Dual ATM and DNA-PK inhibitors for Anti-tumor therapy

Abstract

The present invention relates to dual ATM and DNA-PK inhibitors for use in anti-tumor therapy. Provided herein are compounds of Formula (I): # imgabs0 # and pharmaceutically acceptable salts thereof, wherein the substituents are as described herein. These compounds and their pharmaceutical compositions are useful for the treatment of neoplastic diseases.

Description

[0001] This application is a divisional application of the invention patent application with application number 202080055262.1, application date July 30, 2020, and invention name “Dual ATM and DNA-PK inhibitors for anti-tumor therapy”. Technical Field

[0002] The present invention relates to compounds and pharmaceutically acceptable salts thereof for use in treating cancer as monotherapy or in combination with radiation therapy, chemotherapy and / or immunotherapy, and methods of their use. Background of the Invention

[0004] Several members of the PIKK (PI-3K-like kinase) family of serine-threonine kinases are known mediators of DNA damage signaling.

[0005] Radiation therapy (RT) is used to treat >50% of all cancer patients at some point during their disease course. Despite extensive efforts, previous approaches to developing clinical radiosensitizers have not been very effective, primarily as a result of targeting nonspecific pathways that are not direct regulators of the cellular response to radiation.

[0006] New treatments for neoplastic diseases are needed. Summary of the invention

[0007] Generally speaking, the present invention provides a compound of formula (I):

[0008]

[0009] or a pharmaceutically acceptable salt thereof,

[0010] in

[0011] Z is CH, CR 3 or N;

[0012] Y is CHR 5 or NR 6 ;

[0013] n is 0, 1, 2, or 3;

[0014] R 1 Yes-OLN(R 7 ) 2 or an optionally substituted four-membered saturated N-heterocyclic group;

[0015] R 2 It is C 1-3 alkyl;

[0016] Each R 3 are independently halogen;

[0017] R 4 is optionally substituted alkyl;

[0018] R 5 is hydrogen, optionally substituted C 1-3 Alkyl or benzyloxy;

[0019] R 6 is an optionally substituted C 1-3 alkyl;

[0020] Each R 7 is independently H or optionally substituted C 1-3 alkyl; and

[0021] L is optionally substituted ethylene.

[0022] In some embodiments, n is 1. In certain embodiments, the compound is of formula (IA):

[0023]

[0024] or a pharmaceutically acceptable salt thereof.

[0025] In a specific embodiment, R 3 is halogen (eg, fluorine). In other embodiments, n is 0.

[0026] In yet other embodiments, the compound is of Formula (IB):

[0027]

[0028] or a pharmaceutically acceptable salt thereof.

[0029] In still other embodiments, R 1 Yes-O_L-N(R 7 ) 2 In some embodiments, one R 7 is H, and the rest are R 7 Optionally C 1-3 In certain embodiments, at least one R 7 is isopropyl. In a specific embodiment, R 2 is methyl, ethyl or isopropyl. In other embodiments, R 2 In yet other embodiments, R 4 In yet other embodiments, Y is CHR 5 In still other embodiments, R 5 is hydrogen. In other embodiments, R 5 is an optionally substituted C 1-3 In yet other embodiments, R5 In still other embodiments, Y is NR 6 In some embodiments, R 6 is an optionally substituted C 3 In certain embodiments, R 6 It is isopropyl.

[0030] In specific embodiments, the compound is selected from the group consisting of:

[0031]

[0032]

[0033] and pharmaceutically acceptable salts thereof.

[0034] In other embodiments, the compound has the structure:

[0035]

[0036] or a pharmaceutically acceptable salt thereof.

[0037] In yet other embodiments, the compound has the structure:

[0038]

[0039] or a pharmaceutically acceptable salt thereof.

[0040] In still other embodiments, the compound has the structure:

[0041]

[0042] or a pharmaceutically acceptable salt thereof.

[0043] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0044] In another aspect, the present invention provides a method for treating a tumor disease (e.g., cancer, such as those described herein), which is performed by administering a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present invention to a patient in need thereof. In still another aspect, the present invention provides a pharmaceutical composition, which is used to treat a tumor disease (e.g., cancer, such as those described herein). The pharmaceutical composition comprises a compound of the present invention. In another aspect, the present invention provides the use of a compound of the present invention in the manufacture of a medicament for treating a tumor disease (e.g., cancer, such as those described herein). In some embodiments, the tumor disease is brain cancer, bladder cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gastrointestinal stromal tumor, gastric cancer, head and neck cancer, cheek cancer, oral cancer, hepatocellular carcinoma, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, nasopharyngeal carcinoma, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, salivary gland cancer, sarcoma, testicular cancer, urothelial carcinoma, vulvar cancer or Wilm's tumor. In other embodiments, the tumor disease is breast cancer, lung cancer, head and neck cancer, pancreatic cancer, rectal cancer, glioblastoma, hepatocellular carcinoma, bile duct cancer, metastatic liver lesions, melanoma, osteosarcoma, soft tissue sarcoma, endometrial cancer, cervical cancer, prostate cancer or Merkel cell carcinoma.

[0045] In some embodiments, the patient is receiving radiation therapy. In certain embodiments, the compound or the pharmaceutical composition is administered to the patient simultaneously with the radiation therapy. In specific embodiments, the compound or the pharmaceutical composition is administered to the patient before the radiation therapy. In other embodiments, the compound or the pharmaceutical composition is administered to the patient after the radiation therapy. In yet other embodiments, the radiation therapy comprises external, internal, brachytherapy, or systemic exposure, such as with a radionuclide (e.g., a β-emitting radionuclide (e.g., 32 phosphorus, 67 copper, 77 bromine, 89 strontium, 90 yttrium, 105 rhodium, 131 iodine, 137 cesium, 149 Promethium, 153 samarium, 166 holmium, 177 Lutetium 186 rhenium, 188 Rhenium or 199 gold), alpha-emitting radionuclides (e.g. 211 astatine, 213 bismuth, 223 radium,225 Actinium or 227 Thorium), gamma-ray emitting radionuclides (e.g. 192 Iridium) or electron capture radionuclides (e.g. 67 gallium, 103 Palladium or 125 iodine)), antibody radionuclide conjugates (e.g., 90 Y-ibritumomab tiuxetane, 131 I-Tositumomab, 225 Ac-lintuzumab satetraxetan, 227 Th-anetumab corixetan, 90 Y-epitumomab cituxeta n, 90 Y-clivatuzumab tetraxetan, 177 Lu-Sartattan-Lilotomabsatetraxetan, 90 Y-rosopata mab tetraxetan, 90 Y-tabituximab barzuxetan or 90 y-tetan-tacatuzumab tetraxetan) or another targeted radionuclide conjugate (e.g., 13 1I-PSMA, 90 Y-PSMA, 177 Lu-PSMA or 177 Lu-tetan-satoreotide tetraxetan). Preferably, the radiotherapy comprises administering an antibody radionuclide conjugate. In still other embodiments, the patient is receiving an anti-tumor agent. In other embodiments, the anti-tumor agent is one or more of cisplatin, oxaliplatin, carboplatin, valrubicin, idarubicin, calicheamicin, or a PARP inhibitor. In yet other embodiments, the anti-tumor agent is an anti-tumor biological agent and / or an anti-tumor immunotherapeutic agent. In still other embodiments, the compound or the pharmaceutical composition is administered to the patient simultaneously with the anti-tumor agent. In some embodiments, the compound or the pharmaceutical composition is administered to the patient before the anti-tumor agent. In certain embodiments, the compound or the pharmaceutical composition is administered to the patient after the anti-tumor agent. DETAILED DESCRIPTION

[0046] definition

[0047] It should be understood that the terminology employed herein is for the purpose of describing specific embodiments and is not intended to be limiting. Further, although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods, devices, and materials are now described. In addition to the foregoing, as used in the specification and the appended claims, unless otherwise indicated, the following terms have the meanings indicated:

[0048] "Amino" refers to -NH 2 Group.

[0049] "Cyano" refers to a -CN group.

[0050] "Hydroxy" refers to an -OH group.

[0051] "Imino" refers to a =NH substituent.

[0052] "Nitro" refers to -NO 2 Group.

[0053] "Oxo" refers to a =0 substituent.

[0054] "Thio" refers to a =S substituent.

[0055] "Trifluoromethyl" refers to -CF 3 Group.

[0056] "Alkyl" refers to a straight chain, saturated, acyclic, monovalent hydrocarbon radical or a branched chain, saturated, acyclic, monovalent hydrocarbon radical having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms or 1 to 6 carbon atoms, and attached to the remainder of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Optionally substituted alkyl is an alkyl group optionally substituted with 1, 2, 3, 4 or 5 substituents (valency permitting) independently selected from the group consisting of halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilyl, -OR 14 、-OC(O)-R 14 、-N(R 14 ) 2 、-C(O)R 15 、-C(O)OR 14 、-C(O)N(R 14 ) 2 、-N(R 14 )C(O)OR 16 、-N(R 14 )C(O)R 16 、-N(R14 )S(O) t R 16 (where t is 1 or 2), -S(O) t OR 16 (where t is 1 or 2), -S(O) p R 16 (where p is 0, 1, or 2) and -S(O) t N(R 14 ) 2 (where t is 1 or 2), where each R 14 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl or heteroaryl; each R 15 are independently hydrogen, cycloalkyl, aryl, heterocyclyl or heteroaryl; and each R 16 is independently alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl.

[0057] "Alkenyl" refers to a straight chain, acyclic, monovalent hydrocarbon radical or a branched, acyclic, monovalent hydrocarbon radical containing 1, 2 or 3 carbon-carbon double bonds, having 2 to 12 carbon atoms, preferably 2 to 8 carbon atoms, and connected to the remainder of the molecule by a single bond, such as vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, etc. Optionally substituted alkenyl is alkenyl optionally substituted (valence permitting) with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilyl, -OR 14 、-OC(O)-R 14 、-N(R 14 ) 2 、-C(O)R 15 、-C(O)OR 14 、-C(O)N(R 14 ) 2 、-N(R 14 )C(O)OR 16 、-N(R 14 )C(O)R 16 、-N(R 14 )S(O) t R 16 (where t is 1 or 2), -S(O) t OR 16 (where t is 1 or 2), -S(O) p R 16 (where p is 0, 1, or 2) and -S(O) t N(R 14 ) 2 (where t is 1 or 2), where each R14 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl; each R 15 are independently hydrogen, cycloalkyl, aryl, heterocyclyl or heteroaryl; and each R 16 and R is independently alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, heterocyclyl or heteroaryl.

[0058] "Alkynyl" refers to a straight chain, acyclic, monovalent hydrocarbon radical or a branched chain, acyclic, monovalent hydrocarbon radical containing 1 or 2 carbon-carbon triple bonds and optionally 1, 2 or 3 carbon-carbon double bonds and having 2 to 12 carbon atoms, preferably 2 to 8 carbon atoms, and connected to the rest of the molecule by a single bond, such as ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-1-en-4-ynyl, etc. Optionally substituted alkynyl is alkynyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilyl, -OR 14 、-OC(O)-R 14 、-N(R 14 )2. -C(O)R 15 、-C(O)OR 14 、-C(O)N(R 14 ) 2 、-N(R 14 )C(O)OR 16 、-N(R 14 )C(O)R 16 、-N(R 14 )S(O) t R 16 (where t is 1 or 2), -S(O) t OR 16 (where t is 1 or 2), -S(O) p R 16 (where p is 0, 1, or 2) and -S(O) t N(R 14 ) 2 (where t is 1 or 2), where each R 14 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl; each R 15 are independently hydrogen, cycloalkyl, aryl, heterocyclyl or heteroaryl; and each R 16 is independently alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl.

[0059] "Alkylene" or "alkylene chain" refers to a straight, acyclic, saturated, divalent hydrocarbon chain or a branched, acyclic, saturated, divalent hydrocarbon chain having 1 to 12 carbon atoms, such as methylene, ethylene, propylene, n-butylene, etc. The alkylene chains are connected by single bonds. The connection points of the alkylene chains can be on the same carbon atom or on different carbon atoms within the alkylene chain. An optionally substituted alkylene chain is an alkylene chain optionally substituted with 1, 2, 3, 4 or 5 substituents (valence permitting), the substituents being independently selected from the group consisting of: halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilyl, -OR 14 、-OC(O)-R 14 、-N(R 14 ) 2 、-C(O)R 15 、-C(O)OR 14 、-C(O)N(R 14 ) 2 、-N(R 14 )C(O)OR 16 、-N(R 14 )C(O)R 16 、-N(R 14 )S(O) t R 16 (where t is 1 or 2), -S(O) t OR 16 (where t is 1 or 2), -S(O) p R 16 (where p is 0, 1, or 2) and -S(O) t N(R 14 ) 2 (where t is 1 or 2), where each R 14 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl; each R 15 are independently hydrogen, cycloalkyl, aryl, heterocyclyl or heteroaryl; and each R 16 is independently alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl. In some embodiments, the alkylene group is ethylene.

[0060] "Alkenylene" or "alkenylene chain" refers to a straight chain, acyclic, divalent hydrocarbon chain or a branched, acyclic, divalent hydrocarbon chain containing 1, 2 or 3 carbon-carbon double bonds and having 2 to 12 carbon atoms, for example, vinylene, propenylene, n-butenylene, etc. The alkenylene chain is connected by a single bond. The connection point of the alkenylene chain can be on the same carbon atom or on different carbon atoms within the alkenylene chain. Optionally substituted alkenylene chains are alkenylene chains that are optionally substituted (valence state permitting) with 1, 2, 3, 4 or 5 substituents, the substituents being independently selected from the group consisting of: halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilyl, -OR 14 、-OC(O)-R 14 、-N(R 14 ) 2 、-C(O)R 15 、-C(O)OR 14 、-C(O)N(R 14 ) 2 、-N(R 14 )C(O)OR 16 、-N(R 14 )C(O)R 16 、-N(R 14 )S(O) t R 16 (where t is 1 or 2), -S(O) t OR 16 (where t is 1 or 2), -S(O) p R 16 (where p is 0, 1, or 2) and -S(O) t N(R 14 ) 2 (where t is 1 or 2), where each R 14 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl; each R 15 are independently hydrogen, cycloalkyl, aryl, heterocyclyl or heteroaryl; and each R 16 is independently alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl.

[0061] "Alkynylidene" or "alkynylene chain" refers to a straight chain, acyclic, divalent hydrocarbon chain or a branched, acyclic, divalent hydrocarbon chain containing 1 or 2 carbon-carbon triple bonds and optionally 1, 2 or 3 carbon-carbon double bonds and having 2 to 12 carbon atoms, such as propynylene, n-butynylene, etc. The alkynylene chain is connected by a single bond. The point of attachment of the alkynylene can be on the same carbon atom or on different carbon atoms within the alkynylene chain. An optionally substituted alkynylene chain is an alkynylene chain optionally substituted with 1, 2, 3, 4 or 5 substituents, the substituents being independently selected from the group consisting of halo, cyano, nitro, aryl, cycloalkyl, heterocyclyl, heteroaryl, oxo, trimethylsilyl, -OR 14 、-OC(O)-R 14 、-N(R 14 ) 2 、-C(O)R 15 、-C(O)OR 14 、-C(O)N(R 14 ) 2 、-N(R 14 )C(O)OR 16 、-N(R 14 )C(O)R 16 、-N(R 14 )S(O) t R 16 (where t is 1 or 2), -S(O) t OR 16 (where t is 1 or 2), -S(O) p R 16 (where p is 0, 1, or 2) and -S(O) t N(R 14 ) 2 (where t is 1 to 2), where each R 14 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl; each R 15 are independently hydrogen, cycloalkyl, aryl, heterocyclyl or heteroaryl; and each R 16 is independently alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl.

[0062] "Alkoxy" refers to a group of the formula -OR a A group in which R a is an alkyl group as defined above containing 1 to 12 carbon atoms. The alkyl part of the optionally substituted alkoxy group is optionally substituted as defined above for an alkyl group.

[0063] "Alkoxyalkyl" refers to a group of the formula -R a -OR b A group in which R a is an alkylene group and R bis an alkyl group as defined above. The alkyl and alkylene parts of the optionally substituted alkoxyalkyl group are optionally substituted as defined above for an alkyl and alkylene chain, respectively.

[0064] "Arylalkyl" refers to a group of the formula -R a -R b A group in which R a is an alkylene group and R b and is an aryl group as described herein. The alkylene and aryl portions of the optionally substituted aralkyl are optionally substituted as described herein for alkylene and aryl groups, respectively.

[0065] "Aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon ring system radical containing 6 to 18 carbon atoms, wherein the polycyclic aromatic ring system is a bicyclic, tricyclic or tetracyclic ring system. Aryl includes, but is not limited to, groups such as fluorenyl, phenyl and naphthyl. Optionally substituted aryl is an aryl group optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, nitro, aryl, heteroaryl, heteroarylalkyl, -R 15 -OR 14 , -R 15 -OC(O)-R 14 , -R 15 -N(R 14 ) 2 , -R 15 -C(O)R 14 , -R 15 -C(O)OR 14 , -R 15 -C(O)N(R 14 ) 2 , -R 15 -N(R 14 )C(O)OR 16 , -R 15 -N(R 14 )C(O)R 16 , -R 15 -N(R 14 )S(O) t R 16 (where t is 1 or 2), -R 15 -S(O) t OR 16 (where t is 1 or 2), -R 15 -S(O) p R 16 (where p is 0, 1, or 2) and -R 15 -S(O) t N(R 14 ) 2(where t is 1 or 2), where each R 14 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl; each R 15 is independently a direct bond or a straight or branched alkylene or alkenylene chain; and each R 16 and R is independently alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, heterocyclyl or heteroaryl.

[0066] "Arylalkoxy" refers to a radical of the formula -OR, where R is aralkyl. Optionally substituted arylalkoxy is an optionally substituted arylalkoxy radical as described herein for aralkyl. In some embodiments, arylalkoxy is benzyloxy.

[0067] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, and being saturated or unsaturated and connected to the remainder of the molecule by a single bond. Polycyclic hydrocarbon radicals are bicyclic, tricyclic or tetracyclic ring systems. Unsaturated cycloalkyls contain 1, 2 or 3 carbon-carbon double bonds and / or 1 carbon-carbon triple bond. Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Polycyclic cycloalkyls include, for example, adamantyl, norbornyl, decahydronaphthyl, etc. Optionally substituted cycloalkyls are cycloalkyls optionally substituted by 1, 2, 3, 4 or 5 substituents, the substituents being independently selected from the group consisting of alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, -R 15 -OR 14 , -R 15 -OC(O)-R 14 , -R 15 -N(R 14 ) 2 , -R 15 -C(O)R 14 , -R 15 -C(O)OR 14 , -R 15 -C(O)N(R 14 ) 2 , -R 15 -N(R 14 )C(O)OR 16 , -R 15 -N(R 14 )C(O)R 16 , -R 15 -N(R 14 )S(O) t R 16 (where t is 1 or 2), -R 15 -S(O) tOR 16 (where t is 1 or 2), -R 15 -S(O) p R 16 (where p is 0, 1, or 2) and -R 15 -S(O) t N(R 14 ) 2 (where t is 1 or 2), where each R 14 are independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl; each R 15 is independently a direct bond or a straight or branched alkylene or alkenylene chain; and each R 16 and R is independently alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl or heteroaryl.

[0068] "Fused" refers to any ring system described herein that is fused to an existing ring structure in the compounds of the invention. When the fused ring system is a heterocyclyl or heteroaryl, any carbon atom on the existing ring structure that becomes part of the fused ring system may be replaced by a nitrogen atom.

[0069] "Halo" refers to the halogen substituents: bromo, chloro, fluoro and iodo.

[0070] "Haloalkyl" refers to alkyl as defined above, which is further replaced by one or more halogen substituents. The number of the halogen substituents included in the haloalkyl is 1 to the total number (such as perfluoroalkyl) of the hydrogen atoms that can be replaced by the halogen substituents. The limiting examples of haloalkyl include trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, 1-bromomethyl-2-bromoethyl etc. For the haloalkyl optionally substituted, the hydrogen atom bonded to the carbon atom of the alkyl part of the haloalkyl can optionally be replaced with the substituent defined as above for the alkyl optionally substituted.

[0071] " haloalkenyl " refers to alkenyl as defined above, which is further replaced by one or more halo substituents. The number of the halo substituents included in the haloalkenyl is 1 to the total number (such as perfluoroalkenyl) of the hydrogen atoms that can be replaced by the halo substituents. The limiting examples of haloalkenyl include 2,2-difluorovinyl, 3-chloroprop-1-alkenyl etc. For the haloalkenyl optionally substituted, the hydrogen atom bonded to the carbon atom of the alkenyl part of the haloalkenyl can optionally be replaced with the substituent defined as above for the optionally substituted alkenyl.

[0072] "Haloalkynyl" refers to an alkynyl as defined above, which is further substituted with one or more halo substituents. The number of halo substituents included in the haloalkynyl is 1 to the total number of hydrogen atoms (e.g., perfluoroalkynyl) that can be replaced with halo substituents. Non-limiting examples of haloalkynyl include 3-chloroprop-1-ynyl, etc. The alkynyl moiety of haloalkynyl can additionally be optionally substituted as defined above for alkynyl.

[0073] "Heteroarylalkyl" refers to a group of the formula -R a -R b A group in which R a is an alkylene group and R b and is a heteroaryl group as described herein. The alkylene and heteroaryl portions of the optionally substituted heteroarylalkyl are optionally substituted as described herein for alkylene and heteroaryl, respectively.

[0074] "Heterocyclyl" refers to a stable 3 to 18 membered non-aromatic ring system radical having a carbon number of 2 to 12 and containing a total of 1 to 6 heteroatoms independently selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur. Heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system. Bicyclic, tricyclic or tetracyclic heterocyclyl is a fused ring, spirocyclic and / or bridged ring system. Heterocyclyl can be saturated or unsaturated. Unsaturated heterocyclyl contains 1, 2 or 3 carbon-carbon double bonds and / or 1 carbon-carbon triple bond. Optionally substituted heterocyclyl is a heterocyclyl optionally substituted by 1, 2, 3, 4 or 5 substituents, the substituents being independently selected from the group consisting of alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, oxo, thio, nitro, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, -R 15 -OR 14 , -R 15 -OC(0)-R 14 , -R 15 -N(R 14 ) 2 , -R 15 -C(O)R 14 , -R 15 -C(O)OR 14 , -R 15 -C(O)N(R 14 ) 2 , -R 15 -N(R 14 )C(O)OR 16 , -R 15 -N(R 14 )C(O)R 16 , -R 15 -N(R 14 )S(O) t R 16 (where t is 1 or 2), -R15 -S(O) t OR 16 (where t is 1 or 2), -R 15 -S(O) p R 16 (where p is 0, 1, or 2) and -R 15 -S(O) t N(R 14 ) 2 (where t is 1 or 2), where each R 14 are independently hydrogen, alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl; each R 15 is independently a direct bond or a straight or branched alkylene or alkenylene chain; and each R 16 are independently alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl. The nitrogen, carbon or sulfur atom in the heterocyclyl may be optionally oxidized (when the substituent is oxo and is present on a heteroatom); the nitrogen atom may be optionally quaternized (when the substituent is alkyl, alkenyl, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, -R 15 -OR 14 , -R 15 -OC(O)-R 14 , -R 15 -N(R 14 ) 2 , -R 15 -C(O)R 14 , -R 15 -C(O)OR 14 , -R 15 -C(O)N(R 14 ) 2 , -R 15 -N(R 14 )C(O)OR 16 , -R 15 -N(R 14 )C(O)R 16 , -R 15 -N(R 14 )S(O) t R 16 (where t is 1 or 2), -R 15 -S(O) t OR 16 (where t is 1 or 2), -R 15 -S(O) p R 16 (where p is 0, 1, or 2) and -R 15 -S(O) t N(R 14 ) 2(where t is 1 or 2), where R 15 is a straight or branched alkylene or alkenylene chain, and R 14 and R 16 as defined above). Examples of optionally substituted heterocyclyl include, but are not limited to, azetidinyl, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.

[0075] "Heterocyclylene" refers to a heterocyclyl group in which one hydrogen atom is replaced by a valency. An optionally substituted heterocyclylene group is optionally substituted as described herein for a heterocyclyl group.

[0076] "Heteroaryl" refers to a 5 to 18 membered ring system radical containing at least one aromatic ring, having a carbon number of 1 to 17 carbon atoms, and containing a total of 1 to 10 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Heteroaryl is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system. A bicyclic, tricyclic, or tetracyclic heteroaryl is a fused and / or bridged ring system. An optionally substituted heteroaryl is a heteroaryl optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkoxy, halo, haloalkyl, haloalkenyl, cyano, oxo, thio, nitro, oxo, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, or heteroarylalkyl, -R 15 -OR 14 , -R 15 -OC(O)-R 14 , -R 15 -N(R 14 ) 2 , -R 15 -C(O)R 14 , -R 15 -C(O)OR 14 , -R 15 -C(O)N(R 14 ) 2 , -R 15 -N(R 14 )C(O)OR 16 , -R 15 -N(R 14 )C(O)R 16 , -R 15 -N(R 14 )S(O)t R 16 (where t is 1 or 2), -R 15 -S(O) t OR 16 (where t is 1 or 2), -R 15 -S(O) t R 16 (where p is 0, 1, or 2) and -R 15 -S(O) t N(R 14 ) 2 (where t is 1 or 2), where each R 14 are independently hydrogen, alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl; each R 15 is independently a direct bond or a straight or branched alkylene or alkenylene chain; and each R 16 is alkyl, alkenyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl. The nitrogen, carbon or sulfur atom in the heterocyclyl may be optionally oxidized (when the substituent is oxo and is present on a heteroatom), provided that at least one ring in the heteroaryl remains aromatic; the nitrogen atom may be optionally quaternized (when the substituent is alkyl, alkenyl, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, -R 15 -OR 14 , -R 15 -OC(O)-R 14 , -R 15 -N(R 14 ) 2 , -R 15 -C(O)R 14 , -R 15 -C(O)OR 14 , -R 15 -C(O)N(R 14 ) 2 , -R 15 -N(R 14 )C(O)OR 16 , -R 15 -N(R 14 )C(O)R 16 , -R 15 -N(R 14 )S(O) t R 16 (where t is 1 or 2), -R 15 -S(O) t OR 16 (where t is 1 or 2), -R 15 -S(O) p R 16 (where p is 0, 1, or 2) and -R15 -S(O) t N(R 14 ) 2 (where t is 1 or 2), where R 15 is a straight or branched alkylene or alkenylene chain, and R 14 and R 16 As defined above), provided that at least one ring in the heteroaryl group remains aromatic. Examples of optionally substituted heteroaryl groups include, but are not limited to, aza yl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxolyl 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophenyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolyl, isoindolyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyl, naphthyridinyl, oxadiazolyl, 2-oxoazepine phenyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl).

[0077] The invention disclosed herein is also intended to cover all pharmaceutically acceptable compounds of formula (I) which are isotopically labeled by replacing one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S.18 F. 36 Cl, 123 I and 125 I. These radiolabeled compounds can be used to help determine or measure the effectiveness of compounds by characterizing, for example, the site or mode of action on ATM and DNA-PK enzymes, or the binding affinity to pharmacologically important sites of action on ATM and DNA-PK enzymes. Certain isotopically labeled compounds of formula (I), for example, those incorporating radioisotopes, are useful in drug and / or substrate tissue distribution studies. In view of their ease of incorporation and ease of detection, the radioisotope tritium (i.e., 3 H) and carbon 14 (i.e., 14 C) is particularly suitable for this purpose.

[0078] With heavier isotopes (such as deuterium, i.e. 2 H) substitutions may offer certain therapeutic advantages resulting from greater metabolic stability (eg, increased in vivo half-life) or reduced dosage requirements and, therefore, may be preferred in some circumstances.

[0079] Using positron-emitting isotopes such as 11 C. 18 F. 15 O and 13 N) substitution can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the examples and preparations listed below, using appropriate isotopically labeled reagents in place of the non-labeled reagents previously employed.

[0080] The invention disclosed herein is also intended to cover the in vivo metabolites of the disclosed compounds. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, mainly due to enzymatic processes. Therefore, the present invention includes compounds produced by a method comprising contacting the compounds of the present invention with a mammal for a period of time sufficient to obtain its metabolites. Such products are generally administered to animals (such as rats, mice, guinea pigs, dogs, monkeys) or humans by administering the radiolabeled compounds of the present invention in a detectable dose, allowing sufficient time for metabolism to occur, and separating and identifying their conversion products from urine, blood or other biological samples.

[0081] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0082] "Mammal" includes humans, as well as domestic animals (such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domestic animals (such as wild animals), etc.

[0083] "Optional" or "optionally" means that the subsequently described event of the circumstances may or may not occur, and that the description includes instances where the event or circumstances occur and instances where it does not occur. For example, "optionally substituted aryl" means that the aryl may or may not be substituted, and that the description includes both substituted aryl and aryl without substitution.

[0084] "Patient" means a human or non-human animal (eg, a mammal) having a disease or condition as determined by a qualified professional (eg, a physician, nurse practitioner, or veterinarian) with or without laboratory testing of a sample from the patient as known in the art.

[0085] "Pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration for use in humans or domestic animals.

[0086] As used herein, "pharmaceutically acceptable salts" means those salts which are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, etc., and which are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66: 1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (Editors P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008. Pharmaceutically acceptable salts include acid and base addition salts.

[0087] "Pharmaceutically acceptable acid addition salts" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are prepared with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, , fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.).

[0088] "Pharmaceutically acceptable base addition salts" refer to those salts which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, decanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benzylpenicillin, benzathine penicillin, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0089] Typically crystallization produces a solvate of the compounds of the invention. As used herein, the term "solvate" refers to an aggregate of molecules comprising one or more compounds of the invention and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Therefore, the compounds of the invention may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and corresponding solvated forms. The compounds of the invention may be true solvates, while in other cases, the compounds of the invention may retain only exogenous water or a mixture of water plus some exogenous solvents.

[0090] "Pharmaceutical composition" refers to a preparation of a compound of the invention and a medium generally accepted in the art for delivering biologically active compounds to mammals (eg, humans). For this purpose, such a medium includes all pharmaceutically acceptable carriers, diluents or excipients.

[0091] "Therapeutically effective amount" refers to an amount of a compound of the invention that is sufficient to achieve treatment as defined below in a mammal, preferably a human, when administered to a mammal, preferably a human or a dog. The amount of a compound of the invention or another pharmaceutical agent (e.g., an anti-tumor agent) that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by a person of ordinary skill in the art based on his or her own knowledge and this disclosure.

[0092] As used herein, "Treating" or "treatment" encompasses the treatment of a target disease or condition in a mammal (preferably a human) having the target disease or condition, and includes:

[0093] (i) preventing a disease or condition from occurring in a mammal, particularly where the mammal is susceptible to the condition but has not yet been diagnosed with the condition;

[0094] (ii) inhibiting the disease or condition, i.e., arresting its development;

[0095] (iii) alleviate the disease or condition, that is, cause a remission of the disease or condition; or

[0096] (iv) Alleviate symptoms caused by a disease or condition, i.e., relieve pain, but do not address the underlying disease or condition. The terms "disease" and "condition" as used herein may be used interchangeably, or may be different, in that a particular disease or condition may not have a known causal agent (such that the etiology has not been worked out), and therefore is not yet recognized as a disease, but only as an undesirable condition or syndrome in which clinicians have identified a more or less specific set of symptoms.

[0097] The compounds of the invention, or their pharmaceutically acceptable salts, may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined in terms of absolute stereochemistry as (R)- or (S)-, or as (D)- or (L)- for amino acids. The present invention is intended to include all such possible isomers, as well as racemic and optically pure forms thereof. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques (e.g., chromatography and fractional crystallization). Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0098] "Stereoisomers" refer to compounds made up of the same atoms bonded by the same bonds but with different three-dimensional structures, which are not interchangeable. The present invention encompasses various stereoisomers and mixtures thereof and includes "enantiomers", which refers to two stereoisomers whose molecules are non-superimposable mirror images of each other.

[0099] "Tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any of the described compounds.

[0100] Also within the scope of the present invention are the intermediate compounds of formula (I) as well as all polymorphs of the abovementioned species and their crystal habits. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 are images of immunoblots from a representative experiment evaluating the effects of compound 569 on MCF7 cells with or without irradiation.

[0102] Figure 2 is a graph showing the results of a clonogenic survival assay examining the viability of MCF7 cells following exposure to vehicle (DMSO) or Compound 569 in the presence or absence of radiation.

[0103] Figure 3is an immunoblot image showing that compound 569, a selective ATM inhibitor (ATMi), a selective DNA-PK inhibitor (DNA-PKi), and a combination of a selective ATM inhibitor and a selective DNA-PK inhibitor (Ai+Di) induce TBK1 phosphorylation in HCT116 cells expressing wild-type p53 or HCT116 cells negative for p53 expression. Figure 3 In the figure, p.ATM, p.DNA-PK, p.TBK1 and p.STING represent the phosphorylated forms of ATM, DNA-PK, TBK1 and STING, respectively.

[0104] Figure 4 is an immunoblot showing the inhibition of radiation-induced autophosphorylation of DNA-PK kinase and radiation-induced phosphorylation of KAP1 (ATM substrate) by compound 569 in FADU head and neck squamous cell carcinoma (HNSCC) human tumor xenografts. Figure 4 In the figure, pDNA-PK and pKAP1 represent the phosphorylated forms of DNA-PK and KAP1, respectively.

[0105] Figure 5 is an immunoblot showing the inhibition of radiation-induced autophosphorylation of DNA-PK kinase and radiation-induced phosphorylation of KAP1 (ATM substrate) by compound 569 in MDA-MB-231 breast cancer human tumor xenografts. Figure 5 In the figure, pDNA-PK and pKAP1 represent the phosphorylated forms of DNA-PK and KAP1, respectively.

[0106] Figure 6 Dosing of FADU subcutaneous human xenograft mouse model with Compound 569 and / or IR, qd×3 is shown. The median relative tumor volume of each group over time in the study is shown. Figure 6 In the , "569" means compound 569, "Veh." means vehicle, and "Rad." means radiation.

[0107] Figure 7 Represents the Kaplan-Meier fivefold survival rate for each group dosed with Compound 569 and / or IR, qd×3 in the FADU subcutaneous human xenograft mouse model. Figure 7 In the , "569" means compound 569, "Veh." means vehicle, and "Rad." means radiation.

[0108] Figure 8 Dosing of the MDA-MB-231 subcutaneous human xenograft mouse model with Compound 569 and / or IR, qd x 3, is shown. The median relative tumor volume of each group over time in the study is shown. Figure 8In the , "569" means compound 569, "Veh." means vehicle, and "Rad." means radiation.

[0109] Fig. 9 The Kaplan-Meier data for each group dosing qd×3 with Compound 569 and / or IR in the MDA-MB-231 subcutaneous human xenograft mouse model show no five-fold survival. Fig. 9 In the , "569" means compound 569, "Veh." means vehicle, and "Rad." means radiation.

[0110] Compounds, compositions and methods

[0111] The present invention provides compounds and compositions that can be used, for example, alone or in combination with radiation therapy and / or anti-tumor therapy to treat neoplastic diseases (e.g., cancer, such as those described herein). The compound can be a compound of formula (I):

[0112]

[0113] or a pharmaceutically acceptable salt thereof,

[0114] in

[0115] Z is CH, CR 3 or N;

[0116] Y is CHR 5 or NR 6 ;

[0117] n is 0, 1, 2, or 3;

[0118] R 1 Yes-OLN(R 7 ) 2 or an optionally substituted four-membered saturated N-heterocyclic group;

[0119] R 2 It is C 1-3 alkyl;

[0120] Each R 3 is independently halogen or optionally substituted C 1-3 alkyl;

[0121] R 4 is optionally substituted alkyl;

[0122] R 5 is hydrogen, optionally substituted C 1-3 Alkyl or benzyloxy;

[0123] R 6 is an optionally substituted C 1-3 alkyl;

[0124] Each R 7 is independently H or optionally substituted C 1-3 alkyl; and

[0125] L is optionally substituted ethylene.

[0126] Advantageously, the compounds of the invention (e.g., compounds 568, 569, or 570) may exhibit superior inhibitory activity against ATM and DNA-PK. Advantageously, the compounds of the invention (e.g., compounds 568, 569, or 570) may exhibit superior selectivity, as measured by reduced off-target activity (e.g., mTOR inhibition, PI3Kα / δ inhibition, and / or hERG inhibition). For example, the compounds of the invention (e.g., compounds 568, 569, or 570) may have a lower IC value than ATM. 50 or DNA-PK IC s0 An mTOR IC that is at least 10 times (e.g., at least 20 times) larger 50 The compounds of the invention (eg, compounds 568, 569, or 570) may have an mTOR IC of 10 nM or greater (eg, >100 nM). 50 Additionally or alternatively, the compounds of the invention (eg, compounds 568, 569, or 570) may have a 50 or DNA-PK IC 50 hERG IC at least 100-fold (e.g., at least 500-fold, at least 1000-fold, or at least 3000-fold) greater 50 , when measured at the same compound concentration. The compounds of the invention (eg, compounds 568, 569, or 570) may have a hERG IC of 3 μM or greater (eg, 10 μM or greater). 50 .

[0127] Advantageously, the compounds of the invention (eg, compounds 568, 569, or 570) may exhibit excellent pharmacokinetic properties (eg, C max , AUC and / or t 1 / 2 ).

[0128] In some embodiments, the compound is selected from the group consisting of:

[0129]

[0130] The advantage of the compounds of the present invention is that they can inhibit ATM (ataxia-telangiectasia, mutant) and DNA-PK kinases. ATM (ataxia-telangiectasia, mutant) and DNA-PK kinases are especially important regulators of the response of cells to DNA breaks and the inhibition of any of these molecules significantly increases the sensitivity of cells to ionizing radiation. Therefore, the compounds of the present invention can be effective inhibitors of ATM and DNA-PK effects, with or without radiation and with or without chemotherapy or immunotherapy, to provide effective therapies for treating tumor diseases (e.g., cancers, such as those described herein). Treatment of patients with the compounds of the present invention can delay or eliminate the repair of DNA damage by radiotherapy. Therefore, patients receiving the compounds of the present invention can better respond to anti-tumor therapy. Advantageously, patients receiving the compounds of the present invention can obtain therapeutic benefits by increasing tumor control from standard doses of radiotherapy or by achieving similar levels of tumor control from lower doses of ionizing radiation compared to conventional use of patients who have not received the compounds of the present invention. Advantageously, lower doses of ionizing radiation are less damaging to non-cancerous tissues than the doses required for patients who have not received the compounds of the present invention.

[0131] Humans and mice with loss-of-function mutations in the ATM or PRKDC genes, encoding mutant ataxia telangiectasia (ATM) kinase and DNA-dependent protein kinase (DNA-PK), respectively, are hypersensitive to ionizing radiation. Inhibition of ATM and DNA-PK kinases together can effectively sensitize tumor cells to radiation or DNA damaging agents (e.g., anti-tumor agents). The efficacy of dual inhibition of ATM and DNA-PK kinases may be superior to the efficacy of inhibiting either kinase alone.

[0132] Additionally, the compounds of the invention may advantageously exhibit reduced inhibition of other kinases (ATR and mTOR) and may therefore exhibit reduced toxicity.

[0133] The compounds of the invention may sensitize tumor cells to radiation and / or anti-tumor agents.

[0134] method

[0135] On the other hand, the present invention provides a method for treating a tumor disease (e.g., cancer) in a mammal, preferably a human or a dog, wherein the method comprises administering a therapeutically effective amount of a compound of the present invention to a mammal in need thereof. In some embodiments, the compound is administered to a mammal receiving radiation therapy.

[0136] On the other hand, the present invention provides a method for treating a tumor disease (e.g., cancer) in a mammal, wherein the method comprises administering a therapeutically effective amount of a compound of the present invention to a mammal in need thereof. In some embodiments, the compound is administered to a mammal in combination with a DNA damaging agent. Non-limiting examples of DNA damaging agents include cisplatin, oxaliplatin, carboplatin, valrubicin, idarubicin, calicheamicin, and PARP inhibitors.

[0137] On the other hand, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition comprises a compound of the present invention in a pharmaceutically acceptable carrier in an amount effective to treat a tumor disease in an animal (preferably a mammal).

[0138] The compounds of the invention (when used in combination therapy) can enhance the effectiveness of other radiotherapy or drug therapy, which can reduce the incidence and / or severity of adverse events associated with other drug therapy if it allows the dose of the other therapy to be reduced. For example, in patients receiving a combination therapy comprising a compound of the invention and a dose-reduced radiotherapy, side effects of radiation (e.g., oral or gastrointestinal mucositis, dermatitis, pneumonia, or fatigue) can be reduced (e.g., the incidence of adverse events can be reduced by at least 1%, 5%, 10%, or 20%) compared to patients receiving a standard full-dose radiotherapy without a compound of the invention. In addition, other adverse events that can be reduced (e.g., the incidence of adverse events can be reduced by at least 1%, 5%, 10%, or 20%) in patients receiving a combination therapy comprising a compound of the invention and a dose-reduced radiotherapy compared to patients receiving a standard full-dose radiotherapy without a compound of the invention can be late effects of radiation, such as radiation-induced pulmonary fibrosis, heart damage, intestinal obstruction, nerve damage, vascular damage, lymphedema, brain necrosis, or radiation-induced cancer. Similarly, when the compound is administered in combination therapy with another anticancer drug (e.g., those described herein), the combination therapy may result in the same or even increased tumor cell death, even when the dose of the other anticancer drug is reduced. Thus, reducing the dose of the other anticancer drug can reduce the severity of adverse events caused by the other anticancer drug.

[0139] On the other hand, the present invention relates to the use of the compounds of the present invention as described above in the form of their stereoisomers, enantiomers, tautomers or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, or the use of pharmaceutical compositions comprising pharmaceutically acceptable excipients and compounds of the present invention as described above in the form of their stereoisomers, enantiomers, tautomers or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, which are used to prepare medicaments for treating diseases. In some embodiments, the compounds of the present invention are administered in combination with radiotherapy. In other embodiments, the compounds of the present invention are administered in combination with DNA damaging agents. In other embodiments, the compounds of the invention are administered in combination with an anti-tumor immunotherapeutic agent (e.g., ipilimumab, ofatumumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, obinutuzumab, ocaratuzumab, tesimumab, spartalizumab, carrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, veltuzumab, INCMGA00012, AMP-224, AMP-514, KN035, CK-301, AUNP12, CA-170, or BMS-986189). In other embodiments, the anti-tumor immunotherapy agent is ofatumumab, obinutuzumab, okatumumab or veltuzumab. In other embodiments, the anti-tumor immunotherapy agent is nivolumab, pembrolizumab, cimiprilimab, spartalizumab, carrelizumab, sindilimab, tislelizumab, tereprilimab, dotalimab, INCMGA00012, AMP-224 or AMP-514. In other embodiments, the anti-tumor immunotherapy agent is atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170 or BMS-986189. In certain preferred combination therapy embodiments, the compounds of the present invention are administered in combination with anti-tumor immunotherapy agents.

[0140] The methods of the present invention can be used to treat tumor diseases described herein. The tumor disease can be, for example, a precancerous tumor or a malignant tumor (e.g., a solid tumor or a liquid tumor). Malignant tumors are generally referred to as cancer. In certain embodiments, the tumor disease is cancer.

[0141] In other embodiments, examples of cancers to be treated using the methods and uses disclosed herein include, but are not limited to, blood cancers, such as leukemias and lymphomas. Non-limiting examples of cancer include acute myeloid leukemia, acute lymphoblastic leukemia, acute megakaryocytic leukemia, promyelocytic leukemia, erythroleukemia, lymphocytic T-cell leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, chronic neutrophilic leukemia, plasmacytoma, immunoblastic large cell leukemia, mantle cell leukemia, multiple myeloma, malignant lymphoma, diffuse large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic T-cell lymphoma, Burkitt's lymphoma, and follicular lymphoma.

[0142] In yet other embodiments, examples of cancers to be treated using the methods and uses disclosed herein include, but are not limited to, solid tumors. Non-limiting examples of solid tumors include brain cancer (e.g., astrocytoma, glioma, glioblastoma, medulloblastoma, or ependymoma), bladder cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gastrointestinal stromal tumors, gastric cancer, head and neck cancer, cheek cancer, oral cancer, hepatocellular carcinoma, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, nasopharyngeal carcinoma, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, salivary gland cancer, sarcoma, testicular cancer, urothelial carcinoma, vulvar cancer, and Wilm's tumor. Preferably, the method of the invention is used to treat lung cancer, head and neck cancer, pancreatic cancer, colorectal cancer, glioblastoma, hepatocellular carcinoma, cholangiocarcinoma, metastatic liver lesions, melanoma, osteosarcoma, soft tissue sarcoma, endometrial cancer, cervical cancer, prostate cancer or Merkel cell carcinoma.

[0143] In still other embodiments, examples of cancers to be treated using the methods and uses disclosed herein include, but are not limited to, metastasis and metastatic cancers. For example, the methods and uses disclosed herein for treating cancer may include treatment of primary tumors and metastases.

[0144] In some embodiments, the methods of the present invention can reduce the size of a subject's tumor, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or can eliminate the tumor (e.g., relative to the tumor size at the start of treatment or relative to a reference subject receiving a placebo instead of a compound of the present invention). In some embodiments, the methods of the present invention can reduce the tumor burden of a subject, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or can eliminate the tumor (e.g., relative to the tumor burden at the start of treatment or relative to a reference subject receiving a placebo instead of a compound of the present invention). In some embodiments, the methods of the present invention can increase the average survival time of a subject, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150% or 200% (e.g., relative to a reference subject receiving a placebo instead of a compound of the present invention). In some embodiments, the methods of the invention can enhance the ability of radiation therapy or drug therapy to reduce pain or other symptoms in a subject for a longer average time, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% (e.g., relative to a reference subject receiving a placebo instead of a compound of the invention).

[0145] In some embodiments, the methods and uses disclosed herein include pre-treating the patient with an ATM and DNA-PK dual inhibitor prior to administering radiotherapy or a DNA damaging agent. Pre-treating the patient with an ATM and DNA-PK dual inhibitor can delay or eliminate the repair of DNA damage after radiotherapy.

[0146] Radiation therapy includes but is not limited to the use of X-rays (photons), 60 External beam radiation therapy using gamma rays, neutrons, electrons, protons, carbon ions, helium ions, and other charged particles from cobalt or other radioactive isotopes. Radiation therapy also includes brachytherapy and radiopharmaceuticals that emit gamma rays, alpha particles, beta particles, Auger electrons, or other types of radioactive particles from isotopes including: 32 phosphorus, 67 copper, 77 bromine, 89 strontium, 90 yttrium, 105 rhodium, 131 iodine, 137 cesium, 149 Promethium, 153 samarium, 166 holmium, 177 Lutetium 186 rhenium, 188rhenium, 199 gold, 211 astatine, 213 bismuth, 223 radium, 225 Actinium or 227 thorium, 192 iridium, 67 gallium, 103 palladium, 125 Iodine and other radioactive isotopes (e.g. 192 iridium, 125 iodine, 137 cesium, 103 Palladium, 32-phosphorus, 90 yttrium, 67 gallium, 211 Astatine or 223 Radiation therapy also includes radioimmunotherapy (RIT) using antibodies or small molecules conjugated to radioisotopes, including 131 iodine, 90 yttrium, 225 Actinium, 211 astatine, 67 gallium, 177 Lutetium 227 Thorium and other radioactive isotopes.

[0147] In some embodiments, the combination therapy comprises administering to the patient an ATM and DNA-PK inhibitor and an anti-tumor agent, such as cisplatin, oxaliplatin, carboplatin, topoisomerase I inhibitors, topoisomerase II inhibitors, anthracyclines, valrubicin, idarubicin, calicheamicin, PARP inhibitors (e.g., olaparib, rucaparib, niraparib, veliparib, or talazoparib), as well as other anti-cancer agents known to those skilled in the art.

[0148] In certain embodiments, the combination therapy comprises administering to the patient an ATM and DNA-PK inhibitor and an anti-tumor immunotherapeutic agent, including but not limited to ipilimumab, ofatumumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and the like.

[0149] In the combination therapies described herein, ATM and the DNA-PK inhibitor can be administered to the patient simultaneously or sequentially (eg, before or after the other drug).

[0150] Preparation of the compounds of the present invention

[0151] The compounds of the present invention can be prepared using methods and techniques known in the art. Suitable processes for synthesizing these compounds are provided in the examples. Generally, compounds of formula (I) can be prepared according to the following schemes. The sources of the starting materials for these reactions are also described.

[0152] Protecting groups can be added or removed in the preparation of compounds of the invention according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, TW and PGM Wuts, Greene's Protective Groups in Organic Synthesis (2006), 4th edition, Wiley. The protecting group can also be a polymer resin, such as Wang resin or 2-chlorotrityl chloride resin.

[0153] Those skilled in the art will also understand that, although such protected derivatives of the compounds of the present invention may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in vivo to form pharmacologically active compounds of the present invention.

[0154] All compounds described below that can be present in free base or acid form can be converted into their pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids. Salts of compounds prepared below can be converted into their free base or acid form by standard techniques. It should be understood that all polymorphs, amorphous forms, anhydrates, hydrates, solvates and salts of the compounds of the present invention are within the scope of the present invention. In addition, all compounds of the present invention containing acid or ester groups can be converted into corresponding esters or acids by methods known to those skilled in the art or by methods described herein, respectively.

[0155] A general representation of the preparation of many of these compounds is shown in Scheme 1 below. The compounds are prepared by coupling the various components of the molecule: Suzuki coupling of a halo substituted compound 3 (or 2') with a boronic acid or boronate compound 2 (3'). Further reactions may or may not be required to provide the synthesis of the compounds of the invention. The preparation of specific compounds of the invention is shown in the following schemes.

[0156]

[0157] In this method, the Suzuki coupling reaction conditions can be used to convert halogen substitution into aryl substitution. The conditions of this method are disclosed in many publications, which have been reviewed by A.Suzuki in Modern Arene Chemistry 2002, in a paper entitled "The Suzuki reaction with arylboron compounds in arene chemistry" in 53-106. In carrying out this reaction, any suitable conditions conventional in the Suzuki reaction can be utilized. Usually, the Suzuki coupling reaction is carried out in the presence of a transition metal catalyst such as a palladium catalyst using any conventional organic solvent and a weak inorganic or organic base of this reaction. Wherein preferred organic solvents are polar aprotic solvents. Any conventional polar aprotic solvent can be used to prepare the compounds of the present invention. Suitable solvents are conventional, particularly higher boiling point solvents, for example, dimethoxyethane. The weak inorganic base can be a carbonate or bicarbonate, such as potassium carbonate or cesium carbonate. The organic base can be an amine, such as triethylamine.

[0158]

[0159] Specifically, other spirooxindole intermediates 7 are synthesized as shown in Scheme 2. Cyclic or heterocyclic substituted esters 5 are treated with a strong base such as, but not limited to, lithium diisopropylamide in an anhydrous solvent such as, but not limited to, tetrahydrofuran at low temperature to react with starting material 4 to provide intermediate 6, which is commercially available or prepared by one skilled in the art following methods described in the literature. Intermediate 6 is reduced by a reducing agent such as, but not limited to, iron to give the corresponding amino intermediate, which is cyclized in situ to provide oxindole compound 7. Therefore, compound 7 is then N-alkylated with an alkylating agent in the presence of a base such as, but not limited to, potassium carbonate or sodium hydride in a polar solvent such as, but not limited to, N,N-dimethylformamide or tetrahydrofuran, thereby generating spirooxindole intermediate 8.

[0160]

[0161] Specifically, the compounds of formula (I) in the present invention can be synthesized as shown in Scheme 3. Commercially available 5-bromo-2-chloro-3-nitro-pyridine (9) is reacted with a nucleophile XH (10) in the presence of a strong base such as, but not limited to, sodium hydride to provide intermediate 11. Under palladium catalysis, boronate ester 12 can be prepared, which is then reacted with spiro intermediate 8 to provide the cross-coupled product 13. The nitro group in compound 13 is reduced to an amino group using a reducing agent such as, but not limited to, iron to provide intermediate 14. Reaction of 14 with different sulfonyl chlorides (15) provides the synthesis of compounds of formula (I).

[0162]

[0163]

[0164] Specifically, the compounds of formula (I) in the present invention can also be synthesized as shown in Scheme 4. The nitro group in compound 11 is reduced to an amino group using a reducing agent such as, but not limited to, iron to provide intermediate 16. Reaction of 16 with different sulfonyl chlorides (15) provides sulfonamide intermediate 17, which is converted to its corresponding boronate ester 18 under palladium catalysis. Boronate ester 18 can be coupled with halogenated compound 8 under Suzuki reaction conditions to provide compounds of formula (I).

[0165] In Schemes 3 and 4, cross-coupled compounds can also be synthesized using Suzuki coupling chemistry with components having reversed halogen and boronate / boronic acid substitution patterns, for example, as shown in Scheme 5.

[0166]

[0167] Specifically, the compounds of formula (I) in the present invention can also be synthesized as shown in Scheme 5. The halogenated compound 8 can be converted to its corresponding boronate ester 19 under palladium catalysis. The boronate ester 19 can be coupled with the halogenated compound 17 under Suzuki reaction conditions to provide the compounds of formula (I).

[0168] In the implementation of the inventive method, any one of the compounds of this invention or any combination of the compounds of this invention or its pharmaceutically acceptable salt of effective dose is administered individually or in combination via any commonly used and acceptable method known in the art. Therefore, the compound or composition can be administered orally (e.g., buccal cavity), sublingually, parenterally (e.g., intramuscularly, intravenously or subcutaneously), rectum (e.g., by suppository or lotion), percutaneously (e.g., skin electroporation) or by inhalation (e.g., by aerosol) and in shape or solid, liquid or gas administration (including tablets and suspensions). It can be arbitrarily administered with continuous therapy or with single dose therapy in a single unit dosage form. The therapeutic composition can also be in the form of an oil emulsion or dispersion combined with a lipophilic salt such as pamoic acid, or in the form of a biodegradable sustained-release composition for subcutaneous or intramuscular administration.

[0169] Useful pharmaceutical carriers for preparing compositions thereof may be solid, liquid or gaseous; thus, the compositions may take the form of tablets, pills, capsules, suppositories, powders, enteric coatings or other protective preparations (e.g., bound on ion exchange resins or packaged in lipid-protein vesicles), sustained release preparations, solutions, suspensions, elixirs, aerosols, and the like. The carrier may be selected from a variety of oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water, saline, aqueous dextrose solutions, and glycols are preferred liquid carriers, particularly (when isotonic with blood) for injectable solutions. For example, formulations for intravenous administration comprise a sterile aqueous solution of the active ingredient, which is prepared by dissolving the solid active ingredient in water to prepare an aqueous solution and sterilizing the solution. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, gelatin, malt, rice, flour, chalk, silicon dioxide, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc. The composition can accept conventional pharmaceutical additives such as preservatives, stabilizers, wetting agents or emulsifiers, salts for adjusting osmotic pressure, buffers, etc. Suitable pharmaceutical carriers and their preparations are described in Remington's Pharmaceutical Sciences by EW Martin. In any case, such compositions will contain an effective amount of active compound and a suitable carrier in order to prepare a suitable dosage form for appropriate administration to a recipient.

[0170] The dosage of the compounds of the invention depends on many factors, such as, for example, the mode of administration, the age and weight of the patient, and the condition of the patient to be treated, and will ultimately be determined by the attending physician or veterinarian. This amount of active compound determined by the attending physician or veterinarian is referred to herein and in the claims as an "effective amount".

[0171] The present invention will now be further described in the following examples, which are intended to be illustrative only and not limiting of the scope of the present invention.

[0172] Example

[0173] Reagents were purchased from Aldrich, Sigma, TCI (Shanghai) Development, Chembon Pharmaceutical Co., Ltd, Zhangjiagang Aimate Huaxue Youxiangongsi, Changzhou Qinuo BioTech Co. Ltd and Shanghai Weiyuan Fine Fluorine Technology Development Co., Ltd or other suppliers as shown below and used without further purification. Reactions using microwave radiation for heating were performed using Biotage Initiator +. Multi-milligram to multi-gram scale purification was performed by methods known to those skilled in the art such as elution by silica gel flash column chromatography; in some cases, preparative flash column chromatography purification was also achieved by using a pre-packed silica gel column (Welch / Agela) eluted with a Biotage CombiFlash system.

[0174] For the purpose of determining compound identity and purity, an analytical LC-MS (liquid chromatography / mass spectrometry) system is typically used, which is powered by a Waters ZQ TM The platform is composed of an Agilent 1100 series HPLC with an autosampler. The column is typically a Water Xterra MS C18, 3.0×50 mm, 5 μm. The flow rate is 1 mL / min and the injection volume is 10 μL. UV detection is in the range of 210-400 nm. The mobile phase consists of solvent A (water + 0.06% TFA) and solvent B (acetonitrile + 0.05% TFA) with a gradient of 100% solvent A for 0.7 min, 100% solvent B over 3.75 min, held for 1.1 min, and then returned to 100% solvent A over 0.2 min.

[0175] For some separations, the use of supercritical fluid chromatography may also be useful. Supercritical fluid chromatography separations were performed using a Mettler-Toledo Minigram system with the following typical conditions: 100 bar, 30 °C, 2.0 mL / min, in supercritical fluid CO 2 The 12 mm AD column was eluted with 40% MeOH in 2% CO. In the case of analytes with basic amino groups, 0.2% isopropylamine was added to the methanol modifier.

[0176] Many compounds of formula (I) were also purified by reverse phase HPLC using methods well known to those skilled in the art. In some cases, preparative HPLC purifications were performed using a PE Sciex 150EX Mass Spec attached to a Shimadzu preparative HPLC system and a Leap autoinjector controlling a Gilson 215 collector. Compounds were collected from the elution stream using MS detection in cation detection: Solvent (A) 0.05% TFA / H using an appropriate linear gradient mode over 10 minutes 2 Elution of the compounds from a C-18 column (2.0 X 10 cm, eluted at 20 mL / min) was achieved with 1% O and solvent (B) 0.035% TFA / acetonitrile. For injection onto the HPLC system, the crude sample was dissolved in a mixture of methanol, acetonitrile and DMSO.

[0177] The Bruker ADVANCE III HD 400MHz spectrometer or the Bruker AVANCE 300MHz spectrometer was used. 1 The compounds were characterized by H-NMR.

[0178] List of abbreviations

[0179] DCE 1,2-dichloroethane

[0180] DCM Dichloromethane

[0181] DIPEA Diisopropylethylamine

[0182] DMF N,N-Dimethylformamide

[0183] DMSO Dimethyl sulfoxide

[0184] EtOAc Ethyl acetate

[0185] HOAc Acetic acid

[0186] HPLC High Pressure Liquid Chromatography

[0187] MeI Methyl iodide

[0188] MeOH Methanol

[0189] MW Microwave

[0190] NMP 1-Methyl-2-pyrrolidone

[0191] rt Ambient temperature

[0192] TBDMS tert-butyl-dimethylsilyl

[0193] TEA Triethylamine

[0194] TFA Trifluoroacetic acid

[0195] TEMPO 2,2,6,6-Tetramethyl-1-piperidinyloxy

[0196] THF Tetrahydrofuran

[0197] Preparation of compounds

[0198]

[0199] Methyl 1-(6-bromo-7-fluoro-3-nitroquinolin-4-yl)cyclobutane-1-carboxylate: A solution of methyl cyclobutanecarboxylate (0.73 g, 6.38 mmol) in tetrahydrofuran (5.00 mL) was treated with freshly prepared lithium diisopropylamide (6.38 mmol) in tetrahydrofuran (45.0 mL) at -78 °C under a nitrogen atmosphere for 1 hour, followed by the addition of 6-bromo-4-chloro-7-fluoro-3-nitroquinoline (1.50 g, 4.91 mmol) portionwise over 2 minutes. After stirring for an additional hour at ambient temperature, the reaction was quenched by saturated aqueous ammonium chloride solution (60.0 mL) and diluted with water (120 mL). The resulting mixture was extracted with ethyl acetate (3 x 60.0 mL). The combined organic layers were washed with brine (2 x 50.0 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 1% to 2% ethyl acetate / petroleum ether to give the title compound as a colorless solid (240 mg, 13%): 1 H NMR (400 MHz, CDCl 3 )δ9.14 (s, 1H), 8.20 (d, J = 7.2Hz, 1H), 7.90 (d, J = 8.8Hz, 1H), 3.84 (s, 3H), 3.12-2 .99(m, 1H), 2.58-2.48(m, 3H), 1.91-1.83(m, 1H), 1.45-1.27(m, 1H); MS: [(M+1)] + =383.17,385.17.

[0200]

[0201] 8′-Bromo-7′-fluorospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-2′(3′H)-one: A mixture of methyl 1-(6-bromo-7-fluoro-3-nitroquinolin-4-yl)cyclobutane-1-carboxylate (240 mg, 0.63 mmol) and iron powder (350 mg, 6.26 mmol) in acetic acid (10.0 mL) was stirred at ambient temperature for 18 hours. The resulting mixture was filtered and the filter cake was washed with ethyl acetate (5 x 100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 1% to 2% methanol / dichloromethane to give the title compound as a light yellow solid (100 mg, 50%): 1 H NMR (400 MHz, DMSO-d 6 )δ10.75 (s, 1H), 8.68 (s, 1H), 8.52 (d, J=7.5Hz, 1H), 7.98 (d, J=10.1Hz, 1H), 2.90-2.75 (m, 2H), 2.50-2.37 (m, 4H); MS: [(M+1)] + =321.15,323.15.

[0202]

[0203] 8'-Bromo-7'-fluoro-3'-methylspiro[cyclobutane-1,1'-pyrrolo[2,3-c]quinolin]-2'(3'H)-one: A solution of 8-bromo-7-fluoro-2,3-dihydrospiro[cyclobutane-1,1-pyrrolo[2,3-c]quinolin]-2-one (100 mg, 0.31 mmol) in N,N-dimethylformamide (10.0 mL) was treated with sodium hydride (19.9 mg, 0.50 mmol, 60% dispersion in mineral oil) at 0°C under a nitrogen atmosphere for 30 min followed by the addition of iodomethane (66.3 mg, 0.47 mmol). After stirring for an additional 40 minutes at ambient temperature, the reaction was quenched by saturated aqueous ammonium chloride solution (10.0 mL). The resulting mixture was diluted with water (100 mL) and extracted with ethyl acetate (3 x 30.0 mL). The combined organic layers were washed with brine (2 x 20.0 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC (DCM / MeOH=20 / 1, v:V) to give the title compound (102 mg, 98%) as a colorless solid: 1 H NMR (400 MHz, CD 3OD) δ 8.78 (s, 1H), 8.61 (d, J = 7.4Hz, 1H), 7.85 (d, J = 9.8Hz, 1H), 3.36 (s, 3 H), 2.94-2.85(m, 2H), 2.72-2.61(m, 3H), 2.56-2.48(m, 1H); MS: [(M+1)] + =335.00, 337.00.

[0204]

[0205] N-(2-hydroxyethyl)-N-(propan-2-yl)carbamic acid tert-butyl ester: To a solution of 2-[(propan-2-yl)amino]ethan-1-ol (40.0 g, 388 mmol) in methanol (300 mL) was added di-tert-butyl dicarbonate (127 g, 586 mmol) dropwise at 0°C. The resulting mixture was stirred at ambient temperature for 2 hours and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 0% to 4% ethyl acetate / petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (65.0 g, 82%) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) δ4.17 (s, 1H), 3.71 (t, J = 5.4Hz, 2H), 3.30 (t, J = 5.4Hz, 2H), 1.47 (s, 9H), 1.12 (d, J = 6.8Hz, 6H).

[0206]

[0207] Tert-butyl N-[2-[(5-bromo-3-nitropyridin-2-yl)oxy]ethyl]-N-(propan-2-yl)carbamate: A solution of tert-butyl N-(2-hydroxyethyl)-N-(propan-2-yl)carbamate (15.4 g, 75.8 mmol) in anhydrous tetrahydrofuran (250 mL) was treated with sodium hydride (3.30 g, 82.1 mmol, 60% w / w dispersion in mineral oil) at 0°C for 1 hour under a nitrogen atmosphere, followed by the addition of 5-bromo-2-chloro-3-nitropyridine (15.0 g, 63.2 mmol) over 2 minutes at 0°C. After stirring for an additional 2 hours at 25°C, the reaction was quenched by saturated aqueous ammonium chloride solution (50.0 mL) and diluted with water (500 mL). The aqueous layer was extracted with ethyl acetate (3x150 mL). The combined organic layers were washed with brine (2 x 100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 1% to 18% ethyl acetate / petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (18.0 g, 71%) as a light yellow oil: 1 H NMR (400 MHz, CDCl 3 )δ8.42(d, J=2.4Hz, 1H), 8.37(d, J=2.4Hz, 1H), 4.57(t, J=6.3Hz, 2H), 4.32( s, 1H), 3.51 (t, J=6.3Hz, 2H), 1.47 (s, 9H), 1.15 (d, J=6.9Hz, 6H); MS: [(M+1)] + =404.00,406.00.

[0208]

[0209] N-[2-[(3-amino-5-bromopyridin-2-yl)oxy]ethyl]-N-(propane-2-yl)carbamic acid tert-butyl ester: To a solution of N-[2-[(5-bromo-3-nitropyridin-2-yl)oxy]ethyl]-N-(propane-2-yl)carbamic acid tert-butyl ester (15.0 g, 37.1 mmol) in acetic acid (150 mL) was added iron powder (20.7 g, 371 mmol) at ambient temperature. After stirring for another hour at ambient temperature, the resulting mixture was filtered, and the filter cake was washed with tetrahydrofuran (4 x 100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 20% ethyl acetate / petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (12.0 g, 86%) as a colorless solid: 1 H NMR (400 MHz, CD 3OD) δ7.40 (d, J=2.2Hz, 1H), 7.04 (d, J=2.2Hz, 1H), 4.40 (t, J=6.3Hz, 2H), 4.25-3. 99 (m, 1H), 3.52 (t, J=6.3Hz, 2H), 1.46 (s, 9H), 1.17 (d, J=6.8Hz, 6H); MS: [(M+1)] + =374.10,376.10.

[0210]

[0211] tert-Butyl (2-((5-bromo-3-(methylsulfonylamino)pyridin-2-yl)oxy)ethyl)(isopropyl)carbamate: To a solution of tert-butyl N-[2-[(3-amino-5-bromopyridin-2-yl)oxy]ethyl]-N-(propan-2-yl)carbamate (18.8 g, 50.3 mmol) in pyridine (400 mL) was added methanesulfonyl chloride (8.63 g, 75.4 mmol) dropwise at ambient temperature. After stirring at ambient temperature for 6 hours, the resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 3% to 25% ethyl acetate in petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (16.3 g, 72%) as a colorless solid: 1 H NMR (400 MHz, DMSO-d 6 )δ9.41 (s, 1H), 8.05 (d, J = 2.2Hz, 1H), 7.79 (d, J = 2.2Hz, 1H), 4.35 (t, J = 6.3Hz, 2H), 4.15 ( s, 1H), 3.43 (t, J=6.3Hz, 2H), 3.11 (s, 3H), 1.39 (s, 9H), 1.09 (d, J=6.8Hz, 6H); MS: [(M+1)] + =452.00.454.00.

[0212]

[0213] tert-Butyl (2-((5-bromo-3-(ethylsulfonylamino)pyridin-2-yl)oxy)ethyl)(isopropyl)carbamate: To a stirred solution of tert-butyl (2-((3-amino-5-bromopyridin-2-yl)oxy)ethyl)(isopropyl)carbamate (5.00 g, 13.4 mmol) in pyridine (120 mL) was added ethanesulfonyl chloride (5.15 g, 40.1 mmol) dropwise under nitrogen at ambient temperature. After stirring at ambient temperature for 6 hours under nitrogen, the resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 3% to 25% ethyl acetate in petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (3.78 g, 61%) as a pale yellow solid: 1 H NMR (400MHz, CD3OD) δ7.92 (s, 1H), 7.87 (s, 1H), 4.48 (t, J=6.6Hz, 2H), 4.25-4.20 (m, 1H), 3.54 (t, J=6. 6Hz, 2H), 3.13 (t, J=7.3Hz, 2H), 1.49 (s, 9H), 1.35 (t, J=7.4Hz, 3H), 1.19 (d, J=6.8Hz, 6H); MS: [(M+1)] + =466.10,468.10.

[0214]

[0215] N-[2-[(5-bromo-3-iodopyridin-2-yl)oxy]ethyl]-N-isopropylcarbamic acid tert-butyl ester: To a solution of 5-bromo-3-iodopyridin-2-ol (10.0 g, 33.3 mmol) in anhydrous tetrahydrofuran (300 mL) at 0°C, triphenylphosphine (11.4 g, 43.3 mmol), N-(2-hydroxyethyl)-N-isopropylcarbamic acid tert-butyl ester (8.80 g, 43.3 mmol) and diisopropyl azodicarboxylate (8.80 g, 43.4 mmol) were added dropwise. The resulting mixture was stirred for another 16 hours under a nitrogen atmosphere at ambient temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 2% to 10% ethyl acetate / petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (14.0 g, 87%) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 )δ8.14 (s, 2H), 4.43 (t, J=6.4Hz, 2H), 4.38-3.96 (m, 1H), 3.50 (t, J=6.4Hz, 2H), 1.49 (s, 9H), 1.19 (d, J=6.8Hz, 6H); MS: [(M+1)] +=484.95,486.95.

[0216]

[0217] Tert-butyl (2-((5-bromo-3-((1-methylethyl)sulfonylamino)pyridin-2-yl)oxy)ethyl)(isopropyl)carbamate: To a mixture of tert-butyl (2-((5-bromo-3-iodopyridin-2-yl)oxy)ethyl)(isopropyl)carbamate (5.00 g, 10.3 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (1.80 g, 3.10 mmol) and propane-2-sulfonamide (1.50 g, 12.4 mmol) in toluene (125 mL) was added tripotassium phosphate (10.9 g, 51.5 mmol) and tris(dibenzylideneacetone)dipalladium-chloroform adduct (1.10 g, 1.10 mmol) at ambient temperature. The resulting mixture was stirred at 100 °C for 48 hours under argon atmosphere. After cooling to ambient temperature, the resulting mixture was filtered. The filter cake was washed with ethyl acetate (3 x 20 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 1% to 20% ethyl acetate / petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (1.90 g, 39%) as a colorless solid: 1 HNMR (400 MHz, CDCl 3 )δ7.94 (d, J=2.1Hz, 1H), 7.89 (d, J=2.2Hz, 1H), 4.44 (t, J=6.3Hz, 2H), 4.11 (s, 1H), 3.48 (t, J=6.3 Hz, 2H), 3.24-3.30 (m, 1H), 1.48 (s, 9H), 1.41 (d, J=6.8Hz, 6H), 1.14 (d, J=6.8Hz, 6H); MS: [(M+1)] + =480.20,482.20.

[0218]

[0219] N-(5-bromo-2-[2-[(propane-2-yl)amino]ethoxy]pyridin-3-yl)methanesulfonamide: A solution of tert-butyl N-[2-[(5-bromo-3-methanesulfonylaminopyridin-2-yl)oxy]ethyl]-N-(propane-2-yl)carbamate (3.00 g, 6.63 mmol) in dichloromethane (5.00 mL) was treated with hydrogen chloride (20.0 mL, 4M in 1,4-dioxane) for 40 min at ambient temperature. The resulting mixture was concentrated under reduced pressure. The residue was basified to pH=8 with saturated aqueous sodium bicarbonate solution (30.0 mL). The resulting mixture was extracted with ethyl acetate (6 x 200 mL). The combined organic layers were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 1% to 10% methanol / dichloromethane. The desired fractions were collected and concentrated under reduced pressure to give the title compound as a colorless solid (1.20 g, 50%): 1 H NMR (400 MHz, DMSO-d 6 )δ7.68 (d, J=2.3Hz, 1H), 7.61 (d, J=2.3Hz, 1H), 5.75 (s, 1H), 4.36 (t, J=5.2Hz, 2H), 3.19-3.12 ( m, 1H), 3.07 (t, J=5.1Hz, 2H), 2.84 (s, 3H), 1.15 (d, J=6.4Hz, 6H); MS: [(M+1)]+=352.10, 354.10.

[0220] The following intermediates were prepared according to the procedures described above:

[0221]

[0222]

[0223] N-(2-(2-(isopropylamino)ethoxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methanesulfonamide: To a solution of tert-butyl N-[2-[(5-bromo-3-methanesulfonylaminopyridin-2-yl)oxy]ethyl]-N-(propan-2-yl)carbamate (2.00 g, 5.68 mmol) and bis(pinacolato)diboron (2.88 g, 11.4 mmol) in 1,4-dioxane (50.0 mL) at ambient temperature were added potassium acetate (2.23 g, 22.7 mmol) and bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane adduct (0.46 g, 0.57 mmol). After stirring at 90° C. for 2 hours under a nitrogen atmosphere, the resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with dichloromethane (100 mL). After filtration, the filter cake was washed with dichloromethane (3 x 10.0 mL). The filtrate was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography under the following conditions: Column: Spherical C18, 20-40 μm, 330 g; Mobile phase A: water (plus 10 mM NH 4 HCO 3 ), mobile phase B: acetonitrile; flow rate: 65 mL / min; gradient (B%): 5% to 22%, 6 min; 22% to 40%, 20 min; 40% to 95%; 2 min; 95%, 5 min; detector: UV 254 nm; Rt: 15 min. The fractions containing the desired product were collected and concentrated under reduced pressure to give the title compound (1.57 g, 87%) as an off-white solid: 1 H NMR (400 MHz, CDCl 3 )δ8.27 (d, J=1.7Hz, 1H), 8.06 (d, J=1.7Hz, 1H), 4.51 (t, J=5.2Hz, 2H), 3.05-2. 99(m, 5H), 2.95-2.84(m, 1H), 1.33(s, 12H), 1.11(d, J=6.3Hz, 6H); MS: [(M+1)] + =400.30.

[0224] Compound 568

[0225]

[0226] N-(5-(7′-fluoro-3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinolin]-8′yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)propane-2-sulfonamide: To a solution of N-[5-bromo-2-[2-(isopropylamino)ethoxy]pyridin-3-yl]propane-2-sulfonamide (2.00 g, 5.26 mmol) in 1,4-dioxane (50.0 mL) at ambient temperature were added bis(pinacolato)diboron (4.01 g, 15.8 mmol), potassium acetate (2.06 g, 21.1 mmol) and bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane adduct (0.34 g, 0.42 mmol). The resulting mixture was stirred at 90°C for 3 hours under a nitrogen atmosphere. The resulting mixture was cooled to ambient temperature, followed by the addition of 8-bromo-7-fluoro-3-methylspiro[cyclobutane-1,1-pyrrolo[2,3-c]quinoline]-2-one (1.26 g, 3.76 mmol), water (12.5 mL), sodium carbonate (0.80 g, 7.55 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.43 g, 0.38 mmol). The resulting mixture was stirred at 85°C for 6 hours under a nitrogen atmosphere. After cooling to ambient temperature, the resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography under the following conditions: column: Spherical C18, 20-40 μm, 120 g; mobile phase A: water (plus 10 mM NH 4 HCO 3 ); Mobile phase B: acetonitrile; Flow rate: 45 mL / min; Gradient (B%): 5%, 2 min; 5% to 25%, 8 min; 25% to 39%, 9 min; 39%, 10 min; 39% to 95%; 3 min; 95%, 2 min; Detector: UV 254 nm; Rt: 21 min. The fractions containing the desired product were collected and concentrated under reduced pressure to give the title compound (1.66 g, 80%) as a colorless solid: 1 HNMR (400 MHz, DMSO-d 6 )δ8.88 (s, 1H), 8.35-8.31 (m, 2H), 8.07 (s, 1H), 7.99 (s, 1H), 7.96 (s, 1H), 4.55 (br, 1H), 4.44 (t, J=5.2Hz, 2H), 3 .35-3.30(m, 4H), 2.93-2.79(m, 5H), 2.55-2.38(m, 4H), 1.29(d, J=7.2Hz, 6H), 1.02(d, J=6.0Hz, 6H); MS: [(M+1)] + =556.30.

[0227]

[0228] A solution of N-(5-[7-fluoro-3-methyl-2-oxospiro[cyclobutane-1,1-pyrrolo[2,3-c]quinoline]-8'yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)propane-2-sulfonamide (1.66 g, 2.99 mmol) in dilute aqueous hydrochloric acid (392 mL, 3.14 mmol, 0.008 M) and acetonitrile (79.0 mL) was lyophilized to give the title compound as a yellow solid (1.76 g, 100%): 1 H NMR (400 MHz, DMSO-d 6 )δ9.45 (s, 1H), 9.02 (br, 2H), 8.90 (s, 1H), 8.40 (s, 1H), 8.32 (d, J = 8.4Hz, 1H), 8.16 (s, 1H), 8.00 (d, J = 8.4Hz, 1H), 4.65 (t , J=4.4Hz, 2H), 3.54-3.40 (m, 3H), 3.31 (s, 3H), 2.91 (t, J=11.2Hz, 2H), 2.55-2.45 (m, 5H), 1.34-1.30 (m, 12H); MS: [(M+1)] + =556.30.

[0229] Palladium(II) coupling was achieved in a similar manner or using intermediates CC108 and CC110 to give the following compounds Example 569 and Example 570.

[0230]

[0231]

[0232] Compound 571

[0233]

[0234] 3-(6-bromo-7-fluoro-3-nitroquinolin-4-yl)azetidine-1,3-dicarboxylic acid 1-(tert-butyl) 3-methyl ester: A solution of azetidine-1,3-dicarboxylic acid 1-tert-butyl 3-methyl ester (29.3 g, 137 mmol) in tetrahydrofuran (100 mL) was added to a freshly prepared solution of lithium diisopropylamide (137 mmol) in anhydrous tetrahydrofuran (110 mL) at -78 ° C. After stirring for 1 hour, a solution of 6-bromo-4-chloro-7-fluoro-3-nitroquinoline (32.0 g, 105 mmol) in tetrahydrofuran (100 mL) was added to the reaction mixture over 20 min. The resulting mixture was slowly warmed to 0 ° C and continued for 2 hours. The reaction was quenched by saturated aqueous ammonium chloride solution (20.0 mL) and diluted with water (800 mL). The resulting mixture was extracted with ethyl acetate (3x150mL). The combined organic layers were washed with brine (2 x 100mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with 5% to 20% ethyl acetate / petroleum ether to give the title compound (25.0g, 49%) as a light yellow solid: 1 H NMR (400 MHz, DMSO-d 6 )δ9.35 (s, 1H), 8.21 (d, J = 9.3Hz, 1H), 8.14 (d, J = 7.1Hz, 1 H ), 4.18-4.11(m, 2H), 3.87-3.74(m, 2H), 3.66(s, 3H), 1.37(s, 9H); MS: [(M+1)] + =484.20,486.20.

[0235]

[0236] 8′-bromo-7′-fluoro-2′-oxo-2′,3′-dihydrospiro[azetidine-3,1′-pyrrolo[2,3-c]quinoline]-1-carboxylic acid tert-butyl ester:

[0237] At ambient temperature, iron powder (9.69 g, 174 mmol) was added to a solution of 3-(6-bromo-7-fluoro-3-nitroquinoline-4-yl)azetidine-1,3-dicarboxylic acid 1-tert-butyl ester 3-methyl ester (12.0 g, 24.8 mmol) in acetic acid (300 mL). After stirring for 3 hours at ambient temperature, the resulting mixture was concentrated under reduced pressure. The residue was dissolved in water (200 mL) and extracted with ethyl acetate (4x100 mL). The combined organic layers were washed with brine (2x 100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with 1% to 5% methanol / dichloromethane to give the title compound (10.4 g, 99%) as a colorless solid: 1 H NMR (400 MHz, DMSO-d 6 )δ11.02 (br, 1H), 8.71 (s, 1H), 8.24 (d, J = 7.3Hz, 1H), 8.03 (d, J = 10.1Hz, 1H), 4.29 (d, J = 9.0Hz, 2H), 4.21 (d, J = 9.0Hz, 2H), 1.49 (s, 9H); MS: [(M+1)] + =422.20,424.20.

[0238]

[0239] tert-Butyl 8'-bromo-7'-fluoro-3'-methyl-2'-oxo-2',3'-dihydrospiro[azetidine-3,1'-pyrrolo[2,3-c]quinoline]-1-carboxylate: To a stirred solution of tert-butyl 8-bromo-7-fluoro-2-oxo-2,3-dihydrospiro[azetidine-3,1-pyrrolo[2,3-c]quinoline]-1-carboxylate (4.22 g, 9.99 mmol) in N,N-dimethylformamide (100 mL) was added sodium hydride (0.52 g, 13.0 mmol, 60% dispersion in mineral oil) at 0° C. under a nitrogen atmosphere. The resulting mixture was stirred at ambient temperature for 1 hour followed by the addition of iodomethane (1.70 g, 12.0 mmol). After stirring for another 1 hour at ambient temperature, the reactant was quenched by saturated aqueous ammonium chloride solution (20.0 mL) and diluted with water (1.00 L). The precipitated solid was collected by filtration, washed with water (3 x 30.0 mL) and hexane (2 x 30.0 mL). The resulting solid was dried under infrared light to give the title compound (3.93 g, 90%) as a light yellow solid: 1 H NMR (400 MHz, CDCl 3) δ 8.71 (s, 1H), 8.47 (d, J = 7.0Hz, 1H), 7.94 (d, J = 9.1Hz, 1H), 4.54 (d, J = 9.1Hz, 2H), 4.31 (d, J = 9.0Hz, 2H), 3.40 (s, 3H), 1.56 (s, 9H); MS: [(M+1)] + =436.15,438.15.

[0240]

[0241] 8-Bromo-7-fluoro-3-methyl-2,3-dihydrospiro[azetidine-3,1-pyrrolo[2,3-c]quinoline]-2-one: A solution of tert-butyl 8-bromo-7-fluoro-3-methyl-2-oxo-2,3-dihydrospiro[azetidine-3,1-pyrrolo[2,3-c]quinoline]-1-carboxylate (3.93 g, 9.01 mmol) and trifluoroacetic acid (20.0 mL) in dichloromethane (100 mL) was stirred at ambient temperature for 5 hours. The resulting mixture was concentrated under reduced pressure. The residue was basified to pH=8 with saturated aqueous sodium bicarbonate solution. The resulting mixture was extracted with ethyl acetate (6 x 300 mL). The combined organic layers were washed with brine (2 x 300 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give the title compound (3.00 g, 99%) as a pale yellow solid: 1 H NMR (400 MHz, DMSO-d 6 )δ9.56 (d, J=7.7Hz, 1H), 8.92 (s, 1H), 8.02 (d, J=10.2Hz, 1H), 4.18 (d, J=7.5Hz, 2H), 3.59 (d, J=7.5Hz, 2H), 3.29 (s, 3H); MS: [(M+1)] + =335.95,337.95.

[0242]

[0243] 8′-Bromo-7′-fluoro-1-isopropyl-3′-methylspiro[azetidine-3,1′-pyrrolo[2,3-c]quinolin]-2′(3′H)-one: To a stirred solution of 8′-bromo-7′-fluoro-3′-methylspiro[azetidine-3,1′-pyrrolo[2,3-c]quinolin]-2′(3′H)-one (100 mg, 0.30 mmol) and acetone (4.00 mL) in ethanol (8.00 mL) was added sodium cyanoborohydride (94.0 mg, 1.50 mmol) at ambient temperature. The resulting mixture was stirred at 50° C. for 3 hours under nitrogen atmosphere. The resulting mixture was purified by reverse phase flash chromatography using the following conditions: Column: Spherical C18, 20-40 μm, 120 g; Mobile phase A: Water (plus 10 mM NH 4 HCO 3 ); Mobile phase B: acetonitrile; Flow rate: 50 mL / min; Gradient (B): 5% to 20%, 6 min; 20% to 50%, 30 min; 50% to 95%, 5 min; 95%, 5 min; Detector: UV 254 nm. The desired fractions were collected and concentrated under reduced pressure to give the title compound (85.0 mg, 77%) as a colorless solid: 1 H NMR: 1 H NMR (400 MHz, DMSO-d 6 )δ9.84 (d, J=8.0Hz, 1H), 8.93 (s, 1H), 8.00 (d, J=10.2Hz, 1H), 3.69 (d, J=7.6Hz, 2H), 3. 46 (d, J=7.3Hz, 2H), 3.30 (s, 3H), 2.63-2.56 (m, 1H), 1.01 (d, J=6.1Hz, 6H); MS: [(M+1)] + =378.10,380.10.

[0244]

[0245] N-[5-[7-fluoro-3-methyl-2-oxo-1-(propan-2-yl)-2,3-dihydrospiro[azetidine-3,1-pyrrolo[2,3-c]quinolin]-8-yl]-2-[2-[(propan-2-yl)amino]ethoxy]pyridin-3-yl]methanesulfonamide: 8-bromo-7-fluoro-3-methyl-1-(propan-2-yl)-2,3-dihydrospiro[azetidine-3,1-pyrrolo[2,3-c]quinolin]-2-one (80.0 mg, 0.21 mL) was added. To a solution of 1-[(2-[( ... The residue was purified by Prep-TLC (DCM / MeOH=10 / 1, v / v) to give a crude product, which was further purified by reverse phase flash chromatography using the following conditions: column: Spherical C18, 20-40 μm, 120 g; mobile phase A: water (plus 10 mM NH 4 HCO 3 ), mobile phase B: acetonitrile; flow rate: 45 mL / min; gradient (B%): 5%, 2 min; 5% to 24%, 5 min; 24% to 34%, 9 min; 34%, 8 min; 34% to 95%; 3 min; 95%, 2 min; detector: UV 254 nm; Rt: 21 min. The desired fractions were collected and concentrated under reduced pressure to give the title compound (65.0 mg, 54%) as a colorless solid: 1 H NMR (400 MHz, DMSO-d 6 )δ9.66(d, J=8.9Hz, 1H), 8.89(s, 1H), 8.25(t, J=1.9Hz, 1H), 7.99-7.91( m, 2H), 4.42 (t, J=5.4Hz, 2H), 3.76 (d, J=7.2Hz, 2H), 3.47 (d, J=7.3Hz, 2H ), 3.31 (s, 3H), 3.02 (s, 3H), 2.96 (t, J = 5.4Hz, 2H), 2.92-2.84 (m, 1H), 2. 62-2.53 (m, 1H), 1.05 (d, J = 6.3Hz, 6H), 0.97 (d, J = 6.1Hz, 6H); MS: [(M+1)] + =571.25.

[0246] Compound 572

[0247]

[0248] 1-(6-bromo-7-fluoro-3-nitroquinolin-4-yl)-3-methoxycyclobutane-1-carboxylic acid methyl ester: To a freshly prepared solution of lithium diisopropylamide (10.6 mmol) in anhydrous tetrahydrofuran (100 mL) was added 3-methoxycyclobutane-1-carboxylic acid methyl ester (1.53 g, 10.6 mmol) at -78 ° C. After stirring for another hour, a solution of 6-bromo-4-chloro-7-fluoro-3-nitroquinoline (2.50 g, 8.18 mmol) in tetrahydrofuran (5.00 mL) was added over 5 min. The resulting mixture was slowly warmed to 0 ° C and then quenched by saturated aqueous ammonium chloride solution (100 mL). The resulting mixture was diluted with water (1.00 L) and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (3x200 mL). The combined organic layers were washed with brine (2x200 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 5% to 50% ethyl acetate / petroleum ether to give the title compound (720 mg, 21%) as a brown syrup: 1 H NMR (400 MHz, DMSO-d 6 )δ9.25 (s, 1H), 8.31 (d, J = 7.3Hz, 1H), 8.17 (d, J = 9.3Hz, 1H), 4.16-4.13 (m, 1H ), 3.71(s, 3H), 3.11(s, 3H), 2.47-2.38(m, 2H), 2.00-1.89(m, 2H); MS: [(M+1)] + =413.20,415.20.

[0249]

[0250] 8′-Bromo-7′-fluoro-3-methoxyspiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-2′(3′H)-one: To a solution of methyl 1-(6-bromo-7-fluoro-3-nitroquinolin-4-yl)-3-methoxycyclobutane-1-carboxylate (720 mg, 1.74 mmol) in acetic acid (10.0 mL) was added iron powder (681 mg, 12.2 mmol). The resulting mixture was stirred at ambient temperature for 3 hours. The resulting mixture was diluted with water (150 mL) and extracted with ethyl acetate (4 x 50.0 mL). The combined organic layers were washed with brine (2 x 50.0 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with 5% to 30% ethyl acetate / petroleum ether to give the title compound (550 mg, 89%) as a light yellow solid: 1 H NMR (400MHz, CDCl3) δ8.93 (s, 0.45H), 8.88 (d, J = 7.2Hz, 0.55H), 8.84 (s, 1H), 8.78 (s, 0.55H), 8.27 (d, J = 7.0Hz, 0.45H) 7 .92 (d, J=9.3Hz, 1H), 4.71-4.63 (m, 0.45H), 4.57-4.49 (m, 0.55H), 3.48 (d, J=6.6Hz, 3H), 3.07-2.83 (m, 4H); MS: [(M+1)] + =351.00,353.00.

[0251]

[0252] 8'-Bromo-7'-fluoro-3-methoxy-3'-methylspiro[cyclobutane-1,1'-pyrrolo[2,3-c]quinolin]-2'(3'H)-one: A solution of 8-bromo-7-fluoro-3-methoxy-2,3-dihydrospiro[cyclobutane-1,1-pyrrolo[2,3-c]quinolin]-2-one (550 mg, 1.56 mmol) in N,N-dimethylformamide (10.0 mL) was treated with sodium hydride (81.4 mg, 2.04 mmol, 60% dispersion in mineral oil) at 0°C for 0.5 hours, followed by the addition of iodomethane (265 mg, 1.87 mmol) dropwise over 2 minutes at 0°C. After stirring for an additional hour at ambient temperature, the reaction was quenched by saturated aqueous ammonium chloride solution (20.0 mL) and diluted with water (150 mL). The resulting mixture was extracted with ethyl acetate (3 x 50.0 mL). The combined organic layers were washed with brine (2 x 30.0 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 5% to 10% ethyl acetate / petroleum ether to give the title compound (500 mg, 87%) as a yellow solid: MS: [(M+1)] + =365.10,367.10.

[0253]

[0254] 8'-Bromo-7'-fluoro-3-hydroxy-3'-methylspiro[cyclobutane-1,1'-pyrrolo[2,3-c]quinoline]-2'(3'H)-one: To a stirred solution of 8'-bromo-7'-fluoro-3-methoxy-3'-methylspiro[cyclobutane-1,1'-pyrrolo[2,3-c]quinoline]-2'(3'H)-one (900 mg, 2.46 mmol) in dichloromethane (20.0 mL) was added boron tribromide (24.6 mL, 24.6 mmol, 1 M in dichloromethane) dropwise at -78 °C under nitrogen atmosphere. The resulting mixture was slowly warmed to ambient temperature. The resulting mixture was stirred at ambient temperature for 2 hours under nitrogen atmosphere. The mixture was neutralized with saturated aqueous sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with brine (3 x 100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 1% to 5% methanol / dichloromethane. The desired fractions were collected and concentrated under reduced pressure to give the title compound (530 mg, 62%) as a colorless solid: 1 H NMR (400 MHz, DMSO-d 6)δ8.96-8.89 (m, 1.6H), 8.32 (d, J=7.4Hz, 0.4H), 8.05-7.98 (m, 1H), 5.99 (d, J=6.5Hz, 0.6H), 5.73 (d, J=5.7Hz, 0.4H), 4.97-4.87 (m, 0 .4H), 4.75-4.65(m, 0.6H), 3.31(s, 1.2H), 3.29(s, 1.8H), 2.98-2.87(m, 0.8H), 2.81-2.67(m, 2.4H), 2.65-2.55(m, 0.8H); MS: [(M+1)] + =351.00,353.00.

[0255]

[0256] 3-(Benzyloxy)-8′-bromo-7′-fluoro-3′-methylspiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinolin]-2′(3′H)-one:

[0257] Under nitrogen atmosphere, sodium hydride (13.7 mg, 0.35 mmol, 60%, dispersed in mineral oil) was added to a solution of 8′-bromo-7′-fluoro-3-hydroxy-3′-methylspiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-2′(3′H)-one (100 mg, 0.29 mmol) in N,N-dimethylformamide (5.00 mL) at 0° C. The resulting mixture was stirred at 25° C. for 1 hour, and then benzyl bromide (97.4 mg, 0.57 mmol) was added at 0° C. After stirring for another 1 hour at 25° C., the reaction was quenched by saturated aqueous ammonium chloride solution (10.0 mL). The resulting mixture was diluted with water (100 mL) and extracted with ethyl acetate (3× 50.0 mL). The combined organic layers were washed with brine (2× 50.0 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC (DCM / MeOH=25 / 1, v / v) to give the title compound (64.0 mg, 51%) as a yellow solid: 1 H NMR (400 MHz, DMSO-d 6)δ8.92 (d, J=3.6Hz, 1H), 8.79 (d, J=7.6Hz, 0.5H), 8.25 (d, J=7.2Hz, 0.5H) , 8.03 (d, J=10.1Hz, 1H), 7.50 (d, J=7.0Hz, 1H), 7.46-7.37 (m, 3H), 7.37-7. 30(m, 1H), 4.87-4.79(m, 0.5H), 4.62-4.50(m, 2.5H), 3.30(d, J=5.7Hz, 3H ), 2.98-2.90(m, 1H), 2.80(d, J=6.7Hz, 2H), 2.72-2.64(m, 1H); MS: [(M+1)] + =441.00,443.00.

[0258]

[0259] N-(5-(3-(Benzyloxy)-7′-fluoro-3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinolin]-8′yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide: 3-(Benzyloxy)-8′-bromo-7′-fluoro-3′-methylspiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinolin]-2′(3′H)-one (64.0 mg, 0.15 mm To a solution of 1-[2-[2-(isopropylamino)ethoxy]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl]methanesulfonamide (69.5 mg, 0.17 mmol) in water (1.00 mL) and 1,4-dioxane (4.00 mL) was added sodium carbonate (15.4 mg, 0.15 mmol) and tetrakis(triphenylphosphine)palladium(0) (33.5 mg, 0.029 mmol). After stirring at 80° C. for 2 hours under a nitrogen atmosphere, the resulting mixture was concentrated under reduced pressure. The residue was purified by Prep-TLC (DCM / MeOH=10 / 1, v / v) to give the title compound (55.0 mg, 60%) as a pale yellow solid: MS: [(M+1)] + =634.55.

[0260]

[0261] Cis-N-(5-(3-(Benzyloxy)-7′-fluoro-3′-methyl-2′-oxo-2′, 3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′-yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (Example 572) and trans-N-(5-(3-(benzyloxy)-7′-fluoro-3′-methyl-2′-oxo-2′, 3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′-yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (Example 567). The above N-(5-(3-(benzyloxy)-7′-fluoro-3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinolin]-8′-yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (55.0 mg, 0.087 mmol) was separated by Prep-Chiral-HPLC using the following conditions: (column: CHIRALPAK ID, 2x 25cm (5μm); mobile phase A: methyl tert-butyl ether (plus 0.2% isopropylamine), mobile phase B: EtOH; flow rate: 17mL / min; gradient: 10% B, 13min; detector: UV220 / 254nm) to obtain cis-N-(5-(3-(benzyloxy)-7′-fluoro-3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′-yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (Example 572, RT1: 8.70min) (15.3mg, 28%) as a light yellow solid: 1 H NMR (400 MHz, DMSO-d 6 )δ8.90 (s, 1H), 8.29 (s, 1H), 8.09 (d, J = 8.3Hz, 1H), 8.02 (d, J = 1.9Hz, 1H), 7 .98(d, J=12.1Hz, 1H), 7.42-7.34(m, 4H), 7.32-7.27(m, 1H), 4.90-4.82(m, 1H), 4.53 (s, 2H), 4.47 (t, J=5.4Hz, 2H), 3.32 (s, 3H), 3.06 (s, 3H), 3.05-2. 88 (m, 5H), 2.68 (dd, J=13.9, 6.2Hz, 2H), 1.08 (d, J=6.2Hz, 6H); MS: [(M+1)] +=634.50 and trans-N-(5-(3-(benzyloxy)-7′-fluoro-3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′-yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (Example 567, RT2: 11.2 min) (12.6 mg, 23%) as a colorless solid: 1 H NMR (400 MHz, DMSO-d 6 )δ8.88 (s, 1H), 8.59 (d, J = 8.3Hz, 1H), 8.33 (s, 1H), 8.02-7.94 (m, 2H), 7.36-7.21 (m, 5 H), 4.58 (q, J=6.3Hz, 1H), 4.44 (t, J=5.4Hz, 2H), 4.41-4.33 (m, 1H), 3.28 (s, 3H), 3.05 (s, 3H), 2.00-3.94 (m, 3H), 2.92-2.84 (m, 1H), 2.83-2.74 (m, 1H), 2.69 (dd, J=13.1, 5. 8Hz, 2H), 2.59-2.54 (m, 1H), 1.35 (d, J=6.4Hz, 3H), 1.05 (d, J=6.3Hz, 6H); MS: [(M+1)] + =634.55.

[0262] Compound 573

[0263]

[0264] 3-Oxocyclobutane-1-carboxylic acid decyl ester: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (7.56g, 39.4mmol) and 4-dimethylaminopyridine (0.32g, 2.62mmol) were added to a stirred solution of 3-methylcyclobutane-1-carboxylic acid (3.00g, 26.3mmol) and 1-decanol (4.16g, 26.3mmol) in dichloromethane (90.0mL) at ambient temperature. The resulting mixture was stirred at 25°C for 16 hours and quenched by water (30.0mL). The resulting mixture was extracted with dichloromethane (3x 100mL). The combined organic layer was washed with brine (2x 50.0mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with 1% to 8% ethyl acetate / petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (3.10 g, 47%) as a pale yellow oil: 1 H NMR (400 MHz, CDCl 3)δ4.15 (t, J=6.7Hz, 2H), 3.47-3.35 (m, 2H), 3.35-3.15 (m, 3H), 1.69-1.61 (m, 2H), 1.39-1.22 (m, 14H), 0.88 (t, J=6.7Hz, 3H); MS: [(M+1)] + =252.20.

[0265]

[0266] 3-Ethylidenecyclobutane-1-carboxylic acid decyl ester: Potassium tert-butoxide (4.94 g, 44.1 mmol) was added portionwise to a solution of ethyltriphenylphosphonium bromide (17.3 g, 46.5 mmol) in dimethyl sulfoxide (300 mL) at 14 ° C. Under a nitrogen atmosphere, the resulting mixture was stirred at 25 ° C for 0.5 hours, and then 3-oxocyclobutane-1-carboxylic acid decyl ester (8.00 g, 31.5 mmol) was added dropwise at 14 ° C for 2 min. After stirring at 25 ° C for 4 hours, the reaction was quenched by saturated aqueous ammonium chloride solution (20.0 mL) at 0 ° C. The resulting mixture was diluted with water (1.00 L) and extracted with ethyl acetate (3 x 200 mL). The combined organic layer was washed with brine (2 x 100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 1% to 5% ethyl acetate / petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (1.70 g, 21%) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 )δ5.22-5.14 (m, 1H), 4.09 (t, J=6.7Hz, 2H), 3.10 (tt, J=9.2, 7.2Hz, 1H), 2.96-2.79 (m, 4H) , 1.67-1.58 (m, 2H), 1.49 (dq, J=6.0, 2.0Hz, 3H), 1.39-1.20 (m, 14H), 0.88 (t, J=6.7Hz, 3H).

[0267]

[0268] 3-Ethylcyclobutane-1-carboxylic acid decyl ester: Anhydrous Pd / C (70.0 mg, 10% palladium on carbon) was added to a stirred solution of 3-ethylidenecyclobutane-1-carboxylic acid decyl ester (700 mg, 4.94 mmol) in methanol (10.0 mL) at ambient temperature. After stirring for 16 hours under a hydrogen atmosphere (2 atm.) at ambient temperature, the resulting mixture was filtered. The filter cake was washed with methanol (3x20.0 mL). The filtrate was concentrated under reduced pressure to give the title compound (666 mg, 95%) as a light yellow oil: 1 HNMR (400 MHz, CDCl 3 )δ4.10-4.02(m, 2H), 3.10-2.87(m, 1H), 2.40-2.22(m, 2H), 2.16-2.03(m, 1H) , 1.91-1.76 (m, 2H), 1.67-1.56 (m, 2H), 1.48-1.21 (m, 16H), 0.92-0.75 (m, 6H).

[0269]

[0270] 1-(6-bromo-7-fluoro-3-nitroquinolin-4-yl)-3-ethylcyclobutane-1-carboxylic acid decyl ester: Under nitrogen atmosphere, 3-ethylcyclobutane-1-carboxylic acid decyl ester (657 mg, 2.45 mmol) was added to a solution of freshly prepared lithium diisopropylamide (2.45 mmol) in anhydrous tetrahydrofuran (20.0 mL) at -78 ° C. The resulting mixture was stirred at -78 ° C for 1 hour, and then 6-bromo-4-chloro-7-fluoro-3-nitroquinoline (575 mg, 1.88 mmol) was added. After stirring for another 1 hour at 25 ° C, the reaction was quenched by saturated aqueous ammonium chloride solution (10.0 mL). The resulting mixture was diluted with water (20.0 mL) and separated. The aqueous layer was extracted with ethyl acetate (5 x 30 mL). The combined organic layers were washed with brine (4 x 30.0 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 3% to 9% ethyl acetate / petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (450 mg, crude) as a yellow oil: MS: [(M+1)] + =537.25,539.25.

[0271]

[0272] 8'-Bromo-3-ethyl-7'-fluorospiro[cyclobutane-1,1'-pyrrolo[2,3-c]quinoline]-2'(3'H)-one: A mixture of crude 1-(6-bromo-7-fluoro-3-nitroquinoline-4-yl)-3-ethylcyclobutane-1-carboxylic acid decyl ester (450 mg, 0.84 mmol) and iron powder (327 mg, 5.86 mmol) in acetic acid (8.00 mL) was stirred at ambient temperature for 16 h. The resulting mixture was filtered and the filter cake was washed with tetrahydrofuran (5 x 300 mL). The filtrate was concentrated under reduced pressure. The residue was basified to pH = 8 with saturated aqueous sodium bicarbonate solution. The resulting mixture was extracted with ethyl acetate (5 x 100 mL). The combined organic layers were washed with brine (50.0 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC (DCM / MeOH=15 / 1, v / v) to give the title compound (40.0 mg, 14%) as a yellow solid: 1 H NMR (400 MHz, DMSO-d 6 )δ10.76 (d, J=5.3Hz, 1H), 8.67 (d, J=1.7Hz, 1H), 8.45 (d, J=7.2Hz, 0.4H), 8.39 (d, J=7.4Hz, 0.6H), 7.98 (dd, J=10.1, 3 .4Hz, 1H), 2.88-2.73(m, 2H), 2.55-2.51(m, 2H), 2.29-2.19(m, 1H), 1.78-1.64(m, 2H), 0.99-0.88(m, 3H); MS: [(M+1)] + =349.05,351.05.

[0273]

[0274] 8′-Bromo-3-ethyl-7′-fluoro-3′-methylspiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinolin]-2′(3′H)-one: To a solution of 8′-bromo-3-ethyl-7′-fluorospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinolin]-2′(3′H)-one (35.0 mg, 0.10 mmol) in N,N-dimethylformamide (3.00 mL) was added sodium hydride (5.21 mg, 0.13 mmol, 60% dispersion in mineral oil) at 0° C. under nitrogen atmosphere. The resulting mixture was stirred at 25° C. for 30 minutes, then iodomethane (21.4 mg, 0.15 mmol) was added at 0° C. After stirring for another 40 minutes at 25° C., the reaction was quenched by saturated aqueous ammonium chloride solution (20.0 mL). The resulting mixture was diluted with water (100 mL) and extracted with ethyl acetate (4 x 100 mL). The combined organic layers were washed with brine (4 x 100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC (DCM / MeOH=20 / 1, v / v) to give the title compound (30.0 mg, 83%) as a yellow solid: 1 H NMR (400 MHz, DMSO-d 6 )δ8.90 (d, J=2.9Hz, 1H), 8.49 (d, J=7.4Hz, 0.4H), 8.42 (d, J=7.4Hz, 0.6H), 8.02 (dd, J=10.1, 3.1Hz, 1H), 3.29 (d, J=4 .5Hz, 3H), 2.93-2.75(m, 2H), 2.61-2.52(m, 2H), 2.29-2.21(m, 1H), 1.82-1.67(m, 2H), 0.98-0.89(m, 3H); MS: [(M+1)] + =363.05,365.05.

[0275]

[0276] N-(5-(3-ethyl-7′-fluoro-3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinolin]-8′-yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide: 8′-bromo-3-ethyl-7′-fluoro-3′-methylspiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinolin]-2′(3′H)-one (50.0 mg, 0.14 mmol) was added. ) and N-[2-[2-(isopropylamino)ethoxy]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl]methanesulfonamide (165 mg, 0.41 mmol) in water (0.75 mL) and 1,4-dioxane (3.00 mL) were added sodium carbonate (17.5 mg, 0.17 mmol) and tetrakis(triphenylphosphine)palladium(0) (31.8 mg, 0.028 mmol). After stirring at 80° C. for 2 hours under a nitrogen atmosphere, the resulting mixture was concentrated under reduced pressure. The residue was purified by Prep-TLC (DCM / MeOH=10 / 1, v / v) to give a crude product, which was further purified by reverse phase flash chromatography using the following conditions: column: Spherical C18, 20-40 μm, 120 g; mobile phase A: water (plus 10 mM NH 4 HCO 3 ), mobile phase B: acetonitrile; flow rate: 45 mL / min; gradient (B%): 5% to 25%, 7 min; 25% to 45%, 25 min; 45% to 65%, 8 min; 95%, 5 min; detector: UV 254 nm. The fractions containing the desired product were collected at 17 min and concentrated under reduced pressure to give the title compound (30.0 mg, 40%) as a yellow solid: MS: [(M+1)] + =556.20.

[0277]

[0278] Cis-N-(5-(3-ethyl-7′-fluoro-3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (Example 573) and trans-N-(5-(3-ethyl-7′-fluoro-3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (Example 573) -(2-(Isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (Example 566): The above-mentioned N-(5-(3-ethyl-7′-fluoro-3′-methyl-2′-oxo-2′, 3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′-yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (30.0 mg) was separated by Prep-Chiral-HPLC using the following conditions: Column: Chiralpak IC, 2x 25cm, 5μm; Mobile phase A: hexane / DCM=3 / 1 (plus 0.2% isopropylamine), mobile phase B: EtOH; Flow rate: 20mL / min; Gradient: 30% B, 15min; Detector: UV 220 / 254nm. The desired fractions were collected and concentrated under reduced pressure to give cis-N-(5-(3-ethyl-7′-fluoro-3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′-yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (Example 573, RT1: 10.94 min) (12.8 mg, 43%) as a colorless solid: 1 H NMR (400 MHz, DMSO-d 6 )δ8.87 (s, 1H), 8.32-8.22 (m, 2H), 7.98 (d, J=12.2Hz, 2H), 4.44 (t, J=5.3Hz, 2H), 3.30 (s, 3H), 3.04 (s, 3H), 2.99 (t, J=5.3Hz, 2H), 2.9 4-2.78 (m, 4H), 2.26 (dd, J=12.2, 5.7Hz, 2H), 1.75 (t, J=7.2Hz, 2H), 1.07 (d, J=6.3Hz, 6H), 0.89 (t, J=7.3Hz, 3H); MS: [(M+1)]+=556.25

[0279] And trans-N-(5-(3-ethyl-7′-fluoro-3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′-yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide (Example 566, RT2: 13.63 min) (8.6 mg, 29%) as a colorless solid: 1 H NMR (400 MHz, DMSO-d 6 )δ8.88 (s, lH), 8.34-8.26 (m, 2H), 8.01-7.93 (m, 2H), 4.44 (t, J=5.4Hz, 2H), 3.31 (s, 3H), 3.05 (s, 3H), 3.00 (d, J=5.4Hz, 2H), 2.96-2. 83 (m, 2H), 2.70-2.60 (m, 2H), 1.66 (q, J=7.2, 6.7Hz, 2H), 2.56-2.52 (m, 2H), 1.07 (d, J=6.2Hz, 6H), 0.89 (t, J=7.3Hz, 3H); MS: [(M+1)] + =556.20.

[0280] Compound 574

[0281]

[0282] 1-(5-bromo-3-nitropyridine-2-yl)-N,N-dimethylazetidine-3-amine: To a stirred solution of N,N-dimethylazetidine-3-amine hydrochloride (0.27 g, 2.08 mmol) and 5-bromo-2-chloro-3-nitropyridine (0.49 g, 2.08 mmol) in tetrahydrofuran (40.0 mL) was added diisopropylethylamine (0.67 g, 5.19 mmol) at ambient temperature. The resulting mixture was stirred for 3 hours and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with 3% to 9% ethyl acetate / petroleum ether. The desired fractions were collected and concentrated under reduced pressure to give the title compound (0.50 g, 59%) as a yellow solid: 1 H NMR (400 MHz, CDCl 3 )δ8.37 (d, J=2.2Hz, 1H), 8.29 (d, J=2.2Hz, 1H), 4.20 (ddd, J=10.0, 7.0, 1.2Hz, 2H), 3. 93 (ddd, J=9.9, 5.1, 1.2Hz, 2H), 3.16 (tt, J=7.0, 5.1Hz, 1H), 2.20 (s, 6H); MS: [(M+1)] + =301.00,303.00.

[0283]

[0284] 1- (5- bromo -3- nitropyridine -2- bases) -N, N- dimethylazetidine -3- amine. At ambient temperature, iron powder (11.5g, 206mmol) was added to a solution of 1- (5- bromo -3- nitropyridine -2- bases) -N, N- dimethylpiperidin -4- amine (6.20g, 20.7mmol) in acetic acid (90.0mL). The obtained mixture was stirred at ambient temperature for 2 hours. The obtained mixture was filtered, and the filter cake was washed with tetrahydrofuran (3 x 100mL). The filtrate was concentrated under reduced pressure. The residue was dissolved with saturated sodium carbonate aqueous solution (100mL) and extracted with ethyl acetate (3 x 100mL). The combined organic layer was washed with brine (3 x 100mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 2% to 4% methanol / dichloromethane to give the title compound as a grey solid (5.20 g, 92%): 1 H NMR (400 MHz, CDCl 3 )δ7.74 (d, J=2.0Hz, 1H), 6.93 (d, J=2.0Hz, 1H), 4.19-4.04 (m, 2H), 3.93-3.88 (m, 2H), 3.21-3.14 (m, 1H), 2.24 (s, 6H); MS: [(M+1)] + =271.00, 273.00.

[0285]

[0286] N-(5-bromo-2-(3-(dimethylamino)azetidin-1-yl)pyridin-3-yl)methanesulfonamide: To a stirred solution of 5-bromo-2-[3-(dimethylamino)azetidin-1-yl]pyridin-3-amine (2.10 g, 7.77 mmol) and N,N-4-dimethylaminopyridine (77.0 mg, 0.63 mmol) in pyridine (70.0 mL) was added methanesulfonyl chloride (1.77 g, 15.5 mmol) dropwise at ambient temperature. After stirring for 3 hours under nitrogen atmosphere at ambient temperature, the resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography using the following conditions: Column: Spherical C18, 20-40 μm, 330 g; Mobile phase A: Water (plus 10 mM NH 4 HCO 3), mobile phase B: acetonitrile; flow rate: 65 mL / min; gradient (B%): 5% to 20%, 8 min; 20% to 40%, 20 min; 40% to 95%, 2 min; 95%, 5 min; detector: UV 254 nm. The desired fractions were collected at 19 min and concentrated under reduced pressure to give the title compound (1.40 g, 52%) as a colorless solid: 1 H NMR (400 MHz, CD 3 OD) δ7.92 (d, J=2.2Hz, 1H), 7.61 (d, J=2.2Hz, 1H), 4.27 (dd, J=8.8, 7.2Hz, 2H), 3.9 6(dd, J=9.0, 5.6Hz, 2H), 3.24-3.16(m, 1H), 3.00(s, 3H), 2.20(s, 6H); MS: [(M+1)] + =349.00, 351.00.

[0287]

[0288] N-(2-(3-(dimethylamino)azetidin-1-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methanesulfonamide: To a solution of N-[5-bromo-2-[3-(dimethylamino)azetidin-1-yl]pyridin-3-yl]methanesulfonamide (1.00 g, 2.86 mmol) and 4-bis(pinacolato)diboron (2.18 g, 8.59 mmol) in 1,4-dioxane (30.0 mL) was added potassium acetate (1.12 g, 11.5 mmol) and bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane adduct (351 mg, 0.43 mmol) at ambient temperature. The resulting mixture was stirred at 85 °C for 2 hours under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (plus 10 mM NH 4 HCO 3 ), mobile phase B: acetonitrile; flow rate: 65 mL / min; gradient (B%): 5% to 20%, 7 min; 20% to 40%, 12 min; 40% to 95%; 2 min; 95%, 5 min; detector: UV 254 nm. The desired fractions were collected at 20 min and concentrated under reduced pressure to give the title compound (800 mg, 71%) as a yellow solid: 1 H NMR (400 MHz, DMSO-d 6)δ8.78 (s, 1H), 8.18 (d, J = 1.5Hz, 1H), 7.50 (d, J = 1.6Hz, 1H), 4.19 (t, J = 8.0Hz, 2H), 3.93 (dd , J=8.9, 5.0Hz, 2H), 3.12-3.04(m, 1H), 2.99(s, 3H), 2.09(s, 6H), 1.27(s, 12H); MS: [(M+1)] + =397.20.

[0289]

[0290] N-(2-(3-(dimethylamino)azetidin-1-yl)-5-(3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinolin]-8′-yl)pyridin-3-yl)methanesulfonamide: 8-bromo-3-methyl-2,3-dihydrospiro[cyclobutane-1,1-pyrrolo[2,3-c]quinolin]-2-one (320 mg, 1.01 mmol) and N-[2-[3- To a solution of [(dimethylamino)azetidine-1-yl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl]methanesulfonamide (600 mg, 1.51 mmol) in 1,4-dioxane (13.0 mL) and water (2.00 mL) was added sodium carbonate (160 mg, 1.51 mmol) and tetrakis(triphenylphosphine)palladium(0) (175 mg, 0.15 mmol). After stirring at 85 ° C for 2 hours under a nitrogen atmosphere, the resulting mixture was concentrated under reduced pressure. The residue was purified by Prep-TLC (DCM / MeOH=8 / 1, v / v) to give the title compound as a light yellow solid: 1 H NMR (400 MHz, DMSO-d 6 )δ9.07 (s, 1H), 8.80 (s, 1H), 8.58 (s, 1H), 8.33 (s, 1H), 8.13 (d, J = 8.9Hz, 1H), 7.95 (d, J = 9.2Hz, 1H), 7.91 (d, J = 2.1Hz, 1H), 4. 28-4.21(m, 2H), 4.02-3.95(m, 2H), 3.30(s, 3H), 3.14(s, 4H), 2.98-2.90(m, 2H), 2.60-2.52(m, 4H), 2.13(s, 6H); MS: [(M+1)] + =507.20.

[0291]

[0292] N-(2-(3-(dimethylamino)azetidin-1-yl)-5-(3′-methyl-2′-oxo-2′,3′-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′yl)pyridin-3-yl)methanesulfonamide hydrochloride: N-(2-(3-(dimethylamino)azetidin-1-yl)-5-(3′-methyl-2′-oxo-2′,3 A solution of 1,2-dihydrospiro[cyclobutane-1,1′-pyrrolo[2,3-c]quinoline]-8′-yl)pyridin-3-yl)methanesulfonamide (350 mg, 0.69 mmol) in dilute aqueous hydrochloric acid (115 mL, 0.69 mmol, 0.006 M) and acetonitrile (20.0 mL) was directly lyophilized to give the title compound (375 mg, 100%) as an orange solid: 1 H NMR (400 MHz, DMSO-d 6 )δ10.77 (s, 1H), 9.24 (s, 1H), 8.83 (s, 1H), 8.64 (d, J = 2.1Hz, 1H), 8.35 (s, 1H), 8.17 (d, J = 8.9Hz, 1H), 8.02-7.94 (m, 2H), 4.50-4.41 (m, 2H) , 4.39-4.32 (m, 2H), 4.27-4.16 (m, 1H), 3.31 (s, 3H), 3.18 (s, 3H), 2.99-2.88 (m, 2H), 2.82 (d, J=4.1Hz, 6H), 2.63-2.52 (m, 4H); MS: [(M+1)] + =507.20.

[0293] On-target and off-target inhibitory activity

[0294] ATM in Cell Western Assay:

[0295] In the morning, MCF-7 breast cancer cells were placed in 384-well plates (Corning, #356663) at a density of 10,000 cells / well, 25 μL cells per well. The next day, the compound was added to the plate using a pin tool (Echo 550) to a final concentration of 1 μM via 3-fold serial dilutions (a total of 10 doses). Then, etoposide (Sigma, #E1383) was added to a final concentration of 100 μM. The plate was incubated at 37 ° C for 1 h, and at ambient temperature, the cells were fixed for 20 minutes by adding 25 μL of fixative solution (8% paraformaldehyde). The cells were permeabilized by washing 5 times with 1X PBS (phosphate buffered saline) containing 0.1% Triton X-100; each wash was 5 minutes long. Cells were blocked for 1.5 hours at ambient temperature in a 384-well plate with shaking by adding 50 μL of Odyssey blocking buffer (LI-COR, #927-40000). The blocking buffer was then removed and 20 μL of anti-pKAP1 antibody (Bethyl Laboratories, #A300-767A) (1 / 2000) solution was added to each well of the 384-well plate. The plate was incubated overnight at 4°C and then washed 5 times with 1X PBST (1X PBS containing 0.1% Tween-20). A secondary antibody (IRDye 800CW goat anti-rabbit IgG, LI-COR, #926-32211) (1 / 5,000) solution containing DNA stain DRAQ5 (CST, #4084L) (1 / 5,000) (20 μL) was added to each well of the plate and the plate was incubated for 1 hour in the dark with gentle shaking. Wash cells 5 times with 1X PBST (1X PBS containing 0.1% Tween-20) at ambient temperature with gentle shaking in the dark. After the last wash, remove the wash solution, invert the plate on a thin paper towel and centrifuge at 1000 rpm for 1 min to absorb all the wash buffer. Clean the bottom of the plate with a damp lint-free paper. Scan the plate immediately using ODYSSEY CLx (LI-COR).

[0296] DNA-PK ELISA:

[0297] On the first day, each well was treated with 0.1 M Na 2 CO 3 / NaHCO 33 μg of GST-p53 was diluted in 4% PBS (pH 9.6) to coat a 96-well plate (ThermoFisher, catalog number: 442404) with GST-p53 (1-101) peptide (purified by Pharmaron, BCS department). The plate was incubated overnight at 4°C. The next day, the coating buffer was removed and the plate was washed twice with PBST (1X PBS containing 0.1% Tween-20). DNA-PK enzyme solution (Invitrogen, #PR9107A; final DNA-PK concentration: 0.1 μg / mL) was then added. The compound was serially diluted to a final maximum concentration of 100 nM (3-fold serial dilutions, a total of 10 doses), and ATP solution (final ATP concentration: 20 μM) was added to the plate. The plate was incubated at 25°C for 1 hour. The plate was washed three times with PBST (1X PBS containing 0.1% Tween-20) and blocked overnight at 4°C with a solution of PBST and 1% BSA. On the third day, the plate was washed 4 times with PBST (1X PBS containing 0.1% Tween-20). Anti-phospho-p53 primary antibody (cell signaling Technology, #9286, Phospho-p53 (Ser15) (16G8) mouse mAb) (1 / 1000) was added to each well. The plate was sealed, incubated at 37°C for 1h, and washed 4 times with PBST (1X PBS containing 0.1% Tween-20). HRP-linked secondary antibody (Cell signaling Technology, #7076, anti-mouse IgG, HRP-linked antibody) (1 / 1000) (100 μL) was added to each well. The plate was sealed with tape, incubated at 37°C for 30min, and washed 4 times with PBST (1X PBS containing 0.1% Tween-20). At this time, 100 μL of TMB (Cell signaling Technology, #7004) substrate was added to each well. The plate was sealed with tape and incubated for 10 min at 37° C. Stop solution (Cell signaling Technology, #7002) (100 μL) was added to each well, and the plate was monitored for absorbance at 450 nm.

[0298] mTOR biochemical assay:

[0299] The mTOR kinase reaction was performed in a low volume 384-well plate in a volume of 10 μL. Typically, PerkinElmer model 6008260 plates were used. The composition of the 1x kinase reaction buffer was: 50 mM HEPES pH 7.5, 0.01% Tween 20, 1 mM EGTA, 10 mM MnCl 2and 2mM DTT. A solution of mTOR enzyme (ThermoFisher, #PR8683B; final mTOR concentration: 0.5μg / mL) was added, and the compound was serially diluted to a final maximum concentration of 100nM (3-fold serial dilutions, a total of 10 doses). GFP-4E-BP1 (final concentration: 0.4μM) and ATP solution (final ATP concentration: 3μM) were added to a 384-well plate. The plate was incubated at 25°C for 1 hour, and 10μL of EDTA solution (20mM) and Tb-labeled anti-p4E-BP1 antibody (4nM) in TR-FRET dilution buffer were added to each well. The plate was sealed, incubated at 25°C for 30min, and incubated for LanthaScreen TM Read on a plate reader configured for TRFRET.

[0300] PI3Kα and PI3Kδ biochemical assays:

[0301] PI3Kα and PI3Kδ kinase reactions were performed in low volume 384-well plates in 5 μL volumes. Typically, PerkinElmer model 6008280 plates were used. 1x kinase reaction buffer consisted of: 50 mM HEPES pH 7.5, 3 mM MgCl 2 , 0.03% CHAPS, 1 mM EGTA, 100 mM NaCl and 2 mM DTT. PI3Kα (ThermoFisher, #PV4788; final PI3Kα concentration: 120 ng / mL) or PI3Kδ enzyme solution (ThermoFisher, #PV6451; final PI3Kδ concentration: 250 ng / mL) was added to the plate, the compound was serially diluted to a final maximum concentration of 100 nM (3-fold serial dilutions, a total of 10 doses), and PIP2:3PS (final concentration: 10 μg / mL) and ATP solution (final ATP concentration: 10 μM) were added to the 384-well plate. The plate was incubated at 25°C for 1 hour. ADP-Glo ​​reagent buffer (5 μL) was added to each well. The plate was sealed and incubated at 25°C for 40 min. ADP-Glo ​​detection buffer (10 μL) was added to each well, and the plate was incubated at 25° C. for 40 min and read on a plate reader configured for Luminescence.

[0302] In vitro hERG inhibition assay:

[0303] The hERG coding sequence in the Tet-regulated expression vector pT-Rex-DEST30 was transfected into cells expressing the Tet repressor (T-Rex TMHEK293) cells that can be induced to express high levels of hERG channels to generate hERG-T-REx TM HEK 293 cells (Invitrogen, K1236) were cultured in a medium containing 85% DMEM, 10% dialyzed FBS, 0.1 mM NEAA, 25 mM HEPES, 100 U / mL penicillin-streptomycin, 5 μg / mL blasticidin, and 400 μg / mL geneticin. TrypLE TM The cells were split approximately 3 times using 1 μg / mL doxycycline (Sigma, D9891) and maintained at a confluency of approximately 40% to approximately 80%. Prior to the assay, the cells were induced for 48 hours with 1 μg / mL doxycycline (Sigma, D9891). On the day of the experiment, the induced cells were resuspended and incubated at 5 × 10 5 Cells / 6 cm cell culture dish were plated on coverslips. hERG channel-mediated currents were acquired by a manual patch clamp recording system equipped with an amplifier (HEKA, EPC10 and Molecular Devices, multiclamp 700B) and an inverted phase contrast microscope (Olympus, IX51 / 71 / 73). Glass pipettes were prepared by a micropipette puller (Sutter, P97 and Narishige, PC-10) and qualified by pipette resistance in the range of 2-4 MΩ. The internal pipette solution was 140 mM KCl, 2 mM MgCl 2 , 10 mM EGTA, 5 mM MgATP, and 10 mM HEPES (pH adjusted to 7.35 with KOH), and the external buffer was 132 mM NaCl, 4 mM KCl, 3 mM CaCl 2 , 0.5 mM MgCl 2 , 11.1mM glucose and 10mM HEPES (pH adjusted to 7.35 with NaOH). Maintain full-cell configuration and continuously monitor access resistance (<15MΩ). The hERG current is induced by depolarizing the membrane to +30mV for 4.8 seconds, and the voltage is set back to -50mV for 5.2 seconds to remove inactivation and measure the deactivated tail current. The maximum amount of tail current size is used to measure the hERG current amplitude. In order to evaluate the hERG inhibitory effect, blank vehicle and test articles are perfused into cells by a liquid perfusion system (ALA, VM8 gravity flow delivery system) under the full-cell recording configuration. For dose response determination, the test article is cumulatively applied to the cell from low concentration to high concentration. Positive control (dofetilide) was used in the experiment to ensure the performance of the cell and operation as the main part of the method validation. The hERG current inhibition percentage is fitted for the dose concentration to establish a dose response curve and determine the IC50 .

[0304] In some embodiments, the compounds of the invention are selected from the group consisting of the compounds listed in the following table.

[0305] Table 1 Measurement results

[0306]

[0307]

[0308] Clonogenicity and in vivo efficacy assays

[0309] method

[0310] Cell culture. MCF-7 human breast cancer cell line and HCT116 p53 wild-type and HCT116 p53- / - cell lines were obtained from ATCC. MCF7 and HCT116 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM (Gibco #11995-065). MDA-MB-231 human triple-negative breast cancer cell line and FADU human head and neck squamous cell carcinoma cell line were obtained from Charles River Laboratories (Morrisville, NC). MDA-MB-231 cells were cultured in RPMI1640 medium (Gibco, 11875-093). FADU cells were cultured in RPMI1640 medium (Gibco, 11875-093) supplemented with 1 mM sodium pyruvate (Gibco, 11360-070), 1X MEM non-essential amino acids (Gibco, 11140-050) in minimal essential medium α (MEMα) (Gibco, 12571-063). All cell lines were supplemented with 10% fetal bovine serum (FBS) (Corning, 35-010-CV) and 1X antibiotic-antimycotic (Gibco #15240-062) and grown at 37°C in 5% CO2. All cell lines were authenticated by short tandem repeat profiling and mycoplasma testing was negative.

[0311] Antibodies. Antibodies recognizing phosphorylated / activated ATM (S1981, #ab81292) and DNA-PKcs (S2056, #ab18192) were purchased from Abcam Biotechnology (Cambridge, MA). Antibodies against ATM (#A1106) and DNA-PKcs (#ab1832) were from Sigma and Abcam, respectively. Phospho-KAP1 (S824, #A300-767A) and KAP1 (A700-014) antibodies were from Bethyl Labs (Montgomery, TX). Phospho-TBK1 (#5483), cGAS (#15102), and pSTING (S366, #19781S) antibodies were from Cell Signaling Technology (CST) (Danvers, MA). STING (PA5-23381) antibody was from ThermoFisher Scientific (Waltham, MA), and actin (#A5316) antibody was from Sigma. All antibodies were diluted 1:1000, except for actin, which was diluted 1:3000 in 1X TBST (1X TBS-10X TBS, (Corning, 46-012-CM), 0.05% Tween-20 (P7949) Sigma and 1% BSA. The secondary antibodies used were (a) Li-Cor method - goat anti-rabbit (926-3221) and goat anti-mouse (926-68020) Li-Cor; (b) ECL method: anti-rabbit IgG, HRP-linked (#7074S), CST.

[0312] Immunoblotting. Cells were pretreated with 20, 100, 500 or 1000 nM of compound 569 or DMSO for 30 min and then irradiated with 0 or 10 Gy (XRAD 160, Precision X Ray). Figure 1 As shown. Figure 3 Cells were pretreated for 30 min with 1 μM compound 569, 1 μM ATMi (AZD0156), 1 μM DNA-PKi (peposertib) as single agents or DMSO or with ATMi and DNA-PKi in combination (0.5 μM each or 1 μM each), and then irradiated with 0 or 10 Gy (XRAD160, Precision X Ray), as described above. Figure 1One hour after IR, cells were incubated in RIPA buffer (50 nM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% CA-630 (Sigma, 18896) for 20 min. Cell lysates were centrifuged at 13,200 g for 10 min at 4°C and analyzed using the BCA assay (Pierce TM The protein content of the supernatant was analyzed by BCA protein assay kit, catalog number 23227, ThermoFisher Waltham, MA02451) and equal amounts of protein / lane were used for subsequent immunoblot analysis. The protein was denatured by adding NuPAGE LSD sample buffer [4X] (Invitrogen, NP0007) containing a final concentration of 2.5% β-mercaptoethanol, and the sample was boiled for 5 minutes. The sample was loaded into NuPAGE 3-8% Tris-acetate protein gel (Invitrogen, EA03785BOX) and electrophoresed with 1X Tris-acetate SDS running buffer (Invitrogen, LA0041), or loaded into NuPAGE 4-12% Bis-Tris protein gel (Invitrogen, NP0336BOX) and electrophoresed with 1X MOPS SDS running buffer (Invitrogen, NP0001).

[0313] Protein was transferred to nitrocellulose membrane (Amersham, 10600003) overnight. The membrane was blocked with 5% skim milk powder in 1X TBST for 1 hour and incubated with primary antibody at 4°C overnight. The membrane was washed with 1X TBST for 4x 10min at RT, then incubated with secondary antibody for 1 hour at RT, followed by 3x 10min washing with 1X TBST at RT. The membrane was visualized using ODYSSEY CLX system or ECL (ECL blotting substrate, Pierce, 32209, 34075, 34095).

[0314] Clonogenic survival assay. This assay was performed with compound 569. Cells were plated in 6-well plates at different densities: 250 cells for no IR control; 5000 cells for 2Gy IR; and 10000 cells for 4Gy IR, and cultured overnight. The next day, cells were pre-incubated with 100, 250, 500 or 1000nM of compound 569 or DMSO for 30 minutes and then exposed to increasing doses of IR (0, 2 or 4Gy). After IR, cells were incubated continuously for 5h with DMSO or compound 569, after which the culture medium was removed and the cells were washed with PBS. Cells were cultured in complete medium in the absence of inhibitors for 9 days. The cells were then stained (PBS, 0.0037% v / v formaldehyde, 0, 1% crystal violet), rinsed with water and dried.

[0315] Tumor studies. Female NCr nu / nu mice (Crl:NU(NCr)-Foxnlnu, strain 490) were obtained from Charles River at 5-6 weeks of age. All animal procedures used in these studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Duke University.

[0316] FADU or MDA-MB-231 cells were harvested during logarithmic phase growth for in vivo implantation. Resuspended cells were washed three times in phosphate-buffered saline (PBS) before preparing working dilutions in PBS. FADU cells were diluted to 1x10 7 cells / mL, and dilute MDA-MB-231 cells to 5 x 10 7 100 μL of cell suspension was injected subcutaneously in the right flank of each mouse. Tumor growth was monitored until the tumor reached at least 100 mm 3 The target volume was reached, at which point the mice were randomized into four treatment groups.

[0317] After implantation of the tumor cell lines, mice were monitored weekly for tumor progression. After detection, tumors were measured in two dimensions using a digital caliper. Tumors were measured when they reached at least 100 mm 3 Treatment was initiated after 4-6 months and measurements were taken 2 to 3 times per week after enrollment in one of the treatment groups. The study endpoint was defined as a five-fold increase in tumor volume on treatment.

[0318] The vehicle used in this study was 0.5% (hydroxypropyl) methylcellulose (HPMC) and 0.2% Tween 80 dissolved in deionized water. The pH of the vehicle was adjusted to 7.0-8.0 and the vehicle was stored at 4°C. On each day of in vivo treatment, the appropriate amount of the test compound was added to the vehicle at room temperature. If necessary, the mixture was vortexed to produce a final concentration of 1.6 mg / mL.

[0319] Pharmacodynamic (PD) Assay. Tumor studies were performed as follows. Mice bearing FADU or MDA-231 tumors were pre-administered with vehicle alone or compound 569 alone (3, 6, and 10 mg / kg). Tumors were then irradiated with 10 Gy 45 minutes after administration of vehicle or compound 569 or not irradiated (vehicle and compound 569, 10 mg / kg). Tumors were harvested 1 h after irradiation.

[0320] Tissue homogenates for pharmacodynamic analysis were collected and processed as follows. Tumor tissues from FADU or MDA-MB-231 tumors were snap frozen in liquid nitrogen and pulverized on dry ice using a tissue grinder. A portion of the tumor powder was transferred to a microtube and then 500 μL of RIPA buffer [50 nM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% CA-630 (Sigma, I8896)]. To 10 ml of RIPA buffer, 2 tablets were added, each containing a protease inhibitor and a phosphatase inhibitor (Roche, #04693159001), 200 μL 0.1 M PMSF and 160 μL aprotinin (Sigma, A6279). The suspension was homogenized on ice with a pellet pestle motor (KONTES) and incubated on ice for 30 minutes. The cell lysate was centrifuged at 13,200 rpm for 15 min at 4°C and the supernatant was transferred to a new tube and analyzed for protein concentration using a BCA kit (Pierce, 23227), followed by immunoblotting.

[0321] In vivo tumor growth delay studies were performed as follows. After implantation of the tumor cell lines, mice were monitored weekly for tumor progression. 3 After tumor progression, tumor-bearing mice were divided into one of four groups, with five mice in each group. Tumor volumes ranged between 100 and 500 mm 3Between. Vehicle or compound 569 (10 mg / kg) was administered once a day by oral gavage for three consecutive days (qd×3), alone or in combination with focal radiation. The delivery volume of vehicle or compound 569 was adjusted based on the weight of each animal. Mice assigned to the radiotherapy group received 3Gy every day for three consecutive days (qd×3) 45 minutes after the administration of vehicle or compound 569. Mice were anesthetized with isoflurane during radiotherapy, and radiotherapy was performed using an X-RAD 225Cx small animal image-guided irradiator (Precision X-Ray). By fluoroscopy, the irradiation field of 40mmx 40mm was centered on the leg carrying the tumor. Mice were irradiated with parallel relative front and back fields of 225kVp, 13mA X-rays at an average dose rate of 300cGy / min using a 0.3mm Cu filter.

[0322] Group 1 mice received vehicle, qd×3.

[0323] The mice in group 2 received 10 mg / kg of compound 569, qd×3.

[0324] Mice in group 3 received vehicle 45 min before 3 Gy focal irradiation, qd×3.

[0325] Mice in group 4 received 10 mg / kg of compound 569 qd×3 45 minutes before 3 Gy focal irradiation.

[0326] Statistical and graphical analysis of in vivo data. Prism (GraphPad) was used for graphical representation. SAS 9.4 and R 3.6.1 were used for statistical analysis. Tumor growth rates were compared among the four treatment groups. To adjust for different tumor sizes at the start of treatment, relative tumor volumes were calculated by dividing the tumor volume at each time point by the baseline volume. Line graphs were used to show median relative tumor volumes over time ( Figure 6 and 8 When an animal was withdrawn from the study due to tumor quintuples, the final tumor volume recorded for that animal was included in the data used to calculate the median volume at subsequent time points. Kaplan-Meier plots were used to display the percentage of mice in each treatment group that remained in the study over time ( Figure 7 and 9 ). Mice found dead before reaching the five-fold endpoint were right-censored. The log-rank test was used to assess differences in five-fold survival between the four treatment groups. P values ​​less than 0.05 were considered statistically significant.

[0327] result

[0328] Figure 1The image of the immunoblot from the in vitro experiment of assessing the effect of compound 569 on MCF7 cells with or without radiation is shown. The immunoblot shows the inhibition of the radiation-induced autophosphorylation of ATM and DNA-PK kinases and the radiation-induced phosphorylation of KAP1 (ATM substrate) by compound 569 in tumor cells. MCF7 (a kind of human breast cancer cell line) was pretreated with compound 569 at a concentration of 20, 100, 500 and 1000nM for 30 minutes. DMSO was used as a negative control. The cells were then exposed to ionizing radiation (IR) of 0 or 10Gy, and after one hour, the harvested cells were used for immunoblot analysis. Compound 569 showed effective dose-dependent inhibition of the radiation-induced phosphorylation (both autophosphorylation events) of DNA-PKcs at Ser2056 and ATM at Ser1981 and the radiation-induced phosphorylation of ATM substrate KAP1 (ser824), proving that compound 569 inhibits both DNA-PK and ATM kinases in cells.

[0329] Figure 2 The radiosensitization properties of compound 569 in an in vitro clonogenic survival assay were demonstrated. MCF7 cells were pretreated with vehicle or compound 569 at 100, 250, 500, and 1000 nM for 30 minutes and then irradiated with increasing doses of IR (0, 2, and 4 Gy). After 5 h, the culture medium was removed and fresh culture medium without inhibitor was added to the cells and cultured for 9 days before fixation, staining, and colony counts. In the absence of IR, a 5-hour exposure of cells to compound 569 in MCF7 cells induced a significant degree of radiosensitization without evidence of cytotoxicity ( Figure 2 , Table 1).

[0330] Figure 3It is an immunoblot showing that compound 569, AZD0156 (selective ATM inhibitor; ATMi), peposertib (selective DNA-PK inhibitor; DNA-PKi) and the combination of AZD0156 and peposertib (Ai+Di) induce TBK1 phosphorylation in HCT116 cells expressing wild-type p53 or HCT116 cells with negative p53 expression. Phosphorylation of TBK1 is a marker of type I interferon response activation and is associated with enhanced tumor response to immune checkpoint blockade therapy. Dual inhibition of ATM and DNA-PKcs by combining two selective chemical inhibitors (AZD0156 and peposertib) leads to deeper activation of TBK1 than using any selective inhibitor alone. Similarly, exposing cells to compound 569 (i.e., a dual inhibitor of ATM and DNA-PKcs) is a more effective TBK1 phosphorylation activator than any single-targeted kinase inhibitor and is independent of p53 status. Activation of TBK1 by 569 and dual inhibition of ATM and DNA-PKcs appear to be independent of cGAS and phospho-STING induction.

[0331]

[0332] In order to evaluate the pharmacodynamic activity of compound 569 in vivo, FADU and MDA-MB-231 cells were grown as tumor xenografts in nude mice. Vehicle or compound 569 was administered alone or in combination with focal radiation by oral gavage. Vehicle or compound 569 (3, 6 and 10 mg / kg) was given to mice. Then the tumor received focal irradiation of 10 Gy or was not irradiated (0 Gy, vehicle and 0 Gy+10 mg / kg compound 569) after 45 minutes. Tumors were collected 1 h after radiation. Immunoblotting of lysates from these tumors showed radiation-induced pDNA-PK autophosphorylation and radiation-induced KAP1 (downstream target of ATM) phosphorylation and the dose-dependent inhibition of those phosphorylation events in FADU and MDA231 tumors treated with radiation plus compound 569. At 10 mg / kg, the tumor levels of radiation-induced DNA-PK autophosphorylation and radiation-induced KAP1 phosphorylation were significantly inhibited by approximately 55% and >80%, respectively ( Figure 4 and 5 , Table 2-5). Basal phosphorylation of DNA-PK was also inhibited by compound 569 in tumors.

[0333] Table 1 Clonogenicity determination of compound 569 in MCF7 cells (see Figure 2 )

[0334]

[0335] Table 2 Quantification of pDNA-PK / DNA-PK in FADU tumors

[0336] treat pDNA-PK / DNA-PK Standard Deviation No radiation, medium 1.14 0.263 No radiation, 10mg / kg compound 569 0.343 NA 10 Gy, vehicle 1 0.0565 10Gy, 3mg / kg compound 569 0.938 0.181 10Gy, 6mg / kg compound 569 0.658 0.126 10 Gy, 10 mg / kg compound 569 0.476 0.188

[0337] Table 3 Quantification of pKAP1 / KAP1 in FADU tumors

[0338] treat pKAP1 / KAP1 Standard Deviation No radiation, medium 0.00505 0.000265 No radiation, 10mg / kg compound 569 0.00595 NA 10 Gy, vehicle 1 0.0616 10Gy, 3mg / kg compound 569 0.0903 0.005 10Gy, 6mg / kg compound 569 0.0982 0.0741 10 Gy, 10 mg / kg compound 569 0.156 0.171

[0339] Table 4 Quantification of pDNA-PK / DNA-PK in MDA-231 tumors

[0340] treat pDNA-PK / DNA-PK Standard Deviation No radiation, medium 0.648 0.0948 No radiation, 10mg / kg compound 569 0.223 NA 10 Gy, vehicle 1 0.231 10Gy, 3mg / kg compound 569 0.889 0.414 10Gy, 6mg / kg compound 569 0.670 0.278 10 Gy, 10 mg / kg compound 569 0.454 0.116

[0341] Table 5 Quantification of pKAP1 / KAP1 in MDA-231 tumors

[0342] treat pKAP1 / KAP1 Standard Deviation No radiation, medium 0.00357 0.000826 No radiation, 10mg / kg compound 569 -0.00319 NA 10 Gy, vehicle 1 0.114 10Gy, 3mg / kg compound 569 0.327 0.158 10Gy, 6mg / kg compound 569 0.0130 0.00879 10 Gy, 10 mg / kg compound 569 0.00901 0.00242

[0343] FADU tumors. Mice in Group 1 received vehicle, qd x 3. Aggregate tumor growth in this group was progressive. The median time to endpoint was 14 days, with a range of 11-14 days ( Figure 6 and 7 ). Mice in Group 2 received a 10 mg / kg dose of Compound 569, qd×3. The median time to endpoint was 14 days, ranging from 11 to 17 days. The tumor growth curve and quintuple-free survival of Group 2 animals were almost identical to those of Group 1 ( Figure 6 and 7 ). Mice in Group 3 received vehicle and a focal radiation dose of 3 Gy, delivered 45 minutes after vehicle administration. Both vehicle and radiation were qd×3. One animal was found dead on Day 16 due to unknown reasons. The median time to endpoint was 21 days, ranging from 19 to 25 days. Overall survival was significantly improved compared with Groups 1 and 2 (P<0.01, log rank). The delay in aggregate tumor growth and the increase in five-fold survival in Group 3 relative to Groups 1 and 2 are shown in Figure 6 and 7 Mice in Group 4 received Compound 569 and a focal radiation dose of 3 Gy, delivered 45 minutes after compound administration. Compound 569 and radiation were both administered qd×3. The median time to endpoint was 42 days, ranging from 31 to 51 days. Overall survival was significantly improved compared to Groups 1, 2, and 3 (P<0.01, log rank). The delay in aggregate tumor growth and the increase in five-fold survival in Group 4 relative to all other groups are shown in Figure 6 and 7 All treatments were well tolerated.

[0344] MDA-MB-231 tumors. Mice in Group 1 received vehicle, qd×3. The median time to endpoint was 12 days, ranging from 12 to 17 days. Mice in Group 2 received a 10 mg / kg dose of Compound 569, qd×3. The median time to endpoint was 17 days, ranging from 12 to 22 days. The tumor growth curve and quintuple-free survival of Group 2 animals were almost identical to those of Group 1 ( Figure 8 and 9 ). Mice in Group 3 received vehicle and a focal radiation dose of 3 Gy delivered 45 minutes after vehicle administration. Both vehicle and radiation were given qd×3. The median time to endpoint was 22 days, with a range of 16 to 22 days. Overall survival was significantly improved compared to Group 1 animals treated with vehicle alone (P<0.01, log rank). The five-fold increase in survival in Group 3 relative to Groups 1 and 2 is shown in Fig. 9 Mice in Group 4 received 10 mg / kg of Compound 569 and a focal radiation dose of 3 Gy delivered 45 minutes after compound administration. Compound 569 and radiation were both qd×3. One animal was found dead on day 35 due to unknown reasons. The median time to endpoint was 43 days, ranging from 37 to 58 days. The overall survival rate was significantly improved compared with Groups 1, 2, and 3 (P<0.01, log rank). The delay in aggregate tumor growth and the increase in five-fold survival in Group 4 relative to all other groups are shown in Figure 8 and 9 All treatments were well tolerated.

[0345] Other Implementations

[0346] Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, it will be apparent to those skilled in the art that various modifications may be made to the described manner of the invention within the scope of the invention.

[0347] Other embodiments are within the claims.

Claims

1. A compound of formula (I): or a pharmaceutically acceptable salt thereof, in Y is CHR 5 or NR 6 ; Z is CH, CR 3 or N; n is 0, 1, 2, or 3; R 1 Yes-OLN(R 7 )2 or an optionally substituted four-membered saturated N-heterocyclic group; R 2 It is C 1-3 alkyl; Each R 3 is independently halogen or optionally substituted C 1-3 alkyl; R 4 is optionally substituted alkyl; R 5 is hydrogen, optionally substituted C 1-3 Alkyl or benzyloxy; R 6 is an optionally substituted C 1-3 alkyl; Each R 7 is independently H or optionally substituted C 1-3 alkyl; and L is optionally substituted ethylene.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein n is 1.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (IA): or a pharmaceutically acceptable salt thereof.

4. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein R 3 It's a halogen.

5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein the halogen is fluorine.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein n is 0.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (IB): or a pharmaceutically acceptable salt thereof.

8. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein R 1 Yes-OLN(R 7 )2.

9. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein one R 7 is H, and the rest are R 7 Optionally C 1-3 alkyl.

10. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein at least one R 7 It is isopropyl.

11. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein R 2 It is methyl, ethyl or isopropyl.

12. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein R 2 It's methyl.

13. A compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, wherein R 4 It's methyl.

14. The compound of any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, wherein Y is CHR 5 .

15. The compound of any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, wherein R5 is hydrogen.

16. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, wherein R 5 is an optionally substituted C 1-3 alkyl.

17. A compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof, wherein R 5 It is benzyloxy.

18. A compound as claimed in any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, wherein Y is NR 6 .

19. A compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, wherein R 6 is an optionally substituted C3 alkyl group.

20. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 18, wherein R 6 It is isopropyl.

21. A compound selected from the group consisting of: and pharmaceutically acceptable salts thereof.

22. A compound having the following structure: or a pharmaceutically acceptable salt thereof.

23. A compound having the following structure: or a pharmaceutically acceptable salt thereof.

24. A compound having the following structure: or a pharmaceutically acceptable salt thereof.

25. A pharmaceutical composition comprising a compound according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

26. A method for treating a tumor disease, comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24 or a pharmaceutical composition according to claim 25 to a patient in need thereof.

27. The method of claim 26, wherein the patient is receiving radiation therapy.

28. The method of claim 27, wherein the compound or the pharmaceutical composition is administered to the patient concurrently with the radiation therapy.

29. The method of claim 27, wherein the compound or the pharmaceutical composition is administered to the patient prior to radiation therapy.

30. The method of claim 27, wherein the compound or the pharmaceutical composition is administered to the patient following radiation therapy.

31. The method of any one of claims 27 to 30, wherein the radiation therapy comprises external, internal, brachytherapy, or systemic exposure.

32. The method of any one of claims 27 to 31, wherein the radiation therapy comprises administering an antibody radionuclide conjugate.

33. The method of any one of claims 26 to 32, wherein the patient is receiving an anti-tumor agent.

34. The method of claim 33, wherein the anti-tumor agent is cisplatin, oxaliplatin, carboplatin, valrubicin, idarubicin, calicheamicin, or a PARP inhibitor.

35. The method of claim 34, wherein the anti-tumor agent is an anti-tumor biological agent or an anti-tumor immunotherapeutic agent.

36. The method of any one of claims 33 to 35, wherein the compound or the pharmaceutical composition is administered to the patient simultaneously with the anti-tumor agent.

37. The method of any one of claims 33 to 35, wherein the compound or the pharmaceutical composition is administered to the patient prior to the anti-tumor agent.

38. The method of any one of claims 33 to 35, wherein the compound or the pharmaceutical composition is administered to the patient after the anti-tumor agent.

39. The method of any one of claims 26 to 38, wherein the tumor disease is brain cancer, bladder cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gastrointestinal stromal tumors, gastric cancer, head and neck cancer, cheek cancer, oral cancer, hepatocellular carcinoma, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, nasopharyngeal carcinoma, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, salivary gland cancer, sarcoma, testicular cancer, urothelial carcinoma, vulvar cancer, or Wilm's tumor.

40. The method of any one of claims 26 to 38, wherein the tumor disease is breast cancer, lung cancer, head and neck cancer, pancreatic cancer, colorectal cancer, glioblastoma, hepatocellular carcinoma, cholangiocarcinoma, metastatic liver lesions, melanoma, osteosarcoma, soft tissue sarcoma, endometrial cancer, cervical cancer, prostate cancer or Merkel cell carcinoma.

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