Design and synthesis of novel ROR inverse agonists with a dibenzosilole scaffold as a hydrophobic core structure

Accepted Manuscript Design and synthesis of novel ROR inverse agonists with a dibenzosilole scaffold as a hydrophobic core structure Hirozumi Toyama, Masaharu Nakamura, Yuichi Hashimoto, Shinya Fujii PII: DOI: Reference:

S0968-0896(15)00402-2 http://dx.doi.org/10.1016/j.bmc.2015.05.004 BMC 12298

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

15 April 2015 2 May 2015 5 May 2015

Please cite this article as: Toyama, H., Nakamura, M., Hashimoto, Y., Fujii, S., Design and synthesis of novel ROR inverse agonists with a dibenzosilole scaffold as a hydrophobic core structure, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.05.004

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Design and synthesis of novel ROR inverse agonists with a dibenzosilole scaffold as a hydrophobic core structure Hirozumi Toyama, Masaharu Nakamura, Yuichi Hashimoto, Shinya Fujii*

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-0032 Tokyo, Japan

To whom correspondence should be addressed: E-mail: [email protected] Tel.: +81-3-5841-7848; fax: +81-3-5841-8495

Abstract Molecular structure calculations indicated that the dibenzosilole skeleton could be well superposed on phenanthridinone, which is a structural component of ligands of retinoic acid receptor-related orphan receptors (RORs). Therefore, we designed, synthesized and biologically evaluated a series of novel ROR ligands based on the dibenzosilole scaffold as a hydrophobic core structure. Dibenzosilole derivatives bearing a hexafluoro-2-hydroxypropyl group on the benzene ring exhibited significant ROR-inhibitory activity, comparable to that of the lead phenanthridinone derivative 5. Our results indicate that the dibenzosilole skeleton would be a useful scaffold for developing novel biologically active compounds, and that cis-amide structure can be replaced by an alkylsilyl functionality.

Keywords: retinoic acid receptor-related orphan receptor, ROR, dibenzosilole, sila-substitution

1. Introduction Various silicon-containing bioactive compounds have been developed and some of them, such as alkylsilyl derivatives 1 and 2, have been evaluated in clinical trials (Figure 1).1-4 The design rationale has been that introduction of a silicon atom in place of a carbon atom (sila-substitution) can alter the activity, selectivity and pharmacokinetics of bioactive compounds, owing to the differences of covalent radius, hydrophobicity and electronegativity.5 We previously hypothesized that a silyl group can function as a cis-olefin mimetic, and we designed silyl analogs of combretastatin A-4 (CA-4), such as compound 3 (Figure 1), by replacement of the unstable cis-olefin moiety with a silyl group.6 The resulting compounds were potent inhibitors of tubulin

polymerization and tumor cell proliferation. These results suggested that silyl groups can serve as mimics of two-atom substructure such as cis-olefinic double bond.

Figure 1. Structures of silicon-containing compounds used in clinical trials (1, 2) and our silyl analog of CA-4 (3).

Retinoic acid receptor-related orphan receptor (ROR) is a member of the nuclear receptor superfamily of ligand-dependent factors that regulate DNA transcription; 7 its three subtypes, α, β and γ, are differentiated by alternative promoter usage and alternative splicing. ROR is classified as an orphan receptor, since its endogenous ligands have not been fully identified, and it is thought to be involved in immunity, cellular metabolism and circadian rhythm.8 ROR is constitutively active in the absence of any activating ligand, and increased transcriptional activation of ROR (especially RORα and RORγ) is thought to be involved in autoimmune diseases.9 Therefore, ROR inverse agonists, which inhibit the constitutive activity of ROR, would be candidate drugs for treatment of these diseases. Various ROR inverse agonists have been developed, among which sulfonamide derivative T0901317, which was originally developed as an LXR agonist, is a representative ROR inverse agonist with RORα/γ selectivity (Figure 2A).10

A.

F 3C

O O N S

O

n-Bu

O

N Si

F3C HO CF 3 T0901317

N H

Si R R = Et (4) R = C(CF3) 2OH (5)

6

B.

Figure 2. (A) Chemical structures of T0901317 and our phenanthridinone (4,5) and benzanilide (6) derivatives (B) Binding model of the phenanthridinone derivative 5 at the RORγ LBD

We have also developed ROR inverse agonists such as phenanthridinone derivatives 4 and 5,11 which were derived from T0901317, and silicon-containing benzanilide derivative 6 (Figure 2A).12 In the present work, we further investigated the use of silyl functionalities for structural development of bioactive compounds by designing and synthesizing novel ROR inverse agonists based on the dibenzosilole skeleton, focusing on the replacement of the cis-amide structure of phenanthridinone with an alkylsilyl moiety.

2. Results and discussion 2.1. Molecular design The cocrystal X-ray structure of RORγ ligand-binding domain (LBD) and T0901317 was recently reported.13 Based on this structure, we performed a computational docking study of the phenanthridinone derivative 5 with RORγ LBD. The docking study suggested the amide structure of compound 5 does not form a hydrogen bond to the RORγ LBD, and the ligand-binding site of RORγ is quite hydrophobic (Figure 2B). Therefore, replacement of the amide moiety with a hydrophobic substructure could be a promising strategy for structural development. As mentioned above, we have recently reported that diphenylsilane structure can function as a cis-stilbene

surrogate.6 Therefore, the similar geometry of cis-amide and cis-olefin led us to hypothesize that cis-amide bond in phenanthridinone derivatives could be replaced by a silyl functionality. Figure 3A shows a superimposition of the calculated structures of phenanthridinone and dimethyldibenzosilole, which has a dimethylsilyl group instead of the cis-amide moiety of phenanthridinone. The overall structures, especially the two benzene rings of these compounds, appear to be well superimposed. A.

Side view

Top view

B.

1

R1 R2 Si

2

R , R = Me, Et, n-Bu 3

R = H, Et, C(CF ) OH 3 2

R3

Figure 3. (A) Superposition of phenanthridinone (cyan) and dimethyl dibenzosilole structures (magenta) calculated with MM2 program (B) Structural development of the dibenzosilole skeleton

Therefore, we focused on dibenzosilole derivatives as candidate ROR inverse agonists. Based on our structure-activity relationship (SAR) study on phenanthridinone derivatives,11 we introduced an ethyl group and a hexafluoro-2-hydroxypropyl group on the benzene ring of dibenzosilole, and conducted an SAR study to examine the effect of substituents on the silicon atom (Figure 3B).

2.2. Synthesis

Scheme 1. Synthesis of dibenzosilole derivatives 14-17. Reagents and conditions: (a) SnCl2·2H2O, EtOH/H2O, reflux, 5 h; (b) Ac2O, Na2CO3, H2O, 0°C to rt, 1 h, 42% (2 steps); (c) NBS, DMF, rt, o/n, 74%; (d) HCl, EtOH, reflux, 6 h; (e) i) HCl/NaNO2 , H2O, 0°C, 30 min; ii) KI, H2O, rt, o/n, 34% (2 steps); (f) i) n-BuLi, THF, -78 °C, 30 min; ii) Si(n-Bu)2Cl2, -78°C to rt, o/n, 92%; (g) n-BuLi, THF, -78°C, 30 min; acetaldehyde, THF, -78°C to rt, o/n; (h) H2, Pd/C, MeOH, rt, o/n.

Dibenzosilole derivatives 14-17 were synthesized as shown in Scheme 1. Reduction of 2,2'-dinitrobiphenyl (7) using SnCl2 ･ 2H2O afforded 2,2'-diaminobiphenyl (8). Acetylation of compound 8 with acetic anhydride gave amide 9, and then bromination with NBS gave dibrominated compound 10. Deacetylation of 10 under acidic conditions afforded intermediate diamine 11, which was iodinated by means of Sandmeyer reaction to give diodobiphenyl 12. The 2and 2’-positions of 12 were lithiated with n-butyllithium, and reaction between the lithiated form of 12 and dibutyldichlorosilane gave dibenzosilole 13. The lithiated form of 13 reacted with acetaldehyde to give alcohol 14. The hydroxyl group of 14 was removed to afford 16 by

hydrogenation using Pd/C. Compounds 15 and 17 were isolated as by-products in the preparation of compound 14 and 16, respectively (Scheme 1). .

Scheme 2. Synthesis of dibenzosilole derivatives 24-26, 29. Reagents and conditions: (a) Sc CF3COCF3･1.5H2O, p-TsOH, MS4A, toluene, 100 °C, 3 h, 61%; (b) i) HCl/NaNO2 , H2O, 0°C, 30 min; ii) KI, EtOH/H2O, rt, o/n, 24%; (c) BnBr, NaH, DMF, reflux, o/n, 79%; (d) i) n-BuLi, THF, -78°C, 30 min; ii) SiR2Cl2 (R = Me, Et, n-Bu), THF, -78°C to rt, o/n, 24-68%; (e) H2, Pd/C, MeOH, rt, o/n, 17-71%; (f) i) t-BuLi, THF, -78°C, 20 min → rt, 15 min; ii) Si(n-Bu)Cl3 , THF, -78°C to rt, o/n; (g) MeMgBr, THF, -78°C to rt, 3 h, 32%; (h) H2 , Pd/C, AcOH, MeOH, rt, o/n, 90%

The dibenzosilole derivatives 24-26, and 29 were synthesized as shown in Scheme 2. Reaction of diamine 8 with hexafluoroacetone gave alcohol 18, and then iodination of 18 by means of Sandmeyer reaction gave diiodobiphenyl 19. The hydroxyl group of 19 was protected with a benzyl group to give 20. The lithiated form of 20 was treated with dialkyldichlorosilanes to give the corresponding dibenzosiloles 21, 22 and 23. Removal of the benzyl group of 21, 22 and 23 by hydrogenation using Pd/C afforded the desired compounds 24, 25 and 26, respectively. Lithiation of 20 with t-butyllithium and reaction with butyltrichlorosilane gave a chlorosilane intermediate 27, and then reaction with methylmagnesium bromide gave compound 28. Finally, the benzyl group of

28 was removed by hydrogenation using Pd/C to give 29 (Scheme 2).

2.3. Biological evaluation Table 1. Inhibition of ROR transcriptional activity by dibenzosiloles 14-17. Values are inhibition ratio (percent of control) at the concentration of 10 µM.

compound

R

RORα

RORβ

RORγ

4

-

37

29

37

14

CH(OH)CH3

17

< 5.0

< 5.0

15

H

22

< 5.0

13

16

Et

7.0

< 5.0

18

17

COCH3

13

< 5.0

< 5.0

Inverse agonistic potency of the synthesized compounds was evaluated by assay of inhibitory activity toward constitutive activity of ROR, using luciferase reporter gene assay. Table 1 shows the ROR-transcriptional repression activity of compounds 14-17. Compound 14 bearing a 1-hydroxyethyl group exhibited 17% inhibition of RORα. The simple dibutyldibenzosilole 15 exhibited 22% inhibition of RORα. Compound 16 bearing an ethyl group showed 18% inhibition of RORγ, and acetyl derivative 17 showed 13% inhibition of RORα. However, the potencies of these compounds were fairly low in comparison with that of the lead phenanthridinone derivative 4 (Table 1).

Table 2. Inhibition of ROR-transcriptional activity by dibenzosiloles 24-26 and 29.

2

R

R

RORα IC50 (µM)

RORβ IC50 (µM)

RORγ IC50 (µM)

5

-

-

7.2

5.7

4.7

6.2

6.7

5.5

24

a

1

compound

n-Bu n-Bu

25

Me

Me

9.4

(40%)a

5.4

26

Et

Et

(34%)a

(28%)a

4.2

29

n-Bu

Me

7.7

7.0

4.4

Inhibition ratio at the concentration of 10 µM.

Table 2 shows the ROR-transcriptional repression activity of compound 24-26 and 29 bearing a hexafluoro-2-hydroxypropyl moiety on the benzene ring. In contrast to compounds 14-17, these hexafluoro-2-hydroxypropyl derivatives exhibited significant ROR inverse agonist activity. In particular, they showed potent RORγ-inhibitory activity comparable to that of the lead phenanthridinone 5. Compound 24 and compound 29 also exhibited potent RORα/β-transcriptional repression activity. This result suggests that the n-butyl substituent is preferable to a short alkyl group, such as methyl or ethyl group, for RORα/β inverse agonist activity. As for RORγ, Si,Si-diethyl derivative 26 exhibited the most potent inverse agonistic activity. Overall, dibenzosilole structure appears to function as an effective alternative structure to phenanthridinone for ROR ligands bearing a hexafluoro-2-hydroxypropyl functionality.

2.4. Docking simulation

We performed a docking study of dibenzosilole derivative 26 with the reported cocrystal X-ray structure of the RORγ ligand-binding domain (LBD) and T0901317 (PDB ID: 4NB6),13 using the AutoDock 4.2 program (Figure 4).14 As described above, the ligand-binding site of RORγ is quite hydrophobic and the hydrophobic moiety of dibenzosilole derivative 26 occupied the hydrophobic

space. In addition, the hexafluoro-2-hydroxypropyl group of the dibenzosilole derivative 26 and the main-chain carbonyl group of CYS299 formed a polar interaction. This result suggests that the hexafluoro-2-hydroxypropyl group on the benzene ring can increase RORγ-transcriptional repression activity, and is consistent with the observation that hexafluoro-2-hydroxypropyl derivatives exhibited higher ROR-transcriptional repression activity than dibenzosilole derivatives 14-17 without the hexafluoro-2-hydroxypropyl group. On the other hand, hydroxyl group of T0901317 did not form hydrogen bonding in the crystal structure. The significance of the suggested polar interaction of 26 is under further investigation.

Figure 4. Binding model of the dibenzosilole derivative 26 at the RORγ LBD

3. Conclusion Based on the hypothesis that a silyl group can function as a mimic of cis-amide, as well as olefinic cis-double bond structure, we designed and synthesized novel ROR inverse agonists bearing a dibenzosilole scaffold in place of the phenanthridinone skeleton. The synthesized dibenzosilole derivatives bearing a hexafluoro-2-hydroxypropyl group exhibited similar ROR-transcriptional repression activities to the parent phenanthridinone derivative. These results suggest that the cis-amide moiety can be replaced by a silyl functionality, at least in the case of ROR ligands. Few bioactive compounds containing a dibenzosilole skeleton have so far been reported, but our results indicate that dibenzosilole could be a versatile scaffold for development of novel ligands of ROR and other nuclear receptors.

4. Experimental 4.1 Chemistry General 1

H-NMR and 13C-NMR spectra were recorded on a JEOL JNM-GX500 (500 MHz) spectrometer.

Chemical shifts are expressed in δ (ppm) values with tetramethylsilane (TMS) as an internal reference. The following abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Fast atom bombardment mass spectra (FAB-MS) and high-resolution mass spectra (HRMS) were recorded on a JEOL JMS-DX303 spectrometer with m-nitrobenzyl alcohol. Flash column chromatography was performed on silica gel 60 Kanto Kagaku (40-100 µm).

2,2'-Diaminobiphenyl (8) A mixture of 7 (10.0 g, 40.8 mmol) and SnCl2･2H2O (55.2 g, 245 mmol) in ethanol (100 mL) and H2O (70 mL) was refluxed for 4 h. Ethanol was evaporated and sat. Na2CO3 aq. was added to the remaining solution until the pH reached 10. Excess SnCl2 was filtered off and the solution was extracted several times with EtOAc. The organic layer was washed with brine and H2O, dried over anhydrous MgSO4 and evaporated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to give 8 as a yellow solid (5.30 g, 70%). 1H-NMR (500 MHz, CDCl3) δ 7.19 (td, 2H, J = 7.7, 1.5 Hz), 7.13 (dd, 2H, J = 7.4, 1.7 Hz), 6.85 (td, 2H, J = 7.4, 1.1 Hz), 6.80 (dd, 2H, J = 8.0, 1.1 Hz), 3.31 (s, 4H). MS (FAB [M]) m/z 184, MS (FAB [M+H]+) m/z 185.

2,2'-Bisacetamidobiphenyl (9) A mixture of 7 (10.0 g, 40.8 mmol) and SnCl2･2H2O (55.2 g, 245 mmol) in ethanol (100 mL) and

H2O (70 mL) was refluxed for 6 h. Ethanol was evaporated and sat. Na2CO3 aq. was added to the remaining solution until the pH reached 10. Excess SnCl2 was filtered off and the solution was extracted several times with EtOAc. The organic layer was washed with brine and H2O, dried over anhydrous MgSO4 and evaporated to give 2,2'-diaminobiphenyl (8), which was added to Na2CO3 aq. (7.30 g in 26.2 mL of H2O). The solution was cooled to 0 °C, then acetic anhydride (20 mL) was added, and the mixture was stirred for 1 h. The precipitate was collected by filtration and washed with H2O. Recrystallization from ethanol afforded 9 as a pink-white powder (4.50 g, 42%). 1

H-NMR (500 MHz, CDCl3) δ 8.09 (d, 2H, J = 8.0 Hz), 7.46-7.43 (m, 2H), 7.27-7.20 (m, 2H), 6.93

(s, 2H), 1.97 (s, 6H).

2,2'-Bisacetamido-5,5’-dibromobiphenyl (10)

A mixture of 9 (1.14 g, 4.24 mmol), N-bromosuccinimide (1.66 g, 9.33 mmol) and N,N-dimethylformamide (12 mL) was stirred overnight at room temperature, and then poured into excess H2O. The precipitate was collected by filtration and washed with H2O. Recrystallization from ethanol afforded 10 as a yellow-white powder (1.34 g, 74%). 1H-NMR (500 MHz, CDCl3) δ 7.94 (d, 2H, J = 8.6 Hz), 7.57 (dd, 2H, J = 8.6, 2.3 Hz), 7.36 (d, 2H, J = 2.3 Hz), 1.99 (s, 6H).

5,5'-Dibromo-2,2'-diiodobiphenyl (12) To a solution of 10 (495 mg, 1.16 mmol) in ethanol (9.9 mL) and H2 O (9.9 mL) was added dropwise conc. HCl aq. (4.9 mL). The mixture was refluxed for 6 h, then ethanol was removed by evaporation, and sat. Na2CO3 aq. was added to the remaining solution until the pH reached 10. The precipitate was collected by filtration to give 5,5'-dibromobiphenyl-2,2'-diamine (11), which was added to a mixture of sodium nitrite (0.176 g, 2.55 mmol in 4.0 mL of H2O) and conc. HCl aq. (1.2 mL) at 0 °C. The reaction mixture was stirred for a further 20 min, then added to a vigorously stirred solution of KI (4.24 g, 25.5 mmol) in 9.8 mL of H2O at room temperature. Stirring was continued overnight. Sat. Na2CO3 aq. was added until the pH reached 7, and the solution was extracted several times with EtOAc. The organic layer was washed with H2O, dried over anhydrous MgSO4, and evaporated. The residue was purified by silica gel column chromatography using hexane to give 12 as an off-white solid (225 mg, 34%). 1H-NMR (500 MHz, CDCl3) δ 7.78 (d, 2H, J = 8.0 Hz), 7.32 (d, 2H, J = 2.9 Hz), 7.24 (dd, 2H, J = 8.3, 2.6 Hz). MS (FAB) not found.

2,8-Dibromo-5,5-dibutyl-5H-dibenzosilole (13) To a solution of 12 (233 mg, 0.410 mmol) in THF (3.0 mL) at -78 °C was added dropwise n-BuLi in hexane (1.60 M, 619 µL, 0.990 mmol) under an Ar atmosphere. The resulting mixture was stirred for a further 30 min at the same temperature. Dibutyldichlorosilane (133 µL, 0.620 mmol) was added, and the mixture was allowed to warm to room temperature. Stirring was continued overnight. The reaction was then quenched with H2O, and the whole was extracted with EtOAc. The organic layer was washed with brine and H2O, dried over anhydrous MgSO4 and evaporated. The residue was purified by silica gel column chromatography using hexane to yield 13 as a pale yellow oil (172 mg, 92%). 1H-NMR (500 MHz, CDCl3) δ 7.90 (d, 2H, J = 1.7 Hz), 7.46 (d, 2H, J = 7.4 Hz), 7.42 (dd, 2H, J = 7.7, 1.4 Hz), 1.39-1.24 (m, 8H), 0.91 (tt, 6H, J = 12.0, 3.3 Hz), 0.81 (dd, 2H, J = 9.2, 4.6 Hz). 0.60 (t, 2H, J = 8.0 Hz). 13C-NMR (125 MHz, CDCl3) δ 149.2, 136.8, 134.6, 130.71, 125.3, 124.5, 26.4, 26.3, 26.1, 25.0, 15.9, 13.9, 13.7, 11.9. MS (FAB [M]) m/z 452.

5,5-Dibutyl-2-(1-hyroxyethyl)dibenzosilole (14) To a solution of 13 (19.6 mg, 43.3 mmol) in THF (0.30 mL) at -78 °C was added dropwise n-BuLi in hexane (1.60 M, 64.9 µL, 0.104 mmol) under an Ar atmosphere. The resulting mixture was stirred for a further 30 min at the same temperature. Acetaldehyde (12.0 µL, 0.156 mmol) was added, and the mixture was allowed to warm to room temperature. Stirring was continued overnight. The reaction was quenched with H2O and the whole was extracted with EtOAc. The organic layer was washed with brine and H2O, dried over anhydrous MgSO4 and evaporated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to give 14 as a colorless oil (5.4 mg, 37%). 1H-NMR (500 MHz, CDCl3) δ 7.86 (d, 2H, J = 8.6 Hz), 7.60 (t, 2H, J = 7.4 Hz), 7.43 (td, 1H, J = 7.7, 1.3 Hz), 7.27 (dd, 2H, J = 10.0, 3.7 Hz), 4.97 (q, 1H, J = 6.3 Hz), 1.57 (d, 3H, J = 6.3 Hz), 1.37-1.25 (m, 8H), 0.93 (dd, 4H, J = 9.2, 6.9 Hz), 0.82 (t, 6H, J = 7.2 Hz). 13C-NMR (125 MHz, CDCl3) δ 148.9, 148.2, 147.8, 138.3, 137.1, 133.5, 133.3, 130.1, 127.3, 124.5, 120.9, 117.9, 70.9, 29.8, 26.5, 26.2, 25.2, 13.7, 12.1. HRMS (FAB) calcd for C22H30 NaOSi 361.1964; found: m/z 361.1949 (M+Na)+.

5,5'-Dibutyl-5H-dibenzosilole (15) This product was obtained in the reaction yielding the compound 14 when using reagents containing impurities (54.0 mg, 98%, colorless oil). 1H-NMR (500 MHz, CDCl3) δ 7.82 (d, 2H, J = 8.0 Hz), 7.61 (d, 2H, J = 6.9 Hz), 7.42 (td, 2H, J = 7.7, 1.1 Hz), 7.26 (d, 2H, J = 14.3 Hz), 1.37-1.25 (m, 10H), 0.94 (dd, 4H, J = 8.9, 7.2 Hz), 0.82 (t, 6H, J = 7.2 Hz). 13C-NMR (125 MHz, CDCl3) δ 148.4, 137.9, 133.3, 130.0, 127.2, 120.9, 29.8, 26.5, 26.2, 13.7, 12.1, 1.1. MS (FAB, [M]) m/z 294.

5,5-Dibutyl-2-ethyl-5H-dibenzosilole (16) A mixture of 14 (17.6 mg, 52.0 µmol) and 10% Pd/C (1.8 mg) in MeOH (3.5 mL) was stirred at room temperature. The reaction vessel was evacuated and back-filled with H2, and the mixture was stirred vigorously overnight, then filtered over a pad of Celite (hexane and EtOAc eluent). The filtrate was extracted with hexane, and the organic solution was washed with brine, dried over MgSO4 and concentrated. The residue was purified by silica gel column chromatography using hexane to give 16 (1.8 mg, 11%) as a colorless oil. 1H-NMR (500 MHz, CDCl3) δ 7.83 (d, 1H, J = 8.0 Hz), 7.67 (s, 1H), 7.60 (d, 1H, J = 6.9 Hz), 7.53 (d, 1H, J = 7.4 Hz), 7.41 (td, 1H, J = 7.7, 1.3 Hz), 7.24 (t, 1H, J = 3.7 Hz), 7.12 (t, 1H, J = 4.0 Hz), 2.72 (q, 2H, J = 7.6 Hz), 1.30 (m, 8H), 0.93-0.87 (m, 4H), 0.82 (m, 9H). 13C-NMR (125 MHz, CDCl3) δ 146.3, 138.4, 134.7, 133.3, 130.0,

127.1, 127.0, 120.8, 120.6, 32.0, 29.8, 29.5, 29.3, 26.6, 26.2, 25.0, 22.8, 15.9, 15.6, 14.2, 13.9, 13.7, 12.2. MS (FAB [M]) m/z 322.

2-Acetyl-5,5-dibutyl-5H-dibenzosilole (17) This product was obtained as a by-product (0.4 mg, 4%, colorless oil) in the reaction yielding 16. 1

H-NMR (500MHz, CDCl3) δ 8.37 (s, 1H), 7.94 (d, 1H, J = 7.4 Hz), 7.83 (dd, 1H, J = 7.4, 1.7 Hz),

7.71 (d, 1H, J = 7.4 Hz), 7.63 (d, 1H, J = 6.9 Hz), 7.47 (td, 1H, J = 7.6, 1.3 Hz), 7.31 (t, 1H, J = 6.9 Hz), 2.67 (s, 3H), 1.35-1.24 (m, 8H), 0.96 (t, 4H, J = 8.0 Hz), 0.81 (t, 6H, J = 6.9 Hz). 13C-NMR (125 MHz, CDCl3) δ 198.8, 149.1, 147.5, 144.6, 138.7, 137.8, 133.4, 133.4, 130.4, 127.8, 127.1, 121.3, 119.9, 29.8, 27.0, 26.4, 26.1, 13.7, 11.9. HRMS (FAB) calcd for C22H28NaOSi 359.1807; found: m/z 359.1822 (M+Na)+.

2-(2',6-Diaminobiphenyl-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (18) To a solution of hexafluoroacetone･1.5H2O (4.70 mL, 41.2 mmol) in toluene (137 mL) was added 8 (3.80 g, 20.6 mmol), p-TsOH･H2O (470 mg, 2.50 mmol), and MS 4A (small amount), and the mixture was stirred at 100 °C for 3 h, then allowed to cool to room temperature. The reaction was quenched with H2O, then sat. Na2 CO3 aq. was added until the pH reached 7, and the mixture was extracted several times with EtOAc. The organic layer was washed with brine and H2O, dried over anhydrous MgSO4 and evaporated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 3:1) to give 18 as a yellow oil (4.40 g, 61%).1H-NMR (500 MHz, CDCl3) δ 7.46 (s, 2H), 7.23 (td, 1H, J = 7.7, 1.6 Hz), 7.15 (dd, 1H, J = 7.6, 1.6 Hz), 6.92 (m 2H), 6.79 (d, 1H, J = 9.2 Hz), 4.00 (s, 4H). MS (FAB [M]) m/z 350, MS (FAB [M+H]+) m/z 351.

2-(2',6-Diiodobiphenyl-3-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (19) A solution of 18 (4.40 g, 12.6 mmol in 40 mL of EtOH) at 0 °C was added to a mixture of sodium nitrite (3.81 g, 55.3 mmol in 43 mL of H2O) and conc. HCl aq. (26 mL) at 0 °C. Stirring was continued for a further 30 min, and then the mixture was added to a vigorously stirred solution of KI (45.9 g, 276 mmol in 102 mL of H2O) at room temperature. Stirring was continued overnight, and then sat. Na2CO3 aq. was added until the pH reached 7. The solution was extracted several times with EtOAc. The organic layer was washed with H2O, dried over anhydrous MgSO4, and evaporated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 3:1) to give 19 as a yellow oil (1.70 g, 24%). 1H-NMR (500 MHz, CDCl3) δ 8.04 (dd, 1H, J = 8.6, 2.3 Hz), 7.96 (dd, 1H, J = 8.0, 1.1 Hz), 7.54 (d, 1H, J = 2.3 Hz), 7.47-7.41 (m, 2H), 7.23 (dd, 1H, J =

7.6, 1.6 Hz), 7.12 (td, 1H, J = 7.7, 1.6 Hz), 3.76 (s, 1H). MS (FAB [M]) m/z 572, (FAB [M-I]+) m/z 446.

5-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-2,2'-diiodobiphenyl (20) To a solution of 19 (1.75 g, 3.06 mmol) in N,N-dimethylformamide (3.15 mL) was added sodium hydride (73.3 mg, 3.06 mmol). The resulting mixture was stirred for 30 min at room temperature. Benzyl bromide (0.364 mL, 3.06 mmol) was added and the mixture was allowed to warm to 105 °C. Stirring was continued overnight, then the reaction was quenched with H2O, and the mixture was extracted with EtOAc. The organic layer was washed with brine and H2O, dried over anhydrous MgSO4 and evaporated. The residue was purified by silica gel column chromatography using hexane to give 20 as a colorless paste (1.75 g, 79%). 1H-NMR (500 MHz, DMSO-d6) δ 8.29 (dd, 1H, J = 8.4, 3.3 Hz), 8.02 (dd, 1H, J = 7.9, 1.0 Hz), 7.56 (td, 1H, J = 7.4, 1.1 Hz), 7.44-7.38 (m, 6H), 7.34 (dd, 1H, J = 7.6, 1.6 Hz), 7.30 (d, 1H, J = 2.0 Hz). 7.23 (td, 1H, J = 7.7, 1.6 Hz), 4.76-4.69 (m, 2H). 13

C-NMR (125 MHz, CDCl3) δ 149.8, 148.1, 139.7, 139.2, 136.0, 129.9, 129.0, 128.7, 128.6, 128.4,

128.3, 127.6, 127.4, 102.7, 99.3, 68.5. MS (FAB [M]) m/z 662, (FAB [M-I]+) m/z 535.

2-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-5,5-dibutyl-5H-dibenzosilole (21) To a solution of 20 (146 mg, 0.220 mmol) in THF (2.0 mL) at -78 °C was added dropwise n-BuLi in hexane (1.60 M, 330 µL, 0.530 mmol) under an Ar atmosphere. The resulting mixture was stirred for a further 30 min at the same temperature. Dibutyldichlorosilane (71.1 µL, 0.330 mmol) was added and the mixture was allowed to warm to room temperature. Stirring was continued overnight. The reaction was then quenched with H2O, and sat. Na2CO3 aq. was added to the mixture until the pH reached 7. Extraction with EtOAc afforded an organic layer, which was washed with brine and H2O, dried over anhydrous MgSO4 and evaporated. The residue was purified by silica gel column chromatography using hexane to give 21 as a colorless oil (81.9 mg, 68%). 1H-NMR (500 MHz, CDCl3) δ 8.02 (s, 1H), 7.69 (d, 1H, J = 7.7 Hz), 7.64 (dd, 2H, J = 12.5, 7.3 Hz), 7.50 (d, 1H, J = 7.7 Hz), 7.45-7.35 (m, 6H), 7.29 (td, 1H, J = 7.2, 0.9 Hz), 4.71 (s, 2H), 1.33 (m, 8H). 0.96 (m, 4H), 0.84 (6H, t, J = 7.2 Hz).

13

C-NMR (125 MHz, CDCl3) δ 149.2, 136.8, 134.6, 130.7, 125.3, 124.5, 26.4,

26.3, 26.1, 25.0, 15.9, 13.9, 13.7, 11.9. MS (FAB [M]) m/z 452.

2-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-5,5-dimethyl-5H-dibenzosilole (22) To a solution of 20 (145 mg, 0.220 mmol) in THF (2.0 mL) at -78 °C was added dropwise n-BuLi in hexane (1.60 M, 328 µL, 0.520 mmol) under an Ar atmosphere. The resulting mixture was stirred for

a further 30 min at the same temperature. Dimethyldichlorosilane (39.2 µL, 0.330 mmol) was added and the mixture was allowed to warm to room temperature. Stirring was continued overnight. The reaction was then quenched with H2O, and sat. Na2CO3 aq. was added to the mixture until the pH reached 7. Extraction with EtOAc afforded an organic layer, which was washed with brine and H2O, dried over anhydrous MgSO4 and evaporated. The residue was purified by silica gel column chromatography using hexane to give 22 as a colorless oil (41.5 mg, 41%). 1H-NMR (500 MHz, CDCl3) δ 8.02 (s, 1H), 7.71 (d, 1H, J = 7.4 Hz), 7.64 (d, 2H, J = 6.9 Hz), 7.44-7.37 (m, 7H), 7.31 (dd, 1H, J = 11.2, 4.3 Hz), 4.70 (s, 2H), 0.45 (s, 6H). 13 C-NMR (125 MHz, CDCl3) δ 148.6, 141.8, 136.5, 133.1, 133.0, 130.5,130.0, 128.8, 128.3, 128.1, 127.5, 126.85, 121.1, 120.4, 68.3, -3.3. MS (FAB [M]) m/z 466, (FAB [M-Me]-) m/z 451.

2-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-5,5-diethyl-5H-dibenzosilole (23) To a solution of 20 (55.0 mg, 0.0831 mmol) in THF (0.740 mL) at -78 °C was added dropwise n-BuLi in hexane (1.60 M, 124 µL, 0.199 mmol) under an Ar atmosphere. The resulting mixture was stirred for 30 min at the same temperature. Diethyldichlorosilane (18.6 µL, 0.125 mmol) was added and the mixture was allowed to warm to room temperature. Stirring was continued overnight. The reaction was then quenched with H2 O, and sat. Na2CO3 aq. was added to the mixture until pH reached 7. Extraction with EtOAc afforded an organic layer, which was washed with brine and H2O, dried over anhydrous MgSO4 and evaporated. The residue was purified by silica gel column chromatography using hexane to give 23 as a colorless oil (9.9 mg, 24%). 1H-NMR (500 MHz, CDCl3) δ 8.02 (s, 1H), 7.70 (d, 1H, J = 7.4 Hz), 7.64 (dd, 2H, J = 10.9, 7.4 Hz), 7.50 (d, 1H, J = 8.0 Hz), 7.44-7.36 (m, 6H), 7.29 (t, 1H, J = 7.2 Hz), 4.70 (s, 2H), 1.03 (dt, 6H, J = 9.4, 3.6 Hz), 0.96 (tt, 4H, J = 10.3, 2.8 Hz).

13

C-NMR (125 MHz, CDCl3) δ 149.2, 147.5, 140.4, 137.5, 136.5, 133.6,

133.5, 130.4, 129.9, 128.8, 128.3, 127.9, 127.5, 126.6, 123.8, 121.1, 120.3, 68.3, 7.6, 3.7. MS (FAB [M]) not found.

2-(5,5-Dibutyl-5H-dibenzosilol-2-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (24) A mixture of 21 (76.8 mg, 0.140 mmol) and 10% Pd/C (15.4 mg) in MeOH (9.3 mL) was stirred at room temperature. The reaction vessel was evacuated and back-filled with H2. The mixture was stirred vigorously overnight, and then filtered over a pad of Celite (hexane and EtOAc eluent). The filtrate was extracted with hexane, and the organic solution was washed with brine, dried over MgSO4 and concentrated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 10:1) to give 24 as a colorless oil (45.7 mg, 71%). 1H-NMR (500 MHz, CDCl3)

δ 8.15 (s, 1H), 7.87 (d, 1H, J = 8.0 Hz), 7.68 (d, 1H, J = 7.4 Hz), 7.63 (d, 1H, J = 6.9 Hz), 7.57 (d, 1H, J = 7.4 Hz), 7.46 (dd, 1H, J = 10.9, 4.6 Hz), 7.30 (t, 1H, J = 7.2 Hz), 3.60 (s, 1H), 1.38-1.24 (m, 8H), 0.95 (t, 4H, J = 8.0 Hz), 0.83 (t, 6H, J = 6.9 Hz). 13 C-NMR (125 MHz, CDCl3) δ 149.0, 147.5, 140.7, 138.0, 133.5, 133.4, 131.1, 130.3, 127.8, 125.0, 123.9, 121.7, 121.1, 118.7, 60.6, 26.5, 26.1, 13.7, 11.9. HRMS (FAB) calcd for C23H25 F6OSi 459.1579; found: m/z 459.1587 (M-H)-.

2-(5,5-Dimetyl-5H-dibenzosilol-2-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (25) A mixture of 22 (38.7 mg, 0.0830 mmol) and 10% Pd/C (7.4 mg) in MeOH (5.5 mL) was stirred at room temperature. The reaction vessel was evacuated and back-filled with H2, and the mixture was stirred vigorously. Stirring was continued overnight, and then the mixture was filtered over a pad of Celite (hexane and EtOAc eluent). The filtrate was extracted with hexane, and the organic solution was washed with brine, dried over MgSO4 and concentrated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to give 25 as a colorless oil (8.3 mg, 17%). 1

H-NMR (500 MHz, CDCl3) δ 8.16 (s, 1H), 7.87 (d, 1H, J = 8.0 Hz), 7.70 (d, 1H, J = 7.4 Hz), 7.65

(d, 1H, J = 7.4 Hz), 7.61-7.59 (m, 1H), 7.47 (tt, 1H, J = 7.4, 2.2 Hz), 7.31 (t, 1H, J = 7.2 Hz), 0.44 (s, 6H).

13

C-NMR (125 MHz, CDCl3) δ 147.0, 139.1, 133.0, 131.3, 133.5, 129.1, 129.0, 128.0,

127.4, 125.2, 121.2, 118.7, -3.3. HRMS (FAB) calcd for C17H13F6OSi 375.0640; found: m/z 375.0654 (M-H)-.

2-(5,5-Diethyl-5H-dibenzosilol-2-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (26) A mixture of 23 (2.7 mg, 5.46 µmol) and 10% Pd/C (0.5 mg) in MeOH (0.25 mL) was stirred at room temperature. The reaction vessel was evacuated and back-filled with H2. The mixture was stirred vigorously overnight, and then filtered over a pad of Celite (hexane and EtOAc eluent). The filtrate was extracted with hexane, and the organic solution was washed with brine, dried over MgSO4 and concentrated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 10:1) to give 26 as a colorless oil (0.9 mg, 41%). 1H-NMR (500 MHz, CDCl3) δ 8.15 (s, 1H), 7.87 (d, 1H, J = 7.4 Hz), 7.69 (d, 1H, J = 7.4 Hz), 7.63 (d, 1H, J = 7.4 Hz), 7.57 (d, 1H, J = 7.4 Hz), 7.46 (td, 1H, J = 7.6, 1.3 Hz), 7.30 (t, 1H, J = 6.9 Hz), 1.02-0.99 (m, 6H), 0.97-0.94 (m, 4H).

13

C-NMR (125 MHz, CDCl3) δ133.4, 130.4, 127.8, 121.2, 7.6, 3.7. HRMS

(FAB) calcd for C19H17F6OSi 403.0953; found: m/z 403.0975 (M-H)-.

2-(2-Benzyloxy-1,1,1,3,3,3-hexafluoropropan-2-yl)-5-butyl-5-methyl-5H-dibenzosilole (28) To a solution of 20 (109 mg, 0.165 mmol) in dry tetrahydrofuran (1.5 mL) was added t-BuLi in

n-hexane (1.63 M, 0.304 mL, 0.496 mmol) under an Ar atmosphere at -78 °C. The mixture was stirred at the same temperature for 20 min, and then warmed to room temperature. Stirring was continued at the same temperature for 30 min. The reaction mixture was cooled to -78 °C, n-butyltrichlorosilane (89.2 µL, 0.545 mmol) was added to it, and stirring was continued at room temperature overnight. A solution of methylmagnesium bromide (0.436 mL, 0.397 mmol) in dry tetrahydrofuran was then added under an Ar atmosphere at -78 °C, and stirring was continued at room temperature overnight. The reaction was quenched with H2O and the mixture was extracted with EtOAc. The extract was successively washed with H2O and brine, and dried over MgSO4. The solvent was evaporated, and the residue was purified by silica gel column chromatography using hexane to give 28 (26.5 mg, 32%) as a colorless oil. 1H-NMR (500 MHz, CDCl3) δ 8.02 (s, 1H), 7.70 (d, 1H, J = 8.0 Hz), 7.64 (t, 2H, J = 7.2 Hz), 7.56-7.47 (m, 1H), 7.44-7.36 (m, 6H), 7.30 (t, 1H, J = 7.4 Hz), 4.71 (s, 2H, J = 8.6 Hz), 1.42 (tt, 2H, J = 9.5, 3.6 Hz), 1.37-1.29 (m, 2H), 0.96-0.93 (m, 2H), 0.86 (t, 3H, J = 7.2 Hz), 0.44 (s, 3H). 13C-NMR (125 MHz, CDCl3 ) δ 138.6, 133.4, 133.2, 130.4, 129.0, 128.8, 128.3, 128.0, 127.5, 127.2, 126.8, 121.1, 120.3, 68.3, 26.5, 26.1, 13.7, 13.3, -5.1. MS (FAB [M]) m/z 508, (FAB [M-nBu]-) m/z 451, (FAB [M-nBu-Me+H]-) m/z 437.

2-(5-Butyl-5-methyl-5H-dibenzosilol-2-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (29) A mixture of 28 (15.1 mg, 0.0300 mmol) and 10% Pd/C (3.0 mg) in MeOH (0.149 mL) and acetic acid (15 µL) was stirred at room temperature. The reaction vessel was evacuated and back-filled with H2. The mixture was stirred vigorously overnight, and then filtered over a pad of Celite (hexane and EtOAc eluents). The filtrate was extracted with hexane, and the organic solution was washed with brine, dried over MgSO4 and concentrated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to give 29 as a colorless oil (11.1 mg, 90%). 1

H-NMR (500 MHz, CDCl3) δ 8.15 (s, 1H), 7.87 (d, 1H, J = 8.0 Hz), 7.69 (d, 1H, J = 8.0 Hz), 7.63

(d, 1H, J = 6.3 Hz), 7.58 (d, 1H, J = 7.4 Hz), 7.46 (td, 1H, J = 7.6, 1.3 Hz), 7.31 (t, 1H, J = 7.7 Hz), 1.43-1.36 (m, 2H), 1.34-1.29 (m, 2H), 0.94 (td, 2H, J = 5.6, 4.2 Hz), 0.85 (t, 3H, J = 7.2 Hz), 0.43 (s, 3H). 13C-NMR (125 MHz, CDCl3) δ 148.7, 141.2, 138.6, 133.2, 131.2, 130.4, 127.9, 125.1, 121.1, 118.7, 26.4, 26.1, 13.7, 13.3, -5.1. HRMS (FAB) calcd for C19H17F6OSi 417.1109; found: m/z 417.1120 (M-H)-.

4.2 Reporter gene assay PcDNA3.1(-)-hRORα1, pcDNA3.1(-)-hRORβ1, pcDNA3.1(-)-hRORγ1 and RORE-TK-Luc were provided by Itsuu Laboratory. CMX-β-GAL was provided by Professor Dr. Makoto Makishima

(Nihon University School of Medicine). Human embryonic kidney (HEK) 293 cells were cultured in DMEM containing 5% FBS, penicillin and streptomycin mixture at 37 °C in a humidified atmosphere of 5% CO2 in air. Cells were seeded at a density of 20% confluence/96-well plate 24 h prior to transfection. Cells in each well were cotransfected with 150 ng of a nuclear receptor expression plasmid, 500 ng of a luciferase reporter and 10 ng of CMX-β-GAL expression vector. Transfections were performed by the calcium phosphate co-precipitation method. After 8 h, transfected cells were treated with test compounds or dimethyl sulfoxide (DMSO) for 16 h. Treated cells were assayed for luciferase activity with a luminometer. The luciferase activity of each sample was normalized by the level of β-galactosidase activity. Each transfection was carried out in triplicate.

4.3. Docking simulation Structure of LBD of RORγ was prepared from the Protein Data Bank accession 4NB6. The structure added for polar hydrogens, and partial atomic charges were assigned using AutoDockTools (ADT).14 Molecular docking was performed using AutoDock 4.2 with Genetic Algorithm. Autodock parameters for silicon atom Rii = 4.30 and εii = 0.402 were used.

Acknowledgements The work partially supported by “Platform for Drug Discovery, Informatics, and Structural Life Science”, and “Grants-in-Aid for Scientific Research from The Ministry of Education Culture, Sports, Science and Technology, Japan, and the Japan Society for the Promotion of Science (KAKENHI Grant No. 26293025 (YH) and No. 25460146 (SF))”.

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6. Nakamura, M.; Kajita, D.; Matsumoto, Y.: Hashimoto, Y.; Bioorg. Med. Chem. 2013, 21, 7381. 7. Mangelsdorf, D.J.; Thummel, C.; Beato, M.; Herrlich, P.; Schütz, G.; Umesono, K.; Blumberg, B.; Kastner, P.; Mark, M.; Chambon, P.; Evans, R. M. Cell 1995, 83, 835. 8. Jetten, A. Nucl. Recept. Signal. 2009, 7, e003. 9. Fauber, B. P.; Magnuson, S. J. Med. Chem. 2014, 57, 5871. 10. Solt, L.; Kumar, N.; Nuhant, P.; Wang, Y.; Lauer, J.; Liu, J.; Istrate, M.; Kamenecka, T.; Roush, W.; Vidovic´ , D.; Schürer, S.; Xu, J.; Wagoner, G.; Drew, P.; Griffin, P.; Burris, T. Nature 2011, 472, 491. 11. Nishiyama, Y.; Nakamura, M.; Misawa, T.; Nakagomi, M.; Makishima, M.; Ishikawa, M.; Hashimoto, Y. Bioorg. Med. Chem. 2014, 22, 2799. 12. Toyama, H.; Nakamura, M.; Nakamura, M.; Matsumoto, Y.; Nakagomi, M.; Hashimoto Y. Bioorg. Med. Chem. 2014, 22, 1948. 13. Fauber, B. P.; de Leon Boenig, G.; Burton, B.; Eidenschenk, C.; Everett, C.; Gobbi, A.; Hymowitz, S. G.; Johnson, A. R.; Liimatta, M.; Lockey, P.; Norman, M.; Ouyang, W., Rene, O.; Wong, H. Bioorg. Med. Chem. Lett. 2013, 23, 6604. 14. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. J. Comput. Chem. 2009, 16, 2785.