Synthesis and Liver Microsomal Metabolic Stability Studies of a Fluorine-Substituted δ-Tocotrienol Derivative

Article abstract of DOI:10.1002/cmdc.201900676

A fluoro-substituted δ-tocotrienol derivative, DT3-F2, was synthesized. This compound was designed to stabilize the metabolically labile terminal methyl groups of δ-tocotrienol by replacing one C?H bond on each of the two methyl groups with a C?F bond. However, in vitro metabolic stability studies using mouse liver microsomes revealed an unexpected rapid enzymatic C?F bond hydrolysis of DT3-F2. To the best of our knowledge, this is the first report of an unusual metabolic hydrolysis of allylic C?F bonds.

Full text of DOI:10.1002/cmdc.201900676

Accepted Article
Title: Synthesis and Liver Microsomal Metabolic Stability Studies of a
Fluoro-Substituted δ-Tocotrienol Derivative
Authors: Xingui Liu, Saikat Poddar, Lin Song, Howard Hendrickson,
Xuan Zhang, Daohong Zhou, and Guangrong Zheng
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900676
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900676
A Journal of
www.chemmedchem.org

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
Synthesis and Liver Microsomal Metabolic Stability Studies of a
Fluoro-Substituted δ-Tocotrienol Derivative
Xingui Liu,[a, b] Saikat Poddar,[c] Lin Song,[a] Howard Hendrickson,[a, d] Xuan Zhang,[a, c] Yaxia Yuan,[b]
Daohong Zhou,[a, b] Guangrong Zheng*[a, c]
[a]
Dr. X. Liu, Prof. L. Song, Prof. H. Hendrickson, Prof. D. Zhou, Prof. G. Zheng
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)
E-mail: zhengg@cop.ufl.edu
[b]
[c]
[d]
Dr. X. Liu, Prof. Y. Yuan, Prof. D. Zhou
Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL 32610 (United States)
Mr. S. Poddar, Prof. X. Zhang, Prof. G. Zheng
Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610 (United States)
Prof. H. Hendrickson
Department of Pharmaceutical, Social and Administrative Sciences, School of Pharmacy, Samford University, Birmingham, AL 35229 (United States)
Supporting information for this article is given via a link at the end of the document.
Abstract: A fluoro-substituted δ-tocotrienol derivative, DT3-F2, was
synthesized. This compound was designed to stabilize the
metabolically labile terminal methyl groups of δ-tocotrienol by
replacing one C-H bond on each of the two methyl groups with a C-F
bond. However, in vitro metabolic stability studies using mouse liver
microsomes revealed an unexpected rapid enzymatic C-F bond
hydrolysis of DT3-F2. To the best of our knowledge, this is the first
report of an unusual metabolic hydrolysis of allylic C-F bonds.
HO
R
O
-tocotrienol, R = CH3
-tocotrienol, R = H
-hydroxylation
(rate-limiting step)
HO
OH
R
O
-13'-hydroxychromanol, R = CH3
-13'-hydroxychromanol, R = H
-oxidation
HO
R
O
COOH
-13'-carboxylchromanol, R = CH3
-13'-carboxylchromanol, R = H
Introduction
5 cycles of
-oxidation
HO
R
Naturally occurring Vitamin E has two major forms, tocotrienols
and tocopherols, both of them have α-, β-, γ-, and δ-homologues
and share a common 6-hydroxychroman moiety. The difference
between these two forms is that tocotrienols have an unsaturated
farnesyl side chain while tocopherols bear a saturated phytyl side
chain.[1] Although structurally closely related, tocotrienols,
especially γ- and δ-tocotrienol (Figure 1), are often more potent
than tocopherols in many biological effects and have unique
bioactivities that are not observed with tocopherols.[1] However,
both γ- and δ-tocotrienol have low bioavailability and short plasma
elimination half-lives,[2] which limit their exposure in systemic
circulation and require administration of large doses to be
effective in vivo.[3,4] It is believed that the low bioavailability and
short elimination half-lives of tocotrienols are, at least in part,
caused by rapid liver metabolism.[5] The major metabolic pathway
of tocotrienols is the CYP450 ω-hydroxylase pathway where ω-
hydroxylation is the rate-limiting metabolic step (Figure 1),[6–8]
indicating that the terminal C-H bonds on the farnesyl side chain
are the potential metabolic labile site.
O
COOH
-carboxyethylhydroxychromanol, R = CH3
-carboxyethylhydroxychromanol, R = H
Figure 1. CYP450 ω-hydroxylase metabolic pathway of tocotrienols.
In an attempt to improve the metabolic stability of δ-tocotrienol,
herein, we report the synthesis of DT3-F2 ((R)-2-((3E,7E)-13-
fluoro-12-(fluoromethyl)-4,8-dimethyltrideca-3,7,11-trien-1-yl)-2,8
-dimethylchroman-6-ol, 1), in which two terminal hydrogen atoms
on the side chain of δ-tocotrienol are replaced by two fluorine
atoms (Figure 2). In vitro metabolic stability of 1 was evaluated
using mouse liver microsomes and an unexpected, NADPH-
independent, rapid hydrolysis of one or both C-F bonds of 1 was
observed.
The unique physicochemical properties of fluorine have rendered
it an important element that can be incorporated into bioactive
molecules to improve bioactivity, physicochemical and/or
pharmacokinetic properties.[9,10] Over 150 fluorine-containing
drugs have reached market since the first approval of
fludrocortisone by the FDA in 1955.[11–13] A widely used strategy
in drug design is to replace a metabolically labile C-H bond with a
strong C-F bond to slow down CYP450 mediated metabolism.[13]
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
O
S
HO
F
TBSO
Ph
F
O
a
6'
2
2
3
F
F
O
O
7'
3'
5'
11'
16
1
)
DT3-F2 (
TBSO
R
Sulfone based C-C coupling
b
R
O
F
TBSO
F
17
18
R = F (not detected)
R = H
Cl
PhO2S
O
2
3
HWE olefination
Wittig olefination
HWE olefination
Scheme 2. Reagents and conditions: a) n-BuLi, THF, HMPA, -78 °C, 83%; (b)
Pd(dppp)Cl2, LiBHEt3, THF; or Na(Hg)/MeOH, THF; or Al(Hg)/MeOH, THF.
TBSO
F
O
O
O
OH
P(OEt)2
F
O
O
EtO
O
5
6
7
4
Coupling of 2[14] and 3 yielded 16. However, subsequent attempts
to remove the phenylsulfonyl group of 16 using LiBHEt3/palladium
catalyst, sodium amalgam, or aluminum amalgam failed to
produce the desired product 17. Instead, compound 18 was
obtained, where simultaneous desulfonylation and defluorination
occurred. This is likely due to the two fluorine atoms are residing
at the allylic position, making them good leaving groups under the
reductive desulfonylation conditions (Scheme 2).
Figure 2. Initial retrosynthetic analysis of DT3-F2 (1).
Results and Discussion
Synthesis of DT3-F2
HO
F
MOMO
6'
12'
Several synthetic strategies were investigated for the preparation
of DT3-F2 (1). Our initial retrosynthetic analysis of 1 is delineated
in Figure 2. It was envisioned that bond breakage at C5՛-C6՛ of 1
would lead to segments 2 and 3, which could be connected
through a sulfone based C-C coupling reaction. Segment 3 could
be constructed from commercially available starting materials
using Horner-Wadsworth-Emmons (HWE) and Wittig olefination
reactions. We have previously reported the synthesis of chloride
2 via aldehyde 4 using δ-tocotrienol as a starting material.[14]
F
PPh3I
O
O
11'
5'
DT3-F2 (1)
19
MOMO
OTBS
X
PhO2
S
O
20 X = Cl
21
22
X = Br
Figure 3. The second retrosynthetic analysis of DT3-F2 (1).
O
a
c
b
OH
OTBS
OTBS
With the initial synthetic strategy unsuccessful, a second method
was tested (Figure 3). As we learned from the previous method
that the fluorine atoms at the allylic position cannot tolerate the
reductive desulfonylation conditions, we decided to rearrange the
reaction sequence. Disconnection at C11՛-C12՛ of 1 would lead to
Wittig olefination partners 19 and 7. Disconnection at C5՛-C6՛ of
19 would lead to chloride/bromide 20/21 and sulfone 22.
O
O
EtO
F
6
8
9
F
F
O
g
f
O
R
F
EtO
EtO
R
10
11
12
R = OH
R = Br
13
d
e
14
15
3
R = OH
h
i
R = Br
R = PhSO2
R = PPh3Br
Scheme 1. Reagents and conditions: a) TBSCl, imidazole, DMF, rt, 50%; b) 5,
NaH, THF, 0 °C, 78% for Z/E mixture; c) n-Bu4N+F-, THF, rt, 58% for E isomer;
d) PPh3, CBr4, 0 °C, CH2Cl2, 95%; e) PPh3, toluene, reflux, 90%; f) 7, LiHMDS,
THF, -78 °C-rt, 68%; g) DIBAL-H, CH2Cl2, -78 °C, 54%; h) PBr3, THF 0 °C; i)
sodium benzenesulfinate, DMF, rt, 70% (over 2 steps).
MOMO
MOMO
a
b
-tocotrienol
O
O
O
23
24
MOMO
c
MOMO
26
20
21
R = OH
R = Cl
R = Br
d
e
f
R
O
25
CO2Et
O
Synthesis of sulfone 3 started from commercially available 5-
hydroxypentan-2-one (6). Protection of the hydroxyl group of 6
with tert-butyldimethylsilyl (TBS) gave compound 8, which was
converted to ester 9 as a mixture of Z/E isomers[15] in a ratio of
~1:2 (determined by GC-MS analysis). Removal of the TBS
protection afforded the corresponding alcohols, from which the E-
isomer 10 was isolated. Treating 10 with triphenylphosphine
(PPh3) and carbon tetrabromide (CBr4), followed by heating with
PPh3 in toluene afforded phosphonium bromide 12. Wittig
olefination between ylide generated from 12 and 1,3-
difluoroacetone (7) yielded ester 13. Reduction of 13 with DIBAL-
H to alcohol 14, followed by converting to bromide 15 and treating
with sodium benzenesulfinate in DMF, furnished sulfone 3
(Scheme 1).
Scheme 3. Reagents and conditions: a) MOMCl, NaH, DMF, 0 °C, 92%; b) i.
OsO4 (5 mol%), NMO (4 equiv), 2,6-lutidine (6 equiv), acetone/H2O (10:1); ii.
PhI(OAc)2 (4 equiv), acetone/H2O (10:1), rt, 64%; c) triethyl 2-
phosphonopropionate, NaH, THF, 0 °C, 48% (E-isomer); d) DIBAL-H, CH2Cl2, -
78 °C, 92%; e) Me2S, NCS, CH2Cl2, 0 °C; f) i. MeSO2Cl, TEA, THF; ii. LiBr.
Compounds 20 and 21 were prepared in a similar way to the
synthesis of 2.[14] Briefly, the OH group of δ-tocotrienol was initially
protected with a methoxymethyl (MOM) group to afford 23, which
was followed by double bond cleavage to form aldehyde 24 and
transformation into α,β-unsaturated esters with E-isomer 25 as
the major product (Z/E ratio of ~1:9, according to GC-MS).
Reduction of 25 with DIBAL-H gave alcohol 26, which was
converted into chloride 20 and bromide 21 by standard methods
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
(Scheme 3). Sulfone 22 was synthesized via a three-step process
starting with reduction of 9 (Scheme 1) to alcohol and isolation of
the E-isomer 27, followed by chlorination and treatment with
sodium benzenesulfinate (Scheme 4).
to a fragment that is ready to be coupled with 1,3-difluoroacetone
(7). The major challenge for this synthetic strategy, however, is to
achieve the regioselective cleavage of the C11′-C12′ double bond
with all three double bonds in the farnesyl side chain having
similar chemical environment. To this end, we initially tried the
NMO/OsO4 and m-CPBA oxidative systems, in attempts to
selectively convert C11′-C12′ to a vicinal diol or an epoxide, which
can then be cleaved to aldehyde or ketones by oxidants like HIO4
and Pb(OAc)4.[17] However, neither of these methods gave
satisfactory results. To our delight, when we used a condition
modified from a previously reported method,[17] treatment of
MOM-protected δ-tocotrienol (23) with NBS and H2O in THF at
0 °C, the desired bromohydrin compound 38 was produced
(Scheme 6). The optimal ratio between THF and H2O was studied
in order to maximize the region-selectivity and yield. A THF/H2O
ratio of 10/1 and a substrate 23 concentration at ~9 mmol/L gave
the best result. Treatment of bromohydrin 38 with K2CO3 formed
epoxide 39, which was subsequently treated with HIO4 to give
aldehyde 40. Reduction of 40 afforded alcohol 33, which could be
converted to the final product by following the chemical
transformations in Scheme 5.
a
c
b
9
22
OTBS
OTBS
HO
Cl
27
28
MOMO
SO2Ph
d
OTBS
21
O
29
MOMO
MOMO
c
d
20
OTBS
SO2Ph
O
O
28
SO2Ph
30
31
Scheme 4. Reagents and conditions: a) DIBAL-H, CH2Cl2, -78 °C, 63%; b) i.
MeSO2Cl, TEA, THF; ii. LiCl; c) sodium benzenesulfinate, DMF, rt, (55% over
two steps from alcohol); d) n-BuLi, THF/HMPA, -78 °C, 59%.
Unexpectedly, coupling between chloride 20 and sulfone 22
under the same reaction condition used in Scheme 2 did not yield
any product. However, the reaction proceeded smoothly when 20
was replaced by bromide 21 to form 29. Alternatively, we
converted 20 into sulfone 30 and coupled with 28 to form 31
(Scheme 4).
The preparation of phosphonium salt 36 (Scheme 5) requires
heating 34 with PPh3 in toluene for a long period of time, resulting
in a low yield of compound 37. Alternatively, we converted alcohol
33 to sulfide 41 and selectively oxidized the sulfur atom using
H2O2 and ammonium molybdate to form sulfone 42. The Julia-
Kocienski olefination was carried out to couple 42 with 1,3-
difluoroacetone (7) to afford 37 (Scheme 6). Through this method,
DT3-F2 (1) was obtained in a ~17% overall yield.
MOMO
MOMO
a
b
29/31
OTBS
OH
O
O
32
33
RO
MOMO
c
d
In vitro metabolic stability test
I
P+Ph3I-
O
O
O
36
34 R = MOM
35
R = H
In vitro metabolic stabilities of δ-tocotrienol and synthetic DT3-F2
(1) were tested in mouse liver microsomes using propranolol as a
positive control (Table 1). A control without NADPH was also
performed to reveal any NADPH independent enzymatic
degradation or chemical instability. Both δ-tocotrienol and
propranolol underwent degradation in the presence of NADPH but
remained intact in the absence of NADPH, indicating that NADPH
dependent enzymes (CYP450 enzymes) are responsible for their
degradation. Unexpectedly, DT3-F2 (1) was exhausted (below
detection limit) in the incubation mixture, regardless of the
presence or the absence of NADPH (Table 1), suggesting factors
other than CYP450 enzymes could be also involved in the
decomposition of this compound.
MOMO
F
HO
F
e
f
F
F
O
37
DT3-F2 (1)
Scheme 5. Reagents and conditions: a) LiBH(Et)3, Pd(dppp)Cl2, THF, 90%; b)
n-Bu4N+F-, THF, 94%; c) i. MeSO2Cl, TEA, THF; ii. NaI, acetone, 89% (over 2
steps); (d) PPh3, toluene, reflux; (e) 7, n-BuLi, THF, -78 °C, 51% (over 2 steps);
(f) 1N HCl in 1,4-dioxane, MeOH, 94%.
The assembled intermediates 29 and 31 were subjected to
desulfonylation to afford compound 32 (Scheme 5). Removal of
the TBS protection of 32 gave the alcohol 33, which was then
converted to the corresponding iodide 34. We observed that the
MOM group could be removed during the course of iodization
when the reaction was allowed to stir for an extended period of
time, giving 35 as a byproduct. Indeed, MOM de-protection has
been reported under NaI/acetone in the presence of a catalytic
amount of acid.[16] Treatment of iodide 34 with PPh3 in toluene
under reflux yielded the Wittig salt 36, which was coupled with
1,3-difluoroacetone (7) to afford 37. The final product, DT3-F2 (1)
was obtained upon removal of the MOM group of 37.
To further investigate the rapid decomposition of DT3-F2 (1), we
incubated DT3-F2 (1) with buffer (PBS) alone, inactivated mouse
liver microsomes + buffer, mouse liver microsomes + buffer, or
mouse liver microsomes + NADPH + buffer. As shown in Figure
4, DT3-F2 (1) was stable in buffer or inactivated mouse liver
microsomes over the incubation time period, but decreased by
~50% after incubation with active mouse liver microsomes for 10
min. More degradation was observed when NADPH was present.
It is worth to note that DT3-F2 (1) was stable during synthesis and
purification processes and remained intact when stirred in 15%
aqueous NaOH for 30 min (data not shown), thus ruling out the
chemical instability of this compound during incubation. We also
tested the stability of compound 3 (Scheme 1), which contains the
ω,ω-difluoro function as in DT3-F2 and has good solubility in the
reaction media, in the presence of nucleophilic sulfide
However, the synthetic route for 1 was lengthy with over 10 steps
and an overall yield of ~6%. In addition, highly toxic reagent, OsO4,
was used for the side chain cleavage reaction.[14] These
disadvantages in this synthetic method prompted the
development of an alternative route. We envisioned that selective
cleavage of the C11′-C12′ double bond of δ-tocotrienol would lead
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
MOMO
MOMO
MOMO
b
a
OH
c
O
O
O
O
O
11'
3'
7'
Br
23
40
38
39
MOMO
S
d
e
O
OH
S
N
O
O
33
41
O
MOMO
F
O
g
S
N
h
O
1
DT3-F2 ( )
S
F
O
O
42
37
Scheme 6. Reagents and conditions: a) NBS/H2O, THF, 0 °C, 40-60%; b) K2CO3, DMF, 60 °C; c) HIO4, THF, H2O, 34% (over 3 steps); d) NaBH4, EtOH, 86%; e) i.
MeSO2Cl, TEA, THF 0 °C; ii. then 2-mercaptobenzothiazole, K2CO3, DMF, rt, 91% (over 2 steps); f) H2O2, (NH4)6Mo7O24•4H2O, EtOH, rt, 85%; g) KHMDS, 7, THF,
-78 °C, 82%; h) 1 N HCl in 1,4-dioxane, MeOH, 94%.
Table 1. Metabolic Stability Assay Using Mouse Liver Microsomes.[a]
Compound
% remaining at 30 min[b]
without NADPH
106.8 ± 8.9
In vitro
half-life (min)
Clin,u (μL/min/mg)[c]
with NADPH
14.5 ± 10.4
63.7 ± 6.7
0[d]
Propranolol
δ-Tocotrienol
DT3-F2 (1)
25.0 ± 9.6
107.8 ± 17.2
--
124.5 ± 39.9
5240 ± 819.7
--
100.0 ± 6.6
0[d]
[a] The concentration of the compounds for the assay was 1 μM. [b] Data are presented as Mean ± SD, n ≥ 3. [c]Clin,u calculation follows the literature.[18] [d] Below
instrument detection limit.
Figure 4. Stability of DT3-F2 when incubated under four different conditions at 37 °C. 1 μM of DT3-F2 was incubated with conditions shown in the legend, percent
of compound remaining was determined by comparing with compound concentration at 0 min. Buffer is PBS; compound is DT3-F2; inactivated microsomes was
prepared by heating in boiling water for 10 min. Error bars represent Means ± SD, n = 3.
N-acetyl cysteine (NAC) No reaction between compound 3 and
NAC was observed under our experimental conditions (see
Figure S2, Supporting Information). These observations led us to
believe that NADPH independent enzymatic degradation
processes might play a role in the decomposition of DT3-F2 (1) in
mouse liver microsomes.
grew in intensity after 30 min of incubation, indicating that peak b
is related to DT3-F2 (1). ESI-MS of DT3-F2 (1) (peak a) gave two
major mass peaks at m/z 411 and m/z 391, which correspond to
the [M-H-HF]- ion and [M-H-2HF]- ion, respectively (Figure 5B).
The ESI-MS of peak b gave two mass peaks at m/z 409 and m/z
429, which could belong to M1, a mono-hydrolyzed product of
DT3-F2 (1) (Figure 5B). However, because the two fluorine atoms
of DT3-F2 (1) are not symmetrical, we were interested to explore
1) which C-F bond was hydrolyzed? 2) does peak b contain a pair
of co-eluting Z/E isomers? 3) can both C-F bonds be hydrolyzed
To detect and identify metabolites, we incubated DT3-F2 (1) in
mouse liver microsomes (no NADPH added) and monitored the
reaction with HPLC-UV-MS at 0 min, 10 min, and 30 min (Figure
5A). As shown in the chromatogram, a new peak (peak b) with a
retention time of ~13 min appeared after 10 min of incubation and
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
to form compound M2 (Figure 6) if the incubation time is longer?
and 4) is the current method sensitive enough to detect small
amount of M2?
Figure 5. LC (reversed-phase)-UV-MS analyses of DT3-F2 (1) incubating with
mouse liver microsomes. Peak b is the new peak after incubating DT3-F2 with
mouse liver microsomes; peak a is DT3-F2.
Figure 7. Incubation of DT3-F2 (1) with mouse liver microsomes for indicated
time and the metabolites were monitored by using a normal phase LC-
fluorescence detection system.
HO
F
HO
HO
F
F
OH
O
O
O
DT3-F2 (1)
M1
M2
To address these questions, the proposed C-F bond hydrolysis
products, M1 and M2, of DT3-F2 (1) in mouse liver microsomes
were synthesized (See supporting information for the synthesis).
Interestingly, M2, also known as δ-amplexichromanol, is a natural
product present in the stem bark of Garcinia amplexicaulis, a
shrug found in New Caledonia.[19,20] To the best of our knowledge,
the synthesis of this natural product has never been reported.
Normal phase chromatography and fluorescence detection
(λexcitation = 292 nm, λemission = 330 nm) was used for better
separation of Z/E isomers and improved detection limits. DT3-F2
OH
OH
Figure 6. Proposed hydrolysis pathway of DT3-F2 (1).
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
Nutrition, Inc. THF, CH2Cl2, toluene, and DMF were used from a solvent
purification system. All other reagents and solvents obtained from
commercial sources were used without further purification. If dry and air-
free conditions were required, reactions were performed in oven-dried
(1) was incubated in mouse liver microsomes for a 60 min period
and aliquots of the incubation mixture were analyzed at 0 min, 15
min, 30 min, and 60 min. The DT3-F2 (1) peak (peak c, Figure 7)
decreased over the incubation period and was no longer
detectable at 60 min. Peaks d, e, and f (Figure 7) emerged at 15
min and peak f grew over time, suggesting that these new peaks
correspond to products of DT3-F2 (1). Since these product peaks
were observable by fluorescence, this further suggests that the
chromane ring is still intact with these compounds. Comparing
with our synthetic compounds M1 and M2, we found that M1, a
mixture of Z/E isomers, have the same retention times as peaks
d and e (Figure 7), and M2 has the same retention time as peak
f, confirming that DT3-F2 (1) was initially hydrolyzed to mono-
hydroxyl compounds M1 and then to bis-hydroxyl compound M2
(Figure 6). Normal phase columns appear to have better
separation capability for Z/E isomers,[21] as the mixture of two
mono-hydroxyl compounds was shown as a single peak under
reversed-phase conditions. The two mono-hydroxyl compounds
were in different amounts, suggesting some degree of
stereoselectivity of the hydrolase(s) involved.
glassware (130 °C) under
a positive pressure of argon. Flash
chromatography was performed using silica gel (230-400 mesh) as the
stationary phase. Reaction progress was monitored by TLC (silica-coated
glass plates) and visualized after p-anisaldehyde stain solution (recipe:
135 mL alcohol + 5 mL H2SO4 + 3.7 mL p-anisaldehyde + 1.5 mL glacial
acetic acid) staining, or by GC-MS (Agilent, 5975 series system) or TLC-
MS (Advion). NMR spectra were recorded in CDCl3 at 400 MHz for 1H, 100
MHz for 13C NMR, and 600 MHz for 19F NMR. Chemical shifts δ are given
in ppm using tetramethylsilane as an internal standard. Multiplicities of
NMR signals are designated as singlet (s), broad singlet (br s), doublet (d),
doublet of doublets (dd), triplet (t), quartet (q), and multiplet (m). MS were
recorded on a GC-MS (EI) or Advion Express (APCI or ESI) instrument.
Experimental data for Scheme 1
5-((tert-Butyldimethylsilyl)oxy)pentan-2-one (8): To a stirring solution
of 5-hydroxypentan-2-one (2.65 g, 25.9 mmol) in CH2Cl2 (10 mL) was
added imidazole (2.7 g, 39.7 mmol) and TBSCl (3.0 g, 19.8 mmol). The
resulting mixture was stirred at room temperature overnight, followed by
condensation under vacuum. The crude product was chromatographed on
silica gel to afford the title compound (2.8 g, 65% yield) as colorless oil. 1H
NMR (400 MHz, CDCl3) δ 3.60 (t, J = 6.0 Hz, 2H), 2.50 (t, J = 7.2 Hz, 2H),
2.14 (s, 3H), 1.85-1.70 (m, 2 H), 0.88 (s, 9H), 0.03 (s, 6H).
Conclusions
Fluoro-substituted δ-tocotrienol, DT3-F2 (1), was synthesized and
evaluated for its in vitro metabolic stability using mouse liver
microsomes. We found that the C-F bonds of DT3-F2 (1) were
rapidly hydrolyzed to hydroxyl compounds by microsomes in a
NADPH independent manner. The instability of DT3-F2 (1) could
Ethyl (E)-6-hydroxy-3-methylhex-2-enoate (10): To
a
stirring
suspension of NaH (60%, 833 mg, 20.8 mmol) in THF at 0 °C was added
ethyl 2-(diethoxyphosphoryl)acetate (4 g, 17.9 mmol). The resulting
mixture was stirred at 0 °C for 1 h. Compound 8 (3 g, 13.9 mmol) was
added to the mixture dropwise. After stirring at room temperature overnight,
aqueous NH4Cl solution was added and the mixture was extracted with
ethyl acetate (20 mL × 3). The combined organic layers were washed with
brine, dried over anhydrous Na2SO4, filtered, and condensed to give a
crude product, which was chromatographed on silica gel (hexanes/ethyl
acetate 30:1) to afford compound 9 (3.1 g, 78% yield) as a mixture of Z/E
isomers (~1:2, GC-MS). To a solution of 9 (4 g, 14.0 mol) in THF (15 mL)
was added TBAF•3H2O (8.8 g, 27.9 mmol) at 0 °C. After stirring at 0 °C for
2 h, water was added and the mixture was extracted with ethyl acetate (40
mL × 3). The combined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtered, and condensed. The crude product was
chromatographed on silica gel (hexanes/ethyl acetate 10:1-3:1) to afford
the title compound (1.39 g, 58% yield) as colorless oil. 1H NMR (400 MHz,
CDCl3) δ 5.64 (s, 1H), 4.09 (q, J = 7.1 Hz, 2H), 3.66-3.54 (m, 2H), 2.18 (t,
J = 7.7 Hz, 2H), 2.12 (s, 3H), 1.75-1.63 (m, 2H), 1.22 (t, J = 7.1 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 166.84, 159.39, 115.73, 61.92, 59.54, 37.06,
30.20, 18.70, 14.25; GC-MS (EI) m/z 172.1 (M+).
be due to the fluorine atoms reside at the allylic positions.[22]
A
literature survey failed to find any reports on stability studies of
allylic fluorine derivatives. Thus, to the best of our knowledge, our
study revealed, for the first time, that fluorine atoms at the allylic
position are susceptible to enzymatic hydrolysis. However, it is
unclear whether such hydrolysis could occur in other substrates
that contain C-F bond(s) at the allylic position. Studies on benzylic
fluorides have been shown that hydrogen-bond donor (HBD)
solvents such as water can stabilize the leaving fluoride anion by
strong solvation thus facilitate nucleophilic substitutions.[23] We
tested our model compound 3 under the same conditions and
found that compound 3 was able to react with morpholine in HBD
solvents at 70 °C (see Figure S3, Supporting Information). It is
likely that metabolic enzymes in the microsomes can further
reduce the reaction energy thus favor a rapid hydrolysis at 37 °C.
It would be interesting to find the enzymes that are responsible for
this reaction. It would also be interesting to investigate the stability
of the terminal methyl difluorinated or trifluorinated δ-tocotrienol
derivatives and identify specific enzyme(s) in liver microsomes
that mediate the hydrolysis. Nevertheless, our study provided
another cautionary tale about the potential liability of fluorine-
containing compounds.[22]
(E)-Ethyl-6-bromo-3-methyl-hex-2-enoate (11): CBr4 (2.9 g, 8.7 mmol)
was added to a stirring solution of compound 10 (1.5 g, 8.7 mmol) in
CH2Cl2 (15 mL), followed by the addition of PPh3 at 0 °C. The resulting
mixture was stirred at 0 °C for 0.5 h, and water was added to quench the
reaction. Upon partitioning of the mixture between water and CH2Cl2, the
CH2Cl2 layer was collected, washed with water, dried over anhydrous
Na2SO4, filtered, and condensed under vacuum to dryness. The crude
product was chromatographed on silica gel to afford the title compound
(1.9 g, 95% yield) as colorless oil.1H NMR (400 MHz, CDCl3) δ 5.71 (s,
1H), 4.15 (q, J = 7.1 Hz, 2H), 3.40 (t, J = 6.6 Hz, 2H), 2.31 (t, J = 7.4 Hz,
2H), 2.17 (s, 3H), 2.11-1.99 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR (100
MHz, CDCl3) δ 166.43, 157.49, 116.53, 59.52, 38.91, 32.61, 30.16, 18.57,
14.25; GC-MS (EI) m/z 234.1/236.1 (M+).
Experimental Section
Chemistry
(E)-Ethyl-8-fluoro-7-(fluoromethyl)-3-methylocta-2,6-dienoate (13): To
a stirring solution of compound 11 (178 mg, 0.76 mmol) in toluene (2 mL)
was added PPh3 (219.5 mg, 0.84 mmol). The resulting mixture was stirred
under reflux for 2 days. Solvent was removed under vacuum and the
General methods: δ-Tocotrienol was separated from DeltaGold® using
flash column chromatography with silica gel (230-400 mesh) as the
stationary phase. Gradient elution was performed with ethyl acetate and
hexanes (20/1 to 10/1). DeltaGold® was obtained from American River
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
resulting solid was washed with diethyl ether for 3-5 times to afford
compound 12 (270 mg, yield 72%) as white solid. m.p. 172-175 °C.
Compound 12 (270 mg, 0.544 mmol) was dried under vacuum with heating
and then suspended in THF (5 mL) and cooled down to -78 °C, LiHMDS
(0.54 mL, 1M in THF, 0.544 mmol) was added dropwise. The resulting
mixture was allowed to stir at -78 °C for 1 h, 1,3-difluoroacetone (0.047 mL,
0.65 mmol) was then added. After stirring at r.t. overnight, the reaction was
quenched with aqueous NH4Cl solution and extracted with ethyl acetate.
The combined organic phases were washed with brine, dried over
anhydrous Na2SO4, filtered, and condensed under vacuum. The crude
product was chromatographed (hexanes/ethyl acetate 20:1-15:1) to afford
the title compound (85.9 mg, 68% yield) as colorless oil. 1H NMR (400 MHz,
CDCl3) δ 5.83 (s, 1H), 5.69-5.65 (m, 1H), 4.99 (d, J = 47.7 Hz, 2H), 4.87
(d, J = 47.7 Hz, 2H), 4.15 (q, J = 7.1 Hz, 2H), 2.44-2.32 (m, 2H), 2.28-2.22
(m, 2H), 2.17 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ 166.52, 157.65, 135.71(t, 3JCF = 7 Hz), 131.63 (t, 2JCF = 14 Hz), 116.40,
mL × 3). The combined organic layers were washed with water and brine,
dried over anhydrous Na2SO4, and condensed under vacuum. The residue
was purified by column chromatography on silica gel (hexanes/ethyl
acetate 20:1~10:1) to afford the title compound (7.17 g, 92% yield). 1H
NMR (400 MHz, CDCl3) δ 6.69 (d, J = 2.7 Hz, 1H), 6.61 (d, J = 2.8 Hz, 1H),
5.26-4.96 (m, 5H), 3.50 (d, J = 7.7 Hz, 3H), 2.74 (dd, J = 10.3, 4.2 Hz, 2H),
2.16 (s, 3H), 2.15-2.03 (m, 6H), 1.99 (dd, J = 8.1, 3.8 Hz, 4H), 1.86-1.71
(m, 2H), 1.69 (s, 3H), 1.67-1.63 (m, 1H), 1.63-1.53 (m, 10H), 1.28 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 149.74, 147.24, 135.24, 135.07, 131.35,
127.34, 124.52, 124.41, 124.31, 121.05, 117.38, 114.31, 95.45, 75.55,
55.91, 39.93, 39.84, 39.82, 31.43, 26.89, 26.72, 25.83, 24.21, 22.72, 22.30,
17.81, 16.31, 16.13, 16.02; GC-MS (EI) m/z 440.4 (M+).
(S)-3-(6-(Methoxymethoxy)-2,8-dimethylchroman-2-yl)propanal (24):
To a stirring solution of compound 23 (600 mg, 1.36 mmol) in acetone and
water (15 mL/1.5 mL) was added NMO (718 mg, 6.14 mmol), 2,6-lutidine
(876 mg, 8.19 mmol), and 2% aqueous OsO4 (1.0 mL, 0.0816 mmol). The
resulting mixture was stirred at room temperature overnight. PhI(OAc)2
(2.20 g, 6.8 mmol) was added. After stirring for 2 h, the reaction was
quenched with aqueous NaSO3 solution, and starch KI testing paper was
used to make sure the reaction is fully quenched. The mixture was then
extracted with ethyl acetate (30 mL × 3). The combined organic layers
were washed with brine, dried over anhydrous Na2SO4, and condensed
under vacuum. The crude product was purified by silica gel column
chromatography to afford the title compound (244 mg, 64% yield). 1H NMR
(400 MHz, CDCl3) δ 9.80 (s, 1H), 6.68 (s, 1H), 6.61 (s, 1H), 5.07 (s, 2H),
3.47 (s, 3H), 2.76 (dd, J = 13.7, 7.0 Hz, 2H), 2.61 (t, J = 7.5 Hz, 2H), 2.12
(s, 3H), 2.04 (dt, J = 14.8, 7.5 Hz, 1H), 1.90 (dt, J = 14.5, 7.5 Hz, 1H), 1.84-
1.70 (m, 2H), 1.24 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 202.46, 150.01,
146.63, 127.33, 120.76, 117.52, 114.30, 95.32, 74.63, 55.89, 38.60, 32.42,
31.55, 23.73, 22.51, 16.33; MS (EI) m/z 278.1 (M+).
1
1
84.36 (d, JCF = 165 Hz), 77.36 (d, JCF = 162 Hz), 59.62, 40.00, 25.39,
18.62, 14.27; 19F NMR (600 MHz, CDCl3) δ -213.0 (tm, J = 48.0 Hz), -217.2
(t, J = 48.0 Hz); GC-MS (EI) m/z 232.0 (M+).
(E)-8-Fluoro-7-(fluoromethyl)-3-methylocta-2,6-dien-1-ol (14): To
a
stirring solution of compound 13 (232 mg, 1.0 mmol) in CH2Cl2 (3 mL) was
added DIBAL-H (2.5 mL, 1.2 M in toluene, 3.0 mmol) at -78 °C. The
resulting mixture was stirred at -78 °C for 3 h before quenching with MeOH
(2 mL). The mixture was poured into Rochelle solution (20 mL) and stirred
at room temperature overnight until the mixture layered clearly. The
organic phase was collected, dried over anhydrous Na2SO4, filtered, and
condensed under vacuum. The crude product was chromatographed
(hexanes/ethyl acetate 10:1-5:1) to afford the title compound (102 mg,
54% yield) as colorless oil. 1H NMR (400 MHz, CDCl3) δ 5.90-5.80 (m, 1H),
5.46-5.37 (m, 1H), 4.99 (d, J = 48 Hz, 2H), 4.87 (d, J =48 Hz, 2H), 4.15 (d,
J = 6.7 Hz, 2H), 2.38-2.26 (m, 2H), 2.13 (t, J = 7.5 Hz, 2H), 1.68 (s, 3H);
3
13C NMR (100 MHz, CDCl3) δ 138.13, 137.33 (t, JCF = 5 Hz), 131.25 (t,
Ethyl
(R,E)-5-(6-(methoxymethoxy)-2,8-dimethylchroman-2-yl)-2-
1
1
2JCF = 14 Hz), 124.7, 84.79 (d, JCF = 163 Hz), 77.71(d, JCF = 160 Hz),
59.44(t, 3JCF = 5 Hz), 38.82, 25.95, 16.42; 19F NMR (600 MHz, CDCl3) δ -
212.0 (tm, J = 48.0 Hz), -216.7 (t, J = 48.0 Hz); GC-MS (EI) m/z 190.0 (M+).
methylpent-2-enoate (25): To a stirring suspension of NaH (60% in
mineral oil, 130 mg, 3.24 mmol) in THF was added triethyl 2-
phosphonopropionate (0.56 mL, 2.59 mmol) at 0 °C. The resulting mixture
was stirred at 0 °C for 1 h, followed by adding compound 24 (600 mg, 2.16
mmol) in THF (15 mL). After stirring at 0 °C for an additional hour, the
reaction was quenched by aqueous NH4Cl solution and extracted with
ethyl acetate (15 mL × 3). The combined organic phases were washed
with brine, dried over anhydrous Na2SO4, and condensed under vacuum.
The crude product was purified with column chromatography on silica gel
(hexanes/ethyl acetate 10:1~5:1) to afford the title compound (E isomer,
380 mg, 48% yield) as colorless oil. 1H NMR (400 MHz, CDCl3) δ 6.83-
6.74 (m, 1H), 6.68 (d, J = 2.5 Hz, 1H), 6.60 (d, J = 2.5 Hz, 1H), 5.08 (s,
2H), 4.18 (q, J = 7.2 Hz, 2H), 3.48 (s, 3H), 2.85-2.63 (m, 2H), 2.33 (dd, J
= 15.9, 7.9 Hz, 2H), 2.14 (s, 3H), 1.83 (s, 3H), 1.82-1.71 (m, 3H), 1.70-
1.60 (m, 1H), 1.31-1.25 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 168.23,
149.87, 146.91, 142.10, 127.90, 127.30, 120.84, 117.44, 114.27, 95.36,
75.11, 60.47, 55.87, 38.52, 31.50, 23.98, 23.07, 22.58, 16.27, 14.38,
12.31; GC-MS (EI) m/z 362.2 (M+).
(E)-((8-Fluoro-7-(fluoromethyl)-3-methylocta-2,6-dien-1-yl)sulfonyl)
benzene (3): To a solution of compound 14 (95 mg, 0.5 mmol) in THF (5
mL) was added PBr3 (0.06 mL, 0.6 mmol) at 0 °C. After stirring at room
temperature for 1 h, aqueous NaHCO3 solution was added to quench the
reaction. The resulting mixture was extracted with ethyl acetate for 3 times
and the combined organic phases were washed with brine, dried over
anhydrous Na2SO4, filtered, and condensed under vacuum to give a
residue. The residue was stirred with PhSO2Na (98.4 mg, 0.6 mmol) in
DMF at room temperature overnight, then partitioned between water and
ethyl acetate. The organic phase was collected and washed with brine,
dried over anhydrous Na2SO4, filtered, and condensed under vacuum. The
crude product was chromatographed on silica gel to afford the title
compound (110 mg, 70% yield over 2 steps) as light yellow oil. 1H NMR
(400 MHz, CDCl3) δ 7.92-7.50 (m, 5H), 5.84-5.74 (m, 1H), 5.21 (t, J = 7.9
Hz, 1H), 4.98 (d, J = 41.3 Hz, 2H), 4.86 (d, J = 41.2 Hz, 2H), 3.82 (d, J =
7.9 Hz, 2H), 2.30-2.18 (m, 2H), 2.11 (t, J = 7.4 Hz, 2H), 1.38 (s, 3H); 13
C
(R,E)-5-(6-(Methoxymethoxy)-2,8-dimethylchroman-2-yl)-2-
NMR (100 MHz, CDCl3) δ 144.95, 138.76, 136.29 (t, 3JCF = 9 Hz), 133.65,
131.31(t, 2JCF = 14 Hz), 129.06, 128.41, 111.33 (d, 3JCF = 6 Hz, ), 84.50 (d,
methylpent-2-en-1-ol (26): To a stirring solution of compound 25 (16 mg,
0.044 mmol) in CH2Cl2 (2 mL) was added DIBAL-H (1.0 M in THF, 0.132
mL, 0.132 mmol) at -40 °C. After stirring at -40 °C for 1 h, MeOH was
added dropwise to quench the reaction. The resulting mixture was poured
into Rochelle solution (10 mL) and stirred overnight until the mixture was
clearly layered. The organic phase was separated, dried over anhydrous
Na2SO4, and condensed under vacuum. The crude product was purified
by column chromatography on silica gel (hexanes/ethyl acetate 8:1~3:1)
to afford the title compound (13 mg, 92% yield) as colorless oil. 1H NMR
(400 MHz, CDCl3) δ 6.68 (d, J = 2.6 Hz, 1H), 6.60 (d, J = 2.6 Hz, 1H), 5.42
(t, J = 6.8 Hz, 1H), 5.07 (s, 2H), 3.99 (s, 2H), 3.48 (s, 3H), 2.73 (dd, J =
11.8, 6.4 Hz, 2H), 2.23-2.11 (m, 5H), 1.85-1.73 (m, 2H), 1.66 (s, 3H), 1.64-
1.53 (m, 2H), 1.27 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.80, 147.16,
134.93, 127.34, 126.33, 121.03, 117.43, 114.35, 95.46, 75.41, 69.10,
1JCF = 163 Hz), 77.47 (d, 1JCF = 162 Hz), 55.96, 38.89, 25.60, 16.16; 19
F
NMR (600 MHz, CDCl3) δ -212.7 (tm, J = 48.0 Hz), -216.9 (t, J = 48.0 Hz);
GC-MS (EI) m/z 314.1 (M+).
Experimental data for Scheme 3
(R)-6-(Methoxymethoxy)-2,8-dimethyl-2-((3E,7E)-4,8,12-
trimethyltrideca-3,7,11-trien-1-yl)chromane (23): To
a
stirred
suspension of NaH (60% in mineral oil, 1.1 g, 26.5 mmol) in DMF (20 mL)
at 0 °C was added a solution of δ-tocotrienol (7 g, 17.7 mmol) in DMF.
After bubbles production stopped, MOMCl (1.61 mL, 21.2 mmol) was
added dropwise. After stirring at 0 °C for 2 h, saturated aqueous NH4Cl
was added and the resulting mixture was extracted with ether acetate (50
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
55.95, 39.55, 31.50, 24.17, 22.69, 21.97, 16.32, 13.68; GC-MS (EI) m/z
320.2 (M+).
tert-Butyl(((4E,8E)-11-((R)-6-(methoxymethoxy)-2,8-
dimethylchroman-2-yl)-4,8-dimethyl-7-(phenylsulfonyl)undeca-4,8-
dien-1-yl)oxy)dimethylsilane (29): To a stirred solution of compound 22
(50 mg, 0.136 mmol) in THF/HMPA (5 mL/1mL) was added n-BuLi (1.6 M
in hexane, 0.127 mL, 0.2 mmol) at -78 °C. After stirring at the same
temperature for 45 min, a solution of bromide 21 (prepared from 58.5 mg
of corresponding alcohol) in THF (0.5 mL) was added. After stirring at -
78 °C for 1 h, the reaction was quenched with aqueous NH4Cl solution and
extracted with ethyl acetate (10 mL × 3). The combined organic phases
were washed with brine, dried over anhydrous Na2SO4, and condensed
under vacuum. The crude product was purified by silica gel column
chromatography (hexanes/ethyl acetate 8:1) to afford the title compound
(53 mg, 59% yield) as a mixture of two diastereomers which was subject
to desulfonylation in the next step.
(R,E)-2-(5-Chloro-4-methylpent-3-en-1-yl)-6-(methoxymethoxy)-2,8-
dimethylchromane (20): To a stirring solution of N-chlorosuccinamide
(37.5 mg, 0.28 mmol) in CH2Cl2 (2 mL) was added Me2S (23 μL, 0.309
mmol) at 0 °C. The resulting milky suspension was stirred at 0 °C for 20
min. A solution of alcohol 26 (45 mg, 0.141 mmol) in CH2Cl2 (0.5 mL) was
then added. The resulting mixture was stirring at the same temperature
until the reaction mixture turned clear. The reaction was then quenched
with water and extracted with ethyl acetate (5 mL × 3). The combined
organic layers were washed with brine, dried over anhydrous Na2SO4,
filtered, and condensed under vacuum to dryness. The crude product was
used in the next step without column purification.
(R,E)-6-(Methoxymethoxy)-2,8-dimethyl-2-(4-methyl-5-
(R,E)-2-(5-Bromo-4-methylpent-3-en-1-yl)-6-(methoxymethoxy)-2,8-
dimethylchromane (21): To a stirring solution of alcohol 26 (65 mg, 0.203
mmol) in THF (3 mL) at 0 °C was added triethylamine (51 μL, 0.365 mmol)
and methanesulfonyl chloride (24 μL, 0.305 mmol). The resulting mixture
was stirred at 0 °C for 45 min. LiBr (44 mg, 0.508 mmol) was added to the
mixture and stirred for an additional hour. The reaction was quenched with
water, extracted with ethyl acetate (8 mL × 3). The combined organic
layers were washed with brine, dried over anhydrous Na2SO4, filtered, and
condensed under vacuum. The crude product was used in the next step
without purification.
(phenylsulfonyl)pent-3-en-1-yl)chromane (30): To a stirred solution of
compound 20 (prepared from 56 mg alcohol compound 26) in DMF (6 mL)
was added sodium benzenesulfinate (29 mg, 0.177 mmol). After stirring at
room temperature overnight, water was added and the mixture was
extracted with ethyl acetate (20 mL × 3). The combined organic layers
were washed with water and brine, dried over anhydrous Na2SO4, and
condensed under vacuum. The crude product was purified by column
chromatography on silica gel (hexanes/ethyl acetate 8:1) to afford the title
compound (51 mg, 66% yield) as colorless oil. 1H NMR (400 MHz, CDCl3)
δ 7.84-7.78 (m, 2H), 7.60-7.43 (m, 3H), 6.68 (d, J = 2.6 Hz, 1H), 6.60 (d, J
= 2.6 Hz, 1H), 5.11-5.01 (m, 3H), 3.70 (s, 2H), 3.48 (s, 3H), 2.79-2.58 (m,
2H), 2.13-2.11 (s, 3H), 2.09-2.02 (m, 2H), 1.77-1.73 (m, 3H), 1.73-1.64 (m,
2H), 1.44-1.23 (m, 2H), 1.20 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.84,
146.95, 138.40, 136.32, 133.58, 128.97, 128.60, 127.23, 123.48, 120.88,
117.43, 114.30, 95.38, 75.09, 66.35, 55.92, 38.71, 31.37, 24.06, 22.64,
22.58, 16.56, 16.27; GC-MS (EI) m/z 444.1 (M+).
Experimental data for Scheme 4
(E)-6-((tert-Butyldimethylsilyl)oxy)-3-methylhex-2-en-1-ol (27): To a
stirring solution of compound 9 (a mixture of Z and E isomers, Z/E = 1/2,
4.3 g, 15 mmol) in CH2Cl2 (5 mL) was added DIBAL-H (1.2 M in tolulene,
28 mL, 33 mmol) at -78 °C. The mixture was then warmed to room
temperature and stirred overnight. MeOH was added to quench to the
reaction and the mixture was then poured to Rochelle solution and stirred
overnight until the mixture was clearly layered. The organic phase was
separated, washed with brine, dried over anhydrous Na2SO4, filtered, and
condensed under vacuum. The crude product was purified by column
chromatography on silica gel (hexanes/ethyl acetate 10:1~5:1) to afford
the E isomer 27 (2.3 g, 63% yield) as colorless oil. 1H NMR (400 MHz,
CDCl3) δ 5.40 (t, J = 6.9 Hz, 1H), 4.12 (d, J = 6.9 Hz, 2H), 3.58 (t, J = 6.5
Hz, 2H), 2.08-1.94 (m, 2H), 1.70-1.53 (m, 5H), 0.88 (s, 9H), 0.03 (s, 6H);
13C NMR (100 MHz, CDCl3) δ 139.51, 123.50, 62.86, 59.41, 35.85, 31.00,
26.05, 18.43, 16.36, -5.17; GC-MS (EI) m/z 244.1 (M+).
tert-Butyl(((4E,8E)-11-((R)-6-(methoxymethoxy)-2,8-
dimethylchroman-2-yl)-4,8-dimethyl-7-(phenylsulfonyl)undeca-4,8-
dien-1-yl)oxy)dimethylsilane (31): To a stirred solution of compound 30
(50 mg, 0.112 mmol) in THF/HMPA (5 mL/1mL) was added n-BuLi (1.6 M
in hexane, 0.127 mL) at -78 °C. After stirring at the same temperature for
45 min, a solution of chloride 28 (prepared from 58.5 mg corresponding
alcohol) in THF (0.5 mL) was added dropwise. After stirring at -78 °C for 1
h, the reaction was quenched with aqueous NH4Cl solution and extracted
with ethyl acetate (10 mL × 3). The combined organic phases were washed
with brine, dried over anhydrous Na2SO4, and condensed under vacuum.
The crude product was purified by silica gel column chromatography
(hexanes/ethyl acetate 8:1) to afford the title compound (44 mg, 59% yield)
as a mixture of two diastereomers which was subject to desulfonylation in
the next step.
(E)-tert-Butyldimethyl((4-methyl-6-(phenylsulfonyl)hex-4-en-1-
yl)oxy)silane (22): To a stirring solution of compound 27 (440 mg, 1.80
mmol) in THF (10 mL) was added triethylamine (0.376 mL, 2.70 mmol) and
methanesulfonyl chloride (0.167 mL, 2.16 mmol) at 0 °C. After stirring at
this temperature for 1 h, LiCl (191 mg, 4.5 mmol) was added and the
resulting mixture was allowed to stir for 2 h. The reaction was quenched
with water and extracted with ethyl acetate (15 mL × 3). The combined
organic layers were washed with brine, dried over anhydrous Na2SO4,
filtered, and condensed under vacuum. The crude chloride product 28 was
used in the next step without purification. To a solution of 28 in DMF (10
mL) was added sodium benzenesulfinate (266 mg, 1.62 mmol). The
resulting mixture was stirred at room temperature overnight. Water was
added to the reaction and the mixture was then extracted with ethyl acetate
(20 mL × 3). The combined organic layers were washed with water and
brine, dried over anhydrous Na2SO4, filtered, and condensed under
vacuum. The crude product was purified with column chromatography on
silica gel (hexanes/ethyl acetate 10:1) to afford the title compound (364
mg, 55% yield over 2 steps) as colorless oil. 1H NMR (400 MHz, CDCl3) δ
7.90-7.45 (m, 5H), 5.19 (t, J = 8.0 Hz, 1H), 3.80 (d, J = 8.0 Hz, 2H), 3.55
(t, J = 6.4 Hz, 2H), 2.09-1.95 (m, 2H), 1.57-1.46 (m, 2H), 1.30 (s, 3H), 0.88
(s, 9H), 0.03 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 146.50, 138.78, 133.64,
129.08, 128.64, 110.37, 62.67, 56.21, 36.07, 30.97, 26.05, 18.43, 16.31, -
5.15; GC-MS (EI) m/z 368.1 (M+).
Experimental data for Scheme 5
tert-Butyl(((4E,8E)-11-((R)-6-(methoxymethoxy)-2,8-
dimethylchroman-2-yl)-4,8-dimethylundeca-4,8-dien-1-
yl)oxy)dimethylsilane (32): To a stirring solution of compound 29 or 31
(50 mg, 0.075 mmol) in THF (5 mL) was added Pd(dppp)Cl2 (26 mg, 0.045
mmol). The mixture was cooled to 0 °C and LiBH(Et)3 (0.37 mL, 0.373
mmol) was added dropwise. After stirring at 0 °C for 15 min, the reaction
was quenched with water and extracted with ethyl acetate (8 mL × 3). The
combined organic layers were washed with brine, dried over anhydrous
Na2SO4, and condensed under vacuum. The crude product was purified
by column chromatography on silica gel (hexanes/ethyl acetate 20:1) to
yield the title compound (35.6 mg, 90% yield) as colorless oil. 1H NMR (400
MHz, CDCl3) δ 6.68 (d, J = 2.8 Hz, 1H), 6.60 (d, J = 2.8 Hz, 1H), 5.16-5.09
(m, 2H), 5.08 (s, 2H), 3.58 (t, J = 6.6 Hz, 2H), 3.48 (s, 3H), 2.78-2.64 (m,
2H), 2.15 (s, 3H), 2.13-2.03 (m, 4H), 2.02-1.92 (m, 4H), 1.86-1.71 (m, 2H),
1.68-1.53 (m, 10H), 1.27 (s, 3H), 0.90 (s, 9H), 0.05 (s, 6H); 13C NMR (100
MHz, CDCl3) δ 149.75, 147.26, 135.25, 134.81, 127.36, 124.40, 124.38,
121.07, 117.39, 114.32, 95.47, 75.57, 63.05, 55.94, 39.95, 39.83, 35.93,
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
31.44, 31.32, 26.75, 26.13, 24.22, 22.74, 22.31, 18.51, 16.33, 16.12, 16.04,
-5.10; MS (APCI) m/z 531.0 [M+H]+.
211.8 (tm, J = 48.0 Hz), -216.8 (t, J = 48.0 Hz); MS (APCI) m/z 477.0
[M+H]+.
(4E,8E)-11-((R)-6-(Methoxymethoxy)-2,8-dimethylchroman-2-yl)-4,8-
dimethylundeca-4,8-dien-1-ol (33): Method 1: To a stirring solution of
compound 32 (93 mg, 0.175 mmol) in THF (2 mL) was added TBAF•H2O
(122 mg, 0.39 mmol). After stirring at room temperature overnight, the
mixture was partitioned between ethyl acetate and water. The organic
layer was collected, washed with brine, dried over anhydrous Na2SO4,
filtered, and condensed under vacuum. The crude product was
chromatographed on silica gel to afford the title compound (68 mg, 94%
colorless). 1H NMR (400 MHz, CDCl3) δ 6.68 (d, J = 2.7 Hz, 1H), 6.60 (d,
J = 2.7 Hz, 1H), 5.21-5.10 (m, 2H), 5.08 (s, 2H), 3.61 (t, J = 6.5 Hz, 2H),
3.48 (s, 3H), 2.73 (dd, J = 10.3, 4.0 Hz, 2H), 2.15 (s, 3H), 2.14-1.95(m, 8H),
1.85-1.71 (m, 2H), 1.70-1.62 (m, 3H), 1.60 (s, 3H), 1.59 (s, 3H), 1.58-1.50
(m, 1H), 1.42 (br, 1H), 1.27 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.73,
147.25, 135.08, 134.79, 127.35, 124.85, 124.56, 121.09, 117.40, 114.34,
95.47, 75.58, 62.94, 55.94, 39.87, 39.72, 36.12, 31.43, 30.85, 26.60, 24.23,
22.72, 22.30, 16.32, 16.00, 15.98; GC-MS (EI) m/z 416.3 (M+).
(R)-2-((3E,7E)-13-Fluoro-12-(fluoromethyl)-4,8-dimethyltrideca-
3,7,11 -trien-1-yl)-2,8-dimethylchroman-6-ol (DT3-F2, 1): To a stirring
solution of compound 37 (20 mg, 0.049 mmol) in MeOH (1.5 mL) was
added HCl (4.0 N in 1,4-dioxane, 0.5 mL). The resulting mixture was stirred
at room temperature for 2 h. Saturated aqueous NaHCO3 was added and
the mixture was extracted with ethyl acetate for 3 times. The combined
organic layers were washed with brine, dried over anhydrous Na2SO4,
filtered, and condensed under vacuum to give a residue which was
chromatographed on silica gel to yield the title compound (17 mg, 94%
yield) as colorless oil. 1H NMR (400 MHz, CDCl3) δ 6.47 (s, 1H), 6.38 (s,
1H), 5.88-5.79 (m, 1H), 5.17-5.08 (m, 2H), 4.99 (d, J = 47.7 Hz, 2H), 4.86
(d, J = 47.7 Hz, 2H), 4.13 (br, 1H), 2.73-2.65 (m, 2H), 2.32-2.21 (m, 2H),
2.15-2.00 (m, 9H), 2.00-1.92 (m, 2H), 1.82-1.70 (m, 2H), 1.63-1.50 (m, 6H),
1.57-1.50 (m, 2H), 1.26 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 147.84,
146.11, 137.88, 135.07, 133.62, 130.78, 127.50, 125.52, 124.55, 121.36,
115.75, 112.70, 84.85 (d, J = 166 Hz), 77.71 (d, 162 Hz), 75.44, 39.74,
39.69, 39.01, 31.50, 26.66, 26.24, 24.20, 22.61, 22.30, 16.21, 16.06,
16.00; 19F NMR (600 MHz, CDCl3) δ -211.8 (tm, J = 48.0 Hz), -216.8 (t, J
= 48.0 Hz); MS (APCI) m/z 431.0 [M-H]-.
(R)-2-((3E,7E)-11-Iodo-4,8-dimethylundeca-3,7-dien-1-yl)-6-
(methoxymethoxy)-2,8-dimethylchromane (34): To a solution of alcohol
33 (30 mg, 0.072 mmol) in CH2Cl2 (2 mL) was added triethylamine (0.018
mL, 0.13 mmol) and then MeSO2Cl (8.36 μL, 0.11 mmol) dropwise at 0 °C.
After stirring at 0 °C for 15 min, the reaction was quenched with water,
extracted with ethyl acetate for 3 times. The combined organic phases
were washed with brine, dried over anhydrous Na2SO4, filtered, and
condensed under vacuum to afford a residue. The residue was dissolved
in acetone (3 mL), sodium iodide (32.4 mg, 0.22 mmol) was then added.
The resulting mixture was stirred under reflux for 3 h. After cooling down
to room temperature, the mixture was partitioned between water and ethyl
acetate. The organic layer was collected and washed with brine, dried over
anhydrous Na2SO4, filtered, and condensed under vacuum to give a
residue which was chromatographed on silica gel to afford the title
compound (34 mg, 89% yield over 2 steps) as colorless oil. 1H NMR (400
MHz, CDCl3) δ 6.68 (d, J = 2.9 Hz, 1H), 6.60 (d, J = 2.9 Hz, 1H), 5.14 (dd,
J = 15.5, 7.1 Hz, 2H), 5.08 (s, 2H), 3.48 (s, 3H), 3.13 (dd, J = 8.0, 5.9 Hz,
2H), 2.73 (dd, J = 10.3, 4.1 Hz, 2H), 2.15 (s, 3H), 2.13-2.02 (m, 6H), 2.01-
1.95 (m, 2H), 1.90 (dd, J = 14.3, 7.1 Hz, 2H), 1.85-1.70 (m, 2H), 1.68-1.62
(m, 1H), 1.62-1.55 (m, 7H), 1.27 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
149.75, 147.25, 134.99, 132.93, 127.34, 125.90, 124.64, 121.08, 117.39,
114.33, 95.47, 75.56, 55.94, 40.11, 39.89, 39.69, 31.69, 31.45, 26.60,
24.25, 22.74, 22.31, 16.34, 15.96 (2C), 6.83; GC-MS (EI) m/z 526.2 (M+).
Experimental data for Scheme 6
(4E,8E)-11-((R)-6-(Methoxymethoxy)-2,8-dimethylchroman-2-yl)-4,8-
dimethylundeca-4,8-dienal (40): To a stirring solution of compound 23
(1.28 g, 2.9 mmol) in THF/H2O (30 mL/3 mL) at 0 °C was added NBS (600
mg, 3.2 mmol) in portions within 2 h. The mixture was allowed to stir at
0 °C for an additional hour. Water was added and the resulting mixture
was extracted with ethyl acetate for 3 times. The combined organic phases
were washed with brine, dried over anhydrous Na2SO4, filtered, and
condensed under vacuum to give a residue which was chromatographed
on silica gel to afford 38 (730 mg, 47%). To a stirring solution of 38 (730
mg, 1.4 mmol) in DMF (15 mL) was added K2CO3 (470 mg, 3.4 mmol). The
resulting mixture was stirred at 60 °C overnight. Upon cooling down to
room temperature, the mixture was partitioned between water and ethyl
acetate. The organic layer was collected and washed with brine, dried over
sodium sulfate, filtered, and condensed under vacuum to give the crude
epoxide 39, which was used in the next step without further purification.
To a solution of 39 obtained above in THF (10 mL) was added a solution
of HIO4 (294 mg, 1.29 mmol) in water (1 mL). After stirring at room
temperature for 2 h, aqueous Na2SO3 solution was added and the resulting
mixture was extracted with ethyl acetate for 3 times. The combined organic
layers were washed with brine, dried over anhydrous Na2SO4, filtered, and
condensed under vacuum. The crude product was purified on silica gel to
give the title compound (400 mg, 71% yield over 2 steps). 1H NMR (400
MHz, CDCl3) δ 9.73 (s, 1H), 6.68 (s, 1H), 6.60 (s, 1H), 5.13 (d, J = 6.4 Hz,
2H), 5.07 (s, 2H), 3.48 (s, 3H), 2.72 (t, J = 6.5 Hz, 2H), 2.49 (t, J = 7.5 Hz,
2H), 2.30 (t, J = 7.4 Hz, 2H), 2.15 (s, 3H), 2.13-2.02 (m, 4H), 1.97 (t, J =
7.4 Hz, 2H), 1.88-1.69 (m, 2H), 1.67-1.49 (m, 8H), 1.27 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 202.77, 149.74, 147.22, 134.90, 133.04, 127.31,
125.44, 124.64, 121.05, 117.38, 114.31, 95.44, 75.53, 55.91, 42.25, 39.86,
39.57, 31.95, 31.42, 26.58, 24.22, 22.70, 22.27, 16.30, 16.20, 15.96; GC-
MS (EI) m/z 414.2 (M+).
(R)-2-((3E,7E)-13-Fluoro-12-(fluoromethyl)-4,8-dimethyltrideca-3,7,11
-trien-1-yl)-6-(methoxymethoxy)-2,8-dimethylchromane (37): Method
1: To a stirring solution of iodide 34 (34 mg, 0.065 mmol) in toluene (2 mL)
was added PPh3 (169 mg, 0.65 mmol). The mixture was allowed to stir
under reflux overnight. Removal of the solvent under vacuum afforded an
oil residue which was washed with diethyl ether for 3 times. The residue
(36) was then dried and dissolved in THF (2 mL), upon cooling down to -
78 °C, n-BuLi (1.6 M in hexane, 50 uL, 0.097 mmol) was added and the
mixture was stirred at -78 °C for 45 min. 1,3-Difluoroacetone (7) (12.2 mg,
0.13 mmol) was then added and the resulting mixture was stirred for an
additional 45 min. The reaction was then quenched with aqueous NH4Cl
solution and extracted with ethyl acetate. The combined organic layers
were collected, washed with brine, dried over anhydrous Na2SO4, filtered,
and condensed under vacuum. The crude product was chromatographed
on silica gel to afford the title compound (15.7 mg, 51% yield over 2 steps)
as colorless oil. 1H NMR (400 MHz, CDCl3) δ 6.68 (d, J = 2.6 Hz, 1H), 6.60
(d, J = 2.7 Hz, 1H), 5.88-5.79 (m, 1H), 5.16-5.09 (m, 2H), 5.08 (s, 2H), 4.99
(d, J = 47.9 Hz, 2H), 4.86 (d, J = 47.9 Hz, 2H), 3.48 (s, 3H), 2.73 (dd, J =
8.1, 5.6 Hz, 2H), 2.32-2.20 (m, 2H), 2.17-2.01 (m, 9H), 2.00-1.93 (m, 2H),
1.85-1.69 (m, 2H), 1.66-1.51 (m, 8H), 1.26 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 149.75, 147.25, 137.88, 135.10, 133.61, 132.93, 127.36, 125.54,
(4E,8E)-11-((R)-6-(Methoxymethoxy)-2,8-dimethylchroman-2-yl)-4,8-
dimethylundeca-4,8-dien-1-ol (33): Method 2: To a solution of compound
40 (590 mg, 1.4 mmol) in ethanol was added NaBH4 (191 mg, 5.2 mmol),
the resulting mixture was partitioned between ethyl acetate and water after
stirring at room temperature for 30 min. The organic layer was collected
and washed with brine, dried over anhydrous Na2SO4, filtered, and
condensed under vacuum to afford a residue which was chromatographed
on silica gel to yield the title compound (500 mg, 86% yield). 1H NMR (400
MHz, CDCl3) δ 6.68 (d, J = 2.7 Hz, 1H), 6.60 (d, J = 2.7 Hz, 1H), 5.21-5.10
(m, 2H), 5.08 (s, 2H), 3.61 (t, J = 6.5 Hz, 2H), 3.48 (s, 3H), 2.73 (dd, J =
1
124.53, 121.08, 117.40, 114.33, 95.48, 84.82 (d, JCF = 164 Hz), 77.6
(d,1JCF = 170 Hz), 75.56, 55.96, 39.92, 39.70, 39.01, 31.44, 26.69, 26.23,
24.23, 22.73, 22.31, 16.33, 16.06, 16.02; 19F NMR (600 MHz, CDCl3) δ -
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
10.3, 4.0 Hz, 2H), 2.15 (s, 3H), 2.14-1.95(m, 8H), 1.85-1.71 (m, 2H), 1.70-
1.62 (m, 3H), 1.60 (s, 3H), 1.59 (s, 3H), 1.58-1.50 (m, 1H), 1.42 (br, 1H),
1.27 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.73, 147.25, 135.08, 134.79,
127.35, 124.85, 124.56, 121.09, 117.40, 114.34, 95.47, 75.58, 62.94,
55.94, 39.87, 39.72, 36.12, 31.43, 30.85, 26.60, 24.23, 22.72, 22.30, 16.32,
16.00, 15.98; GC-MS (EI) m/z 416.3 (M+).
1.69 (m, 2H), 1.66-1.51 (m, 8H), 1.26 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 149.75, 147.25, 137.88, 135.10, 133.61, 132.93, 127.36, 125.54, 124.53,
121.08, 117.40, 114.33, 95.48, 84.82 (d, 1JCF = 164 Hz), 77.6 (d,1JCF = 170
Hz), 75.56, 55.96, 39.92, 39.70, 39.01, 31.44, 26.69, 26.23, 24.23, 22.73,
22.31, 16.33, 16.06, 16.02; 19F NMR (600 MHz, CDCl3) δ -211.8 (tm, J =
48.0 Hz), δ -216.8 (t, J = 48.0 Hz); MS (APCI) m/z 477.0 [M+H]+.
2-(((4E,8E)-11-((R)-6-(Methoxymethoxy)-2,8-dimethylchroman-2-yl)-
4,8-dimethylundeca-4,8-dien-1-yl)thio)benzothiazole (41): To a stirring
solution of alcohol 33 (50 mg, 0.095 mmol) in THF (1.5 mL) was added
triethylamine (25 μL, 0.14 mmol) and then MeSO2Cl (11.1 μL, 0.11 mmol).
After stirring at 0 °C for 0.5 h, the mixture was partitioned between water
and ethyl acetate. The organic layer was collected and washed with brine,
dried over anhydrous Na2SO4, filtered, and condensed under vacuum to
give a residue which was dissolved in DMF (2 mL). To the DMF solution
was added 2-mercaptobenzolthiozole (22 mg, 0.13 mmol) and then K2CO3
(33 mg, 0.24 mmol). The mixture was allowed to stir at room temperature
overnight and then partitioned between water and ethyl acetate. The
organic layer was collected, washed with brine, dried over anhydrous
Na2SO4, filtered, and condensed under vacuum to give the crude product
which was purified by chromatography on silica gel to afford the title
compound (62 mg, 91% yield) as colorless oil. 1H NMR (400 MHz, CDCl3)
δ 7.88-7.83 (m, 1H), 7.76-7.72 (m, 1H), 7.40 (ddd, J = 8.3, 7.3, 1.2 Hz, 1H),
7.27 (ddd, J = 8.2, 4.7, 1.0 Hz, 1H), 6.68 (d, J = 2.7 Hz, 1H), 6.59 (d, J =
2.7 Hz, 1H), 5.19-5.10 (m, 2H), 5.07 (s, 2H), 3.47 (s, 3H), 3.31-3.26 (m,
2H), 2.71 (dd, J = 10.5, 4.2 Hz, 2H), 2.19-2.05 (m, 9H), 1.88-2.00 (m, 4H),
1.81-1.67 (m, 2H), 1.66-1.51 (m, 8H), 1.24 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 167.43, 153.50, 149.73, 147.24, 135.30, 135.04, 133.49, 127.34,
126.10, 125.78, 124.59, 124.21, 121.57, 121.07, 121.02, 117.38, 114.32,
95.45, 75.55, 55.93, 39.90, 39.71, 38.59, 33.07, 31.41, 27.42, 26.65, 24.20,
22.72, 22.29, 16.33, 15.98, 15.94; MS (APCI+) m/z 566.1 [M+H]+.
Microsomal metabolic stability assay
Metabolic stability in microsomes with or without NADPH. A solution
of the test compound in DMSO/ACN (V/V, 20/80) (1.64 µL 137.5 µM) was
added to 178.4 µL mouse liver microsome suspension prepared by mixing
mouse liver microsomes (Corning® GentestTM, 20 mg protein/mL) with
EDTA (Corning® GentestTM, 0.5 M) and 758 µL potassium phosphate
buffer (PBS, pH 7.4, 0.1 M) in a 25:2:765 ratio. After incubating at 37 °C
for 30 min, 45 µL NADPH generating solution which was freshly prepared
by mixing solution A (NADP+; Glc-6-PO4; MgCl2, Corning® GentestTM),
solution B (G6PDH, Corning® GentestTM), and PBS (pH 7.4, 0.1 M) in a
5:1:14 ratio, was added to initiate the reaction. For control groups, equal
volume of PBS buffer was added instead of NADPH generating solution.
After incubation for 30 min, the reaction was quenched by adding 450 µL
cold acetonitrile containing internal standard. The sample was then
extracted with 900 µL hexane. The upper layer was transferred to a glass
tube and dried under a stream of nitrogen and then reconstituted with 50
µL acetonitrile. An aliquot of this solution was then analyzed by HPLC-UV-
MS. Propranolol was used as metabolism reference standard.
Comparing stability of DT3-F2 in PBS buffer, microsomes,
microsomes+NADPH, and inactivated microsomes. A solution of DT3-
F2 in DMSO/ACN (V/V, 20/80) (8.2 µL 137.5 µM) was added to 1117 µL
PBS buffer, 28.2 µL microsomes + 1088.8 µL buffer, 28.2 µL microsomes
+ 225 µL NADPH + 863.7 buffer, or 28.2 µL inactivated microsomes + 225
µL NADPH + 863.7 buffer. The resulting mixtures were then incubated at
37 °C. 225 µL aliquots of the mixture were taken at 0 min, 2 min, 5 min,
and 10 min and quenched with cold acetonitrile. The samples were then
extracted with hexane, and the up-layers were transferred to newly labeled
glass tubes and dried under a stream of nitrogen. The pellets were
reconstituted with 50 µL acetonitrile and analyzed with LC-UV.
2-(((4E,8E)-11-((R)-6-(Methoxymethoxy)-2,8-dimethylchroman-2-yl)-
4,8-dimethylundeca-4,8-dien-1-yl)sulfonyl)benzo[d]thiazole (42): To a
stirring solution of compound 41 (20 mg, 0.035 mmol) in EtOH (2 mL) was
added ammonium molybdate (4.3 mg, 0.0035 mmol) and H2O2 (35%,
0.046 mL, 0.47 mmol) at 0 °C. The resulting mixture was stirred at room
temperature for 2-3 h. Saturated aqueous Na2SO3 solution was added and
the mixture was then extracted with ethyl acetate for 3 times. The
combined organic layers were washed with brine, dried over anhydrous
Na2SO4, filtered, and condensed under vacuum to give a residue which
was chromatographed on silica gel to give the title compound (18 mg, 84%
yield) as colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.24-8.19 (m, 1H),
8.03-7.99 (m, 1H), 7.67-7.57 (m, 2H), 6.68 (d, J = 2.7 Hz, 1H), 6.59 (d, J =
2.7 Hz, 1H), 5.11 (m, 2H), 5.07 (s, 2H), 3.48 (s, 3H), 3.46-3.41 (m, 2H),
2.76-2.67 (m, 2H), 2.14 (s, 3H), 2.13-2.01 (m, 6H), 1.96 (dd, J = 15.0, 7.9
Hz, 4H), 1.84-1.68 (m, 2H), 1.67-1.60 (m, 1H), 1.58 (s, 3H), 1.56-1.50 (m,
4H), 1.25 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.07, 152.87, 149.74,
147.25, 136.89, 134.90, 132.39, 128.12, 127.77, 127.35, 126.89, 125.59,
124.69, 122.48, 121.09, 117.39, 114.33, 95.47, 55.95, 54.08, 39.88, 39.61,
37.88, 31.41, 27.00, 26.62, 24.21, 22.73, 22.29, 20.42, 16.34, 15.97,
15.65; MS (APCI+) m/z 598.8 [M+H]+.
Stability of DT3-F2 in microsomes and comparing metabolites with
synthetic compounds. A solution of DT3-F2 in DMSO/ACN (V/V, 20/80)
(8.2 µL 1.375 mM) was added to 1117 µL microsome suspension. The
resulting mixture was incubated at 37 °C and 225 µL aliquots quenched at
0 min, 15 min, 30 min, or 60 min by adding 450 µL cold acetonitrile. The
mixture was extracted with 990 µL hexane and the up-layers were
transferred to a glass tube and dried over nitrogen. The dried residue was
reconstituted with acetonitrile and analyzed with LC-fluorescence. For
comparison, the same analytic condition was applied to synthetic
compounds M1 and M2.
HPLC-UV-MS (Reversed Phase) and HPLC-fluorescence (Normal
Phase) analysis. For LC-UV-MS analysis, WatersTM 2695 HPLC system
and BetabasicTM3.5 μm, 4.6 * 150 mm, C-18 column was used for the
separation, WatersTM 2489 UV/Visible detector was used for UV detection
with absorption wavelength set at 298 nm, WatersTM Q-Tof micro Mass
Spectrometer was used for mass analysis. Mobile phase was 80%
methanol/20% water containing 6 mM NH4OH. Flow rate was set at 1
mL/min, injection volume was 20 µL. For LC-fluorescence analysis,
WatersTM 2695 HPLC system and Astec CYCOBONDTM I 2000 HP-RSP,
5 μm, 25 cm *4.6 mm column were used for separation. Shimadzu® RF-
10AXL fluorescence detector with excitation wavelength set at 292 nm and
emission wavelength set at 330 nm was used for detection. Mobile phase
was isopropanol and n-heptane with isopropanol/n-heptane ratio ranging
from 5/95 to 95/5. Injection volume was 2 µL.
(R)-2-((3E,7E)-13-Fluoro-12-(fluoromethyl)-4,8-dimethyltrideca-3,7,11
-trien-1-yl)-6-(methoxymethoxy)-2,8-dimethylchromane (37) method
2: To a stirring solution of compound 42 (15 mg, 0.025 mmol) in THF at -
78 °C was added KHMDS (0.5 M in hexane, 75 μL, 0.038 mmol). After
stirring at the same temperature for 45 min, 1,3-difluroacetone (7) (4.7 mg,
0.5 mmol) was added, the resulting mixture was stirred at -78 °C for an
additional 45 min, and aqueous NH4Cl solution was then added to quench
the reaction. The mixture was partitioned between water and ethyl acetate.
The organic layer was collected, washed with brine, dried over anhydrous
Na2SO4, filtered, and condensed under vacuum to give crude product
which was purified on silica gel to afford the title compound (9.8 mg,
82%).1H NMR (400 MHz, CDCl3) δ 6.68 (d, J = 2.6 Hz, 1H), 6.60 (d, J =
2.7 Hz, 1H), 5.88-5.79 (m, 1H), 5.16-5.09 (m, 2H), 5.08 (s, 2H), 4.99 (d, J
= 47.9 Hz, 2H), 4.86 (d, J = 47.9 Hz, 2H), 3.48 (s, 3H), 2.73 (dd, J = 8.1,
5.6 Hz, 2H), 2.32-2.20 (m, 2H), 2.17-2.01 (m, 9H), 2.00-1.93 (m, 2H), 1.85-
Acknowledgements
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
[8]
[9]
K. Z. Edson, B. Prasad, J. D. Unadkat, Y. Suhara, T. Okano, F. P.
Guengerich, A. E. Rettie, Biochemistry 2013, 52, 8276–8285.
K. Mϋller, C. Faeh, F. Diederich, Science. 2007, 317, 1881–1886.
This work was supported by the National Institute of General
Medical Sciences of the NIH under grant number P20GM109005.
[10] E. P. Gillis, K. J. Eastman, M. D. Hill, D. J. Donnelly, N. A. Meanwell, J.
Med. Chem. 2015, 58, 8315–8359.
Conflict of interest
[11] J. Fried, E. F. Sabo, J. Am. Chem. Soc. 1954, 76, 1455–1456.
[12] L. H. Sarett, Ann. N. Y. Acad. Sci. 1959, 82, 802–808.
[13] Y. Zhou, J. Wang, Z. Gu, S. Wang, W. Zhu, J. L. Aceña, V. A. Soloshonok,
K. Izawa, H. Liu, Chem. Rev. 2016, 116, 422–518.
The authors declare no conflict of interest.
[14]
X. Liu, S. Gujarathi, X. Zhang, L. Shao, M. Boerma, C. M. Compadre, P.
A. Crooks, M. Hauer-Jensen, D. Zhou, G. Zheng, Tetrahedron 2016, 72,
4001–4006.
Keywords: tocotrienol • fluorination • metabolic stability •
synthesis • C-F bond hydrolysis
[15]
[16]
L. Liu, P. E. Floreancig, Org. Lett. 2009, 11, 3152–3155.
D. R. Williams, B. A. Barner, K. Nishitani, J. G. Phillips, J. Am. Chem.
Soc. 1982, 104, 4708–4710.
References:
[17] P. Spannring, P. C. A. Bruijnincx, B. M. Weckhuysen, R. J. M. K. Gebbink,
[1]
[2]
[3]
[4]
[5]
[6]
[7]
B. Tan, R. R. Watson, V. R. Preedy, Tocotrienols: Vitamin E Beyond
Tocopherols, Second Edition, CRC Press, 2012.
Catal. Sci. Technol. 2014, 4, 2182–2209.
[18]
R. P. Austin, P. Barton, S. L. Cockroft, M. C. Wenlock, R. J. Riley, Drug.
Metab. Dispos. 2002, 30, 1497–1503.
S. P. Yap, K. H. Yuen, J. W. Wong, J. Pharm. Pharmacol. 2001, 53, 67–
71.
[19] A. Lavaud, P. Richomme, M. Litaudon, R. Andriantsitohaina, D. Guilet, J.
Nat. Prod. 2013, 76, 2246–2252.
S. P. Ghosh, S. Kulkarni, K. Hieber, R. Toles, L. Romanyukha, T.-C. Kao,
M. Hauer-Jensen, K. S. Kumar, Int. J. Radiat. Biol. 2009, 85, 598–606.
X. H. Li, D. Fu, N. H. Latif, C. P. Mullaney, P. H. Ney, S. R. Mog, M. H.
Whitnall, V. Srinivasan, M. Xiao, Haematologica 2010, 95, 1996–2004.
Q. Jiang, S. Christen, M. K. Shigenaga, B. N. Ames, Am. J. Clin. Nutr.
2001, 74, 714–722.
[20] A. Lavaud, P. Richomme, J. Gatto, M.-C. Aumond, C. Poullain, M.
Litaudon, R. Andriantsitohaina, D. Guilet, Phytochemistry 2015, 109,
103–110.
[21] S. R. Abbott, J. Chromatogr. Sci. 1980, 18, 540–550.
[22] Y. Pan, ACS Med. Chem. Lett. 2019, 10, 1016–1019.
[23] P. A. Champagne, J. Pomarole, M.-È. Thérien, Y. Benhassine, S. Beaulieu,
C. Y. Legault, J.-F. Paquin, Org. Lett. 2013, 15, 2210–2213.
C. Abe, T. Uchida, M. Ohta, T. Ichikawa, K. Yamashita, S. Ikeda, Lipids
2007, 42, 637–645.
T. J. Sontag, R. S. Parker, J. Biol. Chem. 2002, 277, 25290–25296.
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900676
ChemMedChem
FULL PAPER
Entry for the Table of Contents
HO
H
HO
F
chemical
transformation
H
F
O
O
1
-tocotrienol
DT3-F2 (
)
liver Microsomes
+/- NADPH
HO
OH
OH
HO
F
OH
O
O
In an attempt to improve metabolic stability of δ-tocotrienol, we designed and synthesized DT3-F2 (1). However, subsequent in vitro
metabolic studies using mouse liver microsomes revealed that the C-F bonds of DT3-F2 were rapidly hydrolyzed to form the hydroxyl
metabolites. This unexpected C-F bond hydrolysis appeared to be enzymatic and NADPH-independent.
12
This article is protected by copyright. All rights reserved.