New cytotoxic annonaceous acetogenin mimetics having a nitrogen- heterocyclic terminal and their application to cell imaging

Article abstract of DOI:10.1016/j.tet.2014.05.047

A terminal unsaturated lactone or its equivalent is commonly believed to be essential for the cytotoxicity of natural annonaceous acetogenins and their artificial mimetics. In this work, we discovered a series of new cytotoxic ethylene glycol ether-contai

Full text of DOI:10.1016/j.tet.2014.05.047

Tetrahedron xxx (2014) 1e8
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Tetrahedron
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New cytotoxic annonaceous acetogenin mimetics having
a nitrogen-heterocyclic terminal and their application to cell imaging
*
Yi-Jie Chen, Sheng Jin, Jie Xi, Zhu-Jun Yao
State Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of Microstructures, School of Chemistry and Chemical Engineering,
Nanjing University, 22 Hankou Road, Nanjing, Jiangsu 210093, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A terminal unsaturated lactone or its equivalent is commonly believed to be essential for the cytotoxicity
of natural annonaceous acetogenins and their artificial mimetics. In this work, we discovered a series of
new cytotoxic ethylene glycol ether-containing mimetics, in which a variety of simple aliphatic nitrogen-
heterocycles were introduced to replace the lactone terminal of AA005 (1), a representative bioactive
polyether mimic identified from our previous research, for the first time. Among these, mimic 4 bearing
a terminal piperazine was found to be the most potent compound against the proliferation of three
cancer cells. Based on our new findings, a fluorescent probe 7 was also developed and successfully ap-
plied to the imaging of cancer cells. This work provides a new strategy for developing simpler cytotoxic
mimetics of natural annonaceous acetogenins and molecular tools for biological imaging.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 20 March 2014
Received in revised form 3 May 2014
Accepted 15 May 2014
Available online xxx
Keywords:
Annonaceous acetogenins
Mimicry
Heterocycle
Antitumor agent
Fluorescent probe
1. Introduction
efforts proved that removal of the terminal unsaturated lactone or
its equivalent (lactam) would result in significant loss of the cyto-
Annonaceous acetogenins are a large family of linear fatty acid-
derived natural products.1,2 Many members of this family display
a wide spectrum of biological activities, among which the most
remarkable is the antitumor activity.1,3 Our group has been devoted
to simplify and mimic this class of natural products and further
investigate their antitumor activities for several years. Several no-
table analogues without natural THF functionalities, such as AA0054
and AA019,5 were developed, and they were found to exhibit more
potent or comparable activities as those of natural products. AA005
usually presents an IC50 of nM range against the proliferation of
toxicity both in natural acetogenins and the artificial mimetics.9
Very recently, we also synthesized a number of AA005 analogues
with embedment of a certain nitrogen-heterocycle in the middle
hydrocarbon region (remaining the terminal lactone) to improve
water solubility of these lipophilic compounds, and they were also
found to show low micromolar inhibitory activities against the
proliferation of several cancer cell lines.10 Such elaboration is
commonly applied in development of small-molecule drugs to ei-
ther increase water solubility or provide a new modification site
(on nitrogen atom) without introducing any new chiral center.
various cancer cells, such as 0.276
mM (MCF-7 cells), 0.305 mM (A549
cells), and 0.041
m
M (Bel-7404 cells).6,7 More importantly, some of
them show selective actions predominately on cancerous cells over
normal cells. Our previous works also found that the representative
mimetic AA005 could inhibit NADH:ubiquinone oxidoreductase
(complex I) of the mitochondrial electron transport system, and
therefore induced cancer cell death through autophage processes.8
In order to study the structureeactivity relationship of repre-
sentative bioactive mimic AA005 (Fig. 1, 1), several different series
of AA005 analogues have been designed, synthesized, and evalu-
ated, including inserting additional hydroxyl(s) to the middle
hydrocarbon chain,5,9 and displacing the terminal unsaturated
g
-lactone with equivalent lactam.6 Furthermore, our previous
* Corresponding author. Tel./fax: þ86 25 8359 3732; e-mail addresses: yaoz@nju.
edu.cn, yaoz@sioc.ac.cn (Z.-J. Yao).
Fig. 1. Design of new acetogenin mimics having a nitrogen-heterocyclic terminal.
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.05.047
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Y.-J. Chen et al. / Tetrahedron xxx (2014) 1e8
In parallel to our previous works, several aromatic nitrogen-
TsNHNH2 to give ester 11 in 94% yield. Reduction of ester 11 with
LiAlH4 followed by treatment with MsCl and Et3N provided the
common mesylate 10 in an excellent yield.
heterocycles were introduced by other groups to the natural ace-
togenin skeleton individually.11 In addition, Hocquemiller and co-
workers once applied alicyclic nitrogen-heterocycles to replace the
terminal lactone of natural acetogenin squamocin in an a-aceto-
nylamide form,12 though the definite function of the heterocycles is
unclear yet. As an expansion of our previous findings, we further
explored the biological effects through replacement of the bi-
ologically ‘essential’ lactone terminal with a nitrogen-heterocyclic
functionality (2e9) using AA005 (Fig. 1, 1) as an examination plat-
form. In this study, a molecule without any heterocycles (6) was
designed as the negative control to verify the roles of the newly
introduced terminal heterocycle(s) accordingly, and another N-
bounded florescent derivative (7) was designed as a potential
molecular tool for cell imaging. Herein, we wish to report our dis-
covery of a new series of cytotoxic acetogenin mimetics based on
the structure of AA005, in which the terminal lactone functionality
was replaced with simple nitrogen-heterocycle(s) for the first time.
2. Results and discussion
Scheme 1. Reagents and conditions: (a) n-BuLi, BF3$Et2O, THF, ꢀ78 ꢁC, 93%; (b) (i)
MsCl, Et3N, CH2Cl2, 0 ꢁC to rt; (ii) DBU, THF, 0 ꢁC to rt, 85% for two steps; (c) TsNHNH2,
NaOAc, DME/H2O, reflux, 94%; (d) LiAlH4, Et2O, 0 ꢁC to rt, 76%; (e) MsCl, Et3N, CH2Cl2,
Because they show structural difference only at their right ter-
minals, most of the newly designed mimetics could be synthesized
by a diverse approach from the common mesylate 10 (Fig. 2). Com-
pounds 2e5 could be synthesized via the corresponding nucleo-
philic substitutions,13 and 7e9 could be provided via proper Click
chemistry.10,14 Compound 6 without a terminal heterocycle also
could be prepared from 10 by reduction with NaBH4.15 The common
intermediate, mesylate 10, could be prepared by reduction of ester
11 and mesylation. Further analysis suggested that ester 11 could be
assembled through epoxide-opening, hydroxyl elimination, and
hydrogenation16 from the known alkyne 1217 and epoxide 13.18
0
ꢁC to rt, 96%.
With mesylate 10 in hand, synthesis of the first batch of AA005
analogues including the mimetics 2e5 equipped with a piperazine,
morpholine, piperidine, or pyrrolidine moiety, was then conducted
through parallel reactions with corresponding nitrogen-heterocy-
cles (Scheme 2). Using 2 as a representative, treatmentof mesylate 10
with piperidine inTHFat reflux provided an amine intermediate,13 of
which the two MOM groups were then deprotected in 20% HCl so-
lution in MeOH, giving 2 in 85% yield finally. Other compounds 3e5
were prepared correspondingly, inparallel. In addition, the reference
compound 6 without any heterocycles was also prepared from
mesylate 10 via reduction with NaBH4,15 and deprotection of the
MOM protecting groups with 20% HCl solution in MeOH.
X
R
N
( )n
N
N
N
2: X = CH2 (n=1)
3: X = O (n=1)
7: R = -N(CH2CH2)2N-RhB
8: R = H
9: R = -N(CH2CH2)2NH
4: X = NH (n=1)
5: X = CH2 (n=0)
6
Nucleophilic
substitution
Click
chemistry
Reduction
( )7
( )7
O
O
O
OR
O
OR
O
OMs
OMe
( )11
( )11
11 (R = MOM)
10 (R = MOM)
RO
RO
epoxide opening
O
OMe
( )7
O
O
( )7
+
OMOM
OMOM
O
12
our previous work
13
Scheme 2. Reagents and conditions: (a) 20e23 (in parallel), THF, rt to reflux for 4 h;
(b) 20% HCl/MeOH, rt, 6 h, 46e93% for two steps; (c) NaBH4, DMF, 0e70 ꢁC; (d) 20%
HCl/MeOH, rt, 91% for two steps.
O
( )7
OMs
HO
OH
Cl
OMOM
16
15
14
For the availability of mimetics 2e6, their in vitro cytotoxicities
against cancer cells were evaluated using an MTT assay (MTT¼3-
Fig. 2. Retrosynthetic analysis of the newly designed compounds.
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide).
Starting from alkyne 12 and epoxide 13, a five-step procedure
was applied to synthesize the crucial mesylate 10 with the alkyne
epoxide-opening protocol16 (Scheme 1). Deprotonation of alkyne
12 with n-BuLi, activation with BF3$Et2O, and followed by reaction
with epoxide 13 afforded alcohol 17 as a diastereomeric mixture in
93% yield. Elimination of the newly born hydroxyl group was ac-
complished by conversion of the mixture into the corresponding
mesylate and followed by treatment with DBU, affording eneyne 18
as an E/Z-olefin mixture. Eneyne 18 was then entirely reduced with
Doxorubicin was used as a positive control, and cancer cell lines
Bel-7404, A547, and MCF-7 were chosen as the cell models for the
assessment (Table 1). To our delight, all the compounds bearing
a terminal heterocycle (mimetics 2e5) showed low micromolar
inhibitory activities against the proliferation of all the tested cancer
cell lines, while the compound without a terminal heterocycle
(mimetic 6, designed as a negative control) did not exhibit signif-
icant inhibitory activity (IC50>40
mM). In addition, the small
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Y.-J. Chen et al. / Tetrahedron xxx (2014) 1e8
3
Table 1
As mentioned above, the free piperazine NH-functionality
existing in one terminal of mimic 4 also provided us a potential
opportunity to explore other biological applications. For instance,
proper modification of 4 might enable us to develop a molecular
tool for cell imaging. To trace the behavior of live cells, florescent
probe 7, on the basis of 4, was thus designed employing the widely
applied Click chemistry,10 and rhodamine B was employed as the
fluorophore. Synthesis of fluorescent probe 7 was illustrated in
Scheme 3. Firstly, treatment of mesylate 10 with NaN3 in DMF gave
azide 24, of which the two MOM groups were then removed with
20% HCl in MeOH, affording azido-diol 25. Under mild Click con-
ditions, successful coupling of azide 25 and alkyne 2619,20 provided
7 in 65% yield.
In vitro cytotoxicities of analogues 2e6 against cancer cellsa,b
Compounds
IC50
(
m
M)
A549
MCF-7
Bel-7404
2
3
4
5
2.91ꢂ0.38
11.66ꢂ1.78
2.70ꢂ0.30
2.81ꢂ0.27
>40
3.84ꢂ0.11
13.01ꢂ0.67
2.88ꢂ0.05
2.96ꢂ0.16
>40
3.23ꢂ0.27
25.76ꢂ1.09
2.05ꢂ0.05
2.44ꢂ0.14
>40
6
Doxorubicinc
1.32ꢂ0.11
0.19ꢂ0.02
0.68ꢂ0.08
a
Inhibition of cell growth by the listed compounds was determined using an MTT
assay, and the inhibition time was 48 h, each data represent the meanþSD of three
independent experiments (performed in triplicate).
b
Bel-7404 (human hepatocellular carcinoma cells); A549 (human lung adeno-
carcinoma cells); MCF-7 (human breast adenocarcinoma cells).
c
Doxorubicin was used as a positive control.
heterocycles 20e23 (see Scheme 2 for their structures) had no
cytotoxicity (IC50>40 mM, see Supplementary data). These results
clearly indicated that the newly introduced terminal nitrogen-
heterocycles are important structural factors for the bioactivity of
new mimetics, and combination of the linear hydrocarbon chain
containing an ethylene glycol ether moiety with the heterocycles
was indispensable and very effective for the inhibitory activities.
Among these new mimetics, compounds 2, 4, and 5 all showed
low micromolar inhibitory activities with an IC50 range from 2 to
4
mM approximately. Mimetic 3 (having a terminal morpholine)
also exhibited inhibitory activity, but it was relatively weaker than
other three compounds. The inhibitory difference between 2 and 3
suggests that the oxygen atom of the terminal morpholine moiety
of 3 might be inappropriate for better bioactivity. Furthermore,
little difference of inhibitory activity was observed between 2 (with
a terminal piperidine) and 5 (with a terminal pyrrolidine). It is
deduced that small change of the ring size of terminal heterocycle
doesn’t greatly affect the activity. According to IC50 values, 4 is the
most potent compound among these mimetics. Compared with 2
(with a piperidine), an additional nitrogen atom was introduced
into the terminal ring of mimetic 4 (with a piperazine). It demon-
strates that introduction of the second nitrogen atom into the ter-
minal heterocycle is helpful. It was also observed that the growth of
all the tested cancer cells was inhibited without huge difference,
suggesting that these new mimetics might have the potential of
being inhibitors of a wide range of cancer cells.
Using mimetic 4 as a representative, the corresponding exper-
iments of dose- and time-dependent inhibition were also con-
ducted. To some extent, this compound could inhibit the
proliferation of three cancer cell lines in a dose- and time-
dependent fashion. The difference of inhibition rate was more ob-
vious at lower doses, though the inhibition rate reached a maxi-
mum at higher doses (Fig. 3).
Scheme 3. Reagents and conditions: (a) NaN3, DMF, 70 ꢁC, 81%; (b) 20% HCl/MeOH, rt,
90%; (c) CuSO4$5H2O, sodium ascorbate, THF/H2O: 1:1, 65%.
Because an additional triazole was introduced to the structure of
probe 7 via the above Click approach (Scheme 3), evaluation of its
effects on the biological activities might be necessary. We further
synthesized two fragment molecules 8 and 9 (Scheme 4) for com-
parison in the biological experiments. One of them was only
equipped with a triazole ring at the right end (8), and the other was
devised with both the triazole ring and the piperazine ring at the
end (9). These two compounds were synthesized in parallel with
Fig. 3. Cancer cell lines MCF-7, Bel-7404, and A549 were treated with mimetic 4 at indicated concentrations and time points. Each column represents the meanþSD of three
independent experiments (performed in triplicate).
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4
Y.-J. Chen et al. / Tetrahedron xxx (2014) 1e8
Scheme 4. Reagents and conditions: (a) CuSO4$5H2O, sodium ascorbate, THF/H2O: 1:1,
86%; (b) (i) Et3N, MsCl, DCM, 0 ꢁC, overnight; (ii) NaBH4, DMF, 0e70 ꢁC, 58% for two steps;
(c) 20% HCl/MeOH, rt, 85%; (d) CuSO4$5H2O, sodium ascorbate, THF/H2O: 1:1, 47%.
our previously used Click conditions. Compound 8 was elaborated
from azide 24 in four steps, including Click reaction with propargyl
alcohol, mesylation, NaBH4 reduction,15 and final deprotection of
the two MOM groups. Preparation of compound 9 could be ach-
ieved by a direct Click coupling of azido-diol 25 with acetylene 29.21
The cytotoxicity of compounds 7e9 was then evaluated and the
results were summarized in Table 2. It is found that introduction of
a triazole functionality into the molecules affects little on the cy-
totoxicity. By comparison with piperazine mimetic 4, both 7 and 9
exhibited similar inhibitory activities against the three cancer cell
lines with only slight decreases, while 8 having a triazole terminal
showed significant decrease in its cytotoxicity. Based on such ob-
servation, we thought the fluorescent probe 7 proved itself to be
qualified as a molecular tool for the future cell imaging.
Fig. 4. The intracellular localization of probe 7. The cells were incubated with 7 at
100 nM for 12 h, and analyzed by confocal microscopy using a MitoTrackerÒ Green FM
(invitrogen) to counter-stain mitochondria.
original lactone terminal of AA005 with a simple nitrogen-
heterocycle. Biological evaluation indicated that most of these
compounds exhibited low micromolar cytotoxicity against the
proliferation of all three tested cancer cell lines, and mimetic 4
containing a terminal piperazine was found to exhibit the most
potent anti-proliferation activity. A fluorescent probe 7 was also
developed through further modification of the newly introduced
nitrogen atom of mimetic 4, and applied to cell imaging success-
fully. Direct introduction of simple aliphatic nitrogen-heterocycles
into one terminal of AA005 thus provides a new workable strat-
egy to acquire unnatural anticancer agents derived from natural
annonaceous acetogenins, and it is also helpful for developing
molecular tools in various biomedical researches.
Table 2
Cytotoxicity of newly synthesized compounds 7e9a
Compounds
IC50
(mM)
A549
MCF-7
Bel-7404
7
8
9
1.61ꢂ0.06
8.42ꢂ1.03
4.73ꢂ0.30
1.32ꢂ0.11
2.01ꢂ0.01
>25
1.16ꢂ0.11
21.46ꢂ2.88
5.60ꢂ0.25
0.68ꢂ0.08
4. Experimental section
4.1. General methods
8.27ꢂ0.33
Doxorubicinb
0.19ꢂ0.02
a
Inhibition of cell growth by the listed compounds in cells was determined using
an MTT assay, and the inhibition time was 48 h, each data represent the meanþSD of
three independent experiments (performed in triplicate).
Optical rotations were measured at 25 ꢁC. Infrared spectra (IR)
were recorded on a Fourier transform infrared spectrometer (FTIR).
1H NMR spectra were recorded at 300 MHz or 500 MHz, and are
b
Doxorubicin was used as a positive control.
reported in ppm (
dard, and 13C NMR spectra were recorded at 75 MHz or 100 MHz,
and assigned in ppm ( ). HRMS spectra were recorded on Agilent
G6500 TOF-MS. All melting points were uncorrected. Flash column
chromatography was performed on 300e400 mesh silica gel.
d) downfield relative to CDCl3 as internal stan-
With rhodamine B-labeled probe 7, the corresponding experi-
ments of cell imaging were conducted. All cancer cells A549, MCF-7,
and Bel-7404 were visualized by a confocal fluorescent microscopy
after treatment with 100 nM of compound 7. We obviously iden-
tified the probe in these cancer cells after 12 h of drug treatment.
Furthermore, by co-staining with MitoTrackerÒ Green FM, a specific
mitochondrial probe, we found that the florescent probe 7 localized
at almost exclusively the same area inside cells by the overlapping
patterns of the green and red fluorescences (Fig. 4). This reveals
that probe 7 also localize in the mitochondrial as our previous
conclusions for AA005,17,8a indicating that the cell death induced by
the new mimetics might be due to deficiency of ATP as the same
mechanism by AA005.8
d
4.2. Synthesis of compounds
4.2.1. (5S,12S)-Methyl-5-decyl-17-hydroxy-12-(methoxymethoxy)-
2,4,7,10-tetraoxahexacos-14-yn-26-oate (17). To a stirred solution
of 12 (2.63 g, 6.3 mmol) in 24 mL anhydrous THF was added
2.4 M n-BuLi in hexane (2.92 mL, 7.0 mmol) dropwise at ꢀ78 ꢁC
under N2 atmosphere. After the reaction mixture was stirred for
30 min at ꢀ78 ꢁC, BF3$Et2O (0.81 mL, 6.3 mmol) was added and
the mixture was stirred for an additional 20 min. Epoxide 13
(0.83 g, 3.87 mmol) in 24 mL anhydrous THF was then slowly
added to the reaction mixture. After being stirred at ꢀ78 ꢁC for
2 h, the reaction was quenched by saturated aq NH4Cl solution,
and extracted with ethyl acetate for three times. The combined
3. Conclusion
A new series of cytotoxic mimetics of natural annonaceous
acetogenin have been designed and synthesized by replacing the
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Y.-J. Chen et al. / Tetrahedron xxx (2014) 1e8
5
organic phases were washed with water and brine, dried over
Na2SO4, filtered, and concentrated. The residue was purified by
flash chromatography to give 17 (2.27 g, 93%) as a colorless oil.16
Et2O, H2O, and Na2SO4 at 0 ꢁC. After being stirred for 1 h, the
mixture was filtered, and the filtrate was directly concentrated. The
residue was purified by flash chromatography to give 19 (1.01 g,
1H NMR (300 MHz, CDCl3)
d
4.76 (d, J¼6.8 Hz, 1H), 4.73 (s, 2H),
76%) as a colorless oil. [
a
]
25 ꢀ8.31 (c 1.47, CHCl3). 1H NMR (300 MHz,
D
4.65 (d, J¼6.8 Hz, 1H), 3.88e3.80 (m, 1H), 3.72e3.65 (m, 5H),
3.63e3.59 (m, 6H), 3.49 (d, J¼5.1 Hz, 2H), 3.39 (s, 3H), 3.38 (s,
3H), 2.57e2.44 (m, 2H), 2.34–2.42 (m, 1H), 2.33e2.18 (m, 4H),
1.61 (t, J¼7.2 Hz, 2H), 1.48 (dd, J¼12.8, 6.8 Hz, 4H), 1.29e1.25 (m,
26H), 0.87 (t, J¼6.7 Hz, 3H). IR (film, nmax): 3484, 2927, 2855, 1741,
1464, 1359, 1212, 1150, 1106, 1040, 919, 724 cmꢀ1. MS (ESI, m/z):
653 [MþNa]þ. HRMS (ESI, m/z): [MþNa]þ calcd for C35H66O9Na
653.4599; found 653.4610.
CDCl3)
d
4.75 (d, J¼6.8 Hz, 2H), 4.65 (d, J¼6.8 Hz, 2H), 3.70 (dd,
J¼11.4, 5.5 Hz, 2H), 3.64 (d, J¼6.6 Hz, 2H), 3.61e3.57 (m, 4H), 3.49
(d, J¼5.1 Hz, 4H), 3.37 (s, 6H), 1.58e1.47 (m, 6H), 1.25 (br s, 38H),
0.87 (t, J¼6.7 Hz, 3H). 13C NMR (75 MHz, CDCl3)
d 96.01, 76.26, 74.14,
70.81, 62.83, 55.45, 32.86, 32.05, 31.97, 29.79, 29.68, 29.54, 29.40,
25.87, 25.51, 22.74, 14.17. IR (film, nmax): 3499, 2925, 2854, 1464,
1359,1261,1214,1144,1104,1040, 919, 802, 723 cmꢀ1. MS (ESI, m/z):
613 [MþNa]þ. HRMS (ESI, m/z): [MþNa]þ calcd for C34H70O7Na
613.5014; found 613.5031.
4.2.2. (5S,12S)-Methyl-5-decyl-12-(methoxymethoxy)-2,4,7,10-
tetraoxahexacos-16-en-14-yn-26-oate (18). To a solution of com-
pound 17 (2.27 g, 3.60 mmol) and DMAP (22 mg, 0.18 mmol) in
anhydrous CH2Cl2 (300 mL) were added Et3N (1.06 mL, 7.56 mmol)
and MsCl (0.34 mL, 4.32 mmol) at 0 ꢁC under N2 atmosphere. The
reaction mixture was stirred overnight at room temperature and
quenched with water. The mixture was extracted by CH2Cl2 for
three times. The combined organic phases were washed with 1 N
aq HCl, water, and brine, dried over Na2SO4, filtered, and con-
centrated. The residue was used directly without purification. A
solution of the above residue in anhydrous THF (35 mL) was
treated with DBU (0.60 mL, 4.02 mmol) at 0 ꢁC, and then stirred at
room temperature for 24 h. The mixture was directly distilled in
vacuum, and the residue was purified by flash chromatography to
give 18 (1.87 g, 85% for two steps) as a colorless oil. 1H NMR
4.2.5. (5S,12S)-5-Decyl-12-(methoxymethoxy)-2,4,7,10-
tetraoxahexacosan-26-yl methanesulfonate (10). To a solution of 19
(1.02 g, 1.72 mmol) in anhydrous CH2Cl2 (25 mL) were added Et3N
(0.3 mL, 2.064 mmol) and MsCl (0.17 mL, 2.06 mmol) under N2
atmosphere at 0 ꢁC. The reaction mixture was stirred overnight at
room temperature and quenched by water. The mixture was
extracted by CH2Cl2 for three times. The combined organic phases
were washed with 1 N aq HCl, water and brine, dried over Na2SO4,
filtered, and concentrated. The residue was purified by flash chro-
25
matography to give 10 (1.1 g, 96%) as a colorless oil. [
a
]
D
ꢀ7.14
(c 0.67, CHCl3). 1H NMR (300 MHz, CDCl3)
d
4.74 (d, J¼6.8 Hz, 2H),
4.63 (d, J¼6.8 Hz, 2H), 4.20 (t, J¼6.6 Hz, 2H), 3.72e3.63 (m, 2H),
3.63e3.53 (m, 4H), 3.48 (d, J¼5.0 Hz, 4H), 3.36 (s, 6H), 2.98 (s, 3H),
1.77e1.66 (m, 2H), 1.48 (t, J¼6.6 Hz, 4H), 1.24 (brs, 38H), 0.86
(300 MHz, CDCl3)
d
6.09e5.78 (m, 1H), 5.45e5.39 (m, 1H),
(t, J¼6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3)
d 96.18, 76.37, 74.27,
4.77e4.73 (m, 3H), 4.65 (d, J¼6.8 Hz, 1H), 3.87 (dt, J¼10.8, 5.3 Hz,
1H), 3.75e3.65 (m, 5H), 3.64e3.59 (m, 5H), 3.50 (d, J¼5.1 Hz, 2H),
3.39 (s, 3H), 3.38 (s, 3H), 2.66e2.56 (m, 2H), 2.32e2.23 (m, 3H),
2.04 (t, J¼6.8 Hz, 1H), 1.50 (t, J¼6.6 Hz, 2H), 1.37e1.25 (m, 28H),
0.87 (t, J¼6.7 Hz, 3H). IR (film, nmax): 2926, 2855, 1741, 1464, 1359,
1150, 1106, 1040, 957, 919, 724 cmꢀ1. MS (ESI, m/z): 635 [MþNa]þ.
HRMS (ESI, m/z): [MþNa]þ calcd for C35H64O8Na 635.4493; found
635.4506.
70.94, 70.38, 55.62, 37.52, 32.19, 32.09, 29.93, 29.80, 29.71, 29.60,
29.52, 29.30, 29.21, 25.65, 25.60, 22.86, 14.30. IR (film, nmax): 2925,
2854, 1465, 1358, 1213, 1176, 1145, 1105, 1040, 951, 919, 824, 722,
528 cmꢀ1. MS (ESI, m/z): 691 [MþNa]þ. HRMS (ESI, m/z): [MþNa]þ
calcd for C35H72O9SNa 691.4789; found 691.4803.
4.2.6. Synthesis of 2, 3, 4, 5. General procedure (using 4 as an ex-
ample). To a solution of 10 (89 mg, 0.13 mmol) in anhydrous THF
(5 mL) was added piperazine (57.3 mg, 0.67 mmol) under N2 at-
mosphere. The reaction mixture was heated to reflux for 4 h. After
water (15 mL) was added, the mixture was extracted by CH2Cl2 for
several times. The combined organic phases were washed with
water and brine, dried over Na2SO4, filtered, and concentrated. The
residue was re-dissolved in methanol (4 mL), and concd HCl (1 mL)
was added. The mixture was stirred overnight at room tempera-
ture. The pH value of the mixture was adjusted to 11 using 1 M
aq NaOH solution. The mixture was then extracted with CH2Cl2 for
several times. The combined organic phases were washed with
water and brine, dried over Na2SO4, filtered, and concentrated. The
residue was purified by flash chromatography on silica gel to give 4
(35 mg, 46% for two steps) as a white solid.13
4.2.3. (5S,12S)-Methyl-5-decyl-12-(methoxymethoxy)-2,4,7,10-tetra
oxahexacosan-26-oate (11). A mixture of 18 (1.24 g, 2.02 mmol)
and p-toluenesulfono-hydrazide (45.26 g, 0.24 mol) in 1,2-
dimethoxyethane (200 mL) was heated to reflux. An aqueous so-
lution containing AcONa (23.63 g, 0.29 mol, 200 mL) was then
dropped to the above mixture in 5 h, and then refluxed for addi-
tional 7 h. The mixture was cooled down to room temperature and
extracted with diethyl ether for several times. The combined or-
ganic phases were washed with water and brine, dried over
Na2SO4, filtered, and concentrated. The residue was purified by
flash chromatography to give 11 (1.17 g, 94%) as a colorless oil.9
[
a]
ꢀ9.67 (c 1.08, CHCl3). 1H NMR (300 MHz, CDCl3)
d 4.76 (d,
25
D
J¼6.8 Hz, 2H), 4.65 (d, J¼6.8 Hz, 2H), 3.72e3.64 (m, 5H), 3.64e3.57
(m, 4H), 3.49 (d, J¼5.1 Hz, 4H), 3.38 (s, 6H), 2.30 (t, J¼7.5 Hz, 2H),
1.50 (t, J¼6.6 Hz, 4H), 1.25 (brs, 38H), 0.87 (t, J¼6.7 Hz, 3H). 13C
The procedure for the synthesis of 2, 3, and 5 was analogous.
4.2.6.1. (S)-1-(2-(((S)-2-Hydroxydodecyl)oxy)ethoxy)-16-(piper-
25
NMR (75 MHz, CDCl3)
d
174.51, 96.22, 76.43, 74.32, 70.98, 55.63,
azin-1-yl)hexadecan-2-ol (4). Mp 61e63 ꢁC. [
a
]
D
þ11.00 (c 0.40,
51.60, 34.31, 32.23, 32.11, 29.95, 29.82, 29.65, 29.54, 29.46, 29.36,
25.67, 25.16, 22.88, 14.30. IR (film, nmax): 2925, 2854, 1742, 1465,
1361, 1105, 1040, 919, 723 cmꢀ1. MS (ESI, m/z): 641 [MþNa]þ.
HRMS (ESI, m/z): [MþNa]þ calcd for C35H70O8Na 641.4963; found
641.4975.
CHCl3). 1H NMR (300 MHz, CDCl3)
d 3.80e3.75 (m, 2H), 3.72e3.60
(m, 4H), 3.53 (dd, J¼9.8, 2.4 Hz, 2H), 3.31 (t, J¼9.0 Hz, 2H), 2.92
(t, J¼4.2 Hz, 4H), 2.43 (s, 4H), 2.31 (t, J¼7.6 Hz, 2H), 1.47e1.25 (m,
44H), 0.87 (t, J¼6.3 Hz, 3H). 13C NMR (75 MHz, CDCl3)
d 76.11, 70.64,
70.37, 59.62, 54.54, 46.06, 33.23, 32.09, 29.87, 29.79, 29.76, 29.52,
27.78, 26.78, 25.75, 22.86, 14.30. IR (KBr, nmax): 3490, 3440, 2919,
2850, 1468, 1400, 1375, 1325, 1143, 1116, 959, 909, 885, 860, 831,
724, 597 cmꢀ1. MS (ESI, m/z): 593 [MþNa]þ. HRMS (ESI, m/z):
[MþNa]þ calcd for C34H70N2O4Na 593.5228; found 593.5236.
4.2.4. (5S,12S)-5-Decyl-12-(methoxymethoxy)-2,4,7,10-tetraoxahexa
cosan-26-ol (19). To
a stirred suspension of LiAlH4 (0.20 g,
5.02 mmol) in anhydrous THF (40 mL) was added compound 11
(1.40 g, 2.25 mmol) in THF (20 mL) dropwise under N2 atmosphere
at 0 ꢁC. The reaction mixture was stirred at room temperature
overnight, and the mixture was poured to a suspension containing
4.2.6.2. (S)-1-(2-(((S)-2-Hydroxydodecyl)oxy)ethoxy)-16-(piper-
idin-1-yl)hexadecan-2-ol (2). White solid, yield: 85%. Mp 55e57 ꢁC.
Please cite this article in press as: Chen, Y.-J.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.047

6
Y.-J. Chen et al. / Tetrahedron xxx (2014) 1e8
[
a
]
25 þ12.21 (c 0.34, CHCl3). 1H NMR (300 MHz, CDCl3)
d
3.84e3.73
filtered, and concentrated. The residue was purified by flash chro-
D
25
(m, 2H), 3.69e3.60 (m, 4H), 3.52 (dd, J¼9.6, 2.1 Hz, 2H), 3.30
(t, J¼9.0 Hz, 2H), 2.56 (s, 4H), 2.44 (t, J¼7.9, 2H), 1.79e1.64 (m, 4H),
1.62e1.20 (m, 46H), 0.86 (t, J¼6.1 Hz, 3H). 13C NMR (75 MHz, CDCl3)
matography to give 24 (0.94 g, 81%) as a colorless oil. [
a
]
ꢀ7.50
D
(c 0.48, CHCl3). 1H NMR (300 MHz, CDCl3)
d
4.76 (d, J¼6.8 Hz, 2H),
4.65 (d, J¼6.8 Hz, 2H), 3.73e3.65 (m, 2H), 3.65e3.56 (m, 4H), 3.49
(d, J¼5.1 Hz, 4H), 3.38 (s, 6H), 3.25 (t, J¼7.0 Hz, 2H), 1.63e1.56
d
76.09, 70.65, 70.42, 59.25, 54.36, 33.18, 32.09, 29.86, 29.80, 29.76,
29.62, 29.52, 27.70, 26.06, 25.74, 25.11, 23.99, 22.87, 14.31. IR (KBr,
nmax): 3493, 3436, 2920, 2851, 2803, 2765, 1467, 1327, 1143, 1115,
1095, 959, 725 cmꢀ1. MS (ESI, m/z): 592 [MþNa]þ. HRMS (ESI, m/z):
[MþNa]þ calcd for C35H71NO4Na 592.5275; found 592.5287.
(m, 2H), 1.55e1.47 (m, 4H), 1.25 (brs, 38H), 0.87 (t, J¼6.7 Hz, 3H). 13
C
NMR (75 MHz, CDCl3) d 96.09, 76.30, 74.21, 70.88, 55.51, 51.57, 32.12,
32.03, 29.86, 29.73, 29.66, 29.60, 29.45, 29.27, 28.95, 26.83, 25.57,
22.79, 14.22. IR (film, nmax): 2926, 2855, 2096, 1464, 1355, 1258,
1214, 1145, 1106, 1041, 919, 722 cmꢀ1. MS (ESI, m/z): 638 [MþNa]þ.
HRMS (ESI, m/z): [MþNa]þ calcd for C34H69N3O6Na 638.5079;
found 638.5093.
4.2.6.3. (S)-1-(2-(((S)-2-Hydroxydodecyl)oxy)ethoxy)-16-
morpholinohexadecan-2-ol (3). White solid, yield: 93%. Mp
25
1
58e62 ꢁC. [
d
a]
þ12.25 (c 0.41, CHCl3). H NMR (300 MHz, CDCl3)
D
3.81e3.69 (m, 6H), 3.69e3.57 (m, 4H), 3.53 (dd, J¼9.8, 2.5 Hz, 2H),
4.2.9. (S)-16-Azido-1-(2-(((S)-2-hydroxydodecyl)oxy)ethoxy)hex-
adecan-2-ol (25). To a solution of 24 (0.30 g, 0.49 mmol) in meth-
anol (7 mL) was added concd HCl (1.6 mL). The reaction mixture
was stirred overnight at room temperature. After water (20 mL)
was added, the mixture was extracted with Et2O for several times.
The combined organic phases were washed with saturated
aq NaHCO3 solution, water and brine, dried over Na2SO4, filtered,
3.31 (t, J¼9.0 Hz, 2H), 2.44 (s, 4H), 2.32 (t, J¼7.8 Hz, 2H),1.48e1.22 (m,
44H), 0.87 (t, J¼6.5 Hz, 3H). 13C NMR (75 MHz, CDCl3)
d 76.11, 70.68,
70.46, 67.13, 59.44, 53.95, 33.19, 32.11, 29.81, 29.78, 29.54, 27.72,
26.68, 25.76, 22.89,14.33. IR (KBr, nmax): 3489, 3420, 2955, 2917, 2849,
2805, 2760, 1466, 1401, 1362, 1252, 1118, 1087, 1059, 1014, 917, 889,
868, 723 cmꢀ1. MS (ESI, m/z): 572 [MþH]þ, 594 [MþNa]þ. HRMS (ESI,
m/z): [MþNa]þ calcd for C34H69NO5Na 594.5068; found 594.5081.
and concentrated. The residue was purified by flash chromatogra-
25
phy to give 25 (0.23 g, 90%) as a white solid. Mp 49e51 ꢁC. [
a]
D
4.2.6.4. (S)-1-(2-(((S)-2-Hydroxydodecyl)oxy)ethoxy)-16-(pyrro-
þ5.91 (c 0.88, CHCl3). 1H NMR (300 MHz, CDCl3)
d 3.84e3.74 (m,
lidin-1-yl)hexadecan-2-ol (5). White solid, yield: 87%. Mp 54e57 ꢁC.
[
2H), 3.73e3.58 (m, 4H), 3.53 (dd, J¼9.8, 2.8 Hz, 2H), 3.31 (dd, J¼9.8,
8.3 Hz, 2H), 3.25 (t, J¼7.0 Hz, 2H), 2.79 (s, 2H), 1.65e1.53 (m, 2H),
1.45e1.21 (m, 42H), 0.87 (t, J¼6.7 Hz, 3H). 13C NMR (75 MHz, CDCl3)
a]
25 þ11.65 (c 0.41, CHCl3). 1H NMR (300 MHz, CDCl3)
d 3.83e3.72
D
(m, 2H), 3.72e3.60 (m, 4H), 3.53 (dd, J¼9.8, 2.7 Hz, 2H), 3.32
(t, J¼9.0 Hz, 2H), 2.68 (s, 4H), 2.55 (t, J¼7.9 Hz, 2H), 1.86 (dt, J¼6.6,
3.5 Hz, 4H), 1.65e1.52 (m, 2H), 1.45e1.22 (m, 42H), 0.87 (t, J¼6.6 Hz,
d
76.11, 70.55, 70.28, 51.58, 33.13, 32.03, 29.82, 29.75, 29.60, 29.46,
29.27, 28.95, 26.83, 25.71, 22.80, 14.23. IR (KBr, nmax): 3439, 2917,
2850, 2131, 1743, 1665, 1466, 1377, 1316, 1262, 1159, 1143, 1097, 1021,
957, 884, 864, 802, 725, 612 cmꢀ1. MS (ESI, m/z): 550 [MþNa]þ.
HRMS (ESI, m/z): [MþNa]þ calcd for C30H61N3O4Na 550.4554;
found 550.4566.
3H). 13C NMR (75 MHz, CDCl3)
d 76.09, 70.68, 70.43, 56.64, 54.23,
33.20, 32.10, 29.88, 29.81, 29.77, 29.63, 29.53, 28.27, 27.68, 25.75,
23.57, 22.88, 14.32. IR (KBr, nmax): 3512, 3435, 2919, 2850, 2794,
1467, 1329, 1143, 1118, 1092, 959, 884, 725 cmꢀ1. MS (ESI, m/z): 556
[MþH]þ, 578 [MþNa]þ. HRMS (ESI, m/z): [MþNa]þ calcd for
C
34H69NO4Na 578.5119; found 578.5129.
4.2.10. Synthesis of fluorescent probe 7. A mixture of alkyne 26
(66.5 mg, 0.011 mmol), azide 25 (50 mg, 0.095 mmol), CuSO4$5H2O
(8.3 mg, 0.033 mmol), sodium ascorbate (15.1 mg, 0.076 mmol),
water (4 mL), and THF (4 mL) was stirred at room temperature for
5 h under N2 atmosphere. The mixture was extracted with CH2Cl2
for several times. The combined organic phases were washed with
water and brine, dried over Na2SO4, filtered, and concentrated.
Flash chromatography of the residue on silica gel (DCM/CH3OH,
20:1) yielded 7 (68.5 mg, 65%) as a purple solid. 1H NMR (300 MHz,
4.2.7. (S)-1-(2-(((S)-2-Hydroxydodecyl)oxy)ethoxy)hexadecan-2-ol
(6). To a solution of 10 (20 mg, 0.03 mmol) in DMF (2 mL) was
added NaBH4 (2.3 mg, 0.06 mmol) at 0 ꢁC. The mixture was warmed
to room temperature, and gradually heated to 70 ꢁC. After 7 h, the
reaction was quenched by water. The whole mixture was extracted
with Et2O for several times. The combined organic phases were
washed with water and brine, dried over Na2SO4, filtered, and
concentrated. The residue was re-dissolved in methanol (1.6 mL),
and treated with concd HCl (0.4 mL). The reaction mixture was
stirred overnight at room temperature. After water (20 mL) was
added, the mixture was extracted with CH2Cl2 for several times.
The organic phases were washed with saturated aq NaHCO3 solu-
tion, water and brine, dried over Na2SO4, filtered, and concentrated.
CDCl3)
d 7.73e7.56 (m, 3H), 7.56e7.47 (m, 1H), 7.34e7.27 (m, 1H),
7.23 (d, J¼9.6 Hz, 2H), 6.96 (d, J¼9.3 Hz, 2H), 6.73 (d, J¼2.2 Hz, 2H),
4.31 (t, J¼7.3 Hz, 2H), 3.85e3.73 (m, 2H), 3.73e3.50 (m, 16H),
3.47e3.26 (m, 6H), 2.36 (s, 4H), 1.91e1.82 (m, 2H), 1.48e1.38
(m, 6H), 1.21e1.35 (m, 48H), 0.87 (t, J¼6.4 Hz, 3H). 13C NMR
(100 MHz, CDCl3)
d 167.51, 157.95, 156.51, 155.87, 143.74, 135.85,
The residue was purified by flash chromatography to give 6
132.43, 130.83, 130.29, 129.93, 129.04, 127.92, 123.48, 114.45, 114.00,
96.32, 76.03, 70.63, 70.53, 65.76, 53.29, 52.90, 52.83, 50.54, 47.78,
46.25, 41.87, 33.19, 32.11, 30.77, 30.48, 29.90, 29.88, 29.81, 29.79,
29.76, 29.72, 29.61, 29.53, 29.22, 26.71, 25.74, 22.88, 19.39, 14.32,
13.92, 12.79. IR (KBr, nmax): 3441, 2924, 2853, 1735, 1632, 1597, 1528,
1489, 1467, 1416, 1348, 1277, 1262, 1184, 1133, 1082, 1048, 1017, 843,
684, 662, 557 cmꢀ1. MS (ESI, m/z): 1076 [M]þ. HRMS (ESI, m/z): [M]þ
calcd for C65H102N7O6 1076.7886; found 1076.7906.
25
(13.2 mg, 91% for two steps) as a white solid. Mp 62e65 ꢁC. [
a]
D
þ21.25 (c 0.16, CHCl3). 1H NMR (300 MHz, CDCl3)
d 3.85e3.74
(m, 2H), 3.74e3.58 (m, 4H), 3.54 (dd, J¼9.8, 2.8 Hz, 2H), 3.31 (dd,
J¼9.7, 8.3 Hz, 2H), 1.44e1.24 (m, 44H), 0.88 (t, J¼6.6 Hz, 6H). 13C
NMR (75 MHz, CDCl3)
d 76.10, 70.72, 70.49, 33.20, 32.14, 29.91,
29.84, 29.58, 25.77, 22.91, 14.35. IR (KBr, nmax): 3437, 2957, 2919,
2850, 1716, 1467, 1329, 1158, 1142, 1090, 958, 887, 724, 613 cmꢀ1. MS
(ESI, m/z): 509 [MþNa]þ. HRMS (ESI, m/z): [MþNa]þ calcd for
C
30H62O4Na 509.4540; found 509.4552.
4.2.11. (1-((5S,12S)-5-Decyl-12-(methoxymethoxy)-2,4,7,10-
tetraoxahexacosan-26-yl)-1H-1,2,3-triazol-4-yl)methanol (27). The
same procedure was applied as that for synthesis of 7, using azide 24
4.2.8. (5S,12S)-5-(14-Azidotetradecyl)-12-decyl-2,4,7,10,13,15-
hexaoxahexadecane (24). To a solution of 10 (1.26 g, 1.89 mmol) in
freshly distilled DMF (20 mL) was added NaN3 (0.25 g, 3.78 mmol)
in one portion. The mixture was heated to 70 ꢁC for 8 h. After being
cooled down to room temperature, the reaction was quenched by
water and extracted by Et2O for several times. The combined or-
ganic phases were washed with water and brine, dried over Na2SO4,
(60 mg, 0.097 mmol) and propargyl alcohol (13.8 mL, 0.29 mmol),
yielding 27 (57 mg, 86%) as a brown oil. [
NMR (300 MHz, CDCl3)
a
]
25 ꢀ5.48 (c 0.58, CHCl3).1H
D
d
7.51 (s, 1H), 4.78 (s, 2H), 4.75 (d, J¼6.8 Hz,
2H), 4.64 (d, J¼6.8 Hz, 2H), 4.33 (t, J¼7.2 Hz, 2H), 3.73e3.64 (m, 2H),
3.63e3.56 (m, 4H), 3.48 (d, J¼5.0 Hz, 4H), 3.37 (s, 6H), 1.92e1.84 (m,
2H),1.56e1.45 (m, 4H),1.39e1.21 (m, 38H), 0.86 (t, J¼6.5 Hz, 3H).13
C
Please cite this article in press as: Chen, Y.-J.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.047

Y.-J. Chen et al. / Tetrahedron xxx (2014) 1e8
7
NMR (75 MHz, CDCl3)
d
147.93, 121.66, 96.12, 76.34, 74.22, 70.88,
29.73, 29.70, 29.58, 29.46, 29.08, 26.61, 25.70, 22.80, 14.25. IR (KBr,
nmax): 3468, 2919, 2849, 1466, 1371, 1320, 1273, 1143, 1114, 1098,
1082, 1051, 1006, 874, 831, 797, 721, 574 cmꢀ1. MS (ESI, m/z): 674
[MþNa]þ. HRMS (ESI, m/z): [MþNa]þ calcd for C37H73N5O4Na
674.5555; found 674.5569.
56.45, 55.57, 50.51, 32.14, 32.04, 30.43, 29.88, 29.75, 29.64, 29.51,
29.47, 29.13, 26.62, 25.60, 22.81, 14.25. IR (film, nmax): 3425, 3139,
2924, 2854, 1465, 1359, 1261, 1216, 1144, 1105, 1040, 918, 803,
722 cmꢀ1. MS (ESI, m/z): 672 [MþH]þ. HRMS (ESI, m/z): [MþNa]þ
calcd for C37H73N3O7Na 694.5341; found 694.5351.
4.3. Biological experiments
4.2.12. 1-((5S,12S)-5-Decyl-12-(methoxymethoxy)-2,4,7,10-tetraoxa
hexacosan-26-yl)-4-methyl-1H-1,2,3-triazole (28). To a solution of
27 (250 mg, 0.37 mmol) in anhydrous CH2Cl2 (9 mL) were added
4.3.1. Cell culture conditions. The human lung adenocarcinoma
cells A549, human breast adenocarcinoma cells MCF-7 were cul-
tured in Roswell Park Memorial Institute (RPMI) 1640 medium
containing 10% fetal bovine serum (FBS; Gibco/BRL, Grand Island,
NY), 100 U/mL penicillin and 100 mg/mL streptomycin. Human he-
patocellular carcinoma cells Bel-7404 were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% FBS,
Et3N (0.13 mL, 0.93 mmol) and MsCl (72 m
L, 0.93 mmol) at 0 ꢁC under
N2 atmosphere. The reaction mixture was stirred overnight at 0 ꢁC
and quenched by water. The mixture was extracted by CH2Cl2 for
three times. The combined organic phases were washed with 1 N
aq HCl, water and brine, dried over Na2SO4, filtered, and concen-
trated. The residue was re-dissolved in DMF (3 mL) and treated with
NaBH4 (57 mg, 0.08 mmol) at 0 ꢁC. Thereaction mixturewas warmed
to room temperature, and then heated at 70 ꢁC for 5 h. The reaction
was quenched by water, and extracted by Et2O for several times. The
combined organic phases were washed with water and brine, dried
over Na2SO4, filtered, and concentrated. The residue was purified by
chromatography on silica gel to give 28 (140 mg, 58% for two steps)
100 U/mL penicillin and 100 mg/mL streptomycin. All cell lines were
cultivated in a humidified atmosphere of 5% CO2 at 37 ꢁC.
4.3.2. MTT cell growth inhibition assay.7 A549 cells (9ꢃ103), MCF-7
cells (3ꢃ103), and Bel-7404 (7ꢃ103) cells were plated in flat-
bottomed 96-well microplates per well. Background control wells
lacking the cells but containing the same volume of media were
included in each assay plate. Twelve hours after seeding, new
medium containing increasing concentrations of tested compounds
as a colorless oil. [
a
]
25 ꢀ5.36 (c 1.0, CHCl3).1H NMR (300 MHz, CDCl3)
D
d
7.24 (s, 1H), 4.75 (d, J¼6.8 Hz, 2H), 4.64 (d, J¼6.8 Hz, 2H), 4.28 (t,
J¼7.2 Hz, 2H), 3.68 (dt, J¼11.1, 5.4 Hz, 2H), 3.63e3.57 (m, 4H), 3.48 (d,
J¼5.0 Hz, 4H), 3.37 (s, 6H), 2.34 (s, 3H), 1.89e1.80 (m, 2H), 1.49 (t,
J¼6.2 Hz, 4H), 1.24 (brs, 38H), 0.86 (t, J¼6.4 Hz, 3H). 13C NMR
at 0.10e40
further incubated for corresponding time (e.g., 24 h, 48 h or 72 h)
and then treated with MTT (20 L/well, 5 mg/mL) and incubated for
another 4 h. Then the medium was removed carefully and 150 mL
mM or vehicle control (DMSO) was added. Cells were
m
(75 MHz, CDCl3)
d
121.07, 96.19, 76.39, 74.29, 70.95, 55.63, 50.34,
32.21, 32.10, 30.55, 29.94, 29.81, 29.71, 29.58, 29.53, 29.21, 26.69,
25.66, 22.87,14.31,11.04. IR (film, nmax): 2925, 2855,1557,1464,1359,
1215,1144,1105,1041, 918, 722 cmꢀ1. MS (ESI, m/z): 656 [MþH]þ, 678
[MþNa]þ. HRMS (ESI, m/z): [MþNa]þ calcd for C37H73N3O6Na
678.5392; found 678.5398.
DMSO was added to each well, including controls and blanks. After
the samples were shaken gently for 20 min, the absorbance in each
well at 570 nm in a microtiter plate reader (Varioskan Flash) was
measured with a reference wavelength at 690 nm. Experiments
were independently repeated for three times (performed in tripli-
cate), and growth inhibition rate was calculated as follows: growth
4.2.13. (S)-1-(2-(((S)-2-Hydroxydodecyl)oxy)ethoxy)-16-(4-methyl-
inhibition
rate
(%)¼{1ꢀ[
D
OD(compounds)ꢀ
DOD(blank)]/
1H-1,2,3-triazol-1-yl)hexadecan-2-ol (8). To
a
solution of 28
[
D
OD(controls)ꢀ
DOD(blank)]}ꢃ100%. The growth inhibition (IC50
)
(18.0 mg, 0.027 mmol) in methanol (1.6 mL) was added concd HCl
(0.4 mL). The reaction mixture was stirred overnight at room
temperature, and quenched with water (20 mL). The mixture was
extracted with ethyl acetate for several times. The combined or-
ganic phases were washed with saturated aq NaHCO3 solution,
water and brine, dried over Na2SO4, filtered, and concentrated. The
residue was purified by flash chromatography to give 8 (13.2 mg,
for each compound was defined as the concentration of drug
leading to a 50% reduction in A570 compared with controls.
4.3.3. Confocal microscopy analyses. Cells were grown on V 20 mm
glass bottom cell culture dishes, and incubated at 37 ꢁC for 24 h,
florescent probe 7 sample in DMEM (or RPMI-1640) with 10% fetal
bovine serum (FBS) at a concentration of 100 nM were added to
replace the culture medium. After 12 h of incubation, the medium
was removed and the cells were washed three times with PBS. Af-
terward, new medium containing 200 nM MitoTrackerÒ Green FM
(invitrogen) was added to co-stain mitochondria. After 20 min of
incubation at 37 ꢁC, the cells was againwashed three times with PBS.
Cell culture medium of 1 mL was added and the cells were examined
by a Zeiss LSM 710 microscope equipped with a 63ꢃ oil-immersion
objective. The fluorescence was examined under excitation at
543 nm for probe 7 and 488 nm for MitoTrackerÒ Green FM. Image
processing and analysis were done with ZEN 2008 software.
85%) as a white solid. Mp 79e82 ꢁC. [
NMR (300 MHz, CDCl3)
a
]
25 þ9.09 (c 0.53, CHCl3). 1H
D
d
7.25 (s, 1H), 4.29 (t, J¼7.2 Hz, 2H),
3.83e3.74 (m, 2H), 3.73e3.58 (m, 4H), 3.53 (dd, J¼9.8, 2.7 Hz, 2H),
3.32 (t, J¼9.0 Hz, 2H), 2.35 (s, 3H), 1.94e1.80 (m, 2H), 1.44e1.22
(m, 42H), 0.87 (t, J¼6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3)
d 121.10,
76.09, 70.70, 70.49, 50.37, 33.20, 32.12, 30.58, 29.91, 29.82, 29.72,
29.59, 29.56, 29.23, 26.71, 25.76, 22.90, 14.33, 11.06. IR (KBr, nmax):
3464, 3141, 2920, 2848, 1558, 1463, 1438, 1405, 1373, 1261, 1213,
1143, 1098, 1081, 875, 831, 724, 571 cmꢀ1. MS (ESI, m/z): 590
[MþNa]þ. HRMS (ESI, m/z): [MþNa]þ calcd for C33H65N3O4Na
590.4867; found 590.4880.
Acknowledgements
4.2.14. (S)-1-(2-(((S)-2-Hydroxydodecyl)oxy)ethoxy)-16-(4-(piper-
azin-1-ylmethyl)-1H-1,2,3-triazol-1-yl)hexadecan-2-ol
(9). The
Ministry of Science and Technology of the People’s Republic of
China (2010CB833202, SS2013AA090203), National Natural Science
Foundation of China (91213303, 21032002), and National Science
Fund for Talent Training in Basic Science (J1103310) are greatly
appreciated for the financial support.
same procedure was applied as that of synthesizing compound 7,
using azide 25 (100 mg, 0.19 mmol) and alkyne 2921 (70.6 mg,
0.57 mmol), yielding 9 (58 mg, 47%) as a white solid. Mp 81e83 ꢁC.
[
a
]
25 þ12.96 (c 0.22, CHCl3). 1H NMR (300 MHz, CDCl3)
d 7.46 (s,1H),
D
4.32 (t, J¼7.3 Hz, 2H), 3.83e3.73 (m, 2H), 3.72e3.58 (m, 6H), 3.52
(dd, J¼9.8, 2.6 Hz, 2H), 3.31 (t, J¼9.0 Hz, 2H), 2.89 (t, J¼4.7 Hz, 4H),
2.49 (s, 4H), 1.88 (dt, J¼12.9, 6.4 Hz, 2H), 1.49e1.21 (m, 42H), 0.87 (t,
Supplementary data
J¼6.4 Hz, 3H). 13C NMR (75 MHz, CDCl3)
d
144.23, 122.55, 76.06,
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2014.05.047.
70.57, 70.28, 54.12, 54.00, 50.43, 45.85, 33.20, 32.02, 30.40, 29.81,
Please cite this article in press as: Chen, Y.-J.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.047

8
Y.-J. Chen et al. / Tetrahedron xxx (2014) 1e8
2006, 45, 2721e2728; (d) Kojima, N.; Fushimi, T.; Tatsukawa, T.; Yoshimitsu, T.;
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