Design, Synthesis, and Biological Evaluation of Novel Hydroxamic Acid-Based Organoselenium Hybrids

We report the design and synthesis of novel hydroxamic acid-tethered organoselenium (OSe) hybrids. Their antimicrobial and anticancer activities were assessed against different microbes (e.g., Candida albicans (C. albicans), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus)), as well as liver and breast carcinomas. OSe hybrid 8 showed promising anticancer activity, with IC50 = 7.57 ± 0.5 µM against HepG2 and IC50 = 9.86 ± 0.7 µM against MCF-7 cells. Additionally, OSe compounds 8 and 15 exhibited promising antimicrobial activities, particularly against C. albicans (IA% = 91.7 and 83.3) and S. aureus (IA% = 90.5 and 71.4). The minimum inhibitory concentration (MIC) assay confirmed the potential antimicrobial activity of OSe compound 8. OSe compounds 8 and 16 displayed good antioxidant activities compared to vitamin C in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. These results indicate that hydroxamic acid-based organoselenium hybrids have promising biological activities such as anticancer, antimicrobial, and antioxidant properties, especially compounds 8, 13, 15, and 16, which warrant further studies.


Introduction
Hydroxamic acids (HAs) are among the most important families of organic compounds with general structures R-CO-NHOH and R-CO-NR OH, where R, R represent various organic residues [1,2].
They are distributed throughout nature. Many naturally occurring hydroxamic acids have been isolated and examined for their biological activities. For example, Aspergillic acid and Actinonin have antibiotic activity ( Figure 1) [3].

Introduction
Hydroxamic acids (HAs) are among the most important f pounds with general structures R-CO-NHOH and R-CO-NR′O various organic residues [1,2].
They are distributed throughout nature. Many naturally occ have been isolated and examined for their biological activities. F acid and Actinonin have antibiotic activity ( Figure 1) [3]. Hydroxamic acid moiety has gained significant attention worldwide. Therefore, many hydroxamic derivatives have been ined for their biological activities and chemistry applications [4][5][6] Furthermore, HAs play a distinct role as potent chelating age group that form complexes with various metals, including transi  Hydroxamic acid moiety has gained significant attention from research groups worldwide. Therefore, many hydroxamic derivatives have been synthesized and examined for their biological activities and chemistry applications [4][5][6].
Furthermore, HAs play a distinct role as potent chelating agents, due to the bidentate group that form complexes with various metals, including transition metals [7][8][9].  [20][21][22][23][24]. Surprisingly, there is less focus on hydroxamic acid-bas pounds and their hydroxamic acid derivatives. In fact, and to ou examples related to hydroxamic acid-based Se in the literature a compounds to SeHA (II) and SeHA (III) [25,26]; these studies have ated anticancer activity, and these compounds were found to be m [25,26].
Se is an essential microelement in the human body. It is a no group 16 of the periodic table and lies between sulfur and tellur found in several dietary sources, such as seafood, organ meats, and plements [26,29]. Se deficiency results in severe consequences, suc dence of cancer and heart diseases. On the other hand, elevated Se lethal to the human body. Accordingly, maintaining the Se concen levels is critical in protecting the immune system and preventing c Therefore, scientists have paid much attention to the develop owing to its diverse applications, which are not only limited to c extend to electronics (e.g., optoelectronics, photocells, and solar c OSe compounds have recently become a hot spot in biochemical inorganic Se compounds, since they are less toxic and have higher Many analogous hydroxamic acids have been reported. For example, sulfur-based hydroxamic acids have been investigated, and their applications have been documented [20][21][22][23][24]. Surprisingly, there is less focus on hydroxamic acid-based selenium (Se) compounds and their hydroxamic acid derivatives. In fact, and to our knowledge, the only examples related to hydroxamic acid-based Se in the literature are the modified HA (I) compounds to SeHA (II) and SeHA (III) [25,26]; these studies have developed and evaluated anticancer activity, and these compounds were found to be more potent than HA (I) [25,26].
Se is an essential microelement in the human body. It is a non-metal that belongs to group 16 of the periodic table and lies between sulfur and tellurium [27,28]. Se can be found in several dietary sources, such as seafood, organ meats, and Brazil nuts, or as supplements [26,29]. Se deficiency results in severe consequences, such as increasing the incidence of cancer and heart diseases. On the other hand, elevated Se concentrations may be lethal to the human body. Accordingly, maintaining the Se concentrations at their normal levels is critical in protecting the immune system and preventing cancer [30][31][32].
Therefore, scientists have paid much attention to the development of Se chemistry, owing to its diverse applications, which are not only limited to cancer therapy but also extend to electronics (e.g., optoelectronics, photocells, and solar cells) [31,32]. Moreover, OSe compounds have recently become a hot spot in biochemical processes compared to inorganic Se compounds, since they are less toxic and have higher bioavailability [30][31][32]. As a result, numerous studies have focused on synthesizing new OSe compounds with potential cancer chemopreventive, antioxidant, antitumor, and antiviral activities [26][27][28][29].
In this context, ebselen (IV) has shown anti-inflammatory, antioxidant, and glutathione peroxidase-like activities as a neuroprotective agent ( Figure 3) [27,28]. Furthermore, selenocyanate V has demonstrated good chemoprotective and chemopreventive activities toward various cancers. Moreover, the diselenide compounds (VI) and (VII) synthesized within our laboratory have displayed great cytotoxic potential against liver cancer cells ( Figure 3) [29].  Therefore, combining OSe compounds with HAs is expected to enhance their overall biological activity. Within the context of this paper, we aimed to synthesize novel hydroxamic acid-based Se compounds (Has-Se) and evaluate them for biological activities such as antimicrobial, anticancer, and antioxidant properties.

Synthesis
The combination of Se and HAs affords unique pharmacophores with possible applications in different fields. Hydroxamic acids are generally obtained from the condensation of carboxylic acids and hydroxylamine. Therefore, Se-containing carboxylic acid 3 was used as a key starting material. We synthesized 4-Oxo-4-((4-selenocyanatophenyl)amino)but-2-enoic acid (3) at 92% from the reaction of corresponding 4-selenocyanatoaniline (2) and maleic anhydride (Scheme 1). An esterification reaction activated the Se-containing carboxylic acid 3. Within this context, we obtained a 77% yield of methyl-4-oxo-4-((4-selenocyanatophenyl)amino)but-2-enoate (4) by 3′s reaction with methanol and H2SO4 (catalytic amounts). Unfortunately, the reaction of 4 with o-benzylhydroxylamine failed, and we could not isolate the desired products (Scheme 1). The reaction of o-benzylhydroxylamine hydrochloride's with chloroacetyl chloride created a 73% yield for 4-N-(benzyloxy)-2-chloroacetamide (7). We prepared 2-((4- Therefore, combining OSe compounds with HAs is expected to enhance their overall biological activity. Within the context of this paper, we aimed to synthesize novel hydroxamic acid-based Se compounds (Has-Se) and evaluate them for biological activities such as antimicrobial, anticancer, and antioxidant properties.

Synthesis
The combination of Se and HAs affords unique pharmacophores with possible applications in different fields. Hydroxamic acids are generally obtained from the condensation of carboxylic acids and hydroxylamine. Therefore, Se-containing carboxylic acid 3 was used as a key starting material. We synthesized 4-Oxo-4-((4-selenocyanatophenyl)amino)but-2-enoic acid (3) at 92% from the reaction of corresponding 4-selenocyanatoaniline (2) and maleic anhydride (Scheme 1). An esterification reaction activated the Se-containing carboxylic acid 3. Within this context, we obtained a 77% yield of methyl-4-oxo-4-((4selenocyanatophenyl)amino)but-2-enoate (4) by 3 s reaction with methanol and H 2 SO 4 (catalytic amounts). Unfortunately, the reaction of 4 with o-benzylhydroxylamine failed, and we could not isolate the desired products (Scheme 1). Therefore, combining OSe compounds with HAs is expected to enhance their over biological activity. Within the context of this paper, we aimed to synthesize novel hydro amic acid-based Se compounds (Has-Se) and evaluate them for biological activities su as antimicrobial, anticancer, and antioxidant properties.
The structure of compound 8 was proved by the IR, which showed absorption bands at 3452 and 3343 cm −1 for the NH2 and NH groups and 1648 cm −1 for the C=O group. The 1 H NMR for compound 8 showed two singlet signals at 4.71 ppm and 3.21 ppm for the two methylene groups, i.e., CH2Se and CH2O. The mass spectrum exhibited molecular ion peaks at 336.20 (M + , 10.01) and the base peak at m/z 91 for the benzyl residue. Once the selenium-based HA 8 was prepared, our focus changed to delivering a range of diverse structures via further derivatization. Within this context, the reaction of 8 with acetic anhydride and acetic formic mixed anhydride created the yields of 85% acetanilide 9 and 34% formamide (Scheme 3).
The structure of compound 9 was proved by the IR, which showed absorption bands at 3297 and 3258 cm −1 for the two NH groups and 1697 and 1665 cm −1 for the two C=O groups. The 1 H NMR for compound 9 showed three singlet signals at 4.92, 3.82, and 2.02 ppm for the CH2O, CH2Se, and CH3 groups, respectively. The mass spectrum exhibited molecular ion peaks at 378.20 (M + , 0.04) and the base peak at m/z 91 for the benzyl residue (see the Supporting Materials). Compound 10 was proved by the IR, which showed absorption bands at 3298 and 3193 cm −1 for the two NH groups and 1706 and 1682 cm −1 for the two C=O groups. The 1 H NMR for compound 10 manifested five singlet signals at 11.18, 10.30, 8.31, 4.74, and 3.40 ppm for the NHO, CHO, NH, CH2O, and the CH2Se groups, respectively. The mass spectrum exhibited molecular ion peaks at 364.25 (M + , 1.43) and the base peak at m/z 91 for the benzyl residue (see Supporting Materials).
The structure of compound 8 was proved by the IR, which showed absorption bands at 3452 and 3343 cm −1 for the NH 2 and NH groups and 1648 cm −1 for the C=O group. The 1 H NMR for compound 8 showed two singlet signals at 4.71 ppm and 3.21 ppm for the two methylene groups, i.e., CH 2 Se and CH 2 O. The mass spectrum exhibited molecular ion peaks at 336.20 (M + , 10.01) and the base peak at m/z 91 for the benzyl residue.
Once the selenium-based HA 8 was prepared, our focus changed to delivering a range of diverse structures via further derivatization. Within this context, the reaction of 8 with acetic anhydride and acetic formic mixed anhydride created the yields of 85% acetanilide 9 and 34% formamide (Scheme 3).
Once the selenium-based HA 8 was prepared, our focus changed t range of diverse structures via further derivatization. Within this context, 8 with acetic anhydride and acetic formic mixed anhydride created the yie etanilide 9 and 34% formamide (Scheme 3).
The structure of compound 9 was proved by the IR, which showed abs at 3297 and 3258 cm −1 for the two NH groups and 1697 and 1665 cm −1 fo groups. The 1 H NMR for compound 9 showed three singlet signals at 4.92, ppm for the CH2O, CH2Se, and CH3 groups, respectively. The mass spect molecular ion peaks at 378.20 (M + , 0.04) and the base peak at m/z 91 for the (see the Supporting Materials).  The structure of compound 9 was proved by the IR, which showed absorption bands at 3297 and 3258 cm −1 for the two NH groups and 1697 and 1665 cm −1 for the two C=O groups. The 1 H NMR for compound 9 showed three singlet signals at 4.92, 3.82, and 2.02 ppm for the CH 2 O, CH 2 Se, and CH 3 groups, respectively. The mass spectrum exhibited molecular ion peaks at 378.20 (M + , 0.04) and the base peak at m/z 91 for the benzyl residue (see the Supporting Materials). Compound 10 was proved by the IR, which showed absorption bands at 3298 and 3193 cm −1 for the two NH groups and 1706 and 1682 cm −1 for the two C=O groups. The 1 H NMR for compound 10 manifested five singlet signals at 11.18, 10.30, 8.31, 4.74, and 3.40 ppm for the NHO, CHO, NH, CH 2 O, and the CH 2 Se groups, respectively. The mass spectrum exhibited molecular ion peaks at 364.25 (M + , 1.43) and the base peak at m/z 91 for the benzyl residue (see Supporting Materials).
The reaction of 8 with succinic and maleic anhydrides created the corresponding N-succinalinic and N-mealnilinic acids 11 and 12, giving 76 and 73% yields, respectively (Scheme 4). By contrast, the reaction of 8 with phthalic anhydride produced a 53% yield of phthaloyl derivative 13 (Scheme 4). The reaction of 8 with succinic and maleic anhydrides created the corresponding succinalinic and N-mealnilinic acids 11 and 12, giving 76 and 73% yields, respectiv (Scheme 4). By contrast, the reaction of 8 with phthalic anhydride produced a 53% yi of phthaloyl derivative 13 (Scheme 4).
The structure of compound 11 was proved by the 1 H NMR, which showed five sing signals at 12.18, 11.16, 10.07, 4.73, 3.37 ppm for the COOH, NH, NH, CH2O, and CH2 groups, respectively (see Supporting Materials). Compound 12 showed characteristic t doublets of doublet signals for the two olefinic carbons at 6.47 and 6.35 ppm. In 13 CNMR, the three carbonyl groups signals appeared at 167.39, 166.84, and 163.76 pp (see the Supporting Materials). The phthaloyl derivative 13 manifested two singlet sign at 4.88 and 3.51 ppm for the two methylene groups, CH2O and CH2Se, respective whereas they appeared at 77.31 and 26.70 ppm in the 13 CNMR. The structure of compound 14 was proved by the IR, which showed absorpti

Evaluation of the Cytotoxicity of the OSe-Based HAs Compounds
Recently, OSe agents have attracted interest due to their potential chemoprote and antitumor properties [30][31][32][33]. We have developed several OSe compounds promising antioxidant, antimicrobial, and antitumor properties [34][35][36][37][38]. Therefore, w timated the OSe agents' antiproliferative activities against different cancer cells, i.e., M 7 and HepG2 cells and healthy lung fibroblast WI-38 cells, using the MTT assay. The doxorubicin was employed as the standard. The minimal inhibition dose causing dea 50% of cells was also estimated and is presented in Table 1. The corresponding therap indices (TI) are described as the proportion of the WI38 cells IC50 to the IC50 of the ca cell (e.g., MCF-7 and HepG2) ( Table 1) [10][11][12][13].
Interestingly, OSe hybrids were more cytotoxic to HepG2 cells than MCF-7 cells example, OSe compound 8 exhibited promising anticancer activity with IC50 = 7.57 µM against HepG2 and IC50 = 9.86 ± 0.7 µM against MCF-7 cells, respectively (Tab OSe compound 15 also demonstrated anticancer activity with IC50 = 15.83 ± 1.3 µM ag HepG2 and IC50 = 21.58 ± 1.6 µM against MCF-7 cells, respectively (Table 1). TI values were more evident in the case of HepG2 cells than in MCF-7 cells. Fo The structure of compound 14 was proved by the IR, which showed absorption bands at 3202 and 3176 cm −1 for the two NH groups and 1652 and 1606 cm −1 for the two C=O groups. The 1 H NMR for compound 14 showed three singlet signals at 4.73, 4.27, and 2.52 ppm for the CH 2 O, CH 2 Se, and the CH 2 Cl, respectively. The mass spectrum exhibited molecular ion peaks at 412.20 (M + + 1) and the base peak at m/z 91 for the benzyl residue (see Supporting Materials). The structure of compound 15 was confirmed by the IR, which showed absorption bands at 3218 and 3176 cm −1 for the two NH groups and 1685 and 1645 cm −1 for the two C=O groups. The 1 H NMR for compound 15 showed three singlet signals at 4.75 and 3.39 ppm for the CH 2 O and CH 2 Se, respectively. The ethyl group showed a distinctive pattern in the 1 HNMR as quartet and triplet at 4.29 and 1.31 ppm for the CH 2 and the CH 3 groups, respectively. The mass spectrum exhibited molecular ion peaks at 460.20 (M + + 1) and the base peak at m/z 91 for the benzyl residue (see Supporting Materials).

Evaluation of the Cytotoxicity of the OSe-Based HAs Compounds
Recently, OSe agents have attracted interest due to their potential chemoprotective and antitumor properties [30][31][32][33]. We have developed several OSe compounds with promising antioxidant, antimicrobial, and antitumor properties [34][35][36][37][38]. Therefore, we estimated the OSe agents' antiproliferative activities against different cancer cells, i.e., MCF-7 and HepG2 cells and healthy lung fibroblast WI-38 cells, using the MTT assay. The drug doxorubicin was employed as the standard. The minimal inhibition dose causing death to 50% of cells was also estimated and is presented in Table 1. The corresponding therapeutic indices (TI) are described as the proportion of the WI38 cells IC 50 to the IC 50 of the cancer cell (e.g., MCF-7 and HepG2) ( Interestingly, OSe hybrids were more cytotoxic to HepG2 cells than MCF-7 cells. For example, OSe compound 8 exhibited promising anticancer activity with IC 50 = 7.57 ± 0.5 µM against HepG2 and IC 50 = 9.86 ± 0.7 µM against MCF-7 cells, respectively (Table 1). OSe compound 15 also demonstrated anticancer activity with IC 50 = 15.83 ± 1.3 µM against HepG2 and IC 50 = 21.58 ± 1.6 µM against MCF-7 cells, respectively (Table 1).
TI values were more evident in the case of HepG2 cells than in MCF-7 cells. For instance, OSe compounds 15 and 8 manifested TI values of 5.2 and 3.6 in the case of HepG2 cells and 3.8 and 2.8 in the case of MCF-7, respectively. Ultimately, these promising, selective antitumor properties are worthy of further studies that employ a more comprehensive panel of normal and cancer cells and in vivo experiments.

Estimation of the Antimicrobial Properties of the OSe-Based HAs Compounds
The encouraging antitumor properties of the OSe compounds motivated us to evaluate their corresponding antimicrobial activities against the C. albicans fungal strain, S. aureus Gram-positive bacteria, and E. coli Gram-negative bacteria. Therefore, we applied the agar diffusion and minimum inhibitory concentration (MIC) methods using clotrimazole antifungal and ampicillin antibacterial drugs as standards. The diameters of the inhibition zones (ZID) (mm) and the percentage activity index (IA%) are presented in Table 2. In general, the antimicrobial activities were more pronounced in the case of S. aureus Gram-positive bacteria and C. albicans fungus. For instance, OSe agents 8 and 15 exhibited promising antimicrobial activities against C. albicans (IA% = 91.7 and 83.3) and S. aureus (IA% = 90.5 and 71.4) ( Table 2).
We evaluated the MIC of the most active OSe compound, 8 (Table 3) to further explore its potential activity. OSe compound 8 showed antimicrobial activities with MIC of 4 µM against C. albicans, MIC of 4 µM against S. aureus, and MIC of 8 against E. coli strains, respectively.

Evaluation of the Antioxidant Properties of the OSe-Based HAs Compounds
The antioxidant activities of the OSe compounds were intensively investigated because these are usually the cause of their chemopreventive potency [34,39]. Their redox activities were evaluated via DPPH and ABTS assays, using ascorbic acid as the standard [40,41]. We spectrophotometrically measured the antioxidant efficiency of the OSe compounds by their ability to decolorise the DPPH . and ABTS . radicals at 734 and 517 nm, respectively ( Figure 2).
As shown in Figure 2, products 8, 10, and 13 showed 73.9, 64.7, and 87.0% antioxidant activities, respectively, compared with 88.4% by vitamin C in the ABTS assay. Similarly,  products 8, 10, and 13 exhibited 85.1, 64.2, and 89.4% antioxidant activities, respectively, whereas vitamin C showed 94.5% in the DPPH assay (Figure 4). These findings encouraged us to estimate the activity of the most active OSe compound, 8, via its respective minimum concentrations, which caused a 50% decrease in the absorbance of the ABTS and DPPH assays, respectively (Table 4). Interestingly, OSe compound 8 has antioxidant, antimicrobial, and anticancer activities, suggesting its potential biological activities.  These findings encouraged us to estimate the activity of the most active OSe compound, 8, via its respective minimum concentrations, which caused a 50% decrease in the absorbance of the ABTS and DPPH assays, respectively (Table 4). Interestingly, OSe compound 8 has antioxidant, antimicrobial, and anticancer activities, suggesting its potential biological activities.

Experimental
Melting points were calculated using Gallenkamp apparatus and were uncorrected. Elemental analyses were carried out at Cairo University. The IR spectra were measured using an Agilent Technologies Cary 630 FTIR instrument at King Faisal University. Mass spectra were recorded at Cairo University on a GC-MS-QP-100 EX Shimadzu instrument. The 1H and 13C NMR spectra were recorded on DMSO-d 6 at King Faisal University or Mansoura University using a Varian Spectrophotometer at 400 MHz. Chemical shifts were recorded in parts per million (ppm), and TMS (0.00 ppm) was used as a reference. Coupling constants (J) are reported in hertz (Hz). Biological experiments were conducted at the Faculty of Pharmacy, Mansoura University. Compound numbers 2, 3, and 6, 7 were synthesised according to previously documented procedures [5,[42][43][44][45][46].
This resulted in the formation of a dark brown solid, which yielded 174 mg (40%); Rf = 0. 43

Anticancer Activity
The products' anticancer activity was examined using the MTT assay against breast (MCF-7) carcinoma cells, liver (HepG2), and normal WI-38 cells, following the previously reported method [38,[44][45][46]. Experimental details can be found in the Supplementary Materials.

Antimicrobial Activity
The products' antimicrobial properties were examined according to the previously reported method, via agar well diffusion assay against E. coli, S. aureus, and C. albicans [45][46][47]. A microdilution method was also used to record MICs (M), following the previously reported procedure [29,45]. Experimental details can be found in the Supplementary Materials.

Antioxidant Activity
The compounds' antioxidant properties were assessed by in vitro bioassays using DPPH and ABTS, following the reported method [34,35,[42][43][44]. Details are in the Supplementary Materials.

Conclusions
Novel hydroxamic acid-tethered OSe hybrids were synthesized in good yields, and their chemical structures were confirmed via spectroscopic methods. In addition, their biological activities were examined. Their antimicrobial and antitumor activities were examined against various microbial strains and cancer cells. OSe compound 8 showed promising anticancer activity with IC 50 = 7.57 ± 0.5 µM against HepG2 and IC 50 = 9.86 ± 0.7 µM against MCF-7 cells. Additionally, OSe compounds 8 and 15 exhibited good antimicrobial activities against C. albicans (IA% = 91.7 and 83.3) and S. aureus (IA% = 90.5 and 71.4).
Similarly, OSe compounds 8 and 16 showed 87 and 81.4% scavenging activities in the ABTS assay, compared to 88.4% by vitamin C. OSe compound 8 had 92.3% scavenging activity, compared to 94.5% by vitamin C in the DPPH assay. These results point to the potential antimicrobial, anticancer, and antioxidant properties of OSe compounds 8, 13, 15, and 16, and warrant further investigation of these hydroxamic acids' derivatives.
While it is too early to evaluate why the OSe 8 was the most potential compound in most of the assays performed, one may guess that it might inhibit specific, biological target(s) and modify specific enzymes or proteins causing activation. Indeed, OSe 8 is amphiphilic, and therefore can enter cells without problems. Moreover, it is likely that OSe 8 may be altered in vivo into a metabolically active intermediate. It can also attack the cysteine proteins via its amino group, and this may upregulate the antioxidant pathways. Of course, these speculations require further in-depth research.
This pilot research has introduced simple synthetic avenues to develop tailor-made HA-based OSe compounds. Some of the synthesized HA-based OSe agents showed potential anticancer activities. We believe this provides ample scope for future study at the biology/chemistry interface. Future research might expand and/or refine the synthetic procedures proposed, building upon more and more diverse HA and OSe building blocks. Accordingly, the development of such HA-based OSe will be mainly driven by an interest in antimicrobial and anticancer drugs.