Compound (SMILES): C[C@H]1CC2(CCN(c3ccc(C(=O)N4CC(CN5CCN(c6cccc(NC7CCC(=O)NC7=O)c6)CC5)C4)nn3)CC2)CN1c1ccc(C#N)c(Cl)c1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): AR (UniProt P10275)
POI sequence: MEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQNPGPRHPEAASAAPPGASLLLLQQQQQQQQQQQQQQQQQQQQQQQETSPRQQQQQQGEDGSPQAHRRGPTGYLVLDEEQQPSQPQSALECHPERGCVPEPGAAVAASKGLPQQLPAPPDEDDSAAPSTLSLLGPTFPGLSSCSADLKDILSEASTMQLLQQQQQEAVSEGSSSGRAREASGAPTSSKDNYLGGTSTISDNAKELCKAVSVSMGLGVEALEHLSPGEQLRGDCMYAPLLGVPPAVRPTPCAPLAECKGSLLDDSAGKSTEDTAEYSPFKGGYTKGLEGESLGCSGSAAAGSSGTLELPSTLSLYKSGALDEAAAYQSRDYYNFPLALAGPPPPPPPPHPHARIKLENPLDYGSAWAAAAAQCRYGDLASLHGAGAAGPGSGSPSAAASSSWHTLFTAEEGQLYGPCGGGGGGGGGGGGGGGGGGGGGGGEAGAVAPYGYTRPPQGLAGQESDFTAPDVWYPGGMVSRVPYPSPTCVKSEMGPWMDSYSGPYGDMRLETARDHVLPIDYYFPPQKTCLICGDEASGCHYGALTCGSCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGARKLKKLGNLKLQEEGEASSTTSPTEETTQKLTVSHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALLSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELHQFTFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: LNCaP (Homo sapiens)
Cell line description: Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*01
Instable (MSI).
Metastatic; Left supraclavicular lymph node; 
Caucasian.
Space-flown cell line (cellonaut).
Cancer cell line
Assay: ELISA
DC50:  0.4 nM
Assay description: Degradation of the Androgen Receptor in LNCaP Cells by Compounds of the Disclosure
The following in vitro cell-based assay measures the degradation activity of test compounds against the androgen receptor (AR) in human prostate cancer cell lines. The activity is expressed as a DC50 value. On the first day of the experiment, LNCaP cells were seeded at a density of 20,000 cells/well in 96-well plates using phenol red-free RPMI 1640 medium supplemented with 5% charcoal-stripped fetal bovine serum. 200 μL of cell suspension was added to each well and the cells were incubated at 37°C, 5% CO? in a cell culture incubator for three days. On the fourth day, 22 μL of a serial dilution of the test compound prepared in plating medium containing 1 nM R1881 was added to each well. The final concentration of R1881 was 0.1 nM. The compound concentration was determined by a six-fold serial dilution starting at 2.5 μM over seven concentration points. A blank control well containing 0.5% DMSO was also added. The plates were incubated at 37°C, 5% CO? in a cell culture incubator for 24 hours. On the fifth day, remove the 96-well cell culture plate, aspirate and discard the cell culture supernatant, and wash the cells once with 300 μL of ice-cold PBS. Add 50 μL of 1× lysis buffer containing 1 mM PMSF diluted in ddH2O. Shake on a microplate for 1 minute, then place the plate on ice for 15-20 minutes. Mix thoroughly by pipetting, and centrifuge at 4000 rpm for 10 minutes at 4°C. Detect AR protein levels using an AR ELISA kit according to the manufacturer's instructions. Add 96 μL of sample diluent and 4 μL of sample lysis buffer or blank lysis buffer to the 96-well plate. Seal the plate, shake well, and incubate in a 37°C incubator for 2 hours. Discard the liquid from the wells, wash the plate five times with 300 μL of wash buffer per well, and then add 100 μL of detection antibody working solution. Seal the plate and incubate in a 37°C incubator for 1 hour. Discard the liquid from the wells, wash the plate five times with 300 μL of wash solution per well, then add 100 μL of enzyme conjugate working solution. Seal the plate and incubate in a 37°C incubator for 30 minutes. Discard the liquid from the wells, wash the plate five times with 300 μL of wash solution per well, then add 100 μL of colorimetric solution. Seal the plate and incubate at room temperature in the dark for 5 minutes. Add 100 μL of stop solution to each well. Mix well and immediately read the OD450 and OD540 values on a microplate reader. Subtract the OD540 absorbance of the corresponding well from the OD450 absorbance of all wells to obtain the corrected OD450 absorbance (OD450-correction). Calculate the degradation rate of each compound concentration using the following formula, where the maximum value (Max) represents the maximum degradation rate of the compound against AR. GraphPad Prism was used to fit the curve based on the logarithmic concentration of the compound and the degradation efficiency, and calculate the DC50 value. Degradation rate (%) = (OD450 - calibration blank control - OD450 - calibration compound) / (OD450 - calibration blank control - OD450 - calibration lysate control) × 100%
The biological activity of the disclosed compounds was determined using the above analysis. The calculated DC50 and Max values are shown in Table 1.
Reference: CHIMERIC COMPOUND FOR TARGETED DEGRADATION OF ANDROGEN RECEPTOR PROTEIN, PREPARATION METHOD THEREFOR, AND MEDICAL USE THEREOF (patent)
Source database: TPDdb