Please cite this article in press as: Li et al., Development and Characterization of a Wee1 Kinase Degrader, Cell Chemical Biology (2019), https://
doi.org/10.1016/j.chembiol.2019.10.013
(10 mg, 0.035 mmol) and N, N-diisopropylethylamine (15 mL, 0.09 mmol) were added, and the mixture was stirred at 90ꢁC for 3 h. The
mixture was purified by HPLC (10-90% MeOH in H2O) to give TFA salt ZNL-02-047 (8 mg, 30%) as a yellow solid. LC-MS: m/z
918.46 [M+1]+.
1H NMR (500 MHz, DMSO) d 11.11 (s, 1H), 10.20 (s, 1H), 9.75 (s, 1H), 8.85 (s, 1H), 8.03 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.62
(d, J = 7.6 Hz, 3H), 7.57 (dd, J = 8.4, 7.2 Hz, 1H), 7.13 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.99 (d, J = 9.0 Hz, 2H), 6.60 (s, 1H),
5.67 (ddt, J = 16.3, 10.3, 6.0 Hz, 1H), 5.06 (dd, J = 12.8, 5.4 Hz, 1H), 5.00 (dd, J = 10.2, 1.1 Hz, 1H), 4.83 (dd, J = 17.1, 1.2 Hz, 1H), 4.69
(d, J = 4.8 Hz, 2H), 3.80 – 3.75 (m, 5H), 3.64 –3.57 (m, 12H), 3.49 – 3.44 (m, 2H), 3.39 (s, 2H), 3.22 (d, J = 9.8 Hz, 2H), 3.01 (t, J = 11.9 Hz,
2H), 2.89 (ddd, J = 17.1, 14.0, 5.4 Hz, 1H), 2.65 – 2.53 (m, 1H), 2.06 – 1.98 (m, 1H), 1.47 (s, 6H).
13C NMR (125 MHz, DMSO) d 173.29, 171.19, 170.57, 169.45, 168.12, 167.75, 161.54, 160.90, 158.64, 158.37, 156.56, 147.54,
146.87, 145.90, 139.26, 136.73, 132.66, 132.57, 121.71, 118.73, 117.92, 116.75, 111.20, 109.73, 72.79, 70.15, 69.99, 69.36,
67.29, 53.92, 51.38, 49.04, 47.04, 46.61, 42.17, 40.59, 36.59, 36.10, 31.46, 30.93, 24.45, 22.61.
4-((6-(4-(4-((2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)
piperazin-1-yl)hexyl)amino)-2-(2,6- dioxopiperidin-3-yl)isoindoline-1,3-dione (ZNL-02-040). To a solution of Intermediate (4)
(35 mg, 0.073 mmol) in DMF (1.5mL) was added tert-Butyl (6-bromohexyl) carbamate (24 mg, 0.08 mmol) and K2CO3 (20 mg,
0.15 mmol). The mixture was heated at 60ꢁC for 6 h. Then allowing the reaction to cool to room temperature, the solution was
dissolved in water and ethyl acetate. The mixture extracted with EtOAc (3 x 10 ml). The organic extract was washed with brine
(20 ml), dried over MgSO4, and concentrated under reduced pressure. The residue was purified via silica gel chromatography
(10:1 DCM: MeOH) to give compound (6) (37 mg, 75 % yield). LC-MS: m/z 686.47 [M+1]+. Intermediate (6) (20 mg, 0.03 mmol)
was dissolved in DCM (2 mL). TFA (0.5 mL) was added and the mixture was stirred for 30 minutes. The solvent was removed under
reduced pressure and the residue was dissolved in DMSO (1 mL). 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (8)
(10 mg, 0.035 mmol) and N, N-diisopropylethylamine (15 mL, 0.09 mmol) were added, and the mixture was stirred at 90ꢁC for
3 h. The mixture was purified by HPLC (10-90% MeOH in H2O) to give TFA salt ZNL-02-040 (10 mg, 39%) as a yellow solid.
LC-MS: m/z 842.52 [M+1]+.
1H NMR (500 MHz, DMSO) d 11.08 (s, 1H), 10.13 (s, 1H), 8.82 (s, 1H), 8.04 (s, 1H), 7.75 (s, 1H), 7.59 (dd, J = 14.7, 7.4 Hz, 4H), 7.09 (d,
J = 8.5 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.90 (d, J = 8.5 Hz, 2H), 6.53 (s, 1H), 5.66 (dd, J = 16.6, 10.4 Hz, 1H), 5.33 (s, 1H), 5.04 (dd,
J = 12.7, 5.2 Hz, 1H), 4.99 (d, J = 10.1 Hz, 1H), 4.82 (d, J = 17.2 Hz, 1H), 4.67 (s, 2H), 3.07 (s, 5H), 2.87 (dd, J = 21.4, 8.8 Hz, 1H), 2.60 –
2.53 (m, 1H), 2.29 (d, J = 6.5 Hz, 2H), 2.11 – 1.95 (m, 1H), 1.59 (s, 2H), 1.46 (s, 6H), 1.42 – 1.21 (m, 6H).
13C NMR (125 MHz, DMSO) d 173.28, 170.57, 169.44, 167.78, 161.64, 160.98, 156.47, 147.71, 146.92, 139.29, 136.77, 132.68,
131.25, 121.62, 118.73, 117.68, 116.75, 115.92, 110.86, 109.49, 72.79, 58.30, 53.28, 49.16, 49.02, 47.07, 42.29, 40.59, 40.43,
40.26, 40.09, 31.46, 30.93, 29.12, 27.16, 26.70, 22.64.
4-((3-(4-(4-((2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)
piperazin-1-yl)propyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (ZNL-02-096). To a solution of Intermediate (4)
(100 mg, 0.2 mmol) in DMF (2 mL) was added tert-Butyl (3-bromopropyl) carbamate (60 mg, 0.24 mmol) and K2CO3 (55 mg,
0.4 mmol). The mixture was heated at 60ꢁC for 6 h. Then allowing the reaction to cool to room temperature, the solution
was dissolved in water and ethyl acetate. The mixture extracted with EtOAc (3 x 10 ml). The organic extract was washed
with brine (20 ml), dried over MgSO4, and concentrated under reduced pressure. The residue was purified via silica gel
chromatography (10:1 DCM: MeOH) to give compound (7) (110 mg, 85 % yield). LC-MS: m/z 644.44 [M+1]+. Intermediate (7)
(30 mg, 0.04 mmol) was dissolved in DCM (2 mL). TFA (0.5 mL) was added and the mixture was stirred for 30 minutes. The
solvent was removed under reduced pressure and the residue was dissolved in DMSO (1 mL). 2-(2,6-dioxopiperidin-3-yl)-4-
fluoroisoindoline-1,3-dione (8) (15 mg, 0.055 mmol) and N, N-diisopropylethylamine (20 mL, 0.12 mmol) were added, and the
mixture was stirred at 90ꢁC for 3 h. The mixture was purified by HPLC (10-90% MeOH in H2O) to give TFA salt ZNL-02-096
(11 mg, 35%) as a yellow solid. LC-MS: m/z 800.52 [M+1]+.
1H NMR (500 MHz, DMSO) d 11.10 (s, 1H), 10.72 (s, 1H), 10.21 (s, 1H), 8.84 (s, 1H), 8.04 (t, J = 7.6 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H),
7.67 – 7.57 (m, 4H), 7.18 (d, J = 8.6 Hz, 1H), 7.06 (d, J = 7.0 Hz, 1H), 7.00 (d, J = 9.0 Hz, 2H), 6.78 (s, 1H), 5.66 (ddt, J = 16.4, 10.3,
6.0 Hz, 1H), 5.06 (dd, J = 12.7, 5.4 Hz, 1H), 5.02 – 4.96 (m, 1H), 4.82 (dd, J = 17.1, 1.1 Hz, 1H), 4.73 – 4.61 (m, 2H), 3.82 – 3.64 (m, 3H),
3.62 – 3.52 (m, 2H), 3.52 – 3.36 (m, 3H), 3.22 – 3.08 (m, 6H), 2.89 (ddd, J = 16.9, 13.9, 5.4 Hz, 1H), 2.66 – 2.53 (m, 2H), 2.15 – 1.98 (m,
3H), 1.46 (s, 6H).
13C NMR (125 MHz, DMSO) d 173.29, 170.57, 169.21, 168.11, 167.76, 163.49, 161.53, 161.40, 160.88, 156.51, 147.51, 146.55,
145.96, 139.28, 136.78, 132.77, 132.66, 132.44, 121.69, 118.73, 117.81, 116.80, 116.70, 111.17, 109.94, 72.88, 72.79, 72.63,
70.99, 70.39, 68.08, 65.64, 63.27, 63.20, 60.65, 60.61, 53.72, 51.16, 49.03, 47.03, 46.36, 31.46, 30.93, 23.63, 22.65.
Scheme 3: Synthesis of 3-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy) propanoic acid (11).
e7 Cell Chemical Biology 27, 1–9.e1–e9, January 16, 2020