrimazole antifungal and the ampicillin antibacterial drugs were used as the positive controls. The zones of inhibition diameters (ZID) (in mm) and the activity index percentage (IA%) are shown in Table 2. In general, C. Albicans fungus and S. aureus gram-positive bacteria were more sensitive than gram-negative bacteria toward the OSe compounds. Interestingly, OSe 14 showed similar antifungal activity against C. albicans to the standard drug clotrimazole (IA% = 100%) and exciting antibacterial activity against E. coli (IA% = 91.3%) and S. aureus (IA% = 90.5%). Accordingly, the minimum inhibitory concentration (MIC) of 14 was also estimated (Table 3) to confirm its antimicrobial activity. In this regard, 14 manifested similar antimicrobial activity to the standard drugs clotrimazole and ampicillin drugs with MIC = of 2, 2, and 0.5 µM against C. albicans, S. aureus, and E. coli strains, respectively. Similarly, OSe compounds 15 and 5 have shown excellent antimicrobial activities with A% of 91.7% and 87.5% against C. albicans, 85.7% and 85.7% against S. aureus, and 73.9% and 82.6% against E. coli. Additionally, good antimicrobial activities were also observed in the case of OSe compounds 6 and 4, with IA% of 70.8% and 75% against C. albicans, 76.2% and 76.2% against S. aureus, and 69.6% and 60.9% against E. coli (Table 2). Ultimately, such interesting antimicrobial patterns are worth further research and screening against an extensive panel of bacterial and fungal strains.

The Antioxidant Properties of the OSe Compounds
The redox modulation activities of the OSe compounds were extensively explored over the last decade owing to their chemopreventive and antioxidant potency [7,37]. The latter is usually investigated by the ABTS and DPPH in vitro tests employing vitamin C as the positive control [21,38]. The OSe compound's magnitude estimated the antioxidant potential to decolorize the distinctive colors of the DPPH . and ABTS . radicals at 517 nm and 734 nm, respectively ( Figure 2).

The Antioxidant Properties of the OSe Compounds
The redox modulation activities of the OSe compounds were extensively explored over the last decade owing to their chemopreventive and antioxidant potency [7,37]. The latter is usually investigated by the ABTS and DPPH in vitro tests employing vitamin C as the positive control [21,38]. The OSe compound's magnitude estimated the antioxidant potential to decolorize the distinctive colors of the DPPH . and ABTS . radicals at 517 nm and 734 nm, respectively ( Figure 2).

Conclusions
Sixteen new diselenide-tethered methyl anthranilate hybrids were synthesized in excellent yields (up to 96%), and their chemical structures were elucidated using different spectroscopic techniques. Their antimicrobial and antitumor activities were assessed
Furthermore, OSe compounds 14 and 5 exhibited promising antioxidants with 96%, 92%, 91%, and 86% scavenging activities compared to 95% by vitamin C in the DPPH and ABTS assays, respectively. To this end, these results point to potential antimicrobial, anticancer, and antioxidant activities of the OSe 14 and warrant further studies.