ars.els-cdn.com€¦ · Web view(3.74 g, 7 mmol) in CH 2 Cl 2 (20 mL) was added 3-chloropropanoyl chloride (1.07 g, 8.40 mmol) in an ice bath. The mixture was stirred at room temperature

Supplementary Information

Discovery and biological evaluation of proteolysis targeting chimeras

(PROTACs) as an EGFR degraders based on osimertinib and lenalidomide

Kailun He a, Zhuo Zhang a, Wenbing Wang a, Xiaoliang Zheng b, Xiaoju Wangb,*, Xingxian Zhanga,*

(a College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, Zhejiang 310014)

(b Center for Molecular Medicine, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310032)

Experimental methods1.1 Chemistry and chemical methods

Generally, all of the chemical materials and reagents were obtained from

commercial source. The experiments described in this paper were all performed in

conventional glass instruments and all reactions were monitored and tracked by thin

layer chromatography (TLC) on silica gel plates (GF 254) and visualized with UV

light fluorescence (254 nm). Alkaline potassium permanganate solution,

phosphomolybdic acid, and iodine tank were used for color detection based on the

structure properties of the compounds. The products were separated by silica gel

column chromatography. The silica gel used for the column is 200-300 mesh silica gel

(Qingdao Ocean Chemical Plant). NMR spectra were measured by the Bruker

Avance-500 nuclear magnetic resonance instrument, and HRMS (ESI) was measured

with Thermo LCQTM Deca XP plus detector. Chloroform-d (CDCl3) or Dimethyl

sulfoxide-d6 (DMSO-d6) was used as solvents, and the tetramethylsilane (TMS) was

used as an internal standard. Melting points were measured on a BUCHI B-540 and

uncorrected.

1.1.1 A general method for the preparation of compounds 2a-2c

To a solution of compound 1 (100.00 mmol) in CH2Cl2 (30 mL) was added Et3N

(200.00 mmol) and stirred at room temperature. 4-toluene sulfonyl chloride was

dissolved in CH2Cl2 and was added slowly in an ice bath. The mixture was allowed to

warm to room temperature and stirred for 12 h. The reaction was quenched by 4

mol/L aqueous hydrochloric acid and extracted with CH2Cl2 (3×40 mL). The organic

layer was combined, dried over anhydrous sodium sulfate and concentrated under

reduced pressure to afford the crude product. The residue was purified by column

chromatography on silica gel to give compounds 2a-2c.

2-(2-hydroxyethoxy)ethyl-4-methylbenzenesulfonate (2a).1 colorless liquid. 1H

NMR (500 MHz, CDCl3) δ 7.75 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 4.19 –

4.09 (m, 2H), 3.69 – 3.58 (m, 4H), 3.52 – 3.43 (m, 2H), 2.40 (s, 3H) ppm.

2-(2-(2-hydroxyethoxy)ethoxy)ethyl-4-methylbenzenesulfonate (2b).2 colorless

liquid. 1H NMR (500 MHz, CDCl3): δ 7.79 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 7.7 Hz,

2H), 4.24-4.07 (m, 2H), 3.72-3.66 (m, 4H), 3.65-3.51 (m, 6H), 2.44 (s, 3H) ppm.

2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate (2c).2

colorless liquid. 1H NMR (500 MHz, CDCl3): δ 7.74 (d, J = 8.4 Hz, 2H), 7.48-7.16 (d,

J = 8.0 Hz, 2H), 4.12-4.09 (m, 2H), 3.66-3.62 (m, 4H), 3.60-3.56 (m, 4H), 3.55-3.52

(m, 6H), 2.39 (s, 3H) ppm.


To a solution of compound 2 (100 mmol) in toluene (30 mL) was added tert-butyl

bromoacetate 3 (120 mmol), TBAB (5 mmol) and KOH (160 mmol). The resulting

mixture was stirred at room temperature for 2h. After completion, water (10 mL) was

added. The organic layer was separated, dried over anhydrous sodium sulfate and

concentrated under reduced pressure to afford the crude product. The residue was

purified by column chromatography on silica gel to give compounds 3a-3c.

tert-butyl-2-(2-(2-(tosyloxy)ethoxy)ethoxy)acetate (3a).2 colorless liquid. 1H

NMR (500 MHz, CDCl3): 7.80 (d, J = 8.4 Hz, 2H), 7.37-7.31 (m, 2H), 4.19-4.14 (m,

2H), 3.98 (s, 2H), 3.72- 3.67 (m, 2H), 3.67-3.59 (m, 4H), 2.44 (s, 3H), 1.47 (s, 9H)

ppm.

tert-butyl-2-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)acetate (3b).2 colorless

liquid. 1H NMR (500 MHz, CDCl3) δ 7.80 (d, J = 8.4 Hz, 2H), 7.58 – 7.07 (m, 2H),

4.19 – 4.12 (m, 2H), 4.01 (s, 2H), 3.73 – 3.63 (m, 6H), 3.60 (s, 4H), 2.45 (s, 3H), 1.47

(s, 9H) ppm.

tert-butyl-14-(tosyloxy)-3,6,9,12-tetraoxatetradecanoate (3c).2 colorless liquid. 1H

NMR (500 MHz, CDCl3): δ 7.73 (d, J = 8.3 Hz, 2H), 7.30-7.27 (m, 2H), 4.10-4.08

(m, 2H), 3.96 (s, 2H), 3.66-3.62 (m, 6H), 3.61-3.60 (m, 4H), 3.52-3.50 (m, 4H), 2.39

(s, 3H), 1.41 (s, 9H) ppm.


To a solution of compound 3 (100 mmol) in acetone (20 mL) was added NaI (140

mmol). The reaction mixture was refluxed for 5 h. After removal of the solution, the

reaction was added water (10 mL) and extracted with ethyl acetate (3×20 mL). The

organic layer was dried over anhydrous sodium sulfate and concentrated under

reduced pressure to afford the crude product. The residue was purified by column

chromatography on silica gel to give compounds 4a-4c.

tert-butyl-2-(2-(2-iodoethoxy)ethoxy)acetate (4a). 2 colorless liquid. 1H NMR (500

MHz, CDCl3): δ 4.04 (s, 2H), 3.77 (d, J = 7.1 Hz, 2H), 3.75-3.68 (m, 4H), 3.28 (t, J =

6.9 Hz, 2H), 1.48 (s, 9H) ppm.

tert-butyl-2-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)acetate (4b).2 colorless liquid. 1H

NMR (500 MHz, CDCl3): δ 4.03 (s, 2H), 3.77-3.74 (m, 2H), 3.72-3.69 (m, 4H), 3.67

(s, 4H), 3.28-3.25 (m, 2H), 1.47 (s, 9H) ppm.

tert-butyl-14-iodo-3,6,9,12-tetraoxatetradecanoate (4c).2 colorless liquid. 1H

NMR (500 MHz, CDCl3): δ 4.02 (s, 2H), 3.76 (dd, J = 7.3, 6.5 Hz, 2H), 3.74 - 3.68

(m, 4H), 3.67 (d, J = 4.3 Hz, 8H), 3.27 (dd, J = 7.3, 6.5 Hz, 2H), 1.48 (s, 9H) ppm.


To a solution of compound 4 (100 mmol) in CH2Cl2 (20 mL) was added TFA (1.8

mol). The reaction was stirred at room temperature for 2h. The reaction was

concentrated under reduced pressure and added 2 mol/L NaOH to adjust pH = 7,

washed twice with CH2Cl2. The aqueous phase was acidified by 4 mol/L HCl to adjust

pH = 2 and extracted with CH2Cl2 (3×20 mL). The organic layer was dried over

anhydrous sodium sulfate and concentrated under reduced pressure to afford the crude

product 5, which was directly used to the next step without further purification.

Compound 5 was treated with oxalyl chloride (100 mmol) and catalytic amount of

DMF (0.02 mmol) in an ice bath. The resulting solution was stirred at room

temperature for 2 h, then removed the excess oxalyl chloride under reduced pressure.

The residue was added DMF to be dissolved and pomalidomide (20 mmol). After

stirring for 1 h, the reaction was added water (10 mL) and extracted with ethyl acetate

(3×20 mL). The organic layer was washed with saturated brine, dried over anhydrous

sodium sulfate and concentrated under reduced pressure to afford the crude product.

The residue was purified by column chromatography on silica gel to give compounds

6a-6c.

N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-2-(2-(2-

iodoethoxy)ethoxy)acetamide (6a).2 white solid. m.p 198.8-199.9 ; R℃ f = 0.35 (PE:

EA = 1:2); 1H NMR (500 MHz, CDCl3): 8.77 (s, 1H), 8.55 (s, 1H), 7.76 (d, J = 7.5

Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 5.22 (dd, J = 13.3, 5.1 Hz,

1H), 4.46 (s, 2H), 4.19 (d, J = 1.4 Hz, 2H), 3.85 – 3.80 (m, 2H), 3.79 (t, J = 6.4 Hz,

2H), 3.76 – 3.70 (m, 2H), 3.24 (td, J = 6.5, 1.5 Hz, 2H), 2.92 – 2.71 (m, 2H), 2.37 (qd,

J = 13.0, 5.2 Hz, 1H), 2.20 (dtd, J = 13.0, 5.2, 2.6 Hz, 1H) ppm. 13C NMR (126 MHz,

CDCl3) δ 171.31, 169.72, 168.83, 168.21, 134.62, 132.92, 131.83, 129.15, 126.34,

121.63, 71.74, 71.10, 70.42, 69.63, 51.86, 46.61, 31.52, 23.39, 2.75 ppm.

N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-2-(2-(2-(2-

iodoethoxy)ethoxy)ethoxy)acetamide (6b).2 white solid. m.p 150.9-151.3 ; ℃ 1H

NMR (500 MHz, CDCl3): δ 8.94 (s, 1H), 8.51 (s, 1H), 7.77 (d, J = 7.5 Hz, 1H), 7.66

(d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 5.22 (dd, J = 13.3, 5.1 Hz, 1H), 4.46 (s,

2H), 4.18 (d, J = 2.1 Hz, 2H), 3.82 (ddd, J = 5.0, 2.7, 1.2 Hz, 2H), 3.79 – 3.73 (m,

2H), 3.73 – 3.66 (m, 2H), 3.65 – 3.56 (m, 4H), 3.11 (td, J = 6.7, 3.7 Hz, 2H), 2.92 –

2.73 (m, 2H), 2.36 (td, J = 13.0, 5.2 Hz, 1H), 2.27 – 2.15 (m, 1H) ppm. 13C NMR (126

MHz, CHCl3) δ 171.27, 169.63, 168.80, 168.48, 134.82, 132.92, 131.98, 129.06,

126.45, 121.61, 71.72, 71.39, 70.58, 70.37, 70.20, 69.92, 51.86, 46.65, 31.51, 23.37,

2.66 ppm.

N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-14-iodo-3,6,9,12-

tetraoxatetradecanamide (6c).2 white solid. m.p 142.5-143.7 ; ℃ 1H NMR (500 MHz,

CDCl3): δ 9.10 (s, 1H), 8.99 (s, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 7.9 Hz,

1H), 7.46 (t, J = 7.7 Hz, 1H), 5.17 (dd, J = 13.3, 5.1 Hz, 1H), 4.42 (d, J = 2.1 Hz, 2H),

4.15 (d, J = 3.4 Hz, 2H), 3.78-3.76 (m, 2H), 3.71-3.70 (m, 2H), 3.68 – 3.62 (m, 4H),

3.63 – 3.57 (m, 2H), 3.54 – 3.47 (m, 4H), 3.16 (t, J = 6.8 Hz, 2H), 2.83 – 2.73 (m,

2H), 2.36-2.27 (m, 1H), 2.16-2.11 (m, 1H) ppm. 13C NMR (126 MHz, CDCl3) δ

171.27, 169.60, 168.85, 168.48, 134.67, 132.85, 132.10, 129.08, 126.38, 121.50,

71.84, 71.34, 70.64, 70.41, 70.36, 70.29, 70.10, 70.05, 51.86, 46.57, 31.53, 23.40,

2.83 ppm.

1.1.5 Synthesis of N-(4-fluoro-2-methoxy- 5-nitrophenyl) -4- (1-methyl-1H-indol-3 -

yl)pyrimidin-2-amine (9)3

To a solution of 3-(2-chloropyrimidin-4-yl)-1H-indole (7) (5.00 g, 20.5 mmol) in

1,4-dioxane (30 mL) was added 4-fluoro-2- methoxy-5-nitroaniline (8) (3.80 g, 20.5

mmol) and p-toluene sulfonic acid (4.70 g, 24.6 mmol) at room temperature. The

mixture was refluxed for 4 h, then cooled to room temperature, filtered and washed

with CH2Cl2/EA (v/v 1:3). The yellow solid was dried at 60 C to give compound 9

(4.52 g, 95%). 1H NMR (500 MHz, DMSO-d6) 8.86 (s, 1H), 8.77 (d, J = 8.2 Hz,

1H), 8.55-8.44 (m, 2H), 7.95 (s, 1H), 7.57 (d, J = 7.2 Hz, 1H), 7.41 (d, J =13.3 Hz,

1H), 7.34-7.20 (m, 3H), 3.99 (s, 3H), 2.30 (s, 3H) ppm.

1.1.6 tert-butyl-(2-((5-methoxy-4-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-

yl)amino)-2-nitroPhenyl)(methyl)amino)ethyl)(methyl)carbamate (11)4

To a solution of compound 9 (5.50 g, 13.90 mmol) in DMSO (30 mL) was added

compound 10 (3.14 g, 16.70 mmol) and K2CO3 (2.90 g, 20.90 mmol). The resulting

mixture was stirred at 80 for 8h, then cooled to room temperature, filtered and℃

washed with ethyl acetate. The yellow solid was dried at 60 C to give 11 (6.39, 82%).

m.p 137.9-138.2 ; ℃ 1H NMR (500 MHz, CDCl3): δ 9.52 (s, 1H), 8.34 (d, J = 5.3 Hz,

1H), 8.18 (s, 1H), 8.16-8.09 (m, 1H), 7.51 (s, 1H), 7.39-7.31 (m, 1H), 7.32-7.22 (m,

2H), 7.12 (d, J = 5.3 Hz, 1H), 6.69 (s, 1H), 3.97 (s, 3H), 3.87 (s, 3H), 3.47 (d, J = 7.5

Hz, 2H), 2.96-2.77 (m, 8H), 1.45 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ

161.65, 159.10, 157.66, 155.43, 152.02, 142.66, 137.95, 132.92, 125.65, 123.00,

122.20, 121.18, 120.62, 116.08, 113.53, 109.98, 107.99, 101.92, 101.22, 79.39, 56.08,

52.71, 46.18, 41.11, 34.93, 33.25, 28.36, 28.33, 28.24 ppm.

1.1.7 tert-butyl-(2-((2-amino-5-methoxy-4-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-

yl)amino)phenyl)(methyl)amino)ethyl) (methyl)carbamate (12)4

To a solution of compound 11 (7.10 g, 12.6 mmol) in ethanol/water (40 mL, v/v

3:1) was added iron (4.20 g, 75.8 mmol) and ammonium chloride (0.508 g, 9.50

mmol). The reaction mixture was stirred at 100 for 8 h. The mixture was cooled to℃

room temperature, filtered and the residue was washed with CH2Cl2 (60 mL). The

combined filtrate was concentrated under reduced pressure and added water (20 mL),

extracted with CH2Cl2 (3×30 mL). The organic layer was dried over anhydrous

sodium sulfate and concentrated under reduced pressure to afford a black solid. The

crude product was purified by column chromatography on silica gel to give compound

12 (3.77 g, 57%). m.p 136.4-138.2 ; ℃ 1H NMR (500 MHz, CDCl3): δ 8.60-8.43 (m,

1H), 8.34 (d, J = 5.3 Hz, 1H), 8.18 (s, 1H), 7.79 (s, 1H), 7.60 (s, 1H), 7.44-7.36 (m,

1H), 7.36-7.20 (m, 2H), 7.02 (d, J = 5.3 Hz, 1H), 6.74-6.65 (m, 1H), 3.87 (s, 6H),

3.37 (s, 2H), 3.02 (s, 2H), 2.88 (d, J = 14.6 Hz, 3H), 2.70 (s, 3H), 1.48 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3): δ 162.2, 160.1, 157.1, 155.8, 141.2, 137.9, 131.1,

126.0, 122.6, 122.0, 121.2, 114.2, 109.7, 107.7, 107.0, 105.0, 79.3, 56.8, 54.4, 53.8,

47.1, 42.1, 33.2, 28.4 ppm.

1.1.8 tert-butyl-(2-((2-(3-chloropropanamido)-5-methoxy-4-((4-(1-methyl-1H-indol-

3-yl)pyrimidin-2-yl)amino)phenyl)(methyl) amino)ethyl)(methyl)carbamate (13)4

To a solution of compound 12 (3.74 g, 7 mmol) in CH2Cl2 (20 mL) was added 3-

chloropropanoyl chloride (1.07 g, 8.40 mmol) in an ice bath. The mixture was stirred

at room temperature for 1 h. The reaction was concentrated under reduce pressure and

purified by column chromatography on silica gel to give compound 13 (3.48 g, 80%)

as a yellow solid. m.p 140.6-141.2 ; R℃ f = 0.25 (DCM: MeOH = 60:1); 1H NMR (500

MHz, CDCl3): δ 9.92 (s, 1H), 9.02 (m, 2H), 7.97-7.77 (m, 2H), 7.43 (d, J = 7.9 Hz,

1H), 7.37-7.30 (m, 2H), 7.20-7.10 (m, 1H), 3.98 (s, 5H), 3.93 (t, J = 6.0 Hz, 2H), 3.75

(t, J = 6.7 Hz, 2H), 3.45 (s, 3H), 3.13-2.99 (m, 3H), 2.90 (s, 3H), 2.82 (t, J = 6.6 Hz,

2H), 1.46 (s, 9H) ppm.

1.1.9 tert-butyl-(2-((2-acrylamido-5-methoxy-4-((4-(1-methyl-1H-indol-3-

yl)pyrimidin-2-yl)amino)phenyl)(methyl)amino)ethyl) (methyl)carbamate (14)4

To a solution of compound 13 (4.40 g, 7.10 mmol) in acetonitrile (20 mL) was

added Et3N (2.50 g, 21.30 mmol) at room temperature. The reaction mixture was

stirred at 80 for 7 h. The reaction was filtered and washed with EA (20 mL). ℃ The

yellow solid was dried at 65 C to give compound 14 (0.294 g, 70%). m.p 167.8-168.3 oC; Rf = 0.25 (DCM: MeOH = 60:1); 1H NMR (500 MHz, CDCl3): δ 9.87 (s, 1H), 9.74

(s, 1H), 9.07 (s, 1H), 8.95 (s, 1H), 8.39-8.38 (m, 1H), 8.09-8.07 (m, 1H), 7.75 (d, J =

7.4 Hz, 1H), 7.41-7.40 (m, 1H), 7.32-7.28 (m, 2H), 7.22-7.21 (m, 1H), 6.79 (d, J = 3.5

Hz, 1H), 6.47 (d, J = 5.0 Hz, 1H), 5.78 (d, J = 5.0 Hz, 1H), 3.99 (s, 3H), 3.90 (s, 3H),

3.37 (d, J = 7.6 Hz, 2H), 3.00 (s, 2H), 2.86 (s, 3H), 2.69 (s, 3H), 1.47 (s, 9H) ppm.

HRMS-ESI calculated for C32H40N7O4 [M+H]+: 586.3136, found 586.3151.

1.1.10 N-(4-methoxy-2-(methyl(2-(methylamino)ethyl)amino)-5-((4-(1-methyl-1H-

indol-3-yl)pyrimidin-2-yl)amino)phenyl) acrylamide (15)4

Compound 14 (0.81 g, 1.40 mmol) in CH2Cl2 (20 mL) was added TFA (2.80 g,

24.8 mmol) and stirred at room temperature for 2 h. The reaction was washed with

saturated NaHCO3 and extracted with CH2Cl2 (3×20 mL). The organic layer was dried

over anhydrous sodium sulfate and concentrated under reduced pressure to afford the

crude product. The residue was purified by column chromatography on silica gel to

give compound 15 (0.29 g, 94%) as yellow solid. m.p 175.4-176.2 oC; Rf = 0.2 (DCM:

MeOH = 10:1); 1H NMR (500 MHz, DMSO-d6): δ 9.38 (s, 1H), 8.69 (s, 1H), 8.60 (s,

1H), 8.29 (d, J = 6.0 Hz, 1H), 7.57 (dd, J = 8.3, 4.4 Hz, 1H), 7.35-7.31 (m, 1H), 7.31-

7.26 (m, 1H), 7.18 (t, J = 7.7 Hz, 1H), 7.02 (d, J = 12.0 Hz, 1H), 6.75 (dd, J = 17.0,

10.2 Hz, 1H), 6.29 (dd, J = 16.9, 2.0 Hz, 1H), 5.79 (dd, J = 10.2, 2.0 Hz, 1H), 3.93 (s,

3H), 3.85 (s, 3H), 3.27 (d, J = 5.9 Hz, 2H), 3.16 (d, J = 6.0 Hz, 2H), 2.64 (s, 6H) ppm. 13C NMR (126 MHz, CDCl3) δ 168.46, 161.95, 160.02, 158.76, 158.50, 157.64,

146.71, 138.68, 137.86, 133.82, 133.72, 125.53, 122.20, 121.09, 112.61, 110.59,

107.38, 104.49, 104.43, 56.25, 45.92, 42.98, 41.15, 33.06, 32.51 ppm. HRMS-ESI

calculated for C27H32N7O2 [M+H]+: 486.2612, found 486.2592.

1.1.11 2-(2-acrylamido-5-methoxy-4-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-

yl)amino)phenyl)-N-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-4-yl)-5-methyl-

8,11-dioxa-2,5-diazatridecan-13-amide (16a).

To a solution of compound 15 (186 mg, 0.38 mmol) and compound 6a (188 mg,

0.35 mmol) in DMF (10 mL) was added DIPEA (136 mg, 1.05 mmol). Then the

reaction was stirred at 90 for 8 h. The reaction was added water (10 mL) and℃

extracted with CH2Cl2 (3×20 mL). The organic layer was dried over anhydrous

sodium sulfate and concentrated under reduced pressure to afford the crude product.

The residue was purified by column chromatography on silica gel to give compound

16a (120 mg, 53%). 1H NMR (500 MHz, CDCl3) δ 9.74 (s, 1H), 9.56 (d, J = 12.0 Hz,

1H), 8.91 (s, 1H), 8.78 (d, J = 11.5 Hz, 1H), 8.37 (dd, J = 5.4, 2.6 Hz, 1H), 8.15 –

7.99 (m, 1H), 7.77 (s, 1H), 7.70 (dd, J = 7.6, 4.7 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H),

7.47 – 7.34 (m, 3H), 7.17 (dd, J = 5.3, 3.3 Hz, 1H), 6.72 (s, 1H), 6.42 (dd, J = 16.9,

1.8 Hz, 1H), 5.69 (dd, J = 9.9, 1.8 Hz, 1H), 5.13 (dt, J = 13.4, 4.6 Hz, 1H), 4.38 (d, J

= 5.5 Hz, 2H), 4.07 (d, J = 2.2 Hz, 2H), 3.95 (s, 3H), 3.84 (s, 3H), 3.75 – 3.51 (m,

8H), 2.94 (s, 3H), 2.83 – 2.65 (m, 5H), 2.62 (d, J = 7.1 Hz, 4H), 2.40 – 2.28 (m, 4H),

2.13 – 2.09 (m, 1H) ppm. 13C NMR (126 MHz, CDCl3) δ 171.48, 169.93, 168.78,

168.14, 163.16, 162.15, 159.46, 157.69, 144.61, 138.18, 134.84, 134.46, 133.87,

132.85, 132.82, 131.93, 129.04, 128.47, 127.43, 126.19, 125.88, 121.95, 121.32,

121.05, 120.37, 113.56, 110.08, 108.00, 104.03, 71.04, 70.47, 70.04, 56.89, 56.16,

54.99, 51.90, 46.54, 43.77, 42.68, 33.10, 31.91, 31.46, 29.68, 23.27 ppm. HRMS-ESI




8,11,14,17-tetraoxa-2,5-diazanonadecan-19-amide (16b).

The preparation of 16b was similar to that of 16a described in procedure 4.1.11. 1H NMR (500 MHz, CDCl3) δ 9.79 (s, 1H), 9.59 (s, 1H), 8.98 (d, J = 17.9 Hz, 2H),

8.38 (d, J = 5.3 Hz, 1H), 8.12 – 8.01 (m, 1H), 7.79 (s, 1H), 7.75 – 7.67 (m, 2H), 7.44

(t, J = 7.8 Hz, 1H), 7.41 – 7.35 (m, 1H), 7.29 – 7.25 (m, 3H), 7.18 (d, J = 5.3 Hz, 1H),

6.76 (s, 1H), 6.42 (dd, J = 16.6, 1.9 Hz, 1H), 5.73 – 5.65 (m, 1H), 5.16 (dd, J = 13.3,

5.2 Hz, 1H), 4.39 (s, 2H), 4.08 (d, J = 8.5 Hz, 2H), 3.96 (s, 3H), 3.85 (s, 3H), 3.67 (dt,

J = 5.3, 2.6 Hz, 2H), 3.61 – 3.49 (m, 5H), 3.42 (dt, J = 16.3, 5.6 Hz, 5H), 2.91 (s, 3H),

2.83 – 2.71 (m, 2H), 2.63 (s, 3H), 2.52 (s, 2H), 2.29 – 2.26 (m, 4H), 2.20 – 2.06 (m,

1H) ppm. 13C NMR (126 MHz, CDCl3) δ 171.51, 169.85, 168.78, 168.45, 162.90,

162.10, 159.47, 157.70, 144.44, 138.17, 135.00, 134.34, 132.95, 132.77, 132.14,

128.97, 128.65, 127.36, 126.08, 125.87, 125.60, 121.82, 121.18, 120.95, 120.27,

113.55, 110.03, 107.88, 104.22, 71.19, 70.34, 70.31, 70.00, 69.90, 60.34, 56.92,

56.11, 54.95, 51.77, 46.32, 43.61, 42.88, 33.03, 31.50, 29.65, 23.34 ppm. HRMS-ESI




8,11,14-trioxa-2,5-diazahexadecan-16-amide (16c).

The preparation of 16c was similar to that of 16a described in procedure 4.1.11. 1H NMR (500 MHz, CDCl3) δ 9.80 (s, 1H), 9.63 (t, J = 21.2 Hz, 1H), 9.03 (d, J = 5.1

Hz, 2H), 8.38 (dd, J = 5.4, 1.6 Hz, 1H), 8.11 – 7.99 (m, 1H), 7.81 (s, 1H), 7.72 (dd, J

= 7.7, 2.1 Hz, 2H), 7.46 (td, J = 7.7, 3.3 Hz, 1H), 7.42 – 7.35 (m, 1H), 7.29 – 7.24 (m,

3H), 7.19 (dd, J = 5.4, 2.7 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.44 (dd, J = 16.8, 1.9

Hz, 1H), 5.70 (dd, J = 9.8, 2.1 Hz, 1H), 5.17 (ddd, J = 13.4, 5.2, 2.5 Hz, 1H), 4.41 (d,

J = 2.2 Hz, 2H), 4.19 – 4.03 (m, 2H), 3.97 (s, 3H), 3.86 (s, 3H), 3.69 (q, J = 4.7, 3.1

Hz, 3H), 3.61 (m, 2H), 3.59 – 3.42 (m, 7H), 3.38 (s, 3H), 3.01 – 2.88 (m, 3H), 2.86 –

2.71 (m, 3H), 2.66 (s, 5H), 2.40 – 2.26 (m, 4H), 2.15 (m, 1H) ppm. 13C NMR (126

MHz, CDCl3) δ 171.46, 169.80, 168.85, 168.47, 162.12, 159.47, 157.71, 144.43,

138.19, 135.10, 134.74, 134.42, 133.00, 132.77, 132.15, 129.00, 127.35, 126.23,

125.90, 121.89, 121.81, 121.21, 120.95, 120.33, 120.24, 113.56, 110.05, 107.87,

104.20, 71.21, 70.45, 70.34, 70.19, 70.16, 70.04, 69.93, 56.86, 56.12, 54.92, 51.80,

46.37, 43.45, 42.91, 40.37, 33.06, 31.52, 29.66, 23.34 ppm. HRMS-ESI calculated for

C50H61N10O10 [M+H]+: 961.4567, found 961.4598.

2.1 Biology

2.1.1 Cell Culture and MTT assay

Cell Culture. All cells were purchased from the American Type Culture Collection

(ATCC). The human NSCLC cell lines including PC9(EGFRDel19), HCC827

(EGFRDel19), and H1975 (EGFRL858R/T790 M) were grown as a monolayer respectively.

All of the aforementioned cell lines were cultured in RPMI-1640 medium containing

10% fetal bovine serum (FBS) and grown in a humidified incubator at 37 C, 5% CO2.

MTT assay. The antiproliferative activities of the compounds were determined in

vitro by MTT assay. PC9, H1975(5x103 cells/well) and HCC827(1x104 cells/well)

cells line were added in 100 μL of RPMI-1640 culture medium in 96 wells cell culture

plate, respectively. A series of concentrations of PROTACs 16a-16c and positive drug

osimertinib were dissolved in DMSO and diluted 10 times by sterilized phosphate

buffer saline (PBS). Diluted compounds solution was added to the wells with 10%

DMSO as vehicle control. After cultured for 24 h, the different concentrations of

PROTACs 16a-16c and azd9291 were added and cells were incubated for another 72

h. 20 mL of MTT solution (5 mg/mL in PBS) was added to each well of the 96-well

cell culture plate and incubated at 37 C for 4 h. The supernatant of each well was

removed carefully and the formed blue formazan crystals were dissolved in 150 mL of

DMSO. The optical density at 490 nm wavelength was determined by SpectraMax

Plus Microplate Reader (Molecular Device). Three separate experiments with

triplicate data were performed to obtain mean cell viability. The IC50 value, that is, the

concentration (mM) of a compound was able to cause 50% cell death with respect to

the control culture, was calculated by means of GraphPad Prism 7.0 Software.

2.1.2. Western bolt assay

Cells were seeded in 6-well plates at 1 x 106 per well, incubated at 37 C with 5%

CO2 for 24 h before drug exposure. PC9 Cells were treated with the indicated

concentrations of PROTAC for the specified time and then harvested in cell lysis

buffer (20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100, and sodium

pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin, 1 mM phenyl methyl

sulfonyl fluoride(PMSF), 1 mM protease inhibitor cocktail ). Following shocked for

30 minutes under ice and centrifugation at 13,000 rpm for 20 min at 4 to collect℃

the cell supernatants in lysis buffer (Beyotime) and later removed the insoluble

material. Same amounts of proteins were loaded and separated by 10% SDS-PAGE

and transferred to polyvinylidene fluoride membranes (Millpore) after that. Anti-

EGFR (Cell Signaling Technology#4267, 1:1000), anti- GAPDH (Cell Signaling

Technology#8884, 1:1000) and the secondary antibodies (Cell Signaling

Technology#4267, 1:2000) were diluted, respectively. Immunoblots were developed

using enhanced chemiluminescence and visualized using a Bio-Rad Chemi-Doc MP

Imaging System and quantitated with Image Lab v.5.2 software (Bio-Rad

Laboratories).

2.1.3 Immunofluorescence Microscopy

PC9 cells were plated at a density of 1×105 cells/well in 96 wells cell culture plate,

cultured overnight then treated with 0.3 μM PROTAC 16c for 24 h before washing

with PBS. Cells were fixed with 4% paraformaldehyde for 20 minutes at room

temperature, washed with ice-cold PBS, permeabilized and blocked with 0.3% Triton

X-100, 1% BSA in PBS for 30 minutes. Fixed cells were incubated with EGFR

Antibody (1:50 dilution, Cell Signalling#4267) overnight, washed three times with

PBS for 5 minutes, incubated with Alexa Fluor-488 conjugated anti-rabbit antibody

(1:500 dilution, ThermoFisher A-11008) for 1h washed three times with PBS for 5

minutes and mounted in vectashield containing DAPI. Imaged on ImageXpress®

confocal/Widefield.

2.1.4 Cell health and cycle

PC9 cells were plated in 6-well plates overnight, incubated at 37 C with 5% CO2

for 24 h, and then 0.3μM PROTAC 16c was added. Medium of none FBS was used

for synchronization. Medium with 0.1% DMSO was used as control. After incubated

for 24 h, Hoechst33342 was added in dark for 15 minutes. After that, 50 μL buffer

that containing FITC Annexin V and PI was added into each well, avoiding light

incubation immediately after 15 minutes on microscopic imaging on ImageXpress®

confocal/Widefield with the objective lens for 10x. Fluorescent wavelength channels

are selected as DAPI (blue), FITC (green), and Cy3 (orange). The results of images

were analyzed by High-Content Analysis System to get the condition of cell health

and cycle.

References

1. Wurz RP, Dellamaggiore K, Dou H, et al. A “click chemistry platform” for the rapid synthesis of bispecific molecules for inducing protein degradation. J Med Chem. 2017, 61(2): 453-461.2. Crew AP, Berlin M, Dong H, et al. Alanine-based modulators of proteolysis and associated methods of use. WO2017011590 (2017-1-19).3. Finlay MRV, Anderton M, Susan A, et al. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor. J Med Chem. 2014, 57, 8249−8267.4. Hallberg B, Palmer RH. Mechanistic insight into ALK receptor tyrosine kinasein human cancer biology. Nat Rev Canc. 2013, 13 (10): 685-700.

NMR Spectra for 2a-16c1H NMR of 2a

1H NMR of 2b

1H NMR of 2c

1H NMR of 3a

1H NMR of 3b

1H NMR of 3c

1H NMR of 4a

1H NMR of 4b

1H NMR of 4c

1H NMR of 6a

13C NMR of 6a

1H NMR of 6b

13C NMR of 6b

1H NMR of 6c

13C NMR of 6c

1H NMR of 11

13C NMR of 11

1H NMR of 12

13C NMR of 12

1H NMR of 13

1H NMR of 14

1H NMR of 15

13C NMR of 15

1H NMR of 16a

13C NMR of 16a

1H NMR of 16b

13C NMR of 16b

1H NMR of 16c

13C NMR of 16c

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