An efficient synthesis of LipidGreen and its derivatives via microwave assisted reaction and their live lipid imaging in zebrafish

Article abstract of DOI:10.1016/j.tet.2013.01.090

We have developed an efficient synthesis of LipidGreen. The conversion is achieved by selective methylation with trimethylsilyldiazomethane, selective deprotection by BBr₃ and an improved microwave-assisted C-allylation procedure. Using this ro

Full text of DOI:10.1016/j.tet.2013.01.090

Tetrahedron 69 (2013) 3039e3044
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
An efficient synthesis of LipidGreen and its derivatives via
microwave assisted reaction and their live lipid imaging in zebrafish
,
,
a,b,
Haushabhau S. Pagire a b, Hang-Suk Chun a, Myung Ae Bae a , Jin Hee Ahn
*
*
a Bioorganic Science Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, South Korea
b Medicinal and Pharmaceutical Chemistry Major, University of Science and Technology, 3085-333, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 August 2012
Received in revised form 28 January 2013
Accepted 31 January 2013
Available online 8 February 2013
We have developed an efficient synthesis of LipidGreen. The conversion is achieved by selective
methylation with trimethylsilyldiazomethane, selective deprotection by BBr3 and an improved
microwave-assisted C-allylation procedure. Using this route, we have synthesized novel LipidGreen
derivatives, and evaluated their live imaging abilities in zebrafish. In this series, Compound 7 is the most
active, which is at least 10 fold more potent than LipidGreen.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
LipidGreen
Fluorescent probe
Lipid imaging
Small molecule
Zebrafish
1. Introduction
Lipid droplets (LDs), which are observed in many disease states,
consist of a neutral lipid core (primarily triacylglycerol and cho-
lesteryl ester) surrounded by a phospholipid monolayer.1 LDs have
been considered as inert and static aggregates of neutral lipids.
However, this view has changed dramatically in recent years. Now
LDs are regarded as dynamic and complex subcellular organelles in
adipocytes of fat tissues.2e5 Increased fat tissues have been recog-
nized as highly relevant for widespread and serious human
diseases such as diabetes, obesity, alcoholic and non-alcoholic
hepatosteatosis, and atherosclerosis. In order to study LDs in liv-
ing cells and bodies, fluorescence imaging is an essential tool.
Traditionally, fluorescent dyes such as Oil Red O (ORO) and Nile
Red6e8 have been used extensively for the fluorescent labeling of
LDs. Also, immunofluorescence of LD-associated proteins has also
been used for indirect observation of LDs. However, in general,
these methods are only applicable to fixed samples and cause de-
formation of LD structure. Recently we reported LipidGreen9 as
a new small molecule probe, which stained lipid droplets in 3T3L1
cell lines and fat deposits in zebrafish.
was only w12% or the silyl intermediate was unstable. Therefore,
we tried to develop a more efficient synthetic route with proposed
pathway (Scheme 1), and herein, we wish to report our improved
methodology for the synthesis of LipidGreen and its derivatives and
their live lipid imaging in zebrafish.
2. Results and discussion
Commercially available indole-2-carboxylic acid (1) was
converted to ester, followed by the Vilsmeier reaction afforded
formyl indole, which further underwent BaeyereVilliger oxidation
with m-CPBA afforded compound 2. Next, we studied selective
O-protection with methyl group instead of previous silyl protection.
As shown in Table 1, using dimethylsulfate and methyl iodide
with bases, reactions were not successful. Fortunately, selective
O-methylation was achieved in excellent yield (99%) without
C or N methylation using trimethylsilyldiazomethane at room
temperature.
In our previous study, LipidGreen was synthesized through two
different pathways; one with and one without protecting group.
However, over the course of the synthesis, final C allylation yield
* Corresponding authors. E-mail address: jhahn@krict.re.kr (J.H. Ahn).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.01.090

3040
H.S. Pagire et al. / Tetrahedron 69 (2013) 3039e3044
OH
O- and C-allylated mixtures were obtained in our previous study.
Therefore, we explored the optimization of a selective C-allylation
procedure under several reaction conditions as shown in Table 3.
O
O
O
O
O
a,b,c
N
H
N
H
OH
2
Table 3
1
Optimization of C-allylation for the synthesis of LipidGreen
d
Protection
O
Protection
O
O
O
O
O
e
N
O
N
O
H
3
4
f
O
O
OH
g
Entry Solvent
Base
Heating
method
Temp
(ꢁC)
Time Yield
(h)
O
O
O
O
N
O
N
1
2
3
4
5
6
7
8
Benzene
Acetone
Acetone
Acetone
Acetone
1,4 Dioxane K2CO3
DMF
Acetone
NaH
Oil bath
Oil bath
Microwave 80 ꢁC
Reflux 5 h
Reflux 24 h w40%
w5%
K2CO3
K2CO3
K2CO3
K2CO3
1.5 h w56%
6
Microwave 100 ꢁC 1.5 h w78%
Microwave 130 ꢁC 1.5 h 99%
LipidGreen
5
Microwave 130 ꢁC 1.5 h d (decomposed)
Microwave 130 ꢁC 1.5 h w55%
Scheme 1. Synthesis of LipidGreen; reagents and optimized conditions: (a) H2SO4,
EtOH, 10 h, reflux, 99%; (b) DMF, POCl3, 3 h, room temperature, 99%; (c) m-CPBA,
TsOH$H2O, CH2Cl2, 8 h, room temperature, 70%; (d) 2.0 M TMSCHN2 in hexanes,CH3OH,
THF, 24 h, room temperature, 99%; (e) NaH, DMF, allyl iodide, 3 h, room temperature,
99%; (f) BBr3, CH2Cl2, 45 min, ꢀ30 ꢁC, 99%; (g) allyl iodide, K2CO3, Acetone, 130 ꢁC, MW,
1.5 h, 99%.
K2CO3
Na2CO3 Microwave 130 ꢁC 1.5 h w88%
As can be seen in Table 1, under regular heating conditions
(entries 1 and 2), C-allylated product 6 (LipidGreen) was isolated in
less than 50% yield. In contrast, the microwave assisted conditions
dramatically improved the yield of the C-allylation product. Among
the various solvent, base, and reaction temperature conditions, we
were able to quantitatively obtain LipidGreen (99% yield) from
compound 5 in the presence of K2CO3 in acetone at 130 ꢁC (entry 5).
Using this microwave condition, we further investigated C-ally-
lation (including cinnamylation), C-benzylation, and C-alkylation
reactions of compound 5, and results are summarized in Table 4.
Table 1
Selective O-methylation of compound 2
Table 4
Entry
Solvent
Reagent
Base
Temp (ꢁC)
Time (h)
Yield
Microwave assisted allylation, benzylation, and alkylations
1
2
3
4
H2O
Me2SO4
CH3I
CH3I
KOH
K2CO3
K2CO3
d
rt
24 h
24 h
24 h
24 h
w20%
wtrace
wtrace
99%
Acetone
Acetone
THF
Reflux
Reflux
rt
(CH3)3SiCHN2
The N-allylation reaction was smoothly proceeded at room
temperature to give compound 4 (99% yield). Next, selective
demethylation at the 3-position was required and examined as
shown in Table 2. Without demethylation at the 5-position, this was
successfully accomplished in 99% yield by BBr3 at below ꢀ30 ꢁC.
Compd no
RX
I:IIa
Yield Ib
99%
6 LipidGreen
w100:0
7
8
w100:0
w90:10
96%
88%
Table 2
Selective demethylation of compound 4
9
10
CH3I
CH3CH2I
w85:15
w80:20
81%
77%
a
C/O ratio was determined by NMR and LC.
Isolated yield.
b
As shown in Table 4, compound
5 underwent selective
Entry
Temp (ꢁC)
Time (h/min)
Yield
C-allylation with cinnamyl bromide to produce the corresponding
2-cinnamyl indole derivative (7) in excellent yield (99%) under
microwave condition. Also, the microwave-assisted benzylation
smoothly proceeded to obtain C-benzylated product (8) in good
yield (88%, C/O benzylation ratio 90:10). The structure of 8 was
determined by single crystal X-ray diffraction (Fig. 2). Moreover,
alkylation with methyl and ethyl iodide was examined and the C-
alkylation products (compounds 9 and 10, respectively) were
produced in yields of approximately 80%.
1
2
3
ꢀ78
ꢀ30
0
2 h
45 min
1 h
99%
99%
20%
Finally, allylation of compound 5 to give the desired C-allylated
product (LipidGreen, 6) was investigated. The alkylation reactions
of 3-hydroxyindole-2-carboxylate esters with alkyl halides usually
produce mixture of O- and C-alkylated products. Indeed, such

H.S. Pagire et al. / Tetrahedron 69 (2013) 3039e3044
3041
Table 5
Comparison of lipid imaging efficacies of LipidGreen and its derivatives by
observation of fat accumulation in zebrafish
Compd no
20
m
M
10 mM
LipidGreen 6
7
ND
Fig. 1. Quantitative analysis of the fluorescence intensity (gut area of zebrafish) of
compound 6e10.
8
ND
9
ND
10
ND
Fig. 2. X-ray crystal of 8.
ND: not determined; top panel: fluorescence with bright field image; bottom panel:
fluorescence image.
The lipid imaging abilities of newly synthesized indolinone
derivatives 7e10 were evaluated in live zebrafish.
Zebrafish10,11 are optically transparent from the time of external
fertilization to early adulthood. Therefore the zebrafish model
provides an excellent opportunity to monitor and study fat accu-
mulation through in vivo observation of developmental and phys-
iological processes.
Table 6
We compared LipidGreen to newly synthesized compounds
7e10 to determine whether any of the new derivatives had better
lipid imaging capabilities and the results summarized in Table 5. As
shown in Table 5, compound 7 showed dramatically increased
Dose dependency test and quantitative analysis of the fluorescence changes for
compound 7 and 8
Compound 7
Compound 8
fluorescence in fat deposits of zebrafish at 10 mM. Benzyl derivative
(8) also have better imaging ability than LipidGreen. However,
methyl and ethyl derivatives (compounds 9 and 10) showed weak
lipid imaging efficacy in zebrafish.
5 mM
Next, we stained fat deposits in zebrafish with two active
compounds (compounds 7 and 8) at different doses (Table 6) and
quantitatively analyzed the fluorescence intensity of 6e10 (Fig. 1).
Compound 7 showed good fluorescence intensity even at 1
M and 10 M, saturated fluorescence intensities were shown),
whereas compound 8 showed weak fluorescence in 1 M. As
shown in Fig.1, it was confirmed that the fluorescence of compound
7 makes it an effective stain for lipid deposit in zebrafish. Com-
pound 7 is the most active compound in this series and is at least 10
mM (at
5
m
m
m
1 mM
fold more fluorescent than LipidGreen at 10 mM.

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H.S. Pagire et al. / Tetrahedron 69 (2013) 3039e3044
3. Conclusion
To a solution of 3-formyl-5-methoxy-indole-2-carboxylic acid
ethyl ester (22 g, 88.98 mmol) in CH2Cl2 (176 mL) were added
TsOH$H2O (16.7 g, 88.98 mmol) and m-CPBA (30.8 g, 133.47 mmol)
at room temperature. The reaction mixture was stirred for 8 h at
room temperature and then evaporated. The residue was purified
by silica gel column chromatography using CH2Cl2 to give 3-
hydroxy-5-methoxyindole-2-carboxylic acid ethyl ester (14.6 g,
70%) as a yellow solid. Mp 96 ꢁC; IR (ATR) vmax¼3411, 3273,
1694 cmꢀ1; Rf 0.29 (n-Hexane/EtOAc, 4:1); 1H NMR (300 MHz,
An efficient synthesis of LipidGreen has been developed. Selective
methylation with trimethylsilyldiazomethane, selective depro-
tection using BBr3 and an improved microwave-assisted C-allylation
reaction were investigated and an optimized process was estab-
lished. Using the aforementioned route, novel LipidGreen derivatives
were synthesized, andtheiractivities were evaluated forlive imaging
in zebrafish. In this series, compound 7 was found to be the most
active, having at least 10 fold more potency than LipidGreen.
CDCl3)
d
7.68 (br s, 1H), 7,17 (d, J¼9.0 Hz, 1H), 7.10 (d, J¼1.8 Hz, 1H),
7.01 (dd, J¼8.8, 2.3 Hz, 1H), 4.43 (q, J¼7.1 Hz, 2H), 3.85 (s, 3H), 1.42
4. Experimental section
(t, J¼7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3);
d 163.7, 153.8, 147.8,
130.8, 119.2, 117.5, 113.0, 108.8, 99.5, 60.6, 55.6, 14.52; LC-MS (m/z):
4.1. Materials and methods
236 (MHþ); HRMS (C12H13NO4): calcd, 235.0845 found, 235.0843.
Microwave reactions were performed in a Biotage Initiator 2.5
single mode microwave reactor with a new sealed pressure regu-
lation 20 mL pressurized vial. Melting points were determined on
an MEL-TEMP apparatus and are uncorrected. IR spectra were ob-
tained on a Smith ATR-FT-IR spectrometer and the absorption fre-
quencies are reported in wavelength (cmꢀ1). 1H NMR spectra and
13C NMR spectra were run on Bruker AVANCE-500, Bruker AVANCE-
300 and Varian OXFORD-300 spectrometers at 500 and 300 MHz
4.2.2. 3,5-Dimethoxy-1H-indole-2-carboxylic acid ethyl ester (3). To
a solution of 3-hydroxy-5-methoxy-1H-indole-2-carboxylic acid
ethyl ester 2 (4 g, 17 mmol) in tetrahydrofuran (100 mL) was added
trimethylsilyldiazomethane solution 2 M in n-hexane (40 mL) and
methanol (40 mL). The reaction mixture was stirred for 24 h at room
temperature. The reaction mixture was evaporated, diluted with
H2O and extracted with dichloromethane. Organic layer was sep-
arated, dried over anhydrous MgSO4, and evaporated under re-
duced pressure. The residue was purified by silica gel column
chromatography to give 3,5-dimethoxy-1H-indole-2-carboxylic
acid ethyl ester (4.19 g, 99%) as a yellow solid. Mp 113 ꢁC; IR (ATR)
vmax¼3323,1645 cmꢀ1; Rf 0.27 (n-Hexane/EtOAc, 4:1); 1H NMR
for 1H NMR, and 125 and 75 MHz for 13C NMR. Chemical shifts (
d)
are expressed in parts per million downfield from TMS as internal
standard. The letters s, d, t, q, and m are used to indicate singlet,
doublet, triplet, quadruplet, and multiplet, respectively. High-res-
olution mass spectra were obtained on the Autospec Magnetic
sector mass spectrometer (Micromass, Manchester, UK). All anhy-
drous solvents (stored over molecular sieves) and chemicals were
obtained from standard commercial vendors and were used with-
out any further purification. The reactions were monitored by thin-
layer chromatography (TLC) and column chromatography was
performed using Chromatorex GS60-40/75. X-ray diffraction crystal
structure analysis was obtained on Bruker SMART APEX II.
(300 MHz, CDCl3)
d
8.24 (s, 1H), 7.22 (d, J¼9.0 Hz, 1H), 7.09 (s, 1H),
6.99 (d, J¼9.1 Hz,1H), 4.42 (q, J¼7.1 Hz, 2H), 4.08 (s, 3H), 3.86 (s, 3H),
1.43 (t, J¼7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3);
d 161.3,154.2,144.8,
129.7, 121.0, 118.0, 115.6, 113.1, 99.6, 62.5, 60.7, 55.7, 14.4.
4.2.3. 1-Allyl-3,5-dimethoxy-1H-indole-2-carboxylic acid ethyl ester
(4). To a solution of 3,5-dimethoxy-1H-indole-2-carboxylic acid
ethyl ester 3 (3 g, 12.03 mmol) in DMF (10 mL) was added NaH
(527.3 mg, 13.23 mmol) and allyl iodide (2.2 g, 13.23 mmol). The
reaction mixture was stirred for 3 h at room temperature. The
resulting mixture was diluted with H2O and extracted with ethyl
acetate. Organic layer was separated, dried over anhydrous MgSO4,
and evaporated under reduced pressure. The residue was purified
by silica gel column chromatography to give 1-allyl-3,5-dimethoxy-
1H-indole-2-carboxylic acid ethyl ester (3.47 g, 99%)as an oil. IR
(ATR) vmax¼1695 cmꢀ1; Rf 0.48 (n-Hexane/EtOAc, 4:1); 1H NMR
4.2. General procedure for the synthesis of 2e10
4.2.1. 3-Hydroxy-5-methoxyindole-2-carboxylic acid ethyl ester
(2). A mixture of 5-methoxyindole-2-carboxylic acid 1 (20 g,
104.61 mmol) and sulfuric acid (16 mL) in EtOH (160 mL) was
refluxed for 10 h. The reaction mixture was evaporated, neutralized
to pH 7 with 2 N-NaOH and extracted with ethyl acetate. The or-
ganic layer was dried over anhydrous Na2SO4, and evaporated un-
der reduced pressure. The residue was purified by silica gel column
chromatography using solvent CH2Cl2 and isolated by ether to give
5-methoxyindole-2-carboxylic acid ethyl ester (22.8 g, 99%). Mp
157 ꢁC; IR (ATR) vmax¼3325, 1676 cmꢀ1; Rf 0.39 (n-Hexane/EtOAc,
(300 MHz, CDCl3)
d
7.22 (d, J¼9.0 Hz, 1H), 7.08 (s, 1H), 7.00 (d,
J¼8.97 Hz,1H), 6.03e5.87 (m,1H), 5.11e5.04 (m, 3H), 4.93e4.83 (m,
1H), 4.41 (q, J¼7.2 Hz, 2H), 4.01 (s, 3H), 3.86 (s, 3H), 1.43 (t, J¼7.1 Hz,
3H); 13C NMR (75 MHz, CDCl3);
d 161.5, 154.2, 145.6, 134.2, 131.9,
119.9, 117.6, 116.9, 115.8, 111.6, 99.4, 62.7, 60.4, 55.7, 46.8, 14.33.
4:1); 1H NMR (300 MHz, CDCl3)
d
8.81 (br s, 1H), 7.31 (d, J¼8.9 Hz,
1H), 7.16e7.05 (m, 2H), 7.00 (dd, J¼9.0, 2.1 Hz), 4.40 (q, J¼7.1 Hz, 2H),
4.2.4. 3-Hydroxy-5-methoxy-1-allyl-1H-indole-2-carboxylic
acid
3.85 (s, 3H), 1.41 (t, J¼7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3);
d
162.0,
ethyl ester (5). To a solution of 1-allyl-3,5-dimethoxy-1H-indole-2-
carboxylic acid ethyl ester 4 (2.5 g, 8.64 mmol) in CH2Cl2 (25 mL)
was added 1 M BBr3 solution in hexane (8.72 mL, 8.72 mmol). The
reaction mixture was stirred for 45 min at ꢀ30 ꢁC. After that time,
CH2Cl2 (25 mL) and saturated sodium bicarbonate solution (25 mL)
were added slowly, and reaction mixture was extracted with CH2Cl2
(2ꢂ20 mL). Combined organic phases were washed with brine
(20 mL), dried over anhydrous sodium sulfate and evaporated un-
der reduced pressure to offered the crude product, which was
purified by silica gel column chromatography to give 1-allyl-3-
hydroxy-5-methoxy-1H-indole-2-carboxylic acid ethyl ester
(1.7 g, 99%) as a yellow solid. Mp 56 ꢁC, IR (ATR) vmax¼3331,
1664 cmꢀ1; Rf 0.44 (n-Hexane/EtOAc, 4:1); 1H NMR (300 MHz,
154.7, 132.3, 127.9, 127.8, 116.7, 112.8, 108.2, 102.5, 60.9, 55.7, 14.4.
To a solution of 5-methoxyindole-2-carboxylic acid ethyl ester
(21 g, 95.78 mmol) in DMF (175 mL) was added phosphorus oxy-
chloride (22.32 mL, 239.47 mmol) at room temperature. The re-
action mixture was stirred for 3 h at room temperature, evaporated
excess phosphorus oxychloride and neutralized to pH 7 by 2 N-
NaOH at 0 ꢁC. The resulting solid was collected and washed with
H2O to give 3-formyl-5-methoxy-indole-2-carboxylic acid ethyl
ester (23.6 g, 99%). Mp 241 ꢁC; IR (ATR) vmax¼3110, 1706,
1635 cmꢀ1; Rf 0.23 (n-Hexane/EtOAc, 4:1); 1H NMR (300 MHz,
DMSO-d6)
d
12.66 (s, 1H), 10.58 (s, 1H), 7.69 (d, J¼2.07 Hz, 1H), 7.47
(d, J¼8.9 Hz, 1H), 7.03 (dd, J¼9.0, 2.4 Hz, 1H), 4.43 (q, J¼7.0 Hz, 2H),
3.80 (s, 3H), 1.38 (t, J¼7.1 Hz, 3H); 13C NMR (75 MHz, DMSO-d6);
CDCl3)
d
8.65 (br s, 1H), 7.15 (d, J¼9.0 Hz, 1H), 7.11 (d, J¼2.1 Hz, 1H),
d
187.9, 160.6, 157.1, 132.8, 131.3, 126.1, 118.6, 117.6, 114.7, 102.8, 62.1,
7.03 (dd, J¼9.0, 2.1 Hz, 1H), 5.96e5.81 (m, 1H), 5.10e5.02 (m, 1H),
4.98e4.83 (m, 3H), 4.44 (q, J¼7.1 Hz, 2H), 3.85 (s, 3H), 1.42 (t,
55.7, 14.5. HRMS (C13H13NO4): calcd, 247.0845 found, 247.0841.

H.S. Pagire et al. / Tetrahedron 69 (2013) 3039e3044
3043
J¼7.1 Hz, 3 H); 13C NMR (75 MHz, CDCl3);
d
164.0,153.6,148.3,134.0,
F2, Data/restraints/parameters¼4710/0/244, Goodness-of-fit on
F2¼1.050, Final R indices [I>2sigma(I)]¼R1¼0.0419, wR2¼0.0953, R
indices (all data)¼R1¼0.0497, wR2¼0.0995, Largest diff. peak and
133.0, 119.3, 116.4, 115.8, 111.3, 109.3, 99.6, 60.6, 55.6, 46.9, 14.4;
HRMS (C15H17NO4): calcd, 275.1158 found, 275.1157.
ꢀꢀ3
hole¼0.318 and ꢀ0.197 e.A . These data can be obtained free of
4.2.5. 1,2-Diallyl-5-methoxy-3-oxoindoline-2-carboxylic acid ethyl
ester (6). To a solution of 1-allyl-3-hydroxy-5-methoxy-1H-indole-
2-carboxylic acid ethyl ester 5 (1 g, 3.63 mmol) in acetone (40 mL)
were added K2CO3 (2.5 g, 18.16 mmol) and allyl iodide (1.2 g,
7.2 mmol). The reaction mixture was subsequently irradiated in
a single-mode microwave instrument (Biotage Initiator 2.5) at
130 ꢁC for 1.5 h. The reaction mixture was evaporated, diluted with
H2O and extracted with ethyl acetate. Organic layer was separated,
dried over anhydrous MgSO4, and evaporated under reduced
pressure to offered the crude product, which was purified by silica
gel column chromatography to give 1,2-diallyl-5-methoxy-3-
oxoindoline-2-carboxylic acid ethyl ester (1.12 g, 99%)as an oil. IR
(ATR) vmax¼1739, 1697 cmꢀ1; Rf 0.41 (n-Hexane/EtOAc, 4:1); 1H
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
4.2.8. 1-Allyl-5-methoxy-2-methyl-3-oxo-2,3-dihydro-1H-indole-2-
carboxylic acid ethyl ester (9). Yellow oil 37 mg (yield 81%). IR (ATR)
vmax¼1738, 1698 cmꢀ1; Rf 0.36 (n-Hexane/EtOAc, 4:1); 1H NMR
(500 MHz, CDCl3)
d
7.15 (dd, J¼8.9, 2.5 Hz, 1H), 7.05 (d, J¼2.5 Hz,
1H), 6.76 (d, J¼8.9 Hz, 1H), 5.91e5.82 (m, 1H), 5.30e5.17 (m, 2H),
4.19e4.10 (m, 2H), 4.03e3.82 (m, 2H), 3.76 (s, 3H), 1.55 (s, 3H), 1.20
(t, J¼7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3);
d 196.6, 168.3, 156.8,
152.6, 133.7128.5, 118.4,117.0,110.9,105.3, 74.7, 62.0, 55.8, 46.7,18.4,
14.0; HRMS (C16H19NO4): calcd, 289.1314 found, 289.1311; Ele-
mental analysis for C16H19NO4. Calcd: C, 66.42%, H, 6.62%, N, 4.84%.
Found: C, 66.07%, H, 6.72%, N, 4.93%.
NMR (300 MHz, CDCl3)
d
7.14 (dd, J¼9.0, J¼2.4 Hz, 1H), 7.02 (d,
J¼2.4 Hz, 1H), 6.79 (d, J¼8.9 Hz, 1H), 5.97e5.80 (m, 1H), 5.54e5.38
(m, 1H), 5.35e5.10 (m, 3H), 5.02e4.95 (m, 1H), 4.19e4.10 (m, 2H),
4.05e3.82 (m, 2H), 3.76 (s, 3H), 3.05e2.86 (m, 2H), 1.20 (t, J¼7.1 Hz,
4.2.9. 1-Allyl-2-ethyl-5-methoxy-3-oxo-2,3-dihydro-1H-indole-2-
carboxylic acid ethyl ester (10). Yellow oil 27 mg (yield 77%). IR
(ATR) vmax¼1737, 1695 cmꢀ1; Rf 0.38 (n-Hexane/EtOAc, 4:1); 1H
3H); 13C NMR (75 MHz, CDCl3);
d 194.4, 166.6, 156.7, 151.6, 132.8,
129.9, 127.2, 118.7, 118.4, 116.2, 109.8, 104.0, 76.9, 61.0, 54.7, 46.5,
35.9, 13.0; HRMS (C18H21NO4): calcd, 315.1471 found, 315.1470. El-
emental analysis for C18H21NO4. Calcd: C, 68.55%, H, 6.71%, N, 4.44%.
Found: C, 67.98%, H, 6.71%, N, 4.41%.
The following compounds 7e10 were prepared from the cor-
responding starting materials in a similar manner to that described
for compound 6.
NMR (500 MHz, CDCl3)
d
7.14 (d, J¼6.73 Hz, 1H), 7.03 (s, 1H), 6.81 (d,
J¼8.3 Hz, 1H), 5.95e5.85 (m, 1H), 5.35e5.18 (m, 2H), 4.19e4.07 (m,
2H), 4.02e3.82 (m, 2H), 3.76 (s, 3H), 2.41e2.04 (m, 2H), 1.19 (t,
J¼7.1 Hz, 3H), 0.7 (t, J¼7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3);
d 195.8, 167.5, 156.7, 151.5, 133.6128.4, 118.4, 116.8, 110.8, 104.9, 73.6,
61.8, 55.7, 46.7, 17.6, 14.0, 13.1; HRMS (C17H21NO4): calcd, 303.1471
found, 303.1476. Elemental analysis for C17H21NO4. Calcd: C, 67.31%,
H, 6.98%, N, 4.62%. Found: C, 66.98%, H, 6.78%, N, 4.59%.
4.2.6. 1-Allyl-5-methoxy-3-oxo-2-(3-phenyl-allyl)-2,3-dihydro-1H-
indole-2-carboxylic acid ethyl ester (7). Oil 161 mg (yield 96%). IR
(ATR) vmax¼1737, 1696 cmꢀ1; Rf 0.39 (n-Hexane/EtOAc, 4:1); 1H
4.3. Biological experiment
NMR (300 MHz, CDCl3)
d
7.24e7.08 (m, 5H), 7.02 (d, J¼2.3 Hz, 1H),
4.3.1. Maintenance of fish. Zebrafish were raised and kept under
standard laboratory condition. Zebrafish embryos were obtained
from spontaneous spawning and raised at 28.5 ꢁC in egg water.
Zebrafish are staged and fixed at specific days post fertilization (dpf).
6.78 (d, J¼8.9 Hz, 1H), 6.49 (d, J¼15.7 Hz, 1H), 5.99e5.78 (m, 2H),
5.36e5.18 (m, 2H), 4.23e4.10 (m, 2H), 4.09e3.84 (m, 2H), 3.75 (s,
3H), 3.18e3.02 (m, 2H), 1.22 (t, J¼7.1 Hz, 3H); 13C NMR (75 MHz,
CDCl3);
d 195.3, 167.5, 157.8, 152.6, 137.0, 134.5, 133.8, 128.4 (2CH),
128.3, 127.3, 126.1 (2CH), 122.5, 119.4, 117.2, 111.0, 105.2, 78.3, 62.1,
55.7, 47.7, 36.4, 14.1; HRMS (C24H25NO4): calcd, 391.1784 found,
391.1797. Elemental analysis for C24H25NO4. Calcd: C, 73.64%, H,
6.44%, N, 3.58%. Found: C, 72.96%, H, 6.47%, N, 4.06%.
4.3.2. Treatment of LipidGreen and derivatives with zebrafish. Zebra-
fish embryos were treated with LipidGreen and derivatives that was
diluted in DMSO. Zebrafish were incubated with several concen-
trations (1, 5, 10, 20 mM) LipidGreen or derivatives in the dark for
15 min. After incubation of LipidGreen or derivatives, zebrafish
were washed with egg water for 30 min. After washing, photogra-
phy was performed on a Leica MZ10F.
4.2.7. 1-Allyl-2-benzyl-5-methoxy-3-oxo-2,3-dihydro-1H-indole-2-
carboxylic acid ethyl ester (8). Yellowish solid, 58.4 mg (yield 88%),
mp 73e76 ꢁC, IR (ATR) vmax¼1737,1696 cmꢀ1; Rf 0.33 (n-Hexane/
EtOAc, 4:1); 1H NMR (300 MHz, CDCl3)
d 7.40e7.35 (m, 2H), 7.09 (s,
4.3.3. Imaging and quantitative analysis. LipidGreen or derivatives
stained-embryos were imaged by using Leica MZ10F microscope.
After compound staining and washing, embryos were anaes-
thetized with tricaine and embedded in 3% methylcellulose for
imaging. Laser at 470/40 nm or 545/30 nm filter was used for ex-
citation. Images were acquired with 525/50 nm or 620/60 nm
emission filter. Quantitative analysis was performed by LAS v3.8
Leica Microsystems software (Experimental Details).
3H), 7.00 (dd, J¼9.0, 2.5 Hz, 1H), 6.93 (d, J¼2.43 Hz, 1H), 6.65 (d,
J¼8.9 Hz, 1H), 5.89e5.73 (m, 1H), 5.24e5.15 (m, 1H), 4.73e4.68 (m,
1H), 4.17 (q, J¼7.1 Hz, 2H), 4.07e3.82 (m, 2H), 3.71 (s, 3H), 3.61 (d,
J¼14.4 Hz, 1H), 3.42 (d, J¼14.4 Hz, 1H), 1.22 (t, J¼7.1 Hz, 3H); 13C
NMR (75 MHz, CDCl3);
d 195.8, 167.9, 157.7, 152.4, 134.5, 133.8, 130,
128.5, 127.9, 127.6, 126.9, 126.8, 119.9, 117.4, 110.9, 104.9, 78.9, 62.1,
55.6, 47.8, 37.9, 14.0; HRMS (C22H23NO4): calcd, 365.1627 found,
365.1630. Elemental analysis for C22H23NO4. Calcd: C, 72.31%, H,
6.34%, N, 3.83%. Found: C, 72.17%, H, 6.58%, N, 3.83%.
Acknowledgements
Crystal data of 8. CCDC reference number is 917644, crystal
size0.28ꢂ0.24ꢂ0.08 mm3, crystal system monoclinic, space
This research was supported by the Ministry of Education, Sci-
ence and Technology (2010-0019773), the Ministry of Knowledge
Economy (2012-10033674 and SI-1206), Korea.
ꢀ
ꢀ
ꢀ
groupP2(1)/n, Z¼4,a¼7.93460(10) A, b¼17.7108(3) A, c¼13.7296(2) A,
alpha¼90ꢁ, beta¼101.1640(10)ꢁ, gamma¼90ꢁ, volume¼1892.88-
(5) A , density¼1.282 Mg/m3, T¼100(1) K, Absorption coeffi-
3
ꢀ
cient¼0.088 mmꢀ1
F(000)¼776, Theta range for data collec-
,
tion¼1.90e28.35ꢁ, Index ranges¼ꢀ10ꢃhꢃ10, ꢀ23ꢃkꢃ23, ꢀ18ꢃlꢃ18,
Reflections collected¼50,651, Independent reflections¼4710 [R(int)¼
0.0278], Completeness to theta¼28.35ꢁ¼99.6%, Absorption correc-
tion¼None, Refinement method¼Full-matrix least-squares on
Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2013.01.090.

3044
H.S. Pagire et al. / Tetrahedron 69 (2013) 3039e3044
7. Flynn, E. J., III; Trent, C. M.; Rawls, J. F. J. Lipid Res. 2009, 50, 1641.
References and notes
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