EA042574B1 - COMPOUND BASED ON QUINOLINE OR QUINAZOLINE AND ITS APPLICATION - Google Patents

Description

Technical field

The present invention relates to the field of medical chemistry technology, in particular to a quinoline or quinazoline-based compound and methods of using the same.

Background of the invention

AXL is a member of the class of receptor tyrosine kinases and belongs to the TAM family of receptor tyrosine kinases, which also includes two other members: Mer and Tyro3. For the first time, TAM representatives were found in tumor cells, in which overexpression and ectopic expression are closely associated with immunoregulation and tumor proliferation, growth, and migration. AXL was isolated from patients with chronic myeloid leukemia and patients with chronic myeloproliferative disease in 1988. AXL is widely expressed in the brain, immune cells, platelets, endothelial cells, skeletal muscle, heart, liver, kidney and other tissues. Vitamin K-dependent protein kinase Gas6 (growth arrest protein 6) is the most widely studied AXL ligand found to date, and other ligands in the TAM family include protein S, Tubby, Tulp-1 and Galectin-3. Members of the TAM family are characterized by a similar protein structure, which mainly consists of three parts: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains two N-terminal immunoglobulin-like Ig regions and two repetitive fragments of fibronectin III (FNIII). Gas6, after combining with the extracellular domain of AXL, induces dimerization of AXL, initiating transautophosphorylation of the intracellular domain, thereby activating the intracellular signal transduction pathway and regulating a number of physiological activities, such as regulation of cell growth and proliferation through the Src/MAPK/ERK pathway, stimulation of the expression of anti-apoptotic proteins through the PI3K/AKT pathway, regulation of cell migration and proliferation through the PI3K/p38/MAPK pathway. In addition to Gas6 dependent activation, AXL can be activated in a ligand independent manner. AXL is involved in the process of adhesion and immunoregulation of normal cells. Studies have found that AXL overexpression occurs in a number of tumor cells, and the Gas6/AXL signaling pathway is closely associated with the onset and development of various tumors such as chronic myeloid leukemia, breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, melanoma, glioma and renal cell carcinoma. It has been confirmed that downregulation of AXL expression can lead to a reduction in tumor cell proliferation and growth in pancreatic cancer and suppression of tumor cell invasion and migration in breast cancer. In non-small cell lung cancer, AXL gene silencing can suppress tumor growth. At the same time, high AXL expression is also associated with tumor recurrence and tolerance to other cancer drugs such as imatinib (Gliver), erlotinib (Tarceva), and lapatinib (Tyverb). Such evidence indicates that AXL is an effective target for tumor-targeted therapy.

Although bosutinib (SKI606, PF5208763, Bosulif; Pfizer, 2012), cabozantinib (XL184, Cometriq; Exelixis, 2012), sunitinib (SU11248, Sutent; Pfizer, 2006), and other drugs on the market have activity against AXL, they are multipurpose drugs, but are not specific. BGB324 (R428; Rigel Pharmaceuticals, BergenBio) is currently known as the most specific small molecule AXL inhibitor in phase II clinical trials and was designated an orphan drug for the treatment of AML by the FDA in December 2014. There are currently no small molecule inhibitors on the market that target AXL kinases.

Brief description of the invention

Based on these findings, the present invention provides quinoline or quinazoline based compounds which have good AXL kinase inhibitory activity and have the advantage of good metabolic stability.

Specific technical solutions are as follows: a quinoline or quinazoline-based compound having the structure represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof

(O, where X is selected from CH and N;

each of R1 and R2 is independently selected from -O(CR4R ₅ ) _o R ₃ ;

- 1 042574 where o is an integer from 0 to 6;

each of R ₃ , R4, R ₅ is independently selected from the group consisting of -H, C1-C ₆ alkyl, -OR8 and -NR ₈ R ₉ ;

each of R8 and R ₉ is independently selected from C ₁ -C ₆ alkyl, or R8 and R ₉ together with N attached to them form a saturated or unsaturated 5-6 membered heterocyclic group containing 1-4 heteroatoms selected from O , N and S; where a saturated or unsaturated 5-6 membered heterocyclic group may be independently and optionally substituted with one or more R ₁₀ ; where R ₁₀ is selected from the group consisting of C1-C ₆ alkyl.

In some embodiments, R1 is -O(CH2)oR ₃ ;

o is an integer from 0 to 4;

R ₃ is selected from the group consisting of -H, C1-C ₆ alkyl, C1-C ₃ alkoxy and -NR ₈ R ₉ ;

each of R ₈ and R ₉ is independently selected from the group consisting of C1-C ₃ alkyl, or R ₈ and R ₉ together with N attached to them form a saturated or unsaturated 5-6 membered heterocyclic group containing 1-4 a heteroatom selected from O, N and S; where a saturated or unsaturated 5-6 membered heterocyclic group may be independently and optionally substituted with one or more R ₁₀ ; where R ₁₀ is selected from the group consisting of C 1 -C ₃ alkyl.

In some of these embodiments, R1 is selected from the group consisting of methoxyl, ethoxyl, propoxyl, 2-methoxyethoxyl, 3-methoxypropoxyl, 3-morpholinopropoxyl, 2(pyrrolidin-1-yl)ethoxyl, 3-(pyrrolidin-1-yl) propoxy, (piperidin-1-yl)ethoxy, (piperidin-1-yl)propoxy, 4-methoxybutoxy, 2-morpholinoethoxy, (4-methylpiperazin-1-yl)propoxy, dimethylaminoethoxy and isopentyloxy.

In some of these embodiments, R2 is -O(CH2)oR3;

o is an integer from 0 to 4;

R ₃ is selected from the group consisting of -H, C1-C ₃ alkyl, C1-C ₃ alkoxy and -NR ₈ R ₉ ;

R ₈ and R ₉ together with N attached to them form a saturated 5-6 membered heterocyclic group containing 1-4 heteroatoms selected from O, N and S.

In some of these embodiments, R2 is selected from the group consisting of methoxyl, ethoxyl, propoxyl, 2-methoxyethoxyl, 3-methoxypropoxyl, 2-morpholinoethoxyl, and 3morpholinopropoxyl.

In some of these embodiments, X is N.

In some of these embodiments, the quinoline or quinazoline compound is selected from the group consisting of

N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1 ,4-dihydroquinoline-3-carboxamide,

N-(4-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)oxy)-3-fluorophenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1,4 -dihydroquinoline-3-carboxamide,

N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)1,4-dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((7-methoxy-6-(3-methoxypropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((6-methoxy-7-(4-methoxybutoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((6-methoxy-7-(2-morpholinoethoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo- 6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((6-methoxy-7-(2-(piperidin-1-yl)ethoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4oxo-6- (trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)oxy)phenyl)-1,2dimethyl-4-oxo- 6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((6-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4oxo-6- (trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((6-methoxy-7-(3-methoxypropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl4-oxo-6- (trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide,

N-(4-((7-(2-(dimethylamino)ethoxy)-6-methoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-1,2-dimethyl-4oxo-6-(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((7-(isopentyloxy)-6-methoxyquinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1,4- dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((6-methoxy-7-propoxyquinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline- 3-carboxamide,

N-(4-((7-ethoxy-6-methoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-1,2-dimethyl-4-oxo-6-(trifluorome- 2 042574 oxy)-1,4 -dihydroquinoline-3-carboxamide,

N-(3-fluoro-4-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4oxo-6-(trifluoromethoxy)-1,4 -dihydroquinoline-3-carboxamide and

N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo6-(trifluoromethoxy)-1,4 -dihydroquinoline-3-carboxamide.

The present invention also provides ways of using the above compounds based on quinoline or quinazoline.

Specific technical solutions are as follows:

ways of using the above compounds based on quinoline or quinazoline or their pharmaceutically acceptable salts, stereoisomers thereof, as AXL kinase inhibitors and/or Flt3 kinase inhibitors;

ways of using the above compounds based on quinoline or quinazoline or their pharmaceutically acceptable salts, their stereoisomers, for the preparation of a drug for the prevention or treatment of tumors.

In some embodiments, the tumor is a hematologic tumor, gastrointestinal stromal tumor, histiocytic lymphoma, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, pancreatic cancer, breast cancer, prostate cancer, liver cancer, cancer skin, epithelial cell carcinoma, or nasopharyngeal carcinoma. The hematologic tumor is preferably a leukemia.

The present invention also provides a pharmaceutical composition for preventing or treating a tumor.

Specific technical solutions are as follows: a pharmaceutical composition for the prevention or treatment of a tumor, containing an active ingredient and a pharmaceutically acceptable excipient, where the active ingredient provides the above compound based on quinoline or quinazoline or a pharmaceutically acceptable salt or its stereoisomer.

The quinoline or quinazoline based compounds or pharmaceutically acceptable salts thereof, stereoisomers thereof or pharmaceutical compositions thereof of the present invention can be effective in inhibiting the action of AXL protein kinase and can inhibit the proliferation, migration and invasion of various tumor cells. And based on a large number of ingenious experimental studies, the present inventors unexpectedly found that the introduction of trifluoromethoxy at the 6-position of the 1,4-dihydroquinoline of the quinoline or quinazoline based compounds of the present invention can significantly improve the in vivo metabolic stability indicators of such compounds, thus providing more high levels of antitumor activity of the compounds in vivo, while they have the advantage of less toxicity and less side effects, and such compounds can be used to obtain drugs for the prevention or treatment of hyperproliferative diseases such as tumors in humans and other mammals.

Brief description of graphic materials

In FIG. 1 shows spectra for the results of detection of metabolites associated with the compound TL134 in hepatocytes using the UPLC/Q-TOF MS method; where A represents the spectrum for an inactivated hepatocyte, B represents the spectrum for a human hepatocyte, and C represents the spectrum for a monkey hepatocyte.

In FIG. 2 shows the spectra for the results of detection of metabolites associated with the compound TL134 in hepatocytes using the UPLC/Q-TOF MS method; where D is the spectrum for canine hepatocyte, E is the spectrum for rat hepatocyte, and F is the spectrum for mouse hepatocyte.

In FIG. 3 shows spectra for the results of detection of metabolites associated with TL134 in hepatocytes using the UPLC-UV method (254 nm); where A represents the spectrum for an inactivated hepatocyte, B represents the spectrum for a human hepatocyte and C represents the spectrum for a monkey hepatocyte.

In FIG. 4 shows spectra for the results of detection of metabolites associated with TL134 in hepatocytes using the UPLC-UV method (254 nm); where D is the spectrum for canine hepatocyte, E is the spectrum for rat hepatocyte and F is the spectrum for mouse hepatocyte.

Detailed description of embodiments

The present invention will be described in more detail below with reference to examples and drawings, but the embodiments of the present invention are not limited to them.

In the compounds of the present invention, if any variables (eg, R1, R, etc.) appear more than once in any component, the definition for each occurrence is independent of the definition for every other occurrence. Combinations of substituents and variables are also allowed, provided such combinations provide a stable connection. The drawn line entering the ring system from a substituent means that said bond may be attached to any atom of the ring which may be substituted. If the ring system is polycyclic, this means that such a bond is attached only to any suitable carbon atoms of adjacent rings. It should be understood that substituents and substituted forms of the compounds of the present invention may be selected by those skilled in the art to provide chemically stable compounds that can be easily synthesized from commonly available starting materials using the techniques of the prior art and the methods set forth below. If the substituent itself is substituted with more than one group, it is to be understood that such groups may be attached to the same carbon atom or to different carbon atoms, provided that the structure is stable.

The term alkyl as used herein is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups containing the number of carbon atoms indicated. For example, the definition of C1- _C5 in C1- _C5 alkyl includes groups containing 1, 2, 3, 4, or 5 carbon atoms in a straight or branched chain. For example, C ₁ -C ₅ alkyl specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, and pentyl. The term cycloalkyl refers to a monocyclic saturated aliphatic hydrocarbon group containing the specified number of carbon atoms. For example, cycloalkyl includes cyclopropyl, methylcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

The term heterocycle or heterocyclyl/heterocyclic group as used herein refers to a 5-6 membered aromatic or non-aromatic heterocyclic ring containing 1-4 heteroatoms selected from O, N and S and may include bicyclic groups. Therefore, the term heterocyclyl includes the heteroaryl groups mentioned above as well as their dihydrogenated and tetrahydrogenated analogs. Additional examples of heterocyclyl include, without limitation, imidazolyl, thiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinoxalinyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, and azolyl. The connection of heterocyclic substituents can be provided through carbon atoms or through heteroatoms.

As will be understood by those skilled in the art, the halogen or halogen atom used herein is intended to include chlorine, fluorine, bromine, and iodine.

Alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl substituents may be unsubstituted or substituted unless otherwise indicated. For example, (C1- _C6 )alkyl may be substituted with one, two, or three substituents selected from the group consisting of OH, halogen, nitrile, cyano, alkoxy, dialkylamino, and a heterocyclic group such as morpholinyl, piperidinyl, and the like.

The present invention provides a free form of the compound of formula I, as well as its pharmaceutically acceptable salts and stereoisomers. Some specific illustrative compounds in this document are protonated salts of amino compounds. The term free form refers to amino compounds in non-salt form. Included pharmaceutically acceptable salts include not only exemplary salts of the specific compounds described herein, but also exemplary pharmaceutically acceptable salts of all compounds of formula I in free form. The free form of a particular salt of a compound can be separated using techniques known in the art. For example, the free form can be restored by treating the salt with a suitable dilute basic aqueous solution such as dilute aqueous NaOH, dilute aqueous potassium carbonate, dilute ammonia, and dilute aqueous sodium bicarbonate. The free form differs in part from its corresponding salt form in certain physical properties, such as solubility in polar solvents, but for the purposes of the present invention, such acid salts and base salts are comparable to their corresponding free forms in other pharmaceutical aspects.

Pharmaceutically acceptable salts of the present invention can be synthesized from compounds of the present invention containing a basic or acid moiety using conventional chemical methods. In general, alkali salts are prepared by ion exchange chromatography or by reacting the free base and inorganic or organic acids of the desired salt forms in stoichiometric amount or excess in a suitable solvent or combination of several solvents. Similarly, salts of acidic compounds are formed by reaction with suitable inorganic or organic bases.

Therefore, pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting the alkaline compounds of the present invention with inorganic or organic acids. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc., as well as salts derived from organic acids such as acetic acid. acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid , salicylic acid, paminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, and the like.

When the compound of the present invention is acidic, the corresponding pharmaceutically acceptable salt refers to a salt prepared with pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, ferrous manganese salts, potassium salts, sodium salts, zinc salts, etc. .P. Ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts are particularly preferred. With regard to salts derived from pharmaceutically acceptable organic non-toxic bases, these bases include salts of primary amines, secondary amines and tertiary amines, while substituted amines include naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, glycine betaine, caffeine , choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethanediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, aminoglucose, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine gum, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

Methods for preparing the above pharmaceutically acceptable salts and other typical pharmaceutically acceptable salts are described in more detail in Berg et al., Pharmaceutical Salts, J. Pharm. sci. 1977:66:1-19.

Since a deprotonated acid moiety of a compound, such as a carboxyl group, can be anionic under physiological conditions, and this charge can then be balanced by a protonated or alkylated base moiety with a cation inside, such as a tetravalent nitrogen atom, it should therefore be noted that the compounds of the present of the invention are potential internal salts or zwitterions.

In addition to standard methods known from the literature or methods presented in experimental procedures, the compounds of the present invention can be obtained using the reactions shown in the following schemes. Therefore, the following illustrative schemes are presented for the purpose of illustration and are not limited to the listed compounds or any specific substituents. The number of substituents shown in the diagrams is not necessarily consistent with the number used in the claims, and for clarity it is shown that one substituent is attached to a compound that allows for multiple substituents in accordance with the definition of formula (I) above.

Synthesis schemes

As shown in Scheme A, the compound of formula (I) can be synthesized from 7-benzyloxy-4-chloro-6methoxyquinazoline as a starting material by a 4-step reaction.

Scheme A

The compounds of formula (I) provided in the present invention or their pharmaceutically acceptable salts or their stereoisomers can be used to treat hyperproliferative diseases or symptoms such as tumors in humans or other mammals. In particular, they are used in the preparation of medicaments for the treatment or control of hyperproliferative diseases such as gastrointestinal stromal tumor, histiocytic lymphoma, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, pancreatic cancer, breast cancer, cancer prostate cancer, liver cancer, skin cancer, epithelial cell carcinoma, prostate cancer, nasopharyngeal carcinoma, leukemia, and the like.

- 5 042574

The compounds of the present invention, or their pharmaceutically acceptable salts or stereoisomers thereof, can be used in combination with currently used or under development drugs to enhance their clinical effects, drugs such as estrogen receptor modulators, androgen receptor modulators , retinal receptor modulators, cytotoxins/cytostatics, antiproliferatives, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducer, cytotoxic drugs, tyrosine protein kinase inhibitors, EGFR inhibitors, VEGFR inhibitors, serine-threonine protein kinase inhibitors, Bcr-Abl inhibitors, c-Kit inhibitors, Met inhibitors, inhibitor Raf inhibitors, MEK inhibitors, MMP inhibitors, topoisomerase inhibitors, histidine deacetylase inhibitors, proteasome inhibitors, CDK inhibitors, Bcl-2 family protein inhibitors, MDM2 protein family inhibitors, IAP protein family inhibitors, STAT family protein inhibitors, PI3K inhibitors, AKT inhibitors, blockers integrin, interferon-α, interleukin-12, COX-2 inhibitors, p53 activators, anti-VEGF antibodies, anti-EGF antibodies, etc.

The compounds of formula (I), or their pharmaceutically acceptable salts, or their stereoisomers, or pharmaceutical compositions based on them in accordance with the present invention can be used for the preparation of drugs for the prevention or treatment of the following diseases and other diseases not listed below:

(1) human or other mammalian breast cancers, including, without limitation, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ;

(2) respiratory tract cancers in humans or other mammals, including, without limitation, small cell lung cancer, non-small cell lung cancer, and bronchial adenoma, and pleuropulmonary blastoma;

(3) brain cancers in humans or other mammals, including, without limitation, brainstem glioma and optic nerve glioma, cerebellar astrocytoma and brain astrocytoma, ependymoma and neuroectodermal tumor, and pineal gland tumor;

(4) tumors of the male and female reproductive organs of humans or other mammals, tumors of the male reproductive organs, including, without limitation, prostate cancer and testicular cancer; tumors of the female reproductive organs, including, without limitation, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, and vulvar cancer, and intrauterine tumor;

(5) tumors in the digestive tract of humans or other mammals, including, but not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, stomach cancer, pancreatic cancer, rectal cancer, small intestine cancer, or salivary gland cancer;

(6) tumors in the urethra of humans or other mammals, including, but not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureteral cancer, or urethral cancer;

(7) human or other mammalian eye cancers, including, without limitation, intraocular melanoma and retinocytoma;

(8) liver cancers in humans or other mammals, including, without limitation, hepatocellular carcinoma (stem cell carcinoma with or without fibroblast changes), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma;

(9) skin cancers in humans or other mammals, including, without limitation, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer;

(10) head and neck cancers in humans or other mammals, including, but not limited to, cancers of the larynx, hypopharynx, nasopharynx, mesopharynx, lip cancer, and cancer of the oral cavity;

(11) types of lymphoma in humans or other mammals, including, without limitation, AIDS-associated lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, and lymphoma of the central nervous system;

(12) types of sarcoma in humans or other mammals, including, without limitation, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma;

(13) types of leukemia in humans or other mammals, including, without limitation, acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

Route of administration and dosage range

According to standard pharmaceutical procedures, the compounds of the present invention may be administered to a mammal, preferably a human, alone or in combination with pharmaceutically acceptable receptors, excipients or diluents in pharmaceutical compositions. The compounds can be administered orally or subcutaneously, intramuscularly, intraperitoneally, intravenously, rectally and topically, into the eyes, lungs, nasal cavities and parenterally.

In one embodiment, the dosage range is 0.1 to 500 mg/day/kg body weight orally when the compounds of formula (I) are used in the manufacture of medicaments for the treatment or control of patients with cancer, and the like. A suitable route of administration is a single dose daily, or two, three or four doses daily, or administration using slow release techniques. For a number of large mammals, the preferred dosage range for them is 0.1 to 1500 mg/day/kg body weight, preferably 0.5 to 100 mg/day/kg body weight. For patients with an average weight of 70 kg for them, the daily dosage is from 1 to 500 mg. For some particularly highly potent compounds, the daily dosage for adult patients may be as low as 0.1 mg/day.

Drug metabolites and prodrugs

Metabolites of the compounds of the present invention or their pharmaceutically acceptable salts and prodrugs that can be converted in vivo to the structures of the compounds of the present invention or their pharmaceutically acceptable salts are also included in the claims of the present invention.

Combined introduction

The compounds of formula (I) may be used in combination with known drugs to treat or alleviate such symptoms. With combined administration, the method of administration and dosage of known drugs remained the same, while the compound of formula (I) is administered simultaneously or sequentially. When a compound of formula (I) is used simultaneously with one or more other drugs, a pharmaceutical composition containing one or more other known drugs and a compound of formula (I) is preferably used. Combination drug therapy also involves administering a compound of formula (I) with one or more other known drugs at overlapping time periods. When a compound of formula (I) is used in combination with one or more other drugs, the compound of formula (I) and other known drugs can be used at a lower dosage than when used alone.

Drugs or active ingredients that can be used in combination with compounds of formula (I) include, but are not limited to, estrogen receptor modulators, androgen receptor modulators, retinal receptor modulators, cytotoxins/cytostatics, antiproliferative agents, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducer action, cytotoxic drugs, tyrosine protein kinase inhibitors, EGFR inhibitors, VEGFR inhibitors, serine-threonine inhibitors protein kinases, Bcr-Abl inhibitors, c-Kit inhibitors, Met inhibitors, Raf inhibitors, MEK inhibitors, MMP inhibitors, topoisomerase inhibitors, histidine deacetylase inhibitors, proteasome inhibitors, CDK inhibitors, Bcl-2 family protein inhibitors, ingi MDM2 family protein inhibitors, IAP family protein inhibitors, STAT family protein inhibitors, PI3K inhibitors, AKT inhibitors, integrin, interferon-α, interleukin-12 blockers, COX-2 inhibitors, p53, p53 activators, antibodies to VEGF, antibodies to EGF and etc.

In one embodiment, drugs or active ingredients that can be used in combination with compounds of formula (I) include, without limitation, aldesleukin, alendronic acid, interferon, alitretinoin, allopurinol, allopurinol sodium, palonosetron hydrochloride, altretamine, aminoglutethimide, amifostine, amrubicin, amsacrine, anastrozole, dolasetron, aranesp, arglabin, arsenic trioxide, aromasin, 5-azacytidine, azathioprine, bacillus Calmette Guerin (BCG) or TICE BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate drug, bexarotene, bleomycin sulfate, bromuridine, bortezomib, busulfan , calcitonin, alemtuzumab injection, capecitabine, carboplatin, casodex, cefecon, cellmoleukin, daunorubicin, chlorambucil, cisplatin, cladribine, clodronic acid, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, liposomal daunorubicin, dexamethasone, dexamethasone phosphate, estradioxyl leucotyl phosphate, depo-medrol, deslorelin, dexrazoxane, stilb estrol, diflucan, docetaxel, doxifluridine, adriamycin, dronabinol, Ho-166-chitosan complex, eligard, rasburicase, epirubicin hydrochloride, aprepitant, epirubicin, epoetin alfa, erythropoietin, eptaplatin, levamisole tablets, estradiol preparations, 17-b-estradiol, estramustine sodium phosphate, ethinylestradiol, amifostine, hydroxyphosphoric acid, etoposide phosphate, etoposide, fadrozole, tamoxifen preparations, filgrastim, finasteride, filgrastim, floxuridine, fluconazole, fludarabine, 5-fluoro-2-deoxyuridine monophosphate, 5-fluorouracil, fluoxymesterone, flutamide, formestane, 1 β-O-arabinofuranosylcytosine-5'stearyl phosphate, fotemustine, fulvestrant, gamma globulin, gemcitabine, gemtuzumab, imatinib mesylate, carmustine glutinous rice paper capsules, goserelin, granisetron hydrochloride, hystrelin, hycamtine, hydrocortisone, erythrohydroxynonyladenine, hydroxyurea, ibritumomab tiuxetan,

- 7 042574 idarubicin, ifosfamide, interferon α, interferon-a2, interferon a-2A, interferon a-2B, interferon a-n1, interferon α-η3, interferon β, interferon γ-la, interleukin-2, intron A, press , irinotecan, cytrile, lentinan sulfate, letrozole, formyltetrahydrofolate, leuprorelin, leuprorelin acetate, levamisole, calcium levofolinate, levothyroxine sodium, levothyroxine sodium preparations, lomustine, lonidamine, dronabinol, chlormethine, methylcobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogen, 6-mercaptopurine, mesnu, amethopterin, methylaminolevulinate, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, trilostane, liposomal adriamycin citrate, nedaplatin, pegylated filgrastim, oprelvekin, neupogen, nilutamide, tamoxifen, NSC-631570, recombinant human interleukin 1 , octreotide, ondansetron hydrochloride, oral dehydrocortisone solutions, oxaliplatin, paclitaxel, prednisolone sodium phosphate preparations, pegasparga zu, pegasys, pentostatin, picibanil, pilocarpine hydrochloride, pyrarubicin, plicamycin, sodium porfimer, prednimustine, prednisolone steaglat, prednisone, premarin, procarbazine, recombinant human erythropoietin, raltitrexed, rebif, etidronate rhenium-186, mabtheru, redoxone-A, romurtide, pilocarpine hydrochloride tablets, octreotide, sargramostim, semustine, sizofiran, sobuxane, sodium methylprednisolone, sparfloxacin, stem cell therapy, streptozocin, strontium-89 chloride, levothyroxine sodium, tamoxifen, tamsulosin, tasonermin, tastolactone, taxotere, teceleukin, temozolomide, teniposide, testosterone propionate, methyltestosterone, thioguanine, thiotepa, thyroid-stimulating hormone, tiludronic acid, topotecan, toremifene, tositumomab, trastuzumab, treosulfan, tretinoin, methotrexate tablets, trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, UFT, uridine, valrubicin, vesnarinone, vinblastine, vincristine, vindesine, vinorelbine, virulizin, de xrazoxan, cynostatin, stimalamer, zofran, protein-stabilized paclitaxel, acolbifene, interferon r-1b, affinitac, aminopterin, arzoxifene, azoprisnil, atamestan, atrasentan, BAY 43-9006, avastin, CCI-779, CDC-501, celebrex, cetuximab , crisnatol, cyproterone acetate, decitabine, DN-101, adriamycin-MTS, dSLIM, dutasteride, edotecarin, eflornithine, exatecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant labeled with holmium-166 DOTMP, ibandronic acid, interferon-γ, PEGIntron, ixabepilone, keyhole hemocyanin, L-651582, lanreotide, lasofoxifene, libru, lonafarnib, miproxifen, minocolate, MS-209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, neovastat, nolatrexed, oblimersen, onco-TCS, ozidem, paclitaxel polyglutamate, sodium pamidronate, PN-401, QS-21, quazepam, R-1549, raloxifene, onconase, 13-cis-retinoic acid, satraplatin, seoccalcitol, T-138067, tarceva, docosahexaenoic acid-paclitaxel conjugate, thymosin α1, galazolin , Tipifar nib, tirapazamine, TLK-286, toremifene, trans-MID-lo7R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, and zoledronic acid, or a combination thereof.

The following are specific examples and all starting materials in the form of reagents used in the following examples are commercially available.

Example 1 Preparation of N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-( trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide (named TL134)

a) p-MeCaSCHBO3H.H2O pentane, heating under reflux, overnight

NaOH,THF, _H2O

Mel,K ₂ CO ₃ ,DMF

REFRIGERATOR during the night

Stage a1. Preparation of diethyl 2-(1-((4-(trifluoromethoxy)phenyl)amino)ethylidene)malonate p-Trifluoromethoxyaniline (2.42 g, 20 mmol) and diethylacetylmalonate (2.02 g, 10 mmol) were dissolved in 50 ml of n -pentane, then a catalytic amount of p-toluenesulfonic acid (20 mg) was added and the reaction mixture was heated under reflux overnight. The reaction mixture was cooled to room temperature and a small amount of saturated NaHCO ₃ solution was added, then the mixture was extracted twice with EA. The organic phases were combined, washed once with brine and dried over anhydrous Na ₂ SO ₄ , filtered, and dried by rotary evaporation, and then subjected to column chromatography to obtain 2.68 g (87.8%) of a solid.

Stage a2. Preparation of ethyl 2-methyl-4-oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxylate

Diethyl 2-(1-((4-(trifluoromethoxy)phenyl)amino)ethylidene)malonate (2.5g, 8.2mmol) was dissolved in 25mL of diphenyl ether, heated to 200°C and reacted with stirring in over 2 hours. The reaction mixture was cooled to room temperature and the solid precipitated, then the mixture was filtered, washed with PE and then dried with suction to obtain 2 g (94.3%) of a white solid.

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Stage a3. Preparation of ethyl 1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxylate

Ethyl 2-methyl-4-oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxylate (2 g, 7.7 mmol) and K ₂ CO ₃ (3.18 g, 23.1 mmol) was dissolved in 50 ml DMF, then MeI (0.72 ml, 11.55 mmol) was added with stirring and the mixture was reacted at 50° C. overnight. The reaction mixture was cooled to room temperature, quenched with water, then a solid precipitated. The mixture was washed several times with water and the solid was extracted several times with DCM. The organic phases were combined and dried by rotary evaporation and then subjected to column chromatography to give 1.52 g (72.4%) of a white solid.

Stage a4. Preparation of 1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxylic acid

Ethyl 1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxylate (1.5 g, 5.5 mmol) and NaOH (880 mg, 22 mmol) were dissolved in a mixed solvent containing 30 ml of ethanol and 15 ml of water, then the mixture was subjected to reaction overnight. The reaction mixture was cooled to room temperature, then most of the organic solvent was dried by rotary evaporation, water was added, the pH was adjusted to 7-8 with dilute HCl in an ice bath, then a solid precipitated, the mixture was filtered and then dried with suction to obtain 1. 25 g (93.3%) white solid.

b)

-BuOK, DMF, 80

K ₂ CO ₃ , DMF, 80

HATU, DIPEA, DMF

Stage b1. Preparation of 4-((7-(benzyloxy)-6-methoxyquinazolin-4-yl)oxy)-3-fluoroaniline

To the reaction flask were added 7-benzyloxy-4-chloro-6-methoxyquinazoline (4.5 g, 15 mmol), 4-amino-2-fluorophenol (2.3 g, 18 mmol), potassium tert-butoxide (2.4 g, 21 mmol) and DMF (250 ml) and the mixture was heated to 80°C to react for 2 hours. After the reaction was completed and the solvent was removed under reduced pressure, the mixture was subjected to dry column chromatography to obtain 3.6 g ( 62%) 4-((7-(benzyloxy)-6-methoxyquinazolin-4-yl)oxy)-3-fluoroaniline.

1H NMR (400 MHz, 06-DMSO) δ 8.53 (s, 1H), 7.55 (s, 1H), 7.52 (m, 2H), 7.49 (s, 1H), 7.44 (t, J=7.2 Hz, 2H), 7.37 (t, J=7.2 Hz, 1H), 7.04 (t, J=8.8 Hz, 1H), 6.50 (dd , J=2.4, 13.2 Hz, 1H), 6.42 (dd, J=2.4, 8.8 Hz, 1H), 5.39 (s, 2H), 5.35 (s, 2H), 3.97 (s, 3H). MS (ESI), mass/charge: 391 [M+H]+.

Stage b2. Preparation of 4-(4-amino-2-fluorophenoxy)-6-methoxyquinazolin-7-ol

4-((7-(Benzyloxy)-6-methoxyquinazolin-4-yl)oxy)-3-fluoroaniline (5.2 g, 13.3 mmol), Pd/C (0.4 g) and methanol (250 ml ) was reacted at 0° C. under a hydrogen atmosphere overnight, then the Pd/C was removed by filtration and the filtrate was concentrated and passed through a column to give 2.4 g (60%) 4-(4-amino-2-fluorophenoxy)- 6-methoxyquinazolin-7-ol.

1H NMR (400 MHz, d6-DMSO) δ 10.72 (s, 1H), 8.45 (s, 1H), 7.52 (s, 1H), 7.22 (d, J=3.2 Hz , 1H), 7.02 (t, J=8.8 Hz, 1H), 6.49 (dd, J=2.4, 12.8 Hz, 1H), 6.41 (dd, J=2, 0, 8.8 Hz, 1H), 5.37(s, 2H), 3.97(s, 3H). MS (ESI), mass/charge: 301 [M+H]+.

Stage b3. Preparation of 3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)oxy)aniline

4-(4-Amino-2-fluorophenoxy)-6-methoxyquinazolin-7-ol (400mg, 1.3mmol), 4-(3-chloropropyl)morpholine (3-5a) (640mg, 3.9mmol ) and potassium carbonate (540 mg, 3.9 mmol) were added to DMF (50 ml), the mixture was heated to 80° C. and reacted for two hours, then extracted three times with ethyl acetate. The organic phases were combined, washed with brine and dried by rotary evaporation and then passed through a column to give 380 mg (67%) of 3fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)oxy )aniline.

1H NMR (400 MHz, CDCl ₃ ) δ 8.60 (s, 1H), 7.53 (s, 1H), 7.31 (s, 1H), 7.05 (t, J=8.8 Hz, 1H), 6.49 (dd, J=2.4, 12.0 Hz, 1H), 6.41 (dd, J=2.4, 8.8 Hz, 1H), 4.26 (t, J =6.4 Hz, 2H), 4.02 (s, 3H), 3.71 (t, J=4.4 Hz, 4H), 2.56 (t, J=7.2 Hz, 2H), 2.47 (s, 4H), 2.11 (m, 2H). MS (ESI), mass/charge: 428 [M+H]+.

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Stage b4. Preparation of N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)- 1,4-dihydroquinoline-3-carboxamide (named

TL134)

3-Fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)oxy)aniline (450 mg, 1 mmol), 1,2dimethyl-4-oxo-6-(trifluoromethoxy)- 1,4-dihydroquinoline-3-carboxylic acid (277 mg, 1.2 mmol), HATU (570 mg, 1.5 mmol) and DIEA (0.5 ml, 3 mmol) were dissolved in 30 ml DMF, then the mixture was stirred overnight at room temperature. Ice water was added to the reaction solution, followed by precipitation of a solid. The mixture was filtered and the solid was extracted twice with dichloromethane. The organic phases were combined and washed once with brine, dried over anhydrous Na ₂ SO ₄ , then filtered, and dried by rotary evaporation, and then subjected to column chromatography to give 478 mg (72%) of a white solid.

Example 2 Preparation of N-(4-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)oxy)-3-fluorophenyl)-1,2dimethyl-4-oxo-6-(trifluoromethoxy)-1 ,4-dihydroquinoline-3-carboxamide (named TL197)

The synthesis method is mentioned in example 1.

1H NMR (400 MHz, DMSO-d ₆ ) δ 10.78 (s, 1H), 8.56 (s, 1H), 8.12-8.04 (m, 2H), 7.94 (dd, J =12.8, 2.3Hz, 1H), 7.82(dd, J=9.3, 3.0Hz, 1H), 7.63(s, 1H), 7.53-7.39( m, 3H), 4.35 (ddd, J=9.1, 4.4, 2.7 Hz, 4H), 3.88 (s, 3H), 3.77 (q, J=4.9 Hz , 4H), 3.36 (s, 5H), 2.63 (s, 3H).

Example 3 Preparation of N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-1,2-dimethyl-4-oxo6-(trifluoromethoxy)-1,4-dihydroquinoline-3 -carboxamide (named TL198)

The synthesis method is mentioned in example 1.

' H NMR (400 MHz, DMSO-d ₆ ) δ 10.78 (s, 1H), 8.57 (s, 1H), 8.07 (d, J=12.0 Hz, 2H), 7.94 (d, J=12.7 Hz, 1H), 7.83 (d, J=8.5 Hz, 1H), 7.59 (s, 1H), 7.54-7.34 (m, 3H) , 4.00 (s, 6H), 3.88 (s, 3H), 2.63 (s, 3H).

Example 4 Preparation of N-(3-fluoro-4-((7-methoxy-6-(3-methoxypropoxy)quinazolin-4-yl)oxy)phenyl)1,2-dimethyl-4-oxo-6-(trifluoromethoxy )-1,4-dihydroquinoline-3-carboxamide (named TL199)

The synthesis method is mentioned in example 1.

1H NMR (400 MHz, chloroform-d) δ 12.32 (s, 1H), 8.62 (s, 1H), 8.38 (dd, J=2.7, 1.3 Hz, 1H), 7 .98 (dd, J=12.4, 2.5 Hz, 1H), 7.68 (d, J=9.3 Hz, 1H), 7.64-7.57 (m, 2H), 7. 47-7.41(m, 1H), 7.33(s, 1H), 7.28(d, J=8.6Hz, 1H), 4.32(t, J=6.5Hz, 2H ), 4.05 (s, 3H), 3.95 (s, 3H), 3.62 (t, J=6.1 Hz, 2H), 3.38 (s, 3H), 3.11 (s , 3H), 2.22 (p, J=6.3 Hz, 2H).

Example 5 Preparation of N-(3-fluoro-4-((6-methoxy-7-(4-methoxybutoxy)quinazolin-4-yl)oxy)phenyl)1,2-dimethyl-4-oxo-6-(trifluoromethoxy )-1,4-dihydroquinoline-3-carboxamide (named TL204)

The synthesis method is mentioned in example 1.

1H NMR (400 MHz, chloroform-d) δ 12.32 (s, 1H), 8.62 (s, 1H), 8.37 (dd, J=2.9, 1.4 Hz, 1H), 7 .98 (dd, J=12.4, 2.4 Hz, 1H), 7.67 (d, J=9.4 Hz, 1H), 7.63-7.58 (m, 1H), 7. 56 (s, 1H), 7.43 (ddd, J=8.7, 2.4, 1.2 Hz, 1H), 7.30 (d, J=13.5 Hz, 2H), 4.24 (t, J=6.6 Hz, 2H), 4.05 (s, 3H), 3.95 (s, 3H), 3.48 (t, J=6.4 Hz, 2H), 3.36 (s, 3H), 3.11 (s, 3H), 2.10-1.98 (m, 2H), 1.88-1.76 (m, 2H).

Example 6 Preparation of N-(3-fluoro-4-((6-methoxy-7-(2-morpholinoethoxy)quinazolin-4-yl)oxy)phenyl)1,2-dimethyl-4-oxo-6-(trifluoromethoxy )-1,4-dihydroquinoline-3-carboxamide (named TL209)

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The synthesis method is mentioned in example 1.

1H NMR (400 MHz, chloroform-d) δ 12.34 (s, 1H), 8.63 (s, 1H), 8.39 (dd, J=2.9, 1.3 Hz, 1H), 7 .99 (dd, J=12.4, 2.4 Hz, 1H), 7.68 (d, J=9.3 Hz, 1H), 7.64-7.55 (m, 2H), 7. 44 (ddd, J=8.8, 2.5, 1.3 Hz, 1H), 7.33 (s, 1H), 7.30 (d, J=8.6 Hz, 1H), 4.36 (s, 2H), 4.06 (s, 3H), 3.96 (s, 3H), 3.78 (d, J=5.2 Hz, 4H), 3.12 (s, 3H), 2 .97 (s, 2H), 2.66 (s, 4H).

Example 7 Preparation of N-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4yl)oxy)phenyl)-1,2-dimethyl-4- oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide (named TL212)

I n II 11 Ί _O AJ O o Gb / ^ Ν Ο Ν

The synthesis method is mentioned in example 1.

1H NMR (400 MHz, chloroform-d) δ 12.33 (s, 1H), 8.62 (s, 1H), 8.39 (s, 1H), 7.99 (dd, J=12.3, 2.4 Hz, 1H), 7.68 (d, J=9.3 Hz, 1H), 7.64-7.58 (m, 1H), 7.57 (s, 1H), 7.44 ( d, J=8.9 Hz, 1H), 7.34 (s, 1H), 7.30 (s, 1H), 4.29 (t, J=6.7 Hz, 2H), 4.06 ( s, 3H), 3.96 (s, 3H), 3.11 (s, 2H), 2.69 (t, J=7.5 Hz, 2H), 2.56 (s, 4H), 2, 24-2.11 (m, 2H), 2.05 (s, 1H), 1.80 (t, J=4.8 Hz, 4H), 1.25 (q, J=6.9, 6, 4 Hz, 2H).

Example 8 Preparation of N-(3-fluoro-4-((6-methoxy-7-(2-(piperidin-1-yl)ethoxy)quinazolin-4yl)oxy)phenyl)-1,2-dimethyl-4- oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide (named TL213) ₀ AJ o o

A ⁿ AA- /

The synthesis method is mentioned in example 1.

1H NMR (400 MHz, chloroform-d) δ 12.32 (s, 1H), 8.62 (s, 1H), 8.38 (d, J=2.7 Hz, 1H), 7.99 (dd , J=12.3, 2.4 Hz, 1H), 7.68 (d, J=9.3 Hz, 1H), 7.63-7.54 (m, 2H), 7.44 (dd, J=8.8, 2.0Hz, 1H), 7.33(s, 1H), 7.29(d, J=8.8Hz, 1H), 4.39(t, J=6.1 Hz, 2H), 4.05 (s, 3H), 3.96 (s, 3H), 3.11 (s, 3H), 3.02 (s, 2H), 2.67 (s, 4H), 1.78-1.62 (m, 6H).

Example 9 Preparation of N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4yl)oxy)phenyl)-1,2-dimethyl- 4-oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide (named TL238)

The synthesis method is mentioned in example 1.

1H NMR (400 MHz, DMSO-d ₆ ) δ 10.80 (s, 1H), 8.56 (s, 1H), 8.13-8.03 (m, 2H), 7.94 (dd, J =12.8, 2.4 Hz, 1H), 7.82 (dd, J=9.3, 3.1 Hz, 1H), 7.59 (s, 1H), 7.50 (dd, J= 8.9, 2.3Hz, 1H), 7.44(t, J=8.6Hz, 1H), 7.40(s, 1H), 4.24(t, J=6.4Hz, 2H), 3.99 (s, 3H), 3.88 (s, 3H), 2.63 (s, 3H), 2.45 (t, J=7.0 Hz, 3H), 2.33 ( s, 6H), 2.15 (s, 3H), 1.98 (q, J=6.9, 6.4 Hz, 3H), 1.55 (s, 1H).

Example 10 Preparation of N-(3-fluoro-4-((6-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)quinazolin-4yl)oxy)phenyl)-1,2-dimethyl-4- oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide (named TL231)

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The synthesis method is mentioned in example 1.

Ή NMR (400 MHz, chloroform-d) δ 12.30 (s, 1H), 8.62 (s, 1H), 8.35 (dd, J=2.8, 1.4 Hz, 1H), 7 .97 (dd, J=12.4, 2.4Hz, 1H), 7.66 (d, J=9.4Hz, 1H), 7.58 (d, J=13.6Hz, 2H) , 7.42 (ddd, J=8.8, 2.5, 1.2 Hz, 1H), 7.32 (s, 1H), 7.28 (d, J=8.5 Hz, 1H), 4.35 (t, J=6.1 Hz, 2H), 4.05 (s, 3H), 3.93 (s, 3H), 3.10 (d, J=6.0 Hz, 2H), 3.09 (s, 3H), 2.73 (d, J=6.0 Hz, 4H), 1.85 (p, J=3.3 Hz, 5H).

Example 11 Preparation of N(3-fluoro-4-((6-methoxy-7-(3-methoxypropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy )-1,4-dihydroquinoline-3-carboxamide TL226) (named

The synthesis method is mentioned in example 1.

¹ H NMR (400 MHz, chloroform-d) δ 12.27 (s, 1H), 8.61 (s, 1H), 8.35-8.29 (m, 1H), 7.97 (dd, J =12.3, 2.4 Hz, 1H), 7.64 (d, J=9.3 Hz, 1H), 7.58 (d, J=3.1 Hz, 1H), 7.56 (s , 1H), 7.41 (dt, J=8.8, 1.7 Hz, 1H), 7.34 (s, 1H), 7.30-7.24 (m, 1H), 4.31 ( t, J=6.5 Hz, 2H), 4.05 (s, 3H), 3.92 (s, 3H), 3.61 (t, J=6.1 Hz, 2H), 3.38 ( s, 3H), 3.07 (s, 3H), 2.21 (p, J=6.3 Hz, 2H).

Example 12 Preparation of N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4yl)oxy)phenyl)-1,2-dimethyl-4- oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide (named TL216)

The synthesis method is mentioned in example 1.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.78 (s, 1H), 8.55 (s, 1H), 8.12-8.04 (m, 2H), 7.94 (dd, J=12.9, 2.3Hz, 1H), 7.82(dd, J=9.3, 3.0Hz, 1H), 7.59(s, 1H), 7.53-7.37 (m, 3H), 4.24 (t, J=6.5 Hz, 2H), 4.00 (d, J=2.5 Hz, 3H), 3.88 (s, 3H), 3.33 (s, 3H), 2.63 (s, 3H), 2.42 (t, J=7.1 Hz, 2H), 2.35 (s, 4H), 2.02-1.90 (m, 2H), 1.51 (p, J=5.5 Hz, 4H), 1.39 (q, J=6.7, 6.2 Hz, 2H).

Example 13 Preparation of N-(4-((7-(2-(dimethylamino)ethoxy)-6-methoxyquinazolin-4-yl)oxy)-3fluorophenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy )-1,4-dihydroquinoline-3-carboxamide (named TL233)

The synthesis method is mentioned in example 1.

Ή NMR (400 MHz, chloroform-d) δ 12.30 (s, 1H), 8.62 (s, 1H), 8.35 (d, J=2.3 Hz, 1H), 7.98 (dd , J=12.3, 2.4 Hz, 1H), 7.66 (d, J=9.3 Hz, 1H), 7.63-7.54 (m, 2H), 7.46-7, 39 (m, 1H), 7.32 (s, 1H), 7.29 (d, J=8.5 Hz, 1H), 4.31 (t, J=5.9 Hz, 2H), 4. 04 (s, 3H), 3.94 (s, 3H), 3.09 (s, 3H), 2.92 (t, J=5.8 Hz, 2H), 2.41 (s, 6H).

Example 14 Preparation of N-(3-fluoro-4-((7-(isopentyloxy)-6-methoxyquinazolin-4-yl)oxy)phenyl)-1,2dimethyl-4-oxo-6-(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide (named TL230)

The synthesis method is mentioned in example 1.

¹ H NMR (400 MHz, chloroform-d) δ 12.28 (s, 1H), 8.61 (s, 1H), 8.37-8.31 (m, 1H), 7.97 (dd, J =12.4, 2.5

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Hz, 1H), 7.65 (d, J=9.3 Hz, 1H), 7.58 (d, J=12.1 Hz, 2H), 7.42 (ddd, J=8.8, 2 .5, 1.2 Hz, 1H), 7.32 (s, 1H), 7.307.24 (m, 1H), 4.24 (t, J=6.7 Hz, 2H), 4.05 (s , 3H), 3.93 (s, 3H), 3.08 (s, 3H), 1.94-1.81 (m, 2H), 1.66 (s, 1H),

1.01 (s, 3H), 1.00 (s, 3H).

Example 15 Preparation of N-(3-fluoro-4-((6-methoxy-7-propoxyquinazolin-4-yl)oxy)phenyl)-1,2dimethyl-4-oxo-6-(trifluoromethoxy)-1,4- dihydroquinoline-3-carboxamide (named TL240)

The synthesis method is mentioned in example 1.

¹ H NMR (400 MHz, chloroform-d) δ 12.26 (s, 1H), 8.61 (s, 1H), 8.31 (t, J=1.8 Hz, 1H), 7.97 ( dd, J=12.4, 2.4Hz, 1H), 7.63 (d, J=9.4Hz, 1H), 7.57 (d, J=6.2Hz, 2H), 7, 41 (ddd, J=8.8, 2.5, 1.2 Hz, 1H), 7.30 (s, 1H), 7.29-7.23 (m, 1H), 4.17 (t, J=6.8Hz, 2H), 4.05(s, 3H), 3.91(s, 3H), 3.06(s, 3H), 1.98(h, J=7.2Hz, 2H), 1.10 (t, J=7.4 Hz, 3H).

Example 16 Preparation of N-(4-((7-ethoxy-6-methoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-1,2-dimethyl4-oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline -3-carboxamide (named TL241)

The synthesis method is mentioned in example 1.

Ή NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 8.55 (s, 1H), 8.12-8.03 (m, 2H), 7.94 (dd, J= 12.9, 2.3 Hz, 1H), 7.82 (dd, J=9.3, 3.0 Hz, 1H), 7.59 (s, 1H), 7.50 (dd, J=8 .9, 2.3 Hz, 1H), 7.45 (t, J=8.6 Hz, 1H), 7.39 (s, 1H), 4.27 (q, J=6.9 Hz, 2H ), 4.00 (s, 3H), 3.88 (s, 3H), 3.32 (s, 2H), 2.63 (s, 3H), 1.44 (t, J=6.9 Hz , 3H).

Example 17 Preparation of N-(3-fluoro-4-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-( trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide (named TL236)

The synthesis method is mentioned in example 1.

¹ H NMR (400 MHz, chloroform-d) δ 12.33 (s, 1H), 8.62 (s, 1H), 8.41-8.34 (m, 1H), 7.99 (dd, J =12.4, 2.4Hz, 1H), 7.67 (d, J=9.4Hz, 1H), 7.60 (d, J=8.6Hz, 2H), 7.47-7 .39 (m, 1H), 7.32 (s, 1H), 7.29 (d, J=8.5 Hz, 1H), 4.29 (t, J=6.6 Hz, 2H), 4 .04 (s, 3H), 3.95 (s, 3H), 3.73 (t, J=4.7 Hz, 5H), 3.10 (s, 3H), 2.59 (t, J= 7.1 Hz, 2H), 2.50 (t, J=4.6 Hz, 4H), 2.14 (p, J=6.8 Hz, 2H).

Example 18 Preparation of N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-( trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide (named CCB-310)

With)

Stage c1. Preparation of 7-(benzyloxy)-4-(2-fluoro-4-nitrophenoxy)-6-methoxyquinoline

To the reaction flask were added 7-benzyloxy-4-chloro-6-methoxyquinoline (4.5 g, 15 mmol), 2-fluoro4-nitrophenol (2.4 g, 15 mmol), DIEA (18 ml) and xylene (9 ml ), the mixture was heated to 140° C. and reacted overnight, then cooled to room temperature. The solid was precipitated, then

- 13 042574 the mixture was filtered, washed with ethanol and then dried with suction to obtain 3.7 g (81.0%) of a white solid.

1H NMR (300 MHz, d6-DMSO) δ 8.56 (d, J=5.1 Hz, 1H), 8.45 (dd, J=10.5, 2.5 Hz, 1H), 8.20 (m, 1H), 7.61 (dd, J=8.8, 8.8 Hz, 1H), 7.56 (s, 1H), 7.53 (m, 2H), 7.48 (s, 1H), 7.32-7.47 (m, 3H), 6.78 (d, J=5.1 Hz, 1H), 5.33 (s, 2H), 3.93 (s, 3H). MS (ESI), mass/charge: 421 [M+H]+.

Stage c2. Preparation of 4-(4-amino-2-fluorophenoxy)-6-methoxyquinolin-7-ol

7-(Benzyloxy)-4-(2-fluoro-4-nitrophenoxy)-6-methoxyquinoline (3.0 g, 10 mmol) was dissolved in 10 ml DMF, then 10% Pd/C (0.5 g) was added and 10 ml of ethanol and the mixture were reacted at room temperature under a hydrogen atmosphere overnight. The Pd/C was removed by filtration and the filtrate was concentrated and passed through a column to give 1.8 g (60%) of 4-(4-amino-2-fluorophenoxy)-6methoxyquinolin-7-ol.

1H NMR (400 MHz, d6-DMSO) δ 10.11 (s, 1H), 8.39 (d, J=2.2 Hz, 1H), 7.49 (s, 1H), 7.27 (s , 1H), 7.06 (s, 1H), 6.68-6.40 (m, 2H), 6.32 (s, 1H), 5.49 (s, 2H), 3.95 (s, 3H). MS (ESI), mass/charge: 301 [M+H] ⁺ .

Stage c3. Preparation of 3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)aniline

4-(4-Amino-2-fluorophenoxy)-6-methoxyquinolin-7-ol (600 mg, 2 mmol), 4-(3-chloropropyl)morpholine (3-5a) (982 mg, 6 mmol) and potassium carbonate (828 mg, 6 mmol) was added to DMF (20 ml), the mixture was heated to 80° C. and reacted for two hours and then extracted three times with ethyl acetate. The organic phases were combined, washed with brine and dried by rotary evaporation and then passed through a column to give 615 mg (72%) of 3-fluoro-4-((6methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy )aniline.

1H NMR (300 MHz, d6-DMSO) δ 8.44 (d, J=5.2 Hz, 1H), 7.50 (s, 1H), 7.37 (s, 1H), 7.06 (dd , J=9.2, 8.8 Hz, 1H), 6.55 (dd, J=13.2, 2.4 Hz, 1H), 6.47 (m, 1H), 6.38 (dd, J=5.2, 1.0 Hz, 1H), 5.46 (s, 2H), 4.19 (t, J=6.4 Hz, 2H), 3.94 (s, 3H), 3, 59 (m, 4H), 2.47 (t, J=7.1 Hz, S48 2H), 2.39 (m, 4H), 1.97 (m, 2H). MS (ESI), mass/charge: 428 [M+H] ⁺ .

Stage c4. Preparation of N-(3-fluoro-4-((6-methoxy-7-(3-morpholino-propoxy)quinolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)- 1,4-dihydroquinoline-3-carboxamide (named CCB-310)

3-Fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)aniline (428 mg, 1 mmol), 1,2dimethyl-4-oxo-6-(trifluoromethoxy)- 1,4-dihydroquinoline-3-carboxylic acid (277 mg, 1.2 mmol), HATU (570 mg, 1.5 mmol) and DIEA (0.5 ml, 3 mmol) were dissolved in 5 ml DMF and then the mixture was stirred overnight at room temperature. Ice water was added to the reaction solution, followed by precipitation of a solid. The mixture was filtered and the solid was extracted twice with dichloromethane. The organic phases were combined and washed once with brine, dried over anhydrous Na ₂ SO ₄ , then filtered, and dried by rotary evaporation, and then subjected to column chromatography to give 533 mg (75%) of a white solid.

1H NMR (400 MHz, d6-DMSO) δ 10.82 (s, 1H), 8.47 (d, J=5.2 Hz, 1H), 8.08 (t, J=6.2 Hz, 2H ), 8.01 (dd, J=13.1, 2.3Hz, 1H), 7.83 (dd, J=9.3, 2.8Hz, 1H), 7.54 (d, J= 4.2 Hz, 2H), 7.45 (m, 2H), 6.49 (d, J=5.0 Hz, 1H), 4.21 (t, J=6.4 Hz, 2H), 3 .96 (s, 3H), 3.88 (s, 3H), 3.69-3.50 (m, 4H), 2.63 (s, 3H), 2.47 (s, 2H), 2, 41 (s, 4H), 2.05-1.91 (m, 2H). MS (ESI), mass/charge: 711 [M+H] ⁺ .

Comparative Example 1 Preparation of 6-ethyl-N-(3-fluoro-4-((6-methoxy-7-(3-morpholunopropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4- oxo-1,4-dihydroquinoline-3-carboxamide (named GDL5000123)

The synthesis method is mentioned in example 1.

1H NMR (500 MHz, d6-DMSO) δ 11.01 (s, 1H), 8.56 (s, 1H), 8.08 (d, J=1.5 Hz, 1H), 7.97-7 .94 (dd, J=2.0, 13.0 Hz, 1H), 7.81 (d, J=9.0 Hz, 1H), 7.67-7.65 (dd, J=2.0 , 8.5 Hz, 1H), 7.58 (s, 1H), 7.50 (m, 1H), 7.44 (t, J=9.0 Hz, 1H), 7.40 (s, 1H ), 4.26 (t, J=6.0 Hz, 2H), 3.99 (s, 3H), 3.83 (s, 3H), 3.60 (s, 4H), 2.77 (q , J=7.5 Hz, 2H), 2.65 (s, 3H), 2.50 (m, 2H), 2.41 (s, 4H), 1.99 (t, J=6.5 Hz , 2H), 1.25 (t, J=7.5 Hz, 2H). MS (ESI), mass/charge: 656 [M+H] ⁺ .

Example 19 IC ₅₀ Tests for Quinoline and Quinazoline Based Compounds for AXL Kinase

Detection of kinase activity

Enzyme-linked immunosorbent assay (ELISA) technology was used to detect the kinase inhibitory activity of the compounds. Enzymatic Reaction Substrate Poly (Glu, Tyr) 4:1 was diluted to 20 µg/mL with potassium-free PBS (10 mM sodium phosphate buffer, 150 mM NaCl, pH 7.2-7.4) and plate with enzyme labels were coated with 125 µl/well, then

- 14 042574 were incubated at 37°C for 12-16 hours. After the liquid in the well was removed, the plate was washed three times with T-PBS (containing 0.1% Tween-20 PBS), 200 μl of T-PBS per well , each time for 5 min. The tablet with enzyme labels was dried in an oven at 37°C for 1-2 hours. 50 μl of ATP solution diluted with reaction buffer (50 mM HEPES, pH 7.4, 50 mM MgCl ₂ , 0.5 mM MnCl ₂ , 0.2 mM Na ₃ VO ₄ , 1 mM DTT) was added to each well, with a final concentration of 5 µM. Test compounds were diluted with DMSO to an appropriate concentration, such as 1 μl/well, or to contain the appropriate concentration of DMSO (negative control well), and then recombinant AXL kinase protein (eurofins, 14-512) diluted with 49 µl reaction buffer. Each experiment requires two wells without enzyme as a control. The plate was incubated on a shaker (100 rpm) at 37° C. and reacted for 1 hour. The plate was washed three times with T-PBS. 100 μl/well of diluted PY99 primary antibody was added and the contents of the plate were shaken at 37° C. for 0.5 hours. The plate was washed three times with T-PBS. 100 μl/well of diluted horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody was added and the contents of the plate were shaken at 37° C. for 0.5 h. The plate was washed three times with T-PBS. 2 mg/ml OPD color solution was added at 100 µl/well (diluted with 0.1 M citric acid-sodium citrate buffer (pH=5.4) containing 0.03% H ₂ O ₂ ) and the contents of the plate were subjected to reaction at 25°C in the dark for 1-10 minutes. (Sonication is required to dissolve the OPD and a color solution must be prepared for immediate use). 50 μl/well of 2 M H ₂ SO ₄ was added to stop the reaction and a wave-adjustable SPECTRA MAX 190 enzyme labeled activity microplate reader was used to read data at 490 nm.

The degree of suppression for the sample was obtained using the following formula.

Sample Inhibition Rate (%) = Compound OD Value - Enzyme Control Well OD Value Negative Control Well OD Value - Enzyme Control Well OD Value ⁷

IC ₅₀ values were obtained by four-parameter regression using the software included with the microplate reader for reading activity after labeling with the enzyme.

In competitive experiments with quinoline and ATP quinazoline compounds, all compounds showed strong inhibitory activity against AXL kinase (results are shown in Table 1). With respect to the modification of substituents R ₁ and R 2 in the general formula (I), it was found that if R 1 and R 2 are hydrophilic substituents, the compounds have better inhibitory activity and can withstand more significant substituent modifications.

Table 1 Compound numbers and corresponding results as indicators of kinase inhibitory activity

Comm. IC50 against AXL (nM) TL-209 2.0 TL-212 1.5 TL-199 1.8 TL-213 2.2 TL-197 4.2 TL-198 2.1 TL-204 1.6 TL-134 1.1 SSV-310 1.8 GDL5000123 1.2 R428 4.2

Example 20 IC ₅₀ Tests for Quinoline and Quinazoline Compounds for AXL Kinase (Z'-LYTE™ Technology)

Detection of kinase activity

The AXL kinase inhibitory activity of the compounds (eurofins, 14-500) was determined by a second order reaction using Z'-LYTE™ technology (fluorescence detection, enzymatic binding based on the difference in sensitivity of phosphorylated and non-phosphorylated polypeptides to proteolytic cleavage) based on on the principle of fluorescence resonance energy transfer (FRET), using a peptide substrate for FRET Z'-LYTE™ (peptide substrate tyrosine 6 for Z'-LYTE™, Invitrogen, PV4122).

enzymatic reaction.

5 μl enzyme-substrate system (50 mM HEPES, pH 6.5, 0.01% BRIJ-35, 10 mM MgCl ₂ , 1 mM EGTA, 0.02% NaN ₃ ) was added to a 384-well plate and transferred 5 nl compound (concentration gradient) using the echo520 ultramicro volume liquid handler. After shaking the plate at room temperature for 10-20 min, 25 nl of ATP (final concentration 50 μM) was transferred into the plate using an echo520 ultramicrovolume liquid handler. After shaking and thorough mixing, the mixture was centrifuged and reacted at 30°C in the dark for 1.5 hours.

detection reaction.

2.5 µl of developing solution (1:128 dilution) was added to each well and the plate was incubated at 37° C. in the dark for 1 hour, then 5 µl of Stop Reagent was added.

Reading data from a tablet.

Fluorescent signals were detected using a Perkin Elmer EnVision multi-mode plate reader (excitation wavelength 400 nm, emission wavelengths 460 nm, 535 nm).

Calculation.

The degree of suppression for each well was calculated from wells with full activity and wells with a control signal.

The data analysis method is as follows.

Degree of phosphorylation=1-{(radiance x F100%-C100%)/[C0%-C100%+radiance x(F100%-F0%)]} x 100;

Degree of inhibition = 100 x (1-degree of phosphorylation for compound/degree of phosphorylation for negative control).

IC ₅₀ values were calculated using GraphPad Prism software.

In competitive experiments with quinoline and ATP quinazoline compounds, all compounds showed strong inhibitory activity against AXL kinase (results are shown in Table 2). With respect to the modification of substituents R ₁ and R 2 in the general formula (I), it was found that if R 1 and R 2 are hydrophilic substituents, the compounds have better inhibitory activity and can withstand more significant substituent modifications.

Table 2. Compound numbers and corresponding results as indicators of kinase inhibitory activity

Comm. IC50 against AXL (nM) TL-216 1.5 TL-226 4.7 TL-230 59.4 TL-231 2.3 TL-233 1.9 TL-236 2.4 TL-238 1.2 TL-240 6.1 TL-241 2.6 TL-242 167.0 R428 5.5

Example 21 IC50 Tests for Quinoline and Quinazoline Based Compounds for Flt3 Kinase

Detection of kinase activity.

The inhibitory activity of the compounds against Flt3 kinase (life, PV6253) was determined by a second order reaction using Z'-LYTE™ technology (detection by fluorescence, enzymatic binding, based on the difference in sensitivity of phosphorylated and non-phosphorylated polypeptides to proteolytic cleavage), based on the principle fluorescence resonance energy transfer (FRET), using a peptide substrate for FRET Z'-LYTE™ (peptide substrate tyrosine 2 for Z'-LYTE™, Invitrogen, PV3191).

enzymatic reaction.

To a 384-well plate was added 5 μl of the enzyme-substrate system (50 mM HEPES, pH 7.5, 0.01% BRIJ-35, 10 mM MgCl ₂ , 1 mM EGTA), 2.5 μl of the compound (concentration gradient) and 2.5 µl of ATP mixed solution (final concentration of FRET Z'-LYTE™ tyrosine 2 peptide substrate was 2 µM and final ATP concentration was 500 µM), and then the plate was incubated at 37° C. in the dark for 1 hour

detection reaction.

5 µl of developing solution (1:64 dilution) was added to each well and the plate was incubated at 37° C. in the dark for 1 hour, then 5 µl of stop reagent was added.

Reading data from a tablet.

Fluorescent signals were detected using a Synergy H1 microplate reader (excitation wavelength is 400 nm, emission wavelengths are 445, 535 nm).

- 16 042574

Calculation.

The degree of suppression for each well was calculated from wells with full activity and wells with a control signal. The data analysis method is as follows.

Degree of phosphorylation=1-{(radiance x F100%-C100%)/[C0%-C100%+radiance x(F100%-F0%)]} x 100;

Degree of inhibition=100 x (1-degree of phosphorylation for compound/degree of phosphorylation for negative control).

IC ₅₀ values were calculated using GraphPad Prism software.

In competitive experiments with quinoline and ATP quinazoline compounds, all compounds showed strong inhibitory activity against Flt3 kinase (results are shown in Table 3). With respect to the modification of the R1 and R2 substituents in the general formula (I), it has been found that if R1 and R2 are hydrophilic substituents, the compounds have better inhibitory activity and can withstand more significant substituent modifications.

Table 3. Compound numbers and corresponding results as indicators of kinase inhibitory activity

Comm. IC50 against Flt3 (nM) TL-209 9.2 TL-212 5.2 TL-199 25.8 TL-213 6.4 TL-197 4.7 TL-198 2.3 TL-204 AND 2 TL-134 1.9 SSV-310 9.5 GDL5000123 10.0 AC220 10.3

Example 22 Effects of Quinoline and Quinazoline Based Compounds on Proliferation of MV4-11 Cells

The inhibitory effects of the compounds on MV4-11 cell proliferation were determined using the CCK-8 cell scoring kit (Dojindo). The specific stages are as follows. Logarithmic phase MV4-11 cells were plated in a 96-well culture plate at the appropriate density at 90 μl per well. After culturing overnight, compounds were added at various concentrations for a reaction for 72 h and a solvent control group was established (negative control). After 72 hours of exposure of the compounds to the cells, the effects of the compounds on cell proliferation were determined using the CCK-8 cell scoring kit (Dojindo). 10 µl of CCK-8 reagent was added to each well and the plate was placed in an incubator at 37°C for 2-4 hours. After that, a microplate reader was used for reading activity after enzyme labeling with a full range of wavelengths SpectraMax 190 with a wavelength at measuring 450 nm.

The degree of inhibition (%) for the compound in relation to the growth of tumor cells was calculated using the following formula.

Degree of Inhibition (%)=(OD of control well - OD of well after administration)/OD of control well x 100%

IC50 values were obtained by four-parameter regression using the software included with the microplate reader for reading activity after labeling with the enzyme.

The quinoline and quinazoline based compounds showed strong inhibitory activity against MV4-11 leukemic cells (results are shown in Table 4).

Table 4. IC50 values for compounds regarding inhibition of MV4-11 cell proliferation

Comm. 1С ₅₀ (nM) TL-134 <0.4 AC220 <4

Example 23. Comparative study of the types of metabolism of compounds based on quinoline and quinazoline in hepatocytes

In this example, the UPLC-UV/Q-TOF MS method was used to assess differences in metabolic processes in relation to TL134 in hepatocytes of five species, such as human, monkey, canine, rat and mouse. It provides a basis for selection based on preclinical pharmacokinetic parameters and safety assessment studies in animal species.

- 17 042574

Materials and methods are as follows. 1. Medicines and reagents

TL134 The receipt is described in example 1 Mixed primary human hepatocytes (batch number 1410266) Xenotech, USA Mixed primary hepatocytes of Macas fascicularis (batch number IXCH) RILD Company Mixed primary hepatocytes from a male beagle breed (serial number 1410275) Xenotech, USA Mixed primary hepatocytes of male SD rats (batch number 1210260) Xenotech, USA CD-1 mouse mixed primary hepatocytes (batch number 1510134) Xenotech, USA Ammonium acetate (chromatographically pure) ROE Company, USA Acetonitrile (chromatographically pure) Merck, Germany Formic acid (chromatographically pure) Fluka, Germany

2. Incubation Systems for In Vitro Metabolism Studies

The total volume of each incubation sample was 100 μl, the medium was WME (pH 7.4) and the incubation sample included hepatocytes with a cell density of 1.0x106 cells/ml and TL134 with a final concentration of 3.0 μM; a negative control sample was incubated with thermally inactivated mixed hepatocytes (no cell count) of five animal species and TL134, and a control buffer sample was incubated with WME and TL134. All samples were incubated at 37°C for 180 min and then 100 μl of ice-cold acetonitrile was added to stop the reaction. Samples were stored at -70° C. ready for testing and all incubation samples were duplicate samples.

3. Instruments and conditions

Triple TOF 5600+ (Q-TOF MS) quadrupole time-of-flight tandem mass spectrometer equipped with an electrospray ionization source (ESI source) and CDS automatic calibration system, AB SCIEX, USA; UPLC Acquity liquid chromatography system, including a double infusion pump, autosampler, column thermostat, degasser and TUV ultraviolet radiation sensor, Waters, USA.

Chromatography Conditions: The chromatography column was an HSS T3 C18 ACQUITYTM column (100 x 2.1 mm I.D., particle size 1.8 µm), Waters, USA; the column temperature was 40°C; the flow rate was 0.4 ml/min; the UV detection wavelength was 254 nm; the mobile phase gradient is shown in the table below.

Time (min.) A (5 mM ammonium acetate containing 0.1% formic acid,%) V (acetonitrile, %) 0 90 10 1 90 10 8.5 46 54 9.5 5 95 10.5 5 95 I,5 90 10 15 90 10

Conditions for mass spectrometry: electrospray ionization source (ESI), detection by scanning method in positive ion mode (high sensitivity mode), gas 1: 55 psi, gas 2: 50 psi, gas curtain: 40 psi, source temperature was 500°C, ion sputter voltage (ISVF) was 5500 V with declustering voltage of 80 V, collision energy was 10 eV during full first level scan, and collision energy was 20± 10 eV while scanning the product in positive ion mode, the scanning range was 80-1000 mass/charge, an automatic calibration system (CDS) with an external standard method was used to correct the mass number.

4. Sample pretreatment

Samples in two repetitions of the solution for incubation of hepatocytes of each type were taken in full and combined, mixed on a vortex for 1 min. and centrifuged for 5 min. (14000 rpm), then took the entire amount of the supernatant, transferred into a test tube with a volume of 10 ml and dried in a stream of nitrogen at 40°C. The residue was then dissolved in 150 µl of acetonitrile-water (10:90, v/v), centrifuged for 5 min. (14000 rpm) and 5.0 μl of the supernatant were taken for analysis using UPLC-UV/Q-TOF MS. Negative control samples and samples with control buffer were treated in the same way as samples of the solution for incubation of hepatocytes.

5. Data analysis

The types of software that were used to obtain the data were

- 18 042574

Analyst®TF V1.6 from AB Sciex and Masslynx V4.1 from Waters, and the software types used for data analysis were PeakView® V1.2 and MetabolitePilot V1.5 from AB Sciex.

6. Results of the experiment

The TL134 fluid for incubation of human, monkey, canine, rat and mouse hepatocytes was processed using the MetabolitePilot software to obtain their respective metabolite spectra (FIGS. 1 and 2) and the chromatograms obtained by ultraviolet detection shown in FIG. 3 and 4. Metabolites are named in order of their mass-to-charge ratio from low to large, and metabolites with the same mass-to-charge ratio are named in order of low to high chromatographic retention times, information according to UPLC-UV/Q-TOF MS in relation to TL134 and metabolites in hepatocyte incubation systems is shown in Table. 5.

Metabolic levels of compound GDL5000123 in hepatocytes of both simian and canine species were assessed using the same method and conditions as described above.

The results are shown in table. 6.

Table 5. Information according to UPLC-UV/Q-TOF MS regarding TL134 metabolites in hepatocytes of five species, such as human, monkey, canine, rat and mouse

Metabolic pathway „ Off

Respectively a. ratio Molecular mass to mass formula (ppm)

Peak area according to LC-MS (x10 ³ )

Time Termichesk

UDSRZh I tt gt tg

Human Obesity Pso Rat , , and inactivated Mouse _{z h} century yana high a (min.)

hepatocytes Progogi ^P 712 240 MO drugs C ₃₅ H ₃₃ N ₅ O ₇ F ₄ 2.8 7.37 1750 708 920 832 ISO 1970 ennogo facilities Hydrolysis amide Ml bonds 302.0639 Ci ₃ H ₁₀ NO ₄ F ₃ 1.3 7.53 19.2 8.32 7.95 13.1 10.4 (sour conditions) ABOUT- M2 dealkyl 411.0965 Ci9Hi ₄ N2O ₄ F ₄ 0.6 7.19 16.9 6.71 5.16 12.3 16.2 ing Hydrolysis amide M3 connection 429.1937 C ₂ 2H ₂₅ N ₄ O ₄ F 1.0 4.38 7.93 2.80 2.34 3.70 6.25 (sour conditions) ABOUT- depropyl M4 585.1398 C2 ₈ H ₂ oN ₄ 0 ₆ F ₄ 1.1 8.73 morpholi 33.2 10.5 18.5 20.6 9.03 new rings Oxidize spruce removal M5 ^M °RF ° ^LI 657.1592 C ₃₁ H ₂₄ N ₄ O ₈ F ₄ -1.7 8.74 0.06 3.64 21.7 1.91 0.03 new rings (sour conditions) V- _M6 demethyl _{2249 s} 1.23 ing e One time tnaya M7 ^oxidized 726.2188 C ₃₅ H ₃₁ N ₅ O ₈ F ₄ 0.9 8.98 2.11 2.57 dehydrogen enization One time М8 ™° ^e 728.2345 C ₃₅ H ₃₃ N ₅ O ₈ F ₄ 1.0 7.49 198 19.1 99.7 235 35.5 oxidation e Peak area obtained by UV chromatography Thermally Human Obez Pso Rat Mouse

- 19 042574

MO Prototype drug 7.32 467 406 270 236 315 577

M4 O-depropylation of the morpholine ring/oxidative removal^ * * * * of the morpholine ring (acid conditions)

M8 Single oxidation 7.43 88 13 43 78 18 *: Related metabolites were detected, but peak areas obtained by UV detection could not be accurately integrated due to matrix noise

Table 6. UPLC/Q-TOF MS Information for GDL5000123 Metabolites in Hepatocytes of Both Monkey and Canine Species

Compliant Deviation Time Peak area according to

Metabolic Molecular Niepo Retention LC-MS ( ^x 10 ³ ) Path Massaging Formula Inactivated Monkeys Canids ^J kcharge t g j g ______________________ _________________(ppm) (min.) ragged on e Prototype

MO medicinal product Oxidative 656.2889 SzbN^ObR 1.5 8.07 166 264 218 ml removal of morpholine 601.2078 C32H29N4O7F -2.4 9.43 2.57 5.84 rings M2 Ml Single oxidation 617.2042 C32H29N4O8F 0 7.33 1.19 3.42 M3 Dehydrogenation 654.2721 СзбНзг№О ₆ R -0.2 7.74 1.35 1.01 Single M4 oxidative 670.2673 C36H36N _5O7F 0.2 6.41 1.76 0.92 dehydrogenation M5-1 Single oxidation 672.2835 SzbNzaboch 1.0 5.96 25.8 M5-2 Single oxidation 672.2839 C36H38N5O7F 1.6 6.03 22.4 23.3 M6-1 Double oxidation 688.2780 C36H38N5O8F 0.5 5.20 1.41 M6-2 Double oxidation 688.2778 C36H38N5O8F 0.2 6.19 2.26 M8 oxidative 686.2623 SzbNzbCODE oh, s 6.57 0.88

dehydrogenation Peak area obtained by UV detection Inacti Virova Monkey Canidae MO Prototype medicinal 129 191 144 M5-1 M5-2 products Single oxidation Single oxidation 102 32.3 53.7

Analysis of experimental data

Based on the peak area obtained by UV detection chromatography after incubation of compound GDL5000123 with monkey and canine hepatocytes for 180 min. approximately 41.3% and 27.2% of the prototype drug in each incubation system were metabolized, respectively; based on the peak area obtained by UV detection chromatography after incubation of compound TL134 with human, monkey, canine, rat and mouse hepatocytes for 180 minutes approximately 17.8, 4.6, 15.4, 19.8 and 3 .0% of the prototype drug in each incubation system was metabolized, respectively. It can be seen that the introduction of trifluoromethoxy instead of ethyl in the quinolone molecular moiety can greatly improve the stability of the compound, improve the metabolic stability of the compound and increase the level of effect, thus it can provide an increase in the in vivo drug efficacy of the compound and can provide a reduction in dosage with the same drug effect.

Each of the technical features of the above examples can be combined arbitrarily. To simplify the description, not all possible combinations of each of the technical features in the above examples are described. However, all combinations of such technical features are to be considered to be within the scope of the present invention, provided such combinations do not conflict with each other.

The above examples are merely illustrative of several embodiments of the present invention, which are described specifically and in detail, but should not be construed as limiting the scope of the present invention. It should be noted that several variations and improvements can be made by those skilled in the art without departing from the spirit of the present invention,

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Claims (12)

and all of them are within the scope of legal protection of the present invention. Therefore, the scope of legal protection of the present invention will be determined by the appended claims. CLAIM 1. A quinoline or quinazoline-based compound having the structure represented by formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof: I (I) where X is selected from CH and N; each of R ₁ and R2 is independently selected from -O(CR4R ₅ ) _o R ₃ ; where o is an integer from 0 to 6; each of R ₃ , R4 and R ₅ is independently selected from the group consisting of -H, C ₁ -C ₆ alkyl, -OR ₈ and -NR ₈ R9; each of R ₈ and R ₉ is independently selected from C ₁ -C ₆ alkyl, or R ₈ and R9 together with N attached to them form a saturated or unsaturated 5-6 membered heterocyclic group containing 1-4 heteroatoms selected from O, N and S; where a saturated or unsaturated 5-6 membered heterocyclic group may be independently and optionally substituted with one or more R ₁₀ ; Where R ₁₀ is selected from the group consisting of C ₁ -C ₆ alkyl. 2. Connection based on quinoline or quinazoline according to claim 1 or its pharmaceutically acceptable salt or its stereoisomer, where R1 represents-O(CH ₂ ) _o R ₃ ; o is an integer from 0 to 4; R3 is selected from the group consisting of -H, C ₁ -C ₆ alkyl, C ₁ -C ₃ alkoxy and -NR8R9; each of R ₈ and R 9 is independently selected from the group consisting of C ₁ -C ₃ alkyl, or R ₈ and R 9 together with N connected to them form a saturated or unsaturated 5-6 membered heterocyclic group containing 1-4 heteroatoms selected from O, N and S; where a saturated or unsaturated 5-6 membered heterocyclic group may be independently and optionally substituted with one or more R10; Where R ₁₀ is selected from the group consisting of C ₁ -C ₃ alkyl. 3. A quinoline or quinazoline compound according to claim 2, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R ₁ is selected from the group consisting of methoxyl, ethoxyl, propoxyl, 2methoxyethoxyl, 3-methoxypropoxyl, 3-morpholinopropoxyl, 2-(pyrrolidine -1-yl)ethoxy, 3(pyrrolidin-1-yl)propoxyl, (piperidin-1-yl)ethoxyl, (piperidin-1-yl)propoxyl, 4-methoxybutoxyl, 2-morpholinoethoxyl, (4-methylpiperazin-1- yl) propoxy, dimethylaminoethoxy and isopentyloxy. 4. Compound based on quinoline or quinazoline according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, where R2 is -O(CH ₂ ) _o R ₃ ; o is an integer from 0 to 4; R ₃ is selected from the group consisting of -H, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy and -NR8R9; R ₈ and R 9 together with N attached to them form a saturated 5-6 membered heterocyclic group containing 1-4 heteroatoms selected from O, N and S. 5. A quinoline or quinazoline compound according to claim 4, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein _R2 is selected from the group consisting of methoxyl, ethoxyl, propoxyl, 2methoxyethoxyl, 3-methoxypropoxyl, 2-morpholinoethoxyl and 3-morpholinopropoxyl. 6. A quinoline or quinazoline based compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein X is N. 7. A quinoline or quinazoline based compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the quinoline or quinazoline based compound is selected from the group consisting of N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1 ,4-dihydroquinoline-3-carboxamide, N-(4-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)oxy)-3-fluorophenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1,4 -dihydroquinoline-3-carboxamide, N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)1,4-dihydroquinoline-3-carboxamide, N-(3-fluoro-4-((7-methoxy-6-(3-methoxypropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide, N-(3-fluoro-4-((6-methoxy-7-(4-methoxybutoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide, - 21 042574 N-(3-fluoro-4-((6-methoxy-7-(2-morpholinoethoxy)quinαzolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide, N-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo- 6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide, N-(3-fluoro-4-((6-methoxy-7-(2-(piperidin-1-yl)ethoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4oxo-6- (trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide, N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)oxy)phenyl)-1,2dimethyl-4-oxo- 6-(trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide, N-(3-fluoro-4-((6-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4oxo-6- (trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide, N-(3-fluoro-4-((6-methoxy-7-(3-methoxypropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide, N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl4-oxo-6- (trifluoromethoxy)-1,4-dihydroquinoline-3-carboxamide, N-(4-((7-(2-(dimethylamino)ethoxy)-6-methoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-1,2-dimethyl-4oxo-6-(trifluoromethoxy)-1, 4-dihydroquinoline-3-carboxamide, N-(3-fluoro-4-((7-(isopentyloxy)-6-methoxyquinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1,4- dihydroquinoline-3-carboxamide, N-(3-fluoro-4-((6-methoxy-7-propoxyquinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline- 3-carboxamide, N-(4-((7-ethoxy-6-methoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1,4-dihydroquinoline-3 -carboxamide, N-(3-fluoro-4-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo-6-(trifluoromethoxy)-1 ,4-dihydroquinoline-3-carboxamide and N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-1,2-dimethyl-4-oxo6-(trifluoromethoxy)-1,4 -dihydroquinoline-3-carboxamide. 8. Use of a quinoline or quinazoline based compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt or stereoisomer thereof, as an AXL kinase inhibitor. 9. Use of a quinoline or quinazoline based compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt or stereoisomer thereof, as an inhibitor of Flt3 kinase. 10. The use of a quinoline or quinazoline based compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt or stereoisomer thereof, for the preparation of a medicament for the prevention or treatment of a tumor. 11. Use according to claim 10, wherein the tumor is a hematological tumor, gastrointestinal stromal tumor, histiocytic lymphoma, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, pancreatic cancer, breast cancer, prostate cancer, cancer liver, skin cancer, epithelial cell carcinoma or nasopharyngeal carcinoma. 12. Pharmaceutical composition for the prevention or treatment of a tumor, containing an active ingredient and a pharmaceutically acceptable excipient, where the active ingredient provides a compound based on quinoline or quinazoline according to any one of claims 1 to 7 or a pharmaceutically acceptable salt or its stereoisomer. -