Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8625
(3 ꢀ 15 mL), and the hexane layer was extracted with methanol
(3 ꢀ 20 mL). The combined methanolic layers were evaporated
in vacuo, and the residue was dissolved in CH2Cl2 (40 mL).
The solution was washed with 5% NaHCO3 (20 mL), and the
organic layer was dried over anhydrous Na2SO4, evaporated, and
purified withchromatography (CH3OH:CH2Cl2 = 1:15 andthen
1:3) to obtain 210 mg (46.8% yield) of a white foam solid 78. 1H
NMR (CD3OD, 500 MHz): δ 0.85-0.90 (-CH3, m, 6H), 1.18-
1.27 (-CH2, m, 2H), 1.51-1.56 (-CH, m, 1H), 1.68-1.72
(-CH2, m, 1H), 1.93-2.05 (-CH2, m, 3H), 2.57-2.60 (-CH,
m, 1H), 2.58-2.76 (-CH2, m, 2H), 2.95-3.01 (-CH2, m, 2H),
3.73-3.79 (-CH, m, 1H), 5.41-5.46 (-CH, m, 1H), 6.89-6.92
(-Ph, m, 1H), 6.98-7.00 (-Ph, m, 1H), 7.07-7.14 (-Ph, m, 3H),
7.16-7.18 (-Ph, m, 1H), 7.21-7.23 (-Ph, m, 1H), 7.34-7.40
(-Ph, m, 1H). 13C NMR (CD3OD, 125 MHz): δ 22.1, 23.8, 26.9,
28. 8, 30.2, 37.4, 41.0, 45.5, 47.9, 49.8, 51.0, 114.7, 115.7, 123.6,
127.0, 127.9, 129.9, 130.5, 131.7, 135.3, 138.9, 144.5, 165.4, 176.7,
177.6. MS (ESI) m/z (%) 477.2 (62) [M þ Na]þ, 481.2 (100) [M þ
27]-. HRMS [M þ Na]þ calcd, 477.2336; found, 477.2327.
(R)-1-[3-(2-Naphthamido)-3-(3-fluorophenyl)propanamido]-
3-methylbutyl Boronic Acid (79). A similar procedure was used as
described with 78 with the corresponding starting materials. 1H
NMR (CD3OD, 500 MHz): δ 0.83-0.88 (-CH3, m, 6H), 1.20-
1.25 (-CH2, m, 1H), 1.26-1.30 (-CH2, m, 1H), 1.50-1.58
(-CH, m, 1H), 2.63-2.66 (-CH, m, 1H), 3.12-3.15 (-CH2,
m, 1H), 3.18-3.21 (-CH2, m, 1H), 5.68-5.72 (-CH, m, 1H),
7.02-7.06 (-Ph, m, 1H), 7.26-7.29 (-Ph, m, 1H), 7.31-7.34
(-Ph, m, 1H), 7.38-7.41 (-Ph, m, 1H), 7.54-7.61 (-Ph, m,
2H), 7.88-7.97 (-Ph, m, 3H), 7.98-8.02 (-Ph, m, 1H), 8.41-
8.42 (-Ph, m, 1H). 13C NMR (CD3OD, 125 MHz): δ 22.0, 23.8,
27.0, 37.5, 41.0, 45.3, 51.8, 114.7, 115.8, 123.8, 125.0, 127.9, 128.5,
128.8, 129.0, 129.4, 130.0, 131.7, 132.5, 134.0, 136.4, 144.8, 163.8,
169.7, 177.0. MS (ESI) m/z (%) 473.1 (62) [M þ Na]þ, 477.2 (100)
[M þ 27]-. HRMS [M þ Na]þ calcd, 473.2023; found, 473.2030.
(R)-1-[3-(3-Fluorophenyl)-3-(5,6,7,8-tetrahydronaphthalene-
4-carboxamid)propanamido]-3-methyl Butyl Boronic Acid (80).
A similar procedure was used as described with 78 with the
corresponding starting materials. 1H NMR (CD3OD, 500 MHz):
δ 0.87-0.90 (-CH3, m, 6H), 1.21-1.24 (-CH2, m, 1H), 1.27-
1.31 (-CH2, m, 1H), 1.53-1.59 (-CH, m, 1H), 1.73-1.78
(-CH2, m, 4H), 2.62-2.65 (-CH, m, 1H), 2.70-2.71 (-CH2,
m, 2H), 2.74-2.79 (-CH2, m, 2H), 2.98-3.05 (-CH2, m, 2H),
5.55-5.60 (-CH, m, 1H), 7.02-7.06 (-Ph, m, 1H), 7.11-7.15
(-Ph, m, 3H), 7.19-7.21(-Ph, m, 1H), 7.26-7.27 (-Ph, m, 1H),
7.38-7.42 (-Ph, m, 1H). 13C NMR (CD3OD, 125 MHz): δ 22.1,
23.8, 24.0, 26.9, 27.6, 30. 7, 37.6, 41.0, 45.2, 49.8, 51.5, 114.8,
115.8, 123.8, 125.3, 126.4, 131.9, 135.5, 137.7, 139.2, 144.7, 165.4,
172.8, 176.7. MS (ESI) m/z (%) 477.2 (62) [M þ Na]þ, 467.2 (100)
[M þ 13]-. HRMS [M þ Na]þ calcd, 477.2336; found, 477.2329.
Proteasome Inhibition Assays. A 20S proteasome activity assay
kit was purchased from Chemicon (Chemicon, United States).
Other reagents and solvents were purchased from commercial
sources. In brief, substrates and compounds were previously
dissolved in DMSO, with the final solvent concentration kept
constant at 3% (v/v). The reaction buffers were (pH 7.5) 20 mM
Tris, 1 mM DTT, 10% glycerol, and 0.02% (w/v) DS for CT-L
activities. The proteasome activity was determined by monitoring
the hydrolysis of the fluorogenic substrate, Suc-Leu-Leu-Val-Tyr-
AMC (λexc=360 and λexc=465 nm for AMC substrates), reacting
for 1 h at 37 °C in the presence of untreated (control) or
proteasome that had been incubated with a different concentra-
tion of test compounds. Fluorescence was measured using an
Infinite M200 microplate reader (Tecan, Austria).
and HepG2 (human hepatocellular liver carcinoma cell line) cell
lines were obtained from China Pharmaceutical University. HL-
60 cells were cultured in IMDM supplemented with 20% fetal
bovine serum at 37 °C in 5% CO2. RPMI 8226 cells were
cultured in RPMI 1640 supplemented with 10% fetal bovine
serum at 37 °C in 5% CO2. BXPC-3, HepG2, and SW-480 cells
were grown in RPMI 1640 supplemented with 10% fetal bovine
serum at 37 °C in 5% CO2. PC-3 and A549 cells were cultured in
F12-k supplemented with 10% fetal bovine serum at 37 °C in 5%
CO2. SKOV-3 cells were cultured in McCoy’s 5A supplemented
with 10% fetal bovine serum at 37 °C in 5% CO2.
A standard MTT assay was used to measure cell growth. In
brief, a suspension of 3000 cells/150 μL of medium was added to
each well of 96-well plates and allowed to grow. Twenty-four
hours later, drugs prepared in medium at 10 different concen-
trations were added to the corresponding plates at a volume of
50 μL per well, and the plates were incubated for 72 h with drugs.
Then, 20 μL of a solution of 5 mg/mL MTT was added to each
well and incubated for another 4 h at 37 °C. Plates were then
centrifuged at 1000 rpm at 4 °C for 5 min, and the medium was
carefully discarded. The formazan crystals were dissolved in 100
μL of DMSO, and absorbance was read on an Infinite M200
(Tecan, Austria) microplate reader at 540 nm. The result was
expressed as the mean IC50 value, which was the average from at
least three independent determinations.
Pharmacokinetic Analyses in Rodents. Solutions of 78, 79, 80,
and bortezomib were prepared in 1% ethanol, 1% Tween80,
and 98% buffered saline with the final concentration of both
compounds 0.5 mg/mL for iv and ig administration. Heparin-
ized blood samples collected for PK analyses (n = 4) were
centrifuged at 4000 rpm for 5 min at 4 °C. Plasma samples were
analyzed after protein precipitation with acetonitrile acidified
with 1% formic acid. LC/MS/MS analysis of 78, 79, 80, and
bortezomib was performed under optimized conditions to obtain
the best sensitivity and selectivity of the analyte in selected
reaction monitoring mode (SRM). Selected product ions of 78,
79, and 80 were monitored for the quantification of the com-
pound using bortezomib as an internal standard. Plasma con-
centration-time data were analyzed by a noncompartmental
approach using the software WinNonlin Enterprise version 5.2
(Pharsight Co., Mountain View, CA).
Acknowledgment. We thank Dr. Xin Cao, State Key Lab-
oratory of Bioorganic and Natural Products Chemistry
Shanghai Institute of Organic Chemsitry, Chinese Academy
of Sciences, for his manuscript reading.
Note Added after ASAP Publication. This paper published
on the web on November 15, 2010 with errors in the author affil-
iations. The correct version was published on November 18, 2010.
Supporting Information Available: General and experimental
procedures and characterization data for 4A1-4A48, 6, 9,
10a-10j, 12a-12j, 14-77, and 81-85. This material is available
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