Development of novel silanol-based human pregnane X receptor (PXR) agonists with improved receptor selectivity

Development of novel silanol-based human pregnane X receptor (PXR) agonists

with improved receptor selectivity

1

2

1

1,*

Hirozumi Toyama, Hitoshi Shirakawa, Michio Komai, Yuichi Hashimoto, Shinya Fujii

1

Institute of Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032,

Japan

2

Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku,

Sendai 981-8555, Japan

*

Corresponding Author: fujiis@iam.u-tokyo.ac.jp

Keywords: Pregnane X receptor; Nuclear receptor; Silanol; Sila-substitution; C/Si-Exchange;

Alcohol/silanol-exchange; T0901317

Abstract

Pregnane X receptor (PXR) is a ligand-dependent transcription factor that is considered to be a

potential therapeutic target for multiple diseases. Herein, we report the development and structure-activity

relationship studies of a new series of hPXR agonists. Focusing on our recently developed

silanol-sulfonamide scaffold, we developed the potent hPXR agonist 28, which shows good selectivity

over hLXRα and β, hFXR, and hRORα and γ. Examination of the structure-activity relationship suggested

a possible strategy to manipulate the selectivity. Docking simulation indicated the presence of an

additional binding cavity and polar contacts in the ligand-binding pocket of hPXR. This information

should be helpful for the future development of more potent and selective hPXR ligands.

1

. Introduction

Nuclear receptors (NRs) are ligand-dependent transcription factors that regulate the expression of

1

,2

their target genes. They play roles in many essential physiological systems, including the decision of

3

,4

cell fate, and thus are attractive targets for drug discovery. For example, retinoic acid receptors (RARs),

5

,6

7–9

steroid hormone receptors such as estrogen receptors (ERs), and androgen receptor (AR)

are

promising therapeutic targets for multiple diseases, including cancer, and a number of drugs have entered

clinical use. In addition to these classical NRs, liver X receptor (LXR) α and β, which are sensors of

9

–11

oxysterols, may be therapeutic targets for metabolic syndrome, including atherosclerosis and diabetes.

Farnesoid X receptor (FXR) functions as sensor of bile acids in the intestine and liver, and is also a

1

2

potential target for metabolic syndrome. Further, various ligands of retinoic acid receptor-related orphan

1

3,14

receptor γ (RORγ) have been developed for treatment of inflammatory diseases.

Thus, development of

modulators of so-called orphan NRs is considered a promising approach for drug discovery.

Human pregnane X receptor (hPXR), also known as SXR (steroid and xenobiotic receptor) and PAR

(pregnane activated receptor), is a nuclear receptor identified in 1998 as a human orthologue of murine

1

5-17

PXR.

hPXR is involved in physiological processes such as absorption, distribution, metabolism and

elimination of xenobiotics and endobiotics. It is expressed in kidney and intestine, and representative

target genes of hPXR include drug-metabolizing enzyme CYP3A4 (cytochrome P450) and multidrug

18,19

20

resistance transporter MDR1.

hPXR also plays important roles in bone homeostasis and

2

1

inflammation. Therefore, modulation of hPXR could have potential therapeutic applications. Various

hPXR ligands, including natural hormones, vitamins and therapeutic agents, are known. The antibiotic

rifampicin (1) is a representative hPXR agonist, and steroid hormones and lipophilic vitamins such as

2

0,22

progesterone (2) and menaquinone-4 (vitamin K

2

: 3) are natural PXR agonists.

The antifungal agent

2

3

ketoconazole (4) is a hPXR antagonist. However, most of these compounds have other primary targets,

and hPXR is an off-target. Also, though a few structure-activity relationship (SAR) studies have been

24

reported, the SAR of hPXR ligands is largely obscure at present. Thus, investigation of the SAR of

hPXR ligands and development of more potent and selective hPXR ligands would be helpful to clarify the

therapeutic potential of hPXR.

Recently, we developed 6 (sila-T) as a silanol analog (sila-substitution) of the multi-target nuclear

2

5

receptor modulator T0901317 (5). Benzenesulfonamide derivative 5 was originally developed as an

26

agonist for hLXRα and hLXRβ, but subsequent studies revealed that 5 exhibits agonistic activity toward

2

7,28

multiple nuclear receptors including hFXR and hPXR, as well as hLXRs,

and is also an inverse

2

9

agonist of hRORs. In the field of medicinal chemistry, several series of silanol derivatives, including

3

0

silanediols, have been developed. We previously examined the similarities and differences among

silanols, alcohols and perfluoroalcohols from the viewpoint of physicochemical properties. We found that

conversion of the perfluoroalcohol functionality of 5 into a silanol group altered the selectivity profile,

and silanol 6 exhibited agonistic activity toward hPXR, partial agonistic activity toward hFXR, inverse

agonistic activity toward hRORs, and no activity toward hLXRs. Based on these findings, we aimed to

develop novel PXR-selective agonists using silanol 6 as a lead compound.

Fig.1. Examples of reported natural and synthesized PXR ligands.

2

. Results and discussion

.1. Molecular design

Our previous results indicated that the silanol moiety, which was introduced as an alternative to the

perfluoroalcohol moiety of 5, functions as the polar pharmacophore of hPXR, hFXR and hRORs ligands.

On the other hand, carbinol derivative 7 exhibited no significant activity toward any of these receptors.

We hypothesized that the relatively high acidity of the silanol moiety in comparison to carbinol is

important for the activities. Therefore, we initially set out to investigate the utility of the acidic carboxy

group as a polar functionality for PXR agonists. Then, we planned structural development of the silanol

derivatives to improve the PXR selectivity, with the aid of the reported X-ray co-crystal structures of

hPXR, hFXR, and hRORγ. It is suggested that the hPXR cavity is larger than those of hFXR and

3

1

hRORs. The X-ray structure of hPXR bound to 5 and a docking simulation of 6 bound to hPXR also

indicated that multiple hydrophobic amino acid residues surround the 2,2,2-trifluoroethyl moiety of 5 and

6

. Based on these considerations, we designed silanol derivatives bearing various substituents, including

bulky hydrophobic groups, on the nitrogen atom of sulfonamide as candidate PXR-selective ligands.

Fig.2. Design scheme of selective hPXR ligands.

2

.2 Synthesis

Scheme 1 illustrates the synthesis of carboxy derivative 8. Reaction between ethyl 4-aminobenzoate

9) and benzenesulfonyl chloride gave sulfonamide 10, and then alkylation of the nitrogen atom with

-bromo-2,2,2-trifluoroethane afforded 11. Hydrolysis of the ethyl ester group under basic conditions

(

1

gave the desired carboxylic acid 8 (Scheme 1).

Scheme 1. Synthesis of carboxylic acid 8. Reagents and conditions: (a) Benzenesulfonyl chloride,

pyridine, THF, rt, quant; (b) NaH, CF

3

CH

2

Br, DMF, 80℃, 33%; (c) LiOH, H O-EtOH, 80℃, quant.

2

The synthesis of the designed silanol derivatives 22-28 is illustrated in Scheme 2. Sulfonamide

formation using 4-bromoaniline (12) and benzenesulfonyl chloride gave sulfonamide 13, which was

3

2

converted to dimethylsilanol 14 under Denmark’s conditions. Namely, in the presence of PdCl

2

and

2

1

-(di-tert-butylphosphino)biphenyl

(BPTBP),

the

bromide

reacted

with

,2-diethoxy-1,1,2,2-tetramethyldisilane to afford a ethoxydimethylsilyl intermediate, and then removal

of the ethyl group gave the desired silanol 14. Sulfonamide 13 was also alkylated with alkyl halides or

alkyl triflates to afford compounds 15-21. The bromines of compounds 15-21 were converted to

dimethylsilanol under the same conditions as used for 14. In some cases the yield was not sufficient, since

the conversion of starting material was low and formation of siloxane was also observed.

Scheme 2. Synthesis of the designed silanol derivatives 22-28. Reagents and conditions: (a)

Benzenesulfonyl chloride, pyridine, THF, rt, 93%; (b) NaH, R-X, DMF, 80℃, 3-92%; (c) i, PdCl

i-Pr EtN, NMP, (Me EtOSi) , 60℃; ii, AcOH, Me NCH CH SH, MeCH; 3-26%.

2

, BPTBP,

2

We also designed and synthesized alcohol 31, which is the carbinol derivative corresponding to

silanol 28. Treatment of ester 10 with methylmagnesium bromide gave alcohol 29. Protection of the

hydroxyl group of 29 using trimethylsilyl chloride gave compound 30, and then alkylation with methyl

bromoacetate under basic conditions afforded the desired carbinol 31 (Scheme 3).

Scheme 3. Synthesis of carbinol derivative 31. Reagents and conditions: (a) MeMgBr, THF, 0℃, 28%

yield; (b) TMSCl, imidazole, DMF, 0℃to rt, quant; (c) NaH, BrCH COOMe, DMF, 80℃, 86% yield.

2

.3. Determination of logPo/w values

We firstly determined the hydrophobicity of the synthesized silanol and carbinol derivatives in terms

33

of the octanol–water partition coefficient (Po/w), using an HPLC method. The determined logPo/w values

are listed in Table 1. In this study, silanol 28 exhibited a logPo/w value of 2.79, which is 0.44 larger than

that of the corresponding carbinol 31 (logPo/w: 2.35). We recently reported that sila-T (6) exhibited a

logPo/w value of 4.23, which is 0.53 larger than that of the corresponding carbinol 7 (logPo/w: 3.70). We

also showed that C/Si-exchange of the tertiary butyl group to a trimethylsilyl group increased the logPo/w

34

value by 0.6. These results suggest that the increase of hydrophobicity in silanol/carbinol exchange is

smaller than that in C/Si exchange of the tertiary alkyl group. The differences can be attributed to the

differences of hydrophobic surface area, namely, three methyl groups in t-butyl/TMS alcohol or two

methyl groups in silanol/alcohol.

2

.4. Biological evaluation

We next evaluated the potencies of the compounds toward hPXR by means of luciferase reporter gene

assay in HEK293 cells. The carboxy derivative 8 exhibited no activity toward hPXR. This result suggests

that the acidity of the hydroxyl group is not sufficient, and that suitable hydrophobicity around the acidic

hydroxyl group is important. In contrast to the carboxy (8) or carbinol (7) derivatives, all the synthesized

silanol derivatives exhibited significant hPXR-agonistic activity. Among the silanol derivatives,

trifluoroethyl derivative 6 (sila-T) exhibited the most potent activity, and methoxycarbonylmethyl

derivative 28 was the second most potent compound, with an EC50 value of 1.4 μM. Compounds bearing

comparatively small substituents such as 14, 22 and 23 also exhibited potent activity. On the other hand,

introduction of bulky substituents such as an n-butyl group (24) or a phenethyl group (26) reduced the

activity. Significant correlation between hydrophobicity (LogP value) and PXR agonistic activity was not

observed. We initially considered that introduction of a bulky and hydrophobic functionality on the

nitrogen might improve the PXR activity and selectivity, but the hydrophobic cavity in this region seems

to be restricted. It is interesting that compounds 27 and 28 with hydrogen-bonding capability exhibited

potent hPXR activity. Alcohol 31, which is the carbinol derivative corresponding to the potent silanol

derivative 28, did not exhibit PXR agonistic activity, suggesting that the silanol moiety is important for

PXR activity.

In order to verify the hPXR-selectivity of the silanol derivatives, we also investigated their activities

toward other nuclear receptors, hLXRα and β, hFXR, and hRORα and γ. None of the synthesized silanol

derivatives, as well as sila-T (6), showed significant agonistic activity toward hLXRα and β. This result

suggests that the hLXRs agonistic activity is extinguished by conversion of the hexafluoropropanol

moiety of 5 to a silanol group, and increased hydrophobicity does not compensate for this. Concerning the

T0901317 (5) derivatives, the hexafluoropropanol moiety seems to be indispensable for hLXR activity,

and therefore the silanol-sulfonamide scaffold we developed is a promising core structure for the

development of hPXR agonists without hLXR activity. The silanols 14 and 22-28 synthesized in this

2

5

study did not exhibit hFXR activity, while sila-T (6) exhibited partial hFXR-agonistic activity. The

hFXR activity may depend upon the presence of the 2,2,2-trifluoroethyl moiety. Regarding hRORα and γ,

silanol derivatives bearing ethyl (23), n-butyl (24), benzyl (25) and 2-methoxyethyl groups (27), as well

as sila-T (6), exhibited moderate or weak agonistic activities toward RORα and γ, whereas compounds

bearing a hydrogen atom (14) or a bulky substituent such as 26 did not exhibit activity toward hRORα

and γ. Compound 24 bearing methyl group exhibited agonistic activity toward hRORα and γ. The

structure-activity relationship data for hRORα and γ indicate that there is a “window” of bulkiness or

hydrophobicity for hRORα and γ modulators. These findings support the idea that the silanol-sulfonamide

scaffold we focused on in this study is a versatile core structure for development of hPXR agonists, and

that proper selection of the substituent on the nitrogen atom can improve the selectivity toward hPXR.

Table 1. Hydrophobicity parameters and biological activities of synthesized compounds.

1

2

a

Compd

R

logPo/w

hPXR

hLXRα

hLXRβ

hFXR

-

hRORα

hRORγ

EC50 (μM)

IC50 (μM)

1

5

6

7

8

-

2.0

0.12

0.63

n.a.

2.4

3.4

1.9

6.5

3.2

14

-

b

-C(CF

3

)

2

OH -CH

2

CF

3

CF

3

CF

3

CF

3

5.38

4.23

3.70

0.40

n.a.

0.23

3.4

11

4.4

11

b

-SiMe

-CMe

2

OH

-CH

-H

n.a.

11

>30

n.a.

2

n.a.

>30

n.a.

c

-COOH

-SiMe

-CMe

n.d.

1

2

3

4

2

3

4

5

6

7

8

1

2

OH

2.22

2.90

3.20

4.05

3.92

4.32

2.81

2.79

2.35

d

2

-Me

-Et

(+53%)

>30

n.a.

(+54%)

>30

n.a.

2

-n-Bu

2

-Bn

2

-(CH

2

)

2

Ph

2

OMe

4.2

1.4

n.a.

2

-CH

2

CO

2

Me

n.a.

2

OH

n.a.

>30

a

b

IC50 values were calculated with respect to the activity of 5 (30 μM) as the maximum response.

c

d

Reported values in reference 25. Not determined. Agonistic activity (% activation with respect to the

basal luciferase activity). n.a.: Activation or inhibition in the concentration of 30 μM was less than 11%.

2

.5. Docking simulation

In order to estimate the binding mode of the developed silanol derivatives and to investigate the

structure-activity relationships, we conducted docking simulation of the co-crystal structure of the hPXR

35

LBD with T0901317 (5) (PDB ID: 2O9I). We first investigated docking of the methoxycarbonyl

derivative 28, which exhibited potent and selective PXR-agonistic activity. We obtained two distinct

binding forms. The first (form A) is similar to that of 5 in the co-crystal. In this calculated structure, the

silanol moiety of 28 forms a polar contact with His 407, with which the hexafluoropropanol of 5 forms a

polar contact in the crystal structure. The sulfonyl group also forms a polar contact with Gln285. The

methoxycarbonyl moiety is located the hydrophobic cavity, surrounded by several hydrophobic amino

acid residues, including Leu209, Val211, Trp299, Leu308 and Leu324, where the trifluoroethyl moiety of

5

is located in the crystal structure (Fig. 3, top). On the one hand, the second binding form (form B) is

quite different. In this calculated structure, the phenylsilanol moiety, instead of the methoxycarbonyl

moiety in form A, is located in the hydrophobic cavity, and the hydroxyl group of silanol forms a polar

contact with amide groups of the protein backbone. The sulfonyl group forms a polar contact with Gln285,

similarly to form A, and interestingly, in addition to the sulfonyl group, the ester group also forms a polar

contact with Gln285 (Fig. 3, middle). Although we could not establish which conformation is close to the

actual binding form of compound 28, the additional polar contact at the methoxycarbonyl moiety in form

B could be a possible reason for the high potency of 28.

We also conducted docking simulations of compounds 14 and 22. These compounds have a relatively

small substituent at the nitrogen atom, and exhibited potent PXR-agonistic activity. Generally, both of

3

7-39

secondary and tertiary aromatic sulfonamides have a synclinal structure,

different from the case of

40

aromatic carboxamides. The calculations on these compounds indicated binding forms fairly different

from both form A and form B. In these calculated structures, similarly to the co-crystal of 5 and form A of

2

8, the hydroxyl group of the silanol moiety is located near His407 and forms a polar contact with it.

However, the benzenesulfonyl group is located in a hydrophobic cavity surrounded by amino acid

residues, including Leu206, Lys210, Leu239 and Leu240, and this is distinct from the cavity occupied by

5

or 28. The relatively small substituents on the nitrogen atom of 14 and 22 could allow these compounds

to access this cavity (Fig. 3, bottom).

The docking simulation suggested the existence of multiple binding forms, depending on the

substructure of the compounds. It is possible that the comparatively large binding pocket of hPXR allows

the compounds to bind in multiple binding modes. Although the stability of the methoxycarbonyl group in

the biological system can be problematic for in vivo application, the additional binding cavity and polar

contacts suggested by the docking simulations might provide useful clues for the development of potent

and selective hPXR ligands.

Fig. 3. Docking models of silanol derivatives with hPXR LBD (PDB ID: 2O9I) obtained with

3

6

AutoDock. (Top and middle) Superimposition of the hPXR LBD complex with 5 (gray) and docking

models of 28 (green) (top: form A, middle: form B). (Bottom) Superimposition of the hPXR LBD

complex with 5 (gray) and docking models of 14 (white) and 22 (pink).

3

. Conclusion

In this study, we developed a new series of selective hPXR agonists based on our previously

developed silanol-sulfonamide scaffold. Structure-activity relationship studies of the sulfonamide

derivatives toward hPXR, hLXRα and β, hFXR, and hRORα and γ suggested a possible strategy to

manipulate the selectivity of the compounds. The hexafluoropropanol moiety of 5 is indispensable for

hLXRs activity, whereas the silanol group is acceptable in hPXR ligands. In addition, carbinol and

carboxylic acid resulted in loss of activity as a hPXR ligand. Therefore, the silanol-sulfonamide scaffold

we developed seems to be a promising core structure for hPXR ligands with selectivity over hLXRs. As

regards hFXR and hRORs, there seems to be a “window” of acceptable bulkiness or hydrophobicity for

these receptors. Based on these findings, we developed compound 28 as a potent and selective hPXR

agonist. Though the PXR potency of compound 28 was not potent than the lead compound 5, we

succeeded in reducing the potency toward other NRs to improve the PXR selectivity. Docking simulation

suggested multiple binding modes of the compounds, including an additional binding cavity and polar

contacts. The potent and selective hPXR agonist 28 and the structure−activity relationship data obtained

in this study should be useful to expand the pharmaceutical potential of hPXR modulation and to assist

the development of more potent and selective hPXR modulators.

4

. Experimental

.1. Chemistry

General remarks. All reagents were purchased from SigmaAldrich Chemical Co., Tokyo Kasei Kogyo

Co., Wako Pure Chemical Industries, or Kanto Kagaku Co., Inc. Silica gel for column chromatography

was purchased from Kanto Kagaku Co., Inc. Melting points were taken on a Yanagimoto micro melting

1

13

point apparatus. H-NMR and C-NMR spectra were recorded on a JEOL JNM-GX500 (500 MHz and

25 MHz, respectively) spectrometer. Chemical shifts are expressed in δ (ppm) values with

1

tetramethylsilane (TMS) as an internal reference. The following abbreviations are used: s = singlet, d =

doublet, t = triplet, q = quartet, m = multiplet, br = broad. Electrospray ionization mass spectra (ESI-MS)

and high-resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF spectrometer with Low

Concentration Tuning mix (G1969-85000).

4

.1.1. Ethyl 4-(phenylsulfonamido)benzoate (10). To a solution of 9 (2.00 g, 12.1 mmol) in THF (121

mL) were added benzenesulfonyl chloride (4.65 mL, 36.3 mmol) and pyridine (9.76 mL, 121 mmol), and

the mixture was stirred room temperature for 1.5 h. The reaction was then quenched with H O, and the

whole was extracted with EtOAc. The organic layer was washed with brine and H O, dried over

anhydrous MgSO and evaporated. The residue was purified by silica gel column chromatography

2

4

(

n-hexane/EtOAc = 3:1) to give 10 as a white solid. This material was used in the next step without

1

further purification. H-NMR (500 MHz, CDCl

3

) δ 7.92 (td, 2H, J = 5.6, 3.2 Hz), 7.84-7.82 (m, 2H), 7.56

(

tt, 1H, J = 7.4, 1.3 Hz), 7.46 (td, 2H, J = 7.7, 3.1 Hz), 7.13 (dt, 2H, J = 9.0, 2.3 Hz), 7.08 (1H, s), 4.33 (q,

+

2

H, J = 7.3 Hz), 1.36 (t, 3H, J = 7.2); MS (ESI+) m/z 328 [for (M+Na) ].

4

.1.2. Ethyl 4-(N-(2,2,2-trifluoroethyl)phenylsulfonamido)benzoate (11). To a solution of 10 (1.20 g,

3

.92 mmol) in DMF (18.0 mL) was added sodium hydride (188 mg, 7.84 mmol), and the mixture was

stirred room temperature for 30 min. 2,2,2-Trifluoroethyl triflate (1.70 mL, 11.8 mmol) was added, and

the mixture was refluxed for 1 h. The reaction was then quenched with H O, and the whole was extracted

with EtOAc. The organic layer was washed with brine and H O, dried over anhydrous MgSO and

2

4

evaporated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to

1

give 11 as a yellow solid (767 mg, 33% (2 steps)). H-NMR (500 MHz, CDCl

3

) δ 8.00 (dt, 2H, J = 9.0,

2

7

.3 Hz), 7.63-7.58 (m, 3H), 7.48 (dt, 2H, J = 12.2, 2.9 Hz), 7.13 (dt, 2H, J = 9.0, 2.0 Hz), 4.38 (q, 2H, J =

+

.3 Hz), 4.25 (q, 2H, J = 8.2 Hz), 1.39 (t, 3H, J = 7.2 Hz); MS (ESI+) m/z 410 (for [M+Na] ).

4

.1.3. 4-(N-(2,2,2-Trifluoroethyl)phenylsulfonamido)benzoic acid (8). To a solution of 11 (213 mg,

0

2 2 

.550 mmol) in H O-EtOH (H O:EtOH = 1:3, 45.5 mL) was added LiOH･ mg, 0.825

mmoland the mixture was stirred at 80℃ for 4 h. The reaction was then quenched with HCl, and the

whole was extracted with EtOAc. The organic layer was washed with brine and H O, dried over

and evaporated to give 8 as a white solid (529 mg, quant.). H-NMR (500 MHz,

) δ 8.06 (dt, 2H, J = 9.0, 2.0 Hz), 7.64-7.59 (m, 3H), 7.49 (t, 2H, J = 8.0 Hz), 7.19 (dt, 2H, J = 9.0,

2

1

anhydrous MgSO

4

CDCl

3

1

3

2

1

3

.3 Hz), 4.27 (q, 2H, J = 8.0 Hz); C-NMR (125 MHz, CDCl ) δ 169.86, 143.90, 137.91, 133.72, 131.50,

29.42, 129.31, 129.14, 127.76, 124.73, 122.51, 52.30, 52.02, 51.74, 51.46. HRMS (ESI-) m/z 358.0374

-

[

(M-H) :calcd for C15

H

11

F

3

O

4

NS, 358.0355].

4

.1.4. N-(4-Bromophenyl)benzenesulfonamide (13). To a solution of 12 (1.00 g, 5.81 mmol) in THF

(

58.1 mL) were added benzenesulfonyl chloride (1.49 mL, 11.6 mmol) and pyridine (4.68 mL, 58.1

mmol), and the mixture was stirred at room temperature for 1.5 h. The reaction was then quenched with

O, and the whole was extracted with EtOAc. The organic layer was washed with brine and H O, dried

over anhydrous MgSO and evaporated. The residue was purified by silica gel column chromatography

H

2

4

1

(

3

n-hexane/EtOAc = 3:1) to give 13 as a white solid (1.53 g, 84%). H-NMR (500 MHz, CDCl ) δ

7

6

.79-7.77 (m, 2H), 7.56 (tt, 1H, J = 7.4, 1.3 Hz), 7.46 (t, 2H, J = 8.0 Hz), 7.35 (dt, 2H, J = 9.4, 2.3 Hz),

-

.98-6.94 (m, 3H); MS (ESI-), m/z 311, 309 [(M-H) ].

4

1

2

.1.5. N-(4-(Hydroxydimethylsilyl)phenyl)benzenesulfonamide (14). To a solution of PdCl (7.1 mg,

0.1 mol), 2-(di-t-butylphosphino)biphenyl (23.9 mg, 80.2 mol) and 13 (250 mg, 0.801 mmol) in

-methyl-2-pyrrolidinone (8.00 mL) was added diisopropylethylamine (411 L), and the mixture was

stirred at 60 °C under an argon atmosphere. 1,2-Diethoxy-1,1,2,2-tetramethyldisilane (238 L, 0.961

mmol) was added, and the mixture was stirred at 60 °C for 4 h, then cooled to rt. CH CN (17.7 mL),

-(dimethylamino)ethanethiol hydrochloride (28.4 mg, 0.200 mmol) and 0.1 M acetic acid (23.1 mL)

were added to it, and the resulting mixture was stirred at rt for 2 h. The reaction was then quenched with

3

2

H

2

O, and the whole was extracted with EtOAc. The organic layer was washed with brine and H

2

O, dried

over anhydrous MgSO and evaporated. The residue was purified by silica gel column chromatography

4

1

(

n-hexane/EtOAc = 2:1) to give 14 as a colorless oil (14.2 mg, 6%). H-NMR (500 MHz, DMSO-d6) δ

1

0.38 (s, 1H), 7.79-7.77 (m, 2H), 7.62-7.58 (m, 1H), 7.57-7.53 (m, 2H), 7.37 (2H, d, J = 8.6 Hz), 7.08

1

3

(

2H, d, J = 8.6 Hz), 5.76 (s, 1H), 0.16 (6H, s); C-NMR (125 MHz, CDCl

3

) δ 139.20, 137.31, 136.24,

-

1

34.12, 133.08, 129.07, 127.19, 120.19, 0.62; HRMS (ESI-) m/z 306.0642 [(M-H) : calcd for

14 3

C H16NO SSi, 306.0615].

4

.1.6. N-(4-Bromophenyl)-N-methylbenzenesulfonamide (15). To a solution of 13 (302 mg, 0.966

mmol) in DMF (9.66 mL) was added sodium hydride (27.8 mg, 1.16 mmol), and the mixture was stirred

at room temperature for 30 min. Iodomethane (72.2 L, 1.16 mmol) was added, and stirring was

2

continued at 80 °C. The reaction was then quenched with H O, and the whole was extracted with EtOAc.

The organic layer was washed with brine and H O, dried over anhydrous MgSO

2

4

and evaporated. The

residue was purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to give 15 as a

1

pale-yellow oil (267 mg, 85%). H-NMR (500 MHz, CDCl

3

) δ 7.59 (dq, 1H, J = 11.5, 2.6 Hz), 7.54 (dd,

2

H, J = 8.6, 1.1 Hz), 7.49-7.45 (m, 2H), 7.42 (td, 2H, J = 6.0, 3.4 Hz), 6.97 (td, 2H, J = 5.9, 3.6 Hz), 3.15

+

(

s, 3H); MS (ESI+) m/z 348, 350 [(M+Na) ].

4

.1.7. N-(4-Bromophenyl)-N-ethylbenzenesulfonamide (16). To a solution of 13 (312 mg, 1.00 mmol)

in DMF (9.90 mL) was added sodium hydride (28.8 mg, 1.20 mmol), and the mixture was stirred at room

temperature for 30 min. Iodoethane (124 L, 1.20 mmol) was added, and stirring was continued at 80 °C.

The reaction was then quenched with H

2

O, and the whole was extracted with EtOAc. The organic layer

and evaporated. The residue was purified

was washed with brine and H O, dried over anhydrous MgSO

2

4

by silica gel column chromatography (n-hexane/EtOAc = 5:1) to give 16 as a pale-yellow oil (312 mg,

1

9

5

2%); H-NMR (500 MHz, CDCl

3

) δ 7.58 (dd, 3H, J = 9.2, 7.4 Hz), 7.48-7.43 (m, 4H), 6.91 (td, 2H, J =

+

.9, 3.6 Hz), 3.58 (q, 2H, J = 7.1 Hz), 1.07 (t, 3H, J = 7.2 Hz). MS (ESI+) m/z 362, 364 [(M+Na) ].

4

.1.8. N-(4-Bromophenyl)-N-butylbenzenesulfonamide (17). To a solution of 13 (367 mg, 1.18 mmol)

in DMF (11.8 mL) was added sodium hydride (33.9 mg, 1.41 mmol), and the mixture was stirred room

temperature for 30 min. 1-Iodobutane (162 L, 1.41 mmol) was added, and stirring was continued at

8

0 °C. The reaction was then quenched with H

2

O, and the whole was extracted with EtOAc. The organic

and evaporated. The residue was

layer was washed with brine and H O, dried over anhydrous MgSO

2

4

purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to give 17 as a pale-yellow oil

1

(

273 mg, 63%). H-NMR (500 MHz, CDCl

3

) δ 7.60-7.56 (m, 3H), 7.48-7.42 (m, 4H), 6.91 (dt, 2H, J =

5

3

.9, 3.6 Hz), 3.50 (t, 2H, J = 6.9 Hz), 1.41-1.25 (m, 4H), 0.86 (3H, t, J = 7.2 Hz); MS (ESI+) m/z 390,

+

92 [(M+Na) ].

4

.1.9. N-Benzyl-N-(4-bromophenyl)benzenesulfonamide (18). To a solution of 13 (429 mg, 1.37 mmol)

in DMF (13.7 mL) was added sodium hydride (39.5 mg, 1.65 mmol), and the mixture was stirred room

temperature for 30 min. Benzyl bromide (189 L, 1.65 mmol) was added, and stirring was continued at

8

0 °C. The reaction was then quenched with H

2

O, and the whole was extracted with EtOAc. The organic

and evaporated. The residue was

layer was washed with brine and H O, dried over anhydrous MgSO

2

4

purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to give 18 as a pale-yellow oil

1

(

465 mg, 84%); H-NMR (500 MHz, CDCl

3

) δ 7.66 (td, 2H, J = 4.3, 1.3 Hz), 7.62 (tt, 1H, J = 7.7, 1.7

Hz), 7.51 (t, 2H, J = 7.7 Hz), 7.32 (dt, 2H, J = 9.2, 2.4 Hz), 7.21 (dt, 5H, J =9.4, 4.4 Hz), 6.84 (td, 2H, J =

+

6

.0, 3.4 Hz), 4.70 (s, 2H); MS (ESI) m/z 424, 426 [(M+Na) ].

4

.1.10. N-(4-Bromophenyl)-N-phenethylbenzenesulfonamide (19). To a solution of 13 (327 mg, 1.05

mmol) in DMF (10.5 mL) was added sodium hydride (43.1 mg, 1.26 mmol), and the mixture was stirred

room temperature for 30 min. (2-Bromoethyl)benzene (170 L, 1.26 mmol) was added, and stirring was

2

continued at 80 °C. The reaction was then quenched with H O, and the whole was extracted with EtOAc.

The organic layer was washed with brine and H O, dried over anhydrous MgSO

2

4

and evaporated. The

residue was purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to give 19 as a

1

pale-yellow oil (265 mg, 61%). H-NMR (500 MHz, CDCl

3

) δ 7.56 (tdd, 3H, J = 8.5, 3.8, 2.3 Hz), 7.44

(

dq, 4H, J = 7.4, 2.4 Hz), 7.26 (dq, 2H, J = 11.3, 2.7 Hz), 7.21 (dq, 1H, J = 10.0, 2.3 Hz), 7.10 (t, 2H, J =

4

.3 Hz), 6.89 (td, 2H, J = 6.0, 3.4 Hz), 3.76-3.73 (m, 2H), 2.76 (dd, 2H, J = 8.9, 7.2 Hz); MS (ESI+) m/z

+

38, 440 [(M+Na) ].

4

.1.11. N-(4-Bromophenyl)-N-(2-methoxyethyl)benzenesulfonamide (20). To a solution of 13 (415 mg,

1

.33 mmol) in DMF (13.3 mL) was added sodium hydride (38.3 mg, 1.59 mmol), and the mixture was

stirred room temperature for 30 min. 2-Bromoethyl methyl ether (150 L, 1.59 mmol) was added, and

stirring was continued at 80 °C. The reaction was then quenched with H O, and the whole was extracted

with EtOAc. The organic layer was washed with brine and H O, dried over anhydrous MgSO and

2

4

evaporated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to

give 20 as a crude oil (377 mg, 77%). This material was used in the next step without further purification.

4

.1.12. Methyl N-(4-bromophenyl)-N-(phenylsulfonyl)glycinate (21). To a solution of 13 (304 mg,

0

.972 mmol) in DMF (9.72 mL) was added sodium hydride (40.0 mg, 1.17 mmol), and the mixture was

stirred room temperature for 30 min. Methyl bromoacetate (108 L, 1.17 mmol) was added, and the

stirring was continued at 80 °C. The reaction was then quenched with H O, and the whole was extracted

with EtOAc. The organic layer was washed with brine and H O, dried over anhydrous MgSO and

2

4

evaporated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 5:1) to

give 21 as a crude oil (165 mg, 44%). This material was used in the next step without further purification.

+

MS (ESI+) m/z 405, 407 [(M+Na) ].

4

.1.13. N-(4-(Hydroxydimethylsilyl)phenyl)-N-methylbenzenesulfonamide (22). To a solution of

PdCl (8.2 mg, 46.2 mol), 2-(di-t-butylphosphino)biphenyl (27.6 mg, 92.4 mol) and 15 (302 mg, 0.924

2

mmol) in 1-methyl-2-pyrrolidinone (9.20 mL) was added diisopropylethylamine (474 L) and the

mixture was stirred at 60 °C under an argon atmosphere. 1,2-Diethoxy-1,1,2,2-tetramethyldisilane (274



L, 1.11 mmol) was added to the mixture and stirring was continued at 60 °C for 4 h. The reaction

mixture was cooled to rt and CH CN (20.5 mL), 2-(dimethylamino)ethanethiol hydrochloride (32.7 mg,

.231 mmol) and 0.1 M acetic acid (26.7 mL) were added. The resulting mixture was stirred at rt for 2 h.

The reaction was then quenched with H O, and the whole was extracted with EtOAc. The organic layer

was washed with brine and H O, dried over anhydrous MgSO and evaporated. The residue was purified

3

0

2

4

by silica gel column chromatography (n-hexane/EtOAc = 2:1) to give 22 as a colorless oil (11.3 mg, 4%).

1

6

H-NMR (500 MHz, DMSO-d ) δ 7.71 (tt, 1H, J = 7.2, 1.6 Hz), 7.59 (dt, 2H, J = 12.4, 2.7 Hz), 7.52 (tdd,

4

H, J = 10.6, 5.3, 3.0 Hz), 7.09 (dt, 2H, J = 8.4, 2.0 Hz), 5.93 (1H, s), 3.13 (3H, s), 0.23 (s, 6H);

1

3

C-NMR (125 MHz, DMSO-d

6

) δ 142.38, 139.98, 136.78, 134.12, 133.82, 129.82, 127.86, 125.73, 38.35,

+

1

.09; HRMS (ESI+) m/z 344.0758 [(M+Na) : calcd for C15

H19NO

3

SSiNa, 344.0747].

4

.1.14. N-Ethyl-N-(4-(hydroxydimethylsilyl)phenyl)benzenesulfonamide (23). To a solution of PdCl

2

(8.8 mg, 49.9 mol), 2-(di-t-butylphosphino)biphenyl (29.8 mg, 99.8 mol) and 16 (340 mg, 1.00 mmol)

in 1-methyl-2-pyrrolidinone (9.90 mL) was added diisopropylethylamine (512 L), and the mixture was

stirred at 60 °C under an argon atmosphere. 1,2-diethoxy-1,1,2,2-tetramethyldisilane (296 L, 1.20

mmol) was added and the mixture was stirred at 60 °C for 4 h. The reaction mixture was cooled to rt and

CH

acid (28.8 mL) were added. The resulting mixture was stirred at rt for 2 h. The reaction was then

quenched with H O, and the whole was extracted with EtOAc. The organic layer was washed with brine

and H O, dried over anhydrous MgSO

3

CN (22.1 mL), 2-(dimethylamino)ethanethiol hydrochloride (35.3 mg, 0.250 mmol) and 0.1 M acetic

2

4

and evaporated. The residue was purified by silica gel column

1

chromatography (n-hexane/EtOAc = 2:1) to give 23 as a colorless oil (8.5 mg, 3%). H-NMR (500 MHz,

6

DMSO-d ) δ 7.69 (ddd, 1H, J = 12.7, 5.0, 3.9 Hz), 7.35 (d, 4H, J = 4.6 Hz), 7.28 (dt, 2H, J = 8.2, 1.9 Hz),

1

3

6

.81-6.78 (m, 2H), 5.70 (1H, s), 3.41-3.32 (m, 2H), 0.77-0.70 (m, 3H), 0.00 (s, 6H); C-NMR (125 MHz,

DMSO-d

6

) δ 140.23, 139.15, 138.12, 133.72, 133.05, 129.29, 127.56, 127.14, 45.06, 13.90, 0.51; HRMS

+

(

ESI+) m/z 358.0886 [(M+Na) : calcd for C16

H21NO

3

SSiNa, 358.0904].

4

.1.15. N-Butyl-N-(4-(hydroxydimethylsilyl)phenyl)benzenesulfonamide (24). To a solution of PdCl

2

(4.0 mg, 22.5 mol), 2-(di-t-butylphosphino)biphenyl (13.4 mg, 45.0 mol) and 17 (166 mg, 0.45 mmol)

in 1-methyl-2-pyrrolidinone (4.50 mL) was added diisopropylethylamine (231 L), and the mixture was

stirred at 60 °C under an argon atmosphere. 1,2-Diethoxy-1,1,2,2-tetramethyldisilane (133 L, 0.540

mmol) was added, and the mixture was stirred at 60 °C for 4 h, then cooled to rt. CH

-(dimethylamino)ethanethiol hydrochloride (16.0 mg, 0.113 mmol) and 0.1 M acetic acid (13.0 mL)

were added, and the resulting mixture was stirred at rt for 2 h. The reaction was then quenched with H O,

and the whole was extracted with EtOAc. The organic layer was washed with brine and H O, dried over

anhydrous MgSO and evaporated. The residue was purified by silica gel column chromatography

3

CN (10.0 mL),

2

4

1

(

6

n-hexane/EtOAc = 2:1) to give 17 as a colorless oil (15.8 mg, 10%). H-NMR (500 MHz, DMSO-d ) δ

7

1

.71-7.68 (m, 1H), 7.59 (dq, 4H, J = 12.3, 2.8 Hz), 7.52 (dt, 2H, J = 8.2, 1.9 Hz), 7.04 (dd, 2H, J = 10.3,

.7 Hz), 5.93 (s, 1H), 3.53 (t, 2H, J = 6.6 Hz), 1.28-1.25 (m, 4H), 0.81 (t, 3H, J = 7.2 Hz), 0.25 (s, 6H);

1

3

C-NMR (125 MHz, DMSO-d

6

) δ 140.73, 139.89, 138.48, 134.24, 133.58, 129.82, 128.00, 127.70, 49.94,

+

3

0.22, 19.41, 13.91, 1.04; HRMS (ESI+) m/z 386.1206 [(M+Na) : calcd for C18

H25NO

3

SSiNa, 386.1217].

4

.1.16. N-Benzyl-N-(4-(hydroxydimethylsilyl)phenyl)benzenesulfonamide (25). To a solution of PdCl

2

(10.3 mg, 57.8 mol), 2-(di-t-butylphosphino)biphenyl (34.5 mg, 116 mol) and 18 (465 mg, 1.16 mmol)

in 1-methyl-2-pyrrolidinone (11.6 mL) was added diisopropylethylamine (593 L), and the mixture was

stirred at 60 °C under an argon atmosphere. 1,2-Diethoxy-1,1,2,2-tetramethyldisilane (342 L, 1.39

mmol) was added, and the mixture was stirred at 60 °C for 4 h, then cooled to rt. CH

-(dimethylamino)ethanethiol hydrochloride (40.9 mg, 0.289 mmol) and 0.1 M acetic acid (33.2 mL)

were added, and the resulting mixture was stirred at rt for 2 h. The reaction was then quenched with H O,

and the whole was extracted with EtOAc. The organic layer was washed with brine and H O, dried over

3

CN (25.5 mL),

2

anhydrous MgSO

4

and evaporated. The residue was purified by silica gel column chromatography

1

(

n-hexane/EtOAc = 2:1) to give 25 as a colorless oil (84.5 mg, 18%). H-NMR (500 MHz, DMSO-d

6

) δ

7

.75-7.72 (m, 1H), 7.67 (dd, 2H, J = 5.4, 3.2 Hz), 7.64-7.61 (m, 2H), 7.42 (dd, 2H, J = 6.3, 1.7 Hz), 7.26

(t, 4H, J = 2.3 Hz), 7.20-7.17 (m, 1H), 7.07 (d, 2H, J = 8.6 Hz), 5.86 (s, 1H), 4.81 (s, 2H), 0.19 (s, 6H);

1

3

C-NMR (125 MHz, DMSO-d

6

) δ 140.48, 139.91, 138.58, 136.84, 134.06, 133.78, 130.00, 128.89,

+

1

28.41, 127.96, 127.85, 127.79, 53.85, 0.98; HRMS (ESI+) m/z 420.1083 [(M+Na) :calcd for

21 3

C H23NO SSiNa, 420.1060].

4

.1.17. N-(4-(Hydroxydimethylsilyl)phenyl)-N-phenethylbenzenesulfonamide (26). To a solution of

PdCl (5.6 mg, 31.8 mol), 2-(di-t-butylphosphino)biphenyl (19.0 mg, 63.6 mol) and 19 (265 mg, 0.636

2

mmol) in 1-methyl-2-pyrrolidinone (6.36 mL) was added diisopropylethylamine (0.326 mL), and the

mixture was stirred at 60 °C under an argon atmosphere. 1,2-Diethoxy-1,1,2,2-tetramethyldisilane (0.188

mL, 0.763 mmol) was added, and the mixture was stirred at 60 °C for 4 h. The reaction mixture was

cooled to rt and CH

and 1.0 M acetic acid (18.4 mL) were added to it. The resulting mixture was stirred at rt for 2 h. The

reaction was then quenched with H O, and the whole was extracted with EtOAc. The organic layer was

washed with brine and H O, dried over anhydrous MgSO and evaporated. The residue was purified by

3

CN (14.1 mL), 2-(dimethylamino)ethanethiol hydrochloride (22.5 mg, 0.159 mmol)

2

4

silica gel column chromatography (n-hexane/EtOAc = 2:1) to give 26 as a colorless oil (22.0 mg, 8%).

1

H-NMR (500 MHz, DMSO-d

6

) δ 7.68 (tt, 1H, J = 6.9, 1.9 Hz), 7.58-7.52 (m, 6H), 7.26 (dd, 2H, J = 9.7,

4

3

1

0

.6 Hz), 7.20 (dt, 1H, J = 10.5, 2.6 Hz), 7.13 (2H, t, J = 4.0 Hz), 7.05 (2H, d, J = 8.6 Hz) 5.96 (1H, s),

1

3

6

.79 (t, 2H, J = 7.4 Hz), 2.64 (t, 2H, J = 7.4 Hz), 0.26 (6H, s); C-NMR (125 MHz, DMSO-d ) δ 140.30,

39.29, 138.05, 137.76, 133.73, 133.10, 129.27, 128.71, 128.33, 127.51, 127.19, 126.35, 51.18, 34.19,

+

.53; HRMS (ESI+) m/z 434.1187 [(M+Na) : calcd for C22

H25NO

3

SSiNa, 434.1217].

4

.1.18. N-(4-(Hydroxydimethylsilyl)phenyl)-N-(2-methoxyethyl)benzenesulfonamide (27). To a

solution of PdCl (9.0 mg, 51.0 mol), 2-(di-t-butylphosphino)biphenyl (30.4 mg, 0.102 mmol) and 20

377 mg, 1.02 mmol) in 1-methyl-2-pyrrolidinone (10.2 mL) was added diisopropylethylamine (0.523

mL), and the mixture was stirred at 60 °C under an argon atmosphere.

,2-Diethoxy-1,1,2,2-tetramethyldisilane (0.302 mL, 1.22 mmol) was added to it, and the mixture was

stirred at 60 °C for 4 h, then cooled to rt. CH CN (22.5 mL), 2-(dimethylamino)ethanethiol hydrochloride

36.1 mg, 0.255 mmol) and 0.1 M acetic acid (29.3 mL) were added to it, and the resulting mixture was

stirred at rt for 2 h. The reaction was then quenched with H O, and the whole was extracted with EtOAc.

The organic layer was washed with brine and H O, dried over anhydrous MgSO and evaporated. The

2

(

1

3

(

2

4

residue was purified by silica gel column chromatography (n-hexane/EtOAc = 2:1) to give 27 as a

1

colorless oil (14.0 mg, 4%). H-NMR (500 MHz, DMSO-d

6

) δ 7.69 (dq, 1H, J = 10.3, 2.4 Hz), 7.60-7.58

(

m, 4H), 7.52-7.50 (m, 2H), 7.05-7.03 (m, 2H), 5.95 (1H, s), 3.74-3.70 (m, 2H), 3.29 (t, 2H, J = 5.7 Hz),

1

3

1

.15 (s, 3H), 0.24 (6H, s); C-NMR (125 MHz, DMSO-d

6

) δ 140.36, 139.63, 138.24, 133.73, 129.28,

+

27.68, 127.20, 69.21, 57.85, 49.73, 0.53; HRMS (ESI+) m/z 388.0994 [(M+Na) : calcd for

17 4

C H23NO SSiNa, 388.1009].

4

.1.19. Methyl N-(4-(hydroxydimethylsilyl)phenyl)-N-(phenylsulfonyl)glycinate (28). To a solution of

PdCl (3.5 mg, 0.0401 mmol), 2-(di-t-butylphosphino)biphenyl (11.8 mg, 39.5 mol) and 21 (165 mg,

.395 mmol) in 1-methyl-2-pyrrolidinone (3.95 mL) was added diisopropylethylamine (203 L) and the

mixture was stirred at 60 °C under an argon atmosphere. 1,2-Diethoxy-1,1,2,2-tetramethyldisilane (117

L, 0.474 mmol) was added, and the mixture was stirred at 60 °C for 4 h. The reaction mixture was

cooled to rt and CH CN (8.78 mL), 2-(dimethylamino)ethanethiol hydrochloride (14.0 mg, 98.8 mol)

and 1.0 M acetic acid (11.4 mL) were added to it. The resulting mixture was stirred at rt for 2 h. The

reaction was then quenched with H O, and the whole was extracted with EtOAc. The organic layer was

washed with brine and H O, dried over anhydrous MgSO and evaporated. The residue was purified by

2

0



3

2

4

silica gel column chromatography (n-hexane/EtOAc = 2:1) to give 28 as a colorless oil (4.3 mg, 3%).

1

6

H-NMR (500 MHz, DMSO-d ) δ 7.68 (ddd, 3H, J = 16.2, 10.7, 2.1 Hz), 7.58 (ddd, 2H, J = 10.6, 5.7, 2.0

Hz), 7.49 (dd, 2H, J = 8.3, 2.0 Hz), 7.15 (td, 2H, J = 4.6, 2.9 Hz), 5.93 (1H, s), 4.56 (d, 2H, J = 5.2 Hz),

1

3

1

.61 (d, 3H, J = 1.7 Hz), 0.22 (6H, s); C-NMR (125 MHz, CDCl

3

) δ169.31, 140.75, 140.00, 138.99,

+

34.03, 133.01, 128.89, 127.82, 127.77, 52.62, 52.46, 0.86; HRMS (ESI+) m/z 402.0822 [(M+Na) : calcd

5

for C17H21NO SSiNa 402.0802].

4

.1.20. N-(4-(2-Hydroxypropan-2-yl)phenyl)benzenesulfonamide (29). To a solution of 10 (329 mg,

.08 mmol) in THF was added methyl magnesium bromide (2.60 mL, 2.37 mmol) under an Ar

1

atmosphere at 0°C, and the mixture was stirred at the same temperature for 3 h. The reaction was then

quenched with H O, and the whole was extracted with EtOAc. The organic layer was washed with brine

and H O, dried over anhydrous MgSO

2

4

and evaporated. The residue was purified by silica gel column

1

chromatography (n-hexane/EtOAc = 3:1) to give 29 as an orange solid (87.3 mg, 28%). H-NMR

(

[

3

500MHz, CDCl ) δ 7.78 (dt, 2H, J = 8.4, 1.6 Hz), 7.56-7.52 (m, 1H), 7.44 (dt, 2H, J= 12.8, 2.7 Hz), 7.36

dt, 2H, J= 9.2, 2.3 Hz), 7.02 (dt, 2H, J = 9.2, 2.4 Hz), 6.64 (s, 1H), 1.52 (s, 6H); MS (ESI+) m/z 314

+

(M+Na) ].

4

.1.21. N-(4-(2-((Trimethylsilyl)oxy)propan-2-yl)phenyl)benzenesulfonamide (30). A solution of 29

275 mg, 0.943 mmol) in DMF (9.40 mL) was stirred at 0°C, and imidazole (257 mg, 3.77 mmol) was

added to it. Stirring was continued at 0°C. Trimethylsilyl chloride (239 L, 1.89 mmol) was added, and

the mixture was further stirred at 0°C for 1 h. The reaction was then quenched with H O, and the whole

(

2

was extracted with EtOAc. The organic layer was washed with brine and H

MgSO

500 MHz, CDCl

2

O, dried over anhydrous

1

4

and evaporated. This material was used in the next step without further purification. H-NMR

(

3

) δ 7.81 (s, 1H), 7.75 (d, 2H, J = 8.0 Hz), 7.48 (t, 1H, J = 7.4 Hz), 7.38 (t, 2H, J = 7.7

Hz), 7.26 (t, 2H, J = 4.3 Hz), 7.03 (d, 2H, J = 8.6 Hz), 1.48 (s, 6H), 0.00 (s, 9H); MS (ESI+) m/z 386

+

[

(M+Na) ].

4

.1.22. N-(4-(2-Hydroxypropan-2-yl)phenyl)-N-(phenylsulfonyl)glycinate (31). To a solution of 30 in

DMF (9.40 mL) was added sodium hydride (77.6 mg, 2.26 mmol), and the mixture was stirred at room

temperature for 30 min. Methyl bromoacetate (313 L, 3.39 mmol) was added, and the mixture was

stirred at 80°C. The reaction was then quenched with H

2

O, and the whole was extracted with EtOAc. The

and evaporated. The residue

organic layer was washed with brine and H O, dried over anhydrous MgSO

2

4

was purified by silica gel column chromatography (n-hexane/EtOAc = 2:1), and the trimethylsilyl group

1

was removed to give 31 as a yellow solid (295 mg, 86%). White powder, mp: 96-99°C; H-NMR (500

MHz, CDCl

3

) δ 7.69 (t, 2H, J = 4.3 Hz), 7.58 (t, 1H, J = 7.4 Hz), 7.46 (dd, 2H, J = 10.6, 5.4 Hz), 7.41 (td,

2

H, J = 5.7, 3.4 Hz), 7.17-7.14 (2H, m), 4.40 (2H, s), 3.70 (s, 3H), 1.55 (s, 6H); ¹³C-NMR (125 MHz,

3

CDCl ) δ169.35, 149.36, 139.14, 138.22, 132.95, 128.88, 128.65, 127.83, 125.53, 72.43, 52.82, 52.44,

3

1.83; HRMS (ESI+) m/z 386.1026 [(M+Na): calcd for C18H21NO SNa, 386.1033].

5

4

.2. Determination of logP value by an HPLC method

S1

We calculated of logP values based on the OECD Guideline for Testing Chemicals 117. The

measurements were performed on a Inertsil ODS-4 (5 μM, 4.6 × 150 mm) (GL Science Inc., Japan) by

using an HPLC instrument (detector (MD-2010, JASCO), pump (PU-2080, JASCO)) with acetonitrile

2

and H O (50%:50％). As reference compounds, thiourea, benzyl alcohol, methyl benzoate, 4-phenyl

phenol, benzyl benzoate and dibenzyl were used.

4

.3. Biological evaluation by reporter gene assay.

pcDNA3.1(-)-hRORα1, pcDNA3.1(-)-hRORβ1, pcDNA3.1(-)-hRORγ1 and RORE-TK-Luc were

provided by Itsuu Laboratory. CMX-Gal4N-hLXRα-LBD, CMX-Gal4N-hLXRβ-LBD,

CMX-Gal4N-hFXR-LBD, CMX-β-GAL, and tk-MH100×4 were provided by Professor Dr. Makoto

Makishima (Nihon University School of Medicine). pVP16-PXR was constructed by insertion of

hPXR-ORF into the multiple cloning site of pVP16 plasmid (Clontech). HEK 293 cells were cultured in

DMEM containing 5% FBS, penicillin and streptomycin mixture at 37 °C in a humidified atmosphere of

5

2

% CO in air. Cells were seeded at a density of 20% confluence/96-well plate 24 h prior to transfection.

Cells in each well were co-transfected 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 24 h, transfected cells were treated with test

compounds or DMSO for 24 h. Treated cells were assayed for luciferase activity with a Wallac ARVO

SX 1420 Multilabel Counter (PerkinElmer). The luciferase activity of each sample was normalized by the

β-galactosidase activity. Each transfection was carried out in triplicate. Assays were conducted in the

concentration range of 0.03 - 30 μM of each tested compound, and EC50 values were calculated by

sigmoid fitting using Origin data analysis software.

4. 4. Docking simulation

The structure of the LBD of hPXR was prepared from the Protein Data Bank accession 2O9I, chain

A [35]. Polar hydrogens and partial atomic charges were assigned using AutoDockTools (ADT).

Molecular docking was performed using AutoDock 4.2 with the Genetic Algorithm. AutoDock

parameters for silicon atom were Rii = 4.30 and eii = 0.402.

Acknowledgments

The hPXR reporter gene was given by S. Sato, Ph.D (Tokyo University of Science). This work was

partially supported by the Japan Society for the Promotion of Science (JSPS) Core-to-Core Program, A.

Advanced Research Networks, and Grants in-Aid for Scientific Research from JSPS (KAKENHI Grant

Nos. 20380071 and 23380070 (H.S.), Nos. 17H03996 and 16K15137 (Y.H.), and Nos. 17H03887 and

1

6K15138 (S.F.)).

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Graphical abstract

Article Doi

Development of novel silanol-based human pregnane X receptor (PXR) agonists with improved receptor selectivity

DOI: 10.1016/j.bmc.2018.07.038

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