Replacing the phthalimide core in thalidomide with benzotriazole

Abstract The advent of proteolysis-targeting chimaeras (PROTACs) mandates that new ligands for the recruitment of E3 ligases are discovered. The traditional immunomodulatory drugs (IMiDs) such as thalidomide and its analogues (all based on the phthalimide glutarimide core) bind to Cereblon, the substrate receptor of the CRL4ACRBN E3 ligase. We designed a thalidomide analogue in which the phthalimide moiety was replaced with benzotriazole, using an innovative synthesis strategy. Compared to thalidomide, the resulting “benzotriazolo thalidomide” has a similar binding mode, but improved properties, as revealed in crystallographic analyses, affinity assays and cell culture.


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Experimental procedures and characterization data General considerations.
All reagents were obtained from commercial sources and used without further purification.
Tetrahydrofuran and dichloromethane were distilled over suitable drying agents. Mass spectra were recorded with a Bruker Maxis HRMS-ESI-qTOF spectrometer (electrospray ionization mode). NMR data were recorded with Bruker Avance 400 spectrometer (400.13 MHz for 1 H, 100.61 MHz for 13 C) in CDCl3 and were referenced to residual solvent proton peaks (δH = 7.28) and solvent carbon peaks (δC = 77.0).

Microscale Thermophoresis
Binding affinities of thalidomide (1) and its benzotriazole analog (2) to the thalidomide-binding domain of human CRBN (hTBD) were determined as described previously. 1 In short, a 16-point 1:1 dilution series of the compound in DMSO was diluted 1:100 in ddH2O and then mixed with protein:reporter stock to final concentrations of 10 µM hTBD and 200 nM BODIPY-uracil.
Measurements were performed on a Monolith NT.115 with a Nano BLUE detector (NanoTemper Technologies), using 20% excitation power, MST power set to medium and temperature control at 25 °C. Data at on-time 20 s were analyzed using Prism 9 (GraphPad).  Table S1. The structure was deposited in the Protein Data Bank (PDB) under the accession code 7PJK. Multiple myeloma cell line MOLP-2 and KMS-12-PE were purchased from the DSMZ. Cells were maintained in RPMI-1640 (Gibco, UK) supplemented with 20% fetal bovine serum (FBS, Gibco, UK), penicillin (100 UI mL -1 ), streptomycin (100 µg mL -1 ) and GlutaMax (2 mM, Gibco, UK).

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All cells line cultivation under a humidified atmosphere of 95% air/5% CO2 at 37 °C. The number of viable cells was determined by trypan blue exclusion.

MTT assay
All examined cells were diluted with the growth medium to 3.0×10 5 cells per mL and the aliquots (15×10 3 cells per 50 μL) were placed in individual wells in white 96-multiplates (Nunc, USA).
Triplicate wells were treated with test compounds starting at 500.0 μM concentration and diluted at various concentrations or DMSO (Sigma, USA) as control with final concentration 0.1%. Plates were incubated for 72 h at 37 °C in 5% CO2 atmosphere. After incubation, the cells were then treated with 100 μL CellTiter-Glo ® One Solution (Promega, USA). The plates were shaken for 10 min. The luminescence was determined using a microplate reader GloMax Multi+ (Promega, USA). Each of the tested compounds was evaluated for cytotoxicity in three separate experiments.

Apoptosis assay
For the detection of apoptosis, the cells were plated at 6-well culture plates (Eppendorf, Germany).
The exposed cells were placed at 37 °C in a 5% CO 2 incubator for 48 h. The cultured cells were washed twice with PBS and resuspended in 1× binding buffer (AnnexinV-FITC kit, Invitrogen, USA) at a concentration 1 × 10 6 mL -1 . Annexin-FITC (5 L) and propidium iodide (PI, 2 L) were added to 100 L of the cell suspension and incubated for 15 min at room temperature (25 °C) in the dark. After incubation 400 L of 1× binding buffer was added to each tube and the stained cells were analyzed within 1 h using CytoFlex (Beckham Culture, USA) and CytExpert 2.1 program. Since, Annexin V FITC staining precedes the loss of membrane integrity that accompanies the later stage identified by PI, Annexin FITC positive, PI negative indicates early apoptosis, while the viable cells are Annexin V FITC negative, PI negative. The cells that are in late apoptosis, or dead are both Annexin V FITC and PI positive. Each of the tested compounds was evaluated for induction apoptosis in three separate experiments. S10