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Novel Non-steroidal Progesterone Receptor Ligands Based on m-Carborane Containing a Secondary Alcohol: Effect of Chirality on Ligand Activity
Shuichi MoriRyohei TakagakiShinya FujiiKo UrushibaraAya TanataniHiroyuki Kagechika
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Volume 65 (2017) Issue 11 Pages 1051-1057

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Abstract

The progesterone receptor (PR) controls various physiological processes, including the female reproductive system, and nonsteroidal PR ligands are considered to be drug candidates for treatment of various diseases without significant adverse effects. Here, we designed and synthesized m-carborane-based secondary alcohols and investigated their PR-ligand activity. All the synthesized alcohols exhibited PR-antagonistic activity at subnanomolar concentration. Among them, alcohols having a small alkyl side chain and a 4-cyanophenyl group also exhibited PR-agonistic activity in a relatively high concentration range. Optical resolution of secondary alcohols having a methyl side chain was performed, and the PR-ligand activity and PR-binding affinity of the purified enantiomers were examined. The chirality of the secondary alcohol appears to have a more significant influence on PR-agonistic activity than on antagonistic activity.

The progesterone receptor (PR) is a member of the nuclear receptor superfamily and plays important roles in many physiological processes, especially in the female reproductive system, via ligand-dependent transcriptional activation of various target genes.1) Progesterone (1, Fig. 1) is an endogenous PR agonist that regulates many physiological processes,25) including uterine cell proliferation/differentiation, the ovulation cycle, and implantation. In addition, 1 modulates the activities of major physiological systems such as the cardiovascular,6) immune,7) and nervous systems.8) Several synthetic PR agonists and antagonists have been developed for clinical use911) in contraception, hormone replacement therapy, treatment of gynecological disorders, and abortion. For example, synthetic PR antagonist mifepristone (2, Fig. 1) is a representative abortifacient.12) It has also been suggested that 2 might be effective in the treatment of endometriosis,13) uterine leiomyoma,14) and breast cancer.15,16) However, all PR ligands currently in clinical use, including 2, are steroid derivatives, and may have significant adverse effects because of their cross-activity toward other steroid hormone receptors.17) In order to avoid these adverse effects, non-steroidal PR ligands are required, and indeed, several non-steroidal ligands have been developed, such as tanaproget (3)18) and a sulfonamide derivative (4)19) (Fig. 1).

Fig. 1. Structures of Typical PR Agonists and Antagonists

Results and Discussion

We have reported non-steroidal PR ligands bearing boron-cluster compounds (carborane) as their hydrophobic core structures.2022) Carboranes, more precisely dicarba-closo-dodecaboranes (C2B10H12), are carbon-containing boron clusters with icosahedral cage structure; there are three isomers depending upon the position of the two carbon atoms, that is, ortho-, meta-, and para-carborane2325) (Fig. 2a). Carboranes have high thermal and chemical stability, and exceptionally high hydrophobicity,2629) and we have applied them as hydrophobic core structures of a variety of bioactive compounds, especially ligands for a number of nuclear receptors.3035) We have reported that p-carborane derivatives rac-5 and 6 (Fig. 2b), bearing a 4-cyanophenyl group on one carbon atom of p-carborane and an alcohol or a ketone group on the other carbon atom, exhibited PR-antagonistic activity at sub-nanomolar concentration.21) Docking simulation of 6 with human PR ligand-binding domain (hPR-LBD) suggested that the cyano group and carbonyl group of 6 interact with hPR-LBD amino acid residues Gln725 and Arg766 (the cyano group), and Cys891 and Thr894 (the carbonyl group), which are known to interact with the 3-carbonyl group and 20-carbonyl group of 1, respectively. The p-carborane moiety of 6 is located at the position corresponding to the CD ring of 1 in the hPR-LBD. Furthermore, using alcohol 5 as a lead compound, we designed and synthesized m-carborane derivative 7a, and conducted optical resolution by means of lipase-catalyzed asymmetric acetylation22) (Fig. 2b). Evaluation of the PR-ligand activity of R- and S-7a revealed that both enantiomers exhibited bidirectional ligand activity, that is, they exhibited PR-agonistic activity at high concentration, while at lower concentration, they inhibited the activity of coexisting progesterone (1). The agonistic activity of S-7a was higher than that of R-7a, though the antagonistic potencies of the two enantiomers were similar.

Fig. 2. Structures of (a) Boron-Cluster Carboranes and (b) Carborane-Based PR Ligands

In this study, we designed and synthesized m-carborane-based PR ligands 7be having a secondary alcohol bearing various alkyl chains, in order to clarify the structural requirements for the bidirectional PR-ligand activity of 7a (Chart 1). Compounds 8 and 9 having a 3-cyano group and 3-trifluoromethyl group on the phenyl group, respectively, were also synthesized to evaluate the importance of a polar substituent on the phenyl group.

Chart 1. Synthesis of m-Carborane Derivatives 79

Synthesis of m-carborane derivatives 79 is illustrated in Chart 1. Ullmann-type coupling of m-carborane with substituted iodobenzenes gave phenyl-m-carborane derivatives 1012. The remaining carbon atom of 1012 was lithiated with lithium diisopropylamide and then reacted with several aldehydes, affording the corresponding secondary alcohols 7be, 8a, and 9a as racemic mixtures.

The PR-agonistic and antagonistic activities of m-carborane derivatives rac-79 were evaluated by means of alkaline phosphatase assay using T-47D human breast cancer cell line, in which alkaline phosphatase expression is regulated by PR.36) The increase ratio of alkaline phosphatase activity in the presence of test compound alone is an index of PR-agonistic activity (Fig. 3a), and the test compound-induced reduction ratio of alkaline phosphatase activity in the presence of 1 nM progesterone (1) is an index of PR-antagonistic activity (Fig. 3b).

Fig. 3. PR-Ligand Activity of Racemic m-Carborane Derivatives 79 Evaluated by T-47D Alkaline Phosphatase (AP) Assay

AP activity induced by (a) test compound alone or (b) test compound in the presence of 1 nM progesterone (1). Mifepristone (2) was examined as a positive control.

In the case of rac-7ae, having a 4-cyanophenyl group, the nature of the PR-ligand activity depended on the length of the alkyl group (R group in Chart 1). Rac-7a (Me), 7b (Et), and 7c (c-Pr) with a short alkyl group exhibited PR-agonistic activity. However, the agonistic potency decreased as the alkyl group became longer, and rac-7e having an n-Bu group showed no agonistic activity below 10 µM. In addition, compounds 7ae all inhibited the activity of progesterone (1) (Fig. 3b). Among them, rac-7b and 7c, as well as 7a, having a short alkyl group, showed concentration-dependent bidirectional PR-ligand activity. In other word, they exhibited PR-agonistic activity at relatively high concentrations, while at low concentrations in the presence of progesterone (1) they showed PR-antagonistic activity. On the other hand, rac-7d and 7e having a longer alkyl group exhibited only PR-antagonistic activity in dose-dependent manner, resembling typical PR antagonists such as the p-carborane derivatives 5 and 6.

Compounds rac-8a and 9a having a 3-cyanophenyl or 3-trifluoromethylphenyl group exhibited only antagonistic activity, although they have same 1-hydroxyethyl group as compound rac-7a.

Previously, we have employed lipase-catalyzed asymmetric acetylation of carborane-containing secondary alcohols to separate the enantiomers of 1-carboranylethanols, such as 7a, in high enantiomeric purity.22) However, separation of the enantiomers of carboranyl secondary alcohols with longer alkyl groups was unsuccessful. Based on these results, we tried to separate the enantiomers of 8a and 9a by means of lipase-catalyzed asymmetric acetylation. We obtained unreacted alcohols and acetylated products 13a and 14a with complete enantiomeric excess (ees, eep >99%) (Chart 2). Absolute configurations of unreacted alcohols and the acetylated products were determined as S and R, respectively, based on our previous finding of the lipase selectivity for 1-carboranylethanols.22) After isolation of unreacted S-alcohols and R-acetylated products by silica gel column chromatography, the R-acetylated products were hydrolyzed with K2CO3 aq. to obtain the R-alcohols.

Chart 2. Optical Resolution of Carborane-Containing Secondary Alcohols 8a, and 9a by Means of Lipase-Catalyzed Asymmetric Acetylation

The PR-ligand activity of the purified R- and S-enantiomers of 7a, 8a and 9a was examined by alkaline phosphatase assay, and their EC50 and IC50 values are shown in Table 1. As previously reported,22) both enantiomers of 7a exhibited similar behavior to the racemic mixture. Namely, they exhibited agonistic activity in the high concentration range, and antagonistic activity toward coexisting progesterone (1) in the low concentration range. Both enantiomers of 7a and rac-7a acted as PR partial agonists, whose maximum response was about 60% of that of progesterone (1). S-7a exhibited more potent agonistic activity than R-7a. On the other hand, the chirality of 7a had little influence on the antagonistic activity. Neither of the enantiomers of 8a and 9a exhibited PR-agonistic activity, and both exhibited only antagonistic activity, like the racemic compounds. In contrast, the chirality of 8a and 9a affected the potency of their antagonistic activity. In both cases, the S-enantiomer showed higher PR-antagonistic activity than the R-enantiomer. The position of the polar substituent on the phenyl group might affect the interaction mode with PR, which in turn might alter the influence of the chirality of the alcohol part on the antagonistic activity.

Table 1. Agonistic and Antagonistic Activities of the Enantiomers of 7a, 8a, and 9a toward PR Evaluated by Alkaline Phosphatase Assay Using T-47D Cells
Compoundee (%)EC50 (µM)a)IC50 (µM)b)
rac-7ac)00.790.037
R-7ac)981.050.032
S-7ac)>990.570.039
rac-8a00.26
R-8a>990.32
S-8a>990.18
rac-9a00.61
R-9a980.76
S-9a>990.49
Progesterone0.0015

a) Half-maximum effective concentration of compound. The maximum response of compound 7a was 60% of that induced by progesterone (1). b) Half-maximum inhibitory concentration of test compound toward the activity of coexisting 2 nM progesterone (1). c) See ref. 22.

Next, we selected the potent bidirectional derivatives R-7a and S-7a, as well as rac-7e with moderate antagonistic activity, and examined their PR-binding affinity (Fig. 4). The binding affinities were evaluated by means of competitive binding assay using hPR-LBD and [1,2,6,7-3H]progesterone. All three compounds bound to the hPR-LBD, which suggested that the hPR-ligand activity of the carborane derivatives evaluated by T-47D alkaline phosphatase assay was mediated by PR. R-7a and S-7a, which showed potent antagonistic activity and agonistic activity, bound to hPR-LBD more strongly than rac-7e, which showed moderate antagonistic activity. Both enantiomers of 7a exhibited similar binding potency, in accordance with their similar antagonistic potency. Thus, the binding affinity of the two enantiomers of 7a well reflected their antagonistic activity, whereas the difference of agonistic activity between the two enantiomers of 7a could not be explained by a difference in their binding affinity to hPR-LBD. Generally, agonistic activities toward nuclear receptors, including PR, are thought to involve an appropriate ligand-induced conformational change of the LBD to enable proper interactions of the LBD with transcriptional co-activators.37) Therefore, the chirality of the alcohol part in 7a might influence the conformation of hPR-LBD, resulting in the difference of agonistic activity between the two enantiomers, even though their binding affinities are similar. On the other hand, antagonistic activity toward nuclear receptors is thought to require only the ability to bind to the LBD, and thus to disturb induction of the proper conformation.38,39) This may be the reason why the chirality of 7a has a greater effect on the agonistic activity than on the antagonistic activity.

Fig. 4. Competitive Binding Assay of the Synthesized Alcohols

The concentration of [3H]PG was 4 nM.

In conclusion, we synthesized a series of m-carborane derivatives 79, and evaluated their activities as PR ligands. All of them exhibited PR-antagonistic activity in the subnanomolar concentration range. Among them, compounds having a short alkyl chain and a 4-cyanophenyl group also exhibited PR-agonistic activity in a relatively high concentration range. Separation of enantiomers and evaluation of their PR ligand activities and PR-binding affinity suggested that the chirality of the ligand pharmacophore could be more significant for agonistic activity than for antagonistic activity. This information could be helpful in designing ligand structures, not only for PR, but also for other nuclear receptors.

Experimental

General

All reagents were purchased from Sigma-Aldrich (U.S.A.), Tokyo Kasei (Japan), Wako Pure Chemical Industries, Ltd. (Japan), and Kanto Chemicals (Japan) and were used without further purification. NMR spectra were recorded on BRUCKER AVANCE 400 or BRUCKER AVANCE 500 spectrometers. Chemical shifts for NMR are reported as parts of per million (ppm) relative to chloroform (7.27 ppm for 1H-NMR and 77.23 ppm for 13C-NMR) and DMSO-d6 (2.50 ppm for 1H-NMR and 39.51 ppm for 13C-NMR). Mass spectra were collected on a Bruker Daltonics microTOF-2focus spectrometer in the positive ion modes. Melting points were obtained on a Yanagimoto micro melting point apparatus without correction. Specific rotations were obtained on a JASCO P-2200 polarimeter. HPLC data were recorded with a Hitachi D-2000 Elite type HPLC system manager. Chiral column IC (4.6 mmϕ×250 mm) was purchased from Daicel Chemical Industries (Japan).

General Procedure for Synthesis of Phenyl-m-carborane Derivatives 10–12

A solution of n-butyllithium in n-hexane (1.6 M, 7.4 mL, 11.9 mmol) was added to a solution of m-carborane (1.50 g, 10.8 mmol) in l,2-dimethoxyethane (DME) (12 mL) at 0°C under Ar atmosphere, and the mixture was stirred at 0°C for 30 min. Copper(I) chloride (10 mg, 104 mmol) was added to the reaction mixture at room temperature. After 1 h, pyridine (3.8 mL) and substituted iodobenzene (11.9 mmol) were added, and the mixture was stirred at 80°C for 2.5 h. After cooling, the reaction mixture was diluted with ether, and insoluble materials were filtered off through celite. The filtrate was washed successively with 5% sodium thiosulfate, 2 M hydrochloric acid, and brine, dried over sodium sulfate, and evaporated. The residue was purified by silica gel column chromatography (eluent: n-hexane–ethyl acetate, 20 : 1) to give 1012.

1-(4-Cyanophenyl)-1,7-dicarba-closo-dodecaborane (10)

Fifty-nine percent yield; Colorless solid; mp 49–51°C; 1H-NMR (400 MHz, CDCl3) δ: 7.58–7.53 (m, 4H), 3.5–1.7 (br, 10H), 3.12 (br s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ: 138.9, 132.8, 128.7, 117.9, 112.2, 76.9, 56.9; High-resolution (HR)-MS Calcd for C9H15B10NNa [M+Na]+: 270.2033. Found 270.2035.

1-(3-Cyanophenyl)-1,7-dicarba-closo-dodecaborane (11)

Sixty-six percent yield; Colorless solid; mp 114–115°C; 1H-NMR (400 MHz, CDCl3) δ: 7.71 (t, J=1.6 Hz, 1H), 7.64 (ddd, J=8.2, 1.2, 0.8 Hz, 1H), 7.58 (dt, J=7.8, 1.4 Hz, 1H), 7.37 (t, J=8.0 Hz, 1H), 3.5–1.7 (br, 10H), 3.11 (br s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ: 135.7, 133.1, 132.5, 130.9 130.2, 117.9, 112.1, 76.6, 56.9; HR-MS Calcd for C9H15B10NNa [M+Na]+: 270.2033. Found 270.2040.

1-(3-Trifluoromethylphenyl)-1,7-dicarba-closo-dodecaborane (12)

Forty percent yield; mp 51–52°C; Colorless solid; 1H-NMR (500 MHz, DMSO-d6) δ: 7.79 (m, 2H), 7.63–7.60 (m, 2H), 4.35 (br s, 1H), 3.6–1.6 (br, 10H); 13C-NMR (125 MHz, DMSO-d6) δ: 135.5, 131.9, 130.4, 129.5 (q, J=32.5 Hz), 126.1 (q, J=3.8 Hz), 123.6 (q, J=270.0 Hz), 123.5 (q, J=3.8 Hz), 76.9, 56.9; HR-MS Calcd for C9H14B10F3 [M−H]: 289.1978. Found 289.1978.

General Procedure for Preparation of Secondary Alcohols 7–9

A solution of lithium diisopropylamide in a mixture of n-hexane and tetrahydrofuran (1.1 M, 0.56 mL, 0.62 mmol) was added to a solution of 1012 (0.52 mmol) in tetrahydrofuran (THF) (10 mL) at −78°C under Ar atmosphere, and the mixture was stirred at −78°C for 10 min. Corresponding aldehyde (2.0–10 eq) was added to the reaction mixture at −78°C. The mixture was stirred at −78°C for 30 min, then poured into saturated ammonium chloride, and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over sodium sulfate, and evaporated. The residue was purified by silica gel column chromatography (eluent: n-hexane–ethyl acetate, 8 : 1) to give the racemic alcohols 79.

rac-1-(4-Cyanophenyl)-7-(1-hydroxy-n-propyl)-1,7-dicarba-closo-dodecaborane (rac-7b)

Eighty percent yield; Colorless solid; mp 89–91°C; 1H-NMR (500 MHz, CDCl3) δ: 7.62–7.51 (m, 4H), 3.73 (m, 1H), 3.7–1.8 (br, 10H), 1.88 (d, J=6.5 Hz, 1H), 1.70 (m, 1H), 1.39 (m, 1H), 1.01 (t, J=7.5 Hz, 3H); 13C-NMR (125 MHz, CDCl3) δ: 140.1, 123.3, 128.9, 118.1, 113.0, 82.9, 75.8, 74.3, 30.6, 11.3; HR-MS Calcd for C12H20B10NO [M−H]: 304.2475. Found 304.2472.

rac-1-(4-Cyanophenyl)-7-(cyclopropylhydroxymethyl)-1,7-dicarba-closo-dodecaborane (rac-7c)

Ninety percent yield; Colorless solid; mp 117–119°C; 1H-NMR (500 MHz, CDCl3) δ: 7.57 (s, 4H), 3.8–1.8 (br, 10H), 3.19 (dd, J=8.0, 4.5 Hz, 1H), 2.01 (d, J=4.5 Hz, 1H), 1.00 (m, 1H), 0.69 (m, 1H), 0.63 (m, 1H), 0.41 (m, 2H); 13C-NMR (125 MHz, CDCl3) δ: 140.2, 132.3, 129.0, 118.2, 113.0, 82.1, 76.9, 75.8, 18.6, 5.2, 3.6; HR-MS Calcd for C13H20B10NO [M−H]: 316.2475. Found 316.2479.

rac-1-(4-Cyanophenyl)-7-(1-hydroxy-n-butyl)-1,7-dicarba-closo-dodecaborane (rac-7d)

Ninety-five percent yield; Colorless solid; mp 88–90°C; 1H-NMR (500 MHz, CDCl3) δ: 7.55 (s, 4H), 3.82 (d, J=11.0 Hz, 1H), 3.7–1.8 (br, 10H), 2.03 (br s, 1H), 1.58 (m, 2H), 1.44–1.29 (m, 2H), 0.93 (t, J=7.0 Hz, 3H); 13C-NMR (125 MHz, CDCl3) δ: 140.2, 132.3, 128.9, 118.1, 113.0, 83.2, 75.8, 72.7, 39.6, 19.9, 13.8; HR-MS Calcd for C13H22B10NO [M−H]: 318.2632. Found 318.2654.

rac-1-(4-Cyanophenyl)-7-(1-hydroxy-n-pentyl)-1,7-dicarba-closo-dodecaborane (rac-7e)

Eighty-two percent yield; Colorless oil; 1H-NMR (500 MHz, CDCl3) δ: 7.58–7.54 (m, 4H), 3.81 (dd, J=10.5, 2.0 Hz, 1H), 3.7–1.7 (br, 10H), 2.04 (br s, 1H), 1.63 (m, 1H), 1.51 (m, 1H), 1.44–1.26 (m, 4H), 0.91 (t, J=7.3 Hz, 3H); 13C-NMR (125 MHz, CDCl3) δ: 140.2, 132.3, 128.9, 118.1, 113.0, 83.2, 75.9, 72.9, 37.3, 28.8, 22.4, 14.1; HR-MS Calcd for C14H24B10NO [M−H]: 332.2788. Found 318.22780.

rac-1-(3-Cyanophenyl)-7-(1-hydroxyethyl)-1,7-dicarba-closo-dodecaborane (rac-8a)

Fifty-four percent yield; Colorless solid; mp 64–65°C; 1H-NMR (400 MHz, CDCl3) δ: 7.72 (t, J=1.6 Hz, 1H), 7.67 (ddd, J=8.0, 2.0, 1.2 Hz, 1H), 7.60 (dt, J=7.6, 1.4 Hz, 1H), 7.40 (t, J=7.8 Hz, 1H), 4.09 (br s, 1H), 3.9–1.8 (br, 10H), 1.33 (d, J=6.4 Hz, 3H); 13C-NMR (125 MHz, CDCl3) δ: 136.9, 132.4, 132.4, 131.6, 129.6, 118.2, 113.1, 82.9, 75.7, 69.4, 23.9; HR-MS Calcd for C11H18B10NO [M−H]: 290.2327. Found 290.2326.

rac-1-(3-Trifluorophenyl)-7-(1-hydroxyethyl)-1,7-dicarba-closo-dodecaborane (rac-9a)

Eighty percent yield; Colorless solid; mp 57–58°C; 1H-NMR (500 MHz, CDCl3) δ: 7.68 (br s, 1H), 7.63 (br d, J=8.0, 1H), 7.57 (br d, J=7.8 Hz, 1H), 7.41 (t, J=7.9 Hz, 1H), 4.10 (quit. J=6.4 Hz, 1H), 3.8–1.7 (br, 10H), 1.93 (d, J=6.1 Hz, 1H), 1.34 (d, J=6.4 Hz, 3H); 13C-NMR (125 MHz, CDCl3) δ: 136.4, 131.4, 131.2 (q, J=32.8 Hz), 129.2, 125.8 (q, J=3.6 Hz), 124.8 (q, J=4.0 Hz), 123.8 (q, J=272.6 Hz), 83.0, 76.3, 69.4, 23.9; HR-MS Calcd for C11H18B10F3O [M−H]: 333.2240. Found 333.2248.

General Procedure of the Optical Resolution of Alcohols by Lipase TL-Catalyzed Asymmetric Acetylation

Lipase TL (360 mg) was added to a solution of vinyl acetate (4.8 mmol) and racemic alcohol (0.48 mmol) in diisopropyl ether (5 mL) and the mixture was stirred at 50°C for 3 h. After removal of the lipase by filtration, the filtrate was concentrated. The residue was purified by silica gel column chromatography (eluent: n-hexane–ethyl acetate, 3 : 1) to give S-alcohol as colorless solid and R-acetylated product as colorless liquid. Then, R-acetylated product was dissolved in 0.5 M potassium carbonate in a mixture of methanol and water (4 : 1), and the mixture was stirred for 40 min at room temperature. The reaction mixture was extracted with ethyl acetate, and the organic layer was dried over sodium sulfate, and then concentrated. The residue was purified by silica gel column chromatography (eluent: n-hexane–ethyl acetate, 4 : 1) to give R-alcohol as colorless solid.

S-1-(3-Cyanophenyl)-7-(1-hydroxyethyl)-1,7-dicarba-closo-dodecaborane (S-8a)

Fifty percent from rac-8a; >99% ee; Colorless solid; mp 61–62°C; [α]D25 −9.7 (c=1.0, CHCl3); HPLC: CHIRALPAK IC (25 cm), flow rate 1 mL/min, n-hexane–2-propanol=9 : 1, detection 220 nm, 14.0 min.

R-1-(3-Cyanophenyl)-7-(1-acetoxyethyl)-1,7-dicarba-closo-dodecaborane (R-13a)

Forty-seven percent from rac-8a; >99% ee; Colorless oil; 1H-NMR (500 MHz, CDCl3) δ: 7.71 (t, J=1.7 Hz, 1H), 7.66 (ddd, J=8.2, 2.2, 1.1 Hz, 1H), 7.61 (dt, J=7.7, 1.2 Hz, 1H), 7.40 (t, J=8.1 Hz, 1H), 5.27 (q, J=6.6 Hz, 1H), 3.6–1.6 (br, 10H), 2.10 (s, 3H), 1.31 (d, J=6.6 Hz, 3H); 13C-NMR (125 MHz, CDCl3) δ: 169.4, 136.8, 132.5, 132.4, 131.6, 129.6, 118.2, 113.1, 79.1, 75.5, 69.2, 21.4, 21.1; CHIRALPAK IC (25 cm), flow rate 1 mL/min, n-hexane–2-propanol=9 : 1, detection 220 nm, 10.1 min.

R-1-(3-Cyanophenyl)-7-(1-hydroxyethyl)-1,7-dicarba-closo-dodecaborane (R-8a)

Eighty-eight percent from R-13a; >99% ee; Colorless solid; mp 62–63°C; [α]D25 +10.0 (c=1.0, CHCl3); HPLC: CHIRALPAK IC (25 cm), flow rate 1 mL/min, n-hexane–2-propanol=9 : 1, detection 220 nm, 13.0 min.

S-1-(3-Trifluorophenyl)-7-(1-hydroxyethyl)-1,7-dicarba-closo-dodecaborane (S-9a)

Forty-nine percent from rac-9a; >99% ee; Colorless solid; mp 35–36°C; [α]D25 −8.0 (c=1.0, CHCl3); HPLC: CHIRALPAK IC (25 cm), flow rate 1 mL/min, n-hexane–2-propanol=9 : 1, detection 220 nm, 14.7 min.

R-1-(3-Trifluorophenyl)-7-(1-acetoxyethyl)-1,7-dicarba-closo-dodecaborane (R-14a)

Forty-seven percent from rac-9a; >99% ee; Colorless oil; 1H-NMR (500 MHz, CDCl3) δ: 7.66 (br s, 1H), 7.62 (br d, J=7.9 Hz, 1H), 7.58 (br d, J=7.8 Hz, 1H), 7.40 (t, J=7.9 Hz, 1H), 5.28 (q, J=6.6 Hz, 1H), 3.6–1.6 (br, 10H), 2.09 (s, 3H), 1.32 (d, J=6.6 Hz, 3H); 13C-NMR (125 MHz, CDCl3) δ: 136.2, 131.4, 131.2 (q, J=39.5 Hz), 129.3, 125.9 (q, J=3.8 Hz), 125.4 (q, J=271.3 Hz), 124.8 (q, J=3.8 Hz), 79.0, 76.3, 69.3, 21.4, 21.1; HPLC: CHIRALPAK IC (25 cm), flow rate 1 mL/min, n-hexane–2-propanol=9 : 1, detection 220 nm, 4.6 min.

R-1-(3-Trifluorophenyl)-7-(1-hydroxyethyl)-1,7-dicarba-closo-dodecaborane (R-9a)

Ninety percent from R-14a; 98% ee; Colorless solid; mp 35–36°C; [α]D25 +8.2 (c=1.0, CHCl3); HPLC: CHIRALPAK IC (25 cm), flow rate 1 mL/min, n-hexane–2-propanol=9 : 1, detection 220 nm, 13.6 min.

Alkaline Phosphatase Assay Using T-47D Cells

T-47D breast-carcinoma cells were cultured in RPMI 1640 medium with 10% (v/v) fetal bovine serum. Cells were plated in 96-well plates at 104 cell/well and incubated overnight (37°C, 5% CO2 in air). The next day, cells were treated with fresh medium containing test compound, and further incubated for 24 h. The medium was aspirated and the cells were fixed with 100 µL of 1.8% formalin (in phosphate buffered saline (PBS)). The fixed cells were washed with PBS and 75 µL of assay buffer (1 mg/mL p-nitrophenol phosphate in diethanolamine water solution, pH 9.0, 2 mM MgCl2) was added. The mixture was incubated at room temperature with shielding from light for 2 h, and then the reaction was terminated by the addition of 100 µL of NaOH. The absorbance at 405 nm was measured.

hPR-Binding Assay

hPR-binding assay was performed using recombinant hPR-LBD purchased from Invitrogen (A15672). hPR-LBD was diluted with buffer (20 mM Tris–HCl, 300 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM dithiothreitol (DTT), pH 8.0) to 14 µg/mL of total protein and 300 µL aliquots were incubated in the dark at 4°C with 4 nM [1,2,6,7-3H]progesterone (PerkinElmer, Inc., U.S.A.) and reference or test compounds (dissolved in dimethyl sulfoxide (DMSO); final concentration of DMSO was 3%). Nonspecific binding was assessed by addition of a 200-fold excess of nonradioactive progesterone. After 24 h, 30 µL of Dextran T-70/c-globulin-coated charcoal suspension was added to the ligand/protein mixtures (1% activated charcoal, 0.05% γ-globulin, 0.05% Dextran 70, final concentrations) and incubated at 4°C for 5 min. The charcoal was removed by centrifugation for 5 min at 1300×g, and the radioactivity of the supernatant was measured in Ultima Gold scintillation cocktail (PerkinElmer, Inc.) by using a liquid scintillation counter. All experiments were performed in duplicate.

Acknowledgments

This work was partly supported by Sasagawa Scientific Research Grant from the Japan Science Society, and the Japan Society for the Promotion of Science (JSPS) Core-to-Core Program, A. Advanced Research Networks, and the Platform Project for Supporting Drug Discovery and Life Science Research funded by Japan Agency for Medical Research and Development (AMED).

Conflict of Interest

The authors declare no conflict of interest.

References
 
© 2017 The Pharmaceutical Society of Japan
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