KRAS inhibitors

Abstract

The present invention provides compounds of the formula: wherein A, Z, G, R1, R2 and R4 are as described herein, pharmaceutically acceptable salts thereof, and methods of using these compounds and pharmaceutically acceptable salts thereof for the treatment of cancer patients.

Description

[0001] background

[0002] The MAPK / ERK signaling pathway transmits extracellular stimuli to the cell nucleus, thereby regulating a variety of cellular responses, including cell proliferation, differentiation, and apoptosis. KRas proteins are initiators of the MAPK / ERK signaling pathway, acting as a switch responsible for inducing cell division. In its inactive state, KRas binds to guanosine diphosphate (GDP), effectively sending a negative signal to inhibit cell division. In response to extracellular signals, KRas is allosterically activated, causing GDP to undergo a nucleotide exchange with guanosine triphosphate (GTP). In its GTP-bound active state, KRas recruits and activates proteins essential for growth factor-induced signal transduction and other cell signal transduction receptors. Examples of proteins recruited by KRas-GTP include c-Raf and PI3-kinase. As a GTPase, KRas converts bound GTP back to GDP, restoring itself to an inactive state and re-transmitting the signal to inhibit cell division. KRas gain-of-function mutations exhibit increased GTP binding and decreased ability to convert GTP to GDP. The result was an increase in MAPK / ERK signaling, which promotes cancer cell growth. The KRas missense mutation at codon 12 was the most common mutation, significantly reducing GTPase activity.

[0003] Oncogenic KRas mutations have been found in approximately 30% of human cancers and have been shown to activate multiple downstream signaling pathways. Despite their prevalence, KRas mutations have remained a challenging therapeutic target. (Cox, AD Drugging the Undruggable RAS: Mission Possible? Nat. Rev. Drug Disc. 2014, 13, 828-851; Pylayeva-Gupta, et al. RAS Oncogenes: Weaving a Tumorigenic Web. Nat. Rev. Cancer 2011, 11, 761-774).

[0004] To date, work has primarily focused on KRas G12C mutation inhibitors (e.g., KRas G12C inhibitors are disclosed in WO2019 / 099524, WO2020 / 081282, WO2020 / 101736, WO2020 / 146613 and WO2021 / 118877), while WO2021 / 041671 discloses small molecule inhibitors of KRas G12D, and WO2017 / 011920 discloses small molecule inhibitors of KRas G12C, G12D and G12V.

[0005] There remains a need for alternative small-molecule KRas inhibitors. In particular, there is a need for more potent, orally deliverable KRas inhibitors for the treatment of cancer. More specifically, there is a need for small-molecule inhibitors that specifically inhibit KRas GTP activity. There is also a need for small-molecule KRas inhibitors that exhibit greater potency at the same or reduced KRas inhibitory activity. Furthermore, there is a desire to provide KRas inhibitors with enhanced pharmacokinetic / pharmacodynamic properties. There is also a need for more potent KRas inhibitors that exhibit increased potency and reduced or minimized adverse or undesirable effects. In addition, there is a need for more potent KRas inhibitors that exhibit selective inhibitory preference for KRas G12D mutations relative to KRas wild-type. This invention addresses one or more of these needs by providing novel KRas inhibitors.

[0006] Overview

[0007] This article provides compounds of formula I:

[0008]

[0009] Formula I

[0010] in

[0011] A is -C(H)- or -N-;

[0012] Z is -C(R) 3c - or -N-;

[0013] G is -C(R) 3b - or -N-;

[0014] R1 is a -H group or a group of the following formula.

[0015] , or ;

[0016] R2 is -H, halogen, or methyl;

[0017] R 3b and R 3c Each can be independently -H, halogen, or methyl;

[0018] R4 is a group in the following formula.

[0019] or ;

[0020] R5 is a C-type ... 1-4 alkyl;

[0021] R 5a It is C 1-3 Alkylene;

[0022] R6 is C 1-3 alkyl;

[0023] R7 is -H or C 1-3 Alkyl; and

[0024] R8 is -H, halogen, or C. 1-3 Alkyl groups; or pharmaceutically acceptable salts thereof.

[0025] The present invention also provides methods for treating cancer, particularly lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, ovarian cancer, bile duct cancer, and colorectal cancer, using compounds of Formula I, pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof. The methods comprise administering a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof to a patient in need.

[0026] This document also provides compounds of formula I for therapeutic purposes, as well as pharmaceutically acceptable salts thereof. This document further provides compounds of formula I for the treatment of cancers, particularly lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, ovarian cancer, bile duct cancer, and colorectal cancer, as well as pharmaceutically acceptable salts thereof. Furthermore, this document provides the use of compounds of formula I or their pharmaceutically acceptable salts in the preparation of pharmaceutical agents for the treatment of cancers, particularly lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, ovarian cancer, bile duct cancer, and colorectal cancer. Invention Details

[0028] This article describes novel inhibitors of the KRas gain-of-function mutation G12D. These new compounds address the need for inhibitors of KRas GTP activity in gain-of-function mutants of cancers such as lung, colorectal, pancreatic, bladder, cervical, endometrial, ovarian, bile duct, or esophageal cancers, as mentioned above. Some of these novel KRas inhibitor compounds exhibit selectivity for KRas G12D relative to wild-type KRas. Furthermore, some of these novel KRas inhibitor compounds are nonselective and can simultaneously inhibit both wild-type KRas and KRas G12D mutants (and / or possibly other mutation types such as G12C or G12V).

[0029] This invention provides compounds of formula I:

[0030]

[0031] Formula I

[0032] A, G, Z, R1, R2 and R4 are as defined above, or their pharmaceutically acceptable salts.

[0033] As used herein, the term halogen means fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). As used herein, the term alkyl means a saturated straight-chain or branched monovalent hydrocarbon group of one to a specified number of carbon atoms, such as "C". 1-4 Alkyl or C 1-3 Alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, 1-propyl, isopropyl, butyl, and isobutyl. As used herein, the term alkylene refers to a saturated straight-chain or branched divalent hydrocarbon group of one to a specified number of carbon atoms, such as "C". 1-3 Alkylene. Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, 1-propylene, and isopropylene. C 1-3 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, 1-propoxy, and isopropoxy.

[0034] The following are other aspects of the invention's designation:

[0035] 1. Compounds with the following formula:

[0036]

[0037] in

[0038] A is -C(H)- or -N-;

[0039] Z is -C(R) 3c - or -N-;

[0040] G is -C(R) 3b - or -N-;

[0041] R1 is a -H group or a group of the following formula.

[0042] , or ;

[0043] R2 is -H, halogen, or methyl;

[0044] R 3b and R 3c Each can be independently -H, halogen, or methyl;

[0045] R4 is a group in the following formula.

[0046] or ;

[0047] R5 is a C-type ... 1-4alkyl;

[0048] R 5a It is C 1-3 Alkylene;

[0049] R6 is C 1-3 alkyl;

[0050] R7 is -H or C 1-3 Alkyl; and

[0051] R8 is -H, halogen, or C. 1-3 Alkyl groups; or pharmaceutically acceptable salts thereof.

[0052] 2. A compound as defined in aspect 1 or a pharmaceutically acceptable salt thereof, wherein G is -N-.

[0053] 3. A compound as defined in aspect 1 or a pharmaceutically acceptable salt thereof, wherein G is -C(R 3b )-.

[0054] 4. A compound as defined in aspect 3 or a pharmaceutically acceptable salt thereof, wherein G is -C(R 3b )-, and R 3b It is -H or halogen.

[0055] 5. A compound as defined in aspect 4 or a pharmaceutically acceptable salt thereof, wherein G is -C(F)-.

[0056] 6. A compound as defined in aspect 4 or a pharmaceutically acceptable salt thereof, wherein G is -C(Cl)-.

[0057] 7. A compound as defined in aspect 4 or a pharmaceutically acceptable salt thereof, wherein G is -C(H)-.

[0058] 8. A compound as defined in aspect 3 or a pharmaceutically acceptable salt thereof, wherein G is -C(CH3)-.

[0059] 9. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-8, wherein Z is -N-.

[0060] 10. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-8, wherein Z is -C(R 3c )-.

[0061] 11. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-8, wherein Z is -C(R 3c )-, where R 3c It is either -H or -F.

[0062] 12. A compound as defined in aspect 11 or a pharmaceutically acceptable salt thereof, wherein Z is -C(H)-.

[0063] 13. A compound as defined in aspect 11 or a pharmaceutically acceptable salt thereof, wherein Z is -C(F)-.

[0064] 14. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 1, wherein G is -N- and Z is -C(R) 3c )-, and R 3c It is -H or halogen.

[0065] 15. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 1, wherein G is -N- and Z is -C(H)-.

[0066] 16. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 1, wherein G is -N- and Z is -C(F)-.

[0067] 17. A compound as defined in aspect 1 or a pharmaceutically acceptable salt thereof, wherein G is -C(R 3b )-, and Z is -N-.

[0068] 18. A compound as defined in aspect 1 or a pharmaceutically acceptable salt thereof, wherein G is -C(R 3b )-, R 3b It is -H or halogen, and Z is -N-.

[0069] 19. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 1, wherein G is -C(F)- and Z is -N-.

[0070] 20. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 1, wherein G is -C(H)- and Z is -N-.

[0071] 21. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 1, wherein G is -C(CH3)- and Z is -N-.

[0072] 22. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1, 3 or 10, wherein R 3b and R 3c Each is independently -H or halogen.

[0073] 23. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-22, wherein A is -N-.

[0074] 24. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-22, wherein A is -C(H)-.

[0075] 25. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-24, wherein R2 is F or Cl.

[0076] 26. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-24, wherein R2 is F.

[0077] 27. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-24, wherein R2 is Cl.

[0078] 28. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-27, wherein R1 is -H.

[0079] 29. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-27, wherein R1 is a group of the following formula:

[0080] , or .

[0081] 30. A compound as defined in aspect 29 or a pharmaceutically acceptable salt thereof, wherein R1 is a group of the following formula:

[0082] .

[0083] 31. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 29 or 30, wherein R 5a It is ethylene.

[0084] 32. A compound as defined in aspect 31 or a pharmaceutically acceptable salt thereof, wherein R1 is selected from...

[0085] , , , , or .

[0086] 33. A compound as defined in aspect 32 or a pharmaceutically acceptable salt thereof, wherein R1 is selected from...

[0087] , , or .

[0088] 34. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-33, wherein R4 is a group of the following formula:

[0089] .

[0090] 35. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0091] .

[0092] 36. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0093] .

[0094] 37. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 35 or 36, wherein R4 is a group of the following formula:

[0095] .

[0096] 38. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0097] .

[0098] 39. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 35 or 38, wherein R4 is a group of the following formula:

[0099] .

[0100] 40. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0101] , , , , , , or .

[0102] 41. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0103] .

[0104] 42. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0105] .

[0106] 43. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0107] .

[0108] 44. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0109] .

[0110] 45. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0111] .

[0112] 46. ​​A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0113] .

[0114] 47. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0115] .

[0116] 48. A compound as defined in aspect 34 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0117] .

[0118] 49. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-33, wherein R4 is a group of the following formula:

[0119] .

[0120] 50. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0121] .

[0122] 51. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0123] .

[0124] 52. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 50 or 51, wherein R4 is a group of the following formula:

[0125] .

[0126] 53. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0127] .

[0128] 54. A compound or a pharmaceutically acceptable salt thereof as defined in aspect 50 or 53, wherein R4 is a group of the following formula:

[0129] .

[0130] 55. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0131] , , , , , , or .

[0132] 56. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0133] .

[0134] 57. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0135] .

[0136] 58. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0137] .

[0138] 59. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0139] .

[0140] 60. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0141] .

[0142] 61. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0143] .

[0144] 62. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0145] .

[0146] 63. A compound as defined in aspect 49 or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula:

[0147] .

[0148] 64. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-63, wherein R6 is a methyl group.

[0149] 65. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-64, wherein R7 is methyl.

[0150] 66. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-65, wherein R6 and R7 are methyl groups.

[0151] 67. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-66, wherein R8 is -H, -F or methoxy.

[0152] 68. A compound as defined in aspect 67 or a pharmaceutically acceptable salt thereof, wherein R8 is -H.

[0153] 69. A compound as defined in aspect 67 or a pharmaceutically acceptable salt thereof, wherein R8 is -F.

[0154] 70. A compound as defined in aspect 67 or a pharmaceutically acceptable salt thereof, wherein R8 is a methoxy group.

[0155] 71. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-63, wherein R6 and R7 are methyl and R8 is H.

[0156] 72. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-63, wherein R6 and R7 are methyl and R8 is F.

[0157] 73. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-63, wherein R6 and R7 are methyl and R8 is methoxy.

[0158] 74. A compound or a pharmaceutically acceptable salt thereof as defined in any one of aspects 1-33, wherein R4 is selected from...

[0159] , , or Or, or a pharmaceutically acceptable salt thereof.

[0160] 75. The compound according to aspect 74, wherein R4 is selected from...

[0161] , , or Or, or a pharmaceutically acceptable salt thereof.

[0162] 76. The compound according to aspect 74, wherein R4 is selected from...

[0163] , , or Or, or a pharmaceutically acceptable salt thereof.

[0164] 77. The compound according to aspect 74, wherein R4 is selected from...

[0165] , , or Or, or a pharmaceutically acceptable salt thereof.

[0166] 78. The compound according to aspect 75 or 76, wherein R4 is selected from...

[0167] , , or Or, or a pharmaceutically acceptable salt thereof.

[0168] 79. According to aspect 1, the compounds are selected from:

[0169] , , , , , , , , , , , or Or, or a pharmaceutically acceptable salt thereof.

[0170] 80. According to aspect 1, the compound is selected from:

[0171] , , , , , , , , , or Or, or a pharmaceutically acceptable salt thereof.

[0172] 81. According to aspect 1, the compounds are selected from:

[0173] or Or, or a pharmaceutically acceptable salt thereof.

[0174] 82. According to aspect 1, the compounds are selected from:

[0175] , , , , , , .

[0176] 83. A pharmaceutical composition comprising a compound according to any one of aspects 1-82 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0177] 84. A method of treating a patient with cancer, the method comprising administering to a patient in need an effective amount of a pharmaceutical composition according to aspect 83, wherein the cancer is selected from lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, ovarian cancer, bile duct cancer, and colorectal cancer.

[0178] 85. A method of treating a patient with cancer, the method comprising administering to a patient in need an effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of aspects 1-82, wherein the cancer is selected from lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, ovarian cancer, bile duct cancer, and colorectal cancer.

[0179] 86. The method according to aspect 84 or 85, wherein the patient has cancer prior to administration of the compound or a pharmaceutically acceptable salt thereof, the cancer being identified as cells having one or more KRas G12D mutant proteins.

[0180] 87. The method according to any one of aspects 84-86, wherein the cancer is non-small cell lung cancer.

[0181] 88. The method according to any one of aspects 84-86, wherein the cancer is colorectal cancer.

[0182] 89. The method according to any one of aspects 84-86, wherein the cancer is pancreatic cancer.

[0183] 90. The method according to any one of aspects 84, 85 or 87-89, wherein one or more cells express the KRasG12D mutant protein.

[0184] 91. A method for treating a patient with cancer having a KRas G12D mutation, the method comprising administering to the patient in need an effective amount of a compound according to any one of aspects 1-82 or a pharmaceutically acceptable salt thereof.

[0185] 92. The method according to aspect 91, wherein the cancer is selected from lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, mutant ovarian cancer, bile duct cancer, and colorectal cancer.

[0186] 93. According to the method of aspect 92, the cancer is non-small cell lung cancer.

[0187] 94. According to the method of aspect 92, the cancer mentioned is colorectal cancer.

[0188] 95. According to the method of aspect 92, the cancer mentioned is pancreatic cancer.

[0189] 96. The method according to any one of aspects 84-95, wherein the patient is also administered an effective amount of one or more of the following drugs: PD-1 inhibitor, PD-L1 inhibitor, CDK4 / CDK6 inhibitor, EGFR inhibitor, ERK inhibitor, Aurora A inhibitor, SHP2 inhibitor, platinum-based drugs and pemetrexed, or pharmaceutically acceptable salts thereof.

[0190] 97. A compound or a pharmaceutically acceptable salt thereof according to any one of aspects 1-82, used for therapeutic purposes.

[0191] 98. A compound or a pharmaceutically acceptable salt thereof according to any one of aspects 1-82, used for the treatment of cancer.

[0192] 99. The compound of aspect 98 for the purpose described herein, or a pharmaceutically acceptable salt thereof, wherein the cancer has a KRas G12D mutation.

[0193] 100. A compound or a pharmaceutically acceptable salt thereof for the purpose according to any one of aspects 98 or 99, wherein the cancer is selected from lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, ovarian cancer, bile duct cancer, and colorectal cancer.

[0194] 101. A compound of any one of aspects 1-82 or a pharmaceutically acceptable salt thereof, used in the treatment of cancer in conjunction with, separately or sequentially with, one or more of the following drugs: PD-1 or PD-L1 inhibitors, CDK4 / CDK6 inhibitors, EGFR inhibitors, ERK inhibitors, Aurora A inhibitors, SHP2 inhibitors, platinum-based drugs and pemetrexed, or a pharmaceutically acceptable salt thereof.

[0195] In the above embodiments of the compounds of Formula I, the chemical diagrams are planar and lack chiral information. These compounds typically have multiple chiral centers, and various combinations of chiral centers are included. Furthermore, these compounds can have a wide variety of enantiomers, diastereomers, and transisomers, all of which are included within the scope of this document.

[0196] In embodiments of a compound of Formula I or a pharmaceutically acceptable salt thereof, the compound is an isotopic derivative of any of the compounds described herein or a pharmaceutically acceptable salt thereof.

[0197] It should be understood that isotope derivatives can be prepared using any of a variety of techniques recognized in the art. For example, isotope derivatives can typically be prepared by performing the processes described herein and / or the procedures disclosed in the examples, or by using a reagent containing an isotope instead of a reagent containing a non-isotope.

[0198] In embodiments of a compound of Formula I or a pharmaceutically acceptable salt thereof, said compound is a deuterated compound of any of the compounds described herein or a pharmaceutically acceptable salt thereof.

[0199] In the compounds of this invention, any atom not specifically designated as a particular isotope refers to any stable isotope representing that atom. Unless otherwise stated, when an atom is specifically designated as "H" or "hydrogen," that atom is understood to be hydrogen having its naturally occurring isotopic composition. Furthermore, unless otherwise stated, when an atom is specifically designated as "D" or "deuterium," that atom is understood to be deuterium having an abundance substantially greater than the naturally occurring abundance of deuterium (i.e., 0.015%).

[0200] Compounds of Formula I or pharmaceutically acceptable salts thereof, said compounds being selected from:

[0201] , , , , , , , , , , or Or, or a pharmaceutically acceptable salt thereof.

[0202] The chemical diagrams of the compounds above contain indications of the chiral properties of the specific compounds shown. However, the chemical diagrams of the compounds above do not include all possible chiral characteristics of these compounds, and the chiral indications shown are not intended to exclude variations in the chiral properties shown. Therefore, this document covers and includes alternative chiral forms of the compounds and different combinations of chiral properties.

[0203] This document also provides pharmaceutical compositions comprising a compound of formula I or a pharmaceutically acceptable salt thereof (examples of which include, but are not limited to, the compounds disclosed herein), and a pharmaceutically acceptable carrier, diluent, or excipient.

[0204] This article also provides a method for treating cancer, the method comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. In this method, the cancer may be lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, gastric cancer, or esophageal cancer. In this method, the cancer may more particularly be non-small cell lung cancer, pancreatic cancer, or colorectal cancer. In one embodiment, the cancer may be non-small cell lung cancer. In one embodiment, the cancer may be pancreatic cancer. In one embodiment, the cancer may be colorectal cancer.

[0205] This document also provides a method for treating cancer, the method comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the cancer has one or more cells expressing a mutated KRas G12D protein. In this method, the cancer may be non-small cell lung cancer, pancreatic cancer, or colorectal cancer, wherein the cancer has one or more cells expressing a mutated KRas G12D protein. In one embodiment, the cancer is non-small cell lung cancer, wherein the cancer has one or more cells expressing a mutated KRas G12D protein. In one embodiment, the cancer is mutated pancreatic cancer, wherein the cancer has one or more cells expressing a mutated KRas G12D protein. In one embodiment, the cancer is colorectal cancer, wherein the cancer has one or more cells expressing a mutated KRas G12D protein. The method also includes treating cancers with KRas G12D mutations in other organs.

[0206] This article also provides a method for treating a patient with cancer harboring a KRas G12D mutation, the method comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. In this method, the cancer harboring a KRas G12D mutation may be KRas G12D-mutated lung cancer, KRas G12D-mutated pancreatic cancer, KRas G12D-mutated cervical cancer, KRas G12D-mutated esophageal cancer, KRas G12D-mutated endometrial cancer, KRas G12D-mutated ovarian cancer, KRas G12D-mutated cholangiocarcinoma, or KRas G12D-mutated colorectal cancer. In one embodiment, the cancer harboring a KRas G12D mutation may be KRas G12D-mutated non-small cell lung cancer. In one embodiment, the cancer harboring a KRas G12D mutation may be KRas G12D-mutated pancreatic cancer. In one embodiment, the cancer harboring a KRas G12D mutation may be KRas G12D-mutated colorectal cancer.

[0207] Furthermore, this document provides a method for modulating mutant KRas G12D enzymes in patients in need by administering a compound of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the method comprises inhibiting human mutant KRas G12D enzymes.

[0208] This article also provides methods for treating cancer in patients in need, wherein the patients have cancer identified as expressing the KRas G12D mutant protein. The methods involve administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof to the patient. Typically, one or more biopsies containing one or more cancer cells are obtained and sequenced and / or polymerase chain reaction (PCR) is performed on them. Circulating cell-free DNA may also be used, for example, in advanced cancers. Non-limiting examples of sequencing and PCR techniques used to determine mutation status (e.g., G12D mutation status, in one or more cancer cells or in circulating cell-free DNA) include direct sequencing, next-generation sequencing, reverse transcription polymerase chain reaction (RT-PCR), multiplex PCR, and pyrosequencing and multi-analyte profiling.

[0209] This document also provides compounds of Formula I or pharmaceutically acceptable salts thereof for therapeutic purposes. These compounds or pharmaceutically acceptable salts thereof may be used to treat cancer. For this use in treating cancer, the cancer may be lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, or esophageal cancer. The cancer may more particularly be non-small cell lung cancer, pancreatic cancer, or colorectal cancer. In one embodiment, the cancer is non-small cell lung cancer. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the cancer is colorectal cancer. The cancer may have one or more cancer cells expressing the KRas G12D protein, such as KRas G12D-mutant lung cancer, KRas G12D-mutant pancreatic cancer, KRas G12D-mutant cervical cancer, KRas G12D-mutant esophageal cancer, KRas G12D-mutant endometrial cancer, KRas G12D-mutant ovarian cancer, KRas G12D-mutant bile duct cancer, and KRas G12D-mutant colorectal cancer. In these applications, the cancer is selected from: KRas G12D-mutant non-small cell lung cancer, KRas G12D-mutant colorectal cancer, and KRas G12D-mutant pancreatic cancer. Additionally, the cancer may be non-small cell lung cancer with one or more cells expressing the KRas G12D mutant protein. Furthermore, the cancer may be colorectal cancer with one or more cells expressing the KRas G12D mutant protein. Additionally, the cancer may be pancreatic cancer with one or more cells expressing the KRas G12D mutant protein. The patient had cancer prior to administration of the compound or a pharmaceutically acceptable salt thereof, and the cancer was identified as having one or more cells expressing the KRas G12D mutant protein. The patient may have undergone different treatment procedures prior to receiving the treatment described herein.

[0210] This document provides compounds of Formula I or pharmaceutically acceptable salts thereof, which can be used to prepare agents for treating cancer. When used to prepare the agent, the cancer may be lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, or esophageal cancer. The cancer may be more particularly non-small cell lung cancer, pancreatic cancer, or colorectal cancer. In one embodiment, the cancer is non-small cell lung cancer. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the cancer is colorectal cancer. The cancer may have one or more cancer cells expressing the KRas G12D protein. When the cancer cells express the KRas G12D protein, the cancer may be selected from KRas G12D-mutant non-small cell lung cancer, KRas G12D-mutant colorectal cancer, and KRas G12D-mutant pancreatic cancer.

[0211] This article also provides methods for treating cancer, the methods comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, along with one or more PD-1 inhibitors, PD-L1 inhibitors, CDK4 / CDK6 inhibitors, EGFR inhibitors, ERK inhibitors, Aurora A inhibitors, SHP2 inhibitors, platinum-based drugs, and pemetrexed or pharmaceutically acceptable salts thereof, wherein the cancer has one or more cells expressing a mutant KRas G12D protein. This article also provides compounds of formula I or pharmaceutically acceptable salts thereof for use in cancer treatment in combination, simultaneously, separately, or sequentially, with one or more of PD-1 or PD-L1 inhibitors, CDK4 / CDK6 inhibitors, EGFR inhibitors, ERK inhibitors, Aurora A inhibitors, SHP2 inhibitors, platinum-based drugs, and pemetrexed or pharmaceutically acceptable salts thereof. In addition, combinations are provided comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and one or more of PD-1 or PD-L1 inhibitors, CDK4 / CDK6 inhibitors, EGFR inhibitors, ERK inhibitors, Aurora A inhibitors, SHP2 inhibitors, platinum-based drugs, and pemetrexed or pharmaceutically acceptable salts thereof, for use concurrently, separately, or sequentially in cancer treatment.

[0212] A method of treating cancer is also provided, comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, along with a PD-1 or PD-L1 inhibitor, wherein the cancer has one or more cells expressing a mutant KRas G12D protein. A compound of formula I or a pharmaceutically acceptable salt thereof for use concurrently, separately, or sequentially in combination with a PD-1 or PD-L1 inhibitor is also provided for the treatment of cancer. Furthermore, combinations comprising a compound of formula I or a pharmaceutically acceptable salt thereof, along with a PD-1 or PD-L1 inhibitor, are provided for use concurrently, separately, or sequentially in cancer treatment. As used herein, the PD-1 or PD-L1 inhibitor may be pembrolizumab; the PD-1 or PD-L1 inhibitor may be nivolumab; the PD-1 or PD-L1 inhibitor may be cemiplimab; the PD-1 or PD-L1 inhibitor may be sintilimab; the PD-1 or PD-L1 inhibitor may be atezolizumab; the PD-1 or PD-L1 inhibitor may be avelumab; the PD-1 or PD-L1 inhibitor may be durvalumab; or the PD-1 or PD-L1 inhibitor may be lodapilimab. As used herein, the cancer may be non-small cell lung cancer, wherein the cancer has cells expressing one or more KRas G12D mutant proteins; the cancer may be colorectal cancer, wherein the cancer has cells expressing one or more KRas G12D mutant proteins; or the cancer may be mutant pancreatic cancer, wherein the cancer has cells expressing one or more KRas G12D mutant proteins. The method also includes treating cancers with KRas G12D mutations in other organs.

[0213] A method of treating cancer is also provided, the method comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and a CDK4 / CDK6 inhibitor or a pharmaceutically acceptable salt thereof, wherein the cancer has cells expressing one or more mutant KRas G12D proteins. A compound of formula I or a pharmaceutically acceptable salt thereof is also provided for use simultaneously, separately, or sequentially in combination with a CDK4 / CDK6 inhibitor or a pharmaceutically acceptable salt thereof for treating cancer, wherein the cancer has cells expressing one or more mutant KRas G12D proteins. Furthermore, a combination is provided comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a CDK4 / CDK6 inhibitor or a pharmaceutically acceptable salt thereof, for use simultaneously, separately, or sequentially in cancer treatment, wherein the cancer has cells expressing one or more mutant KRas G12D proteins. As used herein, the CDK4 / CDK6 inhibitor may be abemaciclib; the CDK4 / CDK6 inhibitor may be palbociclib; or the CDK4 / CDK6 inhibitor may be ribociclib. As used herein, the cancer may be non-small cell lung cancer, wherein the cancer has one or more cells expressing a KRas G12D mutant protein; the cancer may be colorectal cancer, wherein the cancer has one or more cells expressing a KRas G12D mutant protein; the cancer may be mutant pancreatic cancer, wherein the cancer has one or more cells expressing a KRas G12D mutant protein. The method also includes treating cancers with KRas G12D mutations in other organs.

[0214] A method of treating cancer is also provided, comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and an EGFR inhibitor or a pharmaceutically acceptable salt thereof, wherein the cancer has one or more cells expressing a mutant KRas G12D protein. A compound of formula I or a pharmaceutically acceptable salt thereof for use concurrently, separately, or sequentially in combination with an EGFR inhibitor or a pharmaceutically acceptable salt thereof is also provided for the treatment of cancer. Combinations comprising a compound of formula I or a pharmaceutically acceptable salt thereof and an EGFR inhibitor or a pharmaceutically acceptable salt thereof are also provided for use concurrently, separately, or sequentially in the treatment of cancer. As used herein, the EGFR inhibitor may be erlotinib; the EGFR inhibitor may be afatinib; the EGFR inhibitor may be gefitinib; the EGFR inhibitor may be cetuximab. As used herein, the cancer may be non-small cell lung cancer, wherein the cancer has cells expressing one or more KRas G12D mutant proteins; the cancer may be colorectal cancer, wherein the cancer has cells expressing one or more KRas G12D mutant proteins; or the cancer may be mutant pancreatic cancer, wherein the cancer has cells expressing one or more KRas G12D mutant proteins. The method also includes treating cancers with KRas G12D mutations in other organs.

[0215] A method of treating cancer is also provided, the method comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and an ERK inhibitor or a pharmaceutically acceptable salt thereof, wherein the cancer has one or more cells expressing a mutant KRas G12D protein. A method of treating cancer is also provided, the method comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and an Aurora A inhibitor thereof, wherein the cancer has one or more cells expressing a mutant KRas G12D protein. A compound of formula I or a pharmaceutically acceptable salt thereof for use simultaneously, separately, or sequentially in combination with an Aurora A inhibitor or a pharmaceutically acceptable salt thereof is also provided for treating cancer, wherein the cancer has one or more cells expressing a mutant KRas G12D protein. A compound of formula I or a pharmaceutically acceptable salt thereof for use simultaneously, separately, or sequentially in combination with an ERK inhibitor or a pharmaceutically acceptable salt thereof is also provided for treating cancer, wherein the cancer has one or more cells expressing a mutant KRas G12D protein. In addition, combinations are provided comprising a compound of formula I or a pharmaceutically acceptable salt thereof and an ERK inhibitor or a pharmaceutically acceptable salt thereof, for use simultaneously, separately, or sequentially in cancer treatment. As used herein, the ERK inhibitor may be LY3214996; the ERK inhibitor may be LTT462; or the ERK inhibitor may be KO-947. As used herein, the cancer may be non-small cell lung cancer having one or more cells expressing a KRasG12D mutant protein; the cancer may be colorectal cancer having one or more cells expressing a KRasG12D mutant protein; the cancer may be mutant pancreatic cancer having one or more cells expressing a KRas G12D mutant protein. The method also includes treating cancers with KRas G12D mutations in other organs.

[0216] A method of treating cancer is also provided, the method comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and an Aurora A inhibitor, wherein the cancer has one or more cells expressing a mutant KRas G12D protein. A compound of formula I or a pharmaceutically acceptable salt thereof for use simultaneously, separately, or sequentially in combination with an Aurora A inhibitor or a pharmaceutically acceptable salt thereof is also provided for treating cancer, wherein the cancer has one or more cells expressing a mutant KRas G12D protein. Furthermore, a combination is provided comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and an Aurora A inhibitor, for use simultaneously, separately, or sequentially in cancer treatment. Furthermore, a combination is provided comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and an Aurora A inhibitor, for use simultaneously, separately, or sequentially in cancer treatment. As used herein, the Aurora A inhibitor may be alisertib, tozasertib, (2R,4R)-1-[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3-fluoro-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]-2-methyl-piperidin-4-carboxylic acid, or (2R,4R)-1-[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3-fluoro-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]-2-methyl-piperidin-4-carboxylic acid. 2-Methylpropyl-2-amine (1:1) salt and (2R,4R)-1-[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3-fluoro-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]-2-methyl-piperidin-4-carboxylic acid:amine (1:1) salt, or a pharmaceutically acceptable salt thereof. In one embodiment, the Aurora A inhibitor is (2R,4R)-1-[(3-chloro-2-fluoro-phenyl)methyl]-4-[[3-fluoro-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyridyl]methyl]-2-methyl-piperidin-4-carboxylic acid. As used herein, the cancer may be non-small cell lung cancer, wherein the cancer has one or more cells expressing the KRas G12D mutant protein; the cancer may be colorectal cancer, wherein the cancer has one or more cells expressing the KRas G12D mutant protein; the cancer may be mutant pancreatic cancer, wherein the cancer has one or more cells expressing the KRas G12D mutant protein. The method also includes treating cancers with KRas G12D mutations in other organs.

[0217] A method of treating cancer is also provided, comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and an SHP2 inhibitor, wherein the cancer has one or more cells expressing a mutant KRasG12D protein. A compound of formula I or a pharmaceutically acceptable salt thereof for use simultaneously, separately, or sequentially in combination with an SHP2 inhibitor or a pharmaceutically acceptable salt thereof is also provided for treating cancer, wherein the cancer has one or more cells expressing a mutant KRasG12D protein. Furthermore, combinations comprising a compound of formula I or a pharmaceutically acceptable salt thereof and an SHP2 inhibitor are provided for use simultaneously, separately, or sequentially in cancer treatment. As used herein, the SHP2 inhibitor or a pharmaceutically acceptable salt thereof may be a type I SHP2 inhibitor or a type II SHP2 inhibitor. Examples of type I SHP2 inhibitors include, but are not limited to, PHPS1, GS-493, NSC-87877, NSC-117199, and cefuroxime, and pharmaceutically acceptable salts thereof. Examples of type II SHP2 inhibitors include, but are not limited to, JAB-3068, JAB-3312, RMC-4550, RMC-4630, SHP099, SHP244, SHP389, SHP394, TNO155, RG-6433, and RLY-1971, and their pharmaceutically acceptable salts. Other examples of SHP2 inhibitors include, but are not limited to, BBP-398, IACS-15509, IACS-13909, X37, ERAS-601, SH3809, HBI-2376, ETS-001, and PCC0208023, and their pharmaceutically acceptable salts. As used herein, the cancer may be non-small cell lung cancer, wherein the cancer has one or more cells expressing the KRasG12D mutant protein; the cancer may be colorectal cancer, wherein the cancer has one or more cells expressing the KRasG12D mutant protein; the cancer may be mutant pancreatic cancer, wherein the cancer has one or more cells expressing the KRasG12D mutant protein. The method also includes treating cancers with KRasG12D mutations in other organs.

[0218] A method of treating cancer is also provided, comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, along with a platinum-based drug, wherein the cancer comprises one or more cells expressing a mutant KRasG12D protein. A compound of formula I or a pharmaceutically acceptable salt thereof for use simultaneously, separately, or sequentially in combination with a platinum-based drug or a pharmaceutically acceptable salt thereof is also provided for treating cancer wherein the cancer comprises one or more cells expressing a mutant KRasG12D protein. Furthermore, combinations comprising a compound of formula I or a pharmaceutically acceptable salt thereof, along with a platinum-based drug, are provided for use simultaneously, separately, or sequentially in cancer treatment. As used herein, the platinum-based drug may be cisplatin; the platinum-based drug may be carboplatin; or the platinum-based drug may be oxaliplatin. As used herein, the cancer may be non-small cell lung cancer, wherein the cancer has cells expressing one or more KRas G12D mutant proteins; the cancer may be colorectal cancer, wherein the cancer has cells expressing one or more KRas G12D mutant proteins; the cancer may be mutant pancreatic cancer, wherein the cancer has cells expressing one or more KRas G12D mutant proteins. The method also includes treating cancers of other organs with KRas G12D mutations. As used herein, the platinum-based agent may be cisplatin; the platinum-based agent may be carboplatin; or the platinum-based agent may be oxaliplatin.

[0219] A method of treating cancer is also provided, comprising administering to a patient in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof and pemetrexed, wherein the cancer has one or more cells expressing a mutant KRasG12D protein. A compound of formula I or a pharmaceutically acceptable salt thereof for use concurrently, separately, or sequentially with pemetrexed is also provided for treating cancer, wherein the cancer has one or more cells expressing a mutant KRasG12D protein. Furthermore, a combination comprising a compound of formula I or a pharmaceutically acceptable salt thereof and pemetrexed is provided for use concurrently, separately, or sequentially in the treatment of cancer, wherein the cancer has one or more cells expressing a mutant KRasG12D protein. As used herein, the cancer has one or more cells expressing a mutant KRasG12D protein. Additionally, platinum-based agents may also be administered to the patient (and the platinum-based agents may be cisplatin, carboplatin, or oxaliplatin). As used herein, the cancer may be colorectal cancer, wherein the cancer has cells expressing one or more KRas G12D mutant proteins, or the cancer may be mutant pancreatic cancer, wherein the cancer has one or more cells expressing KRas G12D mutant proteins. The method also includes treating cancers of other organs with KRas G12D mutations.

[0220] As used in this article, “pharmaceuticalally acceptable salt” refers to a salt of a compound that is considered acceptable for clinical and / or veterinary use. Examples of pharmaceutically acceptable salts and common methods for their preparation can be found in the following literature: “Handbook of Pharmaceutical Salts: Properties, Selection and Use”, P. Stahl et al., 2nd revised edition, Wiley-VCH, 2011, and SM Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Sciences, 1977, 66(1), 1-19.

[0221] Pharmaceutical compositions containing compounds of Formula I described herein can be prepared using pharmaceutically acceptable additives. As used herein, the term "pharmaceutically acceptable additive" for pharmaceutical compositions refers to one or more carriers, diluents, and excipients that are compatible with other additives in the composition or formulation and are harmless to the patient. Examples of pharmaceutical compositions and methods of their preparation can be found in the following literature: “Remington: The Science and Practice of Pharmacy”, edited by Loyd, V. et al., 22nd edition, Mack Publishing Co., 2012. Non-limiting examples of pharmaceutically acceptable carriers, diluents, and excipients include: saline, water, starch, sugars, mannitol, and silica derivatives; binders such as carboxymethyl cellulose, alginate, gelatin, and polyvinylpyrrolidone; kaolin and bentonite; and polyethylene glycol.

[0222] As used herein, the term "effective amount" refers to the amount of medication necessary to effectively achieve the desired therapeutic effect, such as the effective treatment of cancerous lesions or the progression of abnormal cell growth and / or cell division. Factors considered in determining the effective amount or dosage of a compound include: whether the compound or its salts are administered; co-administration of other active agents (if used); the species of the patient being treated; the patient's body type, age, sex, and overall health status; the extent or stage and / or severity of the impairment; the individual patient's response; the route of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of other concomitant medications.

[0223] The treating physician, veterinarian, or other medical personnel will be able to determine the effective amount of the compound for treating patients in need. The pharmaceutical composition can be formulated as tablets or capsules for oral administration, solutions for oral administration, or injectable solutions. Tablets, capsules, or solutions may contain an effective amount of the compound of the present invention for treating patients requiring cancer treatment.

[0224] As used herein, the term "treatment" includes slowing, controlling, delaying, reducing, stopping, reversing, preventing, or improving the progression or severity of existing symptoms, impairments, or conditions, which may specifically include slowing the growth of cancerous lesions or the progression of abnormal cell growth and / or cell division. Treatment does not necessarily mean the complete elimination of all impairments or disease symptoms.

[0225] As used in this article, the term "patient" refers to a mammal in need of treatment. In particular, a patient can be a person who needs treatment for cancer, such as cancer with the KRas G12D mutation.

[0226] Some abbreviations are defined as follows: "ACN" refers to acetonitrile; "AcOH" or "HOAc" refers to acetic acid; "AIBN" refers to azobisisobutyronitrile; "Alloc" refers to allyloxycarbonyl; "aq." refers to aqueous or hydrated; "atm" refers to atmospheric pressure; "Boc-Gly-OH" refers to N-(tert-butoxycarbonyl)glycine; "BrettPhos" refers to 2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl; "BroP" refers to tris(dimethylamino)phosphonium hexafluorophosphate; "Cbz" refers to benzyloxycarbonyl; "Cbz-Cl" refers to benzyl chloroformate; "conc." "CSI" refers to concentrated; "CV" refers to column capacity; "DCM" refers to dichloromethane; "DIAD" refers to diisopropyl azodicarbonate; "DIBAL-H" refers to diisobutylaluminum hydride; "DIEA" and "DIPEA" refer to N,N-diisopropylethylamine; "(dippf)Rh(cod)BF4" refers to [1,4-bis(diphenylphosphine)butane](1,5-cyclooctadiene)rhodium(I)tetrafluoroborate; "DMAP" refers to 4-dimethylaminopyridine; "DMEA" refers to N,N-dimethylethylamine; "DMEM" refers to Dulbecco's modified Eagle's medium. (medium); "DMF" refers to N,N-dimethylformamide; "DMSO" refers to dimethyl sulfoxide; "DNA" refers to deoxynucleotide; "DPEPhosPdCl2" refers to palladium(II) dichlorobis(diphenylphosphine) ether; "DTT" refers to dithiothreitol; "EDTA" refers to ethylenediaminetetraacetic acid; "EGTA" refers to ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; "ELISA" refers to enzyme-linked immunosorbent assay; "ERK" refers to extracellular signal-regulated kinase; "EtOAc" refers to ethyl acetate; "Et2O" refers to diethyl ether; "EtOH" refers to ethanol; "FA" is formic acid; "FBS" refers to fetal bovine serum; "Fmoc" "GTP" refers to fluorenylmethoxycarbonyl; "GDP" refers to guanosine diphosphate; "GTP" refers to guanosine triphosphate; "h" refers to hours; "HATU" refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate; "Hex" or "hex" refers to hexane; "HPLC" refers to high-performance liquid chromatography; "HRP" refers to horseradish peroxidase; "IPA" refers to isopropyl alcohol; "IPAm" refers to isopropylamine; "KOAc" refers to potassium acetate; "LC-ES / MS" refers to liquid chromatography-electrospray mass spectrometry; "LC-MS" refers to liquid chromatography-mass spectrometry; "LiHMDS" refers to lithium bis(trimethylsilyl)amino."L-prolyl" refers to [(2S)-pyrrolidine-2-yl]methanol; "MAPK" refers to mitogen-activated protein kinase; "mCPBA" refers to 3-chloro-peroxybenzoic acid; "Me" refers to methyl; "MeOH" refers to methanol; "min" refers to minutes; "MTBE" refers to methyl tert-butyl ether; "NaBH(OAc)3" refers to sodium triacetoxyborohydride; "NaOMe" refers to sodium methoxide; "NBS" refers to N-bromosuccinate. Imide; "NCS" refers to N-chlorosuccinimide; "N-methyl-L-prolyl" refers to [(2S)-1-methylpyrrolidone-2-yl]methanol; "NMM" refers to N-methylmorpholine; "NMP" refers to 1-methylpyrrolidone-2-one; "NIS" refers to N-iodosuccinimide; "PCR" refers to polymerase chain reaction; "Pd-117" refers to [bis(2-(diphenylphosphino)phenyl)ether]palladium(II) dichloride. CAS 205319-06-8; “Pd-118” refers to 1,1'-bis(di-tert-butylphosphino)ferrocene palladium dichloride, CAS 95408-45-0; “Pd2(dba)3” refers to tris(dibenzylacetone)palladium(0); “Pd(dppf)Cl2” refers to [1,1'-bis(diphenylphosphino)ferrocene]palladium(II); “Pd(OAc)2” refers to palladium(II) acetate; Pd(PPh3)4 refers to tetra(triphenylphosphine)palladium(0); “PE” refers to petroleum ether or diethyl ether; “Ph” refers to phenyl; “RBF” refers to a round-bottom flask; “RPMI” refers to the Roswell Park Memorial Institute; “RT” refers to room temperature; “RuPhos” refers to 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl, CAS 787618-22-8; "sat." refers to saturation; "SCX" refers to strong cation exchange; "Selectfluor"; TM " refers to 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octanebis(tetrafluoroborate)," "SPE" refers to solid phase extraction; "SPhos" refers to 2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl; "TBAF" refers to tetrabutylammonium fluoride; "TBDMSCl" refers to tert-butyldimethylsilyl chloride; "TBDMS" refers to tert-butyldimethylsilyl; "tBu" refers to tert-butyl; "t-BuOH" refers to tert-butanol or tert-butyl alcohol; "TEA" refers to triethylamine; "TES" refers to triethylsilane; "Tf2O" refers to trifluoromethanesulfonic anhydride; "TFA" refers to trifluoroacetic acid; "THF" refers to tetrahydrofuran; "TMEDA" refers to tetramethylethylenediamine; "t R"XantPhos" refers to 4,5-bis(diphenylphosphino)-9,9-dimethylxanthione; "XPhos" refers to 2-(dicyclohexylphosphino)-2',4',6'-tri-isopropyl-1,1'-biphenyl; "XPhos Palladacycle G2" refers to chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II), CAS 1310584-14-5; "XPhos Palladacycle Gen.4" or "XPhos Pd G4" is methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-methylamino-1,1'-biphenyl-2-yl)palladium(II). CAS 1599466-81-5.

[0227] The individual isomers, enantiomers, diastereomers, and transisomers can be separated or resolved at any convenient time point in the synthesis of the compounds listed below, for example by selective crystallization or chiral chromatography (see, for example, J. Jacques et al., "Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc., 1981, and E.L. Eliel and S.H. Wilen, "Stereochemistry of Organic Compounds", Wiley-Interscience, 1994). The molecules described herein include compounds that are transisomers as well as compounds that can exist in different conformations or different rotational isomers. Transisomers are compounds that exist in different conformations, which are produced by restricted rotation around a single bond. If the energy barrier for rotation around the single bond is high enough that the rate of interconversion is slow enough for the individual rotational isomers to be separated from each other, multiple transisomers can be separated into individual chemical substances. This specification is intended to include all possible isomers, enantiomers, diastereomers, and transisomers of compounds disclosed herein or prepared using compounds disclosed herein. Among the molecules described herein, only those with an absolute conformation (or transisomer conformation) of their chiral center known are given a nomenclature or chemical formula indicating chirality or transisomerism. Those skilled in the art will readily understand when other chiral centers are present in the molecules described herein and when these chiral centers can be identified.

[0228] Compounds of any one of formula I that are chemically capable of forming salts are readily converted into pharmaceutically acceptable salts and can be isolated as pharmaceutically acceptable salts. Salt formation can occur by adding a pharmaceutically acceptable acid to form an acid addition salt. Salt formation can occur simultaneously with deprotection of nitrogen or oxygen, i.e., removal of the protecting group. Examples, reactions, and conditions of salt formation can be found in the following literature: Gould, PL, “Salt selection for basic drugs,” International Journal of Pharmaceutics, 33 : 201-217 (1986); Bastin, RJ et al. "SaltSelection and Optimization Procedures for Pharmaceutical New ChemicalEntities," Organic Process Research and Development, 4 : 427-435 (2000); and Berge, SM et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66 : 1-19, (1977).

[0229] The compounds of the present invention or their salts can be prepared by various operations, some of which are described in the schemes, preparation examples, and examples below. The specific synthetic steps of each described route can be combined in different ways or combined with steps from different routes to prepare the compounds or salts of the present invention. The products of each step in the preparation examples below can be recovered by conventional methods including extraction, evaporation, precipitation, chromatography, filtration, grinding, and crystallization.

[0230] Preparation Example 1

[0231] 5-Fluorobenzofuran-1(3H)-one

[0232]

[0233] At room temperature, Pd(OAc)₂ (10.95 g, 48.77 mmol, 0.02 equivalent) and XantPhos (42.33 g, 73.16 mmol, 0.03 equivalent) were added to a stirred mixture of (2-bromo-5-fluorophenyl)methanol (500 g, 2.44 mol) and TEA (474.6 mL, 3.41 mol, 1.4 equivalent) in ACN (2500 mL), and the mixture was stirred at 120 °C for 3 days under 10 atm of carbon monoxide. The reaction was cooled to room temperature and concentrated. The residue was diluted with H₂O (1,000 mL) and then extracted with EtOAc (2 × 1000 mL). The combined organic layers were washed with brine (2 × 1000 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue was ground with 10:1 hexane / EtOAc (1,100 mL) and then filtered. The filter cake was dried at 50°C for about 18 hours to give the title compound as a yellow solid (300 g, 81%). MS (ES) m / z = 153 (M+1).

[0234] Preparation Example 2

[0235] 4-Bromo-5-fluoro-6-nitroisobenzofuran-1(3H)-one

[0236]

[0237] At 65 °C, HNO3 (273.38 g, 4.348 mol, 2.2 equivalents) was added dropwise to a stirred mixture of 5-fluoroisobenzofuran-1(3H)-one (300 g, 1.97 mol) in H2SO4 (1,500 mL). The reaction was stirred for 1 hour and then cooled to room temperature. After 20 minutes, 1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione (2,255.43 g, 7.88 mol, 4 equivalents) was added in portions and stirred at room temperature for about 18 hours. The mixture was poured into ice / water (pretreated with 3 kg Na2SO3) and filtered. The filter cake was dissolved in EtOAc (3,000 mL), washed with saturated Na2CO3 aqueous solution (2 × 1000 mL) and brine (2 × 1000 mL), dried over anhydrous Na2SO4, and concentrated. The residue was ground with 10:1 hexane / EtOAc (660 mL), filtered, and dried at 50 °C for about 18 hours to give the title compound as a yellow solid (270 g, 49%), which was used in subsequent steps without further purification. 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 5.51 (s, 2H).

[0238] Preparation Example 3

[0239] 4-Bromo-5-fluoro-6-nitro-1,3-dihydroisobenzofuran

[0240]

[0241] DIBAL-H (1M, in THF, 1,467 mL, 1.467 mol, 1.5 equivalents) was added dropwise to a stirred mixture of 270 g (978 mmol) of 4-bromo-5-fluoro-6-nitroisobenzofuran-1(3H)-one in DCM (2,500 mL) at -78 °C and N2. The reaction was stirred at -78 °C for 5 h, and then quenched at -78 °C with 5N NaOH (300 mL). The resulting mixture was allowed to cool to room temperature and then concentrated. The residue was diluted with EtOAc (2,500 mL), washed with brine (2 × 1000 mL), dried over anhydrous Na2SO4, and concentrated. The residue was ground with 10:1 hexane / EtOAc (550 mL) and filtered. The solid was dried (190 g, 683.4 mmol) and then dissolved in DCM (1,500 mL). Et3SiH (662 mL, 4.10 mol, 6 equivalents) was added dropwise at 0 °C. The reaction was stirred at 0 °C for 20 min. TFA (152 mL, 2.05 mol, 3 equivalents) was added dropwise at 0 °C. The ice bath was removed, and the reaction was stirred at room temperature for approximately 18 h. The reaction was concentrated to give an oily substance, which was diluted with EtOAc (2,000 mL), washed with saturated aqueous solution of Na2CO3 (2 × 500 mL) and brine (2 × 500 mL), dried over anhydrous Na2SO4, filtered, and concentrated to give the title compound (110 g, 42%), which was used in subsequent steps without further purification. 1 H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J= 6.2 Hz, 1H), 5.18 – 5.15 (m, 2H), 5.11 –5.06 (m, 2H).

[0242] Preparation Example 4

[0243] 7-Bromo-6-fluoro-1,3-dihydroisobenzofuran-5-amine

[0244]

[0245] Fe (117.22 g, 2.09 mol, 5 equivalents) was added fractionally to a mixture of 4-bromo-5-fluoro-6-nitro-1,3-dihydroisobenzofuran (110 g, 420 mmol) and NH4Cl (112.3 g, 2.10 mol, 5 equivalents) in EtOH (1,000 mL) and H2O (200 mL) at room temperature, and then stirred at 80 °C for about 18 hours. The mixture was filtered and concentrated. The mixture was diluted with H2O (500 mL) and extracted with EtOAc (2 × 1000 mL). The combined organic layers were washed with brine (2 × 500 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified with silica gel (25% to 50% EtOAc / Hex) to give the title compound (70 g, 72%) as a yellow solid. MS (ES) m / z = 231 (M+1).

[0246] Preparation Example 5

[0247] N-[(7-bromo-6-fluoro-1,3-dihydroisobenzofuran-5-yl)thiocarbamoyl]carbamate

[0248]

[0249] Ethoxycarbonyl isothiocyanate (9.7 mL, 82 mmol, 0.93 equivalence) was slowly added via a feeding funnel to a solution of 7-bromo-6-fluoro-1,3-dihydroisobenzofuran-5-amine (20.4 g, 87.9 mmol) in DCM (550 mL), followed by stirring at room temperature for approximately 4 hours. The solid was filtered off. The filtrate was concentrated and suspended in DCM (100 mL) and hexane (350 mL) under stirring at room temperature. The resulting filtered solid and the previously filtered solid were dried under vacuum at 50 °C for 2 hours. The batches were combined to give the title compound (32.6 g, quantitative yield) as a white solid. MS (ES) m / z = 363 (M+1).

[0250] Preparation Example 6

[0251] (((7-bromo-6-fluoro-1,3-dihydroisobenzofuran-5-yl)amino)(ethylthio)methylene)carbamate

[0252]

[0253] A suspension of N-[(7-bromo-6-fluoro-1,3-dihydroisobenzofuran-5-yl)aminothiocarbamate]ethyl carbamate (32.6 g, 89.8 mmol) and acetone (450 mL) was added to a 2 L three-necked round-bottom flask equipped with a top stirrer, dropping funnel, and thermocouple. Solid K₂CO₃ (37.2 g, 269 mmol, 3.00 equivalent) was added in portions, followed by dropwise addition of EtI (7.2 mL, 90 mmol, 1.0 equivalent), over a period of 20 min. The mixture was stirred at room temperature for approximately 18 hours. The solid was filtered, and the filtrate was concentrated and partitioned between DCM (500 mL) and H₂O (500 mL). The organic layer was further washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue was purified with silica gel (0 to 30% EtOAc / Hex) to give the title compound (30.9 g, 85.6%) as a white solid. MS (ES) m / z = 391 (M+1).

[0254] Preparation Example 7

[0255] 6-Bromo-3-(ethio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-1-ol

[0256]

[0257] A 2L four-necked round-bottom flask equipped with a top stirrer, dropping funnel, N2 inlet tube, and thermocouple was purged with N2. 300 mL of anhydrous NMP was added. The mixture was heated to 175°C. In a second flask, under N2, 22.63 g (57.83 mmol) of ethyl (7-bromo-6-fluoro-1,3-dihydroisobenzofuran-5-yl)amino)(ethylthio)methylene)carbamate and 100 mL of anhydrous NMP were combined and stirred until a homogeneous solution was obtained. When the first flask reached 175°C, the contents of the second flask were poured into the dropping funnel and rapidly added dropwise to the hot NMP. After 30 min, the heating was turned off and the reaction was cooled to 45°C. 500 mL of H2O was slowly added, and the mixture was stirred at room temperature for 1 hour. The solid was filtered, washed with H2O (300 mL), and dried under vacuum at 50 °C for about 18 hours to give the title compound (15.2 g, 73%) as a creamy white solid. MS (ES) m / z = 363 (M+1).

[0258] Preparation Example 8

[0259] 6-Bromo-1-chloro-3-(ethio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolino

[0260]

[0261] A solution of DMF (50 mL, 646 mmol, 4 equivalents) in DCM (1,000 mL) was added to a 5 L three-necked round-bottom flask equipped with a dropping funnel, thermocouple, and top stirrer, and the flask was placed in an ice / water bath and cooled to approximately 4 °C. Oxaloyl chloride (50.0 mL, 576 mmol, 4 equivalents) was added dropwise through a feeding funnel over approximately 40 min. Once the addition was complete, the reaction mixture was stirred at approximately 4 °C for 15 min. Solid 6-bromo-3-(ethylthio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-1-ol (50.4 g, 140 mmol) was added to the reaction mixture in several portions, and the resulting suspension was stirred at approximately 4 °C for 30 min. The ice bath was removed, and the reaction mixture was warmed to room temperature and stirred for 1 h. Then, H₂O (1 L) was added, and the mixture was stirred for 15 min. The mixture was partitioned, the organic layer was washed with brine (1 L), dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue was purified with silica gel and eluted with DCM / Hex (60% to 90%) to give the title compound (45.1 g, 89%) as a white solid. MS (ES) m / z = 363 (M+1).

[0262] Preparation Example 9

[0263] 6-Bromo-3-(ethio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolino

[0264]

[0265] At room temperature, [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride (0.73 g, 1.00 mmol) and sodium cyanoborohydride (partially, 2.25 g, 35.8 mmol) were added to a mixture of 6-bromo-1-chloro-3-(ethylthio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazoline (6.50 g, 17.9 mmol) and tetramethylethylenediamine (2.28 g, 19.7 mmol) in THF (100 mL). The reaction mixture was stirred overnight under nitrogen, then diluted with water (500 mL) and extracted with EtOAc (3 x 500 mL). The combined organic layers were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by elution with silica gel using a PE solution containing 16-20% EtOAc to give the title compound (5.0 g, 75%) as a yellow solid. MS (ES) m / z = 329 (M+1).

[0266] Preparation Example 10

[0267] 8-(6-bromo-3-(ethio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-1-yl)-3,8-diazabicyclo[3.2.1] tert-butyl oct-3-carboxylic acid ester

[0268]

[0269] To a suspension of 6-bromo-1-chloro-3-(ethylthio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazoline (21.0 g, 57.8 mmol) in ACN (580 mL), tert-butyl 3,8-diazabicyclo[3.2.1]octyl-3-carboxylic acid (15.2 g, 69.5 mmol, 1.20 equivalents) and DIPEA (40 mL, 229 mmol, 4 equivalents) were added, and the mixture was stirred at room temperature for 90 min. H₂O (1 L) was slowly added through a funnel, and the mixture was stirred at room temperature for 1 min. The solid was filtered, washed with H₂O (500 mL), and dried under vacuum at 50 °C to give the title compound (31 g, quantitative) as a white solid, MS (ES) m / z = 539 (M+1).

[0270] Preparation Example 11

[0271] 6-Bromo-1-(3-((R)-2-((tert-butyldimethylsilyl)oxy)propyl)-3,8-diazabicyclo[3.2.1]oct-8-yl)-3-(ethio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin

[0272]

[0273] Using 3-((R)-2-((tert-butyldimethylsilyl)oxy)propyl)-3,8-diazabicyclo[3.2.1]octane in a manner similar to that described in Preparation Example 10, the title compound (4.7 g, 95%) was obtained as a brown solid.

[0274] Preparation Example 12

[0275] (4-Chloro-3-cyano-7-fluorothiopheno[3,2-c]pyridin-2-yl)tert-butyl carbamate

[0276]

[0277] Ethyl (3-cyano-7-fluorothieno[3,2-c]pyridin-2-yl)carbamate. A solution of 2-(4-chloro-5-fluoropyridin-3-yl)acetonitrile (11.8 g, 56.1 mmol) in DMF (112 mL) was cooled to 0 °C. Potassium tert-butoxide (7.00 g, 61.1 mmol) was added. After 15 minutes, ethoxycarbonyl isothiocyanate (7.45 mL, 61.8 mmol) was added dropwise. The reaction mixture was slowly warmed to room temperature overnight. The reaction mixture was poured into an ice / water (1.5 L) mixture, stirred until all the ice melted, and filtered through diatomaceous earth. The solid was dried in a vacuum drying oven (60 °C) overnight and separated from the diatomaceous earth to give ethyl N-(3-cyano-7-fluorothieno[3,2-c]pyridin-2-yl)carbamate (11.9 g, 79%) as a solid. MS (ES) m / z=266(M+1).

[0278] 2-Amino-7-fluorothieno[3,2-c]pyridine-3-carboxylonitrile. A suspension of ethyl (3-cyano-7-fluorothieno[3,2-c]pyridine-2-yl)carbamate (11.9 g, 44.4 mmol) in DMSO (90 mL) was cooled to 0 °C. NaOH (5 M, in water, 90 mL) was added dropwise over 15 minutes. The reaction mixture was heated to 105 °C for 1 hour and then cooled to room temperature. The reaction mixture was poured into an ice / water mixture (1.8 L), stirred until all the ice melted, and filtered through diatomaceous earth. The solid was dried overnight in a vacuum drying oven (50 °C) and separated from the diatomaceous earth to give crude 2-amino-7-fluoro-thieno[3,2-c]pyridine-3-carboxylonitrile.

[0279] (3-Cyano-7-fluorothieno[3,2-c]pyridin-2-yl) tert-butyl carbamate. A mixture of crude 2-amino-7-fluorothieno[3,2-c]pyridin-3-carboxylonitrile (8.6 g, 44.4 mmol), DCM (90 mL), DMF (90 mL), and N,N-diisopropylethylamine (15.5 mL, 88.9 mmol) was cooled to 0 °C. 4-Dimethylaminopyridine (0.54 g, 4.42 mmol) and di-tert-butyl dicarbonate (14.6 g, 66.7 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure, and the remainder was diluted with DCM (400 mL) and 5% citric acid aqueous solution (250 mL). The aqueous phase was washed twice with DCM. The combined organic phases were washed with a saturated aqueous solution of NaHCO3, dried over MgSO4, filtered, and concentrated to give N-(3-cyano-7-fluoro-thieno[3,2-c]pyridin-2-yl)carbamate tert-butyl ester (7.5 g, 58%), as a brown solid. MS (ES) m / z = 294 (M+1).

[0280] 2-((tert-Butoxycarbonyl)amino)-3-cyano-7-fluorothieno[3,2-c]pyridine 5-oxide. 3-Chloroperbenzoic acid (9.00 g, 40.2 mmol) was added to a solution of (3-cyano-7-fluorothieno[3,2-c]pyridin-2-yl)carbamate tert-butyl ester (7.85 g, 26.8 mmol) in DCM (180 mL). The reaction mixture was stirred overnight at room temperature and then cooled to 0 °C for about 15 minutes. The solid was collected by filtration and dried in a vacuum drying oven (60 °C). The filtrate was diluted with MeOH and silica gel, concentrated, and the residue was purified by silica gel elution with 0–6% MeOH in DCM. The fraction containing the desired substance was combined with the solid from filtration and concentrated to give N-(3-cyano-7-fluoro-5-oxo-thieno[3,2-c]pyridin-5-onthiol-2-yl)carbamate tert-butyl ester (7.26 g, 88%), as a creamy white solid. MS (ES) m / z = 310 (M+1).

[0281] (4-Chloro-3-cyano-7-fluorothieno[3,2-c]pyridin-2-yl)carbamate tert-butyl ester. A suspension of 2-((tert-butoxycarbonyl)amino)-3-cyano-7-fluorothieno[3,2-c]pyridine 5-oxide (5.27 g, 17.0 mmol) in 1,2-dichloroethane (34 mL) was cooled to 0 °C. A solution of phosphoryl chloride (32 mL, 344 mmol) in 1,2-dichloroethane (34 mL) was added dropwise. The reaction mixture was stirred at room temperature for 30 min, then at 45 °C for 90 min, and cooled to room temperature. The reaction mixture was diluted with 1,2-dichloroethane (100 mL) and added to a mixture of saturated aqueous NaHCO3 solution (500 mL), NaOH (5 M, in water, 40 mL), and ice. Solid NaHCO3 was added to the stirred mixture to maintain pH ~6-7. Once bubbling stopped, the phases were separated. The aqueous phase was extracted three times with DCM. The combined organic phases were dried over MgSO4 and filtered. The filtrate was diluted with MeOH and silica gel, concentrated, and the residue was eluted with 50-100% DCM in hexane and purified with silica gel. The fractions containing the desired substance were combined to give the title compound (3.87 g, 69%) as a white solid. MS (ES) m / z = 328 (M+1).

[0282] Preparation Example 13

[0283] 8-(6-(2-((tert-butoxycarbonyl)amino)-3-cyano-7-fluorothieno[3,2-c]pyridin-4-yl)-3-(ethio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-1-yl)-3,8-diazabicyclo[3.2.1]tert-butyl oct-3-carboxylate

[0284]

[0285] Potassium acetate (13.6 g, 139 mmol) and palladium dichloride;{2-[2-(diphenylphosphino)phenoxy]phenyl}diphenylphosphine (4.98 g, 6.95 mmol) were added to a solution of 8-(6-bromo-3-(ethylthio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-1-yl)-3,8-diazabicyclo[3.2.1]octyl-3-carboxylic acid tert-butyl ester (25.0 g, 46.3 mmol) and 5,5,5',5'-tetramethyl-2,2'-bi(1,3,2-dioxoborhexane) (20.9 g, 92.7 mmol) in 1,4-dioxane (300 mL) under nitrogen atmosphere. The mixture was stirred overnight at 85 °C and then filtered. The filter cake was washed with 1,4-dioxane (2 x 100 mL). The filtrate was concentrated under reduced pressure and purified by elution with silica gel using 0-20% EtOAc in PE to give 8-(6-(5,5-dimethyl-1,3,2-dioxaborhexane-2-yl)-3-(ethio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-1-yl)-3,8-diazabicyclo[3.2.1]oct-3-carboxylic acid tert-butyl ester (23 g, 87%) as a yellow solid.

[0286] Potassium phosphate (7.77 g, 36.6 mmol) and [2-dicyclohexane-2-yl)-3-(ethylthio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-1-yl)-3,8-diazabicyclo[3.2.1]octyl-3-carboxylic acid tert-butyl ester (6.99 g, 12.2 mmol) and (4-chloro-3-cyano-7-fluorothieno[3,2-c]pyridin-2-yl)carbamate tert-butyl ester (4.00 g, 12.2 mmol) in toluene (150 mL) were added to a stirred mixture of 8-(6-(5,5-dimethyl-1,3,2-dioxaborane-2-yl)-3-(ethylthio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolino-1-yl)-3,8-diazabicyclo[3.2.1]octyl-3-carboxylic acid tert-butyl ester (4.00 g, 12.2 mmol) in toluene (150 mL). 0.026 g, 0.031 mmol) and XPhosPalladacycle Gen 4 (CAS 1599466-81-5; 0.026 g, 0.031 mmol). The mixture was stirred at 80 °C for 4 hours, then diluted with water (300 mL) and extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with brine (2 x 300 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by elution with silica gel using 0–45% EtOAc in PE to give the title compound (6.0 g, 65%) as a yellow solid. MS (ES) m / z = 752 (M+1).

[0287] The following compounds in Table 1 were prepared in a manner similar to that described in Preparation Example 13. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0288] Table 1:

[0289]

[0290] Preparation Example 16

[0291] 8-(6-(2-((tert-butoxycarbonyl)amino)-3-cyano-7-fluorothieno[3,2-c]pyridin-4-yl)-3-(ethylsulfonyl)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-1-yl)-3,8-diazabicyclo[3.2.1]tert-butyl octyl-3-carboxylate

[0292]

[0293] mCPBA (4.05 g, 23.5 mmol) was added in portions to a solution of 8-(6-(2-((tert-butoxycarbonyl)amino)-3-cyano-7-fluorothieno[3,2-c]pyridin-4-yl)-3-(ethylthio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-1-yl)-3,8-diazabicyclo[3.2.1]octyl-3-carboxylic acid tert-butyl ester (8.4 g, 11.2 mmol) in DCM (100 mL). The reaction mixture was stirred at 0 °C for 3 hours. The mixture was diluted with DCM (800 mL) and washed with saturated aqueous solutions of NaHCO3 (3 x 400 mL) and brine (2 x 300 mL). The organic matter was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by elution with silica gel using 0-10% MeOH in DCM to give the title compound (8.3 g, 95%) as a creamy white solid. MS (ES) m / z = 784 (M+1).

[0294] The compounds in Table 2 below were prepared in a manner similar to that described in Preparation Example 16. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0295] Table 2:

[0296]

[0297] Preparation Example 18

[0298] (4-(1-(3-((R)-2-((tert-butyldimethylsilyl)oxy)propyl)-3,8-diazabicyclo[3.2.1]oct-8-yl)-3-(ethylsulfonyl)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-6-yl)-3-cyano-7-fluorothieno[3,2-c]pyridin-2-yl)tert-butyl carbamate

[0299]

[0300] (4-(1-(3-((R)-2-((tert-butyldimethylsilyl)oxy)propyl)-3,8-diazabicyclo[3.2.1]oct-8-yl)-3-(ethio)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-6-yl)-3-cyano-7-fluorothieno[3,2-c]pyridin-2-yl)carbamate (4 g, 5 mmol), hexaammonium heptamolybdate tetrahydrate (1 g, 1 mmol), and hydrogen peroxide (8 mL, 35 wt% aqueous solution, 100 mmol) were combined in DCM (30 mL) and EtOH (30 mL). The mixture was stirred at room temperature for 1 hour, then concentrated under reduced pressure. It was diluted with water (100 mL) and stirred for 30 minutes. The solid obtained was filtered and dried in a vacuum oven (45°C) to give the title compound (2 g, 50%) as a white solid. MS (ES) m / z = 856 (M+1).

[0301] Preparation Example 19

[0302] (2R,3S)-3-fluoro-2-(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0303]

[0304] A solution of 2-methyl (1.02 g, 4.13 mmol) 1-(tert-butyl) 1,2-dicarboxylic acid in THF (20 mL) was treated with lithium chloride (0.385 g, 9.08 mmol) and sodium borohydride (0.390 g, 10.3 mmol). The mixture was cooled to 0 °C and ethanol (40 mL) was added dropwise. The reaction mixture was slowly warmed to room temperature and stirred overnight. The mixture was cooled to 0 °C, acidified to pH ~4 with 1 M aqueous citric acid solution, and concentrated under reduced pressure to remove volatiles. The residue was diluted with water and extracted with EtOAc (3x). The combined organic compounds were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the title compound (0.95 g, 100%) as a colorless oil. MS (ES) m / z = 164 (M+1, -tBu).

[0305] The compounds in Table 3 below were prepared in a manner similar to that described in Preparation Example 19. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0306] Table 3:

[0307]

[0308] Preparation Example 21

[0309] (2R,3S)-3-fluoro-2-carboxypyrrolidine-1-carboxylic acid tert-butyl ester

[0310]

[0311] A mixture of (2R,3S)-3-fluoro-2-(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (1.96 g, 8.94 mmol) in DCM (50 mL) at 0 °C was treated with Dys-Martin periodide (4.74 g, 11.2 mmol). The reaction mixture was slowly warmed to room temperature and stirred overnight. The mixture was cooled to 0 °C, carefully treated with a saturated aqueous sodium bicarbonate solution, and diluted with water. The organic layers were separated. The aqueous layer (pH ~ 8) was extracted with DCM (3x). The combined organic compounds were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by elution with silica gel using 0–40% EtOAc in hexane to give the title compound (1.22 g, 63%) as a pale yellow oil. MS (ES) m / z = 162 (M+1, -tBu).

[0312] The compounds in Table 4 below were prepared in a manner similar to that described in Preparation Example 21. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0313] Table 4:

[0314]

[0315] Preparation Example 23

[0316] (2S,4R)-2-formyl-4-methoxypyrrolidine-1-carboxylic acid tert-butyl ester

[0317]

[0318] A mixture of (2S,4R)-4-methoxypyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) ester 2-methyl ester (9.84 g, 37.9 mmol) in DCM (190 mL) at -75 °C was treated dropwise with diisobutylaluminum hydride (1 M, in DCM; 79.7 mL, 79.7 mmol). The reaction mixture was stirred at -75 °C for 2.5 h, diluted with MeOH (10 mL), and allowed to warm to room temperature. The mixture was treated dropwise with saturated sodium potassium tartrate aqueous solution (100 mL), stirred at room temperature for 1 h, and extracted with DCM (2 x 250 mL). The combined organic matter was washed with brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by elution with silica gel using 0–60% EtOAc in hexane to give the title compound (8.00 g, 92%).

[0319] Preparation Examples 24 and 25

[0320] (2R,3S)-3-fluoro-2-(1-hydroxyethyl)pyrrolidine-1-carboxylic acid tert-butyl ester isomers 1 and 2

[0321]

[0322] A solution of (2R,3S)-3-fluoro-2-formylpyrrolidine-1-carboxylic acid tert-butyl ester (1.22 g, 5.62 mmol) in THF (30 mL) at -70 °C was treated with methyl magnesium bromide (1.4 M, in 3:1 THF:toluene; 6.0 mL, 8.4 mmol), maintaining the internal temperature below -60 °C. The reaction mixture was stirred for 1 hour and then warmed to 0 °C. The reaction mixture was cooled to -30 °C and treated with additional methyl magnesium bromide (1.4 M, in 3:1 THF:toluene; 2.0 mL, 2.8 mmol). The reaction mixture was stirred for 1 hour and then warmed to 15 °C. The reaction mixture was cooled to 0 °C and treated with acetic acid (1.29 mL, 22.5 mmol). The reaction mixture was stirred at room temperature for 15 minutes, diluted with a saturated aqueous solution of ammonium chloride and EtOAc, and stirred for 5 minutes. Add a small amount of water. Separate the organic layer. Extract the aqueous layer (pH ~ 5) with EtOAc (3x). Dry the combined organic matter over anhydrous Na2SO4, filter, and concentrate under reduced pressure. Purify the residue by elution with silica gel using 0–10% MTBE in DCM to give the title compound (isomer 1 was the first eluted, 0.279 g, 21%; isomer 2 was the second eluted, 0.297 g, 23%) as a white solid. Both had MS (ES) m / z of 178 (M+1, -tBu).

[0323] The compounds in Table 5 below were prepared in a manner similar to that described in Preparation Examples 24 and 25. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0324] Table 5:

[0325]

[0326] 1 Silica gel purification; 5% THF in DCM

[0327] 2 Silica gel purification; 2-10% THF in DCM

[0328] Preparation Example 29

[0329] 1-((2R,3S)-3-fluoro-1-methylpyrrolidone-2-yl)ethanol-1-ol isomer 1

[0330]

[0331] A mixture of (2R,3S)-3-fluoro-2-(1-hydroxyethyl)pyrrolidine-1-carboxylic acid tert-butyl ester isomer 1 (a monomethyl isomer from the precursor of Preparation Example 24; 0.279 g, 1.20 mmol) in THF (10 mL) at 0°C was treated with lithium aluminum hydride (1 M, in THF; 3.59 mL, 3.59 mmol). The reaction mixture was heated overnight at 70°C, cooled to 0°C, and quenched with sodium sulfate decahydrate (1.19 g, 3.69 mmol). The mixture was diluted with ether (10 mL), allowed to reach room temperature, and stirred for 2 hours. The mixture was filtered through diatomaceous earth and washed with ether. The combined filtrates were concentrated under reduced pressure. The residue was purified by elution with silica gel using 0–5% 1 M ammonia-methanol in DCM to give the title compound (0.092 g, 52%) as a yellow oil. MS (ES) m / z=148 (M+1).

[0332] The compounds in Table 6 below were prepared in a manner similar to that described in Preparation Example 29. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0333] Table 6:

[0334]

[0335] 1 Monomethyl isomer (from the precursor of Preparation Example 26)

[0336] 2 Monomethyl isomer (from the precursor of Preparation Example 27)

[0337] 3 Monomethyl isomer (from the precursor of Preparation Example 25)

[0338] 4 Monomethyl isomer (from the precursor of Preparation Example 28)

[0339] Preparation Example 34

[0340] 8-(6-(2-((tert-butoxycarbonyl)amino)-3-cyano-7-fluorothieno[3,2-c]pyridin-4-yl)-5-fluoro-3-(1-((2R,3S)-3-fluoro-1-methylpyrrolidine-2-yl)ethoxy)-7,9-dihydrofurano[3,4-f]quinazolin-1-yl)-3,8-diazabicyclo[3.2.1]tert-butyl octanoate isomer 1

[0341]

[0342] A solution of 1-((2R,3S)-3-fluoro-1-methylpyrrolidin-2-yl)ethanol-1-ol isomer 1 (a monomethyl isomer from the precursor of Preparation Example 24; 0.090 g, 0.61 mmol) in THF (5 mL) was treated with lithium bis(trimethylsilyl)amino (1.M, in THF; 1.3 mL, 1.3 mmol). The reaction mixture was stirred for 10 min and 8-(6-(2-((tert-butoxycarbonyl)amino)-3-cyano-7-fluorothieno[3,2-c]pyridin-4-yl)-3-(ethylsulfonyl)-5-fluoro-7,9-dihydrofurano[3,4-f]quinazolin-1-yl)-3,8-diazabicyclo[3.2.1]tert-butyl octyl-3-carboxylate (0.400 g, 0.51 mmol) was added. The reaction mixture was stirred for 1 hour, and lithium bis(trimethylsilyl)amino (1 M, in THF; 1.3 mL, 1.3 mmol) was added. The reaction mixture was stirred for 1 hour, then diluted with a saturated aqueous solution of ammonium chloride and EtOAc. A small amount of water was added. The organic layer was separated. The aqueous layer (pH ~ 7-8) was extracted with EtOAc (2x). The combined organic compounds were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by elution with silica gel using 0-5% 1 M ammonia-methanol in DCM to give the title compound (0.168 g, 39%) as an orange solid. MS (ES) m / z = 837 (M+1).

[0343] The compounds in Table 7 below were prepared in a manner similar to that described in Preparation Example 34. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0344] Table 7:

[0345]

[0346]

[0347] 1 Monomethyl isomer (from the precursor of Preparation Example 26)

[0348] 2 Monomethyl isomer (from the precursor of Preparation Example 27)

[0349] 3 Monomethyl isomer (from the precursor of Preparation Example 25)

[0350] 4 Monomethyl isomer (from the precursor of Preparation Example 28)

[0351] Preparation Example 44

[0352] 4-(1-(3,8-diazabicyclo[3.2.1]oct-8-yl)-5-fluoro-3-(1-((2R,3S)-3-fluoro-1-methylpyrrolidine-2-yl)ethoxy)-7,9-dihydrofurano[3,4-f]quinazolino-6-yl)-2-amino-7-fluorothieno[3,2-c]pyridine-3-carboxylonitrile isomer 1

[0353]

[0354] A solution of 8-(6-(2-((tert-butoxycarbonyl)amino)-3-cyano-7-fluorothieno[3,2-c]pyridin-4-yl)-5-fluoro-3-(1-((2R,3S)-3-fluoro-1-methylpyrrolidine-2-yl)ethoxy)-7,9-dihydrofurano[3,4-f]quinazolin-1-yl)-3,8-diazabicyclo[3.2.1]octyl-3-carboxylic acid tert-butyl ester isomer 1 (from the monomethyl isomer of the precursor of Preparation Example 24; 0.165 g, 0.168 mmol) in DCM (2 mL) was treated with TFA (0.80 mL). The reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere in a sealed vial, and then concentrated under a nitrogen stream. The residue was diluted with DCM and then concentrated under a nitrogen stream. The residue was dissolved in MeOH and purified on a strong cation exchange medium (10 g) by elution first with MeOH and DCM, followed by elution with 1:1 2M ammonia-methanol:DCM. The alkaline fraction was concentrated under reduced pressure. The residue was purified by silica gel elution with 0-13% 1M ammonia-methanol in DCM to give the title compound (0.078 g, 73%) as an orange solid. MS (ES) m / z = 637 (M+1).

[0355] The compounds in Table 8 below were prepared in a manner similar to that described in Preparation Example 44. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0356] Table 8:

[0357]

[0358]

[0359] 1 Monomethyl isomer (from the precursor of Preparation Example 26)

[0360] 2 Monomethyl isomer (from the precursor of Preparation Example 27)

[0361] 3 Monomethyl isomer (from the precursor of Preparation Example 25)

[0362] 4 Monomethyl isomer (from the precursor of Preparation Example 28)

[0363] Example 1

[0364] 2-Amino-7-fluoro-4-(5-fluoro-3-(1-((2R,3S)-3-fluoro-1-methylpyrrolidone-2-yl)ethoxy)-1-(3-((R)-2-hydroxypropyl)-3,8-diazabicyclo[3.2.1]oct-8-yl)-7,9-dihydrofurano[3,4-f]quinazolino-6-yl)thieno[3,2-c]pyridine-3-carboxynitrile isomer 1

[0365]

[0366] A solution of 4-(1-(3,8-diazabicyclo[3.2.1]oct-8-yl)-5-fluoro-3-(1-((2R,3S)-3-fluoro-1-methylpyrrolidone-2-yl)ethoxy)-7,9-dihydrofurano[3,4-f]quinazolino-6-yl)-2-amino-7-fluorothieno[3,2-c]pyridine-3-carboxynitrile isomer 1 (a monomethyl isomer from the precursor of Preparation Example 24; 0.075 g, 0.12 mmol) in THF (1.2 mL) and MeOH (0.3 mL) was treated with sodium triacetoxyborohydride (0.38 g, 1.8 mmol) and (R)-2-hydroxypropanal (0.9 M, in water; 2.0 mL, 1.8 mmol). The reaction mixture was stirred at room temperature for 1 hour and then poured into an ice-cold saturated aqueous solution of sodium bicarbonate. The organic layer was separated. The aqueous layer (pH ~ 8) was extracted with DCM (2x). The combined organic matter was passed through a hydrophobic filter and concentrated under a nitrogen stream. The residue was purified by elution with silica gel using 0–7.5% 1M ammonia-methanol in DCM to give the title compound (0.048 g, 59%) as a white solid. MS (ES) m / z = 695 (M+1).

[0367] The compounds in Table 9 below were prepared in a manner similar to that described in Example 1. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0368] Table 9:

[0369]

[0370]

[0371]

[0372] 1Monomethyl isomer (from the precursor of Preparation Example 26)

[0373] 2 Monomethyl isomer (from the precursor of Preparation Example 27)

[0374] 3 Monomethyl isomer (from the precursor of Preparation Example 25)

[0375] 4 Monomethyl isomer (from the precursor of Preparation Example 28)

[0376] The compounds in Table 10 below were prepared in a manner similar to that described in Preparation Example 44. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0377] Table 10:

[0378]

[0379] 1 Monomethyl isomer (from the precursor of Preparation Example 28)

[0380] Example 15

[0381] 2-Amino-7-fluoro-4-(5-fluoro-1-(3-(3-methoxypropyl)-3,8-diazabicyclo[3.2.1]oct-8-yl)-3-((S)-1-((S)-1-methylpyrrolidine-2-yl)ethoxy)-7,9-dihydrofurano[3,4-f]quinazolino-6-yl)thieno[3,2-c]pyridine-3-carboxynitrile methanesulfonic acid

[0382]

[0383] A solution of 2-amino-7-fluoro-4-(5-fluoro-1-(3-(3-methoxypropyl)-3,8-diazabicyclo[3.2.1]oct-8-yl)-3-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-7,9-dihydrofurano[3,4-f]quinazolino-6-yl)thieno[3,2-c]pyridin-3-carboxynitrile (1.3 g, 1.9 mmol) in DCM (10 mL) and MeOH (10 mL) was treated with methanesulfonic acid (0.13 mL, 1.9 mmol). The reaction mixture was stirred at room temperature for 20 min and then concentrated under reduced pressure. The solid was placed in a vacuum oven (40 °C) overnight to give the title compound (1.5 g, 100%) as a brown solid. MS (ES) m / z = 691 (M+1).

[0384] The compounds in Table 11 below were prepared in a manner similar to that described in Example 15. The compounds were purified using various methods, which will be apparent to those skilled in the art.

[0385] Table 11:

[0386]

[0387] 1 Monomethyl isomer (from the precursor of Preparation Example 26)

[0388] 2 Monomethyl isomer (from the precursor of Preparation Example 28)

[0389] Example 21

[0390] 2-Amino-7-fluoro-4-(5-fluoro-1-(3-(2-methoxyethyl)-3,8-diazabicyclo[3.2.1]oct-8-yl)-3-((S)-1-((S)-1-methylpyrrolidine-2-yl)ethoxy)-7,9-dihydrofurano[3,4-f]quinazolino-6-yl)thieno[3,2-c]pyridine-3-carboxynitrile methanesulfonic acid

[0391]

[0392] In a manner similar to that described in Example 1, 4-(1-(3,8-diazabicyclo[3.2.1]oct-8-yl)-5-fluoro-3-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-7,9-dihydrofurano[3,4-f]quinazolino-6-yl)-2-amino-7-fluorothieno[3,2-c]pyridine-3-carboxylonitrile and 2-methoxyethoxy Aldehydes were extracted to yield crude 2-amino-7-fluoro-4-(5-fluoro-1-(3-(2-methoxyethyl)-3,8-diazabicyclo[3.2.1]oct-8-yl)-3-((S)-1-((S)-1-methylpyrrolidine-2-yl)ethoxy)-7,9-dihydrofurano[3,4-f]quinazolino-6-yl)thieno[3,2-c]pyridin-3-carboxynitrile (1.01 g, 46%) as a brownish-brown solid. MS (ES) m / z = 677 (M+1).

[0393] Following a similar manner to that described in Example 15, 2-amino-7-fluoro-4-(5-fluoro-1-(3-(2-methoxyethyl)-3,8-diazabicyclo[3.2.1]oct-8-yl)-3-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-7,9-dihydrofurano[3,4-f]quinazolino-6-yl)thieno[3,2-c]pyridine-3-carboxynitrile was used to give the title compound (1.11 g, 96%) as a yellow solid. MS (ES) m / z = 677 (M+1).

[0394] Biological assay

[0395] The following assays demonstrate that the example compound is a potent Kras G12D inhibitor and inhibits the growth of certain tumors in vitro and / or in vivo.

[0396] PANC-1 cell viability RAS GTPase ELISA (KRas G12D mutation)

[0397] The purpose of this assay is to measure the ability of the test compound to inhibit constitutive RAS GTPase activity in human PANC-1 (RRID: CVCL_0480) pancreatic ductal adenocarcinoma cells (supplier: ATCC#CRL-1469). The RAS GTPase ELISA kit (Active Motif, catalog number 52097) contains a 96-well capture plate coated with glutathione and a glutathione-S-transferase (GST) fused to the Raf-Ras binding domain (RBD) protein provided with the kit. Activated pan-RAS (GTP-binding) in the cell extract specifically binds to the Raf-RBD. Bound RAS is detected by Ras primary antibodies that recognize human K-Ras (and H-Ras). HRP-conjugated anti-rat IgG secondary antibody recognizes the primary antibody, and the chromogenic substrate solution promotes chemiluminescent reading.

[0398] PANC-1 cells were seeded at a concentration of 75,000 cells / well in 80 µL of complete medium (DMEM, high glucose, L-glutamine, GIBCO; 10% heat-inactivated fetal bovine serum, GIBCO) and incubated overnight at 37°C / 5% CO2. After approximately 24 hours, 20 µL (1:3) serially diluted (in complete medium) of the test compound (1–50 µM maximum concentration) and 20 µL serially diluted (in complete medium) of the control (maximum signal well: 0.5% DMSO, minimum signal well: 10 µM reference positive control compound) were added to the cell plate and incubated for 2 hours at 37°C / 5% CO2. Complete lysis / binding buffer containing a protease inhibitor mixture (PIC) was prepared and stored on ice. One hour before the completion of cell plate incubation, GST-Raf-RBD was diluted in lysis / binding buffer, and 50 µL of the mixture buffer was added to each well of the supplied opaque white ELISA plate. The plate was incubated at 4°C for at least 1 hour with gentle shaking. After 2 hours, the cells were washed with 100 µL of ice-cold, Ca2+ / Mg2+-free PBS and lysed with 100 µL of the kit-supplied lysis / binding buffer (AM11). After vigorous shaking at ambient temperature for 30–50 minutes, the cell plate was centrifuged at 410×g (approximately 1500 rpm) for 10 minutes. During centrifugation, a wash buffer diluted to 1× with ultrapure H2O and filtered through a 0.2 µm filter was prepared at ambient temperature and used to wash the GST-Raf-RBD-coated plate (3×100 µL). Next, 50 µL of cell lysate was added to the GST-Raf-RBD-coated plate and incubated at ambient temperature for 1 hour with gentle shaking. During this incubation cycle, 1× antibody binding buffer was prepared from the thawed concentrate. The assay plate was washed with 1× wash buffer (3×100 µL), and then 50 µL of RAS primary antibody (kit supply #101678) diluted 1:500 in 1× antibody binding buffer was added. After incubating at ambient temperature with gentle shaking for 1 hour, the assay plate was washed with 1× wash buffer (3×100 µL). Subsequently, 50 µL of anti-rat HRP conjugated IgG secondary antibody (0.25 µg / µL) (diluted 1:5000 in 1× antibody binding buffer) was added to each well of the assay plate, and incubated again at ambient temperature with gentle shaking for 1 hour. Finally, the assay plate was washed with 1× wash buffer (4×100 µL), and then 50 µL of a mixed chemiluminescent working solution (a combination of reaction buffer and chemiluminescent substrate) at ambient temperature was added. A luminescence program optimized for the assay plate size was used with EnVision 2104. ™ The reader (Perkin Elmer) records the light emission data from each aperture.

[0399] The signal was converted to inhibition percentage using the following equation: Inhibition% = 100 - [(Test compound signal - Median minimum signal) / (Median maximum signal - Median minimum signal) × 100]. The maximum signal is the control well without inhibitor (DMSO). The minimum signal is the control well containing a reference inhibitor sufficient to completely inhibit activity. GenedataScreener was used. ® v17 determines IC by fitting the percentage of inhibition at each inhibitor concentration to a four-parameter nonlinear logic equation. 50 y = (A + ((BA) / (1 + ((x / C)^D)))), where y = suppression%, A = minimum asymptote, B = maximum asymptote, and C = relative IC. 50 Alternatively, an inhibitor concentration with 50% inhibition can be generated within the fitting range of the two asymptotes, and D = Hill slope.

[0400] In the above assays, compounds from Examples 1, 2, 4, 6-12, and 15-21 were tested. All compounds exhibited the ability to inhibit constitutive RAS GTPase activity, indicating that they have inhibitory effects on KRas G12D mutant enzymes, relative to IC50. 50 <100 nM. This data indicates that the compound of formula I described herein is a potent inhibitor of KRAS-GTP activity in this human pancreatic cancer cell culture, demonstrating its ability to inhibit KRas G12D mutations.

[0401] MKN-45 cell viability RAS GTPase ELISA (KRas wild type)

[0402] The purpose of this assay is to measure the ability of the test compound to inhibit constitutive RAS GTPase activity in human MKN-45 gastric adenocarcinoma cells (supplier: JCRB, supplier ID: JCRB 0254, batch: 05222009). The RAS GTPase ELISA kit (Active Motif, catalog number 52097) comprises a 96-well capture plate coated with glutathione and a glutathione-S-transferase (GST) fused to the Raf-Ras binding domain (RBD) protein provided with the kit. Activated pan-RAS (GTP-binding) in the cell extract specifically binds to the Raf-RBD. RAS binding is detected by a Ras primary antibody that recognizes human K-Ras (and H-Ras). An HRP-conjugated anti-rat IgG secondary antibody recognizes the primary antibody, and a chromogenic substrate solution facilitates chemiluminescent readings.

[0403] MKN-45 cells were seeded at a concentration of 75,000 cells / well in 80 µL of complete medium (DMEM, high glucose, L-glutamine, GIBCO; 10% heat-inactivated fetal bovine serum, GIBCO) and incubated overnight at 37°C / 5% CO2. After approximately 24 hours, 20 µL (1:3) serially diluted (in complete medium) of the test compound (1–10 µM maximum concentration) and 20 µL serially diluted (in complete medium) of the control (maximum signal well: 0.1% DMSO, minimum signal well: 10 µM reference positive control compound) were added to the cell plates and incubated for 2 hours at 37°C / 5% CO2. Complete lysis / binding buffer containing a protease inhibitor mixture (PIC) was prepared and stored on ice. One hour before the completion of cell plate incubation, GST-Raf-RBD was diluted in lysis / binding buffer, and 50 µL of the mixture buffer was added to each well of the supplied opaque white ELISA plate. The plate was incubated at 4°C for at least 1 hour with gentle shaking. After 2 hours, the cells were washed with 100 µL of ice-cold, Ca2+ / Mg2+-free PBS and lysed with 100 µL of the kit-supplied lysis / binding buffer (AM11). After vigorous shaking at ambient temperature for 30–50 minutes, the cell plate was centrifuged at 410×g (approximately 1500 rpm) for 10 minutes. During centrifugation, the wash buffer was diluted to 1× with ultrapure H2O and used to wash the GST-Raf-RBD-coated plate (3×100 µL). Next, 50 µL of cell lysate was added to the GST-Raf-RBD-coated plate and incubated at ambient temperature for 1 hour with gentle shaking. During this incubation cycle, 1× antibody binding buffer was prepared from the thawed concentrate. The assay plate (3×100 µL) was washed with 1× wash buffer, and then 50 µL of RAS primary antibody (kit supply #101678) diluted 1:500 in 1× antibody binding buffer was added. After incubating at ambient temperature with gentle shaking for 1 hour, the assay plate was washed with 1× wash buffer (3×100 µL). Subsequently, 50 µL of anti-rat HRP conjugated IgG secondary antibody (0.25 µg / µL) (diluted 1:5000 in 1× antibody binding buffer) was added to each well of the assay plate, and incubated again at ambient temperature with gentle shaking for 1 hour. Finally, the assay plate was washed with 1× wash buffer (4×100 µL), and then 50 µL of a mixed chemiluminescent working solution (a combination of reaction buffer and chemiluminescent substrate) at ambient temperature was added. A luminescence program optimized for the assay plate size was used with EnVision 2104. ™ The reader (Perkin Elmer) records the light emission data from each aperture.

[0404] The signal was converted to inhibition percentage using the following equation: Inhibition% = 100 - [(Test compound signal - Median minimum signal) / (Median maximum signal - Median minimum signal) × 100]. The maximum signal is the control well without inhibitor (DMSO). The minimum signal is the control well containing a reference inhibitor sufficient to completely inhibit activity. GenedataScreener was used. ® v17 determines IC by fitting the percentage of inhibition at each inhibitor concentration to a four-parameter nonlinear logic equation. 50 y = (A + ((BA) / (1 + ((x / C)^D)))), where y = suppression%, A = minimum asymptote, B = maximum asymptote, and C = relative IC. 50 Alternatively, an inhibitor concentration with 50% inhibition can be generated within the fitting range of the two asymptotes, and D = Hill slope.

[0405] The compounds in Examples 1, 2, 4, 6-8, 10-12, and 15-21 were tested in the two assays described above (PANC-1 cell activity RASGTPase ELISA and MKN-45 cell activity RASGTPase ELISA), and all compounds showed a significant (i.e., greater than 10-fold) selective inhibitory preference for KRas G12D mutants relative to KRas wild-type.

[0406] AlphaLISA targeting KRAS-inhibited cellular phosphorylated ERK ® Measurement

[0407] The purpose of these assays was to quantify the ability of the test compounds to selectively inhibit KRAS signaling in cells with amplified KRAS and those expressing the activating KRAS G12D mutation (Table 12). Cancer cell lines used in this study were selected based on the presence of homozygous activating KRAS G12D mutations or KRAS gene amplification.

[0408] Table 12: Cell line information

[0409]

[0410] The activity of the compound was determined by measuring changes in the phosphorylation levels of downstream effector extracellular signal-regulated kinases-1 and 2 (ERK1 / 2) in compound-treated cells. AlphaLISA was used. ® SureFire ® Ultra™ p-ERK 1 / 2 (Thr202 / Tyr204) Assay Kit (#ALSU-PERK-A50K, PerkinElmer) ®Waltham, MA) measures ERK-1 / 2 phosphorylation levels. The AlphaLISA® assay is a quantitative sandwich immunoassay that can be used to detect phosphorylation of target proteins from cell lysates using bead-based alpha technology. The assay kit contains two antibodies, one that binds to the phosphate-Thr202 / Tyr204 epitope on ERK-1 / 2, and another that recognizes a different site on the protein. One of these antibodies is biotinylated and binds to streptavidin-coated alpha donor beads, and the other antibody is used with AlphaLISA. ® Receptor bead conjugation. When ERK-1 / 2 is phosphorylated in cell lysates, donor and acceptor beads approach each other. When the donor bead is excited by light at a wavelength of 600 nm, a photosensitizer within the bead converts ambient oxygen into excited singlet states. When the acceptor bead is within 200 nm of this reaction, the singlet oxygen reacts with the acceptor, resulting in chemiluminescent emission. The amount of light measured is proportional to the amount of phosphorylated ERK-1 / 2 in the lysate. AlphaLISA ® SureFire ® The Ultra™ p-ERK 1 / 2 (Thr202 / Tyr204) Assay Kit contains AlphaLISA® antibody-conjugated donor and acceptor beads, lysis buffer concentrate, and a suite of proprietary buffers (activation buffer, reaction buffer 1, reaction buffer 2, and dilution buffer).

[0411] For the determination, a 10-point, 3-fold dilution series of test compounds and control acoustics (Labcyte ECHO) will be performed in 30 nL DMSO. ® Cells (from San Jose, CA) were allocated to white 384-well assay plates (Proxiplate-384, PerkinElmer #6008280). Cells were then added to the assay plates at cell line-specific densities (Table 18) in 8 μL of assay medium (HBSS, Sigma #55021C, 10% FBS, GIBCO #10082-147) per well. The final concentration of the compound in each well ranged from 0.5 to 10,000 nM, and the final concentration of DMSO was 0.375%. The maximum signal control well contained only 0.375% DMSO (negative control), and the minimum signal control well contained 10,000 nM of control compound (positive control). The cells in suspension were incubated with the test and reference compounds at 37°C / 5% CO2 for 2 h. After 2 h of incubation, the cells were analyzed by adding 2 μL of AlphaLISA supplemented with a protease / phosphatase inhibitor mixture (Thermo Scientific #78442). ®Cells were lysed using lysis buffer concentrate (5×). The assay plate was covered with an opaque cap and inducing cell lysis by shaking at 750 rpm for 30 minutes at room temperature on a multi-plate shaker (Heidolph, Schwabach, Germany). During lysis, AlphaLISA was used. ® Receptor beads in the prepared buffer mixture (1:1 AlphaLISA) ® Reaction buffers 1 and 2 and AlphaLISA ® Diluted 1:50 in a 1:25 dilution of activation buffer. After cell lysis, the plate was briefly centrifuged, and 5 μL of the prepared receptor beads were added to each well. The plate was then capped and incubated in the dark at room temperature for 2 hours. During receptor bead incubation, the cells were analyzed by AlphaLISA. ® Donor beads were prepared by diluting Alpha streptavidin donor beads 1:50 in dilution buffer. After incubation with recipient beads, 5 μL of the donor bead mixture was added to each well of the plate. The plate was then covered and incubated in the dark at room temperature for 2 hours. After this incubation period, the plates were used with AlphaLISA. ® Pherastar, compatible with optical cubes ® The FSX multi-mode plate reader (BMG Labtech, Ortenberg, Germany) reads AlphaLISA signals.

[0412] The raw signals obtained from the AlphaLISA® assay were analyzed using Genedata Screener® 17.0.3. In the procedure, data were normalized using 32 wells treated with an inhibition control (maximum inhibition / positive control) and 32 wells treated with only 0.375% DMSO (minimum inhibition / negative control) to calculate the % activity of the compound:

[0413] Equation 1

[0414] Using Genedata Screener ® 17.0.3 Fit the activity value (%) to a four-parameter nonlinear logic equation to determine the IC. 50 value%:

[0415] Equation 2

[0416] Where y = % activity, floor = minimum asymptote, ceiling = maximum asymptote, x = compound concentration, IC50 50 = The concentration of the compound that achieves half the maximum activity, and h = Hill coefficient.

[0417] In the above assays, the compounds of Examples 13-21 were tested and showed an ability to reduce the phosphorylation level of ERK-1 / 2 in cells expressing KRAS G12D, indicating that they inhibited constitutive RAS activity in cells expressing KRAS G12D, relative to IC50. 50 <100 nM. This data indicates that the compound of formula I described herein is a potent inhibitor of KRAS human cancer cells expressing KRAS G12D, demonstrating its ability to inhibit KRAS signaling in these cells.

[0418] Table 13: Abbreviations

[0419] .

Claims

1. The following compound: in A is -C(H)- or -N-; Z is -C(R) 3c - or -N-; G is -C(R) 3b - or -N-; R1 is a -H group or a group of the following formula. , or ; R2 is -H, halogen, or methyl; R 3b and R 3c Each can be independently -H, halogen, or methyl; R4 is a group in the following formula. or ; R5 is a C-type ... 1-4 alkyl; R 5a It is C 1-3 Alkylene; R6 is C 1-3 alkyl; R7 is -H or C 1-3 Alkyl; and R8 is -H, halogen, or C. 1-3 Alkyl groups; or pharmaceutically acceptable salts thereof.

2. The compound of claim 1, wherein G is -N- or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, wherein G is -C(R) 3b )-, or its pharmaceutically acceptable salt.

4. The compound according to claim 3, wherein R 3b It is -F, or a pharmaceutically acceptable salt thereof.

5. The compound according to any one of claims 1-4, wherein Z is -N- or a pharmaceutically acceptable salt thereof.

6. The compound according to any one of claims 1-4, wherein Z is -C(R) 3c )-, or its pharmaceutically acceptable salt.

7. The compound according to claim 6, wherein R 3c It is -H or -F, or a pharmaceutically acceptable salt thereof.

8. The compound according to claim 6, wherein R 3c It is -F, or a pharmaceutically acceptable salt thereof.

9. The compound according to any one of claims 1-8, wherein A is -N- or a pharmaceutically acceptable salt thereof.

10. The compound according to any one of claims 1-8, wherein A is -C(H)- or a pharmaceutically acceptable salt thereof.

11. The compound according to any one of claims 1-10, wherein R2 is F or Cl, or a pharmaceutically acceptable salt thereof.

12. The compound according to any one of claims 1-10, wherein R2 is -F, or a pharmaceutically acceptable salt thereof.

13. The compound according to any one of claims 1-10, wherein R2 is Cl, or a pharmaceutically acceptable salt thereof.

14. The compound according to any one of claims 1-13, wherein R1 is -H, or a pharmaceutically acceptable salt thereof.

15. The compound according to any one of claims 1-13, wherein R1 is a group of the following formula: , or Or, or a pharmaceutically acceptable salt thereof.

16. The compound according to claim 15, wherein R1 is a group of the following formula: Or, or a pharmaceutically acceptable salt thereof.

17. The compound according to claim 15 or 16, wherein R 5a It is an ethylene group, or a pharmaceutically acceptable salt thereof.

18. The compound of claim 17, wherein R1 is selected from... , , , , or Or, or a pharmaceutically acceptable salt thereof.

19. The compound of claim 18, wherein R1 is selected from... , , or Or, or a pharmaceutically acceptable salt thereof.

20. The compound according to any one of claims 1-19, wherein R4 is a group of the following formula: Or, or a pharmaceutically acceptable salt thereof.

21. The compound according to claim 20, wherein R4 is a group of the following formula: Or, or a pharmaceutically acceptable salt thereof.

22. The compound of claim 20, wherein R4 is a group of the following formula: Or, or a pharmaceutically acceptable salt thereof.

23. The compound according to any one of claims 1-19, wherein R4 is a group of the following formula: Or, or a pharmaceutically acceptable salt thereof.

24. The compound according to claim 23, wherein R4 is a group of the following formula: Or, or a pharmaceutically acceptable salt thereof.

25. The compound according to claim 23, wherein R4 is a group of the following formula: Or, or a pharmaceutically acceptable salt thereof.

26. The compound according to any one of claims 1-25, wherein R6 is a methyl group or a pharmaceutically acceptable salt thereof.

27. The compound according to any one of claims 1-26, wherein R7 is a methyl group or a pharmaceutically acceptable salt thereof.

28. The compound according to any one of claims 1-27, wherein R8 is -H, -F or methoxy, or a pharmaceutically acceptable salt thereof.

29. The compound of claim 28, wherein R8 is -H, or a pharmaceutically acceptable salt thereof.

30. The compound of claim 28, wherein R8 is -F, or a pharmaceutically acceptable salt thereof.

31. The compound of claim 28, wherein R8 is a methoxy group or a pharmaceutically acceptable salt thereof.

32. The compound according to any one of claims 1-19, wherein R4 is selected from... , , or Or, or a pharmaceutically acceptable salt thereof.

33. The compound of claim 32, wherein R4 is selected from... , , or Or, or a pharmaceutically acceptable salt thereof.

34. The compound of claim 32, wherein R4 is selected from... , , or Or, or a pharmaceutically acceptable salt thereof.

35. The compound of claim 32, wherein R4 is selected from... , , or Or, or a pharmaceutically acceptable salt thereof.

36. The compound according to claim 33 or 34, wherein R4 is selected from... , , or Or, or a pharmaceutically acceptable salt thereof.

37. The compound according to claim 1, wherein the compound is selected from: , , , , , , , , , , , or Or, or a pharmaceutically acceptable salt thereof.

38. A pharmaceutical composition comprising a compound according to any one of claims 1-37 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

39. A method of treating a patient with cancer, the method comprising administering to a patient in need an effective amount of the pharmaceutical composition according to claim 38, wherein the cancer is selected from lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, ovarian cancer, bile duct cancer, and colorectal cancer.

40. A method of treating a patient with cancer, the method comprising administering to a patient in need an effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-37, wherein the cancer is selected from lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, ovarian cancer, bile duct cancer, and colorectal cancer.

41. The method of claim 39 or 40, wherein the patient has cancer prior to administration of the compound or a pharmaceutically acceptable salt thereof, the cancer being identified as having one or more cells expressing the KRas G12D mutant protein.

42. The method according to any one of claims 39-41, wherein the cancer is non-small cell lung cancer.

43. The method according to any one of claims 39-41, wherein the cancer is colorectal cancer.

44. The method according to any one of claims 39-41, wherein the cancer is pancreatic cancer.

45. The method of any one of claims 39, 40 or 42-44, wherein one or more cells express the KRas G12D mutant protein.

46. ​​A method of treating a patient with cancer having a KRas G12D mutation, the method comprising administering to the patient in need an effective amount of a compound of any one of claims 1-37 or a pharmaceutically acceptable salt thereof.

47. The method of claim 46, wherein the cancer is selected from lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, mutant ovarian cancer, bile duct cancer, and colorectal cancer.

48. The method of claim 47, wherein the cancer is non-small cell lung cancer.

49. The method of claim 47, wherein the cancer is colorectal cancer.

50. The method of claim 47, wherein the cancer is pancreatic cancer.

51. The method according to any one of claims 39-50, wherein the patient is further administered an effective amount of one or more drugs selected from: PD-1 inhibitors, PD-L1 inhibitors, CDK4 / CDK6 inhibitors, EGFR inhibitors, ERK inhibitors, Aurora A inhibitors, SHP2 inhibitors, platinum-based drugs and pemetrexed, or pharmaceutically acceptable salts thereof.

52. The compound of any one of claims 1-37 or a pharmaceutically acceptable salt thereof, used for therapeutic purposes.

53. The compound of any one of claims 1-37 or a pharmaceutically acceptable salt thereof, for the treatment of cancer.

54. The compound of claim 53 for the said use, or a pharmaceutically acceptable salt thereof, wherein said cancer has a KRas G12D mutation.

55. The compound or a pharmaceutically acceptable salt thereof for the purpose according to any one of claims 53 or 54, wherein the cancer is selected from lung cancer, pancreatic cancer, cervical cancer, esophageal cancer, endometrial cancer, ovarian cancer, bile duct cancer, and colorectal cancer.

56. A compound of any one of claims 1-37 or a pharmaceutically acceptable salt thereof, for use in cancer treatment in conjunction with, separately or sequentially with, one or more of the following drugs: PD-1 or PD-L1 inhibitors, CDK4 / CDK6 inhibitors, EGFR inhibitors, ERK inhibitors, Aurora A inhibitors, SHP2 inhibitors, platinum-based drugs and pemetrexed, or a pharmaceutically acceptable salt thereof.

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