COTESTA SIMONA (CH)
CUI XIAOMING (US)
DE KANTER RUBEN (CH)
FARAGO ANNA (US)
GERSPACHER MARC (CH)
GRAUS PORTA DIANA (CH)
KIM JAEYEON (US)
LEBLANC CATHERINE (CH)
LORTHIOIS EDWIGE LILIANE JEANNE (CH)
MACHAUER RAINER (CH)
MAH ROBERT (CH)
MURA CHRISTOPHE (CH)
RIGOLLIER PASCAL (CH)
PRAHALLAD ANIRUDH CADAPA (CH)
SCHNEIDER NADINE (CH)
STRINGER ROWAN (CH)
STUTZ STEFAN (CH)
VAUPEL ANDREA (CH)
WARIN NICOLAS (CH)
WILCKEN RAINER (CH)
WEISS ANDREAS (CH)
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What is claimed is: 1. A method of treating a cancer or a tumor in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a KRAS G12C inhibitor, or a pharmaceutically acceptable salt thereof, alone or in combination with at least one additional therapeutically active agent. 2. A method according to claim 1, wherein the KRAS G12C inhibitor is selected from 1-{6- [(4M)-4-(5-Chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H- pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-1-one, (Compound A), sotorasib (Amgen), adagrasib (Mirati), D-1553 (InventisBio), BI1701963 (Boehringer), GDC6036 (Roche), JNJ74699157 (J&J), X-Chem KRAS (X-Chem), LY3537982 (Lilly), BI1823911 (Boehringer), AS KRAS G12C (Ascentage Pharma), SF KRAS G12C (Sanofi), RMC032 (Revolution Medicine), JAB-21822 (Jacobio Pharmaceuticals), AST-KRAS G12C (Allist Pharmaceuticals), AZ KRAS G12C (Astra Zeneca), NYU-12VC1 (New York University), and RMC6291 (Revolution Medicines), or a pharmaceutically acceptable salt thereof. 3. A method according to claim 2, wherein the KRAS G12C inhibitor is selected from 1-{6- [(4M)-4-(5-Chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H- pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-1-one, (Compound A), sotorasib, adagrasib, D-1553, and GDC6036), or a pharmaceutically acceptable salt thereof. 4. A method according to claim 2, wherein the KRAS G12C inhibitor is 1-{6-[(4M)-4-(5- Chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl]-2- azaspiro[3.3]heptan-2-yl}prop-2-en-1-one, (Compound A), or a pharmaceutically acceptable salt thereof. 5. A method according to claim 2, wherein the KRAS G12C inhibitor is 1-{6-[(4M)-4-(5- Chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl]-2- azaspiro[3.3]heptan-2-yl}prop-2-en-1-one, (Compound A). 6. A method according to any one of claims 1 to 5, wherein the additional therapeutically active agent is selected from the group consisting of an EGFR inhibitor, a SHP2 inhibitor, a SOS1 inhibitor, an AKT inhibitor, an EGFR inhibitor, a SHP2 inhibitor (such as TNO155, or a pharmaceutically acceptable salt thereof), a Raf-inhibitor, an ERK inhibitor, a MEK inhibitor, a PI3K inhibitor, an mTOR inhibitor, a CDK4/6 inhibitor, an FGFR inhibitor and combinations thereof. 7. A method according to according to any one of claims 1 to 5 wherein the at least one additional therapeutically active agent is selected from the group consisting of an EGFR inhibitor (such as cetuximab, panitumuab, erlotinib, gefitinib, osimertinib or nazartinib, or a pharmaceutically acceptable salt thereof), a SOS inhibitor (such as BAY-293, BI-3406, or BI- 1701963, or a pharmaceutically acceptable salt thereof), a SHP2 inhibitor (such as NO155 (Novartis), JAB3068 (Jacobio), JAB3312 (Jacobio), RLY1971 (Roche), SAR442720 (Sanofi), RMC4450 (Revolution Medicines), BBP398 (Navire), BR790 (Shanghai Blueray), SH3809 (Nanjing Sanhome), PF0724982 (Pfizer), ERAS601 (Erasca), RX-SHP2 (Redx Pharma), ICP189 (InnoCare), HBI2376 (HUYA Bioscience), ETS001 (Shanghai ETERN Biopharma), TAS- ASTX (Taiho Oncology) and X-37-SHP2 (X-37), or a pharmaceutically acceptable salt thereof), a Raf-inhibitor (e.g. belvarafenib or LXH254 (naporafenib), or a pharmaceutically acceptable salt thereof), an ERK inhibitor (such as LTT462 (rineterkib), GDC-0994, KO-947, Vtx-11e, SCH-772984, MK2853, LY3214996 or ulixertinib, or a pharmaceutically acceptable salt thereof), a MEK inhibitor (such as pimasertib, PD-0325901, selumetinib, trametinib, binimetinib or cobimetinib, or a pharmaceutically acceptable salt or solvate thereof), AKT inhibitor (such as capivasertib (AZD5363) or ipatasertib, or a pharmaceutically acceptable salt thereof), a PI3K inhibitor (such as AMG 511, buparlisib, alpelisib, or a pharmaceutically acceptable salt thereof), an mTOR inhibitor (such as everolimus or temsirolimus, or a pharmaceutically acceptable salt thereof), and a CDK4/6 inhibitor (such as ribociclib, palbociclib or alemaciclib, or a pharmaceutically acceptable salt thereof). 8. A method according to claim 7, wherein the at least one additional therapeutically active agent is an EGFR inhibitor (such as cetuximab, panitumuab, erlotinib, gefitinib, osimertinib or nazartinib, or a pharmaceutically acceptable salt thereof). 9. A method according to claim 7, wherein the at least one additional therapeutically active agent is an a SOS inhibitor (such as BAY-293, BI-3406, or BI-1701963, or a pharmaceutically acceptable salt thereof). 10. A method according to claim 7, wherein the at least one additional therapeutically active agent is a SHP2 inhibitor (such as JAB3068 (Jacobio), JAB3312 (Jacobio), RLY1971 (Roche), SAR442720 (Sanofi), RMC4450 (Revolution Medicines), BBP398 (Navire), BR790 (Shanghai Blueray), SH3809 (Nanjing Sanhome), PF0724982 (Pfizer), ERAS601 (Erasca), RX-SHP2 (Redx Pharma), ICP189 (InnoCare), HBI2376 (HUYA Bioscience), ETS001 (Shanghai ETERN Biopharma), TAS-ASTX (Taiho Oncology) and X-37-SHP2 (X-37), or a pharmaceutically acceptable salt thereof). 11. A method according to claim 7, wherein the at least one additional therapeutically active agent is a Raf-inhibitor (e.g. belvarafenib or LXH254 (naporafenib), or a pharmaceutically acceptable salt thereof). 12. A method according to claim 7, wherein the at least one additional therapeutically active agent is an ERK inhibitor (such as LTT462 (rineterkib), GDC-0994, KO-947, Vtx-11e, SCH- 772984, MK2853, LY3214996 or ulixertinib, or a pharmaceutically acceptable salt thereof). 13. A method according to claim 7, wherein the at least one additional therapeutically active agent is a MEK inhibitor (such as pimasertib, PD-0325901, selumetinib, trametinib, binimetinib or cobimetinib, or a pharmaceutically acceptable salt or solvate thereof) or wherein the at least one additional therapeutically active agent is an AKT inhibitor (such as capivasertib (AZD5363) or ipatasertib, or a pharmaceutically acceptable salt thereof). 14. A method according to claim 7, wherein the at least one additional therapeutically active agent is a PI3K inhibitor (such as AMG 511, buparlisib, alpelisib, or a pharmaceutically acceptable salt thereof). 15. A method according to claim 7, wherein the at least one additional therapeutically active agent is an mTOR inhibitor (such as everolimus or temsirolimus, or a pharmaceutically acceptable salt thereof). 16. A method according to claim 7, wherein the at least one additional therapeutically active agent is a CDK4/6 inhibitor (such as ribociclib, palbociclib or alemaciclib, or a pharmaceutically acceptable salt thereof). 17. A method according to claim 1 or 7, wherein the at least one additional therapeutically active agent is a SHP2 inhibitor (such as TNO155 (Novartis), JAB3068 (Jacobio), JAB3312 (Jacobio), RLY1971 (Roche), SAR442720 (Sanofi), RMC4450 (Revolution Medicines), BBP398 (Navire), BR790 (Shanghai Blueray), SH3809 (Nanjing Sanhome), PF0724982 (Pfizer), ERAS601 (Erasca), RX-SHP2 (Redx Pharma), ICP189 (InnoCare), HBI2376 (HUYA Bioscience), ETS001 (Shanghai ETERN Biopharma), TAS-ASTX (Taiho Oncology) and X-37- SHP2 (X-37), or a pharmaceutically acceptable salt thereof) and wherein the method further comprises administering to the subject a therapeutically effective amount of a third therapeutically active agent which is selected from a Raf-inhibitor (e.g. belvarafenib or LXH254 (naporafenib), or a pharmaceutically acceptable salt thereof), an ERK inhibitor (such as LTT462 (rineterkib), GDC-0994, KO-947, Vtx-11e, SCH-772984, MK2853, LY3214996 or ulixertinib, or a pharmaceutically acceptable salt thereof), a MEK inhibitor (such as pimasertib, PD-0325901, selumetinib, trametinib, binimetinib or cobimetinib, or a pharmaceutically acceptable salt or solvate thereof), AKT inhibitor (such as capivasertib (AZD5363) or ipatasertib, or a pharmaceutically acceptable salt thereof), a PI3K inhibitor (such as AMG 511, buparlisib, alpelisib, or a pharmaceutically acceptable salt thereof), an mTOR inhibitor (such as everolimus or temsirolimus, or a pharmaceutically acceptable salt thereof), and a CDK4/6 inhibitor (such as ribociclib, palbociclib or alemaciclib, or a pharmaceutically acceptable salt thereof). 18. A method according to any one of the previous claims, wherein the cancer or tumor is a cancer or tumor which is selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer and squamous cell lung cancer), colorectal cancer (including colorectal adenocarcinoma), pancreatic cancer (including pancreatic adenocarcinoma), uterine cancer (including uterine endometrial cancer), rectal cancer (including rectal adenocarcinoma), appendiceal cancer, small-bowel cancer, esophageal cancer, hepatobiliary cancer (including liver cancer and bile duct carcinoma), bladder cancer, ovarian cancer and a solid tumor; or wherein the cancer or tumor to be treated may be selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer and squamous cell lung cancer), colorectal cancer (including colorectal adenocarcinoma), pancreatic cancer (including pancreatic adenocarcinoma), uterine cancer (including uterine endometrial cancer), rectal cancer (including rectal adenocarcinoma), appendiceal cancer, small-bowel cancer, esophageal cancer, hepatobiliary cancer (including liver cancer, bile duct cancer and bile duct carcinoma), bladder cancer, ovarian cancer, duodenal papillary cancer and a solid tumor, particularly when the cancer or tumor harbors a KRAS G12C mutation. 19. A method according to any one of the previous claims, wherein the cancer is selected from lung cancer (such as non-small cell lung cancer), colorectal cancer, pancreatic cancer and a solid tumor, or wherein the cancer is selected from non-small cell lung cancer, colorectal cancer, bile duct cancer, ovarian cancer, duodenal papillary cancer and pancreatic cancer, particularly when the cancer or tumor harbors a KRAS G12C mutation. 20. A method according to any one of the previous claims wherein the cancer or tumor is a KRAS G12C mutated cancer or tumor. 21. A method according to any one of the previous claims, wherein the therapeutic agents in the combination therapy are administered simultaneously, separately or over a period of time. 22. A method according to any one of the previous claims, wherein the amount of each therapeutic agent is administered to the subject in need thereof is effective to treat the cancer or tumor. 23. A method according to any one of claims 3, 4, 8, 9, 10, 13 to 17, wherein the SHP2 inhibitor is TNO155, or pharmaceutically acceptable salt thereof, and is administered orally at a total daily dose ranging from 10 to 80 mg, or from 10 to 60 mg. 24. A method according to claim 18, wherein the dose per day of TNO155 is administered on a 21 day cycle of 2 weeks on drug followed by 1 week off drug. 25. A method according to any one of the previous claims, wherein Compound A, or a pharmaceutically acceptable salt thereof, is administered at a therapeutically effective dose ranging from 50 mg to 1600 mg per day, e.g. from 200 to 1600 mg per day, e.g. from 400 to 1600 mg per day. 26. A method according to any one of the previous claims, wherein Compound A, or a pharmaceutically acceptable salt thereof, is administered at a therapeutically effective dose which is selected from 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 and 600 mg per day. 27. A method according to any one of the previous claims, wherein the total daily dose of Compound A is administered once daily or twice daily. 28. A method according to any one of the previous claims, wherein the subject or patient to be treated is selected from: - a patient suffering from a KRAS G12C mutant solid tumor (e.g. advanced (metastatic or unresectable) KRAS G12C mutant solid tumor), optionally wherein the patient has received and failed standard of care therapy or is intolerant or ineligible to approved therapies; - a patient suffering from KRAS G12C mutant NSCLC (e.g., advanced (metastatic or unresectable) KRAS G12C mutant NSCLC), optionally wherein the patient who has received and failed a platinum- based chemotherapy regimen and an immune checkpoint inhibitor therapy either in combination or in sequence; -a patient suffering from KRAS G12C mutant NSCLC (e.g., advanced (metastatic or unresectable) KRAS G12C mutant NSCLC), optionally wherein the patient who has previously been treated with a KRAS G12C inhibitor (e.g. sotorasib, adagrasib, GDC6036 or D-1553); and - a patient suffering from KRAS G12C mutant CRC (e.g., advanced (metastatic or unresectable) KRAS G12C mutant CRC), optionally wherein the patient has received and failed standard of care therapy, including a fluropyrimidine-, oxaliplatin-, and / or irinotecan-based chemotherapy. 29. A pharmaceutical combination comprising a KRAS G12C inhibitor and at least one additional therapeutically active agent which is an agent targeting the MAPK pathway or an agent targeting parallel pathways. 30. A pharmaceutical combination comprising a KRAS G12C inhibitor KRAS G12C inhibitor, such as Compound A, or a pharmaceutically acceptable salt thereof, and a therapeutically active agent which is selected from the group consisting of an EGFR inhibitor, a SOS inhibitor, a SHP2 inhibitor (such as TNO155, or a pharmaceutically acceptable salt thereof), a Raf-inhibitor, an ERK inhibitor, a MEK inhibitor, AKT inhibitor, a PI3K inhibitor, an mTOR inhibitor, a CDK4/6 inhibitor and combinations thereof. 31. A pharmaceutical combination according to claim 29 or 30, wherein the additional agent is selected from an EGFR inhibitor (such as cetuximab, panitumab, afatinib,lapatinib, erlotinib, gefitinib, osimertinib or nazartinib), a SOS inhibitor (such as BAY-293, BI-3406, or BI- 1701963), a Raf-inhibitor (e.g. belvarafenib or LXH254 (naporafenib)), an ERK inhibitor (such as LTT462 (rineterkib), GDC-0994, KO-947, Vtx-11e, SCH-772984, MK2853, LY3214996 or ulixertinib), a MEK inhibitor (such as pimasertib, PD-0325901, selumetinib, trametinib, binimetinib or cobimetinib), AKT inhibitor (such as capivasertib (AZD5363) or ipatasertib), a PI3K inhibitor (such as AMG 511, buparlisib, alpelisib), an mTOR inhibitor (such as everolimus or temsirolimus), and a CDK4/6 inhibitor (such as ribociclib, palbociclib or alemaciclib), or a pharmaceutically acceptable salt thereof. 32. A pharmaceutical combination comprising Compound A, or a pharmaceutically acceptable salt thereof, and a second agent which is selected from: (i) naporafenib (LXH254), or a pharmaceutically acceptable salt thereof,; (ii) trametinib, pharmaceutically acceptable salt or solvate thereof, e.g. the DMSO solvate thereof; (iii) rineterkib (LTT462), or a pharmaceutically acceptable salt thereof, e.g. the HCl salt thereof; (iv) alpelisib (BYL719), or a pharmaceutically acceptable salt thereof; (v) ribociclib (LEE011), or a pharmaceutically acceptable salt thereof, e.g. the succinate salt thereof; and (vi) everolimus (RAD001). or a pharmaceutically acceptable salt thereof. 33. A pharmaceutical combination comprising: (a) Compound A, or a pharmaceutically acceptable salt thereof, (b) TNO 155, or a pharmaceutically acceptable salt thereof, and a third agent which is selected from: (i) naporafenib (LXH254), or a pharmaceutically acceptable salt thereof,; (ii) trametinib, pharmaceutically acceptable salt or solvate thereof, e.g. the DMSO solvate thereof; (iii) rineterkib (LTT462), or a pharmaceutically acceptable salt thereof, e.g. the HCl salt thereof; (iv) alpelisib (BYL719), or a pharmaceutically acceptable salt thereof; (v) ribociclib (LEE011), or a pharmaceutically acceptable salt thereof, e.g. the succinate salt thereof; and (vi) everolimus (RAD001). or a pharmaceutically acceptable salt thereof. 34. A pharmaceutical combination according to any one of claims 29 to 33 for use in a method of treating a cancer or a solid tumor, wherein the method is according to any one of claims 1 to 28. 35. A compound which is 1-{6-[(4M)-4-(5-Chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1- methyl-1H-indazol-5-yl)- 1H-pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-1-one (Compound A), or a pharmaceutically acceptable salt thereof, for use in a method of treating a cancer or a tumor, according to any one of claims 1 to 28. 36. A compound for use according to claim 35 wherein the cancer or tumor is selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer and squamous cell lung cancer), colorectal cancer (including colorectal adenocarcinoma), pancreatic cancer (including pancreatic adenocarcinoma), uterine cancer (including uterine endometrial cancer), rectal cancer (including rectal adenocarcinoma), appendiceal cancer, small-bowel cancer, esophageal cancer, hepatobiliary cancer (including liver cancer and bile duct carcinoma), bladder cancer, ovarian cancer and a solid tumor a cancer of unknown primary site, particularly when the cancer or tumor harbors a KRAS G12C mutation. 37. A compound for use according to claim 36, wherein the compound is administered in combination with one or two additional therapeutically active agents. 38. A compound for use according to any one of claims 35 to 37 for use in a method of treating a cancer or a solid tumor, wherein the additional therapeutically active agent is selected from a SHP2 inhibitor (such as TNO155 (Novartis), JAB3068 (Jacobio), JAB3312 (Jacobio), RLY1971 (Roche), SAR442720 (Sanofi), RMC4450 (Revolution Medicines), BBP398 (Navire), BR790 (Shanghai Blueray), SH3809 (Nanjing Sanhome), PF0724982 (Pfizer), ERAS601 (Erasca), RX-SHP2 (Redx Pharma), ICP189 (InnoCare), HBI2376 (HUYA Bioscience), ETS001 (Shanghai ETERN Biopharma), TAS-ASTX (Taiho Oncology) and X-37-SHP2 (X-37), or a pharmaceutically acceptable salt thereof) and wherein the method further comprises administering to the subject a therapeutically effective amount of a third therapeutically active agent which is selected from a Raf-inhibitor (e.g. belvarafenib or LXH254 (naporafenib), or a pharmaceutically acceptable salt thereof), an ERK inhibitor (such as LTT462 (rineterkib), GDC-0994, KO-947, Vtx-11e, SCH-772984, MK2853, LY3214996 or ulixertinib, or a pharmaceutically acceptable salt thereof), a MEK inhibitor (such as pimasertib, PD-0325901, selumetinib, trametinib, binimetinib or cobimetinib, or a pharmaceutically acceptable salt or solvate thereof), AKT inhibitor (such as capivasertib (AZD5363) or ipatasertib, or a pharmaceutically acceptable salt thereof), a PI3K inhibitor (such as AMG 511, buparlisib, alpelisib, or a pharmaceutically acceptable salt thereof), an mTOR inhibitor (such as everolimus or temsirolimus, or a pharmaceutically acceptable salt thereof), and a CDK4/6 inhibitor (such as ribociclib, palbociclib or alemaciclib, or a pharmaceutically acceptable salt thereof). 39. A compound for use in a method of treating a cancer or a solid tumor, or a combination for use in in a method of treating a cancer or a solid tumor, or a method of treating a cancer or a solid tumor according to any one of the claims, wherein the cancer or a solid tumor is present in a patient who has previously received KRAS G12C inhibitor treatment or who is a KRAS G12C inhibitor naive patient (i.e. has not previously received KRAS G12C inhibitor treatment). |
Step C.1: tert-butyl 6-(tosyloxy)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate C2) To a solution of tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate [1147557-97- 8] (2.92 kg, 12.94 mmol) in DCM (16.5 L) were added DMAP (316.12 g, 2.59 mol) and TsCl (2.96 kg, 15.52 mol) at 20 °C-25 ºC. To the reaction mixture was added dropwise Et 3 N (2.62 kg, 25.88 mol) at 10 ºC-20 °C. The reaction mixture was stirred 0.5 h at 5 ºC-15 °C and then was stirred 1.5 h at 18 ºC - 28 °C. After completion of the reaction, the reaction mixture was concentrated under vacuum. To the residue was added NaCl (5% in water, 23 L) followed by extraction with EtOAc (23 L). The combined aqueous layers were extracted with EtOAc (10 L x 2). The combined organic layers were washed with NaHCO 3 (3% in water, 10 L x 2)) and concentrated under vacuum to give the title compound. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.81 - 7.70 (m, 2H), 7.53 - 7.36 (m, 2H), 4.79 - 4.62 (m, 1H), 3.84 - 3.68 (m, 4H), 2.46 - 2.38 (m, 5H), 2.26 - 2.16 (m, 2H), 1.33 (s, 9H). UPLC-MS-1: Rt = 1.18 min; MS m/z [M+H] + ; 368.2. Step C.2 : 3,5-dibromo-1H-pyrazole To a solution of 3,4,5-tribromo-1H-pyrazole [17635-44-8] (55.0 g, 182.2 mmol) in anhydrous THF (550 mL) was added at -78 ºC n-BuLi (145.8 mL, 364.5 mmol) dropwise over 20 min maintaining the internal temperature at -78 ºC / -60 ºC. The RM was stirred at this temperature for 45 min. Then the reaction mixture was carefully quenched with MeOH (109 mL) at -78 °C and stirred at this temperature for 30 min. The mixture was allowed to reach to 0 °C and stirred for 1 h. Then, the mixture was diluted with EtOAc (750 mL) and HCl (0.5 N, 300 mL) was added. The layers were concentrated under vacuum. The crude residue was dissolved in DCM (100 mL), cooled to -50 ºC and petroleum ether (400 mL) was added. The precipitated solid was filtered and washed with n-hexane (250 mL x2) and dried under vacuum to give the title compound. 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.5 (br s, 1H), 6.58 (s, 1H). Step C.3: tert-butyl 6-(3,5-dibromo-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-car boxylate To a solution of tert-butyl 6-(tosyloxy)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate C2) (Step C.1, 900 g, 2.40 mol) in DMF (10.8 L) was added Cs 2 CO 3 (1988 g, 6.10 mol) and 3,5-dibromo-1H-pyrazole (Step C.2, 606 g, 2.68 mol) at 15 °C. The reaction mixture was stirred at 90 °C for 16 h. The reaction mixture was poured into ice-water/brine (80 L) and extracted with EtOAc (20 L). The aqueous layer was re-extracted with EtOAc (10 L x 2). The combined organic layers were washed with brine (10 L), dried (Na 2 SO 4 ), filtered, and concentrated under vacuum. The residue was triturated with dioxane (1.8 L) and dissolved at 60 °C. To the light yellow solution was slowly added water (2.2 L), and recrystallization started after addition of 900 mL of water. The resulting suspension was cooled down to 0 °C, filtered, and washed with cold water. The filtered cake was triturated with n-heptane, filtered, then dried under vacuum at 40 °C to give the title compound. 1 H NMR (400 MHz, DMSO-d 6 ) δ 6.66 (s, 1H), 4.86 - 4.82 (m, 1H), 3.96 - 3.85 (m, 4H), 2.69 - 2.62 (m, 4H), 1.37 (s, 9H); UPLC-MS-3: Rt = 1.19 min; MS m/z [M+H] + ; 420.0 / 422.0 / 424.0. Step C.4: tert-butyl 6-(3-bromo-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane- 2-carboxylate (Intermediate C3) To a solution of tert-butyl 6-(3,5-dibromo-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2- carboxylate (Step C.3, 960 g, 2.3 mol) in THF (9.6 L) was added n-BuLi (1.2 L, 2.5 mol) dropwise at -80 °C under an inert atmosphere. The reaction mixture was stirred 10 min at -80 °C. To the reaction mixture was then added dropwise iodomethane (1633 g, 11.5 mol) at -80 °C. After stirring for 5 min at -80 °C, the reaction mixture was allowed to warm up to 18 °C. The reaction mixture was poured into sat. aq. NH 4 Cl solution (4 L) and extracted with DCM (10 L). The separated aqueous layer was re-extracted with DCM (5 L) and the combined organic layers were concentrated under vacuum. The crude product was dissolved in 1,4-dioxane (4.8 L) at 60 °C, then water (8.00 L) was added dropwise slowly. The resulting suspension was cooled to 17 °C and stirred for 30 min. The solid was filtered, washed with water, and dried under vacuum to give the title compound. 1 H NMR (400 MHz, DMSO-d 6 ) δ 6.14 (s, 1H), 4.74 - 4.66 (m, 1H), 3.95 - 3.84 (m, 4H), 2.61 - 2.58 (m, 4H), 2.20 (s, 3H), 1.37 (s, 9H); UPLC-MS-1: Rt = 1.18 min; MS m/z [M+H] + ; 356.1 / 358.1. Step C.5: tert-butyl 6-(3-bromo-4-iodo-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]h eptane-2- carboxylate (Intermediate C4) To a solution of tert-butyl 6-(3-bromo-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane- 2-carboxylate (Intermediate C3) (Step C.4, 350 g, 0.980 mol) in acetonitrile (3.5 L) was added NIS (332 g, 1.47 mol) at 15 °C. The reaction mixture was stirred at 40 °C for 6 h. After completion of the reaction, the reaction mixture was diluted with EtOAc (3 L) and washed with water (5 L x 2). The organic layer was washed with Na 2 SO 3 (10% in water, 2 L), with brine (2 L), was dried (Na 2 SO 4 ), filtered, and concentrated under vacuum to give the title compound. 1 H NMR (400 MHz, DMSO-d 6 ) δ 4.81 - 4.77 (m, 1H), 3.94 - 3.83 (m, 4H), 2.61 - 5.59 (m, 4H), 2.26 (s, 3H), 1.37 (s, 9H); UPLC-MS-1: Rt = 1.31 min; MS m/z [M+H] + ; 482.0 / 484.0. Step C.6: tert-butyl 6-(3-bromo-4-(5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl) -1H-indazol- 4-yl)-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-car boxylate (Intermediate C1) To a stirred suspension of tert-butyl 6-(3-bromo-4-iodo-5-methyl-1H-pyrazol-1-yl)-2- azaspiro[3.3]heptane-2-carboxylate (Intermediate C4) (Step C.5, 136 g, 282 mmol) and 5-chloro- 6-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl -1,3,2-dioxaborolan-2-yl)-1H- indazole (Intermediate D1, 116 g, 310 mmol) in 1,4-dioxane (680 mL) was added aqueous K 3 PO 4 (2M, 467 mL, 934 mmol) followed by RuPhos (13.1 g, 28.2 mmol) and RuPhos-Pd-G3 (14.1 g, 16.9 mmol). The reaction mixture was stirred at 80 °C for 1 h under inert atmosphere. After completion of the reaction, the reaction mixture was poured into 1M aqueous NaHCO 3 solution (1 L) and extracted with EtOAc (1L x 3). The combined organic layers were washed with brine (1 L x3), dried (Na 2 SO 4 ), filtered, and concentrated under vacuum. The crude residue was purified by normal phase chromatography (eluent: Petroleum ether / EtOAc from 1/0 to 0/1) to give a yellow oil. The oil was dissolved in petroleum ether (1 L) and MTBE (500 mL), then concentrated in vacuo to give the title compound. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.81 (s, 1H), 7.66 (s, 1H), 5.94 - 5.81 (m, 1H), 4.90 - 4.78 (m, 1H), 3.99 (br s, 2H), 3.93 - 3.84 (m, 3H), 3.81 - 3.70 (m, 1H), 2.81 - 2.64 (m, 4H), 2.52 (s, 3H), 2.46 - 2.31 (m, 1H), 2.11 - 1.92 (m, 5H), 1.82 - 1.67 (m, 1H), 1.64 - 1.52 (m, 2H), 1.38 (s, 9H); UPLC-MS-3: Rt = 1.30 min; MS m/z [M+H] + ; 604.1 / 606.1. Synthesis of Intermediate D1: 5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole
Step D.1: 1-chloro-2,5-dimethyl-4-nitrobenzene To an ice-cooled solution of 2-chloro-1,4-dimethylbenzene (3.40 kg, 24.2 mol) in AcOH (20.0 L) was added H 2 SO 4 (4.74 kg, 48.4.mol, 2.58 L) followed by a dropwise addition (dropping funnel) of a cold solution of HNO 3 (3.41 kg, 36.3 mol, 2.44 L, 67.0% purity) in H 2 SO 4 (19.0 kg, 193.mol, 10.3 L). The reaction mixture was then allowed to stir at 0 - 5 °C for 0.5 h. The reaction mixture was poured slowly into crushed ice (35.0 L) and the yellow solid precipitated out. The suspension was filtered and the cake was washed with water (5.00 L x 5) to give a yellow solid which was suspended in MTBE (2.00 L) for 1 h, filtered, and dried to give the title compound as a yellow solid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.90 (s, 1H), 7.34 (s, 1H), 2.57 (s, 3H), 2.42 (s, 3H). Step D.2: 3-bromo-2-chloro-1,4-dimethyl-5-nitrobenzene To a cooled solution of 1-chloro-2,5-dimethyl-4-nitrobenzene (Step D.1, 2.00 kg, 10.8 mol) in TFA (10.5 L) was slowly added concentrated H 2 SO 4 (4.23 kg, 43.1 mol, 2.30 L) and the reaction mixture was stirred at 20 °C. NBS (1.92 kg, 10.8 mol) was added in small portions and the reaction mixture was heated at 55 °C for 2 h. The reaction mixture was cooled to 25 °C, then poured into crushed ice solution to obtain a pale white precipitate which was filtered through vacuum, washed with cold water and dried under vacuum to give the title compound as a yellow solid which was used without further purification in the next step. 1 H NMR (400 MHz, CDCl 3 ) δ 7.65 (s, 1H), 2.60 (s, 3H), 2.49 (s, 3H). Step D.3: 3-bromo-4-chloro-2,5-dimethylaniline To an ice-cooled solution of 3-bromo-2-chloro-1,4-dimethyl-5-nitrobenzene (Step D.2, 2.75 kg, 10.4 mol) in THF (27.5 L) was added HCl (4M, 15.6 L) then Zn (2.72 kg, 41.6 mol) in small portions. The reaction mixture was allowed to stir at 25 °C for 2 h. The reaction mixture was basified by addition of a sat. aq. NaHCO 3 solution (untill pH = 8). The mixture was diluted with EtOAc (2.50 L) and stirred vigorously for 10 min and then filtered through a pad of celite. The organic layer was separated and the aqueous layer was re-extracted with EtOAc (3.00 L x 4). The combined organic layers were washed with brine (10.0 L), dried (Na 2 SO 4 ), filtered and concentrated under vacuum to give the title compound as a yellow solid which was used without further purification in the next step. 1 H NMR (400 MHz, DMSO-d 6 ) δ 6.59 (s, 1H), 5.23 (s, 2H), 2.22 (s, 3H), 2.18 (s, 3H). Step D.4: 3-bromo-4-chloro-2,5-dimethylbenzenediazonium tetrafluoroborate BF 3 .Et 2 O (2.00 kg, 14.1 mol, 1.74 L) was dissolved in DCM (20.0 L) and cooled to -5 to - 10 °C under nitrogen atmosphere. A solution of 3-bromo-4-chloro-2,5-dimethylaniline (Step D.3, 2.20 kg, 9.38 mol) in DCM (5.00 L) was added to above reaction mixture and stirred for 0.5 h. Tert-butyl nitrite (1.16 kg, 11.3 mol, 1.34 L) was added dropwise and the reaction mixture was stirred at the same temperature for 1.5 h. TLC (petroleum ether:EtOAc = 5:1) showed that starting material (Rf = 0.45) was consumed completely. MTBE (3.00 L) was added to the reaction mixture to give a yellow precipitate, which was filtered through vacuum and washed with cold MTBE (1.50 L x 2) to give the title compound as a yellow solid which was used without further purification in the next step. Step D.5: 4-bromo-5-chloro-6-methyl-1H-indazole To 18-Crown-6 ether (744 g, 2.82 mol) in chloroform (20.0 L) was added KOAc (1.29 kg, 13.2 mol) and the reaction mixture was cooled to 20 °C. Then 3-bromo-4-chloro-2,5- dimethylbenzenediazonium tetrafluoroborate (Step D.4, 3.13 kg, 9.39 mol) was added slowly. The reaction mixture was then allowed to stir at 25 °C for 5 h. After completion of the reaction, the reaction mixture was poured into ice cold water (10.0 L), and the aqueous layer was extracted with DCM (5.00 L x 3). The combined organic layers were washed with a sat. aq. NaHCO 3 solution (5.00 L), brine (5.00 L), dried (Na 2 SO 4 ), filtered and concentrated under vacuum to give the title compound as a yellow solid. 1 H NMR (600 MHz, CDCl 3 ) δ 10.42 (br s, 1H), 8.04 (s, 1H), 7.35 (s, 1H), 2.58 (s, 3H). UPLC-MS-1: Rt = 1.02 min; MS m/z [M+H] + ; 243 / 245 / 247. Step D.6: 4-bromo-5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-in dazole To a solution of PTSA (89.8 g, 521 mmol) and 4-bromo-5-chloro-6-methyl-1H-indazole (Step D.5, 1.28 kg, 5.21 mol) in DCM (12.0 L) was added DHP (658 g, 7.82 mol, 715 mL) dropwise at 25 °C. The mixture was stirred at 25 °C for 1 h. After completion the reaction, the reaction mixture was diluted with water (5.00 L) and the organic layer was separated. The aqueous layer was re-extracted with DCM (2.00 L). The combined organic layers were washed with a sat. aq. NaHCO 3 solution (1.50 L), brine (1.50 L), dried over Na 2 SO 4 , filtered and concentrated under vacuum. The crude residue was purified by normal phase chromatography (eluent: Petroleum ether/ EtOAc from 100/1 to 10/1) to give the title compound as a yellow solid. 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.04 (s, 1H), 7.81 (s, 1H), 5.88 - 5.79 (m, 1H), 3.92 - 3.83 (m, 1H), 3.80 - 3.68 (m, 1H), 2.53 (s, 3H), 2.40 - 2.32 (m, 1H), 2.06 - 1.99 (m, 1H), 1.99 - 1.93 (m, 1H), 1.77 - 1.69 (m, 1H), 1.60 - 1.56 (m, 2H). UPLC-MS-6: Rt = 1.32 min; MS m/z [M+H] + ; 329.0 / 331.0 /333.0 Step D.7: 5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-te tramethyl-1,3,2- dioxaborolan-2-yl)-1H-indazole (Intermediate D.1) A suspension of 4-bromo-5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-in dazole (Step D.6, 450 g, 1.37 mol), KOAc (401 g, 4.10 mol) and B 2 Pin 2 (520 g, 2.05 mol) in 1,4-dioxane (3.60 L) was degassed with nitrogen for 0.5 h. Pd(dppf)Cl 2 .CH 2 Cl 2 (55.7 g, 68.3 mmol) was added and the reaction mixture was stirred at 90 °C for 6 h. The reaction mixture was filtered through diatomite and the filter cake was washed with EtOAc (1.50 L x 3). The mixture was concentrated under vacuum to give a black oil which was purified by normal phase chromatography (eluent: Petroleum ether/ EtOAc from 100/1 to 10/1) to give the desired product as brown oil. The residue was suspended in petroleum ether (250 mL) for 1 h to obtain a white precipitate. The suspension was filtered, dried under vacuum to give the title compound as a white solid. 1 H NMR (400 MHz, CDCl 3 ) δ 8.17 (d, 1H), 7.52 (s, 1H), 5.69 - 5.66 (m, 1H), 3.99 - 3.96 (m, 1H), 3.75 – 3.70 (m, 1H), 2.51 (d, 4H), 2.21 - 2.10 (m, 1H), 2.09 - 1.99 (m, 1H), 1.84 - 1.61 (m, 3H), 1.44 (s, 12H); UPLC- MS-6: Rt = 1.29 min; MS m/z [M+H] + ; 377.1 / 379. Synthesis of Compound A
Step 1: Tert-butyl 6-(4-(5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-inda zol-4-yl)-5- methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl)-2-azasp iro[3.3]heptane-2-carboxylate In a 500 mL flask, tert-butyl 6-(3-bromo-4-(5-chloro-6-methyl-1-(tetrahydro-2H-pyran- 2-yl)-1H-indazol-4-yl)-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[ 3.3]heptane-2-carboxylate (Intermediate C1, 10 g, 16.5 mmol), (1-methyl-1H-indazol-5-yl)boronic acid (6.12 g, 33.1 mmol), RuPhos (1.16 g, 2.48 mmol) and RuPhos-Pd-G3 (1.66 g, 1.98 mmol) were suspended in toluene (165 mL) under argon. K 3 PO 4 (2M, 24.8 mL, 49.6 mmol) was added and the reaction mixture was placed in a preheated oil bath (95 °C) and stirred for 45 min. The reaction mixture was poured into a sat. aq. NH 4 Cl solution and was extracted with EtOAc (x3). The combined organic layers were washed with a sat. aq. NaHCO 3 solution, dried (phase separator) and concentrated under reduced pressure. The crude residue was diluted with THF (50 mL), SiliaMetS®Thiol (15.9 mmol) was added and the mixture swirled for 1 h at 40 °C. The mixture was filtered, the filtrate was concentrated and the crude residue was purified by normal phase chromatography (eluent: MeOH in CH 2 Cl 2 from 0 to 2%), the purified fractions were again purified by normal phase chromatography (eluent: MeOH in CH 2 Cl 2 from 0 to 2%) to give the title compound as a beige foam. UPLC-MS-3: Rt = 1.23 min; MS m/z [M+H] + ; 656.3 / 658.3. Step 2: 5-Chloro-6-methyl-4-(5-methyl-3-(1-methyl-1H-indazol-5-yl)-1 -(2-azaspiro[3.3]heptan- 6-yl)-1H-pyrazol-4-yl)-1H-indazole TFA (19.4 mL, 251 mmol) was added to a solution of tert-butyl 6-(4-(5-chloro-6-methyl- 1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-5-methyl-3-(1- methyl-1H-indazol-5-yl)-1H- pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate (Step 1, 7.17 g, 10.0 mmol) in CH 2 Cl 2 (33 mL). The reaction mixture was stirred at RT under nitrogen for 1.5 h. The RM was concentrated under reduced pressure to give the title compound as a trifluoroacetate salt, which was used without purification in the next step. UPLC-MS-3: Rt = 0.74 min; MS m/z [M+H] + ; 472.3 / 474.3. Step 3: 1-(6-(4-(5-Chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-me thyl-1H-indazol-5-yl)- 1H-pyrazol-1-yl)-2-azaspiro[3.3]heptan-2-yl)prop-2-en-1-one A mixture of acrylic acid (0.69 mL, 10.1 mmol), propylphosphonic anhydride (50% in EtOAc, 5.94 mL, 7.53 mmol) and DIPEA (21.6 mL, 126 mmol) in CH 2 Cl 2 (80 mL) was stirred for 20 min at RT and then added (dropping funnel) to an ice-cooled solution of 5-chloro-6- methyl-4-(5-methyl-3-(1-methyl-1H-indazol-5-yl)-1-(2-azaspir o[3.3]heptan-6-yl)-1H-pyrazol-4- yl)-1H-indazole trifluoroacetate (Step 2, 6.30 mmol) in CH 2 Cl 2 (40 mL). The reaction mixture was stirred at RT under nitrogen for 15 min. The RM was poured into a sat. aq. NaHCO 3 solution and extracted with CH 2 Cl 2 (x3). The combined organic layers were dried (phase separator) and concentrated. The crude residue was diluted with THF (60 mL) and LiOH (2N, 15.7 mL, 31.5 mmol) was added. The mixture was stirred at RT for 30 min until disappearance (UPLC) of the side product resulting from the reaction of the acryloyl chloride with the free NH group of the indazole then was poured into a sat. aq. NaHCO 3 solution and extracted with CH 2 Cl 2 (3x). The combined organic layers were dried (phase separator) and concentrated. The crude residue was purified by normal phase chromatography (eluent: MeOH in CH 2 Cl 2 from 0 to 5%) to give the title compound. The isomers were separated by chiral SFC (C-SFC-1; mobile phase: CO 2 /[IPA+0.1% Et3N]: 69/31) to give Compound A, i.e. a(R)-1-(6-(4-(5-chloro-6-methyl- 1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-py razol-1-yl)-2- azaspiro[3.3]heptan-2-yl)prop-2-en-1-one, as the second eluting peak (white powder): 1 H NMR (600 MHz, DMSO-d 6 ) δ 13.1 (s, 1H), 7.89 (s, 1H), 7.59 (s, 1H), 7.55 (s, 1H), 7.42 (m, 2H), 7.30 (d, 1H), 6.33 (m, 1H), 6.12 (m, 1H), 5.68 (m, 1H), 4.91 (m, 1H), 4.40 (s, 1H), 4.33 (s, 1H), 4.11 (s, 1H), 4.04 (s, 1H), 3.95 (s, 3H), 2.96-2.86 (m, 2H), 2.83-2.78 (m, 2H), 2.49 (s, 3H), 2.04 (s, 3H); UPLC-MS-4: Rt = 4.22 min; MS m/z [M+H] + 526.3 / 528.3; C-SFC-3 (mobile phase: CO 2 /[IPA+0.1% Et 3 N]: 67/33): Rt = 2.23 min. The compound of Example 1 is also referred to as “Compound A”. The atropisomer of Compound A, a(S)-1-(6-(4-(5-chloro-6-methyl-1H-indazol-4-yl)-5- methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl)-2-azasp iro[3.3]heptan-2-yl)prop-2-en-1- one was obtained as the first eluting peak: C-SFC-3 (mobile phase: CO 2 /[IPA+0.1% Et 3 N]: 67/33): Rt = 1.55 min. Example 2: Compound A (JDQ443) shows anti-tumor activity in KRAS G12C-mutated CDX models, driven by target occupancy Single-agent antitumor activity of JDQ443 at daily oral doses of 10 mg/kg, 30 mg/kg and 100 mg/kg, in a panel of KRAS G12C-mutated CDX models across different indications. Cell lines for xenografting were: MIA PaCa-2 (PDAC); NCI-H2122, LU99, HCC-44, NCI-H2030 (NSCLC); and KYSE410 (esophageal cancer). JDQ443 inhibited the growth of all models in a dose-dependent manner (Fig.8A), with model-specific differences in dose-response dynamics and maximal response patterns that ranged from regression (MIA PaCa-2, LU99), to stasis (HCC44, NCI-H2122), to moderate tumor inhibition (NCI-H2030, KYSE410). The largest dynamic range was observed in LU99. In contrast, JDQ443 showed no growth inhibition in a KRASG12V-mutated xenograft model (NCI-H441; Fig.8B), confirming KRASG12C specificity and consistent with the in vitro data. Efficacy was maintained across once- (QD) or twice-daily (BID) administration of the same daily dose: 30 mg/kg QD versus 15 mg/kg BID in MIA PaCa-2 (Fig.8C), or 100 mg/kg QD versus 50 mg/kg BID in NCI-H2122 and LU99 (Fig.8D-E). The efficacy of QD vs BID dosing correlated well with comparable daily area under the concentration-time curve (AUC) in blood. These findings suggested that JDQ443 efficacy is related to target occupancy (TO), and that efficacious AUC exposures can be achieved under both QD and BID dosing. To characterize whether AUC can act as a surrogate for TO, the effect of continuous infusion versus oral dosing in the LU99 xenograft model was investigated. Once-daily oral dosing at 30 mg/kg induced stasis for about one week followed by tumor progression, and 100 mg/kg induced tumor regression (Fig.8F), with approximate steady-state average concentrations (Cav) of 0.3 µM and ~1 µM, respectively. To assess continuous dosing, JDQ443 was delivered intravenously via programmable microinfusion pumps to achieve target concentrations approximating the oral Cav. Continuous infusion and oral dosing resulted in comparable antitumor responses (Fig. 8F,G). PK/PD model simulation showed that efficacy correlates best with TO and the AUC of JDQ443 (Fig.8H, I), rather than other PK metrics. Example 3: Compound A potently inhibits KRAS G12C H95Q, a double mutant mediating resistance to adagrasib in clinical trials GFP-tagged KRASG12C H95Q, KRASG12C Y96D or KRASG12C R68S double mutations were generated by site–directed mutagenesis (QuikChange Lightning Site-Directed Mutagenesis Kit (Catalog # 210518) Template: pcDNA3.1(+)EGFP-T2A-FLAG-KRAS G12C and expressed in Cas9 containing Ba/F3 cells by stable transfection. Cells were treated with a dose response curve starting at 10μM with 1/3 dilution from a 10mM DMSO stock. Cell lines were treated with indicated compounds for 72 hours and the viabilities of the cells were measured with CellTiter-Glo. Results: In contrast to MRTX-849 (adagrasib), JDQ443 (Compound A) and AMG-510 (sotorasib) are potently inhibiting the cellular viability of the KRASG12C H95Q double mutant. KRASG12C Y96D or KRASG12C R68S double mutant are not inhibited by MRTX-849, AMG- 510 or JDQ443 at the indicated concentrations and in the described setting (Ba/F3 system, 3-day proliferation assay) and confer resistance to all three tested KRASG12C inhibitors. Conclusion: Compound A might overcome resistance towards adagrasib in the KRASG12C H95Q setting. In addition, since Compound A has unique binding interactions with mutated KRAS G12C, when compared with sotorasib and adagrasib, Compound A, alone or in combination with one or more therapeutic agent as described herein, may be useful to treat patients suffering from cancer who have previously been treated with other KRAS G12C inhibitors such as sotorasib or adagrasib, or to target resistance after an acquired KRAS resistance mutation emerges on the initial KRAS G12C inhibitor treatment. Example 4: Compound A potently inhibits KRAS G12C double mutants The effect of Compound A and other KRASG12C inhibitors on second-site mutations reported to confer resistance to adagrasib was also investigated as follows. Materials and Methods: Cell lines and KRAS G12C Inhibitors: The Ba/F3 cell line is a murine pro-B-cell line and is cultured in RPMI 1640 (Bioconcept, #1-41F01-I) supplemented with 10 % Fetal Bovine Serum (FBS) (BioConcept, #2- 01F30-I), 2 mM Sodium pyurvate (BioConcept, # 5-60F00-H), 2 mM stable Glutamine (BioConcept, # 5-10K50-H), 10 mM HEPES (BioConcept, # 5-31F00-H) and at 37 °C with 5 % CO2, except as otherwise indicated. The parental Ba/F3 cells were cultured in the presence of 5 ng/ml of recombinant murine IL-3 (Life Technologies, #PMC0035). Ba/F3 cells are normally dependent on IL-3 to survive and proliferate, however, by expressing oncogenes they are able to switch their dependency from IL-3 to the expressed oncogene (Curr Opin Oncology, 2007 Jan;19(1):55-60. doi: 10.1097/CCO.0b013e328011a25f.) Individual plasmid mutagenesis and generation of Ba/F3 stable cell lines: QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent; # 210519) was used to generate the resistant mutations on the pSG5_Flag-(codon optimized) KRAS G12C _puro plasmid template and sequences were confirmed by sanger sequencing. The mutant plasmids were transfected into the Ba/F3 WT cells by electroporation with the NEON transfection kit (Invitrogen, #MPK10025). Therefore, two million Ba/F3 cells have been electroporated with 10 μg pf plasmids with the NEON System (Invitrogen, #MPK5000), using following conditions Voltage (V) 1635, Width (ms) 20, Pulses 1. After 72 h of electroporation, puromycin selection was performed at 1 μg / ml to generate stable cell lines. IL-3 withdrawal Ba/F3 cells are normally dependent on IL-3 to survive and proliferate, however, by expressing oncogenes they are able to switch their dependency from IL-3 to the expressed oncogene. To assess whether the KRAS G12C single and double mutants are able to sustain the proliferation of Ba/F3 cells, the engineered Ba/F3 cells expressing the mutant constructs were cultured in absence of IL-3. Cell number and viability was measured every three days and after seven days the IL-3 withdrawal was completed. The expression of the mutants after the IL-3 withdrawal were confirmed by Western Blot (data not shown, an upwards shift was observed for KRAS G12C/R68S ). Drug response curves for KRASG12C inhibitors and validation of resistance mutations: 1000 Ba/F3 cells/well were seeded at in 96-well plates (Greiner Bio-One, #655098). Treatment was performed on the same day with a Tecan D300e drug dispenser. Viability was detected on the same day of treatment for the start plate (Day 0) and three days post-treatment (Day 3) using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, #G7573) on a Tecan infinitiy M200 Pro reader (Intergration Time 1000ms). To determine the growth, the three days post-treatment (Day 3) readout was normalized to start plate (Day 0). The percentage viability was then calculated by normalizing treated wells to DMSO treated control samples. XLfit was used to make the fitted curve with a Sigmoidal Dose-Response Model (four-parameter curve) (Figure 9). The horizontal red dotted line represents the GI50 value. Tabular data are shown below. Table: Effect of Compound A (JDQ443) on the the proliferation of KRAS G12C/H95 double mutants. («STDEV» indicates the standard deviation for the % growth value)
Table: Effect of sotorasib (AMG510) on the the proliferation of KRAS G12C/H95 double mutants («STDEV» indicates the standard deviation for the % growth value)
Table: Effect of adagrasib (MRTX-849) on the the proliferation of KRAS G12C/H95 double mutants («STDEV» indicates the standard deviation for the % growth value) Western blot After treatment with the different compounds at the indicated concentrations and for the indicated time, the cells were collected, pelleted and snap frozen at - 80 °C. Sixty μL of lysis buffer (50 mM Tris HCl, 120 mM NaCl, 25 mM NaF, 40 mM β-glycerol phosphate disodium salt pentahydrate, 1% NP40, 1 μM microcystin, 0.1 mM Na3VO3, 0.1 mM PMSF, 1 mM DTT and 1 mM benzamidine, supplemented with 1 protease inhibitor cocktail tablet (Roche) for 10 mL of buffer) was added to each sample. The samples were then vortexed, incubated on ice for 10 min, vortexed again and centrifuged at 14000 rpm at 4 °C for 10 min. Protein concentration was determined with the BCA Protein Assay kit (Pierce, 23225). After normalization to the same total volume with lysis buffer, NuPAGE™ LDS Sample buffer 4 X (Invitrogen, NP0007) and NuPAGE™ Sample reducing agent 10 X (Invitrogen, NP0009) was added. The samples were heated at 70 °C for 10 min before loading on a NuPAGE™ Novex™ 4 – 12 % Bis-Tris Midi Protein Gel, 26 - wells (Invitrogen, WG1403A). Gels were run for 45 min at 200 V (PowerPac HC, Biorad) in NuPAGE MES SDS running buffer (Invitrogen, NP0002). The proteins were transferred for 7 min at 135 mA per gel on a Trans-Blot® Turbo™ Midi Nitrocellulose Transfer Packs membrane (Biorad, 1704159) using the Trans-Blot® Turbo™ system (Biorad) before staining the membrane with Ponceau red (Sigma, P7170). The membranes were blocked with TBST with 5 % of milk at RT. Anti-RAS (Abcam, 108602) and anti-phospho-ERK 1/2 p44/42 MAPK (Cell Signaling, 4370) antibodies were incubated overnight at 4 °C, the anti-vinculin (Sigma, V9131) antibody was incubated for 1 h at RT. Membranes were washed 3 X for 5 min with TBST and the anti-rabbit (Cell Signaling, 7074) and anti-mouse (Cell Signaling, 7076) secondary antibodies were incubated for 1 h at RT. All antibodies were diluted in TBST to 1/1000, except of anti-vinculin (1/3000). Revelation was performed with WesternBright ECL (Advansta, K-12045-D20) for Ras and vinculin and with SuperSignal West Femto maximum sensitivity substrate (Thermo Fischer, 34096), on a Fusion FX (Vilber Lourmat) using the FusionCapt Advance FX7 software. (Figure 10). Results Table: Compound A (JDQ443) inhibits the proliferation of KRAS G12C/H95 double mutants. Ba/F3 cells expressing the indicated FLAG-KRAS G12C single or double mutants were treated with JDQ443 (Compound A, AMG-510 (sotorasib) and MRTX-849 (adagrasib) (8-point dilution starting at 1 mM) for 3 days and the inhibtion of proliferation was assessed by Cell titer glo viability assay. The average of GI 50 ± standard deviation (St DV) of 4 independent experiments are shown. Biophysical data Material and methods: Preparation of reagents: Cloning, expression and purification of RAS protein constructs The E. coli expression constructs used in this study were based on the pET system and generated using standard molecular cloning techniques. Following the cleavable N-terminal his affinity purification tag the cDNA encoding KRAS, NRAS, and HRAS comprised aa 1-169 and was codon-optimized and synthesized by GeneArt (Thermo Fisher Scientific). Point mutations were introduced with the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent). All final expression constructs were sequence verified by Sanger sequencing. Two liters of culture medium were inoculated with a pre-culture of E. coli BL21(DE3) freshly transformed with the expression plasmid and protein expression induced with 1 mM isopropyl-β-D-thiogalactopyranoside (Sigma) for 16 hours at 18 °C. Proteins with an avi-tag were transformed into E. coli harboring a compatible plasmid expressing the biotin ligase BirA and the culture medium was supplemented with 135 µM d-biotin (Sigma). Cell pellets were resuspended in buffer A (20 mM Tris, 500 mM NaCl, 5 mM imidazole, 2 mM TCEP, 10 % glycerol, pH 8.0) supplemented with Turbonuclease (Merck) and cOmplete protease inhibitor tablets (Roche). The cells were lysed by three passages through a homogenizer (Avestin) at 800-1000 bar and the lysate clarified by centrifugation at 40000 g for 40 min. The lysate was loaded onto a HisTrap HP 5 ml column (Cytiva) mounted on an ÄKTA Pure 25 chromatography system (Cytiva). Contaminating proteins were washed away with buffer A and bound protein was eluted with a linear gradient to buffer B (buffer A supplemented with 200 mM imidazole). During dialysis O/N the N-terminal His affinity purification tags on the non- tagged and avi-tagged proteins were cleaved off by TEV or HRV3C protease, respectively. The protein solution was re-loaded onto a HisTrap column and the flow through containing the target protein collected. Guanosine 5’-diphosphate sodium salt (GDP, Sigma) or GppNHp-Tetralithium salt (Jena Bioscience) was added to a 24-32x molar excess over protein. EDTA (pH adjusted to 8) was added to a final concentration of 25 mM. After 1 hour at room temperature the buffer was exchanged on a PD-10 desalting column (Cytiva) against 40 mM Tris, 200 mM (NH4)2SO4, 0.1 mM ZnCl2, pH 8.0. GDP (for KRAS G12C resistance mutants H95Q/D/R, Y96D/C and R68S) or GppNHp was added to a 24-32x molar excess over protein to the eluted protein.40 U Shrimp Alkaline Phosphatase (New England Biolabs) was added to GppNHp containing samples only. The sample was then incubated for 1 hour at 5°C. Finally, MgCl2 was added to a concentration of about 30 mM. The protein was then further purified over a HiLoad 16/600 Superdex 200 pg column (Cytiva) pre-equilibrated with 20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 2 mM TCEP, pH 7.5. The purity and concentration of the protein was determined by RP-HPLC, its identity was confirmed by LC-MS. Present nucleotide was determined by ion-pairing chromatography [Eberth et al, 2009]. Determination of covalent rate constants by RapidFire MS Assay and curve fitting Serial dilutions of the test compounds (50 µM, ½ dilutions) were prepared in 384well plates and incubated with 1 µM KRAS G12C (with/without additional mutants) in 20mM Tris pH7.5, 150mM NaCl, 100 µM MgCl 2 , 1% DMSO at room temperature. Reactions were stopped at different time points by addition of formic acid to 1%. MS measurements were carried out using a Agilent 6530 quadrupole time-of-flight (QToF) MS system coupled to an Agilent RapidFire autosampler RF360 device, resulting in % modification values for each well. In parallel, compound solubility was assessed by nephelometry and compound concentrations resulting in measurable turbidity were excluded from curve fitting. Plotting the % modification vs. time allowed for extraction of kobs values for the different compound concentrations. In a second step, the obtained kobs values were plotted against the compound concentrations. Rate constants (i.e. kinact/KI) were derived from the initial linear part of the resulting curves. MS measurements The RapidFire autosampler RF 360 was used to perform the injections. Solvents were delivered by Agilent 1200 pumps. A C18 Solid Phase Extraction (SPE) cartridge was used for all experiments. A volume of 30 μL was aspirated from each well of a 384-well plate. The sample load/wash time was 3000 ms at a flow rate of 1.5 mL/min (H2O, 0.1% formic acid); elution time was 3000 ms (acetonitrile, 0.1% formic acid); reequilibration time was 500 ms at a flow rate of 1.25 mL/min (H2O, 0.1% formic acid). Mass spectrometry (MS) data were acquired on an Agilent 6530 quadrupole time-of- flight (QToF) MS system, coupled to a dual Electrospray (AJS) ion source, in positive mode. The instrument parameters were as follows: gas temperature 350 °C, drying gas 10 L/min, nebulizer 45 psi, sheath gas 350 °C, sheath gas flow 11 L/min, capillary 4000 V, nozzle 1000 V, fragmentor 250 V, skimmer 65 V, octapole RF 750 V. Data were acquired at the rate of 6 spectra/s. The mass calibration was performed over the 300–3200 m/z range. All data processing was performed using a combination of Agilent MassHunter Qualitative Analysis, Agilent Rapid-Fire control software, and the Agilent DA Reprocessor Offline Utilities. A Maximum Entropy algorithm produced zero-charge spectra in separate files per injection. A batch processing generated a single file incorporating all mass spectra in a text format as x,y coordinates. This file was used to calculate the % of protein modification in each well. Results Quantification of the second order rate constants for modification for the indicated constructs (all GDP-loaded) was carried out using kinetic MS experiments, measuring %modification at different time points for a range of compound concentrations. K inact /K I was extrapolated from the initial slope of the k obs vs. compound concentration plot. Activities against KRAS G12D:GDP were set to 1 and relative activities for the resistance mutants are given. Average values of n=4 experiments for KRAS G12C, n=3 for G12C_Y96D and n=2 for other mutants are given in the Table below. Table: Fold change of second order rate constant ( K inact /K I ) for resistance mutants relative to KRAS G12C Quantification of the second order rate constants for modification for the indicated constructs (all GDP-loaded) was carried out using kinetic MS experiments, measuring %modification at different time points for a range of compound concentrations.K inact /KI was extrapolated from the initial slope of the K obs vs. compound concentration plot. Average values of n=4 experiments for KRAS G12C, n=3 for G12C_Y96D and n=2 for other mutants are given. Table: Second order rate constants (K inact /KI [mM-1*s-1]) for Compound A (JDQ443), sotorasib and adagrasib against resistance mutants Conclusions First generation KRAS G12C inhibitors have shown efficacy in clinical trials. However, the emergence of mutations that disrupt inhibitor binding and reactivation in downstream pathways, limits the duration of response. Second-site mutants reported to confer resistance to adagrasib in clinical trials (ref: N Engl J Med.2021 Jun 24;384(25):2382-2393. doi: 10.1056/NEJMoa2105281., Cancer Discov.2021 Aug;11(8):1913-1922. doi: 10.1158/2159- 8290.CD-21-0365. Epub 2021 Apr 6.PMID: 33824136.) were expressed in Ba/F3 cells and analyzed for their sensitivity towards Compound A (JDQ443) in comparison to KRAS G12C (GI 50 = 0.115 ± 0.060 mM). As expected from the binding mode, Compound A inhibited proliferation and signaling of KRAS G12C H95 double mutants. Compound A potently inhibited the proliferation of G12C/H95R and G12C/H95Q (GI 50 = 0.024 ± 0.006 mM, GI 50 = 0.284 ± 0.041 mM, respectively), while expression of G12C/R68S, G12C/Y96C and G12C/Y96D conferred resistance to Compound A (GI 50 >1 mM, all). Surprisingly, expression of G12C/H95D resulted in reduced sensitivity to Compound A (GI 50 = 0.612 ± 0.151 mM) compared to H95R or Q although Compound A is not directly interacting with Histidine 95. Western blot analysis of pERK upon Compound A treatment as well as the analysis of the rate constants of Compound A (biophysical data, above) towards these clinically observed SWII pocket mutations in biophysical settings were in agreement with the cellular growth inhibition data (see table). The difference between H95D compared to H95R or Q could be due the negative charge of the aspartate, which could further increase the negative electrostatic potential of the KRAS G12C surface. This might affect ligand recognition and therefore decrease the specific reactivity and cellular activity of Compound A for this mutant. Another possible explanation is that the H95D mutation could affect KRAS dynamic so that the conformation allowing Compound A binding becomes less accessible. In conclusion, the data show Compound A should overcome adagrasib induced resistance in G12C/Q95R or G12C/H95Q settings. Compound A treatment, particularly in combinations of the invention may still be useful in the G12C/H95Q setting where it has shown activity. Example 5: JDQ443 antitumor efficacy in vivo is enhanced in combination with inhibitors of RAS-upstream and RAS-downstream signaling The antitumor efficacy of JDQ443 ± inhibitors of RAS-upstream or RAS-downstream signaling was evaluated in PDX panels of human KRAS G12C-mutated NSCLC and CRC. Patient-derived xenograft (PDX) models of human NSCLC and CRC were established by direct implantation of patient NSCLC or CRC tumor tissue subcutaneously into nude mice. PDX models were maintained through in vivo serial passaging. A cohort of mice was implanted subcutaneously with tumor fragments from each PDX model (typically passages 4-9). Ten NSCLC and nine CRC PDX models were used. Each model is named with a code, e.g.30580-HX, 30581-HX etc, for identification and tracking purposes. Individual mice were assigned to treatment groups or control groups for dosing once their tumor volume reached 200-250mm 3 (T=0, on the x-axis of the spider plots). One animal per PDX model was assigned to each treatment arm. Once enrolled into treatment arms, tumor volumes were measured twice weekly by caliper, and tumor volume was estimated in mm 3 using the formula: Length x Width 2 /2. The end of study per model was defined as minimum of 28 days treatment, or duration for untreated tumor to reach 1500mm 3 , or duration for 2 doublings of untreated tumor, whichever was slower. Mice were treated orally with KRAS G12C inhibitor (Compound A at 100 mg/kg QD) alone or in combination with the combination partner as described in the Tables below. For example, Compound A was dosed at 100 mg/kg once daily (QD) in combination with LXH254 (naporafenib) at 50 mg/kg twice daily (BID). Dual combinations Triple combinations
Compound A and TNO155 were formulated as a suspension in 0.1% Tween 80 and 0.5% Methylcellulose in water. The Raf inhibitor (LXH254 (naporafenib)) was formulated as a suspension.The MEK inhibitor (trametinib) was formulated as a suspension in 0.2% Tween 80, 0.5% hydroxypropyl methylcellulose (HPMC), pH adjusted to pH ~8. The ERK inhibitor (LTT462 (rineterkib)) was formulated as a suspension in 0.5% hydroxypropyl cellulose (HPC)/0.5% Pluronic in pH 7.4 phosphate-buffered saline (PBS) buffer, pH 4. The CDK4/6 inhibitor (LEE011) was formulated as a suspension in 0.5% methylcellulose. The PI3K inhibitor (BYL719) was formulated as a suspension in 0.5% Tween 80 and 1% carboxymethylcellulose in water. The mTOR inhibitor (RAD001) was formulated in 5% glucose. The control groups were not treated. Results: Tumor volume improvement and objective antitumor responses were greater for all combination treatments than for JDQ443 monotherapy in both the NSCLC and CRC models (Figures 1-6). Similarly, combination treatment benefits were observed for time to tumor volume doubling in both models (Figure 7). In CRC models, Compound A treatment alone caused a moderate anti-tumor response in a few models. Compound A in combination with each of the combination partners improved the anti-tumor response. Triple combinations appeared to improve the response further (Figures 1 and 2). In NSCLC models, Compound A treatment alone caused no to moderate anti-tumor response in half of the models and a good anti-tumor response in the other half of the models. Compound A in combination with each of the combination partners improved the anti-tumor response (Figures 3, 4 and 5). Example 6: PI3K inhibitors in combination with a KRAS G12C inhibitor alone or in the presence of a SHP2 inhibitor show highest synergy scores in a 3-day proliferation assay. Matrix combination proliferation assays (treatment time 3 days, cell titer glow assay) were performed with a KRAS G12C inhibitor (labelled “KRAS G12C i” in Figure 11) as single agent or in combination with 10 μM SHP099, a SHP2 inhibitor, (labelled “SHP2i” in Figure 11) in the presence of either upstream receptor kinase inhibitors BGJ398, an FGFR inhibitor (labelled “FGFRi” in Figure 11), and erlotinib, an EGFR inhibitor (labelled “EGFRi” in Figure 11) or trametinib, a MEK inhibitor (labelled as “MEKi” in Figure 11) or the PI3K effector arm inhibitors alpelisib (labelled “PI3Kαi” in Figure 11) and GDC0941, a pan-PI3K inhibitor (labelled “panPI3Ki” in Figure 11) in a KRAS G12C mutated H23 cell line. Synergy scores (SS) were calculated by Loewe index and are indicated as “SS” values on top of each grid. Values in the grid are growth inhibition (%) values: a value higher than 100% indicates cell death. Growth inhibition %: 0-99 = delayed proliferation, 100= growth arrest/stasis, 101-200= reduction in cell number/cell death. The values on the x-axis of each grid indicate the concentration (in μM) of the KRASG12c inhibitor used. The values on the y-axis of each grid shows the concentration (in μM) of the second agent (i.e the FGFR inhibitor, the EGFR inhibitor, the MEK inhibitor, the PI3αK inhibitor and the pan-PI3K inhibitor respectively). As shown in Figure 11A and Figure 11B, the addition of a SHP2 inhibitor to a dual combination of a KRASG12C inhibitor and a second agent selected from an FGFR inhibitor, an EGFR inhibitor, a MEK inhibitor and a PI3K inhibitor increases the synergy score. For example, the synergy score increases from 1.522 for a dual combination of a KRASG12 C inhibitor and an EGFR inhibitor.to 3.533 for a triple combination of a KRASG12 C inhibitor, an EGFR inhibitor and a SHP2 inhibitor. Highest synergy scores were obtained in the presence of a PI3K inhibitor in combination with a KRAS G12C inhibitor alone or in the presence of a SHP2 inhibitor (Figure 11A and Figure 11 B). Example 7: Beneficial eff Dose response of JDQ443 in combination with Erlotinib or Cetuximab in NSCLC cell linesects of a combination of Compound A and ribociclib on a NSCLC xenograft model. A combination study of Compound A with ribociclib was conducted in a KRAS G12C and CDKN2A-mutated LU99 xenograft model in mice. Compound A single-agent induced tumor regression for approximately two and a half weeks, followed by tumor relapse while treatment was still ongoing. Ribociclib single-agent did not have any effect on tumor growth. The combination significantly improved the sustainability of response and time to relapse seen with Compound A as a single agent. Example 8: Compound A in combination with a SHP2 inhibitor, a PI3K inhibitor or a CDK4/6 inhibitor delays time to progression (TPP) compared to single agent treatment with Compound A in a NSCLC xenograft model. An in vivo efficacy study of Compound A (JDQ443) as single agent or in combination(double, triple, quadruple) with TNO155 (a SHP2 inhibitor), BYL719 (alpelisib, a PI3K inhibitor) and LEE011 (ribociclib, a CDK4/6 inhibitor) was conducted in a KRAS G12C, PIK3CA and CDKN2A-mutated LU99 xenograft model in mice. Daily dosing with JDQ443 at 100 mg/kg induced deep tumor regression for approximately two and a half weeks, followed by tumor relapse while treatment was still ongoing. TNO155 given at 7.5 mg/kg daily did not have any effect on tumor growth compared to the vehicle group. Double combinations of JDQ443 with TNO155, BYL719 or LEE011, triple combinations of JDQ443 and TNO155 with BYL719 or LEE011, and the quadruple combination of JDQ443 with TNO155, BYL719 and LEE011 improved the sustainability of response and time to progression seen with JDQ443 as a single agent in following order: single agent < double combination < triple combination < quadruple combination (Figure 12). Example 9: Dose response of Compound A (JDQ443) in combination with an EGFR inhibitor in NSCLC cell lines and CRC cell lines A combination of cetuximab and Compound A brings additive benefit to Compound A treatment and cetuximab treatment in a CRC cell line ( SW1463) (Figure 13, top panel). The % growth inhibition was also increased with a combination of erlotinib or cetuximab with Compound A in NSCLC (NCI-H358 and NCI-H2122) cell lines (Figure 13 center and bottom panels). Example 10: Effect of Compound A, SOS-inhibitor BI-3406 and a combination of Compound A, SOS- inhibitor BI-3406 on NSCLC and CRC cell lines. Matrix combination proliferation assays were performed as follows. For each of the cell lines, cells were dispensed into tissue culture treated 384-well plates (Greiner #781098) in a final volume of 25 μL per well. Cells were allowed to adhere and begin growth for twenty-four hours. On plate was counted prior treatment (= Day 1), and the other plate was treated with compounds or DMSO using a HP D300 digital dispenser. After seventy-two hours the medium was refreshed by supplementing 25 µl per well of culture medium containing the corresponding compounds or DMSO. All treatments were done in triplicates. Seven days after treatment initiation, cell growth was determined using CellTiter-Glo® (Promega #G7573), which measures the amount of ATP in the well. Plates were equilibrated to room temperature for approximately thirty minutes and one volume of CellTiter-Glo® Reagent equal to the volume of cell culture medium was added. Cell lysis was induced for two minutes on an orbital shaker, the plates were incubated at room temperature for ten minutes, and luminescence was recorded. Cells were treated with the indicated final concentrations of compounds. Dose response curves were derived using XLfit dose response one site, model 205. Reported is the percentage of growth inhibition versus DMSO (percentage GI) after subtracting the reads of Day 1. Low growth inhibition was observed with single agent treatment with SOS-inhibitor BI- 3406. Combination benefit was observed with the addition of a KRAS G12C inhibitor (Figure 14). Example 11: Clinical efficacy of Compound A as monotherapy and combination therapy A phase Ib/II open-label, multi-center, dose escalation study of Compound A (JDQ443) alone and in combination with specific agents is conducted in patients with advanced solid tumors harboring the KRAS G12C mutation, including KRAS G12C-mutated NSCLC and KRAS G12C-mutated colorectal cancer (KontRASt-01 (NCT04699188)). The study is conducted to evaluate the antitumor efficacy, safety and tolerability of JDQ443 as a single agent and JDQ443 in combination with other agents. JDQ443 + TNO155 and JDQ443 + a PD1- inhibitor such as tislelizumab may be used to treat patients suffering KRAS G12C-mutated solid tumors. Patients to be treated include patients with advanced, KRAS G12C-mutated solid tumors who have received standard-of-care therapy, or who are intolerant of or ineligible for approved therapies; or , Eastern Cooperative Oncology Group Performance Status (ECOG PS 0–1); or had no prior treatment with KRAS G12C inhibitors. Key exclusion criteria for the JDQ443 monotherapy arm are: active brain metastases and/or prior KRASG12C inhibitor treatment. Patients with NSCLC include patients previously treated with a platinum-based chemotherapy regimen and an immune checkpoint inhibitor, either in combination or in sequence, unless ineligible to receive such therapy. Patients with CRC include patients who have previously received standard-of-care therapy, including fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy, unless ineligible to receive such therapy. The preliminary data from the monotherapy dose escalation arm study are as follows. At a cut-off date of January 5, 2022, 39 patients were treated with 200 mg QD, 400 mg QD, 200 mg BID or 300 mg BID of Compound A. Compound A was administered with food. Patients had a median of 3 prior lines of anti-neoplastic therapy. The recommended dose for the monotherapy is a dose of 200 mg of Compound A taken orally twice daily (BID). Efficacy data (cutoff of 05 Jan 2022) from the pooled Phase Ib JDQ443 single agent cohort (n=39) showed: • 57% (4/7) confirmed overall response rate (ORR) at 200 mg BID in NSCLC • 45% (9/20) confirmed and unconfirmed ORR across doses in NSCLC • 35% (7/20) confirmed ORR across doses in NSCLC • PD/PK modeling predicted sustained, high-level target occupancy at the recommended dose of 200 mg BID Compound A treatment was generally well tolerated . Most treatment-related adverse events (TRAEs) were Grade (Gr) 1–2. There were no Grade 4–5 TRAEs. Four Grade 3 TRAEs occurred in 4 separate pts;. The most common TRAEs were fatigue, nausea, edema, diarrhea, and vomiting. There was one DLT (Grade 3 fatigue) and one treatment-related serious AE (Grade 3 photosensitivity reaction), each in separate patients treated at 300 mg BID.
At the recommended dose of 200 mg BID, there was prolonged absorption, with a median time to maximum plasma concentration (Tmax) of 3–4 hrs following administration with food. No significant accumulation was observed at steady state, and there was no evidence of auto-induction. The half-life was about 7 hours, and steady-state area under the curve (AUCss) was more than threefold above the exposure required for maximum efficacy in less-sensitive KRAS G12C xenograft models. Figure 15 shows the PK profile at steady state. The predicted target occupancy profile is shown in Figure 15. Patient PK and preclinical target occupancy models were integrated to predict target occupancy in patients at >90% in >82% patients. The models assume that JDQ443 binding and target (KRAS) turn-over rates are the same in mice and humans (~25 hr half-life for KRAS) and that only free drug can bind the target. The best overall response across dose levels and indications is shown in the top half of Figure 16 and in the Table below.
The best overall response across dose levels in all patients with NSCLC is shown in the bottom half of Figure 16 and in the Table below. All patients with a Partial Response or unconfirmed Partial Response were ongoing treatment at the data cut-off. NE, not evaluable; NSCLC, non-small cell lung cancer; ORR, overall response rate; PD, progressive disease; PR, partial response; QD, once daily. Responses are investigator assessed per RECIST v1.1. Two (10.0%) patients had a uPR, which contributed toward the ORR (confirmed and unconfirmed). uPR = unconfirmed PR pending confirmation, treatment ongoing with no PD. One of two patients with a uPR had confirmed PR after the data cut-off. • Figure 17 shows PET scans showing a substantial reduction in the 2-[fluorine-18]-fluoro-2- deoxy-d-glucose (18-F-FDG) avidity of the tumor mass after four cycles of treatment with Compound A administered at 200 mg BID to a patient with NSCLC. The patient had received pemetrexed/pembrolizumab, docetaxel, tegafur/gimeracil/oteracil, and carboplatin/ paclitaxel/atezolizumab. Post-Cycle 2 scan showed a 30.4% reduction in the sum of the longest diameters of target lesions compared with baseline. PR was confirmed on subsequent scans The combination of Compound A and a SHP2 inhibitor such as TNO155 also showed clinical efficacy. Figure 18 shows a post-cycle 2 scan from a patient with KRAS G12C- mutated duodenal papillary cancer and who had previously treated with cisplatin/gemcitabine and tegafur, each with a best response of progressive disease. The patient was treated with with JDQ443200 mg QD continuously and TNO15520 mg QD 2 weeks on/1 week off. The post- cycle 2 scan showed a 44.2% reduction in the sum of the longest diameters of target lesions compared with baseline. Two cases of patients treated in the first-in-human clinical trial are provided here to illustrate the clinical antitumor activity of JDQ443 alone or with TNO155 (Figure 17 and Figure 18). Case 1: a 57 year old male with metastatic KRAS G12C-mutated NSCLC. Local molecular testing using next generation sequencing (NGS) identified no mutations in TP53. Mutation status of STK11, KEAP1 and NRF2 were unknown. The patient had received prior carboplatin/pemetrexed/pembrolizumab, docetaxel, tegafur-gimeracil-oteracil, and carboplatin/paclitaxel/atezolizumab. He was enrolled to the JDQ443 monotherapy dose escalation part of the study at a dose of JDQ443200 mg BID given continuously on a 21-day cycle. Disease assessment after 2 cycles of treatment demonstrated a RECIST 1.1 partial response, with a –30.4% change in the sum of the longest diameters of target lesions compared with baseline. Partial response was confirmed on subsequent scans (Figure 17) and the patient continued on treatment. Positron emission tomography imaging at baseline and after 4 cycles of treatment also showed substantial reduction in 2-[fluorine-18]-fluoro-2-deoxy-d-glucose avidity of the tumor mass. Case 2: a 58 year old female with KRAS G12C-mutated duodenal papillary cancer metastatic to liver. An R175H mutation in TP53 was observed by NGS (Foundation One panel). The patient had received prior treatment with cisplatin/gemcitabine and tegafur, both with a best response of progressive disease. She was enrolled to the dose escalation portion of the study’s JDQ443 + TNO155 arm, and received JDQ443200 mg QD continuously with TNO15520 mg QD 2 weeks on / 2 weeks off. Disease assessment after two cycles of treatment demonstrated a RECIST 1.1 partial response, with a –44.2% change in the sum of the longest diameters of target lesions compared to baseline (Figure 18). Partial response was confirmed on subsequent scans and the patient continued on treatment. Example 12: Clinical study investigating Compound A versus docetaxel in patients with previously treated, locally advanced or metastatic KRAS G12C-mutated NSCLC An open label study which is designed to compare Compound A as monotherapy to docetaxel in participants with advanced non-small cell lung cancer (NSCLC) harboring a KRAS G12C mutation who have been previously treated with a platinum-based chemotherapy and immune checkpoint inhibitor therapy either in sequence or in combination may be carried out. The study consists of 2 parts: -Randomized part will evaluate the efficacy and safety of Compound A as monotherapy in comparison with docetaxel. -Extension part will be open after final progression-free survival (PFS) analysis (if the primary endpoint has met statistical significance) to allow participants randomized to docetaxel treatment to crossover to receive Compound A treatment. The study population include adult participants with locally advanced or metastatic (stage IIIB/IIIC or IV) KRAS G12C mutant non-small cell lung cancer who have received prior platinum-based chemotherapy and prior immune checkpoint inhibitor therapy administered either in sequence or as combination therapy. Participants are treated with Compound A or docetaxel following local guidelines as per standard of care and product labels (docetaxel concentrated solution for infusion, intravenously administered) Primary Outcome Measures include: Progression free survival (PFS) PFS is the time from date of randomization/start of treatment to the date of event defined as the first documented progression or death due to any cause. PFS is based on central assessment and using RECIST 1.1 criteria. Secondary Outcome Measures include: • Overall Survival (OS) • OS is defined as the time from date of randomization to date of death due to any cause • Overall Response Rate (ORR) • ORR is defined as the proportion of patients with best overall response of complete response (CR) or partial response (PR) based on central and local investigator's assessment according to RECIST 1.1. • Disease Control Rate (DCR) • DCR is defined as the proportion of participants with Best Overall Response (BOR) of Complete Response (CR), Partial Response (PR), Stable Disease (SD) or Non-CR/Non- PD. • Time To Response (TTR) • TTR is defined as the time from the date of randomization to the date of first documented response (CR or PR, which must be confirmed subsequently) • Duration of Response (DOR) • DOR is calculated as the time from the date of first documented response (complete response (CR) or partial response (PR)) to the first documented date of progression or death due to underlying cancer. • Progression-Free Survival after next line therapy (PFS2) • PFS2 (based on local investigator assessment) is defined as time from date of randomization to the first documented progression on next line therapy or death from any cause, whichever occurs first. • Concentration of Compound A and its metabolite in plasma • To characterize the pharmacokinetics of Compound A and its metabolite HZC320 • Time to definitive deterioration of Eastern Cooperative Group of Oncology Group (ECOG) performance status • Deterioration of Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) • Time to definitive 10-point deterioration symptom scores of chest pain, cough and dyspnea per QLQ-LC13 • The EORTC QLQ LC13 is a 13-item, lung cancer specific questionnaire module, and it comprises both multi-item and single-item measures of lung cancer-associated symptoms (i.e. coughing, hemoptysis, dyspnea and pain) and side-effects from conventional chemo- and radiotherapy (i.e. hair loss, neuropathy, sore mouth and dysphagia). The time to definitive 10-point deterioration is defined as the time from the date of randomization to the date of event, which is defined as at least 10 points absolute increase from baseline (worsening), with no later change below the threshold or death due to any cause • Time to definitive deterioration in global health status/QoL, shortness of breath and pain per QLQ-C30 • The EORTC QLQ-C30 is a questionnaire developed to assess the health-related quality of life of cancer participants. The questionnaire contains 30 items and is composed of both multi-item scales and single-item measures based on the participants experience over the past week. These include five domains (physical, role, emotional, cognitive and social functioning), three symptom scales (fatigue, nausea/vomiting, and pain), six single items (dyspnea, insomnia, appetite loss, constipation, diarrhea and financial impact) and a global health status/HRQoL scale. The time to definitive 10-point deterioration is defined as the time from the date of randomization to the date of event, which is defined as at least 10 points absolute increase from baseline (worsening) of the corresponding scale score, with no later change below the threshold or death due to any cause • Change from baseline in EORTC-QLQ-C30 • The EORTC QLQ-C30 is a questionnaire developed to assess the health-related quality of life of cancer participants. The questionnaire contains 30 items and is composed of both multi-item scales and single-item measures based on the participants experience over the past week. These include five domains (physical, role, emotional, cognitive and social functioning), three symptom scales (fatigue, nausea/vomiting, and pain), six single items (dyspnea, insomnia, appetite loss, constipation, diarrhea and financial impact) and a global health status/HRQoL scale. A higher score indicates a higher presence of symptoms. • Change from baseline in EORTC-QLQ-LC13 o The EORTC QLQ LC13 is a 13-item, lung cancer specific questionnaire module, and it comprises both multi-item and single-item measures of lung cancer- associated symptoms (i.e. coughing, hemoptysis, dyspnea and pain) and side- effects from conventional chemo- and radiotherapy (i.e. hair loss, neuropathy, sore mouth and dysphagia). A higher score indicates a higher presence of symptoms. • Change from baseline in EORTC-EQ-5D-5L o The EQ-5D-5L is a generic instrument for describing and valuing health. It is based on a descriptive system that defines health in terms of 5 dimensions: Mobility, Self-Care, Usual Activities, Pain/Discomfort, and Anxiety/Depression. • Change from baseline in NSCLC-SAQ o The Non-Small Cell Lung Cancer Symptom Assessment Questionnaire (NSCLC- SAQ) is a 7-item, patient-reported outcome measure which assess patient- reported symptoms associated with advanced NSCLC. It contains five domains and accompanying items that were identified as symptoms of NSCLC: cough (1 item), pain (2 items), dyspnea (1 item), fatigue (2 items), and appetite (1 item). • PFS based on KRAS G12C mutation status in plasma • To compare the clinical outcomes for Compound A vs docetaxel based on KRAS G12C mutation status in plasma • OS based on KRAS G12C mutation status in plasma. • To compare the clinical outcomes for Compound A vs docetaxel based on KRAS G12C mutation status in plasma • ORR based on KRAS G12C mutation status in plasma. • To compare the clinical outcomes for Compound A vs docetaxel based on KRAS G12C mutation status in plasma Example 13: Clinical study of JDQ443 with select combinations in patients with advanced solid tumors harboring the KRAS G12C mutation A Phase Ib/II, multicenter, open-label platform study of JDQ443 with select combinations in patients with advanced solid tumors harboring the KRAS G12C mutation may be conducted. This study aims to characterize the safety, tolerability, pharmacokinetics, pharmacodynamics, and anti-tumor activity of JDQ443 in combination with selected therapies in adult patients with solid tumors harboring KRAS G12C mutations. This study focuses on a single molecular subset of patients whose tumors harbor the KRAS G12C mutation and who have shown or, based on historical data, are predicted to have only modest responsiveness to single-agent KRAS G12C inhibition. The combination of JDQ443 with selected targeted therapies or other antineoplastic therapies may prevent or overcome this resistance in KRAS G12C mutant tumors, and may enable deeper and more durable responses than is historically seen with KRAS G12C inhibitor monotherapy in similar patient populations. Each treatment arm includes a dose escalation part (Phase Ib) and a Phase II part. Dose escalations will be conducted in KRAS G12C mutant solid tumors (JDQ443+cetuximab may be be explored in CRC) to establish safety/efficacy and determine the maximum tolerated doses (MTD) and/or recommended doses (RD). Phase II parts of the study will further explore the RD in selected indications (e.g. NSCLC and CRC for JDQ443 in combination with selected therapies). The purpose of the Phase II is to assess anti-tumor efficacy and further explore safety and tolerability of JDQ443 in combination with selected therapies at the RD(s).
All publications, patents, and Accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. References in this specification to "the invention" are intended to reflect embodiments of the several inventions disclosed in this specification and should not be taken as unnecessarily limiting of the claimed subject matter. It is understood that the Examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.