Microfluidic light-driven synthesis of tetracyclic molecular architectures

Herein we report an effective synthetic method for the direct assembly of highly functionalized tetracyclic pharmacophoric cores. Coumarins and chromones undergo diastereoselective [4 + 2] cycloaddition reactions with light-generated photoenol intermediates. The reactions occur by aid of a microfluidic photoreactor (MFP) in high yield (up to >98%) and virtually complete diastereocontrol (>20:1 dr). The method is easily scaled-up to a parallel setup, furnishing 948 mg of product over a 14 h reaction time. Finally, a series of manipulations of the tetracyclic scaffold obtained gave access to valuable precursors of biologically active molecules.

High resolution mass spectra (HRMS) were obtained using a Waters GCT gas chromatograph coupled with a time-of-flight mass spectrometer (GC/MS-TOF) with electron ionization (EI) or AB SCIEX MALDI-TOF 4800 Plus mass spectrometer (MALDI-TOF).
Chromatographic purification of products was accomplished using flash chromatography on silica gel (SiO 2 , 0.04-0.063 mm) purchased from Machery-Nagel, with the indicated solvent system according to the standard techniques. Thin layer chromatography (TLC) analysis was performed on precoated Merck TLC plates (silica gel 60 GF254, 0.25 mm). Visualization of the developed chromatography was performed by checking UV absorbance (254 nm) as well as with aqueous ceric ammonium molybdate and potassium permanganate solutions. Organic solutions were concentrated under reduced pressure on a Büchi rotary evaporator.

The authors are grateful to the research support area at Department of Chemical Sciences (DiSC) of the University of Padova.
Determination of diastereomeric ratio: The diastereomeric ratio of products 4a-h, 5a-f, 6a, 7a, 8a and 9a was determined by 1 H NMR analysis of the crude reaction mixture through integration of diagnostic signals.

Materials:
Commercial grade reagents and solvents were purchased at the highest commercial quality from Sigma Aldrich and used as received, unless otherwise stated. 2methylbenzophenone (1a), (2,4-dimethylphenyl)(phenyl)methanone (1b), coumarin (2a), 6-methylcoumarin (2b), 7-methylcoumarin (2c), 7-methoxycoumarin (2d), as well as chromone (3a) were purchased from Sigma Aldrich and used as received. 2-MBP 1c-g 1 as well as 2H-chromene-2-thione (2e) 2 were prepared according to literature procedures.  Figure S1 (below) shows a schematic representation of the microfluidic reactor employed in the present study for the photoreactions between 2-MBP 1 and coumarins 2a-e or chromone (3a). In this setup, the solution containing 1a-g (0.06 M in toluene) and the acceptor 2a-e or 3a (1.5 M in toluene) was firstly degassed by bubbling nitrogen for 15 min. Subsequently, the solution under a nitrogen atmosphere was introduced in continuous flow into the microphotoreactor via a double syringe pump (Syrris Atlas, see general information). The microfluidic reactor consists of a transparent TFE capillary (BGB®; internal diameter: 750 µm; inner volume: 1000 µL; microreactor tubing length: 226.5 cm); a 9 W 365 nm bulb lamp ( Figure S2) equipped with a transparent glass-blown adaptor ( Figure S3). Other microreactors used in this study consisted of 12 × 3 365 nm single LEDs ( Figure S4). In both cases, aluminium foil was used to avoid undesired irradiation of the tubing. To maintain a stable reaction temperature, two fans were placed in close proximity to the reactor and the temperature was controlled by a thermometer (25 ± 2 °C). A 0.1 mL aliquot of the product solution was introduced into an NMR tube, diluted with CDCl 3 and analysed by 1 H NMR in order to calculate the reaction conversion at different flow rates for each reaction. To determine the NMR yields, a 0.1 mL aliquot of the same solution was introduced into an NMR tube, using trimethoxybenzene as internal standard.

S8
The photoreactor shown in Figure S3 consisted of a transparent glass-blown adaptor and a FEP tubing reactor wrapped around the support.  The photoreactor shown in Figure S5 comprised a 3D-printed PLA support holding a ring of 12 LEDs pointing toward a FEP tubing reactor wrapped around a cylindrical support. The distance between the LEDs and the FEP tubing is 2 mm.  Figure S6 (below) shows the general setup for the batch reactions. Two reaction vials containing the same degassed solution under N 2 atmosphere were placed in front of the 9 W 365 nm bulb (approximatively 1.5 cm distance). The reactions were stirred vigorously until full conversion was detected by 1 H NMR analysis of the crude reaction mixture. To maintain a stable reaction temperature two fans were placed in close proximity to the reaction vials (25 ± 2 °C) and the temperature was controlled by a thermometer. A 0.1 mL aliquot of the product solution was introduced into an NMR tube, diluted with CDCl 3 and analysed by 1 H NMR in order to calculate the reaction conversion at different times for each reaction. To determine the NMR yields, a 0.1 mL aliquot of the same solution was introduced into an NMR tube, using trimethoxybenzene as internal standard. reaction vials fans S10

D. LIGHT SOURCES AND EMISSION SPECTRA
The following spectra were recorded by an AvaSpec ULS3648 high-resolution fiber-optic spectrometer which was placed at a fixed distance of 0.5 cm from the light source.

GENERAL PROCEDURE FOR THE CONTINUOUS-FLOW REACTIONS
Coumarin (2a, 219.2 mg, 5 equiv, 1.5 mmol) was introduced into a 12 mL vial under nitrogen atmosphere and dissolved in 5 mL of degassed toluene. Then, 2methylbenzophenone (1a, 55 µL, 1 equiv, 0.3 mmol) was added in one portion and the solution was further bubbled with nitrogen for 5 min. The resultant solution was pumped into the MFP and irradiated by a 9 W 365 nm bulb with a residence time of 35 min. The product solution was collected into a 7 mL vial. Subsequently, the solvent was removed by rotary evaporation and the crude subjected to flash column chromatography on silica gel (9:1 hexane/EtOAc) yielding pure 4a (white solid), as a single diastereoisomer in >98% yield (100.1 mg, 0.293 mmol). S12

F. REACTION OPTIMISATION
For the light-driven microfluidic [4 + 2] cycloaddition reaction between 2-MBP 1a and coumarin (2a) investigated in the present work, different reaction conditions were tested in function of the light source and power, solvent, reagent concentration, flow rate, residence time and reactor volume. The reactions performed in batch were irradiated by a 9 W 365 nm bulb as reported in Figure S5, section C2 of this file.

G. ABSORPTION SPECTRA ANALYSIS
All the absorption spectra were recorded using an Agilent Cary 100 UV-Vis spectrophotometer. The spectra were recorded in toluene using the same concentrations as in the reaction conditions. Due to the high concentration of the solutions, short light path cuvettes (1 mm Hellma Quartz SUPRASIL®) were employed to avoid fast signal saturation.   Synthesised following the continuous-flow procedure 1 described in section E using 1 equivalent of 1a (55 µL, 0.3 mmol) and 5 equivalents of coumarin (2a, 219.2 mg, 1.5 mmol) in 5 mL of toluene with a residence time of 35 min. In order to calculate the isolated yield, 4 mL of the product solution of 4a were collected and subjected to flash column chromatography on silica gel (9:1 Hexane/EtOAc) yielding pure 4a (white solid), in >98% yield (82.0 mg, 0.24 mmol).
The obtained data matched with the previously reported in reference 3. Synthesised following the continuous-flow procedure 1 described in section E using 1 equivalent of 1b (63.1 mg, 0.3 mmol) and 5 equivalents of coumarin (2a, 219.2 mg, 1.5 mmol) in 5 mL of toluene with a residence time of 35 min. In order to calculate the isolated yield, 4 mL of the product solution of 4b were collected and subjected to flash column chromatography on silica gel (9:1 Hexane/EtOAc) yielding pure 4b (white solid), in 83% yield, (70.9 mg, 0.199 mmol).

11-
Synthesised following the continuous-flow procedure 1 described in section E using 1 equivalent of 1c (63.1 mg, 0.3 mmol) and 5 equivalents of chromone (3a, 219.2 mg, 1.5 mmol) in 5 mL of toluene with a residence time of 35 min. In order to calculate the isolated yield, 4 mL of the product solution of 5c were collected and subjected to flash column chromatography on silica gel (9:1 Hexane/EtOAc) yielding pure 5c (white solid), in 61% yield (52.2 mg, 0.146 mmol).

11-(3-
Synthesised following the continuous-flow procedure 1 described in section E using 1 equivalent of 1d (64.3 mg, 0.3 mmol) and 5 equivalents of chromone (3a, 219.2 mg, 1.5 mmol) in 5 mL of toluene with a residence time of 35 min. In order to calculate the isolated yield, 4 mL of the product solution of 5f were collected and subjected to flash column chromatography on silica gel (9:1 Hexane/EtOAc) yielding pure 5f (white solid), in 59% yield (51.0 mg, 0.142 mmol).

. GENERAL PROCEDURE FOR THE FORMATION OF 6A
Chromenone 4a (200 mg, 1 equiv, 0.60 mmol) was dissolved in H 2 O (3 mL). To the resulting heterogenous solution, NaOH (120 mg, 5 equiv, 3 mmol) was added portionwise under vigorous stirring at room temperature. The solution was then heated to reflux. After 3 h, full conversion was observed by TLC analysis. The reaction mixture was cooled to room temperature and extracted with 3 × 10 mL of DCM. The combined organic phases were dried over MgSO 4 and the solvent was removed by rotary evaporation. Concentration under high vacuum yielded 2-(4-phenyldihydronaphthalenyl)phenol (6a) as white solid in 95% yield (170 mg, 0.576 mmol).

. GENERAL PROCEDURE FOR THE FORMATION OF 7A
Chromenone 4a (200 mg, 1 equiv, 0.60 mmol) was dissolved in dry toluene (6 mL) under an inert atmosphere. To the resulting solution, triflic acid (160 µL, 3 equiv, 1.8 mmol) was added dropwise under vigorous stirring at 0 °C. The solution was stirred for 1 h at 0 °C and allowed to gradually warm to room temperature. After 3 h, full conversion was observed by TLC analysis and the reaction was quenched with NaHCO 3 . The mixture was extracted with 3 × 10 mL of DCM, the combined organic phases were dried over MgSO 4 and the solvent was removed by rotary evaporation. Concentration under high vacuum yielded 7phenyldihydronaphtho[2,3-c]chromenone (7a) as white solid in 95% yield (170 mg, 0.572 mmol).

J. X-RAY STRUCTURE DETERMINATION AND REFINEMENT FOR COMPOUNDS 4A AND 5A
Colourless, block-shaped single crystals of compounds 4a and 5a were easily obtained by slow evaporation of Et 2 O/hexane solutions of the respective synthetic diasteroisomeric mixtures (see Scheme S1). X-ray quality crystals with approximate dimensions 0.40 × 0.30 × 0.20 mm 3 (4a) and 0.40 × 0.55 × 0.55 mm 4 (5a) were chosen under the microscope for the measurement. Diffraction data were collected at room temperature (296 K) with an Oxford Diffraction Gemini E diffractometer, equipped with a 2 K × 2 K EOS CCD area detector and sealed-tube Enhance (Mo) and (Cu) X-ray sources. MoKa (λ = 0.71073 Å) radiation was used for 4a, while 5a was investigated using CuKa (λ = 1.54184 Å). Data collection, reduction and finalisation were carried out through the CrysAlisPro software.
The structures were solved by direct methods and subsequently completed by Fourier recycling using the SHELXTL-2013 software package 4 and refined by the full-matrix leastsquares refinements based on F 2 with all observed reflections. All non-hydrogen atoms were refined anisotropically; hydrogen atoms were included at geometrically calculated positions and refined using a riding model. Scheme S1. Molecular structure of compounds 4a and 5a with the atom numbering used in this section (only selected atoms). According to this numbering, the synthetic procedures led to diasteroisomers 3RS,4RS,11SR-4a and 2RS,3RS,11SR-5a.
Compound 4a is racemic, as expected, and it crystallizes in the centrosymmetric orthorhombic space group Pbca, with eight molecules in the unit cell. Figure S14 shows S31 the content of the asymmetric unit as the 3R,4R,11S enantiomer (arbitrary choice). In the case of compound 5a, instead, the crystallization process also afforded chiral resolution, as the analyzed crystal of 5a turned out to comprise the pure 2S,3S,11R enantiomer (see Figure S15). This crystallised in the orthorhombic chiral space group P2 1 2 1 2 1 , with four molecules in the unit cell. The final geometrical calculations and graphical manipulations were performed by using the XP utility within SHELX. 4 A summary of the crystallographic data and structure refinement for 4a and 5a is given in Table S5. Crystallographic data have been deposited at the Cambridge Crystallographic Data Centre (CCDC reference numbers 1837120 (4a) and 1851516 (5a)). These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures/.