Journal of Natural Products
Article
6
5% CH CN−H O (A2143G.1; 8.6 mg), 75% CH CN−H O
in THF (0.3 mL) was added dropwise. The reaction was stirred at −78
°C for 30 min, at which time the dry ice bath was removed and the
reaction was stirred for an additional 1 h. The mixture was diluted in
3
2
3
2
(
A2143G.2; 5.8 mg), 85% CH CN−H O (A2143G.3; 0.5 mg), 95%
3
2
CH CN−H O (A2143G.4; 1.0 mg), CH CN (A2143G.5; 0.5 mg),
3
2
3
and MeOH (A2143G.6; 2.8 mg). Fraction A2143G.1 was pooled with
A2143G.2 and subjected to RP-HPLC purification using a
Phenomenex Kinetex C18 column (150 × 10.00 mm, 5 μm) and
gradient solvent system (65% CH CN−H O for 5 min, linear gradient
EtOAc (5 mL) and then quenched with saturated aqueous NH Cl (5
4
mL) and H O (5 mL). The layers were partitioned, and the aqueous
2
phase was extracted with EtOAc (3 × 5 mL). The combined organic
layers were washed with brine (10 mL), dried over MgSO , filtered,
3
2
4
to 80% CH CN in 15 min, linear gradient to 100% CH CN in 5 min)
at 4.0 mL/min to yield compound 2 (t = 17.6 min; 1.8 mg, 0.02%
and concentrated under vacuum. The crude reaction product was
purified via normal-phase silica flash chromatography (0−25% EtOAc
in hexanes) to afford pure 1 (51 mg, 0.162 mmol, 62% yield).
Characterization data of the synthesized compound 1 exactly matched
that of the natural product.
3
3
R
w/w extract yield). Spectroscopic, spectrometric, and specific rotation
data {[α]23 +7.30 (c 0.15, MeOH)]} obtained for compound 2
D
25
matched corresponding literature values {e.g., [α]
+4.73 (c 0.43,
D
CHCl )]} for the isotactic polymethoxy-1-alkene 4(S),6(S),8(S),10-
In Vitro Cytotoxicity Test Protocols. Samples were evaluated for
3
(
S),12(S),14(R),16(R),18(R),20(R),22(R)-decamethoxyheptacos-1-
cytotoxicity to NCI-H460 cells according to previously published
15−17
26
ene.
protocols with the exception of the concentrations studied. Briefly,
Caracolamide A (1): slightly yellow, oily residue; UV (MeOH) λmax
180 μL of suspended cells were added to each well of several 96-well
plates at 3.33 × 10 cells/mL in RPMI 1640 medium with 10% fetal
4
(
1
log ε) 201 (4.31), 280 (2.85) nm; IR (film) ν 3301, 2927, 2857,
max
−1
1
13
643, 1546, 1457, 1369, 879, 740, 698 cm ; H NMR and C NMR,
Table 1; HRESIMS m/z 336.08983 [M + Na] (calcd for
C H Cl NONa, 336.08924); LR MS/MS m/z 313.97 [M + H] ,
bovine serum (FBS) and 1% penicillin−streptomycin. The plates were
+
incubated overnight at 37 °C in a 5% CO chamber before application
2
+
of test samples. Samples were dissolved in DMSO and diluted in
RPMI 1640 medium without FBS to final concentrations of 1 or 10
μg/mL for crude samples, 10 final concentrations with 3-fold serial
dilutions starting from 100 μg/mL, screened in duplicate, for pure
compounds. Doxorubicin (1 μg/mL) and DMSO in RPMI 1640
without FBS were used as positive and negative controls, respectively.
Plates were incubated for 48 h and then stained with MTT (thiazolyl
blue tetrazolium bromide 98%; Sigma-Aldrich) prior to being
measured on a ThermoElectron Multiskan Ascent plate reader
(Thermo) at 630 and 570 nm. Dose−response curves were generated
using nonlinear regression in GraphPad Prism computer software.
Cerebrocortical Neuron Culture. Cerebrocortical neurons were
harvested and cultured according to previously described proto-
1
6
21
2
1
21.81, 105.02.
Synthesis of 7-Oxo-N-phenethylheptanamide (4). To a
solution of 2-phenylethylamine (3; 2.487 g, 20.52 mmol; Sigma-
Aldrich), 7-hydroxyheptanoic acid (1.5 g, 10.26 mmol; OxChem), and
HATU (4.29 g, 11.29 mmol; Sigma-Aldrich) in dimethylformamide
(
(
DMF; 6.84 mL)/CH Cl2 (27.4 mL) was quickly added DIPEA
2
1.792 mL, 10.26 mmol). The reaction mixture was stirred overnight
at room temperature (rt) to generate compound 4. The mixture was
worked up by addition of excess EtOAc, washed three times with 1 M
HCl and then saturated sodium bicarbonate and brine. The organic
layer was dried using anhydrous sodium sulfate, filtered, and
evaporated under reduced pressure. The residue was purified on
normal-phase silica flash chromatography using a gradient of 0−10%
2
7,28
cols.
Briefly, Swiss-Webster gravid dams were euthanized by CO2
MeOH in EtOAc to obtain 4 (515.3 mg; 2.07 mmol; 20% yield).
asphyxiation and embryonic day 16 mice were removed under sterile
conditions. Animal care and handling complied with protocols
approved by the Creighton University Institutional Animal Care and
Use Committee and employed measures to minimize pain and
discomfort. Cerebrocortices were dissected, stripped of meninges,
minced, and incubated with trypsin for 25 min at 37 °C. Cells were
dissociated by trituration in isolation buffer containing soybean trypsin
inhibitor and DNase, centrifuged, and resuspended in Eagle’s minimal
essential medium with Earle’s salt (MEM) supplemented with 1 mM
L-glutamine, 10% FBS, 10% horse serum, 100 IU/mL penicillin, and
0.10 mg/mL streptomycin (pH 7.4). Cells were plated onto poly-L-
lysine-coated 96-well (9 mm), clear-bottomed, black-well culture
1
7
-Hydroxy-N-phenethylheptanamide (4): H NMR (500 MHz) δ
7
.30 (t, J = 7.4 Hz, 2H), 7.22 (m, 1H), 7.19 (m, 2H), 3.60 (t, J = 6.5
Hz, 2H), 3.50 (q, J = 6.9 Hz, 2H), 2.80 (t, J = 6.9 Hz, 2H), 2.12 (t, J =
7
1
1
2
.4 Hz, 2H), 1.59 (pent, J = 7.4 Hz, 2H), 1.54 (pent, J = 7.4 Hz, 2H),
.33 (m, 2H); 13C NMR (125 MHz) δ 173.2, 139.0, 128.9, 128.7,
26.6, 62.8, 40.6, 36.7, 35.8, 32.6, 29.0, 25.7, 25.5; HRESIMS m/z
+
50.1800 [M + H] (calcd for C H NO , 250.1802).
15
24
2
Synthesis of 7-Oxo-N-phenethylheptanamide (5). To a
solution of 4 (68 mg, 0.273 mmol; Sigma-Aldrich) and sodium
bicarbonate (92 mg, 1.091 mmol; Sigma-Aldrich) in CH Cl (1.364
2
2
mL) was added DMP (173 mg, 0.409 mmol) in one portion at rt. The
reaction mixture was stirred at this temperature for 1 h to generate 5.
The reaction was monitored by normal-phase silica TLC for the
conversion of 4 (5:1 CH Cl −acetone; R = 0.33) to 5 (5:1 CH Cl −
5
plates (MidSci) at a density of 1.5 × 10 cells/well. Cells were then
incubated at 37 °C in a 5% CO and 95% humidity atmosphere.
2
Cytosine arabinoside (ARA-C; 10 μM) was added to the culture
medium 24 h after plating, to prevent proliferation of non-neuronal
cells. From day 4 in vitro, culture media was changed every other day
using a serum-free growth medium containing neurobasal medium
supplemented with B-27, 100 IU/mL penicillin, 0.10 mg/mL
streptomycin, and 0.2 mM L-glutamine.
2
2
f
2
2
acetone; R = 0.50). The reaction mixture was worked up by dilution
f
with 15 mL of EtOAc and the addition of 5 mL of a saturated solution
of sodium thiosulfate in H O, then extracted twice with 10 mL of
2
EtOAc, washed with brine, dried over MgSO , filtered, and
4
concentrated under vacuum. After workup, the crude material was
determined to be of high enough purity by NMR and LC-MS to carry
In Vitro Intracellular Ca2 Channel Oscillation Monitoring
+
2
+
forward in the synthesis (67.5 mg; 0.273 mmol; 99% yield).
Protocols. Cerebrocortical neuron cultures were used for [Ca ]
1
28
7
-Oxo-N-phenethylheptanamide (5): H NMR (500 MHz,
measurements at day 12 in vitro (DIV12) as previously described.
CDCl ) δ 9.73 (t, J = 1.6 Hz, 1H), 7.30 (m, 2H), 7.22 (m, 1H),
Briefly, the growth medium was removed and replaced with dye
loading medium (100 μL per well) containing 4 μM fluo-3 AM and
0.04% pluronic acid in Locke’s buffer (8.6 mM HEPES, 5.6 mM KCl,
3
7
.19 (m, 2H), 3.50 (q, J = 7.0 Hz, 2H), 2.80 (t, J = 7.0 Hz, 2H), 2.42
td, J = 7.4, 1.6 Hz, 2H), 2.11 (t, J = 7.4 Hz, 2H), 1.61 (q, J = 7.4 Hz,
H), 1.60 (q, J = 7.4 Hz, 2H), 1.29 (m, 2H); 13C NMR (125 MHz,
CDCl ) δ 202.7, 172.9, 139.0, 128.8, 128.7, 126.6, 43.7, 40.6, 36.5,
(
2
154 mM NaCl, 5.6 mM glucose, 1.0 mM MgCl , 2.3 mM CaCl ,
2
2
0.0001 mM glycine, pH 7.4). After 1 h of incubation in dye loading
medium, the neurons were washed four times in fresh Locke’s buffer
(180 μL per well) using an automated microplate washer (Bio-Tek
Instruments Inc.) and transferred to a FLIPR II (Molecular Devices).
3
+
3
5.7, 28.7, 25.4, 21.7; HRESIMS m/z 248.1642 [M + H] (calcd for
C H NO , 248.1645).
15
22
2
Synthesis of 8,8-Dichloro-N-phenethyloct-7-enamide (Car-
2+
acolamide A; 1). To a solution of PPh (90 mg, 0.342 mmol; Sigma-
Cells were excited at 488 nm, and Ca -bound fluo-3 emission was
recorded at 515−575 nm at 0.5 s intervals. After recording baseline
fluorescence for 2 min, 20 μL of 10× concentrations of compounds
was added to wells at a rate of 20 μL/s, and the fluorescence was
monitored for an additional 2 min. GraphPad Prism 7 software was
used to analyze time and concentration−response relationships. Fluo3
3
Aldrich) and CHCl (0.0274 mL, 0.342 mmol) in tetrahydrofuran
3
(
0
THF; 1.315 mL) at −78 °C was added dropwise n-BuLi (0.147 mL,
.368 mmol) to generate dichloromethylenetriphenylphosphorane.
The reaction turned a dark brown-orange color and was stirred for 15
min at this temperature before a solution of 5 (65.1 mg, 0.263 mmol)
F
J. Nat. Prod. XXXX, XXX, XXX−XXX
Naman, C. Benjamin
