Journal of Natural Products
Note
Alltech Davisil silica 30−40 μm 60 Å was used for preadsorption of the
C-12), 170.3 (C-8); (−)-LRESIMS m/z 292 (100), 294 (30) [M −
−
+
crude reaction products and was packed into an open glass column (35
H] ; (+)-LRESIMS m/z 294 (100), 296 (30) [M + H] ;
−
×
70 mm) for silica gel flash chromatography. X-ray diffraction data
(−)-HRESIMS m/z 292.0548 [M − H] (calcd for C H ClFNO ,
1
5
12
2
were collected on an Oxford-Diffraction Gemini S Ultra CCD
diffractometer utilizing CrysAlis software. All solvents used for
chromatography, UV, and MS were Lab-Scan HPLC grade, and the
292.0546).
2-(3-Chloro-4-hydroxyphenyl)-N-(2-fluorobenzyl)acetamide (14):
off-white solid (11.4 mg, 16%); UV (MeOH) (log ε) 231 (3.86), 282
H O was Millipore Milli-Q PF filtered. All the reagents were
(3.40) nm; IR ν 1609, 1542, 1494, 1419, 1284, 1236, 1199, 1056
2
max
−
1 1
purchased from Sigma-Aldrich and used without further purification.
Synthesis of Methyl (3-Chloro-4-hydroxyphenyl)acetate. 3-
Chloro-4-hydroxyphenylacetic acid (1, 1.5 g, 0.0075 mol, Sigma-
Aldrich, 99%) was dissolved in anhydrous MeOH (1 mL), a crystal of
pTsOH was added, and the mixture was stirred at room temperature
cm ; H NMR (600 MHz, DMSO-d ) δ 3.37 (2H, s, H-7), 4.30
6 H
(2H, d, J = 6.0 Hz, H-9), 6.88 (1H, d, J = 8.2 Hz, H-5), 7.02 (1H, dd, J
= 8.2, 2.2 Hz, H-6), 7.14 (1H, overlap, H-14), 7.15 (1H, overlap, H-
12), 7.23 (1H, d, J = 2.2 Hz, H-2), 7.28 (1H, overlap, H-15), 7.30 (1H,
overlap, H-13), 8.45 (1H, t, J = 6.0 Hz, NH-8), 9.96 (1H, br s, OH-4);
4
0,41
13
3
for 48 h.
Excess solvent was removed under N , and the resulting
C NMR (150 MHz, DMSO-d ) δ 36.0 (d, J = 4.6 Hz, C-9), 40.8
2
6 C CF
2
crude mixture was partitioned between CH Cl (2 × 100 mL) and
(C-7), 115.0 (d, J = 20.6 Hz, C-12), 116.4 (C-5), 119.2 (C-3),
CF
124.2 (d, J = 3.4 Hz, C-14), 125.9 (d, J = 15.2 Hz, C-10), 127.9
(C-1), 128.5 (C-6), 128.7 (d, J = 8.1 Hz, C-13), 130.1 (C-2), 129.5
CF
(d, J = 4.5 Hz, C-15), 151.6 (C-4), 160.0 (d, J = 244.5 Hz, C-
2
2
4
2
saturated NaHCO3 (100 mL). The CH Cl2 layer was dried over
2
CF CF
3
anhydrous MgSO . Then the solvent removed under reduced pressure
4
3
1
to yield methyl (3-chloro-4-hydroxyphenyl)acetate (2, 1.43 g, 95%) as
C
F
C
F
−
a pale brown gum. NMR and MS data were consistent with literature
11), 170.2 (C-8); (−)-LRESIMS m/z 292 (100), 294 (30) [M − H] ;
42
+
values.
(+)-LRESIMS m/z 294 (100), 296 (30) [M + H] ; (−)-HRESIMS
−
Generation of the Amide Library. Methyl (3-chloro-4-
hydroxyphenyl)acetate (2, 50 mg, 0.25 mmol) and the relevant
primary amine (0.5 mL) were added together and stirred for 16 h at
m/z 292.0549 [M − H] (calcd for C H ClFNO , 292.0546).
15
12
2
Cellular Lipid Content Analysis. LNCaP and PC-3 cells were
obtained from the American Type Cell Culture Collection. Both cell
lines were seeded in optical 96-well plates (ibidi) at 4000 cells/well
and 3000 cells/well, respectively, in phenol-red-free RPMI-1640
medium (Thermo Fisher Scientific) supplemented with 5% CSS
(Thermo Fisher Scientific) for 72 h. The evaluation of the cellular lipid
room temperature. The reaction mixture was dried under N and then
2
high vacuum before being redissolved in CH Cl −MeOH (1:1) and
2
2
preadsorbed to silica (∼1 g) overnight. Two different purification
conditions were used during these studies. The first method was
applied to the neutral analogues (3−15) and included the preadsorbed
material being dry packed onto a CH Cl equilibrated silica flash
4
3
content was performed as previously described. Briefly, after
treatment for 48 h with the indicated compounds at 10 μM in 0.3%
DMSO, the medium was removed, and cells were fixed in 4%
paraformaldehyde for 20 min on ice. Cells were then stained for 20
min at room temperature protected from light in 100 μL of PBS
containing 1.0 μg/mL DAPI [2-(4-amidinophenyl)-1H-indole-6-
carboxamidine, Sigma-Aldrich] and 0.25 μg/mL Nile Red (Sigma-
Aldrich). Images in the DAPI, Cy3, and Cy5 channels were acquired
on an INCell Analyzer 2200 system (GE Healthcare) at 10×
magnifications. Image segmentation and quantitation of cellular
mean fluorescence intensities of phospholipids (Cy5) and neutral
lipids (Cy3) of ∼1500 cells per treatment were performed with
2
2
column (35 × 70 mm) and subsequently flushed using CH Cl (100
2
2
mL), 95:5 CH Cl −MeOH (100 mL), 90:10 CH Cl −MeOH (100
2
2
2
2
mL), and 80:20 CH Cl −MeOH (100 mL). The second method was
2
2
applied to the basic analogues (16−22) whereby the preadsorbed
material was dry packed onto a CH Cl equilibrated silica flash column
2
2
and subsequently flushed using 100:1 CH Cl −TEA (101 mL), 95:5:1
2
2
CH Cl −MeOH−TEA (101 mL), 90:10:1 CH Cl −MeOH−TEA
2
2
2
2
(
101 mL), and 80:20:1 CH Cl −MeOH−TEA (101 mL). For both
2
2
purification methods, 10 fractions were collected from each eluent
flush and then analyzed by both TLC and H NMR spectroscopy with
1
4
4,45
only high-purity fractions combined and added to the screening
CellProfiler software (Broad Institute).
Statistical significance was
library. The physical and spectroscopic data of compounds 3−11 and
analyzed with GraphPad Prism (GraphPad Software) by one-way
ANOVA with Dunnett’s multiple comparison test. Control cells were
treated with the equivalent dose of DMSO (negative control) or
TOFA [5-(tetradecyloxy)-2-furoic acid, Sigma-Aldrich] as the positive
control.
1
5−22 are summarized in the Supporting Information (S44).
2
-(3-Chloro-4-hydroxyphenyl)-N-(4-fluorobenzyl)acetamide (12):
light brown needles (45.2 mg, 66%); mp 175−178 °C; UV (MeOH)
(
1
3
log ε) 233 (3.81), 282 (3.39) nm; IR ν 1607, 1592, 1538, 1509,
421, 1210, 1157, 1058 cm ; H NMR (600 MHz, DMSO-d ) δ
6 H
.38 (2H, s, H-7), 4.25 (2H, d, J = 6.0 Hz, H-9), 6.91 (1H, d, J = 8.3
max
−1 1
ASSOCIATED CONTENT
■
Hz, H-5), 7.03 (1H, dd, J = 8.3, 2.1 Hz, H-6), 7.12 (2H, m, H-12, H-
*
S
Supporting Information
1
4), 7.24 (1H, d, J = 2.1 Hz, H-2), 7.26 (2H, dd, J = 8.2, 5.9 Hz, H-11,
1
13
13
H and C NMR spectra for compounds 2−22, physical and
H-15), 8.47 (1H, t, J = 6.0 Hz, NH-8), 9.96 (1H, br s, OH-4);
C
spectroscopic data for compounds 3−11 and 15−22, structures
and physicochemical properties of virtual analogues VA1−
VA25, physicochemical properties of 1−22, X-ray crystallog-
raphy data for 8, 12, and 22, ORTEP drawings for 8 and 22,
cellular lipid content analysis data for PC-3 cells, and protocols
NMR (150 MHz, DMSO-d ) δ 41.0 (C-7), 41.6 (C-9), 114.9 (2C, d,
6
C
2
JCF = 20.8 Hz, C-12, C-14), 116.4 (C-5), 119.3 (C-3), 128.0 (C-1),
3
1
1
28.6 (C-6), 129.2 (2C, d, J = 8.2 Hz, C-11, C-15), 130.2 (C-2),
CF
4
1
35.6 (d, J = 2.3 Hz, C-10), 151.6 (C-4), 161.2 (d, J = 240.8 Hz,
CF
CF
C-13), 170.2 (C-8); (−)-LRESIMS m/z 292 (100), 294 (30) [M −
−
+
H] ; (+)-LRESIMS m/z 294 (100), 296 (30) [M + H] ;
−
(
−)-HRESIMS m/z 292.0550 [M − H] (calcd for C H ClFNO ,
15
12
2
2
92.0546).
-(3-Chloro-4-hydroxyphenyl)-N-(3-fluorobenzyl)acetamide (13):
brown solid (21.4 mg, 34%); UV (MeOH) (log ε) 232 (3.79), 282
2
AUTHOR INFORMATION
■
Corresponding Author
(
1
3.33) nm; IR νmax 1610, 1542, 1510, 1489, 1420, 1338, 1285, 1242,
−1
1
199, 1143, 1056 cm ; H NMR (600 MHz, DMSO-d ) δ 3.39 (2H,
6 H
s, H-7), 4.28 (2H, d, J = 6.0 Hz, H-9), 6.90 (1H, d, J = 8.2 Hz, H-5),
7
.03 (1H, dd, J = 8.2, 2.2 Hz, H-6), 7.03 (2H, overlap, H-11, H-13),
Notes
7
.06 (1H, d, J = 7.9 Hz, H-15), 7.25 (1H, d, J = 2.2 Hz, H-2), 7.33
The authors declare no competing financial interest.
(
1H, ddd, J = 7.9, 7.9, 6.1 Hz, H-14), 8.50 (1H, t, J = 6.0 Hz, NH-8),
9
7
.95 (1H, br s, OH-4); 13C NMR (150 MHz, DMSO-d ) δ 41.0 (C-
6 C
ACKNOWLEDGMENTS
2
2
■
), 41.7 (C-9), 113.4 (d, J = 20.9 Hz, C-13), 113.7 (d, J = 21.6
CF
CF
4
The authors acknowledge the National Health and Medical
Research Council (NHMRC) for financial support (Grant
APP1024314 to R.A.D. and APP1067728 to V.M.A.) and thank
Hz, C-11), 116.4 (C-5), 119.3 (C-3), 123.1 (d, J = 2.9 Hz, C-15),
CF
3
1
1
28.0 (C-1), 128.6 (C-6) 130.1 (d, J = 9.2 Hz, C-14), 130.2 (C-2),
42.5 (d, J = 7.1 Hz, C-10), 151.6 (C-4), 162.2 (d, J = 241.6 Hz,
CF
3
1
CF
CF
D
J. Nat. Prod. XXXX, XXX, XXX−XXX