Design, synthesis and evaluation of novel ¹⁹F magnetic resonance sensitive protein tyrosine phosphatase inhibitors

Abstract

Fluorine is a highly attractive element for both medicinal chemistry and imaging technologies. To facilitate protein tyrosine phosphatases (PTPs)-targeted drug discovery and imaging-guided PTP research with fluorine, several highly potent and ¹⁹F MR sensitive PTP inhibitors were discovered through a structure-based focused library strategy.

Graphical abstract

The first ¹⁹F MRI sensitive PTP inhibitors were discovered through a structure-based focus library strategy. Ortho-bis(trifluoromethyl)carbinol phenol not only successfully mimics the interactions between salicylic acid and PTPs, but also sheds new light on PTPs.

Introduction

PTPs play crucial roles in such fundamental cellular processes as proliferation, differentiation, survival, apoptosis, motility and adhesion.¹ Abnormal PTP activity is well known to be associated with a broad spectrum of human diseases.² As a superfamily of more than 100 signalling enzymes, many PTPs have emerged as attractive drug targets, such as mPTPB for tuberculosis, SHP2 for many types of cancers, LYP for autoimmune diseases, PTP1B for type 2 diabetes, obesity and breast cancer.³ To this end, the discovery of highly potent and specific small-molecule PTP inhibitors and their application in probing the biological and pathological mechanisms of PTPs, especially with the aid of modern imaging and spectroscopy technologies, are the cornerstone for PTPs-targeted drug discovery.

As a versatile element in biomedical research, fluorine has promising utility in PTPs-targeted drug discovery. On one hand, the introduction of fluorine(s) into bioactive molecules is usually accompanied by improved pharmacokinetic properties and protein-ligand binding interactions.⁴ Thus, fluorination has become a routine strategy in drug discovery and fluorinated compounds have made up over 20% of all pharmaceuticals. On the other hand, fluorinated molecules can be monitored in vivo without ionizing radiation and background signals by ¹⁹F magnetic resonance (¹⁹F MR) which provides high-contrast and non-invasive spectroscopy (¹⁹F NMR) and images (¹⁹F MRI). In recent years, ¹⁹F MRI/NMR has been widely used in tracking targets of interest⁵ and monitoring biological reactions.⁶ Therefore, the discovery of fluorinated small-molecule PTP inhibitors with high ¹⁹F MR sensitivity may provide easy access to PTPs-targeted drugs and detailed understanding of PTPs’ biological and pathological mechanisms.

A recent discovery of a ¹⁹F MRI sensitive salinomycin derivative with specific toxicity towards cancer cells⁷ by this group promoted us to develop novel fluorinated PTP inhibitors. Herein, ortho-bis(trifluoromethyl)carbinol phenol was designed as a novel chemical scaffold for ¹⁹F MRI sensitive PTP inhibitors (Scheme 1). Due to the strong electron-withdraw ability of 2 trifluoromethyl groups, the bis(trifluoromethyl)-carbinol is a weak acid and therefore a suitable substitute for the carboxylic group in salicylic acid from which a number of highly potent and selective PTP inhibitors have recently been discovered.⁸ Consequently, the ortho-bis(trifluoromethyl)-carbinol phenols may mimic the well-established binding mode of salicylic acid-based inhibitors at the highly positively charged active site of PTPs.⁸ It is noteworthy that the 6 symmetric fluorines in the bis(trifluoromethyl)carbinol, which were recently employed in the construction of highly ¹⁹F MRI sensitive dendritic drug delivery vehicles,⁹ aggregately provide a strong ¹⁹F MR signal for conveniently probing the mode of interaction and related biological reactions with ¹⁹F NMR and ¹⁹F MRI. Moreover, cell permeability is a challenge for PTP inhibitors. The bis(trifluoromethyl)carbinol-based PTP inhibitors without negative charge may exhibit favorable cell permeability, bioavailability and pharmacokinetic properties by the introduction hydrophobic trifluoromethyl groups.⁴

Scheme 1.

Design of ¹⁹F MR sensitive PTP inhibitors.

Materials and methods

Chemistry general information

¹H, ¹⁹F and ¹³C NMR spectra were recorded at 400 MHz. Chemical shifts (δ) are in ppm and coupling constants (J) are in Hertz (Hz). ¹H NMR spectra were referenced to tetramethylsilane (d, 0.00 ppm) using CDCl₃, Acetone-d₆ or DMSO-d₆ as solvents. ¹³C NMR spectra were referenced to solvent carbons (77.16 ppm for CDCl₃, 29.84, 206.26 ppm for Acetone-d₆ and 39.52 ppm for DMSO-d₆). ¹⁹F NMR spectra were referenced to 2% perfluorobenzene (s, −164.90 ppm). The splitting patterns for ¹H NMR spectra are denoted as follows: s (singlet), d (doublet), q (quartet), m (multiplet), dd (doublet of doublets), td (triplet of doublets). High resolution mass spectra were recorded using Electron Spray Ionization (ESI).

Unless otherwise indicated, all reagents were obtained from commercial supplier and used without prior purification. DCM and DMF were dried and freshly distilled prior to use. Flash chromatography was performed on silica gel (200–300 mesh) with either petroleum ether/EtOAc as eluents.

Synthesis of Compounds

Phenol 1c

Hexafluoroacetone trihydrate (9.71 g, 6.1 mL, 44.1 mmol) was dried over concentrated sulfuric acid and the resulting anhydrous hexafluoroacetone was bubbled into to a solution of 4-phenylphenol (5.00 g, 29.4 mmol) and aluminium chloride (0.39 g, 2.94 mmol) in 1,2-dichloroethane (250 mL) slowly. After the addition, the mixture was heated to reflux at 80°C until 4-phenylphenol was consumed as indicated by TLC. The reaction mixture was then cooled to rt, washed with 2 N HCl (100 mL) and extracted with DCM (50 mL × 2). The combined organic layers were dried over anhydrous Na₂SO₄, concentrated under vacuum and purified by flash chromatography on silica gel (5% EtOAc/petroleum ether) to give 1c as white wax (3.6 g, 85% yield). ¹H NMR (CDCl₃, 400 MHz) δ 7.00 (d, J = 8.5 Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.44 (t, J = 8.0 Hz, 2H), 7.48–7.54 (m, 2H), 7.57 (dd, J = 8.5, 2.1 Hz, 1H), 7.66 (s, 1H); ¹⁹F NMR (CDCl₃, 376 MHz) δ −78.53; ¹³C NMR (Acetone-d₆, 100 MHz) δ 80.0–81.2 (m), 116.0, 119.5, 124.2 (q, J = 286 Hz), 127.4, 127.7, 128.2, 129.9, 131.0, 134.7, 140.7, 156.6; HRMS (ESI) calcd for C₁₅H₁₁F₆O₂⁺ ([M+H]⁺) 337.0658, found 337.0671.

Phenol 1a

was prepared from benzene by following the general procedure as clear oil (2.5 g, 30%). ¹H NMR (CDCl₃, 400 MHz) δ 7.39–7.53 (m, 3H), 7.73 (dd, J = 7.4, 0.9 Hz, 2H); ¹⁹F NMR (CDCl₃, 376 MHz) δ −78.69.

Phenol 1b

was prepared from p-cresol (3.0 g, 27.7 mmol) by following the general procedure as white wax (6.1 g, 80%). ¹H NMR (CDCl₃, 400 MHz) δ 2.31 (s, 3H), 6.16 (s, 1H), 6.81 (d, J = 8.3 Hz, 1H), 6.88 (s, 1H), 7.15 (dd, J = 8.3, 1.6 Hz, 1H), 7.23 (s, 1H); ¹⁹F NMR (CDCl₃, 376 MHz) δ −78.64.

Phenol 1d

Prepared from [1,1'-biphenyl]-3-ol (5.00 g, 29.4 mmol) in the same manner as described for 1c (8.6 g, 87% yield). ¹H NMR (CDCl₃, 400 MHz) δ 7.13 (d, J = 1.8 Hz, 1H), 7.19–7.26 (m, 1H), 7.35–7.47 (m, 3H), 7.47–7.59 (m, 3H); ¹⁹F NMR (CDCl₃, 376 MHz) δ −78.72; ¹³C NMR (Acetone-d₆, 100 MHz) δ 79.9–81.1 (m), 114.5, 117.0, 120.2, 124.2 (q, J = 286 Hz), 128.8, 129.1, 129.9, 140.0, 145.3, 157.5; HRMS (ESI) calcd for C₁₅H₁₁F₆O₂⁺ ([M+H]⁺) 337.0658, found 337.0651.

Phenol 1e

Prepared from [1,1'-biphenyl]-2-ol (5.00 g, 29.4 mmol) in the same manner as described for 1c (3.6 g, 74% yield). ¹H NMR (CDCl₃, 400 MHz) δ 7.11 (t, J = 7.9 Hz, 1H), 7.35 (dd, J = 7.5, 1.5 Hz, 1H), 7.43–7.50 (m, 3H), 7.50–7.59 (m, 3H); ¹⁹F NMR (CDCl₃, 376 MHz) δ −78.41; ¹³C NMR (Acetone-d₆, 100 MHz) δ 80.6–81.8 (m), 115.5, 121.4, 124.1 (q, J = 286 Hz), 128.2, 128.3, 129.2, 130.5, 133.1, 133.8, 138.4, 154.9; HRMS (ESI) calcd for C₁₅H₁₁F₆O₂⁺ ([M+H]⁺) 337.0658, found 337.0654.

Phenol 1f

Prepared from 2-naphthalenol (5.00 g, 34.7 mmol) in the same manner as described for 1c (6.2 g, 57% yield). ¹H NMR (CDCl₃, 400 MHz) δ 7.29 (s, 1H), 7.42 (dd, J = 11.1, 3.9 Hz, 1H), 7.52 (dd, J = 11.1, 4.0 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 8.04 (s, 1H); ¹⁹F NMR (CDCl₃, 376 MHz) δ −78.40; ¹³C NMR (Acetone-d₆, 100 MHz) δ 80.2–81.4 (m), 113.3, 117.7, 124.2 (q, J = 286 Hz), 125.5, 126.6, 128.9, 129.0, 129.5, 130.6, 135.7, 154.0; HRMS (ESI) calcd for C₁₃H₉F₆O₂⁺ ([M+H]⁺) 311.0501, found 311.0489.

Phenol 1g

Prepared from 1-naphthalenol (5.00 g, 34.7 mmol) in the same manner as described for 1c (6.5 g, 60% yield). ¹H NMR (CDCl₃, 400 MHz) δ 7.39 (s, 2H), 7.46–7.63 (m, 2H), 7.71–7.84 (m, 1H), 8.24–8.38 (m, 1H); ¹⁹F NMR (CDCl₃, 376 MHz) δ −78.55; ¹³C NMR (Acetone-d₆, 100 MHz) δ 81.2–82.4 (m), 107.0, 120.6, 123.5, 124.18 (q, J = 287 Hz), 124.19, 126.8, 126.9, 128.2, 129.0, 135.9, 155.5; HRMS (ESI) calcd for ([M+H]⁺) C₁₃H₉F₆O₂⁺ 311.0501, found 311.0498.

Naphthol 3

Prepared from 2,7-naphthalenediol (30.0 g, 187.2 mmol) in the same manner as described for 1c (10.2 g, 17% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 7.01–7.13 (m, 2H), 7.25 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 8.04 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.10; ¹³C NMR (Acetone-d₆, 100 MHz) δ 80.1–81.3 (m), 107.9, 111.7, 114.2, 118.4, 124.1, 124.3 (q, J = 286 Hz), 130.3, 131.5, 137.6, 154.5, 158.2; HRMS (ESI) calcd for C₁₃H₉F₆O₃⁺ ([M+H]⁺) 327.0450, found 327.0444.

Naphthol 4

To an ice-cold suspension of diol 3 (2.40 g, 7.36 mmol) in trifluoroacetic acid was added acetone (2.2 mL, 29.5 mmol), then TFA (10.8 mL, 145.87 mmol) was added to the mixture dropwise. The reaction mixture was warmed slowly to rt and then stirred for 48 h. After evaporation of the solvent, the residue was purified by flash chromatography on silica gel (2% EtOAc/petroleum ether) to give 4 as white wax (0.85 g, 34% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 1.61 (s, 6H), 7.11–7.24 (m, 2H), 7.34 (s, 1H), 7.95 (d, J = 8.8 Hz, 1H), 8.09 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −78.30; ¹³C NMR (Acetone-d₆, 100 MHz) δ 27.0, 76.6–77.8 (m), 102.8, 108.5, 110.0, 113.7, 119.2, 123.2 (q, J = 287 Hz), 125.2, 128.4, 131.6, 138.0, 150.0, 158.5; HRMS (ESI) calcd for C₁₆H₁₃F₆O₃⁺ ([M+H]⁺) 367.0763, found 367.0773.

Ester 8

To a solution of 4 (470.0 mg, 1.28 mmol) and methyl bromoacetate (588.7 mg, 3.85 mmol) in acetone was added K₂CO₃ (381.5 mg, 3.85 mmol), then the reaction mixture was heated at reflux until 4 was consumed as indicated by TLC. After removal of the solvent under reduced pressure, the residue was dissolved in EtOAc (20 mL), and washed with water (50 mL × 2). The organic layer was dried over anhydrous Na₂SO₄, concentrated under vacuum and purified by flash chromatography on silica gel (2% EtOAc/petroleum ether) to give ester 8 as light yellow oil (480.0 mg, 86% yield). ¹H NMR (CDCl₃, 400 MHz) δ 1.60 (s, 6H), 3.83 (s, 3H), 4.76 (s, 2H), 6.96 (d, J = 2.4 Hz, 1H), 7.19 (dd, J = 9.0, 2.5 Hz, 1H), 7.29 (s, 1H), 7.78 (d, J = 9.0 Hz, 1H), 7.99 (s, 1H); ¹⁹F NMR (CDCl₃, 376 MHz) δ −78.24; ¹³C NMR (CDCl₃, 100 MHz) δ 26.8, 52.4, 65.2, 76.0–76.6 (m), 101.8, 105.5, 113.6, 118.3, 122.1 (q, J = 287 Hz), 125.1, 127.6, 130.7, 136.2, 149.5, 157.5, 169.0; HRMS (ESI) calcd for C₁₉H₁₇F₆O₅⁺ ([M+H]⁺) 439.0975, found 439.0981.

Acid 9

Ester 8 (400.0 mg, 0.91 mmol) was dissolved in THF/H₂O (5 mL/5 mL) and the solution was stirred at 0°C. Then NaOH (43.8 mg, 1.10 mmol, 10 N aqueous solution) was added at 0°C. The reaction mixture was stirred at rt until 8 was consumed as indicated by TLC. The solution was acidified to pH 6.0, then extracted with EtOAc (20 mL × 2) and washed with water (10 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated under vacuum to give acid 9 as white wax (370 mg, 96% yield). ¹H NMR (CDCl₃, 400 MHz) δ 1.60 (s, 6H), 4.82 (s, 2H), 6.99 (d, J = 2.4 Hz, 1H), 7.19 (dd, J = 9.0, 2.5 Hz, 1H), 7.29 (s, 1H), 7.79 (d, J = 9.1 Hz, 1H), 7.99 (s, 1H); ¹⁹F NMR (CDCl₃, 376 MHz) δ −78.33; ¹³C NMR (Acetone-d₆, 100 MHz) δ 27.0, 65.4, 76.6–77.8 (m), 102.9, 106.6, 111.0, 114.6, 119.5, 123.2 (q, J = 286 Hz), 126.0, 128.4, 131.5, 137.6, 150.2, 159.0, 170.0; HRMS (ESI) calcd for C₁₈H₁₅F₆O₅⁺ ([M+H]⁺) 425.0818, found 425.0798.

Amide 7a

Potassium carbonate (170.0 mg, 1.23 mmol) was added to a solution of 4 (150.0 mg, 0.41 mmol) and 6a (111.6 mg, 0.62 mmol) in acetone (5 mL), then the result suspension was heated at reflux until 4 was consumed as indicated by TLC. After removal of the solvent under reduced pressure, the residue was dissolved in EtOAc (25 mL), washed with 2N HCl (30 mL) and brine (30 mL × 2). The organic layer was dried over anhydrous Na₂SO₄, concentrated under vacuum and used without purification. The residue was dissolved in TFA/H₂O (9/1, 11.3 mL), then anisole (45 µL) was added and the resulting mixture was stirred overnight. The reaction mixture was concentrated under vacuum and then diluted with EtOAc (25 mL), washed with brine (30 mL × 2), the organic layer was dried over anhydrous Na₂SO₄, concentrated under vacuum and purified by flash chromatography on silica gel (20–80% EtOAc/petroleum ether) to give 7a as clear oil (131 mg, 77% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 0.88 (t, J = 7.4 Hz, 3H), 1.41–1.66 (m, 2H), 3.24–3.31 (m, 2H), 4.64 (s, 2H), 7.09–7.25 (m, 2H), 7.42 (s, 1H), 7.91 (d, J = 9.0 Hz, 1H), 8.09 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.08; ¹³C NMR (Acetone-d₆, 100 MHz) δ 11.1, 23.0, 41.0, 67.4, 79.7–80.3 (m), 105.4, 111.9, 114.8, 117.8, 123.6 (q, J = 286 Hz), 124.1, 129.5, 130.8, 136.6, 154.4, 157.8, 168.6; HRMS (ESI) calcd for C₁₈H₁₈F₆NO₄⁺ ([M+H]⁺) 426.1135, found 426.1118.

Amide 7b

Prepared from 4 (110.0 mg, 0.30 mmol) in the same manner as described for 7a (100 mg, 78% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 0.48–0.64 (m, 2H), 0.66–0.80 (m, 2H), 2.06 (dt, J = 4.4, 2.2 Hz, 1H), 4.61 (s, 2H), 7.08–7.23 (m, 2H), 7.42 (s, 1H), 7.90 (d, J = 9.0 Hz, 1H), 8.08 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.06; ¹³C NMR (Acetone-d₆, 100 MHz) δ 5.9, 22.8, 67.6, 79.9–80.5 (m), 105.5, 112.1, 114.9, 117.9, 123.8 (q, J = 286 Hz), 124.2, 129.7, 131.0, 136.7, 154.5, 158.0; HRMS (ESI) calcd for C₁₈H₁₆F₆NO₄⁺ ([M+H]⁺) 424.0978, found 424.0976.

Amide 7c

Prepared from 4 (150.0 mg, 0.41 mmol) in the same manner as described for 7a (130 mg, 72% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 1.12 (t, J = 7.1 Hz, 3H), 1.26 (t, J = 8.0 Hz, 3H), 3.42 (q, J = 7.0 Hz, 2H), 3.51 (q, J = 7.1 Hz, 2H), 4.95 (s, 2H), 7.02–7.18 (m, 2H), 7.30 (s, 1H), 7.85 (d, J = 8.8 Hz, 1H), 8.02 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.04; ¹³C NMR (Acetone-d₆, 100 MHz) δ 12.8, 14.1, 40.6, 66.0, 78.0–79.2 (m), 104.7, 110.0, 116.5, 122.4, 123.1 (q, J = 288 Hz), 129.6, 130.2, 136.0, 153.9, 157.6, 166.0; HRMS (ESI) calcd for C₁₉H₂₀F₆NO₄⁺ ([M+H]⁺) 440.1291, found 440.1298.

Amide 7d

Prepared from 4 (150.0 mg, 0.41 mmol) in the same manner as described for 7a (90 mg, 42% yield). ¹H NMR (DMSO-d₆, 400 MHz) δ 1.62 (s, 6H), 2.00 (d, J = 10.7 Hz, 10H), 4.53 (s, 2H), 7.06 (d, J = 8.4 Hz, 2H), 7.18 (s, 1H), 7.44 (s, 1H), 7.85 (d, J = 8.7 Hz, 1H); ¹⁹F NMR (DMSO-d₆, 376 MHz) δ −76.01; ¹³C NMR (DMSO-d₆, 100 MHz) δ 28.7, 35.9, 40.9, 51.0, 67.0, 78.0–79.1 (m), 104.6, 110.1, 116.6, 122.5, 123.1 (q, J = 288 Hz), 129.6, 130.2, 136.0, 153.9, 157.3, 166.4; HRMS (ESI) calcd for C₂₅H₂₆F₆NO₄⁺ ([M+H]⁺) 518.1761, found 518.1766.

Amide 7e

Prepared from 4 (100.0 mg, 0.27 mmol) in the same manner as described for 7a (91 mg, 71% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 4.51 (d, J = 8.0 Hz, 2H), 4.71 (s, 2H), 7.10–7.16 (m, 1H), 7.17–7.33 (m, 6H), 7.40 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.03; ¹³C NMR (DMSO-d₆, 100 MHz) δ 41.8, 67.0, 78.0–78.9 (m), 104.8, 110.1, 116.7, 122.6, 123.1 (q, J = 287 Hz), 126.7, 127.1, 128.2, 129.7, 130.2, 136.0, 139.3, 153.9, 157.1, 167.6; HRMS (ESI) calcd for C₂₂H₁₈F₆NO₄⁺ ([M+H]⁺) 474.1135, found 474.1138.

Amide 7f

Prepared from 4 (150.0 mg, 0.41 mmol) in the same manner as described for 7a (200 mg, 99% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 4.50 (d, J = 5.9 Hz, 2H), 4.72 (d, J = 2.0 Hz, 2H), 6.98–7.07 (m, 2H), 7.13 (dd, J = 9.0, 2.5 Hz, 1H), 7.20 (d, J = 2.4 Hz, 1H), 7.30–7.38 (m, 2H), 7.42 (s, 1H), 7.90 (d, J = 9.0 Hz, 1H), 8.09 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −117.73, −76.14; ¹³C NMR (DMSO-d₆, 100 MHz) δ 41.1, 67.0, 78.0–79.2 (m), 104.8, 110.2, 114.8, 115.0, 116.7, 122.6, 123.1 (q, J = 287 Hz), 129.0, 129.1, 129.7, 130.2, 135.5, 136.0, 153.9, 157.1, 159.9, 162.3, 167.6; HRMS (ESI) calcd for C₂₂H₁₇F₇NO₄⁺ ([M+H]⁺) 492.1040, found 492.1041.

Amide 7g

Prepared from 4 (150.0 mg, 0.41 mmol) in the same manner as described for 7a (170 mg, 78% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 3.65 (s, 3H), 3.74 (s, 3H), 4.43 (d, J = 6.2 Hz, 2H), 4.70 (s, 2H), 6.81 (d, J = 1.0 Hz, 2H), 6.89 (s, 1H), 7.08–7.26 (m, 2H), 7.39 (s, 1H), 7.90 (d, J = 9.0 Hz, 1H), 8.09 (s, 2H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.03; ¹³C NMR (Acetone-d₆, 100 MHz) δ 42.7, 55.4, 55.6, 67.5, 79.8–80.4 (m), 105.6, 112.0, 112.1, 115.0, 118.0, 120.2, 123.8 (q, J = 286 Hz), 124.2, 129.7, 131.0, 131.8, 136.7, 149.1, 149.8, 154.4, 158.0, 168.8; HRMS (ESI) calcd for C₂₄H₂₂F₆NO₆⁺ ([M+H]⁺) 534.1346, found 534.1369.

Amide 7h

Prepared from 4 (150.0 mg, 0.41 mmol) in the same manner as described for 7a (210 mg, 95% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 2.98 (t, J = 7.0 Hz, 2H), 3.59 (dd, J = 13.2, 6.8 Hz, 2H), 4.65 (s, 2H), 7.03–7.21 (m, 3H), 7.26 (d, J = 8.2 Hz, 1H), 7.35–7.47 (m, 2H), 7.91 (d, J = 9.0 Hz, 2H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −75.99; ¹³C NMR (Acetone-d₆, 100 MHz) δ 33.2, 39.0, 67.7, 80.0–80.6 (m), 105.7, 112.2, 115.1, 118.2, 123.9 (q, J = 287 Hz), 124.5, 127.7, 129.4, 129.9, 131.2, 132.9, 133.0, 135.2, 136.4, 137.9, 154.5, 158.1, 168.9; HRMS (ESI) calcd for C₂₃H₁₈Cl₂F₆NO₄⁺ ([M+H]⁺) 556.0512, found 556.0510.

Amide 7i

Prepared from 4 (115.1 mg, 0.31 mmol) in the same manner as described for 7a (28 mg, 16% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 4.84 (s, 2H), 6.26 (s, 1H), 7.05 (dd, J = 9.0, 2.5 Hz, 1H), 7.21–7.32 (m, 3H), 7.36–7.43 (m, 3H), 7.51 (d, J = 7.5 Hz, 2H), 7.81 (t, J = 8.4 Hz, 3H), 8.04 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.08; ¹³C NMR (DMSO-d₆, 100 MHz) δ 54.3, 67.3, 78.8–79.4 (m), 105.1, 110.6, 117.1, 120.6, 123.0, 123.7 (q, J = 288 Hz), 125.2, 128.0, 128.9, 130.1, 130.7, 136.5, 140.6, 144.9, 154.9, 157.7, 169.0; HRMS (ESI) calcd for C₂₈H₂₀F₆NO₄⁺ ([M+H]⁺) 548.1291, found 548.1284.

Amide 7j

Prepared from 4 (150.0 mg, 0.41 mmol) in the same manner as described for 7a (88 mg, 95% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 4.81 (s, 2H), 7.11 (t, J = 7.4 Hz, 1H), 7.20–7.39 (m, 4H), 7.42 (s, 1H), 7.72–7.83 (m, 2H), 7.95 (d, J = 9.0 Hz, 1H), 8.12 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.04; ¹³C NMR (Acetone-d₆, 100 MHz) δ 67.7, 79.7–80.3 (m), 105.6, 111.9, 114.8, 117.9, 120.5, 123.6 (q, J = 286 Hz), 124.2, 124.5, 129.0, 129.6, 130.9, 136.6, 138.6, 154.2, 157.9, 166.8; HRMS (ESI) calcd for C₂₁H₁₆F₆NO₄⁺ ([M+H]⁺) 460.0978, found 460.0982.

Amide 7k

Prepared from 4 (150.0 mg, 0.41 mmol) in the same manner as described for 7a (130 mg, 63% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 1.20 (d, J = 6.9 Hz, 6H), 2.71–2.98 (m, 1H), 4.81 (s, 2H), 7.12–7.33 (m, 4H), 7.42 (s, 1H), 7.60–7.73 (m, 2H), 7.93 (d, J = 9.0 Hz, 1H), 8.12 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.11; ¹³C NMR (Acetone-d₆, 100 MHz) δ 23.9, 33.8, 67.9, 79.8–80.4 (m), 105.7, 112.1, 114.9, 118.0, 120.8, 123.7 (q, J = 287 Hz), 124.3, 127.0, 129.7, 131.0, 136.3, 136.7, 145.2, 154.3, 158.0, 166.7; HRMS (ESI) calcd for C₂₄H₂₂F₆NO₄⁺ ([M+H]⁺) 502.1448, found 502.1424.

Amide 7l

Prepared from 4 (120.0 mg, 0.33 mmol) in the same manner as described for 7a (47 mg, 27% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 4.82 (s, 2H), 7.18–7.29 (m, 2H), 7.34 (d, J = 7.4 Hz, 1H), 7.44 (dd, J = 16.7, 8.8 Hz, 3H), 7.60–7.70 (m, 4H), 7.83–7.98 (m, 3H), 8.09 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.04; ¹³C NMR (Acetone-d₆, 100 MHz) δ 68.0, 80.0–80.5 (m), 105.9, 112.2, 115.0, 118.2, 120.9, 121.0, 123.9 (q, J = 286 Hz), 124.4, 127.1, 127.6, 127.7, 127.8, 129.4, 130.0, 131.1, 136.8, 137.2, 138.3, 140.9, 154.4, 158.1, 167.0; HRMS (ESI) calcd for C₂₇H₂₀F₆NO₄⁺ ([M+H]⁺) 536.1291, found 536.1272.

Amide 7m

1,3-diisopropylcarbodiimide (42.8 mg, 0.34 mmol) was added slowly to a solution of acid 9 (120.0 mg, 0.28 mmol) and 1-hydroxytriazole (45.8 mg, 0.34 mmol) in dry DMF (3 mL) at 0°C. After 15 minutes, a solution of 4-morpholinoaniline (60.5 mg, 0.34 mmol) in dry DMF (2 mL) was added and the resulting mixture was stirred at rt overnight. The reaction mixture was diluted with brine (40 mL) and extracted with EtOAc (20 mL × 2). The organic layer was dried over anhydrous Na₂SO₄, concentrated under vacuum and used without purification. The residue was dissolved in TFA/H₂O (v/v, 9/1, 7.6 mL), then anisole (30 µl) was added and the resulting mixture was stirred overnight. The reaction mixture was concentrated under vacuum and then diluted with EtOAc (20 mL), washed with brine (30 mL × 2), the organic layer was dried over anhydrous Na₂SO₄, concentrated under vacuum and purified by flash chromatography on silica gel (20–80% EtOAc/petroleum ether) to give 7m as white wax (115 mg, 75% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 3.03–3.17 (m, 4H), 3.71–3.83 (m, 4H), 4.77 (s, 2H), 6.93 (d, J = 9.0 Hz, 2H), 7.17–7.31 (m, 2H), 7.42 (s, 1H), 7.57–7.69 (m, 2H), 7.94 (d, J = 8.9 Hz, 1H), 8.10 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.06; ¹³C NMR (DMSO-d₆, 100 MHz) δ 48.8, 66.0, 67.2, 78.3–78.9 (m), 104.7, 110.2, 115.3, 116.6, 121.0, 122.6, 123.1 (q, J = 286 Hz), 129.7, 130.3, 136.0, 147.6, 153.9, 157.3, 165.7; HRMS (ESI) calcd for C₂₅H₂₃F₆N₂O₅⁺ ([M+H]⁺) 545.1506, found 545.1490.

Amide 7n

Prepared from 4 (150.0 mg, 0.41 mmol) in the same manner as described for 7a (120 mg, 57% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 4.98 (s, 2H), 7.29–7.41 (m, 2H), 7.41–7.60 (m, 4H), 7.76–8.06 (m, 5H), 8.14 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.07; ¹³C NMR (Acetone-d₆, 100 MHz) δ 68.1, 80.0–80.6 (m), 105.8, 112.1, 115.0, 118.3, 122.6, 122.8, 124.5, 123.9 (q, J = 287 Hz), 126.0, 126.6, 126.7, 126.8, 128.87, 128.90, 129.8, 131.2, 133.1, 134.8, 136.8, 154.4, 158.1, 167.8; HRMS (ESI) calcd for C₂₅H₁₈F₆NO₄⁺ ([M+H]⁺) 510.1135, found 510.1110.

Amide 7o

Prepared from 4 (110.0 mg, 0.30 mmol) in the same manner as described for 7a (110 mg, 68% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 4.79 (s, 2H), 6.98–7.11 (m, 2H), 7.39 (d, J = 45.1 Hz, 11H), 7.81–7.95 (m, 1H), 8.06 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.07; ¹³C NMR (DMSO-d₆, 100 MHz) δ 66.03, 78.4–80.0 (m), 104.5, 110.2, 116.2, 122.5, 123.2 (q, J = 288 Hz), 129.6, 130.3, 136.0, 154.3, 157.3, 166.7; HRMS (ESI) calcd for C₂₇H₂₀F₆NO₄⁺ ([M+H]⁺) 536.1291, found 536.1296.

Amide 7p

Prepared from 4 (150.0 mg, 0.41 mmol) in the same manner as described for 7a (53 mg, 35% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 5.06 (s, 2H), 6.86–7.02 (m, 2H), 7.26–7.49 (m, 4H), 7.52–7.65 (m, 2H), 7.65–7.89 (m, 4H), 8.03 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.12; ¹³C NMR (DMSO-d₆, 100 MHz) δ 65.5, 78.0–79.2 (m), 104.0, 104.1, 110.0, 116.4, 122.5, 123.1 (q, J = 286 Hz), 126.9, 127.5, 128.1, 129.6, 130.3, 132.0, 135.76, 135.83, 136.8, 137.2, 137.5, 153.88, 153.93, 156.0, 157.0, 166.0; HRMS (ESI) calcd for C₂₇H₂₀F₆NO₄⁺ ([M+H]⁺) 536.1291, found 536.1296.

Amide 7q

Prepared from 9 (200.0 mg, 0.47 mmol) in the same manner as described for 7m (126 mg, 76% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 5.09 (s, 2H), 7.24–7.54 (m, 5H), 7.77 (d, J = 8.0 Hz, 1H), 7.96 (dd, J = 12.5, 5.3 Hz, 2H), 8.11 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.12; ¹³C NMR (Acetone-d₆, 100 MHz) δ 67.5, 80.0–81.1 (m), 106.0, 112.4, 115.3, 118.3, 121.8, 122.2, 124.1 (q, J = 286 Hz), 124.70, 124.74, 127.0, 130.1, 131.4, 133.0, 137.0, 149.6, 154.8, 158.1, 158.3, 168.2; HRMS (ESI) calcd for C₂₂H₁₅F₆N₂O₄S⁺ ([M+H]⁺) 517.0651, found 517.0646.

Amide 7r

Prepared from 9 (120.0 mg, 0.28 mmol) in the same manner as described for 7m (95 mg, 61% yield). ¹H NMR (Acetone-d₆, 400 MHz) δ 2.34 (s, 3H), 4.65 (s, 2H), 7.03–7.16 (m, 2H), 7.21 (d, J = 8.1 Hz, 2H), 7.37 (s, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.89 (dd, J = 22.1, 8.7 Hz, 1H), 8.12 (s, 1H); ¹⁹F NMR (Acetone-d₆, 376 MHz) δ −76.03; ¹³C NMR (Acetone-d₆, 100 MHz) δ 21.1, 66.7, 79.9–80.5 (m), 105.7, 112.1, 118.2, 123.8 (q, J = 286 Hz), 124.4, 128.8, 130.0, 131.1, 136.1, 136.8, 144.5, 154.4, 158.0, 167.1; HRMS (ESI) calcd for C₂₂H₁₉F₆N₂O₆S⁺ ([M+H]⁺) 553.0863, found 553.0861.

¹⁹F MRI experiments

¹⁹F MRI experiments were performed on a 9.4 T microimaging system with a 10 mm inner diameter ¹⁹F coil (376.4 MHz) for both radiofrequency transmission and reception. The MSME (Multi Slice Multi Echo) pulse sequence was employed for all MRI acquisitions with single average. FOV = 8 × 8 mm², SI = 40.0 mm TR = 2500 ms and TE = 7.6 ms were used. The data collection time was 160 ms.

Computational analysis

For computational analysis, PDB code 3O5X was used as model structure. Molecular docking was done using AutoDock Vina. Small molecule binding mode was modelled manually using Moloc(Gerber Mulecular Design:Switzerland). The image was produced by PyMOL.

PTPs activity assay

PTP activity was assayed using p-nitrophenyl phosphate (pNPP) as a substrate in 3,3-dimethylglutarate buffer (50 mM 3,3-dimethylglutarate, pH 7.0, 1 mM EDTA, 150 mM NaCl) at 25 °C. The library compounds was screened in a 96-well format. The amount of product p-nitrophenol was determined from the absorbance at 405 nm detected by a Spectra MAX340 microplate spectrophotometer (Molecular Devices). The nonenzymatic hydrolysis of pNPP was corrected by measuring the control without the addition of enzyme. All PTPs used in the study were recombinant proteins prepared in-house.

Results and discussion

To probe the structure-activity relationship of the ortho-bis(trifluoromethyl)carbinol phenol-based inhibitors, a structure-based focused library strategy was employed. Our initial effort involved the construction of a focused library of 7 bis(trifluoromethyl)carbinol-substituted benzene to identify the optimal relative positions for these substituents (Scheme 2). Through the Lewis acid-catalysed Friedel-Crafts reaction, the bis(trifluoromethyl)-carbinol moiety was conveniently anchored to benzene, phenols, and naphthols with good yields. Due to the strong directing effect of phenolic hydroxyl group, the desired ortho-bis(trifluoromethyl)carbinol phenols were isolated as the major products (1b–g).

Scheme 2.

Synthesis of bis(trifluoromethyl)carbinol library.

The ability of library compounds 1a–g to inhibit a selected panel of PTPs of therapeutic interest, including mPTPB, SHP2, PTP1B, CD45, LYP, and FAP-1, was assessed at pH 7 and 25°C (Table 1). The results indicate that the phenolic hydroxyl group plays a crucial role in PTP binding through which the inhibitors may mimic the binding mode of salicylic acid-based inhibitors. No appreciable activity was found for 1a, which lacks phenolic hydroxyl group in the scaffold. The PTP inhibitory activity is also very sensitive to the size and position of substituent. Neither 1c with a para-phenyl group nor 1d with a meta-phenyl group has appreciable activity, while 1b with a small-sized para-methyl group has moderate activity. Among the library compounds 1a–g, 2-naphthol derived 1f is the most potent one for the selected panel of PTPs which was then selected for further optimization.

Table 1.

IC₅₀ (µM) of 1a–g for a selected panel of PTPs.

	1a	1b	1c	1d	1e	1f	1g
mPTPB	-	179.5 ± 19	351.0 ± 154	-	148.4 ± 6	105.6 ± 10	-
SHP2	-	201.8 ± 37	392.2 ± 71	-	136.0 ± 28	114.8 ± 17	-
PTP1B	-	360.2 ± 207	-	-	422.4 ± 311	260.4 ± 47	-
CD45	-	207.1 ± 17	-	-	157.2 ± 14	112.4 ± 9	-
LYP	-	302.9 ± 165	-	-	268.4 ± 149	133.9 ± 34	-
FAP-1	-	454.3 ± 754	-	-	448.8 ± 1506	127.9 ± 85	-

A “-“ indicates IC₅₀ >> 500 µM.

To further improve the potency and selectivity, 1f was modified into a focused library to target both the active site and a peripheral secondary binding site of PTPs (Scheme 3).^8,10 Starting from 2,7-naphthalene-diol 2, a core compound 3, with an extra 7-hydroxyl group compared to 1f, was constructed through Friedel-Crafts reaction with good yield. Then, a panel of amines 5a–r with structural diversity were selected for the side chains 6a–r construction by reacting with bromoacetyl bromide, respectively. After protecting the 2 neighbouring hydroxyl groups in 3 with acetones, side chains 6a–r were anchored to the 7-hydroxyl group in 4 in the presence of K₂CO₃ to give ester intermediates after which the acetonide protecting group was removed with TFA to give amides 7a–p with good yields over 2 steps. However, the preparation of 7m, 7q and 7r was unsuccessful. So, an alternative method was developed by first anchoring an acetic acid side chain to 4 and then coupling amines 5m, 5q, and 5r, respectively, to give the corresponding amides 7m, 7q, and 7r. In these ways, the focused library of 18 ortho-bis(trifluoromethyl)carbinol phenols 7a–r with an amide side chain was conveniently prepared.

Scheme 3.

Synthesis of focused library of PTP inhibitors.

To illustrate the structures of ortho-bis(trifluoro methyl)carbinol phenols 7a–r, a single-crystal X-ray structure of 7c was obtained (Fig. 1). However, many attempts to prepare a single-crystal of 7p were unsuccessful.

As expected, the activities of library compounds 7a–r are much higher than these of 1f (Table 2). Compound 7r with sulfonohyrazide side chain was identified as a highly potent and selective mPTPB inhibitor with an IC₅₀ value of 2.3 µM and more than 7-fold selectivity compared to SHP2, PTP1B, CD45, LYP, and FAP-1. It is interesting to point out that most of aliphatic amine derived compounds 7a–c and 7e–g show no appreciable PTP inhibitory activity, while bulky aliphatic amine derived compounds 7d, 7h, and 7i exhibit moderate activities. In contrast, most of aromatic amine derived compounds 7j–r have good activities and selectivity except for the positively charged 7m. Among them, compound 7p with a bulky aromatic group on the side chain exhibits very high activity toward the selected panel of PTPs with IC₅₀ ranging from 2.2 µM for FAP-1 to 6.6 µM for PTP1B. Based on these observation, it is obvious that the potency of ortho-bis(trifluoromethyl) carbinol phenols-based inhibitors can be considerably optimized up to 58-fold by tethering an amide side chain. A bulky aromatic group containing side chain, i.e. 7l and 7p, can efficiently promote the binding affinity between PTPs and inhibitors by interacting with a peripheral pocket in the vicinity of the PTPs active sites, probably through steric effects and π-π stacking.

Table 2.

IC₅₀ (µM) of 7a–r for a selected panel of PTPs.

	7a–c	7d	7e–f	7g	7h	7i	7j	7k	7l	7m	7n	7o	7p	7q	7r
mPTPB	-	-	-	18.1 ± 9.5	5.1 ± 0.4	9.4 ± 0.2	14.4 ± 1.9	7.3 ± 0.5	4.8 ± 0.1	-	4.7 ± 0.2	-	2.9 ± 0.1	2.6 ± 0.1	2.3 ± 0.1
SHP2	-	9.0 ± 2.2	-	-	6.7 ± 1.2	6.9 ± 1.4	19.0 ± 25.6	8.5 ± 1.4	3.5 ± 0.5	-	-	19.5 ± 47.8	3.2 ± 0.3	12.8 ± 3.6	20.3 ± 18.6
PTP1B	-	14.7 ± 3.0	-	-	12.6 ± 4.8	13.8 ± 2.0		17.2 ± 8.9	-	-	-	-	6.6 ± 1.3	-	-
CD45	-	7.6 ± 0.8	-	-	5.2 ± 0.6	6.6 ± 0.6	-	7.8 ± 0.5	3.4 ± 0.2	-	29.1 ± 38.2	14.9 ± 7.2	2.8 ± 0.2	10.9 ± 1.4	16.1 ± 5.0
LYP	-	10.1 ± 0.8	-	-	6.9 ± 1.1	7.5 ± 0.8	-	8.8 ± 1.1	-	-	-	20.6 ± 35.9	3.4 ± 0.3	14.3 ± 3.1	15.4 ± 3.1
FAP-1	-	7.4 ± 0.8	-	-	4.3 ± 1.8	5.6 ± 0.7	-	6.5 ± 0.7	2.8 ± 0.3	-	-	12.4 ± 8.3	2.2 ± 0.3	9.9 ± 1.5	14.9 ± 5.2

A “-“ indicates IC₅₀ >> 20µM.

Computational analysis of the binding activity of 7p in the highly conservative active site of PTPs provided some insight into the structure-activity relationship between these novel inhibitors and PTPs. Oncogenic SHP2 with known complex structure (PDB ID: 3O5X) was selected as a model. Fig. 2 shows the binding mode of 7p with SHP2 compared to that of a known salicylic acid-based SHP2 inhibitor 10 which has a IC₅₀ of 5.5 µM toward SHP2.^8a As expected, ortho-bis(trifluoromethyl) carbinol phenol moiety can mimic the binding mode of salicylic acid by interacting with the corresponding amino acid residues Trp423, Arg465 and Gln510 (the distances between O of ortho-bis(trifluoromethyl)carbinol and the three hydrogen bonding heavy atoms of the residues are 3.6 Å). However, due to the difference in molecular geometry, the side chains of 7p and 10 interacted with SHP2 in different ways. Instead of interacting with Arg362 and Lys364 of SHP2, the aromatic side chain in 7p has a strong π-π interaction with Tyr-279.

Fig. 2.

Calculated structure of 7p bound to SHP2 with salicylic acid-based inhibitor 10 as a comparison.

Finally, ¹⁹F magnetic resonance properties of PTP inhibitors 7a–r were investigated. As designed, all 6 symmetrical fluorines in 7a–r generated a strong singlet ¹⁹F NMR signal, respectively (Fig. 3). Uniformed ¹⁹F signal dramatically improved the ¹⁹F NMR sensitivity of these fluorinated inhibitors for downstream applications. Then, 7p with high potencies toward a panel of PTPs was selected for ¹⁹F MRI study. It was found that 7p has a very short longitudinal relaxation time T₁ of 299 ms which could further improve its ¹⁹F MRI sensitivity by allowing the collection of more transient signals without prolonging the data acquisition time. ¹⁹F MRI phantom experiment on an array of 7p solution indicated that 7p could be clearly imaged by ¹⁹F MRI with a scan time of 120 seconds at a concentration of as low as 8.3 mM (or 50 mM in ¹⁹F concentration, Fig. 3). Therefore, 7p is a novel PTP inhibitor as well as a highly valuable tool molecule whose local information, such as distribution and concentration, and interactions with PTPs, such as binding mode and affinity, can be conveniently monitored by ¹⁹F MR spectroscopy and imaging without extra modification in the absence of background signals.

Fig. 3.

¹⁹F NMR of selected inhibitors (upper) and ¹⁹F MRI of 7p (lower).

Conclusions and outlook

In summary, we have successfully demonstrated a strategy of developing novel ¹⁹F magnetic resonance sensitive small molecule PTP inhibitors for drug discovery and biomedical research through rational molecular design and symmetrical fluorination. Ortho-bis(trifluoromethyl)carbinol phenol is a valuable substitute for salicylic acid in PTP inhibitor discovery, which successfully integrates the PTP binding ability and high ¹⁹F NMR signal generating ability. As fluorinated drugs are booming in pharmaceutical industry, it is of great importance to utilize their inherent ¹⁹F magnetic resonance properties in target identification, pharmacology study, in vivo drug tracking, image/spectroscopy-guided drug therapy and beyond.

Finally, we want to point out that both ¹⁹F NMR and ¹⁹F MRI are valuable modalities for biomedical research. ¹⁹F MRI provides high-contrast images at ¹⁹F concentrations of mM and above, while ¹⁹F NMR provides sensitive spectroscopies even at sub-µM ¹⁹F concentrations. Improving the PTP inhibition potency and selectivity, ¹⁹F MRI sensitivity of these inhibitors and their application in ¹⁹F magnetic resonance-guided PTP mechanism study are currently in progress and will be published in due course.