Stereochemical Assignment of the Protein–Protein Interaction Inhibitor JBIR-22 by Total Synthesis

Recent reports have highlighted the biological activity associated with a subfamily of the tetramic acid class of natural products. Despite the fact that members of this subfamily act as protein–protein interaction inhibitors that are of relevance to proteasome assembly, no synthetic work has been reported. This may be due to the fact that this subfamily contains an unnatural 4,4-disubstitued glutamic acid, the synthesis of which provides a key challenge. A highly stereoselective route to a masked form of this unnatural amino acid now enabled the synthesis of two of the possible diastereomers of JBIR-22 and allowed the assignment of its relative and absolute stereochemistry.


Additional experimental information 1.Proposed formation of 13
Scheme S1. Lactone 13 likely forms via an in situ aldol reaction of initially formed imine 7 and remaining ethyl pyruvate (8) followed by an intramolecular cyclisation to provide 13. The relative and absolute configuration of 13 is proposed based on X-ray crystallographic analysis of the related compound 12. The observed diastereoselectivity in the formation of 12 and 13 can be rationalized using a similar Zimmerman-Traxler transition state model to that proposed by Ellman for the condensation of N-sulfinyl imines to simple aldehydes. [1,2] Table S1. A selection of the key results obtained from a screen of reaction conditions for the condensation of ethyl pyruvate (8) and (R S )-tert-butanesulfinamide. Reagents and conditions: (a) (R S )-tert-butanesulfinamide, THF. Ratio of 7:13 was determined by 1  [a] (R S )-tert-butanesulfinamide and Ti(OEt) 4 were heated to 65 °C in THF prior to addition of ethyl pyruvate (8) in one portion.

NOE analysis of 12
Figure S1. NOE analysis of 12.

Mechanistic investigation into the tandem deprotection-reduction of 15 to give 12.
Scheme S2. Mechanistic studies were carried out to elucidate the reaction sequence in the tandem deprotection-reduction. The experimental procedure involved the addition of HCl (4N in dioxane) to 15 in THF at 0 C followed by stirring for 10 minutes. Work-up of the reaction prior to the addition of the reducing agent and analysis of the crude reaction mixture by 1 H NMR (data not shown) revealed that the deprotected enamine S1 was formed. Subsequent NaBH 3 CN-mediated reduction of S1 in the presence of HCl (4N in dioxane) provided the desired product 12 with similar diastereoselectivity but diminished yield when compared to the one-pot process. As the chiral auxiliary was cleaved prior to the addition of the reducing agent, the observed diastereoselectivity appears to be purely substrate controlled with the hydride attacking from the same side as the ester substituent. The high level of diastereocontrol would suggest that it may not be purely sterically induced and could be a result of coordination of the reducing agent by the ester, directing the attack of the hydride.

Intramolecular Claisen-like reaction of 3-oxo-AHLs to form tetramic acids
Scheme S3. Previously reported conversion of 3-oxo-AHLs to the corresponding tetramic acids via an intramolecular Claisen-like reaction. [3][4][5] 1.6 NOE analysis of 17 (1) In CDCl 3 at room temperature the 17 exists as a (3 : 1) keto : enol mixture. NOE analysis of the major keto tautomer is shown. (2)   Trienal 23 was subjected to an organocatalytic intramolecular Diels-Alder (IMDA) reaction using MacMillan's conditions. [6] The moderate diastereoselectivity observed in the formation of 24a and 24b is in agreement with the findings of Christmann et al. [7] for the same transformation. This reduction in diastereoselectivity may be due to epimerisation of the C2 position which was also observed by MacMillan for similar substrates. [6] Throughout subsequent steps in the synthesis, the amounts of products resulting from the minor diastereomer reduced (on purification) although it was not possible to remove these minor products completely until the final step. The aldehydes 24a and 24b were converted to the corresponding alcohols S2a and S2b via NaBH 4 mediated reduction for determination of their enantiomeric purity. A racemic standard was obtained by a BF 3 .OEt 2 catalysed cycloaddition of 23 to provide (±)-24 as a single diastereomer, which was subsequently reduced to the corresponding alcohol (±)-S2. Enantiomeric excesses were obtained by chiral GC analysis using an Agilent Cyclosil-B (isotherm, 140 C, see below). The determined enantiomeric purity of 24a (87% ee) and 24b (84% ee) are in agreement with the reported values for this reaction (80-90% ee). [6][7][8] The enantiomeric purity could be improved if required by recrystallization at the alcohol oxidation state and subsequent reoxidation. [9] The minor diastereomers of S2a and S2b can be observed with retention times at approximately 21-22 minutes. (±)-S2

General information
All chemicals and solvents were purchased from Aldrich (UK), Alfa Aesar, or Acros Organics and used without further purification. All reactions involving moisture sensitive reagents were performed in oven or flame dried glassware under a positive pressure of nitrogen. Tetrahydrofuran (THF), dichloromethane (DCM) and hexanes were obtained dry from a solvent purification system (MBraun, SPS-800). Anhydrous N,N-dimethylformamide (DMF) was purchased from Aldrich. Thin layer chromatography (TLC) analysis was performed using glass plates coated with silica gel (with fluorescent indicator UV 254 ). Developed plates were air dried and analysed under a UV lamp Transform infra-red spectra (FT IR) were acquired on a Perkin Elmer paragon 1000 FT spectrophometer (KBr disc) or a Shimadzu IRAffinity-1 FT spectrophotometer with a Pike MIRacle TM (solid or thin film). Absorption maxima are reported in wavenumbers (cm -1 ). Nuclear magnetic resonance (NMR) spectra were recorded at room temperature on Bruker Avance 500 ( 1 H, 499.9 MHz; 13 C, 125.7 MHz), Bruker Avance 400 ( 1 H, 400.1 MHz; 13 C, 100.6 MHz) and Bruker Avance 300 ( 1 H, 300.1 MHz; 13 C, 75.5 MHz) instruments. NMR spectra were recorded in deuterated solvents and internally referenced to the residual solvent peak, chloroform-d (δ C 77.16, δ H 7.26 ppm), acetone-d 6 (δ C 29.8, δ H 2.04 ppm), DMSO-d 6 (δ C 39.52, δ H 2.50 ppm) or methanol-d 4 (δ C 49.0, δ H 3.31 ppm).
Chemical shifts are expressed as δ in units of ppm. 13 C NMR spectra were recorded using the PENDANT sequence mode. Data processing was carried out using the MestReNova 8.1.1 NMR program (Mestrelab Research S.L.). For 1 H NMR, the multiplicity used for assignment is indicated by the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, h = sextet, hept = heptet, m = multiplet, br = broad. Signals of protons and carbons were assigned, as far as possible, by using the following two dimensional NMR spectroscopy techniques: . The concentration is given in g/100 mL. The UPLC-TOFMS data was obtained using a Waters Xevo G2 Q-Tof mass spectrometer with Aquity TM UPLC system 6 Experimental procedures Ethyl (R,E)-2-((tert-butylsulfinyl)imino)propanoate (42) [11] Ethyl pyruvate 8 (4.3 g, 37.1 mmol, 1.5 eq.) was added to a stirred solution of (R)-(+)-2-methyl-2propanesulfinamide (3.0 g, 24.8 mmol, 1.0 eq.) and Ti(OEt) 4 (8.5 g, 37.1 mmol, 1.5 eq.) in THF (100 mL) at 65 °C. The reaction was stirred at the same temperature for 4 hours before cooling to room temperature. The crude reaction mixture was poured into brine (100 mL) whilst being vigorously stirred. The resulting suspension was filtered through Celite, and the filter cake was washed with EtOAc (2  30 mL). The filtrate was washed with brine (30 mL), and the brine layer was back extracted with EtOAc (3  30 mL). The combined organic extracts were dried over MgSO 4 , filtered, concentrated in vacuo and purified via the Biotage SP4 (silica-packed SNAP column 180 g; 10-50% EtOAc/hexanes) to give the title product 7 as a pale yellow oil (2.5 g, 45%) and the enamine tautomer as a pale yellow oil (0.8 g, 15%). 7 was isolated in a 3:1 ratio (by analysis of the crude NMR spectrum) to lactone 13 (1.1 g). The NMR analysis of the imine tautomer of 7 was complicated by the existence of rotamers. 1 H NMR (500 MHz, -125.8). [11]

'S,7'S) -JBIR-22 (2a)
To a solution of 26a (226 mg, 0.50 mmol, 1.0 eq.) in THF (10 mL) at 0 C was added t BuOK (62 mg, 0.56 mmol, 1.1 eq.) and the reaction was stirred for 1 hour. The reaction was slowly warmed to room temperature and stirred at this temperature for 1 hour. The reaction was concentrated in vacuo and the residue partitioned between DCM (10 mL) and an aqueous solution of HCl (10 mL, 1N). The aqueous layer was separated and extracted with DCM (3  10 mL). The organic extracts were combined, washed with an aqueous solution of HCl (5 mL, 1 N), brine (5 mL), dried over Na 2 SO 4 , filtered and concentrated in vacuo to give JBIR-22 ethyl ester S3a as a colourless oil (226 mg, 100%), which was used in the next step without further purification. To a solution of S3a (215 mg, 0.48 mmol, 1.0 eq.) in EtOH (4 mL) was added an aqueous solution of NaOH ( To a solution of 26b (121 mg, 0.27 mmol, 1.0 eq.) in THF (5 mL) at 0 C was added t BuOK (34 mg, 0.30 mmol, 1.1 eq.) and the reaction was stirred for 1 hour. The reaction was slowly warmed to room temperature and stirred at this temperature for 1 hour. The reaction was concentrated and the residue partitioned between DCM (5 mL) and an aqueous solution of HCl (5 mL, 1N). The aqueous layer was separated and extracted with DCM (3  10 mL). The organic extracts were combined, washed with HCl (5 mL, 1 N), brine (5 mL), dried over Na 2 SO 4 , filtered and concentrated in vacuo to give JBIR-22 ethyl ester S3b as a colourless oil (121 mg, 100%), which was used in the next step without further purification. To a solution of S3b (110 mg, 0.25 mmol, 1.0 eq.) in EtOH (3 mL) was added an aqueous solution of NaOH (2 mL, 2N) and the reaction was heated to 110 C under