Journal of Medicinal Chemistry
Article
Compound 11. Colorless oil. tR− HPLC, 11.30 min (97.3%);
[α]10D 37.5 (c 0.39, CH3OH). UV (CH3OH) λmax (log ε) 227.6 (4.14)
nm. IR (KBr) νmax 3434, 2955, 2871, 1726, 1670, 1640, 1466, 1385,
53%) as colorless oil: tR− HPLC, 24.32 min (98.6%); [α]18 37.0 (c
D
0.55, CH3OH). UV (MeOH) λmax (log ε) 235.0 (3.97) nm. IR (KBr)
νmax 3443, 2962, 2929, 2874, 2857, 1724, 1602, 1497, 1468, 1454,
1370, 1272, 1239, 1174, 1120, 1071, 1027, 981, 917, 747, 706 cm−1.
1H NMR (acetone-d6, 500 MHz) δ 8.01 (d, J = 7.0 Hz, 2H), 7.62 (t, J
= 7.0 Hz, 1H), 7.50 (t, J = 7.5 Hz, 2H), 7.45 (d, J = 7.5 Hz, 2H), 7.34
(t, J = 6.5 Hz, 2H), 7.25 (t, J = 7.0 Hz, 1H), 6.37 (d, J = 9.0 Hz,1H),
6.26−6.12 (overlap, 2H), 5.70 (br s, 1H), 5.50 (s, 1H), 5.12 (br s,
1H), 5.03 (s, 1H), 4.96 (br s, 1H), 4.60 (br s, 1H), 3.06 (d, J = 9.0 Hz,
1H), 2.73−1.70 (m, 4H), 2.06 (s, 3H), 1.98 (s, 3H), 1.93 (s, 3H), 1.91
(s, 3H), 1.37 (s, 9H), 1.28 (s, 3H), 1.19 (s, 3H), 1.17 (s, 3H). 13C
NMR (acetone-d6, 125 MHz) δ 206.8 s, 174.4 s, 171.7 s, 170.8 s, 170.4
s, 170.4 s, 166.7 s, 162.1 s, 148.4 s, 141.4 s, 133.9 d, 131.4 s, 130.6 d,
129.4 d, 129.1 d, 128.1 d, 128.0 d, 116.1 t, 76.1 s, 75.3 d, 74.9 d, 71.8 d,
70.8 d, 70.1, d, 69.2 d, 63.3 s, 57.5 d, 55.6 s, 46.4 d, 45.1 s, 44.6 t, 33.5
t, 29.1 q, 28.6 q, 27.3 q, 21.7 q, 21.0 q, 20.9 q, 15.2 q, 9.5 q. HRMS
(ESI, m/z) calcd for C47H57NO15Na [M + Na]+, 898.3625; found,
898.3628.
1
1368, 1272, 1241, 1042, 990, 711 cm−1. H NMR (acetone-d6, 500
MHz) δ 8.02−8.00 (overlap, 3H), 7.97 (d, J = 7.5 Hz, 2H), 7.62 (t, J =
7 Hz, 1H), 7.56−7.48 (overlap, 7H), 7.38 (t, J = 8.0 Hz, 2H), 7.29 (t, J
= 7.5 Hz, 1H), 6.12 (br s, 1H), 5.85 (s, 1H), 5.80 (s, 1H), 5.77 (t, J =
7.5 Hz, 1H), 5.55 (s, 1H), 5.15 (br s, 1H), 5.24 (br s, 1H), 4.82 (s,
1H), 3.37 (d, J = 7.5 Hz, 1H), 2.61−2.54 (overlap, 2H), 2.34 (m, 1H),
2.05 (s, 3H), 1.29 (s, 3H), 1.14 (s, 3H), 1.96 (s, 3H), 1.92 (s, 3H),
2.34−1.74 (m, 4H), 0.96 (s, 3H). 13C NMR (acetone-d6, 125 MHz) δ
198.3 s, 173.2 s, 170.5 s, 170.4 s, 170.4 s, 167.4 s, 166.3 s, 147.7 s,
141.0 s, 141.0 s, 144.9 s, 135.7 s, 133.9 d, 132.4 d, 131.4 s, 130.6 d,
129.4 d, 129.3 d, 129.2 d, 128.8 t, 128.4 d, 128.2 d, 128.2 d, 82.2 d,
75.8 s, 75.4 d, 74.8 d, 71.0 d, 70.6 d, 70.1 d, 69.8 s, 56.5 d, 43.9 s, 42.9
d, 38.3 t, 39.4 t, 28.9 q, 28.2 q, 22.1 q, 21.0 q, 21.0 q, 13.1 q, 12.1 q.
HRMS (ESI, m/z) calcd for C49H53NO14Na, 902.3363 [M + Na]+;
found, 902.3359.
Conversion of Compound 5 into Compounds 12 and 10.
Coupling of compounds 8 (32.8 mg, 0.10 mmol) and 5 (50 mg, 0.08
mmol) using NaHMDS (0.05 mL, 0.10 mmol, 2 M in THF) was
performed with the same procedure as for 9. Subsequent removal of
the protecting groups at C-2′ with 0.5 N HCl (15 mL) gave 12 (18
mg, 25%) and 10 (23 mg, 34%).
Synthesis of Compounds 16 and 17. Compounds 16 and 17 were
prepared from 15 following the procedures described in our previous
paper.14
4.2. NCI-60 Human Tumor Cell Line Screening. Compounds
were evaluated in the NCI’s human tumor 60-cell line screening, and
data calculations were performed as described.15
Compound 12. Colorless oil. tR− HPLC, 19.31 min (98.1%);
[α]18D 16.3 (c 0.24, CH3OH). UV (CH3OH) λmax (log ε) 228.4 (3.60)
nm. IR (KBr) νmax 3433, 2960, 2925, 2874,2853, 1729, 1629, 1466,
4.3. Apoptosis Assay. HCT-116 cells were treated with increasing
amounts of compound 11 or 12 for 24 h, washed with phosphate
buffered saline (PBS), and after trypsinization, apoptosis was evaluated
using annexin V-fluorescein 5-isothiocyanate (annexin V-FITC)/
propidium iodine (PI) staining with an Annexin V-FITC apoptosis
detection kit (BD Biosciences, San Jose, USA). Samples were analyzed
by flow cytometry using FACSCalibur and CellQuest software
(Becton Dickinson). Co-staining with Annexin V and PI allows
classification of viable cells (Annexin V-negative, PI-negative) from
early apoptotic cells (Annexin V-positive, PI-negative) and late
apoptotic or necrotic cells (Annexin V-positive, PI-positive).
4.4. Tubulin Polymerization Assay. The effects of compounds
on tubulin polymerization were determined by tubulin polymerization
assay kit (Cytoskeleton, Denver, CO, USA). Standards and tested
compounds were mixed with tubulin according to manufacturer’s
instructions. Fluorescence was detected by a temperature regulated
fluorimeter capable of reading at 410−460 nm in kinetic mode using
excitation filters of 340−360 nm. The resulting polymerization curve is
representative of the three phases of microtubules polymerization,
namely nucleation, growth, and steady state equilibrium. Compounds
or proteins that interact with tubulin will often alter one or more of the
characteristic phases of polymerization.
4.5. Immunofluorescence Staining of Tubulin. PC-3 cells were
grown on 8-well chamber slide (Lab-Tech) for 36 h prior to treatment
with DMSO (control), 2 or 10 μM compound 11, 5 or 10 μM
compound 12, 200 nM CA4, or 200 nM PXL for 24 h. Cells were fixed
in 4% paraformaldehyde in PBS, and permeabilized with 0.5% Triton
X-100 in PBS. Cells were labeled with mouse monoclonal antibody to
α-tubulin (B5−1−2, Sigma), followed by FITC conjugated secondary
antibody (Sigma).17 Nuclei were labeled with DAPI. Fluorescence
labeled cells were observed using an Olympus BX61 fluorescence
microscope, and images were captured by an ORCA-R2 digital camera
(Hamamatsu Photonics) controlled by Volocity 5.5.2 software
(Perkin-Elmer Inc.) (Microscopy Service Laboratory, UNC−CH).
Final images were prepared using Adobe Photoshop.
1
1369, 1273, 1247, 1124, 1073, 745, 707 cm−1. H NMR (acetone-d6,
500 MHz) δ 8.03 (d, J = 8.0 Hz, 2H), 7.62 (t, J = 7.5 Hz, 1H), 7.50 (t,
J = 7.5 Hz, 2H), 7.45 (d, J = 7.5 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.28
(t, J = 7.0 Hz, 1H), 6.40 (d, J = 10.0 Hz, 1H), 6.15 (br s, 1H), 5.92 (d,
J = 7.5 Hz, 1H), 5.87−5.85 (overlap, 2H), 5.59 (s, 1H), 5.31 (d, J =
10.0 Hz, 1H), 5.24 (m, 1H), 4.53 (br s, 1H), 3.44 (d, J = 7.5 Hz, 1H),
2.57−1.92 (m, 4H), 2.05 (s, 3H), 1.96 (s, 3H), 1.93 (s, 3H), 1.90 (s,
3H), 1.45 (s, 9H), 1.29 (s, 3H), 1.19 (s, 3H), 1.00 (s, 3H). 13C NMR
(acetone-d6, 125 MHz) δ 198.3 s, 173.1 s, 170.7 s, 170.5 s, 170.3 s,
167.7 s, 165.8 s, 147.9 s, 145.2 s, 142.5 s, 136.3 s, 133.9 d, 131.5 s,
130.7 d, 129.4 d, 129.2 d, 128.2 d, 128.0 d, 126.5 t, 82.2 d, 75.8 s, 75.0
d, 74.9 d, 71.1 d, 70.8 d, 70.2 d, 66.9 s, 57.5 d, 55.4 s, 44.0 s, 43.0 d,
39.5 t, 38.2 t, 29.1 q, 28.8 q, 28.3 q, 22.3 q, 21.1 q, 21.0 q, 13.2 q, 12.0
q. HRMS (ESI, m/z) calcd for C47H57NO15Na [M + Na]+, 898.3625;
found, 898.3624.
Synthesis of Compound 13. Using the same procedure as for 9,
coupling of compounds 7 (66 mg, 0.20 mmol) and 2 (100 mg, 0.16
mmol) was performed with NaHMDS (0.10 mL, 0.20 mmol).
Removal of the C-2′ protecting groups with 0.5 N HCl (37 mL) gave
13 (73 mg, 51%) as colorless oil. tR− HPLC, 17.37 min (96.9%);
[α]18 −22.9 (c 0.37, CH3OH). UV (CH3OH) λmax (log ε) 228.4
D
(3.80) nm. IR (KBr) νmax 3430, 2958, 2926, 2872, 2855, 1732, 1465,
1374, 1376, 1270, 1252, 1221, 1173, 1122, 1072, 1029, 746, 711 cm−1.
1H NMR (acetone-d6, 500 MHz) δ 7.91 (d, J = 7.5 Hz, 2H), 7.64 (t, J
= 7.2 Hz,1H), 7.51 (t, J = 7.5 Hz, 2H), 7.45 (d, J = 7.5 Hz, 2H), 7.34
(t, J = 7.0 Hz, 2H), 7.25 (t, J = 7.5 Hz, 1H), 6.63 (d, J = 10.5 Hz,1H),
6.55 (br s, 1H), 6.19 (d, J = 8.0 Hz 1H), 6.07 (d, J = 10.5 Hz,1H), 5.55
(m, 1H), 5.34 (s, 1H), 4.96 (s, 1H), 4.72−4.69 (overlap, 3H), 3.41 (d,
J = 8.0 Hz, 1H), 2.60−2.46 (m, 2H), 2.16 (s, 3H), 2.14−2.08 (m, 2H),
2.05 (s, 3H), 2.02 (s, 3H), 1.78 (s, 3H), 1.40 (s, 9H), 1.29 (s, 3H),
1.19 (s, 3H), 1.07 (s, 3H). 13C NMR (acetone-d6, 125 MHz) δ172.1 s,
171.8 s, 170.4 s, 170.4 s, 170.0 s, 165.0 s, 153.8 s, 141.5 s, 141.5 s,
134.3 d, 134.1 s, 130.3 s, 130.3 d, 129.7 d, 129.1 d, 128.1 d, 128.0 d,
115.7 t, 77.7 d, 77.2 d, 77.0 d, 75.8 s, 75.6 d, 70.9, d, 69.6 d, 69.3 s, 68.4
d, 58.0 d, 55.6 s, 46.0 s, 44.0 d, 41.1 t, 35.6 t, 28.7 q, 28.3 q, 27.1 q, 22.1
q, 21.5 q, 20.9 q 14.0 q, 12.5 q. HRMS (ESI, m/z) calcd for
C47H59NO15Na [M + Na]+, 900.3782; found, 900.3787.
4.6. Cell Cycle Analysis. Cell cycle was evaluated by measurement
of the DNA content by PI (St Louis, MO) staining. Briefly, HCT-116
cells were seeded in six-well plates at a density of 5 × 105 cells/well 24
h prior to treatment with compounds 11 or 12. After 12 h, cells and
supernatants were collected together, centrifuged at 1000 rpm for 5
min, and pelleted. The pellet was resuspended and fixed with 70%
EtOH overnight at 4 °C. After being stained with PI, cells were
subjected to flow cytometric cell cycle analysis. Experiments were
repeated a minimum of two times.
Oxidation of Compound 13 to 14. PCC (78 mg, 0.36 mmol) in
CH2Cl2 (8 mL) was added to a solution of 13 (53 mg, 0.06 mmol) in
CH2Cl2 (10 mL). The reaction was stirred at rt and monitored by
TLC until the starting material was consumed. The mixture was
filtered and concentrated. The residue was purified by silica gel column
chromatography (40% EtOAc in petroleum ether) to afford 14 (28 g,
4.7. Luciferase Reporter Assay. HEK 293T cells were transiently
transfected with pNFκB-luc (Beyotime) and pRL-TK (Promega,
H
dx.doi.org/10.1021/jm400479p | J. Med. Chem. XXXX, XXX, XXX−XXX