Carmaphycins: New Proteasome Inhibitors
(200 mL) and partitioned. The aqueous layer was extracted with
Et2O (3ꢂ100 mL) and the combined organic layers were dried with
Na2SO4. After solvent removal in vacuo, repetitive silica gel column
chromatography (5% and 20% EtOAc/hexanes) provided starting
material 5 (0.12 g) and the desired diastereomer 6 (colorless oil,
0.59 g, 26%) as the major reaction product. [a]2D4 = +79 (c 0.7,
Synthetic carmaphycin A (1): A solution of dipeptide sulfoxide 8
(76.2 mg, 200 mmol) and LiOH·H2O (42.1 mg, 1.0 mmol) in 1,4-diox-
ane/H2O (9 mL, 2:1) was stirred at 258C. After 1 h, TLC (80%
EtOAc/hexanes) showed absence of starting material. Solvents
were evaporated off and the resulting residue was dissolved in
H2O (10 mL), acidified and extracted with n-butanol (3ꢂ15 mL).
The combined organic extracts were dried (Na2SO4) and concen-
trated to obtain the free acid of 8 as a white solid. In a separate re-
action, Boc-l-Leu epoxyketone (6) (31.0 mg, 110 mmol) in CH2Cl2
(1 mL) was treated with TFA (200 mL, 2.6 mmol) and stirred at 258C
until TLC showed the absence of starting material (1 h), whereupon
it was concentrated in vacuo to a reddish oil. This oil was dissolved
in CH2Cl2 (2 mL) and added to a solution of the previously pre-
pared free acid (34.0 mg, 93.0 mmol) and benzotriazol-1-yl-oxytri-
pyrrolidinophosphonium hexafluorophosphate (PyBOP, 50.0 mg,
93.0 mmol) in CH2Cl2 (3 mL) at 258C; followed by addition of DIPEA
(66 mL, 380 mmol). After stirring 20 h at 258C, the reaction mixture
was quenched with saturated NH4Cl, followed by solvent partition
and CH2Cl2 (3ꢂ10 mL) extractions of the aqueous layer. All organic
extracts were combined and dried (Na2SO4), to be then concentrat-
ed in vacuo. Silica gel column chromatography (10% MeOH/
EtOAc), followed by normal-phase HPLC (Phenomenex Luna 5mm
1
CH3CN); H NMR (CDCl3, 500 MHz): d=4.84 (d, J=8.5 Hz, 1H), 4.31
(dt, J=3.5, 8.5 Hz, 1H), 3.28 (d, J=5.0 Hz, 1H), 2.88 (d, J=5.0 Hz,
1H), 1.72 (m, 1H), 1.51 (s, 3H), 1.49 (ddd, J=3.5, 10.0, 14.0 Hz, 1H),
1.40 (s, 9H), 1.16 (ddd, J=3.5, 10.0, 14.0 Hz, 1H), 0.96 (d, J=6.5 Hz,
3H), 0.93 (d, J=7.0 Hz, 3H); 13C NMR (CDCl3, 125 MHz): d=209.5
(C), 155.6 (C), 79.7 (C), 59.0 (C), 52.3 (CH2), 51.4 (CH), 40.4 (CH2),
28.2 (3CH3), 25.1 (CH), 23.3 (CH3), 21.3 (CH3), 16.7 (CH3); HRESIMS
m/z [M+Na]+ 294.1673 (calcd for C14H25NO4Na: 294.1676).
Boc-l-Leucine epoxyketone derivative (7): Boc-l-Leu epoxyketone
(6) (44.0 mg, 160 mmol) in CH2Cl2 (1.5 mL) was treated with TFA
(300 mL, 3.9 mmol) and stirred at 258C until TLC showed the ab-
sence of starting material (1.5 h), whereupon it was concentrated
in vacuo to a reddish oil. This oil was dissolved in dry CH2Cl2
(10 mL), and to this solution were sequentially added 4-bromoben-
zoyl chloride (53.0 mg, 240 mmol) and diisopropylethylamine
(DIPEA, 50 mL, 290 mmol) at 258C. After stirring 16 h at 258C, the re-
action mixture was quenched with saturated NH4Cl, followed by
solvent partition and CH2Cl2 (3ꢂ10 mL) extractions of the aqueous
layer. All organic extracts were combined and dried (Na2SO4), to be
then concentrated in vacuo. Silica gel column chromatography
(20% EtOAc/hexanes) gave 7 as a white solid (35.1 mg, 61%). Addi-
tional purification by normal-phase HPLC (Phenomenex Luna 5mm
Silica 100 ꢁ, 250ꢂ10.0 mm, 50% i-PrOH/hexanes at 3.0 mLminÀ1
,
detection at 211 and 235 nm), yielded synthetic 1 as a colorless oil
comprised of a 1.6:1 diastereomeric mixture (24.4 mg, 50%).
[a]2D4 = +22 (c=0.5, CH3CN), CD: c=0.2 (CH3CN), lmax (De)=290
(+3.36), 250 (+0.17), 230 (+1.13), 200 (À6.99); major diastereomer
1H NMR (CDCl3, 600 MHz): d=7.72 (d, J=7.2 Hz, 1H), 6.08 (d, J=
8.4 Hz, 1H), 4.82 (m, 1H), 4.48 (m, 1H), 4.29 (dd, J=6.0, 8.4 Hz, 1H),
3.39 (ddd, J=4.2, 10.2, 14.4 Hz, 1H), 3.33 (d, J=5.1 Hz, 1H), 2.90 (d,
J=5.1 Hz, 1H), 2.73 (s, 3H), 2.69 (dt, J=5.4, 15.0 Hz, 1H), 2.50 (m,
1H), 2.23 (t, J=7.8 Hz, 2H), 2.14 (m, 1H), 2.12 (m, 1H), 1.70 (m,
1H), 1.64 (m, 2H), 1.55 (m, 1H), 1.52 (s, 3H), 1.34 (m, 1H), 1.31 (m,
2H), 1.29 (m, 2H), 0.94 (d, J=7.8 Hz, 3H), 0.93 (d, J=7.2 Hz, 3H),
0.92 (d, J=6.6 Hz, 3H), 0.91 (d, J=7.2 Hz, 3H), 0.89 (t, J=6.6 Hz,
3H); 13C NMR (CDCl3, 150 MHz): d=208.6 (C), 173.5 (C), 171.5 (C),
170.35 (C), 59.3 (C), 58.33 (CH), 52.5 (CH2) 51.3 (CH), 50.9 (CH), 48.9
(CH2), 39.1 (CH2), 38.8 (CH3), 36.7 (CH2), 31.4 (CH2), 30.9 (CH), 28.2
(CH2), 25.4 (CH2), 25.3 (CH), 23.35 (CH3), 22.4 (CH2), 21.1 (CH3), 19.3
Silica 100 ꢁ, 250ꢂ10.0 mm, 1% i-PrOH/hexanes at 3.0 mLminÀ1
,
detection at 256 and 280 nm), yielded highly pure 7 (15.2 mg),
which was crystallized from EtOAc/i-octane as colorless tridimen-
sional needles. [a]D28 = +41 (c=1.5, CH3CN); 1H NMR (CDCl3,
600 MHz): d=7.61 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H), 6.52
(d, J=8.4 Hz, 1H), 4.83 (ddd, J=3.0, 10.5, 10.8 Hz, 1H), 3.41 (d, J=
5.1 Hz, 1H), 2.94 (d, J=5.1 Hz, 1H), 1.75 (m, 1H), 1.64 (ddd, J=3.6,
13.8, 13.8 Hz, 1H), 1.54 (s, 3H), 1.36 (ddd, J=4.2, 13.8, 13.8, 1H),
1.01 (d, J=6.6 Hz, 3H), 0.97 (d, J=6.6 Hz, 3H); 13C NMR (CDCl3,
150 MHz): d=209.1 (C), 166.4 (C), 132.5 (C), 131.8 (2CH), 128.6
(2CH), 126.5 (C), 59.1 (CH), 52.6 (CH2) 50.8 (CH), 40.4 (CH2), 25.4
(CH), 23.4 (CH3), 21.4 (CH2), 16.7 (CH3); HRESIMS m/z [M+Na]+
376.0520 (calcd for C16H20BrNO3Na: 376.0519).
1
(CH3), 17.9 (CH3), 16.8 (CH3), 13.9 (CH3); minor diastereomer H NMR
(CDCl3, 600 MHz): d=7.93 (d, J=7.2 Hz, 1H), 7.00 (d, J=6.6 Hz,
1H), 6.08 (d, J=8.4 Hz, 1H), 4.82 (m, 1H), 4.48 (m, 1H), 4.27 (dd,
J=6.6, 8.4 Hz, 1H), 3.39 (ddd, J=4.2, 10.2, 14.4 Hz, 1H), 3.36 (d, J=
5.1 Hz, 1H), 2.91 (d, J=5.1 Hz, 1H), 2.77 (dt, J=5.4, 13.8 Hz, 1H),
2.71 (s, 3H), 2.41 (m, 1H), 2.22 (t, J=7.2 Hz, 2H), 2.10 (m, 1H), 2.18
(m, 1H), 1.70 (m, 1H), 1.63 (m, 2H), 1.53 (m, 1H), 1.52 (s, 3H), 1.35
(m, 1H), 1.31 (m, 2H), 1.29 (m, 2H), 0.94 (d, J=7.8 Hz, 3H), 0.93 (d,
J=7.2 Hz, 3H), 0.92 (d, J=6.6 Hz, 3H), 0.91 (d, J=7.2 Hz, 3H), 0.89
(t, J=6.6 Hz, 3H); 13C NMR (CDCl3, 150 MHz): d=209.2 (C), 173.4
(C), 171.1 (C), 170.31 (C), 59.3 (C), 58.28 (CH), 52.6 (CH2) 51.4 (CH),
50.7 (CH), 46.9 (CH2), 39.2 (CH2), 36.7 (CH2), 36.1 (CH3), 31.4 (CH2),
31.1 (CH), 26.0 (CH2), 25.4 (CH2), 25.2 (CH), 23.31 (CH3), 22.4 (CH2),
21.1 (CH3), 19.2 (CH3), 18.0 (CH3), 16.8 (CH3), 13.9 (CH3); HRESIMS m/
z [M+H]+ 516.3100 (calcd for C25H46N3O6S: 516.3102).
CCDC 855711 (compound 7) contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
Dipeptide sulfone (9): Oxone (270 mg, 420 mmol) was added to
a
solution of dipeptide 8 (20.0 mg, 53.0 mmol) and NaHCO3
(150 mg, 1.8 mmol) in a 45% acetone/H2O solution (9.6 mL) at 08C.
The mixture was stirred at 258C for 1 h. After addition of H2O
(10 mL), the resulting solution was extracted with CHCl3 (3ꢂ
10 mL). The organic layer was dried over Na2SO4, filtered, and
evaporated in vacuo to give 9 as a white powder (20.6 mg, 99%).
1
[a]2D4 =À20 (c=0.4, CH3CN); H NMR (CDCl3, 600 MHz): d=7.03 (d,
J=7.8 Hz, 1H), 6.10 (d, J=7.8 Hz, 1H), 4.69 (m, 1H), 4.24 (dd, J=
7.2, 7.8 Hz, 1H), 3.77 (s, 3H), 3.15 (m, 1H), 3.08 (m, 1H), 2.94 (s,
3H), 2.46 (m, 1H), 2.23 (dd, J=7.8, 7.8 Hz, 2H), 2.22 (m, 1H), 2.09
(m, 1H), 1.62 (m, 2H), 1.32 (m, 2H), 1.31 (m, 2H), 0.97 (d, J=6.6 Hz,
3H), 0.96 (d, J=7.2 Hz, 3H), 0.89 (t, J=6.6 Hz, 3H); 13C NMR (CDCl3,
150 MHz): d=173.6 (C), 171.7 (C), 171.0 (C), 58.7 (CH), 52.8 (CH3),
50.7 (CH2), 50.6 (CH), 40.8 (CH3), 36.6 (CH2), 31.4 (CH2), 30.7 (CH),
25.3 (CH2), 24.7 (CH2), 22.3 (CH2), 19.2 (CH3), 18.3 (CH3), 13.9 (CH3);
HRESIMS m/z [M+H]+ 393.2055 (calcd for C17H33N2O6S: 393.2054).
Synthetic carmaphycin B (2): A solution of dipeptide sulfone (9)
(37.6 mg, 96.0 mmol) and LiOH·H2O (21.0 mg, 480 mmol) in 1,4-diox-
ane/H2O (5 mL, 2:1) was stirred at 258C. After 1 h, TLC (80%
EtOAc/hexanes) showed absence of starting material. Solvents
were evaporated off and the resulting residue was dissolved in
H2O (10 mL), acidified and extracted with n-butanol (3ꢂ15 mL).
The combined organic extracts were dried (Na2SO4) and concen-
trated to obtain the free acid of 9 as a white solid. In a separate
reaction, Boc-l-Leu epoxyketone (6) (32.0 mg, 110 mmol) in CH2Cl2
ChemBioChem 2012, 13, 810 – 817
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
815