M. Zheng et al.
Bioorganic Chemistry 110 (2021) 104788
1
2
16.9, 111.6, 110.3, 70.75, 70.72, 70.1, 69.6, 50.7, 48.9, 42.4, 31.5,
2.8.
NMR (100 MHz, CDCl
3
) δ 171.88, 169.75, 169.37, 167.82, 153.36,
150.24, 146.86, 139.25, 136.30, 133.84, 132.74, 132.58, 130.41,
130.33, 128.02, 125.54, 119.32, 116.90, 111.91, 110.51, 70.57, 70.44,
69.79, 69.18, 60.58, 49.21, 42.43, 40.99, 36.24, 31.76, 29.23, 23.20,
4
-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-
1
dioxopiperidin-3-yl)isoindoline-1,3-dione (4B) H NMR (400 MHz,
CDCl
) δ 8.85 (br s, 1H), 7.43 (dd, J = 8.4, 7.2 Hz, 1H), 7.04 (d, J = 7.2
+
3
22.83, 22.80, 21.24, 18.94, 14.30, 11.61. HRMS (ESI ): m/z calculated
+
Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.44 (t, J = 5.6 Hz, 1H), 4.92–4.87 (m,
for C38
H43Cl
N
2 11
O
6
[M+H] , 820.2848; found, 820.2807.
1
2
H), 3.68 (t, J = 5.4 Hz, 2H), 3.64–3.61 (m, 10H), 3.43 (q, J = 5.6 Hz,
13
H), 3.33 (t, J = 5.0 Hz, 2H), 2.83–2.68 (m, 3H), 2.08–2.05 (m, 1H);
) δ 171.7, 169.4, 168.8, 167.7, 146.9, 136.1,
32.5, 116.9, 111.6, 110.3, 70.7, 70.0, 69.5, 50.7, 48.9, 42.4, 31.5, 22.8.
C
4.1.5. Synthesis of 4-((2-(2-(2-(2-(4-(((1-(6-amino-5-(2,3-
dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-1H-
1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-
dioxopiperidin-3-yl)isoindoline-1,3-dione (SP3)
NMR (100 MHz, CDCl
3
1
1
4
C H NMR (400 MHz, CDCl
3
) δ 8.81 (br s, 1H), 7.48 (dd, J = 8.0, 7.2
Hz, 1H), 7.09 (d, J = 7.2 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.49 (t, J =
Yield 20%, 1H NMR (400 MHz, DMSO‑d
6
) δ 11.10 (s, 1H), 7.95 (s,
5
1
3
1
7
.6 Hz, 1H), 4.95–4.90 (m, 1H), 3.72 (t, J = 5.2 Hz, 2H), 3.68–3.66 (m,
1H), 7.75 – 7.45 (m, 3H), 7.45 – 7.19 (m, 2H), 7.07 (dd, J = 37.1, 7.8 Hz,
2H), 6.59 (d, J = 6.1 Hz, 1H), 5.61 (s, 1H), 5.04 (dd, J = 12.8, 5.4 Hz,
1H), 4.47 (t, J = 5.2 Hz, 2H), 4.10 – 3.37 (m, 19H), 3.01 – 2.52 (m, 4H),
4H), 3.48 (q, J = 5.6 Hz, 2H), 3.38 (t, J = 5.0 Hz, 2H), 2.88–2.72 (m,
1
3
3
H), 2.13–2.09 (m, 1H); C NMR (100 MHz, CDCl ) δ 171.6, 169.4,
1
3
68.8, 167.8, 146.9, 136.1, 132.6, 116.9, 111.7, 110.3, 70.8,
0.74,70.71, 70.7, 70.65, 70.62, 70.1, 69.6, 50.8, 48.9, 42.5, 31.5, 22.9.
2.14 – 1.86 (m, 2H), 1.59 (d, J = 47.7 Hz, 5H), 1.29 – 1.12 (m, 3H);
C
3
NMR (100 MHz, CDCl ) δ 172.11, 169.80, 169.29, 168.10, 154.04,
4
-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-
150.92, 147.21, 139.18, 136.52, 134.10, 132.97, 132.85, 130.69,
130.61, 128.71, 125.75, 123.64, 119.53, 117.69, 112.53, 110.74, 71.20,
71.08, 70.99, 70.97, 69.94, 69.88, 60.86, 52.17, 50.63, 49.38, 42.83,
1
dioxopiperidin-3-yl)isoindoline-1,3-dione (4D) H NMR (400 MHz,
CDCl
) δ 8.82 (br s, 1H), 7.48 (dd, J = 8.0, 7.2 Hz, 1H), 7.09 (d, J = 7.2
3
+
Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.49 (t, J = 5.6 Hz, 1H), 4.95–4.90 (m,
41.25, 37.37, 36.48, 31.93, 25.14, 23.21, 21.51, 14.65. HRMS (ESI ):
1
2
H), 3.72 (t, J = 5.2 Hz, 2H), 3.68–3.66 (m, 18H), 3.49 (q, J = 5.6 Hz,
m/z calculated for
C
40
H47Cl
2
N
11
O
7
[M+H]+, 864.3110; found,
1
3
H), 3.38 (t, J = 5.0 Hz, 2H), 2.88–2.72 (m, 3H), 2.13–2.09 (m, 1H);
) δ 171.6, 169.4, 168.7, 167.8, 146.9, 136.1,
32.6, 116.9, 111.7, 110.3, 70.8, 70.72,70.71, 70.6, 70.5, 70.1, 69.6,
C
864.3053.
NMR (100 MHz, CDCl
3
1
5
4.1.6. Synthesis of 4-((17-(4-(((1-(6-amino-5-(2,3-dichlorophenyl)
pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-
yl)-3,6,9,12,15-pentaoxaheptadecyl)amino)-2-(2,6-dioxopiperidin-3-yl)
0.8, 48.9, 42.5, 31.5, 22.9.
4
.1.3. Synthesis of 4-((14-(4-(((1-(6-amino-5-(2,3-dichlorophenyl)
isoindoline-1,3-dione (SP5)
1
pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-
yl)-3,6,9,12-tetraoxatetradecyl)amino)-2-(2,6-dioxopiperidin-3-yl)
isoindoline-1,3-dione (SP4)
Yield 24%, H NMR (400 MHz, CDCl
3
) δ 10.11 (s, 1H), 8.96 (s, 1H),
7.86 – 7.51 (m, 2H), 7.46 (ddd, J = 8.3, 5.1, 3.0 Hz, 2H), 7.30 (td, J =
7.2, 1.9 Hz, 1H), 7.07 (d, J = 7.0 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 6.48
(s, 1H), 5.32 (t, J = 4.9 Hz, 1H), 4.89 (dd, J = 11.6, 5.5 Hz, 1H), 4.63 –
4.17 (m, 4H), 4.09 (q, J = 7.2 Hz, 3H), 3.93 (s, 1H), 3.84 (t, J = 5.2 Hz,
1H), 3.76 – 3.40 (m, 20H), 2.87 – 2.68 (m, 3H), 2.19 (t, J = 7.6 Hz, 4H),
2
(17.4 mg, 0.045 mmol), 4C (23.3 mg, 0.045 mmol), CuSO
.056 mmol) and sodium ascorbate (23.8 mg, 0.135 mmol) were added
to 2 mL THF:H
O (V/V = 1:1), and the mixture was stirred at room
temperature until starting material was completely consumed. 30 mL
O and 30 mL EtOAc were added to the reaction. The organic layer was
separated and dried over anhydrous Na SO , and filtered, and then
4
(9.0 mg,
0
2
1
3
1.68 (d, J = 49.8 Hz, 4H), 1.23 (s, 3H); C NMR (100 MHz, CDCl
3
) δ
H
2
171.68, 169.43, 168.95, 167.80, 153.72, 150.10, 146.95, 138.88,
136.20, 133.79, 132.66, 132.54, 130.38, 130.30, 127.99, 125.45,
123.47, 119.23, 116.95, 111.77, 110.43, 77.43, 70.88, 70.77, 70.69,
70.66, 70.62, 70.58, 70.55, 69.56, 50.35, 49.05, 42.50, 40.94, 37.10,
36.10, 31.60, 29.84, 29.46, 27.35, 24.27, 22.95, 22.83, 14.27. HRMS
2
4
concentrated under reduced pressure. The obtained residue was purified
by silica gel column chromatography with dichloromethane: methanol
(
V/V = 40/1) as an eluent to give SP4 as a yellow solid (9.0 mg, yield
2%). Compounds SP2, SP3 and SP5 were prepared as the same
procedure.
+
+
2
(ESI ): m/z calculated for C44
H
55Cl
2
N
11
O
9
[M+H] , 952.3634; found,
952.3704.
1
SP4 H NMR (400 MHz, DMSO‑d ) δ 11.10 (s, 1H), 8.99 (s, 1H), 8.22
6
(
s, 1H), 7.63 – 7.51 (m, 3H), 7.37 (t, J = 7.8 Hz, 1H), 7.29 (dd, J = 7.7,
4.2. Clone, expression and purification of SHP2
1
.6 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 7.1 Hz, 1H), 6.59 (t, J
=
5.8 Hz, 1H), 5.69 (s, 1H), 5.07 – 5.02 (m, 1H), 4.58 (t, J = 5.2 Hz, 2H),
Human SHP2 (NCBI RefSeq NP_002825.3) containing an N-terminal
6 × His tag followed by tomato etch virus (TEV) cleavage site was cloned
into a pet30a and heterologously expressed in E. coli strain BL21 Star
3
2
.82 (t, J = 5.2 Hz, 2H), 3.59 (t, J = 5.3 Hz, 2H), 3.54 – 3.42 (m, 18H),
.97 – 2.76 (m, 2H), 2.67 – 2.51 (m, 3H), 2.34 (d, J = 9.5 Hz, 2H), 1.94
1
3
◦
(
d, J = 33.1 Hz, 4H), 1.54 (s, 3H); C NMR (100 MHz, CDCl
3
) δ 171.65,
(DE3). Cells were cultured at 37 C for 3–4 h and supplemented with 1
◦
1
1
1
7
3
69.93, 168.49, 167.32, 153.73, 150.12, 146.97, 138.89, 136.25,
mM IPTG to boost protein expression at 18 C. Cells were collected by
◦
33.83, 132.68, 132.56, 130.98, 130.32, 128.03, 125.53, 123.58,
23.32, 119.28, 116.35, 111.83, 110.45, 77.43, 70.89, 70.79, 70.71,
0.59, 69.64, 69.59, 60.58, 52.53, 50.36, 49.06, 42.54, 40.95, 37.10,
centrifugation at 3,000 g for 10 min after overnight growth at 18 C. The
cell pellet was re-suspended in lysis buffer (50 mM Tris-HCl, pH 8.5, 25
2
mM imidazole, 500 mM NaCl, 2.5 mM MgCl , 1 mM TCEP, 1 mM PMSF)
+
6.08, 31.62, 24.60, 22.97, 21.24, 14.37. HRMS (ESI ): m/z calculated
and homogenized by using a homogenizer on ice, followed by centri-
fugation at 40000 g for 1 h. After centrifugation, the supernatant was
collected and incubated with 2 mL Ni-NTA resin (Qiagen) in a gravity
column (Bio-Rad), washed with 30 mL buffer A (50 mM Tris-HCl, 500
mM NaCl, 25 mM imidazole), and then eluted with buffer B (50 mM Tris-
HCl, 500 mM NaCl, 200 mM imidazole). The eluted protein was digested
with TEV protease overnight. Digested SHP2 protein was purified with
Ni-NTA resin gravity column again. Flow-through containing untagged
SHP2 was collected, concentrated, and purified by size-exclusion chro-
matography on a Superdex 200 10/300 GL column.
+
for C42
H51Cl
2
N
11
O
8
[M+H] , 908.3372; found, 908.3246.
4
.1.4. Synthesis of 4-((2-(2-(2-(4-(((1-(6-amino-5-(2,3-dichlorophenyl)
pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)
ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-
dione (SP2)
Yield 24%, 1H NMR (400 MHz, DMSO‑d
) δ 11.08 (s, 1H), 9.01 (s,
6
1
7
H), 8.21 (s, 1H), 7.61 (dd, J = 8.0, 1.7 Hz, 1H), 7.58 – 7.48 (m, 2H),
.38 (t, J = 7.9 Hz, 1H), 7.29 (dd, J = 7.7, 1.6 Hz, 1H), 7.10 (d, J = 8.5
Hz, 1H), 7.01 (d, J = 7.1 Hz, 1H), 6.59 (s, 1H), 5.69 (s, 1H), 5.04 (dd, J =
1
2.8, 5.3 Hz, 1H), 4.59 (d, J = 5.4 Hz, 2H), 3.84 (t, J = 4.9 Hz, 2H), 3.81
13
–
3.39 (m, 17H), 2.31 (s, 2H), 1.95 (d, J = 44.1 Hz, 4H), 1.53 (s, 3H);
C
6