Compound (SMILES): CO[C@@H](C)COc1nc(N2CC[C@H]2C(N)=O)c2cc(C3CC3)c(-c3c(C)c(F)cc4[nH]ncc34)c(OCc3ccc(CN4CCN(Cc5ccc6c(N7CCC(=O)NC7=O)nn(C)c6c5)C[C@@H]4C)cc3)c2n1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): KRAS (UniProt P01116)
POI sequence: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: PaTu 8902 (Homo sapiens)
Cell line description: African=0%; Native American=0.21%; East Asian, North=1.73%; East Asian, South=0%; South Asian=0%; European, North=62.61%; European, South=35.45%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*02
Stable (MSS) (Sanger).
In situ; Pancreas; 
Caucasian.
Cancer cell line
Assay: ELISA
DC50:  26 nM
Assay description: Test Example 1 Evaluation of RAS G12V degrading effect on human KRAS G12V mutation-positive pancreatic cancer line PA-TU-8902 The RAS G12V degrading effect of the test compound was evaluated by measuring the RAS G12V expression level by Cell ELISA method.
36 μL of PA-TU-8902 cells were seeded in a 384-well plate at 1.5×10 4 cells per well. Cell culture was carried out at 37° C. in the presence of 5% CO 2 using DMEM medium containing 10% fetal bovine serum.
The next day, the test compound (10 points in the range from 3 μM final concentration to 0.1 nM), the compound of Example No. 7 at a final concentration of 3 μM as a positive control, and dimethyl sulfoxide (DMSO), the solvent for the test compound, as a negative control were added to a fresh plate. It was diluted 100 times with medium, added 4 μL to each well, and cultured for 24 hours.
The next day, the culture supernatant was removed, 20 μL of 4% paraformaldehyde phosphate buffer was added to each well, and the cells were fixed by standing at room temperature for 30 minutes. Thereafter, the supernatant was removed, and 20 μL of Phosphate buffered saline (PBS) containing 0.1% Triton X-100 was added to each well. After standing at room temperature for 10 minutes, the supernatant was removed, 25 μL of PBS was added to each well, and each well was washed twice by removing the supernatant. Subsequent washing operations were performed in the same manner. Next, the supernatant was removed, 20 μL of PBS containing 0.5% sodium dodecyl sulfate (SDS) was added to each well, and after standing at room temperature for 10 minutes, the supernatant was removed by centrifuging the plate. . After washing with PBS, the supernatant was removed by centrifugation, and 20 μL of blocking solution was added to each well. After standing at room temperature for 30 minutes, remove the supernatant by centrifugation, and use anti-Ras antibody (Ras Rabbit mAb; Cell Signaling Technology; diluted 500 times) as the primary antibody. Add 15 μL of anti-β-Actin antibody (Abcam; 5,000-fold dilution) diluted with Can Get Signal Solution 2 to each well and leave it at 4°C overnight. did.
The next day, the supernatant was removed by centrifugation and washed with PBS. Remove the supernatant by centrifugation, and use anti-rabbit IgG antibody (IRDye 800CW Goat anti-Rabbit IgG) and anti-mouse IgG antibody (IRDye 680RD Donkey anti-Mouse IgG) as secondary antibodies. was diluted 1,000 times with Can Get Signal Solution 2 and 15 μL of the solution was added to each well. After standing at room temperature for 1 hour, the supernatant was removed by centrifugation and washed with PBS. After removing the supernatant, the plate was air-dried at room temperature for 2 hours or more, and fluorescence signals at 700 nm and 800 nm were measured using Aerius.
Degradation rate of RAS G12V corrected by the signal value of β-Actin, with the RAS G12V signal value upon addition of DMSO as 0% and the RAS G12V signal value upon addition of the compound of Example No. 7 at a final concentration of 3 μM as 100%. was calculated. The decomposition rate at the concentration of the compound that showed the highest decomposition effect was set as Dmax, and based on the decomposition rate up to the concentration of the compound that showed Dmax, the 50% decomposition value (DC50) of RAS G12V was subjected to Sigmoid-Emax model nonlinear regression analysis. Calculated. The results for several test compounds of formula (I) are shown in the table below.
Reference: HETEROCYCLIC COMPOUND FOR INDUCING DEGRADATION OF G12V MUTANT KRAS PROTEIN (patent)
Source database: TPDdb