X. Liu, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
4.1.2. triphenyl(propan-2-yl-d7)phosphonium bromide (4)
DT3 was emulsified with PBS containing 33% PEG400 and 2% tween 80
and the concentration was adjusted to give 150 μL/mouse. The emul-
sion was sterilized by filtering through 0.22 μm filter and the first
0.5 mL filtrate was discarded.
A mixture of d7-2-bromopropane (1.5 mL, 15.1 mmol) and triphenyl
phosphine (4.36 g, 16.6 mmol) was stirred in a pressure tube at 150 °C
for 24 h. The resulting mixture was washed with a mixture of hexanes
and ethyl acetate (1:1, v/v) to afford a white solid (3.6 g, 61% yield).36
1H NMR (400 MHz, DMSO) δ 7.94 – 7.86 (m, 9H), 7.80 – 7.75 (m, 6H).
4.3.3. Animal treatment
MS (ESI+) m/z 312 [M−Br]+
.
Mice were randomly assigned to one of the following treatments:
vehicle group (5% tween 80 in PBS for SC injection and PBS containing
33% PEG400 and 2% tween 80 for IV injection), DT3 treatment group,
d6-DT3 treatment group (n = 4). After administration of the drug
(200 mg/Kg subcutaneously or 50 mg/Kg intravenously), blood was
collected into tubes coated with EDTA potassium through the retro-or-
bital vein at the following time points: for SC PK (0 min, 7.5 min,
15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h.); for IV PK (0 min,
5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h.).
4.1.3. (R)-2-((3E,7E)-4,8-dimethyl-12-(methyl-d3)trideca-3,7,11-trien-1-
yl-13,13,13-d3)-2,8-dimethylchroman-6-ol (d6-DT3)
To a stirring solution of 2 (400 mg, 0.90 mmol) in THF (7 mL) was
added HCl (4.0 N in 1,4-dioxane, 1.0 mL). The resulting mixture was
stirred at room temperature for 2 h, aqueous NaHCO3 solution was
added and the mixture was extracted twice with ethyl acetate. The
combined organic layers were washed with brine, dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography to yield the
title compound (335 mg, 95% yield). 1H NMR (400 MHz, CDCl3) δ 6.48
(d, J = 2.7 Hz, 1H), 6.38 (d, J = 2.7 Hz, 1H), 5.18–5.06 (m, 3H), 4.20
(s, 1H), 2.70 (t, J = 6.3 Hz, 2H), 2.17–2.03 (m, 9H), 2.01–1.93 (m, 4H),
1.85–1.70 (m, 2H), 1.69–1.61 (m, 1H), 1.61–1.51 (m, 7H), 1.26 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 147.82, 146.15, 135.28 (2C), 135.12,
127.52, 124.56, 124.41, 124.31, 121.38, 115.75, 112.70, 75.47, 39.87,
39.83 (2C), 31.49, 26.88, 26.73, 24.18 (2C), 22.62, 22.31, 16.21 (2C),
16.15, 16.02; MS (EI) m/z 402.4 (M+).
4.3.4. Plasma collection and plasma drug concentration analysis
Blood collected from mice were kept on ice and centrifuged shortly
after collection. Plasma was obtained by centrifugation (1200 rpm,
15 min, 4 °C) and stored (aliquoted) at −80 °C until analysis. For
plasma drug concentration determination, 50 μL internal standard were
added to each 50 μL of plasma, followed by addition of 100 μL of
methanol (containing 2% (w/v) ascorbic acid) and 300 μL of heptane
(containing 1% (w/v) BHT). The resulting mixture was vortexed and
centrifuged (1200 rpm, 5 min, 25 °C) and the upper layer transferred to
a pre-labeled testing tube and dried under a N2 stream to afford a re-
sidue. The residue was reconstituted with 100 μL of methanol, 10 μL of
which was utilized for HPLC-fluorescence analysis.
4.2. In vitro metabolic stability assay
A stock solution of test compound in DMSO (687.5 μM) was diluted
with acetonitrile to afford a solution in DMSO and acetonitrile (20%
DMSO and 80% acetonitrile) with a concentration of 137.5 μM. Mouse
liver microsome suspension was prepared by mixing mouse liver mi-
crosomes (20 mg protein/mL (Corning® GentestTM)) with 0.5 M EDTA
and 0.2 M potassium phosphate buffer (pH 7.4) at a volume ratio of
25:2:765. NADPH generating solution was freshly prepared by mixing
solution A (NADP+; Glc-6-PO4; MgCl2), solution B (G6PDH) and po-
tassium phosphate buffer (pH 7.4) at a volume ratio of 3:1:14. One
volume of test compound was pre-incubated with 109 volumes of mi-
crosome suspension at 37 °C for 30 min and the metabolic reaction was
initiated by addition of 27.5 volume of NADPH solution. An equal vo-
lume of buffer instead of NADPH solution was added for the control
sample. After incubating for a specific time (5 min, 15 min, or 30 min),
aliquots of the incubating mixture were taken and added to cold acet-
onitrile (2 times the volume of incubating mixture) containing GT3 as
an internal standard. The sample was then extracted with hexanes. The
upper layer was transferred to a glass tube, dried under a stream of
nitrogen and then reconstituted with acetonitrile. Acetonitrile solution
was then analyzed by HPLC-UV. Propranolol were used as metabolism
reference standard. Data were presented and analyzed with Graph Pad
Prism6.
4.4. In vivo G-CSF assay
CD2F1 mice were randomly assigned to cages and treated with
vehicle (5% tween 80 in PBS), DT3, or d6-DT3 subcutaneously at a dose
of 0.5 mM/Kg (200 mg/Kg of DT3 = 0.5 mM DT3/Kg, n = 3).
Ketoconazole was suspended in corn oil and given to mice (5 mg/
250 μL/mice) through oral gavage 3 times (once 26 h prior to admin-
istration of DT3, once 14 h prior to administration of DT3, and once 2 h
prior to administration of DT3). Blood was collected into EDTA po-
tassium-coated tubes at 8 h and 24 h after vehicle/ DT3/ d6-DT3 ad-
ministration. Mice were euthanized after blood collection. Blood col-
lected from mice was kept on ice and centrifuged shortly after
collection. Plasma was prepared by centrifugation (1200 rpm, 15 min,
4 °C) and stored at −80 °C until analysis. For the assay of time-de-
pendent induction of G-CSF by DT3 and d6-DT3, ketoconazole was not
used, and blood was collected at 8 h, 12 h, 24 h, and 48 h after vehicle/
DT3/d6-DT3 administration. Plasma G-CSF concentration was de-
termined using mouse G-CSF ELISA kit (Novex®). Every plasma sample
was analyzed in duplicate and the average value was used in calcula-
tion. Standard curves were constructed for each plate by plotting ab-
sorption versus known concentrations of standard samples.
Concentrations of test samples were determined from the appropriate
standard curve using their absorption values.
4.3. In vivo pharmacokinetics study
4.3.1. Animal housing
The animal protocol was reviewed and approved by the Institutional
Animal Care and Use Committees (IACUC) of University of Arkansas for
medical Sciences (UAMS). CD2F1 male mice aged 8–10 weeks (Charles
River Breeding Labs) with average body weight at 27 g were randomly
housed among cages and were kept under standardized condition with
controlled humidity and temperature with 12/12 day/night cycle. Free
access to water and chow was provided for mice.
Acknowledgement
This work was supported by the National Institute of General
Medical Sciences of the NIH under grant number P20GM109005 and
R01CA122023.
Appendix A. Supplementary data
4.3.2. Preparation
For SC injection, DT3/d6-DT3 was emulsified with PBS containing
5% tween 80. The volume of drug solution was adjusted to give the
desired dose (100 μL/mouse). For IV injection, a mixture of DT3/d6-
Supplementary data (1H NMR and 13C NMR spectra for compound
2, d6-DT3, and DT3 (PDF)) to this article can be found online at https://
5