Tricyclic KRAS G12C inhibitors

Abstract

This disclosure provides compounds of formula (I): wherein R1, R2, R3, R4, R5, A and B are as described herein, pharmaceutically acceptable salts thereof, and methods of treating patients with cancer using these compounds and salts.

Description

[0001] This PCT international application claims the benefit of U.S. Provisional Application No. 63 / 121,272, filed on December 4, 2020, the full text of which is incorporated herein by reference.

[0002] This disclosure relates to novel tricyclic heterocyclic compounds and pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising said tricyclic heterocyclic compounds and salts, and methods of using said compounds and salts to treat cancers such as lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, or esophageal cancer.

[0003] The MAPK / ERK signaling pathway transmits extracellular stimuli to the cell nucleus, thereby regulating various cellular responses, including cell proliferation, differentiation, and apoptosis. KRas proteins are initiators of the MAPK / ERK signaling pathway and act 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 undergoes allosteric activation to allow GDP nucleotide exchange for guanosine triphosphate (GTP). In its GTP-binding active state, KRas recruits and activates proteins essential for the propagation of growth factor-induced signaling, as well as other cellular signaling receptors. Examples of proteins recruited by KRas-GTP include c-Raf and PI3-kinase. KRas, as a GTPase, converts bound GTP back to GDP, thereby returning itself to an inactive state and again propagating the signal to inhibit cell division. Gain-of-function mutations in KRas exhibit increased GTP binding and decreased ability to convert GTP to GDP. The result is increased MAPK / ERK signaling that promotes cancer cell growth. The missense mutation at codon 12 of KRas is the most common mutation and significantly reduces GTPase activity.

[0004] Oncogenic KRas mutations have been identified 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, ADD Rugging 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).

[0005] WO2015 / 054572 and WO2016 / 164675 disclose certain quinazoline derivatives capable of binding to KRas G12C. WO2016 / 044772 also discloses methods using such quinazoline derivatives. WO2020 / 0081282 discloses KRasG12C inhibitors. WO2018 / 206539 and WO2020 / 178282 disclose certain heteroaryl compounds capable of binding to the KRas G12C RAS ​​protein.

[0006] 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 efficacy at the same or reduced KRas inhibitory activity. Furthermore, there is a desire to provide KRas inhibitors that exhibit better pharmacokinetic / pharmacodynamic properties. There is also a need for more potent KRas inhibitors that exhibit improved efficacy and reduced or minimized adverse or undesirable effects. This disclosure addresses one or more of these needs by providing novel KRas inhibitors.

[0007] This disclosure provides compounds of formula I:

[0008]

[0009] Or its pharmaceutically acceptable salt, wherein:

[0010] A is -OCH2-, -N(R6)CH2, -OCH2CH2-, -N(R6)CH2CH2-, -CH2OCH2-, or -CH2N(R6)CH2-;

[0011] B is either -CH2- or -C(O)-;

[0012] R1 is a group of -CN, -C(O)C≡CR8 or the following formula.

[0013]

[0014] R2 is H, methyl, or -CH2CN;

[0015] R3 and R5 are independently H, halogen, cyclopropyl, and -C groups. 1-3 Alkyl-cyclopropyl, optionally R 10 Replaced 1-3 times -C1-6 alkyl or optionally R 10 Substitution of -O-C1-6 alkyl groups (1-3 times);

[0016] R4 is a group in the following formula

[0017]

[0018]

[0019] R is H, halogen, or optionally R 10 Replace -C 1-3 times 1-6 alkyl;

[0020] R′ is H or -C 1-6 alkyl;

[0021] R6 is H or optional R 10 Replace -C 1-3 times 1-6 alkyl;

[0022] R7 is H, halogen, -NR 11 R 12 -CH2NR 11 R 12 , optional R 10 or -NR 13 R 14 Replace -C 1-3 times 2-6 Alkyl, cyclopropyl, -C 1-3 alkylcyclopropyl or optionally R 10 or -NR 13 R 14 Substitution of -O-C1-6 alkyl groups (1-3 times);

[0023] R8 is H, optional R 10 Replaced 1-3 times -C1-4 alkyl or optionally R 10 Substitution of 1-3 times -C3-6 cycloalkyl groups;

[0024] R9 represents H, halogen, -CN, C. 3-6 cycloalkyl, -C 1-3 Alkyl-C 3-6 cycloalkyl or optionally R 10 Replace -C 1-3 times 1-6 alkyl;

[0025] R 10 Each time it appears, it is independently of halogen, oxygen, hydroxyl, -C. 1-4 Alkyl or -OC 1-4 alkyl;

[0026] R 11 and R 12 Each is independently H, -C 1-4 Alkyl or -C 1-4 Heteroalkyl, wherein R 11 and R 12 Can combine to form C 5-6Heterocyclic alkyl groups; and

[0027] R 13 and R 14 Each is independently H or -C 1-4 alkyl.

[0028] As used herein, the term halogen refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). The term alkyl refers to a saturated straight-chain or branched monovalent hydrocarbon group. "-C 1-6 Examples of "alkyl" include, but are not limited to, methyl, ethyl, propyl, 1-propyl, isopropyl, butyl, isobutyl, pentyl, and hexyl. The term "-C" as used herein... 1-4 "Heteroalkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon group containing 1 to 4 carbon atoms and at least one heteroatom. The term "-C" as used herein... 3-6 "Cycloalkyl" refers to a saturated monovalent cyclic molecule having 3 to 6 carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0029] Regarding R 11 and R 12 These two groups can, when chemically permissible, combine with the nitrogen to which they are attached to form C. 5-6 Heterocyclic alkyl groups. The term "-C" as used herein... 5-6 "Heterocyclic alkyl" refers to a saturated monovalent cyclic molecule having 4 to 5 carbon atoms and at least one heteroatom. Examples of heterocyclic alkyl include, but are not limited to, morpholine, pyrrolidine, piperidine, imidazoline, pyrazolidine, and piperazine.

[0030] One embodiment disclosed herein provides a compound of formula Ia or a pharmaceutically acceptable salt thereof.

[0031]

[0032] R1, R2, R3, R4, R5, A, and B are defined as above.

[0033] One embodiment disclosed herein provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2-, -N(R6)CH2-, -OCH2CH2-, or -N(R6)CH2CH2-. Another embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2- or -OCH2CH2-. Yet another embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2CH2-.

[0034] Further embodiments provide a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein B is -C(O)-.

[0035] Further embodiments provide a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R1 is -CN or -C(O)C≡CR8. Another embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R1 is a group of the following formula.

[0036]

[0037] Further embodiments provide a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R2 is H or a methyl group. Another embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R2 is H.

[0038] Further embodiments provide a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R3 is H, a halogen, preferably F or Cl, methyl, methoxy, ethyl, isopropyl, or cyclopropyl. Another embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R3 is a halogen, preferably F or Cl.

[0039] Further embodiments provide a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula

[0040] Another embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula

[0041] Another embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R4 is a group of the following formula

[0042]

[0043] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R is H or a halogen, preferably F.

[0044] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R5 is H, a halogen, CHF2, CH2F, CH2OH, or CH2OCH3. A further embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R5 is a halogen, preferably Cl.

[0045] Further embodiments provide a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R6 is H or CH3.

[0046] Further embodiments provide a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R9 is H, F, Cl, -CH2F, -CF3, or -CH2OH. Further embodiments provide a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R9 is H.

[0047] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R7 is H, -CHF2, -CH2F, -CH2OH, -CH2OCH3, -CH2N(CH3)2, or -CH2-morpholine. Another embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R7 is H.

[0048] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R9 is H and R7 is H, -CHF2, -CH2F, -CH2OH, -CH2OCH3, -CH2N(CH3)2 or -CH2-morpholine.

[0049] Another embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R9 is H, F, Cl, -CH2F, -CF3 or -CH2OH and R7 is H.

[0050] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R7 and R9 are both H.

[0051] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R1 is -CN or -C(O)C≡CR8 and R8 is H, methyl, -CH2F or -CH2OH.

[0052] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R1 is a group of the following formula

[0053]

[0054] And R7 is H, -CHF2, -CH2F, -CH2OH, -CH2OCH3, -CH2N(CH3)2 or -CH2-morpholine.

[0055] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R1 is a group of the following formula

[0056]

[0057] And R9 is H, F, Cl, CHF2, CF3 or CH2OH.

[0058] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R1 is a group of the following formula

[0059]

[0060] Both R7 and R9 are H.

[0061] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R1 is a group of the following formula

[0062]

[0063] Furthermore, R7 is tert-butyl and R9 is -CN.

[0064] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2-, -N(R6)CH2-, -OCH2CH2- or -N(R6)CH2CH2-, and B is -C(O)-.

[0065] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2- or -OCH2CH2- and B is -C(O)-.

[0066] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2CH2- and B is -C(O)-.

[0067] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2-, -N(R6)CH2-, -OCH2CH2- or -N(R6)CH2CH2-; B is C(O); and R2 is H or -CH3.

[0068] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2- or -OCH2CH2-; B is -C(O)-; and R2 is H or methyl.

[0069] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2CH2-, B is -C(O)-, and R2 is H or methyl.

[0070] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2-, -N(R6)CH2-, -OCH2CH2- or -N(R6)CH2CH2-; B is -C(O)-; and R2 is H.

[0071] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2- or -OCH2CH2-; B is -C(O)-; and R2 is H.

[0072] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2CH2-, B is -C(O)-, and R2 is H.

[0073] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2CH2- and R2 is H or methyl.

[0074] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2CH2- and R2 is H.

[0075] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein B is -C(O)- and R2 is H or methyl.

[0076] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein B is -C(O)- and R2 is H.

[0077] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R3 and R5 are each independently selected from H, halogens and methyl groups.

[0078] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein R3 or R5 is a halogen, preferably F or Cl.

[0079] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2- or -OCH2CH2-; B is -C(O)-; and R1 is a group of the following formula.

[0080]

[0081] R2 is H or methyl; R3 and R5 are each H, F, Cl or methyl; R4 is a group of the following formula.

[0082]

[0083] R6 is H or methyl; R7 and R9 are both H; R is H or F; R′ is H or methyl.

[0084] One embodiment provides a compound of formula I or Ia or a pharmaceutically acceptable salt thereof, wherein A is -OCH2CH2-; B is -C(O)-; and R1 is a group of the following formula.

[0085] R2, R7, and R9 are each H; R4 is a group of the following formula.

[0086] R3 and R5 are both halogens.

[0087] One embodiment provides a compound selected from any one of formulas II-VI:

[0088]

[0089]

[0090] Or its pharmaceutically acceptable salt.

[0091] Another embodiment provides a compound of formula II or a pharmaceutically acceptable salt thereof.

[0092] Another embodiment provides a compound of formula III or a pharmaceutically acceptable salt thereof.

[0093] Another embodiment provides a compound of formula IV or a pharmaceutically acceptable salt thereof.

[0094] Another embodiment provides a compound of formula V or a pharmaceutically acceptable salt thereof.

[0095] Another embodiment provides a compound of formula VI or a pharmaceutically acceptable salt thereof.

[0096] Another embodiment provides a pharmaceutical composition comprising a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient.

[0097] Another embodiment provides a method of treating cancer, comprising administering to a patient in need an effective amount of a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof. In various embodiments, the cancer is lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, or esophageal cancer. In a preferred embodiment, the cancer is non-small cell lung cancer, pancreatic cancer, or colorectal cancer. In a more preferred embodiment, the cancer is non-small cell lung cancer.

[0098] Another embodiment provides a method of treating cancer, comprising administering to a patient in need an effective amount of a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. In another embodiment, the cancer is non-small cell lung cancer, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. In another embodiment, the cancer is colorectal cancer, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. In yet another embodiment, the cancer is mutant pancreatic cancer, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. Another embodiment provides a method of treating cancers from other sources carrying a KRas G12C mutation.

[0099] Another embodiment provides a method for treating a patient with cancer having a KRAS G12C mutation, comprising administering to the patient in need an effective amount of a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof.

[0100] Another embodiment provides a method for modulating a mutant KRas G12C enzyme in a patient requiring it by administering a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof. The method preferably comprises inhibiting a human mutant KRas G12C enzyme.

[0101] Another embodiment provides a method for treating cancer in a patient who requires it, wherein the patient has cancer that has been identified as expressing a KRas G12C mutant protein. The method includes administering to the patient an effective amount of a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof. The G12C mutation status of one or more cancer cells can be determined by a number of assays known in the art. Typically, one or more biopsies containing one or more cancer cells are obtained and sequenced and / or polymerase chain reaction (PCR) is performed. 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., the G12C mutation status in one or more cancer cells or cell-free DNA) include direct sequencing, next-generation sequencing, reverse transcription polymerase chain reaction (RT-PCR), multiplex PCR, pyrosequencing, and multi-analyte profiling.

[0102] Another embodiment provides a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof for treatment. Another embodiment provides said compound or a pharmaceutically acceptable salt thereof for treating cancer. Preferably, the cancer is lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, or esophageal cancer. In a preferred embodiment, the cancer is non-small cell lung cancer, pancreatic cancer, or colorectal cancer. In a more preferred embodiment, the cancer is non-small cell lung cancer. In other embodiments, the cancer has one or more cancer cells expressing a mutant KRas G12C protein. Preferably, the cancer is selected from: KRas G12C mutant non-small cell lung cancer, KRas G12C mutant colorectal cancer, and KRas G12C mutant pancreatic cancer. In another embodiment, the cancer is non-small cell lung cancer, and one or more cells express the KRas G12C mutant protein. In another embodiment, the cancer is colorectal cancer, and one or more cells express the KRas G12C mutant protein. In another embodiment, the cancer is pancreatic cancer, and one or more cells express the KRas G12C mutant protein. In another embodiment, the patient has cancer identified as having one or more cells expressing the KRas G12C mutant protein prior to administration of the compound or a pharmaceutically acceptable salt thereof.

[0103] Another embodiment provides the use of a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer. Preferably, the cancer is lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, or esophageal cancer. In a preferred embodiment, the cancer is non-small cell lung cancer, pancreatic cancer, or colorectal cancer. In a more preferred embodiment, the cancer is non-small cell lung cancer. In other embodiments, the cancer has one or more cancer cells expressing a mutant KRas G12C protein. Preferably, the cancer is selected from KRas G12C mutant non-small cell lung cancer, KRas G12C mutant colorectal cancer, and KRas G12C mutant pancreatic cancer.

[0104] One embodiment provides a method of treating cancer, comprising administering to a patient in need an effective amount of a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof, and one or more of the following: a PD-1 inhibitor, a PD-L1 inhibitor, a CDK4 / CDK6 inhibitor or a pharmaceutically acceptable salt thereof, an EGFR inhibitor or a pharmaceutically acceptable salt thereof, an ERK inhibitor or a pharmaceutically acceptable salt thereof, platinum, and pemetrexed or a pharmaceutically acceptable salt thereof, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. Another embodiment also provides a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof, which is used simultaneously, separately, or sequentially in combination with one or more of a PD-1 or PD-L1 inhibitor, a CDK4 / CDK6 inhibitor or a pharmaceutically acceptable salt thereof, an EGFR inhibitor or a pharmaceutically acceptable salt thereof, an ERK inhibitor or a pharmaceutically acceptable salt thereof, platinum, and pemetrexed or a pharmaceutically acceptable salt thereof for the treatment of cancer. Another embodiment provides a combination comprising a compound according to any one of formulas I-VI or a pharmaceutically acceptable salt thereof, and one or more of a PD-1 or PD-L1 inhibitor, a CDK4 / CDK6 inhibitor or a pharmaceutically acceptable salt thereof, an EGFR inhibitor or a pharmaceutically acceptable salt thereof, an ERK inhibitor or a pharmaceutically acceptable salt thereof, a platinum agent, and pemetrexed or a pharmaceutically acceptable salt thereof, for simultaneous, separate, or sequential use in the treatment of cancer.

[0105] One embodiment provides a method of treating cancer, comprising administering to a patient in need an effective amount of a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof and a PD-1 or PD-L1 inhibitor, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. One embodiment also provides a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof, used simultaneously, separately, or sequentially in combination with a PD-1 or PD-L1 inhibitor for treating cancer. One embodiment further provides a combination comprising a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof and a PD-1 or PD-L1 inhibitor for simultaneous, separately, or sequential treatment of cancer. In another embodiment, the PD-1 or PD-L1 inhibitor is pembrolizumab. In another embodiment, the PD-1 or PD-L1 inhibitor is nivolumab. In another embodiment, the PD-1 or PD-L1 inhibitor is cimiprimab. In another embodiment, the PD-1 or PD-L1 inhibitor is sintilimab. In another embodiment, the PD-1 or PD-L1 inhibitor is atezolizumab. In another embodiment, the PD-1 or PD-L1 inhibitor is avelumab. In another embodiment, the PD-1 or PD-L1 inhibitor is durvalumab. In another embodiment, the PD-1 or PD-L1 inhibitor is lodapilimab. In another embodiment, the cancer is non-small cell lung cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. In another embodiment, the cancer is colorectal cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. In another embodiment, the cancer is mutant pancreatic cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. One embodiment includes a method of treating cancers from other sources carrying the KRas G12C mutation.

[0106] One embodiment provides a method of treating cancer, comprising administering to a patient in need an effective amount of a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof and a CDK4 / CDK6 inhibitor or a pharmaceutically acceptable salt thereof, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. One embodiment also provides a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof, used simultaneously, separately, or sequentially in combination with a CDK4 / CDK6 inhibitor or a pharmaceutically acceptable salt thereof for treating cancer, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. One embodiment further provides a combination comprising a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof and a CDK4 / CDK6 inhibitor or a pharmaceutically acceptable salt thereof for simultaneous, separately, or sequentially treating cancer, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. In another embodiment, the CDK4 / CDK6 inhibitor is abemaciclib. In another embodiment, the CDK4 / CDK6 inhibitor is palbociclib. In another embodiment, the CDK4 / CDK6 inhibitor is ribociclib. In another embodiment, the cancer is non-small cell lung cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. In another embodiment, the cancer is colorectal cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. In another embodiment, the cancer is mutant pancreatic cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. One embodiment includes a method of treating cancers from other sources carrying the KRas G12C mutation.

[0107] One embodiment also provides a method of treating cancer, comprising administering to a patient in need an effective amount of a compound of any one of formulas I-VI 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 G12C protein. One embodiment also provides a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof, used simultaneously, separately, or sequentially in combination with an EGFR inhibitor or a pharmaceutically acceptable salt thereof for treating cancer. One embodiment also provides a combination comprising a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof and an EGFR inhibitor or a pharmaceutically acceptable salt thereof for simultaneous, separately, or sequential treatment of cancer. In one embodiment, the compound is a compound of formula I-VI or a pharmaceutically acceptable salt thereof. In another embodiment, the EGFR inhibitor is erlotinib. In another embodiment, the EGFR inhibitor is afatinib. In another embodiment, the EGFR inhibitor is gefitinib. In another embodiment, the EGFR inhibitor is cetuximab. In another embodiment, the cancer is non-small cell lung cancer, wherein the cancer has one or more cells expressing the KRasG12C mutant protein. In another embodiment, the cancer is colorectal cancer, wherein the cancer has one or more cells expressing the KRasG12C mutant protein. In another embodiment, the cancer is mutant pancreatic cancer, wherein the cancer has one or more cells expressing the KRasG12C mutant protein. One embodiment includes a method of treating cancers from other sources carrying the KRasG12C mutation.

[0108] One embodiment also provides a method of treating cancer, comprising administering to a patient in need an effective amount of a compound of any one of formulas I-VI 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 G12C protein. One embodiment provides a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof, used simultaneously, separately, or sequentially in combination with an ERK inhibitor or a pharmaceutically acceptable salt thereof for treating cancer, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. One embodiment provides a combination comprising a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof and an ERK inhibitor or a pharmaceutically acceptable salt thereof for simultaneous, separately, or sequential treatment of cancer. In one embodiment, the compound is a compound of formula I-VI or a pharmaceutically acceptable salt thereof. In another embodiment, the ERK inhibitor is 6,6-dimethyl-2-[2-[(2-methylpyrazol-3-yl)amino]pyrimidin-4-yl]-5-(2-morpholin-4-ylethyl)thieno[2,3-c]pyrrolo-4-one. In another embodiment, the ERK inhibitor is LTT462. In another embodiment, the ERK inhibitor is KO-947. In another embodiment, the cancer is non-small cell lung cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. In another embodiment, the cancer is colorectal cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. In another embodiment, the cancer is mutant pancreatic cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. One embodiment includes a method of treating cancers from other sources carrying the KRas G12C mutation.

[0109] One embodiment provides a method of treating cancer, comprising administering to a patient in need an effective amount of a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof and a platinum agent, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. One embodiment provides a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof, in combination with a platinum agent or a pharmaceutically acceptable salt thereof, simultaneously, separately, or sequentially for treating cancer, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. One embodiment provides a combination comprising a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof and a platinum agent for simultaneous, separately, or sequentially treating cancer. In one embodiment, the compound is a compound of formulas I-VI or a pharmaceutically acceptable salt thereof. In another embodiment, the platinum agent is cisplatin. In another embodiment, the platinum agent is carboplatin. In another embodiment, the platinum agent is oxaliplatin. In another embodiment, the cancer is non-small cell lung cancer, wherein the cancer has one or more cells expressing a mutant KRasG12C protein. In another embodiment, the cancer is colorectal cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. In another embodiment, the cancer is mutant pancreatic cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. One embodiment includes a method for treating cancers from other sources carrying the KRas G12C mutation.

[0110] One embodiment also provides a method of treating cancer, comprising administering to a patient in need an effective amount of a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof, and pemetrexed, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. One embodiment provides a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof, in combination with pemetrexed simultaneously, separately, or sequentially for treating cancer, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. One embodiment also provides a combination comprising a compound of any one of formulas I-VI or a pharmaceutically acceptable salt thereof and pemetrexed for simultaneous, separately, or sequentially treating cancer, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. In one embodiment, the compound is a compound of formula I-VI or a pharmaceutically acceptable salt thereof. In another embodiment, the cancer is non-small cell lung cancer, wherein the cancer has one or more cells expressing a mutant KRas G12C protein. In another embodiment, a platinum agent is also administered to the patient. In another embodiment, the platinum agent is cisplatin. In another embodiment, the platinum agent is carboplatin. In another embodiment, the platinum agent is oxaliplatin. In another embodiment, the cancer is colorectal cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. In another embodiment, the cancer is mutant pancreatic cancer, wherein the cancer has one or more cells expressing the KRas G12C mutant protein. One embodiment includes a method of treating cancers from other sources carrying the KRas G12C mutation.

[0111] 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 “Handbook of Pharmaceutical Salts: Properties, Selection and Use”, P. Stahl et al., 2nd revised edition, Wiley-VCH, 2011, and SMBerge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Sciences, 1977, 66(1), 1-19.

[0112] The pharmaceutical compositions disclosed herein can be prepared using pharmaceutically acceptable additives. As used herein, the term "pharmaceutically acceptable additive" 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 "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, sugar, mannitol, and silica derivatives; binders such as carboxymethyl cellulose, alginate, gelatin, and polyvinylpyrrolidone; kaolin and bentonite; and polyethylene glycol.

[0113] As used herein, the term "effective dose" refers to the amount that is effective as a dosage for treating an impairment or disease, such as cancerous lesions or the progression of abnormal cell growth and / or cell division. An attending physician, as a person skilled in the art, can readily determine the effective dose using conventional techniques and by observing results obtained in similar situations. The daily dose for treatment is typically in the range of approximately 1 mg / day or twice daily to 1000 mg / day or twice daily, more preferably from 100 mg / day or twice daily to 900 mg / day or twice daily. Factors considered in determining the effective dose or dosage of a compound include: administration of the compound or its salt; co-administration of other agents, if used; the species of the patient being treated; the patient's body type, age, and general health condition; the degree or stage and / or severity of the impairment; the individual patient's response; the mode of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of other concomitant medications.

[0114] A treating physician, veterinarian, or other medical professional can determine the effective amount of the compound for treating a patient in need. Preferred pharmaceutical compositions may 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 disclosed herein for treating a patient in need of cancer treatment.

[0115] The term “treatment” as used herein includes slowing, alleviating, stopping, or reversing the progression or severity of existing symptoms, disorders, or conditions, which may include specifically slowing the growth of cancerous lesions or the progression of abnormal cell growth and / or cell division.

[0116] As used herein, the term "patient" refers to a mammal in need of treatment. Preferably, a patient is a human in need of treatment for cancer, such as cancer carrying the KRas G12C mutation.

[0117] Some abbreviations are defined as follows: "ACN" refers to acetonitrile; "Boc-Gly-OH" refers to N-(tert-butoxycarbonyl)glycine; "DCM" refers to dichloromethane; "DIEA" refers to N,N-diisopropylethylamine; "DMAP" refers to 4-dimethylaminopyridine; "DMEM" refers to Dulbecco modified Eagle medium; "DMF" refers to N,N-dimethylformamide; "DMSO" refers to dimethyl sulfoxide; "DNA" refers to deoxyribonucleic acid; "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 acetic acid. Ethyl ester; "EtOH" refers to ethanol; "FBS" refers to fetal bovine serum; "GDP" refers to guanosine diphosphate; "GTP" refers to guanosine triphosphate; "HPLC" refers to high-performance liquid chromatography; "HRP" refers to horseradish peroxidase; "HATU" refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; "IPA" refers to isopropanol; "IPAm" refers to isopropylamine; "LC-ES / MS" refers to liquid chromatography-electrospray ionization mass spectrometry; "LC-MS" refers to liquid chromatography-mass spectrometry; "MAPK" refers to mitogen-activated protein kinase; "MeOH" refers to methanol; "NCS" refers to N-chlorosuccinimide; "PCR" refers to polymerase chain reaction; "RPMI" refers to Roswell. Park Memorial Institute; "TEA" refers to triethylamine; "TFA" refers to trifluoroacetic acid; and "THF" refers to tetrahydrofuran.

[0118] Independent isomers, enantiomers, diastereomers, and transisomers can be separated or resolved at any convenient point in the synthesis of the following compounds by methods such as 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. Leel and S.H. Wilen, "Stereochemistry of Organic Compounds", Wiley-Interscience, 1994). This disclosure includes certain compounds that are transisomers and can exist in different conformations or as different rotational isomers. Transisomers are compounds that exist in different conformations resulting from restricted rotation around a single bond. If the energy barrier for rotation around the single bond is high enough and the rate of interconversion is slow enough to allow the individual rotational isomers to separate from each other, transisomers can be separated as separate chemical classes. This disclosure envisions all isomers, enantiomers, diastereomers and transisomers disclosed herein, or all isomers, enantiomers, diastereomers and transisomers that can be prepared using the compounds disclosed herein.

[0119] Compounds of any one of formulas I-VI are readily convertible into pharmaceutically acceptable salts and can be isolated as pharmaceutically acceptable salts. Salt formation can occur upon the addition of a pharmaceutically acceptable acid to form an acid addition salt. Salts can also be formed simultaneously with nitrogen or oxygen deprotection, i.e., removal of the protecting group. Examples, reactions, and conditions for salt formation can be found in Gould, PL, “Salt selection for basic drugs,” International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, RJ et al., “Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,” Organic Process Research and Development, 4: 427-435 (2000); and Berge, SM et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66: 1-19, (1977).

[0120] Compounds of formulas I-VI or their salts can be prepared by various procedures, some of which are illustrated in the preparations and examples below. The specific synthetic steps of each route can be combined in different ways, or combined with steps from different routes, to prepare the compounds or salts disclosed herein. The products of the steps in the following preparations can be recovered by conventional methods, including extraction, evaporation, precipitation, chromatography, filtration, preparation, and crystallization.

[0121] Preparation and Examples

[0122] The following preparations and examples further illustrate the present disclosure and represent typical synthesis of the compounds disclosed, but should not be construed as limiting the scope of the disclosure in any way. The reagents and starting materials are readily available or can be easily synthesized by known procedures or by various modifications that can be made by those skilled in the art.

[0123] Compounds were characterized by liquid chromatography-electrospray ionization mass spectrometry (LC-ES / MS) performed on an Agilent HP1100 HPLC system. Electrospray ionization mass spectrometry measurements were performed on a quadrupole mass spectrometer with a mass-selective detector connected to the HP1100 HPLC (acquired in positive and / or negative modes). LC-MS conditions (low pH): Column: NX C-182.1×50mm 3.0μm; Gradient: 5-100% B over 3 min, then 100% B for 0.75 min; Column temperature: 50℃ ± 10℃; Flow rate: 1.2 mL / min; Solvent A: Deionized water containing 0.1% HCOOH; Solvent B: ACN containing 0.1% formic acid; Wavelength: 214 nm. Alternative LC-MS conditions (high pH): Column: WATERS TM MS C-18 column, 2.1 × 50 mm, 3.5 m; gradient: 5% solvent A held for 0.25 min, gradient from 5% to 100% solvent B over 3 min, and 100% solvent B held for 0.5 min, or from 10% to 100% solvent B over 3 min, and 100% solvent B held for 0.75 min; column temperature: 50℃ ± 10℃; flow rate: 1.2 mL / min; solvent A: 10 mM NH4HCO3, pH 9; solvent B: ACN; wavelength: 214 nm.

[0124] Preparative reversed-phase chromatography was performed on an Agilent 1200LC-ES / MS instrument equipped with a mass-selective detector and a Leap autosampler / fraction collector. High pH method was performed on a 75 x 30 mm instrument. The system was run on a 5 μm particle size column with a 10 x 20 mm guard column. The flow rate was 85 mL / min. The eluent was 10 mM ammonium bicarbonate (pH 10) / ACN.

[0125] Option 1

[0126]

[0127] Scheme 1, step A depicts the reaction of compound (1) and 2-bromo-5-fluoroaniline in a solvent such as THF to form thiourea, followed by basic deprotection to provide compound (2). Step B shows the bromination and cyclization of compound (2) in a solvent such as sulfuric acid using a suitable brominating agent such as pyridinium tribromide to give compound (3). Step C depicts the deamination of compound (3) by treatment with isoamyl nitrite in a suitable solvent such as 1,4-dioxane under heating to give compound (4). Step D shows the conversion of the bromide of compound (4) to a boronic ester by treatment with bis(pinacolyl)diboron in a solvent such as 1,4-dioxane under heating using a suitable base such as potassium acetate and a catalyst such as [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) to give compound (5).

[0128] Option 2

[0129]

[0130] Scheme 2, step A describes the Curtiss rearrangement of compound (6) with diphenyl azidophosphate in a solvent such as tert-butanol using a suitable base such as DIEA to give compound (7). Step B shows compound (7) being cyaninated with chlorosulfonyl isocyanate in a solvent such as THF, followed by treatment with DMF to give compound (8).

[0131] Option 3

[0132]

[0133] Scheme 3, step A describes the nucleophilic substitution of compound (9) with malononitrile in a solvent such as DMF using a suitable base such as NaH to give compound (10). Step B shows the nitro reduction of compound (10) with zinc and acetic acid and subsequent cyclization to give compound (11). Step C shows the overall protection of compound (11) in a solvent such as THF with di-tert-butyl dicarbonate and a suitable base such as DMAP to give compound (12).

[0134] Option 4

[0135]

[0136] Scheme 4, step A describes the chlorination of compound (13) with NCS in a suitable solvent such as DMF to give compound (14). Step B shows the Sandmeier reaction of converting the aniline nitrogen of compound (14) to iodine under conditions known to those skilled in the art to give compound (15). Step C shows the basic hydrolysis of the ester of compound (15) to the acid of compound (16).

[0137] Option 5

[0138]

[0139] In scheme 5, step A is carried out in a manner substantially similar to that in step A of scheme 4 to obtain compound (18). Step B shows the Sandmeier reaction of converting the aniline nitrogen of compound (18) to bromine to obtain compound (19), under conditions known to those skilled in the art.

[0140] Option 6

[0141]

[0142] Scheme 6, Step A depicts the reductive amination between compound (20) and benzaldehyde in a suitable solvent such as DCM using a suitable reducing agent such as sodium triacetoxyborohydride, to give compound (21). Step B shows the amide coupling between compound (21) and boc-protected glycine in a solvent such as DCM using propylphosphonic anhydride and a suitable base such as TEA, to give compound (22). Step C depicts the acidic deprotection and rearrangement of compound (22) in a solvent such as DCM using TFA, to give compound (23). Step D shows the bulk amide reduction of compound (23) in a solvent such as THF using a reducing agent such as lithium aluminum hydride, to give compound (24). Step E depicts the protection of compound (24) in an aqueous sodium bicarbonate solution using di-tert-butyl dicarbonate, to give compound (25). Step F shows the deprotection of compound (25) by means of hydrogenation, to give compound (26).

[0143] Option 7

[0144]

[0145] In Scheme 7, compound (27) represents benzoic acid from Schemes 4-5 and commercially available benzoic acid. Step A shows the amide coupling between compound (27) and compound (26) in a solvent such as THF using HATU and a suitable base such as DIEA to obtain compound (28). Those skilled in the art will recognize that amide coupling is carried out under various conditions. Step B depicts the intramolecular cyclization of compound (28) into compound (29) in a solvent such as DMF using a suitable base such as sodium hydride.

[0146] Option 8

[0147]

[0148] Scheme 8, step A is carried out in a manner substantially similar to that in step D of Scheme 1 to obtain compound (30). Step B describes the Suzuki cross-coupling between compound (29) and a suitable borate ester in a solvent system such as 1,4-dioxane and water under heating using a suitable catalyst such as [1,1'-bis(diphenylphosphino)ferrocene]palladium dichlorochloride (II) with a suitable base such as tripotassium phosphate, to obtain compound (31). Step C also produces compound (31) by Suzuki cross-coupling between compound (30) and a suitable coupling partner in a solvent system such as 1,4-dioxane and water under heating using a suitable catalyst such as 1,1'-bis(di-tert-butylphosphino)ferrocene palladium dichlorochloride with a suitable base such as potassium carbonate, and compound (30). Step D shows that compound (31) is acidically deprotected with an acid such as TFA in a solvent such as DCM to obtain compound (32). Step E shows the amide formation between compound (32) and acryloyl chloride using a suitable base such as DIEA in a solvent such as DCM to obtain compound (33). This amide can also be formed using potassium carbonate as a base in two-phase solvent systems such as EtOAc, THF, and water.

[0149] Preparation 1

[0150] N-(4-bromo-1,3-benzoxazol-2-yl)tert-butyl carbamate

[0151]

[0152] Di-tert-butyl dicarbonate (724 mg, 3.317 mmol) and DMAP (28 mg, 0.227 mmol) were added to a mixture of 4-bromobenzo[d]oxazol-2-amine (504 mg, 2.248 mmol) in DCM (11 mL). The resulting mixture was stirred at ambient temperature under nitrogen for 18 hours. After this time, an additional 490 mg, 2.245 mmol, of di-tert-butyl dicarbonate was added to the mixture and stirred at ambient temperature for 1 hour, followed by the addition of sodium methoxide (5 M in MeOH) (4.5 mL, 23 mmol) and vigorous stirring for 10 minutes. The mixture was diluted with DCM and water. The aqueous layer was adjusted to pH 9 with saturated ammonium chloride solution, and the layers were separated. The aqueous phase was extracted twice more with DCM. The organic matter was combined, passed through a hydrophobic glass buffer, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography elution with 5-25% acetone / hexane to give the title compound (638 mg, 91%) as a white solid. ES / MS m / z ( 79 Br / 81 Br)256.8 / 258.8[Mt-Bu+H] + .

[0153] Preparation 2

[0154] N-[(2-bromo-5-fluoro-phenyl)thiocarbamoyl]benzamide

[0155]

[0156] A solution of 2-bromo-5-fluoroaniline (250 g, 1289.4 mmol) in THF (400 mL) was stirred with a top-mounted mechanical stirrer. Benzoyl isothiocyanate (130 g, 780.6 mmol) was added to THF (800 mL) over 30 minutes using a feeding funnel. The internal temperature was maintained below 30 °C using a water bath during the addition. After 1.5 hours, the reaction mixture was aliquoted into three 4 L flasks containing 3 L of water. The resulting solid was filtered through a sintered glass funnel under vacuum. The solid was washed with deionized water (8 L) and dried under vacuum to give the title compound (456 g, 99+%) as a brown solid. ES / MS m / z ( 79 Br / 81 Br)353 / 355[MH - .

[0157] Preparation 3

[0158] (2-Bromo-5-fluoro-phenyl)thiourea

[0159]

[0160] A suspension of N-[(2-bromo-5-fluoro-phenyl)thiocarbamoyl]benzamide (1600 g, 4.53 mol), THF (6 L), and MeOH (1.6 L) was added to a 5N NaOH aqueous solution (1 L). After stirring at ambient temperature for 18 hours, the reaction mixture was filtered through a diatomaceous earth mat to remove black particles. The mat was rinsed with THF / MeOH, then with 100% MeOH. The solvent was removed under vacuum to obtain a brown solid. Ice water (4 L) was added to the solid, and using a top-mounted stirrer, 5N HCl (300 mL) was added in 100 mL portions to adjust the pH to 7. Ice water was added again, and the mixture was stirred for 1 hour. Water (4 L) was added, and the suspension was filtered under vacuum through a large sintered glass funnel. The solid was rinsed with deionized water, and after removing most of the water, it was rinsed with hexane (8 L) and dried. The solid was placed in a vacuum oven at 50°C for 24 hours to obtain the title compound (1035 g, 92%) as a grayish-white solid. ES / MS m / z ( 79 Br / 81 Br)249 / 251[MH] - .

[0161] Preparation 4

[0162] 4-Bromo-7-fluoro-1,3-benzothiazol-2-amine

[0163]

[0164] Sulfuric acid (350 mL) cooled in an ice / sodium chloride bath was stirred with a top-mounted mechanical stirrer. (2-Bromo-5-fluoro-phenyl)thiourea (130.5 g, 523.9 mmol) was added aliquots over 5 minutes. After 10 minutes, the internal temperature reached 1 °C. Under nitrogen, pyridinium tribromide (185 g, 549.53 mmol) was added in 8 aliquots over 15 minutes, while maintaining the internal temperature below 5 °C. The generated vapors were bubbled through an ice-cooled NaOH trap. After stirring at 0 °C for 75 minutes, the reaction was heated to ambient temperature. The reaction was then heated to the initial internal temperature of 50 °C, and then gradually increased to 59 °C. After 1.5 hours, the reaction was cooled to ambient temperature. The reaction mixture was poured into a large flask containing ice. The pH was carefully adjusted to 7 with 18.9 N NaOH (620 mL). The solid was filtered through a sintered glass funnel and washed with deionized water until the pH of the filtrate matched that of the deionized water. The solid was air-dried and then placed in a vacuum oven at 50°C for 24 hours to obtain the title compound (129.3 g, 99+%) as a brownish-brown solid. ES / MS m / z ( 79 Br / 81 Br)247 / 249[M+H+ .

[0165] Preparation 5

[0166] 4-Bromo-7-fluoro-1,3-benzothiazole

[0167]

[0168] A solution of 4-bromo-7-fluoro-1,3-benzothiazol-2-amine (626 mg, 2.53 mmol), 1,4-dioxane (10 mL), and isoamyl nitrite (0.50 mL, 3.7 mmol) was stirred at 60 °C for 1 hour. Once cooled, the reaction mixture was concentrated under vacuum. The crude substance was purified by rapid silica gel chromatography eluting with 5–20% EtOAc / hexane to give the title compound (359 mg, 99+%). 1 H NMR (DMSO-d6) δ9.59 (1H, s,), 7.87 (1H, dd, J=7.9, 7.41 (1H, t, J=8.8Hz)).

[0169] Preparation of 6

[0170] N-(4-bromobenzofuran-2-yl)tert-butyl carbamate

[0171]

[0172] A mixture of 4-bromobenzofuran-2-carboxylic acid (1.00 g, 4.15 mmol), diphenyl azidophosphate (1.34 mL, 6.20 mmol), and DIEA (1.09 mL, 6.25 mmol) in tert-butanol (12 mL) was stirred at ambient temperature for 10 min, then heated to 95 °C. After 2.5 h, the heat was removed and the solvent was removed under vacuum. The resulting residue was purified by rapid silica gel chromatography eluting with 0–50% EtOAc / hexane to give the title compound (850 mg, 66%) as a white solid. ES / MS m / z ( 79 Br / 81 Br)255.6 / 257.6[Mt-Bu+H] + .

[0173] Preparation 7

[0174] N′-(4-bromo-3-cyano-benzofuran-2-yl)-N,N-dimethylformamidinium

[0175]

[0176] To a mixture of N-(4-bromobenzofuran-2-yl)carbamate tert-butyl ester (500 mg, 1.60 mmol) in THF (8 mL) stirred at -78 °C, chlorosulfonyl isocyanate (0.21 mL, 2.40 mmol) was added. After 1 hour, chlorosulfonyl isocyanate (0.21 mL, 2.40 mmol) was added again and the mixture was stirred at -78 °C for another 1 hour. After this time, the mixture was poured into DMF (5 mL) and stirred at ambient temperature for 20 minutes. The mixture was then diluted with EtOAc, washed twice with saturated sodium bicarbonate aqueous solution, and once with saturated sodium chloride aqueous solution. The organic matter was dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluting with 0–100% EtOAc / hexane to give the title compound (210 mg, 37%) as a pale yellow solid. ES / MS m / z ( 79 Br / 81 Br)291.6 / 293.6[M+H] + .

[0177] Preparation of 8

[0178] 1-Iodo-7-nitronaphthalene

[0179]

[0180] A suspension of 7-nitronaphthyl-1-amine (1.15 g, 6.11 mmol), tert-butyl nitrite (3.2 mL, 24 mmol), and cuprous iodide (2.39 g, 12.3 mmol) in ACN (12 mL) was stirred at 50 °C for 2 hours. The mixture was concentrated under vacuum, diluted with EtOAc, and washed successively with 1 N HCl, saturated NaHSO3 solution, and saturated sodium chloride aqueous solution. The organic matter was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluting with 0–10% acetone / heptane to give the title compound (745 mg, 41%) as a pale yellow solid. EI / MS m / z 299 [M].

[0181] Preparation 9

[0182] 2-(2-bromo-6-nitro-phenyl)malononitrile

[0183]

[0184] Sodium hydride (60% suspension in mineral oil, 0.32 g, 8.0 mmol) was added to a solution of malononitrile (0.911 g, 13.8 mmol) in DMF (15 mL) at 0 °C. The mixture was stirred for 30 minutes. Solid 1,2-dibromo-3-nitrobenzene (2.0 g, 6.8 mmol) was added in portions, and the mixture was heated at 70 °C. After 5 hours, the mixture was cooled to ambient temperature, diluted with water, and the pH was adjusted to 2 with concentrated HCl. The aqueous solution was extracted with EtOAc, the combined organic matter was washed with 0.2 M LiCl aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. The resulting solid was used as the crude product (2.9 g, 99+%) for the next step. ES / MS m / z ( 79 Br / 81 Br)264 / 266[MH] - .

[0185] Preparation 10

[0186] 2-Amino-4-bromo-1H-indole-3-carboxynitrile

[0187]

[0188] Crude 2-(2-bromo-6-nitro-phenyl)malononitrile (2.9 g) and zinc powder (9.0 g, 117 mmol) were suspended in glacial acetic acid (70 mL) and heated at 75 °C. After 1 hour, the mixture was cooled to ambient temperature and concentrated under vacuum, then diluted with EtOAc and washed with saturated sodium bicarbonate solution. The organic matter was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluting with 0–50% acetone / hexane to give the title compound (550 mg, 32%) as a brown solid. ES / MS m / z ( 79 Br / 81 Br)236 / 238[M+H + .

[0189] Preparation 11

[0190] 2-[bis(tert-butoxycarbonyl)amino]-4-bromo-3-cyano-indole-1-carboxylic acid tert-butyl ester

[0191]

[0192] A solution of 2-amino-4-bromo-1H-indole-3-carboxynitrile (0.320 g, 1.36 mmol) in THF (7 mL) was treated with DMAP (0.033 g, 0.27 mmol) and di-tert-butyl dicarbonate (1.26 g, 5.60 mmol) and stirred at room temperature for 22 hours. The mixture was diluted with EtOAc and washed with saturated ammonium chloride solution and saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluting with 0–30% EtOAc / hexane to give the title compound (327 mg, 45%) as a white solid. ES / MS m / z ( 79 Br / 81 Br)434 / 436[M-Boc-H] - .

[0193] Preparation 12

[0194] 2-(7-fluorobenzothiophene-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentane

[0195]

[0196] 7-Fluorobenzothiophene-4-ol (200 mg, 1.19 mmol) was dissolved in DCM (12 mL) and pyridine (0.2 mL, 2 mmol) was added. The mixture was cooled to 0 °C and trifluoromethanesulfonic anhydride (0.24 mL, 1.4 mmol) was added dropwise. The solution was stirred at 0 °C for 1 hour. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (5 mL) and diluted with EtOAc (70 mL). The organic layer was separated, washed with saturated sodium chloride aqueous solution (30 mL), dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was immediately suspended in 1,4-dioxane (8 mL) and bis(pinacol)diboron (453 mg, 1.78 mmol), 1,1'-bis(diphenylphosphino)ferrocene-palladium(II) dichloride DCM complex (100 mg, 0.120 mmol), and potassium acetate (350 mg, 3.57 mmol) were added successively. The mixture was bubbled with nitrogen for 10 minutes and then heated to 110°C with stirring. After stirring for 1 hour, the mixture was cooled and diluted with EtOAc (150 mL). The organic layer was washed with a saturated aqueous sodium chloride solution (100 mL), dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluting with 10% EtOAc / hexane to give the title compound (76.1 mg, 23%) as a white solid. 1H NMR (CDCl3) δ 8.03 (1H, dd, J=5.5, 4.0Hz), 7.87 (1H, dd, J=7.9, 5.7Hz), 7.52 (1H, d, J=5.4Hz), 7.04 (1H, dd, J=9.9, 7.9Hz), 1.38 (s, 12H).

[0197] Preparation 13

[0198] 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaboranecyclopentan-2-yl)-1,3-benzothiazole

[0199]

[0200] Potassium acetate (500 mg, 5.09 mmol), 4-bromo-1,3-benzothiazole (365 mg, 1.70 mmol), and bis(pinacol)diboron (662 mg, 2.61 mmol) were added to a vial. 1,4-dioxane (11 mL) was added under nitrogen, and the mixture was purged with nitrogen for 10 min. [1,1'-bis(diphenylphosphine)ferrocene]dichloropalladium(II) (134 mg, 0.18 mmol) was added in a single batch, and the vial was sealed. The mixture was stirred at 110 °C for 1 h, then at 80 °C for 18 h. The reaction mixture was cooled to ambient temperature and filtered through a diatomaceous earth mat. The filtrate was concentrated under vacuum and purified by rapid silica gel chromatography eluting with 0–100% EtOAc / hexane to give the title compound (386 mg, 56%). 1 H NMR (DMSO-d6) δ9.43 (1H, s,), 8.30 (dd, J=8.0, 1.0Hz, 1H), 7.83 (dd, J=7.0, 1.0Hz, 1H), 7.48 (t, J=7.5Hz, 1H), 1.34 (s, 12H).

[0201] Table 1: Compounds prepared in a manner substantially similar to that used in preparation 13

[0202]

[0203] NMR data for intermediate 15: 1 H NMR (DMSO-d6) δ 8.15 (1H, d, J = 8.1Hz), 7.89 (1H, d, J = 5.3Hz), 7.83 (1H, d, J = 5.7Hz), 7.76 (1H, dd, J = 6.9, 1.0Hz), 1.35 (12H, s).

[0204] Preparation of 16

[0205] Methyl 4-amino-3,5-dichloro-2-fluorobenzoate

[0206]

[0207] Methyl 4-amino-2-fluorobenzoate (27.0 g, 160 mmol) and NCS (46.3 g, 336 mmol) were dissolved in DMF (300 mL) and heated at 80 °C. After 40 min, the reaction mixture was poured over ice water and extracted twice with EtOAc. The combined organic extracts were washed once with 5 N NaOH and twice with 0.2 N LiCl, dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum to give the title compound (37.0 g, 97%). ES / MS m / z ( 35 Cl / 37 Cl)238 / 240[M+H + .

[0208] Table 2: Compounds prepared in a manner substantially similar to that used in preparation 16

[0209]

[0210] Preparation of 18

[0211] 3,5-Dichloro-2-fluoro-4-iodobenzoic acid

[0212]

[0213] Cuprous iodide (10.0 g, 51.5 mmol), ACN (128 mL), and tert-butyl nitrite (13.6 mL, 103 mmol) were combined and heated at 50 °C for 30 min. Methyl 4-amino-3,5-dichloro-2-fluorobenzoate (6.11 g, 25.7 mmol) was then added to the mixture. Gas escaping was observed. After 1 hour and 40 minutes at 50 °C, the solvent was removed under vacuum. Water, EtOAc, and 1 N HCl were added. The aqueous layer was extracted twice with EtOAc. The combined organic extracts were washed with an aqueous sodium bisulfite solution, dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum. The crude mixture was purified by rapid silica gel chromatography eluting with 3–5% EtOAc / hexane. The cleanest fractions were combined and concentrated under vacuum to give the methyl ester intermediate (5.85 g, 65%).

[0214] To methyl 3,5-dichloro-2-fluoro-4-iodobenzoate (5.85 g, 16.8 mmol), MeOH (170 mL) and 1N NaOH (17 mL) were added. The substance was completely dissolved during stirring at ambient temperature for 40 minutes. The MeOH was removed under vacuum. EtOAc and 1N HCl were added to the residue. The aqueous layer was extracted twice with EtOAc, and the combined organic extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum to give the title compound (5.56 g, 99%). 1 H NMR (CDCl3) δ 8.063-8.08 (d, 1H, J=6.34Hz).

[0215] Preparation of 19

[0216] 4-Bromo-5-chloro-2,3-difluorobenzoic acid

[0217]

[0218] A solution of 4-amino-2,3-difluorobenzoic acid (33.3 g, 192 mmol) in ACN (400 mL) was added to a three-necked flask equipped with a reflux condenser. NCS (34.5 g, 251 mmol) was added in small portions. The mixture was then heated to 80 °C and stirred for 2.5 h. After this time, the reaction was cooled to ambient temperature and then to 10 °C in an ice-water bath. Water (1.2 L) was added dropwise and stirring continued for 1 h. The mixture was then stirred for 3 days while being heated to ambient temperature. The resulting solid was then collected by filtration to obtain 4-amino-5-chloro-2,3-difluorobenzoic acid (39.9 g, 65%). 1 H NMR (DMSO-d6) δ 12.96 (s, 1H), 7.54 (dd, J = 6.9, 2.1 Hz, 1H).

[0219] A solution of copper bromide (2.15 g, 9.63 mmol) and tert-butyl nitrite (2.55 mL, 19.3 mmol) in ACN (25 mL) was added to a flask, and the container was placed in an oil bath preheated to 80 °C. 4-Amino-5-chloro-2,3-difluorobenzoic acid (1.00 g, 4.82 mmol) was added aliquots, and stirring was continued at 80 °C. After 1.5 hours, the mixture was cooled to ambient temperature and concentrated under vacuum. The mixture was diluted with EtOAc (50 mL) and washed with 1N HCl (2 × 50 mL) and a saturated aqueous solution of NaHSO3 (50 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum to give the title compound (1.30 g, 82%). 1H NMR (DMSO-d6) δ7.89 (dd, J=2.2, 6.2Hz, 1H).

[0220] Preparation 20

[0221] (3S)-3-(benzylamino)tetrahydrofuran-2-one

[0222]

[0223] L-homoserine lactone hydrochloride (100g, 726.93mmol), A suspension of powdered molecular sieve (178 g) and benzaldehyde (57 mL, 561.3 mmol) in DCM (2500 mL) was stirred overnight at 35 °C under nitrogen. After this time, the heating was removed and the mixture was cooled to 20 °C. Sodium triacetoxyborohydride (208 g, 981.41 mmol) was added to the mixture, and after 20 minutes, it was heated to ambient temperature and stirred for 2 hours. Subsequently, the mixture was cooled to -10 °C and carefully quenched with a saturated aqueous sodium bicarbonate solution (400 mL). The pH was adjusted to 8 with a saturated aqueous sodium bicarbonate solution and solid sodium bicarbonate. The mixture was filtered through a diatomaceous earth pad and thoroughly washed with DCM. The layers were separated, and the aqueous layer was extracted once more with DCM (1 L). The combined organic matter was dried over sodium sulfate, filtered, and concentrated under vacuum to give the title compound (87 g, 66.5%, 81% by mass) as a clear oil. ES / MS m / z: 192.2 (M+H). For further analysis, a sample (1 g) of the title compound was purified by rapid silica gel chromatography eluting with 10–50% EtOAc / DCM to yield 422 mg of purified title compound. This substance was used... IC, 4.6x150mm, 10% IPA (0.2% IPAm) / CO2, 5mL / min, 225nm analysis, showed >98% ee.

[0224] Preparation 21

[0225] N-[2-[benzyl-[(3S)-2-oxotetrahydrofuran-3-yl]amino]-2-oxo-ethyl]tert-butyl carbamate

[0226]

[0227] A mixture of (3S)-3-(benzylamino)tetrahydrofuran-2-one (86 g, 364.27 mmol, 81% by mass), boc-Gly-OH (97 g, 553.71 mmol), and TEA (103 mL, 739 mmol) in DCM (700 mL) was cooled to 5 °C. Propylphosphonic anhydride (50% by mass in EtOAc) (430 mL, 737 mmol) was added dropwise to the mixture over 1 hour while maintaining the internal temperature at approximately 10 °C. After the addition, the mixture was warmed to ambient temperature. After 7 hours, the mixture was cooled to 10 °C, and boc-Gly-OH (3.1 g, 18 mmol), TEA (5 mL, 35.9 mmol), and propylphosphonic anhydride (50% by mass in EtOAc) (22 mL, 37.7 mmol) were added. The mixture was warmed to ambient temperature and stirred overnight. After this time, the mixture was cooled to 8°C and boc-Gly-OH (9.6 g, 55 mmol), TEA (10 mL, 71.7 mmol), and propylphosphonic anhydride (50% by mass in EtOAc) (42 mL, 71.9 mmol) were added. The mixture was heated to ambient temperature and stirred for 7 hours. After this time, the mixture was poured onto ice and carefully quenched with a saturated aqueous solution of sodium bicarbonate (1 L). The pH was adjusted to 8 with solid sodium bicarbonate and the layers were separated. The aqueous layer was extracted twice more with DCM. The combined organic matter was washed with a saturated aqueous solution of sodium chloride (500 mL). The organic layer was filtered through a diatomaceous earth mat and sodium sulfate. The organic matter was concentrated to a volume of 1 L under vacuum and washed twice with a saturated aqueous solution of sodium chloride. The organic matter was dried over magnesium sulfate, filtered, and concentrated under vacuum to give the title compound (194 g, 53.5%, 35% by mass) for use without further purification. ES / MS m / z: 249.0 (Mt-Bu+H).

[0228] Preparation 22

[0229] (6S)-1-benzyl-6-(2-hydroxyethyl)piperazine-2,5-dione

[0230]

[0231] TFA (100 mL, 1310 mmol) was added to a mixture of N-[2-[benzyl-[(3S)-2-oxotetrahydrofuran-3-yl]amino]-2-oxo-ethyl]carbamate tert-butyl ester (194 g, 194.9 mmol, 35% by mass) in DCM (500 mL). The mixture was stirred overnight at ambient temperature. After this time, TFA (50 mL, 653 mmol) was added and the mixture was stirred at ambient temperature. After 2 hours, TFA (50 mL, 653 mmol) was added and the mixture was stirred overnight at ambient temperature. After this time, the mixture was concentrated under vacuum. The resulting residue was diluted with water (600 mL) and washed twice with diethyl ether. The pH was adjusted to 7 with 1 N NaOH and further adjusted to pH 12 with 5 N NaOH. MeOH (10 mL) was added and the mixture was stirred at ambient temperature. 5N NaOH was added to the mixture at 5-minute intervals to readjust the pH to 12. Overall, the mixture was stirred at pH 12 for 35 minutes. After this time, the pH was adjusted to 8 with a 10% aqueous HCl solution and extracted with DCM (5 × 1 L). The aqueous phase pH was then adjusted to 5 with a 10% aqueous HCl solution and extracted again with DCM (1 L). The combined organics were dried over sodium sulfate, filtered, and concentrated under vacuum to give the title compound (17 g, 33.4%) as a light brown solid. Further extraction of the aqueous phase was performed with alternating 25% IPA / chloroform and DCM until the title product was removed from the aqueous phase according to LC / MS. IPA (150 mL) was added to the combined organics. The organics were washed twice with a saturated aqueous sodium chloride solution, dried over sodium sulfate and magnesium sulfate, filtered, and concentrated under vacuum to give the remaining title compound (17.2 g, 35.5%), with a total yield of 34.2 g (68.9%). ES / MS m / z: 249.0 (M+H).

[0232] Preparation 23

[0233] 2-[(2S)-1-benzylpiperazine-2-yl]ethanol

[0234]

[0235] A solution of (6S)-1-benzyl-6-(2-hydroxyethyl)piperazine-2,5-dione (34 g, 131.5 mmol) in 200 mL of THF was added dropwise to a 45 °C solution of 2 M lithium aluminum hydride in THF (131 mL, 262 mmol). After the addition, the mixture was heated to 60 °C. After 3.5 hours, 33 mL of 2 M lithium aluminum hydride / THF (66 mmol) was added, and the mixture was stirred at 60 °C. After 1 hour, 131 mL of 2 M lithium aluminum hydride / THF (262 mmol) was added, and the mixture was stirred at 60 °C overnight. After this time, 6 mL of 2 M lithium aluminum hydride / THF (12 mmol) was added, and the mixture was stirred at 60 °C. After 4 hours, 6 mL of 2 M lithium aluminum hydride / THF (12 mmol) was added, and the mixture was stirred at 60 °C for 2 hours. The heat was removed, and the mixture was cooled to 10 °C. Water (16 mL) was added dropwise, followed by 3.75 M NaOH aqueous solution (16 mL), then THF (300 mL). Water (48 mL) was added, and the resulting mixture was stirred overnight at ambient temperature. After this time, the mixture was filtered through a diatomaceous earth mat and thoroughly washed with EtOAc. The filtrate was concentrated under vacuum to give the title product (28.8 g, 67.6%, 68% by mass). ES / MSm / z: 221.0 (M+H).

[0236] Preparation 24

[0237] (3S)-4-benzyl-3-(2-hydroxyethyl)piperazine-1-carboxylic acid tert-butyl ester

[0238]

[0239] Sodium bicarbonate (80 g, 952 mmol) in water (500 mL) was added to 2-[(2S)-1-benzylpiperazin-2-yl]ethanol (28 g, 86.43 mmol) in 1,4-dioxane (500 mL). Di-tert-butyl dicarbonate (26.6 g, 122 mmol) was added and the mixture was stirred at ambient temperature for 20 minutes. After this time, ice (400 mL), water (200 mL), and EtOAc (1 L) were added and the layers were separated. The aqueous layer was extracted once more with EtOAc (1 L). The combined organic matter was washed with water (250 mL) and a saturated aqueous sodium chloride solution (250 mL), dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting oil was purified by rapid silica gel chromatography eluting with 10–70% EtOAc / hexane to give the title compound (20.79 g, 74%). ES / MS m / z: 321.2 (M+H). This substance uses... IC, 4.6x150mm, 15% IPA (0.2% IPAm) / CO2, 5mL / min, 225nm analysis, showing 96% ee.

[0240] Preparation 25

[0241] (3S)-3-(2-hydroxyethyl)piperazine-1-carboxylic acid tert-butyl ester

[0242]

[0243] 20% Pd(OH)₂ / carbon (24.39 g, 176.4 mmol) was added to a container purged with nitrogen. EtOH (620 mL) was added to the container, followed by (3S)-4-benzyl-3-(2-hydroxyethyl)piperazine-1-carboxylic acid tert-butyl ester (61.93 g, 193.3 mmol) and EtOH (620 mL). The container was sealed, purged with nitrogen, then with hydrogen, and pressurized under hydrogen (60 psi). The container was placed on a Parr shaker at ambient temperature for 15 hours. After this time, the reaction mixture was filtered and concentrated under vacuum to give the title compound (42.74 g, 98%). ES / MS m / z 231.0:(M+H).

[0244] Preparation 26

[0245] (3S)-4-(3,5-dichloro-2-fluoro-4-iodobenzoyl)-3-(2-hydroxyethyl)piperazine-1-carboxylic acid tert-butyl ester

[0246]

[0247] 3,5-Dichloro-2-fluoro-4-iodobenzoic acid (0.80 g, 2.4 mmol) was added to DIEA (2 mL, 11.5 mmol) in THF (22 mL), followed by HATU (0.84 g, 2.2 mmol) and stirring for 1 hour. Then, tert-butyl (3S)-3-(2-hydroxyethyl)piperazine-1-carboxylate (0.50 g, 2.2 mmol) was added, and the reaction mixture was refluxed overnight. After this time, 5N NaOH was added, and the reaction mixture was stirred for 1 hour. EtOAc and water were added. The aqueous layer was extracted twice with EtOAc. The combined organic extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum. The crude mixture was purified by rapid silica gel chromatography eluting with EtOAc:hexane (30:70) to give the title compound (0.858 g, 72%). ES / MS m / z ( 35 Cl / 37 Cl)491 / 493[Mt-Bu+H] + .

[0248] Table 3: Compounds prepared in a manner substantially similar to that used in preparation 26

[0249]

[0250]

[0251] Preparation 29

[0252] (13aS)-8,10-dichloro-9-iodo-6-oxo-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzoxazine-2-octate tert-butyl ester

[0253]

[0254] (3S)-4-(3,5-dichloro-2-fluoro-4-iodobenzoyl)-3-(2-hydroxyethyl)piperazine-1-carboxylic acid tert-butyl ester (4.438 g, 8.110 mmol) in DMF (100 mL) was cooled to 0 °C, and then NaH (60% by mass in paraffin oil) (0.81 g, 20 mmol) was added to the solution. After 1 hour at 0 °C, the reaction mixture was quenched with a saturated aqueous sodium bicarbonate solution. Water and EtOAc were added. The aqueous layer was extracted twice with EtOAc. The combined organic extracts were washed twice with 0.2 M lithium chloride aqueous solution, dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum. The crude mixture was purified by rapid silica gel chromatography eluting with 30–50% EtOAc / hexane to give the title compound (3.394 g, 79%). ES / MS m / z ( 35 Cl / 37 Cl)471 / 473[Mt-Bu+H] + .

[0255] Table 4: Compounds prepared in a manner substantially similar to that used in preparation 29

[0256]

[0257]

[0258] Preparation of 32

[0259] (13aS)-8-chloro-6-oxo-9-(4,4,5,5-tetramethyl-1,3,2-dioxaboronecyclopentan-2-yl)-1,3,4,12,13,13a-hexahydropyrazine[2,1-d][1,5]benzoxazine-octatetraen-2-carboxylic acid tert-butyl ester

[0260]

[0261] The title compound was prepared from (13aS)-9-bromo-8-chloro-6-oxo-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzoxazine-octatetraen-2-carboxylic acid tert-butyl ester in a manner substantially similar to that used to prepare 13aS. ES / MSm / z( 35 Cl / 37 Cl)391 / 393[M-Boc+H] + .

[0262] Preparation of 33

[0263] (13aS)-9-(1,3-benzothiazo-4-yl)-8,10-dichloro-6-oxo-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzooxazine-2-carboxylic acid tert-butyl ester

[0264]

[0265] Add (13aS)-8,10-dichloro-9-iodo-6-oxo-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzoxazetene-2-carboxylic acid tert-butyl ester (110 mg, 0.21 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1,3-benzothiazole (124 mg, 0.31 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (14 mg, 0.02 mmol), and tripotassium phosphate (69 mg, 0.31 mmol) to a vial in that order. Add 1,4-dioxazetene (2 mL) and water (0.7 mL) under nitrogen atmosphere, and bubble the mixture under nitrogen atmosphere for 10 minutes. The vial was placed in a heating block preheated to 80°C, and the mixture was stirred for 40 minutes. The mixture was combined with a mixture from the same reaction carried out at a scale of 15 mg (0.03 mmol). The combined crude mixture was diluted with DCM and transferred to a separatory funnel. The organic layer was washed with water, and the aqueous layer was extracted again with DCM. The combined organics were dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluting with 0–100% EtOAc / hexane to give the title compound (157 mg, 65% purity, 91%). ES / MS m / z ( 35 Cl / 37 Cl)534.2 / 536.2[M+H] + .

[0266] Table 5: Compounds prepared in a manner substantially similar to that used in preparation 33

[0267]

[0268]

[0269]

[0270] Preparation of 38

[0271] (13aS)-9-(benzothiophene-4-yl)-8-chloro-6-oxo-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzooxazine-2-carboxylic acid tert-butyl ester

[0272]

[0273] (13aS)-8-chloro-6-oxo-9-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzoxazonite-2-carboxylic acid tert-butyl ester (200 mg, 0.340 mmol), 4-bromobenzothiophene (80 mg, 0.375 mmol), 1,1'-bis(di-tert-butylphosphino)ferrocene palladium dichloride (23 mg, 0.035 mmol), and potassium carbonate (141 mg, 1.020 mmol) were suspended in 1,4-dioxane (3.4 mL) and water (1.13 mL). The mixture was bubbled with nitrogen for 10 minutes and then stirred at 50°C for 1 hour. After this time, the mixture was cooled to ambient temperature and diluted with EtOAc (100 mL). The solution was washed with a saturated aqueous sodium chloride solution (100 mL), dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography with hexane followed by elution with 10–50% EtOAc / hexane to give the title compound (137 mg, 80.5%) as a white solid. ES / MS m / z ( 35 Cl / 37 Cl)499 / 501[M+H + .

[0274] Table 6: Compounds prepared in a manner substantially similar to that used to prepare 38

[0275]

[0276]

[0277]

[0278] Preparation of 45

[0279] (13aS)-9-(2-amino-3-cyano-benzofuran-4-yl)-8-chloro-6-oxo-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzooxazine-2-carboxylic acid tert-butyl ester

[0280]

[0281] 1N NaOH (0.5 mL, 0.5 mmol) was added to a mixture of (13aS)-8-chloro-9-[3-cyano-2-[(E)-dimethylaminomethyleneamino]benzofuran-4-yl]-6-oxo-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzooxazetene-2-carboxylic acid tert-butyl ester (65 mg, 0.112 mmol) in MeOH (5 mL) and heated to 60 °C for 3 hours. After this time, the reaction mixture was quenched with 5N HCl (0.1 mL) and then concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluting with 0–100% EtOAc / hexane to give the title compound (59 mg, 77%) as a white solid. ES / MS m / z ( 35 Cl / 37 Cl)522.6 / 524.6[M+H] + .

[0282] Preparation of 46

[0283] (13aS)-9-(7-amino-1-naphthyl)-8-chloro-6-oxo-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzoxazine-octatetraene-2-carboxylic acid tert-butyl ester

[0284]

[0285] Add 5% Pt / C(S) (0.013 g, 0.066 mmol) to a nitrogen-purged container. Add EtOAc (5 mL) to the container, followed by (13aS)-8-chloro-9-(7-nitro-1-naphthyl)-6-oxo-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzoxazonite-2-carboxylate tert-butyl ester (0.050 g, 0.093 mmol). Seal the container, purge with nitrogen, purge with hydrogen, and pressurize under hydrogen (60 psi). Place the container on a Parr shaker at ambient temperature for 1 hour. After this time, filter the reaction mixture and concentrate under vacuum. Reduce another batch of 100 mg of the nitro compound in a similar manner as described above, combine the two batches, and concentrate under vacuum to obtain the title compound, which is used as is for the next step (0.155 g, 99+%). ES / MS m / z 508.2 / 510.2[M+H] + .

[0286] Preparation 47

[0287] (13aS)-9-(1,3-benzothiazo-4-yl)-8,10-dichloro-2,3,4,12,13,13a-hexahydro-1H-pyrazino[2,1-d][1,5]benzooxazine-6-one

[0288]

[0289] A flask was loaded with (13aS)-9-(1,3-benzothiazo-4-yl)-8,10-dichloro-6-oxo-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzoxazonite-octate-2-tert-butyl ester (157 mg, 65% purity, 0.19 mmol) and DCM (2.5 mL) was added, followed by TFA (3 mL). The mixture was stirred at ambient temperature for 30 min, after which the solvent was removed under vacuum. The mixture was co-evaporated three times with DCM to remove excess TFA. The resulting residue was loaded onto an ion exchange column and washed with MeOH, 2N NH3 / MeOH, and again with MeOH. The basic fraction was concentrated under vacuum to give the title compound (86 mg, 99+%). ES / MS m / z ( 35 Cl / 37 Cl)434.2 / 436.2[M+H] + .

[0290] Table 7: Compounds prepared in a manner substantially similar to that used in preparation 47

[0291]

[0292]

[0293]

[0294]

[0295] Preparation 59

[0296] Kras probe

[0297] N-(2-{2-[2-({6-chloro-8-fluoro-7-(3-hydroxynaphthyl-1-yl)-4-[4-(prop-2-enoyl)piperazin-1-yl]quinazolin-2-yl)amino)ethoxy]ethoxy)ethyl)-5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]50enzoxaze-4-yl]pentanamide]

[0298]

[0299] Step A 4-(7-bromo-2,6-dichloro-8-fluoroquinazoline-4-yl)piperazine-1-carboxylic acid tert-butyl ester (0.51 g, 1.1 mmol) and IPA (5 mL) were combined in a microwave-safe container. DIPEA (0.55 mL, 3.3 mmol) and 5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]50enzoxaze-4-yl]-N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]pentanamide (0.48 g, 1.32 mmol) were added, and the mixture was heated to 120 °C in a microwave reactor for 6 hours. After this time, the mixture was diluted with a saturated aqueous ammonium chloride solution and 25% IPA / CHCl3, and the layers were separated. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by normal-phase chromatography using a 50-100% B / A gradient (A: hexane, B: 10% MeOH / DCM) to give tert-butyl 4-{7-bromo-6-chloro-8-fluoro-2-[(2-{2-[2-({5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]51enzoxaze-4-yl]valeryl}amino)ethoxy]ethoxy}ethyl)amino]quinazolin-4-yl}piperazine-1-carboxylic acid ester (0.68 g, 78%) as a yellow solid. ES / MS m / z: 819 (M+H).

[0300] Step BCombine 4-{7-bromo-6-chloro-8-fluoro-2-[(2-{2-[2-({5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]51enzoxaze-4-yl]valeryl}amino)ethoxy]ethoxy}ethyl)amino]quinazolin-4-yl}piperazine-1-carboxylic acid tert-butyl ester (0.30 g, 0.37 mmol), 1,4-dioxane (4 mL), and water (0.75 mL). Potassium carbonate (0.24 g, 1.11 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)naphthyl-2-ol (0.20 g, 0.74 mmol), and tetra(triphenylphosphine)palladium(0) (0.085 g, 0.074 mmol) were added, and the mixture was stirred at 85 °C under nitrogen for 12 hours. After this time, the mixture was cooled to ambient temperature and filtered to remove solids. The filtrate was diluted with a saturated aqueous solution of ammonium chloride and EtOAc, and the layers were separated. The organic layer was washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by normal-phase chromatography with elution using a 90-100% B / A gradient (A: hexane, B: 10% MeOH / DCM) to give tert-butyl 4-{6-chloro-8-fluoro-7-(3-hydroxynaphthyl-1-yl)-2-[(2-{2-[2-({5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]51enzoxaze-4-yl]valeryl}amino)ethoxy]ethoxy}ethyl)amino]quinazolin-4-yl}piperazine-1-carboxylic acid tert-butyl ester (0.31 g, 96%) as a yellow solid. ES / MS m / z: 881 (M+H).

[0301] Step CA solution of 4-{6-chloro-8-fluoro-7-(3-hydroxynaphthyl-1-yl)-2-[(2-{2-[2-({5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]51enzoxaze-4-yl]pentanoyl}amino)ethoxy]ethoxy}ethyl)amino]quinazolin-4-yl}piperazine-1-carboxylic acid tert-butyl ester (0.31 g, 0.35 mmol) in MeOH (4 mL) was cooled to 0 °C. HCl (3 M in MeOH, 6 mL, 17.5 mmol) was added and the mixture was stirred at 0 °C for 30 min, then allowed to warm to ambient temperature. After ~18 hours, the reaction mixture was concentrated under vacuum. The residue was diluted with DCM and concentrated again under vacuum. The resulting residue was diluted with hexane and stirred at ambient temperature for 2 h. The resulting solid was filtered and dried under vacuum to give N-{2-[2-(2-{[6-chloro-8-fluoro-7-(3-hydroxynaphthyl-1-yl)-4-(piperazin-1-yl)quinazolin-2-yl]amino}ethoxy)ethoxy]ethyl}-5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]52enzoxaze-4-yl]pentanamide hydrochloride. This hydrochloride (0.19 g, 0.23 mmol) was neutralized by combining it with DIEA (0.16 mL, 0.92 mmol) in DCM (2.5 mL). The mixture was cooled to -78 °C and acryloyl chloride (0.5 M in DCM, 0.4 mL, 0.21 mmol) was added. After 30 minutes, the mixture was heated to ambient temperature. After 1 hour, the mixture was diluted with MeOH (1 mL) and concentrated under vacuum. The resulting residue was purified by reversed-phase chromatography using a 35–60% B / A gradient (A: 10 mM NH4HCO3 aqueous solution containing 5% MeOH; B: ACN) to give the title compound (0.027 g, 14%) as a white solid. ES / MS m / z: 835 (M+H).

[0302] Example 1

[0303] (13aS)-9-(1,3-benzothiazo-4-yl)-8,10-dichloro-2-prop-2-enoyl-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzooxazine-octatetraen-6-one

[0304]

[0305] A vial was loaded with (13aS)-9-(1,3-benzothiazo-4-yl)-8,10-dichloro-2,3,4,12,13,13a-hexahydro-1H-pyrazino[2,1-d][1,5]benzoxazetene-6-one (86 mg, 0.20 mmol), DCM (3.6 mL), and DIEA (0.07, 0.40 mmol). The mixture was cooled to -78 °C, and acryloyl chloride solution (0.1 mL, 0.1 mmol, 1 M in DCM) was slowly added. After stirring for 10 min, MeOH (1.8 mL) was added, and the mixture was heated to ambient temperature. The mixture was diluted with DCM and washed with a saturated aqueous sodium chloride solution. The aqueous layer was extracted with EtOAc, the organic layers were combined, dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography elution with 50–100% EtOAc / hexane to give the title compound (62 mg, 64%) as a mixture of two rotational isomers. ES / MS m / z ( 35 Cl / 37 Cl)488.2 / 490.2[M+H] + The two isomers were separated using chiral HPLC (Chiralpak AD-H 30 x 250 mm column, 30 mL / min flow rate, 100% MeOH). The desired isomer was eluted at 5.6 min.

[0306] Table 8: Compounds prepared in a manner substantially similar to that of Example 1

[0307]

[0308]

[0309]

[0310] Example 10

[0311] (13aS)-9-(2-amino-1,3-benzoxazol-4-yl)-8-chloro-2-prop-2-enoyl-1,3,4,12,13,13a-hexahydropyrazino[2,1-d][1,5]benzoxazono-octatetraen-6-one

[0312]

[0313] Acryloyl chloride (14.7 μl, 0.175 mmol) was added at 0 °C to a two-phase mixture of (13as)-9-(2-amino-1,3-benzoxazol-4-yl)-8-chloro-2,3,4,12,13,13a-hexahydro-1h-pyrazino[2,1-d][1,5]benzoxazetene-6-one (70 mg, 0.176 mmol) and potassium carbonate (73 mg, 0.528 mmol) in EtOAc (2 mL) and water (2 mL). The resulting mixture was stirred at 0 °C for 15 min. After this time, water and DCM were added, and the layers were separated. The aqueous layer was extracted twice more with DCM. The organics were combined, passed through a hydrophobic glass buffer, and concentrated under vacuum. The residue was purified by reversed-phase chromatography to give the title compound (54 mg, 68%) as a white solid. ES / MS m / z ( 35 Cl / 37 Cl)453 / 455[M+H + .

[0314] Table 9: Compounds prepared in a manner substantially similar to that of Example 10

[0315]

[0316]

[0317] Bioassay

[0318] The following assays demonstrate that the compound in this study is an inhibitor of KRas G12C and inhibits the growth of certain tumors in vitro and / or in vivo.

[0319] KRas G12C probe occupancy TR-FRET measurement

[0320] The purpose of this assay was to measure the ability of the inhibitor to compete with the probe for binding to KRas G12C at codon 12 and to covalently modify KRas G12C. This was achieved by using an antibody bound to KRas G12C, specifically the europium-labeled antihistamine-tagged antibody LanthaScreen (Eu Anti-His antibody), and a fluorescent tracer 647 (Alexa Fluor) bound to KRas G12C via streptavidin and a biotinylated inhibitor (“KRas probe”, see Preparation 59). TM The time-resolved fluorescence transfer between the two points generates a signal.

[0321] Inhibitors were tested in dose-response form using a 10 mM stock solution in 100% DMSO. Labycyte 555 was used to dilute and transfer 100 nL / well of the 10-point 2.8-fold serial dilution to the assay plate. Two copies of the assay plate were prepared to measure the potency of the inhibitor after incubation with KRas G12C for 5 and 60 minutes. His-labeled KRas G12C (20 nM) was added to the plate in assay buffer (20 mM Tris-HCl, pH 7.5, 0.01% TX-100, and 1 mM DTT). After incubation for 5 or 60 minutes, 1 μM KRas probe was added, and it was covalently modified with free KRas G12C for 1 hour. This was diluted 4-fold in a buffer containing EuAnti-His antibody and Streptavidin-Coated Tracer 647 (both from Life Technologies) to achieve KRas G12C (5 nM), Anti-His antibody (2 nM), KRas probe (300 nM), and Streptavidin-Coated Tracer 647 (500 nM). After 30 minutes, the results were obtained from Envision. TM Fluorescence signals were read on a plate reader (excitation at 340 nM, tracer emission (em) at 665 nM, and antibody emission at 615 nM). The largest control well lacked inhibitor, while the smallest control well lacked both inhibitor and KRas G12C. The signal ratio (em at 665 nM / em at 615 nM) was converted to a percentage of inhibition using the following equation: %inhibition = 100 - [(test compound signal - median minimum signal) / (median maximum signal - median minimum signal) x 100]. This was achieved using Genedata. The IC50 is determined by fitting the inhibition percentage at each inhibitor concentration to a four-parameter nonlinear logic equation: y = (A + ((BA) / (1 + ((C / x)^D)))). 50 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 the slope of D = Hill.

[0322] In this assay, compounds within the Formula I range were evaluated essentially as described above, exhibiting KRas G12C inhibitory activity by competitively binding to KRas G12C at codon 12 and covalently modifying KRas G12C with the probe. The example compounds exhibited IC50 values ​​in the range of 20-425 nM. 50 The compound of Example 2 showed a relative IC50 of 34 nM in this assay. 50 .

[0323] H358 cell phosphorylation-ERK

[0324] The purpose of this assay was to measure the ability of the test compound to inhibit the phosphorylation of p-ERK1 / 2, a downstream effector of KRas in human lung cancer cells H358 (ATCC CRL-5807). In short, Ultra TM The p-ERK 1 / 2 (Thr202 / Tyr204) assay is a sandwich immunoassay used to quantitatively detect phosphorylated ERK 1 / 2 (phosphorylated on Thr202 / Tyr204 in ERK1 or Thr185 / Tyr187 in ERK2) in cell lysates using Alpha Technology (Perkin ElmerCat#ALSU-PERK-A50K).

[0325] H358 cells were seeded at 40 cells / well in 100 μL of medium containing 10% FBS (GIBCO Cat#: 10082-147) in a 96-well plate (Costar#3596) (RPMI 1640, GIBCO Cat#22400-071) and incubated overnight at 37°C in a humidified tray with 5% CO2. The following morning, 10 μL of serially diluted (3-fold) test compound (50 μM maximum concentration) and 10 μL of control (maximum signal well: 5% DMSO, minimum signal well: 2 μM N-(3-{3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-D]pyrimidin-1(2H)-yl}phenyl)acetamide (trametinib, as a positive control)) were added to the cell culture plates and incubated in a humidified tray at 37°C / 5% CO2 for 2 hours. A lysis buffer containing a mixture of protease and phosphatase inhibitors was prepared at ambient temperature. The culture medium was removed by inverting and shaking the cell culture plates in a water bath and then aspirating them onto paper towels. The lysis buffer was added to the cell culture plates (50 μL / well) and the plates were incubated on a shaker at ambient temperature for 10 minutes. For p-ERK assays, receptor beads were diluted with buffer to form a suspension mixture. Using a STARlet liquid processor, 5 μL of receptor beads and 2 μL of cell lysis buffer were transferred to a 384-well assay plate as a single-step in-tip dilution. The assay plate was sealed with foil and incubated at ambient temperature for 2 hours. Donor beads were diluted with buffer to form a suspension mixture. Using a STARlet, 5 μL of donor beads were added to the assay plate, which was then sealed and wrapped with foil. The plate was incubated at ambient temperature in the dark for 2 hours. Then, the assay was performed using EnVision. TMThe measurement plate is read using a light-emitting program on the Perkin Elmer plate reader.

[0326] Use the following equation to convert the signal to a suppression percentage:

[0327] %Inhibition = 100 - [(Test compound signal - Median minimum signal) / (Median maximum signal - Median minimum signal) x 100]. The maximum signal is the control well without inhibitor. The minimum signal is the control well containing a reference inhibitor sufficient to completely inhibit activity. (Using Genedata) The IC50 is determined by fitting the inhibition percentage at each inhibitor concentration to a four-parameter nonlinear logic equation: y = (A + ((BA) / (1 + ((C / x)^D)))). 50 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 the slope of D = Hill.

[0328] In this assay, compounds within the Formula I range were evaluated essentially as described above, exhibiting the ability to inhibit the phosphorylation of p-ERK1 / 2. The example compounds showed IC50 values ​​in the range of 89-1430 nM. 50 The compound of Example 2 showed a relative IC50 of 89 nM in this assay. 50 This data indicates that the compounds in this example exhibit KRas G12C inhibitory activity in this cell assay.

[0329] H358 cell viability RAS GTPase ELISA

[0330] The purpose of this assay is to measure the ability of the test compound to inhibit constitutive RAS GTPase activity in human lung cancer cells H358 (ATCC CRL-5807). The RAS GTPase ELISA kit (Active Motif Cat#52097) contains a 96-well plate pre-coated with glutathione to capture the GST-Raf-RBD protein supplied with the kit. Activated RAS (GTP-bound) in the cell extract specifically binds to Raf-RBD. The bound RAS is detected using a primary antibody that recognizes human KRas. A secondary antibody conjugated with HRP recognizes the primary antibody, and a developing solution provides a chemiluminescent reading.

[0331] H358 cells were seeded at 80,000 cells / well in 90 μL of serum-free medium (RPMI 1640, Gibco) and incubated overnight at 37°C / 5% CO2. The next morning, 10 μL of serially diluted (3-fold) test compound (500 μM maximum concentration) and 10 μL of control (maximum signal well: 5% DMSO, minimum signal well: 500 μM 1-[4-[6-chloro-8-fluoro-7-(3-hydroxy-1-naphthyl)quinazolin-4-yl]piperazin-1-yl]propyl-2-en-1-one, WO2015054572, as inhibitors) were added to the cell plate and incubated at 37°C / 5% CO2 for 2 hours. Complete lysis / binding buffer containing a protease inhibitor mixture and GST-Raf-RBD was prepared and stored on ice. One hour before the cell plate incubation was complete, 50 μL of GST-Raf-RBD was diluted in lysis / binding buffer, and the buffer was added to the ELISA plate. The plate was incubated at 4°C with gentle shaking for 1 hour. After 2 hours, the cells were washed with 100 μL of ice-cold PBS and lysed with 100 μL of lysis / binding buffer. The cell plate was then agitated at ambient temperature for 10 minutes. The plate was then centrifuged at 1500 rpm for 10 minutes at ambient temperature. During this time, 1X wash buffer was prepared at ambient temperature and used to wash (3 x 100 μL) of the GST-Raf-RBD-coated plate. After washing, 50 μL of cell lysis buffer was added to the GST-Raf-RBD-coated plate, and the plate was incubated at ambient temperature with gentle shaking for 1 hour. During this incubation, 1X antibody binding buffer was prepared and brought to ambient temperature. Wash the assay plate with 3 x 100 μL of 1X wash buffer, then add 50 μL of primary antibody (1:500 diluted in 1X antibody binding buffer). Incubate the plate at room temperature for 1 hour. Wash the assay plate with 3 x 100 μL of 1X wash buffer, then add 50 μL of secondary antibody (1:5000 diluted in 1X antibody binding buffer) and incubate at room temperature for 1 hour. Wash the assay plate with 4 x 100 μL of 1X wash buffer, then add 50 μL of chemiluminescent working solution at room temperature. Then, in EnVision... TM The measurement plate is read using a light-emitting program on the Perkin Elmer plate reader.

[0332] Use the following equation to convert the signal to a suppression percentage:

[0333] %Inhibition = 100 - [(Test compound signal - Median minimum signal) / (Median maximum signal - Median minimum signal) x 100]. The maximum signal is the control well without inhibitor. The minimum signal is the control well containing a reference inhibitor sufficient to completely inhibit activity. (Using Genedata) The IC50 is determined by fitting the inhibition percentage at each inhibitor concentration to a four-parameter nonlinear logic equation: y = (A + ((BA) / (1 + ((C / x)^D)))). 50 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 the slope of D = Hill.

[0334] In this assay, compounds within the Formula I range were evaluated essentially as described above, exhibiting the ability to inhibit constitutive RASGTPase activity. The example compounds showed IC50 values ​​in the range of 326-4570 nM. 50 The compound of Example 2 showed a relative IC50 of 326 nM in this assay. 50 This data indicates that the compounds in this example exhibit KRas-GTP inhibitory activity in this human lung cancer cell culture.

Claims

1. A compound of the following formula or a pharmaceutically acceptable salt thereof: in: A is -OCH2CH2-; B is -C(O)-; R1 is a group of -C(O)C≡CR8 or the following formula. R2 is H; R3 and R5 are each independently H or halogen; R4 is a group in the following formula R is H or halogen; R' is H or -C 1-6 alkyl; R7 is H; R8 is H; and R9 is H.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is a group of the following formula:

3. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein R3 is H, F or Cl.

4. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein R is H or F.

5. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein R4 is selected from...

6. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein R5 is H or Cl.

7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

8. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, for use in treatment.

9. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, for the treatment of cancer.

10. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein the cancer is selected from lung cancer, pancreatic cancer, esophageal cancer, and colorectal cancer.

11. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, used simultaneously, separately or sequentially in combination with one or more of the following for the treatment of cancer: a PD-1 or PD-L1 inhibitor; a CDK4 inhibitor or a pharmaceutically acceptable salt thereof; a CDK6 inhibitor or a pharmaceutically acceptable salt thereof; an EGFR inhibitor or a pharmaceutically acceptable salt thereof; an ERK inhibitor or a pharmaceutically acceptable salt thereof; platinum; and pemetrexed or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition comprising a compound according to any one of claims 1-7 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

13. Use of the pharmaceutical composition of claim 12 in the preparation of a medicament for treating a patient’s cancer, wherein the cancer is selected from lung cancer, pancreatic cancer, esophageal cancer, and colorectal cancer.

14. Use of a compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a patient’s cancer, wherein the cancer is selected from lung cancer, pancreatic cancer, esophageal cancer, and colorectal cancer.

15. The use according to claim 14, wherein the cancer is non-small cell lung cancer, and wherein one or more cells express the KRas G12C mutant protein.

16. The use according to claim 14, wherein the cancer is colorectal cancer, and wherein one or more cells express the KRas G12C mutant protein.

17. The use according to claim 14, wherein the cancer is pancreatic cancer, and wherein one or more cells express the KRas G12C mutant protein.

18. The use according to claim 14, wherein the patient has cancer identified as having one or more cells expressing the KRas G12C mutant protein prior to administration of the compound or a pharmaceutically acceptable salt thereof.

19. The use according to any one of claims 13 to 18, wherein the drug is administered in combination with one or more of the following: a PD-1 inhibitor, a PD-L1 inhibitor, a CDK4 inhibitor or a pharmaceutically acceptable salt thereof, a CDK6 inhibitor or a pharmaceutically acceptable salt thereof, an EGFR inhibitor or a pharmaceutically acceptable salt thereof, an ERK inhibitor or a pharmaceutically acceptable salt thereof, a platinum preparation, and pemetrexed or a pharmaceutically acceptable salt thereof.

Images