Supporting Information
Inhibitor-based affinity probes for the
investigation of JAK signaling pathways
Michael Höfener1, Fiona Pachl2, Bernhard Kuster2,3, Norbert Sewald1*
1
Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University,
Universitätsstraße 25, 33615 Bielefeld, Germany
2
Chair of Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan,
Technische Universität München, Freising, Germany
3
Center for Integrated Protein Science Munich, Freising, Germany
*Corresponding author: Prof. Dr. Norbert Sewald, norbert.sewald@uni-bielefeld.de, fax:
int+49 (0)521 106 156963
Materials and methods for chemical synthesis
Chemicals and Solvents: Solvents were usually used as purchased in p.a. quality. Solvents of lower
quailty were purified by distillation. If necessary, methylene chloride was dried over calcium hydride.
Toluene and tetrahydrofuran were dried over sodium. Acetonitrile for analytical and preparative
HPLC was purchased in Lichrosolv quality from Merck. Water for this purpose was obtained from a
Millipore MilliQ-Ultrapure water system. Chemicals were purchased at Acros, Sigma Aldrich, VWR
and Alfa Aesar and were used without further treatment. Silica gel (0.04-0.063 mm) Macherey-Nagel
was used for flash chromatography.
NMR-spectroscopy: 1H-NMR and 1H,1H-COSY experiments were recorded using a Bruker DRX 500
instrument (1H-NMR: 500.1 MHz), 13C-NMR, HSQC and HMBC experiments were recorded using a
Bruker Avance 600 instrument (1H-NMR: 600.1 MHz , 13C-NMR: 150.9 MHz). The recorded data was
processed using TopSpin v 2.1, the spectra were evaluated using MestReNova software 6.0.2.
Deuterated chloroform was usually used as solvent for NMR measurements with tetramethylsilane
1
(TMS) as internal standard. Other deuterated solvents were referenced to the solvent peak.
Structures of unpublished molecules were assigned with the help of 1H,1H-COSY, HSQC and HMBC
NMR experiments.
Mass-spectrometry: ESI-MS: ESI/APCI mass spectra were recorded using an Esquire 3000 ion trap
mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with a standard ESI/APCI
source. Samples were introduced by direct infusion with a syringe pump. Nitrogen served both as the
nebulizer gas and the dry gas. Nitrogen was generated by a Bruker nitrogen generator NGM 11.
Helium served as cooling gas for the ion trap and collision gas for MSn experiments. The spectra were
recorded with the Bruker Daltonik esquireNT 5.2 esquireControl software by the accumulation and
averaging of several single spectra. DataAnalysis™ software 3.4 was used for processing the spectra.
MALDI-MS: MALDI experiments were performed using a Fourier Transform Ion Cyclotron
Resonance (FT-ICR) mass spectrometer APEX III (Bruker Daltonik GmbH, Bremen, Germany) equipped
with a 7.0 T, 160 mm bore superconducting magnet (Bruker Analytik GmbH – Magnetics, Karlsruhe,
Germany), infinity cell, and interfaced to an external MALDI ion source. Nitrogen served both as the
nebulizer gas and the dry gas for ESI. Nitrogen was generated by a Bruker nitrogen generator NGM
11. Argon served as cooling gas in the infinity cell and collision gas for MSn experiments. Scan
accumulation and fourier transformation were performed with XMASS NT (7.08) on a PC
Workstation, for further data processing DataAnalysis™ 3.4 was used. LC-MS: LC-MS analysis was
accomplished using a Waters Alliance HT equipped with a Waters Symmetry 3.5 µm column (C8, 100
x 2.1 mm, eluent A H2O/HCOOH = 100:0.1, eluent B CH3CN/HCOOH = 100:0.1, flowrate 0.4 mL⋅min-1
using a gradient from 5-95% B over 10 minutes) coupled with a Waters micromass ZQ2000 ESI-MS.
Analytical Reversed Phase-High Performance Liquid Chromatography (RP-HPLC)
Thermo Separation Products System: Surveyor Autosampler Plus, Surveyor LC Pump Plus, Surveyor
PDA Plus Detector (190 -800 nm, continous); column: ET 125/4 Nucleosil 100-5 C18 PPN; software:
Chromquest 5.0; eluent A: 5 % H2O, 95 % ACN, 0.05 % TFA; eluent B: 95 % H2O, 5 % ACN,
0.05 % TFA.
Supplementary table 1: Analytical HPLC method, λ = 192-480 nm.
time [min]
eluent A
eluent B
flow [mL min-1]
0
0
100
0.75
1
0
100
0.75
4
100
0
0.75
5
0
100
0.75
Preparative Reversed Phase - High Performance Liquid Chromatography (RP-HPLC) Hitachi
MERCK LaChrom system: LC 7150 pump; UV-Vis L 7420 detector; precolumn: Vydac highperformance guard column (C18); column: Phenomenex Jupiter 10 µ 300 Å (C18; 250 x 21.2 mm);
eluent A: 5 % H2O, 95 % ACN, 0.05 % TFA; eluent B: 95 % H2O, 5 % ACN, 0.05 % TFA.
2
Supplementary table 2: Preparative HPLC method, λ = 254 nm.
time [min]
eluent A
eluent B
flow [mL min-1]
0
0
100
10
5
0
100
10
40
100
0
10
50
0
100
10
Microwave assisted synthesis: Reactions were performed with a CEM Discover instrument,
CEM Matthews Inc. (USA), max. 300 W, max. 2455 MHz. The reaction containers were sealed
during the heating progress that was run as stated in each corresponding synthetic
procedure.
Synthesis of probe 1b:
Supplementary Figure 1: Overview of the synthesis of probe 1b.
tert-Butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (1) A mixture of Boc2O (2.9 g, 14 mmol) in
THF (30 mL) was added dropwise over a period of 5 h to a solution of 2-(2-(2aminoethoxy)ethoxy)ethanamine (14.5 g, 98 mmol) in THF (30 mL). The resulting suspension was
stirred overnight at rt prior evaporation of the solvent. The residue was dissolved in water (50 mL)
and extracted with DCM (3x20 mL). The organic phases were washed with water (10 mL) dried over
MgSO4 and the solvent evaporated. The crude product was used without purification for the next
step. Formula: C11H24N2O4. Yield: 3.3 g (13.3 mmol, 95%). 1H-NMR (500 MHz, CDCl3): δ [ppm] = 5.16
(s, 1H, NH), 3.62 (s, 4H, 2xCH2), 3.54 (m, 4H, 2xCH2), 3.32 (m, 2H, CH2), 2.90 (t, 2H, J = 5.1 Hz, CH2),
1.44 (s, 9H, tBu).
tert-Butyl 2-(2-(2-(cyclohexylmethylamino)ethoxy)ethoxy)ethylcarbamate (2) Cyclohexylmethyl
bromide (18 µL, 27.0 mg, 0.15 mmol) was dissolved in dry DCM (1 mL) and added dropwise over 4 h
to a solution of amine 1 (47.2 mg, 0.19 mmol) and TEA (29.1 µL, 21.0 mg, 0.21 mmol) in DCM (1 mL)
at rt. After stirring overnight at rt the solvent was evaporated to give the crude product that was
purified via flash chromatography (eluent: DCM/MeOH, 9.5:0.5 +1% TEA). Formula: C18H36N2O4.
Yield: 37 mg (0.11 mmol, 73%). 1H-NMR (500 MHz, CDCl3): δ [ppm] = 5.18 (s, 1H, NH), 3.61 (m, 6H,
3
3xCH2), 3.53 (t, 2H, J = 5.3 Hz, CH2), 3.31 (m, 2H, CH2), 2.82 (t, 2H, J = 5.2 Hz, CH2), 2.40 (m, 2H, CH2),
1.60-187 (m, 5H, 2xCH2 1xCH), 1.43 (s, 9H, tBu), 1.10-1.20 (m, 6H, 3xCH2).
tert-Butyl2-(2-(2-((cyclohexylmethyl)(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)ethoxy)ethoxy)ethyl
carbamate (3) A microwave tube was charged with amine 2 (37 mg, 0.11 mmol) 6-chloro-7deazapurine (17 mg, 0.11 mmol) and TEA (21 mL, 15 mg, 0.15 mmol). The tube was sealed and
heated for 120 min to 150 °C (max 40 W) in the microwave. The crude product was purified using
flash chromatography (eluent: EE/DCM, 3:1) to give the title compound as an off-white solid.
Formula: C42H54F2N6O6. Yield: 33 mg (0.072 mmol, 65%). 1H-NMR (500 MHz, CDCl3): δ [ppm] = 11.84
(s, 1H, ArNH), 8.27 (s, 1H, ArH), 7.05 (d, 1H, J = 3.4 Hz, ArH), 6.42 (d, 1H, J = 3.4 Hz, ArH), 5.08 (m, 1H,
NH), 3.95 (t, 2H, J = 6.2 Hz, PEG-CH2), 3.75 (t, 2H, J = 6.2 Hz, PEG-CH2), 3.64 (d, 2H, J = 7.4 Hz, CH-CH2),
3.57-3.62 (m, 4H, 2xPEG-CH2), 3.52 (t, 2H, J = 4.7 Hz, PEG-CH2), 3.29 (m, 2H, PEG-CH2), 1.87 (m, 1H,
CH2-CH), 1.61-1.79 (m, 6H, Chex-CH2), 1.43 (s, 9H, tBu), 1.14-1.20 (m, 2H, Chex-CH2), 0.98-1.06 (m, 2H,
Chex-CH2). 13C-NMR (150 MHz, CDCl3) : δ [ppm] = 156.7 (ArC), 156.0 (Boc-C=O), 151.7 (ArC), 150.1
(ArC-H), 120.1 (ArC-H), 102.1 (ArC), 101.7 (ArC-H), 79.1 (Boc-CCH3), 70.6 (PEG-CH2), 70.3 (PEGCH2),69.1 (PEG-CH2), 56.3 (CH2-CH), 50.2 (PEG-CH2), 40.3 (PEG-CH2), 37.5 (CH), 30.8 (PEG-CH2), 28.4
(3xBoc-CH3), 26.4 (2xCH2), 25.9 (2xCH2), 14.1 (CH2).
N-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)-N-(cyclohexylmethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
(4) The carbamate 3 (33 mg, 0.072 mmol) was dissolved in 1 mL of a mixture of TFA, water and TIS
(95:2.5:2.5) and stirred for 1 h at rt. The reaction mixture was coevaporated with isopropanol
(2x10 mL) and dried in vacuum to give the title compound as its corresponding ditrifluoro acetate.
Formula: C42H54F2N6O6. Yield: 33 mg (0.072 mol, quant.). 1H-NMR (500 MHz, CDCl3): δ [ppm] = 11.14
(s, 1H, ArNH), 8.29 (s, 1H, ArH), 7.81 (m, 3H, NH3+), 7.20 (m, 1H, ArH), 6.54 (m, 1H, ArH), 4.01 (m, 2H,
PEG-CH2), 3.55-381 (m, 10H,2xCH2CH+8x PEG-CH2), 3.15 (m, 2H, PEG-CH2), 1.87 (m, 1H, CH2-CH),
1.61-1.79 (m, 6H, Chex-CH2), 1.14-1.20 (m, 2H, Chex-CH2), 0.98-1.06 (m, 2H, Chex-CH2). 13C-NMR
(150 MHz, CDCl3) : δ [ppm] = 142.3 (ArC), 123.2 (ArC), 116.7 (ArC-H), 114.8 (ArC-H), 103.6 (ArC), 102.1
(ArC-H), 70.2 (PEG-CH2), 68.2 (PEG-CH2),66.3 (PEG-CH2), 39.7 (CH2-CH), 30.5 (PEG-CH2), 26.1 (PEGCH2), 25.6 (CH), 17.6 (PEG-CH2), 17.2 (2xCH2), 12.2 (2xCH2), 11.6 (CH2). Exact mass (ESI): mz1
= 362.25490 [M+H]+, calc.: 362.25505.
4
Synthesis of probe 2b:
Supplementary Figure 2: Overview of the synthesis of probe 2b.
2-Bromo-6-fluoroaniline (5) (Bioorganic & Medicinal Chemistry Letters 2010, 20, 2609–2613) 2Bromo-6-fluorobenzoic acid (1 g, 5 mmol) was dissolved in 95% sulfuric acid (12 mL) and heated to
75 °C for 30 min. The reaction mixture was allowed to cool to rt prior to addition of Sodium azide
(429 mg, 6.6 mmol) in three portions over 30 min. After stirring over night at rt the reaction mixture
was cooled to 0-4 °C and neutralized with 25% Ammonia solution. The resulting suspension was
extracted with EE (5x50 mL) and the organic phases washed with brine and dried over Na2So4.
Evaporation of the solvent gave the crude product as viscous oil which was used as obtained in the
next step. Formula: C6H5BrFN. Yield: 918 mg (4.8 mmol, 95%). 1H-NMR (500 MHz, CDCl3): δ
[ppm] = 7.19 (d, 1H, J = 8.1 Hz), 6.95 (ddd, 1H, J = 10.6, 8.2, 1.3 Hz), 6.57 (td, 1H, J = 8.2, 5.5 Hz).
1-Bromo-3-fluoro-2-nitrobenzene (6) (Bioorganic & Medicinal Chemistry Letters 2010, 20, 2609–
2613) 2-Bromo-6-fluoroaniline (5) (950 mg, 5.0 mmol) as obtained in the previous step was dissolved
in DCE (25 mL) and treated with meta-chloroperbenzoic acid (3.9 g, 22.6 mmol). The reaction mixture
was heated to 70 °C overnight. After cooling to rt EE (25 mL) was added and the organic phase was
washed with 0.1M NaOH solution till complete removal of the perbenzoic acid. After washing with
brine and drying over Na2SO4 the solvent was evaporated to give the crude product as a yellow
viscous oil that was used without further purification in the next step. Formula: C6H3BrFNO2. Yield:
1.100 g (5.00 mmol, quantitative) 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.49 (dt, 1H, J = 8.2, 1.3 Hz),
7.38 (td, 1H, J = 8.3, 5.5 Hz), 7.25 (td, 1H, J = 8.7, 1.0 Hz).
5
Ethyl 2-([3-bromo-2-nitrophenyl]amino)acetate (7) (Bioorganic & Medicinal Chemistry Letters 2010,
20, 2609–2613) 1-Bromo-3-fluoro-2-nitrobenzene (6) (1.00 g, 4.5 mmol) as obtained in the previous
step was mixed with DIPEA (3.04 mL, 2.30 g, 18 mmol) in DMF (5 mL) and heated to 80 °C. Glycine
ethyl ester hydrochloride (1.88 g, 13.5 mmol) was added in three portions during 5 h. After allowing
the reaction mixture to cool down to rt the volatile components were evaporated. The crude product
was purified using flash chromatography (eluent: PE/EE 9:1) to give an orange crystalline solid.
Formula: C10H11BrN2O4. Yield: 950 mg (3.1 mmol, 69%). 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.16 (t,
1H, J = 8.2 Hz), 7.02 (dd, 1H, J = 7.9, 1.1 Hz), 6.61 (m, 1H), 6.09 (t, 1H, J = 5.3 Hz), 4.27 (q, 2H,
J = 7.2 Hz), 3.95 (d, 2H, J = 5.0 Hz), 1.30 (t, 3H, J = 7.1 Hz).
8-Bromoquinoxalinone (8) (Bioorganic & Medicinal Chemistry Letters 2010, 20, 2609–2613) Ethyl 2([3-bromo-2-nitrophenyl]amino)acetate (7) (112 mg, 0.37 mmol) was dissolved in EtOH/H2O 95:5
(2 mL) and treated with Fe powder (62 mg, 111 mmol) and CaCl2 (41 mg, 0.37 mmol). The reaction
mixture was heated to reflux for 3 h. After cooling to rt the resulting suspension was filtrated and the
filtrate evaporated to dryness. The residue was dissolved in EtOH/THF 1:1 (2 mL) and treated with
KOtBu (166 mg, 1.4 mmol) Oxygen was bubbled through the solvent for 1 h and the reaction mixture
stirred overnight under oxygen atmosphere at rt. After neutralizing the resulting suspension with 1 M
HCl-solution the reaction mixture was evaporated to dryness resolved in DCM (10 ml) and washed
with brine (2x5 mL). The organic phase was dried over Na2SO4 and the solvent removed under
reduced pressure to give the crude product as a light brown solid that was purified using preparative
RP-HPLC. Formula: C8H6BrN2O. Yield: 49 mg (0.22 mmol, 60% over two steps). 1H-NMR (500 MHz,
(CD3)2SO): δ [ppm] = 11.66 (s, 1H), 8.21 (s, 1H), 7.85 (d, 1H, J = 7.2 Hz), 7.80 (d, 1H, J = 7.6 Hz), 7.37
(m, 1H).
8-Bromo-2-chloroquinoxaline (9) (Bioorganic & Medicinal Chemistry Letters 2010, 20, 2609–2613) 8Bromoquinoxalinone (8) (49 mg, 0.22 mmol) was dissolved in POCl3 (1.5 mL) and heated for 3 h to
80 °C. The reaction mixture was allowed to cool to rt and unreacted POCl3 was removed under
reduced pressure. Water (15 mL) and DCM (15 mL) were added to the residue at 4 °C and the phases
were separated. The organic phase was washed with sat. NaHCO3-solution, brine (10 mL) and dried
over Na2SO4.The solvent was evaporated to give the crude product that was used as obtained for the
next step. Formula: C8H4BrClN2. Yield: 44 mg (0.18 mmol, 82%). 1H-NMR (500 MHz, (CD3)2SO): δ
[ppm] = 9.08 (s, 1H), 8.30 (d, 1H, J = 7.5 Hz), 8.19 (d, 1H, J = 8.3 Hz), 7.84 (dd, 2H, J = 8.3, 7.5 Hz).
4-(4-Bromo-2,6-difluorophenyl)morpholine (10) (WO 2008/148867 A2, PCT/EP2008057058) (4Bromo-2,6-difluorophenyl)methanol (179 mg, 0.80 mmol) was dissolved in dry DCM (8 mL) treated
with phosphorus tribromide (380 µL, 1.08 g, 4.00 mmol) and stirred overnight at rt. Cold methanol
(5 mL) was added and the organic phase was washed with sat. NaHCO3-solution (2x2 mL) before
evaporation of the solvent. The residue was dissolved in dry THF (8 mL) and treated with morpholine
(694 µL, 697 mg, 8 mmol) and TEA (223 µL, 162 mg, 1.6 mmol) before stirring for 3 h at rt. After
adding DCM (20 mL) the reaction mixture was washed with 1M NaOH-solution (2x5 mL) dried over
Na2SO4 and the solvent evaporated to give the crude product that was used as obtained for the next
step. Formula: C11H12BrF2NO. Yield: 165 mg (0.57 mmol, 70%). 1H-NMR (500 MHz, CDCl3): δ
[ppm] = 7.09 (m, 2H, ArH), 3.68 (m, 4H, 2xO-CH2), 3.62 (s, 2H, Ar-CH2), 2.48 (m, 4H, 2xN-CH2).
4-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolanyl)2,6-difluorophenyl)morpholine (11) (Angew. Chem.
2010, 122, 7320-7322) 4-(4-Bromo2,6-difluorophenyl)morpholine (10) (106 mg, 0.36 mmol),
bispinacolatodiboron (120 mg, 0.47 mmol) and KOAc (71.0 mg, 0.72 mmol) were dissolved in DMF
6
(1 mL). After 5 cycles of freeze-pump-thaw PdCl2(dppf) (29 mg, 0.04 mmol) was added prior to
heating the reaction mixture for 60 min at 80 °C in the microwave (max. 20 W). After allowing to cool
to rt EE (15 mL) was added and the organic phase washed with water (1x5 mL) sat. NaHCO3-solution
(1x5 mL) and brine (1x5 mL). After drying over Na2SO4 and evaporation of the solvent the crude
product was purified using flash chromatography (eluent: EE + 1% TEA) to give the desired product as
viscous oil. Formula: C17H24BF2NO3. Yield: 87 mg (0.26 mmol, 72%). 1H-NMR (500 MHz, CDCl3): δ
[ppm] = 7.30 (m, 2H, ArH), 3.65-3.75 (m, 6H, 2xAr-CH2 + 4xO-CH2), 2.51 (m, 4H, N-CH2), 1.34 (s, 12H,
4xCH3).
2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethanol (12) Tetraethyleneglycol (54.3 g, 280 mmol) and TEA
(30.0 mL, 21.8 g, 215 mmol) were mixed in dry THF (200 mL)and cooled down to 0 °C prior to adding
methane sulfonyl chloride (7.9 mL, 11.7 g, 102 mmol) over 45 min. After allowing to warm up to rt
the resulting mixture was stirred at rt overnight. The solvent was evaporated and the residue was
treated with ethanol (200 mL) and NaN3 (13.0 g, 200 mmol). The reaction mixture was heated to
reflux overnight. After evaporation of the solvent the residue was extracted with Et2O (5x100 mL) the
residue treated with H2O (200 mL) and extracted with Et2O (3x30 mL). The combined organic layers
were washed with brine (2x100 mL) dried over MgSO4 and evaporated to give the crude product that
was purified using flash chromatography (eluent: DCM/MeOH 49:1). Formula: C8H17N3O4. Yield: 4.5 g
(20 mol, 20% over two steps). 1H-NMR (500 MHz, CDCl3): δ [ppm] = 3.71 (m, 2H, O-CH2), 3.62-3.68
(m, 10H, 5xO-CH2), 3.59 (m, 2H, O-CH2), 3.38 (m, 2H, N-CH2), 2.62 (s, 1H, OH). 13C-NMR (150 MHz,
CDCl3) : δ [ppm] = 72.5 (HO-CH2), 70.8 (O-CH2), 70.7 (O-CH2), 70.6 (O-CH2), 70.4 (O-CH2), 70.1 (O-CH2),
61.8 (O-CH2CH2N3), 50.7 (O-CH2CH2N3).
2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethanol (13) The azide 12 (4.3 g, 19.6 mmol) was dissolved in
methanol (50 mL) and treated with Pd/C (5m%, 200 mg). The suspension was flushed with argon prior
to flushing with hydrogen. After stirring overnight at rt under hydrogen atmosphere the catalyst was
filtered off and the filtrate was evaporated to give the title compound that was used without further
purification for the next step. Formula: C8H19NO4. Yield: 3.8 g (19.6 mol, quant). 1H-NMR (500 MHz,
CDCl3): δ [ppm] = 3.55-3.75 (m, 12H, CH2), 3.52 (t, 2H, J = 4.9 Hz, CH2), 2.84 (t, 2H, J = 4.9 Hz, CH2).
tert-Butyl 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethylcarbamate (14) The amine 13 (3.4 g,
17.4 mmol) was dissolved in THF (30 mL) and treated with TEA (3.6 mL, 2.6 g, 26.0 mmol). A solution
of boc2O (4.9 g, 22.6 mmol) in THF (10 mL) was added dropwise over 5 h to the reaction mixture that
was subsequently stirred overnight at rt. The solvent was evaporated to give the crude product that
was purified using flash chromatography (eluent: DCM/MeOH, 49:1) to give the title compound as a
colorless viscous oil. Formula: C13H27NO6. Yield: 2.7 g (9.1 mmol, 52%). 1H-NMR (500 MHz, CDCl3): δ
[ppm] = 5.63 (s, 1H, NH), 3.68-3.78 (m, 4H, 2xCH2), 3.58-3.67 (m, 6H, 3xCH2), 3.53 (t, 2H, J = 5 Hz,
CH2), 3.31 (m, 2H, CH2), 1.44 (s, 9H, tBu). ESI-MS: m·z-1 = 316.2 [M+Na]+(calc.: 316.2).
2-(2-(2-(2-(tert-Butoxycarbonyl)ethoxy)ethoxy)ethoxy)ethyl
4-methylbenzenesulfonate
(15)
Alcohol 14 (4.1 g, 14 mmol), TsCl (3.4 g, 18 mmol) and TEA (3.9 mL, 2.8 g, 28 mmol) were mixed in
dry DCM (25 mL) prior to cooling to 0 °C. Trimethylamine hydrochloride (0.7 g, 7 mmol) was added in
one portion to the reaction mixture that was stirred at 0 °C for 3h and at rt overnight. The reaction
mixture was concentrated to dryness and the residue purified using flash chromatography (eluent:
DCM/MeOH, 95:5 +1% TEA). Formula: C20H33NO8S. Yield: 4.2 g (9.5 mmol, 68%). 1H-NMR (500 MHz,
CDCl3): δ [ppm] = 7.80 (d, 2H, J = 8.0 Hz, Ar-H), 7.34 (d, 2H, J = 8.0 Hz, Ar-H), 5.00 (s, 1H, NH), 4.16 (t,
7
2H, J = 4.9 Hz, CH2), 3.69 (t, 2H, J = 4.8 Hz, CH2), 3.57-3.63 (m, 8H, 4xCH2), 3.52 (t, 2H, J = 5.3 Hz, CH2),
3.30 (m, 2H, CH2), 2.45 (s, 3H, CH3), 1.43 (s, 9H, tBu). ESI-MS: m·z-1 = 470.2 [M+Na]+(calc.: 470.2).
tert-Butyl 2-(2-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)ethoxy)ethylcarbamate (16) Piperazin·6H2O
(9.1 g, 47 mmol) and K2CO3 (1 g, 7 mmol) were dissolved in ACN (50 mL) and heated to 80 °C prior to
adding tosylate 15 (2.1 g, 4.7 mmol) in ACN (10 mL) over 4 h to this suspension. The reaction mixture
was stirred at 80 °C overnight, allowed to cool down to rt and the solvent evaporated. The residue
was dissolved in DCM (50 mL) washed with sat. NaHCO3 solution (2x10 mL) dried over MgSO4 and the
solvent evaporated. The crude product was purified using flash chromatography (eluent:
DCM/MeOH, 95:5 +1% TEA) to give the title compound (1.25 g, 3.5 mmol, 74%). Formula: C17H35N3O5.
Yield: 1.25 g (3.5 mmol, 74%). 1H-NMR (500 MHz, CDCl3): δ [ppm] = 5.18 (s, 1H, NH), 3.57-3.70 (m,
10H, 5xCH2), 3.53 (t, 2H, J = 5.1 Hz, CH2), 3.31 (m, 2H, CH2), 2.92 (m, 4H, 2xCH2), 2.59 (t, 2H, J = 5.3 Hz,
CH2), 2.51 (m, 4H, 2xCH2), 1.44 (s, 9H, tBu). ESI-MS: m·z-1 = 362.3 [M+H]+(calc.: 362.3).
tert-Butyl 2-(2-(2-(2-(4-(4-bromophenyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)ethylcarbamate (17)
Secondary amine 16 (3.9 g, 11 mmol), 1-bromo-4-iodo benzene (6.1 g, 22 mmol), K3PO4 (6.9 g,
33 mmol), CuI (3 g, 16 mmol) and ethylene glycol (911 µL, 1011 mg, 16 mmol) were dissolved in
i
PrOH (90 mL) and heated to 80 °C for 3 d. The solvent was evaporated and the residue dissolved in
NaOH solution (100 mL). The aqueous phase was extracted with EE (5x20 mL) the combined organic
layers washed with NaOH solution and dried over MgSO4. The solvent was evaporated to give the
crude product that was purified using flash chromatography (eluent: EE+1% TEA) to give the title
compound (4.4 g, 8.5 mmol, 78%). Formula: C23H38BrN3O5. Yield: 4.4 g (8.5 mmol, 78%). 1H-NMR
(500 MHz, CDCl3): δ [ppm] = 7.33 (d, 2H, J = 9.3 Hz, Ar-H), 6.78 (d, 2H, J = 9.1 Hz, Ar-H), 5.11 (s, 1H,
NH), 3.57-3.70 (m, 12H, 6xCH2), 3.54 (t, 2H, J = 5.2 Hz, CH2), 3.31 (m, 2H, CH2), 3.18 (m, 4H, 2xCH2),
2.67 (m, 4H, 2xCH2), 1.44 (s, 9H, tBu). MALDI-MS: m·z-1 = 516.2 [M+H]+(calc.: 516.2).
tert-Butyl 2-(2-(2-(2-(4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)ethylcarbamate (18) (Angew. Chem. 2010, 122, 7320-7322) Aryl bromide 17
(565 mg, 1.1 mmol), KOAc (215 mg, 2.2 mmol) and bis(pinacolato)diboron (362 mg, 1.4 mmol) were
dissolved in ACN/H2O, 1:1 (1 mL). After 5 cycles of freeze-pump-thaw PdCl2(dppf) (80 mg,
0.11 mmol)was added prior to heating the reaction mixture for 60 min at 80 °C in the microwave
(max. 20 W). After allowing to cool to rt EE (15 mL) was added and the organic phase washed with
water (1x5 mL) sat. NaHCO3 solution (1x5 mL) and brine (1x5 mL). Evaporation of the solvent derived
the crude product that was purified using flash chromatography (eluent: DCM/MeOH, 95:5 +1% TEA)
to give the title compound (200 mg, 0.36 mmol, 32%). Formula: C29H50BN3O7. Yield: 200 mg
(0.36 mmol, 32%). 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.73 (d, 2H, J = 9.0 Hz, Ar-H), 6.87 (d, 2H,
J = 8.5 Hz, Ar-H), 5.08 (s, 1H, NH), 3.91 (m, 2H, CH2), 3.56-3.85 (m, 12H, 6xCH2), 3.51 (m, 2H, CH2),
3.24-3.45 (m, 6H, 3xCH2), 3.10 (m, 2H, CH2), 1.44 (s, 9H, tBu), 1.32 (s, 12H, 4xCH3). 13C-NMR (150 MHz,
CDCl3) : δ [ppm] = 156.1 (C=O), 151.4 (ArC), 136.3 (2xArC), 115.3 (2xArC), 83.5 (2xC3-C-O), 79.3
(Me3C), 70.2 (C-C-O), 70.1 (C-C-O), 65.6 (C-C-O), 60.4 (C-C-O), 56.6 (C-C-O), 52.3 (C-C-O), 56.6 (2xC-CN), 46.0 (2xC-C-N), 40.2 (C-C-O), 27.8 (3xCH3), 24.8 (4xCH3C-O). Exact mass (ESI): mz-1 = 564.38235
[M+H]+ (calc.: 564.38146).
8-Bromo-2(2-(4-(4-(2-(2-(2-(2-(tert-butoxycarbonyl)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1yl)phenyl))quinoxaline (19) (WO 2008/148867 A2, PCT/EP2008057058) 2-Bromo-8chloroquinoxaline (9) (7.0 mg, 0.029 mmol), boronate 18 (16.0 mg, 0.029 mmol) and Na2CO3 (3.0 mg,
0.087 mmol) were dissolved in DMF (100 µL). This suspension was degassed with 5 cycles freeze8
pump-thaw prior to adding 5 mol% Pd(PPh3)4 (3.4 mg, 2.9 nmol). The reaction mixture was heated
for 4 h at 100 °C in the microwave (max. 40 W). After allowing to cool to rt DMF was evaporated and
the residue dissolved in DCM, filtered and the solvent evaporated to give the crude product that was
purified using preparative RP-HPLC to give the desired product as its corresponding trifluoroacetate.
Formula: C31H42BrN5O5. Yield: 5 mg (6.7 µmol, 24%). 1H-NMR (500 MHz, CDCl3): δ [ppm] = 9.31 (s, 1H,
Ar-H), 8.29 (d, 2H, J = 8.6 Hz, Ar-H), 8.07 (d, 1H, J = 8.4 Hz, Ar-H), 8.06 (d, 1H, J = 9.5 Hz, Ar-H), 7.56
(dd, 1H, J = 9.5, 8.4 Hz, Ar-H), 7.07 (d, 2H, J = 8.8 Hz, Ar-H), 5.07 (s, 1H, NH), 3.94 (m, 2H, CH2), 3.583.88 (m, 12H, 6xCH2), 3.53 (m, 2H, CH2), 3.24-3.45 (m, 6H, 3xCH2), 3.10 (m, 2H, CH2), 1.43 (s, 9H, tBu).
13
C-NMR (150 MHz, CDCl3) : δ [ppm] = 156.1 (C=O), 150.8 (ArC), 143.5 (ArC), 141.5 (ArC), 140.0 (ArC),
133.8 (ArC), 133.0 (ArC), 129.1 (2x ArC), 129.0 (ArC), 128.5 (ArC), 124.0 (ArC), 116.4 (2x ArC) 79.3
(Me3C), 70.3 (C-C-O), 69.9 (C-C-O), 69.8 (C-C-O), 69.6 (C-C-O), 65.7 (C-C-O), 65.1 (C-C-O), 56.6 (C-C-O),
51.8 (2xC-C-N), 45.3 (2xC-C-N), 40.3 (C-C-O), 27.8 (3xCH3). Exact mass (ESI): mz-1 = 644.24328 [M+H]+
(calc.: 644.24421).
8-(2,6-Difluorophenyl)morpholine)-2(2-(4-(4-(2-(2-(2-(2-(tert-butoxycarbonyl)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)phenyl))quinoxaline (20) (WO 2008/148867 A2, PCT/EP2008057058)
Bromoquinoxaline 19 (5 mg, 6.7 µmol), boronate 11 (2.7 mg, 8 µmol), SPhos (0.3 mg, 0.7 µmol) and
K3PO4 (5.7 mg, 27 µmol) were dissolved in DMF (500 µL). After 5 cycles of freeze-pump-thaw
Pd(OAc)2 (0.03 mg, 0.1 µmol) was added to the suspension prior to heating 2 h at 100 °C in the
microwave (max. 40 W). After allowing the reaction mixture to cool to rt the solvent was evaporated.
The residue was dissolved in DCM and the solids filtered off. After removal of DCM the crude product
was purified using preparative RP-HPLC to give the title compound as its corresponding trifluoro
acetate. Formula: C42H54F2N6O6. Yield: 1 mg (1.0 µmol, 15%). 1H-NMR (600 MHz, CDCl3): δ
[ppm] = 9.37 (s, 1H, Ar-H), 8.18 (dd, 1H, J = 8.5, 1.7 Hz, Ar-H), 8.10 (d, 2H, J = 8.7 Hz, Ar-H), 7.82 (dd,
1H, J = 7.0, 1.6 Hz, Ar-H), 7.79 (dd, 1H, J = 8.5, 7.0 Hz), 7.54 (d, 2H, J = 9.3 Hz, Ar-H), 5.08 (s, 1H, NH),
4.42 (s, 2H, Benzyl-CH2), 3.99-4.06 (m, 4H, 2xCH2), 3.95 (m, 2H, CH2), 3.22-3.88 (m, 26H, 13xCH2), 1.42
(s, 9H, tBu). 13C-NMR (150 MHz, CDCl3) : δ [ppm] = 161.0 (2xArC), 156.1 (C=O), 151.0 (ArC), 150.6
(ArC), 143.0 (ArC), 142.8 (ArC), 139.2 (ArC), 139.1 (ArC), 136.5 (ArC), 130.7 (2x ArC), 130.6 (ArC),
130.3 (ArC), 128.7 (2xArC), 128.4 (ArC), 116.5 (2x ArC), 114.0 (2x ArC), 103.4 (ArC), 79.3 (Me3C), 70.3
(C-C-O), 69.9 (C-C-O), 69.8 (C-C-O), 69.6 (C-C-O), 65.7 (C-C-O), 65.1 (C-C-O), 63.8 (2xC-C-O), 56.6 (C-CO), 51.8 (2xC-C-N), 50.8 (BnC), 50.4 (2xC-C-N) 45.3 (2xC-C-N), 40.3 (C-C-O), 27.8 (3xCH3). Exact mass
(ESI): mz-1 = 777.41365 [M+H]+ (calc.: 777.41457).
2-(2-(2-(2-(4-(4-(8-(3,5-Difluoro-4-(morpholinomethyl)phenyl)quinoxalin-2-yl)phenyl)piperazin-1yl)ethoxy)ethoxy)ethoxy)ethanamine (21) The carbamate 20 (1 mg, 1.0 µmol) was dissolved in 1 mL
of a mixture of TFA, water and TIS (95:2.5:2.5) and stirred for 1 h at rt. The reaction mixture was
coevaporated with isopropanol (2x10 mL) and dried in vacuum to give the title compound as its
corresponding tritrifluoro acetate. Formula: C37H46F2N6O4, Yield: 0.92 mg (0.9 µmol, 90%) Analytical
HPLC (Rt [min]): 2.25. Exact mass (ESI): mz-1 = 677.36183 [M+H]+ (calc.: 677.36214).
9
Synthesis of probe 3b:
Supplementary Figure 3: Overview of the synthesis of probe 3b and its original inhibitor 4.
4-(4-(Benzyloxy)-3-methoxyphenyl)-2-chloropyrimidine (22) (Bioorganic & Medicinal Chemistry
Letters 2009, 19, 5887–5892) 4-(Benzyloxy)-3-methoxyphenylboronic acid (300 mg, 1.162 mmol) and
2,4-dichloropyrimidine (260 mg, 1.744 mmol) were dissolved in 2M aqueous Na2CO3-solution
(1.5 mL, 2.324 mmol). After addition of toluene (1.5 mL) the reaction mixture was degassed with 4
cycles of freeze-pump-thaw before Pd(PPh3)4 (130 mg, 0.12 mmol) was added to this suspension. The
reaction mixture was heated to 100 °C for 120 min using mw irradiation (max 20 W). DCM (10 mL)
and H2O (10 mL) were added prior to separation of the two layers. The organic phase was washed
with sat. NaHCO3-solution (3x3 mL), brine (3x3 mL), dried over MgSO4 and the solvent evaporated.
Residual dichloropyrimidine was removed via short way distillation. The crude product was used as
obtained in the next step. Formula: C18H15ClN2O2. Yield: 379.7 mg (1.162 mol, quant.). LC-MS (ESI):
Rt = 9.04 min, mz-1 = 327.3 (calc.: 327.1).
tert-Butyl piperazine-1-carboxylate (23) Piperazine (1 g, 11.6 mmol) and amberlyst-15 (15 %,
150 mg) were dissolved in DCM (50 mL) and cooled to 4 °C. A solution of Boc2O (2.3 g, 10.5 mmol) in
DCM (25 mL) was added dropwise over 2 h to this suspension. After allowing to warm up to rt the
reaction mixture was filtrated and evaporated. The crude product was purified using flash
chromatography (eluent: DCM/MeOH 95:5 +1 % TEA) to give the title compound. Formula:
C15H23N3O2. Yield: 1.17 g (6.26 mmol, 54 %). 1H-NMR (500 MHz, CDCl3): δ [ppm] = 3.34-3.46 (m, 6H,
6xH-CH), 2.83 (m, 2H, 2xH-CH), 1.46 (s, 9H, tBu).
tert-Butyl 4-(4-aminophenyl)piperazine-1-carboxylate (24) Secondary amine 23 (1.17 g, 6.26 mmol),
4-iodobenzeneamine (920 mg, 4.2 mmol), K3PO4 (1.78 g, 8.2 mmol), CuI (400 mg, 2.1 mmol) and
ethylene glycol (463 µL, 509 mg, 8.2 mmol) were dissolved in iPrOH (90 mL) and heated to 80 °C for
3 d in a sealed reaction tube. The solvent was evaporated and the residue purified using flash
10
chromatography (eluent: DCM/EE 1:1) to give the title compound. Formula: C15H23N3O2 Yield:
859 mg, 3.1 mmol, 74%, LC-MS (ESI): Rt = 4.32 min, mz-1 = 278.3 (calc.: 278.2).
2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (25) Alcohol 8 (900 mg,
4.1 mmol), TsCl (1010 mg, 5.3 mmol) and TEA (1137 µL, 830 mg, 8.2 mmol) were dissolved in dry
DCM (20 mL) prior to cooling to 0 °C. Trimethylamine hydrochloride (118 mg, 1.23 mmol) was added
in one portion to the reaction mixture that was stirred at 0 °C for 3h and at rt overnight. The reaction
mixture was concentrated to dryness and the residue purified using flash chromatography (eluent:
DCM/EE 1:1). Formula: C15H23N3O6S Yield: 1082 mg (2.9 mmol, 71%). LC-MS (ESI): Rt = 1.09 min,
mz-1 = 373.5 (calc.: .374.1).
tert-Butyl 4-(4-(4-(4-(benzyloxy)-3-methoxyphenyl)pyrimidin-2-ylamino)phenyl)piperazine-1-carboxylate (26) (Bioorganic & Medicinal Chemistry Letters 2009, 19, 5887–5892) Aryl chloride 22 (40 mg,
0.123 mmol) and amine 24 (34 mg, 0.123 mmol) were dissolved in 1,4-dioxane (1 mL) and treated
with pTsOH monohydrate (7 mg, 0.04 mmol). The resulting suspension was heated to 80 °C for 10 h
and the solvent evaporated to give the crude product that was purified via preparative RP-HPLC to
give the title compound. Formula: C33H37N5O4. Yield: 24 mg (0.042 mmol, 34 %). LC-MS (ESI):
Rt = 9.60 min, mz-1 = 568.4 (calc.: 568.3).
2-Methoxy-4-(2-(4-(piperazin-1-yl)phenylamino)pyrimidin-4-yl)phenol (27) The protected aryl
compound 26 (24 mg, 0.042 mmol) was dissolved in a solution of TFA/H2O/TIS 95/2.5/2.5 (1.5 mL)
and stirred at rt for 120 min. Volatile substances were evaporated and the residue purified via
preparative RP-HPLC to give the desired product as its corresponding trifluoroacetate. Formula:
C21H23N5O2. Yield: 9.9 mg (0.0201 mmol, 48 %). LC-MS (ESI): Rt = 3.81 min, mz-1 = 378.5 (calc.: 378.2).
4-(2-(4-(4-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)phenylamino)pyrimidin-4-yl)2-methoxyphenol (28) The secondary amine 27 (3 mg, 0.0061 mmol) and TEA (200 µL, 240 mg,
1.61 mmol) were dissolved in dry DCM (1 mL) and treated with tosylate 25 (301 mg, 0.805 mmol).
The resulting reaction mixture was heated to reflux for 6 h. After evaporating the solvent the crude
product was purified via RP-HPLC to give the title compound as its corresponding trifluoroacetate.
Formula: C29H38N8O5. Yield: 4 mg (0.006 mmol, 95 %). 1H-NMR (600 MHz, MeOD): δ [ppm] = 8.24 (d,
1H, J = 5.91 Hz, Ar-H), 7.84 (d, 1H, J = 1.8 Hz, Ar-H), 7.72 (dd, 1H, J = 2.1, 8.3 Hz, Ar-H), 7.60 (d, 2H,
J = 9.0 Hz, Ar-H), 7.37 (d, 1H, J = 6.3 Hz, Ar-H), 7.11 (d, 2H, J = 9.0 Hz, Ar-H), 6.93 (d, 2H, J = 8.4 Hz, ArH), 3.95 (s, 3H, CH3), 3.90 (m, 2H, CH2), 3.70-375 (m, 4H,2xCH2), 3.65-3.69 (m, 8H, 4xCH2), 3.37-3.50
(m, 8H, 4xCH2), 1.31 (m, 4H, 2xCH2). LC-MS (ESI): Rt = 4.63 min, mz-1 = 579.4 (calc.: 579.3).
4-(2-(4-(4-(2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)phenylamino)pyrimidin-4yl)-2-methoxyphenol (29) The azide 28 (2 mg, 0.0035 mmol) was dissolved in MeOH (1 mL) and
treated with TEA (200 µL, 240 mg, 1.61 mmol) and 1,3-propanedithiol (200 µL, 216 mg, 2.0 mmol).
The reaction mixture was stirred overnight and the solvent evaporated prior to preparative RP-HPLC
purification. The title compound was obtained as its corresponding ditrifluoroacetate. Formula:
C29H40N6O5. Yield: 2.7 mg (0.0035 mmol, quant). 1H-NMR (600 MHz, MeOD): δ [ppm] = 8.26 (d, 1H,
J = 5.9 Hz, Ar-H), 7.83 (d, 1H, J = 2.0 Hz, Ar-H), 7.71 (dd, 1H, J = 8.4, 2.0 Hz, Ar-H), 7.61 (d, 2H,
J = 9.0 Hz, Ar-H), 7.35 (d, 1H, J = 6.0 Hz, Ar-H), 7.09 (d, 2H, J = 9.0 Hz, Ar-H), 6.92 (d, 1H, J = 8.3 Hz, ArH), 3.95 (s, 3H, CH3), 3.90 (m, 2H, CH2), 3.65-3.80 (m, 18H, 9xCH2), 3.47 (m, 2H, CH2), 3.12 (m, 2H,
CH2). 13C-NMR (125 MHz, CDCl3): δ [ppm] = 167.1 (ArC), 157.5 (ArC), 152.1 (ArC), 151.0 (ArC), 147.8
(ArC), 146.1 (ArC), 127.5 (ArC), 122.5 (2xArC), 122.0 (ArC), 121.7 (ArC), 117.3 (2xArC), 115.0 (ArC),
11
110.6 (ArC), 106.2 (ArC), 70.0 (CH2), 69.9 (7xCH2), 66.2 (CH2), 63.8 (CH2), 55.9 (CH2), 55.0 (CH3), 39.2
(CH2). LC-MS (ESI): Rt = 3.42 min, mz-1 = 553.4 (calc.: 553.3). Exact mass (ESI): mz-1 = 553.31317 (calc.:
553.31329).
2-Methoxy-4-(6-(4-morpholinophenylamino)pyrazin-2-yl)phenol (30) (Bioorganic & Medicinal
Chemistry Letters 2009, 19, 5887–5892) Aryl chloride 22 (106 mg, 0.324 mmol), 4morpholinobenzenamine (58 mg, 0.324 mmol) and pTsOH∙H2O (19 mg, 0.097 mmol) were dissolved
in 1,4-dioxane (3 mL) and heated to reflux for 4 h. Volatile substances were subsequently evaporated
to give the crude substitution product that was treated with 95 % aqueous TFA (10 mL) and stirred
for 8 h at 60 °C. After the reaction mixture reached ambient temperature the solvent was evaporated
and the crude product purified using preparative HPLC to give the title compound. Formula:
C21H22N4O3, Yield: 78 mg (0.206 mmol, 64 %, over two steps), 1H-NMR (500 MHz, CDCl3): δ [ppm] =
11.79 (s, 1H, NH), 8.09 (d, 1H, J = 6.3 Hz, ArH), 7.74 (d, 1H, J = 2.1 Hz, ArH), 7.63-7.68 (m, 3H, 3xArH),
7.17 (d, 1H, J = 6.7 Hz, ArH), 7.04 (d, 1H, J = 8.3 Hz, ArH), 6.98 (d, 2H, J = 8.5 Hz, 2xArH), 3.98 (s, 3H,
CH3), 3.90 (m, 4H, 2xCH2), 3.18 (m, 4H, 2xCH2). 13C-NMR (125 MHz, CDCl3): δ [ppm] = 164.3 (ArC),
164.0 (ArC), 153.5 (ArC), 151.2 (ArC), 148.3 (ArC), 147.1 (ArC), 129.9 (ArC), 126.5 (ArC), 123.2 (ArC),
123.0 (2xArC), 116.2 (2xArC), 115.1 (ArC), 110.3 (ArC), 105.1 (ArC), 66.7 (2xCH2), 56.0 (CH3), 49.8
(2xCH2). MS (MALDI): mz-1 = 378.8 (calc.: 379.2).
12
Binding profile of probe 3
Supplementary Table 3: Kinases identified in all replicates in the inhibitor affinity
purification experiment using probe 3b and MV4-11 cell lysate. JAK isoforms JAK1, JAK2 and
Tyk2 are highlighted in red.
13