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

David Sedl á k,^○Tyler A. Wilson,^○Werner Tjarks,^○Hanna S. Radomska, Hongyan Wang,

̌

́

̌

Jayaprakash Narayana Kolla, Zbigniew J. Lesnikowski, Alena Spic á kov á , Tehane Ali, Keisuke Ishita,

Liva Harinantenaina Rakotondraibe, Sandip Vibhute, Dasheng Wang, Pavel Anzenbacher, Chad Bennett,

Petr Bartunek,*^,○and Christopher C. Coss*^,○

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ABSTRACT: 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.

ization of ERα,¹⁴ERβ,¹⁵and GPER1¹⁶genetically null mice

supports distinct biological functions of each receptor

including a very limited role for ERβ in control of

INTRODUCTION

■

Although the physiologic functions of estrogens have been

recognized as clinically important for roughly a century,¹the

steroidogenesis and homeostasis of uterine and breast tissues.

underlying mechanisms of estrogen action were largely

One considerable challenge in understanding ERβ biology

unknown prior to the discovery of a high-aﬃnity estrogen

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 (diarylpropionitrile¹⁹[DPN]) or ERα selective

(propylpyrazole triol²⁰[PPT]) tool compounds, a clearer

role for ERβ emerged.¹⁷The 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

receptor (estrogen receptor α, or ERα) in 1958 and

subsequent cloning of its gene in 1985.²Nonetheless, residual

eﬀects of exogenous estrogens in ERα knockout (ERα-KO)

mice³were not readily explained until the surprising discovery

of the second estrogen receptor (estrogen receptor β, or

ERβ).⁴Mechanistically, 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.^7,8ERα, 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

Received: March 26, 2021

Published: June 28, 2021

All three receptor isoforms bind to the predominate

circulating endogenous estrogen 17β-estradiol (E2) (Figure

1) with very similar high aﬃnities^9,12,13such that genetic

approaches are required to deconvolute the contributions of

each receptor to whole animal estrogen response. Character-

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three-dimensional ﬂexibility for target−ligand interactions

compared to their two-dimensional benzene counterpart, and

their resistance toward conventional enzymatic degrada-

tion.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 metal-

lacarboranes in the 17α-position.³⁹Although 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 carboranyl-

phenols provided the proper molecular geometry and hydro-

phobicity to ﬁt within the ERα LBD while maintaining key

hydrogen bonds required for high aﬃnity.⁴²From 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.⁴³By comparing BE120 to ERB-041, this same group

more recently reported para-carboranylcyclohexanol deriva-

tives with ERβ selective activity.⁴⁴Herein, we describe a novel

series of BE120-based para-carborane derivatives demonstrat-

ing highly selective ERβ agonist activity.

Figure 1. Estrogen receptor ligands.

RESULTS AND DISCUSSION

■

Chemistry. The intermediate hydrocarbon- (pathway 1) or

α-hydroxy-hydrocarbon-substituted para-carboranes 2a−x

(pathway 2) were prepared by lithiation of 1^43,45with 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 3b−x.

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) conditions⁴⁶as seen in Scheme 2.

Subsequent demethylation using boron tribromide in methyl-

ene 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 chroma-

tography (HPLC) of intermediates 5 and 6. The absolute

conﬁguration of chiral alcohol 8 was established by Mosher

ester analysis.^47,48

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.

between DNA-binding domains and 54% between LBDs of

human ERα and ERβ.²²Given the high structural similarity in

human and preclinical species LBDs,²³selective 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.²⁴Clinical development of

ERβ agonists has thus far been limited to two agents. ERB-

041²⁵(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.²⁶LY500307 (SERBA-1, erteberel), a selective

agonist with a benzopyran core,²⁷was later advanced for the

treatment of symptoms associated with benign prostatic

hypertrophy but similarly failed to demonstrate eﬃcacy in

phase II trials.²⁸Though development of ERB-041 has been

terminated, LY500307 has recently been evaluated for

symptoms associated with schizophrenia²⁹(NCT01874756)

and for estrogen withdrawal-induced mood symptoms in

women with past perimenopausal depression³⁰

(NCT03689543). The continued description of novel, iso-

form-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 bioisosteres³²belonging to a structurally

unique class of molecules known as dicarba-closo-dodecabor-

anes (carboranes). Carboranes are perhaps best known for

their use in boron-neutron capture therapy³³⁻³⁵(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

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a

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

a

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

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) fol-

lowed 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.

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]-bicy-

clobromide 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 conditions⁴⁶yielded 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.

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-dimethoxy-

ethane 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 dichloro-

methane to furnish the target meta-substituted phenol

compound 31.

Structure−Activity Relationship (SAR) Study. The

target compounds were analyzed for their ability to interact

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a

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

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.

a

Scheme 3. Synthesis of Phenyl Compound 13

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.

a

Scheme 4. Synthesis of Cyclohexyl Compound 17

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.

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 modu-

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a

Scheme 5. Synthesis of [2.2.2]Bicyclic Compound 26

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.

a

Scheme 6. Synthesis of Compounds 28 and 31

a

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

lation of transcription from the ER-regulated promoters.

Considering this complex mechanism of ligand-induced

transactivation, our SAR was driven using cellular trans-

activation data and binding aﬃnities were primarily used to

conﬁrm the speciﬁcity of the interaction and observed activity

trends.

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,³²was 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 R³position. 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. Reintroduc-

ing 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 3d−g) resulted in reduced

potencies, similar in magnitude between the isoforms with a

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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 EC₅₀values 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 E_maxfor each compound and

b

comparing to the activity of 100 nM 17β-estradiol set to 100%. ERβ selectivity was calculated as the ratio of EC₅₀for ERα/EC₅₀for ERβ.

c

Compound cytotoxicity from parallel experiment to reporter assays determined by measurement of intracellular adenosine 5′-triphosphate (ATP)

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Table 1. continued

d

levels. IC₅₀values are expressed in micromolar. 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

e

f

receptor in a dose response experiment, and K_dvalues were calculated from IC₅₀values using the Cheng−Prusoﬀ equation. n.a., not active. n.c.,

not calculated.

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×).

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 EC₅₀values 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 E_maxfor each compound and

b

comparing to the activity of 100 nM 17β-estradiol set to 100%. ERβ selectivity was calculated as the ratio of EC₅₀for ERα/EC₅₀for ERβ.

c

Compound cytotoxicity from parallel experiment to reporter assays determined by measurement of intracellular adenosine 5′-triphosphate (ATP)

d

levels. IC₅₀values are expressed in micromolar. 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 K_dvalues were calculated from IC₅₀values using the Cheng−Prusoﬀ equation.

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.

Adding a branching methyl at the end of the alkyl chain

(3n−q) 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

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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 E_maxfor each compound and

b

comparing to the activity of 100 nM 17β-estradiol set to 100%. ERβ selectivity was calculated as the ratio of EC₅₀for ERα/EC₅₀for ERβ.

c

Compound cytotoxicity from parallel experiment to reporter assays determined by measurement of intracellular adenosine 5′-triphosphate (ATP)

d

levels. IC₅₀values are expressed in micromolar. 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

e

receptor in a dose response experiment, and K_dvalues were calculated from IC₅₀values using the Cheng−Prusoﬀ equation. n.c., not calculated.

f

n.d., not determined.

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 EC₅₀values 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 E_maxfor each compound and

b

comparing to the activity of 100 nM 17β-estradiol set to 100%. ERβ selectivity was calculated as the ratio of EC₅₀for ERα/EC₅₀for ERβ.

c

Compound cytotoxicity from parallel experiment to reporter assays determined by measurement of intracellular adenosine 5′-triphosphate (ATP)

d

levels. IC₅₀values are expressed in micromolar. 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

e

f

receptor in a dose response experiment, and K_dvalues were calculated from IC₅₀values using the Cheng−Prusoﬀ equation. n.a., not active. n.c.,

not calculated.

3n−q 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

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analogues will be needed to better understand this portion of

the SAR.

(MMTV) harboring response elements for diﬀerent SRs.

Data summarized in Figure 3 and the Supporting Information

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 mean-

ingfully 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.

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.

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.

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.^49,50To 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

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).

(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.

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

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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

b

was detected by HPLC Prominence system (Shimadzu; Kyoto, Japan) with a UV/ﬂuorescence detection. n.a., not active.

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.⁵¹We

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).

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.⁵⁵However, 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 K_ifor full-length recombinant human ERβ

with 14-fold selective ERβ binding in a radio-ligand binding

assay.²⁷Using a similar reporter gene assay system to the

activity data we present, LY500307 was shown to be 30-fold

ERβ selective with an EC₅₀0.66 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.

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.⁵²showing 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^53,54Overall,

our data support limited risk of DDIs with this new class of

ERβ agonists.

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 BE120.⁴³However, 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.³⁷In 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

■

1

Chemicals, Materials, and Methods. H 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

CONCLUSIONS

■

Herein, we describe novel ERβ selective para-carborane

agonists with in vitro isoform selectivity and potency that

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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.⁵⁶Coupling

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 chroma-

tography (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 1,⁴³BE120,⁴³3p,⁵⁷27,⁵⁸and 29⁴³were

synthesized as described previously. Unless speciﬁed otherwise, all

reactions were carried out under argon atmosphere.

(RS)-1-[1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane-

12-yl]ethan-1-ol (2c). To a solution of 1⁴³(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, R_f: 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-dodecabor-

ane (2d). To a solution of 1⁴³(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, R_f: 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 1⁴³(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, R_f: 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-dodecabor-

ane (2f). The method described for the synthesis of 2d was adapted

to synthesize 2f. Starting materials: 1⁴³(500 mg, 2 mmol), 1-

Iodobutane (0.34 mL, 3.0 mmol). Yield: 400 mg (65%) of white solid,

R_f: 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.

Synthesis. 1-(4-Methoxyphenyl)-12-ethyl-1,12-dicarba-closo-

dodecaborane (2b). To a solution of 1⁴³(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, R_f: 0.78 (hexanes/

1

EtOAc, 49/1, v/v), m.p.: 70−80 °C. H 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]butan-1-ol (2g). The method described for the synthesis of 2e

was adapted to synthesize 2g. Starting materials: 1⁴³(500 mg, 2

mmol), 1-butanal (0.27 mL, 3.0 mmol). Yield: 500 mg (78%) of white

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solid, R_f: 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-dodecabor-

ane (2h). The method described for the synthesis of 2d was adapted

to synthesize 2h. Starting materials: 1⁴³(500 mg, 2 mmol), 1-

Iodopentane (0.392 mL, 3.0 mmol). Yield: 520 mg (81%) of white

solid, R_f: 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: 1⁴³(500 mg, 2

mmol), 1-pentanal (0.32 mL, 3 mmol). Yield: 540 mg (80%) of white

solid, R_f: 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-dodecabor-

ane (2j). The method described for the synthesis of 2d was adapted to

synthesize 2j. Starting materials: 1⁴³(500 mg, 2 mmol), 1-

Iodoheptane (0.49 mL, 3.0 mmol). Yield: 550 mg (79%) of white

solid, R_f: 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, C_carborane−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.

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: 1⁴³(0.5 g, 2 mmol),

1-decanal (0.57 mL, 3 mmol). Yield: 680 mg (86%) of a white solid,

R_f: 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: 1⁴³

(0.5 g, 2 mmol), 4-methylpentan-1-al (250 mg, 2.5 mmol). Yield: 580

mg (83%) of a white solid, R_f: 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: 1⁴³

a

(0.5 g, 2 mmol), 5-methylhexan-1-al (370 mg, 3.25 mmol). Yield:

250 mg (34%) of a white solid, R_f: 0.32 (hexanes/EtOAc, 14/1, v/v),

1

m.p.: 64−65 °C. H 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: 1⁴³

b

(1.0 g, 4 mmol), 6-methylheptan-1-al (750 mg, 5.85 mmol). Yield:

1.16 g (77%) of a white solid, R_f: 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-di-

carba-closo-dodecaborane-12-yl]methanol (2r). The method de-

scribed for the synthesis of 2e was adapted to synthesize 2r. Starting

materials: 1⁴³(450 mg, 1.8 mmol), 5-formylindane (100 mg, 0.69

mmol). Yield: 240 mg (34%) of a white solid, R_f: 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: 1⁴³(500 mg, 2

(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: 1⁴³(2.5 g, 10

mmol), 1-heptanal (1.83 mL, 13 mmol). Yield: 3.0 g (82%) of a white

solid, R_f: 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-dodecabor-

ane (2l). The method described for the synthesis of 2d was adapted to

synthesize 2l. Starting materials: 1⁴³(500 mg, 2 mmol), 1-Iododecane

(0.64 mL, 3.0 mmol). Yield: 420 mg (54%) of white solid, R_f: 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, C_carborane−CH₂), 1.85−

3.0 (br. m, 10H, BH), 3.74 (s, 3H, OCH₃), 6.67 (d, 2H, arom., J = 9.0

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mmol), 1-bromo-3-phenylpropane (0.46 mL, 3.0 mmol). Yield: 530

(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, R_f: 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

mg (72%) of white solid, R_f: 0.64 (hexanes/EtOAc, 49/1, v/v), m.p.:

1

93−94 °C. H 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: 1⁴³

(250 mg, 1 mmol), of 3-phenyl-1-propanal (0.17 g, 1.5 mmol). Yield:

344 mg (90%) of a white solid, R_f: 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

99.61%. H 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, R_f: 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,

1-(4-Methoxyphenyl)-12-(4-phenylbutyl)-1,12-dicarba-closo-do-

decaborane (2u). The method described for the synthesis of 2e was

adapted to synthesize 2u. Starting materials: 1⁴³(350 mg, 1.4 mmol),

1-bromo-4-phenylbutane (450 mg, 2.1 mmol). Yield: 490 mg (91%)

of white solid, R_f: 0.57 (hexanes/EtOAc, 49/1, v/v), m.p.: 102−103

1

°C. H 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.

1

retention time: 15.97 min, purity 99.33%. H 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-dodecabor-

ane (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, R_f: 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,

(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: 1⁴³

(500 mg, 2 mmol), of 4-phenyl-1-butanal (0.46 mL, 3 mmol). Yield:

620 mg (78%) of a white solid, R_f: 0.27 (hexanes/EtOAc, 14/1, v/v),

1

m.p.: 123−125 °C. H 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: 1⁴³(440 mg, 1.77

mmol), 1-bromo-5-phenylpentane (600 mg, 2.65 mmol). Yield: 620

mg (88%) of white solid, R_f: 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.

1

wavelength: 280 nm, retention time: 11.87 min, purity 99.29%. H

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,

R_f: 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

(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: 1⁴³

(500 mg, 2 mmol), of 5-phenyl-1-pentanal (0.52 mL, 3 mmol). Yield:

480 mg (58%) of a glasslike compound, R_f: 0.32 (hexanes/EtOAc,

1

14/1, v/v). H 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

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(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.

heptane cooled to −78 °C followed by pentane, cooled to −78 °C.

Yield: 300 mg (65%) of a white solid after column chromatography,

R_f: 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-butyl-1,12-dicarba-closo-dodecabor-

ane (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, R_f: 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,

1-(4-Hydroxyphenyl)-12-heptyl-1,12-dicarba-closo-dodecabor-

ane (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, R_f: 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,

1

wavelength: 280 nm, retention time: 11.733 min, purity 98.83%. H

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, R_f: 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,

1

retention time: 6.860 min, purity 99.52%. H NMR (CDCl₃): δ 0.87

(t, 3H, CH₃), 1.08−1.29 (m, 10H, 5 × CH₂), 1.64 (m, 2H, C_carborane

−

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, R_f: 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

1

wavelength: 254 nm, retention time: 9.87 min, purity 98.10%. H

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

nm, retention time: 9.52 min, purity 99.94%. H 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-pentyl-1,12-dicarba-closo-dodecabor-

ane (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, R_f: 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,

1-(4-Hydroxyphenyl)-12-decyl-1,12-dicarba-closo-dodecabor-

ane (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, R_f: 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,

1

wavelength: 280 nm, retention time: 11.37 min, purity 97.39%. H

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.

1

wavelength: 280 nm, retention time: 10.23 min, purity 100.0%. H

NMR (CDCl₃): δ 0.88 (t, 3H, CH₃), 1.08−1.23 (m, 16H, 8 × CH₂),

1.64 (m, 2H, C_carborane−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-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

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(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,

R_f: 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:

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). ¹³

C

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-di-

carba-closo-dodecaborane-12-yl]methanol (3r). The method de-

scribed 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, R_f: 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,

1

9.41 min, purity 99.73%. H 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, R_f: 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,

1

purity 99.69%. H 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, R_f: 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

1

retention time: 11.07 min, purity 99.60%. H 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, R_f: 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,

1

99.77%. H 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 recrystalliza-

tion 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, R_f: 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

1

retention time: 10.63 min, purity 98.39%. H 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 recrystalliza-

tion 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, R_f: 0.23

(hexanes/EtOAc, 9/1, v/v), m.p.: 120−121 °C. Analytical HPLC:

1

100.00%. H 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-do-

decaborane (3u). The method described for the synthesis of 3b was

adapted to synthesize 3u. Starting materials: 2u (550 mg, 1.44 mmol);

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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, R_f: 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,

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%), R_f: 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, R_f: 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.

1

wavelength: 280 nm, retention time: 12.64 min, purity 99.93%. H

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, R_f: 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,

1

retention time: 12.65 min, purity 99.78%. H 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-do-

decaborane (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, R_f: 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, R_f: 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,

(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, R_f: 0.43 (hexanes/EtOAc, 19/1, v/v), m.p.: 95−96 °C,

20

C

[α]_D

° = −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

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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, R_f: 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,

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 H₂development. 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.

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%), R_f: 0.23 (hexanes/EtOAc, 9/1, v/v), m.p.: 120−121 °C,

20

C

[α]_D

° = −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%. H

1

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, R_f: 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,

4-(4-Methoxyphenyl)cyclohexan-1-one (15). The reaction mix-

ture 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

retention time: 8.89 min, purity 100.00%. H 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-(4-Methoxyphenyl)cyclohexyl)heptan-1-one (16). To a

solution of (methoxymethyl)triphenylphosphonium chloride (3.8 g,

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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).

(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 MgSO₄and 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. H

NMR (300 MHz, DMSO-d₆) δ 1.86−1.91 (m, 6H), 2.17−2.23 (m,

6H), 3.56 (s, 3H).

1

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

zene (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

1

product as a white solid. H 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

1

(S)-4-(4-(1-Hydroxyheptyl)cyclohexyl)phenol (17). Borane−tetra-

hydrofuran 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 H₂development. 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.

405 mg (52% over two steps) of pure product as a white solid. H

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 sol-

ution 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

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

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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

(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,

R_f: 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:

1

135 mg (85%) of pure product as a white waxlike solid. H 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-oxazaboroli-

dine [(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 H₂development. 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).

1

10.27 min, purity 100.00%. H 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.⁵⁹HEK-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 previously⁵⁹

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

(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

1

(RediSep Rf column) to yield a white solid, 98 mg. H 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: 27⁵⁸(0.27 g, 1.3 mmol),

1-heptanal (0.28 mL, 2 mmol). Yield: 280 mg (64%) of a white solid,

R_f: 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:

1

12.09 min, purity 99.61%. H 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: 29⁴³(0.5 g, 2

mmol), 1-heptanal (0.42 mL, 3 mmol). Yield: 580 mg (80%) of a

white solid, R_f: 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.

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384-well plates (Corning Costar) by Multidrop Combi (Thermo

Fisher Scientiﬁc) or Tempest (Formulatrix, Inc.).

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;⁶¹CYP2A6, coumarin 7-

hydroxylation;⁶²CYP2B6, 7-ethoxy-4-(triﬂuoromethyl)coumarin 7-

deethylation;⁶³CYP2C8, paclitaxel 6α-hydroxylation;⁶⁴CYP2C9,

diclofenac 4′-hydroxylation;⁶⁵CYP2C19, mephenytoin 4′-hydroxyla-

tion;⁶⁶CYP2D6, bufuralol 1′-hydroxylation;⁶⁷CYP2E1, chlorzox-

azone 6-hydroxylation,⁶⁸and CYP3A4, midazolam 1′-hydroxyla-

tion.^69,70Incubation mixtures contained 100 mM potassium

phosphate buﬀer (pH 7.4), reduced nicotinamide adenine dinucleo-

tide 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 K_Mdetermined separately as

described previously for each CYP and for its respective substrate.

Activities were measured using HPLC Prominence system (Shimad-

zu; Kyoto, Japan) with a UV/ﬂuorescence detection. Data were

analyzed using Sigma Plot v.12.0 graphing software (Jandel Scientiﬁc,

Chicago, IL).

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 K_dfor the competitor ligand

Fluormone EL Red was determined in a saturation binding

experiment for each receptor independently: K_d(ERα) = 12.4 nM

and K_d(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 K_dvalues for each compound were calculated from

obtained IC₅₀values by Cheng−Prusoﬀ equation.⁶⁰

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00555.

■

sı

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 chromato-

grams (PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

Petr Bartunek − CZ-OPENSCREEN, Institute of Molecular

Genetics of the Czech Academy of Sciences, 142 20 Prague,

Czech Republic; Email: bartunek@img.cas.cz

Christopher C. Coss − Division of Pharmaceutics and

Pharmacology, College of Pharmacy and Drug Development

Institute, Comprehensive Cancer Center, The Ohio State

University, Columbus, Ohio 43210, United States;

orcid.org/0000-0002-8184-9190; Phone: 614-688-1309;

Email: coss.16@osu.edu

Authors

David Sedlák − CZ-OPENSCREEN, Institute of Molecular

Genetics of the Czech Academy of Sciences, 142 20 Prague,

Czech Republic; 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

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Jayaprakash Narayana Kolla − CZ-OPENSCREEN, Institute

of Molecular Genetics of the Czech Academy of Sciences, 142

20 Prague, Czech Republic

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.

́

Zbigniew J. Lesnikowski − Laboratory of Medicinal

Chemistry, Institute of Medical Biology PAS, 93-232 Lodz,

Poland; orcid.org/0000-0003-3158-5796

̌

Alena Spicáková − Department of Pharmacology, Faculty of

Medicine, Palacky University, 77515 Olomouc, Czech

Republic

ABBREVIATIONS

■

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

States; 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

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 trans-

membrane 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

Pavel Anzenbacher − Department of Pharmacology, Faculty

of Medicine, Palacky University, 77515 Olomouc, Czech

Republic

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

ADDITIONAL NOTES

Preparation of 5-Methylhexan-1-al: PCC (3.71 g, 17.21

■

a

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.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00555

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.

b

Preparation of 6-Methylheptan-1-al: PCC (2.89 g, 13.44

ACKNOWLEDGMENTS

■

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.

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

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(70) Kronbach, T.; Mathys, D.; Umeno, M.; Gonzalez, F. J.; Meyer,

U. A. Oxidation of Midazolam and Triazolam by Human Liver

Cytochrome P450IIIA4. Mol. Pharmacol. 1989, 36, 89−96.

9353

https://doi.org/10.1021/acs.jmedchem.1c00555

J. Med. Chem. 2021, 64, 9330−9353

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Structure-Activity Relationship of para-Carborane Selective Estrogen Receptor β Agonists

DOI: 10.1021/acs.jmedchem.1c00555

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Article abstract of DOI:10.1021/acs.jmedchem.1c00555

Full text of DOI:10.1021/acs.jmedchem.1c00555

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