Rational Development of Furoindolizine Core Skeleton Guided by Oscillator Strength

Abstract

Molar absorptivity (ε) is an intrinsic property of every chemical entity, and it represents how well the entity absorbs light at any given wavelength [1]. From a synthetic point of view, the development of a new design strategy that allows for the predictable enhancement of the molar absorptivity of a species is highly valuable—with benefits spanning from aiding in the development of light-harvesting materials, such as dye-sensitized solar cells (DSSC), to various types of fluorescent materials [2]. However, very few papers have reported the successful improvement of molar absorptivity of particular initial compounds [3] because the prime strategy, an extension of the π-conjugated systems of these compounds, does not always guarantee increased molar absorptivity, and some instances have even been shown to have negligible or opposite effects [4]. To the best of my knowledge, there was no scientific report pursuing the rational design of organic fluorophores with enhanced molar absorptivity.

Experimental Section

4.1.1 General Information

a. Basic characterization

¹H and ¹³C NMR spectra were recorded on an Agilent 400-MR (Agilent Technologies) and Varian Inova-500 (Varian Associates), and chemical shifts were measured in ppm downfield from internal tetramethylsilane (TMS) standard. Multiplicity was indicated as follows: s (singlet); d (doublet); t (triplet); q (quartet); m (multiplet); dd (doublet of doublet); dt (doublet of triplet); br s (broad singlet), br d (broad doublet) etc. Coupling constants were reported in Hz. Low resolution mass spectrometry (LRMS) was obtained by LC/MS system, Finnigan MSQ_plus Surveyer (Thermo Scientific) or 6120 Quadrupole LC/MS (Agilent Technologies). High resolution mass spectrometry (HRMS) of furoindolizine fluorescence compounds was further confirmed by Ultra High Resolution ESI Q-TOF mass spectrometer (Bruker).

b. Absorption and fluorescence related properties

Absorption spectra and molar absorption coefficient at the absorption maxima of furoindolizine fluorescence compounds were measured by UV-VIS spectrophotometer UV-1650PC (Shimatzu, Japan). Emission spectra was measured by Cary Eclipse Fluorescence spectrophotometer (Varian Associates) and absolute quantum yield was measured by QE-2000 (Otsuka Electronics).

c. Chemical and bio reagents

3-Bromopropylamine hydrobromide, di-tert-butyl dicarbonate, propargyl amine, triethylamine (TEA), bromoacetyl bromide, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), copper(I) iodide (CuI), furo[3,2-b]pyridine, 2-phenylfuro[3,2-b]pyridine, iodobenzene, 4-iodobenzonitrile, 4-iodoanisole, 4-bromophenyl methyl sulfone, palladium acetate (PdOAc), bis(triphenylphosphine)palladium(II) dichloride (PdCl₂(PPh₃)₂), silver acetate (AgOAc), potassium acetate (KOAc), tetrabutylammonium fluoride (TBAF) solution, silver trifluoromethanesulfonate, and all terminal alkyne derivatives were purchased from Sigma-Aldrich, Tokyo Chemical Industry Co., Ltd or Acros, and used without further purification. The progress of reaction was monitored using thin-layer chromatography (TLC) (silica gel 60, F₂₅₄ 0.25 mm), and components were visualized by observation under UV light (254 and 365 nm) or by treating the TLC plates with anisaldehyde, KMnO₄, and ninhydrin followed by heating. Solvents were purchased from commercial venders and used without further purification. Cell culture reagents including fatal bovine serum, culture media, and antibiotic-antimycotic solution were purchased from GIBCO. MitoTracker Red CMXRos was purchased from Molecular Probes. The culture dish and glass-bottom dish were purchased from CORNING.

d. Quantum mechanical calculations

All quantum mechanical calculations were performed in Gaussin09 W. The ground state structures of furoindolizine compounds, Seoul-Fluor derivatives, and benzocoumarin derivatives were optimized using density functional theory (DFT) at the B3LYP/6-31G^* level. The oscillator strength values were calculated through time dependent density functional theory (TD-DFT) with the optimized structures of the ground state. The Energy gap between the S₀–S₁ states of furoindolizine analogues were calculated based on the optimized structure of the first-excited state to compare with experimental emission properties. Calculation for BODIPY derivatives were performed in the Materials Studio^® 4.2 program (Accelrys Software, Inc.) at the GAA/PBE/DNP level in DMol3 to reduce the calculation cost.

e. Fluorescence microscopy

Fluorescence microscopy studies were carried with DeltaVision Elite imaging system (GE Healthcare) equipped with a sCMOS camera. Objective lenses are supported by Olympus IX-71 (Olympus) inverted microscope equipped with Plan APO 60X/Oil (PLAPON60 × O), 1.42 NA, WD 0.15 mm. DeltaVision Elite uses a solid state illumination system, InSightSSI fluorescence illumination module. Four-color standard filter set (GE Healthcare, 52-852113-003) was used to detect fluorescence signals.

4.1.2 Experimental Procedure for Live Cell Fluorescence Image

1. Cell culture

HeLa cell line (human cervical carcinoma cells) was obtained from American Type Culture Collection (ATCC). HeLa cells were cultured in RPMI 1640 (GIBCO) supplemented with heat-inactivated 10% (v/v) fetal bovine serum (FBS, GIBCO) and 1% (v/v) antibiotic-antimycotic agent (GIBCO). HeLa cell line was maintained in humidified atmosphere of 5% CO₂ and 95% air at 37 °C, and cultured in 100 mm cell culture dish (CORNING).

2. Mitochondria staining experiment with Mito-18 and MitoTracker Red

HeLa cells were seeded on cover glass bottom dish and incubated at 5% CO₂, 37 °C for overnight. Cells are treated with 20 µM Mito-18 in media for 1 h. After 1 h, 20 nM MitoTracker Red CMXRos (Life Technologies) was added to cells, and incubate for 30 min. After the treatment, dyes were washed with PBS buffer for 3 times and then fluorescence images were taken by fluorescence microscopy under PBS buffer with DeltaVision Elite imaging system (GE Healthcare) equipped with 60X/1.42 NA oil lens. Fluorescence signal of each probes were obtained using FITC filter (Mito-18, Ex; 475 nm with 28 nm bandwidth, Em; 525 nm with 48 nm bandwidth) and Cy5 filter (MitoTracker Red CMXRox, Ex; 632 nm with 22 nm bandwidth, Em; 670 nm with 34 nm bandwidth).

Absorption and emission spectra of all furoindolizine compounds

• Horizontal axis: wavelength (nm)

• Vertical axis: normalized intensity (a.u.)

4.1.3 Computational Results of Furoindolizine Analogues

# cpd	Electronic transition	Energy (eV)	f (oscillator strength)
01	S0 → S1	3.7316 eV 332.26 nm	f = 0.2348
	S0 → S2	4.1879 eV 296.06 nm	f = 0.0420
	S0 → S3	4.7567 eV 260.65 nm	f = 0.0001
02	S0 → S1	3.3275 eV 372.60 nm	f = 0.5467
	S0 → S2	3.7156 eV 333.68 nm	f = 0.0712
	S0 → S3	4.0517 eV 306.00 nm	f = 0.0142
03	S0 → S1	3.4905 eV 355.21 nm	f = 0.3259
	S0 → S2	4.0271 eV 307.87 nm	f = 0.0706
	S0 → S3	4.2297 eV 293.13 nm	f = 0.1535
04	S0 → S1	3.2084 eV 386.44 nm	f = 0.2737
	S0 → S2	3.8527 eV 321.81 nm	f = 0.0593
	S0 → S3	4.0629 eV 305.16 nm	f = 0.0159
05	S0 → S1	2.9199 eV 424.62 nm	f = 0.6262
	S0 → S2	3.8066 eV 325.71 nm	f = 0.1642
	S0 → S3	4.0519 eV 305.99 nm	f = 0.0621
06	S0 → S1	2.8076 eV 441.61 nm	f = 0.9179
	S0 → S2	3.4494 eV 359.43 nm	f = 0.2282
	S0 → S3	3.7989 eV 326.37 nm	f = 0.0862
07	S0 → S1	2.7515 eV 450.61 nm	f = 0.7117
	S0 → S2	3.7433 eV 331.21 nm	f = 0.1957
	S0 → S3	3.8626 eV 320.99 nm	f = 0.1392
08	S0 → S1	2.4405 eV 508.02 nm	f = 0.5137
	S0 → S2	3.2408 eV 382.57 nm	f = 0.3306
	S0 → S3	3.5917 eV 345.20 nm	f = 0.1929
09	S0 → S1	3.2300 eV 383.85 nm	f = 0.5929
	S0 → S2	3.9430 eV 314.44 nm	f = 0.0602
	S0 → S3	4.3047 eV 288.02 nm	f = 0.0179
10	S0 → S1	3.0152 eV 411.20 nm	f = 0.9641
	S0 → S2	3.4429 eV 360.11 nm	f = 0.0539
	S0 → S3	3.8750 eV 319.96 nm	f = 0.0503
11	S0 → S1	3.0455 eV 407.11 nm	f = 0.6955
	S0 → S2	3.8669 eV 320.63 nm	f = 0.0636
	S0 → S3	3.9850 eV 311.12 nm	f = 0.2064
12	S0 → S1	2.8039 eV 442.18 nm	f = 0.5454
	S0 → S2	3.6142 eV 343.05 nm	f = 0.2764
	S0 → S3	3.7556 eV 330.13 nm	f = 0.0947
13	S0 → S1	3.1553 eV 392.94 nm	f = 0.9299
	S0 → S2	3.7940 eV 326.79 nm	f = 0.0179
	S0 → S3	4.0536 eV 305.86 nm	f = 0.0444
14	S0 → S1	2.8613 eV 433.32 nm	f = 1.1950
	S0 → S2	3.2935 eV 376.45 nm	f = 0.1272
	S0 → S3	3.6195 eV 342.55 nm	f = 0.0098
15	S0 → S1	3.0058 eV 412.48 nm	f = 1.0864
	S0 → S2	3.6786 eV 337.04 nm	f = 0.0622
	S0 → S3	3.8175 eV 324.78 nm	f = 0.1140
16	S0 → S1	2.8657 eV 432.65 nm	f = 0.8915
	S0 → S2	3.5335 eV 350.89 nm	f = 0.3607
	S0 → S3	3.7179 eV 333.48 nm	f = 0.0784
17	S0 → S1	3.1616 eV 392.15 nm	f = 0.9272
	S0 → S2	3.8097 eV 325.44 nm	f = 0.0533
	S0 → S3	4.0779 eV 304.04 nm	f = 0.0608
18	S0 → S1	2.8862 eV 429.58 nm	f = 0.8972
	S0 → S2	3.6101 eV 343.43 nm	f = 0.2309
	S0 → S3	3.8490 eV 322.12 nm	f = 0.0618
19	S0 → S1	2.6442 eV 468.90 nm	f = 0.6753
	S0 → S2	3.5330 eV 350.93 nm	f = 0.0000
	S0 → S3	3.6495 eV 339.73 nm	f = 0.2998

4.1.4 Prediction of Photophysical Properties for 17, 18 and 19

1. From Fig. 4.6

The equation for ɛ estimation = 30382f + 5221.8

(f: calculated oscillator strength values)

2. From Fig. 4.9c

The equation for λ_em estimation = 1092.9x + 73.263,

(x: calculated 1/eV)

	f (calculated oscillator strength values for S0 → S1)	Estimated ɛ	x (calculated 1/eV)	Estimated λem
17	0.9272	33,392	0.357935	464 nm
18	0.8972	32,480	0.406835	518 nm
19	0.6753	25,738	0.456496	572 nm

4.1.5 Synthetic Procedure and Compound Characterization

Preparation of tert-butyl (3-(prop-2-yn-1-ylamino)propyl)carbamate was conducted through the previous synthetic report.¹

Preparation of tert -butyl (3-(2-bromo- N -(prop-2-yn-1-yl)acetamido)propyl)carbamate

To a stirred solution of bromoacetyl bromide (2.0 equiv.) in anhydrous CH₂Cl₂ (0.1 M) at –78 °C under argon, added dropwise a tert-butyl (3-(prop-2-yn-1-ylamino)propyl)carbamate (1 equiv.) and 3 equiv. of triethylamine (TEA) in CH₂Cl₂ (0.1 M) over a period of 1 h. The solution was stirred at −78 °C for 2 h. When the reaction was completed checked by TLC, saturated NaHCO₃(aq) was added to the solution and the organic material was extracted with CH₂Cl₂. The combined organic extracts were dried over Na₂SO₄(s), and concentrated in vacuo after filtration. The residue was purified by silica-gel flash column chromatography to afford the desired product. (Transparent oil, Y: 83%).

tert -Butyl (3-(2-bromo- N -(prop-2-yn-1-yl)acetamido)propyl)carbamate

¹H NMR (2:1 rotamer ratio, asterisks denote minor rotamer peaks, 400 MHz, CDCl₃) δ 5.23 (br s, 1H), 4.76* (br s, 1H), 4.23* (d, J = 2.0 Hz, 2H), 4.14 (d, J = 2.0 Hz, 2H), 3.96 (s, 2H), 3.89* (s, 2H), 3.56–3.52 (m, 2H), 3.56–3.52* (m, 2H), 3.23–3.18* (m, 2H), 3.13–3.09 (m, 2H), 2.38 (br s, 1H), 2.26* (br s, 1H), 1.94–1.88* (m, 2H), 1.79–1.73 (m, 2H), 1.45* (s, 9H), 1.44 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 167.5, 166.4*, 156.2, 79.1, 78.2*, 78.0, 73.7, 72.6*, 45.9*, 44.2, 38.2, 38.0*, 37.1, 34.9*, 29.3*, 28.5, 28.5*, 27.6, 26.1, 25.9*; LRMS (ESI) m/z calcd for C₁₃H₂₂BrN₂O₃ [M + H]⁺: 333.08; Found: 332.92.

General procedure for preparation of furopyridine derivatives

(a) Terminal alkyne derivatives required to prepare various furopyridine derivatives were commercially available, except 1-(4-ethynylphenyl)ethanone and 4-(4-ethynylphenyl) morpholine, which were synthesized by reported procedure.², ³

(b) Preparation of 2-bromopyridin-3-yl acetate was conducted refer to the reported synthetic procedure.⁴

To a suspension of 2-bromopyridin-3-yl acetate (1.0 equiv.), PdCl₂(PPh₃)₂ (3 mol%), and CuI (6 mol%) in THF (0.1 M), in the presence of TEA (15.0 equiv.), was added a terminal alkyne derivative (2.0 equiv.) under argon atmosphere with vigorous stirring at 50 °C. After the reaction was completed checked by TLC, the resulting solution was filtered through Celite and concentrated in vacuo. After that, to the solution of filtered residue in MeOH (0.1 M), were added potassium carbonate (3.0 equiv.) and silver triflate (10 mol%) with stirring at room temperature. When the reaction was completed checked by TLC, brine was added to the solution and the organic material was extracted with ethyl acetate. The combined organic extracts were dried over Na₂SO₄(s), and concentrated under reduced pressure after filtration. The residue was purified by silica-gel flash column chromatography to afford the desired furopyridine derivatives.

4-(Furo[3,2- b ]pyridin-2-yl)benzonitrile

Yield: 45%; ¹H NMR (400 MHz, CDCl₃) δ 8.59 (d, J = 4.4 Hz, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.37 (s, 1H), 7.28 (dd, J = 4.4, 8.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 157.1, 148.6, 148.4, 146.9, 133.8, 132.8, 125.7, 120.0, 118.6, 118.4. 112.7, 105.4; LRMS (ESI) m/z calcd for C₁₄H₉N₂O [M + H]⁺: 221.07; Found: 220.88.

4-(Furo[3,2-b]pyridin-2-yl)-N,N-dimethylaniline

Yield: 77%; ¹H NMR (400 MHz, CDCl₃) δ 8.46 (br s, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.0 Hz, 1H), 7.09 (dd, J = 4.6, 8.2 Hz, 1H), 6.97 (s, 1H), 6.74 (d, J = 8.8 Hz, 2H), 3.00 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 161.0, 151.2, 150.0, 147.7, 145.6, 126.7, 117.7, 117.5, 117.0, 112.0, 99.0, 40.3; LRMS (ESI) m/z calcd for C₁₅H₁₅N₂O [M + H]⁺: 239.12; Found: 238.98.

2-(4-Morpholinophenyl)furo[3,2- b ]pyridine

Yield: 63%; ¹H NMR (400 MHz, CDCl₃) δ 8.48 (d, J = 4.0 Hz, 1H), 7.81 (d, J = 9.2 Hz, 2H), 7.71 (d, J = 8.0 Hz, 1H), 7.15 (dd, J = 4.6, 8.2 Hz, 1H), 7.05 (s, 1H), 6.98 (d, J = 8.8 Hz, 2H), 3.89 (t, J = 4.8 Hz, 4H), 3.27 (t, J = 5.0 Hz, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 160.2, 152.1, 149.7, 147.9, 145.9, 126.7, 121.0, 118.2, 117.4, 115.1. 100.4, 66.8, 48.5; LRMS (ESI) m/z calcd for C₁₇H₁₇N₂O₂ [M + H]⁺: 281.13; Found: 281.03.

1-(4-(Furo[3,2- b ]pyridin-2-yl)phenyl)ethanone

Yield: 54%; ¹H NMR (400 MHz, CDCl₃) δ 8.57 (br s, 1H), 8.06 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 7.28–7.24 (m, 1H), 2.65 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 197.3, 158.2, 148.6, 148.5, 146.5, 137.4, 133.8, 129.1, 125.4, 119.6, 118.3, 104.6, 26.8; LRMS (ESI) m/z calcd for C₁₅H₁₂NO₂ [M + H]⁺: 238.09; Found: 238.1.

2-(Triisopropylsilyl)furo[3,2- b ]pyridine

Yield: 90%; ¹H NMR (500 MHz, acetone-d₆) δ 8.52 (dd, J = 1.3, 4.8 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.32 (d, J = 1.0 Hz, 1H), 7.28 (dd, J = 4.5, 8.5 Hz, 1H), 1.50–1.44 (m, 3H), 1.22–1.13 (m, 18H); ¹³C NMR (125 MHz, acetone-d₆) δ 166.0, 151.3, 149.0, 146.7, 120.4, 119.8, 118.4, 18.9, 11.7; LRMS (ESI) m/z calcd for C₁₆H₂₆NOSi [M + H]⁺: 276.18; Found: 276.2.

General procedure for preparation of furoindolizine-based core skeletons

To a solution of tert-butyl (3-(2-bromo-N-(prop-2-yn-1-yl)acetamido)propyl)carbamate (1.2 equiv.) in acetonitrile (0.1 M), was added a furopyridine derivative (1.0 equiv.), and the solution was stirred at 80 °C overnight. After the complete consumption of starting materials, copper iodide (1.0 equiv.) was added to a reaction mixture followed by slow addition of 1,8-diazabicycloundec-7-ene (DBU) (3.0 equiv.) at room temperature with vigorous stirring. When the reaction was completed checked by TLC, the resulting mixture was filtered through the short bed of silica gel and concentrated in vacuo. The residue was purified by silica-gel flash column chromatography to afford the desired furoindolizine-based core skeltons. Some furopyridine derivatives used different metal sources instead of copper iodide; AgOTf was used in the case of R² = 4-(acetyl)Ph group, and Ag₂O was used in the case of R² = 4-(NMe₂)Ph and 4-(morpholino)Ph.

tert -Butyl (3-(9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (01)

Yield: 19%; ¹H NMR (400 MHz, CDCl₃) δ 7.85 (d, J = 2.0 Hz, 1H), 7.62 (d, J = 2.4 Hz, 1H), 7.29–7.23 (m, 2H), 6.45 (s, 1H), 5.44 (br s, 1H), 4.35 (s, 2H), 3.66 (t, J = 6.2 Hz, 2H), 3.17 (q, J = 6.1 Hz, 2H), 1.86–1.79 (m, 2H), 1.44 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 162.2, 156.2, 143.3, 143.1, 138.4, 135.8, 125.7, 121.6, 114.3, 109.7, 104.8, 94.9, 79.0, 46.6, 40.1, 37.4, 28.9, 28.5; HRMS (ESI) m/z calcd for C₂₀H₂₃N₃NaO₄ [M + Na]⁺: 392.1581; Found: 392.1581.

tert -Butyl (3-(2-(4-cyanophenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (05)

Yield: 49%; ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 9.6 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 6.51 (s, 1H), 5.36 (br s, 1H), 4.40 (s, 2H), 3.68 (t, J = 6.4 Hz, 2H), 3.21 (br d, J = 5.6 Hz, 2H), 1.87–1.84 (m, 2H), 1.46 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 162.1, 156.2, 151.8, 143.9, 138.3, 136.1, 133.9, 132.7, 127.1, 124.7, 122.3, 118.9, 115.9, 111.3, 109.2, 102.1, 95.9, 79.2, 46.8, 40.3, 37.6, 29.1, 28.6; HRMS (ESI) m/z calcd for C₂₇H₂₆N₄NaO₄ [M + Na]⁺: 493.1846; Found: 493.1847.

tert -Butyl (3-(9-oxo-2-phenyl-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (09)

Yield: 35%; ¹H NMR (400 MHz, CD₂Cl₂) δ 8.08 (s, 1H), 7.92 (d, J = 7.2 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.38–7.31 (m, 3H), 6.51 (s, 1H), 5.50 (br s, 1H), 4.37 (s, 2H), 3.65 (t, J = 6.2 Hz, 2H), 3.13 (q, J = 6.3 Hz, 2H), 1.83–1.76 (m, 2H), 1.44 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 162.3, 156.3, 154.4, 142.9, 138.6, 136.0, 130.1, 128.9, 128.6, 127.5, 124.7, 121.6, 114.1, 109.4, 99.4, 95.1, 79.1, 46.7, 40.3, 37.6, 29.0, 28.6; HRMS (ESI) m/z calcd for C₂₆H₂₇N₃NaO₄ [M + Na]⁺: 468.1894; Found: 468.1893.

tert -Butyl (3-(2-(4-(dimethylamino)phenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (13)

Yield: 26%; ¹H NMR (400 MHz, CDCl₃) δ 7.87 (s, 1H), 7.78 (d, J = 9.2 Hz, 2H), 7.25 (d, J = 6.4 Hz, 1H), 7.19 (d, J = 9.6 Hz, 1H), 6.77 (d, J = 8.8 Hz, 2H), 6.42 (s, 1H), 5.40 (br s, 1H), 4.36 (s, 2H), 3.67 (t, J = 6.2 Hz, 2H), 3.19 (br d, J = 5.6 Hz, 2H), 3.03 (s, 6H), 1.85–1.82 (m, 2H), 1.45 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 162.4, 156.3, 155.8, 150.7, 142.0, 138.9, 136.1, 128.1, 126.1, 121.1, 118.3, 112.5, 112.2, 109.4, 96.4, 94.5, 79.1, 46.7, 40.4, 40.3, 37.5, 29.1, 28.6; HRMS (ESI) m/z calcd for C₂₈H₃₂N₄NaO₄ [M + Na]⁺: 511.2316; Found: 511.2317.

tert -Butyl (3-(2-(4-morpholinophenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (17)

Yield: 22%; ¹H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.28–7.21 (m, 2H), 6.96 (d, J = 8.4 Hz, 2H), 6.44 (s, 1H), 5.38 (br s, 1H), 4.37 (s, 2H), 3.89 (t, J = 4.6 Hz, 4H), 3.67 (t, J = 6.4 Hz, 2H), 3.25 (t, J = 4.6 Hz, 4H), 3.20 (q, J = 5.9 Hz, 2H), 1.87–1.81 (m, 2H), 1.45 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 162.4, 156.3, 155.0, 151.4, 142.4, 138.8, 136.1, 127.9, 126.0, 121.6, 121.3, 115.3, 113.2, 109.4, 97.5, 94.7, 79.1, 66.9, 48.7, 46.7, 40.3, 37.6, 29.1, 28.6; HRMS (ESI) m/z calcd for C₃₀H₃₄N₄NaO₅ [M + Na]⁺: 553.2421; Found: 553.2421.

tert -Butyl (3-(2-(4-acetylphenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate

Yield: 29%; ¹H NMR (400 MHz, CDCl₃) δ 8.07 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 9.6 Hz, 1H), 7.20 (d, J = 9.2 Hz, 1H), 6.42 (s, 1H), 5.41 (br s, 1H), 4.33 (s, 2H), 3.66 (t, J = 6.2 Hz, 2H), 3.21 (br d, J = 6.0 Hz, 2H), 2.59 (s, 3H), 1.87–1.84 (m, 2H), 1.46 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 197.2, 162.0, 156.2, 152.7, 143.5, 138.3, 136.1, 135.9, 133.9, 128.9, 127.1, 124.2, 121.9, 115.2, 109.1, 101.3, 95.6, 79.2, 46.7, 40.3, 37.6, 29.0, 28.5, 26.6; LRMS (ESI) m/z calcd for C₂₈H₃₀N₃O₅ [M + H]⁺: 488.22; Found: 488.2.

tert -Butyl (3-(9-oxo-2-(triisopropylsilyl)-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate

Yield: 70%; ¹H NMR (400 MHz, CDCl₃) δ 8.07 (s, 1H), 7.29–7.23 (m, 2H), 6.44 (s, 1H), 5.31 (br s, 1H), 4.35 (s, 2H), 3.67 (t, J = 6.4 Hz, 2H), 3.19 (q, J = 6.1 Hz, 2H), 1.88–1.83 (m, 2H), 1.48–1.40 (m, 12H), 1.17–1.15 (m, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 162.3, 160.0, 156.3, 146.9, 138.6, 135.7, 126.4, 121.8, 115.2, 114.1, 110.0, 94.8, 79.1, 46.6, 40.3, 37.7, 29.0, 28.5, 18.7, 11.2; LRMS (ESI) m/z calcd for C₂₉H₄₄N₃O₄Si [M + H]⁺: 526.31; Found: 526.3.

Procedure for preparation of R ¹ group embedded furoindolizine compounds (Pd coupling reaction)

Preparation of N,N-diethyl-4-iodoaniline was conducted through the reported procedure.⁵

For general iodo-aryl moieties

Condition A. To a solution of a furoindolizine-based core skeleton in dimethylformamide (0.1 M), were added a iodo-aryl derivative (2.0 equiv.), bis(triphenylphosphine)palladium(II) dichloride (PdCl₂(PPh₃)_2, 10 mol%), silver acetate (1.5 equiv.), and potassium acetate (3.0 equiv.), and the solution was stirred at 100 °C overnight. When the reaction was completed checked by TLC, the reaction mixture was filtered through the short bed of silica gel and concentrated in vacuo. The residue was purified by silica-gel flash column chromatography to afford the desired products. (** at isolated yield indicates 10 mol% of PdCl₂(PPh₃)₂ was applied additionally to completely convert the starting materials)

For N,N-diethyl-4-iodoaniline and 1-iodo-4-methoxybenzene

Condition B. To a solution of a furoindolizine-based core skeleton in dimethylformamide (0.1 M), were added a iodo-aryl derivative (2.0 equiv.), palladium acetate (20 mol%), and potassium acetate (3.0 equiv.), and the solution was stirred at 80 °C overnight. When the reaction was completed checked by TLC, the reaction mixture was filtered through the short bed of silica gel and concentrated in vacuo. The residue was purified by silica-gel flash column chromatography to afford the desired products.

tert -Butyl (3-(2,6-bis(4-cyanophenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (06)

Yield: 80%**; ¹H NMR (400 MHz, CDCl₃) δ 8.29 (s, 1H), 7.96 (d, 8.8 Hz, 2H), 7.76–7.65 (m, 7H), 7.43 (d, J = 9.6 Hz, 1H), 5.24 (br s, 1H), 4.57 (s, 2H), 3.73 (t, J = 6.6 Hz, 2H), 3.23–3.20 (m, 2H), 1.91–1.88 (m, 2H), 1.45 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 161.7, 156.2, 152.8, 144.2, 139.3, 135.1, 134.9, 133.6, 133.1, 132.8, 127.7, 127.6, 125.0, 122.8, 119.1, 118.7, 114.3, 111.9, 111.0, 109.6, 109.4, 102.1, 79.4, 47.0, 40.6, 37.7, 29.1, 28.6; HRMS (ESI) m/z calcd for C₃₄H₂₉N₅NaO₄ [M + Na]⁺: 594.2112; Found: 594.2111.

tert -Butyl (3-(2-(4-cyanophenyl)-9-oxo-6-phenyl-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (07)

Yield: 61%; ¹H NMR (500 MHz, CDCl₃) δ 8.24 (s, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 9.0 Hz, 3H), 7.56 (d, J = 7.0 Hz, 2H), 7.50–7.47 (m, 2H), 7.34–7.29 (m, 2H), 5.33 (br s, 1H), 4.53 (s, 2H), 3.71 (t, J = 6.5 Hz, 2H), 3.21 (br d, J = 6.0 Hz, 2H), 1.88–1.86 (m, 2H), 1.45 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 162.0, 156.2, 152.1, 144.2, 134.8, 134.4, 134.3, 133.8, 132.7, 129.3, 127.6, 127.3, 126.6, 124.8, 122.0, 118.9, 115.1, 111.5, 111.4, 109.7, 102.2, 79.3, 47.0, 40.5, 37.6, 29.1, 28.6; HRMS (ESI) m/z calcd for C₃₃H₃₀N₄NaO₄ [M + Na]⁺: 569.2159; Found: 569.2160.

tert -Butyl (3-(2-(4-cyanophenyl)-6-(4-(diethylamino)phenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (08)

Yield: 27%; ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 10.0 Hz, 1H), 7.41 (d, J = 9.2 Hz, 2H), 7.22 (d, J = 10.0 Hz, 1H), 6.79 (d, J = 8.8 Hz, 2H), 5.37 (br s, 1H), 4.49 (s, 2H), 3.70 (t, J = 6.4 Hz, 2H), 3.42 (q, J = 7.0 Hz, 4H), 3.20 (br d, J = 5.6 Hz, 2H), 1.87–1.84 (m, 2H), 1.45 (s, 9H), 1.22 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 162.2, 156.3, 151.7, 146.6, 144.2, 134.4, 134.0, 133.7, 132.7, 128.7, 127.2, 124.7, 121.6, 121.0, 119.0, 115.6, 112.3, 112.2, 111.2, 108.7, 102.3, 79.3, 47.0, 44.6, 40.5, 37.6, 29.1, 28.6, 12.8; HRMS (ESI) m/z calcd for C₃₇H₄₀N₅O₄ [M + H]⁺: 618.3075; Found: 618.3077.

tert -Butyl (3-(6-(4-cyanophenyl)-9-oxo-2-phenyl-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (10)

Yield: 81%; ¹H NMR (400 MHz, CDCl₃) δ 8.06 (s, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H), 7.61–7.55 (m, 3H), 7.44 (t, J = 7.6 Hz, 2H), 7.38–7.34 (m, 2H), 5.30 (br s, 1H), 4.49 (s, 2H), 3.70 (t, J = 6.2 Hz, 2H), 3.22 (br d, J = 6.0 Hz, 2H), 1.90–1.86 (m, 2H), 1.45 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 161.7, 156.2, 155.3, 143.1, 139.6, 135.3, 134.8, 132.9, 129.6, 129.1, 129.0, 128.0, 127.2, 124.8, 122.2, 119.3, 112.5, 111.1, 108.8, 108.6, 99.3, 79.3, 47.0, 40.5, 37.7, 29.1, 28.6; HRMS (ESI) m/z calcd for C₃₃H₃₀N₄NaO₄ [M + Na]⁺: 569.2159; Found: 569.2158.

tert -Butyl (3-(9-oxo-2,6-diphenyl-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (11)

Yield: 67%; ¹H NMR (400 MHz, CDCl₃) δ 8.13 (s, 1H), 7.90 (d, J = 7.2 Hz, 2H), 7.61 (d, J = 9.6 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.49–7.43 (m, 4H), 7.37–7.28 (m, 3H), 5.37 (br s, 1H), 4.50 (s, 2H), 3.69 (t, J = 6.2 Hz, 2H), 3.21 (br d, J = 6.4 Hz, 2H), 1.87–1.83 (m, 2H), 1.45 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 162.2, 156.3, 154.7, 143.1, 135.1, 134.7, 134.4, 130.0, 129.2, 129.0, 128.8, 127.7, 127.5, 126.3, 124.8, 121.4, 113.3, 110.7, 110.0, 99.5, 79.2, 46.9, 40.4, 37.6, 29.0, 28.6; HRMS (ESI) m/z calcd for C₃₂H₃₁N₃NaO₄ [M + Na]⁺: 544.2207; Found: 544.2207.

tert -Butyl (3-(6-(4-(diethylamino)phenyl)-9-oxo-2-phenyl-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (12)

Yield: 30%; ¹H NMR (400 MHz, CDCl₃) δ 8.12 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 10.0 Hz, 1H), 7.47–7.41(m, 4H), 7.36–7.32 (m, 1H), 7.27–7.25 (m, 1H), 6.79 (d, J = 8.4 Hz, 2H), 5.42 (br s, 1H), 4.48 (s, 2H), 3.69 (t, J = 6.2 Hz, 2H), 3.41 (q, J = 7.0 Hz, 4H), 3.20 (br d, J = 5.6 Hz, 2H), 1.86–1.83 (m, 2H), 1.45 (s, 9H), 1.22 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 162.4, 156.3, 154.3, 146.4, 143.2, 134.7, 133.7, 130.2, 128.9, 128.7, 128.6, 127.6, 124.7, 121.4, 120.9, 113.8, 112.4, 111.3, 109.1, 99.6, 79.2, 46.9, 44.6, 40.4, 37.6, 29.0, 28.6, 12.8; HRMS (ESI) m/z calcd for C₃₆H₄₁N₄O₄ [M + H]⁺: 593.3122; Found: 593.3121.

tert -Butyl (3-(6-(4-cyanophenyl)-2-(4-(dimethylamino)phenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (14)

Yield: 86%; ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.77 (d, J = 9.2 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 10.0 Hz, 1H), 7.40 (d, J = 10.0 Hz, 1H), 6.76 (d, J = 9.2 Hz, 2H), 5.29 (br s, 1H), 4.53 (s, 2H), 3.71 (t, J = 6.4 Hz, 2H), 3.21 (q, J = 6.1 Hz, 2H), 3.05 (s, 6H), 1.90–1.86 (m, 2H), 1.44 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 161.9, 157.0, 156.3, 151.0, 142.4, 140.0, 135.8, 135.0, 133.0, 128.9, 127.2, 126.4, 121.8, 119.4, 117.7, 112.2, 111.2, 110.8, 108.6, 108.0, 96.2, 79.3, 47.0, 40.5, 40.4, 37.7, 29.1, 28.6; HRMS (ESI) m/z calcd for C₃₅H₃₅N₅NaO₄ [M + Na]⁺: 612.2581; Found: 612.2581.

tert -Butyl (3-(2-(4-(dimethylamino)phenyl)-9-oxo-6-phenyl-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (15)

Yield: 81%**; ¹H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.59–7.56 (m, 3H), 7.47 (t, J = 7.8 Hz, 2H), 7.34–7.28 (m, 2H), 6.77 (d, J = 9.2 Hz, 2H), 5.38 (br s, 1H), 4.52 (s, 2H), 3.70 (t, J = 6.2 Hz, 2H), 3.21 (q, J = 6.0 Hz, 2H), 3.04 (s, 6H), 1.88–1.84 (m, 2H), 1.44 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 162.4, 156.3, 156.2, 150.8, 142.4, 135.5, 135.0, 134.5, 129.2, 128.5, 127.5, 126.2, 126.1, 120.9, 118.2, 112.2, 111.6, 110.1, 110.0, 96.4, 79.2, 46.9, 40.5, 40.4, 37.6, 29.0, 28.6; HRMS (ESI) m/z calcd for C₃₄H₃₇N₄O₄ [M + H]⁺: 565.2809; Found: 565.2807.

tert -Butyl (3-(2-(4-acetylphenyl)-6-(4-methoxyphenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (19)

Yield: 17%; ¹H NMR (500 MHz, CD₂Cl₂) δ 8.28 (s, 1H), 8.04 (d, J = 8.5 Hz, 2H), 8.01 (d, J = 9.0 Hz, 2H), 7.66 (d, J = 10.0 Hz, 1H), 7.52 (d, J = 9.0 Hz, 2H), 7.35 (d, J = 10.0 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H), 5.45 (br s, 1H), 4.51 (s, 2H), 3.86 (s, 3H), 3.69 (t, J = 6.5 Hz, 2H), 3.16 (q, J = 6.2 Hz, 2H), 2.62 (s, 3H), 1.85–1.80 (m, 2H), 1.43 (s, 9H); HRMS (ESI) m/z calcd for C₃₅H₃₅N₃NaO₆ [M + Na]⁺: 616.2418; Found: 616.2417.

tert -Butyl (3-(6-(4-cyanophenyl)-9-oxo-2-(triisopropylsilyl)-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate

Yield: 78%; ¹H NMR (400 MHz, CDCl₃) δ 8.17 (s, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.66–7.62 (m, 3H), 7.45 (d, J = 10.0 Hz, 1H), 5.21 (br s, 1H), 4.54 (s, 2H), 3.71 (t, J = 6.6 Hz, 2H), 3.21 (br d, J = 6.4 Hz, 2H), 1.90–1.87 (m, 2H), 1.50–1.42 (m, 12H), 1.17 (d, J = 7.2 Hz, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 161.8, 161.6, 156.2, 147.2, 139.9, 135.3, 134.5, 133.0, 127.4, 127.0, 122.4, 119.3, 115.1, 112.5, 111.8, 108.8, 108.5, 79.2, 46.9, 40.5, 37.8, 29.0, 28.5, 18.7, 11.2; LRMS (ESI) m/z calcd for C₃₆H₄₇N₄O₄Si [M + H]⁺: 627.34; Found: 627.3.

tert -Butyl (3-(9-oxo-6-phenyl-2-(triisopropylsilyl)-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate

Yield: 84%**; ¹H NMR (400 MHz, CDCl₃) δ 8.14 (s, 1H), 7.63 (d, J = 10.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.47 (t, J = 7.8 Hz, 2H), 7.34 (d, J = 9.6 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 5.30 (br s, 1H), 4.51 (s, 2H), 3.70 (t, J = 6.6 Hz, 2H), 3.20 (q, J = 6.0 Hz, 2H), 1.88–1.85 (m, 2H), 1.49–1.42 (m, 12H), 1.17 (d, J = 7.2 Hz, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 162.2, 160.5, 156.3, 147.1, 135.0, 134.9, 134.1, 129.2, 127.5, 126.7, 126.2, 121.5, 115.2, 113.2, 110.6, 110.4, 79.1, 46.8, 40.4, 37.7, 29.0, 28.5, 18.8, 11.2; LRMS (ESI) m/z calcd for C₃₅H₄₈N₃O₄Si [M + H]⁺: 602.34; Found: 602.3.

tert -Butyl (3-(6-(4-(diethylamino)phenyl)-9-oxo-2-(triisopropylsilyl)-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate

Yield: 32%; ¹H NMR (400 MHz, CD₂Cl₂) δ 8.12 (s, 1H), 7.60 (d, J = 9.6 Hz, 1H), 7.42 (d, J = 8.8 Hz, 2H), 7.29 (d, J = 9.8 Hz, 1H), 6.78 (d, J = 9.2 Hz, 2H), 5.47 (br s, 1H), 4.48 (s, 2H), 3.66 (t, J = 6.6 Hz, 2H), 3.40 (q, J = 7.1 Hz, 4H), 3.14 (q, J = 6.1 Hz, 2H), 1.84–1.78 (m, 2H), 1.49–1.42 (m, 12H), 1.21–1.18 (m, 24H); ¹³C NMR (100 MHz, CD₂Cl₂) δ 162.4, 160.1, 156.3, 147.4, 146.7, 134.6, 133.7, 128.7, 126.6, 121.8, 121.5, 115.5, 114.0, 112.6, 111.3, 109.7, 78.9, 47.0, 44.8, 40.5, 37.7, 29.1, 28.5, 18.8, 12.8. 11.5; LRMS (ESI) m/z calcd for C₃₉H₅₇N₄O₄Si [M + H]⁺: 673.41; Found: 673.4.

General procedure for TIPS deprotection reaction (For 02, 03, and 04)

To a stirred solution of TIPS protected furoindolizine-based core skeleton in THF (0.1 M) at –40 °C, was slowly added tetrabutylammonium fluoride solution (1.0 M in THF, 1.5 equiv.) which was cooled down to –10 °C. Stirred reaction mixture was allowed to warm up to 0 °C for 1 h. When the reaction was completed checked by TLC, brine was added to the solution and the organic material was extracted with ethyl acetate. The combined organic extracts were dried over Na₂SO₄(s), and concentrated under reduced pressure after filtration. The residue was purified by silica-gel flash column chromatography to afford the desired product.

tert -Butyl (3-(6-(4-cyanophenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (02)

Yield: 82%; ¹H NMR (400 MHz, CD₂Cl₂) δ 7.91 (d, J = 2.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 2.0 Hz, 1H), 7.65 (d, J = 8.4 Hz, 3H), 7.41 (d, J = 9.6 Hz, 2H), 5.42 (br s, 1H), 4.51 (s, 2H), 3.66 (t, J = 6.6 Hz, 2H), 3.13 (q, J = 6.5 Hz, 2H), 1.85–1.78 (m, 2H), 1.41 (s, 9H); ¹³C NMR (100 MHz, CD₂Cl₂) δ 161.9, 156.2, 144.6, 143.8, 139.9, 135.4, 135.1, 133.2, 127.6, 126.5, 122.7, 119.5, 113.3, 111.6, 109.1, 108.9, 105.1, 79.1, 47.1, 40.5, 37.6, 29.1, 28.5; HRMS (ESI) m/z calcd for C₂₇H₂₆N₄NaO₄ [M + Na]⁺: 493.1846; Found: 493.1845.

tert -Butyl (3-(9-oxo-6-phenyl-7 H -furo[3,2-e]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (03)

Yield: 99%; ¹H NMR (400 MHz, CD₂Cl₂) δ 7.89 (d, J = 1.2 Hz, 1H), 7.68 (d, J = 1.6 Hz, 1H), 7.65 (d, J = 10.0 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.47 (t, J = 8.0 Hz, 2H), 7.33–7.27 (m, 2H), 5.50 (br s, 1H), 4.49 (s, 2H), 3.66 (t, J = 6.4 Hz, 2H), 3.13 (q, J = 6.1 Hz, 2H), 1.83–1.76 (m, 2H), 1.42 (s, 9H); ¹³C NMR (100 MHz, CD₂Cl₂) δ 162.2, 156.2, 144.0, 143.7, 135.1, 135.0, 134.6, 129.4, 127.7, 126.4, 126.2, 121.8, 113.8, 110.7, 110.5, 105.1, 79.0, 47.0, 40.4, 37.6, 29.1, 28.5; HRMS (ESI) m/z calcd for C₂₆H₂₇N₃NaO₄ [M + Na]⁺: 468.1894; Found: 468.1896.

tert -Butyl (3-(6-(4-(diethylamino)phenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (04)

Yield: 99%; ¹H NMR (400 MHz, acetone-d₆) δ 7.90–7.88 (m, 2H), 7.67 (d, J = 9.6 Hz, 1H), 7.45 (d, J = 9.2 Hz, 2H), 7.34 (d, J = 9.6 Hz, 1H), 6.81 (d, J = 8.8 Hz, 2H), 6.14 (br s, 1H), 4.58 (s, 2H), 3.66 (t, J = 6.4 Hz, 2H), 3.43 (q, J = 7.1 Hz, 4H), 3.15 (q, J = 6.4 Hz, 2H), 1.90–1.83 (m, 2H), 1.40 (s, 9H), 1.18 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, acetone-d₆) δ 162.2, 156.6, 147.2, 144.7, 144.0, 134.7, 134.6, 129.2, 126.2, 122.1, 122.0, 114.9, 113.1, 112.1, 109.8, 105.3, 78.5, 47.2, 44.9, 40.9, 38.3, 29.3, 28.6, 13.0; HRMS (ESI) m/z calcd for C₃₀H₃₇N₄O₄ [M + H]⁺: 517.2809; Found: 517.2809.

Procedure for preparation of 18

To a solution of 01 (1.0 equiv.) in dimethylformamide (0.1 M), were added 4-bromophenyl methyl sulfone (10.0 equiv.), bis(triphenylphosphine)palladium(II) dichloride (20 mol%), silver acetate (1.5 equiv.) and potassium acetate (3.0 equiv.), and the solution was stirred at 100 °C. When the reaction was completed checked by TLC, the reaction mixture was filtered through the short bed of silica gel and concentrated in vacuo. The residue was purified by silica-gel flash column chromatography to afford 18.

tert -Butyl (3-(2,6-bis(4-(methylsulfonyl)phenyl)-9-oxo-7 H -furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)carbamate (18)

Yield: 26%; ¹H NMR (400 MHz, CDCl₃) δ 8.31 (s, 1H), 8.05–7.99 (m, 6H), 7.75–7.70 (m, 3H), 7.43 (d, J = 9.6 Hz, 1H), 5.24 (br s, 1H), 4.57 (s, 2H), 3.73 (t, J = 6.6 Hz, 2H), 3.23–3.18 (m, 2H), 3.13 (s, 3H), 3.11 (s, 3H), 1.92–1.89 (m, 2H), 1.45 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 161.7, 156.2, 152.7, 144.2, 140.3, 140.0, 137.7, 135.2, 135.0, 134.6, 128.5, 128.2, 127.7, 127.6, 125.2, 122.8, 114.3, 111.0, 109.4, 102.2, 79.4, 47.0, 44.8, 44.7, 40.6, 37.7, 29.1, 28.6; HRMS (ESI) m/z calcd for C₃₄H₃₅N₃NaO₈S₂ [M + Na]⁺: 700.1758; Found: 700.1759.

Procedure for preparation of Mito-18

To a solution of compound 18 in DCM (0.1 M) was added a 10% v/v of hydrochloric acid (HCl), and the solution was stirred overnight at room temperature. After full consumption of starting material, the solution was diluted with DCM and washed with sodium bicarbonate solution. The organic layer was extracted with DCM, dried over sodium sulfate and filtered through a cotton plug. The resulting solution was evaporated under reduced pressure to afford 18-1. LRMS (ESI) m/z calcd for C₂₉H₂₈N₃O₆S₂ [M + H]⁺: 578.14; Found: 578.1.

After that, 6-Bromohexanoyl chloride (1.5 equiv.) was slowly added to a stirred solution of 18-1 with TEA (3.0 equiv.) in DCM (0.1 M) at room temperature and the solution was stirred for 6 h. After 18-1 was fully consumed, the solvent was removed, and crude mixture was dissolved in acetonitrile (0.1 M) followed by addition of triphenylphosphine (3.0 equiv.). The mixture was stirred under reflux condition for 2 days. The crude product was purified by reverse-phase-HPLC to afford a desired Mito-18. [HPLC solvents consist of water containing 0.1% TFA (trifluoroacetic acid) for solvent A, and acetonitrile containing 0.1% TFA for solvent B]

(6-((3-(2,6-bis(4-(methylsulfonyl)phenyl)-9-oxo-7H-furo[3,2- e ]pyrrolo[3,4- b ]indolizin-8(9 H )-yl)propyl)amino)-6-oxohexyl)triphenylphosphonium (Mito-18)

Yield: 24% (overall yield over 3 steps); ¹H NMR (400 MHz, DMSO-d₆) δ 8.26 (s, 1H), 8.08 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.0 Hz, 2H), 7.94–7.84 (m, 7H), 7.80–7.69 (m, 13H), 4.73 (s, 2H), 3.61–3.51 (m, 4H), 3.29 (s, 3H), 3.27 (s, 3H), 3.13–3.08 (q, J = 6.5 Hz, 2H), 2.06–2.03 (m, 2H), 1,81–1.76 (m, 2H), 1.51–1.47 (m, 6H); LRMS (ESI) m/z calcd for C₅₃H₅₁N₃O₇PS₂ [M]⁺: 936.29; Found: 936.2.