A bioinspired, one-step total synthesis of peshawaraquinone

A concise synthesis of a stereochemically complex meroterpenoid, peshawaraquinone, via the unsymmetrical dimerization of its achiral precursor, dehydro-α-lapachone, is reported. Enabled by reversible oxa-6π-electrocyclizations of 2H-pyran intermediates, the base-catalyzed dimerization sets up an intramolecular (3 + 2) cycloaddition, with the formation of six stereocenters during the cascade. Combining the generation and in situ dimerization of dehydro-α-lapachone allows a one-step total synthesis of peshawaraquinone from lawsone and prenal.


General Methods
All chemicals were purchased from commercial suppliers and used as received. All organic extracts were dried over anhydrous sodium sulfate. Thin layer chromatography was performed using aluminium sheets coated with silica gel F254. Visualization was aided by viewing under a UV lamp and staining with p-anisaldehyde stain followed by heating. All Rf values were measured to the nearest 0.05. Flash column chromatography was performed using 40-63-micron grade silica gel. Infrared spectra were recorded using an FT-IR spectrometer as the neat compounds. High field NMR spectra were recorded using either a 500 MHz spectrometer ( 1 H at 500 MHz, 13 C at 126 MHz) or 600 MHz spectrometer ( 1 H at 600 MHz, 13 C at 151 MHz). The solvent used for NMR spectra was CDCl3 unless otherwise specified. 1 H chemical shifts are reported in ppm on the δ-scale relative to TMS (δ 0.0) or residual CHCl3 (δ 7.26) and 13 C NMR chemical shifts are reported in ppm relative to CDCl3 (δ 77. 16). Multiplicities are reported as (br) broad, (s) singlet, (d) doublet, (t) triplet, (q) quartet, (quin) quintet, (sext) sextet, (hept) heptet and (m) multiplet. All J-values were rounded to the nearest 0.1 Hz. ESI high resolution mass spectra were recorded on an Agilent 6230 TOF LC/MS mass spectrometer.

Synthesis of dehydro-a-lapachone:
Prepared according to a modified literature procedure. 1 To a suspension of lawsone (10.0 g, 57.4 mmol) in H2O (500 mL) was added prenal (11.1 mL, 115 mmol) at room temperature. This mixture was stirred at 80 °C for 6 h. Upon completion, the reaction mixture was cooled with ice and the solid, orange product was filtered and washed rigorously with ice-cold water. Following filtration, the orange solid was dried under vacuum for 24 h affording dehydro-a-lapachone (

Dimerization of dehydro-a-lapachone with DIPEA:
To a solution of dehydro-a-lapachone (2.50 g, 10.4 mmol) in PhMe (100 mL) was added N,Ndiisopropylethylamine (DIPEA) (1.82 mL, 10.4 mmol) and the reaction mixture was heated at 110 °C for 18 h. This mixture was cooled to room temperature and then directly purified by flash chromatography on silica gel with CH2Cl2 as the eluent (prior evaporation of the PhMe solvent is unnecessary) to give a 1:2.2 mixture of peshawaraquinone and 11'-epi-peshawaraquinone (1.34 g, 54%). Recovered dehydro-a-lapachone starting material was also obtained (290 mg, 12%). Analytical samples of 11'-epi-peshawaraquinone were obtained by further flash chromatography on silica gel (1:1 hexanes-CH2Cl2 to neat CH2Cl2, gradient elution) or by preparative TLC with neat CH2Cl2 as the eluent. Single crystals of 11'-epi-peshawaraquinone were obtained by crystallization from PhMe.

General Methods
To a reaction vial containing a solution of dehydro-a-lapachone (1.0 eq.) in solvent was added base. The reaction was heated and stirred for the times and temperatures listed in the appropriate results tables for each variable screened (vide infra). Individual reactions were monitored by TLC using either pure CH2Cl2 or petroleum spirit 60-80 / ethyl acetate (3:1) as eluents. Visualization was aided by viewing under a UV lamp and staining with p-anisaldehyde stain followed by heating. Reactions which did not display product spots were discarded. Successful reactions had their solvents removed under reduced pressure or by work-up (see Solvent Screening for more detail). To the resulting crude mixtures, a known mass (approx. 44 mg or approx. 1.0 eq.) of benzyl benzoate for use as an internal NMR standard was added. These mixtures were then dissolved in CDCl3 until homogenous after which, a small aliquot was submitted for 1 H NMR. Normalized integrals at H-12' (see NMR Assignments) were measured against the -CH2 singlet of benzyl benzoate (5.37 ppm) to calculate crude NMR yields.
Temperature Screening. To a reaction vial containing a solution of dehydro-a-lapachone (50 mg, 0.21 mmol, 1.0 eq.) in PhMe (2 mL) was added Et3N, DIPEA or DMAP (0.21 mmol, 1.0 eq.). The reactions were stirred at room temperature, 50, 70, 90 and 110 °C between 4-25 h. PhMe was removed under reduced pressure. Solvent Screening. To a reaction vial containing a solution of dehydro-a-lapachone (50 mg, 0.21 mmol, 1.0 eq) in solvent (2 mL) was added Et3N, DIPEA or DMAP (0.21 mmol, 1.0 eq.). The reactions were stirred at either 80 or 100 °C between 4-21 h. Reactions in DMF or DMSO were diluted with water and extracted with EtOAc (3 x 10 mL). The organic layers were washed with H2O (3 x 10 mL) and brine (1 x 10 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Reactions in H2O were extracted with EtOAc (3 x 10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. All other solvents tested were removed under reduced pressure on a rotary evaporator, increasing the temperature of the water bath for higher boiling point solvents e.g., xylenes, 1,4-dioxane.
Concentration Screening. To a reaction vial containing, dehydro-a-lapachone (1.0 eq.) was added PhMe (0.5-2 mL) to make up a solution of known concentration (0.1 M -2.0 M). To these solutions was added DIPEA or DMAP (1.0 eq.). The reactions were stirred at 110 °C for 4 h. PhMe was removed under reduced pressure.

Single Crystal X-ray Crystallography
A single crystal of 11'-epi-peshawaraquinone (obtained by slow evaporation of a solution in toluene) was mounted in Paratone-N oil on a MiTeGen micromount. X-ray diffraction data were collected at 100(2) K on a Rigaku-Oxford Diffraction Synergy single crystal diffractometer using Cu Ka radiation. 3 The data set was corrected for absorption using a multi-scan method, and the structure solved by intrinsic phasing (SHELXT) 4 and refined by full-matrix least squares on F2 by SHELXL, 5 interfaced through the programs X-Seed (version 4) 6 and Olex2.3. 7 All non-hydrogen atoms were refined anisotropically and hydrogen atoms were included as invariants at geometrically estimated positions. Disordered solvent (toluene) was modelled over two positions using EXYZ, EADP, SIMU and RIGU restraints. Table S1 lists the X-ray experimental data and refinement parameters for the crystal structures. Perspective views of the structure of 11'-epi-peshawaraquinone are shown in Figure S1.   Figure S1. Perspective views of (a) the partially labelled asymmetric unit, without the toluene solvate molecule, with the ellipsoids shown at 50% probability level, and (b) the interdigitated hydrogen bonded dimers, shown as rods, which are directed along the crystallographic b axis, in the structure of 11'-epi-peshawaraquinone. Carbon -grey, hydrogen -white, oxygen -red. The intermolecular hydrogen bonding parameters are: DO1-H1···O1AA = 2.87 Å, angleO1-H1···O1AA = 168.7° and the centroidcentroid distance from the offset stacked phenyl and phenyl rings (central interaction) and quinone and phenyl rings (outer interactions) between two dimers are 3.73 and 3.96 Å respectively.

Computational Methods
Molecular geometries and energies were calculated by density function theory (DFT) using the ORCA quantum chemistry software package (version 5.0.3.). 8 Geometries were optimized in the gas phase using the ωB97X-D3 functional 9 and def2-SVP basis set. 10 Optimized molecular geometries are given below. Single-point energy calculations (EωB97X-D3/def2-TZVPD,toluene) of the optimized geometries were also carried out using the same functional with the def2-TZVPD basis set 11 and the SMD continuum solvent model 12 with toluene as the solvent. The experimental X-ray crystal structure of 11'-epi-peshawaraquinone was used as the starting point for the geometry optimization of this molecule, while the same X-ray structure with stereochemistry inverted at the appropriate position was used as the starting point for the geometry optimization of peshawaraquinone. The global minimum-energy conformer from a conformer search with a metadynamics-based algorithm 13 and semiempirical tight binding at the GFN2-xTB level, 14 conducted using xtb 15 and the Conformer-Rotamer Ensemble Sampling Tool (CREST) 16 with default parameters, was used as a starting point for the DFT geometry optimization of structures dehydro-a-lapachone (0), 1, 3, and 3'. The transition state connecting each pair of stable states was obtained using the NEB-TS method, 17 which combines the climbing image nudged elastic band (CI-NEB) method with an eigenvector following optimization starting from the climbing image, with the default parameters in ORCA. Stable states and transition states were identified by the number of imaginary vibrational frequencies (0 and 1, respectively) obtained from a harmonic frequency calculation at the ωB97X-D3/def2-SVP level. The thermal correction to the Gibbs free energy (Gthermal,ωB97X-D3/def2-SVP) was calculated at 298.15 K and 1 atm using unscaled harmonic frequencies. The total Gibbs free energy in toluene solution was calculated as Gsoln = EωB97X-D3/def2-TZVPD,toluene + Gthermal,ωB97X-D3/def2-SVP.
A summary of the calculated energies and free energies is given in Table S2, and reaction free-energy profiles for the conversion of dehydro-a-lapachone (0) to 1, 3 to 5, and 3' to 5' are shown in Figures  S2, S3, and S4, respectively. The minimum-energy pathway from the global-minimum energy conformer of 3 to 5 was found to involve interconversion of several different conformers of 3, with the most direct pathway being much higher in energy. Similarly, the minimum-energy pathway from the global-minimum energy conformer of 3' to 5', which consists of an analogous series of steps, was found to involve interconversion of several different conformers of 3'. Scheme S1. Summary of the reactions modelled.  a To enable a direct comparison of the free energies of peshawaraquinone and 11'-epi-peshawaraquinone with those of the structures in the table from which they are formed, the energy or free energy of a H + ion has been subtracted from each table entry for these compounds. The contribution of H + to the electronic energy or solvation free energy is zero, while its contribution to the thermal Gibbs free energy correction and total Gibbs free energy is its translational free energy, which for an ideal gas or solution is -0.009998 a.u. = -26.25 kJ/mol at 1 atm or -18.32 kJ/mol at 1 mol/L. Note that the specified pressure or concentration does not affect the relative free energies of any of the other entries in the table, which only concern single species.
Reaction free-energy profiles Figure S2. Calculated reaction free-energy profile for conversion of dehydro-a-lapachone (0) to 1 (s-trans) in toluene at 298.15 K. Figure S3. Calculated reaction free-energy profile for conversion of 3a to 5 (peshawaraquinone anion) in toluene at 298.15 K. In the simplified reaction free-energy profile in the main paper, only the lowest free-energy conformer of 3 (3b) and the highest free-energy transition state between 3 and 4 (TS 3e → 4) are shown. (TS 3d → 3e has a slightly lower free energy than 3e, which is unphysical, but it should be noted that it has a higher energy at the level of theory used for geometry optimization.) Figure S4. Calculated reaction free-energy profile for conversion of 3a' to 5' (11'-epipeshawaraquinone anion) in toluene at 298.15 K. (TS 3d' → 3e' has a slightly lower free energy than 3e', which is unphysical, but it should be noted that it has a higher energy at the level of theory used for geometry optimization.)