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

Tetrahedron 69 (2013) 3039e3044

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An efficient synthesis of LipidGreen and its derivatives via microwave assisted reaction and their live lipid imaging in zebrafish Haushabhau S. Pagire a, b, Hang-Suk Chun a, Myung Ae Bae a, *, Jin Hee Ahn a, b, * a b

Bioorganic Science Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, South Korea 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 cholesteryl 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 recognized 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 living 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 deformation 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. 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: [email protected] (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

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.

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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.

OH O

O N H

O

a,b,c

O N H

OH

O

2

Table 3 Optimization of C-allylation for the synthesis of LipidGreen

1 d O Protection O

O

N

O Protection O

O

e

O

N H

O

4 3 f O OH O

O

g

O

N

O

O

6 LipidGreen

5

Entry

Solvent

Base

Heating method

Temp ( C)

Time (h)

Yield

1 2 3 4 5 6 7 8

Benzene Acetone Acetone Acetone Acetone 1,4 Dioxane DMF Acetone

NaH K2CO3 K2CO3 K2CO3 K2CO3 K2CO3 K2CO3 Na2CO3

Oil bath Oil bath Microwave Microwave Microwave Microwave Microwave Microwave

Reflux Reflux 80  C 100  C 130  C 130  C 130  C 130  C

5h 24 h 1.5 h 1.5 h 1.5 h 1.5 h 1.5 h 1.5 h

w5% w40% w56% w78% 99% d (decomposed) w55% w88%

O

N

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%.

Table 1 Selective O-methylation of compound 2

Entry

Solvent

Reagent

Base

Temp ( C)

Time (h)

Yield

1 2 3 4

H2O Acetone Acetone THF

Me2SO4 CH3I CH3I (CH3)3SiCHN2

KOH K2CO3 K2CO3 d

rt Reflux Reflux rt

24 24 24 24

w20% wtrace wtrace 99%

h h h h

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. Table 2 Selective demethylation of compound 4

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-allylation (including cinnamylation), C-benzylation, and C-alkylation reactions of compound 5, and results are summarized in Table 4. Table 4 Microwave assisted allylation, benzylation, and alkylations

I:IIa

Yield Ib

6 LipidGreen

w100:0

99%

7

w100:0

96%

Compd no

RX

8 9 10 a b

Entry

Temp ( C)

Time (h/min)

Yield

1 2 3


78
30 0

2h 45 min 1h

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

CH3I CH3CH2I

w90:10

88%

w85:15 w80:20

81% 77%

C/O ratio was determined by NMR and LC. Isolated yield.

As shown in Table 4, compound 5 underwent selective 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 Calkylation products (compounds 9 and 10, respectively) were produced in yields of approximately 80%.

H.S. Pagire et al. / Tetrahedron 69 (2013) 3039e3044

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Table 5 Comparison of lipid imaging efficacies of LipidGreen and its derivatives by observation of fat accumulation in zebrafish Compd no

20 mM

10 mM

LipidGreen 6

Fig. 1. Quantitative analysis of the fluorescence intensity (gut area of zebrafish) of compound 6e10.

7

ND

8

ND

9

ND

10

ND

Fig. 2. X-ray crystal of 8.

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 accumulation through in vivo observation of developmental and physiological processes. 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 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. 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 mM (at 5 mM and 10 mM, saturated fluorescence intensities were shown), whereas compound 8 showed weak fluorescence in 1 mM. As shown in Fig. 1, it was confirmed that the fluorescence of compound 7 makes it an effective stain for lipid deposit in zebrafish. Compound 7 is the most active compound in this series and is at least 10 fold more fluorescent than LipidGreen at 10 mM.

ND: not determined; top panel: fluorescence with bright field image; bottom panel: fluorescence image.

Table 6 Dose dependency test and quantitative analysis of the fluorescence changes for compound 7 and 8 Compound 7

5 mM

1 mM

Compound 8

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3. Conclusion An efficient synthesis of LipidGreen has been developed. Selective methylation with trimethylsilyldiazomethane, selective deprotection using BBr3 and an improved microwave-assisted C-allylation reaction were investigated and an optimized process was established. Using the aforementioned route, novel LipidGreen derivatives were synthesized, and their activities were evaluated for live imaging in zebrafish. In this series, compound 7 was found to be the most active, having at least 10 fold more potency than LipidGreen. 4. Experimental section 4.1. Materials and methods Microwave reactions were performed in a Biotage Initiator 2.5 single mode microwave reactor with a new sealed pressure regulation 20 mL pressurized vial. Melting points were determined on an MEL-TEMP apparatus and are uncorrected. IR spectra were obtained on a Smith ATR-FT-IR spectrometer and the absorption frequencies are reported in wavelength (cm
1). 1H NMR spectra and 13 C NMR spectra were run on Bruker AVANCE-500, Bruker AVANCE300 and Varian OXFORD-300 spectrometers at 500 and 300 MHz 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-resolution mass spectra were obtained on the Autospec Magnetic sector mass spectrometer (Micromass, Manchester, UK). All anhydrous solvents (stored over molecular sieves) and chemicals were obtained from standard commercial vendors and were used without any further purification. The reactions were monitored by thinlayer 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. 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 organic layer was dried over anhydrous Na2SO4, and evaporated under 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 cm
1; Rf 0.39 (n-Hexane/EtOAc, 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), 3.85 (s, 3H), 1.41 (t, J¼7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3); d 162.0, 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 oxychloride (22.32 mL, 239.47 mmol) at room temperature. The reaction mixture was stirred for 3 h at room temperature, evaporated excess phosphorus oxychloride and neutralized to pH 7 by 2 NNaOH 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 cm
1; 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); d 187.9, 160.6, 157.1, 132.8, 131.3, 126.1, 118.6, 117.6, 114.7, 102.8, 62.1, 55.7, 14.5. HRMS (C13H13NO4): calcd, 247.0845 found, 247.0841.

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 3hydroxy-5-methoxyindole-2-carboxylic acid ethyl ester (14.6 g, 70%) as a yellow solid. Mp 96  C; IR (ATR) vmax¼3411, 3273, 1694 cm
1; Rf 0.29 (n-Hexane/EtOAc, 4:1); 1H NMR (300 MHz, 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 (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): 236 (MHþ); HRMS (C12H13NO4): calcd, 235.0845 found, 235.0843. 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 separated, dried over anhydrous MgSO4, and evaporated under reduced 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 cm
1; Rf 0.27 (n-Hexane/EtOAc, 4:1); 1H NMR (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-dimethoxy1H-indole-2-carboxylic acid ethyl ester (3.47 g, 99%)as an oil. IR (ATR) vmax¼1695 cm
1; Rf 0.48 (n-Hexane/EtOAc, 4:1); 1H NMR (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. 4.2.4. 3-Hydroxy-5-methoxy-1-allyl-1H-indole-2-carboxylic acid ethyl ester (5). To a solution of 1-allyl-3,5-dimethoxy-1H-indole-2carboxylic 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 (220 mL). Combined organic phases were washed with brine (20 mL), dried over anhydrous sodium sulfate and evaporated under reduced pressure to offered the crude product, which was purified by silica gel column chromatography to give 1-allyl-3hydroxy-5-methoxy-1H-indole-2-carboxylic acid ethyl ester (1.7 g, 99%) as a yellow solid. Mp 56  C, IR (ATR) vmax¼3331, 1664 cm
1; Rf 0.44 (n-Hexane/EtOAc, 4:1); 1H NMR (300 MHz, CDCl3) d 8.65 (br s, 1H), 7.15 (d, J¼9.0 Hz, 1H), 7.11 (d, J¼2.1 Hz, 1H), 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,

H.S. Pagire et al. / Tetrahedron 69 (2013) 3039e3044

J¼7.1 Hz, 3 H); 13C NMR (75 MHz, CDCl3); d 164.0, 153.6, 148.3, 134.0, 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. 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-indole2-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-3oxoindoline-2-carboxylic acid ethyl ester (1.12 g, 99%)as an oil. IR (ATR) vmax¼1739, 1697 cm
1; Rf 0.41 (n-Hexane/EtOAc, 4:1); 1H 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, 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. Elemental 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 corresponding starting materials in a similar manner to that described for compound 6. 4.2.6. 1-Allyl-5-methoxy-3-oxo-2-(3-phenyl-allyl)-2,3-dihydro-1Hindole-2-carboxylic acid ethyl ester (7). Oil 161 mg (yield 96%). IR (ATR) vmax¼1737, 1696 cm
1; Rf 0.39 (n-Hexane/EtOAc, 4:1); 1H NMR (300 MHz, CDCl3) d 7.24e7.08 (m, 5H), 7.02 (d, J¼2.3 Hz, 1H), 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%. 4.2.7. 1-Allyl-2-benzyl-5-methoxy-3-oxo-2,3-dihydro-1H-indole-2carboxylic acid ethyl ester (8). Yellowish solid, 58.4 mg (yield 88%), mp 73e76  C, IR (ATR) vmax¼1737,1696 cm
1; Rf 0.33 (n-Hexane/ EtOAc, 4:1); 1H NMR (300 MHz, CDCl3) d 7.40e7.35 (m, 2H), 7.09 (s, 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%. Crystal data of 8. CCDC reference number is 917644, crystal size0.280.240.08 mm3, crystal system monoclinic, space group P2(1)/n, Z¼4, a¼7.93460(10)  A, b¼17.7108(3)  A, c¼13.7296(2)  A, alpha¼90 , beta¼101.1640(10) , gamma¼90 , volume¼1892.88(5)  A3, density¼1.282 Mg/m3, T¼100(1) K, Absorption coefficient¼0.088 mm
1, F(000)¼776, Theta range for data collection¼1.90e28.35 , Index ranges¼
10h10,
23k23,
18l18, Reflections collected¼50,651, Independent reflections¼4710 [R(int)¼ 0.0278], Completeness to theta¼28.35 ¼99.6%, Absorption correction¼None, Refinement method¼Full-matrix least-squares on

3043

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 hole¼0.318 and
0.197 e. A
3. These data can be obtained free of 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-2carboxylic acid ethyl ester (9). Yellow oil 37 mg (yield 81%). IR (ATR) vmax¼1738, 1698 cm
1; 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; Elemental analysis for C16H19NO4. Calcd: C, 66.42%, H, 6.62%, N, 4.84%. Found: C, 66.07%, H, 6.72%, N, 4.93%. 4.2.9. 1-Allyl-2-ethyl-5-methoxy-3-oxo-2,3-dihydro-1H-indole-2carboxylic acid ethyl ester (10). Yellow oil 27 mg (yield 77%). IR (ATR) vmax¼1737, 1695 cm
1; Rf 0.38 (n-Hexane/EtOAc, 4:1); 1H 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%. 4.3. Biological experiment 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). 4.3.2. Treatment of LipidGreen and derivatives with zebrafish. Zebrafish embryos were treated with LipidGreen and derivatives that was diluted in DMSO. Zebrafish were incubated with several concentrations (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, photography was performed on a Leica MZ10F. 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 anaesthetized with tricaine and embedded in 3% methylcellulose for imaging. Laser at 470/40 nm or 545/30 nm filter was used for excitation. 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). Acknowledgements This research was supported by the Ministry of Education, Science and Technology (2010-0019773), the Ministry of Knowledge Economy (2012-10033674 and SI-1206), Korea. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tet.2013.01.090.

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