1. Introduction
Besides genetic mutations, epigenetic changes, diet, and lifestyle, chronic inflammatory processes are a significant factor in carcinogenesis [1,2]. The process of cancer formation is a stepwise progress which can be split into three phases, namely initiation, promotion, and progression. Cancer progression relies on the control of tumor microenvironment which is essential to provoke further tumor invasiveness and metastasis [3,4,5]. Hence, there is need for a balance between immunosuppressive and immunostimulatory mediators in the tumor- and inflammatory microenvironment [2,6,7,8]. Inflammatory mediators, such as cytokines, chemokines, and eicosanoids, can stimulate tumor cell proliferation and cancer progression by disrupting homeostatic mechanisms [3,9]. Eicosanoids, in particular prostaglandins (PGs) and leukotrienes (LTs), are hormone-like lipids that play a pivotal part in mediating the crosstalk between cells in the tumor microenvironment [2]. Targeting these inflammatory mediators by interfering with related signaling pathways may offer a rational strategy for the treatment of cancer. Pro-inflammatory PGs and LTs are mainly formed from arachidonic acid (AA) via cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) signaling pathways [10,11,12]. Cyclooxygenases (COX) catalyze the rate-determining step in the biosynthesis of PGs [13,14,15]. The constitutively expressed COX-1 is involved in physiological “housekeeping” functions, such as the homeostasis of the gastrointestinal (GI) tract [16,17]. COX-2 is highly inducible by pro-inflammatory stimuli through macrophage activation or tumor promoters [12,18]. It can be found in macrophages, leukocytes, and fibroblasts, and COX-2 dependent production of pro-inflammatory PGs is upregulated during inflammatory diseases, such as rheumatoid arthritis, cardiovascular diseases, and asthma [10,12,15,19]. Furthermore, COX-2, in particular the downstream product prostaglandin E2 (PGE2), is able to promote carcinogenesis and progression in different cancer types, such as skin, breast, colon, and lung cancer, that are associated with poor prognosis [2,15,20,21,22]. As COX-1 and COX-2 share nearly homologous amino acid sequence identity, selective COX-2 inhibition remains challenging [17,19]. COX-2 is physiologically repressed by the active form of vitamin D and glucocorticoids [23,24]. Artificial COX inhibitors are categorized into two groups, namely non-steroidal anti-inflammatory drugs (NSAIDs) and COXIBs. As a result, of two crucial changes in the amino acid sequence, the COX-2 active site is ca. 17% larger and has a slightly different form than that of COX-1 [25]. Thus, size enlargement of inhibitors has been shown to enable COX-2 selective inhibition [19,20]. While non-selective COX inhibition by NSAIDs leads to the reduction of gastroprotective PGs, COX-2 selective inhibition suffers from adverse cardiovascular effects caused by dysbalanced prostaglandin I2 (PGI2) and thromboxane A2 (TXA2) levels [2,25,26,27,28].
5-LO is a non-heme iron-containing oxidoreductase that belongs to a heterogeneous family of lipid peroxidizing enzymes and catalyzes the conversion of AA into LTs with the help of 5-lipoxygenase-activating protein (FLAP) [10,29,30,31]. These important lipid mediators are involved in host defense, inflammatory processes, and cellular signaling [2,32]. 5-LO is highly inducible and can be stimulated by growth factors and pro-inflammatory stimuli [20,28,33]. Indeed, 5-LO upregulation and LTs overproduction are related to hypersensitivity reactions, inflammatory diseases, and allergic disorders, such as arthritis or psoriasis. It has also been observed in different types of epithelial cancers, such as lung, colon, and prostate cancer [9,12,33,34,35,36]. 5-LO can be downregulated by four different types of inhibitors, namely redox (non-competitive), iron-chelating, non-redox (competitive), and allosteric inhibitors [37,38,39]. 5-LO inhibitors, in general, suffer from having a short half-life and exhibiting hepatic toxicity [10,32].
PGs and LTs have complementary effects on the pathogenesis of cancer and tumor progression, and thus dual COX-2/5-LO inhibition may represent a promising strategy to treat cancer and to prevent the pathogenesis of cancer [33,34,40]. Dual COX-2/5-LO inhibitors block the release of both PGs and LTs from the corresponding COX-2 and 5-LO signaling pathways [10,17]. Dual COX-2/5-LO inhibition may prevent the upregulation of the respective opposed signaling pathway by blocking both targets. Consequently, balanced COX-2/5-LO inhibition may lower the risk for the appearance of severe adverse effects, such as GI injury and hypersensitive reactions [21,38,41,42,43,44].
Di-tert-butylphenols represent the predominant class of dual COX-2/5-LO inhibitors and share a conserved 2,6-di-tert-butylphenol motif substituted at the para (p)-position (Figure 1). Di-tert-butylphenols show a superior therapeutic index, i.e., the ratio of anti-inflammatory effect to GI safety profile, compared to that of classical NSAIDs [10,33,34]. Several di-tert-butylphenols have been synthesized and pharmacologically evaluated. R-830 (3,5-di-tert-butyl-4-(2′-thienoyl)-phenol) produces anti-edemic, analgesic, antipyretic, and anti-erythemic effects in several animal models [45]. It further reduces PGE2 production by stimulated rat synovial cells in the nanomolar range and significantly inhibited leukotriene B4 (LTB4) production by human neutrophils at micromolar concentrations [45,46]. The dual COX-2/5-LO inhibitor KME-4 (α-3,5-di-tert-butyl-4-hydroxybenzylidene)-γ-butyrolactone) inhibits both guinea pig peritoneal polymorphonuclear leukocytes (PMNL) and rabbit platelet COX at low micromolar concentrations [47,48]. Moreover, in vivo experiments revealed that KME-4 possess anti-arthritic activity in a dose-dependent manner, while showing lower ulcerogenic potential compared to other NSAIDs, such as indomethacin or naproxen [49,50].
The pyrrolidone derivative E-5110 (N-methoxy-3-(3,5-di-tert-butyl-4-hydroxybenzylidene)-2-pyrrolidone) inhibits both PGE2 production by cultured rat synovial cells at nanomolar concentrations and LTB4 generation by human leukocytes stimulated in the micromolar range [51,52,53,54]. Further in vivo studies revealed that E-5110 exhibits analgesic activity similar to indomethacin with lower ulcerogenic effect on rat gastric mucosa than indomethacin and piroxicam [54,55]. Both in vitro and in vivo toxicokinetic studies on the metabolism of E-5110 demonstrated induction of cytochrome P450 3A (CYP3A) activity, which is therefore expected to mainly contribute to its metabolism as well as its bioavailability [56]. S-2474 ((E)-5-(3,5-di-tert-butyl-4-hydroxybenzylidene)-2-ethylisothiazo-lidine-1,1-dioxide) is a selective inhibitor of COX-2 and 5-lipoxygenase in vitro with half-maximal inhibitory concentration (IC50) values in the low micromolar and nanomolar range, respectively [57]. Furthermore, it shows in vivo anti-inflammatory activity in rats with low ulcerogenic activity due to its COX-2 selectivity [58].
A promising approach to increase metabolic stability of carbon-based drugs is the incorporation of hydrophobic boron clusters, such as the 12-vertex dicarba-closo-dodecaborane (carborane), as bioisosters of substituted or unsubstituted phenyl rings [59,60,61]. Carboranes are icosahedral boron clusters in which at least one BH− vertex in (B12H12)2− is replaced by an isolobal CH group [61,62,63]. As their distorted icosahedral shape reflects the shape of a rotating phenyl ring, they can be considered benzene analogs [63,64,65,66]. In analogy to the nomenclature of the substitution of a benzene ring, three different regioisomers, namely 1,2-(ortho), 1,7-(meta), or 1,12-carborane (para), are reported [64,67,68]. Due to a slightly larger van-der-Waals diameter compared to a phenyl ring (carborane: 5.25 Å, phenyl ring: 4.72 Å) [59], carboranes are frequently used as phenyl mimetics for biologically active compounds [61,62,65,69]. Multiple non-covalent interactions, like dihydrogen bond formation, may increase the affinity to biological targets, including enzymes or receptor proteins [61,62,65]. Due to their remarkable properties, carboranes offer a wide range of possibilities for drug design in medicinal chemistry [61,70].
Our group has intensively investigated the introduction of carborane moieties into NSAIDs, COXIBs, and 5-LO inhibitors [71,72,73,74,75,76,77]. We have recently reported the first carborane-based dual COX-2/5-LO inhibitors that are derived from RWJ-63556 showing excellent inhibitory potential toward COX-2 and 5-LO, accompanied by high anticancer activity on the A375 melanoma cell line [78].
Herein, we report the synthesis of p-carborane-containing analogs of the di-tert-butylphenol 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.
2. Results and Discussion
2.1. 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 tert-butyldimethylsilyl (TBDMS) ether by a conventional silylation reaction with excess of tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of triethylamine (NEt3) 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 2-ethylisothiazolidine-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-3-pyrrolidinyl)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 NEt3 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.
All compounds were fully characterized by NMR and IR spectroscopy, mass spectrometry and elemental analysis. Three-dimensional structures of intermediates 1, 5, 6, and 7, as well as of p-carborane-containing analogs R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb, were determined by single-crystal X-ray crystallography (Figure 2; for further details, see Supplementary Materials, Tables S3–S6).
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 1H- and 11B{1H}-NMR spectroscopy over four weeks and confirmed that all compounds are stable (see Supplementary Materials, Figure S24).
2.2. 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.
2.3. Evaluation of Inhibitory Potential toward COX
All compounds were tested in vitro at a concentration of 100 µ
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.
2.4. 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 IC50 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.
2.5. 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].
3. Experimental Section
3.1. Materials and Methods
All reactions were carried out under nitrogen using standard Schlenk techniques. All solvents were degassed, dried and purified with the solvent purification system SPS-800 by MBraun. Molecular sieves (3 and 4 Å) were activated at 300 °C in vacuum for a minimum of three hours for storage of dry solvents. Hexane has been used as a mixture of different isomers. For column chromatography, silica gel 60 with a particle size in the range of 0.035–0.070 mm (Acros) was used. A gradient of hexane and ethyl acetate (EtOAc) was usually used as the eluent system. Reactions were monitored with thin-layer chromatography using glass plates coated with silica gel (0.25 mm, silica gel 60, F254, Merck). Carborane-containing areas were stained and identified with a 5% solution of palladium(II) chloride in methanol. All NMR spectra were recorded on a Bruker Avance DRX 400 spectrometer at the following measurement frequencies at 26 °C: 1H-NMR 400.16 MHz, 11B-NMR 128.38 MHz, 13C-NMR 100.63 MHz. Tetramethylsilane (TMS) served as an internal standard for 1H-NMR spectra, and deuterated chloroform (CDCl3) (7.26 ppm) was used for referencing. Signal multiplicities were indicated by the following abbreviations and combinations: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets, td = triplet of doublets, ddd = doublet of doublets of doublets, dtd = doublet of triplets of doublets. The unit of the coupling constant J is given in hertz (Hz). The chemical shifts in the 13C{1H}-NMR spectra could be extracted from the spectra by 1H broadband decoupling in the proton channel. Residual signals of CDCl3, acetone-d6, deuterated dimethyl sulfoxide (DMSO-d6) at 77.2, 29.9, 39.5 ppm served as a reference. All 11B{1H} NMR spectra were also internally referenced to TMS with the aid of the Ξ scale [92]. The analysis of the data of the measurement was carried out using the software Mestrenova 14. ESI mass spectra were measured using a Bruker Daltonics Apex II FT-ICR spectrometer. All IR spectra were obtained using an ATR-IR spectrometer (Nicolet iS5, Thermo Fisher Scientific, Waltham, MA, USA) in the range of 400–4000 cm−1. Elemental analyses were carried out with a Heraeus Vario El analyser. The melting points were determined in glass capillaries using a Gallenkamp MPD350·BM2.5 apparatus and are uncorrected. 1,12-Dicarba-closo-dodecaborane(12) (Katchem, Prague, Czech Republic), 2-ethylisothiazolidine-1,1-dioxide (BLD Pharmatech Ltd., Shanghai, China), 2-thiophenecarbonyl chloride (Alfa Aesar, Ward Hill, MA, USA) are commercially available. All chemicals were used without further purification. 2,6-Bis(1,1-dimethylethyl)-4-[(E)-(2-ethyl-1,1-dioxido-5-isothiazolidinylidene)methyl]phenol (S-2474) [58], 3-([3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene)dihydro-2(3H)-furanone (KME-4) [53], 3-([3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene)-1-methoxy-2-pyrrolidinone (E-5110) [54], 2,6-di-tert-butyl-4-(2’-thenoyl)phenol (R-830) [93], (1-methoxy-2-oxo-3-pyrrolidinyl)triphenyl-phosphonium bromide [54], 1-(tert-butyl-dimethylsiloxy)-1,12-dicarba-closo-dodecaborane(12) [94], and 3-(triphenylphosphoranylidene)-γ-butyrolactone [81] were prepared according to the literature.
3.2. Synthetic Procedures
3.2.1. 1-(tert-Butyl-dimethylsiloxy)-12-(thiophen-2′-carbonyl)-1,12-dicarba-closo-dodecaborane(12) (1)
1-(tert-Butyl-dimethylsiloxy)-1,12-dicarba-closo-dodecaborane(12) (1.00 eq., 0.83 g, 3.02 mmol) was dissolved in 25 mL dry diethyl ether. At 0 °C, nBuLi (1.05 eq., 1.43 M in hexane, 2.32 mL, 3.32 mmol) was added dropwise. Afterwards, the solution was stirred at room temperature for two hours. At 0 °C, a solution of 2-thiophenecarbonyl chloride in 10 mL dry diethyl ether (1.50 eq., 0.48 mL, 4.53 mmol) was added dropwise. The solution was stirred at room temperature for 24 h, then 20 mL of a 1 M HCl was added and the phases were separated. The aqueous phase was extracted three times with 25 mL diethyl ether, the combined organic phases were washed twice with 25 mL brine and then dried over magnesium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (SiO2, hexane → hexane/EtOAc, 30:1, v/v). Colorless solid, yield 64% (0.74 g, 1.92 mmol); m.p.: 102–103 °C; TLC (hexane/EtOAc, 30:1, v/v): Rf = 0.51. 1H-NMR (400.16 MHz, CDCl3): δ [ppm] = 0.10 (s, 6H, 8-CH3), 0.76 (s, 9H, 10-CH3), 1.60–3.52 (br m, 10H, BH), 7.06 (dd, 3JHH = 5.0; 4.0 Hz, 2H, 2-CH), 7.64 (dd, 3JHH = 5.0, 1.1 Hz, 1H, 1-CH), 7.83 (dd, 3JHH = 4.0, 1.1 Hz, 1H, 3-CH); 13C{1H}-NMR (100.63 MHz, CDCl3): δ [ppm] =−4.18 (8-CH3), 17.8 (9-Cq), 25.1 (10-CH3), 71.5 (6-Cq), 113.4 (7-Cq), 128.2 (2-Cq), 135.8 (1-Cq), 135.9 (3-Cq), 140.6 (4-Cq), 179.8 (5-Cq); 11B{1H}-NMR (128.37 MHz, CDCl3): δ [ppm] = −15.4 (s, 5B), −12.8 (s, 5B); FTIR (ATR): [cm−1] = 3126 (m, νC–H), 2959 (m, νC–H), 2929 (m, νC–H), 2857 (m, νC–H), 2620 (s, νB–H), 2599 (s, νB–H), 1644 (s, νC=O), 1515 (m, vc=c), 1470 (m, δC–H), 1462 (m, δC–H), 1409 (s, δC–H), 1355 (m, δC–H), 1247 (s, νC=S), 1115 (s), 1064 (m), 1021 (s), 947 (s), 866 (w), 839 (m), 803 (m), 781 (s), 755 (m), 725 (s, νB-B), 688 (s), 673 (s), 656 (s), 603 (m), 574 (m), 493 (w), 466 (m); HR-ESI-MS (negative mode, CH3CN/CH2Cl2): m/z [M–SiMe2tBu]−, calcd. for C7H13B10O2S: 271.1572, found: 271.1579; elemental analysis: calcd. (%) for C13H28B10O2SSi: C 40.60, H 7.34, found: C 40.75, H 7.14.
3.2.2. 1-Hydroxy-12-(thiophen-2′-carbonyl)-1,12-dicarba-closo-dodecaborane(12) (R-830-Cb)
TBDMS (tert-butyl-dimethylsilyl)-protected 1-hydroxy-1,12-dicarba-closo-dodecaborane derivative (1) (1.00 eq., 0.50 g, 1.11 mmol) was dissolved in 4.00 mL dry tetrahydrofuran (THF). At 0 °C, a tetra-n-butylammonium fluoride (TBAF) solution (1.05 eq., 1.00 M in THF, 1.36 mL, 1.36 mmol) was added dropwise. The reaction mixture was stirred for 20 min at 0 °C. The reaction was then stopped by adding 20 mL distilled water. After dilution with 50 mL EtOAc, the phases were separated. The aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed with 25 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography (SiO2, hexane/EtOAc, 2:1, v/v). Colorless solid, yield 90% (0.32 g, 1.18 mmol); m.p.: 131–132 °C; TLC (hexane/EtOAc, 2:1, v/v): Rf = 0.56. 1H-NMR (400.16 MHz, CDCl3): δ [ppm] = 1.56–3.43 (br m, 10H, BH), 3.56 (br s, 1H, 7-OH), 7.08 (t, 3JHH = 4.5 Hz, 1H, 2-CH), 7.66 (d, 3JHH = 5.0, 1.1 Hz, 1H, 1-CH), 7.83 (d, 3JHH = 4.0 Hz, 1H, 3-CH); 13C{1H}-NMR (100.63 MHz, CDCl3): δ [ppm] = 72.1 (6-Cq), 110.5 (7-Cq), 128.4 (2-Cq), 136.1 (1-Cq), 136.4 (3-Cq), 140.3 (4-Cq), 179.6 (5-Cq); 11B{1H}-NMR (128.37 MHz, CDCl3): δ [ppm] = −15.1 (s, 5B), −13.1 (s, 5B); FTIR (ATR): ṽ [cm−1] = 3328 (b, νO–H), 2615 (s, νB–H), 2601 (s, νB–H), 1646 (s, νC=O), 1629 (s, νC=O), 1510 (m, νC=C), 1449 (w, δC–H), 1403 (s, δC–H), 1355 (s, δC–H), 1345 (s, δC–H), 1261 (s, νC=S), 1215 (s),1192 (s, νC–O), 1115 (s), 1063 (m), 1035 (w), 1010 (s), 935 (m), 865 (m), 741 (s), 722 (s, νB–B), 691 (s), 665 (s), 573 (w), 556 (w), 447 (m); HR-ESI-MS (negative mode, MeOH): m/z [M−H]− calcd. for C7H13B10O2S: 271.1572, found: 271.1570; elemental analysis: calcd. (%) for C7H14B10O2S: C 31.19, H 5.22, found: C 31.04, H 5.21.
3.2.3. 1-(tert-Butyl-dimethylsiloxy)-12-formyl-1,12-dicarba-closo-dodecaborane(12) (2)
1-(tert-Butyl-dimethylsiloxy)-1,12-dicarba-closo-dodecaborane(12) (1.00 eq., 2.14 g, 7.79 mmol) was dissolved in 100 mL dry diethyl ether. At 0 °C, nBuLi (1.05 eq., 1.43 M in hexane, 5.72 mL, 8.18 mmol) was added dropwise. Afterwards, the solution was stirred at room temperature for two hours. At −78 °C, methyl formate (3.50 eq., 2.20 mL, 27.3 mmol) was added dropwise. The solution was first stirred for one hour at −78 °C, then warmed to room temperature and stirred for 24 h. After addition of 30 mL 1 M HCl, the phases were separated. The aqueous phase was extracted three times with 50 mL diethyl ether, and the combined organic phases were washed twice with 25 mL brine and then dried over magnesium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (SiO2, hexane → hexane/EtOAc, 20:1, v/v). Colorless solid, yield 86% (2.02 g, 6.68 mmol); m.p.: 40–41 °C; TLC (hexane): Rf = 0.57. 1H-NMR (400.16 MHz, CDCl3): δ [ppm] = 0.09 (s, 6H, 4-CH3), 0.75 (s, 9H, 6-CH3), 1.30–3.36 (br m, 10H, BH), 8.96 (s, 1H, 1-CH); 13C{1H}-NMR (100.63 MHz, CDCl3): δ [ppm] = −4.20 (4-CH3), 17.7 (5-Cq), 25.1 (6-CH3), 70.7 (2-Cq), 113.1 (3-Cq), 187.2 (1-Cq); 11B{1H}-NMR (128.37 MHz, CDCl3): δ [ppm] = −16.9 (s, 5B), −12.5 (s, 5B); FTIR (ATR): ṽ [cm−1] = 2955 (m, νC–H), 2930 (m, νC–H), 2859 (m, νC–H), 2609 (s, νB–H), 1970 (w), 1735 (s, νC=O), 1471 (m, δC–H), 1463 (w, δC–H),1390 (w, δC–H), 1362 (w, δC–H), 1252 (s, νC–O), 1168 (s, νC–O), 1031 (s, νC–O), 1004 (w), 962 (m), 781 (s), 730 (m, νB-B), 669 (m), 642 (m), 575 (w), 557 (m); HR-ESI-MS (negative mode, CH3CN): m/z [M+Cl]−, calcd. for C9H26B10ClO2Si: 339.2326, found: 339.2302; elemental analysis: calcd. (%) for C9H26B10O2Si: C 35.74, H 8.66, found: C 35.70, H 8.67.
3.2.4. 1-(tert-Butyl-dimethylsiloxy)-12-(hydroxymethyl-[2′-ethylisothiazolidine-1′,1′-dioxide])-1,12-dicarba-closo-dodecaborane(12) (3)
2-Ethylisothiazolidine-1,1-dioxide (1.10 eq., 1.08 g, 7.27 mmol) was dissolved in 15 mL dry THF. At −78 °C, nBuLi (1.15 eq., 1.36 M in hexane, 5.61 mL, 7.63 mmol) was added dropwise. Afterwards, the solution was stirred at −78 °C for 30 min. At −78 °C, aldehyde 2 (1.00 eq., 2.00 g, 6.61 mmol) was added dropwise. The solution was first stirred for 1.5 h at −78 °C, then it was warmed to room temperature and stopped by adding 15 mL 1 M HCl. The phases were separated, and the aqueous phase was extracted three times with 75 mL diethyl ether. The combined organic phases were washed twice with 25 mL brine and then dried over magnesium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (SiO2, hexane/EtOAc 3:1 → 2:1, v/v). Colorless solid, yield 53% (1.58 g, 3.49 mmol); m.p.: 149–150 °C; TLC (hexane/EtOAc 3:1, v/v): Rf = 0.55. 1H-NMR (400.16 MHz, CDCl3): δ [ppm] = 0.09 (s, 6H, 9-CH3), 0.74 (s, 9H, 11-CH3), 1.29–3.33 (br m, 10H, BH), 1.18 (t, 3JHH = 7.2 Hz, 2H, 1-CH3), 2.22 (m, 1H, 4-CH2), 2.37 (m, 1H, 4-CH2), 2.97 (d, 3JHH = 3.9 Hz, 2H, 3-CH2), 3.08 (m, 4H, 2/3-CH2), 4.33 (d, 3JHH = 3.6 Hz, 1H, 6-CH); 13C{1H}-NMR (100.63 MHz, CDCl3): δ [ppm] = −4.3 (9-CH3), 13.2 (1-CH3), 17.7 (10-Cq), 18.1 (4-CH2), 39.4 (5-CH), 44.4 (2-CH2), 60.8 (3-CH2), 68.1 (6-C-OH), 77.3 (7-Cq), 111.6 (8-Cq); 11B{1H}-NMR (128.37 MHz, CDCl3): δ [ppm] = −16.0 (s, 5B), −12.8 (s, 5B); FTIR (ATR): ṽ [cm−1] = 3447 (b, νO–H), 2958 (m, νC–H), 2929 (m, νC-H), 2858 (m, νC–H), 2598 (s, νB–H), 1466 (m, δC–H), 1409 (w, δC–H), 1361 (w, δC–H), 1329 (w, δC–H), 1284 (s, νS=O), 1266 (m), 1216 (s), 1183 (w, νC–O), 1129 (s, νC–O), 1109 (s, νC–O), 1078 (w), 1032 (s), 1004 (w), 989 (m), 953 (w), 940 (m), 887 (w), 837 (w), 783 (s), 741 (s, νB–B), 707 (w), 672 (m), 656 (w), 640 (w), 613 (w), 579 (m), 503 (w), 482 (w), 452 (w); HR-ESI-MS (positive mode, MeOH): m/z [M + H]+, calcd. for C14H38B10NO4SSi: 454.3215, found: 454.3234; elemental analysis: calcd. (%) for C14H37B10NO4SSi: C 37.23, H 8.26, N 3.10, found: C 37.90, H 8.25, N 3.89.
3.2.5. 1-(tert-Butyl-dimethylsiloxy)-12-(p-toluenesulfonylmethyl-[2′-ethylisothiazolidine-1′,1′-dioxide])-1,12-dicarba-closo-dodecaborane(12) (4)
1-(tert-Butyl-dimethylsiloxy)-12-(hydroxymethyl-[2′-ethylisothiazolidine-1′,1′-dioxide])-1,12-dicarba-closo-dodecaborane(12) (3) (1.00 eq., 1.00 g, 2.21 mmol) was dissolved in 7.00 mL dry THF. At 0 °C, NaH (1.05 eq., 60% dispersion in mineral oil, 93.1 mg, 0.69 mmol) was added. The reaction mixture was stirred for two hours at room temperature. At 0 °C, p-toluenesulfonyl chloride (2.00 eq., 0.84 g, 4.42 mmol) was added and the solution was stirred for 24 h at room temperature. The reaction was then stopped by adding 30 mL of a 1 M HCl. After dilution with 75 mL diethyl ether, the phases were separated. The aqueous phase was extracted three times with 25 mL diethyl ether. The combined organic phases were washed with 25 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography (SiO2, hexane/EtOAc, 4:1 → 3:1, v/v). Colorless solid, yield 87% (1.17 g, 1.93 mmol); m.p.: 181–182 °C; TLC (hexane/EtOAc, 4:1, v/v): Rf = 0.54. 1H-NMR (400.16 MHz, CDCl3): δ [ppm] = 0.05 (s, 6H, 13-CH3), 0.72 (s, 9H, 15-CH3), 1.09–3.24 (br m, 10H, BH), 1.00 (t, 3JHH = 7.3 Hz, 2H, 1-CH3), 2.26 (dtd, 3JHH = 13.1, 7.0, 6.2 Hz, 4JHH = 3.2 Hz, 2H, 4-CH2), 2.44 (s, 3H, 10-CH3), 2.71 (m, 1H, 3-CH2), 2.82 (m, 1H, 3-CH2), 2.92 (m, 2H, 2-CH2), 3.21 (ddd, 3JHH = 8.6, 6.4 Hz, 4JHH = 2.3 Hz, 1H, 5-CH), 5.29 (d, 3JHH = 2.3 Hz, 1H, 6-CH), 7.31 (d, 3JHH = 8.1 Hz, 1H, 8-CH), 7.77 (d, 3JHH = 8.2 Hz, 1H, 9-CH); 13C{1H}-NMR (100.63 MHz, CDCl3): δ [ppm] = −4.3 (13-CH3), 12.7 (1-CH3), 17.7 (14-Cq), 20.5 (4-CH2), 21.8 (10-CH3), 25.1 (15-CH3), 38.9 (3-CH2), 43.5 (2-CH2), 60.6 (5-CH), 70.1 (11-Cq), 77.3 (6-CH), 112.6 (12-Cq), 128.5 (9-CH), 129.4 (8-CH), 134.2 (10-CH3), 145.0 (7-Cq); 11B{1H}-NMR (128.37 MHz, CDCl3): δ [ppm] = −15.8 (s, 5B), −12.6 (s, 5B); FTIR (ATR): ṽ [cm−1] = 2956 (m, νC–H), 2930 (m, νC–H), 2857 (m, νC–H), 2619 (s, νB–H), 2592 (s, νB–H), 1595 (w), 1463 (w, δC–H), 1450 (w, δC–H), 1372 (m, δC–H), 1341 (w, δC–H), 1293 (m, δC–H), 1254 (s, νS=O), 1189 (m, νC–O),1172 (s, νC-O), 1132 (s), 1109 (s), 1095 (w), 1076 (w), 1034 (w), 1019 (w), 1005 (w), 972 (s), 894 (m), 870 (w), 838 (s), 810 (s), 783 (s), 751 (s, νB–B), 720 (w), 670 (s), 639 (w), 631 (w), 577 (m), 559 (m), 544 (s), 498 (w), 455 (w), 442 (w), 435 (w), 429 (w); HR-ESI-MS (positive mode, MeOH): m/z [M + H]+, calcd. for C21H44B10NO6S2Si: 608.3304, found: 608.3350; elemental analysis: calcd. (%) for C21H43B10NO6S2Si: C 41.63, H 7.51, N 2.31 found: C 42.18, H 7.28, N 2.22.
3.2.6. (3E)-1-(tert-Butyl-dimethylsiloxy)-12-(methylene-[2′-ethylisothiazolidine-1′,1′-dioxide])-1,12-dicarba-closo-dodecaborane(12) (5)
1-(tert-Butyl-dimethylsiloxy)-12-(p-toluenesulfonylmethyl-[2′-ethylisothiazolidine-1′,1′-dioxide])-1,12-dicarba-closo-dodecaborane(12) (4) (1.00 eq., 0.46 g, 0.75 mmol) was dissolved in 3.80 mL dry THF. At 0 °C, NaH (1.00 eq., 60% dispersion in mineral oil, 30.4 mg, 0.75 mmol) was added. The reaction mixture was stirred for two hours at room temperature. The reaction was then stopped by adding 25 mL distilled water. After dilution with 25 mL EtOAc, the phases were separated. The aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed with 25 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography (SiO2, hexane/EtOAc, 4:1 → 0:1, v/v). Colorless solid, yield 64% (0.21 g, 0.48 mmol); m.p.: 147–148 °C; TLC (hexane/EtOAc, 4:1, v/v): Rf = 0.50. 1H-NMR (400.16 MHz, CDCl3): δ [ppm] = 0.08 (s, 6H, 9-CH3), 0.75 (s, 9H, 11-CH3), 1.14–3.34 (br m, 10H, BH), 1.23 (t, 3JHH = 7.2 Hz, 2H, 1-CH3), 2.89 (td, 3JHH = 6.6 Hz, 4JHH = 2.9 Hz, 2H, 4-CH2), 3.08 (q, 3JHH = 7.3 Hz, 2H, 2-CH2), 3.14 (3JHH = 6.6 Hz, 2H, 3-CH2), 6.09 (t, 4JHH = 2.8 Hz, 1H, 6-CH); 13C{1H}-NMR (100.63 MHz, CDCl3): δ [ppm] = −4.4 (9-CH3), 12.7 (1-CH3), 17.6 (10-Cq), 21.9 (4-CH2), 24.9 (11-CH3), 39.1 (2-CH2), 42.8 (3-CH2), 63.9 (7-Cq), 112.6 (12-Cq), 111.8 (8-Cq), 126.9 (6-CH), 140.5 (5-Cq); 11B{1H}-NMR (128.37 MHz, CDCl3): δ [ppm] = −15.0 (s, 5B), −12.4 (s, 5B); FTIR (ATR): ṽ [cm−1] = 2948 (m, νC–H), 2930 (m, νC–H), 2859 (w, νC–H), 2619 (s, νB–H), 1667 (w, νC=C), 1471 (w, δC–H), 1461 (m, δC–H), 1422 (w, δC–H), 1390 (m, δC–H), 1361 (w, δC–H), 1309 (m, δC–H), 1297 (s, νS=O), 1262 (s, νS=O), 1250 (s, νS=O), 1208 (m, νC–O),1189 (m, νC–O), 1172 (m), 1156 (m), 1136 (w), 1102 (w), 1073 (m), 1037 (s), 1023 (m), 1005 (w), 972 (w), 939 (m), 837 (s), 781 (s), 750 (w), 729 (s, νB-B), 698 (m), 670 (m), 643 (w), 596 (m), 572 (m), 561 (m), 533 (s), 509 (s), 466 (m), 448 (m), 441 (w), 435 (w); HR-ESI-MS (positive mode, MeOH): m/z [M + H]+, calcd. for C14H36B10NO3SSi: 436.3110, found: 436.3120; elemental analysis: calcd. (%) for C14H35B10NO3SSi: C 38.77, H 8.13, N 3.23 found: C 39.49, H 8.27, N 2.98.
3.2.7. (3E)-1-Hydroxy-12-(methylene-[2′-ethylisothiazolidine-1′,1′-dioxide])-1,12-dicarba-closo-dodecaborane(12) (S-2474-Cb)
(3E)-1-(tert-Butyl-dimethylsiloxy)-12-(methylene-[2′-ethylisothiazolidine-1′,1′-dioxide])-1,12-dicarba-closo-dodecaborane(12) (5) (1.00 eq., 100 mg, 0.23 mmol) was dissolved in 0.80 mL dry THF. At 0 °C, a TBAF solution (1.05 eq., 1.00
3.2.8. (3E)-1-(tert-Butyldimethylsiloxy)-12-(methylene-[dihydrofurane-2′(3H)-one])-1,12-dicarba-closo-dodecaborane(12) (6)
Aldehyde 2 (1.00 eq., 0.70 g, 2.31 mmol) and 3-(triphenylphosphoranylidene)-γ-butyrolactone(1.50 eq., 1.20 g, 3.47 mmol) were dissolved in 11 mL dry THF and 1.40 mL dry (N,N)-dimethylformamide (DMF). The reaction mixture was heated under reflux for 48 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was then dissolved in 100 mL EtOAc and 75 mL distilled water. The phases were separated and the aqueous phase was extracted three times with 50 mL EtOAc. The combined organic phases were washed twice with 50 mL brine and then dried over magnesium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (SiO2, hexane/EtOAc, 5:1 → 3:1, v/v). Colorless solid, yield 84% (0.72 g, 1.94 mmol); m.p.: 149–151 °C; TLC (hexane/EtOAc, 5:1, v/v): Rf = 0.62. 1H-NMR (400.16 MHz, CDCl3): δ [ppm] = 0.08 (s, 6H, 8-CH3), 0.74 (s, 9H, 10-CH3), 1.34–3.31 (br m, 10H, BH), 2.95 (td, 3JHH = 7.2 Hz; 4JHH = 3.0 Hz, 2H, 3-CH2), 4.30 (t, 3JHH = 7.2 Hz, 2H, 1-CH2), 6.38 (t, 4JHH = 3.1 Hz, 1H, 5-CH); 13C{1H}-NMR (100.63 MHz, CDCl3): δ [ppm] = −4.2 (8-CH3), 17.7 (9-Cq), 25.1 (3-CH3), 64.7 (6-Cq), 65.5 (1-CH2), 111.8 (7-Cq), 128.6 (4-Cq), 134.0 (5-CH), 170.8 (2-Cq); 11B{1H}-NMR (128.37 MHz, CDCl3): δ [ppm] = −15.1 (s, 5B), −12.5 (s, 5B); FTIR (ATR): ṽ [cm−1] = 2925 (m, νC–H), 2885 (w, νC–H), 2856 (m, νC–H), 2608 (s, νB–H), 1725 (s, νC=O), 1676 (m, vc=c), 1470 (m, δC–H), 1428 (w, δC–H), 1380 (m, δC–H), 1360 (m, δC–H), 1350 (m, δC–H), 1261 (s, νC–O–C), 1261 (s, νC–O–C), 1250 (s, νC–O–C), 1207 (s), 1072 (m), 1029 (s), 965 (m), 939 (w), 836 (s), 779 (s), 781 (s), 736 (m, vB–B), 695 (m), 646 (m), 628 (w), 574 (m), 517 (m), 461 (m); HR-ESI-MS (positive mode, CH3CN): m/z [M + NH4]+, calcd. for C13H34B10NO3Si: 390.3233, found: 390.3228; elemental analysis: calcd. (%) for C13H30B10O3Si: C 42.14, H 8.16, found: C 42.22, H 8.04.
3.2.9. (3E)-1-Hydroxy-12-(methylene-[dihydrofurane-2′(3H)-one])-1,12-dicarba-closo-dodecaborane(12) (KME-4-Cb)
(3E)-1-(tert-Butyl-dimethylsiloxy)-12-(methylene-[dihydrofuran-2′(3H)-one])-1,12-dicarba-closo-dodecaborane(12) (6) (1.00 eq., 0.29 g, 0.78 mmol) was dissolved in 2.50 mL dry THF. At 0 °C, a TBAF solution (1.05 eq., 1.00 M in THF, 0.82 mL, 0.82 mmol) was added dropwise. The reaction mixture was stirred for 25 min at 0 °C. The reaction was then stopped by adding 20 mL distilled water. After dilution with 25 mL EtOAc the phases were separated. The aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed with 20 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography (SiO2, hexane/EtOAc, 1:1, v/v). Colorless solid, yield 99% (0.19 g, 0.77 mmol); m.p.: 210–212 °C; TLC (hexane/EtOAc, 1:1, v/v): Rf = 0.62. 1H-NMR (400.16 MHz, acetone-d6): δ [ppm] = 1.47–3.27 (br m, 10H, BH), 3.03 (td, 3JHH = 7.2 Hz, 4JHH = 3.1 Hz, 2H, 3-CH2), 4.35 (t, 3JHH = 7.1 Hz, 2H, 1-CH2), 6.20 (t, 4JHH = 3.1 Hz, 1H, 5-CH); 13C{1H}-NMR (100.63 MHz, acetone-d6): δ [ppm] = 26.0 (3-CH3), 66.0 (6-Cq), 66.3 (1-CH2), 111.5 (7-Cq), 131.5 (4-Cq), 132.1 (5-CH), 170.9 (2-Cq); 11B{1H}-NMR (128.37 MHz, acetone-d6): δ [ppm] = −14.8 (s, 5B), −12.6 (s, 5B); FTIR (ATR): ṽ [cm−1] = 3157 (b, νO-H), 2977 (w, νC–H), 2920 (w, νC–H), 2603 (s, νB–H), 1727 (s, νC=O), 1672 (m, νC=C), 1577 (w, νC=C), 1480 (m, δC–H), 1452 (w, δC–H), 1427 (m, νC=C), 1387 (s), 1358 (s, δC–H),1282 (w, νC–O), 1209 (s, νC–O–C), 1088 (m), 1032 (s), 976 (m), 866 (w), 776 (w), 754 (w), 732 (w), 715 (w), 693 (m), 634 (m), 588 (w), 572 (w), 524 (w), 483 (w); HR-ESI-MS (positive mode, CH3CN): m/z [M + H]+, calcd. for C7H17B10O3: 257.2102, found: 257.2184; elemental analysis: calcd. (%) for C7H16B10O3: C 32.80, H 6.29, found: C 32.19, H 6.20.
3.2.10. (3E)-1-(tert-Butyl-dimethylsiloxy)-12-(methylene-[1′-methoxypyrrolidine-2′-one])-1,12-dicarba-closo-dodecaborane(12) (7)
(1-Methoxy-2-oxo-3-pyrrolidinyl)triphenyl-phosphonium bromide (1.50 eq., 0.95 g, 2.08 mmol) and NEt3 (1.50 eq., 0.29 mL, 2.08 mmol) were dissolved in 7.00 mL dry THF. The solution was stirred for 45 min at room temperature. Afterwards, a solution of aldehyde 2 (1.00 eq., 0.38 g, 1.38 mmol) in 2.00 mL dry DMF was added dropwise. The reaction mixture was stirred for 24 h at 75 °C. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was then dissolved in 50 mL EtOAc and 30 mL distilled water. The phases were separated, and the aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed twice with 30 mL brine and then dried over magnesium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (SiO2, hexane/EtOAc, 1:1 → 0:1, v/v). Colorless solid, yield 50% (0.28 g, 0.69 mmol); m.p.: 168–170 °C; TLC (hexane/EtOAc, 1:1, v/v): Rf = 0.60. 1H-NMR (400.16 MHz, CDCl3): δ [ppm] = 0.07 (s, 6H, 9-CH3), 0.74 (s, 9H, 11-CH3), 1.42–3.30 (br m, 10H, BH), 2.79 (td, 3JHH = 6.4 Hz, 4JHH = 2.9 Hz, 2H, 4-CH2), 3.50 (t, 3JHH = 6.4 Hz, 2H, 3-CH2), 3,78 (s, 3H, 1-CH3), 6.19 (t, 4JHH = 2,8 Hz, 1H, 6-CH); 13C{1H}-NMR (100.63 MHz, CDCl3): δ [ppm] = −4.2 (9-CH3), 17.7 (10-Cq), 21.1 (4-CH2), 25.1 (11-CH3), 42.8 (3-CH2), 62.2 (1-CH3), 65.2 (7-Cq), 111.3 (8-Cq), 128.5 (6-Cq), 131.3 (5-Cq), 163.7 (2-Cq); 11B{1H}-NMR (128.37 MHz, CDCl3): δ [ppm] = −15.0 (s, 5B), −12.5 (s, 5B); FTIR (ATR): ṽ [cm−1] = 2953 (w, vC–H), 2932 (m, νC–H), 2896 (w, νC–H), 2858 (w, νC–H), 2603 (s, νB–H), 1994 (w), 1708 (s, νC=O), 1672 (m, vC=C), 1472 (m, vC=C), 1461 (w, δC–H), 1428 (w, δC–H), 1399 (w, δC–H), 1362 (w, δC–H), 1344 (m, δC–H), 1249 (s, vC–O–C), 1119 (w), 1038 (s), 1003 (w), 970 (m), 926 (m), 837 (s), 779 (s), 731 (m, vB–B), 694 (m), 666 (w), 628 (w), 603 (m), 521 (w), 482 (m), 466 (m), 449 (m), 431 (m), 461 (m); HR-ESI-MS (positive mode, CH3CN): m/z [M + H]+, calcd. for C14H34B10NO3Si: 402.3233, found: 402.3248; elemental analysis: calcd. (%) for C14H33B10NO3Si: C 42.08, H 8.32, N 3.51, found: C 42.39, H 8.07, N 3.53.
3.2.11. (3E)-1-Hydroxy-12-(methylene-[1′-methoxypyrrolidine-2′-one])-1,12-dicarba-closo-dodecaborane(12) (E-5110-Cb)
(3E)-1-(tert-Butyl-dimethylsiloxy)-12-(methylene-[1′-methoxypyrrolidine-2′-one])-1,12-dicarba-closo-dodecaborane(12) (7) (1.00 eq., 0.18 g, 0.45 mmol) was dissolved in 1.50 mL dry THF. At 0 °C, a TBAF solution (1.05 eq., 1.00 M in THF, 0.47 mL, 0.47 mmol) was added dropwise. The reaction mixture was stirred for 30 min at 0 °C. The reaction was then stopped by adding 20 mL distilled water. After dilution with 25 mL EtOAc the phases were separated. The aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed twice with 20 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography (SiO2, hexane/EtOAc, 2:1, v/v). Colorless solid, yield 93% (0.12 g, 0.42 mmol); m.p.: 222–224 °C; TLC (hexane/EtOAc, 2:1, v/v): Rf = 0.52. 1H-NMR (400.16 MHz, acetone-d6): δ [ppm] = 1.48–3.28 (br m, 10H, BH), 2.84 (ddd, 3JHH = 6.8, 5.9 Hz, 4JHH = 2.9 Hz, 2H, 4-CH2), 3.58 (dd, 3JHH = 6.7, 5.9 Hz, 2H, 3-CH2), 3.73 (s, 3H, 1-CH3), 5.99 (t, 4JHH = 2.9 Hz, 1H, 6-CH); 13C{1H}-NMR (100.63 MHz, acetone-d6): δ [ppm] = 21.9 (4-CH2), 43.3 (3-CH2), 62.1 (1-CH3), 66.4 (7-Cq), 111.1 (8-Cq), 126.8 (6-Cq), 134.5 (5-Cq), 163.2 (2-Cq); 11B{1H}-NMR (128.37 MHz, acetone-d6): δ [ppm] = −14.7 (s, 5B), −12.6 (s, 5B); FTIR (ATR): ṽ [cm−1] = 3165 (b, vO–H), 2941 (w, vC–H), 2603 (s, νB–H), 1686 (s, νC=O), 1662 (s, νC=O), 1487 (w, vC=C), 1458 (w, δC–H), 1439 (m, δC–H), 1393 (w, δC–H), 1355 (w, δC-H), 1286 (m), 1215 (s), 1126 (m), 1036 (s), 1003 (m), 955 (m), 923 (w), 894 (w), 840 (w), 749 (w, vB–B), 692 (s), 618 (s), 571 (w), 522 (w), 489 (w); HR-ESI-MS (positive mode, MeOH): m/z [M + H]+, calcd. for C8H20B10NO3: 288.2368, found: 288.2388; elemental analysis: calcd. (%) for C8H19B10NO3: C 33.67, H 6.71, N 4.91, found: C 33.22, H 6.68, N 4.80.
3.3. HPLC Measurements
3.3.1. Analysis of Purity Ultra Performance Liquid Chromatography (UPLC)
Samples were monitored at 254 nm using the following UPLC system: column Aquity UPLC® BEH C18 column (waters, 150 × 2.1 mm, 1.7 µM, 130 Å), UPLC (waters, Milford, MA, USA): binary solvent manager UPB, sample manager UPA, column manager UPM, and diode array detector PDA, γ detector Gabi Star (Raytest), flow rate 0.4 mL/min, eluent: (A) 0.1% trifluoroacetic acid in H2O, (B) MeCN; gradient: t0 min 95/5—t0.3 min 95/5—t5.3 min 5/95—t6.5 min 5/95—t6.8 min 95/5—t10 min 95/5). All compounds were found to have a purity of >97% (see Supplementary Materials, Figures S21–S23).
3.3.2. Determination of Lipophilicity (logD) by HPLC
The logD7.4, 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 (Raytest); column ODP-50 4B (Shodex Asahipak 50 × 4.6 mm) with C18 pre-column; eluent: MeOH/phosphate buffer (10 mM, pH 7.4), gradient t0 min 30/70—t25 min 95/5—t27 min 95/5—t28 min 30/70—t40 min 30/70, flow rate = 0.6 mL/min. Oxycarboxin (tR 9.02 min, logD7.4 1.13) and triphenylene (tR 29.47 min, logD7.4 5.49) served as references. Toluene was used as control and logD7.4 was found to be 2.72 (literature logD7.4 2.72) [81].
3.4. 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].
3.5. 5-LO Inhibitory Studies
For the determination of 5-LO inhibitory activities in intact cells, freshly isolated polymorphonuclear leukocytes (PMNL) (5 × 106) were re-suspended in phosphate-buffered saline (PBS) (pH 7.4) containing 1 mg/mL glucose and 1 µM CaCl2. After preincubation with R-830, S-2474, KME-4, and E-5110 in DMSO for 15 min at 37 °C, 5-LO product formation was stimulated by addition of calcium ionophore A23187 (2.5 µM in MeOH) and exogenous arachidonic acid (20 µM in EtOH). After 10 min at 37 °C, the reaction was stopped by addition of ice-cold methanol (1 mL). HCl (30 μL, 1 M), prostaglandin B1 (200 ng) and PBS (500 μL) were added and the formed metabolites were extracted and analyzed by HPLC as described previously [96]. 5-LO product formation was determined as the number of 5-LO products produced (nanograms) per 106 cells, which includes leukotriene B4 (LTB4), its all-trans isomers and 5-hydroperoxyicosatetraenoic acid (5-H(P)ETE). Cysteinyl LTs C4, D4, and E4 as well as oxidation products of LTB4 were not detected. Data were normalized to vehicle control (DMSO) and IC50 values and 95% confidence intervals (CIs) of at least three independent measurements were calculated (Table 2, see Supplementary Materials, Table S2, Figures S25 and S26). The selective 5-LO inhibitor BWA4C (0.1 µM) was used as control and inhibited 5-LO product formation by 92.5% ± 0.9.
3.6. 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 λmax = 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 λmax = 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%.
3.7. 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.
4. 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 carborane-based 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 IC50 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.
Conceptualization, S.B. and E.H.-H.; methodology, S.B., S.J., M.L., B.H., P.L., S.M., D.M.-I. and E.H.-H.; software, S.B., S.J., M.L., B.H., P.L., S.M., D.M.-I. and E.H.-H.; validation, S.B., S.J., M.L., S.G., B.H., P.L., D.S., J.P., S.M., D.M.-I. and E.H.-H.; formal analysis, S.B., M.L., B.H., J.P., D.M.-I. and E.H.-H.; investigation, S.B., S.J., M.L., S.G., B.H., P.L., S.M., D.M.-I. and E.H.-H.; resources, D.S., J.P., S.M., D.M.-I. and E.H.-H.; data curation, S.B., S.J., M.L., B.H., P.L., S.M., D.M.-I. and E.H.-H.; writing—original draft preparation, S.B.; writing—review and editing, S.B., S.J., M.L., B.H., P.L., J.P., S.M., D.M.-I. and E.H.-H.; visualization, S.B., S.J., M.L., B.H., P.L., J.P., S.M., D.M.-I. and E.H.-H.; supervision, E.H.-H.; project administration, E.H.-H.; funding acquisition, E.H.-H. All authors have read and agreed to the published version of the manuscript.
Animal handling and all study protocols were in agreement with the European Community guidelines (EEC Directive of 1986; 86/609/EEC) and the local guidelines. Experimental protocols were approved by the local Institutional Animal Care and Use Committee (IACUC) and by the national licensing committee at the Department of Animal Welfare, Veterinary Directorate, Ministry of Agriculture, Forestry and Water Management of the Republic of Serbia (Permission No. 323-07-02147/2023-05).
Not applicable.
The data presented in this study are available in the article or
The authors declare no conflict of interest.
| Abbreviation | Meaning |
| A375 | Human melanoma cell line |
| A549 | Human lung carcinoma cell line |
| AA | Arachidonic acid |
| AnnV | Annexin V |
| AO | Acridine orange |
| Apostat | FITC-conjugated pan-caspase inhibitor |
| ATR-IR | Attenuated total reflection infrared spectroscopy |
| b/br | Broad |
| BWA4C | N-[(E)-3-(3-Phenoxyphenyl)prop-2-enyl]acetohydroxamic acid |
| CDCl3 | Deuterated chloroform |
| CFSE | Carboxyfluorescein N-succinimidyl ester |
| CIs | Confidence intervals |
| COX | Cyclooxygenase |
| COXIBs | COX-2 specific inhibitors |
| CV | Crystal violet |
| CYP | Cytochrome P450 |
| d | Doublet |
| dd | Doublet of doublets |
| ddd | Doublet of doublets of doublets |
| dtd | Doublet of triplets of doublets |
| DHR 123 | Dihydrorhodamine 123 |
| DMAP | 4-Dimethylaminopyridine |
| FLAP | 5-Lipoxygenase-activating protein |
| FT-ICR | Fourier-transform ion cyclotron resonance mass spectrometry |
| GI | Gastrointestinal |
| HCT116 | Human colorectal carcinoma cell line |
| 5-H(p)ETE | 5-Hydroperoxyeicosatetraenoic acid |
| HPLC | High-performance liquid chromatography |
| HR-ESI-MS | High-resolution electrospray ionization mass spectrometry |
| HT29 | Human colorectal adenocarcinoma cell line |
| IC50 | Half-maximal inhibitory concentration |
| logD | Partition coefficient |
| LO | Lipoxygenase |
| LTs | Leukotrienes |
| LTB4 | Leukotriene B4 |
| m | Medium (IR), multiplet (NMR), meta |
| M | Molarity |
| 3-MA | 3-Methyladenine |
| MDA-MB-231 | M. D. Anderson-Metastatic breast-231 triple negative adenocarcinoma cell line |
| m.p. | Melting point |
| MTT | 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide |
| nBuLi | n-Butyllithium |
| NEt3 | Triethylamine |
| n.i. | no inhibition |
| NMR | Nuclear magnetic resonance |
| NSAIDs | Non-steroidal anti-inflammatory drugs |
| p | para |
| PBS | Phosphate-buffered saline |
| PFA | Paraformaldehyde |
| PGs | Prostaglandins |
| PGE2 | Prostaglandin E2 |
| PGI2 | Prostaglandin I2 |
| PI | Propidium iodide |
| PMNL | Polymorphonuclear leukocytes |
| ppm | Parts per million |
| p-Ts | p-Toluenesulfonyl |
| q | Quartet |
| R F | Retention factor |
| RT | Room temperature |
| s | Strong (IR), singlet (NMR) |
| SC-560 | 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole |
| SD | Standard deviation |
| SI | Selectivity index |
| t | Triplet |
| td | Triplet of doublets |
| TBAF | Tetra-n-butylammonium fluoride |
| TBDMSCl | Tert-butyldimethylsilyl chloride |
| TLC | Thin-layer chromatography |
| TMS | Tetramethylsilane |
| t R | Retention time |
| TXA2 | Thromboxane A2 |
| UPLC | Ultra-performance liquid chromatography |
| w | Weak |
Footnotes
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Enlarge this image.Figure 1. Chemical structures of selected di-tert-butylphenols showing the conserved 2,6-di-tert-butylphenol motif (blue).
Enlarge this image.Scheme 1. (A) Synthesis of carborane-based di-tert-butylphenol analogs R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb. (B) Reagents and conditions: (I) Synthetic entry towards p-substituted di-tert-butylphenol analog R-830-Cb. (a) 1. TBDMSCl, NEt3 and 4-dimethylaminopyridine (DMAP), DCM, 0 °C → RT, 48 h; 2. HCl(aq), RT (97% yield); (b) 1. nBuLi, Et2O, 0 °C → RT, 2 h; 2. 2-thiophenecarbonyl chloride, 0 °C → RT, 24 h; 3. HCl(aq), RT (64% yield); (c) 1. TBAF, THF, 0 °C, 20–30 min; 2. H2O, RT (90% yield); Key intermediate: (d) 1. nBuLi, Et2O, 0 °C → RT, 2 h; 2. methyl formate, 0 °C → RT, 24 h; 3. HCl(aq), RT (86% yield); (II) Synthetic entry towards p-substituted di-tert-butylphenol analog S-2474-Cb. (e) 1. 2-ethylisothiazolidine-1,1-dioxide, THF, −78 °C, 30 min; 2. nBuLi, THF, −78 °C, 1.5 h → RT; 3. HCl(aq), RT (53% yield); 4. NaH, THF, RT, 2 h; 5. p-toluenesulfonyl chloride, 0 °C → RT, 24 h; 6. HCl(aq), RT (87% yield); (f) 1. NaH, THF, RT, 2 h; 2. H2O, RT (64% yield); (g) 1. TBAF, THF, 0 °C, 20–30 min; 2. H2O, RT (96% yield); (III) Synthetic entry towards p-substituted di-tert-butylphenol analog KME-4-Cb; (h) 1. 3-(triphenylphosphoranylidene)-γ-butyrolactone, THF/DMF, 120 °C, 48 h; 2. H2O, RT (84% yield); (i) 1. TBAF, THF, 0 °C, 25 min; 2. H2O, RT (99% yield); (IV) Synthetic entry towards p-substituted di-tert-butylphenol analog E-5110-Cb; (j) 1. (1-methoxy-2-oxo-3-pyrrolidinyl)triphenyl-phosphonium bromide, NEt3, THF, RT, 45 min; 2. DMF, 75 °C, 24 h; 3. H2O, RT (50% yield); (k) 1. TBAF, THF, 0 °C, 30 min; 2. H2O, RT (93% yield).
Enlarge this image.Figure 2. Molecular structure of compounds: (A) R-830-Cb, (B) KME-4-Cb, (C) E-5110-Cb, and (D) S-2474-Cb. Hydrogen atoms, except for O-H bonds, are omitted for clarity. Thermal ellipsoids are drawn at 50% probability level (beige = B, grey = C, blue = O, orange = N, yellow = S). Selected bond lengths, distances and bond angles are given in the Supplementary Materials, Tables S3–S6.
Enlarge this image.Figure 3. Mode of action of R-830 and R-830-Cb. HCT116 cells were treated with an IC50 dose of R-830 and R-830-Cb for 72 h and stained with (A) Annexin V (AnnV)/propidium iodide (PI), (B) PI (white arrows mark apoptotic nuclei), (C) Apostat, (D) Carboxyfluorescein diacetate succinimidyl ester (CFSE), (E) Dihydrorhodamine 123 (DHR 123), and (F) Acridine orange (AO). (G) HCT116 cells were co-treated with R-830-Cb and 3-methyladenine (3-MA) or chloroquine and viability was estimated by crystal violet (CV) assay. * p < 0.05; *** p < 0.001 in comparison to relevant control.
Results of COX assay and logD7.4, HPLC determination.
| Compound | % Inhibition at 100 µM (A,B) | logD7.4, HPLC | |
|---|---|---|---|
| COX-1 | COX-2 | ||
| R-830 | 93 | 85 | 3.91 |
| R-830-Cb | 18 | 6 | 2.37 |
| KME-4 | 64 | 97 | 3.79 |
| KME-4-Cb | 26 | n.i. | 1.87 |
| E-5110 | 66 | 105 | 3.35 |
| E-5110-Cb | n.i. | n.i. | 1.50 |
| S-2474 | 38 | 86 | 3.75 |
| S-2474-Cb | n.i. | n.i. | 1.88 |
(A) n.i. = no inhibition (%inhibition below 5%); (B) Celecoxib served as reference for COX-2: pIC50 (pIC50 = −log10 (IC50 [
IC50 values for inhibition of 5-LO product formation in intact PMNL for the carborane derivatives R-830-Cb, KME-4-Cb, E-5110-Cb, S-2474-Cb, and their respective reference compounds R-830, KME-4, E-5110, and S-2474 (see
| Compound | IC50 [µ |
Compound | IC50 [µ |
|---|---|---|---|
| R-830-Cb | 0.65 | R-830 | 0.26 |
| KME-4-Cb | 0.07 | KME-4 | 0.15 |
| E-5110-Cb | 0.22 | E-5110 | 0.12 |
| S-2474-Cb | <0.03 | S-2474 | <0.03 |
(A) The selective 5-LO inhibitor BWA4C (0.1 µM) was used as control and inhibited 5-LO product formation by 92.5% ± 0.9; (B) Data are presented as the mean of at least three independent experiments with 95% CIs (CIs = confidence intervals).
IC50 values [µM] of di-tert-butylphenol derivatives and their carborane analogs for specific cancer cell lines. Data are presented as mean ± SD of three independent experiments for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) tests. * Carbon-based reference compounds.
| Compounds | Assays | A375 | A549 | HCT116 | HT-29 | MDA-MB-231 |
|---|---|---|---|---|---|---|
| R-830 * | MTT | 16.7 ± 3.1 | 19.0 ± 0.7 | 20.6 ± 0.4 | 19.4 ± 0.4 | 33.3 ± 1.2 |
| CV | 29.4 ± 2.3 | 32.6 ± 1.3 | 30.8 ± 3.2 | 24.9 ± 1.3 | 34.0 ± 1.6 | |
| R-830-Cb | MTT | 35.9 ± 0.8 | 66.5 ± 1.7 | 40.9 ± 3.1 | 65.6 ± 5.3 | 46.5 ± 0.1 |
| CV | 47.2 ± 0.7 | 77.3 ± 1.7 | 49.1 ± 6 | 73.3 ± 3.4 | 55.6 ± 0.7 | |
| KME-4 * | MTT | 13.8 ± 0.6 | 21.9 ± 0.7 | 18.4 ± 4.6 | 18.5 ± 1.5 | 80.9 ± 7.3 |
| CV | 36.4 ± 1.3 | 36.3 ± 2.8 | 36.2 ± 1.9 | 36.2 ± 1.9 | 88.6 ± 2.4 | |
| KME-4-Cb | MTT | 83.3 ± 2.3 | 119.7 ± 7.0 | 88.5 ± 4.3 | 139.3 ± 5.4 | 119.7 ± 3.9 |
| CV | 155.2 ± 5.8 | 157.8 ± 3.0 | 127.0 ± 8.0 | 164.7 ± 8.5 | 132.7 ± 4.1 | |
| E-5110 * | MTT | 12.9 ± 1.1 | 22.1 ± 1.6 | 25.1 ± 1.1 | 25.6 ± 2.6 | 34.2 ± 1.2 |
| CV | 33.5 ± 1.7 | 41.1 ± 4.3 | 39.6 ± 2.8 | 39.8 ± 2.1 | 47.3 ± 1.7 | |
| E-5110-Cb | MTT | 69.3 ± 1.7 | 176.6 ± 14.5 | 143.9 ± 10.5 | 119.9 ± 9.7 | 200 ± 0.0 |
| CV | 103.2 ± 10.9 | 197.7 ± 5.7 | 193.1 ± 3.0 | 155.4 ± 6.0 | >200 | |
| S-2474 * | MTT | 13.2 ± 2.7 | 16.2 ± 1.2 | 17.5 ± 1.0 | 16.1 ± 1.1 | 34.8 ± 1.7 |
| CV | 19.2 ± 0.7 | 17.9 ± 1.2 | 23.7 ± 1.9 | 23.8 ± 1.7 | 43.5 ± 1.8 | |
| S-2474-Cb | MTT | 106.0 ± 6.3 | 121.8 ± 10.4 | 108.1 ± 11.5 | 81.8 ± 6.1 | >200 |
| CV | 135.5 ± 6.3 | 149.7 ± 4.3 | 133.4 ± 7.1 | 137.5 ± 7.8 | >200 |
Supplementary Materials
The following Supplementary Materials can be downloaded at
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Details
; Laube, Markus 3
; George, Sven 4 ; Hofmann, Bettina 4 ; Lönnecke, Peter 1
; Steinhilber, Dieter 4 ; Pietzsch, Jens 5
; Mijatović, Sanja 2 ; Maksimović-Ivanić, Danijela 2
; Hey-Hawkins, Evamarie 1
1 Institut für Anorganische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany;
2 Department of Immunology, Institute for Biological Research “Siniša Stanković”, National Institute of Republic of Serbia, University of Belgrade, Bul. Despota Stefana 142, 11060 Belgrade, Serbia;
3 Department of Radiopharmaceutical and Chemical Biology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstrasse 400, 01328 Dresden, Germany;
4 Institute of Pharmaceutical Chemistry, University of Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt, Germany;
5 Department of Radiopharmaceutical and Chemical Biology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstrasse 400, 01328 Dresden, Germany;