Development of a Thyroid Hormone Receptor ... - ACS Publications

May 29, 2008 - The thyroid hormone receptors are important regulators of genes involved in metabolic regulation and homeostasis, as well as critical e...
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Bioconjugate Chem. 2008, 19, 1227–1234

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Development of a Thyroid Hormone Receptor Targeting Conjugate Jianfei Zheng, Atsushi Hashimoto,† Marc Putnam,‡ Katherine Miller, and John T. Koh* Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716. Received January 24, 2008; Revised Manuscript Received March 14, 2008

Molecular conjugates of hormone receptor-ligands with molecular probes or functional domains are finding diverse applications in chemical biology. Whereas many examples of hormone conjugates that target steroid hormone receptors have been reported, practical ligand conjugates that target the nuclear thyroid hormone receptor (TRβ) are lacking. TR-targeting conjugate scaffolds based on the ligands GC-1 and NH-2 and the natural ligand triiodothyronine (T3) were synthesized and evaluated in vitro and in cellular assays. Whereas the T3 or GC-1 based conjugates did not bind TRβ with high affinity, the NH-2 inspired fluorescein-conjugate JZ01 showed low nanomolar affinity for TRβ and could be used as a nonradiometric probe for ligand binding. A related analogue JZ07 was a potent TR antagonist that is 13-fold selective for TRβ over TRR. JZ01 localizes in the nuclei of TRβ expressing cells and may serve as a prototype for other TR-targeting conjugates.

INTRODUCTION Advances in chemical biology have led to many important new applications of hormone conjugates as tools to probe nuclear receptor biology. The high affinity and specificity of small-molecule hormones can be used to selectively direct new bimolecular functions to the sites of nuclear hormone receptors (NHR) action. Notable examples include testosterone-based PROTAC conjugates that can down-regulate AR stability by recruiting ubiqitin ligases (1, 2), or peptoid activation domain conjugates of dexamethasone that can be used to activate transcription from GR (glucocorticoid receptor) independent of its normal coactivator proteins (3). Other examples include fluorescent NHR agonists or ligand-fluorophore conjugates that have been used as nonradiometric methods for detecting NHR binding (4). Yet another new and exciting discovery in this area is hormone conjugates that selectively regulate non-nuclear hormone actions by limiting cellular penetration and localization (5). The development of hormone conjugates for NHRs is not always straightforward, as NHR agonist ligands are generally completely encapsulated by the protein in their bound forms. Many NHR antagonist have been shown to have molecular appendages or “extensions” that displace the active conformation of helix-12, an essential element of the coactivator binding site (6–9). In some cases, these appendages extend to the receptor surface providing a potential handle for generating new hormone conjugates. Structure-activity relationship studies revealed that the design of such molecular extensions is more complicated than simply introducing steric bulk at an appropriate site on the ligand, as subtle structural modifications to these extensions can have a substantial effect on ligand activity (10–12). Therefore, a priori, it is not obvious how to modify an agonist structure in a manner that would allow reporter groups or functional domains to be presented from the receptor surface while maintaining high-affinity binding. The thyroid hormone receptors are important regulators of genes involved in metabolic regulation and homeostasis, as well † Current address: Mitsubishi Pharma Corporation, 2-2-6, Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8405, Japan. ‡ Current Address: Hercules Incorporated, Wilmington, Delaware 19808.

as critical events in development (13). Mutations of TRβ have been implicated in RTH (resistance to thyroid hormone) as well as certain forms of cancer (14–17). New molecular probes of thyroid hormone action may help understand the molecular roles of TR in normal development and in these TR-associated disease states. Examples of thyroxine-fluorophore conjugates have been reported that change properties upon binding antithyroxine (T4) antibodies or detergent; however, no TR-targeting conjugates have been developed that effectively target the nuclear thyroid hormone receptor (TR) (18, 19). Recently, several TR antagonists and partial agonists prompted us to investigate the development of TR-targeting hormone conjugates. Of the recently described antagonists, the compound NH-3 and the closely related agonist NH-2 (Figure 1) were of particular interest because the 5′-arylethynyl appendages could be expected to point toward helix-12 and have demonstrated subnanomolar affinity for TRβ (11). Although NH-2 is reported to be a TR agonist, the additional n-pentyl substitution of NH-2 could be envisioned to access the protein surface from the ligand binding site of TR through displacement of helix-12 or from repositioning of helix-11 as has been observed in some TR-ligand cocrystal structures (20). Scaffold I was therefore proposed as a conjugate scaffold for presentation of molecular probes from TR (Figure 1). To evaluate the properties of scaffold I as a means to synthesize TR-targeting conjugates, we generated the fluorescein conjugated probe JZ01 and evaluated its ability to bind to TRβ.

EXPERIMENTAL PROCEDURES Synthesis of JZ01 and JZ07 5-(2,6-dimethyl-4-(triisopropylsilyloxy)benzyl)-3-isopropyl-2-(methoxymethoxy)benzaldehyde (2). Compound 1was made as previously described (21). To a solution of 1 (2.3 g, 4.9 mmol) in a 2:1 (v/v) mixture of anhydrous THF and hexane (48 mL) at -78 °C under N2 was added 5.4 mL 2 M n-BuLi solution in anhydrous hexane (10.8 mmol). The solution was allowed to warm to room temperature and stirred for 2 h before DMF (1.15 mL, 14.7 mmol) was added dropwise to the stirring solution. After an hour, the solution was poured over 1 N cold HCl solution (100 mL) and the mixture was extracted with ethyl acetate (2 × 50 mL). The organic extracts were washed with water (20 mL) and brine (20 mL), dried over

10.1021/bc8000326 CCC: $40.75  2008 American Chemical Society Published on Web 05/29/2008

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Figure 1. Thyroid hormone, T3, and the synthetic thyroid hormone analogues, GC-1 and NH-2/NH-3 and proposed TR-targeting conjugate scaffolds.

MgSO4, filtered, and evaporated under reduced pressure. The resulting crude product was purified by flash column chromatography on silica gel (1:10 ethyl acetate/hexane) to give compound 2 (1.68 g, 3.37 mmol, 69%). 1H NMR (400 MHz, CDCl3) δ 10.22 (s, 1H), 7.34 (d, J ) 1.6 Hz, 1H), 7.13 (d, J ) 1.6 Hz, 1H), 6.63 (s, 2H), 5.02 (s, 2H), 3.96 (s, 2H), 3.58 (s, 3H), 3.32 (heptet, J ) 6.8 Hz, 1H), 2.16 (s, 6H), 1.27 (heptet, J ) 6.8 Hz, 3H), 1.15-1.11 (m, 24H). 13C NMR (100 MHz, CDCl3) δ 191.09, 155.65, 154.22, 142.80, 138.03, 137.13, 132.24, 129.42, 128.76, 125.96, 119.68, 101.74, 57.76, 33.77, 26.18, 23.49, 20.37, 18.00, 12.54. HRMS (CI) calculated for [C30H46O4Si + H]+ 499.3244, found 499.3209. (4-(3-ethynyl-5-isopropyl-4-(methoxymethoxy)benzyl)-3,5dimethylphenoxy)triisopropylsilane (3). To a solution of diisopropylamine (0.46 g, 4.5 mmol) in anhydrous THF (10 mL) at -78 °C was added 1.8 mL 2.0 M solution of n-BuLi in anhydrous hexane (3.6 mmol). The solution was allowed to warm up to 0 °C and stirred for 30 min before it was cooled to -78 °C. Then 1.8 mL 2.0 M solution of TMS-diazomethane in anhydrous hexane was added dropwise to the solution at -78 °C and the solution was stirred for 30 min. To the reaction solution at -78 °C, 2 (1.5 g, 3.0 mmol) in 10 mL anhydrous THF was added dropwise and the solution was stirred at -78 °C for 1 h. Then the solution was allowed to reflux for 2.5 h, cooled to room temperature, and poured over ice-water. The mixture was extracted with ethyl acetate (2 × 50 mL), and organic extracts were washed with water (20 mL) and brine solution (20 mL), dried over MgSO4, and evaporated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (1:10 ethyl acetate/hexane) to give 3 (0.9 g, 1.8 mmol, 60%). 1H NMR (400 MHz, CDCl3) δ 6.90 (d, J ) 2 Hz, 1H), 6.86 (d, J ) 2 Hz, 1H), 6.60 (s, 2H), 5.17 (s, 2H), 3.88 (s, 2H), 3.60 (s, 3H), 3.36 (heptet, J ) 6.8 Hz, 1H), 3.17 (s, 1H), 2.15 (s, 6H), 1.25 (heptet, J ) 6.8 Hz, 3H), 1.23-1.10 (m, 24H). 13C NMR (100 MHz, CDCl3) δ 154.50, 154.07, 141.95,

138.13, 136.06, 130.52, 128.99, 127.04, 119.55, 115.39, 99.89, 81.21, 80.55, 57.55, 33.65, 26.34, 23.34, 20.36, 18.00, 12.71. HRMS (CI) calculated for [C31H46O4Si + H]+ 495.3294, found 495.3275. Methyl 6-(4-((5-(2,6-dimethyl-4-(triisopropylsilyloxy)benzyl)-3-isopropyl-2-(methoxymethoxy)phenyl)ethynyl)phenyl)hexanoate (5). Compound 4 was prepared as previously described. CuI (17 mg, 0.086 mmol) and bis(triphenylphosphine)palladium(II) chloride (61 mg, 0.086 mmol) were added into a Schlenk flask, and the flask was vacuumed then backfilled with N2 atmosphere 3 times. Compound 3 (850 mg, 1.72 mmol) and compound 4 (630 mg, 1.89 mmol) were dissolved in 20 mL distilled piperidine and the solution was added to the Schlenk flask. The reaction mixture was stirred overnight at 40 °C. The reaction mixture was poured over 100 mL cold 0.1 N HCl solution and extracted ethyl acetate (2 × 50 mL). The organic extracts were washed with water (30 mL) and brine solution (30 mL), dried over MgSO4, and evaporated under reduced pressure. The resulting crude product was purified by flash column chromatography on silica gel (1:10 ethyl acetate/hexane) to give 5 (950 mg, 79%). 1 H NMR (400 MHz, CDCl3) δ 7.39 (d, J ) 8 Hz, 2H), 7.12 (d, J ) 8 Hz, 2H), 6.89 (d, J ) 0.8 Hz, 2H), 6.63 (s, 2H), 5.26 (s, 2H), 3.91 (s, 2H), 3.64 (s, 3H), 3.60 (s, 3H), 3.39 (heptet, J ) 6.8 Hz, 1H), 2.60 (t, J ) 7.6 Hz, 2H), 2.29 (t, J ) 7.6 Hz, 2H), 2.17 (s, 6H), 1.64 (m, 4H), 1.34-1.23 (m, 5H), 1.16 (d, J ) 7.2 Hz, 6H), 1.11 (d, J ) 7.2 Hz, 18H). 13 C NMR (100 MHz, CDCl3) δ 174.12, 154.07, 153.81, 142.93, 141.84, 138.18, 135.99, 131.38, 129.81, 129.20, 128.48, 126.40, 120.73, 119.59, 116.61, 99.78, 92.94, 86.39, 57.55, 51.47, 35.66, 33.97, 33.75, 30.87, 28.70, 26.43, 24.79, 23.40, 20.42, 18.04, 12.74. HRMS (ESI) calculated for [C44H62O5Si + Na]+ 721.4264, found 721.4231.

Thyroid Hormone Receptor Conjugates

Methyl 6-(4-((5-(4-Hydroxy-2,6-dimethylbenzyl)-3-isopropyl-2-(methoxymethoxy) phenyl)ethynyl)phenyl)hexanoate (6). To a solution of compound 5 (140 mg, 0.2 mmol) in 2 mL THF was added 0.4 mL 1 M solution of Bu4NF in THF (0.4 mmol). The solution was stirred for 30 min and poured over 0.1 N HCl (30 mL). The mixture was extracted with ethyl acetate (2 × 30 mL). The organic portions were combined, washed with water (15 mL) and brine solution (15 mL), dried over MgSO4, and evaporated under reduced pressure. The resulting crude product was purified by flash column chromatography on silica gel (1:4 ethyl acetate/hexane) to yield 6 (96 mg, 88%). 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J ) 8.4 Hz, 2H), 7.12 (d, J ) 8.4 Hz, 2H), 6.96 (d, J ) 2 Hz, 1H), 6.86 (d, J ) 2 Hz, 1H), 6.56 (s, 2H), 5.26 (s, 2H), 5.21 (s, 1H), 3.89 (s, 2H), 3.67 (s, 3H), 3.61 (s, 3H), 3.41 (heptet, J ) 6.8 Hz, 1H), 2.60 (t, J ) 8 Hz, 2H), 2.31 (t, J ) 8 Hz, 2H), 2.17 (s, 6H), 1.64 (m, 4H), 1.35 (m, 2H), 1.17 (d, J ) 6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 174.49, 153.78 (2), 142.97, 141.87, 138.67, 136.01, 131.38, 129.54, 128.70, 128.47, 126.64, 120.61, 116.66, 114.90, 99.76, 93.03, 86.30, 57.60, 51.62, 35.63, 34.02, 33.76, 30.83, 28.68, 26.46, 24.77, 23.43, 20.40. HRMS (ESI) calculated for [C35H42O5 + Na]+ 565.2930, found 565.2946. Methyl 6-(4-((5-(4-(2-Tert-butoxy-2-oxoethoxy)-2,6-dimethylbenzyl)-3-isopropyl-2-(methoxymethoxy)phenyl)ethynyl)phenyl)hexanoate (7). To the solution of 6 (49 mg, 0.09 mmol) and cesium carbonate (117 mg, 0.36 mmol) in 4 mL 1:1 (v/v) mixture of anhydrous THF and DMF was added tertbutylbromoacetate (0.029 mL, 0.18 mmol). The reaction mixture was allowed to stir for 6 h at room temperature, then poured over 15 mL of cold 0.1 N HCl and extracted with ethyl acetate (3 × 15 mL). The combined organic extracts were washed with water (10 mL) and brine solution (10 mL) and concentrated under reduced pressure to afford crude product, which was purified by flash column chromatography on silica gel (1:4 ethyl acetate/hexane) to yield 7 (54.1 mg, 92%). 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J ) 8 Hz, 2H), 7.12 (d, J ) 8 Hz, 2H), 6.94 (d, J ) 2 Hz, 1H), 6.85 (d, J ) 2.4 Hz, 1H), 6.63 (s, 2H), 5.25 (s, 2H), 4.52 (s, 2H), 3.91 (s, 2H), 3.67 (s, 3H), 3.61 (s, 3H), 3.41 (heptet, J ) 6.8 Hz, 1H), 2.61 (t, J ) 7.6 Hz, 2H), 2.31 (t, J ) 7.6 Hz, 2H), 2.21 (s, 6H), 1.68-1.61 (m, 4H), 1.49 (s, 9H), 1.35 (m, 2H), 1.17 (d, J ) 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 174.19, 168.35, 156.03, 153.85, 142.95, 141.88, 138.48, 135.76, 131.37, 129.69, 129.56, 128.44, 126.57, 120.63, 116.64, 114.22, 99.76, 92.97, 86.27, 82.18, 65.74, 57.58, 51.51, 35.64, 33.98, 33.83, 30.04, 28.68, 28.06, 26.44, 24.76, 23.40, 20.59. HRMS (ESI) calculated for [C41H52O7 + Na]+ 679.3611, found 679.3644. 6-(4-((5-(4-(2-Tert-butoxy-2-oxoethoxy)-2,6-dimethylbenzyl)-3-isopropyl-2-(methoxymethoxy)phenyl)ethynyl)phenyl)hexanoic acid (8). To the solution of 7 (40 mg, 0.06 mmol) in 0.3 mL of THF was added 0.66 mL of 0.1 N LiOH solution over 2 h. The reaction mixture was stirred for 24 h, poured over 5 mL of cold 0.1 N HCl, and extracted with ethyl acetate (2 × 15 mL). The organic extracts were washed with water (2 × 10 mL) and brine solution (10 mL), dried over MgSO4, and evaporated under reduced pressure. The resulting crude was purified by flash column chromatography on silica gel (1:2 ethyl acetate/hexane) to yield 8 (22.4 mg, 58%). 1H NMR (400 MHz, CDCl3) δ 7.39(d, J ) 8.4 Hz, 2H), 7.12 (d, J ) 8.4 Hz, 2H), 6.94 (d, J ) 2 Hz, 1H), 6.85 (d, J ) 2 Hz, 1H), 6.63 (s, 2H), 5.25 (s, 2H), 4.51 (s, 2H), 3.91 (s, 2H), 3.60 (s, 3H), 3.41 (heptet, J ) 6.8 Hz, 1H), 2.61 (t, J ) 7.6 Hz, 2H), 2.35 (t, J ) 7.6 Hz, 2H), 2.21 (s, 6H), 1.69-1.61 (m, 4H), 1.49 (s, 9H), 1.39-1.37 (m, 2H), 1.80 (d, J ) 6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 179.53, 168.40, 156.03, 153.85, 142.90, 141.88, 138.48, 135.77, 131.39, 129.69, 129.58, 128.31, 126.58, 120.66, 116.65, 114.23, 99.76, 92.98, 86.29, 82.21, 65.75, 57.58, 35.63, 33.88,

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33.84, 30.85, 28.62, 28.06, 26.44, 24.49, 23.41, 20.60. HRMS (ESI) calculated for [C40H50O7+Na]+ 665.3454, found 665.3436. Perfluorophenyl 6-(4-((5-(4-(2-Tert-butoxy-2-oxoethoxy)2,6-dimethylbenzyl)-3-isopropyl-2-(methoxymethoxy)phenyl)ethynyl)phenyl)hexanoate (9). To 8 (18 mg, 0.029 mmol) in anhydrous THF was added triethyl amine (0.0045 mL, 0.032 mmol) and then pentafluorophenyl trifluoroacetate (0.006 mL, 0.035 mmol). The solution was stirred for 30 min and diluted with 3 mL of ethyl acetate before pouring over cold 0.1 N HCl. The mixture was extracted with ethyl acetate (2 × 10 mL), washed with water (2 × 5 mL) and brine solution (5 mL), dried over MgSO4, and concentrated under reduced pressure to yield 9 (22 mg, 94%) without further purification. 1H NMR (400 MHz, CDCl3) δ 7.41 (d, J ) 8 Hz, 2H), 7.13 (d, J ) 8.4 Hz, 2H), 6.94 (d, J ) 2.4 Hz, 1H), 6.85 (d, J ) 2.0 Hz, 1H), 6.63 (s, 2H), 5.25 (s, 2H), 4.51 (s, 2H), 3.91 (s, 2H), 3.61 (s, 3H), 3.41 (heptet, J ) 6.8 Hz, 1H), 2.66 (m, 4H), 2.21 (s, 6H), 1.83-1.63 (m, 4H), 1.49-1.40 (m, 11H), 1.83 (d, J ) 6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 169.48, 168.43, 156.02, 153.85, 142.86, 141.89, 138.48, 135.78, 131.42, 131.34, 129.70, 129.57, 128.43, 126.61, 120.75, 116.62, 114.22, 99.76, 92.92, 86.33, 82.24, 65.73, 57. 58, 35.58, 33.83, 33.23, 30.71, 28.38, 28.05, 26.44, 24.60, 23.39, 20.58. HRMS (ESI) calculated for [C46H49F5O7 + Na]+ 831.3296, found 831.3322. N-Fluoresceinylmethyl 6-(4-((5-(4-(2-Tert-butoxy-2-oxoethoxy)-2,6-dimethylbenzyl)-3-isopropyl-2-(methoxymethoxy)phenyl)ethynyl)phenyl)hexanamide (10). To the solution of 9 (18.5 mg, 0.023 mmol) and 10 mg 5-(aminomethyl)fluorescein, hydrochloride (Invitrogen, catalog number A1353) in 1 mL of 1:1 (v/v) mixture of anhydrous THF and DMF was added TEA (6.8 µL, 0.048 mmol) and the solution was allowed to stir at room temperature for 2 h. The reaction mixture was pour over cold water (5 mL), extracted with ethyl acetate (2 × 10 mL), washed with brine solution (2 × 5 mL), dried over MgSO4, and evaporated under reduced pressure. The resulting crude product was purified by column chromatography on silica gel (1:9 MeOH/CH2Cl2) to yield 10 (7.7 mg, 34%) and recovered 9.0 mg of 9 (49%). 1H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.57 (d, J ) 8 Hz, 1H), 7.26 (d, J ) 8 Hz, 2H), 7.08-7.03 (m, 3H), 6.89 (d, J ) 1.6 Hz, 1H), 6.72 (d, J ) 2 Hz, 1H), 6.57-6.54 (m, 4H), 6.48-6.42 (m, 4H), 5.10 (s, 2H), 4.45 (s, 2H), 4.42 (s, 2H), 3.86 (s, 2H), 3.46 (s, 3H), 3.32 (heptet, J ) 6.8 Hz, 1H), 2.53 (t, J ) 6.4 Hz, 2H), 2.19 (t, J ) 6.8 Hz, 2H), 2.10 (s, 6H), 1.62-1.54 (m, 4H), 1.37 (s, 9H), 1.37-1.30 (m, 2H), 1.07 (d, J ) 6.8 Hz, 6H). HRMS (ESI) calculated for [C73H53NO6 + Na]+ 702.3771, found 702.3759. N-Fluoresceinylmethyl 6-(4-((5-(4-(Carboxymethoxy)-2,6dimethylbenzyl)-2-hydroxy-3-isopropylphenyl)ethynyl)phenyl)hexanamide (JZ01). To the solution of 10 (3.8 mg, 0.0039 mmol) in 0.5 mL of THF was added 1 mL of 6 N HCl (aq). The reaction mixture was allowed to stir at 40 °C for 3 h and then extracted with ethyl acetate (3 × 5 mL). The organic extracts were washed with H2O (2 mL) and brine solution (2 mL), dried over MgSO4, and concentrated under reduced pressure. The resulting residue was purified by reverse-phase HPLC (Higgins, C8, 5 µm, 40-60% acetonitrile/water) to yield JZ01 (0.73 mg, 21%). Purity was assessed by HPLC (UV, 254 nm) to be >98%. 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.51 (d, J ) 7.6 Hz, 1H), 7.31 (d, J ) 8 Hz, 2H), 7.12-7.03 (m, 3H), 6.83 (s, 1H), 7.73-7.20 (m, 2H), 7.57-7.55 (m, 5H), 6.45 (d, J ) 8.8 Hz, 2H), 4.42 (s, 2H), 4.26 (s, 2H), 3.79 (s, 2H), 3.54 (s, 2H), 3.38 (heptet, J ) 6.8 Hz, 1H), 2.54 (t, J ) 7.6 Hz, 2H), 2.19 (t, J ) 7.6 Hz, 2H), 2.10 (s, 6H), 1.63-1.52 (m, 4H), 1.31-1.25 (m, 2H), 1.02 (d, J ) 6.4 Hz, 6H), HRMS (ESI) calculated for [C55H51NO10 + H]+ 886.3591, found 886.3608.

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(Tert-butyl-2-(4-(3-isopropyl-4-(methoxymethoxy)-5-((4-(6oxo-6-(prop-2-ynylamino)hexyl)phenyl)ethynyl)benzyl)-3,5dimethylphenoxy)acetate) (11). To a solution of 39.0 mg of 9 (0.048 mmol) and 26 mg of propargyl amine (0.48 mmol) in 0.9 mL of anhydrous THF was added a solution of 6 mg of TEA in 0.1 mL of anhydrous THF. After 30 min at ambient temperature, the solution was diluted with 15 mL of ethyl acetate and washed with 2 × 5 mL of 0.1 N HCl (aq) and 2 × 5 mL of brine, dried over Mg2SO4, and evaporated under reduced pressure. The residue was purified by flash column chromatography on silica gel (1:1 ethyl acetate/hexane) to yield 29.2 mg of 11 (89%). 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J ) 8.0 Hz, 2H), 7.11 (d, J ) 8.0 Hz, 2H), 6.94 (d, J ) 2.4 Hz, 1H), 6.85 (d, J ) 2.0 Hz, 1H), 6.63 (s, 2H), 5.74 (s, 2H), 4.51 (s, 2H), 4.04-4.02 (m, 2H), 3.91 (s, 2H), 3.41 (heptet, J ) 6.8 Hz, 1H), 2.60 (t, J ) 7.6 Hz, 2H), 2.22-2.15 (m, 9H), 1.70-1.59 (m, 4H), 1.49 (s, 9H), 1.35-1.20 (m, 2H), 1.18 (d, J ) 6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 172.57, 168.37, 156.04, 153.84, 142.95, 141.89, 138.47, 135.77, 131.37, 129.70, 129.56, 128.45, 126.59, 120.63, 116.64, 114.24, 99.75, 92.98, 86.31, 82.19, 79.67, 71.53, 65.75, 57.57, 36.27, 35.63, 33.83, 30.87, 29.13, 28.74, 28.06, 26.44, 25.31, 23.40, 20.58. HRMS (ESI) calculated for [C37H41NO5+Na]+ 602.2882, found 602.2875. (2-(4-(4-Hydroxy-3-isopropyl-5-((4-(6-oxo-6-(prop-2-ynylamino)hexyl)phenyl)ethynyl)benzyl)-3,5-dimethylphenoxy)acetic acid) (JZ07). To a solution of 14.6 mg of 11 in 1 mL of THF was added 1 mL of 6 N HCl (aq) . The reaction mixture was stirred at 40 °C for 8 h. The reaction mixture was cooled, diluted with 15 mL ethyl acetate, washed with 3 × 5 mL H2O (×3) and 5 mL of brine (×2), dried over Mg2SO4, and evaporated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (1:15 MeOH/DCM) to yield 11.6 mg of JZ07 (95%). 1H NMR (400 MHz, CDCl3) δ 7.40 (dd, J ) 7.6 Hz, 1.6 Hz, 2H), 7.12 (d, J ) 8.4 Hz, 2H), 6.94 (d, J ) 1.6 Hz, 1H), 6.69 (d, J ) 1.6 Hz, 1H), 6.65 (s, 2H), 5.68 (bs, 1H), 4.66 (s, 2H), 4.05-4.03 (m, 2H), 3.88 (s, 2H), 3.26 (heptet, d, J ) 6.8 Hz, 1H), 2.60 (t, d, J ) 7.6 Hz, 2H), 2.23-2.14 (m, 9H), 1.69-1.61 (m, 4H), 1.36-1.30 (m, 2H), 1.22 (d, d, J ) 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 172.95, 172.44, 155.51, 152.05, 143.43, 138.75, 134.12, 131.54, 131.36, 130.62, 128.57, 127.31, 127.01, 119.75, 114.18, 109.09, 96.05, 83.32, 79.44, 71.72, 64.91, 36.25, 35.60, 33.64, 30.77, 29.27, 28.64, 27.55, 25.30, 22.34, 20.55. HRMS (ESI) calculated for [C37H41NO5+Na]+ 602.2882, found 602.2875. Plasmid Construction. Expression vectors pET15b-TRβ wild-type, F451∆ and C446∆ mutant was constructed by subcloning the wild-type and mutant sequences for thyroid hormone receptor into the BamH I and Nde I sites of pET15b (Novagen), using the cloning primers 5′-GGAAAATTCCATATGACCCCCAACAGTATGACAGAAAATGGC-3′ and 5′CGCGGATCCCTAATCCTCGAACACTTCCAGGAACAAAGG-3′ (w.t.). Protein Expression and Purification. Full-length histidinetagged recombinant thyroid receptor protein was expressed in E. coli and purified by Ni-NTA affinity chromatography. Rosetta (DE3)pLysS cells (Novagen) were transformed with pET15bTRβ (wt or mutant). The cells were grown at 18 °C in 500 mL Luria Broth containing 100 µg/mL ampicillin and 34 µg/mL chloramphenicol to an OD of 0.3, at which point protein expression was induced with 1 mM IPTG and continued for 20 h at 18 °C. The cells were harvested by centrifugation at 4000 g for 30 min at 4 °C and the cell pellet was immediately frozen at -80 °C. Cells were resuspended with 5 mL lysis buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 12 mM 2-mercaptoethanol, 10% glycerol, and Roche complete EDTA free protease inhibi-

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tors) and sonicated on ice (15 s bursts, 4 times). The lysate was centrifuged for 10 min at 14 000 g (4 °C) and the soluble supernatant was loaded onto Ni-NTA resin (Qiagen) from which thyroid receptor was eluted with 250 mM imidazole. Protein concentration was determined by Bradford assay (BioRad) and purity was assessed by SDS-PAGE and Western blot with thyroid hormone receptor monoclonal antibody C3 (Covance). Yield was approximately 1 mg purified protein per liter of culture. Binding Assay and Competition Assay. A 0.7 mM stock solution of JZ01 in DMSO was prepared, which was diluted with binding buffer containing 50 mM sodium phosphate, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.01% Nonidet P-40, and 10% glycerol (pH ) 7.2) to obtain a 5.0 nM solution. Solutions of TRβ with different concentrations ranging from 1 µM to 0.3 nM were prepared with the same buffer. 90 µL aliquots of the TRβ solutions were mixed with 10 µL of 5.0 nM JZ01 solution in individual triplicate wells of a 96-well plate and incubated at room temperature for 1 h. Fluorescence polarization value (mP) was measured on Fusion plate reader (Perkin-Elmer, excitation ) 485 nM, emission ) 535 nM). As a control, solutions of ovalbumin with concentrations ranging from 1 µM to 1 nM were prepared, then mixed with the solution of JZ01 and measured with fluorescence polarization value similarly to TRβ. The probe JZ01 bound to TRβ with an apparent EC50 ) 21.4 ( 5.6 nM and the dynamic range about 250 mP. Ovalbumin showed only a very small change of fluorescence polarization even at a high concentration of 1 µM. (Note: BSA should not be used as a control as it has known thyronine binding capacity). The ligands T3 and GC-1 were evaluated for their ability to competitively displace the probe JZ01 from TRβ. A solution of TRβ (43 µL, 2.8 µM) was added to 6 mL of 0.1 nM probe JZ01 in the binding buffer containing 50 mM sodium phosphate, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.01% Nonidet P-40, and 10% glycerol (pH ) 7.2) and aliquoted into individual wells of a 96-well plate (90 µL/well). The solutions were incubated at room temperature for 30 min before adding the solutions of ligands. The stock solutions of the ligands were diluted with the same buffer to achieve different concentrations ranging from 100 µM to 1 nM. Then 10 µL of the solutions of ligands were added to the wells containing 90 µL of solution of JZ01 and TRβ and incubated at room temperature for 1 h before taking the FP value. The assay was conducted in triplicate. The measured FP value (mP) was plotted against the concentration of the ligands. The IC50 values for T3 and GC-1 were determined to be 429.3 ( 249.2 nM and 214.23 ( 134.7 nM, respectively. Cellular Reporter Gene Assays. Cellular reporter gene assays were performed as described previously (22). Briefly, HEK293 cells were transiently transfected (Calcium phosphate method) with pSG5-TRβ (or pSG5-TRR), DR4-tk-Luc (reporter), and pRL-CMV (control). Ligand-dependent transcription response was measured by dual-luciferase assay (Promega) following manufacturer’s protocol. Under these assay conditions, T3 is slightly more potent in TRR (EC50 ) 0.61 nM) than in TRβ (EC50 ) 1.4 nM). Cellular Localization Studies. Cellular localization studies were performed with NIH3T3 cells seeded at 45 000 cells per well in 24-well plates. Cells were transfected with pSG5-TRβ using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. After 24 h, the cells were washed and treated with 50 nM of JZ01 in PBS buffer at 37 °C for 1 h. Direct DAPI treatment without fixation/permeabilization was achieved by treatment with 600 nM of DAPI (Invitrogen) at room temperature for 2 min followed by PBS wash (500 µL × 2). Fixation/permeabilization was performed by treatment with 4% formaldehyde in PBS at 37 °C for 15 min, washed 2× in PBS,

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Figure 2. Structures of hormone conjugates MP01 and AH-20. Scheme 1. Synthesis of Scaffold I Based Fluorescent Probe JZ01a

a (a) (1) n-BuLi,THF/Hexane, (2) DMF; (b) TMSCLiN2, THF; (c) (PPh3)2PdCl2, CuI, Piperidine; (d) TBAF,THF; (e) Cs2CO3, tert-butyl bromoacetate, DMF/THF; (f) LiOH, H2O/THF; (g) pentafluorophenyl trifluoroacetate, TEA, THF, 0 °C-RT; (h) 5-(aminomethyl)fluorescein hydrochloride, TEA, THF/DMF; (i) 6 N HCl(aq)/THF, 40°C.

Figure 3. (A) Fluorescence polarization titration of JZ01 (0.5 nM) with TRβ (σ) and ovalbumin control (ϑ). (B) Competitive titration of JZ01 (0.1 nM) and TRβ (20 nM) with T3 (ν) and GC-1 (σ).

followed by treatment with 0.2% Triton X-100 in PBS for 10 min and 2× rinse with PBS. DAPI solution (600 nM in PBS) was then applied to the fixed/permeabilized cells, incubated for 2 min, and washed 2× in PBS according to the Invitrogen protocol.

RESULTS In the course of developing JZ01, we developed two additional scaffolds having flexible tethers attached via the 5′-

position of T3 and the halogen-free thyromimetic GC-1 (Figure 1, scaffolds II and III) (21). Model conjugate AH-20 based on the structure of T3 and the peptide conjugate MP01 based on GC-1 were also conjugated via the 5′-position but showed poor affinity by radioligand displacement assays (Figure 2; see Supporting Information). AH-20 showed no appreciable binding to TRβ wild-type below 1 µM, suggesting that the T3 core itself is not sufficient to provide high-affinity binding to TRβ when it has a flexible alkyl tether at the 5′-position. The GC-1 based

1232 Bioconjugate Chem., Vol. 19, No. 6, 2008

peptide conjugate, MP01, shows submicromolar binding to TRβ wild-type (Ki ) 580 nM) but is over 2500× less potent than T3 or GC-1. To further explore the properties of MP01, we evaluated its binding to truncated forms of the receptor TRβC446∆ and TRβF451∆ in radioligand displacement assays. These nonsense mutants completely lack the c-terminal helix-12 but have previously been shown to retain low nanomolar binding affinity for T3 (23, 24). MP01 had modest but improved binding to the nonsense mutants TRβC446∆, Ki ) 73.8 nM (log Ki ) 1.86 ( 2.37), and TRβF451∆, Ki ) 237 (log Ki ) 2.37 ( 1.27 nM) (25). The improved affinities of MP01 for the these receptors lacking helix-12 imply that this scaffold forms unfavorable interactions with helix-12 in its bound conformation. Together, these examples further illustrate that the development of highaffinity scaffolds for TR targeting conjugates is nontrivial and often depends on subtle aspects of the ligand structure. Synthesis of JZ01, a Fluorescent Conjugate Based on NH-2. The fluorescent probe, JZ01, was synthesized from the differentially protected bisphenol 1 by formylation of the methoxymethyl (MOM) protected phenol followed by CoreyFuchs alkynylation. Sonogoshira coupling afforded the diarylalkyne 5 in good yield. Desilylation and alkylation of the triisopropyl silyl (TiPS) protected phenol followed by selective hydrolysis of the methyl ester afforded compound 8, which served as the protected form of our scaffold. The scaffold 8 can be coupled to amine probes such as 5-(aminomethyl)fluorescein via the pentafluorophenyl ester. Deprotection with 6 N aqueous HCl in THF afforded the target fluorescent probe compound JZ01 (Scheme 1). Fluorescence Polarization Assays with JZ01. The affinity of JZ01 for TRβ can be measured directly by fluoresence polarization (FP) assay (Figure 3A). The polarization of the fluorescent probe JZ01 (0.5 nM) was measured as a function of increasing concentration of full-length TRβ. A sigmoidal dose-response of the FP value (mP) was observed with TRβ but not with ovalbumin control. The titration curve showed that the probe JZ01 bound to TRβ with Kd ) 21.4 ( 5.6 nM. These results suggest that the NH-2-inspired conjugate scaffold was a high-affinity probe for TRβ, that could accommodate even relatively bulky substitutions such as fluorescein. As a probe for binding the conjugate, JZ01 provided a relatively large dynamic range even at probe concentrations below 1 nM. To further explore the utility of JZ01, we evaluated its ability to serve as a nonradiometric probe for TR binding of ligands through competition. Competition assays were conducted with the native TR ligand, T3, and GC-1 as the competing ligands. The measured FP value (mP) was plotted against the concentration of the competing ligands (Figure 3B). T3 and GC-1 have been reported to have similar potencies toward TRβ and were determined to have IC50 values of 152.9 ( 87 nM and 93.5 ( 26 nM (average of two determinations ( min/max), respectively, using fluorescence polarization. JZ01 provides a facile nonradiometric means to compare the potency of TRβ ligands by competition. Subtype Selective Antagonist. The agonist and antagonist activities of JZ01 for TRR and TRβ were further determined by cellular reporter gene assays in HEK293 cells transiently transfected with pSG5TRR or pSG5TRβ and a DR4-luc reporter. JZ01 showed no detectable agonist activity at or below 250 nM (data not shown). JZ01 does have significant antagonist activity at 250 nM (38% reduction in activity) against 1 nM T3 in cells expressing TRβ but only slight antagonist activity (15% reduction) with 1 nM T3 in cells expressing TRR (Figure 4A). Concerns about potential fluorescein aggregation of JZ01 at high concentrations precluded us from establishing an accurate measure of its potency (IC50).

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Figure 4. Antagonist activities of ligands in cellular luciferase reporter gene assays. (A) 250 nM ligands with 1 nM T3 andTRR or TRβ. (B) Structure of JZ07. (C) Cellular dose-response behavior of JZ07 vs 1 nM T3 and TRR (σ) or TRβ (ν).

To assess the generic subtype selectivity of Scaffold I, the analogue JZ07, having a propargyl group in place of the fluorescein of JZ01, was prepared by coupling propargyl amine to intermediate 9 (11, R3dNHCH2CCH) followed by deprotection (Figure 4B). The smaller propargyl amide can be used to assess the intrinsic selectivity of the scaffold in the absence of potential influence of the much larger fluorescein group of JZ01. JZ07 had nearly identical activity to JZ01 at 250 nM in antagonist assays with TRβ wt and T3 (Figure 4A). The analogue JZ07 showed no signs of cellular toxicity at concentrations up to 10 µM and is a full antagonist of TRR (IC50 ) 2.83 ( 0.3 µM) and TRβ (IC50 ) 221 ( 76 nM) (Figure 4C). This is in contrast to the parent compound NH-2 that was reported as a weak TRβ agonist (26). In addition to being more potent

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Figure 5. (a) 3T3 cells transfected with pSG5-TRβ with 50 nM of JZ01 and stained with DAPI; (b) cells from panel viewed under fluorescein filter (Nikon B-2E/C); (c) DAPI-stained pSG5-TRB transfected 3T3 cells view under fluorescein filter; (d) pSG5-TRB transfected 3T3 cells with 50 nM, fixed and DAPI-stained; (e) cells from panel d viewed under fluorescein filter; (f) cells from panel d observed under DAPI-filter; (g) merge of panels e and f; (h) 3T3 cells (no TRβ) with JZ01; (i) same as panel h observed under fluorescein filter.

than the structurally related nitro-substituted antagonist NH-3, the compound JZ07 is as subtype selective as the most selective TR antagonist reported to date (IC50R/IC50β ) 12.8) (27–30). Subcellular localization of JZ01 was further examined in NIH3T3 cells treated with 50 nM JZ01 and visualized by fluorescence microscopy (Figure 5). Strong intracellular JZ01 fluorescence was visible only in NIH3T3 cells transfected with pSG5TRβ (compare Figure 5b and f). This cellular fluorescence is almost completely blocked by the addition of 500 nM T3, further supporting that staining is TR-specific (see Supporting Information). JZ01’s localization was further demonstrated to be nuclear and coincident with DAPI nuclear stain. Whereas efficient DAPI staining requires first fixing the cells, the fixation process caused significant loss of the intracellular but noncovalently bound JZ01 (compare Figure 5b to e). Nonetheless, although the JZ01 fluorescence is diminished JZ01 is clearly coincident with the DAPI nuclear stain (Figure 5g). In the absence of transfected TRβ, JZ01 is more diffuse and less intense, suggesting that JZ01 is indeed binding TRβ in cells (Figure 5i).

DISCUSSION Studies with the fluorescent probe JZ01 and antagonist JZ07 demonstrate that Scaffold I represents, to our knowledge, the first thyroid hormone conjugate capable of high-affinity binding of the nuclear thyroid hormone receptor TRβ. Conceptually similar molecular scaffolds based on the natural hormone T3 or the halogen-free thyromimetic GC-1 were not found to be as effective. The compound JZ01 can serve as an efficient probe for direct visualization of TRβ expression in cells and as a nonradiometric probe for ligand binding in vitro. The analogue JZ07 is a potent and highly β-subtype-selective TR antagonist. These findings suggest that scaffold I may be employed as a

general scaffold for making β-selective TR-targeting hormone conjugates for a variety of applications in chemical biology.

ACKNOWLEDGMENT We thank the National Institutes of Health DK054257 for support of this work. A.H. was supported by Mitsubishi Pharma Corp. Supporting Information Available: Complete synthetic details for the preparation ad characterization of of MP01 and AH-20 along with additional cellular imaging methods are available. This material is available free of charge via the Internet at http://pubs.acs.org.

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