N
A
T
U
R
E
C
O
M
M
UN
I
CA
T
I
O
NS
|
D
OI
:
1
0.
1
0
3
8
/
s
4
1
46
7
-
01
8
-
0
44
6
5
-
5
A R T I C L E
was passed through a plug of silica with EtOAc as the eluent. The organic material
was dried over Na2SO4 and concentrated to give a dark brown solid that was
adsorbed onto silica gel using DCM. Material purified by flash column
chromatography (SiO2, 20% EtOAc/hexanes) to afford 4′-fluoroisoflavone (8.14 g,
67% yield) as a yellow–orange solid that showed minor impurities by 1H NMR
spectroscopy. Slightly impure material was taken onto the next step of the synthesis
without further purification. 1H NMR (500 MHz, CDCl3) δ 8.35 (dd, J = 8.0, 1.6
Hz, 1 H), 8.05 (s, 1 H), 7.73 (ddd, J = 8.7, 7.1, 1.7 Hz, 1 H), 7.61–7.54 (m, 2 H), 7.53
(dd, J = 8.4, 1.1 Hz, 1 H), 7.48 (ddd, J = 8.2, 7.0, 1.1 Hz, 1 H), 7.17 (ap t, J = 8.7 Hz,
2 H); 13C NMR (126 MHz, CDCl3): δ 176.2, 163.8, 161.8, 156.2, 152.9, 133.8, 130.7,
127.8, 126.4, 125.4, 124.5, 118.1, 115.5; UPLCMS: mass calculated for C15H9FO2,
[M + H]+, 241. Found 241.
modulation of the signature of client protein binding to chaper-
one scaffold proteins, in turn resulting in highly selective func-
tional effects at the cellular and systemic level. In parallel, this
approach serves to inform us about novel pathways for regulating
critical biological processes. Finally, our approach that coupled
efficient chemical synthesis routes with a well-designed pheno-
typic screening strategy has the potential to be broadly applied as
a tool to interrogate other critical biological processes.
Methods
Chemical synthesis. Procedure for Large-Scale Production of 4′-fluoroiso-
flavanone (KBU2046):
4′-fluoroisoflavanone (KBU2046).
3-(dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-one.
The reaction conditions to synthesize 4′-fluoroisoflavanone on large scale were
adapted from a procedure reported by Wähälä26. To a flame-dried 500 mL round
bottom flask was added 4′-fluoroisoflavone (25 mmol, 6.01 g), and the solid was
dissolved in dry THF (100 mL). The solution was cooled to −78 °C (dry ice/acetone
bath), monitored by a thermocouple. Once the solution had cooled to the desired
temperature, L-selectride (55 mmol, 55 mL, 1 M solution in THF) was added
dropwise over a period of 30–45 min. The reaction was then allowed to stir at −78 °
C for 2 h, after which time it was quenched with MeOH (55 mL) at −78 °C. The
mixture was then poured into 300 mL of water, and the aqueous layer was adjusted
to pH 7 with 2 M HCl. The aqueous layer was extracted 2 × 200 mL with EtOAc,
then the combined organic layers were dried over Na2SO4 and concentrated to give
a dark brown oily solid. This was purified by flash column chromatography (SiO2,
1:1 hexanes:DCM) to give 4.5 g of crude material that was recrystallized in hexanes
to afford 4′-fluoroisoflavanone (3.4 g, 56%) as a fluffy white solid. It was checked for
purity by both 1H NMR and HPLC analysis, with material that was >98% pure
taken onto animal studies. Analytical data for isoflavanone 4′-fluoroisoflavanone:
1H NMR (500 MHz, CDCl3) δ 7.98 (dd, J = 7.9, 1.7 Hz, 1 H), 7.55 (ddd, J = 8.6, 7.1,
1.7 Hz, 1 H), 7.33–7.24 (m, 1 H), 7.14–6.99 (m, 4 H), 4.77–4.54 (m, 2 H), 4.02 (dd, J
= 9.0, 5.3 Hz, 1 H); 13C NMR (126 MHz, CDCl3) δ 192.0, 163.3, 161.5, 136.2, 130.7,
130.2, 127.8, 121.8, 120.9, 117.9, 115.8, 71.4, 51.5; UPLCMS: mass calculated for
The starting materials 2′-hydroxyacetophenone (50 mmol, 6.02 mL) and N,N-
dimethylformamide dimethyl acetal (50 mmol, 6.64 mL) were added to a 10–20 mL
microwave vial. The vial was capped and heated in a Biotage Initiator microwave
synthesizer at 150 °C and 11 bar for 10 min. The resulting dark orange liquid was
allowed to cool to 23 °C, at which time yellow–orange crystals crashed out of solution.
The crystals were collected and washed with hexanes (50 mL), then dried and weighed
to give 3-(dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-one (9.09 g, 95%) as
orange–yellow needles. Analytical data for 3-(dimethylamino)-1-(2-hydroxyphenyl)
prop-2-en-1-one: 1H nuclear magnetic resonance (NMR) (500 MHz, CDCl3) δ 13.97
(s, 1 H), 7.92 (d, J = 12.1 Hz, 1 H), 7.72 (dd, J = 8.0, 1.6 Hz, 1 H), 7.38 (ddd, J = 8.5,
7.2, 1.6 Hz, 1 H), 6.96 (dd, J = 8.3, 1.2 Hz, 1 H), 6.85 (ddd, J = 8.3, 7.2, 1.2 Hz, 1 H),
5.81 (d, J = 12.1 Hz, 1 H), 3.23 (s, 3 H), 3.01 (s, 3 H); 13C NMR (126 MHz, CDCl3): δ
191.5, 163.0, 154.8, 134.0, 128.2, 120.3, 118.3, 118.0, 90.1, 45.5, 37.5; ultra performance
liquid chromatography/mass spectrometry (UPLCMS): mass calculated for
C
11H13NO2, [M + H]+, 192. Found 192.
3-bromochromone.
C
15H11FO2, [M + H]+, 243. Found 243.
Synthesis of additional compounds. A series of related analog compounds was
3-bromochromone was prepared by a procedure taken from Gammill23. To a
synthesized in addition to the parent 4′-fluoroisoflavanone (KBU2046). These
compounds were prepared in the same general manner of KBU2046 and the
structures of each such compound are depicted in supplementary figures. The
structure and purity of the additional analogs were confirmed by NMR
spectroscopy (1H and 13 C) as well as by UPLCMS (minimal ion fragmentation).
All compounds were isolated and stored in powdered form (in the absence of light)
and were formulated into DSMO stock solutions just prior to use.
flame-dried 250 mL round bottom flask, was added 3-(dimethylamino)-1-(2-
hydroxyphenyl)prop-2-en-1-one (36.6 mmol, 7.0 g), which was dissolved in CHCl3
(70 mL). The reaction flask was cooled to 0 °C in an ice bath, then Br2 (36.6 mmol,
1.87 mL) was added dropwise through an addition funnel. After all of the Br2 was
added, water (70 mL) was added slowly to the reaction and it was stirred at 23 °C
for 10 min. The dark orange–yellow organic layer was then separated from the
aqueous layer, which was back-extracted with 3 × 50 mL CHCl3. The combined
organic layers were then dried over Na2SO4 and concentrated to give a dark orange
oil. This was purified by flash column chromatography (SiO2, 10% EtOAc/hexanes)
to afford 3-bromochromone (5.26 g, 64%) as an off-white solid. Analytical data for
3-bromochromone: 1H NMR (500 MHz, CDCl3) δ 8.31 (dd, J = 8.0, 1.7 Hz, 1 H),
8.27 (s, 1 H), 7.75 (ddd, J = 8.7, 7.1, 1.7 Hz, 1 H), 7.57–7.44 (m, 2 H); 13C NMR
(126 MHz, CDCl3): δ 172.3, 156.1, 153.8, 134.2, 126.5, 125.9, 123.2, 118.2, 110.7;
UPLCMS: mass calculated for C9H5BrO2, [M + H]+, 226. Found 226.
Palladium tetrakis (triphenylphosphine) (Pd(PPh3)4). The catalyst for the
Suzuki-Miyaura cross-coupling reaction to synthesize 4′-fluoroisoflavone was
made using a procedure by Coulson24. To a flame-dried 100 mL Schlenk flask was
added PdCl2 (5 mmol, 890 mg) and triphenylphosphine (25 mmol, 6.56 g). The
solids were dissolved in DMSO (60 mL), then the mixture was purged with N2 and
heated to 145 °C, at which time it turned a bright yellow–orange color. The reaction
was removed from heat and allowed to stir at room temperature for 15 min, then
hydrazine hydrate (20 mmol, 0.972 mL) was added via syringe, with a vent needle
in place to account for the formation of N2 gas. After the hydrazine hydrate had
been added, the reaction was cooled to 23 °C, during which time a yellow solid
crashed out of solution. The solid was washed under Schlenk filtration conditions
with 2 × 50 mL EtOH, then 2 × 50 mL ether to yield Pd(PPh3)4 (5.31 g, 94%) as a
canary yellow solid that was stored under N2 in the glovebox.
Cell culture and reagents. PCa (PC3, LNCaP, and DU145), breast cancer (MDA-
MB-231 and MCF-7), colon cancer (HCT110 and HT29), and lung cancer cells
(H226 and A549) were obtained from American Type Culture Collection and not
further authenticated. The origin, characteristics, for PC3-M, as well as for human
papilloma virus (HPV) transformed primary 1532NPTX (normal), 1532CPTX
(cancer), 1542NPTX (normal), and 1542CP3TX (cancer) cell lines, have previously
been described by us27. The origin of the stable polyclonal HEK293T cell lines
expressing Renilla-HSP90β were previously described16. LM2-4H2N human breast
cancer metastatic variant cells were derived from MDA-MB-231 cells as descri-
bed28, and the tdTomato-Luc2-expressing cell line was established by transduction
of these cells with a lentiviral vector encoding fluorescent (tdTomato) and biolu-
minescent (Luc2) genes as described29. All cells were cultured as described27,28
,
were maintained at 37 °C in a humidified atmosphere of 5% carbon dioxide with
biweekly media changes, were drawn from stored stock cells, and replenished on a
standardized periodic basis and were routinely monitored for Mycoplasma (Plas-
moTest™, InvivoGen, San Diego, CA), at least every 3 months. Cells were
authenticated by the following: they were acquired from the originator of that line,
grown under quarantine conditions, expanded and stored as primary stocks and
not used until following conditions were met: mycoplasma negative; through
morphologic examination; growth characteristics; hormone responsiveness or lack
thereof, when applicable; replenished from primary stocks at least every 3 months;
working with a single primary stock cell line at a time with hood sterilization in
between.
4′-fluoroisoflavone.
Phospho-HSP27 (catalog #2401), phospho-p38 MAPK (#4631), p38 MAPK
(#9212), phospho-CK2 substrate (#8738), CDC37 (#3618), HSP90β (#5087), GST
(#2622), phsopho-c-RAF (ser338) (#9427), SGK3 (#8573), GAPDH (#2118), anti-
mouse IgG-HRP linked secondary (#7076), and anti-rabbit IgG-HRP linked
secondary (#7074) antibodies were purchased from Cell Signaling Technology.
MAP3K6 (#SAB1300114) antibody, estradiol, and 4′,5,7-trihydroxyisoflavone,
genistein, were purchased from Sigma-Aldrich. Pierce ECL western blotting
substrate (#32106) and SuperSignal West Femto maximum sensitivity substrate
(#34096) were purchased from Thermo Scientific. All primary antibodies were used
at a dilution of 1:1000 and corresponding secondary antibodies used at a dilution of
4′-fluoroisoflavone was prepared on large scale according to a procedure from
Suzuki and Miyaura25. To a flame-dried 500 mL round bottom flask was added 3-
bromochromone (50 mmol, 11.25 g), 4-fluorophenylboronic acid (55 mmol, 7.69
g), and Na2CO3 (100 mmol, 10.6 g). The solids were dissolved in a mixture of
benzene (100 mL) and water (50 mL), and the system was purged with N2 for 10-
15 min. The Pd(PPh3)4 catalyst (2.5 mmol, 2.89 g) was then added, at which time
the reaction turned a bright orange. The flask was equipped with a reflux condenser
and the reaction was heated to reflux (80 °C) overnight. After ~16 h, the reaction
was cooled to 23 °C and was diluted with EtOAc (250 mL), then the crude material
N A T U R E C OM M U N I C A T I ON S
( 2 0 1 8 ) 9 :2 4 5 4
|
|
|
Xu, Li
