Supplementary Note 1
General procedure for the synthesis of TZ derivative TZ-md:
Abbreviations:
BrCN: cyanogen bromide;
DCM: dichloromethane;
DIPEA: ethyldiisopropylamine;
DMF: N, N’-dimethylformamide;
EDCI: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride;
Et3N: triethylamine;
HMPA: hexamethylphosphoramide;
LDA: lithium diisopropylamide ;
MeCN: acetonitrile;
NBS: N-bromosuccinimide;
NHS: N-hydroxysuccinimide;
r.t.: room temperature;
THF: tetrahydrofuran
1
Conditions, reagents and reaction yields: a: NBS, MeCN, r.t., 4 hours, 97%; b: CuCN, DMF, reflux, overnight,
63%; c: LDA, HMPA, piperazin-1-yl(tetrahydrofuran-2-yl)methanone, THF, -78 °C, 2 hour, 46%; d: HCl (4.0
N), ethyl acetate, r.t., overnight, 81%; e: Et3N, CHCl3, r.t., 2 hours, 23%; f: BrCN, DIPEA, DCM, r.t., 4 hours,
92%; g: EDCI, NHS, DCM, r.t., 2 hours.
Specific procedures for each step of the synthesis:
Under a nitrogen atmosphere, a mixture of compound 1,2-dimethoxy-4-methylbenzene (9.00
g, 59.2 mmol) and NBS (11.60 g, 65.1 mmol) in 100 mL of MeCN was stirred at room
temperature (r.t.) for 4 hours. The solvent was removed by rotary evaporation, and then 100
mL of CCl4 was added. The solid was removed by filtration. The solution was evaporated in
vacuo. The crude product was purified by flash column chromatography with petroleum
ether/ethyl
acetate
(50:1
to
20:1)
to
afford
the
product
1-bromo-4,5-dimethoxy-2-methylbenzene (13.60 g, 97%) as a colorless liquid. The product
was used for the next reaction without further characterization.
2
Under a nitrogen atmosphere, a mixture of 1-bromo-4,5-dimethoxy-2-methylbenzene (9.3 g,
40.4 mmol) and CuCN (9.0 g, 100 mmol) in 100 mL of anhydrous DMF was refluxed
overnight. To the mixture 200 mL of water and 200 mL of CHCl3 were added, and then the
solution was filtered to remove copper salt and partitioned. The organic layer was washed
with water and brine, dried by Na2SO4, and concentrated with a rotary evaporator. The crude
product was purified by flash column chromatography with petroleum ether / ethyl acetate
(20:1 to 5:1) to furnish the product 4,5-dimethoxy-2-methylbenzonitrile (4.5 g, 25.4 mmol,
63%) as a white solid. The product was used for the next reaction without further
characterization.
Under a nitrogen atmosphere, a solution of 4,5-dimethoxy-2-methylbenzonitrile (1.2 g, 6.8
mmol) in 20 mL of anhydrous THF was added to a solution of LDA (4.1 mL, 2.0 M, 8.1
mmol) and HMPA (1.4 mL, 8.1 mmol) in 25 mL of anhydrous THF drop-wise at -78° C.
After the addition, the mixture was stirred for 10 min. Then, the solution was added to the
mixture drop-wise, and then the mixture was stirred for 1 h at -78° C and for 2 h at room
temperature. 10 mL of water was added to quench the reaction, and then the solution was
partitioned by DCM and water. The inorganic layer was then extracted by DCM twice. The
organic layer was combined, washed by brine, dried by Na2SO4, and concentrated with a
rotary evaporator. The crude product was purified by flash column chromatography with
DCM/methanol (100:1) to furnish the product 1 (1.22 g, 46%) as a yellow solid. 1H NMR
(300 MHz, CDCl3) δ 1.49 (s, 9H), 3.41(dd, J = 5.2, 5.2 Hz, 2H), 3.58 (dd, J = 5.2, 5.2 Hz,
2H), 3.95 (s, 3H), 3.97 (s, 3H), 4.80 (b, 2H), 6.84 (s, 1H), 6.88 (s, 1H). The product was
used for the next reaction without further characterization.
Compound 1 (1.22 g, 3.2 mmol) was dissolved in 50 mL of ethyl acetate at room
temperature. 7.0 mL of 4 M aqueous HCl solution was added to the solution drop-wise, and
then stirred overnight at room temperature. The solution was concentrated to about 10 mL
by rotary evaporator, basified to pH = 10 by 1 M aqueous NaOH solution. The solution was
extracted with CHCl3 three times. The organic solution was dried by Na2SO4 and
concentrated
in
vacuo
to
generate
the
product
6,7-dimethoxy-3-(piperazin-1-yl)isoquinolin-1-amine (0.73 g, 81%). The product was then
used for the next reaction without further characterization.
To a solution of 6,7-dimethoxy-3-(piperazin-1-yl)isoquinolin-1-amine (0.73 g, 2.54 mmol)
and compound 2,5-dioxopyrrolidin-1-yl tetrahydrofuran-2-carboxylate (0.54 g, 2.54 mmol)
in CHCl3, Et3N (513 mg, 5.08 mmol) was added at 0° C. The solution was stirred for 2 hours
3
at room temperature. 30 mL of CHCl3 was then added. The solution was washed by brine,
evaporated in vacuo and purified by flash column chromatography with DCM and methanol
(100:1 to 100:4). To the product, 10 mL of HCl (4.0 N) was added. The solution was
evaporated in vacuo with toluene to obtain the salt. The crude product was recrystallized by
methanol and ethyl acetate, or purified by RP-HPLC to obtain the product TZ-md
(Compound 2, 223 mg, 23.6%). 1H NMR (400 MHz, DMSO-d6) δ 1.79-1.90 (m, 2H),
1.97-2.04 (m, 2H), 3.22-3.39 (m, 4H), 3.59-3.88 (m, 6H), 3.88 (s, 3H), 3.91 (s, 3H), 4.74 (dd,
J = 7.6, 5.6 Hz, 2H), 6.35 (s, 1H), 7.17 (s, 1H), 7.78 (s, 1H), 8.80 (b, 2H), 13.55 (b, 1H); 13C
NMR (133 MHz, DMSO-d6) δ 168.5, 155.4, 153.0, 148.3, 144.7, 136.6, 106.1, 106.0, 104.7,
92.2, 74.9, 68.2, 56.2, 56.9, 48.7, 48.1, 44.1, 40.2, 27.9, 25.2; HRMS (m/z): [M+H]+ calcd
for C20H27N4O4: 387.20268; found 387.20347.
Preparation of compound tert-butyl 4-cyanopiperazine-1-carboxylate: To a stirred solution
of tert-butyl piperazine-1-carboxylate (745 mg, 4.0 mmol) in 20 mL of DCM, DIPEA (836
L, 4.8 mmol) and BrCN (466 mg, 4.4 mmol) in DCM were added at 0° C. After 4 hours’
stirring at r.t., 50 mL of water was added to the solution to quench the reaction. Then the
mixture was extracted by ethyl acetate. The organic layer was washed by water and brine,
dried by Na2SO4, and evaporated. The resulting residue was washed with hexane. The crude
product was purified by flash column chromatography with petroleum ether/ethyl acetate
(20:1 to 5:1) to get product tert-butyl 4-cyanopiperazine-1-carboxylate (582 mg, 92%). The
product was used for the subsequent reaction without further characterization.
Preparation of compound 2,5-dioxopyrrolidin-1-yl tetrahydrofuran-2-carboxylate: A solution
of tetrahydrofuran-2-carboxylic acid (350 mg, 3.0 mmol ), EDCI (200 mg, 4.5 mmol) and
NHS (420 mg, 3.6 mmol) in DCM (10 mL) was stirred at room temperature for 3 hours.
Then the solution was washed with 5% aqueous NaHSO4 solution twice, water and brine.
Dried by Na2SO4 and evaporated to get a white solid (720 mg) without further purification.
The product 2,5-dioxopyrrolidin-1-yl tetrahydrofuran-2-carboxylate was used for the next
reaction without further characterization.
General procedure for the synthesis of the bait molecule TZ-TA:
4
A mixture of 2,4-dichloro-6,7-dimethoxyquinazoline (5.29 g, 20.4 mmol) and
2-(2-aminoethoxy)ethanol (10.76 g, 102.0 mmol) in 100 mL of methanol was stirred at room
temperature for 4 hours. After cooling, diethyl ether (100 mL) was added and the reaction
medium was maintained in a 4 C fridge, leading to the formation of a white solid that was
filtered and recrystallized from petroleum ether and ethyl acetate, yielding
2-(2-((2-chloro-6,7-dimethoxyquinazolin-4-yl)amino)-ethoxy)ethanol (3.9276 g, 41%) as a
white solid, which was directly used without further characterization.
Under an Argon atmosphere, 2-(2-((2-chloro-6,7-dimethoxyquinazolin-4-yl)amino)ethoxy)
ethanol (1.170 g, 3.58 mmol) and piperazin-1-yl(tetrahydrofuran-2-yl)methanone (0.660 g,
3.58 mmol) in 3-methyl-1-butanol (30 mL) were heated under reflux for 4.5 hours while
stirring. The mixture was then cooled to 0–5° C and the solvent was removed by reducing
pressure. The white precipitate was collected by filtration and washed with acetone (2 x 10
mL), before being recrystallized twice from methanol and precipitated using diethyl ether, to
yield the product TZ-TA (3), as a white crystalline solid (1.059 g, 62%). 1H NMR (300 MHz,
DMSO-d6): δ 1.84 (m, 2H), 2.03 (m, 2H), 3.47–4.07 (m, 14H), 3.84 (s, 3H), 3.87 (s, 3H),
4.73 (m, 1H), 7.16 (s, 1H), 7.72 (s, 1H); 13C NMR (300 MHz, DMSO-d6): d 25.2, 27.9, 40.9,
44.1, 44.6, 55.8, 56.2, 60.1, 68.1, 68.2, 72.1, 74.9, 102.4, 104.4, 106.9, 146.2, 154.4, 154.2,
158.6, 169.6; HRMS (m/z): [M+H]+ calcd. for C23H33N5O6, 476.25091; found 476.25120.
General procedure for the immobilization of the bait molecule TZ-TA:
The solid-phase beads used in small molecule immobilization and affinity chromatography
were Affi-Gel 102 (Bio-Rad) with a loading level of 16 μmol/mL in suspension (Bio-Rad
Affi-Gel 102, an agarose-based gel with a 6-atom hydrophilic amino-terminated arm). The
bead suspension (1 mL) was transferred to a 2 mL Eppendorf tube and washed with H2O (3
5
x 1.5 mL) and DMF (3 x 1.5 mL). The beads were then suspended in anhydrous DMF (0.5
mL).
The bait molecule Ta, (30 μmol, 14.2 mg) was dissolved in 0.3 mL of anhydrous DMF.
DSC (22.9 mg, 90 μM, 3 eq.) was dissolved in 0.9 mL of anhydrous DMF and was added to
the bait molecule solution before the addition of TEA (16.65 μL, 120 μM, 4 eq.). The
reaction solution was stirred at 50 ºC for 12 hours and the activation efficiency was
monitored
by
TLC
(DCM,
MeOH).
The
activation
product
1-(2-(2-((6,7-dimethoxy-2-(4-(tetrahydrofuran-2-carbonyl)piperazin-1-yl)quinazolin-4-yl)a
mino)ethoxy)ethoxy)pyrrolidine-2,5-dione in solution was used without further purification.
The activation solution was added to the beads. After addition of the activation solution, the
suspension was vortexed at room temperature for 1 hour. After the immobilization, the vials
were centrifuged, the supernatant was removed and the beads were washed with DMF (10 x
1 mL) and H2O (10 x 1 mL). The beads were subsequently suspended in 1 x PBS (0.8 mL)
and stored at 4º C before use.
6
7