Please cite this article in press as: Li et al., Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11, Cell
CH3), 3.72 (2H, s, SCH2), 3.75 (3H, s, OCH3), 3.98 – 4.02 (2H, m, CH2CH3), 4.59 (1H, d, J 17.4, NCH2), 4.86 (1H, d, J 17.4, NCH2), 5.85
(1H, s, CHS), 6.79 (2H, d, J 8.4, Ar-H), 6.92 (1H, s, CH(OAc)2), 7.16 – 7.27 (7H, m, Ar-H); d C (101 MHz; CDCl3) 13.76 (CH2CH3), 20.20
(C(O)CH3), 20.25 (C(O)CH3), 35.49 (CH2), 48.68 (CH2), 55.18 (OCH3), 61.65 (CHS), 62.21 (CH2CH3), 83.99 (CH(OAc)2), 113.97 (Ar-C-
H), 126.79 (Ar-C-H), 127.66 (Ar-C-H), 128.48 (Ar-C-H), 128.65 (quaternary C), 130.21 (Ar-C-H), 135.72 (quaternary C), 158.87
(quaternary C), 165.76 (CO), 167.16 (CO), 168.34 (CO), 168.62 (CO); m/z 504 (100%, [M+H]+): Found [M+H]+ 504.1688,
C25H30NO8S requires 504.1692.
1,4-Dibenzyl-3-methoxy-6-((4-methoxybenzyl)thio)piperazine-2,5-dione (10).
MeO
MeO
O
O
S
Benzylamine
MeOH, DMAP
S
N
Ph
OMe
O
Me
Ph
N
Ph
N
OAc
OAc
O
O
9
10
Chemical Formula: C25H29NO8S
Molecular Weight: 503.56
Chemical Formula: C27H28N2O4S
Molecular Weight: 476.59
Benzylamine (0.21 mL, 1.93 mmol) was added to a solution of 2-(benzyl(2-ethoxy-1-((4-methoxybenzyl)thio)-2-oxoethyl)amino)-2-
oxoethane-1,1-diyl diacetate (0.65 g, 1.29 mmol) (9) in methanol (15.0 mL) and the resulting mixture stirred for 2 minutes. DMAP
(0.08 g, 0.64 mmol) was added and the resulting mixture stirred for 5 hours at RT and solvent removed under reduced pressure.
The residue was purified via Biotageꢀ Horizon (3:1, petroleum spirit 40-60ꢁC: ethyl acetate; snap 25 g) and further purified by recrys-
tallisation (petroleum spirit 40-60ꢁC: ethyl acetate) to give 1,4-dibenzyl-3-methoxy-6-((4-methoxybenzyl)thio)piperazine-2,5-dione
(0.49 g, 79%) (10) as a colourless solid; m.p. 85-88ꢁC; nmax/cm-1 3030 (C-H), 3005 (C-H), 1671 (C=O), 1357 (CH3), 1002 (C-O-C),
832 (C-H); d H (400 MHz, CDCl3) 3.53 (3H, s, OCH3), 3.62 (1H, d, J 14.5, NCH2), 3.87 – 3.90 (4H, m, SCH2 & OCH3), 4.09 (1H, d, J
13.8, SCH2), 4.25 (1H, d, J 14.9, NCH2), 4.66 (1H, s, CHO), 4.45 (1H, s, CHS), 5.18 (2H, d, J 14.7, NCH2), 6.91 – 6.95 (2H, m,
Ar-H), 7.16 – 7.29 (6H, m, Ar-H), 7.36 (6H, dd, J 16.4 & 9.2, Ar-H); d C (101 MHz, CDCl3) 36.29 (CH2), 45.93 (CH2), 48.07 (CH2),
55.39 (OCH3), 57.66 (OCH3), 85.50 (CHS), 114.29 (CHO), 128.03 (Ar-C-H), 128.19 (Ar-C-H), 128.53 (Ar-C-H), 128.66 (Ar-C-H),
128.75 (Ar-C-H), 129.01 (Ar-C-H), 130.88 (Ar-C-H), 134.98 (quaternary C), 135.30 (quaternary C), 159.27 (quaternary C), 162.73
(CO), 166.29 (CO); m/z 477 (100%, [M+H]+); Found [M+H]+ 477.1841, C27H29N2O4S requires 477.1848.
1,3,4-Tribenzyl-6-methoxy-3-((4-methoxybenzyl)thio)piperazine-2,5-dione (11).
MeO
MeO
O
Ph
S
O
O
S
Benzyl bromide
N
Ph
OMe
N
Ph
OMe
Ph
N
Ph
N
LiHMDS
THF, -78 °C
O
10
11
Chemical Formula: C27H28N2O4S
Molecular Weight: 476.59
Chemical Formula: C34H34N2O4S
Molecular Weight: 566.71
LiHMDS (0.50 mL of a 1M solution in tetrahydrofuran, 0.50 mmol) was added dropwise to a solution of 1,4-dibenzyl-3-methoxy-6-
((4-methoxybenzyl)thio)piperazine-2,5-dione (0.20 g, 0.41 mmol) (10) and benzyl bromide (0.05 mL, 0.41 mmol) in tetrahydrofuran
(2.00 mL) at -78ꢁC and the resulting mixture stirred at this temperature for 1.5 hours and 1 hour at 0ꢁC. Saturated aqueous sodium
hydrogen carbonate (10.0 mL) was added and solvent removed under reduced pressure. The residue was partitioned between water
(10.0 mL) and dichloromethane (20.0 mL). The aqueous phase was extracted with dichloromethane (2 x 10.0 mL) and the combined
extracts were dried (MgSO4), filtered and solvent removed under reduced pressure. The residue was purified via Biotageꢀ Horizon
(3:1, petroleum spirit 40-60ꢁC: ethyl acetate; snap 10 g) to give 1,3,4-tribenzyl-6-methoxy-3-((4-methoxybenzyl)thio)piperazine-2,5-
dione (0.14 g, 59%) (11) as a pale yellow oil; nmax/cm-1 3004 (C-H), 1670 (C=O), 1453 (CH2), 1357 (CH3), 1000 (C-O-C); d H (400 MHz,
CDCl3) 3.18 (1H, d, J 10.9, CH2), 3.32 (1H, d, J 14.2, CH2), 3.38 (1H, d, J 8.8, SCH2), 3.44 (3H, s, OCH3), 3.73 (1H, d, J 9.1, SCH2), 3.78
(3H, s, OCH3), 4.05 (1H, d, J 14.2, NCH2), 4.13 (1H, s, CHO), 4.65 (1H, d, J 14.7, NCH2), 4.75 (1H, d, J 14.7, NCH2), 5.30 (1H, d, J 14.7,
NCH2), 7.01 (6H, dd, J 11.5 & 4.5, Ar-H), 7.14 – 7.17 (3H, m, Ar-H), 7.29 – 7.33 (8H, m, Ar-H), 7.54 – 7.57 (2H, m, Ar-H); d C (101 MHz,
CDCl3) 34.61 (CH2), 43.51 (CH2), 47.21 (CH2), 47.91 (CH2), 55.41 (OCH3), 82.93 (OCH3), 113.98 (CHO), 127.63 (Ar-C-H), 128.13
(Ar-C-H), 128.36 (Ar-C-H), 128.60 (Ar-C-H), 128.82 (Ar-C-H), 129.03 (Ar-C-H), 129.39 (Ar-C-H), 129.56 (Ar-C-H), 130.19 (Ar-C-H),
130.48 (Ar-C-H), 130.65 (Ar-C-H), 133.87 (quaternary C), 134.70 (quaternary C), 135.44 (quaternary C), 137.89 (quaternary C),
159.02 (quaternary C), 164.23 (quaternary C), 165.06 (CO), 166.07 (CO); m/z 589 (100%, [M+Na]+); Found [M+Na]+ 589.2107,
C34H34N2O4SNa requires 589.2137.
Cell Chemical Biology 25, 1–9.e1–e9, November 15, 2018 e6