Total synthesis of (–)-negamycin from a chiral advanced epoxide

Abstract A concise route for the total synthesis of (–)-negamycin is described. Key point features (a) Cu-mediated ring-opening reaction of the advanced epoxide with vinyl magnesium chloride leads to the introduction of the carboxyl synthon; (b) Consecutive placement of diamino groups via azides was designed, including an unexpected cleavage of the silyl protecting group; (c) removal of four hydrogenolytically labile groups in one step. GRAPHICAL ABSTRACT


Introduction
The epic war against bacteria never ends. The widespread introduction of antibiotics since the 1940s, the crisis emerged because of the development of multidrug-resistant bacteria, such as MRSA (methicillin-resistant Staphylococcus aureus), VRE (vancomycinresistant Enterococcus), and MDR-TB (multidrug resistant tuberculosis), means that new antibacterial molecules and targets will need urgently to be identified to combat this crisis. [1] Due to serious antibiotic drug resistance issues, it is urgent for scientists to develop new molecules, new targets, and new treatments to overcome emerging healthy threats.
(þ)-Negamycin, an unusual antibiotic containing a hydrazido peptide linkage was originally isolated from Streptomyces purpeofuscus strains by the team of Umezawa in 1970. [2] This compound exhibits strong inhibitory activity against Gram-positive and Gram-negative bacteria including Pseudomonas aeruginosa and Klebsiella pneumonia. [3] Its potent antibacterial activity has made (þ)-negamycin an attractive synthetic target. Since the confirmation of negamycin structure by Shibahara and coworkers in 1972, [4] and the completion of the first total synthesis of (þ)-negamycin and its antipode, diverse approaches including racemic and asymmetric syntheses have been reported. [5] For instance, Davies [6 ] and Hayashi [7] independently used a chiral lithium amide to react with an oxazolidine containing a,b-unsaturated ester for the introduction of second nitrogen of (þ)-negamycin via asymmetric aza-Michael addition (Fig. 1). Both teams also used the similar strategy to complete the synthesis of (þ)-3-epi-negamycin or (-)-5-epi-negamycin. During our study [8] toward the synthesis of halogenated polyketides, the epoxide 1 was prepared and proved to be a good starting material to obtain the desired targets by choosing different Grignard reagents and benzoate formations. Compound 1 has the main carbon chain of acyl moiety of pseudopeptide negamycin (Fig. 1). Reported herein are our efforts through which the concise synthesis of the levorotatory isomer of (þ)-negamycine from the established epoxide 1 has been developed.

Results and discussion
To build the carboxyl group for the coupling with a hydrazinoacetate, CuI-catalyzed ring-opening [9] of the epoxide 1 with vinyl magnesium chloride furnished homoallylic alcohol 2 regioselectively in 88% yield (Scheme 1). The first (internal) nitrogen was designed to introduce by the treatment of DPPA/DBU or TMSN 3 /DIAD/Ph 3 P. However, the combination (Merck method) [10] stopped at the stage of a diphenyl phosphate (3). [11] Besides, although 2 was consumed under TMSN 3 Mitsunobu conditions, only moderate 60% yield of azide product 4 along with 24% of silyl ether 5 was obtained. Despite the compound 5 can be converted back to the starting secondary alcohol by the acidic workup and to repeat Mitsunobu reaction, but this is not an efficient method to get the desired azide 4.
To obtain satisfactory yields of 4, we decided to add sodium azide as an external azide source to convert diphenyl phosphate 3 to the corresponding azide 4. Initially attempted conversion 3 to 4 had problems under normal S N 2 conditions. And several modifications were attempted as summarized in Table 1.
The formation of 4 was heavily affected by the conditions of the reaction. The reaction was carried out with only sodium azide in DMF at ambient temperature resulting Scheme 1. The azidation of b-hydroxy carbonyl compounds. in no reaction (entry 1, Table 1). When the reaction was performed with large excess (6 eq.) of sodium azide at elevated temperature, two products were obtained, but in a poor yield (entry 2). To our surprise, azido alcohol 6 was obtained, which indicated that the silyl ether (TBDPS) would be deprotected under the reaction conditions. It indicated that the silyl ether would be deprotected under reaction conditions. Encouraged by this finding, the polar solvent was changed to DMSO with prolonged 72 h heating at 65 C, azido alcohol 6 was obtained as the major product (entry 3). When the temperature was raised to 100 C, the reaction time was shortened to 16 h and azido silyl ether 4 was obtained at 71% (entry 4). It should be noted that the selective cleavage of aryl silyl ethers in the presence of alkyl silyl ethers using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) is reported by Kim. [12] With the addition of DBU, azido silyl ether 4 was produced exclusively in moderate 70% yield suggesting DBU suppressed the formation of azido alcohol and no desilylated product was found (entry 5). Although the mechanism is unclear, it was found that the second azide source (DPPA) and DBU were acted as the additives, azido alcohol 6 was isolated in good 95% yield (entry 6). The polarity of solvent system was adjusted by the addition of toluene and the proportion of 4 was increased but the formation of 6 was still favorable (entry 7). However, only two azide additives without the use of DBU were employed, the result gave the sole azido alcohol in poor 50% yield (entry 8).
Completion of the synthesis is illustrated in Scheme 2. The second nitrogen was introduced through TMSN 3 Mitsunobu reaction to get the diazide 7. A OsO 4 -catalyzed oxidative cleavage [13] of the alkene 7 gave diazido acid 8 which was coupling with benzyl (1-methylhydrazino)acetate (9) [14] to obtain compound 10. Tanner/Somfai [15] and Kumar [16] had synthesize similar compounds of diazide 9 with different protecting groups on the hydroxy group. This presented work gave an efficient method to setup diazido moiety continuously at the late stage of the synthesis.
The final global deprotection was carried out via high pressure hydrogenolysis and purified via plate chromatography (silica gel eluting with i-PrOH:H 2 O 1/1 and 10% aqueous NH 4 OH) to furnish (-)-negamycin. All characterization data for this synthetic In the presence of DBU (1.5 eq.). b In the presence of DPPA (1.5 eq.) and DBU (1.5 eq.). c In the presence of DPPA (1.5 eq.). d Not found. enantiomer, except for the direction of optical rotation, matched those reported in the literature for (þ)-negomycin.

Conclusion
In summary, we have developed a concise synthetic method for (-)-negamycin. The method provides a 7-step route starting from chiral epoxy ether 1, where the chirality is generated by Krische enantioselective allylation. Homoallylic alcohol 2 is generated by Cu-mediated ring opening using vinyl Grignard reagents. The desired diazide 7 was provided from the silyloxyphosphate 3 via a series of unusual azidation, spontaneous desilylation, and TMSN 3 Mitsunobu reactions. Application of the given strategy to other natural products containing promising biological activities is ongoing.

General
Unless otherwise noted, all reagents and solvents were obtained commercially and used without further purification. 1 H and 13 C NMR spectra were recorded on a Jeol-400 MHz spectrometer. Chloroform-d (d ¼ 7.24) was used as an internal standard in 1 H NMR spectra. The center peak of deuterochloroform (d ¼ 77.0) was used as an internal standard in 13 C NMR spectra. High-resolution mass spectrometry (HRMS) analyses were determined on a Thermo Scientific Orbitrap LTQ XL mass spectrometer. Optical rotations were measured in CH 2 Cl 2 solution with a cuvette of 1 dm length on a Rudolph Autopol IV automatic polarimeter at k ¼ 589 nm (Na). IR spectra were recorded with a Thermo Scientific Nicolet iS5 FT-IR spectrophotometer and only structurally important peaks are listed. Melting points were measured on a melting point apparatus with a capillary melting point tube. Thin-layer chromatography (TLC) plates were visualized by exposure to ultraviolet light at 254 nm and/or immersion in a staining solution (phosphomolybdic acid, potassium Scheme 2. The introduction of second nitrogen: total synthesis of (-)-negamycin. permanganate, ninhydrin, or p-anisaldehyde) followed by heating on a hot plate. Flash chromatography was carried out utilizing silica gel 60, 70-230 mesh ASTM.

Preparation of compound 2
Under a nitrogen atmosphere a flame-dried round-bottom flask, fitted with a magnetic stirring bar and CuI (258 mg, 1.35 mmol, 0.2 eq.), is charged with vinyl magnesium chloride solution (1.6 M in THF, 11 mL, 16.9 mmol, 2.5 eq.). The resulting mixture was cooled to À5 C and stirred for 10 min. A solution of compound 1 [8] (3.022 g, 6.77 mmol) in dry THF (14 mL) solution was added to the above mixture via a syringe pump over a period of 1 h. The reaction was allowed to stir for another 2.5 h, quenched by the addition of saturated NH 4 Cl (aq) , and diluted with EtOAc (100 mL). The aqueous layer was extracted with EtOAc (2Â), dried over anhydrous MgSO 4 , filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography [silica gel, hexanes/ethyl acetate 20/1 (v/v)] to afford the title compound (2.827 g, 5.96 mmol, 88%) as a colorless oil. (3S,5S)-3,6-diazido-5-(benzyloxy)hexanoic acid (8) The olefin 7 (240 mg, 0.838 mmol) was dissolved in DMF (4 mL), and OsO 4 (1% in H 2 O, 450 mL, 0.0168 mmol, 0.02 eq) was added and the resulting mixture was stirred for 5 min. Oxone V R (2.07 g, 3.35 mmol, 4 eq.) was added in one portion and the reaction was stirred at room temperature for 3 h or until the solution became colorless. The reaction progress was monitored by TLC. Na 2 SO 3 (635 mg, 5.03 mmol, 6 eq.) was added to reduce the remaining Os(VIII), and the mixture was allowed to stir for an additional hour or until the solution became dark brown. EtOAc (10 mL) was added to extract the products and 1 N HCl (aq) was used to dissolve the salts. The organic extract was washed with 1 N HCl (aq) (3Â) and brine, dried over anhydrous MgSO 4 , and the solvent was removed under reduced pressure to obtain the crude product, which was purified by