The Challenge of Antibiotic Resistance

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Chapter Three:Experimental

General Details

Unless otherwise noted, all reactions were performed under an oxygen-free atmosphere of nitrogen or argon. Tetrahydrofuran and diethyl ether were freshly distilled over sodium/benzophenone ketyl. Dichloromethane, acetonitrile, methanol and dimethylsulfoxide were freshly distilled from calcium hydride. Toluene was freshly distilled over sodium. Triethylamine and diisopropylamine were freshly distilled from calcium hydride and stored over potassium hydroxide. All other reagents were used as received unless otherwise noted. Yields refer to chromatographically and spectroscopically (¹H NMR) homogeneous materials, unless otherwise stated. Reactions performed at low temperature were cooled either with an acetone/dry ice bath to reach −78 °C or an ice/water bath to reach 0 °C. Reactions were monitored by thin-layer chromatography (TLC) carried out on E. Merck silica gel plates using UV light as visualizing agent and an ethanolic solution of vanillin and ammonium molybdate and heat as developing agents. Kieselgel S 63-100 μm (Riedel-de-Hahn) silica gel was used for flash chromatography. Preparatory TLC was carried out on 500 μm, 20 × 20 cm UniplateTM (Analtech) silica gel thin layer chromatography plates. NMR spectra were recorded at room temperature in CDCl3, CD3OD, (CD3)3CO, C6D6 or (CD3)SO solutions on either a Bruker DRX300 spectrometer operating at 300 MHz for ¹H nuclei and 75 MHz for ¹³C nuclei or using a Bruker DRX-400 spectrometer operating at 400 MHz for ¹H nuclei and 100 MHz for ¹³C nuclei. Chemical shifts are reported in parts per million (ppm) from tetramethylsiane (δ = 0) and were measure relative to the solvent in which the sample was analysed. Coupling constants, J, are reported in hertz (Hz). Multiplicities are reported as “s” (singlet), “br s” (broad singlet), “d” (doublet), “dd” (doublet of doublets), “ddd” (doublet of doublets of doublets), “t” (triplet) and “m” (multiplets). Where distinguishable from those due to a major rotamer or diastereomer, resonances due to minor rotamers or diastereomers are denoted by an asterix. Optical rotations were measured with an Autopol^® IV automatic polarimeter, using the sodium-D line (589 nm), with the concentration measured in grams per 100 mL. Infrared (IR) spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR spectrometer using a diamond ATR sampling accessory. Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. High-resolution mass spectra (HRMS) were obtained using a VG70SE spectrometer or on a micrOTOF-Q II mass spectrometer.

EXPERIMENTAL PROCEDURES

A cold solution (0-5°C) of nitric acid (4.3 mL, 65.7 mmol) in acetic anhydride (30 mL) was added dropwise over a period of 2 h to a solution of pyrrole (4.0 g, 59.7 mmol) in acetic anhydride (30 mL) at –50 °C. After 1 h, the mixture was poured over ice/water and extracted with dichloromethane (4 × 200 mL). The combined organic extracts were washed with water and saturated sodium bicarbonate, dried over sodium sulfate solution, and concentrated in vacuo. The crude product was purified by flash column chromatography (hexanes/EtOAc 8:1) to afford the title compound (3.26 g, 45%) as a light yellow solid. dH  (400 MHz, CDCl3): d 9.68 (br s, 1H, NH), 7.26-7.13 (m, 1H, H-3), 7.0 (m, 1H, H-4), 6.34 6.31(m, 1H, H-5); dC (CDCl3, 100 MHz): d  123.3, 111.5, 111.3, CNO2 was not observed. Spectroscopic data was consistent with that reported in the literature. ^[153] mixture of 2-nitropyrrole 144 (90 mg, 0.80 mmol), di-tert-butyl dicarbonate (209 mg, 0.96 mmol), DMAP (19.5 mg, 0.12 mmol) in acetonitrile (1.5 mL) was stirred at r.t. overnight. The mixture was diluted with water (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The crude was purified by flash chromatography (hexanes/EtOAc 9:1) to afford the title compound (130 mg, 80%) as a colourless oil. vmax (neat)/cm^–1: 2954, 1324, 1255, 1234, 1026, 7291; dH (400 MHz, CDCl3): d 7.26-7.25 (m, 1H, H-3), 7.08-7.06 (m, 1H, H-4), 6.22-6.20 (m, 1H, H-5), 1.58 (s, 9H, 3×CH3); dC (100 MHz, CDCl3): 147.0 (C=O), 126.8, 116.9, 109.5, 87.0, 27.5, CNO2 was not observed; m/z (ESI+) calcd for [C9H12N2NaO4]⁺ : 235.0689, found: 235.0695 A mixture of 2-nitropyrrole 144 (183 mg, 1.6 mmol), p-toluenesulfonyl chloride (286 mg, 1.6 mmol) and triethyl amine (303 mg, 3.0 mmol) in dichloromethane (10 mL) was refluxed for 1 h. The mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash column chromatography (hexanes/EtOAc 8:1) to afford the title compound (391 mg, 98%) as a brown solid. dH (400 MHz, CDCl3): d 7.91-7.89 (m, 2H, Ar-H), 7.73-7.72 (m, 1H, H-3), 7.38-7.36 (m, 2H, Ar- H), 7.26-7.25 (m, 1H, H-4), 6.37-6.35 (m, 1H, H-5), 2.45 (s, 3H, CH3); dC (100 MHz, CDCl3): d 146.1, 134.6, 129.8, 128.9, 128.7, 118.5, 109.7, 21.8, CNO2 was not observed; Spectroscopic data is consistent with that reported in the literature.^[274] To a solution of 2-nitropyrrole 144 (336 mg, 3.0 mmol) in DMF (4 mL) was added sodium hydride (144 mg, 3.6 mmol) portionwise at 0 °C. The reaction mixture was stirred for 20 min, and benzyl chloride (615 mg, 3.6 mmol) was added dropwise at 0 °C. The reaction mixture was warmed to r.t. and stirred overnight. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (hexanes/EtOAc 10:1) to afford the title compound (582 mg, 96%) as a light yellow solid. dH (400 MHz, CDCl3): d 7.35-7.28 (m, 3H, Ar-H), 7.28-7.25 (m, 1H, Ar-H), 7.13-7.11 (m, 2H, Ar-H), 6.89 (t, 1H, J = 2.5 Hz, Ar-H), 6.23-6.22 (m, 1H, Ar-H), 5.55 (s, 2H, CH2); dC (100 MHz, CDCl3): d 136.1, 129.4, 128.9, 128.1, 127.1, 115.1, 108.8, 53.5, CNO2 was not observed; Spectroscopic data is consistent with that reported in the literature. ^[275] A mixture of 2-nitropyrrole 144 (56 mg, 0.5 mmol), 4-methoxybenzyl chloride (117 mg, 0.8 mmol), potassium carbonate (276 mg, 2.0 mmol) in DMF (2 mL) was stirred at r.t. overnight. The mixture was directly subjected to flash chromatography (hexanes/EtOAc 8:1) to afford the title compound (109 mg, 94%) as a light yellow solid. m.p. 62.3-63.5 °C; vmax (neat)/cm^–1: 2954, 1324, 1255, 1234, 1026, 729. dH (400 MHz, CDCl3): d 7.25-7.23 (m, 1H, Ar-H), 7.13-7.09 (m, 2H, Ar-H), 6.88-6.84 (m, 3H, Ar-H), 6.21-6.19 (m, 1H, Ar-H), 5.48 (s, 2H, H-6) 3.79 (s, 3H, CH3); dC (100 MHz, CDCl3): d 159.5 129.2, 128.9, 127.9, 115.1, 114.3, 108.7, 55.3, 53.0, CNO2 was not observed; m/z (ESI+) calcd for [C12H12N2NaO3]⁺: 255.0740, found: 255.0749. A mixture of 2-nitropyrrole 144 (22 mg, 0.2 mmol), 3,4-dimethoxybenzyl chloride (55.8 mg, 1.5 mmol) and potassium carbonate (110 mg, 0.8 mmol) in DMF (1 mL) was stirred at r.t. overnight. The mixture was directly subjected to flash chromatography (hexanes/EtOAc 8:1) to afford the title compound (50 mg, 92%) as a light yellow solid. m.p. 72.1-73.2 °C; vmax (neat)/cm^–1: 2934, 1325, 1257, 1244, 1020, 739. dH (400 MHz, CDCl3): d 7.24-7.23 (m, 1H, Ar-H), 6.86-6.85 (m, 1H, Ar-H), 6.81 (m, 1H, Ar-H), 6.71-6.69 (m, 2H, Ar-H), 6.21-6.19 (m, 1H, Ar-H), 5.47 (s, 2H, H-6), 3.84 (s, 3H, CH3), 3.82 (s, 3H, CH3); dC  (100 MHz  CDCl3): d 149.3, 149.0, 137.5, 129.2, 128.4, 119.9, 115.1, 111.4, 110.8, 108.7, 55.9, 55.9, 53.3; m/z (ESI+) calcd. for [C13H14N2NaO4]⁺ : 285.0845 , found: 285.0851. To a solution of 2-nitropyrrole 144 (2.01 g, 17.9 mmol) in DMF (5 mL) was added sodium hydride (786 mg, 19.6 mmol, 60% dispersion in mineral oil) portionwise at 0 °C. After 20 min, benzyloxymethyl chloride (3.35 g, 21.4 mmol) was added dropwise at 0 °C. The reaction mixture was then warmed to r.t. and stirred overnight. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (hexanes/EtOAc 10:1) to afford the title compound (3.76 g, 90%) as a light yellow solid. m.p. 49.3-51.6 °C; vmax (neat)/cm^–1: 3137, 3110, 3032, 1523, 1455, 1320, 1277, 1254, 1141, 1076, 1039, 732, 694; dH (CDCl3, 400 MHz) d 7.31-7.27 (m, 5H, Ar-H), 7.25-7.23 (m, 1H, H-3), 6.99 (t, 1H, J = 2.4, Hz, H-5), 6.23-6.21 (m, 1H, H-4), 5.76 (s, 2H, H-6), 4.55 (s, 2H, H-7); dC (100 MHz, CDCl3): d 136.4, 128.8, 128.6, 128.2, 127.8, 115.6, 109.1, 77.7, 71.1, CNO2 was not observed; m/z (ESI+) calcd for [C12H12N2NaO3]⁺ : 255.0740 , found: 255.0749. To a solution of N-Boc-2-nitropyrrole (145 a) (42 mg, 0.2 mmol) in dichloromethane (3 mL), TFA (0.1 mL) was added and the reaction mixture was stirred at r.t. for 2 h. The solution was diluted with dichloromethane (30 mL), washed with NaHCO3 (30 mL, 1 M) and brine, dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (hexanes/EtOAc 8:1) to afford 2-nitropyrrole (20 mg, 90%). A solution of N-tosyl-2-nitropyrrole (145 b) (27 mg, 0.1 mmol), TBAF (0.2 mL, 1 M in THF, 0.2 mmol), and THF (3 mL) was stirred at 60 °C for 2 h. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (hexanes/EtOAc 8:1) to afford 2-nitropyrrole (10 mg, 96%). To a solution of N-BOM-2-nitropyrrole (11a) (46 mg, 0.20 mmol) in dichloromethane (3 mL) was added AlCl3 (33 mg, 0.25 mmol) at 0 °C. The reaction mixture was stirred at r.t. for 1 h. The reaction mixture was diluted with water (20 mL) and extracted with dichloromethane (3 × 20 mL). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (hexanes/EtOAc 8:1) to afford 2-nitropyrrole (18 mg, 80%). A solution of bromine (106 mg, 0.67 mmol) in dichloromethane (2 mL), was added dropwise to a solution of 2-nitropyrrole 144 (68 mg, 0.61 mmol) at 0 °C. The mixture was stirred at 0 °C for 2 h. Upon completion, the reaction was quenched with sodium thiosulfate, extracted with ethyl acetate (3 × 20 mL). The combined organic extracts were washed with brine and dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (hexanes/EtOAc 8:1) to afford 4-bromo-2-nitropyrrole as a pale solid (100 mg, 87%). mixture of 4-bromo-2-nitropyrrole 147 (84 mg, 0.44 mmol), p-toluenesulfonyl chloride (100 mg, 0.53 mmol) and triethyl amine (111 mg, 1.10 mmol) in dichloromethane (5 mL) was refluxed for 1 h. The mixture was diluted with water (30 mL) and extracted with dichloromethane (3 × 30 mL). The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash column chromatography (hexanes/EtOAc 8:1) to afford the title compound (160 mg, 95%) as a brown solid. m.p. 110.6-113.7 °C; νmax  (neat)/cm^–1: 3141, 2919, 1503, 1437, 1362, 1175, 1054, 810, 739; dH (CDCl3, 300 MHz): d 7.92 (d, J =8.3 Hz, 2H, Ar-H), 7.72 (d, J =2.4 Hz, 1H, H-3), 7.39 (d, J =8.3 Hz, 2H, Ar-H), 7.23 (d, J =2.4 Hz, 1H, H-5), 2.46 (s, 3H, CH3); dC  (CDCl3, 75 MHz): d 146.1, 134.6, 129.8, 128.9, 128.7, 118.5, 109.7, 21.8, CNO2 was not observed; m/z (ESI) calculated for [C4H3BrN2NaO2]⁺: 266.2731, found: 266.2736. m.p. 106.4-108.7 °C; νmax (neat)/cm^–1: 3185, 3142, 1503, 1443, 1372, 1331, 1245, 1219, 1089, 967, 911, 846, 701; dH  (CDCl3, 400 MHz): d 9.61 (br s, 1H, NH ), 7.15-7.14 (m, 1H, H-3), 6.99-6.98 (m, 1H, H-5); dC  (CDCl3, 100 MHz): d 122.4, 112.5, 99.0, CNO2  was not observed; m/z (ESI) calculated for [C4H3BrN2NaO2]⁺: 212.9270, found: 212.9272. To a solution of 4-bromo-2-nitropyrrole 147 (270 mg, 1.4 mmol) in DMF (5 mL) was added sodium hydride (62 mg, 1.5 mmol, 60% dispersion in mineral oil) portionwise at 0 °C. The reaction mixture was stirred for 30 min, and benzyloxymethyl chloride (313 mg, 1.8 mmol) was added dropwise at 0 °C. The reaction mixture was warmed to r.t. and stirred overnight. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (hexanes/EtOAc 10:1) to afford the title compound (582 mg, 96%) as a light yellow solid. m.p. 60.3-62.7 °C; °C νmax  (neat)/cm^–1: 3129, 2840, 1721, 1515, 1447, 1332, 1258, 1156, 1117, 1020, 922, 818, 721; dH  (CDCl3, 300 MHz): d 7.38-7.32 (m, 3H, Ar-H), 7.25 (d, J = 2.4 Hz, 1H, H-3), 7.19-7.14 (m, 2H, Ar-H), 6.84 (d, J =2.4 Hz, 1H, H-5), 5.52 (s, 2H, CH2); dH  (CDCl3, 75 MHz): d 135.1, 129.1, 128.5, 128.2, 127.4, 116.1, 96.5, 53.8, CNO2 was not observed; m/z (ESI) calculated for [C11H9BrN2NaO2]⁺: 302.9740, found: 302.9728.

CHAPTER ONE: INTRODUCTION
1.1 The Challenge of Antibiotic Resistance
1.2 Heronapyrroles and Nitropyrrolins: Isolation, Bioactivity and Biosynthesis
1.3 Antimicrobial Activity and Chemotherapeutic Value of Nitroheterocyclic Compounds
1.4 Stereoselective Synthesis of Functionalized Tetrahydrofurans and Its Application In Natural Product Synthesis
1.5 Previous Syntheses of Heronapyrroles
1.6 Aim of Present Research
1.7 Retrosynthetic Analysis of Heronapyrrole C
CHAPTER TWO: DISCUSSION
2.1 Synthesis Towards the Key Intermediate: 4-Farnesyl-2-nitropyrrole via a Transition Metal Catalysed Corss-Coupling Strategy
2.2 Investigation of the Coupling Reactivity of 2-Nitropyrrole
2.3 Synthesis Towards the Key Intermediate 4-Farnesyl-2-nitropyrrole 136 via a Julia- Kocienski Olefination Strategy
2.4 Synthetic Investigation Towards the Proposed Structure of Heronapyrrole C
2.5 Synthesis of BOM-heronapyrrole C
2.6 Revised Protecting Group Strategy and Synthesis of Heronapyrrole C
2.7 Second Generation Synthesis of Heronapyrroles and Future Work
CHAPTER THEREE: EXPERIMENTAL
3.1 General Details
3.2 EXPERIMENTAL PROCEDURES

GET THE COMPLETE PROJECT
Total Synthesis of the 2-Nitropyrrole Natural Product Heronapyrrole C