Synthesis and In Vitro Biological Evaluation of p-Carborane-Based Di-tert-butylphenol Analogs

Targeting inflammatory mediators and related signaling pathways may offer a rational strategy for the treatment of cancer. The incorporation of metabolically stable, sterically demanding, and hydrophobic carboranes in dual cycloxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids is a promising approach. The di-tert-butylphenol derivatives R-830, S-2474, KME-4, and E-5110 represent potent dual COX-2/5-LO inhibitors. The incorporation of p-carborane and further substitution of the p-position resulted in four carborane-based di-tert-butylphenol analogs that showed no or weak COX inhibition but high 5-LO inhibitory activities in vitro. Cell viability studies on five human cancer cell lines revealed that the p-carborane analogs R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb exhibited lower anticancer activity compared to the related di-tert-butylphenols. Interestingly, R-830-Cb did not affect the viability of primary cells and suppressed HCT116 cell proliferation more potently than its carbon-based R-830 counterpart. Considering all the advantages of boron cluster incorporation for enhancement of drug biostability, selectivity, and availability of drugs, R-830-Cb can be tested in further mechanistic and in vivo studies.

Herein, we report the synthesis of p-carborane-containing analogs of the di-tertbutylphenol derivatives R-830, KME-4, E-5110, and S-2474, their inhibitory potential toward COX-1, COX-2, and 5-LO, as well as their cytotoxicity on five human cancer cell lines.

Design and Synthesis of Di-tert-butylphenol Analogs
We recently reported the preparation and biological evaluation of eight meta (m)-and p-carborane analogs of the di-tert-butylphenol derivative tebufelone [79]. There, the incorporation of carborane moieties led to loss of COX inhibition, but retained 5-LO inhibitory potential, and to an overall enhanced anticancer activity. Inspired by these results, in particular by the p-carborane-containing analogs, we have now extended the investigation on the bioisosteric replacement of the bulky di-tert-butylphenyl motif with a p-carborane moiety to the di-tert-butylphenols R-830, KME-4, E-5110, and S-2474. These compounds are potent dual COX-2/5-LO inhibitors sharing a conserved 2,6-di-tert-butylphenol motif, but they differ in the p-position bearing different pharmacophores, such as thiophene, γ-sulfonyl lactam, lactam, and γ-lactones (Scheme 1). Katsumi and co-workers demonstrated that the structural combination of at least one tert-butyl group at the ortho-position and an oxygen atom at the p-position is essential for anti-inflammatory activity [51,53]. Accordingly, four p-carborane-containing di-tert-butylphenol analogs were synthesized by replacing the 2,6-di-tert-butylphenol motif with a 1-hydroxy-p-carborane moiety while keeping the pharmacophoric group (Scheme 1). 1-Hydroxy-p-carborane was prepared according to a published procedure [80] and converted quantitatively into the corresponding tertbutyldimethylsilyl (TBDMS) ether by a conventional silylation reaction with excess of tertbutyldimethylsilyl chloride (TBDMSCl) in the presence of triethylamine (NEt 3 ) catalyzed by 4-dimethylaminopyridine (DMAP). The TBDMS-protected p-carborane-containing R-830 analog 1 was obtained by deprotonation and lithiation of the TBDMS-protected hydroxy-p-carborane with nBuLi followed by reaction with excess of the corresponding 2-thiophenecarbonyl chloride in good yield (64%). 1 was subsequently deprotected with tetra-n-butylammonium fluoride (TBAF) to obtain the desired analog R-830-Cb in almost quantitative yield (90%, Scheme 1, (I)). The key intermediate 1-(tert-butyl-dimethylsiloxy)-12-formyl-1,12-dicarba-closo-dodecaborane(12) (p-carboranylaldehyde 2) was obtained by deprotonation and lithiation of the TBDMS-protected 1-hydroxy-p-carborane with nBuLi followed by addition of excess methyl formate in almost quantitative yield (86%). The p-carborane analog S-2474-Cb was prepared in a four-step sequence starting from 2ethylisothiazolidine-1,1-dioxide and p-carboranylaldehyde 2 (Scheme 1, (II)). Selective α-proton abstraction of the γ-sulfonyl lactam precursor by nBuLi under kinetic control followed by addition of the p-carboranylaldehyde 2 led to formation of the intermediate alcohol 3 in moderate yield (53%). Compound 3 was further converted to the corresponding tosylate 4 by deprotonation of the hydroxyl group with sodium hydride (NaH) and subsequent reaction with excess p-toluenesulfonyl chloride (87% yield) to introduce an excellent leaving group. Reaction with NaH, a strong non-nucleophilic base, gave the TBDMS-protected p-carborane analog of S-2474 5 in good yield (64%). Deprotection of the alcohol 5 with TBAF gave the p-carborane analog S-2474-Cb in excellent yield (96%, Scheme 1). The p-carborane-containing analogs KME-4-Cb and E-5110-Cb were prepared in two steps starting from p-carboranylaldehyde 2 and the corresponding Wittig reagents 3-(triphenylphosphoranylidene)-γ-butyrolactone and (1-methoxy-2-oxo-3pyrrolidinyl)triphenyl-phosphonium bromide, respectively (Scheme 1, (III) and (IV)). The Wittig reagents were prepared according to literature procedures und represent key intermediates in the synthesis of both target compounds KME-4-Cb [81] and E-5110-Cb [54]. The TBDMS-protected analogs 6 and 7 were obtained by reaction of the p-carboranylaldehyde 2 and the corresponding Wittig reagents with or without the presence of NEt 3 in good to excellent yields (50-84%). Finally, the isolated TBDMS-protected compounds 6 and 7 were quantitatively converted to the final products KME-4-Cb (99%) and E-5110-Cb (93%) by TBAF-mediated deprotection of the hydroxyl groups. Solubility and chemical stability in organic solvents, such as aqueous dimethyl sulfoxide (DMSO), are crucial for biological investigations. Therefore, the stability of the p-carborane-containing compounds R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb in aqueous DMSO-d6 at room temperature in air was studied by 1 H-and 11 B{ 1 H}-NMR spectroscopy over four weeks and confirmed that all compounds are stable (see Supplementary Materials, Figure S24).

Determination of Lipophilicity (logD) by HPLC
The lipophilicity was determined as logD7.4,HPLC value by a high-performance liquid chromatography (HPLC) method originally described by Donovan and Pescatore (Table  1) [82]. R-830, KME-4, S-2474 and E-5110 have logD values in the range of 3.35-3.91. Interestingly, the introduction of the lipophilic carborane cluster in R-830-Cb, KME-4-Cb, S-2474-Cb, and E-5110-Cb instead of the bulky but also lipophilic di-tert-butylphenyl motif resulted in a marked decrease in lipophilicity and logD values in the range of 1.50-2.37. Solubility and chemical stability in organic solvents, such as aqueous dimethyl sulfoxide (DMSO), are crucial for biological investigations. Therefore, the stability of the p-carborane-containing compounds R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb in aqueous DMSO-d 6 at room temperature in air was studied by 1 H-and 11 B{ 1 H}-NMR spectroscopy over four weeks and confirmed that all compounds are stable (see Supplementary Materials, Figure S24).

Evaluation of Inhibitory Potential toward COX
All compounds were tested in vitro at a concentration of 100 µM for their inhibitory potential toward ovine COX-1 and human recombinant COX-2 using the COX Fluorescent Inhibitor Screening Assay Kit (Cayman Chemical Company) employing the selective COX-2 inhibitor celecoxib as well as the COX-1 inhibitor SC-560 as references (Table 1). While all di-tert-butylphenol derivatives showed the expected inhibition of COX-2 and COX-1 as reported in the literature [46][47][48][49]57], no or only weak inhibition (<26%) of both isoforms was observed for the respective p-carborane analogs (Table 1).
This finding is in line with previous reports claiming that the presence of at least one tert-butyl and a hydroxyl group is essential for anti-inflammatory activity [51,53] which could not be compensated by the hydroxy-substituted p-carborane cluster.

Evaluation of Inhibitory Potential toward 5-LO
All compounds were tested for their 5-LO inhibitory activity in an intact cell assay (polymorphonuclear leukocytes, PMNL) by using the selective 5-LO inhibitor BWA4C as control ( Table 2). The four carborane-based analogs inhibited 5-LO product formation, with IC 50 values in the nanomolar range, comparable to those of the respective reference compounds. These results confirm that the substitution of the di-tert-butylphenyl motif by p-carborane is well tolerated and leads to strong inhibitors of 5-LO product formation.

In Vitro Determination of Cell Viability
Five human cell lines (A375 melanoma, A549 lung adenocarcinoma, HCT116 and HT29 colorectal carcinoma, MDA-MB-231 triple-negative breast adenocarcinoma) were used to study the antitumor effects of the di-tert-butylphenols and their respective carborane analogs. All selected cell lines were derived from inflammation-associated tumors [83,84]. Culturing cells in the presence of R-830, KME-4, E-5110, and S-2474 resulted in a dose-dependent decrease in cell viability determined after 72 h (Table 3, Supplementary Materials, Figures S27 and S28).
Incorporation of a p-carborane moiety resulted in a significant decrease in cytotoxic potential for all tested compounds, except R-830-Cb, which showed antitumor potential in a similar micromolar range as the parent compound. In general, values obtained with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were significantly lower than those obtained with the crystal violet (CV) assay, as expected, because dual COX-2/5-LO inhibitors affect the redox status of cells [85]. Since HCT116 cells were highly sensitive to the carborane analog R-830-Cb and the di-tert-butylphenols, the antitumor effects of the compounds might be independent of the inhibition of COX-2 and 5-LO [86,87]. Accordingly, R-830-Cb was selected for further in-detail investigation of the mechanism of action. Treatment of primary peritoneal exudate cells isolated from healthy mice with R-830 or its carborane counterpart R-830-Cb showed that the latter one was completely non-toxic in the applied dose range, whereas R-830 diminished the viability of primary cells at 100 µM (selectivity index (SI) ≈ 3, Supplementary Materials, Figure S29). These results suggest that the introduction of p-carborane preserves the cytotoxic potential for the malignant phenotype without affecting normal cells in the tested dose range. Analysis of apoptotic cell death by flow cytometry and fluorescent microscopy showed that the mechanism of antitumor action of R-830 was preserved and even enhanced by the incorporation of p-carborane ( Figure 3A,B). Treatment with R-830-Cb not only resulted in lower cell density, but also revealed the presence of numerous cells with abnormally shaped nuclei and condensed chromatin ( Figure 3B).
Apostat staining confirmed that apoptosis was in correlation with the activation of caspases ( Figure 3C). At the same time, and in parallel with the presence of apoptotic cells, cell division was more severely affected by R-830-Cb ( Figure 3D). The decreased production of reactive oxygen species explains the observed effects of the applied treatments, namely inhibition of proliferation and induction of apoptosis ( Figure 3E). It is widely accepted that drug-mediated hyperproduction of reactive oxygen species that are not buffered by cellular anti-oxidative protection is responsible for cell death induction [88]. However, the mechanisms of action of some therapeutics rely on the fact that tumor cells, especially colon carcinomas, produce a significantly higher amount of reactive oxygen species necessary for their metabolic and proliferative demands [89]. The described feature is acquired during disease progression, and it is tightly connected with abnormal blood supply and a hypoxic microenvironment [90]. Therefore, elimination of these molecules results in a rapid decrease in cell viability. Importantly, the intense autophagy detected by acridine orange (AO) supravital staining of cells exposed to R-830-Cb, revealed cytoprotective character for this compound, but not for R-830 ( Figure 3F,G).
Ultimately, simultaneous treatment of cells with R-830-Cb and specific autophagy inhibitors, chloroquine or 3-methyladenine (3-MA), showed that suppression of the autophagy process caused a drug-induced decrease in cell viability ( Figure 3G). This means that the cytotoxic activity of the tested drug is enhanced by the inhibition of autophagy.
Considering the disadvantages of 2D cell cultures, since the cells are in a monolayer without all the components of the tumor microenvironment, the real improvement and potential of R-830-Cb should be discussed in a more complex experimental setting [91]. Molecules 2023, 28, x FOR PEER REVIEW 9 of 23 Apostat staining confirmed that apoptosis was in correlation with the activation of caspases ( Figure 3C). At the same time, and in parallel with the presence of apoptotic cells, cell division was more severely affected by R-830-Cb ( Figure 3D). The decreased production of reactive oxygen species explains the observed effects of the applied treatments, namely inhibition of proliferation and induction of apoptosis ( Figure 3E). It is widely accepted that drug-mediated hyperproduction of reactive oxygen species that are

Determination of Lipophilicity (logD) by HPLC
The logD 7.4, HPLC value was determined as previously reported by us [95] utilizing an HPLC method originally described by Donovan and Pescatore [82]. The following HPLC system was used: Agilent 1100 HPLC (binary pump G1312A, autosampler G1313A, column oven G1316A, degasser G1322A, UV detector G1314A, γ detector Gabi Star

COX Inhibition Studies
COX inhibition activity against ovine COX-1 and human COX-2 was determined using the fluorescence-based COX assay COX Fluorescent Inhibitor Screening Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer's instructions as previously reported by us. All compounds were screened at a concentration of 100 µM in duplicate [76].

Cell Viability Studies-MTT and CV Assays
All cell lines were seeded overnight and treated with R-830, R-830-Cb, KME-4, KME-4-Cb, E-5110, E-5110-Cb, S-2474, and S-2474-Cb for 72 h. After the incubation period, the supernatant was discarded from the wells, and the cells were washed two times with 200 µL of PBS. Next, MTT solution at a final concentration of 0.5 mg/mL was added and incubated at 37 • C until purple formazan crystals were formed (30 min to one hour). After incubation, the dye was discarded. In order to dissolve the formed formazan crystals DMSO was added, and the absorbance was measured at λ max = 540 nm, with the reference/background wavelength of 670 nm. All results are expressed as a percentage of the control value which was arbitrary set to 100%. After 72 h treatment, cells were washed with 200 µL of PBS and fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). Next, cells were stained with 1% CV solution. After 20 min, cells were washed in tap water and dried on air. Dye was dissolved in 33% acetic acid and the absorbance was measured at λ max = 540 nm, with the reference/background wavelength of 670 nm. Results were expressed as a percentage of the control value which was arbitrary set to 100%.

Statistical Analyses
All experiments were repeated at least three independent times and data presented represents the means ± SD of replicates. To evaluate the significance between groups Student's t-test was used and two-sided p values of less than 0.05 were considered statistically significant.
Further materials, methods, and procedures for physicochemical characterization and biological evaluation: see Supplementary Materials (i.e., Supplementary Materials) for further details.

Conclusions
The substitution of the bulky di-tert-butylphenyl moiety in the potent dual COX-2/5-LO inhibitors R-830, KME-4, E-5110, and S-2474 by p-carborane gave rise to the carboranebased analogs R-830-Cb, KME-4-Cb, E-5110-Cb, and S-2474-Cb. Replacing the bulky but also hydrophobic di-tert-butylphenyl moiety with the hydrophobic boron cluster resulted in a significant decrease in lipophilicity, suggesting that intermolecular interactions, such as hydrogen bonds or dihydrogen bonds, are contributing to the compounds' lipophilicity [97]. The carborane analogs show no or only weak COX inhibition, as anticipated based on previous reports about the anti-inflammatory activity of related mono-or di-tert-butylphenols.
However, in vitro studies on intact cells revealed that all carborane-based analogs are strong inhibitors of 5-LO product formation with IC 50 values in the nanomolar range, comparable to the respective organic counterparts, indicating that the bioisosteric replacement of the di-tert-butylphenyl motif by p-carborane is well tolerated.
Furthermore, the introduction of p-carborane generally decreased the cytotoxic potential of dual inhibitors, except in the case of R-830. Interestingly, the p-carborane analog R-830-Cb was non-toxic to primary cells but effective against tested cell lines derived from various types of inflammation-related tumors. Its cytotoxic potential was mediated by potent inhibition of ROS, resulting in inhibited proliferation and caspase-dependent apoptosis.
Thus, carborane-based analog R-830-Cb is a promising candidate for further assessment and detailed mechanistic as well as in vivo studies.