Synthesis of the pyridinyl analogues of dibenzylideneacetone (pyr-dba) via an improved Claisen-Schmidt condensation, displaying diverse biological activities as curcumin analogues

Article abstract of DOI:10.1039/c1ob06773g

An efficient and easy procedure to synthesize the pyridinyl analogues of dibenzylideneacetone (pyr-dba) was developed by the condensation of substituted nicotinaldehyde and acetone in the presence of K₂CO₃ in toluene-EtOH-H₂O solvent system. Structurally diverse pyr-dba, including quinolinyl dba, can be prepared conveniently in moderate to excellent yields under mild conditions with this method. The resulting pyr-dba functioned as the enone analogs of curcumin and efficiently inhibited the activation of NF-κB and the growth of colorectal carcinoma HCT116 p53+/+ cells as well as the HIV-1 IN-LEDGF/p75 interaction. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c1ob06773g

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry
This journal is © The Royal Society of Chemistry 2011
Synthesis of the pyridinyl analogues of dibenzylideneacetone
(pyr-dba) via an improved Claisen-Schmidt condensation,
displaying diverse biological activities as curcumin analogues
Bin Cao, Yong Wang, Kan Ding, Nouri Neamati and Ya-Qiu Long
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai
Institutes for Biological Sciences, CAS, 555 Zuchongzhi Road, Shanghai 201203, China
yqlong@mail.shcnc.ac.cn
Table of Contents
S1
Title Page
S2-S7
Copies of 1H spectra of the starting material 6b-c, 6e-j,
6m-n.
1
S8-S21
Copies of H and 13C NMR spectra of the final
products 7a-n.
S22-S25
The biological assays including screening inhibitors of
NF-κB based TLR4 signal pathway, cytotoxicity assay,
and the LEDGF/p75-IN AlphaScreen assay.
 To whom correspondence should be addressed. Tel: 86-21-50806876; Fax: 86-21-50807088.
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Screening inhibitors of NF-κB based TLR4 signal pathway
1. Cell lines
293/ NF-κB-Luc: incubated with DMEM medium,10% Gibco FBS
Hek293 stably transfected with NF-κB binding DNA fragment recombinant
luciferase reporter system.
2. Materials
Lipopolysaccharides from Escherichia coli 055:B5 (LPS, Sigma L6529-1MG)
Stock solution:1 mg/ml diluted in sterile PBS, stored at -20℃
Work solution: dilute the stock solution into cell culture medium to 10μg/ml
Compound library
3. Methods
1) Seed 293/ NF-κB-Luc cells in 96 well plate,3×105/ml, 100μl/well, incubate
at 37℃, 5% CO2 overnight
2) Add screening compound solution 100μl to each well, incubate for 15
minutes, set BLANK and CONTROL well in the meanwhile
3) Add LPS solution 20μl to get end concentration 1μg/ml, incubate for 6 hrs
4) Lyse the cells using 1×CCLR buffer 20μl/well according to the luciferase
assay system kit, pipette then transfer to 96 well lumitrac plate
5) Add 40μl assay substrate per well, read RLU immediately among 10
minutes
6) Calculate the inhibition rate of the compound
4. Results：
Fig1-6 shows the inhibition rate of compounds 7a-n (LCX-94, LCX-95, LCX-99,
LCX-100, LCX-101, LCX-102, LCX-103, LCX-104, LCX-105, LCX-109, LCX-110,
LCX-111, LCX-112, LCX-113) to 293/ NF-κB-Luc cells at 100uM, 10uM, 1uM,
0.1uM, 0.01uM working concentration.
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NF-κB assay of 293/NF-κB-luc
120.00
100.00
80.00
60.00
40.00
20.00
0.00
0.01uM
0.1uM
1uM
10uM
100uM
LCX- LCX- LCX- LCX- LCX- LCX- LCX- LCX- LCX- LCX- LCX- LCX- LCX- LCX-
94 95 99 100 101 102 103 104 105 109 110 111 112 113
-20.00
-40.00
-60.00
Fig 1
NF-κB assay of 293/NF-κB-luc
50.00
40.00
30.00
20.00
10.00
0.00
0.01uM
LCX-94 LCX-95 LCX-99 LCX-100 LCX-101 LCX-102 LCX-103 LCX-104 LCX-105 LCX-109 LCX-110 LCX-111 LCX-112 LCX-113
-10.00
-20.00
-30.00
-40.00
Fig 2
NF-κB assay of 293/NF-κB-luc
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0.1uM
LCX-94 LCX-95 LCX-99 LCX-100 LCX-101 LCX-102 LCX-103 LCX-104 LCX-105 LCX-109 LCX-110 LCX-111 LCX-112 LCX-113
-10.00
-20.00
Fig 3
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NF-κB assay of 293/NF-κB-luc
120.00
100.00
80.00
60.00
40.00
20.00
0.00
1uM
LCX-94 LCX-95 LCX-99 LCX-100 LCX-101 LCX-102 LCX-103 LCX-104 LCX-105 LCX-109 LCX-110 LCX-111 LCX-112 LCX-113
-20.00
Fig 4
NF-κB assay of 293/NF-κB-luc
10uM
120.00
100.00
80.00
60.00
40.00
20.00
0.00
LCX-94 LCX-95 LCX-99 LCX-100 LCX-101 LCX-102 LCX-103 LCX-104 LCX-105 LCX-109 LCX-110 LCX-111 LCX-112 LCX-113
Fig 5
NF-κB assay of 293/NF-κB-luc
120.00
100.00
80.00
60.00
40.00
20.00
0.00
100uM
LCX-94 LCX-95 LCX-99 LCX-100 LCX-101 LCX-102 LCX-103 LCX-104 LCX-105 LCX-109 LCX-110 LCX-111 LCX-112 LCX-113
Fig 6
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Cytotoxicity assay. (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), was used to assess cytotoxicity. HCT116 colon cancer cells were seeded in
96-well microtiter plates and allowed to attach overnight. Cells were subsequently
treated with continuous exposure to corresponding drugs for 72 h. An MTT solution
(final concentration of 0.5 mg/mL) was added to each well and cells were incubated
for 4 h at 37°C. After removal of the supernatant, DMSO was added and the
absorbance was read at 570 nm. The IC50 was then determined for each drug from a
plot of log (drug concentration) versus percentage of cells killed.
LEDGF/p75–IN AlphaScreen assay. The AlphaScreen assay was performed
according to the manufacturer’s protocol (Perkin–Elmer, Benelux). Reactions were
performed in 25 μl final volume in 384-well Optiwell microtiter plates (Perkin-Elmer).
The reaction buffer contained 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2,
0.01% (v/v) Tween-20 and 0.1% (w/v) bovine serum albumin. His6-tagged IN (300
nM final concentration) was incubated with the inhibitory compound for 30 min at
4°C. The compounds were added in varying concentrations spanning a wide range
from 0.1 μM up to 100 μM. Afterwards, 100 nM Flag-LEDGF/p75 was added and
incubation was prolonged for an additional hour at 4°C. Subsequently, 5 μl of
Ni-chelate-coated acceptor beads and 5 μl anti-Flag donor beads were added to a final
concentration of 20 μg/ml of both beads. Proteins and beads were incubated for 1 h at
30°C in order to allow association to occur. Exposure of the reaction to direct light
was omitted as much as possible and the emission of light from the acceptor beads
was measured in the EnVision plate reader (Perkin-Elmer) and analyzed using the
EnVision manager software.
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