Tetrazine-Containing Colorful Bioorthogonal Probes Based on the Indolizine Core Skeleton

Abstract

Fluorescent imaging techniques have revolutionized the way to understand biological systems at the nanoscopic, [1] microscopic, [2] and macroscopic levels [3]. To date, fluorescent proteins and immunofluorescence techniques have been most widely used for imaging a target of interest (TOI) [4]. However, the requirement of genetic engineering, fixation, cell membrane penetration or other treatment conditions makes these techniques unsuitable for imaging of innate proteins under a live cell environment, especially for primary cells or clinical samples. An alternative method is making fluorescent conjugate, which binds specifically to the TOI protein [5]. The simplest approach for fluorescent conjugate is the direct coupling of fluorochromes to ligands of TOI proteins, but this approach can reduce the binding affinity of ligand-fluorochrome probes or redirect these probes to off-target proteins in a nonspecific manner [6]. Moreover, extensive washing is required to remove an excess amount of fluorescent conjugates to get a higher signal-to-noise ratio. By taking advantage of bioorthogonal chemistry (highly specific and working in aqueous conditions), [7] researchers now can develop exquisite ligands for protein bioimaging with a minimal perturbation of original binding affinity, due to the much smaller size of bioorthogonal tags for the ligand modification compared to that of direct fluorophore ligation [8].

Experimental Section

3.1.1 General Experimental Information

a. Compound characterization

¹H and ¹³C NMR spectra were recorded on an Agilent 400-NMR [Agilent Technologies], Varian Inova-500 [Varian Associates], and AVANCE^TM 600 [Bruker]. Chemical shifts were reported in parts per million (δ) and calibrated using internal tetramethylsilane (TMS) standard or residual undeuterated solvent for ¹H NMR spectra (CD₂Cl₂ 5.32 ppm; DMSO-d₆ 2.50 ppm; pyridine-d₅ 7.22 ppm) and for ¹³C NMR spectra (DMSO-d₆ 39.52 ppm; CDCl₃ 77.16 ppm, CD₂Cl₂ 53.84 ppm). Multiplicity was indicated as follows: s (singlet); d (doublet); t (triplet); q (quartet); quin (quintet); 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 LCMS-2020 [Shimadzu]. High resolution mass spectrometry (HRMS) of final compounds was further confirmed by Ultra High Resolution ESI Q-TOF mass spectrometer [Bruker] from Organic Chemistry Research Center at Sogang University.

b. Photophysical properties

Absorption spectra were measured by UV-VIS spectrophotometer UV-1650PC [Shimadzu]. Emission spectra and kinetic data were measured by Cary Eclipse Fluorescence spectrophotometer [Varian Associates] and absolute quantum yield was measured by QE-2000 [Otsuka Electronics].

c. Materials

All chemicals were purchased from Sigma-Aldrich, Tokyo Chemical Industry Co., Ltd, ThermoFisher Scientific, or Click Chemistry Tools and used without further purification unless otherwise specified. 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 either with p-anisaldehyde, KMnO₄, or ninhydrin followed by heating. Solvents were purchased from commercial venders and used without further purification. Mouse anti-alpha tubulin monoclonal antibody [#3873] was purchased from Cell Signaling Technology. Rhodamine-tagged goat Anti-mouse IgG (H+L) secondary antibody [31660], TO-PRO™-3 Iodide [T3605], and MitoTracker™ Deep Red FM [M22426] were purchased from Thermo Scientific. RPMI 1640 medium, fetal bovine serum (FBS), and antibiotic-antimycotic solution were purchased from Gibco, Invitrogen. Phosphate-buffered saline (PBS) buffer was purchased from WELGENE. HeLa human cervical cancer cell line was obtained from Korean Cell Line Bank. Dyes, docetaxel-TCO (Dox-TCO), and triphenylphosphonium (TPP)-TCO probe were prepared in dimethyl sulfoxide (DMSO) solution before fluorescence imaging. DMSO, paraformaldehyde, and Triton X-100 were purchased from Sigma-Aldrich. Nunc^TM Lab-Tek^TM II chambered coverglass was purchased from Thermo Scientific. 100-mm culture dish was purchased from CORNING.

d. Quantum mechanical calculations

All quantum mechanical calculations were performed in Gaussin09 W. The ground state structures were optimized using density functional theory (DFT) at the CAM-B3LYP/6-31G^* level. Frequency were checked after ground state geometry optimization to verify the stability of calculated molecular geometry. Energy levels of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) were calculated with the optimized ground state molecular geometry. Vertical transition energy and oscillator strength values (f) were calculated through time-dependent density functional theory (TD-DFT) at the CAM-B3LYP/6-31G^* level, with optimized first-excited state molecular geometry. In all calculations, the substituent at the R³ position of SF_Tzs was fixed as a methyl group to minimize the calculation cost.

e. Fluorescence microscopy

Fluorescence imaging experiment was performed with DeltaVision Elite imaging system from GE Healthcare. Objective lenses were equipped with Olympus IX-71 inverted microscope with PLAN APO 60×/Oil (PLAPON60×O), 1.42 NA, WD 0.15 mm sCMOS camera. InSightSSI fluorescence illumination module was equipped within the system. Imaging was performed with four-color standard filter set [GE Healthcare, 52-852113-003]. For live cell imaging, CO₂ supporting chamber with an objective air heater was installed with the system. Images were analyzed with SoftWorks program supported by GE Healthcare and ImageJ software by National Institutes of Health.

3.1.2 Fluorescence Kinetic Measurements

SF_Tzs and a TCO sample were prepared as 5 mM, and 100 mM DMSO stocks, respectively. To a solution of 20 µM SF_Tzs in a quartz cuvette (2.5 mL, CH₃CN: H₂O = 1:1) at 20 °C, was added 10, 15, or 20 fold excess of TCO-NHS-ester (total DMSO volume was less than 1%), with a vigorous agitation using a Pasteur pipette. SF_Tzs were excited at the corresponding largest λ_ex, and fluorescence signal was recorded over every two seconds at λ_em of each SF_Tz. k_obs values were calculated from GraphPad Prism 5 software, using the one phase exponential association equation.

3.1.3 Experimental Procedure for Live Cell Fluorescence Image

a. Cell culture

HeLa human cervical cancer cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotic-antimycotic solution. Cells were maintained in 100-mm cell culture dish, in 5% CO₂ incubator at 37 ºC with humidified atmosphere.

b. Dox-TCO fluorescence imaging

HeLa cell was seeded in Nunc^TM Lab-Tek^TM II chambered cover-glass and maintained for 1 day. For imaging after fixation, media was aspirated and washed with PBS. Then, cell was incubated with 4% (m/v) paraformaldehyde solution at room temperature for 15 min. 4% (m/v) paraformaldehyde solution was then aspirated and cell was washed with PBS for 3 times. For cell permeabilization, 0.1% (v/v) Triton X-100 in PBS solution were treated at room temperature for 10 min. Solution was then removed and cell was washed with PBS for 3 times. Then, 5~10 μM docetaxel-TCO were treated at room temperature for 1 h, or at 4 ^oC overnight. After brief washing with PBS, TO-PRO-3 Iodide was treated for nucleus staining. Cells were finally washed with PBS for 3 times and imaging was carried out immediately after SF_Tz02^* or SF_Tz08^* (10 μM) treatment.

For the co-localization imaging with alpha-tubulin, media was aspirated and washed with PBS. Then, cell was incubated with 4% (m/v) paraformaldehyde solution at room temperature for 15 min. 4% (m/v) paraformaldehyde solution was then aspirated and cell was washed with PBS for 3 times. For cell permeabilization, 0.1% (v/v) Triton X-100 solution in PBS were treated at room temperature for 10 min. 2% (m/v) BSA solution was treated and incubated at room temperature for 1 h. Solution was then removed and primary antibody diluted in 1% (m/v) BSA solution (1:400) was co-treated with 10 μM docetaxel-TCO at 4 ºC overnight. Primary antibody solution was removed and sample was washed with PBS for 3 times. Secondary antibody diluted in 1% (m/v) BSA solution (1:200) was treated at room temperature for 1 h. For the secondary antibody, rhodamine-tagged goat anti-mouse IgG (H+L) secondary antibody was treated. After 1 h, antibody solution was aspirated and sample was washed with PBS for 3 times. After brief washing with PBS, TO-PRO-3 Iodide was treated for nucleus staining. Cells were finally washed with PBS and imaging was carried out immediately after SF_Tz02^* or SF_Tz08^* (10 μM) treatment. SF_Tz02^* fluorescence was visualized using DAPI/FITC filter set, SF_Tz08^* fluorescence was visualize using DAPI/TRITC filter set, TO-PRO-3 Iodide fluorescence was visualized using Cy5/Cy5 filter set, and alpha-tubulin immunofluorescence was visualized using TRITC/TRITC filter set. DAPI (Excitation: 390/18 nm, Emission: 435/48 nm); FITC (Excitation: 475/28 nm, Emission: 525/48 nm); TRITC (Excitation: 542/27 nm, Emission: 597/45 nm); Cy5 (Excitation: 632/22, Emission: 679/34). Images were analyzed and merged with SoftWorks deconvolution software and ImageJ software.

c. TPP-TCO imaging

HeLa cell was seeded in Nunc^TM Lab-Tek^TM II chambered coverglass and maintained for 1 day. TPP-TCO (10 uM) and MitoTracker™ Deep Red (50 nM) were treated 40 min. Then, cell was washed with PBS, followed by SF_Tz02^* or SF_Tz08^* (10 μM) treatment. For live cell imaging, DeltaVision chamber was maintained in 37 ºC with 5% CO₂ for at least 2 h prior to imaging. Imaging was performed right after SF_Tz02^* or SF_Tz08^* treatment. SF_Tz02^* fluorescence was visualized using DAPI/FITC filter set, SF_Tz08^* fluorescence was visualized using DAPI/TRITC filter set, and MitoTracker™ Deep Red fluorescence was visualized using Cy5/Cy5 filter set. DAPI (Excitation: 390/18 nm, Emission: 435/48 nm); FITC (Excitation: 475/28 nm, Emission: 525/48 nm); TRITC (Excitation: 542/27 nm, Emission: 597/45 nm); Cy5 (Excitation: 632/22, Emission: 679/34). Images were analyzed and merged with SoftWorks deconvolution software and ImageJ software.

3.1.4 Synthetic Procedure and Characterization of New Compounds

Compound SF_Tz01: R1¹ (93.0 mg, 250 µmol), R2² (224 mg, 751 µmol), palladium acetate (11.2 mg, 50.0 µmol), and silver acetate (125 mg, 751 µmol) were dissolved in 2.5 mL of N,N-dimethylformamide (DMF). The reaction mixture was stirred at 80 °C for overnight. The crude reaction mixture was then filtered through Celite and concentrated under reduced pressure. The crude product was purified by silica-gel flash column chromatography [toluene:ethyl acetate(EtOAc) = 1:1] to afford SF_Tz01 (7.7 mg, 5.7% yield) as an orange solid; ¹H NMR (400 MHz, CDCl₃) δ 8.72 (d, J = 8.2 Hz, 2H), 8.62 (d, J = 7.4 Hz, 1H), 8.49 (s, 1H), 7.77 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 7.0 Hz, 1H), 5.32 (br s, 1H, NH), 4.60 (s, 2H), 3.72 (t, J = 6.3 Hz, 2H), 3.20 (m, 2H), 3.13 (s, 3H), 2.66 (s, 3H), 1.88 (t, J = 6.1 Hz, 2H), 1.43 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 195.4, 167.2, 163.8, 161.7, 156.1, 137.9, 135.3, 135.0, 130.0, 129.9, 128.8, 127.9, 125.0, 123.2, 120.9, 112.9, 110.1, 79.2, 46.8, 40.2, 37.3, 28.8, 28.4, 26.2, 21.2; HRMS (ESI) m/z calcd for C₂₉H₃₁N₇NaO₄ [M+Na]⁺: 564.2330, found: 564.2331.

Compound SF_Tz02: A reaction mixture of R3¹ (64.5 mg, 150 µmol), acetonitrile (391 µL, 7.49 mmol), zinc trifluoromethanesulfonate (27.2 mg, 74.9 µmol), and hydrazine monohydrate (728 µL, 15.0 mmol) in microwave reaction vial (10 mL) was irradiated under microwaves at 80 °C (100 W) with magnetic stirring for 0.5 h. After cooled down to room temperature, sodium nitrite (517 mg, 7.49 mmol) in 1 mL of water was added followed by slow addition of 1N HCl until pH value reached at 3 (caution! toxic nitrogen oxide gas evolved!). Then, the resulting residue was washed with saturated NaHCO₃ aqueous solution, extracted with CH₂Cl₂, dried over anhydrous Na₂SO₄, and concentrated. The crude product was purified by normal-phase preparative HPLC using a linear gradient of methanol (1–4%) in CH₂Cl₂ with a flow rate of 5 ml/min to afford SF_Tz02 (16.7 mg, 22.3% yield) as a red solid; ¹H NMR (400 MHz, CDCl₃) δ 9.16 (s, 1H), 8.69 (d, J = 7.4 Hz, 1H), 7.87 (d, J = 7.4 Hz, 1H), 7.59 (d, J = 7.4 Hz, 2H), 7.50 (t, J = 7.6 Hz, 2H), 7.35 (t, J = 7.3 Hz, 1H), 5.39 (br s, 1H, NH), 4.53 (s, 2H), 3.70 (t, J = 6.3 Hz, 2H), 3.20 (m, 2H), 3 s.10 (s, 3H), 1.86 (quin, J = 6.3 Hz, 2H), 1.44 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 167.0, 163.2, 162.0, 156.2, 135.6, 135.2, 133.6, 129.4, 127.6, 127.0, 125.6, 124.2, 122.8, 119.8, 113.8, 109.2, 79.2, 46.8, 40.2, 37.4, 28.9, 28.6, 21.3; HRMS (ESI) m/z calcd for C₂₇H₂₉N₇NaO₃ [M+Na]⁺: 522.2224, found: 522.2225.

Compound S1: R4³ (45.9 mg, 152 µmol) and trifluoroacetic acid (TFA, 300 µL, 3.92 mmol) were dissolved in CH₂Cl₂ (1.2 mL). The reaction mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo to afford S1 (quantitative yield) as a red solid. Resulting product was used for next step without further purification; ¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (d, J = 8.2 Hz, 2H), 8.41 (br s, 3H, NH), 7.74 (d, J = 8.2 Hz, 2H), 4.20 (br s, 2H), 3.01 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.3, 163.0, 138.4, 132.0, 129.8, 127.6, 42.0, 20.9; LRMS (ESI) m/z calcd for C₁₀H₁₂N₅ [M]⁺: 202.1, found: 202.1.

Compound S2: A suspension of R1¹ (114 mg, 308 µmol), methyl 4-iodobenzoate (242 mg, 924 µmol), palladium acetate (13.8 mg, 61.6 µmol), and silver acetate (154 mg, 924 µmol) in DMF (3 mL) was stirred at 80 °C for overnight. The reaction mixture was then filtered through Celite and concentrated under reduced pressure. The crude product was purified by silica-gel flash column chromatography (toluene:EtOAc = 2:1–1:2) to afford S2 (56.1 mg, 36.0% yield) as a yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 8.60 (d, J = 7.0 Hz, 1H), 8.43 (s, 1H), 8.17 (d, J = 8.2 Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.0 Hz, 1H), 5.32 (br s, 1H, NH), 4.55 (s, 2H), 3.97 (s, 3H), 3.70 (t, J = 6.0 Hz, 2H), 3.18 (m, 2H), 2.63 (s, 3H), 1.87–1.84 (m, 2H), 1.43 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 195.5, 166.8, 161.8, 156.2, 138.4, 135.3, 135.1, 130.8, 130.0, 128.4, 127.2, 125.0, 123.3, 121.0, 113.0, 110.2, 79.3, 52.4, 46.8, 40.3, 37.4, 28.9, 28.6, 26.3; LRMS (ESI) m/z calcd for C₂₈H₃₂N₃O₆ [M+H]⁺: 506.2, found: 506.2.

Compound SF_Tz03: A reaction mixture of S2 (43.3 mg, 85.7 µmol), and lithium hydroxide monohydrate (10.8 mg, 257 µmol) in tetrahydrofuran (0.6 mL), methanol (0.3 mL), and water (0.3 mL) was stirred at room temperature for 3 h. The reaction mixture was then washed with saturated NH₄Cl aqueous solution, extracted with CH₂Cl₂, dried over anhydrous Na₂SO₄, and concentrated. The resulting solid, N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methyl4 N-oxide (HATU, 65.1 mg, 171 µmol), and N,N-diisopropylethylamine (DIPEA, 74.6 µL, 428 µmol) were dissolved in DMF (1.2 mL). After the solution was stirred for 0.5 h at room temperature, S1 (40.5 mg, 128 µmol) was added to the solution and stirred at room temperature for additional 2 h. The crude product was purified by silica-gel flash column chromatography (CH₂Cl₂:MeOH = 20:1) followed by washing with EtOAc for several times, and dried in vacuo to afford SF_Tz03 (21 mg, 36% yield) as a red solid; ¹H NMR (500 MHz, CD₂Cl₂) δ 8.59–8.57 (m, 3H), 8.44 (s, 1H), 7.98 (d, J = 8.3 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.32 (dd, J = 7.3, 1.5 Hz, 1H), 6.75 (t, J = 5.8 Hz, 1H, NH), 5.36 (br s, 1H, NH), 4.80 (d, J = 5.9 Hz, 2H), 4.54 (s, 2H), 3.67 (t, J = 6.4 Hz, 2H), 3.10 (m, 2H), 3.07 (s, 3H), 2.60 (s, 3H), 1.81 (quin, J = 6.3 Hz, 2H), 1.41 (s, 9H); ¹³C NMR (100 MHz, CDCl₃:MeOD-d₄ = 10:1, v/v) δ 196.1, 167.6, 167.3, 163.9, 161.9, 156.6, 143.5, 137.0, 135.4, 135.1, 132.2, 130.8, 129.7, 128.5, 128.34, 128.26, 127.4, 124.9, 122.9, 120.9, 113.1, 110.2, 79.4, 46.8, 43.7, 40.4, 37.5, 28.7, 28.4, 26.3, 21.1; HRMS (ESI) m/z calcd for C₃₇H₃₈N₈NaO₅ [M+Na]⁺: 697.2857, found: 697.2860.

Compound SF_Tz04: A reaction mixture of R5¹ (39.2 mg, 84.6 µmol), lithium hydroxide monohydrate (10.6 mg, 254 µmol), tetrahydrofuran (0.6 mL), methanol (0.3 mL), and water (0.3 mL) was stirred at room temperature for 1 h. The reaction mixture was then washed with saturated NH₄Cl aqueous solution, extracted with EtOAc, dried over anhydrous Na₂SO₄, and concentrated. The resulting solid, S1 (40.0 mg, 127 µmol), HATU (64.3 mg, 169 µmol), and DIPEA (73.7 µL, 423 µmol) were dissolved in DMF (0.9 mL). The mixture was stirred at room temperature for 2 h. After the solvent was removed under reduced pressure, the crude product was purified by silica-gel flash column chromatography (CH₂Cl₂:MeOH = 20:1) to afford SF_Tz04 (37.6 mg, 70.2% yield) as a pink solid; ¹H NMR (500 MHz, CD₂Cl₂) δ 8.57 (d, J = 7.3 Hz, 1H), 8.54 (d, J = 8.3 Hz, 2H), 8.34 (s, 1H), 7.60–7.56 (m, 4H), 7.48 (t, J = 7.8 Hz, 2H), 7.31 (t, J = 7.3 Hz, 2H), 7.12 (dd, J = 7.1, 1.7 Hz, 1H), 6.79 (t, J = 5.9 Hz, 1H, NH), 5.41 (br s, 1H, NH), 4.76 (d, J = 5.9 Hz, 2H), 4.49 (s, 2H), 3.64 (t, J = 6.4 Hz, 2H), 3.13–3.10 (m, 2H), 3.06 (s, 3H), 1.79 (quin, J = 6.4 Hz, 2H), 1.40 (s, 9H); ¹³C NMR (100 MHz, CDCl₃:MeOD-d₄ = 10:1, v/v) δ 167.3, 166.4, 163.9, 162.4, 156.6, 143.6, 135.34, 135.29, 133.5, 130.8, 129.2, 128.5, 128.2, 127.5, 126.8, 126.3, 124.7, 121.5, 119.4, 112.7, 109.7, 79.4, 46.8, 43.8, 40.4, 37.5, 28.7, 28.4, 21.1; HRMS (ESI) m/z calcd for C₃₅H₃₆N₈NaO₄ [M+Na]⁺: 655.2752, found: 655.2755.

Compound S3: A suspension of R6¹ (42 mg, 94 µmol) and 12 N HCl (100 µL, 1.2 mmol) in CH₂Cl₂ (1 mL) was stirred at room temperature for 2 h, and solvent was then concentrated under reduced pressure. The resulting crude solid, succinic anhydride (14.1 mg, 141 µmol), and triethylamine (65.4 µL, 469 µmol) were dissolved in CH₂Cl₂ (1 mL). The mixture was stirred at room temperature for overnight. The crude mixture was then directly purified by silica-gel flash column chromatography (CH₂Cl₂:MeOH = 10:1 with 1% acetic acid) to afford S3 (36 mg, 86% yield) as a yellow solid; ¹H NMR (500 MHz, DMSO-d₆) δ 8.55 (d, J = 7.3 Hz, 1H), 8.47 (s, 1H), 7.90 (t, J = 5.3 Hz, 1H, NH), 7.73 (d, J = 7.3 Hz, 2H), 7.54 (t, J = 7.6 Hz, 2H), 7.36 (t, J = 7.2 Hz, 1H), 7.31 (d, J = 7.3 Hz, 1H), 4.70 (s, 2H), 3.52 (t, J = 7.1 Hz, 2H), 3.11–3.08 (m, 2H), 2.64 (s, 3H), 2.42 (t, J = 7.1 Hz, 2H), 2.31 (t, J = 7.1 Hz, 3H), 1.77 (quin, J = 6.8 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 195.7, 174.0, 171.0, 160.3, 135.3, 133.5, 133.2, 129.3, 128.7, 127.3, 126.7, 124.2, 122.6, 121.3, 113.0, 109.5, 46.2, 40.3, 36.3, 30.1, 29.3, 28.2, 26.2; LRMS (ESI) m/z calcd for C₂₅H₂₆N₃O₅ [M+H]⁺: 448.2, found: 448.1.

Compound SF_Tz05: A suspension of S3 (33 mg, 74 µmol), S1 (35 mg, 110 µmol), HATU (56 mg, 150 µmol), and DIPEA (64 µL, 370 µmol) in DMF (1 mL) was stirred at room temperature for 2 h. The crude reaction mixture was directly purified by silica-gel flash column chromatography (CH₂Cl₂:MeOH = 20:1) to afford SF_Tz05 (23 mg, 49% yield) as an orange solid; ¹H NMR (500 MHz, pyridine-d₅) δ 9.41 (t, J = 5.9 Hz, 1H, NH), 8.66 (d, J = 7.3 Hz, 1H), 8.60 (d, J = 7.8 Hz, 2H), 8.57 (s, 1H), 7.77 (d, J = 7.3 Hz, 2H), 7.67 (d, J = 8.3 Hz, 2H), 7.60–7.57 (m, 2H), 7.44–7.40 (m, 2H), 4.78 (d, J = 5.9 Hz, 2H), 4.39 (s, 2H), 3.79 (t, J = 7.0 Hz, 2H), 3.63–3.60 (m, 2H), 3.05–3.03 (m, 2H), 2.99–2.95 (m, 5H), 2.61 (s, 3H), 2.06 (quin, J = 6.8 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃:MeOD-d₄ = 10:1, v/v) δ 196.3, 173.1, 173.0, 167.2, 163.9, 162.2, 143.5, 135.2, 134.9, 133.3, 130.6, 129.43, 129.38, 128.3, 128.1, 127.6, 127.1, 124.7, 122.5, 121.5, 114.4, 109.8, 46.8, 43.1, 40.5, 36.4, 31.73, 31.66, 28.3, 26.1, 21.1; HRMS (ESI) m/z calcd for C₃₅H₃₄N₈NaO₄ [M+Na]⁺: 653.2595, found: 653.2599.

Compound S4: A suspension of R7¹ (181.5 mg, 512 µmol), 4-iodoanisole (360 mg, 1.54 mmol), palladium acetate (23 mg, 102 µmol), and silver acetate (256 mg, 1.54 mmol) in DMF (5 mL) was stirred at 80 °C for 4 h. The crude reaction mixture was then filtered through Celite and concentrated under reduced pressure. The crude product was purified by silica-gel flash column chromatography (toluene:EtOAc = 2:1) to afford S4 (80 mg, 34% yield) as a solid; ¹H NMR (400 MHz, CDCl₃) δ 8.55 (d, J = 7.4 Hz, 1H), 8.09 (s, 1H), 7.41 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 7.0 Hz, 1H), 5.29 (br s, 1H, NH), 4.49 (s, 2H), 3.88 (s, 3H), 3.68 (t, J = 6.3 Hz, 2H), 3.17 (q, J = 6.0 Hz, 2H), 1.84 (quin, J = 6.3 Hz, 2H), 1.42 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 161.6, 159.0, 156.1, 134.7, 133.5, 128.7, 125.8, 125.3, 125.0, 123.3, 118.5, 114.9, 113.6, 111.1, 102.7, 79.1, 55.4, 46.5, 40.2, 37.3, 28.7, 28.4; LRMS (ESI) m/z calcd for C₂₆H₂₉N₄O₄ [M+H]⁺: 461.2, found: 461.1.

Compound SF_Tz06: A reaction mixture of S4 (33.4 mg, 72.5 µmol), acetonitrile (190 µL, 3.63 mmol), zinc trifluoromethanesulfonate (13.2 mg, 36.3 µmol), and hydrazine monohydrate (352 µL, 7.25 mmol) in microwave reaction vial (10 mL) was irradiated under microwaves at 80 °C (100 W) with magnetic stirring for 0.5 h. After cooled down to room temperature, sodium nitrite (250 mg, 3.63 mmol) in 1 ml of water was added followed by slow addition of 1 N HCl until pH value reached at 3 (caution! toxic nitrogen oxide gas evolved!). Then, the resulting residue was washed with brine, extracted with CH₂Cl₂, and dried over anhydrous Na₂SO₄. After concentrated under reduced pressure, the crude product was purified by normal-phase preparative HPLC using a gradient of methanol (1–10%) in CH₂Cl₂ with a flow rate of 5 ml/min to afford SF_Tz06 (7.0 mg, 18% yield) as a red solid; ¹H NMR (400 MHz, CDCl₃) δ 9.12 (s, 1H), 8.69 (d, J = 7.4 Hz, 1H), 7.86 (dd, J = 7.2, 1.2 Hz, 1H), 7.53 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.6 Hz, 2H), 5.39 (br s, 1 H, NH), 4.51 (s, 2H), 3.89 (s, 3H), 3.70 (t, J = 6.3 Hz, 2H), 3.19 (q, J = 5.9 Hz, 2H), 3.09 (s, 3H), 1.85 (quin, J = 6.3 Hz, 2H), 1.43 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 166.8, 163.1, 162.0, 158.6, 156.1, 135.3, 134.6, 128.7, 125.9, 125.4, 123.6, 122.5, 119.8, 114.8, 113.6, 108.9, 79.1, 55.4, 46.6, 40.1, 37.2, 28.7, 28.4, 21.2; HRMS (ESI) m/z calcd for C₂₈H₃₁N₇NaO₄ [M+Na]⁺: 552.2330, found: 552.2330.

Compound S5: A suspension of R7¹ (126 mg, 356 µmol), 1-iodo-4-nitro-benzene (266 mg, 1.07 mmol), palladium acetate (16.0 mg, 71.1 µmol), and silver acetate (178 mg, 1.07 mmol) in DMF (3.6 mL) was stirred at 80 °C for 2 h. The reaction mixture was then filtered through a short bed of silica gel with EtOAc and concentrated under reduced pressure. The crude product was purified by silica-gel flash column chromatography (toluene:EtOAc = 1:1) to afford S5 (134 mg, 283 µmol, 79.5% yield) as a yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 8.68 (d, J = 7.4 Hz, 1H), 8.37 (d, J = 8.6 Hz, 2H), 8.21 (s, 1H), 7.67 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 7.4 Hz, 1H), 5.18 (br s, 1H, NH), 4.59 (s, 2H), 3.71 (t, J = 6.5 Hz, 2H), 3.19 (q, J = 6.1 Hz, 2H), 1.88 (quin, J = 6.3 Hz, 2H), 1.43 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 161.1, 156.0, 146.3, 139.6, 135.7, 134.1, 127.6, 125.9, 125.0, 124.8, 124.3, 117.8, 112.2, 111.2, 105.1, 79.3, 46.6, 40.3, 37.4, 28.8, 28.4; LRMS (ESI) m/z calcd for C₂₅H₂₆N₅O₅ [M+H]⁺: 476.2, found: 476.2.

Compound SF_Tz07: A reaction mixture of S5 (40 mg, 84 µmol), hydrazine monohydrate (409 µL, 8.41 mmol), acetonitrile (220 µL, 4.21 mmol), and zinc trifluoromethansulfonate (15.3 mg, 42.1 µmol) in a microwave reaction vial (10 ml) was irradiated under microwaves at 80 °C (100 W) with magnetic stirring for 1.5 h. After cooled down to room temperature, sodium nitrite (290 mg, 4.21 mmol) in 0.5 mL of water was added followed by slow addition of 1 N HCl until pH value reached 3 (caution! toxic nitrogen oxide gas evolved!). Then, the resulting residue was washed with saturated NaHCO₃ aqueous solution, extracted with CH₂Cl₂, dried over anhydrous Na₂SO₄, and concentrated. The crude product was purified by normal-phase preparative HPLC using a linear gradient of methanol (0.1–10%) in CH₂Cl₂ with a flow rate of 5 ml/min to afford SF_Tz07 (2 mg, 5% yield) as a reddish brown solid; ¹H NMR (400 MHz, CDCl₃) δ 9.13 (s, 1H), 8.67 (d, J = 7.4 Hz, 1H), 7.84 (dd, J = 7.4, 1.6 Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 5.39 (br s, 1H, NH), 4.50 (s, 2H), 3.81 (br s, 2H, NH₂), 3.70 (t, J = 6.3 Hz, 2H), 3.19 (q, J = 6.2 Hz, 2H), 3.09 (s, 3H), 1.84 (quin, J = 6.2 Hz, 2H), 1.43 (s, 9H); ¹³C NMR (150 MHz, CDCl₃) δ 166.7, 163.2, 162.0, 156.1, 145.5, 135.2, 134.4, 128.7, 125.4, 123.6, 123.3, 122.4, 120.1, 115.8, 114.2, 108.8, 79.1, 46.6, 40.1, 37.3, 28.7, 28.4, 21.2; HRMS (ESI) m/z calcd for C₂₇H₃₀N₈NaO₃ [M+Na]⁺: 537.2333, found: 537.2335.

Compound S6: R7¹ (368.4 mg, 895 µmol), N,N-diethyl-4-iodoaniline⁴ (739 mg, 2.69 mmol), palladium acetate (40.2 mg, 179 µmol), and silver acetate (488 mg, 2.69 mmol) were dissolved in DMF (9 mL). The mixture was stirred at 80 °C for overnight. The reaction mixture was filtered through a short bed of silica gel with EtOAc and concentrated under reduced pressure. The crude reaction mixture was purified by silica-gel flash column chromatography (toluene:EtOAc = 10:1) to afford S6 (147.5 mg, 29.5% yield) as a reddish brown solid; ¹H NMR (400 MHz, CDCl₃) δ 8.51 (d, J = 7.4 Hz, 1H), 8.12 (s, 1H), 7.34 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 6.72 (dd, J = 7.0, 1.2 Hz, 1H), 5.34 (br s, 1H, NH), 4.48 (s, 2H), 3.68 (t, J = 6.2 Hz, 2H), 3.42 (q, J = 7.0 Hz, 4H), 3.17 (q, J = 6.0 Hz, 2H), 1.83 (quin, J = 6.3 Hz, 2H), 1.43 (s, 9H), 1.22 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 161.8, 156.2, 147.1, 134.3, 133.3, 128.7, 126.4, 125.2, 123.2, 119.2, 118.9, 114.7, 112.3, 110.9, 101.9, 79.2, 46.7, 44.5, 40.3, 37.4, 28.8, 28.5, 12.7; LRMS (ESI) m/z calcd for C₂₉H₃₆N₅O₃ [M+H]⁺: 502.3, found: 502.2.

Compound SF_Tz08: A reaction mixture of S6 (60.2 mg, 120 µmol), acetonitrile (313 µL, 6.00 mmol), zinc trifluoromethanesulfonate (21.8 mg, 60.0 µmol), and hydrazine monohydrate (583 µL, 12.0 mmol) in microwave reaction vial (10 mL) was irradiated under microwaves at 100 °C (40 W) with magnetic stirring for 1.5 h. After cooled down to room temperature, NaNO₂ was added followed by slow addition of 1 N HCl until pH value reached at 3 (caution! toxic nitrogen oxide gas evolved!). Then, the resulting residue was washed with saturated NaHCO₃ aqueous solution, extracted with CH₂Cl₂, dried over anhydrous Na₂SO₄, and concentrated. The crude product was purified by silica-gel flash column chromatography (CH₂Cl₂:EtOAc = 8:1) to afford SF_Tz08 (11.8 mg, 17.2% yield) as a reddish brown solid; ¹H NMR (400 MHz, CD₂Cl₂) δ 9.15 (s, 1H), 8.65 (d, J = 7.0 Hz, 1H), 7.81 (d, J = 7.4 Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.2 Hz, 2H), 5.46 (br s, 1H, NH), 4.51 (s, 2H), 3.67 (t, J = 6.3 Hz, 2H), 3.43 (q, J = 6.8 Hz, 4H), 3.14 (q, J = 6.0 Hz, 2H), 3.05 (s, 3H), 1.81 (quin, J = 6.3 Hz, 2H), 1.42 (s, 9H), 1.21 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 166.6, 163.2, 162.1, 156.1, 146.7, 135.0, 134.1, 128.6, 125.3, 122.9, 122.4, 120.4, 120.1, 114.6, 112.2, 108.6, 79.0, 46.7, 44.5, 40.0, 37.2, 28.7, 28.4, 21.2, 12.7; HRMS (ESI) m/z calcd for C₃₁H₃₈N₈NaO₃ [M+Na]⁺: 593.2959, found: 593.2960.

Compound S7: A suspension of R8¹ (129 mg, 333 µmol), N,N-diethyl-4-iodo-aniline⁴ (275 mg, 999 µmol), palladium acetate (15.0 mg, 66.6 µmol), and silver acetate (167 mg, 999 µmol) in DMF (3.5 mL) was stirred at 80 °C for overnight. The reaction mixture was then filtered through a short bed of silica gel with EtOAc and concentrated under reduced pressure. The crude product was purified by silica-gel flash column chromatography (toluene:EtOAc = 1:1) to afford S7 (37 mg, 21% yield) as a reddish solid; ¹H NMR (400 MHz, CDCl₃) δ 8.54 (s, 1H), 8.51 (d, J = 7.4 Hz, 1H), 7.41 (d, J = 8.6 Hz, 2H), 7.23 (dd, J = 7.0, 1.6 Hz, 1H), 6.79 (d, J = 8.6 Hz, 2H), 5.41 (br s, 1H, NH), 4.47 (s, 2H), 3.93 (s, 3H), 3.68 (t, J = 6.3 Hz, 2H), 3.42 (q, J = 7.0 Hz, 4H), 3.17 (q, J = 5.9 Hz, 2H), 1.82 (quin, J = 6.2 Hz, 2H), 1.42 (s, 9H), 1.22 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 166.1, 162.1, 156.1, 146.7, 134.4, 133.9, 128.6, 124.2, 122.7, 122.1, 121.1, 120.1, 114.2, 112.2, 110.3, 79.0, 52.3, 46.6, 44.4, 40.0, 37.2, 28.7, 28.4, 12.7; LRMS (ESI) m/z calcd for C₃₀H₃₉N₄O₅ [M+H]⁺: 535.3, found: 535.2.

Compound SF_Tz09: A reaction mixture of S7 (26.7 mg, 49.9 µmol), lithium hydroxide monohydrate (6.3 mg, 150 µmol), tetrahydrofuran (THF, 300 µL), MeOH (150 µL), and H₂O (150 µL) was stirred at room temperature for 2 h. The mixture was then washed with saturated NH₄Cl aqueous solution, extracted with EtOAc, dried over anhydrous Na₂SO₄, and concentrated. The resulting solid, S1 (23.6 mg, 74.9 µmol), HATU (38.0 mg, 100 µmol), and DIPEA (43.5 µL, 250 µmol) were dissolved in DMF (0.5 mL). The mixture was stirred at room temperature for 2 h. After the solvent was removed under reduced pressure, the crude product was purified by silica-gel flash column chromatography (CH₂Cl₂:MeOH = 20:1) to afford SF_Tz09 (32.9 mg, 93.6% yield) as a brown solid; ¹H NMR (400 MHz, CDCl₃) δ 8.54 (d, J = 8.2 Hz, 2H), 8.48 (d, J = 7.4 Hz, 1H), 8.31 (s, 1H), 7.56 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 9.0 Hz, 2H), 7.04 (d, J = 7.0 Hz, 1H), 6.80–6.74 (m, 3H), 5.39 (br s., 1H, NH), 4.77 (d, J = 5.9 Hz, 2H), 4.44 (s, 2H), 3.65 (t, J = 6.1 Hz, 2H), 3.39 (q, J = 7.0 Hz, 4H), 3.15 (d, J = 5.9 Hz, 2H), 3.08 (s, 3H), 1.80 (quin, J = 6.3 Hz, 2H), 1.42 (s, 9H), 1.19 (t, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃:MeOD-d₄ = 10:1, v/v) δ 167.3, 166.7, 164.0, 162.6, 156.7, 146.7, 143.7, 134.8, 134.6, 130.7, 128.6, 128.5, 128.2, 125.2, 124.5, 121.1, 120.3, 119.9, 113.5, 112.5, 109.3, 79.4, 46.9, 44.5, 43.8, 40.4, 37.5, 28.7, 28.4, 21.1, 12.6; HRMS (ESI) m/z calcd for C₃₉H₄₆N₉O₄ [M+H]⁺: 704.3667, found: 704.3669.

Compound S8: A suspension of R9¹ (45 mg, 87 µmol), and TFA (200 µL, 2.61 mmol) in CH₂Cl₂ (0.9 mL) was stirred at room temperature for 3 h. The resulting residue was then washed with saturated NaHCO₃ aqueous solution, extracted with CH₂Cl₂, dried over anhydrous Na₂SO₄, and concentrated. The resulting solid, succinic anhydride (13.0 mg, 130 µmol), and triethylamine (60.5 µL, 433 µmol) were dissolved in CH₂Cl₂ (900 µL). The mixture was stirred at room temperature for 1 h. The crude reaction mixture was directly purified by silica-gel flash column chromatography (CH₂Cl₂:MeOH = 10:1 with 1% acetic acid) to afford S8 (35.3 mg, 78.5% yield) as a red solid; ¹H NMR (400 MHz, CDCl₃) δ 8.49 (d, J = 7.0 Hz, 1H), 8.40 (s, 1H), 7.43 (br s, 2H), 7.25 (d, J = 6.7 Hz, 1H), 6.84 (br s, 2H), 4.51 (s, 2H), 3.66 (t, J = 6.5 Hz, 2H), 3.45 (d, J = 5.9 Hz, 4H), 3.27 (t, J = 6.1 Hz, 2H), 2.68–2.65 (m, 2H), 2.61 (s, 3H), 2.56–2.52 (m, 2H), 1.86 (quin, J = 6.2 Hz, 2H), 1.24 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃:MeOD-d₄ = 10:1, v/v) δ 196.3, 175.3, 173.0, 162.5, 147.1, 134.5, 129.1, 128.8, 128.3, 125.4, 124.6, 122.3, 119.9, 115.4, 112.5, 109.4, 46.9, 44.6, 40.4, 36.2, 31.1, 29.9, 28.2, 26.1, 12.5; LRMS (ESI) m/z calcd for C₂₉H₃₅N₄O₅ [M+H]⁺: 519.3, found: 519.1.

Compound SF_Tz10: A suspension of S8 (26 mg, 50 µmol), S1 (23.7 mg, 75.2 µmol), HATU (38.1 mg, 100 µmol), and DIPEA (43.7 µL, 251 µmol) in DMF (500 µL) was stirred at room temperature for 2 h. Solvent was then removed under reduced pressure and the crude product was purified by silica-gel flash column chromatography (CH₂Cl₂:MeOH = 20:1) to afford SF_Tz10 (26 mg, 74% yield) as a red solid; ¹H NMR (400 MHz, CDCl₃) δ 8.45–8.37 (m, 2H), 8.37 (s, 1H), 7.45 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 7.0 Hz, 1H), 7.03–6.99 (m, 2H), 6.79 (d, J = 8.6 Hz, 2H), 4.55 (d, J = 5.9 Hz, 2H), 4.44 (s, 2H), 3.64 (t, J = 6.1 Hz, 2H), 3.43 (q, J = 7.0 Hz, 4H), 3.29 (q, J = 5.6 Hz, 2H), 3.04 (s, 3H), 2.67 (m, 4H), 2.61 (s, 3H), 1.84–1.78 (m, 2H), 1.23 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 195.5, 172.4, 172.3, 167.1, 163.7, 162.2, 146.7, 143.4, 134.1, 133.9, 130.6, 128.54, 128.46, 128.2, 127.9, 124.3, 122.04, 121.99, 119.8, 115.0, 112.2, 109.2, 46.7, 44.4, 43.2, 39.9, 35.8, 32.0, 28.3, 26.0, 21.1, 12.7; HRMS (ESI) m/z calcd for C₃₉H₄₃N₉NaO₄ [M+Na]⁺: 724.3330, found: 724.3333.

Compound S9: A suspension of R10⁵ (11.7 g, 39.5 mmol), propargylamine (12.7 mL, 198 mmol), and potassium carbonate (10.9 g, 79.0 mmol) in acetonitrile (100 mL) was stirred at 80 °C for overnight. Solvent was then removed under reduced pressure and the resulting residue was washed with saturated NaHCO₃ aqueous solution, extracted with EtOAc, dried over anhydrous Na₂SO₄, and concentrated. The crude product was then purified by silica-gel flash column chromatography (hexane:EtOAc = 2:1–1:1) to afford S9 (8.47 g, 79.5% yield) as a yellow oil; ¹H NMR (400 MHz, CDCl₃) δ 3.79 (t, J = 6.1 Hz, 2H), 3.42 (d, J = 2.3 Hz, 2H), 2.82 (t, J = 6.8 Hz, 2H), 2.19 (t, J = 2.3 Hz, 1H), 1.74 (quin, J = 6.4 Hz, 2H), 1.12–1.03 (m, 21H); ¹³C NMR (100 MHz, CDCl₃) δ 82.5, 71.2, 62.3, 46.4, 38.4, 32.9, 18.2, 12.1; LRMS (ESI) m/z calcd for C₁₅H₃₂NOSi [M+H]⁺: 270.2, found: 270.2.

Compound S10: To a solution of bromoacetyl bromide (5.5 mL, 62 mmol) and dry triethylamine (13 mL, 94 mmol) in dry CH₂Cl₂ (200 mL) was slowly added a dry CH₂Cl₂ (100 mL) containing S9 (8.4 g, 31 mmol) for 5 min at –78 °C under argon atmosphere and the reaction mixture was stirred at –78 °C for 1 h. The crude mixture was then washed with saturated brine, extracted with CH₂Cl₂, dried over anhydrous Na₂SO₄, and concentrated. The crude product was purified by silica-gel flash column chromatography (hexane:EtOAc = 5:1–3:1) to afford S10 (10.3 g, 85% yield) as a yellow oil; ¹H NMR (70:30 mixture of rotamers, ^* denote minor rotamer peaks, 500 MHz, CDCl₃) δ 4.22 (d, J = 2.4 Hz, 2H), 4.20^* (d, J = 2.4 Hz, 2H), 4.00 (s, 2H), 3.94^* (s, 2H), 3.76–3.72 (m, 2H), 3.67 (t, J = 7.1 Hz, 2H), 3.58^* (t, J = 7.1 Hz, 2H), 2.33^* (t, J = 2.4 Hz, 1H), 2.22 (t, J = 2.4 Hz, 1H), 1.89–1.81 (m, 2 H), 1.14–1.05 (m, 21 H); ¹³C NMR (125 MHz, CDCl₃) δ 166.9, 78.5, 73.2^*, 72.3, 60.6^*, 59.6, 45.1, 44.7^*, 38.8^*, 35.1, 31.5, 30.6^*, 26.4^*, 26.1, 18.2, 12.1^*, 12.0; LRMS (ESI) m/z calcd for C₁₇H₃₃BrNO₂Si [M+H]⁺: 390.2, found: 390.2.

Compound S11: A suspension of S10 (3.41 g, 8.73 mmol) and 4-cyanopyridine (1.09 g, 10.5 mmol) in acetonitrile (45 mL) was stirred at 80 °C for overnight. The reaction mixture was then cooled down to room temperature, copper iodide (1.66 g, 8.73 mmol) and potassium carbonate (3.62 g, 26.2 mmol) were added, and reaction mixture was stirred at room temperature for 4 h. The solvent was removed under reduced pressure, and the mixture was filtered through a short bed of silica gel with EtOAc. After concentration, the crude product was purified by silica-gel flash column chromatography (hexane:EtOAc = 4:1) to afford S11 (700 mg, 19.5% yield) as a white solid; ¹H NMR (500 MHz, DMSO-d₆) δ 8.50 (d, J = 7.3 Hz, 1H), 8.33 (s, 1H), 6.98 (dd, J = 7.1, 1.7 Hz, 1H), 6.79 (s, 1 H), 4.44 (s, 2H), 3.72 (t, J = 6.0 Hz, 2H), 3.57 (t, J = 7.3 Hz, 2H), 1.82 (quin, J = 6.8 Hz, 2H), 1.04–0.97 (m, 21 H); ¹³C NMR (125 MHz, DMSO-d₆) δ 160.1, 137.2, 136.5, 126.9, 124.6, 123.4, 118.5, 111.0, 101.0, 98.5, 60.8, 46.1, 39.7, 31.5, 17.8, 11.4; LRMS (ESI) m/z calcd for C₂₃H₃₄N₃O₂Si [M+H]⁺: 412.2, found: 412.2.

Compound S12: A suspension of S11 (300 mg, 729 µmol), iodobenzene (245 µL, 2.19 mmol), palladium acetate (32.7 mg, 146 µmol), and silver acetate (365 mg, 2.19 mmol) in DMF (7.5 mL) was stirred at 80 °C for overnight. The reaction mixture was then filtered through a short bed of silica gel with EtOAc and concentrated under reduced pressure. The crude product was purified by silica-gel flash column chromatography (toluene:EtOAc = 10:1) to afford S12 (195 mg, 54.9% yield) as a yellow solid; ¹H NMR (500 MHz, CDCl₃) δ 8.60 (d, J = 7.3 Hz, 1H), 8.16 (s, 1H), 7.50 (d, J = 4.4 Hz, 4H), 7.39–7.35 (m, 1H), 6.80 (dd, J = 7.1, 1.2 Hz, 1H), 4.57 (s, 2H), 3.81 (t, J = 6.0 Hz, 2H), 3.74 (t, J = 7.2 Hz, 2H), 1.94 (quin, J = 6.7 Hz, 2H), 1.11–1.04 (m, 21H); ¹³C NMR (100 MHz, CDCl₃) δ 161.2, 135.2, 133.6, 133.0, 129.5, 127.7, 127.4, 125.9, 125.5, 124.2, 118.6, 113.7, 111.3, 103.1, 61.0, 47.1, 40.7, 32.1, 18.2, 12.1; LRMS (ESI) m/z calcd for C₂₉H₃₈N₃O₂Si [M+H]⁺: 488.3, found: 488.3.

Compound S13: To a solution of S12 (595 mg, 1.22 mmol) in tetrahydrofuran (3.3 mL), was added tetra-n-butylammonium fluoride (TBAF) solution (1.0 M in THF, 1.8 mL, 1.8 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 1.5 h. The crude product was directly purified by silica-gel flash column chromatography (CH₂Cl₂:MeOH = 20:1) to afford S13 (376.4 mg, 93.1% yield) as a green-yellow solid; ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (d, J = 7.0 Hz, 1H), 8.47 (s, 1H), 7.67 (d, J = 7.4 Hz, 2H), 7.49 (t, J = 7.6 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H), 7.04 (dd, J = 7.0, 1.2 Hz, 1H), 4.68 (s, 2H), 4.53 (t, J = 5.0 Hz, 1H, OH), 3.58 (t, J = 7.0 Hz, 2H), 3.48 (q, J = 5.7 Hz, 2H), 1.79 (quin, J = 6.7 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.0, 135.3, 132.55, 132.53, 129.2, 127.3, 126.8, 125.9, 125.0, 123.3, 118.5, 112.5, 111.5, 102.3, 58.4, 46.3, 39.8, 31.4; LRMS (ESI) m/z calcd for C₂₀H₁₈N₃O₂ [M+H]⁺: 332.1, found: 332.1.

Compound S14: A suspension of S13 (356 mg, 1.07 mmol), methanesulfonic anhydride (281 mg, 1.61 mmol), and anhydrous triethylamine (749 µL, 5.37 mmol) in anhydrous CH₂Cl₂ (4 mL) was stirred at room temperature for 2.5 h under Ar. The crude reaction mixture was then directly purified by silica-gel flash column chromatography (CH₂Cl₂:MeOH = 20:1) to afford S14 (441 mg, 100% yield) as a yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 8.57 (d, J = 7.0 Hz, 1H), 8.15 (s, 1H), 7.53–7.48 (m, 4H), 7.40–7.36 (m, 1H), 6.81 (d, J = 7.4 Hz, 1H), 4.56 (s, 2H), 4.33 (t, J = 6.1 Hz, 2H), 3.76 (t, J = 6.7 Hz, 2H), 3.06 (s, 3H), 2.18 (quin, J = 6.4 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 161.3, 135.1, 133.7, 132.6, 129.4, 127.5, 127.4, 125.7, 125.4, 123.3, 118.3, 113.7, 111.3, 103.3, 67.3, 46.9, 39.7, 37.5, 28.6; LRMS (ESI) m/z calcd for C₂₁H₂₀N₃O₄S [M+H]⁺: 410.1, found: 410.0.

Compound S15: A suspension of S14 (439 mg, 1.07 mmol), 1-Boc-piperazine (399 mg, 2.14 mmol), and triethylamine (449 µL, 3.22 mmol) in DMF (3 mL) was stirred at 50 °C for overnight. The crude reaction mixture was then directly purified by silica-gel flash column chromatography (CH₂Cl₂:MeOH = 5:1) to afford S15 (391 mg, 73% yield) as a yellow solid; ¹H NMR (500 MHz, CDCl₃) δ 8.60 (d, J = 7.4 Hz, 1H), 8.17 (s, 1H), 7.53–7.49 (m, 4 H), 7.39–7.37 (m, 1H), 6.81 (dd, J = 7.1, 1.7 Hz, 1H), 4.53 (s, 2H), 3.67 (t, J = 7.1 Hz, 2H), 3.41 (t, J = 4.8 Hz, 4H), 2.44 (t, J = 7.3 Hz, 2H), 2.38–2.36 (m, 4H), 1.89 (quin, J = 7.2 Hz, 2H), 1.46 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 161.1, 154.7, 135.0, 133.5, 132.8, 129.4, 127.5, 127.3, 125.7, 125.4, 123.8, 118.4, 113.6, 111.2, 103.1, 79.6, 55.7, 53.0, 46.6, 43.9, 41.4, 28.4, 26.1; LRMS (ESI) m/z calcd for C₂₉H₃₄N₅O₃ [M+H]⁺: 500.3, found: 500.1.

Compound S16: A reaction mixture of S15 (80 mg, 160 µmol), acetonitrile (418 µL, 8.01 mmol), hydrazine monohydrate (778 µL, 16.0 mmol), and zinc trifluoromethansulfonate (29.1 mg, 80.1 µmol) in microwave reaction vial (10 mL) was irradiated under microwaves at 90 °C (30 W) with magnetic stirring for 1 h. After cooled down to room temperature, sodium nitrite (552 mg, 8.01 mmol) in 2 mL of water was added followed by slow addition of 1 N HCl until pH value reached at 3 (caution! toxic nitrogen oxide gas evolved!). Then, the resulting residue was washed with saturated NaHCO₃ aqueous solution, extracted with CH₂Cl₂, dried over anhydrous Na₂SO₄, and concentrated. The crude product was purified by normal-phase preparative HPLC using a linear gradient of methanol (0.1–10%) in CH₂Cl₂ with a flow rate of 5.5 ml/min to afford S16 (18 mg, 20% yield) as a red solid; ¹H NMR (400 MHz, CDCl₃) δ 9.15 (s, 1H), 8.70 (d, J = 7.4 Hz, 1H), 7.86 (d, J = 7.4 Hz, 1H), 7.60 (d, J = 7.8 Hz, 2H), 7.51 (t, J = 7.4 Hz, 2H), 7.35 (t, J = 7.2 Hz, 1H), 4.55 (s, 2H), 3.69 (t, J = 6.8 Hz, 2H), 3.45 (br s, 4H), 3.09 (s, 3H), 2.52–2.44 (m, 6H), 1.96–1.92 (m, 2H), 1.45 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 166.8, 163.1, 161.5, 154.7, 135.4, 135.0, 133.5, 129.3, 127.5, 126.8, 125.5, 124.0, 123.0, 119.7, 113.6, 109.0, 79.7, 55.7, 53.0, 46.8, 43.1, 41.3, 28.4, 26.0, 21.2; LRMS (ESI) m/z calcd for C₃₁H₃₇N₈O₃ [M+H]⁺: 569.3, found: 569.2.

Compound SF_Tz02^*: A suspension of S16, 3-bromopropanoic acid (4.8 mg, 32 µmol), and DIPEA (28 µL, 160 µmol,) in DMF (160 µL) was stirred at 50 °C for 2 h. The crude product was directly purified by reverse-phase preparative HPLC using a linear gradient of acetonitrile (5–100%) in water with 1% TFA to afford the TFA salts of SF_Tz02^* (5.7 mg, 47% yield) as a red solid; ¹H NMR (400 MHz, CDCl₃:MeOD-d₄ = 10:1, v/v) δ 9.15 (s, 1H), 8.67 (d, J = 7.4 Hz, 1H), 7.87 (d, J = 7.4 Hz, 1H), 7.64 (d, J = 7.4 Hz, 2H), 7.52 (t, J = 7.6 Hz, 2H), 7.36 (t, J = 7.5 Hz, 1H), 4.60 (s, 2H), 3.72 (t, J = 6.5 Hz, 2H), 3.31 (br s, 8 H), 3.20–3.11 (m, 4H), 3.07 (s, 3H), 2.70 (t, J = 6.8 Hz, 2H), 2.18–2.11 (m, 2H); ¹³C NMR (100 MHz, CDCl₃:MeOD-d₄ = 10:1, v/v) δ 172.8, 167.5, 163.4, 162.5, 136.2, 136.0, 133.8, 129.6, 127.9, 127.2, 125.9, 124.9, 122.5, 119.9, 114.0, 109.6, 54.9, 52.9, 50.5, 49.8, 47.2, 40.5, 30.1, 24.1, 21.3; HRMS (ESI) m/z calcd for C₂₉H₃₃N₈O₃ [M+H]⁺: 541.2670, found: 541.2674.

Compound S17: A suspension of S11 (368.4 mg, 895 µmol), N,N-diethyl-4-iodo-aniline⁴ (739 mg, 2.69 mmol), palladium acetate (40.2 mg, 179 µmol), and silver acetate (448 mg, 2.69 mmol) in DMF (9 mL) was stirred at 80 °C for overnight. The reaction mixture was then filtered through a short bed of silica gel with EtOAc and concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, toluene:EtOAc = 10:1) to afford S17 (147.5 mg, 29.5% yield) as a red solid; ¹H NMR (400 MHz, CDCl₃) δ 8.52 (d, J = 7.0 Hz, 1H), 8.13 (s, 1H), 7.35 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 6.71 (dd, J = 7.2, 1.4 Hz, 1H), 4.52 (s, 2H), 3.81 (t, J = 5.9 Hz, 2H), 3.72 (t, J = 7.2 Hz, 2H), 3.42 (q, J = 7.0 Hz, 4H), 1.93 (quin, J = 6.6 Hz, 2H), 1.22 (t, J = 7.0 Hz, 6H), 1.12–1.03 (m, 21H); ¹³C NMR (100 MHz, CDCl₃) δ 161.3, 146.9, 134.2, 133.0, 128.6, 126.3, 125.1, 123.7, 119.4, 118.9, 114.4, 112.2, 110.6, 101.6, 60.9, 47.0, 44.4, 40.5, 32.1, 18.0, 12.6, 12.0; LRMS (ESI) m/z calcd for C₃₃H₄₇N₄O₂Si [M+H]⁺: 559.4, found: 559.4.

Compound S18: To a solution of S17 (134.4 mg, 240.5 µmol) in tetrahydrofuran (2.5 mL), was added TBAF solution (1.0 M in THF, 361 µL, 361 µmol) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and then washed with saturated brine, extracted with EtOAc, dried over anhydrous Na₂SO₄, and concentrated. The resulting solid, methanesulfonic anhydride (126 mg, 722 µmol), and anhydrous triethylamine (168 µL, 1.20 mmol) were dissolved in anhydrous CH₂Cl₂ (2.5 mL) under Ar. The mixture was stirred at room temperature for 4 h. The crude mixture was directly purified by flash column chromatography (silica gel, CH₂Cl₂:MeOH = 40:1) to afford S18 (116 mg, 100% yield) as a yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 8.50 (d, J = 7.0 Hz, 1H), 8.12 (s, 1H), 7.34 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H), 6.73 (dd, J = 7.2, 1.4 Hz, 1H), 4.52 (s, 2H), 4.33 (t, J = 6.1 Hz, 2H), 3.75 (t, J = 6.7 Hz, 2H), 3.42 (q, J = 7.0 Hz, 4H), 3.05 (s, 3H), 2.17 (quin, J = 6.5 Hz, 2H), 1.22 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 161.7, 147.1, 134.4, 133.4, 128.8, 126.4, 125.3, 123.1, 119.2, 118.8, 114.7, 112.3, 111.0, 102.1, 67.5, 47.1, 44.6, 39.8, 37.6, 28.8, 12.7; LRMS (ESI) m/z calcd for C₂₅H₂₉N₄O₄S [M+H]⁺: 481.2, found: 481.2.

Compound S19: A suspension of S18 (54.0 mg, 112 µmol), 1-methylpiperazine (62 µL, 562 µmol), and potassium carbonate (46.6 mg, 337 µmol) in anhydrous DMF (1.2 mL) was stirred at 50 °C for overnight. The reaction mixture was then directly purified by flash column chromatography (silica gel, CH₂Cl₂:MeOH = 5:1) to afford S19 (38.1 mg, 70% yield) as a yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 8.52 (d, J = 7.4 Hz, 1H), 8.14 (s, 1H), 7.35 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H), 6.72 (d, J = 7.4 Hz, 1H), 4.49 (s, 2H), 3.65 (t, J = 7.0 Hz, 2H), 3.42 (q, J = 7.0 Hz, 4H), 2.47–2.43 (m, 10H), 2.29 (s, 3H), 1.92–1.85 (m, 2H), 1.22 (t, J = 7.0 Hz, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 161.4, 147.1, 134.3, 133.2, 128.7, 126.4, 125.2, 123.6, 119.4, 118.9, 114.5, 112.3, 110.8, 101.8, 55.8, 55.2, 53.2, 46.8, 46.1, 44.5, 41.6, 26.3, 12.8; LRMS (ESI) m/z calcd for C₂₉H₃₇N₆O [M+H]⁺: 485.3, found: 485.3.

Compound SF_Tz08^*: A reaction mixture of S19 (15.2 mg, 31.3 µmol), acetonitrile (82 µL, 1.6 mmol), zinc trifluoromethanesulfonate (5.7 mg, 16 µmol), and hydrazine monohydrate (152 µL, 3.13 mmol) in microwave reaction vial (10 ml) was irradiated under microwaves at 100 °C (40 W) with magnetic stirring for 1.5 h. After cooled down to room temperature, sodium nitrite (43.3 mg, 627 µmol) in 1 mL of water was added followed by slow addition of 1N HCl until pH value reached 3 (caution! toxic nitrogen oxide gas evolved!). Then, the resulting residue was washed with saturated NaHCO₃ aqueous solution, extracted with CH₂Cl₂, dried over anhydrous Na₂SO₄, and concentrated. The crude product was purified by reverse-phase preparative HPLC using a linear gradient of acetonitrile (5–100%) in water with 1% of TFA to afford the TFA salts of SF_Tz08^* (6.5 mg, 31% yield) as a reddish brown solid; NMR spectra were recorded after basic work-up using saturated NaHCO₃ aqueous solution, ¹H NMR (400 MHz, CDCl₃) δ 9.17 (s, 1H), 8.66 (d, J = 7.5 Hz, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.47 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 2H), 4.52 (s, 2H), 3.67 (t, J = 7.0 Hz, 2H), 3.43 (q, J = 7.0 Hz, 4H), 3.07 (s, 3H), 2.47 (m, 10H), 2.28 (s, 3H), 1.90 (quin, J = 7.1 Hz, 2H), 1.23 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 166.5, 163.2, 161.6, 146.7, 134.9, 134.1, 128.6, 125.3, 122.9, 122.7, 120.4, 120.2, 114.5, 112.3, 108.5, 55.8, 55.1, 53.2, 46.8, 46.0, 44.5, 41.4, 26.3, 21.1, 12.7; HRMS (ESI) m/z calcd for C₃₁H₄₀N₉O [M+H]⁺: 554.3350, found: 554.3351.

Compound TPP-TCO: A suspension of R11⁶ (5.0 mg, 12 µmol), TCO-NHS ester (6.7 mg, 25 µmol), and triethylamine (17 µL, 120 µmol) in DMF (200 µL) was stirred at room temperature for 15 min. The crude product was directly purified by reverse-phase preparative HPLC using a linear gradient of acetonitrile (5–100%) in water with 1% TFA to afford TPP-TCO (5.5 mg, 80% yield) as transparent oil; HRMS (ESI) m/z calcd for C₃₀H₃₅NO₂P+ [M]⁺: 472.2400, found: 472.2402

Dox-C8 and Dox-TCO were prepared by a modified procedure of a reported paper.⁷

Compound Dox-C8: Docetaxel (152 mg, 188 µmol) were dissolved in formic acid (6 mL, 159 mmol) and the reaction mixture was stirred at room temperature for 1 h. Formic acid was then removed under reduced pressure. To a resulting solid (52 mg) in DMF (1 mL) was added 8-(tert-butoxycarbonylamino)octanoic acid (35.8 mg, 138 µmol), TSTU (41.5 mg, 138 µmol), and DIPEA (120 µL, 690 µmol). The mixture was stirred at 60 °C for 2 days. The crude product was directly purified by reverse-phase preparative HPLC using a linear gradient of acetonitrile (5–100%) in water with 1% TFA to afford Dox-C8 (19.1 mg, 29.2% yield) as a white solid; LRMS (ESI) m/z calcd for C₅₁H₆₉N₂O₁₅ [M+H]⁺: 949.5, found: 949.2.

Compound Dox-TCO: Dox-C8 (8.1 mg, 8.5 µmol) were dissolved in formic acid (200 µL, 5.30 mmol) and the reaction mixture was stirred at room temperature for 1 h. Formic acid was removed under reduced pressure. To a resulting solid (7.6 mg) in DMF (200 µL), were added TCO-NHS Ester (4.6 mg, 17 µmol), and triethylamine (12 µL, 85 µmol). The mixture was stirred room temperature for 1 h. The crude product was directly purified by reverse-phase preparative HPLC using a linear gradient of acetonitrile (5–100%) in water with 1% TFA to afford Dox-TCO (5.8 mg, 67% yield) as a white solid; HRMS (ESI) m/z calcd for C₅₅H₇₂N₂NaO₁₅ [M+Na]⁺: 1023.4825, found: 1023.4830.