An Enantioselective Total Synthesis of (+)-Duocarmycin SA - The

Mar 20, 2018 - An efficient, concise enantioselective total synthesis of the potent antitumor antibiotic (+)-duocarmycin SA is described. The invented...
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Cite This: J. Org. Chem. 2018, 83, 3928−3940

An Enantioselective Total Synthesis of (+)-Duocarmycin SA Michael A. Schmidt,*,† Eric M. Simmons,† Carolyn S. Wei,† Hyunsoo Park,‡ and Martin D. Eastgate† †

Chemical & Synthetic Development and ‡Drug Product Science Technologies, Material Science and Engineering, Bristol-Myers Squibb Company, 1 Squibb Drive, New Brunswick, New Jersey 08903, United States S Supporting Information *

ABSTRACT: An efficient, concise enantioselective total synthesis of the potent antitumor antibiotic (+)-duocarmycin SA is described. The invented route is based on a disconnection strategy that was devised to facilitate rapid and efficient synthesis of key core compounds to enable preclinical structure−activity relationship investigations. The key tricycle core was constructed with a highly enantioselective indole hydrogenation to set the stereocenter and a subsequent hitherto unexplored vicarious, nucleophilic-substitution/cyclization sequence to effectively forge a final indole ring. Additionally, the development of a stable sulfonamide protecting group capable of mild chemoselective cleavage greatly enhanced sequence yield and throughput. An understanding of key reaction parameters ensured a robust, reproducible sequence easily executable on decagram scales to this highly promising class of compounds.

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INTRODUCTION The duocarmycins represent a subset of potent antitumor antibiotics that were identified from Streptomyces strains in the late 1980s.1 These molecules in general contain, or can form in vitro,2 a cytotoxic 1,1a,2,3,-tetrahydro-5H-cyclopropa[c]indol5-one (Figure 1A, light blue highlight) structural motif. Within this class, (+)-duocarmycin SA (DSA, 1) has an interesting property of being among the most potent in the group yet also among the most stable.3 Recently, there has been much work4 combining the potent cytotoxicity of the duocarmycins and their structural analogues with the cellular specificity of antibodies in the form of antibody−drug conjugate (ADC)based therapeutics. Within ADC development, significant work has been devoted to systematically examining each of the parameters relating to the antibody and the linker attaching the cytotoxin,5 prompting the need for large quantities of materials, which ideally would be accessed by an efficient, flexible total synthesis strategy. Herein, we describe an enantioselective route to 1 that can be carried out straightforwardly on the decagram scale, which enables downstream ADC studies. The first total synthesis of 1 was achieved shortly after its discovery in 19903 by the Boger lab in 1992,6 and in the subsequent years, extensive and thorough work from their lab helped establish a key understanding of the structure−activity relationship and biological association of this general class of alkylating agents.6 Additional, highly innovative syntheses from the Natsume and Muratake,7 Fukuda and Terashima,8 Fukuyama,9 and Tietze10 laboratories were also reported shortly thereafter (Figure 1A). These syntheses differ considerably in how the core tricycle is made (Figure 1A) and pose many challenges. Excluding the creative route by Natsume and Muratake, a unifying strategy across the literature is to begin with an appropriate central © 2018 American Chemical Society

arene then sequentially build the chiral indoline and 2-carboxy ester indole moieties. Approaches that form the indoline ring last are racemic or require sensitive anionic cyclization conditions, outcomes likely complicated by the presence of the fragile indole 2-carboxy ester. Likewise, approaches that form the indole 2-carboxy ester last are lengthy and/or require large loadings of palladium due to the challenges of carbon− carbon bond formation at the indoline C4 position. We sought to develop an enantioselective synthetic strategy that would form the sensitive indole 2-carboxy ester last; a feature that would enable late-stage diversification of ester analogues, overcome the challenges of indoline C4 functionalization, and utilize a rapid and efficient methodology for introduction of the chiral indoline.

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RESULTS AND DISCUSSION Our proposed retrosynthesis (Figure 1B) installs the cytotoxic cyclopropane last and cleaves the trimethoxy indole to reveal the tricyclic core. We desired to forge the indole 2-carboxy ester leveraging an underutilized vicarious nucleophilic substitution (VNS)/cyclization strategy.11 This approach was considered risky as it is absent from the DSA literature, and examples of VNS in complex settings are particularly scarce but notable for their mild, transition metal-free conditions. This strategy was pursued due to the belief that prior introduction of the indole 2-carboxy ester was responsible for several challenges noted in the preceding work. We planned to form the preceding chiral indoline by an enantioselective indole hydrogenation,12 a strategy that obviates the need to synthesize one of the most Received: January 31, 2018 Published: March 20, 2018 3928

DOI: 10.1021/acs.joc.8b00285 J. Org. Chem. 2018, 83, 3928−3940

Article

The Journal of Organic Chemistry

only 0.20 mol % rhodium and 0.22 mol % L1 (96% yield, 98.2% ee on a 30 g scale). The tosyl group was used to enable the hydrogenation, and indoline N-protection of some capacity was necessary to facilitate the remainder of the synthesis; however, we found that it was resistant to cleavage downstream of the eventual indole 2-carboxy ester formation. For example, the Ts analogue of 9 decomposed under a variety of reductive (Mg, SmI2), basic (NaOMe, thiols, and DBU), or nucleophilic (hydrazine, hydroxylamine) conditions. The tosyl group of 4, however, was easily removed with excess magnesium in methanol and toluene and a wide variety of amide-, carbamate-, and sulfonamide14-based protecting groups were screened. Unfortunately, we found none were acceptable; thus, we developed a new amine protecting group by subtly tuning arylsulfonyl electronic properties to enable a mild, functional group-tolerant cleavage. We discovered that the 4-cyanobenzensulfonamide (4-Cs)15 was an excellent protecting group in this context, not only compatible with the downstream chemistry but also rendering intermediates in the sequence crystalline, facilitating purification, handling, and storage. In practice, the tosyl group of 4 was removed with excess magnesium, then the crude stream was quenched into cold (∼0 °C) 3 N HCl containing 10 wt % urea, a finding that greatly enhanced the aqueous solubility of the magnesium salts. The crude indoline was sulfonylated with 4-cyanobenzenesulfonyl chloride and Hunig’s base to afford 5 in 84% overall yield. Unfortunately, the 4-Cs group was not compatible with the initial chiral hydrogenation due to nitrile reduction, prompting this protecting group exchange. The regioselective nitration of 5 was most effectively carried out with red fuming nitric acid with nitroarene 6 being isolated by crystallization from isopropanol in 78% yield. After extensive exploration, the second key transformation, the vicarious nucleophilic substitution of 6, was successfully performed with chloromethyl phenyl sulfone and potassium tert-butoxide affording sulfone 7. By performing the reaction at −25 °C in THF, 7 is obtained as a single regioisomer in 76% yield on a 50 g scale. Other VNS reagents11 failed to afford desired products such as O-methylhydroxylamine, trimethylsulfonium iodide, 1,1,1-trimethylhydrazinium iodide, diethyl (chloromethyl)phosphonate, and ethyl bromopyruvate (Scheme 1); instead, decomposition was observed. The nitro group was cleanly reduced with excess zinc and acetic acid to give the aniline as a foam, which was used directly in the subsequent reaction. The crude aniline was treated with 2.00 equiv of methyl 2-hydroxy-2-methoxyacetate (8) in ethyl acetate, a reaction driven to completion by azeotropic removal of methanol and water below the reagent’s boiling point (∼128 °C at 760 Torr). The resulting imine was highly hydrolytically unstable and as such was immediately used in the next cyclization reaction. Upon extensive screening, cesium carbonate in DMSO was by far the most effective condition to form 9. After neutralization and an aqueous workup, the crude residue is heated in isopropanol, affording beige crystals of 9 that were ∼85 wt % quantitative NMR (73% corrected yield for three steps from 7). The 4-Cs group of 9 was removed under the previously reported conditions (1-dodecanethiol and DBU).15 We found this to be a strategic place to purify the product by proper flash column chromatography, and we isolated the stable, free indoline core 10 in 95% yield. Indoline 10 was treated with acid chloride 11 in the presence of 2,6-lutidine to afford amide 12 as

Figure 1. (A) Previous reports to 1, total yields, and amounts prepared (NG = not given). (B) Proposed retrosynthetic analysis.

challenging heterocyclic rings asymmetrically late in the synthesis. The route to 1 (Scheme 1) begins with a Vilsmeier−Haack formylation of commercially available 6-benzyloxyindole (2). The precipitated aldehyde was tosylated and then reduced with substoichometric LiBH4, and the resulting alcohol was silylated with TBSCl in the presence of imidazole. Product 3 was isolated by crystallization from the DMF reaction stream by the addition of water. The key enantioselective indole hydrogenation was next explored. After surveying a variety of conditions and with multiple catalysts,13 success was only realized with the (S,S)-(R,R)-PhTRAP ligand (L1) developed by Kuwano and co-workers.12 This ligand is not commercially available; however, it can be prepared via the known procedures. For convenience, we have compiled our synthesis in the Experimental Section. We discovered that the precatalyst generated by mixing [Rh(COD)(acac)] with L1 was highly sensitive to air and impurities in 3. However, with purified 3 we were able to lower the catalyst loading while maintaining reaction efficiency, obtaining complete conversion of 3 with 3929

DOI: 10.1021/acs.joc.8b00285 J. Org. Chem. 2018, 83, 3928−3940

Article

The Journal of Organic Chemistry Scheme 1. Total Synthesis of (+)-Duocarmycin SA (1)a

a

Reagents and conditions: (1) 1.25 equiv of POCl3, DMF, then NaOH, H2O; (2) 1.10 equiv of TsCl, 1.50 equiv of Et3N, 5 mol % DMAP, CH2Cl2; (3) 0.5 equiv of LiBH4 in THF, 2-MeTHF; (4) 1.20 equiv of TBSCl, 1.50 equiv of imidazole, DMF; (5) 0.20 mol % [Rh(COD)(acac)], 0.22 mol % L1, 750 psig H2, i-PrOH, 65 °C; (6) 10−15 equiv of Mg, MeOH, PhMe; (7) 1.05 equiv of 4-CsCl, 1.20 equiv of i-Pr2NEt; (8) 1.30 equiv of fuming HNO3, CH2Cl2; (9) 1.15 equiv of chloromethyl phenyl sulfone, 2.30 equiv of KOt-Bu, THF, −25 °C; (10) 10 equiv of Zn, 10 equiv of AcOH, MeCN; (11) 2.00 equiv of methyl 2-hydroxy-2-methoxyacetate (8), EtOAc; (12) 5.00 equiv of Cs2CO3, DMSO, 50 °C; (13) 6.0 equiv of 1dodecanethiol, 5.7 equiv of DBU, DMF; (14) 1.05 equiv of 11, 1.15 equiv of 2,6-lutidine, CH2Cl2; (15) 2.50 mol % Pd(OH)2 on carbon, 5.00 equiv of ammonium formate, MeOH, 50 °C; (16) 1.20 equiv of TBAF, THF, 35 °C; (17) 1.50 equiv of ADDM, 1.50 equiv of PBu3, THF; DMF = N,Ndimethylformamide, TsCl = p-toluenesulfonyl chloride, DMAP = 4-dimethyaminopyridine, THF = tetrahydrofuran, 2-MeTHF = 2methyltetrahydrofuran, TBSCl = tert-butyldimethylsilyl chloride, COD = 1,5-cyclooctadiene, acac = acetylacetonate, 4-CsCl = 4cyanobenzenesulfonyl chloride, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, DMSO = dimethyl sulfoxide, TBAF = tetrabutylammonium fluoride, ADDM = azodicarbonyl dimorpholide. The majority of the hydrogen atoms of the X-ray structures were omitted for clarity; the ellipsoids represent 50% probability.

a crude foam, which after removal of the benzyl group with Pearlman’s catalyst and ammonium formate afforded the crystalline phenol 12 in 95% overall yield. Acid 16 (Scheme 2), the precursor to 11, is commercially available on small scales and is costly. We elected to synthesize 11 by a modification of Fukuyama’s route.9 Specifically, instead of using a phosphonoglycine reagent and tetramethylguanidine base to form a dehydroaminoester derivative (e.g., 15), we elected to use an Erlenmeyer−Plöchl azlactone/methanolysis sequence that employs very inexpensive reagents. Second, we reduced the copper loading in the Ullmann reaction by 97%, which was facilitated by the addition of the ligand phenanthroline, and replaced the 7.00 equiv of cesium acetate with 2.50 equiv of potassium carbonate. This six-step sequence proceeds in 53% total yield and does not employ flash column chromatography. Removal of the TBS group of 12 with TBAF in THF afforded the penultimate 13 in 95% yield after crystallization

from DMF and water. Dehydration of 13 to 1 was effective under a variety of Mitsunobu conditions; however, we chose azodicarbonyl dimorpholide (ADDM)16 and PBu3 based on the ease of byproduct removal. An aqueous wash removed the azoderived products and a hexane wash removed phosphinederived products. A final purification by flash chromatography afforded 1 in 94% yield, identical in all respects to the previously reported characterization data. Because of the highly toxic nature of 1, we limited this reaction to the 1 g scale. The structure of 1 was examined by X-ray diffraction, and consistent with expectations,17 the nitrogen−carbon bond in the vinylogous amide is 0.06−0.07 Å shorter than the average bond length in the pyrrolidine ring, indicating a stabilizing effect. The goal of this work was to develop a synthetic sequence that would be both flexible to allow analogous study but also concise and efficient to facilitate material throughput. This was accomplished by developing a strategy that begins with the conserved pharmacophore (Figure 1A, light blue highlight) and 3930

DOI: 10.1021/acs.joc.8b00285 J. Org. Chem. 2018, 83, 3928−3940

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The Journal of Organic Chemistry Scheme 2. Synthesis of Acid Chloride 11a

on a TOF instrument. Optical rotations were measured on a polarimeter. Synthesis of (+)-Duocarmycin SA (1). 6-Benzyloxy-3-(tertbutyldimethylsilyloxymethyl)-1-tosyl-1H-indole (3). To a three-neck round-bottomed flask equipped with a nitrogen inlet and thermocouple was added DMF (75 mL), and the flask was cooled to an internal temperature of +2.8 °C with an ice water bath. Phosphorus(V) oxychloride (12.7 mL, 137.2 mmol, 1.25 equiv) was added dropwise, keeping the internal temperature below 5 °C (∼1 h). Once the addition was complete, 6-benzyloxyindole (2) (25.0 g, 109.7 mmol, 1.0 equiv) was added in ∼1 g portions keeping the internal temperature below 5 °C (∼1 h). The dark solution was warmed to room temperature and held for 1 h. A solution of sodium hydroxide (50 g) in water (250 mL) was prepared in a Morton flask and cooled to an internal temperature of 2.0 °C. The dark reaction mixture was slowly added to the cold caustic solution under vigorous stirring, keeping the internal temperature between 20 and 30 °C. A light beige solid separates (final pH ∼14). The slurry was heated slowly up to an internal temperature of 70 °C (moderate off-gassing is observed at an internal temperature of ∼70 °C; the use of a large vessel (2× expected volume), strong agitation, adequate venting, and a slow temperature ramp are encouraged) and held for 10−15 min under a light nitrogen sweep to remove evolving dimethylamine; then, it is heated to an internal temperature of 90 °C and held for 15 min. Upon cooling to room temperature, the slurry was diluted with water (40 mL), and the solids were collected via filtration. The cake was washed with water (2 × 100 mL) and dried in a vacuum oven at 50 °C and 23 in Hg with a slight nitrogen sweep to constant weight to afford formylated indole (27.3 g) as a beige/tan solid, which was identical in all respects with the previously reported literature.18 The product was used without further purification and was ground to a fine powder, if clumpy, before use in the next step. Rf (50% ethyl acetate in hexanes, silica gel): 2 0.88 (UV), formylated indole S1 0.35 (UV). Mp (DMF/water): 211−212 °C (dec.). 1H NMR (400 MHz, DMSO-d6) δ: 11.94 (br s), 9.87 (s, 1H), 8.15 (s, 1H), 7.96 (d, J = 8.5 Hz, 1H), 7.47 (d, J = 7.2 Hz, 2H), 7.40 (app t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.0 Hz, 1H), 7.08 (s, 1H), 6.95 (d, J = 8.5 Hz, 1H), 5.15 (s, 2H). 13C NMR (101 MHz, DMSOd6) δ: 184.7, 155.7, 137.9, 137.8, 137.2, 128.4, 127.7, 127.6, 121.4, 118.25, 118.23, 112.4, 96.9, 69.5. IR (KBr) (cm−1): 1634 (s), 1526 (m), 1426 (m), 1385 (m), 1159 (m). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C16H13NO2 252.1019; found 252.1025. To a Morton flask were added the formylated indole S1 (50.00 g, 199.0 mmol, 1.00 equiv), 4-dimethylaminopyridine (1.22 g, 9.95 mmol, 0.05 equiv), and DCM (400 mL) to form a suspension. Triethylamine (42.0 mL, 298.4 mmol, 1.5 equiv) was added followed by para-toluenesulfonyl chloride (42.15 g, 218.9 mmol, 1.10 equiv). A remaining charge of dichloromethane (100 mL) was used to wash any solids down the sides of the flask. The reaction is exothermic; a ΔT of +7−10 °C was observed on this scale. The reaction mixture was vigorously stirred at room temperature for 1 h and had become mostly homogeneous. The mixture was washed with a 1.0 N aqueous solution of hydrochloric acid (500 mL) and then a saturated aqueous solution of brine (500 mL). The mixture was dried over sodium sulfate, filtered, and concentrated in vacuo to afford the tosylated indole-aldehyde as a dark solid (84.06 g) that was used without further purification. Rf (50% ethyl acetate in hexanes, silica gel): formylated indole S1 0.35 (UV), tosylated indole-aldehyde S2 0.73 (UV). The tosylated indole-aldehyde S2 (84.06 g) was slurried in a mixture of 2-methyltetrahydrofuran (375 mL) and THF (125 mL, water content of the system was ∼0.22 wt % by Karl Fischer analysis) under nitrogen and was cooled to an internal temperature of 5 °C. A solution of lithium borohydride (2.0 M in THF, 50.0 mL, 99.48 mmol, 0.50 equiv) was added slowly, keeping the internal temperature below 10 °C (∼30 min). Once the addition was complete, the reaction mixture was warmed to room temperature and stirred for 30 min. The light slurry was cooled to an internal temperature of 5 °C, and acetone (50 mL) was added slowly such that the internal temperature did not exceed 15 °C (∼20 min). The mixture was warmed to room temperature, stirred for 60 min, and washed with a 1.0 M aqueous solution of a pH 7 sodium phosphate buffer (500 mL) and then a

Reagents and conditions: (1) 1.05 equiv of NBS, MeCN, 50 °C; (2) 1.10 equiv of N-Bz-glycine, 0.50 equiv of KOAc, 3.00 equiv of Ac2O, 60 °C; (3) 10 mol % Et3N, MeOH, 60 °C; (4) 5.0 mol % CuI, 7.5 mol % 1,10-phenanthroline, 2.50 equiv of K2CO3, DMF, 80 °C; (5) 3.00 equiv of NaOH, THF, H2O, 60 °C; (6) 2.0 mol % DMF, 1.50 equiv of oxalyl chloride, THF. a

rapidly advances to core 10 using an enantioselective indole 2,3-bond hydrogenation followed by a VNS/cyclization sequence. The synthesis proceeds in 17 chemical transformations (longest linear sequence), and the overall yield from 2 to 1 was 24.1% with an average reaction efficiency of 92%. Four telescoped sequences in the route minimized isolations down 40% (10 out of 17 steps were isolated), a feat greatly enabled using a new nitrogen protecting group, the 4cyanobenzenesulfonyl. The synthetic sequence was developed with late-stage diversification in mind, and compound 10 should find use in structure−activity relationship studies to further develop this important class of compounds.

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EXPERIMENTAL SECTION

All reactions were performed in round-bottom flasks, and stainless steel syringes were used to transfer liquids (unless otherwise noted). Flash column chromatography was performed using silica gel (20−40 μm). Organic solutions were concentrated on a rotary evaporator at ∼20 Torr at 25−35 °C. Commercial reagents and solvents were used as received. All quantities expressed in the individual experiments are corrected for purity as specified by the vendor. Proton nuclear magnetic resonance (1H NMR) spectra were recorded with a 400 MHz spectrophotometer and are recorded in parts per million from internal tetramethylsilane on the δ scale and are referenced from the residual protium in the NMR solvent (CDCl3: CHCl3 δ 7.27, DMSO-d6: C2D5HSO δ 2.50, THF-d8: C4D7HO δ 3.58, 1.73, C6D6: C6D5H δ 7.16). Data are reported as follows: chemical shift [multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant(s) in hertz, integration]. Phosphorus nuclear magnetic resonance (31P NMR) spectra were recorded with a 400 MHz spectrophotometer and are recorded in parts per million from external phosphoric acid on the δ scale (H3PO4: δ 0.00). Data are reported as follows: chemical shift [multiplicity (s = singlet), integration]. Carbon nuclear magnetic resonance (13C NMR) spectra were recorded with a 400 MHz spectrophotometer and are recorded in parts per million from internal tetramethylsilane on the δ scale and are referenced from the carbon resonances in the NMR solvent (CDCl3: δ 77.00, DMSO-d6: δ 39.51, THF-d8: δ 25.37, 67.57, C6D6: δ 128.39). Data are reported as follows: chemical shift. Infrared data (FTIR) were obtained with a Fourier-transform infrared spectrometer and are reported as follows: [frequency of absorption (cm−1), intensity of absorption (a = apparent, s = strong, m = medium, w = weak, br = broad). Melting points were determined with a capillary melting point apparatus. High-resolution mass spectrometry (HRMS) was collected 3931

DOI: 10.1021/acs.joc.8b00285 J. Org. Chem. 2018, 83, 3928−3940

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−5.8. IR (thin film) (cm−1): 1614 (m), 1500 (m), 1349 (s), 1160 (s), 1096 (s). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C29H38NO4SSi 524.2285; found 524.2295. (S)-6-Benzyloxy-3-(tert-butyldimethylsilyloxymethyl)-1-(4cyanobenzenesulfonyl)indoline (5). Caution! This reaction produces hydrogen gas and should be performed in a well-ventilated hood. A threeneck round-bottomed flask was equipped with a nitrogen inlet, thermocouple, and an outlet vent. The flask was charged with indoline 4 (50.00 g, 95.46 mmol, 1.00 equiv), and a mixture of toluene (250 mL) and methanol (250 mL) was added. The solution was sparged with nitrogen for ∼10 min at room temperature and then kept under a small nitrogen sweep. The reaction is sensitive to water; the typical water content ranged between 143.651 and 176.047 ppm by Karl Fischer analysis. Magnesium turnings21 (2.32 g, 95.46 mmol, 1.00 equiv) were added, and the mixture was stirred for 1 h, whereupon an exotherm and off-gassing were noted. The internal temperature was maintained below 35 °C with an ice−water bath. Additional magnesium was added at 1.00 equiv22 at ∼1 h intervals; then, the mixture was held overnight (or until all the solid magnesium reacted). The slurry was poured into a cold (0−5 °C) biphasic solution of toluene (500 mL) and 10 wt % urea in 3 N aqueous HCl (1.00 L)23 and vigorously agitated. The quenching was exothermic; a ΔT of +30− 35 °C was observed on this scale. The pH of the aqueous layer should be 6. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to afford the deprotected indoline as a dark brown solid (37.81 g). Rf (20% ethyl acetate in hexanes, silica gel): 4 0.57 (UV), deprotected indoline S4 0.51 (UV). The crude deprotected indoline S4 was dissolved in ethyl acetate (500 mL, water content ranged between 59.4286 and 86.4664 ppm by Karl Fischer analysis) and cooled to an internal temperature of 0−5 °C. N,N-Diisopropylethylamine (20.0 mL, 114.54 mmol, 1.20 equiv) was added followed by recrystallized15 4-cyanobenzenesulfonyl chloride (20.63 g, 100.23 mmol, 1.05 equiv). A white precipitate formed over 15−20 min, and the slurry was warmed to room temperature. The reaction mixture was stirred for 1 h, washed with a 10 wt % aqueous solution of citric acid (250 mL), and then washed with a 1.0 M aqueous solution of a pH 7 sodium phosphate buffer (375 mL), ensuring the pH of the aqueous layer was >6; proceeding into the crystallization with an acidic stream can lead to cleavage of the TBS group. The red-orange stream was stirred over sodium sulfate and activated carbon (DARCO G-60, 100 mesh, 25.0 g) for 2 h and then filtered over Celite (3.5″ dia, 0.75″ ht), rinsing the pad with ethyl acetate (2 × 25 mL). The light orange solution was concentrated in vacuo to afford a thick orange oil (55.27 g). To the oil was added isopropanol (500 mL), and the solvent level was marked.24 Additional isopropanol (500 mL) was added followed by dimethylethylamine (0.500 mL), and the oil was dissolved with mild heating at an internal temperature of ∼35 °C and then cooled to room temperature under mechanical stirring. The solution was seeded with product (250 mg), and the mixture was stirred for 15 h. The slurry was concentrated slowly (on this scale, 45 min to 1 h, vacuum pressure 8); then, the layers were separated, and the organic layer was washed with a saturated aqueous solution of brine (125 mL) diluted with water (125 mL). The pH of the aqueous layer is typically >7. If the pH of the aqueous layer was 7. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to afford a dark red/brown gel. To the gel was added isopropanol (450 mL), and the mixture was dissolved upon heated to an internal temperature of 70 °C; then, the solution was cooled slowly (20 min) to an internal temperature of 50 °C and held for 30 min whereupon the product began to crystallize, forming a large bed. The slurry was allowed to cool to room temperature and desaturated over 4 h. The crystals were collected by filtration and washed with 20 v % isopropanol in hexanes (2 × 250 mL), and the wet cake was dried in a vacuum oven at 50 °C and 100 Torr with a slight nitrogen sweep to constant weight to afford nitroindoline 6 as dark yellow, thick plates (42.35 g, 78%). Rf (20% ethyl acetate in hexanes, silica gel): 5 0.53 (UV), 6 0.39 (UV). Mp (isopropanol): 84−89 °C. [α]D22 +17.75° (c 9.46 mg/mL, chloroform). 1H NMR (400 MHz, DMSO-d6) δ: 8.03 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 8.6 Hz, 2H), 7.86 (s, 1H), 7.45−7.52 (m, 4H), 7.40 (tt, J = 6.9, 2.0 Hz 1H), 7.34 (s, 1H), 5.44 (s, 2H), 4.08 (app t, J = 9.7 Hz, 1H), 3.75 (dd, J = 10.7, 4.7 Hz, 1H), 3.52−3.57 (m, 1H), 3.35−3.42 (m, 2H), 0.69 (s, 9H), −0.11 (s, 3H), −0.18 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ: 152.8, 146.1, 139.3, 136.0, 134.7, 133.8, 128.7, 128.1, 127.8, 127.0, 124.9, 123.2, 117.3, 116.7, 99.8, 70.5, 63.8, 52.9, 40.4, 25.5, 17.6, −5.6, −5.8. IR (thin film) (cm−1): 2235 (w), 1624 (m), 1515 (s), 1365 (s), 1249 (s). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C29H34N3O6SSi 580.1932; found 580.1931. (S)-6-Benzyloxy-3-(tert-butyldimethylsilyloxymethyl)-1-(4-cyanobenzenesulfonyl)-5-nitro-4-(phenylsulfonylmeth-1-yl)indoline (7). Nitroindoline 6 (50.00 g, 86.25 mmol, 1.00 equiv) and chloromethyl phenyl sulfone (19.10 g, 99.19 mmol, 1.15 equiv) were dissolved in toluene (350 mL) and concentrated in vacuo to afford a dark oil. This was done as a safeguard to remove any residual traces of water or isopropanol from the previous step. The oil was dissolved in anhydrous tetrahydrofuran (300 mL, water content ranged between 31.3467 and 45.3390 ppm by Karl Fischer analysis) and cooled to an internal temperature of −25 ± 2 °C with a dry ice-acetonitrile bath. A solution of potassium tert-butoxide (1.0 M in THF, 198.4 mL, 198.4 mmol, 2.30 equiv) was added dropwise at a rate that maintained the internal temperature at −25 ± 2 °C (∼20 min). The dark solution was stirred at −25 ± 2 °C for 1 h and then quenched by the slow addition

of a solution of acetic acid (14.83 mL, 258.8 mmol, 3.00 equiv) in THF (35.2 mL) at a rate that maintained the internal temperature < −20 °C (∼10 min). The light suspension was warmed to room temperature, poured into a 1.0 M aqueous solution of a pH 7 sodium phosphate buffer (250 mL), and extracted with isopropyl acetate (250 mL). The organic phase was washed with a saturated aqueous solution of brine (250 mL) and then dried over sodium sulfate, filtered, and concentrated in vacuo to afford a dark crude gel. The gel was passed through a plug of silica gel (isocratic: 35% ethyl acetate in hexanes), and the fractions containing the product were pooled and concentrated in vacuo to afford a dark oil. The oil was dissolved in isopropyl acetate (250 mL), and if any solids were present at this point, the solution was polish filtered over a small plug of Celite (0.5″ dia, 0.5″ ht) before proceeding. Solids present at this point caused premature gelling during the crystallization. Heptane (250 mL) was added over 5 min; then, the dark solution was seeded with product (500 mg) and stirred for no less than 1 h. Heptane (50 mL) was added over 1 h, and the mixture was stirred for no less than 1 h (preferably 2 h). This sequence was repeated four more times for a total of 250 mL of heptane added (at any point, the procedure can be held overnight). The mixture was stirred for 14 h, and the slurry thickened. Additional heptane (500 mL) was added slowly over 8 h and then held for an additional 15 h. The crystals were collected by filtration, and the wet cake was dried in a vacuum oven at 50 °C and 100 Torr with a slight nitrogen sweep to constant weight, affording product 7 as very small, beige crystalline rods (48.08 g, 76%). Rf (25% ethyl acetate in hexanes, silica gel): 6 0.50 (UV), 7 0.25 (UV). Mp (isopropyl acetate/heptane): 114−118 °C. [α]D22 +65.04° (c 9.46 mg/mL, chloroform). 1H NMR (400 MHz, DMSO-d6) δ: 8.11 (d, J = 8.3 Hz, 2H), 7.86 (d, J = 8.3 Hz, 2H), 7.65 (app t, J = 7.5 Hz, 1H), 7.57 (d, J = 7.3 Hz, 2H), 7.38−7.51 (m, 8H), 5.43 (ABq, J = 12.8 Hz, ΔδAB = 0.05, 2H), 4.84 (d, J = 14.4 Hz, 1H), 4.59 (d, J = 14.4 Hz, 1H), 3.80 (d, J = 10.9 Hz, 1H), 3.28− 3.38 (m, 2H), 3.01 (dd, J = 10.0, 7.7 Hz, 1H), 2.87 (dd, J = 13.9, 7.1 Hz, 1H), 0.69 (s, 9H), −0.17 (s, 3H), −0.25 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 151.6, 143.2, 139.0, 137.5, 137.4, 135.7, 134.5, 133.9, 129.4, 128.8, 128.2, 128.0, 127.8, 127.0, 126.6, 120.2, 117.3, 116.8, 100.8, 70.6, 65.0, 55.1, 53.1, 40.0,28 25.5, 17.7, −5.8, −6.0. IR (thin film) (cm−1): 2235 (w), 1607 (m), 1534 (s), 1168 (s), 1088 (s). HRMS (ESI-TOF) m/z: [M + NH4]+ calcd for C36H43N4O8S2Si 751.2286; found 751.2289. Methyl (S)-4-Benzyloxy-8-(tert-butyldimethylsilyloxymethyl)-6-(4cyanobenzenesulfonyl)-3,6,7,8-tetrahydropyrrolo[3,2-e]indole-2carboxylate (9). To a solution of the nitroindoline 7 (30.00 g, 40.88 mmol, 1.00 equiv) in anhydrous acetonitrile (300 mL, water content ranged between 39.9137 and 101.5740 ppm by Karl Fischer analysis) was cooled to an internal temperature of 5 °C, and acetic acid29 (23.4 mL, 408.8 mmol, 10.0 equiv) was added. The system was placed under a nitrogen atmosphere and zinc dust30,31 (280 °C; at 260 °C, 13 darkens. [α]D22 +10.19° (c 14.95 mg/mL, DMF). 1H NMR (400 MHz, DMSO-d6) δ: 11.54 (d, J = 1.5 Hz, 1H), 11.32 (d, J = 1.3 Hz, 1H), 9.68 (s, 1H), 7.77 (br s, 1H), 7.13 (d, J = 2.0 Hz, 1H), 6.99 (br d, J = 1.8 Hz, 1H), 6.94 (s, 1H), 4.94 (t, J = 5.2 Hz, 1H), 4.56 (br t, J = 10.0 Hz, 1H), 4.34 (br dd, J = 10.9, 3.8 Hz, 1H), 3.93 (s, 3H), 3.87 (s, 3H), 3.74−3.81 (m, 7H), 3.64−3.70 (m, 1H), 3.43−3.49 (m 1H). 13C NMR (101 MHz, DMSO-d6) δ: 161.7, 159.8, 149.2, 143.0, 139.8, 139.2, 137.9, 131.8, 127.8, 125.9, 125.2, 124.5, 123.4, 114.8, 106.8, 105.7, 100.9, 98.1, 63.4, 61.2, 61.0, 56.0, 54.5, 51.8, 43.2. IR (thin film) (cm−1): 3436 (m), 3342 (br w), 1716 (m), 1583 (m), 1315 (s). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C25H26N3O8 496.1714; found 496.1724. (+)-Duocarmycin SA (1). Caution! (+)-Duocarmycin SA is a potent toxin! Appropriate PPE and laboratory hygiene are necessary when performing this reaction and handling any samples of, reaction streams containing, or equipment/instruments in contact with 1. The following reaction was performed in a laboratory equipped for handling high potency compounds. To a flask containing 13 (1.00 g, 2.02 mmol, 1.00 equiv) and ADDM16 (776 mg, 3.03 mmol, 1.50 equiv) was added THF (15.0 mL) under nitrogen. To the slurry was added tri-n-butylphosphine (770 uL, 3.03 mmol, 1.50 equiv), and after ∼5 min, the mixture became homogeneous. The solution was stirred for 2 h and was then quenched by pouring into water (15 mL). The solution was extracted with dichloromethane (1 × 15 mL, then 2 × 10 mL). The combined organic extracts were concentrated in vacuo to afford a gel. To the gel was added 25 mL of hexanes, which was stirred vigorously until all of the gel had converted into a solid (∼15−20 min). The solids were collected by filtration and washed with additional hexanes (25 mL). The solids were dissolved off the filter back into the original flask with dichloromethane (35 mL) and concentrated in vacuo to afford a solid. The solids were dissolved in the minimum amount of dichloromethane (25 mL) and applied to a column of silica gel prewetted with dichloromethane. Care was taken not to let the column go dry. If the column runs dry, or is locally concentrated, the product may precipitate. If this occurs, the compound will streak heavily on the column, and the product will

appear on the column as a bright yellow band. The product was eluted with a sharp gradient of 0 to 25% acetone in dichloromethane.35 Fractions containing the product were pooled and concentrated in vacuo to afford an orange gel (1.405 g). The gel was stirred vigorously with hexanes (10 mL) until completely converted to a light yelloworange powder. The powder was collected by filtration and dried at ambient temperature under vacuum (100 Torr) until a constant weight was achieved, affording 1 as a light yellow-orange powder (912 mg, 94%) that was identical in all respects with the previously reported literature.6,9 Solid samples of (+)-duocarmycin SA (1) are stored under nitrogen at −20 °C. Rf (30% acetone in dichloromethane, silica gel): 13 0.16 (UV), 1 0.51 (UV, visual, yellow spot). Mp (dichloromethane/acetone/hexanes): >250 °C; at 200 °C, 1 darkens. [α]D22 +188.39° (c 0.34 mg/mL, methanol). 1H NMR (400 MHz, DMSO-d6) δ: 9.95 (br s, 1H), 9.34 (br s, 1H), 7.05 (s, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.80 (s, 1H), 6.62 (d, J = 2.3 Hz, 1H), 4.49 (dd, J = 10.4, 4.8 Hz, 1H), 4.41 (d, J = 10.4 Hz, 1H), 4.09 (s, 3H), 3.96 (s, 3H), 3.93 (s, 3H), 3.91 (s, 3H), 2.81 (app dt, J = 7.6, 4.8 Hz, 1H), 1.76 (dd, J = 7.6, 4.6 Hz, 1H), 1.59 (app t, J = 4.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ: 177.9, 161.5, 161.1, 160.9, 150.3, 141.0, 138.8, 131.5, 129.9, 128.4, 126.7, 126.3, 123.2, 112.5, 107.7, 107.5, 97.5, 61.4, 61.1, 56.2, 54.9, 52.1, 31.3, 26.0, 23.5. IR (thin film) (cm−1): 3415 (br w), 3190 (br w), 1713 (m), 1640 (m), 1271 (s). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C25H24N3O7 478.1609; found 478.1613. Synthesis of 11. 2-Bromo-3,4,5-trimethyoxybenzaldehyde (S8).36 To a three-neck round-bottomed flask equipped with a thermocouple were added 3,4,5-trimethoxybenzaldehyde (14, 50.00 g, 249.7 mmol, 1.00 equiv) and acetonitrile (500 mL), which was then allowed to stir until a homogeneous solution formed (∼5 min). The flask was placed under nitrogen, and N-bromosuccinimide (47.14 g, 262.2 mmol, 1.05 equiv) was added in one portion. The mixture was heated to an internal temperature of 50 °C for 1 h, cooled to room temperature, and diluted with toluene (500 mL). The solution was washed with a half saturated, aqueous solution of sodium thiosulfate (500 mL) and then with a saturated aqueous solution of brine (250 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to afford a dark solid. The solid was dissolved in a 25 v % solution of isopropanol in hexane (200 mL) at 50 °C, cooled to 38−40 °C, and then seeded with product (250 mg). The seed bed was stirred for 1 h, cooled to room temperature (23 °C), and then held for 15 h. The slurry was then further cooled to an internal temperature of 5 °C and held for 1 h. The crystals were isolated by filtration; the cake was washed with a 25 v % solution of isopropanol in hexanes (100 mL) and then dried in a vacuum oven at 50 °C until constant weight to afford bromide S8 as off-white needles (57.62 g, 84%), which were identical in all respects with the previously reported literature.36 Rf (20% ethyl acetate in hexanes, silica gel): 14 0.26 (UV), bromide S8 0.53 (UV). Mp (isopropanol/hexanes): 58.5−59.5 °C. 1H NMR (400 MHz, CDCl3) δ: 10.30 (s, 1H), 7.31 (s, 1H), 3.99 (s, 3H), 3.92 (s, 3H), 3.91 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 191.0, 153.0, 150.8, 148.7, 128.8, 115.6, 107.4, 61.24, 61.17, 56.2. IR (thin film) (cm−1): 2942 (m), 2866 (m), 1691 (s), 1579 (m), 1107 (s). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C10H12O4Br 274.9914; found 274.9919. (Z)-4-(2-Bromo-3,4,5-trimethoxybenzylidene)-2-phenyloxazol-5one (S9). To a three-neck round-bottomed flask equipped with a thermocouple were added 2-bromo-3,4,5-trimethoxybenzaldehyde (S8, 40.00 g, 145.4 mmol, 1.00 equiv), freshly powdered potassium acetate (7.14 g, 72.7 mmol, 0.50 equiv), THF (200 mL), and acetic anhydride (41.2 mL, 436.2 mmol, 3.00 equiv). The light slurry was placed under nitrogen and heated to an internal temperature of 60 °C. Solid N-Bz-glycine (29.54 g, 159.9 mmol, 1.10 equiv) was added in small (∼2.0 g) portions though a funnel against a slight nitrogen flow from the round-bottom flask at a rate that maintained the internal temperature at 60 ± 3 °C (∼5 min, minor delayed exotherm). The light brown slurry became a red/orange slurry and was allowed to stir for 2 h at 60 °C. An addition funnel was attached and filled with a 10 v % solution of water in isopropanol (400 mL), and the solution was added at a rate that maintained the internal temperature at 55 °C (∼1.25 h). The yellow/orange slurry was cooled to room temperature 3935

DOI: 10.1021/acs.joc.8b00285 J. Org. Chem. 2018, 83, 3928−3940

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The Journal of Organic Chemistry and then to an internal temperature of 5 °C and held for 30 min. The crystals were isolated by filtration, then dried in a vacuum oven at 50 °C until constant weight to afford oxazolone S9 as dense, yellow prisms (51.58 g, 85%). Rf (20% ethyl acetate in hexanes, silica gel): bromide S8 0.53 (UV), oxazolone S9 0.39 (UV, visual, yellow spot). Mp (THF/aqueous isopropanol): 167−168 °C. 1H NMR (400 MHz, CDCl3) δ: 8.54 (s, 1H), 8.12 (d, J = 7.8 Hz, 2H), 7.73 (s, 1H), 7.64 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.7 Hz, 2H), 4.04 (s, 3H), 4.00 (s, 3H), 3.92 (s, 3H). 13C NMR (101 MHz, CDCl3) δ:37 167.2, 163.9, 152.5, 151.0, 145.8, 133.6, 129.4, 129.1, 128.3, 128.2, 125.4, 115.6, 111.9, 61.3, 61.0, 56.1. 1H NMR (400 MHz, THF-d8) δ: 8.60 (s, 1H), 8.13− 8.16 (m, 2H), 7.65 (tt, J = 7.3, 1.6 Hz, 1H), 7.59 (s, 1H), 7.53−7.58 (m, 2H), 3.99 (s, 3H), 3.93 (s, 3H), 3.86 (s, 3H). 13C NMR (101 MHz, THF-d8) δ: 167.3, 165.4, 154.0, 152.2, 147.1, 135.1, 134.6, 130.1, 129.24, 129.16, 128.5, 126.9, 115.7, 112.9, 61.4, 61.3, 56.6. IR (thin film) (cm−1): 1797 (s), 1648 (m), 1482 (m), 1327 (s), 1170 (m). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C19H17O5NBr 418.0285; found 418.0290. Methyl (Z)-2-Benzamido-3-(2-bromo-3,4,5-trimethoxyphenyl)acrylate (15). To a three-neck round-bottomed flask equipped with a thermocouple was added the oxazolone S9 (40.00 g, 95.65 mmol, 1.00 equiv), methanol (200 mL), and triethylamine (1.33 mL, 9.57 mmol, 0.10 equiv). The yellow suspension was heated to an internal temperature of 60 °C and then held for 0.5 h, whereupon a light yellow homogeneous solution formed. Heptane (200 mL) was added, maintaining an internal temperature >50 °C. The biphasic mixture was cooled to an internal temperature of 40 °C and seeded with product (200 mg), and the mixture was stirred for 30 min before being cooled to room temperature. The solvents were concentrated in vacuo to ∼200 mL; additional heptane (200 mL) was added, and the solvents were again concentrated to ∼200 mL. The slurry was cooled to an internal temperature of 5 °C and held for 30 min, and then the crystals were isolated by filtration and dried in a vacuum oven at 50 °C until constant weight to afford product 15 as dense, light yellow rods (42.3 g, 98%). Rf (40% ethyl acetate in hexanes, silica gel): oxazolone S9 0.61 (UV, visual, yellow spot), 15 0.41 (UV). Mp (methanol/ heptane): 139−140 °C. 1H NMR (400 MHz, CDCl3) δ: 7.82 (app d, J = 7.3 Hz, 2H), 7.78 (br s, 1H), 7.60 (s, 1H), 7.54 (app tt, J = 7.5, 1.4 Hz, 1H), 7.44 (app t, J = 7.6 Hz, 2H), 6.92 (s, 1H), 3.89 (s, 3H), 3.88 (br s, 6H), 3.55 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 165.8, 165.4, 152.4, 150.9, 143.6, 133.2, 132.3, 130.2, 129.4, 128.8, 127.2, 125.5, 111.9, 108.0, 61.1, 60.9, 55.8, 52.9. IR (thin film) (cm−1): 3393 (m), 1725 (s), 1674 (s), 1470 (s), 1250 (m). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C20H21O6NBr 450.0547; found 450.0550. Methyl 1-Benzoyl-5,6,7-trimethoxyindole-2-carboxylate (S10). To a three-neck round-bottom flask was added the dehydroamino ester 15 (30.00 g, 66.62 mmol, 1.00 equiv), 1,10-phenanthroline (910 mg, 5.00 mmol, 0.075 equiv), potassium carbonate (23.02 g, 166.6 mmol, 2.50 equiv), and anhydrous DMF (150 mL, water content containing from 77.42 to 220.38 ppm was successfully used). The mixture was stirred, and nitrogen was sparged though the suspension for 10 min during which time the supernatant became bright yellow. Copper(I) iodide (634 mg, 3.33 mmol, 0.05 equiv) was added, and the mixture became deep red. The suspension was heated to an internal temperature of 80 °C under nitrogen, held for 2 h, and was then cooled to an internal temperature of 5 °C. An addition funnel filled with water (450 mL) was attached, and water was added at a rate such that the internal temperature did not exceed 10 °C (∼1.25 h). The crystals were isolated by filtration, washed with water (2 × 120 mL), and pulled dry with vacuum thoroughly for ∼30 min to afford dark colored crystals. The crystals were dissolved in THF (300 mL) with mild heating to afford a dark, slightly hazy suspension. Activated carbon (DARCO G-60, 100 mesh, 3.0 g) was added, and the mixture was stirred for 15 min before being filtered over a plug of Celite (1″ dia, 0.5″ ht). The plug was washed with additional THF (2 × 30 mL). The orange/brown filtrate and washings were concentrated in vacuo to afford a brown solid. To the solid was charged methanol (750 mL), and the mixture was heated to reflux for ∼15 min to dissolve the vast majority of the solids. The mixture was allowed to cool slowly over 45 min to room temperature; the volume was carefully reduced in vacuo

to ∼120 mL, and then the slurry was cooled to 5 °C. The crystals were isolated by filtration38 and washed with cold (∼5 °C) methanol (2 × 30 mL). For washing, suction was suspended. Cold methanol was charged to the filter; the cake was agitated caref ully for 5−10 s, and then the wash was removed by suction. The white, granular crystals were then dried in a vacuum oven at 50 °C until constant weight to afford indole S10 (23.19 g, 94%). Rf (40% ethyl acetate in hexanes, silica gel): 15 0.41 (UV), indole S10 0.69 (UV). Mp (methanol): 139−140 °C. 1H NMR (400 MHz, CDCl3) δ: 7.76 (app d, J = 7.1 Hz, 2H), 7.60 (app tt, J = 7.4, 1.1 Hz, 1H), 7.46 (app t, J = 7.8 Hz, 2H), 7.27 (s, 1H),39 6.89 (s, 1H), 3.93 (s, 3H), 3.85 (s, 3H), 3.76 (s, 3H), 3.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 171.0, 161.1, 150.8, 141.8, 139.6, 134.5, 133.8, 129.8, 129.1, 128.7, 128.3, 122.7, 111.6, 97.9, 61.1, 60.4, 56.2, 51.9. IR (thin film) (cm−1): 1724 (s), 1709 (s), 1535 (m), 1454 (m), 1233 (s). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C20H20O6N 370.1285; found 370.1284. 5,6,7-Trimethoxyindole-2-carboxylic Acid (16). To a suspension of indole S10 (55.00 g, 148.9 mmol, 1.00 equiv) in THF (275 mL) was added solution of aqueous sodium hydroxide (2.5 N, 180.0 mL, 446.7 mmol, 3.0 equiv). The headspace was purged with nitrogen,40 and the mixture was heated to an internal temperature of 60 °C and vigorously stirred for 3 h, becoming a homogeneous light yellow solution. The mixture was cooled to room temperature and then poured into an aqueous solution of hydrochloric acid (2.33 M, 275 mL, 640 mmol). The layers were separated, and the aqueous layer was extracted with 2methyltetrahydrofuran (2 × 275 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to afford an off-white solid. To the solid was added methanol (110 mL), and the solvent line was marked.24 Additional methanol (715 mL) was added, and the mixture was brought to reflux to dissolve the solids. The solution was cooled to an internal temperature of 40 °C and seeded with product (550 mg), and the mixture was cooled to room temperature and stirred for 15 h. The volume was carefully reduced in vacuo to ∼110 mL (4.0 mL/g indole), and then the slurry was cooled to 5 °C and held for 30 min. The crystals were isolated by filtration41 and washed with cold (∼5 °C) methanol (2 × 110 mL). For washing, suction was suspended. Cold methanol was charged to the filter; the cake was agitated carefully for 5−10 s, and then the wash was removed by suction. The white, granular crystals were then dried in a vacuum oven at 50 °C until constant weight to afford acid 16 (32.49 g, 87%), which was identical in all respects with the previously reported literature.9 Rf (75% hexanes, 20% ethyl acetate, 5% acetic acid, silica gel): indole S10 0.49 (UV), 16 0.25 (UV). Mp (methanol): 220−221 °C. 1H NMR (400 MHz, DMSO-d6) δ: 12.73 (s, 1H), 11.57 (br s, 1H), 7.00 (d, J = 2.0 Hz, 1H), 6.91 (s, 1H), 3.89 (s, 3H), 3.79 (s, 3H), 3.77 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 162.5, 149.2, 140.2, 139.3, 128.5, 126.7, 123.0, 108.3, 98.0, 61.1, 60.9. 55.9. IR (thin film) (cm−1): 3281 (br s), 1657 (s), 1539 (m), 1504 (m), 1262 (s). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C12H14O5N 252.0866; found 252.0870. 5,6,7-Trimethoxyindole-2-carbonyl Chloride (11). Carboxylic acid 16 (30.00 g, 119.4 mmol, 1.00 equiv) and anhydrous THF (300 mL, water content by KF = 38.6576 ppm) were added to a roundbottomed flask and stirred for 5 min forming a very light slurry. Anhydrous DMF (0.186 mL, 2.388 mmol, 0.020 equiv, water content by KF = 147.901 ppm) was added, and the system was placed under nitrogen with a vent into a solution of 10 wt % aqueous potassium phosphate. Oxalyl chloride (15.5 mL, 179.1 mmol, 1.50 equiv) was added dropwise over 20 min, forming a yellow solution and evolving gas mildly. The solution was held for an additional 1 h and then concentrated in vacuo to afford a deep yellow powder that was dissolved in methyl tert-butyl ether (600 mL) at reflux, leaving behind a small amount of precipitate. The mixture was filtered hot over a 0.45 μm polypropylene filter and concentrated in vacuo to afford a yellow solid. The solids were dissolved in toluene (150 mL) at an internal temperature of 60 °C under mechanical stirring, and heptane (150 mL) was added slowly, maintaining the internal temperature at 60 °C (∼20 min). The internal temperature was cooled to 50 °C, and the product began to crystallize heavily. An additional charge of heptane (150 mL) was added, maintaining the internal temperature at 50 °C 3936

DOI: 10.1021/acs.joc.8b00285 J. Org. Chem. 2018, 83, 3928−3940

Article

The Journal of Organic Chemistry (∼30 min); then, the temperature was lowered to 5 °C over 30 min. The product was isolated by filtration and dried in a vacuum oven at 50 °C and 100 Torr with a slight nitrogen sweep to constant weight to afford acid chloride 11 as bright yellow needles (26.7 g, 92%), which was identical in all respects with the previously reported literature.9 This product was sealed under nitrogen and stored at −17 to −20 °C. Rf (20% ethyl acetate, 75% hexanes, 5% acetic acid, silica gel): 16 0.25 (UV), 11 0.42 (UV). Mp (toluene/heptane): 120.5−121.5 °C. 1H NMR (400 MHz, C6D6)42 δ: 9.00 (s, 1H), 7.24 (d, J = 2.3 Hz, 1H), 6.44 (s, 1H), 3.74 (s, 3H), 3.66 (s, 3H), 3.39 (s, 3H). 13C NMR (101 MHz, C6D6) δ: 159.2, 151.9, 143.4, 139.5, 130.1, 129.7, 123.6, 117.0, 98.5, 61.5, 61.2, 56.1. IR (thin film) (cm−1): 1720 (s), 1495 (m), 1310 (m), 1112 (m), 826 (m). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C12H13O4NCl 270.0528; found 270.0532. Synthesis of L1. (S)-(+)-N,N-Dimethylamino-1-ferrocenylethylammonium L-Tartrate (S14). To a 250 mL round-bottomed flask were added triethylamine (23.5 mL, 166.6 mmol, 4.0 equiv) and dichloromethane (30 mL).43 The solution was cooled to an internal temperature of −20 °C, and formic acid (98%, 17.7 mL, 461.5 mmol, 10.0 equiv) was added dropwise, keeping the internal temperature below −15 °C; then, the solution was warmed to room temperature. Acetylferrocene (S11, 10.0 g, 41.6 mmol, 1.0 equiv) was added followed by RuCl[(S,S)-TsDPEN](mesitylene) (1.38 g, 2.08 mmol, 0.05 equiv). A remaining charge of dichloromethane (20 mL) was used to wash any solids down into the reaction mixture (total volume: 50 mL). The flask was sealed with a septa, and a 16 gauge needle was inserted to allow generated carbon dioxide to escape. The mixture was stirred for 5 days, concentrated in vacuo, then rinsed down with toluene (50 mL) to afford a dark solid mass that was used without further purification. A small aliquot was purified by flash column chromatography over silica gel (0 to 30% ethyl acetate in a hexane gradient) for characterization purposes and was identical in all respects with the previously reported literature.43 The light orange powder was found to have an enantiomeric ratio of 99.26 to 0.74 favoring the (S) configuration as determined by chiral stationary phase HPLC. Rf (20% ethyl acetate in hexanes, silica gel): acetylferrocene 0.39 (UV), (S)-1ferrocenylethanol (S12) 0.34 (UV), Rf (20% ethyl acetate in hexanes, basic alumina): acetylferrocene 0.49 (UV), (S)-1-ferrocenylethanol (S12) 0.21 (UV). Mp (ethyl acetate/hexanes): 77−78 °C. [α]D22 +25.6° (c 10.0 mg/mL, dichloromethane). 1H NMR (400 MHz, CDCl3) δ: 4.50−4.58 (app br s, 1H), 4.15−4.30 (m, 9H), 1.80−1.88 (app br s, 1H), 1.45 (br d, J = 5.6 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ: 95.2, 68.6, 68.22, 68.18, 66.45, 66.35, 65.5, 23.6. IR (thin film) (cm−1): 3340 (br s), 1455 (m), 1409 (m), 1233 (m), 1092 (s). HRMS (ESI-TOF) m/z: [M]+ calcd for C12H14OFe 230.0389; found 230.0393. The crude residue from the previous step was dissolved in dichloromethane (100 mL) and 4-dimethylaminopyridine (514 mg, 4.16 mmol, 0.10 equiv) was added, followed by triethylamine (10.20 mL, 72.89 mmol, 1.75 equiv) and acetic anhydride (6.69 mL, 70.81 mmol, 1.70 equiv). The reaction mixture was allowed to stir for 1 h; then, a solution of 1.0 N aqueous hydrochloric acid (100 mL) was added. The layers were separated, and the aqueous layer was extracted with methyl tert-butyl ether (3 × 100 mL, visualization of difficult-tosee phase splits were aided by using a flashlight or by the addition of small amounts of ice chips). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to afford acylated product S13 as a dark red-brown solid (13.83 g) that was used without further purification. Rf (20% ethyl acetate in hexanes, basic alumina): (S)-1-ferrocenylethanol (S12) 0.25 (UV), acylated product S13 0.78 (UV). Crude acetate44 S13 (13.83 g) was dissolved in methanol (200 mL), and an aqueous solution of 40 wt % dimethylamine (28.0 mL, 221.6 mmol, 5.3 equiv) was added. The mixture was stirred for 24 h, and then the methanol was removed in vacuo to afford a dark aqueous solution. A solution of aqueous phosphoric acid (5.0 mL of an 85 wt % aqueous solution, diluted to a total of 50 mL with water) was added. The pH of the resulting solution was ∼2. The mixture was washed with methyl tert-butyl ether (2 × 100 mL). Solid sodium hydroxide (5.0 g) was added slowly (Caution! Exothermic) and the resulting

solution (pH > 10) was extracted with dichloromethane (3 × 75 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to afford crude amine S14 as a dark red oil (9.37 g). Rf (20% ethyl acetate in hexanes, basic alumina): acylated product S13 0.78 (UV), amine freebase S14-freebase 0.46 (UV, Rf can vary based on loading). The crude amine S14-freebase (9.37 g) was dissolved in methanol (20 mL), and the solution was heated to 60 °C. To a separate flask was dissolved L-(+)-tartaric acid (5.47 g, 36.43 mmol, 1.00 equiv with respect to crude amine) in methanol (20 mL) at 60 °C. The hot acid solution was added to the solution of the amine, maintaining an internal temperature of 60 °C. Additional methanol (6.85 mL) was used to quantitate the transfer of the acid. Once the addition was complete, the mixture was seeded with product (20 mg), cooled to room temperature slowly over 4.5 h, and then held an additional 19.5 h. The crystals were collected by filtration, washed with a minimal amount of methanol (5 mL), and then dried to afford salt S14 (12.45 g, 73% from acetylferrocene) as orange crystals, which were similar to the previously reported literature44 and additionally characterized here. The product was found to have an enantiomeric ratio of 99.92 to 0.08 favoring the (S) configuration as determined by chiral stationary phase HPLC. Mp (methanol): 158−163 °C. [α]D22 +9.0° (c 10.0 mg/mL, DMF). 1H NMR (400 MHz, DMSO-d6) δ: 6.25−10.25 (br s, 4H), 4.43 (br s, 1H), 4.37 (br s, 1H), 4.28 (br s, 2H), 4.18−4.26 (m, 6H), 4.01 (s, 2H), 2.36 (s, 6H), 1.59 (d, J = 6.7 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 174.6, 81.4, 72.1, 70.2, 68.9, 68.58, 68.50, 67.6, 59.8, 38.3, 14.2. IR (KBr) (cm−1): 3494 (s), 3320 (s), 2639 (s), 1715 (m), 1408 (s). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C14H20NFe 258.0940; found 258.0946. (R,R)-2,2″-Bis[(S)-1-diphenylphosphinylethyl]-1,1″-biferrocene Toluene Solvate (S18).45 The tartrate salt S14 (50.0 g, 122.8 mmol, 1.0 equiv) was partitioned between dichloromethane (250 mL), and an aqueous solution of sodium hydroxide (25.0 g dissolved in 250 mL water). The layers were separated, and the aqueous layer was extracted with dichloromethane (2 × 250 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and then concentrated down from benzene (2 × 50 mL) to afford the free base S14-freebase as a dark oil (32.91 g). A sample of S14-freebase was analyzed by polarimetry: [α]D22 −12.9° (c 15.0 mg/mL, absolute ethanol). The oil was dissolved in diethyl ether (298 mL) and cooled to an internal temperature of +1.5 °C using an ice−water bath.46 A 1.4 M cyclohexane solution of sec-butyl lithium (100 mL, 140.0 mmol, 1.14 equiv) was added at a rate maintaining an internal temperature below +5.0 °C (30 min). The solution was allowed to stir for 1 h and then cooled to an internal temperature of −68 °C with a dry ice−acetone bath. A solution of iodine (35.9 g, 141.3 mmol, 1.15 equiv) in THF (298 mL) was added dropwise, maintaining an internal temperature below −50 °C (∼50 min). The suspension was allowed to stir at −60 °C for 1 h and was then warmed to room temperature and washed with a half saturated, aqueous solution of sodium thiosulfate (250 mL). The aqueous layer was extracted with diethyl ether (2 × 100 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to afford a dark red oil. The red oil was passed through a plug of basic alumina (0 to 50% ethyl acetate in a hexane gradient) to afford the iodide as dark red crystals (40.99 g, 87%). Rf (10% ethyl acetate in hexanes, basic alumina): amine freebase S14-freebase 0.29 (UV, Rf can vary based on loading), iodide S15 0.43 (UV, Rf can vary based on loading). Iodide S15 (30.0 g, 78.3 mmol, 1.0 equiv) was dissolved in acetone (150 mL) and then cooled in an ice−water bath to an internal temperature of 1.0 °C, and then iodomethane (22.1 mL, 352.4 mmol, 4.50 equiv) was added dropwise maintaining an internal temperature below 15 °C (∼5 min), whereupon a solid precipitated.47 The heavy slurry was allowed to stir at room temperature for 30 min and concentrated in vacuo (Caution! Iodomethane in headspace) and then down from acetonitrile (2 × 60 mL) to afford crude ammonium iodide salt S16. Separately, diphenylphosphine oxide (20.0 g, 94.0 mmol, 1.2 equiv) was dissolved in anhydrous THF (210 mL) and cooled in an ice− 3937

DOI: 10.1021/acs.joc.8b00285 J. Org. Chem. 2018, 83, 3928−3940

Article

The Journal of Organic Chemistry water bath to an internal temperature of 0.9 °C. A 1.6 M solution of nbutyl lithium in hexanes (58.7 mL, 94.0 mmol, 1.20 equiv) was added dropwise, maintaining an internal temperature below 10 °C (∼45 min). The initially clear solution became cloudy with a white precipitate and slowly developed an orange coloration near the end of the addition. The suspension was allowed to stir for 1.5 h and was then concentrated in vacuo to afford a light yellow residue. Ammonium iodide salt S16 was slurried in acetonitrile (400 mL) and transferred to the residue of the diphenylphosphine oxide anion. A remaining charge of acetonitrile (100 mL, total volume: 16.7 mL/g iodide S15) was used to quantitate the transfer. The slurry was heated to reflux and stirred for 2 h before being cooled to room temperature and concentrated in vacuo (Caution! Trimethylamine in headspace). The mass was partitioned between methyl tert-butyl ether (300 mL) and water (300 mL). The aqueous layer was extracted with methyl tertbutyl ether (300 mL), and the combined organic layers were washed with a solution of 5 wt % citric acid in a saturated aqueous solution of brine (90 mL) and then a saturated aqueous solution of brine (90 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to afford a dark oil that was passed through a plug of silica gel (0 to 10% methanol in dichloromethane gradient) to afford product phosphine oxide S17 as a viscous, dark red oil that was used without further purification (42.4 g). Rf (5% methanol in dichloromethane, silica gel): iodide S15 0.09 (UV, Rf can vary based on loading), phosphine oxide S17 0.45 (UV, Rf can vary based on loading). To an oven-dried round-bottomed flask were added bis(triphenylphosphine)nickel(II) chloride (25.6 g, 39.2 mmol, 0.50 equiv), tetraethylammonium iodine (20.6 g, 78.3 mmol, 1.00 equiv), and freshly washed zinc dust (7.7 g, 117.5 mmol, 1.50 equiv).31 Under a heavy flow of nitrogen, anhydrous DMF (156.5 mL) was added, and the heavy slurry was vigorously stirred under nitrogen for 30 min. Initially, a deep blue suspension was formed that faded to a deep green and then maroon color (all within ∼10 min). Separately, phosphine oxide S17 (42.4 g, 78.3 mmol, 1.00 equiv) was dissolved in anhydrous DMF (78.3 mL) with heating. The dark red solution was cooled to room temperature, degassed by nitrogen sparging for 20 min, and then transferred to the maroon catalyst mixture via cannula and heated in a 120 °C bath for 2 h. The reaction was then cooled to room temperature and partitioned between dichloromethane (600 mL) and water (600 mL), forming a biphasic mixture with heavy precipitates, all of which was passed through a plug of Celite (4″ dia, 3″ ht, gentle agitation of the top layer of Celite with a spatula can maintain a good filtration rate), removing a sticky black film. The two layers of the filtrate were separated, and the aqueous layer was extracted with dichloromethane (2 × 90 mL). The combined organic layers were washed with a saturated aqueous solution of brine (2 × 300 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to afford a dark red oil. The oil was purified by flash column chromatography over silica gel (0 to 40% acetone in toluene) to afford a thick orange paste (42.6 g). The paste was dissolved in toluene (150 mL) at an internal temperature of 80 °C and then allowed to cool to room temperature for 15 h. The orange crystals were collected by filtration, washed with toluene (40 mL), and dried until constant weight to afford dimer S18 as bright orange crystals (17.0 g, 44% from iodide), which was identical in all respects with the previously reported literature.45c The product was found to have an enantiomeric ratio of >99.9% favoring the (S,S)-(R,R) configuration as determined by chiral stationary phase HPLC. Rf (20% acetone in toluene, silica gel): phosphine oxide S17 0.28 (UV, Rf can vary based on loading), dimer S18 0.37 (UV, Rf can vary based on loading). Mp (toluene): 153−156 °C. [α]D22 +138.0° (c 10.0 mg/mL, chloroform). 1H NMR (400 MHz, CDCl3) δ: 7.79−7.91 (m, 4H), 7.68 (app dd, J = 10.1, 8.3 Hz, 4H), 7.42−7.56 (m, 6H), 7.33 (td, J = 7.5, 1.0 Hz, 2H), 7.25−7.31 (m, 6H, PhMe), 7.15−7.24 (m, 6H, partial PhMe), 7.06 (td, J = 7.8, 2.8 Hz, 4H), 4.38 (m, 12H), 4.10 (t, J = 2.5 Hz, 2H), 4.04 (br s, 2H), 3.78−3.91 (m, 2H), 2.37 (s, 5.6 H, PhMe), 1.68 (dd, J = 16.7, 7.3 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ: 137.8, 133.9 (d, J = 94.4 Hz), 132.2 (J = 8.8 Hz), 132.0 (d, J = 93.7 Hz), 131.3 (d, J = 8.8 Hz), 131.2 (d, J = 2.9 Hz), 130.9 (d, J = 2.9 Hz), 129.0, 128.2, 128.16 (d, J = 11.0 Hz), 128.0 (d, J = 11.7 Hz), 125.3,

90.6, 84.4 (d, J = 6.6 Hz), 72.6, 69.3, 68.3 (d, J = 5.1 Hz), 65.4, 30.2 (d, J = 68.1 Hz), 21.4, 19.1. 31P NMR (162.0 MHz, CDCl3) δ: 34.84. IR (thin film) (cm−1): 3413 (br s), 2877 (m), 1618 (w), 1184 (s), 1171 (s). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C48H45O2Fe2P2 827.1588; found 827.1603. (R,R)-2,2″-Bis[(S)-1-diphenylphosphinoethyl]-1,1″-biferrocene Benzene Solvate (L1).48 To an autoclave were added dimer S18 (20.00 g, 20.15 mmol, 1.00 equiv), benzene (54 mL), and triethylamine (23.3 mL, 167.3 mmol, 8.3 equiv). The headspace was blanketed with nitrogen; trichlorosilane (13.0 mL, 127.0 mmol, 6.3 equiv) was added, and the reactor was quickly sealed. The mixture was heated to an internal temperature of 100 °C for 19 h and then cooled to room temperature. The contents of the reactor were transferred cautiously to a room temperature solution of 30 wt % aqueous sodium hydroxide (40 g of sodium hydroxide diluted to 133 g with water) using 400 mL of benzene to aid in the transfer (Caution! Exothermic and will produce wisps of a white precipitate in air, perform quenching in a well-ventilated hood). The mixture was heated to 60 °C for 30 min under nitrogen to dissolve the solid silicon species and then cooled to room temperature, affording a biphasic mixture. The layers were separated, and the organic layer was washed with water (100 mL) and then a saturated aqueous solution of brine (100 mL). The mixture was dried over sodium sulfate, filtered, and concentrated to ∼50% volume in vacuo. The red oil was passed through a plug of basic alumina (3.25″ dia, 1.5″ ht) eluting with benzene until the eluent ran colorless (∼1 L). The solution was concentrated in vacuo to afford an orange paste (16.98 g). The paste was dissolved in boiling benzene (60 mL) under a nitrogen atmosphere and then cooled to room temperature. Absolute ethanol (200 proof, 40 mL) was added, and the mixture was seeded with product (10 mg). Additional ethanol (20 mL) was added, and the slurry was aged for 15 min to develop a seed bed. Additional ethanol (120 mL) was added slowly over 10 min, and the slurry was aged for 1 h. The bright orange crystals were collected by filtration, washed with absolute ethanol (2 × 20 mL), and dried in vacuo at room temperature to afford product L1 as bright orange crystals (10.05 g, 57%) which was identical in all respects with the previously reported literature.48 A sample that was stored on the benchtop in a vial sealed with parafilm showed no signs of aerial oxidation by 31P and 1 H NMR over 15 months; however, we typically store it in a nitrogenfilled glovebox. Rf (20% acetone in toluene, silica gel): dimer 0.37 (UV, Rf can vary based on loading), L1 0.98 (UV, Rf can vary based on loading). Rf (100% benzene, basic alumina): dimer 0.00, L1 0.95. Mp (benzene/ethanol): 133−138 °C. [α]D22 +410.0° (c 5.0 mg/mL, chloroform). 1H NMR (400 MHz, CDCl3) δ: 7.35 (br s, 6H), 7.10− 7.30 (m, 20H), 4.55 (dd, J = 2.3, 1.3 Hz, 2H), 4.29 (s, 10 H), 4.12 (t, J = 2.4 Hz, 2H), 3.79 (m, 2H), 3.49 (q, J = 6.8 Hz, 2H), 1.35 (app t, J = 7.2 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ: 138.9 (app dd, J = 10.2, 7.3 Hz), 136.0 (app dd, J = 12.4, 9.5 Hz) 135.1 (m), 132.1 (m), 128.4, 128.3, 128.0 (t, J = 2.2 Hz), 127.5 (t, J = 3.3 Hz), 127.2, 93.1 (m), 84.4 (t, J = 2.2 Hz), 71.7, 69.3, 68.0 (t, J = 3.7 Hz), 65.4, 29.2 (m), 17.0. 31P NMR (162.0 MHz, CDCl3) δ: 1.52. IR (thin film) (cm−1): 3413 (br s), 1618 (m), 1434 (m), 815 (s), 739 (s). HRMS (ESI-TOF) m/z: [M + H]+ calcd for C48H45Fe2P2 795.1690; found 795.1711.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.8b00285. Reference schemes, an expanded bibliography, analytical data (1H and 13C NMR) for all compounds, chiral HPLC methods and spectra, methods for generating seeds for crystallization, and optimization details (PDF) Crystal structure of duocarmycin (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 3938

DOI: 10.1021/acs.joc.8b00285 J. Org. Chem. 2018, 83, 3928−3940

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The Journal of Organic Chemistry ORCID

equivalent charge of freshly prepared precatalyst solution can restore reactivity and with no loss of enantioselectivity. (20) The reaction can be examined for conversion here by sampling at 65 °C. At temperatures below 45 °C, the product can crystallize. Additionally, if at any point postreaction completion the product crystallizes, it can be dissolved in dichloromethane for further manipulation. (21) A fresh bottle (purchased within 6 months) of ordinary magnesium turnings was used. “Activated” magnesium turnings, for example, prewashed with 0.5% aqueous HCl as in Brown, A. C.; Carpino, L.A. J. Org. Chem. 1985, 50, 1749 or with 5 v % TMSCl in THF (stirred for 30 min) also worked, but no significant difference was observed. (22) Total magnesium added can vary around 11−15 equiv. Initial charges of magnesium are more exothermic than the later charges. A magnesium charge rate that maintains the internal temperature below 35 °C is encouraged. After 10 equiv of magnesium was added, any subsequent charges are accompanied with a 7 mL/g (with respect to Mg (i.e., 7 mL/1 g of magnesium)) charge of methanol. (23) The amount here is for ≤12 equiv of magnesium. If more magnesium is used, increase this quench amount to ensure the pH of the aqueous layer is