Design, Synthesis, and Phenotypic Profiling of Pyrano‐Furo‐Pyridone Pseudo Natural Products

Abstract Natural products (NPs) inspire the design and synthesis of novel biologically relevant chemical matter, for instance through biology‐oriented synthesis (BIOS). However, BIOS is limited by the partial coverage of NP‐like chemical space by the guiding NPs. The design and synthesis of “pseudo NPs” overcomes these limitations by combining NP‐inspired strategies with fragment‐based compound design through de novo combination of NP‐derived fragments to unprecedented compound classes not accessible through biosynthesis. We describe the development and biological evaluation of pyrano‐furo‐pyridone (PFP) pseudo NPs, which combine pyridone‐ and dihydropyran NP fragments in three isomeric arrangements. Cheminformatic analysis indicates that the PFPs reside in an area of NP‐like chemical space not covered by existing NPs but rather by drugs and related compounds. Phenotypic profiling in a target‐agnostic “cell painting” assay revealed that PFPs induce formation of reactive oxygen species and are structurally novel inhibitors of mitochondrial complex I.


General
All reactions were performed in oven dried glassware and under inert Argon atmosphere if not indicated differently. Dry solvents were purchased from Fischer Scientific and/or Acros and used without further treatment. Oxygen and/or moisture sensitive solutions were transferred using syringes and cannulas.
Microwave reactions were carried out in a CEM Discover SP Activent machine.
Batch photoreactor consisted of a 250 mL or 500 mL schlenck flask, magnetic stirrer and two 34W blue LEDs (Kessil H150-Blue LED Lamp). The schlenck flask was placed in a dewar vessel of appropriate size. The dewar vessel was filled with iso-propanol and a constant flow of compressed air over the iso-propanol surface was adjusted. This cooling system maintained a bath temperature between 23-26 °C while irradiating the schlenck flask with two blue LEDs in a 45 °C angle. [2]
The mixture was allowed to warm to room temperature and kept stirring for 2 days.
The reaction was quenched by addition of saturated NaHCO₃ solution (50 mL) and the layers were separated. The aqueous layer was extracted with EtOAc (3 x 50 mL).
Scheme S7. Synthesis of derivatives of general scaffold B by Tsuji-Trost oxa-Michael cascade employing various pyridones 5 and dihydropyranones 8a-c.
[a] Allyl-Pd(II)-chloride dimer + Xanthphos was used as a catalyst. For compound numbering the first digit indicates the general scaffold according to Scheme 1 in the main text, the first letter indicates a specific sub-scaffold (blue) and the second letter indicates consecutive derivatives (red). Scheme S8. Synthesis of Tsuji-Trost oxa-Michael cascade products employing various pyridones 5 and dihydropyranone 8d. 1) Yield for the Tsuji-Trost oxa-Michael cascade step. 2) Yield after the Boc-deprotection over two steps. For compound numbering the first digit indicates the general scaffold according to Scheme 1 in the main text, the first letter indicates a specific sub-scaffold (blue) and the second letter indicates consecutive derivatives (red). Scheme S9. Isolated monopodal connected side products after treatment with HCl of Tsuji-Trost oxa-Michael products. Yields are given over two steps.
[a] Products were isolated as TFA salts when purified by prep. HPLC. Scheme S10. Synthesis of derivatives of general scaffold C by Michael-transacetalization cascade employing various pyridones 5 and dihydropyranones 8a-c. For compound numbering the first digit indicates the general scaffold according to Scheme 1 in the main text, the first letter indicates a specific sub-scaffold (blue) and the second letter indicates consecutive derivatives (red). Scheme S11. Synthesis of Michael-transacetalization cascade products employing various pyridones 5 and dihydropayranone 8d. 1) Yield for the Michael-transacetalization cascade step. 2) Yield after the Boc-deprotection over two steps. [a] Isolated as TFA-salt after purification with prep. HPLC. For compound numbering the first digit indicates the general scaffold according to Scheme 1 in the main text, the first letter indicates a specific subscaffold (blue) and the second letter indicates consecutive derivatives (red).
[a] Pd-tetrakis was used as a catalyst. For compound numbering the first digit indicates the general scaffold according to Scheme 1 in the main text, the first letter indicates indicates consecutive derivatives (red).

General Procedures
General procedure 2 (GP2): An oven-dried microwave vial was loaded with 5 mol% superstable Pd(0) catalyst [54] (Pd[P [3,2C6H3]3]3) and the bis-electrophile under Argon atmosphere. THF (0. 2 M) was added and the vial was sealed. After stirring for 10 minutes the bisnucleophile was added (for pyridones: DMF was added subsequently to afford a 3:1 mixture of THF/DMF with a final concentration of 0. 1 M). The sealed vial was then subjected for microwave irradiation (200 W, 100 to 110 °C, 30 to 60 minutes). The reaction mixture was concentrated in vacuo, immobilized on isolute and purified by FC or MPLC.
General procedure 3 (GP3): [49] An oven-dried schlenck-tube was filled with Argon, charged with 10 mol% Pd-tetrakis, evacuated and then back-filled with Argon. The bis-electrophile was dissolved in toluene (0. 1 M) in a separate vessel under Argon atmosphere and then added to the Pd-catalyst and allowed to stir for 20 min before the bis-nucleophile was added (for pyridones: DMF was added subsequently to afford a 3:1 mixture of toluene/DMF with a final concentration of 0.05 M). After stirring for 3-6 h at room temperature, additional 10 mol% Pd-catalyst were added to the reaction mixture. The mixture was then allowed to stir at room temperature overnight, filtered through celite and concentrated in vacuo. The crude was immobilized on isolute and purified by FC or MPLC.
General procedure 4 (GP4): The glycal substrate was dissolved in DMF (0. 1 M) and Pd(OAc)2 together with boronic acid were added. The mixture was stirred overnight, filtered through a short pad of silica and concentrated under reduced pressure. The crude was purified by MPLC.
General procedure 5 (GP5): The glycal substrate was dissolved in acetonitrile/H2O (1:1, 0.075 M) and NBS (1.5 eq.) was added at room temperature. The mixture was stirred overnight before being quenched by the addition of saturated NaHCO3 and diluted with EtOAc. The layers were separated and the aqueous phase was extracted with EtOAc three times. The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. The crude was immobilized on isolute and purified by FC or MPLC. The hydrobromination product was dissolved in MeOH (0. 1 M) and cooled to 0 °C. NaBH4 (1.2 eq) was added and the reaction mixture was stirred at 0 °C for 30 minutes. The reaction was quenched by addition of acetone (1 mL) and the solvents were removed in vacuo. The crude was immobilized on isolute and purified by MPLC or prep. HPLC.
General procedure 6 (GP6): [112] An oven-dried schlenck-tube was filled with Argon, charged with 5 mol% Pd-tetrakis, evacuated and then back-filled with Argon. The bis-electrophile was dissolved in THF (0. 15 M) in a separate vessel under Argon atmosphere and then added to the Pd-catalyst and allowed to stir for 20 min before the bis-nucleophile was added as a solution in DMF (0. 4 M) and triethylamine (1 equiv). The mixture was then allowed to stir at room temperature overnight, before being quenched by addition of saturated NaHCO₃ solution. The mixture was diluted with EtOAc, the phases were separated, and the aqueous phase was extracted with EtOAc three times. The combined organic layers were washed with brine, dried over MgSO₄ and concentrated in vacuo. The crude was immobilized on isolute and purified by FC or MPLC. General procedure 7 (GP7): [112] To a stirred solution of the bis-electrophile in dry DCM (0.075 M) was added the bis-nucleophile and quinine (1 equiv). After stirring at 60 °C in a sealed vial for 18 h, the solvent was removed under reduced pressure and the crude was purified by MPLC.
A mixture of TMSOTf (0.21 mL, 10 equiv) and 2,6-lutidine (0.20 mL, 15 equiv) in DCM (0.1 mL) was added slowly and the mixture was stirred at 0 °C for 30 min. The reaction was quenched by slow addition of saturated Na₂CO₃ solution at 0 °C and dilution with EtOAc (10 mL). The layers were separated and the aqueous phase was extracted with EtOAc (3 x 10 mL).

HRMS-ESI
The crude was purified by MPLC (EtOAc/MeOH 1:0 to 9:1 + 0.1% DIPEA) to afford the the following only combinations for which natural product examples in the DNP were found are listed in Figure S1. Altogether 121 natural products containing examples for combinations between N-methyl-2-pyridone with DHPs and N-methyl-2-pyridone with THPs were found.
The DNP Version 27.2 has approximately a total of 40 000 entries, giving a coverage of 0.3% of the reported NP chemical space by the shown combinations.

Cell-Painting Assay
The described assay follows closely the method described by Bray et al. [18] and was performed by the Compound Management and Screening Center (COMAS). Initially, 5 µl U2OS medium were added to each well of a 384-well plate (PerkinElmer CellCarrier-384 Ultra). Subsequently, U2OS cells were seeded with a density of 1600 cells per well in 20 µl medium. The plate was incubated for 5 min at the ambient temperature, followed by an additional 4 h incubation (37 °C, 5% CO2 The plates were prepared in triplicates with shifted layouts to reduce plate effects and imaged using a Micro XL High-Content Screening System (Molecular Devices, 5 channels, 9 sites per well, 20x magnification, binning 2).
The generated images were processed with the CellProfiler package (https://cellprofiler.org/) on a computing cluster of the Max Planck Society to extract 1716 cell features (parameters).
In a first step, the data was aggregated as overall medians per well.
A subset of highly reproducible parameters was determined using the procedure described by Woehrmann et al. [19] in the following way: Two biological repeats of one plate containing reference compounds were analyzed. For every parameter, its full profile over each whole plate was calculated. If the profiles from the two repeats showed a similarity >= 0.8 (see below), the parameter was added to the set.
This was carried out once and resulted in a set of 579 parameters that was used for all further analyses.
Z-scores were then calculated for each parameter as how many times the MAD of the controls the measured value deviates from the median of the controls: The phenotypic compound profile is then the list of z-scores of all parameters for one compound.
In addition to the phenotypic profile, an induction value was determined for each compound as the fraction of significantly changed parameters, in percent: Similarities of phenotypic profiles were calculated from the correlation distances between two profiles (https://docs.scipy.org/doc/scipy/reference/generated/scipy.spatial.distance.correlation.html; Similarity = 1 -Correlation Distance) and the compounds with the most similar profiles were determined from a set of 3000 reference compounds that was also measured in the assay.

Clustering
The compounds were sorted by descending induction and therefore the highest inducing compound was put into the first cluster. Compounds exhibiting a fingerprint similarity above 80% to that first compound would be added to this cluster. When no more compounds could be added, a new analysis of the remaining compounds was started. The procedure was repeated until all compounds were distributed into clusters.  Table S3. Fingerprints of entries in the y-axis were individually compared to fingerprints of annotated reference compounds on the x-axis. References targeting GPCRs were excluded for the analysis as they were found to occur ubiquitous in the whole data set for yet unresolved reasons.

Additivity of Profiles
The mathematical addition of the fingerprints of fragments 5r and 8d generates an artificial fingerprint 29dj-art. representing the combination of both fragments. This has a profile similarity of 4% when compared to the experimentally derived fingerprint of 29dj corresponding to the synthesized combination of fragments 5r and 8d. Figure S9. Evaluation of additivity of profiles. The top line is set as reference fingerprint (---% BioSim) to which subjacent fingerprints are compared, respectively; blue indicates a decrease of a specific parameter compared to DMSO control; red indicates an increase of a specific parameter compared to DMSO control.

Cell Culture
HeLa (ACC 57) cells were purchased from DSMZ GmbH (Germany) and cultured in DMEM with 10% FBS, sodium pyruvate, non-essential amino acids, penicillin and streptomycin. Cells were incubated at 37°C, 5% CO2 in a humidified atmosphere. During regular testing for mycoplasma infections, cells were found negative.

MitoSOX Red Assay
Mitochondrial superoxide levels were determined using the indicator dye MitoSOX Red (ThermoFisher, USA). 15,000 Hela cells were seeded per well into black 96 well plates with clear flat bottom and incubated at 37°C, 5% CO2 overnight. Seeding medium was exchanged for staining medium comprising DMEM without additives containing 5 µM MitoSOX Red and 5 µg/µL Hoechst-33342 (ThermoFisher, USA). Cells were incubated for 30 min at 37°C, 5% CO2. Subsequently, the medium was exchanged for DMEM with additives containing test compounds, followed by 60 min of incubation at 37°C, 5% CO2. Cells were fixed in PBS containing 0.5% paraformaldehyde for 10 min at room temperature and washed three times with PBS. Cells were imaged using an Axiovert 200M automated microscope (Carl Zeiss, Germany) at 10x magnification. MetaMorph 7.7.8.0 (Visitron, Germany) was used to quantify the integrated fluorescence intensity of MitoSOX Red per cell. The data was normalized to control cells treated with either DMSO (=0%) or 10 µM CDNB (=100%). Non-linear regression via four-parameter fit was performed using Prism 7 (GraphPad Software, USA) and EC50 values were obtained by interpolating X values for 50% staining intensity. Table S4. Establishment of a structure-phenotype relationship (SPR) for the PFPs by means of the induction parameter delineated from the cell painting assay, and comparison with activity in the MitoSOX Red assay; EC50 determined in HeLa cells (n = 3); Biosimilarity was compared to 14dk if not indicated differently; n.c. means that biosimilarity was not calculated because induction was out of 20-40% comparison window; 1) Biosimilarity was compared to 14df.