Journal of Medicinal Chemistry
ARTICLE
were conducted by Shandong Analysis and Test Center in Ji’nan, China.
ESI-MS spectra were recorded on an API 4000 spectrometer. Melting
points were determined uncorrected on an electrothermal melting point
apparatus. Compound 4 were >95% pure by HPLC analysis, performed
on a Agilent 1100 HPLC instrument using a Phenomenex Synergi 4 μ
Polar-RP 80A column (250 mm ꢁ 4.6 mm), eluted with 50% acetoni-
trile/50% water (containing 0.1% formic acid) over 20 min, with
detection at 254 nm and a flow rate of 1.0 mL/min.
(40 μL) was added, and the mixture was incubated at 37 °C for
30 min and then stopped by addition of 100 μL of developer containing
trypsin and TSA. After incubation at 37 °C for 20 min, fluorescence
intensity was measured using a microplate reader at excitation and
emission wavelengths of 390 and 460 nm, respectively. The inhibition
ratios were calculated from the fluorescence intensity readings of
tested wells relative to those of control wells, and the IC50 values
were calculated using a regression analysis of the concentration/
inhibition data.
(S)-7-Hydroxy-6,8-diiodo-Tic hydrochloride (6), (S)-2-(tert-butoxy-
carbonyl)-7-hydroxy-6,8-diiodo-Tic (7), (S)-2-(tert-butoxycarbonyl)-7-
hydroxy-Tic (8), (S)-tert-butyl 7-hydroxy-3-((4-methoxyphenyl)car-
bamoyl)-3,4-dihydroisoquinoline- 2(1H)-carboxylate (9), and (S)-tert-
butyl 7-(2-methoxy-2-oxoethoxy)-3-((4- methoxyphenyl)carbamoyl)-
3,4-dihydroisoquinoline-2(1H)-carboxylate (10) were synthesized ac-
cording to the methods in our previous work.10 (S)-Methyl 2-((3-((4-
methoxyphenyl)carbamoyl)-1,2,3,4-tetrahydroisoquinolin-7-yl) oxy)
acetate (11), tert-butyl (2S,3S)-1-((S)-7-(2-(hydroxyamino)-2-oxo-
ethoxy)-3-(4-methoxy phenylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-
yl)-3-methyl-1-oxopentan-2-ylcarbamate (1), tert-butyl (2S,3S)-1-((S)-
7-(2-(hydroxyamino)-2-oxoethoxy)-3-(4-methoxyphenylcarbamoyl)-
3,4-dihydroisoquinolin-2(1H)-yl)-3-methylpentan-2-ylcarbamate (2),
and (S)-2-((2S,3S)-2-(3,3-dimethylbutanamido)-3-methylpentyl)-7-(2-
(hydroxyamino)-2-oxoethoxy)-N-(4-methoxyphenyl)-1,2,3,4-tetrahydro-
isoquinoline-3-carboxamide (3) were obtained as described previously.11
(2S,3S)-2-(3,3-Dimethylbutanamido)-3-methylpentanoic
Acid (13). According to the similar procedures described by our
group,28 L-isoleucine 12 was converted to 13 as colorless crystal; mp
143ꢀ145 °C. 1H NMR (DMSO-d6) δ 0.83ꢀ0.86 (m, 6H), 0.95 (s, 9H),
1.16ꢀ1.22 (m, 1H), 1.38ꢀ1.44 (m, 1H), 1.72ꢀ1.78 (m, 1H), 2.03 (d, J =
12.6 Hz, 1H), 2.07 (d, J = 12.6 Hz, 1H), 4.15 (dd, J = 8.4 Hz, J = 6.6 Hz,
1H), 7.83 (d, J = 8.4 Hz, 1H), 12.46 (s, 1H). ESI-MS m/z: 230.2 [M + H]+.
(S)-2-((2S,3S)-2-(3,3-Dimethylbutanamido)-3-methyl-
pentanoyl)-7-(2-(hydroxyamino)-2-oxoethoxy)-N-(4-methoxy-
phenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (4).
At room temperature, to a solution of compound 13 (1.15 g, 5.0 mmol)
in anhydrous THF (30 mL), was added Et3N (0.56 g, 5.5 mmol)
followed by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tet-
rafluoroborate (TBTU, 1.78 g, 5.5 mmol). After 15 min, the amine
compound 11 (1.85 g, 5.0 mmol) was added. Stirring was continued
overnight, and then THF was evaporated with the residue being taken up
in EtOAc (40 mL). The EtOAc solution was washed with saturated
Na2CO3 (3 ꢁ 10 mL), 1 N HCl (3 ꢁ 10 mL), and brine (3 ꢁ 10 mL),
dried over MgSO4, and evaporated under vacuum. The obtained crude
product was treated with a solution of NH2OK in anhydrous methanol
for 1 h, and then the solvent was evaporated under vacuum. The residue
was acidified with 2 N HCl until pH 5ꢀ6 and then extracted with EtOAc
(3 ꢁ 30 mL). The organic layers were combined, washed with brine
(3 ꢁ 20 mL), dried over MgSO4, and evaporated, with the residue being
purified by C18 reversed-phase column chromatography (H2O/MeOH
3:7) to give desired compound 4 (0.93 g, 32% yield) as a white powder;
mp 123ꢀ125 °C. 1H NMR (DMSO-d6) δ 0.87 (t, J = 7.2 Hz, 3H), 0.92
(d, J = 6.0 Hz, 3H), 0.97 (s, 9H), 1.09ꢀ1.15 (m, 1H), 1.33ꢀ1.37 (m,
1H), 1.73ꢀ1.76 (m, 1H), 2.03 (d, J = 12.6 Hz, 1H), 2.18 (d, J = 12.6 Hz,
1H), 2.95ꢀ2.99 (m, 1H), 3.21ꢀ3.26 (m, 1H), 3.69 (s, 3H), 4.42 (s, 2H),
4.83 (d, J = 15.6 Hz, 1H), 4.90 (d, J = 15.6 Hz, 1H), 4.64ꢀ4.67 (m, 1H),
5.14ꢀ5.16 (m, 1H), 6.73ꢀ6.89 (m, 4H), 7.08ꢀ7.09 (m, 1H),
7.50ꢀ7.52 (m, 2H), 8.14 (d, J = 8.4 Hz, 1H), 8.98 (s, 1H), 9.35
(s, 1H), 10.83 (s, 1H). HRMS (AP-ESI) m/z calcd for C31H43N4O7
[M + H]+ 583.3132, found 583.3165. Retention time: 6.5 min.
In Vitro Antiproliferative Assay. In vitro antiproliferative assays
were determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphen-
yl-2H-tetrazolium bromide) method as previously described.11 Briefly,
all cell lines were maintained in RPMI1640 medium containing 10% FBS
at 37 °C in 5% CO2 humidified incubator. Cells were passaged the day
before dosing into a 96-well cell plate and allowed to grow for a minimum
of 4 h prior to addition of compounds. After compounds addition, the
plates were incubated for an additional 48 h, and then 0.5% MTT solution
was added to each well. After further incubation for 4 h, formazan formed
from MTT was extracted by adding 200 μL of DMSO for 15 min.
Absorbance was then determined using an ELISA reader at 570 nm, and
the IC50 values were calculated according to the inhibition ratios.
In Vitro Cell Cycle Analysis. MDA-MB-231 cells were seeded
into six-well plates at a density of 4 ꢁ 105 per well. After overnight
incubation, cells were treated with 4 and SAHA. After 48 h treatment,
cells were harvested and fixed with 70% ethanol phosphate buffer
overnight. Then the cells were washed with PBS twice, incubated with
DNase-free RNase A (1 mg/mL, Solarbio, China) for 30 min, and
stained with propidium iodide (50 mg/mL, Solarbio, China) for 30 min,
avoiding light at room temperature. DNA content was measured by a
fluorescence-activated cell cytometer (FACScan, Becton Dickinson,
USA) and analyzed by MODFit LT for Mac V3.0 software.
In Vivo Human Tumor Xenograft Models. In vivo human
tumor xenograft models were established as previously described.11 In
brief, tumor cell lines (MDA-MB-231 and HCT116) were cultured in
RPMI1640 medium containing 10% FBS and maintained in a 5% CO2
humidified incubator at 37 °C. For in vivo antitumor assays, aforemen-
tioned cells were inoculated subcutaneously in the right flanks of female
athymic nude mice (BALB/c-nu, 5ꢀ6 weeks old, Slac Laboratory
Aniamal, Shanghai, China). About 10 days after injection, tumors were
palpable (about 100 mm3) and mice were randomized into treatment
and control groups (6 mice per group). The treatment groups received
specified concentrations of compounds by oral administration or
intraperitoneal injection, and the blank control group received an equal
volume of PBS solution containing DMSO. During treatment, subcuta-
neous tumors were measured with a vernier caliper every three days, and
body weight was monitoredregularly. After treatment, micewere sacrificed
and dissected to weigh the tumor tissues and to examine the internal organ
injury. All the obtained data were used to evaluate the antitumor potency
and toxicity of compounds. Data were analyzed by Student’s two-tailed
t test. A P level <0.05 was considered statistically significant.
In Vivo Mouse H22 Pulmonary Metastasis Model. The
mouse hepatoma H22 cell line used in this study was a kind gift form
Professor Cui (Shandong Academy of Medical Sciences, China).
Female Kunming mice were purchased from Center for New Drugs
Evaluation of Shandong University, China. For in vivo antimetastasis
assays, H22 cells suspended in saline were injected into female
Kunming mice (7ꢀ8 weeks old) via tail vein on the first day. Mice
were randomly assigned into treatment and control groups (eight
mice per group). After 4 days, tested compounds were dosed orally by
gavage. During treatment, body weight was monitored regularly.
After treatment, mice were sacrificed to examine the abdomen and
chest. The metastatic nodes in lungs were visualized by Bouin’s
solution and counted to evaluate the therapeutic effects of tested
compounds.
In Vitro HDACs Inhibition Fluorescence Assay. In vitro
HDACs inhibition assays were conducted as previously described.11
In brief, 10 μL of enzyme solution (HeLa nuclear extract, HDAC6, or
HDAC8) was mixed with various concentrations of tested compound
(50 μL). Five minutes later, fluorogenic substrate Boc-Lys (acetyl)-AMC
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dx.doi.org/10.1021/jm200577a |J. Med. Chem. 2011, 54, 5532–5539