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Structure–Activity Relationship of para-Carborane Selective Estrogen Receptor β Agonists
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Structure–Activity Relationship of para-Carborane Selective Estrogen Receptor β Agonists
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  • David Sedlák
    David Sedlák
    CZ-OPENSCREEN, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic
  • Tyler A. Wilson
    Tyler A. Wilson
    Medicinal Chemistry Shared Resource, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States
  • Werner Tjarks
    Werner Tjarks
    Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
  • Hanna S. Radomska
    Hanna S. Radomska
    Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
  • Hongyan Wang
    Hongyan Wang
    Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
    More by Hongyan Wang
  • Jayaprakash Narayana Kolla
    Jayaprakash Narayana Kolla
    CZ-OPENSCREEN, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic
  • Zbigniew J. Leśnikowski
    Zbigniew J. Leśnikowski
    Laboratory of Medicinal Chemistry, Institute of Medical Biology PAS, 106 Lodowa Street, 93-232 Lodz, Poland
  • Alena Špičáková
    Alena Špičáková
    Department of Pharmacology, Faculty of Medicine, Palacky University, Hněvotínská 3, 77515 Olomouc, Czech Republic
  • Tehane Ali
    Tehane Ali
    Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
    More by Tehane Ali
  • Keisuke Ishita
    Keisuke Ishita
    Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
  • Liva Harinantenaina Rakotondraibe
    Liva Harinantenaina Rakotondraibe
    Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
  • Sandip Vibhute
    Sandip Vibhute
    Medicinal Chemistry Shared Resource, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States
  • Dasheng Wang
    Dasheng Wang
    Medicinal Chemistry Shared Resource, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States
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  • Pavel Anzenbacher
    Pavel Anzenbacher
    Department of Pharmacology, Faculty of Medicine, Palacky University, Hněvotínská 3, 77515 Olomouc, Czech Republic
  • Chad Bennett
    Chad Bennett
    Medicinal Chemistry Shared Resource, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States
    Drug Development Institute, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States
    More by Chad Bennett
  • Petr Bartunek*
    Petr Bartunek
    CZ-OPENSCREEN, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic
    *Email: [email protected]
  • Christopher C. Coss*
    Christopher C. Coss
    Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
    Drug Development Institute, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States
    *Email: [email protected]. Tel: 614-688-1309.
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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2021, 64, 13, 9330–9353
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https://doi.org/10.1021/acs.jmedchem.1c00555
Published June 28, 2021

Copyright © 2021 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY-NC-ND 4.0 .

Abstract

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Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERβ, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17β-estradiol geometry in the design of ERβ selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERβ selective structure–activity relationship. We report ERβ agonists with low nanomolar potency, greater than 200-fold selectivity for ERβ over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERβ selective agonists measure favorably against clinically developed ERβ agonists and support further evaluation of carborane-based selective estrogen receptor modulators.

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Copyright © 2021 The Authors. Published by American Chemical Society

Introduction

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Although the physiologic functions of estrogens have been recognized as clinically important for roughly a century, (1) the underlying mechanisms of estrogen action were largely unknown prior to the discovery of a high-affinity estrogen receptor (estrogen receptor α, or ERα) in 1958 and subsequent cloning of its gene in 1985. (2) Nonetheless, residual effects of exogenous estrogens in ERα knockout (ERα-KO) mice (3) were not readily explained until the surprising discovery of the second estrogen receptor (estrogen receptor β, or ERβ). (4) Mechanistically, ERα and ERβ function as transcription factors, which upon estrogen stimulation, translocate to the cell nucleus, homo- or hetero-dimerize, and bind to specific DNA sequences present in regulatory elements of target genes. (5,6) Adding to the complexity of estrogen signaling, a G protein-coupled transmembrane receptor, GPER1 (GPR30) localized to endoplasmic reticulum has been recently identified and demonstrated to be involved in acute estrogen-mediated responses. (7,8) ERα, ERβ, and GPER1 are widely expressed in diverse tissues but have distinct distribution patterns, and even opposing functions, which creates a complex picture of composite estrogen action. (9−11)
All three receptor isoforms bind to the predominate circulating endogenous estrogen 17β-estradiol (E2) (Figure 1) with very similar high affinities (9,12,13) such that genetic approaches are required to deconvolute the contributions of each receptor to whole animal estrogen response. Characterization of ERα, (14) ERβ, (15) and GPER1 (16) genetically null mice supports distinct biological functions of each receptor including a very limited role for ERβ in control of steroidogenesis and homeostasis of uterine and breast tissues.

Figure 1

Figure 1. Estrogen receptor ligands.

One considerable challenge in understanding ERβ biology are the well-known differences in phenotypes between the multiple ERβKO mice that have been thus far developed. (17,18) However, when KO mice are simultaneously treated with ERβ selective (diarylpropionitrile (19) [DPN]) or ERα selective (propylpyrazole triol (20) [PPT]) tool compounds, a clearer role for ERβ emerged. (17) The improved understanding of ERβ’s role in nonreproductive tissues and inflammatory response resulted in a heightened interest in developing ERβ-targeted therapeutics that could minimize unwanted ERα-mediated side effects classically associated with estrogen administration. (21)
Structurally, both ERα and ERβ are composed of three major domains: N-terminal transactivation domain, central DNA-binding domain (DBD), and C-terminal ligand-binding domain (LBD). There is 97% amino acid sequence identity between DNA-binding domains and 54% between LBDs of human ERα and ERβ. (22) Given the high structural similarity in human and preclinical species LBDs, (23) selective targeting of ERβ over ERα is challenging. Despite this, numerous chemically diverse selective ERβ agonists have been described with several compounds reporting low nanomolar ERβ activity and ≥100-fold selectivity over ERα. (24) Clinical development of ERβ agonists has thus far been limited to two agents. ERB-041 (25) (prinaberel), a selective agonist with a benzoxazole scaffold, was the first clinical candidate described and was developed to treat rheumatoid arthritis and other inflammatory diseases including Crohn’s disease, endometriosis, and interstitial cystitis, but ultimately failed to demonstrate efficacy in phase II trials. (26) LY500307 (SERBA-1, erteberel), a selective agonist with a benzopyran core, (27) was later advanced for the treatment of symptoms associated with benign prostatic hypertrophy but similarly failed to demonstrate efficacy in phase II trials. (28) Though development of ERB-041 has been terminated, LY500307 has recently been evaluated for symptoms associated with schizophrenia (29) (NCT01874756) and for estrogen withdrawal-induced mood symptoms in women with past perimenopausal depression (30) (NCT03689543). The continued description of novel, isoform-selective, estrogen receptor ligands suggests interest in the development of therapeutic estrogens remains high. (31)
While many groups have focused on “classical” organic scaffolds to develop potent ER modulators, in 1999, Endo and colleagues reported the estrogen receptor activity of a series of novel 17β-estradiol bioisosteres (32) belonging to a structurally unique class of molecules known as dicarba-closo-dodecaboranes (carboranes). Carboranes are perhaps best known for their use in boron-neutron capture therapy (33−35) (BNCT) and also possess multiple attractive features for use in non-BNCT-based medicinal chemistry including, but not limited to, their highly hydrophobic nature, spherical shape, which provides three-dimensional flexibility for target–ligand interactions compared to their two-dimensional benzene counterpart, and their resistance toward conventional enzymatic degradation. (36−38)
To explore the possibility to control ligand selectivity for ERα and ERβ, we prepared a series of 17β-estradiol derivatives substituted by different ortho-, meta-carboranes and metallacarboranes in the 17α-position. (39) Although these compounds acted as partial ER agonists, the selectivity for either isoform was achieved only to a limited extent in contrast to similar efforts carried out with perfluoroalkyl- or aryl-substituted 17β-estradiols. (40,41)
Endo and colleagues explored a different route. Through comprehensive docking studies, they noted that carboranylphenols provided the proper molecular geometry and hydrophobicity to fit within the ERα LBD while maintaining key hydrogen bonds required for high affinity. (42) From this work, the para-carborane BE120 was shown to be an effective 17β-estradiol mimic with sufficient druglike properties to provide in vivo estrogenic effects following daily parenteral dosing in rats. (43) By comparing BE120 to ERB-041, this same group more recently reported para-carboranylcyclohexanol derivatives with ERβ selective activity. (44) Herein, we describe a novel series of BE120-based para-carborane derivatives demonstrating highly selective ERβ agonist activity.

Results and Discussion

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Chemistry

The intermediate hydrocarbon- (pathway 1) or α-hydroxy-hydrocarbon-substituted para-carboranes 2ax (pathway 2) were prepared by lithiation of 1 (43,45) with n-butyllithium in 1,2-dimethoxyethane followed by alkylation with different alkyl halides or treatment with various aldehydes, respectively, as seen in Scheme 1. Subsequent demethylation with boron tribromide in methylene chloride furnished the target carboranes BE120 and 3bx.

Scheme 1

Scheme 1. Synthesis of Target Alkyl- and Hydroxyalkyl-Substituted Carboranesa

a(a) n-BuLi, dimethoxyethane (1,2-DME), −78 or 0 °C; (b) BBr3, CH2CI2, 0 °C to room temperature (RT).

The synthesis of each enantiomer of chiral alcohol 3k and ketone 9 was carried out as described in Scheme 2. Oxidation of 2k using pyridinium chlorochromate (PCC) in methylene chloride provided ketone 4, which was selectively reduced to the corresponding S and R enantiomers 5 and 6 using Corey–Bakshi–Shibata (CBS) conditions (46) as seen in Scheme 2. Subsequent demethylation using boron tribromide in methylene chloride furnished the target S and R enantiomers (7 and 8) and ketone 9. The enantiomeric excesses (ee) of chiral alcohols 7 and 8 were determined to be >80 and >75%, respectively, through chiral high-performance liquid chromatography (HPLC) of intermediates 5 and 6. The absolute configuration of chiral alcohol 8 was established by Mosher ester analysis. (47,48)

Scheme 2

Scheme 2. Synthesis of Ketone 9 and S and R Enantiomers 7 and 8a

a(a) PCC, CH2Cl2; (b) BH3·THF, (S)-2-Me-CBS, RT; (c) BH3·THF, (R)-2-Me-CBS, RT; (d) BBr3, CH2Cl2, 0 °C to RT.

To determine the importance of the para-carborane core to the ERβ activity and selectivity, we made several analogues in which the para-carborane core was replaced with bioisosteric phenyl, cyclohexyl, and [2.2.2]-bicyclo groups. The syntheses of these non-carborane-containing compounds are detailed below.
Commercially available biphenyl aldehyde 10 was treated with n-hexylmagnesium bromide, and then the resulting racemic alcohol was oxidized with PCC, as seen in Scheme 3 to afford ketone 11. Reduction of ketone 11 using borane–tetrahydrofuran (THF) complex and the (R)-2-Me-CBS reagent under CBS conditions yielded chiral alcohol 12. Demethylation of the methyl phenyl ether moiety of 12 afforded bioisosteric phenyl analogue 13.

Scheme 3

Scheme 3. Synthesis of Phenyl Compound 13a

a(a) n-Hexylmagnesium bromide, Et2O, 0 °C; (b) PCC, CH2CI2; (c) BH3·THF, (R)-2-Me-CBS, 0 °C; (d) 1-dodecanethiol, N-methylpyrrolidinone (NMP), NaOH, 100 °C.

Synthesis of the cyclohexyl-containing analogue 17 is depicted in Scheme 4. Commercially available phenol 14 was methylated with iodomethane using cesium carbonate as the base in refluxing acetone to furnish intermediate 15. Ketone 15 was subjected to (methoxymethyl)triphenylphosphonium chloride and lithium hexamethyldisilazane (LiHMDS) followed by subsequent acid hydrolysis. The resulting aldehyde was treated with n-hexylmagnesium bromide, followed by oxidation with PCC to afford ketone 16. Reduction of ketone 16 using borane–THF complex and the (R)-2-Me-CBS reagent under CBS conditions yielded the chiral alcohol, which was subjected to demethylation of the methyl phenyl ether moiety to afford the bioisosteric cyclohexyl analogue 17.

Scheme 4

Scheme 4. Synthesis of Cyclohexyl Compound 17a

a(a) Cs2CO3, CH3I, acetone, reflux; (b) (methoxymethyl)triphenylphosphonium chloride, LiHMDS, THF; 2 N HCI; (c) hexylmagnesium bromide, Et2O, 0 °C; (d) PCC, CH2CI2; (e) BH3·THF, (R)-2-Me-CBS, 0°C; (f) 1-dodecanethiol, NMP, NaOH, 100 °C.

Synthesis of compound 26 started with the asymmetric [2.2.2]-bicyclocarboxylic acid 18, as depicted in Scheme 5. Mercuric oxide-catalyzed bromination provided [2.2.2]-bicyclobromide 19, which was then allowed to alkylate benzene via aluminum trichloride-catalyzed Friedel–Crafts alkylation to provide ester 20. Lithium aluminum hydride reduction of ester 20, followed by PCC promoted oxidation afforded aldehyde 21. Treatment of this aldehyde with n-hexylmagnesium bromide gave racemic alcohol 22, which was then brominated to provide alcohol 23. PCC oxidation followed by reduction of the resulting ketone 24 using borane–THF complex and the (R)-2-Me-CBS reagent under CBS conditions (46) yielded chiral alcohol 25. Palladium-catalyzed substitution of the aryl bromide of 25 to a hydroxyl group by utilizing benzaldehyde oxime afforded the desired [2.2.2]-alcohol 26.

Scheme 5

Scheme 5. Synthesis of [2.2.2]Bicyclic Compound 26a

a(a) Br2, dichloromethane (DCM), HgO; (b) AICI3, benzene, (c) LiAIH4, Et2O; (d) PCC, NaHCO3, NaOAc, CH2CI2; (e) hexylmagnesium bromide, Et2O; (f) AgOAc, Br2, CHCI3; (g) PCC, CH2CI2; (h) BH3·THF, (R)-2-Me-CBS 0 °C; (i) benzaldehyde oxime, Cs2CO3, RockPhos Pd G3.

Finally, to better understand the importance of the para-substituted phenol in relation to its ERβ agonism activity, a series of analogues were prepared in which the aryl group attached directly to the carborane core was replaced with various substituents as seen Scheme 6. Briefly, carboranes 27 and 29 were lithiated with n-butyllithium in 1,2-dimethoxyethane followed by the addition of the resulting lithiated species into 1-heptanal to afford the target phenyl-substituted carborane 28 and intermediate 30, respectively. Intermediate 30 was demethylated utilizing boron tribromide in dichloromethane to furnish the target meta-substituted phenol compound 31.

Scheme 6

Scheme 6. Synthesis of Compounds 28 and 31a

a(a) n-BuLi, 1,2-DME, −78 or 0 °C, 1-heptanal; (b) BBr3, CH2CI2, 0 °C to RT.

Structure–Activity Relationship (SAR) Study

The target compounds were analyzed for their ability to interact with ERα and ERβ by two approaches. We determined binding affinity to both receptors using a competitive biochemical polarized fluorescence-based assay with a fluorescent tracer. We also measured the consequence of receptor binding using luciferase cell-based ER transactivation functional assays in human embryonic kidney (HEK) cells expressing full-length ERα or ERβ. Our results are listed in Table 1. The modulation of ER biological activity is a result of an orchestrated series of ligand–receptor, receptor–DNA, and receptor–transcriptional machinery interactions ultimately concluding in the modulation of transcription from the ER-regulated promoters. Considering this complex mechanism of ligand-induced transactivation, our SAR was driven using cellular transactivation data and binding affinities were primarily used to confirm the specificity of the interaction and observed activity trends.
Table 1. Structure and Biological Activities of Novel para-Carborane-Based Estrogen Receptor Modulators
a

Each compound was tested for ERα and ERβ agonism in luciferase transactivation assay and compared to 17β-estradiol. The EC50 values are shown in nanomolar. Agonism is reported as full (≥67%) colored in green, partial (34−66%) colored in yellow, weak partial (10−33%) colored in red, and no agonism (<10%) not colored. To determine the ranking of agonism, percent agonism was calculated using Emax for each compound and comparing to the activity of 100 nM 17β-estradiol set to 100%.

b

ERβ selectivity was calculated as the ratio of EC50 for ERα/EC50 for ERβ.

c

Compound cytotoxicity from parallel experiment to reporter assays determined by measurement of intracellular adenosine 5′-triphosphate (ATP) levels. IC50 values are expressed in micromolar.

d

Compound affinities for ERα or ERβ proteins were evaluated in polarized fluorescence-based competitive binding assay PolarScreen ER α/β. Compounds were allowed to compete with Fluormone EL Red ligand for the binding to the receptor in a dose response experiment, and Kd values were calculated from IC50 values using the Cheng−Prusoff equation.

e

n.a., not active.

f

n.c., not calculated.

PPT was used as a positive control for ERα-selective agonism, and DPN was used as a positive control for ERβ-selective agonism. 17β-Estradiol (E2) was used as an endogenous ligand benchmark, and it is potent against both isoforms with only 1.3-fold selectivity for ERβ over ERα. For the purposes of this paper, maximal agonism is defined by E2, and all other agonistic effects are reported as either full agonism (≥67%, highlighted in green), partial agonism (34–66%, highlighted in yellow), weak partial agonism (10–33%, highlighted in red), or no agonism (<10%, not highlighted) relative the observed response of E2. Initially, we demonstrated that the highly potent agonist (BE120), originally reported by Endo and colleagues, (32) was nonselective with equimolar potency and full agonism activity for both ERα and ERβ isoforms. In an effort to design a more selective ERβ agonist, we set out to investigate the structure–activity relationship at the R3 position. Interestingly, compound 3b, with a methyl group replacing the hydroxyl group of BE120, reduced the agonist potency for ERα by ∼1000-fold and ERβ by ∼30-fold, resulting in 36-fold selectivity for ERβ over ERα. Reintroducing a hydroxyl at the carbon closest to the carborane cage (compound 3c) restored most of the potency of BE120 but also reduced the selectivity to 8-fold. Based on these analogues, we designed a series of molecules extending and modifying the alkyl chain with and without a hydroxyl adjacent to the carborane core. The addition of one and then two more methylene units (compounds 3dg) resulted in reduced potencies, similar in magnitude between the isoforms with a modest preference for ERβ (Figure 2). By extending by one more methylene to an n-pentyl side chain (3h and 3i), these two series now began to diverge more significantly. Compound 3h retained a very similar profile to that of the n-butyl analogue 3f with good potency but modest selectivity. However, α-hydroxy compound 3i demonstrated a much larger drop in potency for ERα, leading to 92-fold selectivity for ERβ over ERα. Compound 3j with an n-heptyl side chain continued the trend of reduced potency and modest selectivity, while the corresponding α-hydroxylated molecule 3k had improved ERβ potency and selectivity. Importantly, 3k was the first molecule demonstrating full agonism for ERβ, but partial agonism for ERα. Extending the chain length further to n-decyl in 3l and 3m eroded the ERβ potency displayed by 3j and 3k, respectively.

Figure 2

Figure 2. Ligand selectivity for ERβ increases with elongation of the side hydrocarbon chain. BE120, 3g, and 8 were tested in ERα (dashed line) and ERβ (solid line) transactivation assays in the concentration range between 10 μM and 1 pM, in triplicates and activities were expressed relative to 100 nM 17β-estradiol set to 100%. Increasing length of the carboranes’ side hydrocarbon chain is accompanied with decreasing potency in both ERα and ERβ reporter assays and by increasing selectivity for ERβ (blue: nonselective, green: 10–100× selective, red: selectivity >100×).

Adding a branching methyl at the end of the alkyl chain (3nq) followed the SAR described above, with modest decreases in potency (ca. 2- to 10-fold) but with minimal effect on selectivity (compare 3n with 3i, 3p with 3j, and 3q with 3k, 7, and 8.) The single indane-containing molecule (3r) did not exhibit high selectivity. Several molecules were made to explore the effects of replacing the terminal isopropyl substituent of 3nq with a terminal phenyl ring. Overall, the SAR for these analogues is less consistent with regard to potency, selectivity, and agonism. However, it does appear that the α-hydroxylated molecules (3t, 3v, and 3x) are less selective for ERβ than their nonhydroxylated counterparts (3s, 3u, and 3w). Notably, one of the highest ERβ selective activities we observed belonged to this class. Compound 3u showed an EC50 = 87 nM on ERβ and no detectable activity on ERα up to 100 μM, therefore demonstrating selectivity of more than 3 orders of magnitude. This divergence implies that there are favorable aromatic interactions occurring in the binding site and that an α-hydroxyl group reorients the phenyl ring away from these hypothesized favorable interactions. In the future, additional analogues will be needed to better understand this portion of the SAR.
As seen in Table 2, compounds 7 and 8, the individual S and R enantiomers of 3k, each retained good ERβ potency and high selectivity, as well as partial agonism for ERα. While the presence α-hydroxyl group clearly improves ERβ potency and selectivity, its stereo configuration does not seem to meaningfully impact ER-isoform selectivity. Since both enantiomers of 3k are similarly active and selective, it is interesting to note that corresponding ketone 9 displays reduced ERβ potency and selectivity. Based on the overall structure–activity relationship described, compounds 7, 8, and 3q demonstrate a good balance between selectivity and potency for ERβ, warranting additional biological profiling, which will be the subject of future reports.
Table 2. Activity of Enantiomers of 3k and Its Ketone Precursor
a

Each compound was tested for ERα and ERβ agonism in luciferase transactivation assay and compared to 17β-estradiol. The EC50 values are shown in nanomolar. Agonism is reported as full (≥67%) colored in green, partial (34−66%) colored in yellow, weak partial (10−33%) colored in red, and no agonism (<10%) not colored. To determine the ranking of agonism, percent agonism was calculated using Emax for each compound and comparing to the activity of 100 nM 17β-estradiol set to 100%.

b

ERβ selectivity was calculated as the ratio of EC50 for ERα/EC50 for ERβ.

c

Compound cytotoxicity from parallel experiment to reporter assays determined by measurement of intracellular adenosine 5′-triphosphate (ATP) levels. IC50 values are expressed in micromolar.

d

Compound affinities for ERα or ERβ proteins were evaluated in polarized fluorescence-based competitive binding assay PolarScreen ER α/β. Compounds were allowed to compete with Fluormone EL Red ligand for the binding to the receptor in a dose response experiment, and Kd values were calculated from IC50 values using the Cheng−Prusoff equation.

Next, we wanted to evaluate the importance of the para-carborane core to the biological activity of these molecules. To do so, a series of molecules were made in which the para-carborane core was replaced while retaining the left-hand side phenol and the right-hand side 1-hydroxy-n-heptyl side chain of compound 8. The results are detailed in Table 3. Replacing the carborane with either para-phenyl or 1,4-cyclohexyl rings resulted in analogues 13 and 17 that displayed no measurable agonist activity at either ERα or ERβ. We then used a [2.2.2] bridged bicycle 26 as an isostere that would better mimic the three-dimensional shape of the carborane. Bridged bicycle 26 lost ∼100-fold activity for ERβ compared to the benchmark of 8 and had no measurable agonism for ERα. From these data, it seems clear that the carborane core structure plays a unique role in affording ER agonist activity.
Table 3. Activity of Bioisosteric Replacements of para-Carborane Core
a

Each compound was tested for ERα and ERβ agonism in luciferase transactivation assay and compared to 17β-estradiol. The EC50 values are shown in nanomolar. Agonism is reported as full (≥67%) colored in green, partial (34−66%) colored in yellow, weak partial (10−33%) colored in red, and no agonism (<10%) not colored. To determine the ranking of agonism, percent agonism was calculated using Emax for each compound and comparing to the activity of 100 nM 17β-estradiol set to 100%.

b

ERβ selectivity was calculated as the ratio of EC50 for ERα/EC50 for ERβ.

c

Compound cytotoxicity from parallel experiment to reporter assays determined by measurement of intracellular adenosine 5′-triphosphate (ATP) levels. IC50 values are expressed in micromolar.

d

Compound affinities for ERα or ERβ proteins were evaluated in polarized fluorescence-based competitive binding assay PolarScreen ER α/β. Compounds were allowed to compete with Fluormone EL Red ligand for the binding to the receptor in a dose response experiment, and Kd values were calculated from IC50 values using the Cheng−Prusoff equation.

e

n.c., not calculated.

f

n.d., not determined.

Finally, we were interested in probing the importance of the para-substituted phenol in relation to ERβ selectivity and agonism activity. The hydroxyheptyl side chain was kept constant while the phenol moiety was replaced with various aryl groups as seen in Table 4. Replacement of the para-substituted phenol with a phenyl (compound 28) resulted in complete loss of activity. Similarly, methylation of the phenol (compound 2k) resulted in complete loss of activity as well. Interestingly, the meta-substituted phenol compound 31 displayed modest ERβ agonism activity and excellent ERβ selectivity suggesting that there is flexibility in the binding pocket for accommodating phenols with different substitution patterns.
Table 4. Modification of the Phenol
a

Each compound was tested for ERα and ERβ agonism in luciferase transactivation assay and compared to 17β-estradiol. The EC50 values are shown in nanomolar. Agonism is reported as full (≥67%) colored in green, partial (34–66%) colored in yellow, weak partial (10–33%) colored in red, and no agonism (<10%) not colored. To determine the ranking of agonism, percent agonism was calculated using Emax for each compound and comparing to the activity of 100 nM 17β-estradiol set to 100%.

b

ERβ selectivity was calculated as the ratio of EC50 for ERα/EC50 for ERβ.

c

Compound cytotoxicity from parallel experiment to reporter assays determined by measurement of intracellular adenosine 5′-triphosphate (ATP) levels. IC50 values are expressed in micromolar.

d

Compound affinities for ERα or ERβ proteins were evaluated in polarized fluorescence-based competitive binding assay PolarScreen ER α/β. Compounds were allowed to compete with Fluormone EL Red ligand for the binding to the receptor in a dose response experiment, and Kd values were calculated from IC50 values using the Cheng–Prusoff equation.

e

n.a., not active.

f

n.c., not calculated.

Interactions with Other Nuclear Receptors (NRs)

In addition to the successful preparation of different para-carborane series as ligands for ERα and ERβ, Endo and colleagues showed that structurally similar para-carboranes can bind to androgen receptor (AR) and show full antagonistic activities in the micromolar range. (49,50) To investigate the possibility that ERβ and ERα, to a certain extent, are not the sole binding targets of these compounds, we systematically profiled the compound library in a panel of selective luciferase transactivation assays for 20 different nuclear receptors (NRs). All compounds were tested at 1 μM in both agonist and antagonist modes to identify all possible effects mediated through these receptors. Besides ERα and ERβ, we tested the library in the assays for remaining steroid receptors (SRs): androgen (AR), progesterone (PR), glucocorticoid (GR), and mineralocorticoid receptor (MR). These assays consist of clonal lines expressing individual human full-length steroid receptors in U2OS osteosarcoma cells. The reporter luciferase expression is controlled by viral long terminal repeat (LTR) promoter isolated from mouse mammary tumor virus (MMTV) harboring response elements for different SRs. Data summarized in Figure 3 and the Supporting Information (Tables S1 and S2) clearly show the prominent agonist activity on ERβ and, to less extent, on ERα. These data also confirm that chemical diversity represented in the library modulated ERβ selectivity and potency but never switched ER activity into antagonism as we did not detect any ER antagonist activity at 1 μM. Further, we did not find any activity on remaining SRs in any of the tested activity modes. This outcome accents the selectivity of para-carborane derivatives for ERβ.

Figure 3

Figure 3. Activities of the carboranes’ compound library on steroid receptors. (A) Compounds were profiled at 1 μM in the selective luciferase reporter assays for the agonistic and (B) antagonistic activities for steroid receptors. The activity is expressed relative to maximal activity induced by reference compound or as a fold induction compared to untreated cells for control assay (MMTV reporter alone, dark red dots). Cell viability in U2OS cells was used as a control experiment for the antagonistic activities and is expressed in a scale of 0–100% relative to dimethyl sulfoxide (DMSO)-treated cells (green dots).

Then, we extended the profiling further to other members of NR family: ERRγ, farnesoid X receptor (FXR), liver X receptor (LXR)α and β, peroxisome proliferator-activated receptor (PPAR)α, β/δ and γ, vitamin D receptor (VDR), retinoic acid receptor (RAR) α and γ and retinoid X receptor (RXR) α, β and γ. To maintain indispensable selectivity in these assays for individual receptors while keeping unified cellular background of U2OS cells expressing many NRs, we used a Ga4/upstream activating sequences (UAS) reporter system for luciferase transactivation assays. These assays are based on human chimeric NRs generated by the replacement of the N-terminally located DNA-binding domain (DBD) by a DBD from the yeast transcription factor Gal4. The chimeric receptor Gal4-DBD/NR-LBD binds to multiple upstream activating sequences (UAS) in the promoter controlling the expression of the firefly luciferase. The results shown in the Supporting Information (Tables S1 and S2) reveal only a few individual interactions with some receptors. Agonist activity exceeding 25% of the receptor activation was detected only for 3w and 3x in the ERRγ reporter assay (46.1 and 30.8%, respectively). Both efficacy and potency of these compounds are rather low for considering them as true ERRγ ligands, but this finding can be explored in depth in the future. In the antagonist mode, we found only a few compounds with inhibitory activity approaching, but never exceeding 50%. However, these activities appear sporadically in the library and do not show SAR pattern.

Inhibition of Cytochrome 450 Isoforms

As the majority of small molecule drugs (as well as compounds foreign to the organism) are metabolized by liver microsomal cytochromes P450 (CYPs), it is necessary to study interactions of promising active compounds with these enzymes. (51) We characterized five compounds of interest (BE120, 3g, 7, 8, and 3u) showing different levels of ERβ selectivity, with nine forms of CYPs known to participate in drug metabolism to verify whether these compounds may interfere with drug metabolism in human and pose a potential risk for unwanted drug–drug interactions (DDI).
The results summarized in Table 5 indicate a relatively potent inhibition of CYP activities by compound BE120, which has the lowest molecular weight (MW) from the prepared derivatives and highest estrogenic activity with no ER selectivity. This compound interferes most strongly with activity of CYP3A4 exhibiting inhibition in micromolar range (IC50 = 0.5 μM). This observation is in line with study of Lee et al. (52) showing that CYP3A4 enzymes are the major CYPs isoforms responsible for E2 metabolism. Increasing structural complexity and selectivity for ERβ is accompanied with weakening of the CYP inhibition which can be interpreted in light of the structure of CYP3A4’s active site. CYP3A4 has a rather open and flexible active site which allows promiscuous interactions with diverse drugs and xenobiotics as well as endogenous small molecules including steroids (53,54) Overall, our data support limited risk of DDIs with this new class of ERβ agonists.
Table 5. Inhibition of CYP450 Enzyme Isoforms by para-Carboranes Selected for Their Structural Diversity
a

Inhibitory activities of compounds were tested in dose response experiment with specific substrates for individual CYP forms. The enzyme activity was detected by HPLC Prominence system (Shimadzu; Kyoto, Japan) with a UV/fluorescence detection.

b

n.a., not active.

Conclusions

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Herein, we describe novel ERβ selective para-carborane agonists with in vitro isoform selectivity and potency that compare favorably to previously described ERβ agonist clinical candidates. ERB-041 was reported to have striking 222-fold ERβ selective binding to recombinant human ERβ over ERα LBD, with a potency 5.4 nM, which is only 1.5-fold reduced potency compared with 17β-estradiol. (55) However, in a functional assay evaluating the transcription of an ER target gene in cells transformed with either full-length human ERβ or ERα, selectivity was reduced to 14.3-fold with an ERβ potency of 20.0 nM. Similarly, LY500307 has been previously reported to have 0.19 nM Ki for full-length recombinant human ERβ with 14-fold selective ERβ binding in a radio-ligand binding assay. (27) Using a similar reporter gene assay system to the activity data we present, LY500307 was shown to be 30-fold ERβ selective with an EC50 0.66 nM for ERβ. Direct comparisons of LY500307 to 17β-estradiol are not readily ascertained from published data. Though we acknowledge the various assay systems employed limit the utility of directly comparing our data, the potency of compounds 7, 8, and 3q (27, 19, and 61 nM, respectively) and ERβ selectivity (191-, 201-, and 296-fold, respectively) suggest that these ligands may have therapeutically relevant ERβ pharmacological properties and warrant further investigation.
Finally, the general suitability of carboranes’ druglike properties is yet to be described. It is promising that in vivo estrogen effects have been reported following multiple subcutaneous doses of BE120. (43) However, to our knowledge, no comprehensive evaluation of carborane tolerability or drug disposition exists. It has been previously hypothesized that carboranes would likely undergo limited metabolism based on their inorganic nature. (37) In agreement with this, our CYP inhibition data suggests carboranes in our series are poor CYP substrates. We hypothesized that the hydrophobic character of the carboranes may result in problematic solubility and plasma protein binding limitations. To this end, the druglike properties of 7, 8, and 3q were further profiled. The results of these experiments will be the subject of future reports.

Experimental Section

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Chemicals, Materials, and Methods

1H and 13C NMR spectra were recorded at The Ohio State University College of Pharmacy using a Bruker AVIII400HD NMR spectrometer or a Bruker DRX400 NMR spectrometer, or at The Ohio State University Campus Chemical Instrumentation Center using a Bruker Ascend 700 MHz NMR. Chemical shifts (δ) are reported in parts per million (ppm) from chemical reference shifts for internal deuterated chloroform or deuterated acetone reported previously by Fulmer et al. (56) Coupling constants are reported in hertz. 13C NMR spectra are fully decoupled. NMR spectra were analyzed with Mnova Lite SE (Mestrelab Research, Bajo, Spain) or TopSpin. Melting points were obtained on a Thomas Hoover “UNI-MELT” capillary melting apparatus. Optical rotation was measured on a JASCO J-810 spectropolarimeter. Accurate- and high-resolution mass spectra were obtained from Ohio State University Campus Chemical Instrumentation Center using a Waters Micromass LCT mass spectrometer or a Waters Micromass Q-TOF II mass spectrometer, from The Ohio State University College of Pharmacy using a Waters Micromass Q-TOF micro mass spectrometer or a Thermo LTQ Orbitrap mass spectrometer, or from the University of Illinois Urbana–Champaign Mass Spectrometry Laboratory using a Waters Micromass 70-VSE mass spectrometer, or at the University of Michigan College of Literature, Science and the Arts Mass Spectrometry Technical Services on a Micromass Autospec Ultima Magnetic Sector mass spectrometer (HR-EI). For all carborane-containing compounds, the found mass corresponding to the most intense peak of the theoretical isotopic pattern was reported. Measured patterns agreed with calculated patterns.
Silica gel 60 (0.063–0.200 mm), used for gravity column chromatography. Reagent-grade solvents were used for silica gel column chromatography. Precoated glass-backed thin-layer chromatography (TLC) plates with silica gel 60 F254 (0.25-mm layer thickness) from Dynamic Adsorbents (Norcross, GA) were used for TLC. General compound visualization for TLC was achieved by UV light. Carborane-containing compounds were selectively visualized by spraying the plate with a 0.06% PdCl2/1% HCl solution and heating at 120 °C, which caused the slow (15–45 s) formation of a gray spot due to the reduction of Pd2+ to Pd0.
All HPLC studies were carried out using a Hitachi HPLC system (L-2130) equipped with a Windows-based data acquisition and Hitachi Diode array detector (L-2455). HPLC-grade solvents, purchased from Fisher Scientific (Waltham, MA), Acros Organics (Morris Plains, NJ), or Sigma-Aldrich (Milwaukee, WI), were used. The following columns were used: column (I) SMT SAM Analytical Column (250 × 4.6 mm2, particle size: 5 μm, 60 Å pore, normal phase silica, Si, Ref#: 2003P-C0076) from Analtech, Inc., Newark, DE. Column (II) CHIRAL PAK IB-3 column (250 × 4.6 mm2, 3 μm particle size) supplied by Chiral Technologies, PA. Purities of final compounds were determined by HPLC to be >95%. Anhydrous solvents were purchased from Fisher Scientific (Waltham, MA), Acros Organics (Morris Plains, NJ) or Sigma-Aldrich (Milwaukee, WI). closo-p-Carborane was purchased from Katchem Ltd. (Prague, Czech Republic). Other solvents and chemicals were obtained from standard vendors. Compounds 1, (43)BE120, (43)3p, (57)27, (58) and 29 (43) were synthesized as described previously. Unless specified otherwise, all reactions were carried out under argon atmosphere.

Synthesis

1-(4-Methoxyphenyl)-12-ethyl-1,12-dicarba-closo-dodecaborane (2b)

To a solution of 1 (43) (500 mg, 2 mmol) in anhydrous dimethoxyethane (DME, 40 mL) was added n-butyllithium (1 mL, 2.5 mmol, 2.5 M solution in hexanes) at −78 °C. The reaction mixture was stirred for 1.5 h at −78 °C. A quantity of 0.25 mL (3.0 mmol) of bromoethane, precooled to −78 °C, was added at −78 °C. Following stirring at room temperature overnight, the reaction mixture was carefully poured into 150 mL of 1 M HCl and extracted with ethyl acetate. The organic phase was washed with brine and dried over MgSO4. The solvents were evaporated, and the residue was purified by silica gel column chromatography to yield a white solid. Yield: 520 mg (93%) of white solid, Rf: 0.78 (hexanes/EtOAc, 49/1, v/v), m.p.: 70–80 °C. 1H NMR (CDCl3): δ 0.81 (t, 3H, CH3), 1.73 (q, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 3.74 (s, 3H, OCH3), 6.67 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 14.00, 31.31, 55.38, 80.85, 81.60, 113.36, 128.50, 128.96, 159.62. Accurate mass high-resolution mass spectrometry (HRMS) (EI+): m/z calcd for C11H22B10O (M)+ 278.2680, found 278.2675.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]ethan-1-ol (2c)

To a solution of 1 (43) (500 mg, 2 mmol) in anhydrous dimethoxyethane (DME, 40 mL) was added n-butyllithium (1 mL, 2.5 mmol, 2.5 M solution in hexanes) at −78 °C. The reaction mixture was stirred for 1.5 h at −78 °C. A quantity of 0.17 mL (3.0 mmol) of acetaldehyde, precooled to −78 °C, was added at −78 °C. Following stirring at room temperature overnight, the reaction mixture was carefully poured into 150 mL of 1 M HCl and extracted with ethyl acetate. The organic phase was washed with brine and dried over MgSO4. The solvents were evaporated, and the residue was purified by silica gel column chromatography to yield a white solid. Yield: 450 mg (76%) of a white solid, Rf: 0.24 (hexanes/EtOAc, 14/1, v/v), m.p.: 118–119 °C. 1H NMR (CDCl3): δ 1.11 (d, 3H, CH3), 1.66 (d, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.74 (m, 4H, CH and OCH3), 6.68 (d, 2H, arom., J = 8.9 Hz), 7.12 (d, 2H, arom., J = 8.9 Hz). 13C NMR (CDCl3): δ 23.09, 55.40, 69.50, 83.36, 86.26, 113.43, 128.42, 128.80, 159.74. Accurate mass HRMS (EI+): m/z calcd for C11H22B10O2 (M)+ 294.2629, found 294.2632.

1-(4-Methoxyphenyl)-12-propyl-1,12-dicarba-closo-dodecaborane (2d)

To a solution of 1 (43) (500 mg, 2 mmol) in anhydrous dimethoxyethane (DME, 40 mL) was added n-butyllithium (1 mL, 2.5 mmol, 2.5 M solution in hexanes) at 0 °C. The reaction mixture was stirred at room temperature for 1.5 h. A quantity of 0.237 mL (3.0 mmol) of 1-bromopropane was added at 0 °C. Following stirring at room temperature for 4 h, the reaction mixture was carefully poured into 60 mL of 1 M HCl and extracted with ethyl acetate. The organic phase was washed with a 10% aqueous sodium thiosulfate solution and brine and dried over MgSO4. The solvents were evaporated, and the residue was purified by silica gel column chromatography to yield a white solid. Yield: 510 mg (87%) of white solid, Rf: 0.78 (hexanes/EtOAc, 49/1, v/v), m.p.: 95–96 °C. 1H NMR (CDCl3): δ 0.77 (t, 3H, CH3), 1.20 (m, 2H, CH2), 1.63 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 3.74 (s, 3H, OCH3), 6.67 (d, 2H, arom., J = 8.9 Hz), 7.12 (d, 2H, arom., J = 8.9 Hz). 13C NMR (CDCl3): δ 13.82, 22.96, 40.12, 55.38, 80.79, 80.96, 113.36, 128.49, 128.97, 159.62. Accurate mass HRMS (EI+): m/z calcd for C12H24B10O (M)+ 292.2837, found 292.2842.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]propan-1-ol (2e)

To a solution of 1 (43) (0.5 g, 2 mmol) in anhydrous dimethoxyethane DME (40 mL) was added n-butyllithium (1.0 mL, 2.5 mmol, 2.5 M solution in hexanes) at 0 °C. The reaction mixture was stirred at room temperature for 1.5 h. A quantity of 0.22 mL (3 mmol) of 1-propanal was added at 0 °C. Following stirring at room temperature overnight, the reaction mixture was carefully poured into 150 mL of 1 M HCl and extracted with ethyl acetate. The organic phase was washed with brine and dried over MgSO4. The solvents were evaporated, and the residue was purified by column chromatography. Yield: 360 mg (58%) of a white solid, Rf: 0.32 (hexanes/EtOAc, 14/1, v/v), m.p.: 116–117 °C. 1H NMR (CDCl3): δ 0.91 (t, 3H, CH3), 1.17–1.48 (m, 2H, CH2), 1.61 (d, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.39 (m, 1H, CH), 3.74 (s, 3H, OCH3), 6.68 (d, 2H, arom., J = 8.9 Hz), 7.13 (d, 2H, arom., J = 8.9 Hz). 13C NMR (CDCl3): δ 11.29, 29.99, 55.39, 74.56, 83.56, 86.24, 113.42, 128.42, 128.84, 159.73. Accurate mass HRMS (EI+): m/z calcd for C12H24B10O2 (M)+ 308.2786, found 308.2765.

1-(4-Methoxyphenyl)-12-butyl-1,12-dicarba-closo-dodecaborane (2f)

The method described for the synthesis of 2d was adapted to synthesize 2f. Starting materials: 1 (43) (500 mg, 2 mmol), 1-Iodobutane (0.34 mL, 3.0 mmol). Yield: 400 mg (65%) of white solid, Rf: 0.77 (hexanes/EtOAc, 49/1, v/v), m.p.: 55–56 °C. 1H NMR (CDCl3): δ 0.83 (t, 3H, CH3), 1.14–1.17 (m, 4H, 2 × CH2), 1.65 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 3.74 (s, 3H, OCH3), 6.67 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 13.84, 22.41, 31.76, 37.79, 55.38, 80.93, 113.36, 128.49, 128.97, 159.62. Accurate mass HRMS (EI+): m/z calcd for C13H26B10O (M)+ 306.2993, found 306.2992.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]butan-1-ol (2g)

The method described for the synthesis of 2e was adapted to synthesize 2g. Starting materials: 1 (43) (500 mg, 2 mmol), 1-butanal (0.27 mL, 3.0 mmol). Yield: 500 mg (78%) of white solid, Rf: 0.33 (hexanes/EtOAc, 19/1, v/v), m.p.: 96–97 °C. 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.16–1.27 (m, 2H, CH2), 1.35–1.52 (m, 2H, CH2), 1.59 (br. s, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.49 (m, 1H, CH), 3.74 (s, 3H, OCH3), 6.68 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 13.75, 19.82, 38.94, 55.40, 72.84, 83.54, 86.34, 113.42, 128.43, 128.84, 159.73. Accurate mass HRMS (EI+): m/z calcd for C13H26B10O2 (M)+ 322.2943, found 322.2929.

1-(4-Methoxyphenyl)-12-pentyl-1,12-dicarba-closo-dodecaborane (2h)

The method described for the synthesis of 2d was adapted to synthesize 2h. Starting materials: 1 (43) (500 mg, 2 mmol), 1-Iodopentane (0.392 mL, 3.0 mmol). Yield: 520 mg (81%) of white solid, Rf: 0.46 (hexanes/EtOAc, 99/1, v/v), m.p.: 64–65 °C. 1H NMR (CDCl3): δ 0.84 (t, 3H, CH3), 1.08–1.26 (m, 6H, 3 × CH2), 1.64 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 3.74 (s, 3H, OCH3), 6.67 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 14.03, 22.39, 29.32, 31.40, 38.00, 55.38, 80.92, 113.36, 128.49, 128.97, 159.62. Accurate mass HRMS (EI+): m/z calcd for C14H28B10O (M)+ 320.3151, found 320.3147.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]pentan-1-ol (2i)

The method described for the synthesis of 2e was adapted to synthesize 2i. Starting materials: 1 (43) (500 mg, 2 mmol), 1-pentanal (0.32 mL, 3 mmol). Yield: 540 mg (80%) of white solid, Rf: 0.24 (hexanes/EtOAc, 19/1, v/v), m.p.: 92–93 °C. 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.15–1.34 (m, 4H, 2 × CH2), 1.39–1.46 (m, 2H, CH2), 1.59 (br. d, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.47 (m, 1H, CH), 3.74 (s, 3H, OCH3), 6.68 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 14.07, 22.40, 28.75, 36.60, 55.40, 73.09, 83.54, 86.39, 113.42, 128.43, 128.84, 159.73. Accurate mass HRMS (EI+): m/z calcd for C14H28B10O2 (M)+ 336.3100, found 336.3116.

1-(4-Methoxyphenyl)-12-heptyl-1,12-dicarba-closo-dodecaborane (2j)

The method described for the synthesis of 2d was adapted to synthesize 2j. Starting materials: 1 (43) (500 mg, 2 mmol), 1-Iodoheptane (0.49 mL, 3.0 mmol). Yield: 550 mg (79%) of white solid, Rf: 0.38 (hexanes), m.p.: 45–46 °C. Analytical HPLC: column I, solvent system: hexanes (100) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 5.11 min, purity 100.0% 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.08–1.28 (m, 10H, 5 × CH2), 1.64 (m, 2H, Ccarborane–CH2), 1.85–3.0 (br. m, 10H, BH), 3.74 (s, 3H, OCH3), 6.67 (d, 2H, arom., J = 9.0 Hz), 7.11 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 14.21, 22.73, 29.02, 29.24, 29.67, 31.82, 38.05, 55.39, 80.92, 113.36, 128.49, 128.97, 159.61. Accurate mass HRMS (EI+): m/z calcd for C16H32B10O (M)+ 348.3465, found 348.3461.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-ol (2k)

The method described for the synthesis of 2e was adapted to synthesize 2k. Starting materials: 1 (43) (2.5 g, 10 mmol), 1-heptanal (1.83 mL, 13 mmol). Yield: 3.0 g (82%) of a white solid, Rf: 0.43 (hexanes/EtOAc, 19/1, v/v), m.p.: 104–105 °C. Analytical HPLC: column I, solvent system: hexanes/isopropyl alcohol (i-PrOH) (99/1) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 5.32 min, purity 99.87%, analytical HPLC: column II, solvent system: hexanes/CH2Cl2 (9/1) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 9.67 min (area %: 49.99), 10.47 (area %: 49.0). 1H NMR (CDCl3): δ 0.88 (t, 3H, CH3), 1.15–1.30 (m, 8H, 4 × CH2), 1.38–1.47 (m, 2H, CH2), 1.59 (br. s, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.47 (m, 1H, CH), 3.74 (s, 3H, OCH3), 6.68 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 14.20, 22.71, 26.59, 28.98, 31.83, 36.92, 55.39, 73.10, 83.53, 86.39, 113.41, 128.43, 128.84, 159.73. Accurate mass HRMS (EI+): m/z calcd for C16H32B10O2 (M)+ 364.3414, found 364.3423.

1-(4-Methoxyphenyl)-12-decyl-1,12-dicarba-closo-dodecaborane (2l)

The method described for the synthesis of 2d was adapted to synthesize 2l. Starting materials: 1 (43) (500 mg, 2 mmol), 1-Iododecane (0.64 mL, 3.0 mmol). Yield: 420 mg (54%) of white solid, Rf: 0.31 (hexanes), m.p.: 45–46 °C. 1H NMR (CDCl3): δ 0.88 (t, 3H, CH3), 1.08–1.31 (m, 16H, 8 × CH2), 1.64 (m, 2H, Ccarborane–CH2), 1.85–3.0 (br. m, 10H, BH), 3.74 (s, 3H, OCH3), 6.67 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 14.28, 22.84, 29.29, 29.36, 29.44, 29.62, 29.68, 30.04, 38.06, 55.39, 80.93, 113.37, 128.50, 128.99, 159.63. Accurate mass HRMS (EI+): m/z calcd for C19H38B10O (M)+ 390.3936, found 390.3954.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]decyl-1-ol (2m)

The method described for the synthesis of 2e was adapted to synthesize 2m. Starting materials: 1 (43) (0.5 g, 2 mmol), 1-decanal (0.57 mL, 3 mmol). Yield: 680 mg (86%) of a white solid, Rf: 0.31 (hexanes/EtOAc, 19/1, v/v), m.p.: 68–69 °C. 1H NMR (CDCl3): δ 0.88 (t, 3H, CH3), 1.13–1.32 (m, 14H, 7 × CH2), 1.37–1.46 (m, 2H, CH2), 1.58 (d, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.46 (m, 1H, CH), 3.74 (s, 3H, OCH3), 6.68 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 14.26, 22.82, 26.63, 29.31, 29.42, 29.63, 29.65, 32.02, 36.92, 55.40, 73.10, 83.53, 86.39, 113.42, 128.43, 128.85, 159.73.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-4-methylpentan-1-ol (2n)

The method described for the synthesis of 2e was adapted to synthesize 2n. Starting materials: 1 (43) (0.5 g, 2 mmol), 4-methylpentan-1-al (250 mg, 2.5 mmol). Yield: 580 mg (83%) of a white solid, Rf: 0.27 (hexanes/EtOAc, 19/1, v/v), m.p.: 111–112 °C. 1H NMR (CDCl3): δ 0.84 (d, 3H, CH3), 0.85 (d, 3H, CH3), 1.06–1.19 (m, 2H, CH2), 1.30–1.45 (m, 2H, CH2), 1.46–1.52 (m, 1H, CH), 1.58 (d, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.45 (m, 1H, CH), 3.74 (s, 3H, OCH3), 6.68 (d, 2H, arom., J = 8.9 Hz), 7.12 (d, 2H, arom., J = 8.9 Hz). 13C NMR (CDCl3): δ 22.36, 22.91, 27.88, 34.84, 35.72, 55.40, 73.10, 83.54, 86.43, 113.42, 128.43, 128.84, 159.74. Accurate mass HRMS (EI+): m/z calcd for C15H30B10O2 (M)+ 350.3257, found 350.3260.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-5-methylhexan-1-ol (2o)

The method described for the synthesis of 2e was adapted to synthesize 2o. Starting materials: 1 (43) (0.5 g, 2 mmol), 5-methylhexan-1-ala (370 mg, 3.25 mmol). Yield: 250 mg (34%) of a white solid, Rf: 0.32 (hexanes/EtOAc, 14/1, v/v), m.p.: 64–65 °C. 1H NMR (CDCl3): δ 0.85 (d, 3H, CH3), 0.86 (d, 3H, CH3), 1.08–1.28 (m, 4H, 2 × CH2), 1.36–1.46 (m, 2H, CH2), 1.46–1.52 (m, 1H, CH), 1.60 (d, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.47 (m, 1H, CH), 3.74 (s, 3H, OCH3), 6.68 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 22.61, 22.76, 24.45, 28.04, 37.15, 38.58, 55.39, 73.11, 83.54, 86.38, 113.42, 128.43, 128.84, 159.73. Accurate mass HRMS (EI+): m/z calcd for C16H32B10O2 (M)+ 364.3414, found 364.3426.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-6-methylheptan-1-ol (2q)

The method described for the synthesis of 2e was adapted to synthesize 2q. Starting materials: 1 (43) (1.0 g, 4 mmol), 6-methylheptan-1-alb (750 mg, 5.85 mmol). Yield: 1.16 g (77%) of a white solid, Rf: 0.43 (hexanes/EtOAc, 19/1, v/v), m.p.: 95–96 °C. 1H NMR (CDCl3): δ 0.85 (d, 6H, 2 × CH3), 1.11–1.29 (m, 6H, 3 × CH2), 1.39–1.44 (m, 2H, CH2), 1.47–1.53 (m, 1H, CH), 1.60 (br. s, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.47 (br. d, 1H, CH), 3.74 (s, 3H, OCH3), 6.68 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 22.71, 22.78, 26.89, 27.08, 28.04, 36.94, 38.95, 55.40, 73.10, 83.54, 86.39, 113.42, 128.43, 128.84, 159.73. Accurate mass HRMS (EI+): m/z calcd for C17H34B10O2 (M)+ 378.3571, found 378.3576.

(RS)-(2,3-Dihydro-1H-inden-5-yl)-[1-(4-methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]methanol (2r)

The method described for the synthesis of 2e was adapted to synthesize 2r. Starting materials: 1 (43) (450 mg, 1.8 mmol), 5-formylindane (100 mg, 0.69 mmol). Yield: 240 mg (34%) of a white solid, Rf: 0.28 (hexanes/EtOAc, 19/1, v/v), m.p.: 123-124 °C. 1H NMR (CDCl3): δ 1.85–3.0 (br. m, 10H, BH), 2.06–2.10 (m, 3H, CH2, OH), 2.89 (m, 4H, 2 × CH2), 3.74 (s, 3H, OCH3), 4.46 (s, 1H, CH), 6.66 (d, 2H, arom., J = 9.0 Hz), 6.92 (d, 1H, arom.), 7.03 (s, 1H, arom.), 7.09 (d, 2H, arom., J = 9.0 Hz), 7.15 (d, 2H, arom.). 13C NMR (CDCl3): δ 25.56, 32.77, 32.95, 55.39, 76.11, 83.65, 85.84, 113.39, 122.74, 123.95, 124.92, 128.41, 128.86, 138.24, 144.29, 144.95, 159.71. Accurate mass HRMS (EI+): m/z calcd for C19H28B10O2 (M)+ 396.3102, found 396.3096.

1-(4-Methoxyphenyl)-12-(3-phenylpropyl)-1,12-dicarba-closo-dodecaborane (2s)

The method described for the synthesis of 2d was adapted to synthesize 2s. Starting materials: 1 (43) (500 mg, 2 mmol), 1-bromo-3-phenylpropane (0.46 mL, 3.0 mmol). Yield: 530 mg (72%) of white solid, Rf: 0.64 (hexanes/EtOAc, 49/1, v/v), m.p.: 93–94 °C. 1H NMR (CDCl3): δ 1.50 (m, 2H, CH2), 1.98 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 2.57 (app. t, 2H, CH2), 3.72 (s, 3H, OCH3), 6.66–7.25 (m, 9H, arom.). 13C NMR (CDCl3): δ 31.16, 35.38, 37.52, 55.38, 80.42, 81.09, 113.37, 126.12, 128.36, 128.47, 128.53, 128.90, 141.38, 159.63. Accurate mass HRMS (EI+): m/z calcd for C18H28B10O (M)+ 368.3152, found 368.3153.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-3-phenylpropan-1-ol (2t)

The method described for the synthesis of 2e was adapted to synthesize 2t. Starting materials: 1 (43) (250 mg, 1 mmol), of 3-phenyl-1-propanal (0.17 g, 1.5 mmol). Yield: 344 mg (90%) of a white solid, Rf: 0.27 (hexanes/EtOAc, 19/1, v/v), m.p.: 123–124 °C. 1H NMR (CDCl3): δ 01.49–1.77 (m, 2H, CH2), 1.69 (br. s, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 2.51–2.83 (m, 2H, CH2), 3.48 (m, 1H, CH), 3.74 (s, 3H, OCH3), 6.68–7.28 (m, 9H, arom.). 13C NMR (CDCl3): δ 32.69, 38.29, 55.39, 72.31, 83.64, 86.02, 113.42, 126.19, 128.41, 128.52, 128.61, 128.77, 141.15, 159.74. Accurate mass HRMS (EI+): m/z calcd for C18H28B10O2 (M)+ 384.3102, found 384.3101.

1-(4-Methoxyphenyl)-12-(4-phenylbutyl)-1,12-dicarba-closo-dodecaborane (2u)

The method described for the synthesis of 2e was adapted to synthesize 2u. Starting materials: 1 (43) (350 mg, 1.4 mmol), 1-bromo-4-phenylbutane (450 mg, 2.1 mmol). Yield: 490 mg (91%) of white solid, Rf: 0.57 (hexanes/EtOAc, 49/1, v/v), m.p.: 102–103 °C. 1H NMR (CDCl3): δ 1.22 (m, 2H, CH2), 1.44 (m, 2H, CH2), 1.68 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 2.52 (app. t, 2H, CH2), 3.73 (s, 3H, OCH3), 6.67–7.27 (m, 9H, arom.). NMR (CDCl3): δ 29.27, 31.06, 35.62, 37.78, 55.38, 80.62, 81.01, 113.37, 125.92, 128.44, 128.46, 128.48, 128.92, 142.19, 159.63. Accurate mass HRMS (EI+): m/z calcd for C19H30B10O (M)+ 382.3309, found 382.3302.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-4-phenylbutan-1-ol (2v)

The method described for the synthesis of 2e was adapted to synthesize 2v. Starting materials: 1 (43) (500 mg, 2 mmol), of 4-phenyl-1-butanal (0.46 mL, 3 mmol). Yield: 620 mg (78%) of a white solid, Rf: 0.27 (hexanes/EtOAc, 14/1, v/v), m.p.: 123–125 °C. 1H NMR (CDCl3): δ 1.21–1.48 (m, 2H, CH2), 1.51–1.82 (m, 2H, CH2), 1.60 (br. d, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 2.52–2.61 (m, 2H, CH2), 3.50 (m, 1H, CH), 3.73 (s, 3H, OCH3), 6.67–7.28 (m, 9H, arom.). 13C NMR (CDCl3): δ 28.38, 35.51, 36.40, 55.39, 72.87, 83.58, 86.21, 113.42, 125.97, 128.41, 128.48, 128.79, 142.05, 159.74. Accurate mass HRMS (EI+): m/z calcd for C19H30B10O2 (M)+ 398.3259, found 398.3255.

1-(4-Methoxyphenyl)-12-(5-phenylpentyl)-1,12-dicarba-closo-dodecaborane (2w)

The method described for the synthesis of 2d was adapted to synthesize 2w. Starting materials: 1 (43) (440 mg, 1.77 mmol), 1-bromo-5-phenylpentane (600 mg, 2.65 mmol). Yield: 620 mg (88%) of white solid, Rf: 0.62 (hexanes/EtOAc, 49/1, v/v), m.p.: 70-71 °C. 1H NMR (CDCl3): δ 1.17 (m, 4H, 2 × CH2), 1.53 (m, 2H, CH2), 1.63 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 2.52 (app. t, 2H, CH2), 3.73 (s, 3H, OCH3), 6.66–7.27 (m, 9H, arom.). NMR (CDCl3): δ 28.83, 29.50, 31.15, 35.89, 37.91, 55.38, 80.74, 80.97, 113.36, 125.83, 128.41, 128.48, 128.50, 128.93, 142.54, 159.62. Accurate mass HRMS (EI+): m/z calcd for C20H32B10O (M)+ 396.3466, found 396.3483.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-5-phenylpentan-1-ol (2x)

The method described for the synthesis of 2e was adapted to synthesize 2x. Starting materials: 1 (43) (500 mg, 2 mmol), of 5-phenyl-1-pentanal (0.52 mL, 3 mmol). Yield: 480 mg (58%) of a glasslike compound, Rf: 0.32 (hexanes/EtOAc, 14/1, v/v). 1H NMR (CDCl3): δ 1.18–1.28 (m, 2H, CH2), 1.41–1.63 (m, 5H, OH and 2 × CH2), 1.85–3.0 (br. m, 10H, BH), 2.54–2.62 (m, 2H, CH2), 3.46 (m, 1H, CH), 3.77 (s, 3H, OCH3), 6.68–7.28 (m, 9H, arom.). 13C NMR (CDCl3): δ 26.27, 31.13, 35.94, 36.71, 55.40, 72.96, 83.57, 86.26, 113.42, 125.85, 128.43, 128.51, 128.81, 142.48, 159.74. Accurate mass HRMS (EI+): m/z calcd for C20H32B10O2 (M)+ 412.3416, found 412.3417.

1-(4-Hydroxyphenyl)-12-ethyl-1,12-dicarba-closo-dodecaborane (3b)

To a solution of 2b (460 mg, 1.65 mmol) in anhydrous DCM (15 mL) was added boron tribromide (3.3 mL, 3.3 mmol, 1 M solution in DCM) at 0 °C. The reaction mixture was stirred at room temperature overnight, poured carefully into icecold 1 M HCl (15 mL), and extracted with EtOAc. The organic phase was washed with brine and dried over MgSO4. The solvents were evaporated, and the residue was purified by silica gel column chromatography. Further purification was achieved by recrystallization from heptane. The formed crystals were filtered at −20 °C and then washed with heptane cooled to −78 °C followed by pentane cooled to −78 °C. Yield: 360 mg (83%) of a white solid after column chromatography, Rf: 0.38 (hexanes/EtOAc, 6/1, v/v), m.p.: 125-126 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 12.09 min, purity 99.61%. 1H NMR (CDCl3): δ 0.81 (t, 3H, CH3), 1.73 (q, CH2), 1.85–3.0 (br. m, 10H, BH), 4.79 (br. s, 1H, OH), 6.60 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (CDCl3): δ 13.99, 31.30, 81.21, 80.73, 81.66, 114.84, 128.77, 129.28, 155.58. Accurate mass HRMS (EI+): m/z calcd for C10H20B10O (M)+ 264.2523, found 264.2525.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]ethan-1-ol (3c)

The method described for the synthesis of 3b was adapted to synthesize 3c. Starting materials: 2c (400 mg, 1.36 mmol); boron tribromide (4.0 mL, 4 mmol, 1 M solution in DCM). Further purification was achieved by suspending the residue obtained after column chromatography in boiling heptane. The suspension was then cooled to −78 °C, filtered, and the solid residue was washed with heptane precooled to −78 °C followed by pentane, precooled to −78 °C. Yield: 300 mg (79%) of a white solid after column chromatography, Rf: 0.35 (hexanes/EtOAc, 4/1, v/v), m.p.: 176–177 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 15.97 min, purity 99.33%. 1H NMR (acetone-d6): δ 1.07 (d, 3H, CH3), 1.85–3.0 (br. m, 10H, BH), 3.74 (m, 1H, CH), 4.39 (d, 1H, OH), 6.67 (d, 2H, arom., J = 8.9 Hz), 7.07 (d, 2H, arom., J = 8.9 Hz), 8.51 (s, 1H, OH). 13C NMR (acetone-d6): δ 23.74, 69.28, 84.18, 88.18, 115.65, 128.24, 129.09, 158.55. Accurate mass HRMS (EI+): m/z calcd for C10H20B10O2 (M)+ 280.2472, found 280.2471.

1-(4-Hydroxyphenyl)-12-propyl-1,12-dicarba-closo-dodecaborane (3d)

The method described for the synthesis of 3b was adapted to synthesize 3d. Starting materials: 2d (450 mg, 1.54 mmol); boron tribromide (3.0 mL, 3 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from heptane. The formed crystals were filtered at −20 °C and then washed with heptane cooled to −78 °C followed by pentane cooled to −78 °C. Yield: 390 mg (91%) of a white solid after column chromatography, Rf: 0.34 (hexanes/EtOAc, 6/1, v/v), m.p.: 139-140 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 11.87 min, purity 99.29%. 1H NMR (CDCl3): δ 0.77 (t, 3H, CH3), 1.20 (m, 2H, CH2), 1.62 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 4.78 (br. s, 1H, OH), 6.60 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (CDCl3): δ 13.82, 22.95, 40.11, 80.84, 114.84, 128.76, 129.29, 155.58. Accurate mass HRMS (EI+): m/z calcd for C11H22B10O (M)+ 278.2680, found 278.2684.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]propan-1-ol (3e)

The method described for the synthesis of 3b was adapted to synthesize 3e. Starting materials: 2e (250 mg, 0.81 mmol); boron tribromide (2.5 mL, 2.5 mmol, 1 M solution in DCM). Further purification was achieved by suspending the residue obtained after column chromatography in boiling heptane. The suspension was then cooled to −78 °C, filtered, and the solid residue was washed with heptane cooled to −78 °C followed by pentane, cooled to −78 °C. Yield: 180 mg (75%) of a white solid after column chromatography, Rf: 0.35 (hexanes/EtOAc, 4/1, v/v), m.p.: 206–207 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 13.42 min, purity 99.29%. 1H NMR (acetone-d6): δ 0.87 (t, 3H, CH3), 1.15–1.45 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 3.39 (m, 1H, CH), 4.40 (d, 1H, OH), 6.67 (d, 2H, arom., J = 8.9 Hz), 7.07 (d, 2H, arom., J = 8.9 Hz), 8.52 (s, 1H, OH). 13C NMR (acetone-d6): δ 11.52, 30.63, 74.49, 84.41, 88.35, 115.68, 128.30, 129.12, 158.59. Accurate mass HRMS (ESI): m/z calcd for C11H21B10O2 (M – 1) 293.2545, found 293.2544.

1-(4-Hydroxyphenyl)-12-butyl-1,12-dicarba-closo-dodecaborane (3f)

The method described for the synthesis of 3b was adapted to synthesize 3f. Starting materials: 2f (300 mg, 0.97 mmol); boron tribromide (2.0 mL, 2 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from heptane. The formed crystals were filtered at −20 °C and then washed with heptane precooled to −78 °C followed by pentane precooled to −78 °C. Yield: 190 mg (64%) of a white solid after column chromatography, Rf: 0.35 (hexanes/EtOAc, 6/1, v/v), m.p.: 124–126 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 11.733 min, purity 98.83%. 1H NMR (CDCl3): δ 0.82 (t, 3H, CH3), 1.15 (m, 4H, 2 × CH2), 1.65 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 4.78 (br. s, 1H, OH), 6.60 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (CDCl3): δ 13.83, 22.41, 31.76, 37.78, 80.82, 80.91, 114.83, 128.76, 129.29, 155.59. Accurate mass HRMS (ESI): m/z calcd for C12H23B10O (M – 1) 291.2752, found 291.2751.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]butan-1-ol (3g)

To a solution of 2g (450 mg, 1.4 mmol) in anhydrous DCM (40 mL) was added boron tribromide (4.2 mL, 4.2 mmol, 1 M solution in DCM) at 0 °C. The reaction mixture was stirred at room temperature overnight, poured carefully into icecold 1 M HCl (60 mL), and extracted with DCM. The organic phase was washed with a 10% sodium thiosulfate solution and brine and dried over MgSO4. The solvents were evaporated, and the residue was purified by silica gel column chromatography (hexanes/EtOAc, 9/1, v/v) to yield a white solid. Further purification was achieved by recrystallization from hexanes/ isopropanol (24:1) and washing the obtained residue with icecold pentane. Yield: 265 mg (62%) of a white solid after column chromatography, Rf: 0.22 (hexanes/EtOAc, 9/1, v/v), m.p.: 184–185 °C. Analytical HPLC: column I, solvent system: hexanes/i-PrOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 9.87 min, purity 98.10%. 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.15–1.26 (m, 2H, CH2), 1.33–1.51 (m, 2H, CH2), 1.55 (br. s, ∼2H, OH and H2O), 1.85–3.0 (br. m, 10H, BH), 3.48 (m, 1H, CH), 4.69 (br. s, ∼1H, OH), 6.61 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (CDCl3): δ 13.75, 19.82, 38.95, 72.86, 83.41, 86.39, 114.90, 128.71, 129.15, 155.75. Accurate mass HRMS (ESI): m/z calcd for C12H23B10O2 (M – 1) 307.2701, found 307.2700.

1-(4-Hydroxyphenyl)-12-pentyl-1,12-dicarba-closo-dodecaborane (3h)

The method described for the synthesis of 3b was adapted to synthesize 3h. Starting materials: 2h (460 mg, 1.44 mmol); boron tribromide (2.8 mL, 2.8 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from heptane. The formed crystals were filtered at −20 °C and then washed with heptane cooled to −78 °C followed by pentane cooled to −78 °C. Yield: 370 mg (84%) of a white solid after column chromatography, Rf: 0.34 (hexanes/EtOAc, 6/1, v/v), m.p.: 99–100 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 11.37 min, purity 97.39%. 1H NMR (CDCl3): δ 0.84 (t, 3H, CH3), 1.08–1.25 (m, 6H, 3 × CH2), 1.64 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 4.78 (br. s, 1H, OH), 6.60 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (CDCl3): δ 14.03, 22.38, 29.32, 31.39, 37.99, 80.81, 80.97, 114.83, 128.76, 129.30, 155.57. Accurate mass HRMS (EI): m/z calcd for C13H26B10O (M)+ 306.2993, found 306.3001.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]pentan-1-ol (3i)

The method described for the synthesis of 3b was adapted to synthesize 3i. Starting materials: 2i (480 mg, 1.43 mmol); boron tribromide (4.3 mL, 4.3 mmol, 1 M solution in DCM). Further purification was achieved by suspending the residue obtained after column chromatography in boiling heptane. The suspension was then cooled to −78 °C, filtered, and the solid residue was washed with heptane cooled to −78 °C followed by pentane, cooled to −78 °C. Yield: 300 mg (65%) of a white solid after column chromatography, Rf: 0.36 (hexanes/EtOAc, 4/1, v/v), m.p.: 156-157 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 11.83 min, purity 99.77%. 1H NMR (acetone-d6): δ 0.85 (t, 3H, CH3), 1.16–1.46 (m, 6H, 3 × CH2), 1.85–3.0 (br. m, 10H, BH), 3.48 (m, 1H, CH), 4.41 (d, 1H, OH), 6.67 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (acetone-d6): δ 14.24, 22.85, 29.42, 37.33, 72.92, 84.38, 88.49, 115.68, 128.28, 129.12, 158.61. Accurate mass HRMS (ESI): m/z calcd for C13H25B10O2 (M – 1) 321.2858, found 321.2858.

1-(4-Hydroxyphenyl)-12-heptyl-1,12-dicarba-closo-dodecaborane (3j)

The method described for the synthesis of 3g was adapted to synthesize 3j. Starting materials: 2j (600 mg, 1.72 mmol); boron tribromide (3.4 mL, 3.4 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from hexane. The formed crystals were filtered at −20 °C and then washed with pentane precooled to −78 °C. Yield: 380 mg (66%) of a white solid after column chromatograpy, Rf: 0.36 (hexanes/EtOAc, 9/1, v/v), m.p.: 114–115 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99/1) (97/3) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 6.860 min, purity 99.52%. 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.08–1.29 (m, 10H, 5 × CH2), 1.64 (m, 2H, Ccarborane–CH2), 1.85–3.0 (br. m, 10H, BH), 4.68 (br. s, 1H, OH), 6.60 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (CDCl3): δ 14.20, 22.73, 29.02, 29.23, 29.67, 31.87, 38.04, 81.21, 80.82, 80.98, 114.83, 128.76, 129.30, 155.59. Accurate mass HRMS (ESI): m/z calcd for C15H29B10O (M – 1) 333.3216, found 333.3213.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-ol (3k)

The method described for the synthesis of 3g was adapted to synthesize 3k. Starting materials: 2k (570 mg, 1.57 mmol); boron tribromide (4.7 mL, 4.7 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from hexanes/i-PrOH (24/1). The formed crystals were filtered at −20 °C and then washed with pentane precooled to −78 °C. Yield: 400 mg (73%) of a white solid after column chromatography, Rf: 0.23 (hexanes/EtOAc, 9/1, v/v), m.p.: 129–130 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 9.52 min, purity 99.94%. 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.14–1.30 (m, 8H, 4 × CH2), 1.38–1.45 (m, 2H, CH2), 1.62–163 (m, ∼2H, OH and H2O), 1.8.5–3.0 (br. m, 10H, BH), 3.46 (m, 1H, CH), 4.96 (br. s, 1H, OH), 6.61 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (CDCl3): δ 14.19, 22.71, 26.58, 28.97, 31.82, 36.91, 73.14, 83.57, 86.37, 114.90, 128.68, 129.06, 155.82. Accurate mass HRMS (ESI): m/z calcd for C15H31B10O2 (M + 1)+ 351.3329, found 351.3322.

1-(4-Hydroxyphenyl)-12-decyl-1,12-dicarba-closo-dodecaborane (3l)

The method described for the synthesis of 3b was adapted to synthesize 3l. Starting materials: 2l (360 mg, 0.92 mmol); boron tribromide (2.0 mL, 2 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from heptane. The formed crystals were filtered at −20 °C and then washed with heptane precooled to −78 °C followed by pentane precooled to −78 °C. Yield: 270 mg (78%) of a white solid after column chromatography, Rf: 0.38 (hexanes/EtOAc, 6/1, v/v), m.p.: 83–84 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 10.23 min, purity 100.0%. 1H NMR (CDCl3): δ 0.88 (t, 3H, CH3), 1.08–1.23 (m, 16H, 8 × CH2), 1.64 (m, 2H, Ccarborane–CH2), 1.85–3.0 (br. m, 10H, BH), 4.77 (br. s, 1H, OH), 6.60 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (CDCl3): δ 14.27, 22.82, 29.27, 29.34, 29.43, 29.60, 29.67, 32.03, 38.04, 80.81, 80.97, 114.83, 128.76, 129.30, 155.59. Accurate mass HRMS (ESI): m/z calcd for C18H35B10O (M – 1) 375.3725, found 375.3723.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]decan-1-ol (3m)

The method described for the synthesis of 3b was adapted to synthesize 3m. Starting materials: 2m (610 mg, 1.49 mmol); boron tribromide (4.5 mL, 4.5 mmol, 1 M solution in DCM). Further purification was achieved by suspending the residue obtained after column chromatography in boiling heptane. The suspension was then cooled to −78 °C, filtered, and the solid residue was washed with heptane cooled to −78 °C followed by pentane, cooled to −78 °C. Yield: 480 mg (82%) of a white solid after column chromatography, Rf: 0.42 (hexanes/EtOAc, 4/1, v/v), m.p.: 130–131 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 9.41 min, purity 99.73%. 1H NMR (CDCl3): δ 0.88 (t, 3H, CH3), 1.15–1.46 (m, 16H, 7 × CH2), 1.38–1.45 (m, 2H, CH2), 1.58 (m, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.47 (m, 1H, CH), 4.70–4.81 (br. d, 1H, OH), 6.61 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (CDCl3): δ 14.26, 22.82, 26.63, 29.30, 29.42, 29.62, 29.65, 32.02, 36.91, 73.14, 83.45, 86.39, 114.90, 128.70, 129.13, 155.76. Accurate mass HRMS (EI): m/z calcd for C18H36B10O2 (M)+ 392.3728, found 392.3750.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-4-methylpentan-1-ol (3n)

The method described for the synthesis of 3b was adapted to synthesize 3n. Starting materials: 2n (510 mg, 1.44 mmol); boron tribromide (4.4 mL, 4.4 mmol, 1 M solution in DCM). Further purification was achieved by suspending the residue obtained after column chromatography in boiling heptane. The suspension was then cooled to −78 °C, filtered, and the solid residue was washed with heptane cooled to −78 °C followed by pentane, cooled to −78 °C. Yield: 370 mg (76%) of a white solid after column chromatography, Rf: 0.37 (hexanes/EtOAc, 4/1, v/v), m.p.: 175–176 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 11.07 min, purity 99.60%. 1H NMR (acetone-d6): δ 0.83 (d, 3H, CH3), 0.85 (d, 3H, CH3), 1.07–1.45 (m, 4H, 2 × CH2), 1.47–1.52 (m, 1H, CH), 1.85–3.0 (br. m, 10H, BH), 3.46 (m, 1H, CH), 4.41 (d, 1H, OH), 6.67 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz), 8.53 (s, 1H, OH). 13C NMR (acetone-d6): δ 22.51, 23.15, 28.38, 35.59, 36.44, 73.11, 84.39, 88.54, 115.59, 128.28, 129.11, 158.60. Accurate mass HRMS (EI): m/z calcd for C14H28B10O2 (M)+ 336.310, found 336.3112.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-5-methylhexan-1-ol (3o)

The method described for the synthesis of 3b was adapted to synthesize 3o. Starting materials: 2o (200 mg, 0.55 mmol); boron tribromide (1.7 mL, 1.7 mmol, 1 M solution in DCM). Further purification was achieved by suspending the residue obtained after column chromatography in boiling heptane. The suspension was then cooled to −78 °C, filtered, and the solid residue was washed with heptane cooled to −78 °C followed by pentane, cooled to −78 °C. Yield: 125 mg (65%) of a white solid after column chromatography, Rf: 0.36 (hexanes/EtOAc, 4/1, v/v), m.p.: 130–131 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 10.63 min, purity 98.39%. 1H NMR (acetone-d6): δ 0.84 (d, 3H, CH3), 0.85 (d, 3H, CH3), 1.07–1.52 (m, 6H, 3 × CH2, CH), 1.85–3.0 (br. m, 10H, BH), 3.49 (m, 1H, CH), 4.41 (d, 1H, OH), 6.67 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz), 8.52 (s, 1H, OH). 13C NMR (acetone-d6): δ 22.67, 22.93, 25.06, 28.62, 37.89, 39.17, 72.96, 84.40, 88.48, 115.68, 128.30, 129.13, 158.61. Accurate mass HRMS (EI): m/z calcd for C15H30B10O2 (M)+ 350.3257, found 350.3268.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-6-methylheptan-1-ol (3q)

The method described for the synthesis of 3g was adapted to synthesize 3q. Starting materials: 2q (550 mg, 1.46 mmol); boron tribromide (4.4 mL, 4.4 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from hexanes/i-PrOH (24/1). The formed crystals were filtered at −20 °C and then washed with pentane precooled to −78 °C. Yield: 340 mg (72%) of a white solid after column chromatography, Rf: 0.23 (hexanes/EtOAc, 9/1, v/v), m.p.: 120–121 °C. Analytical HPLC: column I, solvent system: hexanes/i-PrOH (97/3) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 12.65 min, purity 99.99%. 1H NMR (CDCl3): δ 0.85 (d, 6H, CH3), 1.14–1.43 (m, 8H, 4 × CH2), 1.46–1.52 (m, 1H, CH), 1.61 (m, ∼2H, OH and H2O), 1.85–3.0 (br. m, 10H, BH), 3.47 (d, 1H, CH), 4.88 (br. s, 1H, OH), 6.61 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.8 Hz). 13C NMR (CDCl3): δ 22.71, 22.78, 26.88, 27.07, 29.67, 28.04, 36.93, 38.94, 73.13, 83.47, 86.38, 114.90, 128.69, 129.09, 155.80. Accurate mass HRMS (ESI): m/z calcd for C16H31B10O2 (M – 1) 363.33217, found 363.33305.

(RS)-(2,3-Dihydro-1H-inden-5-yl)-[1-(4-hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]methanol (3r)

The method described for the synthesis of 3g was adapted to synthesize 3r. Starting materials: 2r (280 mg, 0.63 mmol); boron tribromide (2.0 mL, 2 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from hexanes/i-PrOH (24/1). The formed crystals were filtered at −20 °C and then washed with pentane precooled to −78 °C. Yield: 240 mg (89%) of a white solid after column chromatography, Rf: 0.19 (hexanes/EtOAc, 9/1, v/v), m.p.: 231 °C (decomp.). Analytical HPLC: column I, solvent system: hexanes/i-PrOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 10.20 min, purity 99.69%. 1H NMR (acetone-d6): δ 1.9–3.0 (br. m, 10H, BH), 2.06 (m, ∼2H, CH2), 2.88 (m, ∼4H, 2 × CH2), 4.68 (s, H, OH), 4.99 (m, 1H, CH), 6.66 (d, 2H, arom., J = 8.6 Hz), 6.97 (d, 1H, arom.), 7.05 (d, 2H, arom., J = 8.9 Hz), 7.08 (s, 1H, arom.), 7.13 (d, 2H, arom.), 8.51 (s, 1H, OH). 13C NMR (acetone-d6): δ 26.41, 33.09, 33.31, 75.96, 84.58, 88.01, 115.65, 123.59, 124.21, 125.86, 128.30, 129.09, 140.63, 144.24, 144.71, 158.58. Accurate mass HRMS (ESI): m/z calcd for C18H25B10O2 (M – 1) 381.2852, found 381.2855.

1-(4-Hydroxyphenyl)-12-(3-phenylpropyl)-1,12-dicarba-closo-dodecaborane (3s)

The method described for the synthesis of 3b was adapted to synthesize 3s. Starting materials: 2s (470 mg, 1.28 mmol); boron tribromide (2.6 mL, 2.6 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from heptane. The formed crystals were filtered at −20 °C and then washed with heptane cooled to −78 °C followed by pentane cooled to −78 °C. Yield: 370 mg (84%) of a white solid after column chromatography, Rf: 0.32 (hexanes/EtOAc, 6/1, v/v), m.p.: 143–144 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 12.47 min, purity 99.77%. 1H NMR (CDCl3): δ 1.50–1.55 (m, 2 H, CH2), 1.67–1.76 (m, 2H, OH and CH2) 1.85–3.0 (br. m, 10H, BH), 2.46 (t, 2H, CH2), 4.91 (br. s, 1H, OH), 6.59 – 7.29 (m, 9H, arom.). 13C NMR (CDCl3): δ 31.15, 35.36, 37.50, 80.47, 81.00, 114.84, 126.12, 128.36, 128.53, 128.73, 129.16, 141.38, 155.64. Accurate mass HRMS (EI): m/z calcd for C17H26B10O (M)+ 354.2996, found 354.2975.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-3-phenylpropan-1-ol (3t)

The method described for the synthesis of 3g was adapted to synthesize 3t. Starting materials: 2t (250 mg, 0.65 mmol); boron tribromide (2.0 mL, 2 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from hexanes/i-PrOH (24/1). The formed crystals were filtered at −20 °C and then washed with pentane precooled to −78 °C. Yield: 200 mg (83%) of a white solid after column chromatography, Rf: 0.15 (hexanes/EtOAc, 9/1, v/v), m.p.: 135-136 °C. Analytical HPLC: column I, solvent system: hexanes/i-PrOH (97/3) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 12.87 min, purity 100.00%. 1H NMR (CDCl3): δ 1.49–1.77 (m, 3 H, OH and CH2), 1.85–3.0 (br. m, 10H, BH), 2.50–2.82 (m, 2H, CH2), 1.46–1.52 (m, 1H, CH), 3.47 (d, 1H, CH), 4.81 (br. s, 1H, OH), 6.60–7.29 (m, 9H, arom.). 13C NMR (CDCl3): δ 30.68, 36.29, 70.35, 81.56, 83.85, 112.90, 124.20, 126.52, 126.61, 126.68, 127.04, 139.12, 153.78. Accurate mass HRMS (ESI): m/z calcd for C17H25B10O2 (M – 1) 369.28522, found 369.28508.

1-(4-Hydroxyphenyl)-12-(4-phenylbutyl)-1,12-dicarba-closo-dodecaborane (3u)

The method described for the synthesis of 3b was adapted to synthesize 3u. Starting materials: 2u (550 mg, 1.44 mmol); boron tribromide (2.9 mL, 2.9 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from heptane. The formed crystals were filtered at −20 °C and then washed with heptane cooled to −78 °C followed by pentane cooled to −78 °C. Yield: 340 mg (64%) of a white solid after column chromatography, Rf: 0.32 (hexanes/EtOAc, 6/1, v/v), m.p.: 132–133 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 12.64 min, purity 99.93%. 1H NMR (CDCl3): δ 1.24 (m, 2 H, CH2), 1.46 (m, 2H, CH2), 1.69 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 2.53 (t, 2H, CH2), 4.73 (br. s, 1H, OH), 6.59–7.29 (m, 9H, arom.). 13C NMR (CDCl3): δ 29.26, 31.05, 35.62, 37.77, 80.68, 80.90, 114.84, 125.93, 128.45, 128.46, 128.75, 129.23, 142.20, 155.60. Accurate mass HRMS (ESI): m/z calcd for C18H27B10O (M – 1) 367.3074, found 367.3065.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-4-phenylbutan-1-ol (3v)

The method described for the synthesis of 3b was adapted to synthesize 3v. Starting materials: 2v (550 mg, 1.38 mmol); boron tribromide (4.2 mL, 4.2 mmol, 1 M solution in DCM). Further purification was achieved by suspending the residue obtained after column chromatography in boiling heptane. The suspension was then cooled to −78 °C, filtered, and the solid residue was washed with heptane cooled to −78 °C followed by pentane, cooled to −78 °C. Yield: 400 mg (75%) of a white solid after column chromatography, Rf: 0.35 (hexanes/EtOAc, 4/1, v/v), m.p.: 161–162 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 12.65 min, purity 99.78%. 1H NMR (acetone-d6): δ 1.22–1.47 (m, 2 H, CH2), 1.52–1.81 (m, 2 H, CH2), 1.85–3.0 (br. m, 10H, BH), 2.52–2.63 (m, 2H, CH2), 3.54 (m, 1H, CH), 4.48 (d, 1H, OH), 6.67–7.27 (m, 9H, arom.), 8.52 (s, 1H, OH). 13C NMR (acetone-d6): δ 29.21, 35.87, 37.14, 72.73, 84.42, 88.38, 115.68, 126.51, 128.28, 129.08, 129.12, 128.17, 143.14, 158.60. Accurate mass HRMS (ESI): m/z calcd for C18H27B10O2 (M)+ 384.3102, found 384.3102.

1-(4-Hydroxyphenyl)-12-(-phenylpentyl)-1,12-dicarba-closo-dodecaborane (3w)

The method described for the synthesis of 3b was adapted to synthesize 3w. Starting materials: 2w (430 mg, 1.08 mmol); boron tribromide (2.2 mL, 2.2 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from heptane. The formed crystals were filtered at −20 °C and then washed with heptane cooled to −78 °C followed by pentane cooled to −78 °C. Yield: 340 mg (64%) of a white solid after column chromatography, Rf: 0.36 (hexanes/EtOAc, 6/1, v/v), m.p.: 77–78 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 11.80 min, purity 99.24%. 1H NMR (CDCl3): δ 1.18 (m, 4 H, 2 × CH2), 1.54 (m, 2H, CH2), 1.64 (m, 2H, CH2), 1.85–3.0 (br. m, 10H, BH), 2.55 (t, 2H, CH2), 4.73 (br. s, 1H, OH), 6.59– 7.28 (m, 9H, arom.). 13C NMR (CDCl3): δ 28.83, 29.50, 31.14, 35.88, 37.90, 80.79, 80.86, 114.83, 125.83, 128.41, 128.50, 128.75, 129.24, 142.55, 155.61. Accurate mass HRMS (ESI): m/z calcd for C19H29B10O (M – 1) 381.3256, found 381.3257.

(RS)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]-5-phenylpentan-1-ol (3x)

The method described for the synthesis of 3b was adapted to synthesize 3x. Starting materials: 2x (50 mg, 0.122 mmol); boron tribromide (0.4 mL, 0.4 mmol, 1 M solution in DCM). Further purification was achieved by suspending the residue obtained after column chromatography in boiling heptane. The suspension was then cooled to −78 °C, filtered, and the solid residue was washed with heptane cooled to −78 °C followed by pentane, cooled to −78 °C. Yield: 24 mg (49%) of a white solid after column chromatography, Rf: 0.15 (hexanes/EtOAc, 9/1, v/v), m.p.: 119-120 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 11.89 min, purity 98.56%. 1H NMR (CDCl3): δ 1.18–1.64 (m, 7 H, OH and 3 × CH2), 1.85–3.0 (br. m, 10H, BH), 2.58 (m, 2H, CH2), 3.47 (d, 1H, CH), 4.81 (br. s, 1H, OH), 6.60–7.29 (m, 9H, arom.). 13C NMR (CDCl3): δ 26.25, 31.10, 35.91, 36.69, 70.02, 83.51, 86.21, 114.90, 125.86, 128.43, 128.51, 128.67, 129.04, 142.46, 155.79. Accurate mass HRMS (ESI): m/z calcd for C19H30B10O2 (M)+ 398.3259, found 398.3233.

1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-one (4)

Pyridinium chlorochromate (PCC, 2.0 g, 9.34 mmol) was suspended in anhydrous DCM (50 mL). A solution of (RS)-1-[1-(4-methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-ol (2k) (1.7 g, 4.67 mmol) in anhydrous DCM (15 mL) was then added to give a dark reaction mixture, which was stirred at room temperature overnight. Diethyl ether (60 mL) was added and then molecular sieve followed by stirring for 1 h. The supernatant was decanted, and the insoluble residue was washed with dry ether (3 × 20 mL). The combined organic phases were passed through a short column of florisil followed by evaporation. The residue was purified by silica gel column chromatography to yield a white waxlike solid. Yield: 1.6 g (95%), Rf: 0.13 (hexanes), m.p.: 36–37 °C (waxlike). 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.14–1.46 (m, 8H, 4 × CH2), 1.85–3.0 (br. m, 10H, BH), 2.39 (m, 2H, C(O)–CH2), 3.74 (s, 3H, OCH3), 6.69 (d, 2H, arom., J = 9.0 Hz), 7.10 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 14.14, 22.58, 23.60, 28.51, 31.60, 39.39, 55.41, 83.75, 85.64, 113.50, 128.28, 128.73, 159.92, 195.48. Accurate mass HRMS (EI+): m/z calcd for C16H30B10O2 (M)+ 362.3257, found 362.3254.

(S)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-ol (5)

Borane–tetrahydrofuran complex [16.5 mL, 16.5 mmol, 1.0 M solution in THF, stabilized with 0.005 M N-isopropyl-N-methyl-tert-butylamine (NIMBA)] followed by (S)-2-methyl-CBS-oxazaborolidine [(S)-MeCBS] (1.65 mL, 1.65 mmol, 1.0 M solution in toluene) were added to 15 mL of anhydrous THF. The reaction mixture was stirred at room temperature for 10 min and 1-[1-(4-methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-one (4) (600 mg, 1.65 mmol) in 15 mL of anhydrous THF was added slowly over a period of 2 h at 25 °C. The reaction mixture was stirred for additional 6 h at room temperature and then carefully quenched by the addition of 2.0 M HCl (30 mL) in small portions to control H2 development. Diethyl ether (50 mL) was added, and the organic phase was washed with brine and saturated NaHCO3. The organic phase was dried over MgSO4, filtered, and evaporated. The residue was purified by silica gel column chromatography to yield a white solid. Based on chiral HPLC, the enantiomeric excess (ee) of chiral alcohol 5 was estimated to be >80% (see the Supporting Information). Yield: 440 mg (73%) of a white solid, Rf: 0.43 (hexanes/EtOAc, 19/1, v/v), m.p.: 95–96 °C, [α]D20 °C = +27° (0.1, DCM). Analytical HPLC: column I, solvent system: hexanes/i-PrOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 5.43 min, purity 100.0%, analytical HPLC: column II, solvent system: hexanes/CH2Cl2 (9/1) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 10.01 min (area %: 8.74), 10.52 (area %: 91.26). 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.15–1.31 (m, 8H, 4 × CH2), 1.38–1.48 (m, 2H, CH2), 1.58 (br. s, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.47 (m, 1H, CH), 3.74 (s, 3H, OCH3), 6.68 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 14.20, 22.72, 26.60, 28.98, 31.83, 36.92, 55.40, 73.10, 83.53, 86.39, 113.42, 128.43, 128.85, 159.73. Accurate mass HRMS (EI+): m/z calcd for C16H32B10O2 (M)+ 364.3414, found 364.3417.

(R)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-ol (6)

For the synthesis of 6, the procedure and conditions described for the synthesis of 5 were adapted using 500 mg (1.38 mmol) of 4 and 1.38 mL (1.38 mmol, 1.0 M solution in toluene) of (R)-2-methyl-CBS-oxazaborolidine [(R)-MeCBS]. The residue was purified by silica gel column to yield a white solid. Based on chiral HPLC, the enantiomeric excess (ee) was estimated to be >75% (see the Supporting Information). Yield: 400 mg (80%) of a white solid, Rf: 0.43 (hexanes/EtOAc, 19/1, v/v), m.p.: 95–96 °C, [α]D20 °C = −24° (0.1, DCM). Analytical HPLC: column I, solvent system: hexanes/i-PrOH (99/1) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 5.55 min, purity 99.81%, analytical HPLC: column II, solvent system: hexanes/CH2Cl2 (9/1) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 9.99 min (area %: 87.90), 10.97 (area %: 12.10) 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.15–1.31 (m, 8H, 4 × CH2), 1.38–1.47 (m, 2H, CH2), 1.57 (br. s, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.47 (m, 1H, CH), 3.74 (s, 3H, OCH3), 6.68 (d, 2H, arom., J = 9.0 Hz), 7.12 (d, 2H, arom., J = 9.0 Hz). 13C NMR (CDCl3): δ 14.20, 22.72, 26.60, 28.99, 31.83, 36.92, 55.40, 73.10, 83.54, 86.39, 113.42, 128.43, 128.85, 159.73. Accurate mass HRMS (EI+): m/z calcd for C16H32B10O2 (M)+ 364.3414, found 364.3406.

(S)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-ol (7)

The method described for the synthesis of 3g adapted to synthesize 7. Starting materials: 5 (300 mg, 0.825 mmol); boron tribromide (2.5 mL, 2.5 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from hexanes/isopropyl alcohol [24:1]. The formed crystals were filtered at −20 °C and then washed with pentane precooled to −78 °C. Yield: 220 mg (76%) of a white solid after column chromatography, Rf: 0.23 (hexanes/EtOAc, 9/1, v/v), m.p.: 120–121 °C, [α]D20 °C = +23° (0.1, DCM). Analytical HPLC: column I, solvent system: hexanes/i-PrOH (97/3) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 9.67 min, purity 100%. 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.15–1.30 (m, 8H, 4 × CH2), 1.39–1.45 (m, 2H, CH2), 1.66–1.71 (m, ∼2H, OH and H2O), 1.85–3.0 (br. m, 10H, BH), 3.46 (m, 1H, CH), 5.08 (br. s, 1H, OH), 6.61 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.9 Hz). 13C NMR (CDCl3): δ 14.19, 22.70, 26.58, 28.97, 31.81, 36.90, 73.16, 83.49, 86.33, 114.90, 128.67, 129.03, 155.84. Accurate mass HRMS (ESI): m/z calcd for C15H29B10O2 (M – 1) 349.3165, found 349.3162.

(R)-1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-ol (8)

The method described for the synthesis of 3g was adapted to synthesize 8. Starting materials: 6 (300 mg, 0.825 mmol); boron tribromide (2.5 mL, 2.5 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from hexanes/isopropyl alcohol [24:1]. The formed crystals were filtered at −20 °C and then washed with pentane precooled to −78 °C. The enantiomeric excess of chiral alcohol 8 was enriched to >99% by chiral SFC separation, and then the absolute configuration of resulting enriched chiral alcohol 8 was determined via analysis of the corresponding bis-Mosher esters (see the Supporting Information). Yield: 180 mg (62%), Rf: 0.23 (hexanes/EtOAc, 9/1, v/v), m.p.: 120–121 °C, [α]D20 °C = −28° (0.1, DCM). Analytical HPLC: column I, solvent system: hexanes/i-PrOH (97/3) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 9.52 min, purity 99.84%. 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.15–1.30 (m, 8H, 4 × CH2), 1.39–1.45 (m, 2H, CH2), 1.68–1.76 (m, ∼2H, OH and H2O), 1.9–3.0 (br. m, 10H, BH), 3.47 (m, 1H, CH), 5.17 (br. s, 1H, OH), 6.61 (d, 2H, arom., J = 8.8 Hz), 7.07 (d, 2H, arom., J = 8.9 Hz). 13C NMR (CDCl3): δ 14.19, 22.70, 26.58, 28.96, 31.81, 36.90, 73.17, 83.50, 86.31, 114.90, 128.67, 129.01, 155.86. Accurate mass HRMS (ESI): m/z calcd for C15H29B10O2 (M – 1) 349.3165, found 349.3158.

1-[1-(4-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-one (9)

The method described for the synthesis of 3g was adapted to synthesize 9. Starting materials: 4 (630 mg, 1.74 mmol); boron tribromide (5.2 mL, 5.2 mmol, 1 M solution in DCM). Further purification was achieved by recrystallization of the residue obtained after column chromatography from hexanes. The formed crystals were filtered at −20 °C and then washed with pentane precooled to −78 °C. Yield: 520 mg (86%) of white solid after column chromatography, Rf: 0.31 (hexanes/EtOAc, 9/1, v/v), m.p.: 79–80 °C. Analytical HPLC: column I, solvent system: hexanes/i-PrOH (97/3) [isocratic], flow rate: 1 mL, wavelength: 254 nm, retention time: 8.89 min, purity 100.00%. 1H NMR (CDCl3): δ 0.86 (t, 3H, CH3), 1.12–1.27 (m, 6 H, 3 × CH2), 1.39–1.46 (m, 2 H, CH2), 1.55–3.40 (br. m, 10H, BH), 2.39 (t, 2H, C(O)–CH2), 5.11 (br. s, 1H, OH), 6.62 (d, 2H, arom., J = 8.7 Hz), 7.05 (d, 2H, arom., J = 8.7 Hz). 13C NMR (CDCl3): δ 14.09, 22.52, 23.53, 28.44, 31.54, 39.40, 83.61, 85.83, 114.95, 128.49, 128.87, 155.99, 195.87. Accurate mass HRMS (ESI): m/z calcd for C15H27B10O2 (M – 1) 347.3001, found 347.3014.

1-(4′-Methoxy-[1,1′-biphenyl]-4-yl)heptan-1-one (11)

To a solution of commercially available 4′-methoxy-[1,1′-biphenyl]-4-carbaldehyde (10) (0.69 g, 3.25 mmol) in anhydrous diethyl ether (25 mL) was added dropwise hexylmagnesium bromide (2 M in diethyl ether, 1.95 mL, 3.9 mmol) at 0 °C. The reaction mixture was stirred for another hour after addition and quenched by adding 0.1 N HCl (10 mL), the organic layer was separated, and the aqueous layer was extracted with diethyl ether (2 × 20 mL). The combined organic layers were washed with water, NaHCO3, and brine and dried over Na2SO4. Solvents were evaporated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield a yellow solid (0.85 g), which was taken forward to the next step. Pyridinium chlorochromate (PCC, 0.9 g, 4.1 mmol) was suspended in anhydrous DCM (25 mL). A solution of the yellow solid from the previous step (0.8 g, 2.68 mmol) in DCM (10 mL) was then added, and the reaction mixture was stirred at room temperature overnight. Diethyl ether (25 mL) was added followed by molecular sieves, and then stirred for 1 h. The supernatant was decanted, and the insoluble residue was washed with dry ether (3 × 20 mL). The combined organic phases were passed through a short column of Celite followed by evaporation. The residue was purified by Teledyne Isco (RediSep Rf column) to yield 0.64 g (71% over two steps) of pure product as a white waxlike solid. 1H NMR (400 MHz, CDCl3): δ 0.94 (t, 3H), 1.34–1.45 (m, 6H), 1.74–1.80 (m, 2H), 3.00 (t, 2H), 3.89 (s, 3H), 7.02 (d, 2H), 7.60 (d, 2H), 7.66 (d, 2H), 8.03 (d, 2H).

(S)-1-(4′-Methoxy-[1,1′-biphenyl]-4-yl)heptan-1-ol (12)

Borane–tetrahydrofuran complex (10 mL, 10 mmol, 1.0 M solution in THF, stabilized with 0.005 M N-isopropyl-N-methyl-tert-butylamine (NIMBA)) followed by (R)-2-methyl-CBS-oxazaborolidine [(R)-MeCBS] (1.0 mL, 1.0 mmol, 1.0 M solution in toluene) were added to 10 mL of anhydrous THF. The reaction mixture was stirred at room temperature for 15 min, and 1-(4′-methoxy-[1,1′-biphenyl]-4-yl)heptan-1-one (11) (0.29 g, 1.0 mmol) in 10 mL of anhydrous THF was added slowly over a period of 2 h at 0 °C. The reaction mixture was stirred overnight at room temperature and then carefully quenched by the addition of 2.0 M HCl (15 mL) in small portions to control H2 development. Diethyl ether (15 mL) was added, and the organic phase was washed with brine and saturated NaHCO3. The organic phase was dried over MgSO4, filtered, and evaporated. The residue was purified by Teledyne Isco (RediSep Rf column) to yield 0.21 g (70%) of pure product as a white waxlike solid. 1H NMR (400 MHz, CDCl3) δ 0.90 (t, 3H), 1.26–1.87 (m, 11H, overlap with H2O), 3.88 (s, 3H), 4.71–4.74 (m, 1H), 7.00 (d, 2H), 7.41 (d, 2H), 7.54–7.57 (m, 4H).

(S)-4′-(1-Hydroxyheptyl)-[1,1′-biphenyl]-4-ol (13)

To a mixture of (S)-1-(4′-methoxy-[1,1′-biphenyl]-4-yl)heptan-1-ol (12) (72 mg, 0.24 mmol), 1-dodecanethiol (75 mg, 89 μL, 0.37 mmol) in N-methylpyrrolidinone (NMP, 2 mL), NaOH (29 mg, 0.73 mmol) was added, and the reaction mixture was heated up to 100 °C overnight; cooled to room temperature; diluted with ethyl acetate (15 mL); washed with 1 N HCl (10 mL), water, and brine; and dried over Na2SO4. Solvents were evaporated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 42 mg (62%) of pure product as a white solid. 1H NMR (300 MHz, CDCl3) δ 0.90 (t, 3H), 1.25–1.50 (m, 8H), 1.70–1.82 (m, 3H) 4.74 (m, 2H), 6.92 (d, 2H), 7.41 (d, 2H), 7.48–7.56 (m, 4H). HRMS calcd 283.17708 (M – 1), obsv. 283.17184.

4-(4-Methoxyphenyl)cyclohexan-1-one (15)

The reaction mixture of commercially available 4-(4-hydroxyphenyl)cyclohexan-1-one (14) (2.4 g, 12.62 mmol), Cs2CO3 (6.16 g, 18.91 mmol), and iodomethane (6 mL, 18.91 mmol) in acetone (50 mL) was heated to reflux for 3 h, cooled to room temperature, filtered, and washed with acetone (2 × 20 mL). The combined acetone filtrates were concentrated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 2.58 g (quant.) of pure product as a white solid. 1H NMR (400 MHz, CDCl3) δ 1.88–1.99 (m, 2H), 2.19–2.25 (m, 2H), 2.49–2.54 (m, 4H), 2.98–3.04 (m, 1H), 3.80 (s, 3H), 6.89 (d, 2H), 7.19 (d, 2H).

1-(4-(4-Methoxyphenyl)cyclohexyl)heptan-1-one (16)

To a solution of (methoxymethyl)triphenylphosphonium chloride (3.8 g, 11 mmol) in anhydrous THF (95 mL), lithium bis(trimethylsilyl)amide (1.0 M in THF, 11 mL) was added dropwise at −78 °C. The reaction mixture was stirred for 1 h, and a solution of 4-(4-methoxyphenyl)cyclohexan-1-one (15) (2.04 g, 10 mmol) was added dropwise. This reaction mixture was stirred 30 min after addition, warmed up to room temperature, and stirred overnight. HCl (50 mL, 2N) was added and stirred for 2 h. The reaction mixture was extracted with ethyl acetate (3 × 30 mL), and the combined organic layers were washed with water, NaHCO3, and brine and dried over Na2SO4. Solvents were evaporated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 1.25 g of a yellow solid, which was taken forward to the next step. To a solution of this yellow solid (0.86 g, 3.94 mmol) in anhydrous diethyl ether (50 mL), hexylmagnesium bromide (2 M in diethyl ether, 2.46 mL, 4.52 mmol) was added dropwise at 0 °C. The reaction mixture was stirred for another hour after addition and quenched by adding 0.1 N HCl (20 mL), the organic layer was separated, and the aqueous layer was extracted with diethyl ether (2 × 25 mL). The combined organic layers were washed with water, NaHCO3, and brine and dried over Na2SO4. Solvents were evaporated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 0.99 g of a yellow solid, which was taken forward to the next step. Pyridinium chlorochromate (PCC, 0.97 g, 4.42 mmol) was suspended in anhydrous DCM (25 mL). A solution of the yellow solid from the previous step (0.88 g, 2.89 mmol) in DCM (10 mL) was then added, and the reaction mixture was stirred at room temperature overnight. Diethyl ether (25 mL) was added and followed by molecular sieves, and then stirred for 1 h. The supernatant was decanted, and the insoluble residue was washed with dry ether (3 × 20 mL). The combined organic phases were passed through a short column of Celite followed by evaporation. The residue was purified by Teledyne Isco (RediSep Rf column) to yield 0.72 g (40% over three steps) of pure product as a white waxlike solid. 1H NMR (400 MHz, CDCl3) δ 0.91 (t, 3H), 1.30–1.62 (m, 12H, overlap with H2O), 1.98–2.02 (m, 4H), 2.39–2.51 (m, 4H), 3.81 (s, 3H), 6.87 (d, 2H), 7.14 (d, 2H).

(S)-4-(4-(1-Hydroxyheptyl)cyclohexyl)phenol (17)

Borane–tetrahydrofuran complex (21.5 mL, 21.5 mmol, 1.0 M solution in THF, stabilized with 0.005 M N-isopropyl-N-methyl-tert-butylamine (NIMBA)) followed by (R)-2-methyl-CBS-oxazaborolidine [(R)-MeCBS] (2.15 mL, 2.15 mmol, 1.0 M solution in toluene) were added to 20 mL of anhydrous THF. The reaction mixture was stirred at room temperature for 15 min, and 1-(4-(4-methoxyphenyl)cyclohexyl)heptan-1-one (16) (0.65 g, 2.15 mmol) in 15 mL of anhydrous THF was added slowly over a period of 2 h at 0 °C. The reaction mixture was stirred overnight at room temperature and then carefully quenched by the addition of 2.0 M HCl (25 mL) in small portions to control H2 development. Diethyl ether (25 mL) was added, and the organic phase was washed with brine and saturated NaHCO3. The organic phase was dried over MgSO4, filtered, and evaporated. The residue was purified by Teledyne Isco (RediSep Rf column) to yield 0.50 g of a white waxlike solid, which was taken forward to the next step. To a mixture of the above solid (0.25 g, 0.82 mmol) and 1-dodecanethiol (0.26 g, 0.3 mL, 1.26 mmol) in N-methylpyrrolidinone (NMP, 5 mL), NaOH (100 mg, 2.48 mmol) was added, and the reaction mixture was heated up to 100 °C overnight; cooled to room temperature; diluted with ethyl acetate (15 mL); washed with 1 N HCl (10 mL), water, and brine; and dried over Na2SO4. Solvents were evaporated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 96 mg (31% over two steps) of pure product as a white solid. 1H NMR (400 MHz, CDCl3) δ 0.91 (t, 3H), 1.18–1.60 (m, 16H, overlap with H2O), 1.79–1.87 (m, 1H), 1.93–2.01 (m, 3H), 2.41–2.47 (m, 1H), 3.42–3.46 (m, 1 H), 4.54 (s, 1H), 6.78 (d, 2H), 7.10 (d, 2H). Accurate mass HRMS (ESI): m/z calcd for C19H29O2 (M – 1) 289.21621, found 289.21902.

Methyl 4-Bromobicyclo[2.2.2]octane-1-carboxylate (19)

To a solution of bromine (3.3 g, 20.6 mmol) in dichloromethane (20 mL) was added dropwise over 10 min into a heterogeneous refluxing mixture of commercially available 4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-carboxylic acid (3.0 g, 14.13 mmol) and mercuric oxide (5.12 g) in dichloromethane (60 mL), and heating was continued for 3.5 h. After the reaction mixture was allowed to cool to room temperature, it was filtered, and the resulting light orange filtrate was treated with MgSO4 and filtered again. The volatiles were removed, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 1.93 g (55%) of pure product as a white waxlike solid. 1H NMR (300 MHz, DMSO-d6) δ 1.86–1.91 (m, 6H), 2.17–2.23 (m, 6H), 3.56 (s, 3H).

Methyl 4-Phenylbicyclo[2.2.2]octane-1-carboxylate (20)

A benzene (30 mL) solution of methyl 4-bromobicyclo[2.2.2]octane-1-carboxylate (19) (1.90 g, 7.7 mmol) was added dropwise to a cooled (ca. −12 °C) mixture of benzene (100 mL) and aluminum chloride (5.0 g, 35 mmol) over 15 min. The heterogeneous mixture was stirred for 1 h while allowing the cooling bath to warm gradually to 3 °C, and then stirred at room temperature overnight; diluted with diethyl ether (100 mL); washed with 1 N HCl, water, and brine; and dried over Na2SO4. Solvents were evaporated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 1.6 g (85%) of pure product as a white solid. 1H NMR (400 MHz, CDCl3) δ 1.89–1.96 (m, 12H), 3.70 (s, 3H), 7.15–7.25 (m, 1H), 7.33–7.34 (m, 4H).

4-Phenylbicyclo[2.2.2]octane-1-carbaldehyde (21)

Methyl 4-phenylbicyclo[2.2.2]octane-1-carboxylate (20) (0.51 g, 2.1 mmol) was dissolved in anhydrous diethyl ether (25 mL), treated with LAH (159 mg, 4.2 mmol) at 0 °C for 2 h. NaOH (2 N) was added dropwise until form white precipitation, filtered, washed with diethyl ether (3 × 30 mL). The combined organic layers were dried over Na2SO4. Solvents were evaporated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 0.45 g of a white solid. To a mixture of this white solid (0.43 g, 1.99 mmol), NaHCO3 (166 mg, 1.99 mmol), NaOAc (163 mg, 1.99 mmol) in anhydrous DCM, pyridinium chlorochromate (PCC, 0.43 g, 1.99 mmol) was added. The reaction mixture was stirred at room temperature for 3 h and filtered. The filtrate was washed with 1 N HCl, water, NaHCO3, and brine and dried over Na2SO4. Solvents were evaporated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 405 mg (52% over two steps) of pure product as a white solid. 1H NMR (400 MHz, CDCl3) δ 1.79–1.83 (m, 6H), 1.91–1.95 (m, 6H), 7.19–7.24 (m, 1H), 7.31–7.36 (m, 4H), 9.55 (s, 1H).

1-(4-Phenylbicyclo[2.2.2]octan-1-yl)heptan-1-ol (22)

To a solution of 4-phenylbicyclo[2.2.2]octane-1-carbaldehyde (21) (0.4 g, 1.87 mmol) in anhydrous diethyl ether (25 mL), hexylmagnesium bromide (2 M in diethyl ether, 2.0 mL, 4.0 mmol) was added dropwise at 0 °C. The reaction mixture was stirred for another hour after addition and quenched by adding 0.1 N HCl (10 mL), the organic layer was separated, and the aqueous layer was extracted with diethyl ether (2 × 20 mL). The combined organic layers were washed with water, NaHCO3, and brine and dried over Na2SO4. Solvents were evaporated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 0.48 g (85%) of pure product as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 0.92 (t, 3H), 1.26–1.32 (m, 9H), 1.53–1.68 (m, 8H overlap with H2O), 1.84–1.88 (m, 6H), 3.20–3.24 (m, 1H), 7.17–7.21 (m, 1H), 7.29–7.36 (m, 4H).

1-(4-(4-Bromophenyl)bicyclo[2.2.2]octan-1-yl)heptan-1-ol (23)

To a mixture of 1-(4-phenylbicyclo[2.2.2]octan-1-yl)heptan-1-ol (22) (0.28 g, 0.94 mmol) and silver acetate (0.24 g, 1.09 mmol) in chloroform (25 mL), a solution of bromine (0.16 g, 0.99 mmol) in chloroform (10 mL) was added dropwise at 0 °C and stirred for 3 h; warmed up to room temperature; washed with NaHCO3, water, and brine; and dried over Na2SO4. Solvents were evaporated, and the residue was purified by Teledyne Isco (RediSep Rf column) to yield 0.28 g (79%) of pure product as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 0.92 (t, 3H), 1.26–1.34 (m, 9H, overlap with grease), 1.51–1.64 (m, 8H, overlap with H2O), 1.80–1.84 (m, 6H), 3.20–3.22 (m, 1H), 7.21 (d, 2H), 7.42 (d, 2H).

1-(4-(4-Bromophenyl)bicyclo[2.2.2]octan-1-yl)heptan-1-one (24)

Pyridinium chlorochromate (PCC, 0.27 g, 1.27 mmol) was suspended in anhydrous DCM (25 mL). A solution of 1-(4-(4-bromophenyl)bicyclo[2.2.2]octan-1-yl)heptan-1-ol (23) (0.16 g, 0.42 mmol) in DCM (10 mL) was then added, and the reaction mixture was stirred at room temperature overnight. Diethyl ether (25 mL) was added and followed by molecular sieves, and then stirred for 1 h. The supernatant was decanted, and the insoluble residue was washed with dry ether (3 × 20 mL). The combined organic phases were passed through a short column of Celite followed by evaporation. The residue was purified by Teledyne Isco (RediSep Rf column) to yield 135 mg (85%) of pure product as a white waxlike solid. 1H NMR (400 MHz, CDCl3) δ 0.92 (t, 3H), 1.29–1.30 (m, 6H), 1.50–1.60 (m, 2H, overlap with H2O), 1.86–1.89 (m, 12H), 2.45–2.49 (m, 2H), 7.21 (d, 2H), 7.44 (d, 2H).

(S)-1-(4-(4-Bromophenyl)bicyclo[2.2.2]octan-1-yl)heptan-1-ol (25)

Borane–tetrahydrofuran complex (3.2 mL, 3.2 mmol, 1.0 M solution in THF, stabilized with 0.005 M N-isopropyl-N-methyl-tert-butylamine (NIMBA)) followed by (R)-2-methyl-CBS-oxazaborolidine [(R)-MeCBS] (0.32 mL, 0.32 mmol, 1.0 M solution in toluene) were added to 20 mL of anhydrous THF. The reaction mixture was stirred at room temperature for 15 min, and 1-(4-(4-bromophenyl)bicyclo[2.2.2]octan-1-yl)heptan-1-one (24) (0.12 g, 0.32 mmol) in 10 mL of anhydrous THF was added slowly over a period of 2 h at 0 °C. The reaction mixture was stirred overnight at room temperature and then carefully quenched by the addition of 2.0 M HCl (25 mL) in small portions to control H2 development. Diethyl ether (25 mL) was added, and the organic phase was washed with brine and saturated NaHCO3. The organic phase was dried over MgSO4, filtered, and evaporated. The residue was purified by Teledyne Isco (RediSep Rf column) to yield 98 mg (81%) of product as a white waxlike solid. 1H NMR (400 MHz, CDCl3) δ 0.92 (t, 3H), 1.26–1.34 (m, 9H), 1.51–1.64 (m, 8H, overlap with H2O), 1.80–1.84 (m, 6H), 3.20–3.22 (m, 1H), 7.21 (d, 2H), 7.42 (d, 2H).

(S)-4-(4-(1-Hydroxyheptyl)bicyclo[2.2.2]octan-1-yl)phenol (26)

A mixture of (S)-1-(4-(4-bromophenyl)bicyclo[2.2.2]octan-1-yl)heptan-1-ol (25) (72 mg, 0.19 mmol), benzaldehyde oxime (30 mg, 0.25 mmol), Cs2CO3 (136.2 mg, 0.42 mmol), and RockPhos Pd G3 (8 mg) in DMF (1 mL) was degassed with Ar for 15 min. Then, the mixture was heated to 80 °C for 18 h. The mixture was then cooled down to room temperature; diluted with ethyl acetate (10 mL); washed with 1 N HCl (10 mL), water, and brine; dried over Na2SO4; filtered; and evaporated. The residue was purified by Teledyne Isco (RediSep Rf column) to yield a white solid, 98 mg. 1H NMR (400 MHz, CDCl3) δ 0.92 (t, 3H), 1.26–1.34 (m, 9H, overlap with grease), 1.51–1.64 (m, 8H, overlap with H2O), 1.80–1.84 (m, 6H), 3.20–3.22 (m, 1H), 4.60 (br. s, 1H), 6.78 (d, 2H), 7.21 (d, 2H). Accurate mass HRMS (ESI): m/z calcd for C21H31O2 (M – 1) 315.23186, found 315.23676.

(RS)-1-[(1-Phenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-ol (28)

The method described for the synthesis of 2e was adapted to synthesize 28. Starting materials: 27 (58) (0.27 g, 1.3 mmol), 1-heptanal (0.28 mL, 2 mmol). Yield: 280 mg (64%) of a white solid, Rf: 0.74 (hexanes/EtOAc, 49/1, v/v), m.p.: 106–107 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (99.7/0.3) [isocratic], flow rate: 1 mL, wavelength: 260 nm, retention time: 12.09 min, purity 99.61%. 1H NMR (CDCl3): δ 0.88 (t, 3H, CH3), 1.16–1.32 (m, 8H, 4 × CH2), 1.40–1.47 (m, 2H, CH2), 1.59 (d, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.47 (m, 1H, CH), 7.16- 7.23 (m, 5H, arom.). 13C NMR (CDCl3): δ 14.20, 22.72, 26.59, 28.98, 31.83, 36.91, 55.38, 73.13, 83.63, 87.02, 127.26, 128.18, 128.47, 136.37. Accurate mass HRMS (EI+): m/z calcd for C15H30B10O (M)+ 334.3307, found 334.3302.

(RS)-1-[1-(3-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-ol (30)

The method described for the synthesis of 2e was adapted to synthesize 30. Starting materials: 29 (43) (0.5 g, 2 mmol), 1-heptanal (0.42 mL, 3 mmol). Yield: 580 mg (80%) of a white solid, Rf: 0.39 (hexanes/EtOAc, 14/1, v/v), m.p.: 62–63 °C. 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.15–1.30 (m, 8H, 4 × CH2), 1.38–1.47 (m, 2H, CH2), 1.59 (br. s, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.47 (m, 1H, CH), 3.75 (s, 3H, OCH3), 6.75–7.09 (m, 4H, arom.). 13C NMR (CDCl3): δ 14.20, 22.71, 26.59, 28.98, 31.83, 36.91, 55.38, 73.12, 83.43, 87.02, 113.52, 113.80, 119.73, 129.14, 128.84, 137.77, 159.21. Accurate mass HRMS (EI+): m/z calcd for C16H32B10O2 (M)+ 364.3414, found 364.3412.

(RS)-1-[1-(3-Hydroxyphenyl)-1,12-dicarba-closo-dodecaborane-12-yl]heptan-1-ol (31)

The method described for the synthesis of 3b was adapted to synthesize 31. Starting materials: 30 (500 mg, 1.37 mmol); boron tribromide (4.1 mL, 4.1 mmol, 1 M solution in DCM). Further purification was achieved by suspending the residue obtained after column chromatography in boiling heptane. The suspension was then cooled to −78 °C, filtered, and the solid residue was washed with heptane cooled to −78 °C followed by pentane, cooled to −78 °C. Yield: 270 mg (56%) of a white solid after column chromatography, Rf: 0.48 (hexanes/EtOAc, 4/1, v/v), m.p.: 99–100 °C. Analytical HPLC: column I, solvent system: isooctanol/EtOH (95/5) [isocratic], flow rate: 1 mL, wavelength: 280 nm, retention time: 10.27 min, purity 100.00%. 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.16–1.39 (m, 8H, 4 × CH2), 1.40–1.45 (m, 2H, CH2), 1.67 (br. s, 1H, OH), 1.85–3.0 (br. m, 10H, BH), 3.48 (m, 1H, CH), 4.98 (s, 1H, OH), 6.68–7.04 (m, 4H, arom.). 13C NMR (CDCl3): δ 14.19, 22.70, 26.57, 28.96, 31.80, 36.89, 73.21, 83.13, 86.96, 114.71, 115.45, 119.90, 129.38, 138.02, 155.17. Accurate mass HRMS (ESI): m/z calcd for C15H29B10O2 (M – 1) 349.31653, found 349.31686.

Plasmids and Transfection

Human ERα and ERβ expression vectors (pcDNA3-hERα and pcDNA3-hERβ, respectively), as well as the luciferase reporter vector (pGL4.26-3xERE) were described previously. (59) HEK-293 cells (ATCC CRL-1573) were seeded on 100 mm dishes and grown in Dulbecco’s modified Eagle’s medium (DMEM)/10% fetal bovine serum (FBS) to about 80% confluency. One day before transfection, growth medium was replaced by phenol-red-free DMEM/5% Hyclone charcoal stripped FBS (CS-FBS, GE Healthcare Life Sciences) without penicillin–streptomycin (starvation medium). DNA/polyethylenimine complexes were made by mixing 1.8 μg of pcDNA3-hERα or pcDNA3-hERβ with 3.6 μg of pGL4.26-3xERE and 9.6 μg of pBluescript in 500 μL of DMEM and adding to 30 μL of 1 mg/mL polyethylenimine (linear, MW ∼ 25 000, Cat #23966, Polysciences, Inc., PA) diluted in 500 μL of DMEM. Cells were further cultivated for 24 h and harvested for reporter luciferase assay.

Reporter Luciferase Assays for Steroid Receptors AR, PR, GR, and MR

These assays were performed as described previously (59) and consist of clonal lines created in U2OS cells with stably integrated expression constructs for individual full-length steroid receptors together with reporter vector pGL4.36[luc2P/MMTV/Hygro] (Promega) containing a viral LTR isolated from MMTV. This promoter harbors multiple binding sites for different steroid receptors that regulate the expression of firefly luciferase as a reporter enzyme.

Ga4/UAS Reporter Luciferase Assays for Nuclear Receptors

The panel of selective cell-based luciferase reporter assays is based on chimeric NRs created by the replacement of the N-terminally located DBD by a DBD from the yeast transcription factor Gal4. The chimeric Gal4-DBD/NR-LBD binds to UAS elements in the promoter of the reporter construct controlling the expression of the firefly luciferase from the pGL4.35 reporter vector (Promega). Most of the assays are available in form of clonal stable cell lines established on the unified cellular background in U2OS osteosarcoma cells. These lines were prepared by introducing the expression vector encoding for NR and pGL4.35 reporter vector. Cells that stably integrated both constructs into the genome were isolated with selection medium containing hygromycin and G418 (Geneticin). From these cell cultures, clones of cells were further isolated and screened for optimal response in assays with reference ligands. All lines were extensively validated with a collection of reference ligands for individual NRs.

Luciferase Reporter Assays

Transcription response of different NRs to test compounds was evaluated using transiently transfected HEK293 (ERα and ERβ) or clonal stable reporter U2OS lines (remaining NRs). Cells were cultivated for 24 h in the starvation medium and were harvested, counted, and seeded to cell culture-treated, white, solid 384-well plates (Corning, Inc., NY) 12 h before compound addition. A total of 6000 transiently transfected HEK293 cells (ERα, ERβ reporter assay, dose response compound profiling) or 10 000 U2OS cells (compound selectivity profiling at 1 μM for NR activity) were dispensed in 20 μL of total starvation media volume to 384-well plates (Corning Costar) by Multidrop Combi (Thermo Fisher Scientific) or Tempest (Formulatrix, Inc.).
Test compounds were diluted in DMSO and transferred to cells using contact-free acoustic transfer by ECHO 550 (Labcyte, Inc.) integrated in the fully automated robotic HTS station cell::explorer (PerkinElmer). In ERα and ERβ reporter assays, compounds were tested at 15 concentration points extending from 100 μM to 1 pM, in duplicates. In the Gal4/UAS NR profiling for compound selectivity, they were tested at a single concentration point: 1 μM, in quadruplicates. In the antagonist mode, 30 min incubation of cells with compounds was followed by the addition of 10-fold concentrated agonist to each well on the plate. The concentration of the agonist for the antagonist mode was chosen to induce 80% of the maximal reporter response and specific conditions for all assays are shown in Table S3 (Supporting Information). Luciferase activity was measured 20 h (ERα, ERβ reporter assay, dose response compound profiling) or 14 h (compound selectivity profiling at 1 μM for NR activity) after compound addition, with Bright-Glo Luciferase Assay System (Promega) on the multimode plate reader Envision (PerkinElmer) equipped with enhanced luminescence module. Data were collected, normalized, and processed using proprietary LIMS system ScreenX.
Luciferase activities were normalized for each assay independently in the scale of 0–100%: agonist mode: 0% = activity of untreated cells, 100% = maximal reporter activity achieved with reference agonist ligand (Table S3, Supporting Information). Antagonist mode: 0% = untreated cells, 100% = reporter activity obtained by treating cells with the agonist at concentration inducing 80% of the maximal reporter activity.

Cell Viability Assay

Cells were propagated in the cell growth medium to the amount needed for the experiment. Cells were harvested, counted, and dispensed to 384-well plates at the same number/well as in the reporter assays and were treated with compounds identically as in the reporter assays. Cell viability was assessed after 20 h (ERα, ERβ reporter assay, dose response compound profiling) or 14 h (compound selectivity profiling at 1 μM for NR activity) of incubation with compounds by determining the level of intracellular ATP using CellTiter-Glo (Promega) luminescent assay. Luciferase signal was measured on the multimode plate reader Envision (PerkinElmer). Data were collected, normalized, and processed using proprietary LIMS system ScreenX.

PolarScreen ERα and ERβ Competitor Binding Assay

Compound affinities for ERα and ERβ proteins were assessed in commercial, homogeneous, polarized fluorescence-based PolarScreen ER α/β Competitor Assays, Red (Life Technologies) according to manufacturer′s protocols. First Kd for the competitor ligand Fluormone EL Red was determined in a saturation binding experiment for each receptor independently: Kd (ERα) = 12.4 nM and Kd (ERβ) = 16.7 nM. Then, competitive binding assays with carborane compounds were carried out in a total volume of 5 μL in nontreated, black polystyrene 1536-well plates (Corning, cat. no. 3936). First, 3 μL of reaction buffer was dispensed to each well by a Tempest (Formulatrix) dispenser. Then, dilution series of test compounds prediluted in DMSO in the acoustic dispensing-compliant polypropylene plates (Labcyte) were transferred to the assay plates using contact-free acoustic transfer by ECHO 550 (Labcyte, Inc.) integrated in the fully automated robotic HTS station cell::explorer (PerkinElmer). Compounds were tested in triplicates at 16 different concentration points between 100 μM and 100 pM. The plates were spun down and vigorously shaken for 5 min after which a mixture of 2 μL of ERα (final concentration = 48 nM) or ERβ (final concentration = 138 nM) proteins with Fluormone EL Red (final concentration = 1.4 nM) were dispensed to assay plates. Plates were shaken for 5 min again, spun down, and incubated for 3 h. Fluorescence polarization value (mP) was recorded using a multimode plate reader Envision (PerkinElmer) with optimized set of filters: excitation filter 531/25 nm and two emission filters 595/60 nm (S/P). Data were collected, normalized by proprietary LIMS system ScreenX. Dose response curves were obtained by fitting data with nonlinear regression function and Kd values for each compound were calculated from obtained IC50 values by Cheng–Prusoff equation. (60)

Cytochrome P450 Enzymatic Assays

Enzyme activities of individual CYP forms were measured according to established protocols. The following microsomal CYP activities were tested: CYP1A2, 7-ethoxyresorufin O-deethylation; (61) CYP2A6, coumarin 7-hydroxylation; (62) CYP2B6, 7-ethoxy-4-(trifluoromethyl)coumarin 7-deethylation; (63) CYP2C8, paclitaxel 6α-hydroxylation; (64) CYP2C9, diclofenac 4′-hydroxylation; (65) CYP2C19, mephenytoin 4′-hydroxylation; (66) CYP2D6, bufuralol 1′-hydroxylation; (67) CYP2E1, chlorzoxazone 6-hydroxylation, (68) and CYP3A4, midazolam 1′-hydroxylation. (69,70) Incubation mixtures contained 100 mM potassium phosphate buffer (pH 7.4), reduced nicotinamide adenine dinucleotide phosphate (NADPH)-generating system (0.8 mM NADP+, 5.8 mM isocitrate, 0.3 unit/mL of isocitrate dehydrogenase, and 8 mM MgCl2), human liver microsomes and individual probe substrate. Compounds tested were dissolved in DMSO to a concentration of 200 mM, then diluted with the same phosphate buffer. Compounds were tested at concentrations between 0.01 and 100 μM. Assay conditions are listed in the Supporting Information (Table S4). The concentration of probe substrates for individual CYP forms was chosen in the range of the respective KM determined separately as described previously for each CYP and for its respective substrate. Activities were measured using HPLC Prominence system (Shimadzu; Kyoto, Japan) with a UV/fluorescence detection. Data were analyzed using Sigma Plot v.12.0 graphing software (Jandel Scientific, Chicago, IL).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00555.

  • Activity profiles of library compounds in cell-based reporter assays for nuclear receptors; methods and experimental conditions for cytochrome P450 enzyme assays; and NMR and MS spectra and HPLC chromatograms (PDF)

  • Molecular formula strings (CSV)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
    • Petr Bartunek - CZ-OPENSCREEN, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic Email: [email protected]
    • Christopher C. Coss - Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United StatesDrug Development Institute, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United StatesOrcidhttps://orcid.org/0000-0002-8184-9190 Email: [email protected]
  • Authors
    • David Sedlák - CZ-OPENSCREEN, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech RepublicOrcidhttps://orcid.org/0000-0003-0074-335X
    • Tyler A. Wilson - Medicinal Chemistry Shared Resource, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States
    • Werner Tjarks - Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
    • Hanna S. Radomska - Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
    • Hongyan Wang - Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
    • Jayaprakash Narayana Kolla - CZ-OPENSCREEN, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic
    • Zbigniew J. Leśnikowski - Laboratory of Medicinal Chemistry, Institute of Medical Biology PAS, 106 Lodowa Street, 93-232 Lodz, PolandOrcidhttps://orcid.org/0000-0003-3158-5796
    • Alena Špičáková - Department of Pharmacology, Faculty of Medicine, Palacky University, Hněvotínská 3, 77515 Olomouc, Czech Republic
    • Tehane Ali - Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
    • Keisuke Ishita - Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
    • Liva Harinantenaina Rakotondraibe - Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United StatesOrcidhttps://orcid.org/0000-0003-2166-4992
    • Sandip Vibhute - Medicinal Chemistry Shared Resource, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States
    • Dasheng Wang - Medicinal Chemistry Shared Resource, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States
    • Pavel Anzenbacher - Department of Pharmacology, Faculty of Medicine, Palacky University, Hněvotínská 3, 77515 Olomouc, Czech Republic
    • Chad Bennett - Medicinal Chemistry Shared Resource, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United StatesDrug Development Institute, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States
  • Author Contributions

    D.S., T.A.W., W.T., P.B., and C.C.C. contributed equally to this work.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by the Ministry of Education, Youth and Sports grant: RVO: 68378050-KAV-NPUI, LM2015063, LM2018130, NSC 2014/13/B/NZ1/03989 at IMB PAS, institutional support RVO: 68378050 at IMG and RVO: 61989592 at UPOL. This work was further supported by the College of Pharmacy at The Ohio State University, its instrumentation core and director Dr. Craig McElroy. This study made use of the Campus Chemical Instrument Center NMR facility at The Ohio State University. Compounds 13, 17, and 26 were synthesized by the Medicinal Chemistry Shared Resource, and the corresponding mass spectral data were obtained by the Proteomics Shared Resource, both of which are part of The Ohio State University Comprehensive Cancer Center and supported by NCI/NIH Grant P30CA016058. This work was also supported by the Drug Development Institute within The Ohio State University Comprehensive Cancer Center and Pelotonia. The authors thank the University of Illinois Urbana–Champaign Mass Spectrometry Laboratory and the University of Michigan College of Literature, Science and the Arts Mass Spectrometry Technical Services for instrumentation support. They also thank Olga Martínková for support in cell culture work and transfection experiments, Dr. Martin Popr for support in compound management, Tomas Langammer for automating the experiments on HTS robotics, and Dr. Samuel Kulp for his help in formatting the manuscript.

Abbreviations

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AR

androgen receptor

BNCT

boron-neutron capture therapy

CBS

Corey–Bakshi–Shibata

CDCl3

deuterated chloroform

DBD

DNA-binding domain

DMEM

Dulbecco’s modified Eagle’s medium

DPN

diarylpropionitrile

E2

17β-estradiol

ERRγ

estrogen receptor-related receptor γ

ERα

estrogen receptor α

ERβ

estrogen receptor β

EtOAc

ethyl acetate

EtOH

ethyl alcohol

FBS

fetal bovine serum

FXR

farnesoid X receptor

GPER1

G protein-coupled transmembrane receptor

GR

glucocorticoid receptor

i-PrOH

isopropyl alcohol

KO

knock-out

LTR

long terminal repeat

LXR

liver X receptor

MMTV

mouse mammary tumor virus

MR

mineralocorticoid receptor

NaOAc

sodium acetate

NIMBA

N-isopropyl-N-methyl-tert-butylamine

NR

nuclear receptor

PPAR

peroxisome proliferator-activated receptor

PPT

propylpyrazole triol

PR

progesterone receptor

RAR

retinoic acid receptor

RXR

retinoid X receptor

SR

steroid receptor

UAS

upstream activating sequences

VDR

vitamin D receptor

Additional Notes

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a Preparation of 5-Methylhexan-1-al: PCC (3.71 g, 17.21 mmol) was suspended in dry methylene chloride (40 mL). A solution of 5-methyl-1-hexanol (1 g, 8.61 mmol) in methylene chloride (15 mL) was then added in one portion to the stirred suspension to give a dark reaction mixture, which was stirred at room temperature overnight. Dry ether (60 mL) was added and then molecular sieve followed by stirring for 1 h. The supernatant liquid was decanted, and the insoluble residue was washed with dry ether (3 × 20 mL). The combined organic phases were passed through a short florisil column, and the solvent was removed in vacuo providing 370 mg (38%) of product, which was used without further purification and analysis in the next step.

b Preparation of 6-Methylheptan-1-al: PCC (2.89 g, 13.44 mmol) was suspended in dry methylene chloride (40 mL). A solution of 6-methyl-1-heptanol (1 g, 7.68 mmol) in methylene chloride (15 mL) was then added in one portion to the stirred suspension to give a dark reaction mixture, which was stirred at room temperature overnight. Dry ether (60 mL) was added and then molecular sieve followed by stirring for 1 h. The supernatant liquid was decanted, and the insoluble residue was washed with dry ether (3 × 20 mL). The combined organic phases were passed through a short florisil column, and the solvent was removed in vacuo providing 750 mg (76%) of product, which was used without further purification and analysis in the next step.

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  • Abstract

    Figure 1

    Figure 1. Estrogen receptor ligands.

    Scheme 1

    Scheme 1. Synthesis of Target Alkyl- and Hydroxyalkyl-Substituted Carboranesa

    a(a) n-BuLi, dimethoxyethane (1,2-DME), −78 or 0 °C; (b) BBr3, CH2CI2, 0 °C to room temperature (RT).

    Scheme 2

    Scheme 2. Synthesis of Ketone 9 and S and R Enantiomers 7 and 8a

    a(a) PCC, CH2Cl2; (b) BH3·THF, (S)-2-Me-CBS, RT; (c) BH3·THF, (R)-2-Me-CBS, RT; (d) BBr3, CH2Cl2, 0 °C to RT.

    Scheme 3

    Scheme 3. Synthesis of Phenyl Compound 13a

    a(a) n-Hexylmagnesium bromide, Et2O, 0 °C; (b) PCC, CH2CI2; (c) BH3·THF, (R)-2-Me-CBS, 0 °C; (d) 1-dodecanethiol, N-methylpyrrolidinone (NMP), NaOH, 100 °C.

    Scheme 4

    Scheme 4. Synthesis of Cyclohexyl Compound 17a

    a(a) Cs2CO3, CH3I, acetone, reflux; (b) (methoxymethyl)triphenylphosphonium chloride, LiHMDS, THF; 2 N HCI; (c) hexylmagnesium bromide, Et2O, 0 °C; (d) PCC, CH2CI2; (e) BH3·THF, (R)-2-Me-CBS, 0°C; (f) 1-dodecanethiol, NMP, NaOH, 100 °C.

    Scheme 5

    Scheme 5. Synthesis of [2.2.2]Bicyclic Compound 26a

    a(a) Br2, dichloromethane (DCM), HgO; (b) AICI3, benzene, (c) LiAIH4, Et2O; (d) PCC, NaHCO3, NaOAc, CH2CI2; (e) hexylmagnesium bromide, Et2O; (f) AgOAc, Br2, CHCI3; (g) PCC, CH2CI2; (h) BH3·THF, (R)-2-Me-CBS 0 °C; (i) benzaldehyde oxime, Cs2CO3, RockPhos Pd G3.

    Scheme 6

    Scheme 6. Synthesis of Compounds 28 and 31a

    a(a) n-BuLi, 1,2-DME, −78 or 0 °C, 1-heptanal; (b) BBr3, CH2CI2, 0 °C to RT.

    Figure 2

    Figure 2. Ligand selectivity for ERβ increases with elongation of the side hydrocarbon chain. BE120, 3g, and 8 were tested in ERα (dashed line) and ERβ (solid line) transactivation assays in the concentration range between 10 μM and 1 pM, in triplicates and activities were expressed relative to 100 nM 17β-estradiol set to 100%. Increasing length of the carboranes’ side hydrocarbon chain is accompanied with decreasing potency in both ERα and ERβ reporter assays and by increasing selectivity for ERβ (blue: nonselective, green: 10–100× selective, red: selectivity >100×).

    Figure 3

    Figure 3. Activities of the carboranes’ compound library on steroid receptors. (A) Compounds were profiled at 1 μM in the selective luciferase reporter assays for the agonistic and (B) antagonistic activities for steroid receptors. The activity is expressed relative to maximal activity induced by reference compound or as a fold induction compared to untreated cells for control assay (MMTV reporter alone, dark red dots). Cell viability in U2OS cells was used as a control experiment for the antagonistic activities and is expressed in a scale of 0–100% relative to dimethyl sulfoxide (DMSO)-treated cells (green dots).

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