KRAS-SOS1 ( IC50 = 46 nM )

MRTX0902 (compound 32) (1 μM; 0, 2, 4, 8, 15, and 30 minutes) exhibits a low lipophilicity with a cLogP of 3.4 and a moderate Clint value of 195 mL/min/kg in human liver microsomes[1].
MRTX0902 exhibits strong selectivity for SOS1 (Ki=2 nM) compared to SOS2 and EGFR (both Ki values >10,000 nM). MRTX0902 has an IC50 value of 29 nM, which is sufficient to inhibit MKN1 cells[1].

MRTX0902 (compound 32) (25, 50 mg/kg; p.o. ; twice daily; 25 d), exhibits anti-tumor effect and causes tumor regression in mouse model[1].
MRTX0902 has good brain penetration, low clearance, and high bioavailability at 1-3 mg/kg for intravenous injection and 10-30 mg/kg for oral administration in a single dose[1].

HTRF Displacement Assay to Determine Ki [1]
Measured the ability of a compound to bind to SOS1 was measured using a HTRF displacement assay. A recombinant human SOS1 polypeptide (corresponding to amino acids 560-1049, expressed in E. Coli with N-terminal His-TEV-AviTag-SOS1 (MW=59.4 kDa) and lanthanide labeled streptavidin was incubated with an exemplary compound (in a DMSO stock solution) in buffer (25 mM HEPES pH 7.5, 25 mM NaCl, 1 mM DTT, 0.01% Brij 35, 0.02% BSA, 0.1% DMSO). After a 10-15 minute incubation at room temperature, a solution comprised of a custom-made Cy5 labelled tracer and MAb Anti-6HIS Tb cryptate Gold in buffer was added to the solution containing the SOS1 polypeptide and exemplary compound. After a 1-hour incubation at room temperature, the HTRF signal was measured using Clairostar plate reader according to the manufacturer’s instructions. Excitation filter EX-TR was used, and emission 1 was detected at 650-610 nm and emission 2 detected at 620-610 nm. The HTRF ratio was calculated using the formula: [emission 1/emission 2]*10000. Background signals were calculated from well with a 10µM inhibitor, known to inhibit 100% at that concentration. The background subtracted signals were converted to % binding relative to DMSO controls. Data were analyzed using XLFIT software (IDBS) using a Morrison equation for competitive binding and Ki’s were generated.

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EGFR Selectivity Assay [1]
EGFR selectivity was profiled using Reaction Biology’s radiometric HotSpot kinase assay with human EGFR. Compounds were sent as a powder then suspended as a 10 mM DMSO stock solution. Compounds were tested in 10-dose IC50 mode with a 3-fold serial dilution starting at 10 μM. Control compound, staurosporine, was tested in 10-dose IC50 mode with 4-fold serial dilution starting at 20 μM. Reactions were carried out at 10 μM ATP.

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SOS2 KRAS WT GDP Exchange Assay [1]
The final conditions for the Functional SOS2 Assay are as follows: 50 mM HEPES 7.5, 2nM SOS2, 50nM GTP-CY5, 30nM KRAS, 0.5 nM Tb-SA, 5 mM MgCl2, 1 mM TCEP, 0.2 mg/mL BSA, ~1.0% DMSO. Recombinant human SOS2 polypeptide (corresponding to amino acids 558-1047, expressed in E. coli with a N-terminal HIS-tag MW=59.3 kDa) and Cy5- GTP were added to an exemplary compound (in a DMSO stock solution) at room temperature for 15 minutes. Then recombinant human KRAS polypeptide (amino acids 2-169, expressed in E. coli with a C-terminal Avi-biotinylated tag MW 22.0 kDa) was added with Terbium-StrepAvidin and the Ratio metric data is collected after 30 minutes using a BMG LABTECH CLARIOstar Plus via TR-FRET. 100 percent of control (POC) is determined by using a DMSO control and 0 POC is determined using a concentration of control S101 compound that completely inhibits activity of SOS2. The POC values were fit to an IC50 with Hill equation and the IC50 value reported. Equation 1: Vmax/(1+((x/IC50)^Hill)))+BKD).

MKN1 cells (15,000/w) were seeded in a black clear flat bottom 96-well cell culture plate and incubated at 37oC overnight. Assay day 1, cells were dosed with compounds with a 10 µm starting concentration and serially diluted 3x for a total of 9 concentrations. The cells were incubated for approximately 0.5-1 hour with the compounds solubilized in DMSO at 37 °C. Cells were immediately fixed by adding 50 µL of 4% formaldehyde to all wells in a fume hood and the plates were incubated for 20 minutes at room temperature. The formaldehyde was discarded from the plates and 150 µL of ice-cold methanol was added to permeabilize the cells for 10 minutes at -20 °C. The methanol was discarded from each of the plates and any liquid remaining in the plate by tapping the plate against paper towels. Cells were then blocked with 150 µL of Odyssey blocking buffer using 0.05% Tween for 1 hour at room temperature on a shaker. The blocking buffer was discarded and 50 µL of primary antibodies pERK (Rabbit, 1:500) and GapDH diluted in Odyssey blocking buffer was added. The plates were incubated overnight at 4 °C on a shaker. On Assay day 2, the primary antibody solution was removed. Each plate was washed 3x times with 150 µL of 1x PBST (PBS + 0.1 % Tween 20) and incubated with 50 µL of secondary antibodies: Anti-Rabbit and Anti-Mouse at 1:800 dilution in Odyssey blocking buffer with Tween at room temperature on a shaker for 2 hours (protected from light). The secondary antibody solution as removed and each plate was washed with PBST 3x times. Any liquid remaining was discarded, and the plate was imaged using the Licor Odyssey machine according to the manufacturer’s instruction, using a set focus length at 3mm and both 800nm and 700nm filters. The GAPDH normalized scan values for each well were divided by the average of vehicle wells to get the % of pERK inhibition. The IC50 values were then calculated with the Graph pad Prism software[1].

Mice were maintained under pathogen-free conditions, and food and water was provided ad libitum. 6 – 8-week-old female Hsd:Athymic Nude-Foxn1nu mice were injected subcutaneously with tumor cells in 100 l of PBS and Matrigel matrix in the right hind flank of each mouse with 5.0 x 106 MIA PaCa-2 cells at a ratio of 1:1 in PBS and Matrigel. Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumor volume measurements were determined utilizing the formula 0.5 x L x W2 in which L refers to length and W refers to width of each tumor. When tumors reached the desired average study start tumor volume of 150 mm3 or 200 mm3 for 21–28-day TGI efficacy and 6-day studies, respectively, mice were randomized into treatment groups. MRTX0902 was formulated in 0.5% Methylcellulose (4000cps) + 0.2% Tween80 in water once per week and dosing solution was stored protected from light at 4 °Celsius. MRTX849 was formulated once per week in 10% Captisol in 50 mM Citrate Buffer pH 5.0 and S103 dosing solution was stored protected from light at 4 °Celsius. Mice were orally administered vehicle, MRTX0902, MRTX849 orally (PO) at the indicated doses and schedules. Mice were monitored daily, tumors and body weights were measured 2 or 3 times per week. Percent Tumor Growth Inhibition (% TGI) was calculated using the following formula: (1-(Final Drug Treated Tumor Volume – Initial Drug Treated Tumor Volume) / (Final Vehicle Treated Tumor Volume – Initial Vehicle Treated Tumor Volume))*100. Percent Tumor regression was calculated when the average tumor volume of final treated tumors was less than initial treated tumor volume using the following equation: (-100%)*(1-(Final treated tumor volume)/(Initial treated tumor volume)). Statistical analysis of differences in mean tumor volume between vehicle- and drug-treated cohorts was run using a two-tailed Student t test in GraphPad Prism version 8.2.0. A p value of less than 0.05 was considered to be statistically significant. In studies where tumor collection was performed, mice were humanely sacrificed, and tumors were surgically removed and immediately cut into two pieces. One half piece was transferred to a prefilled homogenizer tube with ceramic beads and the other half piece was transferred to an Eppendorf tube. Both tubes were immediately submerged in liquid nitrogen to snap freeze the tissue and stored at -80°C until further processing was performed[1].

[1]. Design and Discovery of MRTX0902, a Potent, Selective, Brain-Penetrant, and Orally Bioavailable Inhibitor of the SOS1:KRAS Protein-Protein Interaction. J Med Chem. 2022 Jul 28;65(14):9678-9690.

SOS1 Inhibitor MRTX0902 is an orally available, brain-penetrant, phthalazine-based inhibitor of the guanine nucleotide exchange factor (GEF) Son of sevenless homolog 1 (SOS1), with potential antineoplastic activity. Upon oral administration, SOS1 inhibitor MRTX0902 selectively targets and binds to SOS1, thereby preventing the interaction of SOS1 with Kirsten rat sarcoma viral oncogene homolog (KRAS) in the guanosine diphosphate (GDP)-bound 'off' state, which is the inactivated state of KRAS. This abrogates the exchange of RAS-bound GDP for guanosine triphosphate (GTP) and prevents the formation of GTP-loaded KRAS, which is the activated 'on' state of KRAS. This prevents activation of downstream RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway by GTP-loaded KRAS. This inhibits mutant KRAS-dependent signaling and may inhibit growth and survival of KRAS-expressing tumor cells. KRAS is a member of the RAS family of oncogenes that is mutated in many cancer cell types. Mutations of KRAS may induce constitutive signal transduction leading to tumor cell proliferation, survival, invasion, and metastasis. SOS1 regulates the KRAS GDP-GTP cycle and promotes nucleotide exchange and formation of 'active' KRAS-GTP.

C22H24N6O

388.47

388.2

C, 68.02; H, 6.23; N, 21.63; O, 4.12

2654743-22-1

2654743-22-1

156526655

Light yellow to green yellow solid powder

2.8

1

7

4

29

587

1

C12C=NC(N3CCOCC3)=CC1=C(N=NC=2C)N[C@H](C)C1=C(C(=CC=C1)C#N)C

ILPWEAHQRAWJIU-OAHLLOKOSA-N

InChI=1S/C22H24N6O/c1-14-17(12-23)5-4-6-18(14)15(2)25-22-19-11-21(28-7-9-29-10-8-28)24-13-20(19)16(3)26-27-22/h4-6,11,13,15H,7-10H2,1-3H3,(H,25,27)/t15-/m1/s1

2-methyl-3-[(1R)-1-[(4-methyl-7-morpholin-4-ylpyrido[3,4-d]pyridazin-1-yl)amino]ethyl]benzonitrile

MRTX-0902; MRTX0902; CRG69FR93G; UNII-CRG69FR93G; CHEMBL5192659; (R)-2-Methyl-3-(1-((4-methyl-7-morpholinopyrido(3,4-d)pyridazin-1-yl)amino)ethyl)benzonitrile; Benzonitrile, 2-methyl-3-((1R)-1-((4-methyl-7-(4-morpholinyl)pyrido(3,4-d)pyridazin-1-yl)amino)ethyl)-; MRTX 0902

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO 12.5~100 mg/mL (32.2~257.4 mM)
Ethanol: ~100 mg/mL

Solubility in Formulation 1: ≥ 1.25 mg/mL (3.22 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)